Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• the present invention refers to an enhanced process to obtain a standardized pharmaceutical or veterinary product from the skin of fruits of plants from genus Vitis, which is useful for the treatment of symptoms, dysfunctions and other manifestations of the metabolic syndrome, as well as for prophylaxis or treatment of diseases caused by the syndrome.
• Figure 1 shows the absolute reduction of blood pressure with the product of the present invention.
• Figure 2 shows the percentage reduction of blood pressure with the product of the present invention.
• FIGS 3 and 4 show the blood sugar profile with the product of the present invention.
• Figures 5A and 5B show the increase in insulin secretion.
• Figures 6A to 9C show the effect of the product of the present invention over glucose metabolism in the model of genetic blood hypertension (SHR) with 400 mg/kg/day.
• SHR genetic blood hypertension
• Figure 10 shows the effect of the product of the present invention over blood pressure in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day.
• Figure 11 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day.
• Figures 12A to 13C show the effect of the product of the present invention over glucose metabolism in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day.
• Figure 14 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of neuroendocrine obesity with 400 mg/kg/day.
• Figure 15 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of exogenous obesity (hypercaloric coffee shop diet) with 400 mg/kg/day.
• Figures 16A and 16B show blood sugar and insulin curves with 400 mg/kg/day of the product of the present invention in the model of exogenous obesity (hypercaloric coffee shop diet).
• Figures 17A to 17C show the areas over the glucose and insulin curves and insulin sensitivity rate with 400 mg/kg/day of the product of the present invention in the model of exogenous obesity (hypercaloric coffee shop diet). DESCRIPTION OF THE INVENTION
• the present invention refers to an enhanced process to obtain a standardized pharmaceutical or veterinary product from skin of fruits of the plants from genus Vitis.
• the manufacturing process of the present invention comprises the steps of:
• the raw material may comprise, under no limitation, skin of fruits from one or more species of the genus Vitis, particularly Vitis vinifera or Vitis labrusca.
• the ionic exchange resin of the process of the present invention is used to concentrate active principles and withdrawal more polar compounds, remaining more nonpolar compounds.
• a hydroalcoholic solution comprises lower alcohols, particularly methanol, ethanol, propanol, butanol or mixtures thereof.
• the step of recovery of the active principle particularly comprises, in sequence:
• Ratio between water and alcohol as used in the process is preferably 5:1.
• the process of the present invention allows industrial manufacture, since it reduces the processing time, presents suitable yielding and results in a pharmaceutical or veterinary product with standardized amount of polyphenols.
• the pharmaceutical or veterinary product of the present invention contains about 0.01% to about 90% by weight, particularly 16 to 20% by weight, of at least one of the polyphenols of formula (I):
• Ri, R2, R3, R4, R5 and Re are the same or different and each one is independently selected from YX, wherein Y is a chalcogen, preferably having molecular weight of about 16 and valence between 1 and 3 and X is selected from H, CH 3 , COCH 3 , halogens, COOH, alkaline metals, sugars, glucosides, glucuronides or (CH 2 ) n CH 3 , CO(CH) 2n CH 3 , wherein n varies between 0 and 16.
• the pharmaceutical or veterinary product of the present invention is useful both to be directly given to a patient, and to be used to prepare pharmaceutical or veterinary compositions, under contents varying between about 1 and about 5000 mg/kg/day, particularly between about 200 and about 400 mg/kg/day, divided into one or more times per day.
• the present invention refers to pharmaceutical or veterinary compositions containing about 1 to about 5000 mg, particularly about 200 to about 400 mg, of the pharmaceutical or veterinary product of the present invention, as well as pharmaceutically acceptable carriers, or acceptable for veterinary use.
• Suitable carriers for the invention are e. g. and under no limitation the ones as mentioned by the book Remington's Pharmaceutical Sciences, from the U. S. publisher Mack Publishing, European Pharmacopaea or Brazilian Pharmacopaea.
• the pharmaceutical or veterinary product and the pharmaceutical or veterinary compositions of the present invention are useful for the treatment of components of metabolic syndrome, as well as for the prophylaxis and treatment of diseases caused by the syndrome.
• the metabolic syndrome comprises a set of metabolic risk factors manifesting in an individual, predisposing him or her to cardiovascular diseases and atherosclerosis.
• Risk factors or components of the metabolic syndrome as mentioned are abdomen obesity (excess of fat in tissues around the abdomen), dyslipemia (fat dysfunctions in the blood - increase in triglycerides, reduction of HDL cholesterol, increase in LDL cholesterol), increase in blood pressure, resistance to insulin and intolerance to glucose (the organism does not use insulin and sugar from the blood in a suitable manner), increase in fastness blood sugar, hepatic steatosis, pro-thrombotic state and pro-inflammatory state.
• the present invention refers to a method for treatment of responses involved in the metabolic syndrome, as well as for prophylaxis or treatment of diseases caused by the syndrome, consisting in the administration, to a human or animal patient in need of said treatment, of a daily amount of about 1 to about 5000 mg/kg, particularly about 200 to about 400 mg/kg, of the product of the present invention or a pharmaceutical or veterinary composition containing it.
• the resin was sequentially washed with 300 liters of ethanol, 200 liters of an ethanol-water solution (1:1) and 200 liters of water.
• the product as obtained was concentrated under vacuum at 60 0 C and subsequently dried in a spray dryer, not adding adjuvants or carriers, by keeping the inlet temperature at 180 0 C and the outlet temperature at 85 0 C.
• the resulting product is a fine and water soluble powder, with 4% humidity (2 g/105 °C/2 h). Furthermore, by the method of Folin-Ciocalteau, it was verified that the final product as obtained contains 18% polyphenols of formula (I) and pertinent profile by thin layer chromatography (TLC).
• the obtained solution was passed through an ionic (cationic) exchange resin, discharging the water fraction.
• the resin was sequentially washed with 300 liters of ethanol, 200 liters of an ethanol-water solution (1:1) and 200 liters of water.
• the product as obtained was concentrated under vacuum at 60 0 C and subsequently dried in a spray dryer, without adjuvants or carriers, by keeping the inlet temperature at 180 0 C and the outlet temperature at 85 0 C.
• the resulting product is a fine and water soluble powder, with 4% humidity (2 g/105 °C/2 h). Furthermore, by the method of Folin-Ciocalteau, we verified that the final product as obtained contains 30% polyphenols of formula (I) and pertinent profile by high pressure liquid chromatography (HPLC). ACUTE TOXICITY f DLsn) OF THE PRODUCT
• SPF Specific pathogen free mice
• Animals were orally treated with the pharmaceutical or veterinary product of Example 1 under dosages varying between 0 and 5000 mg/kg.
• Control groups received carrier (0.9% NaCI, 10 mi/kg, v. o.).
• mice with the product of the present invention 100 to 1000 mg/kg, daily given for 90 consecutive days orally, caused reduction in plasma levels of triglycerides and total cholesterol in comparison with control animals, as shown by the table below:
• the pharmaceutical or veterinary product obtained as per Example 1 was diluted in distilled water and the volume of solution as orally given varied between 0.8 and 1.3 ml.
• control group was solely treated with aloxane along 25 days.
• the experimental group was treated with aloxane, but on day zero, after measuring sugar blood, animals received 100 mg/kg/day of the product of Example 1 with drinking water and the dosage was later increased to 200 mg/kg/day. Blood sugar was monitored for 25 days.
• FIG. 1 shows the evolution of blood sugar from two groups of mice, control, only treated with aloxane, and experimental, treated with aloxane and the product of Example 1. As we can see, the group treated only with aloxane became diabetic, with blood sugar reaching values above 400 mg/dl.
• Figure 4 shows that, after blood sugar increased with the treatment with aloxane for ten days (control group), treatment of animals with the product of Example 1 orally under the dosage of 200 mg/kg/day produced significant reduction in glucose levels. On the other hand, when the treatment with the product of Example 1 was halted, blood sugar levels increased. EFFECT TO THE SECRETION OF INSULIN BY GLUCOSE IN ISLETS OF LANGERHANS IN
• the removed pancreas was chopped (reduced to small fragments) and washed various times with Krebs solution to separate pancreas tissue from the remaining adipose tissue, which was eliminated after centrifugation.
• Krebs solutions was bubbled with carbogenic gaseous mixture (to keep pH 7.4) and in ice after the end of its use.
• Islets were isolated by the collagenase technique (Lacy & Kostianovsky, 1967). Pancreas fragments were put in a graduated test tube, adding for each one 1 ml of pancreas, 4 mg of collagenase (Collagenase P Boehringer Mannheim Biochemicals) diluted in 500 ⁇ l of Krebs solution. Test tubes with pancreas and collagenase were put under the carbogenic mixture and bath at 37 0 C for eight minutes. After aeration, the tube was well closed to be slowly shaken for four minutes and vigorously for one minute, still in bath at 37 0 C.
• collagenase Collagenase P Boehringer Mannheim Biochemicals
• pancreas was washed with Krebs solution and 1% albumin (bovine serum albumin - SIGMA®) in a test tube and centrifuged at 500 rpm/min for 30 seconds, disposing supernatant. Part of the precipitated material was separated and, with the help of a magnifying glass and silicone pipette, islets were observed and collected. Approximately 120 islets were obtained from each animal.
• the islets were packed in a perfusion chamber with flow kept at 1 ml/min in a peristaltic pump (Bomba Buchler Polystaltic®) at the temperature of 37 0 C.
• Krebs solution was used with different glucose concentrations (2.8 and 16.7 mM for the control group; 2.8 and 16.7 + product of Example 1 (50 ⁇ g/ml) for the experimental group).
• Krebs solution was used with 2.8 mM of glucose (keeping 20 minute rest) and soon afterwards nine perfusates were collected with a five-minute interval (time: -40, -35, -30, -25, -20, -15, -10, -5, 0) to obtain basal values for insulin secretion.
• glucose concentration for Krebs solution was changed only with 16.7 mM glucose for the control group or with 16.7 mM glucose + 50 ⁇ g/ml of the product of Example 1 for the experimental group, and the perfusate was collected each two minutes during thirty minutes. Perfusates were frozen at -70 0 C for subsequent insulin dosage.
• Insulin (as secreted by islets in vitro) contained in the perfusates was analysed in duplicates by the radioimmunoassay method, by using kit Insulin 125 I RIA (MP Biomedicals, LLC - Orangeburg NY 10962).
• Figures 5A and 5B show that the increase in insulin secretion as induced by increasing glucose concentration in the perfusing liquid of Langerhans islets in vitro was not neither reduced nor increased by treating animals with the product of Example 1 within the period of up to 25 minutes of perfusion.
• insulin secretion as observed thirty minutes after the start of perfusion with high glucose and the product of Example 1 (55.5 ⁇ 0.89 ng/ml) was significantly higher than the secretion as observed when the islets were perfused with high glucose and without the product of Example 1 (49.7 ⁇ 1.02 ng/ml) (Figure 5A).
• the analysis of the area under the curve of insulin values, as secreted along thirty minutes, also showed that the product of Example 1 increased insulin secretion (Figure 5B).
• This obesity model consists in the induction of chemical hypothalamic injury (satiety centers) by the subcutaneous administration of monosodium glutamate in the neonatal period. Such model is followed by hyperchortisolism, presents increase in visceral fat content, but low increase in body weight.
• This model consists in giving the animals a hypercaloric diet. Therefore, male rats from the Wistar line, after weaning, started to receive coffe shop diet (hypercaloric and highly palatable) at will until the adult stage (twelve weeks).
• Example 1 Product of Example 1 was given in all experiments under the dosage of 400 mg/kg/day diluted in drinking water, except for a subgroup of SHR animals to which the product was given orally in four daily takings.
• the animals received a cannula at their left femur artery with a PE-10 catheter connected to another PE-50 catheter to collect blood, aiming to determine levels of sugar blood and insulin levels during the Oral Glucose Tolerance Assay (TOTG).
• TOTG Oral Glucose Tolerance Assay
• the first blood collection was made to determine basal glucose and insulin and lipid levels in plasma (total cholesterol, HDL-cholesterol, triglycerides).
• Glucose overcharge (170 g/kg) was orally given and new blood collections were made to determine blood sugar and insulin levels after 15, 30,
• Blood sugar levels were determined in a digital glucose meter and insulin by radioimmunoassay methods. At the end of the oral glucose tolerance test, animals were sacrificed and the fat around epididymis was dissected and weighed.
• Figures 6A to 7C present blood sugar and insulin curves, the areas of said curves and insulin sensitivity rates as obtained for the control and treated groups.
• control SHR rats from blood sugar levels of normal fastness, present a diabetic blood sugar curve to TOTG, reaching values which are much higher than 200 mg/dl.
• treatment with the product of Example 1 no matter which was the mode of administration, provided significant reduction of blood sugar during fastness and an important improvement of the blood sugar profile, which values during TOTG were located within a glucose intolerance level.
• Concerning the insulin curve we can observe that the control group presented normal levels of insulin during fastness, but they did not increase during glucose overcharge, thus suggesting deficiency in insulin release by the pancreas, which even justifies the diabetic blood sugar curve.
• Figures 8A to 9C present the effects of treatment with the product of Example 1 over the metabolism of glucose of SHR glucose, considering the sum of both modes of administration of said product in comparison to the control group receiving just the carrier.
• Figures 12 and 13 present the standards of glucose metabolism in this experimental model of induced blood hypertension and the effects of treatment with the product of Example 1.
• both groups of animals from the L-NAME model presented fastness blood levels within normal, and the chronically treated group with the product of the present invention was slightly lower than the control group.
• Glucose overcharge induced significant and progressive increases in blood sugar in the L-NAME group which surpassed the 200 mg/dl level and is therefore classified as diabetic.
• Treatment with the product of Example 1 significantly reduced the blood sugar curve.
• Concerning insulin curve as presented on the right panel of that same graph we can observe that the insulin levels in fastness were within normal levels, but slightly higher in the group treated with the product of Example 1.
• TOTG we noticed in both groups significant and progressive increases, similar to insulin in response to glucose overcharge.
• Figure 14 presents the relative content of visceral fat (epididymis) in studied groups.
• the relative content of epididymal fat of animals with visceral obesity was very high (about three times the normal content), characterizing visceral obesity.
• Treatment for twelve weeks with the product of Example 1 significantly reduced the deposit of visceral fat in that model, with reduction of about 7% in the relative content of fat as present in animal epididymis and neuroendocrine obesity induced by monosodium glutamate.
• Figure 15 presents the relative content of visceral fat of both groups of obese animals by coffee shop diet. It can be noticed that animals with obesity induced by coffee shop diet present, besides general obesity, an important increase in the relative content of visceral fat as represented by the fat as found around the epididymis. Chronic administration (twelve weeks) of the product of Example 1 significantly reduced (-17.7%) the relative content of epididymal fat in animals fed with coffee shop diet.
• Figures 16A to 17C present standards for glucose metabolism of both groups of animals with obesity induced by the hyper caloric coffee shop diet.
• the chronic administration of the product of the present invention brought important benefits with significant improvement in the sensitivity of peripheral tissues to insulin, resulting in higher tissue collection of glucose and reduction of the deposition of visceral fat which, as we know, is considered an important factor in cardiovascular risk.
• Table 3 summarizes the effects of the product of the present invention over glucose metabolism in these four experimental models presenting resistance to insulin, in comparison to the corresponding control groups.

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