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WO1993009134A1 - Pseudopeptides contenant des esters de phosphonamidate - Google Patents

Pseudopeptides contenant des esters de phosphonamidate Download PDF

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Publication number
WO1993009134A1
WO1993009134A1 PCT/US1992/009834 US9209834W WO9309134A1 WO 1993009134 A1 WO1993009134 A1 WO 1993009134A1 US 9209834 W US9209834 W US 9209834W WO 9309134 A1 WO9309134 A1 WO 9309134A1
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Prior art keywords
amino acid
peptide
sequence
group
acid residue
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Kim Janda
Peter Wirshing
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Scripps Research Institute
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/14—Angiotensins: Related peptides
    • 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 linkage units for joining peptide sequences and to the use of such linkage units for forming pseudopeptides, including pseudopeptides that inhibit aspartic proteinase enzymes. More
  • the invention relates to pseudopeptides that include a "capped” phosphonamidate ester linkage which is acid stable and which is substantially isosteric and isocoulombic with respect to conventional peptides and to the use of such "capped" phosphonamidate ester linkage units in place of amide bonds at the position in a peptide sequence that is cleavable by aspartic proteinase enzymes.
  • Aspartic proteinase enzymes (EC 3.4.23] are a family of related enzymes that cleave (hydrolyze) protein and polypeptide chains. These enzymes have isoelectric points on the acid side of neutrality and molecule masses ranging from 35,000-45,000 Daltons (D) for fungal enzymes and about 35,500 D for pepsin.
  • Exemplary enzymes of this class include pepsin that is a mammalian gastric proteinase, cathepsin D that is the intracellular aspartic proteinase of the lysosomal system and whose level has been positively correlated with
  • angiotensinogen to form angiotensin I, and chymosin
  • Penicillopepsin a microbial enzyme from P. janthinellum is another member of this family, whereas nepenthesin, the digestive proteinase of the pitcher plant is exemplary of the plant aspartic proteinases.
  • hydrolysis of the peptide bond that is hydrolyzed is typically between hydrophobic residues.
  • a covalent intermediate is not thought to be formed between this enzyme and its substrate as is the case with the serine proteinase family.
  • This family of enzymes forms an enzyme-substrate complex as is typical in enzyme-substrate reactions.
  • Binding is often found to be a two-step process even through no covalent bonds are formed.
  • inhibitors include polypeptides similar in sequence to a natural substrate of an enzyme that also include one or more D-amino acids in place of the naturally occurring L-amino acids.
  • Another group of inhibitors contains the surrogate (3S,4R)-4-amino-3-hydroxy-6-methylheptanoic acid, designated AHMHA or statine (Sta), in place of the two residues between which the hydrolysis occurs, such as Leu and Ala.
  • the statine-containing group of inhibitors were first found in the naturally occurring inhibitor known as pepstatin A that inhibits each of pepsin cathepsin D with a K i value of about 10 -10 -10 -11 M and renin with a K i value of 10 -6 M.
  • a phosphonate group has the linkage -P(O)(OH)O-, in which the shown valence of the phosphorus atom is bonded to a carbon and takes the place of an amide carbonyl group, and the free valence of the oxygen is bonded to a carbon atom, taking the place of the amido -NH- group.
  • phosphinate group has the linkage -P(O) (OH)-, so that the phosphorus atom is bonded to two carbon atoms.
  • phosphinamide has the linkage -P(O)(NH 2 )- in which the -NH 2 group replaces the -OH of a phosphinate.
  • the present invention contemplates a linkage unit for joining two peptide sequences, i.e. an amino terminal peptide sequence and a caiboxyl terminal peptide sequence.
  • the linkage unit includes a "capped” surrogate amino acid residue.
  • the "capped” surrogate amino acid residue has a backbone substantially isosteric with a peptide backbone. However, the backbone of the "capped” surrogate amino acid residue lacks a carbonyl group. Instead, "capped"
  • surrogate amino acid residue includes a -P(O)(OR)- group which substitutes for the lacking carbonyl group.
  • R may be a C 1 -C 6 alkyl group, but is preferably a methyl group.
  • the "capped” surrogate amino acid residue lacks an ability to form a backbone peptide bond with the amino end of the carboxyl terminal peptide sequence.
  • the "capped surrogate amino acid residue forms a phosphonamide C 1 -C 6 alkyl ester linkage with the amino end of the carboxyl terminal peptide sequence, i.e. -P(O)(OR)-N(H)-.
  • the phosphonamide C 1 -C 6 alkyl ester linkage is substantially acid stable.
  • the invention also contemplates an improved dipeptide or oligopeptide having a "capped" surrogate amino acid residue as described above and a second amino acid residue.
  • the capped surrogate amino acid residue forming a
  • the oligopeptide or polypeptide may include an amino terminal peptide sequence, a carboxyl terminal peptide sequence, and a "capped” surrogate amino acid residue as described above.
  • the "capped surrogate amino acid residue joins the amino terminal peptide sequence and the carboxyl terminal peptide sequence.
  • the amino terminal peptide sequence may be linked to the "capped” surrogate amino acid residue by means of a conventional peptide bond.
  • the amino end of the carboxyl terminal peptide sequence is linked to the "capped” surrogate amino acid residue by means of the phosphonamide C 1 -C 6 alkyl ester linkage described above.
  • the invention also contemplates a method for linking a "capped” surrogate amino acid residue with a second amino acid residue or to an amino end of a carboxyl terminal peptide sequence.
  • the method includes a step for linking the amino end of the second amino acid residue (or the amino end of a carboxyl terminal peptide sequence) to the capped surrogate amino acid by means of a phosphonamide C 1 -C 6 alkyl ester linkage.
  • the method may also include a step for linking the "capped" surrogate amino acid residue or linkage unit to the carboxyl end of the amino terminal peptide sequence by means of a
  • the present invention also contemplates an inhibitor for an aspartic proteinase enzyme.
  • the inhibitor is a pseudopeptide analog that includes a phosphonamidate ester linkage in place of the amide bond at the position in the pseudopeptide sequence that is cleaved by the enzyme.
  • inhibitor of the invention is cleaved by an aspartic proteinase.
  • a pseudopeptide aspartic proteinase inhibitor has a length of 4 to about 15 amino acid residues, preferably 4 to about 10 residues, and contains a P 1 to P 1 ' bond that is constituted by a phosphonamidate C 1 -C 6 alkyl ester in which the phosphorus atom is bonded to P 1 in place of the carbonyl carbon atom of a peptide bond.
  • the C 1 -C 6 alkyl group is preferably methyl (C 1 ).
  • R 1 -X 1 -Xaa [P(O)(OC 1 -C 6 alkyl)NH]X 2 -Z
  • X 1 is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid
  • Xaa is a surrogate amino acid residue having an amino acid side chain
  • X 2 is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues
  • ⁇ [P(O) (OC 1 -C 6 alkyl)NH] represents the phosphonamide C 1 -C 6 alkyl ester bond between X aa and X 2 .
  • the -P(O) (OC 1 -C 6 alkyl)- portion of the bond belongs to X 1 and constitutes a substitution for a carbonyl group.
  • the -NH- portion of the bond belongs to X 2 and constitutes the amino end of that amino acid residue or oligopeptide;
  • Z is selected from the group consisting of NH 2 , NH-C 1 -C 6 acyl, OH, O-C 1 -C 6 alkyl and 2-amidoindanol;
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 6 acyl, trifluoroacetyl and t-BOC.
  • the pseudopeptide has the length of 4 to about 15 amino acid residues.
  • the pseudopeptide has a length of 4 to about 10 amino acid residues.
  • the C 1 -C 6 alkyl group of the phosphonamide ester is preferably methyl.
  • pseudopeptide preferably competitively inhibits the in vitro activity of an aspartyl proteinase with an inhibition constant of about 10 -6 to about 10 -11 M, and more preferably about 10 -8 to about 10 -10 M.
  • Xaa has the side chain of a
  • a pharmaceutical composition that contains an above pseudopeptide in an amount sufficient to inhibit an
  • aspartic proteinase dissolved or dispersed in a physiologically tolerable carrier or diluent is also contemplated.
  • an aspartic proteinase is admixed in an aqueous medium with the enzyme and a substrate for the enzyme to form an inhibition mixture.
  • the inhibition mixture so formed is maintained for a time sufficient for the aspartic proteinase activity of the enzyme to be inhibited.
  • a compound of the invention is depicted using the "psi bracket" ( ⁇ [ ] ) nomenclature for oligopeptide analogs having backbone modification described in Spatola, Chemistry and Biochemistry of: Amino Acids, Peptides and Proteins,
  • brackets , [ ] the presence of brackets , [ ] , adj acent the ⁇ symbol and between residues, coupled with a structural group within the brackets indicates that the specified structural group within the brackets replaces the peptidyl amide bond;
  • the word "surrogate” refers to an unnatural replacement for a naturally occurring entity, so that a psi-bracketed structural group is a surrogate for the peptidyl amide bond as is the residue containing the bond surrogate a surrogate for an amino acid residue;
  • pseudopeptide refers to a peptide analog having a peptide backbone modification
  • a "pseudodipeptide” is a modified dipeptide structural unit that contains a surrogate bond(s) or amino acid residue(s);
  • a compound of this invention contains a pentavalent, tetrahedral phosphorus atom as part of a phosphonamidate C 1 - C 6 ester surrogate for a peptidyl amide bond. That linkage is thus written ⁇ [P(O) (OC 1 -C 6 alkyl)NH] following the convention of parenthesizing carbonyj. oxygen atoms when depicted in single line notation.
  • oligopeptide is used in its usual sense to mean a peptide containing ten or fewer amino acid residues.
  • oligopseudopeptide refers to a pseudopeptide containing ten or fewer amino acid residues and surrogates therefor.
  • oligopeptide that is cleaved is thus defined as P 1 -P 1 '.
  • the Schechter and Berger nomenclature system is utilized whether or not the bond linking the P 1 and P 1 ' residues is a peptide bond or is capable of hydrolytic cleavage, and is therefore useful with pseudopeptides where P 1 -P 1 '
  • the present invention relates to pseudopeptide
  • a contemplated oligopeptide analog inhibits that enzymatic activity in the presence of a substrate for the enzyme and is therefore a competitive inhibitor.
  • a compound contemplated herein is referred to as a pseudopeptide because although most of the subunit amino acids are linked by peptidyl amide bonds, two such residues are linked by a phosphonamidate C 1 -C 6 ester.
  • Compounds containing peptide bonds and other bonds linking moieties having amino acid side chains are also sometimes referred to as peptidomimetic compounds.
  • a contemplated pseudopeptide reversibly binds to an aspartic proteinase to form an enzyme-inhibitor complex.
  • Such binding and complex formation are familiar to those skilled in enzyme kinetics, and are to be distinguished from the interactions of materials that irreversibly bind to and react with an enzyme that are sometimes referred to as "suicide inhibitors".
  • suicide inhibitors a contemplated
  • pseudopeptide binds to (or is bound by) an aspartic
  • Phosphonamidate-containing compounds have been used as transition state analog inhibitors for metallopeptidases such as carboxypeptidase A, thermolysin and angiotensin converting enzyme (ACE). See, for example Rich, Peptidase Inhibitors. Comprehensive Medicinal Chemistry, Chapter 8.2, Sammes, ed., Pergamon Press, Oxford, Volume 2 (1990).
  • ACE angiotensin converting enzyme
  • a number of phosphonamidate S- and O-esters have been investigated as irreversible phosphorylating agents of serine proteases. See, for example, Sampson et al.,
  • Metallopeptidases and serine proteases act on their substrates in a different manner than do aspartic
  • phosphonamidates are unstable under the acid pH conditions at which aspartic proteinase enzymes act and are therefore poor candidates for use in inhibitors.
  • the phosphonamidate esters contemplated, herein are stable at the acidic pH values at which aspartic proteinases act.
  • a phosphonamidate ester is not susceptible to acid
  • a phosphonamidate ester group contemplated herein is also electrically neutral (is free from ionic charge) at pH values encountered in living organisms and as such contributes to passage of an pseudopeptide through cell membranes.
  • a contemplated pseudopeptide aspartic proteinase inhibitor can have a length of 4 to about 15, and more preferably 4 to about 10, amino acid residues and has a P 1 to P 1 ' bond surrogate that is constituted by a
  • the phosphonamidate C 1 -C 6 alkyl ester in which the phosphorus atom is bonded to a carbon at P 1 in place of the carbonyl carbon atom of a peptide bond.
  • the C 1 -C 6 alkyl ester is preferably methyl (C 1 ).
  • Such an inhibitor typically inhibits the in vitro activity of an aspartic proteinase with an inhibition constant, K i , of about 10 -6 to about 10 -11 M.
  • K i an inhibition constant
  • the inhibition constant is readily ascertained in the presence of a usual, native substrate for the enzyme as discussed hereinafter.
  • the residue length of a contemplated inhibitor is determined as if the P 1 -P 1 ' positions were linked by a peptide bond.
  • the P 1 position surrogate residue is thus considered for this purpose to be an amino acid residue even though the carboxyl group normally present is replaced by a tetrahedral phosphorus-containing moiety.
  • a particularly preferred contemplated pseudopeptide has the formula
  • R 1 -X 1 -Xaa [P(O) (OC 1 -C 6 alkyl)NH]X 2 -Z
  • X 1 is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid
  • Xaa is a surrogate amino acid residue having an amino acid side chain
  • X 2 is an amino acid residue or oligopeptide containing a sequence of up to about ten amino acid residues
  • Z is selected from the group consisting of NH 2 , NH-C 1 -C 6 acyl, OH, O-C 1 -C 6 alkyl and 2-amidoindanol;
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 6 acyl, trifluoroacetyl and t-BOC.
  • a particularly preferred pseudopeptide has a length of 4 to about 15 amino acid residues, and competitively
  • Exemplary C 1 -C 6 acyl groups of R 1 and Z include formyl, acetyl, propionyl, butanoyl, iso-butanoyl, isovaleryl
  • a t-BOC group is a tertiary-butyloxy carbonyl group as is often used as a protecting group in solid phase peptide
  • Exemplary C 1 -C 6 alkyl groups of Z or the C 1 -C 6 alkyl ester portion of the phosphonamide ester include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, 2-methylbutyl, cyclopentyl, hexyl,
  • Methyl is a preferred C 1 -C 6 alkyl ester group.
  • the group Xaa is a surrogate amino acid residue having the side chain of an amino acid.
  • Xaa can have any side chain of one of the twenty naturally occurring amino acids, Xaa preferably has the side chain of a Leu, Tyr or Phe amino acid.
  • the Xaa group is in the P 1 position of a contemplated pseudopeptide.
  • Each of X 1 and X 2 can be an amino acid residue or an oligopeptide of up to about ten, and preferably up to about five amino acid residues.
  • the sequences of X 1 and X 2 can contain any amino acid residues.
  • the amino-terminal residue of X 2 (the P 1 ' position residue) is
  • the overall length of a pse ⁇ dopeptide, aside from an R 1 or Z group, is 4 to about 15 amino acid residues, and more preferably 4 to about 10 amino acid residues.
  • each of X 1 and X 2 can separately be up to about 10 amino acid residues in length, both cannot include 10 amino acid residues .
  • X 1 can include 10 amino acid residues (occupying positions P 2 -P 11 of the sequence) and X 2 can include four residues (occupying positions P 1 '-P 4 ' of the sequence). X 2 can similarly include 10 residues with X 1 including 4 residues.
  • any amino acid residue can be present in X 1 and X 2
  • reactive side chains such as the mercaptan of cysteine, carboxyl groups such as those of aspartic and glutamic acids, and the ⁇ -amino group of a lysine are typically absent from X 1 and X 2 sequences, and from Xaa group side chains.
  • Those reactive side chains are also relatively hydrophilic. This absence of hydrophilic side chains is also a function of the active site of this family of enzymes exhibiting a preference for relatively
  • hydrophobic side chains particularly for the P 1 and P 1 ' positions.
  • An amino acid residue of X 1 and X 2 or side chain of Xaa can be present in an oligopeptide analog sequence in either a D- or L-configuration, as is exemplified below wherein all residues are in the L-configuration unless preceded by a "D-". Not only are both D- and L-amino acid residues contemplated for use in an oligopeptide analog, but
  • carboxylic acids are also contemplated, particularly at or adjacent the amino- and carboxy-termini of an pseudopeptide.
  • 2,2-dimethylglycine residue can be present at either or both termini, or a 2-aminoindanol can be amide-bonded to a carboxy-terminal residue to form a 2-amidoindanol group.
  • a C 1 -C 6 acyl group as discussed previously such as 3-methylbutanoyl (isovaleryl; Iva) can be useful at the amino-terminus, whereas 3-methylbutylamine reacted at the carboxy-terminus to form a 3-methylbutylamide
  • the above terminal substituent groups and modified amino acid residues serve at least two functions. First, their presence removes ionic charge from the pseudopeptide to help facilitate passage through membranes. Second, they help protect the pseudopeptide from degradation by other proteinase and peptidase enzymes such as trypsin and carboxypeptidase A that are present in vivo and can
  • a contemplated pseudopeptide binds to an aspartic proteinase and inhibits the in vitro activity of the enzyme with an inhibitory constant, K i , of about 10 -6 to about 10 -11 , and preferably about 10 -8 to about 10 -10 , in the presence of a usually assayed native substrate.
  • K i inhibitory constant
  • the sequence of a contemplated pseudopeptide is that of an oligopeptide or oligopeptide analog that is reversibly bound by a given aspartic proteinase, except for the phosphonamidate ester linkage as is illustrated before.
  • the K i value of a contemplated inhibitor can also be viewed relative to the dissociation constant of a usual, native substrate for the enzyme, as is angiotensinogen for renin.
  • a contemplated inhibitor binds to its aspartic proteinase about 10 5 to about 10 6 times more tightly than does the usual, native substrate.
  • dissociation constant for the enzyme and its native substrate is about 10 -3
  • dissociation constant for the same enzyme and a contemplated inhibitor is about 10 -8 to about 10 -9 .
  • inhibitor sequences are utilized with different members of the aspartyl proteinase family. Each of those different sequences nevertheless contains a phosphonamidate C 1 -C 6 ester link between the P 1 and P 1 ' residues of the inhibitor.
  • Table 1 below lists illustrative members of the aspartic proteinase enzyme family and exemplary inhibitors for each listed enzyme.
  • molecule's length is constituted by oligopeptides.
  • One preferred method utilizes a first preprepared oligopeptide for the P 2 -containing portion (X 1 -Xaa) and a second
  • a completed pseudopeptide synthesized as described above can also be separated into diastereomers and then resolved.
  • the illustrated t-BOC group is removed, and a salt is formed with R-tartaric acid.
  • the resulting diastereomers can be separated chromatographically and then resolved to enantiomeric purity. Resolution can also be achieved using a brucine salt of a carboxy-terminal
  • the before-described preprepared oligopeptides can be prepared by any well known means.
  • the solid phase techniques pioneered by Merrifield are preferred and are well known by those skilled in the art.
  • R 1 group that is a C 1 -C 6 acyl group can be added to the P 2 -containing peptide portion (X 1 -Xaa) while that peptide is on its synthesis resin.
  • a trifluoroacetyl or t- BOC group is preferably added after cleavage of the peptide from its synthesis resin.
  • a Z group is preferably added to its P 1 '-containing peptide portion (X 2 ) after the peptide is cleaved from the resin.
  • Well known ester- and amide-forming reactions are used for addition of a Z group, and as such reactions are so well known, they will not be dealt, with herein.
  • a pharmaceutical composition is also contemplated that contains a before-described pseudopeptide of the invention as active agent dissolved or dispersed in a physiologically tolerable carrier or diluent.
  • a pseudopeptide inhibitor is present in such a composition in an amount sufficient to inhibit the aspartic proteinase activity of a chosen aspartic proteinase (an effective inhibitory amount).
  • a pharmaceutical composition is prepared by any of the methods well known in the art of pharmacy all of which involve bringing into association the pseudopeptide active agent and the carrier therefor.
  • a pseudopeptide utilized in the present invention can be administered in the form of conventional pharmaceutical compositions.
  • Such compositions can be formulated so as to be suitable for oral or parenteral administration, or as suppositories.
  • the agent is
  • a physiologically tolerable carrier or diluent typically dissolved or dispersed in a physiologically tolerable carrier or diluent.
  • a carrier or diluent is a material useful for
  • physiologically tolerable or “pharmaceutically acceptable” are used interchangeably and refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal.
  • the physiologically tolerable carrier can take a wide variety of forms depending upon the preparation desired for administration and the intended route of administration.
  • a compound of the invention can be utilized, dissolved or dispersed in a liquid composition such as a sterile
  • injectable media constituted by aqueous injectable buffered or unbuffered isotonic and sterile saline or glucose solutions, as well as water alone, or an aqueous ethanol solution.
  • injectable media constituted by aqueous injectable buffered or unbuffered isotonic and sterile saline or glucose solutions, as well as water alone, or an aqueous ethanol solution.
  • Additional liquid forms in which these compounds can be incorporated for administration include flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar
  • liposomes are generally derived from phospholipids or other lipid
  • Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, pharmaceutically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • An active agent can also be used in compositions such as tablets or pills, preferably containing a unit dose of the compound.
  • the agent active ingredient
  • conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic, physiologically tolerable carriers.
  • the tablets or pills can be laminated or
  • the pharmaceutical composition described herein can include, as appropriate, one or more additional carrier ingredients such as
  • diluents diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives
  • the tablets or pills can also be provided with an enteric layer in the form of an envelope that serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the like.
  • enteric coatings including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the like.
  • unit dose refers to physically discrete units suitable as unitary dosages for administration to warm blooded animals, each such unit containing a predetermined quantity of the agent calculated to produce the desired therapeutic effect in association with the pharmaceutically acceptable diluent.
  • suitable unit dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and the like.
  • a pseudopeptide of the invention is present in such a pharmaceutical composition in an amount effective to achieve the desired inhibition.
  • a compound of the invention can be utilized in an amount sufficient to provide a concentration of about 0.01 to about 2,000 nanomolar (nM) with enzyme concentration of about 1 nM to about 1 ⁇ M, and a substrate concentration of about 10 to about 2,000 micromolar ( ⁇ M).
  • the amounts of enzyme, substrate and inhibitor used are largely a function of convenience, with the substrate typically being in large excess over the enzyme (e.g. 100-10,00 fold excess).
  • an effective amount of a compound of the invention is about 0.1 to about 50 mg per kilogram of body weight or an amount sufficient to provide a concentration of about 0.01 to about 100 ⁇ g/mL to the bloodstream.
  • a method of inhibiting an aspartic proteinase is also contemplated.
  • a pharmaceutical composition as discussed before that contains an aspartic proteinase inhibiting amount of a before-discussed pseudopeptide is admixed in an aqueous medium with an aspartic proteinase in the presence of a substrate for the enzyme to form an inhibition mixture.
  • the inhibition mixture is maintained for a time period sufficient for the inhibitor to inhibit the aspartic proteinase, typically five minutes to five hours.
  • the inhibition reaction is typically followed spectrophotometrically.
  • the aqueous medium in such a case is typically a buffer solution.
  • the aqueous medium is constituted by a body fluid such as blood, lymph, stomach fluid or the like, and inhibition of the enzyme is assayed by a body function, as by blood pressure lowering for renin.
  • a body fluid such as blood, lymph, stomach fluid or the like
  • inhibition of the enzyme is assayed by a body function, as by blood pressure lowering for renin.
  • Porcine stomach mucosa pepsin (Sigma Chemical Co.) is chromatographically purified and is dissolved and diluted immediately prior to use in a 0.1 M sodium acetate buffer at pH 3.5.
  • a useful substrate is the octapeptide analog:
  • Substrate hydrolysis is measured by observing the decrease in absorbance at 310 nm. Initial rates are measured from 0.5 minutes until no more than 10 percent of the substrate is hydrolyzed. Inhibition constants are then determined by usual means.
  • incubation mixture (100 ⁇ L) contains 0.135 M maleate buffer at pH 6.0, EDTA at 3 mM, phenylmethanesulfonyl fluoride at 1.4 mM, 0.21 ⁇ M angiotensinogen, 0.24 mGU [Bangham et al., Clin. Sci. Mole. Med., 48:1355 (1975)], BSA 0.44 percent and DMSO at 1 percent.
  • inhibitor pseudopeptide that bracket the IC 50 value (the concentration that inhibits 50 percent activity) are preincubated with renin for about five minutes at 37°C. The substrate is then added and incubation is continued for about 10 minutes. The reaction is stopped by freezing the solution in a methanol/dry ice bath, and after thawing at 4°C, an aliquot is analyzed for angiotensin I by use of a commercial kit (NEN Research). The percent inhibition of the hydrolysis reaction is determined and an IC 50 value is calculated by regression analysis.
  • hypotensive activity of an inhibitor pseudopeptide in anesthetized, sodium depleted marmosets is used in this assay.
  • the fall in mean arterial pressure (MAP) over a two-hour time period is used as the assayed value.
  • An internal standard using a known hypotensive drug such as captopril (at 1.0 mg/kg, iv) or the compound designated CGP 385-60A [Buhlmayer et al., J. Med. Chem., 31:1839 (1988); DeGasparo et al., J. Clin. Pharmac., 27:587 (1989)] as used in this model can also be used.
  • inhibitor is provided in a pharmaceutical composition at about 1-5 mg/kg.
  • amounts on the order of 10-100 mg/kg are utilized.
  • the marmosets used for the study are depleted of sodium for two days by treatment with furosemide at 25 mg/kg/day po and a low sodium diet.
  • a final dose of furosemide is administered one hour prior to anesthesia with Inactin at 120 mg/kg intraperitoneally (ip) followed by a 10 mg/kg/hour iv maintenance infusion.
  • Blood pressure is measured from a catheter in the carotid artery via a Gould Stratham P23 pressure transducer and Lectromed M19 chart recorder.
  • the jugular vein is cannulated for inhibitor injection and anesthetic infusion.
  • Each animal serves as it own blood pressure control.
  • Xaa is a Phe whose carbonyl group is replaced by P(O)(OCH3) group.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Une unité 'coiffée' de liaison à ester d'alkyle C1-C6 de phosphonamide est utile pour relier des peptides. L'unité de liaison est essentiellement isotérique et isocoulombienne par rapport aux squelettes peptidiques classiques. Le fait que le phosphonamide est 'coiffé' par l'ester d'alkyle C1-C6 rend l'unité de liaison stable aux acides. Lorsqu'elle est incorporée à un peptide, l'unité de liaison à ester d'alkyle C1-C6 de phosphonamide devient résistante au clivage par la protéinase aspartique. Par conséquent, l'unité de liaison à ester d'alkyle C1-C6 de phosphonamide améliore et/ou facilite l'activité d'inhibition de la protéinase aspartique des peptides auxquels elle est incorporée.
PCT/US1992/009834 1991-11-06 1992-11-06 Pseudopeptides contenant des esters de phosphonamidate Ceased WO1993009134A1 (fr)

Applications Claiming Priority (2)

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US78991691A 1991-11-06 1991-11-06
US789,916 1991-11-06

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WO1993009134A1 true WO1993009134A1 (fr) 1993-05-13

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PCT/US1992/009834 Ceased WO1993009134A1 (fr) 1991-11-06 1992-11-06 Pseudopeptides contenant des esters de phosphonamidate

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AU (1) AU3136693A (fr)
WO (1) WO1993009134A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0163237A2 (fr) * 1984-05-29 1985-12-04 Merck & Co. Inc. Inhibiteurs di- et tri-peptidiques de la rénine
WO1992007869A1 (fr) * 1990-11-06 1992-05-14 Thrombosis Research Institute Inhibiteurs et substrats de thrombine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0163237A2 (fr) * 1984-05-29 1985-12-04 Merck & Co. Inc. Inhibiteurs di- et tri-peptidiques de la rénine
WO1992007869A1 (fr) * 1990-11-06 1992-05-14 Thrombosis Research Institute Inhibiteurs et substrats de thrombine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Bioorganic & Medical Chemistry Letters, Volume 2, No. 9, September 1992, Nicholas P. Camp et al., "Synthesis of stereochemically defined phosphonamidate-containing peptides: inhibitors for the HIV-1 proteinase" *

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AU3136693A (en) 1993-06-07

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