Preparation of biodegradable microparticles containing a biologically active agent

Disclosed herein is a process for preparing biodegradable microparticles comprising a biodegradable polymeric binder and a biologically active agent, wherein a blend of at least two substantially non-toxic solvents, free of halogenated hydrocarbons, is used to dissolve or disperse the agent and dissolve the polymer. The solvent blend containing the agent and polymer is dispersed in an aqueous solution to form microdroplets. The resulting emulsion is then added to an extraction medium preferably containing at least one of the solvents of the blend, whereby the rate of extraction of each solvent is controlled, whereupon the biodegradable microparticles containing the biologically active agent are formed.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the preparation of microparticles. More 
particularly, the present invention relates to a solvent system useful in 
a method of encapsulating active agents to form controlled-release 
microparticles. By "microparticles" is meant solid particles that contain 
an active agent dispersed or dissolved within a biodegradable polymer that 
serves as the matrix of the particle. 
2. Description of the Related Art 
A variety of methods are known by which compounds can be encapsulated in 
the form of microparticles. It is particularly advantageous to encapsulate 
a biologically active or pharmaceutically active agent within a 
biocompatible, biodegradable, wall forming material (e.g., a polymer) to 
provide sustained or delayed release of drugs or other active agents. In 
these methods, the material to be encapsulated (drugs or other active 
agents) is generally dissolved, dispersed, or emulsified, using stirrers, 
agitators, or other dynamic mixing techniques, in a solvent containing the 
wall forming material. Solvent is then removed from the microparticles and 
thereafter the microparticle product is obtained. 
An example of a conventional microencapsulation process is disclosed in 
U.S. Pat. No. 3,737,337 wherein a solution of a wall or shell forming 
polymeric material in a solvent is prepared. The solvent is only partially 
miscible in water. A solid or core material is dissolved or dispersed in 
the polymer-containing solution and, thereafter, the 
core-material-containing solution, is dispersed in an aqueous liquid that 
is immiscible in the organic solvent in order to remove solvent from the 
microparticles. 
Another example of a process in which solvent is removed from 
microparticles containing a substance is disclosed in U.S. Pat. No. 
3,523,906. In this process, a material to be encapsulated is emulsified in 
a solution of a polymeric material in a solvent that is immiscible in 
water and then the emulsion is emulsified in an aqueous solution 
containing a hydrophilic colloid. Solvent removal from the microparticles 
is then accomplished by evaporation and the product is obtained. 
In still another process, as disclosed in U.S. Pat. No. 3,691,090, organic 
solvent is evaporated from a dispersion of microparticles in an aqueous 
medium, preferably under reduced pressure. 
Similarly, U.S. Pat. No. 3,891,570 discloses a method in which 
microparticles are prepared by dissolving or dispersing a core material in 
a solution of a wall material dissolved in a solvent having a dielectric 
constant of 10 or less and poor miscibility with a polyhydric alcohol, 
then emulsifying in fine droplets through dispersion or solution into the 
polyhydric alcohol and finally evaporating the solvent by the application 
of heat or by subjecting the microparticles to reduced pressure. 
Another example of a process in which an active agent may be encapsulated 
is disclosed in U.S. Pat. No. 3,960,757. Encapsulated medicaments are 
prepared by dissolving a wall material for capsules in at least one 
organic solvent, poorly miscible with water, that has a boiling point of 
less than 100.degree. C., a vapor pressure higher than that of water, and 
a dielectric constant of less than about 10; dissolving or dispersing a 
medicament that is insoluble or slightly soluble in water in the resulting 
solution; dispersing the resulting solution or dispersion to the form of 
fine drops in a liquid vehicle comprising an aqueous solution of a 
hydrophilic colloid or a surface active agent, and then removing the 
organic solvent by evaporation. The size of the fine drops is determined 
according to the stirring speed, the viscosity of the organic solvent 
solution containing the medicament and the wall material, and the 
viscosity and surface tension of the vehicle. 
Tice et al. in U.S. Pat. No. 4,389,330 describe the preparation of 
microparticles containing an active agent by using a two-step solvent 
removal process. This two-step solvent removal process is advantageous 
because it results in microparticles having higher active agent loading 
and a higher quality than techniques in which solvent is removed in a 
single step. In the Tice et al. process, the active agent and the polymer 
are dissolved in a solvent. The mixture of ingredients in the solvent is 
then emulsified in a continuous-phase processing medium that is immiscible 
with the solvent. A dispersion of microparticles containing the indicated 
ingredients is formed in the continuous-phase medium by mechanical 
agitation of the mixed materials. From this dispersion, the organic 
solvent can be partially removed in the first step of the solvent removal 
process. After the first stage, the dispersed microparticles are isolated 
from the continuous-phase processing medium by any convenient means of 
separation. Following the isolation, the remainder of the solvent in the 
microparticles is removed by extraction. After the remainder of the 
solvent has been removed from the microparticles, they are dried by 
exposure to air or by other conventional drying techniques. 
Tice et al., in U.S. Pat. No. 4,530,840, describe the preparation of 
microparticles containing an anti-inflammatory active agent by a method 
comprising: (a) dissolving or dispersing an anti-inflammatory agent in a 
solvent and dissolving a biocompatible and biodegradable wall forming 
material in that solvent; (b) dispersing the solvent containing the 
anti-inflammatory agent and wall forming material in a continuous-phase 
processing medium; (c) evaporating a portion of the solvent from the 
dispersion of step (b), thereby forming microparticles containing the 
anti-inflammatory agent in the suspension; and (d) extracting the 
remainder of the solvent from the microparticles. 
WO 90/13361 discloses a method of microencapsulating an agent to form a 
microencapsulated product, having the steps of dispersing an effective 
amount of the agent in a solvent containing a dissolved wall forming 
material to form a dispersion; combining the dispersion with an effective 
amount of a continuous process medium to form an emulsion that contains 
the process medium and microdroplets having the agent, the solvent, and 
the wall forming material; and adding the emulsion rapidly to an effective 
amount of an extraction medium to extract the solvent from the 
microdroplets to form the microencapsulated product. 
Bodmeier, R. et al., International Journal of Pharmaceutics 43:179-186 
(1988), disclose the preparation of microparticles containing quinidine or 
quinidine sulfate as the active agent and poly(D,L-lactide) as the binder 
using a variety of solvents including methylene chloride, chloroform, and 
benzene as well as mixtures of methylene chloride and a water miscible 
liquid, such as acetone, ethyl acetate, methanol, dimethylsulfoxide, 
chloroform, or benzene to enhance drug content. 
Beck, L. R. et al., Biology of Reproduction 28:186-195 (1983), disclose a 
process for encapsulating norethisterone in a copolymer of D,L-lactide and 
glycolide by dissolving both the copolymer and the norethisterone in a 
mixture of chloroform and acetone that is added to a stirred cold aqueous 
solution of polyvinyl alcohol to form an emulsion and the volatile 
solvents removed under reduced pressure to yield microcapsules. 
Very often the solvents used in the known microencapsulation processes are 
halogenated hydrocarbons, particularly chloroform or methylene chloride, 
which act as solvents for both the active agent and the encapsulating 
polymer. The presence of small, but detectable, halogenated hydrocarbon 
residuals in the final product, however, is undesirable, because of their 
general toxicity and possible carcinogenic activity. Thus, a need exists 
to revise the known microencapsulation processes using safe and acceptable 
alternative solvents. 
SUMMARY OF THE INVENTION 
The present invention relates to a method of preparing microparticles. 
More particularly, the present invention relates to a process for preparing 
biodegradable microparticles comprising a biodegradable polymeric binder 
and a biologically active agent, wherein a blend of at least two 
substantially non-toxic solvents, free of halogenated hydrocarbons, is 
used to dissolve both the agent and the polymer. The solvent blend 
containing the dissolved agent and polymer is dispersed in an aqueous 
solution to form droplets. The resulting emulsion is then added to an 
aqueous extraction medium preferably containing at least one of the 
solvents of the blend, whereby the rate of extraction of each solvent is 
controlled, whereupon the biodegradable microparticles containing the 
biologically active agent are formed. The process has the advantages that 
less extraction medium is required because the solubility of one solvent 
in water is substantially independent of the other and solvent selection 
is increased, especially with solvents that are particularly difficult to 
extract. 
In a preferred embodiment, the present invention relates to a solvent 
system useful in a method of preparing a pharmaceutical composition in 
microparticle form designed for the controlled release of an effective 
amount o of a drug over an extended period of time. This composition 
comprises at least one pharmaceutical agent and at least one 
biocompatible, biodegradable encapsulating polymer. 
More particularly, the present invention relates to a method for preparing 
microparticles comprising: 
A. preparing a first phase comprising a biodegradable polymeric 
encapsulating binder and an active agent dissolved or dispersed in a blend 
of at least two mutually miscible organic solvents free from halogenated 
hydrocarbons and having limited water solubility, 
B. preparing a second phase comprising an aqueous solution of 
(1) a hydrophilic colloid or 
(2) a surfactant, 
C. combining said first phase and said second phase under the influence of 
mixing means to form an emulsion in which said first phase is 
discontinuous and said second phase continuous, and 
D. isolating said discontinuous first phase in the form of microparticles. 
Limited water solubility means having a solubility in water in the range of 
from about 0.1 to about 25 wt. % at 20.degree. C. 
In a preferred embodiment, the present invention relates to a method for 
preparing microparticles comprising preparing a first "oil" phase 
containing from about 5 weight percent to about 50 weight percent solids 
of which from about 5 to about 95 weight percent is a solution of 
biodegradable polymeric encapsulating binder and incorporating from about 
5 to about 95 weight percent, as based on polymeric binder, of an active 
agent in a solvent blend, the blend comprising first and second mutually 
miscible solvents, free from halogenated hydrocarbons, each having a 
solubility in water of from about 0.1 to about 25 weight percent at 
20.degree. C., forming an emulsion containing from 1:1 to 1:10 of the 
first phase in an emulsion process medium to form microdroplets of the 
first phase composition in a continuous aqueous second phase processing 
medium, adding the combined first and second phases to an aqueous 
extraction quench liquid at a level of from about 0.1 to about 20 liters 
of aqueous quench liquid per gram of polymer and active agent, said quench 
liquid containing the solvent of the blend having the greater water 
solubility at a level of from about 20% to about 70% of the saturation 
level of that solvent in the quench liquid at the temperature being used, 
and recovering microparticles from the quench liquid. 
In another aspect, the invention is directed to a method of preparing 
microparticles comprising the steps of: preparing a first phase, said 
first phase comprising a biologically active agent, a biodegradable 
polymer, and a blend of at least two mutually miscible solvents for the 
agent and the polymer free from halogenated hydrocarbons; preparing a 
second phase, wherein said first phase is substantially immiscible in said 
second phase; flowing said first phase through a static mixer at a first 
flow rate; flowing said second phase through said static mixer at a second 
flow rate so that said first phase and said second phase flow 
simultaneously through said static mixer thereby forming microparticles 
containing said active agent; and isolating said microparticles. 
In another aspect, the invention is directed to a method of preparing 
microparticles comprising the steps of: preparing a first phase, said 
first phase comprising a biologically active agent, a biodegradable 
polymer, and a blend of at least two mutually miscible solvents for the 
agent and the polymer free from halogenated hydrocarbons; preparing a 
second phase, wherein said first phase and said second phase are 
substantially immiscible; preparing a quench liquid; pumping said first 
phase and said second phase through a static mixer into said quench liquid 
thereby forming microparticles containing said active agent. 
In further aspects of the invention, the first phase is prepared by (1) 
dissolving the biologically active agent in a solution of the polymer 
dissolved in at least two mutually miscible solvents free from halogenated 
hydrocarbons, or (2) by preparing a dispersion comprising the active agent 
in said solvents, or (3) by preparing an emulsion comprising the active 
agent in said solvents.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention involves the use of a solvent blend, free from 
halogenated hydrocarbons, comprising at least two solvents to produce 
biodegradable microparticles comprising at least one biologically active 
agent. A first solvent component of the solvent blend is a poor solvent 
for the active agent, but is a good solvent for the biodegradable polymer 
used herein. A second solvent component of the solvent blend is a good 
solvent for both the active agent and the polymer. 
The method of the present invention provides advantages over methods known 
in the art. The present method provides, inter alia, a biodegradable 
system, an injectable system that prevents the loss of dose during 
treatment, the ability to mix microparticles containing different drugs, 
microparticles free from halogenated hydrocarbon residues, and the ability 
to program release (multiphasic release patterns) to give faster or slower 
rates of drug release as needed. 
The products prepared by the method of the present invention offer the 
advantage of durations of action ranging from 30 to more than 200 days, 
depending upon the type of microparticle selected. In a preferred 
embodiment, the microparticles are designed to afford treatment to 
patients over a period of 30 to 60 days. The duration of action can be 
easily controlled by manipulation of the polymer composition, polymer:drug 
ratio, and microparticle size. 
Another important advantage of the microparticles prepared by the process 
of the present invention is that practically all of the active agent is 
delivered to the patient because the polymer used in the method of the 
invention is biodegradable, thereby permitting all of the entrapped agent 
to be released into the patient. 
In the process of the present invention, an active agent is dissolved or 
dispersed in a solvent blend free from halogenated hydrocarbons and to the 
agent-containing medium is added the polymeric matrix material in an 
amount relative to the active agent that provides a product having the 
desired loading of active agent. Optionally, all of the ingredients of the 
microparticle product can be blended in the solvent blend medium together. 
The solvent system used herein is a blend of at least two solvents. These 
solvents must be: 
(1) mutually miscible with one another, 
(2) capable, when blended, of dissolving or dispersing the active agent, 
(3) capable, when blended, of dissolving polymeric matrix material, 
(4) chemically inert to the active agent, 
(5) biocompatible, 
(6) substantially immiscible with the quench liquid, e.g., having a 
solubility of no more than about 0.1 to 25%, and 
(7) solvents other than halogenated hydrocarbons. 
By "halogenated hydrocarbons" is meant halogenated organic solvents, i.e., 
C.sub.1 -C.sub.4 halogenated alkanes, e.g., methylene chloride, 
chloroform, methyl chloride, carbon tetrachloride, ethylene dichloride, 
ethylene chloride, 2,2,2-trichloroethane, and the like. 
An ideal solvent blend for encapsulation of an active agent should have a 
high solubility for the polymeric encapsulating agent of generally at 
least about 5 weight percent and, preferably, at least about 20 weight 
percent at 20.degree. C. The upper limit of solubility is not critical, 
but if over about 50 weight percent of the solution is encapsulating 
polymer, the solution may become too viscous to handle effectively and 
conveniently. This is, of course, dependent on the nature of the 
encapsulating polymer and its molecular weight. 
The solvent system, although substantially immiscible with the continuous 
phase process medium and the quenching liquid, which usually are 
water-based, preferably has a limited solubility therein. If the solvent 
system were infinitely soluble in the process medium, microdroplets would 
be unable to form during the emulsion phase; if the solubility of the 
solvent system in the extractive quenching medium is too low, however, 
large quantities of quenching medium are needed. Generally, solvent 
solubilities of from about 0.1 to about 25% in the process medium and 
quench medium are acceptable for use herein. It will often be advantageous 
for the quench medium to contain from about 70 to about 20 weight percent 
of the saturation point of the first solvent, i.e., the solvent of higher 
solubility in the quench medium, to control the rate of loss of the first 
solvent from the microparticles into the quench medium. 
Added considerations in choosing a component of the solvent blend of the 
present invention include boiling point (i.e., the ease with which the 
solvents can be evaporated to form finished product) and specific gravity 
(tendency of the "oil phase" to float during emulsifying and quenching). 
Finally, the solvent system should have low toxicity. 
Generally, the solvent blend composition will contain from about 25 to 
about 75 weight percent of the first solvent and, correspondingly, from 
about 75 to about 25 weight percent of the second solvent. 
The solvent blend of the present invention is preferably a blend of at 
least two of the following: an ester, an alcohol, and a ketone. Preferred 
esters are of the structure R.sup.1 COOR.sup.2 where R.sup.1 and R.sup.2 
are independently selected from the group consisting of alkyl moieties of 
from 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, butyl, and isomers 
thereof. The most preferred ester for use as one component of the solvent 
blend employed in the practice of the present invention is ethyl acetate. 
Preferred alcohols are of the structure R.sup.3 CH.sub.2 OH where R.sup.3 
is selected from the group consisting of hydrogen, alkyl of from 1 to 3 
carbon atoms, and aryl of from 6 to 10 carbon atoms. It is more preferred 
that R.sup.3 be aryl. The most preferred alcohol for use as one component 
of the solvent blend employed in the practice of the present invention is 
benzyl alcohol. Preferred ketones are of the structure R.sup.4 COR.sup.5 
where R.sup.4 is selected from the group consisting of alkyl moieties of 
from 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, butyl, and isomers 
thereof and R.sup.5 is selected from the group consisting of alkyl 
moieties of from 2 to 4 carbon atoms, i.e., ethyl, propyl, butyl, and 
isomers thereof. The most preferred ketone for use as one component of the 
solvent blend employed in the practice of the present invention is methyl 
ethyl ketone. 
The polymeric matrix material of the microparticles prepared by the process 
of the present invention is a biocompatible and biodegradable polymeric 
material. The term "biocompatible" is defined as a polymeric material that 
is not toxic to the human body, is not carcinogenic, and does not 
significantly induce inflammation in body tissues. The matrix material 
should be biodegradable in the sense that the polymeric material should 
degrade by bodily processes to products readily disposable by the body and 
should not accumulate in the body. The products of the biodegradation 
should also be biocompatible with the body in the same sense that the 
polymeric matrix is biocompatible with the body, as should any residual 
solvent that may remain in the microparticles. 
Suitable examples of polymeric matrix materials include poly(glycolic 
acid), poly-D,L-lactic acid, poly-L-lactic acid, copolymers of the 
foregoing, poly(aliphatic carboxylic acids), copolyoxalates, 
polycaprolactone, polydioxanone, poly(ortho carbonates), poly(acetals), 
poly(lactic acid-caprolactone), polyorthoesters, poly(glycolic 
acid-caprolactone), polyanhydrides, polyphosphazines, and natural polymers 
including albumin, casein, and waxes, such as, glycerol mono- and 
distearate,and the like. Various commercially available poly 
(lactide-co-glycolide) materials (PLGA) may be used in the method of the 
present invention. For example, poly (d,1-lactic-co-glycolic acid) is 
commTechnolo available from Medisorb Technologies International L. P. 
(Cincinnati, Ohio). A suitable product commercially available from 
Medisorb is a 50:50 poly (D,L) lactic co-glycolic acid known as 
MEDISORB.RTM. 5050 DL. This product has a mole percent composition of 50% 
lactide and 50% glycolide. Other suitable commercially available products 
are MEDISORB.RTM. 65:35 DL, 75:25 DL, 85:15 DL and poly(d,1-lactic acid) 
(d,1-PLA). Poly(lactide-co-glycolides) are also commercially available 
from Boehringer Ingelheim (Germany) under its Resomer mark, e.g., PLGA 
50:50 (Resomer RG 502), PLGA 75:25 (Resomer RG 752) and d,1-PLA (Resomer 
RG 206), and from Birmingham Polymers (Birmingham, Ala.). These copolymers 
are available in a wide range of molecular weights and ratios of lactic 
acid to glycolic acid. 
The most preferred polymer for use in the practice of this invention is 
poly(d1-lactide-co-glycolide). It is preferred that the molar ratio of 
lactide to glycolide in such a copolymer be in the range of from about 
85:15 to about 50:50. 
The molecular weight of the polymeric matrix material is of some 
importance. The molecular weight should be high enough to permit the 
formation of satisfactory polymer coatings, i.e., the polymer should be a 
good film former. Usually, a satisfactory molecular weight is in the range 
of 5,000 to 500,000 daltons, preferably about 150,000 daltons. However, 
since the properties of the film are also partially dependent on the 
particular polymeric material being used, it is very difficult to specify 
an appropriate molecular weight range for all polymers. The molecular 
weight of a polymer is also important from the point of view of its 
influence upon the biodegradation rate of the polymer. For a diffusional 
mechanism of drug release, the polymer should remain intact until all of 
the drug is released from the microparticles and then degrade. The drug 
can also be released from the microparticles as the polymeric excipient 
bioerodes. By an appropriate selection of polymeric materials a 
microparticle formulation can be made in which the resulting 
microparticles exhibit both diffusional release and biodegradation release 
properties. This is useful in affording multiphasic release patterns. 
The formulation prepared by the process of the present invention contains 
an active agent dispersed in the microparticle polymer matrix material. 
The amount of agent incorporated in the microparticles usually ranges from 
about 1 wt. % to about 90 wt. %, preferably 30 to 50 wt. %, more 
preferably 35 to 40 wt. %. By weight % is meant parts of agent per total 
weight of microparticle. For example, 10 wt. % agent would mean 10 parts 
agent and 90 parts polymer by weight. 
In carrying out the process of the present invention, the encapsulating 
polymer should be essentially 100% dissolved in the solvent blend at the 
time the solution is emulsified. The active agent can be dispersed or 
dissolved in the solvent blend at the time it is added to the continuous 
phase process medium. The content of normally solid material (active agent 
plus encapsulating polymer) in the solvent blend at the time it is first 
emulsified should be at least 5 weight percent and preferably at least 20 
weight percent. Minimizing solvent in the "oil phase" provides a better 
quality microparticle and requires less extraction medium. 
One preferred active agent that can be encapsulated by the process of the 
present invention is norethindrone (NET)--others are risperidone and 
testosterone. 
Ethyl acetate alone is a poor solvent for NET thereby requiring more 
solvent and higher temperatures than the prior art chloroform process. 
Although coreloads of the product microparticles are acceptable, yields, 
especially in the 63-90 .mu.m range, are low. Scanning electron 
micrographs show these larger microparticles to be cracked open (i.e., 
shells) and collapsed. Higher than normal release rates for these 
microparticles corroborate this phenomenon. 
Experiments using benzyl alcohol alone as the solvent resulted in easy 
control of microparticle size as determined by inspection of the quench 
tank contents by optical microscopy. Upon drying, however, generally poor 
quality was found to have resulted. Often, recovery was difficult because 
of stickiness. Also, solvent residuals tended to be elevated. Using a 
solvent system of ethyl acetate and benzyl alcohol for the "oil phase" 
improved the microparticle quality and release characteristics. 
The mixture of ingredients in the "oil phase" solvent system is emulsified 
in a continuous-phase processing medium; the continuous-phase medium being 
such that a dispersion of microdroplets containing the indicated 
ingredients is formed in the continuous-phase medium. 
Although not absolutely necessary, it is preferred to saturate the 
continuous phase process medium with at least one of the solvents forming 
the "oil phase" solvent system. This provides a stable emulsion, 
preventing transport of solvent out of the microdroplets prior to 
quenching. Similarly, a vacuum may be applied as in U.S. Pat. No. 
4,389,330. Where ethyl acetate and benzyl alcohol are the components of 
the solvent system, the aqueous phase of the emulsion preferably contains 
1 to 8 weight percent ethyl acetate and 1 to 4 weight percent benzyl 
alcohol. 
Usually, a surfactant or a hydrophilic colloid is added to the 
continuous-phase processing medium to prevent the solvent microdroplets 
from agglomerating and to control the size of the solvent microdroplets in 
the emulsion. Examples of compounds that can be used as surfactants or 
hydrophilic colloids include, but are not limited to, poly(vinyl alcohol), 
carboxymethyl cellulose, gelatin, poly(vinyl pyrrolidone), Tween 80, Tween 
20, and the like. The concentration of surfactant or hydrophilic colloid 
in the process medium should be sufficient to stabilize the emulsion and 
will affect the final size of the microparticles. Generally the 
concentration of the surfactant or hydrophilic colloid in the process 
medium will be from about 0.1% to about 10% by weight based on the process 
medium, depending upon the surfactant or hydrophilic colloid, the "oil 
phase" solvent system, and the processing medium used. A preferred 
dispersing medium combination is a 0.1 to 10 wt. %, more preferably 0.5 to 
2 wt. %, solution of poly(vinyl alcohol) in water. 
The emulsion can be formed by mechanical agitation of the mixed phases or 
by adding small drops of the organic phase that contains active agent and 
wall forming material to the continuous phase processing medium. The 
temperature during the formation of the emulsion is not especially 
critical, but can influence the size and quality of the microparticles and 
the solubility of the active agent in the continuous phase. Of course, it 
is desirable to have as little of the active agent in the continuous phase 
as possible. Moreover, depending on the solvent blend and continuous-phase 
processing medium employed, the temperature must not be too low or the 
solvent and processing medium may solidify or become too viscous for 
practical purposes. On the other hand, it must not be so high that the 
processing medium will evaporate or that the liquid processing medium will 
not be maintained. Moreover, the temperature of the emulsion cannot be so 
high that the stability of the particular active agent being incorporated 
in the microparticles is adversely affected. Accordingly, the dispersion 
process can be conducted at any temperature that maintains stable 
operating conditions, preferably from about 20.degree. C. to about 
60.degree. C., depending upon the active agent and excipient selected. 
As stated above, in order to create microparticles containing an active 
agent, an organic phase and an aqueous phase are combined. The organic and 
aqueous phases are largely or substantially immiscible, with the aqueous 
phase constituting the continuous phase of the emulsion. The organic phase 
includes the active agent as well as the wall forming polymer, i.e., the 
polymeric matrix material. The organic phase is prepared by dissolving or 
dispersing the active agent(s) in the organic solvent system of the 
present invention. The organic phase and the aqueous phase are combined 
under the influence of mixing means. 
A preferred type of mixing means is a static mixer and a preferred method 
of encapsulating the active agent to form the controlled release 
microparticles of the present invention involves the use of such a static 
mixer. Preferably the combined organic and aqueous phases are pumped 
through a static mixer to form an emulsion and into a large volume of 
quench liquid, to obtain microparticles containing the active agent 
encapsulated in the polymeric matrix material. 
In many of the known techniques for the microencapsulation of biological or 
pharmaceutical agents, the microparticles form when the solvent containing 
the active agent and the polymer is emulsified or dispersed in an 
immiscible second solvent by stirring, agitating, vibrating, or some other 
dynamic mixing technique, often for a relatively long period of time. Such 
dynamic mixing techniques have several drawbacks. For example, it is 
difficult to control the size of the resulting microparticles or the 
distribution of sizes obtained. As a consequence, use of dynamic mixing 
also presents problems when preparing microparticles containing biological 
or pharmaceutical agents on a production or commercial scale. 
Particularly, production equipment includes a costly emulsion tank, 
including equipment to stir or agitate the fluids. One of the controlling 
factors for overall process time is the time required to form a uniform 
emulsion. Increased batch sizes in larger tanks require a longer time to 
form the emulsion, resulting in a longer overall production process time. 
Longer exposure times of the active agent to process solvents and polymers 
in solution can lead to degradation or deactivation of the active agent. 
Scale-up to a production process from a laboratory emulsion process is 
particularly difficult for microencapsulation of biological or 
pharmaceutical agents since, as the batch and tank size are increased, 
stir speeds and viscosities within the larger tank have to be empirically 
determined by trial and error at each stage of the scale-up. This process 
is not only time consuming, but imprecise. 
Accordingly, one advantage of preparing microparticles using a static mixer 
is that accurate and reliable scaling from laboratory to commercial batch 
sizes can be done while achieving a narrow and well defined size 
distribution of microparticles containing biologically or pharmaceutically 
active agents. A further advantage of this method is that the same 
equipment can be used to form microparticles containing active agents of a 
well defined size distribution for varying batch sizes. Yet another 
advantage of the method is that high quality microparticles having a high 
concentration of active agent can be obtained using a single step to 
remove solvent without the need for a two-step solvent removal process as 
described in the above-mentioned Tice et al. patent (U.S. Pat. No. 
4,389,330). In addition to improving process technology, static mixers are 
low maintenance, their small size requires less space than dynamic mixers, 
they have low energy demands, and comparatively low investment costs. 
Static or motionless mixers consist of a conduit or tube in which is 
received a number of static mixing elements. Static mixers provide uniform 
mixing in a relatively short length of conduit, and in a relatively short 
period of time. With static mixers, the fluid moves through the mixer, 
rather than some part of the mixer, such as a blade, moving through the 
fluid. A static mixer is more fully described in U.S. Pat. No. 4,511,258, 
which is incorporated herein by reference. 
When using a static mixer to form an emulsion, a variety of factors 
determine emulsion droplet size. These factors include the density and 
viscosity of the various solutions or phases to be mixed, volume ratio of 
the phases, interfacial tension between the phases, static mixer 
parameters (conduit diameter; length of mixing element; number of mixing 
elements), and fluid velocity through the static mixer. Temperature is a 
variable because it affects density, viscosity, and interfacial tension. 
The primary controlling variable is fluid velocity. Particularly, droplet 
size decreases as fluid velocity increases and alternatively, droplet size 
increases as fluid velocity decreases. Droplets will reach an equilibrium 
size after moving through a fixed number of elements for a given flow 
rate. The higher the flow rate, the fewer elements needed. Because of 
these relationships, scaling from laboratory batch sizes to commercial 
batch sizes is reliable and accurate, and the same equipment can be used 
for laboratory and commercial batch sizes. 
A laboratory set up for carrying out a static mixer process is illustrated 
in FIG. 1. An organic or oil phase 30 is prepared by dissolving an active 
agent and a polymeric matrix material in a stirred pot 32. However, the 
process is not limited to preparing organic phase 30 by dissolving an 
active agent. Alternatively, organic phase 30 may be prepared by 
dispersing an active agent in a solution containing a polymeric matrix 
material. In such a dispersion, the active agent is only slightly soluble 
in organic phase 30. Alternatively, organic phase 30 may be prepared by 
preparing an emulsion containing an active agent and a polymeric matrix 
material (double emulsion process). In the double emulsion process, a 
primary emulsion is prepared that contains an active agent and a polymeric 
matrix material (organic phase 30). The primary emulsion may be a 
water-in-oil emulsion, an oil-in-water emulsion, or any suitable emulsion. 
The primary emulsion (organic phase 30) and an aqueous phase are then 
pumped through a static mixer to form a second emulsion that comprises 
microdroplets containing the active agent encapsulated in the polymeric 
matrix material. 
Organic phase 30 is pumped out of stirred pot 32 by a magnetically driven 
gear pump 34. The discharge of pump 34 feeds a "Y" connection 36. One 
branch 361 of "Y" connection 36 returns to pot 32 for recirculation flow. 
The other branch 362 feeds into an in-line static mixer 10. Aqueous or 
water phase 40 is prepared in like manner, with a stirred pot 42, a 
magnetically driven gear pump 44, and a "Y" connection 46. One branch 461 
of "Y" connection 46 returns to pot 42 for recirculation flow. The other 
branch 462 feeds into in-line static mixer 10. 
Branches 362 and 462 from each solution, which feed in-line static mixer 
10, are joined by another "Y" connection 50 and feed through mixer inlet 
line 51 into static mixer 10. Static mixer 10 discharges through mixer 
outlet line 52 into wash tank 60. Silicone tubing and polypropylene 
fittings are used in the system illustrated in FIG. 1. Silicone tubing 
having 3/8 inch ID is used for all lines except mixer outlet line 52. 
Smaller diameter tubing (3/16 inch ID) is used for mixer outlet line 52 to 
prevent collapse of the emulsion both in mixer outlet line 52 and upon 
entering wash tank 60. 
In one embodiment of the process, pumps 34 and 44 are started in 
recirculation mode and desired flow rates are set for organic phase 30 and 
water phase 40. "Y" connection 46 is then switched so that water phase 40 
flows through branch 462 to static mixer 10. Once water phase 40 fills 
mixer inlet line 51, static mixer 10, and mixer outlet line 52; "Y" 
connection 36 is switched so that organic phase 30 flows through branch 
362 to static mixer 10. When the desired amount of organic phase has been 
pumped to static mixer 10, "Y" connection 36 is switched to recirculation 
through branch 361. Water phase 40 continues to flow for a short time to 
clean out any organic phase remaining in mixer inlet line 51, static mixer 
10, and mixer outlet line 52. "Y" connection 46 is then switched to 
recirculation through branch 461. 
Organic phase 30 and aqueous phase 40 are mixed in static mixer 10 to form 
an emulsion. The emulsion formed comprises microdroplets containing active 
agent encapsulated in a polymeric matrix material. The microdroplets are 
stirred in wash tank 60 which contains a quench solution in order to 
remove the organic solvent from the microdroplets resulting in the 
formation of hardened microparticles. The microparticles are then isolated 
from the aqueous quench solution by any convenient means of separation; 
the fluid can be decanted from the microparticles or the microparticle 
suspension can be filtered or a sieve column can be used. Various other 
combinations of separation techniques can be used, if desired. The 
microparticles are then dried using conventional drying techniques, and 
further size isolation may be carried out. 
Following the movement of the microdroplets from the static mixer and 
entrance into the wash tank, the continuous-phase processing medium is 
diluted and the remainder of the solvent in the microparticles is removed 
by extraction. In this extractive quench step, the microparticles can be 
suspended in the same continuous-phase processing medium used during 
emulsification, with or without hydrophilic colloid or surfactant, or in 
another liquid. The extraction medium removes the solvent from the 
microparticles, but does not dissolve them. During the extraction, the 
extraction medium containing dissolved solvent can, optionally, be removed 
and replaced with fresh extraction medium. This is best done on a 
continual or continuous basis where the rate of extraction medium 
replenishment is critical. If the rate is too slow, active agent crystals 
may protrude from the microparticles or grow in the extraction medium. The 
rate of extraction medium replenishment for a given process is a variable 
that can be determined at the time the process is performed and, 
therefore, no precise limits for the rate need be predetermined. After the 
remainder of the solvent has been removed, the microparticles are isolated 
as stated above and are then dried by exposure to air or by other 
conventional drying techniques, such as, vacuum drying, drying over a 
desiccant, or the like. This process is very efficient in encapsulating an 
active agent since core loadings of up to about 80 wt. %, preferably up to 
about 50 wt. % can be obtained. 
One of the solvents in the blend of solvents used to form the "oil phase" 
droplets in the emulsion will be extracted more quickly than the other 
solvent, e.g., the first solvent, ethyl acetate, in the case of the 
preferred ethyl acetate/benzyl alcohol blend. Thus, high residuals of the 
second solvent (here, benzyl alcohol) are left behind. Owing to the high 
boiling point of benzyl alcohol, it is not easily removed by exposure of 
the microparticles to air or other conventional evaporative means. To 
overcome this, some of the more rapidly extracted solvent is added to the 
extraction medium prior to addition of the emulsion. The concentration of 
the more rapidly extracted solvent in the extraction medium generally is 
from about 20 to about 70% of the saturation point of the solvent in the 
medium at the temperature to be used for the extraction. Thus, when the 
emulsion is added to the quench liquid, extraction of the more rapidly 
extracted solvent is retarded and more of the second, more slowly 
extracted, solvent is removed. 
The exact amount of this more-rapidly-extracted solvent "spike" is of 
importance to final microparticle quality. Too much solvent (i.e., near 
the saturation point) results in porous microparticles with active agent 
visible on the surface, causing what may be an undesirable high rate of 
release. Too little solvent in the extraction medium results in high 
residuals of the more slowly extracted solvent and poor microparticle 
quality. The temperature of the extraction medium is also important as it 
affects solvent solubility and rate of extraction. 
Both temperature and amount of solvent spike may be adjusted to provide the 
final desired product characteristics, i.e., highly porous, quick 
releasing microparticles or slow releasing microparticles having a low 
porosity. 
The quench liquid may be plain water, a water solution, or other suitable 
liquid, the volume, amount, and type of which depends on the solvents used 
in the emulsion phase. The quench liquid preferably is water. Generally, 
the quench liquid volume is on the order of 10 times the saturated volume 
(i.e., 10 times the quench volume needed to absorb completely the volume 
of solvent in the emulsion). Depending on the solvent system, however, 
quench volume can vary from about 2 to about 20 times the saturated 
volume. Additionally, it is convenient to describe the quench volume 
requirement relative to batch size (microparticle product). This ratio is 
an indication of efficiency of the extraction step and, in some cases, 
dictates the batch size for a given set of equipment. The larger the 
ratio, the more volume is required per product weight. On the other hand, 
with a smaller ratio, more product may be obtained from the same amount of 
quench volume. This ratio may vary from about 0.1 to about 10 liters of 
quench volume per gram of microparticles produced. Processes with a ratio 
of less than about 1 liter per gram are preferred. 
When using the preferred solvent combination of benzyl alcohol and ethyl 
acetate, the ethyl acetate of the quench liquid appears to affect the 
residual solvent level in the product microparticles. At low ethyl acetate 
contents in the quench liquid, the benzyl alcohol residuals in the 
microparticles are high while ethyl acetate may be almost non-detectable. 
At high ethyl acetate contents in the quench liquid, 5-7% by weight or 
more, more ethyl acetate may be retained by the microparticles than benzyl 
alcohol. At a quench volume of about 1 liter per gram of active agent and 
polymeric encapsulating material being quenched, about 3-4 weight percent 
ethyl acetate in the quench liquid is optimal at 0.degree.-4.degree. C. 
The coreload of the microparticles varies slightly with changes in ethyl 
acetate concentration in the quench liquid, decreasing with high and low 
concentrations of ethyl acetate. In vitro release rates from the 
microparticles vary substantially as the ethyl acetate content of the 
quench liquid is varied. In the case of NET, quicker release of NET is 
observed at the extreme ethyl acetate contents. Observation with a 
scanning electron microscope shows the presence of NET and pores on the 
microparticle surface when extremes of ethyl acetate are present in the 
quench liquid. 
Altering the volume of the quench liquid also has a profound effect on the 
relative amount of solvent residuals in the microparticles. At low 
volumes, the ratio of benzyl alcohol to ethyl acetate is high and 
decreases to less than one as quench volume is increased to about 1.5 L 
per gram of active agent and polymeric encapsulating material being 
quenched. The rate of active agent release from the product microparticles 
is markedly high. (At 0.125 L quench liquid per gram of solution of NET 
and polymeric encapsulating material, scanning electron micrographs show 
that the product microparticles are extremely porous. From 0.25 to 1.5 L 
quench liquid per gram of solution of NET and polymeric encapsulating 
material, the NET release rate from the product microparticles varies 
slightly with a possible minimum at 1 L quench liquid per gram of NET and 
polymeric encapsulating material being quenched.) 
The microparticle product is usually made up of particles of a spherical 
shape, although sometimes the microparticles may be irregularly shaped. 
The microparticles can vary in size, ranging from submicron to millimeter 
diameters. Preferably, microparticles of 1-500 microns, more preferably, 
25-180 microns, are prepared, whereby administration of the microparticles 
to a patient can be carried out with a standard gauge needle. 
Preferably, the drug loaded microparticles are dispensed to patients in a 
single administration, releasing the drug in a constant or pulsed manner 
into the patient and eliminating the need for repetitive injections. 
The active agent bearing microparticles are obtained and stored as a dry 
material. Prior to administration to a patient, the dry microparticles can 
be suspended in an acceptable pharmaceutical liquid vehicle, such as, a 
2.5 wt. % solution of carboxymethyl cellulose, whereupon the suspension is 
injected into the body. 
The microparticles can be mixed by size or by type so as to provide for the 
delivery of active agent to the patient in a multiphasic manner and/or in 
a manner that provides different active agents to the patient at different 
times, or a mixture of active agents at the same time. For example, 
secondary antibiotics, vaccines, or any desired active agent, either in 
microparticle form or in conventional, unencapsulated form can be blended 
with a primary active agent and provided to the patient. 
Suitable active agents include estrogens such as diethyl stilbestrol, 
17-beta-estradiol, estrone, ethinyl estradiol, mestranol, and the like; 
progestins such as norethindrone, norgestryl, ethynodiol diacetate, 
lynestrenol, medroxyprogesterone acetate, dimesthisterone, megestrol 
acetate, chlormadinoneacetate, norgestimate, norethisterone, ethisterone, 
melengestrol, norethynodrel and the like; and spermicidal compounds such 
as nonylphenoxypolyoxyethyleneglycol, benzethonium chloride, chlorindanol 
and the like. 
Other biologically active agents that can be incorporated using the process 
of the present invention include gastrointestinal therapeutic agents such 
as aluminum hydroxide, calcium carbonate, magnesium carbonate, sodium 
carbonate and the like; non-steroidal antifertility agents; 
parasympathomimetic agents; psychotherapeutic agents; risperidone; major 
tranquilizers such as chlorpromazine HCl, clozapine, mesoridazine, 
metiapine, reserpine, thioridazine and the like; minor tranquilizers such 
as chlordiazepoxide, diazepam, meprobamate, temazepam and the like; 
rhinological decongestants; sedative-hypnotics such as codeine, 
phenobarbital, sodium pentobarbital, sodium secobarbital and the like; 
steroids such as testosterone and testosterone propionate; sulfonamides; 
sympathomimetic agents; vaccines; vitamins and nutrients such as the 
essential amino acids; essential fats and the like; antimalarials such as 
4-aminoquinolines, 8-aminoquinolines, pyrimethamine and the like, 
anti-migraine agents such as mazindol, phentermine and the like; 
anti-Parkinson agents such as L-dopa; anti-spasmodics such as atropine, 
methscopolamine bromide and the like; antispasmodics and anticholinergic 
agents such as bile therapy, digestants, enzymes and the like; 
antitussives such as dextromethorphan, noscapine and the like; 
bronchodilators; cardiovascular agents such as anti-hypertensive 
compounds, Rauwolfia alkaloids, coronary vasodilators, nitroglycerin, 
organic nitrates, pentaerythritotetranitrate and the like; electrolyte 
replacements such as potassium chloride; ergotalkaloids such as ergotamine 
with and without caffeine, hydrogenated ergot alkaloids, 
dihydroergocristine methanesulfate, dihydroergocornine methanesulfonate, 
dihydroergokroyptine methanesulfate and combinations thereof; alkaloids 
such as atropine sulfate, Belladonna, hyoscine hydrobromide and the like; 
analgetics; narcotics such as codeine, dihydrocodienone, meperidine, 
morphine and the like; non-narcotics such as salicylates, aspirin, 
acetaminophen, d-propoxyphene and the like; antibiotics such as the 
cephalosporins, chloranphenical, gentamicin, Kanamycin A, Kanamycin B, the 
penicillins, ampicillin, streptomycin A, antimycin A, chloropamtheniol, 
metromidazole, oxytetracycline penicillin G, the tetracyclines, and the 
like; anti-cancer agents; anti-convulsants such as mephenytoin, 
phenobarbital, trimethadione; antiemetics such as thiethylperazine; 
antihistamines such as chlorophinazine, dimenhydrinate, diphenhydramine, 
perphenazine, tripelennamine and the like; anti-inflammatory agents such 
as hormonal agents, hydrocortisone, prednisolone, prednisone, non-hormonal 
agents, allopurinol, aspirin, indomethacin, phenylbutazone and the like; 
prostaglandins; cytotoxic drugs such as thiotepa, chlorambucil, 
cyclophosphamide, melphalan, nitrogen mustard, methotrexate and the like; 
antigens of such microorganisms as Neisseria gonorrhea, Mycobacterium 
tuberculosis, Herpes virus (humonis, types 1 and 2), Candida albicans, 
Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B 
Streptococcus ecoli, Microplasma hominis, Hemophilus ducreyi, Granuloma 
inguinale, Lymphopathia venereum, Treponema palladium, Brucella abortus, 
Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, 
Campylobacter fetus intestinalis, Leptospira pomona, Listeria 
monocytogenes, Brucella ovis, Equine herpes virus 1, Equine arteritis 
virus, IBR-IBP virus, BVD-MB virus, Chlamydia psittaci, Trichomonas 
foetus, Toxoplasma gondii, Escherichia coil, Actinobadllus equuli, 
Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, 
Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, 
Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, 
Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, and the like; 
antibodies that counteract the above microorganisms; and enzymes such as 
ribonuclease, neuramidinase, trypsin, glycogen phosphorylase, sperm lactic 
dehydrogenase, sperm hyaluronidase, adenosinetriphosphatase, alkaline 
phosphatase, alkaline phosphatase esterase, amino peptidase, trypsin, 
chymotrypsin, amylase, muramidase, acrosomal proteinase, diesterase, 
glutamic acid dehydrogenase, succinic acid dehydrogenase, 
beta-glycophosphatase, lipase, ATP-ase alpha-peptate 
gamma-glutamylotranspeptidase, sterol-3-beta-ol-dehydrogenase, and 
DPN-di-aprorase. 
The following examples further describe tho-materials and methods used in 
carrying out the invention. The examples are not intended to limit the 
invention in any manner. 
EXAMPLE 1 
Preparation of 30%, 33%, and 50% Theoretically Loaded Norethindrone 
Microparticles 
A 1 kg batch of 30% norethindrone loaded microparticles is prepared using a 
3/4" diameter .times.12 element static mixer (Koflo, M/N: 
3/4-TU-3-12RH-11, Koflo Corp., Cary, Ill.). The polymer/drug solution 
(organic phase) is prepared as follows. 329 gm norethindrone USP is 
dissolved in a heated (65.degree.-70.degree. C.) solution of 770 gm 
Medisorb.RTM. 85:15 dl PLGA (Inherent Viscosity (IV)=0.65 dl/gm) in 2.2 kg 
ethyl acetate NF and 2.2 kg benzyl alcohol NF. The solution is filtered 
(0.2 .mu.m) and maintained at 65.degree.-70.degree. C. The process water 
solution (aqueous phase) is prepared as follows. 150 gm of poly(vinyl 
alcohol (PVA-Du Pont Elvanol.RTM. 51-05) is added to 27.27 kg of WFI 
(Water For Injection) and heated (65.degree.-70.degree. C.) until 
dissolved, and then filtered (0.2 .mu.m). To this solution, 810 gm of 
filtered (0.2 .mu.m) benzyl alcohol and 1770 gm of filtered (0.2 .mu.m) 
ethyl acetate are added. The solution is maintained at 
65.degree.-70.degree. C. The quench solution is prepared as follows: 26.25 
kg of ethyl acetate NF (0.2 .mu.m filtered) is dissolved in 750 liters of 
cold WFI and maintained at 2.degree.-4.degree. C. 
The organic phase is pumped through the static mixer at a flow rate of 909 
cc/min, and the aqueous phase at a flow rate of 4500 cc/min into the 
quench solution. After 1 hour of quench, the material is passed through 90 
and 25 .mu.m screens. The 25-90 .mu.m portion is vacuum dried with 
agitation for 36 hours at ambient temperature. The process yield is 650 gm 
of norethindrone loaded microparticles. 
A 1 kg batch of 33% norethindrone loaded microparticles is prepared using a 
3/4" diameter .times.12 element static mixer (Koflo, M/N: 
3/4-TU-3-12RH-11, Koflo Corp., Cary, Ill.). The polymer/drug solution 
(organic phase) is prepared as follows. 363 gm norethindrone USP is 
dissolved in a heated (65.degree.-70.degree. C.) solution of 737 gm 
MEDISORB.RTM. 85:15 dl PLGA (IV=0.62 dl/gm) in 2.2 kg ethyl acetate NF and 
2.2 kg benzyl alcohol NF. The solution is filtered (0.2 .mu.m) and 
maintained at 65.degree.-70.degree. C. The process water solution (aqueous 
phase) is prepared as follows. 150 gm of PVA (Du Pont Elvanol.RTM. 51-05) 
is added to 27.27 kg of WFI and heated (65.degree.-70.degree. C.) until 
dissolved, and then filtered (0.2 .mu.m). To this solution, 810 gm of 
filtered (0.2 .mu.m) benzyl alcohol and 1770 gm of filtered (0.2 .mu.m) 
ethyl acetate are added. The solution is maintained at 
65.degree.-70.degree. C. The quench liquid is prepared as follows. 750 
liters of 3.5% ethyl acetate NF (0.2 .mu.m filtered) is dissolved in WFI 
and maintained at 2.degree.-4.degree. C. 
The organic phase is pumped through the static mixer at a flow rate of 909 
cc/min, and the aqueous phase at a flow rate of 4500 cc/min into the 
quench liquid. After 1 hour of quench, the material is passed through 90 
and 25 .mu.m screens. The 25-90 .mu.m portion is vacuum dried with 
agitation for 36 hours at ambient temperature. The process yield is 630 gm 
of norethindrone loaded microparticles. 
A 1 kg batch of 50% norethindrone loaded microparticles is prepared using a 
3/4" diameter .times.12 element static mixer (Koflo, M/N:3/4-TU-3-12RH-11, 
Koflo Corp., Cary, Ill.). The polymer/drug solution (organic phase) is 
prepared as follows. 546 gm norethindrone USP is dissolved in a heated 
(65.degree.-70.degree. C.) solution of 550 gm MEDISORB.RTM. 85:15 dl PLGA 
(a copolymer of 85 mole % lactic acid and 15 mole % glycolic acid, 
poly(lactide-co-glycolide)) (IV=0.62 dl/gm) in 2.2 kg ethyl acetate NF and 
2.2 kg benzyl alcohol NF. The solution is filtered (0.2 .mu.m) and 
maintained at 65.degree.-70.degree. C. The process water solution (aqueous 
phase) is prepared as follows. 150 gm of PVA (Du Pont Elvanol.RTM. 51-05) 
is added to 27.27 kg of WFI and heated (65.degree.-70.degree. C.) until 
dissolved, and then filtered (0.2 .mu.m). To this solution, 810 gm of 
filtered (0.2 .mu.m) benzyl alcohol and 1770 gm of filtered (0.2 .mu.m) 
ethyl acetate are added. The solution is maintained at 
65.degree.-70.degree. C. The quench solution is prepared as follows: 26.25 
kg of ethyl acetate NF (0.2 .mu.m filtered) is dissolved in 750 liters of 
cold WFI and maintained at 2.degree.-4.degree. C. 
The organic phase is pumped through the static mixer at a flow rate of 909 
cc/min, and the aqueous phase at a flow rate of 4500 cc/min into the 
quench solution. After 1 hour of quench, the material is passed through 90 
and 25 .mu.m screens. The 25-90 .mu.m portion is vacuum dried with 
agitation for 36 hours at ambient temperature. The process yield is 685 gm 
of norethindrone loaded microparticles. 
The 30% and 50% loaded particles were then used to prepare two 65 mg (NET) 
formulations for injecting into baboons. Baboon Formulation 1 consisted of 
35% of the 50% loaded particles and 65% of the 30% loaded particles. 
Baboon Formulation 2 consisted of 50% of each of the 30% loaded and 50% 
loaded particles. Time release data for Baboon Formulations 1 and 2 are 
shown in FIG. 2. 
EXAMPLE 2 
Preparation of 35% Theoretically Loaded Risperidone Microparticles (Batch 
Prodex 2) 
First, the aqueous phase (solution A) is prepared by weighing and mixing 
906.1 g 1% poly(vinyl alcohol) (Vinol 205, Air Products and Chemical Inc., 
Allentown, Pa.), 29.7 g benzyl alcohol (J. T. Baker, Phillipsburg, N.J.) 
and 65.3 g ethyl acetate (Fisher Scientific, Fair Lawn, N.J.). Then the 
organic phase (solution B) is prepared by dissolving 29.3 g of high 
viscosity 75:25 dl (polylactide-co-glycolide), (Medisorb Technologies 
International, L. P., Cincinnati, Ohio) in 108.7 g ethyl acetate and 108.4 
g benzyl alcohol. Once the polymer is completely dissolved, 15.7 g 
risperidone base (Janssen Pharmaceutica, Beerse, Belgium) is added and 
dissolved in the polymer solution. The exposure time of the dissolved 
risperidone with the polymer is kept to a minimum (&lt;10 minutes). Solutions 
A and B are then pumped through a 1/4 inch diameter static mixer (Cole 
Parmer L04667-14) via a gear drive pump and head (Cole-Parmer L07149-04, 
L07002-16) at flow rates of 198 and 24 mL/minute, respectively, into a 
quench medium (wash) composed of 55 liters of water for injection 
containing 1,276.0 g of ethyl acetate, 92.3 g (0.02 Molar) of anhydrous 
sodium bicarbonate, and 116.2 g (0.02 Molar) of anhydrous sodium carbonate 
(Mallinckrodt Specialty Chemicals, Paris, Ky.) at 11.degree. C. The 
microparticles are allowed to stir in this first wash for 1 and 3/4 hours, 
then isolated by sieving with a 25 micron sieve. The product retained by 
the sieve is transferred to a second 20-liter wash of WFI at 13.degree. C. 
After stirring in the second wash for 2 and 1/4 hours, the microparticles 
are isolated and size fractionated by sieving through a stainless-steel 
sieve column composed of 25 and 180 micron mesh sizes. The microparticles 
are dried overnight, then collected and weighed. 
EXAMPLE 3 
Preparation of 40% Theoretically Loaded Risperidone Microparticles (Batch 
Prodex 3) 
First, the aqueous phase (solution A) is prepared by weighing and mixing 
904.4 g 1% poly(vinyl alcohol), (Vinol 205, Air Products and Chemical 
Inc., Allentown, Pa.), 30.1 g benzyl alcohol (J. T. Baker, Phillipsburg, 
N.J.), and 65.8 g ethyl acetate (Fisher Scientific, Fair Lawn, N.J.) Then 
the organic phase (solution B) is prepared by dissolving 27.1 g of high 
viscosity 75:25 dl (polylactide-co-glycolide), (Medisorb Technologies 
International, L. P., Cincinnati, Ohio) in 99.3 g ethyl acetate and 99.1 g 
benzyl alcohol. Once the polymer is completely dissolved, 18.1 g 
risperidone base (Janssen Pharmaceutica, Beerse, Belgium) is added and 
dissolved in the polymer solution. The exposure time of the dissolved 
risperidone with the polymer is kept to a minimum (&lt;10 minutes). Solutions 
A and B are then pumped through a 1/4 inch diameter static mixer 
(Cole-Parmer L04667-14) via a gear drive pump and head (Cole-Parmer 
L07149-04, L07002-16) at flow rates of 198 and 24 mL/minute, respectively, 
and into a quench medium (wash) composed of 55 liters of water for 
injection containing 1,375.6 g of ethyl acetate, 92.4 g (0.02 Molar) of 
anhydrous sodium bicarbonate, and 116.6 g (0.02 Molar) of anhydrous sodium 
carbonate (Mallinckrodt Specialty Chemicals, Paris, Ky.) at 12.degree. C. 
The microparticles are allowed to stir in this first wash for 2 hours, 
then isolated by sieving with a 25 micron sieve. The product retained by 
the sieve is transferred to a second 20-liter wash of WFI at 12.degree. C. 
After stirring in the second wash for 3 hours, the microparticles are 
isolated and size fractionated by sieving through a stainless-steel sieve 
column composed of 25 and 180 micron mesh sizes. The microparticles are 
dried overnight, then collected and weighed. 
EXAMPLE 4 
Lyophilization and Gamma Irradiation of Microparticles from Batches Prodex 
2 and Prodex 3 (Samples Prodex 4A, Prodex 4B, and Prodex 4C) 
Microparticles from batches Prodex 2 and Prodex 3 were lyophilized as 
follows. The microparticles were weighed into 5 cc serum vials. Then an 
aqueous vehicle composed of 0.75% CMC, 5% Mannitol, and 0.1% Tween 80 was 
added to the vials. The microparticles were suspended in the vehicle by 
agitation, then quickly frozen in a dry ice/acetone bath. The vials were 
then lyophilized in a pilot-scale lyophilizer (Dura Stop Microprocessor 
Control, FTS Systems, Inc., Stone Ridge, N.Y.) employing a ramped 
30.degree. C. maximum temperature cycle for 50 hours. Samples Prodex 4A 
and Prodex 4C were lyophilized samples from Prodex 2 and Prodex 3, 
respectively. Sample Prodex 4B was lyophilized from Prodex 2 that had been 
subsequently sterilized by 2.2 MRad gamma irradiation from a .sup.60 Co 
source. 
In Vitro Dissolution Studies 
In vitro dissolution studies were conducted on Prodex 2, Prodex 3, Prodex 
4A, Prodex 4B, and Prodex 4C. Real time and accelerated methodologies were 
used. The equipment consisted of a Hanson research 6-cell USP paddle 
(Method II) dissolution apparatus interfaced with a spectrophotometer and 
data station. Receiving media were continuously recirculated from each 
cell to flow cells inside the spectrophotometer. The absorbance of the 
receiving media was monitored at 236 nm for quantification of risperidone. 
The real time model measured the release rates of microparticles into a 
receiving medium consisting of 50 mM tris buffer at pH 7.4 at 37.degree. 
C. Risperidone was found to have sufficient solubility (.gtoreq.0.5 mg/mL) 
to allow in vitro experiments with this receiving medium. The amount of 
risperidone was kept below 20% of saturation to provide infinite sink 
conditions. Data are shown in FIGS. 3 and 4. 
An accelerated model was also developed. A receiving medium of 27.5 wt% 
ethanol in WFI was used. Results are shown in FIG. 5. 
Animal Dosing and Blood Sampling 
In vivo studies in dogs were conducted on product provided as dry 
microparticles (Prodex 2, Prodex 3) and in lyophilized form (Prodex 4A, 
Prodex 4B, Prodex 4C). The dry microparticles were syringe-loaded and 
resuspended in the syringe with an injection vehicle comprised of 2.5 wt% 
carboxymethyl cellulose (CMC). The lyophilized samples (Prodex 4A, Prodex 
4B, Prodex 4C) were reconstituted in WFI prior to injection. 
Male and female dogs, weighing 11.6.+-.2.3 kg, were divided into groups of 
three dogs each. The dogs were housed in groups of three and fed according 
to standard laboratory conditions. 
The appropriate volumes of the respective depot formulations were dosed 
intramuscularly into the biceps femoralis of the left hind limb at the 
level of the thigh of the dogs at a dose of approximately 2.5 mg/kg 
risperidone. 
Blood samples (5 ml on EDTA) were taken from one of the jugular veins at 0 
(predose), 1, 5, and 24 hours after dosing and also on days 4, 7, 11, 14, 
18, 23, 25, 28, 32, 35, 39, 42, 46, 49, 53, and 56 at the time of the 
apomorphine vomiting test. The apomorphine test was described by P. A. J. 
Janssen and C. J. E. Niemegeers in Arzneim.-Forsch. (Drug Res.), 9:765-767 
(1959). If, during the course of the experiments, each of the three dogs 
of a group no longer showed protection against apomorphine-induced 
vomiting, blood sampling was discontinued. Blood samples were centrifuged 
at 3000 rpm for 10 min and plasma was separated. The plasma samples were 
stored at .ltoreq.20.degree. C. until analysis. 
Plasma samples were analyzed for risperidone (RISP) and for 
9-hydroxyrisperidone (9-OH RISP) using radioimmunoassay (RIA). For the 
plasma samples analyzed with RIA, two different RIA procedures were used, 
one for unchanged risperidone and the other for the active moiety (sum of 
risperidone and 9-hydroxy-risperidone, not to be confused with the term 
"active agent" used elsewhere herein). For the latter plasma samples, the 
concentrations of 9-hydroxy-risperidone were calculated as the difference 
between the concentrations of the active moiety and those of risperidone. 
The quantification limits for the RIA methods were 0.20 ng/ml for 
risperidone and 0.50 ng/ml for the active moiety. 
For each of the formulations, mean (.+-.S. D., n=3) plasma concentrations 
of risperidone, 9-hydroxy-risperidone, and of the active moiety, were 
calculated. Ratios of the plasma concentrations of 9-hydroxy-risperidone 
to those of risperidone were calculated where possible. Peak plasma 
concentrations and peak times of risperidone, 9-hydroxy-risperidone, and 
their sum were determined by visual inspection of the data. AUC ("area 
under the curve") values of risperidone and 9-hydroxy-risperidone were 
calculated between zero time and time using the trapezoidal rule. The time 
t is the last time point at which concentrations of risperidone or 
9-hydroxy-risperidone were higher than the limit of quantification in at 
least 1 out of 3 dogs. For dogs belonging to the same formulation group, 
AUCs were calculated up to the same end-time t, using the value of the 
quantification limit, if one concentration was lower than the 
quantification limit. If two consecutive concentrations were lower than 
the quantification limit, the concentration of the earlier sampling point 
was set equal to the quantification limit, and the concentration of the 
later sampling point was taken as zero. The AUCs were not extrapolated to 
infinity. The AUC of the active moiety was calculated as the sum of the 
AUCs of risperidone and 9-hydroxy-risperidone. 
Mean or median plasma concentrations and/or pharmacokinetic parameters of 
risperidone, 9-hydroxy-risperidone, and the active moiety for formulations 
Prodex 2/3/4A/4B/4C, are given in Table 1. Mean plasma concentration-time 
curves for formulations Prodex 2/3/4A/4B/4C are shown in FIG. 6. For each 
of the formulation groups, results are first discussed for risperidone, 
then for 9-hydroxy-risperidone, and last for the active moiety. For the 
active moiety, plasma concentrations are related to the anti-emetic effect 
in the apomorphine vomiting test. 
After administration of formulations Prodex 2 up to Prodex 4C, mean peak 
plasma levels of risperidone were low. They were attained at largely 
different time points. The further release of risperidone from the 
different formulations proceeded gradually and was long-lasting. This 
resulted in low plasma concentrations of both risperidone and its 
metabolite. Mean peak times for 9-hydroxy-risperidone all ranged from 26 
to 30 days. The plasma concentration-time profile of the active moiety was 
similar for formulations Prodex 2 up to Prodex 4C. At the beginning of the 
experiment, plasma concentrations of the active moiety showed a peak 
within 1 or 2 days, due to a rapid initial release of risperidone. The 
peak was followed by a decrease of the concentrations with a dip at 5-8 
days. From day 8 on, concentrations increased again until day 20, after 
which time they remained at a more or less constant level during a period 
of, on average, 15 days. During this period, for each of the formulations, 
concentrations of the active moiety showed a second peak and 
concentrations were higher than for the first peak. The anti-emetic 
activity lasted 35 to 42 days for formulations Prodex 2, Prodex 4A, and 
Prodex 4B. For formulation Prodex 4C, it lasted 49 days, but without 
interruption in any of the dogs. The longest activity of formulation 
Prodex 4C paralleled the highest C.sub.max, T.sub.max, and AUC.sub.O-t for 
the active moiety, in comparison with the other 4 formulations of the same 
group. 
The duration of action of these microparticle-based risperidone 
formulations in the apomorphine-induced emesis test in dogs was also 
studied. Neuroleptics antagonized apomorphine-induced emesis by blocking 
dopamine D.sub.2 receptors in the area postrema of the fourth ventricle. 
The test is generally used to predict the onset and duration of 
antipsychotic action of neuroleptics in man (Janssen et al., 
Arzneim.-Forsch./Drug Res. 10:1196-1206 (1965); Niemegeers et al., Life 
Sci. 24:2201-2216 (1979)). 9-OH-risperidone has a pharmacological profile 
that is virtually identical to that of its parent compound. Parent 
compound and active metabolite constitute together the "active moiety" 
that determines the biological activity of risperidone. 
Apomorphine was administered subcutaneously at 0.31 mg/kg to the dogs twice 
a week, during the whole course of the experiment. The dogs were observed 
for vomiting during a 1-hour period after the administration of 
apomorphine. Complete absence of emesis for 1 hour after apomorphine 
challenge was considered to reflect significant anti-emetic activity. The 
duration of the anti-emetic action was defined as the time interval during 
which 2 out of 3 dogs were protected from emesis. 
The formulations were injected in a volume of 0.5 mL into the biceps 
femoralis of one of the hind limbs at the level of the thigh. At several 
time intervals after the intramuscular injection, blood samples were taken 
and, immediately thereafter, the dogs were challenged with a dose of 
apomorphine. Complete absence of emesis within 1 h after apomorphine 
challenge (which is never observed in control animals; n&gt;1000) was 
considered to reflect significant antiemetic activity. 
Table 2 indicates whether the dogs were protected (+) or not protected (-) 
from apomorphine-induced emesis at the various time intervals after 
intramuscular injection of the depot formulations. All formulations showed 
an immediate onset of anti-emetic action. 
TABLE 1 
__________________________________________________________________________ 
Mean (.+-.S.D.; n = 3) or median plasma concentrations and mean 
(.+-.S.D.; n = 3) pharmacokinetic parameters 
of risperidone, 9-hydroxy-risperidone, and their sum (= the "active 
moiety") after intramuscular 
administration of risperidone depot formulations at 2.5 mg/kg to beagle 
dogs. 
__________________________________________________________________________ 
Prodex 2 Prodex 3 Prodex 4A 
Time (days) 
RISP 9-OH RISP 
RISP 9-OH RISP 
RISP 9-OH RISP 
__________________________________________________________________________ 
0 .ltoreq.0.20 
.ltoreq.0.50 
.ltoreq.0.20 
.ltoreq.0.50 
.ltoreq.0.20 
.ltoreq.0.50 
0.042 (1 h) 
8.36 .+-. 1.06 
4.17 .+-. 1.71 
21.4 .+-. 8.8 
14.4 .+-. 9.1 
3.25 .+-. 0.57 
1.18 .+-. 0.50 
0.208 (5 h) 
2.87 .+-. 0.20 
7.34 .+-. 2.02 
7.55 .+-. 3.38 
27.4 .+-. 22.0 
2.61 .+-. 0.60 
5.13 .+-. 1.08 
1 1.25 .+-. 0.72 
6.92 .+-. 3.88 
2.90 .+-. 1.70 
23.0 .+-. 17.8 
1.13 .+-. 0.24 
7.82 .+-. 3.55 
4 0.67 .+-. 0.61 
4.36 .+-. 3.32 
1.22 .+-. 0.77 
6.58 .+-. 3.07 
0.74 .+-. 0.38 
2.54 .+-. 1.20 
7 0.35* 
1.65 .+-. 1.24 
1.96 .+-. 1.70 
8.79 .+-. 6.72 
0.39* 1.90 .+-. 1.52 
11 0.41 .+-. 0.15 
1.16 .+-. 0.35 
1.52 .+-. 0.91 
11.2 .+-. 11.7 
2.40 .+-. 3.55 
12.7 .+-. 20.2 
14 --** --** 4.36 .+-. 1.99 
29.4 .+-. 25.0 
2.23 .+-. 1.19 
12.6 .+-. 15.0 
18 -- -- 6.33 .+-. 2.48 
44.1 .+-. 35.4 
4.28 .+-. 1.41 
23.3 .+-. 12.5 
21 -- -- 8.61 .+-. 2.25 
44.8 .+-. 26.3 
6.97 .+-. 1.57 
27.1 .+-. 11.3 
25 6.79 .+-. 1.74 
44.6 .+-. 13.6 
9.08 .+-. 3.95 
47.9 .+-. 19.5 
6.03 .+-. 1.50 
32.3 .+-. 2.8 
29 6.84 .+-. 3.19 
46.0 .+-. 15.1 
9.26 .+-. 5.27 
54.2 .+-. 33.6 
6.52 .+-. 1.40 
40.2 .+-. 3.6 
32 4.97 .+-. 1.89 
39.5 .+-. 36.6 
5.60 .+-. 2.78 
38.8 .+-. 25.2 
3.81 .+-. 1.72 
35.2 .+-. 16.3 
35 3.61 .+-. 1.84 
25.8 .+-. 11.5 
4.70 .+-. 3.39 
28.4 .+-. 21.9 
2.55 .+-. 1.31 
22.1 .+-. 14.4 
39 1.44 .+-. 0.51 
13.0 .+-. 7.1 
2.01 .+-. 1.47 
16.4 .+-. 9.6 
1.13 .+-. 0.82 
10.4 .+-. 6.4 
42 1.05 .+-. 0.45 
7.73 .+-. 3.77 
1.31 .+-. 0.79 
10.7 .+-. 6.5 
0.68* 6.08 .+-. 4.26 
46 .ltoreq.0.20* 
2.94 .+-. 1.35 
0.45* 5.55 .+-. 4.04 
.ltoreq.0.20* 
2.48 .+-. 1.81 
49 -- -- 0.23* 2.13 .+-. 1.34 
.ltoreq.0.20 
1.23* 
53 -- -- -- -- -- -- 
56 -- -- -- -- -- -- 
Cmax (ng/ml) 
8.61 .+-. 1.41 
61.0 .+-. 19.7 
21.4 .+-. 8.8 
56.3 .+-. 32.2 
7.75 .+-. 0.78 
43.9 6.6 
Tmax (days) 
10 .+-. 17 
29 .+-. 4 
0.042 .+-. 
26 .+-. 2 
25 .+-. 4 
30 .+-. 2 
0.000 
AUC0-T (ng.h/ml) 
3212 .+-. 914 
21496 .+-. 4854 
5048 .+-. 2397 
30632 .+-. 
3280 .+-. 576 
19632 .+-. 
19866 8274 
t(days) 46 46 49 49 46 49 
__________________________________________________________________________ 
RISP + 9-OH RISP 
RISP + 9-OH RISP 
RISP + 9-OH RISP 
__________________________________________________________________________ 
Cmax (ng/ml) 
67.3 .+-. 19.8 
66.0 .+-. 37.0 
49.6 .+-. 6.7 
Tmax (days) 
29 .+-. 4 26 .+-. 2 30 .+-. 2 
AUC0-t (ng.h/ml) 
24708 .+-. 5341 
35680 .+-. 22261 
22912 .+-. 8822 
__________________________________________________________________________ 
Prodex 4B Prodex 4C 
Time (days) 
RISP 9-OH RISP RISP 9-OH RISP 
__________________________________________________________________________ 
0 .ltoreq.0.20 
.ltoreq.0.50 
.ltoreq.0.20 
.ltoreq.0.50 
0.042 (1 h) 
3.32 .+-. 0.75 
2.53 .+-. 0.79 
15.5 .+-. 5.2 
3.32 .+-. 2.18 
0.208 (5 h) 
1.52 .+-. 0.33 
5.56 .+-. 2.43 
15.1 .+-. 7.7 
19.2 .+-. 6.2 
1 1.22 .+-. 0.58 
7.10 .+-. 3.40 
4.49 .+-. 1.04 
25.0 .+-. 7.1 
4 0.58* 2.25 .+-. 1.00 
2.00 .+-. 0.42 
12.1 .+-. 2.5 
7 0.35* 1.78* 1.47 .+-. 0.29 
7.96 .+-. 0.74 
11 0.53* 1.87* 3.23 .+-. 1.72 
13.4 .+-. 4.6 
14 4.06 .+-. 3.47 
22.1 .+-. 20.3 
7.67 .+-. 4.54 
30.9 .+-. 17.8 
18 1.41 .+-. 0.14 
5.13 .+-. 0.85 
8.15 .+-. 4.69 
48.5 .+-. 34.5 
21 7.22 .+-. 4.98 
27.1 .+-. 21.1 
13.1 .+-. 9.4 
69.3 .+-. 41.4 
25 5.39 .+-. 3.41 
41.0 .+-. 29.7 
8.37 .+-. 0.88 
67.8 .+-. 28.0 
29 4.66 .+-. 1.47 
31.1 .+-. 13.3 
13.8 .+-. 5.2 
77.9 .+-. 17.7 
32 3.50 .+-. 1.81 
21.4 .+-. 9.8 
10.3 .+-. 4.5 
80.9 .+-. 51.3 
35 1.91 .+-. 0.71 
14.9 .+-. 4.5 
7.58 .+-. 3.49 
61.4 .+-. 15.1 
39 0.67 .+-. 0.16 
7.15 .+-. 2.47 
3.90 .+-. 1.34 
31.2 .+-. 10.7 
42 &lt;0.20* 3.83 .+-. 0.40 
2.97 .+-. 1.35 
23.2 .+-. 13.7 
46 &lt;0.20* 1.08 .+-. 0.53 
0.68 .+-. 0.39 
10.4 .+-. 6.3 
49 -- -- 0.26* 6.04 .+-. 3.75 
53 -- -- .ltoreq.0.20* 
2.98 .+-. 2.39 
56 -- -- .ltoreq.0.20* 
1.89 .+-. 1.40 
Cmax (ng/ml) 
7.71 .+-. 4.23 
42.6 .+-. 27.3 
16.3 .+-. 6.6 
95.4 .+-. 41.7 
Tmax (days) 
24 .+-. 5 
26 .+-. 2 0.097 .+-. 0.096 
30 .+-. 2 
AUC0-t (ng.h/ml) 
2648 .+-. 1199 
15656 .+-. 8104 
7424 .+-. 3018 
46840 .+-. 19125 
t(days) 46 46 56 56 
__________________________________________________________________________ 
RISP + 9-OH RISP RISP + 9-OH RISP 
__________________________________________________________________________ 
Cmax (ng/ml) 
48.5 .+-. 29.8 108 .+-. 44 
Tmax (days) 26 .+-. 2 30 .+-. 2 
AUC0-t (ng.h/ml) 
18311 .+-. 9222 54264 .+-. 22055 
__________________________________________________________________________ 
*Median value. 
**No blood sampling from day 14 until day 25 of the experiment, due to 
absence of protection against apomorphineinduced vomiting. Concentrations 
in italics indicate antiemetic activity in at least 2 out of 3 dogs. 
TABLE 2 
__________________________________________________________________________ 
Protection (+) or no protection (-) from apomorphine-induced emesis in 
dogs at successive time intervals after 
intramuscular administration of microparticle-based depot formulations of 
the antipsychotic risperidone at an 
approximate dose level of 2.5 mg/kg (continued from previous page) 
Form. 
Prodex 2 Prodex 3 Prodex 4A 
Prodex 4B 
Prodex 4C 
__________________________________________________________________________ 
Dog 14.2 
11.5 
9.8 
12.9 
12.4 
13.4 
10.0 
12.3 
9.2 
9.7 
8.6 
10.6 
13.2 
16.4 
16.2 
Weight 
(kg) 
Volume 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
0.53 
(ml/dog) 
Dose 2.5 
2.5 
2.8 
2.5 
2.5 
2.5 
2.5 
2.3 
2.6 
2.5 
2.5 
2.6 
2.4 
2.4 
2.5 
(mg/kg) 
Route 
im im im im im im im im im im im im im im im 
1 h + + - + + + + + + - + + + + + 
5 h + + + + + + + + + + - + + + + 
1 d + + + + + + + + + + + + + + + 
4 d - - + + - + + + - - - + + + + 
7 d - - - - + + - - - - - + + + + 
11 d - - - + + + + + - - - + + + + 
14 d + + + + + + - + + + + + 
18 d + + + + + + + + + + + + 
21 d + + + + + + + + + + + + 
25 d + + + + + + + + + + + + + + + 
29 d + + + + + + + + + + + + + + + 
32 d + + + + + + + + + + + + + + + 
35 d + + + + + + + + + + + + + + + 
39 d - + + + - + + + - - - - + + + 
42 d - - - + - + + - - - - - + + - 
46 d - - - + - - - - - - - - + + - 
49 d Stop - - - - - - Stop + + - 
53 d Stop Stop - + - 
56 d - - - 
Stop 
__________________________________________________________________________ 
3 Injection volume: 0.5 ml/dog; the concentration of the microparticles 
was adapted to the body weight. 
EXAMPLE 5 
An 85:15 D,L-lactide/glycolic acid copolymer (10.6 g) and norethindrone USP 
(9.4 g) were sequentially dissolved in a 50:50 (weight) blend of ethyl 
acetate and benzyl alcohol (80 g) ("oil phase"). Once dissolved, the 
solution was transferred to a 500 g emulsion bath mixture at 
60.degree.-650.degree. C. composed of 0.5 weight percent poly(vinyl 
alcohol) (Vinol 205, Air Products, having a number average molecular 
weight of 15,000 to 27,000 and a degree of hydrolysis of 87-89%), 5.9 
weight percent ethyl acetate, 2.7 weight percent benzyl alcohol, and 90.9 
weight percent water, contained in a 1000 mL jacketed beaker equipped with 
a turbine stirrer and a thermostatic heater. This emulsion bath mixture 
approximated a saturated solution for both ethyl acetate and benzyl 
alcohol at 60.degree. C. During emulsion formation, extraction of solvent 
from the "oil phase" can thus be prevented and any time effect during this 
step minimized. The stir speed was adjusted to provide for an oil droplet 
size of approximately 90 .mu.m. The resulting emulsion was transferred to 
a chilled (2.degree.-4.degree. C.) water tank containing various amounts 
of water and ethyl acetate, as reported in FIGS. 7A-C and 8A-C. After one 
hour, the microparticles were collected on a sieve stack (25, 45, 63 and 
90 .mu.m) and allowed to dry overnight under a hood. The next day the 
microparticles were blended (15%:25-45 .mu.m; 50% :45-63 .mu.m; and 35% : 
63-90 .mu.m) and sampled. The results are reported in FIGS. 7A-C and 8A-C. 
EXAMPLE 6 
Example 5 is repeated except that the size of the "oil phase" solution of 
NET and polymer is 5 g in each case, the emulsification bath is 300 mL of 
water containing 0.5 wt % of the poly(vinyl alcohol) used in Example 5. 
The results are reported in Table 3. 
TABLE 3 
__________________________________________________________________________ 
Emulsion 
Solvent 
Conditions Yield (%) 
Batch Content 
Temp Time 
Quench 
Total 
25-90 
Core Residual 
Scanning Electron 
No. 
Description 
(%) (.degree.C.) 
RPM 
(min) 
Vol (l) 
Recovered 
.mu. 
Load 
In Vitro Dissolution 
Solvent 
Microscopy 
__________________________________________________________________________ 
3151 
Dslvd NET/ 
90.0 
Amb 
230 
15 5 57.2 46 .461 
12% Burst, 
ETAC/BA 
Very porous, out of 
50:50; 60% Release @ 18 hr 
0.0003/ 
round, inconsistent 
ETAC:BA (unblended sample) 
3.77 
3161 
Dslvd NET/ 
84.5 
37 247 
26 5/10 
60.4 35 .416 
40% Release @ 18 hr 
0.83/ 
Inconsistent, less 
50:50; (unblended sample) 
1.5 porous 
ETAC:BA 
3181 
Dslvd NET/ 
89.9 
Amb 
226 
9 5/10 
57.0 32 .456 
No burst, 0.51/ 
Rough surface, 
50:50; 25% Release @ 18 hr 
1.99 inconsistent 
ETAC:BA (unblended sample) 
3251 
Dslvd NET/ 
80.9 
60 250 
7 5/10 
61.4 29 .445 
No burst, 0.38/ 
Round, some irreg., 
50:50; 18% Release @ 18 hr 
2.56 more consistent 
ETAC:BA (unblended sample) 
3261 
Dslvd NET/ 
75.0 
75 199 
7 5/10 
61.0 7.6 .422 
7% Burst, 0.53/ 
Round, some porous, 
50:50; 58% Release @ 18 hr 
2.72 less consistent 
ETAC:BA (unblended sample) 
3301 
Dslvd NET/ 
80.9 
60 250 
11 5 72.0 49 .431 
nd non-det/ 
nd 
50:50; 12.3 
ETAC:BA 
3381 
Dslvd NET/ 
90.1 
25 118 
17 5/10 
39.2 39.2 
.421 
No burst, 0.37/ 
Round, somewhat 
50:50; 20.3% Release @ 18 
3.06 smooth 
ETAC:BA hr (unblended sample) 
3391 
Dslvd NET/ 
80.9 
63 220 
10 5/10 
6.2 2.2 .431 
nd nd Round, uneven 
surface 
50:50; 
ETAC:BA 
3401 
Dslvd NET/ 
80.9 
61 234 
8 5/10 
24.8 24.8 
.427 
No burst, 0.22/ 
Round, uneven 
surface 
50:50; 46.1% Release @ 18 
2.09 
ETAC:BA hr (unblended sample) 
__________________________________________________________________________ 
EXAMPLE 7 
A 20 gram batch of testosterone-loaded microparticles was made as follows: 
10.8 g of the polymer of Example 5 and 9.2 g of testosterone were 
dissolved in 67 g of a 75:25 blend of ethyl acetate and benzyl alcohol and 
heated to approximately 65.degree. C. The solution was then transferred to 
a 500 g aqueous mixture of 0.5% poly(vinyl alcohol) and 6.5% ethyl acetate 
in a 1000 mL jacketed glass reaction vessel equipped with a turbine 
stirrer. Stir speed was adjusted to approximately 245 rpm. After five 
minutes, the emulsion was transferred to a chilled (0.degree.-4.degree. 
C.) tank containing 20 liters of water spiked with ethyl acetate at a 5% 
concentration. After one hour, the microparticles were recovered on a 25 
and 150 micron sieve stack and allowed to dry overnight under a laboratory 
hood. The next day, the microparticles on the 25 micron screen were 
recovered and sampled. The product contained 39.7% testosterone, 3.67% 
ethyl acetate, and 0.89% benzyl alcohol. An accelerated in vitro release 
model indicated 15% of the drug was released after 18 hours in the 
receiving fluid. 
While various embodiments of the present invention have been described 
above, it should be understood that they have been presented by way of 
example only, and not limitation. Thus the breadth and scope of the 
present invention should not be limited by any of the above described 
exemplary embodiments, but should be defined only in accordance with the 
following claims and their equivalents.