MXPA99009384A - Compositions comprising conjugates of stable, active, human ob protein with antibody fc chain and methods - Google Patents

Abstract

The present invention relates to OB protein compositions and related methods. Provided herein are OB protein suspensions which are stable and active at physiologic pH. Such OB protein suspensions are useful for the treatment or modulation of weight adiposity level, diabetes, and other conditions.

Description

COMPOSITIONS THAT INCLUDE CONJUGATES OF THE PROTEIN STABLE HUMAN OB, ACTIVE WITH CHAIN ANTIBODY FC AND METHODS Field of the Invention The present invention relates to compositions of the stable human OB protein, active at high concentrations and at or near physiological pH. Related compositions, methods of making and methods for using such compositions are also provided.

Background Although the molecular basis for obesity is largely unknown, the identification of the "OB" gene and the encoded protein ("OB protein") has provided some clarity in the mechanisms the body uses to regulate the deposition of body fat. Zhang et al., Naturé 372: 425-432 (1994); see also, Correction at Nature 374: 479 (1995). PCT Publication No. WO 96/05309, published February 22, 1996, entitled "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses Thereof" REF .: 31377 completely establishes the OB protein and related compositions and methods, and is incorporated herein by reference. An amino acid sequence for the human OB protein is set forth in WO 96/05309 (incorporated herein by reference) SEQ ID NOS. 4 and 6 (on pages 172 and 174 of such publication), and the residue of the first amino acid of the mature protein is in position 22 and is a valine residue. The mature protein is 146 residues (or 145 if the glutamine at position 49 is absent, SEQ ID NO: 6).

The OB protein is active in vivo in the mutant ob / ob mice (obsessed mice due to a defect in the production of the OB gene product) as well as in normal mice, wild-type mice. The biological activity manifests itself among other things in, weight loss. See in general, Baringa, "Obese Protein Slims Mice, Science 269: 475: 476 (1995) and Fpedman," The Alphabet of Weight Control, "Nature 385: 119-120 (1997) It is known, for example, that in mutant ob / ob mice, administration of the OB protein results in a decrease in serum insulin levels, and serum glucose levels.It is also known that the administration of the OB protein results in ~~ a decrease in body fat, this was observed in the ob / ob mutant mice, as well as in normal non-obese mice. Pelleymount er et al., Science 269: 540-543 (1995); Halaas et al., Science 269: 543-546 (1995). See also, Campfield et al., Science 269: 546-549 (1995) (Peripheral and central admmistrat ion of microgram of OB protein reduced food intake and body weight of ob / ob and diet-induced obese mice but not in db / db obese mice.) In none of these reports have toxicities been observed, even in the highest doses.

For the preparation of a pharmaceutical composition for injection in humans, it has been observed that the human amino acid sequence is insoluble at physiological pH at relatively high concentrations, such as above about 2 mg of the active protein / milliliter of the liquid. Dosages in the milligram range of protein per kilogram of body weight, such as 0.5 or 1.0 mg / kg / day, are desirable for the injection of therapeutically effective amounts in large mammals, such as humans. An increase in the concentration of the protein, is necessary to avoid the injection of large volumes, which can be uncomfortable or possibly painful for the patient.

With advances in recombinant DNA technology, the availability of recombinant proteins for therapeutic use, has engendered advances in the formulation of proteins. A review article describing protein modification and protein fusion is Francis, Fo cus on Growth Fa c t ors 3_: 4-10 (1992).

One such modification is the use of the immunoglobulin Fe region. The antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds the antigen and a constant domain, known as "Fe" that provides the binding to effector functions such as complement or phagocytic cells. The Fe portion of an immunoglobulin has a long plasma half-life, while the Fab is short-lived. Capón et al., Nature 337: 525-531 (1989).

The therapeutic proteins have been constructed using the Fe domain to provide greater half-life or to incorporate functions such as Fe receptor binding, protein A binding, complement fixation and placental transfer, residing in the Fe proteins of the immunoglobulins. Id. For example, the Fe region of an I gG1 antibody has been fused to the N-terminus of CD30-L, a molecule that binds CD30 receptors expressed in tumor cells of Hodgkin's disease, anaplastic lymphoma cells, T cell leukemia cells and other types of malignant cells. See, U.S. Pat. No. 5,480,981. IL-10, an anti-inflammatory agent and an anti-rejection agent, has been fused to murine Fc? 2a to increase the short circulation half-life in the cytokine. Zhang, X. et a l. , The Journal of Immunology, 154: 5590-5600 (1995). Studies on the use of the tumor necrosis factor receptor bound to the human IgC21 Fe protein to treat patients with septic shock have also been evaluated. Fisher, C. et a l. , N. Engl. J. Med., 334: 1697: 1702 (1996); Van Zee, K. et al., The Journal of Imunology, 156: 2221-2230 (1996). Fe has also been fused with the CD4 receptor to produce a therapeutic protein for the treatment of AIDS. See, Capón et al. , Nature, 337: 525-531 (1989). In addition, the N-terminus of interleukin 2 has also been fused to the Fe portion of IgG1 or IgG3 to overcome the short half-life of interleukin-2 and its systemic toxicity. See, Harvill et al? , Immunotechnology, 1 .: 95-105 (1995). _ For insulin, suspension preparations have been reported. But, these conditions that are applicable to insulin are not predictive of conditions that could be applicable to any other protein, including the OB protein. Insulin is a clearly small protein, which has particular physical and chemical characteristics; These characteristics are important to determine the conditions for the formulation. Brange, Galenics of Insulin, Springer-Verlag 1987, describes insulin suspensions (p.36); see also, Schlicht krull et al. Insulin Preparations with Prolonged Effect, pp. 729-777 I_n: Hassellblatt et al., Handbook of Experimental Pharmacology New Series, Vol. XXXII-1/2 Springer-Verlag Berlin, Heidelburg, New York (1975).

To date, there have been no reports of stable preparations of the human OB protein at concentrations of at least about 2 mg / ml at physiological pH, and in addition, there are no reports of stable concentrations of an active human OB protein. at least 50 mg / ml or above. In addition, a frozen or lyophilized form could be used to improve shelf stability but, less desirable than the ready-to-use suspension forms described herein. A frozen form requires storage at a constant freezing temperature that might not be desirable due to the defrosting cycles of the consumer grade refrigerators and freezers. In addition, a lyophilized form must be diluted and mixed which is convenient and could establish the patient's reliability. From a producer's point of view, processing, storage and transport of frozen liquid is expensive and requires more supervision than distribution of a ready-to-use formulation. Also, working up or otherwise making an available or appropriate diluent for a lyophilized formulation is more expensive and less efficient than that which does not require such a diluent. There is a need for the concentrated forms of human pharmaceutical compositions containing the active OB protein that can be released by injection at low volumes. The present invention satisfies these requirements.

Brief Description of the Invention The present invention provides from the observation that certain suspension formulations allow the preparation of the stable OB protein at concentrations of at least about 2 mg / ml at physiological pH. As used herein, the term "physiological pH" refers to a pH of from about 6.0 to about 8.0 The use of such compositions allows the release of relatively low volumes of the therapeutic Ob protein. the Precipitation agents allows the preparation of suspensions of the OB protein that have the characteristics of: 1. Improved stability at physiological pH as compared to the same Ob protein from human in the form of solution. The suspensions of human OB protein at concentrations of 10 mg / ml or higher and at pH 7.0, which, when compared to the same concentration in solution at the highest pH, which allows the solubility (pH 4 for human OB protein in its native form, which is not yet at physiological pH) has better HPLC profiles (high pressure liquid chromatography). A suspension of the Fc-OB fusion protein at a concentration of 5 mg / ml at about pH 7.0 is also shown below, which shows less degradation products in storage relative to a solution of the Fc-OB fusion protein. comparable. 2. The profile of the release time of the prolonged injection compared to the same OB protein of human in the form of solution. The working examples subsequently demonstrate a prolonged release effect using the suspensions of the current human OB protein highly concentrated Such a prolonged release effect is advantageous in that the activity of the material is maintained for a relatively longer period of time, and thus there is a relatively higher potency, and the need for smaller injections as compared to the human OB protein in solution.

Thus, an objective of the present invention is a stable preparation of the active human OB protein at physiological pH, which has a concentration of at least about 2 mg protein / ml. For example, a stable preparation of the active human OB protein at a concentration of at least about 2.0 mg / ml and "a pH of 7.0 is provided." The upper limit of concentration is that the suspension form that is available for administration to a human, and, as described below, working examples have provided a concentration as high as 100 mg / ml at physiological pH (eg, between pH 6.0 and pH 8.0).

In another aspect, the present invention relates to a stable preparation of the human active OB protein within the pH range of about 6.0 to about 8.0 having a concentration of at least about 10 mg / ml.

In yet another aspect, the present invention relates to a stable preparation of the human active OB protein derived by the binding of a Fe region of an immunoglobulin at a concentration of at least about 0.5 mg / ml and a pH of about 6.0 to approximately 8.0. More particularly, stable preparations of the active Fc-OB fusion protein at a concentration of 5 mg / ml to 50 mg / ml at about pH 7.5 are provided.

In still other aspects, the present invention provides the formulations for the stable human OB protein, active at a concentration of 2 mg protein / ml or, preferably, at pH 6.5 to pH 7.5. More particularly, the formulations for the stable human OB protein, active at a concentration of 20 mg / ml to 100 mg / ml at pH 7.0, are provided.

In another aspect, the present invention provides the formulations for stable human O_protein, active at a concentration of 10 mg / ml or above at a pH of 5.0 to 8.0.

In yet another aspect, the present invention provides the formulations for the stable human OB protein, active derived by binding an Fe region of an immunoglobulin to a concentration of 0.5 mg / ml or above and a pH of from about 6.0 to about 8.0. More particularly, formulations are provided for stable Fc-OB fusion protein, active at a concentration of 5 mg / ml to 50 mg / ml at about pH 7.5.

Still other aspects of the present invention include the pharmaceutical compositions of the above methods, for the preparation of such compositions, and the methods of treatment using the present compositions, and the methods of making medicaments containing the present compositions for such treatment.

Brief Description of the Drawings FIGURES 1A and IB are the dose response curves in the mice for the present human OB suspension (1A) of Example 1 and a control of the human OB (OB) protein solution as described in FIG.

Example 1.

FIGURE 2 is a graph illustrating serum OB levels in dogs for the treatment of suspension of the current OB protein and for a solution of the control human OB protein as described in Example 1.

FIGURE 3 is a reverse phase high pressure liquid chromatography (RP-HPLC) which traces the formulations of the human OB protein at 37 ° C for seven weeks: the zinc suspension of the human OB protein of Example 1 is the middle trace, the control of the solution of the human OB protein of Example 1 is the upper line and a suspension of the human OB protein which is maintained at -80 ° C for 56 days is the lower line.

Laf FIGURE 4 is a RP-HPLC trace of human OB protein formulations at 19 ° C for 56 days: the human OB solution of Example 1 is the top line, the human OB crystal clear suspension of Example 2 is the middle line, and the crystalline suspension of human OB of Example 2 at -80 ° C for 56 days is the bottom line.

FIGURES 5A-5C are the DNA sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the human metFc-OB protein.

FIGURES 6A-6C are the DNA sequence (Sec ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) of a variant of the human metFc-OB protein.

FIGURE 7 is a graph representing the rate of iso asp formation in asp 108 (asp 335 using numbering according to SEQ ID NO: 4), as determined by reverse phase HPLC, for the Fc-OB protein of recombinant methionyl human.

Detailed description of the invention The present stable, active OB protein compositions are generally classified as suspensions, in which the protein radical is precipitated and suspended in a liquid radical. The compositions contain a radical of the active OB protein, a precipitating agent, a pH modulating agent and a liquid carrier. The present OB proteins are either amorphous or in the crystalline form.

Preferably, for use as a therapeutic or cosmetic composition in humans, the OB protein is used with the amino acid sequence of the native human OB protein (see Zhang et al., Nature, supra), optionally with an incident N-terminal methionyl residue with the expression bacterial See, PCT Publication WO 96/05309, incorporated herein by reference, for recombinant DNA media to prepare the present OB proteins that could be used. Changes could be made in the selected amino acids, provided that the changes preserve the overall fold or activity of the protein. Table 1 below establishes the conserved amino acid substitutions that could be used, in terms of the particular characteristics (basic, acidic, polar, hydrophobic, aromatic and size (small)). See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference. Small amino terminal spreads may also be present, such as an amino terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an epitope antigenic or a linker domain.

Table 1 Conservative Amino Acid Substitutions In general, human OB proteins that will exhibit increased stability in the present suspensions will be those which, due to exposure to physiological pH, have a hydrophobic region exposed in solution. Additionally, human OB proteins derived by binding an Fe region of an immunoglobulin to the protein moiety will exhibit increased stability in the present suspensions.

In general, a Fe region of an immunoglobulin could be genetically or chemically fused to a human OB protein. Preferably, the Fe region is fused at the N-terminus of the OB protein. See Application U.S. Copendent Serial No. 08 / 770,973, published December 20, 1996, incorporated herein by reference, for preferred Fc-OB fusion proteins.

Preferably, an Fe region with the amino acid sequence of the human IgG-1 immunoglobulin heavy chain is used (see Ellison, JW et al., Nucleics Acids Res. 10: 4071-4079 (1982).) A preferred Fe region is established in SEC IN NO: 2 (see Figure 5) The recombinant Fc-OB sequence of SEQ ID NO: 2 is an Fc-OB protein of 378 amino acids (not counting the methionine residue) The first amino acid residue of the Fc-OB protein in the Figure 5, glutamic acid, is preferred for +1 with methionine in position -1. Variants or analogs of the Fe moiety could be constructed by, for example, making several substitutions of amino acid residues or base pairs.

The cysteine residues can be removed or replaced with other amino acids to prevent cross-disulfide cross-linking of the Fe sequences. In particular, the amino acid at position 5 of SEQ ID NO: 2 is a cysteine residue. You could remove the cysteine residue in position 5 or replace it with one or more amino acids. For example, an alanine residue could be substituted for the alanine residue at position 5 resulting in a variant amino acid sequence. In the same way, the cysteine at position 5 of SEQ ID NO: 2 could be substituted with a serine or other amino acid residue or eliminated.

Variant or analogous nail, could also be prepared by eliminating the amino acids at positions 1, 2, 3, 4 and 5 resulting in a Fe protein of 373 amino acids (not counting the methionine residue). This sequence is set forth in SEQ ID NO: 4 (see Figure 6). Substitutions in these positions can also be be made and are within the scope of this invention Modifications could also be made to introduce four amino acid substitutions to heat remove the Fe receptor binding site and the complement binding site (Clq). According to the * numbering of SEQ ID NO: 4, these variant modifications include leucine at position 15 substituted with glutamic acid, glutamic acid at position 98 is replaced with alanine, and lysine at positions 100 and 102 is replaced with alanines.

Similarly, one p plus tyrosine residues can be replaced by the phenylalanine residues. As described above, changes in the selected amino acids could be made as long as such changes preserve the overall fold or activity of the fusion protein.

In addition, the Fe region could also be linked to the human OB protein of the Fc-OB fusion protein by the "binding" radicals, if the chemicals or amino acids of the variant lengths. Such chemical linkers are well known in the art. The amino acid linker sequences can be included, but limit to (a) wing, wing, wing, wing; (b) wing, wing, wing, wing, wing; (c) wing, wing, wing, wing, wing, wing; (d) gly, gly; (e) gly, gly, gly; Cf) gly, gly, gly, gly, gly; (g) gly, gly, gly, gly, gly, gly, gly; (h) gly-pro-gly; (i) gly, gly, pro, gly, gly; and (j) any combination of subparts (a) to (i) - The present precipitating agents could be a salt having a cationic component, and could be selected from calcium, magnesium, zinc, sodium, iron, cobalt, manganese , potassium and niguel. Preferably, the salt will be compatible for use in a pharmaceutical composition.

Alternatively the precipitating agents could be selected from among the agents which are pharmaceutically acceptable yet known to precipitate proteins, such as polyethylene glycols, or other Water-soluble polymers as set forth in the following paragraphs. A useful precipitation agent will induce precipitation of the OB protein at neutral pH, but is reversible or redissolves due to dilution with physiologically compatible solvents. Without an appropriate precipitating agent, the OB protein precipitates at neutral pH to a form that It is not reversible, by diluting it with physiologically compatible solvents.

The pH range is preferably from about pH 4.0 to about pH 8.0, and more preferably from about 6.5 to about 7.5. The most preferred pH for a pharmaceutical composition is one in which the OB protein using, could retain its maximum biological activity at the concentration of the selected protein. At non-physiological pH, the present suspensions of the OB protein, could also have advantages. At pH lower than 5.0, the present suspensions of the OB protein could be more stable (in terms of shelf life) than the equal concentrations of the Ob protein in solution at equal pH. For example at pH 4.0, and a concentration of 50 mg / ml, the present suspensions could have higher biological activity due to in vivo administration than which would have the equivalent solution.

The buffer could be selected from those containing the desired pH as long as the precipitation characteristics of the composition are not altered. Preferably, the buffers will be acceptable for a pharmaceutical formulation. Tris, MES and PIPES are acceptable for amorphous and crystalline forms. Phosphate is a preferred buffer for crystalline forms.

The final suspension will preferably have a concentration of 5 mg / ml to 100 mg / ml for ease of therapeutic administration.

Methods of Use Therapeutic Therapeutic uses include weight modulation, treatment or prevention of diabetes, reduction of blood lipids (and treatment of related conditions), increase in lean body mass and increase in insulin sensitivity. In addition, the present compositions could be used for the manufacture of one or more medicaments for the treatment or reduction of the above conditions. The administration methods will typically be by injection, aungue other means could be used, such as pulmonary release. See PCT WO 96/05309, incorporated herein by reference to page 83 et seq. , for example. The present suspensions could be spray-dried into particles having an average size of less than 10 microns, or more preferably, 0.5 to 5 microns.

Weight Modulation The present compositions and methods could be used for weight reduction. From another point of view, the present compositions could be used to maintain a desired weight or level of adiposity. As it has been demonstrated, in the murine models (see above), the administration of the present OB protein results in weight loss. The loss of body mass is mainly fat or adipose tissue. Such weight loss can be associated with the treatment of concomitant conditions, such as the previous ones, and therefore constitute a therapeutic application. In addition, the cosmetic uses' ^ are provided here, if the modulation of weight is exclusively for the improvement of the appearance.

Treatment of Diabetes. The present compositions and methods could be used in the prevention or treatment of type II diabetes. As type II diabetes can correlating with obesity, the use of the present invention to reduce weight (or maintain a desired weight or reduce or maintain an adiposity level) may also decrease or prevent the development of diabetes. In addition, even in the absence of sufficient dosages to result in weight loss, the present compositions could be used to prevent or decrease diabetes.

Modulation of Lipids in the Blood. The present compositions and methods could be used in the modulation of lipid levels in the blood. Ideally, in situations where only the reduction of lipid levels in I = Tsangre is desired, or where the maintenance of blood lipid levels is desired, the dosage will be insufficient to result in weight loss. In this way, during an initial course of therapy of an obese patient, the dosages could be administered, whereby the weight loss and the decrease in the level of concomitant blood lipids are achieved. Once sufficient weight loss is achieved, a sufficient dosage could be administered to prevent weight regain, still sufficient to maintain the desired blood lipid levels, or other conditions as set forth herein, for example. These dosages can be determined empirically, since the effects of the OB protein are reversible. E.g . , Camfield et al., Science 269: 546-549 (1995) to 547. Thus, if a dosage is observed that results in weight loss when the loss is not desired, a lower dosage would be administered to achieve the levels of desired blood lipids, while maintaining the desired weight. See, p. e. , WO PCT Publication 97/06816 which is incorporated herein by reference.

Increase in Thin Tissue Mass or Sensitivity to Insulin. Ideally, in situations where an increase in the thin tissue mass is exclusively desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of an obese person, dosages may be administered, whereby weight loss and concomitant fat tissue decrease / thin mass increase is achieved. Once sufficient weight loss is achieved, a sufficient dosage could be administered to prevent weight regain, still sufficient to maintain the desired thin mass increase (or prevention of thin mass reduction). To increase the sensitivity of an individual to insulin, dosing considerations could be taken into account Similar. The increase in the mass of thin tissue without weight loss, could be enough to decrease the amount of insulin (or, potentially, amylin, antagonists or agonists of amylin or iazolidinediones or other potential diabetes treatment drugs), would be administered a individual for the treatment of diabetes. To increase the overall strength, there may be similar dosage considerations. The increase in the mass of thin tissue with the concomitant increase in overall resistance could be achieved with insufficient dosages to result in weight loss. Other benefits, such as an increase in red blood cells (and oxygenation in the blood) and a decrease in bone resorption and osteoporos, could also be achieved in the absence of weight loss. See, p. ei. And PCT Publication No. WO 97/18833 incorporated herein by reference.

Combination Therapies The present compositions and methods could be used in conjunction with other therapies, such as "altered diet and exercise." Other medications, such as those used for the treatment of diabetes (eg, insulin and possibly amylin, antagonists or agonists thereof) , thiazolidinediones (see, e.g., PCT Publication No. WO 98/08512 incorporated herein by reference), or other potential drugs for the treatment of diabetes, cholesterol, and medications for lowering blood pressure (such as those that reduce the levels of lipids in the blood or other cardiovascular drugs), drugs that increase activity (p. eg, amphetamines), diuretics (for fluid elimination) and appetite suppressants (such as agents that act on receptors and neuropeptides or serotonin uptake inhibitors). Such an administration could be simultaneous or it could be in seriat im. further, the present methods could be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (eg, liposuction or laser surgeries designed to reduce body mass or implant surgeries, designed to increase the appearance of body mass). The health benefits of cardiac surgeries, such as bypass surgeries or other surgeries designed to alleviate a harmful condition caused by blockage of blood vessels by fat deposits, such as "arterial" plaque, could be increased with the concomitant use of the present compositions and methods. Methods for removing gallstones, such as ultrasonic or laser methods, could also be used either before, during or after a course of the present methods. therapeutic In addition, the present methods could be used as an additional for surgeries or therapies for broken bones, damaged muscle or other therapies that would be improved by an increase in the mass of thin tissue.

The following examples are offered to more fully illustrate the invention, but are not elaborated as limiting the scope thereof. Example 1 establishes the preparation of a suspension of the human OB protein (as opposed to a crystalline, as infra) at a concentration of 100 mg / ml at pH 7.0. Example 2 establishes the preparation of a suspension of the human crystalline OB protein. Example 3 demonstrates an improved dosage response for the suspensions of the OB protein compared to the OB protein solutions. Example 4 demonstrates the delayed action time profile of the present suspensions in a dog model. Example 5 provides for the preparation of a suspension of the amorphous Fc-OB protein. Examples 6 and 7 demonstrate the improved stability of the present suspensions.

EXAMPLE 1: Preparation of a suspension of the amorphous OB protein.

This example illustrates the preparation of a suspension of the human OB protein of the present invention. A suspension of the amorphous human OB-protein was prepared by precipitation with the zinc salt. At a final pH of 6.0 to 8.0, a concentration of 100 mg of protein / ml of liquid has been obtained. A control composition, pH 4.0, is also established for a solution of the human OB. Protein.

Composition: Protein radical: the recombinant methionyl human OB protein ("rmetHu-lept ina) as set forth in SEQ ID NO: 4 of PCT Publication WO 96/05309, beginning with the amino acid number 22 (Val) and ends with amino acid number 167, which has a methionyl residue at its N-terminus: Precipitant: zinc chloride Shock absorber: Tris, MES and Pipes Final PH: 6.0 - 8.0 Preparation protocol "The solution of recombinant methionyl human OB protein (" rmetHu-lept ina ") was concentrated to approximately 40 mg / ml in water for injection, acidified to pH 3.0, with HCl, zinc chloride was added. and the suspension was formed by adjusting the pH to close the neutrality (approximately pH 7.0) by adding an appropriate buffer.The Tris, MES and PIPES buffers have been successfully used.The final conditions were typically from 10 to 15 mM buffer, zinc 20 to 1000 μM, pH 6.0 to 8.0 and rmetHu-leptin 10 mg / ml The suspensions have been concentrated allowing the particles to settle at 4 ° C for several hours and removing the supernatant, this procedure could be repeated several times until obtain the maximum concentration, and has been used to obtain approximately 100 mg / ml suspensions.

Control Composition: The solution of the recombinant methionyl human OB protein (as above) at 20 mg / ml, pH 4.0 containing 10 mM acetate and 5% w / v sorbitol, was used as a control composition.

EXAMPLE 2: Preparation of a suspension of the crystalline OB protein.

This example illustrates the preparation of a suspension of the crystalline OB protein of the present invention.

Radical protein: The recombinant methionyl human OB protein was used, as in Example 1 above.

Procedure: rmetHu-leptin was mixed at a concentration of 15 mg / ml in 1 mM HCl in a 1: 1 ratio with 4 M NaCl, 100 mM Tris, pH 8.5, 2% v / v ethanol, at 4 ° C. The crystals formed spontaneously by slowly adjusting the temperature for several hours at between 14 ° C and 25 ° C and maintaining this temperature for at least 2 hours, depending on the duration of heating to the final temperature. The "mother liquor" (eg, the liquid in which the crystals grew) was replaced with a more appropriate solvent for injection, harvesting the crystals by centrifugation and resuspension in an appropriate crystalline stabilizing agent. An appropriate replacement solvent is 20-25% polyethylene glycol (which has a molecular weight of about 4000 daltons to about 20,000 daltons, the term "approximately" means the approximate average molecular weight for commercial PEG preparations), buffer-appropriate neutral pH, preferably about pH 6.0 to about pH 8.0, and preferably 10 mM phosphate buffer, pH 6.0 to pH 7.5, and ethanol at "2% v / v. In a typical preparation, some of the commercial salt could be less than 0.25 M.

EXAMPLE 3: Improved dosage response for suspensions of the OB protein compared to the OB protein solutions.

This example demonstrates that the present suspensions of the OB protein are more efficient than the OB protein in solution. The normal thin mice were given daily injections, for 5 days, of the present suspension at 1, 10 and 50 mg of the protein per kg of body weight, or the solution of the OB protein at the same dosage. The mice that were given the suspension lost more weight per unit mass of the OB protein given than those that were not given equal dosages of the solution formulation. This is illustrated in the FIGURE 1A and FIGURE IB. FIGURE 1A shows the percent of the weight change when the suspension was given in Example 1. FIGURE IB shows the percent of the change in weight when the control solution of Example 1 was given.

This illustrates that the present suspension at the same dosage is more efficient than that given in the solution form. While not wishing to be bound by theory, this could be due to the slower absorption rate of the suspension compared to the solution. The suspension must be dissolved before entering the blood, so that this release effect results in greater efficiency. On a mass basis, less protein in suspension needs to be administered than in solution. In addition, in solution there is no difference between the dosage of 10 and 5"0 mg / kg on day 5 (the last day of dosing) (See Table 2, below.) The suspension gives a curve of the dosage response __ more definitive that the formulation of the solution.

TABLE 2 PERCENT OF THE CHANGE OF THE WEIGHT OF THE DAY 0 The results are an average for 5 mice per treatment.

Methods: Animals: Normal CD-1 mice were used. Base weight: Approximately 20 grams. Administration: The animals were injected SC each day in the same place for 5 days. Management: The animáis were housed in groups, and fed ad libitum.

Compositions: Solution: The rmetHu-lept ina solution of Example 1 was used, at a concentration of 20 mg / ml. Suspension: The rmetHu-lept ina suspension of Example 1 was used at pH 7.0, 10 mM MES buffer, 500 mM Zn, 20 mg / ml. PBS: Phosphate-buffered saline was used as a placebo. (Dosage responses for controls using liquid suspension or liquid only, "without protein, were similar to PBS, ... results are not shown).

EXAMPLE 4: Serum levels of the OB protein in dogs.

This example demonstrates the action profile of the delayed time of the present suspensions. Methods: Beagle dogs were given the suspension or solution of rmetHu-lept ina. The serum was extracted, and the levels of the OB protein were measured at the time illustrated in FIGURE 2.

TABLE 3 Compositions used: Solution: The solution was used according to Example 1, at a dosage of 5 mg / kg / day. Suspension: The suspension was used according to Example 2. See Table. Animals: The results presented in Table 3 are for an average-of 3 animals. Animals They were normal beagle dogs. Handling: The animals were housed individually, and fed ad libitum. The good management practices of animals were obeyed. Test An antibody test was used as in Hotta et al, J. Biol. Chem 271: 255327-25331 (1996). Results As can be seen from FIGURE 2, the concentration peaks of the solution 1 to 2 hours after administration of the protein, while the concentration peaks of the suspension much later, after 10-12 hours. This shows that the suspension maintains a minimum effective dosage for a longer period of time.

EXAMPLE 5 Preparation of a suspension of amorphous Fc-OB protein.

This example illustrates the preparation of a suspension of the human Fc-OB protein of the present invention. A suspension of the amorphous human Fc-OB protein was prepared by precipitation with the zinc salt. A control composition, pH 7.5, is also established for a Human protein Fc-OB ~ solution Composition: Radical protein: The recombinant methionyl human Fc-OB protein ("rmetHu-Fc-lept ina") as set forth in SEQ ID NO: 4, a fusion protein of amino acid 373, the Fe portion of the protein which is in amino acids 1-227 and the portion of the human protein OB of the protein that is amino acids 228-373, which has in its N term a methionyl residue: Precipitant: zinc chloride Shock absorber: Tris 100 mM Final PH: 7.5 Preparation protocol: A solution of rmetHu-Fc-leptin was concentrated to approximately 5 mg / ml in 100 mM Tris buffer, pH 7.5. Zinc chloride was added to form the suspension.

Control Composition: A solution of rmetHu-Fc-leptin (as above) at 5 mg / ml, pH 7.5 containing 100 mM Tris, was used as a control composition.

EXAMPLE 6: Stability of the present suspensions of the human OB protein.

This example demonstrates that the amorphous and crystalline forms of the present suspensions are more stable under the conditions of the accelerated stability test than the material in solution.

FIGURE 3 illustrates a RP-HPLC process comparing the amorphous zinc form of the present suspension (as in Example 1) with the solution form (also as in Example 1), at 37 ° C for a period of 7 weeks. As can be seen, the present suspension (middle line) has lower peaks (which indicate lower cut products) than the shape of the solution (upper line). As a comparison, the bottom line presents the amorphous form of zinc that had been stored for 7 weeks at -80 ° C.

FIGURE 4 illustrates a trace of RP-HPLC comparing the "shape of the crystalline suspension of Example 2 with the form of the solution (of Example 1). The materials were stored for 56 days at 19 ° C. 37 ° C was not a re evant condition, as the crystalline form loses its crystalline structure at 37 ° C).

FIGURE 4 shows that there are more peaks, indicating more degradation, for the J-shape solution, (main peak = 86%) than for the shape of the suspension (main peak = 94%). The control in the lower line was the crystalline form stored for 56 days at -80 ° C. Similar results were observed during 4 ° C, although degradation occurred more slowly.

The main degradation product appeared to be aspartate at the amino acid position 108 (according to SEQ ID NO: 4 in WO PCT 96/05309, using the "Val" residue as the 1-position number). A more stable chemical radical in this position, such as another amino acid, could be empirically selected to improve the stability of the molecule. In general, the suspensions of the present invention are more stable than the OB protein in solution.

EXAMPLE 7: Stability of the present suspensions of the human Fc-OB protein.

This example demonstrates that the amorphous form of the present suspensions of the Fc-OB protein is more stable under the conditions of the accelerated stability test than the material in solution. _ The amorphous zinc form of the present suspension (as in Example 5) and the solution form (also as in Example 5) were placed in the stability studies by storing the suspension forms and the solution at 4 ° C. , 29 ° C and 37 ° during a period of 2 weeks. As shown in FIGURE 7, suspensions of Fc-leptin stored at 29 ° C and 37 ° C, show a lower percentage of degradation products relative to aspartate at the amino acid position 108 of the OB protein than the solutions of Fc-leptin stored at the same temperatures, respectively.

While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will be presented to those skilled in the art. Therefore, it is understood that the appended claims cover all equivalent variations that are within the scope of the invention as claimed.It is noted that in relation to this date, the best method known to the applicant to carry out the practice said invention is the conventional one for the manufacture of the objects to which it refers The invention having been described as above, the content of the following is claimed as property.

Claims (13)

1. A suspension of the human OB protein having a pH of about 6.0 to about 8.0 and a concentration of at least 0.5 mg / ml, characterized in that the OB protein is derived by attaching a Fe region of an immunoglobulin to the N terminus. of the radical of the OB protein.

2. A suspension of the human OB protein of claim 1, characterized in that the Fe portion of the human Fc-OB protein is selected from the group consisting of: (a) the amino acid sequences Fe are set forth in SEQ ID NOS: 2 and 4; (b) the amino acid sequences of subpart (a) have a different amino acid substituted or deleted in one or more of the following positions (using numbering according to SEQ ID NO: 2): (i) one or more cysteine residues replaced by a residue of alanine or serine; (ii) one or more tyrosine residues replaced by a phenylalanine residue; (iii) the amino acid at position 5 is replaced with an alanine; (iv) the amino acid at position 20 is replaced with glutamic acid; (v) the amino acid at position 103 is replaced with an alanine; (vi) the amino acid at position 105 is replaced with an alanine; (vii) the amino acid at position 107 is replaced with an alanine; (viii) the amino acids in positions 1, 2, 3, 4 and 5 are eliminated; (ix) one or more residues are replaced or removed to remove the binding site of the Fe receptor; (x) one or more wastes are replaced or disposed of remove the complement binding site (Clq); Y (xi) a combination of subparts i-x; Y (c) the amino acid sequence of subparts (a) or (b) that has a methionyl residue in the term N.

3. A suspension of the human OB protein of claim 1, characterized in that the concentration is at least 5 mg / ml.

4. A suspension of the human OB protein of claim 1, characterized in that the concentration is between 5 mg / ml and 50 mg / ml.

5. A suspension of the human OB protein of claim 1, characterized in that the pH is pH 7.0.

6. A suspension of the human OB protein of claim 2, characterized in that the human OB protein is rmetHu-Fc-lept ina.

7. A suspension of the human OB protein of claim 1, characterized in that it contains a pharmaceutically acceptable precipitating agent.

8. A suspension of the human OB protein of claim 1, characterized in that it contains a precipitating agent selected from a salt and a water-soluble polymer.

9. A suspension of the human OB protein of claim 1, characterized in that it contains a precipitating agent selected from calcium, magnesium, zinc, sodium, iron, cobalt, manganese, potassium and nickel.

10. A method for treating an individual for a condition, by administering an effective dosage of a human OB suspension according to claim 1, characterized in that the condition is selected from: (a) weight modulation, - (b) modulation of adiposity (c) diabetes, (d) modulation of the level of lipids in the blood, (e) increase in thin mass, and (f) increase in sensitivity to insulin.

11. A method for preparing a suspension of the human OB protein, characterized in that it comprises: combining, under the appropriate conditions, a precipitating agent and an OB protein of human in solution, wherein the OB protein is derived by binding an Fe region of an immunoglobulin to the N terminus of the OB protein radical; allow the human OB protein to precipitate; collect the precipitated human OB protein; and _ optionally, resuspending the human OB protein in a diluent.

12. A method of claim 11, characterized in that the human OB protein is resuspended in a diluent having a pH between 6.0 and 8.0.

13. A method of claim 11, characterized in that the human OB protein is rmetHu-Fc-lept ina.