Compositions and methods are provided for achieving in vivo islet cell regeneration in subjects with preexisting diabetes. The methods comprise short term treatment with a composition having a gastrin/cholecystokinin receptor ligand and an EGF receptor ligand. Treatment with such a composition for a short term resulted in a prolonged period of increased insulin release, decreased fasting blood glucose, and improved glucose tolerance, the prolonged efficacy, the period being considered from the time of cessation of treatment.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As used herein, the term "gastrin/CCK receptor ligand" encompasses compounds that stimulate the gastrin/CCK receptor such that when EGF receptors in the same or adjacent tissue or in the same individual are also stimulated, neogenesis of insulin-producing pancreatic islet cells is induced. Examples of such gastrin/CCK receptor ligands are given in U.S. Pat. No. 6,288,301 issued Sep. 11, 2001, and 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 demonstrate the same synergistic activity with EGF receptor ligands, and which can induce differentiation of cells of mature pancreas to form insulin-secreting islet cells, when acting synergistically with an EGF receptor ligand.

Small forms of gastrin such as gastrin 17 are economically prepared by peptide synthesis, and the synthetic peptides are commercially available. Synthetic human gastrin 17, and derivatives such as human gastrin 17 having methionine substituted for leucine at position 15 are also available from Bachem AG, Bubendorf, Switzerland, and from Researchplus.

Gastrin/CCK receptor ligands include also active analogs, fragments and other modifications of the above ligands. 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 soya 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 tissue or in the same individual are also stimulated, neogenesis of insulin-producing pancreatic islet cells is induced. Examples of such EGF receptor ligands include full length EGF which is EGF1-53, and further include EGF1-48, EGF1-49, EGF1-52, and fragments and active analogs thereof. Other examples of EGF receptor ligands are TGFα forms that include 1-48, 1-47, 1-51, and amphiregulin and pox virus growth factor as well as any 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.

EGF has mitogenic activity, i.e., it stimulates the growth of various cells and tissues (Karnes supra; Carpenter, G. et al. J. Cell Physiol 88, 227-237, 1976; Gasslander, T. et al. Eur. Surg. Res. 29, 142-149, 1997). EGF is also found to have a cytoprotective effect, stimulating migration of a cell toward a wound in vivo, or toward a gap introduced in a monolayer of cells in culture, to promote wound healing. These biological activities are specific to a family of structurally related growth factors, including EGF, TGFα, amphiregulin and heparin binding EGF-like growth factor (Karnes, supra). The members of this family of growth factors have identical amino acids at 11 residues of the amino acid sequence, six of which are cysteine residues that form disulfide bonds.

The group of compounds which are EGF receptor ligands further includes "modified EGF", which are variants of normal or wild type EGF. Modifications have been shown to affect one or more biological activity such as the rate of clearance of EGF. The term includes peptides having the an amino acid sequence substantially similar to that of human EGF, for example, with one or a few amino acid substitutions at various residue positions.

Recombinant EGF forms have been genetically engineered to have alterations in structure and activities, for example, EGF having a methionine at position 21 replaced by a leucine residue has been described (U.S. Pat. No. 4,760,023). Recombinant human EGF (hEGF) having 51 residues, i.e., lacking the two C-terminal residues at positions 52 and 53 of hEGF, and having a neutral amino acid substitution at position 51, retain EGF activity and are more resistant to protease degradation during a microbial production process, and following administration to a subject. A series of nucleic acid molecules have been described that encode a family of proteins that have significant similarity to EGF and TGFα (WO 00/29438). EGF muteins (mutated EGF) having histidine at residue 16 replaced with a neutral or acidic amino acid have been described (WO 93/03757), such forms retaining activity at low values of pH. Chemical analogues and fragments of EGF and TGFα retain ability to bind various members of the EGF receptor family (U.S. Pat. No. 4,686,283). Various modifications of EGF or TGFα confer advantageous properties affecting one or more of recombinant protein production, in vitro and in vivo stability, and in vivo activity. A preferred recombinant modified EGF receptor ligand of the embodiments herein retains substantially full I.N.T.™ islet neogenesis therapy activity, and has in vivo and/or in vitro stability that is that is at least about as great or greater than normal or wild type hEGF.

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 an EGF receptor ligand, each in a sufficient amount and in proportion to each other so that the combination of these ligands effects differentiation of pancreatic islet precursor cells to mature insulin-secreting cells, a process known as islet neogenesis. The cells that differentiate are latent islet precursor cells that are present in the pancreatic duct of the mature animal subject. See U.S. Pat. No. 5,885,956, issued Mar. 23, 1999.

The term "mature" as used herein refers exclusively to a post-natal animal, to distinguish the mature animal from a fetal animal. Mature refers to a mammal of chronological age older than 5% of the average lifespan of the species of mammal. This term is unrelated to a stage of the subject's development or to a time of onset of diabetes. In one embodiment, the methods and compositions herein are useful for treating a patient having type I diabetes mellitus, i.e., a form of diabetes characterized by insulin dependency including insulin absence or deficiency, and referred to as "juvenile" diabetes. The method and compositions herein are useful also for treatment of patients with insulin-dependent type II, or adult-onset diabetes, for example, who produce insufficient insulin to maintain blood glucose levels due to pancreatic exhaustion.

One embodiment of a method herein comprises administering, preferably systemically, a composition which is differentiation regenerative amount of a gastrin/CCK receptor ligand, for example, gastrin, and an EGF receptor ligand, for example, EGF or TGFα, to the individual. The gastrin is synthetic, for example, synthetic human gastrin, for example, human gastrin 17, for example, having leucine at residue 15. The EGF receptor ligand is hEGF, for example, recombinantly produced EGF, for example, a recombinant modified EGF, for example, EGF produced recombinantly from a synthetic gene carrying modifications, such as a deletion of two C-terminal amino acids at positions 52 and 53, and encoding an EGF having a length of 51 amino acids. Further EGF modification can include one or more of a variety of substitutions, such as a substitution of a neutral amino acid at position 51 rather than the normal residue at position 51 of hEGF, which is the charged amino acid glutamic acid. A neutral amino acid as defined herein is one that is uncharged at physiological pH, and is preferably hydrophilic, such as by substitution of serine, alanine, and glutamine for the residue normally found at that position.

In another embodiment, the invention relates to a method for effecting the differentiation of pancreatic islet precursor cells of a subject, for example, a mammal, by stimulating such cells with a combination of a gastrin/CCK receptor ligand, for example, a gastrin such as gastrin 17, for example, synthetic gastrin 17 carrying leucine at position 15, and an EGF receptor ligand, for example, a TFGα, or a recombinant modified EGF, according to a particular dosing schedule.

As used herein, a dosing schedule refers to a protocol for administering an I.N.T.™ islet neogenesis therapy composition, and includes the amount of the composition delivered per day, and the duration or period of time over which the composition is administered.

Most insulin dependent diabetic patients require insulin injection at least on a daily basis. Multiple doses per day of insulin are required under certain circumstances of illness or diet for management of diabetes, and the insulin administration is indicated by results of frequent glucose monitoring, another activity which is required of a diabetes patient for optimal management of the disease, which is performed for example as often as five times daily.

In contrast, the compositions and methods herein are used for a dosage schedule of short duration, for example, a dosage period of less than 0.5 percent, or 0.2 percent, or 0.1 percent of a subject species' average lifespan.

Administration over a short duration dosing schedule of the compositions herein is followed by an extended period of efficacy, even complete remission of disease symptoms. Lifespan data used herein include: an average lifespan of a rodent in captivity of about 2.5 years (30 months); an average lifespan of a primate that is a macaque such as a cynomolgus monkey (Macaca fascicularis) in captivity of about 30 years (360 months); an average lifespan of a primate that is a chimpanzee (Pan troglodytes) in captivity of about 45 years (540 months); and an average lifespan of a primate that is a human of about 70-75 years (840-900 months). It is recognized that an average lifespan of a species varies with conditions that are cultural, such as diet, and/or genetic.

The short term of duration of the dosage schedule for a rodent such as a rat is calculated herein as, for example, equal or less than about 28 days, about 18 days, or less than about 9 days. The duration of a dosage schedule for a primate, for example, a cynomolgus monkey, a chimpanzee or a human, is longer than for a rodent, e.g., the treatment period is less than about one year, less than about three months, less than about two months, or even less than about one month.

The short term of duration of the dosage schedule for a human patient represents and even smaller percentage of the lifespan. For example, a four week duration of treatment is 0.11% of a lifespan of 70 years (3640 weeks); a two week treatment is 0.05% of the lifespan. Efficacy of the treatment extends to a period of 26 weeks (0.7%), or 52 Weeks (1.4%), or 104 weeks (2.86%).

As a result of administration of the I.N.T.™ islet neogenesis therapy compositions provided herein according to a dosage schedule of such short duration, the process of islet neogenesis is initiated. Precursor cells in the subject are induced to differentiate, and the differentiating cells then mature into islet cells capable of secreting insulin in response to fluctuations in blood glucose levels, i.e., a subject with diabetes enters a period of remission characterized by a normal response to a blood glucose challenge. As a result of this administration, remission of diabetes is initiated, so that the standard dosage of insulin given to a diabetic patient prior to therapy is reduced, as determined by the level of blood glucose obtained by monitoring, for example, by self-monitoring by the patient, during and following treatment. Remission from diabetes due to successful islet neogenesis therapy is indicated by a decreased fasting blood level of glucose, and by a decreased level and duration of elevated blood glucose in response to a dietary challenge of sugar consumption. Upon achieving successful islet neogenesis, insulin administration is reduced from, for example, five injections to two injections per day; from two injections to one injection per day; and from one to none, as indicated by data obtained from monitoring blood glucose levels. One of ordinary skill in the art of pharmacology, when treating a diabetic patient, is familiar with adjusting insulin dosage to levels of blood glucose following fasting and under other physiological conditions.

Dosages of the I.N.T.™ islet neogenesis therapy compositions to be administered to a subject are adjusted for known variations from species to species in standard data encompassing criteria for absorption, distribution, half-life kinetics in circulation, metabolism, excretion, and toxicology of the receptor ligands of the embodiments herein, for example, for each primate and rodent species. In general, dosages are adjusted to be about 100-fold greater for administration to a rodent species than to a primate species. For example, a dose of an I.N.T.™ islet neogenesis therapy composition for a rat is exemplified by about 3,000 μg/day of a gastrin/CCK receptor ligand and about 100 μg of an EGF receptor ligand, administered for example in three injections per day (for a total of about 9,000 μg of gastrin/CCK receptor ligand and about 300 μg of EGF receptor ligand per day), on a per kg of body weight basis. For a primate such as a cynomolgus monkey, a chimpanzee, or a human, the comparable dose is, for example, about 1 to about 3 μg of EGF receptor ligand, or about 3 to about 10 μg of EGF receptor ligand per kg body weight, and about 30 to about 90 μg, or about 90 to about 300 μg of gastrin/CCK receptor ligand per kg body weight, such daily doses to be administered as a total bolus given once per day, or divided into subdoses to be administered in two or administrations per day.

Modes of administration of the receptor ligands include but are not limited to subcutaneous, transdermal, intramuscular, intraperitoneal, intravenous, intranasal, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by pump, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). The receptor ligands herein may be administered in combination with one or a plurality of 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 sterile carrier or excipient. Such a carrier includes but is not limit to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, such as Tween, 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 formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules and the like.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include additional components, for example, a solubilizing agent. Generally, the ingredients are supplied either separately or pre-mixed in unit dosage form. In addition to solution form, the composition can be supplied as a dry lyophilized powder, or as a non-aqueous concentrate, for example, in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.

The therapeutic agent embodiments of compositions 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, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of each of the therapeutic agents of the compositions herein, and their relative amount with respect to each other which will be 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 a formulation depends also on route of administration, and the extent of the disease or disorder in a given patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous and subcutaneous administration are generally about 0.1 microgram or one microgram, to about one milligram or three milligrams, per kilogram body weight, for example, from about 20 to about 500 micrograms of each active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg per kg body weight, to about 1 mg per kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. A daily dose is administered as a single dose or divided into a plurality of smaller fractional doses, to be administered several times during the day.

Suppositories generally contain active ingredient in the range of about 0.5% to about 10% by weight; oral formulations preferably contain about 10% to about 95% active ingredient by weight.

Embodiments of the invention herein also provide a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, for example, a container having a unit dosage of each or both of a gastrin/CCK receptor ligand and an EGF receptor ligand. Associated with such container(s) 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. The pack or kit can in certain embodiments include one or more containers having insulin, to be administered during the dosing schedule prior to induction of islet neogenesis and remission from diabetes.
 

Claim 1 of 10 Claims

1. A method for treating a subject having insulin-insufficient diabetes, comprising:

administering for a term of treatment which is shorter in duration than about one percent of an average lifespan of the subject species a dose of each of a gastrin 17 having a methionine substituted for leucine at amino acid position 15 relative to the amino acid sequence of wild type gastrin 17, and a recombinant human EGF1-53 having a deletion of two C-terminus amino acids at amino acid positions 52 and 53 and having a neutral amino acid substituted at amino acid position 51 relative to the amino acid sequence of wild type EGF1-53, the treatment resulting in a remission of the diabetes wherein the subject has increased blood insulin and decreased blood glucose; and

repeating administering of said gastrin 17 and said EGF 1-53 at a time corresponding to about the end of the remission, thereby treating the subject having insulin-insufficient diabetes.

____________________________________________
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.