The field of the invention

The present invention relates to pharmaceutical compositions comprising a PPAR agonist and an auris-acceptable gel for preventing or treating hearing loss and/or for preventing or inhibiting hair cell degeneration or hair cell death in a subject.

Background of the invention

Hearing loss is related to damage of auditory hair cells e.g. apoptosis of the hair cells as a consequence of e.g. a continuous stress situation or a traumatic event e.g. leading to the activation of inflammatory pathways. Hearing loss can be caused e.g. by a noise trauma, by a medical intervention, by ischemic injury, by a non specific stress leading to sudden hearing loss or by age or can be chemically induced, wherein the chemical induction is caused e.g. by an antibiotic or a chemotherapeutic agent. Child hearing loss might be caused by pre or post natal deficiencies in the energy homeostasis of the auditory cells. Hearing loss can also be caused by mitochondrial dysfunction. (C. M. Sue PhD, FRACP1, Cochlear origin of hearing loss in MELAS syndrome, Annals of Neurology. Volume 43, Issue 3, pages 350-359, March 1998). In additio,n a link between metabolic syndrome and hearing loss could be shown (Barrenas ML, Jonsson B, Tuvemo T, Hellstrom PA, Lundgren M, J Clin Endocrinol Metab. 2005 Aug;90(8):4452-6. Epub 2005 May 31). Hearing loss can be of sensorineural origin caused by a damage leading to malnutrition of the cells in early brain development.

Hair cells are fully differentiated and are not replaced after cell death (only a few thousand cells from birth). It is well described in the literature that after stress and damage of the hair cells, the cells can enter a resting state with no functionality related to the hearing process but remain viable. Approaches to stimulate development or regeneration of new hair cells e.g. by administering growth factors or by stem cell-based therapies in order to achieve disease modification bear the risk of pro-tumorigenic side-effects.

Hearing impairment is a major global health issue with profound societal and economic impact affecting over 275 million people world- wide. The occurrence of hearing loss is rapidly rising, due to e.g. increasing noise exposure and aging populations. With no approved pharmaceutical therapies available today, the unmet medical need is very high. In particular, there is a need for providing effective methods for prevention and subsequent treatment of hearing loss which allow for immediate as well as long term maintenance of preventive and/or therapeutic effects.

Summary of the invention

The present invention relates generally to pharmaceutical compositions comprising a PPAR agonist and an auris-acceptable gel for use in methods of preventing or treating hearing loss and methods of preventing or inhibiting hair cell degeneration or hair cell death. The present invention provides pharmaceutical compositions comprising a PPAR agonist and an auris- acceptable gel and methods which allow for protection of hair cells from stress e.g. from noise-induced stress, from surgery-induced stress, or from chemically-induced stress such as stress induced by an antibiotic or by a chemotherapeutic agent or from unspecific stress which may cause hearing loss. By using the pharmaceutical compositions and the methods described herein, immediate and subsequent long-term maintenance of preventive and/or therapeutic effect can be achieved. In a standard model established in hearing loss research, it could be shown that treatment with a PPAR agonist protects hair cells, which upon exposure to an antibiotic are normally destroyed within 48 hours. The addition of the PPAR agonist prior to antibiotic challenge was able to prevent hair cells from apoptosis and cell death in a dose- dependent manner. In a further model, it could be shown that a thermoreversible auris- acceptable gel comprising pioglitazone protected hearing from noise trauma.

A disadvantage of liquid pharmaceutical compositions for intratympanic injection is their propensity to drip into the eustachian tube causing rapid clearance of the composition from the inner ear. The pharmaceutical compositons provided herein comprise an auris-acceptable gel, most preferably a thermoreversible auris-acceptable gel, and, by virtue of said auris- acceptable gel, remain in contact with the target auditory surfaces (e.g., the round window) for extended periods of time. Accordingly, the pharmaceutical compositions described herein avoid attenuation of therapeutic benefit due to drainage or leakage of active agents (i.e. PPAR agonists) via the eustachian tube.

Thus, in a first aspect, the present invention relates to a pharmaceutical composition comprising:

i. a PPAR agonist; and

ii. an auris-acceptable gel, wherein the auris-acceptable gel comprises a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, polyesters, colloidal silica, aluminum soaps, zinc soaps, natural gums, starch, cellulose and derivatives thereof, carboxyvinylpolymers, metal silicates and mixtures thereof,

for use in a method of preventing or treating hearing loss in a subject.

In a further aspect, the present invention relates to a pharmaceutical composition comprising:

i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel comprises a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, polyesters, colloidal silica, aluminum soaps, zinc soaps, natural gums, starch, cellulose and derivatives thereof, carboxyvinylpolymers, metal silicates and mixtures thereof,

for use in a method of preventing or inhibiting hair cell degeneration or hair cell death in a subject.

In a further aspect, the present invention relates to a pharmaceutical composition comprising:

i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel comprises a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, polyesters, colloidal silica, aluminum soaps, zinc soaps, natural gums, starch, cellulose and derivatives thereof, carboxyvinylpolymers, metal silicates and mixtures thereof.

In a further aspect, the present invention relates to a kit for preventing or treating hearing loss or preventing or inhibiting hair cell degeneration or hair cell death in a subject comprising a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel described herein and instructions for using the kit.

In a further aspect, the present invention relates to a process for producing a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel described herein, comprising the steps of:

i. dissolving a thermoreversible gel in water for injection; ii. dissolving additional excipients in water for injection;

iii. mixing the solutions thoroughly;

iv. performing a sterile filtration of the resulting solution through an appropriate

sterile filter;

v. preparing a sterile suspension by incorporation of the sterile PPAR agonist in the sterile filtrate, using aseptic procedures; and

vi. filling required quantities of the suspension into an appropriate packaging

configuration.

Brief description of the figures

Figure 1 A-C show quantitation of the average number of hair cells remaining in the apical, basal and middle turns of the organ of Corti (OC). While gentamicin (200mM) treatment resulted in a consistent reduction of hair cell number of approximately 50 - 70% in each segment, Pioglitazone at both concentrations (2mM and 10mM) was able to significantly prevent gentamicin-dependent hair cell loss in all turns. The values for each turn were averaged for the 10 OCs used for each condition. Significant differences between treatment groups in OHC and IHC (OHC = outer hair cell; IHC = inner hair cell) were determined using analysis of variance (ANOVA) followed by the least significant difference (LSD) post-hoc test (Stat View 5.0). Differences associated with P-values of less than 0.05 were considered to be statistically significant. All data are presented as mean ± SD.

Figure 2 shows the change in the average hearing thresholds in guinea pigs determined by auditory brainstem response (ABR) one week or two weeks after noise challenge vs. pre treatment values. Threshold shifts at individual frequencies were calculated for each animal by subtracting post-noise from pre-noise values. Group averages at each frequency were determined. An overall threshold shift was calculated for each treament group and timepoint by averaging the individual frequency shifts over 8 - 20 KHz. Data are mean ± S.D. * p< 0.05.

Figure 3 A-C show quantitation of the average number of hair cells remaining in defined segments in the medio-basal turns of the organ of Corti (OC). While gentamicin (50mM) treatment resulted in a consistent reduction of hair cell number of approximately 50 %, tesaglitazar, muraglitazar and fenofibric acid were all able to significantly prevent gentamicin- dependent hair cell loss. The values were averaged for the 5-7 OCs used for each condition. Significant differences between treatment groups in hair cell numbers were determined using analysis of variance (ANOVA) followed by the least significant difference (LSD) post-hoc test (Stat View 5.0). Differences associated with P-values of less than 0.05 were considered to be statistically significant. All data are presented as mean ± SD. **** = p< 0.001.

Figure 4 A-B show the efficacy of a pharmaceutical composition comprising a thermoreversible gel comprising pioglitazone to protect hearing from noise trauma. Vehicle treated animals showed a frequency-specific threshold shift of about 50-60 dB immediately after noise trauma. Pioglitazone significantly reduced the threshold shift in both experiments (Figure 4 A,B). Animals treated 1 hr. after noise showed the greatest protection (approx. 20 dB across all frequencies). Notably, by day 21, the treated ears had almost fully recovered, as evidenced by the return to pre-noise threshold values. Treatment 48 hr. after noise also reduced threshold shifts, but to a lesser extent compared to animals treated 1 hr. following noise trauma. These data demonstrate the efficacy of pioglitazone to protect hearing in a second species. Early treatment leads to almost full recovery of hearing.

Detailed description of the invention

The present invention provides pharmaceutical compositions comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or treating hearing loss and/or for use in a method of preventing or inhibiting hair cell degeneration or hair cell death.

For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The terms "comprising", "having", and "including" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

The term "PPAR agonist " as used herein refers to a drug that is activating peroxisome proliferator activated receptor (PPAR) such as PPAR gamma receptor, PPAR alpha receptor, PPAR delta receptor or combinations thereof and includes PPAR gamma agonists such as e.g. pioglitazone, troglitazone or rosiglitazone, PPAR alpha agonists such as e.g. fibrates such as fenofibrate (fenofibric acid), clofibrate or gemfibrozil, PPAR dual agonists (PPAR

alpha/gamma or PPAR alpha/delta agonists) such as e.g. aleglitazar, muraglitazar,

tesaglitazar, ragaglitazar, saroglitazar, GFT505 or naveglitazar, PPAR delta agonists such as e.g. GW501516, PPAR pan agonists (PPAR alpha/delta/gamma agonist) or selective PPAR modulators such as e.g. INT131 and the pharmaceutically acceptable salts of these compounds. Usually PPAR gamma agonists, PPAR modulators, PPAR alpha agonists and/or PPAR alpha/gamma dual agonists are used in the pharmaceutical compositions of the present invention, in particular PPAR gamma agonists, PPAR alpha agonists and/or PPAR

alpha/gamma dual agonists are used in the pharmaceutical compositions of the present invention, more particularly PPAR gamma agonists selected from the group consisting of pioglitazone, rosiglitazone, troglitazone and pharmaceutically acceptable salts thereof, preferably pioglitazone or pharmaceutically acceptable salts thereof PPAR alpha agonists used in the pharmaceutical compositions of the present invention are selected from the group consisting of fenofibrate (fenofibric acid), clofibrate, gemfibrozil and pharmaceutically acceptable salts thereof, preferably fenofibrate (fenofibric acid) or pharmaceutically acceptable salts thereof PPAR alpha/gamma dual agonists used in the pharmaceutical compositions of the present invention are selected from the group consisting of aleglitazar, muraglitazar, tesaglitazar, ragaglitazar, saroglitazar, GFT505, naveglitazar or

pharmaceutically acceptable salts thereof, preferably muraglitazar, tesaglitazar or

pharmaceutically acceptable salts thereof Preferably PPAR gamma agonists are used in the pharmaceutical compositions of the present invention, more preferably PPAR gamma agonists or modulators selected from the group consisting of pioglitazone, rosiglitazone, troglitazone, INT131 and pharmaceutically acceptable salts thereof, even more preferably PPAR gamma agonists selected from the group consisting of pioglitazone, rosiglitazone, troglitazone and pharmaceutically acceptable salts thereof are used. Even more preferably, pioglitazone or a pharmaceutically acceptable salt thereof, in particular pioglitazone hydrochloride is used in the pharmaceutical compositions of the present invention. Most preferably, micronized pioglitazone hydrochloride is used. Thus in a preferred embodiment, a micronized PPAR agonist, more preferably a micronized PPAR gamma agonist or modulator, even more preferably a micronized PPAR gamma agonist or modulator selected from the group consisting of micronized pioglitazone, micronized rosiglitazone, micronized troglitazone, micronized INT131 and pharmaceutically acceptable salts thereof, in particular a micronized PPAR gamma agonists selected from the group consisting of micronized pioglitazone, micronized rosiglitazone, micronized troglitazone and pharmaceutically acceptable salts thereof, more particular micronized pioglitazone and pharmaceutically acceptable salts thereof, most particular micronized pioglitazone hydrochloride is used in the pharmaceutical compositions of the present invention. In one embodiment, a thiazolidinedione PPAR agonist is used in the pharmaceutical compositions of the invention. Suitable thiazolidinedione PPAR agonists are for example pioglitazone, troglitazone, rosiglitazone or pharmaceutically acceptable salts thereof. A particularly suitable thiazolidinone PPAR agonist is pioglitazone or a pharmaceutically acceptable salt thereof, in particular pioglitazone hydrochloride.

Pioglitazone is described e.g. in US Patent No. 4,687,777 or in Dormandy JA, Charbonnel B, Eckland DJ, Erdmann E, Massi-Benedetti M, Moules IK, Skene AM, Tan MH, Lefebvre PJ, Murray GD, Standl E, Wilcox RG, Wilhelmsen L, Betteridge J, Birkeland K, Golay A, Heine RJ, Koranyi L, Laakso M, Mokan M, Norkus A, Pirags V, Podar T, Scheen A, Scherbaum W, Schemthaner G, Schmitz O, Skrha J, Smith U, Taton J; PROactive investigators. Lancet. 2005 Oct 8;366(9493): 1279-89, and is represented by the structural formula indicated below:

Troglitazone is described e.g. in Florez JC, Jablonski KA, Sun MW, Bayley N, Kahn SE, Shamoon H, Hamman RF, Knowler WC, Nathan DM, Altshuler D; Diabetes Prevention Program Research Group. J Clin Endocrinol Metab. 2007 Apr;92(4): 1502-9 and is represented by the structural formula indicated below:

Rosiglitazone is described e.g. in Nissen SE, Wolski K. N Engl J Med. 2007 Jun

14;356(24):2457-71. Erratum in: N Engl J Med. 2007 Jul 5;357(l): 100. Fenofibrate is described e.g. in Bonds DE, Craven TE, Buse J, Crouse JR, Cuddihy R, Elam M, Ginsberg HN, Kirchner K, Marcovina S, Mychaleckyj JC, O'Connor PJ, Sperl-Hillen JA. Diabetologia. 2012 Jun;55(6): 1641-50 and is represented by the structural formula indicated below:

Clofibrate is described e.g. in Rabkin SW, Hayden M, Frohlich J. Atherosclerosis. 1988 Oct;73(2-3):233-40 and is represented by the structural formula indicated below:

Fenofibrate (fenofibric acid) is described e.g. in Schima SM, Maciejewski SR, Hilleman DE, Williams MA, Mohiuddin SM. Expert Opin Pharmacother. 2010 Apr;l 1 (5):731 -8 and is represented by the structural formula indicated below:

Gemfibrozil is described e.g. in Adabag AS, Mithani S, Al Aloul B, Collins D, Bertog S, Bloomfield HE; Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. Am Heart J. 2009 May; 157(5):913-8 and is represented by the structural formula indicated below:

Aleglitazar is described e.g. in Lincoff AM, Tardif JC, Schwartz GG, Nicholls SJ, Ryden L, Neal B, Malmberg K, Wedel H, Buse JB, Henry RR, Weichert A, Cannata R, Svensson A, Volz D, Grobbee DE; AleCardio Investigators. JAMA. 2014 Apr 16;311(15): 1515-25 and is represented by the structural formula indicated below:

Muraglitazar is described e.g. in Fernandez M, Gastaldelli A, Triplitt C, Hardies J, Casolaro A, Petz R, Tantiwong P, Musi N, Cersosimo E, Ferrannini E, DeFronzo RA. Diabetes Obes Metab. 2011 Oct;l3(l0):893-902 and is represented by the structural formula indicated below:

Tesaglitazar is described e.g. in Bays H, McElhattan J, Bryzinski BS; GALLANT 6 Study Group. Diab Vase Dis Res. 2007 Sep;4(3): l8l-93 and is represented by the structural formula indicated below:

Ragaglitazar is described e.g. in Saad MF, Greco S, Osei K, Lewin AJ, Edwards C, Nunez M, Reinhardt RR; Ragaglitazar Dose-Ranging Study Group. Diabetes Care. 2004

Jun;27(6): 1324-9 and is represented by the structural formula indicated below:

Saroglitazar is described e.g. in Agrawal R. Curr Drug Targets. 2014 Feb;l5(2):l5l-5. and is represented by the structural formula indicated below:

Naveglitazar is described e.g. in Ahlawat P, Srinivas NR. Eur J Drug Metab Pharmacokinet. 2008 Jul-Sep;33(3): 187-90. GW501516 is described e.g. in Wang X, Sng MK, Foo S, Chong HC, Lee WL, Tang MB, Ng KW, Luo B, Choong C, Wong MT, Tong BM, Chiba S, Loo SC, Zhu P, Tan NS. J Control Release. 2015 Jan 10; 197: 138-47 and is represented by the structural formula indicated below:

GFT505 is described e.g. in Cariou B, Staels B. Expert Opin Investig Drugs. 2014

Oct;23(lO): 1441-8 and is represented by the structural formula indicated below:

INT131 is described e.g. in. Taygerly JP, McGee LR, Rubenstein SM, Houze JB, Cushing TD, Li Y, Motani A, Chen JL, Frankmoelle W, Ye G, Learned MR, Jaen J, Miao S,

Timmermans PB, Thoolen M, Kearney P, Flygare J, Beckmann H, Weiszmann J, Lindstrom M, Walker N, Liu J, Biermann D, Wang Z, Hagiwara A, Iida T, Aramaki H, Kitao Y, Shinkai H, Furukawa N, Nishiu J, Nakamura M. Bioorg Med Chem. 2013 Feb 15;21(4):979-92 and is represented by the structural formula indicated below:

PPAR activation by the PPAR agonist is usually strong in the low nanomolar range to low micromolar range, e.g in a range of 0.1 nM to 100 mM. In some embodiments the PPAR activation is weak or partial, i.e. a PPAR agonist is used in the pharmaceutical compositions of the present invention which yields maximal activation of PPAR-receptor in a reporter assay system of 10% to 100% compared to a reference PPAR agonist which is known to cause a maximum PPAR activation. The preferred target for interaction of the PPAR agonist is the hair cell, which is most preferred; neural cells; and endothelial cells; and further includes adipocytes, hepatocytes, immune cells such as e.g. macrophages or dendritic cells, or skeletal muscle cells.

The term "hearing loss" which is used herein interchangeably with the term“hearing impairment” refers to a diminished sensitivity to the sounds normally heard by a subject. The severity of a hearing loss is categorized according to the increase in volume above the usual level necessary before the listener can detect it. The term "hearing loss" as used herein includes sudden hearing loss (SHL) which is indicated in the literature also as“sudden sensorineural hearing loss (SSHL)”. SHL refers to illness which is characterized by a sudden, rapid sensorineural hearing loss mostly in one ear without obvious causes, normally accompanied with dizziness, and without vestibular symptomatology. SHL is defined as greater than 30 dB hearing reduction, over at least three contiguous frequencies, occurring over a period of 72 hours or less. SHL can be caused e.g. by unspecific stress.

Hearing loss as referred herein is defined as a diminished ability to hear sounds like other people do. This can be caused either by conductive hearing loss, sensorineural hearing loss or a combination of both.

Conductive hearing loss means that the vibrations are not passing through from the outer ear to the inner ear, specifically the cochlea. It can be due to an excessive build-up of earwax, glue ear, an ear infection with inflammation and fluid buildup, a perforated or defective eardrum, or a malfunction of the ossicles (bones in the middle ear). Sensorineural hearing loss is caused by dysfunction of the inner ear, the cochlea, auditory nerve, or brain damage.

Usually, this kind of hearing loss is due to damage of the hair cells in the cochlea.

Hearing loss as referred herein is usually sensorineural hearing loss or a combination of conductive hearing loss and sensorineural hearing loss. Sensorineural hearing loss can be related to age, to an acute or constant exposure to noise or chemicals, to a brain trauma or non specific stress which may lead to sudden hearing loss.

The term "hair cell degeneration" as used herein refers to a gradual loss of hair cell function and integrity and/or leading ultimately to hair cell death.

The term "hair cell death" as used herein refers to apoptosis of the hair cells in the inner ear.

The terms "identification of hair cell damage" or " detection of hair cell damage " which are used interchangeably herein refer to a method by which the degree of hair cell damage in the inner ear can be determined. Such methods are known in the art and comprise for example fluorescent imaging of the hair cells, as described in the examples. An audiogram that demonstrates loss of hearing sensitivity at moderate to high frequencies is also indicative of hair cell damage. A decrease of hearing potential with no subsequent recovery is also diagnostic of hair cell damage.

The term“chemically induced hearing loss” or“hearing loss induced by a chemical” as referred herein refers to hearing loss which is induced and/or caused by chemical agents such as solvents, gases, paints, heavy metals, and/or medicaments which are ototoxic.

The term sound pressure level (SPL) or acoustic pressure level as referred herein is a logarithmic measure of the effective sound pressure of a sound relative to a reference value. Sound pressure level, denoted L _p and measured in dB, above a standard reference level, is given by:

L _p = 10 logio (P _rms ² /po ² ) = 20 logio (P _rmS /po) dB(SPL) where p _rms is the root mean square sound pressure, measured in Pa and po is the reference sound pressure, measured in Pa. The commonly used reference sound pressure in air is po = 20 pPa (Root Mean Squared) or 0.0002 dynes/cm ² , which is usually considered the threshold of human hearing.

The term "individual," "subject" or "patient" are used herein interchangeably. In certain embodiments, the subject is a mammal. Mammals include, but are not limited to primates (including human and non- human primates). In a preferred embodiment, the subject is a human.

The term "about" as used herein refers to +/- 10% of a given measurement.

The term "% w/w" as used herein refers to a mass fraction and is the ratio of one substance with mass rrij to the mass of the total pharmaceutical composition n¾ _0t with a denominator of 100, defined as: 100

The term "% w/V" as used herein refers to a mass fraction and is the ratio of one substance with mass rrij to the volume of the total pharmaceutical composition (i.e. not just the volume of the solvent) V with a denominator of 100, defined as:

% w/w = x 100; wherein the unit of h _¾ is in grams and wherein the unit of V is in mL.

The term "auris-acceptable gel" as used herein includes a gel having no persistent detrimental effect on the auris media (or middle ear) and the auris interna (or inner ear) of the subject being treated.

The term "gel" as used herein refers to semisolid systems consisting of either suspensions made up of small inorganic particles or large organic molecules interpenetrated by a liquid. Gels can consist of a single-phase or a two-phase system. A single-phase gel consists of organic macromolecules distributed uniformly throughout a liquid in such a manner that no apparent boundaries exist between the dispersed macromolecules and the liquid. Single-phase gels are usually prepared from e.g. synthetic macromolecules or from natural gums (e.g.

tragacanth). Gels or jellies are semisolid systems consisting of suspensions of small inorganic particles or large organic molecules interpenetrated by a liquid. Gels are generally classified as a two-phase system, if the particle size of the dispersed phase is large; or as single phase gels, when the organic macromolecules are uniformly distributed throughout a liquid such that no apparent boundaries exist between the dispersed macromolecules and the liquid. Single- phase gels are preferred for use the present invention.

In addition to "single-phase" and "two-phase" (vide supra), gels can also be classified as being hydrophobic, hydrophilic or amphiphilic. Hydrophobic gels usually consist of a non-polar solvent such as liquid paraffin, polyethylene or fatty oils gelled with colloidal silica, or aluminum soaps or zinc soaps. In contrast, hydrophilic gels usually consist of a polar solvent such as water, glycerol, or propylene glycol gelled with a suitable gelling agent (e.g., tragacanth, starch, cellulose and derivatives thereof, carboxyvinylpolymers, and/or magnesium-aluminum silicates). Amphiphilic gels usually consist of an amphiphilic copolymer having both hydrophilic and hydrophobic properties. Amphiphilic copolymers are prone to form micelles when brought in contact with a suitable solvent, e.g. water. Preferably, amphiphilic gels are used in the present invention.

The term "gelling agent" as used herein refers to a compound that can be solubilized, dispersed or mixed with the pharmaceutical composition of the present invention to modify its rheological behaviour, more particularly its viscosity, and can lead to a higher viscosity composition or the formation of a hydrogel or the formation of a thermoreversible gel.

Examples of gelling agents include, but are not limited to, celluloses, cellulose derivatives such as cellulose ethers (e.g., carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose,

methylcellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth, carboxyvinylpolymers, carrageenan, paraffin, petrolatum and any combinations or mixtures thereof. A preferred gelling agent is hydroxypropylmethylcellulose (Methocel®).

The term "thermoreversible gel" as used herein refers to a gel having the ability to change from the liquid state to the gel state at temperatures close to body temperature, therefore allowing useful topical formulations. The terms“thermoreversible gel”,“thermosensitive gel” or“thermogelling” are used herein interchangeably in relation to a material which is liquid at lower temperature, but forms a viscous gel at a higher temperature. The process of thermogelling is fully reversible. Particularly preferred are thermoreversible gels comprising copolymers having a lower critical solution temperature (LCST) below 37°C. Polymers forming such thermoreversible gels are water-soluble below their LCST, also known as gel temperature, due to strong hydrogen bonding between the hydrophilic part of the chains and water, but above the LCST value, hydrogen interactions are weakened and hydrophobic interactions between the hydrophobic domains of the polymer become dominant with consequent precipitation of the polymer resulting in gelation. The LCST value of a polymer depends on the balance of hydrophilic and hydrophobic portions of the copolymer and can be adjusted by varying this balance. It also depends on the concentration of the copolymer in water.

A non-limiting example of a thermoreversible gel is a gel comprising "ReGel™", which is a tradename of MacroMed Inc. for a class of low molecular weight, biodegradable copolymers having reverse thermal gelation properties as described in U.S. Pat. Nos. 6,004,573; 6,117,949; 6,201,072, and 6,287,588. The term "thermoreversible gel" also includes biodegradable copolymers disclosed in U.S. patent application Ser. Nos. 09/906,041, 09/559,799 and 10/919,603. The biodegradable copolymers comprise ABA-type or BAB-type triblock copolymers or mixtures thereof, wherein the A-blocks are hydrophilic and comprise polyethylene glycol (PEG) and the B-b locks are hydrophobic and comprise biodegradable polyesters or poly(ortho ester)s, said copolymers having a hydrophobic content of between 50.1 to 83% by weight and a hydrophilic content of between 17 to 49.9% by weight, and an overall block copolymer molecular weight of between 2000 and 8000 daltons. The copolymers exhibit water solubility at temperatures below normal mammalian body temperatures and undergo reversible thermal gelation to a gel at temperatures equal to physiological mammalian body temperatures. The hydrophilic A-block segment is preferably polyethylene glycol (PEG) having an average molecular weight of between about 500 and 2200 daltons. The biodegradable, hydrophobic B polymer block comprises a polyester or poly(ortho ester), in which the polyester is synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, e-caprolactone, e-hydroxyhexanoic acid, g-butyro lactone, g- hydroxybutyric acid, d-valero lactone, d -hydroxyvaleric acid, hydroxybutyric acids, malic acid, and copolymers thereof and having an average molecular weight of between about 600 and 3000 Daltons.

A further non-limiting example of a thermoreversible gel is a gel comprising a poloxamer.

The term "poloxamer” as used herein refers to a copolymer composed of polyoxyethylene- polyoxypropylene copolymers. Examples of poloxamers include poloxamer 407 e.g.

Kolliphor® P 407 (BASF Carp.), P407, Pluronic® F-127 (BASF Carp.), PF-127, Lutrol® F 127 (BASF Corp.) as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF)), poloxamer 188 e.g. Kolliphor® P 188 BASF Corp., Pluronic® F-68 BASF Corp. as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF)), poloxamer 237 e.g. Kolliphor® P 237 (BASF Corp.), Pluronic® F-87 (BASF Corp.)as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF))) and poloxamer 338 e.g Kolliphor® P 338 BASF Corp., Pluronic® F-108 (BASF Corp.)as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF)).

Poloxamer 407 has normally an Average Molecular Weight of 9,840 - 14,600 Da. Poloxamer 188 has normally an Average Molecular Weight of 7,680 - 9,510 Da. Poloxamer 237 has normally an Average Molecular Weight of 6,840 - 8,830 Da. Poloxamer 338 has normally an Average Molecular Weight of 12,700 - 17,400 Da. Pluronic ® F-127 is a commercially available polyoxyethylene-polyoxypropylene triblock copolymer (Poloxamer 407) with an Average Molecular Weight of 13,000 Da.

Aqueous solutions of poloxamers are stable in the presence of acids, alkalis, and metal ions. Poloxamer 407 is of particular interest since concentrated aqueous solutions (20% w/w) of the copolymer are transformed from low viscosity transparent solutions to solid gels on heating to body temperature. Depending on additional components of a formulation, this transition temperature (LCST, lower critical solution temperature) or transition range may be achieved with distinctly lower concentrations of Poloxamer 407, e.g. with about 10 - 15% w/w. This phenomenon, therefore, suggests that when placed in contact with the body, the gel preparation will form a semi- so lid structure and an extended release depot. Furthermore, Poloxamer 407 has good solubilizing capacity, low toxicity and is, therefore, considered a good medium for drug delivery systems.

A further non-limiting example of a thermoreversible gel is a gel comprising a PEG-PLGA- PEG triblock copolymer (wherein "PLGA" means polylactide-co-glycolide) (Jeong et al, J. Control. Release (2000), 63:155-63; Jeong et al, Adv. Drug Delivery Rev. (2002), 54:37-51). The polymer exhibits sol-gel behavior over a concentration of about 5% w/w to about 40% w/w. Depending on the properties desired, the lactide/glycolide molar ratio in the PLGA copolymer can range from about 1 :1 to about 20:1. The resulting PEG-PLGA-PEG triblock copolymers are soluble in water and form a free-flowing liquid at room temperature, but form a hydrogel at body temperature. A commercially available PEG-PLGA-PEG triblock copolymer is RESOMER RGP t 50106 originally manufactured by Boehringer Ingelheim (now by Evonik Industries). This material is composed of a PLGA copolymer of 50:50 poly(DL-lactideco- glycolide) and is 10% w/w of PEG and has a molecular weight of about 6000 Da. The terms "polyethylene glycol", "PEG" and "polyoxyethylene" are used herein interchangeably.

The term "copolymer" as used herein refers to polymers formed from two, three, four or more monomers. Hence, the term "copolymer" as used herein encompasses not only the basic copolymer of two monomers, but also terpolymers, tetrapolymers, pentapolymers and so forth. Preferably, a copolymer is a copolymer made up of blocks of different polymerized monomers ("block copolymer"). Copolymers in general and block copolymers in particular are commonly known in the art. Preferred copolymers are ABA-type or BAB-type triblock copolymers or mixtures thereof, wherein the A-blocks are hydrophilic and wherein the B- blocks are hydrophobic.

The term "hydrophilic" as used herein refers to the common meaning of this term in the art, and denotes chemical moieties which comprise ionizable, polar, or polarizable atoms, or which otherwise may be solvated by water molecules. Thus, the term "hydrophilic" is typically used to describe materials that are capable of absorbing significant quantities of water, including those that are water-soluble.

The term "hydrophobic" as used herein refers to the common meaning of this term in the art, and denotes non-polar chemical moieties which lack an affinity for water. Thus, the term "hydrophobic" is typically used to describe materials that are insoluble and non-swellable in water or an aqueous fluid.

The term "colloidal silica" as used herein refers to Si0 ₂ particles in the micro-meter to nano- meter size range. Colloidal silica is described in the United States Pharmacopeia - National Formulary (USP/NF) under the designation Colloidal Silicon Dioxide. A hydrophobized version is described under the designation Hydrophobic Colloidal Silica. Commercially available products are AEROSIL™ by Evonik Industries.

The term "soap" as used herein refers to a salt of a fatty acid. Thus, the term "zinc soap" refers to a zinc salt of a fatty acid, such as Zinc Laurate, Zinc Myristate, Zinc Stearate, Zinc

Palmitate, Zinc Decanoate. The term "aluminum soap" refers to an aluminum salt of a fatty acid, such as as Aluminium Laurate, Aluminium Myristate, Aluminium Stearate, Aluminium Palmitate, Aluminium Oleate.

The term "starch" as used herein includes com starch, potato starch, wheat starch, rice starch, partially pregelatinized starch, hydroxypropylstarch and sodium carboxymethyl starch.

The terms "cellulose derivative" and "cellulosic polymer" are used herein interchangeably.

The term "cellulose derivative" as used herein refers to a class of polymers derived from cellulose such as cellulose ethers, e.g. hydroxyalkyl celluloses and alkyl celluloses and cellulose esters. Examples of hydroxyalkyl celluloses include hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (hypromellose, HPMC), hydroxyethyl methyl cellulose (HEMC), ethyl hydroxyethyl cellulose (EHEC) and hydroxyethyl cellulose (HEC). Examples of alkyl celluloses include methyl cellulose and ethyl cellulose. The group of cellulose ethers further comprises carboxymethyl cellulose (carmellose), carboxymethyl hydroxyethyl cellulose (CMHEC) and sodium or calcium carmellose. Examples of cellulose esters include nitro cellulose, cellulose acetate, cellulose butyrate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate and cellulose acetate trimellitate. A cellulosic polymer may also be both a cellulosic ether and a cellulosic ester, e.g. hydroxypropyl methylcellulose phthalate. The term "cellulose derivatives" as used herein further comprises cross-linked cellulosic polymers such as cross-linked carboxymethyl cellulose (croscarmellose) and sodium or calcium croscarmellose. Among cellulose and cellulose derivatives as compound of the Auris-acceptable gel of the present invention, cellulose derivatives are preferred.

The term "carboxyvinylpolymer" as used herein refers to polyacrylic acid and the different Carbomer types (Carbomer Copolymer, Carbomer Homopolymer, Carbomer Interpolymer) as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF).

The term "metal silicate" as used herein inlcudes orthosilicates having the general formula M ₄ Si0 ₄ , condensed noncyclic silicates having the general formula M _2n+2 Si _n 0 _3n+i , and metasilicates having the general formula M _2n Si _n 0 _3n wherein M is hydrogen or an alkali metal and n is an integer equal to or greater than one and preferably from one to three. The silicate additive to the electrolyte may be illustratively, sodium orthosilicate (Na ₄ S1O ₄ ) potassium orthosilicate (R ₄ S1O ₄ ), sodium pyrosilicate (Na ₆ Si ₂ 0 ₇ ), potassium ^py rosilicate (R^S^CE), tetrasodium dilithium pyrosilicate (Na ₄ Li ₂ Si ₂ 0 ₇ ), silicic acid (H ₂ S1O ₃ ), sodium metasilicate (Na ₂ Si0 ₃ ), potassium metasilicate (K ₂ S1O ₃ ), lithium metasilicate (LESiCE), sodium

metadisilicate (Na ₄ Si ₂ 0 ₆ ), potassium metatrisilicate (K ₆ S1 ₃ O ₉ ), or sodium metahexasilicate (Na^S^Ois). Metal silicates useful in the present invention comprise e.g magnesium aluminum silicate which, as used herein, refers to a compound having the chemical formula MgO-AfrCE-SiCE and which is a preferred metal silicate. Metal silicates are usually pharmaceutically acceptable metal silicates. The term "natural gum" as used herein refers to a group of naturally occuring gums comprising tragacanth, gum Arabic (e.g. gum Arabic powder), gum karaya, locust bean gum, carrageens, guar gum and xanthan gum. A particularly useful gum for the pharmaceutical compositions of the invention is tragacanth; a natural gum obtained from the dried sap of several species of Middle Eastern legumes of the genus Astragalus, including A. adscendens, A. gummifer, A. brachycalyx and A. tragacanthus.

The term "pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxy-benzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane- disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4- methylbicyclo[2.2.2]-oct-2-enel-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e. g. an alkaline metal ion, an alkaline earth metal ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. A particularly preferred pharmaceutically acceptable salt is an acid addition salt formed with hydrochloric acid.

Particle size is generally reported on a cumulative distribution by volume basis. The term "micronized" as used herein means particles with a median particle size, expressed by using the cumulative distribution by volume, of between about 1 pm and about 75 pm. Preferably 50 % of the particles (Dv50) of the micronized PPAR agonist as referred herein are smaller than or equal to about about 50 pm, more preferably smaller than or equal to about 10 pm, most preferably smaller than or equal to about 5 pm, in particular about 1 to about 10 pm, more particular about about 2 to about 8 pm, even more particular about 3 to about 5 pm, most particular about 4 pm. Micronization is a process of reducing the average diameter of particles of a solid material, whereby the particles are mostly passed through a jet mill. Other mill types may be used as well, e.g. pin mills. The required particle size specification may as well be achieved without a specific milling step by appropriate process conditions in the final precipitation step of the drug substance chemical production. Size reduction is used to increase the surface area of a drug substance and thereby modulate formulation dissolution properties. Micronization is also used to maintain a narrow and consistent particle size distribution for any formulation described herein. A further purpose of micronization is to allow an easy application of the formulations of the invention by a parenteral syringe. In some embodiments, the needle is wider than a 18 gauge needle. In another embodiment, the needle gauge is from 18 gauge to 30 gauge. In a further embodiment, the needle is a 21 gauge needle. Depending upon the thickness or viscosity of a composition disclosed herein, the gauge level of the syringe or hypodermic needle are varied accordingly. Thus, the formulations of the invention comprising micronized PPAR agonists are ejected e.g. from a 1 mL syringe adapted with a 50 mm length 21 G needle (nominal inner diameter 0.495 mm) without any plugging or clogging.

The term "solvent" as used herein refers to a liquid substance capable of dispersing the other components of the composition. The term "solvent” as used herein further refers to polar and non-polar solvents and mixtures thereof. Solvents with a dielectric constant of less than 15 (at 0 °C) are generally considered to be nonpolar. Non-limiting examples of non-polar solvents are liquid paraffin, polyethylene and fatty oils. Non-limiting examples of polar solvents are water and alcohols, such as ethanol, glycerol, or propylene glycol. Preferably, solvents used in the pharmaceutical composition of the invention are polar solvents such as water.

The term "surfactant" as used herein refers to auris-acceptable compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Non-limiting examples of surfactants are sodium lauryl sulfate, sodium dioctyl sulfosuccinate (Docusate Sodium), polysorbates (e.g. products sold by Croda Int. under the trade name Tween®: Polysorbate 60 (Tween®60), Polysorbate 80 (Tween®80), Polysorbate 20 (Tween® 20)), triacetin, Vitamin E Polyethylene Glycol Succinate (Vitamin E TPGS), phospholipids, lecithins, phosphatidyl cholines (C8-C18), phosphatidylethanolamines (C8-C18),

phosphatidylglycerols (C8-C18), sorbitan monooleate, polyoxyethylene sorbitan monooleate, , bile salts, glyceryl monostearate, and the like. Some further surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Preferred surfactants are polysorbates, e.g. polysorbate 80 and polysorbate 20. The terms Tween™20, Tween®60, Tween®80 as used herein refer to the corresponding Polysorbates as described e.g. in the United States Pharmacopeia - National Formulary (USP/NF).

The term "buffer" as used herein refers to compositions well known to the skilled artisan that act to minimize the change in pH of a solution. Preferred buffers have a pKa that provides effective buffering at a pH of between about 6 and about 8, perferably buffering at a pH of between about 7 and about 8. Suitable buffers for use in the pharmaceutical composition of the invention include, but are not limited to trolamine hydrochloric acid buffer, acetate, bicarbonate, carbonate, ammonium chloride, citrate, phosphate, Tromethamine USP

(tris(hydroxymethyl)aminomethane, TRIS), pharmaceutically acceptable salts thereof and combinations or mixtures thereof. Particularly preferred buffers for use in the pharmaceutical compositions of the invention are trolamine hydrochloric acid buffer and/or tromethamine. The pH of buffers used might be adjusted by adding a water diluted acid or base such as e.g by adding a diluted hydrochloric acid solution (10%) or adiluted sodium hydroxide solution (10%).

The term "trolamine" as used herein refers to triethanolamine.

The term " trolamine hydrochloric acid buffer " refers to a buffer containing trolamine adjusted to a particular pH with a solution containing between 9.5 g and 10.5 g Hydrochloric acid per 100 mL solution, preferably 10 g Hydrochloric acid per 100 mL solution (10% solution) . The pH of a trolamine hydrochloric acid buffer used in the present invention is between about 7 and about 8, preferably about 7.4.

Preferably, the pharmaceutical compositions of the invention have a pH that allows for terminal sterilization (e.g., by heat treatment and/or autoclaving) without degradation of the PPAR agonist and the auris-acceptable gel. For example, in order to reduce hydrolysis and/or degradation of the PPAR agonist and/or the auris-acceptable gel during autoclaving, the buffer pH is designed to maintain pH of the formulation in the 7-8 range at elevated temperatures. Any appropriate buffer is used depending on the particular PPAR agonist used in the formulation. Degradation of an otic agent may be reduced by the use of an appropriate combination of a buffer and polymeric additives (e.g. Poloxamer 407 or carboxymethyl celluose).

The term "permeability enhancer" as used herein refers to chemical entities that facilitate transport of co-administered substances across biological membranes. The terms

"permeability enhancer" and "penetration enhancer" are used herein interchangeably.

Permeability enhancers can be grouped according to chemical structure. Surfactants, both ionic and non-ionic, such as sodium lauryl sulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9, sodium dodecylsulfate, dioctyl sodium sulfo succinate, polyoxyethylene-9- lauryl ether (PLE), Tween 80, nonylphenoxypoly ethylene (NP-POE), polysorbates and the like, function as permeability enhancers. Bile salts (such as sodium glycocholate, sodium deoxycholate, sodium taurocholate, sodium taurodihydrofusidate, sodium

glycodihydrofusidate and the like), fatty acids and derivatives thereof (such as oleic acid, caprylic acid, mono- and di-glycerides, lauric acids, acylcholines, caprylic acids,

acylcamitines, sodium caprates and the like), chelating agents (such as EDTA, citric acid, salicylates and the like), sulfoxides (such as dimethyl sulfoxide (DMSO), decylmethyl sulfoxide and the like), and alcohols (such as ethanol, isopropanol, propylene glycol, polyethylene glycol, glycerol, propanediol, benzyl alcohol and the like) also function as permeability enhancers. Some enzymes, such as hyaluronidase and some glycosaminoglycans, such as hyaluronic acid also function as permeability enhancers. Preferred permeability enhancers are selected from the group consisting of benzyl alcohol, hyaluronic acid and pharmaceutically acceptable salts thereof and DMSO.

Some gelling agents as commonly used in the preparation of hydrophobic and hydrophilic gels (vide supra) may also be used as viscosity increasing agents (thickening agents) and vice versa in the pharmaceutical compositions of the invention. Likewise, some viscosity- increasing agents may be used as mucoadhesives and vice versa. In one embodiment, a combination of a viscosity- increasing agent and a copolymer that is capable of forming a thermoreversible gel is used in the pharmaceutical compositions of the invention. The term "viscosity-increasing agent" as used herein includes, but is not limited to, hydroxypropyl methylcellulose (HPMC, hypromellose), hydroxyethyl cellulose, polyvinylpyrrolidone (PVP: povidone), carboxymethyl cellulose (carmellose), carboxymethylcellulose sodium, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate, acacia (gum arabic), agar, sodium alginate, carbomer, carrageenan, carbopol, xanthan, gelatin, guar gum, maltodextrin, sterculia gum, polyethylene glycol (e.g. PEG 200-4500), tragacanth, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethylmethyl cellulose (hymetellose), hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), pectin, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate),

poly(methoxyethoxyethyl methacrylate), sodium carboxymethyl- cellulose (CMC), silicon dioxide, or combinations thereof. Particularly preferred viscosity- increasing agents are hypromellose and/or carbomer.

The term "carbomer" as used herein refers to certain polyacrylic acid derivatives (vide supra). It includes carbomer homopolymer (types A, B or C), carbomer copolymer (types A, B or C) and carbomer interpolymer (Types A, B or C). According to the USP/NF, the terms“A”,“B” or“C” refer to the viscosity of the corresponding Carbomer as follows:

The term "carbopol" as used herein refers to the different Carbomer products sold under the trade name Carbopol® by The Lubrizol Corporation.

The term "preservative" as used herein is well known to the skilled artisan and refers to compounds that are used to kill or prevent growth of bacteria, yeast, and mold in the dosage form. Accordingly, preservatives are compounds that prevent or delay microbial activity (growth and metabolism). Suitable preservatives for use in the the pharmaceutical compositions of the invention include, but are not limited to, benzoic acid, p- hydroxybenzoates (e.g. methylparabene and propylparabene), benzyl alcohol, lower alkyl alcohols (e.g., ethanol, butanol or the like), Chlorobutanol, quaternary compounds (e.g.

Benzalkonium Chloride, Benzethonium Chloride, Cetylpyridinium Chloride), thimerosal, sorbic acid. Preferred preservatives are selected from the group consisting of benzoic acid, benzyl alcohol, chlorobutanol, p-hydroxybenzoates, methylparabene, and propylparabene, or combinations thereof, most preferably methylparabene, propylparabene, or combinations thereof. Suitable preservatives for use with a pharmaceutical composition of the invention are not ototoxic.

The term "mucoadhesive" as used herein refers to compounds (typically polymers) that bind to the mucin layer of a biological membrane, i.e. increase the interaction of the

pharmaceutical compositions of the invention with a mucosal layer. To serve as

mucoadhesives, compounds should possess some general physiochemical features such as predominantly anionic hydrophilicity with numerous hydrogen bond forming groups, suitable surface property for wetting mucus/mucosal tissue surfaces and sufficient flexibility to penetrate the mucus network. In some embodiments, the pharmaceutical compositions of the invention comprising a mucoadhesive adhere to the round window and/or the oval window and/or any inner ear structure. Mucoadhesive agents include, but are not limited to, soluble polyvinylpyrrolidone polymer (PVP); a water-swellable, but water-insoluble, fibrous, cross- linked carboxy- functional polymer; a crosslinked poly(acrylic acid) (e.g. Carbopol 947P, Carbopol 934P, Majithiya et al, AAPS PharmSciTech (2006), 7(3), p. El; EP0551626); a carbomer homopolymer (e.g. Carbomer 934 (Carbopol™ 934 NF by Lubrizol Corp.), Carbomer 940 (Carbopol™ 940 NF by Lubrizol Corp.), Carbomer 941 (Carbopol™ 941 NF by Lubrizol Corp.)); a carbomer copolymer (e.g. Carbomer Copolymer Type A (Pemulen™ TR-2 NF by Lubrizol Corp.), Carbomer Copolymer Type B (Pemulen™ TR-l NF by Lubrizol Corp.)); a hydrophilic polysaccharide gum, maltodextrin, a cross-linked alginate gum gel, a water-dispersible poly carboxy lated vinyl polymer, or a mixture thereof. Mucoadhesive agents have been described, for example, in U.S. Pat. Nos. 6,638,521, 6,562,363, 6,509,028,

6,348,502, 6,319,513, 6,306,789, 5,814,330, and 4,900,552.

The term "antioxidant" as used herein refers to a substance used as in vitro stabilizers of pharmaceutical preparations to mitigate oxidative processes. Antioxidants delay the onset of and/or significantly reduce the rate of complex oxidative reactions that could otherwise have a detrimental effect on the drug substance. Antioxidants also can be considered for protecting nonactive components such as unsaturated oils, pegylated lipids, flavors, and essential oils. Thus, antioxidants preserve the overall integrity of the dosage form against oxidative stress. Suitable antioxidants include acetylcysteine (N-acetyl-L-cysteine), ascorbic acid and sodium ascorbate, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), glutathione, monothioglycerol, propyl gallate, sodium metabisulfite, sodium sulfite, tocopherol (e.g. dl-alpha-tocopherol) and the like, and suitable combinations of two or more hereof. Preferably the antioxidant is selected from the group consisting of ascorbic acid or sodium ascorbate, benzyl alcohol and monothioglycerol.

The term "liquid paraffin" as used herein refers to a very highly refined mineral oil used in cosmetics and for medical purposes (definition according to the British Pharmacopoeia). Liquid paraffin is also known by its Latin name "paraffinum liquidum".

The term "fatty oils" as used herein refers to at room temperature liquid mixtures of mainly tri- and diglycerides of naturally occurring fatty acids, where the fatty acids are partially unsaturated with cis-configuration. Examples of the unsaturated fatty acids are oleic acid, linoleic acid, palmitoleic acid, and arachidonic acid,

The terms“prolonged release” or“extended release” as used herein refer to release which is not immediate release, but release over a pre-defined, longer time period of up to several days, preferably over a period of more than 1 day, more preferably more than 2 days, more preferably more than 3 days, more preferably more than 4 days, more preferably more than 5 days, more preferably more than 6 days, more preferably 1 to 15 days, more preferably 2 to 12 days, more preferably 3 to 10 days, more preferably 4 to 7 days, more preferably 5 to 7 days, more preferably 6 to 7 days, most preferably 7 days. Accordingly, within this time window, 30-100%, preferably 50-100%, more preferably 70-100%, even more preferably 75-95%, most preferably 80-90% of the content of the PPAR agonist comprised by the pharmaceutical composition of the invention is released from said pharmaceutical composition.

In one aspect, the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or treating hearing loss in a subject. In a further aspect of the invention the present invention provides a method of preventing or treating hearing loss in a subject, which method comprises administering to the subject a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel. In some embodiments, the pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel is administered to the subject in an amount that is sufficient to prevent or treat hearing loss in the subject. In a further aspect, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for the manufacture of a medicament for preventing or treating hearing loss in a subject. In a further aspect, the present invention provides the use of a

pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for preventing or treating hearing loss in a subject.

In some preferred embodiments, hearing loss to be prevented or treated by the pharmaceutical compositions of the present invention is caused by a noise trauma, by a medical intervention, by ischemic injury, by age or is chemically induced. The hearing loss can be thus a consequence of a medical intervention such as e.g. cochlear implantation. The chemical induction is usually caused by a chemical agent e.g. by an antibiotic or a chemotherapeutic agent. In some preferred embodiments, hearing loss is sudden hearing loss. Hearing loss caused by age comprises e.g. presbycusis. Preferably hearing loss caused by a noise trauma, cochlear implantation, or which is chemically induced, preferably by an antibiotic, is prevented or treated by the pharmaceutical compositions of the present invention. More preferably hearing loss caused by a noise trauma or which is chemically induced, preferably by an antibiotic, is prevented or treated by the pharmaceutical compositions of the present invention. In some embodiments, hearing loss is of sensorineural origin caused by a damage leading to malnutrition of the cells in early brain development. In this case early treatment with a PPAR agonist can be disease modifying preventing further damage.

In some embodiments, the pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel is administered before the subject has developed or before it is at risk to develop hearing loss, hair cell degeneration, hair cell death and/or a condition characterized by hair cell damage. In some embodiments, the pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel is administered after the subject has acquired hearing loss, hair cell degeneration, hair cell death and/or a condition characterized by hair cell damage. Further diseases, disorders or conditions which are related to, caused or characterized by hair cell degeneration and/or hair cell death and which can be prevented or treated by

administering the pharmaceutical composition comprising a PPAR agonist and an auris- acceptable gel of the present invention are e.g. Meniere's disease, acute peripheral

vestibulopathy and tinnitus.

Thus, in some embodiments the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or inhibiting hair cell degeneration or hair cell death in a subject, wherein hair cell degeneration or hair cell death is related to and/or caused by Meniere's disease, acute peripheral vestibulopathy and/or tinnitus.

In some embodiments, the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or treating Meniere's disease in a subject.

In some embodiments, the present invention provides a method of preventing or treating Meniere's disease in a subject which method comprises administering to the subject a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel.

In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for the manufacture of a medicament for preventing or treating Meniere's disease in a subject.

In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for preventing or treating Meniere's disease in a subject.

In some embodiments the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or treating acute peripheral vestibulopathy in a subject.

In some embodiments, the present invention provides a method of preventing or treating acute peripheral vestibulopathy in a subject which method comprises administering to the subject a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel.

In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for the manufacture of a medicament for preventing or treating acute peripheral vestibulopathy in a subject. In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for preventing or treating acute peripheral vestibulopathy in a subject.

In some embodiments the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or treating tinnitus in a subject.

In some embodiments, the present invention provides a method of preventing or treating tinnitus in a subject which method comprises administering to the subject a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel.

In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for the manufacture of a medicament for preventing or treating tinnitus in a subject.

In some embodiments, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for preventing or treating tinnitus in a subject.

Hearing loss, hair cell degeneration or hair cell death caused by a noise trauma or by medical intervention

Exposure to loud noise causes noise-induced hearing loss (NIHL) by damaging the organs of Corti. Damage by NIHL depends upon both the level of the noise and the duration of the exposure. Hearing loss may be temporary (temporary threshold shift, TTS) if a repair mechanism is able to restore the organ of the Corti. However, it becomes permanent

(permanent threshold shift, PTS) when hair cells or neurons die. Structural modifications correlated to noise trauma are of two types: (1) mild damage of synapses and or hair cell stereocilia which can be repaired by cellular repair mechanisms and accounts for TTS and recovery and (2) severe damage inducing hair cell and neuronal apoptosis which can not be repaired by cellular repair mechanisms and accounts for PTS.

A noise trauma as referred herein is a noise trauma which is sufficient to cause damage to the organs of corti, in particular a noise trauma causing temporary or permanent hearing loss. A noise trauma can be caused by exposure to a sound pressure level of e.g., at least 70 dB (SPL), at least 90 dB (SPL), at least 100 dB (SPL), at least 120 dB (SPL) or at least 130 dB (SPL). Hearing loss can also be caused by a medical intervention usually by a medical intervention in the ear e.g. by cochlea surgery such as cochlear implantation. In some embodiments, the pharmaceutical composition of the invention is administered before the subject is exposed to a noise trauma or medical intervention. In some embodiments, the pharmaceutical composition of the invention is administered after the subject is exposed to a noise trauma or medical intervention. In a particular embodiment, the pharmaceutical composition of the invention is administered prior to cochlear surgery i.e. before the subject undergoes cochlear surgery.

Hearing loss, hair cell degeneration or hair cell death caused by age

Hearing loss caused by age also referred in the literature as“age-related hearing loss” is the cumulative effect of aging on hearing. It is normally a progressive bilateral symmetrical age- related sensorineural hearing loss. The hearing loss is most marked at higher frequencies. There are four pathological types of hearing loss caused by age:

1) sensory: characterised by degeneration of organs of corti. 2) neural: characterised by degeneration of cells of spiral ganglion. 3) strial/metabolic: characterised by atrophy of stria vascularis in all turns of cochlea. 4) cochlear conductive: due to stiffening of the basilar membrane thus affecting its movement.

Hearing loss caused by age to be prevented or treated by the methods of the present invention is usually related to the first pathological type i.e. hearing loss characterised by degeneration of the organ of Corti. Thus, in some embodiments the pharmaceutical composition of the invention is administered to the subject prior to degeneration of the organ of Corti, e.g. prior to damage or apoptosis of hair cells and/or prior to hair cell degeneration or hair cell death.

Chemically induced hearing loss, hair cell degeneration or hair cell death

Hearing loss, hair cell degeneration or hair cell death can be induced chemically i.e. by a chemical agent e.g. by an antibiotic, a drug, a chemotherapeutic agent, heavy metals or organic agents. Antibiotics which may cause hearing loss include for example cephalosporins such as cephalexin (Keflex), cefaclor (Ceclor), and cefixime (Suprax); aminoglycosides such as gentamicin, tobramycin and streptomycin; macrolides, such as erythromycin, azithromycin (Zithromax) and clarithromycin; sulfonamides such as trimethoprim-sulfamethoxazole or tetracylines such as tetracycline, or doxycycline. In particular hearing loss, hair cell degeneration or hair cell death is effectively prevented or treated by the methods of the present invention in a subject exposed to gentamicin.

Chemotherapeutic agents, e.g. anti-cancer agents which may cause hearing loss, hair cell degeneration or hair cell death include for example platinum-containing agents e.g. cisplatin, and carboplatin, preferably cisplatin. Drugs which may cause hearing loss, hair cell degeneration or hair cell death include for example furosemide, quinine, aspirin and other salicylates. Heavy metals which may cause hearing loss include for example mercury, lead. Organic agents which may cause hearing loss, hair cell degeneration or hair cell death include for example toluene, xylene, or styrene. In some embodiments, the pharmaceutical composition of the invention is administered to the subject before the subject is exposed to a chemical agent, thereby preventing the subject from chemically induced hearing loss, hair cell degeneration or hair cell death. In some embodiments the pharmaceutical composition of the invention is administered to the subject after the subject is exposed to a chemical agent thereby treating the subject having chemically induced hearing loss, hair cell degeneration or hair cell death.

In a preferred embodiment, when hearing loss is caused by a noise trauma or is chemically induced, the pharmaceutical composition of the invention is administered to the subject prior to exposure of the subject to the noise trauma or to the chemical wherein at least 50%, preferably at least 60%, more preferably at least 70%, in particular at least 80%, more particular at least 90% of the cell damage of the hair cells caused by the noise trauma or the chemical agent is prevented.

In one aspect of the invention, the present invention provides a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for use in a method of preventing or inhibiting hair cell degeneration or hair cell death in a subject. In a further aspect of the invention, the present invention provides a method of preventing or inhibiting hair cell degeneration or hair cell death in a subject, which method comprises administering to the subject a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel. In some embodiments, the pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel is administered to the subject in an amount that is sufficient to prevent or inhibit hair cell degeneration or hair cell death in the subject. In a further aspect, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for the manufacture of a medicament for preventing or inhibiting hair cell degeneration or hair cell death in a subject.

In a further aspect, the present invention provides the use of a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel for preventing or inhibiting hair cell degeneration or hair cell death in a subject.

In some embodiments, hair cell degeneration or hair cell death in a subject is caused by a noise trauma, by age, a medical intervention, sudden hearing loss, or ischemic events such as ischemic injury, or is chemically induced wherein the chemical induction is caused by e.g. an antibiotic or a chemotherapeutic agent. Noise trauma, age, a medical intervention, sudden hearing loss, or ischemic events, or chemical induction can cause hair cell degeneration or hair cell death in a subject as described above for methods of preventing or treating hearing loss.

In some embodiments, hearing loss, hair cell degeneration or hair cell death is caused by hair cell damage. In some embodiments, the pharmaceutical composition of the invention is administered to the subject prior to identification of said hair cell damage, i.e. prior to occurrence of hair cell damage. In a preferred embodiment, when hair cell damage is caused by a noise trauma or is chemically induced, the pharmaceutical composition of the invention is administered to the subject prior to exposure of the subject to the noise trauma or to the chemical agent wherein at least 50%, preferably at least 60%, more preferably at least 70%, in particular at least 80%, more particular at least 90% of the cell damage of the hair cells caused by the noise trauma or the chemical agent is prevented. Identification/occurrence of hair cell damage is usually determined by evaluation of the state of the hair cells which can be easily accomplished as described above or as disclosed in the examples.

Pharmaceutical compositions comprising a PPAR agonist and an auris-acceptable gel

As outlined above, the present invention relates to a pharmaceutical composition comprising: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel comprises a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, polyesters, colloidal silica, aluminum soaps, zinc soaps, natural gums, starch, cellulose and derivatives thereof, carboxyvinylpolymers, metal silicates and mixtures thereof; and the use of this composition in a method of preventing or treating hearing loss and/or in a method of preventing or inhibiting hair cell degeneration or hair cell death in a subject.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel, wherein the auris-acceptable gel comprises a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, polyesters, colloidal silica, aluminum soaps, zinc soaps, natural gums, starch, cellulose derivatives, carboxyvinylpolymers, metal silicates and mixtures thereof; and

wherein said PPAR agonist is a PPAR gamma agonist, preferably a PPAR gamma agonist selected from the group consisting of pioglitazone, troglitazone, rosiglitazone and

pharmaceutically acceptable salts thereof, most preferably pioglitazone or a pharmaceutically acceptable salt thereof, in particular pioglitazone hydrochloride.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel comprises a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block.

In one embodiment, said copolymer is present in an amount of about 5% to about 50% w/w, preferably of about 10% to about 30% w/w, more preferably of about 12.5% to about 25% w/w, most preferably of about 15% to about 20% w/w relative to the total weight of the pharmaceutical composition. In one embodiment, said hydrophilic block is selected from the group consisting of polyethylene glycol (PEG), preferably PEG having an average molecular weight of between about 500 and 2200 daltons.

In a further embodiment, said hydrophobic block is selected from the group consisting of a polyester, a poly(orthoester) and a polyoxypropylene. In a preferred embodiment, said hydrophobic blockis a polyoxypropylene. In a further embodiment, said hydrophobic block is a polyester, wherein said polyester is synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, e-caprolactone, e-hydroxyhexanoic acid, g-butyrolactone, g- hydroxybutyric acid, d-valero lactone, d -hydroxyvaleric acid, hydroxybutyric acids, malic acid, and copolymers thereof. In a further embodiment, said hydrophobic block is a polyester having an average molecular weight of between about 600 and 3000 daltons. In one embodiment, both said hydrophobic block and said hydrophilic block are biodegradable. In a preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical

compositions of the invention comprises a copolymer, wherein said copolymer is a PLGA- PEG-PLGA copolymer, preferably a PLGA-PEG-PLGA (1500-1000-1500) copolymer of the following formulas: a) ReGel™

where X = number of ethoxy units of the PEG part; Y = number of the lactic acid units of the PLGA part; and z = number of glycolide units of the PLGA part

The trade name of this polymer is ReGel™ b) PolyVivo AK-Polymers (sold by PolySciTcch, a division of Akina, lnc.)

where X, Y and Z have the same meanings as above

In a further embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a polyester, such as the polyesters comprised by AtriGel™ a polymeric (non-gelatin containing) delivery system consisting of a biodegradable poly (DL-lactide-co-glycolide) (PLGH or PLG) polymer formulation dissolved in a biocompatible solvent, N-methyl-2-pyrrolidone (NMP) and/or those disclosed, e.g. in U.S.

Pat. Nos. 5,324,519; 4,938,763; 5,702,716; 5,744,153; and 5,990,194. Hence, in one embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a polyester selected from the group consisting of polylactides, polyglycolides, polycaprolactones, copolymers thereof, terpolymers thereof, and any combinations thereof.

In a further embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a polyester, wherein said polyester is 50/50 poly(DL- lactide-co-glycolide) having a carboxy terminal group; is present in about 30 wt. % to about 40 wt. % of the composition; and has an average molecular weight of about 23,000 to about 45,000. Alternatively, in another embodiment, the said polyester is 75/25 poly (DL-lactide-co- glycolide) without a carboxy terminal group; is present in about 40 wt. % to about 50 wt. % of the composition; and has an average molecular weight of about 15,000 to about 24,000. In further or alternative embodiments, the terminal groups of the poly(DL-lactide-co-glycolide) are either hydroxyl, carboxyl, or ester depending upon the method of polymerization.

Polycondensation of lactic or glycolic acid provides a polymer with terminal hydroxyl and carboxyl groups. Ring-opening polymerization of the cyclic lactide or glycolide monomers with water, lactic acid, or glycolic acid provides polymers with the same terminal groups. However, ring-opening of the cyclic monomers with a mono functional alcohol such as methanol, ethanol, or l-dodecanol provides a polymer with one hydroxyl group and one ester terminal groups. Ring-opening polymerization of the cyclic monomers with a diol such as 1,6- hexanediol or polyethylene glycol provides a polymer with only hydroxyl terminal groups.

In a preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block, wherein said hydrophilic block is polyethylene glycol and wherein said hydrophobic block is a polypropylene glycol. In a more preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a copolymer, wherein said copolymer is a poloxamer. Preferably, the average molar mass of the poloxamer is between about 6000 to about 18000 Da, more preferably between about 9000 to about 15000 Da.

In an even more preferred embodiment, the auris-acceptable gel comprised by the

pharmaceutical compositions of the invention comprises a copolymer, wherein said copolymer is a poloxamer, preferably a poloxamer selected from the group consisting of poloxamer 407 (PF-127), poloxamer 188 (F-68 grade), poloxamer 237 (F-87 grade) and poloxamer 338 (F-108 grade). In a most preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a copolymer, wherein said copolymer is poloxamer 407 (PF-127).

In a further embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a copolymer, wherein said copolymer is a PEG- PLGA-PEG triblock copolymer, preferably with a lactide/glycolide molar ratio in the PLGA block of from about 1 : 1 to about 20: 1. In a preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical compositions of the invention comprises a copolymer, wherein said copolymer is a PEG-PLGA-PEG triblock copolymer having a 50:50 ratio of lactic acid and glycolic acid in the PLGA block and is 10% w/w of PEG and has a molecular weight of about 6000 ("RESOMER RGP t50l06" originally manufactured by Boehringer Ingelheim (now by Evonik Industries)).

In one embodiment, the pharmaceutical composition of the invention comprises: i. about 0.1% to about 7.5% w/w of PPAR agonist, preferably a micronized PPAR agonist; and

ii. an auris-acceptable gel comprising about 10% w/w to about 30% w/w of a

polyoxyethylene-polyoxypropylene copolymer, preferably of poloxamer, in particular poloxamer 407, or of combinations of different polyoxyethylene- polyoxypropylene copolymers, or of combinations with further polymers.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel is a hydrophobic gel comprising a compound selected from the group consisting of colloidal silica, aluminum soaps and zinc soaps.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel is a hydrophilic gel comprising a compound selected from the group consisting of natural gums, starch, cellulose and derivatives thereof,

carboxyvinylpolymers, metal silicates and mixtures thereof.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel is a thermoreversible gel comprising a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block and polyesters.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel is a thermoreversible gel comprising a compound selected from the group consisting of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block.

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel, wherein the auris-acceptable gel comprises a polyester, preferably a polyester selected from the group consisting of polylactides, polyglycolides, polycaprolactones, copolymers thereof, terpolymers thereof, and any combinations thereof

In a particularly preferred embodiment, the auris-acceptable gel comprised by the

pharmaceutical compositions of the invention is a thermoreversible gel as defined herein.

In one embodiment, the rheology of the pharmaceutical composition of the invention is pseudo plastic, plastic, thixotropic, or diluent.

In a further embodiment, the pharmaceutical composition of the invention comprises a solvent selected from the group consisting of liquid paraffin, polyethylene, fatty oils, water and alcohols (e.g. glycerol or propylene glycol).

In a further embodiment, the pharmaceutical composition of the invention comprises a non polar solvent selected from the group consisting of liquid paraffin, polyethylene and fatty oils. In a preferred embodiment, the pharmaceutical composition of the invention comprises a polar solvent selected from the group consisting of water and alcohols (e.g. glycerol or propylene glycol). In a preferred embodiment, the pharmaceutical composition of the invention comprises a solvent, wherein said solvent is water.

In one embodiment, the pharmaceutical composition of the invention comprises a surfactant selected from the group consisting of polysorbates, cellulosic polymers and mixtures thereof. In one embodiment, the pharmaceutical composition of the invention comprises a surfactant selected from the group consisting of sodium lauryl sulfate, sodium dioctyl sulfosuccinate (Docusate Sodium), polysorbates (e.g. the products sold by Croda Int. under the trade name Tween™: Polysorbate 60 (Tween®60), Polysorbate 80 (Tween®80), Polysorbate 20 (Tween 20)), triacetin, Vitamin E Polyethylene Glycol Succinate, phospholipids, lecithins,

phosphatidyl cholines (C8-C18), phosphatidylethanolamines (C8-cl8), phosphatidylglycerols (C8-C18), sorbitan monooleate, polyoxyethylene sorbitan monooleate, bile salts, glyceryl monostearate, polyoxyethylene fatty acid glycerides, vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil), polyoxyethylene alkylethers and alkylphenyl ethers (e.g., octoxynol 10, octoxynol 40). In a preferred embodiment, said surfactant is a polysorbate, preferably polysorbate 80 or polysorbate 20, most preferably polysorbate 80.

In one embodiment, said surfactant is present in an amount of about 0.1% to about 5% w/w relative to the total weight of the pharmaceutical composition. A preferred range is about 0.75 to about 1.5% w/w relative to the total weight of the pharmaceutical composition, more preferably about 0.75 to about 1.25 % w/w, even more preferably about 1.0 % w/w. In one embodiment, the pharmaceutical composition of the invention comprises a buffer comprising trolamine hydrochloric acid buffer, acetate, bicarbonate, carbonate, ammonium chloride, citrate, phosphate, Tromethamine (tris(hydroxymethyl)aminomethane ("TRIS")), pharmaceutically acceptable salts thereof and/or combinations or mixtures thereof

In a preferred embodiment, said buffer comprises a trolamine hydrochloric acid buffer. In a further preferred embodiment, said buffer is a Tromethamine (TRIS) buffer.

Buffer concentrations as used for parenteral formulations are in the range of 10 - 100 mMol/L. The absolute quantities of a specific buffer used in a parenteral formulation will therefore depend on the molecular weight of the selected buffer.

In one embodiment, said buffer is present in an amount of about 0.15% to about 1.5% w/w relative to the total weight of the pharmaceutical composition. A preferred range is about 0.3 to about 1.3% w/w, more preferred about 0.8 to about 1.1% w/w relative to the total weight of the pharmaceutical composition.

In one embodiment, the pharmaceutical composition of the invention comprises a further excipient selected from the group consisting of a permeability enhancer, a preservative, a viscosity-increasing agent, a mucoadhesive and an antioxidant.

In one embodiment, the pharmaceutical composition of the invention comprises a

permeability enhancer.

In one embodiment, the pharmaceutical composition of the invention comprises a

permeability enhancer selected from the group consisting of surfactants (ionic and non-ionic), bile salts, fatty acids and derivatives thereof, chelating agents, sulfoxides, alcohols, glycosidases and glycosaminoglycans.

In one embodiment, the pharmaceutical composition of the invention comprises a

permeability enhancer selected from the group consisting of surfactants (ionic and non- ionic), bile salts, fatty acids and derivatives thereof, chelating agents, sulfoxides, alcohols, glycosidases and glycosaminoglycans; wherein

said surfactants are selected from the group consisting of sodium lauryl sulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9, sodium dodecylsulfate, dioctyl sodium sulfosuccinate, polyoxyethylene-9-lauryl ether (PLE), Tween 80, nonylphenoxypoly ethylene (NP-POE) and polysorbates; and wherein

said bile salts are selected from the group consisting of sodium glycocholate, sodium deoxycholate, sodium taurocholate, sodium taurodihydrofusidate and sodium

glycodihydrofusidate; and wherein said fatty acids and derivatives thereof are selected from the group consisting of oleic acid, caprylic acid, mono- and di-glycerides, lauric acids, acylcholines, caprylic acids,

acylcamitines and sodium caprates; and wherein

said chelating agents are selected from the group consisting of EDTA, citric acid and salicylates; and wherein

said sulfoxides are selected from the group consisting of dimethyl sulfoxide (DMSO) and decylmethyl sulfoxide; and wherein

said alcohols are selected from the group consisting of ethanol, isopropanol, propylene glycol, polyethylene glycol, glycerol, propanediol and benzyl alcohol; and wherein

said glycosidases are selected from the group consisting of hyaluronidases; and wherein said glycosaminoglycans are selected from the group consisting of hyaluronic acid and pharmaceutically acceptable salts thereof

In a preferred embodiment, said permeability enhancer is selected from the group consisting of benzyl alcohol, hyaluronic acid and pharmaceutically acceptable salts thereof and DMSO. In a particularly preferred embodiment, said permeability enhancer is benzyl alcohol.

In one embodiment, the pharmaceutical composition of the invention comprises a

preservative. In one embodiment, the pharmaceutical composition of the invention comprises a preservative selected from the group consisting of benzoic acid, p-hydroxybenzoates (e.g. methylparabene and propylparabene), benzyl alcohol, lower alkyl alcohols (e.g., ethanol, butanol or the like), Chlorobutanol, quaternary compounds (e.g. Benzalkonium Chloride, Benzethonium Chloride, Cetylpyridinium Chloride), thimerosal, sorbic acid. In a particularly preferred embodiment, said preservative is selected from the group consisting of benzoic acid, benzyl alcohol, chlorobutanol, p-hydroxybenzoates, methylparabene, and propylparabene, or combinations thereof, most preferably methylparabene, propylparabene, or combinations thereof.

In one embodiment, the pharmaceutical composition of the invention comprises a viscosity- increasing agent. In one embodiment, the pharmaceutical composition of the invention comprises a viscosity- increasing agent selected from the group consisting of hydroxypropyl methylcellulose (HPMC, hypromellose), hydroxyethyl cellulose, polyvinylpyrrolidone (PVP: povidone), carboxymethyl cellulose (carmellose), carboxymethylcellulose sodium, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate, acacia (gum arabic), agar, sodium alginate, carbomer, carrageenan, carbopol®, xanthan, gelatin, guar gum, maltodextrin, sterculia gum, polyethylene glycol (e.g. PEG 200-4500), tragacanth, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethylmethyl cellulose (hymetellose), hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), pectin, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), Carboxymethylcellulose Sodium, silicon dioxide and combinations thereof.

In a particularly preferred embodiment, said viscosity- increasing agent is selected from the group consisting of hypromellose and a carbomer (e.g. carbomer homopolymer types A, B or C, carbomer copolymer types A, B or C or carbomer interpolymer types A, B or C).

In one embodiment, viscosity-increasing agents described herein are also utilized as gelling agents for forming the auris-acceptable gel comprised by the pharmaceutical composition of the invention.

In one embodiment, the auris-acceptable gel comprised by the pharmaceutical composition of the invention comprises a combination of a viscosity-increasing agent and a polymer capable of forming a thermoreversible gel as defined supra.

Suitable combinations of viscosity- increasing agents and polymers capable of forming a thermoreversible gel include, by way of non-limiting example, a combination of poloxamer copolymers with cellulose based viscosity- increasing agents described herein. In some instances, the addition of a secondary polymer (e.g., a viscosity-increasing agent) to a polymer capable of forming a thermoreversible gel introduces a diffusional barrier and reduces the rate of release of the PPAR agonist. An appropriate viscosity-increasing agent (e.g., a cellulose based polymer, e.g., Carmellose (carboxymethyl cellulose)) is selected based on the viscosity of a 2% solution of the secondary polymer (e.g., Carmellose (carboxymethyl cellulose)); the selected secondary polymer (e.g., Carmellose (carboxymethyl cellulose)) provides a 2% polymer solution with viscosity less than 15,000 cP. In specific compositions, the selected secondary polymer (e.g., Carmellose (carboxymethyl cellulose)) provides a 2% polymer solution with viscosity from about 4,000 cP to about 10,000 cP. In some

embodiments, the ratio of a thermoreversible poloxamer to a gelling agent is about 50:1, about 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1 or about 10:1. For example, in certain embodiments, the pharmaceutical composition of the invention comprises a combination of poloxamer 407 (pluronic F127) and Carmellose (carboxymethyl cellulose) in a ratio of about 50:1, 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1 or about 10: 1. In one embodiment, the pharmaceutical composition of the invention comprises a mucoadhesive. In one embodiment, the pharmaceutical composition of the invention comprises a mucoadhesive selected from the group consisting of soluble polyvinylpyrrolidone polymer (PVP); a water-swellable, but water-insoluble, fibrous, cross-linked carboxy- functional polymer; a crosslinked poly(acrylic acid) (e.g. Carbopol 947P, Carbopol 934P, Majithiya et al, AAPS PharmSciTech (2006), 7(3), p. El; EP0551626); a carbomer homopolymer (e.g. Carbomer 934 (Carbopol™ 934 NF by Lubrizol Corp.), Carbomer 940 (Carbopol™ 940 NF by Lubrizol Corp.), Carbomer 941 (Carbopol™ 941 NF by Lubrizol Corp.)); a carbomer copolymer (e.g. Carbomer Copolymer Type A (Pemulen™ TR-2 NF by Lubrizol Corp.), Carbomer Copolymer Type B (Pemulen™ TR-l NF by Lubrizol Corp.)); a hydrophilic polysaccharide gum, maltodextrin, a cross-linked alginate gum gel, a water- dispersible polycarboxylated vinyl polymer, or a mixture thereof. In a particularly preferred embodiment, said mucoadhesive is a carbomer selected from the group consisting of Carbopol 947P and Carbopol 934P.

In one embodiment, the pharmaceutical composition of the invention comprises an

antioxidant. In one embodiment, the pharmaceutical composition of the invention comprises an antioxidant selected from the group consisting of sodium meta-bisulfite,

ethylenediaminetetraacetic acid (EDTA) or a pharmaceutically acceptable salt threof (e.g. disodium EDTA), sodium ascorbate, ascorbic acid, ascorbic acid palmitate, butylated hydroxytoluene (BHT), benzyl alcohol, tocopherol (e.g. alpha-tocopherol) and mixtures thereof.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist, preferably a micronized PPAR agonist;

ii. an auris-acceptable gel;

iii. a surfactant;

iv. a buffer; and

v. a solvent;

wherein the auris-acceptable gel comprises a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block; preferably a poloxamer; more preferably P407; and wherein said PPAR agonist is a PPAR gamma agonist, preferably a PPAR gamma agonist selected from the group consisting of pioglitazone, troglitazone, rosiglitazone and

pharmaceutically acceptable salts thereof, most preferably pioglitazone or a pharmaceutically acceptable salt thereof, in particular pioglitazone hydrochloride; and

wherein said surfactant is selected from the group consisting of sodium lauryl sulfate, sodium dioctyl sulfo succinate, polysorbates (e.g. Tween®60, Tween®80, Tween ®20), triacetin, Vitamin E Polyethylene Glycol Succinate (Vitamin E TPGS), phospholipids, lecithins, phosphatidyl cholines (C8-C18), phosphatidylethanolamines (C8-C18), phosphatidylglycerols (C8-C18), sorbitan monooleate, polyoxyethylene sorbitan monooleatebile salts, glyceryl monostearate, polyoxyethylene fatty acid glycerides, vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil), polyoxyethylene alkylethers and alkylphenyl ethers (e.g., octoxynol 10, octoxynol 40); preferably polysorbates; more preferably polysorbate 20 (Tween ®20) and polysorbate 80 (Tween®80); and

wherein said buffer is selected from the group consisting of a trolamine hydrochloric acid buffer, acetate, bicarbonate, carbonate, ammonium chloride, citrate, phosphate (e.g.

phosphate-buffered saline ("PBS")), tris(hydroxymethyl)aminomethane ("TRIS", e.g. TRIS- buffered saline ("TBS")), pharmaceutically acceptable salts thereof and/or combinations or mixtures thereof; preferably a trolamine hydrochloric acid buffer ; and

wherein said solvent is selected from the group consisting of liquid paraffin, polyethylene, fatty oils, water and alcohols (e.g. glycerol or propylene glycol); preferably water.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. about 0.25% w/w to about 2.5% w/w of a PPAR agonist, preferably a micronized PPAR agonist;

ii. an auris-acceptable gel;

iii. about 0.1% w/w to about 5% w/w of a surfactant;

iv. about 0.15% w/w to about 1.5% w/w of a buffer; and

v. about 71% w/w to about 89.5% w/w of a solvent;

wherein the auris-acceptable gel comprises about 10% w/w to about 20% w/w of a copolymer comprising at least one hydrophilic block and/or at least one hydrophobic block relative to the total weight of the pharmaceutical composition; preferably about 12% w/w to about 18% w/w of a poloxamer; more preferably about 14% w/w to about 16% w/w of P407; and wherein said PPAR agonist is a PPAR gamma agonist, preferably a PPAR gamma agonist selected from the group consisting of pioglitazone, troglitazone, rosiglitazone and

pharmaceutically acceptable salts thereof, most preferably pioglitazone or a pharmaceutically acceptable salt thereof, in particular pioglitazone hydrochloride; and

wherein said surfactant is selected from the group consisting of sodium lauryl sulfate, sodium dioctyl sulfo succinate, polysorbates (e.g. Tween®60, Tween®80, Tween ®20), triacetin, Vitamin E Polyethylene Glycol Succinate (Vitamin E TPGS), phospholipids, lecithins, phosphatidyl cholines (C8-C18), phosphatidylethanolamines (C8-C18), phosphatidylglycerols (C8-C18), sorbitan monooleate, polyoxyethylene sorbitan monooleatebile salts, glyceryl monostearate, polyoxyethylene fatty acid glycerides, vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil), polyoxyethylene alkylethers and alkylphenyl ethers (e.g., octoxynol 10, octoxynol 40); preferably polysorbates; more preferably polysorbate 20 (Tween ®20) and polysorbate 80 (Tween®80); and

wherein said buffer is selected from the group consisting of a trolamine hydrochloric acid buffer, acetate, bicarbonate, carbonate, ammonium chloride, citrate, phosphate, Tromethamine USP (tris(hydroxymethyl)aminomethane (TRIS), pharmaceutically acceptable salts thereof and/or combinations or mixtures thereof; preferably a trolamine hydrochloric acid buffer; and wherein said solvent is selected from the group consisting of liquid paraffin, polyethylene, fatty oils, water and alcohols (e.g. glycerol or propylene glycol); preferably water.

In a particularly preferred embodiment, the pharmaceutical composition of the invention comprises:

i. pioglitazone or a pharmaceutically acceptable salt thereof, preferably

micronized pioglitazone or a pharmaceutically acceptable salt thereof;

ii. polysorbate 80;

iii. trolamine hydrochloric acid buffer;

iv. poloxamer 407; and

v. water.

In a preferred embodiment, the auris-acceptable gel comprised by the pharmaceutical composition of the invention provides for an extended release of the PPAR agonist, preferably an extended release over a period of more than 1 day, more preferably more than 2 days, more preferably more than 3 days, more preferably more than 4 days, more preferably more than 5 days, more preferably more than 6 days, more preferably 1 to 15 days, more preferably 2 to 12 days, more preferably 3 to 10 days, more preferably 4 to 7 days, more preferably 5 to 7 days, more preferably 6 to 7 days, most preferably 7 days.

The extended-release aspect is imparted by slow diffusion from the auris-acceptable gel and slow erosion of the auris-acceptable gel comprised by the pharmaceutical compositions of the invention, and by slow diffusion of the PPAR agonist from said auris-acceptable gel.

In one embodiment, the extended release aspect of the pharmaceutical compositions of the invention is imparted by said auris-acceptable gel and/or by an excipient selected from the group consisting of a viscosity- increasing agent, a mucoadhesive excipient and a mixture thereof.

The current standard of care for auris formulations requires multiple administrations of intratympanic injections over several days. Since the pharmaceutical compositions described herein preferably provide for an extended release of a PPAR agonist, they may be

administered at reduced dosing frequency compared to the current standard of care. A reduced frequency of administration alleviates discomfort caused by multiple intratympanic injections in individuals undergoing treatment for a middle and/or inner ear disease, disorder or condition.

A reduced frequency of administration of intratympanic injections further reduces the risk of permanent damage (e.g., perforation) to the ear drum.

The endolymph and the perilymph have a pH that is close to the physiological pH of blood. Thus, the endolymph has a pH range of about 7.2-7.9; the perilymph has a pH range of about 7.2-7.4. The in situ pH of the proximal endolymph is about 7.4 while the pH of distal endolymph is about 7.9.

In some embodiments, the pH of the pharmaceutical composition of the invention is adjusted (e.g., by use of a buffer) to an endolymph-compatible pH range of about 7.0 to 8.0, and a preferred pH range of about 7.2-7.9. In some embodiments, the pH of the pharmaceutical composition of the invention is adjusted (e.g., by use of a buffer) to a perilymph-compatible pH of about 7.0-7.6, and a preferred pH range of about 7.2-7.4.

In one embodiment, the pharmaceutical composition of the invention has at a temperature of 20°C a viscosity of below about 100 centipoise and allows for injection through the tympanic membrane with a 18-24 gauge needle. At 35°C, the same pharmaceutical composition shows a viscosity of between about 750 and 1,000,000 centipoise. In one embodiment, the pharmaceutical composition of the invention is sterile (as determined by the methods described in the European Pharmacopoeia and/or the United States

Pharmacopoeia), and shows an endotoxin level below the threshold for intraveneous application (as defined by the methods described in the European Pharmacopoeia and/or the United States Pharmacopoeia). Accordingly, in one embodiment the pharmaceutical composition of the invention is submitted to a sterilization process. The presence of endotoxins is expressed in“endotoxin units” (EU). Humans can develop a response to as little as 5 EU/kg of body weight. In one embodiment, the pharmaceutical compositions described herein contain lower endotoxin levels (e.g. <4 EU/kg of body weight of a subject) when compared to conventionally acceptable endotoxin levels (e.g., 5 EU/kg of body weight of a subject). In one embodiment, the pharmaceutical composition of the invention has less than about 5 EU/kg of body weight of a subject. In other embodiments, the pharmaceutical composition of the invention has less than about 4 EU/kg of body weight of a subject. In additional embodiments, the pharmaceutical composition of the invention has less than about 3 EU/kg of body weight of a subject. In additional embodiments, the pharmaceutical composition of the invention has less than about 2 EU/kg of body weight of a subject.

In a further aspect, the present invention relates to a process for producing a pharmaceutical composition comprising a PPAR agonist and an auris-acceptable gel described herein, comprising the steps of:

i. dissolving a thermoreversible gel in water for injection;

ii. dissolving additional excipients in water for injection, as for instance, but not being limited to, surfactants and buffers;

iii. mixing the solutions thoroughly;

iv. performing a sterile filtration of the resulting solution through an appropriate

sterile filter e.g a 0.22 pm filter;

v. preparing a sterile suspension by incorporation of the sterile, preferably

micronized PPAR agonist in the sterile filtrate, using aseptic procedures; and vi. filling required quantities of the suspension into an appropriate packaging

configuration, as for instance, but not being limited to, vials, ampoules or pre-filled syringes.

Depending on the aqueous solubility, concentration and thermal stability of the PPAR- agonist, or of a pharmaceutically acceptable salt of the PPAR agonist, the process may be different, e.g. dissolution of the PPAR-agonist or its pharmaceutically acceptable salt in the aqueous phase, filtration of the final solution to remove particles, ampoule filling and closing, and final thermal sterilization.

Composition based on a poloxamer

A pharmaceutical composition according to the invention based on a poloxamer may be prepared as follows:

Poloxamer is dissolved in a solution of a buffer in water for injection at a temperature of not more than lO°C. A solution of a surfactant in water is mixed with the poloxamer - buffer solution. The pH of the mixture may be adjusted by adding dilute hydrochloric acid or dilute sodium hoydroxide. The final solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, micronized, sterile PPAR agonist is suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well-known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a permeability enhancer;

iv. a surfactant;

v. a buffer; and

vi. a solvent;

wherein the auris-acceptable gel comprises a poloxamer.

A suitable poloxamer for use in the auris-acceptable gel (ii) is, for example, poloxamer 407.

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate 80 or polysorbate 20.

A suitable buffer for use in the pharmaceutical composition is, for example, a TRIS or trolamine hydrochloric acid buffer .

A suitable permeability enhancer for use in the pharmaceutical composition is, for example, benzyl alcohol. A pharmaceutical composition according to the invention based on a poloxamer and comprising a permeability enhancer may be prepared as follows:

Poloxamer is dissolved in a solution of a buffer in water for injection at a temperature of not more than lO°C. A solution of a surfactant and a permeability enhancer in water is mixed with the poloxamer - buffer solution. The pH of the mixture may be adjusted by adding dilute hydrochloric acid or dilute sodium hoydroxide. The final solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, micronized, sterile PPAR agonist is suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well- known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

Composition based on a combination of poloxamers

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist; and

ii. an auris-acceptable gel;

wherein the auris-acceptable gel comprises a combination of at least two poloxamers.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a surfactant; and

iv. a buffer; and

v. a solvent;

wherein the auris-acceptable gel comprises a combination of at least two poloxamers.

A suitable combination of poloxamers for use in the auris-acceptable gel (ii) is, for example, a combination of poloxamer 407 and poloxamer 188.

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate 80 or polysorbate 20.

A suitable buffer for use in the pharmaceutical composition is, for example, Tromethamine USP (TRIS) or a trolamine hydrochloric acid buffer USP/NF.

A pharmaceutical composition according to the invention based on a combination of poloxamers may be prepared as follows: A combination of poloxamers is dissolved in a solution of a buffer in water for injection at a temperature of not more than lO°C. A solution of a surfactant in water is mixed with the poloxamer - buffer solution. The pH of the mixture may be adjusted by adding dilute hydrochloric acid or dilute sodium hydroxide. The final solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, micronized, sterile PPAR agonist is suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well- known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

Composition based on a combination of a poloxamer and a cellulosic polymer

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist;

ii. an auris-acceptable gel; and

iii. a cellulosic polymer;

wherein the auris-acceptable gel comprises a poloxamer.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a cellulosic polymer;

iv. a surfactant;

v. a buffer; and

vi. a solvent;

wherein the auris-acceptable gel comprises a poloxamer.

A suitable poloxamer for use in the auris-acceptable gel (ii) is, for example, poloxamer 407. A suitable cellulosic polymer for use in the pharmaceutical composition is, for example, hypromellose.

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate 80 or polysorbate 20.

A suitable buffer for use in the pharmaceutical composition is, for example, a TRIS.

A pharmaceutical composition according to the invention based on a combination of a poloxamer and a cellulosic polymer may be prepared as follows: A cellulosic polymer is dispersed in hot water for injection and dissolved on cooling. After cooling, this solution is added to a solution of a poloxamer in a solution of a buffer in water for injection at a temperature of not more than lO°C. A solution of a surfactant in water is mixed with the poloxamer - buffer solution. The pH of the mixture may be adjusted by adding dilute hydrochloric acid or dilute sodium hoydroxide. The final solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, micronized, sterile PPAR agonist is suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well-known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

Depending on the aqueous solubility, concentration and thermal stability of the PPAR- agonist, the process may be different, e.g. dissolution of the PPAR-agonist in the aqueous phase, filtration of the final solution to remove particles, ampoule filling and closing, and final thermal sterilization.

Composition based on a combination of poloxamers and a carbomer

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist;

ii. an auris-acceptable gel; and

iii. a carbomer

wherein the auris-acceptable gel comprises a combination of at least two poloxamers.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a carbomer;

iv. a surfactant;

v. a buffer; and

vi. a solvent;

wherein the auris-acceptable gel comprises a combination of at least two poloxamer and a carbomer.

The at least two poloxamers for use in the auris-acceptable gel (ii) are preferably poloxamer 407, poloxamer 188. Suitable carbomers for use in the pharmaceutical composition are carbomer homopolymers (types A, B or C), carbomer copolymers (types A, B or C) and carbomer interpolymers (types A, B or C).

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate 80 or polysorbate 20.

A suitable buffer for use in the pharmaceutical composition is, for example, a TRIS buffer.

A pharmaceutical composition according to the invention based on a combination of poloxamers and a carbomer may be prepared as follows:

A solution of poloxamers in water for injection at a temperature of not more than l0°C is prepared. A second solution of a surfactant and a buffer in water for injection is prepared and added to the Poloxamer solution. The combined solution is filtered through an appropriate membrane filter to get a sterile material. In a stepwise procedure, a sterile carbomer and micronized, sterile PPAR agonist are suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well- known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass. Depending on the aqueous solubility and concentration of the selected PPAR-agonist, the process may be adapted, e.g. dissolution of the non-sterile PPAR-agonist in the aqueous phase, followed by sterile filtration and aseptic homogenization of the carbomer in the sterile filtrate.

Composition based on a combination of a poloxamer and a cellulosic polymer and comprising a mucoadhesive

In one embodiment, the pharmaceutical composition according to the invention comprises: i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a cellulosic polymer; and

iv. a mucoadhesive;

wherein the auris-acceptable gel comprises a poloxamer.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a cellulosic polymer; iv. a mucoadhesive;

v. a surfactant;

vi. a buffer;

vii. a preservative; and

viii. a solvent;

wherein the auris-acceptable gel comprises a poloxamer.

A suitable poloxamer for use in the auris-acceptable gel (ii) includes poloxamer 407.

A suitable cellulosic polymer for use in the pharmaceutical composition includes

hypromellose.

Suitable mucoadhesives for use in the pharmaceutical composition are carbomers, in particular carbopol 934P.

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate 80 or polysorbate 20.

A suitable buffer for use in the pharmaceutical composition is, for example, Tromethamine USP (tris(hydroxymethyl)aminomethane,“TRIS") or a trolamine hydrochloric acid buffer .

A suitable preservative for use in the pharmaceutical composition is, for example,

methylparabene, propylparabene, or combinations thereof.

A pharmaceutical composition according to the invention based on a poloxamer and comprising a mucoadhesive may be prepared as follows:

A cellulosic polymer is dispersed in hot water for injection and dissolved on cooling. After cooling, this solution is added to a solution of poloxamer in water for injection, prepared at a temperature of not more than l0°C. A second solution of a surfactant, a preservative, a buffer, and a mucoadhesive is prepared in water for injection at room temperature. The two solutions are mixed. The pH of the mixture may be adjusted by adding, for example, dilute hydrochloric acid. The final solution is filtered through an appropriate membrane filter to get a sterile material. In a stepwise procedure, micronized, sterile PPAR agonist is suspended and homogenized in the sterile filtrate. The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well-known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

Depending on the aqueous solubility and concentration of the selected PPAR-agonist, as well as the selected mucoadhesive, the process may be adapted, e.g. dissolution of the non-sterile PPAR-agonist in the aqueous phase, followed by sterile filtration, and aseptic homogenization of e.g. a carbomer in the sterile filtrate.

Composition based on a combination of a PLGA-PEG- PLGA triblock copolymer

As described above, in one embodiment the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist; and

ii. an auris-acceptable gel,

wherein the auris-acceptable gel comprises a PLGA-PEG- PLGA triblock copolymer.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises:

i. a PPAR agonist;

ii. an auris-acceptable gel;

iii. a surfactant;

iv. a buffer; and

v. a solvent;

wherein the auris-acceptable gel comprises a PLGA-PEG- PLGA triblock copolymer.

Suitable copolymers for use in the auris-acceptable gel (ii) include ReGel™ PLGA-PEG- PLGA triblock copolymer, PolyVivo AK12™, and PolyVivoAK24™ PLGA-PEG- PLGA triblock copolymers.

A suitable surfactant for use in the pharmaceutical composition is, for example, polysorbate

20.

A suitable buffer for use in the pharmaceutical composition is, for example, a Tromethamine (tris(hydroxymethyl)aminomethane, TRIS) buffer.

A pharmaceutical composition according to the invention based on a poloxamer and comprising a mucoadhesive may be prepared as follows:

At a temperature below l5°C, a solution of a surfactant and the PLGA-PEG- PLGA triblock copolymer in water (I ^st part) is prepared. Micronized PPAR agonist is suspended in this solution. A buffer solution in water (2 ^nd part) is added to adjust the pH. Water (3 ^rd part) is added to get the final concentration of 1.5 % of pioglitazone hydrochloride.

Modes of Administration and Treatment The pharmaceutical composition described herein is usually administered topically in the ear or by injection into the inner ear and/or into the middle ear, preferably by injection into the middle ear.

In one embodiment, the auris-acceptable gel comprised by the pharmaceutical compositon of the invention is a thermoreversible gel and is administered by intratympanic injection at or near room temperature.

The pharmaceutical compositions of the invention are liquid as long as stored in the primary container at a temperature below body temperature (typically at or below room temperature) or when handled by the clinician, but rapidly form a gel once injected into e.g. the middle ear cavity (temperature of about 37°C). In one embodiment, the pharmaceutical composition of the invention is injected intratympanically and forms a gel layer over the round window. This has two inter-related effects: a) in contrast to conventional solutions or suspensions, the product remains in the middle ear; it is therefore not rapidly cleared through the Eustachian tube; and b) the diffusion rate through the hydrated gel layer is reduced, thereby providing a sustained release.

Otic administration of the pharmaceutical compositions of the invention avoid certain drawbacks sometimes associated with systemic administration of the PPAR agonist comprised by the compositions (e.g., hepatotoxicity, cardiotoxicity, gastrointestinal side effects, renal toxicity, low bioavailability of the drug in the endolymph or perilymph, variability in concentration of the drug in the middle and/or internal ear). In some instances, localized administration in the ear allows a PPAR agonist to reach a target organ (e.g., inner ear) in the absence of systemic accumulation of said PPAR agonist. In some instances, local

administration to the ear provides a higher therapeutic index for a PPAR agonist that would otherwise have dose-limiting systemic toxicity.

In a particularly preferred embodiment, the pharmaceutical composition of the invention comprises a thermoreversible gel and is suitable for PPAR agonist delivery into the inner ear, including the cochlea and vestibular labyrinth; preferably with little or no systemic release of the PPAR agonist.

For some routes of administration, e.g. for injection into the inner ear and/or into the middle ear a sustained release sytem can be used. In some routes of administration, the penetration of the active ingredient is facilitated by permeability enhancers as e.g. benzyl alcohol, hyaluronic acid or DMSO (vide supra). In some routes of administration, in particular when the pharmaceutical composition of the invention is administered by injection into the inner ear and/or into the middle ear, a thixotropic or thermogeling formulation is used to enable a painless administration and forming a gel or a high viscous composition ensuring prolonged and continuous release of the active ingredient into the inner ear and/or into the middle ear.

The PPAR agonist or the pharmaceutical composition can be located in contact with the crista fenestrae cochlea, the round window, the tympanic cavity, the tympanic membrane, the auris media or the auris externa. In further or alternative embodiments, the pharmaceutical composition of the invention can be administered on or near the round window membrane via intratympanic injection. In other embodiments, the pharmaceutical composition of the invention is administered on or near the round window or the crista fenestrae cochleae through entry via a post-auricular incision and surgical manipulation into or near the round window or the crista fenestrae cochleae area. Alternatively, the pharmaceutical composition of the invention is applied via syringe and needle, wherein the needle is inserted through the tympanic membrane and guided to the area of the round window or crista fenestrae cochleae. The pharmaceutical composition of the invention is then deposited on or near the round window or crista fenestrae cochleae for localized treatment.

Preferably the pharmaceutical composition as described herein is administered by

intratympanic injection into the inner ear and/or into the middle ear, preferably into the middle ear. Intratympanic injection of therapeutic agents is the technique of injecting an agent behind the tympanic membrane into the middle and/or inner ear, preferably into the middle ear.

In one embodiment, the pharmaceutical compositions described herein are administered directly onto the round window membrane via transtympanic injection. In another

embodiment, the pharmaceutical compositions described herein are administered onto the round window membrane via a non-transtympanic approach to the inner ear. In additional embodiments, the pharmaceutical composition described herein is administered onto the round window membrane via a surgical approach to the round window membrane comprising modification of the crista fenestrae cochleae.

In one embodiment the delivery system is a syringe and needle apparatus that is capable of piercing the tympanic membrane and directly accessing the round window membrane or crista fenestrae cochleae of the auris interna.

In some embodiments, the delivery device is an apparatus designed for administration of therapeutic agents to the middle and/or inner ear. Byway of example only: GYRUS Medical Gmbh offers micro-otoscopes for visualization of and drug delivery to the round window niche; Arenberg has described a medical treatment device to deliver fluids to inner ear structures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446. U.S. patent application Ser. No. 08/874,208 describes a surgical method for implanting a fluid transfer conduit to deliver therapeutic agents to the inner ear. U.S. Patent Application Publication 2007/0167918 further describes a combined otic aspirator and medication dispenser for intratympanic fluid sampling and medicament application.

The pharmaceutical composition of the invention is useful in surgical procedures including, by way of non-limiting examples, cochlea surgery, labyrinthotomy, mastoidectomy, stapedectomy, endolymphatic sacculotomy or the like. In a preferred embodiment, the pharmaceutical composition as described herein is administered prior to surgical procedures in particular prior to cochlea surgery.

The pharmaceutical composition described herein is administered for preventive and/or therapeutic treatments. Preventive treatments comprise prophylactic treatments. In preventive applications, the pharmaceutical composition of the invention is administered to a subject suspected of having, or at risk for developing a disease, disorder or condition as described herein. In therapeutic applications, the pharmaceutical composition of the invention is administered to a subject, such as a patient already suffering from a disorder disclosed herein, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition as described herein. Amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the subject's health status and response to the drugs, and the judgment of the treating physician.

In the case wherein the subject's condition does not improve, the pharmaceutical composition of the invention may be administered chronically, which is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

In the case wherein the subject's status does improve, the pharmaceutical composition of the invention may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”).

Once improvement of the patient's otic conditions has occurred, a maintenance dose of the pharmaceutical composition of the invention is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, is optionally reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

In some preferred embodiments the pharmaceutical composition of the invention is administered by a single injection into the inner ear and/or into the middle ear, preferably by a single intratympanic injection into the inner ear, followed by oral administration of a PPAR agonist or by a single intratympanic injection into the middle ear followed by oral

administration of a PPAR agonist, which is preferred, or by administration of a PPAR agonist formulated as ear drops with penetration into the inner ear. Oral administration of a PPAR agonist can be provided chronically, which is, for an extended period of time, including throughout the duration of the subject's life. In some embodiments after long term treatment, e.g. long term treatment using oral administration of a PPAR agonist, hearing capacity is increased based on a reactivation of hair cells from a resting state and/or improvement in neural cell function. In some embodiments after long term treatment, e.g. long term treatment using oral administration of a PPAR agonist, hearing capacity is increased based on an increase of the number of hair cells or hair cell function or improved neural cell function subsequent to PPAR activation.

The amount of the pharmaceutical composition of the invention to be administered will vary depending upon factors such as the disease condition and its severity, according to the particular circumstances surrounding the case, including, e.g., the specific PPAR agonist comprised by said pharmaceutical composition, the condition being treated, the target area being treated, and the subject or host being treated.

In some embodiments, the pharmaceutical composition of the invention comprises a PPAR agonist, usually a PPAR gamma agonist, PPAR alpha agonist and/or PPAR alpha/gamma dual agonist, preferably a PPAR gamma agonist, more preferably pioglitazone or a

pharmaceutically acceptable salt thereof, most preferably pioglitazone hydrochloride and is administered in human by injection into the inner ear and/or into the middle ear, preferably to the middle ear at a concentration of about 0.01% to about 7.5% w/v, preferably about 0.01% to about 5% w/v, more preferably about 0.1% to about 7.5% w/v, more preferably about 0.1% to about 5% w/v, more preferably about 0.1% to about 4% w/v, more preferably about 0.1% to about 3% w/v, more preferably about 0.1% to about 2% w/v, more preferably about 0.5% to about 2% w/v, more preferably about 0.5% to about 1.5% w/v, more preferably about 1% to about 1.5% w/v, most preferably about 1.2% w/v per single injection. Usually 50 mΐ to lml, preferably lml of a solution or suspension containing the PPAR agonist is injected by single injection.

Methods of identification of patients who are suspected of having, or being at risk for developing hearing loss, hair cell degeneration or hair cell death are also comprised by the present invention. In some embodiments patients who are suspected of having, or being at risk for developing hearing loss, hair cell degeneration or hair cell death are identified by measurement of serum and/or plasma adiponectin levels, in particular the measurement of high molecular weight adiponectin levels. In some embodiments the monitoring of the treatment success and/or the identification of the subject e.g. the identification of the subject who is suspected of having, or being at risk for developing hearing loss, hair cell degeneration or hair cell death, is achieved by measurement of serum and/or plasma adiponectin levels.

Kits/ Articles of Manufacture

The disclosure also provides kits for preventing or treating hearing loss and/or

preventing or inhibiting hair cell degeneration or hair cell death in a subject, preferably in human. Such kits generally will comprise the pharmaceutical composition disclosed herein, and instructions for using the kit. The disclosure also contemplates the use of the

pharmaceutical composition disclosed herein, in the manufacture of medicaments for treating, abating, reducing, or ameliorating the symptoms of a disease, dysfunction, or disorder in a mammal, such as a human that has, is suspected of having, or at risk for developing hearing loss, hair cell degeneration or hair cell death.

In some embodiments, kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In other embodiments, the containers are formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein generally will comprise the pharmaceutical composition disclosed herein and packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, tubes, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected composition and intended mode of administration and treatment. Examples

Example 1: Protection against antibiotic-induced hair cell damage

Organs of Corti were obtained from post-natal day 5 Sprague-Dawley rats and placed in organ culture. Gentamicin treatment resulted is 50 - 70% loss of hair cells after 48h in culture. Pioglitazone co-treatment was protective, almost completely preventing gentamicin- dependent hair cell loss, and largely preserving organ morphology.

Methods

Animal procedures

All animal procedures were carried out according to protocols approved by the Kantonales Veterinaramt, Basel, Switzerland. Postnatal day 5 (p5) Sprague-Dawley rats were used for the studies. Studies were performed in vitro, using organ of Corti (OC) explants from p5 animals. Animals were sacrificed and the cochleae carefully dissected to separate the organ of Corti from the spiral ganglion, stria vascularis and Reissner's membrane [Sobkowicz HM, Loftus JM, Slapnick SM. Acta Otolaryngol Suppl. 1993;502:3-36]

Tissue culture

OCs were harvested then placed in culture medium [Dulbecco’s Modified Eagle Medium supplemented with 10% FCS, 25 mM HEPES and 30 U/ml penicillin (Invitrogen, Carlsbad, CA, USA)] and incubated for 24 hours at 37°C in an atmosphere of 95% 02/5% C02. After that period, the culture medium was replaced with fresh medium containing no compound or 200mM gentamicin alone or 200 mM gentamicin with either 2 or 10 mM pioglitazone, and incubated for a further 48 hours at 37°C. Ten OC explants were used for each treatment condition.

Hair cell counting

After incubation with compounds, the OCs were fixed in 4% paraformaldehyde, washed and then stained with a fluorescein (FITC)-conjugated phalloidin to detect inner and outer hair cells. After staining, the OCs were visualized and photographed using a fluorescence microscope (Olympus FSX100). Outer and inner hair cells were separately quantitated for the apical, basal, and middle turn of each organ of Corti. The values for each turn were averaged for the 10 OCs used for each condition. Significant differences between treatment groups in numbers OHC and IHC were determined using analysis of variance (ANOVA) followed by the least significant difference (LSD) post-hoc test (Stat View 5.0). Differences associated with P-values of less than 0.05 were considered to be statistically significant. All data are presented as mean ± SD. Results

Untreated organs of Corti were well preserved after 48 hours in culture presenting with intact ordered rows of outer hair cells (OHC) and inner hair cells (IHC). Pioglitazone treatment alone, at either 2 or 10 mM had no effect on hair cell number or morphology, indicating no direct adverse effect of pioglitazone (Fig 1 A-C). In contrast, 200 mM gentamicin treatment resulted in almost complete destruction and loss of hair cells (Fig 1 A-C). Pioglitazone at both 2 and 10 mM was able to antagonize the effects of gentamicin and to preserve hair cell number and morphology (Fig 1 A-C). Quantative image analysis was performed to count IHC and OHC separately in the apical, basal, and middle turns of each organ of Corti. While gentamicin treatment resulted in a consistent reduction of hair cell number of approximately 50 - 70% in each segment, pioglitazone at both concentrations was able to completely prevent gentamicin-dependent hair cell loss in all turns.

Example 2: Protection against noise-induced hearing loss

A formulation of pioglitazone (composition according to Table 1 of Example 4) or vehicle alone was applied into the middle ears of guinea pigs. The animals were then exposed to a noise trauma (broadband noise 4 - 20 kHz, 115 dB (SPL) and recording of hearing sensitivity over the standard frequency range was performed 7-14 days later. Results obtained in the hearing test were compared to baseline values before injury. Pioglitazone protected hearing, resulting in a reduction of >50% in the threshold shifts in pioglitazone-treated animals vs. vehicle controls.

Methods

Animal Procedures

The guinea pig model is the preferred animal species in hearing research. The agent application as well as the noise trauma was applied under general anesthesia. Upon arrival, animals underwent an acclimatization period of at least one week prior to experiments.

Animals were housed in pairs with ad libitum access to food and water in a temperature and humidity controlled environment on a l2h/l2h light/dark cycle. The protocol was approved by the governmental animal use committee of Berlin, Germany.

Guinea pigs were first anaesthetized and hearing evaluated by a standard ABR method then into treatment groups. Each animal received a single round window application of test substance to both ears. The following day, animals were exposed to 115 dB broad band noise for 2 hrs under anaesthesia. At one and two weeks following noise exposure, the animals underwent a second hearing evaluation.

Animals were dosed with a single 40 mΐ application of pioglitazone formulation or matching vehicle, onto the cochlear round window in both ears the day prior to noise exposure. For this approach, a hole was drilled in the rostral part of the skull to directly access the Bulla which allows drug application to the round window under visual control.

Animals were noise-exposed in a soundproof chamber (0.8x0.8x0.8 m, minimal attenuation 60 dB) for 2 h to broadband white noise (5-20 kHz) at 115 dB sound pressure level (SPL) under anaesthesia (60 mg/kg ketamine and 6 mg/kg xylazine). Noise was delivered binaurally by loudspeakers (HTC 11.19; Visaton, Haan, Germany) placed above the animal’s head. The speakers were connected to an audio amplifier (Tangent AMP-50; Aulum, Denmark) and a DVD player.

Hearing assessment At baseline before the noise exposure and on day 7 and 14 after noise, frequency-specific (2; 4; 8; 12; 16; 20; 24; 28; 32; 36; 40 kHz) auditory brainstem responses (ABR) were recorded in all treated animals and in controls. Auditory stimuli were delivered binaurally at different SPLs with a sinusoid generator (Model SSU2; Werk Femmeldewesen, Berlin, Germany). Frequency output was controlled and adjusted with a digital counter (1941 A Digital Counter; Fluke, Scarborough, Ontario, Canada). Sub-dermal needle electrodes were placed at the vertex (active), mastoid (reference), and in one foot (ground). ABR recordings were carried out with a Viking IV- measurement system (Viasys Healthcare, Conshohocken,

Pennsylvania). The brainstem responses were amplified (IOO,OOOc), filtered (bandpass 0.15-3 kHz), and averaged (300x) by the Viking IV-system. The amplitudes of the ABR waves were measured at different sound intensities by changing the attenuation of signal amplification. The amplitude-growth function was calculated for each tested frequency, and a linear regression was fitted to the linear portion of the data. The hearing threshold could be calculated for each frequency by extrapolating the linear amplitude-grow function of the regression line to zero. From these data, threshold differences (mean threshold shifts) were calculated between the control and the noise-exposed animals using the average values. Results are represented as mean relative hearing loss (±SD) in decibels (dB) of the experimental groups compared to controls.

Results

Vehicle treated animals showed a significant average hearing loss of 31.9 ± 2.2 dB (mean ± SD) over the frequency range of the noise challenge (5 - 20 kHz) at one week. Pioglitazone afforded significant protection of approximately 60 % from noise-induced hearing loss, with only modest threshold shifts of 12.7 ± 1.3 dB (mean ± SD) (Fig 2).

At two weeks, slight recovery in both groups was noted. Vehicle treated animals showed a significant average hearing loss of 27.3 ± 12.6 dB (mean ± SD) at two weeks. Pioglitazone treated animals showed only modest threshold shifts of 6.3 ± 3.9 dB (mean ± SD) at two weeks. (Fig 2). These data demonstrate efficacy of pioglitazone to protect hearing. Example 3: Protection against antibiotic-induced hair cell damage by dual PPARa/g agonists and a PPARa-selective agonist

The experiment was carried out similarly to the experiments in example 1. Gentamicin treatment resulted is 50% loss of hair cells after 24h exposure to mouse OC’s in culture. Treatment with the dual PPARa/g agonists muraglitazar and tesaglitazar and with the

PPARa-selective agonist fenofibric acid protected from gentamicin-dependent hair cell loss.

Methods

Methods were similar to those in Example 1. The main differences were that mouse OC’s were used rather than rat OC’s. Moreover, treatment was performed for 24 hrs with 50mM gentamicin. The number of OC’s used for each experimental condition was 3-5. The concentrations of test substances were 2 mM and 10 mM for tesaglitazar and muraglitazar, and 25 mM and 150 mM for fenofibric acid.

Results

Untreated organs of Corti were well preserved after 24 hours in culture presenting with intact ordered rows of outer hair cells (OHC) and inner hair cells (IHC). None of the test substances alone and any concentration had an effect on hair cell number or morphology, indicating no direct adverse effects (Fig 3 A-C). In contrast, 50 mM gentamicin treatment resulted in approximately 50% loss of hair cells (Fig 3 A-C). Tesaglitazar at both 2 and 10 mM was able to antagonize the effects of gentamicin and to preserve hair cell number and morphology (Fig 3 A). Muraglitazar was not effective at 2 mM but was partially protective at 10 mM (Fig 3 B).

Fenofibric acid was not effective at 25 mM but was completely protective at 150 mM (Fig 3 C).

Example 4: Thermoreversible gel composition comprising pioglitazone hydrochloride Composition according to Table 1 :

Table 1

Process:

a) Polysorbate 80 is dissolved in water for injection

b) Trolamine is dissolved in water for injection; the solution is cooled to a temperature of not more than lO°C. Poloxamer 407 is added and dissolved by stirring

c) The two solutions are mixed dilute hydrochloric acid or dilute sodium hydroxide is added by stirring to the mixture of the two solutions

d) The resulting solution is filtered through an appropriate sterile filter 0.22 pm e) By a stepwise, aseptic process, Pioglitazone is wetted by and suspended in the filtered, sterile solution

f) The required quantities of the resulting suspension are filled into an appropriate

packaging configuration, as for instance, but not being limited to, vials, ampoules or pre-filled syringes

Example 5: Suspension 1.2 % based on Poloxamer 407

Composition according to Table 2.

Table 2

Process is carried out analogously to the process of Example 4

Example 6: Suspension 0.8 % based on Poloxamer 407 and Benzyl Alcohol as

Permeability Enhancer

Composition according to Table 3:

Table 3

Process:

a) Polysorbate 20 is dissolved in water for injection

b) Tromethamine (TRIS) and Benzyl Alcohol are dissolved in water for injection; the solution is cooled to a temperature of not more than l0°C. Poloxamer 407 is added and dissolved by stirring

c) The two solutions are mixed dilute hydrochloric acid or dilute sodium hydroxide is added under continuous stirring to the mixture of the two solutions

d) The resulting solution is filtered through an appropriate sterile filter 0.22 pm e) By a stepwise, aseptic process, Pioglitazone is wetted by and suspended in the filtered, sterile solution

f) The required quantities of the resulting suspension are filled into an appropriate

packaging configuration, as for instance, but not being limited to, vials, ampoules or pre-filled syringes

Example 7: Suspension 1.0 % based on combination of Poloxamer 407 & Poloxamer 188

Composition according to Table 4:

Table 4

Process:

a) Sodium Phosphate Monobasic Dihydrate is dissolved in water for injection. The

solution is cooled to a temperature of not more than lO°C

b) Poloxamer 407 and Poloxamer 188 are added and dissolved by stirring, thereafter Vitamin E Polyethylene Glycol Succinate is added and dissolved by stirring c) Dilute hydrochloric acid is added under continuous stirring to the solution

d) The resulting solution is filtered through an appropriate sterile filter 0.22 pm e) By a stepwise, aseptic process, Pioglitazone is wetted by and suspended in the filtered, sterile solution f) The required quantities of the resulting suspension are filled into an appropriate packaging configuration, as for instance, but not being limited to, vials, ampoules or pre-filled syringes

Example 8: Suspension 0.5 % based on combination of Poloxamer & Hypromellose

Composition according to Table 5:

Table 5

Process:

a) Hypromellose is dispersed in hot water for injection; under continuous stirring, the dispersion is cooled to room temperature to get into solution. Trolamine is dissolved in the resulting solution.

b) The solution is cooled to a temperature of not more than lO°C. Poloxamer 407 is

added and dissolved by stirring, thereafter Vitamin E Polyethylene Glycol Succinate is added and dissolved by stirring

c) Dilute hydrochloric acid is added under continuous stirring to the solution to adjust the pH

d) The resulting solution is filtered through an appropriate sterile filter 0.22 pm e) By a stepwise, aseptic process, Pioglitazone is wetted by and suspended in the filtered, sterile solution

f) The required quantities of the resulting suspension are filled into an appropriate

packaging configuration, as for instance, but not being limited to, vials, ampoules or pre-filled syringes Example 9: Suspension 0.75 % based on combination of Poloxamers & Carbomer

Composition according to Table 6:

Table 6

Process:

a) A solution of Poloxamer 407 and Poloxamer 188 in water for injection at a

temperature of not more than l0°C is prepared

b) A second solution of Polyoxyl 15 Hydroxystearate (Kolliphor™ HS 15 BASF) and Tromethamine (TRIS) in water for injection is prepared and added to the Poloxamer solution. To adjust the pH, dilute hydrochloric acid or dilute sodium hydroxide is added.

c) The combined solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, sterile Carbomer Homopolymer (Type A) and micronized, sterile PPAR agonist are suspended and homogenized in the sterile filtrate

d) The sterile bulk product is filled into appropriate glass ampoules, which are closed by the well-known ampoule closing process based on a flame of mixed gas and oxygen to melt the glass.

Example 10: Mucoadhesive Gel Suspension 1.0 % based on combination of Poloxamer & Carbomer (Carbopol®) Composition according to Table 7:

Table 7

Aqueous preparations which are prepared using aseptic precautions and which cannot be terminally sterilised may contain a suitable antimicrobial preservative in an appropriate concentration. As this formulation is additionally not provided in a unit- dose container, it may contain a preservative

Process:

a) Hypromellose, Methylparabene, and Propylparabene are dispersed in hot water for injection; under continuous stirring, the dispersion is cooled to room temperature to get into solution. Sodium Phosphate Monobasic Dihydrate (NaH2P04 2H20) is dissolved in the resulting solution.

b) A solution of Poloxamer 407 in water for injection at a temperature of not more than lO°C is prepared

c) The two solutions are mixed. To adjust the pH, dilute hydrochloric acid or dilute

sodium hydroxide is added.

d) The combined solution is filtered through an appropriate membrane filter 0.2 pm to get a sterile material. In a stepwise procedure, sterile Carbomer Homopolymer (Type B) and micronized, sterile PPAR agonist are suspended and homogenized in the sterile filtrate e) The sterile bulk product is filled into appropriate crimp neck glass vials with teflonized and siliconized stoppers, with aluminium cap.

Example 11: Experimental Suspension 1.5 % based on PLGA-PEG- PLGA triblock copolymer

Composition according to Table 8:

Table 8

Process:

a) Polysorbate 20 and Tromethamine (TRIS) are dissolved in water for injection b) PLGA-PEG- PLGA triblock copolymer is dissolved in water for injection by stirring c) The two solutions are mixed dilute hydrochloric acid or dilute sodium hydroxide is added by stirring to the mixture of the two solutions

d) The resulting solution is filtered through an appropriate sterile filter 0.22 pm e) By a stepwise, aseptic process, Pioglitazone is wetted by and suspended in the filtered, sterile solution

f) The required quantities of the resulting suspension are filled into an appropriate

packaging configuration, e.g. vials. The material is frozen within the vials and stored at -20°C to guarantee appropriate chemical stability.

Example 12: Pioglitazone protects from pure-tone noise-induced hearing loss in rats

Studies were performed in rats exposed to noise trauma. Animals were injected

intratympanically with pioglitazone 1.2% thermoreversible gel in right ears vs. the vehicle control in left ears. The animals were then exposed to a noise trauma (single 10 kHz pure tone at 120 dB sound pressure level). Hearing assessments were made over the standard frequency range at various times up to 21 days later in each ear. Results obtained in the hearing test were compared to baseline values before injury. Pioglitazone protected hearing, resulting in almost complete recovery of threshold shifts in the right vs. left ears of animals treated 1 hr after noise, and partial recovery when treatment was delayed to 48 hr after the noise.

Methods

Animal Procedures

The effect of a pioglitazone 1.2% thermoreversible gel (composition according to Table 1 of Example 4) vs. matching vehicle was evaluated in rats to assess protection from noise-induced hearing loss (NIHL). Sprague-Dawley rats (age 3 months, weight 200-300g) with normal Preyer’s reflex were used. All work was performed in accordance with the European

Community Council Directive, dating November 24, 1986 (86/609/EEC).

Animals were anesthetized and then placed in a sound-proof room and exposed for 60 min to a 120 dB 10 kHz sound to induce acute acoustic trauma and hearing loss. In one experiment, 1 hr following sound exposure, animals received pioglitazone 1.2% thermoreversible gel in right ears and corresponding vehicle in left ears by intra-tympanic injection (IT) under anesthesia. IT injections of 30 mΐ were performed with a sterile motorized Hamilton 50-m1 syringe into the cochlear round window niche area at a constant rate (2 mΐ/ s) . In a second experiment, animals were treated in the same way but at 48 hr following the noise trauma. Hearing assessment

Hearing function was evaluated in all animals by measuring auditory brainstem responses (ABR) across frequencies. ABRs were measured prior to noise exposure, and up to 21 days after noise exposure. Animals were mildly anesthetized and placed in a sound-proof room. Three electrodes were subcutaneously inserted into the right mastoid (active), vertex

(reference) and left mastoid (ground). A PC-controlled TDT System 3 (Tucker-Davis Technologies, Alachua, Florida, USA) data acquisition system with real time digital signal processing was used to record the ABR and to generate the auditory stimulus. The threshold value was defined as the lowest intensity able to evoke an appropriate ABR response. Hearing function was evaluated in all animals by measuring auditory brainstem responses (ABR) prior to noise exposure, and at multiple time points up to 21 days after noise exposure. The threshold values were the lowest intensity able to evoke an appropriate ABR response. Data are presented by frequency and represent the noise-induced threshold shift at the indicated time point vs. pre-noise exposure baseline levels.

Results

Vehicle treated animals showed a frequency- specific threshold shift of about 50-60 dB immediately after noise trauma. Pioglitazone significantly reduced the threshold shift in both experiments (Figure 4 A and B). Animals treated 1 hr after noise showed the greatest protection (approx. 20 dB across all frequencies). Notably, by day 21, the treated ears had almost fully recovered, as evidenced by the return to pre-noise threshold values. Treatment 48 hr after noise also reduced the threshold shifts, but to a lesser extent compared to animals treated 1 hr following noise trauma. These data demonstrate the efficacy of pioglitazone to protect hearing in a second species. Early treatment after noise exposure leads to almost full recovery of hearing.

Example 13: Stability Data of Drug Product

The formulation of Example 4 (Table 1) was filled into 2.0 mL glass ampoules, stored at 5°C (r.H. not controlled) and 25°C/60% r.H, and thereafter tested on stability. The corresponding data are shown in Tables 9 and 10.

Overall, the stability of this formulation is excellent. Except for some decrease of the viscosity measured at 25°C, no obvious changes in quality can be seen. The decrease in viscosity when measured at 25°C is considered as not relevant for the appropriate functionality of the product.

Table 9: Stability Summary - Pioglitazone 1.2% Thermo-Reversible Suspension - 5°C (r.H. not controlled) Packaging Configuration: 2.0 ml Glass Ampoules

Legend: see below legend Table 10

Table 10: Stability Summary - Pioglitazone 1.2% Thermo-Reversible Suspension - 25°C / 60% r.H. Packaging Configuration: 2.0 ml Glass Ampoules

Legend:

1) Sampling Timepoint (months)

2) TYL = Turbid, whitish, suspension, liquid at < 25 °C, viscous gel at > 035°C

3) compl = complies

4) EP = Ph. Eur., current version, & corresponding chapter ) Parameters related to Pioglitazone hydrochloride) Release: 95 - 105%; Shelf-life: 90 -105%) n.d. = not detected

) n.t. = not tested