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Ob protein compositions and methods

Ob protein compositions and methods description/claims

The present invention relates to OB protein compositions and methods for preparation and use thereof.

Although the molecular basis for obesity is largely unknown, the identification of the “OB gene” and protein encoded by (“OB protein”) has shed some light on mechanisms the body uses to regulate body fat deposition. Zhang et al., Nature 372: 425-432 (1994); see also, the Correction at Nature 374: 479 (1995). The OB protein has been demonstrated to be active in vivo in both ob/ob mutant mice (mice obese due to a defect in the production of the OB gene product) as well as in normal, wild type mice. The biological activity manifests itself in, among other things, weight loss. To date, however, optimum conditions for obtaining the rapid weight loss in normal animals has not been ascertained. In fact, some studies have shown that, when administered by injection, rather large dosages (10 mg of recombinant murine protein/kg body weight/day) are necessary for normal mice to lose 2.6% of their body weight (at the end of a 32 day period). While presently uncertain, one explanation for the necessity of such large dosages is that the optimum weight loss effects are seen predominantly when the protein is in constant circulation, a condition that may not be efficiently achieved by injecting the protein.

The present invention stems from the observation that, as compared to administering OB protein by injection, administering OB protein by continuous pump infusion results in equivalent (or better) weight loss, in a shorter time, and with substantially lower dosages. The working example below demonstrates that a dose of 0.5 mg protein/kg body weight/day, administered via implantable osmotic pump, results in a weight loss of over 4% (as compared to baseline weight). This is in substantial contrast to other studies where similar, or less weight loss (at a comparable time point) was observed with intraperitoneal injection at the relatively high dosage of 10 mg of protein/kg body weight/day.

Thus, one aspect of the present invention is a method of treating excess weight by administering OB protein in a form for constant supply, at a dosage of less than or equal to about 1 mg protein/kg body weight/day. The dosage of less than or equal to about 1 mg protein/kg/day refers to dosages sufficient to result in observable weight loss. This is apparent from the present studies where a dosage of 0.5 mg/kg/day was sufficient to result in observable weight loss when continuously administered. In studies where injection had been the mode of administration, far higher dosages were required for weight loss. At injection dosages of 0.1 and 1 mg/kg/day, substantially no weight loss was observed in wild type (normal) mice. For example, in one study, at a comparable time point (6th day), there was a 0.2% loss at the 1 mg/kg dose (data not shown). Minimal weight loss was observed at the relatively high 10 mg/kg/day dose. (1.9% weight loss at day 6, data not shown). Thus, the present invention provides for dosages of 1 mg/kg/day or less when administered so that the supply of protein is continuous.

Connected with the present studies are the compositions and methods used for production of recombinant murine and human OB protein. The first example below discloses the preparation of recombinant murine protein, and the second example below discloses the preparation of recombinant human protein.

Additional aspects of the present invention, therefore, include the below compositions and methods for preparing recombinant murine methionyl OB protein and recombinant human methionyl OB protein, including DNA sequences, vectors, host cells, methods of fermentation, and methods of purification.

The present invention stems from the observation that continuous administration of OB protein results in the need for much lower dosages for weight loss than those dosages required by acute daily injection. As set forth above, a dosage of 1 mg protein/kg body weight/day or less, continuously administered, resulted in rapid weight loss. When the underivatized protein was administered by acute injection at the 1 mg/kg/day dose, almost no weight loss in wild type (normal) mice.

The OB protein may be selected from the recombinant murine and human methionyl proteins set forth below (SEQ. ID Nos. 2 and 4) or those lacking a glutaminyl residue at position 28. (See Zhang et al, Nature, supra, at page 428.) The recombinant human OB gene product is, as a mature protein, 146 amino acids; some of the DNAs obtained were observed to encode a protein lacking a glutamine residue at position 28. Zhang et al., Nature 372 at 428. The murine protein is substantially homologous to the human protein, particularly as a mature protein, and, further, particularly at the N-terminus. One may prepare an analog of the recombinant human protein by altering (such as substituting amino acid residues), in the recombinant human sequence, the amino acids which diverge from the murine sequence. Because the recombinant human protein has biological activity in mice, such analog would likely be active. Proteins lacking an N-terminal methionyl residue, such as those produced by eukaryotic expression, are also available for use.

In addition, although the present working example involved continuous administration via implantable pump, it is contemplated that other modes of continuous administration may be practiced. For example, chemical derivatization may result in sustained release forms of the protein which have the effect of continuous presence in the blood stream, in predictable amounts. Thus, one may derivatize the above proteins to effectuate such continuous administration. The dosage of 1 mg protein/kg body weight/day or less herein refers to the mass of protein, exclusive of other chemical moieties used to derivatize the protein.

Generally, the present protein (herein the term “protein” is used to include “peptide”, unless otherwise indicated) may be derivatized by the attachment of one or more chemical moieties to the protein moiety. The chemically modified derivatives may be further formulated for intraarterial, intraperitoneal, intramuscular subcutaneous, intravenous, oral, nasal, pulmonary, topical or other routes of administration. Chemical modification of biologically active proteins has been found to provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review, see Abuchowski et al., in Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp. 367-383 (1981)). A review article describing protein modification and fusion proteins is Francis, Focus on Growth Factors 3: 4-10 (May 1992) (published by Mediscript, Mountview Court, Friern Barnet Lane, London N20, OLD, UK). For the present continuous administration, it is preferred that the chemical modification allow for an increase in circulation time of the protein, so that a dosage of about 1 mg protein (exclusive of chemical moiety)/kg body weight of a mammal/day or less will result in weight loss of a mammal. The present continuous administration will provide for weight loss of approximately 5% of body mass in a period of 7 or fewer days.

The chemical moieties suitable for derivatization may be selected from among water soluble polymers. The polymer selected should be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present proteins and peptides, the effectiveness of the derivatization may be ascertained by administering the derivative, in the desired form (i.e., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or nasal delivery, for example), and measuring weight loss.

The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 2 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols). The proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art. E.g., EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s). Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group. Attachment at residues important for receptor binding should be avoided if receptor binding is desired.

One may specifically desire N-terminally chemically modified protein. Using polyethylene glycol as an illustration of the present compositions, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For example, one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa differences between the ε-amino group of the lysine residues and that of the α-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs. Using reductive alkylation, the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein. Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.

In yet another aspect of the present invention, provided are methods of using pharmaceutical compositions of the proteins and derivatives. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal or other forms of administration which allow for the desired circulating dose of about 1 mg protein/kg body weight/day or less. In general, comprehended by the invention are pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, eq., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. The effective amounts are those herein described.

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