FIELD OF THE INVENTION
[0001] The present invention relates in general to the field of drug formulations and more
specifically to controlled release microspheres of fluvastatin and a method of preparation thereof based on response surface methodology.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding
the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Fluvastatin, a statin drug, is used for treating hypercholesterolemia and in
preventing cardiovascular disease. The drug was approved for medical use in 1994. Fluvastatin as a fluvastatin sodium salt (FSS) is used in the treatment of hypercholesterolemia including hyperlipidemia, hyperlipoproteinemia, or hypertriglyceridemia by administering an oral dosage of 20 to 40 mg once daily which can be titrated up to 40 mg twice daily or switched to extended-release Fluvastatin sodium 80 mg once daily. In patients with coronary heart disease, fluvastatin sodium 40 mg twice daily has been demonstrated for effective prevention. However, side effects of FSS include abdominal pain or cramps, blurred vision, nausea, indigestion, insomnia, headaches, dizziness, itching, muscle cramps, rash, yellowing of skin or eyes, and sometimes myalgia or rhabdomyolysis. To avoid the adverse effects to the best possible extent, varied formulations of FSS have been investigated in the art.
[0004] Fluvastatin sodium as extended release formulations and as microspheres has been
synthesized to reduce adverse affects. However, such formulations generally comprise non-biodegradable polymers and other inactive substances such as potassium bicarbonate, povidone, benzyl alcohol, black iron oxide, butylparaben, carboxymethylcellulose sodium, edetate calcium disodium, methylparaben, propylparaben, silicon dioxide, sodium lauryl sulfate, sodium propionate, calcium carbonate, magnesium stearate, red iron oxide, sodium bicarbonate, talc,

titanium dioxide, yellow iron oxide, and other ingredients which are sometimes known to trigger
adverse effects.
[0005] Gelatin microspheres are often used in preparing controlled release formulations as
they are considered to be naturally biocompatible and are low cost polymers. However, gelatin
undergoes degradation as it is a rapidly solubilizable product. To improve its structural integrity
in formulations, cross linkers such as glutaraldehyde and formaldehyde are often used in the
preparation of gelatin microspheres. The use of such cross-linkers in the gelatin formulations
may in turn lead to toxicity and irritation.
[0006] Fluvastatin sodium microcapsules in the art are also manufactured by complexing
the active ingredient with anionic exchange cholestyramine resin Indion-454 and by coating with
ethyl cellulose for achieving controlled release in small intestine. The most often used ethyl
cellulose is Eudragit-RSlOO, a non-biodegradable polymer.
[0007] Further, despite the existence of numerous encapsulated compositions for providing
an extended or controlled release of fluvastatin, the maximum time period achieved by such
formulations is about 24 hours.
[0008] Thus, there is a need to provide controlled release formulations of fluvastatin that
overcomes the deficiencies of the prior art, can provide controlled-release of fluvastatin for a
prolonged duration of time, is non-toxic, has good consistency and diffusion, better tolerance and
bioavailability, and is easy-to-manufacture and cost-effective.
OBJECTS OF THE INVENTION
[0009] An object of the present invention is to provide optimized controlled release
microspheres of fluvastatin or a salt thereof having good entrapment efficiency of fluvastatin
sodium, excellent bioavailability, and exhibits a stable and controlled release of fluvastatin
sodium for a very long duration of time with reduced adverse effects.
[0010] Another object of the present invention is to provide a process for preparing
microspheres of fluvastatin or a salt thereof that can surmount the problems faced in the current
art in the preparation of fluvastatin sodium microspheres in a cost effective manner coupled with
optimal yield by the use of design and optimization tools.
[0011] Another object of the present invention is to provide a process for preparing
optimized microspheres of fluvastatin or a salt thereof having high drug entrapment, excellent

bioavailability over a prolonged duration of time, good consistency and diffusion, better tolerance, reduced adverse effects, and is easy-to-formulate.
SUMMARY OF THE INVENTION
[0012] This summary is provided to introduce a selection of concepts in a simplified form
that are further described below in Detailed Description section. This summary is not intended to
identify key features or essential features of the claimed subject matter, nor is it intended to be
used as an aid in determining the scope of the claimed subject matter.
[0013] The present invention relates to a controlled-release microsphere composition
comprising fluvastatin or a salt thereof having a bilayer shell structure comprising an outer
polymer layer and an inner layer, and a core comprising the fluvastatin or a salt thereof, wherein
the microspheres have a mean size diameter in a range from 100 um to 500 um; and wherein, the
drug to polymer ratio is in the range of 1:1 to 1:5.
[0014] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the shell outer polymer layer is selected from a group
comprising chitosan, starch, poly (lactic-co-glycolic acid), cellulose esters, cellulose acetate,
cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose
propionate butyrate, and combinations thereof.
[0015] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the shell outer polymer is present in an amount ranging from
50%) to 90%o by weight of the composition.
[0016] Another embodiment of the present invention relates to a controlled-release
microsphere composition, wherein the shell inner layer is gelatin or starch, present in an amount
ranging from 8%> to 15% weight of the composition, more preferably in the range from 10%> to
13%o weight of the composition and most preferably in the range from 12.5% to 12.75 % weight
of the composition.
[0017] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the fluvastatin salt is fluvastatin sodium present in a range
from 15%o to 25% by weight of the composition and preferably at 20% by weight of the
composition.

[0018] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the shell outer polymer layer is cellulose acetate butyrate in an amount ranging from 40% to 80% weight of the composition, the shell inner layer is gelatin in the range from 12.5% to 12.75 % weight of the composition and fluvastatin sodium is in a concentration of 20% weight of the composition, wherein the mean size diameter is in a range from 200 um to 250 um; and wherein the ratio of fluvastatin sodium to cellulose acetate butyrate is 1:2.
[0019] Another embodiment of the present invention relates to a controlled-release
microsphere composition comprising one or more pharmaceutically acceptable carriers.
[0020] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the microspheres are optimized for entrapment efficiency, mean size diameter and yield percent by response surface methodology wherein the first independent variable is gelatin in weight percent, the second independent variable is the weight /weight ratio of the drug to the polymer and the third independent variable is the stirring speed of the centrifuge in rpm units.
[0021] Another embodiment of the present invention relates to a controlled-release
microsphere composition wherein the drug is released for an extended period of up to 72 hours.
[0022] Another aspect of the present invention relates to a process for preparing controlled
release microspheres of fluvastatin or a salt thereof comprising the steps of:
(a) mixing gelatin with water at 45°C to obtain an aqueous gelatin solution;
(b) preparing a first emulsifying medium by dissolving fluvastatin or a salt thereof,
cellulose acetate butyrate, and a primary emulsifier in dichloromethane at 40°C and stirring the
solution at 1500 rpm for 20 to 40 minutes;
(c) preparing a second emulsifying medium by dissolving cellulose acetate butyrate and a
secondary emulsifier in dichloromethane at 45°C;
(d) adding the aqueous gelatin solution of step (a) in a drop-wise manner to the first
emulsifying medium of step (b) to obtain microspheres;
(e) transferring the microspheres of step (d) to the second emulsifying medium of step (c) for a period of 10 minutes at 37°C;
(f) hardening the microspheres of step (e) by slowly adding a non-solvent; and

(g) filtering the hardened microspheres of step (f) using an ultra-filtration mesh size
of 0.45 urn to 0.22 urn to obtain the controlled release microspheres of fluvastatin sodium.
[0023] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the concentration of
gelatin solution is in the range from 8% to 15% gelatin, more preferably in the range from 10%
to 13%) gelatin, and most preferably in the range from 12.5% to 12.75 % gelatin.
[0024] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the fluvastatin salt is
fluvastatin sodium present in a concentration range from 15% to 25% by weight of the
composition and preferably in a concentration of 20% by weight of the composition.
[0025] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the concentration of cellulose acetate butyrate in the first emulsifying medium is in the range of 25% to 50% weight of the composition and in the second emulsifying medium is in the range of 15% to 30% weight of the composition.
[0026] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the primary emulsifier is poloxamer-407 and the secondary emulsifier is d-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS).
[0027] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the stirring speed of the centrifuge is in the range of 500-3000 rpm, more preferably in the range of 1000 to 2000 rpm and most preferably at 1500 rpm.
[0028] Another embodiment of the present invention relates to a process for preparing
controlled release microspheres of fluvastatin or a salt thereof, wherein the non-solvent is
selected from a group comprising petroleum ether, acetone, and n-hexane.
[0029] Yet another aspect of the invention relates to cellulose acetate butyrate controlled
release microspheres of fluvastatin sodium having excellent tolerance, enhanced bioavailability and therapeutic efficacy, good consistency and diffusion.

BRIEF DESCRIPTION OF DRAWINGS THE INVENTION
[0030] The following drawings form part of the present specification and are included to
further illustrate aspects of the present disclosure. The disclosure may be better understood by
reference to the drawings in combination with the detailed description of the specific
embodiments presented herein.
Figure 1 is a photomicrograph of a fluvastatin controlled-release microsphere prepared
according to an embodiment of the present invention.
Figure 2 is a Contour plot (2D) showing the effect of independent variables on % EE (Yl) of
CR-FSS according to an embodiment of the present invention.
Figure 3 is a Response surface plot (3D) showing the effect of independent variables on % EE
(Yl) of CR-FSS according to an embodiment of the present invention.
Figure 4 is a Contour plot (2D) showing the effect of independent variables on mean diameter
(um) (Y2) of CR-FSS according to an embodiment of the present invention.
Figure 5 is a Response surface plot (3D) showing the effect of independent variables on mean
diameter (um) (Y2) of CR-FSS according to an embodiment of the present invention.
Figure 6 is a Contour plot (2D) showing the effect of independent variables on % yield (Y3) of
CR-FSS according to an embodiment of the present invention.
Figure 7 is a Response surface plot (3D) showing the effect of independent variables on % yield
(Y3) of CR-FSS according to an embodiment of the present invention.
Figure 8 is a Contour plot (2D) showing desirability value for optimized CR-FSS according to
an embodiment of the present invention.
Figure 9 is a Response surface plot (3D) showing desirability value for optimized CR-FSS
according to an embodiment of the present invention.
Figure 10 is a graph illustrating the burst and extended release of the fluvastatin from CR-FSS
according to an embodiment of the present invention.
DETAILED DESCRIPTION
[0031] The following is a detailed description of embodiments of the disclosure. The
description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. Although each embodiment represents a single combination of

inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0032] Various terms as used herein are shown below. To the extent a term used in a claim
is not defined below, it should be given the broadest definition persons in the pertinent art have
given that term as reflected in printed publications and issued patents at the time of filing. Where
a definition or use of a term in an incorporated reference is inconsistent or contrary to the
definition of that term provided herein, the definition of that term provided herein applies and the
definition of that term in the reference does not apply. The use of any and all examples, or
exemplar language (e.g. "such as") provided with respect to certain embodiments herein is
intended merely to better illuminate the invention and does not pose a limitation on the scope of
the invention otherwise claimed. No language in the specification should be construed as
indicating any non-claimed element essential to the practice of the invention.
[0033] As used in the description herein and throughout the claims that follow, the
meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.
[0034] Unless the context requires otherwise, throughout the specification which follow,
the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense that is as "including, but not limited to."
[0035] As used herein, the term "Biocompatible1: the polymers, solvents and other agents
of the invention must be biocompatible; that is they must not cause irritation or necrosis in the

environment of use. The environment of use is a fluid environment and may comprise a
subcutaneous or intramuscular portion or body cavity of a human or animal.
[0036] As used herein, the term 'microsphere' means a 'matrix microsphere' in which
active ingredient particles are dispersed in direct contact with the polymer matrix. Such a
microsphere is a homogeneous or monolithic particle in which the drug is dissolved or dispersed
throughout the polymer matrix. Release of drugs from a microsphere is a mass transport
phenomenon involving diffusion of drug molecules from the region of high concentration in the
dosage form to a region of low concentration in the surrounding environment.
[0037] As used herein, the term 'emulsify' means to form a stable dispersion of one liquid
in a second immiscible liquid. An 'immiscible' liquid is a liquid which is not soluble in another
substance or liquid, for example, oil in water. In contrast, two substances that are mutually
soluble in all proportions are said to be miscible.
[0038] As used herein, the term 'microsphere composition' is used interchangeably with
the term 'microsphere'. The term 'microsphere composition' also refers to the make-up of the
microsphere which is a composition comprising components, preferably one or more polymers,
drug components, solubilizers and the like.
[0039] The controlled-release microspheres of the present invention are a composition
comprising fluvastatin or a salt thereof and one or more polymers. In an embodiment, the
controlled-release microspheres of fluvastatin or a salt thereof have a bilayer shell structure
comprising an outer polymer layer and an inner layer, and a core comprising the fluvastatin or a
salt thereof.
[0040] In another embodiment of the present invention, the microspheres comprise an
outer layer. Preferably, the outer layer comprises any one or more of chitosan, starch, poly
(lactic-co-glycolic acid) and cellulose esters. Cellulose esters include, but are not limited to
cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate
phthalate, cellulose propionate butyrate, and combinations thereof. In another embodiment, the
polymer of the outer layer is present in an amount ranging from 50% to 90% by weight of the
composition. In a preferred embodiment, the shell outer polymer layer is cellulose acetate
butyrate in an amount ranging from 40% to 80% by weight of the composition.
[0041] In an embodiment of the present invention, the shell inner layer of the
microspheres comprises gelatin or starch. In an embodiment of the present invention, the gelatin

is preferably in the weight percent ranging from 8% to 15% by weight of the composition, more preferably in the range from 10% to 13% by weight of the composition and most preferably in the range from 12.5% to 12.75 % by weight of the composition.
[0042] In an embodiment of the present invention, the microspheres have a core
comprising the active component or drug dispersed in a polymeric matrix. Preferably, the drug includes, but is not limited to, HMG-CoA reductase inhibitors such as atorvastatin, simvastatin, rosuvastatin, pitavastatin, pravastatin, lovastatin, fluvastatin, cerivastatin and salts thereof. In an embodiment, the fluvastatin salt is a sodium salt and the controlled-release microspheres of fluvastatin sodium are also referred to as CR-FSS. Preferably, fluvastatin sodium is present in an amount ranging from 15% to 25% by weight of the composition and more preferably in a constant concentration of 20% by weight of the composition.
[0043] In an embodiment of the present invention, the ratio of the drug to polymer in the
microspheres ranges from 1:1 to 1:5. Preferably, the drug to polymer ratio is 1: 2.
[0044] In an embodiment of the present invention, the mean diameter size of the
microspheres is in a range from 100 um to 500 um. Preferably, the mean diameter size of the microspheres is in a range from 180 um to 320 um and most preferably, the mean diameter size is in a range from 200 um to 250 um. The mean diameter and surface morphology of controlled-release microspheres of fluvastatin sodium were estimated by microscopy method of particle size determination and FIGURE 1 is a photomicrograph of a controlled-release microsphere of fluvastatin sodium according to an embodiment of the present invention.
[0045] In an exemplary embodiment, the microspheres of the present invention comprise
shell outer polymer layer of cellulose acetate butyrate in an amount ranging from 40% to 80% by
weight of the composition, the shell inner layer of gelatin in the range from 12.5% to 12.75 %
weight of the composition and core of fluvastatin sodium in a constant amount of 20% by weight
of the composition, wherein the microspheres have a mean size diameter in a range from 200 um
to 250 um; and wherein the ratio of fluvastatin sodium to cellulose acetate butyrate is 1: 2.
[0046] In an embodiment of the present invention, the controlled-release microspheres of
fluvastatin sodium are optimized for good consistency, good diffusion and enhanced bioavailability. Fluvastatin sodium is a known BCS class II drug having poor aqueous solubility. The controlled-release microspheres of fluvastatin sodium produce initial burst drug release for 1 hour and successive controlled drug release till 72 hours which is almost three times that existent

in the art (FIGURE 10). As the controlled-release microspheres exhibit slow and controlled drug release, formulations comprising these microspheres also exhibit better tolerance in comparison to conventional formulations. In an embodiment of the present invention, the formulations comprising the controlled-release microspheres of fluvastatin sodium exhibit enhanced bioavailability attributed to the smaller particle size of microspheres. Due to the small size of the microspheres, the formulation exhibits higher solubility of the drug resulting in enhanced bioavailability leading to increased therapeutic efficacy of fluvastatin.
[0047] In another embodiment of the present invention, pharmaceutical formulations
comprising controlled-release microspheres of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polylactic acid, polyglycolic acid, polyethylene glycol, Sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, sucrose, lactose and wool fat.
[0048] In another aspect of the invention, there is provided a process for the preparation of
controlled release microspheres of fluvastatin sodium using response surface methodology. Preferably the microspheres of the present invention are optimized using response surface methodology and then prepared based on the optimization results.
[0049] In an embodiment of the present invention, the optimization of the microspheres
using response surface methodology is carried out using the Box-behnken design run on Design-Expert software (Trial Version 11.1.2.0, Stat-Ease Inc., MN). Independent and response variables for fabrication of CR-FSS and 17 batches box-behnken design (BBD) layout are provided in Table 1 and 2, respectively.
[0050] In an embodiment of the present invention, the first independent variable is gelatin
component in weight percent, the second independent variable is the weight /weight ratio of the drug to the polymer and the third independent variable is the stirring speed of the centrifuge in

rpm. These three variables were selected by the inventors for optimizing controlled release
microspheres.
[0051] In an embodiment of the present invention, the first independent variable (A)
gelatin is preferably in the weight percent ranging from 8% to 15%, more preferably in the range
from 10% to 13% and most preferably in the range from 12.5% to 12.75 %.
[0052] In another embodiment of the present invention, the second independent variable
(B) drug to polymer ratio is preferably in the range of 1:1 to 1:5. Preferably, the drug to polymer
ratio is at 1: 2 for a cellulose acetate butyrate (CAB) polymer.
[0053] In another embodiment of the present invention, the third independent variable (C)
Stirring Speed (rpm) is preferably in the range of 500 rpm to 3000 rpm, more preferably in the
range of 1000 to 2000 rpm. Preferably, the stirring speed is about 1500 rpm.
[0054] In a preferred embodiment of the present invention, the BBD model suggested
independent variables for an optimized layout for CR-FSS as gelatin at 12.565%), drug: CAB at a
ratio of 1:2 and a stirring speed of 1500 rpm.
[0055] Optimization is execution of systematic approaches to accomplish the preeminent
amalgamation of product and/or process characteristics under a given set of conditions. Design
of experiments (DOE) is a comprehensive organized methodology to manufacturing that
employs theories and designs at data compilation phase in order to acquire substantiate,
justifiable, and endurable production inferences. Furthermore, this requires least expenditure of
production batches, time, and money. Experimental designs are advantageous in acquiring
significance of numerous factors influencing the properties of formulation. DOE encompass
statistical and diagnostic regression analysis succeeded by response surface analysis.
[0056] In an embodiment of the present invention, the microspheres for controlled release
of fluvastatin sodium (CR-FSS) were successfully optimized as per box-behnken design (BBD)
layout and prepared based on the results. The optimized microspheres were analyzed for three
specific response parameters, namely percent entrapment efficiency or %> EE (Yl), mean
diameter in um (Y2) and yield percent (Y3). FIGURE 2 and FIGURE 3 is the contour plot (2D)
and response surface plot (3D), respectively, showing the effect of independent variables on %>
EE (Yl) of CR-FSS according to an embodiment of the present invention. FIGURE 4 and
FIGURE 5 is the contour plot (2D) and response surface plot (3D), respectively, showing the
effect of independent variables on mean diameter (um) (Y2) of CR-FSS according to an

embodiment of the present invention. FIGURE 6 and FIGURE 7 is the contour plot (2D) and
response surface plot (3D), respectively, showing the effect of independent variables on percent
yield (Y3) of CR-FSS microspheres according to an embodiment of the present invention.
[0057] In a preferred embodiment, the optimization by response surface method for
controlled release microspheres of fluvastatin sodium having entrapment efficiency 82.094% (Yl), mean diameter 220.659 um (Y2), and yield 66.046% (Y3) required independent variables of (XI) gelatin at 12.565%), (X2) drug: polymer ratio of 1:2, and (X3) stirring speed of centrifuge at 1500 rpm. In an embodiment, the optimization of response variables and independent variables generated the highest overall desirability function D of 0.785, a shown in Table 4. FIGURE 8 and FIGURE 9 is a contour plot (2D) and Response surface plot (3D), respectively, showing desirability value for optimized CR-FSS according to an embodiment of the present invention.
[0058] In an aspect of the present invention, a process for preparing controlled release
microspheres of fluvastatin or a salt thereof comprises: (a) mixing gelatin with water at 45°C to obtain an aqueous gelatin solution; (b) preparing a first emulsifying medium by dissolving fluvastatin or a salt thereof, cellulose acetate butyrate, and a primary emulsifier in dichloromethane at 40°C and stirring the solution at 1500 rpm for 20 to 40 minutes; (c) preparing a second emulsifying medium by dissolving cellulose acetate butyrate and a secondary emulsifier in dichloromethane at 44°C; (d) obtaining microspheres by adding the aqueous gelatin solution of step (a) in a drop-wise manner to the first emulsifying medium of step (b); (e) transferring the microspheres of step (d) to the second emulsifying medium of step (c) for a period of 10 minutes at 37°C; (f) hardening the microspheres of step (e) by slowly adding a non-solvent; and (g) filtering the hardened microspheres of step (f) using an ultra-filtration mesh to obtain the controlled release microspheres of fluvastatin sodium.
[0059] In an embodiment, the concentration of gelatin solution is in the range from 8%> to
15%o gelatin, more preferably in the range from 10%> to 13%> gelatin and most preferably in the range from 12.5% to 12.75 % gelatin.
[0060] In an embodiment, the fluvastatin salt is a sodium salt. Preferably, the concentration
of fluvastatin sodium is in the range from 40%> to 80%> by weight of the composition.

[0061] In an embodiment, the concentration of cellulose acetate butyrate in the first
emulsifying medium is in the range of 25% to 50% weight of the composition and in the second
emulsifying medium is in the range of 15% to 30% weight of the composition.
[0062] In an embodiment of the present invention, the primary emulsifier is poloxamer-407
and the secondary emulsifier is d-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS).
Preferably, the emulsifiers help fabricate microspheres having very small size, preferably in the
range of 100 to 500 um.
[0063] In an embodiment of the present invention, the stirring speed of the centrifuge is in
the range of 500 rpm to 3000 rpm, more preferably in the range of 1000 to 2000 rpm and most
preferably at about 1500 rpm.
[0064] In an embodiment of the present invention, the non-solvent is selected from a group
comprising petroleum ether, acetone, and n-hexane. Preferably, the non-solvent is n-hexane.
[0065] In an embodiment of the present invention, the hardened microspheres are collected
by filtering the solution of step (f) through an ultra filtration having a mesh size of 200 to 450
nm.
[0066] In a preferred embodiment, the optimized layout obtained by response surface
methodology for obtaining high entrapment efficiency by the microspheres, high percent yield of
the microspheres and optimum mean size diameter of controlled release microspheres of
fluvastatin sodium has gelatin at 12.565%), the ratio of drug to CAB polymer at 1:2 and the
centrifuge stirring speed of 1500 rpm. In a preferred embodiment, the entrapment efficiency of
optimized microspheres was 82.094%) (Yl), mean size diameter was 220.659 um (Y2), and yield
was 66.046%) (Y3) as seen in Table 4.
[0067] As shown in FIGURE 10, the optimized CR-FSS prepared according to an
embodiment of the present invention comprising 20 mg of the active ingredient exhibited a
controlled release in-vitro drug release profile for a very long duration. Preferably, the profile
exhibits an initial burst release of fluvastatin in the first hour followed by an extended release of
fluvastatin over remaining time. Not wishing to be bound by theory, it is the inventor's belief
that the successful controlled release of the drug occurs due to swelling of hydrophilic core. The
bilayer polymeric coating of gelatin, a natural biodegradable polymer, and cellulose acetate
butyrate, a biocompatible and biodegradable polymer, use of emulsifiers for stability of
fluvastatin, the very low mean size diameter of the microspheres all together contribute to the

advantageous drug release profile of CR-FSS. In an embodiment, the drug release profile
comprises an initial burst drug release in the first hour followed by a controlled drug release up
until 72 hours.
[0068] While the foregoing describes various embodiments of the disclosure, other and
further embodiments of the disclosure may be devised without departing from the basic scope
thereof. The scope of the invention is determined by the claims that follow. The invention is not
limited to the described embodiments, versions or examples, which are included to enable a
person having ordinary skill in the art to make and use the invention when combined with
information and knowledge available to the person having ordinary skill in the art.
Example 1: Designing controlled release microspheres of fluvastatin sodium (CR-FSS) by
response surface methodology
[0069] The Box-Behnken Design (BBD) layout was employed for designing the
independent variables and the response variables. The box-behnken design response surface
methodology was employed using Design-Expert software (Trial Version 11.1.2.0, Stat-Ease
Inc., MN). Table 1 below lists out the independent and response variables employed for
preparing CR-FSS.
Table 1 provides the Independent and response variables for CR-FSS in BBD Design-Expert
software

Independent variables -1 (Low) 0 (Medium) +1 (High)
A = Gelatin (%) 5 10 15
B=D:P(w/w) 1:2 1:3 1:4
C = Stirring Speed (rpm) 500 1000 1500
Response variables Constraint Importance
Yl = % EE Maximize

Y2 = Mean Diameter (urn) Minimize

Y3 = % Yield Maximize

[0070] The D:P ratio refers to the drug: polymer ratio wherein the drug is fluvastatin
sodium and the polymer is cellulose acetate butyrate or CAB. Different batches of BBD layout

were obtained based on the independent variables as presented in Table 1. A total of 17 different layouts obtained by the BBD are presented in Table 2.
Table 2 provides the different layouts obtained by the Box-behnken design (BBD)

Std A
Gelatin
(%) B D:P
(w/w) c
Stirring Speed (rpm)
1 -1 -1 0
2 -1 0
3 -1 1 0
4 1 0
5 -1 0 -1
6 0 -1
7 -1 0 1
8 0 1
9 0 -1 -1
10 0 1 -1
11 0 -1 1
12 0 1 1
13 0 0 0
14 0 0 0
15 0 0 0
16 0 0 0
17 0 0 0
Example 2: Optimization of controlled release microspheres of fluvastatin sodium (CR-FSS) by
response surface methodology
Evaluation of entrapment efficacy of CR-FSS microspheres
[0071] Percent (%) Entrapment efficiency (Yl) was determined by analyzing amount of
un-entrapped drug in supernatant recovered after centrifugation (Remi, RIS-24 BL, Mumbai,
India) of CR-FSS at 5,000 rpm for 10 minutes. Analysis was performed by double beam UV-

visible spectrophotometer (Systronics AU-2701, Ahmedabad, India) at 300 nm. The following formula (Equation 1) was used to determine entrapment efficiency (% EE):
n , __ n , , Total amount of druE-Total amount of un-?nlrapp =d druE ,,-,,-,
% EE. % W/W = -^- — = *100
Total amount DI druE
[0072] Second order polynomial model generated for % EE (Yl) by Design-Expert
software (Trial Version 11.1.2.0, Stat-Ease Inc., MN) through multiple regression analysis was done using Equation 3 provided below:
% EE = 70.86 + 1.36A - 7.33 B + 0.4637 C - 0.8975 AB - 0.0475AC + 0.5850 BC -3.67A2 + 2.14B2+ 1.72 C2 Evaluation of mean diameter o/CR-FSS microspheres
[0073] Mean diameters (Y2) of CR-FSS was determined using APCAM USB2 digital
cameras system (APCAM, India). Measurement was executed in triplicate (n = 3) to obtain mean diameter.
[0074] Second order polynomial model generated for mean diameter (Y2) by Design-
Expert software (Trial Version 11.1.2.0, Stat-Ease Inc., MN) through multiple regression analysis was done using Equation 4 provided below:
Mean Diameter (urn) = 283.01 + 6.29 A + 27.02 B - 30.98 C + 4.89 AB + 5.54
AC+ 15.2 BC- 31.34 A2 + 27.72 B2 - 12.17 C2 Evaluation of percent yield of CR-FSS microspheres
[0075] Percent yield (Y3) was calculated as weight percentage of completely dried
microspheres recovered from each experimental, with respect to initial total weight of FSS, CAB and gelatin using following equation (2):
n , __ , , , Total wsiEht of dri=d microsphacis - -,-
% Yl eld. W, W = =; — ——, X10 0
Initial weight ordrug and polymer
[0076] Second order polynomial model generated for % yield (Y3) by Design-Expert
software (Trial Version 11.1.2.0, Stat-Ease Inc., MN) through multiple regression analysis was done using Equation 5 provided below:
% Yield = 71.65 + 1.37 A + 7.91 B + 0.3687 C + 0.7725 AB - 0.0425 AC - 0.11 BC-3.93 A2+1.31B2+ 1.26 C2 Diagnostic analysis of response parameters (Y1-Y3)

[0077] Negative and positive sign before coefficients of factors A, B and C indicated
antagonistic and synergistic effects on response parameters of CR-FSS. Smaller values of residuals i.e. difference between actual and predicted values of response parameters validated authenticity of response parameters. Table 3 provides Actual, predicted and residuals of response parameters of Y1-Y3

Run Yl Y2 Y3
Order AV PV Residual AV PV Residual AV PV Residual
1 73.44 74.41 -0.96 254.32 250.97 3.35 60.93 60.53 0.39
2 79.34 78.92 0.41 252.89 253.78 -0.88 62.58 61.72 0.85
3 61.12 61.54 -0.41 296.12 295.23 0.88 73.95 74.81 -0.85
4 63.43 62.46 0.96 314.25 317.60 -3.35 78.69 79.09 -0.39
5 67.36 67.04 0.32 268.84 269.72 -0.87 66.76 67.21 -0.45
6 68.79 69.85 -1.06 274.59 271.23 3.36 69.12 70.03 -0.91
7 69.12 68.06 1.06 193.34 196.70 -3.36 68.94 68.03 0.91
8 70.36 70.68 -0.32 221.23 220.35 0.87 71.13 70.68 0.45
9 82.81 82.17 0.64 315.27 317.74 -2.47 65.89 65.84 0.05
10 66.43 66.33 0.09 341.32 341.33 -0.01 83.18 81.88 1.30
11 81.83 81.92 -0.09 225.35 225.34 0.01 65.49 66.80 -1.31
12 67.79 68.43 -0.64 312.29 309.82 2.47 82.34 82.39 -0.05
13 70.33 70.86 -0.52 280.79 283.01 -2.22 71.23 71.65 -0.42
14 71.17 70.86 0.31 283.39 283.01 0.37 70.47 71.65 -1.18
15 70.11 70.86 -0.74 281.39 283.01 -1.62 71.23 71.65 -0.42
16 71.36 70.86 0.50 284.78 283.01 1.77 74.56 71.65 2.91
17 71.32 70.86 0.46 284.71 283.01 1.70 70.78 71.65 -0.87
AV-Actual value; PV- Predicted value
[0078] Application of QbD using Design-Expert software suggests optimized parameters
for preparing CR-FSS as provided below in Table 4. Desirability function analyses performance index of the output responses by obtaining the composite desirability. If a response falls within the unacceptable intervals, the desirability is 0, and if a response falls within the ideal intervals or the response reaches its ideal value, the desirability is 1.
[0079] Optimal values of formulation and process variables generating the highest overall
desirability function D = 0.785 were obtained via numerical optimization process. The composition and process variables of the software-suggested optimized CR-FSS was 12.565%

gelatin (XI), 1:2 D: P ratio (X2), and 1500 rpm stirring speed (X3) for producing CR-FSS having entrapment efficiency 82.094% (Yl), mean diameter 220.659 um (Y2), and yield 66.046% (Y3) which had the highest desirability function D = 0.785.
Table 4 provides Optimized CR-FSS as per the Design-expert® 11.1.2.0 software

Independent variables Criteria Importance Value (Desirability)
Xl= Gelatin (%) In range +++ 12.565
X2 = D:P(w/w) In range +++ 1:2
X3 = Stirring Speed (rpm) In range +++ 1500
Response variables
Yl = % EE Maximize 82.094

Y2 = Mean Diameter (um) Minimize 220.659

Y3 = % Yield Maximize 66.046 0.785

[0080] Figure 8 and Figure 9 show the counter plot and the corresponding response surface
plot for the overall desirability coefficient as a function of the change in gelatin (XI) and D:P (X2) quantities.
Example 3: Preparation of optimized controlled release microspheres of fluvastatin sodium (CR-FSS)
[0081] An aqueous gelatin solution was prepared by mixing 3.141 gm gelatin to 25 mL
water at 45°C. The concentration of this solution was 12.56%) gelatin. A first emulsifying medium was prepared by dissolving 400 mg Fluvastatin sodium, 500 mg cellulose acetate butyrate (CAB), and 1ml poloxamer-407 in 50 mL dichloromethane at 40°C and the mixture stirred at 1500 rpm for 20-40 minutes. A second emulsifying medium was then prepared by dissolving 300 mg CAB and 100 mg d-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS) in 100 ml dichloromethane at 45°C.
[0082] The aqueous gelatin solution was then added to the first emulsifying medium in a
drop-wise manner to obtain microspheres. These microspheres were then transferred to the second emulsifying medium and left therein for 10 minutes at 37°C. After 10 mins in the second emulsifying medium, the microspheres were hardened by the slow addition of 50 ml of n-hexane.

The hardened microspheres were then collected by filtering the solution through ultra-filtration
mesh size of 0.45 urn Millipore filter. The total amount of Fluvastatin sodium used herein was
400 mg and the total amount of CAB used herein was 800 mg leading to a drug: polymer ratio of
1:2.
Example 4: In-vitro release studies of optimized CR-FSS
[0083] The CR-FSS as optimized by the Design-Expert software and prepared
accordingly using the emulsification technique of Example 3 was evaluated for in-vitro
controlled drug release profile.
[0084] In hydrochloric acid buffer pH 1.2, the microspheres of the present invention were
kept initially for 2 h, maintained at 100 rpm and 37±0.5°C. Samples were withdrawn periodically
and reloaded with equivalent volume of dissolution medium. Quantification was performed at
300 nm through UV spectrophotometer.
[0085] This was followed by keeping the microspheres of the present invention in
phosphate buffer of pH 7.4 until the completion of dissolution process using pre-treated dialysis
membrane (Himedia, India) having molecular weight cut-off (MWCO) of -12,000-14,000 Da.
Dialysis bag containing CR-FSS equivalent to 80 mg FSS was dipped in 100 ml of phosphate
buffer and the flask was placed in shaking incubator maintained at 100 rpm and 37±0.5°C.
[0086] Samples were withdrawn periodically and reloaded with equivalent volume of
dissolution medium. Quantification was performed at 300 nm through UV spectrophotometer. It
was found that 20 mg of FSS was burst-released in initial 1 h and successive controlled drug
release till 72 hours due to swelling of hydrophilic core as confirmed from change in physical
state of microspheres product (Figure 10).
[0087] This drug release profile clearly shows that the CR-FSS obtained according to an
embodiment of the present invention could be successfully utilized in treatment of
hypercholesterolemia with reduced side effects.
ADVANTAGES OF THE PRESENT INVENTION
[0088] The present invention provides optimized controlled release microspheres of
fluvastatin sodium (CR-FSS) that is novel and overcomes the deficiencies associated with the existing art as it provides controlled release of the drug for an extended period of time of

resulting in enhanced bioavailability of the drug and completely eliminates the need for toxic
cross linkers.
[0089] The optimized microspheres of the present invention prepared by response surface
methodology exhibit entrapment efficacy of over 80%, yield percent of more than 65% and an
in-vitro release profile of the drug as a burst-release in the first hour followed by an extended
release for over 72 hours.
[0090] The present invention provides novel non-toxic cellulose acetate butyrate (CAB)
and gelatin controlled release (CR) microspheres of fluvastatin sodium (CR-FSS) that surmount
the drawback of rapid solubilisation of gelatin microspheres in aqueous environment by the use
safe, stable, and hydrophobic CAB uniform coating in the outer shell layer.

We Claim

1.A controlled-release microsphere composition comprising fluvastatin or a salt thereof having a bilayer shell structure comprising an outer polymer layer and an inner layer, and a core comprising the fluvastatin or a salt thereof, wherein the microspheres have a mean size diameter in a range from 100 um to 500 um; and wherein, the drug to polymer ratio is in the range of 1:1 to 1:5.
2. The microsphere composition as claimed in claim 1, wherein the shell outer polymer layer is selected from a group comprising chitosan, starch, poly (lactic-co-glycolic acid), cellulose esters, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose propionate butyrate, and combinations thereof.
3. The microsphere composition as claimed in claim 1, wherein the shell outer polymer is present in an amount ranging from 50% to 90% by weight of the composition and preferably in the range of 40%) to 80%) by weight of the composition.
4. The microsphere composition as claimed in claim 1, wherein the shell inner layer is gelatin or starch, present in an amount ranging from 8%> to 15% by weight of the composition, more preferably in the range from 10%> to 13%> by weight of the composition and most preferably in the range from 12.5% to 12.75 % by weight of the composition.
5. The microsphere composition as claimed in claim 1, wherein the fluvastatin salt is fluvastatin sodium in an amount ranging from 15% to 25% by weight of the composition and preferably at a concentration of 20% by weight of the composition.
6. The microsphere composition as claimed in claim 1, wherein the shell outer polymer layer is cellulose acetate butyrate in an amount ranging from 40% to 80% by weight of the composition, the shell inner layer is gelatin in the range from 12.5% to 12.75 % by weight of the composition and fluvastatin sodium is in an amount of 20% by weight of the composition, wherein the mean size diameter is in a range from 200 um to 250 um; and wherein the ratio of fluvastatin sodium to cellulose acetate butyrate is 1: 2.
7. The microsphere composition as claimed in claim 1 comprising one or more pharmaceutically acceptable carriers.
8. The microsphere composition as claimed in claim 1, wherein the microspheres are optimized for entrapment efficiency, mean size diameter and yield percent by response surface

methodology wherein the first independent variable is gelatin in weight percent, the second independent variable is the weight /weight ratio of the drug to the polymer and the third independent variable is the stirring speed of the centrifuge in rpm units.
9. The microsphere composition as claimed in claim 1, wherein the drug is released for an
extended period of up to 72 hours.
10. A process for preparing controlled release microspheres of fluvastatin or a salt thereof as
claimed in claim 1 comprising the steps of:
(a) mixing gelatin with water at 45°C to obtain an aqueous gelatin solution;
(b) preparing a first emulsifying medium by dissolving fluvastatin or a salt thereof, cellulose acetate butyrate, and a primary emulsifier in dichloromethane at 40°C and stirring the solution at 1500 rpm for 20 to 40 minutes;
(c) preparing a second emulsifying medium by dissolving cellulose acetate butyrate and a secondary emulsifier in dichloromethane at 45°C;
(d) adding the aqueous gelatin solution of step (a) in a drop-wise manner to the first emulsifying medium of step (b) to obtain microspheres;
(e) transferring the microspheres of step (d) to the second emulsifying medium of step (c) for a period of 10 minutes at 37°C;
(f) hardening the microspheres of step (e) by slowly adding a non-solvent; and
(g) filtering the hardened microspheres of step (f) using an ultra-filtration mesh size of 0.45 urn to 0.22 urn to obtain the controlled release microspheres of fluvastatin sodium.

11. The process as claimed in claim 1, wherein the concentration of gelatin solution is in the range from 8%> to 15% gelatin, more preferably in the range from 10%> to 13%> gelatin, and most preferably in the range from 12.5% to 12.75 % gelatin.
12. The process as claimed in claim 1, wherein the fluvastatin salt is fluvastatin sodium in an amount ranging from 15% to 25% by weight of the composition and preferably at 20% by weight of the composition.
13. The process as claimed in claim 1, wherein the concentration of cellulose acetate butyrate in the first emulsifying medium is in the range of 25% to 50% by weight of the composition and in the second emulsifying medium is in the range of 15% to 30% by weight of the composition.
14. The process as claimed in claim 1, wherein the primary emulsifier is poloxamer-407 and the secondary emulsifier is d-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS).

15. The process as claimed in claim 1, wherein the stirring speed of the centrifuge is in the range
of 500-3000 rpm, more preferably in the range of 1000 to 2000 rpm and most preferably at 1500
rpm.
16. The process as claimed in claim 1, wherein the non-solvent is selected from a group
comprising petroleum ether, acetone, and n-hexane.