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WO1996039194A1 - Nouvelle cathepsine et procedes et compositions d'inhibition de cathepsine - Google Patents

Nouvelle cathepsine et procedes et compositions d'inhibition de cathepsine Download PDF

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Publication number
WO1996039194A1
WO1996039194A1 PCT/US1996/006211 US9606211W WO9639194A1 WO 1996039194 A1 WO1996039194 A1 WO 1996039194A1 US 9606211 W US9606211 W US 9606211W WO 9639194 A1 WO9639194 A1 WO 9639194A1
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Prior art keywords
carbon atoms
cathepsin
group
compound
nucleic acid
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PCT/US1996/006211
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English (en)
Inventor
Jay S. Tung
Sukanto Sinha
Lisa Mcconlogue
Gwen Tatsuno
John Anderson
Christopher M. F. Semko
Susanna Chrysler
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Athena Neurosciences, Inc.
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Priority claimed from US08/467,607 external-priority patent/US5783434A/en
Priority claimed from US08/469,362 external-priority patent/US5849711A/en
Application filed by Athena Neurosciences, Inc. filed Critical Athena Neurosciences, Inc.
Priority to JP9500507A priority Critical patent/JPH11506923A/ja
Priority to EP96913917A priority patent/EP0831920A4/fr
Publication of WO1996039194A1 publication Critical patent/WO1996039194A1/fr

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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
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to methods and compositions for inhibiting ⁇ -amyloid peptide ( ⁇ AP) production in cells.
  • this invention relates to compounds which are capable of inhibiting the intracellular production of ⁇ AP, and the use of such compounds in methods for inhibiting ⁇ AP production.
  • This invention also relates to an isolated novel protein, Cathepsin Y, which is a novel carboxypeptidase involved in the generation of ⁇ AP. Methods for isolation of this protein are provided. DNA isolates coding for Cathepsin Y and methods of obtaining such DNA are provided, together with expression systems for recombinant production of Cathepsin Y useful in therapeutic or diagnostic compositions.
  • AD Alzheimer's Disease
  • AD is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and, ultimately, death.
  • AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States.
  • AD has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
  • the brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles.
  • senile or amyloid
  • amyloid angiopathy amyloid deposits in blood vessels
  • neurofibrillary tangles Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restricted anatomical distribution are also found in the brains of most aged humans who do not have clinical AD.
  • Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type
  • AD Alzheimer's disease
  • ⁇ AP ⁇ -amyloid peptide
  • APP ⁇ - amyloid precursor protein
  • ⁇ AP is further characterized by its relative mobility in SDS- polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • ⁇ AP can occur in a filamentous polymeric form which exhibits the Congo-red and thioflavin-S dye-binding characteristics of amyloid.
  • ⁇ AP can also occur in a non-filamentous form ("preamyloid" or "amorphous" or
  • APP is normally produced by cells in many tissues of various animals, including humans.
  • APP is encoded by a gene on the long arm of human chromosome 21.
  • Knowledge of the structure of the gene encoding APP has indicated that ⁇ AP arises as a peptide fragment from cleavage of APP by at least one heretofore unidentified protease. This cleavage appears to occur in the lysosomes.
  • the precise biochemical pathway by which the ⁇ AP fragment is cleaved from the APP and subsequently deposited as amyloid plaques is still under investigation.
  • ⁇ AP is released from neural cells grown in culture as well as into cerebral spinal fluid of both normal individuals and AD patients.
  • AD Alzheimer's disease
  • mis-sense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not in unaffected members of several families with a genetically determined (familial) form of AD (Goate et al. , Nature 349:704-706 (1991); Chartier Harlan et al., Nature 353:844-846 (1991); and Murrell et al., (1991) Science 254:97-99).
  • a double mutation changing lysine 595 -methionine 596 to asparagine 595 -leucine 596 (with reference to the 695- amino acid isoform of APP) found in a Swedish family was reported in 1992 (Mullan et al., (1992) Nature Genet 1:345-347) and is referred to as the Swedish variant or mutation.
  • This invention is directed, in part, to methods for inhibiting ⁇ -amyloid peptide production in cells producing ⁇ -amyloid peptide.
  • the methods of this invention are directed, in part, to the discovery that specific compounds, as defined below, are effective in inhibiting ⁇ -amyloid peptide production in cells expressing ⁇ -amyloid peptide. Because ⁇ -amyloid peptide production is associated with deposition of amyloid plaques in mammals and Alzheimer's disease in humans, the compounds described herein are also useful in inhibiting deposition of such plaques and in treating Alzheimer's disease.
  • This invention is further directed, in part, to the identification of a novel protease, Cathepsin Y, and to nucleic acids which encode this protease. This invention is also directed to methods for the recombinant expression of
  • this invention is directed to a method of inhibiting ⁇ -amyloid peptide production in cells producing ⁇ -amyloid peptide, comprising administering to such cells an inhibitory amount of a compound of formula I:
  • R is selected from the group consisting hydrogen, alkyl of from 1 to 6 carbon atoms, and where R and R 2 are joined to form a ring structure of from 4 to 10 carbon atoms,
  • R' is selected from the group consisting hydrogen, alkyl of from 1 to 6 carbon atoms and where R' and R 3 are joined to form a ring structure of from 4 to 10 carbon atoms,
  • R 1 is selected from the group consisting of
  • substituted alkyl group is optionally further substituted with from 1 to 2 hydroxyl groups
  • aryl of from 6 to 10 carbon atoms substituted with 1 to 3 substituents selected from the group consisting of alkyl of from 1 to 6 carbon atoms, aryl of from 6 to 10 carbon atoms, alkoxy of from 1 to 6 carbon atoms, aryloxy of from 6 to 10 carbon atoms, hydroxy, cyano, halo and amino,
  • fluorenyl heterocycles of from 3 to 14 carbon atoms having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur;
  • R 2 and R 3 are independently D- or L-amino acid side chains of at least 2 carbon atoms with the proviso that said amino acid side chains do not include the proline side chain;
  • R 4 is selected from the group consisting of
  • R 5 is hydrogen, alkyl of from 1 to 6 carbon atoms, haloalkyl of from 1 to 6 carbon atoms and 1 to 2 halo groups, alkoxy of from 1 to 6 carbon atoms, -NR 6 R 7 where R 6 and R 7 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 6 carbon atoms, and -N(CH 3 )OCH 3 ;
  • X is selected from the group consisting of -O-, -NR 9 -, and -S- where R 9 is selected from the group consisting of hydrogen, alkyl of from 1 to 6 carbon atoms and aryl of from 6 to 10 carbon atoms;
  • Y is selected from the group consisting of -C(O)- and -C(S)-;
  • n is equal to zero or one
  • n is equal to zero to two
  • R 4 is not -N(CH 3 )OCH 3
  • R 1 is diphenylmethyl, R 2 is p-(benzyloxy)benzyl (L-isomer), Y is -C(O)-, and m and n are zero, then R 4 is not -N(CH 3 )OCH 3
  • R 1 is diphenylmethyl, R 2 is p-(benzyloxy)benzyl (L-isomer)
  • Y is -C(O)-
  • m and n are zero, then R 4 is not -N(CH 3 )OCH 3
  • R 1 is (1,2- diphenyl)ethenyl
  • Y is -C(O)-
  • R 2 is -CR 2 ⁇ (L-isomer)
  • m and n are zero, the R 4 is not -N(CH 3 )OCH 3 .
  • this invention is directed to a method of inhibiting the deposition of amyloid plaque in a mammal, comprising administering to such a mammal an effective amount of a compound of formula I above.
  • this invention is directed to a method of preventing, treating or inhibiting the onset of Alzheimer's disease (AD) in a mammal which comprises administering to such a mammal an effective amount of a compound of formula I above.
  • AD Alzheimer's disease
  • Preferred compounds for use in the methods described herein include, by way of example, the following compounds as defined by formula II below, including all isomers thereof, wherein the amino acid side chain for R 2 and R 3 is indicated beneath the R 2 and R 3 substituent:
  • this invention is directed to an isolated and purified polypeptide having the enzymatic activity of Cathepsin Y protein.
  • this invention is directed to a purified and isolated nucleic acid sequence which sequence encodes for Cathepsin Y.
  • this invention is directed to a purified and isolated nucleic acid sequence capable of hybridizing to Cathepsin Y comprising:
  • this invention is directed to a method for expressing Cathepsin Y which method comprises transfecting a host cell with a nucleic acid sequence which sequence encodes for Cathepsin Y, culturing the transfected cell under conditions which express Cathepsin Y and recovering Cathepsin Y from the cell culture.
  • this invention is directed to a method of detecting the expression of Cathepsin Y comprising
  • RNA from a mammalian tissue or cell a mammalian tissue or cell
  • RNA a labelled nucleic acid sequence capable of hybridizing to Cathepsin Y comprising i) a nucleic acid sequence substantially homologous to the nucleic acid sequence of FIG. 4, wherein T can also be U, ii) nucleic acid sequences substantially complementary to the sequence of FIG. 4, wherein T can also be U, or iii) fragments of the nucleic acid sequence of FIG. 4, wherein T can also be U or nucleic acid sequences
  • FIGs. 1 and 2 illustrate reaction schemes used to prepare some of the compounds described herein.
  • FIGs. 3A-3C illustrate typical purification profiles, analyzed by Western blotting, of Cathepsin Y.
  • FIG. 4 depicts the amino acid (SEQ ID NO:3) and DNA sequence
  • FIG. 5 shows the restriction map of plasmid poCK751.
  • FIG. 6 shows the restriction map of plasmid poCKcatY.
  • FIG. 7 illustrates a standard OP A curve for fluorescence with varying concentrations of valine.
  • This invention is directed, in part, to the inhibition of ⁇ -amyloid peptide production in cells producing ⁇ -amyloid peptide by administering specific compounds to the cells which inhibition can be employed to retard deposition of amyloid plaques and to treat Alzheimer's disease in mammals.
  • This invention is also directed in part to the identification of a novel protein, Cathepsin Y, and to nucleic acids which encode this protein.
  • This invention is also directed to methods for the recombinant expression of Cathepsin Y.
  • ⁇ -amyloid peptide refers to an approximately 4.2 kD protein which, in the brains of subjects suffering from AD, Down's Syndrome, HCHWA-D and some normal aged subjects, forms a subunit of the amyloid filaments comprising the senile (amyloid) plaques and the amyloid deposits in small cerebral and meningeal blood vessels (amyloid angiopathy).
  • ⁇ AP can occur in a filamentous polymeric form (in this form, it exhibits the Congo-red and thioflavin-S dye-binding characteristics of amyloid).
  • ⁇ AP can also occur in a non-filamentous form ("preamyloid” or “amorphous” or “diffuse” deposits) in tissue, in which form no detectable birefringent staining by Congo red occurs.
  • preamyloid or "amorphous” or “diffuse” deposits
  • Congo red forms no detectable birefringent staining by Congo red occurs.
  • ⁇ AP as used herein specifically refers to an approximately 39-43 amino acid peptide that is substantially homologous to the form of the peptide produced by the method described in the '829 patent, but which can also be found in soluble form in the extracellular fluid (conditioned medium) of cultured cells grown in vitro and in body fluids of humans and other mammals, including both normal individuals and individuals suffering from ⁇ AP-related conditions.
  • ⁇ AP also refers to related ⁇ AP sequences that result from mutations in the ⁇ AP region of the normal gene.
  • ⁇ AP is an approximately 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the ⁇ -amyloid precursor protein (APP), encoded by a gene on the long arm of human chromosome 21.
  • APP ⁇ -amyloid precursor protein
  • ⁇ AP is further characterized by its relative mobility in SDS-polyacrylamide gel electrophoresis or in high performance liquid chromatography.
  • lle 43-amino ⁇ AP acid sequence (SEQ ID No. 1) is:
  • ⁇ AP also refers to sequences that are substantially homologous to this 43-amino acid sequence.
  • ⁇ -amyloid precursor protein as used herein is defined as a polypeptide that is encoded by a gene of the same name localized in humans on the long arm of chromosome 21 which includes ⁇ AP within the carboxyl one-third of its length.
  • APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues.
  • Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide described by Kang et al., Nature 325:733-736 (1987) which is designated as the "normal” APP; the 751-amino acid polypeptide described by Ponte et al., Nature 331:525-527 and Tanzi et al., Nature 331:528-530 (1988); and the 770-amino acid polypeptide described by Kitaguchi et al., Nature 331:530-532 (1988).
  • Examples of specific variants of APP include point mutations which can differ in both position and phenotype (for review of known variant mutations see Hardy, Nature Genet. 1:233-234 (1992)).
  • the term " ⁇ AP-related conditions" as used herein is defined as including Alzheimer's disease (which includes familial Alzheimer's disease), Down's Syndrome, HCHWA-D, and advanced aging of the brain.
  • conditioned culture medium and "culture medium” as used herein refer to the aqueous extracellular fluid which su ⁇ ounds cells grown in tissue culture (in vitro) and which contains, among other constituents, proteins and peptides secreted by the cells.
  • body fluid refers to those fluids of a mammalian host which may contain measurable amounts of ⁇ AP and ⁇ AP fragments, specifically including blood, cerebrospinal fluid (CSF), urine, and peritoneal fluid.
  • CSF cerebrospinal fluid
  • blood refers to whole blood, as well as blood plasma and serum.
  • Swedish mutation refers to a mutation in the human gene encoding APP which results in an inherited, familial form of Alzheimer's disease.
  • the mutation occurs at LYS 595 -MET 596 of the normal APP gene, where a substitution to ASN 595 -LEU 596 occurs. It has been found that human cell lines transfected with this mutation will overproduce ⁇ AP, secreting the ⁇ AP into the conditioned culture medium.
  • heterocycles containing from 3 to 14 carbon atoms and 1 to 3 hetereoatoms selected from the group consisting of nitrogen, oxygen and sulfur refers to saturated and unsaturated heterocyclic groups having the requisite number of carbon atoms and heteroatoms.
  • heterocyclic groups include, by way of example, furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, indolyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g.
  • heterocyclic groups can be substituted or unsubstituted. Where the heterocyclic group is substituted, the substituents are selected from alkyl of from 1 to 6 carbon atoms, alkoxy of from 1 to 6 carbon atoms, aryl of from 6 to 10 carbon atoms, aryloxy of from 6 to 10 carbon atoms, and halo.
  • Preferred heterocycles include well known cyclic aromatic groups containing heteroatoms within the cyclic structure. Such groups include, by way of example, furyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, thiazolyl, and triazolyl.
  • alkyl refers to straight and branched chain alkyl groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, sec-butyl, iso-butyl, n-pentyl, n-hexyl, 2-methylpentyl, and the like; whereas the term “alkyl” refers to straight and branched chain alkyl groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, sec-butyl, iso-butyl, n-pentyl, n-hexyl, 2-methylpentyl, and the like; whereas the term “alkyl” refers to straight and branched chain alkyl groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, ter
  • alkoxy refers to -O-alkyl substituents.
  • aryl refers to aromatic substituents comprising carbon and hydrogen such as phenyl, naphthyl and the like whereas the term aryloxy refers to -O-aryl substituents where aryl is as defined above.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine and preferably fluorine and chlorine.
  • pharmaceutically acceptable salts refers to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art.
  • non-toxic acid addition salts which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.
  • Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, and napsylate salts, and the like.
  • the particular salt employed is not critical.
  • DNA refers to deoxyribonucleic acid.
  • RNA refers to ribonucleic acid.
  • Naturally occurring amino acid residues in peptides described herein are abbreviated as recommended by the IUPAC-IUB Biochemical
  • Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Norleucine is Nle; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • Naturally occurring nucleosides in nucleic acids described herein are abbreviated as recommended by the IUPAC-IUB Biological Nomenclature Commission as follows: Adenosine is A; Guanosine is G; Cytidine is C; Thymidine is T and Uridine is U.
  • the abbreviation where the nucleotides are either Cytidine or Thymidine (Uridine) is Y; Adenosine or Guanosine is R; Adenosine or Thymidine (Uridine) is W; Adenosine or Cytidine is M; and Guanosine or Thymidine (Uridine) is K.
  • Cathepsin Y is defined as a polypeptide that is encoded by a gene of the same name. Cathepsin Y is a
  • carboxypeptidase having a molecular weight of approximately 31 kD.
  • Cathepsin Y is able to cleave carboxy-terminal amino acids, with particular activity against aliphatic carboxy-terminal amino acids.
  • Cathepsin Y is a polypeptide having a qualitative biological activity in common with the Cathepsin Y of FIG. 4 and which is greater than about 70% homologous, more preferably greater than 85% homologous and most preferably greater than 90% homologous with the Cathepsin Y sequence of FIG. 4. It is contemplated that the Cathepsin Y of the present invention may be substantially homologous to the sequence of Fig.
  • Cathepsin Y typically being greater than 90% homologous, preferably greater than 95% homologous and sometime greater than 99% homologous provided that the Cathepsin Y retains at least a portion of biological activity of the Cathepsin Y of Fig. 4.
  • Cathepsin Y proteins having the amino acid sequence as set forth in FIG. 4, deglycosylated or unglycosylated derivatives of the sequence in FIG. 4, and homologous generated variants and derivatives of Cathepsin Y, provided that the modifications do not destroy the biological activity in common with the Cathepsin Y of FIG. 4.
  • “Homologous” is defined as the percentage of residues in the candidate sequence that are identical with the residues in the disclosed sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology.
  • a nucleic acid sequence is substantially homologous to the nucleic acid sequence of the disclosed sequence, where it is greater than 80% homologous, preferably greater than 90% homologous and most preferably greater than 95% homologous.
  • “Complementary” is defined as the ability of a nucleic acid sequence to hybridize to a disclosed nucleic acid sequence.
  • a nucleic acid sequence is “substantially complementary” to the dislosed nucleic acid sequence if the sequence is able to hybridize to greater than 80% of the residues, aligning the sequences and introducing gaps if necessary to achieve maximum complementarity.
  • a substantially complementary sequence is greater than 90%, most preferably it is greater than 95% complementary.
  • Cathepsin Y biological activity is defined as the sequential removal of the carboxy-terminal amino acids from a peptide without endopeptidase activity, one amino acid at a time.
  • transformation refers to introducing DNA into an organism or host cell so that the DNA is replicable, either as an
  • transfection refers to the introduction of DNA into a host cell. It is contemplated that coding sequences may be expressed in transfected cells. Numerous methods of transfection are known to the ordinarliy skilled artisan, for example CaPO 4 and electroporation. II. ⁇ - Amyloid Production Suppressors
  • This invention is based, in part, on the discovery of compounds that have been found to inhibit ⁇ -amyloid ( ⁇ AP) secretion in cells.
  • This invention provides methods for inhibiting ⁇ AP secretion in cells, inhibiting the deposition of plaque and treating Alzheimer's disease.
  • this invention provides a method of inhibiting ⁇ -amyloid production in cells producing ⁇ AP, comprising administering to such cells an inhibitory amount of a compound of formula I:
  • R is hydrogen, alkyl of from 1 to 6 carbon atoms or can be joined with R 2 to form a ring structure of from 4 to 10 carbon atoms and R' is hydrogen, alkyl of from 1 to 6 carbon atoms or can be joined with R 3 to form a ring structure of from 4 to 10 carbon atoms.
  • R and R' in formula I are hydrogen.
  • R 1 can be alkyl of from 1 to 4 carbon atoms substituted with from 1 to 5 substituents selected from the group consisting of aryl of from 6 to 10 carbon atoms, aryl of from 6 to 10 carbon atoms substituted with 1 to 3 substituents selected from the group consisting of alkyl of from 1 to 6 carbon atoms, aryl of from 6 to 10 carbon atoms, alkoxy of from 1 to 6 carbon atoms, aryloxy of from 6 to 10 carbon atoms, hydroxy, cyano, halo and amino, cycloalkyl of from 3 to 8 carbon atoms and heterocycles of from 3 to 14 carbon atoms having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur wherein said substituted alkyl group is optionally further substituted with from 1 to 2 hydroxyl groups,
  • aryl of from 6 to 10 carbon atoms substituted with 1 to 3
  • substituents selected from the group consisting of alkyl of from 1 to 6 carbon atoms, aryl of from 6 to 10 carbon atoms, alkoxy of from 1 to 6 carbon atoms, aryloxy of from 6 to 10 carbon atoms, hydroxy, cyano, halo and amino,
  • R 1 Preferred values for R 1 include benzyl, trityl, diphenylmethyl, 4- phenylbutyl, 2-phenylethyl, naphthyl, pyridyl, fluorenyl, xanthanilyl, and the like.
  • R 2 and R 3 are independently side chains of a D- or L- amino acid having at least 2 carbon atoms with the proviso that R 2 and R 3 are not proline.
  • Such side chains refer to the R 8 substituent found on naturally occurring and synthetic amino acids of the formula H 2 NCHR 8 COOH.
  • Side chains of naturally occurring amino acids include, by way of example only, those where R 8 is the L-isomer of (CH 3 ) 2 CH- (valine), (CH 3 ) 2 CHCH 2 - (leucine), CH 3 CH 2 CH(CH 3 )- (isoleucine), ⁇ CH 2 - (phenylalanine), (3- indolyl)-CH 2 - (tryptophan), CH 3 SCH 2 CH 2 - (methionine), CH 3 CH(OH)- (threonine), p-HO- ⁇ -CH 2 - (tyrosine), H 2 NC(O)CH 2 - (asparagine),
  • HOC(O)CH 2 CH 2 - glutamic acid
  • H 2 NCH 2 CH 2 CH 2 CH 2 - lysine
  • H 2 NC(NH)NHCH 2 CH 2 CH 2 - arginine
  • 4-imidazolyl-CH 2 - histidine
  • Side chains of synthetic amino acids include the D-isomer of the above noted naturally occurring amino acids as well as those where R 8 is selected from the group consisting of alkyl of from 2 to 6 carbon atoms (where the alkyl group does not occur in naturally occurring amino acids), cycloalkyl of from 3 to 8 carbon atoms, and alkyl of from 1 to 6 carbon atoms substituted with from 1 to 2 substituents selected from
  • aryl of from 6 to 10 carbon atoms substituted with from 1 to 3 substituents selected from the group consisting of alkyl of from 1 to 6 carbon atoms, alkoxy of from 1 to 6 carbon atoms, aryl of from 6 to 10 carbon atoms, and aryloxy of from 6 to 10 carbon atoms, and
  • heteroaryl of from 3 to 14 carbon atoms having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur (where the substituted alkyl group does not occur in naturally occurring amino acids).
  • Preferred amino acid side chains include the D- and L-isomers of valine, leucine, phenylalanine, tryptophan and isoleucine.
  • X can be -O-, -NR 9 - or -S- where R 9 is selected from the group consisting of hydrogen, alkyl of from 1 to 6 carbon atoms and aryl of from 6 to 10 carbon atoms. Preferably X is -O-.
  • Y can be -C(O)- or -C(S)- and is preferably -C(O)-.
  • m is an integer equal to zero or one and n is an integer equal to zero to two.
  • n is an integer equal to zero or one.
  • the compounds of the invention are synthesized using standard techniques and reagents.
  • the linkages between the various groups in these compounds comprise, for example, a carbon atom linked to a nitrogen atom of an amide, a thioamide, a carbamate, a thiocarbamate, a urea, a thiourea, etc.
  • the methods and reagents for forming such bonds are well known and readily available. See, e.g., March, Advanced Organic Chemistry, 4th Ed. (Wiley 1992), Larock, Comprehensive Organic
  • the synthesis of the compounds of this invention can start with, for example, an amino acid (in the case where n is 0), a dipeptide (in the case where n is 1) or a tripeptide (in the case where n is 2).
  • dipeptide 1 is reacted with at least a stoichiometric amount of R 1 (X) m C(O)Z, where R 1 , X and m are defined as above and Z is suitable leaving group such as a halo group under conditions suitable to form dipeptide 2 terminally N-capped with the R 1 (X) m C(O)- substituent.
  • R 1 (X) m C(S)Z substituents can be used to prepare compounds where Y is -C(S)-.
  • any reactive substituents found on the amino acid side chains R 2 and R 3 of dipeptide 1 will require blocking and subsequent deblocking with a conventional removable blocking group.
  • the reaction is conducted in the presence of a suitable inert diluent typically in the presence of a base to scavenge any acid generated during the reaction, particularly if Z is halo.
  • suitable inert diluents include, by way of example, methylene chloride, chloroform, toluene, pyridine, etc.
  • Suitable bases include triethylamine, diethylisopropylamine, pyridine, and the like.
  • the reaction is typically conducted at from about 0°C to about 25°C and is typically complete in from about 1 to about 12 hours.
  • the resulting dipeptide 2 can be recovered by conventional means such as distillation, chromatography, filtration, etc. or alternatively is converted to aldehyde compound 3 without recovery and/or purification.
  • Dipeptide 2 is then reduced to provide the desired aldehyde 3 via conventional methods such as those described in March or Larock, supra.
  • Such methods include, for example, direct reduction of the carboxyl group of dipeptide 2 to alcohol 4, by, e.g., reaction of the acid with di-(iso- butyl)aluminumhydride (DIBALH) (see, e.g. , J. Gen. Chem. USSR
  • the aldehyde may be formed via the acid chloride using, e.g., thionyl chloride, followed by reduction using, for example, hydrogen and a palladium catalyst, tri-(t-butoxy)lithium aluminum hydride, sodium borohydride (alone or with pyridine or cadmium chloride).
  • the carboxyl group of dipeptide 2 is first converted to the corresponding N,O-dimethylhydroxamide 5 which is then reduced by, for example, lithium aluminum hydride to provide for aldehyde 3.
  • the N,O-dimethylhydroxamide 5 is formed, for example, by reaction of dipeptide 2 with at least a stoichiometric amount of
  • Suitable inert diluents include, by way of example, N,N-dimethyl- formamide, pyridine, etc.
  • the product is preferably not recovered but rather the reaction solution is used to convert the activated ester to the N,O-dimethylhydroxylamide 5.
  • the activated ester is converted to the N,O-dimethylhydroxylamide 5 by reaction with at least a stoichiometric amount of N,O- dimethylhydroxylamine hydrochloride at a temperature of from about 10°C to about 40°C for a period sufficient to form the desired N,O- dimethylhydroxylamide 5.
  • the resulting product can be recovered by conventional methods such as chromatography, distillation, filtration, etc. or, alternatively, used directly in the next step of the synthesis which converts the N,O-dimethylhydroxylamide 5 to aldehyde 3 by conventional reduction using a suitable reducing agent such as lithium aluminum hydride.
  • the first step in this process is illustrated in FIG. 1 and involves conversion of N-protected amino acid 6 to the corresponding N-protected N', O-dimethylhydroxylamide 7.
  • the N-protecting group on such amino acids is of the formula R 1 (X) m C(O)- such that, upon reduction, the resulting compounds are of formula I above and, in the case of FIG.1 is illustrated as ⁇ CH 2 OC(O)-.
  • N-protecting group can be removed by conventional methods and the resulting free amine N', O-dimethylhydroxylamide 8 can be reacted with R 1 (X) m C(O)Z and subsequently reduced to provide for compounds of formula I above (not shown).
  • the free amine 8 is coupled to the free acid of amino acid 9 having a R 1 (X) m C(O)- [e.g., ⁇ CH 2 OC(O)-] group attached to form dimer 10 which, upon subsequent conventional reduction with, for example, lithium aluminum hydride (LAH) forms aldehyde 11.
  • LAH lithium aluminum hydride
  • Conversion of the aldehydes 3 and 11 to the corresponding oximes or the corresponding diazoketones can be accomplished using chemistry known per se in the art.
  • oxime formation is accomplished via reaction of aldehydes 3 and 11 with hydroxylamine whereas
  • diazoketones were prepared using the procedure reported by Shaw (Green, George D.J., Shaw, Elliot (1981) J. Biol. Chem. 256, 1923-1928.
  • the starting materials employed in Reaction Scheme 1 are known in the art.
  • dipeptides 1 may be purchased commercially (e.g.
  • dipeptides are synthesized from amino acids which themselves are commercially available (e.g., from Bachem or Aldrich, Milwaukee, Wl) by, for example, the methods described above or by using known methods such as the Strecker method (see, e.g., March, or Williams, supra).
  • the coupling of the amino acids to form dipeptide 1 requires the blocking of the ⁇ -amino moiety of the N-terminal amino acid, and any other potentially reactive groups present on the side chain, from reaction with the activated carboxyl group of the C-terminal amino acid.
  • Conventional N-terminal amino protecting groups include, by way of example, t-butyloxycarbonyl (BOC) or benzyloxycarbonyl (Cbz).
  • reagents of the formula R 1 (X) m C(O)Z are also known per se in the art and some of these materials are also commercially available.
  • the compounds of formula I above are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • compositions which contain, as the active ingredient, one or more of the compounds of formula I above associated with pharmaceutically acceptable carriers.
  • the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the active compound In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy- benzoates; sweetening agents; and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It, will be understood, however, that the amount of the compound actually is administered.
  • administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • the above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
  • the components are blended and compressed to form tablets, each weighing 240 mg.
  • the active mixture is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • the active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve.
  • the granules so produced are dried at 50° to 60°C and passed through a 16 mesh U.S. sieve.
  • carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
  • the active ingredient, starch and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
  • the medicament, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.
  • the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 560 mg quantities.
  • the white soft paraffin is heated until molten.
  • the liquid paraffin and emulsifying wax are incorporated and stirred until dissolved.
  • the active ingredient is added and stirring is continued until dispersed.
  • the mixture is then cooled until solid.
  • transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference in its entirety.
  • patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier.
  • a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier.
  • One such implantable delivery system used for the transport of biological factors to specific anatomical regions of the body is described in U.S. Patent 5,011,472 which is herein incorporated by reference in its entirety.
  • Indirect techniques which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs.
  • Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier.
  • hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
  • Cathepsin Y is a novel carboxypeptidase having a molecular weight of approximately 31 kD and the amino acid sequence set forth in SEQ ID No. 3. Cathepsin Y is involved in the release of ( ⁇ AP from ⁇ AP-producing cells. The ⁇ AP production inhibition provided by the compounds of formula I described above appears to at least partially occur through inhibition of Cathepsin Y.
  • Cathepsin Y is able to cleave a wide variety of carboxy-terminal amino acids, with particular activity against aliphatic carboxy-terminal amino acids. The ability of Cathepsin Y to cleave the terminal amino acid is strongly affected by the nature of the amino acid residue located two positions away from the terminal amino acid being cleaved. Such specificity is characteristic of the papain super family of cysteine proteases.
  • Cathepsin Y can be obtained from both natural and synthetic sources.
  • Natural Cathepsin Y may be isolated and purified from a variety of mammalian cellular sources, including human 293 cells, human HS683 cells, human brain etc. Cells from these sources may be collected and disrupted to produce a lysate. Cellular and other debris from the resulting lysate may be separated, for example, by centrifugation, and the resulting supernatant subjected to a series of conventional purification steps. Specific methods for isolating and at least partially purifying Cathepsin Y from natural sources are set forth in detail in the Experimental section hereinafter.
  • Cathepsin Y compositions of the present invention will be at least partially purified, typically being at least 10% by weight (w/w) pure and being free from contaminants and substances which would interfere with the enzymatic activity.
  • the Cathepsin Y compositions will be at least 25 % w/w pure, more usually being at least 50% w/w pure, and preferably being at least 75% w/w pure, or higher.
  • substantially pure (homogeneous) compositions of the Cathepsin Y of the present invention typically being greater than 90% w/w pure, preferably being greater than 95% w/w pure and sometimes being 99% w/w pure or higher.
  • compositions having such high purity can be obtained using conventional protein purification techniques in conjunction with assays for the desired Cathepsin Y activity, as described in the Experimental section hereinafter.
  • Synthetic preparation of the Cathepsin Y compositions may be based on either the cDNA sequence (SEQ. ID No. 2) or the amino acid sequence (SEQ.ID No. 3) of the native Cathepsin Y.
  • Cathepsin Y from other mammals, as well as allelic forms of Cathepsin Y may be identified using degenerate oligonucleotide probes to screen suitable human and non-human libraries. Suitable libraries are available from a number of sources.
  • cDNA libraries may be screened by a variety of conventional techniques to identify cDNAs which encode Cathepsin Y of the present invention.
  • Such techniques include direct hybridization, polymerase chain reaction (PCR)- amplified hybridization, the use of anti-Cathepsin Y antibodies, and the like.
  • the identification of other Cathepsin Y cDNA sequences can be confirmed by introducing the identified DNA inserts into an appropriate plasmid vector for expression in an appropriate host, with the resulting recombinant expression vector being mapped by restriction enzyme cleavage and Southern blotting. Internally consistent clones may then be sequenced, with an internally consistent sequence being confirmed for Cathepsin Y.
  • Purified Cathepsin Y compositions of the present invention may be natural, i.e. , including the entire Cathepsin Y enzyme or fragments thereof isolated from the natural source, as described above, or may be synthetic, i.e. , including the entire protein or fragment or analog thereof, prepared by the techniques described below.
  • the fragments and analogs will preferably retain at least a portion of the native biological activity, i.e. , usually retaining at least the native proteolytic activity.
  • Synthetic polypeptides representing intact Cathepsin Y or biologically active fragments or analogs thereof may be prepared by either of two general approaches.
  • polypeptides may be synthesized using conventional solid-phase methods employing automated, commercial systems.
  • Cathepsin Y polypeptides involves the expression in cultured cells of recombinant DNA molecules encoding for the expression of all or a portion of the Cathepsin Y protein.
  • the recombinant DNA molecule may incorporate either a natural or synthetic gene, with natural genes and cDNA being obtainable as described above.
  • Synthetic polynucleotides may be prepared using solid phase techniques and automated commercial synthesizers. Double-stranded fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Natural or synthetic DNA fragments encoding the desired Cathepsin Y protein, fragment, or analog thereof will be incorporated in the DNA construct capable of introduction to and expression in an in vitro cell culture.
  • the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria.
  • the DNA constructs may be suitable for introduction into and integration within mammalian cells, preferably human cells, by a variety of now well known techniques.
  • the expression of the Cathepsin Y protein from such constructs will be performed under conditions wherein the Cathepsin Y is expressed. It is understood that such conditions will depend on the vector and the host cell used and can be determined by a person skilled in the art in light of the circumstances.
  • the nucleic acid may be directly labelled with any detectable label known the art, including radioactive nuclides such as 32 P, 3 H and 24 S, fluorescent markers such as fluorescein, Texas Red, AMCA blue, lucifer yellow, rhodamine and the like or any cyanin dye which is detectable with visible light.
  • the nucleic acid may be directly labelled using methods such as PCR, random priming, end labelling, nick translation and the like.
  • nucleic acids may be indirectly labelled by incorporating a nucleotide covalently linked to a hapten or other molecule such as biotin or digoxigenin (Boehringer Mannheim, Indianapolis, IN) and performing a sandwich hybridization with a labelled antibody or other molecule directed to that hapten.
  • a hapten or other molecule such as biotin or digoxigenin (Boehringer Mannheim, Indianapolis, IN)
  • a sandwich hybridization with a labelled antibody or other molecule directed to that hapten.
  • biotin is incorporated into the nucleic acid avidin conjugated so a detactable label can be used.
  • the isolated and purified Cathepsin Y polypeptides of the present invention may be utilized as proteases in a variety of biological and chemical systems. Additionally, the Cathepsin Y polypeptides may be used in screening assays for identifying test compounds which have ⁇ AP inhibition activity. It is further contemplated that Cathepsin Y can be used diagnostically to evaluate a patient's risk for AD based on the presence and amount of this enzyme present in the patient's body fluid.
  • the isolated and purified nucleic acids substantially homologous to the sequence of FIG. 4, nucleic acids substantially complementary to the sequence of FIG. 4 and fragments of the sequence of FIG. 4 can be used to probe specifically for the presence of Cathepsin Y RNA or DNA in tissues or cloned libraries.
  • the purified nucleic acids can be used to identify those tissues or cells which express RNA encoding for Cathepsin Y.
  • the preferred size of the nucleic acid fragments of FIG. 4 is at least 12 base pairs, preferably the fragments are at least 20 base pairs, more preferably of the fragments are at least 50 base pairs.
  • the nucleic acid may be RNA or DNA.
  • Cathepsin Y can be used in quantitative assays for the identification of compounds having ⁇ AP-production inhibition activity based on inhibition of the carboxypeptidase activity of the Cathepsin Y. Such assays are performed by observing the ability of Cathepsin Y to cleave the carboxy- terminal residue on a suitable oligopeptide substrate. Test compounds which are able to inhibit such carboxypeptidase activity are considered candidates for further testing to determine their ⁇ AP-production inhibition activity. Those test compounds which are unable to inhibit the
  • carboxypeptidase activity are considered less likely candidates for further testing.
  • An exemplary assay for ⁇ AP-production inhibition activity using Cathepsin Y is described in detail in the Experimental section below.
  • BOP Reagent benzotriazol-1-yloxy- tris(dimethylamino)phosphonium
  • dNTP deoxynucleoside triphosphate
  • EDTA ethylene diamine tetraacetic acid
  • LAH lithium aluminum hydride
  • PVDF polyvinylidene difluoride
  • RGW reagent grade water
  • ⁇ M micromolar
  • 293 751 SWE cells were obtained from K293 cells (human kidney cell line) stably transfected with the APP751 CDNA having the Swedish mutation.
  • the N,O-dimethylhydroxyamides illustrated in FIG. 1 were synthesized on a 20 mmol scale according to the following general procedure.
  • the CBZ protected amino acid 6 (20 mmol), BOP Reagent (30 mmol) and 4-methyl morpholine (100 mmol) were added to 100 mL of DMF and all were stirred under an atmosphere of nitrogen for 1 hour at ambient temperature.
  • N,O-dimethylhydroxylamine hydrochloride 24 mmol was added and all were stirred for an additional 12 hours at ambient temperature.
  • the reaction was poured into water (200 mL) then extracted with ethyl acetate (3 X 200 mL).
  • N,O-dimethylhydroxyamide 10 was done on a 10 mmol scale according to the following general procedure to provide for the title compound.
  • the N,O-Dimethylhydroxyamide 10 (10 mmol) was suspended in diethyl ether (65 mL) and cooled to 0°C under an atmosphere of nitrogen. To this vigorously stirred solution was added LAH (40-75 mmol), and the suspension was stirred for 1 hour at 0°C, then 1 hour at ambient temperature. The reaction was quenched with 10% aqueous citric acid (30 mL) and stirred for an additional 30 minutes. This was washed with diethyl ether (3 X 50 mL).
  • FIG. 2 depicts the syntheses of N-substituted amino acid aldehydes.
  • N,O-dimethylhydroxyamides synthesized on a +50 mmol scale were prepared according to the following general procedure to provide for the title compound.
  • the CBZ protected amino acid 6 50 mmol was added to a 2: 1 methylene chloride/tetrahydrofuran solution (500 mL) and all was cooled to -20°C under an atmosphere of nitrogen.
  • N-methylpiperidine (52.5 mmol) was added followed by the dropwise addition of methylchloroformate (52.5 mmol).
  • the removal of the CBZ protecting group was done on a 15 mmol scale according to the following general procedure to provide for the title compound.
  • the CBZ protected amino acid 12 (15 mmol) was suspended in ethanol (20 mL) in a parr bottle. To this was added 10 weight percent of palladium on activated carbon (10% palladium). This mixture was subjected to 50 psi of hydrogen for 3 hours on a parr shaker. The solution was then degassed, filtered through a pad of celite, then concentrated to afford the desired the desired amino N,O-dimethylhydroxyamide 13 (yields were typically 60-68%). This isolated material was used without further purification.
  • N,O-Dimethylhydroxyamide 15 (0.5 mmol) was suspended in tetra-hydrofuran (30 mL) and cooled to 0°C under an atmosphere of nitrogen. To this vigorously stirred solution, LAH (1.3 mmol) was added portion wise over a 30 minute period. Diethyl ether (60 mL) was then added followed by ice-cold 10% aqueous citric acid (80 mmol). After 30 minutes of vigorous stirring the reaction mixture was extracted with diethyl ether (5 X 20 mL).
  • diazomethane was freshly prepared by the slow portion wise addition of 1- methyl-3-nitro-1-nitrosoguanidine (17 mmol) into a solution of diethyl ether (50 mL) that contains a 40% aqueous KOH (15 mL) at 5°C. After allowing the solution to stand for 10 minutes the diethyl ether was decanted off and dried over KOH pellets.
  • C-terminal alcohols were prepared from the corresponding C-terminal aldehydes by conventional reduction with LAH.
  • the aldehyde is combined with LAH (about 4 equivalents) in THF at approximately 0°C. Diethyl ether is then added followed by ice-cold 10% aqueous citric acid. After 30 minutes of vigorous stirring the reaction mixture is extracted with diethyl ether. The combined organic portions are washed with saturated aqueous sodium bicarbonate, water, brine, dried over MgSO 4 , filtered, and concentrated to yield crude alcohol. Chromatography with 50% ethyl acetate/hexanes yields the desired purified alcohol.
  • C-terminal esters were prepared from the corresponding C-terminal carboxyl groups via conventional esterification conditions.
  • the media were again removed and replaced with fresh drug containing media as above and cells were incubated for an additional two hours.
  • plates were centrifuged in a Beckman GPR centrigfuge at 1200 rpm for five minutes at room temperature to pellet cellular debris from the conditioned media. From each well, 100 ⁇ L of conditioned media or appropriate dilutions thereof were transferred into an ELISA plate precoated with antibody 266 against amino acids 13-28 of ⁇ -amyloid peptide as described by Schenk, et al. in
  • results of the ⁇ -amyloid peptide ELISA were fit to a standard curve and expressed as ng/mL ⁇ -amyloid peptide. In order to normalize for cytotoxicity, these results were divided by the MTT results and expressed as a percentage of the results from a drug free control. All results are the mean and standard deviation of at least six replicate assays.
  • test compounds were assayed for ⁇ -amyloid peptide production inhibition activity in cells using this assay.
  • the results of this assay demonstrate that, among others, the compounds of Examples 1-80 each were able to reduce ⁇ -amyloid peptide production as compared to control.
  • the compounds of this invention are useful for reducing ⁇ -amyloid peptide production in cells and, accordingly, would be useful in treating humans in vivo for AD.
  • the binding of an active protease to the aldehyde column involves the equilibrium formation of a reversible covalent bond, between the active site cysteine residue of the protease with the aldehyde or other equivalent functionality. Elution of the protease in this case was achieved by using
  • DPDS which forms a disulfide linkage with the active site cysteine, and thus displaces the enzyme from the column.
  • Recovery of enzymatic activity following elution is achieved by incubating in an excess of reducing agents such as ⁇ -mercaptoethanol or dithiothreitol.
  • Epoxy Sepharose ® (Pharmacia) was swelled in 50 mL of reagent grade distilled water (RGW) for at least 20 minutes at room temperature, and washed on a coarse Buchner filter funnel with one liter of RGW. At no stage in any of the washes was the resin cake allowed to go to complete dryness.
  • Sepharose was sedimented by centrifugation at 550 rpm in a GSR rotor for 5 minutes, then resuspended in 40 mL of 1.0 M ethanolamine, pH 8.2, and incubated as before overnight (16-18 hours).
  • the coupled Sepharose was sedimented by centrifugation at 550 rpm in a GSR rotor for 5 minutes, then resuspended in 40 mL of 1.0 M ethanolamine, pH 8.2, and incubated as before overnight (16-18 hours).
  • ValPheSC-Sepharose was washed on a coarse Buchner filter funnel with 350 mL of 33% DMSO, then with 350 mL of RGW.
  • the semicarbazone was converted to the corresponding aldehyde by incubation in 80 mL of methanol: acetic acid formaldehyde (5:1: 1) at room temperature, with rotation, overnight.
  • the Sepharose was sedimented by centrifugation as above, and incubated with fresh methanokacetic acid formaldehyde as above, for 10 hours.
  • the ValPhe-Sepharose was washed as above with 600 mL of RGW, and stored at 4oC as a 50% slurry in 0.05% sodium azide.
  • the column then received 1.25 volumes of the DPDS solution; the fraction collected at this point contained the bulk of the eluted Cathepsin Y and B.
  • the column was eluted with at least 3 more volumes of DPDS solution, followed by 5 volumes of 6 M urea. Fractions of one column volume were collected.
  • the peak of the DPDS eluted material received 0.1 M NaCl, 1 mM MnCl 2 , and 1 mM CaCl 2 , and was applied to a 1.0 mL column of
  • Concanavalin A-agarose equilibrated with wash buffer (0.1 M NaCl, 50 mM MES, 1 mM MnCl 2 , 1 mM CaCl 2 , pH 6.0). After the starting material had been applied, the column was washed with 5 mL of wash buffer, followed by 1 mL of 0.1 M mannose in the same buffer; this was the first eluted fraction (El). Bound material was eluted with 0.5 M ⁇ -methyl
  • Cathepsin Y was eluted as a broad peak between 0.8 and 6 mL of elution buffer.
  • Enzymatic Activity Purified 31 kD protease (Cathepsin Y) was without effect on various APP preparations, including purified recombinant APP constructs as well as membrane-bound full-length APP. No significant activity was seen also with known synthetic substrates commonly used for assaying Cathepsin B, L or S. These results suggested that Cathepsin Y does not have the standard
  • LFYDQSPTA ⁇ (SEQ ID NO:5)
  • LFYDQSPTAT (aa 1-10 of SEQ ID NO:5)
  • LFYDQSPTA (aa 1-9 of SEQ ID NO:5)
  • LFYDQSPT (aa 1-8 of SEQ ID NO:5)
  • the quantitative analysis of carboxypeptidase activity of Cathepsin Y is based on the detection of the new free amino-terminus generated on cleavage of a selected substrate. This is accomplished by reacting with the reagent o- phthalaldehyde in an alkaline solution in the presence of 2-mercaptoethanol (Simons, et al., JACS, 98:7098-7099, (1976)).
  • EGYYGNYGV (aa 1-9 of SEQ ID NO: 7) was synthesized acetylated on its amino-terminus so that on cleavage by Cathepsin Y, the only free amino-terminus will be present in the reaction mixture will be that of the valine residue, cleaved off by the
  • a standard curve was constructed by incubating varying concentrations of valine (0-20 ⁇ M) in 0.25 M sodium borate, pH 10, containing 0.05% 2- mercaptoethanol and 60 ⁇ g/mL o-phthalaldehyde, in individual wells of a 96 well microtiter plate. The resulting fluorescence is read in a plate reading Cytofluor (ex 340, em 460 nm). There is a linear increase in the signal proportional to the amount of free valine present (FIG. 7).
  • reaction mixtures were set up with enzyme and substrate (0.2 mg/mL) in a total reaction volume of 0.1 mL) in individual wells of 96-well microtiter plates, in 20 mM sodium acetate buffers at different pHs, with 0.1 % 2-mercaptoethanol present. Control wells were set identically except for the presence of enzyme.
  • the reactions were quenched by the addition of an equal volume of 0.45 M sodium borate, pH 10, with 0.25 mg/mL o-phthalaldehyde, and the fluorescence measured. In the absence of added enzyme, no measurable fluorescence is generated above background, even with extended incubations. In the presence of enzyme, there is a time-dependent increase in fluorescence. Based on this analysis, the pH optimum for
  • carboxypeptidase activity was determined to be about 4.5, with the activity dropping off at both lower and higher pHs.
  • cDNA was made from RNA of human HS683 (human glioma cell line: ATCC #HTB138) cells using the Perkin Elmer GeneAmp RNA PCR kit as described by the manufacturer and denatured in a boiling water bath.
  • the PCR reaction contained 1 ⁇ g of poly A+ RNA, lx Perkins-Elmer-Cetus PCR buffer 1, 25 pM of each of oligonucleotides "Acys5" and "I Mer5"
  • PCR reaction 50 ⁇ M each dNTP, and 0.5 units Taq polymerase in the 25 ⁇ L total volume.
  • the PCR reaction was subjected to 30 cycles each consisting of 95°C for 45 seconds, 45°C for 1.5 minutes, a slow 1 minute rise in temperature up to 70°C, and 70°C for 30 seconds followed by 8 minutes at 72°C.
  • Acrylamide gel electrophoresis displayed multiple PCR products so the entire PCR reaction was subcloned and multiple clones sequenced.
  • hCatZ.82 encoded the amino acids of this newly discovered amino-terminal sequence.
  • a larger DNA clone of Cathepsin Y was obtained by degenerate PCR using sequence from hCatZ.82 (nucleotides 299-366 of SEQ ID NO: 2) and conserved sequences of the cy stein proteases of the papain family. PCR oligonucleotides used were
  • LM#4 also called Bcys2 (SEQ ID NO: 13)
  • PCR reactions were performed on phage from a HeLa cell cDNA library (in lambda zap 2 vector from Stratagene). 5 ⁇ L of phage at 1x10 10 phage/mL were boiled for 2 minutes and placed on ice. Hot Start PCR was performed according to the manufacturer (Perkin Elmer/Cetus ) using oligonucleotide
  • RACE31-NC CAG GAG GGT GGA GGG CCA CGC TCC CT
  • oligonucleotide 1705 (nucleotides 301-366 of SEQ ID NO:2) as a probe displayed reactive products and PCR products of the appropriate size. These were subcloned and clones containing a diagnostic Apal site (shown at nucleotides 380- 385 of SEQ ID NO:2) of FIG. 4 were sequenced and the sequence of clone cat Y P3-25 (nucleotides 202-411 of SEQ ID NO:2) is shown on FIG. 4.
  • cDNA was synthesized from polyA+ RNA from human HS683 cells (Frohman et al., (1988) PNAS USA 85:8998-9002) using the cDNA synthesis conditions in Clontech 5'AmpliFINDERTMRACE kit protocol with the following modifications: 2 ⁇ g of RNA was used in 100 ⁇ L volume and the adaptor primer described below was used to prime synthesis.
  • the adapter primers were a modification of that described by Frohman, et.al. , (1988), PNAS USA 85:8998-9002.
  • Hot Start PCR was performed using the adapter primer 1577 and internal primer Nestr31-c (FIG. 4) (nucleotides 343-366 of SEQ ID NO:2) at an annealing temperature of 55°C. Hot Start conditions were modifications of Perkin-Elmer AmpliWaxTM protocol.
  • Lower mix is 1.25X PCR buffer (10X PCR buffer II [Perkin Elmer Cetus, Norwalk, CT] is 500 mM KC l , 100 mM Tris-HCl pH8.3), 2 mM MgCl 2 (Perkin Elmer Cetus), 200 ⁇ M dNTP (Perkin Elmer Cetus), 1 ⁇ M each primer in total volume of 12.5 ⁇ L.
  • Upper mix is 1.25X PCR buffer, 1.25U AmpliTaq ® DNA Polymerase (Perkin Elmer Cetus), 1 ⁇ L template DNA in 37.5 ⁇ L total volume.
  • AmpliwaxTM PCR Gem 50 (Perkin Elmer Cetus) is added to the lower mix which is brought to 80°C for 5 minutes, then held at 25°C until the addition of the upper mix. PCR conditions included initial denaturation at 95°C for 2 minutes followed by 30 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 2 minutes, and then a final single extension at 72°C for 8 minutes. Southern blot analysis of RACE reactions using radiolabelled
  • oligonucleotide 1758 (nucleotides 553-577 of SEQ ID NO:2) (FIG. 4) as probe showed bands at approximately 800 and 1100 bp. These bands were gel-isolated and cloned into pT7Blue (Novagen, La Jolla, CA). Novablue cells (Novagen, LaJolla, CA) were transformed and colonies positive for Cathepsin Y sequence by PCR analysis were sequenced. The larger fragment proved to contain the stop codon and polyA tail, and its sequence is shown on FIG. 4 (indicated in Figure 4 by "3'RACE") (nucleotides 343 to 1558 of SEQ ID NO:2).
  • PCR was done under the above Hot Start conditions with oligonucleotides NestR31-nc (FIG. 4) (reverse complement of nucleotides 385-411 of SEQ ID NO:2) to 1821 (SEQ ID NO: 19) using 65°C annealing temperature.
  • PCR conditions included initial denaturation at 94°C for 2 minutes; then 35 cycles of 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 2 minutes and then a final extension at 72°C for 8 minutes.
  • FIG. 4 DNA of corresponding size was gel-isolated and cloned directly into pT7Blue. Novablue cells (Novagen, LaJolla, CA) were transformed and colonies positive for Cathepsin Y sequence by PCR analysis with the largest inserts were sequenced. One clone of 63 screened contained the sequence identified as region 1 (nucleotides 175-411 of SEQ ID NO: 2).
  • PCR primary PCR was done using the above Hot Start conditions (with the subsitution of 7-deaza-GTP:GTP at a ratio of 3: 1 instead of standard GTP in the dNTP mix) with oligonucleotides 1685 (FIG. 4) (nucleotides 316- 348 of SEQ ID NO:2) and 1821 (SEQ ID NO: 19), annealing at 60°C.
  • PCR conditions included initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 45 seconds, 60°C for 45 seconds, 72°C for 1.5 minutes, and then a single final extension at 72°C for 8 minutes.
  • PCR products run on a 2% agarose gel showed a smear of DNA.
  • DNA greater than 200 bp in size was isolated and purified using GenecleanTM (Bio 101, Madison, Wl). This DNA was used as a template for nested PCR using oligonucleotides 1810 (FIG. 4) (nucleotides 243-270 of SEQ ID NO:2) and 1823 (nucleotides 14-38 of SEQ ID NO: 19) under the above Hot Start conditons with the addition of 1 ⁇ L of single-strand binding protein (USB) in the lower layer along with the template DNA and Hot Start temperature of 95 °C rather than 80°C. The primers were added to the upper mix, and PCR proceeded with annealing at 60°C.
  • FIG. 4 A Southem blot of the PCR product was probed with 32 P-kinased oligonucleotide 1846 (FIG. 4) (nucleotides 175-204 of SEQ ID NO:2) and appropriate sized DNA was isolated from the gel, ligated into pT7Blue, and transformed into SURE cells (Stratagene, La Jolla CA). 7 colonies were positive for Cathepsin Y sequence by PCR analysis out of 27 screened. The one clone sequenced contained the sequence identified as region 2 (FIG. 4) (nucleotides 133-270 of SEQ ID NO:2).
  • Vent PCR Perkin-Elmer PCR buffer and MgCl 2 were replaced by NEB 10X Vent polymerase buffer (100 mM KC l , 200 mM Tris-HCl pH 8.8, 100 mM NH 4 ] 2 SO 4 , 20 mM MgS0 4 , 1.0% Triton X100) at the appropriate
  • Deep Vent R ® DNA Polymerase (NEB) was used at 2U per 50 ⁇ L reaction. Annealing was done at 60°C. The PCR product was blunt ended with Klenow, digested with EcoRl and gel purified. Fragments of 100-150 bp and 150-200 bp were isolated separately and ligated into
  • Cathepsin Y Expression The BamHl fragment containing the entire coding region of the Cathepsin Y was blunt ended and cloned into the vector poCK751 cut with Nrul and Spel (blunt ended) (FIG. 5) so that the ⁇ APP sequences were removed and replaced with those of Cathepsin Y to form the plasmid poCK catY (FIG. 6).
  • Varying amounts of Cathepsin Y cDNA in the poCK expression vector were transfected transiently into 293 cells, in 6 well plates.
  • the cells were washed with cold PBS, then lysed in 1 mL of 20 mM MES, pH 6, 0.1 % Brij-35, 2 mM EDTA. 10 ⁇ L aliquots of the lysate (following centrifugation to remove cell debris) were electrophoresed using SDS-PAGE, followed by transfer of the proteins onto a PVDF membrane. The membrane was probed with the anti-cathepsin B antibody for Western blot analysis. A dose-dependent increase in the ⁇ 31 kDa immunoreactive Cathepsin Y band was seen as a result of transient expression of the

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Abstract

Cette invention concerne des procédés d'inhibition de la sécrétion de peptide β-amyloïde (βAP) par des cellules, lesquels procédés consistent à administrer aux cellules certains composés qui inhibent l'activité d'une protéase d'approximativement 31 kD impliquée dans la sécrétion de βAP. Cette protéase de 31 kD a été désignée sous le nom de Cathepsine Y. Cette invention concerne également des procédés de criblage d'inhibiteurs de βAP, qui consistent à déterminer l'activité de composés tests en présence de la Cathepsine Y et d'un substrat peptidique adéquat. Cette invention concerne enfin une séquence d'acide nucléique codant la Cathepsine Y, ainsi que l'expression et l'isolation de la Cathepsine Y.
PCT/US1996/006211 1995-06-06 1996-04-26 Nouvelle cathepsine et procedes et compositions d'inhibition de cathepsine WO1996039194A1 (fr)

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WO1998022433A1 (fr) * 1996-11-22 1998-05-28 Elan Pharmaceuticals, Inc. AMIDES D'ACIDES AMINES N-(ARYL/HETEROARYL/ALKYLACETYL), COMPOSITIONS PHARMACEUTIQUES LES CONTENANT ET METHODES D'INHIBITION DE LA LIBERATION DU PEPTIDE β-AMYLOIDE ET/OU SA SYNTHESE A L'AIDE DE CES COMPOSES
WO1998023581A1 (fr) * 1996-11-26 1998-06-04 Basf Aktiengesellschaft Benzamidoaldehydes et leur utilisation comme inhibiteurs des cysteine-proteases
WO1998038177A1 (fr) * 1997-02-28 1998-09-03 Elan Pharmaceuticals, Inc. COMPOSES HETEROCYCLIQUES ET LEUR UTILISATION POUR L'INHIBITION DU PEPTIDE β-AMYLOIDE
WO1998022494A3 (fr) * 1996-11-22 1998-11-26 Athena Neurosciences Inc METHODES ET COMPOSES DESTINES A INHIBER LA LIBERATION ET/OU LA SYNTHESE DU PEPTIDE β-AMYLOIDE
US5852007A (en) * 1995-11-28 1998-12-22 Cephalon, Inc. Cysteine and serine protease inhibitors containing D-amino acid at the P2 position, methods of making same, and methods of using same
US5965614A (en) * 1996-11-22 1999-10-12 Athena Neurosciences, Inc. N-(aryl/heteroaryl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
WO1999054320A1 (fr) * 1998-04-20 1999-10-28 Basf Aktiengesellschaft Nouveaux amides heterocycliquement substitues a action de proteases de cysteine
WO1999054294A1 (fr) * 1998-04-20 1999-10-28 Basf Aktiengesellschaft Nouveaux amides substitues, leur production et leur utilisation
WO1999031256A3 (fr) * 1997-12-18 1999-11-04 Immunex Corp Proteines cathepsine dc, adn codant pour ces proteines et utilisation de ces proteines dans l'etablissement de pronostics pour les cancers chez l'homme
WO2000024392A1 (fr) * 1998-10-26 2000-05-04 Sumitomo Pharmaceuticals Company, Limited Inhibiteur de la formation de beta-amyloide
US6117901A (en) * 1996-11-22 2000-09-12 Athena Neurosciences, Inc. N-(aryl/heteroarylacetyl) amino acid esters, pharmaceutical compositions comprising same, and methods for use
US6153652A (en) * 1996-11-22 2000-11-28 Elan Pharmaceuticals, Inc. N-(aryl/heteroaryl/alkylacetyl) amino acid amides, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6207710B1 (en) 1996-11-22 2001-03-27 Elan Pharmaceuticals, Inc. Compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6211235B1 (en) 1996-11-22 2001-04-03 Elan Pharmaceuticals, Inc. Compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6506782B1 (en) 1998-02-27 2003-01-14 Athena Neurosciences, Inc. Heterocyclic compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
EP1336847A1 (fr) * 2002-02-14 2003-08-20 Biofrontera Pharmaceuticals AG Inhibiteurs de la Cathepsine Y pour le dévelopment de médicaments pour le traitement de la douleur
EP1347059A1 (fr) * 2002-03-20 2003-09-24 Biofrontera Pharmaceuticals AG Cathepsin Y, une cible pour le développment de médicaments pour le traitement d'accidents vasculaires cérébraux
US6642261B2 (en) 1997-11-21 2003-11-04 Athena Neurosciences, Inc. N-(aryl/heteroarylacety) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6861558B2 (en) 1996-11-22 2005-03-01 Elan Pharmaceuticals, Inc. Methods and compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6974672B2 (en) 2002-03-19 2005-12-13 Amgen Inc. Gene amplification in cancer
WO2007054776A2 (fr) * 2005-11-10 2007-05-18 Orchid Research Laboratories Limited Composes semblables au stilbene comme inhibiteurs hdac atypiques

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Cited By (32)

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US5852007A (en) * 1995-11-28 1998-12-22 Cephalon, Inc. Cysteine and serine protease inhibitors containing D-amino acid at the P2 position, methods of making same, and methods of using same
US6767918B2 (en) 1996-11-22 2004-07-27 Athena Neurosciences, Inc. N-(aryl/heteroarylacetyl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6153652A (en) * 1996-11-22 2000-11-28 Elan Pharmaceuticals, Inc. N-(aryl/heteroaryl/alkylacetyl) amino acid amides, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
WO1998022494A3 (fr) * 1996-11-22 1998-11-26 Athena Neurosciences Inc METHODES ET COMPOSES DESTINES A INHIBER LA LIBERATION ET/OU LA SYNTHESE DU PEPTIDE β-AMYLOIDE
US6399628B1 (en) 1996-11-22 2002-06-04 Athena Neurosciences, Inc. N-(aryl/heteroaryl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting alpha- amyloid peptide release and/or its synthesis by use of such compounds
US5965614A (en) * 1996-11-22 1999-10-12 Athena Neurosciences, Inc. N-(aryl/heteroaryl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6888022B2 (en) 1996-11-22 2005-05-03 Athena Neurosciences, Inc. Methods and compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6861558B2 (en) 1996-11-22 2005-03-01 Elan Pharmaceuticals, Inc. Methods and compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6476263B1 (en) 1996-11-22 2002-11-05 Elan Pharmaceuticals, Inc. Compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6333351B1 (en) 1996-11-22 2001-12-25 Athena Neurosciences, Inc. N-(aryl/heteroarylacetyl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
WO1998022433A1 (fr) * 1996-11-22 1998-05-28 Elan Pharmaceuticals, Inc. AMIDES D'ACIDES AMINES N-(ARYL/HETEROARYL/ALKYLACETYL), COMPOSITIONS PHARMACEUTIQUES LES CONTENANT ET METHODES D'INHIBITION DE LA LIBERATION DU PEPTIDE β-AMYLOIDE ET/OU SA SYNTHESE A L'AIDE DE CES COMPOSES
US6117901A (en) * 1996-11-22 2000-09-12 Athena Neurosciences, Inc. N-(aryl/heteroarylacetyl) amino acid esters, pharmaceutical compositions comprising same, and methods for use
US6207710B1 (en) 1996-11-22 2001-03-27 Elan Pharmaceuticals, Inc. Compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6211235B1 (en) 1996-11-22 2001-04-03 Elan Pharmaceuticals, Inc. Compounds for inhibiting β-amyloid peptide release and/or its synthesis
US6313152B1 (en) 1996-11-22 2001-11-06 Athena Neurosciences, Inc. N(aryl/heteroarylacetyl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6262302B1 (en) 1996-11-22 2001-07-17 Elan Pharmaceuticals, Inc. N-(aryl/heteroaryl/alkylacetyl) amino acid amides, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6251917B1 (en) 1996-11-26 2001-06-26 Basf Aktiengesellschaft Benzamidoaldehydes and their use as cysteine protease inhibitors
WO1998023581A1 (fr) * 1996-11-26 1998-06-04 Basf Aktiengesellschaft Benzamidoaldehydes et leur utilisation comme inhibiteurs des cysteine-proteases
WO1998038177A1 (fr) * 1997-02-28 1998-09-03 Elan Pharmaceuticals, Inc. COMPOSES HETEROCYCLIQUES ET LEUR UTILISATION POUR L'INHIBITION DU PEPTIDE β-AMYLOIDE
US6642261B2 (en) 1997-11-21 2003-11-04 Athena Neurosciences, Inc. N-(aryl/heteroarylacety) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
WO1999031256A3 (fr) * 1997-12-18 1999-11-04 Immunex Corp Proteines cathepsine dc, adn codant pour ces proteines et utilisation de ces proteines dans l'etablissement de pronostics pour les cancers chez l'homme
US6506782B1 (en) 1998-02-27 2003-01-14 Athena Neurosciences, Inc. Heterocyclic compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6849650B2 (en) 1998-02-27 2005-02-01 Athena Neurosciences, Inc. Heterocyclic compounds, pharmaceutical compositions comprising same, and methods for inhibiting β-amyloid peptide release and/or its synthesis by use of such compounds
US6448254B1 (en) 1998-04-20 2002-09-10 Abbott Laboratories Substituted amides, their production and their use
WO1999054294A1 (fr) * 1998-04-20 1999-10-28 Basf Aktiengesellschaft Nouveaux amides substitues, leur production et leur utilisation
WO1999054320A1 (fr) * 1998-04-20 1999-10-28 Basf Aktiengesellschaft Nouveaux amides heterocycliquement substitues a action de proteases de cysteine
WO2000024392A1 (fr) * 1998-10-26 2000-05-04 Sumitomo Pharmaceuticals Company, Limited Inhibiteur de la formation de beta-amyloide
EP1336847A1 (fr) * 2002-02-14 2003-08-20 Biofrontera Pharmaceuticals AG Inhibiteurs de la Cathepsine Y pour le dévelopment de médicaments pour le traitement de la douleur
US6974672B2 (en) 2002-03-19 2005-12-13 Amgen Inc. Gene amplification in cancer
EP1347059A1 (fr) * 2002-03-20 2003-09-24 Biofrontera Pharmaceuticals AG Cathepsin Y, une cible pour le développment de médicaments pour le traitement d'accidents vasculaires cérébraux
WO2007054776A2 (fr) * 2005-11-10 2007-05-18 Orchid Research Laboratories Limited Composes semblables au stilbene comme inhibiteurs hdac atypiques
WO2007054776A3 (fr) * 2005-11-10 2008-09-04 Orchid Res Lab Ltd Composes semblables au stilbene comme inhibiteurs hdac atypiques

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