AU2020284122B2

AU2020284122B2 - Methods of otoprotection against platinum-based antineoplastic agents

Info

Publication number: AU2020284122B2
Application number: AU2020284122A
Authority: AU
Inventors: Qi-Ying Hu; John Lee; Fuxin Shi; John R. Soglia
Original assignee: Decibel Therapeutics Inc
Current assignee: Decibel Therapeutics Inc
Priority date: 2019-05-31
Filing date: 2020-05-29
Publication date: 2026-02-19
Anticipated expiration: 2040-05-29
Also published as: MX2021014447A; IL288532A; KR20220047217A; CN114302647A; EP3975722A4; JP7585240B2; EP3975722A1; JP2022534617A; WO2020243536A1; AU2020284122A1; BR112021023684A2; SG11202113053RA; CA3142153A1

Abstract

Disclosed herein are methods for otoprotection against platinum-based antineoplastic agents by administering a thiosulfate salt to a subject in need thereof. Typically, the thiosulfate salt is administered to the subject scheduled to be administered a platinum-based antineoplastic agent within 4 hours. Alternatively, the thiosulfate salt is administered within 7 hours after the administration of a platinum- based neoplastic agent.

Description

WO 2020/243536 A1 Published: - with international search report (Art. 21(3))

- wo 2020/243536 WO PCT/US2020/035271

METHODS OF OTOPROTECTION AGAINST PLATINUM-BASED ANTINEOPLASTIC AGENTS

FIELD OF THE INVENTION The present invention provides methods of otoprotection against platinum-based antineoplastic agents.

BACKGROUND Platinum-based antineoplastic agents (e.g., cisplatin) are chemotherapeutic agents widely used to treat

cancers and tumors. These agents are toxic and are known to induce hearing loss both in human and

animal models. Thus, patients undergoing chemotherapy with platinum-based antineoplastic agents can

suffer from hearing loss. There is a need for otoprotective compositions and methods to prevent or

mitigate hearing loss associated with chemotherapeutic regimens including platinum-based antineoplastic

agents.

SUMMARY OF THE INVENTION In general, the invention provides methods for mitigating platinum-induced ototoxicity in a subject in need

thereof. The method involves administering to the subject an effective amount of a thiosulfate salt.

In some embodiments, the subject was administered a platinum-based neoplastic agent not more than 7

hours prior administering the thiosulfate salt or is scheduled to be administered a platinum-based

antineoplastic agent within 4 hours. In certain embodiments, the subject was administered a platinum-

based neoplastic agent not more than 7 hours prior administering the thiosulfate salt. In particular

embodiments, the subject is scheduled to be administered a platinum-based antineoplastic agent within

4.5 hours. In further embodiments, the subject was administered a platinum-based neoplastic agent not

more than 2.5 hours prior to administering the thiosulfate salt. In yet further embodiments, the subject

was administered a platinum-based neoplastic agent not more than 1 hour prior to administering the

thiosulfate salt.

In some embodiments, administration of an effective amount of a thiosulfate salt to the subject produces

a plasma thiosulfate Cmax that is 30 M or less at the time of administration a platinum-based

antineoplastic agent. In certain embodiments, administration of an effective amount of a thiosulfate salt to

the subject produces a cochlear thiosulfate Cmax is at least 30 times greater than a cochlear Cmax of the

platinum-based antineoplastic agent. The cochlear platinum concentrations and the cochlear Cmax are

typically modeled by a pharmacokinetic simulation of intravenous infusion in a two-compartment model.

For example, the pharmacokinetic simulation may be conducted using WinNonlin (Phoenix 64) PK

simulation model 9 (IV infusion, 2 compartment).

In still further embodiments, the thiosulfate salt is administered auricularly. In certain embodiments, the

thiosulfate salt is administered intratympanically, transtympanically, or by inner ear injection. In particular

embodiments, the thiosulfate salt is administered transtympanically or by inner ear injection.

In some embodiments, the method further includes administering the platinum-based antineoplastic

agent.

WO wo 2020/243536 PCT/US2020/035271

In certain embodiments, the thiosulfate salt is an alkaline thiosulfate salt, ammonium thiosulfate salt, or a

solvate thereof. In further embodiments, the effective amount of a thiosulfate salt is administered as a

hypertonic pharmaceutical composition comprising the effective amount of a thiosulfate salt. In yet further

embodiments, 200-1,000 uL (e.g., 200-900 uL, 200-800 uL, 200-700 uL, 200-600 uL, 200-500 uL, 200-

400 uL, 200-300 uL, 300-900 uL, 300-800 uL, 300-700 uL, 300-600 uL, 300-500 uL, 300-400 uL, 400-900

uL, 400-800 uL, 400-700 uL, 400-600 uL, or 400-500 uL) of the hypertonic pharmaceutical composition

are administered to the round window of the subject.

In still further embodiments, the calculated osmolarity of the hypertonic pharmaceutical composition is

500-5,000 mOsm/L (e.g., 600-5,000 mOsm/L, 700-5,000 mOsm/L, 800-5,000 mOsm/L, 900-5,000

mOsm/L, 1,000-5,000 mOsm/L, 1,500-5,000 mOsm/L, 2,000-5,000 mOsm/L, 2,500-5,000 mOsm/L,

3,000-5,000 mOsm/L, 500-4,000 mOsm/L, 600-4,000 mOsm/L, 700-4,000 mOsm/L, 800-4,000 mOsm/L,

900-4,000 mOsm/L, 1,000-4,000 mOsm/L, 1,500-4,000 mOsm/L, 2,000-4,000 mOsm/L, 2,500-4,000

mOsm/L, 3,000-4,000 mOsm/L, 500-3,000 mOsm/L, 600-3,000 mOsm/L, 700-3,000 mOsm/L, 800-3,000

mOsm/L, 900-3,000 mOsm/L, 1,000-3,000 mOsm/L, 1,500-3,000 mOsm/L, 2,000-3,000 mOsm/L, 2,500-

3,000 mOsm/L, 500-2,500 mOsm/L, 600-2,500 mOsm/L, 700-2,500 mOsm/L, 800-2,500 mOsm/L, 900-

2,500 mOsm/L, 1,000-2,500 mOsm/L, 1,500-2,500 mOsm/L, 2,000-2,500 mOsm/L, 500-2,000 mOsm/L,

600-2,000 mOsm/L, 700-2,000 mOsm/L, 800-2,000 mOsm/L, 900-2,000 mOsm/L, 1,000-2,000 mOsm/L,

1,500-2,000 mOsm/L, 500-1,500 mOsm/L, 600-1,500 mOsm/L, 700-1,500 mOsm/L, 800-1,500 mOsm/L,

900-1,500 mOsm/L, or 1,000-1,500 mOsm/L).

In some embodiments, the concentration of the thiosulfate salt in the hypertonic pharmaceutical

composition is 0.5M-2.5M (e.g., about 0.05M to about 1.5 M, about 0.05M to about 0.5M, about 0.05M to

about 0.2M, about 0.05M to about 0.1M, about 0.1M to about 1.5M, about 0.1M to about 0.5M, about

0.1M to about 0.2M, about 0.2M to about 1.5M, about 0.2M to about 0.5M, about 0.5M to about 1.5M,

0.05M to about 1.0 M, about 0.05M to about 0.5M, about 0.05M to about 0.2M, about 0.05M to about

0.1M, about 0.1M to about 1.0M, about 0.1M to about 0.5M, about 0.1M to about 0.2M, about 0.2M to

about 1.0M, about 0.2M to about 0.5M, about 0.5M to about 1.0M, or about 1.0M to about 1.5M).

In certain embodiments, the effective amount is an amount that produces a plasma thiosulfate

concentration that is 30 M or less at the time the platinum-based antineoplastic agent is administered.

In certain embodiments, the effective amount is 0.1-2.5 mmol of the thiosulfate salt. In particular

embodiments, the effective amount is an amount that produces a maximum thiosulfate concentration of

0.6-10 mmol/L by 1h post administration. In further embodiments, the effective amount is an amount that

produces a thiosulfate concentration of 0.1-2 mmol/L by 7 h post administration in the subject's cochlea.

In some embodiments, the subject is scheduled to be administered a platinum-based antineoplastic agent

within about 1 hour to about 6 hours (e.g., within about 1 hour to about 5 hours, about 1 hour to about 4

hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 3 hours,

about 2 hours to about 4 hours, about 2 hours to about 5 hours, about 2 hours to about 6 hours, about 3

hours to about 4 hours, about 3 hours to about 5 hours, about 3 hours to about 6 hours, about 4 hours to

WO wo 2020/243536 PCT/US2020/035271 about 5 hours, about 4 hours to about 6 hours, or about 5 hours to about 6 hours following administration

of the thiosulfate salt).

In some embodiments, the subject is administered a platinum-based antineoplastic agent within about 1

hour to about 6 hours after administration of the thiosulfate salt (e.g., within about 1 hour to about 5

hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about

2 hours to about 3 hours, about 2 hours to about 4 hours, about 2 hours to about 5 hours, about 2 hours

to about 6 hours, about 3 hours to about 4 hours, about 3 hours to about 5 hours, about 3 hours to about

6 hours, about 4 hours to about 5 hours, about 4 hours to about 6 hours, or about 5 hours to about 6

hours after administration of the thiosulfate salt).

hour to about 6 hours before administration of the thiosulfate salt (e.g., within about 1 hour to about 5

hours before administration of the thiosulfate salt).

In yet further embodiments, the invention is described by the following enumerated items.

1. A method of mitigating platinum-induced ototoxicity in a subject in need thereof, the method

comprising administering to the subject an effective amount of a thiosulfate salt, wherein the subject was

administered a platinum-based neoplastic agent not more than 7 hours prior to administering the

thiosulfate salt or is scheduled to be administered a platinum-based antineoplastic agent within 4 hours.

2. The method of item 1, wherein the effective amount is an amount that produces a plasma

thiosulfate concentration that is 30 uM or less at the time the platinum-based antineoplastic agent is

administered.

3. A method of mitigating platinum-induced ototoxicity in a subject in need thereof, the method

comprising administering to the subject an effective amount of a thiosulfate salt to produce (i) a plasma

thiosulfate Cmax that is 30 M or less at the time of administration a platinum-based antineoplastic agent

and (ii) a cochlear thiosulfate Cmax is at least 30 times greater than a cochlear Cmax of the platinum-based

antineoplastic agent, wherein the cochlear platinum concentrations and the cochlear Cmax are modeled by

a pharmacokinetic simulation of intravenous infusion in a two compartment model.

4. The method of item 2, wherein the subject was administered a platinum-based neoplastic agent

not more than 7 hours prior administering the thiosulfate salt or is scheduled to be administered a

platinum-based antineoplastic agent within 4 hours

WO wo 2020/243536 PCT/US2020/035271 5. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 7 hours prior administering the thiosulfate salt.

6. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 6 hours prior administering the thiosulfate salt.

7. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 5 hours prior administering the thiosulfate salt.

8. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 4 hours prior administering the thiosulfate salt.

9. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 3 hours prior administering the thiosulfate salt.

10. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 2.5 hours prior to administering the thiosulfate salt.

11. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 2 hours prior administering the thiosulfate salt.

12. The method of any one of items 1 to 3, wherein the subject was administered a platinum-based

neoplastic agent not more than 1 hour prior administering the thiosulfate salt.

13. The method of any one of items 1 to 3, wherein the subject is scheduled to be administered a

platinum-based antineoplastic agent within 4.5 hours.

14. The method of any one of items 1 to 3, wherein the subject is scheduled to be administered a

platinum-based antineoplastic agent within 4 hours.

15. The method of any one of items 1 to 3, wherein the subject is scheduled to be administered a

platinum-based antineoplastic agent within 3 hours.

16. The method of any one of items 1 to 3, wherein the subject is scheduled to be administered a

platinum-based antineoplastic agent within 2 hours.

17. The method of any one of items 1 to 3, wherein the subject is scheduled to be administered a

platinum-based antineoplastic agent within 1 hour.

18. The method of any one of items 1 to 16, wherein the thiosulfate salt is administered auricularly.

19. The method of item 17, wherein the thiosulfate salt is administered intratympanically.

WO wo 2020/243536 PCT/US2020/035271

20. The method of item 17, wherein the thiosulfate salt is administered transtympanically.

21. The method of item 17, wherein the thiosulfate salt is administered by inner ear injection.

22. The method of any one of items 1 to 20, further comprising administering the platinum-based

antineoplastic agent.

23. The method of any one of items 1 to 21, wherein the thiosulfate salt is an alkaline thiosulfate salt,

ammonium thiosulfate salt, or a solvate thereof

24. The method of any one of items 1 to 22, wherein the effective amount of a thiosulfate salt is

administered as a hypertonic pharmaceutical composition comprising the effective amount of a thiosulfate

salt.

25. The method of item 23, wherein 200-1,000 ul of the hypertonic pharmaceutical composition are

administered to the round window of the subject.

26. The method of item 23 or 24, wherein the calculated osmolarity of the hypertonic pharmaceutical

composition is 500-5,000 mOsm/L.

27. The method of any one of items 23 to 25, wherein the concentration of the thiosulfate salt in the

hypertonic pharmaceutical composition is 0.5M-2.5M.

28. The method of any one of items 23 to 25, wherein the concentration of the thiosulfate salt in the

hypertonic pharmaceutical composition is 0.5M-1.5M.

29. The method of any one of items 23 to 25, wherein the concentration of the thiosulfate salt in the

hypertonic pharmaceutical composition is 0.5M-1.0M.

30. The method of any one of items 1 to 28, wherein the effective amount is at least 0.05 mmol of the

thiosulfate salt.

31. The method of any one of items 1 to 28, wherein the effective amount is at least 0.1 mmol of the

thiosulfate salt.

32. The method of any one of items 1 to 28, wherein the effective amount is at least 0.2 mmol of the

thiosulfate salt.

33. The method of any one of items 1 to 28, wherein the effective amount is at least 0.3 mmol of the

thiosulfate salt.

WO wo 2020/243536 PCT/US2020/035271 34. The method of any one of items 1 to 28, wherein the effective amount is at least 0.4 mmol of the

thiosulfate salt.

35. The method of any one of items 1 to 33, wherein the effective amount is 2.5 mmol or less of the

thiosulfate salt.

36. The method of any one of items 1 to 33, wherein the effective amount is 2.0 mmol or less of the

thiosulfate salt.

37. The method of any one of items 1 to 33, wherein the effective amount is 1.5 mmol or less of the

thiosulfate salt.

38. The method of any one of items 1 to 33, wherein the effective amount is 1.0 mmol or less of the

thiosulfate salt.

39. The method of any one of items 1 to 33, wherein the effective amount is 0.5 mmol or less of the

thiosulfate salt.

40. The method of any one of items 1 to 38, wherein the effective amount is an amount that produces

a maximum thiosulfate concentration of 0.6-10 mmol/L by 1h post administration.

41. The method of any one of items 1 to 39, wherein the effective amount is an amount that produces

a thiosulfate concentration of 0.1-2 mmol/L by 7 h post administration in the subject's cochlea.

25 Definitions The term "about," as used herein, represents a value that is in the range of +10% of the value that follows

the term "about"

The term "alkaline salt," as used herein, represents a sodium or potassium salt of a compound. Alkaline

salts may be monobasic or, if the number of acidic moieties (e.g., -COOH, -SO3H, or

-P(O)(OH)n moieties) permits, dibasic or tribasic.

The term "ammonium salt," as used herein, represents an NH4+ salt of a compound. Ammonium salts

may be monobasic or, if the number of acidic moieties (e.g., -COOH, -SO3H, or -P(O)(OH)n moieties)

permits, dibasic or tribasic.

The term "gelling agent," as used herein, refers to pharmaceutically acceptable excipient known in the art

to produce a gel upon mixing with a solvent (e.g., an aqueous solvent). Non-limiting examples of gelling

agents include hyaluronan, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer),

poly(lactic-co-glycolic) acid, polylactic acid, polycaprolactone, alginic acid or a salt thereof, polyethylene

glycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®), agar-agar, gelatin, glucomannan,

galactomannan (e.g., guar gum, locust bean gum, or tara gum), xanthan gum, chitosan, pectin, starch,

WO wo 2020/243536 PCT/US2020/035271 tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol, paraffin, petrolatum, silicates, fibroin,

and combinations thereof.

The term "hypertonic," as used herein in reference to pharmaceutical compositions, represents a

pharmaceutical composition having a calculated osmolarity of 300 mOsm/L to 7,000 mOsm/L (e.g., 300

mOsm/L to 2,500 mOsm/L), which corresponds to 300 mmol to 7,000 mmol (e.g., 300 mOsm/L to 2,500

mmol) of ions and/or neutral molecules produced by dissolution of platinum-deactivating agent and any

ionic, non-polymeric excipients in 1L of solvent having calculated osmolarity of 0 mOsm/L. For the

purpose of the present disclosure, the calculated osmolarity does not include ions and/or neutral

molecules produced from polymeric excipients (e.g., from a gelling agent). For the purpose of this

disclosure, polymeric excipients (e.g., a gelling agents) are deemed as not contributing to the calculated

osmolarity of the compositions disclosed herein.

The term "intratympanic," as used herein in reference to a route of administration, means delivery to the

round window by injection or infusion through an ear canal with a temporarily removed or lifted tympanic

membrane or through a port created through an auditory bulla into the middle ear of a subject.

The term "pharmaceutical composition," as used herein, represents a composition formulated with a

pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental

regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.

The term "pharmaceutical dosage form," as used herein, represents those pharmaceutical compositions

intended for administration to a subject as is without further modification (e.g., without dilution with,

suspension in, or dissolution in a liquid solvent).

The term "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than the

thiosulfate salts and gelling agents described herein (e.g., a vehicle capable of suspending or dissolving

the active compound) and having the properties of being substantially non-toxic and substantially non-

inflammatory in a patient. Excipients may include, e.g., antioxidants, disintegrants, dyes (colors),

emollients, emulsifiers, fillers (diluents), flavors, fragrances, preservatives, printing inks, sorbents,

suspending or dispersing agents, sweeteners, liquid solvents, and buffering agents.

The term "pharmaceutically acceptable salt," as use herein, represents those salts which are, within the

scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals

without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable

benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example,

pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19,

1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth),

Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the

compounds described herein or separately by reacting the free base group with a suitable organic acid.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate,

benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,

WO wo 2020/243536 PCT/US2020/035271 cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,

glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-

hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate,

methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate,

pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate,

sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative

alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like,

as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to

ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine,

triethylamine, ethylamine, and the like.

The term "pharmaceutically acceptable solvate" as used herein means a compound as described herein

wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is

physiologically tolerable at the dosage administered. For example, solvates may be prepared by

crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a

mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, tri-, tetra-, and

penta-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide

(DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-

tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl

alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When the solvate is water-based, the solvate is

referred to as a hydrate.

The term "platinum-based antineoplastic agent," as used herein, represents a coordination compound of

Pt(II) or Pt(IV). Platinum-based antineoplastic agents are known in the art as platins. Typically, platinum-

based antineoplastic agents include at least two coordination sites at the platinum center that are

occupied by nitrogenous spectator ligand(s). The nitrogenous spectator ligands are monodentate or

bidentate ligands, in which the donor atom is an sp³- or sp2-hybridized nitrogen atom within the ligand.

Non-limiting examples of nitrogenous spectator ligands are ammonia, 1,2-cyclohexanediamine, a

picoline, phenanthrin, or 1,6-hexanediamine. Non-limiting examples of platinum-based antineoplastic

agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,

picoplatin, and satraplatin.

The term "subject," as used herein, refers to an animal (e.g., a mammal, e.g., a human). A subject to be

treated according to the methods described herein may be one who is being treated with a therapeutic

regimen including a platinum-based antineoplastic agent (e.g., for the treatment of a benign tumor,

malignant tumor, or cancer). The subject may have been diagnosed with a benign tumor, malignant

tumor, or cancer by any method or technique known in the art. One skilled in the art will understand that

a subject to be treated according to the invention may have been subjected to standard tests or may have

been identified, without examination, as one at high risk due to receiving a therapeutic regimen including

a platinum-based antineoplastic agent.

WO wo 2020/243536 PCT/US2020/035271 The term "substantially neutral," used herein, refers to a pH level of 5.5 to about 8.5, as measured at 20

°C.

The term "tonicity agent," as used herein, refers to a class of pharmaceutically acceptable excipients that

are used to control osmolarity of pharmaceutical compositions. Non-limiting examples of a tonicity agent

include substantially neutral buffering agents (e.g., phosphate buffered saline, tris buffer, or artificial

perilymph), dextrose, mannitol, glycerin, potassium chloride, and sodium chloride (e.g., as a hypertonic,

isotonic, or hypotonic saline). Artificial perilymph is an aqueous solution containing NaCI (120-130 mM),

KCI (3.5 5 mM), CaCl2 (1.3-1.5 mM), MgCl2 (1.2 mM), glucose (5.0-11 mM), and buffering agents (e.g.,

NaHCO3 (25 mM) and NaH2PO4 (0.75 mM), or HEPES (20 mM) and NaOH (adjusted to pH of about 7.5)).

The term "transtympanic," as used herein, in reference to a route of administration, means delivery to the

round window by injection or infusion across tympanic membrane. A transtympanic injection may be

performed directly through the tympanic membrane or through a tube embedded in the tympanic

membrane (e.g., through a tympanostomy tube or grommet).

The term "inner ear injection," as used herein, refers to the direct injection of drug into the inner ear

space.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart showing the profiles (0-24 h of mean plasma thiosulfate concentrations over time for

each tested human cohort. The X-axis shows time (h), and the Y-axis shows mean plasma thiosulfate

concentrations (ng/mL).

FIG. 2 is a chart showing the profiles (0-4 h) of mean plasma thiosulfate concentrations over time for each

tested human cohort. The error bars shown are standard deviations. The X-axis shows time (h), and the

Y-axis shows mean plasma thiosulfate concentrations (ng/mL).

FIG. 3 is a chart showing the profiles (0-672 h) of mean plasma thiosulfate concentrations over time for

concentrations (ng/mL).

FIG. 4 is a scheme showing the timing of thiosulfate administration relative to cisplatin.

FIG. 5A is a chart showing the average threshold sound pressure levels at 4, 24, and 32 kHz measured

during auditory brainstem response (ABR) tests for the control guinea pigs. The baseline thresholds were

from historic auditory brainstem response tests on cisplatin-naîive guinea pigs (n = 100 ears). The

baseline thresholds are shown as a shaded area curve. The X-axis shows sound frequency in kHz, and

the Y-axis shows the response threshold in decibel of sound pressure level (dB SPL).

FIG. 5B is a chart showing the average threshold sound pressure levels at 4, 24, and 32 kHz measured

during auditory brainstem response (ABR) tests for the guinea pigs administered sodium thiosulfate to

PCT/US2020/035271 one ear each followed by a cisplatin challenge. The baseline thresholds were from historic auditory

brainstem response tests on cisplatin-naîve guinea pigs (n = 100 ears). The baseline thresholds are

shown as a shaded area curve. The X-axis shows sound frequency in kHz, and the Y-axis shows the

response threshold in decibel of sound pressure level (dB SPL). The data shown are Mean + standard

error of the mean (SEM); two way analysis of variance (ANOVA); ** P<0.01; ***P<0.001 Treated ears vs.

Untreated ears (compare to FIG. 5A).

FIG. 6 is a chart showing the concentration dependence for thiosulfate (DB-020) in human tumor cell lines

treated with cisplatin at 15 M. The following tumor cell lines were used; SH-N-AS (brain,

neuroblastoma), SNU899 (larynx, squamous cell carcinoma), NCI-H23 (lung, non-small cell), HLF (Liver,

undifferentiated hepatocellular carcinoma) and A2780 (Ovary, carcinoma).

FIG. 7 is a chart showing the profiles (0-8 h) of mean thiosulfate concentrations (mM) over time in plasma

(human) and perilymph (guinea pig) following administration of Hyaluronan Gel 1 (12% w/v, 0.5M sodium

thiosulfate). The horizontal solid line shows the 30 uM level below which plasma thiosulfate levels should

be. The horizontal dotted line shows the 660 M (0.66mM) level above which perilymph thiosulfate levels

should be.

FIG. 8A is a chart showing the changes in thiosulfate concentrations over time in plasma, perilymph, and

cerebrospinal fluid in guinea pigs administered a gel containing 0.1M sodium thiosulfate and 20% (w/v)

poloxamer 407.

FIG. 8B is a chart showing the changes in thiosulfate concentrations over time in plasma, perilymph, and

cerebrospinal fluid in guinea pigs administered a gel containing 0.5M sodium thiosulfate and 1% (w/v)

hyaluronan.

FIG. 9A is a chart showing the changes in thiosulfate concentrations over time in plasma, perilymph, and

cerebrospinal fluid in guinea pigs administered a gel containing 0.1M sodium thiosulfate and 2% (w/v)

hyaluronan.

FIG. 9B is a chart showing the changes in thiosulfate concentrations over time in plasma, perilymph, and

cerebrospinal fluid in guinea pigs administered a gel containing 0.5M sodium thiosulfate and 2% (w/v)

hyaluronan.

FIG. 10A is a chart showing the threshold sound pressure levels at 4, 24, and 32 kHz measured across

five cohorts of guinea pigs (n = 27 animals) during auditory brainstem response tests. Seven days prior

to the auditory brainstem response tests, all guinea pigs were injected intraperitoneally with a bolus of

cisplatin. The baseline thresholds were from historic auditory brainstem response tests on cisplatin-naîive

guinea pigs (n = 100 ears). The baseline thresholds are shown as a shaded area curve.

FIG. 10B is a chart showing the threshold sound pressure levels at 4, 24, and 32 kHz measured during

auditory brainstem response tests for the guinea pigs deemed to have hearing loss (n = 18 animals).

WO wo 2020/243536 PCT/US2020/035271 Seven days prior to the auditory brainstem response tests, all guinea pigs were injected intraperitoneally

with a bolus of cisplatin. The baseline thresholds were from historic auditory brainstem response tests on

cisplatin-naîve guinea pigs (n = 100 ears). The baseline thresholds are shown as a shaded area curve.

FIG. 11A is a chart showing the average threshold sound pressure levels at 4, 24, and 32 kHz measured

during auditory brainstem response tests for the guinea pigs deemed to have hearing loss (n = 18

animals). Seven days prior to the auditory brainstem response tests, all guinea pigs were injected

intraperitoneally with a bolus of cisplatin. The baseline thresholds were from historic auditory brainstem

response tests on cisplatin-naîve guinea pigs (n = 100 ears). The baseline thresholds are shown as a

shaded area curve.

FIG. 11B is a chart showing the average threshold sound pressure levels at 4, 24, and 32 kHz measured

during auditory brainstem response (ABR) tests for the guinea pigs administered vehicle or sodium

thiosulfate to one ear each followed by a cisplatin challenge. The baseline thresholds were from historic

auditory brainstem response tests on cisplatin-naîive guinea pigs (n = 100 ears). The baseline thresholds

are shown as a shaded area curve.

FIG. 12 is a figure illustrating the cisplatin challenge test following administration of vehicle or sodium

thiosulfate to one ear of a guinea pig.

FIG. 13 is a chart showing the average threshold sound pressure levels at 4, 24, and 32 kHz measured

thiosulfate (0.1M, 0.5M, or 1M sodium thiosulfate gel) to one ear each followed by a cisplatin challenge.

The baseline thresholds were from historic auditory brainstem response tests on cisplatin-naîve guinea

pigs (n = 100 ears). The baseline thresholds are shown as a shaded area curve.

DETAILED DESCRIPTION In general, the invention provides method of mitigating platinum-induced ototoxicity in a subject by

administering to the subject an effective amount of a thiosulfate salt. Preferably, the thiosulfate salt is

administered auricularly (e.g., intratympanically or transtympanically).

Typically, the thiosulfate salt is administered to the subject scheduled to be administered a platinum-

based antineoplastic agent within 4 hours (e.g., within 3 hours, within 2 hours, or within 1 hour).

Alternatively, the thiosulfate salt is administered within 7 hours (e.g., within 6 hours, within 5 hours, within

4 hours, within 3 hours, within 2 hours, or within 1 hour) after the administration of a platinum-based

neoplastic agent. Preferably, the thiosulfate salt is administered to the subject scheduled to be

administered platinum-based antineoplastic agent within 3 hours. Alternatively, the thiosulfate salt is

administered within 4 hours after the administration of a platinum-based neoplastic agent. More

preferably, the thiosulfate salt is administered to the subject scheduled to be administered platinum-based

antineoplastic agent within 1 hour. Alternatively, the thiosulfate salt is administered within 1 hour after the

administration of a platinum-based neoplastic agent.

wo 2020/243536 WO PCT/US2020/035271 An effective amount of a thiosulfate salt typically produces a plasma thiosulfate concentration that is 30

M or less (e.g., 20 M or less, 10 M or less, or near endogenous concentration) at the time of

administration a platinum-based antineoplastic agent. Additionally or alternatively, an effective amount of

a thiosulfate salt typically produces a cochlear thiosulfate concentration that is at least 30 times greater

(e.g., 30 to 1000 times greater, 30 to 500 times greater, or 30 to 150 times greater) than a cochlear Cmax

of the platinum-based antineoplastic agent, wherein the cochlear platinum concentrations and the

cochlear Cmax are modeled by a pharmacokinetic simulation of intravenous infusion in a two compartment

model.

Platinum-induced ototoxicity may occur in subjects receiving a platinum-based antineoplastic agent (e.g.,

a subject having a tumor or cancer). Non-limiting examples of the platinum-based antineoplastic agents

include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and

satraplatin.

A thiosulfate salt may mitigate (e.g., eliminate) hearing loss in a subject receiving a platinum-based

antineoplastic agent, as measured by at least 50% (e.g., at least 60%, at least 70%, or at least 80%)

reduction in the sound pressure level threshold elevation in the subject at a frequency 8 kHz or higher

(e.g., between 8 kHz and 20 kHz) relative to a reference subject that receives the same platinum-based

antineoplastic agent regimen but does not receive the thiosulfate salt.

Thiosulfate salts may exhibit otoprotective properties against platinum-based antineoplastic agents and

may be used in a method of mitigating (e.g., eliminating) platinum-induced ototoxicity in subjects in need

thereof. Typically, a thiosulfate salt is administered to a round window of the subject. The subject may

be undergoing therapy with a platinum-based antineoplastic agent (e.g., cisplatin, carboplatin, oxaliplatin,

nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin).

A thiosulfate salt may be administered to a subject, e.g., before or after the administration of a platinum-

based antineoplastic agent to the subject. Alternatively, a thiosulfate salt may be administered, e.g., at

the same time as the administration of a platinum-based antineoplastic agent. A thiosulfate salt may be

administered to a subject scheduled to be administered a platinum-based antineoplastic agent within 4

hours, e.g., within 3 hour, within 2 hours, or within 1 hour (e.g., at least 5 minute, at least 15 minutes, or at

least 30 minutes prior to platinum-based antineoplastic agent administration). Alternatively, a thiosulfate

salt may be administered, e.g., not more than 7 hours (e.g., not more than 6 hours, not more than 5

hours, not more than 4 hours, not more than 3 hours, not more than 2 hours, or not more than 1 hour)

after platinum-based antineoplastic agent administration (e.g., at least 5 minutes, at least 15 minutes, or

at least 30 minutes after). Administration of a platinum-based antineoplastic agent is typically preceded

by administration of a hydration composition (e.g., saline optionally including, e.g., mannitol or

furosemide). Further, administration of a platinum-based antineoplastic agent typically lasts for a period

of at least 1 hour (e.g., 1 to 24 hours, e.g., 1 to 12 hours, 1 to 6 hours, 1 to 3 hours, or 1 to 2 hours). One

of skill in the art would recognize the timing between the administrations of a thiosulfate salt and platinum-

based neoplastic agent as being the time between the completion of the administration of one and the

commencement of the administration of the other. For example, administration of a platinum-based

WO wo 2020/243536 PCT/US2020/035271 neoplastic agent 30 minutes to 4 hours following the step of administration of a thiosulfate salt indicates

that 30 minutes to 4 hours separate the end of the thiosulfate salt administration and the commencement

of the platinum-based neoplastic agent administration (e.g., infusion of cisplatin).

Typically, the pharmaceutical composition of the invention may be administered by a route different from

the platinum-based antineoplastic agent. The methods of the invention may utilize a local route of

administration, for example, the pharmaceutical composition of the invention may be administered

intratympanically or transtympanically. Transtympanic administration may include injection or infusion of

an effective amount of the pharmaceutical composition of the invention through the tympanic membrane

into the tympanic cavity, thereby providing the anti-platinum chemoprotectant agent to the round window.

In the methods of the invention, typically, a needle is used to pierce the tympanic membrane to instill drug

into the middle ear space or traverse an existing PE tube or perforation of the ear drum to instill drug. A

separate ventilation hole in the tympanic membrane may or may not be created to allow air to escape the

middle ear space. The instilled drug may then target middle ear structures, cells or be designed to enter

the inner ear via the round and oval membranes to affect specific targets. This may be accomplished,

e.g., by instilling drug through the round window membrane, the oval window, a cochleostomy, or

labrinthotomy approach. These surgical procedures may be accomplished by raising a tympanomeatal

flap (lifting up the ear drum) and exposing the round window, stapes/oval window and promontory. A

stapedotomy hole may be created in the footplate and the drug instilled into the vestibule by pump,

injection, or some other method. Alternatively, the bony lip of the round window (RW) is removed

(typically by drill) to expose the RW. The RW may then be pierced with a needle and the drug infused or

the RW may be fenestrated and the drug instilled directly through the fenestration. Finally, an entirely

separate entrance hole to the cochlea may be opened by drilling a cochleostomy hole into the cochlea

and drug instilled.

Alternatively, rather than raising a tympanomeatal flap, a mastoidectomy may be performed and the facial

recess opened to provide direct access to the oval and round windows as well as the promontory and the

semicircular canals. Via this approach, all three sites can be used as just described. In addition, the

labyrinth may be opened, much like a cochleostomy, for the instillation of drug. To dissipate

fluid/pressure buildup in the inner ear, a separate opening into the RW or OW may be created to allow for

excess perilymph to leak out.

Thiosulfate salts may be provided in a pharmaceutical composition. Pharmaceutical compositions may

be, e.g., hypertonic. Without wishing to be bound by theory, the higher tonicity of the pharmaceutical

compositions disclosed herein is believed to improve the bioavailability of thiosulfate at the round window

of a subject, relative to compositions with lower tonicity (e.g., hypotonic or isotonic). The bioavailability is

typically calculated using exposure (AUC) to thiosulfate following its administration to a subject. The

calculated osmolarity of the pharmaceutical composition (e.g., pharmaceutical dosage form) may be, e.g.,

at least 400 mOsm/L (e.g., at least 500 mOsm/L, at least 600 mOsm/L, at least 700 mOsm/L, at least 800

mOsm/L, at least 900 mOsm/L, at least 1,000 mOsm/L, at least 1,500 mOsm/L, at least 2,000 mOsm/L,

at least 2,500 mOsm/L, or at least 3,000 mOsm/L), and/or 5,000 mOsm/L or less (e.g., 4,000 mOsm/L or wo 2020/243536 WO PCT/US2020/035271 less, 3,000 mOsm/L or less, 2,000 mOsm/L or less, 1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/L or less, 1,600 mOsm/L or less, or 1,500 mOsm/L or less). The calculated osmolarity of the pharmaceutical composition (e.g., pharmaceutical dosage form) may be, e.g., 1,500-4,500 mOsm/L. The calculated osmolarity of the pharmaceutical composition (e.g., pharmaceutical dosage forms) may be, e.g., 3,000-4,500 mOsm/L. The measured osmolality of the pharmaceutical composition (e.g., pharmaceutical dosage form) may be, e.g., at least 0.3 Osm/kg (e.g., at least 0.5 Osm/kg, at least 0.6

Osm/kg, at least 0.7 Osm/kg, at least 0.8 Osm/kg, at least 0.9 Osm/kg, at least 1.0 Osm/kg, at least 1.2

Osm/kg, at least 1.4 Osm/kg, or at least 1.8 Osm/kg). The measured osmolality of the pharmaceutical

composition (e.g., pharmaceutical dosage form) may be, e.g., 2.5 Osm/kg or less (e.g., 2.1 Osm/kg or

less). The measured osmolality of the pharmaceutical composition (e.g., pharmaceutical dosage form)

may be, e.g., 0.3-2.5 Osm/kg (e.g., 0.5-2.5 Osm/kg, 0.6-2.5 Osm/kg, 0.7-2.5 Osm/kg, 0.8-2.5 Osm/kg,

0.9-2.5 Osm/kg, 1.0-2.5 Osm/kg, 1.2-2.5 Osm/kg, 1.4-2.5 Osm/kg, 1.8-2.5 Osm/kg, 0.5-2.1 Osm/kg, 0.6-

2.1 Osm/kg, 0.7-2.1 Osm/kg, 0.8-2.1 Osm/kg, 0.9-2.1 Osm/kg, 1.0-2.1 Osm/kg, 1.2-2.1 Osm/kg, 1.4-2.1

Osm/kg, or 1.8-2.1 Osm/kg). "Calculated osmolarity" refers to the number of mmoles of ions and/or

neutral molecules produced by dissolution of one or more compounds in 1L of DI or distilled water;

calculated osmolarity does not include ions and/or neutral molecules produced from polymeric excipients

(e.g., from a gelling agent). "Measured osmolality" refers to the osmolality of a composition, as measured

using an osmometer (typically, a membrane osmometer).

A preferred pharmaceutical dosage form of the invention is a gel.

In some embodiments, at least 50 ul (preferably, at least 100 uL; more preferably, at least 200 uL) of the

pharmaceutical composition are administered to theround windowof the subject. In particular

embodiments, 1 ml or less (e.g., 0.8 ml or less or 0.5 mL or less) of the pharmaceutical composition are

administered to the round window of the subject. In certain embodiments, 100 uL to 1 mL (e.g., 200 ul to

1mL, 100 ul to 0.8 mL, 200 ul to 0.8 mL, 100 uL to 0.5 mL, 200 uL to 0.5 mL, 0.5 mL to 1.0 mL, 0.5 ml

to 0.8 mL, or 0.8 ml to 1.0 mL) of the pharmaceutical composition are administered to the round window

of the subject.

A thiosulfate salt may be, e.g., the sole compound contributing to osmolarity of a pharmaceutical

composition. Alternatively, higher osmolalities than those afforded by the desired concentration of a

thiosulfate salt may be achieved, e.g., through the use of tonicity agents. A tonicity agent may be present

in a hypertonic, isotonic, or hypotonic excipient (e.g., a hypotonic liquid solvent). Non-limiting examples

of tonicity agents include substantially neutral buffering agents (e.g., phosphate buffered saline, tris

buffer, or artificial perilymph), dextrose, mannitol, glycerin, glycerol, potassium chloride, and sodium

chloride (e.g., as a hypertonic, isotonic, or hypotonic saline).

Thiosulfate Salts

Without wishing to be bound by theory, thiosulfate salts are believed to reduce or eliminate the toxicity of

platinum-based antineoplastic agents by competitively ligating and substantially coordinatively saturating

the platinum centers present in the platinum-based antineoplastic agents. The concentration of a

thiosulfate salt in a pharmaceutical composition (e.g., a pharmaceutical dosage form) may be, e.g., at

WO wo 2020/243536 PCT/US2020/035271 least about 0.05M (e.g., at least about 0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M,

at least about 0.5M, or at least about 1M). The concentration of a thiosulfate salt in a pharmaceutical

composition (e.g., a pharmaceutical dosage form) may be, e.g., about 2.5M or less (e.g., 2.0M or less,

1.5M or less, 1.0M or less, 0.5M or less, about 0.3M or less, or about 0.2M or less). Non-limiting

examples of the concentrations of a thiosulfate salt in a pharmaceutical composition (e.g., a

pharmaceutical dosage form) may be, e.g., about 0.05M to about 1.5 M, about 0.05M to about 0.5M,

about 0.05M to about 0.2M, about 0.05M to about 0.1M, about 0.1M to about 1.5M, about 0.1M to about

0.5M, about 0.1M to about 0.2M, about 0.2M to about 1.5M, about 0.2M to about 0.5M, about 0.5M to

about 1.5M, 0.05M to about 1.0 M, about 0.05M to about 0.5M, about 0.05M to about 0.2M, about 0.05M

to about 0.1M, about 0.1M to about 1.0M, about 0.1M to about 0.5M, about 0.1M to about 0.2M, about

0.2M to about 1.0M, about 0.2M to about 0.5M, or about 0.5M to about 1.0M, or about 1.0M to about

1.5M. Preferably, the concentration of a thiosulfate salt agent in a pharmaceutical composition (e.g., a

pharmaceutical dosage form) is about 0.5M to about 1.5M. More preferably, the concentration of a

thiosulfate salt agent in a pharmaceutical composition (e.g., a pharmaceutical dosage form) is about 0.5M

to about 1.0M.

Preferably, the thiosulfate salt is an alkaline or ammonium thiosulfate salt. More preferably, the

thiosulfate salt is sodium thiosulfate.

Gelling Agents

Pharmaceutical compositions disclosed herein include a gelling agent. Gelling agents may be used to

increase the viscosity of the pharmaceutical composition, thereby improving the retention of the

pharmaceutical composition at the targeted site. Pharmaceutical compositions (e.g., pharmaceutical

dosage forms) may contain, e.g., about 0.1% to about 25% (w/v) (e.g., about 0.1% to about 20% (w/v),

about 0.1% to about 10% (w/v), about 0.1% to about 2% (w/v), about 0.5% to about 25% (w/v), about

0.5% to about 20% (w/v), about 0.5% to about 10% (w/v), about 0.5% to about 2% (w/v), about 1% to

about 20% (w/v), about 1% to about 10% (w/v), about 1% to about 2% (w/v), about 5% to about 20%

(w/v), about 5% to about 10% (w/v), or about 7% to about 10% (w/v)) of a gelling agent relative to solvent.

Preferably, pharmaceutical compositions (e.g., pharmaceutical dosage forms) may contain, e.g., about

0.5% to about 25% (w/v) (e.g., about 0.5% to about 20% (w/v), about 0.5% to about 10% (w/v), about

0.5% to about 2% (w/v), about 1% to about 20% (w/v), about 1% to about 10% (w/v), about 1% to about

2% (w/v), about 5% to about 20% (w/v), about 5% to about 10% (w/v), or about 7% to about 10% (w/v)) of

a gelling agent relative to solvent.

Gelling agents that may be used in the pharmaceutical compositions disclosed herein are known in the

art. Non-limiting examples of gelling agents include hyaluronan, a polyoxyethylene-polyoxypropylene

block copolymer (e.g., a poloxamer), poly(lactic-co-glycolic) acid, polylactic acid, polycaprolactone, alginic

acid or a salt thereof, polyethylene glycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®),

agar-agar, gelatin, glucomannan, galactomannan (e.g., guar gum, locust bean gum, or tara gum),

xanthan gum, chitosan, pectin, starch, tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol,

paraffin, petrolatum, silicates, fibroin, and combinations thereof. The gelling agents described herein are

known in the art. Preferably, the gelling agent is hyaluronan.

wo 2020/243536 WO PCT/US2020/035271

A pharmaceutical composition may contain, e.g., about 0.5% to about 2% (w/v) (e.g., about 1% to about

2% (w/v)) of hyaluronan relative to solvent. A pharmaceutical composition may contain, e.g., about 5% to

about 10% (w/v) (e.g., about 6% to about 8% (w/v)) of methylcellulose relative to solvent. A

pharmaceutical composition may contain, e.g., hyaluronan and methylcellulose as a gelling agent (e.g.,

about 0.5% to about 2% (w/v) of hyaluronan and about 5% to about 10% (w/v) of methylcellulose relative

to solvent). A pharmaceutical composition may contain, e.g., a polyoxyethylene-polyoxypropylene block

copolymer (e.g., poloxamer) as a gelling agent. A pharmaceutical composition may contain, e.g., about

1% to about 20% (w/v) (e.g., about 1% to about 15% (w/v), about 1% to about 10% (w/v), about 5% to

about 20% (w/v), about 5% to about 15% (w/v), about 5% to about 10% (w/v), about 10% to about 20%

(w/v), or about 10% to about 15% (w/v)) of a polyoxyethylene-polyoxypropylene block copolymer (e.g.,

poloxamer) relative to solvent. The poloxamer may be poloxamer 407, poloxamer 188, or a combination

thereof. A pharmaceutical composition may contain, e.g., about 0.5% (w/v) to about 20% (w/v) of fibroin

as a gelling agent relative to solvent.

Hyaluronan is a hyaluronic acid or a salt thereof (e.g., sodium hyaluronate). Hyaluronans are known in

the art and are typically isolated from various bacteria (e.g., Streptococcus zooepidemicus, Streptococcus

equi, or Streptococcus pyrogenes) or other sources, e.g., bovine vitreous humor or rooster combs. The

weight-averaged molecular weight (Mw) of hyaluronan is typically about 50 kDa to about 10 MDa.

Preferably, Mw of a hyaluronan (e.g., sodium hyaluronate) is about 500 kDa to 6 MDa (e.g., about 500

kDa to about 750 kDa, about 600 kDa to about 1.1 MDa, about 750 kDa to about 1 MDa, about 1MDa to

about 1.25 MDa, about 1.25 to about 1.5 MDa, about 1.5 MDa to about 1.75 MDa, about 1.75 MDa to

about 2 MDa, about 2 MDa to about 2.2 MDa, about 2 MDa to about 2.4 MDa). More preferably, the Mw

of a hyaluronan (e.g., sodium hyaluronate) is about 620 kDa to about 1.2 MDa or about 1.2 MDa to about

1.9 MDa. Other preferred molecular weight ranges for a hyaluronan include, e.g., about 600 kDa to about

1.2 MDa.

Polyoxyethylene-polyoxypropylene block copolymers are known in the art. A non-limiting example of

polyoxyethylene-polyoxypropylene block copolymers is a poloxamer, in which a single polyoxypropylene

block is flanked by two polyoxyethylene blocks. Poloxamers are commercially available under various

trade names, e.g., Synperonic®, Pluronic®, Kolliphor®, and Lutrol®. A pharmaceutical composition may

contain, e.g., a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) includes a

polyoxypropylene block with a number average molecular weight (Mn) of, e.g., about 100 g/mol to about

17,400 g/mol (e.g., about 2,090 g/mol to about 2,360 g/mol, about 7,680 g/mol to about 9,510 g/mol,

6,830 g/mol to about 8,830 g/mol, about 9,840 g/mol to about 14,600 g/mol, or about 12,700 g/mol to

about 17,400 g/mol). A polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may

include a polyoxypropylene block with a number average molecular weight (Mn) of about 1,100 g/mol to

about 4,000 g/mol and a calculated polyoxyethylene content of about 30% to about 85% (w/w).

Preferably, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may include a

polyoxypropylene block with a calculated molecular weight of, e.g., about 1,800 g/mol to about 4,000

g/mol. Preferably, the calculated polyoxyethylene content a polyoxyethylene-polyoxypropylene block

copolymer (e.g., a poloxamer) may be, e.g., about 70% to about 80% (w/w). Preferably, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may have a number average molecular weight of, e.g., about 7,680 g/mol to about 14,600 g/mol. Non-limiting examples of poloxamers are poloxamer 407 and poloxamer 188.

Celluloses and cellulose ethers are known in the art. Celluloses and cellulose ethers are commercially

available under various tradenames, e.g., Avicel®, MethocelTM, Natrosol®, and Tylose®. Non-limiting

examples of cellulose ethers include methylcellulose, carboxymethylcellulose, ethylcellulose,

hydroxyethylcellulose, methyl hydroxyethylcellulose, hydroxypropyl methylcellulose, or

hydroxypropylcellulose. A cellulose ether (e.g., methylcellulose) may have a number average molecular

weight (Mn) of, e.g., about 5 kDa to about 300 kDa. Methyl-substituted celluloses (e.g., methylcellulose,

hydroxypropyl methyl cellulose, or methyl hydroxyethylcellulose) may have methyl content of, e.g., 19%

to 35% (e.g., 19% to 30%).

Fibroin is a protein present in silk created by numerous insects. Fibroins are known in the art and are

commercially available from various vendors, e.g., Jiangsu SOHO International Group; Simatech,

Suzhou, China; Xi'an Lyphar Biotech, Ltd.; Xi'an Rongsheng Biotechnology; Mulberry Farms, Treenway

Silks, Sharda Group, Maniar Enterprises, and Wild Fibres. The molecular weight of silk fibroin is typically

about 10 kDa to about 500 kDa. Fibroins are described in WO 2017/139684, the disclosure of which is

incorporated herein by reference.

Cross-Linked Gelling Agents

Pharmaceutical compositions may contain non-cross-linked or cross-linked gelling agents. Gelling agents

may be cross-linked using cross-linking agents known in the art. Preferably, the cross-linked gelling

agent is covalently crosslinked. Pharmaceutical compositions (e.g., pharmaceutical dosage forms)

including cross-linked gelling agents may be used to control the release profile of an anti-platinum

chemoprotectant agent. For example, the release of an anti-platinum chemoprotectant agent from a

pharmaceutical composition (e.g., a pharmaceutical dosage form) containing a cross-linked gelling agent

may be extended release relative to a reference composition that differs from the pharmaceutical

composition only by the lack of cross-linking in the gelling agent in the reference composition. The

extension of the release of an anti-platinum chemoprotectant agent may be assessed by comparing Tmax

values for the pharmaceutical composition and the reference composition.

Certain gelling agents, e.g., those having carboxylate moieties (e.g., hyaluronan, alginic acid, and

carboxymethylcellulose), can be cross-linked ionically using ionic cross-linking agents (e.g., a multivalent

metal ion, e.g., Mg2+, Ca2+, or Al³+). Techniques for ionic cross-linking of gelling agents are known in the

art (see, e.g., U.S. Patent No. 6,497,902 and 7,790,699, the disclosures of which are incorporated herein

by reference). Typically, gelling agents can be ionically cross-linked in an aqueous solution using

multivalent metal ions, e.g., Mg2+, Ca2+, or Al³ as ionic cross-linking agents. Without wishing to be

bound by theory, the metal ions are believed to coordinate to different molecules of the gelling agent

(e.g., to pendant carboxylates residing on different molecules of the gelling agent), thereby forming a

linkage between these different molecules of the gelling agent.

WO wo 2020/243536 PCT/US2020/035271 PCT/US2020/035271 Certain gelling agents having reactive functional groups, e.g., -OH, -COOH, or -NH2, may be covalently

cross-linked. Techniques for covalent cross-linking of gelling agents are known in the art (see, e.g.,

Khunmanee et al., J. Tissue Eng., 8: 2041731417726464, 2017, the disclosure of which is incorporated

herein by reference). Non-limiting examples of covalent cross-linking agents include: 1,4-butanediol

diglycidyl ether (BDDE), divinyl sulfone, glutaraldehyde, cyanogen bromide, octeylsuccinic anhydride,

acid chlorides, diisocyanates, methacrylic anhydride, boric acid, and sodium periodate/adipic acid

dihydrazide.

Other Excipients

Pharmaceutical compositions may contain pharmaceutically excipients other than gelling agents. For

example, pharmaceutical compositions may contain, e.g., liquid solvents, tonicity agents, buffering

agents, and/or coloring agents. Certain excipients may perform multiple roles. For example, a liquid

solvent in addition to its function as a carrier may be used as a tonicity agent and/or buffering agent.

Such solvents are known in the art, e.g., salines (e.g., hypertonic saline, hypotonic saline, isotonic saline,

or phosphate-buffered saline) and artificial perilymph.

Liquid solvents may be used in pharmaceutical compositions (e.g., pharmaceutical dosage forms) as a

vehicle. Liquid solvents are known in the art. Non-limiting examples of liquid solvents include water,

salines (e.g., hypertonic saline, hypotonic saline, isotonic saline, or phosphate-buffered saline), artificial

perilymph, and tris buffer. Artificial perilymph is an aqueous solution containing NaCI (120-130 mM), KCI

(3.5 mM), CaCl2 (1.3-1.5 mM), MgCl2 (1.2 mM), glucose (5.0-11 mM), and buffering agents (e.g., NaHCO3

(25 mM) and NaH2PO4 (0.75 mM) or HEPES (20 mM) and NaOH (adjusted to pH of about 7.5)).

Tonicity agents may be included in pharmaceutical compositions (e.g., pharmaceutical dosage forms) to

increase osmolarity relative to that which is afforded by an anti-platinum chemoprotectant agent. Tonicity

agents are known in the art. Non-limiting examples of tonicity agents include substantially neutral

buffering agents (e.g., phosphate buffered saline, tris buffer, or artificial perilymph), dextrose, mannitol,

glycerin, potassium chloride, and sodium chloride (e.g., as a hypertonic, isotonic, or hypotonic saline).

Pharmaceutical compositions (e.g., pharmaceutical dosage forms) include sufficient amount of tonicity

agents to provide for administration to a subject a hypertonic pharmaceutical dosage form (e.g., a

pharmaceutical dosage form having a calculated osmolarity of at least 400 mOsm/L (e.g., at least 500

mOsm/L, at least 600 mOsm/L, or at least 700 mOsm/L), and/or 2,500 mOsm/L or less (e.g., 2,000

mOsm/L, 1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/L or less, 1,600 mOsm/L or less, or

1,500 mOsm/L or less)). For example, the targeted concentration of a tonicity agent in a pharmaceutical

composition (e.g., pharmaceutical dosage form) can be determined, e.g., by (i) subtracting the calculated

osmolarity contributions of an anti-platinum chemoprotectant agent and other non-polymeric excipients

from the total targeted calculated osmolarity to obtain the targeted calculated osmolarity contribution from

the tonicity agent, and (ii) determining the concentration of the tonicity agent by dividing the targeted

calculated osmolarity contribution from the tonicity agent by the number of ions and/or molecules

produced upon dissolution of the tonicity agent in a liquid solvent. An appropriate amount of the tonicity

agent thus can be included in the pharmaceutical composition (e.g., pharmaceutical dosage form).

WO wo 2020/243536 PCT/US2020/035271 Buffering agents may be used to adjust the pH of a pharmaceutical composition (e.g., a pharmaceutical

dosage form) a substantially neutral pH level. Buffering agents are known in the art. Non-limiting

examples of buffering agents include, e.g., phosphate buffers and Good's buffers (e.g., tris, MES, MOPS,

TES, HEPES, HEPPS, tricine, and bicine). In addition to the pH control, buffering agents may be used to

control osmolarity of the pharmaceutical composition (e.g., pharmaceutical dosage form).

Methods of Preparation

A pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention may be prepared

from an anti-platinum chemoprotectant agent, a gelling agent, and a liquid solvent. A method of

preparing a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention includes (i)

providing the anti-platinum chemoprotectant agent and the gelling agent, and (ii) mixing the anti-platinum

chemoprotectant agent and the gelling agent with the liquid solvent to produce the pharmaceutical

composition.

The anti-platinum chemoprotectant agent and the gelling agent may be provided, e.g., as a mixture or as

separate ingredients. When the anti-platinum chemoprotectant agent and the gelling agent are provided

separately, the step (ii) may include, e.g.:

(a) mixing the liquid solvent first with the gelling agent to produce an intermediate mixture and

thereafter mixing the intermediate mixture with the anti-platinum chemoprotectant agent;

(b) mixing the liquid solvent first with the anti-platinum chemoprotectant agent to produce an

intermediate mixture and thereafter mixing the intermediate mixture with the gelling agent; or

(c) mixing a portion of the liquid solvent with the anti-platinum chemoprotectant agent to produce

a first mixture, mixing another portion of the liquid solvent with the gelling agent to produce a

second mixture, and combining the first and second mixtures.

The following examples are meant to illustrate the invention. They are not meant to limit the invention in

any way.

EXAMPLES Example 1. Preparation of Thiosulfate Salt Formulations

Hyaluronan Gel 1 (0.5M STS, 1% (w/v) hyaluronan)

Sodium thiosulfate pentahydrate (619.75 mg) was dissolved in sterile, distilled water (5 mL) in a sterile

vial to produce a clear solution. Hyaluronan (50.30 mg; Pharma Grade 80, Kikkoman Biochemifa

company; 0.6-1.2 mDa) was added to the solution, and the resulting mixture was stirred for 8-10 min at

4°C. The resulting solution was filtered through 0.22 um Millex-GV sterile filter.

Hyaluronan Gel 2 (0.1M STS, 2% (w/v) hyaluronan)

Sodium thiosulfate pentahydrate (124.87 mg) was dissolved in sterile, distilled water (3.031 mL).

Methylcellulose (351.01 mg; Methocel® A15 Premium LV, Dow Chemical Company) was dissolved in

sterile, distilled water (2.0 mL), and the resulting solution was mixed with the sodium thiosulfate solution.

Hyaluronan (100.10 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

the resulting mixture and mixed at 4°C for 10-15 min.

WO wo 2020/243536 PCT/US2020/035271 PCT/US2020/035271

Hyaluronan Gel 3 (0.5M STS, 2% (w/v) hyaluronan)

Sodium thiosulfate pentahydrate (620.35 mg) was dissolved in sterile, distilled water (3 mL).

Methylcellulose (350.23 mg; Methocel® A15 Premium LV, Dow Chemical Company) was dissolved in

Hyaluronan (100.65 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

the resulting mixture and mixed at 4°C for 10-15 min.

Hyaluronan Gel 4 (0.1M STS, 1% (w/v) hyaluronan, manitol)

Hyaluronan (50.09 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

water (5 mL). Sodium thiosulfate pentahydrate (124.9mgs) was added. The pH of the resulting mixture

was adjusted to pH7.12 by addition of sodium hydroxide (1N, ca. 0.5 uL). Add appropriate amount of

mannitol into the vial to adjust the osmolarity to 1.046 Osm/kg. The viscous solution was filtered through

0.22 um Millex-GV filter.

Hyaluronan Gel 5 (0.1M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 5 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.1M

concentration of sodium thiosulfate.

Hyaluronan Gel 6 (0.2M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 6 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.2M

concentration of sodium thiosulfate.

Hyaluronan Gel 7 (0.3M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 7 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.3M

concentration of sodium thiosulfate.

Hyaluronan Gel 8 (0.4M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 8 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.4M

concentration of sodium thiosulfate.

Hyaluronan Gel 9 (0.5M STS, 1% (w/v) hyaluronan, Tris (5x))

Hyaluronan (79.99 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

Tris buffer (8 mL, AMRESCO-0497-500G). The pH of the resulting mixture was adjusted to pH7.13 by

addition of HCI (5N). Sodium thiosulfate pentahydrate (992.60 r mg) was added to the above solution. The

viscous solution was filtered through 0.22 um Millex-GV filter.

WO wo 2020/243536 PCT/US2020/035271 PCT/US2020/035271 Hyaluronan Gel 10 (0.5M STS, 1% (w/v) hyaluronan, phosphate buffered saline (5x))

Hyaluronan (70.38 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

PBS buffer (7 mL, 5x). Sodium thiosulfate pentahydrate (868.46 mg) was added. The pH of the resulting

mixture was adjusted to pH6.99 by addition of NaOH (1N). The viscous solution was filtered through 0.22

M Millex-GV filter.

Hyaluronan Gel 11 (0.8M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 11 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.8M

concentration of sodium thiosulfate.

Hyaluronan Gel 12 (1M STS, 0.8% (w/v) hyaluronan)

Hyaluronan Gel 12 was prepared according to the procedure described for Hyaluronan Gel 1 with the

exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 1M concentration

of sodium thiosulfate, and the amount of hyaluronan was adjusted to provide a 0.8% (w/v) concentration

of hyaluronan.

Hyaluronan Gel 13 (0.5M STS, 0.82% (w/v) hyaluronan (HYALGAN)) Hyaluronan Gel 13 was prepared by mixing of sodium thiosulfate pentahydrate with hyaluronan

(HYALGAN, Fidia Pharma USA, Florham Park, NJ) to afford the final preparation with 0.82% (w/v)

concentration of hyaluronan.

Hyaluronan Gel 14 (0.5M STS, 1% (w/v) hyaluronan (SINGCLEAN))

Hyaluronan Gel 14 was prepared according to the procedure described for Hyaluronan Gel 13 with the

exception that hyaluronan (SINGCLEAN, Hangzhouh Singclean Medical Products Co., Ltd., Hangzhou,

China) was used in the preparation of this gel.

Hyaluronan Gel 15 (0.5M STS, 1% (w/v) hyaluronan (EUFLEXXA)) Hyaluronan Gel 15 was prepared according to the procedure described for Hyaluronan Gel 13 with the

exception that hyaluronan (EUFLEXXA, Ferring Pharmaceuticals Inc., Parsippany, NJ) was used in the

preparation of this gel.

Hyaluronan Gel 16 (0.5M STS, 1% (w/v) hyaluronan (HEALON))

Hyaluronan Gel 16 was prepared according to the procedure described for Hyaluronan Gel 13 with the

exception that hyaluronan (HEALON, Johnson & Johnson, New Brunswick, NJ) was used in the preparation of this gel.

Hyaluronan Gel 17 (1M STS, 1% (w/v) hyaluronan)

Hyaluronan Gel 17 was prepared according to the procedure described for Hyaluronan Gel 1 with the

of sodium thiosulfate.

WO wo 2020/243536 PCT/US2020/035271 Hyaluronan Gel 18 (10% (w/v) N-Acetyl-L-cysteine, 1% (w/v) hyaluronan)

Hyaluronan (39.38 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to

water (4 mL). N-Acetyl-L-cysteine (399.14 mg) was added. The pH of the resulting mixture was adjusted

to pH 7.21 by addition of NaOH (10N, 240 uL). The viscous solution was filtered through 0.22 M Millex-

GV filter. The osmotic pressure was measured as 1.107 Osm/kg.

Other hyaluronan gels may be prepared using the procedures described herein. For example, 1M and

1.5M hyaluronan gels may be prepared according to the same procedure as described for, e.g.,

Hyaluronan Gel 1 and Hyaluronan Gel 12. Additionally, pH levels of the gels may be adjusted to pH 6.5

to 8.5 using Bronsted acids (e.g., hydrochloric acid) and bases (e.g., sodium hydroxide).

Example 2. Pharmacokinetic Modelling of Perilymph Concentrations

Maximum cochlear platinum levels in humans following high dose cisplatin treatment (100 mg/m² were

modeled through the use of pharmacokinetic (PK) simulations of cisplatin distribution in a human. Kinetic

parameters used for simulations were obtained from literature reports on cisplatin population PK (Urien et

al, Br. J. Clin. Pharmacol., 57:756-63, 2004) and PK following high dose cisplatin treatment (Andersson et

al, J. Pharm. Sci., 85:824-27, 1996). The PK simulations were conducted using WinNonlin (Phoenix 64)

PK simulation model 9 (IV infusion, 2 compartment). A plasma-to-cochlea concentration ratio of 1:1 was

assumed based on previous results from animal studies, and this assumption is further supported by

literature reports showing tissue distribution characteristics of cisplatin (Johnsson et al, Cancer

Chemother. Pharmacol., 37:23-31, 1995).

The predicted maximal plasma platinum concentration following cisplatin treatment was determined to be

approximately 22 M. Applying a 30-fold molar stoichiometric ratio results in a concentration of 660 M

(0.66 mM) which, when the molecular weight of thiosulfate is used, results in thiosulfate concentration of

74 ug/mL. Achieving this level of thiosulfate in the human cochlea is expected to provide complete

(maximum) protection against cisplatin induced ototoxicity following high dose (e.g., 100 mg/m² cisplatin

treatment.

Example 3. Pharmacokinetic Studies In Vivo Hyaluronan Gel #1 was administered to male Hartley guinea pigs at a dose of 12% w/v (0.5M, 6.2 mg).

The dose volume was fixed (10 uL). Perilymph was sampled at each timepoint (n=5 animals/timepoint)

and the concentration of thiosulfate was quantified using a liquid chromatography with tandem mass

spectrometry (LC-MS/MS) method.

Hyaluronan Gel 1 achieved maximal perilymph concentration, 868.5 ug/mL (approximately 7.8 mM) at the

first time-point sampled (1h). This maximum perilymph concentration is approximately 10-fold greater

than the Hyaluronan Gel 1 concentration predicted to provide 100% protection (minimal efficacious

concentration; 74 ug/mL, 0.66 mM) from cisplatin induced ototoxicity The perilymph t1/2 ranged from 2.7

to 6.4 hours. The high perilymph Hyaluronan Gel 1 concentration combined with the relatively long half-

life provide a treatment window of 3 h pre to 4 h post cisplatin treatment. In another pharmacokinetic

study, healthy human subjects were divided into 4 dose cohorts: Cohort 1, Cohort 2, Cohort 3, and Cohort wo 2020/243536 WO PCT/US2020/035271 4. Each dose cohort contained 8 human subjects randomized to receive either DB-020 or placebo (6/2 randomization; 6 subjects received DB-020, 2 subjects received placebo). Cohort 1 was administered unilaterally, intratympanically 19 mg of sodium thiosulfate as a 0.15M sodium thiosulfate/hyaluronan gel, prepared as described for Hyaluronan Gel 1. Cohort 2 was administered unilaterally, intratympanically 62 mg of sodium thiosulfate as Hyaluronan Gel 1. Cohort 3 was administered unilaterally, intratympanically

124 mg of sodium thiosulfate as Hyaluronan Gel 17. Cohort 4 was administered unilaterally,

intratympanically 186 mg of sodium thiosulfate as a 1.5M sodium thiosulfate/hyaluronan gel, prepared as

described for Hyaluronan Gel 1. The Placebo subjects were administered 1% w/v aqueous hyaluronan.

The results of the studies are shown in Table 1 and 2 and in FIGS. 1-3.

Table 1

Cmax, 0-24 tmax (h) t1/2 (h) Dose AUC0-24 (ng*h/mL) (ng/mL)

Placebo 37.51 + 8.59 9.06 + 6.31 684.85 + 105.12 ND Cohort 1 38.42 ± + 7.47 11.33 + 2.73 685.99 + ± 97.86 ND Cohort 2 130.32 + 60.26 0.42 + 0.13 1.11 1011.74 + 257.25

Cohort 3 180.00 + 36.59 0.50 + 0.00 0.87 860.90 + ± 219.42

Cohort 4 264.00 + 68.81 0.46 + 0.10 0.63 1167.48 + 204.93

Table 2

Cmax Increase to endogenous a Cohort Cohort C Mean + SD (ng/mL) Mean + SD (pM)b Mean (uM) Range (uM)

2 (12% w/v) 130.32 + 60.26 1.16 + 0.54 0.83 0.04 1.20 3 (25% w/v) 180.00 + 36.59 1.61 + 0.33 1.28 0.36 1.70 4 (37% w/v) 264.00 + 68.81 2.36 + 0.61 2.03 0.49 2.65 a increase to endogenous = (mean thiosulfate Cmax) - (mean thiosulfate Placebo levels)

b MW thiosulfate = 112 g/mol

Example 4. Pharmacodynamic Studies In Vivo

To assess the treatment window for a sodium thiosulfate gel in relation to cisplatin administration, a single

dose of 1.24 mg Hyaluronan Gel 1 was administered intratympanically (IT) to the left ear of guinea pigs

either 24 hours, 6 hours, 3 hours, or 1 hour prior to, or 1 hour, 4 hours or 24 hours following cisplatin

(single 10 mg/kg IV bolus; FIG. 4). All right control ears that were not treated with Hyaluronan Gel 1

demonstrated significant threshold shifts (i.e., hearing loss) compared to naive animals (FIG.

5A). Hyaluronan Gel 1 dosed from 3 hours prior to 4 hours post cisplatin administration, provided

protection from cisplatin-induced hearing loss relative to control ears untreated by Hyaluronan Gel 1 (FIG.

5B). Hyaluronan Gel 1 was moderately less effective at protecting from cisplatin-induced hearing loss

when administered more distally to cisplatin treatment (e.g., > 6 h prior or 24 h post cisplatin dose).

Maximum protection was observed when Hyaluronan Gel 1 was dosed 1 h prior to cisplatin administration

indicating the highest level of protection can be achieved when Hyaluronan Gel 1 is administered prior to

and, preferably, proximally to cisplatin treatment.

WO wo 2020/243536 PCT/US2020/035271

Example 5. Pharmacokinetic Performance of Exemplary Hydrogels

Guinea Pigs, Study 1

Albino guinea pigs (Hartley), body weight at 250-350 g, were used for the studies. For dosing, the animal

was placed on its shoulder with the surgery ear up and auditory bulla was first exposed using

retroauricular approach. A hole of 2-3 mm in diameter was drilled on the bulla to provide direct

visualization of the round window niche. Then, 10 ul of an aqueous composition of 0.5M sodium

thiosulfate / 2% (w/v) hyaluronan (STS Composition) were applied onto the RWM using a 10 ul Hamilton

syringe and a 26-gauge needle. After application, guinea pigs remained at this position for 30 min to

allow compound to diffuse into the cochlea. The bulla opening was sealed with a muscle graft and the

incision closed with sutures.

Sampling procedures are as follows, in brief. All sampling procedures are terminal. Animals were

euthanized with CO2. 0.5 ml samples of blood were collected by cardiac puncture. Plasma was

separated by centrifugation at 5,000 rpm at 4°C for 10 min and collected in a separate tube. 50 ul of

cerebrospinal fluid were collected through the cisterna magma. Perilymph was collected ex vivo to avoid

contamination from the cerebrospinal fluid influx via the cochlear aqueduct. The temporal bone was

rapidly isolated, and the bulla was removed to expose the cochlea. Any visible remaining dosed

compositions were carefully removed with absorbent points under the surgical microscope before

perilymph sampling. A small hole was made at the apex, and then 5-7 ul of perilymph was sampled

using a pulled glass pipette. All samples were frozen immediately on dry ice and stored in -80°C until

analysis. The concentrations of thiosulfate in the samples were measured using the method disclosed in

Togawa et al. Chem. Pharm. Bull., 40:3000-3004, 1992, the disclosure of which is incorporated herein by

reference. The results of this study are shown in FIGs. 8A, 8B, 9A, 9B, and in Table 3.

Cynomolgus Monkey Cynomolgus monkey was administered tolfedine (4 mg/kg) subcutaneously. After 30 minutes, the animal

was anesthetized via intravenous bolus of propofol (5.5 mg/kg). 2-3% isoflurane inhalation was then

used to maintain the animal in anesthetized state. The animal was then immobilized and placed laterally

in reverse Trendelenburg position to ensure the access to the round window. During the surgery process,

the animal was kept on a warm blanket.

Intratympanic injection in right ear was conducted when the animal reaches the anesthetized state. 1.1

mL of epinephrine hydrochloride-saline (0.1 mg in 10 mL saline) and 0.5 mL of lidocaine hydrochloride

(20 mg/mL) were injected subcutaneously into the skin of ear canal posterior wall of each ear respectively

as local anesthetics. An incision was then made in the post-auricular skin, and part of the temporal bone

was drilled to expose middle ear. 50 uL of the STS composition were injected into the round window

membrane using a 25G needle. After dosing, the animal was left on a line with its head up to allow the

dosing solution to settle into the tympanic cavity for 30 mins. The same procedure was then repeated for

the opposite ear.

24

PCT/US2020/035271 Plasma and CSF were collected ca. 2 h after dosing the 1st ear (right). Right ear cochlea perilymph

sampling was conducted ca. 3 h after the right ear dosing. The animal was euthanized by IV

administration of propofol at 11 mg/kg and then exsanguinated via femoral artery. Animal was then

placed in lateral recumbent. A post-auricular skin incision was made and the external ear canal was

extracted to expose middle ear. Part of the temporal bone was then drilled to expose the basal turn of the

cochlea. The remaining dose in the middle ear (if visible) was cleaned with cotton tips. A drop of tissue

glue was spread at the base of the cochlea to minimize the contamination from the dosed compositions.

Using a 0.5-1 mm round-tipped burr or sharped crochet, a hole was made at the basal turn of cochlea.

Perilymph (ca. 10 uL) was then collected using the capillary tube inserted into the cochlear scala tympani.

The same procedure was repeated for the left ear cochlea perilymph sampling ca. 2 h after the left ear

dosing. The results of this study are shown in Table 3.

Table 3

Poloxamer Hyaluronan Hyaluronan Hyaluronan Hyaluronan Hyaluronan

407 gel 1 Gel 2 Gel 3 Gel 1 Gel 1 Gel 1

Cynomolgus Species Guinea Pig Guinea Pig Guinea Pig Guinea Pig Guinea Pig Monkey

Dose 10 ul (IT) 10 ul (IT) 10 ul (IT) 10 ul (IT) 50 ul (TT) 50 ul (IT)

Total Dose 0.11 0.11 0.56 0.56 2.8 2.8 (mg)

Tmax (est.) (h) 1 1 1.8 1 3

Cmax (ng/mL) 59000 104040 712800 1686200 1686200 1930000 1930000 688500 @ 2 h 688500@2h Terminal t(1/2) 2.38 1.53 2.66 2.01 2.73 (h)

AUC last 212523 327512 4634845 9069083 10498230 (h.ng/mL)

AUC inf 255707 377265 4724413 10418954 10703165 (h.ng/mL)

Plasma* 2390 766 766 (ng/mL)

In the above table, IT is intratympanic administration, and TT is transtympanic administration.

* concentration of thiosulfate as measured in plasma samples from the tested animals.

Guinea Pigs, Study 2

Male guinea pigs weighing 200-300 g of approximately 5-7 weeks of age served as subjects (N = 5 per

group). Prior to any procedures, animals were anesthetized using zolazepam hydrochloride (Zoletil 50;

WO wo 2020/243536 PCT/US2020/035271 20 mg/kg) 10 minutes before surgery via the intramuscular route. If needed, an intraoperative booster

was administered intraperitoneally representing a one-tenth of the original dose.

Intratympanic injection:

1. Under microscopic magnification, sharp scissors were used to create a 0.5-1.5 cm postauricular skin

incision, approximately 6-8 mm caudal to the auriculo-cephalic crease. Care was exercised to avoid

cutting deeply to preserve underlying vascular structures.

2. Careful blunt dissection through the subcutaneous fat layer, muscles and tissues was performed with

forceps. The cleidomastoideus muscle body was gently retracted until the shiny dome of the tympanic

bulla periosteum came into view. At the caudal aspect of the bulla, the insertion of a deeper cervical

muscle, the sternomastoideus came into view. The facial nerve, which becomes visible at the dorsal

and rostral aspect of the bulla dome, was preserved during the operation.

3. A self-retaining retractor was placed prior to creating a small hole (0.5mm diameter) either with a

drilling in the posterior part of the bulla. The bulla bone was uncapped in a dorsal and caudal

direction using a pair of jeweler's tip forceps. The bone was removed in a piecemeal fashion under

high magnification. Care was exercised not to puncture the stapedial artery, which lies directly

beneath the bulla cap, as bleeding from this artery may compromise the procedure. The amount of

bone removed was kept to a minimum to prevent excessive fluid entry to the middle ear while still

allowing excellent visualization and access to the round window niche.

4. 10 or 90 ul of a gel formulation was delivered to the round window niche using a sterile glass

Hamilton syringe with 25-26 G blunt needle.

5. The delivered agent was allowed to rest within the round window niche for up to 30 min. The small

hole was covered with muscular tissue and tissue glue.

6. The incision was closed with sutures (4-0 non-absorbable monofilament or 5-0 non-absorbable

nylon) and tissue glue or wound clips. The entire procedure took approximately 3-5 minutes

depending on agent specifications.

7. During the procedure and until recovery, animals were placed on a temperature controlled (38 °C)

heating pad until consciousness was regained, at which time they were returned to the home-cage.

Alternatively, the animals were administered the gel formulations transtympanically.

Sampling collection:

Blood collection:

1. Without preinflating in the euthanasia box, the guinea pig was placed in a box, and 100% carbon

dioxide was introduced to cause the animal unconsciousness and to reduce animal suffering. Carbon

dioxide flow was maintained for a minimum of 1 minute after the breath has stopped. The guinea pig

was removed from the euthanasia box after death was confirmed.

2. Blood was collected immediately after euthanasia.

3. After the operator fixed the animal's back position, the needle was inserted at the front of the sternal

ridge at 4-6 or slightly forward.

4. The needle was pulled back, and the blood was returned.

5. Volume: for each blood collection, ca. 1 mL of blood was collected.

WO wo 2020/243536 PCT/US2020/035271

CSF collection:

CSF was collected after euthanasia. A 0.5*20 intravenous infusion needle was slowly lowered from 90°

to the foramen magnum. The needle reached a distance of 4.5-5 mm under the skin, and 50-200 ul of

clear tissue fluid were withdrawn.

Perilymph collection:

After euthanasia, the animal was stripped excess skin and muscle tissue to obtain a complete auditory

bulla, and the bulla wall was cut with small forceps to expose the cochlea. The basal turn of bulla was

cleaned by using small cotton ball. The cochlear bottom circle and the round window were coated with

bio glue. After drying, a unique microhole was hand-drilled in the top circle of the cochlea. A 2ul volume

of perilymph was then collected using a microcapillary inserted into the cochlear top circle. Perilymph

samples were added to a vial containing 18 ul of bovine serum albumin (BSA, 1M) stored at -80 °C until

analysis.

The results of the Guinea Pig, Study 2 are provided in Tables 4 and 5.

Table 4

Formulation Route Tmax Cmax terminal AUCINE AUClast AUClast Osmol. (h) (ng/mL) T1/2 (h) (hxng/g) (hxng/g) (Osm/kg)

Hyaluronan Gel 1 TT (50 uL) 1 2.73 872360 4837831 4745200 Hyaluronan Gel 4 TT (50 uL) 3 115300 2.3 608881 601694 1.046

TT (50 uL) 1 6.42 Hyaluronan Gel 5 391432 4084991 3529898 0.267

TT (50 uL) 1 8.89 Hyaluronan Gel 6 1069794 5727631 4150041

Hyaluronan Gel 6* TT (50 uL) 3 500900 N/A N/A 3817583 0.491

Hyaluronan Gel 7 TT (50 uL) 7 210933 N/A 2357105 0.657

Hyaluronan Gel 7* TT (50 uL) 3 600420 N/A N/A 3281800 0.66

TT (50 uL) 1 3.48 Hyaluronan Gel 8 508500 4014647 3887550 0.838

TT (50 uL) 1 2.11 Hyaluronan Gel 9 1602936 7029505 6990442 2.048

TT (50 uL) 1 2.2 Hyaluronan Gel 10 1293624 5866461 5825930 Hyaluronan Gel 11 TT (50 uL) 1 2.99 1371800 1371800 5863793 5714356 1.494

TT (50 uL) 1 2.57 1.860 Hyaluronan Gel 12 1892000 1892000 7744025 7644025 1.860

TT (50 uL) 1 2.47 Hyaluronan Gel 17 1044400 1044400 5918627 5843593 TT (50 uL) 1 5.53 Hyaluronan Gel 18 705600 4887980 4876589 1.107

In this table, TT is transtympanic administration,

* This test was a duplicate of the preceding test.

Table 5

Formulation Route Tmax Cmax terminal terminal AUCINE AUCINF AUClast MW Hyaluronan Gel 1 (mDa) 0.6-1.2 IT (10 uL) T C 1 (h) (ng/mL)

625297 T1/2

1.88 (hxng/g)

4112197 (hxng/g)

4099226 0.5-0.73 TT (50 uL) 1 2.82 Hyaluronan Gel 13 815408 4868652 4754315 TT (50 uL) 1 3.27 Hyaluronan Gel 14 unknown 851568 3.27 5178077 4992602 2.4-3.6 TT (50 uL) 1 3.41 Hyaluronan Gel 15 628200 3908302 3761700 ca. 4 TT (50 uL) 1 5.42 Hyaluronan Gel 16 919097 5033151 4407366 In this table, IT is intratympanic administration, and TT is transtympanic administration.

Example 6. Pharmacodynamic Performance of Exemplary Hydrogels

Cisplatin was diluted with 0.9% (w/v) saline to a final concentration of 5 mg/mL. Albino guinea pigs

(Hartley), body weight at 250-350 g were used in the study. After a minimal 3 days acclamation, 28

animals were enrolled into the study. Under aseptic condition, cisplatin was administered

intraperitoneally with a bolus injection. The five cohorts were staggered with different starting dates for

the study.

Seven days after the cisplatin administration, the animals were recorded for their auditory brainstem

responses (ABR) response using TDT RZ6 Multi-I/O processor. Historical ABR data were used to define

a baseline. The animals were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride

(Zoletil). Acoustic stimuli were delivered via an earphone. Needle electrodes were placed near the ear

canal at the causoventral position, the vertex of the skull, and a ground at the lower leg. The stimulus

level was from 10 to 90 dB in 5 dB steps, and the tone-pip frequencies were 4, 24, and 32 kHz. The

ceiling sound pressure level was 90 dB. ABR threshold was observed by visual inspection of stacked

waveforms as the lowest sound pressure level, at which the waveform was above the noise floor.

Prior to the cisplatin study, ABR data from 50 animals were recorded for both ears of each animal (Naîve

n=100). The threshold at 32 kHz in naive animals was 39.8 dB. The range of normal hearing was

defined as a mean + 2SD, 27.9 to 51.6 dB. Cisplatin primarily induces hearing loss at high frequencies.

A clear pattern of hearing loss after cisplatin is defined as a threshold of 60 dB and above at 32 kHz.

In this study, 1 out of 28 animals died before the day 7 measurement. In the remaining 27 animals, 18

animals had hearing loss with threshold >60 dB at 32 kHz (FIG. 10A). The range of hearing loss at 32

kHz was the thresholds from 65 dB to 90 dB (FIG. 10B). 90 dB is the measurement ceiling. Note when

no waveform or waveform only seen at 90 dB, the threshold was defined both as 90 dB. The average

threshold at 32 kHz was 82 dB, which corresponds to an average 42.2 dB shift from the naive threshold

of 39.8 dB (FIG. 11A).

Local Intratympanic Dosing and Cochlear Sampling

Local intratympanic dosing and cochlear sampling were conducted as described in Example 5.

WO wo 2020/243536 PCT/US2020/035271 PCT/US2020/035271 Locally Delivered Anti-platinum Chemoprotectant Agent Provides Hearing Protection

from Platinum-based Antineoplastic Agent

Evaluation of the effect of a locally delivered anti-platinum chemoprotectant agent on hearing protection

from a platinum-based antineoplastic agent was conducted as follows.

An aqueous composition of 0.5M sodium thiosulfate 2% (w/v) hyaluronan (STS Composition) or vehicle

was dosed intratympanically onto the round window in the left ear (LE) as described above, and the right

ear (RE) was left untreated in the guinea pigs (FIG. 12). 60 min after STS Composition or vehicle dosing,

the animals were injected with cisplatin at 10 mg/kg intraperitoneally. ABR at 4, 24, and 32 kHz was

measured in both ears 7 days after cisplatin administration.

Because of heterogenity of hearing loss after cisplatin challenge, the untreated right ears was used to

select the animals with hearing loss. There were 21 animals with right ear threshold >60 dB at 32 kHz.

Of these 21 animals, 3 with otitis media were excluded, leaving 18 animals for the final analysis. 10

animals were dosed with vehicle and 8 animals were dosed with STS Composition (FIG. 11B). In the

untreated right ears of both the STS Composition and vehicle groups, there is no difference in the ABR

thresholds with an average threshold 73 dB at 4 kHz, 71 dB at 24 kHz, and 80 dB at 32 kHz The

vehicle-treated left ears had no significant difference in comparison to their untreated right ears, showing

threholds 74 dB at 4 kHz, 70 dB at 24 kHz, and 74 dB at 32 kHz.

The STS Composition-treated ears had significantly lower thesholds at both 32 kHz and 24 KHz

compared to the vehicle-treated ears and untreated right ears (***P<0.001, two way ANOVA). At 4 kHz

an average threshold was 61 dB in the STS Composition treated ears and 75 dB in their untreated

contralateral right ears; the protection was not statistically significant (P=0.089). The average thresholds

in the STS Composition treated ears were 40 dB and 48 dB at 24 kHz and 32 kHz, respectively, in

contrast to 69 dB and 80 dB in their contralateral untreated right ears. The normal hearing thresholds

were 35 dB and 40 dB at 24 kHz and at 32 kHz, respectively, in the the naive animals. To the naive ears,

the untreated ears after cisplatin had an averange of 34 dB and 40 dB threshold elevation at 24 kHz and

32 kHz, respectively, but STS Composition treated ears only had 5 dB and 8 dB shift. Therefore, sodium

thiosulfate provided, on average, 80% protection at both 24 kHz and 32 kHz.

In a similarly designed study as described hereinabove, sound pressure levels at 4, 24, and 32 kHz were

measured during ABR tests for the guinea pigs administered vehicle or sodium thiosulfate (0.1M, 0.5M, or

1M sodium thiosulfate gel) to one ear each followed by a cisplatin challenge (Cisplatin 10MPK,

intravenous injection). Different doses of hyaluronan gels were administered as a 10 uL IT injection into

the left ear one hour prior to cisplatin administration. The contralateral ear (right ear) of the animal was

untreated. Hyaluronan Gel 5 (0.1M), Hyaluronan Gel 1 (0.5M), and Hyaluronan Gel 17 (1M) was

tested. The untreated ears demonstrated significant threshold shifts compared to naive animals (gray

shaded areas). The groups treated with Hyaluronan Gel 1 (0.5M) and Hyaluronan Gel 17 (1M) showed

hearing protection compared to the untreated contralateral control ears at all tested frequencies. No

protection was seen with the vehicle treated ears. The results are summarized in FIG. 13.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described 5 in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. 2020284122

10 Other embodiments are in the claims.

In this specification, the terms “comprise”, “comprises”, “comprising” or similar terms are intended to mean a non-exclusive inclusion, such that a system, method or apparatus that comprises a list of elements does not include those elements solely, but may well include other elements not listed.

15 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Claims

CLAIMS 30 Jan 2026

1. A method of mitigating platinum-induced hearing loss in a subject in need thereof, the method comprising (i) administering to the subject by intratympanically or transtympanically injecting an effective amount of a hypertonic composition having a calculated osmolarity of 1,500 mOsm/L to 5,000 mOsm/L and comprising 0.5M-1.0M of the thiosulfate salt, and (ii) administering a platinum-based antineoplastic agent to the subject 1 to 3 hours following step (i). 2020284122

2. The method of claim 1, wherein the thiosulfate salt is an alkaline thiosulfate salt, ammonium thiosulfate salt, or a solvate thereof.

3. The method of claim 1 or claim 2, wherein 200-1,000 µL of the hypertonic pharmaceutical composition are administered to the round window of the subject.

4. The method of any one of claims 1 to 3, wherein the effective amount is an amount that produces a plasma thiosulfate concentration that is 30 µM or less at the time the platinum- based antineoplastic agent is administered.

5. The method of any one of claims 1 to 4, wherein the effective amount is an amount that produces a maximum thiosulfate concentration of 0.6-10 mmol/L by 1h post administration.

6. The method of any one of claims 1 to 5, wherein the effective amount is an amount that produces a thiosulfate concentration of 0.1-2 mmol/L by 7 h post administration in the subject’s cochlea.

7. The method of any one of claims 1 to 6, wherein the hypertonic composition produces a cochlear thiosulfate Cmax is at least 30 times greater than a cochlear Cmax of the platinum- based antineoplastic agent, wherein the cochlear Cmax concentrations are modeled by a pharmacokinetic simulation of intravenous infusion in a two compartment model.

8. The thiosulfate salt for use of any one of claims 1 to 7, wherein the platinum-based antineoplastic agent is cisplatin.

9. The thiosulfate salt for use of any one of claims 1 to 8, wherein the hypertonic composition comprises 1.0M of the thiosulfate salt. 30 Jan 2026

10. The thiosulfate salt for use of any one of claims 1 to 9, wherein the thiosulfate salt is sodium thiosulfate.

11. The thiosulfate salt for use of any one of claims 1 to 10, wherein the hypertonic 2020284122

composition further comprises 1%-2% (w/v) hyaluronan.

12. The thiosulfate salt for use of claim 11, wherein the hypertonic composition comprises 1% (w/v) hyaluronan.

13. The thiosulfate salt for use of any one of claims 1 to 12, wherein the hypertonic composition is administered transtympanically.

14. The thiosulfate salt for use of claim 13, wherein the transtympanic administration is by injection directly through a tympanic membrane.

15. The thiosulfate salt for use of claim 14, wherein prior to injection a separate ventilation hole in the tympanic membrane is created to allow air to escape the middle ear space.

16. A method of mitigating platinum-induced hearing loss in a subject in need thereof, the method comprising (i) administering to the subject by intratympanically or transtympanically injecting an effective amount of a hypertonic composition having a calculated osmolarity of 3,000 mOsm/L to 5,000 mOsm/L and comprising 1.0M of the sodium thiosulfate salt and 1% (w/v) hyaluronan, and (ii) administering a platinum-based antineoplastic agent to the subject 1 to 3 hours following step (i).