Cryptic specialized metabolites drive Streptomyces exploration and provide a competitive advantage during growth with other microbes.

Streptomyces bacteria have a complex life cycle that is intricately linked with their remarkable metabolic capabilities. Exploration is a recently discovered developmental innovation of these bacteria, that involves the rapid expansion of a structured colony on solid surfaces. Nutrient availability impacts exploration dynamics, and we have found that glycerol can dramatically increase exploration rates and alter the metabolic output of exploring colonies. We show here that glycerol-mediated growth acceleration is accompanied by distinct transcriptional signatures and by the activation of otherwise cryptic metabolites including the orange-pigmented coproporphyrin, the antibiotic chloramphenicol, and the uncommon, alternative siderophore foroxymithine. Exploring cultures are also known to produce the well-characterized desferrioxamine siderophore. Mutational studies of single and double siderophore mutants revealed functional redundancy when strains were cultured on their own; however, loss of the alternative foroxymithine siderophore imposed a more profound fitness penalty than loss of desferrioxamine during coculture with the yeast Saccharomyces cerevisiae. Notably, the two siderophores displayed distinct localization patterns, with desferrioxamine being confined within the colony area, and foroxymithine diffusing well beyond the colony boundary. The relative fitness advantage conferred by the alternative foroxymithine siderophore was abolished when the siderophore piracy capabilities of S. cerevisiae were eliminated (S. cerevisiae encodes a ferrioxamine-specific transporter). Our work suggests that exploring Streptomyces colonies can engage in nutrient-targeted metabolic arms races, deploying alternative siderophores that allow them to successfully outcompete other microbes for the limited bioavailable iron during coculture.

Rifampin- and Silymarin-Mediated Pharmacokinetic Interactions of Exogenous and Endogenous Substrates in a Transgenic OATP1B Mouse Model.

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3, encoded by the SLCO gene family of the solute carrier superfamily, are involved in the disposition of many exogenous and endogenous compounds. Preclinical rodent models help assess risks of pharmacokinetic interactions, but interspecies differences in transporter orthologs and expression limit direct clinical translation. An OATP1B transgenic mouse model comprising a rodent Slco1a/1b gene cluster knockout and human SLCO1B1 and SLCO1B3 gene insertions provides a potential physiologically relevant preclinical tool to predict pharmacokinetic interactions. Pharmacokinetics of exogenous probe substrates, pitavastatin and pravastatin, and endogenous OATP1B biomarkers, coproporphyrin-I and coproporphyrin-III, were determined in the presence and absence of known OATP/Oatp inhibitors, rifampin or silymarin (an extract of milk thistle [Silybum marianum]), in wild-type FVB mice and humanized OATP1B mice. Rifampin increased exposure of pitavastatin (4.6- and 2.8-fold), pravastatin (3.6- and 2.2-fold), and coproporphyrin-III (1.6- and 2.1-fold) in FVB and OATP1B mice, respectively, but increased coproporphyrin-I AUC0-24h only (1.8-fold) in the OATP1B mice. Silymarin did not significantly affect substrate AUC, likely because the silymarin flavonolignan concentrations were at or below their reported IC50 values for the relevant OATPs/Oatps. Silymarin increased the Cmax of pitavastatin 2.7-fold and pravastatin 1.9-fold in the OATP1B mice. The data of the OATP1B mice were similar to those of the pitavastatin and pravastatin clinical data; however, the FVB mice data more closely recapitulated pitavastatin clinical data than the data of the OATP1B mice, suggesting that the OATP1B mice are a reasonable, though costly, preclinical strain for predicting pharmacokinetic interactions when doses are optimized to achieve clinically relevant plasma concentrations.

The Role of Coproporphyrins As Endogenous Biomarkers for Organic Anion Transporting Polypeptide 1B Inhibition-Progress from 2016 to 2023.

Since the initial clinical study investigating coproporphyrins I and III (CP-I and CP-III) as endogenous biomarkers for organic anion transporting polypeptide (OATP) inhibition drug-drug interactions (DDIs) published in 2016, significant progress has been made in confirming the usefulness of the CPs, particularly CP-I, as biomarkers in assessing OATP functions. CP-I exhibits selectivity toward OATP1B activity in human subjects with genetic variants of OATP1B1. Its sensitivity to a broad spectrum of clinical OATP1B inhibitors has been established from weak to vigorous. Dose-dependent CP-I changes in healthy human subjects show agreement with DDI magnitudes of probe substrates by rifampin treatment. Physiologically based pharmacokinetic models have been established for concentration changes of plasma CP-I with OATP inhibitors, demonstrating the usefulness of supporting the quantitative translation of the effect of CP-I levels into the DDI risk assessment of potential OATP inhibitors. As plasma CP-I's sensitivity, specificity, and selectivity have been validated in humans, monitoring CP-I levels in single and multiple clinical phase I dose escalation studies is recommended for early assessment of DDI risks and understanding the full dose-response of an investigational drug to OATP inhibitions. A decision tree is proposed to preclude the need to conduct a dedicated DDI study by administering a probe substrate drug to human subjects. SIGNIFICANCE STATEMENT: The minireview summarized the validation paths of coproporphyrins I and III (CP-I and CP-III) as biomarkers of organic anion transporting polypeptide 1B (OATP1B) inhibition in humans for their selectivity, specificity, and sensitivity. The utility of monitoring CP-I to assess drug-drug interactions of OATP1B inhibition in early drug development is proposed. Changes in plasma CP-I in phase I dose range studies can be used to frame plans for late-stage development and facilitate the mechanistic understanding of complex drug-drug interactions.

Positive correlation between organic anion transporter 1B function indicated by plasma concentration of coproporphyrin-I and blood concentration of cyclosporin A in real-world patients.

AIMS: Cyclosporin A (CyA) has potent inhibitory activity on organic anion transporting polypeptide 1B (OATP1B), causing drug-drug interactions with its substrate drugs. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a uraemic toxin, has also been suggested to inhibit OATP1B activity. Recent study has identified coproporphyrin-I (CP-I) as a specific endogenous substrate for OATP1B, which is useful to indicate OATP1B activity. We investigated the relationship of CP-I with CyA and CMPF concentrations in patients taking CyA. METHODS: In total, 121 blood samples from 74 patients who took CyA and underwent routine therapeutic drug monitoring were divided into trough and peak samples. RESULTS: CyA and CP-I concentrations were significantly higher in peak samples than in trough samples. A positive correlation between CP-I and CyA concentrations was found in all samples and in trough and peak samples, while no correlation was observed between CP-I and CMPF concentrations. Multiple regression analysis identified CyA and C-reactive protein concentrations as independent factors affecting CP-I concentration, with blood CyA concentration having markedly greater contribution to plasma CP-I concentration. CONCLUSION: The present study suggests that CyA inhibits OATP1B activity in a concentration-dependent manner in clinical setting, and that dose adjustment of OATP1B substrate drugs coadministered with CyA according to plasma CMPF concentration may not be necessary.

Molecular Recognition of Imidazole-Based Drug Molecules by Cobalt(III)- and Zinc(II)-Coproporphyrins in Aqueous Media.

The methods of 1H NMR, spectrophotometric titration, mass spectrometry and elemental analysis are applied to determine the selective binding ability of Co(III)- and Zn(II)-coproporphyrins I towards a series of imidazole-based drug molecules with a wide spectrum of pharmacological activity (metronidazole, histamine, histidine, tinidazole, mercazolil, and pilocarpine) in phosphate buffer (pH 7.4) simulating the blood plasma environment. It is shown that in aqueous buffer media, Co(III)-coproporphyrin I, unlike Zn(II)-coproporphyrin I, binds two imidazole derivatives, and the stability of mono-axial Co-coproporphyrin imidazole complexes is two to three orders of magnitude higher than that of similar complexes of Zn-coproporphyrin I. The studied porphyrinates are found to have the highest binding ability to histamine and histidine due to the formation of two additional hydrogen bonds between the carboxyl groups of the porphyrinate side chains and the binding sites of the ligands in the case of histidine and a hydrogen bond between the amino group of the ligand and the carbonyl oxygen atom of the carboxyl group of the porphyrinate in the case of histamine. The structures of the resulting complexes are optimized by DFT quantum chemical calculations. The results of these studies may be of use in the design of biosensors, including those for the detection, control and verification of various veterinary drug residues in human food samples.

Repression of Organic Anion Transporting Polypeptide (OATP) 1B Expression and Increase of Plasma Coproporphyrin Level as Evidence for OATP1B Downregulation in Cynomolgus Monkeys Treated with Chenodeoxycholic Acid.

Farnesoid X receptor (FXR) is a nuclear receptor known to markedly alter expression of major transporters and enzymes in the liver. However, its effects toward organic anion transporting polypeptides (OATP) 1B1 and 1B3 remain poorly characterized. Therefore, the present study was aimed at determining the effects of chenodeoxycholic acid (CDCA), a naturally occurring FXR agonist, on OATP1B expression in cynomolgus monkeys. Multiple administrations of 50 and 100 mg/kg of CDCA were first shown to significantly repress mRNA expression of SLCO1B1/3 approximately 60% to 80% in monkey livers. It also suppressed cytochrome P450 (CYP)7A1-mRNA and induced OSTα/ß-mRNA, which are well known targets of FXR and determinants of bile acid homeostasis. CDCA concomitantly decreased OATP1B protein abundance by approximately 60% in monkey liver. In contrast, multiple doses of 15 mg/kg rifampin (RIF), a pregnane X receptor agonist, had no effect on hepatic OATP1B protein, although it induced the intestinal P-glycoprotein and MR2 proteins by ∼2-fold. Moreover, multiple doses of CDCA resulted in a steady ∼2- to 10-fold increase of the OATP1B biomarkers coproporphyrins (CPs) in the plasma samples collected prior to each CDCA dose. Additionally, 3.4- to 11.2-fold increases of CPI and CPIII areas under the curve were observed after multiple administrations compared with the single dose and vehicle administration dosing groups. Taken together, these data suggest that CDCA represses the expression of OATP1B1 and OATP1B3 in monkeys. Further investigation of OATP1B downregulation by FXR in humans is warranted, as such downregulation effects may be involved in bile acid homeostasis and potential drug interactions in man. SIGNIFICANCE STATEMENT: Using gene expression and proteomics tools, as well as endogenous biomarker data, for the first time, we have demonstrated that OATP1B expression was suppressed and its activity was reduced in the cynomolgus monkeys following oral administration of 50 and 100 mg/kg/day of chenodeoxycholic acid (CDCA), a Farnesoid X receptor agonist, for 8 days. These results lead to a better understanding of OATP1B downregulation by CDCA and its role on bile acid and drug disposition.

Isotretinoin and its Metabolites Alter mRNA of Multiple Enzyme and Transporter Genes In Vitro, but Downregulation of Organic Anion Transporting Polypeptide Does Not Translate to the Clinic.

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

Quantitative prediction of OATP1B-mediated drug-drug interactions using endogenous biomarker coproporphyrin I.

Evaluation of the organic anion transporting polypeptide (OATP) 1B-mediated drug-drug interaction (DDI) potential is important for drug development. The focus of this study was coproporphyrin I (CP-I), an endogenous OATP1B biomarker.We investigated a new approach to OATP1B-mediated DDI prediction based on the mechanistic static pharmacokinetics (MSPK) model.The ratio of the area under the plasma concentration-time curve (AUCR) with and without co-administration of rifampicin (a typical OATP1B inhibitor) was found for CP-I and OATP1B substrate, respectively, and was then used to derive the correlation curve equation. The AUCR with and without co-administration of another OATP1B inhibitor than rifampicin was then predicted for the OATP1B substrates by substituting the AUCR of CP-I in the correlation curve equation to verify the predictability of the AUCR of the OATP1B substrates.The derived correlation curve equation between CP-I and the OATP1B substrates of the AUCRs with and without co-administration of rifampicin matched the observed AUCRs well. Regarding pitavastatin, rosuvastatin, and pravastatin, 92.9% of the predicted AUCR values were within a two-fold range of the observed values, indicating that this approach may be a good way to quantitatively predict DDI potential.

Evaluation of Ipatasertib Interactions with Itraconazole and Coproporphyrin I and III in a Single Drug Interaction Study in Healthy Subjects.

Ipatasertib is a pan-AKT inhibitor in development for the treatment of cancer. Ipatasertib was metabolized by CYP3A4 to its major metabolite, M1 (G-037720), and was a P-gp substrate and OATP1B1/1B3 inhibitor in vitro. A phase I drug-drug interaction (DDI) study (n = 15) was conducted in healthy subjects to evaluate the effect of itraconazole (200-mg solution QD, 4 days), a strong CYP3A4 and P-gp inhibitor, on pharmacokinetics of ipatasertib (100-mg single dose). Itraconazole increased the Cmax and AUC0 -∞ of ipatasertib by 2.3- and 5.5-fold, respectively, increased the half-life by 53%, and delayed the tmax by 1 hour. The Cmax and AUC0-72h of its metabolite M1 (G-037720) reduced by 91% and 68%, respectively. This study confirmed that CYP3A4 plays a major role in ipatasertib clearance. Furthermore, the interaction of ipatasertib with coproporphyrin (CP) I and CPIII, the two endogenous substrates of OATP1B1/1B3, was evaluated in this study. CPI and CPIII plasma levels were unchanged in the presence of ipatasertib, both at exposures of 100 mg and at higher exposures in combination with itraconazole. This indicated no in vivo inhibition of OATP1B1/1B3 by ipatasertib. Additionally, it was shown that CPI and CPIII were not P-gp substrates in vitro, and itraconazole had no effect on CPI and CPIII concentrations in vivo. The latter is an important finding because it will simplify interpretation of future DDI studies using CPI/CPIII as OATP1B1/1B3 biomarkers. SIGNIFICANCE STATEMENT: This drug-drug interaction study in healthy volunteers demonstrated that CYP3A4 plays a major role in ipatasertib clearance, and that ipatasertib is not an organic anion transporting polypeptide 1B1/1B3 inhibitor. Furthermore, it was demonstrated that itraconazole, an inhibitor of CYP3A4 and several transporters, did not affect CPI/CPIII levels in vivo. This increases the understanding and application of these endogenous substrates as well as itraconazole in complex drug interaction studies.

Transporter Activity Changes in Nonalcoholic Steatohepatitis: Assessment with Plasma Coproporphyrin I and III.

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.

Absence of OATP1B (Organic Anion-Transporting Polypeptide) Induction by Rifampin in Cynomolgus Monkeys: Determination Using the Endogenous OATP1B Marker Coproporphyrin and Tissue Gene Expression.

Organic anion-transporting polypeptide (OATP) 1B induction is an evolving mechanism of drug disposition and interaction. However, there are contradictory reports describing OATP1B expression in hepatocytes and liver biopsies after administration of an inducer. This study investigated the in vivo effects of the common inducer rifampin (RIF) on the activity and expression of cynomolgus monkey OATP1B1 and OATP1B3 transporters, which are structurally and functionally similar their human OATP1B counterparts. Multiple doses of oral RIF (15 mg/kg) resulted in a steady 3.9-fold increase of CYP3A biomarker, 4ß-hydroxycholesterol (4ßHC), in the plasma samples collected before each RIF dose during the treatment period (i.e., predose). In contrast, the predose plasma levels of OATP1B biomarkers coproporphyrin (CP) I and CPIII did not change when compared with RIF treatment. The trough concentration, area under plasma concentration-time curve (AUC), and half-life of RIF decreased markedly during RIF treatment, suggesting that RIF induced its own clearance. Consequently, RIF treatment increased CPI and CPIII AUCs substantially after a single administration and, to a lesser extent, after multiple administrations compared with preadministration AUCs. In addition, OATP1B1 and OATP1B3 mRNA expressions were not modulated by RIF treatment (0.85-1.3-fold), whereas CYP3A8 expression was increased 3.7-5.0-fold, which correlated well with the predose levels of CP and 4ßHC. Rifampin treatment showed 2.0-3.3-fold increases in P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) expression in the small intestine. Collectively, these findings indicate that monkey OATP1B and OATP1B3 are not induced by RIF, and further investigation of OATP1B induction by RIF and other nuclear receptor activators in humans is warranted. SIGNIFICANCE STATEMENT: In this study, combined endogenous biomarker and gene expression data suggested that RIF did not induce OATP1B in cynomolgus monkeys. For the first time, the study determines transporter gene expression in the nonhuman primate liver, gut, and kidney tissues after administration of RIF for 7 days, leading to a better understanding of the induction of OATP1B and other major drug transporters. Finally, it provides evidence to strengthen the claim that coproporphyrin is a suitable endogenous probe of OATP1B activity.

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.

Absorption and Disposition of Coproporphyrin I (CPI) in Cynomolgus Monkeys and Mice: Pharmacokinetic Evidence to Support the Use of CPI to Inform the Potential for Organic Anion-Transporting Polypeptide Inhibition.

Despite a recent expansion in the recognition of the potential utility of coproporphyrin (CP) as an endogenous biomarker of organic anion-transporting polypeptide (OATP) 1B activity, there have been few detailed studies of CP's pharmacokinetic behavior and an overall poor understanding of its pharmacokinetic fate from tissues and excretion. Here, we describe the pharmacokinetics of octadeuterium-labeled coproporphyrin I (CPI-d8) in cynomolgus monkeys following oral and intravenous administration. CPI-d8 has a half-life and bioavailability of 7.6 hours and 3.2%, respectively. Cynomolgus monkeys received oral cyclosporin A (CsA) at 4, 20, and 100 mg/kg which yielded maximum blood concentrations (C max) and area under the plasma concentration-time curve (AUC) values of 0.19, 2.5, and 3.8 µM, and 2.7, 10.5, and 26.6 µM·h, respectively. The apparent CsA-dose dependent increase in the AUC ratio of CPI-d8 (1.8, 6.2, and 10.5), CPI (1.1, 1.4, and 4.4), and CPIII (1.1, 1.8, and 4.6) at 4, 20, and 100 mg, respectively. In contrast, the plasma concentrations of CPI and CPIII were generally not affected by intravenous administration of the renal organic anion and cation transporter inhibitors (probenecid and pyrimethamine, respectively). In addition, tritium-labeled coproporphyrin I ([3H]CPI) showed specific and rapid distribution to the liver, intestine, and kidney after an intravenous dose in mice using quantitative whole-body autoradiography. Rifampin markedly reduced the liver and intestinal uptake of [3H]CPI while increasing the kidney uptake. Taken together, these results suggest that hepatic OATP considerably affects the disposition of CPI in animal models, indicating CPI is a sensitive and selective endogenous biomarker of OATP inhibition. SIGNIFICANCE STATEMENT: This study demonstrated that coproporphyrin I (CPI) has favorable oral absorption, distribution, and elimination profiles in monkeys and mice as an endogenous biomarker. It also demonstrated its sensitivity and selectivity as a probe of organic anion-transporting polypeptide (OATP) 1B activity. The study reports, for the first time, in vivo pharmacokinetics, tissue distribution, sensitivity, and selectivity of CPI as an OATP1B endogenous biomarker in animals. The data provide preclinical support for exploration of its utility as a sensitive and selective circulating OATP biomarker in humans.

A Unique Combination of Two Different Quorum Sensing Systems in the ß-Rhizobium Cupriavidus taiwanensis.

Cupriavidus taiwanensis LMG19424, a ß-rhizobial symbiont of Mimosa pudica, harbors phc and tqs quorum sensing (QS), which are the homologous cell-cell communication systems previously identified from the plant pathogen Ralstonia solanacearum and the human pathogen Vibrio cholerae, respectively. However, there has been no experimental evidence reported that these QS systems function in C. taiwanensis LMG19424. We identified (R)-methyl 3-hydroxymyristate (3-OH MAME) and (S)-3-hydroxypentadecan-4-one (C15-AHK) as phc and tqs QS signals, respectively, and characterized these QS systems. The expression of the signal synthase gene phcB and tqsA in E. coli BL21(DE3) resulted in the high production of 3-OH MAME and C15-AHK, respectively. Their structures were elucidated by comparison of EI-MS data and GC/chiral LC retention times with synthetic standards. The deletion of phcB reduced cell motility and increased biofilm formation, and the double deletion of phcB/tqsA caused the accumulation of the metal chelator coproporphyrin III in its mutant culture. Although the deletion of phcB and tqsA slightly reduced its ability to nodulate on aseptically grown seedlings of M. pudica, there was no significant difference in nodule formation between LMG19424 and its QS mutants when commercial soils were used. Taken together, this is the first example of the simultaneous production of 3-OH MAME/C15-AHK as QS signals in a bacterial species, and the importance of the phc/tqs QS systems in the saprophytic stage of C. taiwanensis LMG19424 is suggested.

Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Organic anion transporting polypeptide 2B1 (OATP2B1), an expanded substrate profile, does it align with OATP2B1's hypothesized function?

An expanded view of the substrate landscape of organic anion transporting polypeptide (OATP) 2B1 was pursued with the goal of understanding if the identification of novel in vitro substrates could shed additional light on the impact of OATP2B1 on intestinal absorption and brain penetration.To examine this hypothesis, a series of experiments measured the cellular accumulation of a diverse array of compounds. Representative angiotensin II receptor blockers (ARBs) and other compounds of interest were subsequently investigated for inhibition, time dependence, and kinetics.The study identified ARBs as a class of OATP2B1 substrates and found balsalazide, olsalzine, and gavestinel to be novel substrates of OATP2B1 too. Some compounds previously reported to be OATP2B1 substrates in the literature, aliskiren, erlotinib, montelukast, fexofenadine, and taurocholate could not be confirmed as substrates.Literature describing in vivo outcomes for OATP2B1 substrates, coproporphyrin III, ARBs, balsalazide, olsalzine, and gavestinel highlight the absence of a substantial impact of OATP2B1 on the oral absorption and/or brain penetration of OATP2B1 substrates. Suggestions of including OATP2B1 assessment as part of the drug approval process are likely premature and further mechanistic work with more robust OATP2B1 substrates, which may include some of those described here, is desirable.

Antibacterial photosensitization through activation of coproporphyrinogen oxidase.

Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of CgoX induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, CgoX activation reduces bacterial burden in murine models of SSTI. Thus, small-molecule activation of CgoX represents an effective strategy for the development of light-based antimicrobial therapies.

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.

Control of Metabolite Flux during the Final Steps of Heme b Biosynthesis in Gram-Positive Bacteria.

The pathway for assembling heme ends with a unique set of enzymes in Gram-positive bacteria. Substrates for these reactions include coproporphyrin III, Fe(II), and H2O2, which are highly reactive and toxic. Because these bacteria lack membranous compartments, we hypothesized that metabolite flux may occur via a transient protein-protein interaction between the final two pathway enzymes, coproporphyrin ferrochelatase (CpfC) and coproheme decarboxylase (ChdC). This hypothesis was tested using enzymes from the pathogen Staphylococcus aureus and a corresponding ΔchdC knockout strain. The ultraviolet-visible spectral features of coproporphyrin III served as an in vitro indicator of a protein-protein interaction. A CpfC-ChdC KD of 17 ± 7 µM was determined, consistent with transient complexation and supported by the observation that the catalytic competence of both enzymes was moderately suppressed in the stable complex. The ΔchdC S. aureus was transformed with plasmids containing single-amino acid mutants in the active site gate of ChdC. The porphyrin content and growth phenotypes of these mutants showed that K129 and Y133 promote the ChdC-CpfC interaction and revealed the importance of E120. Understanding the nature of interactions between these enzymes and those further upstream in the heme biosynthesis pathway could provide new means of specifically targeting pathogenic Gram-positive bacteria such as S. aureus.

Comprehensive Evaluation of the Utility of 20 Endogenous Molecules as Biomarkers of OATP1B Inhibition Compared with Rosuvastatin and Coproporphyrin I.

Endogenous biomarkers can be clinically relevant tools for the assessment of transporter function in vivo and corresponding drug-drug interactions (DDIs). The aim of this study was to perform systematic evaluation of plasma data obtained for 20 endogenous molecules in the same healthy subjects (n = 8-12) in the absence and presence of organic anion transporting polypeptide (OATP) inhibitor rifampicin (600 mg, single dose). The extent of rifampicin DDI magnitude [the ratio of the plasma concentration-time area under the curve (AUCR)], estimated fraction transported (fT), and baseline variability was compared across the biomarkers and relative to rosuvastatin and coproporphyrin I (CPI). Out of the 20 biomarkers investigated tetradecanedioate (TDA), hexadecanedioate (HDA), glycocholic acid, glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), and coproporphyrin III (CPIII) showed the high AUCR (2.1-8.5) and fT (0.5-0.76) values, indicative of substantial OATP1B-mediated transport. A significant positive correlation was observed between the individual GDCA and TDCA AUCRs and the magnitude of rosuvastatin-rifampicin interaction. The CPI and CPIII AUCRs were significantly correlated, but no clear trend was established with the rosuvastatin AUCR. Moderate interindividual variability (15%-62%) in baseline exposure and AUCR was observed for TDA, HDA, and CPIII. In contrast, bile acids demonstrated high interindividual variability (69%-113%) and significant decreases in baseline plasma concentrations during the first 4 hours. This comprehensive analysis in the same individuals confirms that none of the biomarkers supersede CPI in the evaluation of OATP1B-mediated DDI risk. Monitoring of CPI and GDCA/TDCA may be beneficial for dual OATP1B/sodium-taurocholate cotransporting polypeptide inhibitors with consideration of challenges associated with large inter- and intraindividual variability observed for bile acids. Benefit of monitoring combined biomarkers (CPI, one bile acid and one fatty acid) needs to be confirmed with larger data sets and against multiple OATP1B clinical probes and perpetrators.