CA2220060A1 - A method for increasing the electrotransport flux of polypeptides - Google Patents

Abstract

Methods for modifying polypeptide agents to enhance their transdermal electrotransport flux are provided. The polypeptide is modified by reducing the potential of the polypeptide for forming .alpha.-helical or .beta.-sheet segments. In particular, amino acid residues known to stabilize .alpha.-helical and .beta.-sheet segments can be replaced with destabilizing residues and known helix breakers. Modified molecules and compositions including the molecules are also provided.

Description

CA 02220060 l997-ll-03 WO 96/39423 . PCT/US96/09647 A METHOD FOR INCREASING THE ELECTROTRANSPORT
FLUX OF POLYI~tl~ I IL ES

TECHNICAL FIELD
The invention relates generally to ele~;t,ul,~lsport drug delivery. More particularly, the invention relates to a method for increasing ele~ ull a~ ,s,uu, l flux of a polypeptide by reducing the potential of the polypeptide for forming oc-helical or ~-sheet segments. The invention also pertains to molecules which have been so modified.

BACKGROUND ART
Transdermal (ie, through the skin) delivery of therapeutic agents affords a cor"rul lable, convenient and noninvasive technique for administering drugs. The method provides several adva"~ages over conventional modes of drug delivery. For example, variable rates of absorption and (eg, hepatic) metabolism encountered in oral treatment are avoided, and other inherent inconveniences--eg, gastrointestinal irritation and the like are eliminated. Transdermal delivery also allows a high degree of control over blood conce, ILI ~lions of a particular drug and is an especially ~lll ac;live administration route for drugs with narrow therapeutic indexes, short half-lives and potent activities.
Tta"scJe""al delivery can be either passive or active. Many drugs are not suitable for passive transdermal drug delivery bec~use of their size, ionic charge c:hal~cL~I istics and hyd, u~l ~Gbicity. One method of ovel cor"i"g this limitation is the use of low levels of electric current to actively transport drugs into the body through intact skin. This technique is known as "ele_t, ull ~"sport" or "io"to,ul ,or.alic" drug delivery. The technique provides a more controllable process than passive l,d"scler",al drug delivery since the amplitude, timing and polarity of the applied electric current is easily 30 reg~ ted using standard electrical co""~o"ents. In this regard, CA 02220060 l997-ll-03 WO 96/39423 PCT/US!)G/U5647 elecl, ull ~"sport drug flux can be from 50% to several orders of magnitude greater than passive transdermal flux of the same drug.
Elecl, ul, ~"sport devices generally employ at least two electrodes.
Both of these electrodes are positioned in i"Li~"~Le electrical contact with some portion of the skin of the body. One electrode, called the active or donor electrode, is the electrode from which the therapeutic agent is delivered into the body. The other electrode, called the counter or return electrode, serves to close the electrical circuit through the body. In conjunction with the patient's skin, the circuit is completed by connection of the electrodes to a source of electrical energy, eg, a battery, and usuaily to circuitry capable of controlling current passing through the device.
Depending upon the electrical charge of the species to be delivered transdermally, either the anode or c~LI ,Gde may be the active or donor electrode. In this regard, if the ionic sl ~hsPnce to be driven into the body ispositively charged, then the positive elect, u-le (the anode) will be the activeelectrode and the negative electrode (the cathode) will serve as the counter electrode, completing the circuit. On the other hand, if the ionic sl ~hsPnce tobe delivered is negatively charyed, then the c~LI ,odic electrode will be the active electrode and the anodic electrode will be the counter electrode.
Alle" ,dli~/ely, both the anode and the cdlhode may be used to deliver drugs of apprc,p, iate charge into the body. In this case, both ele~;l, udes are considered to be active or donor electrodes. In other words, the anodic Elec~l o.le can deliver positively charged agents into the body while the cdlhG~Jic elec~, ude can deliver negatively charged agents into the body.
Existing elecl, ul, ~"sport devices addiliu, ,ally require a reservoir or source of the therapeutic agent that is to be delivered into the body. Such drug reservoirs are connected to the anode or the cathode of the ele_~l Ull dl ISpGI l device to provide a fixed or renewable source of one or more desired species or agents. Examples of reservoirs and sources include a CA 02220060 l997-ll-03 WO 96/39423 PCT/US!~61'0~6~17 pouch as described in U.S. Patent No. 4 250 878 to Jacobsen; a pre-formed gel body as disclosed in U.S. Patent No. 4 382 529 to Webster; and a glass or plastic container holding a liquid solution of the drug as disclosed in the figures of U.S. Patent No. 4 722,726 to Sanderson et al.
Of particular interest herein is the l~ ~"sder")al elecl~ ul~ d, lsport deliveryof peptides polypeptides and proteins because of the problems encountered with more common drug admi"isl,alion routes such as oral delivery.
Polypeptide and protein molecules are highly susceptible to degradation by proteolytic enzymes in the gastrointestinal tract and are subjected to an 10 extensive hepatic metabolism when taken orally. Polypeptides and proteins usually require parenteral administration to achieve therapeutic levels in the patient's blood. The most conventional pa, ~ r~l administration techniques are hypodermic ir"ec-tions and intravenous admini~l,dlion. Polypeptides and proteins are, however, inherently short acting in their biological activity, 15 requiring frequent injections often several times a day to maintain the therapeutically effective levels needed. Patients frequently find this treatmentregimen to be inconvenient, painful and with an attendant risk of eg, infection.
Much effort has been expended to find other routes (other than 20 ,ual t:l ,ler~l injections) for effective ad" ,i, .isl, ~lion of ,ul ,a" "~ce~ ~tic~l polypeptides and proteins. Ad",i"isl,dlion routes with fewer side effects as well as better patient compliance have been of particular interest. Such ~llel I ,aLi~/e routes have generally incl~ ~rled "shielded" oral adminisl, dLion wherein the polypeptide/protein is released from a capsule or other co, llail ,er 25 after passing through the low pH en~,i,L"""e"l of the stomach, delivery through the m~ ~cos~l tiss~ ~es eg the muGos~l tiss~ ~es of the lung with inhalers or the nasal mucss~l tiss~ ~es with nasal sprays, and implantable pumps.
Unfortunately these alternative routes of polypeptide/protein delivery have met with only limited suc-cess.

Transdermal ele~l, uL,ansport delivery of polypeptides and proteins has also encountered technical difficulties. For example water is the pr~rt:r, ~d liquid solvent for forming the solution of the drug being delivered by elecl,ut,~,,sport due to its excellent biocompatability. The skin colll~ills 5 proteolytic enzymes which may degrade the polypeptide/l lolei,1 as it is delivered transdermally. In addition certain polypeptides/proteins particularly those that are not native to the animal being treated may cause skin reactions eg sensili~alio" or irritation.
A number of investigators have disclosed elecl, ull~, ,sport delivery of polypeptides and proteins. An early study by R. Burnette et al. J. Pharm. Sci.
(1986) 75:738 involved in vitro skin permeation of thyrotropin releasing hormone a small tripeptide molecule. The ele.;tl ull ~n~po, l flux was found to be higher than passive diffusional flux. Chien et al. J. Pharm. Sci. (1988) 78:
376 in both in vifro and in vivo studies showed that l,dnsde""al delivery of 1~; VaSG~ul essin and insulin via ele~;l, ull c,l ~sport was possible. See also, Maulding et al. U.S. Statutory Invention Reyi~l,cliûll No. H1160 which discloses elect, ulra~ ~uort delivery of calcitonin in minipigs.
Several a,cprudches (other than simply increasing the applied levels of elecl, ut, ~, ,spu, L current) have been used to enhance 1, al ,sde",lal 20 clecll ULI al ,s,uort flux of polypeptide and protein drugs. One approach involves the use of flux enhancers such as ionic surfactants. See eg, U.S.
Patent 4 722 726 to Sanderson et al. Another a~ urc,acl, uses cosolvents other than iust water to e"l ,a"ce eleul, ull ~"sport flux. See, eg European Patent Application 278 473. Yet another appru~( l, involves mechanically 25 disrupting the outer layer (ie, the stratum corneum) of the skin prior to ele~ tlullallspull delivery the,~ll"uugh. See eg U.S. Patent 5 250 023 to Lee etal.
Further a~,uruaches to e"l,a"ui,)9 l,~"s.le""al ele~;l,ol,~ poll drug flux involve creating a prodrug or an analog of the drug of interest and CA 02220060 l997-ll-03 WO 96/39423 PCT/US~)6/09G 17 ele~l, ul, ~nsporting the prodrug or modified analog. For example, WO
92/12999 discloses delivery of insulin as an insulin analog having a reduced tendency to self-~ssoci~te (apparently associ~ted forms of insulin present in conventional pharmaceutical compositions reduce transder",al delivery of the 5 insulin). The analogs are created by substituting aspartic acid (Asp) or glutamic acid (Glu) for other amino acid residues at selected positions along the insulin polypeptide chain. WO 93125197 discloses delivery of both peptide and non-peptide drugs as pharmaceutical agent-modifier complexes or prodrugs wherein a chemical modifier (eg, a c hdrged moiety) is covalently 10 bonded to the parent pharmaceutical agent. The covalent bond is broken after the agent is delivered into the body, thereby releasing the parent agent.
Despite the above al.pruaches, some polypeptides still exhibit poor transdermal eleul, ull ~"sport flux. In particular, peptide hydl u~hobicity is known to negatively impact ele~, ull al):,poi l flux in vitro. Various parameters 15 C~ll Ill ibute to hyd, u~hobicity, including the primary structure of a protein, ie, the amino acid sequence of the molecule, as well as the secondary structure of the ~rutein~ namely, the regular, recurring arranyer"enl of the polypeptide chain along three dimensions. Such co, ~rur~dliu~ ~ can take the form of helical structures, such as an a-helix, or a more extended, zigzag 20 COI ~rul Illdliol 1, known as the ~-co~ ~rul Illdlion.
The a-helix has approximately 3.6 residl ~es per turn of the helix. The R groups of the amino acids extend outward from the helix and inl, dCI ,ai"
hydrogen bonds are formed between the backbone ca, bo"yl oxygen of each residue and backbone hydrogen atom attached to the electronegative 25 1 lill Uy~:l I of the fourth residue along the chain. The basic unit of the ,~-co"rur",~lio" is the ,~-strand which exists as a less tightly coiled helix, with2.û residues per turn. The ,B-strand CGI ~rul "~dlion is only stable when il ICCII ~UI d~d into a ~-sheet, where hydl uyen bonds with close to optimal ~eGl, lt:ll y are formed between the peptide groups on ~ cenl ,B-sl~ ~l ~ds, the CA 02220060 l997-ll-03 W096/39423 PCT~S96/09647 dipoie moments of the strands are also aligned favorably. Side chains from adjacent residues of the same strand protrude from opposite sides of the sheet and do not interact with each other, but have significant interactions with their backbone and with the side chains of neiyhLJul i"g strands.
5 For a general description of a-helices and ,~-sheets, see, eg, T.E. Creighton,Proteins: Structures and Molecular ProPerties (W.H. Freeman and Cor",ud"y, 1993); and A.L. Lehninger, Biochemistrv (Worth Publishers, Inc., 1975) The Zimm-Bragg parameters, s and c~ (B.H. Zimm and J.K. Bragg J.
Chem. Phys. (1959) 31:526-535), and Lifson-Roig equations (S. Lifson and 10 A. Roig J. Chem. Phys. (1961) 34:1963-1974) are conventionaliy used to determine the stability of a helical segment in a given polypeptide. S
represents the helix-coil stability constant and c~ is the nucleation factor.
Based on these parameters, the likelihood of certain regions of polypeptide molecules to form a-helices and ,~-sheets can be ~,re~lic~ed using various calcl~l~fions and computer prou,~",s. See, eg, Finkelstein, A.V. Program "ALB" for protein and polypeptide secondary structure calculation and prediction (1983). Deposited at the Brookhaven Protein Data Bank, Upton, N.Y. and at the EMBL, Heidelberg, Germany; Finkelstein, A.V. Biopolymers (1977) 16:525-529; Finkelstein et al. r~O~ S: Sfrucfure, Funcfion and 20 Genetics (1991) 10:287-299. Furthermore, Chou-Fasman ~robabilities (Chou, P.Y. and Fasman, G.D. Ann. Rev. Biochem. (1978) 47:251-276) have been used to ~csess the ,~, o~,el ,sity of a particular amino acid residue to favor or disfavor a-helical and ~-sheet for" ,~LiGn.
However, these principles have not been previously applied to alter 2~ the hydrophobic properties of a given polypeptide in order to increase the elec,L, uLI ~"sport flux thereof.

CA 02220060 l997-ll-03 3 PCT/U' ~G/l~36~17 DISCLOSURE OF THE INVENTION
In accordance with the present invention the ele~l~ ull ansport flux of a given polypeptide is enhanced by disrupting its secondary structure. In particular amino acid residues known to stabilize a-helical and ~-sheet 5 segments can be replaced with destabilizing residues and known helix breakers. In this way the flux of the poly,ueplide through a body surface (eg the skin) may be increased thereby allowing elect~ull~"s~ort delivery of a wider range of polypeptide drugs at therapeutically effective rates.
Acco, di"gly in one embodiment the invention relates to a method of 10 making an analog of a parent polypeptide having an a-helical and/or B-sheet segment the analog exhibiting better/enhanced elecl,c,l,~rlsport- ability through a body surface the method co~"urisi, 19 substituting one or more amino acid resid~ ~es of the parent poiy,ue,ulide in the analog polypeptide to disrupt one or more a-helical and/or ~-sheet segments of the parent ~5 polypeptide. The analog polypeptide exhibits enhanced elecl,~ "sport as compared to the parent polypeptide.
In an additional embodiment the invention pertains to a method for delivering a poly~,e~,lide agent through a body surface. The method co,."~rises (a) providing an analog of a parent polypeptide wherein the 20 parent poly,t,e~lide co"" rises an a-helical and/or ~-sheet segment the analog poly~ e~ lide having one or more amino acid residues substituted relative to the parent polypeptide; and (b) delivering the analog poly~,eplide through the body surface by eleci, ull ~, Isport.
In particularly ,urerer,~d embodi",e"ls the disrupting is done by 25 substituting one or more amino acid resicl~ ~es of the parent polypeptide with one or more amino acid residues having a lower Pa or P~ value than the parent amino acid residue.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US9C/O~C17 In another embodiment the invention is directed to a parathyroid hormone (PTH) analog comprising the amino acid sequence depicted in Figure 1 B.
In yet another embodiment the invention is directed to a parathyroid 5 hormone (PTH) analog comprising the amino acid sequence depicted in Figure 1 C.
These and other embodiments of the subject invention will readily occur to those of skill in the art in light of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the amino acid sequence of various parathyroid I ,u, ",o"e ( PTH ) molecules used to exemplify the invention. Figure 1A
shows the amino acid sequence of the wild-type parent PTH polypeptide.
Figure 1 B shows the amino acid sequence of PTH analog 1 (1-35). Figure 15 1 C showsthe amino acid sequence of PTH analog 2 (1-3~). HSL de"oles homoserine/homoserine lactone. The underlined resid~ ~es in Figures 1 B and 1 C indicate positions of substitution.
Figure 2 shows the amino acid sequences of various hirudin derivative polypeptides used to e,~e:" ".lify the invention. Figure 2A shows the amino 20 acid sequence of a hirulog parent polypeptide. Figure 2B shows the amino acid sequence of hirulog-1. Figure 2C shows the sequence of hirulog-B2.
Figure 3 is a schematic view of a representative elect, ull ~, ISlJU, l drug delivery device which can be used with the present invention.
Figure 4 is a graph of mean residue ellipticity of several parathyroid 25 hormone analogs versus conce, Ill alion of trifluoroethanol.
Figure ~ is a graph of mean residue ellipticity of two hirudin derivative polypeptides versus concentration of trifluoroethanol.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US96tO9647 DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, electrochemistry, molecular biology and recombinant DNA techniques within the skill of the art.
Such techniques are explained fully in the literature. See, eg, T.E.
Creighton, Proteins: Structures and Molecular ProDerties (W.H. Freeman and Co~ dlly, 1993); A.L. Lehninger, Biochemistrv (Worth Publishers, Inc., 1975); J.S. Newman, Electrochemical Svstems (Pr~lice Hall, 1973); A.J.
Bard and L.R. Faulkner, Electrochemical M_lhods, Fundamentals and APPlications (John Wiley & Sons, 1980); Sambrook, et al., Molecular Cloninq: A Labor~lu, ~/ Manual (Cold Spring Harbor Laboratory, 1989).
It must be noted that, as used in this specification and the appended claims, the sinQular forms "a"l "an" and "the'Hnclude plural rerer~l ,ls unless the COI ~lenl clearly dictates otherwise. Thus, for example" ~rel e- ~ce to "a polypeptide" inclndes a mixture of two or more polypeptides, and the like.

I. DEFINITIONS
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
Herein the terms "ele~l, ull ~ansport", "io"topl ,ort:sis", and "iontophoretic" are used to refer to the delivery through a body surface (eg, skin) of one or more pharm~ce~ Itic~lly active polypeptide agents by means of an applied elecl, ur"uli~/e force to an agent-containing reservoir. The agent may be delivered by eleul, ur"iy, ~lion, elec;t, u,uor~lion, elecl, uO5"~0SiS or any - 25 cG",bi"ation ll,er~or. Electroosmosis has also been referred to as elect,uhydrokinesis, electro-convection, and electrically induced osmosis. In ye"eral~ ele.;t, uos",osis of a species into a tissue results from the ",iy, dLiul ~
of solvent in which the species is co"lai"ed, as a result of the application of elecl, u,, ,uli~e force to the therapeutic species reservoir, ie, solvent flow CA 02220060 l997-ll-03 induced by electromigration of other ionic species. During the elecl, ul, dl ,sport process, certain modifications or alterations of the skin may occur such as the formation of transiently existing pores in the skin, also re~r, ed to as "ele~ll upordLion". Any electrically assisted 1, al ,spo, L of species 5 enhanced by modifications or alterations to the body surface (eg, fo""dlion ofpores in the skin) are also included in the term ''elet;l,ul,a, ,sport" as used herein. Thus, as used herein, the terms "ele.;l, ull ~"sport", "iontophoresis"
and "iontophoretic" refer to (1 ) the delivery of charged agents by electromigration, (2) the delivery of uncharged agents by the process of 10 elecl,uos,),osis, (3) the delivery of charged or uncharged agents by ele~;l, uporaliol " (4) the delivery of charged agents by the combined processes of electromigration and electroosmosis, and/or (5) the delivery of a mixture of charged and uncharged agents by the con,~i"ed processes of ele~;l, u, "iyl dlion and electroosmosis.
The terms "polypeptide," "polypeptide agent" and "polypeptide drug"
are used i~ llel cl ,a"geably herein to denote any bioactive polymer of amino acid residues. The terms encompass peptides, oligopeptides, dimers, multimers, and the like. Such polypeptides can be derived from natural sources or can be synthesi~ or recGrllbil Idl Illy produced. The terms also 20 include poslex~ression modifications of the poly~ eulide, for example, glycosylation, acetylation, phosphorylation, etc.
A polypeptide drug or agent as defined herein is generally made up of the 20 natural amino acids Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), lle (I), Leu (L), Lys (K), Met (M), Phe (F), Pro25 (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V) and may also include any of the several known amino acid analogs, both naturally occurring and sy, Ill ,esi~ed analogs, such as but not limited to homoisoleucine, asaleucine, 2-(methylenecyclG~ o,c"/l)glycine, S-methylcysteine, S-(prop-l-enyl)cysteine, I,o",ose,i"e, o",ill,i"e, norleucine, norvaline, homoarginine, 3-(3-CA 02220060 l997-ll-03 carboxyphenyl)alanine, cyclohexylalanine, mimosine, pipecolic acid, 4-methylglutamic acid, canavanine, 2,3-diaminopropionic acid, and the like.
Further examples of polypeptide agents which will find use in the present invention are set forth below.
By "parent" polypeptide, polypeptide agent or polypeptide drug, is meant a polypeptide as defined above, which includes oc-helical or ~-sheet segments which can be modified such that the ele.;ll ull ~"sport flux of the polypeptide is enhanced. In particular, a parent polypeptide will generally include from about 10 to about 50 amino acid residues, more p~ ~rarably from about 10 to about 40 amino acid residues. FullllaIlllore~ the parent polypeptide will be one which is not prone to adopt a stable tertiary folded structure in solution, eg, as a result of a high col Icenll~lion of Cys residues.
The parent polypeptide can be a naturally occurring polypeptide or may itself have structural dirrer~, Ices from a naturally occurring polypeptide such as amino acid substitutions, deletions or additions, as well as post-translational modific~lio,)s as described above.
By polypeptide "analog" is meant a polypeptide as defined above, which results from the modification of the secondary structure of the parent polypeptide. In this regard, the analog differs from the parent by way of substitution of one or more amino acid residues such that one or more oc-helical and/or ~-helical segments present in the parent molecule are disrupted. A~prouriate amino acid substitutions are ~iscl lcsed more fully below. The analog can also co"l~i" additional modifications that do not affect seco".la"/ structure, such as additional amino acid substitutions, - 25 deletions or additions, or post-translational modifications as described above.
The analog can also exist in neutral or salt forms, eg, acid addition salts (fo""ed with the free amino groups of the analog polypeptides) and which are formed with il ,oryd"ic acids such as, for exa" " le, hydrochloric or phosphoricacids, or such organic acids as acetic, succinic, maleic, ta, l~l ic, mandelic, _ and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, al""IG"ium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, l, imell ,ylamine, 2-ethylamino ethanol, histidine, and the like. The polypeptide 5 analog will have at least some of the bioactivity of the parent polypeptide, and more preferably will have the same or greater bioactivity of the parent molecule.
By "enhancing the eleul, ull dl ,sport" of a polypeptide is meant increasing the ele~ll ull dl ,sport flux of the polypeptide through the body 10 surface (eg, the skin or mucosa) as compared to the parent polypeptide.
Transdermal eleull ull ~"sport flux can be determined using a number of in vivo or in vitro methods well known in the art. In vitro methods include clamping a piece of skin of an a~ p, uu, iale animal (eg, human cadaver skin) between the donor and receptor con l,Udl ll l lel IL~ of an ele~, ull dl ,sport flux cell, 15 with the stratum corneum side of the skin piece facing the donor CO"~d~ l",ent. A liquid solution or gel co~ ,tai~1ing the drug to be delivered is placed in contact with the stratum corneum, and electric current is applied to electrodes, one electrode in each compartment. The transdermal flux is Calcl ll~teli by sampling the amount of drug in the receptor compartment. Two 20 Sl Iccessful models used to optimize transdermal eleul, ull dl l~pOI l drug delivery are the isolated pig skin flap model of Riviere, M.C. Heit, et al, J.
Pharm. Sci, 82, 240-243 (1993), and the use of isolated hairless skin from hairless, ude, IL~ or guinea pigs, for example. See B.W. Hadzija et al, J.
Pharm. Phd""acol., 44, 387-390 (1992).

Il. MODES OF CARRYING OUT THE INVENTION
The present invention co"ce~"s substituting amino acids which disrupt a-helical and ~-sheet segments of a polypeptide molecule to enl ,ance the eleCll Ull dl ,:-port flux of that molecule as co" ,par~:d to the flux of the parent CA 02220060 l997-ll-03 polypeptide. The method therefore permits elecl, ul, dl ,s~ort of a large number of substances that would not otherwise be amenable to such delivery. Without being bound by a particular theory, it appears that such secondary changes decrease the ten-le"cy of hydrophobic side chains of amino acid residues such as Leu, Phe and Trp, to become spatially clustered.
Thus, the ability of the molecule to bind to hycJ, u~hobic areas in the skin during ele.;l, ull dl .sport is reduced, thereby facilitating p~ss~3e of the polypeptide through the skin.
The present invention has been exemplified using a parathyroid hormone (PTH) molecule and a hirudin derivative as the parent cor"pounds.
PTH is a peptide hormone which regul~tes homeostatic control of calcium and phosphate metabolism and has been used to treat osteoporosis. Wild-type PTH is shown in Figure 1A. The molecule has 34 amino acid residues and a molecular weight of a~ l o~ ) ldlely 4000 daltons. As shown herein, the molecule has a very large tendency to adopt the oc-helical rc,, Illdliul ~.
Hirulog is a hirudin-based synthetic peptide anticoagulant which effectively inhibits both free and fibrin-bound ll"o",L,in. Hirulog has been shown to inhibit post-operative venous llllolllbosis in pdlie"ls underyoi"g orthopedic surgery. The sequence of the parent hirulog cGr"pound used to exemplify the present invention is shown in Figure 2A.
Although exemplified using the parent s~ ~hsPnces described above, the ,u,-esenl invention will also find use with a wide variety of other parent ~,r-,lei"s and polypeptide agents, such as other polypeptides derived from eucaryotic, prc,ca, yotic and viral sources, as well as synthetic peptides. Such- 25 polypeptides will generally have from about 10 to about 50 amino acids, a seco"da, y structure amenable to manipulation and will not be prone to adopt a stable tertiary folded structure, eg, as a result of a high conce"l, dliGI, ofCys resid~es. Such polypeptides include, without lilllildlion, pe,c,lide drugs which are alllilJiolics and antiviral agents, antineoplastics, CA 02220060 l997-ll-03 immunomodulators, peptide hormones such as ACTH, CRF, GHRH, cholecystokinin, dynorphins, endorphins, endothelin, fibronectin fragments, galanin, gastrin, insulinotropin, glucagon, GTP-binding protein fraylllell~
guanylin, the leukokinins, magainin, maslo~ C,d~15, dermaseptin, s~:,ler"i", 5 neuromedins, neurotensin, pancreastatin, pancreatic polypeptide, suL,~la"ce P, secretin, thymosin, and the like.
The secondary structure of the parent polypeptide is generally manipulated by replacing one or more selected parent amino acid residues with one or more amino acid residues having a lower tendency to form a-helix 10 and/or ~-sheet co"rur",ations. In this regard, each amino acid residue demonstrates cG,,ru,,,,ational ~.r~rere"ces. One measure of the relative tendency of a particular residue to be involved in an a-helix or ,~-strand has been defined by the pa,d",eter:, Pa and P~ (Chou, P.Y. and Fasman, G.D.
Ann. Rev. Biochem. (1978) 47:251-276). An updated and revised list of Pa 15 and P,~ values are depicled in Table 1. The Pa values in Table 1 which are u, ~dler than 1.00 represent amino acids which favor a-helical co~ ,rur'' ~dlions and are defined herein as "high Pa values." Similarly, the P~ values in Table 1 which are greater than 1.00 represent amino acids which favor ,~-strand fo""dlion and are defined herein as "high P,B values." The amino acids with 20 Pa and P~ values of 1.00 or less disfavor or break the secondary cGr,ru""ations and are defined herein as "low Pa values" and "low P~
values," respectively.
Thus, for example, the amino acids Glu, Ala and Leu have high Pa values and favor helix ror",dLion, while Pro, Gly, Ser, Cys, Tyr and Asn have 25 IOW Pa values and therefore disfavor helix formation. Similarly, the amino acid resid~ ~es Val, lle and Tyr have high P~ values and favor B-strand fo",ldliol " while the amino acids Pro, Asp, Asn, Glu and Gly, disfavor such - rC""~al;CJn. CGIIILjlljll9 Pa and P,B values, the amino acids which should CA 02220060 l997-ll-03 WO 96/39423 PCT/US~G~ ;.17 exhibit the lowest tendency to form these types of secondary structures are Gly, Asn, Pro and Ser.

Conformational Preferences of the Amino Acids Amino Acid a-helix ~-strand Residue ~Pa) (PB) Glu 1.59 0.52 Ala 1.41 0.72 Leu 1.34 1.22 Met 1.30 1.14 Gln 1.27 0.98 Lys 1.23 0.69 Arg 1.21 0.84 His 1.05 0.80 Val 0.90 1.87 lle 1.09 1.67 Tyr 0.74 1.45 Cys 0.66 1.40 Trp 1.02 1.35 Phe 1.16 1.33 Thr 0.76 1.17 Gly 0.43 0.58 Asn 0.76 0.48 Pro 0.34 0.31 Ser 0.57 0.96 Asp 0.99 0.39 Data in Table 1 was taken from T.E. Creighton, Proteins: Structures and Molecular Prol~erties (W.H. Freeman and Co"~l,dny, 1993), p 256.

A segment of a particular secondary structure is much more prubable when several ~d; cel ll residues prefer that structure. Thus, an a-helix can be predicted if four out of six ~ cent residues are helix-favoring and if the CA 02220060 l997-ll-03 average Pa value is greater than 1.05 and greater than P,~. Similarly, a ,~-strand is predicted if three out of five adjacent residues are sheet-favoring and if the average value of P~ is greater than 1.04 and greater than Pa. T.E.
Creighton, Proteins: Structures and Molecular ProDerties (W.H. Freeman and Company,1993), pp 25~-257.
Thus, using these principles, amino acid residues in portions of the molecule that are prone to a-helix and ,~-sheet ror",~Lio,l, can be replaced with those residues from Table 1 which disfavor such conru""alions.
r~:r~rably, a residue with a high Pa or P,B value will be replaced with a residue having a low Pa or P,B value, respectively.
Other methods of determining the propensity of a polypeptide to form a-helical and ,~-sheet segments are also known. For example, the a-helix potential of a linear polypeptide can be e:,li",ated by, eg, the Lifson-Roig equations (S. Lifson and A. Roig J. Chem. Phys. (1961) 34:1963-1974) using values for the residue helix formation ~arar"ele, ~i converted from their calcul~ted Zimm-Bragg values (Zimm, B.H. and Bragg, J.K. J. Chem. Phys.
(1959) 31:526-535) employing conversion equations of Qian and Schellman (Qian, H. and Schellman, J.A. J. Chem. Phys. (1992) 96:3987-3994).
More particularly, the Zimm-Bragg parameters, s and ~, represent the helix-coil stability cGnslanl and the n~ le~fion factor, respectively. A set of Zimm-Bragg s and ~s values have been defined for each amino acid residue (see Table 1 of Finkelstein et al. Proteins: Sfructure, Funcfion and Genetics (1991) 10:287-299) and the probability that the entire polypeptide chain (or some defined subsequence) would adopt an a-helix may be computed using the reported equations.
The corresponding Lifson-Roig helix ~I.Jdl allleter::i, U, V and w, can also be used to make a similar determination (Lifson, S and Roig, A. J. Chem.
Phys. (1961) 34:1963-1974). In this regard, w, which is used to define a ,~e~Jlide unit at the interior of an Lll lil llel l upted sequence of helical states, can CA 02220060 l997-ll-03 WO 96/39423 PCT/US!)6/09C~17 be computed from s. Lifson-Roig's v defines a peptide unit at the beginning or at the end of an uninterrupted sequence of helical states and can be computed from the Zimm-Bragg parameter C~1/2 The factor u, defines the statistical weight of the coil region and does not correspond to a Zimm-Bragg 5 parameter.
Using these parameters, there are two calculations generally used to estimate a-helix potential. The first takes the Lifson-Roig helix initiation parameter vto be a constant 0.039 (ie, the Zimm-Bragg cs pa~an,eter is 0.0013 for all residues), with the w parameter for each amino acid c~iC~ ted 10 from the Zimm-Bragg s value found in Table 1 of Finkelstein et al. Proteins:
Strucfure, Function and Genetics (1991 ) 10:287-299. The second computation does not assume that all residues have the same value for the heiix initiation par~"~eter v (ie, the Zimm-Bragg ~ pdra",eter is unique for each amino acid residue). Using this ",ell ,o.-i, the Zimm-Bragg s and ~
15 values for each residue can be determined from Table 1 of Skolnick, J. and Holtzer, A. Macromolecules (1982) 15:812-821. These methods are described further below in the examples.
The polypeptide analogs of the present invention can be produced in any number of ways which are well known in the art. In this regard, since the 20 analogs are relatively small, ie, up to about 50 amino acids in length, they can be conveniently synthesized chemically, by any of several techniques that are known to those skilled in the iJ~plide art. In general, these rlleLllods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or c~, Loxyl group of the first amino 25 acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or I Itiii7~d in solution by adding the next amino acid in the sequence having the comple",entary (amino or carboxyl) group suitably ,urolected, under conditions that allow for the fo"~lion of an amide linkage. The iJI UteCl.il 19 CA 02220060 l997-ll-03 group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques 5 are used) are removed sequentially or concurrently, to render the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral ce"ler~i) a protected l, i~ e~Lide with a properly proLe.;Led dipeptide to form, after 10 deprutection, a pentapeptide. See, eg, J. M. Stewart and J. D. Young, Solid Phase Peptide Svnthesis (Pierce Chemical Co., Rockrord, IL 1984) and G.
Barany and R. B. Merrifield, The PePtides: Analvsis, Svnthesis. Bioloqv, editors E. Gross and J. Meienhofer, Vol. 2, (Acadel"ic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M.
15 Bodansky, PrinciPles of PePtide Svnthesis. (S~,i"yer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The PePtides: Analvsis, Svnthesis, Bioloqv, Vol. 1, for cl~-csic~l solution synthesis.
Typical protecting groups include t-butyloxy~a~L onyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycdlL,on~/l (Cbz); p-toluenesulfonyl 20 (Tx); 2,4--li, liLI o~l ,enyl; benzyl (Bzl); biphenylisopropyloxyca, boxy-ca, Lul Iyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-brur"obe"~yloxycarbonyl, cyclohexyl, isopro,oyl, acetyl, o-nitrophenylsulfonyl and the like.
Typical solid supports are cross-linked polymeric supports. These can include divinylbe"~ene cross-linked-styrene-based polymers, for exc""ple, 25 divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloro",ell"/lstyrene copolymers and divinylbenzene-benzhydryla",i"opolystyrene copolymers.
The polypeptide analogs of the ,~,-esenl invention can also be ~;I,e",ically p~.ared by other methods such as by the method of CA 02220060 l997-ll-03 W096/39423 PCT~S96/09647 simultaneous multiple peptide synthesis. See, eg, Houghten Proc. Natl.
Acad. Sci. USA (1985) 82:5131-5135; U.S. Patent No. 4,631,211.
Alternatively, the peptides can be produced by recombinant techniques, eg, by synthesizing DNA encoding the desired peptide, along 5 with an ATG initiation codon. The nucleotide sequence can be designed with the a~ro,uriate codons for the particular amino acid sequence desired. In general, one selects preferred codons for the intended host in which the sequence is expressed. The complete sequence is generally assembled from overlapping oligonucleotides prepar~:d by standard methods and as-10 sembled into a complete coding sequence. See, eg, Edge Nature (1981)292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem.
(1984) 259:6311. AulumaLed synthetic techniques such as phosphor~l"ide solid-phase synthesis, can be used to generate the nucleotide sequence.
See, eg, Be~ ~c~ge, S.L. et al. Tet. Lett. (1981) 22:1859-1862; ~el lCCi, M.D. et al. J. Am. Chem. Soc. (1981) 103:3185-3191. Next the DNA is cloned into an apprc ~ridle e~,uression vector, either procaryotic or eucaryotic, using conventional methods.
AlLe" ,aLi~ely, recombinant techniques are readily used to clone the parent polypeptide gene which can then be mutagenized in vitro by the 20 repl~ce",e, IL of the a,u,urc,uriate base pair(s) to result in the codon for the desired amino acid. Such a cl Idl ,ge can include as little as one base pair, err~cLi"y a change in a single amino acid, or can encor"pass several base pair cl ,a"yes. Aller"~ /ely, the mulaliuns can be effected using a mismatched primer which hybridizes to the parent nucleotide sequence 25 (yel ~erdlly cDNA corres,uo"di~,g to the RNA sequence), at a temperature below the melting temperature of the mis",~tcl ,ed duplex. The primer can be made specific by keeping primer length and base cor,~uosilion within relatively narrow limits and by keeping the mutant base centrally located.
Zoller and Smith, Methods Enzymol. (1983) 1 û0:468. Primer ekLe"sio" is CA 02220060 l997-ll-03 WO 96/39423 PCT/US~:16/0~ 7 effected using DNA poiymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple 5 point mutations. See, eg, Dalbic McFarland et al. Proc. Natl. Acad. Sci USA
(1982) 79:6409.
Using the above methods, a number of represe"lali~e polypeptide analogs exhibiting e, Iha, ,ced ele~ ull ~"sport flux have been made. In particular, Figure 1 B depicts the sequence of PTH analog 1. This PTH
10 analog differs from the parent sequence (depicted in Figure 1A) as follows:
Met8 has been substituted with Leu; Leu,s has been substituted with Arg;
Met,8 has been substituted with Leu; Glu,9 has been substituted with Arg;
Glu22 has been substituted with Arg; Gln29 has been substituted with Lys;
Phe34 has been substituted with Tyr; and a homoserine lactone is present at 15 the C-terminus of the peptide.
PTH analog 2, depicted in Figure 1 C, differs from the parent sequence (Figure 1A) as follows: Met8 has been sl Ihstih~ted with Leu; Leu" has been substituted with Ser; Leu,5 has been substituted with Arg; Leu,8 has been substituted with Ser; Met,8 has been substituted with Ser; Glu,g has been 20 substituted with Arg; Val2, has been substituted with Ser; Glu22 has been substituted with Arg; Leu24 has been substituted with Ser; Leu28 has been substituted with Ser; Gln29 has been substituted with Lys; Tyr34 has been substituted with Ser; and homoserine lactone is present at the C-terminus of the peptide. Taken together, these substitutions result in a lessened 25 SeCGI Iddl y structure.
The hirulogs are a series of synthetic analogs of hirudin, a natural lhlulllbi~ l i"hiL,iLor. Two of these analogs are shown in Figure 2. Hirulog-1, shown in Figure 2B, is disclosed in Maragnore et al. Biochem. (1990) 29:7095-7101. Hirulog-B2, a twenty amino acid peptide with a molecular CA 02220060 l997-ll-03 weight of about 2186 daltons, has the sequence shown in Figure 2C. As can be seen, the hirulog-B2 analog differs from the hirulog-1 analog shown in Figure 2C, in that it has D-cyclohexylalanine substituted for D-phenylalanine in the first position. Hirulog-B2 is described in Witting et al. Biochem. J.
s (1992) 287:663-664; and International Publication No. WO 92/13952.
Once the desired polypeptide analog is prepared, it can be delivered to the subject using any of several ele~l, ull ~"sport drug delivery systems andis not limited to the use of one particular system. Examples of elecl,uL,~,,s~ort drug delivery systems are described in, eg, U.S. Patent Nos.
5,312,326 to Myers et al., 5,080,646 to Theeuwes et al., 5,387,189 to Gyory et al., and 5,169,383 to Gyory et al., the disclosures of which are incorporated by reference herein.
Figure 3 illustrates a representative ele~,L, uLI ~"spo, L delivery device that may be used in conjunction with the present method. Device 10 co""~rises an upper housing 16, a circuit board assembly 18, a lower housing 20, anode electrode 22, cathode electrode 24, anode reservoir 26, cathode reservoir 28 and skin-compatible adhesive 30. Upper housing 16 has lateral wings 15 which assist in holding device 10 on a patient's skin. Upper housing 16 is prerer~bly composed of an injection moldable elaslor"er (eg, ethylene vinyl acetate). Printed circuit board assembly 18 cor"~,, ises an yldled circuit 19 coupled to cJiscrete co",,uonents 40 and battery 32.
Circuit board assembly 18 is attached to housing 16 by posts (not shown in Figure 3) passing through openings 13a and 13b, the ends of the posts being heatedlmelted in order to heat stake the circuit board asse" l~ly 18 to the housing 16. Lower housing 20 is attached to the upper housing 16 by means of adhesive 30, the upper surface 34 of adhesive 30 being adhered to both lower housing 20 and upper housing 16 including the bottom surfaces of wings 15.

CA 02220060 l997-ll-03 Shown (partially) on the underside of circuit board assembly 18 iS a button cell battery 32. Other types of batteries may also be employed to power device 10.
The device 10 is generally comprised of battery 32, electronic circuitry 19,40, electrodes 22,24, and drug/chemical reservoirs 26,28, all of which are integrated into a self-contained unit. The outputs (not shown in Figure 3) of the circuit board assembly 18 make electrical contact with the electrodes 24 and 22 through openings 23,23' in the depressions 25,2!~' formed in iower housing 20, by means of electrically conductive adhesive strips 42,42'.
Electrodes 22 and 24, in turn are in direct mechanical and electrical co"la~;l with the top sides 44',44 of drug reservoirs 26 and 28. The bottom sides 46',46 of drug reservoirs 26,28 COI ,tacl the patient's skin through the openings 29',29 in adhesive 30.
Device 10 optionally has a feature which allows the patient to self-administer a dose of drug by elec;l, ul, dl ,s,l~ort. Upon depression of push button switch 12, the electronic circuitry on circuit board assembly 18 deliversa predeter",i"ed DC current to the electrodes/reservoirs 22,26 and 24,28 for a delivery interval of predeter",ined length. The push button switch 12 is conveniently located on the top side of device 10 and is easily ~ctl l~e~l through clothing. A double press of the push button switch 12 within a short time period eg, three seconds is prereral,ly used to activate the device for delivery of drug thereby minimizing the likelihood of inadvertent actuation of the device 10. Prererdbly the device lldlls,,,ils to the user a visual and/or audible cG"ri""dlion of the onset of the drug delivery interval by means of LED 14 beco",iny lit and/or an audible sound signal from eg a "beeper".
Drug is delivered through the patient's skin by elec;l, ul, dl ,sport eg, on thearm over the predetermined delivery interval.
Anodic ele~ l,ude 22 is prererably co",prised of silver and cathodic e lect, ude 24 is ,ul ~r~, dbly cor"~ ised of silver chloride. Both reservoirs 26 CA 02220060 l997-ll-03 WO 96/39423 23 PCT/US~G~O~C~7 and 28 are preferably comprised of poiymer hydrogel materials. Electrodes 22,24 and reservoirs 26,28 are retained within the depressions 25',25 in lower housing 20.
The push button switch 12, the electronic circuitry on circuit board 5 assembly 18 and the battery 32 are adhesively"sealed" between upper housing 16 and lower housing 20. Upper housing 16 is ,ur~rerdbly composed of rubber or other elastomeric material. Lower housing 20 is pre~erdbly composed of a plastic or elastomeric sheet material (eg, polyethylene) which can be easily molded to form depressions 25,2~' and cut to form openings 23,23'. The assembled device 10 is pleferably water resistant (ie, splash proof) and is most preferably waler~roof. The system has a low profile that easily co"rc r",s to the body, thereby allowing freedom of movement at, and around, the wearing site. The reservoirs 26 and 28 are located on the skin-contacting side of the device 10 and are sufficiently sepd, dled to prevent ~ccidental electrical shorting during normal handling and use.
The device 10 adheres to the patient's body surface (eg, skin) by means of a peripheral adhesive 30 which has upper side 34 and body-co, llaclir ,y side 36. The adhesive side 36 has adhesive properties which assures that the device 10 remains in place on the body during normal user activity, and yet ,ue, Illils reasonable removal after the ,,r~-lete, I"ined (eg, 24-hour) wear period. Upper adhesive side 34 adheres to lower housing 20 and retains lower housing 20 attached to upper housing 16.
The reservoirs 26 and 28 generally co",,u,ise a gel matrix, with the drug solution uniformly dispersed in at least one of the reservoirs 26 and 28.
Drug co, Icen ll dliGI IS in the range of approximately 1 x 1 O4 M to 1.0 M or more can be used, with drug concel Ill dlions in the lower portion of the range being,urert:r, ~:d. Suitable polymers for the gel matrix may comprise esseulially any"on;o, lic synthetic and/or naturally occurring polymeric ,nalerials. A polar nature is p,er~"~.l when the active agent is polar and/or capable of CA 02220060 l997-ll-03 ionization, so as to enhance agent solubility. Optionally, the gel matrix will be water swellable. Examples of suitable synthetic polymers include, but are not limited to, poly(acrylamide), poly(2-hydroxyethyl acrylate), poly(2-hydroxypropyl acrylate), poly(N-vinyl-2-pyrrolidone), poly(n-methylol 5 acrylamide), poly(diacetone acrylamide), poly(2-hydroxylethyl methacrylate), poly(vinyl alcohol) and poly(allyl alcohol). Hydroxyl functional conde, Isalion polymers (ie, polyesters, polycarbonates, polyurethanes) are also examples of suitable polar synthetic polymers. Polar naturally occurring polymers (or derivatives thereof) suitable for use as the gel matrix are exemplified by 10 cellulose ethers, methyl cellulose ethers, cellulose and hydroxylated cellulose, methyl cellulose and hydroxylated methyl cellulose, gums such as guar, locust, karaya, x~r,ll ,a", gelatin, and derivatives thereof. Ionic polymers can also be used for the matrix provided that the available counterions are either drug ions or other ions that are oppositely cl ~arged relative to the active 15 agent.
Thus, the polypeptide analogs of the present invention will be incorporated into the drug reservoir, eg, a gel matrix as just described, and administered to a patient using an eleul,ul, an s,uort drug delivery system, opliu. ,ally as exemplified hereinabove. I"co, ,uoralion of the drug solution can 20 be done any number of ways, ie, by imbibing the solution into the reservoir matrix, by admixing the drug solution with the matrix " ,alel ial prior to hydrogel ru- . "alion, or the like.
While the invention has been described in conjunction with the p- er~r, ed sl,eciric embodiments thereof, it is to be understood that the 25 foregoing desu- i,ulio" as well as the examples which follow are i"lencJed toillustrate and not limit the scope of the invention. Other aspects, advantages and modificalio"s within the scope of the invention will be a~,ar~nl to those skilled in the art to which the invention pertains.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US96/0~)6~17 Ill. EXPERIMENTAL
In general, standard techniques of recombinant DNA technology are described in various publications7 eg, Sambrook, et al., Molecular Cloninq: A
Laboratorv Manual (Cold Spring Harbor Labor~lo"~, 1989); Ausubel et al., 5 Current Protocols in Molecular Bioloqv, vols. 1 and 2 and supplements (1987); and Wu and Grossman (eds.), Methods in Enzvmoloqv, Vol. 53 (Recombinant DNA Part D, 1987). Restriction enzymes, mammalian cell culture media, and E. coli cell line DH10B (F- mcrA D(mrr-hsdRMS-mcrBC) F8odl~r7nM15 DlacX74 deoR recA1 araD139 D(ara,leu)7697 galU galK 1-10 rpsL endA1 nupG) were purchased from Gibco/BRL (Gaithersburg, MD). Taqpolymerase was from Perkin Elmer Cetus (Norwalk, CT). His-bind resin was purchased from Novagen (Madiso,n, Wl) and used accor~ g to the manufacturer's instructions. DNase and Iysozyme were purchased from Boehringer Mannheim (Indi~, la,c,olis, IN). Cya"oyen bromide was purchased 15 from Aldrich (Milwaukee, Wl). Oligonucleotides were synthesized on an Applied Biosystems Inc. (Foster City, CA), Model 394 DNA synthesizer using ABI chemicals. Synthetic human parathyroid hormone, and synthetic bovine parathyroid hormone were purchased from Bachem (Torrance, CA).

CONSTRUCTION OF EXPRESSION VECTORS FOR
THE PTH POLYPEPTIDES
The PTH e~Jr~ssio, I vectors were constructed in several steps using plasmid pBAD18 (Guzman et al. 1995) as the starting plasmid. Plasmid 25 pBAD18 co, llai- ,s the araB ~I c,n ,oler followed by a polylinker and a te" "i"dlor under the control of the positive/negative regul~tor araC, also specified by the plasmid. Plasmid pBAD18 also contains a modified plasmid pBR322 origin and the bla gene to permit replication and selection in E. coli, as well as the phage M13 intragenic region to permit rescue of single~ , I.Jed DNA.

CA 02220060 l997-ll-03 WO 96/39423 PCT~US~6/096~17 For purposes of the present invention however the actual cloning vector used to construct the expression vectors of the invention is not critical.
For instance plasmid pMC3 could serve as the cloning vector in place of plasmid pBAD18 in the protocols below. Plasmid pMC3 is described in U.S. Patent No. 5 270 170. Plasmid pMC3 differs from plasmid pBAD18 in that plasmid pMC3 encodes a dynorphin B-tailed lac repressor in the region corresponding to the Nhel-Xbal region of the multiple cloning site of pBAD18 and encodes a lac operator sequence in the region corresponding to the Ndel-Clal fragment of plasmid pBAD18. As this latter fragment is not essential for purposes of the present invention one could readily construct suitable vectors for purposes of the present invention from plasmid pMC3.
Plasmid pMC3 is available in strain ARI161 from the American Type Culture Collection under the ~ccession number ATCC 68818.
The PTH vectors were then constructed as follows. The following pair of partially overlapping oligonucleotides were annealed and second strand synthesis was performed with Taq poly,,,e,cse:

S'-GCT CGG GCT AGC TAA CTA ATG GAG GAT ACA TAA ATG A~A GCT ATC TTC
N~e I S&D TrpLe leader GTT CTG A~A GGT TCC CTG GAC CGT GAC CCG GA-3' 3'-AC CTG GCA CTG GGC CTT AAG CAG CTG TAC TAG
Sal I Bcl I
25 TTG GTC TAG AGG GTG GTG GTG GTA GTG GTA ATT ATT TTC GAA GGC AGG-S' Bgl II His6 Hind III

The product was digested with Nhel and HinDIII and inserted into the 30 corres~,o"di"y sites of pBAD18. The duplex col llail ,s the Shine-Dalgarno riboso",e bi.~di~y site (S&D) and the TrpLE leader peptide. This 17 amino acid leader sequence has been previously shown to er,h~"ce ex~rt:ssion of small protei. ,s and may ~rc" "ole the sequestering of fusions into inclusion bodies (Derynck et al. Cell (1984) 38:287-297; Miozari and Ya"uraky J.
35 Bacteriol. (1978) 133:1457-1466). The TrpLe leader peptide is se~.draled .

CA 02220060 l997-ll-03 WO 96/39423 PCTrUS96/09647 from the His6 sequence by a multiple cloning site. The polyhistidine site allows for rapid purification of the recombinant parathyroid hormone molecule (RPTH) and RPTH analogs via nickel chelation chrumalography (see U.S.
Patent No. 4 551 271). Two stop codons (TAATAA) follow the polyhistidine.
An RPTH(1-34) gene with the ~ollowing sequence was designed and synthesized using high-use E. coli codons:

Val-Asp-Met-Ile-Asn-Met-Ser-Val-Ser-Glu-Ile-Gln-Leu-Leu 5'-GGC TGG GTC GAC ATG ATC AAC ATG TCC GTT TCC GAA ATC CAG CTG CTG
3'-CCG ACC CAG CTG TAC TAG TTG TAC AGG CAA AGG CTT TAG GTC GAC GAC
Sal I
9 lO ll 1.2 13 14 15 16 17 18 19 20 21 22 23 24 His-Asn-Leu-Gly Lys-His-Leu-Asn-Ser-Leu-Glu-Arg-Val-Glu-Trp-Leu CAC AAC CTG GGT AAA CAC CTG AAC TCC CTC GAG CGT GTT GAA TGG CTG
GTG TTG GAC CCA TTT GTG GAC TTG AGG GAG CTC GCA CAA CTT ACC GAC

Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Tyr-Met-Gln-Ile-Ser CGT A~A A~A CTG CAG GAC GTC CAC AAC TAC ATG CAG ATC TCC CTC-3' GCA TTT TTT GAC GTC CTG CAG GTG TTG ATG TAC GTC TAG AGG GAG-5' BgI II

This RPTH gene was digested with Sal I and Bgl II and inserted into the modi~ied pBAD vector.

CA 02220060 l997-ll-03 EXPRESSION AND PURIFICATION OF PTH POLYPEPTIDES
E. coli DH10B containing the plasmid described above was grown overnight in LB-media containing ampicillin (50 to 100,ug/mL). Ten mL of this 5 culture were used to inoculate a 500 mL culture of Supelbl ul h (35 g/L Bacto- tryptone, 20 g/L yeast extract, 5 g/L NaCI, and NaOH to pH=7.5) CG~ ing ampicillin. The cells were allowed to grow to an OD600 of about 0.5 to 1.0 and L-(+)-arabinose was added to a final concel,l,~lion of 0.2% The cells were allowed to grow for an additional 3 hours. At the end of this time, the OD600 10 was between 1.5 to 3. The cells were harvested by centrifugation and washed sequentially with 250 mL of WTEK buffer (50 mM Tris, pH=7.5, 10 mM EDTA,100 mM KCI); 250 mL of PBS; and 250 mL of 10 mM Tris, pH=7.5.
The cells were then resuspended in 100 mL of a solution co"" osed of 10 mM Tris, pH=7.5; 0.1 mg/mL of protease inhibitor N-tosyl-L-phenylalanine 15 chloromethyl ketone (TPCK); 0.1 mg/mL of protease inhibitor N-tosyl-L-lysine chloromethyl ketone (TLCK); 0.1 mg/mL of protease inhibitor phenylmethylsulfonyl fluoride (PMSF); and 0.05 mg/mL Iysozyme). The resulting solution was inc~ Ih~terl on ice for 1 hour. The cells were then freeze-thawed; 1 mg of DNAse was added to the freeze-thawed cells; and the 20 resulting mixture was inc~ ~h~ted on ice for an additional hour.
Inclusion bodies from the cells were purified by centrifugation at 10,000Xg for 15 minutes. The inclusion bodies were solubilized in 10% SDS, but in some cases, sonication of the sample was also necessa, y to solubilize all of the protein. Binding buffer (5 mM i",ida,ole, 500 mM NaCI, and 20 mM
25 Tris, pH=7.9) was added to dilute the SDS conce"L, dlio" to 1 %, and the sample was loaded onto a column containing His-bind resin (Novagen). The column was then washed with 15 column volumes of binding buffer, and bound protein was then eluted with 1 column volume of elution buffer (500 CA 02220060 l997-ll-03 WO 96/39423 PCT/US~)6/09C 17 mM NaCI, 100 mM EDTA, and 50 mM Tris, pH=7.9). Two volumes of absolute ethanol were then added to precipitate the protein.
The precipitated PTH polymer was then dissolved in 70% formic acid, and a 500-fold (100 to 1 000-fold excess can be used) molar excess of CNBr 5 was added. A time course of cleavage (conducted at different CNBr concel ,l, dlions to determine the optimal time), as assayed by amino acid analysis, indicated that complete cleavage was achieved in 2 hours at room temperature. After CNBr cleavage, the peptides were Iyophilized and resuspended in distilled water. The peptide was resuspended in Buffer A
10 (0.1% TFA) and further purified by HPLC using a VYDAC C-18 semi-preparative column. Approximately 30 mg of peptide was injected onto the column. The peptide was then eluted with a gradient of 20~0%
acetonitrile/0.1% TFA over 40 minutes. The major peak, eluting at approximately 15 minutes, was collected and Iyophilized to dryness.
After isolation of inclusion bodies and purification as described above, the peptide was greater than 95% pure as determined by SDS-PAGE. The peptide was then desalted and partially purified on a SepPak column.
Individual peaks were isolated and analyzed by electrospray mass s~Jecl,oroetry to deler",i"e the composition. Two minor peaks were pr~senl 20 which were the result of incomplete cyanogen bromide cleavage and represented the PTH-His6 peptide and the l-N-M-PTH peptide. The major peak comprised greater than 80% of the total protein. Cor",uariso" of the "crude" Sep-Pak purified sample with an HPLC purified RPTH showed no significant dirre, e"ce in adenylate cyclase stimulatory ability.

PRODUCTION OF PTH ANALOGS
In order to produce the PTH analogs of the present invention, the PTH
peptide was substituted with the amino acids indicated in Figures 1 B, CA 02220060 l997-ll-03 1 C and 1 D. PTH analogs were recombinantly produced constructed in a manner similar to that described above for the recombinant RPTH-A gene by substituting the appropriate bases. The PTH analogs were expressed and purified as described above.

PTH ANALOG ACTIVITY ASSAY
Synthetic PTH having the wild-type sequence and the recombinant PTH (RPTH) analogs were assayed for their ability to stimulate adenylate 10 cyclase. PTH conce"ll~lions were deter",i"ed by OD28~ using an ekli"~;lio"
coefficient of 6600 for the I eco" ,binant peptide and 5500 for the synthetic peptide. The rat osteosarcor"a cell line UMR106 (ATCC CRL 1661 ) was used for in vitro testing of the peptide's ability to activate the PTH ~ ece~lor.
Activation of the PTH receptor leads to an intracellular rise in cAMP
15 concel ILI ~lion.
The UMR106 cells were seeded at about 2.5 x 1 O5 per well in a 48 well dish and allowed to grow to confluence. Assays were performed on cells 3 -5 days ~o~lco"rluence. The media (Dulbecco's modified Eagle's medium, DMEM with fetal calf serum) was removed and 1 mL of fresh media was 20 applied. PTH (recombinant or synthetic) at various conce"l, ~lions and 3-isobutyl-l-methyl xanthine (IBMX) at 1 mM final col lce~ Liul l were then added to each well of the plate, which was then inc~ ~h~ted for 5 minutes at room te""~alure. The media was then removed and the cells quickly washed with ice cold PBS. The cells were then extracted twice with 1 mL of 25 absolute ethanol. The two exl,~;lions were combined and the ethanol removed by eva~ , dlion. The extract was then redissolved in 1 mL of scintillation ~, u~imil~/ buffer (SPA buffer) (Al "eral Idl 11, Arlington Heights IL).
The cAMP CGI ,ce"l, dlion was detel " ,ined by assay using a scintillation pr~"ci",il~ assay (SPA) kit available from Ame,:,ha",. Analog 1 had a Kd CA 02220060 l997-ll-03 WO 96/39423 PCT/U~i96/0~6 17 (dissociation constant) of 1.5 nM and an EC-50 (effective concentration) of 0.9 nM. Wild-type PTH had a Kd of 8.5 nM and an EC-50 of 4.4 nM. Analogs 2 and 3 have no measurable activity.

E)CAMPLE 5 PREDICTION OF THE EFFECTS OF AMINO ACID
SUBSTITUTIONS IN THE PTH ANALOGS ON a-HELlX POTENTIAL
As explained above, the a-helix potential of linear polypeptides can be estimated using the Lifson-Roig equations (Lifson, S and Roig, A. J. Chem.
Phys. (1961) 34: 1963-1974) and values for the residue helix formation parameters converted from their Zimm-Bragg values (Zimm, B.H. and Bragg, J.K. J. Chem. Phys. (1959) 31 :526-535), employing conversion equations of Qian and Schellman (Qian, H. and Schellman, J.A. J. Chem. Phys. (1992) 96:3987-3994). Once a set of Zimm-Bragg s and ~ values are defined for ~5 each residue, then the probability that the entire polypeptide chain (or some defined subsequence) adopts an a-helix may be computed using equations in the above I erere"ces.
The two calculations yel ~erally used to estimate a-helix potential, described above, were applied to the two PTH analogs generated. The first c~lc~ tion takes the Lifson-Roig helix initiation parameter v to be a co, l~ nl 0.039 (ie, the Zimm Bragg cs parameter is 0.0013 for all residues), with the w ~a, a" ,eler for each amino acid calculated from the Zimm-Bragg s value found in Table 1 of Finkelstein et al. (Finkelstein et al. r~t~ S: Structure, Functionand Genetics (1991) 10:287-299). In applying the c~lclll~tions to the PTH
analogs, the s values in Table 1 of this I ~ference were first converted from their 300 ~K values to the estimated values at 273~K using an assumed ~ enthalpy chc,, ,ge of 1 kcal/mol (i.lel ,lical for all residues). For PTH analog 1 this calculation gives a probability of 0.41 that the entire sequence is a-helical. For PTH analog 2 this calculation gives a probability of 0.18 that the CA 02220060 l997-ll-03 entire sequence is a-helical. For PTH analog 3 this calculation gives a probability of 0.15 that the entire sequence is a-helical.
The second computation does not assume that all residues have the same value for the helix initiation parameter v (ie, the Zimm-Bragg ~
5 parameter is unique for each amino acid residue). In this case the Zimm-Bragg s and c~ values for each residue were taken (except for His, Pro, and Trp) from Table 1 of Skolnick, J. and Holtzer, A. Macromolecules (1982) 15:812-821. The Zimm-Bragg values for His were taken from Sueki et al.
Macromolecules (1984) 17:148-155. The Zimm-Bragg values for Pro and Trp 10 were taken from Finkelstein et al. For this second alternative calculation inevery case the s value was computed at 273~K, and for the resid~ ~es having ionizable side chains, the s value for the state of ionization which wouid predominate at pH 7 was assumed. The s values (at 273~K) and c~ values obtained from Sueki et al. and Skolnick and Holtzer were converted (Qian 15 and Schellman) to their Lifson-Roig v and w values and then used to compute the probability that the entire polypeptide chain (or some subsequence) exists in the a-helical state. For PTH analog 1 this calculation gives a probability of0.07 that the entire sequence is a-helical. For PTH analog 2 this calculation gives a probability of 0.02 that the entire sequence is a-helical. For PTH
20 analog 3 this calculation gives a probability of 0.02 that the entire sequence is a-helical.
Taken together both sets of assumptions regarding the tendency of polypeptides to assume an a-helical co"ru""ation predicts that analog 1 should be two to three times more likely to exist as an a-helix as analog 2.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US~)G/'~!~617 ESTIMATING a-HELlCAL POTENTIAL -ROM CIRCULAR DIC ~ROISM
IN VARYING TRIFLUOROETHA~ OL CONCENTRATIO~ S:
The trifluoroethanol (TFE) circular d chroism studies were cone using 5 an AVIV Associates Inc. Model 62DS Circular Dichroism Spe.;l, u, "eter.
Protein concentrations were determined from absorbance readings using calculated extinction coefficients for the analogs in 1 OmM ionic buffer. TFE
titrations were done from 0 to 50% TFE scanning from 250 to 200nm at 20~C
for each TFE sample. [~3~222 was calcul~ted from:
~o ~Oc5o~ved~t222*(mean residue weight)/(10(cell path length)(protein conce, Ill ~lion)).
Data from the TFE titrations at pH 6.5 1 OmM cacodylate are summarized below in Table 2 for the PTH analogs and plotted in Figure 4.
Figure 4 demonstrates that when the a-helix inducing cosolvent ~5 trifluoroethanol reaches 30% (v/v) co"cer,l, dlion that essenLially the maximum amount of helix is formed for both PTH and analog 1 with the average amount of helicity being about 80%. The parent PTH even in the absence of the helix-inducing agent is about 20% a-helical.
PTH analog 2 in the absence of the helix-inducing TFE is essentially 20 devoid of helix and at 30% TFE is at most about 30% a-helical.
PTH analog 3 in the absence of the helix-inducing TFE is essentially devoid of helix and at 30% TFE is about 15% a-helical.
This 2-3 fold reduction in the observed helical COI ,lenl at 30%
trifluoroethanol was predictable based on the Lifson-Roig equation and 25 c~lc~ tions derived from it as referred to above.

TFE TITRATION DATA

U222fOr U222 for U222 for Uz2 for % TFE hPTH(1-34) analog 1 analog2 analog 3 CA 02220060 l997-ll-03 IN VITRO ELECTROTRANSPORT FLUX
OF THE PTH ANALOGS
In vitro ele~ ll dl ~sport studies were carried out in multi-cor~" d, l",ent custom-built small volume cells using anodic drive. 1 mM PTH in 1 0mM
buffer was placed in the donor compartment and 25mM finai ionic sl, e~ Iyl h receptor was placed in the receptor cor"p~, l" ,ent. The receptor solution for the native PTH and analogs 1 and 2 was 1 5mM NaCI buffered to pH 7 with 10mM imidazole 0.1% bovine serum albumin (BSA) as a blocking agent Tween 20 detergent and sodium azide as a bactericide. The receptor solution for the PTH analog 3 was 15 mM NaCI buffered to pH 7 with 10 mM
i",idi~ole 0.5% dodecyltrimethyl a~ onium bromide (DTAB a surfactant) 15 ard 0.3~g/mL Trp-NIv-A,-g-Phe-amidc a t~ c~J.;d~ us~cd as ar, "aiiclrld~e"
suL,sl, dle for proteolytic enzymes present in the skin.
Heat-stripped human cadaver stratum corneum was placed between the donor and receptor compartments with the epidermis side toward the receptor compa, l" ,e"l. A conslant DC current of 127 ~A mA (ie 0.1 mAlcm2) 20 was applied. Cells were maintained at 32~C during the in vitro studies with receplor samples taken every 2 hours (receptor volume removed and replaced with "fresh" receptor at each sampling). Receptor samples were analyzed by reversed-phase HPLC.
Peak areas of PTH receptor samples were cor"~d, ~:d to ~ "dard 25 curves of the same analog of known concentrations run at the same time using an analytical polymeric column at a flow rate of 1.5 mL/min with a y, ddiel ll from 10-50% B in ten minutes for elution. Buffer A was 0.1%
~ triflu,oacelic acid (TFA) 1% acelor,il,ile; Buffer B was 0.1% TFA 98.9%
acelo, ~ill ile. Analog COI ,ce"l, ~lions were converted to mass flux and plotted 30 VS. time.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US9G1~9617 For the in vitro ele~l, ull al1sport studies described donors cc" ,lai"ed the retro-inverso versions (ie D-amino acid residues were used and were assembled in reverse order than usual) of the PTH analogs 1 and 2 (1 mM) in aqueous solutions at pH 5Ø This was because PTH analogs 1 and 2 were not stable when inc~ Ih~ted with stripped stratum corneum probably due to proteolytic digestion by skin proteases. PTH analog 3 was not a retro-inverso version but instead was recombinantly synthesized. In order to protect analog 3 from proteolytic enzyme digestion the tetrapeptide was added to the receptor solution. The fully assembled ele~,ul,~"sport cells were incubated at 32 + 0.5~C. The skin contact area available for transport was 1.27 cm2. The donor co" ~pa, ll "ent volume was 200 IlL; the receptor compartment volume was 350 ~LL. The receptor solution was separated from the cathode by an anion exchange me",brc"e (lonacTM Eletrosynthesis Co.
La"casler NY). Each cell was connected to a co, l~ldl IL current source at a sla"da, d current density of 100 ~lA cm~2. The voltage drop across each cell was monitored at a~roxi",ately 2-hour intervals for the duration of the experiment.
Hisloy, am s of mass flux for each time point were plotted. The median mass flux (95% co-,ride"ce interval) for PTH analog 1 was 0.3 ~19 cm~2h~' (0.1 to 0.7) for PTH analog 2 was 3.2 ~9 cm~2h~' (2.1 to 4.2) and for PTH analog 3 was 1.1 ~9 cm~2 h-' (0.9-1.7). Average mass flux values + sd for these anaiogs were 1.4 ,ug cm~2h~' + 2.3; 4.1 ~g cm~2h~' + 3.7; and 1.5 ~9 cm~2 h-' +
1.1 for analogs 1 2 and 3 respectively.

ESTIMATING a-HELlCAL POTENTIAL =ROM CIRCULAR DIC ~ROISM
IN VARYING TRlFLUOROETHAi~ OL CONCENTRATIO~ S:
The trifluorut:ll Idl lol (TFE) circular c chroism studies were c one using an AVIV ~soci~les Inc. Model 62DS Circular Dichroism Spectrometer.
30 r, olei" conce, Ill alions were deterl"i"ed from absorbance readings using CA 02220060 l997-ll-03 calculated extinction coefficients for the analogs in 1 OmM ionic buffer. TFE
titrations were done from O to 50% TFE, scanning from 250 to 200nm at 20~C
for each TFE sample. [~]222 was calculated from:
~ObSOlvedat222*(mean residue weight)/(10(cell path length)(protein 5 concer,l, ~lion)).
Data from the TFE titrations at pH 6.5 1 OmM cacodylate are summarized below in Table 3 for the hirulog analogs and plotted in Figure 5.
Figure 5 demonstrates that when the a-helix inducing cosolvent, trifluoroethanol reaches 30% (v/v) conce~ Ill alion that essentially the maximum10 amount of helix is formed for both the parent hirulog and analog B2, with the average amount of helicity being about 10% for the parent hirulog and essentially 0% for the analog B2. The parent hirulog and the analog B2 exhibit no a-helicity in the absence of the helix-inducing TFE. The hirulog-B2 analog had essentially no a-helix even at 80% TFE conce"l, ~lions.

TFE TITRATION DATA

% TFE L~222 for hirulog parent L1222 for analog B2 SU8511TUTE 5HEET tRULE 26) CA 02220060 l997-ll-03 Hirulog-B2 was synthesized using standard techniques. See eg Maraganore et al. Thromb. Haemostasis. (1991) 65:830; Bode et al. EMBO J.
(1989) 8:3467-3475; and International Publication No. WO 92/13952. The transdermal elecl, uL, ansport flux of hirulog-B2 was evaluated using heat separated human epidermis. In addition hydrogel formulations were also investigated using human epidermis as was the effect of current density on transdermal drug flux. Transdermal hirulog-B2 flux was ~ssessed using aqueous solutions prepared with both as received and desalted drug buffered with 10 mM ionic strength acetate buffer pH 5. The hirulog-B2 flux studies were performed at a current density of 0.1 mAJcm2. Using acetate buffered 15 mM NaCI at pH 5 as the receptor solution, steady-state ele~l, ull ansport flux of about 19 ~19 h-' cm-2 and 17 ~9 h-' cm-2 were obtained for the desalted and as received drug solutions respectively.
The variability in hirulog-B2 flux for skin samples from a single donor as well as multiple donors appeared to be about 15 to 40% which is well within the variability ranges observed for peptides and protein co"".ounds.
For the hydrogel studies hydrogel discs were imbibed with drug and asse",bled into foam housings. Either as received (11 mM hirulog-B2 in 20 mM acetate) or desalted drug (4 mM hirulog-B2 in 4 mM acetate) solutions were imbibed into gels. An average steady-state eleul, ul~"sport flux of about 27 ~9 h-1 cm-2 was obtained with hydrogel formulations containing either as received or desalted drug.
Using hydrogels imbibed with as received drug another experiment was u"del lalcen to evaluate the effect of current density on transdermal hirulog-B2 flux. An increase in current density resulted in a corresponding increase in hirulog-B2 flux over the range of current densities exa",i"ed.

WO 96/39423 PCTIUS96/0~6~7 Under passive conditions (zero current), no measurable hirulog-B2 flux was observed.

A. INTRAVENOUS ADMINISTRATION OF HIRULOG-B2 Pigs used for intravenous (IV) studies weighed 11 + 1 kg (n=3), and were fasted overnight before the bolus injections. IV bolus injections were given in the auricle vein or the vena cava. The IV injection was administered 10 over about 30 to 60 seconds and the dose was about 0.3 mg/kg (in 0.5 mL).
Plasma hirulog-B2 levels increased rapidly in all pigs after the IV bolus injection of hirulog-B2, reaching an average peak level of 66 + 6 ng/mL at the 15 minutes sampling point. Plasma hirulog-B2 levels declined to below 2 ng/mL by 2 hours after the bolus injection, and remained relatively constant ~5 from 2 to 8 hours after the bolus i"jection. The elimination half-life and claara,lce calculated for each pig are shown in Table 3. The apparent volume of distribution and average elimination half-life were calcul~t~d from the IV bolus data as 45.4 L and 16 minutes, respectively.

CA 02220060 l997-ll-03 WO 96/39423 PCT/US!~G~5617 Delil,;li~re Pig Pig Pig Mean SD
Phase 303 304 3Q5 lV
BW [kg] 12 11 10 11 Ko [1/h] 2.42 2.01 2.5 2.31 0.26 V [L] 45 5 49.25 41.55 45.43 3.85 V lUKg] 3.8 4.5 4.2 4.2 0.35 CL [Uh] 110 99 104 104 5.5 CL [Uh/Kg] 9.2 9.0 10.4 9.5 0.76 El ~ dliol~ 17.2 20.72 16.6 18.2a 2.23a T.,, [min] 16b SC
Elimination -- -- -- 58b T.,2 lmin]

Elil ";, ldlion -- -- -- 28 T./2 [min]
Accumulation -- -- -- 38b T'A [min]
Screening Pig I.ase SC-3A

Elimination 32 T.,2 lmin]
Accumulation 33 T.,2 [min]
~ Mean caiculated from individuai T~s d~
b Calcubted from linear regression of mean hinulog-B2 plasma ~" ~ vs time culve.

CA 02220060 l997-ll-03 B. SUBCUTANEOUS ADMINISTRATION OF HIRULOG-B2 Pigs were fasted overnight before the subcutaneous (SC) injections.
About 0.5 mg/kg (in 0.5 mL) hirulog-B2 was administered SC over a minute.
Plasma hirulog-B2 levels peaked within the first hour after injection at 57 :: 10 5 ng/mL and declined thereafter. The average elimination half-life following SC
injection was 58 minutes (Table 4). The plasma hirulog-B2 levels were at about 2 ng/mL by 6 hours following the SC injection and were relatively constant from 6 to 8 hours.

10 C. ELECTROTRANSPORT OF HIRULOG-B2 The eleul, ul, ~nsport systems used for hirulog-B2 delivery in vivo included a disposable drug-containing unit in electrical co"tacl with a reusable current controller. The disposable drug-co, ILdil ,ing unit consisted of a laminated medical grade polyethylene adhesive foam tape housing both 15 the silver foil anodic counter electrode and a cathodic AgCI-loaded poiyisobutylene film electrode and the respective gels. The anode gel made contact with the silver foil anode. A cathodic interface gel made contact with the AgCI cathodic electrode. The interface gel was separated from the drug-co"lair,ing donor gel by a cation exchange me",bra"e. The donor gel was 20 placed into the c~LI ,ode cavity of the gel housing just prior to system application.
The drug gels were imbibed the day before eleul, ull ~"sport with hirulog-B2 in acetate buffer (10 mM pH 5) to give a final concel ILI ~lion of 5 mM peptide. The skin contact area for each drug gel was 6 cmZ. The applied 25 current density was 0.1 mA/cm2.
-The ele~L,ul,~llsport systems were ",o,-iloled for current and voltage during elecl, ul, ~"sport to CGI ,ri" " electrical continuity of the system. Voltage values were typical for this animal model and indicated an initial decrease followed by fairly co"slanL values over the study duration. The average elimination half-life following termination of ele.;l, ull dnsport was 28 minutes (Table 5). The average accumulation half-life at the initiation of ele~l~ ull dl Isport was 38 minutes. Plasma hirulog-B2 levels peaked after 4 hours of continuous ele~, ut, dl ,sport at 23 i 6 ng/mL. The plasma hirulog-B2 5 levels remained relatively stable at 21 i 7 ng/mL from 2 to 6 hours of ele.;l, ull dl ,sport and decreased slightly by 8 hours. The apparent decrease in plasma hirulog-B2 levels from 6 to 8 hours of ele~ ull d"sport may be due to hirulog-B2 depletion in the donor hydrogel. A rapid decrease in plasma hirulog-B2 levels occurred after terminating elecl, ull dl l~ ol L By 2 hours after 10 terminating eleul, ull dl ,s,c,ort the hirulog-B2 plasma levels were at or near 2 ng/mL.
The average elimination from three ele~i, ull dl ,sl,ort systems following termination of ele~l, ull dl ,s~ort was 32 minutes while the average accumulation half-life at the initiation of ele~;l, ull dl ISpOI I was 33 minutes 15 (Table 5). The plasma hirulog-B2 level peaked after 2 hours of elecll ull a(l~port at 9 ng/mL after which there was a slow decline in plasma hirulog-B2 levels.
The ll dl IS iar~ 1 Idl flux rates calu 'l~tPd as both an input rate (~lg/h) anda per unit area flux (~g/cm2h) were c~lc~ ted using both one-co" " a, ll l lel ,l 20 and non-co" Ipdl ll I lental analyses. The non-compal ll, lel llal analysis C~iCI liZ~teS the transdermal drug flux from the conce, lll dliol, of drug in the blood of the animal which CGI Icel Ill dliOI I iS measured using standard techniques, and the known rate at which the particular drug is cleared from the blood. The one-compal ll ,lel~l analysis calcl ~l~tes the transde""ai drug 25 flux from the (ie measured) CGI ~cel 1ll dliOI, of drug in the blood of the animal and the assumption that the animal's absollulio" of drug administered by IV
delivery at any particular rate is the same as that achieved through eleCll Ull dl ,sport administration, in order to achieve a particular cc" ,ce, Ill dliGI, of drug in the blood. The one-co" Ipdl 1l I ~el ,l and non-co" Ipdl Ll l lel llal models are ~iscussed in Pharmacokinetics, M. Gibaldi, 2d ed, Marcel Dekker (1982) pps 1 -5 and 319-322. The transdermal input and per unit area fluxes using both analyses are shown in Table 4. The average input rate was calculated to be 1873 i 444 ~g/h, and the flux was calc~ ter~ to be 78 i 18 ~19 h-' cm 2 5 forfour ETS (hirulog-B2) systems using the one com~Jdlllllellt model. Using non-compartmental analysis, the input rate was calculated as 1900 i 523 ~Lg/h, and the flux was calcul~te~l as 79 i 22,ug h-' cm-2 for four ETS (hirulog-B2) systems. An average flux of 22 ~19 h l cm-2 was calculated for the animal in the Screening phase 2 which received three ETS (hirulog-B2) systems.
-CALCULATED ELECTROTRANSPORT INPUT RATES AND

Derinili~re PhasePig 303Pig304Pig 305 Mean SD
4-ETS (one-compd-l...e..l) Ka [1/h] 3.64 2.5 2.94 3.03 0.58 Rate [~g/h] 1361 2096 2161 1873 444 Flux [~Lg/cm2h] 56.7 87.3 90.0 78.0 18.5 4-ETS (non-comp.l-l",e-llal) Rate [,ug/h] 1396 1866 2440 1900 523 Flux ~Lg/cm2h] 58.2 77.8 101.7 79.2 21.8 Screening Phase Pig SC-3-ETS (one-Com, d-l".~
K, [1 /h] 3.15 Rate [~g/h] 392 Flux [,ug/cm2h] 21.8 3-ETS (non-col"~ all".~.,lal) Rate [Ilg/h] 412 Flux,ug/cm2h] 22.9 5UESmUlE SI~E~t~2fS) WO 96/39423 PCT/US96/096 ~7 4~i Thus, methods for enhancing the ele~;t, ull c, ,sport flux of polypeptide agents are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without depa, lil 19 from the spirit and the scope of the5 invention as defined by the appended claims.

Claims (11)

Claims:

1. Use of an analog polypeptide having one or more .alpha.-helical and/or .beta.-sheet segments disrupted, for the manufacture of a composition useful for delivering a polypeptide agent through a body surface using an electrotransport device, said analog polypeptide produced by a method characterized by:
providing a parent polypeptide having an .alpha.-helical and/or .beta.-sheet segment;
substituting one or more amino acid residues of said parent polypeptide with an amino acid residue selected from the group consisting of Pro, Gly and Asn, to disrupt one or more .alpha.-helical and/or .beta.-sheet segments of said parent polypeptide, to render an analog polypeptide, wherein said analog polypeptide exhibits better/enhanced electrotransportability as compared to said parent polypeptide.

2. The use of claim 1, wherein said analog polypeptide exhibits at least about the same biological activity of the parent polypeptide.

3. The use of claim 1, wherein said parent polypeptide is hirulog as depicted in Figure 2A.

4. The use of claim 3, wherein said analog polypeptide is hirulog-B2 as depicted in Figure 2C.

5. Use of a parathyroid hormone (PTH) analog having one or more .alpha.-helical and/or .beta.-sheet segments disrupted, for the manufacture of a composition useful for delivering a polypeptide agent through a body surface using an electrotransport device, said PTH analog produced by a method characterized by:
providing a parent PTH molecule as depicted in Figure 1A;
substituting amino acid residues of said parent molecule with one or more amino acid residues that disrupt one or more .alpha.-helical and/or .beta.-sheet segments of said parent molecule to render a PTH
analog, wherein said PTH analog exhibits better/enhanced electrotransportability as compared to said parent PTH molecule, said PTH analog selected from the group consisting of a PTH analog as depicted in Figure 1C and Figure 1D.

6. A method of making an analog of a parent parathyroid hormone (PTH) molecule as depicted in Figure 1A, said method characterized by:
substituting amino acid residues of said parent molecule with one or more amino acid residues that disrupt one or more .alpha.-helical and/or .beta.-sheet segments of said parent molecule to render a PTH
analog, wherein said PTH analog exhibits better/enhanced electrotransportability as compared to said parent PTH molecule, said PTH analog selected from the group consisting of a PTH analog as depicted in Figure 1C and Figure 1D.

7. A parathyroid hormone (PTH) analog comprising the amino acid sequence depicted in Figures 1C or 1D.

8. A method of making an electrotransport device for delivering a polypeptide agent through a body surface by electrotransport, said method comprising providing a therapeutically effective amount of the polypeptide agent in a donor reservoir of the electrotransport device, the method characterized by:
providing the polypeptide agent in the reservoir as an analog of a parent polypeptide wherein said parent polypeptide comprises an .alpha.-helical and/or .beta.-sheet segment, the analog polypeptide having one or more amino acid residues substituted relative to said parent polypeptide with an amino acid residue selected from the group consisting of Pro, Gly and Asn, to disrupt said .alpha.-helical and/or .beta.-sheet segment.

9. The method of claim 8, wherein said parent polypeptide is hirulog as depicted in Figure 2A.

10. The method of claim 9, wherein said polypeptide analog is hirulog-B2 as depicted in Figure 2C.

11. A method of making an electrotransport device for delivering a polypeptide agent through a body surface by electrotransport, said method comprising providing a therapeutically effective amount of the polypeptide agent in a donor reservoir of the electrotransport device, the method characterized by:
providing the polypeptide agent in the reservoir as an analog of a parent parathyroid hormone (PTH) molecule as depicted in Figure 1A, the analog having amino acid residues substituted relative to said parent molecule with one or more amino acid residues that disrupt one or more .alpha.-helical and/or .beta.-sheet segments present in said parent molecule, said PTH analog selected from the group consisting of a PTH
analog as depicted in Figure 1C and Figure 1D.