Patent 6,558,952

Methods and compositions for treating diabetes mellitus in a patient in need thereof are provided. The methods include administering to a patient a composition providing a gastrin/CCK receptor ligand, e.g., a gastrin, and/or an epidermal growth factor (EGF) receptor ligand, e.g., TGF-.alpha., in an amount sufficient to effect differentiation of pancreatic islet precursor cells to mature insulin-secreting cells. The composition can be administered systemically or expressed in situ by cells transgenically supplemented with one or both of a gastrin/CCK receptor ligand gene, e.g., a preprogastrin peptide precursor gene and an EGF receptor ligand gene, e.g., a TGF-.alpha. gene. The methods also include transplanting into a patient cultured pancreatic islets in which mature insulin-secreting beta cells are proliferated by exposure to a gastrin/CCK receptor ligand and an EGF receptor ligand.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides methods for treating diabetes mellitus in a patient in need thereof by administering a composition providing a gastrin/CCK receptor ligand such as gastrin, an EGF receptor ligand, such as TGF-.alpha., or a combination of both in an amount sufficient to effect differentiation of pancreatic islet precursor cells to mature insulin-secreting cells. When the composition is administered systemically, generally it is provided by injection, preferably intravenously, in a physiologically acceptable carrier. When the composition is expressed in situ, pancreatic islet precursor cells are transformed either in ex vivo or in vivo with one or more nucleic acid expression constructs in an expression vector which provides for expression of the desired receptor ligand(s) in the pancreatic islet precursor cells. As an example, the expression construct includes a coding sequence for a CCK receptor ligand, such as preprogastrin peptide precursor coding sequence which, following expression, is processed to gastrin or a coding sequence for an EGF receptor ligand such as TGF-.alpha., together with transcriptional and translational regulatory regions which provide for expression in the pancreatic islet precursor cells. The transcriptional regulatory region can be constitutive or induced, for example by increasing intracellular glucose concentrations, such as a transcriptional regulatory region from an insulin gene. Transformation is carried out using any suitable expression vector, for example, an adenoviral expression vector. When the transformation is carried out ex vivo, the transformed cells or tissues are implanted in the diabetic patient, for example using a kidney capsule. Alternatively, pancreatic islet tissue containing islet precursor cells are treated ex vivo with a sufficient amount of a gastrin/CCK receptor ligand and/or an EGF receptor ligand to increase the number of pancreatic .beta. cells in the islets prior to implantation into the diabetic patient. As required, the population of insulin-secreting pancreatic .beta. cells is expanded in culture prior to implantation by contacting pancreatic islet precursor cells with the same receptor ligand(s).

The subject invention offers advantages over existing treatment regimens for diabetic patients. By providing a means to stimulate the adult pancreas to regenerate, not only is the need for traditional drug therapy (Type 2) or insulin therapy (Type 1 and Type 2) reduced or even eliminated, but the maintenance of normal blood glucose levels also may reduce some of the more debilitating complications of diabetes. Diabetic complications include those affecting the small blood vessels in the retina, kidney, and nerves, (microvascular complications), and those affecting the large blood vessels supplying the heart, brain, and lower limbs (mascrovascular complications). Diabetic microvascular complications are the leading cause of new blindness in people 20-74 years old, and account for 35% of all new cases of end-stage renal disease. Over 60% of diabetics are affected by neuropathy. Diabetes accounts for 50% of all non-traumatic amputations in the USA, primarily as a result of diabetic macrovascular disease, and diabetics have a death rate from coronary artery disease that is 2.5 times that of non-diabetics. Hyperglycemia is believed to initiate and accelerate progression of diabetic microvascular complications. Use of the various current treatment regimens cannot adequately control hyperglycemia and therefore does not prevent or decrease progression of diabetic complications.

As used herein, the term "gastrin/CCK receptor ligand" encompasses compounds that stimulate the gastrin/CCK receptor. Examples of such gastrin/CCK receptor ligands include various forms of gastrin such as gastrin 34 (big gastrin), gastrin 17 (little gastrin), and gastrin 8 (mini gastrin); various forms of cholecystokinin such as CCK 58, CCK 33, CCK 22, CCK 12 and CCK 8; and other gastrin/CCK receptor ligands that either alone or in combination with EGF receptor ligands can induce differentiation of cells in mature pancreas to form insulin-secreting islet cells. Also contemplated are active analogs, fragments and other modifications of the above. Such ligands also include compounds that increase the secretion of endogenous gastrins, cholecystokinins or similarly active peptides from sites of tissue storage. Examples of these are omeprazole which inhibits gastric acid secretion and soy bean trypsin inhibitor which increases CCK stimulation.

As used herein, the term "EGF receptor ligand" encompasses compounds that stimulate the EGF receptor such that when gastrin/CCK receptors in the same or adjacent tissues or in the same individual also are stimulated, neogenesis of insulin-producing pancreatic islet cells is induced. Examples of such EGF receptor ligands include EGF1-53, and fragments and active analogs thereof, including EGF1-48, EGF1-52, EGF1-49. See, for example, U.S. Pat. No. 5,434,135. Other examples include TGF-.alpha. receptor ligands (1-50) and fragments and active analogs thereof, including 1-48, 1-47 and other EGF receptor ligands such as amphiregulin and pox virus growth factor as well as other EGF receptor ligands that demonstrate the same synergistic activity with gastrin/CCK receptor ligands. These include active analogs, fragments and modifications of the above. For further background, see Carpenter and Wahl, Chapter 4 in Peptide Growth Factors (Eds. Sporn and Roberts), Springer Verlag, (1990).

A principal aspect of the invention is a method for treating diabetes mellitus in an individual in need thereof by administering to the individual a composition including a gastrin/CCK receptor ligand and/or an EGF receptor ligand in an amount sufficient to effect differentiation of pancreatic islet precursor cells to mature insulin-secreting cells. The cells so differentiated are residual latent islet precursor cells in the pancreatic duct. One embodiment comprises administering, preferably systemically, a differentiation regenerative amount of a gastrin/CCK receptor ligand and an EGF receptor ligand, preferably TGF-.alpha., either alone or in combination to the individual.

Another embodiment comprises providing a gastrin/CCK receptor ligand and/or an EGF receptor ligand to pancreatic islet precursor cells of explanted pancreatic tissue of a mammal and reintroducing the pancreatic tissue so stimulated to the mammal.

In another, the invention comprises providing a gastrin/CCK receptor ligand and/or an EGF receptor ligand to pancreatic islet precursor cells of explanted pancreatic tissue from a mammal to expand the population of .beta. cells.

In another embodiment gastrin/CCK receptor ligand stimulation is effected by expression of a chimeric insulin promoter-gastrin fusion gene construct transgenically introduced into such precursor cells. In another embodiment EGF receptor ligand stimulation is effected by expression of an EGF receptor ligand gene transgenically introduced into the mammal. The sequence of the EGF gene is provided in U.S. Pat. No. 5,434,135.

In another embodiment stimulation by a gastrin/CCK receptor ligand and an EGF receptor ligand is effected by coexpression of (i) a preprogastrin peptide precursor gene and (ii) an EGF receptor ligand gene that have been stably introduced into the mammal.

In another aspect the invention relates to a method for effecting the differentiation of pancreatic islet precursor cells of a mammal by stimulating such cells with a combination of a gastrin/CCK receptor ligand and an EGF receptor ligand. In a preferred embodiment of this aspect, gastrin stimulation is effected by expression of a preprogastrin peptide precursor gene stably introduced into the mammal. The expression is under the control of the insulin promoter. EGF receptor ligand, e.g., TGF-.alpha., stimulation is effected by expression of an EGF receptor ligand gene transgenically introduced into the mammal. In furtherance of the above, stimulation by a gastrin and a TGF-.alpha. is preferably effected by co-expression of (i) a preprogastrin peptide precursor gene and (ii) an EGF receptor ligand introduced into the mammal. Appropriate promoters capable of directing transcription of the genes include both viral promoters and cellular promoters. Viral promoters include the immediate early cytomegalovirus (CMV) promoter (Boshart et al (1985) Cell 41:521-530), the SV40 promoter (Subramani et al (1981) Mol. Cell. Biol. 1-854-864) and the major late promoter from Adenovirus 2 (Kaufman and Sharp (1982) Mol. Cell. Biol. 2:1304-13199). Preferably, expression of one or both of the gastrin/CCK receptor ligand gene and the EGF receptor ligand gene is under the control of an insulin promoter.

Another aspect of the invention is a nucleic acid construct. This construct includes a nucleic acid sequence coding for a preprogastrin peptide precursor and an insulin transcriptional regulatory sequence, which is 5' to and effective to support transcription of a sequence encoding the preprogastrin peptide precursor. Preferably, the insulin transcriptional regulatory sequence includes at least an insulin promoter. In a preferred embodiment the nucleic acid sequence coding for the preprogastrin peptide precursor comprises a polynucleotide sequence containing exons 2 and 3 of a human gastrin gene and optionally also including introns 1 and 2.

Another embodiment of the invention is an expression cassette comprising (i) a nucleic acid sequence coding for a mammalian EGF receptor ligand, e.g., TGF-.alpha. and a transcriptional regulatory sequence thereof; and (ii) a nucleic acid sequence coding for the preprogastrin peptide precursor and a transcriptional regulatory sequence thereof. Preferably, the transcriptional regulatory sequence for the EGF receptor ligand is a strong non-tissue specific promoter, such as a metallothionein promoter. Preferably, the transcriptional regulatory sequence for the preprogastrin peptide precursor is an insulin promoter. A preferred form of this embodiment is one wherein the nucleic acid sequence coding for the preprogastrin peptide precursor comprises a polynucleotide sequence containing introns 1 and 2 and exon 2 and 3 of the human gastrin gene.

Another aspect of the invention relates to a vector including the expression cassette comprising the preprogastrin peptide precursor coding sequence. This vector can be a plasmid such as pGem1 or can be a phage which has a transcriptional regulatory sequence including an insulin promoter.

Another aspect of this invention relates to a composition of vectors including (1) having the nucleic acid sequence coding for a mammalian EGF receptor ligand, e.g., TGF-.alpha., under control of a strong non-tissue specific promoter, e.g., a metallothionein promoter; and a preprogastrin peptide precursor coding sequence under control of an insulin promoter. Each vector can be a plasmid, such as plasmid pGem1 or a phage in this aspect. Alternatively, the expression cassette or vector also can be inserted into a viral vector with the appropriate tissue trophism. Examples of viral vectors include adenovirus, Herpes simplex virus, adeno-associated virus, retrovirus, lentivirus, and the like. See Blomer et al (1996) Human Molecular Genetics 5 Spec. No: 1397-404; and Robbins et al (1998) Trends in Biotechnology 16:35-40. Adenovirus-mediated gene therapy has been used successfully to transiently correct the chloride transport defect in nasal epithelia of patients with cystic fibrosis. See Zabner et a. (1993) Cell 75:207-216.

Another aspect of the invention is a non-human mammal or mammalian tissue, including cells, thereof capable of expressing a stably integrated gene which encodes preprogastrin. Another embodiment of this aspect is a non-human mammal capable of coexpressing (i) a preprogastrin peptide precursor gene; and/or (ii) an EGF receptor ligand, e.g., a TGF-.alpha. gene that has been stably integrated into the non-human mammal, mammalian tissue or cells. The mammalian tissue or cells can be human tissue or cells.

Therapeutic Administration and Compositions

Modes of administration include but are not limited to transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The compounds can be administered by any convenient route, for example by infusion or bolus injection by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration is preferably systemic.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. Pharmaceutically acceptable carriers and formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17^th ed. (1985), which is incorporated herein by reference. For a brief review of methods for drug delivery, see Langer (1990) Science 249:1527-1533, which is incorporated herein by reference.

In preparing pharmaceutical compositions of the present invention, it may be desirable to modify the compositions of the present invention to alter their pharmacokinetics and biodistribution. For a general discussion of pharmacokinetics, see Remingtons's Pharmaceutical Sciences, supra, Chapters 37-39. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art (See, e.g., Langer, supra). Examples of such methods include protection of the agents in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. For example, the agents of the present invention can be incorporated into liposomes in order to enhance their pharmacokinetics and biodistribution characteristics. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al (1980) Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, all of which are incorporated herein by reference. Various other delivery systems are known and can be used to administer a therapeutic of the invention, e.g., microparticles, microcapsules and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a preferred embodiment, the composition is formulated in accordance with routine procedures such as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition also can include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quality of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The therapeutics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium and other divalent cations, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the therapeutic of the invention which is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation also will depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective dosages can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% weight; oral formulations preferably contain 10% to 95% active ingredient.

In the gene therapy methods of the invention, transfection in vivo is obtained by introducing a therapeutic transcription or expression vector into the mammalian host, either as naked DNA, complexed to lipid carriers, particularly cationic lipid carriers, or inserted into a viral vector, for example a recombinant adenovirus. The introduction into the mammalian host can be by any of several routes, including intravenous or intraperitoneal injection, intratracheally, intrathecally, parenterally, intraarticularly, intranasally, intramuscularly, topically, transdermally, application to any mucous membrane surface, corneal installation, etc. Of particular interest is the introduction of the therapeutic expression vector into a circulating bodily fluid or into a body orifice or cavity. Thus, intravenous administration and intrathecal administration are of particular interest since the vector may be widely disseminated following such routes of administration, and aerosol administration finds use with introduction into a body orifice or cavity. Particular cells and tissues can be targeted, depending upon the route of administration and the site of administration. For example, a tissue which is closest to the site of injection in the direction of blood flow can be transfected in the absence of any specific targeting. If lipid carriers are used, they can be modified to direct the complexes to particular types of cells using site-directing molecules. Thus, antibodies or ligands for particular receptors or other cell surface proteins may be employed, with a target cell associated with a particular surface protein.

Any physiologically acceptable medium may be employed for administering the DNA, recombinant viral vectors or lipid carriers, such as deionized water, saline, phosphate-buffered saline, 5% dextrose in water, and the like as described above for the pharmaceutical composition, depending upon the route of administration. Other components can be included in the formulation such as buffers, stabilizers, biocides, etc. These components have found extensive exemplification in the literature and need not be described in particular here. Any diluent or components of diluents that would cause aggregation of the complexes should be avoided, including high salt, chelating agents, and the like.

The amount of therapeutic vector used will be an amount sufficient to provide for a therapeutic level of expression in a target tissue. A therapeutic level of expression is a sufficient amount of expression to decrease blood glucose towards normal levels. In addition, the dose of the nucleic acid vector used must be sufficient to produce a desired level of transgene expression in the affected tissues in vivo. Other DNA sequences, such as adenovirus VA genes can be included in the administration medium and be co-transfected with the gene of interest. The presence of genes coding for the adenovirus VA gene product may significantly enhance the translation of mRNA transcribed from the expression cassette if this is desired.

A number of factors can affect the amount of expression in transfected tissue and thus can be used to modify the level of expression to fit a particular purpose. Where a high level of expression is desired, all factors can be optimized, where less expression is desired, one or more parameters can be altered so that the desired level of expression is attained. For example, if high expression would exceed the therapeutic window, then less than optimum conditions can be used.

The level and tissues of expression of the recombinant gene may be determined at the mRNA level as described above, and/or at the level of polypeptide or protein. Gene product may be quantitated by measuring its biological activity in tissues. For example, protein activity can be measured by immunoassay as described above, by biological assay such as blood glucose, or by identifying the gene product in transfected cells by immunostaining techniques such as probing with an antibody which specifically recognizes the gene product or a reporter gene product present in the expression cassette.

Typically, the therapeutic cassette is not integrated into the patient's genome. If necessary, the treatment can be repeated on an ad hoc basis depending upon the results achieved. If the treatment is repeated, the patient can be monitored to ensure that there is no adverse immune or other response to the treatment.

The invention also provides for methods for expanding a population of pancreatic .beta.-cells in vitro. Upon isolation of the pancreas from a suitable donor, cells are isolated and grown in vitro. The cells which are employed are obtained from tissue samples from mammalian donors including human cadavers, porcine fetuses or another suitable source of pancreatic cells. If human cells are used, when possible the cells are major histocompatability matched with the recipient. Purification of the cells can be accomplished by gradient separation after enzymatic (e.g., collagenase) digestion of the isolated pancreas. The purified cells are grown in media containing sufficient nutrients to allow for survival of the cells as well as a sufficient amount of a .beta.-cell proliferation inducing composition containing a gastrin/CCK receptor ligand and/or EGF receptor ligand, to allow for formation of insulin secreting pancreatic .beta. cells. According to the invention, following stimulation the insulin secreting pancreatic .beta. cells can be directly expanded in culture prior to being transplanted into a patient in need thereof, or can be transplanted directly following treatment with .beta.-cell proliferation inducing composition.

According to the invention, following the stimulation of the growth of newly formed insulin secreting pancreatic .beta. islet cells in culture by incubation of pancreatic islet precursor cells with said islet neogenesis-inducing composition, said cells can then be transplanted into a patient in need thereof, or said precursor cells can be transplated directly following treatment with the islet neogenesis-inducing composition.

Methods of transplantation include transplanting insulin secreting pancreatic .beta.-cells obtained into a patient in need thereof in combination with immunosuppressive agents, such as cyclosporin. The insulin producing cells also can be encapsulated in a semi-permeable membrane prior to transplantation. Such membranes permit insulin secretion from the encapsulated cells while protecting the cells from immune attack. The number of cells to be transplanted is estimated to be between 10,000 and 20,000 insulin producing .beta. cells per kg of the patient. Repeated transplants may be required as necessary to maintain an effective therapeutic number of insulin secreting cells. The transplant recipient can also, according to the invention, be provided with a sufficient amount of a gastrin/CCK receptor ligand and an EGF receptor ligand to induce proliferation, from islet precursor cells, of the transplanted insulin secreting .beta. cells.

The effect of treatment of diabetes can be evaluated as follows. Both the biological efficacy of the treatment modality as well as the clinical efficacy are evaluated, if possible. For example, disease manifests itself by increased blood sugar, the biological efficacy of the treatment therefore can be evaluated, for example, by observation of return of the evaluated blood glucose towards normal. The clinical efficacy, i.e. whether treatment of the underlying effect is effective in changing the course of disease, can be more difficult to measure. While the evaluation of the biological efficacy goes a long way as a surrogate endpoint for the clinical efficacy, it is not definitive. Thus, measuring a clinical endpoint which can give an indication of .beta.-cell regeneration alter, for example, a six-month period of time, can give an indication of the clinical efficacy of the treatment regimen.

The subject compositions can be provided as kits for use in one or more procedures. Kits for genetic therapy usually will include the therapeutic DNA construct either as naked DNA with or without mitochondrial targeting sequence peptides, as a recombinant viral vector or complexed to lipid carriers. Additionally, lipid carriers can be provided in separate containers for complexing with the provided DNA. The kits include a composition comprising an effective agent either as concentrates (including lyophilized compositions), which can be diluted further prior to use or they can be provided at the concentration of use, where the vials may include one or more dosages. Conveniently, in the kits single dosages can be provided in sterile vials so that the physician can employ the vials directly, where the vials will have the desired amount and concentration of agents. When the vials contain the formulation for direct use, usually there will be no need for other reagents for use with the method. Associated with such kits can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Claim 1 of 3 Claims

What is claimed is:

1. A method for expanding a population of pancreatic beta cells, said method comprising:

providing said pancreatic beta cells with a sufficient amount of a gastrin/CCK receptor ligand and an epidermal growth factor receptor ligand to induce proliferation of said pancreatic beta cells, whereby an expanded population of pancreatic beta cells is obtained.

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