CA1119955A - Xenobiotic delivery vehicles, method of forming them and method of using them - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A vehicle suitable for delivering a xenobiotic to a mammalian host to beneficially alter the pharmacodynamics (e.g., plasma kinetics, chemotherapeutic effectiveness, toxicity, oral absorption, tissue distribution, metabolism and the like) of the xenobiotic. The delivery vehicle is in the form of micro-reservoirs formed of a phospholipid constituent and a phospho-lipid-immiscible constituent. Xenobiotic binding agents and release agents may be added. Methods for preparing the micro-reservoirs containing the xenobiotic and for administering a xenobiotic to beneficially alter its pharmacodynamics are disclosed.

Description

~.~19~5 This invention relates to the delivery and release of xenobiotics within a mammalian host. More particularly, this invention relates to xenobiotic delivery ~ehicles in the form of circulating microreservoirs, to a method of forming the microreservoirs, and to a method of delivering xenobiotics to a mammalian host which predetermines and controls the pharma~
codynamics of the xenobiotics so delivered and released.
In many different situations and under many varied circumstances it is desirable to introduce into a mammalian host pharmacologically active agents which are foreign to the host, these agents hereinafter being termed ~xenobiotics. n These xenobiotics include/ but are not necessarily limited to, drugs, diagnostic agents, blood substitutes, endogenous biological com-pounds, hormones, immunological adjuvants and the like.
In the administration of any xenobiotic a certain degree of specificity must be attained, and specificity requires that the xenobiotic reach its target selectively and controllably. The absence of specificity associated with the use of many xenobiotics can thus deprive them of an appreciable part, if not essentially all, of their potential effectiveness in attaining the results de-sired from their use. For example, a chemotherapeutic drug which cannot he retained by blood plasma Eor a tlme sufficient for an appreciable amount of the drug to reach the targe~ tissue or an orally administered drug which is destined for the blood stream but which cannot pass through the gastrointestinal tract lacks the degree of specificity which could make it highly effective. Thus in lacking the desired specificity, a xeno-biotic may exhibit essentially none or only a limited degree of the pharmacodynamics desired to realize its full po~ential.
Among such pharmacodynamics may be listed plasma kinetics, S

tissue distribution, degree of tox-city, levels of therapeutic drugs in vivo, solubility of xenobiotics normally incompatible with other pharmaceutical formulations, and metabolic activation of the xenobiotics.
In the prior art it has been recogni~ed that it would be desirable to beneficially alter and contro:L the specificity or pharmacodynamics of many of the xenobiotics found to have desirable properties. One prior art approach to controlling the specificity of drugs involves the use of implant devices located, normally through a surgical procedure, in or near the organ to which ~he drug is to be deliveredO Typicallyl, these implant devices comprise a covalent matrix material containing the drug to be delivered~ These matrix materials may be water-soluble (e.g., carboxymethyl cellulose or poly-vinyl alcohol) r water-swellable (e.g., hydrogels or gelatin), hydrolytic polymers (e.g., polylactic acids, polyglycolic acids or poly-~-amino acids), or nonhydrolytic polymers (e.g., organo-polysiloxane rubber). Generally, although these implant devices can control the rate at which the drug they contain can be deliv-ered through difusion or hydrolysis, they can exercise little if any alteration of the pharmacodynamics of the drug released.
One of the more recent approaches suggested for the alteration and control of the pharmacodynamics of xenobiotics is the use of a carrier capable of delivering and controllably releasing the xenobiotics at the site of action within the host to which the xenobiotics are administered. The use of liposomes has been proposed as carriers for this purpose. (See Gregoriadis, G., "The Carrier Potential of Liposomes in Biology and Medicines,"
The N _ England Journal of Medicine, Vol. 295, No. 13r pp 704-710 [September 23, 1976 and No. 14, pp 765-769 [September 30, 1976~.) ~3-s~

These prior art liposomes are composed of phospholipids, and es-pecially of ph~ophatidyl choline in combination with o~her lipids such as cholesterol, dicetyl phosphate, stearyl amine and other phospholipids such as phosphatidyl serine with which the phospha~
tidyl choline is readily miscible. The lip~somes are character ized by the miscibility of the carrier components, a fac~ which means that there can be little or no phase separation between such components. Furthermore, these carriers lack any high degree of stability, due primarily ~o the oxidation of the phospholipid 19 components and/or their ~hermal instabili~y, especially when they are sonirated to a small size, e.g., about 250 A. Moreover, those liposomes which carry a negative charge, due to the presence of dice~yl phosphate, phosphatidyl serine or other negatively charged components, will aggregate in the presence of divalent cations.
Finally, when liposomes are injected intravenously, they are preferentially concentrated in the liver and spleen, due at least in part to their large size. Thus, although the prior art liposome carriers are compatible with a number of drugs and are biocompatible with the mammalian system, their inherent instability, t~ndency to aggregate materially,and relativèty large size detract from their ability to serve as acceptable drug delivery systems.
Because of th~ ever-increasing role pla~ed by xeno-biotics in the treatment of cancer, diabetes, arthritis, inherited metabolic disorders, metal-storage diseases, and the like, in the prevention of such diseases as anaplasma and of virus infections, and in the study of biological mechanisms the need for improved carriers is an ever~growing one. The desirability of having such improved delivery systems is therefore apparent.
It is therefore a primary object of this invention to provide improved xenobiotic delivery vehicles in the form of microreservoirs capable of circulating w~thin ~he host system.
Another object is to provide xenobiotic delivery vehicles of the character described which are compatible with a wide variety of xenobiotics including hydrophobic~ hydrophilic or a combina-~ion of hydrophobic and hydrophilic compounds and which are non-toxic and biocompatible with the host system. A further object of this invention is to provide xenobiotic delivery vehicles which are stable over extended periods of storage as well as in their use within the host system and amenable to various tech-niques of administration including oral, intravenous, intramuscular, intraperitoneal, subcutaneous, topical and lnhalation.
Another object of this invention is to provide xenobiotic delivery vehicles capable of predeterminably and beneficially altering and controlling the pharmacodynamics of the xenobiotic delivered and released within the host systemO
~nong ~he pharmacodynamics thus beneficially altered and con-trolled are plasma kinetics, tissue distribution, toxicity, oral absorption, chemotherapeutic ability, metabolism and the likeO
It is another primary object of this invention to provide a method of forming xenobiotic delivery vehicles, in the form of microreservoirs, capable o circulating within a mammalian host thereby to deliver the xenobiotic at a pre-determined site by effecting a predetermined beneficial alter-ation in the pharmacodynamics of the xenobiotic.
Yet another primary object of this invention is to provide a method for deliver;ng and releasing a pharmaceutically effective amount of a xenobiotic within a mammalian ho~t in a manner to exercise some predeterminable control over the delivery site, thus enhancing the effectiveness of the xenobiotic.

~5--Other objects of the invention will in part be obvious and will in part be apparent hereinafter.
According to one aspect of this invention there is provided a delivery vehicle biocompa~ible with a mammalian host to deliver and release within the host a xenobiotic, the pharma-codynamics of which are beneficially altered through the use of the vehicle, characteri2ed in that the delivery vehicle is in the form of microreservoirs containing the xenobiotic, the microreservoirs comprising a phospholipid constituent and a phospholipid-immiscible lipid constituent stable in and essentially immiscible with a physiologically-compatible liquid.
According to another aspect of this invention there is provided a method of forming a xenobiotic delivery vehicle for delivering within a manunalian host a xenobiotic, the pharmacodynamics of which are predeterminably altered and controlled, comprising the steps of forming microreservoirs of a phospholipid constituent and a phospholipid-immiscible lipid consitutent which is stable in and essentially immiscible with a physiologically-compatible liquid; and incorporating the xeno-biotic to be delivered within the microreservoirs.
According to yet another aspect of this inventionthere is provided a method of delivering and releasing a xenobiotic within a mammalian host, comprising the step of introducing into the mammalian host a pharmaceutically effective amount of a xenobiotic contained within microreservoirs carried in a physiologically-compatible liquid and formed of a phospho-lipid constituent and a phospholipid-immiscible lipid constituent stable in and essentially immiscible with the physiologically compatible liquid.

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According to still another aspect of this invention there is provided a method of predetermining and controlling the pharmacodynamics under which a xenobiotic is delivered within a mammalian host~ comprising the step of releasing the xenobiotic within the host from circulating microreservoirs formed of a phospholipid constituent and a phospholipid~immis-cible lipid constituent stable in and essentially i~niscible with a physiologically-compatible liquid.
The invention accordingly comprises the several steps and the relation of one or more such steps with respec~ to each of the others, and the composition and arti&le possessing the features, properties, and ~he relation of constituents~ which are exemplified in the follo~ing detailed disclosure, and the scope of the invention will be indicated in the claims.
For a fuller undexstanding of the nature and objects of the invention, reference should be had to the following detailed description taken in connection with the accompanying drawings in which Fig. 1 is a process flow chart illustrating one method ~0 of preparing the xenobiotic delivery vehicles of this invention;
Fig. 2 is a partial process flow chart illustrating a modification of the method of Fig. l;
Fig. 3 is a much-enlarged diagra~natic representation of a microreservoir of this invention in vesicular form;
FigO 4 is a much-enlarged diagrammatic representation of a microreservoir of this invention in nonvesicular form;
Fig. 5 is a much~enlarged diagrammatic representation o~ a drug delivery vehicle of the type disclosed in ~he prior art;

s Fig~ 6 illustrates the i~ vi~o stability of the mircoreservoirs contras~ed with that of liposomes, the *~ vitro stability being measured as the optical density of the micxo-resexvoirs and of the liposomes as a function of t'me;
Fig. 7 illustrates the in vivo stability of the micro-reservoirs plotted as the amount of cholesteryl oleate (cholas-terol ester) ramaining in the blood plasma of a rat as a function of tLme;
Fig. 8 is an elution profile of microreservoirs carrying daunomycin plotted as phosphatidyl choline, cholesteryl oleate and daunomycin concentrations in a series of chromatographic fractions;
Fig, 9 is a diagrammatic representation illustrating the distribution of a typical anthracycline drug shortly after injecting the free drug, showing how little of the drug re-mains in the blood stream for delivery to the target site;
Fig. 10 illustrates the extent to which the circu-lating microreservoirs are capable of beneficially altexing the plasma kinetics of daunomycin, the illustration being in the form of plots of the amount of daunomycin rem~ining in the blood-stream as a function of time after injection of the drug in the microreservoirs and as free daunomycin;
Fig. 11 is a series of plots of the number of sur-vivor~ of a group of mice as a function of time after the in-jection at four dose levels of microreservoirs, daunomYCin in the microreservoirs and d~unomYcin. in free form, the plots illustrating the ability of the microresarvoirs acting as delivexy vehicles to decrease the toxicity of the ~aunomycin;
Fig. 12 is a series of plots of the n~mber of sux~
30 VivQrs of a group of mice injected with tumor cells as a function ~' of time after the injection a~ two dose levels of daunom~cin in the microreservoir~ and in free form, the plots illustrating the beneficial altering of the chemo~herapy o the daunomycin delivered by and released from the microreservoirs;
Fig. 13 comprises a series of plots of efflux rates of d~unomycin as a function of time from microreservoirs of composi-tions containing glycerol trioleate as the phospholipid~immiscible constituen~ and various amounts of cholesterol as a release-rate control agent;
Fig. 14 comprises a s~ries of plots of efflux ratæs of daunomycin as a function of time from microreservoirs of compo-sitions containing cholesteryl ole~te as the phospholipid-immis-cible constituent and various amounts of cholesterol as a release-rate control agent;
Fig~ 15 is an elution proile of microreservoirs car-rying AD 32 plotted as phospholipid, cholesteryl oleate and AD 32 concentrations in a series of chromatographed fractions;
Fig. 16 is a plot of ~he efflux rate o AD 32 from vesicular microreservoirs showing the effect of using a minor amount of phosphatidic acid in the phospholipid constituent of the microreservoir composition;
Fig. 17 illustrates the extent to which the circulat.ing microreservoirs are capable of beneficially altering the plasma kinetics of AD 32;
E~ig. 18 is a series of plots of the number of survivors of a ~roup of mice with tumor cells as a function of time after the injection at four dose levels of AD 32 in the microreservoirs~
the plot~ illustrating the chemotherapeutic ability of AD 32 de-livered by the microreservoirs;

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Fig. 19 is an elution profile o microreservoirs carrying imidocarb plotted as phospholipid, cholesteryl oleate and imidocarb concentratio~s in a series of chromatographed fractions;
Figs. 20 and 21 illustrate the extent to which the circulating microreservoirs are capable of beneficially altering the plasma kinetics of imidocarb at two dose levels, the il-lustrations being in the form of plots of the amount of imidocarb remaining in the bloodstream and the ratios of imidocarb ~o microreservoirs as functions of time after injection of the drug in the microreservoirs and as free imidocarb;
Fig. 22 is an elution profile of microreservoirs car-rying estradiol undecanoate plotted as phosphatidyl choline and estradiol undecanoate concentrations in a series of chromato-graphed fractions;
Fig. 23 is a bar graph showing concentrations in blood plasma at three points in time of estradiol undecanoate given orally as an ethanol solution and in microreservoirs, Fig. 24 is an elution profile of vesicular and non-vesicular microreservoirs carrying estradiol undecanoate; and Fig. ~5 is a plot of the efflux rate of estradiol un~decanoate from vesicular and nonvesicular microreservoirs"
The xenobiotic delivery vehicles of this invention are formed oE a mixture of two or more lipids which are es-sentially immiscible. More specifically, the delivery systems are formed of compositions comprising a phospholipid constituent and a phospholipid-immiscible constituent which may be a cholesterol ester or a triglyceride or a mixture of cholesterol esters, or triglycerides or oE both these constituents. The phospholipid constituent may comprise more than one phospholipid; and the microreservoir composition may also include a binding modifying and/or a release-rate controllin~ cons~ituent. Exemplary of phospholipids suitable for the practice of this invention are phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolaminet phosphatidic acid and sphingomyelin.
Mixtures of two or more of these phospholipids may also be used.
Of these, phosphatidyl choline, either alone or in admixture with other phosphalipids, is preferred.
Phosphatidyl choline is a term applied to compounds which are esters of fatty acids with glycerophosphoric acid and choline. Thus phosphatidyl choline may be represented by the following ormula:

FA'-CHCH2-O P-O-~CH2)2 N-~CH3)3 o wherein FA and FA' are fatty acid residues. The fatty acids used to form these esters may be saturated or unsaturated and may con-tain from about 12 to 20 carbon atoms. Such fatty acids include but are not limited to, palmitic, stearic, oleic and the like.
The phosphatidyl choline used in the drug delivery vehicles of this invention may be isolated from egg yolk by the procedure described by Litman (Biochemistry, 12:2545[1973]) or from other natural sources such as soybeans or it may be synthesized by a suitable procedure such as that described by Robles and vander Berg (B_ochim Biophys Acta, 187:520 [1969]).
In the following detailed description of this inven-tion the phospholipid constituent of the delivery vehicles is illustrated, for convenience, by phosphatidyl choline, a term meant to include thosa esters falling within ~he general formula given. In some of the microreservoir formulations, mlnor amounts of phosphatidic acid are incorporated in the phospholipid con-stituents to improve the binding of the xenobiotic to the micro-reservoirs. It is also to be understood that other phospholipids, including those named, which meet the chemical and physical properties stated below may be used in place of the phosphatidyl choline .
The cholesterol ester or triglyceride constituent used as a component of the delivery vehicle must be essentially immiscible with the phospholipid constituent as well as es-sentially insoluble in an aqueous environment as representedby a physiologically-compatible liquid such as a physiologically-balanced salt solution containing NaCl or KCl. The cholesterol ester or triglyceride constituent must al50 be apolar or nonpolar to the degree that it will not form a monolayer and it must be present in a concentration such that it is essentially immiscible in the phospholipid bilayer.
Cholesterol, C27H15OH, is a monounsaturated, sec-ondary alcohol which readily forms esters with both saturated and unsaturated fatty acids such as oleic, stearic, plamitic and the like. In general, fatty acids having from 10 to 18 carbon atoms are preferred. Suitable procdures for forming these cholesterol esters include the condensation of a fatty acid chloride with cholesterol and the isolation from natural sources.
In choosing the fatty acid to form the cholesterol ester, it is preferable that it is one with a minor degree of unsaturation (e.g., no greater than about two double bonds per fatty acid). In general, the esters formed of fatty acids with higher degrees of saturation form xenobiotic delivery complexes s~

of greater stability than those formed of highly unsaturated fatty acids.
The triglycerides suitable for the practice of this invention are fa~ty acid esters of glycerol having the general formula H C-O-C-R

~ 11 H C-O-C-R
wherein Rl, R2 and R3 of the fatty acids forming ~he esters may have from 10 to 18 carbon atoms. Exemplary of such fatty acids are palmitic, stearic, myristic, oleic and linoleic. These triglycerides are conveniently prepared by the condensation of the fatty acid chloride with glycerol. They may also be isolated from natural sources.
In the following general description it will be assumed, for convenience, that a cholesterol ester is used. It is, of course, to be understood that a triglyceride, a mix~ure of cholesterol esters, a mixture of triglycerides or a mixture of one or more cholesterol esters with one or more triglycerides may also be used in forming the microreservoir delivery vehicles.
Examples are presented in which either a cholesterol ester or a triglyceride is used as the phospholipid~immiscible constituent.
1'he xenobiotic delivery vehicles of this invention are in the form of what are hereinafter termed "microreservoirs."
These microreservoirs may assume one of two forms, namely a vesic-ular form which has a small walled cavity containing the medium used in making the microreservoir, or a nonvesicular form which has the cholesterol ester and/or triglyceride cont~ined within ~9~55 the phospholipid monolayer. As will be described below, the ratio of phosphatidyl choline to cholesterol ester determines which of these forms predominates in any synthetic procedure.
The choice between these forms is principally dependent upon the nature of the xenobiotic to be delivered and released, the pharma-codynamics of the xenobiotic desired and the rnethod of admin-istration.
Two different synthesis routes for Eorming the microreservoirs, along wit~ the incorporation of the xenobiotic therein, are illustrated diagrammatically in Figs. 1 and 2, the route shown in Fig. 1 using sonication to form the microreservoirs representing a preferred process. In this process the phosphatidyl choline and cholesterol ester are mixed in an inert organic liquid which is a solven~ for both of these constituents, e.g., chloro~
form; and, if desired, the xenobiotic is added to this solution as indicated by a dotted line in Fig, 1. The solvent is then removed by evaporation ~n vacuo, leaving a dry residue mixture which is then hydrated by the addition of a physiologically-com-patible liquid, as exemplified by an aqueous solution of NaCl or KCl of suitable concentration and a buffer. If the xenobiotic is not added into the initial mixture, it is added into the re-sultiny liquid suspension prior to the formation of the microre-servoirs.
In forming the liquid suspension it is preferred to use amounts of the residue mixture equivalent to between about one and about five percent by weight of the physiologically-compatible liquid. Although the amount of xenobiotic added to the phospho-lipid/cholesterol ester (or triglyceride) composition may vary, it is preferable to incorporate up to about two percent by residue weight of the xenobiotic in the microreservoirs.

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As illustrated in Fig. 1, the microreservoirs are then formed by sonicating the saline solution under a non-oxidizing, e.g , nitrogen a~mosphere. It has been found that more complete formation of the microreservoirs occurs when the sonicating is carried out at a temperature equivalent to or slightly above the melting point of the cholesterol ester or triglyceride serving as the phospholipid-immiscible constituent.
The sonication is carried out at a suitable power level and for a time to produce the desired microreservoir size. For example, a power input of 120 watts for 20 minutes has been found satis-factory. As an alternative to the sonicating of the saline sus pension it may be forced through a small orfice to form the micro-reservoirs.
As will be seen from Fig. 2, an alternative route to the formation of the microreservoirs in an aqueous medium, e.g., saline solution, lies in the formation of a solution of the phospholipid cholesterol ester and xenobiotic using a water-miscible organic solvent and the subsequent introduction of this solution into an aqueous physiological saline solution. Alter-natively, the xenobiotic may ~e added to the saline solutionprior to the injecting step. Exemplary of this procedure is the injection of an ethanol solution of the lipid mixture into a stirred aqueous solution; or the slow injection of an ether solution of the lipid mixture into an aqueous compartment.
The cloudy liquid resulting from the formation of the microreservoirs in the aqueous suspending medium is then centrifuged to produce a clear phase and an upper cloudy phase, the latter containing larger nonvesicular microreservoirs, o O
eOg., from about 300 A to about 1000 A in diameter. The clear phase is chromatographed to produce a fraction of small (e.g., 3gS5 about 250 A in diarneter~ nonvesicular microreservoirs and vesicular rnicror~servors ~aving diameters of about 1~0 A ~o about 30G A.
The distribution of the microreservoirs between the ~esicular and nonvesicular forms is determined by the mole ratio of phospholipid to phospholipid-irmniscible constituent. Using phosphatidyl choline and cholesterol oleate as illustrative of a microreservoir composition, it may be shown that the use of between ahout 67 and g7 mole % of phosphatidyl choline yields predominantly vesicular microreservoirs; while the use of more than about 50 mole ~ of cholesterol ester yields predominantly nonvesicular microreservoirs~
The microresexvoirs of this invention have unique structural properties due to the use of a mixture of the phospholipid constituent and a lipid constituent (cholesterol ester, triglyceride or mixtures thereof) which is essentially imrniscible with the phospholipid. This structure, in its vesicular and nonvesicular forms, is shown diagrarnrnatically in Figs~ 3 and 4. The insolubility of both the phospholipids (represented by phosphatidyl choline in Figs9 3 and 4) and the immiscible lipid constituent (represented by a cholesterol ester) gives rise to an organized microreservoir structure in which contact between the irNmiscible l;pids and the aqueous environment is avoided or reduced to a minimum. This organiza-tion in turn imparts much greater stability to the rnicroreser-voirs than that attained by a vehicle formed in accordance with the prior art teaching of Gregoriadis previously cited. This may be shown by a comparison of Fig. 3 and 4 on one hand and Fig. 5 on the other, Fi~. 5 representing diagran~atically the structure of the drug delivery system of Gregoriadis. It will 5Si be seen from Fig. 5 that when miscible lipids are used to form the lip~ome the result is a lamellar structure giving rise to an unstable organization due apparently to the oxidation of the phospholipids which destabilizes the lamellar organization; or to crystallization of the phospholipids if the temperature de-creases below their transition temperature.
The stable structure of the microreservoirs of this invention, shown in Figs. 3 and 4, may be attributed, at least in part, to a thermodynamic driving force which prevents the cholesterol ester or triglyceride from being exposed to the aqueous environment. As a result, the structural integri~y of the microreservoirs of this invention is greatly enhanced.
This, in turnl means that the potential for the microreservoirs to control and alter the pharmacodynamics of the xenobiotics incorporated in them is likewise enhanced when compared with other delivery systems. Moreover, the apolarity of the micro-reservoirs, which is a result of the use of cholesterol esters or triglycerides in place of cholesterol and the like, es-sentially prevents aggregation of the microreservoirs. This is, of course, highly desirable since such aggregation would other-wise release the cholesterol esters or triglycerides into an aqueous environment with a resulting large decrease in the free energy oE the system. Thus, the individual microreservoirs, which are similar in many respects to the naturally occurring circulating serum lipoproteins, are particularly well adapted to circulate in the blood plasma of the mammalian host into which they are introduced and to remain in the plasma for maximum effectiveness, The greatly enhanced ~n v~tro stability of the micro-reservoirs of this invention compared with that of the liposomes 5~i sugsested in the prior ar~ is illustrated in Example 1 and Fig. 6.

Various mixtures of 60 ~moles of egg yolk phosphatidyl choline, various amounts of cholesteryl ole~ and 4 mg of daunomycin were taken to dryness under vacuum from a chloroform solution of these microreservoix constituents. To the resulting residue were added S ml of O.lM KCl and 10 mM of trihydroxymethyl-amina (buffer of pH 8~0); and the r sulting suspension was soni-cated at 120 watts for 15 minutes at 51C under a nitrogen at-mosphere. Both of the suspensions were then spun at l00,000g for one hour to remove any undispersed lipid.
Thin layer chromatograms of the centrifugates showed no evidence of degradation of the phosphatidyl choline or dauno-mycin, Aliquo~s of 4 ml of each sample were then placed into stoppered curvettes and the samples were incubated at 37C.
At various points in time the optical densities of the two solu-tions at 400nm and 600nm were determined. The data thus obtained are plotted in Fig. 6 as the ratio of these optical densities, 2Q A400/A600, against time. Since both structures initially had essentially the same molecular size, the ratio of A400/A600 is an indication of any increase in the particle size due to the breakdown of the structural organization of the liposome.
Fig. 6 illustrate~ the dramatic difference in sta~
bility between the microreservoirs o thi~ invention and similarly-si~ed liposomes. Whereas the absorbence o the microreservoirs containing 33~ cholesteryl oleake, as well as lesser amounts of the cholesterul ester, was relatively stable, that of the liposome increased rapidly from the beginning of the period of evaluation and by the end of 10 days the liposomes had de-graded beyond all practical ~se. As previously postulated, the mar~ed degradation of the liposomes i5 believed to be due to the interaction with the aqueous environment and aggregation into larger particles, two factors absent in the microreservoirs by virtue of their structural stability and apolarity.
In addition to in vitro stability, the microreser-voirs of this invention also exhibit a greatly increased %n v~vo stability over the liposomes. This i5 shown in Example 2 and Fig. 7.

~X~MPLE 2 100 ~moles of egg yolk phosphatidyl choline, 10 ~moles of 14C-labeled cholesteryl oleate and 10 ~moles of egg yolk phosphatidic acid were dissolved in chloroform and the solution evaporated in vacuo to dryness. 5 ml of 0.154 M NaCl and 10 mM
of trihydroxymethylamine (pH 8.0) were added to the dry mixed lipid residue. The resulting suspension was sonicated for 15 minutes at 51~C under a nitrogen atmosphere. The sonicated mixture was then chromatographed on a ~.5 X 40cm Sepharose 4B
column. Individual fractions that demonstrated a coincidence in the elution profile of the phospholipids and cholesteryl oleate were pooled and then concentrated by ultrafilatration.
1.1 ml of these concentrated microreservoirs were injected into the tail vein of a rat. Plasma samples were withdrawn at various time periods and assayed for the radioactivity asso ciated with the microreservoirs contained in the plasma sample.
The data thus obtalned are plotted in Fig. 7 as a function of time after injection~
Although the kinetics are complex, it can be seen from Fig~ 7 that at equilibrium, the clearance of the micro-reservoirs has a half-life o 5.5 hours. In contrast~ soni-cated liposomes containing a cimilar negative charge have a plasma half-life of only 8 minutes~ ~R~. Juliano and D. Stamp, Biochem Biophys. Res Comm 53 : 651 11975~) It seems logical to postulate ~hat the nearly 40-fold increase in the lifetime achieved by the microreservoirs is attributable to increased structural stability.
The association of a xenobiotic incorporated in the microreservoirs is illustrated in Example 3 and Fiy. 8.

EXAI~LE 3 2180 ~moles of egg yolk phosphatidyl choline, 242 ~moles of cholesteryl oleate labeled with l4C ~specific activity 1.6 X 104 disintegrations per minute (dpm)~mole and 36 mg of daunomycin were mixed together in chloroform and the solution was evaporated to dryness ~n vaCuo- The mixed dry re-sidue was then hydrated by the addition of llO ml of O.~M KCl and lO mM of trihydroxymethylamine ~pH 8.0). This suspension was then sonicated with a Branson W-185 Sonifer for 15 minutes at 51C under a ni~rogen atmosphere.
The sonicated liquid was then chromatographed on a 205 X 40 cm Sepharose 4B column~ Individual fractions resulting from the chromatographing were then assayed for phosphatidyl choline content by the procedure of Gomori tJ. Lab. Clin. Med, 27: 955 ~l949]), for cholesteryl oleate by radioactivity and for daunomy~in by fluorescence measurements. The results of these analyses are plotted in Fig. 8 with the analytical results for the cholesteryl oleate, phosphatidyl choline and daunomycin being superimposed.

~' The diameter of the vesicular micxoreservoirs was found to be approxima~ely 200 ~ ~o 300 ~ as determined by their elution profile on the gel column which had previously been calibrated using sonicated liposomes of egg yolk phosphatidyl choline.
It can be seen from Fi~P 8 that the elution profiles of the phosphatidyl choline (as monitored by phosphate analysis), of the cholesteryl oleate ~as monitored by radioactivity) and of the daunomycin ~as monitored by fluorescence~ coincide, in-dicating that the xenobiotic (daunomycin) was associated withthe vesicular microreservoirs. Since daunomyCin is a relati~ely hydrophobic drug, with some hydrophilic characteristics, it is reasonable to postulate that ~he site of localization of the drug is within the lipid organization of the microreservoirs, ~ mong the pharmacodynamics of a xenobiotic which can be altered ~o make the xenobiotic more effective are plasma kineticsr degree of toxicity and therapeutical effectiveness~
Daunomycin is being clinically ~valuated as a cancer chemotherapeutic drug; but it i5 relatively hydrophoblc and it is very difficult to maintain in the plasma stream as evidenced by the fact that within two minutes after administration about g5% of it has left the bloodstream and gone to various organs leaving only some 5% to reach the target tissue, i.e," a tumor.
This i~ illustrated in Fig. 9 which illustrates the distribu-tlon of a typical anthracycline druy, given intravenously, minutes after injection. Flnally, daunomycin is known to be to~ic, particularly since it tends to concentrate in the heart tissues, making it necessary not only to carefully monitor its admini~tration but to administer i~ at very low dosage levels.

ss The following Examples 4-6 and Figs. 10-12 illustrate the ability of the microreservoirs of this invention circulating in the bloods~ream to alter the plasma Xinetics, toxicity and chemotherapeutical effectiveness of daunomycin.

200 ~moles of egg yolk phosphatidyl choline, 20 ~moles of cholesteryl olea~e and 4 mg of daun~mYcin were dissolved in chlorofoxm and then taken to dryness under vacuum. To the result-ing dry residue were then added 5 ml of 0.154 M NaCl and 10 mM
of trihydroxymethylamine (pH 7.4). This aqueous suspension was then sonicated for 15 minutes at 51C under a nitrogen atmo-sphere. The daunomycin which was not contai~ed within the vesic-ular microreservoirs formed was separated from the microreservoirs C by passage ~hrough a 2 S X 20 cm Sephadex G-50 gel column. The void volume which contained the daunomycin incorporated in the microreservoirs was concentrated to 3 mg daunomycin/ml.
A control sample of free daunomycin at thP same con-centration and in the same buffer was also prepared.
Three rats were injected intravenously with the micro-reservoirs containing daunomycin; and six other rats were in-jected intravenously with the free daunomycin. The dosage of the daunom~cin contained within the microreservoirs was 4 mg/kg;
and that for the free daunomycin, was 4 mg/kg in three rats and 10 mg/kg in the remaining three ratsu 0O5 ml of blood was taken at various time points from each rat. ~he plasma was separated from the blood samples and assayed for daunomycin fusiny fluor-escence. The data thus obtained are plottPd in Fig. 10 as a function of time after injection~
r~a~ a~1~

~ ~ 1 .= ,. ~

~3L9~55i It is apparent from Fig. 10 that the use of the circulating microreservoirs as a delivery vehicle for the da~nom~cin beneficially alters and Lmproves the plasma kinetics for that drug. From a comparison of Cuxves A and B, represent-ing the same dosage level, it will be seen that after about 15 minutes ~he concentration of daunomycin in the circulating ~icro-reservoirs in the plasma was about 4 ~g~ml; while the concentra-tion of the free daunomycin in the plasma was about 0.4 ~g/ml.
Therefore, at khis time point, the concentration of daunomycin in the bloodstream carried by the circulating ~icroreservoirs ~as some ~en times greater than when this drug was introduced directly into the bloodstream~ Furthermore, as will be seen from Fig. 10, the free daunomycin was virtually absent from the bloodstream after some 3 3~4 hours; while it was still up to a concentration of about 0.4 ~g/ml in the circulating microreser-voirs at this time point. Thus the concentxation of the drug in the bloodstxeam carried in ~he circulating microreservoirs after 3 ~/4 hours was equivalent to that after only 15 minutes when the drug was introduced in the free form.
A fur~her comparison of Curve A with Curve C (free daunomycin in a dosage of 10 mg/kg) shows that after one hour the concentration of the drug in the plasma was about 1.3 ~g/ml when carried by the circulating microreservoirs and about 0.5 ~g/ml when in free form; after 3 hours these igures were about 0.5 ~g/ml and about 0.3 ~g/ml; and after 6 hours they were about 0.21 ~g/ml and ahout 0~14 ~g/ml, respectively. Thus, the use of the circulating microreservoirs of this invention can provide concentrations of ~his drug in the bloodstream which are materially greater than those achieved when the same drug is administered in free form in dosages 2.5 times greater than r.

when administered through the microreservoirs.
The altering of the plasma kinetics of a drug such as daunomycin, and as illustrated in FigO 10, means that it has a less toxic effect since i~ is maintained within the blood-stream longer and is thus prevented from concentrating in such organs as the liver, kidney and heart~ It also means that any given dosage is more effee~ive when carried by the circulating microreservoirs than when circulating freely, since the more drug that remains in the bloodstream the more exposure there will be of the target tissue to the da~nomycin dose~ Since the effec~iveness of this drug in killing tumor cells is based upon the ability of the tumor cells to take it up, maximum contact be~ween the drug and cells is essen~ial for maximum effectiveness.
Finally, altering of the plasma kinetics of a xenobiotic as shown in Fig. 10 offexs the possibility of decreasing dosages;
for if the performance of, say, the 10 mg/kg dose of Curve C
could be considered adequate for chemotherapeutical purposes, it becomes apparent tha~ the dosage level could ~e reduced below 4 mg/kg if ~he drug is delivered and released from the circulat-ing microreservoirs. Such a decrease in dosage woulcl alsomaterially reduce toxicity.
That a marked reduction in daunomycin toxicity is achieved through the use of the circulating microreservvirs is sho~n in Example 5 and F.ig. 11.

1000 ~moles of egg yolk phosphatidyl choline, 100 ~moles of cholesteryl oleate and 20 mg of daunomycin were dissolved in chloroform and the solution was taken to dryness in vacuo~ ~o ~he resulting dry residue mixture were added 20 ml . .

of 0.1 M KC1 and 10 mM of trihydroxymethylamine (pH ~ A O ~ ~ The aqueous su~pension was sonicated for 15 minutes at $1C under a nitrogen atmosphere. The sonicated liquid was chromatographed ;~
on a 2~5 X 40 cm Sepharose 4B gel column. Only those fractions which showed a coincidence of the elution profile of phsopha-tidyl choline, choles~eryl oleate and daunomycin were pooled.
These pooled fractions were then concentrated by ultra~iltra-tion, using an XM-50 membrane, to a final concentration oE 3 mg of daunomycin per ml.
A similar sample of microreservoirs without daunomycin was aiso prepared and concentrated to the same microreservoir concentxation~ A solution of free daunomycin at a concer.tration of 3 mg/ml in 0.1 M KCl and 10 mM of trihydroxymethylamine (pH 8.0) was also prepared as a control to be used as free daun~mycin.
The microreservoirs containing the daunomycin, the microreservoirs ~ithout the drug, and the free drug were in-jected at various drug doses into mice, using a single intra-peritoneal injection. Ten BDFl mice were used for each protocol;
and the resulting data are plotted in Fig. 11. These plots show the survival rate of the mice as a function of time. It will be seen that the microreservoirs themselves, in the ab-sence of daunomycin, exhibited no toxicity. At all of the dosage levels, the use of the circulating microreservoirs to carry the daunomycin decreased the toxic effects of the drug compared to that exhibited by the ree daunomycin. As the dosage levels decreased the ability o the delivery vehicles to reduce toxicity became more marked in comparison with the free drug. It is believed that this reduction in toxicity can 3C be explained, at least in part, by the ability of the circu-k g~SS

lating microxeservoirs to keep the daunomycln in the blood-stream and out vf the tissues and organs~ particularly the heart.
The effect of the chemotherapy of daunomycin using the delivery vehicles of ~his invention is illustrated in Example 6 and Fig. 12.

EX~PIE 5 The procedures of Example 5 were followed to make vesicular microreservoirs with and without daunom~cin and to prepare daunomycin for use as a ree drug. A number of mice were injected intraperitoneally with 1 X 106 P388 tumor cells.
Twenty-four hours later single daily intraperitoneal doses of the vesicular microreservoirs with and without daunomycin and free dauno~ycin were injected for a period of 5 days after the tumor implants. One group of mice, used as a control, received no drug in any form. Five mice were used for each protocol and two different dosage leve~s, 4 mg/kg and 2 mg/kg, were used.
The data obtained from these tests are plotted in Fig 12 as number of survivors as a function of time.
The mice injected with the microreservoirs without daunomycin had a surviva1 rate (not plotted) similar to that of the control mice, Curves C in the plots. At the dosage level o 4 mg/kg the daunomycin delivered by the circulating micro-resexvoirs exhibited chemotherapeutic effect while the free drug at the same dosage level demonstrated a toxicity measurably greater than the tumor cells. At the lower dosage level of

2 mg~kg, the daunomycin delivered by the circulating micro reservoirs exhibited a greater chemotherapeutic response than 30 the free drug.

~,.
C`. _ 1 s~

In formulating the microreservoirs of this invention, it may be ~esirable to include one or more xenobiotic binding modifiers which are capable of either increasing or decreasing the relative amount of the xer.cbiotic picked up by the micro-reservoirs ~uring their formation. For example, it has been found tha~ a minor mole percent, i.e., a~out 5 mole percent or more, cf phosphatidic acid, a charged lipid, added to the phos~ho-lip constituent of the microreservoirs may incr~ase khe affinity of the xenobiotic for the microreservoirs. It is, therefore, ~7ithin the scope of this invention to use one or more different phospholipids to make up the phospholipid constituent of the microreservoir composition. It is, moreover, within the skill of the art to choose a single phospholipid or an optimum com-bination of phospholipids as the phospholipid constituent cf the microreservoirs to obtain a predetermined pickup of the xenobiot c.
It is also possible to modify the binding of a xeno-hiotic to the reservoir by the inclusion of other lipids ~hich are soiuble ir the phospholipid constituent or slightly solu~le in the cholesterol ester cr triglyercide constituent. The modi-fications achieved in a microreservoir/daunomycin system are illustrated in Example 7 and Table l; and the effect such r,odi-fication~ have on the xenobiotic eflux or release rates of the resulting microreservoirs is illustrated in ExaMple 8 znd Figs. 13 and 14.

EXAMP~E 7 A series of formulations of microreservoirs containin~
105 ~mole of 14C-labeled egg yolk phosphatidyl choline (specific activity 4160 dpm~mole phosphatidyl choline), 11~6 ~moles of 355i cholesteryl oleate, or 11.6 ~moles of triolein (glyceryl tri-oleate~, with or wi~hout a second phospholipid as represented by 11.6 ~moles of phosphatidic acid, and with or without choles-terol as a modifying agent present in an amount from 0 tG 75 ~moles was made up. In each formula~ion, 1.72 mg o~ daunomycin was used as the xenobiotic to be carried and released.
The formulations were prepared as solutions of chlorc-form which were taken tG dryness in 10-ml screw-cap vials ard pumped overnightO Each formulation sample was then hydrated with 3 ml of 0.154 M NaCl and 10 mM of trihydroxymethylamine (pH ~.2) and then vortexed for several minutes at room temper-ature. Each sample was then sonicated at the appropriate temperature or 2Q minutes in a stream of nitrogen. ~onication of those samples containing chclesteryl oleate was carried out at 51C; and of those containing triolein at 3C. After soni-CatiGn, each sample was spun in a desktop centrifuge for 10 minutes to remove any titanium ragments.
Each sa~ple was passed down a 2.5 X 15 cn Se~hadex G-50 column using 0.1S4 M NaCl and 10 ~M trihydroxymethylamine as the elutin~ buffer. The void volume of each of the G-5~
columns, which contained the r~icroreservoirs, was collected and assayed for 14C egg yolk phosphatidyl choline radioactivity and fGr the fluorescence from the daunomycin. The results of these ~easurements in terms of ~g daunomycin/~mole phosphatidyl i choline and ~g daunomycin/~mole total phospholipid ar.d the percent of daunomycin piclc up by the microreservoirs are tabulated in Table 1.
Ir. reporting these results, the percent daunomycin encapsulated was normalized tc the sample that hacl the highest degree of encapsulated daunomycin per ~mole of ~ot:al phospho-lipid.

TA~LE 1 EFFECT OF COMPOSITION OF PHOSPHOLIPID CONSTITUENT
AND OF THE ADDITION OF PHOSPHOLIPID-MISCIBI~ LIPIDS
ON THE PICKt,-P OF DAUN0MYCIN BY MICRORESERVOIRS
.

Microreservcir Composition ~g ~.ole~ Daunomycin/
_ _ _ _ _ . __ Sample ~mole ~mole ~ncap-No. PC GTO CO PA Chol PC PL sulated 1 82 9 - 9 - 6.67 6.03 100 2 90 10 - - - 4.90 4.90 81

3 ~2 9 - - 9 4.26 4.26 71

4 72 8 - - 20 4.14 4.14 69 7 - - 28 4.03 4.03 67 6 54 6 - 40 3.91 3.91 65 7 82 - - 9 - 5.83 5.27 87 8 90 - 10 - ~ 4041 4.41 73 9 ~2 - 9 - 9 4.21 4~21 70 72 - 8 - 20 3.43 3.43 57 11 65 - 7 - 28 3.14 3.1~ 52 12 54 - 6 - 40 2.36 2.36 40 PC - Phosphatidyl choline GTO - Glycerol trioleate CO Cholesteryl oleate PA - Phosphatidic acid Chol - Cholesterol PL - ~otal phospholipids (PC and P~) -29 l ~. ~

3~55i From the data in Table 1 it will be seen that the inclusion of a small amount of phospha~idic acid in the phospho-lipid constituent (Samples 1 and 7) increased the uptake of adriamycin by the microreservoirs. In contrast to imidocarb, (examples 13-15~ which re~uires the inclusion of phosphatic acid, daunomycin, which has an ionizable amine ~unctional group profits from the use of phosphatidic acid as a portion of the phospholipid constituent. Thus phosphatidi~ acid is seen to be applicable to xenobiotics of ~arying characteristics.

The incorporation of cholesterol, a phospholipid-miscible lipid, in the microreservoir composition inhibited the binding of the daunomycin to the microreservoirs. The ad-dition of cholesterol to microreservoir compositions containing glycerol trioleate ~Samples 3-6) had a markedly less effect on the binding of the drug than in the case where ~he microreservoir composition contained cholesteryl oleate (Samples ~-12).
Finally, the data of ~able 1 indicate that in the case of daunomycin the use of glycerol trioleate (Samples 1-6) in place of cholesteryl oleate (Samples 7-12) facilitates the binding of this xenobiotic to the microreservoirs. It is ~herefore apparent that through the choice of the phospholipid constituent and the phospholipid-immiscible constituent (with or without an additional xenobiotic binding modifier) it is possible to control and predetermine the degree of xenobiotic uptake in or hinding to the microreservoirs. Such control offers flexibility in the xenobiotic delivery system of ~his invention wi~h re-s~ect to dosage levels, rate of xenobiotic release, and the like.
Not only can the equilibrium binding of a xenobiotic be affected by the microreservoir composition, but also the efflux or release rate of the x~nobiotic can be controlled and 30~

s~

predetermined hy the composition. This is illustrated through the use of a ~odel system which permitted the determination of xenobiotic efflux rates from the microreservoirs in response to nonequilibrium conditions as detailed in Examp~e 8 and shown in Figs. 13 and 14.

EX~YPLE 8 Microreservoirs were formulated as described in Example 7. To establish the required nonequilibrium conditions the microreservoirs containing daunomYCin as the xenobiotic were incuba~ed ~7ith a lar~e excess of unsonicated egg yolk phosphatidyl choline dispersions. These dispersions were formed by adding aliquots of microreservoirs of the various compositions containing ap~roximately 1 ~mole oE egg yolk phosphatidyl chGline to 9 ~moles of unsonicated egg yolk phos-phatidyl choline in l.0 nll of 0.154 ~I NaCl and 10 n~ of tri-hydroxymethylamine (pH 7.2). Comparable control dispersions were made up to contain an equivalent amount of free daunomyCin in place of that carried by the microreservoirs.
~0 In assessing the efflux rate of the daunomycin, one of the mixtures thus formed was used for each time point. At the desi~nated time, the sample was centrifuged at 15,000g for 2 minutes under ~hich ~onditions the unsonicateddispersion was readily separated from the microreservoirs remaining in the re-sulting supernatant. Since ~he daunomycin tends to re-equi librate between the microreservoirs and the unsonicatedphospho-lipid dispersions, the rate of equilibrium can be used as a kinetic parame~er to evaluate the role of various constituer.ts forming the microreservoir composition in determinin~ the efflu~

rate of the daunomycill contained in the microreservoirsO

95~

An aliquot of the resulting supernatant was assayed for fluorescence and the amount o~ fluorescence thus remaining was compared wi~h that ini ially presen~ in the mixture. This amount o daun~m~cin remaining in the supernatant therefore represents the amount of the drug still contained in the microreservoirs.
The data obtained from this series o measurements are plotted in Figs. 13 and 14 which show decrease of fluor-escence as a function of time for the microreservoirs containing glycerol ~rioleate (Fig. 13) and cholesteryl oleate ~Fig. 14).
From the data plotted in Figs. 13 and 14 it will be seen that the free daunomycin was very rapidly removed rom the super-natant, whereas that bound to the microreservoirs was retained to a much greater degree. The inclusion of 9 mole ~ of phos-phatidic acid in the phospholipid constituent of the microre-servoirs materially retarded the efflux rate, while the addition of cholesterol increased it. Finally, the use of a triglyceride in place of a cholesterol ester slightly decreased the efflux rate.
These kinetic evaluations based on efflux rates contribute to the significance of the data of Table 1 coneerning the equilibrium binding of aaunom~cin to the microreservoirs:
and these data confirm the fact that the composition of the microreservoirs can be chosen to predetermine and control the pharmacodynamics of the xenobiotic contained in the microre-servoirs serving a~ the drug delivery vehicle of this invention.
~D 32 i~ a highly hydrophobic analog of adriamycin used in chemotherapy. Since this drug is totally insoluhle in an a~ueous buffer, it is necessary to use a combination of a detergent and an organic liquid to form a solution of AD 32 3~-~, suitable fur clinical use. Exemp~ary of one such solvent presently in use is a mixture o equal volumes of ethanol and a sulfated r ,~,~ ~/~f7~7/ ~
~ ethylene glycol detergent (e~e~.
S~
In spite of the hydrophobic nature vf AD 32, it is possible to incorporate it into the microreservoirs in accordance with this invention and thus to beneficially alter its pharma-codynamics with respect to both plasma kinetics and therapeutic effectiveness as illustrated in Examples 9-12 and Figs. 15~-18.

ExAMæLE 9 1090 ~moles of phosphatidyl choline derived from egg yolk, 121 ~moles o~ 3H-labeled cholesteryl oleate (specific activity of l90,Q00 dpm/~mole) and 17.5 mg of AD 32 were dis-solved in chloroform, then taken to dryness and pumped overnight.

5.5 ml of 0.154 M NaCl and 5 mM of trihydroxymethylamine (pH 7.41) were added to the dry mixture. The liquid was sonicated at 48C
under a nitrogen atmosphere and the resulting sonicated liquid was chromatographed on a 2.5 X 40 cm Sepharose 4B column. In-dividual fractions ~hus obtained were assayed for phosphatidyl choline content, for cholesteryl oleate and or AD 32 as pre-viously describedr The elution profile, a composite of the as-says~ is plotted in Fig. 15. From this plot it can be seen that the AD 32 is associated with both nonvesicular microreservoirs (fractions 2-5) and vesicular microreservoirs (fractions 6-18).
The inclusion of a minor amount of phosphatidic acid in the phospholipid constituent of the microreservoir composition was found to have little, if any, effect on the~efflux rate of AD 32 when a triglyceride was used as the phospholipid-immiscible constituent. This is evident from Example 10 and Fig. 16.
r~

s~

A basic microreservoir formulation containing 104.6 ~moles egg yolk phosphatidyl choline, and 11.6 ~moles of 3H-labelled glycerol ~rioleate (specific activity of 1.5 X 105 dpm/
~mole) was used. 1.73 mg of AD 32 was added and in one formula-tion 11.62 ~moles of egg yolk phosphatidic acid was also addedO
These formulations were taken to dryness from a chloroform 501u-tion and pumped overnight under vacuum. 3 ml of 0.154 M NaCl and 10 m~ trihydroxymethylamine were added to each sample and the hydrated liquids were sonicated for 20 minutes at 3C under a nitrogen atmosphere. Each sample was passed down a 2.5 X 20 cm 9ephadex G 50 column and a quantity of eac~ resulting vesicular microreservoir sample e~uivalent to one ~Imole of phospholipid ~as incubated with g ~moles of phosphatidyl choline liposomes in 0.46 ml of a buffered saline solution to orm a dispersion.
At various time points, the resulting dispersions were centrifuged at 15,000g for 2 minutes and the amount of fluorescence remaining in the supernatant was determined. The data from these measurements of efflux rate are plott~d in Fig. 16. It will be seen that the efflux rate of AD 32 is relatively rapld and essentially unaffected by the inclusion of phosphatidic acid in the rnicroreservoir composition.
Although the efflux rate of AD 32 from the micro reservoirs is comparatively rapid, the use of microreservoirs bring about a marked beneficial alteration of the plasma kinetics of this drug, compared to the presently used dosage forms. ~his is illustrated in Example 11 and Fig. 17.

~X~PLE 11 Fractions 6-18 of Example 9 were concentrated by ultrafiltration to an AD 32 concentration of 3.2 mg/ml. Suf~

i5 ficient quantities of the AD 32 in the microreservoirs were in-jected intravenously into 150-gram rats to provide a aosage level of lS mg/kg; and a clinical formulation of AD 32 dissolved in a 1 to 1 by volume mixture of ethanol/emulphol was ~imilarly injected at the same dosage level in three control rats. Plasma samples were taken at various points in time and A~ 32 concentra-tions in these plasma samples were determined by fluorescence.
The resulting data (average of three rats for each point) are plotted in Fig. 17 as AD 32 equivalents as a function of time from initial injection.
Fig. 17 shows the marked alteration in the plasma kinetics of AD 32 achieved through the use of the microreservoirs of this invention, for the levels of AD 32 in the plasma in which the drug was incorporated in microreservoirs were consistently higher by a factor of between 2 and 3 at all time points than for the free AD 32 introduced as the clinical solution. By allowing the drug a longer period of time to seek out tumor cells, this alteration in its plasma kinetics enhances its effectiveness as a chemotherapeutic agent. This is shown in Example 12 and ~ig.
Fig. 18.

EXA~PLE 12 Vesicular microreservoirs containing AD 32 were formu-lated as in Example 9 and concentrated ~o 3.2 mg AD 32/ml. A
number of rnice were injected intraperitoneall~ with 1 X 106 P388 tumor (leukemic) cells. Twenty-four hours later single daily intraperitoneal doses of ~he vesicular microreservoirs with AD 32 and saline control solutions were injected for a period of 5 days after the tumor implants. Five mice were used ~or each protocol and four different dosage levels, 7.3 mg/kg, 4 mg/kg, 2 mg/kg, and 1 mg/kg were used. The data obtained from ~hese tes~s are plot~ed in ~ig. 18 as number of survivor~ as a function of time. In all cases ~he mice injected with the microreservoirs containing AD 32 had a survivial rate higher than that of the control mice. Moreover, even at the very low dosage level of 1 mg/kg the AD 32 delivered by the circulating microreservoirs exhibiked chemotherapeutic effect and brought about a significant increase in survival of mice given the P388 leukemic cellsO
As in the case of daunomycin, the same plasma con-centrations of A~ 32 can be achieved with lower initial levelsof AD 32 carried by the microreservoir~ than in clinical formula-tions. Furthermore, the microreservoir composition is more biocompatible with blood than ~he mixed solvent of ethanol and detergent now required in clinical formulations of AD 32.
Adriamycin, dauno~ycin and AD 32, as well as carminomycin, are cancer chemotherapeutic agents falling within the general class of anthracycline drugs. The preceding examples illustrate the efectiveness of the delivery ~ehicle o~ this invention in bene-ficially alterin~ the pharmacodynamics of this class of chemo-therapeutic agen~s. The microreservoirs of this invention mayalso be used to deliver other classes of chemotherapeutic agents including, for example, n.itrosoureas and metabolite~
Imidocarb is a drug which has proven very ef:fective as a pa.rasitiaide in the treatment of anaplasma in animals hy killing para~ites in the bloodstream. However, when administered at its most effective dosage levels, traces of the imidocarb tend to remain in the animal tissue, an undesirable situation if the animal is to be used for human consumption. Therefore, it would be desirable to have a delivery vehicle capable of main-taining the imidocarb in the circulaing bloodstream allowing it to perfQrm its t}lerapeutic function without accummulating in the animal tissue.
Imidocarb and its hydrochloric acid salt are hydro-philic compounds, a characteristics which differentiates them from the hydrophobic or hydrophobic~hydrophilic drugs. However, as will be seen from the following Examples :L3 and 14, and Figs.
19-21l the microreservoirs are equally effective in beneficial-ly altering the pharmacodynamics of a hydrophilic xenobiotic such as imidocarb.

900 ~moles of egg yolk phosphatidyl choline, 100 ~moles of phosphatidic acid, 100 ~moles of 3H-labeled cholesteryl oleate (specif~c activity 1.6 X 104 dpm/~mole) and 9.16 mg of 14C-labeled imidocarb were dissolved in chloroform and the solution was evaporated to dryness under vacuum. The resulting mixed dry residue was then hydrated by the addition of 45 ml of 0.154 M
NaCl and 290 mM of trihydroxyethylamine ~pH 8.0) to the mixed lipid/drug residueO The liquid was then sonicated fox 15 minutes at 51C under a nitrogen atmosphere.
The sonicated liquid was then chromatographed on a 2.5 X 40 cm Sepharose 4B column. Individual fractions resulting from the chromatographing were then assayed for phosphatidyl choline and phosphatidic acid by the method of Gomori,and for the 3H-cholesteryl oleate and the 14C-imidocarb by radioactivity.
The results of these analyses are plotted in Fig~ 19 as a func-tion of fraction number, the analytical results being superimposed as in Fig. 8. The diameter of the vesicular microreservoirs was found to range between about 200 A and 300 A.

-~7-9~55 It can be seen that the elution profiles of the three constituents of the micxoreservoirs coincide from fractions 5 through 9, indicating that the imidocarb was associated with the microreservoirs.
The beneficial alteration of the plasma kinetics of imidocarb attained through the use of the delivery vehicles of this invention is illustrated in Example 14 and Figs. 20 and 21.

100 ~moles of phosphatidyl choline and 10 ~moles of phosphatidic acid derived from egg yolk, 10 ~moles of choles-teryl oleate and 2 mg of 14C-labeled imidocarb were dissolved in chloroform and the solution evaporated to dryness under vacuumO
The resulting mixed residue was suspended in 5 ml of 0.154 M NaCl and 10 mM trihydroxymethylamine (pH 8.0) and the resulting suspension was sonicated for 15 minutes at 51C under a nitro-gen atmosphere. The liquid was then chromatographed on a 2O5 X 40 cm Sepharose 4B gel column and the fractions showing a coincidence of the 14C-labeled imidocarb, phospholipids and cholesteryl oleate were pooled and concentrated by ultrafilatra-tion to a final concentration of 0.6 mg of imidocarb per mil.
A solution of free imldocarb at a concentration of O.6 mg/ml in 0.154 M NaCl and 10 mM of trihydroxymethylamine (pH 8.0) was also prepared as a control to be used in free imidocarb admini~tration.
'rhe imidocarb-containing microreservoirs and free imidocarb were injected into the tail veins of rats at two dos-age levels, i e., 5 mg/kg, and 4.4 mg/kg. Plasma samples were taken at various time points and the amount of imidocarb in these samples was determined by measurements of their radio-g~55 activity. The results of these measuremen~s, plotted as imidocarb equivalents as a ~unction of ~ime~ are given iTi FigsO
20 and 21.
The beneficial alteration of the plasma kinetics through the use of the circulating microreservoirs to carry imidocarb in the bloodstream of a living mam~lalian host is clearly evident from Figs. 20 and 21. For example, at a dosage level of 5 mg/kg, the concentration of the imidocarb in the microreservoirs/imidocarb in the bloodstream was about 140 times 10 greater than the free imidocarb after four hours; and at a dos-age level of 4.4 mg~kg about 200 times greater after this same period of time. Moreover, as noted on Fig. 21, the bloodstream still contained a measurable quantity of imidocarb in the micro-reservoir/imidocarb form after 24 hours as contrasted with virtually none in the free form of imidocarb.
The ratio of imidocarb ~o microreservoirs as a function of time is also plotted for each dosage level in Figs.
20 and 21; and these plots clearly show that the drug is released at a rate which can be con~rolled and predetermined for any given 20 set of conditions. From Figs. 20 and 21 it is also apparent that it is possible, if desired, to reduce the dosage levels of the drug to far below tho~e now considered effective in killing the blood-borne parasites which cause anaplasma in animals.
One of the major goals in beneficially altering the pharmacodynamics of a xenobiotics is the ability to alter the tissue distribution of the xenobiotic. That this can be achieved for imidocarb using the microreservoirs of this invention is shown in Example lS and Table 2.

ss EXAM~LE 15 The animals used in Example 14 were sacrificed at 4 hours after the injection of free imidocarb or the microreser-voirs containing imidocarb. Various tissues, such as muscle, spleen, liver and kidney, were taken from the animals. These tissues were combusted in a Searle combustion apparatus and the resulting 14C02 was collected and counted for radioactivity.
The number of counts per gram of tissue was determined and the results are shown in Table 2.

TISSUE DISTRIBUTION OF FREE IMIDOCARB
AND MICRORESERVOIR-BORNE IMIDOCARB

_ dpm/gram of tissue Microreservoir/ Percent Tissue ImidocarbImidocarb Chan~e Muscle 3,985 3,511 -12 Kidney 40,13459,984 f49 Spleen 9,498 15,339 ~61 Liver 27,059 29,991 +11 The ability to maintain a xenobiotic in the bloodstream and within certain organs is particularly significant for a drug such as imidocarb. Thus, the 12% reduction of imidocarb in muscle tissue attained through the use of the microreservoirs is impor-tant to the use of this paraciticide in treating animals to be used for human consumption. Moreover, the greater concentration of the drug in the liver and spleen, organs which contain a greater proportion of the anaplasma microorganism, is another significant parameter in exploiting the use of imidocarb.

--~0--S,5 Among ~he pharmacodynamic~ of a xenobiotic which may be ~eneficially altered hy the delivery vehicle of this invention is oral absorption, the altering being achieved by imparting to the xenobiotic the ability to cross the gastro-intestinal (GI) tract and enter into the bloodstream for ef-fective circulation. At present, several solutions are avail-able for administering xenobiotics which do not cross the GI
tractr or which cross it to a very limited degree, into the bloodstream. One such solution lies in the chemical modification of the xenobiotic, such as forming the undecanoate ester of estradiol which is essentially insoluble in water and suspending it in oil, forming it into a microcrystalline dispersion or making a solution with an organic solvent such as ethanol.
~ he incorporation of estradiol undecanoate (used as a fertility control agent) in microreservoirs in accordance with this invention eliminates the need for such liquid media as oils and organic solvents and at the same time achieves a much more satisfactory discharge of this xenobiotic into the bloodstream.
The incorporation of estradiol undecanoate in the microreservoirs of this invention is shown in Example 16 and Fig. 22.

300 ~moles of egg yolk phosphatidyl choline, 30 ~moles of cholesteryl oleate and 6 ~moles of 3H-labeled estradiol un-decanoate (specific activity 4.6 ~Ci/~mole) were dissolved in benzene and lyophillized. To the resulting dry mixture were then added 5 ml of 0.154 M NaCl and 5 mM trihydroxymethylamine;

and the resulting liquid was sonicated for l7 minutes at 48C
under a nitrogen atmosphere. The resulting sonicated mixture was then fractionated by chromatographing it on a Sepharose 4B

-41~

5~

column ~o produce the elution profile of Fig. 22. Fractions 12-30 constituted the xenobiotic-containing vesicular micro-reservoirs used in making an in vivo evaluation of the delivery vehicle of this invention.
The ability of the microreservoirs of this invention to enhance the oral absorption of estradiol undecanoate is further illus~rated in Example 17, Fig.23 and Table 3.

__ 3H-labeled estradiol undecanoate was encapsulated in microreservoirs composed of egg yolk posphatidyl choline and 14C-labeled cholesteryl oleate. Equivalent amounts of 3~-labeled estradiol undecanoate were dissolved in ethanol. Doses of 1.63 my/kg were administered orally to two sets of 9 rats. After 1, 4 and 24 hours~ three of the rats in each set were killed and the blood collected. The plasma was separated from the red cells by centrifugation and 0.5 ml of each plasma sample was added to 9.5 ml of CHC13/MeOH (2/1 by volume~ containing 1.6 ~moles of unlabeled estradiol and estrone as carriers. The resulting liquid was filtered and sufficient saline was added to the filtrates to make a 2~phase system. The lower phase was taken to dryness and then redissolved in 2.0 ml of methanol.
0.2 ml of this solution was counted for radioactivity. The remaining 1.8 ml was spotted on a TLC plate and developed in benzene/ethyl acetate (3/2 by volume). The plates were exposed briefly to iodine vapor to allow the carrier steroids to become visualized and then iodine vapor was driven off by mild heating.
Those spots corresponding to estradiol and estrone were cut out and counted ~or radioactivity. The equivalents of 3H estradiol undecanoate in the plasma are plotted in Fig. 23. It can be -~2-seen that by the first hour the vesicular microreservoirs enhanced the appearance of 3H-laheled estradiol undecanoate equivalents in the plasma when compared with the ethanol-control solution.
After four hours, the amount of estradiol undecanoate equivalents was decreased for both formulationsO
It is known that estradiol is the active form of the drug, and estrone is inactive. Furthermore, the esters of estradiol are assumed to be inactive until hydrolyzed to estradiol.
Therefore, ~he ratio of estradiol to estrone in plasma should be an index of the ability of the microreservoirs to enhance the active form of the drug.
The results of the TLC analysis of the labeled estradiol derivatives in the plasma are given in Table 3~ It can be seen from these data that the ratio of estradiol to estrone is higher by 60% using the microreservoirs than using an ethanol solution.

RATIO OF H ESTRADIOL TO H ESTRONE IN PLASMA
ONE HOUR AFTER ORAL ADMINISTRATION
OF ESTRADIOL UNDECANOATE

Formulation EstrAdiol~Estrone*
In ethanol solution 1.14 In microreservoirs 1.84 * Ratio is based on average of three rats.

The microreservoir delivery system of this invention also offers the possibility of orally administering xenobiotics heretofore incapable of this mode of administration. By trans-porting such druys across the GI tract it is possible to deliver -~3-them to the bloods~ream indirectly, thus eliminating their metabolism in the GI tract and the need for injections and the trouble and complications attendant on this form of admin-istration.
The microreservoirs may be used in either their vesicular form or nonvesicular form or in a combination of ~hese forms.
This is illustrated by Example 18 and Figs. 24 and 25.

EX~MPLE 18 .
25 ~moles of ~gg phosphatidyl choline, 75 ~moles o 14C-labeled cholesteryl oleate, and 2 ~moles of 3H~labeled estradiol undecanoate were dissolved in ohloroform and taken to drynes6. The lipid mixture was dried overnight under vacuum and ~hen hydrated with 4.4 ml of 0.154 M NaCl and 5 mM trihydroxy-methylamine (pH 7.2). ~he resul~ing liquid was sonicated at 40C
for 20 minutes under a nitrogen atmosphere and passed down a Sepharose 4B Column. The resulting elution profile is shown in Fig. 24. It can be observed that the estradiol undecanoate has a higher affinity for the vesicular form of the microreservoirs than for the nonvesicular form, based on the ratio of estradiol undecanoate to cholesteryl ol~ate in the two forms of the micro-reservoirs. The material in the void volume of the column consist-ed of the nonvesicular form, whereas the material in the internal volume consisted of the vesicular form.
Samples of both the nonvesicular and vesicular ~orms containing estradiol undecanoate and approximately one ~mole of total lipid were incubated with 9 ~moles of an unsonicated egg phosphatidyl choli~e dispersion. At various time points the samples were centrifuged at 15,000g for 2 minutes and the super-natant containing the microreservoirs was counted for radio-5~

activity. Since the cholesteryl oleate is a nonexchangeable species in thls system, the ratio of ~H-labeled estradiol unde-canoate to 14C-laheled cholesteryl oleate is an indication of the rate of efflux of estradiol undecanoate from the nonvesicular or vesicular microreservoirs to the phosphatidyl choline dis-persion. The results are shown in Fig. 25. It will be seen that the estradiol undecanoate remained associated with b~th forms of the microreservoirs, indicating that both are satisfactory for carrying and releasing xenobiotics.
The xenobiotic-containing microreservoirs of this in vention may be formulated into a variety of dosage forms. Thus, for example, they may be dispersed in a physiologically compatible liquid, they may be used dry to form tablets, or they may be contained i~ capsules formed of a suitable biocompatible material.
From the detailed description and examples yiven, it will be seen that the delivery vehicle of this invention is capable of beneficially altering the pharmacodynamics of xenobiotics having a wide range of chemical and physical charac~eristics as well as a wide range of biological uses and properties.
~0 It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in carrying out the above method and in the composition and article set forth without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense~

Claims (71)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A delivery vehicle biocompatible with a mammalian host to delivery and release within said host a xenobiotic the phar-macodinamics of which are beneficially altered through the use of said vehicle, characterized in that said delivery vehicle is in the form of microreservoirs containing said xenobiotic, said microreservoirs comprising a phospholipid constituent and a phospholipid-immiscible lipid constituent stable in and essen-tially immiscible with a physiologically-compatible liquid, said phospholipid-immiscible lipid constituent being a cholesterol ester or a triglyceride or a mixture of cholesterol esters, or triglycerides or of both of these constituents.

2. A delivery vehicle in accordance with claim 1 wherein said microreservoirs are in vesicular form having diameters ranging between about 190 .ANG. and about 300 .ANG..

3. A delivery vehicle in accordance with claim 1 wherein said microreservoirs are in nonvesicular form having diameters ranging between about 250 .ANG. and about 1000 .ANG..

4. A delivery vehicle in accordance with claim 1 wherein said microreservoirs are in both nonvesicular and vesicular forms.

5. A delivery vehicle in accordance with claim 1 wherein said microreservoirs comprise between about 50 mole % and about 97 mole % of said phospholipid constituent.

6. A delivery vehicle in accordance with claim 1 wherein said microreservoirs include a xenobiotic binding modifier.

7. A delivery vehicle in accordance with claim 6 wherein said xenobiotic binding modifier increases the pickup of said xeno-biotic by said microreservoirs.

8. A delivery vehicle in accordance with claim 7 wherein said xenobiotic binding modifier is phosphatidic acid forming a minor portion of said phospholipid constituent.

9. A delivery vehicle in accordance with claim 6 wherein said xenobiotic binding modifier decreases the pickup of said xeno-biotic by said microreservoirs.

10. A delivery vehicle in accordance with claim g wherein said xenobiotic binding modifier is cholesterol.

11. A delivery vehicle in accordance with claim 1 wherein said microreservoirs include a xenobiotic release-rate control agent.

12. A delivery vehicle in accordance with claim 11 where-in said xenobiotic release-rate control agent is a lipid miscible with said phospholipid constituent of said microreservoirs.

13. A delivery vehicle in accordance with claim 12 where-in said lipid is cholesterol.

14. A delivery vehicle in accordance with claim 1 wherein said phospholipid constituent comprises phosphatidyl choline.

15. A delivery vehicle in accordance with claim 1 where-in said phospholipid constituent comprises a mixture of phosphatidyl choline and phsophatidic acid, said phosphatidic acid being present in said mixture in an amount equivalent to at least about 5 mole %.

16. A delivery vehicle in accordance with claim 1 where-in said phopsholipid-immiscible lipid constituent comprises a cholesterol ester of a fatty acid having between 10 and 18 carbon atoms,

17. A delivery vehicle in accordance with claim 16 where-in said cholesterol ester is cholesteryl oleate.

18. A delivery vehicle in accordance with claim 1 where-in said phospholipid immiscible lipid constituent comprises a tri-glyceride.

19. A delivery vehicle in accordance with claim 18 where-in said triglyceride is glycerol trioleate.

20. A delivery vehicle in accordance with claim 1 wherein said xenobiotic is a drug.

21. A delivery vehicle in accordance with claim 20 where-in sAid drug is a chemotherapeutic drug.

22. A delivery vehicle in accordance with claim 21 wherein said drug is a cancer chemotherapeutic drug.

23. A delivery vehicle in accordance with claim 22 wherein said cancer chemotherapeutic drug is an anthracycline.

24. A delivery vehicle in accordance with claim 23 wherein said cancer chemotherapeutic drug is adriamycin.

25. A delivery vehicle in accordance with claim 23 wherein said cancer chemotherapeutic drug is N-trifluoroacetyl adria-mycin-14 valerate.

26. A delivery vehicle in accordance with claim 20 wherein said drug is a parasiticide.

27. A delivery vehicle in accordance with claim 26 wherein said parasiticide is imidocarb.

28. A delivery vehicle in accordance with claim 20 wherein said drug is a fertility control agent.

29. A delivery vehicle in accordance with claim 23 wherein said fertility control agent is estradiol undecanoate.

30. A delivery vehicle in accordance with claim 1 wherein said xenobiotic is a drug, the plasma kinetics of which are beneficially altered.

31. A delivery vehicle in accordance with claim 1 wherein said xenobiotic is a drug, the chemotherapeutic effectiveness of which is beneficially altered.

32. A delivery vehicle in accordance with claim 1 wherein said xenobiotic is a drug, the toxicity of which is beneficial-ly altered.

33. A delivery vehicle in accordance with claim 1 wherein said xenobiotic is a drug, the oral absorption, and hence its ability to pass the gastrointestinal tract, is beneficially altered.

34. A method of forming a delivery vehicle for delivering within a mammalian host a xenobiotic, the pharmacodynamics of whlch are predeterminably altered and controlled, comprising the steps of forming microreservoirs of a composition compris-ing a phospholipid constituent and a phospholipid-immiscible 1 lipid constituent which is stable in and essentially immiscible with a physiologically-compatible liquid, said phospholipid-immiscible lipid constituent being a cholesterol ester or a triglyceride or a mixture of cholesterol esters, or triglycer-ides or of both of these constituents, and incorporating said xenobiotic to be delivered within said microreservoirs.

35. A method in accordance with claim 34 wherein said step of forming said microreservoirs comprises (a) forming with a solvent a solution of said phospho-lipid constituent and of said phospholipid-immiscible lipid constituent;
(b) removing said solvent to produce a dry residue mix-ture of said phospholipid constituent and said phospholipid-immiscible lipid constituent;
(c) hydrating said dry residue mixture with a physio-lodically-compatible liquid to form a suspension;
(d) sonicating said suspension under a nonoxidizing atmosphere at a temperature at least equivalent to the melting point of said phospholipid-immiscible lipid constituent to form said microreservoirs; and (e) separating out said microreservoirs thus formed.

36. A method in accordance with claim 35 wherein said step of incorporating said xenobiotic within said microreservoirs comprises adding said xenobiotic to said solution in step (a).

37. A method in accordance with claim 35 wherein said step of incorporating said xenobiotic within said microreservoirs comprises adding said xenobiotic to said suspension of step (c) prior to said sonicating.

38. A method in accordance with claim 35 wherein said step of separating out said microreservoirs thus formed comprises centri-fuging the sonicated suspension and chromatographing the clear phase resulting from said centrifuging to provide a series of chromatographed fractions.

39. A method in accordance with claim 38 further including the step of separating those of said chromatographed fractions containing said microreservoirs in vesicular form having diameters ranging between about 190 .ANG. and 300 .ANG. from those fractions con-taining said microreservoirs in nonvesicular form having diameter ranging between about 250 .ANG. and about 1000 .ANG..

40. A method in accordance with claim 35 including the step of adding a xenobiotic binding modifier to said solution.

41. A method in accordance with claim 40 wherein said xenobiotic binding modifier increases the pickup of said xenobiotic by said microreservoirs.

42. A method in accordance with claim 41 wherein said xenobiotic binding modifier is phosphatidic acid forming a minor portion of said phospholipid constituent.

43. A method in accordance with claim 40 wherein said xenobiotic binding modifier decreases the pickup of said xenobiotic by said microreservoirs.

44. A method in accordance with claim 43 wherein said xenobiotic binding modifier is cholesterol.

45. A method in accordance with claim 35 including the step of adding a xenobiotic release-rate control agent to said solution.

46. A method in accordance with claim 45 wherein said xenobiotic release-rate control agent is a lipid miscible with said phospholipid-constituent of said microreservoirs.

47. A method in accordance with claim 34 wherein said phospholipid constituent comprises phosphatidyl choline.

48. A method in accordance with claim 34 whereln said phospholipid constituent comprises a mixture of phosphatidyl choline and phosphatidic acid, said phosphatidic acid being present in said mixture in an amount equivalent to at least about 5 mole %.

49. A method in accordance with claim 34 wherein said phospholipid-immiscible lipid constituent comprises a cholesterol ester of a fatty acid having between 10 and 18 carbon atoms.

50. A method in accordance with claim 49 wherein said cholesterol ester is cholesteryl oleate.

51. A method in accordance with claim 34 wherein said phospholipid-immiscible lipid constituent comprises a triglyceride.

52. A method in accordance with claim 51 wherein said triglyceride is glycerol trioleate.

53. A method in accordance with claim 34 wherein said xenobiotic is a drug.

54. A method in accordance with claim 51 wherein said drug is a chemotherapeutic drug.

55. A method in accordance with claim 54 wherein said drug is a cancer chemotherapeutic drug.

56. A method in accordance with claim 55 wherein said cancer chemotherapeutic drug is an anthracycline.

57. A method in accordance with claim 56 wherein said cancer chemotherapeutic drug is adriamycin.

58. A method in accordance with claim 56 wherein said cancer chemotherapeutic drug is N-trifluoroacetyl adriamycin-14 valerate.

59. A method in accordance with claim 53 wherein said drug is a parasiticide.

60. A method in accordance with claim 59 wherein said parasiticide is imidocarb.

61. A method in accordance with claim 53 wherein said drug is a fertility control agent.

62. A method in accordance with claim 61 wherein said f tility control agent is estradiol undecanoate.

63. A method in accordance with claim 34 wherein said xenobiotic is a drug, the plasma kinetics of which are beneficially altered.

64. A mekhod in accordance with claim 34 wherein said xenobiotic is a drug, the chemotherapeutic effectiveness of which is beneficially altered.

65. A method in accordance with claim 34 wherein said xenobiotic is a drug, the toxicity of which is beneficially altered.

66. A method in accordance with claim 34 wherein said xenobiotic is a drug, the oral absorption, and hence its ability to pass the yastrointestinal tract, is beneficially altered.

67. A method in accordance with claim 34 wherein said step of forming said microreservoirs comprises (1) forming in a water-miscible organic solvent a solution of said phospholipid constituent and said phospholipid-immiscible lipid constituent;
(2) injecting said solution into said physiologically-compatible liquid under conditions to form said microreservoirs.

68. A method in accordance with claim S7 wherein said step of incorporating said xenobiotic within said microreservoirs comprises adding said xenobiotic to said solution.

69. A method in accordance with claim 67 wherein said step of incorporating said xenobiotic within said microreservoirs com-prises adding said xenobiotic to said physiologically-compatible liquid.

70. A method in accordance with claim 34 including the step of suspending said microreservoirs containing said xeno-biotic in said physiologically-compatible liquid thereby providing said xenobiotic in liquid dosage form.

71. A method in accordance with claim 34 including the step of encapsulating said microreservoirs containing said xenobiotic in a capsule formed of a physiologically-acceptable material.