USE OF PYRUVATE FOR RETARDING NEUTROPHIL INFILTRATION AND MORPHOLOGIC LOSS IN ISCHEMIC BOWEL

Abstract: Treating mammals having ischemic bowel with a therapeutic quantity of pyruvate enterally or parenterally will retard neutrophil infiltration and retain morphology during and following the bowel ischemia. The pyruvate is introduced into the patient enterally or parenterally during the bowel ischemia or the succeeding bowel reperfusion and, preferably, prior to the bowel ischemia. The pyruvate dosage is from 1 % to 20 % by weight of the patient's caloric intake.

Description: USE OF PYRUVATE FOR RETARDING NEUTROPHIL INFILTRATION AND MORPHOLOGIC LOSS IN ISCHEMIC BOWEL BACKGROUND OF THE INVENTION Field ofthe Invention - The invention concerns treatment of mammals to retard ischemic bowel disease. Bowel ischemia and reperfusion is commonly associated with a sudden increase of generated free-radicals, accompanying neutrophil infiltration and accompanying cell death or loss of morphology which may result in the death of the mammal. Ischemia refers to the stoppage of blood flow to a particular organ or muscle. Reperfusion refers to the restoration of blood flow to the affected organ or muscle. According to the present invention, providing the mammal with pyruvate prior to. and/or during the onset of bowel ischemia will significantly inhibit the neutrophil infiltration to the affected bowel. During reperfusion of the ischemic bowel, the presence of pyruvate in the bowel will significantly retard neutrophil infiltration and villi deterioration. Description of the Prior art - Pyruvate has a number of useful appUcations in medical treatment. Pyruvate has been described for retarding fatty deposits in livers (4.158.057); for diabetes treatment (4,874.790); for retarding weight gain (4.812,879, 4,548.937, 4,351,835); to increase body protein concentration in a mammal (4,415,576); for treating cardiac patients to increase the cardiac output without accompanying increase in cardiac oxygen demand (5,294.641): for extending athletic endurance (4,315.835); for retarding cholesterol increase (5,134, 162); for inhibiting growth and spread of malignancy and retarding DNA breaks (apphcation S.N. 08/194,857, filed February 14, 1994); and for inhibiting generation of free radicals SϋβSTlTUTE SHEET (RULE 26) (apphcation S.N. 08/286,946 filed August 8, 1994). Pyruvate in various forms has been proposed for enteral administration and for parenteral administration. Typically pynivates are available in the form of salts, e.g., calcium pyruvate and sodium pyruvate; pyruvate analogs of amino acids, e.g. pyruvyl-amino acids such as pyruvyl- glycine, pyruvyl-alanine, pyruvyl-leucine, pyruvyl-valine, pyruvyl-isoleucine, pyruvyl- phenyl- alanine, pyruvyl-proline, and their amides. See U.S. Patents 5,283,260 and 5,256,697 for pyruvyl-amino acids. Pyruvate may be administered to a mammal enterally or parenterally to super physiologic levels in the mammal. The amount of admimstered pyruvate preferably is from 1 to 20 per cent ofthe mammal's caloric intake. For enteral dosage, the pyruvate may be dispersed or dissolved in a beverage product or may be included in cookies, candies or other foods. The pyruvate may be introduced as an aqueous solution parenterally. A preferred admimstration procedure is an aqueous energy maintenance drip which includes not only sugars but also the selected pyruvate or pynivates. See U.S. patent apphcation S.N. 08/286,946 filed August 8, 1994. Regardless of the administration procedure, according to this invention, the presence of super physiological pyruvate in a mammalian bowel or circulatory system will retard development of free-radicals during an ischemia incident in the mammal's bowel and during reperfusion. DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation of the measured chemiluminescence resulting from intestinal free-radical generation during ischemia and subsequent reperfusion, comparing a placebo (PL), a pyruvate (PY) treatment and a base line condition. Figure 2 is graphical representation of the neutrophil count for two mammals, one of which has received pyruvate (PY) treatment and the other of which is a placebo (PL). These are compared to a base line condition, prior to the ischemia onset. The neutrophil count was made at 30 minutes reperfusion following bowel ischemia. Figure 3 includes three microphotographs showing intestinal villi under different conditions. DESCRIPTION OF THE PREFERRED EMBODIMENTS Ischemia is a condition in which the flow of blood has been interrupted to a particular region ofthe mammal anatomy, e.g.. muscles or organs. Bowel ischemia is commonly encountered in the course of surgery . mechanical accidents, and intestinal disorders. Restoration ofthe flow of blood to the ischemic anatomy is called reperfusion. At the onset of bowel reperfusion a sudden increase of free-radicals is identified. The free-radical production ofthe mammal remains high for extended periods, e.g., one to two hours following onset of reperfusion. The high level of free-radicals usually causes irreversible damage to the affected anatomy. The elevated free-radical production in the bowel results in cell necrosis (cell death), a decreased morphology and death ofthe mammal. See "Oxygen Free Radicals in Tissue Damage" Merrill Tarr et al.. Birkhauser Boston, 1933. Morphology is the form and structure ofthe mammal A patient treated with pyruvate prior to the onset of bowel ischemia will not display the elevated free-radical development, but instead will maintain free-radical development at a level which is normal or lower than base line level throughout the ischemia and subsequent reperfusion. The neutrophil mfiltration is retard and the morphology is retained. EXAMPLE 1 The small intestine of each of six laboratory rats was tied to closure. The bowel downstream from the closure received directly for ten minutes before and during treatment either: PLACEBO (PL) a Uquid diet preparation containing aqueous polyglucose; or PYRUVATE (PY) a Uquid diet preparation containing aqueous polyglucose with 10% of the energy (caloric) content of the polyglucose being displaced by a mixture of sodium and calcium pyruvate constituting 10% (by energy) of the liquid diet, i.e., 10% ofthe caloric intake. The superior mesenteric artery which supphes blood to the intestines was occluded in each of the six laboratory rats to cause bowel ischemia. The ischemia continued for 45 minutes at which time the mesenteric artery occlusion was released and reperfusion ofthe affected bowel commenced. A portion ofthe intestine of each laboratory rat was removed: (A) Immediately following ischemia; (B) After 30 minutes reperfusion; (C) After 60 minutes reperfusion. Each intestine portion was analyzed for free radicals by chemiluminescence (measured in intensity/mg protein) and examined by hght microscopy. Cherniluminescence measurements are described by Simmonds, N.J. et al., GASTROENTERGOLOGY 1992, Vol. 103, Pages 186-196. The baseline chemiluminescence value for the rats was obtained prior to the testing at 684,000 ± 68,200 chemiluminescence units, a direct correlation to the free- radical population. The values for the Placebo (PL) and for the Pyruvate (PY) treatment are indicated in TABLE I and graphically presented in Figure 1. TABLE I FREE-RADICAL CONTENT OF INTESTINE OF LABORATORY RATS FOLLOWING BOWEL ISCHEMIA (Measured in chemiluminescence units) PLACEBO PYRUVATE TREATMENT Post Ischemia 409,000 ± 76,200 59,700 ± 10,400 (Immediate) Reperfusion 933.000 ± 298.000 69.800 ± 20.400 (30 minutes) Reperfusion 543.000 ± 309.000 62.200 ± 15,900 (60 minutes) The significant reduction of free-radicals (as measured by chemilumi escence ) resulting from Pyruvate treatment (PY) of the mammal is illustrated in Figure 1 wherein the vertical cross-hatch areas representing Pyruvate treatment (PY) are significantly lower than the horizontal cross-hatch areas representing the Placebo (PL). The reduction is apparent at the end ofthe ischemia and throughout the. observed reperfusion. The placebo intestine segment, after reperfusion, was discolored and appeared necrotic. All of the placebo segments displayed petechiae (small hemorrhagic spots) while the pyruvate segments did not show petechiae. In further tests, with bowel ischemia for 30 minutes followed by reperfusion for 30 minutes, laboratory rats were fed as described in EXAMPLE 1. The placebo rats had the described standard diet. The pyruvate treatment rats had the pyruvate- modified standard diet (A) Prior to and during the ischemia; or (B) Only after the onset ofthe ischemia. The neutrophil infiltration ofthe intestine was measured and graphed on Figure 2. The baseline condition in Figure 2 indicates the population of neutrophil in the intestine prior to any evaluations. The two bars labeled indicate the neutrophil population at the end of 30 minutes reperfusion following 30 minutes ischemia. (A) Where pyruvate had been introduced into the rat prior to the onset of ischemia the neutrophil infiltration (PY) was reduced below the base condition, whereas the placebo (PL) showed increased neutrophil infiltration. (B) Where the pyruvate diet was commenced immediately following the onset of ischemia, similar results are observed. From Figure 2 it appears that the pyruvate is beneficial in retarding neutrophil infiltration in both situations i.e., when supphed prior to and during the ischemia and also when suppUed only after onset ofthe ischemia or the onset of reperfusion. For visual evidence ofthe effect of ischemia on intestines, two laboratory rats were evaluated. One rat. PL, received the described standard diet and the other rat, PY, received the described pyruvate modified standard diet. The superior mesenteric arteries of both rats were blocked to create bowel ischemia for 60 minutes. At the end of the 60 minutes of reperfusion, the rats were sacrificed and microphotographs were obtained of a cross-section through the smaU intestine, which is covered with small vilU, extending into the interior of the intestine from the muscle waU. These microphotographs are produced in Figure 3 including: Figure 3a - shows the 200 magnification cross-section of a sUce of smaU intestine of a normal rat without ischemia Ulustrating normal, healthy villi. Figure 3b - shows the 100 magnification cross-section from the intestine ofthe placebo rat (PL) Ulustrating badly deteriorated villi extending from the muscle waU. The morphology is severely diminished. Figure 3c - shows the 100 magnification cross-section from the intestine ofthe pyruvate-treated rat (PY) Ulustrating healthy vitti. The vilU retain morphology. Thus it appears that the pyruvate treated animal maintains healthy villi and retains morphology during intestinal ischemia which is destructive of intestinal villi and reduces morphology in a simUar, untreated animal. Treatment - A patient experiencing bowel ischemia, or about to experience bowel ischemia, should receive a super physiologic dosage of pyruvate by a drip tube extended through the patient's nose to the patient's stomach, or by beverage or food containing pyruvate, or by parenteral intravenous drip. AU of these delivery systems will establish the desired super physiologic level of pyruvate in the patient.

Claims: We claim: 1. A method for retarding neutrophil infiltration and retarding loss of morphology in the ischemic bowel of a mammal experiencing ischemic bowel which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal during said ischemic bowel or during reperfusion. 2. A method for retarding neutrophil infiltration and retarding loss of morphology in the ischemic bowel of a mammal experiencing ischemic bowel exposure which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal prior to and during said ischemic bowel and during reperfusion. 3. The method of claim 1 wherein said pyruvate is one or more pyruvate salts or esters. 4. The method of claim 1 wherein said pyruvate is an organic ester of an amino acid. 5. The method of claim 1 wherein said pyruvate is pyruvyl alanine. 6. The method of claim 1 wherein said therapeutic quantity is from 1% to 20% by weight ofthe mammal's caloric intake. 7. A method for retarding neutroplril infiltration in the ischemic bowel of a mammal experiencing ischemic bowel which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal during said ischemic bowel or during reperfusion. 8. A method for retarding loss of morphology in the bowel of a mammal experiencing ischemic bowel which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal prior to and during said ischemic bowel or during reperfusion. 9. A method for retarding loss of morphology in the bowel of a mammal experiencing ischemic bowel which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal during said ischemic bowel and during reperfusion. 10. A method for retarding neutrophil infiltration in the bowel of a mammal experiencing ischemic bowel which comprises introducing a therapeutic quantity of pyruvate enteraUy or parenteraUy into the mammal during said ischemic bowel or during reperfusion.

Inventors: MCCAMISH MARK A, MILLER ROBERT H, STANKO RONALD T, MCCAMISH, MARK, A., MILLER, ROBERT, H., STANKO, RONALD, T.
Applicants: UNIV PITTSBURGH MEDICAL CENTER, UNIVERSITY OF PITTSBURGH MEDICAL CENTER
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61P9