HK40038382A - Process for the preparation of sterile ophthalmic aqueous fluticasone propionate form a nanocrystals suspensions - Google Patents

Description

Method for preparing sterile ophthalmic aqueous fluticasone propionate A-type nanocrystal suspension

Technical Field

The present invention relates to methods of preparing sterile topical ophthalmic nanosuspensions comprising nanocrystals of fluticasone propionate form a in an aqueous carrier. The process is conveniently suitable for large scale production and produces sterile homogeneous aqueous nanosuspensions having a stable particle size distribution.

A sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals is used to treat an ocular inflammatory disease or an ocular inflammatory disorder by topically applying the nanosuspension (or the nanosuspension suspension) to the eyelids (e.g., upper and lower eyelids), eyelashes, and eyelid margin.

Background

The fluticasone propionate a-type nanocrystals are nanoplates having a [001] crystal axis substantially perpendicular to a surface defining the thickness of the nanoplates. Fluticasone propionate form a nanocrystals were prepared from the commercially available fluticasone propionate polymorph 1 by the antisolvent ultrasonic crystallization (sonocrystallization) method disclosed in WO 2013/16964.

WO 2013/169647 discloses the preparation of nanocrystals of the form of the morphic (morphic) form of fluticasone propionate (form a), their purification, and the preparation of aqueous suspensions comprising said nanocrystals.

Briefly, nanocrystals of fluticasone propionate were prepared according to the continuous ultrasonic flow-through amplification method disclosed in WO 2013/169647 (example 11 and fig. 38), by crystallization using an anti-solvent under ultrasonic treatment, followed by warming and cooking of the nanosuspension; the resulting nanocrystals were purified by continuous flow centrifugation, the carrier of the nanosuspension was centrifuged off, the precipitate was redispersed in a washing solution, and the dispersion was centrifuged again. This washing operation was repeated several times to reach the desired level of purification. The precipitate is then dispersed into the final formulation composition to obtain the desired dosage specification of the final product. However, WO 2013/169647 does not report any data relating to sterility testing of nanosuspensions.

Applicants have found that the continuous ultrasonic flow-through process disclosed in WO 2013/169647 enables the preparation of large quantities of fluticasone propionate form a nanocrystals, but is not suitable for large-scale preparation of sterile ophthalmic aqueous nanosuspensions because dispersing the purified nanocrystals (pellets) into the final aqueous vehicle (see fig. 38) does not produce a final product with sterility requirements that must be met by the pharmaceutical formulation for ocular delivery. Furthermore, it was found that in the large scale production of nanosuspensions, the nanocrystals tend to form aggregates that are difficult to deagglomerate during mixing of the purified nanocrystals with the final aqueous carrier, and thus it is difficult to obtain a uniform nanosuspension. Furthermore, even when some effectively disaggregated and homogeneous nanosuspensions are obtained on the fly, these nanosuspensions show a certain tendency to reaggregate and to be unstable.

As is well known, nanoparticles have a high tendency to aggregate due to their high surface energy, which leads to the formation of aggregates during the preparation and storage of the nanosuspension.

Aggregation of nanoparticles is not only a critical aspect in the suspension manufacturing process; aggregation can also lead to various problems such as inconsistent dosing and patient non-compliance. In particular for nanosuspensions intended for application to the eyelids, eyelashes and eyelid margin, aggregation may affect patient tolerance and potential safety.

Several strategies for ensuring proper physical stability of drug nanosuspensions are well known.

For example, stabilizers are often used, however, selecting a suitable stabilizer for a certain drug can be challenging.

US 2018/0117064 discloses a method for the preparation of an aqueous suspension comprising nanoparticles of a glucocorticoid compound and a dispersion stabilizer. US 2018/0117064 discloses that the primary function of the stabilizer is to wet the drug particles sufficiently to prevent austempering and aggregation of the nanosuspension and to form a physically stable formulation by providing an asteroid (ionic) or ionic barrier. Typical examples of stabilizers used in nanosuspensions are cellulose, poloxamers, polysorbates, lecithin, poly oleates and povidone.

WO 2010/141834 discloses topical ophthalmic formulations of fluticasone propionate for use in the treatment of allergic conjunctivitis and/or allergic rhinoconjunctivitis. WO 2010/141834 discloses various formulations including suspensions having a particle size of not more than 30 μm; examples of carriers include phosphate buffers, propylene glycol, hypromellose, polysorbate 80, disodium edetate and benzalkonium chloride (page 14, lines 7-15).

However, WO 2010/141834 does not report any method for the preparation of ophthalmic formulations and any experimental results relating to the stability of ophthalmic formulations.

WO 2013/025696 discloses the use of high pressure homogenization in a method for preventing the formation of drug aggregates in an ophthalmic formulation comprising an ophthalmic drug suspended in an aqueous carrier comprising at least one wetting agent. In particular, when the drug particles are already present in micronized form having a particle size suitable for topical application, a high pressure homogenization step may be used to prevent the formation of drug aggregates in the ophthalmic formulation; in fact, WO 2013/025696 discloses that high pressure homogenization can be applied to a suspension comprising a pre-micronized drug in an aqueous wetting agent solution, and that high pressure homogenization does not lead to a reduction in particle size, but rather stabilizes the already micronized drug and thereby prevents the formation of drug aggregates.

However, the method of WO 2013/025696 implies the use of high cost instruments, which increases the cost of the dosage form, and furthermore, the application of high pressure homogenization can lead to degradation of the nanocrystals.

Sonication is commonly used to disaggregate and disperse nanomaterials in aqueous-based media, which is essential to improve the homogeneity and stability of the suspension. Despite its widespread use, sonication tested during the set-up of the process for the preparation of nanosuspensions according to the invention did not effectively depolymerise fluticasone propionate nanocrystals.

Summary of The Invention

Accordingly, there is a need to provide a large scale process for preparing ophthalmic nanosuspensions (nanocrystal suspensions) comprising fluticasone propionate type a nanocrystals in an aqueous carrier.

As a result of studies conducted to solve the above problems, it has been surprisingly found that mixing of fluticasone propionate form a nanocrystals with an aqueous carrier comprising glycerol (glycerin) and boric acid under high shear, high speed conditions can stabilize the nanocrystals. Without wishing to be bound by any theory, it is believed that glycerol and boric acid form a complex that acts as a stabilizer for the nanocrystals; the boric acid/glycerol complex prevents nanocrystal aggregate formation during the preparation of the nanosuspension and also stabilizes the nanosuspension during storage. The results of the study also show that the addition of fluticasone propionate form a nanocrystals to a final carrier comprising a pre-formed boronic acid/glycerol complex results in residual aggregation, which is not observed when nanosuspensions are prepared according to the method of the present invention, wherein glycerol is added after the fluticasone propionate nanocrystals are suspended in a carrier comprising boric acid but no glycerol and depolymerised in that carrier. When the nanocrystals of fluticasone propionate are directly suspended in the final carrier containing the pre-formed boric acid/glycerol complex and disaggregated therein, the stabilization is lost and the disaggregation of the nanocrystals is more difficult to achieve and maintain during storage.

Complexes of boric acid with polyhydroxy compounds (borate-polyol complexes) are well known, and the use of borate-polyol complexes in ophthalmic compositions to enhance antimicrobial activity is well known.

WO 93/21903 discloses borate-polyol complexes such as mannitol, glycerol and propylene glycol as co-disinfectants in contact lens disinfecting solutions.

WO 2010/148190 discloses borate-polyol complexes comprising two different polyols to improve the shelf life of multi-dose ophthalmic compositions.

The present invention relates to a method of preparing sterile topical ophthalmic aqueous suspensions comprising fluticasone propionate type a nanocrystals that are readily amenable to large scale preparation for commercial production and produce stable nanosuspensions having a uniform, reproducible particle size distribution.

For the present invention, "fluticasone propionate a-type nanocrystals" refers to fluticasone propionate a-type nanocrystals having: an average particle diameter of 100nm to 1000 nm; an X-ray powder diffraction pattern includes peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ, and further includes peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplate.

Drawings

FIG. 1: XRPD pattern of fluticasone propionate a-form nanocrystals.

FIG. 2: a schematic of the particle size distribution at 40 ℃ at T ═ 1 week; 0.25% fluticasone propionate form a nanocrystals in a vehicle comprising a preformed 1% w/w boric acid/0.25% w/w glycerol complex (comparative).

FIG. 3: a schematic of the particle size distribution at 40 ℃ at T ═ 1 week; 0.25% fluticasone propionate type a nanocrystals in a carrier comprising preformed 1% w/w boric acid 1% w/w glycerol (comparative).

FIG. 4 is a schematic view of: particle size distribution at 40 ℃ at T ═ 1 week; after preliminary analysis at 40 ℃ and storage at 5 ℃ for 20 hours; 0.25% w/w fluticasone propionate form a nanocrystals in a vehicle with 0.25% w/w glycerol (comparative).

Disclosure of Invention

The present invention relates to a method for preparing a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000nm and a fluticasone propionate concentration of 0.001% w/w to 1% w/w, the method comprising:

a) preparing an aqueous carrier 1 comprising: 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and water to 100% w/w;

b) mixing an amount of fluticasone propionate nanocrystal a having an average particle size of 100nm to 1000nm with an amount of aqueous carrier 1 to yield a slurry comprising a fluticasone propionate concentration of 2% w/w;

c) applying high shear high speed mixing to the slurry of step b) for at least 10 minutes;

d) preparing an aqueous carrier 2 comprising: 1.8% w/w glycerol, 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and a suitable amount to 100% w/w water;

e) adding an aliquot of aqueous carrier 2 to the slurry of step c) to give a fluticasone propionate concentration of about 1% w/w;

f) applying high shear, high speed mixing to the slurry of step e) until a target average particle size is obtained;

g) sterilizing the nanosuspension of step f) by autoclaving;

h) preparing an aqueous carrier 3 comprising: 0.9% w/w glycerol, 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and an appropriate amount of water to 100% w/w; and sterilizing the aqueous carrier 3 by filtration;

i) aseptically adding an aliquot of sterile aqueous carrier 3 to the sterile nanosuspension of step g) to prepare a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate nanocrystal form a at the target concentration;

wherein the fluticasone propionate form a nanocrystals have an X-ray powder diffraction pattern of the nanocrystals that includes peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ and further includes peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining a thickness of the nanoplate.

In step c), the slurry comprising fluticasone propionate type a nanocrystals and the glycerol-free aqueous carrier 1 are subjected to high shear, high speed mixing to ensure uniform distribution of the fluticasone propionate nanocrystals, thereby improving the homogeneity of the final nanosuspension.

Preferably, in step c) and step f), the high shear, high speed mixing is preferably carried out at 6000 RPM.

Preferably, the high shear, high speed mixing of step f) is applied for at least 10 minutes.

Preferably, the nanosuspension of step f) comprising a concentration of 1% w/w fluticasone propionate is subjected to sterilization of step g) by autoclaving the nanosuspension in a glass vial at about 122 ℃ for about 40 minutes.

Preferably, the aqueous carriers 1 and 2 are filtered through a 0.2 μm filter before being used in step b) and step e), respectively.

Optionally, an aqueous carrier 2 comprising glycerol 1.8% w/w may be prepared by: an aliquot of the support 1 filtered through a 0.2 μm filter was added with a certain amount of glycerol to give a final concentration of 1.8% w/w glycerol.

Preferably, the concentration of fluticasone propionate in the final sterile topical ophthalmic aqueous nanosuspension prepared according to the method of the present invention is from 0.001% to 0.5% w/w; more preferably, the concentration of fluticasone propionate is 0.5%, 0.25%, 0.20%, 0.10%, 0.05%, 0.03%, 0.01% or 0.005% w/w; most preferably, the concentration of fluticasone propionate is 0.20% w/w or 0.10% w/w or 0.05% w/w.

Alternatively, the method of the present invention may be practiced under completely sterile manufacturing conditions using sterile fluticasone propionate type a nanocrystals and a sterile carrier 1-3, and thus, another embodiment of the present invention is directed to a method for preparing a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000nm and a fluticasone propionate concentration of 0.001% w/w to 1% w/w, the method comprising:

a-1) sterilizing fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000 nm;

b-1) preparing an aqueous carrier 1 comprising: 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and an amount of water to 100% w/w; and sterilizing the aqueous carrier 1 by filtration;

c-1) aseptically mixing an amount of sterilized fluticasone propionate nanocrystal a with an amount of sterilized aqueous carrier 1 to obtain a slurry comprising fluticasone propionate at a concentration of 2% w/w;

d-1) applying high shear, high speed mixing to the slurry of step c-1) for at least 10 minutes;

e-1) preparing an aqueous carrier 2 comprising: 1.8% w/w glycerol, 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and a suitable amount to 100% w/w water; and sterilizing the aqueous carrier 2 by filtration;

f-1) adding an aliquot of the sterilized aqueous carrier 2 to the slurry of step d-1) in a sterile manner to obtain fluticasone propionate at a concentration of about 1% w/w;

g-1) applying high shear and high speed mixing to the slurry obtained in the step f-1) until a target average particle size is obtained;

h-1) preparing an aqueous carrier 3 comprising: 0.9% w/w glycerol, 0.5% w/w methylcellulose 4000cp, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and a suitable amount to 100% w/w water; and sterilizing the aqueous carrier 3 by filtration;

i-1) adding an aliquot of the sterilized aqueous carrier 3 to the nanosuspension of step g-1) in a sterile manner to prepare a sterile topical ophthalmic aqueous nanosuspension comprising the fluticasone propionate nanocrystal form a at the final concentration;

wherein the fluticasone propionate form a nanocrystals have an X-ray powder diffraction pattern comprising peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ, further comprising peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis that is substantially perpendicular to a surface defining the thickness of the nanoplate.

Preferably, the fluticasone propionate type a nanocrystals used in step a-1) are sterilized by autoclaving a suspension of fluticasone propionate type a nanocrystals in water for injection having a concentration of fluticasone 2% -20% w/w; preferably, the suspension of fluticasone propionate form a nanocrystals in water is autoclaved at about 122 ℃ for about 30 minutes.

Preferably, in step d-1) and step g-1), high shear, high speed mixing is performed at 6000 RPM.

Preferably, the high shear, high speed mixing of step g-1) is applied for at least 10 minutes.

Preferably, the concentration of fluticasone propionate in the sterile topical ophthalmic aqueous nanosuspension prepared according to the method of the invention is from 0.001% to 0.5% w/w; more preferably, the concentration of fluticasone propionate is 0.5%, 0.25%, 0.20%, 0.10%, 0.05%, 0.03%, 0.01% or 0.005% w/w; most preferably, 0.20% w/w or 0.10% w/w or 0.05% w/w.

Preferably, the fluticasone propionate type a nanocrystals used in the process of the present invention are prepared according to the general process reported below, which comprises the following steps:

1) preparing a phase I solution comprising: 0.45% w/w fluticasone propionate polymorph 1, 23, 2% w/w polyethylene glycol 400(PEG 400), 68.8% w/w polyethylene glycol 400(PPG 400) and 7.6% w/w polysorbate 80(Tween 80); and filtering the phase I solution through a 0.8/0.2 μm Polyethersulfone (PES) filter;

2) preparing a phase II solution comprising: 0.01% w/w benzalkonium chloride, 0.40% w/w methylcellulose 15cP, 0.1% w/w polyethylene glycol 40 stearate (PEG-40 stearate), citrate buffer to pH 3.4-3.8, and water q.s. to 100% w/w; and filtering the phase II solution through a 0.8/0.2 μm Polyethersulfone (PES) filter;

3) cooling the filtered phase I and phase II solutions to a temperature of 0-4 ℃;

4) mixing the phase I and phase II solutions in a reactor equipped with an ultrasonic transducer (e.g., QSonica Q2000 ultrasonic transducer) to give a phase III suspension of nanocrystals, wherein:

-continuously pumping the phase I solution and the phase II solution into the reactor at a flow rate of 600ml/min (phase I solution) and 2400ml/min (phase II solution), respectively, to obtain a phase III suspension;

-the volume ratio of phase I to phase II is 1: 4;

-applying sonication with 60% output power during mixing;

-the average temperature of the phase III suspension is about 11 ℃;

5) low shear mixing the phase III suspension of step 4) at room temperature in the absence of sonication for a minimum of 30 minutes at a speed sufficient to generate vortexing;

6) annealing (annealing) the phase II I suspension at 40 ℃ over a time period of not less than 16 hours;

7) preparing a buffer solution comprising: 0.2% w/w polyethylene glycol 40 stearate (PEG-40 stearate), 0.2% w/w polysorbate 80(Tween 80), 0.001% w/w benzalkonium chloride, 0.05% w/w sodium dihydrogen phosphate monohydrate, 0.02% w/w disodium hydrogen phosphate dihydrate and water in an amount to 100% w/w, having a pH of 6.3 ± 0.2; and filtering the buffer solution through a 0.8/0.2 μm Polyethersulfone (PES) filter;

8) diluting the phase III suspension of step 6) with filtered buffer solution, wherein the volume ratio of the buffer solution to the phase III is 1: 1;

9) centrifuging the diluted phase III suspension to recover fluticasone propionate nanocrystal a form and washing the recovered nanocrystals;

10) the collected nanocrystals were washed with water for injection.

When the method of the present invention is carried out under completely sterile manufacturing conditions, the washed nanocrystals of step 10) are sterilized prior to use in the preparation of a sterile topical ophthalmic aqueous nanosuspension; for example, the washed nanocrystals were suspended in water for injection such that the concentration of fluticasone propionate was 2% -20% w/w and autoclaved at about 122 ℃ for 30 minutes.

Another embodiment of the present invention relates to an ophthalmic aqueous nanosuspension which can be topically applied to the eyelids, eyelashes or eyelid margin, consisting of:

(a) 0.001% -1% w/w fluticasone propionate type a nanocrystals;

(b) 0.50% w/w methylcellulose 4000 cp;

(c) 0.2% w/w polysorbate 80;

(d) 0.10% w/w disodium edetate dihydrate;

(e) 1.0% w/w boric acid;

(f) 0.9% w/w glycerol;

(g) 0.01% w/w benzalkonium chloride;

(h) 0.055% w/w sodium chloride;

(i) hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to achieve a pH of 7.3-7.5; and

(j) the right amount of water is 100% w/w,

wherein the fluticasone propionate form a nanocrystals have an average particle size of 100nm to 1000nm and an X-ray powder diffraction pattern comprising peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ further comprising peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplate.

Preferably, the concentration of fluticasone propionate in the topical ophthalmic aqueous nanosuspension is 0.001% to 0.5% w/w, more preferably the concentration of fluticasone propionate is 0.5% w/w, 0.25% w/w, 0.20% w/w, 0.1% w/w, 0.05% w/w, 0.03% w/w, 0.01% w/w or 0.005% w/w. Most preferably, the concentration of fluticasone propionate in the topical ophthalmic aqueous nanosuspension is 0.1% w/w, 0.20% w/w or 0.05% w/w.

Another embodiment of the present invention relates to an ophthalmic aqueous nanosuspension which can be topically applied to the eyelids, eyelashes or eyelid margin, consisting of:

(a) 0.1% w/w or 0.5% w/w or 0.25% w/w or 0.20% w/w or 0.05% w/w fluticasone propionate form A nanocrystals;

(b) 0.50% w/w methylcellulose 4000 cp;

(c) 0.2% w/w polysorbate 80;

(d) 0.10% w/w disodium edetate dihydrate;

(e) 1.0% w/w boric acid;

(f) 0.9% w/w glycerol;

(g) 0.01% w/w benzalkonium chloride;

(h) 0.055% w/w sodium chloride;

(i) hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to achieve a pH of 7.3-7.5; and

(j) the proper amount of water is 100% w/w,

wherein the fluticasone propionate form A nanocrystals have an average particle size of 100nm to 1000nm and an X-ray powder diffraction pattern comprising peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 θ, further comprising peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis that is substantially perpendicular to a surface defining the thickness of the nanoplate.

Another preferred embodiment of the present invention relates to an ophthalmic aqueous nanosuspension which can be topically applied to the eyelids, eyelashes or eyelid margin and consists of:

(a) 0.1% w/w or 0.20% w/w or 0.05% w/w fluticasone propionate type a nanocrystals;

(b) 0.50% w/w methylcellulose 4000 cp;

(c) 0.2% w/w polysorbate 80;

(d) 0.10% w/w disodium edetate dihydrate;

(e) 1.0% w/w boric acid;

(f) 0.9% w/w glycerol;

(g) 0.01% w/w benzalkonium chloride;

(h) 0.055% w/w sodium chloride;

(i) hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to achieve a pH of 7.3-7.5; and

(j) the proper amount of water is 100% w/w,

wherein the fluticasone propionate form a nanocrystals have an average particle size of 100nm to 1000nm and an X-ray powder diffraction pattern comprising peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ further comprising peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplate.

Sterile topical ophthalmic aqueous nanosuspensions prepared according to the methods of the present invention have certain advantages such as better tolerability when applied to the eyelids (e.g., upper and lower eyelids), eyelashes, or eyelid margin due to the small particle size and maintenance of drug release over an extended period of time, thereby reducing the amount of active ingredient to be administered, and thus reducing systemic exposure to fluticasone propionate and other structures of the eye. As is well known, corticosteroids have side effects such as elevated intraocular pressure (IOP), increased corneal thickness, mydriasis, ptosis, cataracts, glaucoma, adrenal suppression, decreased bone mineral density; low systemic exposure to the whole eye is therefore an important advantage of ophthalmic aqueous nanosuspensions prepared according to the method of the invention, in particular for therapeutic applications requiring long-term or repetitive corticosteroid therapy.

The sterile topical ophthalmic aqueous nanosuspensions of the present invention have high efficacy and topical tolerability without the adverse side effects associated with systemic absorption of fluticasone propionate active ingredient.

In another embodiment, the nanosuspensions of the invention may be used in a method of treating or alleviating a symptom and/or clinical sign associated with an ocular inflammatory disease or ocular inflammatory disorder (e.g., blepharitis, posterior blepharitis, meibomian gland dysfunction, or dry eye disease) by topically administering the composition to the eyelid, eyelashes, or eyelid margin in a subject in need thereof. Such methods for treating blepharitis, posterior blepharitis, meibomian gland dysfunction, or dry eye disease comprise the step of topically applying to the eyelid, eyelashes, or eyelid margin of a subject an effective amount of a nanosuspension of the invention.

Another embodiment of the present invention provides a method of treating or alleviating the symptoms and/or clinical signs associated with blepharitis, posterior blepharitis, meibomian gland dysfunction, or dry eye disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition is an ophthalmic aqueous nanosuspension consisting of: 0.001% -1% w/w fluticasone propionate A-type nanocrystals, 0.50% w/w methylcellulose 4000cp, 0.2% w/w polysorbate 80, 0.10% w/w ethylenediaminetetraacetic acid disodium dihydrate, 1.0% w/w boric acid, 0.9% w/w glycerin, 0.01% w/w benzalkonium chloride, 0.055% w/w sodium chloride, hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to achieve a pH of 7.3-7.5, and a suitable amount of water to 100% w/w, wherein the fluticasone propionate A-type nanocrystals have an average particle size of 100nm-1000nm and an X-ray powder diffraction pattern including peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 θ, further including peaks at about 9.9, 13.0, 14.6, 16.0, 16.9, 18.9, and 3.34 degrees 2 θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplates, wherein the ophthalmic aqueous nanosuspension is topically applied to the upper and/or lower eyelid margin, meibomian gland ducts, eyelashes, or any region of the eyelid anatomy. Preferably, the ophthalmic aqueous nanosuspension used in the method of the invention comprises a concentration of fluticasone propionate type a nanocrystals of 0.5% w/w, 0.25% w/w, 0.20% w/w, 0.10% w/w, 0.05% w/w, 0.03% w/w, 0.01% w/w or 0.005% w/w; more preferably, the concentration of fluticasone propionate form A nanocrystals in the ophthalmic aqueous nanosuspension is 0.1%, 0.20%, 0.25%, 0.5%, 0.05% or 0.01% w/w; most preferably, the concentration of fluticasone propionate form A nanocrystals is 0.1% w/w, 0.20% w/w or 0.05% w/w.

Signs include eyelid debris, lid edge redness, eyelid swelling, meibomian gland obstruction, and/or qualitative/quantitative changes in meibomian gland secretion.

The most common symptoms associated with dry eye, also known as keratoconjunctivitis sicca, include dry eye, eye discomfort, redness, stinging, burning or stinging, watery eyes, sensitivity to light, blurred vision, pain, or eye fatigue.

Preferably, the methods of the present invention relate to methods for treating non-infectious, inflammatory blepharitis or meibomian gland dysfunction in a subject.

In another embodiment, the topical ophthalmic aqueous nanosuspension of the invention is administered to the subject at least 1 time a day, preferably, the pharmaceutical formulation is administered to the subject 1 time a day.

In another embodiment, the topical ophthalmic aqueous nanosuspension of the invention is administered to the subject at least 1 time daily for at least 2 weeks, more than 2 weeks, at least 3 weeks, or at least 4 weeks.

Another embodiment of the invention relates to a kit comprising: (a) a topically administrable ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals reported above; and (b) applying the nanosuspension to a swab or sponge wipe of the eyelid, eyelashes, or eyelid margin.

Examples

Percentages (%) as used herein in the compositions and examples refer to weight percentages (w/w) unless otherwise indicated. The terms glycerol (glycerin) and glycerol (glycerol) are used as synonyms herein and in the examples.

Example 1

Preparation of fluticasone propionate A-type nanocrystals

Preparation of phase I solution

In a 2L treatment vessel 106.26g polysorbate 80(Tween 80), 963.34g polypropylene glycol 400(PPG 400), 324.10g polyethylene glycol 400(PEG 400) were added at room temperature with stirring until all ingredients were dissolved, then 6.3g fluticasone propionate polymorph 1 was added to the solution with stirring until a clear solution was obtained. The resulting solution was filtered using a 0.8/0.2 μm Polyethersulfone (PES) filter and kept cold at 2-8 ℃ until use. Preparation of phase II solution

In an 8L treatment vessel, an initial amount of about 5276g of pure water was added and stirred with an overhead mixer, thereby generating a vortex.

6.01g of polyethylene glycol 40 stearate (PEG-40 stearate), 5.98g of benzalkonium chloride (10% solution) and 24.02g of methylcellulose 15cP were added and stirred until the methylcellulose was completely dissolved. Citrate buffer was added to adjust the pH to 3.5-4.0, and 585.8g of water were added. The final pH of the phase II solution was 3.83.

Phase II was then filtered using a 0.8/0.2 μm Polyethersulfone (PES) filter and kept cold at 2-8 ℃ until use.

Preparation of a dilution buffer

In a 20L treatment vessel, 12.0g of polysorbate 80(Tween 80) was introduced, 5374g of pure water was added, and stirring was performed with an overhead mixer to generate vortexes.

3.24g of sodium dihydrogen phosphate monohydrate, 1.14g of disodium hydrogen phosphate dihydrate, 12.00g of polyethylene glycol 40 stearate (PEG-40 stearate), 0.6g of benzalkonium chloride (10% solution) were added thereto, and stirred until completely dissolved, followed by addition of 597g of water. The final pH of the buffer solution was 6.4. Filtered using 0.8/0.2 μm Polyethersulfone (PES) filter and kept at 2-8 ℃ for refrigeration until use.

Preparation of fluticasone propionate A-type nanocrystals

480g of phase II were introduced into the reactor chamber. 40g of phase I and 3780g of phase II were cooled to a target temperature of 2-4 ℃ in a jacketed vessel connected to a condenser.

Using peristaltic calibration pumps, phase I and phase II (600 ml/min and 2400ml/min for phase I and phase II pump flow rates, respectively) were pumped through the reactor equipped with its ultrasonic transducers, with the amplitude set at 60% (Q Sonica Q1375W).

The effluent from the reactor (phase III: 5201.7g) was collected in a clean vessel at room temperature. The temperature of phase III was about 11 ℃.

Phase III was then stirred in a collection vessel at room temperature for about 30 minutes to give a homogeneous suspension of fluticasone propionate nanocrystals (final phase III).

Annealing method

The final phase III was transferred to a closed vessel. The container was placed in an incubator and maintained at 40 ℃ for at least 16 hours.

Purification and isolation of nanocrystals

5167g of annealed phase IH was transferred to a treatment vessel and an equal amount of dilution buffer was added so that the ratio of phase III to dilution buffer was 1: 1. The resulting mixture was stirred for 30 minutes using a low shear mixer to give a uniform suspension of nanocrystals (diluted phase III).

The diluted phase III was kept cold at 2-8 ℃ until centrifugation.

The fluticasone propionate form a nanocrystals are first collected by discontinuous centrifugation of the diluted phase III. The nanocrystals were then washed several times with water for injection (4 washing cycles).

The particle size of the isolated nanocrystals was evaluated using a laser scattering particle size distribution analyzer (Horiba LA-950). D ₅₀ 0.2153 μm, D ₉₀ 0.6073 μm.

The nanocrystals were characterized by XRPD and Rietveld refinement. The results are provided in figure 1(XRPD), which shows a typical pattern of fluticasone propionate form a with a strongly inclined crystal habit with the c-axis substantially perpendicular to the surface, as defined in WO 2013/169647, as evidenced by the polar diagram obtained from the Rietveld refinement process.

Example 2

Preparation of fluticasone propionate A type sterile topical ophthalmic aqueous nanosuspension

Step 1) preparation of Carrier 1 (Glycerol-free Carrier)

In a 20L treatment vessel 17600g of water for injection are heated at 80 ℃ and 100.0g of methylcellulose 4000cp are slowly added and the mixture is stirred until the methylcellulose dissolves.

The solution was cooled at 40 ℃, 200.0g of boric acid was added, and the pH was adjusted to 7.4 with sodium hydroxide (1N).

The following excipients were added in the following specific order: 20.0g disodium edetate dihydrate, 11.0g sodium chloride, 4.0g benzalkonium chloride (50% solution), 40.0g polysorbate 80(Tween 80); each excipient is completely dissolved before the next excipient is added and the solution is prepared at a temperature of about 40 c to room temperature. Testing the pH, optionally adjusting it to 7.3-7.5 with hydrochloric acid (1N) or sodium hydroxide (1N); after adjusting the pH, water for injection was added to reach a final weight of 19800 g. The resulting solution was mixed for at least 10 minutes to give a homogeneous solution, which was stored at 2-8 ℃.

Step 2) preparation of 2% fluticasone propionate nanocrystal slurry

Through PES 0.2 μm medium filtration, 4000g of carrier 1 was introduced into a 10L treatment vessel containing a stir bar, and left to stand.

In a 2L beaker, 15.04g fluticasone propionate form a nanocrystals were introduced with some carrier 1. The slurry was mixed well and the fluticasone propionate content (31.6mg/g) was determined and then further diluted with vehicle 1 to achieve a target concentration of 20mg/ml (2% w/w slurry) of fluticasone propionate. The concentrated slurry was subjected to high shear, high speed mixing at 6000. + -.10 RPM for 10 minutes and the particle size distribution was measured by laser diffraction on a Horiba LA-950S2 PSD analyzer.

The results are as follows:

step 3) preparation of Carrier 2 (Carrier containing Glycerol 1.8% w/w)

In a 4L beaker, 1600g of filtered support 1 were introduced and 36g of glycerol were added with stirring until dissolved.

The pH was tested, adjusted to 7.3-7.5 with sodium hydroxide (1N), and carrier 1 was further added to adjust the final weight to 2000g, resulting in a 1.8% w/w glycerol solution.

Step 4) preparation of 1% fluticasone propionate nanocrystal slurry

A slurry of 1% fluticasone propionate nanocrystals was prepared by further diluting the 2% concentrated slurry prepared in step 2) with carrier 2 to a concentration of 1% w/w fluticasone propionate.

A1% w/w slurry of fluticasone propionate form A nanocrystals was subjected to high shear, high speed mixing at 6000. + -.10 RPM for 10 minutes and the particle size distribution was tested by laser diffraction using a Horiba LA-950S2 PSD analyzer.

The results are as follows:

aliquots of the 1% concentrated slurry were filled into 500ml glass bottles containing a stir bar and sterilized by autoclaving at 121.5 ℃ for 40 minutes.

Step 5) preparation of Carrier 3 (Carrier containing Glycerol 0.9% w/w)

In a 20L vessel, 11200g of vehicle 1 was introduced, 126g of glycerol was added with stirring, then the pH was adjusted to 7.3-7.5 with sodium hydroxide and an aliquot of vehicle 1 was added to adjust the final weight to 14000g, yielding a 0.9% w/w glycerol solution. The solution was mixed for at least 10 minutes until homogeneous and sterile filtered into another receiving vessel.

Step 6) preparation of 0.1% w/w fluticasone propionate nanosuspension

The contents of the vial containing the autoclaved sterile 1% w/w fluticasone propionate nanocrystals of step 4) were aseptically transferred in an ISO 5 environment and pooled into the final mixing vessel.

The weight of the 1% fluticasone propionate nanocrystal slurry transferred to the container was recorded (1366.3 g).

A final 0.1% sterile fluticasone propionate nanocrystal suspension was obtained by adding 11774.2g of sterile vehicle 3.

The final sterile nanosuspension was stirred on a stir plate for no more than 15 minutes.

Particle size distribution was measured by laser diffraction using a Horiba LA-950S2 PSD analyzer.

The results were as follows:

the nanosuspension of the invention comprising fluticasone propionate form a at different concentrations, e.g. 0.5% w/w, 0.25% w/w, 0.20% w/w, 0.1% w/w, 0.05% w/w, 0.03% w/w, 0.01% w/w and 0.005% w/w, was obtained by diluting the depolymerised 1% fluticasone propionate nanosuspension of step 4) with an aliquot of the aqueous carrier 3 as disclosed in step 6) to obtain a final fluticasone propionate concentration.

Example 3

Stability evaluation of nanosuspensions of example 2

The nanosuspension compositions prepared in example 2 were tested for storage stability by storing the nanosuspensions under three different conditions of temperature and humidity (5 ℃, 25 ℃/40% RH; 40 ℃/25% RH). The nanosuspension was evaluated for resuspendability, fluticasone propionate content, particle size distribution, and benzalkonium chloride content at 1 month and 3 month time points.

The results reported in tables 2 and 3 indicate that the nanosuspensions are both physically and chemically stable during manufacture and storage. No change in the physical appearance of the nanosuspension was noted on storage. The nanosuspension did not show any sign of chemical degradation, as the chemical analysis of fluticasone propionate on storage was well within the 90% -110% limit required by the label. The related substances and total impurities are kept within the specified limit of not more than 4% during storage.

Example 4 (comparative example)

The results of this study indicate that nanosuspensions of fluticasone propionate type a nanocrystals comprising boric acid, but no glycerol, are unstable and aggregate.

Fluticasone propionate form a nanocrystals are suspended in a vehicle containing exactly the same concentrations of boric acid and methylcellulose as in example 2 above, but without glycerol.

The composition of the nanosuspensions tested is reported in table 4.

Preparation of fluticasone propionate A-type nanocrystal suspension

Suspending a quantity of fluticasone propionate A-type nanocrystals in a carrier aliquot consisting of: 0.50% w/w methylcellulose 4000cp, 0.2% w/w polysorbate 80, 0.10% w/w disodium edetate dihydrate, 1.0% w/w boric acid; 0.01% w/w benzalkonium chloride and an appropriate amount to 100% w/w water (see Table 4) to give the target concentration of fluticasone propionate (see Table 4). Once the nanocrystals were suspended, the suspension was poured into a 500mL glass beaker and depolymerized using a high speed, high shear Silverson mixing device. The suspension was mixed at 6000RPM until the particle size distribution quality criteria were met. The particle size distribution and viscosity of the nanosuspension were measured.

When stable, the nanosuspension was placed at 40 ℃ and the particle size distribution (PDS) was determined at a 2 week time point.

The results reported in tables 5a and 5b show that the average particle size and D are only within a short period of two weeks ₉₀ There was an increase in both, reflecting aggregate formation in the nanosuspension; in contrast, the stability results reported in example 3 show that the nanosuspensions prepared according to the method of the invention are stable for up to 3 months under accelerated conditions.

Example 5 (comparative example)

The results of this study demonstrate that addition of fluticasone propionate type a nanocrystals to a vehicle containing a preformed boric acid/glycerol complex resulted in residual aggregation, a phenomenon not observed when nanosuspensions were prepared according to the method of the present invention in which glycerol was added to a slurry of depolymerised 2% fluticasone propionate nanocrystals under high speed high shear mixing (see example 2-step 4).

Fluticasone propionate form a nanocrystals were suspended in a suspension containing a preformed boronic acid/glycerol complex (boronic acid 1.0% w/w/glycerol 0.25% w/w and boronic acid 1.0% w/w/glycerol 1.0% w/w) and depolymerized therein to give fluticasone propionate at a final concentration of 0.25% w/w.

The vehicle compositions of the two tested nanosuspensions are reported in table 6.

Preparation of fluticasone propionate A type nanocrystal nano suspension

Fluticasone propionate form a nanocrystals were suspended in two vehicles containing preformed boronic acid/glycerol complexes reported in table 6 and stirred overnight on a magnetic stir plate using a stir bar. Once the nanocrystals were suspended, the suspension was poured into a 500mL glass beaker for depolymerization using a high speed, high shear Silverson mixing device. The suspension was mixed at 6000RPM until the particle size distribution quality criteria were met.

Both formulations were held at 40 ℃ for stability testing and the particle size distribution was determined for samples taken at the 1 week time point. Samples were analyzed shortly after removal from the stability chamber. The results showed substantial aggregation in the sample (see figures 2 and 3).

Upon refrigeration of the samples, aggregation was partially reversible, but still with substantial residual levels of aggregation (see FIG. 4); this thermally reversible aggregation is associated with a decrease in the solubility of methylcellulose upon an increase in temperature. It is well known that even though methylcellulose solutions visually clearly show any detectable particles in the range of 30-50 ℃, as the temperature increases, the polymer chains form loosely associated clusters that grow in size. These clusters may cause partial aggregation of the nanocrystals at 40 ℃.

As reported above, the results of this study demonstrate that glycerol acts as a stabilizer by forming a complex with boric acid that prevents the isolated nanocrystals from aggregating. If a nanosuspension is prepared by suspending the nanocrystals in a vehicle comprising the preformed complex boric acid/glycerol, the stabilizing effect of the complex will be reduced.

Example 6

In vitro dissolution test

The dissolution rate of fluticasone propionate type a nanocrystals of the nanosuspension of the invention was evaluated and compared to the dissolution rate of a standard fluticasone propionate form 1 micronized material (reference sample).

Dissolution profiles of two nanosuspensions of the fluticasone propionate form a and micronized fluticasone propionate form 1 of the present invention were achieved using a dissolution method.

The particle size of the fluticasone propionate type a nanocrystals was 0.434 μm (D50-median), while the particle size of the fluticasone propionate form 1 micronized material was 4.64 μm (D50-median).

The study was performed using compartment diffusion analysis through dialysis membrane and immersion conditions. The immersion conditions were achieved by using an acceptor fluid containing 30mM phosphate buffer pH 7.4 and 5% HP β CD cyclodextrin, and by completely exchanging the buffer every 24 hours to stay below the saturation point. Under those experimental conditions, the saturation point was estimated to be 5 μ g/g and the measurements made and the buffer replacement showed a maximum concentration of 1.5 μ g/g. The study was carried out at a temperature of 37 ℃. The details of the dissolution test method are as follows:

a 5-fold dilution with two formulation vehicles (placebo solutions containing all formulation excipients including surfactant) of 0.1% w/w fluticasone propionate type a nanosuspension resulted in a final concentration of 0.02% w/w fluticasone propionate in the dialysis setup.

Micronized fluticasone propionate form 1 was suspended in the same phosphate buffer used in the receiving chamber of the dialysis system.

Release of fluticasone propionate was performed at 1-2 RPM.

1mL sampling aliquots were removed at predetermined time intervals (1, 3, 5, 20, 24, 48, 72 hours) and replaced with an equal volume of dissolution media to maintain a constant total volume of 39mL in a 50mL tube.

These aliquots were immediately centrifuged and measured by HPLC.

Reports in tables 7-9The results of (a) indicate that the dissolution profiles of the fluticasone propionate form a nanocrystals and the fluticasone propionate form 1 micronized material are similar, despite the two test and reference samples having different particle sizes. More specifically, the particle size of the fluticasone propionate form a nanocrystals (D50 ═ 0.434 μm) is larger than the micronized fluticasone propionate form 1 (D) ₅₀ 4.64 μm) is 10 times smaller.

Since the solubility of compounds is generally intrinsically linked to particle size, as particles become smaller, the surface area to volume ratio increases, leading to greater interaction with solvents and hence increased solubility, the results of this study show that the unique properties of the present ophthalmic aqueous fluticasone propionate form nanocrystals, namely small particle size (nanoparticles) on the one hand improves comfort and tolerability of ophthalmic formulations and on the other hand slows the dissolution rate of the fluticasone propionate active ingredient, thereby avoiding rapid and high absorption of fluticasone propionate, which is associated with the undesirable side effects associated with systemic absorption of steroids.

Example 7

14-day repeat dose study of topical ophthalmic aqueous fluticasone propionate type a nanocrystal suspensions in beagle dogs

The objective of this study was to evaluate the toxicity kinetics of fluticasone propionate type a nanosuspensions of the present invention administered by topical direct application to the upper and lower lid margins (rims) of both beagle dogs' eyes (see table 10 below).

Method

At the beginning of the study, fifty naive beagle dogs (25 males, 25 females) aged about 5-6 months and having male and female body weights of 5.7-8.8kg were divided into treatment groups (1-4 groups) and vehicle groups.

Male and female beagle dogs were dosed via an eyelid applicator with fluticasone propionate administered at 1.6, 9.6 and 32 μ g/day (bilateral QD topical application) or 64 μ g/day (bilateral BID topical application) respectively for 14 consecutive days either 1 or 2 times per day (minimum 6 hours interval between doses) directly to the upper and lower eyelids of both eyes.

Blood for pharmacokinetic evaluation was collected from all animals at selected time points on day 1 and day 14.

Ophthalmologic examinations were performed before the start of treatment, during the first and second week of dosing, and during the last week of recovery. The eyes were scored once daily according to the modified Draize scale.

And evaluating the reversible action of the fluticasone propionate nanosuspension according to the 14-day recovery period.

Results and conclusions

The group mean plasma pharmacokinetic parameters are summarized in table 11 below.

The results indicate that exposure to fluticasone propionate is dose dependent with increasing dose. There is no clear evidence of accumulation in male and female animals. No significant gender-related differences were exposed. In a 14-day inhalation toxicity study, the systemic exposure (AUC) at the lowest adverse effect level associated with the corticosteroid-related findings received by dogs was 14-fold and 9-fold higher than that observed in dogs at the ocular dose of 0.1% QD and BID in the 14-day topical ocular toxicity study (Advair-Diskuss-NDA-021077), respectively.

There was no evidence of local or systemic toxicity.

Example 8

Efficacy and safety evaluation of fluticasone propionate A-type nano-suspension in treatment of acute exacerbation of blepharitis

The objective of this study was to compare the efficacy and safety of the fluticasone propionate type a nanosuspension of the invention with aqueous ophthalmic formulations of placebo in reducing the signs and symptoms of blepharitis subjects.

Test preparation (FP-A type-NS hereinafter)

0.1% w/w fluticasone propionate type a nanocrystals (average particle size 100nm-1000nm), 0.50% w/w methylcellulose 4000cp, 0.2% w/w polysorbate 80, 0.10% w/w disodium edetate dihydrate, 1.0% w/w boric acid, 0.9% w/w glycerol, 0.01% w/w benzalkonium chloride, 0.055% w/w sodium chloride, hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to reach pH 7.3-7.5, and an amount of water to 100% w/w.

Placebo preparation

0.50% w/w methylcellulose 4000cp, 0.2% w/w polysorbate 80, 0.10% w/w disodium edetate dihydrate, 1.0% w/w boric acid, 0.9% w/w glycerol, 0.01% w/w benzalkonium chloride, 0.055% w/w sodium chloride, hydrochloric acid (1N) and/or sodium hydroxide (1N) as a regulator in an amount sufficient to achieve a pH of 7.3-7.5, and a suitable amount to 100% w/w water.

Design of research

This was a phase 2 multicenter, randomized, double masked, placebo controlled study that evaluated the safety and efficacy of treatment of blepharitis symptoms and signs with 0.1% fluticasone propionate type a nanosuspension 1 time per day.

The target population for this study was adult males and females with a history of blepharitis, who experienced acute blepharitis exacerbations, defined as the lowest score of "1" for each of lid margin redness, lid debris, and lid discomfort in both eyes at screening and baseline visits (on a 4-point scale). A total of 15 subjects were included in the study. The subjects were between 55 and 80 years of age, with a mean age of 70.8 years.

In three clinical sites in the united states, 15 patients were randomly grouped. 10 patients received FP-type A-NS and 5 patients received placebo 1 time per day for both eyes in the evening.

The study visits were as follows: screening (days-7 to-3), baseline/day 1, day 4 (. + -. 1), day 8 (. + -. 1), day 11 (. + -. 1), day 14 (. + -. 1; last day of treatment) and day 28/withdrawal (. + -. 2; follow-up).

Test nanosuspensions (16 μ g fluticasone propionate per eye) or placebo were administered 1 time daily in the evening. Study drug is self-administered by the subject.

Efficacy and safety evaluation of fluticasone propionate A-type nano-suspension

The efficacy and safety of fluticasone propionate type ｃA nanosuspension (FP-type ｃA-NS) was compared to placebo at each visit of the administration during the study. In addition to the signs and symptoms of blepharitis, the present study also evaluated the characteristic signs and symptoms of dry eye, which are also commonly observed in subjects with blepharitis.

Efficacy of

The study results reported in tables 12 and 13 indicate that fluticasone propionate type a nanosuspension treatment consistently improved the signs and symptoms of patients with blepharitis, while the overall score for ocular debris, redness, and discomfort was reduced in subjects treated with fluticasone propionate type a nanosuspension both from baseline and compared to placebo-treated subjects.

The results presented in tables 12 and 13 are related to signs and symptoms of blepharitis in the study eye assessed 14 days after treatment and performed prior to the daily eyelid scrub procedure.

Subjects also evaluated the following dry eye symptoms using the Visual Analog Scale (VAS): dry eyes, burning-irritation, foreign body sensation, itching, photophobia, pain and blurred vision. The composite VAS scores reported in table 14 (the average of the individual scores for these symptoms) indicate that fluticasone propionate type a nanosuspension treatment is effective in relieving dry eye symptoms relative to placebo.

Safety feature

Treatment with the fluticasone propionate type a nanosuspension of the invention was very well tolerated and all patients completed the treatment. No serious adverse reactions (SAE) occurred, and no clinically relevant changes in intraocular pressure were observed in the subjects, especially during the treatment period or up to 2 weeks after treatment discontinuation.

The research result shows that the fluticasone propionate A-type nanometer suspension can relieve signs and symptoms of blepharitis and dry eye, and the treatment and application method has safety tolerance.

Example 9

Stability evaluation of nanosuspensions of example 2

The nanosuspension compositions prepared in example 2 were tested for storage stability by storing the nanosuspensions under three different conditions of temperature and humidity (5 ℃, 25 ℃/40% RH; 40 ℃/25% RH). Nanosuspension resuspendability, fluticasone propionate content, particle size distribution and benzalkonium chloride content were evaluated at 1 month and 3 month time points, at 40 ℃ for a 5 month time point (table 15), at 5 ℃ and 25 ℃ (table 16) for a time point of up to 12 months (table 16) (see results reported in example 3 and tables 2 and 3).

The results reported in tables 15 and 16 indicate that the nanosuspensions are both physically and chemically stable upon storage. No change in the physical appearance of the nanosuspension was noted on storage. The nanosuspension did not show any sign of chemical degradation, as the results of the chemical analysis of fluticasone propionate were well within the 90% -110% limit required for the label on storage. The related substances and total impurities are kept within a prescribed limit of not more than 4% upon storage.

The particle size distribution was analyzed using a Horiba LA-950 instrument.

Claims (24)

1. A method for preparing a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000nm and fluticasone propionate at a concentration of 0.001% w/w to 1% w/w, the method comprising:

a) preparing an aqueous carrier 1 comprising: 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium ethylenediaminetetraacetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5 and water to 100% w/w;

b) mixing an amount of fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000nm with an aqueous carrier 1 to obtain a slurry comprising fluticasone propionate at a concentration of 2% w/w;

c) applying high shear, high speed mixing to the slurry of step b) for at least 10 minutes;

d) preparing an aqueous carrier 2 comprising: 1.8% w/w glycerol, 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and a suitable amount to 100% w/w water;

e) adding an aliquot of aqueous carrier 2 to the slurry of step c) to give fluticasone propionate at a concentration of 1% w/w;

f) applying high shear, high speed mixing to the slurry of step e) until a target average particle size is obtained;

g) sterilizing the nanosuspension of step f) by autoclaving;

h) preparing an aqueous carrier 3 comprising: 0.9% w/w glycerol, 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH between 7.3-7.5, and water to 100% w/w; and sterilizing the aqueous carrier 3 by filtration;

i) aseptically adding an aliquot of sterile aqueous carrier 3 to the sterilized nanosuspension of step g) to prepare a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate nanocrystal a at the target concentration;

wherein the fluticasone propionate form a nanocrystals have an X-ray powder diffraction pattern of the nanocrystals comprising peaks at 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ, further comprising peaks at 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis that is substantially perpendicular to a surface defining a thickness of the nanoplate.

2. The method according to claim 1, wherein the concentration of fluticasone propionate in the final sterile topical ophthalmic aqueous nanosuspension is from 0.001% to 0.5% w/w.

3. The method according to claim 1, wherein the concentration of fluticasone propionate in the final sterile topical ophthalmic aqueous nanosuspension is 0.5%, 0.25%, 0.2%, 0.1%, 0.05%, 0.03%, 0.01% or 0.005% w/w.

4. A process according to any one of claims 1 to 3 wherein the high shear, high speed mixing of steps c) and f) is carried out at 6000 RPM.

5. The method of claim 4 wherein in step f), the high shear, high speed mixing is applied for at least 10 minutes.

6. A method according to any one of claims 1 to 5, wherein the aqueous carriers 1 and 2 are filtered through a 0.2 μm filter before their use.

7. The method according to any one of claims 1-6, wherein the sterilization of step g) is performed by autoclaving the nanosuspension of step f) at 122 ℃ for 40 minutes.

8. A method for preparing a sterile topical ophthalmic aqueous nanosuspension comprising fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000nm and a fluticasone propionate concentration of 0.001% w/w to 1% w/w, the method comprising:

a-1) sterilizing fluticasone propionate type a nanocrystals having an average particle size of 100nm to 1000 nm;

b-1) preparing an aqueous carrier 1 comprising: 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust the pH at 7.3-7.5, and an amount of water to 100% w/w; and sterilizing the aqueous carrier 1 by filtration;

c-1) aseptically mixing an amount of sterilized fluticasone propionate nanocrystal a with an amount of sterilized aqueous carrier 1 to obtain a slurry comprising fluticasone propionate at a concentration of 2% w/w;

d-1) applying high shear, high speed mixing to the slurry of step c-1) for at least 10 minutes;

e-1) preparing an aqueous carrier 2 comprising: 1.8% w/w glycerol, 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium edetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide to adjust pH at 7.3-7.5, and a suitable amount to 100% w/w water; and sterilizing the aqueous carrier 2 by filtration;

f-1) adding an aliquot of the sterilized aqueous carrier 2 to the slurry of step d-1) in a sterile manner to obtain fluticasone propionate at a concentration of 1% w/w;

g-1) applying high shear and high speed mixing to the slurry obtained in the step f-1) until a target average particle size is obtained;

h-1) preparing an aqueous carrier 3 comprising: 0.9% w/w glycerol, 0.5% w/w methylcellulose 4000cP, 1.0% w/w boric acid, 0.1% w/w disodium ethylenediaminetetraacetate dihydrate, 0.055% w/w sodium chloride, 0.01% w/w benzalkonium chloride, 0.2% w/w polysorbate 80, 1N hydrochloric acid and/or 1N sodium hydroxide adjusted to pH 7.3-7.5, and water to 100% w/w; and sterilizing the aqueous carrier 3 by filtration;

i-1) aseptically adding an aliquot of the sterilized aqueous vehicle 3 to the nanosuspension of step g-1) to prepare a sterile topical ophthalmic aqueous nanosuspension comprising the fluticasone propionate nanocrystal form a at final concentration;

wherein the fluticasone propionate form a nanocrystals have an X-ray powder diffraction pattern comprising peaks at 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ, further comprising peaks at 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplate.

9. The method according to claim 8, wherein the concentration of fluticasone propionate in the final sterile topical ophthalmic aqueous nanosuspension is from 0.001% to 0.5% w/w.

10. The method according to claim 8, wherein the concentration of fluticasone propionate in the final sterile topical ophthalmic aqueous nanosuspension is 0.5%, 0.25%, 0.2%, 0.1%, 0.05%, 0.03%, 0.01% or 0.005% w/w.

11. A method according to any one of claims 8 to 10, wherein in step a-1) the fluticasone propionate type a nanocrystals are sterilized by autoclaving a suspension of fluticasone propionate type a nanocrystals in water for injection having a concentration of 2% to 20% w/w fluticasone propionate.

12. A method according to claim 11, wherein the suspension of fluticasone propionate form a nanocrystals in water is autoclaved for 30 minutes at 122 ℃.

13. A method according to any one of claims 8 to 12, wherein high shear, high speed mixing is carried out at 6000RPM in step d-1) and step g-1).

14. The method according to claim 13, wherein in step g-1), the high shear, high speed mixing is applied for at least 10 minutes.

15. A process according to any one of claims 1 to 14, wherein fluticasone propionate nanocrystal form a is prepared according to the following steps:

1) preparing a phase I solution comprising: 0.45% w/w fluticasone propionate polymorph 1, 23, 2% w/w polyethylene glycol 400(PEG 400), 68.8% w/w polypropylene glycol 400(PPG 400), 7.6% w/w polysorbate 80(Tween 80); and filtering the phase I solution through a 0.8/0.2 μm Polyethersulfone (PES) filter;

2) preparing a phase II solution comprising: 0.01% w/w benzalkonium chloride, 0.40% (w/w) methylcellulose 15cP, 0.1% w/w polyethylene glycol 40 stearate (PEG-40 stearate), citrate buffer to pH 3.4-3.8, and a suitable amount to 100% w/w water; and filtering the phase II solution through a 0.8/0.2 μm Polyethersulfone (PES) filter;

3) cooling the filtered phase I and phase II solutions to a temperature of 0-4 ℃;

4) mixing the phase I and phase II solutions in a reactor equipped with an ultrasonic transducer (QSonica Q2000 ultrasonic transducer) to obtain a phase III suspension of nanocrystals, wherein:

-continuously pumping the phase I solution and the phase II solution into the reactor at a flow rate of 600ml/min (phase I solution) and 2400ml/min (phase II solution), respectively, to obtain a phase III suspension;

-the volume ratio of phase I to phase II is 1: 4;

-applying sonication with 60% output power during mixing;

-the mean temperature of the phase HI suspension is 11 ℃; and

5) low shear mixing the phase III suspension of step 4) at room temperature in the absence of sonication for a minimum of 30 minutes at a speed sufficient to generate vortexing;

6) annealing the phase III suspension at 40 ℃ for a time period of not less than 16 hours;

7) preparing a buffer solution comprising: 0.2% w/w polyethylene glycol 40 stearate (PEG-40 stearate), 0.2% w/w polysorbate 80(Tween 80), 0.001% w/w benzalkonium chloride, 0.05% w/w sodium dihydrogen phosphate monohydrate, 0.02% w/w disodium hydrogen phosphate dihydrate, and a moderate amount of water to 100% w/w, having a pH of 6.3 ± 0.2; and the buffer solution was filtered through a 0.8/0.2 μm Polyethersulfone (PES) filter;

8) diluting the phase III suspension of step 6) with filtered buffer solution, wherein the volume ratio of buffer solution to phase HI is 1: 1;

9) centrifuging the diluted phase III suspension to recover fluticasone propionate nanocrystal a form and washing the recovered nanocrystals;

10) the collected nanocrystals were washed with water for injection.

16. An ophthalmic aqueous nanosuspension that can be topically applied to the eyelids, eyelashes, and eyelid margin, comprising (a) 0.001% -1% w/w fluticasone propionate type a nanocrystals and a carrier consisting of:

(b) 0.50% w/w methylcellulose 4000 cP;

(c) 0.2% w/w polysorbate 80;

(d) 0.10% w/w disodium edetate dihydrate;

(e) 1.0% w/w boric acid;

(f) 0.9% w/w glycerol;

(g) 0.01% w/w benzalkonium chloride;

(h) 0.055% w/w sodium chloride;

(i) 1N hydrochloric acid and/or 1N sodium hydroxide as a regulator in an amount sufficient to achieve a pH of 7.3-7.5; and

(j) the proper amount of water is 100% w/w,

wherein the fluticasone propionate form a nanocrystals have an average particle size of 100nm to 1000nm and an X-ray powder diffraction pattern comprising peaks at 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees 2 Θ, further comprising peaks at 9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2 Θ, and wherein the nanocrystals are nanoplates having a [001] crystallographic axis substantially perpendicular to a surface defining the thickness of the nanoplate, and wherein the ophthalmic aqueous nanosuspension comprises a boric acid/glycerol complex.

17. An ophthalmic aqueous nanosuspension according to claim 16, wherein the boric acid/glycerol complex is formed by addition of glycerol after depolymerization of fluticasone propionate nanocrystals suspended in a carrier comprising boric acid but no glycerol.

18. An ophthalmic aqueous nanosuspension according to claim 16 or 17, wherein the concentration of fluticasone propionate is from 0.001% to 0.5% w/w.

19. An ophthalmic aqueous nanosuspension according to claim 16 or 17, wherein the concentration of fluticasone propionate is 0.5% w/w, 0.25% w/w, 0.2% w/w, 0.1% w/w, 0.05% w/w, 0.03% w/w, 0.01% w/w or 0.005% w/w.

20. An ophthalmic aqueous nanosuspension according to claim 16 or 17, wherein the concentration of fluticasone propionate is 0.2% w/w or 0.1% w/w or 0.05% w/w.

21. An ophthalmic aqueous nanosuspension according to any one of claims 16 to 20, for use in a method of treating blepharitis, posterior blepharitis, meibomian gland dysfunction and dry eye, wherein the method comprises topically applying an effective amount of the ophthalmic aqueous nanosuspension to the eyelids, eyelashes and eyelid margin.

22. An ophthalmic aqueous nanosuspension according to any one of claims 16 to 20, for use in a method of alleviating symptoms and/or clinical signs associated with blepharitis, posterior blepharitis, meibomian gland dysfunction and dry eye disease, wherein the method comprises topically applying an effective amount of the ophthalmic aqueous nanosuspension to the eyelids, eyelashes and eyelid margin.

23. An ophthalmic aqueous nanosuspension according to claim 16 or 17, for use in a method according to claim 21 or 22, wherein the ophthalmic aqueous nanosuspension comprises (a) 0.1% w/w fluticasone propionate type a nanocrystals.

24. An ophthalmic aqueous nanosuspension for use in the method according to any one of claims 21 to 23, wherein the method comprises topically applying the ophthalmic aqueous nanosuspension to the eyelids, eyelashes and eyelid margin at least 1 time per day for at least 2 weeks.