MXPA01003285A - - Google Patents

Description

METHOD FOR DETERMINING A PREDISPOSITION TO LEPTINE TREATMENT Field of the Invention The present invention relates to the field of methods of administration and treatment of a pharmaceutical composition, more specifically, leptin, related analogs and derivatives thereof. More specifically, the present invention relates to methods for determining :: the predisposition of an individual to have a biological response, such as weight loss, to the administration of leptin (or analog or derivative) Background of the Invention Although the molecular basis for obesity is largely known, the identification of the "OB gene" and its encoded protein ("OB protein" or "leptin") has shed some insight into mechanisms the body uses to regulate fat deposition bodily. See WO 96/05309 of the PCT, entitled "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses Thereof ", incorporated herein by reference in its entirety; (128372) Zhang et al., Nature 372: 425-32 (1994); see also; Correction at Nature 374: 479 (1995), both are also incorporated herein by reference. The OB protein is active in vivo in both ob / ob mutant mice (obese mouse due to a defect in the production of the OB gene product) as well as in wild-type and normal mouse mice. The biological activity manifested within itself, among other things, weight loss. See in general, Barinaga, "Obese" Protein Slims Mice ", Science 269: 475-76 (1995) .The OB protein, analogs, derivatives and use thereof as modulators for the control of weight and adiposity of animals, which include mammals and humans, has been described in great detail in WO 96/05309, supra See also, PCT International Publication Numbers WO 96/40912, WO 97/06816, WO 97/18833, WO 97/38014, WO 98/08512 and WO 98/28427 are also incorporated herein by reference, OB protein, or leptin, as it is called here, causes weight loss in humans. "Greenberg et al.," Preliminary safety and efficacy of recombinant methionyl human leptin (rL) administered by SC injection in lean and obese subjects. "Poster presented at: 58th Annual Meeting and Scientific Sessions of the American Diabetes Association, June 14, 1998, Chicago, IL In addition, the weight that is lost is predominantly fat Heymsfield et al., Weight and body composition changes is ean and obese subjects treated with recombinant methionyl human leptin. Poster presented at: International Congress on Obesity; from August 29 to September 3, 1998: Paris, France, incorporated here by reference. Although to date from experience the human clinical test shows that leptin causes weight loss in humans, different humans respond with different levels of sensitivity. The treatment of obese type II diabetics having low levels of endogenous leptin, such as leptin levels between 0 and 80 ng / ml, 0 and 50 ng / ml, 0 and 30 ng / ml and 0 and 15 nm / ml that they are declared in WO 97/02004 of the PCT, published on January 27, 1997 (on page 20) as well as in the US Pat. No. 5,756,461 in column 20, however, it is not stated that such predetermined levels of endogenous leptin would predict the degree of sensitivity of an individual to leptin, particularly for weight loss. It would be desirable to have a prior method of selection for a predisposition of the individual to respond to the administration of leptin, particularly by means of weight loss.

Description of the invention The present invention stems from the observation of a clinical test in humans, which are among individuals with a body mass index (PBMI) of 27.5 or greater, those who have a serum leptin level of baseline approximately below 25% of a population of individuals with BMI similar to, or greater than, for an accepted definition of overweight or obesity (currently a BMI of 27.5%) (who are not genetically deficient for leptin production). more sensitive to the administration of leptin to lose weight The term "approximately" (for "approximately * below 25% of a population) is used here to indicate that there is a fraction of one percent above or below 25% which can be used as a basis of comparison, but less than 1%. Also, as noted below, such a sensitivity of leptin can be prevented by comparing measurements to below 33% of obese individuals as described above, however, higher leptin levels of the comparison group will be likely, and a lower response of sensitivity of the individual to the administration of leptin. Surprisingly and pretentiously, the determination of baseline leptin levels (i.e., leptin levels without leptin treatment) correlates to the predisposition of an individual, particularly an obese individual, to respond to leptin treatment. Previously, it is not known whether the determination of a baseline leptin level results in the prediction of the possibility that an obese individual will respond to leptin. In fact, published reports indicate that because obese individuals may over-express leptin, it is uncertain if obese people respond to leptin treatment. See, "Fat Times for Obesity Research: Tons of New Information, But How Does It All Fit Together?", By Carol Ezzel, The Journal of NIH Research 1_: 39-43 (1995), incorporated herein by reference, eg, in page 43, and see the references cited within this. These observations of clinical results in humans for the first time show that it can be predicted with substantial certainty if an obese individual will respond to the treatment of leptin, and in particular, by means of weight loss (or fat). The "below about 25%" was determined from a population of obese individuals who have a body mass index between 27.5 and 38. Those with known genetic defects (such as individuals known to be ob / ob) were excluded. See, Farooqi IS, Jebb S, Cook G, et al. Treatment of congenital leptin deficiency in man, presented at the 8th International Congress on Obesity; August 29, 1998; Paris, France, incorporated here by reference. More specifically, it was observed that serum leptin levels in untreated males of about 5 ng of endogenous leptin / ml of serum or less, and untreated women with serum leptin levels of about 16 ng / ml or less are substantially more sensitive to the administration of leptin than those with higher levels of serum leptin. Leptin levels were measured after a fast of 8-12 hours, and were measured in the morning (8:00 am -10: 00 am) in individuals with normal sleep cycles (eg, those who are awake during the hours of the day and sleep during the night). Baseline measurements were not taken during the normal sleep cycles, so the peak sleep observation of approximately 3:00 a.m. in serum leptin levels, although one could normalize the present methods to consider such a typical increase in leptin. Accordingly, in one aspect the present invention relates to a method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivatives comprising: (a) the determination of a level of leptin in the individual prior to treatment; and (b) ascertain whether the leptin level is approximately below 25% of the leptin levels of the obese individuals. The invention further relates to the above method, to ascertain whether the level of leptin in the individual is approximately below 33% of the leptin levels of the obese individuals. The invention further relates to methods such as the above, wherein the obese individual has a body mass index greater than 27 The invention further relates to methods such as the above, wherein the obese individual has a body mass index of between 27.5 and 38. The invention further relates to methods such as the above, wherein the level of leptin is determined by means of an antibody test or a nucleic acid hybridization test. The invention further relates to methods such as the above, wherein the level of leptin is a level of leptin in serum. The invention further relates to methods such as the above, wherein the obese individual is a male, and the serum leptin level is equal to or less than 5 ng / ml. The invention further relates to methods such as the above, wherein the obese individual is a woman, and the level of leptin in serum is equal to or less than 16 ng / ml. The invention also relates to a method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivatives comprising: (a) the determination of whether the serum leptin level of the baseline of an obese male is equal to or less than about of 5 ng / ml; or (b) the determination of whether the serum leptin level of an obese female baseline is equal to or less than about 16 ng / ml. In another aspect, the invention relates to a test for determining the predisposition of an individual to respond to treatment by leptin, leptin analog, or leptin derivative comprising: (a) means for detecting the level of endogenous leptin in An individual; (b) means for ascertaining whether said level of endogenous leptin is approximately below 25% of leptin levels for obese individuals. The above invention also refers to tests like the previous ones, which have means to ascertain whether such level of Endogenous leptin is approximately below 33% of leptin levels for obese individuals. In another aspect, the invention relates to an improved esjtuche to determine the predisposition of an obese individual to lose weight in response to treatment using leptin, an analogue of leptin or leptin derivative, the improvement comprises means for determining whether the level of endogenous leptin in the obese individual is approximately below 25%, or even below 33%, of leptin levels of obese individuals. Otherwise, the invention relates to an improved kit for determining the predisposition of an obese individual to lose weight in response to treatment using leptin, an analogue of leptin or leptin derivative, the improvement comprises means for determining whether the level of endogenous leptin in the obese individual is: (a) below about 5 ng / ml in serum, if the obese individual is a male; (b) below about 16 ng / ml in serum, if the obese individual is a woman. The term "around" used here indicates the range of error inherent in the test. Accordingly, for example, if an antibody test is used where the error range is +/- 50%, then the measurements can be 5 ng / ml +/- 50%. If more accurate mechanisms are used, then the range of error is likely to be smaller, and the term "around" applies as such. It is believed that the present measurements (5 ng / ml for males, 16 ng / ml for females, when measured by serum antibody test) represent the upper limit of below 25% of leptin levels in a population of obese individuals like the one described above. In still another form, the present invention relates to methods for determining the predisposition of an individual to respond to treatment with leptin, leptin analog, or leptin derivative comprising: (a) means for detecting the level of endogenous leptin in an individual; (b) means to measure if such a level of endogenous leptin is lower than that which is predicted by the adiposity of that individual; where yes the detected level of endogenous leptin is below the predicted leptin level, the individual may be predisposed to respond to treatment. The present invention relates to the use of algorithms for such determination of the predicted level of leptin of an individual, and the comparison of leptin levels with the level of leptin observed in computer-based methods, and apparatus, in addition to the tests (such as the antibody that was previously recited or tests based on nucleic acids) for such a determination.

Detailed description of the invention As indicated above, the present invention relates to methods for determining the predisposition of an individual with respect to sensitivity to leptin treatment. The present methods provide an effective prediction of who will respond to the administration of leptin to lose weight (and other aspects of the use of leptin) among individuals who have a BMI greater than 27, and more preferable, greater than 27.5. The present clinical trials were conducted among individuals who have a body mass index between 27.5 and 38. As indicated above, the best predictive mechanism for those who would be sensitive, or the degree of sensitivity to leptin, was leptin levels of the baseline. Individuals who have a baseline leptin level in the lowest quartile of the measured population, or (who predicts less effectively) in the lowest tertile lose more weight when administering rmetHu-leptin 1-146 (as established in SEQ ID No. 1), infra The methods used to determine the below 25% or the below 33% of an obese population can be determined empirically, by historical data review or by observation. Presently, the present clinical test information produced the absolute numbers of the following around ("around" means within the error range for the antibody test, here +/- 0.8 ng / ml) the serum leptin levels of the baseline of 5 ng / ml for males, and baseline serum leptin levels of around 16 ng / ml for women. Leptin levels can be measured using a body fluid, most preferably blood or some portion thereof. Here, serum from individuals was used. Other body fluids may also contain leptin, which can be measured, such as whole blood, cerebrospinal fluid, plasma, and possibly urine. The present measurements of 5 ng of leptin / ml of serum (for males) and 16 ng of leptin / ml of serum (for women) can be correlated to corresponding levels in other body fluids, for example, if whole blood is used , the concentration of leptin will be diluted to be counted with respect to the dilution effect of using unfractionated blood The timing to determine leptin levels is important Here, the baseline leptin levels were determined after a fast of 8 -12 hours, during morning hours Baseline leptin levels were not confused by raising levels, such as after meal, or because I observed leptin increase in many individuals through the sleep cycle (eg, at 3:00 a.m. the leptin levels increased.) Such baseline levels can be used, such as observation of nighttime elevation of leptin levels, but those levels should be compared against the levels It is similar to a population of obese humans. A determination to be made is whether the leptin levels measured in an individual are below 25%, or below 33%, or any level up to those upper limits of leptin levels in a population of obese individuals, eg, individuals who have a BMI equal to or greater than 27.5. The population used for comparison purposes should be excluded from those that are deficient in leptin due to profound genetic causes (e.g., human ob / ob, an extremely rare event). Also, because it is the most effective prediction mechanism, it would also be determined if the individual that was tested produces endogenous leptin at all. Occasionally, there may be individuals who produce large amounts of leptin and remain obese, this may be due to defects of the leptin receptor, but it is believed that this is an extremely small proportion of the obese population, who will have a large majority of the receptors available for defective leptin. further, the present methods and compositions are also effective means of prediction when higher levels are used than below 25% of the baseline levels in a population of obese individuals. You can choose to use up to 33% of the measurements of serum leptin level, and continue to have a predictive aspect. As described herein, the present methods are most effectively carried out by the use of an antibody test, any of a number of antibody test formats would typically be used, but all would include some method for determining the amount of antibody bound ( threshold), which would give direct indication of the amount of leptin Typically a test with labeled antibody would be used, see below. The antibody will preferably be able to detect endogenous leptin, and more preferably, amounts in nanograms in the serum, if a serum test is used to determine the baseline leptin levels. Other tests, such as the direct protein test (eg , isolation of the protein leptin from the blood), however, this is not the most feasible. Nucleic acid levels (DNA or RNA) can be correlated to leptin protein levels. Other molecules can also be correlated to leptin levels, such as molecules that are involved in fatty metabolism. A graph can be prepared that correlates the relationships between leptin levels and the levels of other molecules, and therefore use the other molecules to determine an individual's predisposition toward leptin sensitivity. In particular, tests that rely on nucleic acids may be used, such as what is sometimes referred to as "gene-chip" technology (oligonucleotide microarrays, popularly known as gene chips). Such technology is currently known in the art and involves the use of microprocessors (i.e., "chips" (integrated circuits) of the computer) to carry out nucleic acid hybridization techniques. The presence or amount of hybridization is typically determined by automated means, such as an appropriate scanner. In this manner, an additional step can be used to correlate serum leptin levels with nucleic acid transcription, and use nucleic acid hybridization when located on a computer chip (integrated circuit) to ascertain leptin levels of the baseline To really treat the individual, the following leptin proteins, formulations, pharmaceutical compositions and their uses apply.

Leptin proteins. In general, human leptin 1-146, an analogue of leptin (ie, an analog of human leptin 1-146) or a leptin derivative (i.e., human leptin 1-146 or an analogue thereof) have an additional chemical radical that is linked to it, such as dextran, as described below) can be used together with the present methods. Thus, the present methods are referred to determine the predisposition of an individual to respond to treatment with leptin, and to the use of the above "leptins" ie, leptin, an analogue of leptin, or a leptin derivative, as established previously, for such treatment.

The type of leptin used for treatment of the individual can be selected from those described in International Publication Number of WO 96/05309 of PCT, as cited above and which is incorporated herein by reference in its entirety. Figure 3 of that publication (as cited here SEQ ID No. 4) describes the complete deduced sequence of amino acid derivative for human leptin (referred to as the human protein "OB"). The amino acids are numbered from 1 to 167. A cut-off site of the signal sequence is located after amino acid 21 (Ala) so that the mature protein extends from amino acid 22 (Val) to amino acid 167 (Cys). For the present description, a different numbering is used here, where the position of amino acid 1 is the valine residue, which is at the beginning of the mature protein. The amino acid sequence for mature human methionyl recombinant leptin is presented herein as SEQ ID No. 1, where the first amino acid of the mature protein is valine (at position 1) and the methionyl residue is located at position -1 (called here rHu-leptin 1-146, SEQ ID No. 1): V P I Q K V Q D D T K T L I K T I V T R I N D I S H T Q S V S S K Q K V T G L D F I P G L H P I L T L S K M D Q T L A V Y Q Q I L T S M P S R N V I Q I S N Generally, the leptin radical for human pharmaceutical use herein will be capable of therapeutic use in humans (see also, below, animal leptins). In this way, the activity can be tested empirically to determine which radicals of the leptins can be used. As set out in WO 96/05309, the protein leptin in its native form, or fragments (such as enzymatic cleavage products) or other truncated and analogous forms that can retain all biological activity. See also, Los PCT International Publication Numbers WO 97/06816, WO 97/18833, WO 97/38014, WO 98/08512 and WO 98/28427, incorporated herein by reference in their entirety. An analogue of human recombinant leptin can be prepared by altering the amino acid residues in the human recombinant sequence, as substituted: the amino acids that diverge from the murine sequence. The murine leptin is substantially homologous to human leptin, particularly as a mature protein and, additionally, in particular form in the term N. Because the human recombinant protein has biological activity in mouse, such as an analogue would be active in humans. For example, in the native amino acid sequence of human leptin as presented in SEQ ID No. 1, one or more of the amino acids at positions 32, 35, 50, 64, 68 can be substituted with another amino acid, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. The amino acid can be selected at the corresponding position of the murine protein (see Zhang et al., 1994, supra) or another amino acid. Additionally, molecules based on the sequence of the rat OB protein can be prepared by "consensus". Murakami et al., Biochem. Biophys. Res Comm. 209: 944-52 1995) incorporated herein by reference. The rat OB protein differs from the human OB protein in the following positions (using the numbering of SEQ ID No. 1):, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138 and 145. Can be replaced with another amino acid, one or more of the amino acids in these divergent positions. The underlined positions are those in which the murine OB protein as well as the rat OB protein are divergent from the human OB protein and, thus, are particularly suitable for alteration. In one or more of the positions, an amino acid of the corresponding rat OB protein, or other amino acid, may be substituted. The positions of both the rat OB protein and the murine OB protein that diverge from the mature human OB protein are: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. An OB protein according to SEQ ID No. 1 having one may also be effective. or more of the above amino acids replaced with another amino acid, such as the amino acid found in the corresponding murine or rat sequence. In addition, the amino acids found in the rhesus monkey OB protein that differ from the mature human OB protein --- • * '"" are (together with the abbreviated identifications of the amino acid named in the parentheses): 8 (S), 35 (R), 48 (V), 53 (Q), 60 (I), 66 (I), 67 (N), 68 (L), 89 (L), 100 (L), 108 (E), 112 (D) and 118 (L). Since human recombinant OB protein is active in cynomolgus monkeys, a human OB protein according to SEQ ID No. 1 having one or more of the rhesus monkey divergent amino acids replaced with another amino acid, such as the amino acids that are inside the parentheses. It should be noted that certain divergent amino acids of rhesus are also those that are found in the previous murine and rat species (positions 35, 68, 89, 100, 108 and 118). In this way, a molecule can be prepared by urine / rat / rhesus / human consensus (using the numbering of SEQ ID No. 1) having one or more of the amino acids replaced by another amino acid at positions: 4, 8, 32, 33, 3_5, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108 'l l? > 112 'l' 136 '138' 142 and 145- The underlined positions are those in which all three species are divergent from the human OB protein. A particularly preferred human leptin analog is one wherein the amino acids are in the 100 (Trp) or 138 (Trp) position, and more preferably, both positions are substituted with other amino acids, preferably Gln. Other analogs can be prepared by removing a part of the protein from the amino acid sequence, for example, the mature protein lacks a leader sequence (-22 to -1). can prepare the following truncated forms of human OB protein molecules (using the numbering of SEQ ID No. 1 ' (i) amino acids 98-146; (ii) amino acids 1-99 and (connected to) 112-146; (iii) amino acids 1-99 and (connected to) 112-146 having one or more of amino acids 100-111 placed sequentially between amino acids 99 and 112. In addition, truncated forms can also be altered by one or more of the amino acids that are divergent (in the OB protein of resus, rat or murine) from the human OB protein. In addition, any alterations may be in the form of altered amino acids, such as peptidomimetics or amino acids D. Also included are those proteins that were previously established with amino acid substitutions that are "conservative" in accordance with acidity, charge, hydrophobicity, polarity , size or any other feature known to those skilled in the art. These are set forth later in Table 1. See generally, Creighton, Proteins, passim (W.H. Freeman and Company, N.Y., 1984); Ford et al., Protein Expression and Purification 2: 95-107 (1991), which are incorporated herein by reference.

Table 1 Substitutions of Conservative Amino Acids Basic arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic; leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine Accordingly, the leptins used in conjunction with the methods of the present invention can be selected from (in accordance with the amino acid sequence as presented herein in SEQ ID No. 1): [a) the amino acid sequence of SEQ ID NO: 1 No. 1, that 33, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142 and 145 replaced with another amino acid; (vi) the truncated leptin analog of subpart (iv) having one or more of the amino acids 4,:, 32, 33, 35, 48, 50, 53, 60, 64, 66, 66, 67, 68 , 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and 145 replaced with another amino acid; and (vii) the truncated leptin analog of any of subparts (i) - (vl) having an N-terminal methionyl residue; (e) a leptin protein of any of subparts (a) - (d) having one or more of the conservative amino acid substitutions. As stated in SEC ID No. 1, rmetHu-leptin 1-146 is preferred, as it has not been shown to have substantial human toxicity. Other leptins that can be used include rmetHu-leptin 1-146 or rHu-leptin 1-146 (lacking an N-terminal methionyl residue) having a substitution of another amino acid at position 100 (according to SEQ ID NO. 1) or in position 99 (according to SEQ ID No. 2, if leptin 1-145 is used as in SEQ ID No. 2). Such substitution may be selected from alanine, glutamic acid, and glutamine. Also, rmetHulectin 1-146 or rHu-Leptin 1-146 (lacking an N-terminal methionyl residue) having a substitution in one or both of the 100 positions (or 99, if the Q sequence is used) and the position 138 (137 if the sequence Q is used). The substitution can be of any amino acid with the exception of the already present W (tryptophan) and can be selected from alanine, glutamic acid and glutamine. Other leptin derivatives or analog derivatives can be used. For example, co-pending 60/096194, incorporated herein by reference, discloses several modified leptins with dextran. WO 98/28427 PCT (incorporated by reference) discloses several OB fusion protein compositions. A variety of leptin or analogous leptin derivatives may be used in conjunction with the present methods, as described in various publications as cited passim, which are incorporated by reference. Leptin proteins, analogs and related molecules are also reported in the following publications; however, no representation is made regarding the activity of any reported composition: U.S. Patent Numbers 5,521,283; 5.525, '' 05; 5,532,336; 5,552,522; 5,552,523; 5,552,524; 5,554, 727,559,208; 5,563,243; 5,563,244; 5,563,245; 5,567, 678 5,567,803; 5,569,743; 5,569,744; 5,574,133; 5.580, 954.5.594.101; 5,594,104; 5,605,886; 5,614,379; 5,691,309; ,719,266 (Eli Lilly and Company); PCT WO 96/23513; WO 96/23514; WO 96/23515; WO 96/23516; WO 96/23517; WO 96/23518; WO 96/23519; WO 96/23520; WO 96/23815; WO 96/27385; WO 96/34111; WO 96/37517; WO 97/00886; EP 725078; EP 725079; EP 744408; EP 745610; EP 835879 (Eli Lilly and Company); PCT WO 96/22308 (Zymogenetics); PCT WO 96/31526 (Amylin Pharmaceuticals, Inc.); PCT WO 96/34885; WO 97/46585 (Smithkiine Beecham PLC); PCT WO 96/35787 (Chiron Corporation); PCT WO 97/16550 (Bristol-Myers Squibb); PCT WO 97/20933 (Schering Corporation) EP 736599 (Takeda); EP 741187 (F. Hoffman LaRoche).

With the above conditions, to the extent these references provide leptin proteins or useful analogs, or compositions or associated methods, such compositions and / or methods may be used in conjunction with the present methods. These publications are incorporated herein by reference.

Animal Leptins In addition to the previous human therapeutic leptin, certain animal leptins are also suitable for therapeutic use. Canine leptin is described in WO 97 / B2022, which is incorporated herein by reference. Other animal species are described in the following publications: WO 96/36644, EP 743321 (porcine and bovine); WO 98/04288 (bovine); WO 98/04690 (porcine), all are incorporated herein by reference. These are not preferred for human use for reasons of prevention of possible immunogenic response.

Pharmaceutical compositions The present test method can also be used to determine the predisposition of an individual to respond to pharmaceutical compositions for therapeutic uses. Such pharmaceutical compositions may be for administration by bolus injection or infusion (e.g., intravenous or subcutaneous), or orally, pulmonary, nasal, transdermal or other forms of administration. In general, comprised by the invention are pharmaceutical compositions comprising effective amounts of leptin protein compositions, analogs or derivatives together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or vehicles. Such pharmaceutical compositions include diluents of various buffer contents (e.g., tris-HCl, acetate, phosphate), Ph and ionic strength; additives such as detergents and solubility agents ^^ gg ^^ L ^^^^^^^^^^^^^^^^^^^^ (eg, Tween 80, Polysorbate 80), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg , Thimersol, benzyl alcohol) and thickeners (eg lactose, mannitol); the incorporation of the material into 5 particular preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, etc. or in liposomes See, e.g. , WO 96/29989 PCT, incorporated herein by reference. Hyaluronic acid can also be used, and this can have the effect of increasing the sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of release in yjLvo, and evacuation rate of leptins in vivo, analogs or derivatives. See, e.g. , Remington's Pharmaceutical Sciences, l. Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, which are incorporated herein by reference. The compositions may be prepared in liquid form, or may be in dry powder, such as in lyophilized form. Also included are sustained release formulations that are capable of being implemented, such as formulations transdermal. Formulations that are contemplated orally, as described in PCT WO 95/21629, incorporated herein by reference in its entirety. This PCT publication describes the oral delivery of chemically modified proteins, which include proteins modified by dextran radicals. The compositions and methods described herein are applicable to administer compositions of the leptin, analog or derivative protein. Lung delivery is also contemplated, compositions and methods described in WO are also useful herein. 94/20069 of the PCT. WO 94/20069, which describes the pulmonary release of chemically modified G-CSF, is incorporated herein by reference. The particle size for pulmonary administration should advantageously be prepared in particular form with an average particle size of less than 10 microns, more preferably 0.5 to 5 microns. For a more effective release towards the distal lung (from the center). Nasal release is also contemplated. Liberjación allows the entry of the protein into the stream sanefuínea directly after the administration of the therapeutic product to the nose. Without the need for the deposition of the product in the lung. Formulations for nasal release include those with dextran or dextrin, such as cyclodextrin. Release is also contemplated via transport that is through other mucous membranes.

Dosages An expert in the art will be able to determine the effective dosages for the administration and observation of the desired therapeutic effect. Next, the present human clinical results demonstrated efficacy of rmetHu-Leptin (1-146, SEQ ID No. 1) most effectively at a dosage of 0.3 mg / kg body weight / day. See Greenberg et al., Supra.

However, the present subjects who demonstrated that serum leptin base levels correspond to the predisposition to lose weight were administered at either 10 mg / day or 10 mg / twice daily (for 20 mg total protein) of the rmetHu-Leptin 1-146 (SEQ ID No. 1). It is contemplated that other forms of leptin (such as analogs or derivatives) may be advantageous in dosing regimens, or by low administration of protein per unit of body mass. Apart from determining a level of leptin from the baseline to establish if it is predisposed to leptin treatment, it is contemplated that effective dosages can be determined using diagnostic tools outside of the stipulated time. For example, a diagnosis to measure the amount of leptin in the blood (or plasma or serum) can first be used to determine the endogenous levels of leptin protein. Such a diagnostic tool may be - -. t8t .-. * in the form of an antibody test, such as a type "antibody test" used, the amount of endogenous leptin protein is quantified initially, and a baseline is determined.Therapeutic dosages are determined as the quantification The radical of the exogenous and endogenous leptin protein (ie, protein, analog or derivative found within the body, whether produced by itself or administered) is prolonged during the course of therapy. they may vary during the course of the therapy, with, for example, a relatively high dosage that is used initially, until benefit and therapy is observed, and lower dosages are used to maintain the therapeutic benefits.

Methods of Use The present invention can be used to determine a predisposition of the individual to respond to leptin treatment (or treatment using a leptin-related composition, such as an analog or derivative) in the following manner. It is contemplated that the present invention will be used as a screening tool to determine the possibility that an obese individual (ie, an individual with a Body Mass Index greater than 27, preferably greater than 27.5, more preferably in the range of 27.5 to 36 ), that does not have conmorbidos of obesity, such as diabetes, dyslipidemias, such as hyperlepidemia, and hypertension, will respond to the administration of leptin with weight loss. It is further contemplated that such an individual will be selected for predisposition to respond predominantly for fat loss, but may also be selected for predisposition toward leptin response for a variety of uses of leptin. The uses of leptin include: Therapeutic. Therapeutic uses include weight modulation, the treatment or prevention of diabetes, reduction of lipid in the blood (and treatment of related conditions), which increases the body mass without fat and increases the sensitivity to insulin. In addition, the present compositions can be used to make one or more medicaments for the treatment or improvement of the above conditions. Weight Modulation The present compositions and methods can be used for weight reduction. Viewed from another point of view, the present compositions can be used to maintain a desired weight or level of adiposity. The lost body mass is mainly adipose tissue, or fat. Such weight loss may be associated with the treatment of accompanying conditions, such as those mentioned below, therefore constitutes a therapeutic application. In addition, once sufficient weight loss is acquired, sufficient dosage is given to prevent further weight gain, maintaining the desired levels of blood lipid, or other conditions as stated herein. These dosages can be determined empirically. , when the effects of leptin protein are reversible. Eg, Campfield et al., Science 269: 546-549 (1995) in 547. Thus, if a dosage resulting in weight loss is observed when weight loss is not desired, a lower dosage would be administered to acquire : the desired levels of lipid in the blood, maintaining the desired weight. See, e.g. , WO 97/06816 PCT publication incorporated herein by reference. Increase in body mass without fat or insulin sensitivity. Ideally, in situations where only an increase in non-fat body mass is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial therapy course of an obese person, dosages can be administered as long as weight loss is gained and the accompanying fatty tissue / fat-free mass increase is decreased. Once sufficient weight loss is acquired, a sufficient dosage can be administered to prevent further weight gain, maintaining the desired increase in fat-free mass (or prevention of fat-free mass reduction). For - yy -y- &.

To increase an individual's sensitivity to insulin, similar dosing considerations can be taken into account. Sufficient fat-free mass can be acquired, without weight loss to decrease the amount of insulin (or, potentially, amylin, amylin agonists or antagonists, or thiazolidinediones, or other drugs to treat potential diabetes) would be administered to an individual for diabetes treatment. To increase the total force, there may be similar dosage considerations. An increase in body mass without fat can be acquired with an accompanying increase in total strength with insufficient dosages to result in weight loss. Other benefits, such as an increase in red blood cells (and oxygenation in the blood) can also be a decrease in bone resorption or osteoporosis in the absence of weight loss. See, e.g., PCT Publication No. WO 97/18833 incorporated herein by reference.

Combination Therapies The present invention can also be used to determine the predisposition of an individual to respond to leptin, analogs and derivatives, as may be used in conjunction with other therapies, such as altered diet and exercise. Other medicaments, such as those that are useful for the treatment of diabetes (eg, insulin and possibly amylin, antagonists or agonists thereof, thiazolidinediones (see, eg, PCT Publication No. WO 98/08512 incorporated herein by reference) , or other drugs to treat potential diabetes, cholesterol and medications for lowering blood pressure (such as those that reduce blood lipid levels or other cardiovascular medications), medications that increase activity (eg, amphetamines), diuretics (for fluid removal), and appetite suppressants (such as agents that act on neuropeptide receptors and / or serotonin reuptake inhibitors) such administration must be simultaneous or may be in serijatim.

In addition, the present methods can be used in conjunction with surgical intervention procedures, such as cosmetic operations designed to alter the overall appearance of a body (eg, liposuction or laser surgeries designed to reduce body mass, or implants designed to increase the appearance of the body). body mass) . The health benefits of cardiac operations, such as bypass operations (bypass conduit) or other operations designed to alleviate a harmful condition caused by blockage of blood vessels by means of fatty deposits, such as an arterial plaque , may be increased with the accompanying use of the present compositions and methods. Methods to eliminate cholelithiasis (presence of one or more stones in the bile ducts), «^ ¡Tj ^ i ^ 2 = ?? ^^ j ^^^^^^^ > "J ^^^^ * such as laser or ultrasonic methods, may also be used either before, during or after a course of the present therapeutic methods. In addition, the present methods can be used as an adjunct to operations or therapies for broken bones, damaged muscle, or other therapies that would be improved by means of an increase in non-fat tissue mass.

Other uses Other uses of leptin, analog or derivative are contemplated along with the present methods, and these include: administration of leptin (or analog or derivative), according to an endogenous fluctuation of leptin secretion in an individual to advantageously result in effectiveness of leptin; treatment of conditions associated with fertility, treatment of conditions associated with the release of human growth hormones, treatment of associated conditions to heal wounds, hematopoietic conditions, angiogenic conditions for the development of blood vessels in fat deposits, treatment of conditions of chronic stress, treatment of conditions associated with lack of fertility or delayed onset puberty, and other conditions that will be apparent to the skilled artisan for the therapeutic and cosmetic uses of leptin.

Production Methods of Leptin The leptin radicals that are used here can be found in prokaryotic cells or in eukaryotic cells, although, for the leptin radicals that are used later in the operating examples, bacteria are preferred because of the ease of production in the commercial field. . Leptin made in human cells, such as that made by controlling a native or introduced regulatory element that affects the regulation of an endogenous gene encoding the desired protein, can additionally be used. For example, the recombinant expression of portions of leptin has been described in WO 96/40912, incorporated herein by reference, which includes all the vector and reservoirs of the host strain referred to herein.

EXAMPLE The present methods were used to determine leptin levels in humans prior to being subjected to leptin treatment for obesity in a human clinical trial. The study design and protocol of the test; Clinical summary in Greenberg et al., "Preliminary safety and efficacy of recombinant methionyl human leptin (rL) administered by SC injection in lean and obese subjects poster presented at: 58th Annual Meeting and Scientific Sessions of the American Diabetes Association; June 1998; Chicago, IL, incorporated herein by reference, supra An immunosorbent assay linked to a standard enzyme (commonly referred to by the acronym "ELISA") was used to determine leptin levels in the serum of individuals enrolled in The method used a purified rat anti-rmetHu-leptin monoclonal antibody to capture leptin from the whole serum.For affinity the purified rabbit anti-rmetHu-leptin polyclonal antibody conjugated with horseradish peroxidase was used to detect captured leptin, the detection limit of the assay was 0.8 ng / ml, although certain antibodies were used here, antibodies that react specifically with native human leptin, and are sensitive to detect leptin amounts equal to or less than 5 ng / ml in serum (for male patients) and equal to or less than 16 ng / ml in serum (for female patients ) Individuals who have a BMI between about 27.5 and 38 (see Greenberg et al., Infra), will be selected for the absence of co-morbidities of obesity. Subjects were put on a diet and advised to exercise. To determine baseline serum leptin levels, blood was drawn for use after a 8-12 hour fast, between 8:00 a.m. and 10:00 a.m. The serum was obtained, and the above materials were used to carry out an antibody test, which uses standard techniques. The leptin dosages administered were 10 mg / day or 10 mg / 2x / day. (at this level there did not seem to be a response related to the dosage among those who responded). The weight loss was measured after 3 months (12 weeks), and the results of the 3 (third) month are presented later.

Results The results show that there is a large amount of weight loss in individuals who have a baseline leptin level below 33%, in addition to that, below 25% of serum leptin levels measured for obese individuals . As can be seen in Table 2, in a total population of male and female patients who are obese, individuals in the lower quartile (below 25%) of serum leptin levels demonstrate a high response rate than those who they are above 75% leptin in serum leptin levels. Those who are below 25% lost an average of 4.8% of body weight and an average of 5.4 kg after three ir.eses. Those that have a serum leptin level of the baseline above 75%, although they demonstrate weight loss, the loss above the average is substantially lower, 2.2% and 2.0 kg, after the same period of time. Not all in below 25% of serum leptin levels responded as such. Among those who are below 25% of leptin levels in suer 19 had a weight change that was between the average for those above 75% (ie, a percentage less than or equal to -2.2% change in the body weight) . Of those '.9, 6 lost weight, 2 had no change in body weight, and 11 gained weight. For those 19 in the quartile I inferred Fr of serum leptin levels that responded at a higher rate, there was no difference in the response that was dose dependent (10 mg / day or 10 mg / 2x / day). For individuals who are below 33% of serum leptin level 1, 20 individuals did not have a weight loss lower than 1.8%. Of those 20, 12 gained weight, 2 remained unchanged and 6 lost weight, but lost less than 1.8% of total body weight (1.8% that is the approximate average for those who have serum leptin level 1 above 66.6 %). appended claims cover all equivalent variations, which achieve the scope of the invention as claimed. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (12)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for determining the predisposition of an obese individual to respond to treatment by leotine, analogous to leptin or leptin derivative, characterized in that it comprises (a) the determination of a leptin level in the individual prior to treatment; and (b) ascertain whether the level of leptin is approximately below 25% of the leptin levels of the obese individuals.
2. 2. A method of claim 1, characterized in that the obese individual has a body mass index greater than 27.
3. 3. A method of claim 1, characterized in that the obese individual has a body mass index of between 27.5 and 38.
4. 4. A method of claim 1, characterized in that the level of leptin is determined by means of an antibody test.
5. 5. A method of claim 1, characterized in that the level of leptin is a level of serum leptin.
6. 6. A method of claim 4, characterized in that the obese individual is a male, and the serum leptin level is equal to or less than about 5 ng / ml.
7. 7. A method of claim 4, characterized in that the obese individual is a woman, and the level of leptin is equal to or less than about 16 ng / ml. .
8. A method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative, characterized in that it comprises: (a) the determination of a leptin level in the individual prior to treatment; and (b) ascertain whether the level of leptin is approximately below 33% of the leptin levels of the obese individuals.
9. 9. A method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative, characterized in that it comprises: (a) the determination of whether the serum leptin level of the line The basis of an obese male is equal to or less than about 5 ng / ml; or (b) the determination of whether the serum leptin level of an obese woman's baseline is equal to or less than about 16 ng / ml,
10. 10. A test to determine the predisposition for an obese individual to respond to treatment by leptin, leptin analogue, or leptin derivative, characterized in that it comprises: (a) means to detect the level of endogenous leptin in an individual; (b) means to ascertain whether such an endogenous leptin level is approximately below 25% of leptin levels for obese individuals
11. 11. A test to determine the predisposition of an obese individual to respond to treatment by leptin, analogous to leptin, or leptin derivative, characterized in that it comprises: (a) means for detecting the level of endogenous leptin in an individual; (b) means for ascertaining whether such a level of endogenous leptin is approximately below 33% of leptin levels for obese individuals,
12. 12. An improved case, characterized in that it determines the predisposition of an obese individual to lose weight in response to In a treatment using leptin, an analogue of leptin or leptin derivative, the improvement comprises means for determining whether the level of endogenous leptin in the obese individual is approximately below 25% of the leptin levels of the obese individuals.