Metabolite M1 Research Articles

Structure identification of circulating metabolites from somapacitan, a long-acting growth hormone derivative, and pharmacokinetics after single and multiple subcutaneous dosing in rats

Somapacitan is a growth hormone derivative approved for once-weekly treatment of growth hormone deficiency in adults and currently in clinical development for once-weekly dosing in children. The purpose of this study was to obtain non-clinical data from rats to support the safety evaluation of the most abundant metabolites of somapacitan in humans. The aims were to identify somapacitan metabolites and their relative proportions in rat plasma, identify the structure of abundant metabolites and measure the systemic metabolite exposure at the no-observed-adverse-effect level in the rat.After a single dose of radiolabelled somapacitan and analysis by high-performance liquid chromatography with radiochemical detection, seven somapacitan-related metabolites were detected in plasma from male rats, of which six were seen in plasma from female rats. The three most abundant metabolites (M1, M2 and M3) were structurally identified from liquid chromatography and mass spectrometry data, and a fourth metabolite (P1) was characterised from its specific retention time (lacking retention to the stationary phase) in plasma analysis with reversed-phase liquid chromatography and radiochemical detection.The metabolites were products from proteolysis of the peptide backbone in somapacitan. A deamidation product of the M1 metabolite (M1B) was also identified. Following multiple, twice-weekly dosing for 4 weeks, somapacitan was the principal plasma component up to 36 h after dosing. After 36 h, metabolites M1+M1B were the most abundant plasma components. Pharmacokinetic models were developed for somapacitan and metabolite P1 and used for steady-state assessment in the rat.Comparison of our data generated from rats with data from the parallel human study demonstrated that the most abundant metabolites were present in rats at higher levels than in humans. This study has provided non-clinical safety data that contribute to an overall safety assessment of somapacitan.

Validation of enantioseparation and quantitation of an active metabolite of abrocitinib in human plasma.

Aims: A chiral HPLC-MS/MS method for quantitation of an active metabolite (M2) of abrocitinib was validated in human plasma. Methods: Protein precipitation extraction and normal phase LC with baseline separation of five analytes (abrocitinib; isomeric metabolites M1, M2, M3 and M4) were achieved followed by mass spectrometric quantitation of M2 using positive-mode APCI. Results: With a 5-5000ng/ml assay range using 100μl K2EDTA aliquot, the assay provided short (17-min) runtime and robust separation up to approximately 330 injections on one column. Interday and intraday accuracy ranged from -6.80% to 13.4%; between-day and within-day precision was ≤10.4%. Conclusion: The method was used in multiple clinical studies, with excellent run passing rate and incurred sample reproducibility.

Panaxadiol as a major metabolite of AD-1 can significantly inhibit the proliferation and migration of breast cancer cells: In vitro and in vivo study

Previous studies have shown that 20 (R)-25-methoxyl-dammarane-3β, 12β, 20 triol (AD-1) can inhibit various cancer cell lines. This study aimed to explore the effect and mechanism of AD-1 metabolite M2 (Panaxadiol; PD) on breast cancer cells of nude mice. Five AD-1 metabolites were isolated and identified using various chromatographic techniques. PD was the main component. In vitro results showed that PD could inhibit the proliferation and migration of MDA-MB-231 cells by inducing G1-phase arrest. In addition, PD down-regulated the expression of Cyclin D1, cdk2, cdk4, cdk6, P-p38, and MMP9, and up-regulated p21 and p27. In vivo results showed that PD could effectively reduce the volume, weight, and migration of breast cancer Transcriptomics analyzed 491 differentially expressed genes by GO and KEGG enrichment. RT-PCR verification confirmed that the significant down-regulation of MMP9 was consistent with transcriptomics results. In further research showed that PD regulated the protein expression of P-p38 and MMP9 in MAPK pathway. In summary, in vivo and in vitro studies showed that PD significantly inhibit the occurrence and development of breast cancer, possibly through the MAPK pathway.

Pharmacometric analyses of alectinib to facilitate approval of the optimal dose for the first-line treatment of anaplastic lymphoma kinase-positive non-small cell lung cancer.

Alectinib is an anaplastic lymphoma kinase (ALK) inhibitor approved for treatment of ALK‐positive non‐small cell lung cancer. Population pharmacokinetic (PK) models were developed for alectinib and its major active metabolite M4 using phase I/II PK data in crizotinib‐failed patients (N = 138). The PK profiles were best described by two separate models with similar structure for both entities: open one‐compartment models with sequential zero/first‐order input and first‐order elimination rate. Body weight with fixed allometric scaling factor on clearance and volume of both entities was the only significant covariate. Bayesian feedback analyses of the PK data collected from Japanese and global treatment‐naïve patients in phase III studies (N = 334) confirmed the body weight effect. Landmark Cox proportional hazards analyses of progression‐free survival in treatment‐naïve patients identified the average molar concentrations of both entities alectinib and M4 during the first 6 weeks of treatment as a significant covariate, with an optimal response achieved for concentrations above 1040 nmol/L. With 600 mg twice daily (b.i.d.), 92% of global patients are above this threshold concentration, compared with only 43% of patients with 300 mg b.i.d. In Japan, where the body weight distribution is lower, the approved 300 mg b.i.d. dose brings about 70% of Japanese patients above this threshold. Logistic regression analyses found no significant relationship between the combined alectinib–M4 molar concentration and first occurrence of adverse events. These pharmacometric results were used to expedite and facilitate regulatory approvals of 600 mg b.i.d. for first‐line ALK‐positive NSCLC in the United States and European Union in 2017 and in China in 2018.

Physiologically-Based Pharmacokinetic Modelling of Entrectinib Parent and Active Metabolite to Support Regulatory Decision-Making.

Entrectinib is a selective inhibitor of ROS1/TRK/ALK kinases, recently approved for oncology indications. Entrectinib is predominantly cleared by cytochrome P450 (CYP)3A4, and modulation of CYP3A enzyme activity profoundly alters the pharmacokinetics of both entrectinib and its active metabolite M5. We describe development of a combined physiologicallybased pharmacokinetic (PBPK) model for entrectinib and M5 to support dosing recommendations when entrectinib is co-administered with CYP3A4 inhibitors or inducers. A PBPK model was established in Simcyp® Simulator. The initial model based on in vitro-in vivo extrapolation was refined using sensitivity analysis and non-linear mixed effects modeling to optimize parameter estimates and to improve model fit to data from a clinical drug-drug interaction study with the strong CYP3A4 inhibitor, itraconazole. The model was subsequently qualified against clinical data, and the final qualified model used to simulate the effects of moderate to strong CYP3A4 inhibitors and inducers on entrectinib and M5 pharmacokinetics. The final model showed good predictive performance for entrectinib and M5, meeting commonly used predictive performance acceptance criteria in each case. The model predicted that co-administration of various moderate CYP3A4 inhibitors (verapamil, erythromycin, clarithromycin, fluconazole, and diltiazem) would result in an average increase in entrectinib exposure between 2.2- and 3.1-fold, with corresponding average increases for M5 of approximately 2-fold. Co-administration of moderate CYP3A4 inducers (efavirenz, carbamazepine, phenytoin) was predicted to result in an average decrease in entrectinib exposure between 45 and 79%, with corresponding average decreases for M5 of approximately 50%. The model simulations were used to derive dosing recommendations for co-administering entrectinib with CYP3A4 inhibitors or inducers. PBPK modeling has been used in lieu of clinical studies to enable regulatory decision-making.

Insight into the practical models for prediciting the essential role of the cytochrome P450-mediated biotransformation in emodin-associated hepatotoxicity

Emodin is widely present in Chinese herbs with broad application prospects, however, the conflicting reports of its hepatotoxicity have created a concern. It was therefore aimed to develop practical models to elucidate the outcome of CYP450 biotransformation on emodin. HepG2 and rat liver microsomes (RLM) coculture system was first utilized for prediction. It was found that emodin (35 μM)-mediated cytotoxicity was alleviated only when the cofactor of CYP450 NADPH (1 mM) was present. Similarly, both the pan-CYP450 inhibitor 1-aminobenzotriazole (ABT) (2 mM) and the heat-inactivated liver microsomes completely abolished the protective effect of RLM (0.75 mg/mL). Consistently, ABT significantly increased the toxicity of emodin in primary rat liver cells. Along similar lines, only the monohydroxylation metabolite M3 that accounted for neglectable amount of the whole metabolites showed similar toxicity to emodin, both M1 and M2 exhibited far less toxcity than emodin in THLE-2 cells. In vivo study further supported that ABT (50 mg/kg, s.c.) aggravated the hepatotoxicity of emodin (80 mg/kg, i.p.) on mice, as emodin treatment only mediated slight increase of liver index and histological score likely due to the metabolic detoxication of emodin, whereas ABT co-administration resulted in severe liver injury as reflected by the dramatic increase of the liver index value, serum ALT and AST levels, and histopathological score. Moreover, it was explored that ROS generation together with the electrophilicity of emodin contributed to its hepatotoxicity. These findings not only provided a clear evidence of the metabolic detoxification of emodin, but also shed a light on the hepatotoxic mechanisms of emodin, which would lay a solid foundation for the rational application of emodin in the future.

UPLC‐PDA coupled HR‐TOF ESI/MS 2 ‐based identification of derivatives produced by whole‐cell biotransformation of epothilone A using Nocardia sp. CS692 and a cytochrome P450 overexpressing strain

Epothilone A, a microtubule-stabilizing agent used as therapeutics for the treatment of cancers, was biotransformed into three metabolites using Nocardia sp. CS692 and recombinant Nocardia overexpressing a cytochrome P450 from Streptomyces venezuelae (PikC). Among three metabolites produced in the biotransformation reaction mixtures, ESI/MS2 analysis predicted two metabolites (M1 and M2) as novel hydroxylated derivatives (M1 is hydroxylated at the C-8 position and M2 is hydroxylated at C-10 position), each with an opened-epoxide ring in their structure. Interestingly, metabolite M3 lacks an epoxide ring and is known as deoxyepothilone A, which is also called epothilone C. Metabolite M1 was produced only in PikC overexpressing strain. The endogenous enzymes of Nocardia sp. catalyzed hydroxylation of epothilone A to produce metabolite M2 and removed epoxide ring to produce metabolite M3. All the metabolites were identified based on UV-vis analysis and rigorous ESI/MS2 fragmentation based on epothilone A standard. The newly produced metabolites are anticipated to display novel cytotoxic effects and could be subjects of further pharmacological studies.

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite.It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Relationship between Plasma and Intracellular Concentrations of Bedaquiline and Its M2 Metabolite in South African Patients with Rifampin-Resistant Tuberculosis.

ABSTRACTBedaquiline is recommended for the treatment of all patients with rifampin-resistant tuberculosis (RR-TB). Bedaquiline accumulates within cells, but its intracellular pharmacokinetics have not been characterized, which may have implications for dose optimization. We developed a novel assay using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the intracellular concentrations of bedaquiline and its primary metabolite M2 in patients with RR-TB in South Africa. Twenty-one participants were enrolled and underwent sparse sampling of plasma and peripheral blood mononuclear cells (PBMCs) at months 1, 2, and 6 of treatment and at 3 and 6 months after bedaquiline treatment completion. Intensive sampling was performed at month 2. We used noncompartmental analysis to describe plasma and intracellular exposures and a population pharmacokinetic model to explore the relationship between plasma and intracellular pharmacokinetics and the effects of key covariates. Bedaquiline concentrations from month 1 to month 6 of treatment ranged from 94.7 to 2,540 ng/ml in plasma and 16.2 to 5,478 ng/ml in PBMCs, and concentrations of M2 over the 6-month treatment period ranged from 34.3 to 496 ng/ml in plasma and 109.2 to 16,764 ng/ml in PBMCs. Plasma concentrations of bedaquiline were higher than those of M2, but intracellular concentrations of M2 were considerably higher than those of bedaquiline. In the pharmacokinetic modeling, we estimated a linear increase in the intracellular-plasma accumulation ratio for bedaquiline and M2, reaching maximum effect after 2 months of treatment. The typical intracellular-plasma ratios 1 and 2 months after start of treatment were 0.61 (95% confidence interval [CI]: 0.42 to 0.92) and 1.10 (95% CI: 0.74 to 1.63) for bedaquiline and 12.4 (95% CI: 8.8 to 17.8) and 22.2 (95% CI: 15.6 to 32.3) for M2. The intracellular-plasma ratios for both bedaquiline and M2 were decreased by 54% (95% CI: 24 to 72%) in HIV-positive patients compared to HIV-negative patients. Bedaquiline and M2 were detectable in PBMCs 6 months after treatment discontinuation. M2 accumulated at higher concentrations intracellularly than bedaquiline, supporting in vitro evidence that M2 is the main inducer of phospholipidosis.

Insights Into Patient Variability During Ivacaftor-Lumacaftor Therapy in Cystic Fibrosis.

Background: The advent of cystic fibrosis transmembrane conductance regulator protein (CFTR) modulators like ivacaftor have revolutionised the treatment of cystic fibrosis (CF). However, due to the plethora of variances in disease manifestations in CF, there are inherent challenges in unified responses under CFTR modulator treatment arising from variability in patient outcomes. The pharmacokinetic (PK) data available for ivacaftor-lumacaftor cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator drug combination is limited.Methods: Secondary objectives were to identify (1) patient characteristics and (2) the interactions between ivacaftor-lumacaftor responsible for interindividual variability (IIV).Results: Peak plasma concentrations (Cmax) of ivacaftor - lumacaftor were >10 fold lower than expected compared to label information. The one-way ANOVA indicated that the patient site had an effect on Cmax values of ivacaftor metabolites ivacaftor-M1, ivacaftor-M6, and lumacaftor (p < 0.001, p < 0.001, and p < 0.001, respectively). The Spearman’s rho test indicated that patient weight and age have an effect on the Cmax of lumacaftor (p = 0.003 and p < 0.001, respectively) and ivacaftor metabolite M1 (p = 0.020 and p < 0.001, respectively). Age (p < 0.001) was found to effect on Cmax of ivacaftor M6 and on Tmax of ivacaftor M1 (p = 0.026). A large impact of patient characteristics on the IIV of PK parameters Cmax and Tmax, was observed among the CF patients.Conclusion: Understanding the many sources of variability can help reduce this individual patient variability and ensure consistent patient outcomes.

Hepatotoxic Effect of Tramadol and O-desmethyltramadol in HepG2 cells and Potential role of PI3K/AKT/mTOR

: The aim of this study was to compare the in vitro cytotoxic effect of tramadol and M1 metabolite in HepG2 cell line, the underlying mechanism, and PI3K/AKT/mTOR as potential target. Concentrations representing therapeutic level for tramadol (2 µM) and M1 metabolite (0.5 µM) were used. In additions other increasing concentrations representing higher toxic levels were used (6, 10 µM for tramadol and 1.5, 2.5 µM for M1 metabolites). Cytotoxicity was assessed at 24, 48 and 72 hours. Both tramadol and M1 metabolites were able to produce cytotoxicity in dose and time dependent manner. Insignificant difference was detected between cells exposed to tramadol and M1 metabolite at therapeutic concentrations. Tramadol induced apoptotic and autophagic cell death while M1 metabolite induced apoptosis only. For PI3K/AKT/mTOR pathway, therapeutic concentration of tramadol was only able to increase phosphorylation of AKT while higher toxic concentrations were able to increase phosphorylation of whole pathway; Meanwhile, M1 metabolite was able to increase the phosphorylation of the whole pathway significantly in therapeutic and toxic concentrations. In conclusion, both tramadol and M1 are equally cytotoxic. Apoptosis and autophagy both mediate hepatic cell death. PI3K/AKT pathway is involved apoptosis induction while autophagy is regulated through mTOR independent pathway.

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

Effect of Varying Degrees of Hepatic Impairment on the Pharmacokinetics of Omecamtiv Mecarbil.

Omecamtiv mecarbil (OM) is a novel selective cardiac myosin activator under investigation for the treatment of heart failure with reduced ejection fraction. OM is primarily eliminated via metabolism mediated by multiple cytochrome P450 enzymes. This phase 1 single-dose, multicenter, open-label, nonrandomized study evaluated the pharmacokinetics (PK) of OM and major metabolites M3 and M4, safety, and tolerability following oral administration of a single dose of 25-mg MR tablet in subjects with mild (n = 6) or moderate (n = 6) hepatic impairment (according to Child-Pugh classification) versus subjects with normal hepatic function (n = 6). Relative to subjects with normal hepatic function, for subjects with mild or moderate hepatic impairment, OM AUCinf was 103.2% (90%CI, 58.0%-183.6%) and 94.8% (90%CI, 54.7%-164.1%), respectively, and OM Cmax was 126.8% (90%CI, 85.7%-187.7%) and 117.3% (90%CI, 80.7%-170.5%), respectively. Exposures to M3 were similar across groups, whereas slightly lower exposures were observed for M4 with worsening hepatic function. The OM, M3, and M4 tmax and t1/2 values were similar between groups. There were no serious adverse events (AEs) or treatment-related treatment-emergent AEs. Overall, OM, M3, and M4 PK were not meaningfully affected by mild or moderate hepatic impairment, suggesting the same dosing strategy can be used in subjects with mild or moderate hepatic impairment.

A rapid RP-HPLC method for the simultaneous estimation of Ivacaftor and Tezacaftor and in silico study of their metabolitic products

BackgroundThis study was designed to develop a reliable method for estimation of Ivacaftor and Tezacaftor in pure and its pharmaceutical dosage form by RP-HPLC in human plasma. Molecular docking studies were carried out and the results were visualized using PyMol and Discovery studio visualizer (Discovery studio visualizer ver. 2.5). The pharmacokinetic properties such as Swiss ADME and pKCSM of the Ivacaftor and its metabolites Ivacaftor M1, M6 and Tezacaftor and metabolites Tezacaftor M1, M2 were predicted. In admetSAR, web-based query tools incorporating a molecular built-in interface enable the database to be queried by SMILES.ResultsA simple, linear, precise, and accurate RP-HPLC method was developed and validated for the determination of Ivacaftor (IVA) and Tezacaftor (TEZ) in human plasma. Chromatographic separation was achieved isocratically on Inspire C18, (4.6 × 250 mm, 5 μm) column at 30 °C. Mobile phase consisting of methanol and 0.05% formic acid in ratio of 95:5 with flow rate of 1 mL/min with injection volume 20 μl detector used is PDA at 235 nm. The developed method was validated according to ICH guidelines and found to be linearity range was found to be for TEZ (10–50 μg/mL) and IVA (15–75 μg/mL). IVA and TEZ drugs and its metabolites were retrieved from the PubChem database and the 2D chemical structures were generated from SMILES notation by using the Chemsketch Software. The structure was viewed using Swiss-PDB Viewer to form a better understanding of the molecule for toxicity and biological activity prediction.ConclusionThe results obtained by the proposed method from validation parameters and from assay confirmed that the determination of Tezacaftor (TEZ) and Ivacaftor (IVA) in their combined dosage form in human plasma was sensitive and selective method. In silico study has revealed that IVA and its metabolites IVA M1, IVA M6 are according to Lipinski rule. The oral bioactivity of IVA was found to be more when compared to its metabolites (Molinspiration) and TEZ and its metabolites TEZ M1, TEZ M2 even though they have the molecular weight > 500, but all other parameters from Molinspiration revealed better oral bioactivity of TEZ M2. Validation of the developed isocratic RP-HPLC procedure revealed that, regardless of how the sample was purified, the method was characterized by good linearity, sensitivity, reproducibility, specificity, and low values of LOD (0.090 μg/mL) and LOQ (0.275 μg/mL). From the in silico docking results, it is quite evident that metabolites of TEZ and IVA have the great potential against cystic fibrosis.

Toward New Transmission-Blocking Combination Therapies: Pharmacokinetics of 10-Amino-Artemisinins and 11-Aza-Artemisinin and Comparison with Dihydroartemisinin and Artemether.

ABSTRACTAs artemisinin combination therapies (ACTs) are compromised by resistance, we are evaluating triple combination therapies (TACTs) comprising an amino-artemisinin, a redox drug, and a third drug with a different mode of action. Thus, here we briefly review efficacy data on artemisone, artemiside, other amino-artemisinins, and 11-aza-artemisinin and conduct absorption, distribution, and metabolism and excretion (ADME) profiling in vitro and pharmacokinetic (PK) profiling in vivo via intravenous (i.v.) and oral (p.o.) administration to mice. The sulfamide derivative has a notably long murine microsomal half-life (t1/2 > 150 min), low intrinsic liver clearance and total plasma clearance rates (CLint 189.4, CLtot 32.2 ml/min/kg), and high relative bioavailability (F = 59%). Kinetics are somewhat similar for 11-aza-artemisinin (t1/2 > 150 min, CLint = 576.9, CLtot = 75.0 ml/min/kg), although bioavailability is lower (F = 14%). In contrast, artemether is rapidly metabolized to dihydroartemisinin (DHA) (t1/2 = 17.4 min) and eliminated (CLint = 855.0, CLtot = 119.7 ml/min/kg) and has low oral bioavailability (F) of 2%. While artemisone displays low t1/2 of <10 min and high CLint of 302.1, it displays a low CLtot of 42.3 ml/min/kg and moderate bioavailability (F) of 32%. Its active metabolite M1 displays a much-improved t1/2 of >150 min and a reduced CLint of 37.4 ml/min/kg. Artemiside has t1/2 of 12.4 min, CLint of 673.9, and CLtot of 129.7 ml/kg/min, likely a reflection of its surprisingly rapid metabolism to artemisone, reported here for the first time. DHA is not formed from any amino-artemisinin. Overall, the efficacy and PK data strongly support the development of selected amino-artemisinins as components of new TACTs.

In Vitro Assessment of the Drug-Drug Interaction Potential of Verinurad and Its Metabolites as Substrates and Inhibitors of Metabolizing Enzymes and Drug Transporters.

Verinurad is a selective uric acid transporter 1 (URAT1) inhibitor in development for the treatment of chronic kidney disease and heart failure. In humans, two major acyl glucuronide metabolites have been identified: direct glucuronide M1 and N-oxide glucuronide M8. Using in vitro systems recommended by regulatory agencies, we evaluated the interactions of verinurad, M1, and M8 with major drug-metabolizing enzymes and transporters and the potential for clinically relevant drug-drug interactions (DDIs). The IC50 for inhibition of CYP2C8, CYP2C9, and CYP3A4/5 for verinurad was ≥14.5 µM, and maximum free plasma concentration (Iu,max)/IC50 was <0.02 at the anticipated therapeutic Cmax and therefore not considered a DDI risk. Verinurad was not an inducer of CYP1A2, CYP2B6, or CYP3A4/5. Verinurad was identified as a substrate of the hepatic uptake transporter organic anion-transporting polypeptide (OATP) 1B3. Since verinurad hepatic uptake involved both active and passive transport, there is a low risk of clinically relevant DDIs with OATP, and further study is warranted. Verinurad was a substrate of the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and renal transporter organic anion transporter 1 (OAT1), although it is not considered a DDI risk in vivo because of dose-proportional pharmacokinetics (P-gp and BCRP) and limited renal excretion of verinurad (OAT1). M1 and M8 were substrates of multidrug resistance-associated protein (MRP) 2 and MRP4 and inhibitors of MRP2. Apart from verinurad being a substrate of OATP1B3 in vitro, the potential for clinically relevant DDIs involving verinurad and its metabolites as victims or perpetrators of metabolizing enzymes or drug transporters is considered low. SIGNIFICANCE STATEMENT: Drug transporters and metabolizing enzymes have an important role in the absorption and disposition of a drug and its metabolites. Using in vitro systems recommended by regulatory agencies, we determined that, apart from verinurad being a substrate of organic anion-transporting polypeptide 1B3, the potential for clinically relevant drug-drug interactions involving verinurad and its metabolites M1 and M8 as victims or perpetrators of metabolizing enzymes or drug transporters is considered low.

740-P: Proposed Biosimilar Gan &amp; Lee Insulin Glargine (GL-GLA) Shows Pharmacokinetic (PK) and Pharmacodynamic (PD) Bioequivalence (BE) to U.S.- and EU-Licensed Insulin Glargine (IG)

This randomized, double-blind, three-way crossover trial (NCT04236895) compared the PK and PD properties of GL-GLA, a proposed biosimilar IG by Gan & Lee, US- and EU-licensed Lantus® (US-IG and EU-IG) in 114 male subjects with type 1 diabetes (mean±SD age 42±8 years, BMI 25.8±2.0 kg/m²). Each subject received a single 0.5 U/kg dose of either GL-GLA, EU-IG, or US-IG in each of the 3 euglycemic glucose clamp periods (ClampArt, clamp level 100 mg/dl, clamp duration 30h post-dose). PK is presented as concentrations of the mainly absorbed IG metabolite M1. GL-GLA and comparator insulins showed superimposable profiles for PK and glucose infusion rates (GIR, Figure). PK BE was demonstrated between GL-GLA and the comparator insulins as the point estimates for measured metabolite M1 were close to 100% and 90% CIs were well contained within the pre-defined similarity margins of 80-125% (Figure). Likewise, the primary PD endpoints met BE-criteria as did secondary PK/PD-endpoints including AUCM1.0-12h, AUCM1.12-24h, AUCGIR.0-12h, and AUCGIR.12‑24h. All insulins were well tolerated with similar adverse event rates (19% [GL-GLA]; 26% [US-IG]; 21% [EU-IG]) and only few events of injection site reactions per insulin. In conclusion, GL-GLA demonstrated both PK- and PD-bioequivalence to US- and EU-licensed insulin glargine formulations. Disclosure T. Heise: Advisory Panel; Self; Novo Nordisk A/S, Valbiotis, Research Support; Self; ADOCIA, Afon Technology Ltd., AstraZeneca, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestle, Neuraly, Nordic Bioscience, Novo Nordisk, Sanofi, Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk A/S. L. Plum-moerschel: None. G. Andersen: None. J. Lu: Employee; Self; Gan & Lee Pharmaceuticals. M. G. Wilson: Consultant; Self; Gan & Lee Pharmaceuticals, Employee; Spouse/Partner; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company, Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. E. Zijlstra: Speaker’s Bureau; Self; Novo Nordisk A/S. Funding Gan & Lee Pharmaceuticals

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.

Simultaneous quantification of vincristine and its major M1 metabolite from dried blood spot samples of Kenyan pediatric cancer patients by UPLC-MS/MS

Vincristine (VCR) is an integral part of chemotherapy regimens in the US and in developing countries. There is a paucity of information about its disposition and optimal therapeutic dosing. VCR is preferentially metabolized to its major M1 metabolite by the polymorphic CYP3A5 enzyme, which may be clinically significant as CYP3A5 expression varies across populations. Thus, it is important to monitor both VCR and M1 and characterize their dispositions. A previously developed HPLC-MS/MS method for VCR quantification was not sensitive enough to quantify the M1 metabolite beyond 1 h post VCR dose (not published). Establishing a highly sensitive assay is a pre-requisite to simultaneously quantify and monitor VCR and M1, which will enable characterization of drug exposure and dispositions of both analytes in a pediatric cancer population. The addition of formic acid during the extraction process enhanced M1 extraction from DBS samples. A sensitive, accurate, and precise UPLC-MS/MS assay method for the simultaneous quantification of VCR and M1 from human dried blood spots (DBS) was developed and validated. Chromatographic separation was performed on Inertsil ODS-3 C18 column (5 μm, 3.0 × 150 mm). A gradient elution of mobile phase A (methanol-0.2 % formic acid in water, 20:80 v/v) and mobile phase B (methanol-0.2 % formic acid in water, 80:20 v/v) was used with a flow rate of 0.4 mL/min and a total run time of 5 min. The analytes were ionized by electrospray ionization in the positive ion mode. The linearity range for both analytes in DBS were 0.6−100 ng/mL for VCR and 0.4−100 ng/mL for M1. The intra- and inter-day accuracies for VCR and M1 were 93.10–117.17 % and 95.88–111.21 %, respectively. While their intra- and inter-day precisions were 1.05–10.11 % and 5.78–8.91 %, respectively. The extraction recovery of VCR from DBS paper was 35.3–39.4 % and 10.4–13.4 % for M1, with no carryover observed for both analytes. This is the first analytical method to report the simultaneous quantification of VCR and M1 from human DBS. For the first time, concentrations of M1 from DBS patient samples were obtained beyond 1 h post VCR dose. The developed method was successfully employed to monitor both compounds and perform pharmacokinetic analysis in a population of Kenyan pediatric cancer patients.

A Phase 0 ‘trigger’ trial of CDK4/6 plus ERK1/2 inhibitors in recurrent glioblastoma.

2005 Background: The RB-CDK4/6 and MAPK signaling pathways are dysregulated in glioblastoma (GBM). Our recent phase 0 study of ribociclib in recurrent GBM patients suggested that CDK4/6 inhibitor monotherapy is not durable. In this ongoing, dual-drug phase 0 study (NCT04391595), we evaluate the tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of abemaciclib, a selective CDK4/6-inhibitor, plus LY3214996, a selective ERK1/2 inhibitor, in recurrent GBM patients. Methods: Adult patients eligible for this open-label, multi-center phase 0 protocol had recurrent GBM with (1) intact RB expression, (2) &gt;30% pERK expression, and (3) CDKN2A/B deletion or CDK4/6 amplification. Prior to a planned resection, patients received six days of abemaciclib (150mg BID) plus LY3214996 (200mg QD). In a Time-Escalation Arm, ten patients were assigned to 3-5 hour or 7-9 hour intervals from final drug dose to tumor removal. Tumor tissue (gadolinium [Gd]-enhancing and nonenhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. Tumor PD effects, including RB and RSK phosphorylation, were compared to matched archival or pre-treatment biopsied tissue. A PK ‘trigger’ ( i.e., unbound concentration &gt; 5x biochemical IC50) was set for each drug. Gd-nonenhancing tumor tissue exhibiting abemaciclib and LY3214996 concentrations in excess of their trigger threshold qualified patients for postoperative dual-drug therapy. Results: In this interim analysis, no dose-limiting toxicities were observed. In Gd-nonenhancing tumor regions, median unbound concentrations of abemaciclib (including its equipotent M2 and M20 metabolites) were 31.2 nM (3-5 hour cohort) and 25.1 nM (7-9 hour cohort). In the same tissue, median unbound concentrations of LY3214996 were 52.0 nM (3-5 hour cohort) and 10.2 nM (7-9 hour cohort). Tumor RB and RSK phosphorylation decreased in 6/10 and 2/10 patients, respectively. Gd-enhancing tumor proliferation (MIB-1) was decreased in 8/10 patients. 5/10 patients exceeded PK thresholds for both abemaciclib (12 nM) and LY3214996 (25 nM), thereby entering the study’s therapeutic expansion phase. Conclusion: Abemaciclib and LY3214996 achieve pharmacologically-relevant concentrations in Gd-non-enhancing GBM tissue and are associated with suppression of the RB pathway and tumor proliferation. Following 6 days of presurgical drug exposure, the Optimal Time Interval (OTI) for tissue sampling was 3-5 hours after the final drug dose. Based on this interim analysis, the trial will accrue an additional 25 patients at this OTI. Clinical trial information: NCT04391595.