Note: Descriptions are shown in the official language in which they were submitted.
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h'~I~.MZJLA°TI~1~ T~ I~T~EI~ ATE AI~°TIMICIZ~BIAL 1)~IJG
I'~'TEl~'I° AGAINST
~I~GAT~TTI~IdIS I~~T~~MALL~ CGI~~II~E~E~
T~ l~~ l~I~TAI'~TT T~ TFIE 11~~~IJ~
CROSS REFERENCE TO RELATED APPLICATIONS:
This application claims priority from provisional application Ser. No.
60/466,354, filed
on April 29, 2003.
BACKGROUND OF THE INVENTION:
Technical Field
The present invention relates to compositions of antimicrobial agents. More
particularly
the invention relates to formulations of an antimicrobial agent which render
the drug potent
against organisms normally considered to be resistant to the agent.
Background of the Invention
Based upon in vitro microbicidal sensitivity tests, the level of an
antimicrobial drug
considered effective against a particular organism may be determined. This is
referred to as the
MIC (minimum iuubitory concentration) of the drug. On the other hand, safety
studies will
determine the amount of drug that can be safely given to a patient or test
animal. This maximal
amount of drug that can be dosed will determine the maximal biological
exposure to the host
animal, normally measured by the area under the curve (AUC) of the plot of
drug concentration
vs. time, the peak height of the plot of drug concentration vs. time, tissue
levels vs. time, etc.
The instantaneous tissue or plasma level of the in vivo experiment can be
compared with the
MIC value to determine relative efficacy of the attainable drug levels in the
biological fluids.
The actual comparison must be corrected for plasma protein binding, inasmuch
as only the free
drug level is the important parameter because it is in this state that the
drug is freely diffusible to
cross biological membranes.
As a result of such analysis, clinical literature has been established
specifying what drugs
can be used generally for certain strains of organisms, or more precisely, for
certain strains of
organisms with MIC values below certain levels. As an example, the antifungal
agent
itraconazole is not considered effective for strains of CafZdida albicaras
with MIC>8 for this drug
(e.g., for C. albicaras strain c43 (ATCC number 201794), MICRO =16 ~.g/ml for
SPORANOX~
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itraconazole). These strains of Cayadida albicans are considered to be
resistant to itraconazole.
This presupposes the standard dosing level of this drug that can be
administered.
However, if a method were available to substantially increase the amount of
the
antimicrobial drug (e.g., itraconazole) that could be administered, than it
might be possible to
treat infections hithertofore considered untreatable by this agent. Such a
method is available
through formulation of the drug as a nanosuspension. Submicron sized drug
crystals stabilized
by a surfactant coating have been found, in some cases, not to dissolve
immediately upon
injection into the blood stream. Instead, they are captured by fixed
macrophages of the spleen
and liver. From this sanctuary, the drug will be slowly released over a
prolonged period of days.
This is in contrast to conventionally solubilized drugs, which when inj ected,
decrease in blood
concentration at a much faster rate.
An example of an antimicrobial agent which is conventionally formulated to
increase the
solubility of the drug is the triazole antifungal agent itraconazole (FIG. 2).
Itraconazole is
effective against systemic mycoses, particularly aspergillosis and
candidiasis. New oral and
intravenous preparations of itraconazole have been prepared in order to
overcome bioavailability
problems associated with a lack of solubility. For example, the
bioavailability of itraconazole is
increased when it is formulated in hydroxypropyl-beta-cyclodextrin, a carrier
oligosaccharide that
forms an inclusion complex with the drug, thereby increasing its aqueous
solubility. The
commercial preparation is known by the tradename SPORANOX~ Inj ection and was
originated
by JANSSEN PHARMACEUTICAL PRODUCTS, L.P. The drug is currently manufactured by
Abbott Labs and distributed by Ortho Biotech, Inc.
Intravenous itraconazole may be useful in selected clinical situations.
Examples are
achlorhydria in AIDS patients, an inability to effectively absorb oral
medications due to
concurrent treatments with other drugs, or in critical-care patients who
cannot take oral
medications. The current commercial product, SPORANOX~ Inj ection, is made
available in 25
mL glass vials that contain 250 mg of itraconazole, with 10 g of hydroxypropyl-
beta-cyclodextrin
(referenced as "HPBCD"). These vials are diluted prior to use in 50 mL of 0.9%
saline. The
resulting cyclodextrin concentration exceeds 10% (w/v) in the reconstituted
product. Although
HPBCD has been traditionally regarded as safe for inj ection, high
concentrations, such as 10%,
have been reported in animal models to induce significant changes to
endothelial tissues
(Duncker G.; Reichelt J., Effects of the pharmaceutical cosolvent
hydroxypropyl-beta-
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cyclodextrin on porcine corneal endothelium. Graefe's Archive for Clinical and
Experimental
Ophthalmology (Germany) 1998, 236/5, 380-389).
Other excipients are often used to formulate poorly water-soluble drugs for
intravenous
injection. For example, paclitaxel (Taxol~, produced byEristol-Myers Squibb)
contains 52.7%
(w/v) of Cremophor~ EL (polyoxyethylated castor oil) and 49.7% (v/v)
dehydrated alcohol,
USP. Administration of Cremophor~ EL can lead to undesired hypersensitivity
reactions
(Volcheck, G.W., Van Dellen, R.G. Anaphylaxis to intravenous cyclosporine and
tolerance to
oral cyclosporine: case report and review. Annals ofAllergy, Astlama, and
Immunology,1998,
80, 159-163; Singla A.K.; Garg A.; Aggarwal I7., Paclitaxel and its
formulations. Intef~fzatioyaal
.Iou~tZal ofPharmaceutics, 2002, 235/ 1-2, 179-192).
The present invention discloses a composition which renders antimicrobial
drugs more
effective on the basis of their physical and biological properties than in
their unformulated state
or in their existing formulations. The approach used is to formulate the
antimicrobial agents as
nanosupensions. This permits using of the improved formulation to treat
microbes
conventionally thought to be resistant to the unformulated drug. Conventional
formulation
approaches attempt to enhance solubility or bioavailability only. Such methods
include pH
change, modification of the salt form, use of organic modifiers, or
cyclodextrin. The approach
disclosed in the present invention involves altering the pharmacokinetic
characteristic of the
drug, permitting far greater dosing, resulting in improved efficacy over and
above what can be
accomplished by improving solubility and bioavailability only. Acute toxicity
tests have
demonstrated that much more drug, when formulated as a nanosuspension, can be
administered
to animals. More of the drug is therefore available at the target organ to
exert efficacy.
SUMMARY OF THE INVENTION:
The present invention relates to a composition of an aqueous suspension of
submicron- to
micron-size particles of an antimicrobial agent that renders the agent potent
against organisms
normally considered to be resistant to the agent. The composition includes an
aqueous
suspension of submicron- to micron-size particles containing the agent coated
with at least one
surfactant selected from the group consisting of ionic surfactants, non-ionic
surfactants,
biologically derived surfactants, and amino acids and their derivatives. The
particles have a
volume-weighted mean particle size of less than 5 ~,m as measured by light
scattering (HORIBA)
or by microscopic measurements. More preferably the particles should be less
than about 1
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micron and most preferably from about 150 nm to about 1 micron or anyrange or
combination of
ranges therein.
The present invention is suitable for pharmaceutical use.
In an embodiment of the invention, the antimicrobial agent is an antifungal
agent. In a
preferred embodiment, the antifungal agent is a h-iazole antifungal agent. In
yet another
embodiment of the invention, the triazole antifungal agent is selected from
itraconazole,
ketoconazole, miconazole, fluconazole, ravuconazole, voriconazole,
saperconazole,
eberconazole, genaconazole, clotrimazole, econazole, oxiconazole, sulconazole,
terconazole,
tioconazole, and posaconazole. In a preferred embodiment of the invention, the
antifungal agent
is itraconazole.
Suitable surfactants for coating the particles in the present invention can be
selected from
ionic surfactants, nonionic surfactants, biologically derived surfactants, or
amino acids and their
derivatives.
In a further preferred embodiment, the composition of the present invention is
prepared
by a microprecipitation method which includes the steps of (i) dissolving in
the antifungal agent
in a first water-miscible first solvent to form a solution; (ii) mixing the
solution with a second
solvent which is aqueous to define a pre-suspension; and (iii) adding energy
to the pre-suspension
to form particles having an average effective particle size of less than 5
Vim; more preferably less
than about 1 micron, and most preferably from about 150 run to about 1 micron
or any range or
combination of ranges therein, wherein the solubility of the antifungal agent
is greater in the first
solvent than in the second solvent, and the first solvent or the second
solvent comprising one or
more surfactants selected from the group consisting of nonionic surfactants,
ionic surfactants,
biologically derived surfactants, and amino acids and their derivatives.
The present invention also relates to a method of rendering an antimicrobial
agent potent
against organisms normally considered to be resistant to the agent by
formulating the agent as an
aqueous suspension of submicron- to micron-size particles containing the agent
coated with at
least one surfactant selected from the group consisting of ionic surfactants,
non-ionic surfactants,
biologically derived surfactants, and amino acids and their derivatives.
The present invention further relates to a method of treating infection of a
subject by
organisms normally considered to be resistant to an antimicrobial agent by
administering the
agent to the subj ect formulated as an aqueous suspension of submicron- to
micron-size particles
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containing the agent coated with at least one surfactant selected from the
group consisting of
ionic surfactants, non-ionic surfactants, biologically derived surfactants,
and amino acids and
their derivatives.
These and other aspects and attributes of the present invention will be
discussed with
reference to the following drawings and accompanying specification.
BRIEF I~ESC1ZIPTION OF THE I~IZAWINGS:
FIG. 1 is the general molecular structure of a triazole antifungal agent;
FIG. 2 is the molecular structure of itraconazole;
FIG. 3 is a schematic diagram of Method A of the microprecipitation process
used in the
present invention to prepare the suspension;
FIG. 4 is a schematic diagram of Method B of the microprecipitation process
used in the
present invention to prepare the suspension;
FIG. 5 is a graph comparing the pharmacokinetics of SPOR.ANOX~ with
Formulation 1.
suspension of itraconazole of the present invention, wherein ITC = plasma
concentration of
itraconazole measured after bolus injection of Formulation 1 (80 mg/kg), ITC-
OH = plasma
concentration of primary metabolite, hydroxyitraconazole, measured after bolus
injection of
Formulation 1 (80 mg/kg), Total - combined concentration of itraconazole and
hydroxyitraconazole (ITC + ITC-OH) measured after bolus injection of
Formulation 1 (80
mg/kg), Sporanox-ITC = plasma concentration of itraconazole measured after
bolus inj ection of
20 mg/kg Sporanox IV, Sporanox-ITC-OH = plasma concentration of primary
metabolite,
hydroxyitraconazole, measured after bolus inj ection of 20 mg/lcg Sporanox IV,
Sporanox - Total
= combined concentration of itraconazole and hydroxyitraconazole (ITC + ITC-
OH) measured
after bolus injection of 20 mg/kg Sporanox IV;
FIG. 6 is a graph comparing the drug level for the rapidly dissolving
formulation, Form A,
and the slow dissolving (macrophage targeting) formulation, Form B, as
determined in an in vitro
dissolution experiment; the drug level for Form A is much higher than that
attained by Form B;
FIG. 7 is a graph showing the comparison of results for body weight over time
for
immuno-suppressed rats treated with SPORANOX° Injection and
Formulations 14288-1 and
14.288-B;
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FIG. 8 is a graph of kidney drug level vs. dose showing that the greater
dosing that could
be administered permitted greater drug levels to be manifested in the target
organs, in this case,
the kidney;
FIG. 9 is a graph of fungal counts vs. kidney drug level (N = nanosuspension;
S =
Sporanox IV solution) showing that the gr eater drug levels in the target
organ (the kidney) led to
a greater kill of the infectious organisms; and
FIG. 10 is a graph showing the mortality/moribundity profile after daily or
every other day
dosing with antifungal drugs for 10 days in rats systemically infected with
itraconazole resistant
C. albicans.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
While this invention is susceptible of embodiment in many different forms,
there are
shown in the drawing, and will be described herein in detail, specific
embodiments thereof with
the understanding that the present disclosure is to be considered as an
exemplification of the
principles of the invention and is not intended to limit the invention to the
specific embodiments
illustrated.
The present invention relates to a composition of an antimicrobial agent that
renders the
agent potent against organisms normally considered to be resistant to the
agent. The composition
comprises an aqueous suspension of submicron- to micron-size particles
containing the agent
coated with at least one surfactant selected from the group consisting of
ionic surfactants, non-
ionic surfactants, biologically derived surfactants, and amino acids and their
derivatives. The
composition disclosed in the present invention involves altering the
pharmacokinetic
characteristic of the drug, permitting far greater dosing, resulting in
improved efficacy over and
above what can be accomplished by improving solubility and bioavailability
only. Submicron
sized drug crystals stabilized by a surfactant coating have been found, in
some cases, not to
dissolve immediately upon injection into the blood stream. Instead, they are
captured by fixed
macrophages of the spleen and liver. From this sanctuary, the drug can be
slowly released over a
prolonged period of days. Acute toxicity tests have demonstrated that much
more drug, when
formulated as a nanosuspension, can be administered to animals or human
beings. More of the
drug is therefore available at the target organ to exert efficacy.
The particles in the present invention have a volume-weighted mean particle
size of less
than 5 ~,m as measured by light scattering (HORIBA) or by microscopic
measurements. More
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preferably the particles should be less than about 1 micron and most
preferably from about 150
nm to about 1 micron or any range or combination of ranges therein. The
composition can be
administered to a subj ect to treat infection by organisms normally considered
to be resistant to the
agent.
The antimicrobial agent is preferably a poorly water soluble organic compound.
What is
meant by "poorly water soluble" is that the water solubility of the compound
is less than 10
mg/ml, and preferably, less than 1 mg/ml. A preferred class of antimicrobial
agent is an
antifungal agent. A preferred antifungal agent is the triazole antifungal
agents having a general
molecular structure as shown in FIG. 1. Examples of triazole antifungal agents
include, but are
not limited to: itraconazole, ketoconazole, miconazole, fluconazole,
ravuconazole, voriconazole,
saperconazole, eberconazole, genaconazole, clotrimazole, econazole,
oxiconazole, sulconazole,
terconazole, tioconazole, and posaconazole. A preferred antifungal agent for
the present
invention is itraconazole. The molecular structure of itraconazole is shown in
FIG. 2.
The present invention is suitable for pharmaceutical use. The compositions can
be
administered by various routes, including but not limited to, intravenous,
intracerebral,
intrathecal, intralylnphatic, pulmonary, intraarticular, and intraperitoneal.
In an embodiment of
the present invention, the aqueous medium of the composition is removed to
form dry particles.
The method to remove the aqueous medium can be any method known in the art.
One example is
evaporation. Another example is freeze drying or lyophilization. The dry
particles may then be
formulated into any acceptable physical form including, but is not limited to,
solutions, tablets,
capsules, suspensions, creams, lotions, emulsions, aerosols, powders,
incorporation into reservoir
or matrix devices for sustained release (such as implants or transdermal
patches), and the like.
If the particles do not have to be taken up by the macrophages, the particles
can be larger
than 5 ~m (e.g., less than 50 ~,m, or less than 7 yn) or less than 150 nm
(e.g., less than 100 Vim).
These particles can be administered by various routes, including but not
limited to parenteral,
oral, buccal, periodontal, rectal, nasal, pulmonary, transdermal, or topical.
Modes of parenteral
administration include intravenous, infra arterial, intrathecal,
intraperitoneal, intraocular, infra
articular, intrathecal, intracerebral, intramuscular, subcutaneous, and the
like.
The aqueous suspension of the present invention may also be frozen to improve
stability
upon storage. Freezing of an aqueous suspension to improve stability is
disclosed in the
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_g_
commonly assigned and co-pending U.S. Patent Application Serial No.
60/347,548, which is
incorporated herein by reference and made a part hereof.
In an embodiment of the present invention, the antimicrobial agent is present
in an
amount preferably from about 0.01 % to about 50°/~ weight to volume
(w/v), more preferably from
about 0.05% to about 30% w/v, and most preferably from about 0.1% to about
20°/~ w/v.
Suitable surfactants for coating the particles in the present invention can be
selected from
ionic surfactants, nonionic surfactants, biologically derived surfactants or
amino acids and their
derivatives. Ionic surfactants can be anionic, cationic, or zwitterionic.
Suitable aniouc surfactants include but are not limited to: alkyl sulfonates,
alkyl
phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate,
sodium lauryl
sulfate, sodimn dodecylsulfate, alkyl polyoxyethylene sulfates, sodium
alginate, dioctyl sodium
sulfosuccinate, phosphatidylglycerol, phosphatidylinosine,
phosphatidylinositol,
diphosphatidylglycerol, phosphatidylserine, phosphatidic acid and their salts,
sodium
carboxymethylcellulose, cholic acid and other bile acids (e.g., cholic acid,
deoxycholic acid,
glycocholic acid, taurocholic acid, glycodeoxycholic acid) and salts thereof
(e.g., sodium
deoxycholate, etc.). As anionic surfactants, phospholipids may be used.
Suitable phospholipids
include, for example, phosphatidylserine, phosphatidylinositol,
diphosphatidylglycerol,
phosphatidylglycerol, or phosphatidic acid and its salts.
Zwitterionic surfactants are electrically neutral but posses local positive
and negative
charges within the same molecule. Suitable zwitterionic surfactants include
but are not limited to
zwitterionic phospholipids. Suitable phospholipids include
phosphatidylcholine,
phosphatidylethanolamine, diacyl-glycero-phosphoethanolamine (such as
dimyristoyl-glycero
phosphoethanolamine (DMPE), dipalmitoyl-glycero-phosphoethanolamine (DPPE),
distearoyl
glycero-phosphoethanolamine (DSPE), and dioleolyl-glycero-phosphoethanolamine
(DOPE)).
Mixtures ofphospholipids that include anionic and zwitterionic phospholipids
maybe employed
in this invention. Such mixtures include but are not limited to
lysophospholipids, egg or soybean
phospholipid or any combination thereof. The phospholipid, whether anionic,
zwitterionic or a
mixture of phospholipids, may be salted or desalted, hydrogenated or partially
hydrogenated or
natural semisynthetic or synthetic. The phospholipid may also be conjugated
with a water-
soluble or hydrophilic polymer to specifically target the delivery to
macrophages in the present
invention. However, conjugated phospholipids may be used to target other cells
or tissue in other
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applications. A preferred polymer is polyethylene glycol (PEG), which is also
lmown as the
monomethoxy polyethyleneglycol (mPEG). The molecule weights of the PEG can
vary, for
example, from 200 to 50,000. Some commonly used PEG's that are commercially
available
include PEG 350, PEG 550, PEG 750, PEG 1000, PEG 2000, PEG 3000, and PEG 5000.
The
phospholipid or the PEG-phospholipid conjugate may also incorporate a
functional group which
can covalently attach to a ligand including but not limited to proteins,
peptides, carbohydrates,
glycoproteins, antibodies, or pharmaceutically active agents. These functional
groups may
conjugate with the ligands through, for example, amide bond formation,
disulfide or tluoether
formation, or biotiustreptavidin binding. Examples of the ligand-binding
functional groups
include but are not limited to hexanoylamine, dodecanylamine, 1,12-
dodecanedicarboxylate,
thioethanol, 4-(p-maleimidophenyl)butyramide (MPB), 4-(p-
maleimidomethyl)cyclohexane-
carboxamide (MCC), 3-(2-pyridyldithio)propionate (PDP), succinate, glutarate,
dodecanoate, and
biotin.
Suitable cationic surfactants include but are not limited to quaternary
ammonium
compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide,
lauryldimethylbenzylamtnonium chloride, acyl carnitine hydrochlorides,or alkyl
pyridinium
halides, or long-chain alkyl amines such as, for example, n-octylamine and
oleylamine.
Suitable nonionic surfactants include: glyceryl esters, polyoxyethylene fatty
alcohol ethers
(Macrogol and Brij), polyoxyethylene sorbitan fatty acid esters
(Polysorbates), polyoxyethylene
fatty acid esters (Myrj), sorbitan esters (Span), glycerol monostearate,
polyethylene glycols,
polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol,
aryl alkyl polyether
alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers),
poloxamines,
methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, noncrystalline cellulose, polysaccharides
including starch and
starch derivatives such as hydroxyethylstarch (HES), polyvinyl alcohol, and
polyvinylpyrrolidone. In a preferred form of the invention, the nonionic
surfactant is a
polyoxyethylene and polyoxypropylene copolymer and preferably a block
copolymer of
propylene glycol and ethylene glycol. Such polymers are sold under the
tradenasne
POLOXAMER also sometimes referred to as PLURONIC~, and sold by several
suppliers
including Spectrum Chemical and Ruger. Among polyoxyethylene fatty acid esters
is included
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those having short alkyl chains. One example of such a surfactant is SOLUTOL~
HS 15,
polyethylene-660-hydroxystearate, manufactured by BASF Aktiengesellschaft.
Surface-active biological molecules uzclude such molecules as albumin, casein,
hirudin or
other appropriate proteins. Polysaccharide biologics are also included, and
consist of but are not
limited to, starches, heparin and chitosans. Other suitable surfactants
include any amino acids
such as leucine, alanine, valine, isoleucine, lysine, aspartic acid, glutamic
acid, methionine,
phenylalanine, or any derivatives of these amino acids such as, for example,
amide or ester
derivatives and polypeptides formed from these amino acids.
A preferred ionic surfactant is a bile salt, and a preferred bile salt is
deoxycholate. A
preferred nonionic surfactant is a polyalkoxyether, and a preferred
polyalkoxyether is Poloxamer
188. Another preferred nonionic surfactant is Solutol HS 15 (polyethylene-660-
hydroxystearate).
Still yet another preferred nonionic surfactant is hydroxyethylstarch. A
preferred biologically
derived surfactant is albumin.
In another embodiment of the present invention, the surfactants are present in
an amount
of preferably from about 0.001% to 5% w/v, more preferably from about 0.005%
to about 5%
w/v, and most preferably from about 0.01% to 5% w/v.
In a preferred embodiment of the present invention, the particles are
suspended in an
aqueous medium further including a pH adjusting agent. Suitable pH adjusting
agents include,
but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid,
monocarboxylic acids
(such as, for example, acetic acid and lactic acid), dicarboxylic acids (such
as, for example,
succinic acid), tricarboxylic acids (such as, for example, citric acid), THAM
(tris(hydroxymethyl)aminomethane), meglumine (N-methylglucosamine), sodium
hydroxide, and
amino acids such as glycine, arginine, lysine, alanine, histidine and leucine.
The aqueous
medium may additionally include an osmotic pressure adjusting agent, such as
but not limited to
glycerin, a monosaccharide such as dextrose, a disaccharide such as sucrose, a
trisaccharide such
as raffinose, and sugar alcohols such as maimitol, xylitol and sorbitol.
In a preferred embodiment of the present invention, the composition comprises
an
aqueous suspension of particles of itraconazole present at 0.01 to
50°/~ w/v, the particles are
coated with 0.001 to 5% w/v of a bile salt (e.g., deoxycholate) and 0.001 to
5% w/v
polyalkoxyether (for example, Poloxamer 188), and glycerin added to adjust
osmotic pressure of
the formulation.
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In another preferred embodiment of the present invention, the composition
comprises an
aqueous suspension of particles of itraconazole present at about 0.01 to 50%
w/v, the particles
coated with about 0.001 to 5% w/v of a bile salt (for example, deoxycholate)
and 0.001 to 5%
polyethylene-660-hydroxystearate w/v, and glycerin added to adjust osmotic
pressure of the
formulation.
In another preferred embodiment of the present invention, the composition
comprises an
aqueous suspension of itraconazole present at about 0.01 to 50% w/v, the
particles are coated
with about 0.001 to 5% of polyethylene-660-hydroxystearate w/v, and glycerin
added to adjust
osmotic pressure of the formulation.
In still yet another preferred embodiment of the present invention, the
composition
comprises an aqueous suspension of itraconazole present at 0.01 to 50% w/v,
the particles are
coated with about 0.001 to 5% albumin w/v.
The method for preparing the suspension in the present invention is disclosed
in
commonly assigned co-pendingU.S. Patent Applications Serial Nos.
and 60/258,160;
09/874,799;09/874,637;09/874,499;09/964,273; 10/035,821, 60/347,548;
10/021,692;
10/183,035; 10/213,352;10/246,802;10/270,268; 10/270,267, and 10/390,333;
which are
incorporated herein by reference and made a part hereof. A general procedure
for preparing the
suspension useful in the practice of this invention follows.
The processes can be separated into three general categories. Each of the
categories of
processes share the steps of (1) dissolving an antifungal agent in a water
miscible first organic
solvent to create a first solution; (2) mixing the first solution with a
second solvent of water to
precipitate the antifungal agent to create a pre-suspension; and (3) adding
energy to the
presuspension in the form of high-shear mixing or heat to provide a stable
form of the antifungal
agent having the desired size ranges defined above.
The three categories of processes are distinguished based upon the physical
properties of
the antifungal agent as determined through x-ray diffraction studies,
differential scanning
calorimetry (DSC) studies or other suitable study conducted prior to the
energy-addition step and
after the energy-addition step. In the first process category, prior to the
energy-addition step the
antifungal agent in the presuspension takes an amorphous form, a semi-
crystalline form or a
supercooled liquid form and has an average effective particle size. After the
energy-addition
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step, the antifungal agent is in a crystalline form having an average
effective particle size
essentially the same as that of the presuspension (i.e., from less than about
50 ~.m).
hl the second process category, prior to the energy-addition step the
antifungal agent is in
a crystalline form and has an average effective particle size. After the
energy-addition step, the
antifungal agent is in a crystalline form having essentially the same average
effective particle size
as prior to the energy-addition step but the crystals after the energy-
addition step are less likely to
aggregate.
The lower tendency of the organic compound to aggregate is observed by laser
dynamic
light scattering and light microscopy.
In the third process category, prior to the energy-addition step the
antifungal agent is in a
crystalline form that is friable and has an average effective particle size.
What is meant by the
term "friable" is that the particles are fragile and are more easily broken
down into smaller
particles. After the energy-addition step the organic compound is in a
crystalline form having an
average effective particle size smaller than the crystals of the pre-
suspension. By taking the steps
necessary to place the organic compound in a crystalline form that is friable,
the subsequent
energy-addition step can be carried out more quickly and efficiently when
compared to an organic
compound in a less friable crystalline morphology.
The energy-addition step can be carned out in any fashion wherein the pre-
suspension is
exposed to cavitation, shearing or impact forces. In one preferred,form of the
invention, the
energy-addition step is an annealing step. Amlealing is defined in this
invention as the process of
converting matter that is thermodynamically unstable into a more stable form
by single or
repeated application of energy (direct heat or mechanical stress), followed by
thermal relaxation.
This lowering of energy may be achieved by conversion of the solid form from a
less ordered to a
more ordered lattice structure. Alternatively, this stabilization may occur by
a reordering of the
surfactant molecules at the solid-liquid interface.
These three process categories will be discussed separately below. It should
be
understood, however, that the process conditions such as choice of surfactants
or combination of
surfactants, amount of surfactant used, temperature of reaction, rate of
mixing of solutions, rate
of precipitation and the like can be selected to allow for any drug to be
processed under any one
of the categories discussed next.
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The first process category, as well as the second and third process
categories, can be
further divided into two subcategories, Method A, and B shown diagrammatically
in FIG. 3 and
FIG. 4, respectively.
The first solvent according to the present invention is a solvent or mixture
of solvents in
which the organic compound of interest is relatively soluble and which is
miscible with the
second solvent. Such solvents include, but are not limited to water-miscible
erotic compounds,
in which a hydrogen atom in the molecule is bound to an electronegative atom
such as oxygen,
nitrogen, or other Group VA, VIA and VII A in the Periodic Table of elements.
Examples of
such solvents include, but are not limited to, alcohols, amines (primary or
secondary), oximes,
hydroxamic acids, carboxylic acids, sulfonic acids, phosphoric acids,
phosphoric acids, amides
and areas.
Other examples of the first solvent also include aprotic organic solvents.
Some of these
aprotic solvents can form hydrogen bonds with water, but can only act as
proton acceptors
because they lack effective proton donating groups. One class of aprotic
solvents is a Bipolar
aprotic solvent, as defined by the International Union of Pure and Applied
Cliemistry (IUPAC
Compendium of Chemical Terminology, 2nd Ed., 1997):
A solvent with a comparatively high relative permittivity (or
dielectric constant), greater than ca. 15, and a sizable permanent
dipole moment, that cannot donate suitably labile hydrogen atoms
to form strong hydrogen bonds, e.g. dimethyl sulfoxide.
bipolar aprotic solvents can be selected from the group consisting of amides
(fully
substituted, with nitrogen lacking attached hydrogen atoms), areas (fully
substituted, with no
hydrogen atoms attached to nitrogen), ethers, cyclic ethers, nitriles,
ketones, sulfones, sulfoxides,
fully substituted phosphates, phosphonate esters, phosphoramides, vitro
compounds, and the like.
Dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidinone (NMP), 2-pyrrolidinone, 1,3-
dimethylimidazolidinone (DMI), dimethylacetamide (DMA), dimethylformamide
(DMF),
dioxane, acetone, tetrahydrofuran (THF), tetramethylenesulfone (sulfolane),
acetonitrile, and
hexamethylphosphoramide (HMPA), nitromethane, among others, are members of
this class.
Solvents may also be chosen that are generally water-immiscible, but have
sufficient
water solubility at low volumes (less than 10%) to act as a water-miscible
first solvent at these
reduced volumes. Examples include aromatic hydrocarbons, alkenes, alkanes, and
halogenated
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aromatics, halogenated alkenes and halogenated alkanes. Aromatics include, but
are not limited
to, benzene (substituted or unsubstituted), and monocyclic or polycyclic
arenas. Examples of
substituted benzenes include, but are not limited to, xylenes (ortho, mete, or
pare), and toluene.
Examples of alkanes include but are not limited to hexane, neopentane,
heptane, isooctane, and
cyclohexane. Examples of halogenated aromatics include, but are not restricted
to,
chlorobenzene, bromobenzene, and chlorotoluene. Examples of halogenated
alkanes and alkenes
include, but are not restricted to, trichloroethane, methylene chloride,
ethylenedichloride (EI~C),
and the like.
Examples of the all of the above solvent classes include but are not limited
to: N-methyl-
2-pyrrolidinone (also called N-methyl-2-pyrrolidone), 2-pyrrolidinone (also
called 2-pyrrolidone),
1,3-dimethyl-2-imidazolidinone (DMA, dimethylsulfoxide, dimethylacetamide,
carboxylic acids
(such as acetic acid and lactic acid), aliphatic alcohols (such as methanol,
ethanol, isopropanol, 3-
pentanol, and n-propanol), benzyl alcohol, glycerol, butylene glycol
(butanediol), ethylene glycol,
propylene glycol, mono- and diacylated monoglycerides (such as glyceryl
caprylate), dimethyl
isosorbide, acetone, dimethylsulfone, dimethylformamide, 1,4-dioxane,
tetramethylenesulfone
(sulfolane), acetonitrile, nitromethane, tetramethylurea,
hexamethylphosphoramide (HMPA),
tetrahydrofuran (THF), dioxane, diethylether, tart-butyhnethyl ether (TBME),
aromatic
hydrocarbons, alkenes, alkanes, halogenated aromatics, halogenated alkenes,
halogenated
alkanes, xylene, toluene, benzene, substituted benzene, ethyl acetate, methyl
acetate, butyl
acetate, chlorobenzene, bromobenzene, chlorotoluene, trichloroethane,
methylene chloride,
ethylenedichloride (E1~C), hexane, neopentane, heptane, isooctane,
cyclohexane, polyethylene
glycol (PEG, for example, PEG-4, PEG-8, PEG-9, PEG-12, PEG-14, PEG-16, PEG-
120, PEG-
75, PEG-150), polyethylene glycol esters (examples such as PEG-4 dilaurate,
PEG-20 dilaurate,
PEG-6 isostearate, PEG-8 palmitostearate, PEG-150 palinitostearate),
polyethylene glycol
sorbitans (such as PEG-20 sorbitan isostearate), polyethylene glycol monoalkyl
ethers (examples
such as PEG-3 dimethyl ether, PEG-4 dimethyl ether), polypropylene glycol
(PPG),
polypropylene alginate, PPG-10 butanediol, PPG-10 methyl glucose ether, PPG-20
methyl
glucose ether, PPG-15 stearyl ether, propylene glycol dicaprylate/dicaprate,
propylene glycol
laurate, and glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol
ether). A preferred first
solvent is N-methyl-2-pyrrolidinone. Another preferred first solvent is lactic
acid.
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The second solvent is an aqueous solvent. This aqueous solvent may be water by
itself.
This solvent may also contain buffers, salts, surfactant(s), water-soluble
polymers, and
combinations of these excipients.
Method A
In Method A (see FIG. 3), the antimicrobial agent is first dissolved in the
first solvent to
create a first solution. The antimicrobial agent can be added from about 0.01%
(w/v) to about
50% (w/v) depending on the solubility of the antimicrobial agent in the first
solvent. Heating of
the concentrate from about 30°C to about 100°C maybe necessary
to ensure total dissolution of
the antimicrobial agent in the first solvent.
A second aqueous solution is provided with one or more surfactants added
thereto. The
surfactants can be selected from an ionic surfactant, a nonionic surfactant or
a biologically
derived surfactant set forth above.
It may also be desirable to add a pH adjusting agent to the second solution
such as sodium
hydroxide, hydrochloric acid, tris buffer or citrate, acetate, lactate,
meglumine, or the like. The
second solution should have a pH within the range of from about 3 to about 11.
In a preferred form of the invention, the method for preparing submicron sized
particles of
an antimicrobial agent includes the steps of adding the first solution to the
second solution. The
addition rate is dependent on the batch size, and precipitation kinetics for
the antimicrobial agent.
Typically, for a small-scale laboratory process (preparation of 1 liter), the
addition rate is from
about 0.05 cc per minute to about 10 cc per minute. During the addition, the
solutions should be
under constant agitation. It has been observed using light microscopy that
amorphous particles,
semi-crystalline solids, or a supercooled liquid are formed to create a pre-
suspension. The
method further includes the step of subj ecting the pre-suspension to an
annealing step to convert
the amorphous particles, supercooled liquid or semicrystalline solid to a
crystalline more stable
solid state. The resulting particles will have an average effective particles
size as measured by
dynamic light scattering methods (e.g., photocorrelation spectroscopy, laser
diffraction, low-
angle laser light scattering (LALLS), medium-angle laser light scattering
(MALLS), light
obscuration methods (Coulter method, for example), rheology, or microscopy
(light or electron)
within the ranges set forth above).
The energy-addition step involves adding energy through sonication,
homogenization,
counter current flow homogenization (e.g., the Mini DeBEE 2000 homogenizer,
available from
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BEE Incorporated, NC, in which a jet of fluid is directed along a first path,
and a structure is
interposed in the first path to cause the fluid to be redirected in a
controlled flow path along a
new path to cause emulsification or mixing of the fluid), microfluidization,
or other methods of
providing impact, shear or cavitation forces. The sample may be cooled or
heated during this
stage. In one preferred form of the invention the annealing step is effected
by homogenization.
W another preferred form of the invention the annealing may be accomplished by
ultrasonication.
In yet another preferred form of the invention the annealing may be
accomplished by use of an
emulsification apparatus as described in U.S. Patent No. 5,720,551 which is
incorporated herein
by reference and made a part hereof.
Depending upon the rate of annealing, it may be desirable to adjust the
temperature of the
processed sample to within the range of from approximately-30°C to
100°C. Alternatively, in
order to effect a desired phase change in the processed solid, it may also be
necessary to adjust
the temperature of the pre-suspension to a temperature within the range of
from about -30°C to
about 100°C during the annealing step.
Method B
Method B differs from Method A in the following respects. The first difference
is a
surfactant or combination of surfactants are added to the first solution. The
surfactants may be
selected from ionic surfactants, nonionic surfactants, or biologically derived
as set forth above.
A drug suspension resulting from application of the processes described in
this invention
may be administered directly as an injectable solution, provided Water for
Injection is used in
formulation and an appropriate means for solution sterilization is applied.
Sterilization may be
accomplished, by separate sterilization of the drug concentrate (drug,
solvent, and optional
surfactant) and the diluent medium (water, and optional buffers and
surfactants) prior to mixing
to form the pre-suspension. Sterilization methods include pre-filtration first
through a 3.0 micron
filter followed by filtration through a 0.45-micron particle filter, followed
by steam or heat
sterilization or sterile filtration through two redundant 0.2-micron membrane
filters.
~ptionally, a solvent-free suspension may be produced by solvent removal after
precipitation. This can be accomplished by centrifugation, dialysis,
diafiltration, force-field
fractionation, high-pressure filtration or other separation techniques well
known in the art.
Complete removal of N-methyl-2-pyrrolidinone was typically carned out by one
to three
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successive centrifugation runs; after each centrifugation the supernatant was
decanted and
discarded. A fresh volume of the suspension vehicle without the organic
solvent was added to
the remaining solids and the mixture was dispersed by homogenization. It will
be recognized by
others skilled in the art that other high-shear mixing techniques could be
applied in this
reconstitution step.
Furthermore, any undesired excipients such as surfactants may be replaced by a
more
desirable excipient by use of the separation methods described in the above
paragraph. The
solvent and first excipient may be discarded with the supernatant after
centrifugation or filtration.
A fresh volume of the suspension vehicle without the solvent and without the
first excipient may
then be added. Alternatively, a new surfactant may be added. For example, a
suspension
consisting of drug, N-methyl-2-pyrrolidinone (solvent), Poloxamer 188 (first
excipient), sodium
deoxycholate, glycerol and water may be replaced with phospholipids (new
surfactant), glycerol
and water after centrifugation and removal of the supernatant.
I. First Process Category
The methods of the first process category generally include the step of
dissolving the
antimicrobial agent in a water miscible first solvent followed by the step of
mixing tlus solution
with an aqueous solution to form a presuspension wherein the antimicrobial
agent is in an
amorphous form, a semicrystalline form or in a supercooled liquid form as
determined by x-ray
diffraction studies, DSC, light microscopy or other analytical techniques and
has an average
effective particle size within one of the effective particle size ranges set
forth above. The
mixing step is followed by an energy-addition step and, in a preferred form of
the invention is
an annealing step.
II. Second Process Category
The methods of the second processes category include essentially the same
steps as in the
steps of the first processes category but differ in the following respect. An
x-ray diffraction, DSC
or other suitable analytical techniques of the presuspension shows the
antimicrobial agent in a
crystalline form and having an average effective particle size. The
antimicrobial agent after the
energy-addition step has essentially the same average effective particle size
as prior to the energy-
addition step but has less of a tendency to aggregate into larger particles
when compared to that
of the particles of the presuspension. Without being bound to a theory, it is
believed the
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differences in the particle stability may be due to a reordering of the
surfactant molecules at the
solid-liquid interface.
III. Third Process Category
The methods of the third category modify the first two steps of those of the
first and
second processes categories to ensure the antimicrobial agent in the
presuspension is in a friable
form having an average effective particle size (e.g., such as slender needles
and thin plates).
Friable particles can be formed by selecting suitable solvents, surfactants or
combination of
surfactants, the temperature of the individual solutions, the rate of mixing
and rate of
precipitation and the like. Friability may also be enhanced by the
introduction of lattice defects
(e.g., cleavage planes) during the steps of mixing the first solution with the
aqueous solution.
This would arise by rapid crystallization such as that afforded in the
precipitation step. In the
energy-addition step these friable crystals are converted to crystals that are
kinetically stabilized
and having an average effective particle size smaller than those of the
presuspension. Kinetically
stabilized means particles have a reduced tendency to aggregate when compared
to particles that
are not kinetically stabilized. In such instance the energy-addition step
results in a breaking up of
the friable particles. By ensuring the particles of the presuspension are in a
friable state, the
organic compound can more easily and more quickly be prepared into a particle
within the
desired size ranges when compared to processing an organic compound where the
steps have not
been taken to render it in a friable form.
In addition to the microprecipitation methods described above, any other known
precipitation methods for preparing submicron sized particles or nanoparticles
in the art can be
used in conjunction with the present invention. The following is a description
of examples of
other precipitation methods. The examples are for illustration purposes, and
are not intended to
limit the scope of the present invention.
Emulsion Precipitation Methods
~ne suitable emulsion precipitation technique is disclosed in the co-pending
and
commonly assigned U.S. Ser. lVo. 09/964,273, which is incorporated herein by
reference and is
made a part hereof. In this approach, the process includes the steps of: (1)
providing a multiphase
system having an organic phase and an aqueous phase, the organic phase having
a
pharmaceutically effective compound therein; and (2) sonicating the system to
evaporate a
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portion of the organic phase to cause precipitation of the compound in the
aqueous phase and
having an average effective particle size of less than about 2 ~,m. The step
of providing a
multiphase system includes the steps of: (1) mixing a water immiscible solvent
with the
pharmaceutically effective compound to define an organic solution, (2)
preparing an aqueous
based solution with one or more surface active compounds, and (3) mixing the
organic solution
with the aqueous solution to form the multiphase system. The step of mixing
the organic phase
and the aqueous phase can include the use of piston gap homogenizers,
colloidal mills, high
speed stirring equipment, extrusion equipment, manual agitation or shaking
equipment,
microfluidizer, or other equipment or techniques for providing high shear
conditions. The crude
emulsion will have oil droplets in the water of a size of approximately less
than 1 ~m in
diameter. The crude emulsion is sonicated to define a microemulsion and
eventually to define a
submicron sized particle suspension.
Another approach to preparing submicron sized particles is disclosed in co-
pending and
commonly assigned U.S. Ser. No. 10/183,035, which is incorporated herein by
reference and
made a part hereof. The process includes the steps of: (1) providing a crude
dispersion of a
multiphase system having an organic phase and an aqueous phase, the organic
phase having a
pharmaceutical compound therein; (2) providing energy to the crude dispersion
to form a fine
dispersion; (3) freezing the fine dispersion; and (4) lyophilizing the fine
dispersion to obtain
submicron sized particles of the pharmaceutical compound. The step of
providing a multiphase
system includes the steps of (1) mixing a water immiscible solvent with the
pharmaceutically
effective compound to define an organic solution; (2) preparing an aqueous
based solution with
one or more surface active compounds; and (3) mixing the organic solution with
the aqueous
solution to form the multiphase system. The step of mixing the organic phase
and the aqueous
phase includes the use of piston gap homogenizers, colloidal mills, high speed
stirring
equipment, extrusion equipment, manual agitation or shaking equipment,
microfluidizer, or other
equipment or techniques for providing high shear conditions.
Solvent Anti-Solvent Precipitation
Suitable solvent anti-solvent precipitation technique is disclosed in U.S.
Pat. Nos.
5,118,528 and 5,100,591 which are incorporated herein by reference and made a
part hereof. The
process includes the steps of (1) preparing a liquid phase of a biologically
active substance in a
solvent or a mixture of solvents to which may be added one or more
surfactants; (2) preparing a
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second liquid phase of a non-solvent or a mixture of non-solvents, the non-
solvent is miscible
with the solvent or mixture of solvents for the substance; (3) adding together
the solutions of (1)
and (2) with stirring; and (4) removing of unwanted solvents to produce a
colloidal suspension of
nanoparticles. The'S28 Patent discloses that it produces particles of the
substance smaller than
500 nm without the supply of energy.
Phase Inversion Precipitation
One suitable phase inversion precipitation is disclosed in U.S. Pat. Nos.
6,235,224,
6,143,211 and U.S. patent application No. 2001/0042932 which are incorporated
herein by
reference and made a part hereof. Phase inversion is a term used to describe
the physical
phenomena by which a polymer dissolved in a continuous phase solvent system
inverts into a
solid macromolecular network in which the polymer is the continuous phase. One
method to
induce phase inversion is by the addition of a nonsolvent to the continuous
phase. The polymer
undergoes a transition from a single phase to an unstable two phase mixture:
polymer rich and
polymer poor fractions. Micellar droplets of nonsolvent in the polymer rich
phase serve as
nucleation sites and become coated with polymer. The '224 patent discloses
that phase inversion
of polymer solutions under certain conditions can bring about spontaneous
formation of discrete
microparticles, including nanoparticles. The '224 patent discloses dissolving
or dispersing a
polymer in a solvent. A pharmaceutical agent is also dissolved or dispersed in
the solvent. For the
crystal seeding step to be effective in this process it is desirable the agent
is dissolved in the
solvent. The polymer, the agent and the solvent together form a mixture having
a continuous
phase, wherein the solvent is the continuous phase. The mixture is then
introduced into at least
tenfold excess of a miscible nonsolvent to cause the spontaneous formation of
the
microencapsulated microparticles of the agent having an average particle size
of between 10 nm
and 10 yn. The particle size is influenced by the solvent:nonsolvent volume
ratio, polymer
concentration, the viscosity of the polymer-solvent solution, the molecular
weight of the polymer,
and the characteristics of the solvent-nonsolvent pair. The process eliminates
the step of creating
microdroplets, such as by forming an emulsion, of the solvent. The process
also avoids the
agitation and/or shear forces.
pH Shift Precipitation
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pH shift precipitation techniques typically include a step of dissolving a
drug in a
solution having a pH where the drug is soluble, followed by the step of
changing the pH to a
point where the drug is no longer soluble. The pH can be acidic or basic,
depending on the
particular pharmaceutical compound. The solution is then neutralized to form a
presuspension of
submicron sized particles of the pharmaceutcially active compound. One
suitable pH shifting
precipitation process is disclosed in U.S. Pat. No. 5,665,331, which is
incorporated herein by
reference and made a part hereof. The process includes the step of dissolving
of the
pharmaceutical agent together with a crystal growth modifier (CGM) in an
alkaline solution and
then neutralizing the solution with an acid in the presence of suitable
surface-modifying surface-
active agent or agents to form a fine particle dispersion of the
pharmaceutical agent. The
precipitation step can be followed by steps of diafiltration clean-up of the
dispersion and then
adjusting the concentration of the dispersion to a desired level. This process
of reportedly leads to
microcrystalline particles of Z-average diameters smaller than 400 nm as
measured by photon
correlation spectroscopy.
Other examples of pH shifting precipitation methods are disclosed in U.S. Pat.
Nos.
5,716,642; 5,662,883; 5,560,932; and 4,608,278, which are
incorporatedhereinbyreference and
are made a part hereof.
Infusion Precipitation Method
Suitable infusion precipitation techniques are disclosed in the U.S. Pat. Nos.
4,997,454
and 4,826,689, which are incorporated herein by reference and made a part
hereof. First, a
suitable solid compound is dissolved in a suitable organic solvent to form a
solvent mixture.
Then, a precipitating nonsolvent miscible with the organic solvent is infused
into the solvent
mixture at a temperature between about -10°C and about 100°C and
at an infusion rate of from
about 0.01 ml per minute to about 1000 ml per minute per volume of 50 ml to
produce a
suspension of precipitated non-aggregated solid particles of the compound with
a substantially
uniform mean diameter of less than 10 ~,m. Agitation (e.g., by stirring) of
the solution being
infused with the precipitating nonsolvent is preferred. The nonsolvent may
contain a surfactant to
stabilize the particles against aggregation. The particles are then separated
from the solvent.
Depending on the solid compound and the desired particle size, the parameters
of temperature,
ratio of nonsolvent to solvent, infusion rate, stir rate, and volume can be
varied according to the
invention. The particle size is proportional to the ratio of nonsolventaolvent
volumes and the
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temperature of infusion and is inversely proportional to the infusion rate and
the stirring rate. The
pr ecipitating nonsolvent may be aqueous or non-aqueous, depending upon the
relative solubility
of the compound and the desired suspending vehicle.
Temperature Shift Precipitation
Temperature shift precipitation technique, also known as the hot-melt
technique, is
disclosed in U.S. Pat. No. 5,188,837 to Lomb, which is incorporated herein by
reference and
made a part hereof. In an embodiment of the invention, lipospheres are
prepared by the steps of
(1) melting or dissolving a substance such as a drug to be delivered in a
molten vehiche to form a
liquid of the substance to be delivered; (2,) adding a phosphohipid along with
an aqueous medium
to the melted substance or vehicle at a temperature higher than the melting
temperature of the
substance or vehicle; (3) mixing the suspension at a temperature above the
melting temperature
of the vehicle until a homogenous fine preparation is obtained; and then (4)
rapidly cooling the
preparation to room temperature or below.
Solvent Evaporation Precipitation
Solvent evaporation precipitation techniques are disclosed in U.S. Pat. No.
4,973,465
which is incorporated herein by reference and made a part hereof. The '465
Patent discloses
methods for preparing microcrystals including the steps of: (1) providing a
solution of a
pharmaceutical composition and a phospholipid dissolved in a common organic
solvent or
combination of solvents, (2) evaporating the solvent or solvents and (3)
suspending the film
obtained by evaporation of the solvent or solvents in an aqueous solution by
vigorous stirring.
The solvent can be removed by adding energy to the solution to evaporate a
sufficient quantity of
the solvent to cause precipitation of the compound. The solvent can also be
removed by other
well known techniques such as applying a vacuum to the solution or blowing
nitrogen over the
solution.
Reaction Precipitation
Reaction precipitation includes the steps of dissolving the pharmaceutical
compound into
a suitable solvent to form a solution. The compound should be added in an
amount at or below
the saturation point of the compound in the solvent. The compound is modified
by reacting with a
chemical agent or by modification in response to adding energy such as heat or
UV light or the
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like to such that the modified compound has a lower solubility in the solvent
and precipitates
from the solution.
Com~aressed Fluid Precipitation
A suitable technique for precipitating by compressed fluid is disclosed in ~O
97/14407
to Johnston, which is incorporated herein by reference and made a part hereof.
The method
includes the steps of dissolving a water-insoluble drug in a solvent to form a
solution. The
solution is then sprayed into a compressed fluid, which can be a gas, liquid
or supercritical fluid.
The addition of the compressed fluid to a solution of a solute in a solvent
causes the solute to
attain or approach supersaturated state and to precipitate out as fme
particles. In this case, the
compressed fluid acts as an anti-solvent which lowers the cohesive energy
density of the solvent
in which the drug is dissolved.
Alternatively, the drug can be dissolved in the compressed fluid which is then
sprayed
into an aqueous phase. The rapid expansion of the compressed fluid reduces the
solvent power of
the fluid, which in turn causes the solute to precipitate out as fine
particles in the aqueous phase.
In this case, the compressed fluid acts as a solvent.
Other Methods for Preparin Particles
The particles of the present invention can also be prepared by mechanical
grinding of the
active agent. Mechanical grinding include such techniques as jet milling,
pearl milling, ball
milling, hammer milling, fluid energy milling or wet grinding techniques such
as those disclosed
in U.S. Pat. No. 5,145,684, which is incorporated herein by reference and made
a part hereof.
Another method to prepare the particles of the present invention is by
suspending an
active agent. In this method, particles of the active agent are dispersed in
an aqueous medium by
adding the particles directly into the aqueous medium to derive a pre-
suspension. The particles
are normally coated with a surface modifier to inhibit the aggregation of the
particles. One or
more other excipients can be added either to the active agent or to the
aqueous medium.
Example 1: Preparation of 1% Itraconazole Suspension with deoxycholic acid
coating.
Each 100 mL of suspension contains:
Itraconazole 1.0 g (1.0% w/v)
Deoxycholic Acid, Sodium Salt, Monohydrate 0.1 g (0.1% w/v)
Poloxamer 18~, NF 0.1 g (0.1% w/v)
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Glycerin, USP 2.2 g (2.2% w/v)
Sodium Hydroxide, NF (0.1 N or 1.0 N) for pH Adjustment
Hydrochloric Acid, NF (0.1 N or 1.0 N) for pH Adjustment
Sterile Water for Injection, USP QS
Target pH (range) 8.0 (6 to 9)
Preparation of Surfactant Solution (2 Liters) for Microprecipitation
Fill a properly cleaned tank with Sterile Water for hlj action and agitate.
Add the required
amount of glycerin and stir until dissolution. Add the required amount of
deoxycholic acid,
sodium salt monohydrate and agitate until dissolution. If necessary, adjust
the pH of the
surfactant solution with minimum amount of sodium hydroxide and/or
hydrochloric acid to a pH
of 8Ø Filter the surfactant solution through a 0.2 pm filter. Quantitatively
transfer the surfactant
solution to the vessel supplying the homogenizer. Chill the surfactant
solution in the hopper with
mixing.
Preparation of Replacement Solution
Preparation of 4 liters of replacement solution. Fill a properly cleaned tank
with WFI and
agitate. Add the weighed Poloxamer 188 (Spectrum Chemical) to the measured
volume ofwater.
Begin mixing the Poloxamer 188/ water mixture until the Poloxamer 188 has
completely
dissolved. Add the required amount of glycerin and agitate until dissolved.
Once the glycerin
has completely dissolved, add the required amount of deoxycholic acid, sodium
salt monohydrate
and stir until dissolution. If necessary, adjust the pH of the wash solution
with the minimum
amount sodium hydroxide and/or hydrochloric acid to a pH of 8Ø Filter the
replacement
solution through a 0.2 pm membrane filter.
Preparation of Drug Concentrate
For a 2-L batch, add 120.0 mL of N-methyl-2-pyrrolidinone into a 250-mL
beaker.
Weigh 2.0 g Poloxamer 188. Weigh 20.0 g of itraconazole (Wyckoff). Transfer
the weighed
Poloxamer 188 to the 250 mL beaker with N-methyl-2-pyrrolidinone. Stir until
dissolved, then
add the itraconazole. Heat and stir until dissolved. Cool the drug concentrate
to room
temperature and filter through a 0.2-micron filter.
Microprecipitation
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Add sufficient WFI to the surfactant solution already in the vessel supplying
the
homogenizes so that the desired target concentration is reached. When the
surfactant solution is
cooled, start adding the drag concentrate into the surfactant solution with
continuous mixing.
Homogenization
Slowly increase the pressure of the homogenizes until the operating pressure
10,000 psi
has been reached. Homogenize the suspension with recirculation while mixing.
For 2,000 mL of
suspension at SOHz, one pass should require approximately 54 seconds.
Following
homogenization, collect a 20-mL sample for particle size analysis. Cool the
suspension.
Wash Replacement
The suspension is then divided and filled into 500-mL centrifuge bottles.
Centrifuge until
clean separation of sediment is observed. Measure the volume of supernatant
and replace with
fresh replacement solution, prepared earlier. Quantitatively transfer the
precipitate from each
centrifuge bottle into a properly cleaned and labeled container for
resuspension (pooled sample).
Resuspension of the pooled sample is performed with a high shear mixer until
no visible clumps
are observed. Collect a 20-mL sample for particle size analysis.
The suspension is then divided and filled into 500-mL centrifuge bottles.
Centrifuge until
clean separation of sediment is observed. Measure the volume of supernatant
and replace with
fresh replacement solution, prepared earlier. Quantitatively transfer the
precipitate from each
centrifuge bottle into a properly cleaned and labeled container for
resuspension (pooled sample).
Resuspension of the pooled sample is performed with a high shear mixer until
no visible clumps
are observed. Collect a 20-mL sample for particle size analysis.
Second Homogenization
Transfer the above suspension to the hopper of the homogenizes and chill the
suspension
with mixing. Slowly increase the homogenizes pressure until an operating
pressure 10,000 psi
has been reached. Homogenize while monitoring the solution temperature.
Following
homogenization, cool the suspension and collect three 30-mL samples for
particle analysis.
Collect the remaining suspension in a 2-liter bottle.
Filling
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Based on acceptable particle size determination testing (mean volume-weighted
diameter
of 50 nm to 5 microns), collect 30 mL samples in 50 mL glass vials with rubber
stoppers.
Example 2: Preparation of 1% Itraconazole Nanosuspension with phospholipid
coatings
Each 100 mL of suspension contains:
Itraconazole 1.0 g (1.0% w/v)
Phospholipids (Lipoid E 80) 1.2 g (1.2% w/v)
Glycerin, USP 2.2 g (2.2°/~ w/v)
Sodium Hydroxide, NF (0.1 N or 1.0 N) for pH Adjustment
Hydrochloric Acid, NF (0.1 N or 1.0 N) for pH Adjustment
Sterile Water for Injection, USP QS
Target pH (range) 8.0 (7.5 to 8.5)
Preparation of Surfactant Solution (2 Liters) for Microprecipitation
The surfactant solution is prepared in two phases. Phase 1 is dispersed
phospholipids,
whereas Phase 2 includes filtered glycerin. The two fractions are combined
prior to pH
adjustment.
Phase 1: Fill a properly cleaned vessel with approximately 700 mL of Sterile
Water for W j ection,
USP (WFI) with agitation at 50 - 500 rpm. Increase the temperature of the
filtrate to 50°C - 70°C
and add the required amount of phospholipids with mixing at 50 - 500 rpm until
complete
dispersion is achieved. Document the time and temperature at which the
phospholipids were
added and at which it was dispersed. Determine the total mixing time required
to disperse the
phospholipids. Cool the surfactant solution to 18°C - 30°C prior
to the addition of glycerin.
Phase 2: Fill a properly cleaned vessel with approximately 700 mL of WFI with
agitation at 50 -
500 rpm. Add the required amount of glycerin at 18°C - 30°C and
agitate at 50 - 500 rpm until
dissolution.
Combined Phases: Filter the glycerin solution through a 0.2 ~,m filter set-up
into Phase 1 (at
18°C - 30°C) while mixing at 50 - 500 rpm. Volume is
approximately 1.4 liters. Record the pH
of the surfactant solution. If necessary, adjust the pH of the surfactant
solution with a minimum
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amount of sodium hydroxide and/or hydrochloric acid to a pH of 8.0 ~ 0.5.
Measure the volume
of the surfactant solution at 18°C - 30°C using a 2-L graduated
cylinder.
Quantitatively transfer the surfactant solution to the vessel supplying the
homogenizer (Avestin
C-160). Chill the surfactant solution in the hopper with mixing at a speed
with an observable
solution vortex until the temperature is not more than 10°C.
Preparation of Replacement Solution (4 L)
The replacement solution is prepared in two phases. Phase 1 includes dispersed
phospholipids,
whereas Phase 2 includes filtered glycerin. The two fractions are combined
prior to pH
adjustment.
Phase 1: Fill a properly cleaned vessel with approximately 1.4 liters of WFI
with agitation at 50 -
500 rpm. Increase the temperature of the water to 50°C - 70°C
and add the required amount of
phospholipids with mixing at 50 - 500 rpm until complete dispersion is
achieved. Cool the
surfactant solution to 18°C - 30°C prior to the addition of
glycerin.
Phase 2: Fill a properly cleaned vessel with approximately 1.4 L of WFI with
agitation at 50 -
500 rpm. Add the required amount of glycerin and agitate at 50 - 500 rpm until
dissolution.
Combined Phases: Filter the glycerin solution through a 0.2 ~,m filter set-up
into Phase 1 (at
18°C - 30°C) while mixing at 50 - 500 rpm. Dilute to volume with
Water for Injection to 4.0 L
using a graduated cylinder. Record the pH of the wash solution. If necessary,
adjust the pH of
the wash solution with the minimum amount sodium hydroxide and/or hydrochloric
acid to a pH
of8.0~0.5.
Preparation of Drug Concentrate
For a 2-L batch, add 120.0 mL of N-methyl-2-pyrrolidinone (Pharmasolve~, ISP)
into a
250-mL beaker. Weigh 20.0 g of itraconazole (Wyckoff). Transfer the weighed
itraconazole to
the 250-mL beaker with NMP at NMT 70°C. Maintain below 70°C and
stir at 100 -1000 rpm
until dissolved. Cool the drug concentrate to 18°C - 30°C.
Filter the drug concentrate through a
prefilter and filter set-up. Use one polypropylene prefilter SBPP and two 0.2
~,m filters at 15 psi
and ambient temperature. Transfer the drug concentrate to three 60-rnL
syringes and attach
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syringe needles to the luer connections of the syringes. Using the syringes,
determine the volume
of drug concentrate.
Microprecipitation
Add Water for Injection to the surfactant solution already in the vessel
supplying the
homogenizes. The amount of water at 18°C - 30°C added should be
calculated as:
V = 2,000 mL - Volume of Drug Concentrate - Volume of Surfactant Solution
Mount each syringe needle assembly using a syringe pump. Position the outlet
of the needle on
top of the vessel. When the surfactant solution is not more than 10°C,
start adding the drug
concentrate into the surfactant solution with continuous mixing at a speed
needed to create a
distinctive solution vortex. The concentrate should be added so that the drops
hit the point of
highest shear, at the bottom of the vortex. The rate of addition should be
approximately 2.5
mL/min.
Homogenization
An Avestin C160 homogenizes was used. Slowly increase the pressure of the
homogenizes until the operating pressure 10,000 psi has been reached.
Homogenize the
suspension for 20 passes (18 minutes) with recirculation while mixing at 100 -
300 rpm and
maintaining the suspension temperature below 70°C. For 2,000 mL of
suspension at 50 Hz, one
pass requires approximately 54 seconds. Following homogenization, collect a 20
mL sample in a
50 mL glass vial for particle size analysis. Cool the suspension to not more
than 10°C.
Wash Replacement
The suspension is then divided and filled into 500-mL centn-ifuge bottles. Set
the speed
for the centrifuge at 11,000 rpm using the rotor SLA-3000, Superlite
equivalent to approximately
20,434 g. The total centrifuge time is 60 min at not more than 10°C.
Measure the volume of
supernatant and replace with fresh replacement solution. Using spatula(s),
quantitatively transfer
the precipitant from each centrifuge bottle into a properly cleaned and
labeled container for
resuspension (pooled sample). Resuspension of the pooled sample is performed
with a high
shear mixing until no visible clumps are observed.
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Second Washing and Centrifuging Step
The suspension is then divided and filled into 500-mL centrifuge bottles. Set
the speed
for the centrifuge at 11,000 rpm using the rotor SLA-3000, Superlite
equivalent to approximately
20,434 g. The total centrifuge time is 60 min at not more than 10°C.
Measure the volume of
supernatant and replace with fresh replacement solution. Using spatula(s),
quantitatively transfer
the precipitant from each centrifuge bottle into a properly cleaned and
labeled container for
resuspension (pooled sample). Resuspension of the pooled sample is performed
under high-shear
mixing until no visible clumps are observed. Record the pH of the suspension.
If necessary,
adjust the pH of the suspension with the minimum amount sodium hydroxide
andlor hydrochloric
acid to a pH of 8.0 ~ 0.5.
Second Homogenization
Transfer the above suspension to the hopper of the homogenizes. Chill the
suspension
with mixing until the temperature is less than 10°C. Slowly increase
the pressure until an
operating pressure of 10,000 psi has been reached. Homogenize for 20 passes
(18 minutes) while
maintaining the solution temperature below 70°C. Following
homogenization, cool the
suspension to less than 10°C and collect three 30-mL samples for
particle-size analysis. Collect
the remaining suspension in a 2-liter bottle. Spaxge the suspension with
nitrogen gas for 10 min.
Ensure the nitrogen gas is filtered through a 0.2 ~,m filter.
Filling
Based on acceptable particle size determination testing (mean volume-weighted
diameter
of 50 to 1000 nm), collect 30-mL samples in 50-mL glass vials with PTFE~-
coated stoppers.
Purge the headspace of each vial with nitrogen prior to sealing.
Example 3: Other formulations of Itraconazole Suspensions
Other formulations of itraconazole suspensions with different combinations of
the
surfactants can also be prepared using the method described in Example 1 or
Example 2. Table 1
summarizes the compositions of the surfactants of the various itraconazole
suspensions.
Table 1: Summary of the compositions of the various 1~/o itraconazole
suspensions
Formulation No. ~ Surfactants in the formulation Amount*
1 Poloxamer 188 0.1%
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Deoxycholate 0.1%
Gl cerin 2.2%
2 Poloxamer 188 0.1%
Deoxycholate 0.5%
Gl cerin 2.2%
3 Poloxamer 188 2.2%
Deoxycholate 0.1
Gl cerin 2.2%
4 Poloxamer 188 2.2%
Deoxycholate 0.5%
Gl cerin 2.2%
9 Solutol 0.3%
Deoxycholate 0.5%
Gl cerin 2.2%
14331-1 Solutol 1.5%
Gl cerin 2.2%
14443-1 Albumin S%
14 Phospholipid 2.2%
Deoxycholate 0.5%
Glycerin 2.2%
NazP04
0.14%
A6 Phospholipid 1.2%
Gl cerin 2.2%
B Phospholipid 1.2%
Glycerin 2.2%
N-meth 1-2- olidinone trace
C Phospholipid 1.2%
Glycerin 2.2%
Lactic acid trace
14412-3 Phospholipid 1.2%
Hydroxyethyl starch 1.0%
Glycerin 2.2%
TRIS 0.06%
* % by weight of the final volume of the suspension (w/v)
Example 4: Comparison of the acute toxicity between commercially available
itraconazole
formulation (SPORANOX~) and the suspension compositions of the present
invention.
The acute toxicity of the commercially available itraconazole formulation
(SPORANOX~) is compared to that of the various 1 % itraconazole formulations
in the present
invention as listed in Table 1. SPORANOX~ is available from Janssen
Pharmaceutical
Products, L.P. It is available as a 1% intravenous (LV.) solution
solubilizedbyhydroxypropyl-(3-
cyclodextrin. The results are shown in Table 2 with the maximum tolerated dose
(MTD)
indicated for each formulation.
Table 2: Comparison of the acute toxicity of various formulations of
itraconazole
Formulation Number Results and Conclusions
SPORANOX'~ LV. LD,o=30 mg/kg
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MTD=20 m /k sli ht ataxia
MTD=320 mg/kg; NOEL=80 mg/kg
Spleen obsb: 320 mg/kg
Red ears/feet: >_160 m /k
2 MTD=320 mg/kg
Spleen obs~: 320 mg/kg
Slight lethargy: 320 mg/kg
Red urine: >80 mg/kg
Tail obs': >_40m /k
3 MTD=160 mg/kg; NOEL=80 mg/kg
Spleen obsb: 320 mg/lcg
Red ears/feet: >_160 m /k
4 MTD=160 mg/kg
LDZO= 320 mg/kg
Spleen obsb: 320 mg/kg
Slight lethargy: 320 mg/kg
Red urine: >_40 mg/kg
Tail obs: >_40 m /k
9 LD6o=320 mg/kg; MTD=160 mg/kg
Spleen obsb: 320 mg/kg
Tail obs: 320 mg/kg
Red ears/feet: >_160 mg/kg
Red urine: >_40 m /k
14331-1 MTD=40 mg/kg; NOEL=40 mg/kg
LDP=80 m /k
14443-1 LD4o=80 m ; NOEL=40 m /k
14 MTD=320 mg/kg; NOEL=40-80 mg/kg
Spleen obsb: 320 mg/kg
Ataxia=320 mg/kg
Tail obs=320 mg/kg
A6 MTD=320 mg/kg; NOEL=160 mg/kg
Spleen obsb: 320 m g
B MTD=320 mg/kg; NOEL=80 mg/kg
Spleen obsb: 160 mg/kg
Red earslfeet: >_160 mg/kg
C MTD=320 mg/kg; NOEL=80 mg/kg
Spleen obsb: >_160 mg/kg
Red ears/feet: >_160 m /k
14412-3 MTD=320 mg/kg; NOEL=80 mg/kg
Spleen obsb: >_160 m /kg
acyclodextrin = hydroxypropyl-~3-cyclodextrin
bSpleen obs = Enlarged and/or pale
°Tail obs = gray to black and/or necrosis
LDIO = Lethal dose resulting in 10% mortality
LD4o = Lethal dose resulting in 40% mortality
LDSO = Lethal dose resulting in 50°/~ mortality
NOEL = No effect level
MTD = Maximum tolerated dose
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The data in Table 2 indicated that the animals tolerated a much higher level
of the
antifungal agent itraconazole when formulated in a nanosuspension than when
formulated as a
solution with cyclodextrin. It may be thought that the reason for the
increased tolerability is
associated with not using cyclodextrin. However, cyclodextrin, by itself, at
the levels used in
Sporanox would not cause the degree of toxicity observed. Father, it is
believe, the reason lies iiz
alteration of the phannacokinetic profile caused by the nanosuspension.
Example 5: Pharmacokinetic comparison of SPOFANOX~ vs. suspension formulation
of
itraconazole.
Young adult, male Sprague Dawley rats were treated intravenously (IV) via a
caudal tail
vein with a single injection at a rate of 1 ml/min. with either SPORANOX~
hljection, or
Formulations 1 and B at 20, 40, and 80 mg/kg, or Formulations 3, 14, A6 and C
at 80 mg/kg.
Following administration, the animals were anesthetized and retro-orbital
blood was
collected at different time points (n=3). The time points were as follows:
0.03, 0.25, 0.5, 1, 2, 4,
6, 8, 24, 48, 96, 144, 192, 288, and 360 hours (SPORANOX~ Injection only to
192 hours).
Blood was collected into tubes with EDTA and centrifuged at 3200 rpm for 15
minutes to
separate plasma. The plasma was stored frozen at -70°C until analysis.
The concentration of the
parent itraconazole and the metabolite hydroxy-itraconazole were determined by
high-
performance liquid chromatography (HPLC). Pharmacokinetic (PIE) parameters for
itraconazole
(ITC) and hydroxy-itraconazole (OH-ITC) were derived using noncompartmental
methods with
WinNonlin° Professional Version 3.1 (Pharsight Corp., Mountain
View, CA).
Table 3 provides a comparison of the plasma pharmacokinetic parameters
determined for
each itraconazole formulation. Plasma itraconazole was no longer detected at
24 hours for
SPORANOX~ Injection at 5 mg/kg, at 48 hours for SPORANOX~ Injection at 20
mg/kg, and at
96 hours for Formulations 1 and B. Plasma hydroxy-itraconazole was initially
detected at 0.25
hours for SPORANOX~ Inj action and Formulations 1 and B. Plasma hydroxy-
itraconazole was
initially detected at 0.25 hours for SPORANOX~ Inj action at 5 and 20 mg/kg
and Formulations
1 and ;B at 20 mg/kg, Hydroxy-itraconazole was no longer detected at 48 hours
for
SPORANOX~ Injection at Smg/kg, at 96 hours for SPORANOX° hzjection at
20 mg/kg, and at
144 hours for Formulations 1 and B.
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WO 2004/096180 PCT/US2004/013268
33
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34
THIS PAGE WAS NOT FURNISHED UPON FILING
THE INTERNATIONAL APPLICATION
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FIG. 5 compares the pharmacokinetics (PK) of SPORANOX~ with Formulation 1
suspension of itraconazole particles. Eecause, as shown above, the present
suspension
formulation is less toxic than Sporanox~, it was administered at higher
amounts in this equitoxic
experiment. Sporanox was dosed at 20 mglkg and Formulation 1 at ~0 mg/kg. The
SPORANOX~ decreases in plasma concentration relatively quickly, over 20 hours.
The
nanosuspension plasma levels remain elevated for approximately 3-4 times
longer. The
nanosuspension exhibits an initial minimum at 30 minutes in the plasma level.
This corresponds
to a nadir in plasma concentration due to sequestration of the drug
nanocrystals by the
macrophages ofthe spleen and liver, thus temporarilyremoving drug from
circulation. However,
the drug levels rebound quickly, as the macrophages apparently release the
drug into the
circulation. Furthermore, the nanosuspension drug is metabolized effectively,
as is shownbythe
PK curve for the hydroxy itraconazole metabolite. The rate of appearance of
the metabolite for
the nanosuspension is delayed, compared with the PK curve for the metabolite
for the
SPORANOX~ formulation. However, as with the case of the parent molecule for
the
nanosuspension, the metabolite persists in circulation for a much longer time
than is the case with
the metabolite for the SPORANOX~ formulation. When the AUC (area under the
blood
concentration vs time curve) is normalized by the dose, the nanosuspension is
at least as
bioavailable as SPORANOX~.
Example 6: Acute Toxicity Of Fast Dissolving Nanosuspensions
Additional experiments were performed. Itraconazole nanosuspensions were
formulated
differently, so as to dissolve much more readily in blood. This was
accomplished by making the
particles either smaller or amorphous, or both. These acute toxicity of these
formulations is
described for formulation entries 14331-1 and 14443-1 in Table 1. In contrast
to the slowly
dissolving nanosuspensions, the fast dissolving nanosuspension caused death in
the animals at
much lower levels, similar to what was found with SPORANOX~. Since these fast
dissolving
nanosuspensions did not contain cyclodextrin, it is clear that this excipient
was not responsible
for the toxicity. Rather the rapid dissolution, resulting in immediate
availability of the drug in the
blood was the causative factor. The drug level for the rapidly dissolving
formulation, Form A, is
much higher than that attained by the slow dissolving (macrophage targeting)
formulation, Form
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B, as determined in an in vitro dissolution experiment. This involved a plasma
simulating media
consisting of 5% albumin/ Sorenson's buffer. Results are shown in Figure 6.
Example 7: Antifungal Efficacy Studies
Normal and inununo-suppressed (prednisolone administered twice daily on the
day before
and on the day of inoculation) rats inoculated with 9.5 x 106 or 3 x 106 cfu
C. albicaraslml saline
once intravenously were intravenously treated with SPORANOX~ Injection once
daily for ten
consecutive days, with the first dose given 4 to 5 hours after inoculation.
SPOR~TOX~
Injection rats were dosed at 5 or 20 mg/kg for the first 2 days, then at 5 or
10 mg/kg for the
remaining 8 days, due to toxicity at 20 mg/kg after 2 days of dosing.
Similarly, immuno-
suppressed rats inoculated with 1 x 106'5 cfu C. albicanslml saline were
intravenously treated
with Formulation 1 or B each at 20, 40, or 80 mg/kg once every other day for
ten days, beginning
the day of inoculation. The SPORANOX~ Injection, Formulation 1, and
Formulation B
treatment rats were terminated 11 days after the C. albieans inoculation and
the kidneys were
collected, weighed and cultured for determination of C. albicafzs colony
counts and itraconazole
and hydroxy-itraconazole concentration. Kidneys were collected from untreated
control rats
when a moribund condition was observed or when an animal had a 20% body
weight. In
addition, body weights were measured periodically during the course of each
study.
Comparison of results for immuno-suppressed rats treated with SPORANOX~ Inj
ection
and Formulations 1 and B are shown in Table 4 and Figure 7. Daily SPORANOX~
Injection
treatment at 10 - 20 mg/kg appeared to be slightly more effective than daily
treatment with
SPOR.ANOX~ Injection at 5 mg/kg. Based on kidney colony counts, every other
day dosing at
20 mg/kg of Formulation 1 or B appeared to be as effective as every day dosing
with
SPORANOX~ Injection at 20 mg/kg and possiblymore effective than SPORANOX~
Injection at
5 mg/kg (i.e., the recommended clinical dose), whereas the higher doses for
both Formulation 1
and B appeared to most effective, based on kidney colony counts (i.e., C.
albicans not detected)
and increased kidney itraconazole concentration.
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Table 4. Mean C. albicahs Colony Count and Itraconazole and Hydroxy-
Itraconazole
Concentration in Kidney
C. albiearts Concentration
Titer in
Kidney
Count IncidenceITC OI=I-ITC
Treatment (cfu/g) (~g/g) (~.g/g)
No Treatment (3x 106cfu/xnl) 6.9 x 6/6 -- --
104
SPORANOX~, 5 mg/kg, (3x 106cfu/ml)96.5 6/6 1.2 1.5
SPORANOX~, 10-20 mglkg, (3 12.4 4/6 8.5 8.0
x 106 cfu/ml)
No Treatment (2.Sx 106cfu/ml)3.5 x 6/6 -- --
105
Formulation 1, 20 mg/kg, (2.55.3 4/6 6.1 5.7
x 106 cfu/ml)
Formulation 1, 40 mg/kg, (2.50 0/6 18.5 6.0
x 106 cfu/ml)
Formulation 1, 80 mglkg, (2.50 O/6 41.2 6.2
x 106 cfu/ml)
No Treatment (2.Sx 106cfu/ml)8.0 x 6/6 -- --
104
Formulation B; 20 mg/kg, (2.58.9 4/6 2. S 2. S
x 106 cfu/ml)
Formulation B, 40 mglkg, (2.50 0/6 7.8 4.0
x 106 cfu/ml)
Formulation B, 80 mg/kg, (2.50 0/6 21.3 4.6
x 106 cfu/ml)
In the examples above, a rianosuspension formulation of an anti-fungal agent
was shown
to be less toxic than a conventional totally soluble formulation of the same
drug. Thus, more of
the drug could be administered without eliciting adverse effects. Because the
nanoparticles of the
drug did not immediately dissolve upon inj ection, they were trapped in a
depot store in the liver
and spleen. These acted as prolonged release sanctuaries, permitting less
frequent dosing. The
greater dosing that could be administered permitted greater drug levels to be
manifested in the
target organs, in this case, the kidney (Figure 8). The greater drug levels in
this organ led to a
greater kill of infectious organisms. (Figure 9).
Example 8: Resistant Strain Anti-fiuzgal Efficacy Test
A lethal dose of a C. albicans strain c43 (ATCC number 201794) (MICRO=16
~,g/ml for
SPORANOX~ itraconazole; 8-16 for Vfend, and 0.1 for Cancidas) was administered
to an
immunocompromised rat model (prednisolone qd). 24h later, test groups (n=6)
were treated q2d
with 20, 40, or 80 mg/kg NANOEI~GET"" itraconazole nanosuspension. Control
groups included a
no treatment arm, Sporanox~ (10 mg/kg/d), Vfend~ (10 mg/kg/d), and Cancidas~
(lmg/kg/d).
Treatment was continued for 10 days. Survival and kidney cfulg were assessed.
CA 02523151 2005-10-21
WO 2004/096180 PCT/US2004/013268
-38-
The number of surviving animals after 6 and 10 days, were respectively:
Sporanox (3,0),
20 and 40 mg/kg nanosuspension (5,3), 80 mg/kg nanosuspension (6,4), Vfend
(0,0), Cancidas
(0,0). FIG. 10.
It can be concluded that the greater dosing possible with the itraconazole
nanosuspension
can effectively treat infections of C. czlbicczns strains, conventionally
assumed to be resistant to
itraconazole, resulting in increased survival in an immunocompromised rat
model.
Current definitions of sensitive and resistant fungal strains presume a
specified dose of
itraconazole that is administered, using conventional dosage forms. Greater
drug loading,
attendant with nanosuspension inj ections, may permit treatment of what are
currently considered
itraconazole-resistant C. albicaras infections.
Example 9: Prophetic examples of other triazole antifungal agents
The present invention contemplates preparing a 1 % suspension of submicron- or
micron
size of a triazole antifungal agent using the method described in Example 1 or
Example 2 and the
formulations described in Example 3 with the exception that the antifungal
agent is a triazole
antifungal agent other than itraconazole. Examples of triazole antifungal
agents that can be used
include, but are not limited to, ketoconazole, miconazole, fluconazole,
ravuconazole,
voriconazole, saperconazole, eberconazole, genaconazole, clotrimazole,
econazole, oxiconazole,
sulconazole, tercoriazole, tioconazole, and posaconazole.
Example 10: Prophetic example of a non-triazole antifungal agent
The present invention contemplates preparing a 1 % suspension of submicron- or
micron
size non-triazole antifungal agent using the method described in Example 1 or
Example 2 and the
formulations described in Example 3 with the exception that the antifungal
agent is amphotericin
B, nystatin, terbinafine, anidulafungin, or flucytosine instead of
itraconazole.
From the foregoing, it will be observed that numerous variations and
modifications may
be effected without departing from the spirit and scope of the invention. It
is to be understood
that no limitation with respect to the specific apparatus illustrated herein
is intended or should be
inferred. It is, of course, intended to cover by the appended claims all such
modifications as fall
within the scope of the claims.
