WO2015193673A1

WO2015193673A1 - Novel pharmaceutical formulations and their use in the treatment of periodontal disease

Info

Publication number: WO2015193673A1
Application number: PCT/GB2015/051785
Authority: WO
Inventors: Thomas Kissel; Ching Pong Mak; Matthias Rischer; Ansgar BÖGERSHAUSEN; Lena FRANZISKA KURZ
Original assignee: Perioc Limited
Priority date: 2014-06-18
Filing date: 2015-06-18
Publication date: 2015-12-23
Also published as: GB201410869D0

Abstract

The present invention relates to novel treatments of periodontal disease by administering a suitable formulation of a cyclophilin inhibitor. The present invention further relates to novel solid state compositions containing cyclophilin inhibitor compounds and methods for their reconstitution and use.

Description

Novel pharmaceutical formulations and their use in the treatment of periodontal disease

Field of the invention

The present invention relates to a new treatment of periodontal disease by administering a suitable topical formulation of a cyclophilin inhibitor into the gingival pockets. The method includes a new in-situ formulation and use of compounds. Further disclosed is a micro- formulation or nano-formulation of stabilised micro or nanoparticles which is stored as a solid composition and can be made into a suspension before use. The resuspended nanoparticle formulations may be used in the treatment of periodontal disease. The resuspended formulation can be an in-situ forming system which is applied as a liquid, but forms a gel under physiological conditions.

Background of the invention

Periodontal diseases range from simple gum inflammation to serious disease that results in major damage to the soft tissue and bone that surround and support the teeth, ultimately resulting in tooth loss. The diseases are caused by bacterial colonization of the tooth surface, followed initially by a response of the innate immune system and manifest as gum inflammation (gingivitis). This gum inflammation then progresses to become periodontitis, in which the gums pull away from the teeth and form pockets in which the infection further thrives.

The innate immune response is followed by an adaptive immune response, in which antigen- presenting cells (mainly dendritic cells) accumulate in the gingiva and orchestrate a T cell response which in turn activates B cells to produce specific antibodies. CD4+ T cells have been shown to be the predominant population in adult periodontitis and, via recruitment and activation of osteoclasts function as a major source of bone loss. Thus, periodontal disease, while initially triggered by infection, is essentially an immunopathology, in which it is the immune response ensuing the infection that is responsible for the damage to tissue and bone. Presently, the treatment of periodontal diseases consists primarily in eliminating the infection, mainly by mechanical removal of the plaque by scaling, debridement and root planing. The mechanical treatment can be supported by antimicrobial measures, such as mouth rinses or locally applied gels containing an antiseptic such as chlorhexidine. Tetracycline antibiotics (doxycycline, minocycline) are also used to combat the infection, either in form of locally applied preparations or in form of tablets. Tetracyclines act not only as antimicrobials but have in addition anti-inflammatory properties, which are poorly understood. Both minocycline and doxycycline have been shown to inhibit the release and the activity of matrix metallo proteinases (MMPs), a large group of enzymes that can be released from a variety of cells and are the main culprits of degrading tissue, cartilage and bone in many chronic inflammatory diseases, including periodontitis.

There is presently no treatment that addresses the events of the inflammatory response in a comprehensive way, notably its chronic manifestations, such that it would halt or reverse the tissue and bone destructive process. The drug compositions subject of this invention represent such a treatment.

Role of cyclophilin in the inflammatory process

Cyclophilin was first discovered as binding protein of the immunosuppressant cyclosporin, normally resident within cells. The re-discovery of cyclophilin as intracellular peptidyl-prolyl cis-trans isomerase (PPIase) was reported several years later. Exposure of cells to inflammatory stimuli such as bacterial cell wall components (e.g. lipopolysaccharide, LPS) triggers cyclophilin to be secreted from cells into the extracellular space where it acts as a chemoattractant for inflammatory leukocytes. Leukocyte chemotaxis is mediated by a widely expressed membrane glycoprotein called CD 147 or EMMPRTN (Extracellular Matrix Metallo Proteinase INducer) due to its ability to induce the production and release of MMPs from these cells. Both MMP release as well as leukocyte chemotaxis are triggered by the interaction between CD147 and cyclophilin, which occurs via the PPIase catalytic site which is also the cyclosporin binding site. Cyclosporin and other compounds that inhibit the PPIase catalytic activity of cyclophilin therefore block several key events involved in the bone and tissue destructive process of periodontal disease:

(1) They inhibit the infiltration of inflammatory leukocytes

(2) they inhibit the formation of antibody-secreting plasma cells resident in the gingiva

(3) they prevent the production and release of matrix metallo proteinases.

Cyclophilin inhibitors therefore represent a novel modality to treat the underlying mechanisms causing the immunopathology of periodontal disease.

Cyclosporin, when administered subcutaneously, has a positive effect on the formation of new alveolar bone (Toxicologic Pathology, Vol. 34(6), 2006, (Cetinkaya, Burcu Ozkan et al), "The effect of cyclosporin A on alveolar bone in rats subjected to experimental periodontal disease", pages 716-722). The cyclosporin is administered as a subcutaneous injection. The effect on bone growth can only be seen using systemic treatment. The reference does not disclose formulations for topical applications into the inflamed gingival pocket to act as a localised anti-inflammatory agent.

It is known that induction of gingival overgrowth is a major undesired effect of systemic cyclosporin in transplant patients (Journal of Periodontology, Vol. 82(10), 2011, (Becerik, Sema et al), "Gingival crevicular fluid osteocalcin, N-terminal telopeptides, and calprotectin levels in cyclosporin A-induced gingival overgrowth", pages 1490-1497). The side effect of gingival overgrowth is not unique to cyclosporin, other compound classes associated with gingival hyperplasia are anticonvulsants and calcium channel blockers, neither of which has anti-inflammatory activity. Gingival hyperplasia associated with all these medications consists of an excess deposit of extracellular matrix and is fundamentally different from physiological tissue, which consists primarily of cells (e.g. Kataoka et al., "Drug-induced gingival overgrowth-a review", Biol Pharm Bull. 2005 Oct; 28(10): 1817-21).

The undesired side effect of systemic treatment can be overcome by using localised topical formulations. Ongoing gingival inflammation, as seen in periodontitis, is a prerequisite of the onset of gingival overgrowth. Agents inhibiting the inflammatory processes of periodontitis could be expected to antagonise gingival overgrowth (Subramani et al., "The possible potential therapeutic targets for drug induced gingival overgrowth", Mediators Inflamm. 2013). Gingival overgrowth induced by cyclosporin has been shown to be correlated with a certain threshold in cyclosporin blood levels (Webb et al., "Correlation between finger-prick and venous cyclosporin levels: association with gingival overgrowth and hypertrichosis", Pediatr Nephrol. 2007 Dec;22(12):2111 ; Thomas et al., "Risk factors in the development of cyclosporine-induced gingival overgrowth", Transplantation. 2000 Feb 27;69(4): 522-6). However the topical treatments described herein will slow and/or stop the pathophysiology of chronic inflammation which must precede the physiological healing process, without causing the side effects induced by systemic treatment. It is the onset of the physiological healing process, that initiates the re-growth of physiological tissue, and the tissue which is formed by natural healing is fundamentally different from the overgrowth induced by cyclosporin and other drug classes. DE 102008062373 describes the use of compounds known to induce gingival hypertrophy to fill the interdental gaps created by tissue erosion in periodontitis. There is no evidence of topical administration of any of the compounds mentioned in this document, nor is there any evidence of localised anti-inflammatory activity.

JPH0597697 describes the provision of an alveolar bone-regenerating agent containing cyclosporin A. The document lists a long list of possible compounds, including many that do not have any anti-inflammatory activity. There is no evidence of topical administration of cyclosporin A, nor is there any evidence of localised anti-inflammatory activity.

WO 03/033010 mentions periodontal disease as one condition among an exhaustive list of inflammatory and autoimmune diseases that can be treated with the compounds of WO 03/033010. This reference does not disclose any evidence supporting the claim that periodontal disease could be treated by cyclosporins. Evidence is given for inhibition of the Nuclear Factor of Activated T cells (NFAT), which is relevant for immunosuppression and for use of inhibitory compounds in transplantation. Furthermore, WO 03/033010 describes activity of compounds in test systems such as mixed lymphocyte reaction, plaque forming cell assay (Mishell-Dutton test), or delayed type hypersensitivity. All these test systems detect inhibitory activity of compounds on T cells (i.e. immunosuppressive activity). As outlined above, the role of cyclophilin in the inflammatory process of periodontitis is fundamentally different from that of immunosuppression.

The difference between immunosuppressive and anti-inflammatory activity is best illustrated by the fact that the well-known compound FK506 (Tacrolimus), is an immunosuppressant acting by a mechanism identical to that of cyclosporin but is not known to have antiinflammatory activity (see e.g. Mattila et al., "The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes", EMBO J. 1990 Dec;9(13):4425-33; Liu J et al., "Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes", Cell. 1991 Aug 23;66(4):807-15).

WO 03/033010 A teaches that compounds can be administered by parenteral injection in the form of liquid dosage forms, be given by mouth (perorally) in the form of solid dosage forms, or be administered topically to the lung, eye, or vagina. However the document contains no evidence of topical administration of cyclosporin A, nor is there any evidence of localised anti-inflammatory activity.

In order to apply the compositions, the cyclosporin formulations are made as liquid suspensions. However liquid suspensions of micro or nanoparticle formulations are frequently not physically stable over a period of weeks or months as the formulations tend to aggregate forming ever larger particles. Also size increase by Ostwald ripening can be observed. The micro or nano formulations can therefore be dried to solids for long term physical stability, and reconstituted prior to use. Reconstitution is especially advantageous in cases where temperature sensitive gels are required, as the temperature sensitive gel, which is adversely affected by the lyophilisation process, can be prepared after reconstitution.

Summary of the invention

According to one aspect of the invention, cyclophilin inhibitors may be formulated as solid state micro or nanoparticle formulations. These formulations may be resuspended and used to treat periodontitis. According to another aspect, the cyclophilin inhibitors belong to the chemical classes of cyclosporins, sanglifehrins or cycloundecadepsipeptides. According to another aspect, the cyclophilin inhibitors may be applied locally into the gingival pocket. According to another aspect, the micro- or nano-formulation is mucoadhesive in use. According to another aspect, the micro- or nano-formulation allows the cyclophilin inhibitors to exhibit activity over a period of several days or weeks. The nano compositions can be formulated with non-ionic surfactants, for example TPGS and/or poloxamer 407. The resuspended composition may be applied as a liquid, which forms a gel in-situ. The in-situ forming system can be a suspension of cyclosporin nanoparticles which is applied into inflamed gingival pockets as a liquid, where it forms a gel upon exposure to physiological conditions. The in-situ forming systems allow the activity of the cyclosporin to be maintained over a period of several days or weeks. The long acting nature of the composition means that the treatment only needs to be applied once or twice in order to be effective. The liquid formulations of cyclosporins are described in PCT/GB2013/053283. The shelf life of these compositions is in the regions of weeks to months, and the compositions need to be kept refrigerated. The solid state formulations described herein are physically stable at room temperature for prolonged periods. The shelf life of the dried solid state compositions is more than one year at room temperature. The dried compositions of the present invention may be stable at room temperature for at least 12 months. The compositions may also be stable at elevated temperature, for example 37 °C for at least 12 months. The compositions may be stable for at least 2 years at room temperature. The stability of the formulation refers to the prevention of Ostwald ripening, where the co-agglomeration of the particles gradually forms larger and larger particles over time. The dried materials prevent the micro or nanoparticles from aggregating as they would do if left in solution or suspension.

The solid state formulations contain a bulking excipient or matrix forming excipient. There are many bulking excipients known to experts skilled in the art. The bulking excipient may be a polymer, such as for example PVP or methylcellulose. The bulking excipient may be a carbohydrate or polyol. The carbohydrate or polyol may one or more of mannitol, saccharose, raffinose, lactose, glucose, sucrose, sorbitol, trehalose, glycerol and/or polyethylene glycol or other polyols or carbohydrates. The bulking excipient may be a polymeric carbohydrate such as cellulose or starch, carbohydrate ethers such as carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, or other polymeric materials such as poly-N- vinyl-pyrrolidone (PVP), crosspovidone and copolymers of N-vinyl-pyrrolidone with other polymerisable compounds such as acrylic acid derivatives. The solid state formulations are obtained by the removal of solvent. The solvent removal can be achieved by spray-drying or freeze-drying.

Disclosed herein is an aqueous composition comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a carbohydrate or polyol, wherein the total concentration of gel forming material in the composition is 10 % or lower by weight of the solution. The solution may be dried to produce a solid state composition. The composition may be resuspended for pharmaceutical use. The gel forming material can be poloxamer, which can be present at a concentration of 2.5 to 10 % by weight of the solution before drying.

Disclosed herein is an aqueous composition comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a bulking excipient, wherein the total concentration of gel forming material in the composition is 10 % or lower by weight of the solution. The solution may be dried to produce a solid state composition. The composition may be resuspended for pharmaceutical use. The gel forming material can be poloxamer, which can be present at a concentration of 2.5 to 10 % by weight of the solution before drying.

The resuspended composition may be a liquid material which forms a gel in-situ. However such materials are difficult to formulate as solid state compositions. The high levels of gel forming material (for example 10-20 % by weight of solution) makes drying difficult as the resultant dried material is either not a powder, is not capable of re-suspension or causes aggregation of the microparticles. High levels of gel forming materials require high levels of bulking excipients in order to form stable dried compositions. The large amounts of dried powder produced then requires a large volume of solution for resuspension, meaning that the level of the active pharmaceutical agent is too low in the resuspended material. The inventors herein have evaluated the conditions and compositions required for effective drying and resuspension and identified both improved compositions for forming stable dried products and improved methods and reagents for the reconstitution thereof.

Included herein is a solid state formulation comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a carbohydrate or polyol, wherein the concentration of gel forming material in the solution which is dried to form the solid state formulation is 10 % or lower by weight of the solution. The gel forming material can be poloxamer, which can be present at a concentration of 2.5 to 10 % by weight of the solution before drying.

Included herein is a solid state formulation comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a bulking excipient, wherein the concentration of gel forming material in the solution which is dried to form the solid state formulation is 10 % or lower by weight of the solution. The gel forming material can be poloxamer, which can be present at a concentration of 2.5 to 10 % by weight of the solution before drying. The methods described herein are advantageous in producing temperature sensitive gel compositions. Gel compositions having greater than 10 % gel forming material are either difficult to dry to a stable powder or are hard to re-constitute without particle aggregation or using a large volume of solution. Compositions not having any, or merely a low level of gel forming material can be easily dried, but are then difficult to re-constitute into gel forming products as the resuspension solution, which requires a high concentration of gel forming material is too viscous. The inventors have appreciated that a certain level of gel forming material can be included in the dried products, with the extra gel forming material being added during the reconstitution step.

Disclosed herein is a method of producing a temperature sensitive gel comprising a resuspended formulation as described herein, the method comprising resuspending the material in an aqueous solution, the aqueous solution having further gel forming material. The resuspension can be done via the addition of a single solution containing the gel forming material, or by addition of a first solution, then adding a further solution containing the gel forming material.

Figures

Figure 1 shows the formulation and stability of a formulation of the active pharmaceutical ingredient (API) Cyclosporin (5 %) with 1 % TPGS in water. The formulation is stable after 8 weeks at 2-8 °C, and shows only a limited amount of aggregation at 25 °C.

Figure 2 shows the formulation and stability of a formulation of the active pharmaceutical ingredient (API) Cyclosporin (5 %) with 1 % TPGS and 1 % Poloxamer 407 in water. The formulation is stable after 8 weeks at 2-8 °C. The amount of aggregation at 25 °C is reduced by the presence of the poloxamer.

Figure 3 shows the formulation and stability of a formulation of the active pharmaceutical ingredient (API) Cyclosporin (5 %) with 0.8 % sodium glycocholate and 2 % Poloxamer 407 in water. The formulation is stable after 8 weeks at 2-8 °C. The amount of aggregation at 25 °C is substantial. Sodium glycocholate does not appear to confer long term stability to the same level as TPGS.

Figure 4 shows the formulation and stability of a formulation of the active pharmaceutical ingredient (API) Cyclosporin (5 %) with 0.02 % chitosan and 1 % Poloxamer 407 in water. The formulation is not stable, and substantial aggregation of the particles occurs. Chitosan does not appear to confer long term stability to the same level as TPGS. Figure 5 shows the formulation and stability of a formulation of the active pharmaceutical ingredient (API) Cyclosporin (5 %) with 1% TPGS in water. The crystalline cyclosporin is micronized before formulation. Comparison with figure 1 shows improved long term stability at 25 °C.

Figure 6 shows that the microparticle products are destroyed by lyophilisation in the absence of bulking excipients.

Figures 7 to 11 show that the stability of the microparticles after lyophilisation can be maintained by the use of mannitol and poloxamer (2.5 % each of the solution pre-drying). Figure 7 shows that the particles are re-suspended in pure water, no further poloxamer is added. The particle size distribution (PSD) has not significantly altered by the drying and re- suspension. Figure 8 shows that re-constitution of the lyophilised microparticles in a solution of 5 % poloxamer (rather than water) causes a degree of aggregation of the particles.

Figure 9 shows that re-constitution of the lyophilised microparticles in a solution of 14 % poloxamer (rather than water) causes complete aggregation of the particles.

Figure 10 shows the particles sizes following a successful two step reconstitution. The lyophilised products are resuspended in water, and a high concentration solution of poloxamer (25 %) added. Figure 11 shows the particles sizes following an improved two step reconstitution. The lyophilised products are resuspended in water further containing TPGS, and a high concentration solution of poloxamer (25 %) added. The particle size distribution in Fig 1 1 is improved over Fig 10. Figure 12-16 show the effects of higher levels of mannitol and poloxamer (5 % each of the pre-dried solution). Figure 12 (as 7) shows that the material can be resuspended and the PSD has not been affected. Figure 13 (as Fig 8) shows that re-constitution of the lyophilised microparticles in a solution of 5 % poloxamer (rather than water) causes a degree of aggregation of the particles.

Figure 14 shows that re-constitution of the lyophilised microparticles in a solution of 10 % poloxamer (rather than water) causes complete aggregation of the particles.

Figure 15 shows the particles sizes following an unsuccessful two step reconstitution. The lyophilised products are resuspended in water, and a high concentration solution of poloxamer (25 %) added. The particles are destroyed by the process (unlike Fig 10, where the process works)

Figure 16 shows the particles sizes following an improved two step reconstitution. The lyophilised products are resuspended in water further containing TPGS, and a high concentration solution of poloxamer (25 %) added.

From the data shown in the figures, it can be concluded that the wettability depends on both the poloxamer amount in the lyophilisate and in the reconstitution solution. The best reconstitution results could be achieved with pure water. Reconstitution times as well as particle size distributions were better in water than with Poloxamer solutions in different concentrations. Additional TPGS to the water part of the 2 step addition significantly improved the particle size distribution, but that no satisfying PSD could be achieved by addition of TPGS to the concentrated poloxamer solutions.

Figure 17 shows the effect of raising the poloxamer concentration in the lyophilisate. Higher levels of poloxamer still allow the material to be dried, stored and re-constituted, but the re- suspension can be carried out using a lower concentration of poloxamer solution as a single solution, rather than a two-step process. Thus there is a key balance between the level of poloxamer in the lyophilisate and the re-constitution solution. Detailed Description

Described herein is the use of cyclophilin inhibitors in the treatment of periodontal disease. The cyclophilin inhibitor may be a cyclosporin, a sanglifehrin or a cycloundecadepsipeptide. Any compound as described herein may be used in the treatment of periodontal disease and may be formulated into a micro- or nano-formulation, which may be dried to a solid state to allow long term storage. Any compound as described herein may be formulated with a mucoadhesive. Any compound as described herein may be formulated into a micro- or nano- formulation for use as an in-situ forming gel. The micro- or nano-formulation allows the cyclophilin inhibitors to exhibit activity over a period of several days or weeks once resuspended for use in-vivo. The nano compositions can be formulated with non-ionic surfactants, for example TPGS and/or poloxamer 407. The composition may be applied as liquids, and which form gels in-situ. The in-situ forming system can be a suspension of cyclosporin nanoparticles which is applied into inflamed gingival pockets as a liquid, where it forms a gel upon exposure to physiological conditions. The in-situ forming systems allow the activity of the cyclosporin to be maintained over a period of several days or weeks. The long acting nature of the composition means that the treatment only needs to be applied once or twice in order to be effective.

The resuspended composition may be a liquid material which forms a gel in-situ. However such materials are difficult to formulate as solid state compositions. The high levels of gel forming material (for example 10-20% by weight of solution) makes drying difficult as the resultant dried material is either not a powder, is not capable of re-suspension or causes aggregation of the microparticles. High levels of gel forming materials require high levels of bulking excipients in order to form stable dried compositions. The large amounts of dried powder produced then requires a large volume of solution for resuspension, meaning that the level of the active pharmaceutical agent is too low in the resuspended material. The inventors herein have evaluated the conditions and compositions required for effective drying and resuspension and identified both improved compositions for forming stable dried products and improved methods and reagents for the reconstitution thereof.

The methods described herein are advantageous in producing temperature sensitive gel compositions. Gel compositions having greater than 10 % gel forming material are either difficult to dry to a stable powder or are hard to re-constitute without particle aggregation. Compositions having only a low level of gel forming material can be easily dried, but are then difficult to re-constitute into gel forming products. The inventors have appreciated that a certain level of gel forming material can be included in the dried products, with the extra material being added during the reconstitution step.

The first cycloundecadepsipeptide to be identified to be a potent inhibitor of cyclophilins has the structure shown in formula A.

formula A

In accordance with WO 2011/141891, this compound can also be described as

Cyclo-(MeBmt-Thre-Sar-MeLeu-Leu-MeLeu-Ala-D-Hiv-MeLeu-Leu-MeVal). Compounds of this family can generally be designated as Cyclo (AXXi AXX₂ AXX₃ AXX₄ AXX₅ AXX₆ AXX₇ D-Hiv AXX₉ AXX₁₀ AXX_n)

1 2 3 4 5 6 7 8 9 10 11

In which AXXi is N-methyl-(4R)-4-but-2E-en-l-yl-4-methyl-(L)-threonine, and D-Hiv is (D)-2-hydroxyisovalerianic acid.

The compound of formula A may be used in the treatment of periodontal disease or may be formulated into a micro- or nano-formulation as described herein. More recently, new cycloundecadepsipeptides which retain their ability to bind cyclophilins but with significantly reduced immunosuppressive properties have been disclosed (WO2010/052559 Al). This application claims the use of certain cycloundecadepsipeptides as compounds for treating viral infections, notably by Hepatitis C. The application does not describe the use in the treatment of periodontal disease or the formulation of micro or nanoparticles. Any compound described in WO2010052559 is within the scope of the invention herein. The compounds for use in the treatment of periodontal disease therefore include compounds which can generally be designated as Cyclo (AXXi AXX₂ AXX₃ AXX₄ AXX₅ AXX₆ AXX₇ D-Hiv AXX₉ AXX₁₀ AXX_n)

1 2 3 4 5 6 7 8 9 10 11

In which AXXi is MeBmt, 4-fluoro-MeBmt, dihydro-MeBmt, 8-hydroxy-MeBmt, O-acetyl- MeBmt;

AXX₂ is Abu, Val, Thr, Thr(OMe), Thr(OAc), Thr(OCOCH₂CH₂CH₂OH), Nva, 5-hydroxy- Nva (Hnv);

AXX₃ is D-MeAla, D-3-fluoro-MeAla, D-MeSer, D-MeSer(OAc), D-MeSer(OCH₂CH₂OH), D-MeSer(OCH₂CH₂ Et₂), D-MeAsp(OMe);

AXX₄ is MeLeu, Melle, MeMet, Me Val, MeThr, MeThr(OAc), MeAla, EtVal, Etlle, EtPhe, EtTyr, EtThr(OAc), MeThr(OAc), MeTyr, MeTyr(OAc), MeTyr(OMe), MePhe, MeMet(Ox) wherein the sulphur atom of methionine is sulphoxide or sulphone;

AXX₅ is Leu, Val, He, Gly, Abu;

AXX₆ is MeAla, Sar, MeLeu;

AXX₇ is Gly, Ala;

D-Hiv is (D)-2-hydroxyisovalerianic acid;

AXX₉ is MeLeu;

AXXio is Leu; and

AXXn is Me Val. A cyclophilin inhibitor according to the present invention wherein the inhibitor is a cycloundecadepsipeptide can be designated as

Cyclo (AXXi AXX₂ AXX₃ AXX₄ AXX₅ AXX₆ AXX₇ D-Hiv AXX₉ AXX₁₀ AXX_n)

1 2 3 4 5 6 7 8 9 10 11 In which AXXi is MeBmt, 4-fluoro-MeBmt, dihydro-MeBmt, 8-hydroxy-MeBmt, O-acetyl- MeBmt or AXXi contains a nitrogen atom in the side chain;

AXX₂ is Abu, Val, Thr, Thr(OMe), Thr(OAc), Thr(OCOCH₂CH₂CH₂OH) or an alternative threonine ester or threonine-O-alkyl or substituted O-alkyl moiety, Nva, 5-hydroxy-Nva (Hnv) or a moiety of type C(=0)C¾ or C(=N-Y)C¾ where Y is OH, NH₂ or O- or N-alkyl or substituted alkyl versions thereof;

AXX₃ is optionally substituted alkylene, D-MeAla, D-3-fluoro-MeAla, D-MeSer, D- MeSer(OAc), D-MeSer(OCH₂CH₂OH), D-MeSer(OCH₂CH₂NEt₂), D-MeAsp(OMe) or a D- amino acid with a side chain selected from hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylthio or substituted alkylthio;

AXXs is Leu, Val, He, Gly, Abu;

AXX₆ is MeAla, Sar, MeLeu;

AXX₇ is Gly, Ala;

D-Hiv is (D)-2-hydroxyisovalerianic acid;

AXX₉ is MeLeu;

AXXio is Leu; and

AXXn is MeVal.

Where AXXi contains a nitrogen atom, the cyclophilin inhibitor may be a cycloundecadepsipeptide having the formula (1);

or a pharmaceutically acceptable salt, tautomer or N-oxide thereof, wherein L represents an optionally substituted, optionally partially unsaturated chain of 1-6 carbon atoms with optional additional heteroatoms atoms in the chain, and may be optionally branched and optionally linked to Ri to form a ring structure containing one or more nitrogen atoms,

Q represents a primary, secondary or tertiary covalent bond, a carbonyl group and optionally a linking group to Rl,

Rl and R2 may be absent or independently represent H, alkyl, substituted alkyl, -COR₃, - CO2R3, -OR4, -NR4R5, CO R4R5,

and optionally Rl and R2 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted,

R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl,

R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted,

R6 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl,

X represents OH, OC(=0)-alkyl, OC(=0)-substituted alkyl, O-alkyl, O-substituted alkyl, carbonyl (=0) or imine (=N-Y) where Y is -OR4 or -NR4R5,

R_a represents hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or optionally substituted alkylene, and

R_b represents hydrogen or is absent.

The group L-Q-NR1R2 may comprise a primary, secondary or tertiary amino group attached via an optionally substituted alkyl linker.

The group L-Q-NR1R2 may comprise a primary or secondary amide, urea, amidine, guanidine or carbamate group attached via an optionally substituted alkyl linker. Q may be a carbonyl group such that amide may be of orientation -C(=0)N as well as -NC(=0).

The group L-Q- R1R2 may comprise a C=N double bond moiety, for example C=N-OH, C=N-OR, C=N-NH2, C=N-NHR or C=N- RR. The group L-Q-NR1R2 may comprise a nitrogen containing heterocyclic ring. The heterocyclic ring may be a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted.

Linking moiety L may be 1-6 carbon atoms. L may contain one or more heteroatoms in the chain. L may contain O, N or S atoms interspersed between the carbon atoms. L may contain a branch point. L may contain one or more double or triple bonds such that L may be partially unsaturated. L may link with Rl or R2 to form a ring containing one or more nitrogen atoms.

Moiety Q may be a covalent bond. Q may be a primary (single) covalent bond, where both Rl and R2 are present. Q may be a secondary covalent (double) bond, where only a single Rl group is present. Q may be a tertiary covalent (triple) bond to make a cyano (CN) group where Rl and R2 are absent. Q may be a carbonyl group such that Q-N is a C(=0)-N amide group. Q may link with Rl or R2 to form a ring containing one or more nitrogen atoms.

Exemplary compounds may be where Rl and R2 are together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted or optionally partially unsaturated. Exemplary rings include optionally substituted morpholinyl, optionally substituted piperazinyl, optionally substituted oxazepinyl, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted fused pyrrolidinyl, optionally substituted thiomorpholinyl or the S oxides thereof. The ring may be fused to form a bicyclic system. Rl and R2 may be absent or independently represent H, alkyl, substituted alkyl, -COR₃, - CO2R3, -OR4, -NR4R5, CONR4R5,

and optionally Rl and R2 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Rl and/or R2 may be H. Rl and/or R2 may be alkyl or substituted alkyl. Rl and/or R2 may be methyl or ethyl. Rl or R2 may represent an amide COR₃ where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl. Rl or R2 may represent a carbamate CO2R3 where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl. Rl or R2 may represent an oxime or hydroxylamine OR₄ where R4 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl. Rl or R2 may represent an hydrazone NR₄R₅ where R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Rl or R2 may represent

where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted and R6 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl.

X represents OH, OC(=0)-alkyl, OC(=0)-substituted alkyl, O-alkyl, O-substituted alkyl, carbonyl (=0) or imine (=N-Y) where Y is -OR₄ or - R₄R₅. Where X is OH, the amino acid is threonine. The hydroxyl moiety of the threonine can be in the form of an ester or O-alkyl group where the ester or alkyl group is optionally substituted. For example, the amino acid may be Thr(OMe), Thr(OAc), Thr(OCOCH₂CH₂CH₂OH) or an alternative threonine ester or threonine-O-alkyl or substituted O-alkyl moiety. The ester can be in the form OC(=0)-alkyl or OC(=0)-substituted alkyl. X can represent a group of type -OCOR₃ or -OCO₂R_3, where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl. X can represent a group of type -OR4 where R4 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl. X can be present as a carbonyl group (=0). X can be present as an imine (=N-Y) where Y is - ORt or -NR4R5 where R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted.

R_a represents hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylthio, substituted alkylthio or optionally substituted alkylene. Ra includes substituted alkyl groups of type -S-R7, -CH2-S-R7 and the sulfoxide and sulfone analogues thereof where R7 represents H, alkyl or substituted alkyl.

Exemplary groups for Ra include: =CH₂, -CH₂SH, -CH₂-S-(CH₂)nN-R4R5, where R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted and n is 1-4, -CH₂-S-(CH₂)_n-aryl where n is 1-4, -CH₂-S-(CH₂)_n-hereroaryl where n is 1-4, -CH₂-S-C¾, -CH₂-S-cycloalkyl, CH₂-S- heterocycloalkyl, -CH₂-S-(CH₂)_nCOOR4 where R4 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and n is 1-4, -CH₂- S-(CH₂)_n-CH=CH₂ where n is 1-4, -CH₂-S-(CH₂)_nN-C(=NH)-NH₂ where n is 1-4. In each example given above, the sulphur may be oxidised to the sulfoxide or sulfone, and formulas can be represented as -CH₂-S(=0)_m-(CH₂)- where m is 0-2. Further exemplary groups for Ra can be found in publication US2012/0088734, the contents of which are incorporated herein.

R_b represents hydrogen or is absent where Ra is alkylene. Exemplary compounds may include a compound of formula 1 wherein L is a chain of 1-6 carbon atoms, Q is a primary covalent bond or a carbonyl group and Rl and R2 are together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Exemplary compounds may include a compound of formula 1 wherein L is a chain of 1-6 carbon atoms, Q is a primary covalent bond or a carbonyl group and Rl and R2 are together with the nitrogen atom to which they are attached form a 5-7 membered cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Exemplary compounds may include a compound of formula 1 wherein the group L-Q- R1- R2 is selected from -(CH₂)_n-NRlR2 where n is 1-4 and Rl and R2 may independently represent H, alkyl, substituted alkyl or may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Exemplary compounds may include a compound of formula 1 wherein the group L-Q- Rl- R2 is selected from -(CH₂)n-S-(CH₂)m-NRlR2 where n is 1-4, m is 1-4 and Rl and R2 may independently represent H, alkyl, substituted alkyl or may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted.

Exemplary compounds may include a compound of formula 1 wherein the group L-Q- Rl- R2 is selected from -(CH₂)_n-CO- RlR2 where n is 1-4 and Rl and R2 may independently represent H, alkyl, substituted alkyl or may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted.

Exemplary compounds may include a compound of formula 1 wherein the group L-Q- R1- R2 is selected from -(CH₂)„-S-(CH₂)_m-CO- RlR2 where n is 1-4, m is 1-4 and Rl and R2 may independently represent H, alkyl, substituted alkyl or may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Exemplary structures for - R1R2 include

Exemplary compounds include a compound of formula 1 wherein L is a CI -6 alkyl group with 0-1 heteroatom substituents, Q is a primary covalent bond and Rl and R2 are independently H, alkyl or substituted alkyl groups.

Exemplary compounds include a compound of formula 1 wherein L or Q is linked to Ri to form a ring structure containing one or more nitrogen atoms. Exemplary compounds may include a compound of formula 1 wherein the group L-Q- Rl- R2 is selected from -(CH₂)_n-NRlR2 where n is 1-4, Rl is H or alkyl, and R2 represents - COR₃, -CO2R3_, -CONR4R5,

where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl, R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted, and R6 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl.

Exemplary compounds may include a compound of formula 1 wherein the group L-Q- Rl- R2 is selected from -(CH₂)n-S-(CH₂)_m-N R1R2 where n is 1-4, m is 1-4, Rl is H or alkyl, and R2 represents -COR3, -CO2R3, -CO R4R5,

where R3 represents alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl, R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted, and R6 represents H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl.

Exemplary structures for N-COR3, -C0₂R3, -CO R4R5,

include:

II - O , N , N ^ O -

\ N ^A O -' A \, A +s ' ^' ^ J '*^'

I I ' ' I where the arrows indicated positions which can be further substituted. Groups may include

^ N'' N- N-\ O r^O

I I I I

Exemplary compounds include a compound of formula 1 wherein Q is a secondary covalent bond, RI is absent and R2 is -OR₄ or -NR₄R₅, where R4 and R5 independently represent H, alkyl, substituted alkyl, cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl and optionally R4 and R5 may together with the nitrogen atom to which they are attached form a 4-7 membered aryl, cycloalkyl or heterocyclic ring which may be further fused or optionally substituted. Exemplary compounds include those shown below. In the diagram below, the L-Q- R1-R2 group is depicted from the cyclic peptide ring.

Exemplary amide structures of type CONR1R2 include



Where R is one or more optional substituents on the aromatic rin^

Alternative structures

Where R is independently H, alkyl or substituted alkyl.

Alternatively the cyclophilin inhibitor may be a sanglifehrin, or an analogue thereof.

Alternatively, the cyclophilin inhibitor may be a cyclosporin, or a cyclosporin analogue which can be designated as a compound having the formula (2);

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, -CH=CH-CH=CHR or -CH2CH2R, wherein R is -C¾, -CH₂SH, -CH₂S-C„ wherein n is 1, 2, 3, 4, 5 or 6, - (CH₂)_mCOOR_a wherein m is 0 or 1 and R_a is H or Ci-Ce alkyl;

B is methyl, ethyl, 1 -hydroxy ethyl, isopropyl or n-propyl;

C is isobutyl, 2-hydroxyisobutyl, isopropyl or 1-methylpropyl;

D is -CH₃, -CH2OH or -CH₂OCH₂CH₂OH;

Ri is H or a group X-Rd or CRbR_c-X-Rd where Rb and R_c, which are identical or different, each represents hydrogen or C1-C4 alkyl or together represent C3-C7 cycloalkyl;

R₂ is methyl or ethyl;

X is bond, sulfur or -S(0)_n, wherein n is 1 or 2;

Rd is hydrogen, straight or branched C1-C6 alkyl, straight or branched C₂-C6 alkenyl, straight or branched C₂-C6 alkynyl, C3-C7 cycloalkyl, C4-C7 heterocyclyl having 1-3 heteroatoms selected from nitrogen, oxygen, and sulfur, aryl, heteroaryl or Rd contains a carboxyl, amino, amido group and wherein Rd may be optionally substituted with one or more groups, identical or different, of Ci-C alkyl, halogen, hydroxyl, alkoxycarbonyl, carboxyl, cycloalkyl, saturated or partially unsaturated 5-6 member heterocyclyl having 1 -3 heteroatoms selected from nitrogen, oxygen, and sulfur, which heterocyclyl is optionally substituted by one or more groups of C1-C6 alkyl, aryl, heteroaryl, amino, monoalkylamino, dialkylamino, amidino, guanidine or urea.

Certain cyclosporin analogues are described in application US20120088734. The new use, or new formulation of any compounds described therein is within the scope of this invention. The disclosures herein include any pharmaceutically acceptable salts. Where compounds are isomers, all chiral forms and racemates are included. The disclosures include all solvates, hydrates and crystal forms.

The cyclosporin may be cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin G, (D)-serine-8-cyclosporin, (D)-[0-hydroxyethylserine]-cyclosporin (TMM- 125), MeIle-(4)-cyclosporin (NIM-81 1), Aliosporivir (Debio-025), SCY-635, or SCY-641.

The cyclosporin may be cyclosporin A. Cyclosporin A can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -C¾; B is ethyl;

C is isobutyl;

D is -C¾;

Ri is H; and

R₂ is methyl. The cyclosporin may be cyclosporin B. Cyclosporin B can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -CH₃; B is methyl; C is isobutyl;

D is -C¾;

Ri is H; and

R₂ is methyl.

The cyclosporin may be cyclosporin C. Cyclosporin C can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is CH=CHR, wherein R is -C¾; B is 1-hydroxyethyl;

C is isobutyl;

D is -CH₃;

Ri is H; and

R₂ is methyl.

The cyclosporin may be cyclosporin D. Cyclosporin D can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -C¾; B is isopropyl;

C is isobutyl;

D is -C¾;

Ri is H; and

R₂ is methyl. The cyclosporin may be cyclosporin G. Cyclosporin G can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -CH₃; B is n-propyl;

C is isobutyl; D is -C¾;

Ri is H; and

R2 is methyl.

The cyclosporin may be (D)-serine-8-cyclosporin. (D)-serine-8-cyclosporin represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -

B is ethyl;

C is isobutyl;

D is -CH₂OH;

Ri is H; and

R2 is methyl.

The cyclosporin may be (D)-[0-hydroxyethylserine] -cyclosporin (IMM-125). hydroxy ethylserine]-cyclosporin (IMM-125) can be represented by formula:

or a pharmaceutically acceptable salt thereof wherein A is -CH=CHR, wherein R is -CH₃; B is ethyl;

C is isobutyl;

D is -CH₂OCH₂CH₂OH;

Ri is H; and

R₂ is methyl.

The cyclosporin may be MeIle-(4)-cyclosporin (NIM-811). MeIle-(4)-cyclosporin (NIM-811) can be represented by formula:

C is 1-methylpropyl; D is -CH₃;

Ri is H; and

R2 is methyl.

The cyclosporin may be Aliosporivir (Debio-025). Aliosporivir (Debio-025) represented by formula:

C is isopropyl;

D is -CH₃;

Ri is CR_bRc-X-R_d where R and R_c each represents hydrogen; X is bond, and R_d is hydrog (i.e. Ri is methyl); and

R2 is ethyl.

The cyclosporin may be SCY-635 or SCY-641. SCY-635 can be represented by formula:

C is 2-hydroxyisobutyl;

D is -CH₃;

Ri is X-Rci where X is sulphur and R_d is CH₂CH₂NMe₂

R₂ is methyl.

To the extent that any of the compounds described have chiral centres, the present invention extends to all isomers of such compounds, whether in the form of diastereomeric mixtures or or separated diastereomers. The invention described herein relates to all crystal forms, solvates and hydrates of any of the disclosed compounds however so prepared. To the extent that any of the compounds disclosed herein have acid or basic centres such as carboxylates or amino groups, then all salt forms of said compounds are included herein. In the case of pharmaceutical uses, the salt should be seen as being a pharmaceutically acceptable salt.

Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium or organic bases such as ethanolamine, N,N- dialkylethanolamines, morpholine, etc.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, citric, lactic, mandelic, glycolic, adipic, alginic, aryl sulfonic acids (e.g., benzenesulfonic, naphthalene-2-sulfonic, naphthalene-l,5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(l S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane- 1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), , (±)-DL-mandelic, metaphosphoric, methanesulfonic, l-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulfonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

Also encompassed are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography. The solvates can be stoichiometric or non-stoichiometric solvates. Particular solvates may be hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

Preparation of Suspensions

Included herein are novel formulations of the cyclophilin inhibitors. The preparation of certain micro-formulations of cyclosporin is disclosed in application US8202540. US8202540 does not disclose the co-formulation of mucoadhesives and cyclophilin inhibitors. Furthermore, US8202540 does not disclose the preparation of in situ gel forming systems of cyclosporin. The use of these cyclophilin inhibitor formulations in the treatment of periodontal disease is disclosed herein. In order to prolong bioavailability in the oral cavity, the cyclophilin inhibitor may be formulated into a suspension of microparticles or nanoparticles. Microparticles have a size range in the micrometer scale, and nanoparticles have a size range in the nanometer scale. Suitable formulations may have a particle size of around 1 μηι. For example, at least 50 % of the particles in the formulation may be less than 1 μηι in size. At least 50 % of the particles may be of size 200 nm to 1 μιη in size.

The cyclophilin inhibitor may be dispersed as a powder by stirring into a mechanically agitated dispersion medium to prepare a pre-suspension. For the mechanical agitation a variety of devices can be used, such as e.g. a propeller mixer, dissolver discs, or rotor-stator mixers. The dispersion medium may be water containing a suitable surfactant or non-aqueous liquid to act as a stabilising substance. Alternatively, the dispersion medium may be a non aqueous liquid. All liquids except water can be used as dispersion media, such as polyols (e.g. ethylene glycol, propylene glycol, glycerol), polyethylene glycols, medium chain triglycerides, vegetable oils, liquid hydrocarbons, or alcohols. Water may be admixed to the dispersion media up to amounts of 1-20 %, preferably 1-10 %. The cyclophilin inhibitor, in amorphous or crystalline form, may be dispersed as a powder by stirring into a mechanically agitated dispersion medium to prepare a foam-free pre- suspension. For the mechanical agitation a variety of devices can be used, such as e.g. a propeller mixer, dissolver discs, or rotor-stator mixers. As dispersion medium water containing stabilizers can be used.

To stabilise the suspension, one or more stabilising substances can be added. Examples of stabilising substances are poloxamers and poloxamines (polyoxyethylene-polyoxypropylene block copolymers), polysorbates, ethoxylated fatty alcohols or fatty acids. A particularly preferred stabilising substance is Vitamin E TPGS (d-alpha tocopheryl polyethylene glycol 1000 succinate). Stabilising substances can also be charged, such as phosphatidyl glycerol, lecithins of various origins, phospholipids, sphingolipids, cholates, or amino acids; amphoteric ionic surfactants such as CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]- 2-hydroxy-l-propanesulfonate), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate); or cationic surfactants, in particular substances used as preservatives such as cetylpyridinium chloride, benzalkonium chloride, chlorhexidine, or methyl-benzethonium chloride.

To achieve mucoadhesive properties of the formulation, a number of bioadhesive polymers can be used. Bioadhesive polymers have numerous hydrophilic groups such as hydroxyl, carboxyl, amide, phosphate or sulfate groups. These hydrophilic groups cause the polymer to swell in water and attach to mucosal surfaces by a combination of hydrogen bonding, electrostatic and hydrophobic interactions. Examples of such polymers are lectins, carbopol (polyacrylic acid), chitosan, hydroxyethyl cellulose, hydroxypropyl cellulose, or sodium carboxymethyl cellulose.

The pre-suspension can be further dispersed in a high-pressure homogeniser such as a French press, piston-gap homogeniser, jet stream homogeniser, bead mills, rotor-stator systems, or ultrasound-based systems. High-pressure homogenisation can be carried out at pressures between 100 and 2,000 bar using one, several or many cycles.

The micro or nanoparticles can be characterised in terms of particle size by laser diffractometry and photon correlation (PCS) spectroscopy. A particle size stated as D50 % of 1 μΜ means that 50 % of the particles have a diameter of 1 μΜ. Any of the cyclophilin inhibitor compounds disclosed herein may be suspended as microparticle or nanoparticle formulations. Disclosed herein are formulations of cyclosporin and mucoadhesives as microparticles or nanoparticles. Exemplary compounds may include cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin G, (D)-serine-8- cyclosporin, (D)-[0-hydroxyethylserine]-cyclosporin (EVIM-125), MeHe-(4)-cyclosporin (NIM-811), Aliosporivir (Debio-025), SCY-635, or SCY-641.

Preferred active agents include cyclosporin A (CyA). Suitable formulations of CyA include those with a neutral surfactant TPGS (Tocopheryl Polyethylene Glycol Succinate). Further surfactants may include poloxamers, for example Poloxamer 407 (Pluronic F 127). The composition may include poloxamer 407 and TPGS. Suitable compositions may include CyA (5 %), TPGS (1 %) and Poloxamer 407 (1 %). The cyclosporin used can be amorphous or crystalline, and can be micronised before suspension. The use of micronised agents avoids the need for precipitation of the drug from organic solvents, thus avoiding organic solvent residues in the final composition. The use of crystalline CyA avoids any problems with insoluble polymorphs and leads to controlled drug release rates.

The micro or nanosuspensions can be further formulated. The viscosity of the formulation can be increased to form a gel. For example a high concentration (15-20 %) of poloxamer can be used. The gel can be thermosensitive such that it is liquid at room temperature, but can solidify at 37 °C or similar physiological conditions. Suitable gels may contain 15-20 % poloxamer 407. Suitable gels may contain 17 % poloxamer 407. Alternatively suitable gels may contain hydroxypropyl methylcellulose (HPMC).

The formulation may contain a preservative such as an anti-mi crobial formulation. The preservative may be chlorhexidine gluconate. The use of chlorhexidine has no negative effect on the physic-chemical properties of the nanosuspension. Preparation of dried formulations

In order for the formulations to be stored for prolonged periods, the compositions may be made as solid state formulations by removal of liquid. The solid state formulations may contain bulking excipients such as polymers, carbohydrates or polyols. The formulations may contain two or more carbohydrates or polyols in combination. The one or more carbohydrate or polyol may be selected from mannitol, saccharose, raffinose, lactose, glucose, sucrose, sorbitol, trehalose, glycerol and/or polyethylene glycol. Preferably the carbohydrate selected is not one known to cause tooth decay. The formulation may contain trehalose or mannitol. The dried compositions incorporate at least one bulking excipient. The term "bulking excipient" or "matrix forming excipient" refers to a compound or composition that can protect the physical stability of a formulation or reagent during freezing, drying, and/or reconstitution of the dried substance. In the context of the present invention the term refers to a substance that, when included in aqueous solutions, protects the microparticles or nanoparticles suspended in the aqueous solutions from aggregation or rupture due to drying or freezing of the aqueous solutions. The term "lyophilised" or "lyophilisation" refers to drying a substance by freezing it and afterwards applying a high vacuum to remove water from the frozen substance by sublimation under lowered pressure. Examples of bulking excipients or matrix forming excipients include polymers, carbohydrates and polyols, such as PVP, methylcellulose, mannitol, saccharose, raffinose, lactose, glycerol, trehalose, glucose, sucrose, glycerol, polyethylene glycol, and sorbitol. A preferred bulking agent is trehalose, more particularly trehalose di-hydrate. A preferred bulking agent is mannitol. Particularly the at least one bulking excipient is present at between about 0.05 % w/v to about 20 % w/v, of the pre-dried solution, more particularly between about 2 % w/v and 20 % w/v, yet more particularly at about 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 % or 20 % w/v or any range therebetween. The level of bulking agent may be 5 % or less. The bulking agent may be mannitol or trehalose, which may be present at 5 % or less by weight of the pre-dried solution. Compositions of the present invention may include other optional components such as salts, oils, buffers or detergents. A particular bulking excipient is a high molecular weight polyethylene glycol such as carbowax 20M.

Lyophilised compositions of the present invention may be prepared by freeze-drying aqueous suspensions comprising the micro or nanoparticles containing the cyclophilin inhibitor. Lyophilisation is an effective process for preserving biological reagents without loss of activity of the biological reagent. The dried compositions are substantially lacking in water such that they are in the solid, rather than the liquid state. The level of water in the solid state composition may be less than 5 % by weight of the solid material. The level of water in the solid state composition may be less than 1 % by weight of the solid material.

The solid state may contain micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a bulking excipient, wherein the concentration of gel forming material in the solution which is dried to form the solid state formulation is 10 % or lower by weight of the solution.

The solid state composition contain low levels of water, typically less than 5 %, or even less than 2 %. The components of the solid composition are therefore the non-evaporatable consitiuents present in the aqueous solution before drying. Typically the highest volume component in the dried composition is the gel-forming material, followed by the bulking excipient. Typical solution ratios of the mixtures may be 2.5 % to 5 % bulking excipient, 2.5 % to 10 % gel forming material, 1-5 % of the active pharmaceutical cyclophilin inhibitor and less than 1% of the non-ionic surfactant, the rest being water. Once the water is removed, the ratios between the components remains the same, but the %'s alter. Therefore typical examples of solid compositions may be for example 20 % to 40 % bulking excipient, 20 to 80 % gel forming material, 8-40 % of the active pharmaceutical cyclophilin inhibitor and less than 5% of the non-ionic surfactant.

Particularly advantageous ratios include that the amount of gel forming material should be less than 50 % of the solidified mass. The percentage of gel forming material can be 25 % to 50 % of the solidified mass. The percentage of bulking excipient can be less than 30 % of the solidified mass.

The dried solid state formulations of the present invention are physically stable at room temperature for at least 12 months. Room temperature stable, means that the compositions are stable without refrigeration. In some cases they may be stable at temperatures of up to 37 °C for at least 12 months. This has great advantages in terms of reagent storage and transport. Compositions of the present invention may be rehydrated prior to use in the treatment of periodontal disease. Physically stable means that the average particle size before and after the drying and resuspension process is substantially the same. The average particle size is not affected by the drying process, and the dried material shows less particle ripening than would be present if the liquid phase were not removed.

Particularly such rehydration is by addition of water or an aqueous solution containing further surfactants. The addition of an aqueous solution rehydrates the dried compositions of the invention and dilutes the components of that composition (which will generally be in concentrated form) to their working concentration. The resuspended material may contain for example a poloxamer to allow the formation of a temperature sensitive gel. It is not possible to lyophilise and resuspend effectively a solution having greater than 10% poloxamer. Where the solution has a lower level of poloxamer, the material can be dried and reconstituted. However reconstitution with a solution having a high level of poloxamer either causes aggregation of the particles or requires too high a volume of solution; hence causing over-dilution of the active agent. Thus there a need to have the correct balance between the level of poloxamer in the lyophilisate and the re-suspension solution. Alternatively the particles can be suspended in a solution having little or no extra poloxamer (less than 5 %) with a further high concentration solution of poloxamer added. However such high concentration poloxamer solutions are viscous and difficult to handle, and therefore the optimal method is to use a level of poloxamer in the lyophilisate which is high, but not high enough to cause problems with the drying and resuspension process, along with a poloxamer solution that is low enough concentration to be handleable and can reconstitute the dried material directly, thereby allowing preparation of a poloxamer solution in a single resuspension step, the final re-suspension still having the desired gel forming properties (i.e. containing 15-20% poloxamer by weight). Such compositions and methods are described herein and claimed below.

The resuspension can be aided by using a temperature lower than room temperature. The viscosity of the gel forming solution is lower at lower temperatures. Thus the resuspension can be carried out at lower than 20 °C. The resuspension can be carried out at lower than 15 °C. The resuspension can be carried out at 0-15 °C. The resuspension can be carried out at 2- 10 °C. Typical lyophilisation conditions may include freezing over approx. 5 h at -45 °C at normal pressure followed by primary drying over approx. 35 h at -35 °C to -10 °C with a pressure decrease from normal pressure to 160 μbar and finally secondary drying over approx. 18 h at 25 °C at a pressure of to 14 μbar. Lyophilisation conditions may be optimised to improve the general behaviour of the nanoparticles in the lyophilisate matrix.

Chemical Definitions

Amino

Amino means ¾ and substituted amino. Substituted amino means NHR or R²R³ where R² and R³ are independent substituents or where R²R³ forms an optionally substituted 4 to 7 membered non-aromatic heterocyclic ring optionally containing a second heteroatom ring member selected from O, N and S and oxidised forms thereof. Exemplary substituted amino groups include Me2, NEt₂, piperidinyl, piperazinyl, morpholino, N-cyclohexyl, where the rings may be further substituted.

Alkyl

Alkyl means an aliphatic hydrocarbon group. The alkyl group may be straight or branched or cyclic. "Branched" means that at least one carbon branch point is present in the group. Thus, for example, fert-butyl and isopropyl are both branched groups. The alkyl group may be a lower alkyl group. "Lower alkyl" means an alkyl group, straight or branched, having 1 to about 6 carbon atoms, e.g. 2, 3, 4, 5 or 6 carbon atoms. Exemplary alkyl groups include methyl, ethyl, ^-propyl, /-propyl, «-butyl, /-butyl, s-butyl, n- pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, n-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 2- methyl-but-l-yl, 2-methyl-but-3-yl, 2-methyl-pent- 1 -yl, 2-methyl-pent-3-yl.

The alkyl group may be optionally substituted, e.g. as exemplified below.

The term alkyl also includes aliphatic hydrocarbon groups such as alkenyl, and alkylidene and cycloalkyl, cycloalkylidene, heterocycloalkyl and heterocycloalkylidene groups, which may be further substituted. Alkenyl

Alkenyl means an unsaturated aliphatic hydrocarbon group. The unsaturation may include one or more double bond, one or more triple bond or any combination thereof. The alkenyl group may be straight or branched. "Branched" means that at least one carbon branch point is present in the group. Any double bond may, independently of any other double bond in the group, be in either the (E) or the (Z) configuration.

The alkenyl group may be a lower alkenyl group. "Lower alkenyl" means an alkenyl group, straight or branched, having 2 to 6 carbon atoms, e.g. 2, 3, 4, 5 or 6 carbon atoms.

Exemplary alkenyl groups include ethenyl, rc-propenyl, /^'-propenyl, but-l-en-l-yl, but-2-en-l- yl, but-3-en-l-yl, pent-l-en-l-yl, pent-2-en-l-yl, pent-3-en-l-yl, pent-4-en-l-yl, pent-l-en-2- yl, pent-2-en-2-yl, pent-3-en-2-yl, pent-4-en-2-yl, pent-l-en-3-yl, pent-2-en-3-yl, pentadien- 1-yl, pentadien-2-yl, pentadien-3-yl. Where alternative (E) and (Z) forms are possible, each is to be considered as individually identified.

The alkenyl group may be optionally substituted, e.g. as exemplified below. Alkenyl includes cyano. Alkylidene

Alkylidene means any alkyl or alkenyl group linked to the remainder of the molecule via a double bond. The definitions and illustrations provided herein for alkyl and alkenyl groups apply with appropriate modification also to alkylidene groups. Alkylthio

Alkylthio means any alkyl group containing a sulfur atom in the carbon chain. The sulphur atom may be in the form of a thioether (C-S-C), a sulfoxide (C-S(=0)-C) or sulfone (C- S(=0)₂-C). Alkylthio groups may be further substituted. Alkylthio groups include CH₂-S-R where R is a further alkyl, cycloalkyl or substituted alkyl group.

Cycloalkyl

Cycloalkyl means a cyclic non-aromatic hydrocarbon group. The cycloalkyl group may include non-aromatic unsaturation. The cycloalkyl group may have 3 to 6 carbon atoms, e.g. 3, 4, 5 or 6 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl.

The cycloalkyl group may be optionally substituted, as defined below, e.g. as exemplified below. Exemplary substituted cycloalkyl groups include mono- or poly- alkyl- substituted cycloalkyl groups such as 1 -methyl cyclopropyl, 1-methylcyclobutyl, 1 -methy Icy clop entyl, 1- methylcyclohexyl, 2-methylcyclopropyl, 2-methylcyclobutyl, 2-methylcyclopentyl, 2- methylcyclohexyl, 1 ,2-dimethylcyclohexyl or 1,3-dimethylcyclohexyl.

Cycloalkylidene group

Cycloalkylidene means any cycloalkyl group linked to the remainder of the molecule via a double bond. The definitions and illustrations provided herein for cycloalkyl groups apply with appropriate modification also to cycloalkylidene groups.

Heterocycloalkyl

Heterocycloalkyl group means a non-aromatic cyclic group which contains one or more heteroatoms in the ring. The heterocycloalkyl group may contain O, N or S atoms. The heterocycloalkyl group may be fully saturated or partially unsaturated. The heterocycloalkyl group is typically monocyclic or bicyclic, and more usually is monocyclic.

Exemplary heterocycloalkyl groups include azetidinyl, pyrrolidinyl, piperidinyl, azepinyl, diazepinyl, dihydrofuranyl (e.g. 2,3-dihydrofuranyl, 2,5-dihydrofuranyl), 4,5-dihydro-lH- maleimido, dioxolanyl, 2-imidazolinyl, imidazolidinyl, isoxazolidinyl, morpholinyl, oxazolidinyl, piperazinyl, pyrrolidinonyl, 2-pyrrolinyl, 3-pyrrolinyl, sulfolanyl, 3-sulfolenyl, tetrahydrofuranyl, thiomorpholinyl, dihydropyranyl (e.g. 3,4-dihydropyranyl, 3,6- dihydropyranyl), dioxanyl, hexahydropyrimidinyl, 2-pyrazolinyl, pyrazolidinyl, pyridazinyl, 4H-quinolizinyl, quinuclinyl, tetrahydropyranyl, 3,4,5,6-tetrahydropyridinyl, 1,2,3,4- tetrahydropyrimidinyl, 3,4,5,6-tetrahydropyrimidinyl, tetrahydrothiophenyl, tetramethylenesulfoxide, thiazolidinyl, 1,3,5-triazinanyl, 1,2,4-triazinanyl, hydantoinyl, and the like. The point of attachment may be via any atom of the ring system.

Heterocycloalkylidene group

Heterocycloalkylidene means any heterocycloalkyl group linked to the remainder of the molecule via a double bond. The definitions and illustrations provided herein for heterocycloalkyl groups apply with appropriate modification also to heterocycloalkylidene groups.

Optionally substituted

"Optionally substituted" as applied to any group means that the said group may if desired be substituted with one or more substituents, which may be the same or different. Optionally substituted alkyl' includes both 'alkyl' and ' substituted alkyl' .

Examples of suitable substituents for "substituted" and "optionally substituted" moieties include halo (fluoro, chloro, bromo or iodo), Ci_6 alkyl, C3.6 cycloalkyl, hydroxy, Ci_6 alkoxy, cyano, amino, nitro, Ci_6 alkylamino, C2-6 alkenylamino, di-Ci-6 alkylamino, Ci_6 acylamino, di-Ci-6 acylamino, Ci-6 aryl, Ci-s arylamino, Ci-6 aroylamino, benzylamino, Ci-6 arylamido, carboxy, Ci_6 alkoxycarbonyl or (Ci_6 aryl)(Ci.io alkoxy)carbonyl, carbamoyl, mono-Ci.6 carbamoyl, di-Ci-6 carbamoyl or any of the above in which a hydrocarbyl moiety is itself substituted by halo, cyano, hydroxy, C1-2 alkoxy, amino, nitro, carbamoyl, carboxy or C_1-2 alkoxycarbonyl. In groups containing an oxygen atom such as hydroxy and alkoxy, the oxygen atom can be replaced with sulphur to make groups such as thio (SH) and thio-alkyl (S-alkyl). Optional substituents therefore includes groups such as S-methyl. In thio-alkyl groups, the sulphur atom may be further oxidised to make a sulfoxide or sulfone, and thus optional substituents therefore includes groups such as S(0)-alkyl and S(0)2-alkyl.

Substitution may take the form of double bonds, and may include heteroatoms. Thus an alkyl group with a carbonyl (C=0) instead of a CH₂ can be considered a substituted alkyl group.

Substituted groups thus include for example CFH₂, CF₂H, CF₃, CH₂NH₂, CH₂OH, CH₂CN, CH₂SCH₃, CH₂OCH₃, OMe, OEt, Me, Et, -OCH₂0-, C0₂Me, C(0)Me, z-Pr, SCF₃, S0₂Me, NMe₂ , CO H₂, CO Me₂ etc. In the case of aryl groups, the substitutions may be in the form of rings from adjacent carbon atoms in the aryl ring, for example cyclic acetals such as O- CH₂-0. The term "pharmaceutical composition" in the context of this invention means a re-suspended composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may take the form, for example, of tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.

The dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with the smaller dosages which are less than the optimum dose of the compound. Thereafter the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

The magnitude of an effective dose of a compound will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound and its route of administration. The selection of appropriate dosages is within the ability of one of ordinary skill in this art, without undue burden. In general, the daily dose range may be from about 0.1 mg to about 100 mg per kg body weight of a human and non-human animal, preferably from about 1 mg to about 50 mg per kg of body weight of a human and non-human animal, and most preferably from about 3 mg to about 30 mg per kg of body weight of a human and non- human animal. Biological applications

The cyclophilin inhibitors of the invention may be used to treat periodontitis. As described herein, the invention includes the use of the resuspended pharmaceutical compositions containing cyclosporins, sanglifehrins or cycloundecadepsipeptides for the treatment, or the manufacture of medicaments for use in the treatment of periodontitis or periodontal disease. The cyclophilin inhibitors may be prepared as compositions with mucoadhesives. Disclosed herein are pharmaceutical formulations containing a mucoadhesive and one or more agents selected from cyclosporins, sanglifehrins or cycloundecadepsipeptides. The mucoadhesives may be present in the dried solid state formulations, or may be added as part of the resuspension. The term resuspension may take the form of the preparation of a gel or paste, rather than a dilute solution.

The cyclophilin inhibitors or pharmaceutical formulations may be applied locally into the gingival pocket. The cyclophilin inhibitors or formulations may be applied as a micro- or nano-formulations. According to another aspect, the micro- or nano-formulation is or contains a mucoadhesive. The micro- or nano-formulation may be optimised to release the cyclophilin inhibitor over a period of several days or weeks. The cyclophilin inhibitors may be used in combination with other agents. Two or more cyclophilin inhibitors may be used together, or the composition may consist of a cyclophilin inhibitor and a further agent, which may be anti-bacterial or immunosuppressant.

The cyclophilin inhibitors or pharmaceutical formulations may be administered orally or by injection into the gums. The cyclophilin inhibitors or pharmaceutical formulations may be administered via a mouthwash containing the active ingredients. The cyclophilin inhibitors or pharmaceutical formulations may be administered as liquid formulations which become gels in- situ.

The cyclophilin inhibitors or pharmaceutical formulations may be used in humans or in veterinary products. The cyclophilin inhibitors or pharmaceutical formulations may be used in canines to treat canine periodontitis.

Methods for the preparation of Suspensions of Cyclosporin

Example 1

To a 5 % suspension of cyclosporin in glycerol were added under mechanical agitation (rotor- stator mixer, Ultra Turrax T25) 1 % of TPGS, 0.01 % chlorhexidine, 7 % gelatin, and 10 % hydroxypropyl cellulose. The resulting pre-dispersion was then homogenised using 3 cycles at 500 bar and 10 cycles at 1,500 bar in a Gaulin Micron Lab40 high pressure homogeniser at room temperature. Particle size analysis showed a D50 % of 1.8 μΜ, a D75 % of 4.6 μΜ and a D95 % of 5.6μΜ.

Example 2 The same experimental protocol as in example 1 but using 20 high pressure homogenisation cycles gave cyclosporin nanoparticles with a D95 % of 960 nM.

Example 3

The experimental protocol of example 2 but substituting Polyoxamer 407 for TPGS and 20 high pressure homogenisation cycles gave cyclosporin nanoparticles with a D50 % of 890 nM and a D95 % of 1.7 μΜ.

General nanomilling procedure (pilot scale)

The indicated amount of purified water was weighed into a glass beaker of suitable size. Afterwards the listed amounts of surfactant and stabilising polymer were added under magnetic stirring until the components fully dissolved. The described amount of cyclosporin was slowly added under stirring to give an almost homogenous suspension. The suspension was transferred into the milling system (e.g. Netzsch, Delta Vita). The milling system has been previously loaded with milling beads with approx. 0.2 mm diameter. The milling is performed under controlled temperature conditions (< 40 °C) over a suitable time (2-5 h) using an appropriate milling speed (2000 - 3000 rpm).

Characterisation

The isolated nano-suspensions have been measured on particle size distribution (PSD) by static laser diffraction (e.g. Malvern Mastersizer). In addition the stability of the nano- suspensions have been measured after storage at 2-8 °C and 25 °C/60 % humidity.

Additives to the nanosuspension

The surfactants selected from the neutral surfactants TPGS, cationic system (chitosan) or anionic system (sodium glycocholate). Data using the different surfactants is shown in figures 1-4. Figure 5 shows the benefits of micronized cyclosporin for particle stability.

In addition, the polymers like the Poloxamer 407 can also be added to the final nanosuspension to increase the gel forming at higher temperatures without any impact on the particles size distribution, as shown in figure 6. The stability of the formulation is shown below:

G0637N016 with added Poloxamer 407 to a final concentration of IS % Poloxamer:

Preservatives like the Chlorhexidine gluconate can be added without any impact on nanoparticles in the nanosuspension. The data for this is shown below: taipact of the addition of Chlorhexidine gluconate (0.2% CHG) on the neutra! nanosus ension G0637N016:

Lyophilsation, storage and reconstitution have been studied using the processes described below. In all cases the microparticles contained milled cyclosporin A, TPGS and poloxamer 407.

The lyophilisation process is shown below:

Lyophilisation in the absence of any cryprotectants, followed by constitution in water shows that the microparticle products are destroyed by lyophilisation in the absence of cryoprotectants (Fig 6).

Experiments with mannitol and poloxamer

2,3 Lyopit &ation tria! 2 ~ • G0637L002

Figures 7 to 1 1 show the stability of the microparticles after lyophilisation can be maintained by the use of mannitol and poloxamer (2.5 % each of the solution pre-drying). Figure 7 shows the particles are re-suspended in pure water, no further poloxamer is added. The particle size distribution (PSD) has not significantly altered by the drying and re-suspension. To achieve a total amount of 17 % poloxamer in the reconstituted lyophilisate, a 14 % poloxamer solution is needed. Figure 8 shows that re-constitution of the lyophilised microparticles in a solution of 5 % poloxamer (rather than water) causes a degree of aggregation of the particles.

Figure 10 shows the particles sizes following a successful two step reconstitution. The lyophilised products are resuspended in water, and a high concentration solution of poloxamer (25 %) added.

Figure 11 shows the particles sizes following an improved two step reconstitution. The lyophilised products are resuspended in water further containing TPGS, and a high concentration solution of poloxamer (25 %) added. The particle size distribution in Fig 11 is improved over Fig 10.

Data from the experiments is shown below:

Batch No. Particle Size Distribution [nm] Reconstitutio Reconst. data n remarks Time Fig

[min]

D(0.1) D(0.5) D(0.9) D(1.0)

G0637N026.2 82 210 615 1,609 origin n/a

suspension

G0637L002 80 205 784 2,121 with water (2 1-2 7 g)

G0637L002 86 237 3,349 10,884 with 5 % PLX 4-5 8 solution -pure

G0637L002 2,341 5,295 12,38 38,429 with 14 % 40 9

7 PLX solution

-pure

G0637L002 82 220 2,192 26,955 2 step: 1st: 10 10 water; 2nd: 25

% PLX -pure G0637L002 73 163 699 7,684 2 step: 1 st: 40 11 water +

TPGS; 2nd:

25 % PLX - pure

Figure 12-16 show the effects of higher levels of mannitol and poloxamer (5 % each of the pre-dried solution). Figure 12 (as 7) shows that the material can be resuspended and the PSD has not been affected. To achieve the total amount of 17% poloxamer in the reconstituted lyophilisate N003, a 10% poloxamer solution is needed.

Figure 13 (as Fig 8) shows that re-constitution of the lyophilised microparticles in a solution of 5 % poloxamer (rather than water) causes a degree of aggregation of the particles.

Figure 14 shows that re-constitution of the lyophilised microparticles in a solution of 10 % poloxamer (rather than water) causes complete aggregation of the particles. Figure 15 shows the particles sizes following an unsuccessful two step reconstitution. The lyophilised products are resuspended in water, and a high concentration solution of poloxamer (25 %) added. The particles are destroyed by the process (unlike Fig 10, where the process works) Figure 16 shows the particles sizes following an improved two step reconstitution. The lyophilised products are resuspended in water further containing TPGS, and a high concentration solution of poloxamer (25 %) added.

Data from the experiments is shown below

Batch No. Particle Size Distribution [nm] Reconstitution Reconst dat remarks Time a

[min] Fig G0637N026. 75 172 477 1,384 origin suspension n/a

3

G0637L003 71 155 51 1 1,613 with water (2 g) 1-2 12

G0637L003 91 299 4,562 11,548 with 5 % PLX 50 13 solution -pure

G0637L003 2,81 1, 123,96 1,548, 14 1,999,37 with 10 % PLX 240 - 14

4 9 1 1 solution -pure 360

G0637L003 157 2,629 5,851 27,663 2 step: 1 st: water; 30 -45 15

2nd: 25 % PLX - pure

G0637L003 74 168 753 7,317 2 step: 1 st: water + 30 -45 16

TPGS; 2nd: 25 %

PLX -pure

Experiments to increase the amount of of poloxamer

3.3 Lyopfii!Ssattort ti tal S ~ OQ63?LD05

Components V f

Of mpom A 3.5 85

Pii mi® 407 10.2

T ~i?

4.1 100

__ 2008 < 104 PLX -f 183S W¾t«f )

3 S3

Figure 17 shows the effect of raising the poloxamer concentration in the lyophilisate. The additional poloxamer is added to the microparticles, to which a further 5% poloxamer solution is added. Higher levels of poloxamer still allow the material to be dried, stored and re-constituted, but the re-suspension can be carried out using a lower concentration of poloxamer solution as a single solution, rather than a two-step process. To achieve the total amount of 17% poloxamer in the reconstituted lyophilisate, an 8.2 % poloxamer solution is needed. Thus there a key balance between the level of poloxamer in the lyophilisate and the re-constitution solution.

Data is shown below

Batch No. Particle Size Distribution [nm] Reconstitution remarks Reconst data

. Time Fig [min]

G0637L005 70 149 397 1,382 with water (2 g) 1-2

G0637L005 71 154 506 6,900 With 8.2 % PLX solution Over 17

(16.94 % PLX in total) night Experiments with higher levels of mannitol (greater than 5%) were unable to be reconstituted with the poloxamer solution. For use with high levels of poloxamer solution, the mannitol should be less than 5 % by weight of the solution weight.

Claims

1. A solid state formulation comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a bulking excipient, wherein the concentration of gel forming material in the solution which is dried to form the solid state formulation is 10 % or lower by weight of the solution.

2. The formulation according to claim 1 wherein the non-ionic surfactant is TPGS.

3. The formulation according to claims 1 or 2 wherein the gel forming material is a poloxamer.

4. The formulation according to any one of claims 1 to 3 wherein the cyclophilin inhibitor is selected from cyclosporins, sanglifehrins or cycloundecadepsipeptides.

5. The formulation according to any one of claims 1 to 4 wherein the inhibitor is a cycloundecadepsipeptide which can be designated as

1 2 3 4 5 6 7 8 9 10 1 1

In which AXXi is MeBmt, 4-fluoro-MeBmt, dihydro-MeBmt, 8-hydroxy-MeBmt, O-acetyl- MeBmt or AXXi contains a nitrogen atom in the side chain;

AXX₂ is Abu, Val, Thr, Thr(OMe), Thr(OAc), Thr(OCOCH₂CH₂CH₂OH) or an alternative threonine ester or threonine-O-alkyl or substituted O-alkyl moiety, Nva, 5-hydroxy-Nva (Hnv) or a moiety of type C(=0)CH₃ or C(=N-Y)CH₃ where Y is OH, NH₂ or O- or N-alkyl or substituted alkyl versions thereof;

AXX₄ is MeLeu, Melle, MeMet, Me Val, MeThr, MeThr(OAc), MeAla, EtVal, Etlle, EtPhe, EtTyr, EtThr(OAc), MeThr(OAc), MeTyr, MeTyr(OAc), MeTyr(OMe), MePhe, MeMet(Ox) wherein the sulphur atom of methionine is sulphoxide or sulphone; AXX₅ is Leu, Val, He, Gly, Abu;

AXX₆ is MeAla, Sar, MeLeu;

AXX₇ is Gly, Ala;

D-Hiv is (D)-2-hydroxyisovalerianic acid;

AXX₉ is MeLeu;

AXXio is Leu; and

AXXn is Me Val.

6. The formulation according to any one of claims 1 to 4 wherein the inhibitor is cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin G, (D)-serine-8- cyclosporin, (D)-[0-hydroxyethylserine]-cyclosporin (IMM-125), MeIle-(4)-cyclosporin (NIM-811), Aliosporivir (Debio-025), SCY-635, or SCY-641.

7. The formulation according to claims 3 to 6 wherein the bulking excipient is PVP, methylcellulose, mannitol, saccharose, raffinose, lactose, glucose, sucrose, sorbitol, trehalose, glycerol and/or polyethylene glycol.

8. The formulation according to claim 7 wherein the formulation contains trehalose.

9. The formulation according to claim 7 wherein the formulation contains mannitol.

10. The formulation according to any one of claims 3 to 9 wherein the formulation contains two or more carbohydrates or polyols.

11. The formulation according to any one of claims 3 to 10 wherein the poloxamer concentration is 2.5 to 10 % by weight of the solution.

12. The formulation according to any one of claims 1 to 1 1 wherein the composition is produced by spray-drying or freeze-drying.

13. The formulation according to any of claims 1 to 12 wherein the level of water in the solid state composition is less than 5 % by weight of the solid material.

14. The formulation according to claims 1 to 13 comprising cyclosporin A.

15. The formulation according to claim 14 wherein the cyclosporin A is crystalline.

16. The formulation according to claim 14 wherein the cyclosporin A is amorphous.

17. The formulation according to claims 14 to 16 wherein the cyclosporin A is micronized before formulation.

18. The formulation according to any one of claims 1 to 17 wherein the formulation consists of particles where greater than 50 % of the particles are less than 1 micrometer in diameter.

19. The formulation according to any one of claims 1 to 18 further comprising a mucoadhesive.

20. The formulation according to claim 19 wherein the mucoadhesive is selected from lectins, carbopol (polyacrylic acid), chitosan, hydroxyethyl cellulose, hydroxypropyl cellulose, or sodium carboxymethyl cellulose.

21. The formulation according to any one of claims 1 to 20 further comprising an antimicrobial preservative.

22. A composition comprising a re-suspended solution of the formulation according to any one of claims 3 to 21 wherein the concentration of poloxamer in the resuspended solution is 5-25 % by weight.

23. Use of a resuspended formulation according to any one of claims 1 to 22 in the treatment of periodontal disease.

24. Use according to claim 23 wherein the periodontal disease affects humans or canines.

25. A method of producing a temperature sensitive gel comprising a resuspended formulation according to any one of claims 1 to 21, the method comprising resuspending the material of any preceding claim in an aqueous solution.

26. The method according to claim 25 wherein the aqueous solution used for resuspension contains further gel forming material.

27. The method according to claim 25 wherein the formulation according to any one of claims 1 to 20 is suspended in a first solution which does not contain further gel forming material to form a first resuspension and a second solution having the further gel forming material is added to the first resuspension.

28. The method according to claim 26 wherein the aqueous solution used for resuspension contains 5-25 % poloxamer by weight.

29. The method according to claim 27 wherein the first resuspension solution is water, optionally containing TPGS, and the second solution contains poloxamer at a concentration of 25 % or greater.

30. An aqueous composition comprising micro or nanoparticles of a cyclophilin inhibitor, a non-ionic surfactant, a gel forming material and a bulking excipient, wherein the concentration of gel forming material is 10 % or lower by weight.

31. The composition according to claim 30 wherein the non-ionic surfactant is TPGS.

32. The composition according to claims 30 or 31 wherein the gel forming material is a poloxamer.

33. The composition according to any one of claims 30 to 32 wherein the bulking excipient is trehalose or mannitol.