Method of making prolonged release body

In a method of making a biologically compatible, water-insoluble polymeric body for the controlled, prolonged release of a biologically active substance to a surrounding aqueous environment, the method including forming a liquid mixture containing the polymer, the active substance, and an organic solvent capable of dissolving the polymer, and solidifying the liquid mixture to form the polymeric body, the improvement wherein the liquid mixture further comprises water, the solidification is carried out by cooling the liquid mixture to a temperature sufficiently low to cause the water in the mixture to freeze, thereby creating channels in the body for the release of the active substance therefrom, and removing the organic solvent and the water from the body.

BACKGROUND OF THE INVENTION 
This inventin relates to manufacturing biologically compatible, 
water-insoluble polymeric bodies for controlled, prolonged release of a 
biologically active substance to a surrounding aqueous environment. 
Folkman et al, U.S. Pat. No. 4,164,560, hereby incorporated by reference, 
describes a method of making such a body by forming a liquid mixture 
containing the polymer, the active substance, and an organic solvent 
capable of dissolving the polymer, and solidifying the liquid mixture to 
form the polymeric body. 
SUMMARY OF THE INVENTION 
In general, the invention features an improved method of making such a 
body, in which the liquid mixture further includes water; solidification 
is carried out by cooling the mixture to a temperature sufficiently low to 
cause the water in the mixture to freeze, thereby creating channels in the 
body for the release of the active substance therefrom; and removing the 
organic solvent and the water from the body. 
In preferred embodiments, the organic solvent is immiscible with water, so 
that the mixture is an emulsion; the removing of water and solvent is 
facilitated by subjecting the body to reduced pressure; the polymer is an 
ethylene-vinyl ester copolymer of the general formula: 
##STR1## 
wherein R is hydrogen, lower alkyl of 1 to 7 carbons, or aryl, m is 10 to 
40% by weight, and n is (100-m)% by weight; and the biologically active 
substance is an enzyme, a hormone, an enzyme inhibitor, an antigen, or a 
drug. 
The new method provides improved drug release kinetics because of channel 
formation caused by freezing of the water. Adjustment of release rate to 
fit a given applicaion can be achieved by varying the water:polymer ratio 
or the concentration of the active substance. Furthermore, because many 
active substances are available only as aqueous solutions or dispersions, 
the new method obviates complicated and potentially destructive (e.g., 
denaturing) procedures for isolating and purifying the active substance. 
Additional advantages and features of the invention will be apparent from 
the following description of the preferred embodiments thereof, and from 
the claims. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
We turn now to a description of the preferred embodiments, first briefly 
describing the drawings.

MANUFACTURE 
EVA (available as ELVAX 40 from the DuPont Chemical Company, Wilmington, 
Del.), 40% vinyl acetate by weight, in pellet form, is washed ten times in 
distilled water, and then 50 times in absolute ethanol, and is then 
dissolved in methylene chloride to produce a 10% w/v solution. An aqueous 
protein solution (5% w/v) of type I calf intestine alkaline phosphatese is 
then prepared, and 0.3 ml of this solution is combined with 2.0 ml of EVA 
solution. The resultant casting mixture is shaken in a 3 ml scintillation 
vial, silicated with Prosil-28 (PCR Research Chemicals) for 60 sec. on a 
Vortex-Genie mixer at speed "10" to yield a uniform solution, which is 
quickly poured into a 2 cm.times.2 cm.times.0.5 cm glass mold which has 
been previously cooled by placing it on dry ice for 10 minutes. After 5.0 
min., the frozen slab is removed by encircling the inside wall of the mold 
with a cold spatula tip and then prying the slab loose. The slab is 
transferred onto a cold wire screen in a freezer and kept at -20.degree. 
C. for two days. 
Water is then removed from the slab by lowering its temperature to 
-80.degree. C. The slab is then lyophilized by placing it in a glass petri 
dish enclosed in a cardboard box, which is enclosed in a dessicator 
connected to a Vir-Tis Model 10-148 continuous MRBA lyophilizer. 
Lyophilizaton is carried out for three days at a pressure below 50 
millitors; the lowered pressure increases the rate of water removal. The 
dried slab weighs 168.+-.7 mg and is about 13 mm square and 1 mm thick. 
The casting procedure described above is repeated, using, in place of 0.3 
ml 5% alkaline phosphotase, the following aqueous solutions of 
biologically active, proteinaceous substances: 0.1 ml, 0.2 ml, and 0.3 ml 
5% (w/v) bovine serum albumin (BSA); 0.3 ml of 1%, 10%, and 25% (w/v) BSA; 
0.3 ml 5% (w/v) type II bovine pancreas alph-chymotrypsin; 0.3 ml 5% (w/v) 
porcine stomach mucosa pepsin. 
Operation 
The release of proteins from the slabs into 0.9 w/v saline is measured by 
placing each slab in a scintillation vial with 10 ml of saline and 
rotating the vials on a Thomas Rotating Apparatus set at speed 4. Each 
slab is periodically transferred to a new vial containing fresh saline. 
The released protein concentrations are determined spectrophotometrically 
by measuring absorbance at 220 .mu.m. 
FIG. 1 shows that in the case of BSA, increasing the water:polymer ratio 
increases the cumulative percentage of protein released over time. This is 
consistent with the observation that increasing the water:polymer ratio 
also produces a slab with a greater volume; the increased volume probably 
is a reflection of greater porosity and channel tortuosity resulting from 
the expansion of a greater amount of ice. 
FIG. 2 shows that cumulative protein release increases with increased 
protein concentration in BSA slabs. 
Other Embodiments 
Other embodiments are within the following claims. For example, the active 
substance concentration and water:polymer ratio can be varied to obtain 
the release rate desired for a given application. The details of the 
method can also be varied. For example, drying need not be carried out 
under reduced pressure. 
Any biologically active substance can be used, in conjunction with any 
biologically compatible, water-insoluble polymer and any organic solvent 
capable of dissolving the polymer. The active substance can be protein or 
it can be non-proteinaceous, it can be a macromolecule (M.W. over 1000 
daltons) or a smaller molecule, and it can be soluble or insoluble in 
water. Examples of suitable active substances are insoluble in water. 
Examples of suitable active substances are interferon, anti-angiogenesis 
factors, antibodies, antigens, polysaccharides, growth factors, hormones, 
including insulin, glucagan, parathyroid, and pituatary hormones, 
calcitonin, vasopressin, renin, prolactin, growth hormones, thyroid 
stimulating hormone, corticotrophin, follicle stimulating hormone, 
luteinisinng hormone, and chorionic gonadatrophin; enzymes, including 
soybean trypsin inhibitor, lysozyme, catalase, tumor angiogenesis factor, 
cartilege factor, transfereses, hydrolases, lysases, isomerases, 
proteases, ligases, and oxidoreductases such as esterases, phosphatases, 
glycosidases, and peptidases; enzyme inhibitors such as leupeptin, 
antipain, chymostatin, and pepstatin; and drugs. 
Suitable polymers include acyl substituted cellulose acetates and alkyl 
derivatives thereof; partially and completely hydrolyzed alkylene-vinyl 
acetate copolymers; unplasticized polyvinyl chloride; crosslinked homo- 
and co-polymers of polyvinyl acetate; crosslinked polyesters of acrylic 
and methacrylate; polyvinyl alkyl ethers; polyvinyl fluoride; silicone; 
polycarbonate; polyurethane; polyamide; polysulphones; styrene 
acrylonitrile copolymers; crosslinked poly(ethylene oxide); 
poly(alkylenes); poly(vinyl imidazole); poly(esters); poly(ethylene 
terephthalate); and chlorosulphonated polyolefins. 
EVA, the most preferred polymer, is a member of a class of suitable 
polymers of the general formula 
##STR2## 
wherein R is hydrogen, lower alkyl of 1 to 7 carbons, or aryl, and m is 
(10 to 40)% by weight and n is (100-m)% by weight. Typical alkyl groups 
include ethyl, propyl, isopropyl, tert-butyl, pentyl, and hexyl. Suitable 
ethylene-vinyl ester copolymers are the acetates, include ethylene-vinyl 
acetate, ethylene-vinyl methylacetate, ethylene-vinyl ethylacetate, 
ethylene-vinyl propylacetate, and the like. 
Polymer bodies can be made, according to the method of the invention, in 
any desired shape. The shape will sometimes be determined by the body's 
location. Alternatively, the shape can be chosen to affect release 
kinetics. For example, zero order release kinetics (a release rate 
independent of active substance concentration) can be obtained by casting 
the body in the shape of a hemisphere which is sealed everywhere except in 
a depression on its flat surface, through which the active substance is 
released. 
Polymer bodies made according to the method of the invention are implanted 
in animals, including humans, to provide controlled, prolonged release of 
the desired biologically active substance. Some medical applications of 
these polymer bodies are the prolonged release of insulin for the control 
of diabetes, immunizations (the active substance is an antigen), the 
delivery of informational macromolecules for assays for biological 
molecules such as tumor angiogenisis factor, and the prolonged treatment 
of a variety of medical disorders with the appropriate drugs.