CA2163539C - N-morpholino-n-nitrosaminoacetonitrile cyclodextrin inclusion complexes - Google Patents
N-morpholino-n-nitrosaminoacetonitrile cyclodextrin inclusion complexes Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
- C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
Abstract
New inclusion complexes which are stable in their solid state formed of SIN-1A and cyclodextrins or cyclodextrin derivatives, and optionally also containing ions as catalyst or stabilizer. The complexes release nitric oxide at room temperature upon dissolving in water or aqueous systems. Pharmaceutical compositions as well as kits containing the complexes. The kits are to be used as NO-liberating standards to release NO in a predictable amount and rate on dissolving in aqueous media. Processes for the preparation of the complexes by subjecting at a suitable pH SIN-1 to the catalytic action of ions to shift the equilibrium towards formation of SIN-IA in the presence of cyclodextrins or cyclodextrin derivatives capable of forming inclusion complexes, whereby the SIN-1A formed is immediately complexed and stabilized and isolating in the solid state the obtained new complexes optionally containing ions.
Description
N-MORPHOLINO-N-NITROSAMINOACETONITRILE
CYCLODEXTRIN INCLUSION COMPLEXES
The invention relates to physiologically active nitric oxide releasing agents, processes for preparation thereof, composi-tions containing as well as methods to use the same.
More particularly the invention relates to new SIN-lA in-clusion complexes which are stable in their solid state and which are formed with cyclodextrins or with cyclodextrin de-rivatives and which are releasing nitric oxide at room temperature upon dissolving in water or aqueous systems and which optionally also contain ions as catalyst or stabilizer.
The invention also relates to processes for their preparati-on, compositions containing the same and methods for their use.
The following abbreviations are used in this specification:
SIN-1 3-morpholino - sydnonimine SIN-lA N-morpholino-N-nitrosoaminoacetonitrile SIN-1C cyanomethylene-amino-morpholine CDPSI ionic soluble B-cyclodextrin polymer DIMEB heptakis-2,6-di-0-methyl-B-cyclodextrin EDRF endothelium- derived relaxing factor HPBCD hydroxypropyl-H-cyclodextrin 2,8 hydroxypropyl group per CD-unit (average) Molsidomin N-ethoxycarbonyl-3-morpholino-sydnonimine RAMEB randomly methylated-BCD, ~ 12 methoxyl group per CD-unit (average) TRIMEB heptakis 2,3,6-tri-O-methyl-B-cyclodextrin.
It is known, that nitrogen monoxide may have a reduced form (N0~) which is designated as nitric oxide, and an ' oxidized form (NO+) which is called nitrosonium ion. Nitric oxide (NO-) is implicated in numerous important bioregulatory pro cesses.
The utility of a NO releasing donor depends on both the depth and duration of the mean arterial pressure lowering effect.
Longer acting NO-donors are needed, which release the NO
without metabolic transformation of the donors, i.e. which are not depending on the liver functions. Furthermore, a NO-donor should have some lipophilic character, to be able to cross cell-membranes to exert its action also in the targeted organs, tissues. Therefore relatively simple, inorganic compounds are not adequate for this purpose.
The product design may follow three different routes:
a. The NO-donor prodrugs contain the -NO-group, which is re-leased either directly by a metabolic process or after remo-ving by enzymatic hydrolysis of some protecting group. These processes are bound largely to the liver (e. g. MolsidorniriM).
The sydnonimine-type prodrugs (e.g. MolsidomiriM) depend on the liver to remove the protecting ethoxy-carbonyl-group from the molecule to produce first SIN-1 and then (in a se-cond, pH dependent process catalysed by OH- ions) the very instable SIN-lA is formed which independently of the pH spon-taneously decomposes with the release of NO.
b. Preparation of adducts or complexes of nitric oxide with various nucleophiles.
Generally the synthesis of the secondary amine NO complexes is as follows: The secondary amine e.g. anhydrous diethylamine is dissolved in anhydrous ether, oxygen is removed from the system by using aceton dry-ice bath and dry NO is bubbled through the ether solution at -78"C for 3 hours, preferably at high pressure (100 psi).
The half-life of the prior art diethylamine-nitric oxide adduct Et2-N-NH-(ONa)-N=O (DEANO) amounts to about 2~minutes, while the nitric oxide addition product of polyamine-spermine (SPNO) has a half-life of 39 minutes.
c. The prodrug is stabilized by cyclodextrin inclusion-com-plex formation while NO is released spontaneously under phy-siological conditions. The known cyclodextrin complexed pro-drug is stable in the solid state.
It is known, that SIN-1. is a stable compound in solid state, however, its open-chain tautomeric form SIN-lA is extremely unstable. It is highly difficult to isolate the yellow cry-stalline product in pure form and it can be stored only at -SO~C, under nitrogen. The SIN-lA form rapidly releases one mole NO in solid state through photolysis, and in aqueous so-lution even in darkness it is converted to cyanomethylene-amino-morpholine (SIN-1C).
SIN-1 is thus considered to be a prodrug and SIN-1C a biolo-gically inactive degradation product. The equilibrium between the stable SIN-1 and the active SIN-lA (which is the real drug) depends on ambient factors (pH, temperature). However because of the extreme instability of SIN-lA - especially to oxygen - it is practically a short-lived intermediate in the decomposition process of SIN-1.
Presently SIN-1 is marketed only for intravenous administra-tion in powder-ampoules, to be dissolved before injection.
' The orally administered SIN-1 prodrug is ineffective, because the hydrolytic step which is required for the formation of the NO generating SIN-1A form can not take place under acidic pH conditions, and before absorption it is rapidly and completely decomposed to SIN-1C at the higher pH of the gas-trointestinal tract.
It is also public, that SIN-1 can be stabilized by CD comple-xation and the equilibrium SIN-1 -~ SIN-lA -~ SIN-1C can be shifted by means of complexation with cyclodextrin derivati-ves (PCT Publication WO 91/14681). It was disclosed that com-plexati~on with certain CD derivatives inhibit SIN-1C
formation. Thus more stable complexes of SIN-1/CDPSI inclu-ding those containing an increased amount of SIN-lA (showing biological activity based on higher NO release) were prepared by short time heat-treatment of the SIN-1/CDPSI complex product obtained by lyophilisation. Similarly a SIN-1/DIMEB
complex was exemplified. These CD derivatives were proposed to be used in pharmaceuticals.
This document disclosed SIN-1/CD complexes or SIN-1/lactose mixture with 4,5~ (w/w) SIN-1 content where the SIN-lA con-tent in the obtained products were as follows:
in CDPSI complex 1.27 in J3CD complex 0.08 in DIMEB complex 0.06 in HPBCD complex 0.115 in mixture with lactose 0.00 Practically complete conversion of SIN-1 to SIN-1A was not disclosed to take place even when complexation was brought about with CDPSI which was disclosed to be the most effective complexation agent for this purpose.
Moreover: CDPSI and DIMEB are not approved to be used in pharmaceuticals as yet. Thus the preparation of a stable, marketable form of pure SIN-lA is an unsolved problem.
Because SIN-1 can be applied only intravenously, its prodrug Molsidomin is used for the oral treatment of heart insuffi-ciency: Molsidomin is hydrolysed enzymatically in the liver to SIN-1.
WO 95/29172 . PCT/HU95/00011 It was the ultimate goal of this invention to stabilize the NO-donor SIN-1A so as to ensure a formulation which can be administered both orally and parenterally, the innocuousness of which is supported with complete (even i.v.) toxicologi-cal documentation. Presently two types of cyclodextrins ful-fill this fundamental requirement: gammaCD and HPf3CD.
Though the SIN-lA stabilizing effect of CDPSI and DIMEB in aqueous solutions is known it does not however follow, that also the non-ionic gammaCD is effective in this respect, par-ticularly because of its much wider cavity diameter. The ex-cellent stabilizing effect of CDPSI has been attributed to its polymeric structure, i.e. to the cooperative effects of various cyclodextrin rings anchored in sterical vicinity.
The subjects of this invention are new SIN-lA inclusion complexes which are stable in their solid state formed of SIN-1A and cyclodextrins or cyclodextrin derivatives and re-leasing nitric oxide at room temperature upon dissolving in water or aqueous systems and optionally also containing an ion as a catalyst or stabilizer.
According to a preferred embodiment they contain a phy-siologically acceptable anion as a catalyst or stabilizer such as carboxylic acid anions i.a. acetate, formiate, pro-pionate, ascorbinate, tartarate and/or lactate and/or inorga-nic acid anions such as phosphate, phosphite, borate, carbo-nate, hydrocarbonate, sulfate and/or sulfite. Acetate was found to be an excellent anion for the purpose. The anions may be present as salts and the corresponding cations may be preferably ammonium or alkali ions however other cations may be used as well.
As cyclodextrin component they contain BCD, gammaCD or aCD, especially for pharmaceutical use. They also may contain cyclodextrin derivatives namely hydroxy-propylated or methyla-ted cyclodextrins such as HPf3CD, DIMEB, RAMEB, TRIMEB or CDPSI.
Another feature of the invention are biologically active compositions containing as their active ingredient the new SIN-1A/ cyclodextrin complexes along with auxiliary and additive ingredients facilitating their use. These include but are not limited to pharmaceutical compositions contai-ning as an active ingredient SIN-1A inclusion complexes op-WO 95/29f12 PCT/HU95/00011 -5- r.
tionally with usual auxiliary and additional materials used in pharmaceuticals for oral, parenteral or other medical uses. The formulations are preferably powders dissolved di-rectly before medication takes place. Thus the parenteral ' formulations are preferably powders dissolved prior to injec-tion.
Pharmaceutical compositions of preference are those con-taining as an active ingredient SIN-1A inclusion complexes which are stable in their solid state and which are formed with f~CD, gammaCD or aCD and containing a physiologically acceptable anion as a catalyst or stabilizer, such as car-boxylic acid anions including acetate, formiate, propionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions comprising phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and/or sulfite. The anions may be optionally in the form of their salts e.g. the ammonium or alkali salts. Ammonium acetate is a preferred salt for the purpose.
Further objects of the present invention are kits to be used as NO-liberating standards to release NO in a predictable amount and rate on dissolving in aqueous media containing as an active ingredient SIN-lA inclusion complexes according to the present invention.
This is possible because the present invention relates to the preparation and stabilization of the extremely labile SIN-lA, which when released from the cyclodextrin cavity - even after simple dissolving in distilled water - immediately generates the NO in a predictable amount and rate without the need of any further enzyme or reactant.
The present invention includes processes for the preparation of new SIN-lA inclusion complexes which are stable in their solid state and which are formed with cyclodextrins or with cyclodextrin derivatives by way of subjecting at a suitable pH SIN--1 to the catalytic action of ions to shift the equi-librium towards formation of SIN-lA in the presence of cyclo-dextrins or cyclodextrin derivatives capable to form in-clusion complexes, whereby the SIN-lA formed is immediately complexed and stabilized by formation of SIN-lA/ cyclodextrin inclusion complexes. The process includes isolating in the solid state the obtained SIN-1A/CD complexes optionally containing the ions. The ions may be contained in the form of their salts.
WO 95/29172 ~ PCT/HU95/00011 It is a preferred process according to the invention to react SIN-1 and a cyclodextrin or cyclodextrin derivative in the solid state in the presence of an ion preferably in the form of its salt as a catalyst by thoroughly admixing or milling the components together. Another route according to the in vention follows freeze drying an aqueous, oxygen-free so-lution containing the components, followed preferably by "second drying" in vacuo.
It is preferred to use ammonium or alkali salts formed with carboxylic acid anions such as acetate, formiate, propionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions such as borate, carbonate, hydrocarbonate, phosphate, phosphite, sulfate, and/or sulfite as catalyst of the pro-cess. The salts used may contain volatile anions or cations which are eliminated partly or totally during the process such as ammonium or carbonate ions.
When accomplishing the above it is advantageous to carry out the reaction at pH values between 6 to 10 and - when water was used as a reaction medium - applying "second drying" at 40 to 100°C preferably 50 - 70~C.
As it is seen from the NO-release tests, SIN-lA/cyclodextrin complexes immediately produce nitric oxide after dissolution of the solid complexes in aqueous systems. Thus one way to obtain a SIN-1A cyclodextrin complex of controlled compositi-on (having the lowest SIN-1C content) consists in tautomeri-zation of SIN-1 and the simultaneous complexation of the for-med SIN-1A in solid state.
The situation is similar when the complex preparation is per-formed by freeze-drying. In this case the second-drying step (following lyophilization) is able to ensure the conditions for the solid-state catalytic complex formation.
Another subject of the invention consists in methods of nitric oxide treatment of living cells. This includes but is not limited to the treatment of nitric oxide dependent symptoms in humans or animals like anginic and ischemic heart failures, physiological control of blood pressure, platelet aggregation, mediation of relaxation of peristalsis, penile erection and others. This is accomplished by administering ' preferably in oral or parenteral application to the patients in need of such treatment an effective amount of a new SIN-lA inclusion complex which is stable in its solid state and WO 95/291 l2 PCT/HU95/00011 which is formed with cyclodextrins or with cyclodextrin deri-vatives and which contains at least one ion as a catalyst or stabilizer and which upon dissolving in water or aqueous sy-stems at room temperature releases nitric oxide.
The preferred embodiment relies in administering the complexes of SIN-1A formed with f3CD, gammaCD or aCD and con-taining optionally in the form of their ammonium or alkali salts carboxylic acid anions such as acetate, formiate, pro-pionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions such as phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and/or sulfite.
The invention includes the method of treatment of nitric oxide dependent symptoms in humans or animals by treating the patient with the product of the quantitative conversion of SIN-1 into SIN-1A accomplished by way of an ion- catalysed and cyclodextrin-stabilized solid state conversion in the presence of a cyclodextrin or a cyclodextrin derivative ca-pable to immediately form inclusion complexes.
The major advantages of SIN-1A/CD complexes as compared with SIN-1 or other NO -donors known hitherto are thus the following:
- stability at the long term, - rapidity of action, - increased half-life, - independence from the liver, - independence from the pH, - eventually a greater ability to reach their targets (tissues).
Having the stable composition in hands makes it possible to open a new phase for treatments with nitric oxide which is called by some authors as "biochemistry's unexpected new superstar" (Chem.Ing.News Dec.1993.page 26-38).
-g-The following Examples serve illustration and not limitation of the invention.
I. CHEMICAL EXAMPLES
Example I.1.
Preparation of SIN-lA/gammaCD complex 2 g of gammaCD and 0,8 g of ammonium acetate were dissolved in 25 ml of distilled water by ultrasonication. The solution was deoxygenated by bubbling with helium gas, thereafter 200 mg of SIN- 1 substance were dissolved. The solution was imme-diately freeze-dried for isolation of the solid complex. Se-cond drying at 40-50°C for 2 hours was applied to remove the water content of the complex almost completely. Both solution and the substance were protected from light.
The complex is a light yellow powder. Yield: 2.6~0.1 g Loss on drying is less than 1%.
HPLC analysis: SIN-1 content: not detectable SIN-1A content: 11.7 ~0.2%
SIN-1C content: 0.36~O.ls In all examples of this document the following method was used for simultaneous determination of SIN-1, SIN-lA and SIN-1C by HPLC .
Column: UltrasphereTM I.P. analytical column (Beckman-Astec) 4.6~250 mm, particle size 5 um.
Mobile phase: 0.01 M phosphate buffer pH=6.0 800 ml tetrahydrofuran 200 ml sodium-1-dodecanesulfonate 0.405 g/dm3 Ionic strength: 0.05 gion/dm3 (corrected with sodium sul-phate) Flow rate: 1 ml/min., p=190 bar. Sample size: 20 ~1 Wavelength of detection:
-230 nm BW: 6 (ref. 350 nm BW: 80) for SIN-lA and SIN-1C
between 0-8 minutes -292 nm BW: 6 (ref. 400 nm BW:80) for SIN-1 after 8 minutes Retention times: SIN-1 9:6 min.
SIN-1C 4.5 min.
SIN-lA 4.8 min.
Calibration was performed with freshly prepared SIN-1 and SIN-1C solutions, SIN-lA content was expressed in~ SIN-1C
equivalent.
Example I.2 Preparation of SIN-lA/BCD complex -g-16 g of l3CD (water content 14~) and 7 g of ammonium acetate were dissolved in 1000 ml of distilled water by ul-trasonication. The solution was deoxygenated by bubbling with helium gas, thereafter 3 g of SIN-1 substance were dissolved.
The solid complex was isolated by immediate freeze-drying and water content was removed at 40-50°C. Both solution and sub-stance 'were protected from light. The complex is a yellowish very light powder. Yield: 20~1 g. Loss on drying < 1~
HPLC analysis: SIN-1 content: not detectable SIN-1A content: 12~1~
SIN-1C content: 0.30~0.2~
Example I.3 Preparation of SIN-lA/HPBCD complex 2 g of :HPBCD (DS=2.8) and 0.8 g of ammonium acetate were dis-solved in 25 ml of distilled water by ultrasonication. On deoxygenation by bubbling with helium gas; 200 mg of SIN-1 were dissolved. The solid complex solution was isolated by immediate freeze-drying. The water content was removed at 40-50°C. Both solution and substance were protected from light. 2,8 ~ 0,1 g of SIN-lA / HPBCD complex as a light yel-low powder were obtained. Loss on drying <1~.
HPLC analysis: SIN-1 . not detectable SIN-1A: 11.6~0.2~
SIN-1C: 0.36~0,1~.
Example I.4 Preparation of SIN-lA/RAMEB complex 2 g of ItAMEB (DS=1,8) and 0,8 g of ammonium acetate were dis-solved in 25 ml of distilled water by ultrasonication. On deoxygenation by bubbling with helium gas 200 mg of SIN-1 substance were dissolved, the solution was freeze-dried and the isolated solid complex dried at 40-50°C for 2 hours. So-lution .and substance were protected from light.
2.8~0,1 g SIN-lA RAMEB complex were obtained as a light yellow powder. Loss on drying < 1~
HPLC analysis: SIN-1 . not detectable SIN-lA . 12.0~0,2~
SIN-1C . 0.6~0,1~.
Example I. 5.
Preparation of SIN-1A l3CD complex in phosphate buffer solution 1 g of f3CD (water content: 14~) was dissolved in 55 ml of a pH - 8.0 phosphate buffer solution according to USP XXII by WO 95/29172 ~ ' PCT/HU95/00011 ultrasonication. On deoxygenation with helium gas 100 mg of SIN-1 were dissolved. The solution was immediately freeze-dried. Solution and substance Mere protected from light.
1 ~ 0.1 g of SIN-lA/f3CD were obtained as a yellowish coloured very light powder. Loss on drying < l~
HPLC analysis: SIN-1 content: not detectable SIN-lA content: 8.3~
SIN-1C content: 0.24 Example I.6 1 g of f3CD (water content 14~), 100 mg of SIN-1 hydrochloride and 100 mg of ammonium acetate were thoroughly mixed in a mortar for 15 minutes. After a short time of rubbing the mix-ture became visibly yellow. After 2 days of storage in a clo-sed container protected from light, HPLC analysis according to Example I.1 was taken to give the following results:
SIN-1 content: not detectable SIN-lA content: 7.9~0.2~
SIN-1C content: 0.41~0.1~
Example I.7 Using the process of Example I.6. but taking 50 mg of ammo-nium acetate the visible formation of SIN-1A (yellow colora-tion) is slower, it took several hours.
HPLC analysis of the SIN-lA/l3CD after 2 days of storage:
SIN-1 0.5~0,1~
SIN-1A 6.9~0,2~
SIN-1C 0.6~0,1~
Example I.8 Following the procedure as described in Example I.6. but ap-plying 300 mg of ammonium acetate the yellow coloration of the mixture occurs practically immediately after mixing the components.
HPLC analysis of the fiCD complex same day after preparation:
SIN-1 . not detectable SIN-lA : 6.9~0.2$
SIN-1C . 0.5~0,1~
Example I.9 1 g of 13CD (water content 14~), 100 mg of SIN-1 hyrochloride and 100 mg of sodium acetate.3H20 were thoroughly mixed in a mortar for 15 minutes, followed by heat treatment of the mix- ' ture at 70'C for 1 hour.
HPLC analysis of the heat-treated sample:
SIN-1: ' not detectable -11- .
SIN-lA: 7.1 ~ 0.2~
SIN-1C: 0.8 ~ 0.1~
II. COMPARATIVE EXAMPLES
' Example II.1 Attempted preparation of SIN-lA/ l3CD with acetic acid Preparation was carried out as in Example I.2 but instead of water and ammonium acetate the f3CD was dissolved in a 0.1 mo-le acetic acid solution.
HPLC analysis of the product: SIN-1 . 8.4~
SIN-lA: 0.28 SIN-1C: not detectable.
Thus the solid SIN-lA/f3CD complex was not isolated.
Example II.2 Attempted preparation of SIN-1A/f3CD with sodium hydroxide Complexation was carried out as described in Example I.S, but the l3CD was dissolved in 55 ml of pH 8.4 aqueous sodium hydr-oxide istead of using ammonium acetate. HPLC analysis of the obtained solid product:
SIN-1: 10.5$
SIN-lA: 0.40 SIN-1C: not detectable.
No SIN-1A/f3CD complex was isolated.
III. ANALYTICAL AND BIOLOGICAL EXAMPLES
Example III.1 The effectivity of conversion and stability of the SIN-lA
complexes are illustrated by Table 1. The samples were stored at room temperature for 11 months in glass containers under air atmosphere, protected from light. The SIN-1A con-tent is given in SIN-1C equivalent.
Table 1.
SIN-1 contents SIN-lA contents SIN-1C content after after after after after after prepa- storing prepa- storing prepa- storing ration for 11 ration for 11 ration for 11 months months months SIN-1/aCD 3.59 2.46 0.68 SIN-1/J3CD 0 0 10.66 9.59 0.68 0.50 SIN-1/~"CD 0 0 11.71 7.23 0.32 0.49 ~.~~~~~9 HPBCD 0.03 0 11.61 7.53 0.36 0.39 RAMEB 0 0 12.11 9.43 0.68 0.87 Example III.2 A SIN-1A/l3CD sample contained immediately after preparation -11.47~ SIN-1A. When stored at room temperature for 12 months it contained 8.36 SIN-1A, and after 23 months it contained 6.59 SIN-1A. The SIN-iC contents were 0.18, 1.31 and 1.57 respectively.
The complexes contained approx. 5-8~ inclusion water, mainly bound to the cyclodextrin cavity.
Example III.3 The stability enhancing effect of heat-treatment ("second drying") after freeze- drying is illustrated by Table 2.
Table 2.
SIN-1l3CD SIN-1 contents SIN-lA contents SIN-1C content after after after after after after preps- storing preps- storing preps- storing ration for 11 ration for 11 ration for 11 months months months SIN-1/13CD 0 0 12.7 10.89 0.40 0.79 SIN-1/f3CD
heat treated 0 0 12.7 12.50 0.40 0.56 after after after after after after preps- storing preps- storing preps- storing ration for 19 ration for 19 ration for 19 months months months SIN-1/l3CD 0.04 0 14.81 7.41 0.29 1.75 heat treated 0.040 14.81 12.41 0.29 1.15 Example III.4 Rate and extent of NO release from SIN-lA/f3CD complex.
Release of NO in SIN-lA/CD solutions was examined by chemic-al and biological methods.
250 mg of complex (corresponds to approximately 25 mg of SIN-WO 95/29172 ~ . PCT/HU95/00011 1A) were dissolved in 100 ml of distilled water. Right after dissolution liberation of NO was indirectly detected by si-multaneously measuring decrease of the SIN-1A content and formation of the SIN-1C metabolite by HPLC as a function of ' time. The measurements were repeated in different time inter-vals, while protecting from light.
Table 3 illustrates the kinetics of decomposition of SIN-lA
in distilled water at room temperature in presence of f3CD, gammaCD and HPBCD.
Table 3 time, min. 13CD complex BCD complex HPBCD complex SIN-lA SIN-1C SIN-lA SIN-1C SIN-lA SIN-1C
~g/ml ~g/ml ~cg/ml ~Cg/ml ~Cg/ml ~g/ml 70 ~ 86 65 - - - -The estimated T1/2 (half-life) of the complexes calculated from the decrease of SIN-1A content are 53 min. for 13CD, 24 min. for gammaCD and 70 min. for HPBCD.
Example III.S
Release of NO in a more diluted SIN-1A/BCD solution (100 ~g/ml of SIN-1A/f3CD complex corresponds to 10 ~ug/ml of SIN-1A) was detected by W-spectrophotometry measuring the formation of SIN-1C metabolite as a function of time. SIN-1A
SIN-1C transition is accompanied by a very characteristic W-spectrum change, as SIN-lA has an UV maximum at 230~1 nm and shows no absorbance at JL max SIN-1C at 277~1 nm.
mg of the SIN-lA/13CD complex (corresponds to approximate-" 1y 1 mg of SIN-1A) were dissolved in 100 ml of distilled wa-ter. UV-spectrum of the solution was registered between 200-350 nm without dilution immediately after dissolution and registration was repeated in different time intervals, pro-tected from light . The formed SIN-1C amount was calculated from the absorbance values at 277 ~ 1 nm using the cali-bration curve taken with a SIN-1C standard. SIN-1C concentra-tion was less than 0,7 ~Cg/ml right after dissolution and ab-out 7.8 ~g/ml after 270 minutes. Presumably the equivalent amount of NO was released. Estimated half-life of the complex:
90-100 minutes. ,.
Figure 1. illustrates how the UV spectra change as a function of time: UV spectra of a SIN 1A/f3CD complex at 10 minute time intervals up to 160 minutes after dissolution of 10 mg of the complex in 100 ml of distilled water are shown. Absorbance is spotted against wavelength.
Example III.6 Formation of NO was determined directly, using NO specific porphyrinic microsensor detection. The method (Nature, Vol.
358 p. 675, 1992) is able to monitor the NO release up to 10-20 moles, in a single cell in biological microsystems in amperometric or voltametric mode .
In a pH= 7.4 phosphate buffer at 37°C 1 mM SIN-lA/BCD relea-sed NO at a rate of 2.37 ~CM/min. in the first 5 minutes, and between the 10th and 30th minute at a rate of 0.17 ~tM/min.
Dissolving 0,1 mM of substance the relevant values were 0.42 ~uM and 0.07 ~M respectively.
Example III.7 Inhibition of platelet aggregation by SIN-1A/l3CD complex:
Nitric oxide had been identified as a natural messenger mole-cule in the inhibition of platelet aggregation via the gua-nylate-cyclase/cyclic GMP system (Blood, 57. 946, 1981). We studied the biological effects of SIN-1 and SIN-lA/BCD com-plex by comparing their platelet aggregation inhibiting ef-fects. The platelet aggregation was studied in rabbit and hu-man platelet rich plasma (J. Cardiovasc. Pharmacol. ,14 suppl.
11; page 120, 1989). In each batch of platelet rich rabbit plasma 8 dose response curves were registered with the throm-boxane mimetic U-46619 (0.25-4.0 ~M) in the presence of 0, 10-7, 10-6 or 10-5 moles of SIN-1A/BCD complex. The dose re-sponse curves were performed in random sequence. Thereafter a fixed concentration of U46619 (4~CM) was used to make full concentration-inhibition curves to SIN-1 and to SIN-1A/f3CD. "
U46619 induced a concentration-related aggregation. The ag-gregation inhibiting effect of SIN-lA/BCD complex at identi-cal molar concentration in all cases was significantly higher than that of SIN-1. The pD2 (negative logarithm of con-centration producing 50~ inhibition) values were 5.57 ~ 0.11 (n=7) for SIN-1 and 6.36~0.07 (n=7) for SIN-lA/13CD complex, indicating that the complex was about 6-fold more potent than ' SIN-1 in this test.
In some similar experiments the SIN-1A/BCD complex was found to be about 10-fold as potent as SIN-1.
Example III.8 The experiments described in Example III.7 were repeated using human citrate platelet rich plasma with SIN-1, freshly prepared SIN-lA/f3CD complex (SIN-1A content 14.80 and with a SIN-1A/l3CD complex, that has been stored at room temperature for 23 months (SIN-1 content originally was 11.47, after 23 months it was 6.590 . Table 4 illustrates the results showing the negative logarithms of the concentration (pD2) of SIN-1 and SIN-1A f3CD complex causing 50~ inhibition of U46610 in-duced aggregation in human platelet rich plasma.
Both amplitude and slope of the aggregation curve were eva-luated. The difference in the pD2 values indicate that fresh-ly prepared SIN-lA/BCD was about 8-fold more potent than SIN-1. The complex stored for 23 months was found to be 3-fold less active than the freshly prepared complex, but 3-fold more potent than SIN-1 itself.
Table 4.
Substance a g g r a g a t i o n n amplitude slope SIN-1 10 4.8~0.05 4.85~0.13 SIN-1A 13CD freshly prepared (SIN-1A content:14.81~) 10 5.54~0.10 5.85~0.11 SIN-lA l3CD (stored for 23 months, SIN-1A content: 6.59 ~) 3 5.80~0.16 5.42~0.06 The results of in vivo tests suggest that the SIN-lA/BCD
complexes disclosed here are useful inhibitors of platelet aggregation iv vivo.
As is reflected from the half-life values, SIN-lA/cyclodex-trin complexes are able to continuously produce nitric oxide generation at a more nearly constant rate for much longer time after administration.
Example III.9 Commercially available SIN-1 (CORVASALj was compared with the SIN-lA/SCD complex on carotid artery of rabbits. The carotid artery of nembutal anesthesized rabbits was dissected free, 3 mm long rings were immobilized in a clamp and placed.in the organ baths. Contraction was induced with phenylephrine 3x10- M three times with following rinsing. The dose response curves were registered for 3x10-9 M to 3x10-5 M
CORVASAL (referred to SIN-1 content) and for 3x10-9 to 3x10-5 moles SIN-lA/SCD (referred to SIN-lA content).
Figure 2. shows the contraction versus drug concentration curve. As it is seen, the SCD complexed SIN-lA drug was about 6-fold more effective than free SIN-1. The illustrated points represent the average of 6 measurements.
CYCLODEXTRIN INCLUSION COMPLEXES
The invention relates to physiologically active nitric oxide releasing agents, processes for preparation thereof, composi-tions containing as well as methods to use the same.
More particularly the invention relates to new SIN-lA in-clusion complexes which are stable in their solid state and which are formed with cyclodextrins or with cyclodextrin de-rivatives and which are releasing nitric oxide at room temperature upon dissolving in water or aqueous systems and which optionally also contain ions as catalyst or stabilizer.
The invention also relates to processes for their preparati-on, compositions containing the same and methods for their use.
The following abbreviations are used in this specification:
SIN-1 3-morpholino - sydnonimine SIN-lA N-morpholino-N-nitrosoaminoacetonitrile SIN-1C cyanomethylene-amino-morpholine CDPSI ionic soluble B-cyclodextrin polymer DIMEB heptakis-2,6-di-0-methyl-B-cyclodextrin EDRF endothelium- derived relaxing factor HPBCD hydroxypropyl-H-cyclodextrin 2,8 hydroxypropyl group per CD-unit (average) Molsidomin N-ethoxycarbonyl-3-morpholino-sydnonimine RAMEB randomly methylated-BCD, ~ 12 methoxyl group per CD-unit (average) TRIMEB heptakis 2,3,6-tri-O-methyl-B-cyclodextrin.
It is known, that nitrogen monoxide may have a reduced form (N0~) which is designated as nitric oxide, and an ' oxidized form (NO+) which is called nitrosonium ion. Nitric oxide (NO-) is implicated in numerous important bioregulatory pro cesses.
The utility of a NO releasing donor depends on both the depth and duration of the mean arterial pressure lowering effect.
Longer acting NO-donors are needed, which release the NO
without metabolic transformation of the donors, i.e. which are not depending on the liver functions. Furthermore, a NO-donor should have some lipophilic character, to be able to cross cell-membranes to exert its action also in the targeted organs, tissues. Therefore relatively simple, inorganic compounds are not adequate for this purpose.
The product design may follow three different routes:
a. The NO-donor prodrugs contain the -NO-group, which is re-leased either directly by a metabolic process or after remo-ving by enzymatic hydrolysis of some protecting group. These processes are bound largely to the liver (e. g. MolsidorniriM).
The sydnonimine-type prodrugs (e.g. MolsidomiriM) depend on the liver to remove the protecting ethoxy-carbonyl-group from the molecule to produce first SIN-1 and then (in a se-cond, pH dependent process catalysed by OH- ions) the very instable SIN-lA is formed which independently of the pH spon-taneously decomposes with the release of NO.
b. Preparation of adducts or complexes of nitric oxide with various nucleophiles.
Generally the synthesis of the secondary amine NO complexes is as follows: The secondary amine e.g. anhydrous diethylamine is dissolved in anhydrous ether, oxygen is removed from the system by using aceton dry-ice bath and dry NO is bubbled through the ether solution at -78"C for 3 hours, preferably at high pressure (100 psi).
The half-life of the prior art diethylamine-nitric oxide adduct Et2-N-NH-(ONa)-N=O (DEANO) amounts to about 2~minutes, while the nitric oxide addition product of polyamine-spermine (SPNO) has a half-life of 39 minutes.
c. The prodrug is stabilized by cyclodextrin inclusion-com-plex formation while NO is released spontaneously under phy-siological conditions. The known cyclodextrin complexed pro-drug is stable in the solid state.
It is known, that SIN-1. is a stable compound in solid state, however, its open-chain tautomeric form SIN-lA is extremely unstable. It is highly difficult to isolate the yellow cry-stalline product in pure form and it can be stored only at -SO~C, under nitrogen. The SIN-lA form rapidly releases one mole NO in solid state through photolysis, and in aqueous so-lution even in darkness it is converted to cyanomethylene-amino-morpholine (SIN-1C).
SIN-1 is thus considered to be a prodrug and SIN-1C a biolo-gically inactive degradation product. The equilibrium between the stable SIN-1 and the active SIN-lA (which is the real drug) depends on ambient factors (pH, temperature). However because of the extreme instability of SIN-lA - especially to oxygen - it is practically a short-lived intermediate in the decomposition process of SIN-1.
Presently SIN-1 is marketed only for intravenous administra-tion in powder-ampoules, to be dissolved before injection.
' The orally administered SIN-1 prodrug is ineffective, because the hydrolytic step which is required for the formation of the NO generating SIN-1A form can not take place under acidic pH conditions, and before absorption it is rapidly and completely decomposed to SIN-1C at the higher pH of the gas-trointestinal tract.
It is also public, that SIN-1 can be stabilized by CD comple-xation and the equilibrium SIN-1 -~ SIN-lA -~ SIN-1C can be shifted by means of complexation with cyclodextrin derivati-ves (PCT Publication WO 91/14681). It was disclosed that com-plexati~on with certain CD derivatives inhibit SIN-1C
formation. Thus more stable complexes of SIN-1/CDPSI inclu-ding those containing an increased amount of SIN-lA (showing biological activity based on higher NO release) were prepared by short time heat-treatment of the SIN-1/CDPSI complex product obtained by lyophilisation. Similarly a SIN-1/DIMEB
complex was exemplified. These CD derivatives were proposed to be used in pharmaceuticals.
This document disclosed SIN-1/CD complexes or SIN-1/lactose mixture with 4,5~ (w/w) SIN-1 content where the SIN-lA con-tent in the obtained products were as follows:
in CDPSI complex 1.27 in J3CD complex 0.08 in DIMEB complex 0.06 in HPBCD complex 0.115 in mixture with lactose 0.00 Practically complete conversion of SIN-1 to SIN-1A was not disclosed to take place even when complexation was brought about with CDPSI which was disclosed to be the most effective complexation agent for this purpose.
Moreover: CDPSI and DIMEB are not approved to be used in pharmaceuticals as yet. Thus the preparation of a stable, marketable form of pure SIN-lA is an unsolved problem.
Because SIN-1 can be applied only intravenously, its prodrug Molsidomin is used for the oral treatment of heart insuffi-ciency: Molsidomin is hydrolysed enzymatically in the liver to SIN-1.
WO 95/29172 . PCT/HU95/00011 It was the ultimate goal of this invention to stabilize the NO-donor SIN-1A so as to ensure a formulation which can be administered both orally and parenterally, the innocuousness of which is supported with complete (even i.v.) toxicologi-cal documentation. Presently two types of cyclodextrins ful-fill this fundamental requirement: gammaCD and HPf3CD.
Though the SIN-lA stabilizing effect of CDPSI and DIMEB in aqueous solutions is known it does not however follow, that also the non-ionic gammaCD is effective in this respect, par-ticularly because of its much wider cavity diameter. The ex-cellent stabilizing effect of CDPSI has been attributed to its polymeric structure, i.e. to the cooperative effects of various cyclodextrin rings anchored in sterical vicinity.
The subjects of this invention are new SIN-lA inclusion complexes which are stable in their solid state formed of SIN-1A and cyclodextrins or cyclodextrin derivatives and re-leasing nitric oxide at room temperature upon dissolving in water or aqueous systems and optionally also containing an ion as a catalyst or stabilizer.
According to a preferred embodiment they contain a phy-siologically acceptable anion as a catalyst or stabilizer such as carboxylic acid anions i.a. acetate, formiate, pro-pionate, ascorbinate, tartarate and/or lactate and/or inorga-nic acid anions such as phosphate, phosphite, borate, carbo-nate, hydrocarbonate, sulfate and/or sulfite. Acetate was found to be an excellent anion for the purpose. The anions may be present as salts and the corresponding cations may be preferably ammonium or alkali ions however other cations may be used as well.
As cyclodextrin component they contain BCD, gammaCD or aCD, especially for pharmaceutical use. They also may contain cyclodextrin derivatives namely hydroxy-propylated or methyla-ted cyclodextrins such as HPf3CD, DIMEB, RAMEB, TRIMEB or CDPSI.
Another feature of the invention are biologically active compositions containing as their active ingredient the new SIN-1A/ cyclodextrin complexes along with auxiliary and additive ingredients facilitating their use. These include but are not limited to pharmaceutical compositions contai-ning as an active ingredient SIN-1A inclusion complexes op-WO 95/29f12 PCT/HU95/00011 -5- r.
tionally with usual auxiliary and additional materials used in pharmaceuticals for oral, parenteral or other medical uses. The formulations are preferably powders dissolved di-rectly before medication takes place. Thus the parenteral ' formulations are preferably powders dissolved prior to injec-tion.
Pharmaceutical compositions of preference are those con-taining as an active ingredient SIN-1A inclusion complexes which are stable in their solid state and which are formed with f~CD, gammaCD or aCD and containing a physiologically acceptable anion as a catalyst or stabilizer, such as car-boxylic acid anions including acetate, formiate, propionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions comprising phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and/or sulfite. The anions may be optionally in the form of their salts e.g. the ammonium or alkali salts. Ammonium acetate is a preferred salt for the purpose.
Further objects of the present invention are kits to be used as NO-liberating standards to release NO in a predictable amount and rate on dissolving in aqueous media containing as an active ingredient SIN-lA inclusion complexes according to the present invention.
This is possible because the present invention relates to the preparation and stabilization of the extremely labile SIN-lA, which when released from the cyclodextrin cavity - even after simple dissolving in distilled water - immediately generates the NO in a predictable amount and rate without the need of any further enzyme or reactant.
The present invention includes processes for the preparation of new SIN-lA inclusion complexes which are stable in their solid state and which are formed with cyclodextrins or with cyclodextrin derivatives by way of subjecting at a suitable pH SIN--1 to the catalytic action of ions to shift the equi-librium towards formation of SIN-lA in the presence of cyclo-dextrins or cyclodextrin derivatives capable to form in-clusion complexes, whereby the SIN-lA formed is immediately complexed and stabilized by formation of SIN-lA/ cyclodextrin inclusion complexes. The process includes isolating in the solid state the obtained SIN-1A/CD complexes optionally containing the ions. The ions may be contained in the form of their salts.
WO 95/29172 ~ PCT/HU95/00011 It is a preferred process according to the invention to react SIN-1 and a cyclodextrin or cyclodextrin derivative in the solid state in the presence of an ion preferably in the form of its salt as a catalyst by thoroughly admixing or milling the components together. Another route according to the in vention follows freeze drying an aqueous, oxygen-free so-lution containing the components, followed preferably by "second drying" in vacuo.
It is preferred to use ammonium or alkali salts formed with carboxylic acid anions such as acetate, formiate, propionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions such as borate, carbonate, hydrocarbonate, phosphate, phosphite, sulfate, and/or sulfite as catalyst of the pro-cess. The salts used may contain volatile anions or cations which are eliminated partly or totally during the process such as ammonium or carbonate ions.
When accomplishing the above it is advantageous to carry out the reaction at pH values between 6 to 10 and - when water was used as a reaction medium - applying "second drying" at 40 to 100°C preferably 50 - 70~C.
As it is seen from the NO-release tests, SIN-lA/cyclodextrin complexes immediately produce nitric oxide after dissolution of the solid complexes in aqueous systems. Thus one way to obtain a SIN-1A cyclodextrin complex of controlled compositi-on (having the lowest SIN-1C content) consists in tautomeri-zation of SIN-1 and the simultaneous complexation of the for-med SIN-1A in solid state.
The situation is similar when the complex preparation is per-formed by freeze-drying. In this case the second-drying step (following lyophilization) is able to ensure the conditions for the solid-state catalytic complex formation.
Another subject of the invention consists in methods of nitric oxide treatment of living cells. This includes but is not limited to the treatment of nitric oxide dependent symptoms in humans or animals like anginic and ischemic heart failures, physiological control of blood pressure, platelet aggregation, mediation of relaxation of peristalsis, penile erection and others. This is accomplished by administering ' preferably in oral or parenteral application to the patients in need of such treatment an effective amount of a new SIN-lA inclusion complex which is stable in its solid state and WO 95/291 l2 PCT/HU95/00011 which is formed with cyclodextrins or with cyclodextrin deri-vatives and which contains at least one ion as a catalyst or stabilizer and which upon dissolving in water or aqueous sy-stems at room temperature releases nitric oxide.
The preferred embodiment relies in administering the complexes of SIN-1A formed with f3CD, gammaCD or aCD and con-taining optionally in the form of their ammonium or alkali salts carboxylic acid anions such as acetate, formiate, pro-pionate, ascorbinate, tartarate and/or lactate and/or inorganic acid anions such as phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and/or sulfite.
The invention includes the method of treatment of nitric oxide dependent symptoms in humans or animals by treating the patient with the product of the quantitative conversion of SIN-1 into SIN-1A accomplished by way of an ion- catalysed and cyclodextrin-stabilized solid state conversion in the presence of a cyclodextrin or a cyclodextrin derivative ca-pable to immediately form inclusion complexes.
The major advantages of SIN-1A/CD complexes as compared with SIN-1 or other NO -donors known hitherto are thus the following:
- stability at the long term, - rapidity of action, - increased half-life, - independence from the liver, - independence from the pH, - eventually a greater ability to reach their targets (tissues).
Having the stable composition in hands makes it possible to open a new phase for treatments with nitric oxide which is called by some authors as "biochemistry's unexpected new superstar" (Chem.Ing.News Dec.1993.page 26-38).
-g-The following Examples serve illustration and not limitation of the invention.
I. CHEMICAL EXAMPLES
Example I.1.
Preparation of SIN-lA/gammaCD complex 2 g of gammaCD and 0,8 g of ammonium acetate were dissolved in 25 ml of distilled water by ultrasonication. The solution was deoxygenated by bubbling with helium gas, thereafter 200 mg of SIN- 1 substance were dissolved. The solution was imme-diately freeze-dried for isolation of the solid complex. Se-cond drying at 40-50°C for 2 hours was applied to remove the water content of the complex almost completely. Both solution and the substance were protected from light.
The complex is a light yellow powder. Yield: 2.6~0.1 g Loss on drying is less than 1%.
HPLC analysis: SIN-1 content: not detectable SIN-1A content: 11.7 ~0.2%
SIN-1C content: 0.36~O.ls In all examples of this document the following method was used for simultaneous determination of SIN-1, SIN-lA and SIN-1C by HPLC .
Column: UltrasphereTM I.P. analytical column (Beckman-Astec) 4.6~250 mm, particle size 5 um.
Mobile phase: 0.01 M phosphate buffer pH=6.0 800 ml tetrahydrofuran 200 ml sodium-1-dodecanesulfonate 0.405 g/dm3 Ionic strength: 0.05 gion/dm3 (corrected with sodium sul-phate) Flow rate: 1 ml/min., p=190 bar. Sample size: 20 ~1 Wavelength of detection:
-230 nm BW: 6 (ref. 350 nm BW: 80) for SIN-lA and SIN-1C
between 0-8 minutes -292 nm BW: 6 (ref. 400 nm BW:80) for SIN-1 after 8 minutes Retention times: SIN-1 9:6 min.
SIN-1C 4.5 min.
SIN-lA 4.8 min.
Calibration was performed with freshly prepared SIN-1 and SIN-1C solutions, SIN-lA content was expressed in~ SIN-1C
equivalent.
Example I.2 Preparation of SIN-lA/BCD complex -g-16 g of l3CD (water content 14~) and 7 g of ammonium acetate were dissolved in 1000 ml of distilled water by ul-trasonication. The solution was deoxygenated by bubbling with helium gas, thereafter 3 g of SIN-1 substance were dissolved.
The solid complex was isolated by immediate freeze-drying and water content was removed at 40-50°C. Both solution and sub-stance 'were protected from light. The complex is a yellowish very light powder. Yield: 20~1 g. Loss on drying < 1~
HPLC analysis: SIN-1 content: not detectable SIN-1A content: 12~1~
SIN-1C content: 0.30~0.2~
Example I.3 Preparation of SIN-lA/HPBCD complex 2 g of :HPBCD (DS=2.8) and 0.8 g of ammonium acetate were dis-solved in 25 ml of distilled water by ultrasonication. On deoxygenation by bubbling with helium gas; 200 mg of SIN-1 were dissolved. The solid complex solution was isolated by immediate freeze-drying. The water content was removed at 40-50°C. Both solution and substance were protected from light. 2,8 ~ 0,1 g of SIN-lA / HPBCD complex as a light yel-low powder were obtained. Loss on drying <1~.
HPLC analysis: SIN-1 . not detectable SIN-1A: 11.6~0.2~
SIN-1C: 0.36~0,1~.
Example I.4 Preparation of SIN-lA/RAMEB complex 2 g of ItAMEB (DS=1,8) and 0,8 g of ammonium acetate were dis-solved in 25 ml of distilled water by ultrasonication. On deoxygenation by bubbling with helium gas 200 mg of SIN-1 substance were dissolved, the solution was freeze-dried and the isolated solid complex dried at 40-50°C for 2 hours. So-lution .and substance were protected from light.
2.8~0,1 g SIN-lA RAMEB complex were obtained as a light yellow powder. Loss on drying < 1~
HPLC analysis: SIN-1 . not detectable SIN-lA . 12.0~0,2~
SIN-1C . 0.6~0,1~.
Example I. 5.
Preparation of SIN-1A l3CD complex in phosphate buffer solution 1 g of f3CD (water content: 14~) was dissolved in 55 ml of a pH - 8.0 phosphate buffer solution according to USP XXII by WO 95/29172 ~ ' PCT/HU95/00011 ultrasonication. On deoxygenation with helium gas 100 mg of SIN-1 were dissolved. The solution was immediately freeze-dried. Solution and substance Mere protected from light.
1 ~ 0.1 g of SIN-lA/f3CD were obtained as a yellowish coloured very light powder. Loss on drying < l~
HPLC analysis: SIN-1 content: not detectable SIN-lA content: 8.3~
SIN-1C content: 0.24 Example I.6 1 g of f3CD (water content 14~), 100 mg of SIN-1 hydrochloride and 100 mg of ammonium acetate were thoroughly mixed in a mortar for 15 minutes. After a short time of rubbing the mix-ture became visibly yellow. After 2 days of storage in a clo-sed container protected from light, HPLC analysis according to Example I.1 was taken to give the following results:
SIN-1 content: not detectable SIN-lA content: 7.9~0.2~
SIN-1C content: 0.41~0.1~
Example I.7 Using the process of Example I.6. but taking 50 mg of ammo-nium acetate the visible formation of SIN-1A (yellow colora-tion) is slower, it took several hours.
HPLC analysis of the SIN-lA/l3CD after 2 days of storage:
SIN-1 0.5~0,1~
SIN-1A 6.9~0,2~
SIN-1C 0.6~0,1~
Example I.8 Following the procedure as described in Example I.6. but ap-plying 300 mg of ammonium acetate the yellow coloration of the mixture occurs practically immediately after mixing the components.
HPLC analysis of the fiCD complex same day after preparation:
SIN-1 . not detectable SIN-lA : 6.9~0.2$
SIN-1C . 0.5~0,1~
Example I.9 1 g of 13CD (water content 14~), 100 mg of SIN-1 hyrochloride and 100 mg of sodium acetate.3H20 were thoroughly mixed in a mortar for 15 minutes, followed by heat treatment of the mix- ' ture at 70'C for 1 hour.
HPLC analysis of the heat-treated sample:
SIN-1: ' not detectable -11- .
SIN-lA: 7.1 ~ 0.2~
SIN-1C: 0.8 ~ 0.1~
II. COMPARATIVE EXAMPLES
' Example II.1 Attempted preparation of SIN-lA/ l3CD with acetic acid Preparation was carried out as in Example I.2 but instead of water and ammonium acetate the f3CD was dissolved in a 0.1 mo-le acetic acid solution.
HPLC analysis of the product: SIN-1 . 8.4~
SIN-lA: 0.28 SIN-1C: not detectable.
Thus the solid SIN-lA/f3CD complex was not isolated.
Example II.2 Attempted preparation of SIN-1A/f3CD with sodium hydroxide Complexation was carried out as described in Example I.S, but the l3CD was dissolved in 55 ml of pH 8.4 aqueous sodium hydr-oxide istead of using ammonium acetate. HPLC analysis of the obtained solid product:
SIN-1: 10.5$
SIN-lA: 0.40 SIN-1C: not detectable.
No SIN-1A/f3CD complex was isolated.
III. ANALYTICAL AND BIOLOGICAL EXAMPLES
Example III.1 The effectivity of conversion and stability of the SIN-lA
complexes are illustrated by Table 1. The samples were stored at room temperature for 11 months in glass containers under air atmosphere, protected from light. The SIN-1A con-tent is given in SIN-1C equivalent.
Table 1.
SIN-1 contents SIN-lA contents SIN-1C content after after after after after after prepa- storing prepa- storing prepa- storing ration for 11 ration for 11 ration for 11 months months months SIN-1/aCD 3.59 2.46 0.68 SIN-1/J3CD 0 0 10.66 9.59 0.68 0.50 SIN-1/~"CD 0 0 11.71 7.23 0.32 0.49 ~.~~~~~9 HPBCD 0.03 0 11.61 7.53 0.36 0.39 RAMEB 0 0 12.11 9.43 0.68 0.87 Example III.2 A SIN-1A/l3CD sample contained immediately after preparation -11.47~ SIN-1A. When stored at room temperature for 12 months it contained 8.36 SIN-1A, and after 23 months it contained 6.59 SIN-1A. The SIN-iC contents were 0.18, 1.31 and 1.57 respectively.
The complexes contained approx. 5-8~ inclusion water, mainly bound to the cyclodextrin cavity.
Example III.3 The stability enhancing effect of heat-treatment ("second drying") after freeze- drying is illustrated by Table 2.
Table 2.
SIN-1l3CD SIN-1 contents SIN-lA contents SIN-1C content after after after after after after preps- storing preps- storing preps- storing ration for 11 ration for 11 ration for 11 months months months SIN-1/13CD 0 0 12.7 10.89 0.40 0.79 SIN-1/f3CD
heat treated 0 0 12.7 12.50 0.40 0.56 after after after after after after preps- storing preps- storing preps- storing ration for 19 ration for 19 ration for 19 months months months SIN-1/l3CD 0.04 0 14.81 7.41 0.29 1.75 heat treated 0.040 14.81 12.41 0.29 1.15 Example III.4 Rate and extent of NO release from SIN-lA/f3CD complex.
Release of NO in SIN-lA/CD solutions was examined by chemic-al and biological methods.
250 mg of complex (corresponds to approximately 25 mg of SIN-WO 95/29172 ~ . PCT/HU95/00011 1A) were dissolved in 100 ml of distilled water. Right after dissolution liberation of NO was indirectly detected by si-multaneously measuring decrease of the SIN-1A content and formation of the SIN-1C metabolite by HPLC as a function of ' time. The measurements were repeated in different time inter-vals, while protecting from light.
Table 3 illustrates the kinetics of decomposition of SIN-lA
in distilled water at room temperature in presence of f3CD, gammaCD and HPBCD.
Table 3 time, min. 13CD complex BCD complex HPBCD complex SIN-lA SIN-1C SIN-lA SIN-1C SIN-lA SIN-1C
~g/ml ~g/ml ~cg/ml ~Cg/ml ~Cg/ml ~g/ml 70 ~ 86 65 - - - -The estimated T1/2 (half-life) of the complexes calculated from the decrease of SIN-1A content are 53 min. for 13CD, 24 min. for gammaCD and 70 min. for HPBCD.
Example III.S
Release of NO in a more diluted SIN-1A/BCD solution (100 ~g/ml of SIN-1A/f3CD complex corresponds to 10 ~ug/ml of SIN-1A) was detected by W-spectrophotometry measuring the formation of SIN-1C metabolite as a function of time. SIN-1A
SIN-1C transition is accompanied by a very characteristic W-spectrum change, as SIN-lA has an UV maximum at 230~1 nm and shows no absorbance at JL max SIN-1C at 277~1 nm.
mg of the SIN-lA/13CD complex (corresponds to approximate-" 1y 1 mg of SIN-1A) were dissolved in 100 ml of distilled wa-ter. UV-spectrum of the solution was registered between 200-350 nm without dilution immediately after dissolution and registration was repeated in different time intervals, pro-tected from light . The formed SIN-1C amount was calculated from the absorbance values at 277 ~ 1 nm using the cali-bration curve taken with a SIN-1C standard. SIN-1C concentra-tion was less than 0,7 ~Cg/ml right after dissolution and ab-out 7.8 ~g/ml after 270 minutes. Presumably the equivalent amount of NO was released. Estimated half-life of the complex:
90-100 minutes. ,.
Figure 1. illustrates how the UV spectra change as a function of time: UV spectra of a SIN 1A/f3CD complex at 10 minute time intervals up to 160 minutes after dissolution of 10 mg of the complex in 100 ml of distilled water are shown. Absorbance is spotted against wavelength.
Example III.6 Formation of NO was determined directly, using NO specific porphyrinic microsensor detection. The method (Nature, Vol.
358 p. 675, 1992) is able to monitor the NO release up to 10-20 moles, in a single cell in biological microsystems in amperometric or voltametric mode .
In a pH= 7.4 phosphate buffer at 37°C 1 mM SIN-lA/BCD relea-sed NO at a rate of 2.37 ~CM/min. in the first 5 minutes, and between the 10th and 30th minute at a rate of 0.17 ~tM/min.
Dissolving 0,1 mM of substance the relevant values were 0.42 ~uM and 0.07 ~M respectively.
Example III.7 Inhibition of platelet aggregation by SIN-1A/l3CD complex:
Nitric oxide had been identified as a natural messenger mole-cule in the inhibition of platelet aggregation via the gua-nylate-cyclase/cyclic GMP system (Blood, 57. 946, 1981). We studied the biological effects of SIN-1 and SIN-lA/BCD com-plex by comparing their platelet aggregation inhibiting ef-fects. The platelet aggregation was studied in rabbit and hu-man platelet rich plasma (J. Cardiovasc. Pharmacol. ,14 suppl.
11; page 120, 1989). In each batch of platelet rich rabbit plasma 8 dose response curves were registered with the throm-boxane mimetic U-46619 (0.25-4.0 ~M) in the presence of 0, 10-7, 10-6 or 10-5 moles of SIN-1A/BCD complex. The dose re-sponse curves were performed in random sequence. Thereafter a fixed concentration of U46619 (4~CM) was used to make full concentration-inhibition curves to SIN-1 and to SIN-1A/f3CD. "
U46619 induced a concentration-related aggregation. The ag-gregation inhibiting effect of SIN-lA/BCD complex at identi-cal molar concentration in all cases was significantly higher than that of SIN-1. The pD2 (negative logarithm of con-centration producing 50~ inhibition) values were 5.57 ~ 0.11 (n=7) for SIN-1 and 6.36~0.07 (n=7) for SIN-lA/13CD complex, indicating that the complex was about 6-fold more potent than ' SIN-1 in this test.
In some similar experiments the SIN-1A/BCD complex was found to be about 10-fold as potent as SIN-1.
Example III.8 The experiments described in Example III.7 were repeated using human citrate platelet rich plasma with SIN-1, freshly prepared SIN-lA/f3CD complex (SIN-1A content 14.80 and with a SIN-1A/l3CD complex, that has been stored at room temperature for 23 months (SIN-1 content originally was 11.47, after 23 months it was 6.590 . Table 4 illustrates the results showing the negative logarithms of the concentration (pD2) of SIN-1 and SIN-1A f3CD complex causing 50~ inhibition of U46610 in-duced aggregation in human platelet rich plasma.
Both amplitude and slope of the aggregation curve were eva-luated. The difference in the pD2 values indicate that fresh-ly prepared SIN-lA/BCD was about 8-fold more potent than SIN-1. The complex stored for 23 months was found to be 3-fold less active than the freshly prepared complex, but 3-fold more potent than SIN-1 itself.
Table 4.
Substance a g g r a g a t i o n n amplitude slope SIN-1 10 4.8~0.05 4.85~0.13 SIN-1A 13CD freshly prepared (SIN-1A content:14.81~) 10 5.54~0.10 5.85~0.11 SIN-lA l3CD (stored for 23 months, SIN-1A content: 6.59 ~) 3 5.80~0.16 5.42~0.06 The results of in vivo tests suggest that the SIN-lA/BCD
complexes disclosed here are useful inhibitors of platelet aggregation iv vivo.
As is reflected from the half-life values, SIN-lA/cyclodex-trin complexes are able to continuously produce nitric oxide generation at a more nearly constant rate for much longer time after administration.
Example III.9 Commercially available SIN-1 (CORVASALj was compared with the SIN-lA/SCD complex on carotid artery of rabbits. The carotid artery of nembutal anesthesized rabbits was dissected free, 3 mm long rings were immobilized in a clamp and placed.in the organ baths. Contraction was induced with phenylephrine 3x10- M three times with following rinsing. The dose response curves were registered for 3x10-9 M to 3x10-5 M
CORVASAL (referred to SIN-1 content) and for 3x10-9 to 3x10-5 moles SIN-lA/SCD (referred to SIN-lA content).
Figure 2. shows the contraction versus drug concentration curve. As it is seen, the SCD complexed SIN-lA drug was about 6-fold more effective than free SIN-1. The illustrated points represent the average of 6 measurements.
Claims (20)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An inclusion complex which is stable in its solid state and formed of N-morpholino-N-nitrosoamino-acetonitrile and .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin or a hydroxy-propylated or methylated cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin derivative and which releases nitric oxide at room temperature upon dissolution in water or an aqueous system.
2. An inclusion complex according to claim 1, and which contains a physiologically acceptable ion as a catalyst or stabilizer.
3. An inclusion complex according to claim 2 containing one or more ions selected from the group consisting of acetate, formiate, propionate, ascorbinate, tartrate, lactate, phosphate, phosphate, borate, carbonate, hydrocarbonate, sulfate, and sulfite or one or more ions selected from the group consisting of alkali metal and ammonium.
4. An N-morpholino-N-nitrosoaminoacetonitrile inclusion complex according to any one of claims 1 to 3, containing as cyclodextrin .beta.CD, gammaCD or .alpha.CD.
5. An N-morpholino-N-nitrosoaminoacetonitrile inclusion complex according to any one of claims 1 to 3, containing as cyclodextrin derivative a hydroxy-propylated or methylated cyclodextrin selected from: hydroxypropyl-.beta.-cyclodextrin, heptakis-2,6-di-O-methyl-.beta.-cyclodextrin, randomly methylated-.beta.CD, and heptakis 2,3,6-tri-O-methyl-.beta.-cyclodextrin.
6. A pharmaceutical composition containing as active ingredient an inclusion complex according to any one of claims 1 to 5, and one or more conventional auxiliary or additional materials used in pharmaceuticals for oral, parenteral or other medical use.
7. A pharmaceutical composition according to claim 6, wherein the formulation is a powder dissolved directly before medication takes place.
8. A pharmaceutical composition containing as active ingredient a N-morpholino-N-nitrosoaminoacetonitrile inclusion complex which is stable in its solid state and which is formed with .beta.CD, gammaCD, or .alpha.CD and contains a physiologically acceptable anion as a catalyst or stabilizer, selected from those listed in claim 3.
9. A pharmaceutical composition according to claim 8, wherein the composition includes an ammonium or alkali metal salt.
10. A pharmaceutical composition according to claim 9, wherein the salt is ammonium acetate or an alkali metal acetate.
11. A kit for use as an NO-liberating standard for the release of NO in a predictable amount and rate on dissolution in an aqueous medium containing as active ingredient an N-morpholino-N-nitrosoaminoacetonitrile inclusion complex according to any one of claims 1 to 5.
12. A process for the preparation of a N-morpholino-N-nitrosoaminoacetonitrile inclusion complex which is stable in its solid state and which is formed with .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin or a hydroxy-propylated or methylated .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin derivative, which comprises subjecting at a suitable pH 3-morpholino-sydnonimine to the catalytic action of ions to shift the equilibrium towards formation of N-morpholino-N-nitrosoaminoacetonitrile in the presence of cyclodextrin or a cyclodextrin derivative capable of forming an inclusion complex, whereby the N-morpholino-N-nitrosoaminoacetonitrile formed is immediately complexed and stabilized by formation of an N-morpholino-N-nitrosoaminoacetonitrile/ cyclodextrin inclusion complex, and isolating in the solid state the obtained N-morpholino-N-nitrosoaminoacetonitrile /CD complex or a salt thereof.
13. A process according to claim 12, which comprises reacting 3-morpholino-sydnonimine and a cyclodextrin or cyclodextrin derivative in the solid state in the presence of an ion or a salt thereof as a catalyst by thoroughly admixing or milling the components together or by freeze drying an aqueous, oxygen-free solution containing the components.
14. A process according to claims 12 or 13, which comprises using as catalyst or stabilizer an ammonium or alkali metal salt formed with a carboxylic acid anion selected from the group consisting of acetate, formiate, propionate, ascorbinate, tartrate and lactate or with an inorganic acid anion selected from the group consisting of borate, carbonate, hydrocarbonate, phosphate, phosphate, sulfate and sulfite.
15. A process according to claim 14, wherein the complex is thereafter subjected to a "second drying" in vacuo.
16. A process according to claim 15 which comprises carrying out the reaction at a pH value of between 6 to 10 and, when water is used as a reaction medium, applying the "second drying" at 40 to 100°C.
17. The use of an effective amount of a new N-morpholino-N-nitrosoaminoacetonitrile inclusion complex which is stable in its solid state and which is formed with .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin or with a hydroxy-propylated or methylated .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin derivative, for nitric oxide dependent symptoms of humans or animals, which complex contains at least one ion as a catalyst or stabilizer, and which, upon dissolution in water or an aqueous system at room temperature, releases nitric oxide.
18. The use according to claim 17, wherein the nitric oxide dependent symptom comprises anginic or ischemic heart failure, physiological control of blood pressure, platelet aggregation, mediation of relaxation of peristalsis or penile erection.
19. The use, according to claim 17 or 18, of an effective amount of a complex of N-morpholino-N-nitrosoamino-acetonitrile formed with BCD, gammaCD, or aCD
or such a complex in combination with an ammonium or alkali metal salt with a carboxylic acid anion selected from the group consisting of acetate, formiate, propionate, ascorbinate, tartrate and lactate or an inorganic acid anion selected from the group consisting of phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and sulfite, or of a pharmaceutical composition containing such a complex for nitric oxide dependent symptoms in living cells or humans or animals.
or such a complex in combination with an ammonium or alkali metal salt with a carboxylic acid anion selected from the group consisting of acetate, formiate, propionate, ascorbinate, tartrate and lactate or an inorganic acid anion selected from the group consisting of phosphate, phosphite, borate, carbonate, hydrocarbonate, sulfate and sulfite, or of a pharmaceutical composition containing such a complex for nitric oxide dependent symptoms in living cells or humans or animals.
20. The use of an effective amount of the product of the quantitative conversion of 3-morpholino-sydnonimine into N-morpholino-N-nitrosoaminoacetonitrile accomplished by way of an ion- catalysed and cyclodextrin-stabilized solid state conversion in the presence of a .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin or a hydroxy-propylated or methylated .beta.-cyclodextrin, .gamma.-cyclodextrin, or .alpha.-cyclodextrin derivative capable of immediately forming an inclusion complex for nitric oxide dependent symptoms in humans or animals.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HUP9401183 | 1994-04-26 | ||
| HU9401183A HU218280B (en) | 1994-04-26 | 1994-04-26 | Cyclodextrin inclusion complexes containing sin-1a which are stable intheir solid state, process for their preparation and pharmaceutical compositions containing the comlexes |
| PCT/HU1995/000011 WO1995029172A1 (en) | 1994-04-26 | 1995-04-25 | N-morpholino-n-nitrosaminoacetonitril cyclodextrin inclusion complexes |
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| US6143746A (en) * | 1994-01-21 | 2000-11-07 | Icos Corporation | Tetracyclic cyclic GMP-specific phosphodiesterase inhibitors, process of preparation and use |
| MXPA02004743A (en) * | 1999-11-12 | 2003-10-14 | Roquette Freres | Crystalline mixtures of partial methyl ethers of beta cyclodextrin and related compounds. |
| FR2805462B1 (en) * | 2000-02-24 | 2003-08-15 | Therabel Res | NEW ORAL GALENIC FORM WITH EXTENDED RELEASE OF MOLSIDOMINE |
| TWI321054B (en) | 2000-12-19 | 2010-03-01 | California Inst Of Techn | Compositions containing inclusion complexes |
| EP1219306A1 (en) | 2000-12-29 | 2002-07-03 | Nicox S.A. | Compositions comprising cyclodextrins and NO- releasing drugs |
| ES2377318T3 (en) | 2002-09-06 | 2012-03-26 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for the delivery of covalently bound therapeutic agents to them |
| US8357377B2 (en) * | 2002-10-09 | 2013-01-22 | Suzie Hwang Pun | Cyclodextrin-based materials, compositions and uses related thereto |
| JP2010516625A (en) | 2007-01-24 | 2010-05-20 | インサート セラピューティクス， インコーポレイテッド | Polymer-drug conjugates with tether groups for controlled drug delivery |
| AU2010321533A1 (en) * | 2009-11-23 | 2012-05-31 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutic delivery |
| WO2014055493A1 (en) | 2012-10-02 | 2014-04-10 | Cerulean Pharma Inc. | Methods and systems for polymer precipitation and generation of particles |
| US10793327B2 (en) | 2017-10-09 | 2020-10-06 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN114127501B (en) | 2019-03-14 | 2023-02-17 | 泰尔茂比司特生物技术有限公司 | Multi-part lyophilization container and method of use |
| CN116249522A (en) * | 2020-07-20 | 2023-06-09 | 深圳迈瑞生物医疗电子股份有限公司 | Application of composition in platelet depolymerization, depolymerization reagent and depolymerization method |
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| US5208233A (en) * | 1989-09-15 | 1993-05-04 | The United States Of America As Represented By The Department Of Health And Human Services | Anti-hypertensive compositions of secondary amine-nitric oxide adducts and use thereof |
| US5212204A (en) * | 1989-10-18 | 1993-05-18 | The United States Of America As Represented By The Department Of Health And Human Services | Antihypertensive compositions and use thereof |
| HU212730B (en) * | 1990-03-28 | 1996-10-28 | Chinoin Gyogyszer Es Vegyeszet | Process for producing inclusion complexes of cyclodextrine or its derivative and 3-morpholino-sydnonimine or its salt or its tautomer and pharmaceutical compositions containing them |
| DE69223157T2 (en) * | 1991-09-24 | 1998-06-10 | Us Health | OXYGEN-SUBSTITUTED DERIVATIVES OF NUCLEOPHIL NITROGEN OXIDE ADDUCTS AND THEIR USE AS NITROGEN OXIDE DONOR PRODRUGS |
| US5389675A (en) * | 1992-03-27 | 1995-02-14 | The United States Of America As Represented By The Department Of Health And Human Services | Mixed ligand metal complexes of nitric oxide-nucleophile adducts useful as cardiovascular agents |
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