U.S. patent application number 12/281018 was filed with the patent office on 2009-09-03 for pharmaceutical composition containing ghrp-6 to prevent and eliminate fibrosis and other pathological deposits in tissues.
Invention is credited to Jorge Berlanga Acosta, Danay Cibrian Vera, Diana Garcia Del Barco Herrera, Gerardo Enrique Guillen Nieto, Ernesto Lopez Mola, Manuel Selman-Housein Sosa, Jose Suarez Alba, Mariela Vazquez Castillo.
Application Number | 20090221512 12/281018 |
Document ID | / |
Family ID | 37964643 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090221512 |
Kind Code |
A1 |
Acosta; Jorge Berlanga ; et
al. |
September 3, 2009 |
PHARMACEUTICAL COMPOSITION CONTAINING GHRP-6 TO PREVENT AND
ELIMINATE FIBROSIS AND OTHER PATHOLOGICAL DEPOSITS IN TISSUES
Abstract
The present invention is related to the use of secretagogue
peptides repeatedly administered as part of a pharmaceutical
composition that prevent and eradicate the deposition of
pathological fibrotic material in parenchymal tissues of internal
organs like the liver, lungs, esophagus, small intestine, kidneys,
blood vessels, joints, and other systemic forms of cutaneous
fibrosis of any etiopathogenesis. Additionally, these peptides
prevent and eradicate deposition of amiloid and hyaline materials
in any of their correspondent chemical forms and tissue
manifestations in the brain, cerebellum, blood vessels, liver,
intestines, kidneys, spleen, pancreas, joints and the skin, among
others. By this way, cellular, tissular and organ dysfunctions
generated by these depositions are corrected. The peptides of the
present invention are infiltrated or topically applied,
contributing to prevent and eradicate keloids and hypertrophic
scars in the skin, derived as sequelae of burns and other cutaneous
trauma.
Inventors: |
Acosta; Jorge Berlanga;
(Ciudad De La Habana, CU) ; Cibrian Vera; Danay;
(Ciudad De La Habana, CU) ; Del Barco Herrera; Diana
Garcia; (Ciudad De La Habana, CU) ; Guillen Nieto;
Gerardo Enrique; (Ciudad De La Habana, CU) ; Suarez
Alba; Jose; (Ciudad De La Habana, CU) ; Lopez Mola;
Ernesto; (Ciudad De La Habana, CU) ; Selman-Housein
Sosa; Manuel; (Ciudad De La Habana, CU) ; Vazquez
Castillo; Mariela; (Ciudad De La Habana, CU) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Family ID: |
37964643 |
Appl. No.: |
12/281018 |
Filed: |
February 23, 2007 |
PCT Filed: |
February 23, 2007 |
PCT NO: |
PCT/CU2007/000005 |
371 Date: |
January 14, 2009 |
Current U.S.
Class: |
514/6.9 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 25/28 20180101; A61P 1/16 20180101; A61P 27/02 20180101; A61K
38/25 20130101; A61P 27/16 20180101; A61P 43/00 20180101; A61P
13/12 20180101; A61P 17/02 20180101; A61P 19/04 20180101; A61P
25/00 20180101; A61K 38/05 20130101; A61P 17/00 20180101; A61K
38/08 20130101; A61P 21/00 20180101 |
Class at
Publication: |
514/17 |
International
Class: |
A61K 38/08 20060101
A61K038/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2006 |
CU |
2006-048 |
Claims
1. A pharmaceutical composition characterized by containing GHRP-6
and capable of preventing, controlling and eliminating the
pathological deposits of fibrotic material and the excess of
extracellular matrix from internal organs and the skin of the
recipient organism.
2. A composition according to claim 1, wherein said composition
eliminates the intracellular and extracellular deposits of hyaline
and amiloid materials, granular forms of eosinophilic and/or
osmophilic material from internal and external organs and the
vascular network.
3. A composition according to claim 1, wherein said composition
restores the normal functionality of any internal or external organ
or tissue affected by excessive deposits of fibrotic, amiloid,
osmophilic, eosinophilic or hyaline material.
4. A composition according to claim 1, wherein said composition is
administered to animals or patients.
5. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by parenteral route,
including intravenous, intramuscular, subcutaneous,
intraperitoneal, intrathecal; or locally infiltrated into the skin,
on mucosa, epithelia or organs, or more precisely
intralesionally.
6. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by intra-rectal
route.
7. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by topical route as a
liquid, compress, or solid or semi-solid formulations.
8. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal intra-rectally: or locally
infiltrated into the skin, on mucosa, epithelia or organs or more
precisely intralesionally, able to eliminate the deposits of
fibrotic material and the excess of extracellular matrix in any
hepatic etiopathogenesis, like viral hepatitis sequelae,
alcoholism, intoxications, autoimmune or idiopathic, in
general.
9. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal intra-rectally; or locally
infiltrated into the skin, on mucosa, epithelia or organs or more
precisely intralesionally, able to eliminate the deposits of
fibrotic material and the excess of extracellular matrix of toxic,
professional, drug-related, radioactive, autoimmune, asthmatic
sequelae, allergic or idiopathic origin, in general, in organs like
lungs.
10. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal intra-rectally: or locally
infiltrated into the skin, on mucosa, epithelia or organs or more
precisely intralesionally, able to eliminate the deposits of
fibrotic, fibro-hyaline or amiloid material and the excess of
extracellular matrix in organs like kidneys, being the
nephrosclerosis and/or tubulosclerosis of diabetic, toxic,
professional, drug-related, autoimmune or idiopathic origin in
general, or sequelae of repeated infections.
11. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, able to eliminate the deposits
of fibrotic, fibro-hyaline or amiloid material and the excess of
extracellular matrix of autoimmune or idiopathic origin in general
in the skin.
12. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, as a liquid, compress or solid
or semi-solid formulation, able to eliminate the deposits of
fibrotic, fibro-hyaline or amiloid material and the excess of
extracellular matrix from skin, more precisely keloids,
hypertrophic scars or other forms of exuberant scars.
13. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, as a liquid, compress or solid
or semi-solid formulation, able to correct the aesthetic appearance
of skin after reconstructive, aesthetic or similar surgery.
14. The use of the GHRP-6 peptide according to claim 1 for
manufacturing a pharmaceutical composition to be administered to
animals or patients by any of the routes selected from the group
consisting of intravenous intramuscular subcutaneous,
intraperitoneal, intrathecal, intra-rectally; or locally
infiltrated into the skin, on mucosa, epithelia or organs, or more
precisely intralesionally, as a liquid, compress or solid or
semi-solid formulation, in a manner to be able to eliminate the
deposits of fibrotic, fibro-hyaline or amiloid material and the
excess of extracellular matrix from skin, more precisely fibrotic
sequelae of any form of acne.
15. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, able to eliminate the deposits
of fibrotic, fibro-hyaline or amiloid material and the excess of
extracellular matrix from the pancreas and the digestive tube from
the esophagus to the rectum.
16. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, to eliminate the deposits of
fibrotic, fibro-hyaline or amiloid material and the excess of
extracellular matrix in the vascular network.
17. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, to eliminate the deposits of
fibrotic or amiloid, and/or osmophilic or eosinophilic material in
the brain and brain cells.
18. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, able to reduce and/or eliminate
the deposition of amiloid and/or hyaline, and/or granular forms of
eosinophilic and/or osmophilic material in the vessel network of
the body, including the central nervous system and the
meninges.
19. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs,
or more precisely intralesionally, able to reduce and/or prevent
the deposition of fibrotic, amiloid and/or hyaline, and/or granular
forms of eosinophilic and/or osmophilic material in the walls of
blood vessels in general.
20. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal intrathecal intra-rectally; or locally
infiltrated into the skin, on mucosa, epithelia or organs or more
precisely intralesionally, able to eliminate the deposits of
fibrotic, amiloid and/or hyaline, and/or granular forms of
eosinophilic or osmophilic material from cranial, extra-cranial,
sensitive, motor or mixed nerves and/or those from the
neuro-vegetative system.
21. A composition according to claim 1, wherein said composition is
to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous intramuscular
subcutaneous intraperitoneal, intrathecal intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs
or more precisely intralesionally, able to reduce and/or prevent
the deposition of fibrotic, amiloid and/or hyaline, and/or granular
forms of eosinophilic and/or osmophilic material in the walls of
blood vessels in general.
22. A composition according to claim 1 characterized by containing
GHRP-6 or GHRP-2 or Ghrelin or Hexarelin, and able to stop and
control the excessive lipid peroxidation and oxidative stress
processes in cells, tissues and systemic organs, to be administered
to animals or patients by any of the routes selected from the group
consisting of intravenous, intramuscular subcutaneous,
intraperitoneal, intrathecal intra-rectally; or locally infiltrated
into the skin, on mucosa, epithelia or organs or more precisely
intralesionally.
23. A composition according to claim 22, characterized by being
able to stop and control the excessive lipid peroxidation and
oxidative stress processes; particularly in the central nervous
system and peripheral nerves, to be administered to animals or
patients by any of the routes selected from the group consisting of
intravenous intramuscular subcutaneous intraperitoneal intrathecal
intra-rectally; or locally infiltrated into the skin, on mucosa,
epithelia or organs or more precisely intralesionally.
24. The use of peptides GHRP-6 or GHRP-2 or Ghrelin or Hexarelin in
a pharmaceutical composition useful to stop and control the process
of brain aging and deterioration of brain functions because of
excessive lipid peroxidation and oxidative stress processes, or
damage in the brain vascular system, to be administered to animals
or patients by any of the routes selected from the group consisting
of intravenous intramuscular subcutaneous intraperitoneal
intrathecal intra-rectally; or locally infiltrated into the skin,
on mucosa, epithelia or organs or more precisely
intralesionally.
25. The use of peptides GHRP-6 or GHRP-2 or Ghrelin or Hexarelin
according to claim 1 for manufacturing a pharmaceutical composition
useful for stopping and controlling the process of brain aging and
deterioration of brain functions because of excessive lipid
peroxidation and oxidative stress processes or damage in the brain
vascular system, to be administered to animals or patients by any
of the routes selected from the group consisting of intravenous
intramuscular subcutaneous intraperitoneal intrathecal
intra-rectally; or locally infiltrated into the skin, on mucosa,
epithelia or organs or more precisely intralesionally.
26. The use of peptides GHRP-6 or GHRP-2 or Ghrelin or Hexarelin
according to claim 1 for manufacturing a pharmaceutical composition
useful for stopping and controlling the deposition process of
fibrotic, amiloid and/or hyaline, and/or granular forms of
eosinophilic and/or osmophilic material in cells or the tissue
extracellular space of the central and peripheral nervous systems,
and to be administered to animals or patients by any of the routes
selected from the group consisting of intravenous, intramuscular,
subcutaneous, intraperitoneal, intrathecal, intra-rectally; or
locally infiltrated into the skin, on mucosa, epithelia or organs
or more precisely intralesionally.
Description
FIELD OF TECHNIQUE
[0001] The present invention relates to the field of the
pharmaceutical industry and medicine, and more precisely to the use
of secretagogue peptides that repeatedly administered in a
pharmaceutical composition prevent and eliminate pathological
depots of fibrotic material in parenchymatous internal tissues,
like in the liver, lungs, esophagus, small intestine, kidneys,
blood vessels, joints and other systemic forms of cutaneous
fibrosis of any etiopathogenesis.
DESCRIPTION OF THE PRIOR ART
[0002] Fibrosis events comprise a group of mono-organic or systemic
pathological entities characterized by the abnormal depot of
extracellular matrix in parenchyma of almost every internal organ,
blood vessels or the skin. They are considered as consequence of
complex autoimmune-based events or interstitial responses to
prolonged mimicking and inflammatory events. In general, an excess
of collagenous material is deposited in the parenchyma by
interstitial effector cells or expanded stromal material that
obliterates the functional tissue. The effector cells mediating
these events are of mesenchymal origin and seem to be quite
specific according to the tissue affected. In general,
myofibroblasts have been implicated in causing pathological
fibroses. Mechanisms mediating fibrosis establishment are complex,
and remain to be fully comprehended. Whichever the case, the
transforming growth factor .beta. (TGF-.beta.), the connective
tissue-derived growth factor (CTGF) and the platelet-derived growth
factor (PDGF) are considered as involved in these events
irrespective of the target organ. Long-term fibroses are generally
fatal with no cure available so far. Now we shall bring some
technical aspects about fibrosis events in some organs (Ding J, Yu
J, Wang C, Hu W, Li D, Luo Y, Luo H, Yu H. Ginkgo biloba extract
alleviates liver fibrosis induced by CCl.sub.4 in rats. Liver
International 2005: 25: 1224-1232.) (Friedman SL. Reversal of
hepatic fibrosis--Fact or fantasy? Hepatology. 2006 Jan. 30;
43(S1):S82-S88).
Hepatic Fibrosis
[0003] When the liver is damaged, the inflammatory response and the
remodeling of the extracellular matrix (EM) restore the normal
function and architecture in this organ. However, when the damaged
is sustained, a misbalance of factors involved in repairing and
resolving the problem alters the EM regulation and promotes the
excessive synthesis of its components. The liver is the main organ
involved in metabolism regulation, blood filtration and hormone
regulation. The hepatic stellate cells (HSC) are allocated in the
space between endothelial cells and hepatocytes, named the Disse's
or sinusoidal space, surrounding the endothelial cells with long
cytoplasmic processes. HSC are capable of synthesizing and
secreting EM components and represent an important source of
fibrotic events. They store retynil esters, synthesize growth
factors and other cytokines, with an additional role in regulating
the sinusoidal blood flow. The HSC can transit from a quiescent
state into an active one induced by the paracrine secretion of
pro-inflammatory cytokines, the production of reactive oxygen
species or by changes in the EM structure affecting the cellular
phenotype. During their activation, HSC differentiate into
myofibroblasts elongated in shape, expressing the smooth muscle
.alpha.-actin and loosing all the retinol stored. In this state,
HSC acquire new properties helping them to keep and amplify the
inflammatory response: capacity to proliferate, contractility,
cytokine production and mainly the synthesis and secretion of EM
components. Among the main factors promoting the EM protein
production in activated HSC are the TGF-.beta., synthesized mainly
from Kupffer cells in the activated phase; the TGF-.beta. is mainly
produced by HSC in the perpetuation phase, supporting the continued
activation. In general, all the fibrotic indurations of the liver
are incompatible with life (Hepatic Failure. Last Updated: Sep. 3,
2004. Editor(s): David Eric Bernstein, MD, Chief, Section of
Hepatology, North Shore University Hospital, Director, Associate
Professor, Department of Internal Medicine, Division of Hepatology,
New York University School of Medicine; Francisco Talayera, PharmD,
PhD, Senior Pharmacy Editor, eMedicine; Oscar S Brann, MD,
Associate Clinical Professor, UCSD School of Medicine; Program
Director of Gastroenterology Fellowship, Department of Internal
Medicine, Naval Medical Center San Diego; Alex J Mechaber, MD,
FACP, Director of Clinical Skills Program, Assistant Professor,
Department of Internal Medicine, Division of General Internal
Medicine, University of Miami School of Medicine; and Julian Katz,
MD, Professor, Department of Internal Medicine, Division of
Gastroenterology, MCP Hahnemann University) (Sarem M. Hepatic
stellate cells: it's role in normal and pathological conditions.
Gastroenterol Hepatol. 2006 February; 29(2):93-101).
Pulmonary Fibrosis
[0004] Pulmonary fibrosis is a relatively slow progressing disease
also including several entities. Histologically, it is
characterized by a temporal heterogeneity of the lesions,
predominating fibroblasts. Even with the sequence of events in the
pathogenesis of pulmonary fibrosis well documented, there is little
information about the precise mechanisms mediating the main damage.
Immunological factors, mainly autoimmune, are regarded as relevant.
Genetic factors have been also implicated. Microscopically, a
reticent property comprises the hyperplasic change in alveolar
epithelial cells (type 11 pneumocytes) with prominent nucleoli and
cytologically atypical, commonly mimicking a viral infecting. It is
possible to find ultra-structural intra-nuclear tubular inclusions.
The deposition process of EM and particularly collagen in the
pulmonary parenchyma insidiously deteriorates lung architecture,
collapsing bronchioles and alveoli, ultimately becoming
dysfunctional and compromising the pulmonary ventilation. The
TGF-.alpha. is also involved as one of the main cytokines
orchestrating this process. Myofibroblasts are the EM-producing
cells (Medranda Gomez M A, Paricio Nunez P, Tovar Martinez A,
Ferrer Marin F, Gonzalez Martinez P, Garcia Puche M J. Pulmonary
fibrosis. Rev Esp Enferm Dig. 2005 November; 97(11):843-4).
Systemic Cutaneous Fibrosis or Scleroderma
[0005] Systemic sclerosis (SS) is an extremely complex disease.
Until now there is no plausible theory of explaining its
pathogenesis. However, fundamental abnormalities in fibroblasts,
endothelial cells and cells of the immune system, particularly B
and T lymphocytes, have been documented. Functional alterations in
these cells promote the typical triad of pathological changes in
SS: cutaneous and visceral progressive fibrosis, small artery and
arteriole lumen obliteration and immune abnormalities. Alterations
in the humoral and cellular immunity trigger the secretion of large
amounts of antibodies, some of them very specific for the disease,
the infiltration of mononuclear cells into the tissues affected and
the deregulated production of cytokines and growth factors. So far,
there has not been clarified which of these alterations is the main
triggering event of the disease or if all of them are inter-twinned
for causing the progressive SS fibrotic process. However, a key
pathogenic component comprises the deregulated and persistent
activation of genes coding for several types of collagen and other
EM proteins in fibroblasts of SS patients. This is the main
difference between normal fibroblasts capable of normal wound
healing and SS fibroblasts with uncontrolled production and
deposition of collagen, resulting in pathological fibrosis in the
organs affected. Once again, the TGF-.beta. is one of the growth
factors that seem to be pronouncedly involved in SS tissue
fibrosis. One of the most important effects comprises the synthesis
of several types of collagen and other EM proteins like fibronectin
(31). Fibroblasts of patients with SS express high levels of the
TCF-.beta. receptor on their surfaces, plausibly responsible for
the increment in the signal induced by the TGF-.beta. and the
increased collagen production (30). This disease is also
irreversibly fatal (Steen V. Targeted therapy for systemic
sclerosis. Autoimmun Rev. 2006 February; 5(2): 122-4).
Diabetic Nephrosclerosis or Diabetic Nephropathy
[0006] Almost every diabetic patient develops glomerular and
tubular basement membrane swelling after 2 to 3 years from disease
diagnosis. Some of them will also develop expansion of the
glomerular mesangia and interstitial fibrosis, the pathological
markers of the progressive diabetic nephropathy. This nephropathy
clinically progresses also developing proteinuria, hypertension and
renal insufficiency. There is a good correlation between the
expansion of the mesangial region, the severity of interstitial
fibrosis and atherosclerosis, also diminishing the glomerular
filtration rate. Ultimately, the mesangial expansion reduces the
glomerular expansion by occluding the glomerular capillaries and
diminishing the effective filtration area. In the same manner, the
tubulointerstitial fibrosis alters the tubular architecture and
function, leading to renal insufficiency. In this disease, the role
of the TGF-.beta. has been elucidated in orchestrating the fibrotic
processes occurring in the kidneys of diabetic patients. This
disease is progressive, insidious and end with patient life by
renal insufficiency (Cohen, M. P., Ziyadeh, F. N., Hong, S. W.,
Shearman, C. W., Hud, E., Lautenslager, G. T., Iglesias-de la Cruz,
M. C., & Chen, S. (2002). Inhibiting albumin glycation in vivo
ameliorates glomerular overexpression of TGF-beta eta1. Kidney Int,
61: 2025-2032), (Ziyadeh, F. N., Hoffman, B. B., Han, D. C.,
Iglesias-de la Cruz, M. C., Hong, S. W., Isono, M., Chen, S.,
McGowan, T. A., & Sharma, K. (2000). Long-term prevention of
renal insufficiency, excess matrix gene expression and glomerular
mesangial matrix expression by treatment with monoclonal
anti-TGF-beta antibody in db/db diabetic mice. Proc. Natl. Acad.
Sci. USA, 97: 8015-8020).
Penis Fibrosis or Peyronie's Disease
[0007] Conceptually, the disease comprises a pathological scar of
the penile elastic covering (tunica albuginea) of the erectile
tissue, causing organ retraction in the resting state and during
erection curvature and retraction. Although difficult to document
the onset of the disease, most of the authors coincide in that
fibrotic degeneration of the tunica albuginea is preceded by an
inflammatory process triggering a vasculitic, immunological or
traumatic process, or collagenopathy. A first period of invasion is
described, when the fibrotic plaque can silently progress denoting
the curve or retraction of the penis with pain during erection or
penetration. Predominant symptoms are caused by fibrosis. Some
patients can also present additional associated fibrosis in the ear
lobule cartilage. Although not fatal, this disease seriously
compromises the life quality of patients. Once again the TGF-.beta.
is involved as triggering or amplifying factor of the molecular
basis of the disease (Jakut M. New discoveries in the basic science
understanding of Peyronie's disease. Curr Urol Rep. 2004 December;
5(6):478-84).
Brain Microvascular Disease
[0008] Vasculopathy has been identified in brains of patients
suffering Alzheimer's disease, as a marker of pathogenesis of this
and other dementias. The laminar and regional distribution of
vascular lesions is correlated to the appearance of neurofibrillary
tangles and senile plaques. More than 100 years ago were formerly
documented physiological anomalies in brain vessels of elder
people, including rigidity, indirection and curling. Less than 20
years go these evidences were confirmed, additionally describing
further distorted hippocampal senile capillaries with the aging of
major vessels. Ultrastructurally, can be distinguished: (a)
membranous inclusions into the basement membrane; and (b)
microvascular collagen depots (fibrosis) or swelling of basement
membrane components. Capillary pericytes markedly degenerate with
age. Deposition of collagen fibers in the inner basement membrane
has being observed in the brain of mammals. The 64 nm-wide turn in
these fibers allowed the identification of collagen composition of
this microvascular fibrosis, allocated between the endothelium and
pericytes or in the inner face of the basement membrane. The
basement membrane swelling and collagen depots similarly increase
with age in rats and humans, and these fibrotic sclerosis
degeneration processes of the brain microvasculature are considered
the anatomical bases of the processes leading to dementia in
general. These diseases clinically worsen, with progressive
occlusion of arteriole networks, ultimately abrogating every social
relationship and cognitive activity. The role of vascular disease
in the pathogenesis of dementia is currently being resettled, with
more than 50% of dementia patients suggested as having any brain
vascular disease stigma. There are other neuro-brain
non-Alzheimer's dementia processes mediating serious decline of
memory, learning and general skills. The most relevant example is
the autonomic brain artery leukoencephalopathy with sub-cortical
infarction (CADASIL). This disease remains to be elucidated at
molecular and cellular levels. Nevertheless, arteriopathies
resulting from the progressive deposition of osmophilic granular
materials subsequently occlude the arterial lumen, establishing
ischemic foci in the brain, followed by infarction episodes. The
loss of neuronal viability by microinfarction deteriorates the
major nervous activity in the brain, leading to a state of senility
and dementia (Nakanao I. The function and integrity of the
neurovascular unit rests upon the integration of the vascular and
inflammatory cell systems. Curr Neurovasc Res. 2005 December;
2(5):409-23.; Mott R T. Neuropathology of Alzheimer's disease.
Neuroimaging Clin N Am. 2005 November; 15(4):755-65).
Other Degenerative Processes. Beta-Amiloid Deposition.
[0009] Alzheimer's disease is the most prevalent dementia and one
of the main death causes in elder people over 65 years old. With
the origin of the disease unknown, the brains of Alzheimer's
disease patients show disease-related deposition of several types
of proteins inside and outside neurons. The beta-amiloid is one of
the proteins forming the extracellular depots in the brain and the
brain stem. The plaques consist of a compact core of beta-amiloid
protein, derived from its precursor protein. The risk for suffering
dementia is strongly associated to polymorphisms in apolipoprotein
E (Apo-E). In the brain, the Apo-E interacts with the beta-amiloid
protein, with Apo-E4 linked to increased beta-amiloid depots and
increased numbers of neurofibrillary tangles.
[0010] Attention, learning and memory processes are among the brain
capacities affected in Alzheimer's disease, this condition
established as a model among dementia diseases characterized by
beta-amiloid deposition. Until now, there is only one drug approved
by the FDA for treating the cognitive deficit during Alzheimer's
disease. The beta-amiloid protein develops necrogenic effects
mediated by free radicals in brain cells. Deposition of
beta-amiloid in the brain parenchyma is a distinctive pathogenesis
marker in Alzheimer's disease, although at a lower rate in normal
physiological aging.
[0011] The diminished synthesis of the beta-amiloid neurotoxic
protein variant can attenuate processes supporting neuronal damage
in Alzheimer's disease. In the same manner, its elimination from
the brain and further excretion could contribute to the recovery of
major nervous functions in patients. Alzheimer's disease severely
compromises the quality of life in patients and a cure is
unavailable yet (Gurol M E. Plasma beta-amyloid and white matter
lesions in AD, MCI, and cerebral amyloid angiopathy. Neurology.
2006 Jan. 10; 66(1):23-9.; Han HS. The function and integrity of
the neurovascular unit rests upon the integration of the vascular
and inflammatory cell systems. Curr Neurovasc Res. 2005 December;
2(5):409-23; Anderson E. The Organic Brain Syndrome (OBS) scale: a
systematic review. Int J Geriatr Psychiatry. 2006 Jan. 27).
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention relates to the use of secretagogue
peptides in a pharmaceutical composition containing GRHP-6, GRHP-2,
Hexarelin and/or Ghrelin, wherein the said pharmaceutical
composition prevents, controls and eradicates the pathological
intra- and extra-cellular depots of hyaline material, amiloid,
granular forms of eosinophilic and osmophilic materials in internal
organs, external and vascular network organs, like the liver,
lungs, esophagus, intestine, kidneys, blood vessels, joints and the
rest of systemic cutaneous, fibrosis variants of any
etiopathogenesis when said composition is applied to the recipient
organism. The pharmaceutical composition of the present invention
is a liquid, semisolid or solid composition, able to be
administered by intravenous, intramuscular, intraperitoneal,
subcutaneous, intrathecal, rectal and topic routes, by local
infiltration into the skin or mucosa, epithelia or organs, more
precisely intralesionally and/or perilesionally. The pharmaceutical
composition of the present invention contains peptides GHRP-6,
GHRP-2, Hexarelin and or Ghrelin at 5 micrograms-1 milligram
concentrations, more precisely at 30-500 micrograms per dose, and
an acceptable pharmacological vehicle.
BRIEF DESCRIPTION OF FIGURES
[0013] FIGURE. Percent of animals with renal fibrosis compromise
per group at the end of the treatment with GHRP-6. Notice existing
differences between the placebo group receiving saline solution and
those receiving GHRP-6. The highest difference was observed when
comparing the placebo group with the group receiving the 400
.mu.g/kg dosage, suggesting a dosage-dependent effect. The
histological evaluation of the fibroproliferative reaction in the
renal interstitium includes the fibrotic tubule encapsulation and
also the fibrotic glomeruli. In this manner is established the
grade of total renal fibrosis employed to determine the percent of
animals affected or unaffected at the end of the treatment.
(a)-Statistical differences of p<0.001 between the group
receiving the 400 .mu.g/kg GHRP-6 dosage and the placebo group.
EXAMPLES
Example 1
Reverting Hepatic Fibrosis in Rats
[0014] The present experiment was conducted to evaluate the effect
of the GHRP-6-based pharmaceutical composition on reverting the
hepatic fibrosis in rats. Hepatic fibrosis was induced in male
Wistar rats of 250 g of average body weight by ligating the
external bile duct. For this purpose, rats were anesthetized with a
ketamine/xylazine combination and subjected to laparotomy to expose
the common bile duct. The duct was double-ligated with chromium
catgut 4-0. After surgery, animals were randomly distributed into 3
experimental groups of 20 rats each: (1) Control placebo group,
receiving physiological saline solution, (2) Group receiving the
GHRP-6 at a 100 .mu.g/kg dosage, and (3) Group receiving the GHRP-6
at a 400 .mu.g/kg dosage. Treatments were daily administered during
three weeks after inducing the fibrosis of the liver parenchyma.
All the treatments were started three weeks after the appearance of
fibrosis. The follow up of the hepatic damage was conducted by
weekly ultrasound examinations of the projection area of this
organ, the progress of serum levels of GOT and GPT transaminases,
gamma glutamyl transferase (GGT) levels and the volume of ascitis.
Treatments with GHRP-6 or placebo were applied by the
intraperitoneal route once daily. When treatments concluded,
animals were sacrificed and blood serum and the liver were
collected. Liver fragments were fixed in formalin neutral and
processed by hematoxylin/eosin staining, or by Masson's trichromic
staining, to evaluate the general damage and the severity of
fibrotic indurations. Other fragments of liver tissues were stored
at -70.degree. C. until processing to determine the content of
hydroxiproline by acid hydrolysis in 1N HCl, and also the
intrahepatic levels of redox metabolism markers. Biochemical
determinations were carried out by calculating the total protein
content by the Bradford's method.
TABLE-US-00001 TABLE 1 Gradation scale of the process: (0)-*null,
(1)-moderate, (2)-limited, (3)-severe, (4) very severe.
Experimental Group Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Placebo
(N = 14) 0 0 2 5* 7 (50%)* Dosage I (N = 16) 5 9 2 0 0 Dosage II (N
= 20) 8 11 1 0 0 *p < 0.001. Chi-squared test.
TABLE-US-00002 TABLE 2 Evaluation of hydroxiproline levels in liver
at the end of three weeks of treatment. Experimental Content of
OH--P (.mu.g/g of total Group proteins) Placebo (N = 14) 133.25
.+-. 21.69** Dosage I (N = 16) 56.71 .+-. 8.11* Dosage II (N = 20)
16.15 .+-. 1.025 **p = 0.00021. Placebo vs. Treated. *p = 0.001
Dosage I vs. Dosage II. Student's two-tailed t test.
TABLE-US-00003 TABLE 3 Serum levels of GOT, GPT and GGT in all the
groups at the end of three weeks of treatment. Experimental Group
GOT (IU/I) GPT (IU/I) GGT (U/I) Intact animals 32.56 .+-. 9.27
20.93 .+-. 7.74 23.62 .+-. 5.21 Placebo 115.84 .+-. 27.80** 155.30
.+-. 11.63** 143.18 .+-. 25.36** (N = 14) Dosage I 61.58 .+-.
16.10* 81.71 .+-. 30.90* 71.53 .+-. 22.14* (N = 16) Dosage II 30.41
.+-. 6.11 60.64 .+-. 19.87 25.56 .+-. 8.63 (N = 20) **p = 0.0003.
Placebo vs. Treated Dosage II and Intact animals. *p = 0.0002
[0015] Dosage I vs. Dosage II and Intact animals. No differences
were observed between Intact animals and Dosage II animals.
Student's two-tailed t test.
TABLE-US-00004 TABLE 4 Levels of oxidative stress markers in liver
samples at the end of the third week of treatment. Experimental
Groups SODt Catalase HPT MDA Intact group 28261.08 .+-. 1260.94
16.40 .+-. 3.95 27.25 .+-. 2.47 0.06. .+-. 0.01 Saline - Placebo
group 573.83 .+-. 645.93** 580.58 .+-. 57.39** 108.66 .+-. 15.82**
0.25 .+-. 0.04** Dosage I Group 11058.07 .+-. 744.61* 68.50 .+-.
12.73* 43.06 .+-. 1.83* 0.14 .+-. 0.02* Dosage II Group 21029.87
.+-. 498.28 31.50 .+-. 4.3 21.16 .+-. 1.71 0.08 .+-. 0.01 **p =
0.0001. Placebo vs. Treated Dosage II and Intact. *p = 0.0003
Dosage I vs. Dosage II and Intact. No differences were observed
between Intact and Dosage II. Student's two-tailed t test.
[0016] Treatment with GHRP-6 clearly demonstrates the capacity of
the peptide to eradicate and control the deposit of collagenous and
extracellular materials in liver parenchyma, produced by ligation
of the common bile duct. The relevance of the treatment is
demonstrated by the convergence of morphological and biochemical
data, supporting the correction of the severely compromised
periductal and periportal fibrotic state. It is important to notice
that animals in the placebo group did not show spontaneous
remission.
Example 2
Reverting Hepatic Fibrosis in Rats
[0017] This experiment was conducted to evaluate the effect of the
pharmaceutical composition containing the GHRP-6 on reverting
hepatic fibrosis in rats, wherein said hepatic fibrosis was induced
by carbon tetrachloride (CCl.sub.4). This is a hepatotoxic agent
that causes chronic hepatitis and fibrosis after long-term
administration. Hepatic fibrosis was induced in male Wistar rats of
250 g of body weight by intraperitoneally administered CCl.sub.4
50%/50% (v/v) in olive oil, twice per week during four weeks. After
that time, 25% of the rats were sacrificed and subjected to blood
biochemistry and pathological anatomy studies. The process of
hepatic fibrosis was demonstrated in all the animals studied. The
animals remaining were randomly distributed into three experimental
groups of 15 rats each: (1) Control placebo group, receiving
physiological saline solution, (2) Group receiving the GHRP-6 at a
100 .mu.g/kg dosage, and (3) Group receiving the GHRP-6 at a 400
.mu.g/kg dosage. Treatments were applied once daily during four
weeks after detecting the fibrotic process in the liver parenchyma.
Treatments were started immediately after the fibrosis established
and the suspension of CCl.sub.4 administration. When treatments
concluded, the animals were sacrificed and blood serum and the
liver were collected. Liver fragments were fixed in formalin
neutral and processed by hematoxylin/eosin staining, or by Masson's
trichromic staining, to evaluate the general damage and the
severity of fibrotic indurations. Other fragments of liver tissues
were stored at -70.degree. C. until processing to determine the
content of hydroxiproline by acid hydrolysis in 1N HCl, and also
the intrahepatic levels of redox metabolism markers. Biochemical
determinations were carried out by calculating the total protein
content by the Bradford's method. The response to the treatment
with the pharmaceutical composition was characterized by
determining the serum levels of GOT and GPT transaminases,
histological criteria in quantitative scale and some markers
distinctive of the levels of lipid peroxidation.
TABLE-US-00005 TABLE 5 Gradation scale of the process: (0)- null,
(1)- moderate, up to 25% of the slide, (2)- limited, up to 50% of
the slide, (3)- severe, up to the 75% of the slide, (4)- very
severe, over 75% of the slide. Experimental Group (n = 15) Grade 0
Grade 1 Grade 2 Grade 3 Grade 4 Placebo 0 0 0 5* 10** Dosage I 0 9
6* 0 0 Dosage II 8* 7 0 0 0 Histological evaluation of the bridging
patterns fibro-proliferative reaction, including concurrent
necrosis in zone III. Animals per group, according to the
compromise grade at the end of treatment. *p < 0.001.
Chi-squared test. Grade 0. Group II vs. Placebo and Dosage I. Grade
1. Dosage I and II vs. Placebo. Placebo, grades 3 and 4 vs. Animals
treated.
TABLE-US-00006 TABLE 6 Evaluation of hydroxiproline levels in the
liver at the end of treatment. Experimental Group OH-P Content (N =
15) (.mu.g/g of total proteins) Placebo 86.19 .+-. 11.43** Dosage I
40.21 .+-. 3.54* Dosage II 10.22 .+-. 4.33 **p = 0.00021. Placebo
vs. Treated. *p = 0.001 Dosage I vs. Dosage II. Student's
two-tailed t test.
TABLE-US-00007 TABLE 7 Serum levels of GOT and GPT in all the
groups at the end of the treatment. Experimental group GOT (IU/I)
GPT (IU/I) Intact animals 31.56 .+-. 6.55 18.77 .+-. 6.53 Placebo
111.97 .+-. 36.50** 274.14 .+-. 21.75** Dosage I 56.31 .+-. 12.19*
77.15 .+-. 22.66* Dosage II 28.18 .+-. 4.71 26.94 .+-. 12.42 **p =
0.0005. Placebo vs. Treated with Dosage II and Intact animals. *p =
0.001 Dosage I vs. Dosage II and Intact animals. No differences
were observed between Intact animals and Dosage II animals.
Student's two-tailed t test.
TABLE-US-00008 TABLE 8 Levels of oxidative stress markers in liver
samples at the end of the third week of treatment. Experimental
Groups SODt Catalase HPT MDA Intact group 28261.08 .+-. 1260.94
14.84 .+-. 1.24 18.72 .+-. 3.22 0.09 .+-. 0.02 Placebo- saline
289.2 .+-. 116.1** 560.59 .+-. 44.28** 257.84 .+-. 86.14** 0.56
.+-. 1.04** group Dosage I group 17632.08 .+-. 321.55* 60.43 .+-.
11.81* 55.11 .+-. 2.77 0.16 .+-. 1.16* Dosage II group 20187.87
.+-. 245.13 22.67 .+-. 3.56 26.44 .+-. 2.43 0.09 .+-. 0.01 **p =
0.0002. Placebo vs. Treated: Dosage I and II and Intact animals. *p
= 0.0005 Dosage I vs. Dosage II, Placebo and Intact animals. No
differences were observed between Intact animals and Dosage II
animals. Student's two-tailed t test.
[0018] The treatment with GHRP-6 demonstrates the capacity of this
peptide to eradicate and control deposition of collagenous and
extracellular matrix materials in general, in the liver parenchyma
as consequence of repeated CCl.sub.4 administration. The treatment
also prevents individual, focal and pericentrolobular hepatocytes
death. The relevance of the treatment is demonstrated by convergent
morphological, enzymatic and biochemical data, supporting the
reversion of an established and compromising severe diffuse liver
fibrosis with a confluent bridge pattern, to almost undetectable
levels without remission. Once again, animals of the placebo group
did not show spontaneous remissions.
Example 3
[0019] Reverting renal fibrosis of Nephrosclerosis in rats. Third
experiment. This experiment was conducted to evaluate the effect of
the pharmaceutical composition containing GHRP-6 in reverting renal
fibrosis in rats. In this case, the process was induced by
sustained administration of the anti-neoplasic agent Doxorubicin
(DX) at a 2.5 mg/kg dosage twice a week during 8 weeks. The
occurrence of fibrosis deposition in the periportal, peri-bronchial
and over the entire renal interstitium with a cystic-nodular
pattern was demonstrated by histopathological studies of these
organs in 25% of the rat population intoxicated with Doxorubicin.
This point forward, DX administration was interrupted and the
treatment with the pharmaceutical composition containing the GHRP-6
was started. The treatment was applied once daily at 100 and 400
.mu.g/kg in a volume of 1 ml by the intraperitoneal route, during 4
weeks. The animals remaining were randomly distributed into three
experimental groups of 20 rats each: (1) Control placebo group,
receiving physiological saline solution, (2) Group receiving the
GHRP-6 at a 100 .mu.g/kg dosage, and (3) Group receiving the GHRP-6
at a 400 .mu.g/kg dosage. When treatments concluded, the animals
were sacrificed by anesthesia overdose and livers, kidneys, lungs
and blood sera were collected. Tissue fragments were fixed in
formalin neutral and processed by hematoxylin/eosin staining, or by
Masson's trichromic staining, to evaluate the general damage and
the severity of fibrotic indurations. Other fragments were stored
at -70.degree. C. until processing to determine the content of
hydroxiproline by acid hydrolysis in 1N HCl, and also the levels of
creatinine and oxidative stress markers. Biochemical determinations
were carried out by calculating the total protein content by the
Bradford's method.
TABLE-US-00009 TABLE 9 Numbers of animals per group in each
severity level of fibrotic compromise. Gradation scale of the
fibrosis process in kidneys: Grade (0)- null, Grade (1)- Moderate,
affecting the interstitium without deforming or encapsulating the
tubules or glomeruli, Grade (2)- Intense, affecting the entire
interstitium, deforming the tubules, obliterating their lumen and
externally encapsulating glomeruli, Grade (3)- Very intense,
encapsulating and collapsing tubules and collapsing glomeruli. Also
comprises mesangial depots. Grupo Experimental (N = 20) Grado 0
Grado 1 Grado 2 Grado 3 Placebo 0 1 4 15* Dosis I 6 9 5 0 Dosis II
5 15* 0 0 *p < 0.001. Chi-squared test.
TABLE-US-00010 TABLE 10 Evaluation of hydroxiproline levels in
kidneys at the end of treatment. Experimental Group (20 OH-P
Content fragments per group) (.mu.g/g of total proteins) Placebo
65.21 .+-. 22.16** Dosage I 46.15 .+-. 2.73* Dosage II 8.66 .+-.
1.02 **p = 0.0001. Placebo vs. Treated. *p = 0.05 Dosage I vs.
Dosage II. Student's two-tailed t test.
TABLE-US-00011 TABLE 11 Serum levels of GOT and GPT in all the
groups at the end of the treatment. Integrity of the hepatic
parenchyma. Experimental Group GOT (IU/I) GPT (IU/I) Placebo 124.12
.+-. 28.3** 188.77 .+-. 16.98* Dosage I 64.82 .+-. 23.71* 81.0 .+-.
10.25* Dosage II 26.22 .+-. 4.1 28.25 .+-. 5.66 **p = 0.0005.
Placebo vs. Treated. *p = 0.042 Dosage I vs. Dosage II Student's
two-tailed t test.
TABLE-US-00012 TABLE 12 Levels of oxidative stress markers in renal
tissue samples at the end of the fourth week of treatment.
Experimental groups SODt Catalase HPT MDA Placebo-saline group
199.7 .+-. 6.81** 356.2 .+-. 18.15** 287.11 .+-. 20.02** 0.981 .+-.
1.1** Dosage I group 665.08 .+-. 28.42* 126.02 .+-. 12.23* 73.2
.+-. 6.92 0.56 .+-. 2.23* Dosage II group 1287.64 .+-. 112.63 45.38
.+-. 8.27 16.14 .+-. 3.67 0.087 .+-. 0.02 **p = 0.0002. Placebo vs.
Treated: Dosages I and II and Intact animals. *p = 0.0005 Dosage I
vs. Dosage II, Placebo and Intact animals. No differences were
observed between Intact animals and Dosage II animals. Student's
two-tailed t test.
Example 4
Control of Pulmonary Fibrosis
[0020] This experiment was conducted to evaluate the effect of the
pharmaceutical composition containing GHRP-6 in reverting renal
fibrosis in rats. In this case, the process as induced by sustained
administration of the anti-neoplasic agent Bleomycin (Ble) at a 2.5
U/kg dosage twice a week during 4 weeks. Fibrosis was demonstrated
in lungs of 25% of the Ble intoxicated animals by histopathological
analysis. This point forward, the administration of Ble was
suspended and treatment with the pharmaceutical composition
containing the GHRP-6 was started. The treatment was applied once
daily, at 100 and 400 .mu.g/kg in a 1 ml volume by intraperitoneal
route during 4 weeks. The animals were randomly distributed into
three experimental groups of 10 rats each: (1) Control placebo
group receiving saline physiological solution, (2) Group receiving
GHRP-6 at 100 .mu.g/kg dosage, (3) Group receiving GHRP-6 at 400
.mu.g/kg. When treatments concluded, the animals were sacrificed by
anesthesia overdose and lungs and blood serum were collected.
Tissue fragments were fixed in formalin neutral and processed by
hematoxylin/eosin staining, or by Masson's trichromic staining, to
evaluate the general damage and the severity of fibrotic
indurations. Other fragments of lung tissues were stored at
-70.degree. C. until processing to determine the content of
hydroxiproline by acid hydrolysis in 1N HCl, and also the
intrahepatic levels of redox metabolism markers. Biochemical
determinations were carried out by calculating the total protein
content by the Bradford's method. Histological evaluation of the
fibro-proliferative reaction in lungs includes the process of
peri-vascular, peri-bronchial and septal fibrosis. The overall
grade of pulmonary fibrosis was established according to the
extension and intensity of the process in these three segments, to
determine the percent of animals affected or unaffected at the end
of the treatment with the GHRP-6. The numbers of animals in every
group with fibrotic lungs according to the severity of fibrosis
are:
Grade 0--No evidences of fibrosis or only thin and diffuse fiber or
areolar material foci present, without respiratory compromise.
Grade 1--Fibrosis predominantly vascular in more than 75% of
arterioles and capillaries. Grade 2--Fibrosis predominantly
vascular in more than 75% of arterioles and capillaries, with
additional peri-bronchial compromise.
[0021] Grade 3--Fibrosis predominantly vascular in more than 75% of
arterioles and capillaries, with additional peri-bronchial
compromise. Fibrotic material is also detected in the interalveolar
septum.
TABLE-US-00013 TABLE 13 Animals classified according to the
severity of pulmonary fibrosis in each group. Experimental Group (N
= 15) Grade 0 Grade 1 Grade 2 Grade 3 Placebo 0 0 5 10* Dosage I 3
10 2 0 Dosage II 8 7 0 0 *p < 0.05. Exact Fisher's test.
[0022] As can be seen, there were no animals in the placebo group
included in grade 0 or grade 1 scales. Their majority were
classified as grade 3 in severity. By the contrary, dosage II
demonstrated a potent protecting effect, with more than the 50% of
the animals classified as grade 0.
TABLE-US-00014 TABLE 14 Evaluation of hydroxiproline levels in
lungs at the end of treatment with saline or GHRP-6. Experimental
group (15 OH-P content fragments per group) (.mu.g/g of total
proteins) Placebo 178.53 .+-. 42.77** Dosage I 91.24 .+-. 16.84*
Dosage II 12.75 .+-. 3.61
[0023] The effect on eradicating or reverting the pulmonary
fibrosis generated by Ble is also evidenced by the hydroxiproline
content in dry samples of pulmonary tissues, coinciding with the
histopathological results described above.
[0024] So far, evidences have been shown supporting the potent
antifibrotic effect of the pharmaceutical composition containing
the GHRP-6 in four independent experiments, including: two liver
fibrosis, one kidney fibrosis and one pulmonary studies,
respectively. Their results are repeatable and reproducible,
indicating the efficacy of the treatment in controlling these
processes in more than one internal organ, irrespective of their
etiopathogenic origin.
Example 5
Effect of the Pharmaceutical Composition Containing the GHRP-6 in
Controlling and Eradicating the Beta-Amiloid Protein Deposition in
Brain
[0025] This study was conducted to evaluate the influence of the
long-term administration (8 weeks) of the GHRP-6 on the biochemical
and morphological markers in the brain of transgenic mice
expressing the beta-amiloid precursor protein, these markers also
indicating the progression of the central nervous system damage.
For the present study, 20-25 g in body weight male APP transgenic
mice were acquired, expressing the beta-amiloid precursor protein.
Animals (N=30) were randomly distributed in:
Placebo group--Physiological saline solution 0.9%. Dosage I
group--GHRP-6 at 50 .mu.g/kg of body weight in saline solution.
Dosage II group--GRHP-6 at 100 .mu.g/kg of body weight in saline
solution.
[0026] Treatments were applied by the intraperitoneal route in 1
ml, five days a week during 8 weeks, with animals receiving 40
administrations of GHRP-6. We knew from previous exploratory pilot
studies that this period of time was sufficient for improving
cognitive and motor skills in animals under stress.
[0027] After 8 weeks of treatment, mice were sacrificed by
anesthesia overdose, and perfused in situ with saline solution.
Encephala were extracted, one encephalon was frozen in dry-ice and
the other was fixed in 4% para-formaldehyde neutral. Samples were
cryo-sliced at 10 .mu.m and slices were stained with
hematoxylin/eosin, Congo Red, or incubated with an antibody
specific for the beta-amiloid protein. Morphometric procedures were
carried out by microscopic imaging capture by a camera connected to
the microscope, and the images were analyzed with the DIGIPAT
software.
Markers Studied
[0028] The number of fibrillar deposits of the beta-amiloid protein
positive to Congo Red staining.
[0029] The number of foci immunoreactive to the antibody that
recognizes the beta-amiloid protein.
[0030] Size of the beta-amiloid plaques in the brain, recognized at
200.times. and 400.times. magnifications (.mu.m.sup.2).
[0031] Brain concentration of myo-inositol as indicator of aging
and brain metabolism deterioration (.mu.mol/g of tissue).
TABLE-US-00015 TABLE 15 Effect of the treatment with the GHRP-6 on
amiloid deposit removal in the brain. No. of deposits No. of
deposits Myo-inositol Experimental positive to positive to beta-
Size of amiloid concentration in group Congo Red amiloid plaques
the brain. Placebo 35.64 .+-. 11.33** 31.27 .+-. 8.9** 48.5 .+-.
2.03** 61.28 .+-. 16.33** Dosage I. GHRP6 21.78 .+-. 6.57* 15.11
.+-. 3.27 15.51 .+-. 6.44 49.35 .+-. 10* Dosage II. GHRP6 14.52
.+-. 4.18 13.58 .+-. 4.61 10.88 .+-. 4.1 27.45 .+-. 8.61 **p =
0.0001. *p = 0.0023. Mann Whitney U's test.
[0032] All the results corresponding to parameters under study are
shown in table 15, characterizing the effect of the long-term
treatment of pharmaceutical composition containing GHRP-6. Notice
that results are referred to the count of digital images of one
encephalon. To overcome this limitation, counts were carried out
blindly by three independent individuals and results shown
correspond to 5 slide observations. Table 15 shows the effect of
the pharmaceutical composition containing the GHRP-6 in controlling
the beta-amiloid accumulation and the brain biochemistry. As can be
seen, after 8 weeks of treatment with the pharmaceutical
composition containing GHRP-6, a positive impact is plausible in
controlling the beta-amiloid accumulation in its different forms
and also in correcting the metabolism of this organ. A marked
effect characterized by reducing the accumulation of myo-inositol
evidences the correction of biochemical pathways of higher energy
assimilation and nutrition of neurons. These could have a favorable
clinical impact on controlling the brain aging process.
[0033] In the following table 16, the favorable effect of the
pharmaceutical composition containing the GHRP-6 on controlling
lipid peroxidation in the brain of Alzheimer's disease transgenic
mice is demonstrated. One again, these evidences suggest the
favorable effect of this pharmaceutical composition to control one
of the processes responsible for the deterioration of the nervous
tissue in disease and aging.
TABLE-US-00016 TABLE 16 Oxidative stress markers in brain tissues.
Experimental groups SODt Catalase HPT MDA Placebo-saline group
475.9 .+-. 60.32** 118.6 .+-. 26.33** 105.6 .+-. 22.1** 1.232 .+-.
1.14** Dosage I group 611.17 .+-. 44.79* 81.6 .+-. 15.25 54.3 .+-.
11.87* 0.77 .+-. 1.56* Dosage II group 863.22 .+-. 50.3 60.18 .+-.
13.67 21.25 .+-. 5.44 0.4 .+-. 0.02 **p = 0.00014. Placebo vs.
Treated: Dosage I and II. *p = 0.025 Dosage I vs. Dosage II. Mann
Whitney's U test.
Example 6
[0034] Effect of the pharmaceutical composition based on GHRP-6 and
other peptides on controlling dementia of vascular origin.
Eradication of osmophilic material in the brain cortex. Prevention
and control of the brain aging process.
[0035] This experiment was conducted to evaluate the efficacy of
pharmaceutical compositions indistinctly containing one of the
peptides GHRP-6, GHRP-2, hexarelin or ghrelin on the central
neurofunctional involution process in transgenic mice
over-expressing one mutated form of the NOTCH 3 gene in blood
vessel smooth muscle cells. These animals develop in terms of
months an arteriopathy similar to that of the CASADIL disease,
referred to the descriptive memory, and occurring as one of the
main causes of vascular dementia. In these animals, vascular
lesions also include retinal-cerebral, cerebral and cochlear
vasculopathies. The beta-amiloid material present in the brain and
blood vessels, the deposit of osmophilic granular material in brain
and meningeal artery walls and their reduced lumen are
histopathologically relevant. White-pale zones, micro-infarction
and hemorrhagic foci zones appear in the brain and its main nervous
trunks.
[0036] Eighteen- to twenty-months-old male mice were employed when
evidenced the symptoms of the disease. The animals were randomly
assigned to the following experimental treatment groups:
[0037] A--Placebo group receiving the physiological saline
solution.
[0038] B--Group receiving GHRP-6 at a 100 .mu.g/kg dosage.
[0039] C--Group receiving GHRP-2 at a 100 .mu.g/kg dosage.
[0040] D--Group receiving Ghrelin at a 100 .mu.g/kg dosage.
[0041] E--Group receiving Hexarelin at a 100 .mu.g/kg dosage.
[0042] Treatments were applied twice a week during 16 weeks by
intraperitoneal route. When treatments concluded, autopsy studies
were conducted irrespective of the clinical improvement evidenced
in a great number of animals treated. Brain tissue samples
including meningeal tissues were collected for biochemical and
histopathological determinations. The animals received anesthesia
overdosed and subjected to intra-cardiac perfusion with cold
physiological saline solution to wash off the blood present in the
encephala. The encephala were extracted and one encephalon was
frozen in dry-ice and the other was fixed in 4% para-formaldehyde
neutral. Samples were cryo-sliced at 10 .mu.m and slices were
stained with hematoxylin/eosin, Congo Red, or incubated with an
antibody specific for the beta-amiloid protein. Morphometric
procedures were carried out by microscopic imaging capture by a
camera connected to the microscope, and the images were analyzed
with the DIGIPAT software.
Markers Studied
[0043] Number of fibrillar deposits of beta-amiloid in blood
vessels.
[0044] Number of sub-cortical infarctions.
[0045] Number of sub-cortical hemorrhages.
[0046] Brain concentration of myo-inositol as indicator of aging
and brain metabolism deterioration (.mu.mol/g of tissue).
[0047] Brain oxidative stress markers.
TABLE-US-00017 TABLE 17 Results of morphometric determinations in
cerebral tissues. No. of blood No. of blood No. of No. of
Experimental vessels positive vessels positive subcortical
hemorrhagic groups to Congo Red to Nissl infarctions. foci. Placebo
35.64 .+-. 11.33** 31.27 .+-. 8.9** 48.5 .+-. 2.03** 41.28 .+-.
16.33** GHRP-6 16.31 .+-. 5.33 14.22 .+-. 8.15 26.79 .+-. 4.19
19.05 .+-. 5.14 GHRP-2 18.26 .+-. 4.57 13.8 .+-. 6.76 30.13 .+-.
5.72 11.79 .+-. 4.25 Ghrelin 17.67 .+-. 2.26 11.27 .+-. 4.61 26.9
.+-. 4.27 13.06 .+-. 2.77 Hexarelin 15.24 .+-. 1.24 11.36 .+-. 6.4
27.11 .+-. 3.55 14.61 .+-. 3.31 **p < 0.0002 between placebo and
the rest of the groups treated with each of the pharmaceutical
compositions containing each of the substances under study.
[0048] As can be seen, treatment with each of the secretagogue
peptides significantly reduces the number of arteries, arterioles
and capillaries positive to fibrillar amiloid (Congo Red) and
granular depositions of osmophilic material (Nissl's staining).
Consequently, the presence of leukoencephalopathy-associated
sub-cortical infarction and hemorrhagic foci also significantly
diminished in each of the groups treated with pharmaceutical
compositions.
TABLE-US-00018 TABLE 18 Oxidative stress markers in the brain.
Experimental Cerebral Groups SODt Cabalase HPT MDA Myo-Inositol
Saline Group 360.9 .+-. 42.48** 274.6 .+-. 30.14** 265.4 .+-.
18.7** 0.984 .+-. 0.04** 44.2 .+-. 10.08** GHRP-6 12327.31 .+-.
55.1 51.5 .+-. 16.01 101.7 .+-. 22.09 0.23 .+-. 0.05 12.36 .+-.
5.64 GHRP-2 11065.49 .+-. 46.29 48.23 .+-. 14.25 117 .+-. 13.55
0.302 .+-. 0.01 14.65 .+-. 4.38 Ghrelin 12290.9 .+-. 60.32 44.67
.+-. 11.58 142.16 .+-. 10.01 0.261 .+-. 0.03 14.04 .+-. 3.85
Hexarelin 10814.68 .+-. 33.08 51.19 .+-. 7.84 54.3 .+-. 11.87 0.284
.+-. 0.01 11.88 .+-. 2.71 **Represents differences of p < 0.01
between animals of the placebo group receiving physiological saline
solution and the rest of the groups treated with individual
pharmaceutical compositions containing the respective peptides.
[0049] The effect of the peptides is also remarkably evidenced when
studying the process of lipid peroxidation in the brain of
transgenic mice as a model of human CADASIL disease. As
demonstrated in reducing the vascular damage and infarctions, the
secretagogue peptides studied inhere show the capacity to reduce or
attenuate the neurotoxicity associated to the increased production
of reactive oxygen species in the human disease, this increased
production also demonstrated in the animals receiving the saline
solution. This effect extends the concept of general
neuroprotection by using these substances into contexts where brain
aging is mediated by vascular damage and excessive lipid
peroxidation.
Example 7
[0050] Effect of peptides GHRP-6, GHRP-2, Hexarelin and/or Ghrelin
in eradicating the pathological deposits of physiological material
in the skin. To study the effect of peptides GHRP-6, GHRP-2,
Hexarelin and/or Ghrelin in eradicating the pathological deposits
of physiological material in the skin, human keloid fragments were
xeno-transplanted into the dorsal region in athymic mice. After 72
hrs of evolution, to corroborate grafting and viability of
xeno-transplants, the animals (N=6) were randomly distributed into
the following experimental groups: [0051] A--Saline control group
(vehicle of the active principles). [0052] B--Group receiving
GHRP-6. [0053] C--Group receiving GHRP-2. [0054] D--Group receiving
Ghrelin. [0055] E--Group receiving Hexarelin. [0056] Treatments
were applied once every 24 hrs during 7 days. The substances were
infiltrated at the edges in the implants, for local bioavailability
of the active principles, at dosages from 5 micrograms to 1
milligram. After the treatment period, the animals were sacrificed
and the implants extracted to evaluate the pharmacological response
to every substance. Samples were weighed and fragmented for
histological studies and biochemical determinations of collagen.
The fragments for histological studies were fixed in 10% formalin
neutral, and those fragments for biochemical analyses were stored
at -70.degree. C. [0057] The parameters studied were: [0058] a--Wet
weight of the graft collected. [0059] b--Hydroxiproline content in
the tissue. [0060] c--Number of microscopic fields with tissue
positive to Picrosirius red staining and trichromic Masson's
staining. Images were taken with 4.times. and 10.times.
magnifications, with data averaged for each magnification. [0061]
d--Number of mast cells by microscopic field positive to aniline
blue staining, with 20.times. magnifications. [0062] As illustrated
in table 19, all the peptides under study exerted a significant
anti-fibrotic effect when compared to the animals receiving the
vehicle as control.
TABLE-US-00019 [0062] TABLE 19 Anti-fibrotic effect of treatments
in the skin. No. of No. of fields microscopic Hydroxiproline
positive to fields with mast Group of Wet weight content
collagen-specific cells (20X treatment (grams) (.mu.g/g) staining
magnif.) Saline control 13.55 .+-. 2.31 258.61 .+-. 12.53 37.2 .+-.
22.44 25.31 .+-. 6.18 GHRP-6 6.71 .+-. 1.18 a 88.27 .+-. 4.61 11.45
.+-. 5.98 9.54 .+-. 2.15 GHRP-2 7.14 .+-. 1.02 a 84.17 .+-. 7.75
18.12 .+-. 6.75 11.17 .+-. 3.33 Hexarelin 6.25 .+-. 1.73 a 88.59
.+-. 6.58 12.35 .+-. 8.94 10.18 .+-. 2.98 Ghrelin 6.67 .+-. 1.53 a
85.23 .+-. 4.11 11.27 .+-. 4.67 10.71 .+-. 4.57 a Represents the
significant difference of p < 0.01 between groups receiving
peptides and the control saline group. Student's two-tailed
t-test.
[0063] As evidenced, all the peptides exerted at the assayed
dosages a potent anti fibrotic effect in the experimental system
established, characterized by an acute, rapid decrease of the
excessive collagen material and extracellular matrix, the reduction
of inductor cells (mast cells) and effector cells (fibroblasts and
myofibroblasts). Noteworthy, since the third infiltration all the
implants receiving any of the peptides showed a marked reduction in
size and became pale and devitalized.
* * * * *