U.S. patent application number 10/822020 was filed with the patent office on 2005-01-06 for prevention of hyperinsulinemia in subjects undergoing growth hormone (gh) treatment.
Invention is credited to Johansen, Thue, Malmlof, Kjell.
Application Number | 20050004023 10/822020 |
Document ID | / |
Family ID | 33555914 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050004023 |
Kind Code |
A1 |
Johansen, Thue ; et
al. |
January 6, 2005 |
Prevention of hyperinsulinemia in subjects undergoing growth
hormone (GH) treatment
Abstract
The present invention provides methods and compositions for
preventing hyperinsulinemia that can result from therapeutic
administration of growth hormone. The methods are carried out by
imposing a restricted high-fat diet and/or by administering one or
more drugs that lower serum lipids.
Inventors: |
Johansen, Thue; (Copenahgen
O, DK) ; Malmlof, Kjell; (Kalmar, SE) |
Correspondence
Address: |
NOVO NORDISK PHARMACEUTICALS, INC
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Family ID: |
33555914 |
Appl. No.: |
10/822020 |
Filed: |
April 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60467079 |
May 1, 2003 |
|
|
|
Current U.S.
Class: |
514/6.7 ;
514/11.4; 514/7.4 |
Current CPC
Class: |
A23L 33/30 20160801;
A61K 38/27 20130101; A61K 31/4965 20130101; A61K 38/553 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/27 20130101;
A61K 38/553 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/27 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2003 |
DK |
PA 2003 00548 |
Claims
1. A method for preventing hyperinsulinemia in an animal or human
subject undergoing treatment with growth hormone (GH), the method
comprising subjecting said subject, during the growth hormone
treatment period, to a measure that causes a reduction in blood
lipid levels.
2. A method according to claim 1, wherein said measure is selected
from the group consisting of a diet regimen, a drug treatment, or a
combination thereof.
3. A method according to claim 2, wherein the diet regimen
comprises a restricted amount of a high-fat (HF) diet as a sole
food source.
4. A method according to claim 3, wherein the energy content of the
diet does not exceed the theoretical maintenance level for the
subject.
5. A method according to claim 2, wherein the drug treatment
comprises administering a compound selected from the group
consisting of: 5-methylpyrazinecarboxylic acid 4-oxide, a statin, a
fibrate, or a combination of any of the foregoing, wherein said
administering is effective in reducing blood lipid levels in the
subject.
6. A method according to claim 1, wherein the subject is a
human.
7. A method according to claim 6, wherein the human is obese.
8. A method for catabolizing adipose tissue in an animal or human
subject without induction of hyperinsulinemia, the method
comprising (i) administering a growth hormone (GH) to the subject
and (ii) subjecting the subject to a measure that causes a
reduction in blood lipid levels.
9. A method according to claim 8, wherein said measure is selected
from the group consisting of a diet regimen, a drug treatment, or a
combination thereof.
10. A method according to claim 9, wherein the diet regimen
comprises a restricted amount of a high-fat (HF) diet as a sole
food source.
11. A method according to claim 10, wherein the energy content of
the diet does not exceed the theoretical maintenance level for the
subject.
12. A method according to claim 9, wherein the drug treatment
comprises administering a compound selected from the group
consisting of: 5-methylpyrazinecarboxylic acid 4-oxide, a statin, a
fibrate, or a combination of any of the foregoing, wherein said
administering is effective in reducing blood lipid levels in the
subject.
13. A method for reducing blood lipid levels in an animal or human
subject without induction of hyperinsulinemia in the subject, the
method comprising (i) administering a growth hormone (GH) to the
subject and (ii) providing said subject with restricted amounts of
a high-fat (HF) diet as a sole food source.
14. A method for reducing blood lipid levels in an animal or human
subject without induction of hyperinsulinemia in the subject, the
method comprising (i) administering a growth hormone (GH) to said
subject and (ii) inhibiting lipolysis in said subject.
15. A pharmaceutical composition comprising, as active ingredients,
(i) a growth hormone and (ii) an agent capable of reducing blood
lipid levels.
16. A pharmaceutical composition comprising, as active ingredients,
(i) a growth hormone and (ii) a lipolysis-inhibiting agent.
17. A pharmaceutical composition according to claim 16, wherein
said agent is an HSL inhibitor.
18. A medical kit comprising (i) a growth hormone preparation and
(ii) one or more agents capable of causing a reduction in blood
lipid levels.
19. A medical kit according to claim 18, wherein said agent (ii) is
a lipolysis-inhibiting agent.
20. A medical kit according to claim 19, wherein said
lipolysis-inhibiting agent is an HSL inhibitor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119 of
Danish application no. PA 2003 00548 filed Apr. 9, 2003 and U.S.
application Ser. No. 60/467,079 filed May 1, 2003, the contents of
which are fully incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Obesity is known to be associated with serious risk factors,
and there is currently intense interest in identifying new
principles for treatment of this condition. These efforts have
hitherto resulted in identification of a substantial number of
potential central and peripheral targets for treatment. It has also
been shown that growth hormone (GH), more specifically human growth
hormone (hGH) in human beings, acts as a potent regulator of body
fat storage, and thus promotes breakdown of adipose tissue in obese
humans while preserving lean tissues. Since a large proportion of
glucose disposal and energy expenditure is thought to take place in
lean tissues, preservation of such tissues combined with a
selective loss of adiposity appears to be a highly desirable
objective.
[0003] Although it is known that the protein anabolic aspect of the
GH-insulin like growth factor-1 axis is influenced by diet
composition, there does not appear to have been any focus on the
question of whether this is also true for GH-stimulated loss of
adipose tissue. The effect of GH during restriction of energy
intake has also been unclear. There have been some reports
indicating no additional effects of GH administration compared to
the effect of energy restriction alone, whilst other reports have
indicated the such effects.
[0004] A recently reported study by the present inventors and
co-workers indicated GH-stimulated breakdown of adipose tissue in
genetically intact old rats that had become obese while receiving a
high-fat diet. However, despite normalisation of body fat stores
following GH injection, basal insulin levels were significantly
elevated.
[0005] It has been suggested that an excessive hyperinsulinemic
response to GH injections would decrease the net effect of GH on
adipose tissue. This would not be surprising since GH and insulin
have been suggested to have opposing effects in adipose tissue.
Moreover, sustained hyperinsulinemia would also increase the risk
of development of overt Type 2 diabetes in susceptible obese
individuals that already are at risk.
[0006] There is thus a clear need to identify factors determining
the insulin response to administration of GH, both from a
mechanistic and a safety point of view. The present inventors have
now obtained clear indications that the macronutrient composition
of the diet received by obese individuals constitutes one such
factor, and that total energy intake constitutes another.
SUMMARY OF THE INVENTION
[0007] The present inventors have found that the insulin response
in a subject to administration of GH can be modulated, for example,
by varying diet composition and caloric intake, and/or by
administering a drug which brings about a reduction in blood lipid
levels (more precisely a reduction in the level of one or more
blood lipid components), and that this influences adipose tissue
loss and serum leptin levels. Among blood lipid components, free
fatty acids (FFA) are of particular interest since high FFA levels
often are associated with a decrease in insulin sensitivity leading
to a compensatory hyper-secretion of insulin. A broad aspect of the
invention thus relates to a method for substantially preventing
hyperinsulinemia in an animal or human subject undergoing treatment
with growth hormone (GH), the method comprising subjecting the
subject, during the growth hormone treatment period, to one or more
measures (such a diet regimen and/or a drug treatment) which cause
a reduction in blood lipid levels (more precisely a reduction in
the level of one or more blood lipid components). Further aspects
of the invention include, inter alia:
[0008] (i) a method for achieving breakdown of adipose tissue in an
animal or human subject substantially without induction of
hyperinsulinemia in the subject, the method comprising
administering a growth hormone (GH) to the subject whilst
subjecting the subject to one or more measures which cause a
reduction in blood lipid levels (more precisely a reduction in the
level of one or more blood lipid components);
[0009] (ii) a method for reducing blood lipid levels (more
precisely reducing the level of one or more blood lipid components)
in an animal or human subject substantially without induction of
hyperinsulinemia in the subject, the method comprising
administering a growth hormone (GH) to the subject whilst
inhibiting lipolysis in the subject; and
[0010] (iii) a method for reducing blood lipid levels (more
precisely reducing the level of one or more blood lipid components)
in an animal or human subject substantially without induction of
hyperinsulinemia in the subject, the method comprising
administering a growth hormone (GH) to the subject whilst
stimulating lipid clearance from circulation.
[0011] Other aspects of the invention include medical kits suitable
for use in methods according to the invention.
[0012] Further detailed aspects of the invention are described
below.
LIST OF FIGURES
[0013] FIG. 1. Intakes of food and metabolizable energy, and body
weight development during the "fattening" period before GH dosing
in old rats fed a high-fat (HF) diet (.quadrature.-.quadrature.,
n=58) or a low-fat (LF) diet (.smallcircle.-.smallcircle., n=23).
Data represent means.+-.SE.
[0014] FIG. 2. Adipose tissue weight in relation to body weight and
plasma concentrations of leptin and insulin, in old rats injected
with growth hormone (GH) or saline (Sal). LF/LF signifies that
animals were fed a low-fat (LF) diet both in the "fattening" period
before GH dosing and during a 3-week GH-dosing period. By analogy,
HF/HF signifies that a high-fat (HF) diet was provided in both
periods, whereas HF/LF signifies that rats were shifted from the HF
diet to the LF diet as GH dosing began. The suffixes/subscripts
"re" and "pf" denote diet restriction and pair-feeding,
respectively (for details, see Table 2). Data represent means.+-.SE
(n=11-12).
[0015] FIG. 3. Plasma insulin-like growth factor-1 (IGF-1) levels
in old rats injected with growth hormone (GH) or with saline (Sal).
LF/LF signifies that animals were fed a low-fat (LF) diet both in
the "fattening" period before GH dosing and during a 3-week
GH-dosing period. By analogy, HF/HF signifies that a high-fat (HF)
diet was provided in both periods, whereas HF/LF signifies that
rats were shifted from the HF diet to the LF diet as GH dosing
began. The suffixes/subscripts "re" and "pf" denote diet
restriction and pair-feeding, respectively (for details, see Table
2). Data represent means.+-.SE (n=11-12).
[0016] FIG. 4. Plasma concentrations of glucose in old rats
injected with growth hormone (GH) or with saline (Sal). LF/LF
signifies that animals were fed a low-fat (LF) diet both in the
"fattening" period before GH dosing and during a 3-week GH-dosing
period. By analogy, HF/HF signifies that a high-fat (HF) diet was
provided in both periods, whereas HF/LF signifies that rats were
shifted from the HF diet to the LF diet as GH dosing began. The
suffixes/subscripts "re" and "pf" denote diet restriction and
pair-feeding, respectively (for details, see Table 2). Data
represent means.+-.SE (n=11-12).
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides methods and compositions for
minimizing the risk of development of diabetes as a consequence of
excessive overloading of insulin-producing beta-cells.
[0018] One aspect of the present invention relates to a method for
substantially preventing hyperinsulinemia in an animal or human
subject undergoing treatment with growth hormone (GH), the method
comprising subjecting the subject, during the growth hormone
treatment period, to one or more measures which cause a reduction
in blood lipid levels. The method in question is believed to be of
general applicability, irrespective of the underlying rationale for
treatment of the subject with growth hormone. Thus, for example,
the method of the invention may be used in the context of
established GH treatments of immature humans (children or
adolescents), such as for the purpose of stimulating growth to
counteract development of short stature or dwarfism, as well as of
mature (adult) humans.
[0019] A closely related further aspect of the invention relates to
a method for achieving breakdown of adipose tissue in an animal or
human subject--particularly an obese human subject--substantially
without induction of hyperinsulinemia in the subject, the method
comprising administering a growth hormone (GH) to the subject
whilst subjecting the subject to one or more measures which cause a
reduction in blood lipid levels.
[0020] Measures of the type referred to in relation to methods of
the invention include, without limitation, diet regimens or drug
treatments. In one embodiment, the subject is provided with
restricted amounts of a high-fat (HF) diet as sole food source
(nutrition source). Appropriate drug treatments include, without
limitation, treatment with agents such as the
antihyperlipoproteinemic Acipimox.TM. (Olbetam.TM.), i.e.
5-methylpyrazinecarboxylic acid 4-oxide, and related compounds (see
U.S. Pat. No. 4,002,750); statins, such as Fluvastatin.TM.,
Lovastatin.TM., Pravastatin.TM. or Simvastatin.TM.; and fibrates,
such as Bezafibrat.TM., Clofibrat.TM. or Gemfibrozil.TM., in
amounts and dosage regimens that are effective in lowering blood
lipid levels.
[0021] Another, related aspect of the present invention relates to
a method for reducing blood lipid levels in an animal or human
subject substantially without induction of hyperinsulinemia in the
subject, the method comprising administering a growth hormone (GH)
to the subject whilst providing the subject with restricted amounts
of a high-fat (HF) diet as sole food source.
[0022] A still further aspect of the invention provides another
method for reducing blood lipid levels in an animal or human
subject substantially without induction of hyperinsulinemia in the
subject, the method comprising administering a growth hormone (GH)
to the subject whilst inhibiting lipolysis in the subject. In this
connection, inhibition of lipolysis may be reflected in inhibition
of the lipase known as Hormone-Sensitive Lipase (HSL), although
inhibition of other families of lipases may also be of relevance in
the context of the method in question according to the invention.
Non-limiting examples of substances capable of inhibiting the
lipolytic effect of HSL include those disclosed in U.S. Pat. No.
6,596,742 B1 (corresponding to WO 01/17981), WO 01/66531, WO
03/051841, WO 03/051842 and WO 03/105860.
[0023] Yet another aspect of the invention relates to a method for
reducing blood lipid levels in an animal or human subject
substantially without induction of hyperinsulinemia in the subject,
the method comprising administering a growth hormone (GH) to the
subject whilst stimulating lipid clearance from the circulation. In
this connection, stimulation of clearance of lipid from the
circulation may, for example, be achieved by administration of a
substance which acts to stimulate, activate or potentiate a lipase
such as Lipoprotein Lipase (LPL). Non-limiting examples of
LPL-potentiating substances include those described in WO
01/27088.
[0024] With regard to what constitutes "restricted" amounts of a HF
diet in the context of the invention, it is generally preferable
that the energy content (caloric content) of the amount of HF diet
with which the subject is provided does not exceed (i.e. is below
or is equal to or at least approximately equal to) the theoretical
maintenance level for the subject in question. In the case of human
subjects, there is an extensive body of published data which
enables the establishment of the theoretical maintenance level for
an individual on the basis of parameters such as age, gender,
weight, height, ethnicity and level of physical activity. Published
sources of such data include: Ritz, P., Factors affecting energy
and macronutrient requirements in elderly people, Public Health
Nutrition 4 (2001) pp. 561-569; and Lin et al., Estimation of
energy requirements in a controlled feeding trial, Am. J. Clin.
Nutr. 77 (2003) pp. 639-645.
[0025] With regard to animal species, particularly "farm" animals
(animals of importance in relation to production of meat products,
dairy products, eggs and the like, such as cattle, pigs, goats and
poultry), and other domestic animals, such as horses, data are
available from sources such as the UK Agricultural Research Council
(ARC), the Commonwealth Agricultural Bureau, and the US National
Research Council (NRC; e.g. data from 1988 and 1998, published by
National Academy Press, Washington D.C.).
[0026] In some embodiments, the present invention relates to the
treatment of humans, in particular obese humans. In these
embodiments, the growth hormone to be employed will preferably be
human growth hormone (hGH).
[0027] In the light of the above methods of the invention, still
further aspects of the present invention include the following:
[0028] (i) Pharmaceutical compositions comprising, as active
ingredients, a growth hormone and an agent selected from: agents
capable of reducing blood lipid levels; lipolysis-inhibiting agents
(e.g. HSL inhibitors); and lipase-activating or -potentiating
agents (e.g. LPL activators or potentiators);
[0029] (ii) Medical kits suitable for use in methods according to
the invention and comprising a growth hormone preparation and one
or more measures which cause a reduction in blood lipid levels,
such as, e.g., a medical kit comprising a growth hormone
preparation and a high-fat diet, a medical kit comprising a growth
hormone preparation and a drug which causes a reduction in blood
lipid levels, a medical kit comprising a growth hormone preparation
and a lipolysis-inhibiting agent (such as an HSL inhibitor), or a
medical kit comprising a growth hormone preparation and a
lipolysis-activating or -potentiating agent (such as a LPL
activator or potentiator).
[0030] Also encompassed by the invention is the use of a substance
which acts as a growth hormone secretagogue (GHS; also known, inter
alia, as a growth hormone releasing substance), i.e. a substance
which, when administered to a subject by an appropriate route, is
capable of stimulating the release of growth hormone from the
pituitary of the subject, as an alternative to a GH per se in the
various aspects of the invention (i.e. as an active ingredient in
methods, pharmaceutical compositions, medical kits etc. as
described above). Non-limiting examples include the synthetic
hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH- .sub.2, also known as
GHRP-6 (see, e.g., Bowers et al. in Endocrinology 114 (1984) pp.
1537-1545 and in Endocrinology 128 (1991) pp. 2027-2035) and the
peptide derivatives described in WO 95/17423. Naturally occurring
growth hormone releasing substances of potential relevance in the
context in question include so-called "growth hormone releasing
hormone" (often abbreviated GHRH or GHRH(1-44)NH.sub.2) and
truncated forms thereof (see, e.g., Guillemin et al., Science 218
(1982) pp. 585-587 and Rivier et al., Nature 300 (1982) pp.
276-278).
Pharmaceutical Administration
[0031] The regimen for treatment of a given subject/patient with
growth hormone and, where appropriate, with another drug, in the
manner described herein, may be determined by one skilled in the
art. The daily dose to be administered can be determined by a
physician and will depend on the particular substance employed, on
the route of administration and on the age and the condition of the
subject or patient. A convenient daily dosage of GH is typically in
the range of from about 0.001 mg/kg body weight to about 2.0 mg/kg
body weight, often from about 0.01 mg/kg body weight to about 1.0
mg/kg body weight. The therapeutic dose of the substance will
depend upon the frequency and mode of administration, the sex, age,
weight and general condition of the subject treated, the nature and
severity of the condition treated and any concomitant diseases to
be treated and other factors evident to those skilled in the
art.
[0032] GH may be administered in a single dose or in repeated doses
during the day. Administration in the manner described herein
should continue until the treated individual is no longer in need
of such treatment, for example, until an initially obese individual
is no longer obese.
[0033] The route of GH administration may be any route that
effectively transports the active compound to the appropriate or
desired site of action, such as by infusion (continuous or
pulsatile), injection, pulmonary inhalation, or by oral or nasal
administration. Presently preferred routes include parenteral
routes (e.g. via intramuscular, intraperitoneal, intravenous or
subcutaneous injection, or by implant). The growth hormone can be
formulated in dosage forms appropriate for each route of
administration. The compositions or dosage forms may be in
conventional forms, e.g. aerosols, solutions or suspensions.
[0034] A GH composition may be in a form suited for systemic
injection or infusion, and may, as such, be formulated with a
suitable liquid vehicle such as sterile water or an isotonic saline
or glucose solution. The compositions may be sterilized by
conventional sterilization techniques which are well known in the
art. The resulting aqueous solutions may be packaged for use as
such, or they may be filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with the
appropriate sterile aqueous vehicle prior to administration. The
composition may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as buffering agents, tonicity-adjusting agents and the like.
Non-limiting examples of buffering agents include citrate salts and
histidine; non-limiting examples of tonicity adjusting agents
include sugars, such as sucrose and mannitol, and salts, such as
alkali metal and alkaline earth metal chlorides, e.g. sodium,
potassium or calcium chloride, and the like. Examples of liquid
carriers are syrup, peanut oil, olive oil, phospholipids, fatty
acids, fatty acid amines, polyoxyethylene and water. Aqueous liquid
formulations, in particular, may advantageously contain a non-ionic
surfactant, e.g. a polysorbate [such as polysorbate 20 (e.g.
Tween.TM. 20) or a poloxamer [such as poloxamer 188 (e.g.
Pluronic.TM. F68) or poloxamer 407 (e.g. Lutrol.TM. F127)], and a
preservative, such as benzyl alcohol, phenol or a cresol (e.g.
m-cresol), will often be incorporated.
[0035] It may be advantageous to provide GH in the form of a
sustained release formulation. As such, the composition may be
formulated as microcapsules or microparticles containing the growth
hormone encapsulated in, or dispersed in, a suitable
pharmaceutically acceptable biodegradable polymer, such as
polylactic acid, polyglycolic acid or a lactic acid/glycolic acid
copolymer.
[0036] For nasal administration, the GH preparation may contain
growth hormone dissolved or suspended in a liquid carrier, in
particular an aqueous carrier, for aerosol application. The carrier
may contain additives such as solubilizing agents (e.g. propylene
glycol), surfactants, absorption-enhancers such as lecithin
(phosphatidylcholine) or cyclodextrin, or preservatives such as
parabenes.
[0037] Growth hormone may be formulated by any of the established
methods of formulating pharmaceutical compositions, e.g. as
described in Remington: The Science and Practice of Pharmacy
(1995).
[0038] A liquid hGH formulation which is well suited, in
particular, for administration by injection in the context of the
present invention is Norditropin.TM. SimpleXx.TM. (Novo
Nordisk).
Definitions
[0039] High-fat (HF) diet: A high-fat diet for humans, includes,
without limitation, that given by M. R. Freedman et al. in a review
article in Obesity Research 9, Suppl. 1 (March 2001) pp. 1S-40S.
With reference to humans, the following table provides appropriate
definitions not only of high-fat (HF) diets in the context of the
present invention, but also of moderate-fat (MF) and low-fat (LF)
diets:
1TABLE Caloric composition of diets* Fat Carbohydrate Protein Diet
type (% kcals) (% kcals) (% kcals) High-fat (HF) 55-65 <20%
25-30 Moderate-fat (MF) 20-30 55-60 15-20 Low-fat (LF) 11-19 >65
10-20 *M. R. Freedman, J. King and E. Kennedy, Popular diets: a
scientfic review, Obesity Research 9, Suppl. 1 (March 2001) pp.
1S-40S
[0040] Obesity: the terms "obesity" and "obese" when employed in
the context of the present invention imply an excess of adipose
tissue. In this context obesity is best viewed as any degree of
excess adiposity that imparts a health risk. The distinction
between normal and obese individuals can only be approximated, but
the health risk imparted by obesity is probably a continuum with
increasing adiposity. However, in the context of the present
invention, human individuals with a body mass index (BMI=body
weight in kilograms divided by the square of the height in meters)
above 25 are to be regarded as obese.
[0041] Growth hormone (GH): Growth hormone is a hormone that
stimulates growth of all tissues capable of growing. Growth hormone
is released from the pituitary. The release is under tight control
of a number of hormones and neurotransmitters, either directly or
indirectly. Growth hormone release can be stimulated by growth
hormone releasing hormone (GHRH; vide supra) and inhibited by
somatostatin. In both cases, the hormones are released from the
hypothalamus, but their action is mediated primarily via specific
receptors located in the pituitary. In the present context "growth
hormone" may be growth hormone of any origin, e.g. avian, bovine,
equine, human, ovine, porcine, salmon, trout or tuna growth
hormone. Human growth hormone (hGH) will normally be preferred for
the treatment of humans. The growth hormone used in accordance with
the invention may be native growth hormone isolated from a natural
source, e.g. by extracting pituitary glands in a conventional
manner, or a growth hormone produced by recombinant techniques,
e.g. as described in E. B. Jensen and S. Carlsen in Biotech and
Bioeng. 36 1990) pp. 1-11. The term growth hormone (GH) in the
context of the invention also encompasses: truncated forms of GH,
i.e. truncated forms of a growth hormone wherein one or more amino
acid residues has/have been deleted; GH analogues, wherein one or
more amino acid residues in the native molecule has/have been
substituted with another amino acid residue, preferably a residue
of a naturally occurring amino acid, as long as the substitution
does not lead to any adverse effect such as antigenicity or reduced
activity; and GH derivatives, e.g. deamidated or sulfoxidated forms
of the growth hormone, or forms having an N- or C-terminal
extension (such as Met-hGH, Met-Glu-Ala-Glu-hGH or Ala-Glu-hGH).
Other GH derivatives of relevance include those in which a GH (or
biologically active fragment thereof) is conjugated to a molecule
such as an albumin, e.g. human serum albumin (see, e.g., WO
97/24445), or a water-soluble polymer such as a polyethyleneglycol
(PEG) (see, e.g., WO 03/044056), in order to achieve, e.g.,
protracted duration of GH activity. As mentioned above, among
growth hormones per se, the preferred growth hormone in relation to
treatment of humans is normally hGH (i.e. natural human growth
hormone, or recombinantly produced human growth hormone which is
identical to the natural hormone). Methionylated human growth
hormone may, however, often be employed.
[0042] Blood lipids: Lipids are generally defined as being a group
of fats, fat-derived substances and fatlike substances that are of
low solubility in water, are soluble in organic solvents such as
benzene, chloroform and ether, and are utilizable by animal
organisms. They are easily stored in the body, serve as a source of
fuel (energy), are important constituents of cell structure, and
serve other physiological functions. Lipids may be considered to
include neutral fats, fatty acids, certain steroidal substances
(e.g. sterols) and certain waxes.
[0043] Fats: Fats (neutral fats) are glyceryl esters of higher
fatty acids (such as stearic and palmitic acid), and include, in
particular, triglycerides (glyceryl esters in which each of the
three hydroxy groups of glycerol is esterified with a fatty
acid).
[0044] Fatty acids: Fatty acids are generally defined as being
straight-chain, saturated and unsaturated monocarboxylic acids in
which the carboxylic acid group is terminal, and which have a total
(usually even-numbered) of from 4 to 22 (sometimes 24) carbon
atoms.
[0045] Steroidal substances: These include, e.g., cholesterol (a
sterol). In blood, cholesterol is transported primarily in the form
of various lipoproteins, e.g. the so-called low-density
lipoproteins (LDL) and high-density lipoproteins (HDL). In analyses
of lipid levels, e.g. in blood, cholesterol is often determined as
total cholesterol, i.e. the sum total level of all forms of
cholesterol present in the blood.
[0046] In blood, lipids of major importance include triglycerides,
fatty acids [i.e. free fatty acids (FFA)], and cholesterol.
[0047] Usage of the terms "lipid", "fat", "fatty acid",
"triglyceride", "cholesterol" and "total cholesterol" in relation
to blood lipids and levels thereof in the context of the present
invention is in accordance with the definitions and explanation
given above.
[0048] Hyperinsulinemia
[0049] As used herein, hyperinsulinemia encompasses a fasting
plasma level of insulin greater than the normal range, which may
be, e.g., about 30 .mu.IU (micro international units) per ml.
Insulin may be measured by any conventional method, including,
e.g., radioimmunoassay (Pharmacia, Medical Research Laboratories
International).
[0050] Prevention of hyperinsulinemia encompasses the clinical
observation that a patient receiving GH therapeutically does not
exhibit a significant rise in fasting plasma insulin levels over
that exhibited by the same patient prior to GH treatment. It also
encompasses the clinical observation that a patient receiving GH
therapy, who exhibits hyperinsulinemia consequent to the GH
treatment, exhibits a detectable reduction in fasting plasma
insulin levels once the measures of the invention (e.g., restricted
high-fat diet and/or lipid-lowering drugs) have been added to the
treatment regimen.
[0051] The following are intended as non-limiting examples of the
present invention.
EXAMPLE 1
Effects of Diet and GH on Body Composition, Blood Lipids, and
Insulin
[0052] The following experiments were performed in order to
evaluate the effect of varying diet on the effect of GH
administration on insulin levels and other metabolic
parameters.
Materials and Methods
[0053] Animals and Test Substances
[0054] Female Wistar rats weighing about 260 g were purchased 1
month before the start of experiments from M.o slashed.lleg.ang.rd
Breeding and Research Centre (Lille Skensved, Denmark). On arrival,
rats were placed in conventional rat cages housing 2-3 animals.
They were weighed weekly, and had free access to drinking water and
a standard rat feed. All diets, including the experimental diets
(see Table 1, below), were purchased from a local feed supplier
(Brogaarden, Gentofte, Denmark). The experimental protocol was
approved by the Danish national ethical committee for animal
experiments (Dyrefors.o slashed.gstilsynet, Copenhagen, Denmark).
The growth hormone (GH) used in the present study was recombinantly
produced human GH (hGH) from Novo Nordisk A/S (Bagsv.ae butted.rd,
Denmark).
[0055] Experimental Procedures
[0056] At an age of 12 months, rats were randomly assigned to
receive either a high-fat (HF) diet [number of rats (n)=58] or a
low-fat (LF) diet (n=23) (Table 1).
2TABLE 1 Composition of high-fat (HF) and low-fat (LF) diets with
theoretical energy content (percent) in parentheses. Diets
Ingredients (g/kg) HF LF Maize meal 493 818 Wheat bran 27 27 Casein
148 110 Animal fat 300 13 Vitamins and minerals 32 32 Chemical
composition (g/kg) Crude protein (energy %) 170 (18%) 170 (25%)
Crude fat (energy %) 320 (55%) 50 (12%) Carbohydrates, total 344
(27%) 565 (63%) Crude fibre 17 26 Disaccharides 11 18
Polysaccharides 316 521 Metabolizable energy (Mcal/kg) 4.8 3.2
Fatty acids Total (% of crude fat) 94 92 Saturated (% of total) 49
25 Mono-unsaturated (% of total) 39 30 Poly-unsaturated (% of
total) 12 45
[0057] The diet in question was continued for 14 weeks, during
which body weight development was recorded (FIG. 1). During this
time, rats were assigned to 8 groups for dosing of GH thereafter,
as indicated in Table 2.
3TABLE 2 Allocation of rats to high-fat (HF) and low-fat (LF) diets
during a 14-week period before dosing and during a 3-week period of
dosing with either saline or growth hormone (GH). GH was
administered in a total dose of 4 mg/kg/day, divided into two
injections. Diet before dosing Diet during dosing Dosing n
Denomination Low Fat Low Fat Saline 11 LF/LF-Sal GH 12 LF/LF-GH
High Fat High Fat Saline 12 HF/HF-Sal GH 11 HF/HF-GH Energy
restricted* GH 12 HF/HF-GHre Low Fat Saline 11 HF/LF-Sal GH 12
HF/LF-GH Pair fed* Saline 12 HF/LF-Salpf *Age- and weight-matched
rats were fed the same amount of metabolizable energy consumed by
HF/LF-GH-group. In the HF/HF-GHre group, this was achieved by
restricting food intake (energy-restricted), whereas rats in the
HF/LF-Salpf group were pair-fed with the HF/LF-GH group. Both these
groups were run behind the others.
[0058] Injection of GH continued for 3 weeks, after which the rats
were sacrificed by decapitation. After bleeding, serum and plasma
were prepared and frozen at -80.degree. C.
[0059] The large fat pad embedding the kidneys (denoted peri-renal
fat), and fat pads around the uterus, ovaries and intestines
(denoted body fat or adipose tissue) were quickly dissected. After
dissection the adipose tissue was weighed and frozen at -80
.degree. C. One thigh muscle, the quadriceps femoris, was also
dissected and weighed.
[0060] With a view, inter alia, to supplementing measurements of
changes in levels of blood lipids, glycerol and
.beta.-hydroxy-butyrate (BHBT) as a consequence of the
administration of GH or saline in combination with the various diet
regimens, a series of similar experiments of shorter duration (4
days) was conducted using 45 rats (n=15 in each group) that were
all obese due to 10 weeks of feeding with high-fat (HF) diet. All
the animals in question received a low-fat (LF) diet during the
4-day treatment period. They were divided into three groups
receiving (a) saline alone, (b) GH and (c) saline in combination
with pair-feeding with the GH group.
[0061] Chemical Analyses
[0062] The content of fat in muscle tissue was analysed by
Bioteknologisk Institut (Kolding, Denmark) using gas chromatography
and employing standardized methods. Plasma concentrations of
metabolites were determined with a Synchron.TM. CX5 auto-analyzer
system (Beckman Instruments, Fullerton, USA).
[0063] Blood Analyses
[0064] Concentrations of lipids, glycerol and
.beta.-hydroxybutyrate (BHBT) were likewise determined using a
Synchron.TM. CX5 auto-analyzer system (Beckman Instruments,
Fullerton, USA).
[0065] Hormone Assays
[0066] Total plasma IGF-I was measured after acid-ethanol
extraction as previously described (19); the intra-assay and
inter-assay coefficients of variation were 6% and 13%,
respectively. Plasma insulin was determined using an assay
described previously (20); the intra- and inter-assay coefficients
of variation for this assay were 5.4% and 8.4% respectively. Plasma
leptin was determined using a commercial kit from Linco research,
Inc. (St Charles, USA); the intra- and inter-assay coefficients of
variation were 4.6% and 5.7%, respectively.
[0067] Statistical Analyses
[0068] All experimental data were entered into the 6.11 version of
the SAS statistical software program, whereby descriptive
statistics were calculated using the univariate procedure (SAS Inc,
Cary, USA). Before further analysis, data were checked for normal
distribution. In some cases where deviations were found, data were
logarithmically transformed to achieve such a distribution.
Potential differences between treatment groups were tested with a
one-way analysis of variance (GLM procedure of SAS) followed by
Duncan's multiple-range test. In most cases these tests were
performed with an .alpha.-value of 0.05, but where applicable an
.alpha.-value of 0.01 was used. Data are presented as
means.+-.standard error (SE).
Results
[0069] Body Weight Gain and Feed Consumption in the Period Before
GH Dosing
[0070] At the start of the 14-week "fattening" period, rats in the
HF group and the LF group had body weights of 279.+-.3 and 280.+-.6
g, respectively. Rats in the HF group gained 111.+-.5 g in body
weight during the 14 week period. Rats receiving the LF diet gained
63.+-.5 g, which was significantly (p<0.01) lower. This resulted
in significant (p<0.01) differences in live weights, viz.
390.+-.6.4 (HF diet group) vs. 343.+-.8.6 g (LF diet group).
[0071] As can be seen from FIG. 1, rats given the HF diet had a
comparatively high intake of food, and consequently a comparatively
high metabolizable energy intake in the first phase of the
"fattening" period. This was followed by a clear decline, so that
in the last phase, intakes of metabolizable energy in the two
groups were similar.
[0072] Food Consumption During GH Dosing
[0073] As was the case during the "fattening" period, rats fed the
LF diet during GH dosing consumed significantly (p<0.05) more
food than did rats fed the HF diet (see Table 3, below), but due to
the lower energy content of the LF diet the intakes of
metabolizable energy were not significantly different between the
two groups.
[0074] There was a general decrease in food intake in the first
phase after commencement of GH injections (data not shown), thus
confirming our previous observations (18). Measured over the
three-week dosing period, this effect remained statistically
significant for rats fed the LF diet (Table 3). GH-treated rats
which were switched from the HF diet to the LF diet successively
increased their consumption between the second and third week of
treatment, ending up with about the same total amount of food
ingested as the corresponding saline-control rats. During this
phase the pair-fed group could not follow the GH-treated group, and
small food refusals were occasionally observed, resulting in a
slightly lower total consumption of food (Table 3).
4TABLE 3 Effects of growth hormone (GH) dosing on: consumption of
high-fat (HF) and low- fat (LF) diets, body weights, adipose
tissue, skeletal muscle weight and muscle content of fat. Food
intake was registered per cage unit containing 2-3 animals (n = 6),
and fat in muscle was analysed on a randomly selected number (n =
5) of animals from each group. In other cases, means .+-. SE are
based on 11-12 observations. Intakes Body weight Diet Diet
Metaboliza- Change in before during Food ble energy Protein Start
weight Final weight weight dosing dosing Dosing (g/kg/d) (Cal/kg/d)
(g/kg/d) (g) (g) (g) Low-fat Low-fat Saline 46.sup.a .+-.2.0
149.sup.ab .+-.6.5 7.8.sup.a .+-.0.34 345.sup.bc .+-.13 355.sup.c
.+-.11 9.7.sup.d .+-.3.6 GH 40.sup.b .+-.1.9 129.sup.bc .+-.6.1
6.8.sup.b .+-.0.32 341.sup.c .+-.12 419.sup.abc .+-.11 78.sup.a
.+-.3.7 High-fat High-fat Saline 33.sup.cd .+-.1.7 158.sup.a
.+-.8.0 5.6.sup.cd .+-.0.28 391.sup.ab .+-.17 400.sup.bcd .+-.16
9.0.sup.d .+-.3.7 GH 29.sup.d .+-.1.5 142.sup.ab .+-.7.2 5.0.sup.d
.+-.0.25 390.sup.ab .+-.15 452.sup.a .+-.13 62.sup.b .+-.4.9 Energy
GH 22* .+-.0.0 106.sup.* .+-.0.12 3.7.sup.* .+-.0.00 389.sup.ab
.+-.9 429.sup.ab .+-.9 40.sup.c .+-.7.6 restricted* High-fat
Low-fat Saline 37.sup.bc .+-.2.4 119.sup.cd .+-.7.9 6.2.sup.bc
.+-.0.42 388.sup.ab .+-.17 381.sup.cde .+-.16 -7.sup.e .+-.3.6 GH
33.sup.cd .+-.2.8 106.sup.d .+-.9.0 5.6.sup.cd .+-.0.47 389.sup.ab
.+-.16 431.sup.ab .+-.10 41.sup.c .+-.8.2 Pair fed* Saline 30.sup.*
.+-.2.1 97.sup.* .+-.6.7 5.1.sup.* .+-.0.35 393.sup.a .+-.17
371.sup.de .+-.15 -22.sup.e .+-.5.4 Tissue weights and composition
Diet Diet Adipose Skeletal Fat in before during tissue Muscle
Muscle dosing dosing Dosing (g) (g) (%) Low-fat Low-fat Saline
41.sup.bc .+-.3.8 2.3.sup.b .+-.0.10 4.4.sup.a .+-.1.6 GH 30.sup.de
.+-.2.3 3.0.sup.a .+-.0.07 1.7.sup.c .+-.0.2 High-fat High-fat
Saline 60.sup.a .+-.4.7 2.3.sup.b .+-.0.08 3.1.sup.abc .+-.0.9 GH
38.sup.cd .+-.2.7 2.8.sup.a .+-.0.11 1.9.sup.bc .+-.0.4 Energy GH
26.sup.e .+-.1.6 3.0.sup.a .+-.0.16 1.8.sup.bc .+-.0.1 restricted*
High-fat Low-fat Saline 50.sup.ab .+-.4.5 2.2.sup.b .+-.0.09
3.8.sup.ab .+-.0.6 GH 31.sup.cde .+-.2.7 2.9.sup.a .+-.0.07
1.6.sup.c .+-.0.3 Pair fed* Saline 49.sup.b .+-.3.8 2.2.sup.b
.+-.0.05 2.8.sup.abc .+-.0.4 *Groups that did not have free access
to food were excluded from statistical analyses of food intake
variables. .sup.a,b,c,d,eDifferences between groups were tested
with a one-way analysis of variance followed by Duncan's multiple
range-test. Values within columns not sharing a common letter
superscript differ significantly (p < 0.05).
[0075]
5TABLE 4 Effect of GH and diet on levels of blood lipids and
related substances. Duration of Diet before Diet during Experiment
dosing dosing Dosing FFA Glycerol BHBT Triglyceride Cholesterol 4
Days High-fat Low-fat Saline 0.56 .+-. 0.05 0.33 .+-. 0.01 0.60
.+-. 0.07 0.69 .+-. 0.09 1.17 .+-. 0.09 4 Days High-fat Low-fat GH
0.63 .+-. 0.07 0.34 .+-. 0.01 .sup. 1.32 .+-. 0.24.sup.4 .sup. 0.48
.+-. 0.03.sup.5 .sup. 0.64 .+-. 0.05.sup.7 4 Days High-fat Pair-fed
Saline 0.68 .+-. 0.07 0.30 .+-. 0.01 .sup. 1.17 .+-. 0.11.sup.4
0.39 .+-. 0.03 1.03 .+-. 0.08 21 Days Low-fat Low-fat Saline 0.75
.+-. 0.08 0.39 .+-. 0.03 0.25 .+-. 0.04 1.41 .+-. 0.21 1.39 .+-.
0.10 21 Days Low-fat Low-fat GH .sup. 0.43 .+-. 0.04.sup.1 .sup.
0.33 .+-. 0.02.sup.3 0.24 .+-. 0.02 1.40 .+-. 0.14 .sup. 1.01 .+-.
0.08.sup.7 21 Days High-fat High-fat Saline 0.49 .+-. 0.03 0.35
.+-. 0.03 0.51 .+-. 0.04 0.88 .+-. 0.09 1.27 .+-. 0.06 21 Days
High-fat High-fat GH .sup. 0.36 .+-. 0.04.sup.1 0.39 .+-. 0.02 0.30
.+-. 0.04 .sup. 1.54 .+-. 0.12.sup.6 .sup. 0.90 .+-. 0.07.sup.7 21
Days High-fat Energy-restricted GH .sup. 0.20 .+-. 0.01.sup.2 .sup.
0.20 .+-. 0.01.sup.2 .sup. 0.92 .+-. 0.09.sup.2 .sup. 0.53 .+-.
0.04.sup.2 0.82 .+-. 0.03 21 Days High-fat Low-fat Saline 0.60 .+-.
0.06 0.38 .+-. 0.03 0.17 .+-. 0.03 1.58 .+-. 0.25 1.21 .+-. 0.10 21
Days High-fat Low-fat GH .sup. 0.38 .+-. 0.04.sup.1 .sup. 0.30 .+-.
0.01.sup.3 0.22 .+-. 0.03 1.27 .+-. 0.13 .sup. 0.92 .+-. 0.05.sup.7
21 Days High-fat Pair-fed Saline 0.74 .+-. 0.07 0.41 .+-. 0.03
.sup. 0.50 .+-. 0.07.sup.4 1.00 .+-. 0.13 1.28 .+-. 0.06 FFA = free
fatty acids; BHBT = .beta.-hydroxybutyrate; Cholesterol = total
cholesterol. All values in mM. Superscript numbers indicate
significant effects as follows: .sup.1GH lowered FFA level after 21
days compared to saline; .sup.2FFA, glycerol and triglyceride
levels in the high-fat/energy restricted group were lower, while
BHBT level was elevated, compared to the other GH-treated groups
initially fed high-fat diet; .sup.3GH lowered glycerol levels in
animals on low-fat diet
[0076] Superscript numbers indicate significant effects as follows:
.sup.1GH lowered FFA level after 21 days compared to saline;
.sup.2FFA, glycerol and triglyceride levels in the high-fat/energy
restricted group were lower, while BHBT level was elevated,
compared to the other GH-treated groups initially fed high-fat
diet; .sup.3GH lowered glycerol levels in animals on low-fat diet
compared to saline; .sup.4BHBT level elevated compared to saline;
.sup.5Triglyceride decreased after 4 days of GH treatment compared
to saline, but not with the pair-fed group; .sup.6After 21 days,
the combination of GH and high-fat diet caused elevated
triglyceride levels compared to saline; .sup.7GH decreased the
cholesterol level after both 4 and 21 days compared to saline
[0077] Body Weight During Dosing
[0078] Irrespective of diet, three weeks of GH treatment generally
increased body weight (Table 3). A change of diet from HF to LF,
combined with saline alone, produced a significant (p<0.05)
reduction of live weight. Rats with the same dietary record which
were pair fed with GH-treated counterparts also lost weight (Table
3).
[0079] Effects of Diet and GH on Body Composition
[0080] GH treatment significantly (p<0.05) decreased the weight
of adipose tissue excised from rats fed HF or LF diet, or rats that
were switched from HF to LF, both in absolute numbers (Table 3) and
in relation to their body weight (FIG. 2). Pair-feeding alone did
not significantly affect the weight of adipose tissue. In parallel
with the reductions of adipose tissue weight seen in animals
injected with GH, fresh muscle (Quadriceps femoris) weights
generally increased significantly (p<0.05) (Table 3). When
expressed in relation to body weight, this effect was not
statistically significant in rats that were on the HF diet
throughout the entire study. The fat content of muscle tissue was
generally decreased in all groups injected with GH, although the
decrease was not statistically significant in all instances (Table
3).
[0081] Effects of Diet and GH on Blood Lipids
[0082] The blood concentrations (levels) of free fatty acids (FFA),
glycerol, .beta.-hydroxybutyrate (BHBT), triglyceride and
cholesterol (total cholesterol) are shown in Table 4. Serum FFA
levels and total cholesterol levels were lowered significantly
after 21 days of GH treatment, as was plasma glycerol, except
during HF diet feeding. Triglyceride levels were elevated at that
time, but only when GH was combined with HF diet. In contrast,
triglycerides were markedly lowered by feeding restricted amounts
of HF diet in combination with GH treatment, whilst no changes were
seen in the other groups. Cholesterol levels were already low after
4 days of GH injection. BHBT increased initially following GH
injection and pair-feeding. Only the group which received GH and
restricted access to HF diet, and the pair-fed group, exhibited
elevated BHBT levels after 21 days.
[0083] Effects of GH on Plasma Variables
[0084] Injection with GH produced a significant (p<0.05)
increase of plasma IGF-1 concentrations irrespective of dietary
regimen (FIG. 3). However, the increase was significantly
(p<0.05) lower in animals with restricted access to the HF diet.
Exceptionally low insulin and leptin levels were also observed in
this group (FIG. 2), and glucose levels in this group were
concomitantly significantly decreased in comparison to any other
group (FIG. 4). In contrast, GH administration to animals with free
access to the HF diet produced a marked hyperinsulinemia, and no
fall in leptin levels was seen; glucose levels in these animals
were likewise not significantly changed compared with levels in
saline-control animals.
Discussion of Results
[0085] At the conclusion of the experiments described above, rats
that had received the HF diet throughout contained about 30% more
adipose tissue than rats fed the LF diet. This difference is likely
founded in the phase when rats first were introduced to a HF diet,
since in that phase their daily intake of metabolizable energy was
clearly elevated for several weeks. After about 6 weeks, their
caloric intake had fallen to the same level as that of rats fed the
LF diet, indicating that although this adaptation is not as fast as
has been observed in young animals, the old rats employed in the
present work still retain some ability to regulate their caloric
intake.
[0086] Injections of GH produced an increase in skeletal muscle
weight, loss of adipose tissue and a transient decrease in food
intake in those groups of animals that had free access to food.
These effects were observed irrespective of whether rats were
maintained on their habitual diets or were shifted from the HF to
the LF diet. Shift of diet without GH injection, but with
essentially the same caloric intake (pair-fed), produced a fall in
body weight, but only marginal effects on adipose tissue weight. In
view of this, it can be concluded that treatment with GH produces a
significant and specific loss of adipose tissue. It also appears
that promotion of fat loss is a consistent effect of GH under a
variety of dietary conditions, but that modulation of this effect
by the amount of diet eaten occurs. Thus, if the HF diet is fed in
restricted amounts, the fat loss after GH injections is greater
than if there is free access to the same diet (and thereby is
greater consumption of metabolizable energy). This observation
shows that caloric intake either directly or indirectly modulates
the effect of GH on fat loss.
[0087] Surpisingly low plasma insulin levels were found in blood
from animals which had been fed restricted amounts of the HF diet
during GH treatment, whereas hyperinsulinemia was especially marked
in the case of animals which had had free access to the same diet.
The degree of adipose tissue breakdown was significantly different
between these groups. The present inventors thus believe that they
have identified a dietary situation whereby the hyperinsulinemic
response to GH administration may be avoided, thus presenting the
possibility of increasing both the efficacy and safety aspects of
GH treatment of obese humans. It can be seen from Table 4 that this
dietary situation is associated with significant reductions in
blood lipid levels, especially in the case of FFA levels. This can
most probably explain the low insulin levels since it is widely
established that high FFA levels are associated with a decrease in
insulin sensitivity, leading to a compensatory hyper-secretion of
insulin. There is little doubt that prolonged and uncontrolled
hyperinsulinemia represents a real hazard to the patient and must
be taken seriously. For this reason the present invention is
believed to be of considerable importance.
[0088] To summarize, the results reported herein demonstrate not
only that GH mediates breakdown of adipose tissue under a variety
of dietary conditions, but--very importantly and surprisingly--that
induction of hyperinsulinemia can be prevented if GH treatment is
combined with restricted feeding of a diet which is relatively low
in carbohydrates and rich in fat, and that same treatment regimen
also promotes a fall in plasma leptin levels.
* * * * *