Porfiria hepato eritropoyética asociada a diabetes mellitus tipo 1: a propósito de un caso clínico / A clinical case of hepato-erythropoietic porphyria associated with type 1 diabetes mellitus / Relato de caso de porfiria hepato-eritropoiética associada a diabetes mellitus tipo 1

Las porfirias son un grupo complejo y heterogéneo de defectos en la vía de la síntesis del hemo. La porfiria hepato eritropoyética es un subtipo muy poco frecuente y de presentación en la infancia, con compromiso cutáneo predominante. Describimos el caso clínico de una paciente de 5 años, que se presenta con lesiones cutáneas e hipertricosis, se confirma el diagnóstico por elevación de uroporfirinas en orina y secuenciación del gen UROD.

Porphyria is a complex and heterogeneous group of heme synthesis disorder. Hepato-erythropoietic porphyria is a very rare subtype that onsets in childhood, and shows predominant skin involvement. We describe the clinical case of a 5-year-old patient who showed skin lesions and hypertrichosis and whose diagnosis was confirmed due to increased uroporphyrins in urine and UROD gene sequencing

A porfiria é um grupo complexo e heterogêneo de distúrbios da síntese do grupo heme. A porfiria hepato-eritropoiética é um subtipo muito raro que se inicia na infância e mostra envolvimento predominante da pele. Descrevemos o caso clínico de uma paciente de 5 anos que apresentou lesões cutâneas e hipertricose e cujo diagnóstico foi confirmado por aumento de uroporfirinas na urina e sequenciamento do gene UROD.

Further Evaluation of Coproporphyrins as Clinical Endogenous Markers for OATP1B.

Coproporphyrins (CP-I and CP-III) in plasma are considered potential markers for assessing liver organic anion-transporting polypeptide transporter OATP1B activity and monitoring OATP1B-mediated drug-drug interactions (DDIs) in clinical settings. However, the effect of altered renal clearance (CLrenal ) on CP-I and CP-III plasma exposure has rarely been examined. Therefore, the purpose of this study is to further evaluate CP-I and CP-III as clinical endogenous markers for OATP1B activity and to investigate the impact of CLrenal on DDI assessments for the first time. In this study, 18 healthy participants were recruited to receive RO7049389 (a potential inhibitor of OATP1B) 800 mg twice daily for 6 days and a single dose of pitavastatin (a probe drug of OATP1B) before and after RO7049389 treatment. Plasma concentrations of pitavastatin, CP I, CP III, and the amounts of CP-I and CP-III excreted in urine were measured. Seventeen healthy participants completed the study. After multiple doses of RO7049389, the area under the plasma concentration-time curve from time 0 to 12 hours of pitavastatin increased 1.95-fold (90% confidence interval [CI], 1.58-2.41), while for CP-I and CP-III it increased 3.00-fold (90%CI, 2.35-3.82) and 2.84-fold (90%CI, 2.22-3.65), respectively. Concurrently, the CLrenal of CP-I decreased by 31% (90%CI, 23%-39%), and that of CP-III decreased by 70% (90%CI, 61%-77%). In conclusion, CP-I and CP-III in plasma display the potential to be applied as endogenous markers for the evaluation of OATP1B inhibition in clinical trials. While renal transporters contribute significantly to the CLrenal of CP-III, it would be better to investigate the impact of the CLrenal on plasma exposure of CP-III during clinical DDI assessments.

PBPK Model of Coproporphyrin I: Evaluation of the Impact of SLCO1B1 Genotype, Ethnicity, and Sex on its Inter-Individual Variability.

Coproporphyrin I (CPI) is an endogenous biomarker of OATP1B activity and associated drug-drug interactions. In this study, a minimal physiologically-based pharmacokinetic model was developed to investigate the impact of OATP1B1 genotype (c.521T>C), ethnicity, and sex on CPI pharmacokinetics and interindividual variability in its baseline. The model implemented mechanistic descriptions of CPI hepatic transport between liver blood and liver tissue and renal excretion. Key model parameters (e.g., endogenous CPI synthesis rate, and CPI hepatic uptake clearance) were estimated by fitting the model simultaneously to three independent CPI clinical datasets (plasma and urine data) obtained from white (n = 16, men and women) and Asian-Indian (n = 26, all men) subjects, with c.521 variants (TT, TC, and CC). The optimized CPI model successfully described the observed data using c.521T>C genotype, ethnicity, and sex as covariates. CPI hepatic active was 79% lower in 521CC relative to the wild type and 42% lower in Asian-Indians relative to white subjects, whereas CPI synthesis was 23% higher in male relative to female subjects. Parameter sensitivity analysis showed marginal impact of the assumption of CPI synthesis site (blood or liver), resulting in comparable recovery of plasma and urine CPI data. Lower magnitude of CPI-drug interaction was simulated in 521CC subjects, suggesting the risk of underestimation of CPI-drug interaction without prior OATP1B1 genotyping. The CPI model incorporates key covariates contributing to interindividual variability in its baseline and highlights the utility of the CPI modeling to facilitate the design of prospective clinical studies to maximize the sensitivity of this biomarker.

Detection of Weak Organic Anion-Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans.

Probenecid (PROB) is a clinical probe inhibitor of renal organic anion transporter (OAT) 1 and OAT3 that inhibits in vitro activity of hepatic drug transporters OATP1B1 and OATP1B3. It was hypothesized that PROB could potentially affect the disposition of OATP1B drug substrates. The plasma levels of the OATP1B endogenous biomarker candidates, including coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA), were examined in 14 healthy subjects treated with PROB. After oral administration with 1000 mg PROB alone and in combination with furosemide (FSM), AUC (0-24 h) values were 1.39 ± 0.21-fold and 1.57 ± 0.41-fold higher than predose levels for CPI and 1.34 ± 0.16-fold and 1.45 ± 0.57-fold higher for CPIII. Despite increased systemic exposures, no decreases in CPI and CPIII renal clearance were observed (0.97 ± 0.38-fold and 1.16 ± 0.51-fold for CPI, and 1.34 ± 0.53-fold and 1.50 ± 0.69-fold for CPIII, respectively). These results suggest that the increase of CP systemic exposure is caused by OATP1B inhibition. Consistent with this hypothesis, PROB inhibited OATP1B1- and OATP1B3-mediated transport of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively, in transporter-overexpressing human embryonic kidney cell assay. The inhibition potential was further confirmed by CPI and CPIII hepatocyte uptake experiments. In contrast, administration of PROB alone did not change AUC (0-24 h) of HDA and TDA relative to prestudy levels, although the administration of PROB in combination with FSM increased HDA and TDA levels compared with FSM alone (1.02 ± 0.18-fold and 0.90 ± 0.20-fold vs. 1.71 ± 0.43-fold and 1.62 ± 0.40-fold). Taken together, these findings indicate that PROB displays weak OATP1B inhibitory effects in vivo and that coproporphyrin is a sensitive endogenous probe of OATP1B inhibition. This study provides an explanation for the heretofore unknown mechanism responsible for PROB's interaction with other xenobiotics. SIGNIFICANCE STATEMENT: This study suggested that PROB is a weak clinical inhibitor of OATP1B based on the totality of evidence from the clinical interaction between PROB and CP and the in vitro inhibitory effect of PROB on OATP1B-mediated CP uptake. It demonstrates a new methodology of utilizing endogenous biomarkers to evaluate complex drug-drug interaction, providing explanation for the heretofore unknown mechanism responsible for PROB's inhibition. It provides evidence to strengthen the claim that CP is a sensitive circulating endogenous biomarker of OATP1B inhibition.

Developmental changes in urinary coproporphyrin ratio in premature infants.

BACKGROUND: Premature infants have a high concentration of conjugated bilirubin in their blood, although they have a poor glucuronide conjugation of bilirubin. This may be due to developmental changes in the function of adenosine triphosphate binding cassette subfamily C member 2, which is involved in the cellular export of conjugated bilirubin. In the present study, we examined the developmental changes in the urinary coproporphyrin I/(urinary coproporphyrin I+ urinary coproporphyrin III) ratio (UCP (I/ [I + III])), a known biomarker for adenosine triphosphate binding cassette subfamily C member 2 function, in premature infants. METHOD: Twenty-one premature infants born between 25 and 32 weeks of gestation were included in the study. Urine samples were collected within 24 h of birth, and at 1 week and 3-4 weeks after birth. The samples were analyzed by high-performance liquid chromatography to calculate UCP (I/ [I + III]) to examine its association with postnatal age and corrected gestational age. Subjects were excluded if they had liver dysfunction, cholestasis, urinary tract infection, or chromosomal abnormalities. RESULTS: The average UCP (I/ [I + III]) within 24 h of birth, at 1 week, and at 3-4 weeks after birth was 0.84, 0.61, and 0.65, respectively. The UCP (I/ [I + III]) within 24 h of birth was significantly higher than that measured at 1 week or 3-4 weeks after birth. There was no significant correlation between UCP (I/ [I + III]) and the corrected gestational age. CONCLUSION: The UCP (I/ [I + III]) was higher within 24 h of birth. It decreased 1 week after birth and remained low without any significant changes for up to 4 weeks after birth.

Gaining Mechanistic Insight Into Coproporphyrin I as Endogenous Biomarker for OATP1B-Mediated Drug-Drug Interactions Using Population Pharmacokinetic Modeling and Simulation.

This study evaluated coproporphyrin I (CPI) as a selective endogenous biomarker of OATP1B-mediated drug-drug interactions (DDIs) relative to clinical probe rosuvastatin using nonlinear mixed-effect modeling. Plasma and urine CPI data in the presence/absence of rifampicin were modeled to describe CPI synthesis, elimination clearances, and obtain rifampicin in vivo OATP Ki. The biomarker showed stable interoccasion baseline concentrations and low interindividual variability (<25%) in subjects with wildtype SLCO1B1. Biliary excretion was the dominant CPI elimination route (maximal >85%). Estimated rifampicin in vivo unbound OATP Ki (0.13 µM) using CPI data was 2-fold lower relative to rosuvastatin. Model-based simulations and power calculations confirmed sensitivity of CPI to identify moderate and weak OATP1B inhibitors in an adequately powered clinical study. Current analysis provides the most detailed evaluation of CPI as an endogenous OATP1B biomarker to support optimal DDI study design; further pharmacogenomic and DDI data with a panel of inhibitors are required.

A highly selective and sensitive LC-MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting peptide biomarkers.

AIM: Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug-drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials & methods: Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer. Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.14) were used as precursor ions to improve the assay sensitivity and selectivity over the singly charged precursor ions (m/z 655.28). Levels of CP-I and CP-III measured ranged 0.45-1.1 and 0.050-0.50 ng/ml in plasma and 5-35 and 1-35 ng/ml in urine, respectively. CONCLUSION: The described highly selective and sensitive CP-I/CP-III LC-HRMS assay offers options for earlier characterization and clinical safety projections for OATP1B1/3-mediated drug-drug interactions along with pharmacokinetic analyses of a new chemical entity as part of first-in-human clinical studies.

Coproporphyrins in Plasma and Urine Can Be Appropriate Clinical Biomarkers to Recapitulate Drug-Drug Interactions Mediated by Organic Anion Transporting Polypeptide Inhibition.

In the present study, an open-label, three-treatment, three-period clinical study of rosuvastatin (RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12 male healthy subjects to determine if coproporphyrin I (CP-I) and coproporphyrin III (CP-III) could serve as clinical biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 that belong to the solute carrier organic anion gene subfamily. Genotyping of the human OATP1B1 gene was performed in all 12 subjects and confirmed absence of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III prior to drug administration were 0.91 ± 0.21 and 0.15 ± 0.04 nM, respectively, with minimum fluctuation over the three periods. CP-I was passively eliminated, whereas CP-III was actively secreted from urine. Administration of RSV caused no significant changes in the plasma and urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration (Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as compared with the predose values. The area under the plasma concentration curves from time 0 to 24 h (AUC0-24h) of CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively, when compared with RSV alone. In agreement with this finding, Cmax and AUC0-24h of RSV increased by 13.2- and 5.0-fold, respectively, when RIF was coadministered. Collectively, we conclude that CP-I and CP-III in plasma and urine can be appropriate endogenous biomarkers specifically and reliably reflecting OATP inhibition, and thus the measurement of these molecules can serve as a useful tool to assess OATP drug-drug interaction liabilities in early clinical studies.

Developmental characteristics of urinary coproporphyrin I/(I + III) ratio.

BACKGROUND: The ratio of urinary coproporphyrin (UCP) I to total urinary coproporphyrin I and III [UCP {I/(I + III)]] serves as a biomarker of the ATP-binding cassette, sub-family C, member 2 (ABCC2) function. The aim of this study was to clarify the characteristics of the developmental pattern of UCP [I/(I + III)] in order to estimate ABCC2 function in children, especially in the neonatal period, by measuring it throughout the entirety of childhood. METHOD: Measurement of UCP [I/(I + III)] was done high-performance liquid chromatography, using urine samples collected from children from 1 day to 15 years old, involving one sample per child. Urine samples from children with liver and kidney disease and urinary tract infection were excluded. RESULTS: UCP [I/(I + III)] varied widely in infants younger than 6 months old, and was ≥0.3 in 80% of the infants. In contrast, it decreased to <0.30, the lowest, at 1-2 years old. In the 0-6-month-old group, no significant correlation was noted between postnatal age and UCP [I/(I + III)], but a moderate inverse correlation was noted between corrected gestational age and UCP [I/(I + III)]. CONCLUSION: UCP [I/(I + III)] is inversely correlated with corrected gestational age and is lowest at 1-2 years old. This suggests that ABCC2 activity is correlated with corrected gestational age and is highest at 1-2 years old.

Genetic and biochemical study of dual hereditary jaundice: Dubin-Johnson and Gilbert's syndromes. Haplotyping and founder effect of deletion in ABCC2.

Dual hereditary jaundice, a combination of Dubin-Johnson and Gilbert's syndromes, is a rare clinical entity resulting from the compound defects of bilirubin conjugation and transport. We aimed to study the hereditary jaundice in 56 members from seven seemingly unrelated Roma families, to find the causal genetic defect and to estimate its origin in Roma population. On the basis of biochemical results of total and conjugated serum bilirubin and clinical observations, ABCC2 gene, TATA box and phenobarbital enhancer (PBREM) of UGT1A1 gene were analyzed by sequencing, RFLP and fragment analysis. We found a novel variant c.1013_1014delTG in the eighth exon of ABCC2 gene in 17 individuals in homozygous state. Dual defect NG_011798.1:c.[1013_1014delTG]; NG_002601.2:g.[175492_175493insTA] in homozygous state was found in four subjects. Biochemical analyses of porphyrins and coproporphyrin isomers in urine performed by HPLC showed inverted ratio of excreted coproporphyrin, with the predominance of coproporphyrin I (up to 100%), typical for patients with Dubin-Johnson syndrome. Pursuant cultural and social specifics of the population led us to suspect a founder effect; therefore, we performed a haplotype study using genotyping data from Affymetrix Genome-Wide Human SNP Array 6.0. As a result, we detected a common 86 kbp haplotype encompassing promoter and part of the ABCC2 coding region among all families, and estimated the age of the ancestral variant to 178-185 years. In this study, we found a novel deletion in ABCC2 gene, described genetic and biochemical features of dual hereditary jaundice and confirmed the existence of founder effect and common haplotype among seven Roma families.

Direct assay of uroporphyrin and coproporphyrin in human urine by reverse-mode field amplified sample injection-sweeping and micellar electrokinetic chromatography.

In this study, an on-line stacking capillary electrophoresis (CE) method, reverse-mode field amplified sample injection equipped with sweeping micellar electrokinetic chromatography (RMFASI-sweeping MEKC), was established for direct determination of uroporphyrin and coproporphyrin in human urine. Porphyrins playing a very important role in the biosynthesis of heme, chlorophyll and other important enzymes are a series of important molecules in organism. Therefore, determination of porphyrin metabolites, uroporphyrin and coproporphyrin, was very important for clinical survey of some diseases. In this study, the urine sample after simple dilution could be directly analyzed by this on-line stacking CE method. The optimal CE separation buffer was 70 mM phosphate buffer at pH 3. Before sample injection, a water plug was introduced (2.5 psi for 10s), and then the samples were loaded by electrokinetic injection (-10 kV, 200 s). Finally, the phosphate buffer (70 mM, pH 3) containing 100mM SDS was served as the sweeping buffer to stack and separate the analytes at -20 kV. The calibration curves were linear over a range of 15-200 ng/ml for uroporphyrin, and 300-1000 ng/ml for coproporphyrin. The coefficient of correlation (r) in intra-batch (n=5) and inter-batch (n=5) analysis was above 0.983. The LODs (S/N=3) were 5 ng/ml for uroporphyrin, and 100ng/ml for coproporphyrin. The absolute values of relative standard deviation (RSD) and relative error (RE) in intra-batch (n=5) and inter-batch (n=5) assays were less than 8.6% showing the good precision and accuracy. The stacking method was successfully applied in real urine sample and feasible for serving as a tool for detection of uroporphyrin and coproporphyrin in clinical.

Urinary coproporphyrin I/(I + III) ratio as a surrogate for MRP2 or other transporter activities involved in methotrexate clearance.

AIMS: The urinary coproporphyrin I/(I + III) ratio may be a surrogate for MRP2 activity. We conducted a prospective study in patients receiving methotrexate (MTX) to examine the relationship between this ratio and the pharmacokinetics of a MRP2 substrate. METHODS: Three urine samples were collected from 81 patients for UCP I/(I + III) ratio determination: one before (P1), one at the end of MTX infusion (P2), and one on the day of hospital discharge (P3). Three polymorphisms of ABCC2 were analysed and their relationships with basal UCP I/(I + III) ratio values assessed. All associated drugs were recorded and a drug interaction score (DIS) was assigned. Population pharmacokinetic analysis was conducted to assess whether MTX clearance (MTXCL) was associated with the basal UCP I/(I + III) ratio, its variation during MTX infusion, the DIS or other common covariates. RESULTS: The basal UCP I/(I + III) ratio was not associated with ABCC2 polymorphisms and did not differ according to the DIS. Significant changes in the ratio were observed over time, with an increase between P1 and P2 and a decrease at P3 (P < 0.001). No association was found between basal UCP I/(I + III) ratio and MTXCL. The final model indicates that MTXCL was dependent on the change in the ratio between P1 and P3, DIS and creatinine clearance. CONCLUSION: The basal UCP I/(I + III) ratio is not predictive of MTXCL. However, it is sensitive to the presence of MTX, so it is plausible that it reflects a function modified in response to the drug.

A quantitative evaluation of brain dysfunction and body-burden of toxic metals.

BACKGROUND: Toxic metal exposure (e.g. Hg, Pb, As) exposure is known to induce significant adverse effects on human brain function. The aim this study was to assess toxic metal body-burden in relation to potential brain dysfunction in patients diagnosed with neurological disorders (NDs). MATERIAL/METHODS: The Liberty Institutional Review Board (Deland, FL) approved the present study. Quantitative, fractionated, random urinary porphyrin testing (µg/L) from the Clinical Laboratory Improvement Act/Amendment (CLIA)-approved Laboratory Corporation of America (LabCorp) and cortical perfusion index (CPi) values from single-photon-emission-computed-tomography (SPECT) brain scans were employed to evaluate a prospective cohort of qualifying patients with diagnosed NDs (n=52) presenting for medical care at an endocrinology practice in the Cincinnati, OH area. RESULTS: Patients with more severe in comparison to mild brain dysfunction had significant increases in the mean urinary concentration of uroporphyrins (uP), coproporphyrins I (cP I), and total cP (cP I + III), as well as a trend towards significantly increased mean urinary concentration of pentacarboxyporphyins (5cxP) and cP III. A significant positive correlation between Hg body-burden associated porphyrins (5cxP + cP I + cP III) and increased brain dysfunction was observed. CONCLUSIONS: The present study associated brain dysfunction with Hg body-burden in a cohort of patients diagnosed with NDs, but the contributions of other heavy metals or genetic factors cannot be ruled-out. Additional studies should be conducted to evaluate the consistency of the present findings with examinations of other populations.

Disordered porphyrin metabolism: a potential biological marker for autism risk assessment.

Autism (AUT) is a complex neurodevelopmental disorder that, together with Asperger's syndrome and Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS), comprises the expanded classification of autistic spectrum disorder (ASD). The heterogeneity of ASD underlies the need to identify biomarkers or clinical features that can be employed to identify meaningful subtypes of ASD, define specific etiologies, and inform intervention and treatment options. Previous studies have shown that disordered porphyrin metabolism, manifested principally as significantly elevated urinary concentrations of pentacarboxyl (penta) and coproporphyrins, is commonly observed among some children with ASD. Here, we extend these observations by specifically evaluating penta and coproporphyrins as biological indicators of ASD among 76 male children comprising 30 with validated AUT, 14 with PDD-NOS, and 32 neurotypical (NT) controls. ASD children (AUT and PDD-NOS) had higher mean urinary penta (P < 0.006) and copro (P < 0.006) concentrations compared with same-aged NT children, each characterized by a number of extreme values. Using Receiver Operating Characteristic curve analysis, we evaluated the sensitivity and specificity of penta, copro, and their combined Z-scores in ASD detection. The penta sensitivity was 30% for AUT and 36% for PDD-NOS, with 94% specificity. The copro sensitivity was 33% and 14%, respectively, with 94% specificity. The combined Z-score measure had 33% and 21% sensitivity for AUT and PDD-NOS, respectively, with 100% specificity. These findings demonstrate that porphyrin measures are strong predictors of both AUT and PDD-NOS, and support the potential clinical utility of urinary porphyrin measures for identifying a subgroup of ASD subjects in whom disordered porphyrin metabolism may be a salient characteristic.

Urinary elimination of coproporphyrins is dependent on ABCC2 polymorphisms and represents a potential biomarker of MRP2 activity in humans.

MRP2 encoded by ABCC2 gene is involved in the secretion of numerous drugs and endogenous substrates. Patients with Dubin-Johnson syndrome due to mutation in ABCC2 gene have elevated urinary coproporphyrin ratio (UCP I/(I + III)). Here we investigated whether this ratio could serve as a biomarker of MRP2 function. Phenotype-genotype relationships were studied in 74 healthy subjects by measuring individual UCP I/(I + III) ratio obtained on 24-hour urine and by analyzing five common SNPs in ABCC2 gene. The UCP I/(I + III) ratio varied from 14.7% to 46.0% in our population. Subjects with 3972TT genotype had a higher ratio (P = .04) than those carrying the C allele. This higher UCP I/(I + III) ratio was correlated with a higher level of isomer I excretion. This study provides a proof of concept that UCP I/(I + III) ratio can be used as a biomarker of MRP2 function in clinical studies as it provides quantitative information about the in vivo activity of MRP2 in a given patient.