Encapsulated ionophore growth factors

Encapsulated ionophore growth factors and methods of encapsulating these growth factors in lipid vesicles, particularly nonphospholipid paucilamellar lipid vesicles, have been discovered. These methods allow aqueous-based formulations of water-insoluble growth factors to be made.

BACKGROUND OF THE INVENTION 
The present invention relates to aqueous-based formulations of ionophore 
growth factors. More particularly, disclosed are encapsulated ionophore 
growth factors, a method of their production, and methods of treatment 
using encapsulated ionophores. The ionophore growth factors are 
encapsulated within nonphospholipid lipid vesicles which are themselves 
dispersed or suspended in an aqueous-based solution. 
Ionophore growth factors are primarily macrolide antibiotics that attack a 
variety of Gram-negative bacteria and increase growth rate, particularly 
of ruminant animals. The ionophores are active in ruminants by reducing 
the proportion of methane produced by ruminal fermentation and increasing 
the proportion of proprionic acid in the bovine rumen fluid. Most of the 
ionophore growth factors are substantially water-insoluble so that a 
formulation which allows the ionophore to be administered in the drinking 
water of poultry, cattle, or pigs would be highly beneficial. The 
water-insoluble nature of these ionophore growth factors makes it 
difficult to encourage the animals to obtain sufficient concentrations of 
the growth factors by current administration methods, e.g., as a coating 
on foodstuffs. Even if a sufficient level is attained, this will vary 
widely on a day-to-day basis depending on the amount of fluid consumed by 
the animal. However, most animals drink substantially constant amounts of 
liquid so that an aqueous-based formulation which can be incorporated into 
the drinking water would give a more constant level of the ionophore. The 
constant level is of some importance when the macrolides are used as a 
growth enhancer, e.g., in the treatment of animal growth but is of 
paramount importance when the antibiotic properties, e.g., anti-swine 
dysentery activity, are most significant. 
Current ionophore growth factor compositions are primarily coating on feed, 
e.g., cereal products, as well as other grains and grasses. In addition, 
slow release, intra-ruminal pellets or boluses have been tried to give 
proper dosages to animals. For example, see U.S. Pat. No. 4,279,894, 
issued July 21, 1981. However, although the use of liquid carriers have 
been contemplated, the water-insoluble properties of most ionophore growth 
factors have made this an unrealistic possibility. Therefore, a carrier of 
the ionophore growth factor is necessary for aqueous formulations. 
Lipid vesicles have not been considered particularly good carriers for 
water-insoluble materials such as ionophore growth factors because their 
cost has been too high for use in animal feed and the instability of the 
lipid vesicles when carrying large quantities of lipophilic material. 
Lipid vesicles are substantially spherical structures made of materials 
having a high lipid content, e.g., surfactants or phospholipids. The 
lipids of these spherical vesicles are organized in the form of lipid 
bilayers. The lipid bilayers encapsulate an aqueous volume which is either 
interspersed between multiple onion-like shells of lipid bilayers (forming 
multilamellar lipid vesicles or "MLV") or the aqueous volume is contained 
within an amorphous central cavity. The most commonly known lipid vesicles 
having an amorphous central cavity filled with aqueous medium are the 
unilamellar lipid vesicles. Large unilamellar vesicles ("LUV") generally 
have a diameter greater than about 1.mu. while small unilamellar lipid 
vesicles ("SUV") generally have a diameter of less than 0.2.mu.. There are 
a variety of uses for lipid vesicles including the use as adjuvants or as 
carriers for a wide variety of materials. 
Although substantially all the investigation of lipid vesicles in recent 
years has centered on multilamellar and the two types of unilamellar lipid 
vesicles, a fourth type of lipid vesicle, the paucilamellar lipid vesicle 
("PLV"), exists. This lipid vesicle has barely been studied heretofore and 
has only been manufactured previously with phospholipids. PLV's consist of 
about 2 to 10 peripheral bilayers surrounding a large, unstructured 
central cavity. In all the previously described PLV's, this central cavity 
was filled with an aqueous solution. See Callo and McGrath, Cryobiology 
1985, 22(3), pp. 251-267. 
Each type of lipid vesicle appears to have certain uses for which it is 
best adapted. For example, MLV's have a higher lipid content than any of 
the other lipid vesicles so to the extent that a lipid vesicle can carry a 
lipophilic material in the bilayers without degradation, MLV's have been 
deemed more advantageous then LUV's or SUV's for carrying lipophilic 
materials. In contrast, the amount of water encapsulated in the aqueous 
shells between the lipid bilayers of the MLV's is much smaller than the 
water which can be encapsulated in the central cavity of LUV's, so LUV's 
have been considered advantageous in transport of aqueous material. 
However, LUV's, because of their single lipid bilayer structure, are not 
as physically durable as MLV's and are more subject to enzymatic 
degradation. SUV's have neither the lipid or aqueous volumes of the MLV's 
or LUV's but because of their small size have easiest access to cells in 
tissues. 
PLV's, which can be considered a sub-class of the MLV's, are a hybrid 
having features of both MLV's and LUV's. PLV's appear to have advantages 
as transport vehicles for many uses as compared with the other types of 
lipid vesicles. In particular, because of the large unstructured central 
cavity, PLV's are easily adaptable for transport of large quantities of 
aqueous-based materials. Also as illustrated in previously cited U.S. 
patent application Ser. No. 157,571 now U.S. Pat. No. 4,911,928, the 
aqueous cavity of the PLV's can be filled wholly or in part with an apolar 
oil or wax and then can be used as a vehicle for the transport or storage 
of hydrophobic materials. The amount of hydrophobic material which can be 
transported by the PLV's with an apolar core is much greater than can be 
transported by MLV's. The multiple lipid bilayers of the PLV's provides 
PLV's with additional capacity to transport lipophilic material in their 
bilayers as well as with additional physical strength and resistance to 
degradation as compared with the single lipid bilayer of the LUV's. 
All of the early lipid vesicle or liposome studies used phospholipids as 
the lipid source for the bilayers. The reason for this choice was that 
phospholipids are the principal structural components of natural 
membranes. However, there are many problems using phospholipids as 
artificial membranes. First, isolated phospholipids are subject to 
degradation by a large variety of enzymes. Second, the most easily 
available phospholipids are those from natural sources, e.g., egg yolk 
lecithin, which contain polyunsaturated acyl chains that are subject to 
autocatalyzed peroxidation. When peroxidation occurs, the lipid structure 
breaks down, causing premature release of encapsulated materials and the 
formation of toxic peroxidation byproducts. This problem can be avoided by 
hydrogenation but hydrogenation is an expensive process, thereby raising 
the cost of the starting materials. Cost is a third problem associated 
with the use of phospholipids on a large scale. A kilogram of egg yolk 
lecithin pure enough for pharmacological liposome production presently 
costs in excess of $1,000. This is much to high a cost for a starting 
material for most applications. Even less highly purified phospholipids 
are too expensive for most animal uses. 
Recently, there has been some indication, particularly from L'Oreal and 
Micro Vesicular Systems, Inc., that commercially available surfactants 
might be used to form the lipid bilayer in liposome-like multilamellar 
lipid vesicles. Both surfactants and phospholipids are amphiphiles, having 
at least one lipophilic acyl or alkyl group attached to a hydrophilic head 
group. The head groups are attached to one or more lipophilic chains by 
ester or ether linkages. Commercially available surfactants include the 
Brij family of polyoxyethylene acyl ethers, the SPAN sorbitan alkyl 
esters, and the TWEEN polyoxyethylene sorbitan fatty acid esters, all 
available from ICI Americas, Inc. of Wilmington, Del. 
The methods and materials disclosed herein for producing the paucilamellar 
lipid vesicles all yield vesicles with a high aqueous or oil volume. 
Electron micrographs confirm that the paucilamellar lipid vesicles are 
distinct from the LUV's and the classic MLV's. 
Accordingly, an object of the invention is to provide an aqueous-based 
formulation of an ionophore growth factor. 
Another object of the invention is to provide a formulation having factor 
encapsulated within a nonphospholipid vesicle. 
A further object of the invention is to provide a method of preparing an 
aqueous-based formulation of a substantially water-insoluble ionophore 
growth factor which exhibit both growth promoting and antibiotic action. 
A still further object of the invention is to provide a method of treatment 
of animals to enhance growth and provide antibiotic action. 
These and other objects and features of the invention will be apparent from 
the following description. 
SUMMARY OF THE INVENTION 
The present invention features aqueous-based formulations having an 
ionophore growth factor encapsulated in nonphospholipid lipid vesicles. 
The invention further features a method of preparing the vesicles and a 
method of treating animals to enhance growth and provide antibiotic action 
using the vesicles of the invention. 
The aqueous-based formulation of the invention contains at least one active 
agent selected from a group consisting of ionophore growth factors, and 
mixtures, derivatives and analogs thereof, encapsulated in lipid vesicles 
which have nonphospholipid materials as their primary lipid source. The 
lipid vesicles are dispersed in an aqueous-based carrier. As used herein, 
the term "disperse" means, includes and implies dispersions, suspensions, 
colloids, and other similar non-dissolved states. Preferred 
nonphospholipid materials include lipid vesicle forming polyoxyethylene 
fatty esters, polyoxyethylene fatty acid ethers, diethanolamines, 
long-chain acyl amides, long-chain acyl amino acid amides, long-chain acyl 
amides, polyoxyethylene sorbitan oleates, polyoxyethylene glycerol 
monostearates, glycerol monostearates, and mixtures, analogs, and 
derivatives thereof. The vesicles may also include a steroid, and a charge 
producing agent. Preferred steroids include cholesterol, hydrocortisone, 
and analogs, derivatives, and mixtures thereof. Preferred negative charge 
producing materials are oleic acid, dicetyl phosphate, palmitic acid, 
cetyl sulphate, retinoic acid, phosphatidic acid, phosphatidyl serine, and 
mixtures thereof. In order to provide a net positive charge to the 
vesicles, long chain amines, e.g., stearyl amines or oleyl amines, long 
chain pyridinium compounds, e.g., cetyl pyridinium chloride, quaternary 
ammonium compounds, or mixtures of these can be used. A preferred positive 
charge producing material is hexadecyl trimethylammonium bromide, a potent 
disinfectant. The use of this disinfectant as the positive charge 
producing material within the vesicles provides a secondary advantage as 
the vesicles deteriorate; they act as a sustained release germicide 
carriers. 
Although any type of lipid vesicle which could carry sufficient quantities 
of the water-insoluble ionophore growth factor could be used, 
paucilamellar lipid vesicles are the most practical choice. These vesicles 
provide a large, amorphous central transport cavity. The ionophore growth 
factor can be dissolved or dispersed in a water immiscible oily material 
which can be used as a carrier for the ionophore. As used herein, the term 
"water immiscible oily material" means, includes and comprises oils and 
waxy-like material preferably selected from a group consisting of oils, 
waxes, natural and synthetic triglycerides, acyl esters, and petroleum 
derivatives, and their analogs and derivatives. 
Preferred ionophore growth factors are water-insoluble, preferably 
macrolide antibiotics or mixtures, derivatives, or analogs thereof. 
Preferred water-insoluble ionophore growth factors are selected from a 
group consisting of tetronasin, monensin, salinomycin, lasolocids, 
lysocellin, ladlomycin, narosin, and mixtures, derivatives, and analogs 
thereof. The formulation can include a single ionophore growth factor or a 
plurality of ionophore growth factors, each encapsulated in lipid 
vesicles, can be mixed to form a formulation having broad spectrum 
antibiotic properties. 
The formulation is made by dispersing the ionophore containing vesicle in 
an aqueous-based solution. A substantially water-insoluble ionophore 
growth factor is encapsulated in a nonphospholipid lipid vesicles. The 
lipid vesicles are made by forming a lipophilic phase of nonphospholipid 
materials combined with any other lipophilic materials which are to be 
encapsulated, combining the water-insoluble ionophore growth factor with a 
water immiscible oily material, dispersing the water immiscible oily 
material containing the ionophore growth factor in the lipophilic phase, 
forming an aqueous phase of aqueous soluble materials to be encapsulated 
in the lipid vesicle by dispersing the aqueous soluble materials in an 
aqueous carrier, and shear mixing the lipophilic phase and the aqueous 
phase to form lipid vesicles. "Shear mixing" means, includes and implies 
the mixing of the lipophilic phase with the aqueous phase under turbulent 
or shear conditions which provide adequate mixing to hydrate the lipid and 
form lipid vesicles. In many instances, calibrated metering pumps are used 
to drive the phases to form the vesicles. The pump speeds for mixing the 
phases are modified depending on the viscosity of the materials and the 
size of the orifices selected. "Shear mixing" is achieved by liquid shear 
which is substantially equivalent to a relative flow rate for the combined 
phases of about 5-30 m/s through a 1 mm radius orifice. 
All of the lipid materials useful in forming the vesicles of the invention 
can be classified as surfactants. However, standard methods of 
manufacture, although they may be used, are not as efficient as those set 
forth herein. In order to achieve the proper blending necessary to form 
the paucilamellar lipid vesicles, all of the materials are normally in a 
flowable state. However, in the process of the present invention, use of a 
solvent for the surfactant (the classic method of producing multilamellar 
lipid vesicles) is not only unnecessary; it is counter-productive. Many of 
the surfactants useful in the invention are liquids at room temperature or 
at slightly elevated temperatures so only gentle heating is necessary for 
flowability. Even the most difficult surfactants of the group to use, 
e.g., glycerol monostearate, can be easily handled at approximately 
70.degree. C. Therefore, one standard procedure of the invention is to 
elevate the temperature of the lipophilic phase in order to make it 
flowable followed by carrying out the shear mixing between the lipophilic 
phase and the aqueous phase at a temperature such that both phases are 
liquids. While it is often desirable to use the same temperature for both 
phases, this is not always necessary. 
The formulation of the invention can be used as a treatment to enhance 
growth and to provide antibiotic action in animals. The ionophore growth 
factors exhibit both actions simultaneously, making them efficient 
materials to use on animals. However, the invention is not limited to 
cases where both effects of the ionophore growth factors are equally 
pertinent but rather all circumstances where the ionophore growth factors 
are used for treatment as either an antibiotic or a growth promoter. 
The invention will be further understood by the following description and 
the Examples.

DESCRIPTION OF THE INVENTION 
The present invention solves a number of the problems of using ionophore 
growth factors because of the ability to make aqueous-based formulations. 
The ability to add ionophore growth factors to drinking water yields a 
much easier method of treating the animals than those presently used. 
The preferred lipid vesicles are paucilamellar lipid vesicles having a 
water immiscible oily material within the amorphous central cavity. The 
water immiscible oily material can act as a carrier for the 
water-insoluble ionophore. 
Although any lipid vesicle forming material could, theoretically, be used 
to form the lipid vesicles of the invention, the most preferred 
surfactants useful in the invention are selected from a group consisting 
of polyoxyethylene fatty esters having the formula 
EQU R.sub.1 --COO(C.sub.2 H.sub.4 O).sub.n H 
where R.sub.1 is lauric, myristic, cetyl, stearic, or oleic acid, or their 
derivatives and n=2-10; 
polyoxyethylene fatty acid ethers, having the formula 
EQU R.sub.2 --CO(C.sub.2 H.sub.4 O).sub.m H 
where R.sub.2 is lauric, myristic, or cetyl acids or their derivatives, 
single or double unsaturated octadecyl acids or their derivative, or 
double unsaturated eicodienoic acids or their derivatives and m ranges 
from 2-4; 
diethanolamines, having the formula 
EQU (HOCH.sub.2 --CH.sub.2).sub.2 NCO--R.sub.3 
where R.sub.3 is caprylic, lauric, myristic or linoleic acids or their 
derivatives; 
long chain acyl hexosamides having the formula 
EQU R.sub.4 --NOCO--(CH.sub.2).sub.b --CH.sub.3 
where b ranges from 10-18 and R.sub.4 is a sugar molecule selected from a 
group consisting of glucosamine, galactosamine, and N-methylglucamine; 
long chain acyl amino acid amides having the formula 
EQU R.sub.5 --CHCOOH--NOC--(CH.sub.2).sub.c --CH.sub.3 
where c ranges from 10-18 and R.sub.5 is an amino acid side chain; 
long chain acyl amides having the formula 
EQU HOOC--(CH.sub.2).sub.d --N(CH.sub.3).sub.2 --(CH.sub.2).sub.3 
--NCO--R.sub.6 
where R.sub.6 is an acyl chain having 12-20 carbons and not more than two 
unsaturations, and d ranges from 1-3; 
polyoxyethylene (20) sorbitan mono- or trioleate; 
polyoxyethylene glyceryl monostearate with 1-10 polyoxyethylene groups; and 
glycerol monostearate. 
The paucilamellar lipid vesicles can be made by a variety of devices which 
provides sufficiently high shear for shear mixing. There are a large 
variety of these devices available on the market including a 
microfluidizer such as is made by Biotechnology Development Corporation, a 
"French"-type press, or some other device which provides a high enough 
shear force and the ability to handle heated, semiviscous lipids. If a 
very high shear device is used, it may be possible to microemulsify 
powdered lipids, under pressure, at a temperature below their normal 
melting points and still form the lipid vesicles of the present invention. 
A device which is particularly useful for making the lipid vesicles of the 
present invention has been developed by Micro Vesicular Systems, Inc., 
Vineland, N.J. and is further described in previously cited U.S. patent 
application Ser. No. 163,806, and now U.S. Pat. No. 4,895,452. Briefly, 
this device has a substantially cylindrical mixing chamber with at least 
one tangentially located inlet orifice. One or more orifices lead to a 
reservoir for the lipophilic phase, mixed with an oil phase if lipid-core 
PLV's are to be formed, and at least one of the other orifices is attached 
to a reservoir for the aqueous phase. The different phases are driven into 
the cylindrical chamber through pumps, e.g., positive displacement pumps, 
and intersect in such a manner as to form a turbulent flow within the 
chamber. The paucilamellar lipid vesicles form rapidly, e.g., less than 1 
second, and are removed from the chamber through an axially located 
discharge orifice. In a preferred embodiment, there are four tangentially 
located inlet orifices and the lipid and aqueous phases are drawn from 
reservoirs, through positive displacement pumps, to alternating orifices. 
The fluid stream through the tangential orifices is guided in a spiral 
flow path from each inlet or injection orifice to the discharge orifice. 
The flow paths are controlled by the orientation or placement of the inlet 
or injection orifices so as to create a mixing zone by the intersection of 
the streams of liquid. The pump speeds, as well as the orifice and feed 
line diameters, are selected to achieve proper shear mixing for lipid 
vesicle formation. As noted, in most circumstances, turbulent flow is 
selected to provide adequate mixing. 
The invention, and its uses, will be further explained by the following 
Examples. 
EXAMPLE 1 
A series of formulations were made by encapsulating tetronasin, an 
ionophore growth factor, in lipid vesicles and suspending the tetronasin 
filled vesicles in water to make the formulation of the invention. Two 
methods were used to make the vesicles; one using relatively large scale 
amounts uses the Micro Vesicular Systems previously described and the 
second, a test system, used two syringes connected by a stopcock. The same 
proportions of material were used in all the formulations, but 
modifications were made in certain of the ingredients. 
The first formulation tested was a test system made by a syringe method. 
Approximately 0.5 g of tetronasin is dissolved in 5 ml of Drakeol 19 
(mineral oil USP) which is heated to approximately 40.degree. C. until a 
clear solution develops. The tetronasin forms approximately 2% of the 
final preparation while the mineral oil forms approximately 27%. The 
mineral oil can be replaced by a different oil, e.g., peanut oil, which 
work equally as well. The water-immiscible oil-tetronasin is blended with 
a lipid vesicle-forming stock solution containing polyoxyethylene 2-cetyl 
ether (Brij 52-ICI Americas, Inc.), cholesterol, and oleic or palmitic 
acid in a ratio of 33/11/1.5. The total amount of the lipid 
vesicle-forming stock solution used is approximately one-half of the 
mineral oil volume. At 40.degree. C., the lipid solution and the oil 
solution form a clear solution. The solution may then be filtered through 
a 5.mu. filter for further clarification. The lipid phase is then 
hydrated, using approximately an eight fold excess of water. Hydration is 
carried out by shooting the solutions back and forth from syringes through 
a stopcock for about two minutes. Alternatively, a device such as the 
Micro Vesicular Systems lipid vesicle forming machine or could be used. 
Uniform lipid vesicles having a diameter of approximately 1-2.mu. having 
an oily center filling the amorphous center cavity are formed. 
To test stability, the lipid vesicles were centrifuged for approximately 20 
minutes at 3,000 rpm without a dextran layer. No sediment was seen. 
Approximately five days later, the same centrifuge test was carried out, 
also yielding no sediment. No sediment developed after a 10,000 rpm 
centrifuge test. 
The final proportions used are set forth in Table 1. 
TABLE 1 
______________________________________ 
% w/v 
______________________________________ 
TETRONASIN 2.00 
Brij 52 16.24 
CHOLESTEROL 5.06 
OLEIC ACID 0.70 
MINERAL OIL 27.20 
WATER TO 100 cm.sup.3 
______________________________________ 
The final concentration of tetronasin was approximately 2% by weight. 
EXAMPLE 2 
Tetronasin is known to have antibiotic effect against Treponema 
hyodysenteriae infection in pigs, the causative organism in swine 
dysentery. Although aqueous-based solutions of tetronasin have been tried, 
the very low aqueous solubility has made this an ineffective method of 
treatment. Accordingly, a lipid vesicle preparation made as described in 
Example 1 was tried for efficacy against swine dysentery. A series of pigs 
were offered water containing approximately 60 mg/l tetronasin in the 
formulation of Example 1, using mineral oil as the internal carrier. There 
was a slow regression of the dysentery symptoms but the treatment was 
generally effective. However, water intake of the pigs was approximately 
half that of the control group, showing that the pigs either did not like 
the lipid vesicles or the tetronasin itself. Addition of an artificial 
flavor to the water assisted somewhat in increasing water uptake and 
improve the response of the pigs, but water uptake was still lower than a 
control group. 
In order to determine whether the pigs objected to the flavor of the lipid 
vesicle or the tetronasin, one group of pigs were offered water containing 
approximately 2.9 g/l of lipid vesicles without tetronasin. Water uptake 
was compared with a control group given water without the vesicles. There 
was substantially no difference in water uptake, showing that the 
tetronasin was the material objected to by the pigs. To confirm this, the 
pigs were offered water containing lipid vesicles without tetronasin and 
with tetronasin. The pigs receiving the tetronasin drank only about 40% of 
the others, confirming that the taste or smell of the tetronasin was 
objected to by the pigs. 
Replacing the mineral oil with a peanut oil to improve palatability of the 
tetronasin solution showed little improvement. However, it is believed 
formulations can be made which mask the tetronasin taste problem and 
provide adequate protection to the test animals. All the formulations 
showed antibiotic action. 
The foregoing Examples are expressly non-limiting and are merely to show 
the efficacy of the present invention. The invention is defined by the 
following claims.