CA1245557A - Vesicle formulations for the controlled release of therapeutic agents - Google Patents

Abstract

ABSTRACT

Disclosed herein are vesicle compositions for controlled sustained release of an encapsulated therapeutic agent after parenteral administration. By adjusting the osmolarity between the suspending solution and the solution within the vesicles by adjusting the concentration of the suspending solution, the rate of release of the therapeutic agent after parenteral administration can be varied. The compositions can be effec-tively administered by intramuscular, subcutaneous injection or other means.

Description

The present invention relates to formulations for the controlled release in vivo of therapeutic agents. In another aspect, it relates to vesicles and, particularly, to phospholipid vesicles.
Numerous conditions in both man and lower animals are responsive to drugs or other therapeutic agents adminis-tered ln vivo. To be useful, these agents must be adminis-tered at a dosage level which is high enough to cause the desired effect, and that dosage level must be maintained for a sufficient period of time to achieve that effect.
Many such therapeutic agents are routinely administered by intra-muscular injection at dosage levels calculated to produce a concentration of the agent in the circulatory system which is effective. Thereafter, injections are repeated as necessary to maintain the -therapeutically effective level.
It is often the case that the therapeutically effec-tive level of the agent in circulation is main-tained only a short time after injection because of breakdown of the agent by the host's defensive mechanisms against foreign substances.

~5557 ~oreover, the agent itself may have intolerable side effects, even lethal ones, if administered in amounts which substantial-ly exceed the therapeutically useful level. Therefore, pro-longing the effective concentration of the agent in the body by increasing the dosage is always limited by the amount of toxicity. Even in cases where toxi.city is low, the size of an injection can be limited by the size of the bolus which can be administered safely.
In view of such problems, efforts have been made to develop delivery systems for therapeutic agents which may be administered _ vivo and which, after administration, gradually, release the agent into its environment in order to prolong the interval over which the effective concentration of the agent is maintained in that environment. In this way, the interval between administrations of the agent can be increased and, in some instances, the need for further administration can be eliminated.
One such approach to obtaining prolonged or sustained release has been to encapsulate the therapeutic agent in a "vesicle". As used herein, the term vesicle refers to a micellular particle which is usually spherical in form and which is frequently obtained from a lipid which forms a bi-layered membrane and is referred to as a "liposome". ~ethods for making such vesicles are well known in the art. Typcially, they are prepared from a phospholipid such as distearoyl phos-phatidylcholine or lecithin, and may include other materials such as positively or negatively charged compounds. Vesicles made from phospholipids are commonly referred to simply as "phospholipid vesicles". Depending on the techniques for its preparation, a vesicle may form as a simple bilayered shell (a unilamellar vesicle) or it may form in multiple layers (multilamellar vesicle).
After administration, typcially as a suspension in physiological saline, the vesicles gradually release the encap-sulated therapeutic agent which then displays its expected effect. Mowever, prior to its release, the agent exhibits no pharmacokinetic properties and is protected by the vesicle from metabolic degradation or other attack by the host's defense mechanisms against foreign substances. Accordingly, the agent can be safely administered in an encapsulated form in dosages which are high enough that, if directly given the host, could result in severe side effects or even death.
The time interval over which an effective concentra-tion of the therapeutic agent is maintained after administra-tion as a vesicle encapsulant is generally thought to be a function of the rate at which it is released from the vesicle and the rate at which it is absorbed from the point of admin-istration after release. Since the former may vary withvesicle structure and the latter by reason of the properties of the agent, the interval over which the useful concentration of an agent in circulation is maintained can vary widely. General-ly, the rate of release from the vesicle is though-t to be cont-rolling for most compounds and sustained release of encapsu-lated drugs over a period of 6-8 hours is a common observation.
See F.J.T. Fields, (1981) Liposomes: From Physical Structure to Therapeutic Applications; Research Monographs in ~24SS57 Cell and Tissue Physioloqy, Vol. 7, C.G. ~night, ed., Elsevier/
North Holland, N.Y., p. 51ff and R.W. Stevenson et al, Diabeto-ogia, 19, 217 (1980~. However, intramuscular injections in mice of vesicle encapsulated interferon resulted in localized levels of interferon which, after three days, were equivalent to levels observed over 2-4 hours ~ter injection of free inter-feron. D.A. Eppstein et al, J. Virol., 41, 575. This longer time of sustained release likely reflects the lower mobility of this biomacromolecule from the injection site.
Notwithstanding the advance in sustained release which has been achieved using vesicles as encapsulants, further improvements in sustained release composi~ions are desirable to reduce still further the interval between administrations of the therapeutic agents. Even a 6-8 hour period of sustained release makes out-patient treatment difficult, if not impossible, and longer intervals would reduce the workload of hospital or other medical personnel, not to mention reducing the patient's discomfort. Furthermore, although by vesicle encapsulation the period over which an effective concentration of therapeutic agents could be maintained is extended, no effective means,:~o control the rate of release results from encapsulation itself.
Accordingly, there remains as yet unmet, a desire for sustained release formulations of therapeutic agents which extend even further the interval over which an effective concentration of the agent is maintained.

SU~lARY OF THE INVENTION
According to the present invention, a process and a composition are provided for controlling the rate at which a therapeutic encapsulated in a vesicle is released from the vesicle after _ vivo administration. This is achieved by lZ~iS~

suspending the vesicles, which encapsulate a solution of the therapeutic agent, in a solution containing sufficient solute that its osmolarity, relative to that of the solution within the vesicles, is at least substantially isotonic, that is, at least approximately 25% of the osmolarity inside the vesicles and is of greater osmolarity than physiological saline. The suspension can then be administered parenterally, for example by subcutaneous or intramuscular injection.
The rate of release of the therapeutic agent from the vesicles after administration is a function of the initial osmotic pressure. Thus, as the osmolarity of the suspending solution becomes less hypotonic, relative to the solution with-in the vesicles, the rate of release of the therapeutic agent is slowed. Slowest releases are obtained when the suspending solution approaches an isotonic, or even hypertonic, relation-ship with respect to the solution within the vesicles. The compositions of the present invention exhibit a longer inter-val over which the sustained release of the agent is maintained compared to agents encapsulated in vesicles and administered as suspensions in physiological saline as described in the prior art.
BRIEF DESCRIPTION OF THE FIGURES
... _ . . ... .
Figure 1 illustrates the effect of temperature on the stability of vesicle formulations.
Figure 2 illustrates the effec-t of vesicle membrane fluidity on sustained release of 2-PAMCl.
Figure 3 demonstrates the effect of vesicle compos-ition on sustained release of 2-P~Cl.

Figure 4 illustrates the effect of cholesterol cont-ent of phospholipid vesicles on sustained release of 2-PAMCl.

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Figure 5 illustrates the effect of vesicle physical structure on sustained release of 2-PAMCl.
Figure 6 illustrates the effect of suspending 2-PAMCl loaded vesicles in a solution substantially isotonic to the vesicles.
DET~ILED DESCRIPTION
As noted above, the present invention relates to method for controlling the rate of release n vivo of a therapeutic agent from a vesicle encapsulant by adjusting the osmotic pressure between the solution of therapeutic agent within the vesicle and the solution in which the vesicles are suspended for parenteral administration.
Thus in an aspect, the present invention provides a composition suitable for parenteral administration to animals including human beings, said composition comprising a solution of a therapeutic agent encapsulated in vesicles, the vesicles being pharmaceutically acceptable and being suspended in a solution containing sufficient solute to provide an osmolarity which is at least about 25% of the osmolarity of the solution within the vesicles and which is of greater osmolarity than physiological saline.
In another aspect, the present invention provides a process for producing such a composition. The process comprises encapsulating a solution of the agent in pharmaceu-tically acceptable vesicles and suspending the vesicles in a solution containing sufficient solute to provide an osmolarity which is at least 25~ of the osmolarity of the solution of the therapeu-tic agent within the vesicles and which is of greater osmolari-ty than physiological saline.
When such vesicles are parenterally administered to " ~2~5S~

a host, for example, intramuscularly, the interval over which sustained release is maintained is substantially increased.
Although we do not wish to be bound byany particular theory, the increase in the interval of sustained release obtained as the osmolarity of the suspending solution is made less hypotonic, relative to the solution within the vesicles, may result from the fact that, so long as the concentration of solute in the suspending solution is such that the solu-tion is not hypotonic, that is the osmolarity approaches at least a substantially isotonic relationship with the solution within the vesicles, the osmotic pressure is not great enough to cause the solvent of the suspending solution to migrate into the vesicles which would increase the hydraulic pressure with-in the vesicles and cause them to break down or release part of their contents. After administration, however, the concentration of liquid medium around the vesicles gradually becomes more hypotonic with respect to the solution within the vesicles, for example, by absorption of the solute from the - suspending solution. As this occurs, the osmotic pressure between the vesicles and the hypotonic liquid medium causes liquid to migrate into the vesicles, causing release of the therapeutic agent. By contrast, the prior art practice of administering the therapeutic agent in vesicles suspended in physiological saline which is already hypotonic to the vesicles results in a much more rapid release of the vesicle contents.
Accordingly, the therapeutic agen-t is also more rapidly re-leased. By adjusting the osmolarity between the solution of therapeutic agent and the suspending solution, however, the rate of release can be varled giving a degree of control over this rate not hitherto attained.
In the practice of the present invention, the thera-peutic agent i~ dissolved in a suitable solvent, for example, physiological saline, usually at or near the saturation point in the case of agents of limited solubility, and encapsulated in a suitable vesicle, Techn;ques to do this are well known in the art and need not be described in detail here. Presently preferred for use in the invention are multilamellar phospho-lipid vesicles although unilamellar vesicles and vesicles ofother than phospholipid can be used, the basic essential criterion being that the material of the vesicles be tolerable by the host in the amounts to be administered.
The solution used for suspending the vesicles i~
preferably physiological saline ~-O.15M NaCl) to which has been added a second solute to adjust the concentration of this solution to a level that gives the desired rate of release.
This concentration will be adjusted ~o that the osmoloarity of the suspending solution is substantially isotonic with the solution within the vesicles, that is, the suspending solution contains sufficient solute to provide an osmolarity that is at least approximately 25%, preferably at least 40 to 50~
of the osmolarity ol the solution wi~thin the vesicles. The usually desired result of the invention, i.e., lengthening the interval of sustained rele2se, can be achieved if the osmolarity of the suspending solution is within the range from substantially isotonic to hypertonic with respect to the vesicles, with it being especially ~esirable for the suspending solution to be at least about 75~ to about 90~ of being isotonic. Since solute is thus added to the suspending solution, it will be clear that the suspending solution will have an osmolarity greater than that of physiological saline.

~SSS7 Suitable for use as solutes in the suspending agents are any solutes which are well tolerated by the host. Among these may be mentioned the sugars such as dextrose and the hexoses such as glucose and polypeptides which do not exhibit significant biological effects. Presently preferred is glucose as it is readily obtained as a sterile substance for admini-stration to humans and is readily absorbed by the body.
Any of a wide variety of therapeutic agents may be used as part of the invention. Among these may be mentioned antibiotics, metabolic regulators, immune modulators, toxin anti-dotes, etc. For example, the invention is well suited for the controlled release of antidotes to cholinesterase inhibitors.
In order to demonstrate the advantages of the present invention, there follows a description of experiments carried out with vesicle encapsulated 2-pralidoxime chloride (2-PAMCl), an agent which is a well known and thoroughly studied antidote to toxic organophosphates which inhibit cholinesterase. Persons exposed to such intoxicants in lethal amounts suffer cardiac insufficiency or respiratory paralysis which results in death.
~ Agents such as 2-PAMCl reactivate cholinesterase if administered in a timely fashion. However, dosages of 2-PAMCl high enough to maintain the therapeutic level for a long period of time cannot be administered because undesirable side effects, even death, can result.
EXPERIMENTAL RESULTS
.
A. Materials L- a-distearoyl phosphatidylcholine (DSPC), L- a-dipalmitoyl phosphatidylcholine (DPPC) from Calbiochem, and _g_ 12~557 cholesterol (Chol), stearylamine (SA), and dicetylphosphate (DCP) from Sigma Chemical Cornpany were used without further purifica-tion to prepare vesicles. 2-Pyridinealdoxime (2-PAM), pralido-xime chloride (2-PAMCl) and Iodomethane were purchased from Aldrich Chemical Company and AG lx8 ion exchange resin was from BioRad Laboratories (Richmond, CA). Ultrapure InC13 was purchased from Ventron Corporation (Danvers, MA). [ H]-cholesterol oleate (specific activity: 52 Ci/mole) and [14C]- Iodomethane (specific activity: 10 Ci/mole) were purchased from New England Nuclear.
Carrier-free InC13 was purchased from Medi-Physics (Glendale, CA) and purified according to the me-thod of Hwang and Mauk, Proc.
Natl. Acad. Sci. USA, 74, 4991 (1977)~ The ionophore A23187 was from Eli Lilly and Co. Sprague-Dawley rats in the range of 200-250 g were purchased from Charles River Laboratories and kept in an AAALAC approved laboratory for one week before use in expe-riments.
B. Methods Preparation of Vesicles Small unilamellar vesicles (SUV's) were prepared by probe sonication of the lipid mixture in phosphate buffered saline (PBS) containing either nitrilotriacetic acid (NTA) or 2-PAMC1.
See Mauk et al, Anal. Biochem., 94, 302, 307 (1979). A trace amount of [3H] cholesterol oleate was included in the lipid mix-ture as a marker for the lipid ~hase. Following sonication, annealing, and low speed centrifugation, the NTA ex-ternal to the vesicle was removed by passage of the preparation over a Sephadex G-50 column, equilibrated with PBS.

~ r~r ~;~455S7 6072g~1519 Large unilamellar vesicles (LUV's) were prepared by the reverse phase evaporation (REV) method described by Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA, 75, 4194 (1978). REV vesicles are formed when an aqueous buffer con-taining the material to be encapsulated is introduced into a mixture of phospholipid and organic solvent, and the organic solvent is subsequently removed by evaporation under reduced pressure. The REV vesicles are then passed through a gel permeation column to remove the solvent residue and the unencapsulated drug.
Multilamellar vesicles (MLV's) were prepared by stirring the dry lipid film with the material to be encap-sulated. Free unencapsulated materials can be separated from MLV encapsulated material by centrifugation at 12,000 xg. In our preparation of MLV's for in v o injection, 40 mg of DSPC:Chol (2:1 molar ratio) (or other compositions as indicated) were stirred for 1/2 hour in a round bottom flask with 1-2 ml of PBA containing 0.5 M 2-PAMCl, or 3M 2-PAMCl as indicated.
Synthesis of [ C] 2-PAMCl Radiolabeled 2-PAMCl for use in the studies could not be obtained from any source. Therefore, the radiolabeled drug was synthesized. [14C] labeled 2-Pralidoxime Iodide (2-PAMI) was first synthesized by refluxing 2-pyridine aldoxime (2-PAM) with [14C]-methyl iodide in nitrobenzene for three hours at 75-80C. The reaction was then stopped, and the yellow preci-pitate of 2-PAMI was filtered and recrystallized from methanol.
These yellow crystals of 2-PAMI were then dissolved in a minimal amount of water and passed through an anionic exchange column (BioRad AG lx8). The chloride salt of 2-PAM was isolated by drying the solution with a rotary evaporator followed by ~2~5~

recrystallization from ethanol. Approximately 1.5 gm of pure [14C] labeled 2-PAMCl was obtained with specific activity of ~Ci/mole. The chemical identity of this material was confirmed by (1) the melting point of the compound which was found to be 232-234C (literature value 235C) and (2) the characteristic absorption of an acidic solution of 2-PAMCl at approximately 295, 245, and 21~ nm; and ~3) co-migration during thin layer chromatography of the l~C labeled compound with 99~ pùre 2-PAMCl.

In Vitro Vesicle Studies The chemical structure of vesicles was altered by varying the length of their phosphatidyl holine carbon chain, cholesterol content, and surface charge. The stability of the vesicle formulations ln vitro was studied by gamma-ray perturbed angular correlation spectroscopy (PAC) as described by Hwang and Mauk, Proc. Natl. Acad. Sci. USA, 74, 4991 (1977). Prior to PAC measurements the vesicles were loaded with llInC13.
Typcially 1.0 mg of vesicle withtheionophore A23187 incorporated within the bilayer was incubated with InC13 at 80C for 45 minutes. During incubation, the lllIn passed through the ionophore and complexed with NTA inside the vesicles. The remaining In outside the vesicle was subsequently complexed to EDTA and separated from the loaded vesicles by chromatograph-ing the mixture on a Sephadex G-50 column equilibrated with PBS.
The vesicles, now loaded with In-NTA, were then suspended in a 1:1 solution of physiological saline and rat plasma. Gamma-ray PAC spectroscopy was then used to monitor the structural ~integrity of vesicles by measuring the tumbling rate of lllIn 3.

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In chelated to nitrilotriacetic acid exhibits a fast tumbling rate. However, upon disruption of the vesicle, the released lllIn+3 rapidly binds to macromolecules in the surrounding medium and exhibits a markedly decreased tumhling rate. Repeated PAC measurements of each vesicle formulation were used to estimate vesicle stability based on the time course for the release of In.
Similarly, identical vesicle formulations loaded with [ C]-2-PAMCl were used to measure the release rate of 2-PAMCl.
Aliquots were periodically withdrawn from each preparation and free 2-PAMCl separated from the microencapsulated drug by gel filtration chromatography as described by Hwàng, Biochem, 8, 344 (1969). The amount of ~ 4C]-2-PA~lCl remaining within the vesicles was measured by liquid scintillation counting.
In Vi~o Vesicle Studies The rate of release of [ 4C]-2-PAMCl from the various vesicle formulations was measured in vivo using male Sprague-Dawley rats obtained from Charles River Inc. Dosages ranging from 5-240mg/kg body weight (BW) of free and encapsulated [ C]-2-PAMCl were injected into the thigh muscle. Individual injection volumes never exceeded 0.15 ml. At scheduled times after injection, the rats were either sacrificed or bled through the eye orbit. Radiolabeled 2-PAMCl was measured in blood and plasma by liquid scintillation counting using the New England Nuclear procedure for counting labeled biological material. See L.S.C. Note, #44, New England Nuclear Applications Laboratory, Boston, MA.

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1~45S57 60724-1519 C. Results _ . _ In Vitro Vesicle Stability As indicated in the previous section, the rate of In release from vesicles in the presence of plasma can be monitored by PAC spectroscopy. Figure 1 shows the percent llIn remaining encapsulated with seven vesicle formulations varying in cholesterol concentration, carbon chain length, and surface charge. With the exception of the negatively-charged DCP vesicle, all vesicle formulations with 33 mole percent or more cholesterol exhibited the same transition temperature as monitored by 1 lIn release. The DCP formulation produced vesicles with a transition temperature ( In release) approximately 10C higher than other vesicle formulations containing the same amount of cholesterol.
In Vivo Results In the following results, the data are presented as the concentration of 2-PAMCl as a function of time for various vesicle formulations. The time dependence for a standard in~ection of free 2-PAM Cl shows that the blood concentration drops below therapeutic level (TL) in 2-3 hours (Figure 6A). As will be shown, all vesicle formulations with encapsulated

2-PAMCl exhibited extended blood levels. Therapeutic levels of drug were maintained typically 6-8 hours for those formulations having only buffered saline as the suspending medium. Vesicles suspended in a high osmolar medium showed dramatically longer therapeutic levels.
The Effect of Altering the Chemical Composition of the Vesicle Membrane Altering membrane fluidity by changing lipid composi-tion did not affect the extended release period achieved wi-th ",~, ~, ,~ . .

~ 557 60724-1519 all vesicle formulations (Figure 2). Similarly, altering the lipid composition and charge of the vesicle membrane did not significantly alter the release period either. (Figure 3).
Among the vesicle composition and charge variables studied, the only factor affecting the release rate for 2-PAMCl was cholesterol content. Increasing the cholesterol content of vesicle membranes from 12.5 - 50.0 mole percent appeared to slightly lengthen the time therapeutic levels of 2-PAMCl remained in circulation (Figure 4).
The Effects of Altering the Physical Structure of Vesicles The effect of vesicle structure on the extended release of 2-PAMCl was examined using multilamellar vesicles (MLV's) and large unilamellar vesicles (LUV-s) prepared by reverse phase evaporation (REV) vesicles. As shown in Figure 5, DSPC: Cholesterol vesicles possessing the two distinct struct-ures exhibited essentially identical 2-PAMCl release properties.
Consequently MLV's containing 30 mole percent cholesterol were used in the high osmolarity studies as described below.
The Effects of Altering Encapsulated Volume and using High Osmolar Suspending Solu-tion The above data show that there are comparatively small changes in the time dependency of 2-PAMCl blood-levels for the formulations tested. The results indicate that simple mani-pulation of vesicle composition and morphology are not likely to provide extended release beyond 6-8 hours. These results are consistent with other published observations which show modest extended release times.

12~5557 Presented below are results which show that blood levels of 2-PAMCl can be dramatically extended by increasing the osmolarity of the medium in which the vesicles are susp-ended. Also, proportionately higher concentrations of drug can be encapsulated without leakage from the vesicles.
The most effective vesicle formulations for extend-ing the therapeutic plasma levels (4.0 ~g/ml plasma) in rats was found to be a 2:1 DSPC: Cholesterol lipid mixture, formed as MLV's and encapsulating a 3 molarsolution of[ C]-2-PAMCl which was suspended in a isomolar glucose-physiological saline solution 3M in glucose, after working to remove mother liquor.
In this case, the osmolarity of the suspending medium is about 60% of the osmolarity of the encapsulated drug. Intramuscular injections (0.15 ml) of this vesicle formulation extended the therapeutic plasma drug level from 2-1/4 hours, obtained with the conventional saline formulation, (Figure 6A), to at least 40 hours (Figure 6B). Animals receiving the isomolar vesicle formulation exhibited no toxic symptoms and their blood drug levels never exceeded the 20 ~g/ml level achieved by control animals receiving the standard 12 mg 2-PAMCl saline formu-lation (Fi~ure 6A).
The extension of therapeutic blood levels is related to the amount of drug encapsulated. All encapsulation techniques extended the maintenance of therapeutic drug levels.
Doubling the encapsulating lipid material from 2.5 mg to 5.0 mg by doubling the quantity of lipid (and thereby increasing encapsulated volume of drug solution) increased the time that therapeutic levels were maintained from 2.5 to 7.0 hours (Figure 6, C,D). Similar:Ly increasing the amount of drug encapsulated from a saline solution containing 60 mg of 2-PAMCl prevented t~

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all acute toxic symptoms while extending the therapeutic blood titers to 15 hours (Figure 6 E). All animals receiving similar 60 mg injections of 2-PAMCl unencapsulated in saline solution, or encapsulated using a technique which reduces the encapsula-tion ef~iciency died within 30 minutes a~ter injection.
These data suggest that encapsulated drug acts as a third compartment ~rom which its slow release lowers the peak blood levels seen when equal, ~mencapsulated dosages are injected.

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~45557 This delayed release prevents toxic blood levels from being attained and conserves drug for later therapeutic use. All encapsulation systems tested extended the time therapeutic blood levels of drug were maintained from 2.25 hours to about 6.0 hours. However, the chemical structure of the lipid membrane and the physical structure of the vesicle (whether MLV, SUV, or L W) had little effect on extending therapeutic blood levels.
The foregoing demonstrates that suspension of vesicle formulations containing a therapeutic agent in a solution that approaches being isotonic or even hypertonic with respect -to the vesicle greatly extends the interval over which sustained release occurs. It further demonstrates that dose levels of a therapeutic agent which are toxic when the agent is adminis-tered alone or in less effective sustained release formulations can be safely given using a composition of the present invention.
It will be understood by those skilled in the art that the foregoing merely illustrates the presently preferred embodiments of the invention and that modifications may be made in order to accomplish specific ends which donot depart fromlthe spirit of the present invention which is to be limited only by the appended claims

Claims (61)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition suitable for parenteral administration to animals including human beings, said composition comprising a solution of a therapeutic agent encapsulated in vesicles, the vesicles being pharmaceutically acceptable and being suspended in a solution containing sufficient solute to provide an osmolarity which is at least about 25% of the osmolarity of the solution within the vesicles and which is of greater osmolarity than physiological saline.

2. A composition according to claim 1, wherein the osmolarity of the suspending solution is at least about 40 to 50% of the osmolarity of the solution within the vesicles.

3. A composition according to claim 1, wherein the osmolarity of the suspending solution is at least about 75 to about 90% of the isotonic concentration.

4. A composition according to claim 1, wherein the suspending solution is hypertonic with respect to the solution within the vesicles.

5. A composition according to claim 1, wherein the osmolarity of the suspending solution is about 60% of the osmolarity of the solution within the vesicles.

6. A composition according to claim 1, 2 or 3, wherein the suspending solution is physiological saline to which has been added a second solute.

7. A composition according to claim 4 or 5, wherein the suspending solution is physiological saline to which has been added a second solute.

3. A composition according to Claim 1, 2 or 3 wherein the suspending solution is physiological saline to which has been added a second solute which is selected from sugars and polypeptides.

9. A composition according to Claim 4 or 5 wherein the suspending solution is physiological saline to which has been added a second solute which is selected from sugars and polypeptides.

10. A composition according to Claim 1, 2 or 3 wherein the suspending solution is physiological saline to which has been added a second solute, which is a hexose.

11. A composition according to Claim 4 or 5 wherein the suspending solution is physiological saline to which has been added a second solute, which is a hexose.

12. A composition according to Claim 1, 2 or 3 wherein the suspending solution is physiological saline to which has been added a second solute which is glucose.

13. A composition according to Claim 4 or 5 wherein the suspending solution is physiological saline to which has been added a second solute which is glucose.

14. A composition according to Claim 1, 2 or 3 wherein the suspending solution is a glucose solution.

15. A composition according to Claim 4 or 5 wherein the suspending solution is a glucose solution.

16. A composition according to Claim 1, 2 or 3 wherein the suspending solution is a solution of glucose in physiological saline.

17. A composition according to Claim 4 or 5 wherein the suspending solution is a solution of glucose in physiological saline.

18. A composition according to Claim 1, 2 or 3 wherein the vesicle is a phospholipid vesicle.

19. A composition according to Claim 4 or 5 wherein the vesicle is a phospholipid vesicle.

20. A composition according to Claim 1, 2 or 3 wherein the vesicle is a unilamellar phospholipid vesicle.

21. A composition according to Claim 4 or 5 wherein the vesicle is a unilamellar phospholipid vesicle.

22. A composition according to Claim 1, 2 or 3 wherein the vesicle is a multilamellar phospholipid vesicle.

23. A composition according to Claim 4 or 5 wherein the vesicle is a multilamellar phospholipid vesicle.

24. A composition according to Claim 1, 2 or 3 wherein the vesicle is a phospholipid vesicle and the sus-pending solution is a solution of glucose in physiological saline.

25. A composition according to Claim 4 or 5 wherein the vesicle is a phospholipid vesicle and the sus-pending solution is a solution of glucose in physiological saline.

26. A composition according to Claim 1, 2 or 3 wherein the therapeutic agent is selected from antibiotics, metabolic regulators, immune modulators and toxin antidotes.

27. A composition according to Claim 4 or 5 wherein the therapeutic agent is selected from antibiotics, metabolic regulators, immune modulators and toxin antidotes.

28. A composition according to Claim 1, 2 or 3 wherein the therapeutic agent is a cholinesterase inhibitor.

29. A composition according to Claim 4 or 5 wherein the therapeutic agent is a cholinesterase inhibitor.

30. A composition according to Claim 1, 2 or 3 wherein the therapeutic agent is a 2-PAMCl.

31. A composition according to Claim 4 or 5 wherein the therapeutic agent is a 2-PAMCl.

32. A process for producing a composition suitable for parenteral administration to animals including human beings containing a solution of a therapeutic agent encapsulated in vesicles, which process comprises encapsulating a solution of the agent in pharmaceutically acceptable vesicles and suspend-ing the vesicles in a solution containing sufficient solute to provide an osmolarity which is at least 25% of the osmolarity of the solution of the therapeutic agent within the vesicles and which is of greater osmolarity than physiological saline.

33. A process according to claim 32, wherein the osmolarity of said suspending solution is at least about 40-50% of the osmolarity of said solution within said vesicles.

34. A process according to claim 32, wherein the osmolarity of the suspending solution is at least about 75 to about 90% of the isotonic concentration.

35. A process according to claim 32, 33 or 34, wherein the suspending solution is hypertonic with respect to the solution within the vesicles.

36. A process according to claim 32, wherein the suspending solution is physiological saline to which has been added a second solute.

37. A process according to claim 36, wherein the second solute is selected from sugars and polypeptides.

38. A process according to claim 36, wherein the second solute is a hexose.

39. A process according to claim 32, 33 or 34, wherein the suspending solution is a glucose solution.

40. A process according to claim 32, wherein the osmolar-ity of the suspending solution is about 60% of the osmolarity of the solution within the vesicles.

41. A process according to Claim 32, 33 or 37, wherein the suspending solution is a solution of glucose in physiological saline.

42. A process according to Claim 32, 33 or 37, wherein the vesicle is a phospholipid vesicle.

43. A process according to Claim 32, 33 or 37, wherein the vesicle is a unilamellar phospholipid vesicle.

44. A process according to Claim 32, 33 or 37, wherein the vesicle is a multilamellar phospholipid vesicle.

45. A process according to Claim 32, 33 or 37, wherein the therapeutic agent is selected from antibiotics, matabolic regulators, immune modulators and toxin antidotes.

46. A process according to Claim 32, 33 or 37, wherein the therapeutic agent is an antidote for a cholin-esterase inhibitor.

47. A process according to Claim 32, 33 or 37, wherein the therapeutic agent is 2-PAMCl.

48. A pharmaceutical composition which is injectable to animals including human beings and releases a therapeutic agent contained therein for a prolonged interval after administration, wherein said composition comprises physiological saline containing said therapeutic agent dissolved therein and encapsulated in pharmaceutically acceptable vesicles, and said vesicles are suspended in physiological saline containing a second pharmaceutically acceptable solute to provide an osmolarity which is at least 40 to 50% of the osmolarity of the solution within the vesicles and which is of greater osmolarity than physiological saline.

49. A composition according to claim 48, wherein the vesicles are multilamellar vesicles.

50. A composition according to claim 48, wherein the second solute is a sugar.

51. A composition according to claim 48, wherein the vesicles are composed of a phospholipid optionally in combin-ation with cholesterol.

52. A composition according to claim 48, wherein the vesicles are composed of a mixture of a phosphatidylchlorine and cholesterol.

53. A composition according to claim 48, wherein the second solute is glucose.

54. A composition according to claim 50, 51 or 52, wherein the vesicles are multilamellar vesicles.

55. A composition according to claim 51 or 52, wherein the second solute is a sugar.

56. A composition comprising a solution of a therapeutic agent encapsulated in vesicles, the vesicles being suspended in a solution containing sufficient solute to provide an osmolarity which is at least about 25% of the osmolarity of the solution within the vesicles and which is of greater osmolarity than physiological saline, said composition being capable of controlling the release in vivo of said therapeutic agent and capable of subcutaneous, intramuscular and intra-peritoneal administration to animals including human beings.

57. A composition according to claim 56, wherein the osmolarity of the suspending solution is at least about 40 to 50% of the osmolarity of the solution within the vesicles.

58. A composition according to claim 56, wherein the osmolarity of the suspending solution is at least about 75 to about 90% of the isotonic concentration.

59. A composition according to claim 56, wherein the suspending solution is hypertonic with respect to the solution within the vesicles.

60. A composition according to claim 56, 57 or 58,wherein the suspending solution is physiological saline to which has been added a second solute which is selected from sugars and polypeptides.

61. A composition according to claim 56, 57 or 58, wherein the vesicle is a phospholipid vesicle and the suspending solution is a solution of glucose in physlological saline.