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1. (WO2019032469) TREATMENT OF OVERWEIGHT AND OBESITY ASSOCIATED WITH LEPTIN DEFICIENCY
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TITLE

TREATMENT OF OVERWEIGHT AND OBESITY ASSOCIATED WITH LEPTIN DEFICIENCY

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application Serial No. 62/542, 175 filed on August 7, 2017, and of U.S. Provisional Application Serial No. 62/579,819 filed on October 31, 2017, which are both hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 2, 2018, is named Al 19-24_WO_SL.txt and is 3,302 bytes in size.

FIELD OF THE DISCLOSURE

[003] The present invention relates to methods of treating overweight or obesity associated with leptin deficiency and, in some embodiments, one or more weight-related comorbidities. The methods may comprise administration of leptin to a subject in need thereof having said underlying leptin deficiency, including those subjects who are refractory to other methods of treatment.

BACKGROUND

[004] Obesity is a serious and costly health issue in the U.S. In 2015-2016, it was estimated that 93.3 million adults are affected by obesity (Hales et al., NCHS Data Brief, 2017, 288). The high prevalence of obesity among adults— estimated to be about 39.8 %— generates a burden on the health care system, as there are several obesity-related medical conditions, including heart disease, stroke, type 2 diabetes, and certain types of cancer. The estimated annual medical cost of obesity in the U.S. was $147 billion in 2008, and the medical cost for people who have obesity was $1,429 higher than those of normal weight (Finkelstein et al., Health Affairs, 2009;

https://doi.Org/10.1377/hlthaff.28.5.w822).

[005] Leptin, an adipose tissue-secreted hormone, was once viewed as a potential anti-obesity agent (Friedman et al., Metab Clin Exp, 2015, 64: 1-4), as it is not produced in the leptin-deficient oblob mouse model of obesity. Studies in leptin-deficient mice showed that increases in plasma leptin levels reduced food intake and induced weight loss (Halaas et al., Proc Natl AcadSci USA, 1997, 94: 8878-83). Further, treatment with leptin in a human with congenital leptin deficiency (CLD), which is marked by a frame-shift mutation in the leptin gene, led to a sustained reduction in weight (Farooqi et al., N Engl J Med, 1999, 341 : 879-84).

[006] However, further studies on the effects of leptin on obesity were less successful. In particular, clinical trials demonstrated that, for the treatment of common obesity, which is associated with hyperleptinemia and central leptin resistance (Mark, Am J Physiol Regul Integr Comp Physiol, 2013, 305: R566-81), leptin exhibited no or minimal effects. (Flier et al., Cell Metab, 2017, 26: 1-3; Heymsfield et al., JAMA, 1999, 282: 1568-75).

[007] Thus, while leptin has provided a framework for studying the pathogenesis of obesity, the efficacy of leptin on obesity is uncertain at best.

SUMMARY OF INVENTION

[008] The present invention relates to a method of treating an above-normal weight condition in a subject with a leptin deficiency, in which the method comprises administering a therapeutically effective amount of leptin to the subject.

[009] In some embodiments, the above-normal weight condition comprises overweight.

Overweight may comprise a BMI of 25 kg/m2 to 29.9 kg/m2, or a BMI of 25 kg/m2 to less than 30 kg/m2, or a BMI of 27 kg/m2 to less than 30 kg/m2, or a BMI of 27.5 kg/m2 to less than 30 kg/m2.

[010] In some embodiments, the above-normal weight condition comprises obesity. Obesity may comprise a BMI of 30 kg/m2 or greater.

[011] In certain embodiments, the leptin deficiency comprises a pre-treatment serum leptin concentration of < about 16 ng/mL if the subject is female, and < about 5 ng/mL if the subject is male; or < about 8 ng/mL if the subject is female, and < about 3 ng/mL if the subject is male; or < about 5 ng/mL if the subject is female, and < about 2 ng/mL if the subject is male.

[012] In some embodiments, the leptin deficiency comprises a pre-treatment serum leptin concentration that is in about the 10th percentile, or about the 2nd percentile, or about the 1st percentile, according to the third National Health and Nutrition Examination Survey

(NHANES III) (Ruhl et al., Am J Clin Nutr, 2001, 74: 295-301).

[013] In some embodiments, the subject has a detectable pre-treatment serum leptin

concentration.

[014] In certain embodiments, the subject has a weight-related comorbidity. The weight-related comorbidity may be prediabetes, type 2 diabetes mellitus, dyslipidemia, hypertension, cardiovascular disease, cancer, breathing disorders, gallbladder disease, gynecological problems, erectile dysfunction and sexual health issues, liver dysfunction, liver inflammation, liver damage, osteoarthritis, or a combination thereof. In some embodiments, the weight-related comorbidity is type 2 diabetes mellitus, hypertension, dyslipidemia, or a combination thereof.

[015] In some embodiments, the subject is human. In certain embodiments, the subject is an adult human.

[016] In some embodiments, the subject pre-treatment does not have high insulin levels. In some embodiments, the subject pre-treatment does not have impaired glucose tolerance. In certain embodiments, the subject pre-treatment does not have two or more of the following: high triglycerides or high HDL cholesterol, high blood pressure, or high fasting glucose level. In certain embodiments, the subject does not have metabolic syndrome.

[017] In some embodiments, the subject does not have congenital leptin deficiency.

[018] In certain embodiments, the leptin is methionyl-human leptin.

[019] In certain embodiments, the therapeutically effective amount of leptin is about 1-50 mg/day, or about 5-20 mg/day. The leptin may be administered once daily as a single or divided dose, or twice daily, to the subject. In some embodiments the leptin is administered in an amount of about 20 mg once daily or about 10 mg once daily.

[020] In some embodiments, the leptin is administered parenterally. In certain embodiments, the leptin is administered subcutaneously.

[021] In certain embodiments, the administration of the leptin decreases body weight of the subject by about 5 % or greater, or by about 10 % or greater, from pre-treatment body weight. In some embodiments, the decrease in body weight is maintained for about a week or longer, or about a month or longer, or about a year or longer.

[022] In certain embodiments, the administration of the leptin reduces BMI of the subject by about 5 % or greater, or about 10 % or greater, from pre-treatment BMI. In some embodiments, the reduction in BMI is maintained for about a week or longer, or about a month or longer, or about 6 months or longer, or about a year or longer.

[023] In certain embodiments, the present invention relates to a method of chronic weight maintenance in a subject having: (a) pre-treatment low leptin, and (b) either (i) a pre-treatment BMI of >30 kg/m2 (obese), or (ii) a pre-treatment BMI of >27 kg/m2 (overweight) in the presence of at least one weight-related comorbid condition (e.g., hypertension, type 2 diabetes mellitus, or dyslipidemia). In certain embodiments, the pre-treatment low leptin comprises a pre-treatment leptin level of <16 ng/mL for females and <5 ng/mL for males, or a pre-treatment leptin level of <8 ng/mL for females and <3 ng/mL for males. In certain embodiments, the subject has a detectable pre-treatment serum leptin concentration.

BRIEF DESCRIPTION OF THE FIGURES

[024] Reference is made to the following description of an exemplary embodiment of the present invention, and to the accompanying drawings, wherein:

[025] Figure 1 presents results from a post hoc analysis of five studies that involved subjects who were overweight or obese, as described in Example 1. This figure shows the difference in mean percent change in weight between the metreleptin dose groups and placebo, from baseline to Week 12.

[026] Figure 2 presents results from a post hoc analysis of five studies that involved subjects who were overweight or obese, as described in Example 1. This figure shows body weight percent change in subjects administered 20 mg/day metreleptin and who were in the lowest baseline leptin level group (5 ng/mL or less for females, and 2 ng/mL or less for males).

[027] Figure 3 presents results from a post hoc analysis of five studies that involved subjects who were overweight or obese, as described in Example 1. This figure shows body weight percent change in subjects administered 20 mg/day metreleptin and who were in the lower baseline leptin level group (8 ng/mL or less for females, and 3 ng/mL or less for males).

[028] Figure 4 presents results from a post hoc analysis of five studies that involved subjects who were overweight or obese, as described in Example 1. This figure shows body weight percent change in subjects administered 20 mg/day metreleptin or placebo, who were in the lowest baseline leptin level group (5 ng/mL or less for females, and 2 ng/mL or less for males) or who had a pre-treatment leptin concentration above 5 ng/mL for females, and above 2 ng/mL for males.

[029] Figure 5 presents results from a post hoc analysis of five studies that involved subjects who were overweight or obese, as described in Example 1. This figure shows body weight percent change in subjects administered 20 mg/day metreleptin or placebo, who were in the lower baseline leptin level group (8 ng/mL or less for females, and 3 ng/mL or less for males) or who had a pre-treatment leptin concentration above 8 ng/mL for females and above 3 ng/mL for males.

[030] Figure 6 presents results from a post hoc analysis of four studies that involved subjects who were overweight or obese, as described in Example 2. This figure shows mean percent change from baseline in weight for subjects administered 20 mg/day metreleptin or placebo.

[031] Figure 7 presents results from a post hoc analysis of a study that involved subjects who were overweight or obese, as described in Example 3. This figure shows percent change in weight from baseline to Weeks 12, 16, and 24 for subjects having a lower baseline leptin level and administered metreleptin (20 mg/day or 10 mg/day) or placebo.

[032] Figure 8 presents results from a post hoc analysis of a study that involved subjects who were overweight or obese, as described in Example 3. This figure shows difference in mean percent changes in weight between subjects administered 20 mg/day and placebo, over time.

[033] Figure 9 presents results from a post hoc analysis of a study that involved subjects who were overweight or obese, as described in Example 4. This figure shows mean percent changes

in weight from baseline between subjects administered 20 mg/day, 10 mg/day, and placebo, over time.

[034] Figure 10 presents results from a post hoc analysis of five studies that involved pooled subjects who were overweight or obese, as described in Example 4. This figure shows proportion of subjects with greater than/equal to 5 % weight loss among subjects administered 20 mg/day, 10 mg/day, and placebo, at 12 weeks and 24 weeks.

DETAILED DESCRIPTION

[035] The present invention is based, in part, on studies that assessed the effects of metreleptin on subjects who are overweight or obese (unpublished). The studies found that the subjects did not experience a clinically significant loss of weight, with the exception of one subpopulation. This subpopulation, which achieved clinically significant weight loss at 12 and 24 weeks, was found to have subjects with low leptin.

[036] Leptin, an adipose tissue-secreted hormone, is involved in regulating energy homeostasis, including metabolism of glucose, fatty acids and triglycerides, and other physiological functions. (Friedman et al., Nature, 1998, 395: 763-770). One of the most critical roles of the leptin signaling pathway is protection from reductions in fat stores that could impact reproductive capacity, fertility, and therefore overall survival (Rosenbaum et al., J Endocrinol, 2014, 223: T83-96). Without being bound by theory, a subject with low leptin may experience a centrally controlled starvation signal resulting in heightened appetite and restricted energy expenditure, causing the body to believe that it is starving and to seek to preserve body fat. Consequently, the body increases calorie consumption (hyperphagia) and restricts energy expenditure by limiting high energy functions such as immune system surveillance, reproduction and exercise. Thus, administration of leptin may be an effective treatment for subjects who are overweight or obese and who are leptin-deficient.

[037] Obesity or overweight in combination leptin deficiency may be characteristic of an emerging condition called hypoleptinemic metabolic disorder ("HMD"). HMD, which may also be referred to as "Hypoleptinemic Dysmetabolic Disorder," "Hypoleptinemic Dysmetabolic Disorder Obesity," "HDD Obesity," "Low Leptin Metabolic Dysfunction Spectrum," or "LMDS," is a recently-identified disorder that is characterized by leptin deficiency and

metabolic abnormalities; in some cases, HMD is also characterized by not having lipodystrophy. HMD is distinguished from other obesity-related conditions such as CLD, as CLD is associated with a specific genetic etiology, i.e., a frame-shift mutation in the leptin gene; subjects with HMD do not have this particular leptin gene mutation.

[038] HMD is also different than metabolic syndrome. Metabolic syndrome, also known as syndrome X, insulin resistance syndrome, or dysmetabolic syndrome, is a term used for a cluster of metabolic conditions associated with an increase risk of adverse outcomes, including cardiovascular disease and diabetes. There is no precise definition for metabolic syndrome, as various organizations such as the World Health Organization (WHO), the European the

European Group for the Study of Insulin Resistance (EGIR), the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP:ATPIII), the American Association for Clinical Endocrinology (AACE), and the International Diabetes Federation (IDF) have established different criteria, shown in the table below.

[039] Metabolic syndrome includes a requirement of multiple metabolic abnormalities without a single etiological cause. In contrast, HMD has a single common etiological factor (low leptin) potentially mediating the metabolic abnormalities observed, such as obesity, hyperlipidemia, and Type 2 diabetes.

Atty. Docket No. Al 19-24

Table 1. Criteria for defining metabolic syndrome for different medical organizations.


BMI=body mass index; BP=blood pressure; HDL=high density lipoprotein; T2DM=type 2 diabetes mellitus; WC=waist circumference (O'Neill et al., Obes Res, 2015, 16: 1-12).

Overweight or Obesity

[040] The present invention relates to methods of treating an above-normal weight condition in a subject who has a leptin deficiency. As used herein, "above-normal weight condition" refers to "overweight" or "obesity." "Overweight" or "obesity" may be defined using various metrics. For example, "overweight" and "obesity" may be defined based on BMI, in which "overweight" refers to a BMI of 25 kg/m2 to 29.9 kg/m2, or in certain embodiments a BMI of 25 kg/m2 to less than 30 kg/m2, or in some embodiments 27.0 kg/m2 to less than 30 kg/m2, or in other embodiments 27.5 kg/m2 to less than 30 kg/m2. "Obesity" may refer to a BMI of 30 kg/m2 or higher. Under certain classification systems, "obesity" may also refer to obesity class 1

(30 kg/m2 to 34.9 kg/m2), obesity class 2 (35 kg/m2 to 39.9 kg/m2), and/or extreme obesity class 3 (>40 kg/m2).

[041] Alternatively, "overweight" and "obesity" may be defined using other techniques known in the art, including body volume index ("BVI"), skinfold calipers, bioelectrical impedance analysis, hydrostatic weighing, dual-energy X-ray absorptiometry ("DEXA"), computerized tomography ("CT"), and magnetic resonance imaging ("MRI"). In the methods of the invention, subjects may be identified as being overweight or obese based on a pre-treatment or baseline measurements. In certain embodiments, measurements of overweight or obesity may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[042] As used herein, "pre-treatment"— or "baseline" or "endogenous"— refers to a time point prior to the first treatment (i.e. first administration) with leptin. This time point may be 8 weeks or less prior to the first treatment, such as about 6 weeks prior to the first treatment, about four weeks or about 1 month prior to the first treatment, about 2 weeks prior to the first treatment, about 1 week prior to the first treatment, or about 6, about 5, about 4, about 3, about 2, or about 1 day prior to the first treatment. In certain embodiments, the time point may be within about 24 hours prior to the first treatment, including immediately before the first treatment.

Treatment of Overweight or Obesity

[043] In certain embodiments, "treatment of or "treating" overweight or obesity may be defined as a lowering of the metric by which overweight and obesity is being measured as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken

after treatment with leptin was initiated. For instance, in embodiments in which overweight and obesity are based on BMI, "treatment of or "treating" refers to a reduction in BMI. Similarly, in embodiments in which overweight and obesity are based on BVI, "treatment of or "treating" refers to a reduction in BVI. The reduction of the metric for measuring overweight or obesity may be a percent reduction from a pre-treatment measurement, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any other percent therebetween. In certain embodiments, the reduction of the metric for measuring overweight or obesity may be a unit reduction from a pre-treatment measurement; for example, if overweight and obesity is determined using the BMI metric, the reduction may be about 0.1 kg/m2 to about 20 kg/m2, such as about 0.1 kg/m2, or about 0.2 kg/m , or about 0.3 kg/m , or about 0.4 kg/m , or about 0.5 kg/m , or about 0.6 kg/m , or about 0.7 kg/m , or about 0.8 kg/m , or about 0.9 kg/m , or about 1 kg/m , or about 2 kg/m , or about 3 kg/m2, or about 4 kg/m2, or about 5 kg/m2, or about 7 kg/m2, or about 10 kg/m2, or about 15 kg/m2, or about 20 kg/m2, or any other number therebetween.

[044] In certain embodiments, "treatment of or "treating" overweight or obesity may also be defined as a lowering of the body weight of the subject as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction in weight may be a percent reduction from a pre-treatment measurement, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any other percent therebetween. In certain embodiments, the reduction of body weight may also be a unit reduction from a pre-treatment measurement such as a reduction of about 0.5 lbs to about 400 lbs, such as about 1 lb, or about 2 lbs, or about 3 lbs, or about 4 lbs, or about 5 lbs, or about 6 lbs, or about 7 lbs, or about 8 lbs, or about 9 lbs, or about 10 lbs, or about 15 lbs, or about 20 lbs, or about 25 lbs, or about 30 lbs, or about 40 lbs, or about 50 lbs, or about 60 lbs, or about 70 lbs, or about 80 lbs, or about 90 lbs, or about 100 lbs, or about 110 lbs, or about 120 lbs, or about 130 lbs, or about 140 lbs, or about 150 lbs, or about 160 lbs, or about 170 lbs, or about 180 lbs, or about 190 lbs, or about 200 lbs, or about 300 lbs, or about 400 lbs, or any other number

therebetween. The reduction of body weight may also be a reduction from a pre-treatment measurement of about 0.1 kg to about 200 kg, such as about 0.1 kg, or about 0.5 kg, or about

1 kg, or about 2 kg, or about 3 kg, or about 4 kg, or about 5 kg, or about 6 kg, or about 7 kg, or about 8 kg, or about 9 kg, or about 10 kg, or about 11 kg, or about 12 kg, or about 13 kg, or about 14 kg, or about 15 kg, or about 16 kg, or about 17 kg, or about 18 kg, or about 19 kg, or about 20 kg, or about 25 kg, or about 30 kg, or about 35 kg, or about 40 kg, or about 45 kg, or about 50 kg, or about 55 kg, or about 60 kg, or about 65 kg, or about 70 kg, or about 75 kg, or about 80 kg, or about 85 kg, or about 90 kg, or about 95 kg, or about 100 kg, or about 150 kg, or about 200 kg, or any other number therebetween

[045] In certain embodiments, "treatment of or "treating" overweight or obesity may also include a reduction of the metric used to measure overweight or obesity that is maintained for a specified period of time. For instance, the reduction of the metric may be maintained for a period of about 1 day to about 5 years, such as a period of about 1 day, or about 1 week, or about

2 weeks, or about 3 weeks, or about 4 weeks, or about 1 month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about 6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about 11 months, or about 12 months or about

1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about

2 years, or about 3 years, or about 4 years, or about 5 years, or any other duration therebetween. Alternatively or in addition, the reduction of the metric may be at a time point that is about 1 day to about 5 years after the first treatment, such as about 1 day, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 1 month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about 6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about 11 months, or about 12 months or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years, or any other time point therebetween.

[046] In some embodiments, "treatment of or "treating" overweight or obesity may refer to chronic weight management of the subject. The administration of leptin may continue for over two years, up to the rest of the subject's life.

[047] In some embodiments, "treatment of or "treating" overweight or obesity may refer to a reduction in weight or BMI in subjects administered with leptin as compared to a reduction in

weight or BMI (if any) to subjects not administered with leptin. The subjects who are not administered with leptin may be administered a placebo, may be administered or exposed to a different overweight/obesity treatment, or may not be administered or exposed to any type of treatment. For example, "treatment of or "treating" may refer to a reduction in weight or BMI in subjects administered with leptin that is greater than the reduction in weight or BMI in subjects not administered with leptin by about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 50 %, or any other percent therebetween. Alternatively, there may be a difference in the reduction in weight or BMI in subjects administered with leptin as compared to a reduction in weight or BMI in subjects not administered with leptin of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any other percent therebetween.

[048] In some embodiments, "treatment of or "treating" overweight or obesity may be assessed by comparing the proportion of subjects administered leptin and who experienced a reduction in weight or BMI to the proportion of subjects not administered leptin and who experienced a reduction in weight or BMI. The proportion of subjects administered leptin and who experienced a reduction in weight or BMI may be about 5 % to about 90 % of those subjects, such as about 5 %, or about 10%, or about 15 %, or about 20 %, or about 25 %, or about 30 % or about 35 %, or about 40 %, or about 45 %, or about 50 %, or about 55 %, or about 60 %, or about 65 %, or about 70 %, or about 75 %, or about 80 %, or about 85 %, or about 90 %, or any other percent therebetween. The reduction in weight or BMI may be a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any other percent therebetween. For any particular reduction in weight or BMI (e.g., about 5 % reduction or more, about 10 % reduction or more, etc.), the proportion of subjects administered leptin and who experienced that reduction in weight or BMI may be greater than the proportion of subjects not administered leptin and who experienced that reduction in weight or BMI by about 25 % greater, or about 50 % or greater, or about 75 % or greater, or about 100 % or greater, or about 150 % or greater, or about 200 % (double) or greater, or about 250 % or greater, or about 300 % (triple) or greater, or about 350 % or greater, or about 400 % (quadruple) or greater, or about 450 % or greater, or about 500 % (5X) or greater, or about 600 % (6X) or greater, or about 700 % (7X) or greater, or about 800 % (8X) or greater or about 900 % (9X) or greater, or about 1000 % (10X) or greater, or any percent therebetween. In some embodiments, the difference between the proportion of subjects administered leptin and who experienced a particular reduction in weight or BMI and the proportion of subjects not administered leptin and who experienced that same reduction in weight or BMI is assessed at a certain time point after the first treatment, including about 1 day to about 5 years after the first treatment, such as about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about

I month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about

6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about

I I months, or about 12 months or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years, or any other time point therebetween.

[049] In certain embodiments, "treatment of or "treating" overweight or obesity may refer to effective weight management if after one year of treatment either of the following occurs: (a) the difference in weight loss between subjects administered leptin and subjects not administered leptin is at least about 5 % and the difference is statistically significant; or (b) the proportion of subjects administered leptin who lose greater than or equal to 5 % of baseline body weight is at least about 35 % and is approximately double the proportion of subjects not administered leptin and who lose greater than or equal to 5 % of baseline body weight, and the difference in weight loss between the subjects administered leptin and the subjects not administered leptin is statistically significant.

[050] For the definitions of "treatment of or "treating" described herein, the metrics used to assess reductions in weight or BMI may be mean values.

Low Leptin

[051] As used herein, a "leptin deficiency," "leptin-deficient," "hypoleptinemia," or

"hypoleptinemic" may refer to a condition in which the endogenous concentration of leptin in the subject is considered to be below normal, i.e., a low leptin concentration. In some embodiments, the concentration may be defined by a serum concentration below a specified threshold such as,

for example, a serum concentration within a specified percentile based on the NHANES III. In some embodiments, some concentrations may be described by those skilled in the art as "very low" or "ultra-low" leptin levels.

[052] In certain embodiments, a low leptin concentration may be defined by a serum concentration that is about 16 ng/mL or below for a female and about 5 ng/mL or below for a male; a serum concentration that is about 8 ng/mL or below for a female and about 3 ng/mL or below for a male; or a serum concentration that is about 5 ng/mL or below for a female, and about 2 ng/mL or below for a male. In some embodiments, the serum concentration may additionally be non-zero, i.e., a detectable amount or above a specified threshold, such as above about 0.1 ng/mL, or about 0.5 ng/mL, or about 1 ng/mL, or about 1.5 ng/mL, or about 2 ng/mL. A "detectable" amount of leptin refers to an amount or concentration, such as in the serum, that can be measured using methods known in the art.

[053] In certain embodiments, a low leptin concentration may be defined by a serum concentration within about the 15th percentile, or about the 10th percentile, or about the 8th percentile, or about the 5th percentile, or about the second percentile, or about the first percentile per NHANES III. In some embodiments, the serum concentration may additionally be above a specified percentile, such as above a half percentile, or the first percentile, or the second percentile, per NHANES III.

[054] In certain embodiments, a low leptin concentration may be defined by a serum concentration that is within a specified percentile adjusted for BMI, gender, and/or other factors. For instance, a low leptin concentration may be defined by a serum concentration within about the 10th percentile, or about the 5th percentile, or about the 3rd percentile, or about the second percentile, or about the first percentile per NHANES III, adjusted for BMI. As an example, about the 10th percentile of leptin concentration may refer to subjects whose leptin concentration is in the 10th percentile according to NHANES III for subjects with a BMI of 30-35.

[055] Leptin concentration can be measured using methods and techniques known in the art. In some embodiments, leptin concentration may be measured in body fluids other than serum, such as urine, whole blood, cerebral spinal fluid, and plasma. Serum leptin may be measured using

Enzyme-Linked Immunosorbent Assay ("ELISA") using commercially-available kits, such as Millipore ELISA. In other embodiments, leptin levels may be measured using a

radioimmunoassay technique.

[056] In some embodiments, leptin concentration may be assessed prior to the start of the treatment with leptin, such as a pre-treatment leptin concentration measurement. It is recognized by a person of ordinary skill in the art that the levels of leptin in a person may change based on the time of the day and, in certain embodiments, the leptin levels are determined in the morning after fasting.

Weight-Related Comorbidities

[057] The present invention may relate to methods of treating overweight or obesity in subjects who have a leptin deficiency and who have one or more weight-related comorbidities.

[058] The comorbidities may include, but are not limited to, prediabetes or type 2 diabetes mellitus; dyslipidemia; hypertension; cardiovascular disease, based on standardized MedDRA queries (SMQs), ischemic heart disease, cardiac failure, central nervous system hemorrhages, cerebrovascular conditions, embolic and thrombotic events; cancer, including but not limited to cancer of the uterus, cervix, endometrium, ovaries, breast, colon, rectum, esophagus, liver, gallbladder, pancreas, kidney and prostate; breathing disorders, including but not limited to sleep apnea; gallbladder disease; gynecological problems, such as infertility and irregular periods; erectile dysfunction and sexual health issues, liver dysfunction, liver inflammation, and/or liver damage, including nonalcoholic fatty liver disease; osteoarthritis; or a combination thereof.

[059] In embodiments of the invention, the comorbidity is, or includes, type 2 diabetes mellitus. The type 2 diabetes mellitus may be associated with an elevated hemoglobin Ale, which is hemoglobin coated with sugar, i.e., glycated, and is typically tested to diagnose type 1 and type 2 diabetes and/or to gauge how well the diabetes is being managed. Methods for measuring hemoglobin Ale are known in the art, and the test results are typically presented as a percentage of hemoglobin that is glycated. In some embodiments, an "elevated" level of hemoglobin Ale may be about 6 % or higher, such as about 6.5 % or higher, or about 7 % or higher, or about 7.5 % or higher, or about 8 % or higher, or any percentage therebetween. In the methods of the invention, subjects may be identified as having elevated hemoglobin Ale based on a pre-

treatment measurement. In certain embodiments, measurements of hemoglobin Ale may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[060] In certain embodiments, administration of leptin may reduce hemoglobin Ale as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 35 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 35 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction, i.e., a decrease in the percent of glycated hemoglobin, of about 0.1 % to about 5 %, such as about 0.1 %, or about 0.2 %, or about 0.3 %, or about 0.4 %, or about 0.5 %, or about 0.6 %, or about 0.7 %, or about 0.8 %, or about 0.9 %, or about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or any other percent therebetween.

Administration of leptin may also result in a reduction in hemoglobin Ale that is maintained during treatment for a specified period of time, such as a period of about 1 day to about 5 years, for example, a period of about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 1 month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about 6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about 11 months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[061] In certain embodiments, the present invention relates to a method of treating prediabetes or type 2 diabetes mellitus in an overweight or obese subject who has a leptin deficiency, comprising administering leptin. Administration of leptin can lower hemoglobin Ale, therefore treating prediabetes or type 2 diabetes mellitus.

[062] In embodiments of the invention, the comorbidity is, or includes, dyslipidemia. The dyslipidemia may be associated with elevated triglyceride level, elevated low-density lipoprotein (LDL) cholesterol level, elevated total cholesterol level, or a combination thereof.

[063] Methods for measuring triglycerides are known in the art, and are typically performed as a blood test after the subject has fasted (for example, 8 to 12 hours). In some embodiments, an "elevated" level of triglycerides may be about 150 mg/dL or higher, such as about 200 mg/dL or higher, or about 250 mg/dL or higher, or about 300 mg/dL or higher, or about 350 mg/dL or higher, or about 400 mg/dL or higher. In the methods of the invention, a subject may be identified as having an elevated triglyceride level based on a pre-treatment measurement. In certain embodiments, measurements of triglyceride level may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[064] Methods for measuring LDL cholesterol are known in the art, and are typically performed as a blood test after the subject has fasted. In some embodiments, an "elevated" level of LDL cholesterol may be about 130 mg/dL or higher, such as about 140 mg/dL or higher, or about 150 mg/dL or higher, or about 160 mg/dL or higher, or about 170 mg/dL or higher, or about 180 mg/dL or higher, or about 190 mg/dL or higher, or about 200 mg/dL or higher. In the methods of the invention, a subject may be identified as having an elevated LDL cholesterol level based on a pre-treatment measurement. In certain embodiments, measurements of LDL cholesterol level may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[065] Methods for testing total cholesterol are known in the art, and are typically performed as a blood test after the subject has fasted. In some embodiments, an "elevated" level of total cholesterol may be about 200 mg/dL or higher, such as about 200 mg/dL or higher, or about 240 mg/dL or higher, or about 260 mg/dL or higher, or about 280 mg/dL or higher, or about 300 mg/dL or higher, or about 320 mg/dL or higher, or about 340 mg/dL or higher, or about 350 mg/dL or higher. In the methods of the invention, a subject may be identified as having an elevated total cholesterol level based on a pre-treatment measurement. In certain embodiments, measurements of total cholesterol level may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[066] In certain embodiments, administration of leptin may reduce the triglyceride level as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction of about 1 mg/dL to about 300 mg/dL, such as about 10 mg/dL, or about 20 mg/dL, or about 30 mg/dL, or about 40 mg/dL, or about 50 mg/dL, or about 60 mg/dL, or about 70 mg/dL, or about 80 mg/dL, or about 90 mg/dL, or about 100 mg/dL, or about 110 mg/dL, or about 120 mg/dL, or about 130 mg/dL, or about 140 mg/dL, or about 150 mg/dL, or about 160 mg/dL, or about 170 mg/dL, or about 180 mg/dL, or about 190 mg/dL, or about 200 mg/dL, or about 220 mg/dL, or about 240 mg/dL, or about 260 mg/dL, or about 280 mg/dL, or about 300 mg/dL, or any other value therebetween. Administration of leptin may also result in a reduction of triglyceride level that is maintained during treatment for a specified period of time, such as a period of about 1 day to about 5 years, for example, a period of about 1 day, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about

I month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about

6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about

I I months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[067] In certain embodiments, administration of leptin may reduce the LDL cholesterol level as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction of about 1 mg/dL to about 100 mg/dL, such as about 1 mg/dL, or about 5 mg/dL, or about 10 mg/dL, or about 15 mg/dL, or about 20 mg/dL, or about 25 mg/dL, or about 30 mg/dL, or about 35 mg/dL, or about 40 mg/dL, or about

45 mg/dL, or about 50 mg/dL, or about 55 mg/dL, or about 60 mg/dL, or about 65 mg/dL, or about 70 mg/dL, or about 75 mg/dL, or about 80 mg/dL, or about 85 mg/dL, or about 90 mg/dL, or about 95 mg/dL, or about 100 mg/dL, or any other value therebetween. Administration of

leptin may also result in a reduction of LDL cholesterol level that is maintained during treatment for a specified period of time, such as a period of about 1 day to about 5 years, such as a period of about 1 day, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about

I month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about

6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about

I I months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[068] In certain embodiments, administration of leptin may reduce the total cholesterol level as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction of about 1 mg/dL to about 200 mg/dL, such as about 1 mg/dL, or about 5 mg/dL, or about 10 mg/dL, or about 20 mg/dL, or about 30 mg/dL, or about 40 mg/dL, or about 50 mg/dL, or about 60 mg/dL, or about 70 mg/dL, or about 80 mg/dL, or about 90 mg/dL, or about 100 mg/dL, or about 1 10 mg/dL, or about 120 mg/dL, or about 130 mg/dL, or about 140 mg/dL, or about 150 mg/dL, or about 160 mg/dL, or about 170 mg/dL, or about 180 mg/dL, or about 190 mg/dL, or about 200 mg/dL, or any other value therebetween. Administration of leptin may also result in a reduction of total cholesterol level that is maintained during treatment for a specified period of time, such as period of about 1 day to about 5 years, such as a period of about 1 day, or about 1 week, or about 2 weeks, or about

3 weeks, or about 4 weeks, or about 1 month, or about 2 months, or about 3 months, or about

4 months, or about 5 months, or about 6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about 11 months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[069] In certain embodiments, the present invention relates to a method of treating

atherosclerosis and other associated negative cardiovascular outcomes (e.g. stroke) in an

overweight or obese subject who has a leptin deficiency, comprising administering leptin.

Administration of leptin can lower triglyceride level, lower LDL cholesterol level, lower total cholesterol level, or a combination thereof, therefore treating atherosclerosis and other associated negative cardiovascular outcomes.

[070J In embodiments of the invention, the comorbidity is, or includes, liver dysfunction. Poor liver health may be associated with elevated alanine aminotransferase (ALT) level, elevated aspartate aminotransferase (AST) level, or a combination thereof. In particular, nonalcoholic fatty liver disease may be associated with an AST:ALT ratio of greater than about 1.

[071] ALT is an enzyme involved in amino acid metabolism and is found predominantly in the liver. ALT is most commonly used to evaluate hepatocellular injury and to determine liver health. Methods for testing ALT are known in the art, and are typically performed as a blood test. In some embodiments, an "elevated" level of ALT may be about 25 U/L or higher for females, such as about 35 U/L or higher; and about 25 U/L or higher for males, such as about 35 U/L or higher. In the methods of the invention, a subject may be identified as having an elevated ALT level based on a pre-treatment or baseline measurement. In certain embodiments, measurements of ALT level may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[072] AST is an enzyme involved in amino acid metabolism and is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, and red blood cells. AST is regarded as an indicator of liver health, although AST levels may be elevated in diseases affecting other organs, such as myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, musculoskeletal diseases, and trauma. Methods for testing AST are known in the art, and are typically performed as a blood test. In some embodiments, an "elevated" level of AST may be about 20 U/L or higher, such as about 30 U/L or higher, or about 40 U/L or higher, or about 0 U/L or higher, or about 60 U/L or higher. In the methods of the invention, a subject may be identified as having an elevated AST level based on a pre-treatment measurement. In certain embodiments, measurements of AST level may be taken at one or more time points after treatment with leptin is initiated, for example, at about 1 week, at about 2 weeks, at about 3 weeks, etc.

[073] In certain embodiments, administration of leptin may reduce the ALT level as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction of about 1 U/L to about 20 U/L, such as about 1 U/L, or about 2 U/L, or about 3 U/L, or about 4 U/L, or about 5 U/L, or about 6 U/L, or about 7 U/L, or about 8 U/L, or about 9 U/L, or about 10 U/L, or about 11 U/L, or about 12 U/L, or about 13 U/L, or about 14 U/L, or about 15 U/L, or about 16 U/L, or about 17 U/L, or about 18 U/L, or about 19 U/L, or about 20 U/L, or any other value therebetween. Administration of leptin may also result in a reduction of ALT level that is maintained during treatment for a specified period of time, such as a period of about 1 day to about 5 years, such as a period of about 1 day, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about

I month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about

6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about

I I months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[074] In certain embodiments, administration of leptin may reduce the AST level as compared to a pre-treatment measurement or, in some embodiments, a prior measurement taken after treatment with leptin was initiated. The reduction may be a percent reduction, for example, a percent reduction of about 1 % to about 50 %, such as about 1 %, or about 2 %, or about 3 %, or about 4 %, or about 5 %, or about 10 %, or about 15 %, or about 20 %, or about 25 %, or about 30 %, or about 40 %, or about 50 %, or any percent reduction therebetween. In certain embodiments, the reduction may be a unit reduction of about 1 U/L to about 20 U/L, such as about 1 U/L, or about 2 U/L, or about 3 U/L, or about 4 U/L, or about 5 U/L, or about 6 U/L, or about 7 U/L, or about 8 U/L, or about 9 U/L, or about 10 U/L, or about 11 U/L, or about 12 U/L, or about 13 U/L, or about 14 U/L, or about 15 U/L, or about 16 U/L, or about 17 U/L, or about 18 U/L, or about 19 U/L, or about 20 U/L, or any other value therebetween. Administration of

leptin may also result in a reduction of ALT level that is maintained during treatment for a specified period of time, such as a period of about 1 day to about 5 years, such as a period of about 1 day, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 1 month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about 6 months, or about 7 months, or about 8 months, or about 9 months, or about 10 months, or about 11 months, or about 12 months, or about 1 year, or about 15 months, or about 18 months, or about 24 months, or about 1.5 years, or about 2 years, or about 3 years, or about 4 years, or about 5 years.

[075] In certain embodiments, the present invention relates to a method of treating liver dysfunction, liver inflammation, and/or liver damage in an overweight or obese subject who has a leptin deficiency, comprising administering leptin. Administration of leptin can lower ALT level, AST level, or a combination thereof, which may be indicative of treatment of liver dysfunction, liver inflammation, and/or liver damage.

[076] In some embodiments, the present invention relates to a method of treating nonalcoholic fatty liver disease in an overweight or obese subject who has a leptin deficiency, comprising administering leptin. Administration of leptin can lower AST level, which may be indicative of treatment of nonalcoholic fatty liver disease.

Leptin for Administration to Subjects

[077] As used herein, the "leptin" used to administer to subjects according to the invention encompasses naturally occurring human, mouse, rat, and other heterologous species leptins, as well as recombinantly produced mature leptin. For example, mature human leptin is a 146-amino acid polypeptide hormone, which may be represented by the following amino acid sequence (SEQ ID NO: 1):

VPIQKVQDDT TLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIP

GLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFS KSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLS PGC, wherein: Xaa at position 27 is T or A; and Xaa at position 28 is Q or absent.

[078] "Leptin" may also encompass analogs of leptin, which as used herein refers to a compound that has insertions, deletions and/or substitutions of amino acids relative to leptin. In some embodiments, an analog can have at least 50%, for example 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 %, or even higher, sequence identity to leptin. An example is metreleptin (i.e., r-metHuLeptin), which is a 147-amino acid leptin analog generated by the genetically engineered N-terminal addition of a methionine to the N-terminal amino acid of the 146-amino acid, mature, circulating, human leptin. Metreleptin may be represented by the following amino acid sequence (SEQ ID NO: 2):

MVPIQKVQDDTKTLI TIVTRINDISH-Xaa-Xaa-SVSSKQ VTGLDFI

PGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAF S SCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDL SPGC, wherein: Xaa at position 28 is T or A; and Xaa at position 29 is Q or absent.

[079] In addition, "leptin" may encompass biologically active fragments, agonist, agonist analogs, variants, fusion proteins, and other derivatives thereof, such as those compounds disclosed in U.S. Patent No. 5,521,283, U.S. Patent No. 5,532,336, U.S. Patent No. 5,552,522, U.S. Patent No. 5,552,523, U.S. Patent No. 5,552,524, U.S. Patent No. 5,554,727, U.S. Patent No. 5,559,208, U.S. Patent No. 5,580,954, U.S. Patent No. 5,594,101, U.S. Patent No. 5,691,309, U.S. Patent No. 5,756,461, U.S. Patent No. 5,851,995, U.S. Patent No. 5,935,810, U.S. Patent No. 6,001,968, U.S. Patent No. 6,309,853, U.S. Patent No. 6,309,853, U.S. Patent No. 6,350,730, U.S. Patent No. 6,420,339, U.S. Patent No. 6,429,290, U.S. Patent No. 6,541,033, U.S. Patent No. 6,936,439, U.S. Patent No. 7,112,659, U.S. Patent No. 7,183,254, U.S. Patent No. 7,208,577, U.S. Patent No. 8,080,254, U.S. Patent No. 8,394,765, U.S. Publication No. 2016/0083446, PCT Publication No. WO 00/09165, PCT Publication No. WO 00/20872, PCT Publication No.

WO 00/21574, PCT Publication No. WO 00/47741, PCT Publication No. WO 04/39832, PCT Publication No. WO 09/64298, PCT Publication No. WO 96/05309, PCT Publication No.

WO 96/22308, PCT Publication No. WO 96/23517, PCT Publication No. WO 96/40912, PCT Publication No. WO 97/02004, PCT Publication No. WO 97/06816, PCT Publication No.

WO 97/18833, PCT Publication No. WO 97/38014, PCT Publication No. WO 98/08512, PCT Publication No. WO 98/12224, PCT Publication No. WO 98/28427, PCT Publication No.

WO 98/46257, and PCT Publication No. WO 98/55139; each of which is hereby incorporated by reference in its entirety and for all purposes. Leptin is the polypeptide product, for example, of the ob gene as described in the International Publication No. WO 96/05309, and U.S. Patent No. 6,309,853, each of which is incorporated herein by reference in its entirety.

[080] In some embodiments, the present invention may relate to leptin as a conjugate, such as those conjugates disclosed in U.S. Publication No. 2012/0149636, U.S. Publication No.

2016/0137709, and U.S. Publication No. 2016/0207974, each of which is incorporated herein by reference in its entirety.

Leptin Pharmaceutical Compositions

[081] The leptin administered to subjects according to the invention may be in a pharmaceutical composition. The pharmaceutical composition may comprise, in addition to leptin, one or more pharmaceutically acceptable excipients or additives that modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable excipients or additives include, but are not limited to, amino acids (including, but not limited to, glycine, glutamine, asparagine, arginine, or lysine); antimicrobials; antioxidants (including, but not limited to, ascorbic acid, sodium sulfite, or sodium hydrogen sulfite); buffers (including, but not limited to, borate, bicarbonate, Tris HC1, citrates, phosphates, or other organic acids); bulking agents (including, but not limited to, mannitol or glycine), chelating agents (including, but not limited to, ethylenediamine tetraacetic acid (EDTA)); complexing agents (including, but not limited to, caffeine, polyvinylpyrrolidone, beta cyclodextrin, or hydroxypropyl beta

cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (including, but not limited to, glucose, mannose, or dextrins); proteins (including, but not limited to, serum albumin, gelatin, or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (including, but not limited to, polyvinylpyrrolidone); low molecular weight polypeptides; salt forming counterions (including, but not limited to, sodium); preservatives (including, but not limited to, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (including, but not limited to, glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (including, but not limited to, mannitol or sorbitol);

suspending agents; surfactants or wetting agents (including, but not limited to, pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine,

lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (including, but not limited to, alkali metal halides (in one aspect, sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; and/or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990).

[082] In embodiments of the invention, leptin may be formulated in a composition

comprisinglO mM glutamic acid, 2% glycine, 1% sucrose, and 0.01% polysorbate 20, and a pH of 4.25. The composition may be in the form of a lyophilized cake containing 11.3 mg of leptin, and stored or housed in a suitable container or packaging as known in the art. In some embodiments, the composition may be stored in glass vials, such as a 5 mL USP Type I glass vials with bromobutyl rubber stoppers, and aluminum seals with plastic flip-off caps. The leptin may be stored refrigerated (between 36 °F and 46 °F, i.e., 2 °C and 8 °C) and protected from light.

[083] Upon reconstitution with 2.2 mL of bacteriostatic-water-for-injection (BWFI) or water-for-injection (WFI), the cake yields a 5 mg/mL concentration of leptin. Once reconstituted, the drug may be administered immediately (e.g., within three hours after reconstitution).

Alternatively, the drug may be used within three days when stored in the refrigerator between 36 °F and 46 °F (2 °C and 8 °C) and protected from light. Before injection, study medication may be allowed to reach room temperature (15 °C to 30 °C).

Treatments with Leptin

[084] Aspects of the present invention relate to methods of (a) treating overweight or obesity in subjects who have a leptin deficiency; or (b) treating overweight or obesity in subjects who have a leptin deficiency and who have one or more weight-related comorbidities; or (c) treating prediabetes or type 2 diabetes mellitus in an overweight or obese subject who has a leptin deficiency; or (d) treating atherosclerosis and other associated negative cardiovascular outcomes (e.g. stroke) in an overweight or obese subject who has a leptin deficiency; or (e) treating liver dysfunction, liver inflammation, and/or liver damage in an overweight or obese subject who has a leptin deficiency; or (f) treating overweight or obese subjects with HMD; or (g) treating HMD; or (h) a combination thereof. The methods may comprise administering leptin or a

pharmaceutical composition comprising leptin, as described herein.

[085] Aspects of the present invention relate to the use of leptin, or a pharmaceutical composition comprising leptin, for the preparation of a medicament for (a) treating overweight or obesity in subjects who have a leptin deficiency; or (b) treating overweight or obesity in subjects who have a leptin deficiency and who have one or more weight-related comorbidities; or (c) treating prediabetes or type 2 diabetes mellitus in an overweight or obese subject who has a leptin deficiency; or (d) treating atherosclerosis and other associated negative cardiovascular outcomes (e.g. stroke) in an overweight or obese subject who has a leptin deficiency; or (e) treating liver dysfunction, liver inflammation, and/or liver damage in an overweight or obese subject who has a leptin deficiency; or (f) treating overweight or obese subjects with HMD; or (g) treating HMD; or (h) a combination thereof. The treatments may comprise administering leptin or a pharmaceutical composition comprising leptin, as described herein.

[086] Aspects of the present invention also relate to leptin, or a pharmaceutical composition comprising leptin, for use in: (a) treating overweight or obesity in subjects who have a leptin deficiency; or (b) treating overweight or obesity in subjects who have a leptin deficiency and who have one or more weight-related comorbidities; or (c) treating prediabetes or type 2 diabetes mellitus in an overweight or obese subject who has a leptin deficiency; or (d) treating atherosclerosis and other associated negative cardiovascular outcomes (e.g. stroke) in an overweight or obese subject who has a leptin deficiency; or (e) treating liver dysfunction, liver inflammation, and/or liver damage in an overweight or obese subject who has a leptin deficiency; or (f) treating overweight or obese subjects with HMD; or (g) treating HMD; or (h) a combination thereof. The treatments may comprise administering leptin or a pharmaceutical composition comprising leptin, as described herein.

[087] Further, aspects of the present invention relate to the use of leptin, or a pharmaceutical composition comprising leptin, for: (a) treating overweight or obesity in subjects who have a leptin deficiency; or (b) treating overweight or obesity in subjects who have a leptin deficiency and who have one or more weight-related comorbidities; or (c) treating prediabetes or type 2 diabetes mellitus in an overweight or obese subject who has a leptin deficiency; or (d) treating atherosclerosis and other associated negative cardiovascular outcomes (e.g. stroke) in an overweight or obese subject who has a leptin deficiency; or (e) treating liver dysfunction, liver inflammation, and/or liver damage in an overweight or obese subject who has a leptin

deficiency; or (f) treating overweight or obese subjects with HMD; or (g) treating HMD; or (h) a combination thereof. The treatments may comprise administering leptin or a pharmaceutical composition comprising leptin, as described herein.

[088] In some embodiments, the methods of the invention are to provide chronic weight maintenance in subjects who have (a) pre-treatment low leptin, and (b) either:

(i) pre-treatment BMI of >30 kg/m2 (obese), or

(ii) pre-treatment BMI of >27 kg/m2 (overweight) in the presence of at least one weight- related comorbid condition.

In certain embodiments, the low leptin comprise a leptin level of <16 ng/mL for females and <5 ng/mL for males, or by a leptin level of <8 ng/mL for females and <3 ng/mL for males.

[089] In some embodiments, the methods of the invention are to achieve and/or maintain weight loss in subjects with HMD, wherein the subjects have an initial BMI of (i) >30 kg/m2 (obese), or (ii) >27 kg/m2 (overweight) in the presence of at least one weight-related comorbid condition (e.g., hypertension, type 2 diabetes mellitus, or dyslipidemia); wherein the subjects have a leptin level of <16 ng/mL for females and <5 ng/mL for males, or a leptin level of <8 ng/mL for females and <3 ng/mL for males; and wherein the leptin level is a detectable amount.

[090] In some embodiments, HMD may be characterized as having elevated (above-normal) serum levels of apolipoprotein B (apo-B), follicle stimulating hormone (FSH), or a combination thereof. For males, HMD may be further characterized as having high cholesterol and/or hypertension. For females, HMD may be further characterized as having a higher likelihood of type 2 diabetes.

Administration of Leptin

[091] In the methods of the present invention, the leptin may be administered in amounts that are therapeutically effective. A therapeutically effective amount may include a quantity of leptin administered at a particular frequency. In some embodiments, the quantity of leptin may be about 0.001 mg/kg body weight to about 1000 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 50 mg/kg, or from about 1 mg/kg to about 20 mg/kg. In certain embodiments, the quantity of leptin may be measured in international units (IU) ranging from about 0.001 IU/kg body weight to about 1000 IU/kg, from about 0.01 IU/kg to about 100 IU/kg, from about 0.1 IU/kg to about 100 IU/kg, from about 1 IU/kg to about 100 IU/kg, from about 1 IU/kg to about 50 IU/kg, or from about 1 IU/kg to about 20 IU/kg. In other embodiments, the quantity of leptin may be a fixed dose, such as about 1 to about 200 mg, or any dose therebetween, such as about 1 mg, or about 5 mg, or about 10 mg, or about 15 mg, or about 20 mg, or about 25 mg, or about 30 mg, or about 35 mg, or about 40 mg, or about 45 mg, or about 50 mg, or about 75 mg, or about 100 mg, or about 125 mg, or about 150 mg, or about 175 mg, or about 200 mg.

[092] Regarding frequency, leptin may be administered as daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, thrice weekly, four times a week, five times a week, six times a week, weekly, monthly, semi-annually, or even twice or three times daily. On the days that the dose is administered, it may be given as a single or divided dose.

[093] A therapeutically effective amount of leptin may also be in accordance to a titration schedule that involves modifying the quantity or frequency of dosing based on a set schedule or based on the response of the subject.

[094] Administration of leptin may be oral, intravenous, subcutaneous, intranasal, inhalation, transdermal, transmucosal, or by any other route known in the art.

[095] In certain embodiments, leptin may be administered parenterally, such as intravenous, intramuscular, of subcutaneous. For intravenous administration, a composition as disclosed herein can be drawn into a syringe or filled in an intravenous infusion bottle or bag and administered to the human or animal as a bolus and/or continuous infusion. Leptin may also be administered via pen injector, auto injector, dual chamber injector, and the like.

[096] In some embodiments, leptin may be administered orally. Pharmaceutical compositions that are administered orally may comprise carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre systemic degradation is minimized. Additional agents can be included to facilitate absorption of the composition. Diluents, flavorings, low melting point waxes,

vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[097] In some embodiments, leptin may be administered by inhalation. For inhalation, leptin may be in a composition formulated as a dry powder for inhalation. Pharmaceutical composition inhalation solutions may also be formulated with a propellant for aerosol delivery or the solutions may be nebulized.

[098] Other routes of administering leptin include intraperitoneal, intracerebral

(intraparenchymal), intracerebroventricular, intraocular, intraarterial, intraportal, intralesional routes, intraarticular, intratumor, cerebrospinal, intrarectal and colon, topical, subconjunctival, intrabladder, intravaginal, epidural, intracostal, intradermal, transdermal, transserosal, intrabuccal, intranasal, dissolution in the mouth or other body cavities, instillation to the airway, insufflation through the airway, injection into vessels, tumors, organ and the like, and injection or deposition into cavities in the body of a mammal.

Subjects

[099] As described above, the methods of the invention involve administering leptin to subjects in need thereof. The subjects may be overweight or obese, and have a leptin deficiency. In some embodiments, the subjects may have a comorbidity.

[0100] In some embodiments, the subjects of the present invention may have HMD.

[0101] In some embodiments, subjects of the present invention may have previously undergone other treatments to lower weight and/or reduce BMI. Such treatments include, but are not limited to, diets (e.g., volumetrics, vegan, etc., or commercial diets such as Weight Watchers®, Jenny Craig®, etc.), exercise, drugs (e.g., diethylpropion (Tenuate®), phentermine (Adipex-P®), benzphetamine
phendimetrazine, orlistat (Xenical®), lorcaserin (Belviq®), phentermine-topiramate (Qsymia®), naltrexone-bupropion (Contrave®), liraglutide (Saxenda®, etc.), and bariatric surgery (e.g., gastric bypass, sleeve gastrectomy, adjustable gastric band, and biliopancreatic diversion with duodenal switch, etc.).

[0102] In some embodiments, the subject does not have other medical conditions that have been associated with leptin deficiency, including, but not limited to, lipodystrophy syndromes, hypothalamic amenorrhea, and congenital leptin deficiency.

[0103] In certain embodiments, the subject does not have common obesity.

[0104] In some embodiments, the subject pre-treatment does not have high insulin levels. High insulin levels may be regarded as a fasting insulin level of greater than about 8 μΐυ/mL, or greater than about 10 μΐυ/mL, or greater than about 12 μΐυ/mL.

[0105] In certain embodiments, the subject pre-treatment does not have impaired glucose tolerance. Impaired glucose tolerance may be regarded as having a plasma glucose concentration of 140 to 200 mg/dL (7.8 mmol/1 to 1 1.1 mmol/1) two hours after the 75-g oral glucose tolerance test, and/or a fasting blood glucose of less than 7 mmol/L. Impaired glucose tolerance may also be regarded as having a glycated haemoglobin (HbAlc) blood test level of 42 to 47 mmol/mol (6.0 % to 6.5 %).

[0106] In some embodiments, then subject pre-treatment does not have does not have two or more of the following: (i) high triglycerides or high HDL cholesterol, (ii) high blood pressure, or (iii) high fasting glucose level. High triglycerides may be as discussed above (e.g., fasting level of about 150 mg/dL or higher). High HDL cholesterol may be regarded as a fasting level of greater than about 35 mg/dL. In certain embodiments, high HDL cholesterol may differ between genders. For example, high HDL cholesterol for a male may be greater than about 35 mg/dL, or greater than about 40 mg/dL. High HDL cholesterol for a female may be greater than about 39 mg/dL, or greater than about 50 mg/dL. High blood pressure may be regarded as greater than about 120 mm Hg over about 80 mm Hg, or greater than about 130 mm Hg over about 85 mm Hg, or greater than about 140 mm Hg over about 90 mm Hg.

[0107] In some embodiments, the subject does not have metabolic syndrome, which, as described above, has been defined using different criteria by different health organizations (see Table 1) (O'Neill et al., Obes Rev, 2015, 16: 1-12).

[0108] In some embodiments, the subject does not exhibit leptin resistance. Leptin resistance may be caused by a variety of factors, including (i) a failure of circulating leptin to cross the blood-brain-barrier and reach its neuron targets in the brain, (ii) an inhibition of the leptin signaling cascade within neurons in specific brain areas, (iii) a "defensive" decrease in the expression of leptin receptors, and (iv) a desensitization of cellular downstream signaling at central and peripheral level; and may be affected by a variety of factors including inflammation or oxidative stress processes, and the type of diet. (Sainz et al., Metab Clin Exp, 2015, 64: 35-46). Leptin resistance can be assessed based on knowledge in the art. (Id.)

[0109] In embodiments of the invention, the subject is an animal, such as a mammal. In some embodiments, the subject is a human, for example, an adult human.

EXAMPLES

Example 1

[0110] The following describes a post hoc analysis of five Phase 2 studies that involved subjects who were overweight or obese and were administered metreleptin as 20 mg or 10 mg daily doses. The analysis compared mean percent loss of body weight and the proportion of subjects who lost >5 % and >10 % of baseline body weight between the metreleptin and placebo groups. Based on NHANES III, 3 groups of overweight/obese subjects with the following baseline leptin levels were analyzed:

• NHANES III ~10th percentile: <16 ng/mL for females, <5 ng/mL for males

("low baseline leptin level");

• NHANES III -2nd percentile: <8 ng/mL for females, <3 ng/mL for males

("lower baseline leptin level"); and

• NHANES III -1st percentile: <5 ng/mL for females, <2 ng/mL for males

("lowest baseline leptin level").

[0111] These analyses included data from 319 of the 1135 subjects treated across the 5 studies. The majority of the analyses were conducted to Week 12 of treatment with limited data available for Weeks 16 and 24.

[0112] The analysis did not include an imputation method to account for any missing data points. If a subject did not have a value at a specific visit (for example, at week 12 or week 24,) then no data was analyzed for that subject at that visit.

[0113] Table 2 presents mean weight at baseline and at Week 12, and the percent change in weight from baseline to Week 12, grouped by baseline leptin level, across the 5 studies. Results are presented separately for subjects who received 20 mg metreleptin, 10 mg metreleptin, or

placebo. The difference in percent change between the metreleptin dose groups and placebo is displayed graphically in Figure 1.

[0114] The data show that treatment with metreleptin at a dose of 20 mg for 12 weeks led to greater weight loss than placebo across all categories of baseline leptin levels. Mean percent changes in weight to Week 12 in the 20 mg metreleptin group increased as the baseline leptin levels decreased: -3.7 %, -6.5 % and -10.5 % for the baseline leptin in the 10th, 2nd, and 1 st percentile groups, respectively. The difference between the 20 mg metreleptin group and placebo was statistically significant for the low (10th percentile) and lowest (1st percentile) baseline leptin level groups (see Figure 2), while the difference in the lower (2nd percentile) baseline leptin level group (-6.5 % and -3.4 % for 20 mg and placebo, respectively) did not reach statistical significance (see Figure 3). Notably, these effects from administering metreleptin to subjects in the lowest (1 st percentile) baseline leptin level groups were not evident in subjects with a "normal" pre-treatment leptin concentration above 5 ng/mL for females, 2 ng/mL for males (see Figure 4). Moreover, these effects from administering metreleptin to subjects in the lower (2nd percentile) baseline leptin level groups also were not evident in subjects with a "normal" pre-treatment leptin concentration above 8 ng/mL for females, 3 ng/mL for males (see Figure 5).

[0115] Treatment with metreleptin at a dose of 10 mg led to significantly greater weight loss than placebo for the low (10th percentile) and lowest (1st percentile) baseline leptin level groups. A dose response was also apparent in the lower (2nd percentile) baseline leptin level group, although the percent reduction in weight for subjects receiving the 10-mg metreleptin dose was not as high as that observed in subjects receiving the 20-mg metreleptin dose.

[0116] Table 3 shows a responder analysis across the five studies. The analysis reveals that, across all baseline leptin levels, the proportion of subjects who achieved a >5 % reduction in weight at Week 12 was greater for subjects who received metreleptin 20 mg compared to those who received placebo. Consistent results were noted for a >10 % reduction in weight at this time point. Further, the 10 mg-dose group also showed a higher proportion of subjects who achieved these weight-loss goals compared to placebo at both time points in most baseline leptin levels. The number of subjects with data at Week 24 was limited; however, in general, a higher

proportion of subjects who received 20 mg daily of metreleptin achieved these weight loss goals at this time point compared to Week 12.

[0117] Table 4 shows the change in weight at Week 12 by BMI, which suggests that subjects with lower BMI may be more responsive to the metreleptin treatment. In addition, Table 5 shows the change in weight at Week 12 by gender, which suggests that females may be more responsive to metreleptin than males.

[0118] The results of the analysis described above were surprising, as it was not expected that daily administration of 20 mg metreleptin or 10 mg metreleptin would result in a significantly greater reduction in body weight of overweight and obese subjects with low leptin levels as compared to placebo, especially in light of the knowledge in the art that suggested leptin may not be helpful in treating subjects who are overweight/obese.

Table 2. Summary of Change in Weight from Baseline to Week 12 by Baseline Leptin Levels and Treatment Group Across the 5 Studies of Overweight and Obese Subjects.


Abbreviations: MET = metreleptin; PBO = placebo; SD = standard deviation; Wt = weight

aP -value from a 2-sample t-test comparing mean percent change from baseline between placebo and metreleptin.

Table 3. Responder Analysis: Subjects with >5% and >10% Loss in Weight from Baseline to Weeks 12 and 24 by Baseline Leptin Levels and Treatment Group Across the 5 Studies of Overweight and Obese Subjects.


Abbreviations: MET = metreleptin; Wt = weight

Table 4. Summary of Change in Weight from Baseline to Week 12 by Baseline Leptin Levels and BMI Across the 5 Studies of Overweight and Obese Subjects.


Table 5. Summary of Change in Weight from Baseline to Week 12 by Baseline Leptin Levels. Treatment Group, and Gender Across the 5 Studies of Overweight and Obese Subjects.

Example 2

[0119] The following describes a post hoc analysis of four of the five studies described in Example 1 involving subjects who were overweight or obese and were administered metreleptin. The analysis compared mean percent loss of body weight in subjects administered either 20 mg or 10 mg of metreleptin and having low (~10th percentile), lower (-2nd percentile), or lowest (-1st percentile) baseline leptin levels.

[0120] A total of 1064 subjects participated in the four studies, of which 396 subjects met the threshold baseline leptin levels and were included in this analysis. Baseline weight was generally comparable among leptin levels and treatment groups, as shown in Table 6.

[0121] Change from baseline in weight was analyzed using a mixed-effects model for repeated measures, with the percent change from baseline in weight as a dependent variable, treatment groups as factors, and baseline leptin levels as fixed covariates. Missing data at all visits were imputed with the Markov Chain Monte Carlo multiple imputation method to create 100 imputed data sets. For instances in which weight was missing, the median value across the imputed datasets for each subject was used in the summary.

[0122] Administration of metreleptin 10 mg and 20 mg daily decreased weight in adults with low leptin levels, with weight loss being greater at the higher versus lower metreleptin dose. Subjects with the lowest baseline leptin levels (-1st percentile) achieved the greatest weight loss.

[0123] Figure 6 compares weight loss in subject administered the 20 mg metreleptin dose among the three threshold baseline leptin levels. Statistically significant decreases were observed after week 10 for subjects having low (~10th percentile) and lowest (-1st percentile) baseline leptin levels.

Table 6. Baseline weight of subjects from the post hoc analysis of Example 2.


Data are shown as mean (standard deviation)

Example 3

[0124] The following describes a post hoc analysis of a study that involved subjects who were overweight or obese and were administered metreleptin. This study, which was assigned the name LEPT-980236, was one of the five studies analyzed in Example 1. The analysis compared mean percent loss of body weight in subjects administered either 20 mg or 10 mg of metreleptin and having baseline leptin levels in the NHANES III ~10th percentile (<16 ng/mL for females, <5 ng/mL for males). The study was conducted in overweight and obese subjects (BMI of 27.5-29.9 kg/m2 with a history of obesity-related risk factors, or a BMI of 30.0-38.0 kg/m2).

[0125] The analysis did not include an imputation method to account for any missing data points. If a subject did not have a value at a specific visit (for example, at week 12 or week 24,) then no data was analyzed for that subject at that visit.

[0126] A total of 267 subjects were enrolled, and 67 % and 44 % of subjects completed 12 and 24 weeks of treatment, respectively. Demographics and baseline characteristics were similar among treatment groups as shown in Table 7. The mean (standard deviation) baseline leptin level was 14.2 (13.3) ng/mL across all subjects.

[0127] Results to Weeks 12, 16 and 24 are provided in Table 8 and Figure 7. Consistent with the results presented in Example 1, treatment with metreleptin 20 mg daily led to significantly greater weight loss compared to placebo at Week 12 (-4.0 % vs -2.2 %) with continued weight loss that was significantly better than placebo observed at Week 16 (-4.5 % vs -2.1 %) and Week 24 (-7.6 % vs -2.6 %). The weight loss over time is also demonstrated in Figure 8, in which the difference in mean percent change versus placebo was over 7 % at Weeks 28, 32, and 36. At the 10 mg metreleptin dose level, subjects achieved a larger percent reduction in weight than placebo at each time point, and the results reached statistical significance at Week 24 (-6.2 % vs. -2.6 %). Both a dose response and improvement in weight reduction over time on treatment at both dose levels over placebo are apparent in Figure 7.

[0128] These results demonstrate that (i) the daily administration of metreleptin at 20 mg or 10 mg results in a significantly greater reduction in weight in body weight of overweight and obese subjects with low leptin levels as compared to placebo, (ii) the reduction in weight is not only maintained but is elevated over time, and (iii) the difference in weight loss between the raetreleptin-treated groups and the placebo subjects is increased over time. These results are unexpected, especially given what is known about the effects of leptin on weight.

Table 7. Baseline demographics for the Phase 2 Amgen Study LEPT-980236.

SD = standard deviation

Table 8. Summary of Change in Weight from Baseline to Weeks 12, 16, and 24 for Subjects with Low Leptin Levels by Treatment Group (Phase 2 Amgen Study LEPT-980236).

Abbreviations: MET = metreleptin; PBO = placebo; SD = standard deviation; Wt = weight

aP-value from a 2-sample t-test comparing mean percent change from baseline between placebo and metreleptin.

Example 4

[0129] The following describes a post hoc analysis of the same study described in Example 3 above (LEPT-980236), but a different statistical analysis was used to make allowances for missing data and to determine if overall results were consistent with the statistical method used in Example 3 above.

[0130] Change from baseline in weight was analyzed using a mixed-effects model for repeated measures, with the percent change from baseline in weight as a dependent variable, treatment groups as factors, and baseline leptin levels as fixed covariates. Missing data at all visits were imputed with the Markov Chain Monte Carlo multiple imputation method to create 100 imputed data sets. For instances in which weight was missing, the median value across the imputed datasets for each subject was used in the summary

[0131] The results showed that metreleptin decreased weight in adults with low baseline leptin levels in a dose-dependent manner, with statistically significant decreases observed after week 8 for subjects administered the 20 mg dose (see Figure 9).

[0132] In a secondary analysis among the subjects with low leptin levels pooled from this Example and Example 2, a subset of subjects achieved a clinically relevant (>5 %) change in body weight, as shown in Figure 10.

[0133] Moreover, the most common adverse events (AEs) across pooled subjects in this Example and the subjects of Example 2 (all subjects within the four-study pool safety population, irrespective of baseline leptin level) were injection-site reactions, headache, fatigue, gastrointestinal events, and upper respiratory tract infection (see Table 9). Most AEs were mild to moderate in severity, and there was one death among the subjects receiving the 20-mg dose due to lymphocytic leukemia, which was considered unrelated to the study drug.

Table 9. Summary of Common (Incidence >5%) Treatment-Emergent Adverse Events by Preferred Term Among the Subjects Analyzed in Examples 2 and 4.

Example 5

[0134] A post hoc analysis was conducted of the five studies introduced in Example 1 to assess the effects of metreleptin on reducing levels of various parameters that are associated with comorbidities. This analysis focused on subjects who had elevated pre-treatment levels of HbAlc, triglyceride level, LDL cholesterol level, total cholesterol level, ALT level, or AST level, as defined in Table 10. In addition, the analysis targeted subjects who were administered 20 mg metreleptin daily and had the following baseline leptin levels:

• NHANES III -2nd percentile: <8 ng/mL for females, <3 ng/mL for males ("lower baseline leptin level"); and

• NHANES III -1st percentile: <5 ng/mL for females, <2 ng/mL for males ("lowest baseline leptin level").

[0135] Note: the parameters were not assessed at each time point for all subjects. In addition, there was not a placebo group for the elevated hemoglobin Ale and elevated triglyceride level analyses.

[0136] For subjects who had a lower (2nd percentile) baseline leptin level and elevated hemoglobin Ale, administration of metreleptin lowered hemoglobin Ale at 12 weeks and at 16 weeks, as shown in Table 11. There was only one subject who had a lowest (1st percentile) baseline leptin level and elevated hemoglobin Ale, and this subject experienced an increase in hemoglobin Ale after administration of metreleptin.

[0137] For subjects who had a lower (2nd percentile) baseline leptin level and an elevated triglyceride level of 200 mg/dL or greater, administration of metreleptin lowered triglyceride level at 12 and 16 weeks (see Table 12 below). In the one subject with a lowest (1st percentile) baseline leptin level and an elevated triglyceride level of > 350 mg/dL, treatment with metreleptin resulted in about a 65 % decrease in triglyceride level at 36 weeks.

[0138] Subjects who had a lower (2nd percentile) baseline leptin level and an elevated LDL cholesterol level (> 130 mg/dL or > 160 mg/dL) generally experienced a greater decrease in LDL cholesterol level from treatment with metreleptin, as compared to subjects who were not treated with metreleptin (see Table 13 below). In fact, at the 12 weeks and 16 weeks, subjects with an LDL cholesterol level of > 130 mg/dL who did not receive metreleptin treatment had an increase in LDL cholesterol level.

[0139] Subjects who had a lower (2nd percentile) baseline leptin level and an elevated total cholesterol level (> 200 mg/dL or > 240 mg/dL) experienced a greater decrease in total cholesterol level from treatment with metreleptin, as compared to subjects who were not treated with metreleptin (see Table 14 below). Of two subjects who had a lowest (1st percentile) baseline leptin level and a total cholesterol level of > 240 mg/dL, one subject was administered metreleptin and experienced a much greater decrease in total cholesterol level as compared to the other subject who did not receive metreleptin.

[0140] Subjects who had a lower (2nd percentile) baseline leptin level or a lowest (1st percentile) baseline leptin level, and an elevated HDL cholesterol level (> 40 mg/dL or > 60 mg/dL), experienced little, if any, change to their HDL cholesterol level from treatment with metreleptin (see Table 15 below).

[0141] Subjects who had a lower (2nd percentile) baseline leptin level or a lowest (1st percentile) baseline leptin level, and an elevated ALT level, experienced a greater decrease in ALT level from treatment with metreleptin, as compared to subjects who were not treated with metreleptin (see Table 16 below).

[0142] Finally, subjects who had a lower (2nd percentile) baseline leptin level and an elevated AST level experienced a greater decrease in AST level from treatment with metreleptin, as compared to subjects who were not treated with metreleptin (see Table 17 below).

[0143] These results demonstrate that daily administration of metreleptin at 20 mg generally results in a greater reduction of various parameters that are associated with comorbidities as compared to placebo.

Table 10. Pre-treatment Levels of Metabolic and Liver Parameters Assessed in the Study

Table 11. Average Change in Hemoglobin Ale Between Pre-Treatment Level and 12-Week and 16-Week Time Points Obese Subjects with Low Leptin Concentration and Elevated Hemoglobin Ale Treated with Metreleptin.

Table 12. Average Change in Triglyceride Level Between Pre-Treatment Level and 12-Week, 16-Week, and Last-Observed Time Points in Obese Subjects with Low Leptin Concentration and an Elevated Triglyceride Level Treated with Metreleptin.

Table 13. Average change in LDL cholesterol level between pre-treatment level and 12-week, 16-week, and last-observed time points in obese subjects with low leptin concentration and an elevated LDL cholesterol level treated with metreleptin.


Table 14. Average Change in Total Cholesterol Level Between Pre-Treatment Level and 12-Week, 16-Week, and Last- Observed Time Points in Obese Subjects with Low Leptin Concentration and an Elevated Total Cholesterol Level Treated with Metreleptin.


Table 15. Average Change in HDL Cholesterol Level Between Pre-Treatment Level and 12-Week, 16-Week, and Last- Observed Time Points in Obese Subjects with Low Leptin Concentration and an Elevated HDL Cholesterol Level Treated with Metreleptin.


Table 16. Average Change in ALT Level Between Pre-Treatment Level and 12-Week, 16- Week, and Last-Observed Time Points in Obese Subjects with Low Leptin and an Elevated ALT Level Treated with Metreleptin.

Table 17. Average Change in AST Level Between Pre-Treatment Level and 12-Week, 16-Week, and Last-Observed Time Points in Obese Subjects with Low Leptin and an Elevated AST Level Treated with Metreleptin.

Example 6

[0144] The efficacy of metreleptin to treat overweight and obesity in subjects with leptin deficiency and one or more weight-related comorbidities will be further investigated in a clinical study.

[0145] The primary objectives of the study are to (1) assess weight reduction of obese or overweight leptin-deficient subjects in Stratum 1 (leptin levels <8 ng/mL for females, <3 ng/mL for males) after 6 months of 20 mg metreleptin treatment, compared to placebo, by calculating the difference in mean percent loss of baseline body weight; and (2) assess weight reduction of obese or overweight leptin-deficient subjects in Stratum 1 (leptin levels <8 ng/mL for females, <3 ng/mL for males) after 6 months of 20 mg metreleptin treatment, compared to placebo, by determining the proportion of subjects who lose >5 % of baseline body weight.

[0146] The secondary objectives of the study is to (a) assess weight reduction of obese or overweight leptin-deficient patients in Stratum 1 (leptin levels <8 ng/mL for females, <3 ng/mL for males) and Stratum 2 (leptin levels >8 and <16 ng/mL for females, >3 and <5 ng/mL for males) after 6 months of 20 mg metreleptin treatment, compared to placebo, by calculating the difference in mean percent loss of baseline body weight; (b) assess weight reduction of obese or overweight leptin-deficient subjects in Stratum 1 (leptin levels <8 ng/mL for females, <3 ng/mL for males) and Stratum 2 (leptin levels >8 and <16 ng/mL for females, >3 and <5 ng/mL for males) after 6 months of 20 mg metreleptin treatment, compared to placebo, by determining the proportion of subjects who lose >5 % of baseline body weight; and (c) assess reduction in the use of concomitant medications to treat type 2 diabetes, hypertension, and/or hyperlipidemia.

[0147] Exploratory objectives of the study are to (i) identify improvements from baseline in the following parameters: hemoglobin Ale, liver enzymes (AST, ALT), lipid parameters

(triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol), insulin resistance using the homeostatic model assessment-insulin resistance (HOMA-IR) calculator, waist circumference (inclusive of waist:hip ratio), blood pressure, and heart rate; (ii) determine the proportion of subjects who do not gain weight; (iii) determine the proportion of subjects who achieve >10% weight loss; (iv) analyze percent fat loss versus lean muscle by dual energy X-ray absorptiometry (DXA); (v) analyze resting metabolic rate (RMR) by indirect calorimetry; (vi) assess improvement by relevant Quality of Life assessment tools; (vii) determine the effective dose for weight loss and weight maintenance based on baseline

leptin levels; (viii) determine weight loss based on the metreleptin dose and/or achieved leptin levels; and (ix) determine the time to maximum weight loss.

[0148] The study will be a randomized, dose-finding study to determine the safety and efficacy of 5 mg, 10 mg, and 20 mg metreleptin in subjects with HMD.

[0149] Randomization will be stratified based on screening leptin levels:

• Strata 1 : subjects with screening leptin levels <8 ng/mL for females and <3 ng/mL for males;

• Strata 2: subjects with screening leptin levels >8 and <16 ng/mL for females and >3 and <5 ng/mL for males.

[0150] Subjects in Stratum 1 will be randomized 1 : 1 to either 20 mg metreleptin or matching placebo. The arms in Stratum 1 will open sequentially once the 20 mg metreleptin placebo arm has been filled, subjects in Stratum 1 will be enrolled in the open-label 10 mg metreleptin arm. Once the 10 mg metreleptin arm has been filled, subjects will be enrolled in the open-label 5 mg metreleptin arm.

[0151] Subjects in Stratum 2 will be randomized 3: 1 to metreleptin or placebo in 1 of 3 treatment arms: 20 mg metreleptin/placebo, 10 mg metreleptin/placebo, or 5 mg metreleptin/placebo.

[0152] Starting at Month 12, all subjects will receive open-label metreleptin; patients who were receiving placebo will be switched to metreleptin.

[0153] All potentially eligible subjects will undergo prescreening to assess serum leptin levels. Screening assessments to determine eligibility for the study will be conducted within 2 weeks of randomization and will include demographics, medical and surgical history, physical examination (including height and weight), 12-lead electrocardiogram (ECG), vital signs, urine pregnancy test for women of child-bearing potential, drug and alcohol screens, viral (e.g., HIV, HBV, and HCV) testing (and follow-up testing as needed), complete blood count (CBC), clinical chemistry, thyroid panel, and assessment of prior and concomitant medications. Following screening, eligible subjects will be randomized to study treatment on Day 0 based on their baseline leptin levels. Subjects will undergo baseline assessments on Day 0. Each subject will be instructed on self-administration of study treatment and initiate treatment with metreleptin or placebo on Day 0. Subjects will be asked to return to the clinic at Day 7, Day 14, Day 30, Day 90, Day 180, Day 270, Day 360, Day 367, Day 374, Day 390, Day 450, Day 540, Day 630, and Day 720. The focus of the Day 360 (Month 12) to Day 720 (Month 24) assessments will be to review safety, weight management, and metabolic control procedures. The treatment duration will be approximately 720 days (24 months). A follow-up safety visit will be conducted 30 days after the last dose of study treatment.

[0154] Subjects will be placed on a daily 500-calorie deficient diet and will be provided with nutrition counseling by a dietician at the start of the study (Day 0). Subjects will also meet with a dietician every 6 months thereafter until Day 720 (Month 24)

[0155] Subjects must meet all of the following criteria to be enrolled in the study:

• Male or female subjects must be >18 years of age and <75 years of age.

• Subjects must have a leptin level <16 ng/mL females; <5 ng/mL males.

• Subjects must have a BMI >30 kg/m2 (obese) or BMI >27 kg/m2 (overweight) with at least one weight-related comorbidity, such as type II diabetes, hypertension, or dyslipidemia.

• Subjects must have an estimated glomerular filtration rate (eGFR) of >45

mL/min/1.73m2.

• Subjects must have a life expectancy >24 months.

• Subjects with known endocrine disorders (i.e., hypothyroidism, type II diabetes,

hypogonadism) must be stably medicated without dose adjustment for a minimum of 3 months prior to screening.

• Diabetic subjects taking oral hypoglycemic such as dipeptidyl peptidase-4 (DPP-4), glucagon-like peptide 1 (GPL-1), or metformin must be stably medicated without dose adjustment for a minimum of 3 months prior to screening.

• Subjects on antidepressant medications must be stably medicated without dose

adjustment for a minimum of 3 months prior to screening.

• Subjects on antihypertensive and/or dyslipidemia medications must be on stable

medication without dose adjustments for a minimum of 8 weeks prior to screening.

• Subjects on medications that can suppress appetite, including agents that block serotonin reuptake or release, such as fluoxetine, sertraline, or paroxetine, must be stably medicated without dose adjustment for a minimum of 3 months prior to screening.

Female subjects of childbearing potential (i.e., ovulating, premenopausal, and not surgically sterile) and all male subjects not surgically sterilized or partners of either (not surgically sterilized) must use a medically accepted contraceptive regimen, as defined in this protocol, during the study and for 30 days after the last administration of study drug, o Note: female subjects are considered to be of childbearing potential unless they are at least 50 years of age with a minimum of 12 months of consecutive amenorrhea in the absence of another biological/physiological cause and have not undergone a hysterectomy, bilateral oophorectomy, or tubal ligation within a minimum of 26 weeks prior to screening,

o Acceptable methods of contraception for female subjects enrolled in the study include the following:

Surgical sterilization of subject at least 26 weeks prior to screening

(includes hysterectomy or bilateral tubal ligation, oophorectomy, or salpingectomy).

■ Intrauterine device at least 30 days prior to screening in combination with a barrier method.

Hormonal contraception (oral, implant, injection, ring, or patch) for at least 30 days prior to screening in combination with a barrier method.

Diaphragm used in combination with spermicide in combination with a barrier method.

Male subjects must agree to abstain from sperm donation through 90 days after administration of the last dose of study drug.

Subjects must be willing to halt blood donation during the study and for 3 months following administration of the last dose of study drug.

Subjects must be willing and able to give informed consent for participation in the study. Subjects must be willing and able to comply with all scheduled study visits,

recommended diet, treatment plan, laboratory tests, and other study procedures.

Subjects who meet any of the following criteria will not be enrolled in the study:

Subjects with a clinically confirmed diagnosis of lipodystrophy.

Subjects who have had previous treatment with metreleptin.

Subjects who have used any investigational medication or device for any indication within the 30 days or 5 half-lives (whichever is longer) prior to screening.

Subjects with an AST and/or ALT value >3 χ the upper limit of normal (ULN) and a total bilirubin value >1.5 χ the ULN.

Subjects with uncontrolled type 2 diabetes, defined as HbAlc >7.5 %, or type 1 diabetes mellitus.

Subjects with blood pressure > 160/100 mmHg at screening.

Subjects with abnormal and/or uncontrolled thyroid function, defined as any 1 of the following:

o Thyroid-stimulating hormone (TSH) >1.5 the ULN.

o TSH <1.5 x the lower limit of normal (except if the subject has diagnosed and treated central hypothyroidism with a normal free thyroxine [T4]).

o Free T4 outside of the reference range, unless the case is reviewed and approved by the Medical Monitor,

o Thyroid hormone replacement therapy that has not been stable for a minimum of

12 weeks prior to screening,

o A history of hyperthyroidism not definitively treated with surgery and/or

radioiodine ablation.

Subjects who use any weight loss drug or supplement within 3 months prior to screening. Subjects with self-reported or clinically documented history of significant fluctuations (>5 % change) in weight or participation in a formal drug-free weight loss program (investigational or otherwise) not in the maintenance phase within 3 months prior to screening.

o Note: Subjects involved in the drug-free maintenance phase of a weight loss

program are permitted as long as the subject agrees to comply with the study diet. Subjects with chronic pancreatitis/acute pancreatitis within the last 5 years.

Subjects with a known hypersensitivity to Escherichia coli-derived products.

Subjects with a positive result for human immunodeficiency virus (HIV) at screening. Subjects with an acute hepatitis B virus (HBV) infection, as determined by a positive (immunoglobulin m [IgM] antibody against hepatitis B core antigen) IgM anti-HBc result, and/or clinical symptoms of acute viral hepatitis (e.g., anorexia, nausea, vomiting, abdominal pain, acholic stool, fatigue, fever, or jaundice) at screening. Note: Subjects that are chronically infected with HBV (positive hepatitis B surface antigen and negative IgM anti HBc) may qualify if they meet the following:

o Have an undetectable viral load at screening.

o Are hepatitis B e-antigen negative at screening.

o Do not use or has not used anti-viral therapy.

o Do not have cirrhosis.

Subjects with acute hepatitis C virus (HCV) infection, as determined by a positive HCV ribonucleic acid result and a positive HCV antibody result, and/or clinical symptoms of acute viral hepatitis (e.g., anorexia, nausea, vomiting, abdominal pain, acholic stool, fatigue, fever, or jaundice) at screening. Note: Subjects who are chronically infected with HCV may qualify if they meet the following:

o Have an undetectable viral load at screening.

o Are not on or planning to initiate anti-viral therapy. Note: If a subject was

previously on anti-viral therapy, they must have completed therapy with a documented sustained viral response at 12 weeks following completion, o Do not have cirrhosis.

Subjects with a history of liver disease (with exceptions noted in exclusion criterion #14) or nonalcoholic steatohepatitis.

Subjects with a history of malignancy within the past 5 years other than surgically excised basal or squamous cell carcinoma of the skin or noninvasive cervical cancer. Subjects with a history of Cushing's Syndrome, acromegaly, or other endocrine disorders associated with severe metabolic disturbances.

Subjects who have a clinically significant disorder that in the opinion of the Investigator, could contraindicate the administration of study drug, affect compliance, interfere with study evaluations, or confound the interpretation of study results.

o Note: Conditions associated with type 2 diabetes are an exception,

o Note: Subjects with stroke, myocardial infarction, life-threatening arrhythmia, or coronary re vascularization within the 6 months prior to screening are not permitted to participate.

Subjects with a positive urine drug screen or history of substance abuse of legal or illegal drugs and/or alcohol.

o Note: Any use of marijuana within the 3 months prior to screening or during the study is prohibited.

Female subjects of childbearing potential who are pregnant, breastfeeding, <6 months postpartum, intend to become pregnant, or are not using adequate contraceptive methods defined by this protocol.

Subjects who have received a blood transfusion within 3 months prior to screening. Subjects with unstable nicotine (including tobacco, nicotine patch, and/or other nicotine replacement) use, per the Investigator's discretion, or who stopped nicotine use within the 12 months prior to screening.

Subjects with untreated/inadequately-treated sleep apnea (i.e., suspected to have sleep apnea and not yet evaluated or noncompliant with Continuous Positive Airway Pressure treatment).

Subjects with a psychiatric disorder unless stably medicated with medications permitted in this protocol and without dose adjustment for a minimum of 3 months prior to screening.

Subjects with a history of anorexia nervosa, bulimia, or clinical binge-eating disorder. Subjects with a history of laxative abuse (within the past 2 years).

Subjects who have had previous bariatric surgery.

o Note: Subjects who have had a gastric band removed are permitted in the study, as long as the gastric band was removed a minimum of 6 months prior to screening.

Subjects who plan to use or have used chronic systemic steroids (inclusive of oral, intramuscular, intravenous, and intraarticular) (other than sex-hormone replacement on stable medication without dose adjustment for a minimum of 3 months prior to screening) within 3 months prior to screening.

Subjects who have taken within the 3 months prior or are currently taking any of the following over-the-counter or prescription medications, unless approved by the Medical Monitor and Sponsor:

o Thiazolidinediones.

o Insulin.

o Antianxiety agents other than selective serotonin reuptake inhibitors and serotonin norepinephrine reuptake inhibitors.

o Chronic systemic steroids (inclusive of oral, intramuscular, intravenous, and intraarticular) (other than sex-hormone replacement on stable medication without dose adjustment for a minimum of 3 months prior to screening).

o Astemizole.

o Antipsychotics.

o Note: The following medications are not permitted, unless stably medicated

without dose adjustment for a minimum of 3 months prior to screening, and the Investigator does not anticipate dose changes:

o Anticonvulsants.

o Anticoagulants.

• Subjects who are unsuitable candidates for participation in this study, as determined by the Investigator or Sponsor-designated physician.

[0157] The primary efficacy parameters include the following:

• Percent weight loss at Month 6 in obese or overweight hypoleptinemic subjects in

Stratum 1 (leptin levels <8 ng/mL [females]; <3 ng/mL [males])

• Percentage of subjects with at least 5% weight loss at Month 6 in obese or overweight hypoleptinemic subjects in Stratum 1 (leptin levels <8 ng/mL [females]; <3 ng/mL

[males])

[0158] The primary efficacy parameters include the following:

• The secondary efficacy parameters include the following:

• Percent weight loss at Month 6 in obese or overweight hypoleptinemic subjects in

Stratum 1 (leptin levels <8 ng/mL [females] and <3 ng/mL [males]) and Stratum 2 (leptin levels >8 and <16 ng/mL [females] and >3 and <5 ng/mL [males])

• Percentage of subjects with at least 5% weight loss at Month 6 in obese or overweight hypoleptinemic subjects in Stratum 1 (leptin levels <8 ng/mL [females] and <3 ng/mL [males]) and Stratum 2 (leptin levels >8 and <16 ng/mL [females] and >3 and <5 ng/mL [males])

• Clinically meaningful reductions (per Investigator's discretion) in concomitant medications used to treat hypertension, type 2 diabetes, and dyslipidemia over time

[0159] The other efficacy parameters include the following:

• Changes in HbAlc from baseline to Month 3, Month 6, and every 3 months thereafter until Month 24

• Changes in liver enzymes (AST and ALT) from baseline to Day 7, Day 14, Month 1, Month 3, Month 6, and every 3 months thereafter until Month 24

• Changes in lipid parameters (triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol) from baseline to Month 6 and every 3 months thereafter until Month 24

• Changes in insulin resistance (using HOMA-IR) from baseline to Month 6 and every 6 months thereafter until Month 24

• Changes in waist circumference, inclusive of waist:hip ratio, from baseline to Month 3, Month 6, and every 3 months thereafter until Month 24

• Percentage of subjects with at least 10% weight loss at Month 3, Month 6, and every 3 months thereafter until Month 24

• Changes in percent adiposity and percent lean body mass, as measured by DXA, from baseline to Month 6, and every 6 months thereafter until Month 24

• Changes in RMR, measured by indirect calorimetry, from baseline to Month 6 and every 6 months thereafter until Month 24

• Changes in the Impact of Weight on Quality of Life-Lite scores from baseline to Month 6 and every 6 months thereafter until Month 24

• Changes in the Three-Factor Eating Questionnaire (revised 18-item) from baseline to Month 6 and every 6 months thereafter until Month 24

• Changes in the Visual Analog Scale assessments of hunger and satiety from baseline to Month 6 and every 6 months thereafter until Month 24

• Changes in weight from baseline to Month 3, Month 6, and every 3 months thereafter until Month 24

[0160] The study sample size is determined by the primary efficacy analyses based on the Stratum 1 population. Assuming a 7.5 % decrease in weight from baseline at Month 6 in the metreleptin arms and a 2.5 % decrease in weight from baseline at Month 6 in the placebo arm in Stratum 1, and 6 % as the common standard deviation, 24 completed subjects per group will provide 80 % power to this study. With 15 % dropout before Month 6, 28 randomized subjects per group (N=l 12 in total) for Stratum 1 is designed for this study.

[0161] It is desired to have an equal number of subjects for Stratum 2 and Stratum 1. Therefore, the study will randomize approximately 224 subjects, approximately 1 12 subjects in Stratum 1 and approximately 112 subjects in Stratum 2.

[0162] Metreleptin for injection or matching placebo will be supplied in vials. The vials will contain a sterile, white, solid, lyophilized cake of either 11.3 mg metreleptin or matching placebo to be reconstituted with 2.2 mL of sterile water for injection in order to deliver 5 mg/mL of metreleptin. The other components of the reconstituted product are 10 mM glutamic acid, 2 % glycine, 1 % sucrose, 0.01 % polysorbate 20, pH 4.25.

[0163] Study drug is packaged in 5 mL United States Pharmacopeia Type I glass vials with chlorobutyl rubber stoppers, and aluminum seals with plastic flip-off caps. The study drug will be labeled according to the requirements of local law and legislation, as well as current GMP and GCP guidelines. Proof labels, detailing actual label text, will be available in the study files.

[0164] Subjects will receive vials with cake of either metreleptin or matching placebo to be reconstituted with 2.2 mL of sterile water for injection in order to deliver 5 mg/mL of metreleptin. A separate guidance document will be provided to subjects, which will detail the process for reconstitution.

[0165] Subjects receiving 20 mg metreleptin or placebo will receive 2 vials of 10 mg metreleptin or matching placebo to be taken subcutaneously (SC) twice daily.

[0166] Subjects receiving 10 mg metreleptin or placebo will receive 1 vial of 10 mg metreleptin or matching placebo to be taken SC once daily (QD).

[0167] Subjects receiving 5 mg metreleptin or placebo will receive 1 vial of 10 mg metreleptin or matching placebo and be instructed to take half (5 mg) of the vial SC QD.

[0168] Subjects will self-administer SC injections of study drug. The amount of study drug administered is based on the treatment arm to which the subject is assigned (5, 10, 20 mg). A separate guidance document will be provided to subjects that will detail the process for

reconstitution and administration, including how to reconstitute the study drug, how to prepare the injection site, and how to administer the injection.

[0169] At Month 6, a subject's dose will be increased if the subject's weight loss from baseline is <5 %. At Month 12, a subject's dose will be increased if the subject's weight loss from baseline is <10 % for subjects whose baseline BMI was >30 kg/m2; if the subject's baseline BMI was <30 kg/m2, the dose may be increased per Investigator discretion.

[0170] At Month 6, or anytime thereafter, the dose will be increased if the subject has a >2 % body weight increase over 6 months.

[0171] The results of this study will show that administration of metreleptin will result in weight loss at month 6 in obese and overweight or overweight hypoleptinemic subjects in Stratum 1 (leptin levels <8 ng/mL for females, <3 ng/mL for males) and Stratum 2 (leptin levels >8 and <16 ng/mL for females, and >3 and <5 ng/mL for males), and the percentage of subjects of both Stratums with at least 5 % weight loss at month 6 will be greater in subjects administered metreleptin as compared to subjects administered the placebo.

[0172] In addition, the results may show that subjects who taking concomitant medications to treat type 2 diabetes, hypertension, and/or hyperlipidemia will experience a reduction in taking those medications. Subjects will exhibit improvements from baseline in one or more of the following parameters: hemoglobin Ale, liver enzymes (AST, ALT), lipid parameters

(triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol), insulin resistance using the HOMA-IR calculator, waist circumference (inclusive of waist:hip ratio), blood pressure, and heart rate. The percentage of subjects with at least 10% weight loss at month 3, month 6, and every 3 months thereafter until month 24, will be greater for subjects administered leptin as compared to subjects administered the placebo.

[0173] Further, as compared to subjects administered the placebo, subjects administered metreleptin will demonstrate greater positive changes in:

• percent adiposity and percent lean body mass, as measured by DXA, from baseline to month 6, and every 6 months thereafter until month 24;

• RMR, measured by indirect calorimetry, from baseline to month 6 and every 6 months thereafter until month 24;

• IWQOL-Lite scores from baseline to month 6 and every 6 months thereafter until month 24;

• TFEQ (revised 18-item) from baseline to month 6 and every 6 months thereafter until month 24;

• VAS assessments of hunger and satiety from baseline to month 6 and every 6 months thereafter until month 24; and

• weight from baseline to month 3, month 6, and every 3 months thereafter until month 24.

* * * * *

[0174] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

[0175] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0176] Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[0177] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and/or with the aid of or automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various steps may be changed without

departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0178] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

SEQUENCE LISTING

SEQ ID NO: 1

VPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQ VTGLDFIP

GLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFS KSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLS PGC, wherein: Xaa at position 27 is T or A; and Xaa at position 28 is Q or absent.

SEQ ID NO: 2

MVPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFI

PGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAF SKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDL SPGC, wherein: Xaa at position 28 is T or A; and Xaa at position 29 is Q or absent.