WO1987007150A1 - Receptor-targeted drug delivery systems - Google Patents

Receptor-targeted drug delivery systems Download PDF

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Publication number
WO1987007150A1
WO1987007150A1 PCT/US1987/000965 US8700965W WO8707150A1 WO 1987007150 A1 WO1987007150 A1 WO 1987007150A1 US 8700965 W US8700965 W US 8700965W WO 8707150 A1 WO8707150 A1 WO 8707150A1
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receptor
drug
ligand
delivery system
drug delivery
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PCT/US1987/000965
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French (fr)
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Jeffrey S. Nye
Solomon H. Snyder
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The Johns Hopkins University
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542Carboxylic acids, e.g. a fatty acid or an amino acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544Phospholipids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome

Definitions

  • the present invention relates to the field of human diagnostics and therapeutics. Specifically, the invention relates to the targeting of drugs to particular body locations. BACKGROUND OF THE INVENTION
  • Liposomes are synthetic lipid vesicles, which can encapsulate a variety of sizes of molecules in their internal spaces. Liposomes have been widely studied as carriers for delivering substances to cells and tissues in the body. (Gregoriadis, Liposome Technology, Vol. I, II, III, CRC Press, Boca Raton, 1985.) Nanoparticles, like liposomes, also have been studied for use as drug carriers. Nanoparticles are made of synthetic polymeric substances such as polycyanoacrylate, polyanhydrides of aromatic and aliphatic dicarboxylic acids, polymethylcyanoacrylate and the like. They may also be formed of proteins such as albumin and gelatin. (Polymeric Nanoparticles and Microspheres, Boca Raton, CRC Press, 1986.) Nanoparticles may entrap or adsorb the drugs which they carry.
  • the present invention provides an alternative means of targeting drug carrying agents which does not require tissue-specific antibodies.
  • the present invention utilizes the specific interactions of therapeutic and functional pharmacological agents and their cell surface receptors.
  • the receptors for betaadrenergic ligands are predominantly found in the lung, heart and adipose tissue, whereas opiate receptors are found in the gut, spinal cord, and on lymphocytes.
  • cancerous tissues characteristically express receptors, for example, brain glioma expresses beta-adrenergic receptors (Receptor Binding Techniques, Society for Neuroscience, Washington, D.C. 1980) and bronchogenic carcinoma expresses receptors for certain biologically active peptides (Peptides, Vol. 4, pages 683-686, 1983.) SUMMARY OF THE INVENTION
  • a receptor-targeted drug delivery system comprising: a polymeric drug carrying agent selected from the group consisting of liposomes and nanoparticles; a ligand capable of binding a receptor, said ligand being covalently attached to said carrying agent and being a therapeutic and functional pharmacological agent; and a drug, said drug being associated with said drug carrying agent.
  • a therapeutic method of treating a human having a diseased organ or tissue comprising: administering an effective amount of the receptor-targeted drug delivery system of the present invention to the human to affect the metabolism of cells expressing receptors for the ligand on their surfaces or nearby cells, said cells being among those of the diseased organ or tissue.
  • a diagnostic method for determining whether a human has a diseased organ or tissue comprising: administering the receptor-targeted drug delivery system to the human; and noninvasively detecting the localization of said drug.
  • Figure 1 depicts schemes for the synthesis of ligand-lipid conjugates.
  • Figure 2 shows examples of ligands with reactive functional groups and ligand-lipid conjugates made therefrom.
  • Figure 3 shows the affinity of ligand-lipid conjugates for their receptors.
  • Figure 4 reports data demonstrating the formation of stearylpindolol-containing liposomes and the encapsulation of a fluorescent solute.
  • Figure 5 shows the stability of encapsulated solutes within liposomes composed of ligand-lipid conjugates, as measured by 45 Ca-EDTA release.
  • Figure 6 reports data showing that ligand-lipid conjugates are incorporated into the structure of the liposomes.
  • Figure 7 shows the affinity of adenosine phosphatidyl ethanolamine-bearing liposomes for adenosine receptors.
  • Figure 8 shows the affinity of stearyl-pindolol-bearing liposomes for beta-adrenergic receptors.
  • Figure 9 reports data showing the delivery of methotrexate to cells with beta-adrenergic receptors by ligand targeted liposomes containing methotrexate.
  • ligands having therapeutic and functional pharmacologic activity and capable of binding a receptor can be covalently joined to polymeric carrying agents to form ligand-polymeric carrying agent conjugates which retain their ability to bind to cell surface receptors. Drugs thus associated with these ligand-polymeric carrying agents are thereby delivered to cells bearing the corresponding cell surface receptor.
  • Carrying agents contemplated for use in the present invention are polymeric substances, although the polymers need not be covalent polymers.
  • liposomes are suitable polymeric carrying agents whose monomer molecules are generally lipids or phospholipids which are noncovalently joined.
  • nanoparticles may be used as polymeric carrying agents. Nanoparticles are covalently linked polymers of monomers such as acrylates, cyanoacrylates, aromatic and aliphatic dicarboxylic acids and the like. The nanoparticles may be either homopolymers or heteropolymers.
  • the size of the polymeric carrying agents may range from about 20 nm to about 10 u in diameter. Any size which would enable the polymeric carrying agents to circulate in the vasculature or other body compartment is suitable for most applications. However, for topical and other means of administration, even larger particles could be used.
  • Liposomes may be formed using standard methods such as the reverse evaporation method (REV) of Papahadjopolous (Proceedings of the National Academy of Sciences U.S.A., Vo. 75, pgs. 4194-4198, 1978), the dehydration-rehydration method (DRV) of Gregoriadas (U.K. Patent Application 8321012, 1983), sonication to form small unilamellar vesicles (SUV) (Biochemistry Vol. 8, pg. 344, 1967) or other suitable methods.
  • the lipids employed in such liposomes will generally be naturally occurring lipids such as phosphatidylcholine, phosphatidylserine, sphingomyelin, cholesterol, their synthetic analogs, and mixtures thereof.
  • Nanoparticles may be formed using standard procedures taught in the art. For example, Brasseur et al. European Journal of Cancer, vol. 16, pgs. 1441-1445, 1980, teaches preparation of nanoparticles from methylcyanoacrylate by means of polymerization and filtration through a fritted glass filter. (See, for other examples, Couvreur, et al. Journal of Pharmacy and Pharmacology, Vol. 31, pgs. 331-332, 1979, and Leong et al. Journal of Biomedical Materials Research, Vol. 20, pgs. 51-64, 1986)
  • the ligands which are covalently attached to the polymeric carrying agents may be any that are therapeutic and functional pharmacologic agents, and which bind to receptors on the surface of cells.
  • the terra therapeutic and functional pharmacologic agent means natural or synthetic substances which produce biological effects by stimulating or blocking specific cell surface receptors.
  • the substances may be of varied chemical structures, such as peptides, or other organic chemical structures. Desirably the avidity of such binding is K D less than 10 -5 M and preferably the binding would be even stronger, with K D less than 10 -8 M.
  • the ligands may be, for example, hormones, drugs, neurotransmitters or biologically active peptides or their chemical derivatives or analogs.
  • analogs of naturally occurring or synthetic ligands which retain the properties of specificity of binding to the ligand receptors and functionality as pharmacologic agents are used.
  • Such analogs may be designed with appropriate reactive groups for coupling to lipids or to other carrying agent monomers. See, for example, Snyder, Journal of Medicinal Chemistry, Vol. 26, pgs. 1667-1672, 1973.
  • conjugates formed by covalently joining the ligand and polymeric carrying agent retain binding and pharmacologic functionality characteristics. Covalently joined ligands can be routinely tested to insure that these properties are retained.
  • the preferred way to insure retention of receptor-binding and pharamcologic activity is to provide a ligand having a unique functional group for joining to the polymeric carrying agent.
  • Such functional groups should not be constituents of the binding or active portion of the ligand.
  • Covalent ligand-monomer conjugates of defined chemical structure, composition and purity are synthesized according to techniques known in the art.
  • the ligand should preferably have no more than about 10, and more preferably no more than about 2, identical functional groups. If there are multiple similarly reactive groups, then means must be taken to separate the reaction products. Once separated, each can be chemically characterized and tested for retention of binding to cell surface receptors. Such means of separation and testing are standard procedures which are known in the art, and certain techniques will be preferred depending on the compound. As the number of similarly reactive functional groups increases, however, such separation and testing become more difficult, and obtaining substantially pure receptor-targeted carriers becomes significantly more complicated.
  • the ligands may be covalently attached to the polymeric carrying agents either before or after polymerization, that is to say the ligand may be attached to carrying agent monomer molecules or to the subsequently prepared polymeric carrying agent. If first attached to monomers, then the ligand-monomer conjugates may be used alone or preferably are admixed with other free monomers and processed to form the polymeric carrying agent. Of course, the monomers used to prepare the conjugate need not be the same as the free monomers used when subsequently polymerizing the carrying agent. In fact, other monomers generally will be preferred in order to permit separate optimization of the preparation and properties of conjugates and polymeric carrying agents. The ratio of free monomers to ligand-monomer conjugates may vary.
  • At least one ligand-monomer will be incorporated per subsequently prepared carrying agent polymer.
  • the amount of ligand- monomer conjugates be from about 0.5 mol% to about 10 mol%.
  • a ligand bearing a carboxylic acid functional group can be coupled to a lipid bearing a primary amino group, resulting in an amide ligand-lipid conjugate; or the carboxyl group- containing ligand can be coupled to an hydroxyl-containing lipid, resulting in an ester ligand-lipid conjugate.
  • lipids which may be coupled to carboxylic acid-containing ligands include the naturally occurring stearylamine, phosphatidyl ethanolamine, sphinganine, and ceramide, as well as paraffin alcohols and other synthetic lipids.
  • an amino group-containing ligand can be coupled to a fatty acid or its active ester or acid chloride, again yielding an amide ligand-lipid conjugate; or a ligand bearing an hydroxyl group can be reacted with a fatty acid to yield an ester linked ligand-lipid conjugate.
  • Another means of coupling would involve nucleophilic substitution of electrophilic ligands, for example by thiol derivatives of lipids or phospholipids.
  • Lipids which may be coupled to nucleophilic ligands include phosphatidyl serine, fatty acids preferably having from 12 to 18 carbon atoms, and their active esters or acid chlorides (X). Standard reaction conditions are used depending upon the reactants.
  • Lipids for coupling to electrophilic ligands include thiol derivatives of phosphatidyl ethanolamine or other lipid. Such lipids have been described (Nature 288, 602-604, 1980).
  • polyalkyl cyanoacrylate or aerylamide nanoparticles may be attached to ligands by copolymerizing the acrylate monomer with a small amount (0.1 to 5%) of an active ester of acrylic acid, as described in Methods in Enzymology, vol. LXXXIII, pp. 306-310, 1982.
  • an amine containing ligand is reacted with the matrix and the ligand-polymer is purified by standard techiques.
  • a ligand-monomer conjugate is prepared using an amine containing ligand and the active acryloyl ester. This compound is then copolymerized with the acrylic acid monomer to form targeted nanoparticles.
  • the specific reactions to be employed will vary with the compounds used and are known in the art.
  • the drug which is chosen to be associated with (carried by) the carrying agent may or may not interact with the same cell surface receptor as does the ligand which is covalently bound to the outer surface of the carrying agent.
  • insulin could be used to target liposomes, by being covalently bound to the liposomes, and could also be delivered by the liposomes to the target tissue or organ.
  • Association of the drug to the carrying agent can occur, for example, by the drug filling interstitial spaces of the carrying agent, such that the carrying agent physically entraps the drug, or by covalent, ionic, or hydrogen bonding, or by means of adsorption by non-specific bonds. Whatever the mode of association, the drug must retain its therapeutic or diagnostic properties.
  • the drugs may be solid or liquid, hydrophobic, hydrophilic or both.
  • the dosages of drug associated with the carrying agent which are to be administered are those required to deliver an effective amount of the drug to the target tissue or organ. Such amounts are determined clinically. Such amounts are determined clinically.
  • the same considerations of toxicity and efficacy apply for the carrying agent- associated drug as for the free drug. In general lower dosages may suffice as compared to administration of free drug, because the carrying agent will stabilize the drug in the body, making it more resistant to degradation.
  • the enhanced delivery efficiency due to the targeting of the drug will provide the same effective concentration of a drug to a localized area of the body even when using a lower total dosage as computed per body weight.
  • two classes of drugs are contemplated for use in the present invention: bioaffecting molecules and diagnostic molecules.
  • Bioaffecting molecules are any which affect cell and body functions, either positively or negatively. This class includes cytotoxics, cytostatics, hormones, neurotransmitters, biologically active peptides and the like. Diagnostic molecules are those which can be detected in the body without recourse to invasive procedures such as surgery. Such molecules include fluorescent compounds, radiolabeled compounds, X-ray opaque dyes, ferromagnetic compounds, and the like. A compendium of drugs which may be used is found in Gilmah et al., Goodman and Gilman's The Pharmacologic Basis of Therapeutics, MacMillan, New York, 1980.
  • Administration of the receptor-targeted drug delivery system may be by any of the various means known in the art. Such means include but are not limited to: nebulizers, eye drops, topically, by implant, intraperitoneally, intramuscularly, intravenously, and orally. The following examples are not meant to limit the scope of the invention, but only to more fully illustrate the invention.
  • N 6 -(4-carboxymethyl)phen ⁇ ladenosine (la in figure 2) and distearyl phosphatidyl ethanolamine (DSPE) was performed as follows. To la (0.123 mmol) in dimethyl formamide (DMF) (10 ml) at 4°C were added sequentially with stirring hydroxybenzyltriazole and 1-ethyl-3-(3-dimethylaminopro ⁇ yl)carbodiimide (0.15 mmol of each). After 30 min the solution was warmed to 23°C and DSPE (0.125 mmol) and triethylamine (15 mmol) dissolved in CHCI 3 were added. The reaction was stirred under an argon atmosphere for 16 h.
  • DMF dimethyl formamide
  • DSPE 1-ethyl-3-(3-dimethylaminopro ⁇ yl)carbodiimide
  • the product was a ninhydrin negative, phosphate positive spot (R f 0.44) on thin-layer chromatography using silica gel developed in CHCl 3 /MeOH/water (60/30/5.) It forms a white emulsion in aqueous solutions.
  • SUV small unilamellar vesicles
  • 9.5 umol phosphatidyl choline labeleled with tritium
  • 10 umol cholesterol and 0.5 umol of conjugate 1b
  • Figure 2. The resulting lipsomes were purified by chromatography over SephadexTM G25. Fractions containing 3 H-phos ⁇ hatidyl choline were extracted with CHCl 3 /MeOH (2/1) and water. The organic layer was dried and resuspended in 95% ethanol and the UV spectrum was obtained. It revealed a peak at 303 nm corresponding to quantitative recovery of (1b.)
  • the affinity of ligand-carrying agent monomers conjugates for membrane receptors was determined by comparing their ability to inhibit radiolabeled ligands from binding to their specific receptors.
  • Stearyl-pindolol (3b) shows an IC 50 (concentration which produces 50% inhibition) for the beta-adrenergic receptor of about 500 nM and in the presence of the solubilizer HPBCD about 20nM.
  • the adenosine-lipid conjugate shows an IC 50 of ahout 50 nM for adenosine (A 1 ) receptors.
  • Standard receptor binding assays were performed with 125 I- cyanopindolol (50 pM) (compound A) and rat lung membrane homogenates and with 3 H-1-PIA phenyl isopropyl adenosine (compound B) (2 nM) and calf cortex membrane homogenates. The results are shown in Figures 3A and 3B, respectively. Varying concentrations of ligand-lipid conjugates or other compounds were mixed with the radio-ligand before the addition of membranes. Specific binding is defined by the binding inhibited by the addition of 1uM pindolol in assays of compound A and 10uM 1-PIA (phenylisopropyl adenosine) in assays of compound B. Percent binding is defined as the binding at a given concentration of inhibitor compared to specific binding.
  • Small unilamellar vesicles as described above were prepared using distearyl phosphatidyl choline (20 umol) and stearyl pindolol (0.2 umol) with cholesterol (20 umol). Trace quantities of 3 H- phosphatidyl choline were used as a marker of the lipid membranes. Carboxyfluorescein (0.15 M) was included in the aqueous phase. Equal quanities of liposomes were loaded on a molecular sieve column (Sephadex G25, 1.5 x 30 cm). Fractions were assayed for 3 H and fluorescence. The fluorescence measurements plotted in Figure 4 were obtained after releasing encapsulated dye with Triton X-100 (1%). Over 90% of the fluorescence was quenched for both types of liposomes.
  • Phosphatidyl choline cholesterol REV liposomes (as described by Papahadjopolous, Proceedings of the National Academy of Science, USA. 75, pp. 4194-4198, 1978) with or without 1 mol% stearyl pindolol were prepared containing 45 Ca-EDTA.
  • the liposomes were purified by repeated centrifugation and then dialyzed to remove unencapsulated 45 Ca-EDTA. Aliquots of the liposomes were placed into dialysis bags at 4°C or 37°C and transferred to new dialysate at the intervals plotted in Figure 5. Released 45 Ca is defined as the radioactivity measured in the dialysate.
  • SUV liposomes prepared as described above composed of distearyl phosphatidyl choline and cholesterol and supplemented with either adenosine-phosphtidyl ethanolamine (adenosine-PE) 1b, biotin-PE or stearylpindolol (3b) at 1 mol% were prepared and purified.
  • the liposomes were added to standard receptor binding assays using 3 H-1-PIA (phenyl isopropyl adenosine) and calf cortex membranes with 1-PIA (10 mM) used as blank.
  • Figure 7 shows the inhibition of receptor binding to 3 H-1-PIA which is caused by the targeted liposomes.
  • Liposomes containing adenosine-PE but not stearyl-pindolol or biotin-PE compete effectively for adenosine A 1 receptors.
  • Example 10 Liposomes containing adenosine-PE but not stearyl-pindolol or biotin-PE compete effectively for adenosine A 1 receptors.
  • SUV liposomes were prepared as described above with egg PC (phosphatidyl choline) and cholesterol and varying concentrations of stearyl-pindolol (0-10 mol%) as indicated. Varying quantities of liposomes were added to standard receptor binding assays using 125 I- cyanopindolol and rat lung membranes. Liposomes containing stearylpindolol inhibit binding in a dose-dependent manner whereas liposomes lacking stearyl-pindolol do not. Maximal inhibition obtained by the liposomes was only approximately 50% of specific 125 I-cyanopindolol binding, perhaps due to geometric constraints on the accessibility of liposomes to membrane receptors. Example 11.
  • Distearyl phosphatidyl choline (DSPC) and cholesterol liposomes containing methotrexate supplemented with nothing, stearyl-pindolol or adenosine-PE at 1 mol% were added to N18 tg2 mouse neuroblastoma for 48 h. In control experiments free methotrexate was used. In two experiments, before addition of methotrexate targeted stearyl-pindolol liposomes, either free pindolol (10 mM) or empty stearyl-pindolol liposomes were added. Figure 9 shows the results.

Abstract

Drug carrying such as liposomes and nanoparticles may be targeted to specific tissues or organs of the body by covalent attachment of therapeutic and pharmacologic ligands which retain high affinity for their receptors as well as retaining pharmacological activity. The ligands may be conjugated to monomers before they are assembled into the liposome or nanoparticle type structure. Drugs which may be carried by these carrying agents may be therapeutic or diagnostic materials.

Description

RECEPTOR-TARGETED DRUG DELIVERY SYSTEMS
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the field of human diagnostics and therapeutics. Specifically, the invention relates to the targeting of drugs to particular body locations. BACKGROUND OF THE INVENTION
Liposomes are synthetic lipid vesicles, which can encapsulate a variety of sizes of molecules in their internal spaces. Liposomes have been widely studied as carriers for delivering substances to cells and tissues in the body. (Gregoriadis, Liposome Technology, Vol. I, II, III, CRC Press, Boca Raton, 1985.) Nanoparticles, like liposomes, also have been studied for use as drug carriers. Nanoparticles are made of synthetic polymeric substances such as polycyanoacrylate, polyanhydrides of aromatic and aliphatic dicarboxylic acids, polymethylcyanoacrylate and the like. They may also be formed of proteins such as albumin and gelatin. (Polymeric Nanoparticles and Microspheres, Boca Raton, CRC Press, 1986.) Nanoparticles may entrap or adsorb the drugs which they carry.
Because it is often desired that a substance be localized within the body, means have been sought to target liposomes to particular tissues or organs of the body. One targeting means which has been explored employs antibodies attached to liposomal surfaces. This approach requires that there be tissue or organ-specific antibodies available as reagents. However, in many cases such antibodies are not available. The present invention provides an alternative means of targeting drug carrying agents which does not require tissue-specific antibodies. The present invention utilizes the specific interactions of therapeutic and functional pharmacological agents and their cell surface receptors.
Cells of many types, diseased as well as healthy, display recpectors on their surfaces which specifically interact with hormones, drugs, neurotransmitters and biologically active peptides. These receptors are expressed in a characteristic but heterogenous pattern among the cells of an organism. (Handbook of Chemical Anatomy, Vol. VIII, Classical Neurotransmitter and Neurotransmitter Receptors in the CNS, Elsevier, Amsterdam 1984.) For example, the receptors for betaadrenergic ligands are predominantly found in the lung, heart and adipose tissue, whereas opiate receptors are found in the gut, spinal cord, and on lymphocytes. Furthermore, certain cancerous tissues characteristically express receptors, for example, brain glioma expresses beta-adrenergic receptors (Receptor Binding Techniques, Society for Neuroscience, Washington, D.C. 1980) and bronchogenic carcinoma expresses receptors for certain biologically active peptides (Peptides, Vol. 4, pages 683-686, 1983.) SUMMARY OF THE INVENTION
It is an object of the present invention to provide a receptor- targeted drug delivery system.
It is an object of the present invention to provide a therapeutic method of treating humans having diseased organs or tissues.
It is an object of the present invention to provide a diagnostic method for determining whether a human has diseased organs or tissues.
It is another object of the invention to provide processes for making the receptor-targeted drug delivery system. These and other objects are achieved by the present invention which provides in one embodiment a receptor-targeted drug delivery system comprising: a polymeric drug carrying agent selected from the group consisting of liposomes and nanoparticles; a ligand capable of binding a receptor, said ligand being covalently attached to said carrying agent and being a therapeutic and functional pharmacological agent; and a drug, said drug being associated with said drug carrying agent.
In another embodiment a therapeutic method of treating a human having a diseased organ or tissue is provided, comprising: administering an effective amount of the receptor-targeted drug delivery system of the present invention to the human to affect the metabolism of cells expressing receptors for the ligand on their surfaces or nearby cells, said cells being among those of the diseased organ or tissue. In yet another embodiment a diagnostic method for determining whether a human has a diseased organ or tissue is provided, comprising: administering the receptor-targeted drug delivery system to the human; and noninvasively detecting the localization of said drug.
In addition, processes are taught for making the receptor- targeted drug delivery system of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts schemes for the synthesis of ligand-lipid conjugates.
Figure 2 shows examples of ligands with reactive functional groups and ligand-lipid conjugates made therefrom.
Figure 3 shows the affinity of ligand-lipid conjugates for their receptors. Figure 4 reports data demonstrating the formation of stearylpindolol-containing liposomes and the encapsulation of a fluorescent solute.
Figure 5 shows the stability of encapsulated solutes within liposomes composed of ligand-lipid conjugates, as measured by 45 Ca-EDTA release.
Figure 6 reports data showing that ligand-lipid conjugates are incorporated into the structure of the liposomes.
Figure 7 shows the affinity of adenosine phosphatidyl ethanolamine-bearing liposomes for adenosine receptors.
Figure 8 shows the affinity of stearyl-pindolol-bearing liposomes for beta-adrenergic receptors.
Figure 9 reports data showing the delivery of methotrexate to cells with beta-adrenergic receptors by ligand targeted liposomes containing methotrexate. DETAILED DESCRIPTION OF THE INVENTION
It is a discovery of the present invention that ligands having therapeutic and functional pharmacologic activity and capable of binding a receptor can be covalently joined to polymeric carrying agents to form ligand-polymeric carrying agent conjugates which retain their ability to bind to cell surface receptors. Drugs thus associated with these ligand-polymeric carrying agents are thereby delivered to cells bearing the corresponding cell surface receptor.
Carrying agents contemplated for use in the present invention are polymeric substances, although the polymers need not be covalent polymers. For example, liposomes are suitable polymeric carrying agents whose monomer molecules are generally lipids or phospholipids which are noncovalently joined. Alternatively, nanoparticles may be used as polymeric carrying agents. Nanoparticles are covalently linked polymers of monomers such as acrylates, cyanoacrylates, aromatic and aliphatic dicarboxylic acids and the like. The nanoparticles may be either homopolymers or heteropolymers. The size of the polymeric carrying agents may range from about 20 nm to about 10 u in diameter. Any size which would enable the polymeric carrying agents to circulate in the vasculature or other body compartment is suitable for most applications. However, for topical and other means of administration, even larger particles could be used.
Liposomes may be formed using standard methods such as the reverse evaporation method (REV) of Papahadjopolous (Proceedings of the National Academy of Sciences U.S.A., Vo. 75, pgs. 4194-4198, 1978), the dehydration-rehydration method (DRV) of Gregoriadas (U.K. Patent Application 8321012, 1983), sonication to form small unilamellar vesicles (SUV) (Biochemistry Vol. 8, pg. 344, 1967) or other suitable methods. The lipids employed in such liposomes will generally be naturally occurring lipids such as phosphatidylcholine, phosphatidylserine, sphingomyelin, cholesterol, their synthetic analogs, and mixtures thereof.
Nanoparticles may be formed using standard procedures taught in the art. For example, Brasseur et al. European Journal of Cancer, vol. 16, pgs. 1441-1445, 1980, teaches preparation of nanoparticles from methylcyanoacrylate by means of polymerization and filtration through a fritted glass filter. (See, for other examples, Couvreur, et al. Journal of Pharmacy and Pharmacology, Vol. 31, pgs. 331-332, 1979, and Leong et al. Journal of Biomedical Materials Research, Vol. 20, pgs. 51-64, 1986)
The ligands which are covalently attached to the polymeric carrying agents may be any that are therapeutic and functional pharmacologic agents, and which bind to receptors on the surface of cells. As used herein, the terra therapeutic and functional pharmacologic agent means natural or synthetic substances which produce biological effects by stimulating or blocking specific cell surface receptors. The substances may be of varied chemical structures, such as peptides, or other organic chemical structures. Desirably the avidity of such binding is KD less than 10-5M and preferably the binding would be even stronger, with KD less than 10-8M. The ligands may be, for example, hormones, drugs, neurotransmitters or biologically active peptides or their chemical derivatives or analogs. (See, Burger, A., Medicinal Chemistry, Third Ed., Vol. 2, John Wiley & Sons, New York 1970 and Snyder, S. Science, Vol. 209, pgs. 976-983, 1980.) Specific examples are beta-adrenergic compounds, insulin, growth hormone, analogs of adenosine, and opiate drugs. Receptors for these ligands are known, and some are discussed, for example, in Snyder, Science, Vol. 224, pgs. 22-31 (1984).
Typically, analogs of naturally occurring or synthetic ligands which retain the properties of specificity of binding to the ligand receptors and functionality as pharmacologic agents are used. Such analogs may be designed with appropriate reactive groups for coupling to lipids or to other carrying agent monomers. See, for example, Snyder, Journal of Medicinal Chemistry, Vol. 26, pgs. 1667-1672, 1973.
It is important that conjugates formed by covalently joining the ligand and polymeric carrying agent retain binding and pharmacologic functionality characteristics. Covalently joined ligands can be routinely tested to insure that these properties are retained.
The preferred way to insure retention of receptor-binding and pharamcologic activity is to provide a ligand having a unique functional group for joining to the polymeric carrying agent. Such functional groups should not be constituents of the binding or active portion of the ligand. Covalent ligand-monomer conjugates of defined chemical structure, composition and purity are synthesized according to techniques known in the art. In any event, the ligand should preferably have no more than about 10, and more preferably no more than about 2, identical functional groups. If there are multiple similarly reactive groups, then means must be taken to separate the reaction products. Once separated, each can be chemically characterized and tested for retention of binding to cell surface receptors. Such means of separation and testing are standard procedures which are known in the art, and certain techniques will be preferred depending on the compound. As the number of similarly reactive functional groups increases, however, such separation and testing become more difficult, and obtaining substantially pure receptor-targeted carriers becomes significantly more complicated.
The ligands may be covalently attached to the polymeric carrying agents either before or after polymerization, that is to say the ligand may be attached to carrying agent monomer molecules or to the subsequently prepared polymeric carrying agent. If first attached to monomers, then the ligand-monomer conjugates may be used alone or preferably are admixed with other free monomers and processed to form the polymeric carrying agent. Of course, the monomers used to prepare the conjugate need not be the same as the free monomers used when subsequently polymerizing the carrying agent. In fact, other monomers generally will be preferred in order to permit separate optimization of the preparation and properties of conjugates and polymeric carrying agents. The ratio of free monomers to ligand-monomer conjugates may vary. Preferably, at least one ligand-monomer will be incorporated per subsequently prepared carrying agent polymer. In the case of liposomes as the carrying agent, it is preferred that the amount of ligand- monomer conjugates be from about 0.5 mol% to about 10 mol%.
Generally, covalent coupling of ligands to polymeric carrying agents is accomplished using standard techniques of organic chemistry (See Figure 1). For example, a ligand bearing a carboxylic acid functional group can be coupled to a lipid bearing a primary amino group, resulting in an amide ligand-lipid conjugate; or the carboxyl group- containing ligand can be coupled to an hydroxyl-containing lipid, resulting in an ester ligand-lipid conjugate. For example, lipids which may be coupled to carboxylic acid-containing ligands include the naturally occurring stearylamine, phosphatidyl ethanolamine, sphinganine, and ceramide, as well as paraffin alcohols and other synthetic lipids. Generally such reactions require a carbodiimide reagent as a stoichiometric reagent. Conditions for these reactions are well known in organic chemistry and vary with the specific reactants. Alternatively, an amino group-containing ligand can be coupled to a fatty acid or its active ester or acid chloride, again yielding an amide ligand-lipid conjugate; or a ligand bearing an hydroxyl group can be reacted with a fatty acid to yield an ester linked ligand-lipid conjugate. Yet another means of coupling would involve nucleophilic substitution of electrophilic ligands, for example by thiol derivatives of lipids or phospholipids. Lipids which may be coupled to nucleophilic ligands include phosphatidyl serine, fatty acids preferably having from 12 to 18 carbon atoms, and their active esters or acid chlorides (X). Standard reaction conditions are used depending upon the reactants. Lipids for coupling to electrophilic ligands include thiol derivatives of phosphatidyl ethanolamine or other lipid. Such lipids have been described (Nature 288, 602-604, 1980).
As recognized by one skilled in the art, similar reactions also are used to join ligands to synthetic monomers to form nanoparticles and to join ligands to pre-formed liposomes and nanoparticles. As a specific example of ligand-targeted nanoparticles, polyalkyl cyanoacrylate or aerylamide nanoparticles may be attached to ligands by copolymerizing the acrylate monomer with a small amount (0.1 to 5%) of an active ester of acrylic acid, as described in Methods in Enzymology, vol. LXXXIII, pp. 306-310, 1982. After formation of polymer and processing to form nanoparticles, an amine containing ligand is reacted with the matrix and the ligand-polymer is purified by standard techiques. Alternatively, a ligand-monomer conjugate is prepared using an amine containing ligand and the active acryloyl ester. This compound is then copolymerized with the acrylic acid monomer to form targeted nanoparticles. The specific reactions to be employed will vary with the compounds used and are known in the art.
The drug which is chosen to be associated with (carried by) the carrying agent may or may not interact with the same cell surface receptor as does the ligand which is covalently bound to the outer surface of the carrying agent. For example, insulin could be used to target liposomes, by being covalently bound to the liposomes, and could also be delivered by the liposomes to the target tissue or organ. Association of the drug to the carrying agent can occur, for example, by the drug filling interstitial spaces of the carrying agent, such that the carrying agent physically entraps the drug, or by covalent, ionic, or hydrogen bonding, or by means of adsorption by non-specific bonds. Whatever the mode of association, the drug must retain its therapeutic or diagnostic properties. The drugs may be solid or liquid, hydrophobic, hydrophilic or both.
The dosages of drug associated with the carrying agent which are to be administered are those required to deliver an effective amount of the drug to the target tissue or organ. Such amounts are determined clinically. Such amounts are determined clinically. The same considerations of toxicity and efficacy apply for the carrying agent- associated drug as for the free drug. In general lower dosages may suffice as compared to administration of free drug, because the carrying agent will stabilize the drug in the body, making it more resistant to degradation. In addition, the enhanced delivery efficiency due to the targeting of the drug will provide the same effective concentration of a drug to a localized area of the body even when using a lower total dosage as computed per body weight. In general, two classes of drugs are contemplated for use in the present invention: bioaffecting molecules and diagnostic molecules. Bioaffecting molecules are any which affect cell and body functions, either positively or negatively. This class includes cytotoxics, cytostatics, hormones, neurotransmitters, biologically active peptides and the like. Diagnostic molecules are those which can be detected in the body without recourse to invasive procedures such as surgery. Such molecules include fluorescent compounds, radiolabeled compounds, X-ray opaque dyes, ferromagnetic compounds, and the like. A compendium of drugs which may be used is found in Gilmah et al., Goodman and Gilman's The Pharmacologic Basis of Therapeutics, MacMillan, New York, 1980.
Administration of the receptor-targeted drug delivery system may be by any of the various means known in the art. Such means include but are not limited to: nebulizers, eye drops, topically, by implant, intraperitoneally, intramuscularly, intravenously, and orally. The following examples are not meant to limit the scope of the invention, but only to more fully illustrate the invention.
Example 1. Synthesis of Adenosine-Phosphatidyl Ethanolamine (Adenosine-PE)
Coupling of N6-(4-carboxymethyl)phenγladenosine (la in figure 2) and distearyl phosphatidyl ethanolamine (DSPE) was performed as follows. To la (0.123 mmol) in dimethyl formamide (DMF) (10 ml) at 4°C were added sequentially with stirring hydroxybenzyltriazole and 1-ethyl-3-(3-dimethylaminoproρyl)carbodiimide (0.15 mmol of each). After 30 min the solution was warmed to 23°C and DSPE (0.125 mmol) and triethylamine (15 mmol) dissolved in CHCI3 were added. The reaction was stirred under an argon atmosphere for 16 h. The solvents were evaporated, and the isolated oil product was dissolved in a mixture of CHCl3/MeOH (2/1) and extracted three times with ice-cold one-tenth saturated NaHCO3, and then with water. The resulting white powder was chromatographed over silica gel using CHCl3/MeOH/water (60/30/5) as solvent to yield 33 mg pure adenosine-phosphatidylethanolamine (1b) (23% final yield). The product was a ninhydrin negative, phosphate positive spot (Rf 0.44) on thin-layer chromatography using silica gel developed in CHCl3/MeOH/water (60/30/5.) It forms a white emulsion in aqueous solutions. Proton nmr (dgDMSO) PPm 8.54 and 8.39 (each
Figure imgf000024_0001
s, 1H,adenine), 8.21 (t,1H, amide), 7.82 and 7.20 (each d, 2H,aromatic), 5.93 (d,1H, ribose-C1), 0.85-1.49 (m,alkyl). UV (95% ethanol) 303C(log e = 4.36). C55H99O13N6P1, predicted 1130, [M+H]+ (FAB) 1131.
Example 2.
Synthesis of Palmityl-naltrexamine
Acylation of 6-beta -naltrexamine (2a.) with palmityl chloride was performed as follows. Naltrexamine free base (0.24 mmol) in tetrahydrofuran (THF) (2 ml) was cooled to -10°C and palmityl. chloride. (0.26 mmol) was added. The reaction was stirred for 2 h and then at 23ºC for 16h. After evaporation of the solvent and dissolution in methanol, NH4OH (2 drops) was added and stirred 16 h. The solvents were evaporated and the oil was chromatographed on silica gel with a CHCI3 wash and then eluted wth ethylacetate/methanol/ammonia (100/10/3). The peak fractions contained about 80% yield of palmitylnaltrexamine (2b in figure 2.) C34H51N2O4 calculated 551.8. Example 3. Synthesis of Stearyl-Pindolol
Alkylation of stearyl mercaptan with bromoacetylindolementhane (3a in Figure 2) was performed as follows. The Na-salt of stearyl mercaptan (0.12 mmol) was prepared in NaOCH3 in methanol (5 ml). Toluene (5 ml) was added and 3a in methanol (0.12 mmol) was added. The reaction mixture was stirred at room temperature under an argon atmosphere for 24 h. After evaporation and dissolution in CHCI3, the material was extracted with water, dried over Na2SO3 and evaporated to yield a crude oil. The product was isolated from silica column chromatography using CHCl3/methanol (9/1) solvent. Proton nmr (CDCI3 ppm 7.17 - 6.55 (m,6H, aromatic), 4.12-4.03 (m,3H,-CH2CH(OH)-), 3.60 (broad s, 2H,-NH), 3.05 (s, 2H, -COCH2S-), 1.24-0.85 (m, 55H, cycloheaxane and -CH2- and -CH3). UV (MeOH) 265 (6865), 287 (3430). C41H71O3N2S1, predicted 672.07, [M+H]+ (FAB), 687. Example 4.
Ligand-Lipid Incorporated into Liposomes
SUV (small unilamellar vesicles) were formed by the technique of Biochemistry, 8 , pg. 344, 1967, using 9.5 umol phosphatidyl choline (labeled with tritium), 10 umol cholesterol and 0.5 umol of conjugate 1b (Figure 2.) The resulting lipsomes were purified by chromatography over Sephadex™ G25. Fractions containing 3H-phosρhatidyl choline were extracted with CHCl3/MeOH (2/1) and water. The organic layer was dried and resuspended in 95% ethanol and the UV spectrum was obtained. It revealed a peak at 303 nm corresponding to quantitative recovery of (1b.)
Example 5
Affinity of Ligand-Carrying Agent Monomer Conjugates for Receptors
The affinity of ligand-carrying agent monomers conjugates for membrane receptors was determined by comparing their ability to inhibit radiolabeled ligands from binding to their specific receptors. Stearyl-pindolol (3b) shows an IC50 (concentration which produces 50% inhibition) for the beta-adrenergic receptor of about 500 nM and in the presence of the solubilizer HPBCD about 20nM. The adenosine-lipid conjugate shows an IC50 of ahout 50 nM for adenosine (A1) receptors.
Standard receptor binding assays were performed with 125I- cyanopindolol (50 pM) (compound A) and rat lung membrane homogenates and with 3H-1-PIA phenyl isopropyl adenosine (compound B) (2 nM) and calf cortex membrane homogenates. The results are shown in Figures 3A and 3B, respectively. Varying concentrations of ligand-lipid conjugates or other compounds were mixed with the radio-ligand before the addition of membranes. Specific binding is defined by the binding inhibited by the addition of 1uM pindolol in assays of compound A and 10uM 1-PIA (phenylisopropyl adenosine) in assays of compound B. Percent binding is defined as the binding at a given concentration of inhibitor compared to specific binding.
Example 6.
Formation of Stearyl-Pindolol Liposomes and Encapsulation of Solute
Small unilamellar vesicles (SUV) as described above were prepared using distearyl phosphatidyl choline (20 umol) and stearyl pindolol (0.2 umol) with cholesterol (20 umol). Trace quantities of 3H- phosphatidyl choline were used as a marker of the lipid membranes. Carboxyfluorescein (0.15 M) was included in the aqueous phase. Equal quanities of liposomes were loaded on a molecular sieve column (Sephadex G25, 1.5 x 30 cm). Fractions were assayed for 3H and fluorescence. The fluorescence measurements plotted in Figure 4 were obtained after releasing encapsulated dye with Triton X-100 (1%). Over 90% of the fluorescence was quenched for both types of liposomes.
Example 7.
Stable Encapsulation of Solutes into Liposomes Composed of Ligand-Lipid Conjugates .
Phosphatidyl choline: cholesterol REV liposomes (as described by Papahadjopolous, Proceedings of the National Academy of Science, USA. 75, pp. 4194-4198, 1978) with or without 1 mol% stearyl pindolol were prepared containing 45Ca-EDTA. The liposomes were purified by repeated centrifugation and then dialyzed to remove unencapsulated 45Ca-EDTA. Aliquots of the liposomes were placed into dialysis bags at 4°C or 37°C and transferred to new dialysate at the intervals plotted in Figure 5. Released 45Ca is defined as the radioactivity measured in the dialysate. Example 8. Incorporation of Stearyl-pindolol Conjugate into Liposomes Phosphatidyl choline: cholesterol SUV liposomes were prepared using 5 mol% stearyl-pindolol and trace 3H-phosphatidyl choline and chromatographed over Sephadex G25. The fractions containing the liposomes were pooled and rechromatographed and the resulting fractions were assayed for 3H and quantities of stearyl-pindolol using a radioreceptor assay (Life Sciences 23, 2031-2038, 1978). Results are shown in Figure 6. Example 9.
Affinity of the Adenosine-Targeted Liposomes for the Adenosine (A1) Receptor
SUV liposomes prepared as described above composed of distearyl phosphatidyl choline and cholesterol and supplemented with either adenosine-phosphtidyl ethanolamine (adenosine-PE) 1b, biotin-PE or stearylpindolol (3b) at 1 mol% were prepared and purified. The liposomes were added to standard receptor binding assays using 3H-1-PIA (phenyl isopropyl adenosine) and calf cortex membranes with 1-PIA (10 mM) used as blank. Figure 7 shows the inhibition of receptor binding to 3H-1-PIA which is caused by the targeted liposomes.
Liposomes containing adenosine-PE but not stearyl-pindolol or biotin-PE compete effectively for adenosine A1 receptors. Example 10.
Affinity of Stearyl-Pindolol Liposomes for the Beta-Adrenergic Receptor
SUV liposomes were prepared as described above with egg PC (phosphatidyl choline) and cholesterol and varying concentrations of stearyl-pindolol (0-10 mol%) as indicated. Varying quantities of liposomes were added to standard receptor binding assays using 125I- cyanopindolol and rat lung membranes. Liposomes containing stearylpindolol inhibit binding in a dose-dependent manner whereas liposomes lacking stearyl-pindolol do not. Maximal inhibition obtained by the liposomes was only approximately 50% of specific 125I-cyanopindolol binding, perhaps due to geometric constraints on the accessibility of liposomes to membrane receptors. Example 11.
Delivery of Methotrexate to Cells with B-Adrenergic Receptors by Targeted Liposomes.
Distearyl phosphatidyl choline (DSPC) and cholesterol liposomes containing methotrexate supplemented with nothing, stearyl-pindolol or adenosine-PE at 1 mol% were added to N18 tg2 mouse neuroblastoma for 48 h. In control experiments free methotrexate was used. In two experiments, before addition of methotrexate targeted stearyl-pindolol liposomes, either free pindolol (10 mM) or empty stearyl-pindolol liposomes were added. Figure 9 shows the results.
While specific embodiments have been given above, they do not limit the scope of the invention which is defined by the appended claims.

Claims

CLAIMS 1. A receptor-targeted drug system, comprising: a polymeric drug carrying agent selected from the group consisting of liposomes and nanoparticles. a ligand capable of binding a receptor, said ligand being covalently attached to said carrying agent, and being a therapeutic and functional pharmacological agent; and a drug, said drug being associated with said drug carrying agent.
2. The receptor-targeted drug delivery system of claim 1, wherein said drug affects the cell and body functions.
3. The receptor-targeted drug delivery system of claim 1, wherein the localization of said drug can be detected noninvasively.
4. The receptor-targeted drug delivery system of claim 2, wherein the drug is cytotoxic.
5. The receptor-targeted drug delivery system of claim 2, wherein the drug is cytostatic.
6. The receptor-targeted drug delivery system of claim 2, wherein the drug is a hormone.
7. The receptor-targeted drug delivery system of claim 2, wherein the drug is a neurotransmitter.
8. The receptor-targeted drug delivery system of claim 2, wherein the drug is a neuropeptide.
9. The receptor-targeted drug delivery system of claim 3, wherein the drug is fluorescent-labelled.
10. The receptor-targeted drug delivery system of claim 3, wherein the drug is radio-labelled.
11. The receptor-targeted drug delivery system of claim 3, wherein the drug is opaque to X-rays.
12. The receptor-targeted drug delivery system of claim 1, wherein said ligand and said drug are both capable of binding the same receptor.
13. The receptor-targeted drug delivery system of claim 1, wherein the drug carrying agent is a liposome.
14. The receptor-targeted drug delivery system of claim 1, wherein the drug carrying agent is a nanoparticle.
15. A therapeutic method of treating a human having a diseased organ or tissue, comprising: administering an effective amount of the drug delivery system of claim 2 to the human to affect the metabolism of the cells expressing the receptors on their surfaces, said cells being in the diseased organ or tissue.
16. A diagnostic method for determining whether a human has a diseased organ or tissue, comprising: administering the drug delivery system of claim 3 to the human; and noninvasively detecting the localization of said drug.
17. A process for making the receptor-targeted drug delivery system of claim 1, comprising: covalently joining the ligand to the polymeric drug carrying agent, to form ligand-carrying agent conjugates wherein substantially all of said conjugates retain the ability to bind receptors; mixing the ligand-carrying agent conjugates with the drug under conditions to associate the drug with the carrying agent.
18. The process of claim 17, wherein said drug becomes associated with said carrying agent by means of physical entrapment.
19. The process of claim 17, wherein said drug becomes associated with said carrying agent by means of adsorption.
20. A process for making the receptor-targeted drug delivery system of claim 1, comprising: covalently joining the ligand to drug carrying agent monomer molecules to form ligand-monomer conjugates, wherein substantially all of said conjugates retain the ability to bind receptors; assembling said ligand-monomer conjugates in the presence of drug under conditions to form drug-associated ligand-polymeric carrying agents.
21. The process of claim 20, wherein said drug is associated with said ligand-polymer carrying agents by means of entrapment.
22. The process of claim 20, wherein said drug is associated with said ligand-polymer carrying agents by means of adsorption.
23. The receptor-targeted drug delivery system of claim 13 wherein the ligand is N6-p-carboxymethyl phenyladenosine.
24. The receptor-targeted drug delivery system of claim 13 wherein the ligand is 6-beta-naltrexamine.
25. The receptor-targeted drug delivery system of claim 13 wherein the ligand is bromoacetyl indole menthane.
26. The receptor-targeted drug delivery system of claim 13 wherein the ligand is capable of binding to the beta-adrenergic receptor.
27. The receptor-targeted drug delivery system of claim 13 wherein the ligand is capable of binding to the adenosine receptor.
28. The receptor-targeted drug delivery system of claim 13 wherein the ligand is capable of binding to the opiate receptor.
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