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Title:
METHODS, COMPOSITIONS, AND KITS FOR TREATING POLYCYSTIC KIDNEY DISEASE
Document Type and Number:
WIPO Patent Application WO/2021/061873
Kind Code:
A1
Abstract:
Provided herein are methods of treating polycystic kidney disease (PKD) associated with one or more nonsense mutations in a PKD2 gene in a subject using one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, as well as pharmaceutical formulations and kits for use in these methods.

Inventors:
GODDEERIS MATTHEW (US)
Application Number:
PCT/US2020/052338
Publication Date:
April 01, 2021
Filing Date:
September 23, 2020
Export Citation:
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Assignee:
ELOXX PHARMACEUTICALS INC (US)
International Classes:
A61K31/70; A61P13/12; C07H15/20
Domestic Patent References:
WO2018225065A12018-12-13
WO2018167794A12018-09-20
Foreign References:
US20160194630A12016-07-07
US20180265535A12018-09-20
US20170182078A12017-06-29
Other References:
VIRIBAY MIGUEL, TOMOHITO HAYASHI, DOLORES TELLERIA, TOSHIO MOCHIZUKI, DAVID M. REYNOLDS, RAFAEL ALONSO, XOSE M. LENS, FELIPE MOREN: "Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene", HUM GENET, vol. 101, no. 2, 1 November 1997 (1997-11-01), pages 229 - 234, XP055809036
AGRAWAL S.: "Autosomal Dominant Polycystic Kidney Disease and Genetic Counseling", JSM UROLOGY AND RESEARCH, vol. 5, no. 9, 29 July 2019 (2019-07-29), pages 1 - 9, XP055809038
Attorney, Agent or Firm:
GALLAGHER, Audrey et al. (US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A method of treating polycystic kidney disease (PKD) associated with one or more nonsense mutations of a polycystin 2 (PKD2) gene in a subject, comprising administering to said subject a therapeutically effective amount of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157.

2. The method of claim 1, wherein the PKD associated with one or more nonsense mutations is autosomal dominate polycystic kidney disease (ADPKD).

3. The method of claim 1, wherein the PKD associated with one or more nonsense mutations is autosomal recessive polycystic kidney disease (ARPKD).

4. The method of any one of claims 1-3, wherein the one or more NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 are formulated into a pharmaceutical formulation.

5. The method of claim 4, wherein the pharmaceutical formulation further comprises one or more pharmaceutically acceptable carriers.

6. The method of any one of claims 1-5, wherein the subject exhibits a reduction in count or size of liver or kidney cysts.

7. The method of any one of claims 1-5, wherein administration of the one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 slows a progression of liver or kidney cyst growth in the subject.

8. A pharmaceutical formulation comprising one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 for treating polycystic kidney disease (PKD) associated with one or more nonsense mutations of a polycystin 2 (PKD2) gene in a subject.

9. The pharmaceutical formulation of claim 8, further comprising one or more pharmaceutically acceptable carriers.

10. The pharmaceutical formulation of claim 8, wherein the PKD associated with one or more nonsense mutations is autosomal dominate polycystic kidney disease (ADPKD).

11. The pharmaceutical formulation of claim 8, wherein the PKD associated with one or more nonsense mutations is autosomal recessive polycystic kidney disease (ARPKD).

12. A kit comprising one or more NB 122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 for use in treating polycystic kidney disease (PKD) with one or more nonsense mutations of a polycystin 2 (PKD2) gene in a subject.

13. The kit of claim 12, further comprising instructions for use.

14. The method of claim 12, wherein the PKD associated with one or more nonsense mutations is autosomal dominate polycystic kidney disease (ADPKD).

15. The method of claim 12, wherein the PKD associated with one or more nonsense mutations is autosomal recessive polycystic kidney disease (ARPKD).

16. One or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 for use in formulating a medicament for use in treating polycystic kidney disease (PKD) with one or more nonsense mutations of a polycystin 2 (PKD2) gene in a subject.

Description:
METHODS, COMPOSITIONS, AND KITS FOR TREATING POLYCYSTIC KIDNEY

DISEASE

BACKGROUND

[0001] Polycystic kidney disease (PKD) is a genetic disorder associated with nonsense mutations that results in the growth of fluid-filled cysts within the kidneys. As the cysts grow, the kidneys enlarge and lose function overtime, leading to kidney failure.

[0002] PKD is classified into two types, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). ADPKD is the most commonly inherited kidney disease, with a prevalence of approximately 1 per 5,000 people affected.

SUMMARY

[0003] Provided herein methods, formulations, and kits for treating polycystic kidney disease (PKD) in a subject in need thereof using one or more aminoglycosides, including NB122 (ELX- 01), NB84 (ELX-03), NB127 (ELX-04), NB118 (ELX-05), NB128 (ELX-06), NB124-MeS (ELX-07), and NB157 (ELX-10).

[0004] Provided herein in certain embodiments are methods of treating PKD associated with one or more nonsense mutations of a PKD2 gene in a subject comprising administering to said subject one or more aminoglycosides selected from NB 122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof. In certain of these embodiments, the one or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof are administered as part of a pharmaceutical formulation comprising the one or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof and, optionally, one or more pharmaceutically acceptable carriers.

[0005] Provided herein in certain embodiments are methods of increasing gene expression or gene readthrough in a kidney cell comprising administering to said subject one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof. In certain of these embodiments, the gene whose expression or readthrough is being increased comprises one or more nonsense mutations of a PKD2 gene. In certain embodiments, the one or more aminoglycosides or stereoisomers or pharmaceutically acceptable salts thereof are administered as part of a pharmaceutical formulation comprising the one or more aminoglycosides or stereoisomers or pharmaceutically acceptable salts thereof and, optionally, one or more pharmaceutically acceptable carriers.

[0006] Provided herein in certain embodiments are one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical formulations comprising one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124- MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, to treat PKD associated with one or more nonsense mutations of a PKD2 gene.

[0007] Provided herein in certain embodiments are one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or pharmaceutical formulations comprising one or more aminoglycosides or stereoisomers or pharmaceutically acceptable salts thereof, to increase gene expression or gene readthrough in a kidney cell.

[0008] Provided herein in certain embodiments is the use of one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, or of a pharmaceutical formulation comprising one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, to treat a PKD associated with one or more nonsense mutations of a PKD2 gene.

[0009] Provided herein in certain embodiments is the use of one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, or of a pharmaceutical formulation comprising one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, to increase gene expression or gene readthrough in a kidney cell.

[0010] Provided herein in certain embodiments is the use of one or more aminoglycosides selected from NB 122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof to formulate a medicament for treating an PKD associated with one or more nonsense mutations of a PKD2 gene.

[0011] Provided herein in certain embodiments is the use of one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof to formulate a medicament for increasing gene expression or gene readthrough in a kidney cell.

[0012] Provided herein in certain embodiments are kits comprising one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof or pharmaceutical formulations comprising one or more aminoglycosides or derivatives thereof for use in treating PKD associated with one or more nonsense mutations. In certain of these embodiments, the kits further comprise instructions for use.

[0013] Provided herein in certain embodiments are kits comprising one or more aminoglycosides selected from NB 122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof or pharmaceutical formulations comprising one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or stereoisomers or pharmaceutically acceptable salts thereof for use in increasing gene expression or gene readthrough in a kidney cell. In certain of these embodiments, the kits further comprise instructions for use.

[0014] In certain of embodiments of the methods and uses provided herein, the one or more aminoglycosides selected from NB 122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical formulations thereof, are administered in vitro or in vivo. In certain embodiments, the one or more aminoglycosides or stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical formulations thereof, are administered to a subject via parenteral administration. [0015] In certain embodiments of the methods provided herein, the methods further comprise administering one or more additional therapeutic agents in combination with the one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical formulations thereof. These additional therapeutic agents may be administered in the same or in different formulations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.

[0017] FIG. 1 shows the dose-dependent readthrough activity of NB122, NB124, NB84, NB127, NB118, NB128, and NB124-MeS on the nonsense mutation R2430X.

[0018] FIG. 2 shows the dose-dependent readthrough activity of NB122, NB124, NB84, NB127, NB118, NB128, and NB124-MeS on the nonsense mutation Q4042X.

[0019] FIG. 3 shows the dose-dependent readthrough activity of NB122, NB124, NB84, NB127, NB118, NB128, and NB124-MeS on the nonsense mutation R4228X.

[0020] FIGs. 4-6 show the comparative readthrough activity of NB122, NB124, NB84, NB127, NB118, NB128, and NB124-MeS across each of the nonsense mutations R2430X, Q4042X, and R4228X.

[0021] FIG. 7 shows the dose-dependent readthrough activity of NB157 on the nonsense mutations Q4042X, R4228X, and R2430X.

[0022] FIGs. 8A-8B show images the cyst growth for cells with a confirmed nonsense mutation of the PKD2 gene treated with ELX-06 at various concentrations.

[0023] FIGs. 9A-9B show images the cyst growth for cells with a confirmed nonsense mutation of the PKD2 gene treated with ELX-10 at various concentrations.

[0024] FIGs. 10A-10B show images the cyst growth for cells with a confirmed nonsense mutation of the PKD2 gene treated with gentamicin at various concentrations.

[0025] FIGs. 11 A-l IB show images the cyst growth for cells with a confirmed nonsense mutation of the PKD2 gene treated with tolvaptan at various concentrations.

[0026] FIGs. 12A-12B show plots providing a direct comparison between the tested compounds (ELX-06 and ELX-10) and standards/controls (FhO, DMSO, gentamicin and tolvaptan) at two concentrations: 10 mM and 100 mM as a function of RLUs.

[0027] FIGs. 13A-13B show images of the confluent cultures of mixed Cyst 5 and Cyst 8 cells having a confirmed nonsense mutation in the PKD2 gene before lifting and seeding on 3D biogels and one day after seeding cells in the biogel, respectively.

[0028] FIG. 14 shows images of the confluent Cyst 5 and Cyst 8 cells having a confirmed nonsense mutation in the PKD2 gene 3 days after seeding.

[0029] FIG. 15 shows images of water vehicle control treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations.

[0030] FIG. 16 shows images of DMSO treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations.

[0031] FIGs. 17A-17B show plots with data expressed as RLUs and cyst number, respectively for cells treated with either water vehicle control or DMSO.

[0032] FIG. 18 shows images of tolvaptan treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations.

[0033] FIGs. 19A-19B show plots with data expressed as RLUs and cyst number, respectively for cells treated with tolvaptan.

[0034] FIG. 20 shows images of ELX-06 treated ADPKD cells with the confirmed mutation in the PKD2 gene at various concentrations.

[0035] FIGs. 21 A-21B show plots with data expressed as RLUs and cyst number, respectively for cells treated with ELX-06.

[0036] FIG. 22 shows images of ELX-10 treated ADPKD cells with the confirmed mutation in the PKD2 gene at various concentrations.

[0037] FIGs. 23 A-23B show plots with data expressed as RLUs and cyst number, respectively for cells treated with ELX-10.

[0038] FIG. 24 shows images of Mongo Cysts present in the ADPKD cells with the confirmed mutation in the PKD2 treated with water vehicle control, DMSO, tolvaptan, ELX-06, ELX-10, and gentamicin.

DETAILED DESCRIPTION

[0039] The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.

[0040] Nonsense mutations cause premature translational termination, which leads to truncated protein products that are typically unstable. In the context of the PKD being treated herein, these nonsense mutations result in various deleterious effects, including the loss of function in either the polycystin 1 (PKD1) or polycystin 2 (PKD2) proteins. The aminoglycosides and derivatives thereof disclosed herein can be used as small molecule drugs to facilitate readthrough of premature stop codons associated with PKD2 proteins, extending mRNA half-life and restoring full-length functional proteins.

[0041] Previous studies have shown that the aminoglycosides disclosed herein induce readthrough of premature stop codons associated with genetic diseases. As disclosed herein, these aminoglycosides were evaluated for their ability to induce readthrough of premature stop codons associated with PKD.

[0042] Initial proof-of-concept studies show that the aminoglycosides NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 promote readthrough of multiple common nonsense mutations associated with the PKD1 gene in a dual-luciferase reporter assay. With the R2430X, G4042X, and R4228X mutations, NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 have demonstrated significant, dose-dependent readthrough activity. [0043] Following these proof-of-concept studies with PKD1 mutations, select aminoglycosides were evaluated for their effect on nonsense mutations associated with PKD2. NB128 and NB157 were found to induce read-through of premature stop codons associated with previously untested PKD2 genes associated with ADPKD. Significantly, NB128 and NB 157 were found to be particularly effective at reducing formation and slowing progression of cyst formation in ADPKD cells with the PKD2 gene. Based on these findings, methods, formulations, and kits for treating PKD2 are provided herein.

Definitions

[0044] The terms "treat," "treating," and "treatment" as used herein with regard to a polycystic kidney disease (PKD) associated with one or more nonsense mutations may refer to eliminating this disease; preventing, delaying, or reducing the likelihood of development or progression of this disease or of one or more symptoms associated with this disease; reducing or eliminating one or more symptoms associated with this disease; reducing the severity or occurrence of one or more symptoms associated with this disease; or some combination thereof.

[0045] A "subject" as used herein refers to a mammalian subject, preferably a human, who has been diagnosed with, is suspected of having, is at risk of developing, or is exhibiting or has exhibited one or more symptoms associated with a PKD associated with one or more nonsense mutations. In certain embodiments, the subject suffers from ADPKD or ADPKD.

[0046] A "therapeutically effective amount" of an aminoglycoside or a derivative thereof as used herein is an amount of the aminoglycoside or a derivative thereof that produces a desired therapeutic effect in a subject. In certain embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. The precise therapeutically effective amount for a particular aminoglycoside or a derivative thereof will vary based on a variety of factors, including but not limited to the characteristics of the aminoglycoside or a derivative thereof (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications), the nature of any pharmaceutically acceptable carriers present in the aminoglycoside composition, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of the aminoglycoside or a derivative thereof and adjusting the dosage accordingly. For additional guidance, see, e.g., Remington: The Science and Practice of Pharmacy, 22 nd Edition, Pharmaceutical Press, London, 2012, and Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12 th Edition, McGraw-Hill, New York, NY, 2011, the entire disclosures of which are incorporated by reference herein.

Aminoglycosides and Stereoisomers and Pharmaceutically Acceptable Salts Thereof [0047] In certain embodiments, aminoglycosides for use in the methods, formulations, and kits provided herein are selected from the group consisting of NB122 (ELX-01), NB124 (ELX-02), NB84 (ELX-03), NB127 (ELX-04), NB118 (ELX-05), NB128 (ELX-06), NB124-MeS (ELX- 07), and NB 157 (ELX-10), or stereoisomers or pharmaceutically acceptable salts thereof.

[0048] The aminoglycoside NB 122 (PCT Publ. No. W012/66546) has the structure: Publ. No. W012/66546).

[0049] The aminoglycoside NB 124 (PCT Publ. No. W012/66546) has the structure:

(PCT Publ. No. W012/66546). [0050] The aminoglycoside NB84 (PCT Publ. No. WO07/113841) has the structure: [0051] The aminoglycoside NBl 27 (PCT Publ. No. W012/66546) has the structure:

(PCT Publ. No. W012/66546).

[0052] The aminoglycoside NBl 18 (PCT Publ. No. W012/66546) has the structure: Publ. No. W012/66546).

[0053] The aminoglycoside NBl 28 (PCT Publ. No. W012/66546) has the structure:

(PCT Publ. No. W012/66546).

[0054] The aminoglycoside NBl 24-MeS (PCT Publ. No. WO18/225065) has the structure: Publ. No. WO18/225065).

[0055] The aminoglycoside NB157 (PCT Publ. No. W017/37717; W017/37718;

WO17/037719; and W017/118698) has the structure: Publ. No. W017/37717; W017/37718; WO17/037719; and W017/118698).

[0056] In certain embodiments, the methods, formulations, and kits provided herein utilize of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157, or stereoisomers or pharmaceutically acceptable salts thereof. In other embodiments, these methods, formulations, and kits may alternatively use one or more structurally related aminoglycosides, including aminoglycosides of Formula III:

Formula III or stereoisomers or pharmaceutically acceptable salts thereof, wherein:

Ri is selected from the group consisting of hydrogen, alkyl, cycloalkyl, and aryl, and is preferably hydrogen or alkyl;

Ri is hydrogen, sulfonyl, or AHB;

Its is selected from the group consisting of hydrogen, alkyl, cycloalkyl, hydroxyalkyl, and aryl, and is preferably hydrogen, hydroxyalkyl, or alkyl; and a stereo-configuration of each of position 6' and position 5" is independently an R configuration or an S configuration.

[0057] In some embodiments, the sulfonyl is an alkyl sulfonyl or aryl sulfonyl. In some embodiments, the sulfonyl has the chemical formula: -CH 3 O 2 S.

[0058] In some embodiments, the hydroxyalkyl, for example, an alkyl substituted with one or more hydroxy group(s), e.g., hydroxymethyl, 2- hydroxyethyl and 4-hydroxypentyl. In some embodiments, the hydroxyalkyl is an hydroxymethyl having the chemical formula: -CH2OH. [0059] In some embodiments, cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cyclododecyl.

[0060] In some embodiments, the alkyl groups include, for example, an aliphatic hydrocarbon including straight chain and branched chain groups. The alkyl may have 1 to 20 carbon atoms, or 1-10 carbon atoms, and may be branched or unbranched. According to some embodiments of the present invention, the alkyl is a low (or lower) alkyl, having 1-4 carbon atoms (e.g., methyl, ethyl, propyl and butyl). In some embodiments, the alkyl is a methyl. [0061] In some embodiments, the aryl groups include, for example, an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be unsubstituted or substituted by one or more substituents. When substituted, the substituent can be, for example, one or more of an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heteroalicyclic, a halo, a trihaloalkyl, a hydroxy, an alkoxy and a hydroxyalkyl.

Treatment of Polycystic Kidney Disease (PKD)

[0062] In certain embodiments, methods are provided for inducing read-through of a premature stop codon associated with mutations of the PKD2 gene in a subject, comprising administering one or more of NB122, NB124 NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside.

[0063] In certain embodiments, methods are provided for treating PKD in a subject having a nonsense mutation in a PKD2 gene comprising administering to the subject a therapeutically effective amount of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124- MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside.

[0064] In certain embodiments, methods are provided for treating PKD in a subject having a nonsense mutation in a PKD2 gene comprising administering to the subject a therapeutically effective amount of one or more ofNB122, NB124, NB84, NB127, NB118, NB128, NB124- MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside by slowing progression of cyst growth, inhibiting cyst growth, and/or reducing cyst size and cyst count in the subject.

[0065] In certain embodiments, methods are provided for slowing a progression of cyst growth in a subject having a nonsense mutation in a PKD2 gene comprising administering to the subject a therapeutically effective amount of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside. In some embodiments, the cysts are located in the kidney, liver, or both. [0066] In certain embodiments, methods are provided for inhibiting cyst growth in a subject having a nonsense mutation in a PKD2 gene comprising administering to the subject a therapeutically effective amount of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside. In some embodiments, the cysts are located in the kidney, liver, or both. In some embodiments, cyst growth is inhibited at an early stage of cyst formation.

[0067] In certain embodiments, methods are provided for reducing cyst size and count in a subject having a nonsense mutation in a PKD2 gene comprising administering to the subject a therapeutically effective amount of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside. In some embodiments, the cysts are located in the kidney, liver, or both.

[0068] In some embodiments, administration of one or more of NB122, NB124, NB84,

NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof to a subject having a nonsense mutation in a PKD2 gene slows progression of cyst growth, inhibits cyst growth, and/or reduces cyst size and cyst count in the subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 10%, about 15%, about 20%, about 30 %, or more.

[0069] In certain embodiments of the treatment methods provided herein, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside, is administered in combination with one more additional therapeutic agents, including one or more structurally related or structurally unrelated aminoglycosides, either sequentially or simultaneously.

[0070] In certain embodiments of the treatment methods provided herein, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside, may be administered daily, two or more times per week, weekly, bi-weekly (i.e., every other week), every third week, or monthly. [0071] In certain embodiments of the treatment methods provided herein, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside, may be administered more frequently at or near the start of the treatment period. For example, the compound may be administered twice per week at the start of treatment, then once per week for the remainder of the treatment period. In certain of these embodiments, the initial higher frequency dosing may continue for a period of time determined in advance. For example, the compound may be administered at a certain frequency for a specific number of weeks or months, at which point administration frequency is reduced. Alternatively, the initial higher frequency dosing may continue until a specific benchmark is achieved. In certain embodiments, this benchmark is the achievement of a steady-state blood concentration. In these embodiments, the initial higher frequency dosing constitutes a loading phase designed to achieve steady-state, and the ensuing lower frequency dosing constitutes a maintenance phase designed to sustain steady- state. In other embodiments, the benchmark may be the achievement of a specific therapeutic outcome, for example a reduction or cessation in one or more symptoms.

[0072] In certain embodiments, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside, may be administered at a higher dosage at or near the start of the treatment period, and a lower dosage later in the treatment period. In certain of these embodiments, the initial higher dosage may continue for a period of time determined in advance. For example, the compound may be administered at an initial higher dosage for a specific number of weeks or months, at which point dosage is reduced. Alternatively, the initial higher dosage may continue until a specific benchmark is achieved. In certain embodiments, this benchmark is the achievement of a steady-state blood concentration. In these embodiments, the initial higher dosage constitutes a loading phase designed to achieve steady-state, and the ensuing lower dosage constitutes a maintenance phase designed to sustain steady-state. In other embodiments, the benchmark may be the achievement of a specific therapeutic outcome, for example a reduction or cessation in one or more symptoms.

[0073] In certain embodiments of the treatment methods provided herein, both the administration frequency and the dosage of one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or a structurally related aminoglycoside, may be adjusted over the course of treatment. [0074] In certain embodiments of the methods provided herein, a single aminoglycoside or derivative thereof, or a pharmaceutical formulation comprising a single aminoglycoside or a stereoisomer or pharmaceutically acceptable salt thereof, is administered. In other embodiments, two or more aminoglycosides or derivatives thereof, or a pharmaceutical formulation comprising two or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof, are administered. In certain embodiments, one or more additional therapeutic agents are administered in addition to the one or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof. The one or more additional therapeutic agents may be administered as part of the same formulation as the one or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof, or they may be administered separately. In some embodiments, the single aminoglycoside or a stereoisomer or pharmaceutically acceptable salt thereof is selected from NB 122, NB124, NB84, NB127,

NB118, NB128, NB124-MeS, and NB 157. In some embodiments, the two or more aminoglycosides a stereoisomer or pharmaceutically acceptable salt thereof are selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157.

[0075] In certain embodiments of the methods provided herein, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof may be administered for a specific time course determined in advance. For example, the one or more aminoglycosides or derivatives thereof may be administered for a time course of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 36 weeks, 48 weeks, 1 year, 18 months, 2 years, or more than 2 years. In other embodiments, one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 a stereoisomer or pharmaceutically acceptable salt thereof may be administered indefinitely, or until a specific therapeutic benchmark is reached. For example, NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof may be administered until one or more symptoms of the PKD have been eliminated or reduced to a desired level. [0076] Provided herein in certain embodiments is the use of one or more aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof for formulating a medicament for PKD associated with one or more nonsense mutations of the PKD2 gene.

[0077] Provided herein in certain embodiments one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and/or NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or pharmaceutical formulations comprising one or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and/or NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, for use in treating a PKD associated with one or more nonsense mutations of the PKD2 gene.

[0078] One or more of NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB157 or a stereoisomer or pharmaceutically acceptable salt thereof, or pharmaceutical formulations thereof, may be delivered to a subject by any suitable administration pathway known in the art. In certain embodiments, NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or pharmaceutical formulations thereof, may be administered by a parenteral administration route, including intravenous (IV), subcutaneous (SC), or intramuscular administration routes.

[0079] One of ordinary skill in the art will recognize that the various embodiments described herein can be combined. For example, steps from the various methods of treatment disclosed herein may be combined in order to achieve a satisfactory or improved level of treatment.

Kits

[0080] Provided herein in certain embodiments are kits for carrying out the methods disclosed herein. In certain embodiments, the kits comprise one or more aminoglycosides selected from NB122, NB124, NB84, NB127, NB118, NB128, NB124-MeS, and NB 157 or a stereoisomer or pharmaceutically acceptable salt thereof, or one or more pharmaceutical formulations comprising such aminoglycosides or a stereoisomer or pharmaceutically acceptable salt thereof. In certain embodiments, the kits further comprise one or more additional therapeutic agents or pharmaceutical formulations thereof. In those embodiments wherein the kits comprise two or more compounds, e.g., two or more aminoglycosides or stereoisomers or pharmaceutically acceptable salts thereof or an aminoglycoside or stereoisomer or pharmaceutically acceptable salt thereof and an additional therapeutic agent, the two or more compounds may be present in the kit in a single composition or in separate compositions. In certain embodiments, the kits comprise instructions in a tangible medium.

[0081] The foregoing and the following working examples are merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

Examples

[0082] The foregoing and the following working examples are merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

Example 1 : Readthrough Activity for Nonsense Mutations Associated with Polycystic Kidney Disease

[0083] Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic renal disorder that is caused by mutations in either the PKD1 gene (polycystin 1) or the PKD2 gene (polycystin 2). The symptoms of ADPKD are caused by the growth of fluid-filled sacs (e.g., cysts) in the kidneys leading eventually to end-stage renal disease. For the purposes of demonstrating the ability of aminoglycosides disclosed herein to induce readthrough in of ADPKD nonsense mutations, this study focused on PKD1 gene nonsense mutations as proof of principle. NB122 (ELX-01), NB124 (ELX-02), NB-84 (ELX-03), NB127 (ELX-04), NB118 (ELX-05), NB128 (ELX-06), NB124-MeS (ELX-07), and NB157 (ELX-10) were evaluated for their ability to induce readthrough of the ADPKD nonsense mutations R2430X, Q4042X, and R4228X. [0084] A dual-luciferase report assay was used to evaluate the dose-dependence of the compounds to promote readthrough of multiple common nonsense mutations associated with PKD1 as shown in FIGs. 1-3. FIG. 1 shows that each of ELX-01, ELX-02, ELX-03, ELX-04, ELX-05, ELX-06, and ELX-07 exhibited a significant, dose-dependent increase in readthrough activity in cells having the specific ADPKD nonsense mutation R2430X, with the efficacy of each compound ranked in the following order: ELX-02 > ELX-06 > ELX-04 > ELX-01 > ELX- 03 > ELX-05 > ELX-07. Table 1 provides the relevant data for the controls: vehicle, gentamicin, and G418 (LC50 for G418 = 0.7 mM).

Table 1. Readthrough activity for control compounds on PKD1 nonsense mutation R2430X

*two-tailed, unpaired t-test versus vehicle control [0085] FIG. 2 shows that each of ELX-01, ELX-02, ELX-03, ELX-04, ELX-05, ELX-06, and ELX-07 exhibited a significant, dose-dependent increase in readthrough activity in cells having the specific ADPKD nonsense mutation Q4042X, with the efficacy of each compound ranked in the following order: ELX-02 > ELX-06 > ELX-04 > ELX-01 > ELX-03 > ELX-05 > ELX-07. Table 2 provides the relevant data for the controls: vehicle, gentamicin, and G418 (LC50 for G418 = 0.7 mM).

Table 2 Readthrough activity for control compounds on PKD1 nonsense mutation Q4042X

*two-tailed, unpaired t-test versus vehicle control [0086] Lastly, FIG. 3 shows that each of ELX-01, ELX-02, ELX-03, ELX-04, ELX-05, ELX- 06, and ELX-07 exhibited a significant, dose-dependent increase in readthrough activity in cells having the specific ADPKD nonsense mutation R4228X, with the efficacy of each compound ranked in the following order: ELX-02 > ELX-06 > ELX-04 > ELX-01 > ELX-03 > ELX-05 > ELX-07. Table 3 provides the relevant data for the controls: vehicle, gentamicin, and G418 (LC50 for G418 = 0.7 mM).

Table 3. Readthrough activity for control compounds on PKD1 nonsense mutation R4228X

*two-tailed, unpaired t-test versus vehicle control

[0087] ELX-01, ELX-02, ELX-03, ELX-04, ELX-05, ELX-06, and ELX-07 exhibited similar readthrough activity across each of the nonsense mutations R2430X (“UGA”), Q4042X (“El AG”), and R4228X (“E1GA”) as shown in FIGs. 4-6. The readthrough activity in FIGs. 4-6 is represented as a percentage of G418 at 0.8 mM, the highest concentration tolerated in the tested cell line. As shown in FIGs. 4-6, the activity of each ELX-01, ELX-02, ELX-03, ELX-04, ELX- 05, ELX-06, and ELX-07 was similar regardless of the identity of the nonsense mutation.

[0088] FIG. 7 shows the dose-dependent readthrough activity for ADPKD nonsense mutations R2430X, Q4042X, and R4228X for the compound ELX-10. As demonstrated in FIG. 10, ELX- 10 exhibited a significant dose-dependent readthrough activity for each of the ADPKD nonsense mutations R2430X, Q4042X, and R4228X as was similarly demonstrated for each of ELX-01, ELX-02, ELX-03, ELX-04, ELX-05, ELX-06, and ELX-07.

[0089] Taken together, these data show that each of the tested compounds, NB122 (ELX-01), NB124 (ELX-02), NB-84 (ELX-03), NB127 (ELX-04), NB118 (ELX-05), NB128 (ELX-06), NB124-MeS (ELX-07), and NB157 (ELX-10) promote dose-dependent readthrough in nonsense mutations associated with ADPKD.

Example 2: Cyst Reduction in ADPKD Cells with a Nonsense Mutation of the PKD2 Gene [0090] This example provides data demonstrating that exposing human ADPKD cells with the PKD2 gene to exemplary aminoglycosides disclosed herein slows the progression of cyst expansion and/or growth, validating the therapeutic efficacy the aminoglycosides. In specific, this study showed that NB128 (ELX-06) and NB157 (ELX-10) were particularly effective in slowing the progression of cyst expansion and/or growth in human ADPKD cells with a nonsense mutation of the PKD2 gene.

[0091] The cells used in this study were donor ADPKD cells having a confirmed PKD2 mutation. Cells are collected from different cysts from each donor. Cells within a cyst are clonal and have the same PKD2 genotype. However, according to the two-hit ADPKD hypothesis, different cysts from the donor will have one common PKD2 mutation and a second mutation that is likely unique. ADPKD cells derived from each cyst were sequence confirmed for PKD2 mutations and exposed to the standards/controls and experimental compounds in the presence or absence of ImM Forskolin as a stimulus. The standards/controls included DMSO, H2O, gentamicin, and tolvaptan, a therapeutic known to slow down the progression of cyst production in individuals with ADPKD, and the experimental compounds included ELX-06 and ELX-10.

[0092] The treatment schedule for the cyst prevention assay is as enumerated below in Table 4. Table 4: Treatment schedule for cyst prevention assays

[0093] FIGs. 8A-8B and FIGs. 9A-9B show the cyst growth for cells treated with either ELX- 06 or ELX-10, respectively at various concentrations (e.g., 0.3 mM, 1 mM, 3 mM, 10 mM, 30 mM, and 100 mM). As demonstrated, cyst growth significantly decreased with increasing concentrations of ELX-06 or ELX-10. This contrasts with cells treated with gentamicin where cyst growth did not change upon treatment with gentamicin at any concentration (FIGs. 10A- 10B). As an additional point of comparison, cells were also treated with tolvaptan (FIGs. 11 A- 1 IB). The comparison indicates that ELX-06 and ELX-10, similar to tolvaptan, slow the progression of cyst growth in cells with confirmed PKD2. However, unlike both ELX-06 and ELX-10, tolvaptan precipitated at concentrations of 30 mM or greater whereas no precipitation was observed for either ELX-06 and ELX-10 at the higher concentration of 100 mM.

[0094] FIGs. 12A-12B show plots providing direct comparisons between the tested compounds at two concentrations: 10 mM and 100 mM. Due to precipitation issues, neither tolvaptan nor DMSO were included in the 100 mM comparison (FIG. 12B). Treatments were compared by a 1-way ANOVA followed by Tukey’s HSD to respective controls. As shown in FIGs. 12A-12B, both ELX-06 and ELX-10 reduced cyst growth as compared to the controls (DMSO, FhO, and Gentamicin). In particular, at 10 mM, ELX-06 showed a modest but significant decrease in cyst growth and both ELX-06 and ELX-10 showed significant reductions at 100 mM.

[0095] Image analyses were next performed to confirm alterations to cyst number and cyst size upon exposure of human ADPKD cells with a nonsense mutation of the PKD2 gene to ELX-06 and ELX-10 and the standards/controls.

[0096] Cyst 5 and Cyst 8 Donor 3 cells with a confirmed PKD2 mutation were grown on permeable filter supports to confluence before lifting them and seeding them into 3D Biogels (FIGs. 13A-13B). FIGs. 13A-13B show images of the confluent cultures of mixed Cyst 5 and Cyst 8 cells before lifting and seeding on 3D biogels and one day after seeding 35,000 cells per gel, respectively. These single cyst-derived primary cultures had no second mutation or ‘second hit,’ while other single cyst-derived primary cultures were found to have them. The treatment day began at Day 7 with treatments occurring every 2 days through the end of the experiment on Day 15. Typical images on Day 3 of the cell cultures prior to treatment are shown in FIG. 14. The images show elongated structures as well as small spheroids that may grow into translucent cysts. It was determined that an 8-day treatment window was sufficient to determine cysts counts and obtain images demonstrating cystogenesis.

[0097] FIGs. 15 and 16 show images of water vehicle control and DMSO treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations (e.g., 0.3 mM,

1 mM, 3 mM, 10 mM, 30 mM, and 100 mM) with the arrow pointing to the cysts in the images at days 0, 2, 4, 6, and 8, respectively. The images show that the cysts in cells treated with the water vehicle control or DMSO do not decrease in size from Day 0 to Day 8. This is further exemplified by FIGs. 17A-17B with data expressed as relative light units (RLUs) from the CellTiter Glo assessment of cell number and, alternatively, direct cyst counts, for cells treated with either water or DMSO.

[0098] FIG. 18 shows images of tolvaptan treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations (e.g., 0.3 mM, 1 mM, 3 mM, 10 mM, 30 mM, and 100 mM) with the arrow pointing to the cysts in the images at days 0, 2, 4, 6, and 8, respectively. The data show that the effective concentration required for limiting cyst expansion is 30 mM at Day 8 of treatment and 100 mM at Day 8 of treatment. (FIG. 18). This is further exemplified by FIGs. 19A-19B with data expressed as relative RLUs from the CellTiter Glo assessment of cell number and, alternatively, direct cyst counts, for cells treated with tolvaptan. [0099] FIG. 20 shows images of ELX-06 treated ADPKD cells with the confirmed mutation in the PKD2 gene at various concentrations (e.g., 0.3 mM, 1 mM, 3 mM, 10 mM, 30 mM, and 100 mM) with the arrow pointing to the cysts in the images at days 0, 2, 4, 6, and 8. The data show that the effective concentration required for limiting cyst expansion is 3 mM at Day 8 of treatment. (FIG. 20). This is further exemplified by FIGs. 21 A-21B with data expressed as relative RLUs and cyst number, respectively for cells treated with ELX-06. As shown in FIGs.

21 A-21B, the RLUs and cyst number decreases with increasing concentration of ELX-06. However, an increase in cyst number at the highest concentration of 100 mM (reflexive effect) suggesting a bell shaped response in this experiment.

[00100] Similar to ELX-06, ELX-10 was also tested for its efficacy in slowing the progression of cyst growth with FIG. 22 showing images of ELX-10 treated ADPKD cells with the confirmed nonsense mutation in the PKD2 gene at various concentrations (e.g., 0.3 mM, 1 mM, 3 mM, 10 mM, 30 mM, and 100 mM) with the arrow pointing to the cysts in the images at days 0, 2, 4, 6, and 8. The data show that the effective concentration required for limiting cyst expansion is 30 mM at Day 8 of treatment. (FIG. 22). In addition, FIGs. 23A-23B show data expressed as RLUs and cyst number, respectively for cells treated with ELX-10. The RLUs and cyst number decreases with increasing concentration of ELX-10.

[00101] For each standard/control (e.g., water vehicle control, DMSO, tolvaptan, and gentamicin) as well as experimental compounds (e.g., ELX-06 and ELX-10), the largest cysts present in the ADPKD cells (also referred to as “Mongo Cysts”) were identified and imaged at key concentrations. FIG. 24 shows the images of the treated Mongo Cysts, where the Mongo Cysts are attenuated at the effective concentration of 30 mM for ELX-06 and ELX-10.

[00102] This study demonstrates that ELX-06 and ELX-10 are effective in slowing the progression of cyst expansion and/or growth in human ADPKD cells with nonsense mutation in the PKD2 gene. The results show potential for treating patients with ADPKD caused by nonsense mutations in the PKD2 gene.