EP0932614A1 - Peptidische medikament-vorstufen, eine alpha-hydroxysäureartige kopplungsgruppe enthaltend - Google Patents

Peptidische medikament-vorstufen, eine alpha-hydroxysäureartige kopplungsgruppe enthaltend

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Publication number
EP0932614A1
EP0932614A1 EP97939975A EP97939975A EP0932614A1 EP 0932614 A1 EP0932614 A1 EP 0932614A1 EP 97939975 A EP97939975 A EP 97939975A EP 97939975 A EP97939975 A EP 97939975A EP 0932614 A1 EP0932614 A1 EP 0932614A1
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EP
European Patent Office
Prior art keywords
peptide
glu
alkyl
lys
prodrug
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Application number
EP97939975A
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English (en)
French (fr)
Inventor
Bjarne Due Larsen
Arne Holm
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Zealand Pharma AS
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Zealand Pharma AS
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Publication of EP0932614A1 publication Critical patent/EP0932614A1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/62—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 non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to prodrugs of pharmaceutically active peptides having reduced tendency towards hydrolysis.
  • proteases and other proteolytic enzymes are ubiquitous, particularly in the gastro- intestinal tract, therefore peptides are usually susceptible to degradation in multiple sites upon oral administration, and to some extent in the blood, the liver, the kidney, and the vascular endothelia. Furthermore, a given peptide is usually susceptible to degradation at more than one linkage within the backbone; each locus of hydrolysis is mediated by a certain protease .
  • the invention concerns prodrugs of pharmaceutically active peptides (X-OH) , peptide amides (X-NH) , or peptide esters (X-OR) , wherein the prodrug has the general formula I
  • L is a linking group, comprising from 3 to 9 backbone atoms, wherein the bond between the C-termmal carbonyl of X and L is different from a C-N amide bond;
  • R 3 and R 4 independently are selected from C ⁇ _ 6 -alkyl, phenyl, and phenyl-methyl, wherein C ⁇ - 6 -alkyl is optionally substituted with from one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is optionally substituted with from one to three substituents selected from C ⁇ -6-alkyl, C 2 -6 ⁇ alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or R 3 and R 4 together with the carbon atom to which they are bound form a cyclopentyl, cyclohexyl, or cycloheptyl ring:
  • the present invention also relates to the use of a prodrug of the general formula I in therapy, and the use of a prodrug of the general formula I in the preparation of a composition for use in therapy, and a pharmaceutical composition comprising a prodrug of the general formula I and a pharmaceutically acceptable carrier.
  • Another aspect of the present invention relates to an immobilised linker-peptide sequence L-Z-SSM, wherein L and Z are as defined above, and SSM designates a solid support material, the use of an immobilised linker-peptide sequence L- Z-SSM for the preparation of a prodrug of the general formula I, and methods for preparation of prodrugs of the general formula I comprising the use of an immobilised linker-peptide sequence L-Z-SSM.
  • Peptides are utilised in a number of processes, e.g., cell-to- cell communication, some being present the autonomic and central nervous system. Some of the latter peptides, and a number of other peptides, exert important effects on vascular and other smooth muscles.
  • These peptides include, e.g., the vasoconstrictors angiotensin II, vasopress , endothel , neuropeptide Y, vasoactive intestinal peptide, substance P, neurotens , and calcitonm, calcitonin gene-related peptide, and calcitonm gene-related peptide II.
  • peptides may be mentioned analgetic, antidiabetic, antibiotic, and anaesthetic peptides, etc. and, thus, the peptide may be or be pronounced of endorph s, enkephal s, insulin, gramicidin, paracels , delta-sleep inducing peptide, ANF, vasotoc , bradyk m, dynorphm, endothelm, growth hormone release factor, growth hormone release peptide, oxytocm, tachyk in, ACTH, brain natriuretic polypeptide, cholecystokmm, corticotrop releasing factor, diazepam binding inhibitor fragment, FMRF- amide, galan , gastric releasing polypeptide, gastrm, gastrin releasing peptide, glucagon, glucagon-like pept ⁇ de-1, glucagon- like pept ⁇ de-2, LHR
  • the term "pharmaceutically active peptide sequence” as applied to X is intended to mean any peptide or peptide-contain g structure, either naturally occurring or synthetic, having two or more amino acid units (preferably three or more ammo acid units) and exerting a pharmaceutical effect in mammals such as humans.
  • the term "ammo acid unit” as used in connection with X means any naturally occurring or synthetic ⁇ , ⁇ , and ⁇ -ammo acid, as well as side-chain modified amino acids such as modified tyrosines wherein the aromatic ring is further substituted with e.g.
  • halogens, sulfono groups, nitro groups etc., and/or the phenol group is converted into an ester group, etc, including side-chain protected am o acids, wherein the ammo acid side-chams are protected in accordance with methods known to the person skilled m peptide chemistry, such as described m, e.g., M. Bodanszky and A. Bodanszky, "The Practice of Peptide Synthesis", 2. Ed, Sp ⁇ nger-Verlag, 1994, and J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press, 1991, whether m the L-form or the corresponding D-form.
  • the pharmaceutically active peptide sequence X preferably consists of 2-200 am o acid units, more preferably 2-100 ammo acid units (e.g. 3-100), even more preferably 2-50 ammo acid units (e.g. 3-50 or 4-30), in particular 2-20 amino acid units (e.g. 3-20 or 4-20), especially 2-10 ammo acid units (e.g. 3- 10 or 4-10), such as 2-8 ammo acid units (e.g. 3-8 or 4-8).
  • a pharmaceutically active peptide sequence X which the native form is present as the C- term al free carboxylic acid, such as Leu-enkephalm (H-Tyr- Gly-Gly-Phe-Leu-OH) , is denoted X-OH.
  • a pharmaceutically active peptide sequence X with a C-termmal amide group such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu- Gly-NH 2 )
  • X-NH? a pharmaceutically active peptide sequence X with a C-terminal ester groups
  • X-OR a pharmaceutically active peptide sequence X with a C-terminal ester groups
  • OR e.g. is the alkoxy moiety of the alcohol which, together with the pharmaceutically active peptide sequence X, constitutes the ester.
  • R may designate C._ 6 -alkyl, aryl such as phenyl, aryl-C ⁇ - 6 -alkyl such as benzyl, etc.
  • any peptide sequences corresponding to pharmaceutically active peptides having a free C-termmal carboxy group (i.e. X-OH) as well as peptides corresponding to pharmaceutically active peptides having a C-termmal amide (i.e. X- NH 2 ) or ester group (i.e. X-OR) may be used in the compounds and prodrugs of the invention.
  • porcine insulin differ from human msulm by only one ammo acid unit, the B30 ammo acid in porcine msulm being Ala and the B30 ammo acid in human insulin being Thr .
  • porcine insulin has been used as an effective diabetes drug for many years.
  • the essential features for activity in the heptadecanepeptide Porcine gastrin I are all contained in the C-terminal tetrapeptide and that essentially all biological effects of neurotensin are associated with the C-termmal hexapeptide.
  • pharmaceutically active peptides wherein one or more amide bonds have been modified, e.g. reduced, often exhibit a similar or even enhanced biological activity; for example the Cys 2 ⁇ [CH 2 NH] Tyr 3 analogue of somatostat was found to be an even more potent growth hormone releasing agent than somatostatm itself, and also the transition state analogue Leu 10 ⁇ [CH (OH) CH 2 ] Val 11 of angiotensm has been found to show strong inhibitory effect against the aspartic acid protease Renin.
  • the peptide sequence question should preferably comprise at least one amide bond (preferably two amide bonds (this naturally does not apply for a dipeptide) ) susceptible to enzymatic degradation in order to fully take advantage of the present invention.
  • the most interesting prospect of the present invention is that it is possible to prepare "peptide prodrugs" for the treatment of mammals, such as humans, which are stabilised towards degradation by proteases and which subsequently are able to be released in an environment m which the peptide or the pharmaceutically active peptide sequence (X-OH) will exhibit a pharmaceutical action or will be transported to the desired location.
  • the pharmaceutically active peptide sequence X preferably are released as a free acid (due to splitting of e.g. an ester bond between X and L) it is envisaged that the free acid may also posses a pharmaceutical relevant effect m cases where the native expertmaceutically active peptide sequence X is an amide (X-NH 2 ) or ester (X-OR) .
  • the bond between the C- termmal carbonyl function of X and L is capable of being cleaved by blood plasma enzymes such as e.g. butyryl cholinesterase, acetyl cholinesterase, etc.
  • the bond between the C-terminal carbonyl function of X and L is a thiolester bond or an ester bond, preferably an ester bond.
  • the bond between X and L m the peptide prodrugs of the invention must be capable of being cleaved m vivo .
  • the bond between X and L (which is preferably an ester bond) is capable of being cleaved by the enzyme butyryl cholinesterase.
  • the peptide prodrugs of the invention is capable of being cleaved by e.g. esterases present in the blood plasma and thereby releasing the desired pharmaceutically active peptide X-OH at a desired location.
  • the rate of enzymatic cleavage of the peptide may be adjusted in order for a medicament comprising the prodrug I to have a prolonged or retarded effect. Adjustment of the cleavage rate may, e.g., be carried out by increasing or decreasing the bulkmess and/or the electron-donating effect of substituents on L.
  • C ⁇ _ 6 -alkyl used alone or as part of another group designates a straight, branched or cyclic saturated hydrocarbon group having from one to six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.butyl, tert.butyl, n-pentyl, n-hexyl, cyclohexyl, etc.
  • C_ 5 -alkyl covers a straight, branched or cyclic saturated hydrocarbon group having from one to five carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl, tert. butyl, n-pentyl, isopentyl, cyclopentyl, etc.
  • C T i-alkyl covers a straight or branched saturated hydrocarbon group having from one to three carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.
  • C 2 _ 6 -alkenyl designates a hydrocarbon group having from two to six carbon atoms, which may be straight, branched, or cyclic and may contain one or more double bonds, such as vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-l- butenyl, 2-hexenyl, 5-hexenyl, cyclohexenyl, 2, 3-d ⁇ methyl-2- butenyl etc., which may have ci s and/or trans configuration.
  • C 2 -s-alkenyl designates a hydrocarbon group having from two to five carbon atoms, which may be straight, branched, or cyclic and may contain one or more double bonds, such as vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-l- butenyl, cyclopentenyl, etc., which may have cis and/or trans configuration
  • C 2 -.,-alkenyl designates a hydrocarbon group having from two to four carbon atoms, which may be straight or branched and may contain one or more double bonds, such as vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, etc., which may have cis and/or trans configuration.
  • alkoxy means alkyl-oxy.
  • aryl is intended to mean an aromatic, carbocyclic group such as phenyl or naphthyl .
  • halogen includes fluorine, chlorine, bromine, and iodine.
  • heteroaryl includes 5- or 6-membered aromatic monocyclic heterocyclic groups containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, such as pyrrolyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, pyridyl, and aromatic bicyclic heterocyclic groups containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur, such as qu olmyl .
  • the peptide sequence Z is the part of the compound I respon- sible for introduction and/or stabilisation of a certain secondary structure into the molecule which will render the compound more stable towards degradation by proteases, thus, it is believed that Z needs to include at least 2 ammo acid units (preferably at least 3 ammo acid units) order to introduce such a structural element either alone or in combination with the linker L. On the other hand it is also believed that a sequence of more than around 20 am o acid units will not improved the stability further.
  • Z is a peptide sequence of 2-20 am o acid units (preferably 3-20), preferably in the range of 3-15, more preferably 3-9 (such as 4-9), in particular 3-6 ammo acid units, such as 4-6 ammo acid units.
  • Each of the ammo acid units m the peptide sequence Z are independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and ammo acids of the formula II as defined herein.
  • the ammo acid units are selected from Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, and Met, more preferably from Glu, Lys, and Met, especially Glu and Lys.
  • the above-mentioned ammo acids may have either D- or L-configuration, but preferably the above- mentioned am o acids have L-configuration.
  • a peptide sequence Z consisting only or principally of L-amino acids will be advantageous compared to a peptide sequence Z consisting only or principally of D-amino acids.
  • a peptide sequence Z consisting only or principally of D-amino acids may exert toxicological effects due to the resistance of D-peptides and D-amino acids towards biodegradation .
  • the ammo acid units of Z may of course all be different or all be identical.
  • the ammo acid units Z are selected from three different ammo acids or from two different ammo acids, or are identical am o acids, preferably the ammo acid units m Z are identical such as (Lys) n or (Glu) n , wherein n is an integer in the range from 4 to 6, or a combination of two ammo acid units such as (LysGlu) 2 , (LysGlu) 3 , (GluLys) , or (GluLys) 3 , or a combination of three amino acid units, e.g.
  • ammo acid units mentioned as constituents of the peptide sequence Z i.e. Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and ammo acid units of the formula II, are ammo acid units which, due to their ste ⁇ cal arrangement around the ⁇ -carbon atom, and probably also due to a specific electronic configuration, have certain preferences for participating m, or even stabilising or initiating, helix- like structures.
  • the Chou-Fasman approach Chou, P.Y. & Fasman, G.D. Ann . Rev. Biochem .
  • peptide sequence Z it is considered possible to include a small proportion of ammo acid units which are not among the amino acid units selected above as constituents of Z, and still obtain the desired effect from the peptide sequence Z, in that the selected ammo acid units are believed to compensate for any negative or neutral effect of such an alternative ammo acid unit.
  • Such alternative am o acid units may be selected from Val, lie, Pro, Phe, Gly, Trp, as well as N-methyl amino acid units, however, preferably not Pro, Gly and N-methyl ammo acid units.
  • Illustrative examples of the peptide sequences Z are: Lys-Lys-Lys-Lys, Glu-Lys-Lys-Lys, Lys-Glu-Lys-Lys, Lys-Lys-Glu- Lys, Lys-Lys-Lys-Glu, Glu-Glu-Lys-Lys, Glu-Lys-Gly-Lys, Glu- Lys-Lys-Glu, Lys-Glu-Glu-Lys, Lys-Glu-Lys-Glu, Lys-Lys-Glu-Glu, Glu-Glu-Glu-Lys, Glu-Glu-Glu-Glu, Glu-Lys-Glu, Glu-Lys-Glu, Lys-Glu-Glu-Glu-Glu, Lys-Glu-Glu-Glu-Glu, Lys-Glu-Glu-Glu, Lys-Glu-Glu-Glu, Lys-Glu-Glu-Glu, Lys-Glu-Glu-Glu, Lys-
  • C-terminal of Z may be presented in the form of the free acid, the amide, or the ester, e.g. depending on the type of solid support material and cleavage conditions used in connection with the syntheses as will be clear to the person skilled in the art.
  • L is bound at the N-terminal nitrogen atom of Z, i.e. the possible bond types between L and Z are those which involve a nitrogen atom, e.g.
  • the linker L should preferably be able to participate in a helix-like structure initiated or stabilised by Z.
  • the geometry of L should preferably correspond to the geometry of an amino acid (or two or more ammo acids), i.e. the linker L preferably comprises 3 backbone atoms or a multiple thereof such as 6 or 9 backbone atoms.
  • the term "backbone atoms" when used connection with the linker L therefore refers to the atoms the linker L directly linking the pharmaceutically active peptide sequence X and the pre- sequence Z.
  • L is preferably derived from a hydroxy-carboxylic acid, m particular an ⁇ -hydroxy-carboxylic acid. More specifically, L is derived from an ⁇ -hydroxy-carboxylic acid of the general formula HO-CfR 1 ) (R 2 ) -C00H wherein R and R independently is selected from H, C ⁇ - 6 -alkyl, C- 6 -alkenyl, aryl, aryl-C ⁇ -alkyl, heteroaryl, heteroaryl-C ⁇ - 4 -alkyl, or R 1 and R 2 together with the carbon atom to which they are bound form a cyclopentyl, cyclohexyl, or cycloheptyl ring, where an alkyl or alkenyl group may be substituted with from one to three substituents selected from ammo, cyano, halogen, lsocyano, isothiocyano, thiocyano, sulfamyl, C ⁇ -
  • the linker L is derived from an ⁇ -hydroxy-carboxylic acid which also bears a methylene (-CH 2 -) group in the ⁇ -position.
  • the linker L is derived from an ⁇ - hydroxy-carboxylic acid with the general formula HO-C(CH 2 - R 5 ) (R 2 )-COOH, wherein R 5 is selected from H, C _ 5 -alkyl, C 2 -_- alkenyl, aryl, aryl-C ⁇ - 3 -alkyl, heteroaryl, heteroaryl-C ⁇ _ 3 - alkyl, where an alkyl or alkenyl group may be substituted with from one to three substituents selected from amino, halogen, mono- or d ⁇ -C.-4-alkyl-am ⁇ no, hydroxy, d- 4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C ⁇ -4-alkoxycarbonyl, C1-4- alkylcarbonyloxy, and am ocarbonyl and where an aryl or heteroaryl may be substituted with from one to three substituents selected from C -
  • R 2 is as defined above, preferably H, C ⁇ - 6 - alkyl, C 2 - 6 -alkenyl, aryl, aryl-C ⁇ - 4 -alkyl, heteroaryl, heteroaryl-C ⁇ - 4 -alkyl, where an alkyl or alkenyl group may be substituted with from one to three substituents selected from amino, halogen, mono- or di-C 1 _ 4 -alkyl-ammo, hydroxy, Ci_ 4 - alkoxy, aryl, heteroaryl, aryloxy, carboxy, C ⁇ - 4 -alkoxycarbonyl, C ⁇ -
  • the above mentioned adjustment of the cleavage rate by increasing or decreasing the bulkiness and/or the electron- donating effect of substituents on L may e.g. be carried out by increasing or decreasing the bulkiness and/or the electron- donating effect of R 1 and/or R 2 (or R 5 ) .
  • L is derived from hydroxyacetic acid, (S) - (+) -mandelic acid, L-lactic acid ((S)- (+ ) -2-hydroxypropanoic acid), L- ⁇ -hydroxy-butyric acid ((S)-2- hydroxybutanoic acid) , and ⁇ -hydroxy-isobuty ⁇ c acid.
  • the prodrugs of the invention may also be in the form of a salt thereof.
  • Salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts.
  • acid addition salts are hydrochloride slats, sodium salts, calcium salts, potassium salts, etc..
  • basic salts are salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium, and ammonium ions + N(R 6 ) 3 (R 1 ) , where R 6 and R' independently designates optionally substituted C:- ⁇ -alkyl, C 2 - 6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl.
  • X-L-Z The tendency of X-L-Z to resist degradation can for example be expressed as a pseudo-first-order rate constant and/or as the half-life of said prodrugs, which may be compared to the corresponding values of X-OH, X-NH 2 , and/or X-OR.
  • the ability of the prodrugs of the invention to exert the desired biological effect was tested in various m vi tro and m vi vo assay procedures. A detailed description of the above-mentioned tests are given in the examples.
  • the ratio between the half-life of the prodrug in question in the "Hydrolysis in enzyme solution test”, as defined herein, and the half-life of the corresponding peptide (X-OH) , in the "Hydrolysis in enzyme solution test”, is at least 2, preferably at least 5, and even more preferably at least 10, especially at least 20, when using one of the enzymes carboxypeptidase A and leuc e ammopeptidase .
  • the invention also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a prodrug of the general formula I as defined above in combination with a pharmaceutically acceptable carrier.
  • compositions may be in a form adapted to oral, parenteral (intravenous, intraperitoneal), rectal, mtranasal, dermal, vaginal, buccal, ocularly, or pulmonary administration, and such compositions may be prepared in a manner well-known to the person skilled in the art, e.g. as generally described in "Remington's Pharmaceutical Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985 and more recent editions and in the monographs in the
  • the invention also concerns use of a prodrug of the general formula I as defined above or a salt thereof m the preparation of a composition for use in therapy, e.g. in the treatment of disorders the central nerveous system, vaccine therapy, and m the treatment of HIV, cancer, diabetes, incontinence, hypertension, and as analgesics and contraceptives, and such indications known to be treated by therapy comprising administration of pharmaceutically active peptides.
  • the prodrugs of the invention may be prepared by methods known per se in the art.
  • the peptide sequences X and Z may be prepared by standard peptide-preparation techniques such as solution synthesis or Merrifleld-type solid phase synthesis. It is believed that the Boc (tert .butyloxycarbonyl ) as well as the F oc (9-fluorenylmethyloxycarbonyl) strategies are applicable.
  • the prodrugs of the invention may be prepared by solid phase synthesis by first constructing the peptide sequence Z using well-known standard protection, coupling and deprotection procedures, subsequently coupling the linking group L, thereafter sequentially coupling the pharmaceutically active sequence X on the linking group L in a manner similar to the construction of Z, and finally clea- v g off the entire prodrug X-L-Z from the carrier.
  • Another possible strategy is to prepare one or both of the two sequences X and Z separately by solution synthesis, solid phase synthesis, recombinant techniques, or enzymatic synthesis, followed by coupling of the two sequences and the linking group L by well-known segment condensation procedures, either in solution or using solid phase techniques or a combination thereof .
  • a combination of the above- mentioned strategies may be especially applicable where a modified peptide sequence, e.g. from a biologically active peptide comprising reduced peptide bonds, is to be coupled to a peptide sequence Z via a linker L.
  • a linker L it may be advantageous to prepare the immobilised fragment L-Z by successive coupling of amino acids (and the linker) first and then couple a complete biologically active peptide sequence X (prepared in solution or fully or partially using solid phase techniques) to the fragment L-Z.
  • the C-terminal am o acid of the pre-sequence Z is attached to the solid support material by means of a common linker such as 2, 4-d ⁇ methoxy-4 ' -hydroxy-benzophenone, 4- (4- hydroxy-methyl-3-methoxyphenoxy) -butyric acid, 4-hydroxy- methylbenzoic acid, 4-hydroxymethyl-phenoxyacet ⁇ c acid, 3- (4- hydroxymethylphenoxy) propionic acid, and p- [ (R, S) -a [1- (9H- fluoren-9-yl ) methoxyformamido] -2 , -d ⁇ methoxybenzyl ] -phenoxy- acetic acid.
  • a common linker such as 2, 4-d ⁇ methoxy-4 ' -hydroxy-benzophenone, 4- (4- hydroxy-methyl-3-methoxyphenoxy) -butyric acid, 4-hydroxy- methylbenzoic acid, 4-hydroxymethyl-phenoxyacet ⁇ c acid, 3- (4- hydroxymethylphenoxy
  • the present invention also relates to the use of an immobilised l ker-peptide sequence Prot-L-Z-SSM for the preparation of a prodrug according to the invention, and to a method for the preparation of a prodrug of a peptide (X-OH) , a peptide amide (X-NH ? ) , or a peptide ester (X-OR) comprising coupling the corresponding peptide in a C-termmal activated form (X-Act) to an immobilised lmker-peptide sequence H-L-Z- SSM.
  • a peptide X-OH
  • a peptide amide X-NH ?
  • X-OR a peptide ester
  • the present invention also relates to a further method for the preparation of a prodrug of a peptide (X-OH) , a peptide amide (X-NH 2 ) , or a peptide ester (X-OR) comprising the steps of:
  • step b) repeating the removal/coupling procedure in step b) and c) until the desired peptide sequence X is obtained, and then
  • the coupling, removal and cleavage step is performed by methods known to the person skilled in the art taking into consideration the protection strategy and the selected solid phase material.
  • bonds on the one hand between X and L, and on the other hand between L and Z, of the types indicated above may be established by methods known per se for establishing thiol ester, ester, carboxamide, sulfonamide, alkylamine, carbamate, thiocarbamate, urea, thiourea, thioamide, cyanomethyleneamino, or N-methylamide bonds or groupings, see e.g. J. March, "Advanced Organic Chemistry", 3rd edition, John Wiley & Sons, 1985 as well as references cited therein. Thus, e.g.
  • an ester may be formed from an activated derivative (acid halide, acid anhydride, activated ester e.g. HObt-ester etc.) of the appropriate carboxylic acid by reaction with the relevant hydroxy compound.
  • a carboxamide may be formed by reacting an activated derivative (acid halide, acid anhydride, activated ester e.g.
  • a sulfonamide may be formed by reacting a sulfonyl chloride with the appropriate ammo compound
  • an alkyl amme bond or grouping may be formed by reacting the appropriate compound carrying a leaving group such as tosyl, halogen, and mesityl on the carbon atom in question with the relevant ammo compound in a nucleophilic substitution reaction
  • a carbamate bond or grouping may be formed by treating the appropriate alcohol with phosgene to afford the corresponding chlorocarbonate which is then reacted with the relevant amino compound
  • a thiocarbamate bond or grouping may be formed by treating the appropriate alcohol with thiophosgene to afford the corresponding chlorothiocarbonate which is then reacted with the relevant am o compound
  • a urea bond or grouping may be formed by reacting the appropriate compound carrying a iso
  • the peptide prodrug of the invention may be cleaved from the solid support material by means of an acid such as trifluoracetic acid, trifluoromethanesulfonic acid, hydrogenbromide, hydrogenchloride, hydrogenfluoride, etc. or a base such as ammonia, hydrazine, an alkoxide, such as sodium ethoxide, an hydroxide, such as sodium hydroxide, etc.
  • an acid such as trifluoracetic acid, trifluoromethanesulfonic acid, hydrogenbromide, hydrogenchloride, hydrogenfluoride, etc.
  • a base such as ammonia, hydrazine, an alkoxide, such as sodium ethoxide, an hydroxide, such as sodium hydroxide, etc.
  • X is a peptide sequence which is bound to L at the C- terminal carbonyl function of X;
  • L is a linking group, comprising from 3 to 9 backbone atoms, wherein the bond between the C-terminal carbonyl of X and L is different from an C-N amide bond;
  • Z is a peptide sequence of 2-20 amino acid units and bound to L at the N-terminal nitrogen atom of Z, each amino acid unit being independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of the formula II
  • R 3 and R 4 independently are selected from C . - 6 - alkyl, phenyl, and phenyl-methyl, wherein C ⁇ - 6 -alkyl is optionally substituted with from one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is optionally substituted with from one to three substituents selected from Ci-e-alkyl, C 2 - b -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or R 3 and R 4 together with the carbon atom to which they are bound form a cyclopentyl, cyclohexyl, or cycloheptyl ring;
  • DIEA N, N-diisopropylethylamine
  • DMAP 4- (N,N-dimethylamino) -pyridine
  • HAA hydroxyacetic acid
  • HMPA 4-hydroxymethylphenoxyacetic acid
  • HObt 1-hydroxybenzotriazole
  • PEG-PS polyethyleneglycol grafted on polystyrene
  • Peptides were synthesized batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration using 9- fluorenylmethyloxycarbonyl (Fmoc) as N- ⁇ -amino protecting group and suitable common protection groups for side-chain functionalities (Dryland, A. and Sheppard, R.C. (1986) J. Chem. Soc, Perkin Trans. 1, 125-137).
  • Fmoc 9- fluorenylmethyloxycarbonyl
  • Solvents Solvent DMF (N, W-dimethylformamide, Riedel de-Haen, Germany) was purified by passing through a column packed with a strong cation exchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen, Germany) and analyzed for free amines prior to use by addition of 3, -dihydro-3-hydroxy-4-oxo-l , 2, 3-benzotriazine (Dhbt-OH) giving rise to a yellow colour (Dhbt-O-anion) if free amines are present.
  • Solvent DCM (dichloromethane, analytical grade, Riedel de-Haen, Germany) was used directly without purification.
  • Fmoc-protected amino acids were purchased from MilliGen (UK) in suitable side-chain protected forms. Otherwise protected amino acids (H-Glu(OtBu) -OtBu; H-Glu (cHex) -OH; ⁇ -Glu (OtBu) -OH) and the dipeptides Fmoc-Phe-Gly-OH and H-Phe-Leu-OH were purchased from Bachem (Switzerland) . Coupling reagents
  • DIC Coupling reagent diisopropylcarbodnmide
  • DCC dicyclohexylcarbodiimide
  • Linkers (4-hydroxymethylphenoxy) acetic acid (HMPA) , Novabio- chem, Switzerland; hydroxyacetic acid, (S) - (+) -mandelic acid 99°o pure, Aldrich, Germany, and (R) -(-) -mandelic acid 98.5° pure, M&R, England, were coupled to the res or to the N- term al of the pre-sequence Z as a preformed 1- hydroxybenzotriazole (HObt) ester generated by means of DIC.
  • HMPA 4-hydroxymethylphenoxy) acetic acid
  • Peptides synthesised according to the Fmoc-strategy were synthesised on two different types of solid support using 0.05 M or higher concentrations of Fmoc-protected activated ammo acid in DMF: 1) PEG-PS (polyethyleneglycol grafted on polystyrene; NovaSyn TG resm, 0.29 mmol/g, Novabiochem,
  • Dusopropylethylamine (DIEA) was purchased from Aldrich, Germany, and ethylenediam e from Fluka, piperid e and py ⁇ dme from Riedel-de Haen, Frankfurt, Germany. 4-(N,N- d ⁇ methylammo)pyr ⁇ dme (DMAP) was purchased from Fluka, Switzerland and used as a catalyst in coupling reactions involving symmetrical anhydrides. Ethanedithiol was purchased from Riedel-de Haen, Frankfurt, Germany. 3, -d ⁇ hydro-3-hydroxy- 4-oxo-l, 2, 3-benzotr ⁇ az ⁇ ne (Dhbt-OH) and 1-hydroxybenzotr ⁇ azole (HObt) were obtained from Fluka, Switzerland. FmocNHS was purchased from Aldrich, Germany. Enz ymes
  • Carboxypeptidase A (EC 3.4.17.1) type I from Bovine Pancreas, leuc e ammopeptidase (EC 3.4.11.1) type III-CP from Porcine Kidney, butyryl cholinesterase (EC 3.1.1.8) from Horse Serum, ⁇ -chymotrypsm (EC 4.4.21.1) from Bovine Pancreas, and pepsin A (EC 3.4.23.1) from Porcine Stomarch Mucosa Bovine Pancreas were obtained from Sigma, UK.
  • Deprotection of the N- ⁇ -ammo protecting group was performed by treatment with 20% pipe ⁇ dme DMF (1x3 and 1x7 mm.), followed by wash with DMF until no yellow colour (Dhbt-O-) could be detected after addition of Dhbt-OH to the drained DMF.
  • Peptides were cleaved from the resins by treatment with 95% trifluoroacetic acid (TFA, Riedel-de Haen, Frankfurt, Germany) - water v/v or with 95% TFA and 5% ethanedithiol v/v at r.t. for 2 h.
  • TFA trifluoroacetic acid
  • the filtered resins were washed with 95% TFA-water and filtrates and washings evaporated under reduced pressure.
  • the residue was washed with ether and freeze dried from acetic acid-water.
  • the crude freeze dried product was analysed by high-performance liquid chromatography (HPLC) and identified by electrospray lonisation mass spectrometry (ESMS) .
  • HPLC high-performance liquid chromatography
  • ESMS electrospray lonisation mass spectrometry
  • DIC was dissolved in 10 ml DMF and left for activation for 10 mm, after which the mixture was added to the res and the coupling continued for 24 h.
  • the resm was drained and washed with DMF (10 x 15 ml, 5 mm each) , and the acylation was checked by the nmhydrm test.
  • the first am o acid was coupled as the preformed symmetrical anhydride (see above), and the coupling yields estimated as described above. It was m all cases better than 70%.
  • the synthesis was then continued as "jbatc iv-.se ".
  • NovaSyn TG res 250 mg, 0.27-0.29 mmol/g was placed in a polyethylene vessel equipped with a polypropylene filter for filtration.
  • the resm was swelled n DMF (5 ml) , and treated with 20% piperid e DMF to secure the presence of non- protonated ammo groups on the resm.
  • the resm was drained and washed with DMF until no yellow colour could be detected after addition of Dhbt-OH to the drained DMF.
  • HMPA (3 eq.) was coupled as a preformed HObt-ester as described above and the coupling was continued for 24 h.
  • the resm was drained and washed with DMF (5 x 5 ml, 5 mm each) and the acylation checked by the mnhydrin test.
  • the first amino acid was coupled as a preformed symmetrical anhydride as described above.
  • the coupling yields of the first Fmoc-protected ammo acids were estimated as described above. It was m all cases better than 60%.
  • the following ammo acids according to the sequence were coupled as preformed Fmoc-protected, if necessary side-chain protected, HObt esters (3 eq.) as described above. The couplings were continued for 2 h, unless otherwise specified.
  • the resm was drained and washed with DMF (5 x 5 ml, 5 mm each) in order to remove excess reagent. All acylations were checked by the mnhydrin test performed at 80°C. After completed synthesis the peptide-resm was washed with DMF (3x5 ml, 5 mm each), DCM (3x5 ml, 1 mm each) and finally diethyl ether (3x5 ml, 1 mm each) and dried m vac ⁇ o .
  • Isocratic HPLC analysis was preformed on a Shimadzu system consisting of an LC-6A pump, an MERCK HITACHI L-4000 UV detector operated at 215 nm and a Rheodyne 7125 injection valve with a 2, 20, or 100 ⁇ l loop.
  • the column used for isocratic analysis was a Spherisorb ODS-2 (100 x 3 mm; 5- ⁇ m particles).
  • HPLC analysis using gradients was performed on a MERCK-HITACHI L-6200 Intelligent pump, an MERCK HITACHI L-4000 UV detector operated at 215 nm and a Rheodyne 7125 injection valve with a 20 ⁇ l loop.
  • the column used was a RescorceTM RPC 1 ml.
  • Buffer A was 0.1 vol % TFA in water and buffer B 90 vol% acetonit ⁇ le, 9.9 vol" water and 0.1 vol% TFA.
  • the Buffers were pumped through the column at a flow rate of 1.3-1.5 ml/mm using the following gradient for peptide analysis 1. Linear gradient from 0% - 100% B (30 mm), for enzymatic studies 2. Linear gradient from 40 - 100% B (15 min), 3. Linear gradient from 10 - 40% B (15 mm), or 4. Linear gradient from 0 - 50% B (15 mm) .
  • the mobile phase used for isocratic analysis will be mentioned under the description of the individual experiments.
  • the peptide was cleaved from the resm as described above and freeze dried from acetic ac d.
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 90% and the identity of the peptide was confirmed by ES-MS . Yield 76%.
  • Dry PepSyn K (ca 500 mg, 0.1 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated with ethylenediamme as earlier described.
  • the first 6 glutamic acid units forming the pre- sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq. ) .
  • the acylations were checked by the mnhydrin test performed at 80°C as described above.
  • the Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resm was reacted with 6 eq.
  • the remaining ammo acids according to the sequence were coupled as preformed Fmoc-protected, if necessary side-chain protected, HObt esters (3 eq.) with the addition of 1 eq Dhbt- OH DMF (2 ml) for 2 h.
  • the acylation was checked by the mnhydrin test performed as described above.
  • the remaining ammo acids according to the sequence were coupled as Fmoc- protected Pfp esters (3 eq. ) with the addition of Dhbt-OH (1 eq.) in DMF (2 ml) .
  • the peptide was cleaved from the resin as described above and freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous, the purity was found to be better than 80%, the identity of the peptide was confirmed by ESMS, and the yield 58%.
  • Dry PepSyn K (ca 500 mg, 0.1 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated with ethylenediamine as earlier described.
  • the first 6 glutamic acids forming the pre-sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq.) .
  • the acylations were checked by the ninhydrin test performed at 80°C as described above.
  • the Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resin was reacted with 6 eq.
  • Fmoc-Gly-Phe-OH was coupled as a preformed HObt ester (3 eq.) m DMF (5 ml) prepared as described above for 2 h.
  • Excess reagent was then removed by DMF washing (12 mm flow rate 1 ml/mm) and the acylation was checked by the mnhydrin test performed at 80°C as described above.
  • the Fmoc group was then removed by treatment with 20% piperidine in DMF as described above.
  • the remaining ammo acids according to the sequence were coupled preformed Fmoc-protected HObt esters (3 eq.) with the addition of 1 eq Dhbt-OH in DMF (2 ml) for 2 h.
  • the acylation was checked by the nhydrin test performed as described above.
  • the remaining am o acids according to the sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq.) DMF (2 ml).
  • Excess reagent was removed by DMF washing (12 mm flow rate 1 ml/mm) and acylations were checked by the mnhydrin test performed 80°C as described above.
  • the Fmoc group was deprotected as described above. After completed synthesis the peptide-res was washed with DMF (10 mm, flow rate 1 ml/mm), DCM (3x5 ml, 1 mm each) , diethyl ether (3x5 ml, 1 mm each) and dried m vacuo .
  • the peptide was cleaved from the resm as described above and freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous, the purity was found to be better than 80%, the identity of the peptide was confirmed by ESMS, and the yield 57%.
  • the prodrug H-Tyr-Gly-Gly-Phe-Leu- ( (R) - (- ) -Ma) -Glu 6 -0H was prepared as described above for 3.
  • the acylations were checked by the ninhydrin test performed at 80°C as described above.
  • the Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resm was reacted with 6 eq. (S) -(+) -mandelic acid as a preactivated HObt-ester as described above and the coupling was continued for 24 h. Excess reagent was removed by DMF washing (12 mm flow rate 1 ml/mm) . The acylation was checked by the mnhydrin test. The next ammo acid according to the sequence (leucine) was coupled as preformed symmetrical anhydride as described above and the reaction was continued for 2 h.
  • the remaining am o acids according to the sequence were coupled preformed Fmoc-protected HObt esters (3 eq.) with the addition of 1 eq Dhbt-OH m DMF (2 ml) for 2 h.
  • the acylation was checked by the mnhydrin test performed as described above.
  • the remaining amino acids according to the sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq.) in DMF (2 ml).
  • Excess reagent was removed by DMF washing (12 mm flow rate 1 ml/min) and acylations were checked by the mnhydrin test performed at 80°C as described above.
  • the Fmoc group was deprotected as described above. After completed synthesis the peptide-resm was washed with DMF (10 mm, flow rate 1 ml/mm), DCM (3x5 ml, 1 m each), diethyl ether (3x5 ml, 1 mm each) and dried in vacuo .
  • the peptide was cleaved from the resm as described above and freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous, the purity was found to be better than 80%, the identity of the peptide was confirmed by ESMS, and the yield 57%.
  • the prodrug H-Tyr-Gly-Gly-Phe-Leu- ( (R) - (-) -Ma) -Lys b -OH was prepared as described above for 4.
  • Dry PepSyn K (ca 500 mg, 0.1 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated with ethylenediamme as earlier described.
  • the first 6 ammo acids forming the pre-sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq.).
  • the acylations were checked by the mnhydrin test performed at 80 ⁇ C as described above.
  • the Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resm was reacted with 6 eq.
  • the remaining amino acids according to the sequence were coupled preformed Fmoc-protected HObt esters (3 eq.) with the addition of 1 eq Dhbt-OH in DMF (2 ml) for 2 h.
  • the acylation was checked by the ninhydrin test performed as described above.
  • the remaining amino acids according to the sequence were coupled as Fmoc-protected Pfp esters (3 eq.) with the addition of Dhbt-OH (1 eq.) in DMF (2 ml). Excess reagent was removed by DMF washing (12 min flow rate 1 ml/min) and acylations were checked by the ninhydrin test performed at 80°C as described above.
  • the Fmoc group was deprotected as described above. After completed synthesis the peptide-resin was washed with DMF (10 min, flow rate 1 ml/min), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried in vacuo .
  • the peptide was cleaved from the resin as described above and freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous, the purity was found to be better than 80%, the identity of the peptide was confirmed by ESMS, and the yield 63%.
  • the prodrug H-Tyr-Gly-Gly-Phe-Leu- ( (R) - (- ) -Ma) - (LysGlu) 3 -OH was prepared as described above for 5. 6. Peptide synthesis of Fmoc-Phe-Leu-HAA-Glu ⁇ -OH on NovaSyn TentaGel.
  • Dry NovaSyn TG res (0.29 mmol/g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batchwise peptide synthesis on PEG-PS" until finishing the pre-sequence Glu 6 .
  • the peptide-resm was then reacted with 6 eq. hydroxyacetic acid as a preactivated HObt-ester as described above and the coupling was continued for 24 h. Excess reagent was removed by DMF washing (12 min flow rate 1 ml/mm). The acylation was checked by the ninhydrin test.
  • ammo acid according to the sequence (leucine) was coupled as preformed symmetrical anhydride as described above and the reaction was continued for 2 h. Excess reagent was then removed by DMF washing (12 min flow rate 1 ml/mm) . A small resm-sample was removed m order to check the coupling yield, which was estimated as described above, and found to be -100%.
  • the following ammo acid according to the sequence was coupled as preformed Fmoc- protected HObt esters (3 eq.) in DMF (5 ml) generated by means of DIC.
  • the resm was washed with a solution of Dhbt-OH (80 mg in 25 ml), in order to follow the disappearance of the yellow colour as the coupling reaction proceeded. When the yellow colour was no longer visible the couplings were interrupted by washing the res with DMF (5 5 ml, 5 mm each) . The acylations were then checked by the ninhydrin test performed at 80°C as earlier described. After completed synthesis the peptide-res was washed with DMF (3x5 ml, 1 mm each), DCM (3x5 ml, 1 mm each), diethyl ether (3x5 ml, 1 mm each) and dried m vacuo .
  • DMF 3x5 ml, 1 mm each
  • DCM 3x5 ml, 1 mm each
  • diethyl ether 3x5 ml, 1 mm each
  • the peptide was cleaved from the resm as described above freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 90% and the identity of the peptide was confirmed by ES-MS. Yield 83%. 7.
  • the next amino acid according to the sequence (leucine) was coupled as preformed symmetrical anhydride as described above and the reaction was continued for 2 h. Excess reagent was then removed by DMF washing (12 mm flow rate 1 ml/mm) . A small resm-sample was removed in order to check the coupling yield, which was estimated as described above, and found to be 90%.
  • the following ammo acid according to the sequence was coupled as preformed Fmoc-protected HObt esters (3 eq.) in DMF (5 ml) generated by means of DIC.
  • the resm was washed with a solution of Dhbt-OH (80 mg in 25 ml) , in order to follow the disappearance of the yellow colour as the coupling reaction proceeded. When the yellow colour was no longer visible the couplings were interrupted by washing the resm with DMF (5 x 5 ml, 5 mm each) . The acylations were then checked by the ninhydrin test performed at 80°C as earlier described. After completed synthesis the peptide-resm was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 mm each), diethyl ether (3x5 ml, 1 mm each) and dried m vacuo.
  • DMF 3x5 ml, 1 min each
  • DCM 3x5 ml, 1 mm each
  • diethyl ether 3x5 ml, 1 mm each
  • the peptide was cleaved from the resm as described above freeze dried from ammonium hydrogencarbonate (0.1 M) .
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 90% and the identity of the peptide was confirmed by ES-MS. Yield 71%. 8. Peptide synthesis of H-Tyr-Gly-Gly-Phe-Leu-Lys 6 -OH on NovaSyn TentaGel.
  • Dry NovaSyn TG resin (0.29 mmol/g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batchwise peptide synthesis on PEG-PS" until finishing the pre-sequence Lys 6 .
  • the following amino acids forming the Leu-enkephalin sequence were coupled as preformed Fmoc-protected HObt esters (3 eq.) in DMF (5 ml) generated by means of DIC. Before each of the last five couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), in order to follow the disappearance of the yellow colour as the coupling reaction proceed.
  • the peptide was cleaved from the resin as described above and freeze dried from acetic acid.
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 98% and the identity of the peptide was confirmed by ES-MS. Yield 84%.
  • Dry NovaSyn TG resin (0.29 mmol/g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batchwise peptide synthesis on PEG-PS" until finishing the pre-sequence Glu fe .
  • the following amino acids forming the DSIP sequence were coupled as preformed Fmoc-protected HObt esters (3 eq.) in DMF (5 ml) generated by means of DIC. Before each of the last nine couplings the resm was washed with a solution of Dhbt-OH (80 mg m 25 ml ) , in order to follow the disappearance of the yellow colour as the coupling reaction proceeds.
  • the peptide was cleaved from the resm as described above and freeze dried from acetic acid.
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 98% and the identity of the peptide was confirmed by ES-MS . Yield 80%.
  • the peptide-resm was washed with DMF (3x5 ml, 1 mm each), DCM (3x5 ml, 1 mm each), diethyl ether (3x5 ml, 1 mm each) , and dried m vacuo .
  • the peptide was cleaved from the resm as described above and freeze dried from acetic acid.
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 98% and the identity of the peptide was confirmed by ES-MS. Yield 91%.
  • the peptide-resm was washed with DMF (3x5 ml, 1 mm each) , DCM (3x5 ml, 1 mm each) , diethyl ether (3x5 ml, 1 mm each) , and dried m vacuo .
  • the peptide was cleaved from the resm as described above and freeze dried from acetic acid.
  • the crude freeze dried product was analysed by HPLC and found to be homogeneous without deletion and Fmoc-protected sequences. The purity was found to be better than 98% and the identity of the peptide was confirmed by ES-MS . Yield 78%.
  • the decomposition of the peptide prodrug (X-L-Z) and the corresponding peptide (X-OH) is studied at 37°C in a 0.05 M phosphate buffer solution.
  • the buffer solutions contains leucine ammopeptidase (25 u/ml) at pH 7.4, or carboxypeptidase A (25 u/ml) at pH 7.4.
  • the decomposition is initiated by addition of an aliquot ( ⁇ 10 ⁇ 7 -10 ⁇ 8 mol) from a stock solution of the peptide or peptide prodrug, respectively, to the test solution giving a total volume of ⁇ 5 ml reaction mixture which is kept in a water-bath at 37°C.
  • the rates of decomposition were determined by using reversed phase HPLC.
  • the mobile phase systems used for isocratic separation were 20% acetonitrile 79.9% water 0.1% trifluoro- acetic acid or 10% acetonitrile 89.9% water 0.1% trifluoro- acetic acid.
  • buffer A was 0.1% TFA in water v/v and buffer B was 90% acetonitrile 9.9% water 0.1% TFA v/v.
  • the decomposition of the peptides and the peptide prodrugs were studied at 37°C in a 0.05 M phosphate buffer solution containing leucine a inopeptidase (25 u/ml) at pH 7.4, carboxypeptidase A (25 u/ml) at pH 7.4, ⁇ -chymotrypsin (25 u/ml) at pH 7.4, pepsin A (25 u/ml) at pH 2.0, or butyryl cholinesterase (at two concentrations: 25 and 50 u/ml) at pH 7.4.
  • the decomposition was initiated by adding an aliquot ( ⁇ 10 ⁇ '-10 ⁇ 8 ol) from a stock solution of the peptide or peptide prodrug to the test solution giving a total volume of ⁇ 5 ml reaction mixture which was kept in a water-bath at 37°C and at appropriate intervals samples of ⁇ 50 ⁇ l were withdrawn and analysed by reversed phase HPLC as described above without previous precipitation of proteins. Pseudo-first-order rate constants for the degradations were determined from the slopes (i.e.
  • the decomposition of the peptides and the peptide prodrugs were studied at 37°C in 80% human plasma.
  • the decomposition was initiated by adding an aliquot ( ⁇ 10 " '-10 ⁇ 8 mol) from a stock solution of the peptide to the test solution giving a total volume of ⁇ 5 ml reaction mixture which was kept in a water-bath at 37°C and at appropriate intervals samples of ⁇ 50 ⁇ l were withdrawn and the samples were treated with 50 ⁇ l of 2% (w/v) solution of zinc sulphate in methanol-water (1:1 v/v) to deproteinize the samples and stop the reactions. After immediate centrifugation for 3 min. at 13000 rpm.
  • the peptide was characterised as stable. Approximately 15% of the peptide was degraded over a period of 24 h.
  • H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH ⁇ 10 "5 M
  • the half-life was calculated to be less than 20 min.
  • ⁇ -opioid receptor acti vi ty - 1 The affinity of the prodrugs of the invention for the ⁇ -opioid receptor in calf brain was determined as described by Kristensen et al . (1994) [K. Kristensen, C.B. Christensen, L.L. Christrup, and L.C. Nielsen (1994) . The mu ⁇ , mu ? , delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci . 56, PL45-PL50.]. The activity of the prodrugs was determined in freshly made solutions and m solutions stored for 20 h at room temperature. The experimental data are summarised in Table 1.
  • the affinity of the prodrugs of the invention as ⁇ -opioid receptor agonists was determined using the isolated mouse vas deferens m vi tro model described by Kramer et al . (1997) [T.H.
  • % reduction at 100 nM a: ⁇ 25%; aa: ⁇ 50%; aaa: ⁇ 75%
  • NT Not tested.
  • NA Not active.
  • WA Weakly active at 20 mg/kg.
  • the prodrugs of the invention have a reduced affinity towards the ⁇ -opoid receptor compared to native Leu-enkephalm.
  • the prodrug in this case binding to the ⁇ -opoid receptor, the prodrug must be hydrolysed by e.g. blood plasma enzymes such as butyryl cholinesterase in order to release the native pharmaceutically active peptide.
  • the difference between the results obtained from freshly prepared solutions and solutions kept at room temperature for 20 or 48 h, may be due to two different factors:
  • the solutions containing the prodrugs of the invention may be hydrolysed to some extent when stored for 20 or 48 h thereby releasing the native Leu-enkaphalin.
  • the prodrugs of the invention are stable over the entire pH range. The most pronounced effect on standing is observed when applying the pre-sequence (Glu) 6 .
  • Glu pre-sequence
  • a more plausible explanation is the rather low water-solubility of the (Glu) 6 -contammg prodrugs: It is very likely that due to slow solution kinetics only a fraction of the prodrug is dissolved in the freshly prepared solutions. However, when left for 20 or 48 h in solution the compounds will slowly dissolve and thereby increasing the available amount of active substance in the assays .
  • the pre-sequence as well as the linker is of importance.
  • the positively charged pre-sequence (Lys) m combination with the (S) enantiomer of mandelic acid exhibited the desired effect whereas enkephalin containing the pre- sequence (Lys) 6 in combination with the (R) enantiomer of mandelic acid did not show any activity.
  • enkephalin prodrugs with the electroneutral pre-sequence (LysGlu) 3 whether in combination with (R) or (S) mandelic acid did not show the desired effect.
  • the combination of linker and pre-sequence is of importance in e.g. the ability of the prodrugs of the invention to cross biological barriers such as the blood-brain-barrier, and the present invention opens up the prospect of transporting prodrugs to the desired region by selecting an appropriate combination of linker and pre-sequence .

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  • Animal Behavior & Ethology (AREA)
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  • Veterinary Medicine (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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EP97939975A 1996-09-09 1997-09-09 Peptidische medikament-vorstufen, eine alpha-hydroxysäureartige kopplungsgruppe enthaltend Withdrawn EP0932614A1 (de)

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PCT/DK1997/000376 WO1998011126A1 (de) 1996-09-09 1997-09-09 Peptide prodrugs

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IL128828A0 (en) 2000-01-31
AU724326B2 (en) 2000-09-14
CA2265454A1 (en) 1998-03-19
JP2001505872A (ja) 2001-05-08
WO1998011126A1 (de) 1998-03-19
AU4199497A (en) 1998-04-02
KR20000036015A (ko) 2000-06-26
NZ334595A (en) 2000-08-25

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