Title of Invention

PROCESS FOR PRODUCING NANOPARTICLES OF PACLITAXEL AND ALBUMIN

Abstract A process for producing a sterile lyophilized powder consisting of nanoparticles of paclitaxel and human serum albumin, by which an aqueous mixture containing paclitaxel and albumin at a temperature between 0°C and 40°C is subjected to homogenization treatment at high pressure between 9000 and 40000 psi, to give a nanoemulsion which is frozen between -20°C and -80°C and is finally lyophilized by heating to between +20°C and +35°C, characterized in that said aqueous mixture is obtained under sterile conditions by dissolving between 2% and 3% (w/v) of said albumin in sterile water, then adding to said albumin solution between 2% and 4% (v/v) of sterile chloroform and then paclitaxel in sterile powder form in a quantity between 5.4% and 20.0% by weight on the weight of the albumin present in the solution.
Full Text PROCESS FOR PRODUCING NANOPARTICLES OF PACLITAXEL AND
ALBUMIN
The present invention relates to a process for producing nanoparti-
cles of paclitaxel and albumin, usable for obtaining antitumor compositions.
Paclitaxel is a natural substance well known in literature, with im-
portant antitumor activity: its poor water solubility makes it difficult to ad-
minister to man, for which reason various systems have been developed to
render it injectable.
According to one of these systems, paclitaxel is combined with hu-
man serum albumin (HSA) which is biocompatible and has considerable
capacity to bind to the paclitaxel and form injectable emulsions therewith
by known ultrasonication, high pressure homogenization and microfluidi-
zation techniques (Allemann et a1., Eur. J. Pharm. Biopharm. 39 (5), 173-
191 (1993)).
On the basis of these elements and by using the aforestated ultra-
sonication and high pressure homogenization techniques, the American
company VivoRx Pharmaceuticals Inc. has developed the formulation
CAPXOL(R) containing paclitaxel and HSA.
In US 5439686, US 5498421, US 5560933 and the corresponding
WO 94/18954, VivoRx claims microparticles of paclitaxel and HSA prepared
using ultrasonication techniques, to give particles of mean size (MPS) microns. The preparation methods described in these patents cannot be
used on an industrial scale, and moreover the microparticles thus obtained
have too high an MPS, which makes them unsuitable and unusable for
administration to patients.
This was well known to the said VivoRx, which then in US 5916596
and US 6096331 and in WO 98/14174 and WO 99/00113 described and
claimed sterile nanoemulsions of paclitaxel and HSA obtained by reconsti-
tuting with sterile aqueous 0.9% NaCl solution lyophilized powders with
MPS pressure homogenization, as described in the cited patents, are stated to
have high stability, where the term "stability" means both that the MPS is
constant with time and that nanoparticle precipitation is absent (US
6096331, Ex. 11).
According to the teachings of the aforecited VivoRx patents (see Ex-
amples 1, 5 and 6 of US 5916596), a solution of paclitaxel and an aqueous

solution containing HSA are firstly prepared separately, then these phases
are mixed together and the mixture so obtained is subjected to homogeni-
zation treatment at high pressure between 9000 and 40000 psi at room
temperature (between 0°C and +40°C).
After evaporating the solvents and filtering through a sterile filter
(0.22 microns), this mixture is frozen between -20° C and -80°C and is fi-
nally lyophilized by heating at a temperature between +20°C and +35°C to
give a powder usable for preparing injectable formulations having antitumor
properties.
Two separate phases (one containing paclitaxel and the other con-
taining HSA) must therefore be prepared, to be then mixed together before
their high pressure homogenization. This requires the use of at least two
separate reactors and the preparation of two separate solutions with relative
mixing, all to be effected under sterile conditions, involving high plant costs,
lengthy times for completing the mixing operations and the need for ro-
tavapor evaporation of the solvents (at the end of homogenization treatment)
followed by filtration through a sterile filter, with consequent low overall
yields.
The main object of the present invention is therefore to provide a
process for producing sterile lyophilized powder of nanoparticles of pacli-
taxel and HSA, which requires the use of a single reactor for forming the
liquid mixture containing paclitaxel and HSA to be subjected to homogeni-
zation treatment, and which can be completed in a very short time at lower
cost than that of the known art.
These and further objects are attained by a process by which an
aqueous mixture containing paclitaxel and albumin at a temperature be-
tween 0°C and 40°C is subjected to homogenization treatment at high pres-
sure between 9000 and 40000 psi, to give a nanoemulsion which is frozen
between -20°C and -80°C and is finally lyophilized by heating at a tempera-
ture between +20°C and +35°C, wherein said aqueous mixture is obtained
under sterile conditions by dissolving said albumin in sterile water to a con-
centration between 2% and 3% (w/v), then adding to said albumin solution
between 2% and 4% (v/v) of chloroform and then paclitaxel in sterile powder
form in a quantity between 5.4% and 20.0% by weight on the weight of the
albumin present in the solution. The quantity of paclitaxel in sterile powder
form added to the liquid mixture is preferably between 5.6% and 19.4% by
weight on the albumin.

It is important to note that the use of paclitaxel in sterile powder form
in the process not only greatly simplifies the plant itself and the process
compared with the known art and enables the time required to complete the
mixing of the various components before the homogenization treatment to
be considerably shortened, but also enables better final yields to be ob-
tained and simplifies the conditions to be observed in order to obtain the
desired sterile lyophilized powders.
By operating in the aforestated manner powders formed by mixtures
of nanoparticles of paclitaxel and HSA are obtained which are totally similar
or equal to those obtainable by the more complex, laborious and costly
methods described in the aforestated prior patents.
From these mixtures, when processed with an Avestin homogenizer
within the pressure range recommended in US 5916596, nanoemulsions at
pH=6.7 are obtained which, when evaporated in a rotavapor as reported in
the said patent, provide nanoemulsions with MPS of about 0.2 microns
(increase of MPS > 0.02 microns after evaporation) which are poorly stable
in their formulations in injectable physiological solutions (increase in MPS
of about 0.05 microns and tendency to sediment in about 12 hours) and
difficult to filter through 0.22 microns filters for their sterilization, these
filters being easily clogged and reducing the paclitaxel yield to a very low
value (down to or less than 30%).
The stability (evaluated in accordance with the teachings of Example
11 of US 6096331) of the products prepared both by the method of the pre-
sent invention as hereinbefore defined and in accordance with the prior
patents, when lyophilized and reconstituted as reported in US 5916596 and
US 6096331, is acceptable but never exceeds 24 hours.
It has been surprisingly found that if at least one biocompatible acid
is added to the liquid mixture containing the HSA (before the paclitaxel in
powder form is added to it) in a quantity sufficient to bring to between 5.4
and 5.8 (preferably between 5.5 and 5.7) the pH of a reconstituted aqueous
injectable mixture of the nanoparticles in powder form, the stability of the
lyophilized and reconstituted mixture in injectable form considerably in-
creases, beyond 24 hours.
The addition of the aforesaid acid or also forms part of the present
invention.

Preferably, said acid is chosen from the group consisting of HC1, citric
acid, phosphoric acid, acetic acid, biocompatible organic and inorganic ac-
ids, but citric acid is the most preferred one.
To clarify the characteristics of the present invention, some non-
limiting examples of its implementation will now be described, some with
liquid mixtures at physiological pH and some acidified to highlight the dif-
ferences consequent on the use of the acids.
EXAMPLE 1
Preparation of formulation at pH 6.7
An injectable aqueous 20% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 3% (w/v) with sterile demin-
eralized water.
41.4 ml of said solution are mixed under energetic agitation with 1.25
ml of sterile chloroform and with 73.6 mg (5.9% by weight on the weight of
the albumin in the solution) of sterile paclitaxel (titre > 99%) in powder
form, then the mixture is processed in a high pressure homogenizer
(suitably sterilized) until a nanoemulsion (MPS about 0,2 microns) is ob-
tained, this being evaporated under vacuum to remove the solvent, frozen
and lyophilized under sterile conditions for 48 hours.
The powder obtained, containing 4.60 % (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 2 mg/ml. The formulation obtained has an MPS of 0.486 microns,
pH=6.7, and a stability The product obtained has the same characteristics as that prepared
by the method used in Example 1 of US 5916596.
EXAMPLE 2
Preparation of formulation at pH 6.7
An injectable aqueous 25% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 2% (w/v) with sterile demin-
eralized water.
49.0 ml of said solution are mixed with 1.0 ml of sterile chloroform
and with 72.5 mg (7.4% to albumin) of sterile paclitaxel (titre > 99%) in
powder form, then the mixture is processed in a high pressure homogenizer
(suitably sterilized) until a nanoemulsion (MPS about 0,2 microns) is ob-
tained, this being evaporated under vacuum to remove the solvents, filtered
through a sterile filter (0.2 microns), frozen and lyophilized under sterile
conditions for 48 hours.

The powder obtained, containing 0.60% (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 2 mg/ml. The formulation obtained has an MPS of 0,25 microns,
pH=6.7, and a stability EXAMPLE 3
Preparation of formulation at pH 6.7
An injectable aqueous 20% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 3% (w/v) with sterile demin-
eralized water.
46.7 ml of said solution are mixed with 1.40 ml of sterile CHCI3 and
with 108.5 mg (7.7% to albumin) of sterile paclitaxel (titre > 99%) in powder
form, then the mixture is processed in a high pressure homogenizer
(suitably sterilized) until a nanoemulsion (MPS about 0.2 microns) is ob-
tained, this being evaporated under vacuum to remove the solvents, filtered
through a sterile filter (0.2 microns), frozen and lyophilized under sterile
conditions for 48 hours.
The powder obtained, containing 0.77 % (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 2 mg/ml. The formulation obtained has an MPS of 0,12 microns,
pH=6.7, and a stability As already stated in the initial descriptive part, the filtration resulted
in a considerable loss of paclitaxel (the lyophilized powder contains 0.55%
of paclitaxel instead of the 5.2% of Example 2). This enabled a formulation
to be obtained with MPS EXAMPLE 4
Preparation of formulation at pH 6.7
An injectable aqueous 25% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 3% (w/v) with sterile demin-
eralized water.
29.1 ml of said solution are mixed with 0.90 ml of sterile CHCl3 and
67.0 mg (7.7% to albumin) of sterile paclitaxel (titre > 99%) in powder form,
then the mixture is processed in a high pressure homogenizer (suitably
sterilized) until a nanoemulsion (MPS about 0.2 microns) is obtained, this
being evaporated under vacuum to remove the solvents, filtered through a
sterile filter (0.2 microns), frozen and lyophilized under sterile conditions for
48 hours.

The powder obtained, containing 0.70% (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 1.5 mg/ml. The formulation obtained has an MPS of 0.25 microns,
pH=6.7, and a stability The product obtained has the same characteristics as that prepared
by the method used in Example 5 of US 5916596.
EXAMPLE 5
Preparation of formulation at pH 6.7
An injectable aqueous 20% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 2.5% (w/v) with sterile demin-
eralized water and presaturated with chloroform (1% v/v).
48.5 ml of said solution are mixed with 1.0 ml of sterile CHC13 and
with 75 mg (6.2% to albumin) of sterile paclitaxel (titre > 99%) in powder
form, then the mixture is processed in a high pressure homogenizer
(suitably sterilized) until a nanoemulsion (MPS about 0.2 microns) is ob-
tained, this being evaporated under vacuum to remove the solvent, filtered
through a sterile filter (0.2 microns), frozen and lyophilized under sterile
conditions for 48 hours.
The powder obtained, containing 0.70 % (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 2.2 mg/ml. The formulation obtained has an MPS of 0.18 microns,
pH=6.7, and a stability Again in this case the observations made at the end of Example 3 are
valid.
EXAMPLE 6
Preparation of formulation at pH=5.6
An injectable aqueous 25% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 3% (w/v) with sterile demin-
eralized water, the pH being corrected to a value of 5.6 with 1M HC1 which
salifies some basic groups present in albumin.
57 ml of said solution, previously sterilized, are mixed under vigorous
stirring for at least 30 minutes, with 1.40 ml of sterile chloroform and with
108 mg (6.3% to albumin) of sterile paclitaxel (titre > 99%) in powder form.
The mixture is processed in a homogenizer (suitably sterilized) at
high pressure (9000-40000 psi) until a nanoemulsion (MPS is obtained, this being rapidly frozen to -80°C and lyophilized for 55 hours
under sterile conditions, while raising the temperature to +30°C.

The powder obtained, containing 4.83 % (w/w) of paclitaxel and 4%
(w/w) of water, is reconstituted with an aqueous 0.9% NaCl solution to a
paclitaxel concentration of 2 mg/ml. The formulation obtained has an MPS
of 0.175 microns, pH=5.6, and a stability > 24 hours.
Equivalent results are obtained by using phosphoric acid instead of
hydrochloric acid.
EXAMPLE 7
Preparation of formulation at pH=5.4
An injectable aqueous 25% (w/v) HSA solution in accordance with
FDA specifications is diluted to 3% (w/v) with sterile demineralized water,
the pH being corrected to a value of 5.4 with citric acid which salifies some
basic groups present in albumin.
50 ml of said solution, previously sterilized, are mixed under vigorous
stirring for at least 40 minutes, with 1.23 ml of sterile chloroform and with
98 mg (6.5% to albumin) of sterile paclitaxel (titre > 99%) in powder form.
The mixture is processed in a homogenizer (suitably sterilized) at
high pressure (9000-40000 psi) until a nanoemulsion (MPS is obtained, this being rapidly frozen to -30°C and lyophilized for 57 hours
under sterile conditions, while raising the temperature to +35°C.
The powder obtained, containing 4.80 % (w/w) of paclitaxel and 3.8%
(w/w) of water, is reconstituted with an aqueous 0.9% NaCl solution to a
paclitaxel concentration of 2 mg/ml. The formulation obtained has an MPS
of 0.19 microns, pH=5.4, and a stability > 24 hours.
Equivalent results are obtained by using acetic acid instead of citric
acid.
EXAMPLE 8
Preparation of formulation at pH=5.5
An injectable aqueous 25% (w/v) HSA solution in accordance with
FDA specifications is diluted to 3% (w/v) with sterile demineralized water,
the pH being corrected to a value of 5.5 with sterile citric acid which salifies
some basic groups present in albumin.
37 ml of said solution are mixed under vigorous stirring for at least
40 minutes, with 0.91 ml of sterile chloroform and 71 mg (6.4% to albumin)
of sterile paclitaxel (titre > 99%) in powder form, after which the mixture is
cooled to 5-8°C.
The mixture is processed in a homogenizer (suitably sterilised) at
high pressure (9000-40000 psi) until a nanoemulsion (MPS
is obtained, this being rapidly frozen to -80°C and lyophilized for 58 hours
under sterile conditions, while raising the temperature to +30°C.
The powder obtained, containing 4.70 % (w/w) of paclitaxel and 4.5%
(w/w) of water, is reconstituted with an aqueous 0.9% NaCl solution to a
paclitaxel concentration of 2 mg/ml. The formulation obtained has an MPS
of 0.185 microns, pH=5.5, and a stability > 24 hours.
EXAMPLE 9
Preparation of formulation at pH 5.5
An injectable aqueous 20% (w/v) HSA solution in accordance with
FDA specifications (pH=6.9±0.5) is diluted to 3% (w/v) with sterile demin-
eralized water, the pH being corrected to a value of 5.5 with citric acid
which salifies some basic groups present in albumin.
110 ml of said solution are mixed with 4.10 ml of sterile CHCl3 and
with 639 mg (19.4% to albumin) of sterile paclitaxel (titre > 99%) in powder
form, then the mixture is processed in a high pressure homogenizer
(suitably sterilized) until a nanoemulsion (MPS about 0.2 microns) is ob-
tained, this being filtered through a sterile filter (0.2 microns), evaporated
under vacuum to remove the solvents, frozen and lyophilized under sterile
conditions for 48 hours.
The powder obtained, containing 10.8 % (w/w) of paclitaxel, is re-
constituted with an aqueous 0.9% NaCl solution to a paclitaxel concentra-
tion of 2 mg/ml. The formulation obtained has an MPS of 0.15 microns and
a stability > 24 hours.

We clame
1. A process for producing a sterile lyophilized powder consisting of
nanoparticles of paclitaxel and human serum albumin, by which an aque-
ous mixture containing paclitaxel and albumin at a temperature between
0°C and 40°C is subjected to homogenization treatment at high pressure
between 9000 and 40000 psi, to give a nanoemulsion which is frozen be-
tween -20°C and -80°C and is finally lyophilized by heating to between
+20°C and +35°C, characterized in that said aqueous mixture is obtained
under sterile conditions by dissolving between 2% and 3% (w/v) of said al-
bumin in sterile water, then adding to said albumin solution between 2%
and 4% (v/v) of sterile chloroform and then paclitaxel in sterile powder form
in a quantity between 5.4% and 20.0% by weight on the weight of the al-
bumin present in the solution.
2. A process as claimed in claim 1, characterized in that the quantity of
paclitaxel in sterile powder form added to said albumin solution is between
5.6% and 19.4% by weight on the albumin weight.
3. A process as claimed in claims 1 and 2, characterized by adding to
said albumin solution, before adding the paclitaxel, at least one biocom-
patible acid in a quantity sufficient to bring to between 5.4 and 5.8 the pH
of a reconstituted aqueous injectable mixture of the nanoparticles in powder
form.
4. A process as claimed in claim 3, characterized in that the quantity of
said acid is such as to bring the pH of said reconstituted aqueous solution
to between 5.5 and 5.7.
5. A process as claimed in claims 3 and 4, characterized in that said
acid is chosen from the group consisting of HC1, citric acid, phosphoric acid,
acetic acid, biocompatible organic and inorganic acids.
6. A process as claimed in claim 5, characterized in that said acid is cit-
ric acid.

A process for producing a sterile lyophilized powder consisting of
nanoparticles of paclitaxel and human serum albumin, by which an aqueous
mixture containing paclitaxel and albumin at a temperature between 0°C and
40°C is subjected to homogenization treatment at high pressure between 9000
and 40000 psi, to give a nanoemulsion which is frozen between -20°C and -80°C
and is finally lyophilized by heating to between +20°C and +35°C, characterized
in that said aqueous mixture is obtained under sterile conditions by dissolving
between 2% and 3% (w/v) of said albumin in sterile water, then adding to said
albumin solution between 2% and 4% (v/v) of sterile chloroform and then
paclitaxel in sterile powder form in a quantity between 5.4% and 20.0% by
weight on the weight of the albumin present in the solution.

Documents:

140-KOL-2003-CORRESPONDENCE 1.1.pdf

140-KOL-2003-CORRESPONDENCE 1.3.pdf

140-KOL-2003-CORRESPONDENCE-1.2.pdf

140-KOL-2003-CORRESPONDENCE.1.4.pdf

140-KOL-2003-CORRESPONDENCE.pdf

140-KOL-2003-FORM 13.pdf

140-KOL-2003-FORM 27.pdf

140-KOL-2003-FORM 3.1.1.pdf

140-KOL-2003-FORM-1.pdf

140-KOL-2003-FORM-2.pdf

140-KOL-2003-FORM-27.pdf

140-kol-2003-granted-abstract.pdf

140-kol-2003-granted-assignment.pdf

140-kol-2003-granted-claims.pdf

140-kol-2003-granted-correspondence.pdf

140-kol-2003-granted-description (complete).pdf

140-kol-2003-granted-examination report.pdf

140-kol-2003-granted-form 1.pdf

140-kol-2003-granted-form 18.pdf

140-kol-2003-granted-form 2.pdf

140-kol-2003-granted-form 3.pdf

140-kol-2003-granted-form 5.pdf

140-kol-2003-granted-form 6.pdf

140-kol-2003-granted-pa.pdf

140-kol-2003-granted-reply to examination report.pdf

140-kol-2003-granted-specification.pdf

140-kol-2003-granted-translated copy of priority document.pdf

140-KOL-2003-OTHERS 1.1.pdf

140-KOL-2003-OTHERS.1.2.pdf

140-KOL-2003-OTHERS.pdf

140-KOL-2003-PA.pdf


Patent Number 228780
Indian Patent Application Number 140/KOL/2003
PG Journal Number 07/2009
Publication Date 13-Feb-2009
Grant Date 10-Feb-2009
Date of Filing 05-Mar-2003
Name of Patentee ABRAIXIS BIOSCIENCE, INC
Applicant Address 2730 WILLSHIRE BONLEVARD, SNIFE 500, SANTA MOMICA, CALIFORNIA
Inventors:
# Inventor's Name Inventor's Address
1 MAURIZIO ZENONI VIA FLEMING, 7 20067 PAULLO (MI)
2 SIMONE MASCHIO VIA PICCINNI, 7 00199 ROME
PCT International Classification Number A61K 31/335
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 MI2002A000681 2002-03-29 Italy