CA1262862A - Tetrapyrrole therapeutic agents - Google Patents
Tetrapyrrole therapeutic agentsInfo
- Publication number
- CA1262862A CA1262862A CA000487040A CA487040A CA1262862A CA 1262862 A CA1262862 A CA 1262862A CA 000487040 A CA000487040 A CA 000487040A CA 487040 A CA487040 A CA 487040A CA 1262862 A CA1262862 A CA 1262862A
- Authority
- CA
- Canada
- Prior art keywords
- porphyrin
- amino acid
- therapeutic composition
- process according
- composition according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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- 229940124597 therapeutic agent Drugs 0.000 title description 3
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- 230000001225 therapeutic effect Effects 0.000 claims abstract description 50
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- 239000002253 acid Substances 0.000 claims abstract description 27
- 125000003277 amino group Chemical group 0.000 claims abstract description 10
- 239000004952 Polyamide Substances 0.000 claims abstract description 6
- 239000003937 drug carrier Substances 0.000 claims abstract description 6
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- 238000001514 detection method Methods 0.000 claims abstract description 5
- -1 -CHCH3 Chemical group 0.000 claims description 101
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- 229950003776 protoporphyrin Drugs 0.000 claims description 20
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- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 150000002680 magnesium Chemical class 0.000 description 1
- 239000000391 magnesium silicate Substances 0.000 description 1
- 229940099273 magnesium trisilicate Drugs 0.000 description 1
- 229910000386 magnesium trisilicate Inorganic materials 0.000 description 1
- 235000019793 magnesium trisilicate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 206010028320 muscle necrosis Diseases 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 208000018389 neoplasm of cerebral hemisphere Diseases 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000037311 normal skin Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000007968 orange flavor Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 201000001219 parotid gland cancer Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 150000004033 porphyrin derivatives Chemical class 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- UHSGPDMIQQYNAX-UHFFFAOYSA-N protoporphyrinogen Chemical compound C1C(=C(C=2C=C)C)NC=2CC(=C(C=2CCC(O)=O)C)NC=2CC(N2)=C(CCC(O)=O)C(C)=C2CC2=C(C)C(C=C)=C1N2 UHSGPDMIQQYNAX-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- JLUIOEOHOOLSJC-UHFFFAOYSA-N pyrrole-2,5-dicarboxylic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)N1 JLUIOEOHOOLSJC-UHFFFAOYSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- ZLGIYFNHBLSMPS-ATJNOEHPSA-N shellac Chemical compound OCCCCCC(O)C(O)CCCCCCCC(O)=O.C1C23[C@H](C(O)=O)CCC2[C@](C)(CO)[C@@H]1C(C(O)=O)=C[C@@H]3O ZLGIYFNHBLSMPS-ATJNOEHPSA-N 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- 235000020374 simple syrup Nutrition 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical class S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
- 238000010518 undesired secondary reaction Methods 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 239000009637 wintergreen oil Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0036—Porphyrins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
- C07K5/06113—Asp- or Asn-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0819—Tripeptides with the first amino acid being acidic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
ABSTRACT OF THE DISCLOSURE
A therapeutic composition for detection and/or treatment of mammalian tumors which comprises a fluorescent mono- or polyamide of an amino dicarboxylic acid and a tetrapyrrole containing at least one carboxy group of the structure:
A therapeutic composition for detection and/or treatment of mammalian tumors which comprises a fluorescent mono- or polyamide of an amino dicarboxylic acid and a tetrapyrrole containing at least one carboxy group of the structure:
Description
NEW TETRAPYRROLE T~IERAPEUTIC AGENTS
This invention relates to new therapeutic compositions which are useful in photodiagnosis and phototherapy, especially in the detection and treatment of tumors and cancerous tissues in the human or animal body.
It is ~nown to irradiate tumors and cancerous tissues in the human body with intensive light ~ollowing administration of a hematoporphyrin derivative in the wavelength range of 626 to 636 namometers to reduce and, at times, destroy the cancerous cells (see PCT published specification WO 83/00811). It is also known that porphyrins, especially the sodium salt of protoporphyrins, can maintain or promote the normal functions of cells and are useful for preventing the genesis, growth, metastasis, and relapse of malignant tumors. Japanese Published Patent Application No. 125737/76 describes the use of porphyrins as tumor inhibiting agents, exemplifying etioporphyrin, mesoporphyrin, protoporphyrin, deuteroporphyrin, hematoporphyrin, coprophyrin, and uroporphyrin.
In Tetrahedron Letters No. 23, pp. 2017-2020 (1978), there is described an amino monocarboxylic acid adduct of the piyment bonellin obtained by extraction of principally the body wall of the marine echuroid B. viridis. The structure o~
these adducts is presumed to be an amide formed through either of the free carboxy ~roups of bonellin and the amino mono-carboxylic acid. Hydrolysis of the adduct yielded a mixture ~2~
oE valine, isoleucine, leucine and alloisoleucine. Mo use for these amino acid adducts is described in this reference.
That the tetrapyrroles cause intense photo-sensitivity in animals is well-known and has been documented in numerous articles in literature, e.y., J. Intr. Sci.
Vitaminol, 27, 521-527 (1981); Agric. Biol. Chem., 46(9), 2183-2193 (1982); Chem. Abst. 98, 276 (1983) and 88, 69764m (1928).
The therapeutic agents contemplated by this invention are cyclic and acyclic tetrapyrroles derived by various procedures from naturally-occurring tetrapyrroles.
The cyclic tetrapyrroles have as their common parent tetrapyrrole, uroporphyrinogen, and posses the following ring structure:
lg 20 ~ 3 6 ~ 7 14 l~l C 11 9 12 ll ~z~
in which the positions in the molecule are numbered 1-20, and the rings identified by letters A, B, C and D, and also include perhydro-, e.g., dihydro- and tetrahydro-, derivatives of the said ring structure, e.g.l compounds in which one or more double bonds are absent. There are present in the ring system four pyrrole rings joinecl through the alpha positions of the respective pyrrole rings by a methine group, i.e., -CH=. The compounds of the present invention are designated as derivatives of the tetrapyrroles for convenience in the disclosure and the appended claims and it will be understood that the term "tetrapyrrole" will designate compounds of the characteristic ring structure designated hereinbe~ore as well as the corresponding perhydro derivatives, and the corresponding non-cyclic pyrroles, i.e., the linear tetrapyrroles, commonly known as the bile pigments.
The tetrapyrroles employed in the present invention are all derived by various means and various alteration procedures from natural tetrapyrroles. The naturally occurring tetrapyrroles have as their common ancestor uroporphyrinogen III, a hexahydroporphyrin reduced at the bridge positions. For example, synthetic or biosynthetic derivatives or products of protoporphyrins IX or proto-porphyrinogen IX are well-known in the art (see, for example, Porphyrins and Metalloporphyrins, K. Smith Elsivier; The Porphyrins (Vols. 1-7) D. Dolphin, Academic Press; and Biosynthetic ~athways~ Vol. III, Chapter by B. Burnham, editor D.M~ Greenberg, Academic Press~.
'`~.
~26;Z;~36~
The non-cyclic tetrapyrroles are commonly known as bile pigments and include, for example, bilirubin and biliverdin. These tetrapyrroles are also derived from protoporphyrin, e.g., as metabolic products in animals.
A ~urther characteristic of the present new therapeutic composition is the presence of at least one amide linkage in a substituent at any of the numbered positions of the ring structure. These are present in the instant new compounds together with other substituents as defined hereinafter~
Thus, the present invention contemplates the therapeutic compositions comprising of amino acid or peptide derivatives of compounds which contain chromosphore of porphyrins, chlorins or bacteriochlorins, as well as related porphyrin compounds. The peptide linkage involves a carboxy group of the chromophore-bearing compound and the amino group of the specified amino acid. The present new compounds embrace, inter alia, derivatives of the tetrapyrroles which contain a free carboxy group. These derivatives include the major classes of tetrapyrroles: carboxy containing porphyrins, chlorins, and bacteriochlorins, which are well-known to those skilled in this art.
The amino acid employed in the present invention to form the aforesaid peptide linkage are amino-dicarboxylic acids in which the amino group, of course, is located z~z --5~
on a carbon atom of the dicarboxylic acid. The specific position of the amino group in the carbon atom chain is not critical, the only requirement being that the amino group be available to form the requisite peptide linkage with the carboxyl group o~ the selected porphyrin. Thus, a variety of amino dicarboxylic acids are useful in the composition of the present invention, including ~-aminosuccinic (aspartic),~C -aminoglutaric (glutamic), beta-aminoglutaric~ beta-aminosebacic, 2,6-piperidinedicarboxylic, 2,5 pyrrole-dicarboxylic, 2-carboxypyrrole-5-acetic, 2-carboxy-piperidine-6-propionic, oC-aminoadipic, ~-aminoazelaic, and similar such acids. These amino acids may be substituted with angular alkyl groups such as methyl and ethyl groups, as well as other groups which do not adversely affect the capability of the amino group to form the peptide linkage, e.g., alkoxy groups or acylo~y groups, and may also include additional amino groups. The preferred amino acids are the naturally occurring cc-amino acids, glutamic and aspartic acids, which are readily available and, up to the present, have provided the best results.
Exemplary compounds of the tetrapyrrole classes are illustrated in Table I in which the numbered positions of the tetrapyrrole ring structure are used to designate the position of the indicated substituent. The absence of double bonds in the ring system is designated under "dihydro" with each set o~
numbers (ring position~ indicating the absence of a double bond between the designated positions.
~iZ8~%
a ,c' c~ ~, ,a~ ci c~J a~ c' c~ c u~ L~ L~ Ll L~ L~ L~ Ll L~ L~ L~ .
~ ~; ~ 8 r~ ~ ~ h ~ ~ C)=o P~ =O
O C) 'v O ~1 ~ ~ ~ Q ~ Q\ _.
.z r~ S~ v J~
~ i~ --o a~
H ~C) C`; C) a) ~ Q'; '-- Q`; C) C'; ~ e~
~ ~ ~ 0 ~
~ r~
~ LI Q, ~ _ ~
I~ O
~1 s~ Q
. ~ .
H H X ,~ H ~ .
' '~ ,~ ~,, O h ~ H Q~
7. ~, o L~ ~ v ,~ '~ O O R
' C)- o h O ~ O tlJ tr) h ~ r~ 0 1~4 0 ) I ~ Q
:~ ~i. L~ o U, ~ O ~ ~ l~i li j-- o cJ nS v J~ Qo~ o tl) ~ ~ 5 ~ h ~ ~
86~2 Q
_ , W
~ ~ ~ ~ c. X C~
_ ~ ~ ~ ~~
~ ~ _a~ ~
~ ~ o ~
c ~ 8 8 ~ ~ 8 -u=o 8 o C~
o ~ ~
' ~
n~
~! n ~ n ~ ~D
_ a) O
a~ u e _t o o u ~ ~ ~ o 6Z~
-'d~
r I a) 9 --, I ~ ~ "
_ ~ C. 3: G
q~ ~ ,~
_~
, 8 3 ~, ~ .
o ~ - 5 G
~ C~ ~_ ~J ~ ' Q.
_ T ~ I '~
9 ~ ~ ~ ~ , H ~ 4 ~ Q~ O 'O ~ ~
O
r~
~i~ i5~ rr' ~ t~l 11 ~`I ~ ~
~ y ~ ~ ~
a~ -41 r~ O ~ ~ ~
Z
~ O O
3: . 4 .
~ ~ G Z
,: _9 2B6;Z:
Thc Prcscnt ncw ttlerapeutic composition is comprised of mono~ or polyamides of an aminodicarboxylicAand a tetrapyrrole containirlg at lcast one~ carbo~:yl grou? of the st~ucture ~ 0~
H )n ~hercin Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive.
The particularly preferred com?ounds are fluorescent mono- or polyamides of an aminodicarboxylic acid and a tetrapyrrole com?ound of the formula:
~ \tl ~ ~ R3 ~ NH HN ~ ~ R5 ¦ _ I Rg .
or the eorr2sponding di~ or tetrahydrotetrapyrroles wherein -H -OH
Rl is methyl;{ CH { -CH3; H
~-H
R2 is H, vinyl, ethyl, -CHCH3, acetyl,~ -C=O, CH2CH2CO2H, or =CHCHO;
OH
~ r -CH
R is methyl ~r or 3 ~ -CH3 -OH;
R4 is H, vinyl, ethyl, -CHCH3 , CH2CH2CO2H, =CHCHO; or OH
R5 is methyl;
R6 is H, CH2CH2CO2Ht CH2CH2CO2R or CO2H;
-cH2cH2co2H
~7 is CH2CH2CO2H, CH2CH2CO2R, or { H
. -CH
4~ R8 is methyl or ~ 3 Rg is H, COO~, C~2COOH or methyl;
-lOA-provided that when R1 , R2 r R3 , R4 , R7 and R~ represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
--C=O -C=O
R6 and Rg, taken together are -CH2 or -CHC02CH3 with the proviso that at least one of Rl-Rg includes a free carboxyl group; and salts thereof.
~6;~8~;~
1 The especially preferred therapeutic compositions of the invention are comprised of amides which are derived from tetrapyrroles of the formula:
~ \ N ~ ~ ~ R3 R ~ ~ ~r ~R4 Rg ¦ C ¦
~6 ~R5 or the corresponding di- or tetrahydrotetrapyrroles and salts thereof, wherein Rl - Rg are as previously defined.
;
X!.
:~2~ ;2 Particularly preferred therapeutic agents of this invention include the following compounds:
Chlorin Derivatives Mono and diaspartyl trans-mesochlorin IX
Mono and diglutamyl trans-mesochlorin IX
Mono, di and triaspartyl chlorin e6 Mono, di and triaspartyl mesochlorin e6 Mono, di and triglutamyl chlorin e6 Mono, di and triglutamyl mesochlorin e6 Mono and diaspartyl chlorin e4 Mono and dia~oartyl mesochlorin e4 Mono and diaspartyl isochlorin e~
Mono and diaspartyl mesochlorin e4 Mono and diglutamyl chlorin e4 Mono and diglutamyl mesochlorin e4 Mono and diglutamyl isochlorin e~
Mono and diglutamyl mesoisochlorin e4 Monoaspartyl pyropheophorbide a Monoglutamylpyropheophorbide a Monoaspartylpheophorbide a Monoglutamylpheophorbide a Mono and diaspartylphotoprotoporphyrin IX
Mono and diglutamylphotoprotoporphyrin IX
Mono and di-L-alpha-aminoadipyl trans-mesochlorin IX
Porphyrins Derivatives Mono and diaspartylmesoporphyrin IX
Mono and diglutamylmesoporphyrin IX
Mono and diaspartylprotoporphyrin IX
Mono and diglutamyl protoporphyrin IX
Mono and diaspartyldeuteroporphyrin IX
Mono and diglutamyldeuteroporphyrin IX
Mono, di, tri and tetraaspartylcoproporphyrin III (isomer mixture) Mono, di, tri and tetraglutamylcoporphyrin III
Mono and diaspartylhematoporphyrin IX
Mono and diglutamylhematoporphyin IX
Bacteriochlorin Derivatives Mono and diaspartylbacteriochlorin e4.
Mono and diglutamylbactcriochlorin e4 Mono and diaspartylbactcrioisochlorin e4 Mono and diglutamylbactcrioisoch:Lori.n e4.
Mono, di and triaspartylbacteriochlorin e6 Mono, di and triglutamylbacteriochlorin e6 Monoaspartylpyrobacteriopheoohorbide a Monoglutamylpyrobacteriopheophorbide a Monoaspartylbacteriopheophorbide a Monoglutamylbacteriopheophorbide a ~2l~6~
The aforesaid compounds form salts with either acids or bases. The acid salts are particularly useful for purification and/or separation of the final amide products as are the salts formed with base. The base salts, however, are particularly preferred for diagnostic and therapeutic use as hereindescribed.
The acid salts are formed with a variety of acids such as the mineral acidsv hydrochloric, hydrobromic, nitric and sulfuric acids, organic acids such as toluenesulfonic and benezenesulfonic acids.
The base salts include, for example, sodium, potassium, calcium, magnesium, ammonium, triethylammonium, trimethylammonium, morpholine and piperidine salts and similar such salts.
The acid and base salts are formed by the simple expediency of dissolving the selected amino acid tetrapyrrole amide in an aqueous solution of the acid or base and evaporation of the solution to dryness. The use of a water-miscible solvent for the amide can assist in dissolving the amide.
The final amide products can also be converted to metal complexes for example b~ reaction with metal salts.
The magnesium complexes may be useful for the same purpose as the adduct product. Other metal complexes, as well as the magnesium complex, including, for example, iron and zinc, are useful to preclude contamination during processing of the adduct product by metals such as nickel, cobalt and copper, which are difficult to remove. Zinc and magnesium are readily removed fro~ the final adduct product after prccessing is completed.
~L2~
Since many of the aminodicarboxylic acids exist in both the D- and L-forms, and also are employed in mixtures of these forms as well as the D,L-form, the selection of the starting amino acid will, of course, result in products in which the respective isomer or mixture of isomers exist. The present invention contemplates l:he use of all such isomers, but the L-form is particularly pre~erred.
The aforesaid compounds are prepared by the usual peptide synthetic routes which generally include any amide-forming reaction between the selected amino acid and the specific tetrapyrrole. Thus, any amide-forming derivative of the tetra-pyrrole carboxylic acid can be employed in producing the present new peptides, e.g., lower alkyl esters, anhydrides and mixed anhydrides.
The preferred preparative methods use mixed anhydrides of the carboxylic acid or carb~diimides. The reactants are merely contacted in a suitable solvent therefor and allowed to react. Temperatures up to the reflux temperature can be used, with the higher temperatures merely reducing the reaction time. Excessively high temperatures are usually not preferred so as to avoid unwanted secondary reactions however.
The procedures for forming the instant peptides are well known in this art and are provided in detail in the accompanying examples.
When the selected tetrapyrrole contains more than one carboxyl group, then mixtures of products can be formed including isomeric monopeptide products and di and even tri-or higher peptide products, depending on the number of '.~
carboxyl groups and depending on the selected stoichiometry.
Thus, when equimolar mixtures of amino acid and tetrapyrrole are reacted, not only monopeptides but also dipeptides are obtained, although the monopeptide would predominate. With higher molar ratios, the nature of the products will similarly vary. It is generally possible to separate the monopeptides and higher peptides using Icnown chromatographic techniques.
However, such separations are not necessary since the mixed peptides are usually comparable to the separated products in their ultimate use. Thus, mixtures of the mono-, di~ and tri~
peptides of the same tetrapyrrole can be used.
Usually, unreacted tetrapyrrole is separated from the peptide products of the invention during puri-fication as, for example, by chromatographic techniques.
Photodiagnosis and Phototherapy The compositions of the present invention are useful for the photodiagnosis and phototherapy of tumor, cancer and malignant tissue (hereinater referred to as "tumor").
When a man or animal having tumor is treated with doses of a compound of the present invention and when appropriate light rays or electromagnetic waves are applied, the compound emits light, i.e., fluorescence. Thereby the existence, position and size of tumor can be detected, i~e., photodiagnosis.
When the tumor is irradiated with light of proper wavelength and intensity, the compound i9 activated to exert a cell killing effect against the tumor. This is called "phototherapy".
'.~'~, 86;~
Compounds intended for photodiagnosis and phototherapy ideally should have the fol~owing properties:
(a) non-toxic at normal therapeutic dosage unless and until activated by light;
(b)should be selectively photoactive;
(c) when light rays or electromagnetic waves are applied, they should emit characteristic and detectable fluorescence;
(d) when irradiated with light rays or electromagnetic waves are applied, they are activated to an extent to exert a cell killing effect against tumor; and (e) easily metabolized or excreted after treatment.
In accordance with testing up to the present, the compounds of the present new therapeutic compositions have the foregoing properties and are also characterized by reasonable solubility in water at physiological pH.
The aforesaid compounds possess greater fluorescence in tumors than do the corresponding basic tetrapyrroles, and even peptides formed with amino monocarboxylic acias, e.g., alanine and epsilon aminocaproic acid. Their use provides the best contrast in tumors compared to normal tissue around the tumor. The instant compounds absorb activating energy for phototherapy in the convenient range of 600 to 800 nanometers, with the preferred compounds absorbing in the 620-760 nanometer range, i.e., light of longer wavelengths which more readily permits penetration of energy into the tumor for phototherapeutic purpose.
In present experience, the present compounds more uniformly distribute throughout the tumor than the basic ~etrapyrrole permitting the use of considerably lower ~2~ 62 dosage (to about l/lOth of the required normal dose of the basic tetrapyrrole) which lessens, if not eliminates, photosensitization in the host. They also possess a more consistent fluorescence whereas some of the corresponding tetrapyrroles show inconsistent fluorescence or the fluorescence varies from day to day in the host.
A particularly advantageous property of the present compounds resides in the ease with which they are excreted by the host. Generally, within 48 to 72 hours of intravenous or intraperitonal administration, there are little or no detectable amounts in normal muscle tissue. The present compounds which are excreted with their chromosphore intact are recovered from -the feces of the host within 48-72 hours of injection. Under equivalent circumstances, substantial amounts of the corresponding tetrapyrroles remain, as compared with only minor amounts of peptides formed with the amino monocarboxylic acids remain in the host, e.g., up to about 20%. This Property is extremely important in that it contributes to minimization of photosensitization of the host.
The instant composition can be used for diagnosis and therapeutic treatment of a broad range of tumors.
Examples of tumors are gastric cancer, enteric cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcomas, hepatic cancer, cancer of the urinary bladder, cancer of the upper jaw, cancer of the bile duct, cancer of the tongue, cerebral tumor, skin cancer, malignant goiter, prostatic cancer~ cancer of the parotid gland, Hodgkins's disease, multiple myeloma, renal cancer, leukemia, and malignant lymphocytoma.
~Z6~`Z~36~
For diagnosis, the sole requirement is that the tumor be capable of selectively fluorescing when exposed to proper light. For treatment, the tumor must be penetrable by the activation energy. For diagnosis, light of shorter wavelength is used whereas for therapeutic purposes light of longer wavelength is used to permit read~ penetration of the tumor tissue. Thus, for diagnosis, light of from 360-760 nanometers can be used, and for treatment, from 620 to 760, depending on the individual characteristics of the tetrapyrrole. The absorption characteristics of the present new compounds are substantially the same as the tetrapyrrole from which derived.
It is necessary that the light rays be so intense as to cause the compounds to emit fluorescence for diagnosis and to exert a cell killing effect for therapy.
The source of irradiation for photodiagnosis and phototherapy is not restricted, however, but the laser beam is preferable because intensive light rays in a desired wavelength range can be selectively applied. For example, in photodiagnosis, the compound of the invention is administered to a human or animal body, and after a certain period of time, light rays are applied to the part to be examined. When an endoscope can be used for the affected part, such as lungs, gullet, stomach, womb, urinary bladder or rectum, it is irradiated using the endoscope, and the tumor portion selectively emits fluorescence. This portion is observed visually, or observed through an adapted fiber scope by eye or on a CRT screen.
`~ ' In photothe~apy, a~ter administration of the dosage, the irradiation is carried out by laser beams from the tip of quartz fibers. sesides the irradiation of the sur~ace of tumor, the internal part of the tumor can be irradiated by inserting the tip of quartz ~ibers into the tumor. The irradiation can be visually observed or imaged on a CRT screen.
For photodiagnosis, light of wavelengths between 360 and 760 nm. is suitable for activating the present tetrapyrrole compounds. Of course, each compound has a specific optimal wavelength of activation. A long wavelength ultraviolet lamp is particularly suitable Eor photodiagnosis. Similar methods for viewing of the treated tumor can be used as already described for phototherapy.
The dosages of compounds having the present new composition will vary depending on the desired effect, whether for diagnosis or or treatment. For diagnosis, doses of as little as 1 mg/kg will be effective, and up to about 20 mg/kg can be used. For treatmen-t, the dose will usually approximate about 0.5 mg/kg. Of course, the dosage for either diagnosis or treatment can be varied widely in view of aforesaid advantageous properties of the present compounds, e.gO, the ease of elimination from the host, for one.
The present compounds are apparently nontoxic at the dosage levels employed for diagnosis or treatment. No mortality of test animals due the present compounds has been noted in studies employing dosage levels up to 20mg/kg.
For both diagnosis and treatment, the present compounds can be administered by the oral, intravenous, or intramuscular routes. They can be formulated as lyophilized sterile, pyrogen-free compounds, preferably in the form of basic salts, e.g., sodium salt. The preferred dosage forms are provided as injectable solutions (isotonic) ..~
.~.
z~
The irradiation source used in treatment of tUMOrs containing compounds o this invention is a filtered, high-intensity, continuous source or pumped dye, or other laser andlight delivery system, which is capable of performing within the following limits: power intensity 20-500 mw/cm2 at wavelengths between 620 and 760 nm. and a total output of at least 500 mw or greater. Several currently commercially available lasers meet these criteria.
The tetrapyrroles can be prepared by various synthetic methods which are found in the literature, e.g., Pheophorbides Willstatter, R., Stoll, A.; Investigations on Chlorophyll, (Transl. Schertz, FM.M., Merz, A.R.) p. 249. Science Printing Press, Lancaster, Pennsylvania, 1928.
Pennington, F.C., Strain, H.H., Svec, W.~., Katz, J.J.; J.
Amer. Chem. Soc., 86, 1418 ~1964).
-Chlorin e6 Willstatter, R., Stoll, A.; Investigations on Chlorophyll, ~Trans., Schertz, F.M., Merz, A.R.,) p. 176. Science Printing Press, Lancaster, Pennsylvania, 1928.
Willstatter, R., Isler, M.; Ann. Chem., 390, 269 (1912).
Fisher, H., Baumler, R.; Ann. Chem., 474, 65 (1929).
Fisher, H., Siebel, H.; Ann. Chem., 499, 84 (1932).
-Conant, J.B., Mayer, W.W.; J. Amer. Chem. Soc.l 52, 3013(1930) ~2~
Chlorin e4 Fisher, H., Heckmaier, J., Plotz, E.; Justus Leibigs Ann.
Chem., 500 215 (1933).
Chlorin e6, e4, isochlorin e~, mesochlorin e6, bacterio-.
pheophorbide, bacteriochlorin e6 Fischer and Orth, "Des Chemie des Pyrrole" Akademische Verlazsgesellschaft, Leipzig, 1940, Vol. II, Part 2.
General Reference for Porphyrirls "Porphyrins and Metalloporphyrins" ed. Kevin M. Smith, Elsevier 1975 N.Y.
:~2~ %
-23~
The compounds of the present invention can be administered to the host in a variety of forms adapted to the chosen route of administration, i.e., orally, intraveneously, intramuscularly or subcutaneous routes.
The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1~ of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
Pre~erred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 50 and 300 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
:~2~i2~3~%
When the dosage unit ~orm is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a cye and flavoring such as cherry or orange flavor~ Of course~ any material used in preparing any dosage unit Eorm should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and Eormulations.
The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability existsO It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, ~or example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the .
~;i2~3f;;~
use of surfactants. rrhe pre~7ention of the action of micro-organisms can be brought about by varlous antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid~ thimerosal, and the like. ln many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption o~ the injectable compositions can be brought about by the use in the compositions of a~ents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile ~0 injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
The present new compounds may also be applied directly to tumors, whether internal or external, in the host in topical compositions. Exemplary compositions include solutions of the new compounds in solvents, par~icularly aqueous solvents, most preferably water. Alternatively, for topical application particularly to skin tumors, the present new compounds may be dispersed in the usual cream or salve formulations commonly used for this purpose or may be provided in the form of spray solutions or suspensions which may include a propellant usually employed in aerosol preparations.
~o~
As used herein, "pharmaceutically acceptable carrier" ineludes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known the art. Except insofar as any conventional media or agent is incompatable with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into ~he compositions.
It is espeeially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjeets to be treated; eaeh unit eontaining a predetermined quantity of active material ealeulated to produee the desired therapeutie effeet in assoeiation with the required pharmaeeutieal earrier. The speeifieation for the novel dosage unit forms of the invention are dietated by and direetly dependent on (a) the unique eharaeteristies of the aetive material and the partieular therapeutie effeet to be aehieved, and (b) the limitations - inherent in the art of eompounding sueh an aetive material for the treatment of tumors in living subjeets.
......
.28~
Di ~D,L) aspart~l transmesochlorin IX_(Carbodiimide Method) 1~0 mg of transmesochlorin and 200 mg of (D,L) aspartic acid dimethyl ester hydrochloride were dissolved in 30 ml of dimethyl formamide. 300 mg of N,NI~dicyclohe~yl-carbodiimide was added. The reaction was allowed to stand for one hour, then another 300 mg of carbodiimide was added. This procedure was repeated twice and then the reaction mixture was allowed to stand overnight. The reaction may be monitored by thin layer chromatography on silica, using solvent benzene/methanol/88% formic acid 8.5/1.5/0.13 V/V/V.
The disubstituted chlorin has the highest Rf value, the unsubstituted chlorin has the lowest, with the mono-substituted isomers in between and unresolved.
After standing overnight, the reaction rnixture appeared to contain at least 50% of the disubstituted chlorin.
The solvent was removed under vacuum and the remaining solid dissolved in 50 ml of 3N ~Cl.
The solution was allowed to stand at room temperature for 48 hours to hydrolyze the ester groups, then the chlorin mixture was precipitated at pH 2.5-3 and collected and washed with water at the centrifuge.
The chlorin mixture was purified by dissolving in 0.05 M NH40H and applying to a reverse phase (C-18 silica) column 2.5 cm X 30 cm. The elution procedure is a linear gradient from 40 to 70% methanol in 0.01 M KP04 buffer pH 6.85 (1 liter tctal volume).
The leading green band (di D~ L aspartyl trans-mesochlorin IX) was collected and flash evaporated to remove the methyl alcohol, the solution then precipitated at pH 2.5-3 and collected and washed 3 times at the centrifuge with dilute acetic acid. The product was dried under vacuum. The yield was 67 mg of cli (D,L) aspartyl transmesochlorin IX.
~2~i2~
1 EXA~PLE 2 yl tr~nsmesochlolin IX (mixed anhvdride methocl) _ 50 m~ (0.000087 moles) of transmesochlorin IX
~as dissolvcd in l00 ml of tetrahydrofuran (THF). 210 ~1 (0.002 moles) of triethylamine was added wi-th stirring.
After 10 minutes, 195 ~1 (0.00179 moles) of ethyl-chloroforma-te was added. After stirring 10 minu-tes, 50 ml (0.01 moles) oE 0.2 ~I KOH containing 250 mg (0.00169 moles) of (L) glutamic: acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0.13) was used to de~,elop tl;e chromatogram.
Af~er checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mi~ture was resolved using a linear gradient of 40-80% methanol in 0.01 M KPO4 buffer pH 6.85 ~1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (L) glutamyl transmesochlorin IX, mono (L) glutamyl transmesochlorin IX, and unsubstituted transmesochlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
~2~ 2
This invention relates to new therapeutic compositions which are useful in photodiagnosis and phototherapy, especially in the detection and treatment of tumors and cancerous tissues in the human or animal body.
It is ~nown to irradiate tumors and cancerous tissues in the human body with intensive light ~ollowing administration of a hematoporphyrin derivative in the wavelength range of 626 to 636 namometers to reduce and, at times, destroy the cancerous cells (see PCT published specification WO 83/00811). It is also known that porphyrins, especially the sodium salt of protoporphyrins, can maintain or promote the normal functions of cells and are useful for preventing the genesis, growth, metastasis, and relapse of malignant tumors. Japanese Published Patent Application No. 125737/76 describes the use of porphyrins as tumor inhibiting agents, exemplifying etioporphyrin, mesoporphyrin, protoporphyrin, deuteroporphyrin, hematoporphyrin, coprophyrin, and uroporphyrin.
In Tetrahedron Letters No. 23, pp. 2017-2020 (1978), there is described an amino monocarboxylic acid adduct of the piyment bonellin obtained by extraction of principally the body wall of the marine echuroid B. viridis. The structure o~
these adducts is presumed to be an amide formed through either of the free carboxy ~roups of bonellin and the amino mono-carboxylic acid. Hydrolysis of the adduct yielded a mixture ~2~
oE valine, isoleucine, leucine and alloisoleucine. Mo use for these amino acid adducts is described in this reference.
That the tetrapyrroles cause intense photo-sensitivity in animals is well-known and has been documented in numerous articles in literature, e.y., J. Intr. Sci.
Vitaminol, 27, 521-527 (1981); Agric. Biol. Chem., 46(9), 2183-2193 (1982); Chem. Abst. 98, 276 (1983) and 88, 69764m (1928).
The therapeutic agents contemplated by this invention are cyclic and acyclic tetrapyrroles derived by various procedures from naturally-occurring tetrapyrroles.
The cyclic tetrapyrroles have as their common parent tetrapyrrole, uroporphyrinogen, and posses the following ring structure:
lg 20 ~ 3 6 ~ 7 14 l~l C 11 9 12 ll ~z~
in which the positions in the molecule are numbered 1-20, and the rings identified by letters A, B, C and D, and also include perhydro-, e.g., dihydro- and tetrahydro-, derivatives of the said ring structure, e.g.l compounds in which one or more double bonds are absent. There are present in the ring system four pyrrole rings joinecl through the alpha positions of the respective pyrrole rings by a methine group, i.e., -CH=. The compounds of the present invention are designated as derivatives of the tetrapyrroles for convenience in the disclosure and the appended claims and it will be understood that the term "tetrapyrrole" will designate compounds of the characteristic ring structure designated hereinbe~ore as well as the corresponding perhydro derivatives, and the corresponding non-cyclic pyrroles, i.e., the linear tetrapyrroles, commonly known as the bile pigments.
The tetrapyrroles employed in the present invention are all derived by various means and various alteration procedures from natural tetrapyrroles. The naturally occurring tetrapyrroles have as their common ancestor uroporphyrinogen III, a hexahydroporphyrin reduced at the bridge positions. For example, synthetic or biosynthetic derivatives or products of protoporphyrins IX or proto-porphyrinogen IX are well-known in the art (see, for example, Porphyrins and Metalloporphyrins, K. Smith Elsivier; The Porphyrins (Vols. 1-7) D. Dolphin, Academic Press; and Biosynthetic ~athways~ Vol. III, Chapter by B. Burnham, editor D.M~ Greenberg, Academic Press~.
'`~.
~26;Z;~36~
The non-cyclic tetrapyrroles are commonly known as bile pigments and include, for example, bilirubin and biliverdin. These tetrapyrroles are also derived from protoporphyrin, e.g., as metabolic products in animals.
A ~urther characteristic of the present new therapeutic composition is the presence of at least one amide linkage in a substituent at any of the numbered positions of the ring structure. These are present in the instant new compounds together with other substituents as defined hereinafter~
Thus, the present invention contemplates the therapeutic compositions comprising of amino acid or peptide derivatives of compounds which contain chromosphore of porphyrins, chlorins or bacteriochlorins, as well as related porphyrin compounds. The peptide linkage involves a carboxy group of the chromophore-bearing compound and the amino group of the specified amino acid. The present new compounds embrace, inter alia, derivatives of the tetrapyrroles which contain a free carboxy group. These derivatives include the major classes of tetrapyrroles: carboxy containing porphyrins, chlorins, and bacteriochlorins, which are well-known to those skilled in this art.
The amino acid employed in the present invention to form the aforesaid peptide linkage are amino-dicarboxylic acids in which the amino group, of course, is located z~z --5~
on a carbon atom of the dicarboxylic acid. The specific position of the amino group in the carbon atom chain is not critical, the only requirement being that the amino group be available to form the requisite peptide linkage with the carboxyl group o~ the selected porphyrin. Thus, a variety of amino dicarboxylic acids are useful in the composition of the present invention, including ~-aminosuccinic (aspartic),~C -aminoglutaric (glutamic), beta-aminoglutaric~ beta-aminosebacic, 2,6-piperidinedicarboxylic, 2,5 pyrrole-dicarboxylic, 2-carboxypyrrole-5-acetic, 2-carboxy-piperidine-6-propionic, oC-aminoadipic, ~-aminoazelaic, and similar such acids. These amino acids may be substituted with angular alkyl groups such as methyl and ethyl groups, as well as other groups which do not adversely affect the capability of the amino group to form the peptide linkage, e.g., alkoxy groups or acylo~y groups, and may also include additional amino groups. The preferred amino acids are the naturally occurring cc-amino acids, glutamic and aspartic acids, which are readily available and, up to the present, have provided the best results.
Exemplary compounds of the tetrapyrrole classes are illustrated in Table I in which the numbered positions of the tetrapyrrole ring structure are used to designate the position of the indicated substituent. The absence of double bonds in the ring system is designated under "dihydro" with each set o~
numbers (ring position~ indicating the absence of a double bond between the designated positions.
~iZ8~%
a ,c' c~ ~, ,a~ ci c~J a~ c' c~ c u~ L~ L~ Ll L~ L~ L~ Ll L~ L~ L~ .
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Thc Prcscnt ncw ttlerapeutic composition is comprised of mono~ or polyamides of an aminodicarboxylicAand a tetrapyrrole containirlg at lcast one~ carbo~:yl grou? of the st~ucture ~ 0~
H )n ~hercin Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive.
The particularly preferred com?ounds are fluorescent mono- or polyamides of an aminodicarboxylic acid and a tetrapyrrole com?ound of the formula:
~ \tl ~ ~ R3 ~ NH HN ~ ~ R5 ¦ _ I Rg .
or the eorr2sponding di~ or tetrahydrotetrapyrroles wherein -H -OH
Rl is methyl;{ CH { -CH3; H
~-H
R2 is H, vinyl, ethyl, -CHCH3, acetyl,~ -C=O, CH2CH2CO2H, or =CHCHO;
OH
~ r -CH
R is methyl ~r or 3 ~ -CH3 -OH;
R4 is H, vinyl, ethyl, -CHCH3 , CH2CH2CO2H, =CHCHO; or OH
R5 is methyl;
R6 is H, CH2CH2CO2Ht CH2CH2CO2R or CO2H;
-cH2cH2co2H
~7 is CH2CH2CO2H, CH2CH2CO2R, or { H
. -CH
4~ R8 is methyl or ~ 3 Rg is H, COO~, C~2COOH or methyl;
-lOA-provided that when R1 , R2 r R3 , R4 , R7 and R~ represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
--C=O -C=O
R6 and Rg, taken together are -CH2 or -CHC02CH3 with the proviso that at least one of Rl-Rg includes a free carboxyl group; and salts thereof.
~6;~8~;~
1 The especially preferred therapeutic compositions of the invention are comprised of amides which are derived from tetrapyrroles of the formula:
~ \ N ~ ~ ~ R3 R ~ ~ ~r ~R4 Rg ¦ C ¦
~6 ~R5 or the corresponding di- or tetrahydrotetrapyrroles and salts thereof, wherein Rl - Rg are as previously defined.
;
X!.
:~2~ ;2 Particularly preferred therapeutic agents of this invention include the following compounds:
Chlorin Derivatives Mono and diaspartyl trans-mesochlorin IX
Mono and diglutamyl trans-mesochlorin IX
Mono, di and triaspartyl chlorin e6 Mono, di and triaspartyl mesochlorin e6 Mono, di and triglutamyl chlorin e6 Mono, di and triglutamyl mesochlorin e6 Mono and diaspartyl chlorin e4 Mono and dia~oartyl mesochlorin e4 Mono and diaspartyl isochlorin e~
Mono and diaspartyl mesochlorin e4 Mono and diglutamyl chlorin e4 Mono and diglutamyl mesochlorin e4 Mono and diglutamyl isochlorin e~
Mono and diglutamyl mesoisochlorin e4 Monoaspartyl pyropheophorbide a Monoglutamylpyropheophorbide a Monoaspartylpheophorbide a Monoglutamylpheophorbide a Mono and diaspartylphotoprotoporphyrin IX
Mono and diglutamylphotoprotoporphyrin IX
Mono and di-L-alpha-aminoadipyl trans-mesochlorin IX
Porphyrins Derivatives Mono and diaspartylmesoporphyrin IX
Mono and diglutamylmesoporphyrin IX
Mono and diaspartylprotoporphyrin IX
Mono and diglutamyl protoporphyrin IX
Mono and diaspartyldeuteroporphyrin IX
Mono and diglutamyldeuteroporphyrin IX
Mono, di, tri and tetraaspartylcoproporphyrin III (isomer mixture) Mono, di, tri and tetraglutamylcoporphyrin III
Mono and diaspartylhematoporphyrin IX
Mono and diglutamylhematoporphyin IX
Bacteriochlorin Derivatives Mono and diaspartylbacteriochlorin e4.
Mono and diglutamylbactcriochlorin e4 Mono and diaspartylbactcrioisochlorin e4 Mono and diglutamylbactcrioisoch:Lori.n e4.
Mono, di and triaspartylbacteriochlorin e6 Mono, di and triglutamylbacteriochlorin e6 Monoaspartylpyrobacteriopheoohorbide a Monoglutamylpyrobacteriopheophorbide a Monoaspartylbacteriopheophorbide a Monoglutamylbacteriopheophorbide a ~2l~6~
The aforesaid compounds form salts with either acids or bases. The acid salts are particularly useful for purification and/or separation of the final amide products as are the salts formed with base. The base salts, however, are particularly preferred for diagnostic and therapeutic use as hereindescribed.
The acid salts are formed with a variety of acids such as the mineral acidsv hydrochloric, hydrobromic, nitric and sulfuric acids, organic acids such as toluenesulfonic and benezenesulfonic acids.
The base salts include, for example, sodium, potassium, calcium, magnesium, ammonium, triethylammonium, trimethylammonium, morpholine and piperidine salts and similar such salts.
The acid and base salts are formed by the simple expediency of dissolving the selected amino acid tetrapyrrole amide in an aqueous solution of the acid or base and evaporation of the solution to dryness. The use of a water-miscible solvent for the amide can assist in dissolving the amide.
The final amide products can also be converted to metal complexes for example b~ reaction with metal salts.
The magnesium complexes may be useful for the same purpose as the adduct product. Other metal complexes, as well as the magnesium complex, including, for example, iron and zinc, are useful to preclude contamination during processing of the adduct product by metals such as nickel, cobalt and copper, which are difficult to remove. Zinc and magnesium are readily removed fro~ the final adduct product after prccessing is completed.
~L2~
Since many of the aminodicarboxylic acids exist in both the D- and L-forms, and also are employed in mixtures of these forms as well as the D,L-form, the selection of the starting amino acid will, of course, result in products in which the respective isomer or mixture of isomers exist. The present invention contemplates l:he use of all such isomers, but the L-form is particularly pre~erred.
The aforesaid compounds are prepared by the usual peptide synthetic routes which generally include any amide-forming reaction between the selected amino acid and the specific tetrapyrrole. Thus, any amide-forming derivative of the tetra-pyrrole carboxylic acid can be employed in producing the present new peptides, e.g., lower alkyl esters, anhydrides and mixed anhydrides.
The preferred preparative methods use mixed anhydrides of the carboxylic acid or carb~diimides. The reactants are merely contacted in a suitable solvent therefor and allowed to react. Temperatures up to the reflux temperature can be used, with the higher temperatures merely reducing the reaction time. Excessively high temperatures are usually not preferred so as to avoid unwanted secondary reactions however.
The procedures for forming the instant peptides are well known in this art and are provided in detail in the accompanying examples.
When the selected tetrapyrrole contains more than one carboxyl group, then mixtures of products can be formed including isomeric monopeptide products and di and even tri-or higher peptide products, depending on the number of '.~
carboxyl groups and depending on the selected stoichiometry.
Thus, when equimolar mixtures of amino acid and tetrapyrrole are reacted, not only monopeptides but also dipeptides are obtained, although the monopeptide would predominate. With higher molar ratios, the nature of the products will similarly vary. It is generally possible to separate the monopeptides and higher peptides using Icnown chromatographic techniques.
However, such separations are not necessary since the mixed peptides are usually comparable to the separated products in their ultimate use. Thus, mixtures of the mono-, di~ and tri~
peptides of the same tetrapyrrole can be used.
Usually, unreacted tetrapyrrole is separated from the peptide products of the invention during puri-fication as, for example, by chromatographic techniques.
Photodiagnosis and Phototherapy The compositions of the present invention are useful for the photodiagnosis and phototherapy of tumor, cancer and malignant tissue (hereinater referred to as "tumor").
When a man or animal having tumor is treated with doses of a compound of the present invention and when appropriate light rays or electromagnetic waves are applied, the compound emits light, i.e., fluorescence. Thereby the existence, position and size of tumor can be detected, i~e., photodiagnosis.
When the tumor is irradiated with light of proper wavelength and intensity, the compound i9 activated to exert a cell killing effect against the tumor. This is called "phototherapy".
'.~'~, 86;~
Compounds intended for photodiagnosis and phototherapy ideally should have the fol~owing properties:
(a) non-toxic at normal therapeutic dosage unless and until activated by light;
(b)should be selectively photoactive;
(c) when light rays or electromagnetic waves are applied, they should emit characteristic and detectable fluorescence;
(d) when irradiated with light rays or electromagnetic waves are applied, they are activated to an extent to exert a cell killing effect against tumor; and (e) easily metabolized or excreted after treatment.
In accordance with testing up to the present, the compounds of the present new therapeutic compositions have the foregoing properties and are also characterized by reasonable solubility in water at physiological pH.
The aforesaid compounds possess greater fluorescence in tumors than do the corresponding basic tetrapyrroles, and even peptides formed with amino monocarboxylic acias, e.g., alanine and epsilon aminocaproic acid. Their use provides the best contrast in tumors compared to normal tissue around the tumor. The instant compounds absorb activating energy for phototherapy in the convenient range of 600 to 800 nanometers, with the preferred compounds absorbing in the 620-760 nanometer range, i.e., light of longer wavelengths which more readily permits penetration of energy into the tumor for phototherapeutic purpose.
In present experience, the present compounds more uniformly distribute throughout the tumor than the basic ~etrapyrrole permitting the use of considerably lower ~2~ 62 dosage (to about l/lOth of the required normal dose of the basic tetrapyrrole) which lessens, if not eliminates, photosensitization in the host. They also possess a more consistent fluorescence whereas some of the corresponding tetrapyrroles show inconsistent fluorescence or the fluorescence varies from day to day in the host.
A particularly advantageous property of the present compounds resides in the ease with which they are excreted by the host. Generally, within 48 to 72 hours of intravenous or intraperitonal administration, there are little or no detectable amounts in normal muscle tissue. The present compounds which are excreted with their chromosphore intact are recovered from -the feces of the host within 48-72 hours of injection. Under equivalent circumstances, substantial amounts of the corresponding tetrapyrroles remain, as compared with only minor amounts of peptides formed with the amino monocarboxylic acids remain in the host, e.g., up to about 20%. This Property is extremely important in that it contributes to minimization of photosensitization of the host.
The instant composition can be used for diagnosis and therapeutic treatment of a broad range of tumors.
Examples of tumors are gastric cancer, enteric cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcomas, hepatic cancer, cancer of the urinary bladder, cancer of the upper jaw, cancer of the bile duct, cancer of the tongue, cerebral tumor, skin cancer, malignant goiter, prostatic cancer~ cancer of the parotid gland, Hodgkins's disease, multiple myeloma, renal cancer, leukemia, and malignant lymphocytoma.
~Z6~`Z~36~
For diagnosis, the sole requirement is that the tumor be capable of selectively fluorescing when exposed to proper light. For treatment, the tumor must be penetrable by the activation energy. For diagnosis, light of shorter wavelength is used whereas for therapeutic purposes light of longer wavelength is used to permit read~ penetration of the tumor tissue. Thus, for diagnosis, light of from 360-760 nanometers can be used, and for treatment, from 620 to 760, depending on the individual characteristics of the tetrapyrrole. The absorption characteristics of the present new compounds are substantially the same as the tetrapyrrole from which derived.
It is necessary that the light rays be so intense as to cause the compounds to emit fluorescence for diagnosis and to exert a cell killing effect for therapy.
The source of irradiation for photodiagnosis and phototherapy is not restricted, however, but the laser beam is preferable because intensive light rays in a desired wavelength range can be selectively applied. For example, in photodiagnosis, the compound of the invention is administered to a human or animal body, and after a certain period of time, light rays are applied to the part to be examined. When an endoscope can be used for the affected part, such as lungs, gullet, stomach, womb, urinary bladder or rectum, it is irradiated using the endoscope, and the tumor portion selectively emits fluorescence. This portion is observed visually, or observed through an adapted fiber scope by eye or on a CRT screen.
`~ ' In photothe~apy, a~ter administration of the dosage, the irradiation is carried out by laser beams from the tip of quartz fibers. sesides the irradiation of the sur~ace of tumor, the internal part of the tumor can be irradiated by inserting the tip of quartz ~ibers into the tumor. The irradiation can be visually observed or imaged on a CRT screen.
For photodiagnosis, light of wavelengths between 360 and 760 nm. is suitable for activating the present tetrapyrrole compounds. Of course, each compound has a specific optimal wavelength of activation. A long wavelength ultraviolet lamp is particularly suitable Eor photodiagnosis. Similar methods for viewing of the treated tumor can be used as already described for phototherapy.
The dosages of compounds having the present new composition will vary depending on the desired effect, whether for diagnosis or or treatment. For diagnosis, doses of as little as 1 mg/kg will be effective, and up to about 20 mg/kg can be used. For treatmen-t, the dose will usually approximate about 0.5 mg/kg. Of course, the dosage for either diagnosis or treatment can be varied widely in view of aforesaid advantageous properties of the present compounds, e.gO, the ease of elimination from the host, for one.
The present compounds are apparently nontoxic at the dosage levels employed for diagnosis or treatment. No mortality of test animals due the present compounds has been noted in studies employing dosage levels up to 20mg/kg.
For both diagnosis and treatment, the present compounds can be administered by the oral, intravenous, or intramuscular routes. They can be formulated as lyophilized sterile, pyrogen-free compounds, preferably in the form of basic salts, e.g., sodium salt. The preferred dosage forms are provided as injectable solutions (isotonic) ..~
.~.
z~
The irradiation source used in treatment of tUMOrs containing compounds o this invention is a filtered, high-intensity, continuous source or pumped dye, or other laser andlight delivery system, which is capable of performing within the following limits: power intensity 20-500 mw/cm2 at wavelengths between 620 and 760 nm. and a total output of at least 500 mw or greater. Several currently commercially available lasers meet these criteria.
The tetrapyrroles can be prepared by various synthetic methods which are found in the literature, e.g., Pheophorbides Willstatter, R., Stoll, A.; Investigations on Chlorophyll, (Transl. Schertz, FM.M., Merz, A.R.) p. 249. Science Printing Press, Lancaster, Pennsylvania, 1928.
Pennington, F.C., Strain, H.H., Svec, W.~., Katz, J.J.; J.
Amer. Chem. Soc., 86, 1418 ~1964).
-Chlorin e6 Willstatter, R., Stoll, A.; Investigations on Chlorophyll, ~Trans., Schertz, F.M., Merz, A.R.,) p. 176. Science Printing Press, Lancaster, Pennsylvania, 1928.
Willstatter, R., Isler, M.; Ann. Chem., 390, 269 (1912).
Fisher, H., Baumler, R.; Ann. Chem., 474, 65 (1929).
Fisher, H., Siebel, H.; Ann. Chem., 499, 84 (1932).
-Conant, J.B., Mayer, W.W.; J. Amer. Chem. Soc.l 52, 3013(1930) ~2~
Chlorin e4 Fisher, H., Heckmaier, J., Plotz, E.; Justus Leibigs Ann.
Chem., 500 215 (1933).
Chlorin e6, e4, isochlorin e~, mesochlorin e6, bacterio-.
pheophorbide, bacteriochlorin e6 Fischer and Orth, "Des Chemie des Pyrrole" Akademische Verlazsgesellschaft, Leipzig, 1940, Vol. II, Part 2.
General Reference for Porphyrirls "Porphyrins and Metalloporphyrins" ed. Kevin M. Smith, Elsevier 1975 N.Y.
:~2~ %
-23~
The compounds of the present invention can be administered to the host in a variety of forms adapted to the chosen route of administration, i.e., orally, intraveneously, intramuscularly or subcutaneous routes.
The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1~ of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
Pre~erred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 50 and 300 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
:~2~i2~3~%
When the dosage unit ~orm is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a cye and flavoring such as cherry or orange flavor~ Of course~ any material used in preparing any dosage unit Eorm should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and Eormulations.
The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability existsO It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, ~or example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the .
~;i2~3f;;~
use of surfactants. rrhe pre~7ention of the action of micro-organisms can be brought about by varlous antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid~ thimerosal, and the like. ln many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption o~ the injectable compositions can be brought about by the use in the compositions of a~ents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile ~0 injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
The present new compounds may also be applied directly to tumors, whether internal or external, in the host in topical compositions. Exemplary compositions include solutions of the new compounds in solvents, par~icularly aqueous solvents, most preferably water. Alternatively, for topical application particularly to skin tumors, the present new compounds may be dispersed in the usual cream or salve formulations commonly used for this purpose or may be provided in the form of spray solutions or suspensions which may include a propellant usually employed in aerosol preparations.
~o~
As used herein, "pharmaceutically acceptable carrier" ineludes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known the art. Except insofar as any conventional media or agent is incompatable with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into ~he compositions.
It is espeeially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjeets to be treated; eaeh unit eontaining a predetermined quantity of active material ealeulated to produee the desired therapeutie effeet in assoeiation with the required pharmaeeutieal earrier. The speeifieation for the novel dosage unit forms of the invention are dietated by and direetly dependent on (a) the unique eharaeteristies of the aetive material and the partieular therapeutie effeet to be aehieved, and (b) the limitations - inherent in the art of eompounding sueh an aetive material for the treatment of tumors in living subjeets.
......
.28~
Di ~D,L) aspart~l transmesochlorin IX_(Carbodiimide Method) 1~0 mg of transmesochlorin and 200 mg of (D,L) aspartic acid dimethyl ester hydrochloride were dissolved in 30 ml of dimethyl formamide. 300 mg of N,NI~dicyclohe~yl-carbodiimide was added. The reaction was allowed to stand for one hour, then another 300 mg of carbodiimide was added. This procedure was repeated twice and then the reaction mixture was allowed to stand overnight. The reaction may be monitored by thin layer chromatography on silica, using solvent benzene/methanol/88% formic acid 8.5/1.5/0.13 V/V/V.
The disubstituted chlorin has the highest Rf value, the unsubstituted chlorin has the lowest, with the mono-substituted isomers in between and unresolved.
After standing overnight, the reaction rnixture appeared to contain at least 50% of the disubstituted chlorin.
The solvent was removed under vacuum and the remaining solid dissolved in 50 ml of 3N ~Cl.
The solution was allowed to stand at room temperature for 48 hours to hydrolyze the ester groups, then the chlorin mixture was precipitated at pH 2.5-3 and collected and washed with water at the centrifuge.
The chlorin mixture was purified by dissolving in 0.05 M NH40H and applying to a reverse phase (C-18 silica) column 2.5 cm X 30 cm. The elution procedure is a linear gradient from 40 to 70% methanol in 0.01 M KP04 buffer pH 6.85 (1 liter tctal volume).
The leading green band (di D~ L aspartyl trans-mesochlorin IX) was collected and flash evaporated to remove the methyl alcohol, the solution then precipitated at pH 2.5-3 and collected and washed 3 times at the centrifuge with dilute acetic acid. The product was dried under vacuum. The yield was 67 mg of cli (D,L) aspartyl transmesochlorin IX.
~2~i2~
1 EXA~PLE 2 yl tr~nsmesochlolin IX (mixed anhvdride methocl) _ 50 m~ (0.000087 moles) of transmesochlorin IX
~as dissolvcd in l00 ml of tetrahydrofuran (THF). 210 ~1 (0.002 moles) of triethylamine was added wi-th stirring.
After 10 minutes, 195 ~1 (0.00179 moles) of ethyl-chloroforma-te was added. After stirring 10 minu-tes, 50 ml (0.01 moles) oE 0.2 ~I KOH containing 250 mg (0.00169 moles) of (L) glutamic: acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0.13) was used to de~,elop tl;e chromatogram.
Af~er checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mi~ture was resolved using a linear gradient of 40-80% methanol in 0.01 M KPO4 buffer pH 6.85 ~1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (L) glutamyl transmesochlorin IX, mono (L) glutamyl transmesochlorin IX, and unsubstituted transmesochlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
~2~ 2
-2~-1 FXA~IrI.E 3 .
Di and mono (D,L) aspartvl photoDro ~or~hyin IX
(mixed anhyclride mc~hod) 313.4 rng of photoproto?orphyrin IX (isomer mixture) ~as dissolved in 100 mls of tetrahydrofuran (THF). 210 ~ll of triethylamine was added with stirring.
After 10 minutes, 210 ul of ethyl chloroEormate was added. After stirring for 10 minutes, 50 mls of 0.2 m KOH, containing 450 mgs of (D,L) aspartic acid, were added to the T~IF solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mi~ture was checked by silica TLC. Benzene/
methanol/38% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the pH of the mix-ture was adjusted to 7.5-8.0 and -the solution was placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reac-tion mixture was resolved using a linear 20 gradient of 40/80% MeOH in 0.01 _ KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the ma-terial was precipitated at pH 3.0 3.5. The ppt was washed 3 times with dilute acetic acid in H2O. The product was dried under vacuum. The yield of mono(D,L) aspartyl photoprotoporphyrin I~ was 54 mg. The yield
Di and mono (D,L) aspartvl photoDro ~or~hyin IX
(mixed anhyclride mc~hod) 313.4 rng of photoproto?orphyrin IX (isomer mixture) ~as dissolved in 100 mls of tetrahydrofuran (THF). 210 ~ll of triethylamine was added with stirring.
After 10 minutes, 210 ul of ethyl chloroEormate was added. After stirring for 10 minutes, 50 mls of 0.2 m KOH, containing 450 mgs of (D,L) aspartic acid, were added to the T~IF solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mi~ture was checked by silica TLC. Benzene/
methanol/38% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the pH of the mix-ture was adjusted to 7.5-8.0 and -the solution was placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reac-tion mixture was resolved using a linear 20 gradient of 40/80% MeOH in 0.01 _ KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the ma-terial was precipitated at pH 3.0 3.5. The ppt was washed 3 times with dilute acetic acid in H2O. The product was dried under vacuum. The yield of mono(D,L) aspartyl photoprotoporphyrin I~ was 54 mg. The yield
3 of di (D,L) aspartyl photoprotoporphyrin IX was 227.8 mg.
~6~
1 ~AMPL~ 4 Di and Mono (I,) as~ roto~or~h~rin IX
. ~
(mixed anhvdridc mcthod) 100 mg of protoporphyrin ~X was dissolved in 100 ml of P-dioxane. 210 ~1 of triethylamine was added. After stirring 10 minutes, 50 ul of 0.2 M KOH
containing 500 mg of (L) aspartic acid was added to the dioxane solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0~13) was used to develop the chromatogram.
After checking for product, the pH of the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm.
The reaction mixture was resolved using a linear gradient of 40-70% methanol in 0.01 ~ KpO4 buffer pH
6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in H20. The product was then dried under vacuum. The yield of mon (L) aspartyl protoporphyrin IX was 12.3 mg and di (L) aspartyl proto-porphyrin IX was 54 mg.
.
3o ~2G~f''S~62 1 E~iPLE 5 ___ Di and_mo~o (I,) aspa_tyl mcso~?orD ~rin IX
~mi~ed anhvdr:ide mctnocl) __ . .~ _..
200 m~ of mesoporp~lyrin I~ was dissolved in 100 ml of tetr2'lydrofuran (T~F). 21() ~1 of triethylamine was added to the THF solution. After 10 minutes of stirring 210 ~1 et~lyl chloroLormate was added and stirred 10 minutes. 50 ml of 0.2 M KOi~ containing 500 mg of (L) aspartic acid was added to the TIIF solution and allowed to stir one hour at room temperature.
The organic solvent was flashed off and the reaction mi~ture was checked for product by silica TLC
using ben~ene/methanol/88~ formic acid (8.5/1.5/0.13) to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80% methanol in 0.01 ~ K~04 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 3.0-3.5. The ppt was washed 3 times with dilute acetic acid in H20. The product was dried under vacuum with a yield of 41.5 mg mono (L) aspartyl mesoporphyrin and 175.1 mg di (L) aspartyl mesoporphyrin.
3o ~Z6ZB~2 1 EX~IPLE 6 Di and ~Iono (L) as~artvl dcuteroDor~hvrin ~X (rnixed _rydride met}lod) 100 mg deuteroporphyrin IX was dissolved in 50 ml of p-dioxane. 210 ~ul of trlethylamine was added with stirring. After 10 minutes, 210~ul of isobutyl chloroformate was added. After stirring lo minutes, 50 ml of 0.2 M KOH containing 500 mg of L aspartic acid was added to the dioxane solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC Benzene/
methanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-70%
methanol in 0.01 M KP04 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components.
The MeOH was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in H20. The product was then dried under vacuum. The yield of mono (L) aspartyl deuteroporphyrin IX was 10 mg.
3o z ~33 1 ~X~I'LE 7 (L) As~artv] ~vro~heoDhorbide a (mi~ed anhvdride rnethod) .
80 m~ of p~ro?hcoDhorbide a was dissolved in 100 ml of te~rahydroruran (THF) 210/ul of triethyl-amine was to the 'r~lF solution. ~fter 10 minutes ofs-tirring, 210/ul of ethylchloroformate was added and stirred 10 minutes. 50 ml of 0.2 ~1 ~OH containing 500 mg of (L) aspartic acid was added to the THF solution and allowed to stir one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked for product by silica TLC
using benzene (methanol) 88% formic acid (8.5/1.5/0.13) to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80%
methanol in 0.01 M KOH buffer pH 6.85 (1 liter total volume).
The column effluent was co]lected via fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 3.0-3.5. The ppt was washed 3 times with dilute acetic acid in H2O. The product was dried under vacuum to produce a yield of 62 mg (h) aspartyl pyropheophorbide a.
3o l EV~lPL~ 8 -Tetra, ~rl, and di (D,L) as~artyl coproporphvrin III
.
(mixed an~.~dricle method) ..
150 mg of coproporphyrin III was dissolved 5 in 100 ml of tetrahydrofuran (T~F). 210 ~1 of tri~
ethylamine was added and stirring was continued at 20C
for ten minutes. 210~ul of ethylchloroformate was next added and stirred for ten minutes.
50 ml of 0.2 M KOH containing 250 mg of (D,L) lO aspartic acid was added to the THF solution. This mixture ~as then stirred for one hour.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC using the following solvent system: (benzene/methanol/88% formic 15 acid (8.5/~.0/0.2).
The pH of this mixture was then adjusted to 7.5-8.0 and chromatographed on a reverse phase (C-18 silica)2.5x30 cm column. The reaction mixture was resolved using 5-50% methanol in 0.01 in ~P04 buffer 20 pH 6.85 (l liter total volume).
The column effluent was collected via a fraction collector an~ the tube contents were pooled according to individual components. The methanol was flashed off and the material t~as precipitated at pH 3.0-3.5. The ppt was 25 washed 3 times with dilute acetic acid in water. The products were dried under vacuum and the yields were as follows: Tetra (D,L) aspartyl coproporphyrin III 94 mg, Tri (D,L) aspartyl coproporphyrin III 77.2 mg, Di (D,L) aspartyl coproporphyrin III, 28.4 mg.
3o ~26~B6~
1 EXAMPL,~ 9 D:i and mono (DL) aspartyl deutero?or~hvrin IX
(mi~;ed anh~dride metllod) 175 mg (0.00195 moles) of deuteroporphyrin IX
was dissolv~d in 200 ml of tctrahydrofuran (THF).
210 ul (0.00~ moles) of triethylamine was added with stirring. t~fter 10 minutes, 210 ul (0.0019 moles) of ethylchloroformate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH con-taining 200 mg (0.003 moles) of (DL) aspartic acid was added dropwise with stlrring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solven-t was flashed off and the reaction mixture was checked by silica TLC
for product. Benzene/methanol/88% formic acid (8.5/1.5/01.3) was used to develop the chromatogram.
After chec~ing for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-65%
methanol in 0.01 M KPO~ buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (DL) aspartyl deuteroporphyrin lX, mono (DL) aspartyl deuteroporphyrin IX, and unsubstituteddeuteroporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
~Z~ 62 1 E~AMPLE 10 Di and mono (DL) ast~art~ emato~orDhvrin IX (mixed _ _ _ ~ _ anh~dride metllocl_ ~00 mg (0.0059 moles) of hematoporphyrin IX
5 ~.~as dissolvcd in 50 ml of tetrahydrofuran (TH~). 3~0 ~1 (0.0034 moles) of tricthylamine was added with stirring.
After 10 minutes~ 3~0 ~1 ~0.0031 moles) of ethyl-chloroformate was added. A~ter stirring 10 minutes, 10 ml (0.01 moles) of 1 M KOH containing 600 mg (0.0045 moles) of (DL) aspartic acid was added to the THF solution. This mixture was stirred 90 minutes at room temperature.
The c;rganic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88~ formic acid 18-5/1-5/0-13) WclS
used to develop the chromatogram.
~ fter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 20-70~ methanol in 0.01 M KPOa buffer pH 6.85 (1 liter total volume).
The column ef~luent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (DL) aspartyl hematoporphyrin IX, mono(DL) aspartyl hematoporphyrin IX, and unsubstituted hematoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid-in water. The product 3 was dried under vacuum.
.
:
~L2~
~37-1 EXAMPLE ll Di and mono (D,L) asparty] protoporphyrin I~ (mixed anhyAride metIlocl) 300 mg (0.00053 moles) of protoporphyrin XI
was dissolved in lO0 ml of tetrahydrofuran (THF). 210 ~l (0.002 moles) of trlethylamine was added with stirring.
After lO minutes, 210 ~l (0.0019 moles) of ethylchloro-formate was added. ~fter stirring lO minutes, 50 ml (0.01 moles) of 0.2~I KO~-I con-taining 450 mg (0.0033 moles) f (D,L) aspartic acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.-Benzene/me-thanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
~ fter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5x30 cm. The reaction mixture was resolved using a linear gradient of 40-65~ methanol in O.Ol~KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D,L) aspartyl protoporphyrin IX, mono (D,L) aspartyl proto-porphyrin IX, and unsubstituted protoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was ;~ 3O dried under vacuum.
~: :
2~
1 rX~M~LE 12 ~Iono ~DL) aspcrtyl pyropll---e-o~horbide a (mixed aIlh~dride method) . .
100 mg (0.000187 moles) of pyropheophorbide a was dissolved in ]00 ml of te~rahvdrofuran (THF).
210 ~1 (0.002 moles) of triethylamine was added with stirring. After 10 miniutes, 210 ~1 (0.0019 moles) of ethylchloroformate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH contalning 200 mg (0.0015 moles) of (DL) aspartic acid was added to the THF solution. This mixture was stirred 60 minu-tes at room tempera-ture.
The organic solvent was flashed off and the reaction mixture was checked by ilica TLC for product.
Benzene/methanol/88% fcrmic acid (8.5/1.5/0.13) was used to develop ~he chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80~ methanol in 0.01 M XPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was colleeted via fraction collector and the tube contents were pooled according to individual components. The order of elution was mono (DL) aspartyl pyropheophorbide a, and then unsubstituted pyropheophorbide-The methanol was flashed off and the materialwas precipitated at pH 2.5-3Ø The ppt was washéd 3 times with dilute acetic acid in water. The product was dried under vacuum.
3o ~ .
`Z8~i2 1 EX~IPLE 13 Di and mono L-alph~-aminoadipy] transmesochlorin IX
~mixed anhvdride method) 500 mg (0.000087 moles) of transmesochlorin IX
was dissolved in 100 ml of tetrahydrofuran (T~F). 210 ~1 (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210 ~1 (0.00:l9 moles) of ethylchloro-formate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH containing 250 mg (0.00155 moles) of L-alpha-aminoadipic acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by cilica TLC for product.
Ben7ene/methanol/88~ formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a re~erse phase (C-18 silica) column 2.5 x 30 cm. The ~eaction mixture was resolved using a linear gradient of ~0-80~ methanol in 0.01 M KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di L-alpha-aminoadipyl transmesochlorin IX, and unsubstituted transmesochlorin IX~
The methanol was flashed off and the material was precipitated at pl; 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
:
~Z~2X36~
--~o 1 E~lPL~ 1~
Di and mono (D) aspartyl mesoporphyrin IX (mixed anh~dride _ _ _ _ .
method) 200 m~ (0.00035 moles of mesoporphyrin IX was dissolved in 100 ml of tet~ahydrofuran (THF). 210Jul (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210~ul ~0.0019 moles) of ethylchloro-formate was added. Af-ter stirring 10 minutes, 50 ml (0.01 moles) of 0.2M KOH containing 500 mg (0.0038 moles) of (D) aspartic acid was added dropwise wi-th stirring to the THF solution. This mixturewasstirred 60 minu-tes a~
room temperature.
The organic solvent was flashed off and -the reaction mixture was checked by silica TLC for produc-t.
Benzene/methanol/88~ formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
A~ter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5x30 cm. The reaction mix-ture was resolved using a linear gradient of 40-48~ methanol in 0.01M KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D) aspartyl mesoporphyrin IX, mono (D) aspartyl mesoporphyrin IX, and unsubstituted mesoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times , ; with dilute acetic acid in water. The product was dried under vacuum.
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12~iZ~
1 EX~IPLE 15 ._ Di and mono (L) glu amyl mesoporphyrin IX (mixed anh~dride method ~ 00 mg (0.007 moles) of mesoporphyrin IX ~as dissolved in 50 ml of tetrahydrofuran (THF). 360 ~1 (0.0035 moles) oE triethylamine was added with stirring.
After 10 minutes, 3~0~ul (0.0031 moles) ethylchloro-formate was added. After s-tirring 10 minutes, 10 ml (0.01 moles) of 1 _ KOH containing 543 mg (0.00369 moles) of (L) glutamic acid was added to the THF
solution. Thls mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 a.Ad placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mi~ture was resolved using a linear gradient of ~5-60% methanol in 0.01 M KPO buffer pH 6.85 (1 liter total volume).
~6~
1 ~AMPL~ 4 Di and Mono (I,) as~ roto~or~h~rin IX
. ~
(mixed anhvdridc mcthod) 100 mg of protoporphyrin ~X was dissolved in 100 ml of P-dioxane. 210 ~1 of triethylamine was added. After stirring 10 minutes, 50 ul of 0.2 M KOH
containing 500 mg of (L) aspartic acid was added to the dioxane solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0~13) was used to develop the chromatogram.
After checking for product, the pH of the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm.
The reaction mixture was resolved using a linear gradient of 40-70% methanol in 0.01 ~ KpO4 buffer pH
6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in H20. The product was then dried under vacuum. The yield of mon (L) aspartyl protoporphyrin IX was 12.3 mg and di (L) aspartyl proto-porphyrin IX was 54 mg.
.
3o ~2G~f''S~62 1 E~iPLE 5 ___ Di and_mo~o (I,) aspa_tyl mcso~?orD ~rin IX
~mi~ed anhvdr:ide mctnocl) __ . .~ _..
200 m~ of mesoporp~lyrin I~ was dissolved in 100 ml of tetr2'lydrofuran (T~F). 21() ~1 of triethylamine was added to the THF solution. After 10 minutes of stirring 210 ~1 et~lyl chloroLormate was added and stirred 10 minutes. 50 ml of 0.2 M KOi~ containing 500 mg of (L) aspartic acid was added to the TIIF solution and allowed to stir one hour at room temperature.
The organic solvent was flashed off and the reaction mi~ture was checked for product by silica TLC
using ben~ene/methanol/88~ formic acid (8.5/1.5/0.13) to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80% methanol in 0.01 ~ K~04 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 3.0-3.5. The ppt was washed 3 times with dilute acetic acid in H20. The product was dried under vacuum with a yield of 41.5 mg mono (L) aspartyl mesoporphyrin and 175.1 mg di (L) aspartyl mesoporphyrin.
3o ~Z6ZB~2 1 EX~IPLE 6 Di and ~Iono (L) as~artvl dcuteroDor~hvrin ~X (rnixed _rydride met}lod) 100 mg deuteroporphyrin IX was dissolved in 50 ml of p-dioxane. 210 ~ul of trlethylamine was added with stirring. After 10 minutes, 210~ul of isobutyl chloroformate was added. After stirring lo minutes, 50 ml of 0.2 M KOH containing 500 mg of L aspartic acid was added to the dioxane solution. This mixture was stirred for one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC Benzene/
methanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-70%
methanol in 0.01 M KP04 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components.
The MeOH was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in H20. The product was then dried under vacuum. The yield of mono (L) aspartyl deuteroporphyrin IX was 10 mg.
3o z ~33 1 ~X~I'LE 7 (L) As~artv] ~vro~heoDhorbide a (mi~ed anhvdride rnethod) .
80 m~ of p~ro?hcoDhorbide a was dissolved in 100 ml of te~rahydroruran (THF) 210/ul of triethyl-amine was to the 'r~lF solution. ~fter 10 minutes ofs-tirring, 210/ul of ethylchloroformate was added and stirred 10 minutes. 50 ml of 0.2 ~1 ~OH containing 500 mg of (L) aspartic acid was added to the THF solution and allowed to stir one hour at room temperature.
The organic solvent was flashed off and the reaction mixture was checked for product by silica TLC
using benzene (methanol) 88% formic acid (8.5/1.5/0.13) to develop the chromatogram.
After checking for product, the pH of the mixture was adjusted to 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80%
methanol in 0.01 M KOH buffer pH 6.85 (1 liter total volume).
The column effluent was co]lected via fraction collector and the tube contents were pooled according to individual components.
The methanol was flashed off and the material was precipitated at pH 3.0-3.5. The ppt was washed 3 times with dilute acetic acid in H2O. The product was dried under vacuum to produce a yield of 62 mg (h) aspartyl pyropheophorbide a.
3o l EV~lPL~ 8 -Tetra, ~rl, and di (D,L) as~artyl coproporphvrin III
.
(mixed an~.~dricle method) ..
150 mg of coproporphyrin III was dissolved 5 in 100 ml of tetrahydrofuran (T~F). 210 ~1 of tri~
ethylamine was added and stirring was continued at 20C
for ten minutes. 210~ul of ethylchloroformate was next added and stirred for ten minutes.
50 ml of 0.2 M KOH containing 250 mg of (D,L) lO aspartic acid was added to the THF solution. This mixture ~as then stirred for one hour.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC using the following solvent system: (benzene/methanol/88% formic 15 acid (8.5/~.0/0.2).
The pH of this mixture was then adjusted to 7.5-8.0 and chromatographed on a reverse phase (C-18 silica)2.5x30 cm column. The reaction mixture was resolved using 5-50% methanol in 0.01 in ~P04 buffer 20 pH 6.85 (l liter total volume).
The column effluent was collected via a fraction collector an~ the tube contents were pooled according to individual components. The methanol was flashed off and the material t~as precipitated at pH 3.0-3.5. The ppt was 25 washed 3 times with dilute acetic acid in water. The products were dried under vacuum and the yields were as follows: Tetra (D,L) aspartyl coproporphyrin III 94 mg, Tri (D,L) aspartyl coproporphyrin III 77.2 mg, Di (D,L) aspartyl coproporphyrin III, 28.4 mg.
3o ~26~B6~
1 EXAMPL,~ 9 D:i and mono (DL) aspartyl deutero?or~hvrin IX
(mi~;ed anh~dride metllod) 175 mg (0.00195 moles) of deuteroporphyrin IX
was dissolv~d in 200 ml of tctrahydrofuran (THF).
210 ul (0.00~ moles) of triethylamine was added with stirring. t~fter 10 minutes, 210 ul (0.0019 moles) of ethylchloroformate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH con-taining 200 mg (0.003 moles) of (DL) aspartic acid was added dropwise with stlrring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solven-t was flashed off and the reaction mixture was checked by silica TLC
for product. Benzene/methanol/88% formic acid (8.5/1.5/01.3) was used to develop the chromatogram.
After chec~ing for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-65%
methanol in 0.01 M KPO~ buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (DL) aspartyl deuteroporphyrin lX, mono (DL) aspartyl deuteroporphyrin IX, and unsubstituteddeuteroporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
~Z~ 62 1 E~AMPLE 10 Di and mono (DL) ast~art~ emato~orDhvrin IX (mixed _ _ _ ~ _ anh~dride metllocl_ ~00 mg (0.0059 moles) of hematoporphyrin IX
5 ~.~as dissolvcd in 50 ml of tetrahydrofuran (TH~). 3~0 ~1 (0.0034 moles) of tricthylamine was added with stirring.
After 10 minutes~ 3~0 ~1 ~0.0031 moles) of ethyl-chloroformate was added. A~ter stirring 10 minutes, 10 ml (0.01 moles) of 1 M KOH containing 600 mg (0.0045 moles) of (DL) aspartic acid was added to the THF solution. This mixture was stirred 90 minutes at room temperature.
The c;rganic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88~ formic acid 18-5/1-5/0-13) WclS
used to develop the chromatogram.
~ fter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 20-70~ methanol in 0.01 M KPOa buffer pH 6.85 (1 liter total volume).
The column ef~luent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (DL) aspartyl hematoporphyrin IX, mono(DL) aspartyl hematoporphyrin IX, and unsubstituted hematoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid-in water. The product 3 was dried under vacuum.
.
:
~L2~
~37-1 EXAMPLE ll Di and mono (D,L) asparty] protoporphyrin I~ (mixed anhyAride metIlocl) 300 mg (0.00053 moles) of protoporphyrin XI
was dissolved in lO0 ml of tetrahydrofuran (THF). 210 ~l (0.002 moles) of trlethylamine was added with stirring.
After lO minutes, 210 ~l (0.0019 moles) of ethylchloro-formate was added. ~fter stirring lO minutes, 50 ml (0.01 moles) of 0.2~I KO~-I con-taining 450 mg (0.0033 moles) f (D,L) aspartic acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.-Benzene/me-thanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
~ fter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5x30 cm. The reaction mixture was resolved using a linear gradient of 40-65~ methanol in O.Ol~KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D,L) aspartyl protoporphyrin IX, mono (D,L) aspartyl proto-porphyrin IX, and unsubstituted protoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was ;~ 3O dried under vacuum.
~: :
2~
1 rX~M~LE 12 ~Iono ~DL) aspcrtyl pyropll---e-o~horbide a (mixed aIlh~dride method) . .
100 mg (0.000187 moles) of pyropheophorbide a was dissolved in ]00 ml of te~rahvdrofuran (THF).
210 ~1 (0.002 moles) of triethylamine was added with stirring. After 10 miniutes, 210 ~1 (0.0019 moles) of ethylchloroformate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH contalning 200 mg (0.0015 moles) of (DL) aspartic acid was added to the THF solution. This mixture was stirred 60 minu-tes at room tempera-ture.
The organic solvent was flashed off and the reaction mixture was checked by ilica TLC for product.
Benzene/methanol/88% fcrmic acid (8.5/1.5/0.13) was used to develop ~he chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mixture was resolved using a linear gradient of 40-80~ methanol in 0.01 M XPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was colleeted via fraction collector and the tube contents were pooled according to individual components. The order of elution was mono (DL) aspartyl pyropheophorbide a, and then unsubstituted pyropheophorbide-The methanol was flashed off and the materialwas precipitated at pH 2.5-3Ø The ppt was washéd 3 times with dilute acetic acid in water. The product was dried under vacuum.
3o ~ .
`Z8~i2 1 EX~IPLE 13 Di and mono L-alph~-aminoadipy] transmesochlorin IX
~mixed anhvdride method) 500 mg (0.000087 moles) of transmesochlorin IX
was dissolved in 100 ml of tetrahydrofuran (T~F). 210 ~1 (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210 ~1 (0.00:l9 moles) of ethylchloro-formate was added. After stirring 10 minutes, 50 ml (0.01 moles) of 0.2 M KOH containing 250 mg (0.00155 moles) of L-alpha-aminoadipic acid was added dropwise with stirring to the THF solution. This mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by cilica TLC for product.
Ben7ene/methanol/88~ formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a re~erse phase (C-18 silica) column 2.5 x 30 cm. The ~eaction mixture was resolved using a linear gradient of ~0-80~ methanol in 0.01 M KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di L-alpha-aminoadipyl transmesochlorin IX, and unsubstituted transmesochlorin IX~
The methanol was flashed off and the material was precipitated at pl; 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
:
~Z~2X36~
--~o 1 E~lPL~ 1~
Di and mono (D) aspartyl mesoporphyrin IX (mixed anh~dride _ _ _ _ .
method) 200 m~ (0.00035 moles of mesoporphyrin IX was dissolved in 100 ml of tet~ahydrofuran (THF). 210Jul (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210~ul ~0.0019 moles) of ethylchloro-formate was added. Af-ter stirring 10 minutes, 50 ml (0.01 moles) of 0.2M KOH containing 500 mg (0.0038 moles) of (D) aspartic acid was added dropwise wi-th stirring to the THF solution. This mixturewasstirred 60 minu-tes a~
room temperature.
The organic solvent was flashed off and -the reaction mixture was checked by silica TLC for produc-t.
Benzene/methanol/88~ formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
A~ter checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5x30 cm. The reaction mix-ture was resolved using a linear gradient of 40-48~ methanol in 0.01M KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D) aspartyl mesoporphyrin IX, mono (D) aspartyl mesoporphyrin IX, and unsubstituted mesoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times , ; with dilute acetic acid in water. The product was dried under vacuum.
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12~iZ~
1 EX~IPLE 15 ._ Di and mono (L) glu amyl mesoporphyrin IX (mixed anh~dride method ~ 00 mg (0.007 moles) of mesoporphyrin IX ~as dissolved in 50 ml of tetrahydrofuran (THF). 360 ~1 (0.0035 moles) oE triethylamine was added with stirring.
After 10 minutes, 3~0~ul (0.0031 moles) ethylchloro-formate was added. After s-tirring 10 minutes, 10 ml (0.01 moles) of 1 _ KOH containing 543 mg (0.00369 moles) of (L) glutamic acid was added to the THF
solution. Thls mixture was stirred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 a.Ad placed on a reverse phase (C-18 silica) column 2.5 x 30 cm. The reaction mi~ture was resolved using a linear gradient of ~5-60% methanol in 0.01 M KPO buffer pH 6.85 (1 liter total volume).
- 4 The column effluent was collected via fraction collector and the tube contents were pooled according to individual components. The order of elution was di (L) glutamyl mesoporphyrin IX, mono (L) glutamyl mesoporphyrin IX, and unsubstituted mesoporphyrin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dFied under vacuum.
~:
~Z~ 2 -~2-1 EX~ilPLE 16 Di and mono (D) asp~rtyl transmesochlorin IX (mixed anhvdride method in ],9 dioxane) , 50 m~ (0.000087 moles) of transmesochlorin IX
was dissolved in 50 ml of 1,4 dioxane. 210~ul (0.002 moles) of triethylamine was added wlth stirring. After 10 minutes, 210,ul (0.0019 moles) of ethylchloroformate was added.
~fter stirring 10 minutes, 50 ml (0.01 moles) of 0.2~1 KOH
containing 500 mg (0.0038 moles) of (D) aspartic acid was added dropwise wikh stirring to the THF solution. This mixture was sti.rred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid ~8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse pha.se (C-18 silica) column 2.5x30 cm. The reaction mixture was resolved usinga linear gradient of 40-80% methanol in O.OlM KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D) aspartyl transmesochlorin IX, mono (D) aspartyl trans-25 mesochlorin IX, and unsubstituted transmesochlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
.
~fi 3~862 1 ~X~II'LE 17 Di ancl mono (L) aspartyl transmesochlorin IX (mixed anhydride m~thocl in tetrall~drofuran) 135 mcJ (0.00023 moles) of trans~esochlorin IX
was dissolved in 100 ml of tetrah~drofuran (THF). 210~ul (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210 ~1 (0.0019 moles) of ethylchloro~
formate was added. After stirring 10 minutes, 50 ml (0.015 moles) of 0.3~ KOll contailling 750 mg (0.0056 moles) of (L) aspartic acid was added dropwise with stirring to the T}IF solution. This mixture was stirred 60 minutes at room temperature.
The organie solvent was flashed off and the reaetion mixture was eheeked by siliea TLC for produet.
senzene/methanol/88~ formic aeid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) eolumn 2.5x30 cm. The reaetion mixture was resolved using a linear gradient of 40-80~ methanol in 0.01M KPO4 buffer pH 6.85 (1 liter total volume).
The eolumn effluent was eolleeted via a fraetion eollector and the tube eontents were pooled aeeording to individual eomponents. The order of elution was di (L) aspartyl transmesoehlorin IX, mono (L) aspart~l trans-mesoehlorin IX, and unsubstituted transmesoehlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5~3Ø Thepptwas washed 3 times with dilute aeetie acid in water. The produet was dried -3O under vaeuum.
:
~lZ~ 162 --i,,~--1 EX~MPI,E 18 (D,I.)Aspart:~lvileophorbide a (carbodiimide method) 55 mg pheophorbide a was dissolved in 10 ml dimethylformamide. 50 mg (D,L) aspartic acid dimethyl ester dihydrochloride was added, then 100 mg of N,N'-dicyclohe~:yl-carbodiimide was added. The reaction was allowed to stand in the dark at room temperature for 1 hour, then 50 mg more carbodiimide was added.
After standing for 1 additional hour, SO mg more carbodiimide was added and the reaction allowed to stand in the dark for 12 hours at room temperature.
The solvent was removed under vacuum and the product dissolved in 50 ml 1~ KOH in methanol with 0.5 ml H20 and allowed to stand in the dark at room temperature.
The course of the hydrolysis is followed by thin layer chromatography (C-18 plates with solvent 75/25 MeOH/.OlM pH 6.85 KP04 buffer).
~ hen hydrolysis of the ester groups is essen-tially complete, the reaction is terminated by addition of a few drops of glacial acetic acid. The methanol is removed under vacuum and the product is dissolved in 20 ml 0.1 M NH40H. This solution is placed on a reverse phase (C-18 silica) column (1.5 cm x 30 cm).
The elution procedure was a linear gradient from 50 to 80~
methanol in 0.01 M KPO~ buffer pH 6.85 (500 ml total volume).
; The leading green-gray band contained the (D,L) aspartylpheophorbide a which was collected, flash evaporated to remove methyl alcohol, and precipitated at pH 3. The precipitate was collected and washed 3 times at the centrifuge with dilute acetic acid. The yield of dry product was 27 mg.
~z~
-~5-1 EX~PLE 19 . _ ~
L-Monoaspartyl chlorin e6 (carbodiimide method) . _ . . .
150 mg of chlorin e6 and 250 mg of L aspartic acid di-t.butyl ester hydrochloride were dissolved in 20 ml of dimethyl formamide. There was made a total of 3-100 mg additions of N,N'-dicyclohe~yl-carbodiimide at one hour intervals. After 4 hours, the reaction mi~ture was diluted with 300 ml ether, washed twice with 200 ml ll2O
then extracted with 40 ml 1 ~ XOH. The KOH solution was allowed to hydrolyze overniyht, then heated to 70C. for 10 minutes.
The pll oE the solution was adjusted to 7, then any residual ether was removed by flash evaporation.
The solution was then applied to a reverse phase (C-18 silica) column (1.5 cm x 30 cm). The product was purified by a stepwise elution of methanol/.01 M pH 6.85 KPO4 buffer. Eluted with 5~ methanol until unwanted polar pigments were removed. Monoaspartyl chlorin e6 was eluted off with 5-8~ methanol, and unreacted chlorin e was removed with 25% methanol. 6 The product was precipitated at pH 3 after flash evaporating briefly to remove methanol, then washed at the centrifuge 3 times with dilute acetic acid.
The product was dried under vacuum. Yield of L-monoaspartylchlorin e6 was 50 mg.
.
.~
~ ~ 35 : ::
, ~
i2 -~6-1 I`X~PL~ 20 L Glutamyl chlorin e~ (carbodiimide method) 110 mg chlorin e~ and 220 mg L-glutamic acid dimethyl cster hydrochloride were dlssolved in 15 ml of dimethyl formamide. 85 mg of N,N'--dicyclohexyl carbodiimide was then added, and the solu-tion stirred for 1 hour at room temperature. 42 mg more carbodiimide was then added, then 50 mg of carbodiimide was added at 1 hour intervals for two more additions. The reaction mixture was then allowed to stand for 12 hours, one more 50 mg carbodiimide addition was made, and the reaction allowed to stand for 3 hours. Progress of the reaction was followed by reverse phase thin layer chromatography 80% methanol, 20% KP04 buffer (.OlM pH 6.85). A further addition of 50 mg of carbodlimide, with standing, showed no further product formation.
200 ml of ether was added to the reaction mixture, and the ether solution was washed 4 times with water, approximately 100 ml per wash. The ether was then removed by flash evaporation, and the product was dissolved in approximately 25 ml of 3N Hcl. After 48 hours at room temperature, the solution was adjusted to pH3 with NH40H, and the precipitate was collected and washed at the centrifuge. The product was dissolved in 20% methanol/
water with a little NH40H, and applied to a reverse phase (C-18 sillca) column (1.5x30 cm). Elution was continued with 20% MeOH, KP04 buffer (O.OlM pH 6.5).
This removed the product (L-Glutamyl chlorin e4). The methanol concentration was increased to remove the un-reacted chlorin e4.
The solution was flash evaporated until themethanol was substantially removed, then the products were precipitated at pH3 by addition of Hcl, collected and washed at the centrifuge with dilute acetic acid and dried under vacuum. Yield of mono-L-glutamyl chlorin e4 21 mg.
Yield of recovered chlorin e4 59 mg.
~2~;2~
l EXAM~LE 21 L-Monoglutamyl chlorin eG lcarbodiimide method) . . . _ . ;
130 mg of chlorin e6 and 260 mg L glutamic acid dimethyl ester hydrochloride was dissolved in l~ ml of dimethylformamide. 100 mg of N,N'-dicyclohexyl-carbodiimide was added and the reaction mixture stirred for l hour. 50 mg more carbodiimide was then added.
After 1 hour, the reaction mixture appeared to contain 75-80~ of the monosubstituted product by reverse phase TLC (C-18 plates with 70~ MeOH, 30~O .01 M KPO~ pH 6.85).
200 ml Di.ethyl ether was added, washed twice with 100 ml H2O, then extracted with 30 ml l M KOH.
The product was allowed to hydrolyze in the dark in the XOH solution for 12 hours, then was heated to 70C for 10 minutes, to complete the hydrolysis of the ester groups. The product was then separated by reverse phase column chromatography (C-18 reverse phase silica 1.5 cm x 30 cm), using stepwise gradient elution with methanol in buffer .01 M KPO pH 6.85. 5% ~ethanol removed polar impurities. The monoglutamyl chlorin e6 was eluted with 6-8% methanol~ Chlorin e6 was eluted off the column with 25~ methanol. The methanol was removed by flash evaporation and the L-monoaspartyl chlorin e6 was precipitated at pH 3, collected and washed 3 times at the centrifuge with dilute acetic acid, and dried under vacuum. Yield 40 mg.
.
- .
-~8-1 EX~MPL,E 2 ?
Mono and Di (L) Aspartyl Chlorin eG Carbodiimide Method) , 400 mg of chlorin e6 and 1 g of L-aspartic acid diben~yl ester p--tosylate were dissolved in 75 ml of dimethy] formamide. Temperature or the solution was maintained at 65-70C. with stirring and 100 mg of N,N'-dicyclohexyl carbodiimide was added. (A total of 3 additions were made at 2 hour intervals). The solution was allowed to stir at this tmperature for a total of 20 hrs., then checked by TLC (reverse phase) (C-18 silica) plater 70% methanol, 30~ .01 M pH 6.85 KPO4 buffer. The rLC showed greater khan 50~ monosub-stitution with some di-substitution.
150 ml of ether was added, and agitated with 100 ml of wa-ter and several drops of glacial acetic acid. I'he ether phase was separated and the aqueous phase extracted several more times with 100 ml of ether.
The ether extracts were combined and washed with water (100 ml) four times to remove dimethyl formamide.
The aspartyl chlorin e6 esters were then extracted into 100 ml of 1 _ KOH (~ extractions of 25 ml each).
1'he KOH solution was allowed to stand at ambient tem-perature for 24 hours to hydrolyze. The components were separated by neutralizing the solution of pH 7 and applying to a reverse phase (C-18 silica) column (1.5 cm x 30 cm)~ The elution was performed using a 1 liter gradient of 30 % methanol to 80% methanol with 0.1 M pH 6.85 KPO4 buffer. Fractions were collected and characterized by TLC. The order of elution was di (L) 3 diaspartyl chlorin e6, L-monoaspartyl chlorin e6 and -fi9-1 chlorin e6. ~cthanol was removed was flash evaporation and the indivldual components precipitated at pH 3, using ilCl.
The products werc collected by centrifugation, washed several times with very dilute acetic acid and drived under vacuum. Yield was 23.3 mg.
.
:
~ ' ~2~ 2 1 Physical characteristics of representative compounds (relative polarity) is measured by a standard chromatographic system.
TLC Plat:3 D.~kcr si{~l8 20 ~rn pa~ticlo size 200 m~ coating thickncss Solvcn Systom 75~ mcthanol 25~ 0.01 .~; NO4 h~frcr pH G.85 Con~1ow~DGrivative Rf Ccr~oun~ Dcrivativo ~f .
Trans nosochlorin IX
~esoporphyrin IX - .32 " n~no~L)glutamyl .54 n~no~D,L)c~spartyl .53 " di(L)glutamyl .72 0 di~D,L)aspartyl .67 deutcropor2hyrin IX - .55 d i ~D ) aspartyl .66 " mono (D, L) asi~rtyl .75 nr~no ( L) asp~-tyl .5; " di (D, L) aspartyl .85 di (L) asoartyl .66 ~' n~no (L) aspartyl .75 " m~no (D, L) glutamyl .55 " di (L) aspartyl .84 " di~D,L)glutamyl .72 protoporp)lyrin IX - .33 " nDno ~L) aspa-tyl . .56 Trans-mesochlorin IX - .28 ~ di(L)aspartyl .73 nr~no (D) as;~artyl .52 photoprotoxrp.'lyrin IX - .58 lisc~Ter nuxture) di ~ D ) aspartyl .64 " mono ( D, L) asplrtyl .78 mono(L)aspartyl .53 " di(D,L)aspartyl .85 di (L)aspartyl .64 " mono(L)aspartyl .76 Hematoporphyrin IX - .78 " di (L) aspartyl .85 n~no(D,L)aspartyl .88 pyropheophorbide a - .07 di (D,L) a:,paLtyl .89 " (D,L) aspartyl .22 Chlorin e6 ~ .66 " (Llaspartyl .23 Mesoporphyrin IX
" m~no (L) aspartyl .77 " di (L) glutamyl .68 " di (L) asp~-tyl .84 ., m~no (Ll glutamyl .55 ~ono(L)glutamyl .79 protoporphyrin iX
Chlori n o - .57 " di (D, L) c ~partyl .70 n~no (L) glutamyl .74 " mono (D, L) aspartyl .57 Trans-nesochlorin IX - Coproporphyrin III .91 di (D, L) aspartyl .67 " ~no (D, L ) aspartyl .92 di (D, L) aspartyl .93 " tri (D, L) aspartyl .9;
~ tetra(D,L) aspartyl .97 The visible absorption spectrum in pyridine ~; for all of the aminodicarboxylic acid derivatives are identical to the parent porphyrin, chlorin or bacteriochlorin.
:
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~2~
The preparation of pharmacological dosages for the 1 administration of the active ingredient, that is the amino acid porph~rin adducts, which were prepared in Examples 1-22 hereinabove, is as follows:
EXAMPL~E 23 A tablet base was prepared by blending the following ingredient in the proportion by weight indicated:
Grams Sucrose, USP 80.3 Tapioca Starch 13. 2 Magnesium Stearate 4.4 Into this base, there was blended sufficient amino acid porphyrin adducts to provide tablets each containing 100 mg. of active indgredient.
;
A blend was prepared containing the following ingredients:
Grams Calcium phosphate 17.6 Dicalcium phosphate18.8 Magnesium trisilicate, USP 5 . 2 Lactose, U.S.P. 5. 2 Potato Starch 5.2 Magnesium Stearate A0.8 Magnesium Stearate B0.32 Porphyrin Amino Acid Adducts 20 This blend was divided and formed into capsules each containing 25 mg of active ingredient.
~2~i2~36~
EX~IPLE 25 1 To a commercially available raspberry flavored sugar syrup is added to the equivalent of 40 mg of the amino acid porphyrin adduct per milliliter and the mix.ture is homogenized in a mechanical dcvice for this purpose. This mixture is especially suitable for oral administration containing 200 mg of the active ingredient.
EXA~IPLE 26 A sterile solution of the following composition is prepared: 200 mg of the sodium salt of the amino acid porphyrln adduct is dissolved in a 0.9% NaCl solution so that the final concentration is 20 mgtml.
This solution is suitable for I.V. and I.M.
administration.
EX~PLE 27 The sodium salt of the amino acid porphyrin adduct is dissolved in 0.9% ~aCl solution so that the final concentration is 5 mg/ml. This is place in an aerosal dispenser with a hydrocarbon propellant. This preparation is suitable for topical application.
PREPAP~TIO~ OF A METAL SALT
.. .. ..
The sodium salt of the porphyrin amino acid adduct is prepared by dissolving said adduct in water containing an equimolar amount of sodium hydroxide and freeze drying the resulting mixture.
In this fashion, other metal salts are prepared including potassium, calcium, and lithium salts.
PREPARATION OF AN ACI~ SALT
The amino acid porphyrin adduct described in the preceding examples are converted to acid salts, e.g., hydro-chloride, by dissolving in an aqueous solution containing 6~
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dFied under vacuum.
~:
~Z~ 2 -~2-1 EX~ilPLE 16 Di and mono (D) asp~rtyl transmesochlorin IX (mixed anhvdride method in ],9 dioxane) , 50 m~ (0.000087 moles) of transmesochlorin IX
was dissolved in 50 ml of 1,4 dioxane. 210~ul (0.002 moles) of triethylamine was added wlth stirring. After 10 minutes, 210,ul (0.0019 moles) of ethylchloroformate was added.
~fter stirring 10 minutes, 50 ml (0.01 moles) of 0.2~1 KOH
containing 500 mg (0.0038 moles) of (D) aspartic acid was added dropwise wikh stirring to the THF solution. This mixture was sti.rred 60 minutes at room temperature.
The organic solvent was flashed off and the reaction mixture was checked by silica TLC for product.
Benzene/methanol/88% formic acid ~8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse pha.se (C-18 silica) column 2.5x30 cm. The reaction mixture was resolved usinga linear gradient of 40-80% methanol in O.OlM KPO4 buffer pH 6.85 (1 liter total volume).
The column effluent was collected via a fraction collector and the tube contents were pooled according to individual components. The order of elution was di (D) aspartyl transmesochlorin IX, mono (D) aspartyl trans-25 mesochlorin IX, and unsubstituted transmesochlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5-3Ø The ppt was washed 3 times with dilute acetic acid in water. The product was dried under vacuum.
.
~fi 3~862 1 ~X~II'LE 17 Di ancl mono (L) aspartyl transmesochlorin IX (mixed anhydride m~thocl in tetrall~drofuran) 135 mcJ (0.00023 moles) of trans~esochlorin IX
was dissolved in 100 ml of tetrah~drofuran (THF). 210~ul (0.002 moles) of triethylamine was added with stirring.
After 10 minutes, 210 ~1 (0.0019 moles) of ethylchloro~
formate was added. After stirring 10 minutes, 50 ml (0.015 moles) of 0.3~ KOll contailling 750 mg (0.0056 moles) of (L) aspartic acid was added dropwise with stirring to the T}IF solution. This mixture was stirred 60 minutes at room temperature.
The organie solvent was flashed off and the reaetion mixture was eheeked by siliea TLC for produet.
senzene/methanol/88~ formic aeid (8.5/1.5/0.13) was used to develop the chromatogram.
After checking for product, the solution was adjusted to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) eolumn 2.5x30 cm. The reaetion mixture was resolved using a linear gradient of 40-80~ methanol in 0.01M KPO4 buffer pH 6.85 (1 liter total volume).
The eolumn effluent was eolleeted via a fraetion eollector and the tube eontents were pooled aeeording to individual eomponents. The order of elution was di (L) aspartyl transmesoehlorin IX, mono (L) aspart~l trans-mesoehlorin IX, and unsubstituted transmesoehlorin IX.
The methanol was flashed off and the material was precipitated at pH 2.5~3Ø Thepptwas washed 3 times with dilute aeetie acid in water. The produet was dried -3O under vaeuum.
:
~lZ~ 162 --i,,~--1 EX~MPI,E 18 (D,I.)Aspart:~lvileophorbide a (carbodiimide method) 55 mg pheophorbide a was dissolved in 10 ml dimethylformamide. 50 mg (D,L) aspartic acid dimethyl ester dihydrochloride was added, then 100 mg of N,N'-dicyclohe~:yl-carbodiimide was added. The reaction was allowed to stand in the dark at room temperature for 1 hour, then 50 mg more carbodiimide was added.
After standing for 1 additional hour, SO mg more carbodiimide was added and the reaction allowed to stand in the dark for 12 hours at room temperature.
The solvent was removed under vacuum and the product dissolved in 50 ml 1~ KOH in methanol with 0.5 ml H20 and allowed to stand in the dark at room temperature.
The course of the hydrolysis is followed by thin layer chromatography (C-18 plates with solvent 75/25 MeOH/.OlM pH 6.85 KP04 buffer).
~ hen hydrolysis of the ester groups is essen-tially complete, the reaction is terminated by addition of a few drops of glacial acetic acid. The methanol is removed under vacuum and the product is dissolved in 20 ml 0.1 M NH40H. This solution is placed on a reverse phase (C-18 silica) column (1.5 cm x 30 cm).
The elution procedure was a linear gradient from 50 to 80~
methanol in 0.01 M KPO~ buffer pH 6.85 (500 ml total volume).
; The leading green-gray band contained the (D,L) aspartylpheophorbide a which was collected, flash evaporated to remove methyl alcohol, and precipitated at pH 3. The precipitate was collected and washed 3 times at the centrifuge with dilute acetic acid. The yield of dry product was 27 mg.
~z~
-~5-1 EX~PLE 19 . _ ~
L-Monoaspartyl chlorin e6 (carbodiimide method) . _ . . .
150 mg of chlorin e6 and 250 mg of L aspartic acid di-t.butyl ester hydrochloride were dissolved in 20 ml of dimethyl formamide. There was made a total of 3-100 mg additions of N,N'-dicyclohe~yl-carbodiimide at one hour intervals. After 4 hours, the reaction mi~ture was diluted with 300 ml ether, washed twice with 200 ml ll2O
then extracted with 40 ml 1 ~ XOH. The KOH solution was allowed to hydrolyze overniyht, then heated to 70C. for 10 minutes.
The pll oE the solution was adjusted to 7, then any residual ether was removed by flash evaporation.
The solution was then applied to a reverse phase (C-18 silica) column (1.5 cm x 30 cm). The product was purified by a stepwise elution of methanol/.01 M pH 6.85 KPO4 buffer. Eluted with 5~ methanol until unwanted polar pigments were removed. Monoaspartyl chlorin e6 was eluted off with 5-8~ methanol, and unreacted chlorin e was removed with 25% methanol. 6 The product was precipitated at pH 3 after flash evaporating briefly to remove methanol, then washed at the centrifuge 3 times with dilute acetic acid.
The product was dried under vacuum. Yield of L-monoaspartylchlorin e6 was 50 mg.
.
.~
~ ~ 35 : ::
, ~
i2 -~6-1 I`X~PL~ 20 L Glutamyl chlorin e~ (carbodiimide method) 110 mg chlorin e~ and 220 mg L-glutamic acid dimethyl cster hydrochloride were dlssolved in 15 ml of dimethyl formamide. 85 mg of N,N'--dicyclohexyl carbodiimide was then added, and the solu-tion stirred for 1 hour at room temperature. 42 mg more carbodiimide was then added, then 50 mg of carbodiimide was added at 1 hour intervals for two more additions. The reaction mixture was then allowed to stand for 12 hours, one more 50 mg carbodiimide addition was made, and the reaction allowed to stand for 3 hours. Progress of the reaction was followed by reverse phase thin layer chromatography 80% methanol, 20% KP04 buffer (.OlM pH 6.85). A further addition of 50 mg of carbodlimide, with standing, showed no further product formation.
200 ml of ether was added to the reaction mixture, and the ether solution was washed 4 times with water, approximately 100 ml per wash. The ether was then removed by flash evaporation, and the product was dissolved in approximately 25 ml of 3N Hcl. After 48 hours at room temperature, the solution was adjusted to pH3 with NH40H, and the precipitate was collected and washed at the centrifuge. The product was dissolved in 20% methanol/
water with a little NH40H, and applied to a reverse phase (C-18 sillca) column (1.5x30 cm). Elution was continued with 20% MeOH, KP04 buffer (O.OlM pH 6.5).
This removed the product (L-Glutamyl chlorin e4). The methanol concentration was increased to remove the un-reacted chlorin e4.
The solution was flash evaporated until themethanol was substantially removed, then the products were precipitated at pH3 by addition of Hcl, collected and washed at the centrifuge with dilute acetic acid and dried under vacuum. Yield of mono-L-glutamyl chlorin e4 21 mg.
Yield of recovered chlorin e4 59 mg.
~2~;2~
l EXAM~LE 21 L-Monoglutamyl chlorin eG lcarbodiimide method) . . . _ . ;
130 mg of chlorin e6 and 260 mg L glutamic acid dimethyl ester hydrochloride was dissolved in l~ ml of dimethylformamide. 100 mg of N,N'-dicyclohexyl-carbodiimide was added and the reaction mixture stirred for l hour. 50 mg more carbodiimide was then added.
After 1 hour, the reaction mixture appeared to contain 75-80~ of the monosubstituted product by reverse phase TLC (C-18 plates with 70~ MeOH, 30~O .01 M KPO~ pH 6.85).
200 ml Di.ethyl ether was added, washed twice with 100 ml H2O, then extracted with 30 ml l M KOH.
The product was allowed to hydrolyze in the dark in the XOH solution for 12 hours, then was heated to 70C for 10 minutes, to complete the hydrolysis of the ester groups. The product was then separated by reverse phase column chromatography (C-18 reverse phase silica 1.5 cm x 30 cm), using stepwise gradient elution with methanol in buffer .01 M KPO pH 6.85. 5% ~ethanol removed polar impurities. The monoglutamyl chlorin e6 was eluted with 6-8% methanol~ Chlorin e6 was eluted off the column with 25~ methanol. The methanol was removed by flash evaporation and the L-monoaspartyl chlorin e6 was precipitated at pH 3, collected and washed 3 times at the centrifuge with dilute acetic acid, and dried under vacuum. Yield 40 mg.
.
- .
-~8-1 EX~MPL,E 2 ?
Mono and Di (L) Aspartyl Chlorin eG Carbodiimide Method) , 400 mg of chlorin e6 and 1 g of L-aspartic acid diben~yl ester p--tosylate were dissolved in 75 ml of dimethy] formamide. Temperature or the solution was maintained at 65-70C. with stirring and 100 mg of N,N'-dicyclohexyl carbodiimide was added. (A total of 3 additions were made at 2 hour intervals). The solution was allowed to stir at this tmperature for a total of 20 hrs., then checked by TLC (reverse phase) (C-18 silica) plater 70% methanol, 30~ .01 M pH 6.85 KPO4 buffer. The rLC showed greater khan 50~ monosub-stitution with some di-substitution.
150 ml of ether was added, and agitated with 100 ml of wa-ter and several drops of glacial acetic acid. I'he ether phase was separated and the aqueous phase extracted several more times with 100 ml of ether.
The ether extracts were combined and washed with water (100 ml) four times to remove dimethyl formamide.
The aspartyl chlorin e6 esters were then extracted into 100 ml of 1 _ KOH (~ extractions of 25 ml each).
1'he KOH solution was allowed to stand at ambient tem-perature for 24 hours to hydrolyze. The components were separated by neutralizing the solution of pH 7 and applying to a reverse phase (C-18 silica) column (1.5 cm x 30 cm)~ The elution was performed using a 1 liter gradient of 30 % methanol to 80% methanol with 0.1 M pH 6.85 KPO4 buffer. Fractions were collected and characterized by TLC. The order of elution was di (L) 3 diaspartyl chlorin e6, L-monoaspartyl chlorin e6 and -fi9-1 chlorin e6. ~cthanol was removed was flash evaporation and the indivldual components precipitated at pH 3, using ilCl.
The products werc collected by centrifugation, washed several times with very dilute acetic acid and drived under vacuum. Yield was 23.3 mg.
.
:
~ ' ~2~ 2 1 Physical characteristics of representative compounds (relative polarity) is measured by a standard chromatographic system.
TLC Plat:3 D.~kcr si{~l8 20 ~rn pa~ticlo size 200 m~ coating thickncss Solvcn Systom 75~ mcthanol 25~ 0.01 .~; NO4 h~frcr pH G.85 Con~1ow~DGrivative Rf Ccr~oun~ Dcrivativo ~f .
Trans nosochlorin IX
~esoporphyrin IX - .32 " n~no~L)glutamyl .54 n~no~D,L)c~spartyl .53 " di(L)glutamyl .72 0 di~D,L)aspartyl .67 deutcropor2hyrin IX - .55 d i ~D ) aspartyl .66 " mono (D, L) asi~rtyl .75 nr~no ( L) asp~-tyl .5; " di (D, L) aspartyl .85 di (L) asoartyl .66 ~' n~no (L) aspartyl .75 " m~no (D, L) glutamyl .55 " di (L) aspartyl .84 " di~D,L)glutamyl .72 protoporp)lyrin IX - .33 " nDno ~L) aspa-tyl . .56 Trans-mesochlorin IX - .28 ~ di(L)aspartyl .73 nr~no (D) as;~artyl .52 photoprotoxrp.'lyrin IX - .58 lisc~Ter nuxture) di ~ D ) aspartyl .64 " mono ( D, L) asplrtyl .78 mono(L)aspartyl .53 " di(D,L)aspartyl .85 di (L)aspartyl .64 " mono(L)aspartyl .76 Hematoporphyrin IX - .78 " di (L) aspartyl .85 n~no(D,L)aspartyl .88 pyropheophorbide a - .07 di (D,L) a:,paLtyl .89 " (D,L) aspartyl .22 Chlorin e6 ~ .66 " (Llaspartyl .23 Mesoporphyrin IX
" m~no (L) aspartyl .77 " di (L) glutamyl .68 " di (L) asp~-tyl .84 ., m~no (Ll glutamyl .55 ~ono(L)glutamyl .79 protoporphyrin iX
Chlori n o - .57 " di (D, L) c ~partyl .70 n~no (L) glutamyl .74 " mono (D, L) aspartyl .57 Trans-nesochlorin IX - Coproporphyrin III .91 di (D, L) aspartyl .67 " ~no (D, L ) aspartyl .92 di (D, L) aspartyl .93 " tri (D, L) aspartyl .9;
~ tetra(D,L) aspartyl .97 The visible absorption spectrum in pyridine ~; for all of the aminodicarboxylic acid derivatives are identical to the parent porphyrin, chlorin or bacteriochlorin.
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The preparation of pharmacological dosages for the 1 administration of the active ingredient, that is the amino acid porph~rin adducts, which were prepared in Examples 1-22 hereinabove, is as follows:
EXAMPL~E 23 A tablet base was prepared by blending the following ingredient in the proportion by weight indicated:
Grams Sucrose, USP 80.3 Tapioca Starch 13. 2 Magnesium Stearate 4.4 Into this base, there was blended sufficient amino acid porphyrin adducts to provide tablets each containing 100 mg. of active indgredient.
;
A blend was prepared containing the following ingredients:
Grams Calcium phosphate 17.6 Dicalcium phosphate18.8 Magnesium trisilicate, USP 5 . 2 Lactose, U.S.P. 5. 2 Potato Starch 5.2 Magnesium Stearate A0.8 Magnesium Stearate B0.32 Porphyrin Amino Acid Adducts 20 This blend was divided and formed into capsules each containing 25 mg of active ingredient.
~2~i2~36~
EX~IPLE 25 1 To a commercially available raspberry flavored sugar syrup is added to the equivalent of 40 mg of the amino acid porphyrin adduct per milliliter and the mix.ture is homogenized in a mechanical dcvice for this purpose. This mixture is especially suitable for oral administration containing 200 mg of the active ingredient.
EXA~IPLE 26 A sterile solution of the following composition is prepared: 200 mg of the sodium salt of the amino acid porphyrln adduct is dissolved in a 0.9% NaCl solution so that the final concentration is 20 mgtml.
This solution is suitable for I.V. and I.M.
administration.
EX~PLE 27 The sodium salt of the amino acid porphyrin adduct is dissolved in 0.9% ~aCl solution so that the final concentration is 5 mg/ml. This is place in an aerosal dispenser with a hydrocarbon propellant. This preparation is suitable for topical application.
PREPAP~TIO~ OF A METAL SALT
.. .. ..
The sodium salt of the porphyrin amino acid adduct is prepared by dissolving said adduct in water containing an equimolar amount of sodium hydroxide and freeze drying the resulting mixture.
In this fashion, other metal salts are prepared including potassium, calcium, and lithium salts.
PREPARATION OF AN ACI~ SALT
The amino acid porphyrin adduct described in the preceding examples are converted to acid salts, e.g., hydro-chloride, by dissolving in an aqueous solution containing 6~
-5~-an equivalent amount of acid, c.g., hydrochloric acid, and 1 tile solution is evaporated to dryness to obtain the solid salt. ~lternately, alcoholic solutions of hydrogen ch].oride gas, dissolved in ethanol can be used in lieu of the aqueous acid solution and the acid salt is obtained by evaporation of the solvent or crystallization from the alcohol, e.g., by addition of a non-solvent.
.
.
' iZ81E`2 l The ~ollowinc~ Erotocols desc~ib~ th~ pr~c~dure for the utili~ation of -these new compounds of the present invention in the treatment of rat tumors.
~XAMPL~ 29 The photodynamic therapy experiments have been carried out using the compound mono- (L)-aspartyl chloxin e6.
Two transplantable tumor lines in Buffalo rats have been used, Morris Hepatoma 7777 and ~lorris Elepatoma 5123 tc. The tumors were transplanted subcutaneously on the outside of the thigh.
During treatment, the tumors ranyed in size between 1 and 2~5 cm in diameter.
The general treatment regime is as follows. The rats are injected with a solution of the c~llorin prepared as -ollo~s: 20 mg of the sodium salt of the chlorin was dissolved in 1 ml of 0.9~ NaCl. The chlorin solution was then injected intravenously through t:he external jugular while the rat was anesthetized with ether. The volume of solu~ion injected was calculated based upon the weiqht of the animal and the dosage, on a weight to weight basis, for the particular experiment. A specified time interval was then allo~ed to elapse before light treatment was instigated~
Light treatment of the rats was without anesthesia.
The rats were restrained, the hair removed in the treatment area and treated with laser light from a Cooper Aurora argon pumped, tunable dye laser.
The laser was equipped with a fiber optic light delivery system coupled to a microlens system developed by Dr. ~aniel Doiron, D.R.D. Consultiny, Santa Barbara, California.
The lens disperses the laser beam, providing a circular distribution of light with homogenous light intensity throughout the area~of the incident light beam. The wave-lensth of light was ad~usted using a Hartridge reversion speclroscope. The light intensity was determined using a Yellow Sprinss Instrument, ~;odel 65A, radiometer.
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1 The micro lcns was positioned at such a distance from the s~;in of the animal so as to provide an illumination diamcter o 1.5cm, and the li~ht flu~ was varied by control of the laser output.' Subsequent to illumination, the ani~al was returned to its cage and, 24 hours later, it was treated intravenousl'y in the e~ternal jugular vein with 14 mg of Evans 31ue dye, dissolved in 250 ~1 of 0.9~ NaC1. Two hours after injection, the rat was sacrificed and the tumor' cross-sectioned. The e~tent of tumor necrosis was assessed by the lack of dye uptake (l~, and the depth of the necrotic cross section of the tumor was recorded in millimeters.
Table II summarizes the e'fects of these drugs ; on tumors and includes a range of wavelengths, dosages, in-tensities, and time intervals for treatment. This has been necessary, in order to attempt to establish the optimal conditions for phototherapy u~ilizing this new drug.' The conditions described result in measurable and significant damage to the tumors.
In all cases except where noted, tissue damage occurred selectively to the tumor tissue as assayed by the Evans Blue method, even though, in nearly all'cases, normal skin overlayed the tumor and the treatment'area overlapped significant areas of normal muscle tissue.
I.C. Berenbaum~ Br. J. Cancer 45: 571(1982) ~2G~ Z
~57-1 ~he photodynamic therapy date is presented in tab-ular form. Column No. 2 is the total light dose adrninistered in terms of Joules per square centimeter. Column No. 3 is the dose of mono(L)aspa~^tyl chiorin e6 administered in tcrms of mg of drug per ~ilogram of rat body weight. Column No. 4 is the time lapse between administration of drug and treatment with laser light. Column No. 5 is the wavelength of treatment light in nanometers. Column No~6 is the intenslty of the treatment light in milliwatts per square centimeter. In Column No. 7, x is the mean depth of necrosis in millimeters of ~he tumor tissue, i.e., the distance from the necrotic top of the turnor next to the skin to the necrotic edge of the tumor most distant from the skin.
S.D. is the standard deviation of x.
(N) is the number of turnors or legs involved in the experiment.
Column No. 8 is the range of depth of necrosis in millimeters within the group.
~Z~2536Z
TA3Lr I
~i~e in hrs.
d.ugbcwn wave-joules/ dosed.ug &lnch incensicy range curno; c~ m~/kg li~hc nm mW/cm2 x s.d. (n) mm _ _ _ _ _ _ _ 77l7 10 20 24 655 100 2.8 + 1.6 (10) 1-6 24 665 200 2.8 ~ 1.0 (3) 2-4 2~ 650 700 2.9 + 1.1 (5) 1.5-4.5 24 660 100 4.6 + 1. (7) 2,5-8 24 660 200 3.6 + 1.4 (6) 1-6 24 665 100 5.9 + 2.4 (7) 2.5-9 48 655 7.00 7.5 + 4.2 (2) 4.5-10.5 .0 24 655 100 4.1 + 1.3 (17) 2-6 24 660 100 5 4 + 1 9 (8) 2-7.5 Z8.4 20 24 655 200 5 4 ~ 1 6 (29) 2.5-11 28.4 15 24 655 200 4,0 ~ (2) 56,8 15 24 655 200 ~,5 + 0.7 (2) 4-5 56.8 20 2~ 655 200 4,5 + 0,7 (2) 4-5 113 15 24 6S5 200 Damage Non S~ecific ~3) 113 20 24 655 200 4.0 ~ 1.2 (4) 3-5 113 5 48 655 200 3,8 + 1,2 (6) 2-5 169 15 24 655 200 Damage Mon Specific ~5) 169 20 24 655 200 5,0 (2) 169 5 48 655 200 4,8 + 0,8 (6) 3.5-6 5123CC 10 20 24 655 100 4.0 ~ (1) 24 655 100 2.7 ~ 1.0 (7) 1-4 48 6S5 200 2.0 ~ (2) 24 665 100 4.9 ~ 1.0 (8) 3.5-4 24 655 200 4.3 + 1.1 (8) 2,5-6 2/10*
28,4 20 24 655 100 4,1 + 1,3 (4) 3-6 1/5':
28.4 20 24 655 200 3.2 ~ 0.3 (3) 3-3.5 4/7' 24 65S 100 4.4 + 1.0 (4) 3.5-5.5 ~~ 56.8 20 24 655 200 3.0 ~1) 56.8 5 48 655 200 No effecc ~9) 113 5 48 655 200 No effec~ (8) '::No efFecc Con;rol Racs: No Tumor 10 20 24 665 100 ~10) 24 h_ ~valuation: 5 showed some increased dye uptake in the skin at point of treatment.
24 665 100 (10) 24 hr ~valuation: 6 showed some increased dve uptake in the sk n at point of t-ea'ment.
24 665 100 (10) 14 dav ~valuation: none showed signs or skin or tumo~ necrosis and hair had regrown normally.
24 665 100 (10) 14 Evaluation: one leg of one animal showed some sign of muscle necrosis. Skin appeared normal and hair regrew normally on all animals.
~2G~136;2 . .
PDT Experiments with ~lice and mono-L-Aspartyl Chlorin e6 .
PDT with the mono-L-aspartyl chlorin e6 tetrasodium salt was evaluated in another animal/tumor model system.
The tumor, SMT-F, was transplanted subcutaneously into the shoulder/rib area (one side only) of DBA/2 Ha ROS D~
Ha mice. The treatment regime was started when the tumors had reached a dimension of approximately 1.5 - 2 cm long by 1 cm wide and 0.7 to 1 cm deep, (approximately 7 to 8 days after transplant). The drug was administered ~hrough intraperitoneal injection at a concentration of 4 mg/ml.
Specific parameters and results are listed in the ~ollowiny table. The e~aluation was done 24 hours af-ter light treatment ~
using the vital stain Evans B1UQ in a procedure similar to that which was used for evaluating tumor necrosis in the Buffalo rats, the only difference being the intraperitoneal injection of the dye at a dose of 5 mg per mouse. The headings of each column are the same as the rat system.
Jo~les/ Drug Dosa Timein Hrs Wave- Inten~ty ~ s.d. [n~ D s.~.
cm mg/kg btwn drug length mW/c (mm) (mm) ~Iiaht nm 24 665 I00 6.6~ 2.0 7 I0.3~ 3.
No indication of necrosis of normal tissue (muscle or skin) was observed.
Similar results are obtained when the compounds in Examples 1-22 are administered to similarly pretreated mice.
.~
126~l36;2 PDT Experiments with Rats and mono-L-Glutamyl Chlorin e6 Buffalo rats with Morris Hepatoma 7777 transplanted subcutaneously on the outside of each hind leg were subjected to photodynamic therapy, using mono-L-glutamyl chlorin e6 tetrasodium salt as the drug.
The experimental procedure was the same as is employed for testing of the mono-L-aspartyl chlorin e6.
Specific parameters and results are listed in the table below.
No visible damage - as assessed by the Evans Blue method - to the overlying skin or normal muscle tissue surrounding the tumor was observed, although the 1.5 cm diameter area of light treatment overlapped normal tissue in several cases.
Column No. 1 is the total light dose administered in terms of Joules per square centimeter. Column No. 2 is the dose of chlorin administered in terms of mg of drug per kilogram of rat body weight. Column No. 3 is the time lapse between administration of drug and treatment with laser light.
Column No. 4 is the wavelength of treatment light in nanometers. Column No. 5 is the intensity of the treatment light in milliwatts, per square centimeter. In Column No. 6, X is the mean depth of necrosis in millimeters of the tumor tissue, i.e., the distance from the necrotic top of the tumor next to the skin to the necrotic edge of the tumor most distant from the skin. s~d. is the standard deviation of X, (n) is the number of tumors or legs invoIved in the experiment. D is the mean diameter of tumor necrosis with the following s.d. the standard deviation for D.
Joules/cm2 Drug Dose Timein Hrs Wave- Intens~y X s.d. (n) D s.d mg/kg btwn drug length mWIcm lmm) (mm) ~ light nm ; 35 20 20 24 665 lO0 3.4-l.3 l7 9.6~ 7.
Similar results are obtained when Compounds 1-22 of the preceding examples are administered to similarly pretreated rats.
~,, -61- lZG2862 EX~lPLE, 32 PDT EXPERII~lENTS ~ITH ~IICE AND ~.lONO-L~ SPARTYL CHLORIN e6 Thc Sl~lT-F tumor in DBA/2 Ha ROS D+ Ha mouse system was used to evaluate thc photodynamic effcct of mono-L-glutamyl chlorin e6 tetrasodium salt.
The protocol is the same as the experiment involving mono - L - aspartyl chlorin e6 and the column heaclings are the same as those used in this systcm and -- the rat system.
Joules/ Drug Dose Time in Hrs Wave- Intensity X s.d. ~ (n) D s.d.
g g bt w n d l-u~ I ength mW /cm 2 ( mm ) _ _ ( mm ) 24 665 100 7.9 + 2.9 8 13.9 -3.5 * A ninth mouse showed no response and was not included in the above statistical analysis. This is because of the possibility that drug was injected into the gut instead of the peritoneum.
~2G~8~Z
EXAr~1'LE33 l 11uman cells (lleLa, strain D~8/A112) were incubated in 25 cm2 plastic culture flasks for 2~ h to permit attachment.
The~ were then rinsed, incubated for lO minute periods in ~1am's F-l2 medium containing porphyrins, rinsed again in ~1am's F-l2 meclium without porphyrins for 5 minutes, then illuminated for various periods, and cultured at 37C in complete medium for 2~ h. Cell counts were then made using a phase contrast microscope of the fraction of the surviving cells. The broad band incandescent light source used was adjusted to give an incident light intensity of 5 x 105 ery cm 2 sec l. A positioning device permitted illuminating each of five areas of a flask for different times; one area was not illuminated and served as a dar}c control. This gave a - four light dose survival curve from a single flask; the technique is thus suitable for the rapid and econ-omical screening of large numbers of potential photosensitizing agents. The results of this experiment are shown in Table III.
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Sensitizcr ~eriod or Illumlnnt~on (mln ) Di aspartyl mesoporphyrin IX 100 100 100 9~ 5 0 Aspartyl pyropheophorbide a 100 1000 0 0 0 Aspartyl pyropheophorbide a (same solution as above, kept in refrigerator) 100 100 û O O O
Two tenths ml of 4xlO 4 M solution (or suspension) of the sensitizer were mixed with 1.~ ml of Ham's medium for the experiments - thus the cells were treated with 4xlO 5 M of sensitizer. The cells were incubated for 10 minutes in the presence of sensitizer, then washed for 5 minutes in Ham's without sensitizer and then illuminated in Ham's for the time indicated.
~2~862 EX~IPLE 3~
1 SCRE~NING O~ PORPIIYRIN FLUORESC~NCE ~S A
Ft~NCTION_OF MOLECULAR STRUCTURE
Two -transplantable tumor lines in Buffalo rats were used, Morris Hepatoma 7777 and ~lorris ~-lepatoma 5123tc. The tumors were transplanted intrarnuscularly on the rear of the thigh of -the rats. After 10-1~ days, when the tumors reached the appropriate size, 2 mg (0.5 ml~ of an amino acid porphyrin adduct solu-tion were introduced intraperitoneall~ into the rats. The amino acid porphyrin adduct solution was prepared as follows: 4 mg of the amino acid porphyrin was dissolved in 0.1 M ~aOH and adjusted to physiological pH with 1 M HCl.
The rats were killed 24 hours af-ter the injection.
The tumor was bisected ln situ. The porphyrin fluorescence was determined under a constant intensity UV light source.
Tables IV, V, VI and VII list the porphyrin derivatives tested. The compounds are grouped alphabetically.
Following the name of the porphyrin is a number that indicates the total number of tumors examined. The next column of figures (A) is a number calculated as follows: the porphyrin fluorescence within the tumor was ran~ed visually ;~ by one person under a constant intensity U.V. light source according to the scale 0, +~, 1, 2, 3, 4. This number was then multiplied by the percent of the tumor demonstra-ting this fluorescence, i.e. (+~) (80%) + (~1) (10%) = 50. More often than not, the A value in the table represent averages obtained in several series of separa-te experiments conducted at different times.
The "C value" for each tumor is the "A value" for that tumor divided by the average diameter of the tumor, in cm.
A time study of 12-72 hours was also conducted on some of the tumors. The procedure is the same as above, e~cept 1 mg of the amino acid adduct was utilized. The results are also indicated in Table IV.
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' iZ81E`2 l The ~ollowinc~ Erotocols desc~ib~ th~ pr~c~dure for the utili~ation of -these new compounds of the present invention in the treatment of rat tumors.
~XAMPL~ 29 The photodynamic therapy experiments have been carried out using the compound mono- (L)-aspartyl chloxin e6.
Two transplantable tumor lines in Buffalo rats have been used, Morris Hepatoma 7777 and ~lorris Elepatoma 5123 tc. The tumors were transplanted subcutaneously on the outside of the thigh.
During treatment, the tumors ranyed in size between 1 and 2~5 cm in diameter.
The general treatment regime is as follows. The rats are injected with a solution of the c~llorin prepared as -ollo~s: 20 mg of the sodium salt of the chlorin was dissolved in 1 ml of 0.9~ NaCl. The chlorin solution was then injected intravenously through t:he external jugular while the rat was anesthetized with ether. The volume of solu~ion injected was calculated based upon the weiqht of the animal and the dosage, on a weight to weight basis, for the particular experiment. A specified time interval was then allo~ed to elapse before light treatment was instigated~
Light treatment of the rats was without anesthesia.
The rats were restrained, the hair removed in the treatment area and treated with laser light from a Cooper Aurora argon pumped, tunable dye laser.
The laser was equipped with a fiber optic light delivery system coupled to a microlens system developed by Dr. ~aniel Doiron, D.R.D. Consultiny, Santa Barbara, California.
The lens disperses the laser beam, providing a circular distribution of light with homogenous light intensity throughout the area~of the incident light beam. The wave-lensth of light was ad~usted using a Hartridge reversion speclroscope. The light intensity was determined using a Yellow Sprinss Instrument, ~;odel 65A, radiometer.
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~Z~i~1362 -56~
1 The micro lcns was positioned at such a distance from the s~;in of the animal so as to provide an illumination diamcter o 1.5cm, and the li~ht flu~ was varied by control of the laser output.' Subsequent to illumination, the ani~al was returned to its cage and, 24 hours later, it was treated intravenousl'y in the e~ternal jugular vein with 14 mg of Evans 31ue dye, dissolved in 250 ~1 of 0.9~ NaC1. Two hours after injection, the rat was sacrificed and the tumor' cross-sectioned. The e~tent of tumor necrosis was assessed by the lack of dye uptake (l~, and the depth of the necrotic cross section of the tumor was recorded in millimeters.
Table II summarizes the e'fects of these drugs ; on tumors and includes a range of wavelengths, dosages, in-tensities, and time intervals for treatment. This has been necessary, in order to attempt to establish the optimal conditions for phototherapy u~ilizing this new drug.' The conditions described result in measurable and significant damage to the tumors.
In all cases except where noted, tissue damage occurred selectively to the tumor tissue as assayed by the Evans Blue method, even though, in nearly all'cases, normal skin overlayed the tumor and the treatment'area overlapped significant areas of normal muscle tissue.
I.C. Berenbaum~ Br. J. Cancer 45: 571(1982) ~2G~ Z
~57-1 ~he photodynamic therapy date is presented in tab-ular form. Column No. 2 is the total light dose adrninistered in terms of Joules per square centimeter. Column No. 3 is the dose of mono(L)aspa~^tyl chiorin e6 administered in tcrms of mg of drug per ~ilogram of rat body weight. Column No. 4 is the time lapse between administration of drug and treatment with laser light. Column No. 5 is the wavelength of treatment light in nanometers. Column No~6 is the intenslty of the treatment light in milliwatts per square centimeter. In Column No. 7, x is the mean depth of necrosis in millimeters of ~he tumor tissue, i.e., the distance from the necrotic top of the turnor next to the skin to the necrotic edge of the tumor most distant from the skin.
S.D. is the standard deviation of x.
(N) is the number of turnors or legs involved in the experiment.
Column No. 8 is the range of depth of necrosis in millimeters within the group.
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~i~e in hrs.
d.ugbcwn wave-joules/ dosed.ug &lnch incensicy range curno; c~ m~/kg li~hc nm mW/cm2 x s.d. (n) mm _ _ _ _ _ _ _ 77l7 10 20 24 655 100 2.8 + 1.6 (10) 1-6 24 665 200 2.8 ~ 1.0 (3) 2-4 2~ 650 700 2.9 + 1.1 (5) 1.5-4.5 24 660 100 4.6 + 1. (7) 2,5-8 24 660 200 3.6 + 1.4 (6) 1-6 24 665 100 5.9 + 2.4 (7) 2.5-9 48 655 7.00 7.5 + 4.2 (2) 4.5-10.5 .0 24 655 100 4.1 + 1.3 (17) 2-6 24 660 100 5 4 + 1 9 (8) 2-7.5 Z8.4 20 24 655 200 5 4 ~ 1 6 (29) 2.5-11 28.4 15 24 655 200 4,0 ~ (2) 56,8 15 24 655 200 ~,5 + 0.7 (2) 4-5 56.8 20 2~ 655 200 4,5 + 0,7 (2) 4-5 113 15 24 6S5 200 Damage Non S~ecific ~3) 113 20 24 655 200 4.0 ~ 1.2 (4) 3-5 113 5 48 655 200 3,8 + 1,2 (6) 2-5 169 15 24 655 200 Damage Mon Specific ~5) 169 20 24 655 200 5,0 (2) 169 5 48 655 200 4,8 + 0,8 (6) 3.5-6 5123CC 10 20 24 655 100 4.0 ~ (1) 24 655 100 2.7 ~ 1.0 (7) 1-4 48 6S5 200 2.0 ~ (2) 24 665 100 4.9 ~ 1.0 (8) 3.5-4 24 655 200 4.3 + 1.1 (8) 2,5-6 2/10*
28,4 20 24 655 100 4,1 + 1,3 (4) 3-6 1/5':
28.4 20 24 655 200 3.2 ~ 0.3 (3) 3-3.5 4/7' 24 65S 100 4.4 + 1.0 (4) 3.5-5.5 ~~ 56.8 20 24 655 200 3.0 ~1) 56.8 5 48 655 200 No effecc ~9) 113 5 48 655 200 No effec~ (8) '::No efFecc Con;rol Racs: No Tumor 10 20 24 665 100 ~10) 24 h_ ~valuation: 5 showed some increased dye uptake in the skin at point of treatment.
24 665 100 (10) 24 hr ~valuation: 6 showed some increased dve uptake in the sk n at point of t-ea'ment.
24 665 100 (10) 14 dav ~valuation: none showed signs or skin or tumo~ necrosis and hair had regrown normally.
24 665 100 (10) 14 Evaluation: one leg of one animal showed some sign of muscle necrosis. Skin appeared normal and hair regrew normally on all animals.
~2G~136;2 . .
PDT Experiments with ~lice and mono-L-Aspartyl Chlorin e6 .
PDT with the mono-L-aspartyl chlorin e6 tetrasodium salt was evaluated in another animal/tumor model system.
The tumor, SMT-F, was transplanted subcutaneously into the shoulder/rib area (one side only) of DBA/2 Ha ROS D~
Ha mice. The treatment regime was started when the tumors had reached a dimension of approximately 1.5 - 2 cm long by 1 cm wide and 0.7 to 1 cm deep, (approximately 7 to 8 days after transplant). The drug was administered ~hrough intraperitoneal injection at a concentration of 4 mg/ml.
Specific parameters and results are listed in the ~ollowiny table. The e~aluation was done 24 hours af-ter light treatment ~
using the vital stain Evans B1UQ in a procedure similar to that which was used for evaluating tumor necrosis in the Buffalo rats, the only difference being the intraperitoneal injection of the dye at a dose of 5 mg per mouse. The headings of each column are the same as the rat system.
Jo~les/ Drug Dosa Timein Hrs Wave- Inten~ty ~ s.d. [n~ D s.~.
cm mg/kg btwn drug length mW/c (mm) (mm) ~Iiaht nm 24 665 I00 6.6~ 2.0 7 I0.3~ 3.
No indication of necrosis of normal tissue (muscle or skin) was observed.
Similar results are obtained when the compounds in Examples 1-22 are administered to similarly pretreated mice.
.~
126~l36;2 PDT Experiments with Rats and mono-L-Glutamyl Chlorin e6 Buffalo rats with Morris Hepatoma 7777 transplanted subcutaneously on the outside of each hind leg were subjected to photodynamic therapy, using mono-L-glutamyl chlorin e6 tetrasodium salt as the drug.
The experimental procedure was the same as is employed for testing of the mono-L-aspartyl chlorin e6.
Specific parameters and results are listed in the table below.
No visible damage - as assessed by the Evans Blue method - to the overlying skin or normal muscle tissue surrounding the tumor was observed, although the 1.5 cm diameter area of light treatment overlapped normal tissue in several cases.
Column No. 1 is the total light dose administered in terms of Joules per square centimeter. Column No. 2 is the dose of chlorin administered in terms of mg of drug per kilogram of rat body weight. Column No. 3 is the time lapse between administration of drug and treatment with laser light.
Column No. 4 is the wavelength of treatment light in nanometers. Column No. 5 is the intensity of the treatment light in milliwatts, per square centimeter. In Column No. 6, X is the mean depth of necrosis in millimeters of the tumor tissue, i.e., the distance from the necrotic top of the tumor next to the skin to the necrotic edge of the tumor most distant from the skin. s~d. is the standard deviation of X, (n) is the number of tumors or legs invoIved in the experiment. D is the mean diameter of tumor necrosis with the following s.d. the standard deviation for D.
Joules/cm2 Drug Dose Timein Hrs Wave- Intens~y X s.d. (n) D s.d mg/kg btwn drug length mWIcm lmm) (mm) ~ light nm ; 35 20 20 24 665 lO0 3.4-l.3 l7 9.6~ 7.
Similar results are obtained when Compounds 1-22 of the preceding examples are administered to similarly pretreated rats.
~,, -61- lZG2862 EX~lPLE, 32 PDT EXPERII~lENTS ~ITH ~IICE AND ~.lONO-L~ SPARTYL CHLORIN e6 Thc Sl~lT-F tumor in DBA/2 Ha ROS D+ Ha mouse system was used to evaluate thc photodynamic effcct of mono-L-glutamyl chlorin e6 tetrasodium salt.
The protocol is the same as the experiment involving mono - L - aspartyl chlorin e6 and the column heaclings are the same as those used in this systcm and -- the rat system.
Joules/ Drug Dose Time in Hrs Wave- Intensity X s.d. ~ (n) D s.d.
g g bt w n d l-u~ I ength mW /cm 2 ( mm ) _ _ ( mm ) 24 665 100 7.9 + 2.9 8 13.9 -3.5 * A ninth mouse showed no response and was not included in the above statistical analysis. This is because of the possibility that drug was injected into the gut instead of the peritoneum.
~2G~8~Z
EXAr~1'LE33 l 11uman cells (lleLa, strain D~8/A112) were incubated in 25 cm2 plastic culture flasks for 2~ h to permit attachment.
The~ were then rinsed, incubated for lO minute periods in ~1am's F-l2 medium containing porphyrins, rinsed again in ~1am's F-l2 meclium without porphyrins for 5 minutes, then illuminated for various periods, and cultured at 37C in complete medium for 2~ h. Cell counts were then made using a phase contrast microscope of the fraction of the surviving cells. The broad band incandescent light source used was adjusted to give an incident light intensity of 5 x 105 ery cm 2 sec l. A positioning device permitted illuminating each of five areas of a flask for different times; one area was not illuminated and served as a dar}c control. This gave a - four light dose survival curve from a single flask; the technique is thus suitable for the rapid and econ-omical screening of large numbers of potential photosensitizing agents. The results of this experiment are shown in Table III.
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~2~862 EX~IPLE 3~
1 SCRE~NING O~ PORPIIYRIN FLUORESC~NCE ~S A
Ft~NCTION_OF MOLECULAR STRUCTURE
Two -transplantable tumor lines in Buffalo rats were used, Morris Hepatoma 7777 and ~lorris ~-lepatoma 5123tc. The tumors were transplanted intrarnuscularly on the rear of the thigh of -the rats. After 10-1~ days, when the tumors reached the appropriate size, 2 mg (0.5 ml~ of an amino acid porphyrin adduct solu-tion were introduced intraperitoneall~ into the rats. The amino acid porphyrin adduct solution was prepared as follows: 4 mg of the amino acid porphyrin was dissolved in 0.1 M ~aOH and adjusted to physiological pH with 1 M HCl.
The rats were killed 24 hours af-ter the injection.
The tumor was bisected ln situ. The porphyrin fluorescence was determined under a constant intensity UV light source.
Tables IV, V, VI and VII list the porphyrin derivatives tested. The compounds are grouped alphabetically.
Following the name of the porphyrin is a number that indicates the total number of tumors examined. The next column of figures (A) is a number calculated as follows: the porphyrin fluorescence within the tumor was ran~ed visually ;~ by one person under a constant intensity U.V. light source according to the scale 0, +~, 1, 2, 3, 4. This number was then multiplied by the percent of the tumor demonstra-ting this fluorescence, i.e. (+~) (80%) + (~1) (10%) = 50. More often than not, the A value in the table represent averages obtained in several series of separa-te experiments conducted at different times.
The "C value" for each tumor is the "A value" for that tumor divided by the average diameter of the tumor, in cm.
A time study of 12-72 hours was also conducted on some of the tumors. The procedure is the same as above, e~cept 1 mg of the amino acid adduct was utilized. The results are also indicated in Table IV.
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Claims (76)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A therapeutic composition for detection and/or treatment of mammalian tumors which comprises a fluorescent mono- or polyamide of an amino- dicarboxylic acid and a tetrapyrrole containing at least one carboxy group of the structure:
wherein Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive, and a pharmaceutical carrier therefor.
wherein Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive, and a pharmaceutical carrier therefor.
2. The therapeutic composition according to Claim 1 wherein the amino acid is an alpha aminodicarboxylic acid.
3. A therapeutic composition for detection and/or treatment of mammalian tumors which comprises a fluorescent mono- or polyamide of an aminodicarboxylic acid and a tetrapyrrole compound of the formula:
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or , R2 is H, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or R4 is H, vinyl, ethyl, , CH2CH2CO2H, =CHCHO; or , R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydro-pyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or , R2 is H, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or R4 is H, vinyl, ethyl, , CH2CH2CO2H, =CHCHO; or , R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydro-pyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
4. A therapeutic composition for detection and/or treatment of mammalian tumors which comprises a fluorescent mono- or polyamide of an aminodicarboxylic acid and fluorescent tetrapyrrole compound of the formula:
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or ;
R2 is H, vinyl, ethyl, -CHCH3, acetyl, , OH
, CH2CH2CO2H, or =CHCHO;
R3 is methyl or ;
R4 is H, vinyl, ethyl, , CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R or R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydro-pyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or ;
R2 is H, vinyl, ethyl, -CHCH3, acetyl, , OH
, CH2CH2CO2H, or =CHCHO;
R3 is methyl or ;
R4 is H, vinyl, ethyl, , CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R or R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydro-pyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
5. The therapeutic composition according to Claim 4 wherein the tetrapyrrole is a porphyrin.
6. The therapeutic composition according to Claim 4 wherein the tetrapyrrole is a chlorin.
7. The therapeutic composition according to Claim 4 wherein the tetrapyrrole is a bacteriochlorin.
8. The therapeutic composition according to Claim 4 wherein the amino acid is an alpha aminodi-carboxylic acid.
9. The therapeutic composition according to Claim 4 wherein the amino acid is aspartic acid.
10. The therapeutic composition according to Claim 4 wherein the amino acid is glutamic acid.
11. The therapeutic composition according to Claim 4 wherein the amide is monoaspartyl trans-mesochlorin IX.
12. The therapeutic composition according to Claim 4 wherein the amide is diaspartyl trans-mesochlorin IX.
13. The therapeutic composition according to Claim 4 wherein the amide is monoglutamyl trans-mesochlorin IX.
14. The therapeutic composition according to Claim 4 wherein the amide is diglutamyl trans-mesochlorin IX.
15. The therapeutic composition according to Claim 4 wherein the amide is monoaspartyl chlorin e6.
16. The therapeutic composition according to Claim 4 wherein the amide is triaspartyl chlorin e6.
17. The therapeutic composition according to Claim 4 wherein the amide is monoglutamyl chlorin e6.
18. The therapeutic composition according to Claim 4 wherein the amide is diglutamyl protoporphyrin IX.
19. The therapeutic composition according to Claim 4 wherein the amide is monoaspartyl mesochlorin e6.
20. The therapeutic composition according to Claim 4 wherein the monoglutamyl protoporphyrin IX.
21. The therapeutic composition according to Claim 4 wherein the amide is monoaspartyl mesoporphyrin IX.
22. The therapeutic composition according to Claim 4 wherein the amide is diaspartyl mesoporphyrin IX.
23. The therapeutic composition according to Claim 4 wherein the amide is diglutamyl mesoporphyrin IX.
24. The therapeutic composition according to Claim 4 wherein the amide is diaspartyl protoporphyrin IX.
25. The therapeutic composition according to Claim 4 wherein the amide is monoaspartylbacteriochlorin e4.
26. The therapeutic composition according to Claim 4 wherein the amide is diaspartyl deuteroporphyrin IX.
27. The therapeutic composition according to Claim 4 wherein the amide is monoaspartyl deuteroporphyrin IX.
28. The therapeutic composition according to Claim 4 wherein the amide is monoglutamylbacterioisochlorin e4.
29. The therapeutic composition according to Claim 4 wherein the amide is diglutamyl deuteroporphyrin IX.
30. The therapeutic composition according to Claim 4 wherein the amide is mono- or diaspartyl photoproto-porphyrin IX.
31. The therapeutic composition according to Claim 4 wherein the amide is mono- or diglutamyl photoproto-porphyrin IX.
32. The therapeutic composition according to Claim 4 wherein the amide is mono-, di-, tri- or tetraglutamyl coporphyrin III.
33. The therapeutic composition according to Claim 4 wherein the amide is mono- or diaspartyl hematopor-phyrin IX.
34. The therapeutic composition according to Claim 4 wherein the amide is mono- or diglutamyl hematopor-phyrin IX.
35. The therapeutic composition according to Claim 4 wherein the amide is mono- or diglutamyl chlorin e4.
36. The therapeutic composition according to Claim 4 wherein the amide is mono- or diglutamyl mesochlorin e4.
37. The therapeutic composition according to Claim 4 wherein the amide is mono- or diaspartyl chlorin e4.
38. The therapeutic composition according to Claim 4 wherein the amide is monoglutamyl deuteroporphyrin IX.
39. A process for preparing a porphyrin amino acid adduct which comprises reacting an amino dicarboxylic acid with a tetrapyrrole containing at least one carboxy group in a suitable solvent to form a compound of the structure:
wherein Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive, and optionally converting the product to a salt thereof.
wherein Z is the aminodicarboxylic acid residue less the amino group and X is the tetrapyrrole residue less the carboxy group and "n" is an integer from 1 to 4 inclusive, and optionally converting the product to a salt thereof.
40. The process according to Claim 39, wherein the amino acid is an alpha aminodicarboxylic acid.
41. The process according to Claim 39 wherein the tetrapyrrole has the formula:
41. The process according to Claim 39 wherein the tetrapyrrole has the formula:
Claim 41 (cont'd) or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or ;
R2 is H, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or ;
R4 is H, vinyl, ethyl, CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or ;
R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, 7 8 two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1 - R9 includes a free carboxyl group; and optionally converting the product to a salt thereof.
R2 is H, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or ;
R4 is H, vinyl, ethyl, CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or ;
R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, 7 8 two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1 - R9 includes a free carboxyl group; and optionally converting the product to a salt thereof.
42. The process according to Claim 39 wherein the tetrapyrrole has the formula:
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or ;
R2 is a, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or R4 is H, vinyl, ethyl, CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or ;
R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring -to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
or the corresponding di- or tetrahydrotetrapyrroles wherein R1 is methyl; or ;
R2 is a, vinyl, ethyl, , acetyl, , , CH2CH2CO2H, or =CHCHO;
R3 is methyl or R4 is H, vinyl, ethyl, CH2CH2CO2H, =CHCHO; or ;
R5 is methyl;
R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H;
R7 is CH2CH2CO2H, CH2CH2CO2R, or ;
R8 is methyl or R9 is H, COOH, CH2COOH or methyl;
provided that when R1, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring -to which attached is a dihydropyrrole;
R is lower alkyl or benzyl;
R6 and R9, taken together are or with the proviso that at least one of R1-R9 includes a free carboxyl group; and salts thereof, and a pharmaceutically acceptable carrier therefor.
43. The process according to Claim 42, wherein the tetrapyrrole is a porphyrin.
44. The process according to Claim 42, wherein the tetrapyrrole is a chlorin.
45. The process according to Claim 42, wherein the tetrapyrrole is a bacteriochlorin.
46. The process according to Claim 42, wherein the amino acid is an alpha aminodicarboxylic acid.
47. The process according to Claim 42, wherein the amino acid is aspartic acid.
48. The process according to Claim 42, wherein the amino acid is glutamic acid.
49. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartyl trans-mesochlorin IX.
50. The process according to Claim 39 wherein the porphyrin amino acid adduct is diaspartyl trans-mesochlorin IX.
51. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoglutamyl trans-mesochlorin IX.
52. The process according to Claim 39 wherein the porphyrin amino acid addut is diglutamyl trans-mesochlorin IX.
53. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartyl chlorin e6.
54. The process according to Claim 39 wherein the porphyrin amino adduct is triaspartyl chlorin e6.
55. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoglutamyl chlorin e6.
56; The process according to Claim 39 wherein the porphyrin amino acid adduct is diglutamyl proto-porphyrin IX.
57 The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartyl meso-chlorin e6.
58. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoglutamyl proto-porphyrin IX.
59. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartyl meso-porphyrin IX.
60. The process according to Claim 39 wherein the porphyrin amino acid adduct is diaspartyl meso-porphyrin IX.
61. The process according to Claim 39 wherein the porphyrin amino acid adduct is diglutamyl meso-porphyrin IX.
62. The process according to Claim 39 wherein the porphyrin amino acid adduct is diaspartyl proto-porphyrin IX.
63. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartylbacterio-chlorin e4.
64. The process according to Claim 39 wherein the porphyrin amino acid adduct is diaspartyl deutero-porphyrin IX.
65. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoaspartyl deutero-porphyrin IX.
66. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoglutamylbacteriois-chlorin e4.
67. The process according to Claim 39 wherein the porphyrin amino acid adduct is diglutamyl deutero-porphyrin IX.
68. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diaspartyl photo-protoporphyrin IX.
69. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diglutamyl photo-protoporphyrin IX.
70. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono-, di-, tri- or tetraglutamyl coporphyrin III.
71 The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diaspartyl hematoporphyrin IX.
72. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diglutamyl hematoporphyrin IX.
73, The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diglutamyl chlorin e4.
74. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diglutamyl meso-chlorin e4.
75. The process according to Claim 39 wherein the porphyrin amino acid adduct is mono- or diaspartyl chlorin e4.
76. The process according to Claim 39 wherein the porphyrin amino acid adduct is monoglutamyl deutero-porphyrin IX.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA487040A CA1262862C (en) | 1984-07-18 | 1985-07-18 | Tetrapyrrole therapeutic agents |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US631,925 | 1984-07-18 | ||
US06/631,925 US4675338A (en) | 1984-07-18 | 1984-07-18 | Tetrapyrrole therapeutic agents |
CA487040A CA1262862C (en) | 1984-07-18 | 1985-07-18 | Tetrapyrrole therapeutic agents |
Publications (2)
Publication Number | Publication Date |
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CA1262862A true CA1262862A (en) | 1989-11-14 |
CA1262862C CA1262862C (en) | 1989-11-14 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA487040A Expired CA1262862C (en) | 1984-07-18 | 1985-07-18 | Tetrapyrrole therapeutic agents |
Country Status (11)
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US (1) | US4675338A (en) |
EP (1) | EP0168832B1 (en) |
JP (2) | JPH0688902B2 (en) |
AT (1) | ATE94547T1 (en) |
AU (1) | AU579387B2 (en) |
CA (1) | CA1262862C (en) |
DE (1) | DE3587579T2 (en) |
DK (1) | DK167768B1 (en) |
HK (1) | HK135994A (en) |
NO (1) | NO169290C (en) |
SG (1) | SG103194G (en) |
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CN102724951A (en) | 2009-11-09 | 2012-10-10 | 阿勒根公司 | Compositions and methods for stimulating hair growth |
WO2014071138A1 (en) | 2012-11-02 | 2014-05-08 | Lewis Thomas J | Disease detection and treatment through activation of compounds using external energy |
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US4393071A (en) * | 1975-03-17 | 1983-07-12 | Naoharu Fujii | Method of treating gastric, mammary, lung and uterus tumors |
JPS53112900A (en) * | 1977-03-14 | 1978-10-02 | Ajinomoto Co Inc | Porphyrin derivative |
JPS58201791A (en) * | 1982-05-19 | 1983-11-24 | Katsuo Unno | Hematoporphyrin derivative |
WO1984001382A1 (en) * | 1982-09-27 | 1984-04-12 | Oncology Research & Dev Inc | Purified hematoporphyrin derivative for diagnosis and treatment of tumors, and method |
US4649151A (en) * | 1982-09-27 | 1987-03-10 | Health Research, Inc. | Drugs comprising porphyrins |
JPS60158687A (en) * | 1984-01-27 | 1985-08-20 | Matsushita Electric Ind Co Ltd | Semiconductor laser |
JPS60158686A (en) * | 1984-01-27 | 1985-08-20 | Matsushita Electric Ind Co Ltd | Semiconductor laser |
US4675338A (en) * | 1984-07-18 | 1987-06-23 | Nippon Petrochemicals Co., Ltd. | Tetrapyrrole therapeutic agents |
US4693885A (en) * | 1984-07-18 | 1987-09-15 | Nippon Petrochemicals Co., Ltd. | Tetrapyrrole therapeutic agents |
-
1984
- 1984-07-18 US US06/631,925 patent/US4675338A/en not_active Expired - Lifetime
-
1985
- 1985-07-18 JP JP60158686A patent/JPH0688902B2/en not_active Expired - Lifetime
- 1985-07-18 AT AT85108987T patent/ATE94547T1/en not_active IP Right Cessation
- 1985-07-18 EP EP85108987A patent/EP0168832B1/en not_active Expired - Lifetime
- 1985-07-18 CA CA487040A patent/CA1262862C/en not_active Expired
- 1985-07-18 DK DK326785A patent/DK167768B1/en not_active IP Right Cessation
- 1985-07-18 AU AU45158/85A patent/AU579387B2/en not_active Expired
- 1985-07-18 DE DE85108987T patent/DE3587579T2/en not_active Expired - Lifetime
- 1985-07-18 JP JP60158687A patent/JPH0689000B2/en not_active Expired - Lifetime
- 1985-07-18 NO NO852871A patent/NO169290C/en not_active IP Right Cessation
-
1994
- 1994-07-26 SG SG103194A patent/SG103194G/en unknown
- 1994-12-01 HK HK135994A patent/HK135994A/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
AU579387B2 (en) | 1988-11-24 |
NO852871L (en) | 1986-01-20 |
JPS6183186A (en) | 1986-04-26 |
EP0168832B1 (en) | 1993-09-15 |
NO169290B (en) | 1992-02-24 |
SG103194G (en) | 1994-10-28 |
DE3587579D1 (en) | 1993-10-21 |
CA1262862C (en) | 1989-11-14 |
ATE94547T1 (en) | 1993-10-15 |
HK135994A (en) | 1994-12-09 |
US4675338A (en) | 1987-06-23 |
NO169290C (en) | 1992-06-03 |
DK326785D0 (en) | 1985-07-18 |
DK167768B1 (en) | 1993-12-13 |
DK326785A (en) | 1986-01-19 |
EP0168832A2 (en) | 1986-01-22 |
EP0168832A3 (en) | 1988-05-04 |
JPH0688902B2 (en) | 1994-11-09 |
JPH0689000B2 (en) | 1994-11-09 |
DE3587579T2 (en) | 1994-02-17 |
AU4515885A (en) | 1986-03-27 |
JPS61129163A (en) | 1986-06-17 |
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