WO2020180249A1

WO2020180249A1 - Therapy for dry eye (de) and meibomian gland dysfunction (mgd) based on the replacement of o-acyl-ω-hydroxy fatty acids (oahfa)

Info

Publication number: WO2020180249A1
Application number: PCT/SG2020/050102
Authority: WO
Inventors: Hak Tien Louis-Marie Grignion Tong; Aihua HOU; Richard David Webster; Sher Li GAN; Roderick Wayland Bates
Original assignee: Singapore Health Services Pte Ltd; Nanyang Technological University
Priority date: 2019-03-05
Filing date: 2020-03-05
Publication date: 2020-09-10

Abstract

The present invention relates to a process for preparing a fatty acid, a fatty acid obtainable by said process, a composition comprising said fatty acid, an eye drop comprising said fatty acid and a method for treating dry eye disease or Meibomian gland dysfunction using said fatty acid.

Description

THERAPY FOR DRY EYE (DE) AND MEIBOMIAN GLAND DYSFUNCTION (MGD) BASED ON THE REPLACEMENT OF O-ACYL-Q-HYDROXY FATTY ACIDS (OAHFA)

FIELD OF INVENTION

BACKGROUND OF INVENTION

The lipid layer of the human tear film is the outermost coating separating the eye from the environment. It is largely secreted by so-called Meibomian glands and generally consists of two main classes of lipid molecules: polar and nonpolar. Defects of this lipid layer are related to various eye conditions, in particular, the dry eye disease. One type of polar lipids, OAHFA, is suggested to play a vital role in maintaining the lipid layer stability.

The meibomian lipid profiles of dry eye disease (DED) patients indicate that (O-acyl)-omega- hydroxy fatty acids (OAHFA), a relatively new class of polar lipids discovered in the eye, display a distinct inverse relationship with DED severity. Furthermore, an appreciable increase in OAHFAs has been observed in the meibomian lipid profiles of dry eye patients upon eyelid warming treatment, alongside significant correlation with reduced evaporation rate and ocular discomfort. It is speculated that such observations may be due to the OAHFAs playing an essential role maintaining the structural integrity of the tear film based on its amphiphilic properties. Hence, a supplement of OAHFAs onto the ocular surface of the eye via topical application, may be feasible in bringing about an improvement in tear film stability and dry eye conditions.

Replacement of these naturally occurring OAHFAs is an improved and specific treatment approach compared to topical artificial tears and are expected to have minimal side effects when applied topically to the eye in appropriate amounts.

Reported synthesis of monoun saturated fatty acids generally involve classical methods that lead to stereoselective access to Z-alkenes such as the Wittig reaction, olefin metathesis, as well as transformation from alkynes via partial hydrogenation. With the exception of ring-closing metathesis, the lack of stereoselective access to Z-alkenes has been a shortcoming of olefin- metathesis reactions as most reactions mainly afford the thermodynamically stable E-alkene or a mixture of Z- and E- isomers with poor cis- selectivity. A lack of distinct difference in electronic properties of two alkene coupling substrates also more than often leads to a significant proportion of undesired homocoupling side products. Wittig reaction, though being a convenient and widely used method of synthesizing cis- olefins, requires stoichiometric amount of phosphine ylide and hence generates extensive waste due to high mass of phosphine oxide side product that may not be easily separated from the desired product.

Furthermore, adequately low temperatures are usually essential to attain c/s-selectivity which can be problematic in the industrial scale.

There is therefore a need for a process of preparing a fatty acid that at least partially ameliorates the shortcomings above.

SUMMARY

In an aspect, there is provided a process for preparing a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkene or optionally substituted alkyne; the process comprising the steps of: a) providing a compound having the following formula (I):

wherein n is an integer from 1 to 20; b) converting at least one of the hydroxyl groups of the compound of formula (I) to a terminal alkyne group; c) reacting the terminal alkyne group with a haloalkene having the following formula (II):

wherein X¹ is a halogen; and r is an integer from 1 to 20; d) contacting the compound of step c) with a compound having the following formula

wherein X² is a halogen; m is an integer from 1 to 20; and R¹ is an alkyl group; and e) reacting the compound of step d) with a fatty acid.

Advantageously, the process provides a facile, versatile and efficient way of producing a fatty acid, in particular OAHFA ((O-acyl)-omega-hydroxy fatty acids).

In another aspect, there is provided a fatty acid obtainable by the process as defined above.

In another aspect, there is provided a composition comprising a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

In another aspect, there is provided an eye drop comprising a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

In another aspect, there is provided a method for treating dry eye disease or Meibomian gland dysfunction, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. Advantageously, the fatty acid of formula (A) incorporates into the tear film lipid layers of the eye, in particular into the layers deficient of polar-lipids, and takes on the role of polar lipids. The fatty acid of formula (A) may therefore act by itself as the polar lipid component required for stabilization of the tear film lipid layer at the water-air interface.

In another aspect, there is provided a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, for use as a medicament.

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, for use in the treatment of dry eye disease or Meibomian gland dysfunction.

In another aspect, there is provided the use of a fatty acid having the following formula (A): wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, in the manufacture of a medicament for the treatment of dry eye disease or Meibomian gland dysfunction.

DEFINITIONS

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C -C alkyl, C -C alkyl, more preferably a C Ci₀ alkyl, most preferably Ci-C₆ unless otherwise noted. Examples of suitable straight and branched CrC₆ alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, f-butyl, hexyl, and the like. The group may be a terminal group or a bridging group.

"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-20 carbon atoms, preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be a terminal group or a bridging group.

"Alkynyl” as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having 2-20 carbon atoms, preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group.

Halogen" represents chlorine, fluorine, bromine or iodine. The term“optionally substituted” as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkylalkenyl, heterocycloalkyl, cycloalkylheteroalkyl, cycloalkyloxy, cycloalkenyloxy, cycloamino, halo, carboxyl, haloalkyl, haloalkenyl, haloalkynyl, alkynyloxy, heteroalkyl, heteroalkyloxy, hydroxyl, hydroxyalkyl, alkoxy, alkenyloxy, nitro, amino, alkylamino, dialkylamino, alkenylamine, aminoalkyl, alkynylamino, acyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxycarbonyl, alkyloxycycloalkyl, alkyloxyheteroaryl, alkyloxyheterocycloalkyl, acylamino, alkylsulfonyloxy, heterocyclic, heterocycloalkenyl, heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkenyl, heterocycloalkylheteroalkyl, heterocycloalkyloxy, heterocycloalkenyloxy, heterocycloamino, haloheterocycloalkyl, alkylsulfinyl, alkylsulfonyl, aminosulfonyl, sulfinyl, sulfinylamino, sulfonyl, sulfonylamino, aryl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroalkyl, heteroarylamino, heteroaryloxy, arylalkenyl, arylalkyl, aryloxy, arylsulfonyl, cyano, cyanate, isocyanate, -C(0)NH(alkyl), and -C(0)N(alkyl) .

It is understood that included in the family of disclosed compounds are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "F or "Z¹ configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.

Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and /or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the subject matter described and claimed.

Additionally, the disclosed compounds are intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1 % of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

DETAILED DESCRIPTION OF OPTIONAL EMBODIMENTS

This disclosure involves the total synthesis of OAHFA 18:1/34:1 and other similar fatty acids that are naturally present in tear films, which would subsequently be formulated into a novel eye drop containing an optimized concentration of OAFIFA and accompanying pharmaceutical excipients to make it suitable for topical application. OAHFA 18:1/34:1 eye drop is the first natural lipid eye drop, whereby the active pharmaceutical ingredient is a compound that is found endogenously in the eye. To address compliance issues and the purpose of serving as a supplement eye drop, a dosing regimen of twice a day is expected to be safe and sufficient to maintain increased levels of OAHFA in the eye relative to baseline levels. This would be largely dependent on the concentration of OAHFA in the eye drop as well as the vehicle that is selected to deliver the OAFHA on to the eye.

Amongst the range of species present in the OAHFA lipid class, which comprises of various carbon chain lengths and number of double bonds in both the fatty acid and omega-hydroxy fatty acid components (Table 1 ), OAHFA 18:1/34:1 was selected as the active pharmaceutical ingredient as it displayed a significant correlation with reduction of ocular symptoms and improved corneal staining after eyelid-warming treatment. Table 1. Summary of distinct OAHFA species detected.

Similar to that of OAHFA 18:1/34:1 , the total synthesis of the other naturally occurring OAHFAs listed in Table 1 would be focused on the extremely long-chain omega-hydroxy fatty acid, since palmitoleic acid (16:1 ), linoleic acid (18:2) and stearic acid (18:0) are commercially available.

A known total synthesis of OAHFA 18:1/34:1 (Fig. 1 ) requires a total of 19 steps with four convergent routes before arriving at the final product. This is mainly due to the necessary Cadiot-Chodkiewicz coupling, which requires prior synthesis of a terminal alkyne 4 and alkynyl bromide 9 as reaction substrates, and subsequent hydrogenation in order for the long saturated 24-carbon alkyl chain 11 to be obtained. However, this synthetic route might be deemed as a costly process if it were to be translated to an industrial scale, hence suggesting the need for a shorter synthetic route without compromising the stereoselectivity, yields, and ease of purification that are achievable with the proposed classical method.

With reference to Fig. 1 , a moderate yield was also observed in the classical alkylation of terminal alkyne 17 with alkyl halide 13, owing to the need for a strong base to pre-generate the metal acetylide. The carboxylic acid group present in 13, though protected as a tert- butyl ester, has a labile cr-proton that is easily deprotonated under highly basic conditions and hence leading to the formation of undesirable side products of significant yield. More robust protecting groups (eg. oxazoline) can be employed, but they often involve harsh conditions for the deprotection step which may lead to the hydrolysis of the ester bond in the OAHFA.

Pre-functionalisation steps that are essential for Cadiot-Chodkiewcz coupling could be eliminated by adopting a direct transition metal-catalyzed sp³-sp³ cross-coupling reaction. However, unlike cross-coupling reactions with reactive sp and sp²-hybridised species, the sp³- sp³ bond formation with unactivated alkyl halides is greatly hampered by deleterious side reactions such as slow oxidative addition of and facile /3-hydride elimination of the alkyl halide. These side reactions can possibly be circumvented by bulky, electron-rich phosphine ligands which was employed in the palladium-catalysed Suzuki alkyl-alkyl cross-coupling reaction. Pd(OAc)2/PCy₃, in the presence of K₃P0₄, was established by Fu and co-workers to be an efficient catalyst for the coupling of /3-hydrogen-bearing alkyl bromides with alkyl boranes under room temperature conditions, offering tolerance of a wide array of functional groups such as esters, alkynes, etc. Hence, the feasibility of a straightforward Fu-type Suzuki coupling was evaluated in the synthesis of OAHFA, which would bring about a significant improvement in atom and step economy of the synthetic route as illustrated in Fig. 2.

To avoid side reactions associated with the labile cr-proton adjacent to the carboxylic acid group, the internal alkyne was first introduced via alkylation of terminal alkyne 4 (Fig. 2) with a simple and more stable electrophile 8-bromooctene, resulting in a cleaner reaction with higher product yield. The alkyl borane coupling partner was then generated in situ by reacting it with 9- borabicyclo[3.3.1 ]nonane (9-BBN), which selectively hydroborates the terminal alkene in the presence of an internal alkyne. The alkyl borane intermediate was then subjected to Suzuki cross-coupling with 16-bromohexadecanoic acid (protected as a tert- butyl ester group) to obtain the 34-carbon chain. Formation of the cis- olefin was still based on partial hydrogenation of the internal alkyne, which afforded high stereoselectivity. The synthesis was then completed with esterification between the deprotected 34-carbon OHFA and oleic acid, and subsequent removal of the ferf-butyl ester protecting group to provide OAHFA 18:1/34:1 in a total of 10 steps.

However, challenges were faced in the Suzuki cross-coupling step with sluggish reactions of < 20% conversion obtained despite the use of higher catalyst loading (up to 20 mol%) and prolonged stirring for 48h at elevated temperatures. This could be attributed to 3 possible reasons: (1 ) low solubility of K₃P0₄ in the solvent (THF) which might hinder the transmetallation step of the catalytic cycle and consequently the progress of the reaction; (2) quenching of the alkyl-9-BBN derivative during the syringe-transfer process of the intermediate; (3) deactivation of the active catalyst Pd(PCy₃)2 to Pd(0) prior to completion of the reaction. (2) and (3) are likely due to exposure to oxygen as both the alkyl-9-BBN derivative and Pd(PCy₃)₂ are known to be air-sensitive. The use of a more soluble base KOH, though provided better conversion rates, was still unable to afford a complete reaction hence suggesting that oxygen could be the main culprit of sluggish reactions observed. Indeed, when the reaction was performed under a controlled glovebox environment of inert atmosphere with properly degassed solvents, 100% conversion was obtained with 10 mol% catalyst loading, affording a good yield of -80%. Hence, a more concise and efficient synthesis of OAHFA 18:1 /34:1 is proven to be achievable, albeit more stringent conditions required as compared to the classical method in Fig. 1 .

This disclosure further involves investigating the role of long-chain OAHFA in systems mimicking human tear film lipid layer employing a combination of experimental techniques with state-of-the-art computer simulations. It was shown that OAHFA incorporates in the lipid layer but has a tendency to promote inhomogeneities. Still, because of its specific localization in the film, OAHFA forms a stable sublayer allowing nonpolar lipids to be spread atop of water. The findings support the hypothesis that OAHFA can solely act as the polar lipid component needed to stabilization of lipid layer in the tear film.

There is provided a process for preparing a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; the process comprising the steps of: a) providing a compound having the following formula (I):

wherein n may be an integer from 1 to 20; b) converting at least one of the hydroxyl groups of the compound of formula (I) to a terminal alkyne group; c) reacting the terminal alkyne group with a haloalkene having the following formula (II):

wherein X¹ may be a halogen; and r may be an integer from 1 to 20; d) contacting the compound of step c) with a compound having the following formula (III):

wherein X² may be a halogen; m may be an integer from 1 to 20; and

R¹ may be an alkyl group; and e) reacting the compound of step d) with a fatty acid. m, n and r may be independently an integer from 1 to 20, from 1 to 2, 1 to 5, 1 to 10, 1 to 15, 2 to 5, 2 to 10, 2 to 15, 2 to 20, 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20 or 15 to 20. m, n and r may be independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20.

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. R’ may be an optionally substituted C1 to C20 alkyl, optionally substituted C2 to C20 alkenyl, or an optionally substituted C2 to C20 alkynyl. R’ may be a C1 to C20 alkyl, a C2 to C20 alkenyl, or a C2 to C20 alkynyl.

X¹ and X² may independently be a halogen, or fluorine, chlorine, bromine or iodine.

R¹ may be an alkyl group, methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, tert-butyl, 1 -pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl or active pentyl.

In step b), the compound of formula (I) may be reacted with a protecting group compound, whereby the protecting group compound after reacting with the compound of formula (I), may form a protecting group selected from the group consisting of tetrahydropyranyl ether, methyl ether, methoxymethyl ether, methylthomethyl ether, 2-methoxyethoxymethyl ether, 4- methoxytetrahydropyranyl ether, tetrahydrofuranyl ether, 1 -ethoxyethyl ether, 1 -methyl-1 - methoxyethyl ether, t- butyl ether, isopropyldimethylsilyl ether, f-butyldimethylsilyl ether, t- butyldiphenylsilyl ether, tribenzylsilyl ether and triisopropylsilyl ether.

In step b), the compound of formula (I) may be reacted with 3,4-dihydro-2/-/-pyran to form a compound having the following formula (IV): Other forms of protecting groups other than tetrahydropyranyl ether (OTHP) group may be methyl ether (OMe), methoxymethyl ether (OMOM), methylthiomethyl ether (OMTM), 2- methoxyethoxymethyl ether (OMEM), 4-methoxytetrahydropyranyl ether, tetrahydrofuranyl ether, 1 -ethoxyethyl ether, 1 -methyl-1 -methoxyethyl ether, f-butyl ether, isopropyldimethylsilyl ether, f-butyldimethylsilyl ether (OTBDMS), f-butyldiphenylsilyl ether, tribenzylsilyl ether, or triisopropylsilyl ether. The protecting groups may be stable against basic conditions and nucleophilic reagents.

The 3,4-dihydro-2/-/-pyran may be provided as solution in hexane in a range of about 3 % to 10% by volume, about 3% to about 5% by volume, about 3% to about 7% by volume, about 5% to about 7% by volume or about 7% to about 10% by volume.

The molar ratio of the compound of formula (I) to 3,4-dihydro-2/-/-pyran may be in the range of about 5:1 to about 5:2, about 5:1 to about 5:1 .5 or about 5:1 .5 to about 5:2.

The process may further comprise an aqueous acid selected from the group consisting of sodium hydrogen sulfate, potassium hydrogen sulfate, ammonium hydrogen sulfate, iron (III) sulfate and hydrochloric acid.

Other aqueous acid that may be employed include aqueous NH₄HS0₄, KHS0₄, Fe₂(S0₄)₃ and/or diluted hydrochloric acid (0.2 M).

The molar ratio of the compound of formula (I) to sodium hydrogen sulfate may be in the range of about 10:1 to about 10:2.5, about 10:1 to about 10:1.5, about 10:1 to about 10:2, about 10:1 .5 to about 10:2, about 10:1 .5 to about 10:2.5 or about 10:2 to about 10:2.5.

The reaction may be performed in a solvent of dimethylsulfoxide, toluene, hexane, aqueous acid and any mixture thereof. The solvent may be a biphasic mixture of hexane and aqueous sodium hydrogen sulfate, with a small amount of dimethyl sulfoxide (DMSO) to aid in the solubility of the 1 ,8-octanediol in the aqueous phase.

The reaction may be performed at a temperature in the range of about 30 °C to about 50 °C, about 30 °C to about 40 °C or about 40 °C to about 50 °C, for a duration in the range of about 12 hours to about 20 hours, about 12 hours to about 16 hours or about 16 hours to about 20 hours. The compound of formula (IV) may be reacted with a leaving group compound, whereby the leaving group compound after reacting with the compound of formula (IV), forms a leaving group selected from the group consisting of tosylate, mesylate, triflate, bromide and iodide.

The compound of formula (IV) may be reacted with 4-toluenesulfonyl chloride to from a compound having the following formula (V):

The molar ratio between the compound of formula (IV) and 4-toluenesulfonyl chloride may be in the range of about 10:9 to about 9:10, about 10:9 to about 1 :1 or about 1 :1 to about 9:10.

The process may further comprise triethylamine, 4-dimethylaminopyridine, pyridine and any mixture thereof.

The molar ratio of the compound formula (IV) and triethylamine may be in the range of about 1 :3 and about 2:3, about 1 :3 to about 1 :1 or about 1 :1 to about 2:3, and the molar ratio of the compound of formula (IV) and 4-dimethylaminopyridine may be in the range of about 10:9 to about 9:10, about 10:9 to about 1 :1 or about 1 :1 to about 9:10.

The reaction may be performed in a solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, pyridine and any mixture thereof.

The reaction may be performed at a temperature in the range of about 0 °C to about 28 °C, about 0 °C to about 10 °C, about 0 °C to about 20 °C, about 10 °C to about 20 °C, about 10 °C to about 28 °C or about 20 °C to about 28 °C, for a duration in the range of about 2 to about 4 hours, about 2 to about 3 hours, or about 3 hours to about 4 hours.

The compound of formula (V) may be reacted with a reagent selected from the group consisting of i) a lithium acetylide/ethylene diamine complex at a ratio in the range of 2:1 to about 1 :2, about 2:1 to about 1 :1 or about 1 :1 to about 1 :2, ii) a mixture of acetylene gas with elemental sodium or elemental lithium and liquid ammonia, or iii) a mixture of acetylene gas, n- butylllithium, tetrahydrofuran and dimethylsulfoxide to form a compound having the following formula (VI): The molar ratio of the compound of formula (V) to lithium acetylide/ethylene diamine complex may be in the range of about 1 :1 to about 7:10, about 1 :1 to about 7:8, about 1 :1 to about 7:9, about 7:8 to about 7:9, about 7:8 to about 7:10 or about 7:9 to about 7:10.

The reaction may be performed in a solvent of dimethylsulfoxide or hexamethylphosphoramide.

Polar DMSO or HMPT (hexamethylphosphoramide) may be present for reaction to proceed. Other solvents such as THF, diethyl ether and/or hexane may be present to assist in solvation of the starting material.

The reaction may be performed at a temperature in the range of about 0 °C to about 28 °C, about 0 °C to about 10 °C, about 0 °C to about 20 °C, about 10 °C to about 20 °C, about 10 °C to about 28 °C or about 20 °C to about 28 °C, for a duration in the range of about 10 minutes to about 60 minutes, about 10 minutes to about 30 minutes or about 30 minutes to about 60 minutes.

In step c), the terminal alkyne group may be reacted with an organolithium reagent before reacting with the haloalkene.

The organolithium reagent may be selected from the group consisting of n-butyllthium, sec- butyllithium, ferf-butyllithium, methyl lithium, phenyl lithium, lithium dialkyl amides, lithium diisopropylamide, bistrimethylsilylamide and their respective sodium or potassium salts.

The molar ratio between the terminal alkyne group and the n-butyllithium may be in the range of about 10:9 to about 9:10, 10:9 to about 1 :1 ; or 1 :1 to about 9:10.

The reaction may be performed in a solvent of tetrahydrofuran, diethyl ether or a mixture thereof.

The reaction may be performed at a temperature in the range of about -3 °C to about 5 °C. about -3 °C to about 0 °C or about 0 °C to about 5 °C, for a duration in the range of about 1 hour to about 3 hours, about 1 hour to about 2 hours or about 2 hours to about 3 hours.

In step c), the molar ratio between the terminal alkyne and haloalkene may be in the range of about 5:4 to about 5:8, about 5:4 to about 5:6 or about 5:6 to about 5:8. The terminal alkyne may be reacted with the haloalkene in the presence of N,N'- dimethylpropyleneurea, hexamethylphosphoramide or any mixture thereof.

The L/,L/'-dimethylpropyleneurea may be present at a range of about 40 % to 60 % by volume, about 40 % to about 50 % by volume or about 50 % to about 60 % by volume.

The process may be performed at a temperature in the range of about 0 °C to about 28 °C, about 0 °C to about 10 °C, about 0 °C to about 20 °C, about 10 °C to about 20 °C, about 10 °C to about 28 °C or about 20 °C to about 28 °C, for a duration in the range of about 12 hours to about 20 hours, about 12 hours to about 16 hours or about 16 hours to about 20 hours.

The compound of step c) may have the following formula (VII):

In step d), the compound of formula (III) may be formed by reacting a compound having the following formula (IIG) first with an activating reagent and second with a protecting reagent.

The activating reagent may be selected from the group consisting of trifluoroacetic anhydride, concentrated sulfuric acid, A/-(3-Dimethylaminopropyl)-/V -ethylcarbodiimide hydrochloride, 4- (dimethylamino)pyridine, triethylamine, 4-(dimethylamino)pyridine and any mixture thereof.

The activating agent may be trifluoroacetic anhydride, and the molar ratio between the compound of formula (III·) and trifluoroacetic anhydride may be in the range of about 3:10 to about 3:5, about 3:10 to about 3:7 or 3:7 to about 3:5, the reaction may be performed in tetrahydrofuran, or the reaction may be performed at a temperature in the range of about -3 °C to about 5 °C, about -3 °C to about 0 °C or about 0 °C to about 5 °C, for a duration in the range of about 20 minutes to about 40 minutes, about 20 minutes to about 30 minutes or about 30 minutes to about 40 minutes. The protecting reagent may be selected from the group consisting of fert-butyl alcohol, isobutylene, tert-butyl fluoromormate, A/,A/-dimethylamide, A/-7-nitroindoylamide, hydrazides. N- phenylhydrazides and L/,L/’-diisopropylhydrazide.

The protecting reagent may be tert- butyl alcohol and the fert-butyl alcohol may be provided in over 13 mole, 15 mole, 17 mole or 20 moles in excess of the compound of formula (IN’), and the reaction may be performed at a temperature in the range of about 20 °C to about 27 °C, about 20 °C to about 24 °C or about 24 °C to about 27 °C, for a duration in the range of about 1.5 hours to about 3.5 hours, 1 .5 hours to about 2.5 hours or about 2.5 hours to about 3.5 hours.

R¹ may be fert-butyl.

In step d), the contacting step may be performed in an inert atmosphere first in the presence of 9-borabicyclo[3.3.1 ]nonane, then additionally in the presence of palladium (II) acetate, tricyclohexylphosphine or triisopropylphosphine and potassium hydroxide or potassium phosphate.

The molar ratio between the compound of step c) to 9-borabicyclo[3.3.1]nonane may be in the range of about 9:10 to about 10:9, about 9:10 to about 1 :1 or about 1 :1 to about 10:9, the molar ratio between the compound of step c) and palladium (II) acetate may be in the range of 15:1 to about 10:1 , about 15:1 to about 12:1 or about 12:1 to about 10:1 , the molar ratio between the compound of step c) to tricyclohexylphosphine may be in the range of about 3:1 to about 12:1 , about 3:1 to about 6:1 , about 3:1 to about 9:1 , about 6:1 to about 9:1 , about 6:1 to about 12:1 or about 9:1 to about 12:1 , or the molar ratio between the compound of step c) and potassium hydroxide may be in the range of about 3:2 to about 3:4, about 3:2 to about 1 :1 or about 1 :1 to about 3:4.

The reaction may be performed in a solvent selected from the group consisting of tetrahydrofuran, dioxane, dimethoxyethane and any mixture thereof.

The reaction may be performed under inert atmosphere at a temperature in the range of about 20 °C to about 27 °C, about 20 °C to about 24 °C or about 24 °C to about 27 °C, for a duration in the range of about 2 hours to about 30 hours, about 2 hours to about 5 hours, about 2 hours to about 10 hours, about 2 hours to about 20 hours, about 5 hours to about 10 hours, about 5 hours to about 20 hours, about 5 hours to about 30 hours, about 10 hours to about 20 hours, about 10 hours to about 30 hours or about 20 hours to about 30 hours.

The compound of step d) may have the following formula (VIII): The compound of formula (VIII) may be deprotected of the tetrahydropryranyl acetyl protecting group to form a compound having the following formula (IX):

The deprotection may be done by reacting the compound of formula (VIII) with p-toluene sulfonic acid, pyridinium p-toluenesulfonate, a mixture of acetic acid, water and tetrahydrofuran, a mixture of Amberlyst H-15 and methanol or a mixture of iodine and methanol.

The pyridinium p-toluenesulfonate may be reacted with at least an 8 molar, 10 molar, 12 molar or 14 molar excess of the compound of formula (VIII).

The reaction may be performed in a solvent selected from the group consisting of methanol, ethanol, 2-propanol and any mixture thereof.

The reaction may be performed at a temperature in the range of 35 °C to about 60 °C, about 35 °C to about 40 °C, about 35 °C to about 50 °C, about 40 °C to about 50 °C, about 40 °C to about 60 °C or about 50 °C to about 60 °C, for a duration in the range of about 2 hours to about 5 hours, about 2 hours to about 3.5 hours or about 3.5 hours to about 5 hours.

The compound of formula (IX) may be reduced to form a compound having the following formula (X): The reduction may be performed in the presence of hydrogen gas, Lindlar’s catalyst and pyridine at a pressure of about 1 atm and a temperature in the range of about 20 °C to about 27 °C, about 20 °C to about 24 °C or about 24 °C to about 27 °C, for a duration in the range of about 45 minutes to about 1 .5 hours, about 45 minutes to about 1 hour, about 45 minutes to about 1 .25 hours, about 1 hour to about 1 .25 hours, about 1 hour to about 1.5 hours or about 1 .25 hours to about 1 .5 hours.

The process may be performed in a 1 :1 ethyl acetate-hexane solvent mixture, whereby the compound of formula (IX) is dissolved at a concentration in the range of about 0.02 M to about 0.05 M, about 0.02 M to about 0.03 M, about 0.02 M to about 0.04 M, about 0.03 M to about 0.04 M, about 0.03 M to about 0.05 M or about 0.04 M to about 0.05 M.

The Lindlar’s catalyst may be present in a range of about 15 wt% to about 20 wt%, about 15 wt% to about 17 wt% or about 17 wt% to about 20 wt %, with respect to the compound of formula (IX).

The pyridine may be present in a range of about 0.2 vol% to about 0.7 vol%, about 0.2 vol% to about 0.5 vol%, about 0.5 vol% to about 0.7 vol%, with respect to the total volume of solvent.

In step e), the fatty acid may be selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, capyrlyic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-Linolenic acid, arachidonic acid and eicosapentaenoic acid.

The fatty acid may be oleic acid.

The molar ratio between the compound of step d) and the fatty acid may be in the range of about 1 :1 to about 1 :2, about 1 :1 to about 1 :1 .5 or about 1 :1 .5 to about 1 :2.

The process may further comprise reagents selected from the group consisting of i) a mixture of /V-ethyl-A/-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, ii) a mixture of L/,L/'-Dicyclohexylcarbodiimide and -dimethylaminopyridine, iii) thionyl chloride or phosphoryl chloride followed by a mixture of alcohol, pyridine or triethylamine and 4- dimethylaminopyridine, and iii) a mixture of oxalyl chloride with a catalytic amount of dimethylformamide followed by a mixture of alcohol, pyridine or triethylamine and 4- dimethylaminopyridine.

The molar ratio between the compound of step d) and A/-Ethyl-A/-(3- dimethylaminopropyl)carbodiimide hydrochloride may be in the range of about 4:5 to about 2:5, or the molar ratio between the compound of step d) and 4-dimethylaminopyridine in the range of about 3:2 to about 1 :1 , about 3:2 to about 3:2.5, or about 3:2.5 to about 1 :1 .

The reaction may be performed in a solvent selected from the group consisting of dichloromethane, chloroform, acetonitrile and any mixture thereof

The reaction may be performed at a temperature in the range of about 0 °C to about 27 °C, about 0 °C to about 10 °C, about 0 °C to about 20 °C, about 10 °C to about 20 °C, about 10 °C to about 27 °C or about 20 °C to about 27 °C, and for a duration in the range of about 2 hours to about 5 hours, about 2 hours to about 3 hours, about 2 hours to about 4 hours, about 3 hours to about 4 hours, about 3 hours to about 5 hours or about 4 hours to about 5 hours.

The process may further comprise the step of replacing the R¹ group with hydrogen after step e)·

The R¹ group may be replaced with hydrogen by reacting with a reagent selected from the group consisting of trifluoroacetic acid, formic acid, a mixture of hydrochloric acid and acetic acid, a mixture of trimethylsilyl trifluoromethanesulfonate and triethylamine, and a mixture of CeCI₃-7H₂0 and sodium iodide.

The reagent may be trifluoroacetic acid and the trifluoroacetic acid may be present in molar excess.

The reaction may be performed in a solvent that may be dichloromethane, chloroform or a mixture thereof.

The reaction may be performed at a temperature in the range of about 20 °C to about 27 °C, about 20 °C to about 24 °C or about 24 °C to about 27 °C, for a duration in the range of about 20 minutes to about 40 minutes, about 20 minutes to about 30 minutes, or about 30 minutes to about 40 minutes.

The compound of formula (A) may be selected from the group consisting of the following formula (A1 ), (A2) and (A3): wherein p, q, r’, s and t may be independently integers in the range of 1 to 20. p, q, r\ s and t may be independently an integer from 1 to 20, from 1 to 2, 1 to 5, 1 to 10, 1 to 15, 2 to 5, 2 to 10, 2 to 15, 2 to 20, 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20 or 15 to 20. m, n and r may be independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20. In formula (A2), m may be 15, r may be 8, n may be 8, q may be 7 and r’ may be 7.

There is also provided a fatty acid obtainable by the process as defined above.

There is also provided a composition comprising a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

The fatty acid may be present at a concentration in the range of 0.5%w/v to about 2%w/v, about 0.5% w/v to about 1 % w/v, about 0.5 w/v% to about 1.5 w/v%, about 1% w/v to about 1 .5% w/v, about 1 % w/v to about 2% w/v or about 1 .5 w/v% to about 2% w/v.

The composition may be a pharmaceutical composition, comprising a suitable excipient.

The excipient may be selected from the group consisting of oil, water, solubilizers, surfactants, cosurfactants, antioxidants, preservatives, viscosity modifying agents, electrolytes and any mixture thereof.

The oil may comprise triglycerides of capric acid or capric acid, at a concentration in a range of about 0.4%w/v to about 1 .0%w/v, about 0.4% w/v to about 0.7% w/v, about 0.7% w/v to about 1% w/v.

The solubilizer may be glycerol, at a concentration in a range of about 2%w/v to about 3%w/v, about 2% w/v to about 2.5% w/v or about 2.5% w/v to about 3% w/v.

The surfactant may be selected from polyoxyethylene (20) sorbitan monolaurate at a concentration in a range of about 1 .5%w/v to about 2.5%w/v, about 1.5%w/v to about 2% w/v or about 2% w/v to about 2.5% w/v, polyethoxylated castor oil at a concentration in a range of about 0.5%w/v to about 2%w/v, about 0.5% w/v to about 1% w/v, about 0.5% w/v to about 1 .5 % w/v, about 1 % w/v to about 1 .5% w/v, about 1% w/v to about 2% w/v or about 1.5% w/v to about 2% w/v, or any mixture thereof.

The cosurfactant may be sorbitan monooleate at a concentration in a range of about 0.3%w/v to about 1 .0%w/v, about 0.3% w/v to about 0.5% w/v, about 0.3% w/v to about 0.7% w/v, about 0.5% w/v to about 0.7% w/v, about 0.5% w/v to about 1% w/v or about 0.7% w/v to about 1% w/v .

The composition may be an oil-in-water microemulsion or an oil-in-water nanoemulsion.

There is also provided an eye drop comprising a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

There is also provided a method for treating dry eye disease or Meibomian gland dysfunction, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

The fatty acid may be administered to the eye of a subject.

The fatty acid may be administered at a concentration in the range of about 0.5%w/v to about 1 .5%w/v, about 0.5% w/v to about 1 % w/v or about 1 % w/v to about 1 .5% w/v, at a volume in the range of about 30 mI_ to about 75 mI_, 30 mI_ to about 50 mI_, or about 50 mI_ to about 75 mI_.

The fatty acid may be administered twice a day at 12 hour intervals.

The fatty acid may be administered thrice a day at 8 hour intervals.

The fatty acid may be administered every 18 hours or every 24 hours.

The eye drop may be used for dry eye disease or any other forms of eye diseases that may destabilize the tears/tear film.

The dry eye disease may be allergic conjunctivitis, infective keratoconjunctivitis, or allergic conjunctivitis after infective keratoconjunctivitis.

There is also provided a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, for use as a medicament.

There is also provided a fatty acid having the following formula (A): wherein m, n and r may be independently an integer from 1 to 20; and

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, for use in the treatment of dry eye disease or Meibomian gland dysfunction.

There is also provided the use of a fatty acid having the following formula (A):

wherein m, n and r may be independently an integer from 1 to 20; and

R’ may be selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl in the manufacture of a medicament for the treatment of dry eye disease or Meibomian gland dysfunction.

BRIEF DESCRIPTION OF THE DRAWINGS

[Fig. 1 ] depicts a chemical synthetic scheme showing the total synthesis of OAHFA 18:1/34:1 (Prior Art) [Fig. 2] depicts a chemical synthetic scheme showing the 10-steps synthesis scheme of OAHFA 18:1/34:1

[Fig. 3] refers to (A) a photograph showing the appearance of 3 repeated batches of OAFIFA nanoemulsion and (B) TEM image of OAHFA nanoemulsion (x 80,000) that was negatively stained with 2% phosphotungstic acid.

[Fig. 4] refers to graphs showing (A) Droplet size, (B) PDI and (C) OAHFA concentration of the optimised nanoemulsions of OAHFA stored at 4 ± 2 °C and 25 ± 2 °C for 1 , 2, 3 and 6 months.

[Fig. 5] is a graph showing the concentration of OAHFA in tear fluid versus time plot which reflects the elimination profile of OAHFA in tears post instillation. The corrected concentration of OAHFA was calculated by subtracting the baseline concentration of OAHFA from the measured concentration of OAHFA at t hours.

[Fig. 6] refers to images of the eyes of rabbit A1 , where (A1 ) is the right eye under bright light at day 0, (A2) is the fluorescein-stained right eye under cobalt-blue light at day 0, (B1 ) is the left eye under bright light at day 0, (B2) is the fluorescein-stained left eye under cobalt-blue light at day 0, (C1 ) is the right eye under bright light at day 30, (C2) is the fluorescein-stained right eye under cobalt-blue light at day 30, (D1 ) is the left eye under bright light at day 30 and (D2) is the fluorescein-stained left eye under cobalt-blue light at day 30.

[Fig. 7] refers to images of the eyes of rabbit A2, where (A1 ) is the right eye under bright light at day 0, (A2) is the fluorescein-stained right eye under cobalt-blue light at day 0, (B1 ) is the left eye under bright light at day 0, (B2) is the fluorescein-stained left eye under cobalt-blue light at day 0, (C1 ) is the right eye under bright light at day 30, (C2) is the fluorescein-stained right eye under cobalt-blue light at day 30, (D1 ) is the left eye under bright light at day 30 and (D2) is the fluorescein-stained left eye under cobalt-blue light at day 30.

[Fig. 8] refers to images of the eyes of rabbit A3, where (A1 ) is the right eye under bright light at day 0, (A2) is the fluorescein-stained right eye under cobalt-blue light at day 0, (B1 ) is the left eye under bright light at day 0, (B2) is the fluorescein-stained left eye under cobalt-blue light at day 0, (C1 ) is the right eye under bright light at day 30, (C2) is the fluorescein-stained right eye under cobalt-blue light at day 30, (D1 ) is the left eye under bright light at day 30 and (D2) is the fluorescein-stained left eye under cobalt-blue light at day 30.

[Fig. 9] refers to images of the eyes of rabbit A4, where (A1 ) is the right eye under bright light at day 0, (A2) is the fluorescein-stained right eye under cobalt-blue light at day 0, (B1 ) is the left eye under bright light at day 0, (B2) is the fluorescein-stained left eye under cobalt-blue light at day 0, (C1 ) is the right eye under bright light at day 30, (C2) is the fluorescein-stained right eye under cobalt-blue light at day 30, (D1 ) is the left eye under bright light at day 30 and (D2) is the fluorescein-stained left eye under cobalt-blue light at day 30.

[Fig. 10] refers to optical coherence tomography images of the eyes of rabbit A1 , where (A) is the right eye at day 0, (B) is the left eye at day 0, (C) is the right eye at day 30 and (D) is the left eye at day 30.

[Fig. 1 1 ] refers to optical coherence tomography images of the eyes of rabbit A2, where (A) is the right eye at day 0, (B) is the left eye at day 0, (C) is the right eye at day 30 and (D) is the left eye at day 30.

[Fig. 12] refers to optical coherence tomography images of the eyes of rabbit A3, where (A) is the right eye at day 0, (B) is the left eye at day 0, (C) is the right eye at day 30 and (D) is the left eye at day 30.

[Fig. 13] refers to optical coherence tomography images of the eyes of rabbit A4, where (A) is the right eye at day 0, (B) is the left eye at day 0, (C) is the right eye at day 30 and (D) is the left eye at day 30.

[Fig. 14] refers to ring illuminator images of the eyes of the rabbits on day 30 of (A1 ) rabbit A1 right eye, (A2) rabbit A2 left eye, (B1 ) rabbit A2 right eye, (B2) rabbit A2 left eye, (C1 ) rabbit A3 right eye, (C2) rabbit A3 left eye, (D1 ) rabbit A4 right eye and (D2) rabbit A4 left eye.

[Fig. 15] refers to in-vivo confocal microscopy images on day 30 of the right eye of rabbit A1 , where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 16] refers to in-vivo confocal microscopy images on day 30 of the left eye of rabbit A1 , where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 17] refers to in-vivo confocal microscopy images on day 30 of the right eye of rabbit A2, where (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 18] refers to in-vivo confocal microscopy images on day 30 of the left eye of rabbit A2, where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 19] refers to in-vivo confocal microscopy images on day 30 of the right eye of rabbit A3, where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 20] refers to in-vivo confocal microscopy images on day 30 of the left eye of rabbit A3, where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 21] refers to in-vivo confocal microscopy images on day 30 of the right eye of rabbit A4, where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 22] refers to in-vivo confocal microscopy images on day 30 of the left eye of rabbit A4, where (A) indicates the superficial epithelium, (B) indicates the epithelial wing cell layer, (C) indicates the Bowman’s layer, (D) indicates the anterior stroma, (E) indicates the posterior stroma, and (F) indicates the endothelium.

[Fig. 23] refers to images showing the FI and E staining of the cornea of rabbit A2, wherein (A) is the right eye at 4 x magnification, (B) is the left eye at 4 x magnification, (C) is the right eye at 10 x magnification and (D) is the left eye at 10 x magnification.

[Fig. 24] refers to images showing the FI and E staining of the cornea of rabbit A3, wherein (A) is the right eye at 4 x magnification, (B) is the left eye at 4 x magnification, (C) is the right eye at 10 x magnification and (D) is the left eye at 10 x magnification.

[Fig. 25] refers to a graph of surface pressure against area per polar molecule isotherm obtained for a Langmuir film composed of POPC/OAFIFA mixed at different ratios. Measurements were performed at 34.2 °C. All MD simulations were performed at the value of the area/polar lipid indicated by the vertical line.

[Fig. 26] refers to representative wide-field images taken during the compression of a Langmuir film composed of POPC/OAFIFA at 8/2 ratio. A graph showing the surface pressure against area per polar molecule isotherm for this system is shown below the images. Measurements were performed at 34.2 °C. [Fig. 27] refers to representative wide-field images taken during compression of a surface film composed of POPC/OAHFA at a ratio of 5/5. A graph showing the surface pressure against area per polar molecule isotherm for this system is shown below the images. Measurements were performed at 34.2 °C.

[Fig. 28] refers to representative wide-field images taken during compression of a Langmuir film composed of POPC/OAFIFA at a ratio of 1/9. A graph showing the surface pressure against area per polar molecule isotherm for this system is shown below the images. Measurements were performed at 34.2 °C.

[Fig. 29] refers to representative wide-field images taken during compression of a Langmuir film composed of pure OAHFA. A graph showing the surface pressure against area per polar molecule isotherm for this system is shown below the images. Measurements were performed at 34.2 °C.

[Fig. 30] refers to a graph of Surface pressure against area per polar molecule isotherm obtained for a Langmuir film composed of POPC/OAFIFA mixed at different ratios and for the same systems in the presence of nonpolar TO. Measurements were performed at 34.2 °C. All MD simulations were performed at the value of the area/polar lipid indicated by the vertical line.

[Fig. 31] refers to representative wide-field images taken during the compression of a Langmuir film composed of POPC/OAFIFA at a 8/2 ratio with TO. A graph showing the surface pressure against area per polar molecule isotherm for the studied system is presented in the bottom right corner. Measurements were performed at 34.2 °C. Frames labelled as “a” correspond to images obtained with the nonpolar fluorescent probe, frames labelled as“b” correspond to the images obtained with the polar probe.

[Fig. 32] refers to representative wide-field images taken during compression of a Langmuir film composed of POPC/OAFIFA at a 5/5 ratio with TO. A graph showing the surface pressure against area per polar molecule isotherm for the studied system is presented in the bottom right corner. Measurements were performed at 34.2 °C. Frames labelled as

“a” correspond to images obtained with the nonpolar fluorescent probe, frames labelled as“b” correspond to the images obtained with the polar probe.

[Fig. 33] refers to representative wide-field images taken during compression of a Langmuir film composed of POPC/OAFIFA at a 1/9 ratio with TO. A graph showing the surface pressure against area per polar molecule isotherm for the studied system is presented in the bottom right corner. Measurements were performed at 34.2 °C. Frames labelled as “a” correspond to images obtained with the nonpolar fluorescent probe, frames labelled as“b” correspond to the images obtained with the polar probe. [Fig. 34] refers to representative wide-field images taken during compression of a Langmuir film composed of OAHFA and TO. A graph showing the surface pressure against area per polar molecule isotherm for the studied system is presented in the bottom right corner. Measurements were performed at 34.2 °C. Frames labelled as “a” correspond to images obtained with the nonpolar fluorescent probe, frames labelled as“b” correspond to the images obtained with the polar probe.

[Fig. 35] refers to density profiles of selected atoms in five tear film lipid layer (TFLL) model systems studied by molecular dynamics (MD) simulations: a) water, b) Polar Lipid Layer (PLL), c) TFLL, d) TFLL - 90% PLL, and e) TFLL - 90%. The density profiles quantify relative probability of finding specific atoms or groups of atoms in the system as a function of the distance along the normal to the interface.

[Fig. 36] refers to representative snapshots of five TFLL model systems studied by MD simulations: a) water, b) PLL, c) TFLL, d) TFLL - 90% PLL, and e) TFLL - 90%. Color coding: OAHFA molecules (silver), terminal polar group of OAHFA (dark blue), inner polar part of OAHFA (violet), POPC lipids with their P04 groups (light blue). Molecules of water subphase located below each lipid film are omitted in all snapshots for presentation purposes.

EXPERIMENTAL AND METHODS

Examplel : Synthesis Of OAHFA

Materials and Methods

The following chemicals were used as received: 15-pentadecanolide (98%, Alfa Aesar), hydriodic acid (57% wt. in H₂0, Sigma-Aldrich), dichloromethane (HPLC, VWR), sodium thiosulfate (99%, Alfa Aesar), sodium sulfate (anhydrous for analysis EMSURE® ACS, ISO, Reag. Ph Eur, Merck), tetrahydrofuran (ACS, Merck), trifluoroacetic anhydride (99+%, Alfa Aesar), f-butanol (99+%, Alfa Aesar), sodium hydrogen carbonate (for analysis EMSURE® ACS, Reag. Ph Eur, Merck), ethyl acetate (HPLC, VWR), lithium acetylide, ethylenediamine complex (tech. 90%, Alfa Aesar), dimethyl sulfoxide (anhydrous, ³99.9%, Sigma-Aldrich), 1 ,5- pentanediol (97%, Alfa Aesar), sodium hydride (60% w/w in mineral oil, Sigma-Aldrich), benzyl bromide (reagent grade, 98%, Sigma-Aldrich), diethyl ether (ACS, Merck), ammonium chloride (for analysis EMSURE® ACS, ISO, Reag. Ph Eur, Merck), triethylamine (99+%, Alfa Aesar), p- toluenesulfonyl chloride (reagent grade, ³98%, Sigma-Aldrich), 4-(dimethylamino)pyridine (ReagentPlus®, >99%, Sigma-Aldrich), acetone (AR grade, Fischer), silver nitrate (ACS, >99.9% (metals basis), Alfa Aesar), /V-bromosuccinimide (99%, Alfa Aesar), n-butylamine (99%, Alfa Aesar), copper(l) iodide (purum, ³99.5%, Sigma-Aldrich), hydroxylamine hydrochloride (99%, Alfa Aesar), palladium on carbon (10 wt. % loading, matrix activated carbon support, Sigma- Aldrich), pyridine (99+%, Alfa Aesar, p-Toluenesulfonyl chloride (98%, Alfa Aesar), sodium iodide (ACS, >99.5%, Sigma-Aldrich), 1 ,8-octanediol (98+%, Alfa Aesar), sodium hydrogen sulfate (90+%, Alfa Aesar), 3,4-dihydro-2H-pyran (99%, Alfa Aesar), hexane (HPLC, VWR), n- BuLi (1 .6 M in hexane, Sigma-Aldrich), L/,/V’-dimethylpropylene urea (DMPU) (98%, Alfa Aesar), ethanol (ACS, Merck), pyridinium p-toluenesulfonate (puriss., ³99.0%, Sigma-Aldrich), Lindlar’s catalyst (Sigma-Aldrich), oleic acid (>99% (GC), Sigma-Aldrich), A/-ethyl-/V -(3- dimethylaminopropyl)carbodiimide hydrochloride (>98%, TCI), trifluoroacetic acid (>99.0%, TCI), 16-hexadecanolide (97%, Sigma-Aldrich), 8-bromo-1 -octene (97%, Alfa Aesar), 9- borabicyclo[3.3.1 ]nonane dimer (Sigma-Aldrich), palladium(ll) acetate (>98.0%, TCI), potassium hydroxide (ACS AR Pellets, Fischer Scientific), tricyclohexylphosphine (> 94.0 %, Sigma- Aldrich).

All air and moisture sensitive reactions were performed under argon or nitrogen atmosphere using oven-dried glassware (120 °C) that were cooled under vacuum upon removal.

For anhydrous solvents, THF was dried by distillation from sodium metal and benzophenone prior to use under nitrogen. DMSO, DMPU and CH₂CI₂ were dried by distillation from CaH₂ prior to use under nitrogen atmosphere.

¹ FI NMR spectra were recorded using the Bruker Advance DPX at 400 or 500 MHz in deuterated solvents (chloroform-d, dichloromethane-d₂) and the same instruments recorded the ¹³C NMR spectra at 75, 100 or 125 MHz.

Flash chromatography on silica gel was performed with silica gel 230 - 400 mesh.

High Resolution Mass Spectroscopy (HRMS) was performed using Waters Q-Tof premier Mass Spectrometer.

Synthesis of 8-((tetrahydro-2H-pyran-2-yl)oxy)octan-1 -ol (2)

A mixture of 1 ,8-octanediol (1 ) (1 .46 g, 10 mmol), aqueous 5 M NaHS0₄ (2 ml_), 5:95 (vol/vol) dihydropyran-hexane (57.6 mL) and DMSO (2 ml.) was stirred at 40 °C for 16h. The reaction mixture was washed with H₂0 and extracted 3 times with hexane. The organic layer was separated from the aqueous layer, dried with brine and Na₂S0₄ and concentrated under reduced pressure. Purification was done with flash chromatography (40% ethyl acetate/hexane) to give a clear oil of 2 (82%).

¹H NMR (400 MHz, CDCI₃) d 4.56 (d, J = 4.1 Hz, 1 H), 3.94 - 3.80 (m, 1 H), 3.80 - 3.68 (m, 1 H), 3.63 (t, J = 6.2 Hz, 2H), 3.49 (dd, J = 10.9, 4.7 Hz, 1 H), 3.38 (dt, J = 9.5, 6.7 Hz, 1 H), 1.90 - 1 .76 (m, 1 H), 1.76 - 1.45 (m, 10H), 1.33 (m, 8H). ¹³C NMR (100 MHz, CDCI₃) d 98.86, 67.64, 63.02, 62.35, 32.75, 30.77, 29.70, 29.39, 29.32, 26.14, 25.66, 25.49, 19.68.

Synthesis of 8-((tetrahydro-2H-pyran-2-yl)oxy)octyl toluenesulfonate (2)

To 8-((tetrahydro-2/-/-pyran-2-yl)oxy)octan-1 -ol (2) (2.20 g, 9.55 mmol) in CH₂CI₂ (22 mL) was added triethylamine (1.42 mL, 19.1 mmol) followed by addition of TsCI (1 .79 g, 9.38 mmol) and DMAP (0.1 13 g, 9.25 mmol) at 0 °C. The solution was allowed to warm to room temperature and continued stirring. After 3h, the reaction mixture was diluted with CH₂CI₂ and the aqueous layer was extracted two times with CH₂CI₂. The organic layer was separated from the aqueous layer and dried with brine and Na₂S0₄. The solvent was then evaporated and purified by flash chromatography (10% ethyl acetate/hexane) to give 3 (89%).

¹H NMR (400 MHz, CDCI₃) d 7.79 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.56 (t, J = 3.5 Hz, 1 H), 4.01 (t, J = 6.5 Hz, 2H), 3.86 (ddd, J = 11 .0, 7.3, 3.4 Hz, 1 H), 3.71 (dt, J = 9.5, 6.9 Hz,

1 H), 3.49 (dd, J = 10.8, 5.1 Hz, 1 H), 3.36 (dt, J = 9.6, 6.6 Hz, 1 H), 2.45 (s, 3H), 1 .82 (dt, J = 9.8, 6.0 Hz, 1 H), 1.76 - 1 .43 (m, 9H), 1 .43 - 1 .12 (m, 8H). ¹³C NMR (100 MHz, CDCI₃) d 144.59, 133.25, 129.77, 127.86, 98.88, 70.63, 67.57, 62.38, 30.77, 29.65, 29.18, 28.83, 28.77, 26.06, 25.47, 25.25, 21.60, 19.71 .

Synthesis of 2-(dec-9-yn-1-yloxy)tetrahydro-2H-pyran (4)

To lithium acetylide, ethylenediamine complex (1.45 g, 16 mmol) was added anhydrous DMSO to form a ~2 M slurry-solution, which was stirred at room temperature for 15mins. 8- ((tetrahydro-2T/-pyran-2-yl)oxy)octyl toluenesulfonate (3) (4.47g, 13 mmol) in minimal volume of DMSO was then added dropwise at 0 °C. The reaction mixture was then warmed to room temperature and stirred for 1 h. 10 ml. of water was added dropwise at 0 °C to quench the reaction before it is poured into water and extracted three times with ethyl acetate. The organic layer was separated and washed with additional water to remove residual DMSO before it was dried with brine and Na₂S0₄. The solvent was then evaporated under reduced pressure and purified by flash chromatography (40% CH₂CI₂/hexane) to give a clear oil of 4 (85%).

¹H NMR (500 MHz, CDCI₃) d 4.62 - 4.54 (m, 1 H), 3.93 - 3.81 (m, 1 H), 3.73 (dt, J = 9.4, 6.9 Hz,

1 H), 3.58 - 3.44 (m, 1 H), 3.38 (dt, J = 9.5, 6.7 Hz, 1 H), 2.18 (td, J = 7.1 , 2.6 Hz, 2H), 1 .93 (t, J = 2.6 Hz, 1 H), 1 .83 (dd, J = 12.6, 6.8 Hz, 1 H), 1.78 - 1.67 (m, 1 H), 1 .67 - 1 .47 (m, 8H), 1.47 - 1 .26 (m, 8H). ¹³C NMR (100 MHz, CDCI₃) d 98.86, 84.75, 68.03, 67.64, 62.34, 30.79, 29.72, 29.31 , 29.02, 28.68, 28.46, 26.18, 25.51 , 19.70, 18.38.

Synthesis of f-butyl 16-bromohexadecanoate (6)

16-bromohexadecanoic acid (2) (0.091 g, 0.27 mmol) was dissolved in anhydrous THF (1 ml.) and cooled to 0 °C before trifluoroacetic anhydride (0.085 ml_, 0.60 mmol) was added slowly. After 30mins of stirring, 5 ml. of f-BuOH was added all at once and the mixture was stirred at room temperature for 2h. The reaction was then quenched with saturated NaHC0₃ solution and the aqueous layer was then extracted twice with CH₂CI₂. The organic layer was separated from the aqueous layer and dried with brine and Na₂S0₄. The solvent was then evaporated under reduced pressure and purified by flash chromatography (3% ethyl acetate/hexane) to give a clear oil of 3 (85%). ¹H NMR (400 MHz, CDCI₃) d 3.40 (t, J = 6.9 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 1 .90 - 1 .80 (quint, J= 7.1 Hz, 2H), 1 .63 - 1.51 (m, 2H), 1.47 - 1 .35 (m, 1 1 H), 1.26 (d, J = 8.4 Hz, 20H). ¹³C NMR (101 MHz, CDCI₃) d 173.49, 80.01 , 77.48, 77.16, 76.84, 35.79, 34.14, 33.00, 29.77, 29.68, 29.62, 29.58, 29.45, 29.25, 28.91 , 28.33, 28.28, 25.27.

Synthesis of 2-(octadec-17-en-9-yn-1-yloxy)tetrahydro-2H-pyran (7)

To alkyne solution of 2-(dec-9-yn-1 -yloxy)tetrahydro-2H-pyran (4) (0.1 19 g, 0.5 mmol) in 1 mL THF was added n-BuLi (1.6 M in hexane) (0.3 mL, 0.475 mmol) dropwise at 0 °C and the solution was stirred for 2 hrs. N,N-Dimethylpropylene urea (DMPU) (50 vol. %) was then added at 0 °C. The reaction mixture was stirred for another 10 mins before it was added dropwise to a solution of 8-bromooct-1 -ene (0.1 15 g, 0.6 mmol) in 3 mL THF at 0 °C, and slowly raised to room temperature and stirred for another 16 hours. It was quenched with H₂0 and the bulk of THF was removed under reduced pressure. The mixture was then washed with water to remove DMPU and extracted 3 times with Et₂0. It is then dried with brine and Na₂S0₄, and concentrated under reduced pressure. Purification was performed with flash chromatography (2% diethyl ether/hexane) to obtain a clear oil of the product 7 (83%), which solidified under room temperature.

¹H NMR (500 MHz, CDCI₃) d 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1 H), 5.04 - 4.88 (m, 2H), 4.62 - 4.51 (m, 1 H), 3.87 (ddd, J = 1 1 .1 , 7.5, 3.2 Hz, 1 H), 3.73 (dt, J = 9.6, 6.9 Hz, 1 H), 3.55 - 3.43 (m,

1 H), 3.38 (dt, J = 9.6, 6.7 Hz, 1 H), 2.19 - 2.09 (m, 4H), 2.04 (dd, J = 14.4, 6.9 Hz, 2H), 1.83 (dt, J = 1 1 .3, 6.7 Hz, 1 H), 1 .71 (ddd, J = 12.0, 6.0, 3.2 Hz, 1 H), 1.65 - 1.19 (m, 26H). ¹³C NMR (100 MHz, CDCI₃) d 139.12, 114.17, 98.85, 80.25, 80.18, 67.67, 62.34, 33.73, 30.79, 29.74, 29.38, 29.14, 29.09, 28.81 , 28.67, 28.62, 26.20, 25.50, 19.70, 18.74.

Synthesis of f-butyl 34-((tetrahydro-2F/-pyran-2-yl)oxy)tetratriacont-25-ynoate (8)

In a glovebox under Ar atmosphere, (0.24 mL, 0.12 mmol) of 0.5M 9-BBN solution was added to (42 mg, 0.12 mmol) of 7. The resulting solution was left to stir for 3 hours prior to the cross coupling reaction. Under the same environment, Pd(OAc)₂ (2.2 mg, 0.01 mmol), KOH (5.61 mg, 0.1 mmol), and PCy₃ (5.6 mg, 0.02 mmol) were weighed into a vial equipped with a stir bar, followed by addition of the pre-generated 9-BBN-alkyl borane solution and alkyl bromide 6 (38 mg, 0.1 mmol) in the mentioned sequence. The heterogeneous reaction mixture was then stirred vigorously for 24h under ambient temperature. After completion, the mixture was diluted with Et₂0 and filtered through a silica plug with repeated washing (Et₂0). The filtrate was concentrated under reduced pressure and purification was performed with flash chromatography (4% diethyl ether/hexane) to obtain a clear oil of the coupled product 8, which solidified under room temperature. ¹H NMR (400 MHz, CDCI₃) d 4.63 - 4.53 (m, 1 H), 3.87 (ddd, J = 1 1.1 , 7.4, 3.4 Hz, 1 H), 3.73 (dt, J = 9.6, 6.9 Hz, 1 H), 3.49 (dt, J = 5.1 , 4.5 Hz, 1 H), 3.38 (dt, J = 9.6, 6.7 Hz, 1 H), 2.19 (t, J = 7.5 Hz, 2H), 2.13 (dd, J = 9.6, 4.4 Hz, 4H), 1 .89 - 1 .77 (m, 1 H), 1 .71 (ddd,

8.7, 5.9, 3.2 Hz, 1 H), 1 .65 - 1 .19 (m, 67H). ¹³C NMR (100 MHz, CDCI₃) d 173.24, 98.77, 80.19, 80.10, 79.77, 67.60, 62.24, 35.58, 30.75, 29.71 , 29.66, 29.60, 29.56, 29.53, 29.44, 29.34, 29.26, 29.14, 29.1 1 , 29.06,

28.83, 28.76, 28.07, 26.17, 25.49, 25.08, 19.64, 18.71 .

Synthesis of f-butyl 34-hydroxytetratriacont-25-ynoate (9)

To a suspension of f-butyl 34-((tetrahydro-2H-pyran-2-yl)oxy)tetratriacont-25-ynoate (8) (0.18 g, 0.27 mmol) in EtOH (4 ml.) was added PPTS (0.0068 g, 0.027 mmol). The resulting mixture was stirred at 55 °C for 3h. EtOH was then removed under reduced pressure before it was diluted with Et₂0 and washed with water. The organic layer was dried with brine and Na₂S0₄, and concentrated under reduced pressure. Purification was performed with flash chromatography (30% diethyl ether/hexane) to obtain a white solid of 9 (88%).

¹H NMR (400 MHz, CDCI₃) d 3.62 (t, J = 6.6 Hz, 2H), 2.18 (t, J = 7.5 Hz, 2H), 2.12 (t, J = 6.6 Hz, 4H), 1.73 (s, 1 H), 1.61 - 1 .50 (m, 4H), 1.50 - 1 .19 (m, 59H). ¹³C NMR (100 MHz, CDCI₃) d 173.34, 80.26, 80.12, 79.85, 62.99, 35.62, 32.77, 29.68, 29.62, 29.58, 29.55, 29.46, 29.30, 29.28, 29.16, 29.13, 29.09, 29.08, 28.85, 28.76, 28.10, 25.69, 25.1 1 , 18.74, 18.73.

Synthesis of ferf-butyl (Z)-34-hydroxytetratriacont-25-enoate (10)

A slurry of Lindlar’s catalyst (20% wt., 0.115 g) in a mixture of 1 :1 (vol/vol) of ethyl acetate- hexane (6.45 ml_, 0.03 M with respect to 9), containing f-butyl 34-hydroxytetratriacont-25- ynoate (9) (0.577 g, 1 mmol) and pyridine (0.5% vol., 0.032 ml_), was vigorously stirred for 1 h under H₂ atmosphere (1 atm, balloon). The reaction filtered through celite and concentrated under reduced pressure. Purification was performed with flash chromatography (10% ethyl acetate/hexane) to obtain a white solid of 10 (91%, Z-isomer >99%).

¹H NMR (400 MHz, CDCI₃) d 5.40 - 5.25 (m, 2H), 3.62 (t, J = 6.6 Hz, 2H), 2.18 (t, J = 7.5 Hz, 2H), 2.07 - 1.92 (m, 4H), 1.55 (dt, J = 8.7, 6.7 Hz, 4H), 1.43 (s, 9H), 1 .39 - 1 .13 (m, 52H). ¹³C NMR (100 MHz, CDCI₃) d 173.50, 130.07, 129.93, 80.00, 63.13, 35.76, 32.93, 29.90, 29.88,

29.83, 29.79, 29.74, 29.70, 29.64, 29.62, 29.55, 29.45, 29.43, 29.36, 29.23, 28.24, 27.34, 27.32, 25.89, 25.25.

Synthesis of ferf-butyl (Z)-34-(oleoyloxy)tetratriacont-25-enoate (11)

To a stirred solution of oleic acid (0.05 ml_, 0.16 mmol) in 1 ml. anhydrous CH₂CI₂, was added ferf-butyl (Z)-34-hydroxytetratriacont-25-enoate (10) (0.07 g, 0.12 mmol) and DMAP (0.012 g, 0.098 mmol). The resulting suspension was stirred at RT for 10 mins. N-Ethyl-N-(3- dimethylaminopropyl)carbodiimide hydrochloride (0.0345 g, 0.18 mmol) was then added at 0 °C, stirred for 5 mins, followed by another 3h at room temperature. The solution was then washed with H₂0 and extracted 3 times with CH₂CI₂, dried over brine and Na₂S0₄ and evaporated under reduced pressure. Flash chromatography was performed (3% ethyl acetate/hexane) to obtain a pure clear oil of 21 (96%), which solidified at room temperature.

¹H NMR (500 MHz, CDCI₃) d 5.35 (d, J = 5.6 Hz, 4H), 4.05 (t, J = 6.7 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 2.01 (dd, J = 12.2, 6.2 Hz, 4H), 1 .69 - 1 .51 (m, 6H), 1 .43 (d, J = 7.5 Hz, 9H), 1 .39 - 1.20 (m, 74H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCI₃) d 174.13, 173.51 , 130.14, 129.93, 129.90, 80.01 , 64.54, 35.79, 34.55, 32.06, 29.92, 29.86, 29.77, 29.73, 29.68, 29.64, 29.57, 29.47, 29.38, 29.36, 29.33, 29.29, 29.26, 28.81 , 28.27, 27.37, 27.34, 27.32, 26.08, 25.28, 25.17, 22.83, 14.26.

Synthesis of (Z)-34-(oleoyloxy)tetratriacont-25-enoic acid (12)

To tert- butyl (Z)-34-(oleoyloxy)tetratriacont-25-enoate (11 ) (0.168 g, 0.2 mmol) in 0.6 mL CH2CI2 was added 0.4 mL (40% vol.) of trifluoroacetic acid, and the solution was stirred at room temperature for 30 mins. The volatiles were then removed under reduced pressure and flash chromatography was performed (15% diethyl ether/hexane) to obtained a white solid of 12 (93%).

¹H NMR (400 MHz, CDCI₃): d = 5.38-5.33 (m, 4H), 4.05 (t, J= 6.8 Hz, 2H), 2.34 (t, J= 7.6 Hz, 2H), 2.28 (t, J= 7.6 Hz, 2H), 2.01 (q, J= 6.2 Hz, 8H), 1.612 (quint., J= 7 Hz, 6H), 1.25-1.30 (m, 71 H), 0.88 (t, J= 6.6 Hz, 3H) ppm.

¹³C NMR (100 MHz, CDCI₃): d = 180.01 , 174.00, 130.44, 130.16, 129.74, 129.72, 64.40, 34.38, 34.08, 31.92, 29.78, 29.61 , 29.54, 29.45, 29.43, 29.33, 29.25, 29.24, 29.21 , 29.18, 29.14, 29.1 1 , 29.08, 28.66, 27.22, 27.19, 25.63, 25.01 , 22.69, 14.10 ppm.

HRMS (ESI): m/z [M + H]⁺ calculated for Ci₂H₂₃0₂: 787.7543; found: 787.7574.

Analytical Calculation for C₅2H9₈0₄: C, 79.33; H, 12.55. Found: C, 76.40; H, 12.56.

Example 2: OAHFA formulation design

OAHFAs are high molecular weight lipids characterised by a long carbon chain fatty acid esterified with an omega-hydroxy fatty acid containing a very long carbon chain. The presence of a terminal carboxylic acid group provides OAHFA with its amphiphilic and surfactant properties. Regardless, the largely hydrophobic straight-chain structure of the carbon chains causes the lipid to have poor aqueous solubility (<1 pg/mL). Hence, it is essential to design an alternative ophthalmic formulation with adequate concentration of OAHFA (1% w/v) for topical ocular administration as such cannot be attained with common aqueous vehicles.

Colloidal delivery systems such as microemulsions/nanomemulsions that generally consist of oil, water, surfactants and cosurfactants are well known for their simplicity in preparation, natural biodegradability, thermodynamic stability, nanometer droplet sized range and marked improvement in solubility of many lipophilic drugs. Furthermore, in vivo studies have shown potential sustained drug release of microemulsions/nanoemulsions which may increase the retention time of OAFIFA in the eye, allowing for lower frequency of application. Therefore, oil- in-water microemulsion/nanoemulsion is a promising vehicle of OAFIFA to achieve its desired concentration delivered onto the ocular surface.

An oil-in-water nanoemulsion of OAHFA made up of Miglyol 812, Polysorbate 20, Kolliphor EL, Span 80 and glycerin as inactive ingredients, has been formed to attain a desirable droplet size and PDI value with the respective compositions listed in Table 2. The selected excipients are proven to pose minimal or no harm to the ocular surface when used at appropriate concentrations and hence are safe for medical application. Detailed chemical structures of the inactive ingredients are shown in Table 3.

Table 2. Ingredients and their respective composition of OAHFA formulation

Table 3. Excipients selected for ophthalmic formulation of OAHFA

The OAHFA formulation has a clear/translucent appearance (Fig . 3A), with particle size ideally below 100 nm (Fig. 3B) and pH and osmolality values well within the acceptable range for an ideal eye drop (Table 4). Table 4. pH, osmolality, particle size and polydispersity index values of 1 % OAHFA formulations

PH Particle size Polydispersity Osmolality (mOsm/kg)

Example 3: Stability tests

Three batches of the optimised formulation were subjected to stability studies by storing them at 4 ± 2 °C and 25 ± 2 °C for 6 months under light-free conditions to exclude the possibility of degradation via photooxidation. The droplet size, PDI and OAFHA concentration of the nanoemulsions were measured on day 0 and on the 1 ^st, 2^nd, 3^rd and 6^th month after storing the formulations at different temperature conditions. Results of the measured parameters under two sets of temperature conditions at the various time points are listed in Table 5, with the graphical representation of changes in the droplet size, PDI and OAHFA concentration illustrated in Fig. 4A, 4B and 4C respectively.

Table 5. Particle size, PDI and OAHFA concentration after storage at 4 °C and 25 °C for 6 months.

Storage conditions Droplet size (nm) PDI OAHFA concentration

(mg/ml)

Day O 35.14 0.227 10.87

1 month 4 ± 2 °C 33.28 0.213 10.75

25 ± 2 °C 30.41 0.217 10.20

2 months 4 ± 2 °C 32.17 0.177 12.41

25 ± 2 °C 32.09 0.186 1 1 .98

3 months 4 ± 2 °C 37.87 0.123 1 1 .70

25 ± 2 °C 32.66 0.205 1 1 .73

6 months 4 ± 2 °C 33.57 0.224 1 1 .04

25 ± 2 °C 32.55 0.210 10.65

Over a 6-month period of stability studies, neither a visible phase separation nor a considerable increase in droplet sizes and PDI values were observed for both temperature conditions (4 °C and 25 °C). Therefore, concentrations of OAHFA in the respective nanoemulsions were determined to be stable when stored at 4 ± 2 °C and 25 ± 2 °C for 6 months. Example 4: In-vivo release profile of OAHFA eve drop

During this study period, observations of the rabbits’ eyes revealed no signs of conjunctival redness, lid swelling or excessive blinking after each instillation of the eye drop at predetermined time-points, thus indicating good tolerance of the overall formulation.

The in vivo release profile of OAHFA post instillation provided an estimation of the elimination rate of supplemented OAHFA and hence allowing the gross retention time of the instilled OAHFA to be determined, particularly in the tear film region in where OAHFA is expected to be located. The retention time end-point was set to be reached when the concentration of OAHFA was within 2.5 times that of the baseline (preinstallation) concentration. The 2.5-fold change was employed as that would mean that the end-point concentration was more than three standard deviations above the baseline concentration, which accounted for measurement errors, thus providing a conservative estimation of the gross retention time. With that, the gross retention time of the OAHFA was defined as the time taken for the OAHFA concentration to reach from the point of instillation to the first time-point of two consecutive near-baseline OAHFA concentrations. The change in tear fluid concentration of OAHFA over time is listed in Table 6 and with its corresponding elimination profile depicted in Fig. 5.

To account for the intrinsic concentration of OAHFA that was reported to be present in the meibomian lipidome of rabbits, Schirmer’s tears were collected from all the rabbits prior to the instillation of eye drop to determine the baseline concentration of OAHFA. The average inherent concentration of OAHFA was then subtracted from the measured OAHFA concentrations at different time-points to attain a corrected concentration versus time plot that is a more accurate representation of the elimination profile of supplemented OAHFA.

Table 6. Concentration of OAFIFA at the baseline (pre-installation) and different time-points post instillation.

Time No. of tear Concentration of

samples OAHFA (mg/g)

Baseline 4 0.35 (± 0.24)

5 min 4 13.56 (± 2.60)

30 min 4 3.48 (± 1.05)

1 h 4 3.29 (± 1.27)

2 h 4 1 .33 (± 0.59)

4 h 4 2.04 (± 0.67)

6 h 4 0.91 (± 0.24)

8 h 4 1 .25 (± 0.99)

10 h 4 1 .23 (± 0.67)

24 h 4 0.71 (± 0.51 )

From Fig. 5, it can be observed that tear fluid concentration of OAFIFA reached its first near baseline mark at the 24-hour time-point and thereby implying that the gross retention time of supplemented OAFIFA should lie within the 10-hour to 24-hour interval. Flowever, as tear samples were not collected within this time frame due to time constraints, it was not possible to assign a specific value to the gross retention time of OAFIFA which was defined to be first time- point of two consecutive near-baseline OAFIFA concentrations. Nevertheless, it is relatively safe to conclude that the supplemented OAFIFA was still present in the eye 10 hours post instillation, whereby the concentration of OAFIFA was approximately 3.5 times more than the baseline concentration.

Due to significant fluctuations and variability in the elimination profile of the instilled OAFIFA, it was difficult to obtain a reasonably good fit using the first-order single or multiple compartmental model that is typically employed to determine the pharmacokinetic parameters of ocular drugs. Nevertheless, as the aim of this study was to determine a general elimination profile and how long the supplemented OAFIFA was retained in the tear fluid, by which acceptable pilot data could be obtained from the above results, an elaborated pharmacokinetic profile of OAFIFA was not established at this point.

In summary, from the preliminary elimination profile of supplemented OAFIFA, it is safe to conclude that the 1 % w/v OAFIFA nanaoemulsion eye drop is relatively well-retained in the preocular tear film, in which a significant amount of supplemented OAFIFA relative to baseline levels was still present up to the 10-hour time-point. Flence, a dosage regime of twice a day was decided for the subsequent ocular toxicity test of which results are elaborated in the following section.

Example 5: OAHFA formulation ocular surface safety test

A preliminary 29-days ocular surface tolerance study of the eye drop formulation was performed on four rabbits (labelled A1 , A2, A3 and A4 respectively) to observe for any toxic effects before proceeding to a larger rabbit population. Given that the formulation offers relatively good residence time of OAHFA on the ocular surface, with enhanced levels of OAHFA observed 10 hours after instillation. Flence for the preliminary study, 30mI_ of the eye drop was applied to the right eye twice a day, while the left eye was instilled with 30mI_ of Phosphate buffered saline (PBS) solution and served as a control. Slit-lamp (Fig. 6, Fig. 7, Fig, 8 and Fig. 9), Optical Coherence Tomography (Fig. 10, Fig. 1 1 , Fig. 12 and Fig. 13), Ring illuminator (Fig. 14) and in-vivo Confocal Microscopy (Fig. 15, Fig. 16, Fig. 17, Fig. 18, Fig. 19, Fig. 20, Fig. 21 and Fig. 22) images were taken to observe for any damages to the ocular surface upon application of the OAFIFA eye drop. Observations with all these methods showed that there is no obvious difference between the right and left eye of each rabbit.

Slit-lamp images (Fig. 6 to 9) indicated that there were no noticeable signs of corneal opacity, corneal vascularization, conjunctival congestion (redness), conjunctival swelling or conjunctival drainage. There was also no observable signs of fluorescein staining in the upper, middle lower area of the cornea, implying that there was negligible corneal epithelial damage during the study period, in accordance to the Oxford grading scheme.

Corneal staining was used to detect any signs of corneal epithelial damage, in which case the area would appear green under cobalt-blue light. Flowever, no such green colour was observed.

At the end of the study, two rabbits were sacrificed, and both eyeballs were removed and sent to Advanced Molecular Pathology Laboratory, IMCB for pathological evaluation. There were no significant degenerative, inflammatory or toxicity related changes present in all the eyes (Fig. 23 and Fig. 24), hence supporting in vivo observations.

Based on the results obtained from our preliminary ocular surface toxicity studies, it was concluded that the application of 1 % OAFIFA eye drop (in the form of an oil-in-water emulsion with Miglyol 812, Tween 20, Kolliphor EL, Span 80 and glycerin as inactive ingredients) twice a day is well-tolerated among the 4 studied subjects at least in a 29-days timeframe. This data provides a positive outlook on the safety of the proposed OAFIFA eyedrop, which has to be further clarified with a more elaborated toxicity study (in terms of varied dosage frequency or concentration of OAFIFA) on a larger rabbit population and a longer timeframe. Example 6: Effect OF OAHFA On Tear Film Lipid Laver (TFLU

The influence of OAHFA on the tear film lipid layer (TFLL) model system and its stability was studied by employing a Langmuir trough combined with fluorescence microscopy imaging. More specifically, measurements of surface pressure-molecular area isotherms on a Langmuir trough were performed in conjunction with wide-field fluorescence imaging. Here, a two- component model of human TFLL was employed. This consisted of POPC (1 -palmitoyl-2- oleylsn-glycero-3-phosphocholine) polar lipids in an amount that roughly corresponds to a monolayer film and an abundance of TO (glyceryl trioleate) nonpolar lipids. The studies involving the full TFLL model (PO PC/O AH FA/TO with varying ratios of POPC to OAHFA) were preceded by investigations using control systems not comprising any nonpolar lipids (i.e. consisting of POPC/OAHFA with varying ratio of both lipids).

Composition

Table 7. Compositions of experimentally studied models of the tear film lipid layer

Compositions further comprising glyceryl trioleate (TO) with the same molar ratio of POPC to OAHFA in Table 7 were additionally tested.

• 1 -palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) - (Avanti Polar Lipids, Alabaster, AL).

• O-acyl-omega-hydroxy fatty acid (OAHFA)

• Glyceryl trioleate (TO) - (Sigma-Aldrich, St. Louis, MO, USA).

• Phosphate buffered saline (PBS) and chloroform of spectroscopic grade (Sigma-Aldrich, St. Louis, MO, USA).

• Mili Q water - (Milipore, USA).

Langmuir balance experiments

Langmuir monolayer measurements were performed on a commercially available MicroT rough setup with SS/PTFE trough (maximum surface area of 12331 mm²) (ptrough XS, Kibron; Helsinki, Finland). The system was equipped with an ultra-sensitive surface pressure sensor (KBN 315; Kibron) with the DyneProbe. 1 mM solution of POPC and/or OAHFA in chloroform was spread onto a trough filled with 10 mM PBS (0.137 M NaCI, 0.0027 M KCI, pH 7.4) with a Hamilton microsyringe. Surface pressure-molecular area (TT-A) isotherms were collected during the symmetrical movement of two Delrin barriers controlled by software (FilmWare) provided by the equipment manufacturer. The compression speed was 10 mm/min (3.92 A2/chain/min). Measurements were done at the temperature of 34.2°C that was controlled with a temperature control plate (connected to a water-circulating thermostat; ± 0.5 K accuracy) placed under the trough. To slow down subphase evaporation and protect from dust and additional surface disruptions, an acrylic cover box over the trough was used. Before each measurement, the monolayer was left uncovered for 8 minutes to allow chloroform to evaporate and then for 5 minutes covered with the acrylic box to allow the temperature to equilibrate. To balance the evaporation of the buffer in the trough, water was slowly but continuously pumped in with the peristaltic pump from outside of the barriers.

To study the morphology of the lipid films, wide-field fluorescence images were collected. For this purpose, SS/PTFE trough was substituted with a metal alloy trough with a quartz-glass window mounted on the inverted OLYMPUS fluorescence microscope. For fluorescence measurement purposes, Atto633-DOPE (polar) and Bodipy 495/503 (nonpolar) probes were introduced to the lipid mixtures. A mercury lamp was used as an excitation source and an XY camera as a detector. Two dichroic cubes (“blue” and“red”) were implemented in the light pathway in the microscope body. All measurements were performed with the 60x objective. Results

A) Purely polar sublayer - POPC/OAHFA systems

Before studying the properties of full TFLL model (POPC/OAHFA/TO), only the polar part, i.e. POPC/OAHFA at different molar ratios, was investigated. As can be seen in Fig. 25, the exchange of POPC molecules with OAHFA shifts the isotherms to the left (lower area per polar lipid at the particular pressure, which corresponds to increasing surface tension). Firstly, the isotherm obtained for pure OAHFA that resembles that of typical surfactants (such as POPC) demonstrates that OAHFA is surface active and forms a surface film at the water-air interface. Secondly, the shift toward lower areas in the mixed POPC/OAHFA system means that OAHFA are incorporated into the polar layer but on average, an OAHFA molecule occupies a smaller area per lipid when compared with POPC. Interestingly, at 20 mol% OAHFA, a phase transition at around 80 A² per polar molecule can be observed. This may be rationalized by some restructuring of the mixed film, e.g., related to a conformational change in OAHFA molecules. Such phase transition is not observed for other mixed systems or for pure OAHFA alone. In summary, the exchange of POPC with OAHFA caused an increase in surface tension, OAHFA molecules occupied a smaller area per molecule and pure OAHFA was found to be surface active.

POPC monolayers richer in OAHFA were further analyzed with the help of wide-field fluorescence images. For the purpose of fluorescence measurements, the fluorescent probe DOPEAtto633 (for labelling the polar sublayer) was added to the lipid mixture. Chloroform solutions of POPC and OAHFA mixed at different ratios were deposited on the subphase and widefield images were collected during compression of the lipid film.

In Fig. 26, the images obtained for the POPC/OAHFA 8/2 system are shown. It can be seen that up to the phase transition at ~80 A² per polar molecule, the mixed lipid film is homogenous. Later, however, when the area per polar molecule becomes smaller and surface pressure increases, dark structures with complex shapes start to appear (Fig. 26b, 26c, 26d). These dark structures can be identified as OAHFA“pools” that have little fluorescent probe surrounded by POPC rich in homogenously distributed probe molecules. Such images of the mixed polar film are observable until the film collapses. Just before the collapse, small additional inhomogeneities (bright spots) begin to appear within the homogenous regions of the film. These small structures typically occur, for example, in pure phospholipid films that are close to collapse due to formation of high-pressure defects caused by polar lipids. Interestingly, on the edges of the black OAHFA-related regions, close to the collapse (Fig. 26e), many bright structures attributed to POPC begin to appear. This may be rationalized by the enhancement of POPC film collapse at the boundaries of the OAHFA domains. It can by concluded that for the composition OAHFA/POPC 8/2, the film is homogenous only at very low surface pressure, OAHFA typically forms domains in the POPC film and POPC collapse is promoted at domain boundaries.

In Fig. 27, microscopy images collected during compression of the POPC/OAHFA 5/5 system are presented. At this ratio, dark structures of complex shape (“wing”-like) are visible from the beginning of the compression (even in the fully relaxed state) (Fig. 27a, 27b, 27c). No phase transition is observed in the isotherm. The structures are also larger and more abundant than in the case of composition OAFIFA/POPC 8/2. The inhomogeneous character of the system is evident over the entire range of surface pressures tested. At film collapse, numerous bright structures appear at the boundary between the phases. In conclusion, for the composition OAFIFA/POPC 5/5, it is non-homogenous for all ranges of area per polar molecule, i.e. from the fully relaxed state to the collapse. Many“wing”-like domains can be seen in this system.

The film composition that was studied next was POPC/OAFIFA 1/9, as shown in Fig. 28. In this example, because of the large amount of OAFIFA, phase separation that could be seen in the other systems with lower OAFIFA ratio was expected. Indeed, large dark areas poor in the content of polar probe were present (Fig. 28I). Next to these dark areas, large areas of “mountain”-like patterns were observed. The latter are presumed to be probe-rich POPC areas enclosed between phase-separated OAFIFA structures (Fig. 28II, 28III, 28IV). These dark and bright patterns appeared randomly during most of the compression time, which is why numbers I, II, III, and IV in Fig. 28 are not characteristic of a particular area per polar molecule values. The POPC/OAFIFA film at 1/9 ratio is inhomogeneous in the whole range of area per polar molecule values tested.

In the case of pure OAFIFA film, significant inhomogeneities were also observed (Fig. 29). In this system, a small quantity of polar fluorescent probe is present to facilitate the imaging. Large, dark areas (Fig. 29I) are interlaced with brighter regions (Fig. 29II, 29III, 29IV, 29V). The bright structures are formed by OAFIFA having probe molecules entrapped within them.

B) Mixed polar/nonpolar TFLL model - POPC/OAHFA + TO

In order to study the full TFLL model, POPC/OAFIFA (polar lipids) at different molar ratios were mixed with an abundance of TO (nonpolar lipids). As it can be seen in Fig. 30, addition of excess TO to a purely polar film causes an increase in pressure to ~12mN/m. Such effect has been previously observed for the systems without OAHFA. It is presumed that some of the TO molecules aggregate in-between the polar sublayer molecules, while the rest create a multilayer or aggregate on top of the polar sublayer molecules. The pressure, however, does not increase further with increasing amount of nonpolar sublayer molecules but only during compression. All the isotherms representing POPC/OAHFA+TO systems tend to meet the isotherms of corresponding pure polar POPC/OAHFA films. This means that TO molecules are squeezed out continuously from the polar sublayer and the characteristics of the increase of surface pressure during compression depends exclusively on the polar sublayer composition. Again, substitution of the molecules of POPC with OAHFA in POPC+TO model system also shifts isotherms to the left (lower area per polar lipid at the particular pressure), as it was observed in the case of the purely polar sublayer.

Analyzing POPC/OAHFA 8/2 + TO isotherm in Fig. 30, phase transition is not observed. This is believed to be because the starting surface pressure (~12mN/m) is already higher than that observed previously at around 80 A² per polar molecule, where the phase transition occurred. Therefore, it appears that substitution of POPC with OAHFA increases surface tension in the full TFLL model system (POPC+TO), and the manner at which the change of surface pressure occurs during film compression is dependent on the composition of the polar sublayer.

For the purpose of fluorescence imaging of the full TFLL model (POPC+TO) in the presence of OAHFA, two probes, DOPE-Atto633 (polar sublayer label) and Bodipy 496/503 (nonpolar sublayer probe), were added to the lipid mixtures. Chloroform solutions of POPC and OAHFA mixed at different ratios were deposited on the subphase and wide-field images recorded during compression of the lipid film.

The structure of the full TFLL model system at 20 mol % of OAHFA in a polar sublayer appears to be, to some extent, the same as that without OAHFA. Phase la, lla, Ilia and IVa correspond to images obtained for the nonpolar fluorescent probe, while Phase lb, Mb, II lc and IVb correspond to the polar probe. In polar and nonpolar sublayers, there are circular structures present (Fig. 31 II) that correspond to aggregates of TO (Fig. 31 lla, bright structures)“swimming” in the polar sublayer (Fig. 31 11b), The difference is the presence of large, dark complex structures, which may be interpreted as domains caused by a phase separated film (Fig. 31 1, 31 III, 31 1V). It should be apparent that TO molecules/aggregates preferentially come into contact with the phase separated OAHFA structures (Fig. 31 111a, bright spots on the edge of OAHFA domains). Further, at higher surface pressure, mixed structures can be observed at the edges of OAHFA domains (Fig. 31 IVa and 31 IVb, bright spots on the edge of OAHFA domains). Therefore, it appears that within the whole range of surface pressure tested during compression of the composition OAHFA/POPC 8/2 + TO, domains of OAHFA are present, and TO molecules/aggregates appear to have an affinity toward the domain boundaries.

The POPC/OAHFA 5/5 + TO composition, as shown in Fig. 32, displays a similar characteristic to that of POPC/OAHFA 8/2 + TO as described above. However, the range of inhomogeneities observed is even larger. Otherwise, the description of the composition POPC/OAHFA 8/2 + TO applies to the composition POPC/OAHFA 5/5 + TO. That is, for the composition OAFIFA/POPC 5/5 + TO, domains/inhomogeneities of OAHFA are present over the whole range of surface pressure tested during compression, and TO molecules/aggregates appear to have affinity toward the domain boundaries.

In the case of the TFLL model with a polar sublayer composed of POPC/OAHFA mixed in the ratio 1/9 with TO present, an inhomogeneous polar sublayer distributed in “mountain”-like patterns (Fig. 33lb and 33I lb) is observed over the entire surface area of the film.

However, during complete compression of the film, no large aggregates of TO are visible. That is, TO molecules are more homogenously distributed within and above the polar sublayer than in the case of other POPC+TO systems described above. Therefore, it appears that for the composition OAHFA/POPC 1 /9 + TO, an inhomogeneous polar sublayer with a mountain-like pattern is observed over the whole range of surface pressure tested during compression, but no large aggregates of TO are present.

In the case of the film composed of OAHFA and nonpolar TO (no POPC), significant inhomogeneities are observed. Most of the surface is covered by“islands” of OAHFA (Fig. 34lb), which are complementary to areas that are richer in TO (Fig. 34la). On the borders of the two phases, aggregates of TO are present (bright spots in Fig. 34la). With increasing pressure, formation of large TO aggregates entrapped between OAHFA structures (Fig. 34II, 34III) are observed. Therefore, it appears that OAHFA film in the presence of nonpolar TO has a significantly inhomogeneous structure.

Example 7: In Silico Simulations

The effect of OAHFA on the structural properties of a TFLL model was studied by coarse grained molecular dynamics (MD) simulations. The model consisted of a mixture of five different polar lipids mimicking human tear film lipidomics and an abundance of glyceryl trioleate and cholesteryl esters to model nonpolar TFLL components. Overall, five different in silico systems were investigated, including systems deficient in polar lipids, as outlined in Table 8 System compositions

Table 8: Compositions of TFLL models studied in the presence of OAHFA employing MD simulations.

• POPC (1 -palmitoyl-2-oleyl-sn-glycero-3-phosphocholine)

• POPE (1 -palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine)

• SM (N-palmitoyl-D-erythrosphingosylphosphorylcholine)

• Cer (N-palmitoyl-D-erythrosphingosine)

• OAHFA (O-acyl-omega-hydroxy fatty acid)

• TO (glyceryl trioleate)

• CO (cholesteryl oleate)

Methodology

Coarse grained classical molecular dynamics simulations (MD) of TFLL models in the presence of OAPIFA molecules were performed. For all molecules, MARTINI force field was used. In house parameterization for OAFIFA was prepared, following the MARTINI approach. Deprotonated form of OAHFA carboxylic group was assumed. All systems containing OAHFA were neutralized by addition of the appropriate number of positively charged sodium ions to each simulation box. MD simulations were performed at 310 K and 1 bar pressure. The dimension of simulation boxes was equal to 22x22x104 nm³ and the number of added polar lipids corresponded to 0.67 nm² area per polar molecule. Periodic boundary conditions were used for all MD simulations boxes. Overall, the simulation procedure is as described. Non- bonded interactions were cut off at 1 .2 nm with a shift function from 0.9 nm. This is followed by cutting off short-range Coulomb interactions at 1 .2 nm with a shift function from 0.0 nm. Integration of the equations of motion was recorded at a time-step of 20 fs. The temperature of both the lipid and water phases was controlled independently using the Berendsen thermostat algorithm with a relaxation time of 0.3 ps. For non-equilibrium simulations of lateral compression and decompression, lateral pressure was applied using semi-isotropic Parrinello- Rahman barostat with a compressibility of 3x10-5 and relaxation time of 3 ps. The simulated trajectory length was equal to 500 ns, with last 300 ns used for analysis. The first 200 ns was regarded as the equilibration duration and hence was not taken into account during analyses. Simulations were performed using GROMACS 2016.1 package using a specialized computational cluster.

Results

Five TFLL model systems were studied by MD simulations. In the following paragraphs, each of the investigated system is described in points to highlight the most important findings.

A) OAFIFA at water-air interface

Before studying the full TFLL model in presence of OAFIFA, a pure OAFIFA film on water-air interface was investigated. The corresponding results are presented in Fig. 35a and 36a.

• OAFIFA behaves as a surfactant at the air-water interface with the carboxylic group oriented toward the water phase.

• C4A atoms (uncharged terminal) are exposed toward the air phase, while NST atoms (polar ether moiety in the middle of the molecule) display an intermediate behaviour.

• Simulation snapshot (Fig. 36a) demonstrates that OAFIFA molecules form micelle-like structures on the water surface. Flence, they do not form a full film at the water surface. This may be, however, the effect of a small number of OAFIFA in the system. In other words, it may be possible that at a higher concentration of OAFIFA, a full film may be formed

B) Pure Polar Lipid Layer (PLL)

The second system analyzed was a model composed of pure polar lipid layer (PLL) (mix of four polar lipids) and OAFIFA molecules. The results described below correspond to Fig. 35b and Fig. 36b.

• POPC mostly behaves like surfactant molecules. That is, their heads are oriented towards water and their tails towards the air. Flowever, there is a small population of POPC in reverse configuration. This is caused by the presence of OAFIFA as such behaviour was not observed in the previous studies.

• Some of OAFIFA molecules behave similarly to POPC, as their acidic terminal groups are oriented towards the water and nonpolar tails towards air. Flowever, surprisingly, a majority of OAFIFA is in a reverse configuration with their carboxylic groups oriented towards air. • NST and C4A atoms of OAHFA are localized in the middle of the system.

• A bilayer-like structure is formed by OAHFA together with a monolayer film of POPC (see Fig. 36b)

C) Tear Film Lipid Layer (TFLL)

Full TFLL model (PLL plus thick layer of nonpolar molecules atop) in the presence of OAHFA was investigated. The results described corresponds to the data presented in Fig. 35c and Fig. 36c.

• POPCs behaves like it does in the pure PLL system as described above. That is, the heads are located on the surface of the water and the lipid tails are located towards air.

• Near the POPC chains, there is a small peak of TO, then the TO density increases and finally decreases towards air.

• OAHFAs are present in the mixture with TO and cholesteryl oleate (CO) but predominantly it orients with its carboxylic groups towards air.

• In simulation snapshots, as shown in Fig. 36c, a small group of OAHFA molecules penetrating into the POPC layer is occasionally observed.

• Some of the carboxylic OAHFA groups are also found in the middle of TFLL as“interlayer”- like structures

• Overall, the carboxylic OAHFA groups are localized in three layers with the major fraction concentrated at the TFLL/air interface

D) TFLL - 90% PLL

A 90% polar lipid-deficient TFLL model in the presence of OAHFA was investigated. The results correspond to the data presented in Fig. 35d and 36d.

• Empty spaces within the POPC layer are formed and carboxylic groups of OAHFA can easily occupy these film regions. As a result, these polar groups replace the P0₄ atoms of POPC in the polar sublayer. Hence, a considerable number of these groups are in contact with water molecules.

• An“interlayer”-like structure is created (charged carboxylic atoms (000) of OAHFA).

• Carboxylic OAHFA groups are concentrated on the surface with water. They adjust the surface and replace POPC heads. E) TFLL - 100% PLL

TFLL model with no polar lipids in the presence of OAFIFA was studied. The results described below correspond to Fig. 35e and 36e.

• A significant fraction of the carboxylic groups of OAHFA takes the role of POPC headgroups observed in the other systems.

• Overall, the charged groups of OAHFA form three layers (at the water/TFLL interface, at the TFLL/air interface and in the middle of system).

Example 8: Toxicity Testing

The objective of this study was to evaluate the ocular toxicity of OAHFA topically administered twice daily to New Zealand White Rabbits for 13 weeks.

Abbreviations

Summary

Methods

A total of 12 adult male New Zealand White rabbits were randomly and evenly assigned into 3 groups. Animals in each group were bilaterally treated twice daily with topical ocular instillation of 50 mI of vehicle control, 0.75% or 1% OAHFA, respectively. The dosing treatment lasted for 89 days in vehicle group and 91 days in OAHFA groups. Clinical observations were conducted twice daily, body weights were measured once a week, general ocular examinations were performed before dosing (Day -1 ) and twice daily on Day 1 , 3, 7, 14, 28, 56, 89 (group 1 ), 91 (group 2 and 3), prior to the first and post the last daily dose. Tear samples were collected on Day -1 , Day 7 (before first dosing) and 16 hours after final dosing, and the OAHFA concentration was analyzed. Blood samples were collected before dosing and 16 hours after final dosing, aqueous humor and urine samples were collected after final dosing. All the animals were euthanized on Day 92, macroscopic and ocular histopathological examinations was performed.

Results

No test article-related changes were observed during clinical observations and the measurements of body weight. Before dosing on Day 28, minor (score 1 ) conjunctival congestion was observed on the left eye of animal number 1917436 in the low dose group. On Day 56, 89 and 91 , mild corneal fluorescein staining (score 1 ) was seen unilaterally or bilaterally in some of the animals of all groups. All the abnormalities were recovered on the same day or 24 hours later. Compared with the baseline, the OAHFA concentration in the tear samples rose in low and high dose groups on Day 7 and after final dosing, while no change was observed in the vehicle group.

Conclusions

During the 13-week study of twice daily topical ocular administration of OAHFA (0.75% or 1 .0%) in New Zealand White Rabbits, there is no noticeable ocular irritation that was identified in general ocular examinations.

Experimental Design

Randomization and Group Assignment

Any animal with any ocular defect found in the general ophthalmology examination was excluded before grouping. Twelve animals with normal eyes and in similar body weight were selected to enter the experiment. Through a computer-generated randomization procedure, the twelve animals were randomly assigned to respective treatment groups based on the body weight measured within 24 hours pre 1 ^st dosing (Day -1 ), as shown in the following Table 9.

Table 9. Animal assignment

Dosing Delivery

Dosing Eye: Both eyes

Dosing Route: Topical Administration

Frequency and Duration: twice daily (about 10-hour interval), for 91 consecutive days. The vehicle control group was not administered on Day 90 and 91 .

Dosing Volume: 50 pL/eye/time

Delivery Method: 50 mI_ test/control article was drawn with a pipette, the lower eyelid of the animal was pulled out, 50 mI_ article was dropped into the conjunctiva sac, the lower eyelid was closed gently and remained so for 10 seconds.

Justification for Route of Administration: This route of administration selected for this study was the intended route of administration in humans.

Justification for Dose Level, Frequency, and Duration: The dose level, frequency, and duration selected for this study were selected based on relevant guidelines to support the subsequent toxicity studies and/or clinical trials.

In-Life Examinations

Clinical Observations

Daily Observations: Rabbits were observed twice daily (a.m. and p.m.) during the quarantine and the study periods for signs of mortality, morbidity, respiration, secretion, feces, and for the availability of water and food.

Detailed Observations: Each rabbit in Group 1 to 3 were removed from its cage and examined closely for clinical signs of toxicity once weekly after the first dosing, or more often as clinical signs warranted. The observations included, but were not limited to, evaluation of behavior, the skin, fur, eyes, ears, nose, abdomen, external genitalia, anus, limbs, feet and respiration.

Body Weights

Body weights were obtained before group assignment, prior to the first dosing (Day -1 ), and once weekly during study. Ophthalmic Examinations

General Ocular Examination

The instillation site of each animal was scored according to the Modified MacDonald-Shadduck Scoring System before grouping (Day -1 ) and twice daily on Day 1 , 3, 8, 14, 28, 56 and 89 (group 1 ), and Day 91 (groups 2 and 3) (prior to the first daily dose and following the last daily dose). The Corneal Staining (% Area) was not scored in this section. The abnormal findings in the eyelid, iris, sclera and pupil were noted but not scored in the examination.

Ocular fundus: Ocular fundus of each animal were observed using the ophthalmoscopy before grouping (Day -1 ) and twice daily on Day 1 , 3, 8, 14, 28, 56 and 89 (group 1 ) and Day 91 (groups 2 and 3) (prior to the first daily dose and following the last daily dose).

Cornea with Fluorescein staining: Cornea with Fluorescein staining was conducted using the fluorescein sodium ophthalmic strips before grouping (Day -1 ) and twice daily on Day 1 , 3, 8, 14, 28, 56 and 89 (group 1 ), and Day 91 (groups 2 and 3) (prior to the first daily dose and following the last daily dose). If fluorescein staining was noted in any animal, slit-lamp examination was performed every 24 hours until resolution. The Corneal Staining (% Area) was scored according to the Modified MacDonald-Shadduck Scoring System.

Sample Collection and Analysis

Tears: Test strips were used to collect tears on Day -1 , Day 7 (before first dosing) and 16 hours after final dosing, the lower eyelid was lifted gently and the strip was inserted into the conjunctival sac for about 1 min, and put into 500 pl_ n-butanol and methanol solution(50:50,v/v) in a 1.5 ml_ Eppendorf tube. All samples were shipped on dry ice to Bioanalysis Department of JOINN LABORATORIES (CHINA) Co., LTD. for testing (See below “Example 9: Analysis Results” for details).

Animal Euthanasia and General Terminal Examination

Animal Euthanasia

In accordance with the A VMA Guidelines for the Euthanasia of Animals: 2013 Edition (the American Veterinary Medical Association, 2013), all animals were euthanized using Zoletil 50 (12 mg/kg, 50 mg/mL) with intramuscular injection followed by femoral artery exsanguinations on Day 92. Macroscopic and Microscopic Observations

Macroscopic Observations:

All animals euthanized at scheduled necropsy, received a complete macroscopic examination. Carcasses were disposed as medical waste following macroscopic examination and requested tissue collection.

The macroscopic examination included, but not limited to, the external surfaces of the body, all orifices of the body, and the cranial, thoracic, abdominal, and pelvic and their contents. Necropsy and gross lesion were recorded. The eye ball was saved in Davidson’s fixative for histopathological evaluation.

Results

1 . Clinical Observations

The individual data of clinical observation are present in Tables 10A to 10G.

During the study period, no systemic abnormality was noted except ophthalmic abnormalities.

Table 10A: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9 Day10 Day1 1 Day12 Day13

ose

1917437 Oi

CD

1917438 (5

1917439 S

High 1917440 (5

Dose

1917441 S

1917442 (5

Note: indicates no abnormality was observed.

Table 10B: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day14 Day15 Day16 Day17 Day18 Day19 Day20 Day21 Day22 Day23 Day24 Day25 Day26

1917431 a

Vehicle 1917432 a

Control

1917433 a

1917434 a

1917435 a

Low 1917436 a

Dose

1917437 a Oi

1917438 a

1917439 a

High 1917440 a

Dose

1917441 a

1917442 a

Note: indicates no abnormality was observed.

Table 10C: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day27 Day28 Day29 Day30 Day31 Day32 Day33 Day34 Day35 Day36 Day37 Day38 Day39

1917431 S

Vehicle 1917432 S

Control

1917433 s

Low 1917436 s

Dose

1917437 s

1917438 s ¥

1917439 s

High 1917440 s

Dose

1917441 s

1917442 s

Note: indicates no abnormality was observed.

Table 10D: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day40 Day41 Day42 Day43 Day44 Day45 Day46 Day47 Day48 Day49 Day50 Day51 Day52

1917431 S

Vehicle 1917432 8

Control

1917433 8

1917434 8

1917435 8

Low 1917436 8

Dose cn

1917437 8 co

1917438 8

1917439 8

High 1917440 8

Dose

1917441 8

1917442 8

Note: indicates no abnormality was observed.

Table 10E: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day53 Day54 Day55 Day56 Day57 Day58 Day59 Day60 Day61 Day62 Day63 Day64 Day65

1917431 S

Vehicle 1917432 s

1917442 s

Note: indicates no abnormality was observed.

Table 10F: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day66 Day67 Day68 Day69 Day70 Day71 Day72 Day73 Day74 Day75 Day76 Day77 Day78

1917431 S

Vehicle 1917432 S

y .

Table 10G: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Clinical Observations

Group Animal Sex Day79 Day80 Day81 Day82 Day83 Day84 Day85 Day86 Day87 Day88 Day89 Day90 Day91 Day92

1917431 S

Vehicle 1917432 $

^Contro1 1917433 ¹

1917434 - 1917435 S

Low 1917436 ⁷

^Dose 1917437 S

1917438 S

1917439 S

High 1917440 S

D^ose 1917441 S

1917442 "

Note: indicates no abnormality was observed.

2. Body Weights

Body weights are summarized in Tables 1 1 A and 1 1 B and individual data are presented in Table 12.

No test article related changes were noted in body weights at all dose levels after dosing throughout the study.

Table 11 A. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Summary of Body Weight (kg)

Group Treatment Day -1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42

MeanlSD 3.23±0.06 3.26±0.10 3.37±0.09 3.50±0.09 3.5110.14 3.5910.08 3.5910.09

Vehicle

Control

n 4 4 4 4 4 4 4

MeanlSD 3.21 ±0.1 1 3.22±0.07 3.30±0.06 3.3610.08 3.3610.13 3.4210.13 3.4510.19

Low °-^75%

D^0Se OAHFA n 4 4 4 4 4 4 4

1 % MeanlSD 3.27±0.02 3.25±0.04 3.31 ±0.09 3.3710.10 3.3610.14 3.4210.15 3.4510.14

High s>

Dose 4^

OAHFA n 4 4 4 4 4 4 4

Note: n indicates the quantity involved in statistical analysis.

Table 11 B. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Summary of Body Weight (kg)

Group Treatment Day 49 Day 56 Day 63 Day 70 Day 77 Day 84 Day 91

MeaniSD 3.71 ±0.08 3.68±0.08 3.79±0.14 3.77±0.10 3.83±0.12 3.85±0.07 3.83±0.13

Vehicle

Control

n 4 4 4 4 4 4 4

Mean±SD 3.63±0.23 3.51 ±0.21 3.68±0.21 3.63±0.27 3.61 ±0.31 3.67±0.30 376+034

Low °-^75%

D^ose OAHFA n 4 4 4 4 4 4 4

1% MeaniSD 3.55±0.15 3.51 ±0.20 3.61 ±0.22 3.59±0.21 3.60±0.19 3.61 ±0.19 068+022

High O)

Dose cn

OAHFA n 4 4 4 4 4 4 4

Note: n indicates the quantity involved in statistical analysis

Table 12. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of Body Weight (kg)

Group Animal Sex Day- Day7 Day14 Day21 Day28 Day35 Day42 Day49 Day56 Day63 Day70 Day77 Day84 Day91

O)

ose G)

1917437 3.10 3.28 375 342 370 376 376 372 376 378 372 470 474 4.16

1917438 - 3.13 3.12 372 378 374 370 374 342 378 342 372 376 372 3.34

1917439 ⁷ 3.27 3.20 37Ϊ 378 372 378 372 378 372 346 340 344 346 348

High 1917440 " 3.27 3.30 377 345 372 370 372 372 378 372 376 376 378 3.98

Dose

1917441 7 3.28 3.26 340 345 342 348 370 370 374 370 372 372 370 372

1917442 " 3.24 3.22 375 379 378 372 376 348 340 346 346 346 370 3.54

Note:“-’’indicates no measurement at this time point.

3. General Ocular Examination

Individual data of ophthalmic examinations are presented in Tables 13A to 130.

e 13A. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day -1 , re dosing)

Cornea Conjunctivae Corneal

Animal Staining oup ^^ex Opacity Opacity (% Neovascular- Congestion Chemosis and Discharge

No. % Area area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 c^ O O O O O O O O 0 0 0 0 0 0 h^icle 1917432 S 0 0 0 0 0 0 0 0^~ 0 0 0 0 0 0 ntrol

1917433 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917434 _<S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

gh 1917440 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 se

1917441 S o 0 0 0 0 0 0 0 0 0 0 0 0 0

1917442 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 :“-’’indicates no abnormality was observed.

13B: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 1 , e first dosing)

Cornea Conjunctivae Corneal

Staining

Animal

oup Sex

No. Opacity Opacity (% Neovascular- Congestion Chemosis and Discharge % Area area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917442 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 :“-’’indicates no abnormality was observed.

le 13C. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 1,r second dosing)

Cornea Conjunctivae Corneal

Staining

Animal

roup Sex Opacity (% Neo vascular- Chemosis and

No. Opacity Congestion Discharge % Area

area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 (^ 0 0 0 0 0 0 0 0 0 0 0 0 0 0^ehicle 1917432 8 0 0 0 6 0 0 0 0 0 0 6 0 0 O^"ontrol

1917433 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917434 (¹ 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917435 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 w 1917436 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0ose

1917437 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917438 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917439 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0igh 1917440 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0ose

1917441 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917442 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 :“-’’indicates no abnormality was observed.

e 13D. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 3,e first dosing)

Cornea Conjunctivae Corneal

Staining

Animal

oup Sex Opacity (% Neovascular- Chemosis and

No. Opacity Congestion Discharge % Area

area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 ₍5 0 0 0 0 0 0 0 0 0 0 0 0 0 0^hicle 1917432 $ 0 0 0 0 0 0 0 0 0 0 0 (G 0

ntrol

1917434 (5 0 0 0 0 0 0 0 0 0

1917435 (5

w 1917436 (5 0 0 0 0 0 0 0 0 0 0 0 0 0 0se

1917437 (5 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917438 c5 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917439 (5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 h 1917440 (5 0 0 0 0 0 0 0 0 0 0 0 0 0 0se

1917441 (5 o 0 0 0 0 0 0 0 0 0 0 0 0 0

1917442 (5 0 0 0 0 0 0 0 0 0 0 0 0 0 0:“-’’indicates no abnormality was observed.

le 13E. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 3, r second dosing)

Cornea Conjunctivae Corneal

Staining

Animal

roup Sex

No. Opacity Opacity (% Neo vascular- Congestion Chemosis and Discharge % Area area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917442 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 e:“-’’indicates no abnormality was observed.

13F. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 8, e first dosing)

Cornea Conjunctivae Corneal

Staining

Animal

up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917442 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “-’’indicates no abnormality was observed.

13G. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 8, second dosing)

Cornea Conjunctivae Corneal

Staining

Animal

up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917442 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 “-’’indicates no abnormality was observed.

13H. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 14, first dosing)

Cornea Conjunctivae Corneal

Staining

Animal

up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

131: An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 14, second dosing)

Cornea Conjunctivae Corneal

Staining

Animal

up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

13J. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 28, e first dosing)

Cornea Conjunctivae Corneal

Staining

Animal

up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

13K. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 28, econd dosing)

Cornea Conjunctivae Corneal

Animal Staining up Sex

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

13L. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 56, first dosing)

Cornea Conjunctivae Corneal

Animal Stainingup ^^ex Opacity Opacity (% Neovascular- Congestion Chemosis and Discharge % Area

No.

area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 ^ 0 0 0 0 0 0 0 0 0 0 0 0 0 0^icle 1917432 s 6 o 6 6 6 o 6 6 o 6 6 o o o^"trol

1917433 S 0

1917434 (¹ 0 0 0 0 0 0 0 0 0

1917435 S

0 0 0 0 0 0 0 0 0 0 0 0 0

1917436 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0e

1917437 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917438 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 h

e

1917441 S o 0 0 0 0 0 0 0 0 0 0 0 0

e 13M. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 56, second dosing)

Cornea Conjunctivae Corneal

:“-’’indicates no abnormality was observed.

ble 13N. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 91 , before first dosing)

Cornea Conjunctivae Corneal

Animal

roup Sex Opacity Opacity (% Neovascular- Congestion Chemosis and Discharge % Area

No.

area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 c^ O O O O O O O O 0 0 0 0 0 1 e^hicle 1917432 S 0 0 0 0 6 0 0 (G 0 0 0 0 0 0 ontrol

1917433 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917434 ( ¹ 0 0 0 0 0 0 0 0 0 0 0 0 0 T

1917438 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917439 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 igh 1917440 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ose

1917441 8 o 0 0 0 0 0 0 0 0 0 0 0 0 T

1917442 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 te:“-’’indicates no abnormality was observed.

le 130. An Eye Toxicity Study of OAHFA with 13-Week Instillation in New Zealand White Rabbits: Individual Data of General Ocular Examination (Day 91 , after second dosing)

Cornea Conjunctivae Corneal

Animal

No.

area) ization Swelling

OD OS OD OS OD OS OD OS OD OS OD OS OD OS

1917431 c^ O O O O O O O O O O O O O I e^hicle 1917432 S 0 0 0 0 0 6 6 0 0 0 0 0 0 (G ontrol

1917433 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917434 ( ¹ 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917438 S 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1917439 $ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 igh 1917440 $ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ose

1917441 S o 0 0 0 0 0 0 0 0 0 0 0 0 0

1917442 ( ¹ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 e:“-’’indicates no abnormality was observed.

Before dosing on Day 28, conjunctival congestion was observed on the left eye of animal number 1917436 in low dose group. The score was 1 and it recovered after second dosing on that day. After the second dosing on Day 56, corneal fluorescein staining was seen on both the eyes of animal number 1917437 in the low dose group and the right eye of animal number 1917439 in the high dose group. The score was 1 and they recovered 24 hours later. Before the first dosing on Day 89, fluorescein staining was observed on the left eye of animal number 1917431 and animal number 1917434 in the vehicle control group. The score was 1 and they recovered 24 hours after the second dosing on that day. Before the first dosing on Day 91 , fluorescein staining was observed on the left eye of animal number 1917441 in the high dose group. The score is 1 and it recovered after the second dosing on that day. All the abnormality could be recovered after the second dosing or 24 hours later, hence the test article was classified as no causing irritation according to the scoring system.

4. Bioanalvsis

The tear content of OAHFA in each group after administration is presented in Table 14.

Compared with the baseline, the OAHFA concentration of tear samples rose in the low and high dose group on Day 7 and after final dosing, while no change was observed in vehicle group. (See below“Example 9: Analysis Results” for details).

Table 14 OAHFA concentration in tears by bioanalysis

Concentration/ Content (ng)

Note: CV(%)=SD/Mean^*100%.

5. Macroscopic and Microscopic Observations

There was no macroscopic finding in animals sacrificed at the end of the study period (Day 92).

Conclusions

During the 13-week study of twice daily topical ocular administration of OAHFA (0.75% or 1.0%) in New Zealand White Rabbits, no noticeable ocular irritation was identified in general ocular examinations.

Example 9: Analysis Results

The following is a summary of the analysis results for analyzing eye toxicity samples of OAHFA in the above study. The content of OAHFA in New Zealand white rabbit tear samples were determined by the validated LC-MS/MS method. This report provided the data of tear drug content, calibration curves and quality controls. Details of analysis records were kept in raw data.

Materials

The following reference standard (Table 15), internal standard (Table 16), major reagents (Table 17) and major equipment (Table 18) were used.

Table 15. Reference Standard

Code: OAHFA

Lot Number: LipidA082018

Characterization: White Solid

Storage Condition: Cool, dry and well-ventilated

Expiration Date: 8/30/2019

Content: 100%

Storage Condition after opening: Below the -20°C

Expiration Date after opening: 8/30/2019

Supplier: Singapore Eye Research Institute

Producer: Singapore Eye Research Institute

Special Handling: Standard safety precautions (use of protective clothing, gloves, and mask) were taken when handling the reference substance.

Disposal: The reference standard was not reserved and archived.

The quantity, date and the use person was registered. The unused reference standard would be disposed as recommended after the completion of all relevant studies. Table 16. Internal Standard (IS)

Code: 13C-derivative of OAHFA

Lot Number: CA082018

Characterization: White Solid

Storage Condition: Cool, dry and well-ventilated

Expiration Date: 8/30/2019

Content: 100%

Storage Condition after Below the -20°C

opening:

Expiration Date after opening: 8/30/2019

Supplier: Singapore Eye Research Institute

Producer: Singapore Eye Research Institute

Special Handling: Standard safety precautions (use of protective clothing, gloves, and mask) were taken when handling the internal standard.

Disposal: The internal standard was not reserved and archived. The quantity, date and the use person was registered. The unused internal standard would be disposed as recommended after the completion of all relevant studies.

Table 17. Major Reagents

Table 18. Major Equipment

Sample Reception

After collection, all samples were shipped on dry ice to bioanalysis department of JOINN LABORATORIES (CHINA) Co., LTD. Upon reception, the samples were stored below -70°C.

Sample Analysis

The content of OAHFA in tears were determined by a validated LC-MS/MS method as described below.

40 pL of the sample was taken and added into 10 pL deuterated internal standard (IS) work solution (20 ng/mL) and 160 pL of a methanohn-butyl alcohol (50:50, v/v) solution. After mixed them well, the samples were analyzed directly. The Shiseido column (Capcell Pak C18, 2.1 mm I.D^*100 mm, 2 pm) was used to achieve chromatographic separation, and each injection was run for 7 min. The mobile phases were ultrapure wateracetonitrile (40:60, v/v) solution and isopropanol:acetonitrile:water (90:5:5, v/v/v) solution, both of which contained 10 mM ammonium formate. Negative ion scanning mode was adopted and multiple reaction monitoring (MRM) mode was used for analysis. Monitor ion pair of OAHFA and IS were 785.8 281.2 and 803.8 299.3, respectively. The linearity range of OAHFA in this method was 0.05-25 ng. The LLOQ was 0.05 ng.

Results

Acceptable Criteria of Analysis Batch:

The calibration curve should be constructed with at least six points (including nonzero values at two ends) meeting the following requirements: The bias (%) of each point should be within -15 to 15% except the LLOQ point, for which the bias (%) could be within -20 to 20%. Correlation coefficient (R2) should be more than 0.98. At least 67% (2 out of 3) of total QC samples should have a back-calculated concentration bias within -15 to 15% of their respective nominal value in all the concentration levels. . In addition, at least 50% QC samples per concentration needs to meet the above criterion of the bias (%). The mean bias of QC samples in all the concentration levels should be within -15 to 15% of their respective nominal value. The inter-run precision CVs of QC samples should be no more than 15%.

Calibration Curves and Quality Controls Samples:

There were four batches of analysis in this test. The data of calibration curves and QC samples which accompany with sample analysis are shown in Tables 19A and 19B. The results showed that all analytical runs met the acceptance criteria. Eight points (including nonzero value at two ends) in calibration curve with the bias (%) ranged from -10.00% to 10.00%. The correlation coefficients (R²) were all greater than 0.98. The bias (%) of QC samples ranged from -13.33% to 13.33%. The inter-run precision CVs of QC samples were from 5.82% to 13.33%, the mean bias (%) was from 0.00% to 1 .50%.

Tear Drug Content:

The individual tear content of OAHFA from each group of animals after administration was presented in Table 20A to 20C. When the responses were lower than that of LLOQ, the contents of OAHFA were recorded as 0 in Day -1 samples and pre-dose samples for first dosing, while in all other samples, the contents of OAHFA were recorded as BQL.

The result of sample reanalysis (ISR) shows that the bias of 1 1 samples was within 20% between ISR result and its report result among 12 ISR samples totally, which met the acceptance criteria and confirmed the reliability of the analysis results, as shown in Table 21.

Deviations

No deviation was observed during the test.

Table 19A. Calibration Curves and Quality Controls Samples of OAHFA: LC-MS/MS Method for Quantitation of OAHFA in Rabbit Tears- Calibration Curve

Note: The Bias (%) should be within ±15%; LLOQ should be within ±20%.R >0.98

Table 19B. LC-MS/MS Method for Quantitation of OAHFA in Rabbit Tears: Quality Controls

Curve

0.15 (ng) Bias (%) 2 (ng) Bias (%) 20 (ng) Bias (%)

Number

0.14 -6.67 2.1 1 5.50 20.24 1 .20

0.14 -6.67 2.1 1 5.50 21.84 9.20

1

0.15 0.00 1.84 8.00 19.97 -0.15

0.17 13.33 2.19 9.50 18.21 -8.95

0.16 6^67 å05 2.50 Ϊ8TΪ5 4T25

0.16 6.67 2.1 1 5.50 20.65 3.25

2

0.16 6.67 2.09 4.50 19.36 -3.20

0.16 6.67 1.97 -1 .50 20.80 4.00

0.17 13.33 247 8.50 20.85 4.25

0.16 6.67 1.96 2.00 21.94 9.70

3

#0.18 20.00 2.05 2.50 20.74 3.70

#0.18 20.00 2.21 10.50 22.55 12.75

0.14 -6.67 05 2.50 19.46 -2.70

0.13 -13.33 1.86 -7.00 19.26 -3.70

4

0.13 -13.33 1.80 10.00 19.30 -3.50

0.14 -6.67 1.98 1 .00 20.16 0.80

Mean 0.15 2XK3 20.27

SD 0.02 0.12 1 .18

CV (%) 13.33 5.91 5.82

Mean Bias (%) 0.00 1.50 1 .35

n 16 16 16

Note: C V (%) =SD/Mean^*100%.Day -1 shows the day before administration. OS shows left eye and OD showed right eye.

Table 20A. Individual Tear Content of OAHFA: LC-MS/MS Method for Quantitation of OAHFA in Rabbit Tears

Content (ng)

Sex Date Animal ID

OS OD

1917431 0.27 0.16

1917432 0.07 0.10

1917433 0.05 0.07

1917434 0.25 0.17

Mean 0.16 0.13

SD 0.12 0.05

CV(%) 72.53 38.37

1917435 0.13 0.00

1917436 0.07 0.14

1917437 0.00 0.00

Male Day -1 1917438 0.09 0.1 1

Mean 0.07 0.06

SD 0.05 0.07

CV(%) 75.02 1 17.12

1917439 0.13 0.06

1917440 0.10 0.16

1917441 0.09 0.14

1917442 0.08 0.00

Mean 0.10 0.09

SD 0.02 0.07

CV(%) 21.60 82.15

Note: CV (%) =SD/Mean^*100%. OS shows left eye and OD showed right eye.

Table 20B. LC-MS/MS Method for Quantitation of OAHFA in Rabbit Tears

Concentration/ Content (ng)

Sex Date Animal ID

Content OS OD

1917431 0TΪ6 0.30

1917432 0.05 0.10

1917433 0.05 0.07

0 1917434 0.15 0.1 1

Mean OΪ0 0.15 SD 0.06 0.10

CV(%) 59.28 72.22

1917435 10.07 10.95

1917436 24.14 4.1 1

1917437 3.17 23.72

Male Day 7 0.75% 1917438 37.44 2.28

Mean 18.71 10.27

SD 15.24 9.72

CV(%) 81.46 94.64

1917439 140.88 14.01

1917440 53.10 14.16

1917441 8.58 14.00

1% 1917442 22.1 1 15.66

Mean 56.17 14.46

SD 59.47 0.81

CV(%) 105.88 5.57

Note: CV (%) =SD/Mean^*100%. OS shows left eye and OD showed right eye.

Table 20C. LC-MS/MS Method for Quantitation of OAHFA in Rabbit Tears

Concentration/ Content (ng)

Sex Date Animal ID

Content OS OD

1917431 0.18 BQL

1917432 0.09 0.05

1917433 0.10 BQL

Day 89 0 1917434 0.08 0.05

Mean 0.1 1 0.03

SD 0.05 0.03

CV(%) 40.65 1 15.47

1917435 10.59 1 .59

1917436 0.39 1 .32

1917437 5.52 2.22

Male 0.75% 1917438 9.91 12.92

Mean 6.60 4.51

SD 4.71 5.62

CV(%) 71.37 124.49

Day 91

1917439 62.68 19.92

1917440 104.55 39.58

1917441 17.1 1 6.44

1% 1917442 32.37 12.03

Mean 54.18 19.49

SD 38.55 14.49

CV(%) 71.16 74.33

Note: CV (%) =SD/Mean^*100%.BQL shows below the low limit of quantitation. OS shows left eye and OD showed right eye.

Table 21. Sample Reanalysis (ISR)

(Reported Data-ISR

Animal ID Date Reported Data (ng) ISR Data (ng) Mean (ng)

Data)/Mean (%)

1917431 Day -1 OS 0.27 0.25 0.26 7.69

1917434 Day -1 OS 0.25 0.24 0.25 4.08

1917436 Day 7 OS 24.14 21.20 22.67 12.97

1917438 Day 7 OD 2.28 2.24 2.26 1 .77

1917439 Day 7 OD 14.01 15.35 14.68 -9.13

1917441 Day 7 OS 8.58 8.80 8.69 -2.53

1917435 Day 91 OS 10.59 1 1.70 1 1 .15 -9.96

1917437 Day 91 OD 2.22 3.29 2.76 -38.84^*

1917438 Day 91 OS 9.91 10.69 10.30 -7.57

1917439 Day 91 OD 19.92 21.90 20.91 -9.47 CO

1917441 Day 91 OS 17.1 1 18.44 17.78 -7.48

1917442 Day 91 OD 12.03 13.35 12.69 -10.40

Note: Difference (%) = (Reported data -ISR data)x 100%/Mean; Difference (%) of at least 2/3 ISR sample should be within ±20% and the result is reliable. ^*means the difference out of ±20%. OS shows left eye and OD showed right eye.

INDUSTRIAL APPLICABILITY

The disclosed process may be useful in facile, versatile and efficient preparation of a fatty acid, specifically OAHFA ((O-acyl)-omega-hydroxy fatty acids). The fatty acid as prepared using the disclosed process may be useful in preparing a composition or eye drop. The fatty acid as prepared using the disclosed process may be useful in treating dry eye disease or Meibomian gland dysfunction. The fatty acid as prepared using the disclosed process may useful as a medicament or for use in treatment of dry eye disease or Meibomian gland dysfunction. The fatty acid as prepared using the disclosed process may be useful in the manufacture of a medicament for the treatment of dry eye disease or Meibomian gland dysfunction. The fatty acid as prepared using the disclosed process may be safe to use in the treatment of dry eye disease or Meibomian gland dysfunction.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1 . A process for preparing a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; the process comprising the steps of: a) providing a compound having the following formula (I):

wherein n is an integer from 1 to 20; b) converting at least one of the hydroxyl groups of the compound of formula (I) to a terminal alkyne group; c) reacting the terminal alkyne group with a haloalkene having the following formula

(I I):

(Ill):

2. The process according to claim 1 , wherein in step b), the compound of formula (I) is reacted with a protecting group compound, whereby the protecting group compound after reacting with the compound of formula (I), forms a protecting group selected from the group consisting of tetrahydropyranyl ether, methyl ether, methoxymethyl ether, methylthomethyl ether, 2-methoxyethoxymethyl ether, 4- methoxytetrahydropyranyl ether, tetrahydrofuranyl ether, 1 -ethoxyethyl ether, 1 - methyl-1 -methoxyethyl ether, f-butyl ether, isopropyldimethylsilyl ether, t- butyldimethylsilyl ether, f-butyldiphenylsilyl ether, tribenzylsilyl ether and triisopropylsilyl ether.

3. The process according to claim 2, wherein in step b), the compound of formula (I) is reacted with 3,4-dihydro-2A7-pyran to form a compound having the following formula (IV):

4. The process according to claim 3, wherein the 3,4-dihydro-2/-/-pyran is provided as solution in hexane in a range of about 3 % to 10% by volume.

5. The process according to claim 3 or 4, wherein the molar ratio of the compound of formula (I) to 3,4-dihydro-2H-pyran is in the range of about 5:1 to about 5:2.

6. The process according to any one of claims 3 to 5, further comprising an aqueous acid selected from the group consisting of sodium hydrogen sulfate, potassium hydrogen sulfate, ammonium hydrogen sulfate, iron (III) sulfate and hydrochloric acid.

7. The process according to claim 6, wherein the molar ratio of the compound of formula (I) to sodium hydrogen sulfate is in the range of about 10:1 to about 10:2.5.

8. The process according to any one of claims 3 to 7, wherein the reaction is performed in a solvent of dimethylsulfoxide, hexane, or toluene, aqueous acid as defined in claim 6 and any mixture thereof.

9. The process according to any one of claims 3 to 8, wherein the reaction is performed at a temperature in the range of about 30 °C to about 50 °C, for a duration in the range of about 12 hours to about 20 hours.

10. The process according to any one of claims 3 to 9, wherein the compound of formula (IV) is reacted with a leaving group compound, whereby the leaving group compound after reacting with the compound of formula (IV), forms a leaving group selected from the group consisting of tosylate, mesylate, triflate, bromide and iodide.

1 1 . The process according to claim 10, wherein the compound of formula (IV) is reacted with 4-toluenesulfonyl chloride to from a compound having the following formula (V):

12. The process according to claim 1 1 , wherein the molar ratio between the compound of formula (IV) and 4-toluenesulfonyl chloride is in the range of about 10:9 to about 9:10.

13. The process according to claim 1 1 or 12, further comprising triethylamine, 4- dimethylaminopyridine, pyridine and any mixture thereof.

14. The process according to claim 13, wherein the molar ratio of the compound formula (IV) and triethylamine is in the range of about 1 :3 and about 2:3, and the molar ratio of the compound of formula (IV) and 4-dimethylaminopyridine is in the range of about 10:9 to about 9:10.

15. The process according to any one of claims 1 1 to 14, wherein the reaction is performed in a solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, pyridine and any mixture thereof.

16. The process according to any one of claims 1 1 to 15, wherein the reaction is performed at a temperature in the range of about 0 °C to about 28 °C, for a duration in the range of about 2 to about 4 hours.

17. The process according to any one of claims 1 1 to 16, wherein the compound of formula (V) is reacted with a reagent selected from the group consisting of i) a lithium acetylide/ethylene diamine complex at a ratio in the range of 2:1 to about 1 :2, ii) a mixture of acetylene gas with elemental sodium or elemental lithium and liquid ammonia, or iii) a mixture of acetylene gas, n-butylllithium, tetrahydrofuran and dimethylsulfoxide to form a compound having the following formula (VI):

18. The process according to claim 17, wherein the molar ratio of the compound of formula (V) to lithium acetylide/ethylene diamine complex is in the range of about 1 :1 to about 7:10.

19. The process according to claim 17 or 18 wherein the reaction is performed in a solvent of dimethylsulfoxide or hexamethylphosphoramide.

20. The process according to any one of claims 17 to 19, wherein the reaction is performed at a temperature in the range of about 0 °C to about 28 °C, for a duration in the range of about 10 minutes to about 60 minutes.

21 . The process according to any one of the preceding claims, wherein in step c), the terminal alkyne group is reacted with an organolithium reagent before reacting with the haloalkene.

22. The process according to claim 21 , wherein the organolithium reagent is selected from the group consisting of n-butyllthium, sec-butyllithium, ferf-butyllithium, methyl lithium, phenyl lithium, lithium dialkyl amides, lithium diisopropylamide, bistrimethylsilylamide and their respective sodium or potassium salts.

23. The process according to claim 17, wherein the molar ratio between the terminal alkyne group and the /i-butyllithium is in the range of about 10:9 to about 9:10.

24. The process according to any one of claims 21 to 23, wherein the reaction is performed in a solvent of tetrahydrofuran, diethyl ether or a mixture thereof.

25. The process according to any one of claims 21 to 24, wherein the reaction is performed at a temperature in the range of about -3 °C to about 5 °C, for a duration in the range of about 1 hour to about 3 hours.

26. The process according to any one of claims 21 to 25, wherein in step c), the molar ratio between the terminal alkyne and haloalkene is in the range of about 5:4 to about 5:8.

27. The process according to any one of claims 21 to 26, wherein the terminal alkyne is reacted with the haloalkene in the presence of L/,L/'-dimethylpropyleneurea, hexamethylphosphoramide or any mixture thereof.

28. The process according to claim 27, wherein the L/,L/'-dimethylpropyleneurea is present at a range of about 40 % to 60 % by volume.

29. The process according to claim 27 or 28, wherein the process is performed at a temperature in the range of about 0 °C to about 28 °C, for a duration in the range of about 12 hours to about 20 hours.

30. The process according to any one of claims 21 to 29, wherein the compound of step c) has the following formula (VII):

(vii).

31 . The process of any one of the preceding claims, wherein in step d), the compound of formula (III) is formed by reacting a compound having the following formula (III’) first with an activating reagent and second with a protecting reagent

32. The process according to claim 31 , wherein the activating reagent is selected from the group consisting of trifluoroacetic anhydride, concentrated sulfuric acid, A/-(3-Dimethylaminopropyl)-/\/ ' -ethylcarbodiimide hydrochloride, 4- (dimethylamino)pyridine, triethylamine, 4-(dimethylamino)pyridine and any mixture thereof.

33. The process according to claim 32, wherein the activating agent is trifluoroacetic anhydride, and the molar ratio between the compound of formula (III·) and trifluoroacetic anhydride is in the range of about 3:10 to about 3:5, the reaction is performed in tetrahydrofuran, or the reaction is performed at a temperature in the range of about -3 °C to about 5 °C, for a duration in the range of about 20 minutes to about 40 minutes.

34. The process according to any one of claims 31 to 33, wherein the protecting reagent is selected from the group consisting of tert- butyl alcohol, isobutylene, tert- butyl fluoromormate, A/,A/-dimethylamide, A/-7-nitroindoylamide, hydrazides. A/-phenylhydrazides and L/,/V-diisopropylhydrazide.

35. The process according to claim 34, wherein the protecting reagent is fert-butyl alcohol and the tert- butyl alcohol is provided in over 13 mole excess of the compound of formula (III’), and the reaction is performed at a temperature in the range of about 20 °C to about 27 °C for a duration in the range of about 1 .5 hours to about 3.5 hours.

36. The process according to any one of claims 31 to 35, wherein R¹ is fert-butyl.

37. The process according to any one of the preceding claims, wherein in step d), the contacting step is performed in an inert atmosphere first in the presence of 9- borabicyclo[3.3.1 ]nonane, then additionally in the presence of palladium (II) acetate, tricyclohexylphosphine or triisopropylphosphine and potassium hydroxide or potassium phosphate.

38. The process according to claim 37, wherein the molar ratio between the compound of step c) to 9-borabicyclo[3.3.1 ]nonane is in the range of about 9:10 to about 10:9, the molar ratio between the compound of step c) and palladium (II) acetate is in the range of 15:1 to about 10:1 , the molar ratio between the compound of step c) to tricyclohexylphosphine is in the range of about 3:1 to about 12:1 , or the molar ratio between the compound of step c) and potassium hydroxide is in the range of about 3:2 to about 3:4.

39. The process according to claim 37 or 38, wherein the reaction is performed in a solvent selected from the group consisting of tetrahydrofuran, dioxane, dimethoxyethane and any mixture thereof.

40. The process according to any one of claims 37 to 39, wherein the reaction is performed under inert atmosphere at a temperature in the range of about 20 °C to about 27 °C for a duration in the range of about 2 hours to about 30 hours.

41 . The process according to any one of claims 37 to 40, wherein the compound of step d) has the following formula (VIII):

42. The process according to claim 41 , wherein the compound of formula (VIII) is deprotected of the tetrahydropyranyl acetyl protecting group to form a compound having the following formula (IX):

43. The process according to claim 42, wherein the deprotection is done by reacting the compound of formula (VIII) with p-toluene sulfonic acid, pyridinium p- toluenesulfonate, a mixture of acetic acid, water and tetrahydrofuran, a mixture of Amberlyst H-15 and methanol or a mixture of iodine and methanol.

44. The process according to claim 42 or 43, wherein the pyridinium p- toluenesulfonate is reacted at at least an 8 molar excess of the compound of formula (VIII).

45. The process according to any one of claims 42 to 44, wherein the reaction is performed in a solvent selected from the group consisting of methanol, ethanol, 2-propanol and any mixture thereof.

46. The process according to any one of claims 42 to 45, wherein the reaction is performed at a temperature in the range of 35 °C to about 60 °C for a duration in the range of about 2 hours to about 5 hours.

47. The process according to any one of claims 42 to 46, wherein the compound of formula (IX) is reduced to form a compound having the following formula (X):

48. The process according to claim 47, wherein the reduction is performed in the presence of hydrogen gas, Lindlar’s catalyst and pyridine at a pressure of about 1 atm and a temperature in the range of about 20 °C to about 27 °C for a duration in the range of about 45 minutes to about 1.5 hours.

49. The process according to claim 48, wherein process is performed in a 1 :1 ethyl acetate-hexane solvent mixture, whereby the compound of formula (IX) is dissolved at a concentration in the range of about 0.02 M to about 0.05 M.

50. The process according to claim 48 or 49, wherein the Lindlar’s catalyst is present in a range of about 15 wt% to about 20 wt% with respect to the compound of formula (IX).

51 . The process according to any one of claims 48 to 50, wherein the pyridine is present in a range of about 0.2 vol% to about 0.7 vol% with respect to the total volume of solvent.

52. The process according to any one of the preceding claims, wherein in step e), the fatty acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, capyrlyic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-Linolenic acid, arachidonic acid and eicosapentaenoic acid.

53. The process according to claim 52, wherein the fatty acid is oleic acid.

54. The process according to claim 52 or 53, wherein the molar ratio between the compound of step d) and the fatty acid is in the range of about 1 :1 to about 1 :2.

55. The process according to claim 52 or 53, further comprising reagents selected from the group consisting of i) a mixture of A/-ethyl-A/-(3- dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, ii) a mixture of L/,/V-dicyclohexylcarbodiimide and dimethylaminopyridine, ii) thionyl chloride or phosphoryl chloride followed by a mixture of alcohol, pyridine or triethylamine and 4-dimethylaminopyridine, and iii) a mixture of oxalyl chloride with a catalytic amount of dimethylformamide followed by a mixture of alcohol, pyridine or triethylamine and 4-dimethylaminopyridine.

56. The process according to any one of claims 52 to 55, wherein the molar ratio between the compound of step d) and A/-ethyl-A/-(3- dimethylaminopropyl)carbodiimide hydrochloride is in the range of about 4:5 to about 2:5, or the molar ratio between the compound of step d) and 4- dimethylaminopyridine in the range of about 3:2 to about 1 :1.

57. The process according to any one of claims 52 to 56, wherein the reaction is performed in a solvent selected from the group consisting of dichloromethane, chloroform, acetonitrile and any mixture thereof

58. The process according to any one of claims 52 to 57, wherein the reaction is performed at a temperature in the range of about 0 °C to about 27 °C and for a duration in the range of about 2 hours to about 5 hours.

59. The process according to any one of the preceding claims, further comprising the step of replacing the R¹ group with hydrogen after step e).

60. The process according to claim 59, wherein the R¹ group is replaced with hydrogen by reacting with a reagent selected from the group consisting of trifluoroacetic acid, formic acid, a mixture of hydrochloric acid and acetic acid, a mixture of trimethylsilyl trifluoromethanesulfonate and triethylamine, and a mixture of CeCI₃-7H₂0 and sodium iodide.

61 . The process according to claim 60, wherein the reagent is trifluoroacetic acid and the trifluoroacetic acid is present in molar excess.

62. The process according to any one of claims 59 to 61 , wherein the reaction is performed in a solvent that is dichloromethane, chloroform or a mixture thereof.

63. The process according to any one of claims 59 to 62, wherein the reaction is performed at a temperature in the range of about 20 °C to about 27 °C for a duration in the range of about 20 minutes to about 40 minutes.

64. The process of any one of the preceding claims, wherein the compound of formula (A) is selected from the group consisting of the following formula (A1 ), (A2) and (A3):

wherein p, q, r’, s and t are independently integers in the range of 1 to 20.

65. The process according to claim 64, wherein in formula (A2), m is 15, r is 8, n is 8, q is 7 and r’ is 7.

66. A fatty acid obtainable by the process of any one of claims 1 to 65.

67. A composition comprising a fatty acid having the following formula (A): wherein m, n and r are independently an integer from 1 to 20; and

68. The composition according to claim 67, wherein the fatty acid is present at a concentration in the range of 0.5%w/v to about 2%w/v.

69. The composition according to claim 68, wherein the composition is a pharmaceutical composition, comprising a suitable excipient.

70. The composition according to claim 69, wherein the excipient is selected from the group consisting of oil, water, solubilizers, surfactants, cosurfactants, antioxidants, preservatives, viscosity modifying agents, electrolytes and any mixture thereof.

71 . The composition according to claim 70, wherein the oil comprises triglycerides of capric acid or capric acid, at a concentration in a range of about 0.4%w/v to about 1.0%w/v.

72. The composition according to claim 70, wherein the solubilizer is glycerol, at a concentration in a range of about 2%w/v to about 3%w/v.

73. The composition according to claim 70, wherein the surfactant is selected from polyoxyethylene (20) sorbitan monolaurate at a concentration in a range of about 1.5%w/v to about 2.5%w/v, polyethoxylated castor oil at a concentration in a range of about 0.5%w/v to about 2%w/v or any mixture thereof.

74. The composition according to claim 70, wherein the cosurfactant is sorbitan monooleate at a concentration in a range of about 0.3%w/v to about 1 .0%w/v.

75. The composition according to any one of claims 67 to 74, wherein the composition is an oil-in-water microemulsion or an oil-in-water nanoemulsion.

76. An eye drop comprising a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

77. A method for treating dry eye disease or Meibomian gland dysfunction, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

78. The method according to claim 77, wherein the fatty acid is administered to the eye of a subject.

79. The method according to claim 77 or 78, wherein the fatty acid is administered at a concentration in the range of about 0.5%w/v to about 1.5%w/v, at a volume in the range of about 30 mI_ to about 75 mI_.

80. The method according to any one of claims 77 to 80, wherein the fatty acid is administered twice a day at 12-hour intervals.

81 . The method according to any one of claims 77 to 80, wherein the dry eye disease is allergic conjunctivitis, infective keratoconjunctivitis or allergic conjunctivitis after infective keratoconjunctivitis.

82. A fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

83. A fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

84. Use of a fatty acid having the following formula (A):

wherein m, n and r are independently an integer from 1 to 20; and

R’ is selected from an optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl in the manufacture of a medicament for the treatment of dry eye disease or Meibomian gland dysfunction.