WO1993013768A1

WO1993013768A1 - Therapeutic uses of thiol-oxidizing agents and sulfhydryl-alkylating agents

Info

Publication number: WO1993013768A1
Application number: PCT/US1993/000045
Authority: WO
Inventors: Manfred Weigele; Richard D. Klausner
Original assignee: Ariad Pharmaceuticals, Inc.
Priority date: 1992-01-06
Filing date: 1993-01-05
Publication date: 1993-07-22

Abstract

The present invention relates to the therapeutic uses of thiol-oxidizing agents and of sulfhydryl-alkylating agents. The sulfhydryl-alkylating agents are maleimide and its derivatives. Therapeutic compositions comprising such agents are also provided. In a specific embodiment, the therapeutic agent is a thiol-oxidizing agent such as a diazene dicarbonyl compound. In another embodiment, the therapeutic agent is maleimide or a derivative thereof. In yet another embodiment, the invention provides methods of treating cystic fibrosis, by administering a therapeutic agent of the invention.

Description

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THERAPEUTIC USES OF THIOL-OXIDIZING AGENTS AND SULFHYDRYL-ALKYLATING AGENTS

1. INTRODUCTION The present invention relates to thiol- oxidizing agents and sulfhydryl-alkylating agents, and iff use of such agents for treatment of diseases and disorders, particularly those involving a defective cell membrane, lysosomal, or secretory protein.

10

2. BACKGROUND OF THE INVENTION The synthesis and maturation of secretory, lysosomal, and membrane proteins in vertebrate cells involves the participation of various subcellular

15 organelles. After synthesis in the rough endoplasmic reticulum, the protein is moved to the Golgi complex, and then sorted to lysosomes or the plasma membrane or secretory vesicles.

Ribosomes are complexes that carry out

20 protein synthesis within the cell by reading the three letter genetic code (codon) of each messenger RNA. The endoplasmic reticulum (ER) is an interconnected series of flattened, generally layered, sacs within the cell. Ribosomes that are synthesizing secretory

25 and integral membrane (ER, Golgi, and plasma membrane) proteins are tightly bound to the membrane of the ER (which is termed the rough ER with such bound ribosomes) . Secretory proteins are transported across

30 the ER membrane into the lumen, or cisterna, of the ER during synthesis; membrane proteins become inserted into the ER membrane during synthesis. A signal seguence, characteristically near the N-terminus of the newly synthesized protein and consisting of one or

35 more positively charged amino acids followed by 6-12 continuous hydrophobic residues, directs a protein to d the ER, and inserts itself into the ER membrane, with the aid of the signal recognition particle. The signal seguence is cleaved off by signal peptidase, localized in the lumen of the ER. Other topogenic seguences within membrane proteins, e.g., stop- transfer membrane anchor seguences, function to orient the protein within the membrane. The protein traverses the ER membrane in an unfolded state. After insertion into or traversal of the ER membrane, the newly synthesized proteins can undergo additional maturation modifications in the ER lumen, including formation of disulfide bonds and proper folding of the protein, formation into oligomers, and addition and modification of carbohydrates. Disulfide bonding stabilizes the tertiary structure of proteins, and is important for proper maturation and activity of the protein. Formation of multi-chain oligomeric proteins from their subunit constituents also occurs in the ER. Polypeptides that are misfolded are prevented from moving out of the ER and proceeding along their normal •maturation pathway; such proteins either accumulate or are degraded in the ER via an active degradative pathway (Stafford and Bonifacino, 1991, J. Cell Biol. 115(5) .1225-1236; Klausner and Sitia, 1990, Cell 62:611-614; Bonifacino and Lippincott-Schwartz, 1991, Curr. Opin. Cell Biol. 3:592-600).

In eukaryotes, glycosylation of proteins can be classified as O-linked (linked to the hydroxyl group oxygen of serine, threonine, and in collagen, hydroxylysine) or N-linked (linked to the amide nitrogen of asparagine) . Glycosyltransferases are enzymes that catalyze the transfer of sugar to newly synthesized proteins; a different type of glycosyltransferase catalyzes the addition of specific sugars. All known glycosyltransferases are integral membrane proteins with their active sites within the lumen of the ER or Golgi, where sugar transfer thus occurs.

All N-linked oligosaccharides are 5 synthesized from a common precursor in the ER. In the i* lumen of the ER, the complete branched oligosaccharide, consisting of three glucose, nine mannose, and two N-acetylglucosamine molecules, is transferred by the enzyme oligosaccharyltransferase

₁₀ from oligosaccharylpyrophosphoryldolichol to an asparagine residue in an -Asn-X-Ser/Thr- acceptor site (where X is any amino acid except proline) on the nascent protein (Czichi et al. , 1977, J. Biol. Chem. 252:7901-7904; Hart et al., 1979, J. Biol. Chem.

15 254:9747-9753). Oligosaccharyltransferase is a luminally oriented integral membrane protein of the ER, and the glycosylated protein formed by transfer of the oligosaccharide is seguestered within the endoplasmic reticulum (Hanover and Lennarz, 1980, J.

20 Biol. Chem. 255:3600-3604). An .in vitro study has shown that amino-terminal derivatives of Asn-Leu-Thr can act as substrates for oligosaccharyltransferase, while asparagine derivatives of the tripeptide were inactive as substrates or inhibitors of the enzyme

²⁵ (Welply et al., 1983, J. Biol. Chem. 258:11856-11863). A study has suggested that transport from the ER to the cell surface is an unselective process, by comparing the rate of transport of exported proteins with that of an intracellular bulk phase marker; the

30 bulk phase marker used was a tripeptide derivative containing the Asn-X-Ser/Thr acceptor site for glycosylation ( ieland et al. , 1987, Cell 50:289-300).

Immediately after transfer of the oligosaccharide to the protein, catalyzed by

35 oligosaccharyltransferase, certain sugar residues are 1 removed by different enzymes. Further processing of the N-linked oligosaccharide, to the high-mannose or complex form, is completed in the Golgi vesicles. The glycoprotein is transported via transport vesicles from the cis Golgi to the trans Golgi to the trans Golgi reticulum, from where it is sorted to lysosomes or to transport vesicles, or secretory vesicles which eventually fuse with the plasma membrane.

For a general discussion of the foregoing, see Darnell et al., 1990, Molecular Cell Biology. 2d Ed., .H. Freeman & Co., New York, pp. 639-680; Pfeffer and Rothman, 1987, Ann. Rev. Bioche . 56:829- 852.

3. SUMMARY OF THE INVENTION

The present invention relates to the therapeutic uses of thiol-oxidizing agents, and of sulfhydryl-alkylating agents such as maleimide and its derivatives. Therapeutic compositions comprising such agents are also provided. In a specific embodiment, the therapeutic agent is a^"thiol-oxidizing agent such as a diazene dicarbonyl compound. In another embodiment, the therapeutic agent is maleimide or a derivative thereof. In yet another embodiment, the invention provides methods of treating cystic fibrosis, by administering an effective amount of a therapeutic agent of the invention.

4. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to thiol- oxidizing agents and to sulfhydryl-alkylating agents, and therapeutic uses cf the foregoing. Therapeutic methods and compositions are provided. The therapeutic agents of the invention are biocompatible (nontoxic and not highly immunogenic) , and permeable to cell membranes. In a preferred aspect, the therapeutic agent is used in the treatment of a disease or disorder involving a defective cell membrane (plasma, ER, or Golgi) , lysosomal, or secretory protein. In a specific embodiment, the therapeutic agent is an agent that prevents the abnormal misfolding, assembly or increased levels of degradation in the ER lumen of a defective lysosomal or secretory or cell membrane (e.g., plasma, Golgi, ER) protein associated with a disease or disorder, thus allowing the protein to proceed along its normal maturation pathway to secretion or to the plasma membrane or a lysosome.

The invention is further detailed in the subsections below.

4.1 THE THIOL-OXIDIZING AGENTS OF THE INVENTION

The thiol-oxidizing agents of the invention are mild oxidants that are cell membrane-permeable. in a preferred aspect, the oxidizing agent inhibits degradation in the ER of a genetically defective protein.

As an example, a thiol-oxidizing agent for use as a therapeutic agent of the invention has the following structure:

0 0 R ,'-C«-N=N-C"-R² _ (I)

Compound (I) is a diazene dicarbonyl compound, in which R¹ and R² are each independently:

1. straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; 2. mono- or di-substituted amino , e. g. , the following:

R . R _^

N- N- H R " in which R and R¹ are each independently an alkyl, aryl, or aralkyl group;

3. alkoxy, aryloxy, aralkoxy, e.g., R-O- in which R is an alkyl, aryl or aralkyl group.

In addition, in compound I,

O 9

II and/or

R'-C- R²-C- can be the derivatized C-terminus of a (preferably N- protected) peptide or an amino acid. For example, the oxidizing agent can have the structure

R³ R⁴ O O peptide-NH I-CH-CONHI-CHII-C-N=NII-C-R₂ ² in which R³ and R⁴ are each a side chain of an amino acid. Preferably, in order to maintain cell membrane permeability, the peptide is not larger than a tripeptide.

In compound I, R¹ and R² together can also form a ring structure, e.g. , compound (II) or (III) :

in which n is an integer of l or more; R⁵ and R⁶ are each independently H, an alkyl, aryl, aralkyl, or the like; where if n > 1, each R^s and R⁶ can be the same or different.

in which R⁷ is H, an alkyl, aryl, or aralkyl or the like.

In a preferred embodiment, the oxidizing agent is diazene dicarboxylic acid bis(N,N- dimethylamide) [diamide; (CH₃)-NCON=NCON(CH₃)₂; Kosower et al, 1969, Biochem. Biophys. Res. Comm. 37(4) :593- 596].

In a specific aspect, the therapeutic agent is a covalent combination or conjugate of a second therapeutic agent with a diazene dicarbonyl compound (I) . For example, in the treatment of cystic fibrosis, such a second therapeutic agent can be a iloride (see Nowak, 1991, J. NIH Research 3:40-44) . Amiloride has the following structure:

NH

A representative active agent comprising a covalent combination of amiloride with compound (I) can have the structure of the compound (V) or (VI) , shown below:

NH

R CO-N

?

4.2 THE SULFHYDRYL-ALKYLATING AGENTS OF THE INVENTION

The sulfhydryl-alkylating agents of the invention are cell membrane-permeable reagents that can alkylate the sulfur atom in the thiol groups of proteins. In a preferred aspect, this alkylation inhibits the degradation in the ER of a genetically defective protein.

The sulfhydryl-alkylating agents are maleimide and its derivatives having the formula (VII) :

in which R^s, R⁹, and R¹⁰ are each independently H; or a branched or straight chain, substituted or unsubstituted alkyl, aryl, or aralkyl. In a specific embodiment, in which R^β, R⁹, and R¹⁰ are each H, the sulfhydryl-alkylating agent is maleimide. In another specific aspect, in which R⁸ and R⁹ are each H, and R¹⁰ is ethyl, the alkylating agent is N-ethyl-maleimide.

4.3 SYNTHESIS OF THE THERAPEUTIC AGENTS

OF THE INVENTION

The therapeutic agents of the invention can be synthesized by methods known in the art, or where available, purchased from a commercial vendor.

For example, CH₃CH₂0₂C-N=N-CO₂CH₂CH₃ is commercially available, or alternatively, can be synthesized as described by Rabjohn (1955, in Organic Syntheses. Collective Vol. 3, Organic Syntheses, Inc., p. 375) or Kauer (1963, in Organic Syntheses. Collective Vol. 4, Organic Syntheses, Inc., p. 411).

Diazene dicarbonyl compounds of formula (VIII)

R-C-N=N-C-R (VIII) in which R is an alkyl group, can be synthesized as described by Cramer (1957, J. Am. Chem. Soc. 79:6215) Where R is a dimethylamino, or- where R is

the synthesis can -be coarried out as described by Smissman and Makriyannis (1973, J. Org. Chem. 38:1652). The compound of formula (VIII) in which R is NH₂, is commercially available (and used as a maturing and bleaching agent in cereal flour) (Oser et al., 1965, Toxicol. Appl. Pharmacol. 7:445).

The compound 4-phenyl-l,2,4-triazoline-3,5- dione (IX) is commercially available or can be synthesized as described by Cookson et al. (1967, J. Am. Chem. Soc. (C):1905; 1962, Tetrahedron Lett. 615). ID

Many other examples of the therapeutic agents of the invention can be synthesized by the skilled artisan using methods analogous to those described in the art for the foregoing structures.

In a specific embodiment where the therapeutic agent is diazenedicarboxylic acid bis(N,N- di ethylamide) , such agent can be easily made as described by Crawford et al. (1963, J. Org. Chem. 28:2419), or purchased from a commercial vendor (e.g., Sigma) .

Maleimides can be synthesized by methods known in the art (see, e.g., U.S. Patent No. 4,623,734 granted November 18, 1986 by Masao et al.) or purchased from a commercial vendor.

4.4 IN VITRO ASSAYS The therapeutic agent of the invention is preferably tested in vitro to ensure that it is permeable to cell membranes. Such assays can be carried out by any method known in the art. In preferred aspects, the assay is carried out by exposing intact cells or rough microsomes or plasma membrane preparations to the agent (which is preferably labeled) , and detecting passage through the cell membranes, by methods known in the art (see, // e.g., Stafford and Bonifacino, 1991, J. Cell. Biol. 115(5) :1225-1236) ; Welply et al., 1983, J. Biol. Chem. 258:11856-11863; Wieland et al., 1987, Cel] 20:289- 300) . Rough microsomes are small closed vesicles formed by fragments of the rough ER produced upon homogenization of cells; microsomes have the same orientation (ribosomes on the outside of the vesicles) as that of the ER within the cell (Darnell et al., 1990, Molecular Cell Biology. 2d Ed., W.H. Freeman & Co., New York, p. 646).

4.5 UTILITY OF THE INVENTION

The oxidizing and alkylating agents of the invention can be administered therapeutically, where a therapeutic effect is mediated by the agent upon oxidation or alkylation, as the case may be, of a protein in the body of a subject. In a preferred aspect, the protein is a genetically defective protein, in particular, a lysosomal, secretory, or cell membrane protein.

The therapeutic methods of the invention are carried out by administration to a subject of a therapeutically effective amount of an agent of the invention. The subject is preferably a mammal, including but not limited to animals such as cows, pigs, etc. , and is most preferably human.

Methods for prevention of disorders, by administering a therapeutic agent of the invention, are also provided.

In a preferred embodiment, the agent is administered to a patient for treatment of a disorder involving a genetically mutated lysosomal or secretory or plasma, ER, or Golgi membrane protein. Although Applicants do not intend to be limited to any specific mechanism, it is believed that delivery of such an agent to the ER lumen allows oxidation or alkylation, as the case may be, of cysteine sulfhydryl groups therein, thereby permitting proper folding and cellular targeting of the protein that otherwise would not occur, or preventing its degradation. Diseases or disorders which can be treated in this manner include but are not limited to cystic fibrosis, emphysema, Tay-Sachs, lysosomal storage diseases, insulin receptor deficiency, familial hypercholesterolemia, Hunter's syndrome, and Hurler's syndrome. As discussed infra, cystic fibrosis is associated with a mutation in the transmembrane protein CFTR. The major genetic cause of emphysema and difficulty in breathing is due to a mutation in the secretory protein α,- antiprotease (α,-antitrypsin) (Darnell et al., 1990, Molecular Cell Biology, 2d Ed. , W.H._* Freeman & Co. , New York, p. 660) . Tay-Sachs disease is caused by a defect in the lysosomal enzyme beta-N-hexosaminidase A (id., p. 671). Other lysosomal storage diseases are caused by defective lysosomal enzymes. Insulin receptor deficiency results from a mutant (plasma membrane) insulin receptor, while familial hypercholesterolemia results from a mutant LDL (low density lipoprotein) (plasma membrane) receptor. Hunter's syndrome and Hurler's syndrome are caused by genetic defects in the lysosomal enzymes which catabolize sulfated mucopolysaccharides (Darnell et al. , supra at p. 671).

In a specific embodiment, the therapeutic agent of the invention is administered to treat cystic fibrosis. Cells from cystic fibrosis patients show a defect in the putative protein prod t of the cystic fibrosis gene (Rommens et al., 1989, Science 245:1059- 1065; Riordan et al. , 1989, Science 245:1066-1073; Kere et al. , 1989, Science 245:1073-1080) designated iS the CFTR (cystic fibrosis transmembrane conductance regulator; Riordan et al., 1989, Science 245:1066- 1073). CFTR is an integral membrane protein that appears to act as a chloride channel (Anderson et al. , 1991, Cell 67:775-784; Rich et al. , 1990, Nature

347:358-363; Drumm et al., 1990, Cell 62:1277-1233). The present invention provides for treatment of cystic fibrosis by exposure of mutant CFTR in the lumen of the ER, to the oxidizing or alkylating agent that is the therapeutic agent of the invention. Although Applicants do not intend to be limited to a specific mechanism, it is believed that the oxidizing or alkylating agent inhibits degradation and/or promotes the correct folding/assembly in the ER lumen of the mutant CFTR that otherwise would be abnormally processed and never reach the plasma membrane, thus achieving proper processing of CFTR to the cell membrane. The agent is administered so as to allow, or preferably target, delivery to the jLn vivo cellular location of CFTR, (Crawford et al., 1991, Proc. Natl. Acad. Sci. USA 88:9262-9266), namely epithelial cells, such as those lining sweat ducts, small pancreatic ducts, and intestinal crypts, and in the kidney, and in the lung.

In another embodiment, directed to the treatment of cystic fibrosis, the therapeutic agent is a covalent combination of amiloride and a diazene dicarbonyl compound (I), e.g., diamide. In a most preferred aspect, such an agent is administered in combination with adenosine triphosphate (ATP) and uridine triphosphate (UTP) (see Nowak, 1991, J. NIH Research 3:40-44).

Suitable in vitro and in vivo assays can be used to demonstrate therapeutic utility of the conjugates of the invention. For in vivo testing, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free' concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. y

The agents of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. The amount of the oxidizing or alkylating agent of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.

The present invention is not to be limited in scope by the specific embodiments described herein since such embodiments are intended as but single illustrations of one aspect of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference herein in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I)

0 0

(I)

R'-C-N=N-C-R²

in which R¹ and R² are each independently a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy; and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I)

O 0

R^l-C-N=N-C-R² (I)

in which R'-C- and R²-C- are each independently the derivatized carboxy-terminus of an amino acid or a peptide; and a pharmaceutically acceptable carrier.

3. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (II)

(II)

in which n is an integer of 1 or more; R⁵ and R⁶ are each independently H, and alkyl, aryl, or aralkyl; where if n is greater than 1, each R⁵ can be the same or different, and each R⁶ can be the same or different; and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (III)

in which R⁷ is an alkyl, aryl or aralkyl; and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising an effective amount of a compound of formula (V)

NH

II

in which R¹ is a straight chain or branched alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy; and a pharmaceutically acceptable carrier.

6. -.harmaceutical composition comprising a therapeutic:- effective amount of a compound of formula (VI) NH

in which R² is a straight chain or branched alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy; and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising a therapeutically effective amount of diamide, of formula (CH₃)₂NCON=NCON(CH₃)₂; and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (VII)

in which R⁸, R⁹, and R¹⁰ are each independently H; a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; and a pharmaceutically acceptable carrier.

9. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (I)

in which R¹ and R² are each independently a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, arylcxy, or aralkoxy.

10. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (I) 0 0

R'-C-N=N-C-R² (I)

in which R'-C- and R²-C- are each independently the derivatized carboxy-terminus of an amino acid or a peptide.

11. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (II)

in which n is an integer of 1 or more; R⁵ and R⁶ are each independently H, and alkyl, aryl, or aralkyl; where if n is greater than 1, each R⁵ can be the same or different, and each R^δ can be the same or different.

12. A method of treating a disease or disorder in a mammal comprising administering to the til mammal a therapeutically effective amount of a compound of formula (III)

in which R⁷ is an alkyl, aryl or aralkyl,

13. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (V)

NH

II

in which R¹ is a straight chain or branched alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy.

14. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (VI)

fi t-

in which R² is a straight chain or branched alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy.

15. A method of treating a disease or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of diamide, of formula (CH₃)₂NCON=NCON(CH₃)₂.

16. A method of treating a disease, or disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (VII)

in which R^s, R⁹, and R¹⁰ are each independently H; a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl.

17. The method according to claim 9 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

18. The method according to claim 10 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder. A3

19. The method according to claim 11 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

20. The method according to claim 12 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

21. The method according to claim 13 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

22. The method according to claim 15 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

23. The method according to claim 16 in which the disease or disorder involves a genetically defective lysosomal, secretory, or cell membrane protein, and the mammal has or is suspected of having the disease or disorder.

24. The method according to claim 9 in which the disease or disorder is cystic fibrosis, and the mammal has or is suspected of having cystic fibrosis.

25. The method according to claim 10 in which the disease or disorder is cystic fibrosis, and the mammal has or is suspected of having cystic fibrosis.

26. The method according to claim 11 in which the disease or disorder is cystic fibrosis, and the mammal has or is suspected of having cystic fibrosis.

27. The method according to claim 12 in which the disease or disorder is cystic fibrosis, and the mammal has or is suspected of having cystic fibrosis.

28. The method according to claim 15 in which the disease or disorder is cystic fibrosis, and the mammal has or is suspected of having cystic fibrosis.

29. A compound of formula (I) O 0

II - II in which R'-C- and R²-C- are each independently the derivatized carboxy-ter inus of an amino acid or a peptide. &f

30 , A compound of formula ( II)

in which n is an integer of 1 or more; R^s and R⁶ are each independently H, and alkyl, aryl, or aralkyl; where if n is greater than 1, each R⁵ can be the same or different, and each R⁶ can be the same or different.

31. A compound of formula (V)

NH

32. A compound of formula (VI)

33. The method according to claim 9 in which the mammal is a human.

34. The method according to claim 10 in which the mammal is a human.

35. The method according to claim 11 in which the mammal is a human.

36. The method according to claim 12 in which the mammal is a human.