Isomers of phosphosulindac

Abstract

The invention features enantiomers of phosphosulindac, in particular (S)-phosphosulindac, and their compositions, methods of preparation and use in therapeutic methods.

Description

ISOMERS OF PHOSPHOSULINDACThis application claims the benefit of United States provisional application 63 / 601366, filed 21st November 2023, United States provisional application 63 / 601362, filed 21st November 2023, United States provisional application 63 / 650558, filed 22nd May 2024, as well as PCT application PCT / US2023 / 080646, filed 21st November 2023, and PCT application PCT / US2023 / 080649, filed 21st November 2023, the complete contents of which are incorporated herein by reference for all purposes.FIELD OF THE INVENTION

[0001] The invention relates to the enantiomers of phosphosulindac and compositions thereof, methods of preparing the enantiomers of phosphosulindac, and uses of the enantiomers of phosphosulindac in therapy, for example in dry eye disease.BACKGROUND OF THE INVENTION

[0002] Dry eye disease (DED) is a common disorder, affecting about 1 in 6 humans (15% of the population). In the world it is estimated there are over 600 million patients with moderate and severe DED. Although DED has a variety of unrelated pathogenic causes, all causes share as a common effect the breakdown of the ocular tear film, with dehydration of and subsequent damage to the exposed outer ocular surfaces.

[0003] Therapies for DED have included palliative agents (such as artificial tear formulations) and active agents (such as corticosteroids, retinoids, oral pilocarpine, cyclosporin and non-steroidal antiinflammatory drugs (NSAIDs)).

[0004] W02009 / 023631 discloses phosphosulindac but not optically active forms thereof. Phosphosulindac has efficacy in a dry eye disease rabbit model, see Examples 1-5 and 8 of WO2018 / 064354.

[0005] There remains a need for active agents with improved efficacy in the treatment of DED.SUMMARY OF THE INVENTION

[0006] Phosphosulindac contains a stereogenic sulfur atom as part of its sulfoxide group and therefore it is a chiral molecule. The inventor has surprisingly found that (5)-phosphosulindac, (7?)-phosphosulindac, and racemic phosphosulindac provide the same effect in a disease model of neuropathic pain associated with chemotherapy induced peripheral neuropathy upon topical administration (see Example 3 herein and Basu, A. et al., Front Neurosci (2024); 17:1240372). While this finding suggests that each enantiomer of phosphosulindac is therapeutically active, the lack of a differential effect observed for the enantiomers was surprising.

[0007] In view of these results, the inventor hypothesised that (S)-phosphosulindac and (R)-phosphosulindac would provide the same efficacy in other disease models. However, the inventor has further surprisingly found that (S’)-phosphosulindac is more efficacious than ( / ^-phosphosulindac in a dry eye disease model (see Example 4 herein). These data support that (5)-phosphosulindac would provide improved efficacy in the treatment of DED.

[0008] Therefore, in one aspect, the invention provides a compound having the following structure:-phosphosulindac)).

[0009] In an aspect, the invention provides a composition comprising (S)-phosphosulindac.

[0010] In an aspect, the invention provides a method of preparing enantio-enriched (S)-phosphosulindac by chromatography, the method comprising: a) separating a mixture comprising the enantiomers of phosphosulindac with a chiral stationary phase; and b) isolating enantio-enriched (S)-phosphosulindac.

[0011] In an aspect, the invention provides (5)-phosphosulindac for use in therapy. In an aspect, the invention provides (^-phosphosulindac for use in treating a disease described herein, e.g. dry eye disease or neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN). In an aspect, the invention provides a method of treating a disease described herein (e.g. dry eye disease or neuropathic pain associated with CIPN) in a patient in need thereof, the method comprising administering a therapeutically effective amount of (S)-phosphosul indac to the patient. In an aspect, the invention provides use of (5)-phosphosulindac for the manufacture of a medicament for treating a disease described herein.

[0012] Tire Examples described herein also identify (7?)-phosphosulindac as a compound useful in therapy. Therefore, in another aspect, the invention provides a compound having the following structure:-phosphosulindac))

[0013] In an aspect, the invention provides a composition comprising (A)-phosphosulindac.

[0014] In an aspect, the invention provides a method of preparing enantio-enriched (7?)-phosphosulindac by chromatography, the method comprising: a) separating a mixture comprising the enantiomers of phosphosulindac with a chiral stationary phase; and b) isolating enantio-enriched (7?)-phosphosulindac .

[0015] In an aspect, the invention provides ( / ?)-phosphosulindac for use in therapy. In an aspect, the invention provides ( / ?)-phosphosulindac for use in treating a disease described herein, e.g. dry eye disease or neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN). In an aspect, the invention provides a method of treating a disease described herein (e.g. dry eye disease or neuropathic pain associated with CIPN) in a patient in need thereof, the method comprising administering a therapeutically effective amount of (Z?)-phosphosulindac to the patient. In an aspect, the invention provides use of ( / ?)-phosphosul indac for the manufacture of a medicament for treating a disease described herein.

[0016] Furthermore, the inventor has surprisingly demonstrated that (S)-phosphosulindac is more efficacious than (7?)-phosphosulindac in treating neuropathic pain associated with CIPN, post- traumatic peripheral neuropathy (PTPN), and migraine pain when administered orally. Therefore, in a further aspect, the invention provides a method of treating a disease in a patient in need thereof, comprising administering a therapeutically effective amount of (S)-phosphosulindac to the patient, wherein the (5)-phosphosulindac is administered orally. Tire invention also provides (S)-phosphosulindac for use in therapy, wherein the (S)-phosphosulindac is administered orally.

[0017] In another aspect, the invention provides a method of treating and / or preventing neuropathic pain associated with CIPN in a patient in need thereof, the method comprising administering a therapeutically effective amount of (5)-phosphosulindac to the patient, wherein the (S)-phosphosulindac is administered orally. In another aspect, the invention provides a method of treating and / or preventing neuropathic pain associated with PTPN in a patient in need thereof, the method comprising administering a therapeutically effective amount of (S)-phosphosulindac to the patient, wherein the (5)-phosphosulindac is administered orally. In another aspect, the invention provides a method of treating and / or preventing migraine pain in a patient in need thereof, the method comprising administering a therapeutically effective amount of (5)-phosphosul indac to the patient, wherein the (S)-phosphosulindac is administered orally. In a further aspect, the invention provides a method of treating and / or preventing neuropathic pain associated with central sensitization in a patient in need thereof, the method comprising administering a therapeutically effective amount of (5)-phosphosulindac to the patient, wherein the (5)-phosphosul indac is administered orally.BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1A - A chromatogram of racemic phosphosulindac; x-axis is time (minutes), y-axis is mAU (milli-absorbance units). The top line is absorbance at 220 nm, the middle line is absorbance at 254 nm, and the bottom line is absorbance at 280 nm. Peak-1 has a % area of 49.833, and peak-2 has a % area of 50.167.

[0019] Figure IB - A chromatogram of an enantiomer of phosphosulindac, referred to as Peak 1 or Isomer 1; x-axis is time (minutes), y-axis is mAU. The lines show absorbance at 220 nm (top), 254 nm (middle), and 280 nm (bottom). The peak is at 1.91 minutes.

[0020] Figure 1C - A chromatogram of an enantiomer of phosphosulindac, referred to as Peak 2 or Isomer 2; x-axis is time (minutes), y-axis is mAU. The lines show absorbance at 220 nm (top), 254 nm (middle), and 280 nm (bottom). The peak is at 2.58 minutes.

[0021] Figure 2A - The experimental infrared (IR) and vibrational circular dichroism (VCD) spectra of Peak 2 and the calculated IR and VCD spectra for the compound of model system 1. In these plots the frequency scaling factor is not applied. The calculated VCD spectrum for the compound of model system 1 ( (7? (-stereochemistry) is shown bottom right on the figure. The VCD spectrum of the (S (-enantiomer of the compound of model system 1 (obtained by reversing the sign of the peaks calculated for the (R)-enantiomer) is overlaid. Figure 2F shows the calculated VCD spectrum for the compound of model system 1 ((R)-stereochemistry), see the top line in the upper frame, without the spectrum for the opposite enantiomer overlaid.

[0022] Figure 2B - The experimental IR and VCD spectra of Peak 2 and the calculated IR and VCD spectra for the compound of model system 1 . The calculated VCD spectrum for the compound of model system 2 ((Z?)-stereochemistry) is shown bottom right on the figure. The VCD spectrum of the t.S')-enantiomer of the compound of model system 2 (obtained by reversing the sign of the peaks calculated for the (R)-enantiomer) is overlaid. Figure 2G shows the calculated VCD spectrum for the compound of model system 2 ((R)-stereochemistry), see the top line in the upper frame, without the spectrum for the opposite enantiomer overlaid.

[0023] Figure 2C - IR (lower frame) and VCD (upper frame) spectra of Peak 2 in CDCh; 100pm path-length cell with BaF2 windows; 18 h collection for each enantiomer; instrument optimized at 1400 cm1. Solvent subtracted IR and enantiomer subtracted VCD spectra are shown. Uppermost trace is the VCD noise spectrum.

[0024] Figure 2D - IR (lower frame) and VCD (upper frame) spectra of Peak 1 in CDCh; 100pm path-length cell with BaF2 windows; 18 h collection for each enantiomer; instrument optimized at 1400 cm1. Solvent subtracted IR and enantiomer subtracted VCD spectra are shown. Uppermost trace is the VCD noise spectrum.

[0025] Figure 2E - Overlay of both enantiomers, Peak 2 and Peak 1. The IR are nearly identical as expected. The VCD are mirror images due to the half difference processing (El - E2) / 2.

[0026] Figure 2F - IR (lower frame) and VCD (upper frame) spectra observed for Peak 2 (left axes) compared with Boltzmann-averaged spectra of the calculated conformations for the ( / - sulfur) configuration of the compound of model system 1, (right axes). The top line in each frame (graph) is the calculated spectrum. The lower line in each frame is the experimentally obtained spectrum for Peak 2.

[0027] Figure 2G - IR (lower frame) and VCD (upper frame) spectra observed for Peak 2 (left axes) compared with Boltzmann-averaged spectra of the calculated conformations for the ( / Usulfur) configuration of the compound of model system 2, (right axes). The top line in each frame (graph) is the calculated spectrum, and the lower line is the experimentally obtained spectrum for Peak 2.

[0028] Figure 2H - The four lowest energy conformers (of 83 from Boltzmann average) for model system 1 ((R)-configuration).

[0029] Figure 21 - The four lowest energy conformers (of 10 from Boltzmann average) for model system 2 ((R)-configuration).

[0030] Figure 2J - Plot of EST (similarity of correct enantiomer minus incorrect enantiomer to calculated) vs. SNS (overall similarity of correct enantiomer to calculated) for a library of correct assignments verified independently by X-Ray other methods. The arrow to the indicated “X” is the data point for Peak 2 and Model System 1. Upper right corner is strongest assignment and lower left is weakest assignment.

[0031] Figure 3 - Effect of Isomer 1, Isomer 2, the racemate PS, and vehicle on neuropathic pain associated with chemotherapy induced peripheral neuropathy. Values: mean+SEM. *, p<0.005 vs. vehicle.

[0032] Figure 4 - Bar graphs for Corneal sensitivity (cm); Osmolarity (mOsm / Kg H2O); Tear Break Up Time (seconds); and Corneal fluorescence staining (score) for entries Con A, Vehicle, Isomer 1 and Isomer 2. D5 and D7 refer to day 5 and day 7 respectively.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0033] Tire term “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, treating, preventing, ameliorating and / or reducing symptoms of the disease. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art.

[0034] As used herein, the terms “treat,” “treatment,” and / or “treating” may refer to the management of a disease, disorder, or pathological condition, or symptom thereof with the intent to cure, ameliorate, stabilize, and / or control the disease, disorder, pathological condition or symptom thereof. Regarding control of the disease, disorder, or pathological condition more specifically, “control” may include the absence of condition progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g., placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the condition). The term “treating” includes prophylactic (e.g. preventative) and / or therapeutic treatments. In other words, term “treating” can include treating and / or preventing.

[0035] “Pharmaceutical compositions” and their preparations are well-known in the art. See, Tovey (ed, 2018) Pharmacal Formulation: The Science and Technology of Dosage Forms; 2018, Remington The Science and Practice of Pharmacy (23rd ed, 2020), Handbook of Pharmaceutical Excipients (eds. Sheskey, Cook & Cable; 8th edition 2016), all of which are incorporated by reference herein in their entirety.

[0036] “Pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients included in pharmaceutical compositions. The use of such pharmaceutically acceptable excipients for formulating active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable excipient is incompatible with PS, its use in the therapeutic compositions of the invention is contemplated.

[0037] Use of the term “about” when referring to a number is optional and can mean + / - 5%, + / - 2%, or + / - 1%.

[0038] Tire term “comprising” encompasses “including”, “consisting essentially of’ and “consisting”. For example, for chemical compounds or compositions, the use of "consisting essentially of" means that specific further components can be present, namely those not materially affecting the essential characteristics of the compound or composition.

[0039] “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space - i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1 : 1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(+)” can be used to designate a racemic mixture where appropriate. The absolute stereochemistry on an enantiomer is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral atom can be specified by either (R) or (.S'). Resolved compounds can also be designated (+) or (-) depending on the direction (dextro- or levo-rotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Dextrorotatory is positive or clockwise rotation and is designated (+). Levorotatory is negative or anticlockwise rotation and is designated (-).

[0040] The term “enantiomeric excess” (ee) is a measurement of purity used for chiral substances. It reflects the degree to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an ee of 0%, while a single completely pure enantiomer has an ee of 100%. A sample with 70% of one enantiomer and 30% of the other has an ee of 40% (70% - 30%). Tire term “enantiomeric ratio” (er) is another measurement of purity used for chiral substances. A racemic mixture has an er of 50:50, while a single completely pure enantiomer has an er of 100:0. A sample with 70% of one enantiomer and 30% of the other has an er of 70:30. The enantiomeric excess or ratio of a compound can be determined in a number of ways known in the art, including but not limited to chromatography using a chiral support, polarimetric measurement of the rotation of polarized light, nuclear magnetic resonance spectroscopy using chiral shift reagents which include but are not limited to lanthanide containing chiral complexes or Pirkle’s reagents, or derivatization of a compounds using a chiral compound such as Mosher’s acid followed by chromatography or nuclear magnetic resonance spectroscopy. In some embodiments, enantiomeric excess is determined by chromatography, for example supercritical fluid chromatography or high-pressure liquid chromatography.

[0041] A sulfoxide group can ben generally represented with the structural formula R-S(=O)-R', where R and R' are organic groups. A lone pair of electrons resides on the sulfur atom, giving it tetrahedral electron-pair geometry and trigonal pyramidal shape. The S=O bond in a sulfoxide group can be structurally displayed as a single S-O bond with a formal negative charge on the oxygen atom and a formal positive charge on the sulfur atom.

[0042] Tire following definitions of types of pain are according to the International Association for the Study of Pain (IASP). “Pain” is an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage. “Central sensitization” refers toincreased responsiveness of nociceptive neurons in the central nervous system to their normal or subthreshold afferent input. “Peripheral sensitization” refers to increased responsiveness and reduced threshold of nociceptive neurons in the periphery to the stimulation of their receptive fields. The precise etiology of central and peripheral sensitization differs between neuropathic pain and other forms of pain, such as inflammatory pain. “Allodynia” is pain due to a stimulus that does not normally provoke pain. “Hyperalgesia” is increased pain from a stimulus that normally provokes pain. Pain associated with central sensitization can be either generalized or in multiple locations in the body.

[0043] “Neuropathic pain” is caused by a lesion or disease of the somatosensory nervous system. Neuropathic pain is a clinical description (and not a diagnosis) which requires a demonstrable lesion or a disease that satisfies established neurological diagnostic criteria. Patients with neuropathic pain may experience one or more sensations described as heat, burning, throbbing, shooting, stabbing, sharpness, cramping, aching, tingling, numbness, or pins and needles. The term “lesion of the somatosensory nervous system” is commonly used when diagnostic investigations (e.g., imaging, neurophysiology, biopsies, lab tests) reveal an abnormality or when there was obvious trauma. The term “disease of the somatosensory nervous system” is commonly used when the underlying cause of the lesion is known (e.g., stroke, vasculitis, diabetes mellitus, genetic abnormality). “Peripheral neuropathic pain” is pain caused by a lesion or disease of the peripheral somatosensory nervous system. “Central neuropathic pain” is pain caused by a lesion or disease of the central somatosensory nervous system.

[0044] The following definitions of types of headache are according to the International Classification of Headache Disorders (ICHD) 3rd Edition (ICHD-3). Migraine has two major types: “migraine without aura”, a clinical syndrome characterized by headache with specific features and associated symptoms; and “migraine with aura”, primarily characterized by the transient focal neurological symptoms that usually precede or sometimes accompany the headache. “Migraine without aura” (i.e., common migraine; hemicrania simplex) is a recurrent headache disorder manifesting in attacks lasting 4-72 hours. The headache typically has a unilateral location, pulsating quality, moderate or severe intensity, aggravation by routine physical activity and association with nausea and / or photophobia and phonophobia. “Migraine with aura” (i.e., classic or classical migraine) involves recurrent attacks, lasting minutes, of unilateral fully reversible visual, sensory or other CNS symptoms that usually develop gradually and are usually followed by headache and associated migraine symptoms. “Episodic migraine” commonly involves about 1-2 migraine / headaches per month. “Chronic migraine” is a headache occurring on 15 or more days / month for more than three months, which, on at least 8 days / month, has features of migraine headache.Racemic phosphosulindac (PS)

[0045] Phosphosulindac is disclosed in W02009 / 023631 (see paragraph

[0123] ). Phosphosulindac may also be referred to as PS, PS-I, or OXT-328. A chemical name of phosphosulindac is: 4- ((diethoxyphosphoryl)oxy)butyl-(Z)-2-(5-fluoro-2-methyl-l-(4-(methylsulfinyl)benzylidene)-l / / - inden-3-yl)acetate. Phosphosulindac has the following structure. The ‘-C2H5’ moiety is an ethyl, i.e. a ‘-CH2CH3’, group.Enantiomers of PS

[0046] The enantiomers of PS have each been isolated in an enantiopure form and have had their absolute stereochemistry assigned - see Examples 1 and 2 described herein.

[0047] Therefore, in an aspect, the invention provides a compound having the following structure:

[0048] The above structure is the (S)-enantiomer of phosphosulindac, i.e. (5)-phosphosulindac. The Examples refer to (5)-phosphosulindac as peak 1 or isomer 1. An enantiomer of phosphosulindac can be defined without reference to its absolute stereochemistry. For example, an enantiomer of phosphosulindac has a retention time of about 1.8-2.0 minutes, for example about 1.91 minutes, measurable by supercritical fluid chromatography, for example using an amylose tris(3 ,5- dimethylphenylcarbamate) based chiral stationary phase, a mobile phase of about 40% methanol / CC) . a flow rate of about 3 mL / min and a pressure of about 100 bar, and optionally UV detection can be performed at about 220 nm.

[0049] In an additional aspect, the invention provides a compound having the following structure:

[0050] Tire above structure is the ( )-enantiomer of phosphosulindac, i.e. (A)-phosphosulindac. The Examples refer to ( )-phosphosulindac as peak 2 or isomer 2. An enantiomer of phosphosulindac canbe defined without reference to its absolute stereochemistry. For example, an enantiomer of phosphosulindac has a retention time of about 2.5-2.7 minutes, for example about 2.58 minutes, measurable by supercritical fluid chromatography, for example using an amylose tris(3,5- dimethylphenylcarbamate) based chiral stationary phase, a mobile phase of about 40% methanol / CCh, a flow rate of about 3 mL / min and a pressure of about 100 bar, optionally UV detection can be performed at about 220 nm.

[0051] (S)-Phosphosulindac and ( )-phosphosulindac are examples of compounds provided herein or compounds of the invention. An enantiomer of phosphosulindac can also be defined by the direction the enantiomer rotates plane polarised light, e.g. clockwise or anticlockwise. Therefore, an enantiomer of phosphosulindac is (+)-phosphosulindac. In addition, an enantiomer of phosphosulindac is (-)-phosphosulindac.Pharmaceutically acceptable forms

[0052] Tire compounds provided herein can be provided in a pharmaceutically acceptable form. Therefore, (S)-phosphosulindac can be in a pharmaceutically acceptable form. In addition, ( / / (-phosphosulindac can be in a pharmaceutically acceptable form. Examples of pharmaceutically acceptable forms include a solvate, derivative, and / or prodrug.

[0053] Solvate. As used herein, the term “solvate” refers to a compound that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. Therefore, the pharmaceutically acceptable form of (5)-phosphosulindac can be a solvate, such as a hydrate. In addition, the pharmaceutically acceptable form of ( / / (-phosphosulindac can be a solvate, such as a hydrate. The solvate can include at least 1 molecule of solvent. Alternatively, the solvate can include less than 1 molecule of solvent.

[0054] Isotopes. An isotopically labelled derivative is a compound that is identical to a compound provided herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Therefore, the pharmaceutically acceptable form of (S)-phosphosulindac can be an isotopically labelled derivative. In addition, the pharmaceutically acceptable form of (7?)-phosphosulindac can be an isotopically labelled derivative. The isotopically labelled derivative can include one or more isotopes of hydrogen, carbon, oxygen, phosphorus, sulfur, and fluorine. For example, the isotopically labelled derivative can include one or more isotopes of2H,3H,13C,14C,18O,170,31P,32P,35S, and18F, respectively. In particular, the isotopically labelled derivative can include one or more isotopes of2H (deuterium). In particular, the isotopically labelled derivative includes one or more isotopes of3H (tritium). In particular, the isotopically labelled derivative includes one or more isotopes of14C.

[0055] Derivatives and prodrugs. A derivative of phosphosulindac can include a tautomer such as enol or enolate. Therefore, the pharmaceutically acceptable form of (^-phosphosulindac can include a derivative of (5)-phosphosulindac such as a tautomer. In addition, the pharmaceutically acceptable form of ( / / (-phosphosulindac can include a derivative of ( / / (-phosphosulindac such as a tautomer. In some embodiments, the derivative is a metabolite. In some embodiments, the pharmaceutically acceptable form of (.S'j-phosphosulindac is a prodrug of (5)-phosphosulindac. In some embodiments, the pharmaceutically acceptable form of ( / / (-phosphosulindac is a prodrug of ( / (-phosphosulindac.

[0056] The activity of the compounds provided herein would be shared by their pharmaceutically acceptable forms.Compositions

[0057] Tire compounds provided herein can be included in a composition, for example a pharmaceutical composition. In an aspect, the invention provides a composition comprising (S)-phosphosulindac. In an additional aspect, the invention provides a composition comprising (7?)-phosphosulindac. The composition comprising the enantiomer of phosphosulindac may be enantio-enriched with respect to that enantiomer. For example, the composition may consist essentially of the enantiomer of phosphosulindac. This does not preclude the presence of other (non-phosphosulindac) ingredients in the composition. It may also be useful to define the ratio of the enantiomers of phosphosulindac present in the composition. For example, the enantiomeric excess of the enantiomer of phosphosulindac can be at least: 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or about 100%. Compositions which are highly enantio-enriched are preferred. Therefore, in some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is: at least 80%, 90%, 96% or 98%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 80%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 90%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 96%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 98%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 99%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is about 100%.

[0058] As mentioned above, the enantiomers of phosphosulindac are useful in therapy. Therefore, the composition provided herein can be a pharmaceutical composition. Pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a compound provided herein (e.g. (S)-phosphosulindac or I-phosphosulindac).

[0059] In some embodiments, the composition (e.g. the pharmaceutical composition) further comprises an excipient. When the composition is a pharmaceutically acceptable composition, the excipient is a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may comprise one or more: carriers, diluents, fillers, aqueous solutions, organic solvents, solubilizers and adjuvants.

[0060] Tire compounds provided herein are useful in treating diseases such as dry eye disease. In particular, (Sj-phosphosulindac is useful in treating dry eye disease. Thus, in some embodiments, the pharmaceutical compositions provided herein are formulated for topical administration. In particular, topical administration can be administration to the eye or a tissue surrounding the eye. In some embodiments, the tissue surrounding the eye is an eyelid.

[0061] The pharmaceutical compositions provided herein may be formulated for their route of administration, e.g. topical administration. In some embodiments, the pharmaceutical composition comprising the enantiomer of phosphosulindac (e.g. (Sj-phosphosulindac) may be formulated as a semi-solid or liquid. Therefore, the pharmaceutical composition comprising the enantiomer of phosphosulindac may be formulated as a solution, cream, gel (e.g., a hydrogel), lotion, ointment,foam, and / or spray. In some embodiments, the pharmaceutical composition comprising the enantiomer of phosphosulindac is formulated as a cream. In some embodiments, the pharmaceutical composition comprising the enantiomer of phosphosulindac Is formulated as a gel (e.g. a hydrogel). In some embodiments, the pharmaceutical composition comprising the enantiomer of phosphosulindac is formulated as an ointment. These compositions differ in their relative concentrations of oils and water, which causes the compositions to have different densities. Altering the density of the formulation is a way in which exposure of the affected area to the pharmaceutical composition can be controlled. For example, a less dense formulation, which requires rubbing in until it has been absorbed, may result in a shorter exposure time. Alternatively, a denser formulation, which is not readily absorbed, may allow prolonged exposure of the area to the pharmaceutical composition. The skilled person is aware of formulating topical pharmaceutical compositions so as to modify the relative exposure of the area to the active pharmaceutical ingredient.

[0062] Pharmaceutical compositions suitable for topical administration and appropriate pharmaceutically acceptable excipients are well-known in the art. Exemplary formulations for topical administration are provided in WO 2019 / 067919, which is hereby incorporated by reference in its entirety.

[0063] Tire pharmaceutical composition comprising the enantiomer of phosphosulindac (e.g. (S)-phosphosulindac), wherein the pharmaceutical composition is suitable for topical administration may comprise the enantiomer at a concentration of about 0.05% w / w to about 15% w / w of the pharmaceutical composition. Accordingly, the enantiomer of phosphosulindac may be at a concentration of about 15%. 14.5%, 14%, 13.5%, 13%, 12.5%, 12%, 11.5%, 11%, 10.5%, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%. 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1% or 0.05% w / w of the pharmaceutical composition. As an illustrative example, when formulated as a topical cream, the enantiomer of phosphosulindac may be at a concentration of less than or equal to about 8% w / w of the pharmaceutical composition, for example less than or equal to about 6% w / w of the pharmaceutical composition, in particular less than or equal to about 3% w / w of the pharmaceutical composition. As a further illustrative example, when formulated as a gel, the enantiomer of phosphosulindac may be at a concentration of less than or equal to 8% w / w of the pharmaceutical composition, for example less than or equal to 6% w / w of the pharmaceutical composition, in particular less than or equal to 3% w / w of the pharmaceutical composition, for example about 2% or about 1% w / w of the pharmaceutical composition. In particular formulations, for example when formulated as a hydrogel or an ointment, the enantiomer of phosphosulindac may be at a concentration of less than or equal to about 6% w / w of the pharmaceutical composition.

[0064] Tire pharmaceutical composition comprising the enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) may alternatively be formulated for any other form of administration suitable for treating dry eye disease. For example, the pharmaceutical composition may be formulated as eye drops.

[0065] The pharmaceutical composition comprising the enantiomer of phosphosulindac (e.g. (5)- phosphosulindac) may alternatively be formulated for parenteral administration, for example intravenously, intramuscularly, or subcutaneously. In particular' embodiments, the pharmaceuticalcomposition comprising the enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) is formulated for oral administration.

[0066] The enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) for oral administration may be formulated as a liquid or solid dosage form.

[0067] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.

[0068] Solid dosage forms for oral administration include but are not limited to capsules, tablets, pills, powders, and granules. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. The solid dosage forms of capsules, tablers and pills, may be such that they release the enantiomer of phosphosulindac only, or preferentially, in a certain part of the intestinal tract, for example the stomach, optionally, in a delayed manner.

[0069] In some embodiments, the formulation for oral administration comprises one or more fillers, disintigrants, lubricants, glidants, anti-adherents and / or anti-statics.

[0070] The formulations suitable for oral administration may comprise the enantiomer of phosphosulindac (e.g. (5)-phosphosulindac) at a concentration of 15%, 14.5%, 14%, 13.5%, 13%, 12.5%, 12%, 11.5%, 11%, 10.5%, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, or 0.5% w / w of the pharmaceutical composition.

[0071] Tire compositions or pharmaceutical compositions described herein may be sterile. Methods to sterilise a composition are known in the art, e.g. by steam or UV radiation.Methods of preparation / analysis

[0072] The enantiomers of PS can be prepared and / or analysed - see Example 1 described herein. Therefore, in an aspect, the invention provides a method of preparing enantio-enriched (S)-phosphosulindac, for example by chromatography. In an additional aspect, the invention provides a method of preparing enantio-enriched I-phosphosulindac, for example by chromatography. The method can comprise: a) separating a mixture comprising the enantiomers of phosphosulindac with a chiral stationary phase; and b) isolating the enantio-enriched enantiomer of phosphosulindac.

[0073] In an aspect, the invention provides a method of determining the enantiomeric excess of an enantiomer of phosphosulindac, for example by chromatography. The method can comprise: (a) separ ating a mixture comprising the enantiomers of phosphosulindac with a chiral stationary phase; and (b) determining the enantiomeric excess of an enantiomer of phosphosulindac. In some embodiments, the enantiomer is (5)-phosphosulindac. In some embodiments, the enantiomer is I-phosphosulindac.

[0074] In the methods described herein, the enantiomeric excess of the enantio-enriched enantiomer of phosphosulindac can be increased relative to the mixture. Therefore, the enantiomeric excess of the enantiomer of phosphosulindac can be at least: 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or about 100%. High enantio-enrichment is preferred. Therefore, the enantiomeric excess of the enantiomer of phosphosulindac can be at least: 80%, 90%, 96% or 98%.In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 80%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 90%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 96%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 98%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is at least 99%. In some embodiments, the enantiomeric excess of the enantiomer of phosphosulindac is about 100%.

[0075] Example 1 relies on supercritical fluid chromatography to separate the enantiomers of phosphosulindac. Therefore, in the methods provided herein, the chromatography can be supercritical fluid chromatography. The supercritical fluid chromatography methods described herein can use a mobile phase comprising methanol and CO2. For example, the mobile phase can be 25-45% w / w methanol and the remainder is CO2. The methods described herein can be performed at pressure of about 100 bar.

[0076] Methods described herein of preparing an enantio-enriched enantiomer of phosphosulindac (e.g. preparative methods) tend to utilise larger chiral stationary phases which may require increased flow rates of the mobile phase. Therefore, the supercritical fluid chromatography method of preparing an enantio-enriched enantiomer of phosphosulindac can be performed at a flow rate of 50- 70 mL / min, for example about 60 mL / min. A solution of the mixture comprising the enantiomers of phosphosulindac can be used to load the enantiomers on to the chiral stationary phase. In some embodiments, the injection volume is about 2 mL of about a 20 mg / mL solution of the mixture in a solvent, for example methanol.

[0077] The supercritical fluid chromatography method of determining the enantiomeric excess of an enantiomer of phosphosulindac (e.g. the analytical method) is performed at a flow rate of 2-4 mL / min, for example about 3 mL / min. This is because the chiral stationary phase may be smaller.

[0078] Instruments to detect a compound eluting from a stationary phase can aid their isolation. Therefore, the chromatography methods described herein may further comprise detecting the enantiomer with a UV detector. In some embodiments, the wavelength of the UV detector is in the range of about 200 nm to about 300 nm. In some embodiments, the wavelength of the UV detector is about 220, about 254 and / or about 280 nm.

[0079] In the methods provided herein, separation of each enantiomer can be achieved by a chiral stationary phase. For example, the chiral stationary phase can be silica gel coated with a polysaccharide. Polysaccharides are chiral compounds and therefore each enantiomer will typically have a different interaction with the polysaccharide coating, altering each enantiomer’s elution time and thus providing separation. The chiral stationary phase may be a amylose derivative coated on silica gel. In particular', the amylose derivative is amylose-tris(3,5-dimethylphenylcarbamate).

[0080] As shown in Example 1 , chromatographic methods can be used to prepare each enantiomer of phosphosulindac in an enantio-enriched form. Therefore, an enantiomer of phosphosulindac can be obtained by a method provided herein.Methods of treatment

[0081] Phosphosulindac is useful for various therapeutic applications. WO2018 / 064354 discloses that phosphosulindac is effective in a rabbit model of dry eye disease (e.g. see Examples 1-8).WO2022 / 251805 discloses that phosphosulindac is effective in the treatment of neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN).

[0082] Compounds provided herein (e.g. (^-phosphosulindac and (7?)-phosphosulindac) and compositions thereof are also surprisingly useful in therapy, such as in treating dry eye disease or neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN) - e.g. see Examples 3 and 4 described herein.

[0083] Therefore, in an aspect the invention provides an enantiomer of phosphosulindac (e.g. (5)- phosphosulindac) or a composition thereof for use in therapy. The invention also provides an enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) for use in a method of treating neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN). The invention also provides an enantiomer of phosphosulindac (e.g. (5)-phosphosulindac) for use in a method of treating dry eye disease. The invention also provides a composition comprising an enantiomer of phosphosulindac (e.g. ( / >)-phosphosulindac) for use in a method of treating a disease described herein (e.g. dry eye disease or neuropathic pain associated with CIPN). In some embodiments, the enantiomer of phosphosulindac is ( / ^-phosphosulindac. In some embodiments, the enantiomer of phosphosulindac is ( )-phosphosulindac.

[0084] The invention also provides a method of treating a disease in a patient in need thereof, the method comprising administering a therapeutically effective amount of an enantiomer of phosphosulindac (e.g. (5)-phosphosulindac) or a composition thereof to the patient. The invention also provides a method of treating dry eye disease in a patient in need thereof, the method comprising administering a therapeutically effective amount of an enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) to the patient. The invention also provides a method of treating neuropathic pain associated with CIPN in a patient in need thereof, the method comprising administering a therapeutically effective amount of an enantiomer of phosphosulindac (e.g. (5)-phosphosulindac) to the patient. The invention also provides a method of treating a disease described herein (e.g. dry eye disease or neuropathic pain associated with CIPN), the method comprising administering a therapeutically effective amount of a composition comprising an enantiomer of phosphosulindac (e.g. (ri)-phosphosulindac) to the patient. In some embodiments, the enantiomer of phosphosulindac is (5)-phosphosulindac. In some embodiments, the enantiomer of phosphosulindac is ( / ^-phosphosulindac.

[0085] The invention also provides use of an enantiomer of phosphosulindac (e.g. ( / ^-phosphosulindac) or composition thereof for the manufacture of a medicament for treating a disease. The invention also provides use of an enantiomer of phosphosulindac (e.g. (5)-phosphosulindac) for the manufacture of a medicament for treating dry eye disease. The invention also provides use of an enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) for the manufacture of a medicament for treating neuropathic pain associated with CIPN. The invention also provides use of a composition comprising an enantiomer of phosphosulindac (e.g. (S)-phosphosulindac) for the manufacture of a medicament for treating a disease described herein (e.g. dry eye disease or neuropathic pain associated with CIPN). In some embodiments, the enantiomer of phosphosulindac is ( / ^-phosphosulindac; in other embodiments, the enantiomer of phosphosulindac is ( / ?)-phosphosulindac.

[0086] In some embodiments, the disease to be treated (e.g., with (S)-phosphosulindac or a composition comprising (S)-phosphosulindac) is selected from one or more of the following: pain associated with central sensitization (e.g., inflammatory pain; neuropathic pain; fibromyalgia; chronic pain; chronic regional pain syndrome; rheumatoid arthritis; psoriatic arthritis; osteoarthritis; spondyloarthritis; lupus; temporomandibular disorders; and / or idiopathic low back pain); neuropathic pain associated with diabetic peripheral neuropathy (DPN); neuropathic pain associated with post- traumatic peripheral neuropathy (PTPN); neuropathic pain associated with post-herpetic neuropathy (PHN); migraine pain (or pain associated with other headache disorders); and corneal neuropathic pain. In some embodiments, the pain associated with central sensitization is not pain associated with central sensitization caused by chemotherapy-induced peripheral neuropathy (CIPN).

[0087] The pain may be a consequence of central sensitization resulting in allodynia (e.g. , mechanical allodynia) and / or hyperalgesia. The enantiomer of phosphosulindac (e.g., (S)- phosphosulindac) may reduce the neuronal signalling involved in the sensation of pain in a subject. The enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may reduce pain generated via central sensitization. In some instances, the reduction may be complete such that the pain generation is eliminated. Thus, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain signalling occurring centrally. For example, the enantiomer of phosphosulindac (e.g., (S)- phosphosulindac) may reduce pain generation from centrally located neurons, for example neurons in one or more areas of the brain implicated in pain generation and / or sensation, as disclosed herein.

[0088] In particular embodiments, the disease to be treated is neuropathic pain associated with CIPN. In particular embodiments, the disease to be treated is neuropathic pain associated with PTPN. In another preferred embodiment, the disease to be treated is migraine pain. In such embodiments, the enantiomer of phosphosulindac, in particular (S)-phosphosulindac, may be administered orally.

[0089] The neuropathic pain associated with CIPN may be a consequence of central sensitization resulting in allodynia (e.g., mechanical allodynia) and / or hyperalgesia. The enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may reduce the neuronal signalling involved in the sensation of pain in a subject undergoing or following chemotherapy. The enantiomer of phosphosulindac (e.g., ( j-phosphosulindac) may reduce pain generated via central sensitization. In some instances, the reduction may be complete such that the pain generation is eliminated. Thus, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain signalling occurring centrally. For example, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain generation from centrally located neurons, for example neurons in one or more areas of the brain implicated in pain generation and / or sensation, as disclosed herein.

[0090] The neuropathic pain associated with PTPN may be a consequence of central sensitization resulting in allodynia (e.g., mechanical allodynia) and / or hyperalgesia. The enantiomer of phosphosulindac (e.g., ( j-phosphosulindac) may reduce the neuronal signalling involved in the sensation of pain in a subject with PTPN. The enantiomer of phosphosulindac (e.g., (S)- phosphosulindac) may reduce pain generated via central sensitization. In some instances, the reduction may be complete such that the pain generation is eliminated. Thus, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain signalling occurring centrally. Forexample, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain generation from centrally located neurons, for example neurons in one or more areas of the brain implicated in pain generation and / or sensation, as disclosed herein.

[0091] The migraine may be episodic migraine. In certain instances, the migraine may be chronic migraine. The migraine may be migraine without aura or migraine with aura. The migraine with aura may be migraine with typical aura or migraine with brainstem aura. The migraine may be hemiplegic migraine (e.g., familial hemiplegic migraine or sporadic hemiplegic migraine); retinal migraine; chronic migraine; or probable migraine (with and without aura). Migraine pain may be a consequence of central sensitisation resulting in allodynia, for example cutaneous allodynia, and / or hyperalgesia. The enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may reduce the neuronal signalling involved in the sensation of pain in a subject with migraine. In some instances, the reduction may be complete such that the pain is eliminated. Furthermore, the enantiomer of phosphosulindac (e.g., (.ST phosphosulindac) may reduce pain generated via central sensitisation. The reduction may be complete such that the pain generation is eliminated. Accordingly, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain signalling occurring centrally. In some embodiments, the pain is allodynia, for example, cutaneous allodynia. The allodynia may be in response to mechanical and / or thermal stimuli. In addition, in some embodiments, the pain is hyperalgesia. The migraine pain may be neuropathic pain.

[0092] A patient with migraine can be diagnosed using the well-known ICHD-3 guidelines. Migraine pain can be measured on a visual analogue pain scale or using any other appropriate method in the art.

[0093] The methods described herein can comprise administering a therapeutically effective amount of the enantiomer of phosphosulindac to a patient. In such methods, the therapeutically effective amount of the enantiomer of phosphosulindac is typically not administered as racemic phosphosulindac .

[0094] In the treatment of the diseases described herein, the enantiomer of phosphosulindac can be administered topically, for example to the eye or a tissue surrounding the eye such as an eyelid. In some embodiments, the enantiomer of phosphosulindac is topically administered to the outer surface of one or more eyelids. The one or more eyelids may be one or both of the upper eyelids and / or one or both of the lower eyelids. The enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously.

[0095] In particular embodiments, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) is administered orally. In some embodiments, oral administration of the enantiomer of phosphosulindac (e.g., (.Sj-phosphosulindac) reduces pain signalling occurring in the brain. Upon oral administration, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may accumulate (via the vagus nerve) at therapeutically relevant levels in the primary somatosensory cortex, secondary somatosensory cortex, anterior cingulate cortex (ACC), prefrontal cortex (PFC), insular cortex, amygdala, thalamus, cerebellum, and periaqueductal gray matter (PAG). In particular embodiments, upon oral administration, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) accumulates at therapeutically relevant levels in the medulla and / or cerebellum. The enantiomer of phosphosulindac(e.g., (S')-phosphosulindac), for example upon oral administration, may reduce pain signalling in the somatosensory cortex, for example the primary somatosensory cortex. The orally administered enantiomer of phosphosulindac (e.g., (S’)-phosphosulindac) may reduce pain signalling in one or more of the following areas of the brain: primary somatosensory cortex, secondary somatosensory cortex, anterior cingulate cortex, prefrontal cortex, insular cortex, amygdala, thalamus, cerebellum, and periaqueductal gray matter. In particular- embodiments, the orally administered enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may reduce pain signalling in the medulla and / or cerebellum.

[0096] Tire enantiomer of phosphosulindac (e.g., (^ / -phosphosulindac) may be administered orally at dosage levels of about 0.01 mg / kg to about 100 mg / kg, about 0.05 mg / kg to about 50 mg / kg, or about 0.1 mg / kg to about 10 mg / kg of subject body weight. In particular embodiments, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may be administered at dosage levels of about 1 mg / kg to about 5 mg / kg, for example about 3 mg / kg of subject body weight.

[0097] The enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may be administered orally at a dosage of about 1 mg to about 2000 mg. In some embodiments, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may be administered orally at a dosage of about 100 mg to 1500 mg, for example about 200 mg to about 1000 mg. In some embodiments, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may be administered orally at a dosage of about 50 mg to about 400 mg, for example about 100 mg to about 350 mg, for example about 150 mg to about 300 mg, for example about 150mg to about 250 mg. In particular embodiments, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) is administered orally at a dosage of about 250 mg to about 300 mg, preferably about 250 mg. In some embodiments of multiple dosing, equal amount of the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may be administered in each dose. In other embodiments, a higher initial dose may be administered, followed by low maintenance doses.

[0098] In some embodiments, the enantiomer of phosphosulindac (e.g., (.Sj-phosphosulindac) may be administered orally once a day, or more frequently. For example, the enantiomer of phosphosulindac (e.g., (Sj-phosphosulindac) may be administered twice a day, three times a day, four times a day, or more often as necessary. In particular embodiments, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) may be administered orally two or three times a day.

[0099] In particular embodiments, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) is administered orally at a dosage of about 150 mg to about 200 mg twice a day. Accordingly, a subject may be administered the enantiomer of phosphosulindac (e.g., (5)-phosphosulindac) orally at a daily dosage of about 300 mg to about 400 mg.

[0100] In particular embodiments, the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) is administered orally at a dosage of about 250 mg to about 300 mg (for example, about 250 mg) two or three times a day. Accordingly, a subject may be administered the enantiomer of phosphosulindac (e.g., (.Sj-phosphosulindac), for example PS, orally at a daily dosage of about 500 mg to up to about 900 mg a day.

[0101] Tire administration of the enantiomer of phosphosulindac may continue as long as necessary. In some embodiments, the enantiomer of phosphosulindac is administered for more than 1, 2, 3, 4, 5,6, 7, 14, 28, 56, or 84 days. In some embodiments, the enantiomer of phosphosulindac is administered chronically on an ongoing basis for the treatment of chronic effects, for example for at least 3 months. In some embodiments, continuous dosing is achieved and maintained as long as necessary. In some embodiments, the enantiomer of phosphosulindac is administered intermittently according to the recurrence of the symptoms of a disease described herein (e.g. dry eye disease).

[0102] The administration of the enantiomer of phosphosulindac may be performed multiple times a day. In particular, the enantiomer of phosphosulindac may be administered one, two, three, four, five or six times daily, or as often as required in a 24 hour period.

[0103] The administration of the enantiomer of phosphosulindac may be performed multiple times a day and may continue as long as necessary in accordance with the paragraphs above.

[0104] A person having ordinary skill in the art understands that, in certain embodiments, dosages of such compounds may be adjusted depending upon the mammal to be treated. For example, the treatment of mice is described herein and such dosages may or may not be revised upon the administration of the enantiomer of phosphosulindac (e.g., (S)-phosphosulindac) to a human. However, a person having ordinary skill in the art may, if necessary, convert the dosages provided herein as set forth in Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005. A human equivalent dose (HED) may be determined from an animal dose, the animal dose may be multiplied by the following conversion factors, to provide units in mg / kg: mouse = 0.08, hamster = 0.13, rat = 0.16, ferret = 0.19, guinea pig = 0.22, rabbit = 0.32, dog = 0.54, monkey = 0.32, marmoset = 0.16, squirrel monkey = 0.19, baboon = 0.54, micro-pig = 0.73, and mini-pig = 0.95.

[0105] Tire patient in the methods described herein can be a mammal. In a preferred embodiment, the patient is a human.EXAMPLES

[0106] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.Example 1: The separation of the enantiomers of racemic phosphosulindac by SFC

[0107] Phosphosulindac is disclosed in W02009 / 023631 (paragraph

[0123] ). Phosphosulindac can be analysed by the following analytical method: employing a CHIRALPAK ® AD-H (25 x 0.46 cm) column as the chiral stationary phase, a mobile phase of 40% methanol / CCh (100 bar) at a flow rate of 3 mL / min with UV detection at 220, 254 and 280 nm. Figure 1A is a chromatograph of racemic phosphosulindac showing separation of the enantiomers. The CHIRALPAK ® AD-H column is manufactured by the Daicel Corporation (Japan) and is a chiral column containing silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate). Separation of each enantiomer is achieved with peaks having a retention time of 1.91 and 2.58 minutes.

[0108] The enantiomers of racemic phosphosulindac were separated by preparative supercritical fluid chromatography (SFC) using the following method: employing a CHIRALPAK ® AD-H (25 x 3 cm) column as the chiral stationary phase, a mobile phase of 30% methanol / CCh (100 bar) at a flow rate of 60 niL / min with UV detection at 220 nm. The injection volume was 2 mL (of a 20 mg / mL solution of racemic phosphosulindac in methanol). In a typical preparative SFC run, 7 g of phosphosulindac yielded about 3.6 g of isomer 1 (peak 1) and about 3.4 g of isomer 2 (peak 2). The slight difference between yield of the two isomers is likely due to residual solvent in the isomer 1 sample. Figure IB is a chromatograph of a sample of isomer 1 (an enantiomer) of phosphosulindac having a retention time of 1.91 minutes. Figure 1C is a chromatograph of a sample of isomer 2 (the other enantiomer) of phosphosulindac having retention time of 2.58 minutes. In Figures IB and 1C, none of the opposite enantiomer is detected in the chromatographs. Therefore, the preparative SFC method provides each enantiomer of phosphosulindac in an enantiomerically pure form.Example 2: Absolute configuration determination by vibrational circular dichroism (VCD)Methods

[0109] For an overview of the method, see Kellenbach et al. Spectroscopy Europe / World Vol. 19 Issue 4 (2007), pages 15-18. Absolute configuration determination by VCD involved the following steps. The experimental infrared (IR) and VCD spectra were measured for each enantiomer of phosphosulindac. IR and VCD spectra of the enantiomers of model systems 1 and 2 (structures below) were simulated using ab initio density functional theory (DFT) methods. The experimental spectra were then compared with the calculated spectra to see which enantiomer gave the best correlation the model system where the absolute configuration is known.Results:[HO] Table 1: Measurement parameters for experimentally obtained spectra:

[0111] Table 2: Calculation details for calculated spectra:

[0112] Table 3: Model systems for calculated spectra:

[0113] Table 4: Numerical comparison describing the similarity in the range of 945- 1900 cm'1between the calculated IR and VCD spectra for the (R - sulfur) enantiomer at the 6-31G(d) / B3PW91 w / CPCM (Chloroform) level for Model System 1 and the observed IR and VCD spectra for Peak 2:

[0114] ''Z: single VCD similarity, gives the similarity between the calculated and observed VCD spectra.bA: enatiomeric similarity index, gives the difference between the values of Z for both enantiomers of a given diastereoisomer.

[0115] The confidence level is a measure of the degree of congruence between a calculated and measured spectrum. If identical spectra are being compared the confidence level is 100%. The confidence level (CL) is not the likelihood that the assignment is correct, but is a measure of quality or degree of agreement between calculated and measured spectra. With a CL of 99% for this molecule, the visual agreement between measured and calculated spectra is excellent - this is a very high confidence assignment. The high flexibility of the molecule required the use of model systems to ensure a reasonable number of conformations for calculation. Two different systems were calculated, the first (model system 1) was closer in structure to the actual molecule with 610 initial calculated conformations and was limited to the small basis set 6-31G(d). The second model system (model system 2) was a more aggressive truncation and had only 10 conformers, this enabled the use of larger basis sets 6-31 lG(3df,2pd) and cc-pVTZ. These were reasonable approximations for VCDsince the chiral sulfoxide center is all the way at one end of the molecule — the atoms far removed from chirality will contribute very little to the VCD signal — particularly because they are separated by a long chain of methylene groups. Results from both model systems are presented and show excellent agreement with experimental data.

[0116] Figure 2 A shows the experimental and calculated IR and VCD results for Peak 2 and Model System 1. In particular, the calculated IR and VCD spectra are well predicted. The experimentally obtained VCD spectrum for Peak 2 agrees with the calculated VCD spectrum for the compound of model system 1 ((R)-configuration at the sulfur atom), supporting that the absolute stereochemistry of the sulfur atom in Peak 2 is ( / ?). In general, any graphs of overlaid (R) and (S) VCD spectrums can be deciphered by referring to the VCD spectrum for an individual enantiomer since the (R) and (S) VCD spectrums are approximate mirror images (e.g. see Figures 2C-E).

[0117] Figure 2B shows the shows the experimental and calculated IR and VCD results for Peak 2 and Model System 2. While the calculated IR spectrum is less well predicted because the phosphate ester stretches are missing, the calculated VCD spectrum is well predicted. The experimentally obtained VCD spectrum for Peak 2 agrees with the calculated VCD spectrum for the compound of model system 2 ((R)-configuration at the sulfur atom), supporting that the absolute stereochemistry of the sulfur atom Peak 2 is ( / ).

[0118] Figures 2C and 2D show the experimental IR and VCD spectra for Peaks 2 and 1, respectively. The VCD spectrum for Peak 1 appears to be the mirror image of the VCD spectrum for Peak 2. The VCD spectra for Peaks 1 and 2 are overlayed in Figure 2E (top frame), further demonstrating the mirror like symmetry.

[0119] Figure 2F compares the experimental and calculated IR and VCD spectra for Peak 2 and the compound of model system 1 t( / ))-cx>n figuration). The top graph is VCD spectra, and the top line is the calculated VCD spectrum and the bottom line is the experimental VCD spectrum. The sign and magnitude of the VCD spectra are in agreement, indicating that Peak 2 is the (R)-enantiomer of phosphosulindac. The bottom graph is IR spectra, and the top line is the calculated IR spectrum and the bottom line is the experimental IR spectra.

[0120] Figure 2G compares the experimental and calculated IR and VCD spectra for Peak 2 and the compound of model system 2 ((R))-configuration). The top graph is VCD spectra, and the top line is the calculated VCD spectrum and the bottom line is the experimental VCD spectrum. The sign and magnitude of the VCD spectra are in agreement, indicating that Peak 2 is the (R)-enantiomer of phosphosulindac. The bottom graph is IR spectra, and the top line is the calculated IR spectrum and the bottom line is the experimental IR spectra.

[0121] Figure 2H shows the calculated four lowest energy conformers of the compound of model system 1. Figure 21 shows the calculated four lowest energy conformers of the compound of model system 2. Figure 2J further indicates that there is strong assignment of the absolute stereochemistry of the enantiomers of phosphosulindac.Conclusions

[0122] The absolute configuration of peak 1 is (S') at the sulfur atom (confidence level: 99%). Therefore, isomer 1 (peak 1) is (5)-phosphosulindac. The absolute configuration of peak 2 is (7?) at the sulfur atom (confidence level: 99%). Therefore, isomer 2 (peak 2) is (R)-phosphosulindac.Example 3: The effect of topically administered racemic PS, and enantiomers thereof, in the treatment of neuropathic pain in a mouse model of CIPN

[0123] Examples 1-4 of WO2022 / 251805 support that PS can effectively treat and / or prevent neuropathic pain associated with chemotherapy induced neuropathy.

[0124] In this example, the enantiomers of PS were evaluated in the treatment of neuropathic pain in a mouse model of CIPN upon topical administration.Methods

[0125] Induction of CIPN: CIPN was induced in mice with paclitaxel using established protocols (Carozzi et al., Exp Neurol (2010); 226:301-309; Currie et al., PLoS Biol (2019); 17:e3000243; Eldridge et al., Toxicol Pathol (2020); 48:190-201).

[0126] Paclitaxel was dissolved in a mixture of 1 volume ethanol / 1 volume Cremophor EL / 18 volumes distilled water. Paclitaxel was administered as four intraperitoneal injections of 8 mg / kg paclitaxel (in a volume of 1 ml / 100 g body weight) every other day, resulting in a cumulative dose of 32 mg / kg.

[0127] Animals: Adult male C57BL / 6J mice, 8 weeks of age at the beginning of the experiments and weighing 20-30 g, were purchased from The Jackson Laboratory. Mice were housed in an AAALAC-accredited facility in groups of four. Food and water were available ad libitum. The mice in each cage were randomly allocated to treatment groups. All studies were conducted by experimenters blinded to the identity of the treatment groups. Experiments were performed during the light cycle (7 :00 am to 7 :00 pm) and animals were euthanized with CO2 asphyxiation. Studies were approved by the relevant Institutional Animal Care and Use Committee and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animal studies are reported in compliance with the ARRIVE guidelines.

[0128] Ointment: Racemic phosphosulindac, isomer 1 or isomer 2 were formulated as an 5% ointment for topical administration.

[0129] Protocol for the treatment of established neuropathic pain associated with CIPN: Once CIPN was established documented by reduced mechanical allodynia threshold, a 5% ointment of racemic PS, isomer 1 or isomer 2, or vehicle ointment was applied three times daily to the hind paws of the mice for 12 days. Mechanical allodynia was measured at the time points recorded in the figures (Le., day 0 and day 12).

[0130] Assessment of mechanical allodynia (von Frey test): Mechanical allodynia thresholds were determined using von Frey filaments according to an established method (Chaplan et al., J Neurosci Methods (1994); 53:55-63; Bagdas et al., Biochem Pharmacol (2015); 97:590-600). Briefly, mice were placed in a quiet room for 30 min and then were put in a Plexiglas cage with mesh metal flooring and allowed to acclimatise for 30 min before testing. A series of calibrated von Freyfilaments with incremental stiffness were applied perpendicularly to the paw with sufficient force to cause slight bending and held 2-3 s. This process was repeated at each level of stiffness 5 times, a few seconds apart. Paw withdrawn, licking or shaking were considered positive responses. The mechanical threshold, expressed as g, indicates the force of the von Frey filament to which the animal reacted.

[0131] Statistical analysis: Results are expressed as mean ± SEM. PK parameters were calculated by Microsoft Excel and PKSolver. Non-compartmental analyses were employed. Analysis of variance (ANOVA) tests were conducted and followed by the Bonferroni post hoc test. Differences were determined to be significant at P<0.05.Results

[0132] The effect of racemic PS, isomer 1 and isomer 2, compared to vehicle, in mice with neuropathic pain associated with paclitaxel was assessed. This reflects a clinical situation in which patients present with neuropathic pain after their chemotherapy is initiated or completed.

[0133] As shown in Figure 3, paclitaxel induced significant neuropathic pain, evidenced by changes in mechanical allodynia. Topical treatment with either enantiomer, or PS, as a 5% gel 3x / day for 12 days was started after the neuropathic pain was established.

[0134] Paclitaxel administered to the study groups greatly reduced their mechanical allodynia scores (average 56%; range: 51% - 61%) indicative of neuropathic pain associated with CIPN.

[0135] Treatment with either enantiomer for 12 days after the induction of CIPN increased the allodynia score to a similar extent: Isomer 1: day 0 = 1.13±0.14 g vs. day 12 = 1.64±0.09 g (p<0.005); and Isomer 2: day 0 = 0.96+0.07 g vs. day 12 = 1.56+0.18 g (p<0.001). The racemate PS 5% gel had a similar effect: day 0 = 1.04+0.09g vs. day 12 = 1.59+0.19 g (p<0.005). Each of these effects was statistically different from that of the corresponding vehicle value on day 12 = 0.88+0.08 g (p<0.005 to 0.001), demonstrating that the enantiomers when compared to racemate PS are equally efficacious against allodynia. These results are presented in Figure 3.

[0136] Safety of PS and enantiomers thereof. During all the studies, no topical or systemic side effects of PS or enantiomer ointment were observed when applied thrice daily to the hind paws of mice for the study duration. This finding is in keeping with the known safety profile of PS.Conclusions

[0137] The topical administration of racemic PS, isomer 1 and isomer 2 significantly improves the mechanical allodynia score compared to that induced by paclitaxel (i.e., CIPN). Therefore, racemic PS and both enantiomers of PS treat the neuropathic pain associated with CIPN. Consistent with the results in Examples 1-4 of WO2022 / 251805, PS and each enantiomer thereof demonstrate a pain- relieving effect in a treatment model of neuropathic pain associated with CIPN. However, it was unexpected that the effect of each enantiomer of PS and the racemate was identical in the model. These results indicate that each enantiomer of PS is at least useful in therapy. In particular, when the therapy is neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN).Example 4: The effect of each enantiomer of PS on corneal sensitivity and dry eye disease (DED)

[0138] PS is efficacious in a rabbit model of DED (see WO2018 / 064354). In this example, the efficacy of each enantiomer of PS on corneal sensitivity and DED parameters were determined in male Dutch-belted rabbits with DED induced by injecting Concanavalin A (Con A) into the periorbital lacrimal glands (see: Honkanen et al. Transl Res 2018;198:58-72).Methods

[0139] Induction of acute aqueous-deficient dry eye disease (DED) by Con A. Male Dutch-belted rabbits (DB) around 2kg were used in this study. Two weeks after removal of the nictitating membranes, we obtained the baseline values of tear breakup time (TBUT) and Schirmer Tear- Test (STT). Dry eye was induced the next day by injecting Con A dissolved in PBS into all lacrimal glands of the rabbits under ultrasound guidance with the rabbits under deep anesthesia with isoflurane. Tire palpebral (1,000 pg Con A, 0.2 mL) and orbital (500 pg Con A, 0.1 mL) portions of the superior lacrimal gland and the inferior lacrimal gland (1,000 pg Con A, 0.2 mL) were injected bilaterally. The success of the injection into the inferior lacrimal gland was confirmed by ultrasonography immediately after the injection. Repeat assessment of TBUT and STT was performed on day 5 after Con A injection, confirming the induction of dry eye. Each of these parameters was determined 16 h after the last dose of PS or Isomer 1 or Isomer 2.

[0140] The effect of each enantiomer of PS was assessed by measuring the following parameters attributable to DED: tear osmolarity, tear breakup time (TBUT) and corneal fluorescence staining (CFS). Tear osmolarity and TBUT reflect the quality of the tears, and CFS reflects the structural integrity of the ocular surface.

[0141] Tear breakup time (TBUT). Following topical anesthesia and the placement of a wire lid speculum, a 50 pL drop of 0.2% fluorescein was evenly distributed over the eye, and the precorneal tear film was observed under blue light with surgical loupes. The time taken to develop black dots, lines, or obvious disruption of the fluorescein film was measured up to 1 min. If the break up was not seen by 1 min, the observation was halted and TBUT was recorded as 60 s (even if it was actually longer).

[0142] Tear osmolarity (TOsm). Before application of any drops, the eyelids were mechanically blinked 5-10 times (gently) to better distribute the tear layer on the ocular surface. After gently retracting the lower lid, tears were sampled with a TearLab osmometer (TearLab Corporation, San Diego, Calif) at the junction of the palpebral and bulbar conjunctivas along the lower fornix just posterior to the base of the truncated nictitating membrane. Osmolarity was measured using the TearLab Osmolarity Test following the manufacturer's instructions.

[0143] Corneal fluorescein staining (CFS) evaluation. Fluorescein staining was performed by instilling 20 pL 2% fluorescein solution on the ocular surface. After removing excess fluorescein, the cornea and the superior bulbar- conjunctiva were photographed with a digital camera under blue light. The extent of fluorescein staining of the cornea and superior conjunctiva was scored using a modified NEI scoring method. The cornea was divided into five sections, with the superior conjunctiva regarded as one section. Each section was scored from 0 (absent) to 3 (severe) based on the amount, size, and punctate epithelial erosions.

[0144] Corneal sensitivity was measured with the Cochet-Bonnet Esthesiometer (Luneau, France) in a quiet examination room and without anesthesia. For esthesiometry, each rabbit was placed in a restraining bag 16 h after the last dose of PS and before any sedation. The nylon filament was applied to the central cornea, its most sensitive area. Corneal touch threshold testing started at the full filament length of 6 cm, and the filament was incrementally retracted by 0.5 cm until a positive response (full blink) was noted in 3-5 attempts. The length of filament simulating a positive response was the corneal sensitivity score.

[0145] Each enantiomer was formulated in a gel at a concentration of 0.2% and applied as eye drops topically to the surface of the eye. Rabbits were treated with a single eye drop of PS or vehicle applied to the surface of both eyes once daily for 5 days. Assays were performed at baseline (1 day prior to injecting Concanavalin A) and 5 and 7 days after the initiation of treatment with the exception of corneal fluorescence staining that was only assayed at day 5.Results

[0146] Table 5. Effect of PS isomers 1 and 2 on corneal sensitivity and DED parameters at day 5:

[0147] Table 6. The effect of PS isomers 1 and 2 on corneal sensitivity and DED parameters at day 7:

[0148] These data are also displayed in Figure 4.Discussion

[0149] Increased TBUT was observed for isomer 1 ((S)-phosphosulindac) and isomer 2 (( / ?)- phosphosulindac) relative to the ConA and Vehicle entries in Tables 5 and 6, indicating a therapeutic effect for each enantiomer in the treatment of DED. TBUT was greater for isomer 1 ((5)- phosphosulindac) suggesting this enantiomer is more efficacious in the treatment of DED. Similarly, each enantiomer of PS improved CFS, with the lowest score being observed with isomer 1 ((S1)- phosphosulindac). A differential effect of each enantiomer of PS on osmolarity was not observed, asexpected. An effect on comeal sensitivity was observed for isomer 1 ((S)-phosphosulindac) only - see Tables 5 and 6. This result suggests isomer 1 improves corneal sensitivity.Conclusions

[0150] Tire results in connection with corneal sensitivity, osmolarity, TBUT and CFS support that isomer 1 ((S)-phosphosulindac) is more efficacious than isomer 2 in the model. This supports that isomer 1 ((S)-phosphosulindac) may be more efficacious in the treatment of DED.

[0151] Observing a differential effect of isomers 1 and 2 in corneal sensitivity and dry eye disease related experiments (see Tables 5 and 6) is surprising because isomers 1 and 2 did not display a differential effect in a CIPN model (Example 3).Example 5: The effect of orally administered racemic PS, and enantiomers thereof, in the treatment of neuropathic pain in a mouse model of CIPN

[0152] In this example, the oral administration of enantiomers of PS was evaluated in the treatment of neuropathic pain in a mouse model of CIPN.Methods

[0153] CIPN was induced in mice with paclitaxel, in line with the experiments disclosed previously herein. Once CIPN was established, documented by reduced mechanical allodynia threshold, racemic phosphosulindac, isomer 1 ((S)-phosphosulindac) and isomer 2 (( )-phosphosulindac) were administered by oral gavage, once a day for three days. The dose of racemic PS was 50 mg / kg, the dose of isomer 2 was 50 mg / kg, and the dose of isomer 1 was 25 mg / kg or 50 mg / kg as indicated. For determining mechanical allodynia, pain threshold responses were measured after three days of treatment using the well-established method of von Frey filaments in line with that perfoimed in the earlier Examples.Results

[0154] Table 7. The effect of orally administered racemic PS, isomers 1 and 2 on CIPN:Discussion

[0155] A significant increase in PWT was observed for isomer 1 ((S)-phosphosulindac) at both doses compared to vehicle control and for isomer 2 ((R)-phosphosulindac) at the dose tested. Interestingly, isomer 1 showed a significant difference in recovery of PWT compared to isomer 2 at an equivalent dose (and even at the lower dose). This result demonstrates that both isomers are ableto treat pain associated with CIPN when given by oral administration, similar to the PS racemate, but that isomer 1 ((S)-phosphosulindac) appears to be more effective in this regard.Conclusions

[0156] Tire results in this example support the previous observations that the topical administration of either isomer of PS can treat neuropathic pain associated with CIPN. However, the observation that oral administration of isomer 1 ((S)-phosphosulindac) is more effective than isomer 2 ((R)- phosphosulindac) is surprising, given their similar effects after topical administration in a mouse model of CIPN. This supports that isomer 1 ((5)-phosphosulindac) may be more efficacious in the treatment of CIPN, when orally administered.Example 6: The effect of orally administered racemic PS, and enantiomers thereof in a mouse model of migraineMethods

[0157] The nitroglycerin (NTG)-induced migraine mouse model (Bates et al, 2010) is a particularly useful and clinically relevant animal model for migraine. NTG causes activation and sensitization of primary afferent and second-order trigeminovascular neurons. These pathophysiologies are thought to be the underlying mechanism of migraine headache (with the repeated activation of dural afferents believed to occur in patients with recurrent migraine headache), and many craniofacial nociceptive symptoms. NTG also mediates facial cutaneous and hind-paw hypersensitivity to non-noxious peripheral stimuli, indicative of allodynia, a manifestation of central sensitization. Accordingly, the ability of NTG to trigger central sensitization renders this model applicable for both migraine and other pain disorders associated with central sensitization more generally.

[0158] In this Example, the enantiomers of PS were evaluated in a mouse model of migraine.

[0159] Animals: Adult male C57BL / 6J mice, 8 weeks of age at the beginning of the experiments and weighing 20-30 g, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in an AAALAC-accredited facility in groups of four. Food and water were available ad libitum. Experiments were performed during the light cycle (7:00 am to 7:00 pm) and animals were euthanized with CO2 asphyxiation. The mice in each cage were randomly allocated to treatment groups. All studies were conducted by experimenters blinded to the identity of the treatment groups.

[0160] Studies were approved by the Institutional Animal Care and Use Committee of Stony Brook University and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al, 2010).

[0161] Induction of migraine: An NTG-induced migraine mouse model similar' to that described in Bates et al, 2010 was used. Specifically, instead of the intraperitoneal injection of NTG, a finely crushed 0.4 mg NTG tablet (Greenstone Brand) was administered sublingually. As a control, a Practi-Nitroglycerin Sublingual 0.4 mg Simulated Medication tablet was used. The mechanical nociceptive threshold was reduced as early as 30 minutes post NTG administration and persisted for about 24 hours.

[0162] Protocol for the treatment of established neuropathic pain associated with migraine: Once migraine was established, documented by reduced mechanical allodynia threshold, racemic phosphosulindac, isomer 1 ((S)-phosphosulindac) and isomer 2 ((R)-phosphosulindac) were administered orally 30 minutes after establishment of migraine. The dose of PS was 50 mg / kg, the dose of isomer 2 was 50 mg / kg, and the dose of isomer 1 was 25 mg / kg or 50 mg / kg as indicated.

[0163] Assessment of mechanical allodynia (von Frey test): For determining mechanical allodynia, pain threshold responses were measured using the well-established method of von Frey filaments in line with that performed in the earlier Examples. The PWT test, assessing mechanical allodynia, was performed 30 minutes after treatment. The mechanical threshold, expressed as g, indicates the force of the von Frey filament to which the animal reacted.

[0164] Statistical analysis: Results are expressed as mean ± SEM. Differences were determined to be significant at P<0.05.Results

[0165] Initially, we confirmed the NTG model of migraine followed by an assessment of the effect of the enantiomers of PS, in treating migraine pain. In the first study, we used 6 separate groups of mice, 3 for the 30 min time point and 3 for the 60 minute time point, as determination of mechanical allodynia on the same animals within 30 minutes may affect the results. As shown in the table below, the administration of NTG generates allodynia within 30 minutes of NTG administration, in line with the understanding that this compound rapidly triggers neuronal activity corresponding to central sensitization. For completeness, the oral NTG placebo control had no effect on PWT compared to baseline (z.e., did not generate allodynia) (see Table 8A).

[0166] Table 8A. Confirmation of NTG model:

[0167] Table 8B. The effect of orally administered racemic PS, isomers 1 and 2 on migraine pain:Discussion

[0168] The administration of NTG generates allodynia within 30 minutes, in line with the understanding that this compound rapidly triggers central sensitization. For completeness, the oral NTG placebo control had no effect on PWT compared to baseline (i.e., did not generate allodynia) (see Table 8 A).

[0169] As shown in Table 8B, A significant increase in PWT was observed for isomer 1 ((S)- phosphosulindac) at both doses compared to vehicle control. In contrast, isomer 2 ((R)- phosphosulindac) showed no significant difference compared to vehicle control. Indeed, isomer 1 ((S)-phosphosulindac) was significantly improved compared to isomer 2 ((R)-phosphosulindac) at both doses tested. This result suggests that isomer 1 ((S)-phosphosulindac) is distinguished in its ability to treat migraine pain upon oral administration.Conclusions

[0170] Tire results support that isomer 1 ((S)-phosphosulindac) is efficacious in this model, whilst isomer 2 is not. This demonstrates that isomer 1 ((S)-phosphosulindac) is effective in the treatment of migraine pain upon oral administration.Example 7 : The effect of orally administered racemic PS, and enantiomers thereof in treating neuropathic pain in a mouse model of PTPN

[0171] In light of the striking efficacy of orally administered isomer 1 ((S)-phosphosulindac) in the treatment of neuropathic pain associated with CIPN and migraine pain, the oral administration of enantiomers of PS was evaluated in the treatment of neuropathic pain in a mouse model of PTPN.Methods

[0172] Animals: Adult male C57BL / 6J mice, 8 weeks of age at the beginning of the experiments and weighing 20-30 g, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in an AAALAC-accredited facility in groups of four. Food and water were available ad libitum. Experiments were performed during the light cycle (7:00 am to 7:00 pm) and animals were euthanized with CO2 asphyxiation. The mice in each cage were randomly allocated to treatment groups. All studies were conducted by experimenters blinded to the identity of the treatment groups.

[0173] Studies were approved by the Institutional Animal Care and Use Committee of Stony Brook University and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al, 2010).

[0174] Induction of PTPN: Chronic constriction injury was exploited to induce neuropathic pain associated with PTPN. The neuropathic pain was induced in 8 weeks old male C57BL / 6J mice (~25 g) under anesthesia. The left sciatic nerve was exposed by a skin incision and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Two chronic gut ligatures with a 7-0 suture were tied loosely around the sciatic nerve 1 mm apart, to just occlude butnot arrest epineurial blood flow. For the control sham mice, a similar skin incision was made on the left side but without ligating the sciatic nerve. In either case, the surgical wound was closed with sutures in the muscle and staples in the skin. Pain hypersensitivity testing was performed after 5-7 days of recovery from surgery.

[0175] Protocol for the treatment of established neuropathic pain associated with PTPN: Once PTPN was established, documented by reduced mechanical allodynia threshold, racemic phosphosulindac, isomer 1 ((5)-phosphosulindac) and isomer 2 ((R)-phosphosulindac) were administered orally once a day for 10 days. The dose of PS was 50 mg / kg, the dose of isomer 2 was 50 mg / kg, and the dose of isomer 1 was 25 mg / kg.

[0176] Assessment of mechanical allodynia (von Frey test): For determining mechanical allodynia, pain threshold responses were measured using the well-established method of von Frey filaments in line with that performed in the earlier Examples. The PWT test, assessing mechanical allodynia, was performed 30 minutes after treatment. The mechanical threshold, expressed as g, indicates the force of the von Frey filament to which the animal reacted.

[0177] Statistical analysis: Results are expressed as mean + SEM. Differences were determined to be significant at P<0.05.Results

[0178] Table 9. The effect of orally administered racemic PS, and isomers 1 and 2 on pain associated with PTPN:Discussion

[0179] A significant increase in PWT was observed for both isomer 1 ((S)-phosphosulindac) and isomer 2 ((R) -phosphosulindac) at the dose tested. Interestingly, isomer 1 showed a similar, significant recovery of PWT compared to isomer 2 at a lower dose. This result demonstrates that both isomers are able to treat pain associated with PTPN when given by oral administration, similar to the PS racemate, but that isomer 1 ((S)-phosphosulindac) appears to be more effective in this regard.Conclusions

[0180] The results support that both isomer 1 ((S)-phosphosulindac) and isomer 2 are efficacious in this further model of challenging pain with central sites of action, when orally administered.However, in line with the oral administration data presented herein for CIPN and migraine pain, isomer 1 ((S)-phosphosulindac) is more efficacious in the treatment of PTPN, upon oral administration, further pointing to a broad applicability of this compound in the treatment ofchallenging pain indications. Without wishing to be bound by theory, the improved effects may be a consequence of an improved ability of S-phosphosulindac to access key sites of action in therapeutically relevant concentrations.Example 8: Comments on the therapeutic utility of the enantiomers of PS.

[0181] The results herein demonstrate the utility of the enantiomers of PS in the treatment of DED and corneal sensitivity. In particular, (S)-phosphosulindac is particularly effective in treating these indications.

[0182] Additionally, the results herein demonstrate an activity of the enantiomers of PS in the treatment of neuropathic pain indications and pain indications associated with central sensitization. This activity is mechanistically distinct from the ability to treat DED and improve corneal sensitivity. Indeed, treatment of the latter requires an increase in neuronal activity while the treatment of pain, especially neuropathic pain, requires an ability to reduce neuronal pain signalling, in particular that generated at central sites of action. Without wishing to be bound by theory, these observations suggest a bifunctionality of the enantiomers of PS on neuronal activity (similar to that observed previously for the PS racemate), for example depending on the pathology of or site of action involved in the specific indication.

[0183] The results herein with respect to the efficacy of the enantiomers of PS in treating neuropathic pain conditions are unprecedented. A demonstration of the efficacy of a compound in the treatment of challenging pain indications, including neuropathic pain, typically requires confirmation in a specific animal model of the neuropathic pain, as provided herein. However, given the wealth of data generated by the inventor demonstrating the efficacy of PS in multiple different animal models of neuropathic pain, along with observations that orally administered PS accumulates in regions of the brain implicated in pain signalling, the data presented herein regarding the efficacy of the enantiomers of PS in challenging pain indications suggests an equally broad applicability of the PS enantiomers in treating neuropathic pain indications as demonstrated for the PS racemate. Indeed, the results herein with respect to neuropathic pain are supported by earlier observations of the PS racemate in treating neuropathic pain associated with CIPN (see, WO2022 / 251805, which is incorporated by reference in its entirety). Furthermore, the PS racemate has been demonstrated to be effective in pain associated with diabetic peripheral neuropathy (DPN) (see WO2022 / 251806, which is incorporated by reference in its entirety). Additionally, the PS racemate has been shown to be therapeutically effective, via a direct action on neuronal signalling, in the treatment of neuropathic pain associated with post-traumatic peripheral neuropathy (PTPN), neuropathic pain associated with post-herpetic neuropathy (PHN), and corneal neuropathic pain (see the data and observations in WO2024 / 112725 and WO2024 / 112727, the contents of which are hereby incorporated by reference in their entirety). Accordingly, this combination of unprecedented observations permits an extrapolation of the therapeutic applicability of the enantiomers of PS in treatment of neuropathic pain indications, in particular S-phosphosulindac on the basis of observations herein.

[0184] Beyond the shared ability of the PS racemate and enantiomers in the treatment of conditions herein, the improved therapeutic activity of (5)-phosphosulindac is particularly striking and unexpected. Indeed, the improved activity of (Sf-phosphosulindac in the treatment of DED and re-establishment of corneal sensitivity is surprising in light of the equivalence of the PS enantiomers in other settings, for example upon topical administration in the C1PN model.

[0185] The improved therapeutic activity of ( )-phosphosulindac in particular pain indications upon oral administration is further striking. Indeed, (S)-phosphosulindac shows improved therapeutic efficacy in treating neuropathic pain associated with CIPN and PTPN, when administered orally, compared to ( )-phosphosulindac. Furthermore, (5)-phosphosulindac shows improved therapeutic effects in treating migraine pain, when administered orally, compared to (7?)-phosphosulindac. The migraine model used herein exploits NTG which is known to establish central sensitization. Therefore, the efficacy of (S)-phosphosulindac in this model of migraine pain suggests potential therapeutic activity of (S)-phosphosulindac in the treatment of pain associated with central sensitization.

[0186] The therapeutic efficacy of orally administered PS racemate in the challenging pain indications studied herein is in itself a surprise. Indeed, previous observations of the racemate of PS in the treatment of specific forms of neuropathic pain exploited topical administration, and fail to demonstrate the efficacy of the orally administered racemate of PS in treating such indications. With topical administration, PS is administered to areas with a high concentration of sensory neurons in the periphery, permitting its uptake and movement to central sites of action in high enough quantities to achieve an analgesic effect. Upon oral administration, PS is exposed to gut endothelial surfaces distinct to those of the skin epithelium exposed to PS upon topical administration, in terms of environment, structure and neuronal physiology and concentration. As PS is unstable in the blood, the oral administration would typically not be appropriate given the potential for the active compound to be destroyed either in the gut environment or in the bloodstream after absorption, and administering the concentrations of PS required to overcome these issues would have been considered to be inappropriate. Accordingly, it could not have been predicted that oral administration of PS would also achieve an analgesic effect for indications known to have a central site of pain generation.

[0187] Beyond the surprising activity of the PS racemate upon oral administration, the observations herein demonstrate that (S)-phosphosulindac is improved compared to (7?)-phosphosulindac in models of neuropathic pain associated with CIPN, PTPN, and migraine pain (linked to central sensitization), when (S)-phosphosulindac is administered orally. This is particularly surprising given the functional similarity of the enantiomers when topically administered in the CIPN model. Without wishing to be bound by theory, the evidence herein suggests that (S)-phosphosulindac may be more able to accumulate at key central sites of action involved in pain generation upon oral administration. Indeed, orally administered PS is shown to accumulate in regions of the brain involved in pain signalling and / or sensation, even upon oral administration (via traversing the vagus nerve from the stomach) (as provided in the data and observations in WO2024 / 112725 and WO2024Z 112727). Therefore, (S’)-phosphosulindac may reach key sites of pain generation in the brain in more therapeutically effective concentrations after oral administration compared to ( )-phosphosulindac, rendering i.S'j-phosphosulindac potentially more broadly applicable for distinct challenging pain indications upon oral administration as observed herein. Irrespective of the mechanism, the improved ability of (.S')-phosphosul indac after oral administration is unexpected.

[0188] It will be understood that the inventor’s work has been described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

Claims

CLAIMS1. A compound having the following structure:-phosphosulindac).

2. A composition comprising (.S'j-phosphosulindac.

3. The composition of claim 2, wherein the enantiomeric excess of (S)-phosphosulindac is at least: 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or 100%.

4. The composition of claim 2 or 3, wherein the enantiomeric excess of (5)-phosphosulindac is at least: 80%, 90%, 96% or 98%.

5. The composition of any of claims 2-4, wherein the enantiomeric excess of (S)-phosphosulindac is at least 96%.

6. The composition of any of claims 2-5, wherein the enantiomeric excess of (5)-phosphosulindac is at least 98%.

7. The composition of any of claims 2-6, wherein the composition is a pharmaceutical composition.

8. The pharmaceutical composition of claim 7, further comprising at least one excipient.

9. The pharmaceutical composition of claim 7 or 8, wherein the composition is formulated for topical administration.

10. The pharmaceutical composition of any of claims 7-9, wherein the composition is formulated as a solution, cream, gel (e.g., a hydrogel), lotion, ointment, foam, and / or spray.

11. The pharmaceutical composition of any of claims 7-10, wherein the concentration of t.S'J-phosphosulindac is about 0.05% w / w to about 15% w / w of the pharmaceutical composition.

12. A method of preparing enantio-enriched (i')-phosphosulindac by chromatography, the method comprising: a. separating a mixture comprising the enantiomers of phosphosulindac with a chiral stationary phase; and b. isolating enantio-enriched (S)-phosphosulindac.

13. The method of claim 12, wherein the enantiomeric excess of the isolated t.S')-phosphosLilindac is increased relative to the mixture.

14. The method of claim 12 or 13, wherein the enantiomeric excess of the isolated (5)-phosphosulindac is at least: 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or 100%.

15. The method of any of claims 12-14, wherein the enantiomeric excess of the isolated (5)- phosphosulindac is at least 96%16. The method of any of claims 12-15, wherein the enantiomeric excess of the isolated (S)- phosphosulindac is at least 98%17. The method of any of claims 12-16, wherein the chromatography is supercritical fluid chromatography.

18. The method of any of claims 12-17, wherein step b) further comprises detecting the enantiomer with a UV detector.

19. The method of any of claims 12-18, wherein the chiral stationary phase is an amylose derivative coated on silica gel, for example wherein the amylose derivative is amylose-tris(3,5- dimethylphenylcarbamate).

20. The method of any of claims 12-19, wherein the method uses a mobile phase comprising methanol and CO2.

21. The method of any of claims 12-20, wherein the enantiomeric excess is determined by chromatography, for example supercritical fluid chromatography.

22. (5)-Phosphosulindac obtainable by the method of any of claims 12-21.

23. (5)-Phosphosulindac for use in therapy.

24. (S)-Phosphosulindac for use in a method of treating dry eye disease.

25. (5)-Phosphosulindac for use in a method of treating neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN).

26. t.S'j-Phosphosulindac for use in a method of treating neuropathic pain associated with post-traumatic peripheral neuropathy (PTPN).

27. (5)-Phosphosulindac for use in a method of treating migraine pain.

28. A method of treating dry eye disease in a patient in need thereof, the method comprising administering a therapeutically effective amount of (S)-phosphosulindac to the patient.

29. A method of treating neuropathic pain associated with chemotherapy induced peripheral neuropathy in a patient in need thereof, the method comprising administering a therapeutically effective amount of (5)-phosphosulindac to the patient.

30. A method of treating neuropathic pain associated with post-traumatic peripheral neuropathy in a patient in need thereof, the method comprising administering a therapeutically effective amount of (S)-phosphosulindac to the patient.

31. A method of treating migraine pain in a patient in need thereof, the method comprising administering a therapeutically effective amount of (Sj-phosphosu lindac to the patient.

32. Use of (Sj-phosphosulindac for the manufacture of a medicament for treating dry eye disease.

33. Use of (Sj-phosphosulindac for the manufacture of a medicament for treating neuropathic pain associated with chemotherapy induced peripheral neuropathy (CIPN).

34. Use of (Sj-phosphosu lindac for the manufacture of a medicament for treating neuropathic pain associated with post-traumatic peripheral neuropathy.

35. Use of (Sj-phosphosulindac for the manufacture of a medicament for migraine pain.

36. (Sj-phosphosulindac for use according to claim 23-27, the method of claim 28-31, or the use of claim 32-35, wherein (Sj-phosphosulindac is administered topically.

37. (Sj-phosphosulindac for use according to claim 23, 24 or 36, the method of claim 28 or 36, or the use of claim 32 or 36, wherein (Sj-phosphosulindac is administered to the eye or a tissue surrounding the eye such as the eyelid.

38. (Sj-phosphosulindac for use according to claim 23-27, the method of claim 28-31, or the use of claim 32-35, wherein (Sj-phosphosulindac is administered orally.

39. The (Sj-phosphosulindac for use, method, or use of claim 38, wherein the (S)-phosphosulindac is formulated as a liquid or solid dosage form.

40. The (Sj-phosphosulindac for use, method, or use of claim 39, wherein the liquid dosage form is a pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup or and elixir.

41. The (Sj-phosphosulindac for use, method, or use of claim 39, wherein the solid dosage form is a capsule, tablet, pill, powder, or granule.

42. The (Sj-phosphosu lindac for use, method, or use of any one of claims 38-41, wherein the (S)- phosphosulindac is administered orally at dosage levels of about 0.01 mg / kg to about 100 mg / kg, from about 0.05 mg / kg to about 50 mg / kg, or from about 0.1 mg / kg to about 10 mg / kg of subject body weight, for example about 1 mg / kg to about 5 mg / kg, for example about 3 mg / kg of subject body weight.

43. The (Sj-phosphosulindac for use, method, or use of any one of claims 38-42, wherein the (Sj- phosphosulindac is administered orally at a dosage of about 1 mg to about 2000 mg, of about 100 mg to 1500 mg, of about 200 mg to about 100 mg, of about 50 mg to about 400 mg, for example about 100 mg to about 350 mg, for example about 150 mg to about 300 mg, for example about 150mg to about 250 mg.

44. The (.S')-phosphosLilindac for use, method, or use of any one of claims 38-43, wherein the (S)- phosphosulindac is administered orally at a dosage of about 250 mg to about 300 mg, preferably about 250 mg.

45. The (5)-phosphosulindac for use, method, or use of any one of claims 38-44, wherein the (S)- phosphosulindac is administered orally once a day.

46. The (Sf-phosphosulindac for use, method, or use of any one of claims 38-45, wherein the (S)- phosphosulindac is administered orally at least twice a day, at least three times a day, or at least four times a day.

47. The (5)-phosphosulindac for use, method, or use of any one of claims 38-45, wherein the (S)- phosphosulindac is administered orally two or three times a day.

48. The (Sf-phosphosulindac for use, method, or use of any one of claims 38-47, wherein the (S)- phosphosulindac is administered orally at a dosage of from about 150 mg to about 200 mg twice a day.

49. The (5)-phosphosulindac for use, method, or use of any one of claims 38-48, wherein the (S)- phosphosulindac is administered orally at a daily dosage of about 300 mg to about 400 mg.

50. The (5)-phosphosulindac for use, method, or use of any one of claims 38-47, wherein the (5)- phosphosulindac is administered orally at a daily dosage of about 250 mg to about 300 mg (for example, about 250 mg) two or three times a day.

51. The (5)-phosphosulindac for use, method, or use of any one of claims 38-47 or 50, wherein the (S)-phosphosulindac is administered orally at a daily dosage of about 500 mg to up to about 900 mg a day.

52. The (5)-phosphosulindac for use, method, or use of any one of claims 38-51, wherein the (S)- phosphosulindac is administered orally in a pharmaceutical composition.

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