METHOD OF DECREASING THE RATE OF HEME METABOLISM AND COMPOSITION USED THEREFOR

Abstract: Methods for decreasing the rate of metabolism of heme in mammals by administration of tin or chromium protoporphyrins IX, and compositions useful therefore.

Description: METHODOFDECREASINGTHERATEOFHEMEMETABOLISM ANDCOMPOSITIONUSEDTHEREFOR BACKGBOUND OF THE INVENTION This invention relates to methods and composi¬ tions useful for decreasing the rate of metabolism of heme in mammals. More specifically, the invention relates to the use of metal protoporphyrins IX to decrease the rate at which heme is metabolized in living mairmals. Iron protoporphyrin, also called heme, or more accurately ferroprotoporphyrin IX, is the specific por- phyrin isomer found in mammalian whole blood. Heme is an essential component in the respiratory chain and in the energy transfer reactions that take place in the mammal¬ ian body. Heme is synthesized and degraded by known metabolic routes involving known enzymatic reactions. In the normal course of degradation, heme first undergoes a ring opening reaction to form biliverdin which then converts to bilirubin. Bϊlirubϊn is toxic, but normally this toxicity is not manifested since bi¬ liverdin rapidly combines with albumin and is transported to the liver. In the liver, the protein is released and the bilirubin is converted to the corresponding diglu- curonide by reaction with uridine diphosphoglucuronate. The diglucuronϊde is soluble and excreted. A number of synthetic analogs of iron proto- porphyrin IX are known. They are commercially available or are readily synthesized by known methods. These in¬ clude, for example, platinum, zinc, nickel, cobalt, copper, silver, manganese, chromium and tin protoporphy- rϊn IX. For convenience herein, these compounds are referred to generically as Me-protoporphyrin, where Me stands for metal, and specifically by utilizing the chem¬ ical symbol for the metal such as Cr-protophorphyrin and Sn-protophorphyrin for the chromium and tin compounds respectively. O P One of the more difficult aspects of the toxic- ity of bilirubin is the so-called "jaundice of the newborn" which arises from an undesirably high concentra¬ tion of bilirubin in the blood of newborn mammals. In the period immediately after birth, there is a high concentration of heme in mammalian blood. There is also a high degree of heme oxygenase activity. This enzyme is essential to the metabolism of heme to bilirubin. The combination of factors leads to rapid metabolism of large amounts of heme and results in high concentrations of bilirubin in the body fluids of neonates. Since bilirubin is yellow, the infant appears yellow. Hence, the name "jaundice of the newborn". Free bilirubin is fat-soluble and readily crosses the blood-brain carrier causing extensive and serious brain damage. One manifestation of the toxϊcϊty of bilirubin is kernicterus or bilirubin eneephalopathy. It may be present clinically as lethargy, rigidity, opisthotonus, high-pitched cry, fever, and convulsions. Death may result. Survivors often have cerebral palsy, frequently of the choreoathetoid type, deafness, mental retardation, and other neurologic defects in infancy or early childhood. The principal treatments for hyperbϊlirubinemia have been phototherapy and exchange transfusions. In the former treatment, the patient is exposed to increased in¬ tensities of light in the visible range particularly between 420 and 470 nm. This results in increased oxida¬ tion of bilirubin or conversion by other mechanisms to products that are not yet fully identified. The treat¬ ment is widespread, particularly in the United States, but is not regarded as fully satisfactory since there are many as yet unanswered questions concerning its safety and effectiveness. The more traumatic procedure of exchange trans¬ fusion is indicated with full-term infants when there is -^U E OMP an unconjugated bilirubin level of 20 mg/dl. With prema¬ ture infants, the transfusion may be considered necessary at concentrations as low as 9 mg/dl. It is now generally accepted that concentrations that do not exceed 2.0 mg/dl can be tolerated without unnecessary damage to the in¬ fant. However, it appears clearly to be desirable to keep the level of this highly toxic chemical lower than 2 mg/dl if possible. Increased concentrations of bilirubin often appear in the blood of adult humans and other mammals with sickle cell anemia, thalassemia, and other congen¬ ital anemias as well as with animals and humans with acquired liver diseases. With such individuals, the concentration of bilirubin rarely reaches the high levels observed with neonates. It does, however, reach levels that may be toxic and should be controlled. It is desirable, therefore, to have available methods and materials for inhibiting the metabolism of heme so as to decrease the rate at which it degrades and the rate at which bilirubin accumulates in the blood. Disclosure of Invention It has now been discovered that the rate at which heme metabolizes can be decreased in mammals by administration of an effective amount of a synthetic Me- protoporphyrin. The presently preferred compounds are Sn-protoporphyrin and Cr-protoporphyrin. Both of these compounds are readily synthesized in pure form at reason¬ able costs and are therefore readily available. They are water-soluble and may be compounded with a variety of pharmaceutically acceptable carriers. The tin compound appears to be the more active. To determine the efficacy of Sn-protoporphyrin for controlling the rate of heme metabolism, neonatal rats were injected subcutaneously at birth, and at 12, 24, 48, and 72 hours postnatally with 0.1 ml solutions of Sn-protoporphyrin. To prepare the solutions for paren- teral injection, the material was taken up in a small volume of 0.2 N sodium hydroxide, adjusted to pH 7.4 with one normal hydrochloric acid and made up to final volume with 0.9% sodium chloride. The compositions as prepared and used contained sufficient Sn-protoporphyrin to pro¬ vide about 100 umol/Kg body weight in each 0.1 ml injec¬ tion. Control neonates received an equivalent volume of saline at each time point. Groups of neonates (6 - 30 animals per group) were sacrificed at selected time in¬ tervals. The treated animals and the controls were assayed for heme oxygenase activity and bilirubin concen¬ tration. Since heme oxygenase is an essential component in the catabolϊsm of heme, an increase in heme oxygenase activity is indicative of an increase in the rate of heme catabolism. Heme oxygenase was assayed as described by Maines and Kappas in J. Biol. Chem. , 253, 2321-2326 (1978). Total bilirubin in serum was estimated by the method of Roth, Clin. Chem. Acta., 17, 487-492 (1967). In a six-week study, it was found that, with controls, hepatic heme oxygenase levels in neonates rose rapidly, reaching levels four-fold above those charac¬ teristic of adult liver before gradually declining to normal adult levels between days 21 and 42. In the Sn- protoporphyrin-treated neonates, the administration of the active agent completely prevented the normal marked increase in hepatic heme oxygenase activity after birth. Indeed, in these neonates, the heme oxygenase activity declined immediately (1 day) to levels less than half the levels of the controls. They remained well below the control levels for about three weeks, and then rose gradually to finally attain the normal control levels at about day 42. Sn-protoporphyrin administration resulted in an immediate and significant lowering of serum bilirubin - - levels in treated animals. This low level of bilirubin concentration continued until about the fourth day and then remained substantially the same as the controls throughout the balance of the 42-day study period. Similar studies were conducted with neonatal rats treated subcutaneously with 2 and 10 micromoles per kilogram of Cr-protoporphyrin soon after birth. Various organs were analyzed for heme oxygenase activity and bilirubin concentration. The results were compared with controls each of which were treated subcutaneously with 0.1 ml of saline. The results for heme oxygenase activity in liver, kidney and spleen in nanamoles heme oxygenase per milligram microsomal protein per hour are shown in Table 1 at page 6. It is clear from a review of Table 1 that the effect of the administration of Cr-protoporphyrin is to cause an immediate decrease in heme oxygenase. Similar results were observed with serum bilirubin as shown in Table 2 at page 7 in which the concentration is expressed in milligrams per deciliter. CM co ^r CO ■H ■ *r r-l r~ VO o «* co CM r- CO r- ** • • • rH • • • • * • eo eo o ■ ^r CM o -i o o r-i rH rH 00 r^ eo CΛ o CM r-» oo eo en co CΛ - ~ - TABLE 2 Serum Bi1iru in 1 2 3 5 7 10 14 Control 0.39 0.48 0.42 0.31 0.33 0.46 0.38 2 μmol/Kg — 0.39 0.33 0.28 0.32 0.33 0.40 10 μmol/Kg — 0.43 0.33 0.24 0.30 0.33 0.28 Studies were carried out with adult rats treated with Sr-protoporphyrin to determine the effect on heme oxygenase activity and bilirubin production. Three rats weighing about 230 grams each were injected subcu¬ taneously with 50 μmol/Kg of Sn-protoporphyrin in iso- tonic saline at pH 8. Three controls were similarly treated with isotonic saline. The rats were sacrificed sixteen hours after treatment. The microsomal protein of the separate livers and of the three combined kidneys was estimated and heme oxygenase activity determined accord¬ ing to the method of Maines and Kappas. This procedure measures the heme oxygenase activity by determining the -amount of bilirubin produded. The results are shown in Table 3. The concentration unit for bilirubin is nana- moles of bilirubin formed per milligram protein per hour. TABLE 3 LIVER KIDNEY (COMB INED) Control 1 2.19 Control 2 3.29 1 . 24 Control 3 2.39 Experiment 1 0.95 Experiment 2 0.95 0 . 055 Experiment 3 1.53 It is readily apparent from the above that Sn- protoporphyrin is extremely effective for reducing bili¬ rubin production and heme oxygenase activity in adult mammals. With neonate mammals, the therapeutic composi¬ tions of this invention will be administered promptly after birth at a dosage of from 10 to 25 μmol/kg of body weight in animals and 0.1 to 2.5 ymol/kg of body weight in humans. While any of the usual parenteral routes may be employed, intramuscular or intravenous injection is preferred. Normally, one injection will suffice to main¬ tain the bilirubin concentration at a desired low level until the neonate reaches the age where the metabolism of heme is in balance. It is preferred, however, to monitor the serum bilirubin concentration and to utilize a booster dose, if necessary. The physician or veteri¬ narian will select the appropriate dosage. It will nor¬ mally be within the above range, but may be varied to deal with specific conditions. With adults afflicted with sickle cell anemia or another condition resulting in increased bilirubin concentration, the dosage unit is normally smaller, since in all but the most acute situations, the bilirubin con¬ centration is not as high as in neonates. The standard " dosage with adults will normally be from about 0.05 to 1 μmol/Kg of body weight. In order to have appropriate dosage units available, the compositions of this invention will normally be prepared in bulk at concentrations of from 100 to 2500 μmol/liter of buffered isotonϊc aqueous solution at pH 7 to 8 and subdivided into dosage units containing approximately 0.1 to 2.5 ymol/ml of solution. The usual pharmaceutical carriers are isotonic aqueous salt or glucose solutions buffered to a pH of about 7 to 8, normally 7.4. Therapeutic compositions can also be provided in lyophϊlized powder form to be reconstituted with water to the .clinical dosage. For example, the selected Me- protoporphyrin can be taken up in 0.1 molar phosphate or other pharmaceutically acceptable buffer containing suf¬ ficient saline or other solute to form isotonic solu¬ tions, and freeze-dried to form a powder. The powder ean OMPI 9 - be reconstituted with distilled water or saline solution to provide solutions containing 0.1 to 2.5 μmol/ml of Me- protoporphyrin per milliliter of solution.

Claims: CLAIMS 1. A method of decreasing the rate of meta¬ bolism of heme in mammals in need of such treatment by administering to the mammal an amount of Sn-protoporphy¬ rin or Cr-protoporphyrϊn which is effective to decrease said rate. 2. A method as in Claim 1 wherein Sn-protopor¬ phyrin is administered. 3. A method as in Claim 1 wherein Cr-protopor- phyrin is administered. 4. A method as in Claim 2 wherein the mammal is a human. 5. A method as in Claim 3 wherein the mammal is a human. 6. A method of decreasing the rate of bili¬ rubin accumulation in neonatal mammals which comprises administering to the neonate promptly after birth an amount of Sn-protoporphyrin or Cr-protoporphyrin which is effective to decrease said rate. 7. A method as in Claim 6 wherein Sn-protopor¬ phyrin is administered. 8. A method as in Claim 6 wherein Cr-protopor¬ phyrin is administered. 9. A method as in Claim 7 wherein the neonate is human. 10. A method as in Claim 8 wherein the neonate is huma . 11. A pharmaceutical composition containing 100 to 2500 μmol of Sn-protoporphyrin or Cr-protoporphyrin per liter of buffered isotonic aqueous solution at pH 7 to 8. 12. A composition of Claim 11 containing Sn- protoporphyrin. OMPI lS. A composition of Claim 11 containing Cr- protoporphyrin. 14. A pharmaceutical composition in dosage unit form containing 0.1 to 2.5 μmol of Sn-protoporphyrin or Cr-protoporphyrin per milliliter of buffered isotonic aqueous solution at pH 7 to 8. 15. A composition of Claim 14 containing Sn- protoporphyrin. 16. A composition of Claim 14 containing Cr- protoporphyrin.

Inventors: DRUMMOND GEORGE, KAPPAS ATTALAH, DRUMMOND, GEORGE, KAPPAS, ATTALAH
Applicants: DRUMMOND GEORGE, KAPPAS ATTALAH, DRUMMOND, GEORGE, KAPPAS, ATTALAH
Application year: 1982
Main classification: A61K31
IPC codes: A61K31, A61P3, A61P7, C07D487