CN117729913A

CN117729913A - Novel formulations comprising azacytidine

Info

Publication number: CN117729913A
Application number: CN202280052570.8A
Authority: CN
Inventors: A·乔纳森; M·鲁斯; E·林达尔; J·海尔拉普; D·韦斯特博格
Original assignee: CANDIX AB
Current assignee: CANDIX AB
Priority date: 2021-06-10
Filing date: 2022-06-10
Publication date: 2024-03-19
Also published as: GB202117701D0

Abstract

The present disclosure provides a pharmaceutical formulation useful for treating myelodysplastic syndrome, the pharmaceutical formulation comprising a plurality of particles suspended in an aqueous carrier system, the particles: (a) Having an average diameter between an amount of 10nm and about 700 μm on a weight, number or volume basis; and (b) comprising a solid core comprising azacytidine, or a pharmaceutically acceptable salt thereof, the solid core being at least partially coated with a coating of an inorganic material comprising a mixture of: (i) zinc oxide; and (ii) one or more other metal and/or metalloid oxides wherein the atomic ratio ((i): (ii)) is between at least 1:6 and up to and including about 6:1. The mixed oxide coated particles are preferably synthesized by vapor phase coating techniques such as atomic layer deposition. The formulations may provide delayed or sustained release of azacitidine to treat myelodysplastic syndrome without an abrupt release effect.

Description

Novel formulations comprising azacytidine

Technical Field

The present invention relates to a novel formulation for use in the field of, for example, drug delivery, and in particular for the treatment of cancer, in particular myelodysplastic syndrome (MDS).

Prior art and background

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

MDS is a cancer in which immature blood cells in bone marrow fail to mature into healthy blood cells. The problem of hematopoiesis can result in a combination of low red blood cell, platelet and white blood cell counts. Some types of MDS also manifest as an increase in immature blood cell count (primordial cells) in bone marrow or blood.

The incidence of MDS is approximately seven of the hundred thousand patients, with a typical age of onset being 70 years. MDS has a number of subtypes, which are characterized by specific changes in blood cells and bone marrow.

Current treatments for MDS include supportive therapy (blood transfusion, drugs and/or antibiotics known to increase red blood cell count), chemotherapy by cytotoxic agents, hematopoietic stem cell transplantation, and/or combinations thereof.

The chemotherapeutic agent azacytidine (4-amino-1-beta-D-ribofuranosyl-s-triazin-2 (1H) -one; also known as 5-azacytidine or simply "azacytidine") is known under the trade nameAre sold and are commonly prescribed for the treatment of MDS. It is a chemical analogue of cytidine (nucleosides in DNA and RNA).

At low doses, the antitumor activity of azacitidine is thought to be achieved by inhibiting DNA methyltransferase, resulting in DNA hypomethylation. At high doses, azacytidine is thought to produce direct cytotoxicity to abnormal hematopoietic cells in bone marrow by incorporation into DNA and RNA, resulting in cell death.

Incorporation of azacytidine into RNA leads to polysome decomposition, methylation of metastatic RNA, and inhibition of receptor function defects, and protein production. Incorporation of azacitidine into DNA results in covalent binding to DNA methyltransferases, thereby preventing DNA synthesis and subsequently resulting in cytotoxicity.

Azacitidine is administered subcutaneously or intravenously. These injections require frequent administration because they do not form a long-term exuded reservoir for the active ingredient. Specifically, the standard treatment cycle for azacitidine to treat MDS is seven consecutive days of azacitidine injection, with a cycle of 28 days.

In conditions such as MDS, it would be advantageous to provide a delayed release composition in which the active ingredient is released at a desired and predictable rate in the body after injection to ensure a better pharmacokinetic profile.

In the case of any sustained release composition, it is crucial that its release profile shows minimal initial rapid release of the active ingredient, i.e. high drug concentration in the plasma shortly after administration. Such "sudden" release would result in an undesirably high concentration of active ingredient and may be dangerous for drugs with a narrow therapeutic window or drugs that are toxic at high plasma concentrations. This is particularly problematic in view of the cytotoxicity of the drug in question.

In the case of injectable suspensions of active ingredients, it is also important to control the size of the suspended particles so that they can be injected through a needle. If large agglomerated particles are present, they will not only clog the needle from which the suspension is injected, but will not form a stable suspension within the injection (i.e. they will instead sink into the bottom of the injection).

Thus, there is an unmet clinical need for more durable, more effective, and/or improved drug delivery systems comprising azacytidine in the treatment of conditions such as MDS.

Atomic Layer Deposition (ALD) is a technique for depositing thin films comprising a variety of materials, including organic materials, biological materials, polymeric materials, and especially inorganic materials, such as metal oxides, on solid substrates. It is an enabling technology for atomic and near atomic scale fabrication (ACSM) of materials, structures, devices and systems in a variety of applications (see, e.g., zhang et al, nanominuf. Metrol.2022, https:// doi. Org/10.1007/s 41871-022-00136-8). Based on its self-limiting properties, ALD can achieve atomic-scale thicknesses that can only be controlled by adjusting the number of growth cycles. In addition, multiple layers may be deposited and the properties of each layer may be tailored at the atomic level.

ALD is used as a key technology in the manufacture of, for example, atomic scale synthesis of next generation semiconductors, or advanced catalysts, as well as in the precise manufacture of nanostructures, nanoclusters, and single atoms (see, e.g., zhang et al, supra).

This technique is typically performed at low pressure and high temperature. The film coating is produced by alternately exposing solid substrates within the ALD reactor chamber to vaporized reactants in the gas phase. The substrate may be a silicon wafer, a granular material, or small particles (e.g., microparticles or nanoparticles).

The coated substrate is protected from chemical reactions (decomposition) and physical changes by the solid coating. ALD can also potentially be used to control the release rate of substrate materials in solvents, which makes it potentially useful in the formulation of active pharmaceutical ingredients.

In ALD, a first precursor (which may be metal-containing) is fed into an ALD reactor chamber (in the form of a so-called "precursor pulse") and forms an adsorbed monolayer of atoms or molecules on the surface of a substrate. Excess first precursor is then purged from the reactor, and then a second precursor (such as water) is pulsed into the reactor. The second precursor reacts with the first precursor, resulting in the formation of a monolayer, such as a metal oxide, on the substrate surface. After the subsequent purge pulse, a further pulse of the first precursor is performed, starting a new cycle of the same event (the so-called "ALD cycle").

The thickness of the film coating is controlled, inter alia, by the number of ALD cycles performed.

In a normal ALD process, since only atomic or molecular monolayers are generated during any one cycle, no discernable physical interface is formed between the monolayers, which are essentially a continuum of the substrate surface.

In international patent application WO 2014/187995, a method is described in which a plurality of ALD cycles are performed, after which the resulting coated substrate is periodically removed from the reactor and subjected to a redispersion/agitation step to present a new surface available for precursor adsorption.

The agitation step is performed primarily to solve the problems observed for nanoparticles and microparticles, i.e., particle aggregation occurs during ALD coating, resulting in the formation of "pinholes" from the points of contact between particles. The redispersion/agitation step is performed by: the coated substrate is placed in water and sonicated, which results in deagglomeration and destruction of the points of contact between the individual particles of the coated active.

The particles were then returned to the reactor and the steps of ALD coating of the powder and deagglomeration of the powder were repeated 3 times for a total of 4 series of cycles. It has been found that this method allows the formation of coated particles that are largely pinhole free (see also Hellrup et al, int. J. Pharm.,529,116 (2017)).

We have prepared a novel injectable azacitidine composition in which ALD is used to coat azacitidine microparticles with a specific mixed oxide coating, the coated particles being suspended in an aqueous vehicle. The composition produces an advantageous pharmacokinetic profile by: the active ingredient is released over an extended period of time to provide a therapeutically effective level of the drug in the systemic circulation without any significant initial burst effect.

Disclosure of Invention

According to a first aspect of the present invention, there is provided a pharmaceutical formulation useful for treating MDS, the pharmaceutical formulation comprising a plurality of particles suspended in an aqueous carrier system, the particles:

(a) Having an average diameter between an amount of 10nm and about 700 μm on a weight, number or volume basis;

and is also provided with

(b) Comprising a solid core comprising azacitidine or a pharmaceutically acceptable salt thereof, the solid core being at least partially coated with a coating of an inorganic material comprising a mixture of:

(i) Zinc oxide (ZnO); and

(ii) One or more other metal and/or metalloid oxides,

wherein the atomic ratio ((i): ii)) is at least about 1:6 and up to and including about 6:1,

This formulation is hereinafter referred to as "formulation of the present invention".

Preferably, the atomic ratio ((i): ii)) is at least about 1:1 and up to and including about 6:1.

Coatings comprising mixtures of zinc oxide and one or more other metal and/or metalloid oxides are hereinafter referred to as "mixed oxide" coatings or coating materials.

Those skilled in the art will well understand that the term "solid" includes any form of material that retains its shape and density without limitation, and/or in which the molecules are generally compressed as tightly as possible within the range allowed by the repulsive forces between them. The solid core has at least a solid outer surface upon which a layer of coating material can be deposited. The interior of the solid core may also be solid or may be hollow. For example, if the particles are spray dried before being placed in the reactor vessel, the particles may be hollow due to the spray drying technique.

The solid core of the formulation of the invention comprises azacitidine or a pharmaceutically acceptable salt thereof, and in this regard may consist essentially of, or may include azacitidine or the salt thereof, as well as other excipients or other active ingredients.

"consisting essentially of azacitidine or a pharmaceutically acceptable salt thereof" includes that the solid core consists essentially of only azacitidine or a salt thereof, i.e., the solid core is free of non-biologically active substances such as excipients, carriers, and the like (see below), as well as other active substances. This means that the core may contain less than about 5%, such as less than about 3%, including less than about 2%, for example less than about 1%, of such other excipients and/or active substances.

In the alternative, the core comprising azacitidine or a pharmaceutically acceptable salt thereof may comprise the active ingredient admixed with one or more pharmaceutical ingredients, which may comprise pharmaceutically acceptable excipients such as adjuvants, diluents or carriers, and/or may comprise other bioactive ingredients.

Non-bioactive adjuvants, diluents and carriers that can be used in cores to be coated according to the present invention can include water-soluble pharmaceutically acceptable substances such as carbohydrates, e.g., sugars such as lactose and/or trehalose, and sugar alcohols such as mannitol, sorbitol and xylitol; or a pharmaceutically acceptable inorganic salt such as sodium chloride. Preferred carrier/excipient materials include sugars and sugar alcohols.

Azacitidine or a pharmaceutically acceptable salt thereof may be present in crystalline, partially crystalline and/or amorphous state. Azacytidine, or a pharmaceutically acceptable salt thereof, can be in a solid state or can be converted to a solid state at about room temperature (e.g., about 18 ℃) and about atmospheric pressure, regardless of the physical form. The active agent (and optionally the other pharmaceutical ingredients mentioned previously) should also remain in solid form when coated in, for example, an ALD reactor, and should not physically or chemically decompose to a perceptible extent (i.e., not more than about 10% w/w) either when coated with the mixed metal oxide coating material or after being coated.

Pharmaceutically acceptable salts of azacitidine include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reacting the free acid or free base form of the active ingredient with one or more equivalents of the appropriate acid or base, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (e.g., in vacuo, by freeze drying or by filtration). Salts may also be prepared using techniques known to those skilled in the art, such as by exchanging the counterion of the active ingredient in salt form with another counterion (e.g., using a suitable ion exchange resin).

Specific salts which may be mentioned include acid addition salts such as hydrochloric acid, L-lactic acid, acetic acid, phosphoric acid, (+) -L-tartaric acid, citric acid, propionic acid, butyric acid, caproic acid, L-aspartic acid, L-glutamic acid, succinic acid, ethylenediamine tetraacetic acid (EDTA), maleic acid, methanesulfonic acid, and the like.

The formulations of the present invention comprise a pharmacologically effective amount of azacitidine or a pharmaceutically acceptable salt thereof. Preferably, the solid core of the formulation of the invention comprises said pharmacologically effective amount of azacytidine or salt thereof.

The term "pharmacologically effective amount" refers to an amount of azacitidine or salt thereof that is capable of imparting a desired physiological change (such as a therapeutic effect) to a patient receiving treatment, whether administered alone or in combination with another active ingredient. Such biological or medical response or such effect of the patient may be subjective (i.e., the subject gives an indication of the effect or perceives the effect) and include at least partial alleviation of symptoms of the disease or disorder being treated, or cure or prevent the disease or disorder, or may be objective (i.e., measurable by some test or marker).

Thus, the dose of azacitidine/its salts that can be administered to a patient should be sufficient to affect the therapeutic response within a reasonable and/or relevant time frame. Those skilled in the art will recognize that the precise dosage and selection of the composition and the most appropriate delivery regimen will be affected not only by the pharmacological properties of the formulation, but also by the route of administration, the nature and severity of the condition being treated and the physical condition and mental acuity of the recipient, as well as the age, condition, weight, sex and response of the patient to be treated and the stage/severity of the disease and genetic differences between patients.

The dose of azacitidine/its salts can also be determined by the time and frequency of administration. In any event, the practitioner or other technician will be able to routinely determine the actual dose of azacitidine/its salt that is most appropriate for the individual patient.

When injected, the formulations of the present invention provide a depot formulation from which azacitidine is released over an extended period of time. The period of time may be a period of at least about 3 days, such as about 5 days or about 7 days, and up to about 4 weeks, such as about 3 weeks (e.g., about 2 weeks).

Thus, a suitable dose of azacitidine or pharmaceutically acceptable salt thereof in the formulation of the invention may provide a plasma concentration-time curve that provides exposure (AUC, defined as, for example, AUC _Finally (area under the plasma concentration versus time curve up to the last detectable concentration over an extended period of time), or more preferably, AUC _∞ (area under plasma concentration versus time curve up to infinite time)), which provides a therapeutic effect at least as great as that obtained by subcutaneous and/or intravenous injection of current commercial azacytidine used in clinical practice.

The formulations of the invention may be capable of providing exposure of azacitidine in plasma for any of the above periods of time, in terms of AUC _∞ The exposure does not exceed the total exposure (AUC) obtained from the current standard of care/dosing regimen administered by injection or infusion of azacitidine for seven consecutive days _∞ ) The dosing regimen is that 75mg of azacytidine (calculated as free compound) per m are currently administered subcutaneously or intravenously per day over 7 days ² Body Surface Area (BSA).

More preferably, the total exposure of azacitidine over any of the above periods (e.g., AUC _∞ ) May be the total exposure (e.g., AUC) obtained from the current standard of care/dosing regimen of seven consecutive days of administration by injection or infusion of azacitidine _∞ ) At least about 50% (e.g., at least about 65%), at least about 75% (e.g., at least about 80%), such as at least about 85% (in this dosing regimen)，525mg/m ² Average AUC obtained for BSA _∞ 960.+ -. 458ng h/mL).

This achieves a dose of azacitidine or a pharmaceutically acceptable salt thereof within the formulation of the invention that provides or is capable of providing a daily dose (i.e., the average dose released from the formulation per day after injection over any of the time periods described above) ranging between about 10% (e.g., about 15%) and about 80% (e.g., about 70%, such as about 65%) of the daily dose administered within the current standard of care (i.e., daily injection or infusion therapy) (each dose calculated as free compound).

Thus, the total dose injectable into a patient by the formulation of the present invention may be in the range of about 200mg (such as about 300 mg) to about 1000mg per m ² BSA range.

Since the formulation of the invention provides steady state release of azacitidine after injection, this means an average C _{Maximum value} (maximum concentration observed in plasma concentration versus time curve) will be less than the average maximum concentration (750±403 ng/mL) obtained from the current standard of care/dosing regimen administered by injection or infusion of azacitidine for seven consecutive days. For the formulations of the present invention, average C _{Maximum value} May be between about 200 and about 700 ng/mL.

The solid azacitidine-containing core of the formulation of the present invention is provided in the form of nanoparticles (or more preferably microparticles). Preferred average diameters on a weight, number or volume basis are between about 50nm (e.g., about 100nm, such as about 250 nm) and about 30 μm, for example between about 500nm and about 100 μm, more particularly between about 1 μm and about 50 μm, such as about 25 μm, for example about 20 μm.

As used herein, the term "weight-based average diameter" will be understood by the skilled person to include that the average particle size is characterized and defined by a particle size distribution by weight, i.e. wherein the existing fraction (relative amount) in each size class is defined as the distribution of weight fractions as obtained by e.g. sieving (e.g. wet sieving). As used herein, the term "number-based average diameter" will be understood by the skilled person to include that the average particle size is characterized and defined by a number-by-number particle size distribution, i.e. wherein the existing fraction (relative amount) in each size class is defined as the distribution of the number fraction as measured by e.g. microscopy. As used herein, the term "volume-based average diameter" will be understood by the skilled person to include that the average particle size is characterized and defined by a particle size distribution by volume, i.e. wherein the existing fraction (relative amount) in each size class is defined as the distribution of volume fractions as measured by e.g. laser diffraction. Those skilled in the art will also appreciate that other suitable means of expressing the average diameter exist, such as an average diameter based on area, and that these other means of expression of average diameter may be interchanged with those used herein. Other instruments well known in the art may be employed to measure particle size, such as equipment sold by, for example, malvern Instruments, ltd (Worcestershire, UK) and Shimadzu (Kyoto, japan).

The particles may be spherical, i.e. the aspect ratio of these particles is less than about 20, more preferably less than about 10, such as less than about 4, and especially less than about 2, and/or the variation in radius (measured from the centre of gravity to the particle surface) may not exceed about 50% of the average value, such as not more than about 30%, for example not more than about 20% of said value, in at least about 90% of the particles.

However, the particles may also be coated in any shape according to the invention. For example, irregularly shaped (e.g., "raisin" -shaped), needle-shaped, sheet-shaped, or rectangular parallelepiped-shaped particles may be coated. For non-spherical particles, the size may be expressed as the size of a corresponding spherical particle of the same weight, volume or surface area, for example. According to the invention, hollow particles as well as particles with holes, cracks, etc., such as fibrous or "entangled" particles, may also be coated.

The particles may be obtained in their form suitable for coating, or in that form, for example by a particle size reduction process (e.g. crushing, cutting, milling or grinding) to a specified weight-based average diameter (as defined above), for example by wet milling, dry milling, air jet milling (including cryogenic micronization), ball milling, such as planetary ball milling, and with end roller mills, vibratory mills, hammer mills, roller mills, fluid energy mills, pin mills, and the like. Alternatively, the particles may be directly prepared to the appropriate size and shape, e.g., by spray drying, freeze drying, spray freeze drying, vacuum drying, precipitation, including the use of supercritical fluids or other top-down methods (i.e., reducing the size of large particles by, e.g., grinding, etc.) or bottom-up methods (i.e., increasing the size of the particles by, e.g., sol-gel techniques, crystallization, etc.). The nanoparticles may alternatively be fabricated by well known techniques such as gas condensation, friction, chemical precipitation, ion implantation, pyrolysis, hydrothermal synthesis, and the like.

It may be necessary (depending on the manner in which the particles comprising the core were initially provided) to wash and/or clean the particles to remove impurities that may come from their production, and then dry them. Drying may be performed by a variety of techniques known to those skilled in the art, including evaporation, spray drying, vacuum drying, freeze drying, fluid bed drying, microwave drying, IR radiation, drum drying, and the like. If dried, the particles may be deagglomerated by milling, screening, grinding and/or dry sonication. Alternatively, the wick may be treated to remove any volatile material that may adsorb onto its surface, for example by exposing the particles to vacuum and/or elevated temperature.

The surface of the core may be chemically activated prior to application of the first layer of coating material, for example by treatment with hydrogen peroxide, ozone, a radical-containing reactant, or by application of a plasma treatment, to generate oxygen radicals at the surface of the core. This in turn can create advantageous adsorption/nucleation sites on the core for the ALD precursors.

The azacytidine-containing core is coated with a coating material comprising a mixture of zinc oxide and one or more other metal and/or metalloid oxides, the atomic ratio of zinc oxide to other oxides being at least about 1:6 (e.g., at least about 1:4, such as at least about 1:2), preferably at least about 1:1 (e.g., at least about 1.5:1, such as at least about 2:1), including at least about 2.25:1, such as at least about 2.5:1 (e.g., at least about 3.25:1 or at least about 2.75:1 (including 3:1)), and up to (i.e., not exceeding) and including about 6:1, including up to about 5.5:1, or up to about 5:1, such as up to about 4.5:1, including up to about 4:1 (e.g., up to about 3.75:1).

Preferred methods of applying the coating to the core containing the bioactive agent include vapor phase techniques such as ALD or related techniques such as Atomic Layer Epitaxy (ALE), molecular layer deposition (MLD; similar techniques to ALD, except that molecules (typically organic molecules) are deposited in each pulse instead of atoms), molecular Layer Epitaxy (MLE), chemical Vapor Deposition (CVD), atomic layer CVD, molecular layer CVD, physical Vapor Deposition (PVD), sputter PVD, reactive sputter PVD, evaporative PVD, and binary reaction sequence chemistry. ALD is a preferred coating method according to the present invention.

When ALD is used, the above mixed oxide coating may be prepared by: a first zinc, other metal or metalloid-containing precursor is fed into the ALD reactor chamber (in the form of so-called "precursor pulses") to form a monolayer of adsorbed atoms or molecules of zinc, other metal or metalloid on the surface of the particles. A second precursor (e.g., water) is then pulsed into the reactor and reacted with the first precursor, resulting in the formation of a monolayer of zinc, metal or metalloid oxide, respectively, on the substrate surface. After the subsequent purge pulse, a further pulse of the first precursor is performed, starting a new cycle of the same event, which is an ALD cycle.

In most cases, the first reaction in the continuous reaction will involve some functional groups or free electron pairs or radicals, such as hydroxyl (-OH) or primary or secondary amino (-NH), of the surface to be coated ₂ or-NHR, wherein R is for example an aliphatic group such as alkyl). The separate reactions are advantageously carried out separately and under conditions such that all excess reagents and reaction products are substantially removed prior to carrying out the subsequent reactions.

To prepare a mixed oxide coating having an atomic ratio of zinc oxide to one or more other metal and/or metalloid oxides of between, for example, at least about 1:1 and up to and including about 6:1, it will be understood by those skilled in the art that between about 1 and about 6 ALD cycles of zinc oxide must also be deposited for each ALD cycle (i.e., monolayer) of the other oxides. For example, for a 3:1 atomic (zinc: other oxide) mixed oxide coating to be formed, 3 pulses of zinc-containing precursor may be followed by a second precursor pulse, respectively, to form 3 monolayers of zinc oxide, followed by 1 pulse of other metal and/or metalloid-containing precursor, followed by a second precursor pulse to form 1 monolayer of other metal and/or metalloid oxide. Alternatively, 6 monolayers of zinc oxide may be followed by 2 monolayers of other oxides, or any other combination, to provide a total atomic ratio of about 3:1. In this respect, the order of the pulses to produce the relevant oxides is not critical, as long as the resulting atomic ratio is within the relevant range.

Metals and/or metalloid elements other than zinc that may be mentioned include alkali metals, alkaline earth metals, noble metals, transition metals, post-transition metals, lanthanides, and the like. Metallic and/or metalloid elements which may be mentioned include aluminum, titanium, magnesium, iron, gallium, zirconium, niobium, hafnium, tantalum, lanthanum and/or silicon; more preferably aluminum, titanium, magnesium, iron, gallium and/or zirconium. Specific metallic and/or metalloid elements which may be mentioned include aluminum and silicon.

In this regard, the mixed oxide coating material preferably comprises alumina (Al ₂ O ₃ ) And/or silicon dioxide (SiO) ₂ ) One or the other or both.

According to the present invention there is provided a method of preparing a plurality of coated particles, wherein the coated particles are prepared by applying at least two metals forming a mixed oxide and/or precursors of metal oxides to a solid core and/or a previously coated core via a vapour deposition technique. Precursors for forming metal oxides or metalloid oxides typically include oxygen precursors such as water, oxygen, ozone, and/or hydrogen peroxide; and metal and/or metalloid compounds, typically organometallic compounds or organometalloid compounds.

Non-limiting examples of precursors are as follows: the zinc oxide precursor may be water and di-C ₁ -C ₅ Alkyl zinc, such as diethyl zinc. The precursor of alumina may be water and tric ₁ -C ₅ Alkylaluminum such as trimethylaluminum. Of silicon oxide (silicon dioxide)The precursor may be water as an oxygen precursor, as well as silane, alkylsilane, aminosilane and tetraethylorthosilicate. The precursor of iron oxide includes oxygen, ozone, and water as oxygen precursors; two C ₁ -C ₅ Alkyl iron, dicyclohexyl iron and FeCl ₃ . It will be appreciated that those skilled in the art will know which precursors are suitable for the purposes disclosed herein.

In ALD, the layer of coating material may be applied at a process temperature of from about 20 ℃ to about 800 ℃, or from about 40 ℃ to about 200 ℃, such as from about 40 ℃ to about 150 ℃, such as from about 50 ℃ to about 100 ℃. The optimum process temperature depends on the reactivity of the precursors and/or materials (including bioactive agents such as azacytidine/salts) employed in the core and/or the melting point of the core material. Preferably, a lower temperature is employed, such as from about 30 ℃ to about 100 ℃. Specifically, in one embodiment of the method, a temperature of about 20 ℃ to about 80 ℃, such as about 30 ℃ to about 70 ℃, such as about 40 ℃ to about 60 ℃, such as about 50 ℃, is employed.

We have found that when ALD is used to apply a coating comprising zinc oxide at a relatively low temperature (such as about 50 ℃ to about 100 ℃), the coating material is predominantly crystalline in nature (unlike other coating materials that form amorphous layers, such as aluminum oxide and titanium oxide).

Without being bound by theory, since zinc oxide is crystalline, if zinc oxide alone is used as the coating material, it is understood that an interface may be formed between adjacent zinc oxide crystals deposited by ALD through which the carrier system, medium or solvent (e.g., aqueous solvent system) in which the zinc oxide is partially dissolved may enter after being suspended therein. It is believed that this may result in too rapid dissolution for the depot-forming composition it is intended to prepare.

Furthermore, previous studies have shown that zinc oxide coated azacitidine formulations have lower relative bioavailability when suspended in aqueous media than uncoated azacitidine. It is believed that this lower relative bioavailability is due to degradation of azacitidine prior to release into the systemic circulation. As mentioned above, penetration of water through the crystalline interface within the zinc oxide coating is believed to result in hydrolysis of azacitidine within the coated particles.

We have now found that these problems can be alleviated by preparing a mixed oxide coating as described herein. In particular, by forming a mixed oxide coating consisting essentially but not entirely of zinc oxide as described herein, we have been able to coat the active ingredient with a coating that appears to be substantially amorphous, or a composite between crystalline and amorphous materials and/or in which ingress of injection vehicles (such as water) can be reduced. In this regard, it appears that by employing the mixed oxide aspect of the present invention, the presence of the above-described interfaces can be reduced or completely avoided, whether in a heterogeneous manner (wherein other oxides "fill" the gaps formed by the interfaces) or in a homogeneous manner (wherein a true composite of mixed oxide materials is formed during deposition, in a manner that potentially avoids the interfaces in the first place). As described below, the formulations of the present invention exhibit relative bioavailability comparable to uncoated azacitidine.

The vapor deposition reactor chamber used may optionally and/or preferably be a stationary vapor deposition reactor chamber. In the context of a vapor deposition reactor chamber, the term "stationary" is understood to mean that the reactor chamber remains stationary when used to perform vapor deposition techniques, excluding negligible movements and/or vibrations, such as those caused by, for example, associated machinery.

In addition, a so-called "stop-flow" process may be employed. Using a stop-flow process, once the first precursor has been fed into the reactor chamber and before purging the first precursor from the reactor chamber, the first precursor may be allowed to contact the core in the reactor chamber for a predetermined period of time (which may be considered a soak time). During the predetermined period of time, there is preferably substantially no pumping that can cause gas flow and/or substantially no mechanical agitation of the core.

The use of a stopped flow process may improve coating uniformity by allowing each gas to diffuse conformally in a high aspect ratio substrate, such as a powder. The benefits may be more pronounced when using a slow reactive precursor, as the precursor has more time to react on the surface. This is particularly evident when depositing the mixed oxide coating according to the invention. For example, when depositing a mixed zinc oxide/aluminum oxide coating as described herein, we find that zinc-containing precursors, such as diethyl zinc (DEZ), have a lower probability of reacting toward the substrate surface than aluminum-containing precursors, such as Trimethylaluminum (TMA).

In addition to producing coatings with good shell integrity and a more controlled release profile, the ability to obtain specific coating compositions can be improved with such a stop-flow process.

For example, when attempting to produce a coating with an atomic ratio of 3:1 between zinc and aluminum in the resulting shell as described above using gas phase techniques, we have found that a ratio closer to 3:1 can be achieved using a stopped flow process than using a precursor continuous flow deposition material.

Preferably and/or optionally, the first precursor, the second precursor, or both precursors may also be fed into the reactor chamber using a "multipulse" technique.

Using this multipulse technique, the corresponding precursor may be fed into the reactor chamber as a plurality of "sub-pulses", each sub-pulse lasting for a short period of time, such as 1 second to about one minute (depending on the size and nature of the vapor deposition reactor), rather than as one continuous pulse. After each sub-pulse, the precursor may be allowed to contact the core in the reactor chamber for a predetermined period of time, such as about 1 to 500 seconds, about 2 to 250 seconds, about 3 to 100 seconds, about 4 to 50 seconds, or about 5 to 10 seconds, such as 9 seconds. Also, depending on the size and nature of the vapor deposition reactor, this time may extend to several minutes (e.g., up to about 30 minutes). The introduction of the sub-pulses and the subsequent soaking period may be repeated a predetermined number of times, such as about 5 to 1000 times, about 10 to 250 times, or about 20 to 50 times in a single step.

The core may be coated with one or more separate discrete layers of mixed oxide coating as defined herein. Preferably, more than one separate, discrete mixed oxide layer, coating or shell (these terms are used interchangeably herein) is applied (i.e., "applied alone") to the solid core comprising azacitidine.

By "individual application of a separate layer, coating or shell" is meant coating a solid core with a first layer of coating material, the layer being formed by more than one (e.g., multiple or a set) of cycles as described herein, each cycle producing a monolayer of zinc oxide or other metal and/or metalloid oxide (as the case may be), and then subjecting the resulting coated core to some form of deagglomeration process.

In other words, a "vapor deposition (e.g., ALD) cycle" may be repeated multiple times to provide a "set of vapor deposition (e.g., ALD) cycles" that may consist of, for example, 10, 25, or 100 cycles. However, after this set of cycles, the clad core is subjected to some form of deagglomeration process followed by another set of cycles.

This process may be repeated as many times as desired, so that the number of discrete layers of coating material produced by the multiple sets of cycles in the final coating corresponds to the number of these intermittent deagglomeration steps, with the final mechanical deagglomeration being performed before the final layer (one set of cycles) of coating material is applied.

When referring to coated particles, the terms "break up" and "deagglomeration" are used interchangeably, and the break up of the coated particle aggregates is preferably accomplished by mechanical sieving techniques.

The clad core may be internally subjected to the deagglomeration process described above, rather than being removed from the apparatus by a continuous process. Such a process would involve forcing the solid product mass formed by cladding the core through a screen positioned within the reactor and configured to deagglomerate any particle aggregates upon forcing the cladding core by forcing means applied within the reactor, followed by a second and/or further cladding. This process is continued as many times as needed and/or appropriate before the final coating as described herein is applied.

Positioning the screen within the reactor vessel means that the coating can be applied by a continuous process that does not require removal of the particles from the reactor. Thus, no manual handling of the particles is required, nor is an external machine required to deagglomerate the aggregated particles. This not only greatly reduces the time to perform the coating process, but is also more convenient and reduces the risk of personnel handling hazardous (e.g., toxic) materials. It also improves process repeatability by limiting manual labor and reducing the risk of contamination.

Alternatively, and/or preferably, the coating core may be removed from a coating apparatus such as an ALD reactor, and then subjected to an external deagglomeration step, for example as described in international patent application WO 2014/187995. Such an external deagglomeration step may comprise agitation, such as sonication in a wet or dry state, or may preferably comprise subjecting the resulting solid product mass that has been discharged from the reactor to sieving, for example by forcing it through a sieve or mesh to deagglomerate the particles, for example as described below, before the particles are returned to the coating apparatus for the next coating step. Also, this process can be continued as many times as needed and/or appropriate before the final coating is applied.

In an external deagglomeration process, deagglomeration may alternatively (in addition to and/or instead of) be achieved by subjecting the coated particles in wet or dry form to one or more of nozzle aerosol generation, milling, grinding, stirring, high shear mixing, and/or homogenization. If the deagglomeration step is performed on the particles in the wet state, the deagglomerated particles should be dried (as described above with respect to the core) before the next coating step.

However, we prefer that in such external processes, the deagglomeration step comprises one or more sieving steps, which may comprise jet sieving, manual sieving, vibrating sieve shaking, horizontal sieve shaking, slapping sieving, or (preferably) sonic sieving or similar methods as described below, including any combination of these sieving steps. Suitable manufacturers of acoustic screens include Advantech Manufacturing, endecott and Tsutsui.

The vibratory screening technique may involve means for vibrating to force a solid product mass formed by cladding the core through a screen positioned inside or (preferably) outside (i.e. outside the reactor) and configured to deagglomerate any particle aggregates upon said vibrating forcing of the cladding core, and then subject it to a second and/or further layer of coating material. This process is repeated as many times as necessary and/or appropriate before the final layer of coating material is applied.

The vibration forcing device includes a vibration motor coupled to the screen. The vibration motor is configured to vibrate and/or gyrate when power is supplied thereto. For example, the vibration motor may be a piezoelectric vibration motor including a piezoelectric material that changes shape due to an inverse piezoelectric effect when an electric field is applied. The change in shape of the piezoelectric material causes acoustic or ultrasonic vibration of the piezoelectric vibration motor.

Alternatively, the vibration motor may be an Eccentric Rotating Mass (ERM) vibration motor that includes a mass that rotates when power is supplied to the motor. The mass is offset from the axis of rotation, resulting in motor imbalance and vibration and/or gyration due to rotation of the mass. Further, the ERM vibration motor may include a plurality of masses positioned at different locations relative to the motor. For example, an ERM vibration motor may include a top mass and a bottom mass, each positioned at opposite ends of the motor. By varying each mass and its angle relative to the other mass, the vibration and/or gyration of the ERM vibration motor may be varied.

The vibrating motor is coupled to the screen in such a way that: when power is supplied to the motor, vibration and/or gyration of the motor is transferred to the screen.

The screen and vibration motor may be suspended from a mount (such as a frame positionable on a floor) by a suspension device such that the screen and motor vibrate freely with respect to the mount without the vibration being substantially transferred to or damped by the mount. This allows the vibrating motor and screen to vibrate and/or turn in an unobstructed manner and also reduces noise generated during vibratory screening. The suspension means may comprise one or more springs or bellows (i.e. air cushions or equivalent cushioning means) coupling the screen and/or motor to the mount. Manufacturers of shakers or screens suitable for performing this process include, for example, russell Finex, SWECO, filter VibraScreen, gough Engineering, and Farley Greene.

Preferably, the vibratory screening technique further includes controlling a vibratory probe coupled to the screen. The vibration probe may be controlled to vibrate the screen at a frequency different from the frequency of vibration caused by the vibration motor. Preferably, the vibrating probe vibrates the screen at a higher frequency than the vibration caused by the vibrating motor, and more preferably, the frequency is in the ultrasonic range.

Providing additional vibration to the screen through the vibrating probe reduces the occurrence of clogging in the screen, reduces the likelihood of screen overload and reduces the amount of time required to clean the mesh of the screen.

Preferably, the aforementioned vibratory screening technique comprises screening the coated particles at a throughput of at least 1 g/min. More preferably, the vibratory screening technique includes screening the coated particles at a throughput of 4 g/min or greater.

Throughput depends on the area of the screen, the mesh size of the screen, the particle size, the viscosity of the particles, the static nature of the particles. By combining some of these features, higher throughput can be achieved. Thus, vibratory screening techniques may more preferably include screening coated particles at throughput of up to 1 kg/min or even higher.

Any of the above-described throughput represents a significant improvement over the use of known mechanical screening or screening techniques. For example, we have found that sonic screening requires 15 minutes of screening time, with 15 minutes of cooling time in between, which is necessary for the protection device. To screen 20g of coated particles, 9 sets of 15 minutes of active screening time were required, i.e. the total time (including cooling) was 255 minutes. In contrast, by using the aforementioned vibratory screening technique, 20g of coated particles can be continuously screened for up to 20 minutes or more preferably only 5 minutes or less.

The screen size may be determined such that the ratio of the size of the particles after sieving or sonic sieving to the screen size is about 1:1, preferably about 1:2, and optionally about 1:4. The screen size may range from about 20 μm to about 100 μm, preferably from about 20 μm to about 60 μm.

Suitable screens may include perforated plates, microplates, grids, diamond, wire, polymer or wire (woven wire screen), but are preferably formed of metal such as stainless steel.

Surprisingly, the use of stainless steel mesh in vibratory screening techniques has as gentle an effect on particle coating as the use of softer polymer screens in mechanical screening techniques such as sonic screening.

Furthermore, a known problem of sieving powders is the potential risk of static electricity generation. The advantage of steel mesh is that static electricity in the powder can be removed, whereas polymer mesh is not, since polymer mesh must be used in a sonicator.

Further, the mesh size of the known acoustic screen is limited to about 100 μm because the acoustic waves pass through the mesh rather than vibrating the mesh. There is no such limitation to the vibratory screening technique because it does not rely on sound waves to create vibrations in the screen. Thus, the vibratory screening techniques described herein allow for screening of larger particles as compared to using alternative mechanical screening techniques.

If, for example, the vibrating screen is located outside the reactor (i.e., outside the reactor), the method for preparing the coated cores of the formulation of the present invention includes discharging the coated particles from the vapor deposition reactor prior to agitating the coated particles, then reintroducing the deagglomerated coated particles into the vapor deposition reactor, and then applying another layer of at least one coating material to the reintroduced particles.

We have found that applying a separate layer of coating material after external deagglomeration results in a visible and discernable interface that can be observed by analysis of the coated particles according to the present invention and can be observed as a region of higher electron permeability by e.g. TEM. In this regard, the thickness of the layers between the interfaces directly corresponds to the number of cycles in each series that are performed within the ALD reactor and between the various external agitation steps.

Since cladding occurs at the atomic level in an ALD cladding process, such a clear physical interface is generally more difficult to observe.

Without being bound by theory, it is believed that removing the coated particles from the vacuum conditions of the ALD reactor and exposing the freshly coated surface to the atmosphere results in structural rearrangements due to relaxation and reconstruction of the outermost atomic layer. This process is believed to involve rearrangement of surface (and near-surface) atoms, driven by thermodynamic trends that reduce the free energy of the surface.

In addition, the surface adsorption of substances, such as hydrocarbons that are always present in the air, may cause such a phenomenon due to the reaction of the coating formed with hydrocarbons, atmospheric oxygen, and the like, and the surface modification may also cause such a phenomenon. Thus, if such interfaces are subjected to chemical analysis, they may contain trace amounts of contaminants or core materials, such as active ingredients forming part of the core, which are not derived from the cladding process, e.g., ALD.

Whether inside or outside the reactor, the particle aggregates are preferably broken up by forcing them through a sieving forcing means, thus separating the aggregates into individual particles or aggregates of the desired and predetermined size (and thus achieving deagglomeration). In the latter case, the individual primary particle sizes are so small (i.e., <1 μm) that "complete" deagglomeration (i.e., the case of the decomposition of aggregates into individual particles) is not possible. In contrast, deagglomeration is achieved by breaking up the larger aggregates into smaller aggregates of secondary particles of the desired size (dictated by the size of the screen). The smaller aggregates are then coated by gas phase techniques to form fully coated "particles" in the form of small aggregate particles. In this way, when referring to particles that have been deagglomerated and coated in the context of the present invention, the term "particles" refers to both individual (primary) particles and aggregate (secondary) particles having the desired size.

In any event, the desired particle size (whether individual particles of the desired size or aggregates) remains unchanged, and further, after such deagglomeration by sieving, continuing to apply the gas phase coating mechanism to the particles means that a complete coating is formed on the particles, thereby forming fully coated particles (individual particles or aggregates of the desired size).

Whether carried out internally or externally to the reactor, the repeated coating and deagglomeration process described above may be carried out at least 1, preferably 2, more preferably 3, such as 4, including 5, more particularly 6, for example 7, and not more than about 100, for example not more than about 50, such as not more than about 40, including not more than about 30, such as between 2 and 20, for example between 3 and 15, such as 10, for example 9 or 8, more preferably 6 or 7, and in particular 4 or 5.

Whether performed internally or externally to the reactor, it is preferred that at least one sieving step is performed and preferably comprises a vibratory sieving step as described above. It is further preferred that at least the final screening step comprises a vibratory screening step performed before the last layer (a set of cycles) of coating material is applied. However, it is further preferred that more than one (including each) screening step include the vibratory screening techniques, steps or processes described herein.

Preferably these steps are repeated to further facilitate the improvement in throughput of any vibratory screening technique.

The total thickness of the coating (meaning all individual layers/coating/shell) will be on average in the range between about 0.5nm and about 2 μm.

The minimum thickness of each individual layer/coating/shell will be on average in the range of about 0.1nm (including about 0.5nm, e.g., about 0.75nm, such as about 1 nm).

The maximum thickness of each individual layer/coating/shell will depend on the size of the core (initially) and then on the size of the previously coated core, and may be on average about one percent of the average diameter (i.e., average diameter based on weight, number or volume) of the core or previously coated core.

Preferably, for particles having an average diameter between about 100nm and about 1 μm, the total coating thickness should be between about 1nm and about 5nm on average; for particles having an average diameter of between about 1 μm and about 20 μm, the coating thickness should be between about 1nm and about 10nm on average; for particles having an average diameter of between about 20 μm and about 700 μm, the coating thickness should be between about 1nm and about 100nm on average.

We have found that applying a coating/shell followed by one or more deagglomeration steps, such as sonication, can create wear, pinholes, breaks, gaps, cracks and/or voids (hereinafter "cracks") in the layer/coating, as the coated particles "bond" or "glue" together substantially more tightly directly after the thicker coating is applied. Once deagglomeration occurs, this may expose the core containing the bioactive ingredient (i.e., azacytidine/salt) to the outside world.

Since it is intended to provide particles in an aqueous suspension prior to administration to a patient, it is necessary to provide deagglomerated primary particles that are free of pinholes or cracks in the coating. Such cracking will result in undesirable initial peaks (bursts) in the concentration of active ingredient in the plasma directly after administration.

We have found that by performing one or more of the deagglomeration steps described herein, this significantly reduces pinholes, gaps or cracks in the final layer of coating material, such that the particles are not only completely covered by the layer/coating, but also in a manner that allows the particles to be readily deagglomerated (e.g., using non-aggressive techniques such as vortexing) and without damaging the coating material layer formed prior to and/or during drug formulation.

In this regard, the mixed oxide coating typically completely surrounds, encapsulates and/or encapsulates the solid core comprising the active ingredient. In this way, the risk of an initial burst of drug concentration due to direct contact of the drug with the solvent in which the relevant active ingredient is soluble is minimized. This may include not only body fluids, but also any medium in which such coated particles may be suspended prior to injection.

Thus, in another embodiment of the invention, a particle as disclosed hereinbefore is provided wherein the coating surrounding, wrapping and/or encapsulating the core covers at least about 50%, such as at least about 65%, including at least about 75%, such as at least about 80%, more particularly at least about 90%, such as at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about or about 100%, of the surface of the solid core such that the coating substantially completely surrounds, wraps and/or encapsulates the core.

As used herein, the term "substantially completely surrounding, encasing, and/or encapsulating the core" means covering at least about 98%, or at least about 99% of the surface of the solid core.

In the alternative, the methods described herein may result in the deagglomerated coated particles being substantially free of such cracks through which the active ingredient may be released in an uncontrolled manner.

Although some microcracking may occur in the coating without affecting its basic function in controlling release, in another embodiment, particles as disclosed previously are provided wherein at least about 90% of the particles are free of cracking in the coating surrounding, wrapping and/or encapsulating the core. In one embodiment, at least about 91%, such as at least about 92%, such as at least about 93%, such as at least about 94%, such as at least about 95%, such as at least about 96%, such as at least about 97%, such as at least about 98%, such as at least about 99%, such as about 100% of the particles do not exhibit such cracking.

Alternatively, "substantially free of such cracks" in the coating also means that less than about 1% of the surface of the coated particles comprises wear, pinholes, breaks, gaps, cracks and/or voids through which the active substance is potentially exposed (e.g., exposed to the outside).

The coating materials may be laminated together to have a substantially uniform thickness over the surface area of the particles. By "substantially uniform" thickness is meant that the coating thickness of the coated particles present in at least about 10% (such as about 25%, e.g., about 50%) of the formulation of the present invention does not vary by more than about + -20%, including + -50%, of the average thickness, as measured by TEM.

In addition to the basic mixed oxide coating employed in the formulation of the present invention, other coating materials, which may be pharmaceutically acceptable and substantially non-toxic, may be additionally applied between the individual mixed oxide coatings (e.g., between the individual deagglomeration steps) and/or concurrently with the application of the mixed oxide coatings herein. Such materials may comprise a multilayer or composite of the mixed oxide and one or more different inorganic or organic materials to alter the properties of the layers.

The additional coating material may comprise an organic or polymeric material such as polyamide, polyimide, polyurea, polyurethane, polythiourea, polyester, or polyimide. The additional coating material may also comprise a mixed material (e.g., between an organic material and an inorganic material), including a material that is a combination of a metal or another element and an alcohol, carboxylic acid, amine, or nitrile. However, it is preferred that the coating material comprises an inorganic material.

The additional inorganic coating material may comprise other compounds of metals and/or metalloids, such as oxides, nitrides, sulfides, selenides, carbonates, other ternary compounds, and the like. Metals and metalloids, hydroxides, and especially oxides are preferred, especially metal oxides.

In addition, oxides of elements other than zinc, aluminum or silicon include alkali metals, alkaline earth metals, noble metals, transition metals, post-transition metals, lanthanoids, and the like. Metals and metalloids which may be mentioned include titanium, magnesium, iron, gallium, zirconium, niobium, hafnium, tantalum and/or lanthanum; more preferably titanium, magnesium, iron, gallium and/or zirconium.

Thus, additional coating materials that may be mentioned include those comprising titanium dioxide (TiO ₂ ) Iron oxide (Fe) _x O _y For example FeO and/or Fe ₂ O ₃ And/or Fe ₃ O ₄ ) Gallium oxide (Ga) ₂ O ₃ ) Magnesium oxide (MgO), niobium oxide (Nb) ₂ O ₅ ) Hafnium oxide (HfO) ₂ ) Tantalum oxide (Ta) ₂ O ₅ ) Lanthanum oxide (La) ₂ O ₃ ) And/or zirconium dioxide (ZrO ₂ ) Is used for the coating material.

Although the plurality of mixed oxide coated particles according to the present invention are substantially free of the above-described cracks in the applied coating through which the active ingredient is potentially exposed (e.g., to the outside), two further optional steps may be applied to the plurality of coated particles prior to subjecting the plurality of coated particles to further pharmaceutical formulation processing.

The first optional step may include, after the final deagglomeration step as described previously, applying a final outer coating layer, the outer "overcoat" layer/coating, or "containment shell" (these terms are used interchangeably herein) having to be thinner in thickness than the individual layers/coatings/shells (or "sub-shells") previously applied.

Thus, the thickness may not exceed about 0.7 times (e.g., about 0.6 times) the thickness of the previously applied widest sub-shell on average. Alternatively, the thickness may not average more than about 0.7 times (e.g., about 0.6 times) the thickness of the last applied sub-shell, and/or may not average more than about 0.7 times (e.g., about 0.6 times) the average thickness of all previously applied sub-shells. For particles up to about 20 μm, the thickness average may be in the range of about 0.3nm to about 10 nm. For larger particles, the thickness may average no more than about 1/1000 of the average diameter of the coated particles on a weight, number or volume basis.

The effect of such a containment vessel is to provide a "sealing" outer coating over the particles to cover those cracks so that the particles are not only completely covered by the containment vessel, but also in a manner that allows the particles to be readily deagglomerated (e.g., using non-aggressive techniques such as vortexing) and without damaging the sub-shells formed thereunder prior to and/or during drug formulation.

For the reasons described herein, it is preferred that the containment vessel does not contain zinc oxide. On the other hand, the sealing shell may comprise silica, or more preferably, alumina.

The second optional step may include ensuring that a few remaining particles with broken and/or ruptured shells/coatings are treated, wherein all particles are suspended in a solvent in which azacitidine or a salt thereof is soluble (e.g., at least about 0.1 mg/mL), but the most insoluble material in the mixed oxide coating is insoluble (e.g., at a solubility of no more than about 0.1 μg/mL), and then the solid matter particles are separated from the solvent by, for example, centrifugation, sedimentation, flocculation, and/or filtration, resulting in leaving predominantly intact particles.

As discussed previously, the above optional steps provide a method of potentially further reducing the likelihood of (potentially) undesirable initial peaks (bursts) in the plasma concentration of the active ingredient.

At the end of the process, the coated particles may be dried using one or more of the techniques described previously for drying cores. Drying may be performed in the absence or presence of one or more pharmaceutically acceptable excipients (e.g., sugar or sugar alcohol).

Alternatively, at the end of the method, the isolated particles may be resuspended in a solvent (e.g., water, with or without one or more pharmaceutically acceptable excipients as defined herein) for subsequent storage and/or administration to a patient.

The core and/or partially coated particles may be subjected to one or more alternative and/or preparatory surface treatments prior to application of the first layer of coating material or between successive coatings. In this regard, one or more intermediate layers comprising different materials (i.e., other than inorganic materials) may be applied to the relevant surfaces, for example, to protect the core or partially coated particles from undesired reactions with the precursor during the coating step/deposition process, to increase coating efficiency or reduce agglomeration.

For example, the intermediate layer may contain one or more surfactants in order to reduce agglomeration of the particles to be coated and provide a hydrophilic surface suitable for subsequent coating. Suitable surfactants in this regard include the well known nonionic, anionic, cationic or zwitterionic surfactants such as the tween series, for example tween 80. Alternatively, the core may be subjected to a preliminary surface treatment if the active ingredient used as part of the core (or as core) is susceptible to reaction with one or more precursor compounds that may be present in the gas phase during the cladding (e.g. ALD) process.

The application of the "intermediate" layer/surface treatment of this nature may alternatively be achieved by liquid phase non-coating techniques, followed by lyophilization, spray drying or other drying methods to provide particles having a surface layer to which the coating material may be subsequently applied.

The outer surface of the particles of the formulation of the invention may also be derivatized or functionalized, for example by attaching one or more compounds or moieties to the outer surface of the final layer of coating material, for example with a compound or moiety that enhances targeted delivery of the particles in the patient to whom the nanoparticle is administered. Such compounds may be organic molecules (such as PEG) polymers, antibodies or antibody fragments, or receptor binding proteins or peptides, and the like.

Alternatively, the moiety may be an anchor group, such as a moiety comprising a silane functionality (see, e.g., herrera et al, j.mater.chem.,18,3650 (2008) and US 8,097,742). Another compound, such as a desired targeting compound, may be attached to such an anchoring group by covalent or non-covalent bonds, including hydrogen bonding, or van der waals bonds, or combinations thereof.

The presence of such anchoring groups may provide a versatile tool for targeted delivery to specific sites in the body. Alternatively, the use of compounds such as PEG may result in the particles circulating in the blood stream for a longer duration, ensuring that they do not accumulate in the liver or spleen (the human body eliminates the natural mechanism of particles, which may prevent delivery to diseased tissue).

The cores coated with the mixed oxide coating, whether in separate, discrete layers, coatings or shells or other forms, are hereinafter referred to as "coated particles of the formulation of the present invention" as defined herein.

The pharmaceutical (or veterinary) formulation of the present invention may comprise different types of particles, for example comprising differently functionalized (as described previously) particles, differently sized particles and/or layers of mixed oxide coating material of different thickness, or combinations thereof. By combining particles having different coating thicknesses and/or different core sizes in a single pharmaceutical formulation, drug release after administration to a patient can be controlled (e.g., altered or prolonged) over a specific period of time.

The formulations of the invention may be administered systemically, e.g., by intravenous or intra-arterial (including by intravascular or other perivascular devices/dosage forms (e.g., stents)), intramuscular, intraosseous, intracerebral, intraventricular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, intratumoral, intradermal, subcutaneous, transdermal injection or infusion in a pharmaceutically (or veterinarily) acceptable dosage form.

The preparation of the formulations of the present invention comprises incorporating coated particles as described herein into a suitable pharmaceutically acceptable aqueous carrier system, and may be accomplished with appropriate consideration of the intended route of administration and standard pharmaceutical practice. Thus, suitable excipients should be chemically inert to the active agent being used and have no deleterious side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers may also confer immediate or modified release of the active agent (i.e., azacitidine/salt) from the particles of the formulation of the present invention.

Sterile aqueous suspensions of particles of the formulations of the present invention can be formulated according to techniques known in the art. The aqueous medium should contain at least about 50% water, but may also contain other aqueous excipients such as ringer's solution, and may also include polar cosolvents (e.g., ethanol, glycerol, propylene glycol, 1, 3-butanediol, polyethylene glycols of various molecular weights, and tetraethylene glycol); viscosity increasing agents or thickening agents (e.g., carboxymethyl cellulose, microcrystalline cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, sodium starch glycolate, poloxamers such as poloxamer 407, polyvinylpyrrolidone, cyclodextrins such as hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidone, and polyethylene glycols of various molecular weights); surfactants/wetting agents to obtain homogeneous suspensions (e.g. sorbitol esters, sodium lauryl sulphate; monoglycerides, polyoxyethylene esters, polyoxyethylene alkyl ethers, polyoxyglycerides, and preferably tween (polysorbate), such as tween 80 and tween 20). Preferred ingredients include isotonic regulators (e.g., sodium lactate, glucose, and especially sodium chloride); pH adjusting agents and/or buffers (e.g., citric acid, sodium citrate, and in particular phosphate buffers such as disodium hydrogen phosphate dihydrate, sodium acid phosphate, sodium dihydrogen phosphate monohydrate, and combinations thereof, which can be used in combination with standard inorganic acids and bases such as hydrochloric acid and sodium hydroxide); and other ingredients such as mannitol, croscarmellose sodium and hyaluronic acid.

The formulations of the present invention may be further formulated in the form of an injectable suspension of coated particles which is both uniform in size distribution and capable of forming a stable suspension in the injection (i.e., without sedimentation) and which may be injected through a needle. In this regard, the formulations of the present invention may comprise an aqueous medium comprising inactive ingredients that may prevent premature gelation of the formulations of the present invention and or are sufficiently viscous to prevent sedimentation that results in a non- "homogenous" suspension and thus risk under-dosing or overdosing of the active ingredient.

Thus, the formulation may be stored under normal storage conditions and maintain its physical and/or chemical integrity. The phrase "maintaining physical and chemical integrity" essentially means chemical and physical stability.

"chemical stability" includes that any formulation of the present invention can be stored under normal storage conditions (with or without appropriate pharmaceutical packaging) with insignificant levels of chemical degradation or decomposition.

"physical stability" includes that any formulation of the invention can be stored under normal storage conditions (with or without appropriate pharmaceutical packaging), the degree of physical transformation is not significant, such as sedimentation as described above, or the nature and/or integrity of the particles of the coating, e.g., the coating itself or the degree of change in the active ingredient (including dissolution, solvation, solid state phase change, etc.).

Examples of "normal storage conditions" of the formulations of the present invention include temperatures between about-50 ℃ and about +80℃ (preferably between about-25 ℃ and about +75 ℃, such as about 50 ℃), and/or pressures between about 0.1 bar and about 2 bar (preferably atmospheric pressure), and/or exposure to UV/visible light of about 460 lux, and/or relative humidity of about 5% and about 95% (preferably about 10% to about 40%) for a long period of time (i.e., greater than or equal to about twelve months, such as about six months).

Under such conditions, chemical and/or physical degradation/decomposition of the formulations of the present invention may be found suitably to be less than about 15%, more preferably less than about 10%, especially less than about 5%. The skilled person will appreciate that the above-mentioned upper and lower limits of temperature and pressure represent extreme values of normal storage conditions, and that certain combinations of these extreme values will not be experienced during normal storage (e.g. a temperature of 50 ℃ and a pressure of 0.1 bar).

The formulations of the present invention may comprise from about 1% to about 99% by weight, such as from about 10% (such as about 20%, e.g., about 50%) to about 90% by weight of the coated particles, the remainder being composed of the carrier system and/or other pharmaceutically acceptable excipients.

The formulations of the present invention may be in the form of a liquid, sol, or gel, which may be administered by a surgical applicator (e.g., needle, catheter, etc.) to form a depot formulation.

In any event, the preparation of a suitable formulation may be achieved non-inventively by the skilled artisan using conventional techniques. Thus, the formulations of the present invention and dosage forms containing them may be formulated with conventional pharmaceutical additives and/or excipients used in the art to prepare pharmaceutical formulations and then incorporated into various pharmaceutical formulations and/or dosage forms using standard techniques (see, e.g., lachman et al, "The Theory and Practice of Industrial Pharmacy", lea & Febiger, 3 rd edition (1986); "Remington: the Science and Practice of Pharmacy", troy (eds.), university of the Sciences in Philadelphia, 21 st edition (2006); and/or "Aulton's pharmaceuticals: the Design and Manufacture of Medicines", aulton and Taylor (edits), elsevier, 4 th edition, 2013), and documents mentioned therein, the relevant disclosures of all of which are incorporated herein by reference.

According to a further aspect of the present invention there is provided a method for preparing a formulation of the present invention, the method comprising mixing coated particles as described herein with an aqueous carrier system as described herein, for example.

For subcutaneous and/or intramuscular injection, the formulations of the invention may be presented in the form of a sterile injectable and/or infusible dosage form that can be administered by a surgical applicator (e.g., syringe with an injection needle, catheter, etc.) to form a depot formulation.

Further provided is an injectable and/or infusible dosage form comprising the formulation of the invention, wherein the formulation is contained within a reservoir connected to and/or associated with an injection or infusion device (e.g., syringe with injection needle, catheter, etc.).

Alternatively, the formulations of the present invention may be stored prior to loading into a suitable injectable and/or infusible administration device (e.g., a syringe with an injection needle), or may even be prepared immediately prior to loading into such an administration device.

Thus, a sterile injectable and/or infusible dosage form may comprise a container or reservoir in communication with an injection or infusion device into which the formulation of the present invention may be pre-loaded, or may be loaded at some point prior to use, or may comprise one or more reservoirs within which coated particles of the formulation of the present invention and an aqueous carrier system, respectively, are contained and mixed prior to and/or during injection or infusion.

Accordingly, there is further provided a kit of parts comprising:

(a) Coated particles of the formulation of the present invention; and

(b) The carrier system of the formulation of the present invention,

as well as coated particles comprising the formulation of the invention and a kit of parts of instructions directing the end user to mix these particles with the carrier system according to the invention.

There is further provided a preloaded injectable and/or infusible dosage form as described above, but modified by including at least two compartments, one containing the coated particles of the formulation of the present invention and the other containing the aqueous carrier system of the formulation of the present invention, wherein mixing is performed prior to and/or during injection or infusion to form a suspension or other substance.

The formulations of the present invention are useful in human medicine. The formulations of the present invention are particularly useful for any indication for which azacitidine is approved or known to be useful. In particular, the formulations of the invention are useful for treating cancers, such as MDS.

The term "MDS" is understood to include any cancer condition characterized by immature blood cells in the bone marrow (which means that they do not become healthy blood cells), the symptoms of which may initially manifest as fatigue, shortness of breath, bleeding, anemia, frequent infections, and combinations of one or more of the following: red blood cell count decreases, platelet count decreases and/or white blood cell count decreases, and/or the percentage of primary cells in bone marrow or blood increases.

The term "MDS" also includes different subtypes, which can be routinely determined by a physician by evaluating the aforementioned blood cells, platelets, and/or primary cell counts. Thus, the term includes Refractory Anemia (RA), refractory anemia with cyclic iron granulocytes (RARS), refractory cytopenia with multiple dysplasia (RCMD), refractory cytopenia with multiple dysplasia and cyclic iron granulocytes (RCMD-RS), refractory anemia with primitive cell excess (RAEB), refractory anemia with primitive cell excess in transformation (RAEB-T), unclassified MDS (MDS-U), and MDS associated with isolated del (5 q).

In the context of the present invention, MDS may also be classified as a disease that has progressed to leukemia and/or is progressing to leukemia, such as Acute Myelogenous Leukemia (AML), as well as chronic myelomonocytic leukemia (CMML) and myeloless monocytic leukemia (JMML), both of which may be classified as mixed myelodysplastic/myeloproliferative diseases.

The formulations of the present invention are suitable for therapeutic, palliative and/or diagnostic treatment of any of the conditions described above, as well as indications in prophylactic treatment (including preventing and/or eliminating exacerbations and/or exacerbations of the condition).

In the treatment of any of the conditions described above, azacitidine may be combined with other therapies known to be useful in the treatment of the relevant conditions. This includes known anti-cancer drugs, particularly drugs known to be useful in the treatment of MDS, AML, CMML and/or JMML, such as decitabine, sitaglycone and/or lenalidomide, as well as non-chemotherapeutic treatments, such as iron, all-trans retinoic acid, allogeneic stem cell transplantation and/or platelet infusion.

In addition, as described below, we have found that injection of the formulations of the present invention may cause a mild inflammatory response. This response can be alleviated by co-administration with an anti-inflammatory agent suitable for injection.

Suitable anti-inflammatory agents that may be employed in this regard include butylpyrazolidines (such as phenylbutazone, mofebuzon, oxybuprzone, clofezon, kebuzon, and succinum buzon); acetic acid derivatives and related substances (indomethacin, sulindac, tolmetin, zomepirac, diclofenac, alclofenac, ibuprofen Ma Dezong, etodolac, chlorfenazoic, fenticonic acid, acemetacin, bipyramine, oxametacin, proglumide, ketorolac, aceclofenac and carbostyril); oxicams (such as piroxicam, tenoxicam, droxikang, lornoxicam and meloxicam); propionic acid derivatives (such as ibuprofen, naproxen, ketoprofen, fenoprofen, fenbufen, phenoxyibuprofen, superibuprofen, pyriproxyfen, flurbiprofen, indoprofen, tioprofen acid, oxaprozin, ibuprofen, dexibuprofen, fluoronoprofen, amoprofen, dexketoprofen, veraprofen, carprofen and tenipoxalin); fenamic acids (such as mefenamic acid, tolfenamic acid, flufenamic acid, meclofenamic acid, and flunixin), oxicams (such as celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, luminoxib, fe Luo Xibu, luo Beina, ma Faxi, and cimicixib); other non-steroidal anti-inflammatory drugs (such as nabumetone, niflumic acid, azapropimidazolone, glucosamine, benzydamine, polyglucosamine, procasone, oxgliptin, nimesulide, non-prazone, decapril, mo Nifu meter, tenidap, oseltamizole, chondroitin sulfate, pentosan polysulfate, and aminopropionitrile); corticosteroids (such as 11-dehydrocorticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 11-ketoprogesterone, 11 beta-hydroxy pregnenolone, 11 beta-hydroxy progesterone, 11 beta, 17 alpha, 21-trihydroxy pregnenolone, 17 alpha, 21-dihydroxypregnenolone, 17α -hydroxypregnenolone, 17α -hydroxyprogesterone, 18-hydroxy-11-deoxycorticosterone, 18-hydroxycortigesterone, 21-deoxycortisol, 21-hydroxypregnenolone (pregnenolone), aldosterone, corticosterone (17-deoxycortisol), cortisol (hydrocortisone), corticosterone, pregnenolone, progesterone, fluoroacetone (fluoroprogesterone), fluorometholone, medroxyprogesterone (medroxyprogesterone), acetoxypregnenolone (21-acetoxypregnenolone), prednisone, cloprednisone, difluoro pregnenolone butyl ester, fludrocortisone fluocinolone, fluopelone, fluprednisone, loteprednol, methylprednisolone, prednisolide, prednisolone, prednisone, teconone, triamcinolone, alclomethasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, dexamethasone, triamcinolone, dexamethasone, and triamcinolone diflupraised, diflurocort, flucorone, fluorometholone, flucortin, fluprednisodine, fluticasone furoate, halometasone, methylprednisone, mometasone furoate, palatinose, prednisolide, rimexolone, zatadine (halobetasol), fluprednisolide, halobetasol, and halobetasol, and the like, and their uses, anxinide, budesonide, ciclesonide, deflazacort, deanenide, fumcrata, fluclonide (fluclorolone acetonide, flucloronide), fludrolide (fludroxycortide, flurandrenolone, flurandrenolide), flunisolide, fluocinolone acetonide (fluocinolone acetonide, fluocinonide), halcinonide and triamcinolone acetonide); quinolines (such as hydroxy Xin Kefen; gold preparations (such as gold thiosuccinyl, gold sodium sulfite, gold nofin, gold thioglucose and gold thiopropanol); penicillamine and similar agents (such as busiramine); and antihistamines such as atorvastatin, acipimazine, cinazazoline, azazoline, azatadine, dacarbazine, dexanabine, brom Mi Pin, bilastine, benazetin, benazetidine, benzodiazepine, oxygenoline, oxyline, chlorpheniramine (klorfiramine), carboplatin (klorfiramine), casporamine (kloropyramine), levocetirizine (levocetirizine), loratadine (loadadin), mehydralin (mebhydrolin), mekitazepine (mekitazein), mizoribine (meklozin), mepiramine (mepyramin), me Sha Bilin (metarilen), metiracin (metdilizin), mizostatin (mizostatin), oxazamide (aturtimibe), oxazamate (oxazidine), pimazepine (metizine), prazidine (procaterodine), pyrrolbutamine (pyrramin), rupatadine (rupatadin), celecoxafadine (sekunzadin), tastatin (62astin), tenascin (62), trimeprazidine (valadin), and trimethoprim (62, trimethoprim (trimethoprim). Combinations of any one or more of the above anti-inflammatory agents may be used.

Preferred anti-inflammatory agents include non-steroidal anti-inflammatory agents such as diclofenac, ketoprofen, meloxicam, aceclofenac, flurbiprofen, parecoxib, ketorolac tromethamine, or indomethacin.

The subject may receive (or may have received) one or more of the co-therapeutic agents and/or anti-inflammatory agents described above separately from the formulation of the invention, meaning that one or more of those other therapeutic agents are received at prescribed doses before, in addition to, and/or after treatment with the compound of the invention.

When azacytidine/salts thereof are "combined" with such other therapeutic agents, the active ingredients may be administered together in the same formulation, or separately (simultaneously or sequentially) in different formulations (hereinafter referred to as "combination product").

Such combination products provide for the co-administration of azacytidine with other therapeutic agents and, thus, may be presented as separate formulations (wherein at least one of those formulations is a formulation of the present invention and at least one comprises the other therapeutic agent in a separate formulation), or may be presented as a combined formulation (i.e., formulated) (i.e., presented as a single formulation comprising azacytidine/salt and the other therapeutic agent).

In this regard, another therapeutic agent may be co-presented with azacytidine in an appropriate dose in one or more cores forming part of the formulation of the present invention as described previously, or may be formulated using the same or similar coating process as previously described for azacytidine, which may allow release of the other therapeutic agent on the same or different time scales.

Accordingly, there is further provided a pharmaceutical formulation of the invention, further comprising a therapeutic agent useful in the treatment of cancer, such as MDS as defined previously, and/or an anti-inflammatory agent;

in such formulations of the invention, additional therapeutic agents may be included by:

(1) Formulated with azacytidine within the solid core of the formulation of the present invention (this formulation is hereinafter referred to as "combination core formulation"); or (b)

(2) Which is dissolved and/or suspended in the aqueous carrier system of the formulation of the present invention (this formulation is hereinafter referred to as "combined formulation").

In example (2) above, the other therapeutic agent may be present in the formulation of the invention in any form, wherein it is separate from the azacytidine-containing core. This can be achieved, for example, by dissolving or suspending the active ingredient directly in the aqueous medium of the formulation according to the invention, or in a form which also allows control of the release of azacitidine after injection.

The latter option may be achieved, for example, by providing the additional therapeutic agent in the form of additional particles suspended in the aqueous carrier system of the formulation of the invention, the additional particles having a weight, number or volume-based average diameter of between 10nm and about 700 μm, and comprising a core comprising the therapeutic agent and/or anti-inflammatory agent useful in the treatment of cancer, such as MDS as defined above, the core being at least partially coated with one or more coating materials as described hereinbefore (the formulation is hereinafter referred to as a "combined suspension").

Further provided is a pharmaceutical formulation of the invention in the form of a kit of parts comprising the following components:

(A) The pharmaceutical formulation of the present invention; and

(B) Pharmaceutical formulations comprising therapeutic and/or anti-inflammatory agents useful for the treatment of cancer, such as MDS as defined previously,

wherein components (a) and (B) are each provided in a form suitable for administration in combination with the other component.

While component (B) in a multipart kit as described above may differ from component (a) (i.e. a formulation of the invention) in its chemical composition and/or physical form, it may also be in a form substantially identical or at least similar to the azacytidine-containing formulation of the invention, i.e. in the form of a plurality of particles suspended in (e.g. an aqueous) carrier system:

(a) Having an average diameter between an amount of 10nm and about 700 μm on a weight, number or volume basis; and is also provided with

(b) Comprising a solid core comprising the other therapeutic agent, the core being at least partially coated with a coating of one or more (e.g., inorganic) materials.

Furthermore, while in such preferred multipart kit and combined suspension as set forth in example (2) above, the coated cores comprising the other therapeutic agent may differ in their chemical composition and/or physical form, it is preferred that the inorganic material coating employed is the same or similar to the coating employed in the azacytidine-containing formulation of the present invention, meaning that the other therapeutic agent is coated with one or more inorganic coatings as described hereinbefore, e.g., one or more inorganic coating paints comprising one or more metal-containing or metalloid-containing compounds such as metals or metalloid oxides, e.g., iron oxide, titanium dioxide, zinc sulfide, more preferably zinc oxide, silicon dioxide and/or aluminum oxide, which coating materials may consist essentially (on an individual or collective basis) of (e.g., greater than about 80%, such as greater than about 90%, e.g., about 95%, such as about 98%) of such oxides, and more particularly inorganic coatings comprising mixtures of:

(i) Zinc oxide; and

(ii) One or more other metal and/or metalloid oxides,

wherein the atomic ratio ((i): ii)) is at least about 1:6 and up to and including about 6:1.

In any event, and for the avoidance of doubt, all aspects, including preferred aspects, of the azacytidine-containing formulations of the present invention disclosed and/or claimed herein are equally applicable to aspects and/or preferences of a clad core comprising one or more additional therapeutic agents as described above. For the avoidance of doubt, these aspects, preferences and features, individually or in combination, are incorporated by reference.

Thus, all combination products, including the combination core formulations, combination suspensions and multipart kits described above, can be used in human medicine, and in particular, azacitidine is approved or known for use in any indication therein, such as cancer and MDS as defined previously.

In certain instances, some of such additional therapeutic agents, including those useful in treating, for example, MDS, may be referred to as "standard of care" associated with a particular condition. The term "standard of care" will be understood by the skilled artisan to include the therapeutic procedures that a clinician should follow and/or expects to follow in treating certain types of patients, diseases, and/or clinical conditions. In some new or less known cases, standard care may change and/or develop over time.

According to another aspect of the present invention there is provided a method of preparing a kit of parts as defined above, the method comprising combining component (a) as defined above with component (B) as defined above, thereby rendering the two components suitable for administration in combination with each other.

The "binding" of the two components to each other includes that components (a) and (B) of the kit of parts may:

(i) Provided as separate formulations (i.e., independent of each other) which are then brought together for use in combination with each other in combination therapy; or alternatively

(ii) The individual components as a "combination package" are packaged and presented together for use in combination with one another in combination therapy.

Thus, there is further provided a kit of parts as defined hereinbefore, wherein components (a) and (B) are packaged and presented together as separate components of a combination package for use in combination with each other in combination therapy, and a kit of parts comprising:

(I) One of components (a) and (B) as defined herein; and

(II) instructions for use of one of the two components in combination with the other component.

As noted above, the kit of parts described herein may include more than one formulation comprising an appropriate amount/dose of azacitidine/salt, and/or more than one formulation comprising an appropriate amount/dose of other therapeutic agent, for repeated administration as previously described.

In this regard, with respect to the multipart kit described herein, "co-administration" includes sequential, separate and/or simultaneous administration of components (a) and (B) of the kit during treatment of a condition.

Further, the term "in combination" includes that one or the other of the two formulations may be administered (optionally repeated) before, after, and/or at the same time as the other components are administered. The terms "concurrently administered" and "administered at the same time" as used in this context include administration of individual doses of azacitidine and other therapeutic agents within 48 hours (e.g., 24 hours) of each other.

For any of the above-described combination products according to the invention, the corresponding formulation is applied as follows (or, in the case of a multipart kit, the two components are applied in combination with each other, optionally repeatedly): this approach may produce greater beneficial effects to the subject during the same course of treatment than a formulation comprising azacitidine/salt (e.g., a formulation of the invention) administered alone (e.g., repeatedly, as described herein) without other components during the course of treatment of the condition.

Determining whether a combination product provides a greater benefit in terms of treatment and during treatment will depend on the condition to be treated and/or its severity, but may be routinely accomplished by the skilled person.

For example, a physician may initially administer a formulation of the invention comprising azacitidine/salt alone to treat a patient suffering from MDS, and then find that the patient exhibits an inflammatory response (which may be caused by the active ingredient itself and/or by any other component of the formulation).

The physician may then administer one or more of the following:

component (B) of the multipart kit as described above,

a combination core formulation of the present invention,

combination preparations, and/or

A combined suspension as described above, any of which comprises an anti-inflammatory agent as described hereinbefore.

The other active ingredients/therapeutic agents described above that may be employed in the combination product according to the invention may be provided in the form of (e.g. pharmaceutically acceptable) salts, including any such salts known in the art and described for the relevant drugs in the medical literature (such as Martindale-The Complete Drug Reference, 38 th edition, pharmaceutical Press, london (2014) and the references mentioned therein), the relevant disclosures of all of which are incorporated herein by reference.

The amount of other active ingredients/therapeutic agents that can be employed in the combination product according to the invention must be sufficient in order to exert their pharmacological effect.

Thus, the dosage of such other active ingredients that can be administered to a patient should be sufficient to affect the therapeutic response over a reasonable and/or relevant time frame. Those skilled in the art will recognize that the precise dosage and selection of the composition and the most appropriate delivery regimen will be influenced not only by the nature of the other active ingredients, but also by, inter alia, the pharmacological nature of the formulation, the route of administration, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the age, condition, weight, sex and response of the patient to be treated, and the stage/severity of the disease, and the genetic differences between patients.

Since the administration of the formulations of the invention may be continuous or intermittent (e.g., by bolus injection), the dosage of such other active ingredients may also be determined by the timing and frequency of administration.

In any event, the practitioner or other technician will be able to routinely determine the actual dosage of any particular additional active ingredient that is most appropriate for the individual patient, and the dosages of the relevant additional active ingredient described above include those dosages known in the art and described for the relevant drugs in the medical literature (such as Martindale-The Complete Drug Reference, 38 th edition, pharmaceutical Press, london (2014) and the references mentioned therein), the relevant disclosures of all of which are incorporated herein by reference.

The use of the formulation of the invention allows to control the dissolution rate of azacytidine and by reducing any burst effect as defined hereinbefore (e.g. concentration maximum shortly after administration) and/or by reducing C in the plasma concentration-time curve _{Maximum value} To influence the pharmacokinetic profile.

The formulations of the present invention may also provide release and/or pharmacokinetic profiles that increase the length of azacitidine released from the formulation.

These factors not only reduce the frequency or time that the MDS patient needs to administer the formulation, but also allow the patient more time to be an outpatient, resulting in better quality of life.

The formulation of the invention also has the following advantages: by controlling the release of the active ingredient at a steady rate over an extended period of time, a lower daily exposure of, for example, cytotoxic drugs is provided, which is expected to reduce unwanted side effects.

The formulations and methods described herein may also have the following advantages: in the treatment of the relevant condition, the formulations and methods described herein may be more convenient, more effective, less toxic, more active, more potent, less adverse effects, or it may have other useful pharmacological properties for the physician and/or patient than any similar treatment known in the art.

Whenever the word "about" is used herein, for example in the context of amounts (e.g. amount, concentration, dimensions (size and/or weight), dose, time period, pharmacokinetic parameters, etc.), relative amounts (percentage, weight ratio, size ratio, atomic ratio, aspect ratio, scale, coefficient, fraction, etc.), relative humidity, lux, temperature or pressure, it should be understood that these variables are approximate and thus may differ from the numbers specified herein by ± 15%, such as ± 10%, e.g. 5%, and preferably ± 2% (e.g., ± 1%). This is the case even if such numbers are first presented in percent form (e.g., "about 15%" may mean ± 15% with respect to the number 10, i.e., any value between 8.5% and 11.5%).

The invention is illustrated by the following examples and with reference to the accompanying drawings, in no way limited thereto, in which figures 1 and 2 show dose normalized plasma concentrations of azacytidine over different time periods after subcutaneous administration to various rats prepared as described below; figures 3 and 4 show plasma concentration versus time curves for two patients administered azacitidine according to the treatment regimen in a clinical trial as described in example 10 below; FIG. 5 shows the plasma concentration versus time curve of azacitidine after subcutaneous administration of the formulation of the present invention in small pigs; fig. 6 shows the positive effect of subcutaneous co-administration of the formulation of the present invention with mixed oxide coated microparticles comprising the anti-inflammatory agent indomethacin on the local inflammatory response in terms of swelling size.

Examples

Example 1

Mixed oxide coated azacitidine microparticles I

Azacytidine microparticle samples (MSN Labs, india) were prepared by jet milling. The particle size distribution determined by laser diffraction is as follows: d (D) ₁₀ 2.9μm；D ₅₀ 7.9μm；D ₉₀ 23.2μm。

The powder was charged into an ALD reactor (Picosun, sun ^TM R series, espoo, finland), 24 ALD cycles were performed at a reactor temperature of 50 ℃. The coating sequence is as follows: three ALD cycles, diethyl zinc and water were used as precursors for the three ALD cycles, followed by one cycle of trimethylaluminum and water, repeated six times to form a mixed oxide layer with a zinc to aluminum atomic ratio of 3:1. The thickness of the first layer is between about 4 to about 8nm (estimated from the number of ALD cycles).

The powder was removed from the reactor and deagglomerated by forcing the powder through a polymer screen having a mesh size of 20 μm using a sonic screen.

The resulting deagglomerated powder was reloaded into an ALD reactor and subjected to another 24 ALD cycles as previously described to form a second layer of mixed oxide of the aforementioned ratio, which was then extracted from the reactor and deagglomerated by sonic screening as previously described, then reloaded to form a third layer, deagglomerated, and then reloaded to form a final fourth layer.

To determine the drug loading (i.e. w/w% of azacytidine in the powder) HPLC (prominace-i (Shimadzu, japan) was used, equipped with a diode array detector (Shimadzu, japan) set at 223nm, using 4.6x250 mm,3 μm particles, C18 chromatographic column (Luna, phenomenex, USA)). The nanoshell coating was dissolved in 5M phosphoric acid in DMSO and the slurry diluted with DMSO, then filtered (0.2 μm RC, lab Logistics Group, germany) and further analyzed by HPLC (n=2). The drug loading was determined to be 88.9%.

Example 2

Mixed oxide coated azacitidine microparticles II

The same procedure as described in example 1 was performed to produce particles coated with a mixed oxide coating having a zinc to aluminum atomic ratio of 2:1.

The coating sequence is as follows: two ALD cycles using diethyl zinc and water as precursors followed by one cycle of trimethylaluminum and water were repeated ten times, the coated powder was removed from the reactor, deagglomerated, reloaded and repeated the same coating sequence, removed, deagglomerated until a total of 30 cycles of 4 groups were completed.

The drug loading was determined to be 86.6%.

Example 3

Mixed oxide coated azacitidine microparticles III

The same procedure as described in example 1 was followed, but this time by performing 6 sets of 24 ALD cycles to produce a thicker mixed oxide coating with a zinc to aluminum atomic ratio of 3:1.

The drug loading was determined to be 80.7%.

Comparative example 4

Alumina coated azacitidine microparticles

The same particles coated with mixed oxide coating as described in example 1 were coated with a pure alumina coating. 7 ALD cycles were performed prior to removal and deagglomeration of the coated powder from the reactor, as described in example 1. The resulting deagglomerated powder was reloaded into the ALD reactor and subjected to another 7 ALD cycles, followed by 7 cycles of extraction, deagglomeration, and reloading, after which the process was repeated twice, followed by 2 reloads and 14 cycles.

The drug loading was determined to be 91.8%.

Comparative example 5

Suspension of azacitidine microparticles in vehicle

Suspending the same microparticles coated with a mixed oxide coating as described in example 1 in a commercially available aqueous vehicleIn vet, the vehicle is a veterinary product for injection into animals such as horses, comprising 10mg/mL sodium hyaluronate in a sterile, isotonic, phosphate buffer solution (pH 7.4).

The concentration of azacytidine in the formulation was 10mg/mL, which corresponds to 5mg/kg body weight Sprague-Dawley rats.

Comparative example 6

Suspension of alumina-coated azacitidine microparticles in vehicle

The coated microparticles from comparative example 4 were suspended in a commercially available aqueous vehicle In vet, the concentration of azacytidine in the formulation was brought to 27mg/mL, which corresponds to 13.5mg/kg body weight Sprague-Dawley rats.

Example 7

Formulation I of the invention

Three suspensions of coated microparticles of azacitidine (prepared according to the methods described in examples 1, 2 and 3 above) were suspended in Hyonate vet.

Another suspension of coated microparticles of azacitidine (prepared according to the method described in example 3 above) was suspended in an aqueous vehicle comprising 0.1% (w/w) polysorbate 20 and 0.25% (w/w) sodium carboxymethylcellulose in phosphate buffered saline (pH 7.4).

In each case, the concentration of azacytidine in the formulation was 27mg/mL, which corresponds to 13.5mg/kg body weight Sprague-Dawley rats.

Example 8

In vivo rat study

Thirty-eight male Sprague Dawley rats weighing between 266 and 302g on the day of administration were supplied by Charles River (UK). Animals were randomly divided into six animals per group.

The hair of the intended application area is cut off before injection and the injection site is marked. Suspensions described in comparative example 5 (group 1), comparative example 6 (group 2), and example 7 (groups 3 to 6 identified in table 1 below) were drawn into 1mL BD syringes and a single subcutaneous injection (about 0.15 mL) was administered to the flank of each rat through a 20G needle (BD microinject). Administration is performed no more than 10 minutes after the formulation is prepared.

TABLE 1

Group of	Description of the invention	Dosage of
			1	Uncoated particles in Hyonate (example 5)	5.0±0.08
2	Al in Hyonate ₂ O ₃ Coated particles (examples 4 and 6)	13.5±0.23
			3	Mixed (2:1) coated particles in Hyonate (examples 2 and 7)	13.6±0.40
4	HyoMixing in State (3:1) coated particles (examples 1 and 7)	13.5±0.33
			5	Thicker mix (3:1) coated particles in Hyonate (examples 3 and 7)	13.7±0.17
6	Thicker mix (3:1) coated particles in PBS (examples 3 and 7)	13.7±0.30

From tail vein blood sample (about 0.2 mL) to K at the following time points ₂ EDTA (dipotassium ethylenediamine tetraacetate) tube containing 5. Mu.L THU (25. Mu.L/mL blood; tetrahydrouridine, a competitive cytidine deaminase inhibitor) stabilizer (1 mg/mL aqueous solution): 0.5, 1, 3, 6, 12, 24, 48, 72, 120, 168, 240 and 336h post-administration. The actual sampling time is recorded. Plasma was separated by centrifugation (1500 g, at 4 ℃ for 10 minutes) as soon as possible after collection and stored at-80 ℃ until analysis.

After completion of the study, all plasma samples were transported for analysis after deep freezing on dry ice. Animals were sacrificed on the last day of the study.

Plasma concentrations of azacitidine were determined using LC-MS/MS. Study samples were prepared using a TECAN Genesis liquid handling robot by transferring 25 μl of rat plasma into a 96-well plate and adding 25 μl of 5% DMF/acetonitrile and 75 μl of internal standard working solution. The 96-well plates were then shaken for 15 minutes and centrifuged. All samples were then injected into the UPLC-MS/MS system. The separation was carried out using a ACQUITY BEH Amide column, 2.5 μm, 2.1X100 mM,25℃water, using 10mM ammonium formate aqueous solution (pH 3.4) and 0.125. Mu.M lithium acetate as mobile phase A (MP A) and acetonitrile as mobile phase B (MP B).

Using Microsoft Excel for Mac (16.43, microsoft)ft, redmond, washington, USA), the pharmacokinetic analysis of azacitidine in plasma was performed according to standard non-atrioventricular methods. Maximum concentration C _{Maximum value} Time t of correlation _{Maximum value} Is the coordinate of the highest concentration during time. t is t _Finally Is the time at which the concentration was last detectable. Calculating the area AUC under the concentration versus time curve up to the last detectable concentration using a linear trapezoidal rule _Finally 。

The site of administration of each animal was labeled after administration and kept free of hair for the duration of the study. Site observations were made 24, 120, 168 and 336 hours post-dose.

Results

Figure 1 presents the dose normalized plasma concentration of azacitidine within two weeks after a single subcutaneous administration of the various formulations and figure 2 presents the same plasma concentration profile over the first six hours. Plasma pharmacokinetic parameters are also presented in table 2 below as the mean value (standard deviation provided in parentheses) of a group of 6 rats, wherein:

the dose is expressed in mg/kg of rat body weight

·“t _{Maximum value} "is the time to peak concentration expressed in hours

·“C _{Maximum value} "is the maximum concentration found in the assay expressed in μg/mL

·“t _Finally "is the time of last detectable concentration in hours

·“t _1/2,z "is the terminal half-life expressed in hours

·“AUC _∞ "is the area under the concentration versus time curve up to infinite time, expressed in μg h/mL

F is the relative bioavailability expressed as a percentage

·“C _{Maximum value} "D" is the maximum concentration normalized to 1mg/kg, expressed in μg/mL/mg/kg rat body weight

·“AUC _Finally "D" is the area under the normalized blood concentration versus time curve relative to 1mg/kg up to the final detectable concentration expressed in μg h/mL/mg/kg rat body weight

·“AUC _∞ "D" is the area under the concentration versus time curve normalized to 1mg/kg up to infinite time expressed in μg h/mL/mg/kg rat body weight

·“Fr.Rel. _0-12h "is the fraction released in percent over the first twelve hours of the area under the concentration versus time curve up to infinity.

TABLE 2

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It can be seen that the plasma concentration curves of groups 3 to 6 are comparable, wherein the maximum plasma concentration (C _{Maximum value} ) The concentration then slowly but steadily decreased over the 14 day study period.

Group 2 had a higher initial drug release than the mixed oxide coating formulation.

Comparing groups 5 and 6, it can be seen that the PBS-based vehicle resulted in a slightly lower AUC than when using the Hyonate vet, although the corresponding pharmacokinetic profiles were largely comparable.

Groups 2 to 6 also show comparable relative bioavailability (F) to uncoated azacitidine, in sharp contrast to our previous study of aqueous suspensions of zinc oxide coated azacitidine, which showed lower F values.

Group 2 to group 6C compared to uncoated azacytidine (group 1) _{Maximum value} Lower, wherein about one fifth of the dose is released on the first day. When normalized for dose, the difference is about an order of magnitude. In addition, the residual area (related to unreleased drug) after the last sampling time (336 hours after administration)<12％。

The results of group 2 show slightly different curvesCharacterized in that the dose fraction released during the first day is large, C _{Maximum value} Higher and shorter in duration.

In summary, groups 3 to 6 show an extended release profile, unlike the case of a rapid decrease after administration of uncoated azacitidine. Similarly, favorable plasma concentration-time profiles were observed for all formulations of the invention.

Example 9

Mixed oxide coated azacitidine microparticle IV

The same procedure as described in example 1 was carried out, except that the particles of azacitidine had the following particle size distribution: d (D) ₁₀ 1.2μm；D ₅₀ 3.8μm；D ₉₀ 11.3 μm. The drug loading was determined to be 81.3%.

Example 10

Phase Ia clinical trial

An open-test phase Ia clinical study was conducted to evaluate the pharmacokinetics, tolerability and safety of coated azacitidine microparticles from example 9 above, suspended in Hyonate vet (Boehringer Ingelheim Animal Health; an aqueous solution for pH adjustment comprising sodium hyaluronate (10 mg/mL), sodium chloride (8.5 mg/mL), disodium hydrogen phosphate (0.223 mg/mL), sodium dihydrogen phosphate monohydrate (40 μg/mL), HCl and NaOH), and administered as a subcutaneous injection for treating moderate grade 2 or higher risk MDS, CMML or AML in patients who have received azacitidine treatment.

Measuring pharmacokinetic parameters, including AUC _0-24h 、AUC _0-last 、AUC _0-∞) 、C _{Maximum value} 、C _Finally Terminal t _1/2 Volume of distribution V _d And clearance rate.

Local tolerability was measured by examining the injection site. Pain, tenderness erythema/redness and induration/swelling were assessed by a four-level scale, with a level 1 being considered mild and a level 4 being considered potentially life threatening.

Study planning was included in 6 patients, including screening period, treatment period, metaphase analysis, and follow-up period.

Inclusion criteria included:

Written informed consent was signed before any study-specific procedure.

Patient age is greater than or equal to 18 years

The Body Mass Index (BMI) during screening is more than or equal to 19 and less than or equal to 32kg/m ² BSA

Currently undergoing azacitidine treatment at a dose of 100mg/m per treatment cycle ² BSA x 5 or x 4, at least six cycles of diagnosis:

a. diagnosis of intermediate-2 and high risk myelodysplastic syndrome (MDS) based on the International Prognostic Scoring System (IPSS)

b. Has chronic myelomonocytic leukemia (CMML), and the primary cell rate of marrow is 10% -29%

c. Has Acute Myelogenous Leukemia (AML) classified according to World Health Organization (WHO)

Eastern tumor cooperative group (ECOG) expression status of 0, 1 or 2

Restoring hematological and clinical chemistry assessments according to clinical practice at the beginning of the last azacitidine treatment cycle prior to screening visit

Female subjects without fertility (defined as pre-menopausal women with records of tubal ligation or hysterectomy or bilateral tubectomy; or postmenopausal women with 12 months amenorrhea)

Male patients agree to use appropriate contraceptive methods

Willingness and ability to follow study procedures, visit schedules, study restrictions and requirements

The exclusion criteria included:

Patients were enrolled in any other study/intervention trial including study drug 30 days prior to screening (or five half-lives of study drug prior to screening, based on longer time)

Malignant diseases (excluding uncomplicated basal cell carcinoma of the skin, carcinoma of the cervix or breast in situ, or other local malignant tumors with a high possibility of healing after excision or radiotherapy) were diagnosed within 5 years

Any major medical condition, laboratory abnormality or mental illness that would prevent the patient from participating in the study

History of alcoholism or drug taking within 12 months

Any condition that would expose the patient(s) (he/she) to unacceptable risk when participating in the study, including the presence of laboratory abnormalities

Other reasons that researchers consider unsuitable for participation in the study.

The time for patients to participate in the study was approximately 2-3 months. The time range consists of a 3-4 week screening period followed by a treatment period of about four weeks, including from day 1 to day 4, daily injections of uncoated azacitidine @Or universal azacytidine (Mylan), lyophilized powder for injection suspended in water for injection, 100mg/m ² BSA,25mg/mL)。

Samples were collected on day 4 (before study drug start) for pharmacokinetic analysis. Average maximum plasma concentration (C _{Maximum value} ) 562ng/mL, and occurs at a t of 0.433 hours _{Maximum value} And then, the method is carried out. The average half-life was 6.82 hours. Average AUC _{Infinite number of cases} 1120ng h/mL.

On day 5, study drug suspensions (100 mg/m ² BSA,100 mg/mL). Samples were collected for pharmacokinetic analysis on days 5 through 8, and on each of days 10, 12, 15, 17, and 19.

It is also contemplated that after the end of the treatment period, the last dose of azacitidine is replaced with a single dose of azacitidine comparator (as described above) and follow-up on the same day is planned.

However, after two enrolled patients entered the treatment period, the study was shelved after an internal safety committee conference responsible for reviewing the safety data.

Notably, both patients developed moderate induration and inflammation (redness and mild pain) at the injection site. It was therefore decided not to recruit more patients to participate in the study and required an expert to conduct additional reviews/analyses of the biopsy results of both patients.

However, the plasma concentration time profiles of both patients after administration of study drug are shown in figures 3 and 4, respectively, which show a significant steady-state sustained release of azacitidine from the injected study drug formulation. Plasma concentration time curves are shown in the graph in semilogarithmic scale (squares). Average maximum plasma concentration (C _{Maximum value} ) 94.8ng/mL, and occurs at 1.02 hours (T _{Maximum value} ) And then, the method is carried out. The average half-life was 15.2 hours. Average AUC _{Infinite number of cases} Is 495ng h/mL.

Example 11

The combination formulation of the present invention

Various formulations of the invention comprising a combination of azacitidine and indomethacin were prepared as follows:

(A) A blend mixture of azacitidine and indomethacin microparticles in a weight ratio between 100:1 and 1:10 is prepared by jet milling. The particle size distribution determined by laser diffraction is an average particle size between 0.1 and 100 μm.

The resulting powder was coated by ALD as described in example 1 above and formulated in a vehicle and used to treat patients with MDS as described in example 10 above.

(B) Microparticles are prepared comprising a co-precipitated mixture of azacitidine and indomethacin, for example, in a weight ratio of between 100:1 and 1:10. The particle size distribution determined by laser diffraction is an average particle size between 0.1 and 100 μm.

Microparticles were coated by ALD as described in example 1 above and formulated in a vehicle and used to treat patients with MDS as described in example 2 above.

(C) Two sets of microparticle samples were prepared separately by jet milling. The first group comprises azacytidine and the second group comprises indomethacin. The particle size distribution of the two groups of samples, determined by laser diffraction, was between 0.1 and 100 μm.

The two groups of samples were individually coated by ALD as described in example 1 above and mixed in the formulation with a weight ratio between the first and second groups between 100:1 and 1:10.

The mixed powder was formulated in a vehicle and used to treat patients with MDS as described in example 10 above.

(D) Azacytidine samples were prepared as described in example 1 and formulated in vehicle as described in example 10 above. Additional formulations comprising indomethacin particles were prepared in the same manner.

As described in example 10 above, the two formulations are injected at substantially the same time to different sites to treat patients with MDS. The dose to weight ratio of azacitidine and indomethacin two different injections is between 100:1 and 1:10.

(E) Coated granules of azacitidine were prepared essentially as described in example 1 above and formulated as described in example 10 above in a vehicle further comprising dissolved and/or suspended indomethacin.

The formulation is used to treat patients with MDS, as described in example 10 above.

In all of the above cases (a) to (E), any inflammatory response at the site of subcutaneous administration (and the reservoir formed) is inhibited by the anti-inflammatory properties of indomethacin.

Example 12

Mixed oxide coated azacitidine microparticles V

The same procedure as described in examples 1 and 9 was performed to produce coated azacitidine microparticles with a drug loading of 80.1%.

Example 13

Mixed oxide coated azacitidine microparticles VI

Essentially the same procedure as described in examples 1 and 9 was performed, except that 30 ALD cycles were performed at a reactor temperature of 50 ℃ and the coating sequence was: two ALD cycles using diethyl zinc and water as precursors followed by one cycle of trimethylaluminum and water were repeated ten times to form a mixed oxide layer with a zinc to aluminum atomic ratio of 2:1. The thickness of the first layer is estimated to be between about 5 and about 10 nm.

The powder was removed from the reactor and deagglomerated by forcing the powder through a polymer screen having a mesh size of 20 μm using a sonic screen, then the deagglomerated powder was reloaded into the ALD reactor and another 30 ALD cycles were performed as before, forming a second layer of mixed oxide of the same ratio, extracted from the reactor and deagglomerated by sonic screening as described above, and the process was repeated to form a total of eight layers.

The drug loading was determined to be 69.1%.

Comparative example 7

Mixed oxide coated indomethacin Xin Weili

Indometate Xin Weili samples (Recce Pharmaceuticals, australia) were prepared by jet milling. The particle size distribution determined by laser diffraction is as follows: d (D) ₁₀ 1.2μm；D ₅₀ 3.8μm；D ₉₀ 11.3μm。

The same ALD coating and batch deagglomeration process as described in example 1 was performed to form coated indomethacin Xin Weili having four separate mixed oxide layers with a zinc to aluminum atomic ratio of 3:1.

The drug loading was determined to be 81%.

Comparative example 8

Mixed oxide coated lactose microparticles

Sample of lactose particles400, meggle, germany). The nominal particle size distribution is as follows: d (D) ₁₀ 0.8-1.6μm；D ₅₀ 4.0-11.0μm；D ₉₀ 15-35.0μm。

The powder was charged into an ALD reactor (Picosun, sun ^TM R series, espoo, finland), 48 ALD cycles at a reactor temperature of 50 ℃. The coating sequence is as follows: three ALD cycles, with diethyl zinc andwater was used as a precursor for three ALD cycles, followed by one cycle of trimethylaluminum and water, repeated twelve times, to form a mixed oxide layer with a zinc to aluminum atomic ratio of 3:1. The thickness of the first layer is between about 8 to about 16nm (estimated from the number of ALD cycles).

The resulting deagglomerated powder was reloaded into the ALD reactor and subjected to another 48 ALD cycles as previously described, forming a second layer of mixed oxide in the ratio described above, and then extracted from the reactor.

The particle size distribution of the coated lactose particles determined by laser diffraction is as follows: d (D) ₁₀ 2.1μm；D ₅₀ 7.6μm；D ₉₀ 23.4μm。

Example 14

Formulation II of the invention

Coated microparticles from example 12 above were suspended in Hyonate vet in glass vials to give final concentrations of 100mg/mL and 200mg/mL, respectively (hereinafter referred to as "formulation B" and "formulation E", respectively).

The coated microparticles from example 12 above were suspended in a glass bottle with coated indomethacin Xin Weili from comparative example 7 above in a Hyonate vet to give the final concentration of each coated active ingredient as set forth in table 3 below, with the formulation also identified in the manner set forth below.

TABLE 3 Table 3

Comparative example 9

Formulations comprising mixed oxide coated indomethacin and lactose microparticles

Following the procedure described in example 14 above, a suspension of indomethacin type Xin Baofu microparticles (from comparative example 7 above) and lactose coated microparticles (from comparative example 8 above) in 2.2mL of Hyonate vet was produced at a final concentration of 100 mg/mL.

These suspensions were labeled "formulation C" (indomethacin) and "formulation a" (lactose), respectively.

Example 14

Pig study I

The purpose of this study (carried out at the company Scantox a/S in denmark) was to evaluate the local tolerability and pharmacokinetics of azacitidine formulated according to the invention administered to minipigs by subcutaneous injection, as well as the local tolerability after administration of azacitidine formulated according to the invention and indomethacin formulated as described herein.

Minipigs were chosen as the test model because of their wide acceptance in such studies and because of the very similar skin physiology of humans and minipigs. The staggered dose regimen is selected to start with two doses corresponding to 1/4 and 1/2 of the human clinically equivalent dose and then to increase to full dose to reduce the risk of severe local reactions.

The animals were 24.9kg in weight at the time of allocation to the study and were housed according to the EU directive 2010/63/EU on protecting animals for scientific purposes at 9/22 of 2010. Briefly, standard mini-pig diets (morning and afternoon) were provided twice daily, approximately 350g per meal. During the course of the study, the diet may be adjusted to allow reasonable growth of the animals. Dehydrated grass (Compact Gras, hartog b.v., netherlands) is also provided daily, and animals are free to drink quality drinking water for home use.

One week before the start of treatment, animals were anesthetized by neck intramuscular injection (1.0 mL/10kg body weight) and a total of 6 injection sites (approximately 2 x 2 cm) were mapped behind their neck.

Animals were then re-anesthetized 3 days prior to surgery and an ear vein catheter was implanted to collect blood samples during the study. To relieve pain during the study, animals were injected intramuscularly with meloxicam 5mg/mL (0.08 mL/kg) in the hind legs prior to implantation and once daily for the next two days.

Animals received intravenous injection of 200mg ampicillin/mL (0.05 mL/kg). The catheter was rinsed with 10mL sterile saline and locked using 0.5mL TauroLock Hep500 (taurolidine citrate with 500IE/mL heparin). A plug, such as a Bionector IV access system, is applied to the luer fitting (luer).

Between two blood draws, tauroLockTM Hep500 will create a heparin lock in the catheter.

A single dose of the study formulation was administered by subcutaneous injection at each of the six tagged injection sites listed in table 4 below.

TABLE 4 Table 4

Injection site	Formulation of	Dosage volume (mL)	Dosage (mg)
				1	A	1.9	About 200 x
2	B	0.5	50
				3	B	1.0	100
4	B	0.5	50
				5	D	0.5	50+50
6	C	0.5	50

* Estimated value

Local tolerance

On study day 1, animals were anesthetized and formulations a and B were administered subcutaneously at relevant dose volumes at injection sites 1, 2 and 3, as listed in table 4 above. On study day 41, animals were anesthetized and formulations D and C were administered subcutaneously at relevant dose volumes at injection sites 5 and 6, as listed in table 4 above.

The local tolerance of these injections was assessed.

In each case, the relevant sample vial was inverted 3 times prior to injection, and then the sample was withdrawn for each injection to avoid precipitation of test material and thus deviation from the correct dose.

All clinical symptoms of poor health and any behavioral changes were recorded daily. Furthermore, dose-related observations were made before/at the time of administration and not earlier than 30 minutes after administration, and any deviations from normal values were recorded.

The injection sites were photographed, scored and recorded 30 minutes and 2 and 6 hours after dosing, and then daily until no scoring occurred. From day 10, no more photographs were taken and the injection sites were scored once every other day until no score or the study ended.

Injection sites 5 and 6 were photographed, scored and recorded 30 minutes and 2 and 6 hours after dosing and daily until day 48. Thereafter, no more photographs were taken and the injection sites were scored twice weekly until no scoring or the study ended.

Special attention was paid to bleeding, erythema, swelling (indicating/measuring size) and hardness/induration and necrosis, as well as any other signs of inflammation or allergic reaction. The parameters were scored according to the following grading system: 0 (no), 1 (minimum), 2 (slight), 3 (medium) and 4 (clear).

On day 2, blood samples were collected from animals to assess clinical pathology parameters. Additional samples were taken on day 5. Animals were fasted overnight, but were allowed to drink before blood samples were collected.

For hematology, at least 2.5ml K3 EDTA-stabilized blood was collected. A spare smear was prepared from the sample and stained with May-Grunwald and Giemsa (Giemsa) to allow for manual differential white blood cell count at a later date. The smear was not analyzed and was discarded after the study was completed. For the clotting test, 1.8mL of citrate stabilized blood was collected. The parameters, methods and units of the laboratory study are shown in table 5 below.

TABLE 5

Instrument laboratory, automated coagulation laboratory

At the discretion of the skilled person, the individual smears can be counted manually

Approximately 3mL of blood was drawn in a tube containing serum clotting activator for clinical chemistry analysis. The parameters, methods and units of the laboratory study are shown in table 6 below.

TABLE 6

Full thickness biopsies were taken from injection site 1 on days 3 and 7 and from injection sites 2 and 3 on days 2 and 6. A single control biopsy was taken outside the injection site for comparison at the time of histopathological evaluation. Full thickness biopsies were taken from injection sites 5 and 6 on day 43 and day 47.

Animals were anesthetized prior to biopsy collection and 10mg/mL (0.02 mL/kg) of methadone was injected intramuscularly to prevent painful reactions approximately 30 minutes prior to the first biopsy sampling.

Biopsies were collected using an 8mm punch and fixed in phosphate buffered neutral 4% formaldehyde. After fixation, the specimens are trimmed and processed. Specimens were embedded in paraffin and cut to a nominal thickness of about 5 μm, stained with hematoxylin and eosin and examined under an optical microscope. All pathology results were directly input to the Intem(version 9.3.0.0). Histological changes were graded on 5 grades (minimal, mild, moderate, overt and severe).

Pharmacokinetic (PK)

On study day 22, animals were anesthetized and formulation B was administered subcutaneously at injection site 4 at the relevant dose volume, as shown in table 4 above. PK parameters for this injection were assessed.

Prior to injection, the relevant sample vials were inverted 3 times and then the samples were retrieved as described above for each injection to avoid precipitation of the test material and thus deviation from the correct dose.

On the day of dosing, blood samples were collected at the following time points: pre-treatment, as well as 30 minutes, 2, 6, 10, 24, 48, 72, 120, and 168 hours post-treatment.

Approximately 3mL of blood sample was withdrawn from the jugular/jugular vein stem. Blood was sampled into a vacuum blood collection tube containing K2-EDTA as an anticoagulant. The evacuated blood collection tube was placed in ice water until centrifugation (10 min, 1270G, +4℃). Each plasma sample was split into two approximately 0.5mL aliquots and transferred to a sponsor provided freezer tube 90 minutes after collection and frozen at-18 ℃ or less. The first set of samples were transported to dry ice (approximately-70 ℃) for analysis and shipped without a thermal recorder (thermologger). The second set of samples was stored as backup samples at-18 ℃ or lower. The primary samples were shipped for a few days after receipt.

Azacitidine in plasma was determined by UPLC-MS/MS. Azacitidine was extracted from plasma by protein precipitation using DMF: acetonitrile (5:95). After injection into the straight phase column, the material was eluted with an acetonitrile and water gradient and detected by MS/MS.

Non-compartmental pharmacokinetic analysis was performed on individual plasma concentration curves using software PKanalix (version 2020).

When the plasma concentration obtained in the pre-dose sample was lower than LLOQ, the data point was entered as zero. All other concentrations below LLOQ were input at half the LLOQ value (1/2×lloq). Will T _{Maximum value} Subsequent data points below LLOQ are excluded from modeling and analysis.

Estimating maximum plasma concentration (C by visual inspection of data _{Maximum value} ) And its time of occurrence (T) _{Maximum value} )。

Calculating the area under the curve (AUC (0-t)) from the time zero point to the last quantifiable concentration time point and the area under the curve (AUC) from the time zero point to infinity according to the linear/logarithmic trapezoidal method _{Infinite number of cases} ). If the extrapolated area (AUC (% extrapolation)) exceeds 20%, then the AUC is considered _{Infinite number of cases} Less reliable.

Half-life T1/2 is calculated as ln2/1z, where 1z is the elimination rate constant. Half-life was only calculated when at least 3 data points were included. If the regression line yields a Rsq of less than 0.80, the results are considered unreliable.

After the last blood sample/procedure was collected, the animal was no longer part of the study and was terminated.

Results

At injection site 1, no reaction was initially observed after administration of formulation a (lactose). However, on day 14, soft swelling (up to 10×15mm, barely noticeable) was observed.

The next day following administration of formulation B (azacytidine, 50 mg) at injection site 2, a hard swelling (up to 40 x 20 mm) was observed, which lasted for at least 28 days. Minimal to mild erythema was observed on days 2 and 3, and again on day 7. The next day following administration of formulation B (also azacytidine, 50 mg) at injection site 4, a hard swelling (up to 10 x 10mm, barely noticeable) occurred, which continued until 11 days (day 23 to day 33) after administration. Minimal erythema was observed from day 23 to day 28.

However, the next day after administration of formulation B (azacytidine, 100 mg) at injection site 3, a hard, well-defined swelling (up to 55 x 30 mm) occurred at the injection site, which lasted for at least 28 days. Slight erythema was observed on days 2 and 3, and again on day 7.

In contrast, the following day after administration of formulation D (azacytidine 50mg plus indomethacin 50 mg) at injection site 5, no injection site reaction was observed.

For PK parameters, a single subcutaneous administration of 50mg of coated azacitidine (injection site 4) showed systemic exposure and prolonged release profile as shown in figure 5. The duration was 120h and 47% exposure was observed during the first 12h after application.

Histopathological results showed that:

injection site 2: moderate inflammation and necrosis occurred on day 3, and mild inflammation and necrosis occurred on day 7.

Injection site 5: moderate inflammation and mild necrosis occurred on day 45 (3 days post injection) and minimal inflammation and minimal necrosis occurred on day 49 (7 days post injection).

It was concluded that subcutaneous administration of formulation D (combination of mixed oxide-coated azacitidine and mixed oxide-coated indomethacin) caused less skin reaction than subcutaneous administration of formulation B (mixed oxide-coated azacitidine alone).

Example 16

Pig study II

Five minipigs were studied similarly to the study described in example 15 above. On the day of arrival of animals, they were given a final number using a randomization protocol. These animals received a chip with a unique digital code.

Two injection sites were marked on the neck of the minipig approximately one week before the start of treatment in the same manner as described in example 15 above.

The treatment regimen for the animals is shown in table 7 below.(Mylan) is a commercially injectable formulation of azacitidine at a concentration of 25mg/mL, which can be considered to provide an equivalent dose of "uncoated" azacitidine.

TABLE 7

Substantially the same treatment regimen as described in example 14 above was followed. For each animal, subcutaneous injections of the relevant formulation were administered at the dose volumes described above on day 1 (injection site 1 for each animal) and on day 8 (injection site 2 for each animal).

Observations were made in essentially the same manner as described in example 14 above, and necropsies were performed on day 29.

FIG. 6 presentsInflammation swelling size (in mm of lower animals ³ Units):

animal 1/injection site 1 (50 mg azacytidine, lower concentration particles; diamonds);

Animal 3/injection site 1 (50 mg azacytidine, higher concentration particles; triangle);

animal 2/injection site 2 (50 mg azacytidine, lower concentration particles +50mg indomethacin; cross); and

animal 5/injection site 2 (50 mg azacytidine, higher concentration of particles +25mg indomethacin; square).

Figure 6 clearly shows the dramatic effect of azacitidine administration with indomethacin.

Claims

1. A pharmaceutical formulation useful for treating myelodysplastic syndrome, the pharmaceutical formulation comprising a plurality of particles suspended in an aqueous carrier system, the particles:

(b) Comprising a solid core comprising azacytidine or a pharmaceutically acceptable salt thereof, the solid core being at least partially coated with a coating of an inorganic material comprising a mixture of:

(i) Zinc oxide; and

(ii) One or more other metal and/or metalloid oxides,

2. The formulation of claim 1, wherein the atomic ratio ((i): ii)) is at least about 1:1 and up to and including about 6:1.

3. The formulation of claim 1, wherein coated particles comprise:

(a) A solid core comprising azacitidine or a pharmaceutically acceptable salt thereof; and

(b) One or more discrete layers surrounding the core, each comprising at least one separate mixture of zinc oxide and one or more other metal and/or metalloid oxides in an atomic ratio between about 1:1 and about 6:1.

4. The formulation of claim 3, wherein the core consists essentially of azacitidine or a pharmaceutically acceptable salt thereof.

5. The formulation of any one of the preceding claims, wherein the average diameter of the particles on a weight, number, or volume basis is between an amount of 1 μιη and about 50 μιη.

6. The formulation of any one of the preceding claims, wherein more than one discrete layer of oxide mixture is applied to the core sequentially.

7. The formulation of claim 6, wherein between 3 and 10 discrete layers of oxide mixture are applied.

8. The formulation of any one of the preceding claims, wherein the total thickness of the mixed oxide coating is between about 0.5nm and about 2 μιη.

9. The formulation of any one of claims 6 to 8, wherein a maximum thickness of a single discrete layer of mixed oxide coating is about one percent of the weight, number, or volume based average diameter of the core, including any other discrete layers that have been previously applied to the core.

10. The formulation of any one of the preceding claims, wherein the ratio of zinc oxide to other metal and/or metalloid oxides is between about 2:1 and about 5:1.

11. The formulation of any one of the preceding claims, wherein the one or more other metal and/or metalloid oxides are selected from alumina and/or silica.

12. The formulation of any one of the preceding claims, in the form of a sterile injectable and/or infusible dosage form.

13. The formulation of claim 12, in a form that can be administered by a surgical applicator that forms a reservoir formulation.

14. A method for preparing a formulation as defined in any one of the preceding claims, wherein the coated particles are made by applying a layer of mixed oxide coating material to the core and/or previously coated core via atomic layer deposition.

15. The method according to claim 14, wherein:

(i) Coating the solid core with a first discrete layer of mixed oxide coating material;

(ii) A deagglomeration process step is then carried out on the clad core from step (i);

(iii) Coating the deagglomerated coating core from step (ii) with a second discrete layer of mixed oxide coating material;

(iv) Repeating steps (ii) and (iii) to obtain the desired number of discrete layers.

16. The method of claim 15, wherein the deagglomerating step performed between application of the coating comprises sieving.

17. The method of claim 16, wherein the screening comprises vibratory screening.

18. The method of claim 17, wherein the vibratory screening includes controlling a vibratory probe coupled to a screen.

19. The method of claim 16, wherein the screening comprises sonic screening.

20. A process for preparing a formulation as defined in any one of claims 1 to 13, wherein the coated particles are mixed with the carrier system after coating.

21. An injectable and/or infusible dosage form comprising a formulation as defined in any one of claims 1 to 13 contained within a reservoir connected to and/or associated with an injection or infusion device.

22. The dosage form of claim 21, which is a surgical applicator forming a reservoir formulation.

23. The dosage form according to claim 21 or claim 22, wherein the coated particles as defined in any one of claims 1 to 12 and the carrier system are separately housed, and wherein mixing occurs before and/or during injection or infusion.

24. A formulation as defined in any one of claims 1 to 13 or a dosage form as defined in any one of claims 21 to 23 for use in the treatment of myelodysplastic syndrome.

25. Use of a formulation as defined in any one of claims 1 to 13 or a dosage form as defined in any one of claims 20 to 22 for the manufacture of a medicament for the treatment of myelodysplastic syndrome.

26. A method of treating myelodysplastic syndrome, which comprises administering to a patient in need of such treatment a formulation as defined in any one of claims 1 to 13 or a dosage form as defined in any one of claims 21 to 23.

27. The formulation for use according to claim 24, the use according to claim 25 or the method according to claim 26, wherein the myelodysplastic syndrome is selected from the group consisting of: refractory anemia, refractory anemia with annular iron granulocytes, refractory thrombocytopenia with multiple dysplasia and annular iron granulocytes, refractory anemia with excessive primitive cells in conversion, unclassified myelodysplastic syndrome and myelodysplastic syndrome associated with isolated del (5 q).

28. The formulation for use, the use or the method of any one of claims 24 to 27, wherein the formulation provides a depot formulation from which azacitidine is released over a period of between 3 days and about 3 weeks after injection.

29. The formulation for use, the use or the method of claim 28, wherein the total exposure to azacitidine is from the time comprising administration of 75mg/m by injection or infusion of azacitidine for seven consecutive days ² Dosing regimens of body surface areas achieve at least about 50% of the total exposure.

30. The formulation for use, the use or the method according to claim 29, wherein the exposure to the mean area under the concentration versus time curve up to infinity time is 960±458ng h/mL.

31. The formulation for use, the use or the method according to any one of claims 28 to 30, wherein the average maximum concentration observed in plasma is less than 75mg/m from seven consecutive days comprising administration of azacitidine by injection or infusion ² Dosing regimen of body surface area achieves the average maximum concentration.

32. The formulation for use, the use or the method of claim 31, wherein the average maximum concentration observed in plasma is between about 200 and about 700 ng/mL.

33. The formulation according to any one of claims 1 to 13, the dosage form according to any one of claims 21 to 23, or the formulation for use according to any one of claims 24 to 32, the use or the method, wherein the dose (calculated as free compound) of azacytidine, or a pharmaceutically acceptable salt thereof, is between about 200mg and about 1000mg per m ² The body surface area is within the range of the body surface area.