Therapeutic strategies for acute intermittent porphyria.
ABSTRACT: Acute intermittent porphyria (AIP) is an autosomal dominant disease caused by mutations in porphobilinogen deaminase (PBGD), the third enzyme of the heme synthesis pathway. Symptoms of AIP usually manifest as intermittent acute attacks with occasional neuropsychiatric crises. The management of AIP includes treatment of acute attacks, prevention of attacks, long-term monitoring and treatment of chronic complications. Intravenous injection of heme is the most effective method of treating acute attacks. Carbohydrate loading is used when heme is unavailable or in the event of mild attacks. Symptomatic treatment is also needed during attacks. Prevention of attacks includes eliminating precipitating factors, heme prophylaxis and liver transplantation. New treatment options include givosiran (siRNA) to down-regulate ALA synthase-1 (ALAS1) and the messenger RNA of PBGD (PBGD mRNA) delivered to the liver cells of patients with AIP. Long-term monitoring of chronic complications includes regular liver-kidney function and hepatocellular carcinoma (HCC) screening.
Project description: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.
Project description:Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.
Project description: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.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.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.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.
Project description:Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.
Project description:The acute hepatic porphyrias are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks. Factors that induce the expression of hepatic 5-aminolevulinic acid synthase 1 (ALAS1) result in the accumulation of the neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which recent studies indicate are primarily responsible for the acute attacks. Current treatment of these attacks involves i.v. administration of hemin, but a faster-acting, more effective, and safer therapy is needed. Here, we describe preclinical studies of liver-directed small interfering RNAs (siRNAs) targeting Alas1 (Alas1-siRNAs) in a mouse model of acute intermittent porphyria, the most common acute hepatic porphyria. A single i.v. dose of Alas1-siRNA prevented the phenobarbital-induced biochemical acute attacks for approximately 2 wk. Injection of Alas1-siRNA during an induced acute attack significantly decreased plasma ALA and PBG levels within 8 h, more rapidly and effectively than a single hemin infusion. Alas1-siRNA was well tolerated and a therapeutic dose did not cause hepatic heme deficiency. These studies provide proof-of-concept for the clinical development of RNA interference therapy for the prevention and treatment of the acute attacks of the acute hepatic porphyrias.
Project description:The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.
Project description:Porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis, catalyzes the sequential coupling of four porphobilinogen (PBG) molecules into a heme precursor. Mutations in PBGD are associated with acute intermittent porphyria (AIP), a rare metabolic disorder. We used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to demonstrate that wild-type PBGD and AIP-associated mutant R167W both existed as holoenzymes (E<sub>holo</sub>) covalently attached to the dipyrromethane cofactor, and three intermediate complexes, ES, ES<sub>2</sub>, and ES<sub>3</sub>, where S represents PBG. In contrast, only ES<sub>2</sub> was detected in AIP-associated mutant R173W, indicating that the formation of ES<sub>3</sub> is inhibited. The R173W crystal structure in the ES<sub>2</sub>-state revealed major rearrangements of the loops around the active site, compared to wild-type PBGD in the E<sub>holo</sub>-state. These results contribute to elucidating the structural pathogenesis of two common AIP-associated mutations and reveal the important structural role of Arg173 in the polypyrrole elongation mechanism.
Project description:Acute intermittent porphyria (AIP) is an autosomal dominant disorder of heme biosynthesis due to a mutation in the porphobilinogen deaminase gene. The mutation causes a deficiency in the porphobilinogen deaminase enzyme, thereby causing an accumulation of heme precursors (δ-aminolevulinic acid and porphobilinogen). These neurotoxic heme precursors elicit acute neurovisceral attacks, which can be treated with heme-arginate infusions. Some patients require heme-arginate infusions on a regular basis for many years, which ultimately leads to an iron accumulation (increased serum ferritin and iron accumulation in the liver, spleen, and bone marrow on MRI). We report three AIP patients, who developed iron accumulation (with serum ferritin up to 7,850 microgram/liter) due to multiple heme-arginate infusions. We report for the first time that the iron accumulation in these patients was associated with fibrosis on liver histology. CONCLUSION:Regular heme-arginate treatment in AIP does not only lead to increased serum ferritin but may also induce liver fibrosis. This should be taken into account, when weighing the risks and benefits of repeated heme-arginate treatment against the risk and benefits of treating refractory AIP by liver transplantation.
Project description:Acute Intermittent Porphyria (AIP), an autosomal dominant hepatic disorder, results from hydroxymethylbilane synthase (HMBS) mutations that decrease the encoded enzymatic activity, thereby predisposing patients to life-threatening acute neurovisceral attacks. The ~1% penetrance of AIP suggests that other genetic factors modulate the onset and severity of the acute attacks. Here, we characterized the hepatic transcriptomic response to phenobarbital (PB) administration in AIP mice, which mimics the biochemical attacks of AIP. At baseline, the mRNA profiles of 14,138 hepatic genes prior to treatment were remarkably similar between AIP and the congenic wild-type (WT) mice. After PB treatment (~120?mg/kg x 3d), 1347 and 1120 genes in AIP mice and 422 and 404 genes in WT mice were uniquely up- and down-regulated, respectively, at a False Discovery Rate?<?0.05. As expected, the ALAS1 expression increased 4.5-fold and 15.9-fold in the WT and AIP mice, respectively. ALA-dehydrogenase also was induced ~1.7-fold in PB-induced AIP mice, but was unchanged in PB-induced WT mice. There was no statistically significant difference in the overall expression of 155 hepatic cytochrome P450 enzymes, although Cyp2c40, Cyp2c68, Cyp2c69, Mgst3 were upregulated only in PB-induced AIP mice (>1.9-fold) and Cyp21a1 was upregulated only in PB-induced WT mice (>9-fold). Notably, the genes differentially expressed in induced AIP mice were enriched in circadian rhythm, mitochondria biogenesis and electron transport, suggesting these pathways were involved in AIP mice responding to PB treatment. These results advance our understanding of the hepatic metabolic changes in PB-induced AIP mice and have implications in the pathogenesis of AIP acute attacks.
Project description:INTRODUCTION:A small proportion of patients with acute intermittent porphyria (AIP) suffer from recurrent porphyric attacks, with a severely diminished quality of life. In this retrospective case-control study, the burden of disease is quantified and compared among three AIP patient subgroups: cases with recurrent attacks, cases with one or occasional attacks and asymptomatic carriers. METHODS:Data from patient records and questionnaires were collected in patients between 1960 and 2016 at the Erasmus Medical Center, Rotterdam, the Netherlands. We collected symptoms related to porphyria, porphyria related complications, attack frequency, hospitalisation frequency, hospitalisation days related to acute porphyric attacks, frequency of heme infusions and medical healthcare costs based on hospitalisations and heme therapy. RESULTS:In total 11 recurrent AIP cases, 24 symptomatic AIP cases and 53 AIP carriers as controls were included. All recurrent patients reported porphyria related symptoms, such as pain, neurological and/or psychiatric disorders, and nearly all developed complications, such as hypertension and chronic kidney disease. In the recurrent cases group, the median lifelong number of hospitalisation days related to porphyric attacks was 82 days per patient (range 10-374), and they spent a median of 346 days (range 34-945) at a day-care facility for prophylactic heme therapy; total follow-up time was 243 person-years (PYRS). In the symptomatic non-recurrent group the median lifelong number of hospitalisation days related to porphyric attacks was 7 days per patient (range 1-78), total follow-up time was 528 PYRS. The calculated total medical healthcare cost for recurrent cases group was €5.8 million versus €0.3 million for the symptomatic cases group.