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Title:  Orally active fraction of momordica charantia, active peptides thereof, and their use in the treatment of diabetes

United States Patent:  6,391,854

Inventors:  Nag; Bishwajit (Fremont, CA); Medicherla; Satyanarayana (Sunnyvale, CA); Sharma; Somesh D. (Los Altos, CA)

Assignee:  Calyx Therapeutics, Inc. (Hayward, CA)

Appl. No.:  628588

Filed:  July 31, 2000


A water soluble extract of M.charantia named MC6, methods for its preparation and methods for its use in the treatment of hyperglycemic disorders are provided. The active MC6 is characterized by moving as a single band on SDS-PAGE having a molecular weight of less than 10 kDal, and by comprising tree peptides. Also provided is a peptide component of MC6 named MC6.1, as well as analogues and mimetics thereof. The active MC6, MC6.1, MC6.2, and MC6.3 exhibit hypoglycemic activity, even following oral administration. Also provided are methods of using the active agents to treat hyperglycemic disorders, particularly diabetes, where the active agents are preferably orally administered.


A water soluble fraction of M.charantia named MC6, an active peptide component thereof named MC6.1, and peptide derivatives thereof, MC6.2 and MC6.3, methods for their preparation and use in the treatment of hyperglycemic disorders are provided. MC6 is characterized by comprising three peptides that move together as a single band on SDS-PAGE with a molecular weight of 10 kDal and has a size smaller than recombinant insulin. MC6 and MC6.1 exhibit hypoglycemic activity and are orally active. In further describing the subject invention, the characteristics of MC6 and MC6.1 will be further described in greater detail, followed by a description of methods for its preparation and use in the treatment of hyperglycemic disorders, particularly in the treatment of humans suffering from diabetes.

The derivatives, MC6.2 and MC6.3 are, respectively, 11-a.a. and 7-a.a. peptide derivatives of the 18-a.a. peptide, MC6.1.

Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

It must be noted that as used in this specification and the appended claims, the singular forms "a;" "an" and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

MC6 of the subject invention is a water soluble fraction or extract of the plant species M.charantia. Although MC6 may be derived from one or more tissues or components of the M.charantia, including the leaves, stems, roots, fruit, seeds and the like, including the whole M.charantia plant, MC6 is usually derived from M.charantia fruit, preferably unripe M.charantia fruit that has been separated from seeds.

MC6 is characterized by moving as a single band on SDS-20% PAGE, where the molecular weight of the single band is less than 10 kDal, where the strength of the electric field applied during SDS-20% PAGE is 100 V. The MC6 has a size smaller than recombinant insulin, and is therefore less than about 6 kDal. MC6 is further characterized by eluting as three peaks following High Performance Liquid Chromatography (HPLC) where the conditions of HPLC are reverse phase on C8 or C18 columns. Under these conditions, the first peak elutes at 11.33 min, the second peak elutes at 25.35 min and the third peak elutes at 34.67 min.

The MC6 of the subject invention is a preparation free of high molecular weight contaminants, e.g. proteins, where high molecular weight contaminants is used in this application to denote molecules having a molecular weight in excess of about 50 kDal. Usually, the MC6 is free of any contaminants having a molecular weight in excess of 10 kDal.

MC6 exhibits hypoglycemic activity in mammals. By hypoglycemic activity is meant that upon administration of MC6 to the mammal, the blood glucose level of the mammal decreases, where the amount of decrease in the blood glucose level is proportional to the amount of MC6 administered to the mammal. MC6 exhibits hypoglycemic activity whether it is administered orally or intravenously, and thus is orally active or orally bioavailable, by which is meant that it is not inactivated by passing from the gastrointestinal tract into the mammal.

The MC6 may be prepared using any convenient means which provide for separation of the MC6 from the source M.charantia tissue. One means of obtaining MC6 from M.charantia tissue is to first make a raw suspension of the tissue by macerating the tissue in the presence of a solvent, e.g. phosphate buffered saline (PBS), water and the like, where maceration may be accomplished using a blender or other maceration means. The particulate matter of the resultant suspension is then separated and discarded from the liquid phase, where this step may be accomplished by centrifuging, usually at a speed between about 10,000 and 16,000 rpm, followed by filtration of the supernatant, where filtration will generally be through filters having pore sizes ranging from 0.5 to, usually from about 0.45 to, where the filtration may be under vacuum. The resultant filtrate is then passed sequentially through 30 kDal and 10 kDal cutoff membranes, where such membranes are known in the art and representative membranes include Amicon M.W. cut off filters, and the like, to obtain a water soluble fraction of the initial M.charantia tissue which contains MC6. The resultant water soluble fraction may be used as is or flier processes for subsequent use, where further processing may include dehydration, e.g. lyophilization, and the like. The resultant water soluble fraction can also be stored in liquid form at from 1 to 5, usually 2 to 4oC. in the presence of preservatives, such as sodium benzoate, and the like.

MC6.1 is a specific peptide component of MC6 that migrates as a single band in SDS-PAGE analysis (4-20% tris-glycine gradient gel) and has a molecular weight as determined by SDS-PAGE of less than 2.5 kD, MC6.1 has an isolectric point of 8.2. MC6.1 is 18 amino acid residues in length, where the amino acid sequence of MC6.1 is:

K-T-N-M-K-H-M-A-G-A-A-A-A-G-A-V-V-G (SEQ ID NO:01)

MC6.1 exhibits hypoglycemic activity in mammals. The MC6.1 of the present invention is MC6.1 which has been separated from its natural environment, e.g. is present in MC6, is in isolated or pure form, and the like.

Also provided are peptide analogues and mimetics of MC6.1 which exhibit hypoglycemic activity. The MC6.1 analogues and mimetics will comprise, as an active motif sequence, at least 8 amino acids, usually at least about 12 amino acids, more usually at least about 18 amino acids, and fewer than about 40 amino acids, more usually fewer than 30 amino acids. Specific peptides are MC6.2, an 11-a.a. derivative of MC6.1 having the sequence:

K-T-N-M-K-H-M-A-G-A-A (SEQ ID NO:02)

and a 7-a.a. derivative having the sequence:

K-T-N-M-K-H-M (SEQ ID NQ:03)

It is understood that up to about three substitutions or deletions may be made in the subject sequences, where the change will not be more than about 20 number %, usually not more than about 10 number % of the number of amino acids in the active motif. Preferred are conservative substitutions, as known in the art, including substitutions within the large hydrophobic group: isoleucine, leucine, valine and phenylalanine; between serine and threonine; glycine and alanine; asparagine and glutamine; aspartic acid and glutamic acid; or lysine, arginine and histidine.

In addition to purification from its natural source, the MC6.1, as well as peptide analogues and mimetics thereof, such as MC6.2 and MC6.3, may be prepared in accordance with conventional techniques, such as synthesis (for example, use of a Beckman Model 990 peptide synthesizer or other commercial synthesizer). Peptides may be produced directly by recombinant methods (see Sambrook et al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y., 1989) or as a fusion protein, for example to a protein that is one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at a site engineered to yield the desired peptide (see for example Driscoll et al. (1993) J. Mol. Bio. 232:342-350).

The oligopeptides (i.e. MC6.1, MC6.1 analogues, MC6.1 mimetics) may be extended to provide convenient linking sites, e.g. cysteine or lysine, to enhance stability, to bind to particular receptors, to provide for site-directed action, to provide for ease of purification, to alter the physical characteristics (e.g. solubility, charge, etc.), to stabilize the conformation, etc. The oligopeptides may be joined to non-wild-type flanking regions as fused proteins, joined either by linking groups or covalently linked through cysteine (disulfide) or peptide linkages. The oligopeptide may be linked through a variety of bifunctional agents, such as maleimidobenzoic acid, methyldithioacetic acid, mercaptobenzoic acid, S-pyridyl dithiopropionate, etc. The oligopeptides may be joined to a single amino acid at the N- or C-terminus of a chain of amino acids, or may be internally joined. For example, the subject peptides may be covalently linked to an immunogenic protein, such as keyhole limpet hemocyanin, ovalbumin, etc. to facilitate antibody production to the subject oligopeptides.

Alternatively, the subject oligopeptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the chain, either internal, or at the N- or C-terminus. Various post-expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation, such that the subject peptide will be bound to a lipid group at one terminus, and will be able to be inserted into a lipid membrane, such as a liposome.

The subject oligopeptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The subject oligopeptides may also be combined with other proteins, such as the Fc of an IgG isotype to enhance complement binding, or with a toxin, such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain. The oligopeptides may be linked to antibodies for site directed action. For conjugation techniques, see, for example, U.S. Pat. Nos. 3,817,837, 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105; and 4,043,989, which are incorporated herein by reference.

MC6. MC6.1, MC6.2 and MC6.3 (as well as analogues and mimetics thereof) of the subject invention find use in the treatment of diseases characterized by the presence of elevated blood glucose levels, e.g. hyperglycemic disorders, such as diabetes mellitus, including both Type I and Type II diabetes, as well as other hyperglycemic related disorders, such as obesity, increased cholesterol kidney related disorders, and the like. By "treatment" is meant that the MC6, MC6.1, MC6,2 and MC6.3 (as well as analogues and mimetics thereof) are administered to at least reduce the blood glucose level in the host suffering from the hyperglycemic disorder. In treating hyperglycemic disorders with MC6 and the subject oligopeptides, the MC6 or oligopeptides are administered to the host in an amount sufficient to reduce the blood glucose level in the host to an acceptable range, wherein acceptable range means .+-.10%, usually .+-.8%, and more usually .+-.5% of the normal average blood glucose level for the host.

A variety of hosts may be treated according to the subject invention to reduce their blood glucose levels, where such hosts are mammalian and include livestock, valuable or rare animals, pets, such as dogs and cats, and humans.

Of particular interest are methods of treating human hyperglycemic disorders, such as diabetes, including both Type I and Type II diabetes, where MC6, MC6.1, MC6.2, MC6.3 and analogues and mimetics thereof, are administered to the human suffering from the hyperglycemic disorder to at least reduce the blood glucose level of the human, where the blood glucose level is reduced to about the normal blood glucose range for the human. Although, the active agents may be administered to the human using any of the convenient techniques described above, of particular interest is the oral administration of active agents.

For therapy with the fraction MC6 or the MC6.1, 6.2 and/or 6.3 peptides, the active compounds may be administered to the host suffering from the hyperglycemic disorder using any convenient administration technique, where such techniques include intravenous, intradermal, intramuscular, subcutaneous, oral, and the like, where oral routes of administration are of particular interest. The dosage delivered to the host will necessarily depend on the route by which the dosage is administered, but will generally range from about 50 to 500 mg/70 kb human body weight, usually from about 100 to 200 mg/70 kg human body weight. In treating human hyperglycemic disorders with MC6, the dosage of active fraction or compound administered to the human will generally range from about 50 to 500, usually from about 100 to 200 mg/70 kg of human body weight.

In using the active fraction or compounds according to the subject invention, they may be combined with a physiologically acceptable vehicle to produce a pharmaceutical composition. The nature of the physiologically acceptable vehicle with which the active fraction or compound is combined to produce the pharmaceutical composition will necessarily depend on the method by which the pharmaceutical composition is intended to be administered. Illustrative vehicles include water, e.g. sterile water for injection, saline, and the like. Of particular interest will be physiologically acceptable vehicles suitable for use in the oral administration. Such vehicles are known in the art and include water, e.g. deionized water; saline, e.g. phosphate buffered saline, lyophylized powder in the form of tablets and capsules, where such forms may include various fillers, binders etc., and the like. The amount of active ingredient present in the pharmaceutical composition will be selected in view of the method by which the pharmaceutical composition is to be administered, and may be determined empirically by those of skill in the art.

For therapy, MC6.2 and/or MC6.3, and oligopeptide analogues or mimetics thereof may be administered orally, topically or parenterally, e.g. by injection at a particular site, for example, subcutaneously, intraperitoneally, intravascularly, intranasally, transdermally or the like. Formulations for oral administration include those listed above suitable for use with MC6. Formulations for injection will comprise physiologically-acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like. Water soluble preservatives which may be employed include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in individual amounts of from about 0.001 to about 5% by weight and preferably about 0.01 to about 2%. Suitable water soluble buffering agents that may be employed are alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives such as carboxymethylcellulose may be used as a carrier in amounts of from about 0.01 to about 5% by weight. The formulation will vary depending upon the purpose of the formulation, the particular mode employed for modulating the receptor activity, the intended treatment, and the like The formulation may involve patches, capsules, liposomes, time delayed coatings, pills, or may be formulated in pumps for continuous administration. The specific dosage can be determined empirically in accordance with known ways. See, for example Harrison's, Principles of Internal Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.

Generally, a therapeutically effective dose of the active fraction or peptides, analogues and mimetics thereof, will be in the range of about 0.005-10, more usually from about 0.01-1 mg/kg of host weight. Such a dose will be sufficient to achieve the desired hypoglycemic activity. Administration may be as often as daily; usually not more than one or more times daily, or as infrequent as weekly, depending upon the level of drug which is administered. The amount oligopeptide administered will generally be adjusted depending upon the half life of the peptide, where dosages in the lower portion of the range may be employed where the peptide has an enhanced half life or is provided as a depot, such as a slow release composition comprising particles, introduced in a matrix which maintains the peptide over an extended period of time, e.g., a collagen matrix, use of a pump which continuously infuses the peptide over an extended period of time over a substantially continuous rate, or the like. Heller, Biodegradable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 1, CRC Press Boca Baton, Fla., 1987, pp 39-90, describes encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13:850-856 describes controlled drug release from hydrophobic polymers.

Claim 1 of 17 Claims

What is claimed is:

1. The water-soluble composition obtained from Momordica Charantia MC6 characterized by migrating as a single band of less than 10 kDal on SDS-20% PAGE comprising three peptides, exhibiting hypoglycemic activity and being active by oral administration.

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