Link:  Pharm/Biotech Resources

Title:  Intracellular delivery of biological effectors

United States Patent:   6,960,648

Issued:  November 1, 2005

Inventors:  Bonny; Christophe (Lausanne, CH)

Assignee:  Universite de Lausanne (Lausanne, CH)

Appl. No.:  977831

Filed:  October 15, 2001

The invention relates to a sequence of amino acids with the capacity to facilitate transport of an effector across a biological membrane. More specifically, the present invention relates to novel peptide transporters that specifically target certain cell types for the intracellular delivery of drugs and therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides transporter peptides which are capable of translocating across a biological membrane. The invention also relates to methods of using such transporter peptides to translocate an effector across a biological membrane.

In one aspect, the invention involves transporter peptides having at least one amino acid sequence selected from:(X_mRX_oRX_n); (X_mRRRX_n); (X_mRRXRX_n); and(X_mRXRRX_n), where X is a non-basic amino acid; m is an integer from zero to fourteen; n is an integer, independent of m, between zero and fourteen; o is an integer, independent of m and n, between zero and five; and wherein the transporter peptide is capable of translocating across a biological membrane.

In one embodiment, the invention provides a transporter peptide having the amino acid sequence R-X-X-R. In other embodiments, the invention provides a transporter peptide having an amino acid sequence of any one of SEQ ID NOS: 1-34. In various other embodiments, the transporter peptides are derived from protein convertase ligands. In still other embodiments, the transporter peptides are derived from protein convertase cleavage sites.

As used herein, a transporter peptide is a peptide that facilitates the translocation of a substance across a biological membrane.

In some embodiments, the transporter peptide is fused to an effector. The "effector" can be any suitable molecule, including DNA, RNA, a protein, a peptide, or a pharmaceutically active agent, such as, for example, a toxin, an antibiotic, an antipathogenic agent, an antigen, an antibody, an antibody fragment, an immunomodulator, an enzyme, or a therapeutic agent.

The term "fusion" or "fused" is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic, and hydrogen bonding, but does not include non-specific associations such as solvent preferences.

In various embodiments, the transporter peptide can be less than fifty (50), less than twenty-five (25), or less than fifteen (15) amino acids in length.

In further embodiments, translocation occurs within pancreatic B-cells, hepatocytes, colon cells, muscle cells and/or lung cells.

In another embodiment, the invention involves a method of translocating a transporter peptide across a biological membrane. For example, peptides of SEQ ID NOS: 1-6 can be translocated across a membrane of pancreatic B-cells; peptides of SEQ ID NOS: 7-10 can be translocated across a membrane of hepatocytes; the peptide of SEQ ID NO:11 can be translocated across a membrane of colon cells; peptides of SEQ ID NOS: 12-20 can be translocated across a membrane of muscle cells; and peptides of SEQ ID NOS: 21-34 can be translocated across a membrane of lung cells.

In yet another embodiment, the invention involves a transporter unit that is a transporter peptide conjugated to an effector. In various other embodiments, the effector may be a nucleic acid, a peptide, or a pharmaceutically active agent.

In still a further embodiment, the invention includes a method of producing a translocatable conjugate between a transporter peptide and an effector, forming a transporter peptide-effector conjugate. As used herein, "conjugate" or "conjugation" means any type of interaction enabling a physical association between an effector and a transporter peptide. The association may be covalent or a non-covalent in nature, and it must be sufficiently strong so that the vector does not disassociate before or during cellular penetration. Conjugation may be achieved using any chemical, biochemical, enzymatic or genetic coupling known to those skilled in the art. The effector of interest may be coupled to the N-terminal or C-terminal end of the transporter peptide.

In another embodiment, the invention includes a method of translocating an effector into the cytoplasm and nucleus of a eukaryotic cell, whereby the effector is conjugated to a transporter peptide and introduced into the eukaryotic cell. For example, the transporter peptide-effector conjugate can be introduced into the cell by incubating a cell culture in the presence of the conjugate or injecting the conjugate into the cell.

In various other embodiments, the invention includes a method of increasing the cellular concentration of an effector within a eukaryotic cell, whereby an effector is conjugated to a transporter peptide and incubated in a cell under conditions promoting active metabolism of the cell. A preferred embodiment of the invention includes use of a human cell as a eukaryotic cell.

In yet further embodiments, the invention includes a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a transporter unit and a pharmaceutically acceptable carrier.

Preferred "pharmaceutical compositions" are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

In yet still further embodiments, the invention includes a kit in which one or more containers containing a therapeutically or prophylactically effective amount of a pharmaceutical composition.

Another embodiment of the invention involves a method of treating or preventing a disease by administering to a subject in which such treatment or prevention is desired, a pharmaceutical composition in an amount sufficient to treat or prevent a disease. For example, the disease to be treated may include diabetes, colon cancer, respiratory ailments, neurodegenerative disorders, cardioplegia and/or viral infections.

In another aspect, the invention involves a method of screening a phage library for transporter peptides, whereby a phage library is screened against specific cell types and it is then determined which cells have internalized phages.

In another embodiment, the invention includes identifying the DNA of an internalized phage and deducing an expressed peptide.

In yet a further embodiment, the invention includes a screening step whereby a phage library is panned for at least three cycles.

In still a further embodiment, the invention includes a phage having a multivalent display of peptides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a peptide transport system that specifically targets various cell types for the intracellular delivery of drugs and therapeutic agents. Existing transport systems in the art are too limited to be of general application because they are either inefficient, affect the host genome, alter the biological properties of the active substance, kill the target cell, or pose too high a risk to be used in a human subject due to the use of viral conjugates. The peptide transport system of the present invention uses proprotein convertases and specific ligands for the intracellular delivery of potential therapeutics, in order to overcome the limitations of transport systems in the art. The present system exhibits efficient delivery of an unaltered biologically active substance that does not affect the host's genome and that is otherwise non-invasive.

For example, transporter peptides have a use in treating diabetes. β-cell mass is tightly regulated so that insulin secretion maintains normoglycemia. Fitting β-cell mass to the needs of the infant or the adult organism, particularly in certain physiological and physiopathological conditions, is essentially attained by a dynamic balance between β-cell death and regeneration that occurs from differentiation of immature β-cells and from the proliferation of preexisting insulin-secreting cells (54;55). In Type I diabetes, impaired balance results from accelerated β-cell destruction, a process initiated by the specific attack of the immune system that targets pancreatic β-cells. Preventing or decreasing the rate of β-cell destruction may therefore not only help stabilize diabetes, but may also allow for islet regeneration to correct β-cell mass insuffisance.

Several molecules have been established as potent tools to decrease the rate of β-cell loss in experimental models of Type I diabetes. Many of these molecules are peptidyl in nature, and thus easily linked to peptide carriers. The peptides described herein serve as basis for the design of therapeutic "cargos", namely the coupling of the carriers ("transporter peptide") with therapeutic agents ("effectors").

Thus, a preferred embodiment of the transport system of the present invention targets β-cell intracellular mechanisms for the treatment of Type I diabetes. Type I diabetes is secondary to the destruction of the pancreatic β-cells by secretion of the immune system (1). Conclusive data, both in human and rodents, indicate that the cytokines interleukin-1β(IL-1β) in conjunction with TNFα and IFNγ, secreted by macrophages and T-cells, are major components responsible for the final outcome that leads to β-cell dysfunction and destruction and Type I diabetes (2-4). These secreted cytokines engage in a highly complex network of signaling and effector molecules in pancreatic β-cells. The signaling modifies the comportment of the cells and has a decisive impact on the cell fate. Accumulating evidence indicates that this regulatory intracellular network represents a promising target for the development of novel therapeutic approaches (5-11). Each of the molecules involved in the treatment and integration of intracellular cytokine signaling may represent a target for transporter-drug design.

Among the most prominent signaling molecules recruited by IL-1β in β-cells are ceramides, prostaglandins, heat-shock proteins, the inducible NO synthase enzyme (iNOS), the transcription factor NF-κB, and the three MAP kinases ERK1/2, p38 and JNK. Many of these molecules are targets for blockage with existing inhibitors that have led to improvement of β-cell survival and function. iNOS KO mice are resistant to IL-1β cytotoxicity (12) and blockers of iNOS activity prevent different aspects of NO cytotoxicity (reviewed in (6)). Islets and cell-lines studies have indicated that blockers of Ca²⁺ channels or caspase inhibitors prevent rodent β-cell death (13;14). p38 inhibitors attenuate IL-1β-mediated inhibition of glucose-stimulated insulin release (15). β-cell specific suppression of GAD expression in antisense GAD transgenic NOD mice prevented autoinimune diabetes (16). Expression of bc1-2, IL-1Ra as do JBD (a dominant inhibitor of the c-Jun N-terminal Kinase JNK) in pancreatic β-cell lines had lead to the generation of cells that resist apoptosis (17-20). Together, these data indicate that the manipulation of intracellular events with specific tools holds great promise for the treatment of Type I diabetes.

One major challenge for disease treatment is to convert biologically important molecules into bioactive, cell-permeable compounds which are usable in vivo (21). For example, the most promising tools for the prevention of β-cell loss are a number of large proteins (e.g., Bc1-2 (8), inhibitors of cytokine signaling such as dominant negative versions of MyD88, TRAF, FADD or IRAK (22;23), or the JNK inhibitor JBD₂₈₀ (24)) that cannot be currently delivered in vivo to tissues and cell-types including pancreatic β-cells.

Recent work indicates progress in attempts to convert large proteins into small bioactive compounds which can be easily delivered to cells and organs (25). These techniques essentially require two conditions: 1) a specific transporter or a chemical modification thereof is linked to the molecules for efficient delivery inside cells (see, for example, efficient short peptide transporters described in (26-28)); and 2) the active portion of of the protein has to be narrowed down so that small peptides sequence might be linked to the transporter. In short, these conditions generally define 3-30 amino acid-long, bi-partite peptides that are able to enter cells while conserving the essential biological properties of the proteins from which they are derived. As in cancer research (32), there are numerous intracellular events in the β-cells whose manipulations protect β-cells from cytokine-induced apoptosis-manipulations which appear to be promising targets for drug design.

Receptor-mediated endocytosis is widely exploited in experimental systems for the targeted delivery of therapeutic agents into cells (36). Endocytotic activity is a common property that has been described for many receptors including IgG Fc, somatostatin, insulin, IGF-I and II, transferrin, EGF, GLP-1, VLDL or integrin receptors (35;37-43). Recently, the isolation of peptide sequences that direct efficient receptor-mediated endocytosis has been profoundly boosted by the use of phage display technologies (44). Phage display libraries are extremely powerful tools that provide for a practically unlimited source of molecule variants including modifications of natural ligands to cell receptors (45) and short peptides (46). Using this technology, evidence that cell-type specific receptors mediate endocytosis has been reported (47). Similar libraries have been injected directly into mice and peptide sequences that show a 13-fold selectivity for brain and kidney have been successfully isolated (48;49).

Although strong experimental background indicates that transporter peptides which selectively target pancreatic β-cells might be derived from large phage display libraries, no such attempts have been reported. The advantages of small peptide carriers such as those obtained using phage display libraries are numerous and include ease of generation by chemical synthesis, high quality and purity, low immunogenicity and potential for highly efficient delivery to all cells in an organism (26). Accordingly, the peptide carriers of the invention have the potential to perform better than more conventional transporters such as liposomes or viruses in the efficient delivery of many macromolecules (see for example (50;51)).

Phage peptide libraries are traditionally constructed in derivatives of the filamentous phage M13. Peptide libraries are fused to the minor coat protein III of the capsid that displays 1-5 copies of the peptide motif (46). Alternatively, high-valent display is attained by using the major coat protein pVIII.

These types of libraries have not been optimized for the isolation of receptor-mediated endocytotic peptide sequences, and the following considerations are relevant for the recovery of carriers with the highest efficiencies of internalization:

The large size of the M13 derivatives (1-1.5 μm) (53) exceeds the typical size of classical clathrin-coated pits (150 nM). Clathrin-coated pits are invaginated structures on the plasma membrane that occupy approximately 2% of the membrane surface. These specialized structures direct the highly efficient receptor-mediated internalization process that clears extracellular proteins or peptides such as insulin or EGF at the extremely rapid rate of 10-50%/min (43). Thus, receptor-mediated internalization by these specialized and highly efficient structures is not expected to occur with the conventional M13 phages.

Accordingly, published attempts have failed to produce peptides that display a high internalization rate of peptide bearing phages. To date, no consensus internalization motif specific for a particular cell-type has emerged from these studies (44;47-49).

In certain aspects, the invention described herein relates to the identification of transporter peptides which promote the internalization of peptide-bearing phages. Once the peptide sequences are determined, that are bound to effector molecules in order to transport the effector molecules across a biological membrane.

As used herein, the terms "bound" or "binds" or "associates" or "interacts" are meant to include all specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic, and hydrogen bonding, but does not include non-specific associations such as solvent preferences.

A transporter peptide is a peptide that facilitates the passage, or translocation, of a substance across a biological membrane, particularly into the cytoplasm or nucleus, of the cell. Translocation may be detected by various procedures, including a cellular penetration assay as described in, for example, PCT application No. WO 97/02840. Generally, a cellular penetration assay is performed by: a) incubating a cell culture with a translocating peptide; b) fixing and permeabilizing the cells; and c) detection of the presence of the peptides inside the cell. The detection step may be carried out by incubating the fixed, permeabilized cells with labeled antibodies directed to the peptide, followed by detection of an immunological reaction between the peptide and the labeled antibody. Alternatively, detection may also be achieved by using a detectably labeled peptide, and directly detecting the presence of the label in cellular compartments. The label may be, for example, a radioactive label, or a fluorescent label, or a dye.

The invention further includes transport units, which are complexes of the translocation peptide coupled to an effector. As used herein, "coupled" means any type of interaction enabling a physical association between an effector and the peptide. The association may be covalent or a non-covalent in nature, and it must be sufficiently strong so that the vector does not disassociate before or during translocation. Coupling may be achieved using any chemical, biochemical, enzymatic or genetic coupling known to those skilled in the art. The effector of interest may be coupled to the N-terminal or C-terminal end of the peptide vector.

An "effector" refers to any molecule or compound of, for example, biological, pharmaceutical, diagnosis, tracing, or food processing interest. It may consist of nucleic acids (ribonucleic acid, deoxyribonucleic acid) from various origins, and particularly of human, viral, animal, eukaryotic or prokaryotic, plant, synthetic origin, etc. A nucleic acid of interest may be of a variety of sizes, ranging from, for example, a simple trace nucleotide to a genome fragment, or an entire genome. It may a viral genome or a plasmid. Alternatively, the effector of interest may also be a protein, such as, for example, an enzyme, a hormone, a cytokine, an apolipoprotein, a growth factor, an antigen, or an antibody, etc. Furthermore, the effector may be a pharmaceutically active agent, such as, for example, a toxin, a therapeutic agent or an antipathogenic agent, such as an antibiotic, an antiviral, an antifungal, or an anti-parasitic agent. The effector of interest may itself be directly active or may be activated in situ by the peptide, by a distinct substance, or by environmental conditions.

The term "pharmaceutically active agent" is used herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a detectable pharmacologic and/or physiologic effect.

The term "therapeutic agent" is used herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect.

The transporter peptides according to the present invention are characterized by the fact that their penetration capacity is virtually independent of the nature of the substance of the interest (the effector) that is coupled to it.

The invention also includes a method of introducing an substance of interest into a cell or a cell nucleus. The method includes contacting the cell with a transporter peptide-effector conjugate in an amount sufficient to enable efficient penetration into the cells. In general, the method may be used for in vivo or in vitro internalization of the conjugate. For example, the conjugate can be provided in vitro, ex vivo, or in vivo. Furthermore, it has been shown that a transporter peptide according to this invention is capable of potentializing the biological activity of the coupled substance. Therefore, another purpose of this invention is a method of using a transporter peptide that increases the biological activity of the effector to which it is coupled. According to the in vitro method, an effector is first coupled to a transporter, and the conjugate is incubated with cells at a temperature which enables active metabolism of the cells. In some cases, the transporter-effector conjugate is injected into particular cells. Those skilled in the art will recognize that any other method of introducing the conjugate into the cells can also be used.

In addition to the peptide-effector conjugates, the invention also provides a pharmaceutically acceptable base or acid addition salt, hydrate, ester, solvate, prodrug, metabolite, stereoisomer, or mixture thereof. The invention also includes pharmaceutical formulations comprising a peptide-effector conjugate in association with a pharmaceutically acceptable carrier, diluent, or excipient.

Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid to produce "pharmaceutically-acceptable acid addition salts" of the compounds described herein. These compounds retain the biological effectiveness and properties of the free bases. Representative of such salts are the water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2′-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, embonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

According to the methods of the invention, a human patient can be treated with a pharmacologically effective amount of a peptide or conjugate. The term "pharmacologically effective amount" means that amount of a drug or pharmaceutical agent (the effector) that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician.

The invention also includes pharmaceutical compositions suitable for introducing an effector of interest into a cell or cell nucleus. The compositions are preferably suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers. The compounds are especially useful in that they have very low, if any toxicity.

Preferred pharmaceutical compositions are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, nasal, parenteral, transdermal, subcutaneous, or topical administration modes.

Depending on the intended mode of administration, the compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, preferably in unit dosages. The compositions will include an effective amount of active compound or the pharmaceutically acceptable salt thereof, and in addition, and may also include any conventional pharmaceutical excipients and other medicinal or pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as are customarily used in the pharmaceutical sciences.

For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound defined above, may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.

Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.

One approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.

The compounds of the present invention can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions. Likewise, they may also be administered in intravenous (both bolus and infusion) intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an antiandrogenic agent.

The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicated effects, may be provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 or 1000.0 mg of active ingredient.

Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.

The compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.

The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

Any of the above pharmaceutical compositions may contain 0.1-99%, preferably 1-70% of the active compounds, especially compounds of the Formula I as active ingredients.

Equivalents. From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that unique methods of translocation across a biological membrane have been described. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventor that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. For instance, the choice of the particular type of cell, or the particular effector to be translocated is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.

The details of one or more embodiments of the invention have been set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated by reference.
 

Claim 1 of 4 Claims

1. A method of translocating a transporter peptide into a pancreatic B-cell, comprising contacting a pancreatic B-cell with a transporter peptide for a time and under conditions sufficient to allow a transporter peptide to translocate across a membrane of the B-cell, wherein the transporter peptide is SEQ ID NO:1.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.