United States Patent:  6,054,133

Assignee:  The Regents of the University of California (Oakland, CA)

Filed:  July 10, 1997

A method and composition are provided for treating intracellular pathogens that reside in phagosomes. The compositions include antibiotics which are conjugated with transferrin or other ligands to form conjugates that target membrane-bound pathogens. The conjugates are selectively taken up by infected phagosomes. Conjugates are provided which utilize transferrin as the targeting ligand for treating mycobacterium which reside in membrane-bound phagosomes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the use of transferrin as a targeting vehicle to provide selective delivery of antibiotics to phagosomes which are infected with a pathogen. Transferrin is a serum beta-globulin that transports iron. Transferrin is widely available and can be obtained commercially from a number of sources. For example, transferrin can be obtained from Sigma Chemical Co. (St. Louis, Mo.). Human holo-transferrin is preferred. It is also preferred that multimeric aggregates be removed by centrifuging prior to coupling with antibiotic. Human holotransferrin can also be obtained from Boehringer Mannheim Corporation (Indianapolis, Ind.), Calbiochem (LaJolla, Calif.), or Life Technologies (Gaithersburg, Md.).

In addition, recombinant human transferrin can be expressed by genetic engineering techniques in eukaryotic or prokaryotic cells, purified and loaded with iron. Such recombinant human transferrin can be designed to have other features which will facilitate subsequent coupling of antibiotic drugs to the transferrin. For example, such a recombinant transferrin hybrid molecule can be designed to include an antibody binding moiety or other form of binding moiety capable of binding an antibiotic, or modified antibiotic (e.g. dansylated antibiotic), or the hybrid molecule could be designed to contain a peptide antibiotic.

Transferrin receptors are synthesized in the host cell endoplasmic reticulum (ER) and transported via the golgi to the host cell surface. There, they bind their natural ligand, iron-saturated transferrin, after which the receptor-ligand complexes are internalized in coated vesicles that fuse with early endosomes. Acidification of the early endosome to pH 6.5 to 6.0 causes release of iron from the transferrin. From the endosomal compartment, the apotransferrin-transferrin receptor complexes are rapidly recycled to the plasma membrane (9,10). Transferrin and the transferrin receptor do not traffic through the lysosomal compartment (9,10).

With respect to M. tuberculosis, it is believed that there are three possible sources of the transferrin receptors observed on the phagosome. First, the receptors may be derived by fusion of the phagosome with vesicles coming from the trans-golgi network. Second, the receptors may be derived from the plasma membrane during phagocytosis, and be retained on the phagosome. Third, the receptors may be derived from phagosome interaction with early endosomes. To distinguish the first two from the third possibilities, we administered transferrin exogenously to M. tuberculosis-infected macrophages and examined phagosomes for the presence or absence of transferrin. If transferrin receptors are derived from trans-golgi network or the plasma membrane during phagocytosis, then a pulse of transferrin administered after phagocytosis should not be delivered to the phagosome. On the other hand, if transferrin receptors are derived from phagosome-endosome interaction, exogenously administered transferrin should be delivered to the M. tuberculosis phagosome. In accordance with the present invention, it was found that exogenously administered transferrin is delivered to the M. tuberculosis phagosome, hence demonstrating directly that the M. tuberculosis phagosome interacts with endosomes.

Transferrin is coupled to the antibiotic to form a antibiotic-transferrin conjugate. Antibiotics which may be coupled to transferrin include any of the known antibiotics which are effective in treating a wide variety of diseases in humans and animals including tuberculosis (caused by Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria), leprosy, Mycobacterium avium complex (MAC) infections, Mycobacterium marinum infection, Mycobacterium fortuitum infection, Mycobacterium Kansaii infection, brucellosis, Q fever, tularemia, salmonellosis, typhoid fever, Yersinia infections (including Y. pestis, Y. enterocolitica and Y. pseudotuberculosis), ehrlichiosis, chlamydiosis (including C. psittaci, C. trachomatis), histoplasmosis, toxoplasmosis and leishmaniasis.

Exemplary antibiotics include rifampin, rifabutin, isoniazid, ethambutol, pyrazinamide, thiacetazone, para-aminosalicylic acid, aminoglycosides (including gentamycin, streptomycin, amikacin, kanamycin, viomycin, capreomycin, quinolones (including ciprofloxacin, ofloxacin), ethionamide, prothionamide, cycloserine, dapsone, clofazimine, sparfloxacin, minocycline, clarithromycin, azithromycin, doxycycline, cefoxitin, tetracyclines, cefotaxime, fluoroquinolones, ceftriaxone, chloramphenicol, trimethaprim-sulfamethoxazole, ampicillin, sulfonamides, amoxicillin, ketoconazole, itraconazole, fluconazole, pyrimethamine sulfadiazine, clindamycin, atovaquone sodium stibogluconate, antimonials, amphotericin B, pentamidine, polymixin definsins and other peptide antibiotics used to treat intracellular pathogens. Preferred antibiotics are those which are designed to treat diseases that are caused by intracellular pathogens, especially those that reside within phagosomes during their life cycle in host cells.

The antibiotic is coupled to the native or recombinant transferrins by any of the known coupling mechanisms including antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, and particulate surface adsorption (e.g. colloidal gold).

The resulting antibiotic-transferrin conjugant is delivered to the patient in accordance with any of the conventional drug delivery systems. Exemplary delivery methods include intravenous injection, intramuscular injection, subcutaneous injection, intracutaneous injection, intrathecal injection, intravesicular injection, intraocular injection, intraarticular injection, intraperitoneal injection, intrapleural injection, intraarterial injection, intraumbilical injection, oral ingestion, intranasal administration, inhalation (e.g. aerosol inhalation), intrarectal administration, and subconjunctival administration. The antibiotic-transferrin conjugant is preferably mixed with a pharmaceutically acceptable carrier in accordance with well-known procedures to place the drug in a form which can be administered effectively to the patient. Buffered saline and other similar carriers are preferred.

The amount of antibiotic transferrin conjugant which is administered will vary depending upon the particular conjugant and the disease being treated. The dosage levels can be determined by routine experimentation in accordance with well-known protocols for establishing dosage levels.

The studies described herein demonstrate that endosomes interact with the M. tuberculosis phagosome and, more importantly, deliver ligands bound to endosomal receptors to the M. tuberculosis phagosome. In the example described, the receptor is the transferrin receptor and the ligand is transferrin. However, endosomes have other receptors that bind other ligands. For example, endosomes have LDL (low density lipoprotein) receptors that bind LDL. Such ligands also can be delivered to the M. tuberculosis phagosome. Hence, as with transferrin, antibiotics can be coupled to LDL or other ligands that target endosomes. The same procedures described herein for coupling antibiotics to transferrin may be used to couple antibiotics to LDL or other ligands that target endosomes.

Claim 1 of 15 Claims

1. A composition for use in delivering an antiboitic to a phagosome which contains a pathogen, said composition comprising an antibiotic coupled to a ligand selected from the group consisting of transferrin and low density lipoprotein.

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