WO2000032167A1

WO2000032167A1 - A polysome

Info

Publication number: WO2000032167A1
Application number: PCT/US1999/027980
Authority: WO
Inventors: John R. Lau
Original assignee: SDG Technology Inc
Current assignee: SDG Technology Inc
Priority date: 1998-12-01
Filing date: 1999-11-26
Publication date: 2000-06-08
Anticipated expiration: 2001-06-01
Also published as: WO2000032167A9; AU1921900A

Abstract

A polysome is disclosed comprising a polymer linked via a binding agent to a lipid matrix. The polysome can be pharmaceutical polysome which contains a medicament bound to the polymer. Where the polymer is a poly(maleic anhydride-1-octadecene), a suitable binding agent is phosphatidyl ethanolamine. Typical medicaments include biogenic primary amines.

Description

A POLYSOME

This application claims priority from U.S. provisional application Serial No. 60/110,338 filed December 1, 1998. BACKGROUND OF THE INVENTION

This invention relates to a pharmaceutical polysome. More particularly, it relates to a medicament delivery system comprising a liposome matrix and a medicament-polymer complex which is associated with or incorporated into the liposome matrix.

A problem in administering therapeutic agents or medicaments, such as biogenic primary amines, e.g., 5-hydroxytryptamine hydrochloride (5-HT HC1) and y- amino-n-butyric acid (GABA) and cytokines, hormones, biologically active proteins and enzymes for replacement therapy contained in a pharmaceutical delivery system to a patient in need thereof, is the leakage of the therapeutic from such a system into the external media of a patient's body. This leaking results in undesirable physiological responses, pharmacological side effects and an adverse effect on the therapeutic index of such therapeutic agent or medicament. Accordingly a need exists for a pharmaceutical delivery system which will deliver a desired medicament in a pharmacologically effective and safe dose to a patient in need thereof, whereby such system prevents or substantially minimizes the interaction of the medicament in the body of the patient with non-target receptor sites. Heretofore, there have been no known effective means of retaining effective amounts of a selected medicament, e.g., a biogenic amine, in a drug delivery vehicle which enhances the direction of the medicament to a target site or desired receptor in the body of a patient, thus enhancing the therapeutic index of such medicament. SUMMARY OF THE INVENTION This invention relates to a polysome and, more particularly, to a polysome comprising a liposome matrix and a medicament-polymer complex associated therewith or incorporated thereinto. The liposome matrix is obtained by reacting or combining a lipid binding agent with a selected lipid or a mixture of lipids. The resultant liposome then binds, via the binding agent, the medicament-polymer complex to the liposome. Also preferred is a means to attach cellular target molecules and reticulo-endothelial system (R.E.S.) avoidance molecules that either naturally manifest primary amines or that have been subsequently derivatized with synthetic terminal primary amines. In this manner, appropriate combinations of polysome lipid, lipid binding agents, target molecules, R.E.S. avoidance molecules and therapeutic molecules may be formulated by using a stoichiometric single-step addition procedure into the designated pharmaceutical polysome carrier system. BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying drawings in which,

Figure 1 is a depiction of poly(maleic anhydride- 1-octadecene) derivatized with 5-HT HC1; and

Figure 2 is a plot of a dose response of portal serotonin.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a polysome. The term "polysome" has been coined to designate a novel composition of matter which is a reaction product of a selected liposome and a selected polymer. A "pharmaceutical polysome" is a polysome having a selected medicament associated with or incorporated into the reaction product of the liposome and the polymer and optionally contains natural or amine derivatized target molecules and R.E.S. avoidance molecules derivatized in a similar fashion. A suitable polymer is selected. A suitable polymer is one which can react with and bind a suitable selected medicament thereto as well as react with and be bound to a lipid binding agent, e.g. phosphatidylethanoleamine (PE), contained in the liposome. The polymer can also react and bind biologically active proteins and pharmaceuticals which express primary amines. In addition, the polymer provides multiple attachment sites for regulating the stoichiometric binding relationships between target molecules, reticulo-endothelial system avoidance molecules, medicaments or therapeutics and lipid membrane binding agents.

Where the medicament contains a primary amine group, a suitable polymer is a poly (maleic anhydride- 1-octadecene), poly (maleic anhydride- 1-hexadecene), poly (maleic anhydride- 1-tetradecene), poly (maleic anhydride- 1-dodecene). Preferably, the poly (maleic anhydride- 1-octadecene) polymer is employed. Most preferred is a poly(maleic anhydride- 1-octadecene) polymer having a molecular weight of 30,000 to 50,000, where the molecular weight of a single functional polymeric unit thereof, which includes the anhydride functional unit and the octadecylhydrocarbon chain, is 350 as shown in Figure 1. Accordingly, in this polymer there are approximately 86 to 143 functional units available in a single polymer chain which can participate in chemical reactions.

Where the medicament contains a primary amine functional group, a suitable polymer is one which reacts by means of an anhydride functional group. Other nucleophilic addition reactions may be obvious to those skilled in the art. A suitable medicament is any medicament which reacts with and binds to the selected polymer. Such medicaments include pharmacologically active compounds containing a primary amine molecule. Preferably, the medicament is selected from the class of neuro transmitters designated as biogenic primary amines. Such amines

include 5-hydroxytryptamine hydrochloride or serotonin (5-HT HC1), L-β-3,4-

dihydroxyphenylalanine (L-DOPA), 2-(4-imidazole)ethylamine (histamine), l-[3,4-

dihydroxyphenyl]-2-aminoethanol (norepinephrine), γ-amino-n-butyric acid (GABA),

l-(aminomethyl) cyclohexane acetic acid (gabapentin), biologically active cytokines,

e.g. , β, γ, interferon, interleukins 1, 2, and 3, and tumor necrosis factor (TNF);

hormones, e.g., insulin, somatostatin, glucagon, and serotonin; proteins and enzymes, e.g., L-asparaginase indicated for cancer chemotherapy, and other enzymes for replacement therapies. In addition, the medicament can be selected from hematopoietic growth factors, e.g., granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, erythropoietin and also chemotherapeutic agents, e.g., doxorubicin and daunorubicin, antisense oligonucleotides and other medicaments, such as antimicrobials, antivirals and antibiotics.

It is to be noted that other desired medicaments which are reactive with a diamine, such as ethylenediamine and target molecules which contain one or more free acid groups, such as N(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid (DID A), etc. can be reacted with such a diamine to form a monoamide or multiple amine functionalities, whereby an appropriate derivative thereof is obtained. For example, such medicaments as proteins, hormones, and enzymes can be reacted with a diamine, e.g., ethylenediamine, using an appropriate carbodiimide coupling agent whereby the monoamine derivative is obtained. This monoamine derivative can then be reacted with the polymer, e.g., poly(maleic anhydride- 1-octadecene) to become bound thereto to form a desired medicament-polymer complex or reaction product. The same principle applies for attaching other carboxylic acid derivatives of the iminodiacetic acid class of targeting molecules as well as R.E.S. avoidance molecules, e.g., pegylated amino terminal derivatives of the polyethylene glycol class of molecules which can be used to selectively coat the surface of a circulating polysome by molecular attachment to a polymer of the poly(maleic anhydride) class of hydrocarbon polymers.

The selected medicament, e.g. 5-HT HC1, is reacted with a molar excess of the polymer, e.g. poly(maleic anydride- 1-octadecene), whereby the medicament reacts only partially with the polymer to form a medicament-polymer product or complex. Optionally, for 5-HT HC1, the molar ratio of a poly (maleic anydride- 1-octadecene) polymer, having a molecular weight of 30,000 to 50,000, to the 5-HT HC1 is 3:1. The reaction of the medicament and polymer is typically carried out at a temperature ranging from 20° C to 60° C for 0J5 to 1.5 hours until all the medicament has reacted with the limited portion of the polymer, e.g., to form a monoamide or multiple amide bonding patterns as determined by a fluorescamine analytical assay for primary amino group functionalities. Other acceptable analytical assays for primary amines groups can be also employed. The amount of the polymer which is employed is sufficient to partially react with the medicament and also to react partially with the binding agent, e.g. PE, which is incorporated in the liposome to which the medicament polymer complex is destined to be associated, i.e. bound to or incorporated into. The poly(maleic anhydride- 1- octodecene) polymer is present at a preferred amount ranging from 11.5% to 34.7% by weight of the total lipid concentration of the liposome to which the medicament- polymer complex is destined to be bound or to be incorporated into. Higher or lower concentrations of polymer may be used depending on the chemical conditions of the reaction process and the desired amount of medicament.

Typically, the therapeutic agent or medicament is first added to the polymer in order to achieve the desired stoichiometry in the reaction mixture. For example, for

5-HT HC1 the stoichiometry is a 3:1 mole ratio of poly(maleic anhydride- 1- octadecene) polymer to 5-HT HC1. This represents on a mole basis 117 μ moles of the polymer for every 39J μ moles of 5-HT HC1 as illustrated in EXAMPLE 1.

A binding agent is selected. A suitable binding agent is one which (1) will react with or combine with or be mixed with a lipid or a mixture of lipids to form a liposome incorporating this agent, and (2) upon such incorporation is capable of reacting with the medicament-polymer complex to bind the complex to or to incorporate the complex into the liposome. Some suitable agents include phosphatidylethanolamine (PE), stearylamine, phosphatidylserine and additional phosphatidyl amino acids expressing a primary amine. Preferably, where the polymer of the complex is a poly(maleic anhydride- 1-octadecene), phosphatidylethanolamine (PE) is employed as the binding agent.

The selected binding agent, e.g., PE, is reacted or combined with a lipid or a lipid mixture at a temperature of 20° C to 80° C for 0J5 to 2.0 hours to form a liposome following hydration of the lipid. A suitable lipid is selected from 1,2- distearoyl-sn-glycerol-3-phosphocholine (DSL), cholesterol (CHOL), a dicetyl phosphate (DCP) and the chromium (bis) [N-(2,6- diisopropylphenylcarbamoylmethyl) iminodiacetic acid] target molecule or a mixture of any of the forgoing. Preferably a mixture of all of the foregoing lipids is employed. Most preferably, for the primary amine containing medicaments or medicaments that have been derivatized with amines, the lipid mixture comprises 61.9 mole percent of

DSL, 14.1 mole percent CHOL, 19.4 mole percent DCP, 0.98 mole percent of the target molecule combined with 3.6 mole percent PE. Collectively, this represents a total lipid concentration of 28J mgs/ml. When the PE is added as a phospholipid binding agent, it contributes 1J mgs by weight or 1.7 μ moles of PE/ml of liposomal lipid.

Where the binding agent is PE, combined therewith is a lipid selected from either l,2-distearoyl-sn-glycerol-3-phosphocholine (DSL), 1,2-dipalmitoyl-sn- glycerol-3-phosphocholine (DPL), 1 J-dimyristoyl-sn-glycerol-3-phosphocholine (DML) or other such similar compounds.

The desired polysome construct is formulated by initiating a reaction sequence emphasizing competition reactions between poly(maleic anhydride- 1-octadecene), 5- HT HC1 and phosphatidylethanolamine. Experimentally, 5-HT HC1 is first reacted with a limited portion of the available maleic anhydride residues residing in the poly(maleic anhydride- 1-octadecene) polymer. Then in a subsequent step, after a covalent bond has been established between the polymer and 5-HT HC1, the newly formed polymeric construct, which is suspended in aqueous media, is mixed with the dried lipid constituents to initiate a reaction between membrane-bound phosphatidylethanolamine and some of the remaining maleic anhydride functional groups in the polymer. The product of this maleylation reaction creates another amide linkage

this time between the polymer and the membrane-bound phosphatidylethanolamine.

The ultimate purpose of these particular chemical interactions is to retard the movement or desorption of the polymer containing covalently bound 5-HT HCl from the lipid matrix. This goal is accomplished by establishing new covalent bonds between the polymer and 5-HT HCl in conjunction with a number of non-specific

ionic associations.

The resultant medicament-polymer complex, comprising medicament, e.g., 5-

HT HCl, covalently bound through an amide linkage to the polymer, e.g., poly(maleic anhydride- 1-octadecene) is suspended in a suitable aqueous solvent, e.g., deionized water, a physiologically acceptable buffer or phosphate buffered saline at pH 7.4 to form a complex suspension. The specified lipids, e.g., the PE combined with a lipid or a mixture of the designated lipids is dried typically at 40° C to 80° C for 0.5 to 2.5 hours and the suspension is hydrated therewith at 25° C to 80° C for 0.5 to 2.0 hours to react the incorporated binding agent with the polymer moiety of the previously referenced complex in order to bind or incorporate the complex to or into the

liposome matrix to form the polysome. Such binding or incorporation results as a

reaction between some of the remaining available reaction sites of the polymer with the binding agent e.g., PE, of the liposome. For PE this occurs via another amide linkage, as determined by an analytical assay using either fluorescamine or another methodology for detecting primary amine functionalities.

The resultant pharmaceutical polysome provides a pharmaceutical delivery system which retards the movement or desorption of the polymer covalently bound to the medicament, e.g. 5-HT HCl, from the liposome or lipid containing matrix, via additional bonding between the medicament-polymer complex and the incorporated

binding agent. Where the medicament selected to be used is one not having an amine group in its structural formula, but which is reactive with an amine group, such medicament is first partially reacted with a suitable diamine, e.g. ethylene diamine, as previously discussed, to obtain a free monamine derivative of the medicament. This derivative is then reacted with the polymer and the resultant complex is then reacted with the liposome, as previously described, to form the desired pharmaceutical liposome.

The incorporated or lipid linked binding agent, e.g. phosphatidylethanolamine or PE, provides a good anchoring site for the polymer, e.g. poly(maleic anhydride- 1- octadecene), typically through the amide bond and secondarily through van der

Waal's and hydrogen bonds. For example, it has been found that 5.54 μ moles (59%)

of the starting 5-HT HCl reacted with poly(maleic anhydride- 1-octadecene) polymer (30,000 - 50,000 molecular weight) is retained by the liposomal matrix when PE is

present as compared to only 1J8 μ moles (13%) when PE is absent.

Effective amounts of the polysomes of the present invention may be administered to a subject by one of various methods, for example, orally as in capsules or tablets, parenterally in the form of sterile solutions or suspensions, and in some cases intravenously in the form of sterile solutions.

The polysomes of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the polysomes may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 0.01% of the medicament, the active ingredient, but may be varied depending upon the particular form and may conveniently be between 0.01% to about 70% of the weight of the unit. The amount of the medicament present in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between 0J to 300 miligrams of the medicament. The tablets, pills, capsules, troches and the like may also contain the following adjuvants: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid,

Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotex; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl sahcylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes, and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, the polysomes of the present invention may be incorporated into a solution or suspension. These preparations should contain at least 0.01% of the medicament, but may be varied to be between 10% and about 50% of the weight thereof. The amount of the medicament present in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between 5.0 to 100 milligrams of the medicament contained in the polysome. The solutions or suspensions may also include the following adjuvants: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustments of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

The following EXAMPLES are provided to illustrate the invention in terms of evaluating the loading capacity and leakage rates of 5-HT HCl from the resultant polysome. TABLE 1 indicates that PE was employed with the indicated lipids in the formation of the liposome.

Table I - Liposomal Consituents

* weight and μ moles are not included in total column.

In addition, 5-HT HCl was incorporated, via the complex with the poly(maleic anhydride -1-octadecene) polymer, into the liposome at a concentration of 2.0 mg/ml

or 9.4 μ moles/ml of the liposome. EXAMPLE 1

113.2 mgs of the dry lipid components (TABLE I) plus 5.0 mgs (7.0 μ moles) of phosphatidylethanolamine totaling 118J mgs were added to a first container and dissolved in 1.0 ml of a stock solution of chloroforπumethanol 2:1 (v/v) solvent that had been previously dried with molecular sieves of sodium alumino-silicate, 8 to 12 mesh beads, nominal pore diameter of 4 A, to eliminate water. The liposomal constituents were then evaporated under water aspiration by employing slow turning of the sample using a Bϋchi Rotoevaporator for 30 minutes at 60° C and then under high vacuum for 2 hours at 60° C using a Jouan Vacuum Pump equipped with a 105°C cold trap to remove any residual chloroforrmmethanol solvent. Then 41.0 mgs (117 μ moles of poly(maleic anhydride- 1-octadecene) (molecular weight 30,000 to 50,000, a product of Polysciences, Inc.) was added to a second container and dissolved with 10 mis of the chloroform:methanol 2:1 (v/v) stock solution. In order to create a dried polymer film, the polymer was evaporated under water aspiration for 30 minutes at 60° C by employing slow turning of the sample using the Bϋchi

Rotoevaporator and then dried under high vacuum for 30 minutes at 60° C. Next 4J8 mis of a 5-HT HCl solution, obtained from Sigma Chemical Co., was prepared at a concentration of 2.0 mgs 5-HT HCl/ml in 50 mM Tris-HCl buffer pH 8.8. 4J8 mis of this solution contained 8J6 mgs (39J μ moles) of 5-HT HCl and was spiked with 50

μl of ¹⁴C-5-hydroxytryptamine creatinine sulfate (¹⁴C 5-HT CS). The resultant radiolabeled solution of 5-HT HCl was then transferred to the first container containing the poly(maleic anhydride- 1-octadecene). The resulting polymer suspension along with 5-HT HCl was hydrated for 30 minutes at 60° C at high rpm using the Bϋchi Rotoevaporator to form polymeric fibers. The polymer suspension was sonicated in one-minute intervals five separate times at 60° C on a setting #4 of a Tekmar #TM375 Sonicator to reduce the size of the polymeric fibers. Following sonication, the polymer and 5-HT HCl suspension were transferred to a separate container containing 5.0 mgs (7.0 μ moles) of PE along with 113J mgs of the remaining lipid constituents (TABLE I). This mixture which contained all the lipid components, the polymer and the radiolabeled 5-HT HCl was then hydrated for 45 minutes at 60° C at high rpm using the Bϋchi Rotoevaporator. Following hydration, four separate 1.0 ml aliquots of the suspension containing lipid, polymer and 2,000 μg of 5-HT HCl/ml were then sonicated at 60° C for one minute each on setting #4 of the

Tekmar #TM375 Sonicator. The resultant suspension exhibited a milky white appearance. Following sonication, 1.0 ml of the resultant polysome suspension was placed in a Slide- A-Lyzer® 10K dialysis cassette, 10,000 MWCO from Pierce Chemical Co., and was dialyzed against 300 mis of 50 mM Tris HCl buffer pH 8.8 for about 24 hours. The dialysate was changed several times until a negligible number of radiolabeled 5-HT HCl molecules were observed in the external bulk phase media or dialysate. The particle size of the resultant polysome was 89.5 nm as determined by a Coulter N4 Plus Particle Size Analyzer. The value of the free 5-HT HCl and the 5- HT HCl containing (bound) polysome was then measured to yield 1J79 μg (59%) of the 5-HT HCl in the lipid fraction and 934.5 μ g (47%) in the dialysate free pool for a total recovery of 106%. EXAMPLE 2

The procedure of EXAMPLE 1 was repeated except that the following reactants were utilized:

108J mgs of the dry lipid components (TABLE 1);

5.0 mgs (7.0 μ moles) of the PE;

4.0 mis of 1.90 mg 5-HT HCl/ml; and 15.8 mgs (45 J μ moles) of the poly (maleic anhydride- 1-octadecene). The resultant suspension was then chromatographed using a Sephadex G-75 column (2.0 x 20 cm) whereby 66% was retained in the lipid fraction and 30% (563.7 mgs of 5-HT HCl) was in the dialysate free pool. EXAMPLE 3

The procedure of EXAMPLE 2 was repeated except that the following reactants were utilized:

108J mgs of the dried lipids components (TABLE 1);

5.0 mgs (7.0 μ moles) of the PE;

16.8 mgs (48 μ moles) of the poly(maleic anhydride- 1-octadecene);

8 mgs (37.6 μ moles) of 5-HT HCl in 3.995 ml of 50 mM Tris buffer

pH 8.7; and

The ratio of polymer to 5-HT HC was 48.9 μ moles to 37.6 μ moles (1.28:1).

The respective μ molar ratios of the polymer, 5-HT HCl and PE was 48.0:37.6:7.0.

The chromatography of the resultant suspension yielded the following results,

TABLE II

EXAMPLE 4 The procedure of EXAMPLE 3 was employed, except that 1.0 ml of the suspension from EXAMPLE 3 was put in a Slide-a-Lyzer cassette, as discussed in EXAMPLE 1. The sample was then dialyzed against 300 mis of 50 mM Tris buffer pH 8.8. The dialysate was changed three times, using 300 mis of fresh buffer each

time. Analysis yielded 34% (681.1 μg) of 5-HT HCl in the lipid fraction and 64%

(1276.85 μg) of 5-HT HCl in the dialysate free pool.

EXAMPLE 5

The procedure of EXAMPLE 3 was employed except that the following reactants were utilized:

108J mgs of the lipid components (TABLE I);

5.0 mgs (7.0 μ moles) of PE;

15.8 mg (45J μ moles) of the poly(maleιc anhydride- 1-octadecene); and

4.76 mgs/ml (22.4 μ moles/4.04 mis) of 5-HT HCl in 50 mM Tris

buffer pH 8.8, tagged with ¹⁴C 5-HT CS. The resultant suspension was then chromatographed giving the following results,

TABLE III

The procedure of EXAMPLE 4 was also repeated with the Slide-a-Lyser cassette and the dialysis results obtained gave 35% (424.7 mg) of 5-HT HCl in the lipid fraction and 60% (833 J mg of 5-HT HCl in the dialysate free pool. EXAMPLE 6

The procedure of EXAMPLE 1 was repeated using the following reactants:

113J mgs of the dry lipid components (TABLE 1);

5.0 mgs (7.0 μ moles) of PE;

13.7 mgs (39J μ moles) of the poly(maleic anhydride- 1-octadecene);

13.8 mg (39.4 μ moles) of 5-HT HCl in 50 mM Tris buffer pH 8.8,

tagged with ¹⁴C 5-HT CS.

The resultant suspension was dialized to yield 36% (710.4 μg) of 5-HT HCl in the

lipid fraction and 72% (1443.3 μg) of 5-HT HCl in the dialysate free pool.

EXAMPLE 7

113J mgs of the dry lipid components (TABLE I);

5.0 mgs (7.0 μ moles) of PE;

27.3 mgs (78 μ moles) of the poly(maleic anhydride- 1-octadecene);

and

8.36 mg (39.3 μ moles) of 5-HT HCl in 50 mM Tris buffer pH 8.8,

tagged with

¹ C 5-HT CS.

Dialysis of the resultant suspension yielded 43% (860.5 μg) of 5-HT HCl in the lipid

fraction and 72% (1444 μg) of 5-HT HCl in the dialysate free pool.

EXAMPLE 8

The procedure of EXAMPLE 1 was repeated except that the following reactants were utilized: 113J mgs of the dry lipid components (TABLE I);

5.0 mgs (7.0 μ moles) of PE;

15.8 mgs (45J μ moles) of the poly(maleic anhydride- 1-octadecene);

and

3.2 mgs (15.0 μ moles) of 5-HT HCl in 50 mM Tris buffer pH 10.1,

tagged with ¹⁴C 5- HT CS.

The resultant suspension was dialized to yield 59% (455J μg) of 5-HT HCl in the

lipid fraction and 43 %> (332J μg) of 5-HT HCl in the dialysate free pool. EXAMPLE 9

The procedure of EXAMPLE 1 was repeated except that no polymer was

employed. The dialysis results were 2.7% (54 μg) of 5-HT HCl retained in the lipid

fraction and 93.7% (1,874 μg) of 5-HT HCl in the dialysate free pool.

EXAMPLE 10 In this example the weight amounts of the individual lipid constituents were scaled up 1.2 times for expediency in obtaining a larger sample size. All the lipid mole ratios remained constant.

136.0 mgs of the dry lipid components (TABLE I) plus 6.0 mgs (8.0 μ moles) of phosphatidylethanolamine totaling 142.0 mgs were added to a first container and dissolved in 3.0 mis of a stock solution of chloroform:methanol 2:1 (v/v) solvent that had been previously dried with molecular sieves of sodium alumino-silicate, 8 to 12 mesh beads, nominal pore diameter of 4 A, to eliminate water. The liposomal constituents were then evaporated under water aspiration by employing slow turning of the sample using a Bϋchi Rotoevaporator for 30 minutes at 60° C and then under high vacuum for 2 hours at 60° C using a Jouan Vacuum Pump equipped with a -

105° C cold trap to remove any residual chloroforrmmethanol solvent. Then 17.15 mgs (49 μ moles) of poly(maleic anhydride- 1-octadecene) (molecular weight 30,000 to 50,000, a product of Polysciences, Inc.) was added to a second container and dissolved with 10 mis of chloroform:methanol 2:1 (v/v) stock solution. In order to create a dried polymer film, the polymer was evaporated under water aspiration for 30 minutes at 60° C by employing slow turning of the sample using the Bϋchi

Rotoevaporator and then dried under high vacuum for 30 minutes at 60° C. Next 10.0 mis of a 5-HT HCl solution, obtained from Sigma Chemical Co., was prepared at a concentration of 0.349 mgs of 5-HT HCl/ml in 50 mM phosphate (dibasic) buffer pH

8.8. 10 mis of this solution contained 3.49 mgs (16 μ moles) of 5-HT HCl and was

spiked with 50 μl of l⁴C-5-hydroxytryptamine creatinine sulfate (^l4C-5-HT CS). The resultant radiolabeled solution of 5-HT HCl was then transferred to the first container containing the poly(maleic anhydride- 1-octadecene). This mixture of the polymer and 5-HT HCl solution was hydrated for 60 minutes at 60° C at high rpm using the Bϋchi Rotoevaporator. The polymer mixed well with the buffer solution and no globules or fibers were visible. This polymer and 5-HT HCl suspension of 10 mis was transferred to the first container which contained the dried lipids of 6 mgs (8 μ moles) of PE and 136.0 mgs of the remaining lipid constituents (TABLE I). This mixture of lipids, polymer and the radiolabeled 5-HT HCl was then hydrated for 45 minutes at 60° C at high rpm using the Bϋchi Rotoevaporator. Following hydration, 10 mis of the mixture was sonicated in 1.0 ml aliquots for one minute on setting #4 of the Tekmar #TM375 Sonicator at 60° C. The aliquots were pooled and the polysome suspension appeared milky- white with no visible particles. 1.0 ml of the polysome suspension containing 350 μg of 5-HT HCl was placed in a Spectra/Por CE Dialysis Tube (MWCO 10K) from Spectrum, and dialyzed against 1,000 mis of 50 mM phosphate buffer pH 7.0. The dialysate was changed after 24 hours, 72 hours and 35 days and analyzed for radiolabeled 5-HT HCl in the bulk phase media. The particle size of the resultant polysome was 113J nm as determined by a Coulter N4 Plus Particle Size Analyzer. The free 5-HT HCl in the dialysate was measured to be 299 μg (85%) and the polysome-bound 5-HT HCl in the retentate was 28 μg (8%>) for a total recovery of 93%.

ANIMAL STUDIES The 5-HT HCl polysome of EXAMPLE 10 was tested in a normal dog to observe the effect on portal vs hepatic glucose balance as a function of the dose of polysomal serotonin administered. The dose is calculated as the amount of free base 5-HT delivered. There is approximately a full log shift into a lower 5-HT concentration range in the dose response curve indicating that polymeric bound 5-HT can be administered as an effective therapeutic for altering the portal hepatic glucose balance in a normal dog. The graph in Figure 2 illustrates the results.

Claims

CLAIMS:

1. A pharmaceutical polysome comprising (a) a liposome of a binding- agent lipid matrix, and (b) a medicament-polymer complex bound to said matrix.

2. The pharmaceutical polysome of claim 1 wherein said matrix is obtained by reacting phosphatidyl ethanolamine with a lipid selected from (a') distearoyl-sn-glycerol-3-phosphocholine (b') cholesterol, (c') a dicetyl phosphate, (d') chromium(bis)[N(2,6-diisopropyl-phenylcarbamylmethyl)imonodiacetic acid], and (e') a mixture of the foregoing lipids, and wherein said medicament complex is formed by reaction of a selected medicament and a poly(maleic anhydride- 1- octadecene) polymer.

3. The polysome as defined in claim 2 wherein said medicament is a biogenic primary amine neurotransmitter.

4. The polysome as defined in claim 3 wherein said amine neurotransmitter is selected from the group consisting of 5-hydroxytryptaimine

hydrochloride, L-β-3,4-dihydroxyphenylalanine, 2-4(-imindazole)ethylamine, l-[3,4-

dihydroxyphenyl]-2-aminoethanol, γ-amino-n-butyric acid, 1-

(aminomethyl)cyclohexane acetic acid, a cytokine, a hormone, a protein, an enzyme, a hematopoietic growth factor, a chemotherapeutic agent, an antimicrobial, an antiviral, an antibiotic and a mixture of any of the foregoing.

5. The polysome of claim 4 wherein said medicament is 5- hydroxytryptamine hydrochloride.

6. The polysome of claim 6, wherein said medicament complex is formed by reaction of said amine with an amount of said polymer which corresponds to

11.5% to 34.7%) by weight of the total weight of said lipid.

7. The polysome of claim 6 wherein said medicament complex is formed by reacting said amine with said polymer in a mole ratio of 1 :3 of said amine to said polymer.

8. The polysome of claim 7 wherein said lipid comprises said mixture (e').

9. The polysome of claim 8 wherein 4J3 weight percent of phosphatidylethanolamine is reacted with said mixture (e'), which comprises 68J weight percent of (a'), 8.97 weight percent of (b'), 17.4 weight percent of (c'), 1J8 weight percent of (d').

10. A method for delivering a therapeutic agent to a mammal which comprises administering an effective amount of a pharmaceutical polysome to said mammal.

11. The method as defined in claim 10 wherein said polysome comprises (a) a liposome having a binding agent - lipid containing matrix and (b) a medicament- polymer complex bound to said matrix.

12. The method as defined in claim 11 wherein said matrix is obtained by reacting phosphatidylethanolamine with a lipid selected from (a') distearoyl-sn- glycerol-3-phosphocholine (b') cholesterol, (c') dicetyl phosphate, (d') chromium(bis)[N(2,6-diisopropyl-phenylcarbamylmethyl)imonodiacetic acid], and (e') a mixture of the foregoing lipids, and wherein said medicament complex is formed by reaction of a selected medicament and a poly(maleic anhydride- 1- octadecene) polymer.

13. The method as defined in claim 12 wherein said medicament is a biogenic primary amine neurotransmitter.

14. The method as defined in claim 13 wherein said amine

neurotransmitter is selected from the group consisting of 5-hydroxytryptaimine, L-β-

3,4-dihydroxyphenylalanine, 2-4(-imindazole)ethylamine, l-[3,4-dihydroxyphenyl]-2-

aminoethanol, γ-amino-n-butyric acid, l-(aminomethyl)cyclohexane acetic acid, a

cytokine, a hormone, a protein, an enzyme, a hematopoietic growth factor, a chemotherapeutic agent, an antimicrobial, an antiviral, an antibiotic and a mixture of any of the foregoing.

15. The method as defined in claim 14 wherein said medicament is 5- hydroxytryptamine.

16. The method as defined in claim 15, wherein said medicament complex is formed by reaction of said amine with an amount of said polymer which corresponds to 36.3% by weight of the total weight of said lipid.

17. The method as defined in claim 16 wherein said medicament complex is formed by reacting said amine with said polymer in a mole ratio of 1 :3 of said amine to said polymer

18. The method as defined in claim 17, wherein said lipid comprises said mixture (e').

19. The method of claim 17 wherein 4J3 weight percent of phosphatidyl ethanolamine is reacted with said mixture (e') which comprises 68J weight percent of (a'), 8.97 weight percent of (b'), 17.4 weight percent of (c'), 1 J 8 weight percent of

(d')

20. A pharmaceutical composition for delivering a therapeutic agent to a target site in a patient in need thereof which comprises,

(a) the therapeutic agent bound to a first reactive site of a polymer having

a plurality of reactive sites; and (b) a liposome matrix having a binding agent bound thereto where said binding agent is bound to at least a second reactive site of said polymer, to associate said therapeutic agent with said matrix and to prevent said therapeutic agent from associating from said matrix.

21. The composition as defined in claim 20 wherein said binding agent is phosphatidyl ethanolamine.

22. The composition as defined in claim 21 wherein said binding agent is bound to said matrix by combining said binding agent with a lipid selected from (a') distearoyl-sn-glycerol-3-phosphocholine (b') cholesterol, (c') dicetyl phosphate, (d') chromium(bis)[N(2,6-diisopropyl-phenylcarbamylmethyl)imonodiacetic acid, (e') phosphatidylethanolamine and (f ) a mixture of the foregoing lipids, and wherein said medicament complex is formed by reaction of a selected medicament and a poly(maleic anhydride- 1-octadecene) polymer.

23. The composition as defined in claim 22 wherein said polymer is a poly(maleic anhydride- 1-octadecene).

24. The composition as defined in claim 22, wherein said polymer is a poly(maleic anhydride- 1-octadecene) and said therapeutic agent is a biogenic primary amine neurotransmitter.

25. The composition as defined in claim 24 wherein said therapeutic agent

is selected from the group consisting of 5-hydroxytryptaimine, L-β-3,4- dihydroxyphenylalanine, 2-4(-imindazole)ethylamine, 1 -[3 ,4-dihydroxyphenyl]-2-

26. The composition as defined in claim 25 wherein said medicament is 5- hydroxytryptamine hydrochloride.

27. The composition of claim 26, wherein said medicament complex is formed by reaction of said amine with an amount of said polymer which corresponds to 36.3%) by weight of the total weight of said lipid.

28. The composition of claim 27, wherein said medicament complex is formed by reacting said amine with said polymer in a mole ratio of 1 :3 of said amine to said polymer.

29. The composition of claim 28 wherein said lipid comprises said mixture (f).

30. The composition of claim 27 wherein 4J3 weight percent of said phosphatidyl ehtanolamine is combined with said mixture (f ) which comprises 68J weight percent of (a'), 8.97 weight percent of (b'), 17.4 weight percent of (c'), 1J8 weight percent of (d') and 4J3 weight percent of (e') of the total weight of said liposome matrix.

31. A method for making a polysome which comprises reacting a polymer with a binding agent bound to a lipid matrix.

32. A method for making a pharmaceutical polysome which comprises, (a) reacting a medicament with a polymer having a plurality of reactive sites to bind said medicament to at least a first binding site; and

(b) reacting said medicament bound polymer with a binding agent linked to a lipid matrix to bind said linked binding agent to at least a second binding site of said polymer.