Pharmaceutical formulations containing tacrolimus, methods for their preparation, and uses

Abstract

The present invention relates to a long-acting injectable formulation based on the combination of different PLGA polymers and biodegradable poly(D,L-lactide-co-glycolide) microparticles containing tacrolimus. It also relates to a method for the preparation of the microparticles and their use.

Description

[Technical field]

[0001] The present invention relates to a stable sustained release injectable pharmaceutical formulation containing a therapeutically effective amount of tacrolimus, a method for its preparation, and use of the formulation for the treatment and prevention of organ rejection after transplantation, graft-versus-host disease due to bone marrow transplantation, autoimmune diseases, infectious diseases, and the like. [Background technology]

[0002] Transplant rejection is the process by which the transplant recipient's immune system attacks the transplanted organ or tissue. The more similar the antigens are between donor and recipient, the less likely the organ will be rejected. Tissue matching ensures that the organ or tissue is as similar as possible to the recipient's tissue. It is usually not a perfect match. No two people have identical tissue antigens except for identical twins.

[0003] The following transplant drugs are used to suppress the recipient's immune system. The purpose is to prevent the immune system from attacking the newly transplanted organ. Without these drugs, the body will almost always mount an immune response and reject the foreign tissue.

[0004] Immunosuppressants or anti-rejection drugs reduce the body's ability to reject transplanted organs. There are two types of immunosuppressants: induction drugs, which are potent anti-rejection drugs used at the time of transplant, and maintenance drugs, which are anti-rejection drugs typically used immediately after transplant and in the long term. The maintenance drugs commonly used are calcineurin inhibitors (CNIs). Tacrolimus is the CNI immunosuppressant used by the majority of transplant patients based on reports from the Systematic Records of Transplant Recipients (SRTR).

[0005] The immunosuppressive activity of tacrolimus is mediated through the inhibition of calcineurin, a protein phosphatase found in the cytoplasm of T cells, and the subsequent blockade of interleukin-2 production, resulting in a decrease in T-cell proliferation.

[0006] The molecular formula of tacrolimus monohydrate is C 44 H 69 NO 12 .H2O, which corresponds to a molecular weight of 822. It is a white or almost white crystalline powder. It is freely soluble in ethanol and practically insoluble in heptane and water.

[0007] Tacrolimus in aqueous solution epimerizes to intermediate tacrolimus compound I, which is converted to tacrolimus compound II, reaching an equilibrium containing three forms, which is an inherent property of the molecule.

[0008] Tacrolimus is currently available under the trade name PROGRAF™ in oral tablets, capsules and suspensions, as well as concentrates for infusion solutions for hospitalized patients only. Infusion solutions contain polyoxyl 60 hydrogenated castor oil or polysorbate 80 as solubilizing agents whose presence can result in anaphylactic shock (i.e., severe allergic reaction) and death in patients. Instructions for using intravenous infusions recommend that patients should convert from intravenous to oral medication as soon as the individual's circumstances allow to avoid anaphylactic reactions, and that intravenous therapy should not be continued for longer than 7 days. Oral medication is administered at least once a day.

[0009] WO2006 / 002365A2 discloses a formulation comprising microparticles, the microparticles comprising a polymer and a drug such as tacrolimus, the drug being present in the microparticles at a concentration (weight of drug / weight of microparticle) of more than 50% and preferably more than 75%, suggesting that a formulation with a high loading rate may facilitate less frequent dosing.Unfortunately, the disclosed formulation has the drawback that at least 15% of the drug is released almost immediately, and the release of the drug lasts for only a maximum of about 3 weeks.

[0010] EP1868576A2 discloses an injectable nanoparticle formulation free of hydrogenated castor oil, comprising (a) particles of tacrolimus having an effective average particle size of less than about 2000 nm; and (b) at least one surface stabilizer, with the aim of avoiding the anaphylaxis problem.Unfortunately, the use of the disclosed particle size has the drawback that such particles are phagocytosed by immune cells (Dawes GJS et al., Mater Sci: Mater Med (2009) 20:1089-1094).

[0011] While each of the above represents an attempt to overcome the problems associated with existing treatment regimens, without providing a suitable controlled release product, there remains a need for a controlled release injectable formulation that avoids plasma fluctuations, avoids high initial release of drug, provides a satisfactory level of release, reduces the risk of associated side effects such as anaphylaxis, and improves patient compliance by avoiding having to remember to take a daily dose of an oral product. Summary of the Invention

[0012] The present invention provides a pharmaceutical formulation comprising microparticles, the microparticles comprising two different polymers and tacrolimus, each of the polymers being a poly(D,L-lactide-co-glycolide) polymer having the same lactide to glycolide ratio and different molecular weights.

[0013] Tacrolimus according to the present invention may include tacrolimus base or any salt, or derivative thereof, in any crystalline or amorphous form. Two types of conformational heterogeneity of tacrolimus have been reported: 1) Cis-trans conformational isomerization involving restricted rotation of the amide bond in the pipecolic acid moiety. 2) The cis isomer and tacrolimus exist in the cis conformation in the solid state.

[0014] The present invention is directed to injectable pharmaceutical formulations for the controlled release of tacrolimus, optionally in combination with other immunosuppressants, for parenteral administration used to prevent or treat organ rejection after transplantation, more specifically for the prevention of organ rejection in adult and pediatric patients who have received allogeneic liver, kidney or heart transplants.

[0015] It is an object of the present invention to provide tacrolimus encapsulated in polymeric microparticles to control the release of the drug and reduce the frequency of administration. Such a formulation would ensure better medication adherence, reduce the need for therapeutic drug monitoring, reduce the potential for anaphylactic problems associated with current injected tacrolimus formulations, and avoid the need for daily dosing of an oral product.

[0016] A further advantage of the present invention is to provide an injectable formulation of tacrolimus that has a substantially linear release profile for a period of up to two months without any release lag phase or burst, which is achieved by combining two microparticle types made of different PLGA polymers.

[0017] It is a further object of the present invention to provide an injectable formulation that can be administered subcutaneously or intramuscularly to form a depot that provides long-term controlled release of the drug.

[0018] It is a further object of the present invention to provide an injectable controlled release formulation comprising tacrolimus as an active ingredient, which exhibits good syringability, injectability, no clogging or blocking of the syringe needle, good drainage, sterility, and resuspension in the case of suspensions.

[0019] A further object of the present invention is to provide a method for preparing injectable polymeric microparticles in powder form containing tacrolimus. The method involves emulsification (o / w) (single or double) followed by solvent extraction / evaporation. An aqueous vehicle is also provided for reconstitution of the powder prior to administration.

[0020] The microparticles with diluent can be present as a kit with a dual chamber syringe or a syringe pre-filled with the diluent and the microparticles present in separate vials.

[0021] Other objects and advantages of the present invention will become apparent to those skilled in the art in view of the following detailed description. [Brief description of the drawings]

[0022] [Figure 1] The effect of Mw and lactide to glycolide (L:G) ratio on the degradation of the films is presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] For purposes of the present invention, a pharmaceutical formulation containing an active ingredient is considered to be stable if the active ingredient degrades more or less slowly than it does by itself and / or in known pharmaceutical formulations. Words such as controlled release, sustained release, extended release and long acting release are used interchangeably unless otherwise specified.

[0024] As already mentioned, the primary objective of the present invention is to provide a controlled release injectable formulation of tacrolimus in the form of drug-loaded microparticles that contributes to optimizing the pharmacokinetics of tacrolimus and improving medication adherence.

[0025] Despite its success in ensuring graft survival, the therapeutic use of tacrolimus is challenging due to its narrow therapeutic index (5-15 ng / ml). Tacrolimus has high inter- / intra-patient variability in its pharmacokinetic profile and poor oral bioavailability due to its low solubility. Sub-therapeutic levels of tacrolimus can result in acute rejection of xenografts. Furthermore, systemically delivered tacrolimus can cause severe side effects including nephrotoxicity and systemic immunosuppression due to non-selective distribution of the drug. In fact, drug-induced nephrotoxicity is the main dose-limiting side effect of TAC with a reported overall incidence as high as 44%. Unfortunately, nephrotoxicity can result in severe complications such as adverse effects on graft survival and life expectancy of patients. Indeed, nephrotoxic effects present a challenge during treatment regimens involving these drugs (Randhawa, PS, Starzl, TE & Demetris, AJ Tacrolimus (FK506) - Associated Renal Pathology. Adv Anat Pathol 4, 265-276 (1997)).

[0026] Tacrolimus is currently available in oral dosage forms, including immediate release capsules, sustained release capsules and sustained release tablets.Low water solubility, site-dependent permeability, extensive first-pass metabolism in the gastrointestinal tract and liver, P-gp-mediated drug excretion and food effects are the most important reasons for the variously low oral bioavailability of tacrolimus.Tacrolimus is also available as a concentrate for a solution for infusion, but intravenous administration is limited only to the early stages of organ transplantation, when oral administration is not feasible, and it is recommended that intravenous infusion must be discontinued as soon as the patient can tolerate oral administration when the subject is in hospital care (USA Prescribing Information for Prograf® Injection).

[0027] The present invention provides a controlled release drug delivery system for parenteral administration of tacrolimus in biodegradable polymers as microparticles that controls drug release and reduces associated toxicity while maintaining the immunosuppressant activity of tacrolimus, and allows sustained release of the active ingredient after a residence time in the polymer that avoids the poor oral bioavailability problems mentioned above.

[0028] Adherence to treatment is a key determinant of clinical outcome for patients in a wide range of clinical settings. Adherence is particularly important in severe illnesses where patients often require months or years of treatment and premature cessation of treatment can have serious consequences for the patient's health and quality of life.

[0029] Regardless of the specific reason for treatment non-adherence, patient failure to consistently take prescribed medications contributes to high rates of relapse, hospitalization, and, in some patients, an increased risk of death.

[0030] Recent advances in drug delivery technology have led to the development of innovative delivery systems designed to improve therapeutic outcomes. One possible solution to the problem of poor adherence to drug therapy is the development of new long-acting drug delivery systems that slowly release drug over a period of days or weeks with a single application. Long-acting injectable technologies can provide advantages over conventional products by improving safety and efficacy through a long duration of action, and reducing adherence issues as well as side effects. By allowing patients to take drugs less frequently, these technologies create drugs that can be particularly beneficial in treating severe diseases where adherence is closely correlated to improved outcomes.

[0031] The formulations of the present invention have improved solubility characteristics, which in turn improve bioavailability and reduce variability in absorption upon administration to a patient. By meeting these needs, the present invention eliminates the need to use polyoxyl 60 hydrogenated castor oil (HCO-60) and / or polysorbate 80 as solubilizing agents. This is beneficial because conventional injectable tacrolimus formulations contain polyoxyl 60 hydrogenated castor oil or polysorbate 80 as solubilizing agents. The presence of such solubilizing agents can result in anaphylactic shock (i.e., severe allergic reaction) and death in the patient.

[0032] The present invention is used for preventing transplant rejection in adult kidney, liver or heart allograft recipients.A therapeutically effective amount of the injectable formulation of the present invention is administered to a subject to form a depot subcutaneously or intramuscularly in the patient.The depot slowly releases tacrolimus over time to provide long-term treatment to the allogeneic organ recipient.

[0033] Biodegradable materials are of natural or synthetic origin and are degraded in vivo, either enzymatically or non-enzymatically, or both, to produce biocompatible and toxicologically safe by-products that are further eliminated by normal metabolic pathways. The number of such materials used for controlled drug delivery has increased dramatically over the past decade. The basic categories of biomaterials used in drug delivery can be broadly classified as: (1) synthetic biodegradable polymers, including relatively hydrophobic materials such as α-hydroxy acids (a family that includes polylactic-co-glycolic acid, PLGA), polyanhydrides, etc., and (2) naturally occurring polymers, e.g., complex sugars (hyaluronan, chitosan) and minerals (hydroxyapatite).

[0034] The polyester PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). It is the most defined biomaterial available for drug delivery in terms of design and implementation. Polylactic acid contains an asymmetric α-carbon, typically described as D or L in classical stereochemical terms, and sometimes as R and S, respectively. The enantiomeric forms of the polymer PLA are poly D-lactic acid (PDLA) and poly L-lactic acid (PLLA). PLGA is commonly an acronym for poly D,L-lactic-co-glycolic acid, where the D- and L-lactic forms exist in equal ratios.

[0035] Injectable biodegradable and biocompatible PLGA particles (microparticles, microcapsules, nanocapsules, nanospheres) can be utilized in controlled release dosage forms. Drugs formulated in such polymeric devices are released either by diffusion through the polymer barrier, or by erosion of the polymeric material, or by a combination of both diffusion and erosion mechanisms. In addition to its biocompatibility, drug compatibility, favorable biodegradation kinetics, and mechanical properties, PLGA can be easily processed and manufactured in a variety of forms and sizes. The polymer formulation is the most important factor that determines the hydrophilicity and degradation rate of the delivery matrix, which affects the rate of degradation. Increasing the glycolic acid percentage in the oligomer generally accelerates the weight loss of the polymer. PLGA 50:50 exhibits faster degradation than PLGA 65:35 due to the favorable degradation glycolic acid percentage assigned by the higher hydrophilicity. Subsequently, PLGA 65:35 exhibits faster degradation than PLGA 75:25, which in turn exhibits faster degradation than PLGA 85:15. Therefore, the absolute value of the degradation rate increases with the percentage of glycolic acid. The amount of glycolic acid is a critical parameter in controlling the hydrophilicity of the matrix and therefore the degradation and drug release rate. Higher molecular weight polymers generally exhibit slower degradation rates. Molecular weight is directly related to the size of the polymer chain. Polymers with higher molecular weight have longer polymer chains and take longer to degrade than smaller polymer chains.

[0036] The drug release rate and release time can be controlled by adjusting the type of polymer, the polymer molecular weight, and the microsphere size and morphology, making it possible to produce drug-loaded microparticles according to therapeutic needs. There are two expected effects of the application of PLGA microsphere technology to tacrolimus. One is the reduction of adverse effects related to the change in pharmacokinetic profile. The other is the improvement of medication adherence.

[0037] Commercially available polymers suitable for use in preparing PLGA microparticles according to the present invention include, but are not limited to, RESOMER® from Evonik Industries AG and LAKESHORE BIOMATERIALS, Expansorb® from PCAS., PURASORB® from PURAC Biochem BV.

[0038] The objectives of the present invention are particularly aided by the use of PLGA polymers having a lactide to glycolide ratio of 50:50. Such polymers, preferably those having a molecular weight of 15,000-80,000 Da, more preferably 15,000-58,000 Da, especially those having a molecular weight of approximately 17,000 Da to 50,000 Da, are particularly relevant for achieving a linear release profile over a period of at least two months.

[0039] In a preferred embodiment of the present invention, the molecular weight of the first polymer is 15,000 to 30,000 Da, and the molecular weight of the second polymer is 30,000 to 80,000 Da. In a further preferred embodiment of the present invention, the molecular weights of the two polymers are 17,000 Da and 50,000 Da, respectively.

[0040] While the use of a single PLGA polymer does not yield the desired release profile, we have now surprisingly found that a linear profile of tacrolimus release that is controlled to result in a low initial burst release of tacrolimus, over a period of at least two months, is achieved when combining two different PLGA microparticle types. Both types of PLGA polymer have a 50:50 lactide to glycolide ratio, but each microparticle type is prepared with a different molecular weight polymer. When the two microparticle types are combined in a ratio of 70:30 to 30:70, the required release rate is achieved.

[0041] Nevertheless, it remains to be seen whether the appropriate selection and combination of polymers can function in a similar manner.It can be demonstrated that the combination of microparticles made with three or more different polymers of the same or different nature, different molecular weight and / or lactide to glycolide ratio can present behavior that is equivalent to that of the present invention.In addition, the present invention can find application to other pharmacoactive ingredients with low solubility and high membrane permeability, such as tacrolimus.Such pharmacoactive ingredients can be flurbiprofen, naproxen, cyclosporine, ketoprofen, rifampicin, carbamazepine ribenclamide, bicalutamide, ezetimibe, aceclofenac, etc.

[0042] The amount of drug loading as well as the polymer concentration in the drug delivery matrix plays a significant role in the rate and duration of drug release. A matrix with a higher drug loading has a larger initial burst release than one with a lower loading due to a smaller polymer to drug ratio. However, the effect of this drug loading weakens when the drug loading reaches a certain level depending on the type of drug. In the present invention, the drug loading in the microparticles is preferably less than 30% w / w of tacrolimus, especially 20% w / w to 30% w / w. A polymer concentration of 5% w / w to 13% w / w is also preferred in the present invention.

[0043] Several methods are known for making PLGA microparticles. Preferably, the microparticles of the present invention are produced by a single emulsion solvent evaporation method, which is the easiest, fastest and most cost-effective method. The preferred method is described in more detail below: a) the steps of: - dissolving two different molecular weight PLGA polymers in a suitable solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), one or more surfactants, and one or more buffers and maintaining it under controlled temperature, the continuous phase being thermostated at a temperature below 20°C, more preferably between 5 and 10°C; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., an in-line homogenizer) or an overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of organic solvent and microparticle coagulation; - sieving the formed microparticles and washing them with water; - drying the microparticles under reduced pressure 23. A method for the preparation of microparticles comprising: b) the steps of: i) dissolving a first PLGA polymer in a suitable solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), one or more surfactants, and one or more buffers and maintaining it under controlled temperature, the continuous phase being thermostated at a temperature below 20°C, more preferably between 5 and 10°C; mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., an in-line homogenizer) or an overhead stirrer to form a suspension; ii) dissolving a second PLGA polymer comprising a different molecular weight than the first polymer in a suitable solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), one or more surfactants, and one or more buffers and maintaining it under controlled temperature, the continuous phase being thermostated at a temperature below 20°C, more preferably between 5 and 10°C; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., an in-line homogenizer) or an overhead stirrer to form a suspension; iii) mixing the suspension containing the first PLGA polymer and the second PLGA polymer together and subjecting it to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of the organic solvent and the microparticles coagulation; - sieving the formed microparticles and washing them with water; - drying the microparticles under reduced pressure 23. A method for the preparation of microparticles comprising: c) the steps of: i) dissolving a first PLGA polymer in a suitable solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), one or more surfactants, and one or more buffers and maintaining it under controlled temperature, the continuous phase being thermostated at a temperature below 20°C, more preferably between 5 and 10°C; mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., an in-line homogenizer) or an overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of organic solvent and microparticle coagulation; - sieving the formed microparticles and washing them with water; - drying the microparticles under reduced pressure ii) dissolving a second PLGA polymer comprising a different molecular weight than the first polymer in a suitable solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), one or more surfactants, and one or more buffers and maintaining it under controlled temperature, the continuous phase being thermostated at a temperature below 20°C, more preferably between 5 and 10°C; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., an in-line homogenizer) or an overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of organic solvent and microparticle coagulation; - sieving the formed microparticles and washing them with water; - drying the microparticles under reduced pressure iii) physically mixing the microparticles made from the first PLGA polymer and the microparticles made from the second PLGA polymer after drying. 2. A method for the preparation of the microparticles of claim 1, comprising:

[0044] The molar ratio of the PLGA polymers may be from 70:30 to 30:70, preferably 50:50.

[0045] Suitable solvents for PLGA that can be used in the above method include, but are not limited to, organic solvents such as ethyl acetate, tetrahydrofuran, acetonitrile, dichloromethane (DCM) and chloroform, with the preferred solvent being dichloromethane.

[0046] The continuous phase consists of an aqueous solution containing one or more surfactants selected from anionic surfactants (e.g. sodium stearate, sodium lauryl sulfate), non-ionic surfactants (e.g. tweens), polyvinylpyrrolidone, sodium carboxymethylcellulose and gelatin, used independently or in combination. It is preferred to use one surfactant. The preferred surfactant is polyvinyl alcohol (PVA).

[0047] Suitable buffering agents include sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate monobasic, and potassium phosphate dibasic, and combinations thereof, with preferred buffering agents being sodium carbonate and sodium bicarbonate, and combinations thereof.

[0048] The process described in this invention results in the formation of small particles with a size distribution between 10 and 200 microns as measured by laser light diffraction.

[0049] The formulation is preferably administered subcutaneously or intramuscularly after reconstitution with a suitable diluent. More specifically, the diluent can be packed into a pre-filled syringe, and the powder can be contained in a vial containing microparticles. Immediately after use, the contents of the pre-filled syringe (solvent) and the vial (powder) are mixed to prepare a suspension that is injected into the patient. Alternatively, a dual-chamber pen can be used; the powder in one chamber is mixed with the solvent into the other chamber of the pre-filled pen before use, and the resulting suspension is injected into the patient. The formulation is preferably administered once every two months.

[0050] Suitable diluents include pharma- ceutically acceptable additives selected from the group consisting of suspending agents / viscosity enhancers, buffers and / or pH adjusters, surfactants, and tonicity adjusters. Suitable viscosity enhancers include mannitol, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP), such as PLASDONE, and hydroxypropylmethylcellulose (HPMC), such as methocel, preferably sodium carboxymethylcellulose and mannitol. Commonly used buffer additives include citric acid monohydrate, glycine, maleic acid, methionine, sodium acetate, sodium citrate dihydrate, sodium dihydrogen phosphate monohydrate, and sodium disodium phosphate heptahydrate, preferably sodium dihydrogen phosphate monohydrate and sodium disodium phosphate heptahydrate, and / or citric acid monohydrate. Tonicity adjusters, such as dextrose, mannitol, potassium chloride, sodium chloride, preferably sodium chloride, can be used. Surfactants such as polysorbate 20 and 80, Da-tocopheryl polyethylene glycol 1000 succinate, polyoxyethylated castor oil, preferably polysorbate 20 and 80, may also be used. The pH regulator is selected from acetic acid, sodium hydroxide, sodium chloride, preferably sodium hydroxide and / or sodium chloride. Aqueous diluents having a pH range of 6 to 7.5 and a viscosity range of 3 to 90 cP are particularly preferred. EXAMPLES

[0051] Example 1 The suitability of the materials was tested by dissolving the polymers in various solvents (i.e., DCM, THF) and slowly adding the API material to the resulting solution. Different polymers were used as presented in Table 1. Clear solutions were obtained in all cases with tacrolimus concentrations up to 30% w / w.

[0052] For degradation studies, films of each polymer containing 30% w / w tacrolimus were synthesized and compared to a placebo film (without the presence of tacrolimus). After evaporating the solvent, films were prepared from solutions containing appropriate amounts of polymer and tacrolimus in DCM solvent. The separated films were immersed in phosphate buffer saline (PBS, pH=7.4) solution and kept at 37° C. for approximately one month. At regular time intervals, samples of the films were removed and the Mw was measured by GPC to test for mass loss. TIFF2024536879000001.tif33170

[0053] The films were homogeneous and no phase separation was observed. The polymer MW loss over time was measured and the results are presented in Figure 1 below.

[0054] Polymers with the same lactide:glycolide ratio of 50:50 are most suitable for the purposes of the present invention. Furthermore, all films were tested for their compatibility with tacrolimus and exhibited very similar degradation profiles indicating the absence of API-induced biodegradation.

[0055] Example 2 Microparticles of the two polymers with the same lactide to glycolide ratio were prepared using a simple emulsion solvent evaporation method as follows.

[0056] Poly(D,L-lactide-co-glycolide) (Mw=17,000 or Mw=50,000) with a 50:50 molar ratio was dissolved in dichloromethane under stirring. Subsequently, tacrolimus was dissolved in the polymer solution to form the dispersed phase (DP). Poly(vinyl alcohol) was dissolved in water for injection at 80°C, followed by the addition of sodium bicarbonate and sodium carbonate. The solution was cooled to 25°C to form the continuous phase (CP). Microparticles of desired particle size distribution were prepared by delivering the CP and DP to an in-line disperser. The suspension was subjected to solvent extraction and evaporation by stirring under a controlled temperature of 20°C and airflow to ensure the removal of organic solvent and particle coagulation. After 3-4 hours, the microparticles were transferred to a glass filter dryer, washed with excess water at room temperature, and left to dry under reduced pressure for 24 hours.

[0057] The effect of the Mw of the polymer used is shown in Table 2 on the quality attributes of the produced microparticles (ie, particle size and release profile). TIFF2024536879000002.tif55170

[0058] The results show that higher molecular weight polymers lead to larger microparticles for the same set of process parameters. This may be due to the higher viscosity of the dispersed phase during emulsification. The earlier release profile obtained with the smaller microparticles may be due to the higher ratio of surface area to unit volume.

[0059] Example 3 The effect of polymer concentration in the dispersed (oil) phase during microparticle formulation was also examined with low MW PLGA polymers. Formulations are tested to evaluate the effect of PLGA concentration on the quality attributes of the microparticles.

[0060] The effect of polymer concentration in the dispersion (oil phase) is shown in Table 3 on the quality attributes of the produced microparticles (ie, particle size and release profile). TIFF2024536879000003.tif38170

[0061] As shown in Table 3 above, by increasing the polymer concentration of the dispersed phase, the particle size also increases due to the higher viscosity during emulsification. The dissolution profiles of the two formulations were almost similar with slightly higher linearity for the microparticles obtained with the lower PLGA concentration. Thus, it is clear that PLGA concentrations between 5% w / w and 13% w / w create microparticles of acceptable size and are suitable for the purposes of the present invention.

[0062] Example 4 As shown in the previous formulation, particle size plays an important role in dissolution profile. Besides particle size, surface area per unit volume can also be increased by creating porosity in microparticles. The most common way to create porous microparticles using solvent extraction / evaporation method is to apply double emulsion method. To evaluate the effect of porosity on release rate, double emulsion solvent extraction and evaporation method are applied, and the resulting formulation is compared with that made using single emulsion method.

[0063] The effect of emulsion type is tabulated on the quality attributes of the microparticles produced (ie, particle size and release profile). TIFF2024536879000004.tif38170

[0064] The double emulsion method results in larger microparticles compared to single emulsion with the same set of process parameters. From the results obtained based on size, we expected to find a higher release rate for the smaller particles. The results show an earlier release profile for the larger microparticles obtained with the double emulsion method. This indicates that the presence of porosity dominates the size effect, with larger porous microparticles exhibiting a higher release rate due to their larger specific surface area.

[0065] To more conclusively evaluate the effect of porosity on release rate, microparticles exhibiting approximately the same size must be tested. To separate microparticles of the same size, an in-line homogenizer was utilized.

[0066] The effect of emulsion type is shown in Table 5 on the quality attributes of the produced microparticles (ie, particle size and release profile). TIFF2024536879000005.tif38170

[0067] From the results presented it is clear that for microparticles of the same size, the dissolution profile is much more rapid for the microparticles obtained by the double emulsion method, due to the obvious effect of the porosity of the microparticles and their higher specific surface area.

[0068] Example 5 To achieve a linear profile over a two month period according to the present invention, microparticles were prepared with two PLGAs of different MW but using the same (50:50) lactide to glycolide ratio. A single emulsification method was used as described in Example 2. The concentration of PLGA was at an optimal ratio of 5% w / w to 13% w / w, the concentration of tacrolimus was less than 30% w / w, and the microparticles created have a particle size of 10 to 200 microns.

[0069] The separately created microparticles were physically mixed in different mass ratios ranging from 70:30 to 30:70 and their dissolution profiles were measured and are presented in Table 6 below. JPEG2024536879000006.jpg140170

[0070] The release profile exhibits an approximately linear release for at least two months according to the present invention.

[0071] The effect of PLGA mixtures in which the polymers were mixed in situ was also evaluated. This could be done either by dissolving the two polymers in DCM prior to emulsification (one dispersed phase) or by emulsifying one dispersed phase containing one of the polymers followed by emulsifying a second dispersed phase containing the second polymer in the same continuous phase (two different dispersed phases).

[0072] The dissolution profiles of these formulations again resemble those presented in Table 6.

Claims

1. 1. A pharmaceutical formulation comprising microparticles, the microparticles comprising two different polymers and tacrolimus, each of the polymers being a poly(D,L-lactide-co-glycolide) polymer and having the same lactide to glycolide ratio, the first poly(D,L-lactide-co-glycolide) polymer having a molecular weight of about 17,000 Da and the second poly(D,L-lactide-co-glycolide) polymer having a molecular weight of about 50,000 Da, and the pharmaceutical formulation being administered once every two months.

2. 10. The pharmaceutical formulation of claim 1, wherein each of the poly(D,L-lactide-co-glycolide) polymers has a 50:50 ratio of lactide to glycolide.

3. 3. The pharmaceutical formulation of claim 1 or 2, wherein the poly(D,L-lactide-co-glycolide) polymers each have a different weight average molecular weight in the range of 15,000 to 80,000 Da.

4. 4. The pharmaceutical formulation of claim 3, wherein the poly(D,L-lactide-co-glycolide) polymers each have a different weight average molecular weight ranging from 15,000 to 58,000 Da.

5. 4. The pharmaceutical formulation of claim 3, wherein the poly(D,L-lactide-co-glycolide) polymers each have a different weight average molecular weight ranging from 17,000 to 50,000 Da.

6. 3. The pharmaceutical formulation of claim 1, wherein the first poly(D,L-lactide-co-glycolide) polymer has a molecular weight of 15,000 to 30,000 Da and the second poly(D,L-lactide-co-glycolide) polymer has a molecular weight of 30,000 to 80,000 Da.

7. 7. The pharmaceutical formulation of claim 6, wherein the molecular weights of the two poly(D,L-lactide-co-glycolide) polymers are 17,000 Da and 50,000 Da, respectively.

8. 3. The pharmaceutical formulation of claim 1, comprising two different microparticle types in a ratio of 70:30 to 30:

70.

9. 3. The pharmaceutical formulation of claim 1 or 2, wherein the two different microparticles have particle sizes as measured by laser light diffraction of 10 to 200 microns.

10. 3. The pharmaceutical formulation of claim 1 or 2, wherein the concentration of polymer in the microparticles is 5 to 13% w / w.

11. 3. The pharmaceutical formulation of claim 1 or 2, which is reconstituted with a diluent prior to intramuscular or subcutaneous administration.

12. 12. The pharmaceutical formulation of claim 11, wherein the diluent comprises one or more of sodium carboxymethylcellulose, mannitol, sodium chloride, sodium hydroxide, polysorbate, acetic acid, sodium dihydrogen phosphate monohydrate, disodium phosphate heptahydrate.

13. 3. The pharmaceutical formulation of claim 1 or 2, which is administered by intramuscular or subcutaneous injection.

14. 10. The pharmaceutical formulation of claim 1, wherein the tacrolimus drug loading of the microparticles is 20% w / w to 30% w / w.

15. The following steps: - dissolving two different molecular weight PLGA polymers in a solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), poly(vinyl alcohol) (PVA) and a buffer and maintaining it under controlled temperature; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., in-line homogenizer) or overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of the organic solvent and solidification of the microparticles; - collecting the formed microparticles on a sieve and washing them with water; - drying the microparticles under reduced pressure 10. A method for preparing the microparticles of claim 1, comprising:

16. The following steps: i) dissolving a first PLGA polymer in a solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), poly(vinyl alcohol) (PVA) and a buffer and maintaining it under controlled temperature; mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., in-line homogenizer) or overhead stirrer to form a suspension; ii) dissolving a second PLGA polymer comprising a different molecular weight than the first polymer in dichloromethane (DCM) with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), poly(vinyl alcohol) (PVA) and a buffer and maintaining it under controlled temperature; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., in-line homogenizer) or overhead stirrer to form a suspension; iii) mixing the suspension containing the first PLGA polymer and the second PLGA polymer together and subjecting it to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of the organic solvent and solidification of the microparticles; - collecting the formed microparticles on a sieve and washing them with water; - drying the microparticles under reduced pressure 10. A method for preparing the microparticles of claim 1, comprising:

17. The following steps: i) dissolving a first PLGA polymer in a solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), poly(vinyl alcohol) (PVA) and a buffer and maintaining it under controlled temperature; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., in-line homogenizer) or overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of the organic solvent and solidification of the microparticles; - collecting the formed microparticles on a sieve and washing them with water; - drying the microparticles under reduced pressure; ii) dissolving a second PLGA polymer comprising a different molecular weight than the first polymer in a solvent with stirring; - adding tacrolimus to the polymer solution with stirring to form a dispersed oil phase (DP); - preparing a continuous phase (CP) comprising water for injection (WFI), poly(vinyl alcohol) (PVA) and a buffer and maintaining it under controlled temperature; - mixing and emulsifying the dispersed and continuous phases using a high shear rotor-stator continuous flow disperser (i.e., in-line homogenizer) or overhead stirrer to form a suspension; - subjecting the suspension to solvent extraction and evaporation by stirring under controlled temperature and airflow to ensure satisfactory removal of the organic solvent and solidification of the microparticles; - collecting the formed microparticles on a sieve and washing them with water; - drying the microparticles under reduced pressure; iii) After drying, physically mixing the microparticles made from the first PLGA polymer and the microparticles made from the second PLGA polymer.

10. A method for preparing the microparticles of claim 1, comprising:

18. 18. The method of any one of claims 15, 16 and 17, wherein the ratio of the first PLGA polymer to the second PLGA polymer is from 70:30 to 30:

70.

19. 18. The method of any one of claims 15, 16 and 17, wherein the solvent for the PLGA polymer is an organic solvent.

20. 18. The method of any one of claims 15, 16 and 17, wherein the solvent is selected from ethyl acetate, tetrahydrofuran, acetonitrile, dichloromethane (DCM), chloroform and acetone.

21. 18. The method of any one of claims 15, 16 and 17, wherein the solvent is dichloromethane (DCM).

22. 18. The method of any one of claims 15, 16 and 17, wherein the controlled temperature of the continuous phase is less than 20°C.

23. 18. The method of any one of claims 15, 16 and 17, wherein the controlled temperature of the continuous phase is 5 to 10°C.

24. 10. The pharmaceutical formulation of claim 1 for use in preventing graft rejection in adult kidney, liver or heart allograft recipients for the treatment and prevention of organ rejection after transplantation, graft-versus-host disease due to bone marrow transplantation, autoimmune diseases, infectious diseases, etc.

25. 10. The pharmaceutical formulation of claim 1, which is administered intramuscularly or subcutaneously in a dual-chamber syringe or a kit having a syringe pre-filled with the diluent and microparticles present in separate vials.

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