Phase 1 Trial of an RNA Interference Therapy for Acute Intermittent Porphyria.

BACKGROUND: Induction of delta aminolevulinic acid synthase 1 ( ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations in patients with acute intermittent porphyria, a rare inherited disease of heme biosynthesis. Givosiran is an investigational RNA interference therapeutic agent that inhibits hepatic ALAS1 synthesis. METHODS: We conducted a phase 1 trial of givosiran in patients with acute intermittent porphyria. In part A of the trial, patients without recent porphyria attacks (i.e., no attacks in the 6 months before baseline) were randomly assigned to receive a single subcutaneous injection of one of five ascending doses of givosiran (0.035, 0.10, 0.35, 1.0, or 2.5 mg per kilogram of body weight) or placebo. In part B, patients without recent attacks were randomly assigned to receive once-monthly injections of one of two doses of givosiran (0.35 or 1.0 mg per kilogram) or placebo (total of two injections 28 days apart). In part C, patients who had recurrent attacks were randomly assigned to receive injections of one of two doses of givosiran (2.5 or 5.0 mg per kilogram) or placebo once monthly (total of four injections) or once quarterly (total of two injections) during a 12-week period, starting on day 0. Safety, pharmacokinetic, pharmacodynamic, and exploratory efficacy outcomes were evaluated. RESULTS: A total of 23 patients in parts A and B and 17 patients in part C underwent randomization. Common adverse events included nasopharyngitis, abdominal pain, and diarrhea. Serious adverse events occurred in 6 patients who received givosiran in parts A through C combined. In part C, all 6 patients who were assigned to receive once-monthly injections of givosiran had sustained reductions in ALAS1 messenger RNA (mRNA), delta aminolevulinic acid, and porphobilinogen levels to near normal. These reductions were associated with a 79% lower mean annualized attack rate than that observed with placebo (exploratory efficacy end point). CONCLUSIONS: Once-monthly injections of givosiran in patients who had recurrent porphyria attacks resulted in mainly low-grade adverse events, reductions in induced ALAS1 mRNA levels, nearly normalized levels of the neurotoxic intermediates delta aminolevulinic acid and porphobilinogen, and a lower attack rate than that observed with placebo. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02452372 .).

Homozygous hydroxymethylbilane synthase knock-in mice provide pathogenic insights into the severe neurological impairments present in human homozygous dominant acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q+/+) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q+/+ mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood-brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.

Lifestyle factors including diet and biochemical biomarkers in acute intermittent porphyria: Results from a case-control study in northern Norway.

BACKGROUND: Lifestyle factors, including a low intake of carbohydrates, dieting, alcohol consumption, cigarette smoking and stress are some of the possible triggers of attacks in acute intermittent porphyria (AIP). The influence of lifestyle factors, including energy intake, diet and alcohol consumption on the biochemical disease activity in AIP and biochemical nutritional markers were examined. METHODS: A case-control study with 50 AIP cases and 50 controls matched for age, sex and place of residence was performed. Dietary intake was registered using a food diary in 46 matched pairs. Symptoms, alcohol intake, stress and other triggering factors of the last AIP attack were recorded on questionnaires. Porphyrin precursors, liver and kidney function markers, vitamins, diabetogenic hormones and other nutritional biomarkers were analyzed by routine methods. The Wilcoxon matched-pairs signed rank test was used to compare the cases vs. controls. The Spearman's rank correlation coefficient was used on the cases. RESULTS: Increasing total energy intake was negatively correlated with the biochemical disease activity. The intake of carbohydrates was lower than recommended, i.e., 40 and 39% of total energy intake in the AIP cases and controls, respectively. The plasma resistin level was significantly higher (p = .03) in the symptomatic than asymptomatic cases. Plasma insulin was lower in those with high porphobilinogen levels. The intake of sugar and candies were higher in the AIP cases with low U-delta aminolevulinic acid (ALA) levels (p = .04). Attacks were triggered by psychological stress (62%), physical strain (38%), food items (24%) and alcohol (32%) in the 34 symptomatic cases. Alcohol was used regularly by 88% of the cases (3.2 g ethanol/day) and 90% of the controls (6.3 g/day), but the intake was significantly lower in symptomatic than in asymptomatic cases (p = .045). CONCLUSION: A high intake of energy, sugar and candies and a higher insulin level were associated with a lower biochemical disease activity. The resistin level was higher in the symptomatic than the asymptomatic cases. AIP patients drink alcohol regularly, but the intake was significantly lower in the symptomatic cases. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01617642.

Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver.

Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

Biochemical, radiologic, ultrastructural, and genetic evaluation of iron overload in acute leukemia and iron-chelation therapy.

Iron overload in hereditary hemochromatosis and hematologic malignancy has unfavorable effects on morbidity. Herein, 53 children (age 108.4±58.3 mo, 25 girls and 28 boys) with acute myeloblastic and lymphoblastic leukemia, who received 4 different chemotherapy protocols, were evaluated for iron overload throughout chemotherapy. Iron overload arose: (1) before chemotherapy, which was dependent on neither chemotherapy nor packed red blood cell transfusions and (2) after chemotherapy, which was dependent on the duration and nature of chemotherapy and partially on transfusion of packed red blood cells. Iron overload was documented in 75% of patients with a ferritin level >1000 ng/mL, by liver and heart magnetic resonance imaging, and they were administered iron-chelation therapy with success. Three of 10 radiologically iron-overloaded patients were heterozygous for H63D mutation. Aminolevulinic acid and porphobilinogen levels were normal. Light microscopic examination of the bone marrow revealed increased iron granules in erythroblasts, platelets, and megakaryocytes, iron-laden macrophages, free iron in the matrix, dyshematopoiesis, and apoptotic cells. Electron microscopic examination revealed iron-laden secondary lysosomes and autolysosomes in normoblasts and iron-laden primary granules in promyelocytes, irrelevant to the ferritin level, implying autophagia due to chemotherapy as a source of the excess iron. We think that iron overload, which is an important complication of acute leukemia, should be evaluated separately from "transfusion overload," and the management principles specific to leukemia should be implemented.

Sudden bilateral reversible vision loss: a rare presentation of acute intermittent porphyria.

Acute intermittent porphyria is a rare disorder, characterised clinically by variable patterns of neurological and metabolic disturbances. We report a rare presentation with sudden onset painless bilateral reversible vision loss of cerebral origin along with a brief review of the underlying pathophysiology.

Stability of porphyrins and porphyrin precursors in urine and plasma samples: implications for sample handling and storage.

The porphyrias are rare disorders of haem biosynthesis. Diagnosis requires demonstrating increased porphyrins or porphyrin precursors in blood, urine and faeces. Patients may only be investigated once, and therefore, understanding the preanalytical factors affecting the reliability of results is crucial. Guidance for sample handling exists, but published evidence regarding the stability of porphyrins and their precursors is limited. The aim of this study was to evaluate the effect of light exposure and different storage temperatures on analyte stability for measurement of urinary aminolaevulinic acid and porphobilinogen, total urine porphyrin and plasma porphyrin. Our results confirm that all samples should be protected from light. Results from samples exposed to light for greater than 4 hours should be interpreted with caution and repeat samples requested. If transported to a specialist laboratory, samples should be stored at 4°C before transport. Transit time at ambient temperatures should be less than 24 hours.

Circadian rhythms in acute intermittent porphyria--a pilot study.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem synthesis wherein a partial deficiency of porphobilinogen (PBG) deaminase (PBGD) with other factors may give rise to biochemical and clinical manifestations of disease. The biochemical hallmarks of active AIP are relative hepatic haem deficiency and uncontrolled up-regulation of hepatic 5-aminolevulinic acid (ALA) synthase-1 (ALAS1) with over-production of ALA and PBG. The treatment of choice is intravenous haem, which restores the deficient regulatory haem pool of the liver and represses ALAS1. Recently, haem has been shown to influence circadian rhythms by controlling their negative feedback loops. We evaluated whether subjects with AIP exhibited an altered circadian profile. MATERIALS AND METHODS: Over a 21-h period, we measured levels of serum cortisol, melatonin, ALA, PBG and mRNA levels (in peripheral blood mononuclear cells) of selected clock-controlled genes and genes involved in haem synthesis in 10 Caucasian (European-American) women who were either postmenopausal or had been receiving female hormone therapy, six of whom have AIP and four do not and are considered controls. RESULTS: Four AIP subjects with biochemical activity exhibited higher levels of PBG and lower levels and dampened oscillation of serum cortisol, and a trend for lower levels of serum melatonin, than controls or AIP subjects without biochemical activity. Levels of clock-controlled gene mRNAs showed significant increases over baseline in all subjects at 5 a.m. and 11 p.m., whereas mRNA levels of ALAS1, ALAS2 and PBGD were increased only at 11 p.m. in subjects with active AIP. CONCLUSIONS: This pilot study provides evidence for disturbances of circadian markers in women with active AIP that may trigger or sustain some common clinical features of AIP.

Direct and simultaneous quantitation of 5-aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry.

Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Haem arginate as effective maintenance therapy for hereditary coproporphyria.

A 35-year-old woman presented with skin fragility and photosensitivity with blisters affecting her face and hands. Other symptoms included intermittent headache, fatigue, abdominal pain and nausea. Porphyrin studies were markedly raised, with features consistent with hereditary coproporphyria (HCP). Despite strict precautions, symptoms remained significantly problematic. Regular haem arginate infusions of 3 mg/kg per day over 4 days on a monthly basis were commenced and resulted in significant improvement of the patient's symptoms and a reduction in urinary porphobilinogen. Although haem arginate infusion is known as a treatment for severe acute attacks of HCP, the effectiveness of regular infusions as maintenance therapy has not been established. This is the first report of effective symptom control correlating with normalization of biochemical markers in a patient receiving regular haem arginate infusions for the treatment of HCP.

Combined liver and kidney transplantation in acute intermittent porphyria.

We report two patients with acute intermittent porphyria (AIP) who were successfully treated with combined liver and kidney transplantation. Both had a very poor quality of life as a result of years of frequent acute porphyria symptoms, chronic peripheral neuropathy and renal failure requiring dialysis. After transplantation, clinical and biochemical signs of porphyria disappeared. The excretion pattern of porphyrin precursors normalized within the first day and plasma porphyrins returned to normal within a week. These and other recent cases have clarified previous concerns and have helped to formulate the indications for and the timing of transplantation in AIP.

Porphyrin precursors and porphyrins in three patients with acute intermittent porphyria and end-stage renal disease under different therapy regimes.

Porphyrin precursors and porphyrins were measured in three patients with recurrent attacks of acute intermittent porphyria and end-stage renal disease (ESRD): two patients on hemodialysis and one on peritoneal dialysis. Plasma porphobilinogen (PBG) and porphyrins were considerably increased in the three patients. In a previous study, the mean urinary and plasma PBG/ALA ratio in biochemically active AIP patients with conserved renal function was 2.0 (normal 0.3) and plasma porphyrin levels were normal (< 10 nmol/L). In this study we show that the progression to ESRD was paralleled by an increase in urinary and plasma PBG/ALA ratio reaching levels above 6 and higher. Plasma porphyrin increased to levels above 1000 nmol/L causing cutaneous lesions resembling porphyria cutanea tarda. The porphyrin precursors were readily filtered by dialysis membranes but not the porphyrins. The development of kidney failure was a devastating complication in these AIP patients with chronic active disease, leading to unavoidable deterioration of peripheral veins, progression of peripheral neuropathy, dialysis treatment and secondary cutaneous lesions. The clinical course could not be reversed by medical treatment in any of the cases. Today, combined liver and kidney transplantation should be considered as a therapeutic option.

Safety, pharmacokinetics and pharmocodynamics of recombinant human porphobilinogen deaminase in healthy subjects and asymptomatic carriers of the acute intermittent porphyria gene who have increased porphyrin precursor excretion.

BACKGROUND AND OBJECTIVE: Acute intermittent porphyria is an autosomal dominant disorder caused by deficient activity of the third enzyme in the haem biosynthetic pathway, porphobilinogen deaminase. It is characterised by acute, potentially life-threatening neurological attacks that are precipitated by various drugs, reproductive hormones and other factors. During acute attacks, the porphyrin precursors 5-aminolevulinic acid and porphobilinogen accumulate and are excreted at high concentrations in the urine. Current treatment is based on glucose loading and parenteral haem replenishment, which reduce the accumulation of 5-aminolevulinic acid and porphobilinogen. Recently, a new form of treatment based on porphobilinogen deaminase enzyme replacement therapy has been shown to be effective in an acute intermittent porphyria mouse model which, during phenobarbital (phenobarbitone) induction of haem biosynthesis, mimics the biochemical pattern of acute porphyric attacks. The objective of the present study was to investigate the safety, pharmacokinetics and pharmacodynamics of recombinant human porphobilinogen deaminase (P 9808), administered to healthy subjects and asymptomatic porphobilinogen deaminase-deficient subjects with high concentrations of porphobilinogen, the substrate of porphobilinogen deaminase. STUDY DESIGN: Forty individuals participated in this two-part study: 20 asymptomatic porphobilinogen deaminase-deficient subjects (both male and female) with > or =4 times the upper reference urinary porphobilinogen level, and 20 healthy male subjects. Four different doses of recombinant human porphobilinogen deaminase were studied (0.5, 1, 2 and 4 mg/kg bodyweight). Part A included 12 asymptomatic porphobilinogen deaminase-deficient subjects, and the enzyme was administered in an open-label, single-dose design. Part B included 20 asymptomatic porphobilinogen deaminase-deficient subjects and 20 healthy subjects. The same enzyme dosages were administered as divided doses every 12 hours for 4 consecutive days in a randomised, double-blinded, placebo-controlled design. The washout period between Parts A and B was 2 weeks. METHODS: The concentrations of recombinant human porphobilinogen deaminase and titres of antibodies against recombinant human porphobilinogen deaminase were analysed by ELISA. Plasma porphobilinogen and 5-aminolevulinic acid concentrations were analysed using a novel liquid chromatography-tandem mass spectrometry method. Urinary porphobilinogen, 5-aminolevulinic acid and porphyrin concentrations, as well as plasma porphyrin concentrations, were analysed using standard methods. The pharmacodynamic effect of the enzyme was studied through changes in plasma porphobilinogen concentrations. RESULTS: No serious adverse events were observed. Seven subjects (four healthy men and three asymptomatic porphobilinogen deaminase-deficient subjects) developed antibodies against recombinant human porphobilinogen deaminase but did not experience allergic manifestations. The mean elimination half-lives of the highest doses of recombinant human porphobilinogen deaminase ranged between 1.7 and 2.5 hours for both healthy men and asymptomatic porphobilinogen deaminase-deficient subjects. The area under the plasma concentration-time curve was proportional to the respective dose. In asymptomatic porphobilinogen deaminase-deficient subjects, plasma porphobilinogen concentrations decreased below measurable levels almost instantaneously after administration of any dose of the enzyme. The effect lasted for approximately 2 hours, after which the plasma porphobilinogen concentration slowly increased, reaching about 70% of the initial values 12 hours after administration. There was no effect on plasma 5-aminolevulinic acid concentrations, and there was a transitory increment in porphyrin concentrations. The corresponding concentrations of metabolites in the urine reflected the pattern observed in the plasma. CONCLUSIONS: The recombinant human porphobilinogen deaminase enzyme preparation was found to be safe to administer and effective for removal of the accumulated metabolite porphobilinogen from plasma and urine. The pharmacokinetic profile of recombinant human porphobilinogen deaminase showed dose proportionality, and the elimination half-life was about 2.0 hours for the two highest doses. Thus, clinical grounds were established for investigation of the therapeutic efficacy of the enzyme during periods of overt disease in patients with acute intermittent porphyria.

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress.

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.

Altered 5-aminolevulinic acid metabolism leading to pseudoporphyria in hemodialysed patients.

Hemodialysed patients with no history of porphyria may present neurological symptoms similar to those seen in acute porphyrias. Porphyria has been associated with an increase in plasma levels of 5-aminolevulinic acid and porphobilinogen. Our aim was to evaluate these parameters and the activities of the enzymes involved in the first steps of heme metabolism in non-porphyric hemodialysed patients. The activities of 5-aminolevulinate dehydratase and deaminase were determined in red blood cells (RBC) from 78 hemodialysed patients, before and after dialysis. Plasma levels of 5-aminolevulinic acid, porphobilinogen and zinc were also measured. These parameters were also measured in 40 volunteers to obtain controls levels. The levels of 5-aminolevulinic acid (0.98 +/- 0.09 microgram/ml) and porphobilinogen (1.32 +/- 0.13 micrograms/ml) were raised in non-porphyric patients prior to hemodialysis (P < 0.001) compared with controls (5-aminolevulinic acid 0.13 +/- 0.02 microgram/ml; porphobilinogen 0.90 +/- 0.09 microgram/ml). After dialysis there was a decrease in both 5-aminolevulinic acid (to 0.61 +/- 0.05 microgram/ml) and porphobilinogen (to 1.10 +/- 0.16 micrograms/ml) although both parameters remained higher than controls (P < 0.001). The activities of both 5-aminolevulinate dehydratase (0.550 +/- 0.095 U/ml RBC), and deaminase (54.13 +/- 9.13 U/ml RBC) were diminished in blood samples of patients before dialysis (P < 0.001) compared to controls (dehydratase 0.975 +/- 0.115 U/ml RBC; deaminase 77.32 +/- 10.00 U/ml RBC). After dialysis 5-aminolevulinate dehydratase activity was partially recovered (to 0.666 +/- 0.100 U/ml RBC) while deaminase returned to normal values (73.45 +/- 9.46 U/ml RBC). The plasma zinc concentration in hemodialysed patients (44 +/- 12 micrograms/100 ml) was significantly lower than controls (105 +/- 30 micrograms/100 ml, P < 0.001). Addition of 22.5 mM zinc to the dehydratase reaction mixture raised the activity of 5-aminolevulinate dehydratase in blood samples of hemodialysed patients taken before and after dialysis. The study reports a partial loss of activity of 5-aminolevulinate dehydratase and deaminase activities in red blood cells from non-porphyric patients undergoing hemodialysis. Since plasma zinc levels were below normal in hemodialysed patients, and the activity of 5-aminolevulinate dehydratase could be restored by the addition of zinc, it is suggested that these abnormalities in heme metabolism may be explained by altered zinc and associated antioxidant status following dialysis.

Aminolaevulinic acid-induced protoporphyrin IX pharmacokinetics in central and peripheral arteries of the rat.

Photodynamic therapy (PDT) based on the photosensitive protoporphyrin IX (PpIX) may prevent restenosis after transluminal angioplasty. PpIX is synthesized in mitochondria, which differ in number and activity among various tissues. Therefore, we questioned whether the course of PpIX concentration after systemic aminolaevulinic acid (ALA) administration differed among various arteries. ALA was administered intravenously (200 mg/kg) to male Wistar rats (n = 21). At varying time intervals (0, 1, 2, 3, 6, 12 and 24 h) both central and peripheral arteries were isolated and homogenized, and the concentration of the various heme intermediates was determined by a fluorometric extraction method. The maximal PpIX concentration was more than two-fold higher in peripheral arteries (20.49 +/- 3.0 to 24.0 +/- 7.5 pmol/mg protein) than in central arteries (0-9.46 +/- 0.01 pmol/mg protein) (P < 0.004). However, the amount of citrate synthase, reflecting the mitochondrial mass, was lower (0.14-0.61 and 1.87-2.32 U/mg protein, respectively). Apparently, the level of PpIX cannot simply be explained by the mitochondrial content of the arteries. The time interval of maximal PpIX accumulation was similar in peripheral and central arteries (2 h and 27 min vs. 2 h and 8 min) (P = 0.13). Thus, if the efficacy of PDT in vivo is directly related to the tissue concentration of PpIX, more effect can be expected in peripheral arteries than in central arteries.

Sorbent therapy of the porphyrias. III. Comparative efficacy of experimental plasma perfusion with several commercial hemoperfusion cartridges.

The manifestations of the acute attack of an hepatic porphyria may result from the actions of the excessive amounts of delta-aminolevulinic acid (ALA) or porphobilinogen (PBG) that accumulate during the event. Adsorption and removal of these materials by a solid phase sorbent may be an effective rapid treatment of the attack. We have compared the rate at which 5 commercial charcoal hemoperfusion cartridges remove ALA and PBG from an in vitro system, to ascertain if any of these may be useful in the treatment of the porphyric attack. Solutions of ALA (3.0 mg/L in plasma) or PBG (1.5 mg/L in saline) were circulated from a reservoir through the cartridge and back to the reservoir. Clearances (ml/min +/- SD) of ALA or PBG were determined from their concentration in both the reservoir (kept nearly constant by periodic addition of solute) and the cartridge effluent. For ALA, clearances decreased in the order Hemosorba (118 +/- 15), two Detoxyl 2 devices in series (93 +/- 17), one Detoxyl 2 (72 +/- 16), Adsorba 300 C (52 +/- 12), Haemocol 100 (48 +/- 14) and Hemodetoxifier (46 +/- 17). Differences in ALA clearance between the Hemosorba, one or two Detoxyl 2 cartridges and all other cartridges were of high statistical significance. For PBG, clearances decreased in the order Hemosorba (1462 +/- 23), one Detoxyl 2 (719 +/- 89), Adsorba 300 C (477 +/- 131), Hemodetoxifier (376 +/- 75) and Haemocol 100 (48 +/- 13). Differences in PBG clearance among all cartridges were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorbent therapy of the porphyrias. II. Experimental plasma or hemoperfusion with a commercial charcoal cartridge.

We studied the ability of a commercial charcoal hemoperfusion cartridge (Hemosorba Hemoperfusion Cartridge, Asahi Medical Company) to adsorb the porphyrin precursors delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in vitro. For measurement of clearances or decreases in concentration of porphyrin precursor, indices of adsorptivity, solutions of porphyrin precursor were circulated from a reservoir through the cartridge and back to the reservoir. For determination of change in concentration of porphyrin precursor with time, the concentration of material in the reservoir was determined periodically. Clearances of porphyrin precursor were determined from their concentration in both the reservoir (maintained nearly constant by the periodic addition of solute) and the cartridge effluent. Clearances were independent of the nature of the medium (saline, 0.1 M phosphate buffer, pH 7.4, or human plasma), time (through 2 hr) and initial concentration of porphyrin precursor (3.0-10.3 mg/L for ALA, 1.5-3.6 mg/L for PBG). ALA clearances (ml/min) were 106 +/- 22, while maximal PBG clearances were never ascertained (greater than or equal to 310). The concentration of porphyrin precursor decreased rapidly during perfusion: about 82% and 98% of ALA and PBG, respectively, were removed in 5 minutes. Neither the clearances nor the concentration decreases were affected by the simultaneous presence in the solution of hematin (initial concentration of 50 mg/L), which itself had a clearance of 10.7 +/- 12.6 and only a modest concentration change with time (60-68% decrease in 40 min). Hemosorba cartridges adsorbed 650 mg ALA and 85 mg PBG without any appreciable change in adsorptivity. Hemoperfusion may be an appropriate adjunctive therapy, in combination with infusions of hematin, of particularly severe or refractory acute porphyric crises.

Hepatocyte transplantation ameliorates the metabolic abnormality in a mouse model of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder characterized by insufficient porphobilinogen deaminase (PBGD) activity. When hepatic heme synthesis is induced, porphobilinogen (PBG) and 5-aminolevulinic acid (ALA) accumulate, which causes clinical symptoms such as abdominal pain, neuropathy, and psychiatric disturbances. Our aim was to investigate if hepatocyte transplantation can prevent or minimize the metabolic alterations in an AIP mouse model. We transplanted wild-type hepatocytes into PBGD-deficient mice and induced heme synthesis with phenobarbital. ALA and PBG concentrations in plasma were monitored, and the gene transcriptions of hepatic enzymes ALAS1, PBGD, and CYP2A5 were analyzed. Results were compared with controls and correlated to the percentage of engrafted hepatocytes. The accumulation of ALA and PBG was reduced by approximately 50% after the second hepatocyte transplantation. We detected no difference in mRNA levels of PBGD, ALAS1, or CYP2A5. Engraftment corresponding to 2.7% of the total hepatocyte mass was achieved following two hepatocyte transplantations. A lack of precursor production in less than 3% of the hepatocytes resulted in a 50% reduction in plasma precursor concentrations. This disproportional finding suggests that ALA and PBG produced in PBGD-deficient hepatocytes crossed cellular membranes and was metabolized by transplanted cells. The lack of effect on enzyme mRNA levels suggests that no significant efflux of heme from normal to PBGD-deficient hepatocytes takes place. Further studies are needed to establish the minimal number of engrafted hepatocytes needed to completely correct the metabolic abnormality in AIP and whether amelioration of the metabolic defect by partial restoration of PBGD enzyme activity translates into a clinical effect in human AIP.