Field of the Invention

The present invention relates to pharmaceutical formulations, and the use of such formulations in medicine. In particular, the present invention relates to oral formulations which have been obtained by a process which involves wet milling of the active ingredient and which comprise an enteric coating.

Background of the Invention

AMP-activated protein kinase (AMPK) is a protein kinase enzyme that consists of three protein sub-units and is activated by hormones, cytokines, exercise, and stresses that diminish cellular energy state (e.g. glucose deprivation). Activation of AMPK increases processes that generate adenosine 5'-triphosphate (ATP) (e.g., fatty-acid oxidation) and restrains others such as fatty acid-, glycerolipid- and protein-synthesis that consume ATP, but are not acutely necessary for survival. Conversely, when cells are presented with a sustained excess of glucose, AMPK activity diminishes and fatty acid-, glycerolipid- and protein-synthesis are enhanced. AMPK thus is a protein kinase enzyme that plays an important role in cellular energy homeostasis. Therefore, the activation of AMPK is coupled to glucose lowering effects and triggers several other biological effects, including the inhibition of cholesterol synthesis, lipogenesis, triglyceride synthesis, and the reduction of hyperinsulinemia.

Given the above, AMPK is a preferred target for the treatment of the metabolic syndrome and especially type 2 diabetes. AMPK is also involved in a number of pathways that are important for many different diseases (e.g. AMPK is also involved in a number of pathways that are important in CNS disorders, fibrosis, osteoporosis, heart failure and sexual dysfunction).

AMPK is also involved in a number of pathways that are important in cancer. Several tumour suppressors are part of the AMP pathway. AMPK acts as a negative regulator of the mammalian TOR (mTOR) and EF2 pathway, which are key regulators of cell growth and proliferation. The deregulation may therefore be linked to diseases such as cancer (as well as diabetes). AMPK activators may therefore be of utility as anti-cancer drugs. 4-Chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1 ,2,4-thiadiazol-5-yl]benzamide (i.e. the compound of formula I) was first disclosed in WO 201 1/004162 and was found to be useful as an AMPK agonist.

As an AMPK agonist (i.e. an AMPK activator), the compound of formula I was found to be useful in the treatment of disorders or conditions which are ameliorated by the activation of AMPK. The compound of formula I may therefore be useful in the treatment of cancer, diabetes, hyperinsulinemia and associated conditions, a condition/disorder where fibrosis plays a role, sexual dysfunction, osteoporosis and neurodegenerative diseases.

Processes for wet milling pharmaceutically active ingredients are disclosed in e.g. Loh et ai, Asian Journal of Pharmaceutical Sciences, 10 (2015), 255-274.

An enteric coating is a barrier that is applied to an oral medication to prevent dissolution or disintegration of the medication in the gastric environment. Enteric coatings help to protect drugs from the acidity of the stomach, to protect the stomach from any detrimental effects of the drug (e.g. irritation of the stomach lining), and delay release of the drug until it reaches the upper tract of the intestine. Enteric coatings are discussed in MD. Saiful Islam et al. IJPPR. Human, 2016; Vol. 6 (3): 141-159.

The inventors have now found a formulation of the compound of formula I that is surprisingly effective in increasing the bioavailability (with reduced variation) of said compound in vivo.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Detailed Description of the Invention

According to a first aspect of the invention, there is provided a pharmaceutical formulation containing the compound of formula I,

wherein the pharmaceutical formulation is obtained by a process which involves wet milling to produce particles comprising said compound or a mixture containing said compound; and wherein the formulation comprises an enteric coating on said formulation or on said particles.

As mentioned above, the compound of formula I may be referred to as 4-chloro-N-[2-[(4- chlorophenyl)methyl]-3-oxo-1 ,2,4-thiadiazol-5-yl]benzamide. This compound name was derived using the commercially available software package Autonom (brand of nomenclature software provided as an add-on for use in the Symyx Draw 2.1 (TM) office suite marketed by MDL Information Systems).

Throughout this specification, structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails. Where it is possible for the compound to exist as a tautomer the depicted structure represents one of the possible tautomeric forms, wherein the actual tautomeric form(s) observed may vary depending on environmental factors such as solvent, temperature or pH. All tautomeric forms and mixtures thereof are included within the scope of the invention. For example, the following tautomers are included within the scope of the invention:

The compound of formula I may be prepared in accordance with techniques that are well known to those skilled in the art. For example, the compound of formula I may be made in accordance with the techniques described in international patent application WO 2011/004162, and all of its content is hereby incorporated by reference.

For the avoidance of doubt, the compound of formula I is a solid under ambient conditions, and thus the scope of the invention includes all amorphous, crystalline and part crystalline forms thereof.

Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.

The term“milling” (which may be used interchangeably with other terms of the art such as “size reduction”,“comminution”,“grinding” and“pulverization”), as used herein, refers to an operation where mechanical energy is applied (e.g. through grinding) to reduce the particle size of a solid sample (e.g. granules). For example, coarse particles may be broken down to finer ones, such that the average particle size is reduced.

Milling is regarded as a ‘top-down’ approach to the production of fine particles. For example, a drug solid may be cut by sharp blades (e.g. cutter mill), impacted by hammers, subjected to high pressure homogenisation, or crushed or compressed by the application of pressure (e.g. roller-mill or pestle and mortar). As a limited amount of energy is typically imparted, particles produced by such methods remain relatively coarse. Technological advancements in milling equipment have enabled the production of ultrafine drug particles down to the micron (i.e. the pm unit range) or even sub-micron (e.g. the nm unit range) dimensions.

Generally, a milling process may be broadly characterised as being either a dry milling process or a wet milling process.

‘Dry milling’ refers to a process in which a drug is milled in its dry state, i.e. in the absence of a liquid medium (e.g. in the substantial absence of water). In the dry state, the drug can be milled alone, or in the presence of pharmaceutically acceptable excipients. Other abrasive materials, such as salts, may be present during the milling process to aid in the particle size reduction. The mechanical energy imparted by dry milling fosters interactions between the drug (and/or excipient) particles via van der Waals forces or hydrogen bonding. The term‘wet milling’ refers to a process in which a drug is milled in the presence of a liquid medium (e.g. water). The drug particles are dispersed in a solution (often containing a surfactant and/or stabilizer) and the subsequent suspension is then subjected to milling energy. The drug concentration in the suspension typcially ranges from about 5% to about 40% (weight per volume of the suspension).

The drug particle size is reduced by the shear forces generated by the movement of the milling apparatus. The drug particles are ground between moving pearls (i.e. balls or beads), moved by an agitator, resulting in a nanosuspension. Different sized coated mill ing pearls of glass, stainless steel, zirconium dioxide or highly crosslinked polystyrene resin-coated beads may be used.

Pharmaceutically acceptable excipients (e.g. surfactants, polymers, and the like) are often added to aid the dispersion of particles by helping to minimize the agglomeration of suspended particles via electrostatic and steric mechanisms. The selection of suitable excipients is important for stabilising smaller sized particles and for maintaining the shelf- life of the final product. Commonly used excipients that may be mentioned include sugar alcohols (e.g. mannitol), amino acids (e.g. L-leucine), cellulose derivatives (e.g. hydroxypropylmethyl cellulose (HMPC) , hydroxyethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose and carboxymethylcellulose sodium salt), polymers (e.g. PVP (polyvinylpyrrolidone), polyvinyl alcohol and Carbopol® 981), vitamin derivatives (e.g. vitamin E-TPGS (D-a-tocopherol polyethylene glycol 1000 succinate), surfactants (e.g. sodium lauryl sulfate, docusate, Tween® 80 and Plantacare® 2000 UP), poloxamers (e.g. Pluronic® F68 and Pluronic® F127) and polysaccharides (e.g. sodium alginate).

The wet milling time depends on many factors such as solid content, surfactant/stablizer concentration, hardness, suspension viscosity, temperature, energy inputs and size of the milling media. The wet milling time may vary from minutes to hours or days depending on the particle size desired.

In the context of the present invention, wet milling may be performed using techniques, apparatus and conditions that are known in the art, including, for example, using a planetary mill. A planetary mill (such as a Pilverisette 7 Premium Line) usually comprises a vessel filled with (e.g. zirconia) beads. The material to be milled is placed inside the vessel, which is made to rotate or vibrate at a particular speed or frequency. The movement of the vessel causes the balls to collide with each other and the matrial to be milled. Size reduction of the drug particles is caused by impact and attritive forces received from the beads. The skilled person would appreciate that various aspects of the millling process, such as the milling time, milling speed (rotation speed), excipient misture, suspension volumne, etc. may be varied in order to achieve the desired particle size. For example, the wet milling is performed in a planetary mill at about 700 rpm for about 20 minutes.

As mentioned above, the formulation of the invention is obtained by a process which involves wet milling.

The term“a process which involves wet milling” will be understood to mean a process which involves a step of wet milling particles (comprising or consisting of the compound of formula I) so that the average particle size of the wet milled product is less than the average particle size of any product that has been produced by processes known in the art but which has not been milled.

Thus, the term“pharmaceutical formulation is obtained by a process which involves wet milling to produce particles comprising said compound” refers to a pharmaceutical formulation per se of the compound of formula I which has been prepared using any process which involves a step of wet milling particles of said compound of formula I, or a mixture containing said compound e.g. according to the processes described therein.

Solidification techniques transform drug particle suspensions, e.g. as obtained directly from a wet-milling process, into solid dosage forms such as tablets, capsules, and pellets. For example, the solvent from drug particle suspension can be removed using drying processes such as fluid bed coating / granulation, spray drying and freeze drying. Transformation into a solid dosage form helps increase the storage stability of the drug. Matrix formers (e.g., mannitol and cellulose derivatives) are usually added to suspensions before solidification to prevent destabilisation of particles due to additional thermal stresses (e.g heating during spray drying or freezing during lyophilisation).

A review of wet (and dry) milling processes for pharmaceutical products may be found in e.g. Loh et ai, Asian Journal of Pharmaceutical Sciences, 10 (2015), 255-274. Excipients suitable for inclusion in drug particles are known in the art, e.g. as described in Peltonen et ai, in Handbook of Polymers for Pharmaceutical Technologies, ed Thakur and Thakur, Wiley, volume 4, chapter 3, 67-87, and Nekkanti etal, in Drug Nanoparticles - An Overview, The Delivery of Nanoparticles, IntechOpen. The content of these documents are incorporated by reference. Wet milling reduces the average size of the particles containing the compound of formula I. The extent and effectiveness of the wet milling may be determined by measuring the particle size distribution of said particles before and after the wet milling process.

The term “particle size distribution” refers to the relative number of particles present according to size in a solid sample, such as a powder, a granular material, or particles dispersed in a fluid. Particle size distribution affects the properties of a solid sample (e.g. a powder, and the like) in many ways. We have found that the reduction of the average particle size results in a surprising improvement in the bioavailability of the resulting pharmaceutical product when the compound of formula I is combined with an enteric coating.

The particle size distribution of a solid sample may be measured using techniques that are well known in the art. For example, the particle size distribution of a solid sample may be measured by laser diffraction, dynamic light scattering, image analysis (e.g. dynamic image analysis), sieve analysis, air elutriation analysis, optical counting, electro-resistance counting, sedimentation, laser obscuration and acoustic (e.g. ultrasound attenuation) spectroscopy. Particular methods that may be mentioned for measuring the particle size distribution of particles of the formulation of the invention are dynamic light scattering and laser diffraction.

Particle size distributions also may be determined based on results from sieve analysis. Sieve analysis presents particle size information in the form of an S-curve of cumulative mass retained on each sieve versus the sieve mesh size. The most commonly used metrics when describing particle size distributions are D-values (D10, D50 & D90) which are the intercepts for 10%, 50% and 90% of the cumulative mass. The particle size distribution of the present invention is preferably defined using one or more of such values. D-values essentially represent the diameter of the sphere which divides the sample's mass into a specified percentage when the particles are arranged on an ascending mass basis. For example, the D10 value is the diameter at which 10% of the sample's mass is comprised of particles with a diameter of less than this value. The D50 value is the diameter of the particle that 50% of a sample's mass is smaller than and 50% of a sample's mass is larger than.

For volume weighted particle size distributions, such as those measured by laser diffraction, it is often convenient to report parameters based upon the maximum particle size for a given percentage volume of the sample. For example, the D _v 50 is the maximum particle diameter below which 50% of the sample volume exists - also known as the median particle size by volume. Similarly, the D _v 10 and D _v 90 are the maximum particle diameter below which 10% and 90% of the sample volume exists. Commonly reported percentiles are the D _v 10, D _v 50 and D _v 90.

Formulations of the invention may further be characterised by the average diameter (also referred to as the Z-average diameter) of the wet milled particles of the compound of formula I. The Z-average diameter of the particles may be measured in the suspension comprising the wet milled particles, e.g. by dynamic light scattering.

The term“Z-average diameter” refers to the harmonic intensity averaged particle diameter of a sample of particles measured by dynamic light scattering. The Z-average is derived from a Cumulants analysis of the measured correlation curve, wherein a single particle size is assumed and a single exponential fit is applied to the autocorrelation function.

As described herein, there is provided a formulation comprising particles containing the compound of formula I, wherein said particles have a Z-average diameter of less than about 1000 nm, and wherein the formulation comprises an enteric coating on said formulation or on said particles.

The particle size distribution parameters mentioned above may be applicable, individually or in combination, to any given formulation.

The particle size distribution of a formulation comprising particles containing the compound of formula I may be measured after the wet-milling by dynamic light scattering, using, for example a particle size analyser (such as a Malvern ZetaSizer Nano). Where such a process involves the dispersion of the substance to be analysed, a suitable amount of a dispersion agent may be used, for example water.

The present invention also encompasses a pharmaceutical formulation comprising particles containing the compound of formula I with any of the particle size distributions defined herein, regardless of the process by which the formulation is produced.

Thus, according to a second aspect of the invention there is provided a formulation comprising particles containing the compound of formula I, wherein said particles have a particle size distribution defined by a Z-average diameter of less than about 1000 nm; and wherein the formulation comprises an enteric coating on said formulation or on said particles, preferably where the particle size distribution has been measured after the particles have been wet-milled.

Preferably, the pharmaceutical formulation of the second aspect of the invention comprises particles of the compound of formula I with any of the particular particle size distributions described herein, wherein the particles are obtained by a process which involves wet milling said compound or a mixture containing said compound. Therefore, in a particular embodiment of the second aspect of the invention, the particles are obtained by a process which involves wet milling said compound or a mixture containing said compound.

Formulations according to the first and second aspects of the invention are herein referred to as“formulations of the invention”. The pharmaceutical formulations of the invention may be prepared in accordance with standard and/or accepted pharmaceutical practice.

In an embodiment of the first and second aspects of the invention, the compound of formula I is the sole active pharmaceutical ingredient in the particles, or in the formulations of the invention.

Wet milling is useful for producing particles that are generally smaller in size than those obtained from dry milling. Particular particle size distributions that may be mentioned in the context of the present invention include those with a Z-average diameter value of from about 20 nm to about 800 nm; a Z-average diameter of from about 20 nm to about 600 nm; a Z-average diameter of from about 25 nm to about 200 nm; or a Z-average diameter of from about 25 nm to about 100 nm. More specifically, the particles comprising of the compound of formula I present in a formulation of the invention may have a Z-average diameter of about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 550 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm or about 1000 nm. Said Z-average diameter values preferably relate to particles that have been wet milled and that comprise the compound of formula I and further excipients (e.g. PVP K30, Na-docusate and/or mannitol).

In a particular embodiment of the formulation of the invention (i.e. formulations according to any embodiment of the first or second aspects of the invention) the particles comprising of the compound of formula I particles have a particle size distribution defined by a Z- average diameter of from about 20 nm to about 800 nm.

Preferably, the particles comprising the compound of formula I may have a Z-average diameter of from about 25 nm to about 200 nm.

More preferably, the particles comprising the compound of formula I may have a Z-average diameter of from about 25 nm to about 100 nm.

The product obtained by the wet milling process may be converted into a solid form, e.g. using any of the solidification processes disclosed herein, including fluid bed coating / granulation, spray drying and freeze drying. Other techniques for removing the liquid medium known to the person skilled in the art may be used. Where solidification is achieved using freeze-drying, for example, the particle size distribution may change. Such freeze-dried products, and products that have been solidified by other processes, are included within the scope of the invention. Where the wet-milled product is freeze-dried, the particle size distribution may be characterised by a D _v 50 of from about 5 pm to about 100 pm, from about 10 pm to about 90 pm, from about 20 pm to about 80 pm, from about 30 pm to about 70 pm, or from about 40 pm to about 60 pm. More specifically, the particles consisting of the compound of formula I present in a formulation of the invention may have a particle size distribution defined by a D _v 50 of about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm. Said particle size distributions preferably relate to particles that comprise of the compound of formula I and suitable excipients known in the art. These particle size distributions relate to particles that have been subjected to a freeze-thaw cycle after they have been wet milled.

Formulations of the invention may also be defined by the average Pdl (polydispersity index) of the particles. The polydispersity index of the particles indicates the degree of particle size homogeneity in a suspension. On the nanoscale, the Pdl is always less than 1. A Pdl of less than 0.2 indicates a mono-dispersed suspension, whereas a Pdl greater than 0.7 is an indication of a broad particle size distribution or of particle aggregation.

Particular Pdl values that may be mentioned in the context of the present invention include those that are less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6 or less than about 0.5. More specifically, the particles comprising of the compound of formula I present in a formulation of the invention may have a particle size homogeneity defined by Pdl of about 0.5, about 0.6 or about 0.7. Said Pdl values preferably relate to particles that have been wet milled and that comprise the compound of formula I and further excipients (e.g. PVP K30, Na-docusate and/or mannitol).

The compound of formula I is not considered to be acid labile, and protection from exposure to stomach acid has therefore not been found to be necessary for oral formulations containing the compound of formula I. Nevertheless, it has surprisingly been found that the use of an enteric coat can improve the pharmacokinetic properties of formulations containing this compound, particularly when the compound has been wet milled to produce a particle size distribution as described herein. The pharmaceutical formulations of the invention therefore comprise an enteric coating. An“enteric coating” is a substance (e.g. a polymer) that is applied on the surface of an oral medication (e.g. on the surface of a tablet or a capsule, or on the surfaces of particles or pellets comprised in an oral formulation) that inhibits dissolution or disintegration of the medication in the gastric environment. Enteric coatings are stable at the highly acidic pH found in the stomach, but break down rapidly in the relatively basic pH of the small intestine. Therefore, enteric coatings prevent release of the active ingredient in the medication until the medication reaches the small intestine e.g. the duodenum, the jejunum and the ileum. The use of an enteric coating may therefore allow for targeted release of an active ingredient in the small intestine.

For the avoidance of doubt, the enteric coating is present on surface of the formulation of the invention (e.g. on the surface of a tablet, a capsule or pellets), or the particles comprising said compound of formula I are coated with the enteric coating.

In a particular embodiment, the formulation comprises enteric coated particles of the compound of formula I and the formulation is in a form that does not necessarily have (but preferably does have) an enteric coating on the overall formulation, e.g. an uncoated tablet containing coated particles. Any enteric coating known to the skilled person may be used in the present invention. Particular enteric coating materials that may be mentioned include those which comprise beeswax, shellac, an alkylcellulose polymer resin (e.g. ethylcellulose polymers, carboxymethylethylcellulose, or hydroxypropyl methylcellulose phthalate) or an acrylic polymer resin (e.g. acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, methyl methacrylate, copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, methyl methacrylate copolymers, methacrylate copolymers, methacrylic acid copolymer, aminoalkyl methacrylate copolymer, methacrylic acid copolymers, methyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), methyl methacrylate, polymethacrylate, methyl methacrylate copolymer, poly(methyl methacrylate), poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, poly(methacrylic acid anhydride), glycidyl methacrylate copolymers), cellulose acetate phthalate, and polyvinyl acetate phthalate. A particular polyacrylic resin that may be mentioned is polyacrylic resin HB-50.

For the avoidance of doubt, the skilled person will understand that references herein to particular aspects of the invention (such as the first aspect of the invention, i.e. referring to a pharmaceutical formulation of the compound of formula I obtained by wet milling particles of said compound, and wherein the formulation comprises an enteric coation on said formuation or on said particles) will include references to all embodiments and particular features thereof, which embodiments and particular features may be taken in combination to form further embodiments and features of the invention.

The formulations of the invention (as defined by the first aspect of the invention, as well as the second aspect of the invention described elsewhere herein, and all embodiments of those aspects) have been found to be surprisingly effective at improving (e.g. increasing) the bioavailability of the compound of formula I in vivo compared to a pharmaceutical formulation in which the compound of formula I has not been wet milled. Improvement in bioavailability may be demonstrated by measuring the C _max or the area under the curve (AUC) following administration of the pharmaceutical formulation to a subject. Preferably the subject is a human.

The terms“Cmax” and“AUC” will be well understood by the person skilled in the art to refer, in this context, to the peak plasma concentration of the compound of formula I after administration (e.g. to a human subject) and the integral of the concentration/time curve post administration of said compound, respectively.

The data in the examples show that the use of wet-milling for the active ingredient coupled with administration via a formulation comprising an enteric coated capsule surprisingly resulted in substantial (several-fold) increase in the systemic exposure of compound of formula I as compared to a non-milled product in a non-coated formulation. Moreover, the variation in the bioavailability between subjects who have been administered wet milled compound of formula I in an enteric coated capsule has been found to be surprisingly low compared to the variation in the bioavailability between subjects who have been administered wet milled compound of formula I in a non-coated capsule.

The effect of micronisation of any small molecule active ingredient on dissolution and bioavailability is not always easily predicted. Aggregation of finely ground particles may occur which can ultimately slow down the dissolution process. Stabilisers, such as polymers and surfactants, may need to be added in order to increase the repulsion between particles and inhibit aggregation.

Thus, the formulation of the invention results in the bioavailability of the compound of formula I being increased compared to the bioavailability of the compound of formula I in a pharmaceutical formulation in which the compound of formula I has not been wet milled or the formulation does not comprise an enteric coating. The variation in the bioavailability of the compound of formula I may additionally or alternatively be reduced compared to the variation in the bioavailability of the compound of formula I in a pharmaceutical formulation in which the compound of formula I has not been wet milled or that does not comprise an enteric coating.

When we state“the bioavailability of the compound of formula I is increased compared to the bioavailability of the compound of formula I in a pharmaceutical formulation in which the compound of formula I has not been wet milled”, we mean that administration of the formulation of the invention results in a larger systemically available fraction of the compound of formula I in vivo compared to administration of a formulation in which the compound of formula I has not been wet milled. The increase in the amount of compound of formula I that is systemically available following administration of the formulation of the invention as compared to administration of a formulation in which the compound of formula I has not been wet milled (e.g. as is represented by the C _max or AUC) may be at least about 10%, (at least) about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% (i.e. 2-fold), about 150%, about 200% (i.e. 3-fold), about 250%, about 300% (i.e. 4-fold), about 350%, about 400% (i.e. 5-fold), about 450%, about 500% (i.e. 6-fold), about 600% (i.e. 7-fold), about 700% (i.e. 8-fold), about 800% (i.e. 9-fold) or about 900% (i.e. 10-fold).

The improvement of the bioavailability provided by the formulations of the invention may be demonstrated using suitable methods known in the art. For example, the improvement in bioavailability may be demonstrated by comparing the AUC data of a subject who has been administered a formulation of the invention with the AUC data of a subject who has been administered a pharmaceutical formulation in which the compound of formula I has not been wet milled or that does not comprise an enteric coating.

When we state“the variation in the bioavailability of the compound of formula I is reduced compared to the variation in the bioavailability of the compound of formula I in a pharmaceutical formulation in which the compound of formula I has not been wet milled or that does not comprise an enteric coating”, we mean that the inter-individual variation in bioavailability between subjects who have been administered a formulation of the invention is at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% less compared to the inter-individual variation in the bioavailability of the compound of formula I between subjects who have been administered a formulation in which the compound of formula I has not been wet milled or that does not comprise an enteric coating. In one embodiment, the difference between the bioavailability (as represented by the C _max or AUC) value for a subject that has been administered a formulation of the invention and the median bioavailability (or the median C _max or AUC) value for a population of subjects that have been administered said formulation is not more than 30%, e.g. not more than 25%, not more than 20%, not more than 15%, not more than 10%, not more than 5% of said median bioavailability (or the median C _max or AUC) value.

The reduction in the variability of bioavailability provided by the formulations of the invention may be determined by suitable methods known in the art. For example, the reduction may be assessed comparing the C _max and AUC values of subjects following administration of the formulation.

The formulation of the invention will generally be administered as a mixture comprising the compound of formula I and one or more pharmaceutically acceptable excipients. The one or more pharmaceutically acceptable excipients may be selected with due regard to the intended route of administration in accordance with standard pharmaceutical practice. Such pharmaceutically acceptable excipients are preferably chemically inert to the active compound and are preferably have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). A brief review of methods of drug delivery may also be found in e.g. Langer, Science 249, 1527 (1990).

Thus, according to particular embodiments, the formulation further comprises at least one pharmaceutically acceptable excipient. In particular, the at least one pharmaceutically acceptable excipient may be a lubricant, a binder, a filler, a surfactant, a diluent, an anti adherent, a coating, a flavouring, a colourant, a glidant, a preservative, a sweetener, a disintegrant, an adsorbent, a buffering agent, an antioxidant, a chelating agent, a dissolution enhancer, a dissolution retardant or a wetting agent.

Particular pharmaceutically acceptable excipients that may be mentioned include mannitol, PVP (polyvinylpyrrolidone) K30, lactose, saccharose, sorbitol, starch, amylopectin, cellulose derivatives, gelatin, or another suitable ingredients, as well as disintegrating agents and lubricating agents such as Na-docusate, magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes. In the preparation of a pharmaceutical formulation of the invention for oral administration, particles comprising of the compound of formula I may be wet milled, either together or separately, with mannitol, PVP (polyvinylpyrrolidone) K30 and Na-docusate. Thus, in particular embodiments, the formulation comprises PVP K30, Na-docusate and mannitol.

Such mixtures may then be processed into blocks, pellets, granules a powder, or compressed into tablets or mini-tablets.

The skilled person will understand that formulations of the invention may act systemically, and may therefore be administered accordingly using suitable techniques known to those skilled in the art. Formulations as described herein will normally be administered orally, in a pharmaceutically acceptable dosage form. Thus, the pharmaceutical formulation of the present invention is preferably an oral pharmaceutical formulation.

Formulations of the invention may also be prepared for oral administration in the form of a capsule. For example, capsules such as soft gelatin capsules may be prepared containing a formulation of the invention alone, or together with a suitable vehicle, e.g. vegetable oil, fat etc. Similarly, hard gelatin capsules may contain the formulation of the invention alone, or in combination with solid powdered ingredients such as a disaccharide (e.g. lactose or saccharose), an alcohol sugar (e.g. sorbitol or mannitol), a vegetable starch (e.g. potato starch or corn starch), a polysaccharide (e.g. amylopectin or cellulose derivatives) or gelling agent (e.g. gelatin).

Thus, pharmaceutical formulations of the invention include formulations that are provided in the form of a tablet or capsule e.g. for oral administration.

As mentioned above, the formulation of the invention comprises an enteric coating. The enteric coating is present on surface of the formulation (e.g. on the surface of a tablet or a capsule) or each of the particles comprising the compound of formula I are coated with the enteric coating.

Thus, in particular embodiments, the formulation is provided in the form of a capsule or tablet, and the enteric coating is present as an outer layer on said capsule or tablet. For example, the formulation may be in the form of a capsule with an enteric coating, the capsule containing wet milled compound of formula I, or alternatively the wet milled compound of formula I may be compressed into a tablet and the tablet is subsequently coated with an enteric coating.

Alternatively, the formulation is provided in the form of a capsule or tablet containing particles comprising the compound of formula I, wherein the particles are coated with the enteric coating. Optionally, said particles may have a particle size distribution defined by a D _v 50 of less than about 100 pm; a D _v 50 of from about 20 pm to about 100 pm; a D _v 50 of from about 20 pm to about 90 pm; a D _v 50 of from about 20 pm to about 50 pm; a D _v 50 of from about 50 pm to about 100 pm; or a D _v 50 of from about 70 pm to about 90 pm. In one embodiment, particles comprising the compound of formula I are first wet milled to obtain the desired particle size distribution. They are then may be coated with an enteric coating. Said coated particles may then be compressed into a tablet or loaded into a (e.g. non- coated) capsule.

As described herein, formulations of the invention may be prepared by a process that involves wet milling the compound of formula I, or a mixture containing said compound, to produce particles with a particle size distribution defined by a D _v 50 of less than about 100 pm. Thus, according to a third aspect of the invention, there is provided a process for preparing a capsule or tablet as defined herein above, which process comprises:

(i) wet milling the compound of formula I, optionally together with one or more excipients, to produce particles having a particle size distribution defined by a Z-average diameter of less than about 1000 nm;

(ii) drying the product of step (i); and

(iii) incorporating the particles obtained in step (ii) into a capsule or a tablet.

Optionally, the compound of formula I, is mixed with PVP K30, Na-docusate and mannitol prior to wet-milling.

In the process of the third aspect of the invention, the enteric coating may be introduced at any time point, provided the compound of formula I in the resulting formulation is encompassed with the enteric coating. For example, the process may further comprise the step of coating said capsule or tablet with the enteric coating before or after (preferably before) the particles are incorporated into said capsule or tablet. Alternatively, the process may further comprise the step of applying the enteric coating onto the particles prior to the incorporation of said particles into a capsule or tablet.

Pharmaceutical formulations that may be mentioned include those in which the compound of formula I is present in a total amount that is at least 1 % (or at least 10%, at least 30% or at least 50%) by weight of the formulation. That is, the weight ratio of the compound of formula I to the totality of the components (i.e. the compound of formula I and all pharmaceutical excipients, e.g. adjuvants, diluents and carriers) of the pharmaceutical formulation is at least 1 :99 (or at least 10:90, at least 30:70 or at least 50:50).

A“therapeutically effective amount”, an“effective amount” or a“dosage” as used herein refers to an amount of a formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount or dosage will vary with the age or general condition of the subject, the severity of the condition being treated, the particular agents administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, a“therapeutically effective amount”,“effective amount” or“dosage” in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. The skilled person will understand that formulations of the invention may be administered (for example, by way of one or more preparations as described herein) at varying doses, with suitable doses being readily determined by one of skill in the art. The total dosage of the compound of formula I that is to be administered to a subject in need thereof may range from about 0.01 to about 2000 mg/kg of body weight per day (mg/kg/day), about 0.1 to about 500 mg/kg/day, and about 1 to about 100 mg/kg/day. Such dosages may be, for example, oral dosages of the formulations of the invention.

When administered orally, treatment with such formulations (including capsules containing such formulations) may comprise administration of a unit dose formulation containing from about 0.01 mg to about 3000 mg of the compound of formula I, for example from about 0.1 mg to about 2000 mg, or from about 1 mg to about 1000 mg (e.g. from about 10 mg to about 500 mg), of the active ingredient. Advantageously, treatment may comprise administration of the formulation of the invention (including capsules containing said formulation) using a single daily dose. Alternatively, the total daily dosage of the compound of formula I may be administered in divided doses two, three or four times daily (e.g. twice daily with reference to the doses described herein, such as a dose of 100 mg, 250 mg, 500 mg or 1000 mg twice daily). The skilled physician will recognise that the dosage will vary from subject to subject.

In particular embodiments, the formulation of the invention is administered to a subject where the daily dose of the compound of formula I is in the range of from about 1 to about 3000 mg, preferably from about 1 to about 1000 mg.

The term "about," as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, refers to variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount. It is contemplated that, at each instance, such terms may be replaced with the notation“±10%”, or the like (or by indicating a variance of a specific amount calculated based on the relevant value). It is also contemplated that, at each instance, such terms may be deleted.

For the avoidance of doubt, the dose administered to a subject, particularly a human subject, in the context of the present invention should be sufficient to effect a therapeutic response in the subject over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the subject to be treated, and the stage/severity of the disease.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage which will be most suitable for an individual subject. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Medical uses

As indicated herein, the formulations of the invention are useful as pharmaceuticals.

Formulations of the invention (i.e. a formulation as defined in the first or second aspect of the invention, including all embodiments and particular features thereof) may be particularly useful in treating a disorder or condition ameliorated by the activation of AMP- activated protein kinase (AMPK). Thus, in a fourth aspect of the invention, there is provided the use of a formulation of the invention, or a capsule containing said formulation, as defined herein, in the manufacture of a medicament for the treatment of a disorder or condition ameliorated by the activation of AMPK.

Similarly, there is provided a method of treating a disorder or condition ameliorated by the activation of AMPK comprising administering to a subject in need thereof a therapeutically effective amount of a formulation of the invention or a capsule containing said formulation. Likewise, there is provided the formulation of the invention (or a capsule containing said formulation) for use in a method of treating a disorder or condition ameliorated by the activation of AMPK.

By 'activate AMPK’, we mean that the steady state level of phosphorylation of the Thr-172 moiety of the AMPK-a subunit is increased compared to the steady state level of phosphorylation in the absence of the compound of formula I. Alternatively, or in addition, we mean that there is a higher steady state level of phosphorylation of any other proteins downstream of AMPK, such as acetyl-CoA carboxylase (ACC). The terms“disorder or condition ameliorated by the activation of AMPK” will be understood by those skilled in the art to include cancer, diabetes, hyperinsulinemia and associated conditions, a condition/disorder where fibrosis plays a role, sexual dysfunction, osteoporosis and neurodegenerative diseases.

The term "cancer" will be understood by those skilled in the art to include one or more diseases in the class of disorders that is characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion, proliferation or by implantation into distant sites by metastasis.

The compound of formula I is capable of inhibiting the proliferation of cancer cells and the metastasis of cancer cells. By "proliferation" we include an increase in the number and/or size of cancer cells. By "metastasis" we mean the movement or migration (e.g. invasiveness) of cancer cells from a primary tumor site in the body of a subject to one or more other areas within the subject's body (where the cells can then form secondary tumors).

Thus, formulations of the invention may be suitable for use in the treatment of any cancer type, including all tumors (non-solid and, preferably, solid tumors, such as carcinoma, adenoma, adenocarcinoma, blood cancer, irrespective of the organ). For example, the cancer cells may be selected from the group consisting of cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, thyroid, hematopoietic system, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract. Preferably, the cancer is selected from the group of colon cancer (including colorectal adenomas), breast cancer (e.g. postmenopausal breast cancer), endometrial cancer, cancers of the hematopoietic system (e.g. leukemia, lymphoma, etc.) thyroid cancer, kidney cancer, oesophageal adenocarcinoma, ovarian cancer, prostate cancer, pancreatic cancer, gallbladder cancer, liver cancer and cervical cancer. More preferably, the cancer is selected from the group of colon, prostate and, particularly, breast cancer. Where the cancer is a non-solid tumor, it is preferably a hematopoietic tumor such as a leukemia (e.g. Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL).

The term "diabetes" (i.e. diabetes mellitus) will be understood by those skilled in the art to refer to both type 1 (insulin-dependent) diabetes and type 2 (insulin-independent) diabetes, both of which involve the malfunction of glucose homeostasis. Thus, formulations of the invention may be suitable for use in the treatment of type 1 diabetes and/or type 2 diabetes.

The term“hyperinsulinemia or an associated condition” will be understood by those skilled in the art to include hyperinsulinemia, type 2 diabetes, glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hyperinsulinism in childhood, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions such as stroke, systemic lupus erythematosus, neurodegenerative diseases such as Alzheimer’s disease, and polycystic ovary syndrome. Other disease states include progressive renal disease such as chronic renal failure.

A condition/disorder where fibrosis plays a role includes (but is not limited to) scar healing, keloids, scleroderma, pulmonary fibrosis (including idiopathic pulmonary fibrosis), nephrogenic systemic fibrosis, and cardiovascular fibrosis (including endomyocardial fibrosis), systemic sclerosis, liver cirrhosis, eye macular degeneration, retinal and vitreal retinopathy, Crohn’s/inflammatory bowel disease, post-surgical scar tissue formation, radiation and chemotherapeutic-drug induced fibrosis, and cardiovascular fibrosis.

Formulations of the invention may also be useful in the treatment of sexual dysfunction (e.g. the treatment of erectile dysfunction).

Neurodegenerative diseases that may be mentioned include Alzheimer ^' s disease, Parkinson ^' s disease and Huntington ^' s disease, amyotrophic lateral sclerosis, polyglutamine disorders, such as spinal and bulbar muscular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), and a number of spinocerebellar ataxias (SCA).

The skilled person will understand that references to the “treatment” of a particular condition (or, similarly, to“treating” that condition) will take their normal meanings in the field of medicine. In particular, the terms may refer to achieving a reduction in the severity and/or frequency of occurrence of one or more clinical symptom associated with the condition, as judged by a physician attending a subject having or being susceptible to such symptoms. As used herein, references to a subject (or to subjects) refer to a living subject being treated, or receiving preventative medicine, including mammalian (e.g. human) subjects. In particular, references to a subject refer to a human subject.

The skilled person will understand that such treatment or prevention will be performed in a subject in need thereof. The need of a subject for such treatment or prevention may be assessed by those skilled the art using routine techniques.

In the context of the present invention, a“subject in need” of the formulation of the invention includes a subject that is suffering a disorder or condition ameliorated by the activation of AMPK.

As used herein, the terms“disease” and“disorder” (and, similarly, the terms condition, illness, medical problem, and the like) may be used interchangeably.

Without wishing to be bound by theory, it is believed that the formulations of the invention as described herein are capable of improving the bioavailability of the compound of formula I, i.e. by increasing the amount of the compound in the systemic circulation following administration of the formulation to a subject. Formulations of the invention may provide at least a three-fold, a four-fold, a five-fold, six-fold, eight-fold or ten-fold (or greater) increase in the bioavailability of the compound of formula I compared to a formulation that was not obtained using a wet milling process. In particular, a surprising increase in bioavailability has been obtained for wet milled products (wherein an enteric coating is present) in comparison to dry milled products.

Formulations of the invention may have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, other therapies known in the prior art, whether for use in the above-stated indications or otherwise. In particular, formulations of the invention may have the advantage that they are more efficacious and/or exhibit advantageous properties in vivo.

Figures The following figures are provided to illustrate various aspects of the present inventive concept and are not intended to limit the scope of the invention unless specified herein.

Figure 1 shows comparative results of oral pharmacokinetic studies of non-milled (without excipients), dry milled (without excipients) and wet milled (with excipients) compound of formula I, as a suspension and in non-coated and enteric coated capsules.

Figure 2 shows comparative results of oral pharmacokinetic studies of non-milled and to dry milled compound of formula I (with excipients) in non-coated and enteric coated capsules.

Figures 3 and 4 show absolute and relative C _max results for formulations (without excipients) of non-milled and dry milled compound of formula I in non-coated and enteric coated capsules.

Figures 5 and 6 show absolute and relative AUC results for formulations (without excipients) of non-milled and dry milled compound of formula I in non-coated and enteric coated capsules.

Figures 7 and 8 show absolute and relative C _max results for formulations (with excipients) of non-milled, dry milled and wet milled compound of formula I in non-coated and enteric coated capsules.

Figures 9 and 10 show absolute and relative AUC results for formulations (with excipients) of non-milled, dry milled and wet milled compound of formula I in non-coated and enteric coated capsules.

Figure 11 shows results of oral pharmacokinetic studies of wet-milled compound of formula I administered in non-coated capsules.

Figure 12 shows results of oral pharmacokinetic studies of wet-milled compound of formula I administered in enteric coated capsules.

Figure 13 shows results of oral pharmacokinetic studies of dry-milled compound of formula I administered in enteric coated capsules. Examples

Abbreviations

AUCo-t: Area under the concentration-time curve from time zero to last quantifiable concentration

AUCo- : Area under the concentration-time curve from time zero to infinity b.w.: Body weight

CE: Collision energy

Cmax- Peak plasma concentration.

CXP: Collision exit potential

DLS: Dynamic light scattering

DP: Declustering potential

EP: Entrance potential

h: Hours

HPLC: High performance liquid chromatography

ISD: Internal standard

LC: Liquid chromatography

LC-MS/MS: Liquid chromatography - (tandem) mass spectrometry

LS: Light scattering

MRT: Mean residence time.

min: Minutes

PVP K30: Polyvinylpyrrolidone K 30

Pdl: Polydispersity index

Rl: Refractive index

rpm: Revolutions per minute

RT: Room temperature

Sdv Z-avg: Standard deviation in Z-average

T1/2: Half-life

T max- Time to reach the peak plasma concentration

w/v: Weight per volume

The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.

Materials

4-Chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1 ,2,4-thiadiazol-5-yl]benzamide (compound 1) was prepared by Anthem Biosciences. Sodium-docusate, PVP K30 and mannitol were supplied by Sigma-Aldrich.

Example 1 - dry milled compound of formula I Dry milling was performed for Examples 1a to 1c using an air jet mill (Equipment code: CP-AJM-01 ; Promas engineers) with the following parameters:

Micronisation was repeated in Example 1c by performing the air jet milling for a second time using the same parameters. This allowed the particle size D90 to be reduced to less than 10 pm.

Particle size analysis

Reagents

Instrument

Method parameter

Preparations

Four drops of Tween 20 were added to 25m L of water and the mixture was sonicated for 3 minutes. 0.05 g of milled material was transferred into a 250ml_ glass beaker. 25 ml_ of the dispersant solution (Tween 20/water mixture) was added to the beaker with continuous swirling for 2 to 3 minutes. The suspension was transferred into the measuring unit and the particle size distribution measurements were conducted in triplicate.

^# Dry milling performed once on sample

* Dry milling performed twice on sample

Table 1 : Average particle size results for non- and dry-milled compound of formula I

Example 2 - wet milled compound of formula I

Formulations

Four different formulation examples were prepared and tested. All of the formulations contained sodium-docusate and PVP K30 in the same proportion . Examples 2c and 2d further comprised mannitol.

Milling

Wet-milling was performed using a Pulverisette 7 Premium Line planetary mill. Two milling bowls were filled with 0.65 mm zirconia beads and approximately 15 g of suspension in each bowl. The amounts of zirconia beads were adjusted to the size of the bowls (45 mL) according to the recommendations in the instrument manual (approximately 60 g of beads in each bowl). The suspensions were milled using the following milling program:

Speed: 700 rpm

Milling time: 20 min

Pause in between cycles: 30 min

Number of cycles: 3

The suspensions were then sampled for particle size distribution measurements.

Particle size measurements

The particle size distribution of each formulated example was measured at the following time points and storage conditions after milling:

Table 3. Time points and storage conditions of the formulated solutions for particle size measurements. Particle size analysis

The suspensions were analysed with a Malvern ZetaSizer Nano (Dynamic Light Scattering (DLS) equipment). Approximately 1 mL of nanosuspension was used to fill a disposable micro cuvette. The measurements were run in duplicate. The freeze/thaw samples were analysed with a Malvern Mastersizer 2000 equipped with a Hydro2000pP (A) unit (light scattering (LS) equipment) due to larger particle size. The suspensions were added directly to the volume dispersion unit until an obscuration value between 5-10% was obtained. Measurements were also run in duplicate. The following instrument settings were used for both the DLS and LS:

Dispersant Rl: 1.330

Particle Rl: 1.690

Particle Absorption: 0.05

Measurements per sample: 3

Runs recorded per measurement: 11

Results

The particle size distribution of each of the formulated nanosuspensions was evaluated after milling, after 24 h and 7 days stored at 2-8 °C. Table 4 shows the average results of the particle size measurements performed with Malvern ZetaSizer (dynamic light scattering) for each of the formulated examples after milling.

Table 4. Particle size measurements results at different time points for Examples 2a-d.

After evaluation of the results obtained for the formulated systems, Example 2c was chosen for use in PK studies.

All the formulations contained Na-Docusate and PVP K30 in the same proportion (see table 2). The systems with a higher concentration of the active substance (100 mg/ml - example 2a and example 2c) showed a smaller particle size after milling. Changes in particles size after storage for 7 days were not more than ±10 %.

Despite the similarities in particle sizes between example 2a and example 2c, example 2c was selected for the manufacturing of the nanosuspension for having mannitol in the formulation. Mannitol is usually added as co-milling agent to increase coalition between particles favouring particle size reduction during milling. Its effect is not significant at this scale for the formulated example 2a and example 2c, but it might help to reduce the particle size if scale up of the process is necessary at later stages. The effect of mannitol is however noticeable for example 2b and example 2d which have a lower concentration of the active substance (50 mg/ml). A higher concentration of the compound of formula I also increases the mechanical forces during wet-milling thus reducing the particle size of the suspension.

Polydispersity index (Pdl) indicates the degree of particle size homogeneity in a suspension. In nanoscale systems Pdl is always lower than 1. An index of 0.2 or lower indicates a mono dispersed suspension while a Pdl higher than 0.7 is an indication of a broad particle size distribution or particle aggregation. The Pdl index of the formulated systems is in the range of 0.6±0.1 which shows a certain degree of particle size variation increasing the tendency of particle aggregate formation (see table 4).

Examples 3 to 13 - Single Dose Oral Pharmacokinetic Studies in Rabbits

Formulation Preparation

1. Capsules

Ready to use enteric coated and non-coated capsules were used. Capsules were obtained from CapsulCN International Co., Ltd.

For non-milled compound of formula I, the compound of formula I was made using the process described in WO 201 1/004162. The non-coated or enteric coated gelatin capsules were individually filled with 180 mg of non-milled compound of formula I, or a mixture containing said compound and accompanying excipients as indicated. Both capsule types were filled with either compound of formula I alone or compound of formula I together with 1.8 mg of sodium docusate, 0.18 mg of PVP K30 and 9 mg of mannitol.

Similarly, for dry-milled compound of formula I, non-coated or enteric coated gelatin capsules were individually filled with 180 mg of dry-milled compound of formula I (as obtained in Example 1 c). Each capsule type was filled with either compound of formula I alone or compound of formula I together with 1.8 mg of sodium docusate, 0.18 mg of PVP K30 and 9 mg of mannitol.

For wet-milled compound of formula I, each gelatin capsule was individually filled with 55.9 mg of the wet-milled compound together with accompanying excipients (using the product obtained in Example 2c). Once prepared, the capsules were stored in a desiccator at between 19 and 25 °C prior to administration to the animals.

Details of the formulations are summarized in Table 5.

2. Suspensions

A 2% w/v methylcellulose, in 4 mM phosphate buffer pH 7.4 was prepared.

40ml_ of the 2% w/v methyl cellulose solution was added to a 250 ml_ conical flask together with 10 g of 2 mm glass beads, and vigorously stirred. Compound of formula I (720 mg) was slowly added to the solution and the mixture was with continuously stirred for 1 h. The homogenate was transferred to separate flask and its pH recorded. The suspension formulation was prepared before administration to the animals.

Animal Husbandry

Rabbits (New Zealand white; male) were housed under standard laboratory conditions, in environmentally monitored air-conditioned room with adequate fresh air supply (10-15 air changes per hour), room temperature (22 ± 3°C) and relative humidity 30 to 70 %, with 12- hour light and 12-hour dark cycle. The temperature and relative humidity were recorded once daily.

Each animal was housed in a standard stainless steel rabbit cage SS-304 (Size: L 24” x B 18” x H 18”) with stainless steel mesh and removable bottom tray for refuse disposal, food hopper for holding pelleted food, holder for drinking water bottle and siphon tube and label holder. Clean, sterilized corncob was provided as bedding material.

The animals were fed ad libitum throughout the acclimatization and experimental periods, with Krishna Valley Agrotech rabbit feed.

Water was provided ad libitum throughout the acclimatization and experimental periods. Water from an Aqua guard water filter cum purifier was autoclaved and provided in polypropylene water bottles with stainless steel sipper tubes.

Acclimatization

The animals were acclimatized for a minimum period of 1 weeks (7 days) to facility room conditions and observed for clinical signs daily. Veterinary examination of all the animals were performed on the day of receipt, daily and on the day of randomization. Grouping

Animal grouping was done by the method of body weight stratification and randomization. The animals selected for the study were weighed and grouped in to body weight ranges. These body weight stratified rabbits were distributed to all the study groups in equal numbers if possible, such that body weight variation of animals used does not exceed ±20% of the mean body weight. The grouping was done one day prior to the initiation of treatment.

Study Design

* 1% sodium docusate, 0.1% PVP K30 and 5% mannitol

Table 5.

Dose Administration

Adult healthy male New Zealand white rabbits aged approximately 2 to 3 months old were used for experimentation after 7 days of acclimatization.

1. Capsules

To dose the filled capsules, a soft plastic dosing tube was used. The filled capsule was inserted into the dosing tube so that the short end of the capsule protrudes slightly from the tip of the tube. The tip of the capsule was dipped in mineral oil to aid swallowing.

The head was grasped firmly with one hand about the maxilla. The dosing tube containing the capsule was inserted behind the incisors. The dosing tube was slid straight into the back of the mouth. The capsule was ejected by pushing the plunger on the dosing tube. The dosing tube was removed, and the rabbit's mouth was closed. The neck was stroked gently to facilitate swallowing.

2. Suspension

To dose the suspension, an infant feeding tube was used. The feeding tube was inserted through the mouth of the rabbit to the oesophagus and the stomach, and ascertained that it has not been placed in the trachea before dosing to the animals. The suspension of the compound of formula I was administered through the feeding tube. After the administration of the suspension, drinking water of approximately 2.0-2.5 ml_ was administered to flush out the contents in the feeding tube.

The animals were restrained in a rabbit restrainer and blood samples (400-500 pl_ /time point) were collected via the central ear artery at 0.16, 0.25, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0, 24.0, 48.0 and 72 hours post-dosing. Collected blood specimens were centrifuged at 4000 rpm, 4°C for 10 minutes and plasma samples were separated and stored at -80°C until analysis.

Bioanalysis

Concentrations of the analyte of the compound of formula I in New Zealand white rabbits was determined using an API 3200 Q-trap LC-MS/MS system.

Method Summary

Chromatographic separation was achieved on Zorbax C18, 50 x 4.6 mm, 5pm column with methanol-0.1 % formic acid as mobile phase with gradient elution. The flow rate was set at 1.0 ml_ min ¹ . Detection was accomplished by a triple- quadrupole tandem mass spectrometer in multiple-reaction monitoring (MRM) scanning via electrospray ionization source, applied in the positive mode. The optimised mass transition ion-pairs for quantitation were m/z 379.999 125.000 for the compound of formula I and m/z 376.165 165.00 for the ISD (Haloperidol). Calibration plots were linear over the range of 11.073 to 50620.625 ng/ml_.

Buffer (0. 1% Formic Acid)

About 1.0 ml_ of formic acid was added to 999 ml_ of ultrapure water type-1 to make the buffer. This solution was stored at room temperature and used within two days from the date of preparation.

Dilution solvent (Methanol: Water, 80:20 % v/v)

Exactly 800 mL of methanol and 200 mL of ultrapure water type-1 were added to a reagent bottle, mixed well, and sonicated. This solution was stored at room temperature and used within seven days from the date of preparation.

Rinsing Solvent (Methanol: Water, 50:50 % v/v)

Exactly 500 mL of methanol and 500 mL of ultrapure water type-1 were added to a reagent bottle, mixed well, and sonicated. This solution was stored at room temperature and used within three days from the date of preparation.

Preparation of Compound of Formula I stock solution

3.010 mg of the compound of formula I was weighed, transferred into a 5.0 mL volumetric flask and dissolved in 5 mL of methanol to obtain a 598.99 pg/mL stock solution. The final concentration of the compound of formula I was corrected according to the potency of the standard and the actual amount weighed.

Internal standard stock solution

About 2.00 mg of Haloperidol (Sigma-Aldrich) was weighed, transferred into a 5.0mL volumetric flask and dissolved in methanol to obtain a 400 pg/mL internal standard stock solution. The final concentration of Haloperidol was corrected according to the potency of the standard.

Preparation of Internal Standard (Haloperidol) Working Solution

About 0.250mL of the internal standard stock solution was diluted to 10 mL using a dilution solvent (methanol/water, 80:20) to obtain about approximately 10 pg/mL solution. Preparation of Calibration curve for compound of formula I in plasma

Calibration curve standards were prepared in a range between 11.073 - 50620.63 ng/mL (Prepared concentrations: 11.073, 13.842, 27.683, 55.366, 110.733, 221.465, 442.930, 885.861 , 1771.722, 3543.444, 7086.887, 14173.78, 28347.55, 40496.50 and

50620.625ng/ml_) in plasma by spiking blank plasma with aqueous analyte standards.

Quality control samples LQC, MQC and HQC falling in the calibration curve range were prepared for the compound of formula I (prepared concentrations: 27.683, 28347.550 and 50620.625ng/ml_) in plasma by spiking blank plasma with suitable aqueous analyte standards.

Liquid-Liquid extraction method for plasma samples

• 10pL of ISD working solution (approx. 10 pg/mL) was added to an RIA vial.

• Exactly 50 pL of rabbit plasma was added from a polypropylene capped tube/vial into the RIA vial and vortexed for 30-40 seconds.

• 2.5 mL of TBME was added to the RIA vial and then vortexed for 10 minutes at 2000 rpm using a vibramax shaker.

• The samples were centrifuged at 4000 rpm for 10 minutes at 4°C.

• 2 mL of organic layer was separated and evaporated to dryness for 20 minutes at 50°C using a turbo evaporator.

• The sample residues were reconstituted with 200 pL of reconstitution solvent (methanol).

• The reconstituted samples were then transferred into auto sampler vials.

• 10 pL of the reconstituted sample was injected into the API 3200 Q Trap LC-MS/MS system.

Instrumentation Conditions

LC Parameters

Column: Zorbax C18, 50 x 4.6 mm, 5pm

Mobile phase: Methanol (A) : 0.1 % Formic acid (B)

Separation mode: Gradient Mode

Flow rate: 1.0ml_/min

Injection volume: 10 pL

Auto sampler temperature: 15 °C

Column oven temperature: 40 °C

LC-MS/MS API 3200 QTRAP

Source Turbo Ion Spray

Polarity Positive

Scan type MRM

m/z of analyte (compound of formula I) 379.999/125.000

[M+1]

m/z of internal standard (Haloperidol) 376.165/165.000

[M+1]

Ion Spray Voltage (IS) 5500

Temperature (TEM) 500

Data Analysis

The data of plasma concentration to respective time points for the analyte the compound of formula I were used for the pharmacokinetic analysis. Pharmacokinetic analysis was performed using non-compartmental analysis (NCA) module of Phoenix WinNonlin 6.3 software to determine the following pharmacokinetic parameters:

Chromatograms of data acquired using the Analyst software version 1.6.1. were processed by the peak area ratio method. 1/x2 was used as the weighting factor. The concentration of the unknown is calculated from the following equation.

y = mx + c

Where, x = concentration of drug;

m = slope of calibration curve;

y = peak area ratio; and

c = intercept of the calibration curve. Results

The results for the single dose oral pharmacokinetic study in rabbits are tabulated in T ables 7 to 12 below and are shown graphically in Figures 1 to 10 and 12 to 14.

The results show that there is no significant difference in the systematic exposure of non- milled compound of formula I (without excipients) when administered in either non- or enteric-coated capsules (Examples 3 and 6; Figures 3 to 6).

The addition of excipients to the non-milled formulations resulted in a moderate increase in the systemic exposure of compound of formula I when administered in non-coated capsules (Example 10 vs Example 3; Table 6). A larger increase in the systemic exposure of compound of formula I was observed when administered with excipients in enteric- coated capsules (Example 1 1 vs Example 6; Table 6).

The results also show that wet milled compound of formula I administered in enteric-coated capsules generated a substantial (several-fold) increase in systemic exposure of the compound compared to a non-milled (with excipients) formulation administered in non- coated tablets (Examples 8 and 10; Figures 7 to 10). In addition, compared to non-milled (with excipients) formulations administered in enteric-coated tablets, a formulation of wet milled compound of formula I administered in enteric-coated capsules generated a smaller yet still substantial (several-fold) increase in systemic exposure of the compound.

Importantly, when the compound of formula I has been wet milled, it has been found that the use of enteric coated capsules in place of non-coated capsules resulted in substantially higher Cmax (increase of 53%) and AUC values (increase of 63%). See Example 8 (enteric coated capsule), Example 5 (non-coated capsule) and Figures 7 to 10. These results suggest that there is a synergistic effect arising from the combination of wet milling and an enteric coating.

In non-coated capsules, wet-milled compound of formula I generated a similar increase in bioavailability compared to a dry milled formulation in a non-coated capsule (Examples 5 and 12; Figures 7 to 10).

Moreover, the variation in the bioavailability between subjects who have been administered wet milled compound of formula I in an enteric coated capsule (Figure 12) is much less compared to the variation in the bioavailability between subjects who have been administered wet milled compound of formula I in a non-coated capsule (Figure 11). Similarly, the variation in the bioavailability between subjects who have been administered wet milled compound of formula I in an enteric coated capsule (Figure 12) is much less compared to the variation in the bioavailability between subjects who have been administered dry milled compound of formula I in an enteric-coated capsule (Figure 13)

Thus, administration of wet milled compound of formula I in an enteric-coated capsule very surprisingly results in a significant increase in the systemic exposure of the compound of formula I.

Table 7. Plasma Concentration of Non-milled Compound of Formula I

Table 8. Mean Plasma Pharmacokinetic Parameters For Dry Milled Compound of Formula I

Table 9. Plasma Concentration of Dry Milled Compound of Formula I

Table 10. Mean Plasma Pharmacokinetic Parameters For Wet Milled Compound of Formula I

Table 11. Plasma Concentration of Wet Milled Compound of Formula I