Patent Application
20080227806
This invention relates generally to chemistry, medicinal chemistry, enzymology, biochemistry, molecular and cellular biology, genetics, pharmacology, pharmacy, neurobiology, medicine, and drug therapy. In particular, the present invention relates to novel prodrugs, pharmaceutical compositions containing them and methods of making and using them. Specifically, the invention includes prodrugs having improved properties for affecting dementia.
Proteolytic enzymes catalyse the hydrolytic cleavage of peptide bonds in proteins and peptides. They can be classified as exopeptidases and endopeptidases (called proteinases or proteases) that are further subdivided into protease classes, the serine-, aspartic-, metallo- and cysteine proteases. Cysteine proteases are found in viruses and prokaryotes as well as plants and mammals, including man.
Cysteine proteases have a common catalytic mechanism arising from the presence of the catalytic amino acids cysteine and histidine in the active site of the enzyme, the site where the peptide bond is cleaved. Cleavage involves a nucleophilic cysteine thiol in a catalytic triad. The first step is deprotonation of a thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue. The next step is nucleophilic attack by the deprotonated cysteine's anionic sulfur on the substrate carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. The thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate fragment, while regenerating the free enzyme.
Examples of cysteine proteases include the cathepsins, caspases, calpains and papain. Cathepsins are a large family that are distinguished by their substrate specificities and cathepsin B enzyme in the cathepsin class.
Cysteine proteases, cathepsins, and cathepsin B, specifically, are known to be involved in neurological pathologies. In particular, these enzymes are known to be important in the development of dementia, especially dementia due to Alzheimer's disease (AD) and vascular dementia (VD).
Many cysteine protease, cathepsin protease and cathepsin B inhibitors are known in the art. However, prodrug forms are needed having improved pharmacological and toxicological properties. In particular, new prodrugs are needed having superior properties for treating neurological pathologies, especially dementia. The present invention provides these prodrugs, compositions and method of using the same to treat dementia.
The invention is directed to novel prodrugs containing a chemical delivery system (CDS) moiety and a cysteine protease inhibitor moiety. The CDS moiety targets the cysteine protease inhibitor to the brain or central nervous system. The cysteine protease inhibitor moiety is inactive in the prodrug but active upon release. Cysteine protease inhibitors are useful for treating dementia, Alzheimer's disease (AD) and vascular dementia (VD) and, thus, CDS prodrugs of the invention that target the brain and central nervous system offer significant advantages for treating those conditions and diseases. A preferred CDS prodrug contains a dihydrotrigoneline CDS moiety and an epoxysuccinyl peptide cysteine protease inhibitor moiety. The invention includes pharmaceutical compositions containing a CDS prodrug and methods of using such compositions to treat those conditions and diseases.
(A) Loxistatin reduces brain Ab levels. E64d treatment for 7 or 30 days reduced the total Aβ by 31% and 55% relative to control, respectively (diagonal striped bars). Results are shown as mean ±SEM, with * indicating significance at p<0.05 (by student's t-test).
(B) CA074Me reduces brain Ab levels. In vivo CA074Me treatment for 7 or 30 days reduced total Aβ by 57% and 62% relative to control, respectively (solid bars). After 7 days of treatment, CA074Me significantly reduced total Aβ. Data are shown as mean ±SEM, with statistical significance of *p<0.05 (by student's t-test).
Aβ40 and Aβ42 were individually measured in brain extracts from animals treated with CA074Me or saline control for 7 days. Results are expressed as the mean ±SEM, with statistical significance of * p<0.05 by the student's t-test.
(A) Reduction of Ab40 by CA074Me. CA074Me treatment (solid bars) significantly decreased Aβ40 in brain extracts, with a 47% reduction.
(B) Reduction of Ab42 by CA074Me. CA074Me treatment (solid bars) significantly reduced Aβ42 in brain by 56%. Importantly, the mean Aβ40 and Aβ42 levels in control samples did not significantly differ from each other, nor did the mean Aβ40 and Aβ42 levels from the CA074Me-treated animals. These data show that the CA074Me treatment reduced each Aβ40 and Aβ042 peptide form to a similar extent.
CTFβ was analyzed in brain extracts from animals treated with CA074Me or control for 7 days by western blots.
(A) CTFb analyses by western blots. Brain extracts from animals treated with CA074Me were subjected to western blots using antibody MAB 1560. The 12 kDa band (indicated by arrow) represents CTF{tilde over (b)}
(B) Quantitative evaluation of CTFb. Relative levels of CTFb were assessed by densitometric scanning of western blots from
Levels of sAPPα were analyzed in brain extracts from animals treated with CA074Me and saline control for 7 days.
(A) Analyses of sAPPa by western blots. Brain extracts were subjected to western blots using antibody directed to the NH2-terminal domain of APP, antibody MAB 343. The 90 kDa band represents sAPPa.
(B) Quantitative evaluation of sAPPa. Relative levels of sAPPa were determined by densitometric scanning of western blots from
A prodrug is a pharmacological substance, which is administered in an inactive or less active form. Once administered, the prodrug is metabolized in the body in vivo to release a biologically active compound (drug). In the context of this invention, a prodrug is also one that can be metabolized by cells or in vitro to release a drug. A prodrug has improved absorption, distribution, metabolism or excretion properties (ADME) relative to the drug. The prodrug of this invention has improved ADME for delivery to the brain and central nervous system
A prodrug of the invention contains a moiety that reduces the enzymatic activity of any cysteine protease after being metabolically released from the prodrug. As such, a prodrug of the invention contains a cysteine protease inhibitor moiety that is active upon release from the prodrug. In one embodiment, the prodrug contains a moiety that inhibits the enzymatic activity of some, most or all cysteine proteases upon release. The invention also includes a prodrug wherein the moiety inhibits some, most or all cathepsin proteases upon liberation. In another preferred form, the moiety selectively inhibits cathepsin B after metabolism. Cysteine protease inhibitor moieties of the invention are detailed below.
A prodrug of the invention is one having the following structure:
where
R1 is a cysteine protease inhibitor moiety;
R2 is an O, NH, or N substituted with any group that results in cysteine protease inhibition;
R3, is absent when X is 0 (absent), or a H, 1-6 alkyl, optionally containing one or more aryl, heteroaryl, or heteroatoms, an aryl, or heteroaryl, when X is 1 or 2;
R4 is absent when X is 0, or an O, NH, or N substituted with a 1-6 alkyl, optionally containing one or more aryl, heteroaryl, or heteroatoms, an aryl, or heteroaryl, when X is 1 or 2; and
R5 is a dihydropyridine ring attached via a dihydropyridine ring C, where:
the dihydropyridine ring C are each optionally substituted by a 1-6 alkyl optionally containing one or more aryl, heteroaryl, or heteroatoms, an aryl, heteroaryl, or, taken together, form a 5, 6, or 7 member aromatic or acyclic fused ring optionally containing one or more heteroatoms; and
the dihydropyridine ring N is substituted by a 1-6 alkyl, which optionally contains one or more aryl, heteroaryl, or heteroatoms, aryl or heteroaryl.
The prodrug is metabolized to produce a biologically active moiety having the following structure:
Thus, metabolism of the prodrug results in the release of the cysteine protease inhibitor moiety, R1, containing the carbonyl-R2 group. As such, a CDS prodrug contains any cysteine protease inhibitor moiety such that, upon release from the prodrug, a functional cysteine protease containing the carbonyl-R2 group is liberated. Thus, a preferred R2 is or NH as these form the carboxylic acid or amide moieties, respectively, found on many cysteine protease inhibitors (see Example 1). But functional cysteine protease inhibitors also have at the R2 position a substituted N. Thus, R2 also includes a N having a substituent that is any group known in the art to result in a cysteine protease inhibitor. A R2 also includes a N substituted with a 1-6 alkyl, optionally substituted with one or more aryl, heteroaryl or heteroatoms, which can be optionally substituted with a 1-6 alkyl, optionally containing one or more aryl, heteroaryl, or heteroatoms.
Prodrugs generally and their metabolism specifically is well known in the art (see for example, Testa, B., and J. M. Mayer, “Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry and Enzymology”, Wiley Pub., 2003, ISBN 3-90639-025-X). Hydrolysis is the means of metabolism by which the cysteine protease inhibitor is released from the prodrug.
The prodrug of the invention selectively targets the cysteine protease inhibitor to the brain or central nervous system. Using a chemical delivery system (CDS) coupled to the cysteine protease inhibitor, the ratio of brain to periphery drug levels can be increased. This offers significant advantages in the treatment of dementia because the drug is concentrated in the brain thereby reducing the dose and toxicity (see for example, Bodor and Buchwald, Barriers to remember: brain-targeting chemical delivery systems and Alzheimer's disease, Drug Discov Today, 7(14):766-74, 2002).
The prodrug of the invention is a CDS prodrug, which exploits the bidirectional properties of the blood-brain barrier to lock-in the CDS prodrug in the brain and allow sustained release of the cysteine protease inhibitor. CDS moiety as broadly described above and includes 1,4-dihydrotrigoneline esters and amides linked to cysteine protease inhibitor moieties (see for example, Bodor, Farag, Barros, Wu and Buchwald, In vitro and in vivo evaluations of dihydroquinoline- and dihydroisoquinoline-based targetor moieties for brain-specific chemical delivery systems, J Drug Target, 10(1):63-71, 2002). The 1,4 dihydrotrigoneline esters and amides are hydrophobic and allow the CDS prodrug to readily cross the blood-brain barrier. But the hydrophobic 1,4 dihydrotrigoneline is converted in vivo to a hydrophilic quaternary form. This conversion takes place throughout the body via oxidation but without generating active or reactive radical intermediates. In the periphery the charged quaternary form is rapidly eliminated from the body but in the brain the charged form is locked behind the blood-brain barrier. This results in the brain being the only site containing the CDS prodrug and drug after a relatively short period of time. There, the CDS prodrug continues to be metabolized and release the cysteine protease inhibitor. As a result, there is a sustained release of the drug in the brain. Thus, the CDS prodrug not only delivers the cysteine protease inhibitor moiety to the brain, it also provides preferential delivery there, thus targeting the brain (Buchwald and Bodor, Physicochemical aspects of the enzymatic hydrolysis of carboxylic esters, Pharmazie, 57(2):87-93, 2002). CDS prodrugs have been applied to deliver antibiotics to the brain (Pop, Wu, Shek and Bodor, Brain-specific chemical delivery systems for beta-lactam antibiotics. Synthesis and properties of some dihydropyridine and dihydroisoquinoline derivatives of benzylpenicillin, J Med Chem, 32(8):1774-81, 1989) (Wu, Pop, Shek and Bodor, Brain-specific chemical delivery systems for beta-lactam antibiotics. In vitro and in vivo studies of some dihydropyridine and dihydroisoquinoline derivatives of benzylpenicillin in rats, J Med Chem, 32(8):1782-8, 1989).
Broadly, the CDS prodrugs of the invention include those in which structure (I) is attached to any of the dihydropyridine ring C of R5. In select CDS prodrugs, structure (I) is attached to any unsaturated dihydropyridine ring C of R5.
A CDS prodrug includes those having the following structure:
where:
R5 is a H, a 1-6 alkyl, optionally containing one or more aryl, heteroaryl or heteroatoms, an aryl or heteroaryl;
R6 is a 1-6 alkyl, optionally containing one or more aryl, heteroaryl, or heteroatoms, aryl, heteroaryl, aryl or heteroaryl; and
R7, R8, and R9 are each a H, 1-6 alkyl, optionally containing one or more aryl, heteroaryl or heteroatoms, an aryl, heteroaryl, or, taken together, form a 5, 6, or 7 member aromatic or acyclic fused ring containing one or more heteroatoms.
In another preferred form, the CDS prodrugs include those having this structure:
where the variables are as defined above.
Another form of CDS prodrug has the following structure:
where:
R7 and R8 together form the 5, 6, or 7 member aromatic or acyclic fused ring containing one or more heteroatoms and the other variables are as defined above.
A CDS prodrug also has the following structure:
where:
R8 and R9 together form the 5, 6, or 7 member aromatic or acyclic fused ring containing one or more heteroatoms and the other variables are as defined above.
A R1 cysteine protease inhibitor moiety is any compound that contains the carbonyl-R2 group described above that, upon liberation from the prodrug, is capable of inhibiting a cysteine protease, cathepsin protease or cathepsin B. Large proteinaceous cysteine protease inhibitors are known (see, for example, Rzychon, Chmiel and Stec-Niemczyk, Modes of inhibition of cysteine proteases, Acta Biochim Pol, 51(4):861-73, 2004) but generally not suitable as drugs. Rather, small molecule protease inhibitors generally, and cysteine protease inhibitors specifically are useful as therapeutics (see for example, Abbenante and Fairlie, Protease inhibitors in the clinic, Med Chem, 1(1):71-104, 2005) (Turk, Targeting proteases: successes, failures and future prospects, Nat Rev Drug Discov, 5(9):785-99, 2006) (Turk and Guncar, Lysosomal cysteine proteases (cathepsins): promising drug targets, Acta Crystallogr D Biol Crystallogr, 59(Pt 2):203-13, 2003). Thus, preferred cysteine protease inhibitor of the invention is a small molecule suitable for drug development.
Many small molecule cysteine protease inhibitors are known in the art and are preferred for use as the cysteine protease inhibitor moiety in the CDS prodrug (see, for example, Frlan and Gobec, Inhibitors of cathepsin B, Curr Med Chem, 13(19):2309-27, 2006). Among such known small molecule cysteine protease inhibitors, the epoxysuccinyl peptides are preferred. These inhibitors are the most extensively developed small molecule cysteine protease and cathepsin B specific inhibitors. E64, [2S,3S)-3-(N-{(S)-1-[N(4-guanidinobutyl)carbamoyl]3-methylbutyl}carbamoyl) oxirane-2-carboxylic acid], was originally isolated from Aspergillus japonicus. Derivatives of e64 have been described, (for example, in U.S. Pat. Nos. 6,689,785, 6,387,908, 6,110,967, 5,843,992, 5,679,708, 5,556,853, 4,418,075, and 4,333,879 and Frian, ibid.).
Thus, a preferred R1 has the following structure:
where:
R10, R11, and R12 are each, separately, any group that results in cysteine protease inhibition, and the “•” in structure VI represents the attachment site of R1 to the prodrug.
In a preferred prodrug, R10, R11, and R12 are each, separately, any group known in the art to result in a cysteine protease inhibitor. R10 in preferred prodrug is also a 1-6 alkyl, optionally containing one or more aryl, heteroaryl or heteroatoms, an aryl or heteroaryl. In another preferred prodrug, R10 is a 1-6 alkyl, optionally containing a benzyl or phenol. In a preferred prodrug, R10 is a butyl group. Another preferred R10 forms in structure VI a leucine or isoleucine group. R11 is a 1-10 alkyl, optionally containing one or more aryl, heteroaryl or heteroatoms in a selected prodrug. In another preferred prodrug, R11 is 1-6 alkyl, optionally containing guadinium, imidazol amine group, or a propionylamino-acid methyl ester group. R11 is a pentane group, preferably an isopentane group, in a preferred prodrug. In a preferred prodrug, R12 is H. The SS conformer (trans) of the epoxide of structure VI is preferred.
A preferred prodrug is where the cysteine protease inhibitor moiety is e64c (3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxirane-2-carboxylic acid) with R2 the O, coupled to structures I through V. A preferred CDS prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure III, where R6 is a methyl. A preferred CDS prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure IV, where R6 is a methyl and R7 and R8 together form a fused benzyl or pyridyl ring. A preferred CDS prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure V, where R6 is a methyl and R8 and R9 together form a fused benzyl or pyridyl ring.
A CDS preferred prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure III, where X is 0, and R6 is a methyl. A preferred CDS prodrug also is a 1-Methyl-1,4-dihydro-pyridine-3-carboxylic acid 3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxiranecarbonyloxymethyl ester, 1-methyl-1,4-dihydro-pyridine-3-carboxylic acid 1-{3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxiranecarbonyloxy}-ethyl ester, and 3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxirane-2-carboxylic acid 2-[(1-methyl-1,4-dihydro-pyridine-3-carbonyl)-amino]-ethyl ester.
A preferred CDS prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure IV, where X is 0, R6 is a methyl, and R7 and R8 together form a fused benzyl ring. A preferred CDS prodrug is also 3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxirane-2-carboxylic acid 2-[(1-methyl-1,4-dihydro-quinoline-3-carbonyl)-amino]-ethyl ester, 1-ethyl-1,4-dihydro-[1,7]naphthyridine-3-carboxylic acid ({3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxiranecarbonyl}-amino)-methyl ester, and 1-methyl-1,4-dihydro-quinoline-3-carboxylic acid {3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxiranecarbonyl}-amide.
A preferred CDS prodrug is where R1 is the e64c moiety, R2 the O, coupled to structure V, where X is 0, R6 is a methyl, and R8 and R9 together form a fused benzyl ring. A preferred CDS prodrug is also 3-[3-Methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxirane-2-carboxylic acid 2-[(2-methyl-1,2-dihydro-isoquinoline-4-carbonyl)-amino]-ethyl ester.
A derivative of e64, CA074, also referred to as CA-074 (1-{3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carboxylic acid), has been developed having increased specificity for inhibiting cathepsin B. As such, prodrugs of CA074 derivatives are a preferred embodiment. CA074 and its derivatives have been described (see for example, in U.S. Pat. No. 5,281,717, Yamamoto, Tomoo, Matsugi, Hara, In, Murata, Kitamura and Ishida, Structural basis for development of cathepsin B-specific noncovalent-type inhibitor: crystal structure of cathepsin B-E64c complex, Biochim Biophys Acta, 1597(2):244-51, 2002, and Frlan, ibid).
Thus, another preferred R1 has the following structure:
where:
R10 is as defined above,
R13 is any group that results in cysteine protease inhibition; and the “•” in structure VII represents the attachment site of R1 to the prodrug.
In a preferred prodrug, R13 is any group known in the art to result in a cysteine protease inhibitor. Also preferred is when R10 and R13 are each any group known in the art to result in a cathepsin B inhibitor. R13 in preferred prodrug is also a 1-6 alkyl, optionally containing one or more aryl, heteroaryl or heteroatoms, an aryl or heteroaryl. In a preferred prodrug, R13 is propyl group, preferably an n-propyl group. The SS conformer (trans) of the epoxide of structure VII is preferred.
A preferred prodrug is where the cysteine protease inhibitor moiety is CA074 with R2 the O, coupled to structures I through V. A preferred CDS prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure III, where R6 is a methyl. A preferred CDS prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure IV, where R6 is a methyl and R7 and R8 together form a fused benzyl or pyridyl ring. A preferred CDS prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure V, where R6 is a methyl and R8 and R9 together form a fused benzyl or pyridyl ring.
A CDS preferred prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure III, where X is 0, and R6 is a methyl. A preferred CDS prodrug also is a 1-{3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carboxylic acid 1-[(1-methyl-1,6-dihydro-pyridine-3-carbonyl)-amino]-ethyl ester, 1-{3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carboxylic acid 2-[(1-methyl-1,4-dihydro-pyridine-3-carbonyl)-amino]-ethyl ester, and 1-methyl-1,4-dihydro-pyridine-3-carboxylic acid 1-{3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carbonyloxymethyl ester.
A preferred CDS prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure IV, where X is 0, R6 is a methyl, and R7 and R8 together form a fused benzyl ring. A preferred CDS prodrug is also oxirane-2,3-dicarboxylic acid 2-({1-[2-(1-isopropyl-1,4-dihydro-quinoline-3-carbonyl)-aminocarbonyl]-pyrrolidine-1-carbonyl}-2-methyl-butyl)-amide) 3-propylamide, 1-{3-Methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carboxylic acid 2-[(1-methyl-1,4-dihydro-quinoline-3-carbonyl)-amino]-ethyl ester, 1-Propyl-1,4-dihydro-[1,7]naphthyridine-3-carboxylic acid [(1-{(3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carbonyl)-amino]-methyl ester.
A preferred CDS prodrug is where R1 is the CA074 moiety, R2 the O, coupled to structure V, where X is 0, R6 is a methyl, and R8 and R9 together form a fused benzyl ring. A preferred CDS prodrug is also 1-propyl-1,4-dihydro-[1,7]naphthyridine-3-carboxylic acid [(1-{3-methyl-2-[(3-propylcarbamoyl-oxiranecarbonyl)-amino]-pentanoyl}-pyrrolidine-2-carbonyl)-amino]-methyl ester.
Another preferred CDS prodrug of the invention contains a cysteine protease inhibitor moiety that is an azirdine (see for example, Martichonok, Plouffe, Storer, Menard and Jones, Aziridine analogs of [[trans-(epoxysuccinyl)-L-leucyl]amino]-4-guanidinobutane (E-64) as inhibitors of cysteine proteases, J Med Chem, 38(16):3078-85, 1995, Schirmeister, New peptidic cysteine protease inhibitors derived from the electrophilic alpha-amino acid aziridine-2,3-dicarboxylic acid, J Med Chem, 42(4):560-72, 1999, and Schirmeister and Peric, Aziridinyl peptides as inhibitors of cysteine proteases: effect of a free carboxylic acid function on inhibition, Bioorg Med Chem, 8(6):1281-91, 2000). These compounds have been described as cathepsin protease and cathepsin B inhibitors.
Thus, a preferred R1 has the following structure:
where:
R14 and R15 are each any group that results in cysteine protease inhibition; and the “•” in structure VII represents the attachment site of R1 to the prodrug.
In a preferred CDS prodrug, R14 and R15 are each, separately, any group known in the art to result a cysteine protease inhibitor. R14 in a preferred prodrug is H, Boc-phenylalanine or Boc-phenylalaninealanine. R15 in a preferred prodrug is a OH, benzyl ester, isoamyl ester, or methyl ester or leucine-proline, leucine, each optionally containing a benzyl ester, isoamyl ester or methyl ester. In a preferred prodrug in which R1 is structure VIII, R2 is the O. In a preferred prodrug the SS conformer (trans) or the RR conformer (cis) of the aziridine structure is preferred.
A preferred CDS prodrug has the aziridine moiety, structure VIII, with R2 the O, coupled to structures I through V. A preferred CDS prodrug is where R1 is the VIII moiety, R2 the O, coupled to structure III, where R6 is a methyl. A preferred CDS prodrug is where R1 is the structure VIII moiety, R2 the O, coupled to structure IV, where R6 is a methyl and R7 and R8 together form a fused benzyl or pyridyl ring. A preferred CDS prodrug is where R1 is the structure VIII moiety, R2 the O, coupled to structure V, where R6 is a methyl and R8 and R9 together form a fused benzyl or pyridyl ring. A preferred CDS prodrug is where R1 is the VIII moiety, R2 the O, coupled to structure III, where X is 0, R6 is a methyl, R14 is Boc-phenylalanine, and R15 is leucine-proline. A CDS preferred prodrug is where R1 is the VIII moiety, R2 the O, coupled to structure IV, where X is 0, R6 is a methyl, R14 is Boc-phenylalanine, and R15 is leucine-proline. A CDS preferred prodrug is where R1 is the VIII moiety, R2 the O, coupled to structure V, where X is 0, R6 is a methyl, R14 is Boc-phenylalanine, and R15 is leucine-proline.
Other CDS prodrugs include those containing other natural products that are cysteine protease inhibitors, such as imizaridine, tokaramide A, or leupeptin.
In addition to known cysteine protease inhibitors, new compounds previously not known as cysteine protease inhibitor can be identified and used in the CDS prodrugs of the invention. New compounds that are small molecules are preferred with new derivatives of known small molecule cysteine protease inhibitors also preferred. Any assay known in the art can be used to identify new compounds that are cysteine protease inhibitors, cathepsin protease inhibitors or cathepsin B inhibitors (see for example, Hook, Toneff, Aaron, Yasothornsrikul, Bundey and Reisine, β-Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β-secretase activity in chromaffin vesicles, Journal of Neurochemistry, 81:237-256, 2002, and U.S. Pat. Nos. 6,245,884, and 6,627,409). New compounds that reduce activity in such assays can thus be identified as cysteine protease, cathepsin protease, or cathepsin B inhibitors. Moreover, similar assays can be used to determine a compound's affect, or lack thereof, on proteases that are not cysteine proteases in order to determine the specificity of a compound for inhibiting cysteine proteases, cathepsin proteases or cathepsin B. It is understood that such assays would contain the necessary components for enzymatic activity. For example, assays would include reducing conditions because such conditions are known to be required for cysteine protease activity. Moreover, in the case of prodrug testing, assays would include the components necessary for liberation of the active inhibitor from the inactive prodrug.
Assays for measuring enzymatic activity commonly employ a substrate that the protease can cleave and detection of a cleavage product produced by that cleavage. Assays include those in which the substrate contains a structure preferred for cleavage by a particular protease or class of proteases. Such substrates are generally known in the art. The cleavage product detected after cleavage of the substrate is a measure of the enzymatic activity of the protease. Detection of the cleavage product can be by any method known in the art, such as, for example, detection of a compound that fluoresces after cleavage from the substrate. Alternatively, the cleavage product can be determined by mass spectrometry. The cleavage product can also be detected by radioactive immune methods.
Assays include those in which a protease is present as a purified or recombinant cysteine or cathepsin B protease. Cysteine proteases, cathepsin proteases for use in such assays can be identified using various data bases such as, for example, at the web site merops.sanger.ac.uk. Alternatively, assays include those in which other proteases or other biological molecules are present in addition to a cysteine or cathepsin B. In the latter case, the specificity the cleavage can be determined by means known in the art such as, for example, a known preference by the protease for the substrate producing the cleavage product. The inhibition by compounds known to selectively inhibit cysteine proteases, cathepsin proteases, or cathepsin B can also be compared to new compounds to identify appropriate inhibitors. Further, assays in which peptides known to be produced by proteolytic processing by cysteine or cathepsin B activity can be detected and used to screen for compounds that inhibit that activity and thereby select for biologically active moieties of the present invention. Such assays can be cell based assays in which a proteolytic product known to be produced by cysteine protease cleavage is assayed and are well known in the art.
A new compound is considered a cysteine protease inhibitor, cathepsin protease or cathepsin B inhibitor, if the 50% inhibitory concentration (IC50) of the compound is 10 μM or less for cleavage of an appropriate substrate by such a cysteine, cathepsin or cathepsin B protease in an in vitro assay containing the purified or recombinant protease. A better inhibitor has an IC50 of 100 nM in such an assay. The selectivity of a new compound for inhibiting cysteine proteases can be determined by methods known in the art. For example in inhibitory activity of such new compounds on non-cysteine proteases, serine-, aspartic-, and metallo proteases, can be determined. A preferred new selective cysteine protease inhibitor has an IC50 concentration of more than 10 μM for cleavage of an appropriate substrate by a non-cysteine protease in an in vitro assay containing the purified or recombinant protease. But it is understood that cysteine protease inhibitors may inhibit some non-cysteine proteases and still be selective. A new compound that inhibits cysteine proteases is considered selective if does not inhibit the preponderance of non-cysteine proteases. A preferred new selective cysteine protease inhibitor does not inhibit 95% of known non-cysteine proteases. Similarly, a new cathepsin protease inhibitor is selective if it does not inhibit non-cathepsin proteases at the same level. Likewise a new cathepsin B inhibitor does not inhibit non-cathepsin B proteases at that level.
As used here, the term “alkyl” includes a univalent radical containing only carbon and hydrogen atoms arranged in a chain. The term “alkyl,” including “1-6 alkyl”, or “1-10 alkyl”, includes both straight and branched chained alkyl groups. Preferred alkyl groups include 1,1-2,1-3,1-4,1-5,1-6 and 1-10 alkyl groups.
The term “aryl” includes any functional or substituent derived from a simple aromatic ring. A preferred alkyl is a phenyl group.
A term including the phrase “hetero,” such as in “heteroaryl,” or the phase “substituted heteroatoms” means that the compound includes atoms other just than carbon and hydrogen. Preferred heteroatoms include O, N, and S. Thus, for example, a preferred heteroaryl would include a pyridine group.
The phrase “one or more” includes at least one but not more than the maximum possible based on chemistry. Preferred among the phrase “one or more” is 1,1-2,1-3, and 1-4.
The phrase “any group that results in cysteine protease inhibition” includes any substituent, which results in a compound that inhibits cysteine protease activity when metabolized from the prodrug. Substituents that result in such inhibitors can be readily determined by any means known the art for doing so, such as by testing a compound containing the substituent for inhibition in an appropriate protease activity assay such as those described herein. Such substitutents are those that result in compounds, which can be synthesized using known methods or obvious variants of those methods to make them.
The phrase “any group known in the art to result a cysteine protease inhibitor” includes a substituent, which is known in the art to result in a compound or a structurally similar compound that inhibits a cysteine protease. Such compounds include those cited in references herein. A “structurally similar compound” is one which would be expected to inhibit cysteine proteases based on its structural similarity to compounds known to inhibit cysteine proteases using any means known and especially those in the fields of medicinal chemistry or pharmacology.
The epoxy and aziridine structures described herein include the stereochemistry inherent in them, that results in various conforms of the structures, including configurations of (R,R), (S,R), (R,S), and (S,S). The present invention includes all of these conformations. In addition, some cysteine protease inhibitors contain an asymmetric carbon atom at positions in addition to the epoxy and aziridine structures. In those cases, both R and S conforms are included in the invention.
Methods of making the CDS prodrugs of the invention are well known in the art (see for example, references cited above in relation to CDS) and various CDS prodrug synthesis is outlined in Schemes I through IV. But it is understood that modifications can be made to this general approach in order to obtain the desired CDS prodrug. Further, the synthesis of CDS prodrug species is provided in Examples 2 and 3.
Methods of making the cysteine protease inhibitor moiety of the prodrug are also well known in the art. For example, chemical synthesis of many small molecule cysteine protease inhibitors, especially the expoxide and aziridine inhibitors, are known and provided in the references cited above. In addition, the synthesis of e64c and CA074 moieties is described in Examples 2 and 3. Methods of making natural product cysteine protease inhibitors, such as the aziridine mizaridine, and aldehyde inhibitors, tokaramide A, and leupeptin, include their production by an organism that makes such inhibitors and the subsequent purification of the inhibitor. Such organisms can be those found in nature or those genetically engineered to enhance production or modify the structure of the natural product. Methods of purifying the natural product inhibitor are very well known in the art.
In Scheme 1, the carboxyl group of the cysteine protease inhibitor moiety is reacted with chloromethyl chlorosulfate to produce the chloromethyl ester. Reaction of this ester with the potassium salt of pyridine-3-carboxylic acid provides the pyridine ester which in turn is methylated and reduced with sodium dithionite to provide the CDS prodrug.
Scheme II depicts the synthesis of an ethyl-bridged CDS prodrug. Reaction of the carboxyl group of the cysteine protease inhibitor moiety with iodoethanol provides the hydroxyethyl ester. Reaction with nicotinic anhydride provides the pyridine compound which is processed to the CDS prodrug via alkylation and reduction.
In scheme III, nicotinic amides are prepared by reacting the carboxyl group of the cysteine protease inhibitor moiety with various known (hydroxyalkyl)nicotinamides followed by methylation of the pyridine nitrogen and reduction to the CDS ester.
A similar process is followed to prepare the 4-isoquinolinecarboxamides as depicted in scheme IV.
The invention is also directed to pharmaceutical compositions containing the prodrugs described above. Pharmaceutical compositions suitable for prodrugs of the invention are generally well known in the art (see, for example, Allen, L. V., Popovich, N. G., and Ansel, H., “Ansel's Pharmaceutical Dosages Form and Drug Delivery Systems, Lippincott, William and Wilkins, Pub., 2004, ISBN 0-7817-4612-4, and Gennaro, A., Remington: The Science and Practice of Pharmacy,” 20th ed., Lippincott, Williams and Wilkins, Pub., 2000, ISBN 0-7817-4673-6). Moreover, the special considerations relating to prodrug pharmaceutical compositions are specifically known in the art (see for example, Testa, B., and J. M. Mayer, ibid.).
The prodrug of the invention may be administered orally, topically, parenterally, rectally or by inhalation spray in dosage forms or formulations containing conventional, non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The formulation and preparation of any of this broad spectrum of dosage forms into which the subject prodrugs can be disposed is well-known to those skilled in the art of pharmaceutical formulation.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide a pharmaceutically elegant and palatable preparation.
Formulations for oral use include tablets which contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium chloride, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, potato starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions usually contain the active materials in admixture with appropriate excipients. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally-occurring phosphatide, for example, lecithin; a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol; a condensation product of ethylene oxide with a partial ester derived from fatty acids and a hexitol such as polyxyethylene sorbitol monooleate; or a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate; one or more colouring agents; one or more flavouring agents; and one or more sweetening agents such as sucrose or saccharin.
Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example, liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soybean lecithin; and esters including partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents, The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic monoor diglycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
The pharmaceutical compositions may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable nonirritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug, for example, cocoa butter, or adeps solidus polyethylene glycols.
From the foregoing description, one of ordinary skill in the art can easily ascertain the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and/or modifications of the invention to adapt it to various usages and conditions. As such, these changes and/or modifications are properly, equitably and intended to be within the full range of equivalence of the following claims.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 5 mg to 5 gm of the active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 to about 95% of the total composition. Other dosage forms such as ophthalmic dosage forms contain less active ingredient such as for example from 0.1 mg to 5 mg. Dosage unit forms will generally contain between from about 0.1 mg to about 500 mg of active ingredient.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general heath, sex, diet, time of administration, route of administration, rate of excretion, drug combination and severity of the dementia.
Dementia is the progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging. Particularly affected areas may be memory, attention, language and problem solving, although particularly in the later stages of the condition, affected persons may be disoriented in time (not knowing what day, week, month or year it is), place (not knowing where they are) and person (not knowing who they are).
The cost of caring for dementia patients is estimated to be $156 billion by 2003 world-wide. That cost is expected to increase substantially as the population of industrialized nations ages, because dementia is a disease of the aged.
Dementia is a non-specific term that encompasses many disease processes with the leading cause of dementia due to Alzheimer's disease (AD). AD is a neurodegenerative disease characterized by progressive cognitive deterioration together with declining activities of daily living and neuropsychiatric symptoms or behavioral changes. The most striking early symptom is short term memory loss (amnesia), which usually manifests as minor forgetfulness that becomes steadily more pronounced with illness progression, with relative preservation of older memories. As the disorder progresses, cognitive (intellectual) impairment extends to the domains of language (aphasia), skilled movements (apraxia), recognition (agnosia), and those functions (such as decision-making and planning) closely related to the frontal and temporal lobes of the brain as they become disconnected from the limbic system, reflecting extension of the underlying pathological process. The AD pathological process consists principally of neuronal loss or atrophy, principally in the temporoparietal cortex, but also in the frontal cortex, together with an inflammatory response to the deposition of amyloid plaques and neurofibrillary tangles.
There is currently no cure for AD, although there are drugs that offer symptomatic benefit, specifically with respect to short-term memory impairment. Unfortunately current drugs produce only temporary effects and do not stop the ultimate progression of the disease. Current FDA approved drugs for AD include, for example, acetylcholinesterase inhibitors and NMDA antagonists.
AD, and the resulting dementia, is thought to be caused by the abnormal accumulation of β-amyloid peptides (Aβ) in the brain. Aβconsist of 3 peptides, Aβ040, Aβ42 and Aβ043, with Aβ40 and Aβ042 the more abundant and important forms. AD are a primary component of senile plaques that form in AD brain and which are a hallmark of the disease. Aβ are neurotoxic and result in neuronal cell death and the pathological neuronal structures seen in AD brain, called neurofibrillary tangles, which are also characteristic of the disease. Moreover, individuals carrying genetic mutations, which result in increased brain Aβ, develop AD at earlier age than the AD patient population generally. As such, reducing brain Aβ are thought to be effective at stopping the progression of AD and may reverse dementia that has already occurred.
Recently, cysteine protease inhibitors, generally, and cathepsin B inhibitors, specifically, have been shown to reduce Aβ production in vitro and by cells secreting Aβ (see, for example, U.S. Patent Application Publication 2004/0248232, Hook and Reisine, Cysteine Proteases Are the Major β-Secretase in the Regulated Secretory Pathway That Provides Most of the β-Amyloid in Alzheimer's Disease: Role of BACE 1 in the Constitutive Secretory Pathway, Journal of Neuroscience Research, 74:393-405, 2003, and Hook, Toneff, Bogyo, Medzihradszky, Nevenu, Lane, Hook and Reisine, Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer's disease Biological Chemistry, 386:931-940, 2005). Aβ are proteolytically produced by cleavage of a precursor protein called amyloid precursor protein (APP). Cysteine proteases and cathepsin B, specifically, are thought to be, in part, responsible for that proteolytic processing and thus cysteine protease and cathepsin B inhibitors reduce Aβ by reducing proteolytic production. As such, cysteine protease and cathepsin B inhibitors are potentially effective AD agents. In particular, cathepsin B specifically is thought to cut the Aβ amino terminal end and is thus, thought to be a β-secretase, the term used in the art to refer to those proteases that cleave the amino terminal end of Aβ. All three Aβ have the same amino terminal end and differ only at the carboxyl terminal end. Moreover, cysteine proteases and cathepsin B specifically is thought to act as a β-secretase in the neuronal regulated secretory pathway, the pathway by which most Aβ is thought to be produced.
But the effectiveness of cysteine protease inhibitors or cathepsin B inhibitors at reducing brain Aβ had not been validated in vivo. As such, there was some question as to whether cysteine protease inhibitors or cathepsin B inhibitors would be useful for treating AD. Here, cathepsin inhibitors and cathepsin B inhibitors, specifically, are validated as effective at reducing brain Aβ in vivo and, as such, are effective for treating AD. Using the guinea pig animal model of APP processing, the general cysteine protease inhibitor, e64d (also known as loxistatin, EST, and oxiranecarboxylic acid, 3-[[[(1S)-3-methyl-1-[[(3-methylbutyl)amino]carbonyl]butyl]amino]carbonyl], ethyl ester) and ethyl ester of e64c L-Proline, N[[(2S,3S)3[(propylamino)carbonyl]oxiranyl]carbonyl]-L-isoleucyl-, methyl ester) significantly reduces brain Aβ after direct infusion into the brain (icv) for 7 or 30 days. E64d and CA074Me are the ethyl and methyl esters of e64c and CA074, respectively. These esters are metabolically converted to e64c and CA074 upon administration. The results are shown in Examples 4 through 8.
The guinea pig is an excellent model of human APP processing and human Aβ production (see, for example, Beck, Bigl and Rossner, Guinea pigs as a nontransgenic model for APP processing in vitro and in vivo, Neurochem Res, 28(3-4):637-44, 2003). Using that model, cholesterol lowering statins drugs were found to reduce brain Aβ by similar amount shown here for cysteine potease and cathepsin B inhibitors. Based on the results from the guinea pig model, statins have subsequently gone into clinical trials for AD treatment (see for example Fassbender, Simons, Bergmann, Stroick, Lutjohann, Keller, Runz, Kuhl, Bertsch, von Bergmann, Hennerici, Beyreuther and Hartmann, Simvastatin strongly reduces levels of Alzheimer's disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo, Proc Natl Acad Sci USA, 98(10):5856-61, 2001 and www.clinicaltrials.gov, Lipitor, or Simvastatin). As such, the results here reasonably predict that cysteine protease inhibitors and cathepsin B inhibitors are effective AD therapeutics.
The second leading cause of dementia is vascular dementia (VD), also known as ischemic vascular dementia or multi-infarct dementia. These terms refer to a group of syndromes caused by different mechanisms all resulting in vascular lesions in the brain. The main subtypes of VD described at the moment are: vascular mild cognitive impairment, multi-infarct dementia, vascular dementia due to a strategic single infarct, vascular dementia due to hemorrhagic lesions, small vessel disease (which includes vascular dementia due to lacunar lesions and Binswanger disease), and mixed AD and VD. Vascular lesions can be the result of diffuse cerebrovascular disease or focal lesions (or a combination of both, which is what is observed in the majority of cases). Mixed dementia is diagnosed when patients have evidence of AD and cerebrovascular disease, either clinically or based on neuroimaging evidence of ischemic lesions. In fact VD and AD often coexist, especially in older patients with dementia.
For VD, the prevention of further cerebrovascular lesions is the primary therapy. This includes administering antiplatelet drugs and controlling major vascular risk factors (hypertension, hypercholesterolemia, smoking and diabetes mellitus to mention a few). The ischemia that results from VD causes neuronal cell death and resulting dementia. Reducing the neurological damage caused by the ischemia is thought to be effective for treating dementia.
Cysteine protease inhibitors and cathepsin B inhibitors have been shown effective in protecting brain neurons during ischemia. In particular, e64c and CA074 have been shown to protect hippocampus neurons from damage in a primate ischemic animal model (see for example, Tsuchiya, Kohda, Yoshida, Zhao, Ueno, Yamashita, Yoshioka, Kominami and Yamashima, Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors, Exp Neurol, 155(2):187-94, 1999, and Yoshida, Yamashima, Zhao, Tsuchiya, Kohda, Tonchev, Matsuda and Kominami, Primate neurons show different vulnerability to transient ischemia and response to cathepsin inhibition, Acta Neuropathol (Berl), 104(3):267-72, 2002). Thus, these results reasonably predict that cysteine protease inhibitors and cathepsin B inhibitors are effective for treating neuronal cell death due to ischemia and VD.
Moreover, cysteine proteases and cathepsin B are useful therapeutic targets because their inhibition is not lethal. For example, e64d, a general cysteine protease inhibitor also known as loxistatin and EST, successfully passed Phase I clinical trials in Japan (see for example Miyahara, Shimojo, Toyohara, Imai, Miyajima, Honda, Kamegai, Ohzeki and Kokatsu, Phase I tudy of EST, a new thiol protease inhibitor—the 2nd report: safety and pharmacokinetics in continuous administration, Rinsho Yakuri, 16(3):537-546, 1985). Those studies were in preparation for its use in treating muscular dystrophy. However, after a Phase III study, further testing was halted due, in part, to a low efficacy for muscular dystrophy (see for example, Scrip, 1765:11, 1992). Cathepsin B inhibitors are also not likely to be lethal, because mice in which cathepsin B has been knocked out appear and behave normally (see for example, Reinheckel, Deussing, Roth and Peters, Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L, Biol Chem, 382(5):735-41, 2001).
Nonetheless, there are concerns about the toxicity of cysteine protease inhibitors, especially those such as the expoxide and arizidine compounds that covalently bind to the protease. Such covalent binding is thought to make the compound more likely to cause toxic side effects, especially in the liver. And indeed, at high dose, liver toxicity was found in rats using e64d (see for example Fukushima, Arai, Kohno, Suwa and Satoh, An epoxysuccinic acid derivative (loxistatin)-induced hepatic injury in rats and hamsters, Toxicol Appl Pharmacol, 105(1):1-12, 1990). Such concerns are magnified in the treatment of AD and VD patients because these patients are elderly and often frail.
The CDS prodrugs of the present invention provide significant advantages in the treatment of these patients because of significantly lower systemic toxicity. As discussed above, the CDS prodrugs of the invention are not reactive in and rapidly cleared from the systemic system, thereby minimizing hepatic exposure. Moreover, CDS prodrugs are targeted to the central nervous system where they provide sustained release of the inhibitors further reducing toxic concerns because of reduced effective doses in the circulation.
The CDS prodrugs of the invention thus, can be used to treat, which includes to ameliorate or prevent, dementia, AD and VD. It is understood that in these conditions and diseases, even temporary reduction in symptoms or a delay in the progression of the disease is significant. Moreover, it is also appreciated that these conditions take a long time to develop and thus methods that prevent the condition or disease are also with the scope of the invention.
The effectiveness of the CDS prodrugs in treating these conditions and diseases can be assessed by any means known in field (see for example www.clinicaltrials.gov). For example, cognitive tests have been developed for such, including, for example, the Mattis Dementia Rating Scale and the Mini-Mental State Examination. Neuroimaging methods have also been developed for evaluating dementia, AD and VD and can be used such as, magnetic resonance imaging and positron emission tomography. Further, biomarkers associated with these conditions and diseases can be monitored by any means known, such as imaging or chemical analysis, to determine the effect of the CDS prodrugs. For example, Aβ is a biomarker, which can be monitored. Of course, brain cysteine protease activity and cathepsin B activity can also be monitored to assess effectiveness. Moreover, methods such as bioinformatics and proteomics can be used determine other substances that are biomarkers. For example, in AD, proteomic analysis has found that cerebral spinal fluid levels of cathepsin B are elevated in AD (see for example Zhang, Goodlett, Quinn, Peskind, Kaye, Zhou, Pan, Yi, Eng, Wang, Aebersold and Montine, Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease, J Alzheimers Dis, 7(2):125-33; discussion 173-80, 2005). A ventriculoperitonial shunt is useful for obtaining cerebrospinal fluid in order to assess such biomarkers directly.
This example demonstrates the release of a cysteine protease inhibitor from the prodrug. In this case, the cysteine the protease inhibitor is e64c (3-[3-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-oxirane-2-carboxylic acid). This inhibitor contains a carboxylic acid group at the epoxide ring. A prodrug form of e64c as incorporated into structure I has the following structure:
In this case, R2 is O and, upon metabolism, the carbonyl-R2 group becomes the carboxylic acid group of e64c and a functional inhibitor having the following structure is released:
This example provides a synthesis of the protease inhibitor e64c and various exemplary CDS prodrugs of e64c.
This example provides a synthesis of the protease inhibitor CA074 and various exemplary CDS prodrugs of CA074.
Synthesis of:
Dipeptide Cbz-Ile-Pro-Et (3)
To a stirred solution of triethylamine (26.9 mL, 195 mmol) in CH2Cl2 (350 mL), ethyl L-proline 1(18.276 g), Cbz-Ile 2 (34.49 g, 130 mmol), DMAP (1.612 g, 13.0 mmol), HOBt (26.34 g, 195 mmol) and EDC (37.2 g, 195 mmol) were added. After stirring overnight, the solvent was removed in vacuo. The residue obtained was redissolved in EtOAc and washed with citric acid aq (10%) and then saturated NaHCO3 and brine. The organic phase was dried over anhydrous MgSO4 and concentrated. Purification by flash chromatography with petroleum ether/EtOAc (8:1, v/v) gave the desired product 3 18.37 g (83%) as yellow oil, ES-MS: 391.2 [M+H+].
Ile-Pro-Et (4)
The dipeptide product 3 obtained above (13.0 g, 33.3 mmol) was hydrogenated in MeOH (150 mL) over 10% Pd/C (1.3 g). The catalyst was filtered off though a celite column and the solvent was removed in vacuo to provide compound 4, ES-MS: 257.2 [M+H+], which was used directly and rapidly in the next step without further purification.
Diethyl 2-acetoxy-3-bromosuccinate (6)
30% HBr/AcOH (440.5 mL) was added dropwise with stirring to D-(−)-tartrate 5 (150 g, 727.87 mmol) in an ice bath. Then the mixture was stirred overnight at room temperature. Ice water 700 mL was added to the mixture carefully. The aqueous phase was extracted with ether (8×300 mL). The combined ether layer was washed with water (2×200 mL) and brine (2×200 mL), dried over anhydrous MgSO4, concentrated in vacuo to provide compound 6 as an oil (194 g, 94%), ES-MS: 311.1 [M+H+], which was pure enough for the next step.
Diethyl 2-bromo-3-hydroxysuccinate (7)
To a solution of compound 6 (194 g, 626 mmol) in EtOH (730 mL), 30% HBr/AcOH (71 mL) was added dropwise. Then the reaction mixture was stirred under reflux for 4 h. After concentration in vacuo, the crude oil was distilled under reduced pressure. The desire product was collected with bp 125-134° C./4 mmHg (133.25 g, 79%), ES-MS: 269.0 [M+H+].
(2S,3S)-diethyl oxirane-2,3-dicarboxylate (8)
Mental sodium (13.334 g, 579 mmol) was dissolved carefully in an absolute EtOH (332 mL) and the solution was cooled to 0-5° C. in an ice bath. A solution of compound 7 (133.25 g, 497 mmol) in EtOH (115 mL) was added dropwise. After stirring at room temperature for 2 h, HOAc (100 mL) was added. After concentration in vacuo at room temperature, ice water (500 mL) was added and then extracted with ether (6×300 mL). The combined organic phase was washed with brine, dried over anhydrous MgSO4, concentrated in vacuo. Distillation under reduced pressure gave the pure product with bp 100-124° C./3 mmHg (60.7 g, 65%), ES-MS: 189.2 [M+H+].
(2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (9)
KOH (18.061 g, 322 mmol) was dissolved in absolute EtOH (300 mL). With stirring, a solution of compound 8 (60.659 g, 322 mmol) in absolute EtOH (466 mL) was added dropwise over 2 h at 0-5° C. After continuous stirring at room temperature for 2 h, the reaction mixture was concentrated in vacuo at room temperature and the residue was diluted with ether (2000 mL). The undissolved solid was filtered off and the solvent was evaporated to dryness to give a white solid (53 g). 5% KHSO4 was added to acidify the white solid and the solution was saturated with solid NaCl. The free acid was extracted with EtOAc (6×300 mL) and the combined organic phase was washed with water (2×300 mL), dried over anhydrous MgSO4. After concentration, the crude product was obtained as an oil (40.69 g, 79%), ES-MS: 161.1 [M+H+], which was used directly in the next step without further purification.
(2S,3S)-ethyl 3-(propylcarbamoyl)oxirane-2-carboxylate (10)
Monoethyl (2S,3S)-trans-epoxysuccinate 9 (10 g, 62 mmol) and n-PrNH2 (7.387 g, 10.361 mL, 62 mmol) was mixed in CHCl3 (350 mL). The mixture was cooled to 0-5° C. in ice bath. DMAP (0.775 g, 6 mmol), HOBt (8.435 g, 62 mmol), NMM (6.953 g, 7.616 mL, 69 mmol) and EDC·HCl (13.176 g, 69 mmol) were added. After stirring overnight, the reaction mixture was concentrated in vacuo. The residue was redissolved in EtOAc (600 mL) and washed with 5% KHSO4 aq., saturated NaHCO3 (2×100 mL) and brine (2×100 mL), dried over anhydrous MgSO4. Concentration and purification by recrystallization from petroleum ether/EtOAc afforded the desired compound 11 (10.6 g, 35%) as white solid, Mp 84-85° C. ES-MS: 202.1 [M+H+]; 1H NMR (CDCl3) δ: 6.10 (br s, 1 H), 4.26 (qd, J=7.5, 1.8 Hz, 2H), 3.68 (d, J=2.1 Hz, 1H), 3.47 (d, J=2.1 Hz, 1H), 3.23 (q, J=6.9 Hz, 2H), 1.59-1.47 (m, 2H), 1.31 (t, J=7.5 Hz, 3H), 0.92 (t, J=7.5 Hz, 3H).
(2S,3S)-3-(propylcarbamoyl)oxirane-2-carboxylic acid (11)
A solution of KOH (3.028 g, 54 mmol) in EtOH (250 mL) was added dropwise to the solution of compound 10 (5.437 g, 27 mmol) in EtOH (185 mL) at 0-5° C. with stirring. After completion, the solution was warmed to room temperature and stir was continued for 1 h. The solvent was evaporated in vacuo at room temperature and diluted with water (300 mL). The solution was acidified with 5% KHSO4 and saturated with NaCl, then extracted with EtOAc (5×120 mL). The combined organic phase was washed with brine (2×100 mL), dried over anhydrous MgSO4. After concentration, the desired product was obtained as an oil (4.4 g, 95%), ES-MS: 174.0 [M+H+], which was used directly in the next step without further purification.
Coupling with EDC/HOBt: Synthesis of Compound 12
To a solution of the mono-amidated epoxysuccinate (1.687 g, 19 mmol) and dipeptide 4 (5 g, 19 mmol) in CHCl3 (200 mL), HOBt (1.318 g, 19 mmol), Et3N (1.086 g, 1.496 mL, 21 mmol) and DMAP (0.121 g, 2 mmol) were added. After cooling in an ice bath EDC (2.057 g, 21 mmol) was added and the reaction mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the residue was dissolved in EtOAc (600 mL). The organic phase was washed with 5% KHSO4 aq., saturated NaHCO3, brine (2×100 mL), and then dried over anhydrous MgSO4. After concentration, the residue was purification by flash chromatography with petroleum ether/EtOAc afforded compound 12 as an oil (3.878 g, 75%), ES-MS: 412.2 [M+H+].
Synthesis of Compound 13
To a solution of Compound 12 (2.305 g, 5.6 mmol) in THF (200 mL) and water (100 mL), LiOH·H2O (0.471 g, 11.2 mmol) was added. After stirred for 5 h, the reaction solution was acidified with 5% KHSO4 to Ph 2 and then saturated with NaCl. The aqueous phase was extracted with EtOAc, washed with water and brine, dried over anhydrous MgSO4 and evaporated to afford compound 13 (1.8 g, 90%), ES-MS: 384.1 [M+H+].
As shown in
The following procedures are those of used in obtaining the data of the Examples 4-8.
In vivo icv administration of protease inhibitors into guinea pig brains. Guinea pigs (Harley strain, 300-500 grams) were obtained from Charles River (Wilmington, Mass.). The animals were given free access to food and water before and during the experiment. An ALZET osmotic mini-pump (ALZET company, Cupertino, Calif.) was implanted subcutaneously in the mid-scapular area of the back of each animal. A catheter was connected to the osmotic mini-pump and inserted into the lateral ventricle of the brain (using a brain infusion kit manufactured by ALZET).
E64d or CA074Me (from CalBiochem, San Diego, Calif.) were each administered to guinea pig brains by icv minipump infusion at a rate of 2.5 ml/hr with each compound at 1 mg/ml in saline (with 1.5% DMSO). Each e64d and control group consisted of five animals; each CA074Me and control group consisted of ten animals. Continuous infusion was conducted for 7 days and 30 days. At the end of each infusion period, animals were sacrificed and half the brain was prepared as extracts for ELISA measurement of Aβ peptide levels conducted in triplicate with Ab ELISAs (from Biosource International). The other half brain was used for isolation of synaptosome preparations and Ab analyses.
Brain Extracts. Brain extracts were prepared by subjecting fresh tissues to homogenization at 1:10 w/v in guanidine buffer consisting of 5 M guanidine-HCl in 50 mM Tris-HCl, pH 8.3 (buffer reagents from Sigma-Aldrich, St. Louis, Mo.). Homogenates were agitated at room temperature for 3-4 hours, then stored at −20° C. The day before the ELISA assay, the tissue samples were diluted further by 1:10 for a final guanidine concentration of 0.5 M. These diluted homogenates were then centrifuged at 14,000 rpm for 20 minutes at 4° C. and supernatants were collected for measurement of total Ab, and Aβ40 and Aβ42 separately, by ELISA assay. Isolation of synaptosomes from brain. Synaptosomes were prepared by homogenization of fresh brain tissue in isotonic sucrose (320 mM), pH 7.4, 1 mM EDTA, 0.25 mM dithiothreitol, and 30 U/ml RNAse inhibitor. The homogenate was centrifuged at 1000×g for 10 min. The supernatant was loaded on a Sucrose-Percoll discontinuous gradient and then centrifuged at 32,000×g for 5 min. Enriched synaptosomes were washed in PBS, and centrifuged at 12,000×g for 4 min. The supernatant was carefully removed, and the synaptosome-containing pellet was resuspended in 50% OptiPrep (Accurate Chemical, Westbury, N.Y.), loaded to an OptiPrep discontinuous flotation gradient (9, 12.5, 25, and 35%) and centrifuged at 10,000×g for 20 min. The synaptosomes were obtained at the 15-25% interface. Synaptosomes were washed in PBS and then extracted as described for brain extracts. Aβ analyses by ELISA assays. For quantitation of Aβ peptides, ELISA assays were used to measure Aβ levels. For measurement of total Aβ, consisting of Aβ40 and Aβ42, ELISA assay was used (88-344/88-348 from Biosource International, Camarillo, Calif.). For measurement of Aβ40 and Aβ42, specific ELISA assays were used respectively (88-348 and 88-344 from Biosource International). Brain and synaptosome extracts were subjected to ELISA assays as samples diluted in 0.5 M guanidine. Analyses of CTFβ and sAPPa fragments. CTFβ fragments derived from APP were analyzed by western blots with antibody MAB 1560 (Chemicon, Temecula, Calif.). The sAPPα fragment derived from APP was analyzed by western blots with antibody MAB 343 (Chemicon, Temecula, Calif.). Relative amounts of CTFb and sAPPa bands on western blots were achieved densitometry.
Data Analyses. Data were analyzed by one-way analysis of variance for analyses of statistical significance of the effects of compounds on Ab levels, with p<0.05 indicating statistical significance, using GraphPad Prism software program. Following ANOVA analyses among treatment groups, individual group values for Ab levels were analyzed using Dunnett's multiple comparison test. To facilitate comparisons among all studies, data are presented as percent of the corresponding vehicle control ±standard error of the mean (SEM).
In
CTFβ is a proteolytic product of β-secretase cleavage of APP and is direct measure of P-secretase activity.
sAPPα is a proteolytic of α-secretase cleavage of APP. The α-secretase cleaves APP with the Aβ sequence and thereby precludes Aβ formation. α-secretase and β-secretase can be, therefore, seen as competing for the same APP substrate.
Each of the references cited above is expressly incorporated herein in its entirety. Although the invention has been described with references to the examples provided above, modifications can be made without departing from the invention. Accordingly, the invention is limited only by the claims.
This invention was made with United States Federal government support under grant number 2 R44AG18044, awarded by the National Institutes of Health. The Federal government may have certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/38387 | 9/28/2006 | WO | 00 | 3/26/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60722066 | Sep 2005 | US | |
| 60772631 | Feb 2006 | US |
