Pharmacological agent-lipid solution preparation

A pharmacological agent-lipid solution preparation comprising a lipophilic pharmacological agent, a desalted charged lipid and an aqueous-miscible lipid solvent such that upon introduction into an aqueous medium a suspension of lipid aggregates associated with the pharmacological agent are formed, and methods of manufacture and use.

FIELD OF THE INVENTION 
This invention discloses a pharmacological agent-lipid solution preparation 
comprising a lipophilic pharmacological agent, a desalted charged lipid, 
and an aqueous-miscible lipid solvent such that upon introduction into an 
aqueous medium a suspension of lipid associated with the pharmacological 
agent is formed. Further disclosed are preparation and methods of 
manufacture and use of the pharmacological agent-lipid solution 
preparation, and the co-solubilizing of lipid associating pharmocological 
agent in lipid and ethanol solution. 
BACKGROUND OF THE INVENTION 
Lipids are known to be useful as carriers for the delivery of drugs to 
mammals including humans. In pharmaceutical preparations lipids are 
variously used as admixtures with drugs or in the form of liposomes. 
Liposomes are vesicles comprising closed bilayer membranes containing an 
entrapped aqueous phase. Liposomes may be any variety of unilamellar 
vesicles (possessing a single membrane bilayer) or multilamellar vesicles 
(e.g. onion-like structures characterized by concentric membrane bilayers, 
each separated from the next by an aqueous layer). 
Liposomes are formed by methods well known in the art. The original 
liposome preparation of Bangham et al. (1965, J. Mol. Biol. 13:238-252) 
involves suspending phospholipids in an organic solvent which is then 
evaporated to dryness leaving a phospholipid film on the reaction vessel. 
Then an appropriate amount of aqueous phase is added, the mixture is 
allowed to "swell", and the resulting liposomes which consist of 
multilamellar vesicles are dispersed by mechanical means. The structure of 
the resulting membrane bilayer is such that the hydrophobic (nonpolar) 
"tails" of the lipid orient toward the center of the bilayer while the 
hydrophilic (polar) "heads" orient toward the aqueous phase. This 
technique provides the basis for the development of the small sonicated 
unilamellar vesicles described by Papahadjopoulos and Miller (1967, 
Biochim. Biophys. Acta. 135:624-638) and large unilamellar vesicles. 
Another class of liposomes is characterized as having substantially equal 
interlamellar solute distribution. This class of liposomes is denominated 
as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 
4,522,803 to Lenk et al. and includes monophasic vesicles as described in 
U.S. Pat. No. 4,588,578 to Fountain et al. and frozen and thawed 
multilamellar vesicles (FATMLV) as described in "Solute Distributions and 
Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles," 
Mayer et al., Biochima et Biophysica Acta. 817:193-196 (1985). 
Another method of liposome formation is by the infusion of lipid solvent 
such as diethyl ether or ethanol which contains phospholipids into an 
aqueous solution containing a pharmacological agent resulting in the 
formation of liposomes which entrap a portion of the aqueous solution. 
This procedure cannot be used to entrap lipid soluble pharmacological 
agents soluble in fat or fat solvents due to the very limited solubility 
of such agents in an aqueous solution. 
Lipid soluble pharmacological agents include anti-neoplastics such as 
doxorubicin; antifungals such as miconazole, terconazole and amphotericin 
B; immunomodulators such as cyclosporin A; derivatives of muramyl 
dipeptides such as muramyl tripeptide phosphatidylethanolamine; and, 
hormones such as glucocorticoids, mineralocorticoids and estrogens; 
anti-inflammatories such as the steroidals, prednisone, dexamethasone and 
fluromethasone and the nonsteroidals indomethacin, salicylic acid acetate 
(aspirin) and ibuprofen, further including analgesic agents such as 
acemetacin and flurobiprofen; and other agents such as lipoxygenase 
inhibitors, prostaglandins, neuroleptics, antidepressants, fat-soluble 
vitamins, contrast materials and antivirals. Pharmacological agents as 
used herein includes agents administered to animals including mammals, 
particularly humans, in the course of treatment or diagnosis. Biologically 
active materials such as drugs as well as diagnostic agents and contrast 
materials which are usually nonreactive are all to be understood to be 
pharmacological agents. 
Solubilization of lipid soluble pharmacological agent-lipid suspension 
preparation in water is usually done with the help of solubilizing agents 
such as polyethylene glycols and propylene glycol, or via surfactants 
including such well known surfactants as polysorbates, poloxamers, and 
polyethoxylated castor oil. Upon administration, however, these agents may 
be present in concentrations sufficient to induce undesirable side 
effects. 
To avoid the use of such agents, D. Schmidt (U.S. Pat. No. 4,271,196) 
proposed colloidal suspensions formed by solubilization of lipids in 
ethanol, removal of the solvent by evaporation and addition of water or 
buffer with the drug added before water or in the colloidal suspension of 
lipids. Similarly, J. Schrank and H. Steffen (U.S. Pat. No. 4,411,894) 
solubilized both lipids and drug in ethanol, then ethanol was removed and 
buffer was added to form liposomes. 
These and other procedures involving the removal of ethanol and liposome 
formation have two major disadvantages. First, ethanol cannot solubilize 
certain lipids; in particular, salt forms of acidic, or basic phosphatides 
("charged phosphatides") such as phosphatidic acid, dicetylphosphate, 
phosphatidylethanolamine, and phosphatidylserine. Second, the entrapment 
of lipophilic drug in liposomes is limited such that the drug/lipid ratio 
(wt/wt) is usually less than 0.2. 
It is to be understood that neutral lipids are those which do not present a 
charge at neutral pH. Phospatidylcholine having a zwitterionic group is 
termed a neutral polar lipid and compounds such as cholesterol or 
triglycerides are nonionizable at physiological pH's and are termed 
neutral nonpolar lipids. 
To increase the efficacy of drug solubilization by the lipids, F. Tsunekazu 
et al. (European Pat. No. 0161445A1) proposed the solubilization of lipids 
and drug in an organic solvent, removal of the organic solvent, 
homogenization of the resulting film in aqueous solution by ultrasonic 
treatment, centrifugation of the suspension and recovery of the lower most 
layer of the sediment, to yield a particular drug-phospholipid complex. In 
this publication, particular reference is made to drugs having a molecular 
weight below 1,000. 
Lipid preparations such as liposomes carrying pharmacological agent-lipid 
solution agents are often characterized by having insufficient shelf life. 
Dried liposome preparations have been offered to overcome this problem 
however such preparations must be reconstituted at the time of use. 
Reconstitution may be associated with problems of clumping and uncertainty 
as to the liposomal size of the reconstituted preparation, and uncertainty 
as to the strength of an aliquot. These preparations are also associated 
with rapid sedimentation. 
It is an object of this invention to provide a pharmacological agent-lipid 
solution preparation in high drug to lipid ratio. 
It is a further object of this invention to provide a pharmacological 
agent-lipid solution preparation wherein the pharmacological agent is of a 
molecular weight of greater than about 1000. 
It is another object of this invention to provide a pharmacological 
agent-lipid solution preparation sterilizable by filtration. 
It is an additional object of this invention to provide a pharmacological 
agent-lipid solution preparation of lipophilic pharmacological agent. 
It is another object of this invention to provide a pharmacological 
agent-lipid solution preparation that will form a suspension of lipid 
associated with said pharmacological agent upon introduction into an 
aqueous medium and further that such suspension exhibit a stability of at 
about least 0.25 to 6 hours or longer without sedimentation and preferably 
at least about 2 hours. 
It is a further object of this invention to provide a method of forming 
such suspension. 
It is another object of this invention to provide a method of treating 
mammals, including humans, with therapeutically effective amounts of such 
suspension. 
SUMMARY OF THE INVENTION 
This invention comprises a pharmacological agent-lipid solution preparation 
comprising a lipophilic pharmacological agent, a desalted charged lipid 
and an aqueous miscible lipid solvent. This preparation, upon introduction 
into an aqueous medium, forms a suspension of lipid associated with the 
pharmacological agent. In some embodiments the pharmacological agent-lipid 
solution preparation may be in an oral dosage form such as a unit oral 
dosage form including tablets, capsules, dragees, and troches which is to 
include methods of treating subjects employing such dosage forms. The 
suspended lipid associated with pharmacological agent will be termed an 
aggregate. 
This invention further comprises such preparation wherein the desalted 
charged lipids are desalted charged phosphatides such as phosphatidic 
acid, dicetylphosphate, phosphatidylethanolamine, and phosphatidylserine. 
Further encompassed are pharmacological agent-lipid solution preparations 
which are both pharmaceutically acceptable and of limited flammability 
preferably by use of less volatile lipid solvents or by admixture of a 
first lipid solvent with secondary less flammable solvents such as 
polyethylene glycol (about 400-800 mw being preferred) and propylene 
glycol. Particularly preferred are preparations of at least about 10% 
(wt/wt) polyethylene glycol (about 800 mw) with about 30% most preferred. 
Additionally encompassed by this invention is the nonaqueous water-miscible 
lipid solvent being absolute ethanol. 
Further entailed in this invention is the lipid soluble pharmacological 
agent being an immunomodulator such as cyclosporin A; an anti-neoplastic 
such as doxorubicin; an antifungal such as miconazole, terconazole, and 
amphotericin B; an anti-inflammatory such as the steroidal 
anti-inflammatories prednisone, doxamethasone, fluoromethasone and the 
nonsteroidal anti-inflammatories such as indomethacin, salicylic acid 
acetate, ibuprofen; and the derivatives of muramyl dipeptide such as 
muramyl tripeptide phosphatidylethanolamine, and hormones such as 
glucocorticoids, mineralocorticoids and estrogens. 
Particularly included in this invention are anti-inflammatories in unit 
oral dosage form including tablet, capsule, dragee, or troche, and methods 
of treating subjects employing such dosage forms. 
Included in this invention is a preparation wherein the pharmacological 
agent is indomethacin is present from about 0.5% to about 30% by weight, 
and more particularly present at from about 10 to about 20% by weight, 
perticularly in unit dosage forms, and also wherein the lipid is 
additionally comprised of at least about 70% by weight 
phosphatidylcholine. 
In another embodiment polypeptides having a molecular weight of greater 
than about 1000, such as cyclosporin A or insulin, are the pharmacological 
agents. 
A further embodiment of the invention is the method of preparing a 
suspension from the pharmaceutical agent-lipid liquid solution preparation 
by adding the preparation to pharmaceutically acceptable aqueous medium. 
This is preferably added at a ratio of at least about 0.1:1 v/v. This 
method utilizes the pharmaceutical agent-lipid liquid preparations with 
all of the above noted lipids, solvents, and pharmaceutical agents. 
The aqueous media used in the method of preparation include water, 5% 
dextrose in water (wt/v), 0.9% saline, physiological phosphate buffer, and 
physiological citrate buffer. 
Yet further this invention encompasses suspensions of the pharmacological 
agent-lipid solution preparation in aqueous media wherein the 
pharmacological agent to lipid ratio is at least about 20 moles percent. 
This invention further includes a method of treating animals (including 
humans) in need of such treatment comprising the step of administering to 
the animal a therapeutically effective amount of pharmacological 
agent-lipid solution preparation added to a pharmaceutically acceptable 
aqueous medium thus forming a suspension. This administration is 
preferably parenteral, intramuscularly, intraperitioneally, intravenously, 
subcutaneously, or topically, via inhalation, or oral administration 
including suppository, or ingestion. The pharmacological agents of this 
method of administration will be any of those noted above and others. The 
lipids of this method of administration will be any of those noted above 
and others. 
An included method of treatment comprises treating animals (including 
humans) in need of such treatment comprising the step of administering to 
the animal a therapeutically effective amount of the pharmacological 
agent-lipid solution preparation such as by oral administration of such 
preparation, particularly in unit dosage form such as tablets, capsules, 
troches or dragees. 
Further included is a method of increasing the solubility of a lipid 
soluble pharmacological agent in lipid solvent (particularly ethanol) and 
lipid by the process of co-solubilizing said agent in a co-solution of 
lipid and lipid solvent. The invention includes the lipid comprising at 
least about 10% by weight of the ethanol:lipid co-solution. In one 
embodiment the lipid comprises phosphatidylcholine. The method also 
includes the agent being a nonsteroidal anti-inflammatory, such as 
indomethacin or salicylic acid acetate. 
Also included is pharmacological preparation comprising absolute ethanol, 
lipid, and nonsteroidal anti-inflammatory, particularly wherein the 
nonsteroidal anti-inflammatory is indomethacin, and wherein the 
indomethacin is present from about 0.5% to about 30% by weight and wherein 
the indomethacin is present at from about 10 to about 20% by weight.

DETAILED DESCRIPTION OF THE INVENTION 
The "pharmacological agent-lipid solution preparations" of this invention 
first comprise at least one lipophilic pharmacological agent, as well as a 
lipid solvent, and at least one lipid. Lipophilic (or lipid soluble) as 
defined herein includes along with true lipid solubility, the capacity to 
closely associate with lipids. 
Lipid soluble pharmacological agents include respiratory agents such as 
theophyllin, anti-epileptics such as diphenylhydantoin, anti-neoplastics 
such as doxorubicin; antifungals such as miconazole, terconazole and 
amphotericin B, (some antifungals will require desalting and or a 
acidification of the lipid solvent to increase solubility); 
immunomodulators such as cyclosporin A; derivatives of muramyl dipeptides 
such as muramyl tripeptide phosphatidylethanolamine; and, hormones such as 
glucocorticoids, mineralocorticoids and estrogens; anti-inflammatories 
such as the steroidals, prednisone, dexamethasone and fluromethasone and 
the nonsteroidals such as indomethacin, salicylic acid acetate and 
ibuprofen, further including analgesic agents such as acemetacin and 
flurobiprofen, and other agents such as lipoxygenase inhibitors, 
prostaglandins, neuroleptics, antidepressants, fat-soluble vitamins, 
contrast materials and antivirals. 
Other preferred nonsteroidal anti-inflammatories are: 
carboxylic acids 
salicylates 
Acetylsalicylic Acid (i.e., Salicyclic Acid Acetate) 
Salsalate 
Diflunisal 
Fendosal 
Acetic Acids 
Indomethacin 
Acemetacin 
Cinmetacin 
Sulindac 
Tolmetin 
Zomepirac 
Diclofenac 
Fenclofenac 
Isoxepac 
Furofenac 
Fentiazac 
Clidanac 
Oxepinac 
Fenclorac 
Lonazolac 
Metiazinic Acid 
Clopirac 
Amfenac 
Benzofenac 
Clometacine 
Etodolac 
Bumidazone 
Clamidoxic Acid 
Propionic Acids 
Ibuprofen 
Flurobiprofen 
Naproxen 
Ketoprofen 
Fenoprofen 
Benoxaprofen 
Indoprofen 
Pirprofen 
Carprofen 
Oxaprozin 
Pranoprofen 
Suprofen 
Microprofen 
Tioxaprofen 
Alminoprofen 
Cicloprofen 
Tiaprofenic Acid 
Furaprofen 
Butibufen 
Fenbufen 
Furobufen 
Bucloxic Acid 
Protizinic Acid 
Fenamates 
Mefanamic Acid 
Flufenamic Acid 
Meclofenamate 
Niflumic Acid 
Tolfenamic Acid 
Flunixin 
Clonixin 
Pyrazoles 
Phenylbutazone and Analogs 
Peprazone (Prenazone) 
Apazone (Azapropazone) 
Trimethazone 
Mofebutazone 
Kebuzone 
Suxibuzone 
Oxicams 
Piroxicam 
Isoxicam 
Tenoxicam 
Indomethacin is a most preferred nonsteroidal anti-inflammatory. In the 
preparations of this invention, indomethacin preferably comprises from 
about 0.5% to about 30% of the final weight of the pharmacological 
agent-lipid solution preparation, and particularly from about 10 to about 
20%, and most particularly about 15%. 
It is a limitation of this invention that at least one lipid be charged and 
desalted, and such desalted charged lipids are (a) soluble in the water 
miscible lipid solvents of this invention and (b) exhibit only a limited 
tendency for sedimentation upon dispersal in the aqueous medium of 
suspension formation. 
Lipid materials used in this invention are amphipathic in character. 
Amphipathic as defined herein is a moiety with a hydrophobic portion and a 
hydrophilic portion. Hydrophilic character will be imparted to a molecule 
through the presence of phosphatidic, carboxylic, sulphatic, amino, 
sulfhydryl, nitro, and other groups such as carbohydrates. Hydrophobicity 
will be conferred by the inclusion of groups that include, but are not 
limited to, long straight and branched chain saturated and unsaturated 
aliphatic hydrocarbon groups and such groups substituted by one or more 
aromatic, cycloaliphatic or heterocyclic group. The preferred amphipathic 
compounds are phosphoglycerides, representative examples of which include 
phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylcholines, 
lysophosphatidylethanoloamines, phosphatidylserines, 
phosphytidylinositols, phosphatidic acids, phosphatidylglycerols and 
diphosphatidylglycerols as well as sphingomyelins. Synthetic saturated 
compounds such as dimyristoylphosphatidylcholine and 
dimyristoylphosphatidylglycerol or unsaturated species such as 
dioleoylphosphatidylcholine or dilinoleoylphospatidylcholine are also 
usable. Other compounds lacking phosphorous, such as members of the 
glycolipids, and glycosphingolipid, ganglioside and cerebroside families, 
are also within the group designated as amphipathic lipids. Salts of acid 
derivatives of sterols and tocopherols such as cholesterol or tocopherol 
hemisuccinate are also amphipathic. Ionic detergents such as 
octadecanylsulfonate are also included. 
Salts of acidic or basic lipids (i.e., charged lipids) that otherwise are 
not soluble in ethanol can be rendered soluble by desalting, that is by 
removal of the counterion. For example phosphatidic acid, 
phosphatidylserine, dicetylphosphate, phosphatidylglycerol and 
phosphatidylethanolamine may be desalted. Natural soy or egg phosphatides 
may be desalted and the resulting desalted mixture of various lipids will 
contain sufficient desalted charged lipids in the form of acidic 
phospholipids for use in this invention. Thus in the practice of this 
invention, the presence of an amount of neutral lipids, polar or nonpolar, 
along with desalted charged lipids will not adversely affect the 
preparation. The tolerable amount of neutral lipid is limited by the 
solubility of the various lipids in water-miscible lipid solvent system 
and the required stability of the suspension formed upon mixture with the 
aqueous medium. Thus, a critical element of this invention is the presence 
of a desalted charged lipid. The minimum amount of desalted charged lipid 
will vary with the system. However, at minimum at least sufficient 
desalted charged lipid must be present to form a stable suspension. Each 
system will present a different minimum amount of charged lipid necessary 
for stability but, by way of example, desalted phosphatidic acid will be 
effective in a presence as low as about 0.5 mole percent relative to total 
lipid in a cyclosporin A-ethanol system. The desirable amount of desalted 
charged lipid for other systems is easily determined with reference to the 
solubility of proposed lipid in the solvent system and the required 
stability of the final aqueous suspension. 
Desalting of lipids is accomplished by exchanging the counterion from the 
acidic or basic moiety of the amphipathic lipid for a proton or hydroxide, 
respectively. This is done by any of a number of methods well known in the 
art including ion exchange resin column elution. 
The typical ion exchange resin procedure employs commercially available 
resins such as those of the Biorad Company of Richmond, Va. A typical 
cation exchange resin is Biorad AG50-X8 and Biorad AG1-X8 is a typical 
anion exchange resin. These procedures, performed in the aqueous-soluble 
lipid solvent itself, are relatively insensitive to temperature and 
pressure and are conveniently performed at ambient or room temperature 
(i.e., about 22.5.degree. C. +/- about 2.5.degree. C.) and atmospheric 
pressure. 
Lipids used in this invention are obtainable from a number of sources. 
Natural phosphatide mixtures from egg or soy containing more than 70% 
phosphatidylcholine are obtained from a number of commercial sources such 
as Sigma Chemical of St. Louis, MO, and Lipoid KG, Ludwigshafen, West 
Ger., Hepar of Franklin, Ohio. Hepar supplies egg phosphatidylcholine. 
Other sources of lipid such as soy phosphatidylcholine are American 
Lecithin, Woodside, L.I., NY, and Riceland Foods, Little Rock, Ark. 
Phosphatidic acid of 99% purity is obtained from Avanti Chemical of 
Birmingham, AL. 
A method for desalting utilizes trichlorofluromethane (CCl.sub.3 F) (E.I. 
du Pont de Nemours & Co., Wilmington, Del., under the trademark Freon 11). 
In this method phosphatides are added to a mixture of absolute ethanol and 
CCl.sub.3 F at a ratio of from about 0.5:1 to 1:0.5 with 1:1 being most 
preferred to form a solution. A temperature of 15.degree. C. is preferred 
at atmospheric pressure but any temperature below the boiling point of 
CCl.sub.3 F at the operating pressure is suitable, particularly 
20.degree.-35.degree. C. under pressure. 
About 5 g of phosphatide may be added to about 40 ml of the CCl.sub.3 
F/ethanol mixture but this proportion may be increased and is limited only 
by the formation of emulsion upon making of two phases. Up to about a 20 
wt % mixture of phosphatide:CCl.sub.3 F/ethanol solvent may be used with 
about 1-10 wt % being preferred. The resulting solution is titrated with a 
slight excess of dilute acid such as HCl and the solution is mixed by any 
convenient method including stirring, shaking and sonication. The slight 
emulsion formed is permitted to separate and usually this requires only a 
matter of minutes. 
The CCl.sub.3 F layer is removed and an ethanol:water (about 2:1-1:2 
ethanol:water v/v) mixture is then added to the CCl.sub.3 F solution for 
repeated washing and removal of excess acid, until the upper phase is 
neutral. The lower CCl.sub.3 F solution is then warmed to about room 
temperature (i.e., about 22.5.degree. C. +/- about 2.5.degree. C.) to 
evaporate the CCl.sub.3 F leaving the desalted lipid residue. Care is 
required in warming so that frothing does not occur. The solvent is then 
removed. This is conveniently accomplished first by evaporation with a 
thin stream of nitrogen at about 22.5.degree. C. +/- about 2.5.degree. C. 
and then by rotoevaporation. Again care is taken so that frothing/bumping 
does not occur. 
The lipid solvents of this invention must be (1) dissolving of lipids, (2) 
substantially soluble (termed herein "miscible") in water, and (3) 
pharmaceutically acceptable. As the lipid solvent will only appear in the 
administered dose upon dilution by the aqueous phase and the dilution 
would ordinarily constitute about a 5 to 50 times reduction in lipid 
solvent concentration a number of pharmaceutically acceptable lipid 
solvents are available. These include ethanol, polyethylene glycol and 
propylene glycol. The preferred polyethylene glycols have molecular 
weights of about 400 to 800 with about 800 most preferred. Absolute 
ethanol is the preferred lipid solvent, but any pharmaceutically 
acceptable lipid solvent may be used. 
The lipid solvent must be miscible or at least significantly soluble with 
the aqueous solution in order to deliver simultaneously both the 
pharmacological agent and lipid to this solution as well as for the 
purpose of diluting the lipid solvent in the aqueous solution. 
The lipid solvents for the solution preparation must be substantially water 
free but water miscible. The presence of excess water in the preparation 
will cause the lipophilic pharmacological agents to be insoluble and can 
adversely affect the storage stability of the preparation through 
hydrolysis. The maximum amount of water will vary with the specific agent 
but generally will be less than about 1% and not greater than about 5% or 
10% (w/w). In practice the maximum amount of water tolerable in a system 
is easily determined in that if excess water is present the solution 
becomes cloudy indicating the presence of precipitate or liposomes. 
Certain pharmacological agents susceptable to hydrolysis such as salicylic 
acid acetate do not tolerate the presence of water even at about 5% in 
ethanol, though lesser amounts of water are tolerable in this system. 
Substantially water-free lipid solvents wherein the pharmacological 
agent-lipid is not appreciably hydrolized or rendered insoluble will be 
termed nonaqueous. 
Due to the high flammability of absolute ethanol, admixing with at least 
about 10% lipid solvent diluent such as polyethylene glycol 400 or 800 is 
preferred and up to about 30% polyethylene glycol 800 most preferred in 
reducing flammability of the preparation while maintaining pharmaceutical 
acceptability. Other weights of polyethylene glycol and other lipid 
solvent diluents are also acceptable. 
Sterility of drug-lipid solution is necessary both for a prolonged shelf 
life as well as subsequent use of this solution. Therefore, drug-lipid 
solution is conveniently terminally sterilized by filtration. This is 
preferably done through a 0.2 micron polycarbonate filter, 
cellulose-containing filter or other inert filter that does not interact 
either with lipid solvent or with the solubilized drug or lipid. 
Filtration also removes undissolved particles from the preparation. 
Sterilization by filtration is a particular advantage of this preparation. 
Storage stability of the pharmocological agent-lipid solution preparation 
will vary but is directly related to the stability of the lipids. The 
storage stability is enhanced by storing at reduced temperatures. 
The pharmacological agent-lipid solution may be advantageously employed by 
direct administration wherein a suspension will be formed in vivo wherein 
the aqueous medium is the physiological environment. In such application a 
pharmacological agent-lipid solution, such as indomethacin mixed with a 
non-aqueous water-miscible lipid solvent such as ethanol and a desalted 
charged lipid, when ingested, conveniently in capsule or liquid form, 
becomes liposomal in the gastric environment. Other suitable oral dosage 
forms are dragees, troches, lozenges, tablets and additional forms known 
to those skilled in the art. Oral dosage forms configured and adapted for 
oral administration to subjects in need of such dosages shall be termed 
unit oral dosage forms. 
Aggregate suspensions prepared from the pharmacological agent-lipid 
solution preparation are made by agitating the preparation in an aqueous 
medium. Agitation is accomplished by any convenient method, but is most 
easily accomplished by loading the solution preparation into a syringe and 
then injecting the preparation into the aqueous medium as contained within 
an ampoule or container. The exact rate of injection is not critical. 
Injection of the solution preparation should be accompanied by rapid 
mixing such that the water miscible lipid solvent and the aqueous medium 
rapidly mix. Beyond the syringe method other convenient methods of 
preparing the suspension are pouring, dropping, or spraying in while hand 
mixing, vortexing, stirring or sonicating. 
Any pharmaceutically acceptable aqueous medium may be used. Examples of 
suitable aqueous media are water, 5% dextrose in water, physiological 
citrate buffer, physiological phosphate buffer and 0.9% saline. As used 
here in referring to physiological buffers indicates pharmaceutical 
acceptability in view of the intended use in animals such as mammmals 
(including humans). The use of such medium will be both for formation of 
the suspension and as a pharmaceutical diluent. For oral administration 
the preferred aqueous medium is water or palatable fluids such as fruit 
juices and syrups or infusions such as teas and coffee. 
At moderate pharmacological agent to lipid ratios pharmacological 
agent-lipid solution will generally form aggregates in the structure of 
liposomes upon mixing with aqueous medium. 
At higher pharmacological agent to lipid ratios, the aggregate structure 
becomes nonliposomal. For example, at pharmacological agent to lipid 
ratios of about 1:1 (wt/wt), the aggregates are spherical particles of 
about 0.2 .mu.m or higher, have minimal entrapped water, appear to be 
temporarily closely associated with the water miscible lipid solvent, and 
upon centrifugation appear denser than liposomes of similar 
pharmacological agent and lipid composition. Higher pharmacological agent 
to lipid ratios will be understood to mean from about 20 moles percent 
pharmacological agent to lipid ratio up to about 60 moles percent or more. 
The formation of aggregates upon formation of suspension is strongly 
related to the pharmacological agent/lipid ratio (wt/wt) of the solution 
preparations. Examination of this ratio was done from about 10:1 to about 
0.5:1. In general, aggregate diameter and suspension opacity decreased as 
less lipid in relation to pharmacological agent was utilized. 
At the higher pharmacological agent to lipid ratios, the aggregates were of 
submicron size and the suspensions colloidal, thus the physical parameters 
of the suspenson are adjustable by varying the ratios of the 
pharmacological agent to lipid. 
Preparations of pharmacological agent-lipid solution such as those with 
cyclosporin A used in this invention are preferably begun by the 
dissolving of the agent and/or the lipid into lipid solvent. While this 
can conveniently be accomplished at about 22.5.degree. C.+/- about 
2.5.degree. C., using a heated water bath, facilitates dissolving. For 
cyclosporin A, a water bath at about 40.degree. C.-50.degree. C. 
facilitates the dissolution. 
Generally, lipids and pharmacological agent are separately solubilized into 
lipid solvent as stock solutions at convenient concentrations. Stock 
solutions can be maintained at convenient and nondegrading temperature, 
for example 4.degree. C. When preparing a particular pharmacological 
agent-lipid solution preparation of the present invention appropriate 
aliquots of stock solutions were combined to achieve desired final 
concentrations of lipid and agent. 
However, it is quite acceptable to add pharmacological agent and lipid 
directly to the lipid solvent of a preparation. In such circumstance it is 
preferable to add the lipid to the lipid solvent first as the lipid in 
some circumstances increases the solubility of a pharmacological agent. 
This co-solubilizing or "salting-in" may be up to about a 50% increase in 
solubility or apparent solubility with agents such as salicylic acid 
acetate and indomethacin. In the context of salting in the term "apparent 
solubility" recognizes that a pharmacological agent salted in may be in 
the form of a complex with dissolved lipid rather than truly solubilized, 
such as is the case with amphotericin B. 
The co-solubilizing or salting in is a surprising aspect of this invention 
as to pharmacological agents that will associate with lipid. Lipid soluble 
pharmacological agents include the nonsteroidal anti-inflammatories such 
as salicylic acid acetate and indomethacin as noted above. To practice 
this aspect of the invention requires dissolving lipid in the lipid 
solvent, such as ethanol, forming a co-solution prior to addition of the 
pharmacological agent to be co-solubilized. From about 10% lipid by weight 
up to the solubility limit of the particular lipid (or lipids) in the 
lipid solvent may be used as the solution in which to co-solubilize the 
lipid soluble (or apparently soluble) pharmacological agent. 
After the solution of lipid and lipid solvent is made diluents such as 
polyethylene glycol (which may also be a lipid solvent) can be added to 
reduce flammability. Antioxidants may also be added then. The final 
pharmacological agent-lipid solution preparation is conveniently stored in 
an ampoule and preferably at about 4.degree. C. 
The pharmacological agent-lipid solution preparation concentration of 
constituents will only be limited by the relative solubility of such 
constituents with a view to the desired final concentration. A typical 
preparation will be comprised of up to about 0.5 gm of drug/ml of lipid 
solvent with about 0.5 gm of lipid. 
In the preferred pharmacological agent-lipid solution preparation storage 
stability is enhanced by the inclusion of an antioxidant such as 
alpha-tocopherol. This is present in amounts at about 0.1 to about 1% 
(wt/wt) relative to the amount of lipid. 
To determine whether phospholipids can increase the solubility or apparent 
solubility of a pharmacological agent such as salicylic acid acetate, both 
drug and lipid were co-solubilized in a lipid solvent such as absolute 
ethanol (USP). Five ml of this lipid solvent was able to dissolve about a 
maximum of 1 gr. salicylic acid acetate. A lipid such as egg 
phosphatidylcholine was completely dissolved in 5 ml of ethanol in a 
series of test tubes and to this solution crystals of the test 
pharmacological agent salicylic acid acetate were added gradually and 
dissolved. The maximum amount of salicylic acid acetate dissolved in 5 ml 
ethanol containing 1.5 gm egg phosphatidylcholine was about 1.5 gm 
indicating about a 50% increase in solubility of drug under these 
conditions. Any convenient temperature and pressur may be used for this 
procedure that will dot adversely affect the pharmacological agent or boil 
the lipid solvent. 
This "salting in" illustrates for one skilled in the art the use of lipids 
for increasing the solubility of lipid soluble drugs up to 50% or more. 
Salicylic acid acetate solubility in an egg phosphatidylcholine ethanol is 
seen to increase about 50% and the solubility of indomethacin in a similar 
system is seen to increase about 50%. Amphoteracin B is similarly salted 
in but appears to do so in the form of complexes with lipid rather than 
simple solubilizing. 
A number of analytical steps known in the art are useful in selecting those 
lipid/lipid solvent systems which upon mixture with an aqueous medium, 
generate pharmaceutically acceptable suspensions. Such analytical steps 
include assessment by visual inspection for appearance, opacity and the 
presence of crystals, precipitates or sediment; (b) light microscopic 
examination such as in a Neubauer cytometer with a micrometer scale at 100 
times and 400 times magnification; (c) turbidimetric measurements by 
assessing transmission at 520 nm; (d) electron microscopy of negatively 
stained preparations; (e) quasielastic light scattering (QELS) for 
determination of mean particle dimension; (f) ultracentrifugation; (g) 
organ distribution after intravenous innoculation of aggregate suspension 
having aggregates labeled with a reporting group such as a radioactive 
tracer, (e.g., .sup.3 H-cyclosporin when using cyclosporin); and (h) 
bioactivity in cell culture of a particular cell type (e.g., for 
cyclosporin, spleen lymphocytes stimulated with concanavaline A and 
labeled with .sup.3 H-thymidine). 
In addition, such suspensions must be without large aggregations, 
precipitates or crystals. The suspension must remain free of large 
aggregations precipitates or crystals during the time necessary for 
preparing and administering injections under hospital conditions. This 
time was presumed to be at minimum from about 15 minutes to about two 
hours. For the suspensions intended for intravenous administration, 
selection for small aggregate size is necessary as suspensions containing 
bodies, such as aggregates, over 5 microns in diameter are not usually 
suitable for intravenous administration. Thus, those skilled in the art 
may rapidly define a lipid solvent system suspension suitable for internal 
and particularly intravenous administration. 
The results of organ distribution of aggregates showed that the aggregates 
do not accumulate in liver and spleen as would be predicted for liposomes. 
The pharmacological agent-lipid solution preparations of this invention are 
useful for treating animals (including humans) in need of such treatment. 
Treatment as used herein includes administration of any pharmacological 
agents such as diagnostic materials, biologically active agents and 
contrast materials. 
Treatment is frequently accomplished by preparing a suspension from the 
solution preparation and administering the suspension in therapeutically 
effective amounts. However, as noted, the pharmacological agent-lipid 
solution may be directly administered for treating mammals. A 
therapeutically effective amount will be understood to mean a sufficient 
amount to achieve a physical or physiological response, and for known 
drugs will generally be the same dose for the existing dosage forms of the 
drug. 
The therapeutically effective amount of a given pharmacological agent will 
vary with the purpose of the administration, the particularities of the 
recipient and other factors well known in the art. 
In a liposome-drug delivery system, a pharmacological agent such as a drug 
is entrapped in or associated with the liposome and then administered to 
the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 
3,993,754; Sears, U.S. Pat. No. 4,145,410; Paphadjopoulos et al., U.S. 
Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,114,179; Lenk et al., U.S. 
Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. 
The mode of administration of the preparation may determine the sites and 
cells in the organism to which the compound will be delivered. Aggergates 
of this invention can be administered alone but will generally be 
administered in admixture with a pharmaceutical carrier selected with 
regard to the intended route of administration and standard pharmaceutical 
practice. The preparations may be injected parenterally, for example, 
intra-arterically or intravenously. The preparations may also be 
administered via oral, subcutaneous, or intramuscular routes, or by 
inhalation. For parenteral administration, they can be used, for example, 
in the form of a sterile aqueous solution which may contain other solutes, 
for example, enough salts or glucose to make the solution isotonic. Other 
uses, depending upon the particular properties of the preparation, may be 
envisioned by those skilled in the art. 
For administration to humans in the curative treatment of disease states, 
the prescribing physician will ultimately determine the appropriate dosage 
for a given human subject, and this can be expected to vary according to 
the age, weight, and response of the individual as well as the nature and 
severity of the patient's disease. The dosage of the drug in aggregate 
form will generally be about that employed for the free drug. In some 
cases, however, it may be necessary to administer doses outside these 
limits. 
This invention will be better understood by reference to the following 
examples which are merely illustrative of the invention. 
EXAMPLE 1 
Cyclosporin A Solution Preparation:Desalted Lipids 
Stock solution of cyclosporin A (Sandoz Pharmaceuticals Corporation, East 
Hanover, NJ) at 200 mg/ml and stock solution of desalted egg phosphatides 
(Hepar Industrial, Inc., Franklin, Ohio) at 250 mg/ml were prepared in 
advance and kept at 4.degree. C. To prepare 10 ml of cyclosporin-lipid 
solution 2 ml of lipid stock solution was brought up to 8.75 ml with 
absolute ethanol and mixed by hand at about 22.5.degree. C.+/- about 
2.5.degree. C. and atmospheric pressure. To this solution 1.25 ml of 
cyclosporin A stock was added and mixed again. The final solution 
containing cyclosporin 25 mg/ml and desalted egg phosphatides 50 mg/ml was 
filtered through 0.2 micron polycarbonate filter. The solution was then 
bubbled through with oxygen free nitrogen for 10 seconds, overlayed with 
nitrogen and tightly enclosed. 
EXAMPLE 2 
Cyclosporin A Solution Preparation:Desalted and Neutral Lipids 
1.25 gm of dry powdered egg yolk phosphatides (nondesalted) (Hepar) were 
dissolved in 2 ml absolute ethanol by heating at 56.degree. C. for 10 min. 
in a water bath. The solution was then cooled in a ice basket and filtered 
through a 1.0 micron polycarbonate filter (Nucleopore, Pleasanton, CA). 
The resulting lipid solution was adjusted to a concentration of 400 mg 
lipids/ml with absolute ethanol. Desalted phosphatidic acid was dissolved 
in ethanol at a concentration of 200 mg/ml. 0.63 ml of the egg yolk 
phosphatide solution was mixed with 0.63 ml of a cyclosporin A-ethanol 
solution containing 200 mg cyclosporin/ml drug to which was then added 
0.06 ml of desalted phosphatidic acid ethanol solution followed by mixing. 
All mixing steps took only a matter of minutes. The resulting mixture was 
adjusted to 5 ml with absolute ethanol to a final concentration of 50 mg 
egg phosphatides, 6.25 mg desalted phosphatidic acid and 25 mg cyclosporin 
A per ml ethanol. This solution was sterilized by filtration through 0.2 
micron polycarbonate filter (Nucleopore, Pleasanton, CA), bubbled through 
with oxygen-free nitrogen and sealed in an ampoule. 
EXAMPLE 3 
Cyclosporin A Solution Preparation and Suspension 
Soy phosphatidylcholine after removal of absolute ethanol insoluble 
impurities and being desalted was dissolved in absolute ethanol at about 
22.5.degree. C.+/- about 2.5.degree. C. and atmosphereic pressure at a 
concentration of 382 mg/ml. Cyclosporin A was dissolved in a separate 
aliquot of absolute ethanol at a concentration of 200 mg/ml. 
The cyclosporin A-lipid solution was prepared by adding sequentially in a 
glass tube 0.327 ml lipid solution (125 mg), 1.25 ml cyclosporin A 
Solution (250 mg), absolute ethanol 1.87 ml (1.47 gm) and 6.50 ml (7.34 
gm) polyethylene glycol 400. 
After each addition solutions were briefly shaken and the final solution 
was vortexed. This solution contained 50 mg cyclosporin A, and 25 mg lipid 
in 1 ml of 65% polyethylene glycol 400 in absolute ethanol. 
A suspension was formed by adding 20 mg of this solution by injection into 
500 ml 5% dextrose in water. The aggregates in the suspension thus formed 
were 1.0 micron or smaller. 
The aggregates including the aggregates in suspension were assessed by: (a) 
visual inspection for appearance, opacity and presence of concretions, 
crystals, precipitates or sediment; (b) light microscopic examination in a 
Neubauer cytometer with a micrometer scale at 100 times and 400 times 
magnification; (c) turbidimetric measurements by assessing transmission at 
520 nm; (d) electron microscopy of negatively stained preparations; (e) 
quasielastic light scattering (QELS) for determination of mean particle 
dimension; (f) ultracentrifugation; (g) organ distribution after 
intravenous innoculation of aggregate suspension having aggregates labeled 
with .sup.3 H-cyclosporin; and (h) bioactivity in cell culture of spleen 
lymphocytes stimulated with concanavaline A and labeled with .sup.3 
H-thymidine. 
QELS analysis indicated that the mean diameter of aggregates was below 0.3 
microns (Table 1). Turbidimetric measurements of suspensions kept at about 
22.5.degree. C.+/- about 2.5.degree. C. without agitation showed that the 
transmission at 520 nm gave similar value at "zero" time (shortly after 
suspension was formed) and at subsequent time points (FIG. 1) indicating a 
rapidly forming and stable suspension. 
Electron microscopy revealed round droplet-like particles having a diameter 
below 1.0 micron in agreement with light microscopy examination and QELS 
results (FIG. 2). All three measurements clearly showed that the dimension 
of aggregates can be modulated by changing the pharmaceutical agent/lipid 
ratio and the nature of the lipids used. 
Ultracentrifugation analysis showed that the aggregates sediment when the 
suspension is placed over a Hypaque-Ficoll solution diluted one-third with 
distilled water and having a density higher than 1.0. Under the same 
preparation and centrifugation conditions, liposomes formed by dispersion 
of lipids alone in aqueous medium, do not sediment. 
EXAMPLE 4 
Cyclosporin A Suspension 
A sample for parenteral administration from the solution of Example 1 was 
prepared by taking the content of an ampuole (10 ml ethanol containing 500 
mg egg phosphatides which were desalted and 250 mg cyclosporin A with a 
#23G needle (1.5 in) adapted to a 10 ml syringe. The needle was inserted 
through the rubber stopper of a 500 ml bottle containing 5% dextrose in 
water. The bottle was kept upside down and mixed by hand to create a 
vortex, while the contents of the syringe was infused over 20 seconds 
continuously into the aqueous solution. After injection was completed, the 
needle was retracted and the bottle shaken by hand for 10 sec. The 
resulting aggregates, the cyclosporin A-lipid suspension, was allowed to 
stand for 15 minutes to allow disappearance of the gas bubbles formed 
during shaking. This suspension was intended for use within 15 min. to 6 
hrs after preparation. 
EXAMPLE 5 
Cyclosporin Behavior in vivo 
Cyclosporin A aggregates were slightly more efficacious in suppressing 
lymphocyte proliferation as measured by .sup.3 H-thymidine uptake, than 
the cyclosporin solubilized in polyethoxylated castor oil and ethanol. 
The dimensions of the nonliposomal lipid aggregates wherein the 
pharmacological agent is cyclosporin A are relatively uniform of a size 
about 250 nm +/- 20 nm. 
The results of organ distribution of aggregates showed that the aggregates 
do not accumulate in liver and spleen as would be predicted for liposomes. 
EXAMPLE 6 
Aspirin Solution Preparation 
Desalted egg phosphatides (Hepar) were dissolved in ethanol at a 
concentration of 250 mg/ml in a 1000 ml flask. To 400 mg of salicylic acid 
acetate, 1.6 ml of the above solution containing 400 mg of phosphatides 
was added and the solution adjusted with absolute ethanol to 4 ml. The 
final concentration of both ingredients in this pharmacological 
agent-lipid solution was 100 mg/ml. This procedure was performed at about 
22.5.degree. C.+/- about 2.5.degree. C. and atmospheric pressure. 
EXAMPLE 7 
Aspirin Suspension 
To prepare a suspension, desalted egg phosphatides (Hepar) were dissolved 
in ethanol at a concentration of 250 mg/ml in a 1000 ml flask. To 400 mg 
of salicylic acid acetate, 1.6 ml of the above solution containing 400 mg 
of phosphatides was added and the solution adjusted with absolute ethanol 
to 4 ml. The final concentration of both ingredients in this 
pharmacological agent-lipid solution was 100 mg/ml. This procedure was 
performed at about 22.5.degree. C.+/- about 2.5.degree. C. and atmospheric 
pressure. Then 0.5 ml of the salicylic acid acetate-lipid solution was 
added to 9.5 ml of distilled water and briefly shaken by hand, at about 
22.5.degree. C.+/- about 2.5.degree. C. and atmospheric pressure forming a 
suspension. The resulting suspension was milky in appearance and did not 
contain visable crystals or aggregates after a 30 minute period. Light 
microscopy of the suspension revealed aggregates, primarily with a 
diameter below 5 microns and no crystal characteristic for salicylic acid 
acetate. This suspension contained 0.5 mg salicylic acid acetate/ml. 
EXAMPLE 8 
Topical/Oral Aspirin Suspension 
A dosage form of salicylic acid acetate for topical or oral use was 
prepared as follows: Desalted egg phosphatides (Hepar) were dissolved in 
absolute ethanol at a concentration of 250 mg/ml in a 1000 ml flask. To 
400 mg of salicylic acid acetate, 1.6 ml of the above solution containing 
400 mg of phosphatides was added and the solution adjusted with absolute 
ethanol to 4 ml. The final concentration of both ingredients in this 
pharmacological agent-lipid solution was 100 mg/ml. This procedure was 
performed at about 22.5.degree. C.+/- about 2.5.degree. C. and atmospheric 
pressure. Next, the topical/oral administration dosage form was prepared 
by adding the pharmacological agent-lipid solution, to water at about 
22.5.degree. C.+/- about 2.5.degree. C. and briefly agitating the mixture. 
A cloudy suspension promptly formed. This salicylic acid acetate 
formulation may then be ingested or used topically. 
EXAMPLE 9 
Indomethacin Preparation 
15 g of egg phosphatides (Lipoid E80, Lipoid KG, Ludwigshafen, West Ger.) 
containing 80% phosphatidylcholine was solubilized in 3 ml absolute 
ethanol. The resulting co-mixture then solubilized indomethacin, 1 g of 
which was then added to the co-mixture. The final preparation contained 25 
mg of indomethacin and 375 mg of lipid per 0.4 ml. This precedure was 
performed at about 22.5.degree. C.+/- about 2.5.degree. C. at and 
atmospheric pressure. The ethanol concentration of the preparation was 
0.075 ml/0.4 ml. The preparation was encapsulated in a soft gelatin 
capsule as a unit oral dosage form. 
PREATION 1 
Desalting of Lipids: Hexane-Ethanol-Hydrochloric Acid Procedure 
Natural phosphatide mixtures from soy containing more than 70% 
phosphatidylcholine (PC) were dissolved in hexane at 1 gm lipid/10 ml 
solvent. To this solution 6.6 ml of absolute ethanol and 3.3 ml of 0.2N 
HCl was added and mixed thoroughly at atmospheric pressure and at about 
22.5.degree. C.+/- about 2.5.degree. C. Phase formed and were permitted to 
separate and the lower aqueous phase discarded. The hexane phase was 
repeatedly washed with ethanol-water, 1:1 (v/v) until the pH in the lower 
aqueous phase was neutral. The resulting desalted lipids were recovered 
from the hexane phase by removal of the hexane by rotoevaporation at 
35.degree. C. and 100 mm Hg. 
PREATION 2 
Desalting of Lipids:CCl.sub.3 F 
At atmospheric pressure and in a cold room (4.degree.-10.degree. C.) 5 
grams of egg phosphatides (Hepar) were dissolved in a mixture of absolute 
ethanol:CCl.sub.3 F (Freon 11, du Pont), 1:1 (40 ml) at 15.degree. C.; 25 
ml of 0.5N aqueous HCl was added and the mixture shaken The lower 
CCl.sub.3 F phase was removed after the emulsion was broken and mixed with 
20 ml of absolute ethanol and 25 ml of water. The CCl.sub.3 F lower phase 
was again removed and the ethanol/water wash was repeated until the upper 
aqueous phase was neutral. The lower CCl.sub.3 F solution was allowed to 
warm to about 22.5.degree. C.+/- about 2.5.degree. C. and the solvent 
driven off with a stream of nitrogen and finally, on a rotoevaporator. 
Yield=4.9 grams of desalted lipids. 
PREATION 3 
Desalted Lipids:Ionic Exchange Resin 
20 gm of egg phosphatides (Hepar) dissolved in 100 ml of absolute ethanol 
was passed through 200 g of the cation exchange resin (Biorad of Richmond, 
Va) (AG 50 WX8) in the hydrogen form and in ethanol. The column was 
further diluted with 50 ml of ethanol, at about 22.5.degree. C.+/- about 
2.5.degree. C. and atmospheric pressure. The total eluant of the first 
volume was passed through 200 g column of anion exchange resin (Biorad 
AG1-X8) in the hydroxyl form and in ethanol. The columns were washed with 
50 ml of absolute ethanol and the total 200 ml of eluant contained 
desalted phospholipids at a concentration of 10 g/100 ml of ethanol. This 
was useable directly or diluted further with ethanol. 
PREATION 4 
Salting In 
Five ml of absolute ethanol was able to dissolve maximum 1 gr. salicylic 
acid acetate. Egg phosphatidylcholine (Hepar) 1.5 gm was completely 
dissolved in 5 ml of ethanol in a series of test tubes and to this 
solution crystals of salicylic acid acetate were added gradually and 
dissolved. The maximum amount of salicylic acid acetate dissolved in 5 ml 
ethanol containing 1.5 gm egg phosphatidylcholine was 1.5 gm indicating a 
50% increase in solubility of drug. To accelerate the dissolving process 
all test tubes containing crystals of salicylic acid acetate and lipid 
solvent were agitated gently in a water bath of 40.degree. C. and cooled 
to about 22.5.degree. C.+/- about 2.5.degree. C. after crystals were 
completely dissolved. 
TABLE 1 
______________________________________ 
QELS ANALYSIS OF AGGREGATE DIMENSION 
MEAN DIAMETER (nm) 
SAMPLE NICOMP ANALYSIS* 
GAUSSIAN ANALYSIS 
______________________________________ 
HDrrC4-33 
129.0 150.0 
HDrrC5-34 
183.00 144.0 
HDrrC6-35 
258.00 176.7 
HDrrC7-36 
209.00 143.4 
JDrrC1-37 
166.0 151.4 
.sup.-- X .+-. S: 
189.0 .+-. 48 
153.0 .+-. 13 
______________________________________ 
*NICOMP analysis is a data reduction extracting the component sizes 
contributing to the exponential curve.