US20090221512A1 - Pharmaceutical composition containing ghrp-6 to prevent and eliminate fibrosis and other pathological deposits in tissues - Google Patents

Pharmaceutical composition containing ghrp-6 to prevent and eliminate fibrosis and other pathological deposits in tissues Download PDF

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US20090221512A1
US20090221512A1 US12/281,018 US28101807A US2009221512A1 US 20090221512 A1 US20090221512 A1 US 20090221512A1 US 28101807 A US28101807 A US 28101807A US 2009221512 A1 US2009221512 A1 US 2009221512A1
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Jorge Berlanga Acosta
Danay Cibrian Vera
Diana Garcia Del Barco Herrera
Gerardo Enrique Guillen Nieto
Jose Suarez Alba
Ernesto Lopez Mola
Manuel Selman-Housein Sosa
Mariela Vazquez Castillo
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P11/00—Drugs for disorders of the respiratory system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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.
  • 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.
  • 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.
  • myofibroblasts have been implicated in causing pathological fibroses. Mechanisms mediating fibrosis establishment are complex, and remain to be fully comprehended.
  • TGF- ⁇ transforming growth factor ⁇
  • CTGF connective tissue-derived growth factor
  • PDGF platelet-derived growth factor
  • the liver is the main organ involved in metabolism regulation, blood filtration and hormone regulation.
  • the hepatic stellate cells 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.
  • 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.
  • HSC differentiate into myofibroblasts elongated in shape, expressing the smooth muscle ⁇ -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.
  • the TGF- ⁇ synthesized mainly from Kupffer cells in the activated phase
  • the TGF- ⁇ is mainly produced by HSC in the perpetuation phase, supporting the continued activation.
  • all the fibrotic indurations of the liver are incompatible with life (Hepatic Failure. Last Updated: Sep. 3, 2004.
  • 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 TGF- ⁇ 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).
  • SS Systemic sclerosis
  • 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.
  • the TGF- ⁇ is one of the growth factors that seem to be pronouncedly involved in SS tissue fibrosis.
  • Fibroblasts of patients with SS express high levels of the TCF- ⁇ receptor on their surfaces, plausibly responsible for the increment in the signal induced by the TGF- ⁇ 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).
  • tubulointerstitial fibrosis alters the tubular architecture and function, leading to renal insufficiency.
  • the role of the TGF- ⁇ 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).
  • 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.
  • penile elastic covering titanium albuginea
  • 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.
  • TGF- ⁇ 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).
  • 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.
  • CADASIL autonomic brain artery leukoencephalopathy with sub-cortical infarction
  • Beta-Amiloid Deposition Other Degenerative Processes. Beta-Amiloid Deposition.
  • 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).
  • Apo-E interacts with the beta-amiloid protein, with Apo-E4 linked to increased beta-amiloid depots and increased numbers of neurofibrillary tangles.
  • beta-amiloid deposition a condition established as a model among dementia diseases characterized by beta-amiloid deposition.
  • 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.
  • 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).
  • OBS Organic Brain Syndrome
  • 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.
  • 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 ⁇ 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 ⁇ g/kg GHRP-6 dosage and the placebo group.
  • 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.
  • 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.
  • mice 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 ⁇ g/kg dosage, and (3) Group receiving the GHRP-6 at a 400 ⁇ 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.
  • GTT gamma glutamyl transferase
  • 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° 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.
  • 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.
  • 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 4 ).
  • CCl 4 carbon tetrachloride
  • Hepatic fibrosis was induced in male Wistar rats of 250 g of body weight by intraperitoneally administered CCl 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.
  • mice 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 ⁇ g/kg dosage, and (3) Group receiving the GHRP-6 at a 400 ⁇ 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 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.
  • liver tissues were stored at ⁇ 70° 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.
  • 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 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.
  • 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 ⁇ 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 ⁇ g/kg dosage, and (3) Group receiving the GHRP-6 at a 400 ⁇ g/kg dosage.
  • 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° 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.
  • mice 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 ⁇ g/kg dosage, (3) Group receiving GHRP-6 at 400 ⁇ g/kg.
  • 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° 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.
  • 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.
  • Placebo group Physiological saline solution 0.9%.
  • Dosage I group GHRP-6 at 50 ⁇ g/kg of body weight in saline solution.
  • Dosage II group GRHP-6 at 100 ⁇ g/kg of body weight in saline solution.
  • 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.
  • 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 ⁇ 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.
  • the number of foci immunoreactive to the antibody that recognizes the beta-amiloid protein is the number of foci immunoreactive to the antibody that recognizes the beta-amiloid protein.
  • Brain concentration of myo-inositol as indicator of aging and brain metabolism deterioration ( ⁇ mol/g of tissue).
  • 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.
  • mice 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:
  • A—Placebo group receiving the physiological saline solution A—Placebo group receiving the physiological saline solution.
  • 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 ⁇ 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.
  • Brain concentration of myo-inositol as indicator of aging and brain metabolism deterioration ( ⁇ mol/g of tissue).
  • 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.
  • 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.
  • 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.

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US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9180082B2 (en) 2012-03-28 2015-11-10 Incospharm Corporation Biotin-conjugated hexapeptide-2 derivative and use thereof
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10709783B2 (en) 2015-12-30 2020-07-14 Neuboron Medtech Ltd. Neutron capture therapy system for eliminating amyloid β-protein

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US9119832B2 (en) * 2014-02-05 2015-09-01 The Regents Of The University Of California Methods of treating mild brain injury
FR3034662B1 (fr) * 2015-04-10 2020-08-28 Isp Investments Inc Nouvelles utilisations du peptide de sequence his-d-trp-ala-trp-d-phe-lys-nh2 pour diminuer ou retarder l'apparition de la senescence cellulaire et des signes du vieillissement cutane
CN109908331A (zh) * 2019-04-30 2019-06-21 青岛大学附属医院 海沙瑞林在制备保护肾脏缺血再灌注损伤药物/药物组合物中的应用

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Cited By (15)

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Publication number Priority date Publication date Assignee Title
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US11008366B2 (en) 2010-08-13 2021-05-18 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10703780B2 (en) 2010-08-13 2020-07-07 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9522947B2 (en) 2011-10-18 2016-12-20 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10308699B2 (en) 2011-10-18 2019-06-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9180082B2 (en) 2012-03-28 2015-11-10 Incospharm Corporation Biotin-conjugated hexapeptide-2 derivative and use thereof
US10669230B2 (en) 2012-11-01 2020-06-02 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10709783B2 (en) 2015-12-30 2020-07-14 Neuboron Medtech Ltd. Neutron capture therapy system for eliminating amyloid β-protein

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KR101462077B1 (ko) 2014-11-20
KR20080100378A (ko) 2008-11-17
BRPI0708349A2 (pt) 2011-05-24
US8722626B2 (en) 2014-05-13
RU2465913C2 (ru) 2012-11-10
RU2008138561A (ru) 2010-04-10
JP5426175B2 (ja) 2014-02-26
DK1994939T3 (da) 2012-09-10
US20110230415A1 (en) 2011-09-22
CU23592A1 (es) 2010-11-11
CA2644431C (fr) 2016-08-30
AR059644A1 (es) 2008-04-16
ZA200807600B (en) 2009-06-24
MX2008011140A (es) 2008-09-08
AU2007219568A1 (en) 2007-09-07
JP2009528302A (ja) 2009-08-06
AU2007219568B2 (en) 2012-10-04
EP2368563A1 (fr) 2011-09-28
BRPI0708349B8 (pt) 2021-05-25
ES2388879T3 (es) 2012-10-19
WO2007098715A1 (fr) 2007-09-07
CA2644431A1 (fr) 2007-09-07
CN101426517A (zh) 2009-05-06
EP1994939B1 (fr) 2012-05-30
CN101426517B (zh) 2013-04-03
BRPI0708349B1 (pt) 2019-04-24

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