Methods of neuroendocrine regulation of affective disorders

Methods of treating an affective disorder in an individual are disclosed. Affective disorders include major depression, melancholic and atypical subtypes, and dysthymia.

BACKGROUND OF THE INVENTION 
Affective and mood disorders are included in a group of mental disorders 
characterized by neuroendocrine dysregulation and are characterized by a 
disturbance in the regulation of mood, behavior, and affect. Affective and 
mood disorders can have serious impact on an individual's functional 
ability, interpersonal relationships and behavior. Major depression and 
dysthymia are examples such disorders. 
Major depression is a syndromal, episodic and recurrent illness with both 
psychological and biological components. A diagnosis of bipolar disorder 
is given to those patients with recurring depression and mania. Those 
patients with recurrent depression alone have a unipolar pattern. Within 
the spectrum of depressive illness, there are two distinct subtypes: 
melancholic depression and atypical depression (Gold et al., N. Engl. J. 
Med., 319:348-353 (1988); and Gold et al., N. Engl. J. Med., 319:413-420 
(1988)). 
Melancholic depression is equally common among those with a pattern of 
unipolar and bipolar depression. Melancholic depression is characterized 
by hyposomnia (early morning awakening), anorexia and diurnal variation in 
mood, and is associated with a state of hyperarousal in which patients are 
painfully preoccupied with personal inadequacy, loss, feelings of 
worthlessness, guilt and suicidal ideation (Licinio et al., Bailliere's 
Clin. Endocrin. Met., 5(1):51-58 (1991)). 
Atypical depression is more common in bipolar patients than in unipolar 
depressed patients. Atypical depression is characterized by a state which 
seems to be opposite to that of melancholic depression. Patients with 
atypical depression have a syndrome of hypoarousal with hypersomnia, 
hyperphagia, weight gain and mood liability (Licinio et al., Bailliere's 
Clin. Endocrin. Met., 5 (1):51-58 (1991)). 
Neuroendocrine dysregulation, specifically changes in the 
hypothalamic-pituitary-adrenal (HPA) axis, has been investigated as a 
biological correlate of depression. Overall, the HPA axis regulates 
physiologic responses to stress. The hypothalamus controls endocrine 
functions and the autonomic nervous system. It is involved in behaviors 
related to fight, flight, feeding and mating, many of which are altered 
during episodes of depression. The hypothalamus releases 
corticotrophic-releasing hormone (CRH) in response to stress, which then 
stimulates the anterior pituitary to secrete 
adrenocorticotrophic-releasing hormone (ACTH). ACTH prompts the adrenal 
cortex to release cortisol which, through elaborate feedback mechanisms 
signals the hypothalamus to increase or decrease CRH production. 
Under ordinary circumstances, activation of hypothalamic CRH is terminated 
rapidly by the negative feedback of rising glucocorticoid levels. However, 
in melancholic depression, hypercortisolism does not adequately restrain 
the production of CRH in the hypothalamus. Thus, in melancholic 
depression, CRH levels are chronically elevated causing hyperactivity of 
the HPA axis, hypercortisolism and ACTH levels that are numerically 
normal, but excessive in the context of high levels of circulating 
cortisol (Gold et al., N. Engl. J. Med., 314:1329-1335 (1986)). 
Antidepressant treatments consistently lower HPA axis activity in 
individuals with melancholic depression. In contrast, atypical depression 
is associated with hyposecretion of hypothalamic CRH. Thus, in atypical 
depression, hypothalamic CRH levels are lower than normal causing 
hypoactivity of the HPA axis. For a review, see Gold et al., Mol. 
Psychiatry, 1:257-264 (1996). 
Dysthymia is a chronic disorder characterized by symptoms that include poor 
appetite or overeating, low energy (decreased arousal), insomnia or 
hypersomnia, and poor concentration. These functions are modulated by 
neuropeptides in the brain, such as CRH (Vale, W. et al., Science, 
213:1394-1397 (1981)). Generally, dysthymia is characterized by 
hypothalamic CRH levels that are higher than normal, thereby causing 
hyperactivity of the HPA axis. However, in dysthymia, hypothalamic CRH 
levels can be lower than normal, causing hypoactivity of the HPA axis, in 
individuals with a higher than normal body mass index (BMI). Thus, in 
dysthymia, hypothalamic CRH levels are inversely related to the BMI of the 
individual. 
Effective disorders are extremely common in general medical practice, as 
well as in psychiatry. The severity of these conditions covers an 
extraordinarily broad range, from normal grief reactions to severe, 
incapacitating, and sometimes fatal psychosis. The lifetime risk of 
suicide in major affective disorders is about 10 to 15%, but this 
statistic does not begin to represent the morbidity and cost of this group 
of under-diagnosed illnesses. Typically these disorders are treated with 
antidepressant agents or lithium salts (GOODMAN AND GILMAN'S THE 
PHARMACOLOGICAL BASIS OF THERAPEUTICS, Eight Ed., 1990; Pergramon Press, 
New York, N.Y.). Nevertheless, many shortcomings and problems continue to 
be associated with all drugs used to treat affective disorders. In 
addition to less than-dramatic efficacy in some cases, virtually all the 
drugs used to treat disorders of mood are potentially lethal when acute 
over dosage occurs and can cause appreciable morbidity even with careful 
clinical use. 
SUMMARY OF THE INVENTION 
The present invention relates to the discovery that the symptoms of an 
affective or mood disorder can be alleviated by altering or modifying 
leptin levels in the cerebrospinal fluid (CSF) of an individual. In a 
particular embodiment, CSF leptin levels in an individual with melancholic 
depression can be increased from the endogenous CSF leptin levels that are 
present in the individual to decrease or alleviate symptoms of melancholic 
depression. 
In another embodiment, CSF or plasma leptin levels in an individual with 
atypical depression can be lowered, or decreased, from the endogenous CSF 
or plasma leptin levels in the individual to decrease or alleviate 
symptoms of atypical depression. Also encompassed by the present invention 
are methods of treating atypical depression associated with Cushing's 
Disease and Chronic Fatigue Syndrome. 
In yet another embodiment, CSF or plasma leptin levels in an individual 
with dysthymia can be lowered, or decreased, from endogenous CSF or plasma 
leptin levels present in the individual to decrease or alleviate symptoms 
of dysthymia. 
CSF leptin levels in an individual can be altered or modified by altering 
or modifying endogenous plasma leptin levels and/or endogenous CSF leptin 
levels in the individual. Increased leptin levels can be achieved by 
administering to an individual a leptin compound such as exogenous leptin, 
a leptin analog, biologically active leptin fragment or leptin fusion 
protein, in an amount sufficient to increase CSF leptin concentration, 
resulting in a decrease or alleviation of symptoms of depression, e.g., 
melancholic depression. Decreased leptin levels can be achieved by 
administering to an individual a leptin antagonist, in an amount 
sufficient to decrease, or lower, blood or CSF leptin levels, thereby 
resulting in a decrease or alleviation of symptoms of depression, e.g., 
atypical depression. 
In one embodiment, the invention relates to the use of leptin to treat 
individuals with forms of affective disorders characterized by higher than 
normal levels of hypothalamic-pituitary-adrenal (HPA) axis activity. An 
affective disorder characterized by higher than normal levels of HPA axis 
activity in accordance with the present invention is treated by 
administering to an individual in need thereof an effective amount of 
exogenous leptin, biologically active leptin fragment, or leptin analog or 
leptin fusion protein. Administration of an effective amount of exogenous 
leptin, leptin analog, biologically active leptin fragment or leptin 
fusion protein increases cerebrospinal fluid (brain) leptin levels in the 
individual. As a result, HPA activity is suppressed in the individual, 
alleviating symptoms of the disorder. 
In still another embodiment, methods for treating dysthymia are provided. 
In accordance with the invention, dysthymia is treated by administering to 
an individual in need thereof an effective amount of leptin antagonist. 
Administering leptin antagonists to the individual increases HPA activity 
and reduces or alleviates the symptoms of dysthymia. 
The invention also relates to methods of treating an affective disorder in 
an individual in need thereof comprising determining the level of HPA axis 
activity in the individual and comparing that level with a control level. 
In accordance with the invention, if the level of HPA axis activity 
determined in the individual is higher than the control level, an 
effective amount of exogenous leptin, leptin analog, biologically active 
leptin fragment or leptin fusion protein is administered to the 
individual. If the level of HPA axis activity determined in the individual 
is lower than the control level, an effective amount of leptin antagonist 
is administered to the individual. 
The invention further relates to methods of treating an affective disorder 
in an individual in need thereof comprising determining the level of 
corticotrophic-releasing hormone (CRH) production in the individual and 
comparing that level with a control level. In accordance with the 
invention, if the level of CRH production determined in the individual is 
higher than the control level, an effective amount of exogenous leptin, 
leptin analog, biologically active leptin fragment or leptin fusion 
protein is administered to the individual. If the level of CRH production 
determined in the individual is lower than the control level, an effective 
amount of leptin antagonist is administered to the individual.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the discovery that leptin levels are 
increased from normal levels in the blood of patients with an affective 
disorder characterized by higher than normal levels of HPA axis activity, 
but are not increased from normal levels in their cerebrospinal fluid. 
Therefore, even though leptin levels are higher than normal levels in the 
plasma of these patients, these levels are not high enough to result in 
increased cerebrospinal fluid leptin levels, which reflect leptin levels 
in the brain. High cerebrospinal fluid (brain) leptin levels are needed to 
suppress the increased activity of the HPA axis in these patients. 
As defined herein, "normal" levels of leptin means levels of leptin in 
blood or CSF obtained from individuals not affected by a mood disorder. 
Typically levels of blood and CSF leptin are determined by assay as 
described herein. "Normal" levels of HPA axis activity is defined herein 
as to mean levels of HPA axis activity in individuals not affected by a 
mood disorder. Typically physiological parameters of HPA axis activity 
include measurements of corticotrophin-releasing hormone (CRH) and ACTH, 
and may also include measurement of other hormones, e.g., corticosteroids. 
Assays for measuring these hormones are well known to those of skill in 
the art. HPA axis activity can also be evaluated by behavioral parameters 
such as sleep disturbances, appetite, libido and psychomotor alterations. 
The present invention also relates to the discovery that plasma leptin 
levels are higher than normal in patients with an affective disorder 
characterized by lower than normal levels of HPA axis activity. It is 
reasonable to believe that high levels of leptin excessively suppress HPA 
activity in these patients and contribute to the symptoms of the disorder. 
Leptin is inversely related to pituitary-adrenal function, and that in 
humans, high leptin levels decrease activity of the 
hypothalamic-pituitary-adrenal (HPA) axis, while low leptin levels 
increase activity of the HPA axis. Human leptin levels are pulsitile and 
inversely related to pituitary-adrenal functions (U.S. Ser. No. 
08/749,534, the teachings of which are herein incorporated by reference). 
Thus, the present invention encompasses methods of treating an affective 
disorder in an individual comprising administering an effective amount of 
exogenous leptin, leptin analog, biologically active leptin fragment, 
leptin fusion protein, or leptin antagonist to the individual. The 
exogenous leptin, leptin analog, biologically active leptin fragment, 
leptin fusion protein or leptin antagonist can be administered in single 
or multiple doses. 
As used herein, the term "affective disorder" refers to a mental disorder 
characterized by neuroendocrine dysregulation and by a disturbance in the 
regulation of mood, behavior and affect. Affective disorders are also 
referred to herein as mood disorders and include major depressive 
disorders, such as melancholic depression, atypical depression as well as 
chronic mood disorder such as dysthymia. 
The present invention relates to the use of leptin, leptin analogs, 
biologically active leptin fragments and leptin fusion proteins in the 
treatment of affective disorders characterized by higher than normal 
levels of HPA axis activity. An affective disorder characterized by higher 
than normal levels of HPA axis activity includes forms of depression 
characterized by higher than normal levels of HPA axis activity, such as 
melancholic depression. 
The present invention also relates to the use of leptin antagonists in the 
treatment of affective disorders characterized by lower than normal levels 
of HPA axis activity. An affective disorder characterized by lower than 
normal levels of HPA axis activity includes forms of depression 
characterized by lower than normal levels of HPA axis activity, such as 
atypical depression. 
In a particular embodiment, methods of treating an affective disorder in an 
individual comprise determining the level of HPA axis activity in the 
individual and comparing that level with a control level. In accordance 
with the invention, if the level of HPA axis activity determined in the 
individual is higher than the control level, an effective amount of 
exogenous leptin, leptin analog, biologically active leptin fragment or 
leptin fusion protein is administered to the individual. If the level of 
HPA axis activity determined in the individual is lower than the control 
level, an effective amount of leptin antagonist is administered to the 
individual. As used herein, the control level of HPA axis activity refers 
to the level of HPA axis activity in a healthy individual not suffering 
from an affective disorder or experiencing neuroendocrine dysfunction, 
matched for age, sex and BMI with the individual to be treated. 
In another embodiment, methods of treating affective disorders in an 
individual comprise determining the level of CRH production in the 
individual and comparing that level with a control level. In accordance 
with the invention, if the level of CRH production determined in the 
individual is higher than the control level, an effective amount of 
exogenous leptin, leptin analog, biologically active leptin fragment or 
leptin fusion protein is administered to the individual. If the level of 
CRH production determined in the individual is lower than the control 
level, an effective amount of leptin antagonist is administered to the 
individual. As used herein, the control level of CRH refers to the level 
of CRH in a healthy individual not suffering from an affective disorder or 
experiencing neuroendocrine dysfunction, matched for age, sex and BMI with 
the individual to be treated. 
CRH can be evaluated in an individual at one or more time points using 
suitable techniques. CRH levels can be measured in blood (e.g., plasma) or 
CSF. For example, level of CRH production can be determined by 
immunoassay. A variety of suitable immunoassays are known to those of 
skill in the art. For example, the level of CRH production in an 
individual can be determined by radioimmunoassay (RIA) or enzyme-linked 
immunosorbent assay (ELISA). 
Leptin is the product of the obese (ob) gene and is secreted by adipose 
cells (Zhang et al., Nature, 372:425-432 (1994)). Leptin receptors are 
found in the choroid plexus and the hypothalamus. (Tartaglia, L. A., Coll, 
83:1263-1271 (1995)). The action of leptin to regulate energy balance 
appears to be primarily through effects in the brain, in particular the 
hypothalamus. A rising level of leptin, as triglyceride stores increase, 
is proposed to serve as a negative feedback signal to the brain, resulting 
in decreased food intake, increased energy expenditure and resistance to 
obesity. In addition, circulating leptin appears to play an important role 
in the neuroendocrine axis (Ahima, R. S. et al., Nature, 382:250-252 
(1996)). 
It has recently been shown that leptin levels are inversely related to 
pituitary-adrenal function and that in humans, high leptin levels seem to 
decrease HPA axis activity, while low leptin levels seem to increase HPA 
axis activity (Licinio et al., Nature Med., 3:575-579 (1997)). It has also 
been shown that in humans, serum leptin concentrations vary with the 
percentage of body fat, and that during weight loss, serum leptin 
concentrations initially decline, but increase again during maintenance of 
the lower weight (Considine et al., N. Eng. J. Med., 334:292-295 (1996)). 
Leptin can be recombinantly produced as described in e.g., WO 96/05309; 
U.S. Pat. No. 5,552,522; U.S. Pat. No. 5,552,523; and U.S. Pat. No. 
5,552,524, the teachings of which are incorporated by reference. Leptin 
can also be produced by chemical synthesis, or isolated from mammalian 
plasma using methods well-known to those of skill in the art. The 
exogenous leptin administered in the present methods can be intact 
protein, e.g., the full-length 167 amino acid polypeptide as described in 
Zhang, Y. et al., Nature, 372:425-432 (1994). 
Leptin used in the present methods can also be a functional or biologically 
active equivalent of the leptin protein described above. A "functional or 
biologically active" equivalent is defined herein as a protein which 
shares significant amino acid sequence identity with the corresponding 
sequence of the endogenous, or naturally-occurring protein (e.g., 
typically about 80% sequence identity, more typically about 90% sequence 
identity and most typically about 95% sequence identity) and possesses at 
least one, or more, of the biological functions thereof. Biological 
functions include antigenic, immunogenic, and structural properties, 
anti-obesity activity, such as activity to reduce weight, appetite and 
mobilize fat, and hypothalamic-regulating activity. Antigenicity is 
defined herein as the ability of the protein or analog to bind to 
anti-leptin antibodies. Immunogenicity is defined herein as the ability of 
the protein or analog to induce the production of antibodies that 
specifically react with endogenous leptin. These properties and activities 
can be a measure of biological function. 
Specifically included in the present invention are leptin analogs, or 
derivatives, defined herein as proteins having amino acid sequences 
analogous to endogenous leptin. Analogous amino acid sequences are defined 
herein to mean amino acid sequences with sufficient identity of amino acid 
sequence of endogenous leptin to possess the biological activity of 
endogenous leptin, but with one or more "silent changes" in the amino acid 
sequence. A "silent" amino acid change means that one, or more residues 
differ from the endogenous amino acid sequence. Examples of such 
differences include additions, deletions, or substitutions of residues, as 
well as analogous proteins that exhibit greater, or lesser activity than 
edogenous leptin. 
Also encompassed by the present invention is the administration of 
biologically active polypeptide fragments of leptin. Such fragments can 
include only a part of the full-length amino acid sequence of leptin, yet 
possess biological activity. Such fragments can be produced by carboxyl or 
amino terminal deletions, as well as internal deletions. Such polypeptides 
can be tested for biological activity as described herein. Biologically 
active analogs and fragments of leptin useful in the present invention are 
described, e.g., in WO 96/05309; U.S. Pat. No. 5,552,522; U.S. Pat. No. 
5,552,523; and U.S. Pat. No. 5,552,524, the teachings of which are 
incorporated by reference. 
The present invention also encompasses the administration of fusion 
proteins comprising leptin proteins as described herein, referred to as a 
first moiety, linked to a second moiety not occurring in the leptin 
protein. The second moiety can be a single amino acid, peptide or 
polypeptide or other organic moiety, such as a carbohydrateor, a lipid, or 
an inorganic molecule. Examples of a second moiety include maltose or 
glutathione-S-transferase. 
Also encompassed by the present invention are biologically active 
derivatives or analogs of leptin referred to herein as leptin peptide 
mimetics. These mimetics can be designed and produced by techniques known 
to those of skill in the art. (See e.g., U.S. Pat. Nos. 4,612,132; 
5,643,873 and 5,654,276, the teachings of which are herein incorporated by 
reference). These mimetics are based on the leptin sequence, and peptide 
mimetics possess biologically activity (i.e., leptin activity) similar to 
the biological activity of the corresponding peptide compound, but possess 
a "biological advantage" over the corresponding peptide inhibitor with 
respect to one, or more, of the following properties: solubility, 
stability, and susceptibility to hydrolysis and proteolysis. 
Methods for preparing peptide mimetics include modifying the N-terminal 
amino group, the C-terminal carboxyl group, and/or changing one or more of 
the amino linkages in the peptide to a non-amino linkage. Two or more such 
modifications can be coupled in one peptide mimetic inhibitor. The 
following are examples of modifications of peptides to produce peptide 
mimetics as described in U.S Pat. Nos: 5,643,873 and 5,654,276, the 
teachings of which are incorporated herein by reference. 
Increased leptin levels can be achieved by the administration of exogenous 
leptin or, alternatively, by increasing endogenous leptin production, for 
example by stimulating the endogenous gene to produce increased amount of 
leptin. In some individuals the amount of leptin being produced can be of 
sufficient quantity, but the leptin is abnormal in some way and, thus, 
cannot exert its biological effect. In this instance, providing copies of 
normal leptin genes to adipocytes, using techniques of gene transfer 
well-known to those of skill in the art, can increase leptin levels. 
The present invention further encompasses the administration of leptin 
antagonists. As used herein, a leptin antagonist decreases, blocks, 
inhibits, abrogates or interferes with leptin biological function or 
activity or receptor signaling in vivo. Leptin antagonists include 
anti-leptin antibodies, receptor molecules and derivatives which bind 
specifically to leptin and prevent leptin from binding to its cognate 
receptor. 
Leptin antagonists also include agents, or drugs, which decrease, inhibit, 
block, abrogate or interfere with binding of leptin to its receptors or 
extracellular domains thereof; agents which decrease, inhibit, block, 
abrogate or interfere with leptin production or activation; and agents 
which are antagonists of signals that drive leptin production or 
synthesis. Such an agent can be any organic molecule that inhibits or 
presents the interaction of leptin with its receptor, or leptin 
production. 
Candidate receptor antagonists can be identified by evaluating the binding 
of leptin to its receptor in the presence of, and absence of, the 
candidate antagonist. Such techniques are well-known to those of skill in 
the art. Leptin antagonists so identified can be further tested in-vivo 
for leptin antagonist activity. 
The methods of the present invention can be accomplished by the 
administration of leptin, leptin analogs, biologically active leptin 
fragments, leptin fusion proteins or leptin antagonists by parenteral or 
enteral means. Routes of administration encompassed by the present 
invention include intravenous, intraarterial, intraperitoneal, 
intramuscular or subcutaneous routes, as well as oral and nasal 
administration. Suppositories or transdermal patches can also be used. 
Leptin, leptin analogs, biologically active fragments, fusion proteins and 
leptin antagonists can be administered as leptin compositions in admixture 
with conventional excipients, i.e., pharmaceutically, or physiologically, 
acceptable organic, or inorganic carrier substances suitable for 
parenteral or enteral application which do not deleteriously react with 
the active leptin. Suitable pharmaceutically acceptable carriers include, 
e.g., water, salt solutions, alcohols, oils, gelatins and carbohydrates. 
Leptin, leptin analogs, biologically active fragments, fusion proteins and 
leptin antagonists can also be incorporated into liposomes or administered 
via transdermal patches. Pharmaceutical compositions suitable for use in 
the present invention are well-known to those of skill in the art and are 
described e.g., in WO 96/05309, the teachings of which are hereby 
incorporated by reference. 
An "effective amount" of leptin, leptin analog, biologically active 
fragment, leptin fusion protein or leptin antagonist is defined herein as 
that amount, or dose, of leptin, leptin analog, biologically active 
fragment, leptin fusion protein or leptin antagonist that, when 
administered to a mammal, is sufficient for therapeutic efficacy (e.g., an 
amount sufficient for significantly reducing or eliminating signs or 
symptoms associated with a particular affective disorder). 
Plasma and CSF leptin levels are strongly correlated in a non-linear 
manner, and CSF leptin levels can be modified by peripheral administration 
of exogneous leptin. For example, Schwartz, M. W., et al., (Nature 
Medicine 2:589 (1996)) describes the relationship between plasma and CSF 
leptin as the following: y=0.0047x+0.0404Log.sub.e x+0.0486, where y=CSF 
leptin, and x=plasma leptin. 
The dosage administered to an individual will vary depending upon a variety 
of factors, including the pharmacodynamic characteristics of the 
particular leptin analog, biologically active fragment, leptin fusion 
protein or leptin antagonist, and its mode and route of administration; 
size, age, sex, health, body weight, body mass index (BMI), and diet of 
the recipient; nature and extent of symptoms of the disorder being 
treated, kind of concurrent treatment, frequency of treatment, and the 
effect desired. 
Leptin, leptin analogs, biologically active fragments, leptin fusion 
proteins or leptin antagonists can be administered in single or multiple 
doses depending upon factors such as nature and extent of symptoms, kind 
of concurrent treatment and the effect desired. Other therapeutic regimens 
or agents can be used in conjunction with the methods and leptin, leptin 
analogs, biologically active fragments, leptin fusion proteins and leptin 
antagonists of the present invention. Adjustment and manipulation of 
established dosage ranges are well within the ability of those skilled in 
the art. 
The present invention is further illustrated by the following examples, 
which are not intended to be limiting in any way. 
EXAMPLE 1 
Plasma and Cerebrospinal Fluid Leptin Levels in Patients with Depression 
Four depressed female patients, with an average age of 30 years were 
enrolled in this study. A control group of three healthy female adults, 
matched for age and BMI, were also enrolled in the study. None of the 
subjects were morbidly obese. 
All subjects underwent a 30 hour blood and spinal fluid sampling period 
starting at 10:00 am. Blood and spinal fluid were simultaneously drawn 
through an indwelling catheter at one hour intervals. 
Leptin was measured by radioimmunoassay (RIA). 50 .mu.l of test plasma or 
50 .mu.l of test cerebrospinal fluid (concentrated 5.times.) was incubated 
for 24 hour at 4.degree. C. with phosphate-buffered saline (PBS) 
containing 0.1% Triton X-100 and a 1:2000 dilution of anti-leptin 
antiserum. Twenty-four (24) hours later .sup.125 I-labeled leptin 
(approximately 15,000 cpm) was added to the tube and the reagents were 
incubated for an additional 24 hours. Antibody-bound leptin was 
precipitated by addition of 500 .mu.l of precipitating reagent. Tubes were 
centrifuged for 45 minutes at 2,500 rpm, after which supernatants were 
decanted and pellets counted in a gamma counter. For this leptin assay, 
the limit of detection is 0.2 ng/ml; within-assay coefficient of variation 
(CV) was 4.4% for low levels (2.9 ng/ml) and 5.7% for high levels (14.1 
ng/ml). Interassay CV was 6.9% and 9% for low and high levels, 
respectively. Values were analyzed by ANOVA. 
Results 
As a group, the patients with depression had significantly increased plasma 
leptin levels as compared to the healthy controls (FIGS. 1A and 1C). 
Cerebrospinal fluid leptin levels, which reflect leptin levels in the 
brain, did not differ significantly between the depressed patients and 
healthy controls (FIGS. 1B and 1D). Thus, although plasma leptin levels 
were higher in the depressed patients than in the healthy controls, these 
levels were not high enough to result in increased cerebrospinal fluid 
(brain) leptin levels. 
EXAMPLE 2 
Plasma Leptin Levels in Patients with Atypical Depression 
One female patient diagnosed with atypical depression and three female 
patients diagnosed with depression (non-atypical) (average age of 30 
years), were enrolled in this study. One healthy female adult was also 
enrolled as a control subject. None of the subjects were morbidly obese. 
Diagnosis of depression (atypical and non-atypical) was made using the 
DSM-IV. The patient with atypical depression suffered from hypersomnia, 
increased food intake, weight gain and fatigue. 
All subjects underwent a 24 hour blood sampling period starting at 08:00 
am. Blood was drawn through an indwelling forearm catheter. In the patient 
with atypical depression, blood was drawn at 7 minute intervals. For all 
other subjects, blood was drawn at one hour intervals. 
Leptin was measured by RIA. 50 .mu.l of test plasma was incubated for 24 
hour at 4.degree. C. with PBS containing 0.1% Triton X-100 and a 1:2000 
dilution of anti-leptin antiserum. Twenty-four (24) hours later .sup.125 
I-labeled leptin (approximately 15,000 cpm) was added to the tube and the 
reagents were incubated for an additional 24 hours. Antibody-bound leptin 
was precipitated by addition of 500 .mu.l of precipitating reagent. Tubes 
were centrifuged for 45 minutes at 2,500 rpm, after which supernatants 
were decanted and pellets counted in a gamma counter. For this leptin 
assay, the limit of detection is 0.2 ng/ml; within-assay coefficient of 
variation (CV) was 4.4% for low levels (2.9 ng/ml) and 5.7% for high 
levels (14.1 ng/ml). Interassay CV was 6.9% and 9% for low and high 
levels, respectively. Values were analyzed by ANOVA. 
Results 
The patient with atypical depression had significantly higher plasma leptin 
levels as compared to the healthy control and the patients with 
non-atypical depression (FIG. 2). This suggests that high leptin levels in 
patients with atypical depression contribute to their symptoms of 
hypersomnia, increased food intake, weight gain and fatigue. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.