U.S. patent application number 15/482592 was filed with the patent office on 2018-10-11 for growth hormone releasing factor analogs and uses.
The applicant listed for this patent is EZ IP, LLC. Invention is credited to Joey D. Edge, Chanda Zaveri.
Application Number | 20180289774 15/482592 |
Document ID | / |
Family ID | 63710551 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180289774 |
Kind Code |
A1 |
Zaveri; Chanda ; et
al. |
October 11, 2018 |
Growth Hormone Releasing Factor Analogs and Uses
Abstract
Growth hormone releasing factor (GHRF) analogs that can be used
to induce endogenous growth hormone secretion are disclosed; as
well as pharmaceutical compositions comprising the GHRF analogs and
methods of using the GHRF analogs.
Inventors: |
Zaveri; Chanda; (Rancho
Palos Verdes, CA) ; Edge; Joey D.; (Castle Rock,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EZ IP, LLC |
Castle Rock |
CO |
US |
|
|
Family ID: |
63710551 |
Appl. No.: |
15/482592 |
Filed: |
April 7, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/61 20170801;
A61K 38/00 20130101; A61K 38/25 20130101; C07K 14/60 20130101 |
International
Class: |
A61K 38/25 20060101
A61K038/25; C07K 14/60 20060101 C07K014/60 |
Claims
1. A growth hormone releasing factor (GHRF) analog comprising a
sequence of Xaa.sub.1-D-2-Nal-Trp-His-Trp-D-Phe-Xaa.sub.2, wherein
Xaa.sub.1 is an amino acid residue selected from D-Ala, D-Val and
Gly and Xaa.sub.2 is an amino acid residue selected from Lys and
Arg, or a pharmaceutically acceptable salt thereof.
2. The GHRF analog of claim 1, wherein Xaa.sub.1 is an amino acid
residue selected from D-Ala and D-Val and Xaa.sub.2 is an amino
acid residue selected from Lys and Arg.
3. The GHRF analog of claim 1, wherein Xaa.sub.1 is D-Ala and
Xaa.sub.2 is an amino acid residue selected from Lys and Arg.
4. The GHRF analog of claim 1, wherein Xaa.sub.1 is D-Ala and
Xaa.sub.2 is Lys.
5. The GHRF analog of claim 1 having a weight average molecular
weight of 700 Daltons to 1000 Daltons.
6. The GHRF analog of claim 1 having an optical purity of at least
90%.
7. The GHRF analog of claim 1, wherein the GHRF analog is
formulated as a conjugate.
8. The GHRF analog of claim 7, wherein the GHRF analog is
conjugated to a compound selected from the group consisting of
glycosaminoglycans (GAGs), glycosaminoglycan-like (GAG-like)
polysaccharides, oligosaccharides and combinations thereof.
9. A pharmaceutical composition comprising the GHRF analog of claim
1 and one or more pharmaceutically acceptable carriers, excipients
and/or diluents.
10. The pharmaceutical composition of claim 9, further comprising
one or more amino acids.
11. The pharmaceutical composition of claim 10, wherein the amino
acid(s) is/are present in a concentration from 300 mg to 600
mg.
12. The pharmaceutical composition of claim 10, wherein the amino
acid(s) is/are selected from the group consisting of lysine,
argenine, ornithine, glutamine and combinations thereof.
13. A pharmaceutical composition comprising the GHRF analog of
claim 7 and one or more pharmaceutically acceptable carriers,
excipients and/or diluents.
14. The pharmaceutical composition of claim 13, further comprising
one or more amino acids.
15. The pharmaceutical composition of claim 14, wherein the amino
acid(s) is/are present in a concentration from 100 mg to 500
mg.
16. The pharmaceutical composition of claim 14, wherein the amino
acid(s) is/are selected from the group consisting of lysine,
argenine, ornithine, glutamine and combinations thereof.
17. A method of inducing growth hormone secretion in a subject in
need thereof, said method comprising administering to said subject
an therapeutically effective amount of the GHRF analog of claim 1
or a conjugate or pharmaceutically acceptable salt thereof.
18. The method of claim 17, wherein said GHRF analog, conjugate or
pharmaceutically acceptable salt is administered at a daily dose of
100 ng/Kg to 500 ng/Kg.
19. The method of claim 17, wherein said GHRF analog, conjugate or
pharmaceutically acceptable salt is administered orally or
mucosally.
20. The method of claim 17, wherein the GHRF analog, conjugate or
pharmaceutically acceptable salt is administered as an oral spray
or effervescent tablet.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] None.
BACKGROUND
[0002] Established anti-aging therapies have dearly shown that
science can intercede in the aging process to maximize health and
longevity. And indeed, many scientists now regard aging as both a
treatable and reversible condition.
[0003] Central to the popular acceptance of anti-aging therapy has
been the creation of a practical working theory on aging. This
theory associates the body's progressive inability at self-repair
with hormonal deficiency. The application of this idea, and the
unprecedented popularity of hormonal supplementation, testifies to
the validity of hormone replacement therapy as an effective means
of restoring the body's resilience and ability to self-repair.
[0004] Human Growth Hormone, or HGH, has emerged at the forefront
of anti-aging therapy. The hormone's ascension has been buttressed
by an impressive collection of basic science and clinical findings.
The hormone's ability to promote cell regeneration, stimulate
tissue repair and bolster the immune system is critical in this
forum, since virtually all of the complications of aging stem from
the body's inability to repair and replace cells as it loses
them.
[0005] Theories of Aging
[0006] The precise reason why Growth Hormone (GH) levels decrease
with advancing age is unknown. However, there are a number of
theories that begin to explain this depression within the context
of an organism's natural aging process. The theories are as
follows: [0007] Oxidative Stress Theory asserts that the body's
absorption of oxygen is intricately related to the aging process.
The more food a person or animal consumes, the more oxygen the body
needs to break it down into energy, and the more rapidly the animal
ages due to the creation of free radicals that impair cell
function. [0008] The Genetic Theory of Aging asserts that as
genetic damage accumulates simply as a consequence of living (i.e.
via ongoing cell repair and division), the body's overall
efficiency decreases, with aging as a consequence. [0009] The
Theory of Somatopause (or Cellular Senescence) asserts that cells
are pre-programmed to either die after a finite number of divisions
or simply go dormant. As the cells lapse into this phase of their
cycle, the efficiency of the organism to maintain its vital
functions decreases, until ultimately the organism itself dies.
[0010] The Hormonal Theory of Aging asserts that aging is linked to
a decline in the body's secretion of a variety of hormones without
losing the ability to respond to these hormones. This is the
principle basis for hormone replacement therapy with GH.
[0011] Growth Hormone
[0012] Growth hormone (GH) is a 191 amino acid long protein that is
synthesized and secreted by the pituitary gland found at the base
of the brain. The hormone enters the bloodstream in pulses,
predominantly at night, and is then quickly metabolized by the
liver into the insulin-like growth factors, IGF-1 and IGF-2. (See
FIG. 1)
[0013] The natural depression of GH secretion with age starts in
the mid-twenties and continues as we grow older. By the age of 60,
GH levels are typically 15-20% of what they were when a person was
in their twenties, sometimes even less.
[0014] People that have taken HGH by injection have noticed an
overall improvement in their general well-being and vitality, with
the following specific reports: [0015] Stronger bones [0016]
Enhanced immune system [0017] Accelerated wound healing/tissue
regeneration [0018] Decrease in total cholesterol [0019] Increase
in muscle mass without exercise [0020] Loss of body fat without
exercise [0021] Weight Loss [0022] Improved blood pressure [0023]
Younger, more durable skin with fewer wrinkles [0024] Increased
energy [0025] Enhanced sexual performance and libido [0026]
Increased cardic output [0027] Enhanced exercise performance [0028]
Improved mood [0029] Improved memory [0030] Improved sleep pattern
[0031] Organ regeneration [0032] Regrowth of lost hair
[0033] Insulin-Like Growth Factors, IGF-1 & IGF-2
[0034] GH is released into the bloodstream in small pulsatile
bursts during the day that peak during deep sleep. Since the
longevity of GH in the bloodstream is very short after its
secretion, accurately measuring GH levels directly can be very
cumbersome. Consequently, measurement of GH levels is typically
achieved indirectly, by measuring for levels of another hormone
called Insulin-like Growth Factor 1 (IGF-1).
[0035] IGF-1 is produced by the liver in response to circulating GH
and remains relatively constant throughout the day in contrast to
GH. As such, IGF-1 levels are the standard means to evaluate how
much GH the pituitary is releasing, especially when looking for a
change over time.
[0036] The goal of GH replacement therapy by injection for
individuals over 50 is to raise IGF-1 levels to within the range of
healthy young adults, which is around 350 .mu.g/m L.
[0037] HGH Replacement Supplementation Vs. GH Releasing Factors
[0038] It is helpful to understand the relative benefits and risks
of HGH replacement supplementation by injection versus the use of
an agent that stimulates the release of GH.
[0039] GH replacement supplementation by injection can offer
substantial benefits, as presented earlier. As with any hormone
replacement or supplementation, however, the converse is also true.
Poorly monitored HGH supplementation may be associated with
allergic reactions, carpal tunnel syndrome, irregularities of bone
growth (acromegaly), diabetes and swelling.
[0040] Use of a GH Releasing Factor (GHRF), or secretagogue,
minimizes the risk of complications associated with GH injections.
This decrease in risk is achieved by eliminating the shock of
pharmacologic GH injections and the resultant steep increase in
IGF-1 levels. By using a GHRF, IGF-1 levels increase more slowly
and plateau at levels far below the extremes of GH replacement
therapy. Furthermore, healthy advocates of GH supplementation can
reasonably use an oral GHRF agent without a physician's
involvement.
SUMMARY
[0041] The present invention relates to synthetic growth hormone
releasing factors (GHRFs) or GHRF analogs that are novel GH
secretagogues, which promote the release of endogenous growth
hormone (GH) in mammals, particularly humans, needing elevation of
serum growth hormone levels.
[0042] In an aspect, a growth hormone releasing factor (GHRF)
analog comprises a sequence of
Xaa.sub.1-D-2-Nal-Trp-His-Trp-D-Phe-Xaa.sub.2, wherein Xaa.sub.1 is
an amino acid residue selected from D-Ala, D-Val and Gly and
Xaa.sub.2 is an amino acid residue selected from Lys and Arg, or a
pharmaceutically acceptable salt thereof. In an embodiment,
Xaa.sub.1 is an amino acid residue selected from D-Ala and D-Val
and Xaa.sub.2 is an amino acid residue selected from Lys and Arg.
In an embodiment, Xaa.sub.1 is D-Ala and Xaa.sub.2 is an amino acid
residue selected from Lys and Arg. In an embodiment, Xaa.sub.1 is
D-Ala and Xaa.sub.2 is Lys.
[0043] In an aspect, a growth hormone releasing factor (GHRF)
analog comprises a sequence of
D-Ala-D-2-Nal-Trp-His-Trp-D-Phe-Lys.
[0044] In an aspect, a growth hormone releasing factor (GHRF)
analog consists of a sequence of
H-D-Ala-D-2-Nal-Trp-His-Trp-D-Phe-Lys-NH.sub.2.
[0045] In an embodiment, a GHRF analog has a weight average
molecular weight of 700 Daltons to 1000 Daltons, or 750 Daltons to
950 Daltons, or 790 Daltons to 900 Daltons.
[0046] In an embodiment, a GHRF analog has an optical purity of at
least 90%, or at least 95%, or at least 98%, or at least 99%, or at
least 99.5%, or at least 99.9%.
[0047] In some embodiments, a GHRF analog of the present invention
is formulated as a conjugate or proteoglycan. In the conjugate or
proteoglycan, the GHRF analog is chemically bound (e.g., covalently
bound or ionically bound) to a molecule or moiety to provide one or
more chemical or physical properties differing from those of the
GHRF analog alone. For example, a GHRF analog-conjugate may provide
a higher level of absorption in a subject or improved storage
stability relative to the GHRF analog alone.
[0048] In an embodiment, the GHRF analog is conjugated to a
compound selected from the group consisting of glycosaminoglycans
(GAGS), glycosaminoglycan-like (GAG-like) polysaccharides,
oligosaccharides and combinations thereof. In an embodiment, the
GAG is selected from the group consisting of chondroitin sulfate,
dermatan sulfate, keratin sulfate, heparin, heparin sulfate,
hyaluronan (hyaluronic acid) and pharmaceutically acceptable salts
thereof.
[0049] In some embodiments, the GHRF analog is formulated as a
pharmaceutical composition comprising a GHRF analog, a GHRF
analog-conjugate, or a pharmaceutically acceptable salt of the GHRF
analog and one or more pharmaceutically acceptable carriers,
excipients and/or diluents.
[0050] In some embodiments, a pharmaceutical composition of the
present invention comprises a GHRF analog, a GHRF analog-conjugate,
or a pharmaceutically acceptable salt of the GHRF analog; one or
more pharmaceutically acceptable carriers, excipients and/or
diluents; and one or more amino acids, which may be present in a
concentration from 300 mg to 600 mg, or 350 mg to 550 mg, or 380 mg
to 500 mg. Suitable amino acids for use in the pharmaceutical
compositions include, but are not limited to, amino acids selected
from the group consisting of lysine, argenine, ornithine, glutamine
and combinations thereof.
[0051] In an embodiment, a pharmaceutical composition is
administered to a subject 1-12 times per day, or 1-6 times per day,
or 1-3 times per day, or twice daily, or once daily.
[0052] In an aspect, a method of inducing growth hormone secretion
in a subject in need thereof comprises administering to the subject
a therapeutically effective amount of a GHRF analog, a GHRF
analog-conjugate, or a pharmaceutical salt of the GHRF analog. In
an embodiment, the GHRF analog, conjugate or pharmaceutically
acceptable salt is administered at a daily dose of 50 ng/Kg to 750
ng/Kg, or 100 ng/Kg to 500 ng/Kg, or 150 ng/Kg to 350 ng/Kg.
[0053] In an embodiment, the GHRF analog, conjugate or
pharmaceutically acceptable salt is administered, for example as a
pharmaceutical composition, intravenously, orally, sublingually,
transdermally, subcutaneously, mucosally, intramuscularly,
intranasally, intrapulmonary, parenterally, intrarectally,
intravaginally or topically. In an embodiment, the GHRF analog,
conjugate or pharmaceutically acceptable salt is administered, for
example as a pharmaceutical composition, orally or mucosally. In an
embodiment, the GHRF analog, conjugate or pharmaceutically
acceptable salt is administered, for example as a pharmaceutical
composition, as an oral spray, solution or suspension.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 is a schematic of the growth hormone mechanism.
[0055] FIG. 2 is a schematic of the HTA-8 mechanism.
[0056] FIG. 3 shows graphs of average percent change of IGF-1 by
age group and sex during Trial #1 and Trial #2.
[0057] FIG. 4 is a graph showing decreases in total cholesterol
during Trial #2.
[0058] FIG. 5 is a graph of average percent increase in radial bone
density by age group and sex during Trial #2.
[0059] FIG. 6 shows graphs of average percent change of lean body
mass (LBM) and adipose tissue mass (ATM) by age group and sex
during Trial #2.
DETAILED DESCRIPTION
[0060] In general, the terms and phrases used herein have their
art-recognized meaning, which can be found by reference to standard
texts, journal references and contexts known to those skilled in
the art. The following definitions are provided to clarify their
specific use in the context of this description.
[0061] An "amino acid" is a molecular building block of protein. An
"amino acid residue" is the simplest discreet unit or monomer of a
protein chain.
[0062] A "hormone" is a chemical messenger that initiates a
reaction in or near cells.
[0063] "Human growth hormone" (HGH) is a hormone comprising 191
amino acid residues. HGH is secreted by the anterior pituitary, is
responsible for somatic growth, and contributes to the regulation
of metabolism, the regeneration of tissues and immune system
function.
[0064] A "growth hormone releasing factor" (GHRF) refers to the
endogenous hypothalamic GH secretagogue, from any species.
[0065] A "secretagogue" is a releasing factor that stimulates the
secretion of a chemical, usually a hormone.
[0066] A "growth hormone releasing factor (GHRF) analog" is a
synthetic, non-naturally occurring (exogenous) secretagogue.
[0067] A "conjugate" is a chemical entity formed by coupling or
bonding two chemical moieties. In an embodiment, a conjugate is a
chemical molecule formed by covalently or ionically bonding two
chemical moieties. In an embodiment, a proteoglycan is a conjugate
formed by covalently or ionically bonding a protein/peptide and a
glycan.
[0068] "Pharmaceutically acceptable salts" include both acid and
base addition salts.
[0069] "Pharmaceutically acceptable acid addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free bases and which are not biologically or
otherwise undesirable, formed with inorganic acids including but
not limited to hydrochloric acid, hydrobromic acid, sulfuric acid,
sulfamic nitric acid, phosphoric acid and the like, and organic
acids such as acetic acid, propionic acid, glycolic acid, pyruvic
acid, lactic acid, oxalic acid, maleic acid, malic acid, maloneic
acid, succinic acid, fumaric acid, tartaric acid, citric acid,
stearic acid, ascorbic acid, benzoic acid, cinnamic acid, mandelic
acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic
acid, salicyclic acid, naturally occurring amino acids and the
like.
[0070] "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particularly preferred are
the ammonium, potassium, sodium, calcium and magnesium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine,
dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
methylglucamine, theobromine, purines, piperizine, piperidine,
N-ethylpiperidine, polyamine resins and the like. Particularly
preferred organic non-toxic bases are isopropylamine, diethylamine,
ethanolamine, trimethamine, dicyclohexylamine, choline, and
caffeine.
[0071] "Optical isomers", "diastereomers", and "geometric isomers"
of some of the compounds represented by the formulae described
herein are comprehended to be within the scope of the instant
invention, as well as racemic and resolved enantiomerically pure
forms and pharmaceutically acceptable salts thereof.
[0072] "Optical purity" is a comparison of the optical rotation of
a pure sample of unknown stereochemistry versus the optical
rotation of a sample of pure enantiomer. It is expressed as a
percentage, where 0% indicates a 50/50 racemic mixture and 100%
indicates an enantiomerically pure sample.
[0073] Methods of Peptide Synthesis
[0074] One method of producing GHRF analogs involves chemical
synthesis of the peptide. This can be accomplished using solid
phase methodologies well known to those skilled in the art. (See,
e.g., Stewart, J. M. & Young, J. D. "Solid Phase Peptide
Synthesis" Pierce Chemical Co, Rockford, Ill., 1984; Merrifield, J.
Am. Chem. Soc., 85:2149 1964; Houghten, Proc. Natl. Acad. Sci. USA
82:5132 1985; and U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067;
and 3,862,925).
[0075] Solid phase synthesis begins at the carboxy-terminus of the
putative peptide by coupling a protected amino acid to a suitable
resin (e.g. chloromethylated polystyrene resin). After removal of
the .alpha.-amino protecting group with, for example,
trifluoroacetic acid (TFA) in methylene chloride and neutralizing
in, for example TEA, the next .alpha.-amino- and sidechain
protected amino acid in the synthesis is added. The remaining
.alpha.-amino- and, if necessary, side-chain-protected amino acids
are then coupled sequentially in the desired order by condensation
to obtain an intermediate compound connected to the resin.
Alternatively, some amino acids may be coupled to one another
forming a peptide prior to addition of the peptide to the growing
solid phase polypeptide chain.
[0076] The condensation between two amino acids, or an amino acid
and a peptide, or a peptide and a peptide can be carried out
according to the usual condensation methods such as the azide
method, mixed acid anhydride method, DCC
(N,N'-dicyclohexylcarbodiimide) or DIPC
(N,N'-diisopropylcarbodiimide) methods, active ester method
(p-nitrophenyl ester method), BOP benzotriazole-1-yl-oxy-tris
(dimethylamino) phosphonium hexafluorophosphate method,
N-hydroxysuccinic acid imido ester method, etc., and Woodward
reagent K method.
[0077] Common to chemical syntheses of peptides is the protection
of any reactive side-chain groups of the amino acids with suitable
protecting groups. Ultimately, these protecting groups are removed
after the desired polypeptide chain has been sequentially
assembled. Also common is the protection of the .alpha.-amino group
on an amino acid or a fragment while that entity reacts at the
carboxyl group followed by the selective removal of the
.alpha.-amino-protecting group to allow subsequent reaction to take
place at that location. Accordingly, it is common in polypeptide
synthesis that an intermediate compound is produced which contains
each of the amino acid residues located in the desired sequence in
the peptide chain with various of these residues having side-chain
protecting groups attached. These protecting groups are then
commonly removed substantially at the same time so as to produce
the desired resultant product following removal from the resin.
[0078] Suitable protective groups for protecting the .alpha.- and
.epsilon.-amino side chain groups are exemplified by
benzyloxycarbonyl (CBZ), isonicotinyloxycarbonyl (iNOC),
O-chlorobenzyloxycarbonyl (2-Cl-CBZ), p-nitrobenzyloxycarbonyl
Z(NO.sub.2, p-methoxybenzyloxycarbonyl Z(OMe), t-butoxycarbonyl,
(BOC), t-amyloxycarbonyl (AOC), isoborrnyloxycarbonyl,
adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (BPOC),
9-fluorenylmethoxycarbonyl (FMOC), methylsulfonyiethoxycarbonyl
(Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl
(NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl
(Mpt) and the like.
[0079] Protective groups for the carboxy functional group are
exemplified by benzyl ester (OBzl), cyclohexyl ester (Chx),
4-nitrobenzyl ester (ONb), t-butyl ester (OtBu), 4-pyridylmethyl
ester (OPic), and the like. It is often desirable that specific
amino acids such as arginine, cysteine, and serine possessing a
functional group other than amino and carboxyl groups are protected
by a suitable protective group. For example, the guanidino group of
arginine may be protected with nitro, p-toluenesulfonyl,
benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl,
4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),
1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group
of cysteine may be protected with p-methoxybenzyl, triphenylmethyl,
acetylaminomethyl ethylcarbamoyle, 4-methylbenzyl,
2,4,6-trimethy-benzyl (Tmb) etc., and the hydroxyl group of serine
can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl
and the like.
[0080] After the desired amino acid sequence has been completed,
the intermediate peptide is removed from the resin support by
treatment with a reagent, such as liquid HF and one or more
thio-containing scavengers, which not only cleaves the peptide from
the resin, but also cleaves all the remaining side-chain protecting
groups. Following HF cleavage, the peptide residue is washed with
ether, and extracted from the resin by washing with aqueous
acetonitrile and acetic acid.
[0081] Amino Acid Substitutions
[0082] It is a well-established principle of protein and peptide
chemistry that certain amino acid substitutions, entitled
"conservative" amino acid substitutions, can frequently be made in
a protein or a peptide without altering either the conformation or
the function of the protein or peptide. Conservative substitutions
may be employed in the synthesis of proteins, peptides or analogs
disclosed herein. Accordingly, peptides having conservative amino
acid substitutions are within the scope of the present invention.
Such changes include substituting any of isoleucine (I), valine
(V), and leucine (L) for any other of these amino acids; aspartic
acid (D) for glutamic acid (E) and vice versa; glutamine (O) for
asparagine (N) and vice versa; and serine (S) for threonine (T) and
vice versa. The above-mentioned substitutions are not the only
amino acid substitutions that can be considered "conservative."
Other substitutions can also be considered conservative, depending
on the environment of the particular amino acid. For example,
glycine (G) and alanine (A) can frequently be interchangeable, as
can be alanine and valine. Methionine (M), which is relatively
hydrophobic, can frequently be interchanged with leucine and
isoleucine, and sometimes with valine. Lysine (K) and arginine (R)
are frequently interchangeable in locations in which the
significant feature of the amino acid residue is its charge and the
differing pKs of these two amino acid residues are not significant.
Cysteine (C) can frequently be replaced by serine (S) when
cysteine's capacity to form disulfide bonds is either undesirable
or unneeded. Still other changes can be considered "conservative"
in particular environments.
[0083] Pharmaceutical Compositions
[0084] Another aspect of the present invention is pharmaceutical
compositions that include GHRF analogs according to the present
invention. In general, a pharmaceutical composition of the present
invention comprises a GHRF analog, conjugate or pharmaceutically
acceptable salt in a therapeutically effective amount and a
pharmaceutically acceptable carrier, excipient or diluent.
[0085] The GHRF analogs may be formulated into the pharmaceutical
compositions as neutral or salt forms. Pharmaceutically acceptable
nontoxic salts include the acid addition salts (formed with the
free amino groups) and which are formed with inorganic acids such
as, for example, hydrochloric or phosphoric acids, or organic acids
such as acetic, oxalic, tartaric, mandelic, and the like. Base
addition salts may be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropyl amine, 2-ethylamino
ethanol, histidine, procaine, and the like.
[0086] The therapeutically effective amount can be determined by
one of ordinary skill in the art, with reference to the dosages
described herein.
[0087] Conventional pharmaceutically acceptable carriers known in
the art can include alcohols, e.g., ethyl alcohol, serum proteins,
cholesterol, human serum albumin, liposomes, buffers such as
phosphates, water, sterile saline or other salts, electrolytes,
glycerol, hydroxymethylcellulose, propylene glycol, polyethylene
glycol, polyexyethylenesorbitan, other surface active agents,
vegetable oils, and conventional anti-bacterial or anti-fungal
agents, such as parabens, chlorobutanol, phenol, sorbic acid, and
the like. A pharmaceutically acceptable carrier within the scope of
the present invention meets industry standards for sterility,
isotonicity, stability, and non-pyrogenicity.
[0088] The active ingredient is often mixed with diluents or
excipients that are physiologically tolerable and compatible with
the active ingredient. Suitable diluents and excipients are, for
example, water, saline, dextrose, glycerol, or the like, and
combinations thereof. In addition, if desired the compositions may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, stabilizing or pH-buffering agents, and the
like. For a more detailed description of the foregoing see a
standard pharmaceutical text such as Remington's Pharmaceutical
Sciences, Mack Publishing Co. Easton, Pa. (1970).
[0089] Oral formulations may include excipients such as
pharmaceutical grade mannitol, lactose, starch, magnesium stearate,
sodium saccharin, cellulose; magnesium carbonate, and the like.
These compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained-release formulations, or
powders, and contain 0.5% to 15% of active ingredient, or 1% to 12%
of active ingredient or 2%-10% of active ingredient, or 2.5% to 8%
of active ingredient.
[0090] In an embodiment, a solution or suspension may be formed by
the combination of an effervescent tablet or powder with a liquid.
An effervescent tablet or powder is designed to dissolve and
produce bubbling when the tablet or powder comes into contact with
water or another liquid. The bubbling is due to release of carbon
dioxide when sodium bicarbonate and citric acid, or other
appropriate chemicals, in the tablet or powder dissolve in the
liquid and react.
[0091] Methods of Use
[0092] The compounds of this invention are shown to induce release
of growth hormone and IGF-1. It is known to those skilled in the
art that there are many uses for growth hormone and the IGFs.
Therefore administration of the GHRF analogs and pharmaceutical
compositions of this invention for purposes of stimulating the
release of endogenous growth hormone or IGF-1 can have the same
effects or uses as growth hormone or the IGFs themselves. These
uses of growth hormone and IGF-1 include, but are not limited to,
slowing, stopping or reversing aging; strengthening bones; treating
osteoporosis; accelerating bone fracture repair; stimulating the
immune system; accelerating wound healing; decreasing total
cholesterol; increasing muscle mass; stimulating weight loss;
treating obesity; treating high blood pressure; improving skin
texture; increasing energy; treating fatigue; enhancing sexual
performance; increasing libido; increasing cardiac output;
improving exercise performance; improving mood; treating
depression; improving memory; treating cognitive degeneration;
improving sleep patterns; regenerating tissue; and stimulating hair
growth.
[0093] Administration
[0094] GHRF analogs and pharmaceutical compositions according to
the present invention can be administered by a number of routes.
When used for wound healing, they are typically administered
topically to the skin or other mucous membranes. However, when they
are administered as immunostimulants or as growth factors to
stimulate pituitary or hypothalamus function, they can be
administered by other routes, such as intravenously, orally,
sublingually, transdermally, subcutaneously, mucosally,
intramuscularly, intranasally, intrapulmonary, parenterally,
intrarectally, intravaginally, intradermally or topically. In an
embodiment, oral administration may be accomplished by an oral
spray, solution or suspension, e.g., where the solution or
suspension is formed by the combination of an effervescent tablet
or powder with a liquid.
[0095] The dosages to be administered can be determined by one of
ordinary skill in the art depending on the clinical severity of the
disease, the age, sex and weight of the patient, the exposure of
the patient to conditions that may affect the course of wound
healing, the existence or nonexistence of underlying systemic
problems such as diabetes, impaired circulation, and
immunocompromised status, and other pharmacokinetic factors
generally understood in the art, such as liver and kidney
metabolism. The interrelationship of dosages for animals of various
sizes and species and humans based on mg/m.sup.3 of surface area is
described by E. J. Freireich et al., "Quantitative Comparison of
Toxicity of Anticancer Agents in Mouse, Rat, Hamster; Dog, Monkey
and Man," Cancer Chemother. Rep. 50:219-244 (1966). Adjustments in
the dosage regimen can be made to optimize the therapeutic
response. Doses can be divided and administered on a daily basis or
the dose can be reduced proportionally depending on the therapeutic
situation.
[0096] Methods according to the present invention can be used to
treat humans or socially or economically important animal species
such as dogs, cats, horses, sheep, cows, goats, or pigs. Methods
according to the present invention are not limited to use in
humans.
[0097] The invention is illustrated by the following Examples.
These Examples are for illustrative purposes only and are not
intended to limit the invention.
Example 1: Development of a New Growth Hormone Releasing Factor
[0098] This Example describes the development of a new growth
hormone releasing factor analog, particularly, an oral GHRF analog
that could safely offer a viable alternative to HGH injections. The
new GHRF analog is referred to herein as HTA-8.
[0099] HTA-8
[0100] HTA-8 peptide sequence: [0101] H-D-Ala-D-2-Nal-Trp-H
is-Trp-D-Phe-Lys-NH.sub.2
[0102] Standard three letter codes are used to designate the amino
acids with a "D" placed before the three-letter code to signify the
dextrorotatory enantiomer. Accordingly, HTA-8 contains the
following seven amino acids: [0103]
(D-Alanine)-(D-2-Naphthylalanine)-Tryptophan-Histidine-Tryptophan-(D-Phen-
ylalanine)-Lysine.
[0104] HTA-8 is a small protein chain that behaves like GHRF1 on
the pituitary to stimulate the release of GH. HTA-8 also binds to
GH receptors in the liver. (See FIG. 2) The end result is that a
low dose of HTA-8 has a similar efficacy to higher doses of HGH
injection. HTA-8 is not a GH precursor.
Example 2: Human Clinical Testing
[0105] Two challenges were being considered at the beginning of the
human clinical trial. The first challenge was to document the
stand-alone efficacy of HTA-8 as a new growth hormone releasing
factor. The second was to determine if HTA-8 could be combined with
other common releasing factors to create an even better result. As
such, two separate trials were conducted. [0106] Trial #1/HTA-8:
Ten individuals ranging in age from 30 to 69 years were given 30 ng
of HTA-8 in the form of an aerosolized oral spray before bed, over
a 30-32 day period. [0107] Trial #2/HTA-8+: Fifteen individuals
ranging in age from 38 to 70 years of age were given an
effervescent tablet to take once daily over a 25-36 day period that
contained 20 ng of HTA-8 in combination with 1200 mg each of the
amino acids lysine and arginine. (Previous published
experimentation has shown that the combination of lysine and
arginine in the doses used in Trial #2 is an effective GH releasing
factor.)
[0108] HTA-8 & IGF-1
[0109] The principal hormone measured in order to establish whether
there is an increase in growth hormone secretion is IGF-1. IGF-1
concentrations and total cholesterol levels were determined by
collecting participant blood samples at the beginning of the
studies and at their conclusion.
[0110] The results of Trial #1 confirm that HTA-8 alone is a bona
fide, stand-alone GHRF. Furthermore, it also appears that HTA-8 in
combination with the amino acids lysine and arginine is capable of
increasing IGF-1 levels in excess of HTA-8 alone (Trial #2). These
results confirm that GH secretion is increased with HTA-8.
[0111] IGF-1 levels in HTA-8 Trial #2 increased from 169.8 .mu.g/mL
to 202.4 .mu.g/mL on average. The maximum IGF-1 level measured in
all clinical trial participants after the study period was 410
.mu.g/mL in a 30 year-old male in HTA-8 Trial #1.
[0112] In addition, the following observations may be made from the
IGF-1 data in FIG. 3: [0113] The change in IGF-1 concentrations in
the presence of HTA-8 alone or in combination with amino acids was
found to be both age and sex dependent. A more substantial change
in IGF-1 concentrations was noted with advancing age groups,
regardless of sex. [0114] A woman's IGF-1 response to HTA-8 with or
without amino acids was calculated to be greater than a man's
response. This difference in IGF did not result in significant body
mass changes between the sexes. [0115] It should be noted that
across all clinical trial participants, the greatest percent change
in IGF-1 concentrations was observed in participants that started
with very low baseline IGF-1 concentrations (e.g. 21 ng/mL).
[0116] Total Cholesterol
[0117] The National Cholesterol Education Program (NCEP) has been
advocating that people maintain a low total blood cholesterol level
as a means to minimize the potential for cardiovascular disease.
Current NCEP guidelines recommend that adult individuals work
toward reducing total cholesterol levels to at least 200 mg/mL as
part of a healthful lifestyle. The majority of clinical trial
participants using HTA-8 (with or without amino acids) demonstrated
a clinically significant decrease in their total cholesterol levels
over the study period, in the absence of additional measures. Study
results for Trial #2 participants are presented in FIG. 4, where
the change in total cholesterol for each participant is represented
as a shift from the top (darker) area of the bar to the bottom
(lighter) area of the bar.
[0118] Bone Density
[0119] An increase in bone density decreases the chance of fracture
and osteoarthritis. Bone density is a particularly important issue
as the body ages, regardless of sex. During the Clinical Trials,
radial bone density was measured using an ultrasonographic
technique.
[0120] The average increase in bone density for subjects taking
HTA-8 alone was 6.8%. The average increase in bone density for
subjects taking HTA-8 plus amino acids was 12.6%.
[0121] FIG. 5 illustrates the bone density increases measured for
men and women in Clinical Trial #2. Note that neither sex nor age
group has a significant influence on the activity of HTA-8 to
affect bone density.
[0122] Lean Body Mass/Adipose Tissue Mass
[0123] One of the basic roles of growth hormone and IGF-1 is in the
regulation of insulin levels, glucose metabolism and fatty tissue
metabolism.
[0124] An increase in lean muscle mass and a decrease in adipose
tissue mass was recorded for all study participants in both
clinical trials using an ultrasound-based body composition
analysis. The noted increase in muscle mass and decrease in adipose
tissue mass at the conclusion of the study period was slightly more
than doubled for study participant's given HTA-8 with amino acids.
Participants lost an average of 13.2% of their body weight in
conjunction with this change in body composition--and this was
achieved without diet or exercise. (See FIG. 6.)
Example 3: Pharmaceutical Compositions Containing HTA-8
[0125] This Example describes the development of a pharmaceutical
composition containing HTA-8. In addition to HTA-8, lysine and
arginine, the pharmaceutical composition of this Example may
contain several other key ingredients with additional,
complementary functions, as well as excipients, diluents,
preservatives and the like. [0126] Ornithine. Ornithine is an amino
acid acting as a powerful releasing factor for GH, that is known to
work well in combination with lysine and arginine. [0127]
GHRF.sub.2. GHRF.sub.2 is predominantly responsible for stimulating
the liver's synthesis and secretion of IGF-2. This second
insulin-like growth factor has a greater affinity than IGF-1 for
bone marrow receptors that stimulate immune cell function and
osteoblast receptors that stimulate an increase in bone density.
The competition between IGF-2 and IGF-1 actually releases more
IGF-1 to target cells that IGF-1 has a greater affinity for than
IGF-2, such as the skeletal muscle mass and the skin. [0128]
Melatonin. The principal accepted application of melatonin is to
decrease the amount of time that it takes to fall asleep and to
increase the duration and quality of restful sleep. By including
melatonin in the HTA-8 formulation, there is an improvement in
sleep patterns which also assists the natural nocturnal release of
GH. [0129] DHEA. DHEA is the adrenal gland precursor to the sex
hormones testosterone, estrogen and progesterone. Proponents of
DHEA report that the steroid has a basic anti-aging influence.
Recent studies have shown that DHEA supplementation helps to
restore muscle mass and decrease adipose tissue mass, and decrease
total serum cholesterol. [0130] L-Glutamine. L-Glutamine has
excellent anti-aging and stress reduction properties. As an oral
supplement, glutamine has been shown to work as an effective
anti-oxidant, strengthen immunity, cut illness short, aid in
recovery from illness, and rejuvenate muscles weakened by stress
and illness. [0131] Calcium, Iron, Magnesium coenzyme minerals.
These minerals need to be present as cofactors that permit the
secretion of GH.
TABLE-US-00001 [0131] HTA-8 Formulation Ingredients Quantity Range
Deioinized Water 97,807.840 gm L-Lysine 380.000 gm 200-500 gm
L-ArginineHCl 380.000 gm 200-500 gm L-Ornithine 380.000 gm 200-500
gm Pyridoxine HCl 380.000 gm 200-500 gm Melatonin 10.000 gm 1-20 gm
Sodium Chondroitin Sulfate 10.000 gm 1-20 gm L-Glutathione 380.000
gm 200-500 gm HTA-8 0.005 gm 0.001-0.010 gm Potassium Sorbate
22.680 gm Sodium Benzoate 22.680 gm Benzyl Alcohol 113.400 gm
Methylparaben 113.400 gm
[0132] Recommended Dosage & Instructions for Administration
[0133] Oral Spray: Administer one spray of the HTA-8 pharmaceutical
corn position to the back of the mouth in the morning, and two
sprays in the evening prior to bed. Abstain from drinking or eating
for at least 10 minutes after administration.
Example 4: Oral Absorption of HTA-8 Versus HGH in an Oral Spray
[0134] Oral absorption of HTA-8 is based on the peptide's small
size and its ability to be readily absorbed through the oral
mucosa. HGH, on the other hand, is not readily absorbed through the
intact oral mucosa because HGH is a 191 amino acid protein, and the
upper threshold of trans-mucosal absorption for peptides is around
60 amino acids.
[0135] In addition to HGH's large size as a prohibitive factor for
an oral spray formulation, it should also be understood that HGH in
liquid form is extremely unstable. Consequently, although the
concentration of HGH placed in solution at the time of manufacture
may be substantial, final levels of HGH by the time the product
gets into the hands of the end consumer will be very low. At this
time, there is no reliable technology available to stabilize HGH in
solution.
[0136] Including a relatively small sugar/protein transport
molecule called a glycosaminoglycan, or GAG molecule, in the
formulation, assists HTA-8 transmucosal absorption, and also helps
to stabilize HTA-8 in the formulation. HTA-8 was attached to a GAG
molecule(s) to form a proteoglycan in all clinical trials.
Proteoglycan formation is discussed, for example, in Hileman et
al., Glycosaminoglycan-protein interactions: definition of
consensus sites in glycosaminoglycan binding proteins, BioEssays,
20, 156-167 (1998), which is hereby incorporated by reference in
its entirety.
Example 5: HTA-8 Pharmaceutical Composition Versus HGH
Supplementation
[0137] The HTA-8 pharmaceutical composition is ideal for those
individuals whose objective is to benefit from an increase in their
body's own release of GH without the hassles of typical HGH
supplementation. In particular: [0138] HTA-8 formulation users will
never have to be concerned by the need for injections that are
required with HGH. A simple intra-oral spray or effervescent tablet
releases enough HTA-8 to stimulate the body's own synthesis and
secretion of GH. [0139] HTA-8 formulation use will not suppress the
body's natural cyclical release of GH, as is commonly the case with
HGH supplementation. Consequently, a subject may start and stop the
use of the HTA-8 formulation without risk. [0140] HTA-8 formulation
users can comfortably use HTA-8 in the absence of physician
supervision because HTA-8 does not expose the body to the
pharmacologic concentrations of GH commonly used with HGH
injections.
STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS
[0141] All references cited throughout this application, for
example patent documents including issued or granted patents or
equivalents; patent application publications; and non-patent
literature documents or other source material; are hereby
incorporated by reference herein in their entireties, as though
individually incorporated by reference.
[0142] The terms and expressions which have been employed herein
are used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the invention has been
specifically disclosed by preferred embodiments, exemplary
embodiments and optional features, modification and variation of
the concepts herein disclosed can be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims. The specific embodiments provided herein are
examples of useful embodiments of the invention and it will be
apparent to one skilled in the art that the invention can be
carried out using a large number of variations of the devices,
device components, and method steps set forth in the present
description. As will be apparent to one of skill in the art,
methods and devices useful for the present methods and devices can
include a large number of optional composition and processing
elements and steps.
[0143] When a group of substituents is disclosed herein, it is
understood that all individual members of that group and all
subgroups are disclosed separately. When a Markush group or other
grouping is used herein, all individual members of the group and
all combinations and subcombinations possible of the group are
intended to be individually included in the disclosure.
[0144] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide" includes a plurality of such
peptides and equivalents thereof known to those skilled in the art,
and so forth. As well, the terms "a" (or "an"), "one or more" and
"at least one" can be used interchangeably herein. It is also to be
noted that the terms "comprising", "including", and "having" can be
used interchangeably. The expression "of any of claims XX-YY"
(wherein XX and YY refer to claim numbers) is intended to provide a
multiple dependent claim in the alternative form, and in some
embodiments is interchangeable with the expression "as in any one
of claims XX-YY."
[0145] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0146] Whenever a range is given in the specification, for example,
a range of integers, a temperature range, a time range, a
composition range, or concentration range, all intermediate ranges
and subranges, as well as all individual values included in the
ranges given are intended to be included in the disclosure. As used
herein, ranges specifically include the values provided as endpoint
values of the range. As used herein, ranges specifically include
all the integer values of the range. For example, a range of 1 to
100 specifically includes the end point values of 1 and 100. It
will be understood that any subranges or individual values in a
range or subrange that are included in the description herein can
be excluded from the claims herein.
[0147] As used herein, "comprising" is synonymous and can be used
interchangeably with "including," "containing," or "characterized
by," and is inclusive or open-ended and does not exclude
additional, unrecited elements or method steps. As used herein,
"consisting of" excludes any element, step, or ingredient not
specified in the claim element. As used herein, "consisting
essentially of" does not exclude materials or steps that do not
materially affect the basic and novel characteristics of the claim.
In each instance herein any of the terms "comprising", "consisting
essentially of" and "consisting of" can be replaced with either of
the other two terms. The invention illustratively described herein
suitably can be practiced in the absence of any element or
elements, limitation or limitations which is/are not specifically
disclosed herein.
[0148] All art-known functional equivalents of materials and
methods are intended to be included in this disclosure. The terms
and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed can be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
* * * * *