AU6396999A - Method to determine a predisposition to leptin treatment - Google Patents

Description

WO 00/20872 PCTIUS99/21903 METHOD TO DETERMINE A PREDISPOSITION TO LEPTIN TREATMENT Field The present invention relates to the field of 5 methods of administration and treatment of a pharmaceutical composition, more specifically, leptin and related analogs and derivatives thereof. More specifically, the present invention relates to methods for determining the predisposition of an individual to 10 have a biological response, such as weight loss, to administration of leptin (or analog or derivative). Background Although the molecular basis for obesity is largely unknown, the identification of the "OB gene" 15 and its encoded protein ("OB protein" or "leptin") has shed some light on mechanisms the body uses to regulate body fat deposition. See PCT WO 96/05309, entitled "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses 20 Thereof," herein incorporated by reference in its entirety; Zhang et al., Nature 372:425-32 (1994); see also, the Correction at Nature 374:479 (1995), both of which are also herein incorporated by reference. The OB protein is active in vivo in both ob/ob mutant mice 25 (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. See generally, Barinaga, "Obese" Protein Slims Mice," Science 269:475-76 (1995). 30 The OB protein, analogs, derivatives and use thereof as modulators for the control of weight and adiposity of animals, including mammals and humans, has been disclosed in greater detail in WO 96/05309, supra. See also, PCT International Publication Numbers WO 00/20872 PCT/US99/21903 -2 WO 96/40912, WO 97/06816, WO 97/18833, WO 97/38014, WO 98/08512 and WO 98/28427 also herein incorporated by reference. The OB protein, or leptin, as it is called herein, causes weight loss in humans. Greenberg et 5 al., "Preliminary safety and efficacy of recombinant methionyl human leptin (rL) administered by SC injection in lean and obese subjects." Poster presented at: 5 8 th Annual Meeting and Scientific Sessions of the American Diabetes Association; June 14, 10 1998; Chicago, IL. Further, the weight that is lost is predominantly fat. Heymsfield et al., Weight and body composition changes in lean and obese subjects treated with recombinant methionyl human leptin. Poster presented at: International congress on Obesity; 15 August 29-September 3, 1998: Paris, France, herein incorporated by reference. Although human clinical trial experience to date demonstrates that leptin causes weight loss in humans, different humans respond with different levels of 20 sensitivity. Treatment of obese type II diabetics having low endogenous leptin levels, such as leptin levels between 0 and 80 ng/ml, 0 and 50 ng/ml, 0 and 30 ng/ml and 0 and 15 ng/ml is stated in PCT publication WO 97/02004, published January 27, 1997 25 (at page 20) as well as U.S. Patent No. 5,756,461 at column 20, however, it is not stated that such predetermined endogenous leptin levels would forecast the degree of responsiveness of an individual to leptin, particularly for weight loss. It would be 30 desirable to have a method to prescreen for an individual's predisposition to respond to leptin administration, particularly by weight loss.

WO 00/20872 PCT/US99/21903 -3 Summary of the Invention The present invention stems from the human clinical trial observation that among individuals with a Body Mass Index ("BMI") of 27.5 or greater, those 5 having a baseline serum leptin level in approximately the bottom 25% of a population of individuals with BMIs equal to or greater than an accepted definition of overweight or obese (currently BMI 27.5%) (who are not genetically deficient for leptin production) were most 10 responsive to leptin administration for weight loss. The term "approximately" (for "approximately the bottom 25% of a population) is used herein to indicate that there a fraction of a percent above or below 25% which may be used as a basis of comparison, but less than 1%. 15 Also, as indicated below, one may forecast such leptin responsiveness by comparing measurements up to the lowest 33% of obese individuals as described above, however, the higher the leptin levels of the comparison group, the lower a subject individual's responsiveness 20 to leptin administration will likely be. Surprisingly, and importantly, determining the baseline leptin levels (i.e., the leptin levels without leptin treatment) correlates to the predisposition of an individual, particularly an obese individual, to 25 respond to leptin treatment. Previously, it was not known if determining a baseline leptin level would result in prediction of the likelihood of an obese individual to respond to leptin. In fact, published reports indicated that because obese individuals may 30 over express leptin, it was uncertain if obese people would 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 WO 00/20872 PCT/US99/21903 -4 of NIH Research 7: 39-43 (1995), herein incorporated by reference, e.g., at page 43, and see the references cited therein. These observations of human clinical data for the first time demonstrate that one may 5 predict with substantial certainty whether an obese individual will respond to leptin treatment, and in particular, by weight (or fat) loss. The "approximate bottom 25%" was determined from a population of obese individuals having a body mass 10 index of 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; 15 August 29 September 3, 1998; Paris, France, incorporated herein by reference. More specifically, it was seen that untreated male serum leptin levels of about 5 ng endogenous leptin/ml serum or less and untreated females with serum leptin 20 levels of about 16 ng/ml or less are substantially more responsive to leptin administration than those with higher serum leptin levels. The leptin levels were measured after an 8-12 hour fast, and were measured in the morning (8:00 a.m.-10:00 a.m.) in individuals with 25 normal sleep-wake cycles (e.g., those who are awake during daytime hours and sleep during the nighttime). Baseline measurements were not taken during the normal sleep cycles, and thus the observation of the approximately 3:00 a.m. "spike" in serum leptin levels 30 was not used, although one could normalize the present methods to account for such typical leptin increase. Thus, in one aspect the present invention relates to a method for determining the predisposition of an WO 00/20872 PCTIUS99/21903 -5 obese individual to respond to treatment by leptin, leptin analog, or leptin derivative comprised of: (a) determining a leptin level in said individual prior to said treatment; and 5 (b) ascertaining if said leptin level is in approximately the bottom 25% of leptin levels of obese individuals. The invention further relates to the above method, but ascertaining if the leptin level in the individual 10 is in approximately the bottom 33% of leptin levels of obese individuals. The invention further relates to methods as above wherein said obese individual has a body mass index of greater than 27. 15 The invention further relates to methods as above wherein said obese individual has a body mass index of between 27.5 and 38. The invention relates furthermore to methods as above wherein said leptin level is determined by an 20 antibody assay or a nucleic acid hybridization assay. The invention relates furthermore to methods as above wherein said leptin level is a serum leptin level. The invention relates furthermore to methods as 25 above wherein said obese individual is a male and said serum leptin level is equal to or less than 5 ng/ml. The invention relates further to a method as above wherein said obese individual is a female and said serum leptin level is equal to or less than 16 ng/ml. 30 The invention relates also to a method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative comprised of: WO 00/20872 PCT/US99/21903 -6 (a) determining if the baseline serum leptin level of an obese male is equal to or less than about 5 ng/ml; or (b) determining if the baseline serum leptin 5 level of an obese female is equal to or less than about 16 ng/ml. In another aspect, the invention relates to an assay for determining the predisposition of an individual to respond to leptin, leptin analog, or 10 leptin derivative treatment comprising: (a) means to detect the endogenous leptin level in an individual; (b) means to ascertain if such endogenous leptin level is in approximately the bottom 25% of 15 leptin levels for obese individuals. The above invention also relates to assays as above having means to ascertain if such endogenous leptin level is in approximately the bottom 33% of leptin levels for obese individuals. 20 In another aspect, the invention relates to an improved kit for determining the predisposition of an obese individual to lose weight in response to treatment using leptin, a leptin analog or leptin derivative, said improvement comprising means for 25 determining whether the endogenous leptin level in said obese individual is in approximately the bottom 25%, or up to the bottom 33%, of leptin levels of obese individuals. In another way, the invention relates to improved 30 kit for determining the predisposition of an obese individual to lose weight in response to treatment using leptin, a leptin analog or leptin derivative, said improvement comprising means for determining WO 00/20872 PCT/US99/21903 -7 whether the endogenous leptin level in said obese individual is: (a) below about 5 ng/ml serum if said obese individual is a male; 5 (b) below about 16 ng/ml serum if said obese individual is a female. The term "about", used above, indicates the range of error inherent in the assay. Thus, for example, if an antibody assay is used wherein the range of error is 10 +/- 50%, then the measurements may be 5 ng/ml +/- 50%. If more accurate means are used, then the range of error is likely to be lower, and the term "about" applies as such. It is believed that the present measurements (5 ng/ml for males, 16 ng/ml for females, 15 as measured by antibody assay on serum) represents the upper limit of the bottom 25% of leptin levels in a population of obese individuals as described above. And in yet another way, the present invention relates to methods for determining the predisposition 20 of an individual to respond to leptin, leptin analog, or leptin derivative treatment comprising: (a) means to detect the endogenous leptin level in an individual; (b) means to measure if such endogenous 25 leptin level is less than that would be predicted for the adiposity of that individual; wherein if said detected endogenous leptin level is below said predicted leptin level, said individual may be predisposed to respond to said treatment. 30 The present invention relates to the use of algorithms for such determination of the predicted leptin level of an individual, and the comparison of leptin levels with the observed leptin level in WO 00/20872 PCT/US99/21903 -8 computer-based methods, and apparatus, in addition to assays (such as the above recited antibody or nucleic acid based assays) for such determination. Detailed Description of the Invention 5 As indicated above, present invention relates to methods for determining the predisposition of an individual for responsiveness to leptin administration. The present methods provide an effective prediction of who will respond to leptin administration 10 for weight loss (and other aspects of leptin use) among individuals having a BMI of greater than 27, and more preferred, greater than 27.5. The present clinical trials were conducted among individuals having a body mass index of between 27.5 and 38. As indicated above, 15 the best predictor as to who would be responsive, or the degree of responsiveness to leptin, was baseline leptin levels. Individuals having a baseline leptin level in the lowest quartile of the measured population, or (less effectively predictive) in the 20 lowest tertile lost the most weight when administered rmetHu-leptin 1-146 (as set forth in SEQ ID NO. 1, infra. Methods used to determine the lowest 25% or the lowest 33% of an obese population may be determined 25 empirically, by historical review of data or by observation. Currently, the present clinical trial information yielded the absolute numbers of below about ("about" meaning within the range of error for the antibody assay, here +/- 0.8ng/ml) 5 ng/ml baseline 30 serum leptin levels for males, and about 16 ng/ml baseline serum leptin levels for females. Leptin levels may be measured using a body fluid, most preferably blood or some portion thereof. Here, WO 00/20872 PCT/US99/21903 -9 serum from individuals was used. Other body fluids may also contain leptin which may be measured, such as whole blood, cerebral spinal fluid, plasma, and possibly urine. The present measurements of 5 ng 5 leptin/ml of serum (for males) and 16 ng leptin/ml serum (for females) may be correlated to corresponding levels in other body fluids. For example, if whole blood is used, the leptin concentration will be diluted to account for the diluting effect of using 10 unfractionated blood. The timing for determining the leptin levels is important. Here, the baseline leptin levels were determined after an 8-12 hour fast, during morning hours. Baseline leptin levels were not confounded by 15 raising levels, such as after a meal, or due to sleep cycle rise in leptin seen in most individuals (e.g., 3:00 a.m. rise in leptin levels). Such baseline levels may be used, such as observation of nocturnal elevation of leptin levels, but those levels should be compared 20 against similar levels in a population of obese humans. One determination to be made is if the leptin levels measured in an individual is in the bottom 25%, or bottom 33%, or any level up to those upper limits, of leptin levels in a population of obese individuals, 25 e.g., individuals having a BMI of equal to or greater than 27.5. The population used for comparison purposes should exclude those who are leptin deficient due to profound genetic causes (e.g., ob/ob humans, an exceedingly rare 30 event). Also, to be the most effective predictor, one would also determine if the individual being tested produces endogenous leptin at all. From time to time, there may be individuals who produce large amounts of WO 00/20872 PCT/US99/21903 - 10 leptin yet remain obese, and this may be due to leptin receptor defects, but it is thought that this is a vanishingly small proportion of the obese population who will have a large majority of the available leptin 5 receptors defective. Further, the present methods and compositions are also effective predictors when greater than the lowest 25% of baseline levels in a population of obese individuals is used. One may choose to use up to the 10 lowest 33% of serum leptin level measurements, and continue to have a predictive aspect. The present methods are most effectively carried out by use of an antibody assay, as described herein. One would typically use any of a number of antibody 15 assay formats, but all would include some method of determining the amount of antibody bound, which would give direct indication of the amount of leptin. Typically a labeled antibody assay would be used, see below. The antibody should preferably be able to detect 20 endogenous leptin, and most preferably, to nanogram amounts in serum, if a serum assay is to be used to determine baseline leptin levels. Other assays may be used, such as direct protein assay (e.g., isolation of leptin protein from blood), 25 however this is not the most practicable. Nucleic acid (DNA or RNA) levels may be correlated to leptin protein levels. Other molecules may also be correlated to leptin levels, such as molecules involved in fat metabolism. One may prepare a graph correlating the 30 relationship between leptin levels and the levels of the other molecules, and therefore use the other molecule to determine the predisposition of an individual to leptin responsiveness.

WO 00/20872 PCTIUS99/21903 - 11 In particular, one may use nucleic-acid based assays, such as what is sometimes referred to as "gene chip" technology. Such technology is currently known in the art and involves the use of microprocessors 5 (i.e., computer "chips") to perform nucleic acid hybridization techniques. The presence or amount of hybridization is typically determined by automated means, such as a suitable scanner. Thus, one may use an additional step of correlating the serum leptin 10 levels with nucleic acid transcription, and use nucleic acid hybridization as localized on a computer chip to ascertain baseline leptin levels. For actually treating the individual, the following leptin proteins, formulations, pharmaceutical 15 compositions and uses apply. Leptin Proteins. In general, human leptin 1-146, a leptin analog (that is, an analog of human leptin 1-146) or a leptin derivative (that is, human leptin 1-146 or an analog thereof having an additional chemical moiety 20 attached thereto, such as dextran, as described below) may be used in conjunction with the present methods. Thus, the present methods relate to determining the predisposition of an individual for responding to leptin treatment, and use of one of the above "leptins" i.e., 25 leptin, a leptin analog, or a leptin derivative, as set forth above, for such treatment. The type of leptin used for the treating the individual may be selected from those described in PCT International Publication Number WO 96/05309, as cited 30 above and herein incorporated by reference in its entirety. Figure 3 of that publication (as cited therein SEQ ID NO. 4) depicts the full deduced amino acid sequence derived for human leptin (referred to as WO 00/20872 PCT/US99/21903 - 12 the human "OB" protein). The amino acids are numbered from 1 to 167. A signal sequence cleavage site is located after amino acid 21 (Ala) so that the mature protein extends from amino acid 22 (Val) to amino acid 5 167 (Cys) . For the present disclosure, a different numbering is used herein, where the amino acid position 1 is the valine residue which is at the beginning of the mature protein. The amino acid sequence for mature, recombinant methionyl human leptin 10 is presented herein as SEQ ID NO. 1, where the first amino acid of the mature protein is valine (at position 1) and a methionyl residue is located at position -1 (herein called rHu-Leptin 1-146, SEQ ID No. 1): 15 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 D L E N L R D L L H V L A F S K S C H L 20 P W A S G L E T L D S L G G V L E A S G Y S T E V V A L S R L Q G S L Q D M L W Q L D L S P G C Alternatively, one may use a natural variant of human leptin, which has 145 amino acids, and, as compared 25 to rHu-Leptin 1-146, has a glutamine absent at position 28, presented below (herein called rHu-Leptin 1-145, SEQ ID NO. 2, wherein the blank ("_") indicates no amino acid): V P I Q K V Q D D T K T L I K T I V 30 T R I N D I S H T _ 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 D L E N L R D L L H V L A F S K S C H L P W A S G L E T L D S L G G V L E A S G WO 00/20872 PCTIUS99/21903 - 13 Y S T E V V A L S R L Q G S L Q D M L W Q L D L S P G C Generally, the leptin moiety for human pharmaceutical use herein will be capable of 5 therapeutic use in humans (see also, animal leptins, below). Thus, one may empirically test activity to determine which leptin moieties may be used. As set forth in WO 96/05309, leptin protein in its native form, or fragments (such as enzyme cleavage products) 10 or other truncated forms and analogs may all retain biological activity. 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, herein incorporated by reference in their entireties. 15 One may prepare an analog of recombinant human leptin by altering amino acid residues in the recombinant human sequence, such as substituting the amino acids which diverge from the murine sequence. Murine leptin is substantially homologous to human 20 leptin, particularly as a mature protein and, further, particularly at the N-terminus. Because the recombinant human protein has biological activity in mice, such an analog would likely be active in humans. For example, in the amino acid sequence of native human 25 leptin as presented in SEQ ID NO. 1, one may substitute with another amino acid one or more of the amino acids at positions 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. One may select the amino acid at the 30 corresponding position of the murine protein (see Zhang et al., 1994, supra) or another amino acid. One may further prepare "consensus" molecules based on the rat OB protein sequence. Murakami et al., Biochem. Biophys. Res. Comm. 209: 944-52 (1995) herein 35 incorporated by reference. Rat OB protein differs from WO 00/20872 PCT/US99/21903 - 14 human OB protein at the following positions (using the numbering of SEQ ID NO. 1): 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138 and 145. One may substitute with 5 another amino acid one or more of the amino acids at these divergent positions. The positions underlined 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. 10 At one or more of the positions, one may substitute an amino acid from the corresponding rat OB protein, or another amino acid. The positions from both rat and murine OB protein which diverge from the mature human OB protein are: 4, 15 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 or more of the above amino acids replaced with another amino acid, such as the amino acid found 20 in the corresponding rat or murine sequence, may also be effective. In addition, the amino acids found in rhesus monkey OB protein which diverge from the mature human OB protein are (with identities noted in parentheses in 25 one letter amino acid abbreviation): 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 the recombinant human OB protein is active in cynomolgus monkeys, a human OB protein according to SEQ ID NO. 1 30 having one or more of the rhesus monkey divergent amino acids replaced with another amino acid, such as the amino acids in parentheses, may be effective. It should be noted that certain rhesus divergent amino acids are also those found in the above murine and rat 35 species (positions 35, 68, 89, 100, 108 and 118). Thus, one may prepare a murine/rat/rhesus/human WO 00/20872 PCT/US99/21903 - 15 consensus molecule (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, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 5 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and 145. The positions underlined are those in which all three species are divergent from human OB protein. A particularly preferred human leptin analog is one wherein the amino acids at position 100 (Trp) or 10 138 (Trp), and more preferably, both positions are substituted with another amino acid, preferably Gln. Other analogs may be prepared by deleting a part of the protein amino acid sequence. For example, the mature protein lacks a leader sequence (-22 to -1). 15 One may 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) 20 112-146; (iii) amino acids 1-99 and (connected to) 112-146 having one or more of amino acids 100-111 sequentially placed between amino acids 99 and 112. In addition, the truncated forms may also have 25 altered one or more of the amino acids which are divergent (in the murine, rat or rhesus OB protein) from human OB protein. Furthermore, any alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids. 30 Also included are those proteins as set forth above with amino acid substitutions which are "conservative" according to acidity, charge, hydrophobicity, polarity, size or any other characteristic known to those skilled in the art. 35 These are set forth in Table 1, below. See generally, Creighton, Proteins, passim (W.H. Freeman and Company, WO 00/20872 PCTIUS99/2190 3 - 16 N.Y., 1984); Ford et al., Protein Expression and Purification 2:95-107 (1991), which are herein incorporated by reference. 5 Table 1 Conservative Amino Acid Substitutions 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 Therefore, the leptins used in conjunction with 10 the methods of the present invention may be selected from among (according to the amino acid sequence as presented in SEQ ID NO. 1 herein): (a) the amino acid sequence of SEQ ID NO. 1, optionally lacking a glutaminyl residue at position 28, 15 and further optionally having a methionyl residue at the N-terminus; (b) an amino acid sequence of subpart (a) having a different amino acid substituted in one or more of the following positions: 4, 8, 32, 33, 35, 48, 20 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and 145; WO 00/20872 PCT/US99/21903 - 17 (c) an amino acid sequence of subpart (b) wherein the amino acids at positions 100 and 138 are substituted with Gln; (d) a truncated leptin protein analog 5 selected from among: (i) amino acids 98-146 (ii) amino acids 1-99 and 112-146 (iii) amino acids 1-99 and 112-146 having one or more of amino acids 100-111 10 sequentially placed between amino acids 99 and 112; and, (iv) the truncated leptin analog of subpart (i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 15 142 and 145 substituted with another amino acid; (v) the truncated leptin analog of subpart (iii) having one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142 and 20 145 replaced with another amino acid; (vi) the truncated leptin analog of subpart (iv) having one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 25 111, 112, 118, 136, 138, 142 and 145 replaced with another amino acid; and (vii) the truncated leptin analog of any of subparts (i)-(vi) having an N-terminal methionyl residue; 30 (e) a leptin protein of any of subparts (a)-(d) having one or more conserved amino acid substitutions. Preferred is rmetHu-leptinl-1 46 as set forth in SEQ ID No. 1, as this has been demonstrated in humans 35 to have substantially no toxicity. Other leptins which may be used include rmetHu-leptin 1-146 or rHu-leptin WO 00/20872 PCT/US99/21903 - 18 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 position 99 (accord to SEQ ID NO. 2, if leptin 1-145 as in SEQ ID NO. 2 is 5 used). Such substitution may be selected from among alanine, glutamic acid, and glutamine. Also, rmetHu-leptin 1-146 or rHu-leptin 1-146 (lacking an N-terminal methionyl residue) having a substitution at one or both position 100 (or 99, if the Q- sequence is 10 used) and position 138 (137 if the Q- sequence is used). Thesubstitution may of any amino acid apart from the already present W (tryptophan) and may be selected from among alanine, glutamic acid, and glutamine. 15 Other leptin derivatives or derivatives of analogs may be use. For example, co-pending 60/096194, herein incorporated by reference, discloses various dextran modified leptins. PCT publication WO 98/28427 (incorporated by reference)discloses various OB fusion 20 protein compositions. A variety of leptin or leptin analog derivatives may be used in conjunction with the present methods, as disclosed in the various publications as cited passim, which are incorporated by reference. 25 Leptin proteins, analogs and related molecules are also reported in the following publications; however, no representation is made with regard to the activity of any composition reported: U.S. Patent Nos. 5,521,283; 5,525,705; 30 5,532,336; 5,552,522; 5,552,523; 5,552,524; 5,554,727; 5,559,208; 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; 35 5,719,266 (Eli Lilly and Company); WO 00/20872 PCT/US99/21903 - 19 PCT W096/23513; W096/23514; W096/23515; W096/23516; W096/23517; W096/23518; W096/23519; W096/23520; W096/23815; W096/27385; W096/34111; W096/37517; 5 W097/00886; EP 725078; EP 725079; EP 744408; EP 745610; EP 835879 (Eli Lilly and Company); PCT W096/22308 (Zymogenetics); PCT W096/31526 (Amylin Pharmaceuticals, Inc.); 10 PCT W096/34885; W097/46585 (Smithkline Beecham PLC); PCT W096/35787 (Chiron Corporation); PCT W097/16550 (Bristol-Myers Squibb); PCT W097/20933 (Schering Corporation) 15 EP 736599 (Takeda); EP 741187 (F. Hoffman LaRoche). To the extent these references provide for useful leptin proteins or analogs, or associated compositions 20 or methods, such compositions and/or methods may be used in conjunction with the present methods. With the above provisos, these publications are herein incorporated by reference. Animal Leptins 25 In addition to the above human therapeutic leptin, certain animal leptins are also available for therapeutic use. Canine leptin is disclosed in WO 97/32022, herein incorporated by reference. Other animal species are disclosed in the following 30 publications: WO 96/36644, EP 743321 (porcine and bovine); WO 98/04288 (bovine); WO 98/04690 (porcine), all of which are herein incorporated by reference. These are not preferred for human use for reasons of avoidance of possibly immunogenic response.

WO 00/20872 PCTIUS99/21903 - 20 Pharmaceutical Compositions The present assay method may also be used to determine the predisposition of an individual to respond to pharmaceutical compositions for therapeutic 5 uses. Such pharmaceutical compositions may be for administration by bolus injection or by infusion (e.g., intravenous or subcutaneous), or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, comprehended by the invention are 10 pharmaceutical compositions comprising effective amounts of leptin protein, analog or derivative compositions together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such pharmaceutical 15 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), 20 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. See, 25 e.g., PCT WO 96/29989, herein incorporated by reference. Hylauronic acid may also be used, and this may have the effect of increasing sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and 30 rate of in vivo clearance leptins or analogs or derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated WO 00/20872 PCT/US99/21903 - 21 by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal 5 formulations. Contemplated are oral formulations, as described in PCT WO 95/21629, herein incorporated by reference in its entirety. This PCT publication describes oral delivery of chemically modified proteins, including 10 proteins modified by dextran moieties. The compositions and methods disclosed therein are applicable here to administer leptin protein, analog or derivative compositions. Pulmonary delivery is also contemplated, and 15 compositions and methods disclosed in PCT WO 94/20069 are useful herein. WO 94/20069, which discloses the pulmonary delivery of chemically-modified G-CSF, is herein incorporated by reference. The particle size for pulmonary administration 20 should most advantageously be prepared in particulate form with an average particle size of less than 10 microns, most preferably 0.5 to 5 microns, for most effective delivery to the distal lung. Nasal delivery is also contemplated. Nasal 25 delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or 30 dextrin, such as cyclodextrin. Delivery via transport across other mucus membranes is also contemplated.

WO 00/20872 PCT/US99/21903 - 22 Dosages One skilled in the art will be able to ascertain effective dosages by administration and observing the desired therapeutic effect. Presently, the present 5 human clinical data demonstrated efficacy of rmetHu-leptin (1-146, SEQ ID NO. 1) most effectively at a dose of 0.3 mg/kg body weight/day. See Greenberg et al., supra. However, the present subjects who demonstrated that the base serum leptin levels 10 correspond to predisposition to weight loss were administered either 10 mg/day or 10 mg/twice a day (for 20 mg total protein) of rmetHu-Leptin 1-146 (SEQ ID NO. 1). It is contemplated that other forms of leptin (such as analogs or derivatives) may result in 15 advantageous dosing regimes, or lower protein administration per unit of body mass. Apart from determine a baseline leptin level to determine if one is predisposed to leptin treatment, it is contemplated that the effective dosages may be 20 determined using diagnostic tools over time. For example, a diagnostic for measuring the amount of leptin in the blood (or plasma or serum) may first be used to determine endogenous levels of leptin protein. Such diagnostic tool may be in the form of an antibody 25 assay, such as an antibody sandwich assay. The amount of endogenous leptin protein is quantified initially, and a baseline is determined. The therapeutic dosages are determined as the quantification of endogenous and exogenous leptin protein moiety (that is, protein, 30 analog or derivative found within the body, either self-produced or administered) is continued over the course of therapy. The dosages may therefore vary over the course of therapy, with, for example, a relatively WO 00/20872 PCT/US99/21903 - 23 high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits. Methods of Use 5 The present invention may be used to determine an individual's predisposition to respond to leptin treatment (or treatment using a leptin related composition such as an analog or derivative) in the following way. It is contemplated that the present 10 invention will be used as a screening tool to determine the likelihood that an obese individual (i.e., an individual with a Body Mass Index of greater than 27, and preferably greater than 27.5, most preferably in the range of 27.5 to 36), not having comorbidities of 15 obesity, such as diabetes, dislipidemias, such as hyperlepidemias, and hypertension, will respond to leptin administration with weight loss. It is further contemplated that such individual will be screened for predisposition to respond predominantly by fat loss, 20 but also may be screened for predisposition to leptin response for a variety of leptin uses. Uses of leptin include: Therapeutic. Therapeutic uses include weight modulation, the treatment or prevention of diabetes, 25 blood lipid reduction (and treatment of related conditions), increasing lean body mass and increasing insulin sensitivity. In addition, the present compositions may be used for manufacture of one or more medicaments for treatment or amelioration of the above 30 conditions. Weight Modulation. The present compositions and methods may be used for weight reduction. Viewed another way, the present compositions may be used for maintenance of a desired weight or level of adiposity.

WO 00/20872 PCTIUS99/21903 - 24 The body mass lost is primarily of adipose tissue, or fat. Such weight loss can be associated with the treatment of concomitant conditions, such as those below, and therefore constitute a therapeutic 5 application. In addition, cosmetic uses are provided herein if weight modulation is solely for improvement in appearance. Treatment of Diabetes. The present compositions and methods may be used in the prevention or treatment 10 of Type II diabetes. As Type II diabetes can be correlated with obesity, use of the present invention to reduce weight (or maintain a desired weight, or reduce or maintain an adiposity level) can also alleviate or prevent the development of diabetes. 15 Moreover, even in the absence of dosages sufficient to result in weight loss, the present compositions may be used to prevent or ameliorate diabetes. Blood Lipid Modulation. The present compositions and methods may be used in the modulation of blood 20 lipid levels. Hyperlipidemia (also called lipemia; dyslipidemia) is the presence of an abnormally large amount of lipids in the circulating blood. Ideally, in situations where solely reduction in blood lipid levels is desired, or where maintenance of blood lipid levels 25 is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of an obese patient, dosages may be administered whereby weight loss and concomitant blood lipid level lowering is achieved. Once sufficient 30 weight loss is achieved, a dosage sufficient to prevent re-gaining weight, yet sufficient to maintain desired blood lipid levels, or other conditions as set forth herein, for example, may be administered. These dosages can be determined empirically, as the effects 35 of leptin protein are reversible. E.g., Campfield et al., Science 269: 546-549 (1995) at 547. Thus, if a WO 00/20872 PCT/US99/21903 - 25 dosage resulting in weight loss is observed when weight loss is not desired, one would administer a lower dose in order to achieve the desired blood lipid levels, yet maintain the desired weight. See, e.g., PCT 5 Publication WO 97/06816 herein incorporated by reference. Increasing Lean Mass or Insulin Sensitivity. Ideally, in situations where solely an increase in lean body mass is desired, the dosage will be insufficient 10 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/lean mass increase is achieved. Once sufficient weight loss is achieved, a dosage sufficient 15 to prevent regaining weight, yet sufficient to maintain desired lean mass increase (or prevention of lean mass depletion) may be administered. For increasing an individual's sensitivity to insulin, similar dosage considerations may be taken into account. Lean mass 20 increase without weight loss may be achieved sufficient to decrease the amount of insulin (or, potentially, amylin, amylin antagonists or agonists, or thiazolidinediones, or other potential diabetes treating drugs) an individual would be administered for 25 the treatment of diabetes. For increasing overall strength, there may be similar dosage considerations. Lean mass increase with concomitant increase in overall strength may be achieved with doses insufficient to result in weight loss. Other benefits, such as an 30 increase in red blood cells (and oxygenation in the blood) and a decrease in bone resorption or osteoporosis may also be achieved in the absence of weight loss. See, e.g., PCT Publication No. WO 97/18833 herein incorporated by reference. 35 Combination Therapies. The present invention may also be used to determine the predisposition of an WO 00/20872 PCT/US99/21903 - 26 individual to respond to leptin, analogs and derivatives, such as may be used in conjunction with other therapies, such as altered diet and exercise. Other medicaments, such as those useful for the 5 treatment of diabetes (e.g., insulin and possibly amylin, antagonists or agonists thereof, thiazolidinediones (see, e.g., PCT Publication No. WO 98/08512 herein incorporated by reference), or other potential diabetes treating drugs), cholesterol and 10 blood pressure lowering medicaments (such as those which reduce blood lipid levels or other cardiovascular medicaments), activity increasing medicaments (e.g., amphetamines), diuretics (for liquid elimination), and appetite suppressants (such as agents which act on 15 neuropeptide Y receptors or serotonin reuptake inhibitors). Such administration may be simultaneous or may be in seriatim. In addition, the present methods may be used in conjunction with surgical procedures, such as cosmetic surgeries designed to 20 alter the overall appearance of a body (e.g., 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 25 surgeries designed to relieve a deleterious condition caused by blockage of blood vessels by fatty deposits, such as arterial plaque, may be increased with concomitant use of the present compositions and methods. Methods to eliminate gall stones, such as 30 ultrasonic or laser methods, may also be used either prior to, during or after a course of the present therapeutic methods. Furthermore, the present methods may be used as an adjunct to surgeries or therapies for broken bones, damaged muscle, or other therapies which 35 would be improved by an increase in lean tissue mass.

WO 00/20872 PCT/US99/21903 - 27 Other Uses In conjunction with the present methods other leptin or analog or derivative uses are contemplated, and these include: administration of leptin (or analog 5 or derivative), in accordance with an endogenous fluctuation of leptin secretion in an individual to most advantageously result in leptin effectiveness; treatment of conditions associated with fertility, treatment of conditions associated with release of 10 human growth hormone, treatment of conditions associated with wound healing, hematopoietic conditions, angiogenic conditions for growing blood vessels in fat deposits, treatment of the chronic stress conditions, treatment of conditions associated 15 with lack of fertility or delayed onset puberty, and other conditions as will be apparent to one skilled in the art of therapeutic or cosmetic leptin uses. Methods of Leptin Production The leptin moieties used herein may be made in 20 prokaryotic or in eukaryotic cells, although, for the leptin moieties used in the working examples below, bacteria is preferred for ease in commercial manufacture. One may further use leptin made in human cells, such as that made by controlling a native or 25 introduced regulatory element which affects the regulation of an endogenous gene encoding the desired protein. Recombinant expression of leptin moieties has been described, for example, in WO 96/40912, herein incorporated by reference, including all vector and 30 host strain deposits cited therein.

WO 00/20872 PCTIUS99/21903 - 28 EXAMPLE The present methods were used to determine the leptin levels in humans prior to undergoing leptin 5 treatment for obesity in a human clinical trial. The study design and clinical trial protocol are summarized as in Greenberg et al., "Preliminary safety and efficacy of recombinant methionyl human leptin (rL) administered by SC injection in lean and obese 10 subjects." Poster presented at: 5 8 th Annual Meeting and Scientific Sessions of the American Diabetes Association; June 14, 1998; Chicago, IL, incorporated by reference, supra. A standard enzyme-linked immunosorbent assay 15 (commonly referred to by the acronym, "ELISA") was used to determine leptin levels in the serum of individuals enrolled in the above clinical trial. The method used a purified rat monoclonal anti-rmetHu-Leptin antibody for capturing leptin from whole serum. Affinity 20 purified rabbit anti- rmetHu-leptin polyclonal antibody conjugated to horse-radish peroxidase was used to detect captured leptin. The limit of detection of the assay was 0.8 ng/ml. Although certain antibodies were used herein, preferred antibodies are those which 25 specifically react with native human leptin, and are sensitive to detect leptin quantities of equal to or below 5 ng/ml serum (for male patients) and equal to or below 16 ng/ml serum (for female patients). Individuals having a BMI of between about 27.5 30 and 38 (see Greenberg et al., infra), were screened for the absence of co-morbidities of obesity. Subjects were placed on dietary and exercise counseling. To determine baseline serum leptin WO 00/20872 PCTIUS99/21903 - 29 levels, blood was drawn after an 8-12 hour fast, between 8:00 a.m. and 10:00 a.m. Serum was obtained, and the above materials were used to perform an antibody assay, using standard techniqes. Doses of 5 leptin administered were either 10mg/day or 10mg/2x/day. (At this level, there did not appear to be a dose related response among those who responded). Weight loss was measured after 3 months (12 weeks), and the 3 month data is presented below. 10 Results Results demonstrate that there is a greater amount of weight loss in individuals who have a baseline leptin level in the lowest 33%, and further, in the lowest 25% of serum leptin levels measured for 15 obese individuals. As can be seen in Table 2, in an overall population of male and female obese patients, individuals in the bottom quartile (bottom 25%) of leptin serum levels demonstrated a larger response 20 rate than those in the upper 75% of leptin serum leptin levels. Those in the bottom 25% lost an average of 4.8% body weight and an average of 5.4 kg after three months. Those having a baseline serum leptin level in the upper 75%,although demonstrating 25 weight loss, lost on average substantially less, 2.2% and 2.0kg, after the same time period. Not all in the bottom 25% of serum leptin levels responded as such. Among those in the bottom 25% of serum leptin levels, 19 had a weight change which was 30 within the average for the upper 75% (i.e., a lower percentage or equal to -2.2% change in body weight. Of those 19, 6 lost weight, 2 had no change in body weight, and 11 gained weight. For those 19 in the WO 00/20872 PCT/US99/21903 - 30 bottom quartile of serum leptin levels who responded at the higher rate, there was no difference in response which was dose dependent (10mg/dy or 10mg/2x/dy). For the bottom 33.3% of serum leptin 5 level individuals, 20 individuals did not have a weight loss less 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% being the approximate average for the those having a serum 10 leptin level in the upper 66.6%). Table 2. % Leptin Level as Compared to Obese Population and Weight Loss Patients No of patients Average weight loss (percent/kg) Bottom 25% 74 -4.8%/-5.4kg Top 75% 211 -2.2%/-2.Okg Bottom 33.3% 94 -5.0%/-4.8kg Top 66.6% 191 -1.8%/-1.6kg 15 As can be seen from Table 3, below, these results hold true when separated for gender. If the serum leptin levels are determined from either only females or only from males, not from a mixed population, the bottom 33% and further the bottom 25% also have a 20 greater average response rate than those who have higher serum leptin levels. Table 3. Serum Leptin Levels and Weight Loss Patients No of patients Average weight loss (percent/kg) Female - Bottom 25% 46 -4.6%/-3.7kg Female - Bottom 33.3% 64 -4.6%/-3.8kg Male - Bottom 25% 21 -5.4%/5.2kg Male - Bottom 33% 29 -5.1%/5.1kg WO 00/20872 PCT/US99/21903 - 31 When the data were reviewed, it became apparent that these obese individuals who had endogenous, fasting leptin levels in the bottom 25% of the obese population (excluding known ob/ob and db/db equivalent 5 humans, both rare events) responded to leptin administration of fixed doses of 10mg per day or 10 mg twice a day (20 mg total protein). Actual serum leptin levels were below about 5 ng/ml (for male patients) and below about 16 ng/ml for female 10 patients. * * * While the present invention has been described in terms of the preferred embodiments, it is understood 15 that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed. 20

Claims (3)

1. A method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative comprised of: 5 (a) determining a leptin level in said individual prior to said treatment; and (b) ascertaining if said leptin level is in approximately the bottom 25% of leptin levels of obese individuals. 10 2. A method of claim 1 wherein said obese individual has a body mass index of greater than 27.

3. A method of claim 1 wherein said obese individual has a body mass index of between 27.5 and

38. 15 4. A method of claim 1 wherein said leptin level is determined by an antibody assay. 5. A method of claim 1 wherein said leptin level is a serum leptin level. 6. A method of claim 4 wherein said obese 20 individual is a male and said serum leptin level is equal to or less than about 5 ng/ml. 7. A method of claim 4 wherein said obese individual is a female and said serum leptin level is equal to or less than about 16 ng/ml. 25 8. A method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative comprised of: (a) determining a leptin level in said individual prior to said treatment; and 30 (b) ascertaining if said leptin level is in approximately the bottom 33% of leptin levels of obese individuals. WO 00/20872 PCTIUS99/21903 - 33 9. A method for determining the predisposition of an obese individual to respond to treatment by leptin, leptin analog, or leptin derivative comprised of: (a) determining if the baseline serum leptin 5 level of an obese male is equal to or less than about 5 ng/ml; or (b) determining if the baseline serum leptin level of an obese female is equal to or less than about 16 ng/ml. 10 10. An assay for determining the predisposition of an individual to respond to leptin, leptin analog, or leptin derivative treatment comprising: (a) means to detect the endogenous leptin level in an individual; 15 (b) means to ascertain if such endogenous leptin level is in approximately the bottom 25% of leptin levels for obese individuals. 11. An assay for determining the predisposition of an individual to respond to leptin, leptin analog, 20 or leptin derivative treatment comprising: (a) means to detect the endogenous leptin level in an individual; (b) means to ascertain if such endogenous leptin level is in approximately the bottom 33% of 25 leptin levels for obese individuals. 12. An improved kit for determining the predisposition of an obese individual to lose weight in response to treatment using leptin, a leptin analog or leptin derivative, said improvement comprising means 30 for determining whether the endogenous leptin level in said obese individual is in approximately the bottom 25% of leptin levels of obese individuals. WO 00/20872 PCT/US99/21903 - 34 13. An improved kit for determining the predisposition of an obese individual to lose weight in response to treatment using leptin, a leptin analog or leptin derivative, said improvement comprising means 5 for determining whether the endogenous leptin level in said obese individual is: (a) below about 5 ng/ml serum if said obese individual is a male; (b) below about 16 ng/ml serum if said obese 10 individual is a female.