Methods for in vivo delivery of substantially water insoluble pharmacologically active agents and compositions useful therefor

In accordance with the present invention, there are provided compositions for the in vivo delivery of substantially water insoluble pharmacologically active agents (such as the anticancer drug taxol) in which the pharmacologically active agent is delivered in a soluble form or in the form of suspended particles. In particular, the soluble form may comprise a solution of pharmacologically active agent in a biocompatible dispersing agent contained within a protein walled shell. Alternatively, the protein walled shell may contain particles of taxol. In another aspect, the suspended form comprises particles of pharmacologically active agent in a biocompatible aqueous liquid.

The present invention relates to in vivo delivery of substantially water 
insoluble pharmacologically active agents (e.g., the anticancer drug 
taxol). In one aspect, the agent is dispersed as a suspension suitable for 
administration to a subject, or is dissolved in a suitable biocompatible 
liquid. In another aspect, water insoluble pharmacologically active agents 
(e.g., taxol) are encased in a polymeric shell formulated from a 
biocompatible polymer. The polymeric shell contains particles of 
pharmacologically active agent, and optionally a biocompatible dispersing 
agent in which pharmacologically active agent can be either dissolved or 
suspended. 
BACKGROUND OF THE INVENTION 
Taxol is a natural product first isolated from the Pacific Yew tree, Taxus 
brevifolia, by Wani et al. [J. Am. Chem. Soc. Vol. 93:2325 (1971)]. Among 
the antimitotic agents, taxol, which contains a diterpene carbon skeleton, 
exhibits a unique mode of action on microtubule proteins responsible for 
the formation of the mitotic spindle. In contrast with other antimitotic 
agents such as vinblastine or colchicine, which prevent the assembly of 
tubulin, taxol is the only plant product known to inhibit the 
depolymerization process of tubulin, thus preventing the cell replication 
process. 
Taxol, a naturally occurring diterpenoid, has been shown to have 
significant antineoplastic and anticancer effects in drug-refractory 
ovarian cancer. Taxol has shown excellent antitumor activity in a wide 
variety of tumor models such as the B16 melanoma, L1210 leukemias, MX-1 
mammary tumors, and CS-1 colon tumor xenografts. Several recent press 
releases have termed taxol as the new anticancer wonder-drug. Indeed, 
taxol has recently been approved by the Federal Drug Administration for 
treatment of ovarian cancer. The poor aqueous solubility of taxol, 
however, presents a problem for human administration. Indeed, the delivery 
of drugs that are inherently insoluble or poorly soluble in an aqueous 
medium can be seriously impaired if oral delivery is not effective. 
Accordingly, currently used taxol formulations require a cremaphore to 
solubilize the drug. The human clinical dose range is 200-500 mg. This 
dose is dissolved in a 1:1 solution of ethanol:cremaphore and diluted to 
one liter of fluid given intravenously. The cremaphore currently used is 
polyethoxylated castor oil. 
In phase I clinical trials, taxol itself did not show excessive toxic 
effects, but severe allergic reactions were caused by the emulsifiers 
employed to solubilize the drug. The current regimen of administration 
involves treatment of the patient with antihistamines and steroids prior 
to injection of the drug to reduce the allergic side effects of the 
cremaphore. 
In an effort to improve the water solubility of taxol, several 
investigators have modified its chemical structure with functional groups 
that impart enhanced water-solubility. Among them are the sulfonated 
derivatives [Kingston et al., U.S. Pat. No. 5,059,699 (1991)], and amino 
acid esters [Mathew et al., J. Med. Chem. Vol. 35:145-151 (1992)] which 
show significant biological activity. Modifications to produce a 
water-soluble derivative facilitate the intravenous delivery of taxol 
dissolved in an innocuous carrier such as normal saline. Such 
modifications, however, add to the cost of drug preparation, may induce 
undesired side-reactions and/or allergic reactions, and/or may decrease 
the efficiency of the drug. 
Microparticles and foreign bodies present in the blood are generally 
cleared from the circulation by the `blood filtering organs`, namely the 
spleen, lungs and liver. The particulate matter contained in normal whole 
blood comprises red blood cells (typically 8 microns in diameter), white 
blood cells (typically 6-8 microns in diameter), and platelets (typically 
1-3 microns in diameter). The microcirculation in most organs and tissues 
allows the free passage of these blood cells. When microthrombii (blood 
clots) of size greater than 10-15 microns are present in circulation, a 
risk of infarction or blockage of the capillaries results, leading to 
ischemia or oxygen deprivation and possible tissue death. Injection into 
the circulation of particles greater than 10-15 microns in diameter, 
therefore, must be avoided. A suspension of particles less than 7-8 
microns, is however, relatively safe and has been used for the delivery of 
pharmacologically active agents in the form of liposomes and emulsions, 
nutritional agents, and contrast media for imaging applications. 
The size of particles and their mode of delivery determines their 
biological behavior. Strand et al. [in Microspheres-Biomedical 
Applications, ed. A. Rembaum, pp 193-227, CRC Press (1988)] have described 
the fate of particles to be dependent on their size. Particles in the size 
range of a few nanometers (nm) to 100 nm enter the lymphatic capillaries 
following interstitial injection, and phagocytosis may occur within the 
lymph nodes. After intravenous/intraarterial injection, particles less 
than about 2 microns will be rapidly cleared from the blood stream by the 
reticuloendothelial system (RES), also known as the mononuclear phagocyte 
system (MPS). Particles larger than about 7 microns will, after 
intravenous injection, be trapped in the lung capillaries. After 
intraarterial injection, particles are trapped in the first capillary bed 
reached. Inhaled particles are trapped by the alveolar macrophages. 
Pharmaceuticals that are water-insoluble or poorly water-soluble and 
sensitive to acid environments in the stomach cannot be conventionally 
administered (e.g., by intravenous injection or oral administration). The 
parenteral administration of such pharmaceuticals has been achieved by 
emulsification of the oil solubilized drug with an aqueous liquid (such as 
normal saline) in the presence of surfactants or emulsion stabilizers to 
produce stable microemulsions. These emulsions may be injected 
intravenously, provided the components of the emulsion are 
pharmacologically inert. U.S. Pat. No. 4,073,943 describes the 
administration of water-insoluble pharmacologically active agents 
dissolved in oils and emulsified with water in the presence of surfactants 
such as egg phosphatides, pluronics (copolymers of polypropylene glycol 
and polyethylene glycol), polyglycerol oleate, etc. PCT International 
Publication No. W085/00011 describes pharmaceutical microdroplets of an 
anaesthetic coated with a phospholipid such as dimyristoyl 
phosphatidylcholine having suitable dimensions for intradermal or 
intravenous injection. 
Protein microspheres have been reported in the literature as carriers of 
pharmacological or diagnostic agents. Microspheres of albumin have been 
prepared by either heat denaturation or chemical crosslinking. Heat 
denatured microspheres are produced from an emulsified mixture (e.g., 
albumin, the agent to be incorporated, and a suitable oil) at temperatures 
between 100.degree. C. and 150.degree. C. The microspheres are then washed 
with a suitable solvent and stored. Leucuta et al. [International Journal 
of Pharmaceutics Vol. 41:213-217 (1988)] describe the method of 
preparation of heat denatured microspheres. 
The procedure for preparing chemically crosslinked microspheres involves 
treating the emulsion with glutaraldehyde to crosslink the protein, 
followed by washing and storage. Lee et al. [Science Vol. 213:233-235 
(1981)] and U.S. Pat. No. 4,671,954 teach this method of preparation. 
The above techniques for the preparation of protein microspheres as 
carriers of pharmacologically active agents, although suitable for the 
delivery of water-soluble agents, are incapable of entrapping 
water-insoluble ones. This limitation is inherent in the technique of 
preparation which relies on crosslinking or heat denaturation of the 
protein component in the aqueous phase of a water-in-oil emulsion. Any 
aqueous-soluble agent dissolved in the protein-containing aqueous phase 
may be entrapped within the resultant crosslinked or heat-denatured 
protein matrix, but a poorly aqueous-soluble or oil-soluble agent cannot 
be incorporated into a protein matrix formed by these techniques.

The invention will now be described in greater detail by reference to the 
following non-limiting examples. 
EXAMPLE 1 
Preparation of Taxol Particles 
Crystals of taxol (Sigma Chemical) were ground in a ball mill until 
particles of solid taxol were obtained having a size less than 10 microns. 
Size of particles were determined by suspending the particles in isotonic 
saline and counting with the aid of a particle counter (Elzone, Particle 
Data). Grinding was continued until 100% of the particles had a size less 
than 5 microns. The preferred particle size for intravenous delivery is 
less than 5 microns and most preferably less than 1 micron. 
Alternatively, particles of taxol were obtained by sonicating a suspension 
of taxol in water until all particles were below 10 microns. 
Taxol particles less than 10 microns can also be obtained by precipitating 
taxol from a solution of taxol in ethanol by adding water until a cloudy 
suspension is obtained. Optionally, the solution of taxol can be sonicated 
during the water addition, until a cloudy suspension is obtained. The 
resulting suspension is then filtered and dried to obtain pure taxol 
particles in the desired size range. 
Fine particles of taxol were prepared by spray drying a solution of taxol 
in a volatile organic such as ethanol. The solution was passed through an 
ultrasonic nozzle that formed droplets of ethanol containing taxol. As the 
ethanol evaporated in the spray drier, fine particles of taxol were 
obtained. Particle size can be varied by changing the concentration of 
taxol in ethanol, adjusting the flow rate of liquid through the nozzle and 
power of sonication. 
EXAMPLE 2 
Preparation of Protein Shell Containing Oil 
Three ml of a USP (United States Pharmacopeia) 5% human serum albumin 
solution (Alpha Therapeutic Corporation) were taken in a cylindrical 
vessel that could be attached to a sonicating probe (Heat Systems, Model 
XL2020). The albumin solution was overlayered with 6.5 ml of USP grade 
soybean oil (soya oil). The tip of the sonicator probe was brought to the 
interface between the two solutions and the assembly was maintained in a 
cooling bath at 20.degree. C. The system was allowed to equilibriate and 
the sonicator turned on for 30 seconds. Vigorous mixing occurred and a 
white milky suspension was obtained. The suspension was diluted 1:5 with 
normal saline. A particle counter (Particle Data Systems, Elzone, Model 
280 PC) was utilized to determine size distribution and concentration of 
oil-containing protein shells. The resulting protein shells were 
determined to have a maximum cross-sectional dimension of about 
1.35.+-.0.73 microns, and the total concentration determined to be 
.about.10.sup.9 shells/ml in the original suspension. 
EXAMPLE 3 
Parameters Affecting Polymeric Shell Formation 
Several variables such as protein concentration, temperature, sonication 
time, concentration of pharmacologically active agent, and acoustic 
intensity were tested to optimize formation of polymeric shell. These 
parameters were determined for crosslinked bovine serum albumin shells 
containing toluene. 
Polymeric shells made from solutions having protein concentrations of 1%, 
2.5%, 5% and 10% were counted with the particle counter to determine a 
change in the size and number of polymeric shells produced. The size of 
the polymeric shells was found not to vary with protein concentration, but 
the number of polymeric shells per ml of "milky suspension" formed 
increased with the increase in concentration of the protein up to 5%. No 
significant change in the number of polymeric shells was found to occur 
above that concentration. 
Initial vessel temperatures were found to be important for optimal 
preparation of polymeric shells. Typically, initial vessel temperatures 
were maintained between 0.degree. C. and 45.degree. C. The aqueous-oil 
interfacial tension of the oils used for formation of the polymeric shell 
was an important parameter, which also varied as a function of 
temperature. The concentration of pharmacologically active agent was found 
not to significantly effect the yield of protein shells. It is relatively 
unimportant if the pharmacologically active agent is incorporated in the 
dissolved state, or suspended in the dispersing medium. 
Sonication time was an important factor determining the number of polymeric 
shells produced per ml. It was found that a sonication time greater than 
three minutes produced a decrease in the overall count of polymeric 
shells, indicating possible destruction of polymeric shells due to 
excessive sonication. Sonication times less than three minutes were found 
to produce adequate numbers of polymeric shells. 
According to the nomograph provided by the manufacturer of the sonicator, 
the acoustic power rating of the sonicator employed herein is 
approximately 150 watts/cm.sup.2. Three power settings in order of 
increasing power were used, and it was found that the maximum number of 
polymeric shells were produced at the highest power setting. 
EXAMPLE 4 
Preparation of Polymeric Shells Containing Dissolved Taxol 
Taxol was dissolved in USP grade soybean oil at a concentration of 2 mg/ml. 
3 ml of a USP 5% human serum albumin solution was taken in a cylindrical 
vessel that could be attached to a sonicating probe. The albumin solution 
was overlayered with 6.5 ml of soybean oil/taxol solution. The tip of the 
sonicator probe was brought to the interface between the two solutions and 
the assembly was maintained in equilibrium and the sonicator turned on for 
30 seconds. Vigorous mixing occurred and a stable white milky suspension 
was obtained which contained protein-walled polymeric shells enclosing the 
oil/taxol solution. 
In order to obtain a higher loading of drug into the crosslinked protein 
shell, a mutual solvent for the oil and the drug (in which the drug has a 
considerably higher solubility) can be mixed with the oil. Provided this 
solvent is relatively non-toxic (e.g., ethyl acetate), it may be injected 
along with the original carrier. In other cases, it may be removed by 
evaporation of the liquid under vacuum following preparation of the 
polymeric shells. 
EXAMPLE 5 
Stability of Polymeric Shells 
Suspensions of polymeric shells at a known concentration were analyzed for 
stability at three different temperatures (i.e., 4.degree. C., 25.degree. 
C., and 38.degree. C.) Stability was measured by the change in particle 
counts over time. Crosslinked protein (albumin) shells containing soybean 
oil (SBO) were prepared as described above (see Example 2), diluted in 
saline to a final oil concentration of 20% and stored at the above 
temperatures. Particle counts (Elzone) obtained for each of the samples as 
a function of time are summarized in Table 1. 
TABLE 1 
______________________________________ 
Protein Shells (#/ml .multidot. 10.sup.10) 
in saline 
Day 4.degree. C. 25.degree. C. 
38.degree. C. 
______________________________________ 
0 7.9 8.9 8.1 
1 7.4 6.9 6.8 
7 7.3 8.3 5.0 
9 7.8 8.1 5.8 
17 7.8 8.3 6.1 
23 6.9 7.8 7.4 
27 7.2 8.8 7.1 
______________________________________ 
As demonstrated by the above data, the concentration of counted particles 
(i.e., polymeric shells) remains fairly constant over the duration of the 
experiment. The range is fairly constant and remains between about 
7-9.10.sup.10 /ml, indicating good polymeric shell stability under a 
variety of temperature conditions over almost four weeks. 
EXAMPLE 6 
In Vivo Biodistribution-Crosslinked Protein Shells Containing a Fluorophore 
To determine the fate of crosslinked albumin shells following intravenous 
injection, a fluorescent dye (rubrene, obtained from Aldrich) was 
dissolved in toluene, and crosslinked albumin shells containing 
toluene/rubrene were prepared as described above by sonication. The 
resulting milky suspension was diluted five times in normal saline. Two ml 
of the diluted suspension was then injected into the tail vein of a rat 
over 10 minutes. One animal was sacrificed an hour after injection and 
another 24 hours after injection. 
Frozen lung, liver, kidney, spleen, and bone marrow sections were examined 
under fluorescence for the presence of polymeric shells containing 
fluorescent dye. At one hour, most of the polymeric shells were intact and 
found in the lungs and liver as brightly fluorescing particles of about 1 
micron diameter. At 24 hours, polymeric shells were found in the liver, 
lungs, spleen, and bone marrow. A general staining of the tissue was also 
observed, indicating that the polymeric shells had been digested, and the 
dye liberated from within. This result was consistent with expectations 
and demonstrates the potential use of invention compositions for delayed 
or controlled release of entrapped pharmaceutical agent such as taxol. 
EXAMPLE 7 
Toxicity of Polymeric Shells Containing Soybean Oil (SBO) 
Polymeric shells containing soybean oil were prepared as described in 
Example 2. The resulting suspension was diluted in normal saline to 
produce two different solutions, one containing 20% SBO and the other 
containing 30% SBO. 
Intralipid, a commercially available TPN agent, contains 20% SBO. The 
LD.sub.50 for Intralipid in mice is 120 ml/kg, or about 4 ml for a 30 g 
mouse, when injected at 1 cc/min. 
Two groups of mice (three mice in each group; each mouse weighing about 30 
g) were treated with invention composition containing SBO as follows. Each 
mouse was injected with 4 ml of the prepared suspension of SBO-containing 
polymeric shells. Each member of one group received the suspension 
containing 20% SBO, while each member of the other group receive the 
suspension containing 30% SBO. 
All three mice in the group receiving the suspension containing 20% SBO 
survived such treatment, and showed no gross toxicity in any tissues or 
organs when observed one week after SBO treatment. Only one of the three 
mice in the group receiving suspension containing 30% SBO died after 
injection. These results clearly demonstrate that oil contained within 
polymeric shells according to the present invention is not toxic at its 
LD.sub.50 dose, as compared to a commercially available SBO formulation 
(Intralipid). This effect can be attributed to the slow release (i.e., 
controlled rate of becoming bioavailable) of the oil from within the 
polymeric shell. Such slow release prevents the attainment of a lethal 
dose of oil, in contrast to the high oil dosages attained with 
commercially available emulsions. 
EXAMPLE 8 
In vivo Bioavailability of Soybean Oil Released from Polymeric Shells 
A test was performed to determine the slow or sustained release of 
polymeric shell-enclosed material following the injection of a suspension 
of polymeric shells into the blood stream of rats. Crosslinked protein 
(albumin) walled polymeric shells containing soybean oil (SBO) were 
prepared by sonication as described above. The resulting suspension of 
oil-containing polymeric shells was diluted in saline to a final 
suspension containing 20% oil. Five ml of this suspension was injected 
into the cannulated external jugular vein of rats over a 10 minute period. 
Blood was collected from these rats at several time points following the 
injection and the level of triglycerides (soybean oil is predominantly 
triglyceride) in the blood determined by routine analysis. 
Five ml of a commercially available fat emulsion (Intralipid, an aqueous 
parenteral nutrition agent--containing 20% soybean oil, 1.2% egg yolk 
phospholipids, and 2.25% glycerin) was used as a control. The control 
utilizes egg phosphatide as an emulsifier to stabilize the emulsion. A 
comparison of serum levels of the triglycerides in the two cases would 
give a direct comparison of the bioavailability of the oil as a function 
of time. In addition to the suspension of polymeric shells containing 20% 
oil, five ml of a sample of oil-containing polymeric shells in saline at a 
final concentration of 30% oil was also injected. Two rats were used in 
each of the three groups. The blood levels of triglycerides in each case 
are tabulated in Table 2, given in units of mg/dl. 
TABLE 2 
______________________________________ 
SERUM TRIGLYCERIDES (mg/dl) 
GROUP Pre 1 hr 4 hr 24 hr 48 hr 72 hr 
______________________________________ 
Intralipid Control 
11.4 941.9 382.9 
15.0 8.8 23.8 
(20% SBO) 
Polymeric Shells 
24.8 46.7 43.8 
29.3 24.2 43.4 
(20% SBO) 
Polymeric Shells 
33.4 56.1 134.5 
83.2 34.3 33.9 
(30% SBO) 
______________________________________ 
Blood levels before injection are shown in the column marked `Pre`. 
Clearly, for the Intralipid control, very high triglyceride levels are 
seen following injection. Triglyceride levels are then seen to take about 
24 hours to come down to preinjection levels. Thus the oil is seen to be 
immediately available for metabolism following injection. 
The suspension of oil-containing polymeric shells containing the same 
amount of total oil as Intralipid (20%) show a dramatically different 
availability of detectible triglyceride in the serum. The level rises to 
about twice its normal value and is maintained at this level for many 
hours, indicating a slow or sustained release of triglyceride into the 
blood at levels fairly close to normal. The group receiving oil-containing 
polymeric shells having 30% oil shows a higher level of triglycerides 
(concomitant with the higher administered dose) that falls to normal 
within 48 hours. Once again, the blood levels of triglyceride do not rise 
astronomically in this group, compared to the control group receiving 
Intralipid. This again, indicates the slow and sustained availability of 
the oil from invention composition, which has the advantages of avoiding 
dangerously high blood levels of material contained within the polymeric 
shells and availability over an extended period at acceptable levels. 
Clearly, drugs delivered within polymeric shells of the present invention 
would achieve these same advantages. 
Such a system of soybean oil-containing polymeric shells could be suspended 
in an aqueous solution of amino acids, essential electrolytes, vitamins, 
and sugars to form a total parenteral nutrition (TPN) agent. Such a TPN 
cannot be formulated from currently available fat emulsions (e.g., 
Intralipid) due to the instability of the emulsion in the presence of 
electrolytes. 
EXAMPLE 9 
Preparation of Crosslinked Protein-walled Polymeric Shells Containing a 
Solid Core of Pharmaceutically Active Agent 
Another method of delivering a poorly water-soluble drug such as taxol 
within a polymeric shell is to prepare a shell of polymeric material 
around a solid drug core. Such a `protein coated` drug particle may be 
obtained as follows. The procedure described in Example 4 is repeated 
using an organic solvent to dissolve taxol at a relatively high 
concentration. Solvents generally used are organics such as benzene, 
toluene, hexane, ethyl ether, and the like. Polymeric shells are produced 
as described in Example 4. Five ml of the milky suspension of polymeric 
shells containing dissolved taxol are diluted to 10 ml in normal saline. 
This suspension is placed in a rotary evaporator at room temperature and 
the volatile organic removed by vacuum. After about 2 hours in the rotary 
evaporator, these polymeric shells are examined under a microscope to 
reveal opaque cores, indicating removal of substantially all organic 
solvent, and the presence of solid taxol within a shell of protein. 
Alternatively, the polymeric shells with cores of organic 
solvent-containing dissolved drug are freeze-dried to obtain a dry crumbly 
powder that can be resuspended in saline (or other suitable liquid) at the 
time of use. In case of other drugs that may not be in the solid phase at 
room temperature, a liquid core polymeric shell is obtained. This method 
allows for the preparation of a crosslinked protein-walled shell 
containing undiluted drug within it. Particle size analysis shows these 
polymeric shells to be smaller than those containing oil. Although the 
presently preferred protein for use in the formation of the polymeric 
shell is albumin, other proteins such as .alpha.-2-macroglobulin, a known 
opsonin, could be used to enhance uptake of the polymeric shells by 
macrophage-like cells. Alternatively, a PEG-sulfhydryl (described below) 
could be added during formation of the polymeric shell to produce a 
polymeric shell with increased circulation time in vivo. 
EXAMPLE 10 
In vivo Circulation and Release Kinetics of Polymeric Shells 
Solid core polymeric shells containing taxol were prepared as described 
above (see, for example, Example 4) and suspended in normal saline. The 
concentration of taxol in the suspension was measured by HPLC as follows. 
First, the taxol within the polymeric shell was liberated by the addition 
of 0.1M mercaptoethanol (resulting in exchange of protein disulfide 
crosslinkages, and breakdown of the crosslinking of the polymeric shell), 
then the liberated taxol was extracted from the suspension with 
acetonitrile. The resulting mixture was centrifuged and the supernatant 
freeze-dried. The lyophilate was dissolved in methanol and injected onto 
an HPLC to determine the concentration of taxol in the suspension. The 
taxol concentration was found to be about 1.6 mg/ml. 
Rats were injected with 2 ml of this suspension through a jugular catheter. 
The animal was sacrificed at two hours, and the amount of taxol present in 
the liver determined by HPLC. This required homogenization of the liver, 
followed by extraction with acetonitrile and lyophilization of the 
supernatant following centrifugation. The lyophilate was dissolved in 
methanol and injected onto an HPLC. Approximately 15% of the administered 
dose of taxol was recovered from the liver at two hours, indicating a 
significant dosage to the liver. This result is consistent with the known 
function of the reticuloendothelial system of the liver in clearing small 
particles from the blood. 
EXAMPLE 11 
Preparation of Crosslinked PEG-walled Polymeric Shells 
As an alternative to the use of thiol (sulfhydryl) containing proteins in 
the formation of, or as an additive to polymeric shells of the invention, 
a thiol-containing PEG was prepared. PEG is known to be nontoxic, 
noninflammatory, nonadhesive to cells, and in general biologically inert. 
It has been bound to proteins to reduce their antigenicity and to liposome 
forming lipids to increase their circulation time in vivo. Thus 
incorporation of PEG into an essentially protein shell would be expected 
to increase circulation time as well as stability of the polymeric shell. 
By varying the concentration of PEG-thiol added to the 5% albumin 
solution, it was possible to obtain polymeric shells with varying 
stabilities in vivo. PEG-thiol was prepared by techniques available in the 
literature (such as the technique of Harris and Herati, as described in 
Polymer Preprints Vol. 32:154-155 (1991)). 
PEG-thiol of molecular weight 2000 g/mol was dissolved at a concentration 
of 1% (0.1 g added to 10 ml) in a 5% albumin solution. This protein/PEG 
solution was overlayered with oil as described in Example 2 and sonicated 
to produce oil-containing polymeric shells with walls comprising 
crosslinked protein and PEG. These Polymeric shells were tested for 
stability as described in Example 5. 
Other synthetic water-soluble polymers that may be modified with thiol 
groups and utilized in lieu of PEG include, for example, polyvinyl 
alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, 
polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidinone, 
polysaccharides (such as chitosan, alginates, hyaluronic acid, dextrans, 
starch, pectin, etc), and the like. 
EXAMPLE 12 
Targeting of Immunosuppressive Agent to Transplanted Organs using 
Intravenous Delivery of Polymeric Shells Containing Such Agents 
Immunosuppressive agents are extensively used following organ 
transplantation for the prevention of rejection episodes. In particular, 
cyclosporine, a potent immunosuppressive agent, prolongs the survival of 
allogeneic transplants involving skin, heart, kidney, pancreas, bone 
marrow, small intestine, and lung in animals. Cyclosporine has been 
demonstrated to suppress some humoral immunity and to a greater extent, 
cell mediated reactions such as allograft rejection, delayed 
hypersensitivity, experimental allergic encephalomyelitis, Freund's 
adjuvant arthritis, and graft versus host disease in many animal species 
for a variety of organs. Successful kidney, liver and heart allogeneic 
transplants have been performed in humans using cyclosporine. 
Cyclosporine is currently delivered in oral form either as capsules 
containing a solution of cyclosporine in alcohol, and oils such as corn 
oil, polyoxyethylated glycerides and the like, or as a solution in olive 
oil, polyoxyethylated glycerides, etc. It is also administered by 
intravenous injection, in which case it is dissolved in a solution of 
ethanol (approximately 30%) and Cremaphor (polyoxyethylated castor oil) 
which must be diluted 1:20 to 1:100 in normal saline or 5% dextrose prior 
to injection. Compared to an intravenous (i.v.) infusion, the absolute 
bioavailibility of the oral solution is approximately 30% (Sandoz 
Pharmaceutical Corporation, Publication SDI-Z10 (A4), 1990). In general, 
the i.v. delivery of cyclosporine suffers from similar problems as the 
currently practiced i.v. delivery of taxol, i.e., anaphylactic and 
allergic reactions believed to be due to the Cremaphor, the delivery 
vehicle employed for the i.v. formulation. 
In order to avoid problems associated with the Cremaphor, cyclosporine 
contained within polymeric shells as described above may be delivered by 
i.v. injection. It may be dissolved in a biocompatible oil or a number of 
other solvents following which it may be dispersed into polymeric shells 
by sonication as described above. In addition, an important advantage to 
delivering cyclosporine (or other immunosuppressive agent) in polymeric 
shells has the advantage of local targeting due to uptake of the injected 
material by the RES system in the liver. This may, to some extent, avoid 
systemic toxicity and reduce effective dosages due to local targeting. The 
effectiveness of delivery and targeting to the liver of taxol contained 
within polymeric shells following intravenous injection is demonstrated in 
Example 9. A similar result would be expected for the delivery of 
cyclosporine (or other putative immunosuppressive agent) in accordance 
with the present invention. 
While the invention has been described in detail with reference to certain 
preferred embodiments thereof, it will be understood that modifications 
and variations are within the spirit and scope of that which is described 
and claimed.