Stabilized pharmaceutical composition and stabilizing solvent

A stabilized pharmaceutical composition containing paclitaxel, teniposide, camptothecin or other antineoplastic agent susceptible to degradation during storage is produced using a solvent system containing a low carboxylate anion content. The solvent system is a mixture of ethanol and a polyoxyethylated castor oil. The polyoxyethylated castor oil is treated with an acid or contacted with alumina to reduce the carboxylate anion content of the solvent. The low carboxylate anion content of the solvent provides extended shelf life and lower amounts of degradation by-products.

FIELD OF THE INVENTION 
The present invention is directed to a solvent system suitable for 
preparing a stabilized injection concentrate containing a pharmaceutical 
agent. More particularly, the invention is directed to a stabilized 
injection concentrate using a treated solvent system having a reduced 
carboxylate anion content and a method for stabilizing a pharmaceutical 
composition. 
BACKGROUND OF THE INVENTION 
Pharmaceutical compositions usually require a suitable solvent or carrier 
system to disperse the active agent to enable the composition to be 
administered to a patient. The solvent must typically be capable of 
solubilizing or dispersing a therapeutically effective amount of the 
active agent to produce an effective injection composition. Moreover, the 
solvent system must be compatible with the active agent and be non-toxic 
to the patient. 
Numerous pharmaceutical agents are not sufficiently soluble in any one 
solvent to enable the resulting composition to be effective. To overcome 
the disadvantages of the limitations of the solvent to solubilize the 
active agent, mixtures of two or more solvents are sometimes used. These 
co-solvent systems are suitable for solubilizing many pharmaceutical 
agents which cannot otherwise be solubilized or dispersed in a carrier 
system. 
One example of a co-solvent system incorporates a mixture of a polar 
solvent and a non-ionic solvent, such as a mixture of a polyethylene 
glycol and Cremophor EL. Cremophor EL is a condensation product of castor 
oil and ethylene oxide sold by BASF. Another suitable co-solvent system 
for many pharmaceutical agents is a 50:50 mixture of ethanol and Cremophor 
EL. Although these co-solvent systems can be effective in solubilizing 
many compounds, they are not without their disadvantages. For example, 
co-solvents of ethanol and Cremophor are known to result in particulates 
forming upon dilution with infusion solutions. In addition, fibrous 
precipitates of unknown composition form in some compositions during 
storage for extended periods of time. It is generally believed that the 
precipitates are decomposition byproducts of either components in the 
solvent or the solubilized agent. 
In WO91/02531, published Mar. 7, 1991, Cremophor is disclosed as reversing 
the multi-drug resistance phenotype of a tumor cell without altering the 
drug sensitivity of the parent cell line. Cremophor is also disclosed to 
increase hemopoiesis reconstituting capacity and/or maintain hemopoiesis 
reconstituting capacity following perturbation of bone marrow to protect a 
patient during radiation and/or chemotherapy cancer treatments. 
Another example of a pharmaceutical composition including a co-solvent 
system is Taxol which contains paclitaxel in a 50:50 mixture of ethanol 
and Cremophor EL. Paclitaxel is isolated from the Pacific yew bark and has 
been used to treat cancer patients. Although the ethanol and Cremophor EL 
co-solvent system is effective in solubilizing sufficient amounts of the 
paclitaxel, the resulting composition has been shown to have a limited 
shelf life. During storage for extended periods of time, the potency or 
pharmaceutical activity of the composition can decrease as much as 60%. 
It has been discovered that commercial grade Cremophor EL with ethanol as a 
co-solvent, although effective in solubilizing pharmaceutical agents, 
produces injection compositions that exhibit instability over extended 
periods of time. In particular, pharmaceutical compositions of Taxol in a 
co-solvent of 50:50 by volume of dehydrated ethyl alcohol and commercial 
grade Cremophor EL exhibit a loss of potency of greater than 60% after 
storage for 12 weeks at 50.degree. C. The loss of potency is attributed to 
the decomposition of paclitaxel during storage. 
Previous efforts to develop a shelf stable composition of some 
pharmaceutical compositions in various co-solvent systems have not been 
entirely successful. Thus, there is a continuing need in the art for a 
co-solvent system capable of being used for preparing stabilized 
compositions and, in particular, stabilized injection compositions 
containing a pharmaceutical agent. 
SUMMARY OF THE INVENTION 
The invention is directed to a solvent system and in particular a 
co-solvent system suitable for preparing stabilized injection compositions 
containing at least one pharmaceutical agent. Accordingly, it is a primary 
object of the invention to provide a method of preparing a solvent and a 
method of preparing stabilized pharmaceutical compositions including the 
novel solvent. 
The stabilized pharmaceutical composition produced from the solvent system 
of the invention has been shown to have a shelf life greater than the 
previous compositions. The co-solvent system of the invention is 
particularly suitable for use with pharmaceutical compounds that exhibit 
decomposition which is catalyzed by carboxylate anions. Of particular 
interest are the antineoplastic agents such as paclitaxel, teniposide, 
camptothecin and derivatives thereof. 
The solvent system of the invention includes a non-ionic solubilizing 
agent. The solvent system typically includes a solvent and a solubilizing 
agent. In preferred forms of the invention, the solubilizing agents are 
polyoxyalkylene modified lipids and alkylene oxides such as polyethylene 
glycol and derivatives thereof. The solubilizing agent can be a 
condensation product of an alkylene oxide and a lipid or fatty acid. The 
preferred solvent system includes a polyoxyethylated castor oil such as 
that sold under the tradename Cremophor EL. The Cremophor EL is treated to 
reduce the carboxylate anion content to a sufficiently low concentration 
to minimize the decomposition of the pharmaceutical agent that is 
catalyzed by the carboxylate anions. The carboxylate anion content of the 
Cremophor EL is lowered by either contacting the Cremophor EL with an 
aluminum oxide bed to separate the carboxylate anions as well as other 
impurities or by the addition of an acid and particularly a mineral acid 
such as HCl or HNO.sub.3. In further embodiments, the solvent is treated 
with a reactant to reduce the carboxylate anion or convert the carboxylate 
anion to a non-reactive form. 
The advantages of the invention are also attained by producing a stabilized 
pharmaceutical composition comprising at least one antineoplastic compound 
and a solvent capable of dispersing the antineoplastic compound, the 
solvent comprising a solubilizing amount of a polyoxyethylated castor oil 
having a carboxylate anion content sufficiently low to substantially 
prevent carboxylate anion catalyzed degradation of the antineoplastic 
compound. 
Further advantages of the invention are attained by providing a method of 
stabilizing a pharmaceutical composition containing a pharmaceutical agent 
selected from the group consisting of paclitaxel and teniposide, and a 
solvent containing ethanol and a solubilizing amount of at least one 
solubilizing agent, the method comprising treating the solvent to reduce 
the carboxylate content to a sufficiently low level to substantially 
prevent carboxylate anion catalyzed degradation of the pharmaceutical 
agent. 
DETAILED DESCRIPTION OF THE INVENTION 
The disadvantages and limitations of the previous injection composition and 
solvent systems are overcome by the present invention while providing a 
convenient and efficient method of producing a solvent system and a method 
of stabilizing pharmaceutical compositions suitable for injection. The 
present invention is primarily directed to a solvent system suitable for 
producing a stabilized pharmaceutical composition and to a method of 
producing and stabilizing a pharmaceutical composition. 
It has been surprisingly discovered that paclitaxel reacts with ethanol 
during storage and that the decomposition of paclitaxel is catalyzed by 
the carboxylate anions in the solvent. Lowering the carboxylate 
concentration of the solvent has been found to produce a stabilizing 
effect in the pharmaceutical composition. The lower carboxylate 
concentration extends the shelf life of the composition by reducing the 
rate of decomposition of the pharmaceutical agent and reducing the 
formation of decomposition byproducts. 
The solvent system has a sufficiently low impurity content to provide a 
stabilizing effect in pharmaceutical compositions. The resulting 
pharmaceutical composition produced by the method can be used to prepare 
injection compositions by diluting with a pharmaceutically acceptable 
carrier or diluent. The composition is sufficiently stable to minimize 
degradation of the active compound and to reduce the loss of potency 
during storage. The diluted composition does not exhibit the formation of 
precipitates so that the pharmaceutically active agent can be stored in a 
readily usable form. 
The pharmaceutical composition in preferred embodiments include at least 
one pharmaceutical compound having a tendency to degrade during storage 
where the decomposition reaction is catalyzed by carboxylate anions. The 
preferred pharmaceutical compounds have antineoplastic activity such as, 
for example, paclitaxel sold under the tradename Taxol. The various 
analogs and derivatives of Taxol may also be used. 
Taxol is prepared as an injection concentrate containing 6 mg/ml paclitaxel 
in 50:50 by volume ethanol and polyoxyethylated castor oil. The 
polyoxyethylated castor oil is sold under the tradename Cremophor EL. 
Paclitaxel is an active component isolated from the bark of Pacific yew 
trees. Recently, paclitaxel has been found to be produced in small amounts 
from a fungus found in the bark of yew trees. Paclitaxel is known to have 
antineoplastic activity. 
Another compound suitable for use in the invention having antineoplastic 
activity is Teniposide. Teniposide is a semi-synthetic derivative of 
podophyllotoxin having the chemical name 4'-demethylepiodophyllotoxin 
9-(4,6-O-2-thenylidene-.beta.-D-glucopyranoside). Teniposide and the 
analogs thereof are available from commercial sources and may be prepared 
by the method disclosed in U.S. Pat. No. 3,524,844. 
Another suitable pharmaceutical agent is Camptothecin having the chemical 
name 
4-ethyl-hydroxy-1H-pyrano- 3',4':6,7!indolizino 1,2-b!quinoline-3,14(4H, 
12H)dione. Camptothecin is isolated from the stem wood of the Chinese tree 
and has been shown to exhibit antileukemic and antitumor activity. 
The pharmaceutical agents of particular interest are those which exhibit 
degradation and loss of activity during storage. The solvent system and 
process of the invention are particularly advantageous for use with 
pharmaceutical agents that react with or are unstable in the solvent. In 
particular, the pharmaceutical agents of interest are those having an 
ester linkage that can be cleaved by an alcohol in the presence of 
carboxylate anions. Several known pharmaceutical agents when diluted form 
precipitates after extended periods of time. Although the antineoplastic 
agents are of particular interest, other pharmaceutical agents which are 
subject to degradation during storage are also suitable. The 
pharmaceutical agent alternatively may, for example, be an antifungal or 
antibacterial. 
Paclitaxel is typically produced as a concentrate or solution in a vehicle 
suitable for injection in the amount of 6 mg/ml. The vehicle is usually a 
mixture of ethanol and Cremophor EL in the amount of 50:50 by volume. 
During storage, the activity of the paclitaxel is known to decrease. 
Paclitaxel has the Formula I, which HPLC shows to degrade into Baccatin 
III of Formula II and the ethyl ester side chain of Formula III. 
##STR1## 
In preferred embodiments, the solvent is a co-solvent mixture of at least 
one solvent and a solubilizing agent. The preferred solvents include the 
alcohols such as dehydrated ethanol and the pharmaceutically acceptable 
polyols such as polyethyleneglycol. The solubilizing agent in preferred 
embodiments is a polyoxyethylated castor oil such as that sold under the 
tradename Cremophor EL by BASF. The co-solvent in preferred embodiments 
contains about 40% to 60% by volume of the polyoxyethylated castor oil 
with the balance being an alcohol or polyol. In a particularly preferred 
embodiment of the invention, the co-solvent comprises about 50:50 by 
volume of dehydrated ethanol and Cremophor EL. 
The co-solvent of the invention preferably includes a non-ionic surfactant 
as the solubilizing agent with Cremophor EL being the most preferred. 
Cremophor EL is a condensation product of castor oil and about 20 to 40 
moles and preferably 30 to 35 moles of ethylene oxide per mole of castor 
oil. Cremophor EL may be prepared by the method disclosed in U.S. Pat. No. 
3,070,499. Cremophor EL is also known by its common names 
polyoxyethylene-glycerol triricinoleate and glycerol-polyethyleneglycol 
ricinoleate. The biological and chemical equivalents or derivatives of 
Cremophor EL may also be used. 
In alternative embodiments, the non-ionic surfactant or solubilizing agent 
may include other ethylene oxide modified lipids, hydroxylated tallow 
oils, polysorbate 80, also known as Tween 80, polyethoxylated 12-hydroxy 
stearic acid, polyoxyethylene sorbitan fatty acid esters, polyethylene 
glycol esters, polyethylene fatty acid esters, block co-polymers of 
ethylene oxide and propylene oxide, ethylated fatty alcohol ethers, and 
octyl-phenoxy polyethoxy ethanol compounds. These non-ionic, solubilizing 
agents can be produced by methods well known in the art or can be obtained 
from commercial suppliers. The solubilizer may include other condensation 
products of alkylene oxides, although the alkylene oxide modified lipids 
are generally preferred. Examples of suitable solubilizing agents are. PEG 
400 and PEG 40 castor oil. 
The carboxylate anion content of the solvent can be lowered by a number of 
methods. In a first embodiment of the invention, the Cremophor EL or other 
solvent is passed through a standard chromatography column of aluminum 
oxide. The aluminum oxide adsorbs the carboxylate anions as well as other 
impurities to reduce the carboxylate anion content of the solvent. 
In an alternative embodiment of the invention, the solvent is treated by 
the addition of an acid in a stabilizing amount to reduce the carboxylate 
anion content to a sufficiently low level to substantially prevent 
catalyzed degradation of the pharmaceutical compound. The acid may be 
added to the solvent before or after admixing with the pharmaceutical 
compound. Generally, mineral acids such as, for example, HCl, HBr, HF, HI, 
H.sub.2 SO.sub.4 and HNO.sub.3 are used. Alternatively, organic acids such 
as acetic may be used. The organic acids are generally less preferred 
since they can provide a source of carboxylate anions to hinder the 
stabilizing effect of the acid treatment. The acid is preferably added in 
amounts to provide 5.6.times.10.sup.-6 to 8.4.times.10.sup.-6 grams of 
H.sup.+ per ml of the solvent. Effective stabilization of the composition 
is obtained by the addition of the acid to provide about 
7.0.times.10.sup.-6 grams of H.sup.+ per ml. The acid is added to lower 
the carboxylate anion content to less than or equal to about 
0.6.times.10.sup.-6 gram equivalents of carboxylate anion per ml of 
solvent. 
Taxol solutions containing 0.6.times.10.sup.-6 gram equivalents of 
carboxylate anion have exhibited 94% paclitaxel remaining after storage at 
50.degree. C. for 28 days. 1.0 gram equivalents of anions is neutralized 
by 1.0 gram H.sup.+ per equivalent. 
The amount of acid added to the solvent will depend on the amount of the 
non-ionic surfactant in the co-solvent system and the type of the 
surfactant. The non-ionic surfactants which by their method of preparation 
inherently contain greater amounts of carboxylate anion impurities will 
typically require larger amounts of acid to lower the carboxylate anion 
content. 
In further embodiments the solvent may be treated with other reactants 
which are capable of lowering the carboxylate anion concentration of the 
solution. For example, insoluble salts of the carboxylates or derivatives 
of carboxylates may be formed. It is generally preferred to remove the 
carboxylate anions using a reagent which does not form precipitates in the 
composition. The addition of a strong mineral acid is generally preferred 
since the mineral acids and the resulting carboxylic acids are non-toxic 
and readily solubilized in the solvent. When the carboxylate anions are 
reacted in a manner which form an insoluble precipitate, the precipitate 
should be separated from the composition prior to administering the 
composition to a patient.

The following non-limiting examples are intended to demonstrate the 
preferred embodiments of the invention. One skilled in the art will 
readily recognize that numerous embodiments of the invention can be 
practiced to achieve the stabilizing effect. 
EXAMPLE 1 
This example analyzes the properties of cleaned and unprocessed Cremophor 
EL. A first group of samples 1-4 were different batches of processed 
Cremophor EL and a second group of samples 5-7 were different batches of 
un-processed commercial grade Cremophor EL. The processed Cremophor EL was 
prepared by passing 100 Kg of the Cremophor EL through a standard 
chromatography column containing 19.5 Kg of aluminum oxide sold under the 
tradename CAMAG. In order to understand the cause of the Taxol instability 
in unprocessed Cremophor EL, each sample was analyzed by observing the 
Karl-Fischer moisture, potassium ion content, acid value, and peroxide 
value. The results of these observations are presented in Table 1. 
TABLE 1 
______________________________________ 
Type of KF Potassium 
Cremophor 
Sample Moisture level Acid Peroxide 
EL No. % (ppm) Value Value 
______________________________________ 
Processed 
Sample 1 0.2 &lt;1 0.2 8 
Sample 2 0.3 2 0.3 8 
Sample 3 0.4 2 0.2 9 
Sample 4 0.8 4 0.3 3 
Un- Sample 5 2.7 463 3.4 18 
Processed 
Sample 6 2.6 482 1.6 n.d. 
Sample 7 2.6 n.d. 1.4 15 
______________________________________ 
n.d = not determined 
The data of Table 1 show consistency of moisture content, potassium 
content, acid value and peroxide value between the batches of processed 
Cremophor EL used to make samples 1-4. The data further shows consistently 
higher levels of moisture, potassium content, acid values and peroxide 
values for the unprocessed Cremophor EL. 
EXAMPLE 2 
This example was carried out to demonstrate the instability of Taxol and 
paclitaxel in a co-solvent containing Cremophor EL and to determine the 
nature of the decomposition products. Specifically, this example was 
carried out to determine whether high levels of moisture, potassium, acid 
value and peroxide value of unprocessed Cremophor EL as determined in 
Example 1 are related to the rate of decomposition and loss of potency of 
Taxol. 
Samples 8-21 were prepared by dissolving 6 mg/ml of paclitaxel in 50:50 v/v 
mixture of processed Cremophor EL and dehydrated ethanol. The Cremophor EL 
of samples 8-21 was processed as discussed above in Example 1. Sample 22 
was prepared as a control sample from unprocessed Cremophor EL in a 50:50 
v/v mixture with paclitaxel in the amount of 6 mg/ml. Each of the samples 
8-20 were mixed with the components listed in Tables 2 and 3. Three ml 
aliquots of the test samples were placed in 6 cc Type I flint glass vials. 
The vials were stoppered with West 4455/45 Teflon-faced stoppers, sealed 
and stored for four weeks at 50.degree. C., and then analyzed by HPLC for 
Taxol concentration. The control parameters of the HPLC are as follows: 
______________________________________ 
Column Jones Cyano RP 5.mu., 25 cm .times. 4.6 mm ID 
Detector Wavelength 
227 nm 
Mobile phase 35% acetonitrile: 65% 20 mM acetate 
buffer (pH 4) 
Flow rate 1.5 mL/min. 
Diluent Mobile phase 
Sample Concentration 
0.05 mg/mL 
Injection volume 
20 microliters 
Retention time 
Taxol 10.5 mins. 
______________________________________ 
The pH value for each of the samples was noted following 1:10 dilution with 
water. Peroxide value (EP method), acid value (USP method), moisture 
content and potassium levels were determined as shown in Tables 2 and 3. 
TABLE 2 
______________________________________ 
Following storage 
for 28 days at 
50.degree. C. 
Component % KF Initial 
% taxol 
Sample No. 
Added Moisture pH.sup.4 
remaining 
pH.sup.4 
______________________________________ 
Sample 8.sup.b 
4% H.sub.2 O.sub.2 
5.5 4.6 94.9 3.6 
solution 
Sample 9.sup.c 
10% H.sub.2 O.sub.2 
9.6 4.6 96.8 3.5 
solution 
Sample 10.sup.d 
4% water 5.8 5.0 94.5 4.2 
Sample 11.sup.e 
Acetic 2.6 3.8 94.1 4.1 
acid (1.5 
mg/mL) 
Sample 12.sup.f 
None 2.6 4.4 95.5 4.5 
(Control) 
______________________________________ 
.sup.a Following 1:10 dilution with Water for Injection. 
.sup.b Peroxide value = 232. 
.sup.c Peroxide value = 652. 
.sup.d Peroxide value = 14. 
.sup.e Acid value = 1.2. 
.sup.f Acid value = 0.5, Peroxide value = 4. 
As shown in Table 2, the stability of Taxol as a solution of a 50:50 
mixture of dehydrated ethanol and Cremophor EL is not affected by the 
addition of hydrogen peroxide, water, or acetic acid, thereby 
demonstrating that the Taxol instability is not related to peroxide value, 
moisture content or acid value of the Cremophor EL. Each of samples 8-11 
exhibited comparable loss of Taxol potency regardless of the reagent 
added. In each of samples 8-11, the percent of Taxol remaining reflecting 
the amount of paclitaxel in the samples ranged from 94.1% to 96.8% 
compared to 95.5% of the control Sample 12. 
TABLE 3 
______________________________________ 
Following 
Storage 
for 28 Days at 
K.sup.- 50.degree. C. 
Component level Initial 
% Taxol 
Sample No. 
Added (ppm) pH.sup.4 
Remaining 
pH.sup.a 
______________________________________ 
Sample 13 
CH.COOK 530 5.6 19.3 5.1 
0.126% w/v 
Sample 14 
KCl 551 6.0 98.8 4.7 
0.10% w/v 
Sample 15 
NaCl 4 6.3 96.8 4.9 
0.125% w/v 
Sample 16 
CH.COONH.sub.4 
4 5.8 63.7 4.7 
0.10% w/v 
Sample 17 
CH.sub.3 COONa 
5 6.1 5.8 5.5 
0.104% w/v 
Sample 18 
CH.sub.3 CH.sub.2 COOK 
468 5.4 15.2 5.1 
0.144% w/v 
Sample 19 
Linolelaidic 
5 6.4 99.5 5.4 
acid (LA) 
0.2% W/V 
Sample 20 
K salt of LA 
150 6.3 52.8 6.2 
0.228% w/v 
Sample 21 
None &gt;2 4.4 95.5 4.5 
(Control) 
Sample 22 
prepared with 
.sup.- 240.sup. 
4.6 86.5 4.8 
unprocessed 
Cremophor EL 
______________________________________ 
.sup.a Following 1:10 dilution with Water for Injection. 
Sample 13 of Table 3 containing potassium acetate is shown to be less 
stable than control Sample 21 which contained no added component. Sample 
14 containing potassium chloride is shown to be comparable with the 
control Sample 21. These data suggest that the stability of the Taxol is 
not affected by the presence of potassium ions but rather are adversely 
affected by the anionic form of the carboxylic acids. Sample 13 containing 
potassium acetate, Sample 16 containing ammonium acetate, Sample 17 
containing sodium acetate, Sample 18 containing potassium propionate and 
Sample 20 containing potassium linolelaidate are all less stable than the 
control Sample 22. However, the stability of Sample 11 containing acetic 
acid and Sample 19 containing linolelaidic acid is comparable to control 
Sample 22 indicating that the carboxylic acids in the non-ionized form do 
not affect Taxol stability. Sample 22 containing unprocessed Cremophor EL 
is also shown to be less stable than control Sample 21 containing 
processed Cremophor EL. HPLC of each of the samples indicated the 
degradation products to be Baccatin III and the ethyl ester of the 
paclitaxel side chain. 
EXAMPLE 3 
This example demonstrates the stabilizing effect of acids added to Taxol 
samples containing unprocessed Cremophor EL. The samples in this example 
were prepared from Taxol containing 6 mg/ml paclitaxel in a 50:50 v/v 
mixture of dehydrated ethanol and unprocessed commercial grade Cremophor 
EL. As indicated in Table 4, Samples 23-28 were mixed with HCl, Sample 29 
was mixed with acetic acid and Sample 30 was mixed with nitric acid. 
Sample 31 was the control sample containing no added acid. Sample 32 
containing acetic acid and control Sample 33 containing no added acid were 
also prepared from unprocessed Cremophor EL. The samples were separated 
into 3 ml aliquots and placed into 5 cc Type 1 flint gloss vials and 
stoppered with Daikyo #713 fluoresin coated closures. The vials were 
stored at 50.degree. C. for 56 days and analyzed by HPLC for paclitaxel 
(Taxol) concentration. The HPLC parameters were as follows: 
______________________________________ 
Column Jones Cyano RP 5.mu., 25 cm .times. 4.6 mm ID 
Detector Wavelength 
227 nm 
Mobile phase 35% acetonitrile: 65% 20 mM acetate 
buffer (pH 4) 
Flow rate 1.5 mL/min. 
Diluent Mobile phase 
Sample Concentration 
.sup..about. 0.05 mg/mL 
Injection volume 
20 microliters 
Retention time 
paclitaxel .sup..about. 10.5 mins. 
______________________________________ 
The data in Table 4 demonstrate that the stabilizing effect of the acid 
increased with increasing amounts of the acid. These results are 
consistent with the premise that the degradation of Taxol is due to the 
presence of carboxylate anions rather than the acid. The data further 
demonstrate that the mineral acids such as HCl and HNO.sub.3 are better 
stabilizing agents with greater than 97% of the Taxol remaining after 56 
days when the acid is added at levels to provide 5.6.times.10.sup.-6 to 
8.4.times.10.sup.-6 grams of H.sup.+ per ml. The mineral acids showed a 
greater stabilizing effect than acetic acid even when the acetic acid was 
added at a higher level of 70.times.10.sup.-6 grams of H.sup.+ per ml. 
These results are due to the acid strength of the mineral acids compared 
to organic acids to neutralize the carboxylate anions. In addition, the 
acetic acid as a source of carboxylate anions can contribute to some 
paclitaxel degradation. The amount of paclitaxel remaining in samples 26 
and 30 being greater than 100% is due to analytical variations in the 
measurements. 
TABLE 4 
______________________________________ 
Following 
Storage for 
TAXOL Acid added 56 Days at 50.degree. C. 
Injection 
(Gms of H.sup.+ 
Initial % Paclitaxel 
Sample No. 
added per mL) pH.sup.c Remaining 
pH.sup.c 
______________________________________ 
Sample 23.sup.a 
HCl (3.5 .times. 10.sup.-6) 
5.1 62.8 4.5 
Sample 24.sup.a 
HCl (5.6 .times. 10.sup.-6) 
3.9 98.1 3.8 
Sample 25.sup.a 
HCl (6.3 .times. 10.sup.-6) 
3.8 97.9 3.8 
Sample 26.sup.a 
HCl (7.0 .times. 10.sup.-6) 
3.8 100.9 3.6 
Sample 27.sup.a 
HCl (7.7 .times. 10.sup.-6) 
3.6 99.3 3.6 
Sample 28.sup.a 
HCl (8.4 .times. 10.sup.-6) 
3.6 99.1 3.6 
Sample 29.sup.a 
CH.sub.3 COOH 4.4 69.5 4.5 
(7.0 .times. 10.sup.-6) 
Sample 30.sup.a 
HNO.sub.3 (7.0 .times. 10.sup.-6) 
3.7 100.4 3.8 
Sample 31.sup.a 
Control (No 5.5 49.0 5.0 
acid added) 
Sample 32.sup.b 
CH.sub.3 COOH 3.7 87.8 3.7 
(70 .times. 10.sup.-6) 
Sample 33.sup. b 
Control (No 6.3 22.6 5.5 
acid added) 
______________________________________ 
.sup.a Unprocessed Cremophor EL, BASF Lot No. 982384, was used in these 
solutions. 
.sup.b Unprocessed Cremophor EL, BASF Lot No. 141213, was used in these 
solutions. 
.sup.c Following 1:10 dilution with water for Injection. 
Samples 34A, 34B, 35A, 35B, 36A and 36B as shown in Table 5 were prepared 
using different lots of Cremophor to show the consistency of the 
stabilizing effect of mineral acids. Each sample was prepared containing 6 
mg/ml paclitaxel in a 50:50 mixture of dehydrated ethanol and unprocessed 
Cremophor EL. Samples 34A, 35A and 36A were prepared in the manner of 
Samples 23-28 and to contain HCl in an amount to provide 
7.0.times.10.sup.-6 of H.sup.+ per ml. Control Samples 34B, 35B and 36B 
did not contain added acid. The samples were stored in closed vials for 56 
days at 50.degree. C. The amount of paclitaxel remaining was determined by 
HPLC as shown in Table 5. 
TABLE 5 
______________________________________ 
Following Storage 
Cremophor Initi- 56 Days at 50.degree. C. 
EL Lot No. - 
Acid al % Paclitaxel 
unprocessed 
added pH.sup.a Remaining 
pH.sup.a 
______________________________________ 
Sample 34A 
HCl 4.0 98.4 3.9 
Sample 34B 
Control.sup.b 
6.1 25.0 5.4 
Sample 35A 
HCl 3.9 97.8 4.0 
Sample 35B 
Control.sup.b 
6.3 22.6 5.5 
Sample 36A 
HCl 3.8 100.9 3.6 
Sample 36B 
Control.sup.b 
5.5 49.0 5.0 
______________________________________ 
.sup.a Following 1:10 dilution with Water for Injection. 
.sup.b No acid was added in the control solutions. 
The data of Table 5 demonstrate the consistent stabilizing effect of 
mineral acids with greater than 97% of the paclitaxel remaining after 56 
days compared to less than 50% remaining in the control samples. 
The data of Examples 2 and 3 demonstrate the stabilizing effect of 
processing the Cremophor EL by contacting with aluminum oxide or treating 
with an acid to reduce the carboxylate content of the solvent. It will be 
appreciated by those skilled in the art that the process of reducing the 
carboxylate anion level can be used with other solvents and solubilizers. 
Taxol is disclosed in the preferred embodiment of the invention which has 
been demonstrated to exhibit increased shelf life from the stabilizing 
effect of the acids and the reduction in carboxylate anion content. In 
further embodiments of the invention, other bioactive agents that are 
sensitive to carboxylate anion degradation may be used.