Immunosuppression by succinylacetone. II. Prevention of graft-vs-host disease.

Succinylacetone is a seven-carbon organic ketoacid that we have previously shown to inhibit tumor allograft rejection as well as the primary antibody response to sheep erythrocytes in rats. Because it appeared to be such a potent immunosuppressive agent in our initial studies, we evaluated succinylacetone for its ability to block graft-vs-host disease (GVHD) in adult F1 rats injected with parental strain spleen cells. Untreated ACE X Lewis F1 rats given Lewis strain spleen cells died of GVHD, with a mean survival of 24 days. By contrast, 62% of F1 rats in the group treated with succinylacetone survived, and the deaths that occurred in this group occurred significantly later. To confirm this observation and determine whether succinylacetone would interfere with bone marrow engraftment, lethally irradiated adult Wistar-Furth rats were reconstituted with syngeneic or totally allogeneic Fisher 344 lymphohemopoietic cells consisting of equal numbers of bone marrow and spleen cells. All animals given allogeneic cells without additional immunosuppressive treatment developed severe GVHD and died by day 42. By contrast 92% of the allogeneic cell recipients that had been treated with succinylacetone were long term survivors. Cytotoxicity typing of peripheral lymphocytes demonstrated greater than 90% donor-type lymphocytes persisting in the succinylacetone-treated recipients as long as 255 days post-transplantation. No chronic or late developing acute GVHD was observed even though the animals received succinylacetone as the sole immunosuppressant for only 28 days after transplantation. Furthermore, hemopoietic reconstitution in recipients of syngeneic cells was essentially identical between the control group and the group treated with succinylacetone. In addition, no gross or histologic evidence for renal, hepatic, cardiovascular, endocrine, or neurologic toxicity was observed in the long term transplant survivors treated with succinylacetone. These data indicate that succinylacetone treatment is effective in preventing lethal GVHD in this allogeneic bone marrow transplantation system while permitting normal hemopoietic reconstitution in the absence of significant chronic toxicity to other major organ systems.

Augmentation of hematoporphyrin uptake and in vitro-growth inhibition of L1210 leukemia cells by succinylacetone.

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

The effects of metalloporphyrins, porphyrins and metals on the activity of delta-aminolevulinic acid synthase in monolayers of chick embryo liver cells.

The effect of various metals, porphyrins and metalloporphyrins on the activity of delta-aminolevulinate synthase (ALAS) was measured in monolayers of chick embryo liver cells in order to determine whether the metal moiety of heme or heme itself is the regulator of ALAS activity. Iron, magnesium, zinc, copper, manganese and nickel did not decrease ALAS activity in non-induced and in cells induced by allyl-isopropylacetamide (AIA). Cobalt decreased both non-induced and induced activity. Porphyrins inhibited ALAS, apparently only after having been converted into metalloporphyrins. Almost all the metalloporphyrins examined decreased ALAS activity. None of the substances, at the concentrations used, was toxic to the cells. These observations indicate that probably heme and not iron is the regulator of ALAS activity in monolayers of chick embryo liver cells.

Evidence relating the inhibitory effect of cobalt on the activity of delta-aminolevulinate synthase to the intracellular concentration of porphyrins.

This study shows that the inhibition of ALAS activity caused by cobalt is directly correlated with the intracellular porphyrin concentration, thus indicating that cobalt exerts its inhibitory effect on ALAS activity as a result of the formation of cobalt-protoporphyrin.

Characteristics of hematin uptake in malignant, embryonic and normal cells.

The characteristics of hematin uptake were examined in three malignant cell lines [L1210 leukemia, 745 murine erythroleukemia (MEL) and Walker carcinoma (W256)], a cell line derived from normal rat liver (BRL-3A) and a normal embryonic cell, chick embryo fibroblasts (CEF). Uptake in the normal liver cell line was slight and occurred at a slow rate in contrast to the rapid uptake, which was more rapid and of greater magnitude in the three tumor cell lines, Saturation of the heme uptake mechanism was observed in MEL cells at an extra-cellular hematin concentration of 160 micro M and in L1210 cells at 300 micro M. At saturation L1210 cells achieved a cellular heme concentration nine times as high as MEL cells. Hematin uptake in MEL cells was markedly augmented by pretreatment with DMSO, procaine, detergent or proteolytic enzymes or by increases in the pH of the medium from 8 to 9.5. In contrast to MEL cells where SA inhibits growth by lowering cellular heme, the inhibition of growth of L1210 cells by SA appears to operate by a mechanism independent of heme. In gradual increase in hematin uptake capacity in MEL cells over a period of days. Afer exposure of MEL cells to a high concentration of hematin in the medium, the egress of heme was followed under various conditions. Of the various agents studied, only cyanide produced a loss of heme from MEL cells.

Growth inhibitory activity of succinylacetone: studies with Walker 256 carcinosarcoma, Novikoff hepatoma and L1210 leukemia.

Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.

Immunosuppressive activity of succinylacetone.

SA, an inhibitor of ALA dehydrase, the second enzyme of the heme biosynthetic pathway, has been shown to exert immunosuppressive activity in several systems. When the Walker 256 tumor was grown subcutaneously in outbred Sprague-Dawley rats, SA prevented rejection of the tumor by the host. Human peripheral blood lymphocyte transformation in response to mitogens and antigens in vitro was markedly impaired by addition of SA to the medium. Addition of hematin to the medium did not reverse the SA-mediated inhibition of transformation. This finding suggests that SA may exert immunosuppressive activity by a mechanism separate from inhibition of heme biosynthesis. Continuous administration of SA to Fisher 344 rats profoundly suppressed hemolytic antibody production after immunization with SRBC. Administration of large doses (equivalent to the highest dose of Sa given to tumor-bearing animals) for a month produced a 20% decrease of hemoglobin concentration, a 12% decrease in hematocrit, and no significant effect on leukocyte or erythrocyte concentration. There were no significant changes in tissue histology, including marrow cellularity.

Uptake of hematin and growth of malignant murine erythroleukemia cells depleted of endogenous heme by succinylacetone.

Heme levels and growth of malignant murine erythroleukemia cells in heme-free medium are drastically reduced by incubation of these cells in the presence of 4,6-dioxoheptanoic acid [succinylacetone (SA)]. When hematin was added to the culture medium of heme-depleted cells, the intracellular heme levels returned to normal, and growth inhibition produced by SA was also reversed. However, when cells depleted of heme by growth in heme-free medium containing SA were placed in heme-free medium without SA, heme levels were restored to normal, and growth was resumed. Hematin uptake in both untreated and heme-depleted malignant murine erythroleukemia cells exhibited biphasic kinetics, with a rapid phase of about 2 min followed by a slower uptake. The rate of uptake of exogenous hematin was slightly greater at 37 degrees than at 20 degrees. Although supplementation of heme-free medium with exogenous hematin increased total cellular heme in both untreated and heme-depleted malignant murine erythroleukemia cells, the fraction of heme in the 20,000 X g sediment was unaffected. A nonmalignant fibroblastic cell line, 3T3, exhibited little or no capacity to take up exogenous hematin.

Hematin administration to an adult with lead intoxication.

Lead poisoning and acute intermittent porphyria (AIP) may exhibit similar neurologic manifestations, and they have in common elevated excretion of urinary aminolevulinic acid (ALA). Despite their similarities, the possible pathophysiologic connection between AIP and lead poisoning in not known. Because intravenous hematin administration has produced biochemical improvement in AIP, a hematin trial in lead intoxication was of interest with respect to some of the heme metabolism abnormalities observed in the condition. Significant diminution of urinary ALA and coproporphyrin excretion occurred in association with intravenous hematin administration.

Hematin therapy for acute porphyria.

1. A therapeutic trial of intravenous hematin is presented. Eleven cases of AIP and one of VP who did not improve with conventional treatment (high carbohydrate intake) received this new agent. 2. Urinary ALA, PBG and, when possible, uroporphyrin and coproporphyrin were used to monitor the chemical response to the treatment. Objective clinical parameters of hypertension and tachycardia were followed when present in addition to subjective estimates of acute porphyric symptomatology (abdominal pain, backache, extremity pain and paresthesias, weakness, depression, etc.). 3. At a dosage of approximately 3 mg/kg, diminution of urinary ALA and PBG excretion was achieved in every patients. Hypertension and tachycardia improved in those instances where they were observed in association with the attack. Also, subjective improvements in the clinical status of the patients were observed frequently. 4. Hematin appears to be a promising therapeutic agent for the treatment of acute attack forms of porphyria.

Family evaluations in acute intermittent porphyria using red cell uroporphyrinogen I synthetase.

Acute intermittent porphyria (AIP) is a primary disorder of haem biosynthesis that is chemically characterised by raised urinary porphobilinogen (PBG). A defect in the biochemical pathway at the step of PBG conversion to uroporphyrinogen has been shown to be a result of a partial deficiency of the enzyme uroporphyrinogen I synthetase (uro I syn). The ascertainment rate of latent AIP (that is, chemically manifest but clinically asymptomatic) was examined in 185 individuals from 12 AIP kindreds using three parameters: red cell uro I syn, quantitative urinary PBG, and pedigree analysis with respect to uro I syn. Approximately 80% of individuals could be assigned as normal or latent AIP on the basis of the uro I syn assay alone. The remaining 20% could not be assigned because of an intermediate range of activity for the red cell assay in which the diagnosis cannot be certain. When the pedigree was used in the evaluation of the uro I syn data, the number of uncertain individuals, with respect to AIP, decreased to 10%. The enzyme method detected latent AIP in 37.5% of blood relatives, whereas quantitative urinary PBG alone detected only 15.2%. The pattern of inheritance for the uro I syn deficiency is consistent with Mendelian dominant inheritance, and it is likely that it is the basic inherited defect in AIP.

Danazol administration to females with menses-associated exacerbations of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a disorder of porphyrin metabolism in which the basic defect is a partial deficiency of uroporphyrinogen I synthase. The clinical disorder is more common in women, and some experience acute attacks before menstrual periods. Oral contraceptives have prevented menstrual-associated attacks in some cases, but exogenous estrogens and progestins are otherwise contraindicated in this disease. Danazol, a new synthetic steroid with weak androgenic activity, was thought to be of potential therapeutic benefit in AIP because of its effect of decreasing gonadotropin secretion without exposure to estrogen or progesterone. The drug was administered at a dosage of 200 mg t.i.d. to two adult females with AIP who were experiencing frequent exacerbations of their disease in association with their menstrual periods. Symptomatic and chemical evidence for exacerbation of porphyria occurred within 10 days of commencing danazol treatment in both patients.

Prevention of acute porphyric attacks by intravenous haematin.

A thirty-three-year-old female with acute intermittent porphyria (A.I.P.) was having regular attacks of the disease with her menstrual periods. During several of these attacks she received intravenous haematin, which was followed by chemical and clinical remissions. Hormones failed to prevent the regular attacks, which were completely prevented by 200 mg of haematin, given approximately once a week for six months. There were no changes in menstruation. The monthly attacks recurred on withdrawal of haematin.

Screening tests in acute porphyria.

Two recognized screening procedures for rapid evaluation of urinary porphobilinogen (PBG) concentrations are compared with quantitative PBG determinations (expressed as mg/24 hours and as a concentration in urine, mg/liter). One hundred ninety-one 24-hour urine specimens from 74 patients with suspected or documented acute type porphyria are included in this investigation. The two screening tests are compared with regard to sensitivity and method of performance. In this study, the Watson-Schwartz and Hoesch tests are positive at PBG concentrations in urine greater than 9 mg/liter. Both methods produced the characteristic pink color only once at a concentration below 3 mg/liter. Both methods are useful in detecting the abnormal concentration of urinary PBG observed during an acute porphyric attack. The Hoesch procedure provides a simple and rapid evaluation of urinary PBG. Positive results from either screening test demand quantitative urinary PBG determinations to confirm the suspected abnormality.

Comparative effects of glycerol and dextrose on porphyrin precursor excretion in acute intermittent porphyria.

The effects of dietary manipulations on excretion of the porphyrin precursors, delta-aminolevulinic acid (ALA), and porphobilinogen (PBG) were studied in eight patients with acute intermittent porphyria. Three diet periods of 9-17 days comprised each study. In each patient, a "baseline" protein, fat, and carbohydrate intake was kept constant throughout. In addition, during the first diet period each patient received 150 g dextrose; during the second, this was replaced by an isocaloric amount of neutral fat; and during the third, the fat was replaced by 150 g glycerol. In each of the patients, three comparisons of the effect of diet on both ALA and PBG excretion were made: (1) 300 g carbohydrate versus 150 g carbohydrate (dextrose versus fat), (2) 150 g carbohydrate + 150 g glycerol versus 150 g carbohydrate (glycerol versus fat), and (3) 300 g carbohydrate versus 150 g carbohydrate + 150 g glycerol (dextrose versus glycerol). For each of these three diet comparisons, there are sixteen individual comparisons possible for the effect of diet on porphyrin precursor excretion, eight for ALA and eight for PBG. Thus, the mean values for ALA and PBG excretions during each of the diet periods are statistically compared internally within each individual patient. Increasing carbohydrate intake from 150 g to 300 g by isocaloric substitution of dextrose for fat produced a significant (p less than 0.05) decline in eight of the sixteen comparisons of ALA and PBG excretion. Addition of 150 g glycerol by isocaloric substitution for fat caused a significant (p less than 0.05) decline in nine of the sixteen possible comparisons. In the sixteen comparisons of isocaloric dextrose and isocaloric glycerol-substituted diets, dextrose produced significantly (p less than 0.05) lower porphyrin precursor excretion in four cases and glycerol produced significantly (p less than 0.05) lower values in five. One patient showed no significant change on any of the diets. Of the four patients having symptoms believed referrable to porphyria during the study, three reported an improvement in symptoms during the high glycerol intake. The effects of dietary perturbations on porphyrin precursor excretion in acute intermittent porphyria are variable, but glycerol appears to be capable of decreasing the excretions and may prove useful in treating some of these patients.