Release mechanism and pharmacodynamics of entecavir micro spheres.

Entecavir, an effective anti-hepatitis B drug with low resistance rate, was designed as sustained-release micro spheres in our previous study. Here, we aimed to reveal the drug-release mechanism by observing the drug distribution and degradation behavior of poly (lactic-co-glycolic acid) and to investigate the pharmacodynamics of entecavir micro spheres. Raman spectroscopy was used to analyze the distribution of active pharmaceutical ingredients in the micro spheres. The results showed that there was little entecavir near the micro sphere surface. With increasing micro sphere depth, the drug distribution gradually increased and larger-size entecavir crystals were mainly distributed near the spherical center. The degradation behavior of poly (lactic-co-glycolic acid) was investigated using gel permeation chromatography. Changes in poly (lactic-co-glycolic acid) molecular weights during micro sphere degradation revealed that dissolution dominated the release process, which proved our previous research results. Pharmacodynamics studies on transgenic mice indicated that the anti-hepatitis B virus replication effect was maintained for 42 days after a single injection of entecavir micro spheres, similar to the effect of daily oral administration of entecavir tablets for 28 days. The entecavir micro spheres prepared in this study had a good anti-hepatitis B virus replication effect and it is expected to be used in anti hepatitis B virus treatment against hepatitis B virus.

Passive Influx and Ion Trapping Are More Relevant to the Cellular Accumulation of Highly Permeable Low-Molecular-Weight Acidic Drugs than Is Organic Anion Transporter 2.

Recently published work suggests that highly permeable low-molecular-weight (LMW) acidic drugs are transported by organic anion transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g., lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV)-dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in Madin-Darby canine kidney (MDCK) cells, which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines, however, demonstrated sensitivity to the mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling, rather than the result of OAT2-mediated transport. SIGNIFICANCE STATEMENT: The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low-molecular-weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g., the mitochondrial matrix) that may be confused for transporter-mediated uptake.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

An LC-MS/MS method for determination of O6 -benzylguanine and its metabolite O6 -benzyl-8-oxoguanine in human plasma.

O6 -benzylguanine (O6 BG) is an inhibitor of O6 -alkylguanine-DNA alkyltransferase (AGT). It binds to AGT by transferring its benzyl moiety to the cysteine residue at the active site of the enzyme. O6 BG synergizes the cytotoxic effects of alkylating agents by halting AGT-mediated DNA repair. O6 -benzyl-8-oxoguanine (8-oxo-O6 BG) is a metabolite of O6 BG, which is an equally potent inhibitor of AGT. In this work, we report the development and validation of an LC-MS/MS method for simultaneous determination of O6 BG and 8-oxo-O6 BG in human plasma. O6 BG and 8-oxo-O6 BG along with the analog internal standard, pCl-O6 BG, were extracted from alkalinized human plasma by liquid-liquid extraction using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 ml/min. Quantification was performed using multiple-reaction-monitoring mode with positive ion-spray ionization. The linear calibration ranges of the method for O6 BG and 8-oxo-O6 BG were 1.25-250 ng/ml and 5.00-1.00 × 103 ng/ml, respectively, with acceptable assay accuracy, precision, recovery and matrix factor. This method was applied to the measurement of O6 BG and 8-oxo-O6 BG in patient plasma samples from a prior phase I clinical trial.

In Situ Stimulated Raman Scattering (SRS) Microscopy Study of the Dissolution of Sustained-Release Implant Formulation.

Localized drug delivery systems (DDSs) provide therapeutic levels of drug agent while mitigating side effects of systemic delivery. These systems offer controlled release over extended periods of time making them attractive therapies. Monitoring drug dissolution is vital for developing safe and effective means of drug delivery. Currently, dissolution characterization methods are limited to bulk analysis and cannot provide dissolution kinetics at high spatial resolution. However, dissolution rates of drug particles can be heterogeneous with influences from many factors. Insights into finer spatiotemporal dynamics of single particle dissolution could potentially improve pharmacokinetic modeling of dissolution for future drug development. In this work, we demonstrate high-resolution chemical mapping of entecavir, a hepatitis B antiviral drug, embedded in a slow release poly(d,l-lactic acid) formulation with stimulated Raman scattering (SRS) microscopy. By tracking the volume change of individual micron-sized drug particles within the polymer matrix, we establish an analytical protocol for quantitatively profiling dissolution of single crystalline particles in implant formulations in an in situ manner.

Liver-Targeted Co-delivery of Entecavir and Glycyrrhetinic Acid Based on Albumin Nanoparticle To Enhance the Accumulation of Entecavir.

Hepatitis B, one of the most common contagious viral hepatitis with high infection rate, is challenging to treat. Although the treatment for hepatitis B has been improved over the years, many therapeutic drugs still have either severe adverse effects or insufficient effectiveness via systemic administration. In this study, we confirmed that glycyrrhetinic acid can enhance the accumulation of entecavir in HepaRG cell and liver. Then we constructed a novel albumin nanoparticle co-loading entecavir and glycyrrhetinic acid (ETV-GA-AN) to improve liver accumulation of entecavir and investigated its ability to deliver both drugs to liver. In vitro cellular uptake study and in vivo tissue distribution experiment showed that these negatively charged ETV-GA-AN (112 ± 2 nm in diameter) can increase the accumulation of entecavir in hepatic HepaRG cells and improve entecavir distribution in liver. We also revealed the mechanism that glycyrrhetinic acid enhances intracellular accumulation of entecavir by inhibiting the activity of specific efflux transporters. Our delivery system is the first liver-targeted albumin nanoparticle that utilizes the site-specific co-delivery strategy to delivery entecavir and glycyrrhetinic acid. As it combines high efficiency and low toxicity, it possess great potential for treating hepatitis B.

A novel in vitro allometric scaling methodology for aldehyde oxidase substrates to enable selection of appropriate species for traditional allometry.

1. Failure to predict human pharmacokinetics of aldehyde oxidase (AO) substrates using traditional allometry has been attributed to species differences in AO metabolism. 2. To identify appropriate species for predicting human in vivo clearance by single-species scaling (SSS) or multispecies allometry (MA), we scaled in vitro intrinsic clearance (CLint) of five AO substrates obtained from hepatic S9 of mouse, rat, guinea pig, monkey and minipig to human in vitro CLint. 3. When predicting human in vitro CLint, average absolute fold-error was ≤2.0 by SSS with monkey, minipig and guinea pig (rat/mouse >3.0) and was <3.0 by most MA species combinations (including rat/mouse combinations). 4. Interspecies variables, including fraction metabolized by AO (Fm,AO) and hepatic extraction ratios (E) were estimated in vitro. SSS prediction fold-errors correlated with the animal:human ratio of E (r2 = 0.6488), but not Fm,AO (r2 = 0.0051). 5. Using plasma clearance (CLp) from the literature, SSS with monkey was superior to rat or mouse at predicting human CLp of BIBX1382 and zoniporide, consistent with in vitro SSS assessments. 6. Evaluation of in vitro allometry, Fm,AO and E may prove useful to guide selection of suitable species for traditional allometry and prediction of human pharmacokinetics of AO substrates.

Simple and rapid UPLC-MS/MS method for quantification of entecavir in human plasma: Application to a bioequivalence study
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A simple and fast ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated to determine entecavir in human plasma with the stable isotopically labeled internal standard entecavir-13C215N. Samples (100 µL each) were pretreated by protein precipitation with methanol, and then separated on an ACQUITY UPLC BEH C18 analytical column (2.1 × 50 mm, 1.7 µm) with a simple isocratic elution. The detection was operated by a positive ionization electrospray mass spectrometry in multiple reaction monitoring mode. The method had a short chromatographic run time of 2 minutes, and obtained sharp peaks of entecavir and the internal standard. Good linearity was found within 0.1 - 20 ng/mL. The intra- and inter-day precision and accuracy met the acceptance criteria, and no matrix effect was observed. This method was successfully applied in a bioequivalence study of two kinds of entecavir tablets in healthy Chinese volunteers. And the results showed that no significant differences were found between the test and reference preparations in pharmacokinetic parameters (p > 0.05) by ANOVA. The 90% confidence intervals for the geometric mean ratios (test/reference) of Cmax, AUC0-tlast, and AUC0-∞ fell within the bioequivalence acceptance criteria (80 - 125%). No significant difference was found in tmax between the two preparations. The two one-sided t-tests showed that these two products were bioequivalent.
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Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir.

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na+ free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.

Multiple Drug Transporters Are Involved in Renal Secretion of Entecavir.

Entecavir (ETV) is a first-line antiviral agent for the treatment of chronic hepatitis B virus infection. Renal excretion is the major elimination path of ETV, in which tubular secretion plays the key role. However, the secretion mechanism has not been clarified. We speculated that renal transporters mediated the secretion of ETV. Therefore, the aim of our study was to elucidate which transporters contribute to the renal disposition of ETV. Our results revealed that ETV (50 µM) remarkably reduced the accumulation of probe substrates in MDCK cells stably expressing human multidrug and toxin efflux extrusion proteins (hMATE1/2-K), organic cation transporter 2 (hOCT2), and carnitine/organic cation transporters (hOCTNs) and increased the substrate accumulation in cells transfected with multidrug resistance-associated protein 2 (hMRP2) or multidrug resistance protein 1 (hMDR1). Moreover, ETV was proved to be a substrate of the above-described transporters. In transwell studies, the transport of ETV in MDCK-hOCT2-hMATE1 showed a distinct directionality from BL (hOCT2) to AP (hMATE1), and the cellular accumulation of ETV in cells expressing hMATE1 was dramatically lower than that of the mock-treated cells. The accumulation of ETV in mouse primary renal tubular cells was obviously affected by inhibitors of organic anion transporter 1/3 (Oat1/3), Oct2, Octn1/2, and Mrp2. Therefore, the renal uptake of ETV is likely mediated by OAT1/3 and OCT2 while the efflux is mediated by MATEs, MDR1, and MRP2, and OCTN1/2 may participate in both renal secretion and reabsorption.

[Effect of Huangqi decoction on entecavir pharmacokinetics in rat plasma].

Entecavir (ETV), a guanosine nucleotide antiviral agent with activity against hepatitis B virus (HBV) and Huangqi decoction (HQD) that exerts significant therapeutic effects in liver cirrhosis are used as an effective drug combination in the treatment of liver cirrhosis with HBV. Therefore, this study was designed to assess the effect of HQD on ETV pharmacokinetics in rat plasma. Spraque-Dawley (SD) rats were randomized into single- and 7-day-dose experimental groups. The ETV and ETV-HQD groups were administered ETV and a simultaneous combination of ETV and HQD, respectively while the ETV-HQD-2h group received HQD 2 h after ETV treatment, all administered via intragastric (i.g.) gavage. A rapid, sensitive, and efficient ultra-high- performance liquid chromatography-linear trap quadrupole (UHPLC-LTQ)-Orbitrap method was developed and validated to determine ETV in rat plasma from blood samples collected at different time points following treatment. The linearity, accuracy, precision, recovery, matrix effects and stability of ETV were all satisfactory. The ETV-HQD group exhibited a decrease in the maximum plasma concentration (Cmax), and a delay in time to achieve Cmax (tmax) following single- and multi-dose administrations, and decreased area under the concentration- time curve (AUC0­t) following single dosing. ETV pharmacokinetics did not change significantly between the ETV and ETV-HQD-2h groups. In vitro everted intestinal sac models experiments indicated that HQD decreased the absorption of ETV. HQD prevented ETV from accessing the intestinal mucosa epithelial surface, thereby decreasing its absorption in rats.

Prediction of renal transporter mediated drug-drug interactions for pemetrexed using physiologically based pharmacokinetic modeling.

Pemetrexed, an anionic anticancer drug with a narrow therapeutic index, is eliminated mainly by active renal tubular secretion. The in vitro to in vivo extrapolation approach used in this work was developed to predict possible drug-drug interactions (DDIs) that may occur after coadministration of pemetrexed and nonsteroidal anti-inflammatory drugs (NSAIDs), and it included in vitro assays, risk assessment models, and physiologically based pharmacokinetic (PBPK) models. The pemetrexed transport and its inhibition parameters by several NSAIDs were quantified using HEK-PEAK cells expressing organic anion transporter (OAT) 3 or OAT4. The NSAIDs were ranked according to their DDI index, calculated as the ratio of their maximum unbound concentration in plasma over the concentration inhibiting 50% (IC50) of active pemetrexed transport. A PBPK model for ibuprofen, the NSAID with the highest DDI index, was built incorporating active renal secretion in Simcyp Simulator. The bottom-up model for pemetrexed underpredicted the clearance by 2-fold. The model we built using a scaling factor of 5.3 for the maximal uptake rate (Vmax) of OAT3, which estimated using plasma concentration profiles from patients given a 10-minute infusion of 500 mg/m(2) of pemetrexed supplemented with folic acid and vitamin B12, recovered the clinical data adequately. The observed/predicted increases in Cmax and the area under the plasma-concentration time curve (AUC0-inf) of pemetrexed when ibuprofen was coadministered were 1.1 and 1.0, respectively. The coadministration of all other NSAIDs was predicted to have no significant impact on the AUC0-inf based on their DDI indexes. The PBPK model reasonably reproduced pemetrexed concentration time profiles in cancer patients and its interaction with ibuprofen.

Dihydromethysticin from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis and differentially reduces DNA damage in A/J mice.

We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kava's hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.

A phase I, dose-escalation trial of continuous- and pulsed-dose afatinib combined with pemetrexed in patients with advanced solid tumors.

INTRODUCTION: Afatinib, an irreversible ErbB family blocker, demonstrated synergistic inhibition of epidermal growth factor receptor-mutant cell growth with pemetrexed. This phase I study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics, and antitumor activity of afatinib plus pemetrexed in patients with advanced solid tumors. METHODS: In a 3 + 3 dose-escalation design, patients were given intravenous pemetrexed (500 mg/m(2)) on day 1 of a 21-day cycle (maximum 6 cycles), combined with continuous daily oral afatinib (schedule A [SA]; starting dose 30 mg, escalation to 50 mg) or pulsed-dose daily oral afatinib (schedule B [SB]; starting dose 50 mg, escalation to 70 mg) on days 1-6 of each 21-day cycle. Primary endpoint was determination of MTD based on dose-limiting toxicities (DLTs) in cycle 1. RESULTS: Fifty-three patients were treated (SA: n = 23; SB: n = 30). Eight patients had DLTs in SA, 11 patients in SB; diarrhea and fatigue were the most common. MTD of afatinib was 30 mg in SA and 50 mg in SB. Six patients in SA and eight in SB completed 6 treatment cycles. One patient in each schedule had confirmed objective response; 18/53 patients had disease control (SA: n = 7; SB: n = 11). Most frequent drug-related adverse events were diarrhea, rash, fatigue, and stomatitis. No relevant pharmacokinetic interactions were observed. CONCLUSIONS: Continuous- or pulsed-dose afatinib combined with pemetrexed exhibited a manageable safety profile. Pulsed dosing conferred no apparent safety or dose advantage. Continuous-dose afatinib 30 mg/day with pemetrexed is recommended for phase II studies.

Preclinical analyses and phase I evaluation of LY2603618 administered in combination with pemetrexed and cisplatin in patients with advanced cancer.

LY2603618 is an inhibitor of checkpoint kinase 1 (CHK1), an important regulator of the DNA damage checkpoints. Preclinical experiments analyzed NCI-H2122 and NCI-H441 NSCLC cell lines and in vitro/in vivo models treated with pemetrexed and LY2603618 to provide rationale for evaluating this combination in a clinical setting. Combination treatment of LY2603618 with pemetrexed arrested DNA synthesis following initiation of S-phase in cells. Experiments with tumor-bearing mice administered the combination of LY2603618 and pemetrexed demonstrated a significant increase of growth inhibition of NCI-H2122 (H2122) and NCI-H441 (H441) xenograft tumors. These data informed the clinical assessment of LY2603618 in a seamless phase I/II study, which administered pemetrexed (500 mg/m(2)) and cisplatin (75 mg/m(2)) and escalating doses of LY2603618: 130-275 mg. Patients were assessed for safety, toxicity, and pharmacokinetics. In phase I, 14 patients were enrolled, and the most frequently reported adverse events included fatigue, nausea, pyrexia, neutropenia, and vomiting. No DLTs were reported at the tested doses. The systemic exposure of LY2603618 increased in a dose-dependent manner. Pharmacokinetic parameters that correlate with the maximal pharmacodynamic effect in nonclinical xenograft models were achieved at doses ≥240 mg. The pharmacokinetics of LY2603618, pemetrexed, and cisplatin were not altered when used in combination. Two patients achieved a confirmed partial response (both non-small cell lung cancer), and 8 patients had stable disease. LY2603618 administered in combination with pemetrexed and cisplatin demonstrated an acceptable safety profile. The recommended phase II dose of LY2603618 was 275 mg.

Penciclovir pharmacokinetics after oral and rectal administration of famciclovir in African elephants (Loxodonta africana) shows that effective concentrations can be achieved from rectal administration, despite lower absorption.

OBJECTIVE: To evaluate the pharmacokinetics of famciclovir and its metabolite penciclovir following a single dose administered orally and rectally in African elephants (Loxodonta africana). ANIMALS: 15 African elephants (6 males and 9 females) of various ages. METHODS: Famciclovir (15 mg/kg) was administered orally or per rectum once, with at least a three-week washout period between administrations. Blood was collected at 13 different timepoints per administration for 6 elephants, occurring between February and March 2020. An additional 9 elephants were sampled at variable timepoints per administration utilizing a sparse sampling design between July 2020 and January 2021. Plasma famciclovir and penciclovir levels were measured via HPLC and fluorescence detection. Pharmacokinetic analysis was completed in the summer of 2021 using noncompartmental analysis and nonlinear mixed-effects modeling. RESULTS: Famciclovir was not detected in any sample, suggesting complete metabolism. Key pharmacokinetic parameters for penciclovir following oral administration were time to maximum concentration (tmax; 2.12 hours), area under the concentration-versus-time curve (AUC; 33.93 µg·h/mL), maximum observed concentration (Cmax; 3.73 µg/mL), and absorption half-life (t1/2; 0.65 hours). Following rectal administration, the values were: tmax, 0.65 hours; AUC, 15.62 µg·h/mL; Cmax, 2.52 µg/mL; and absorption t1/2, 0.13 hours. CONCLUSIONS: Famciclovir was rapidly metabolized to penciclovir. Oral administration resulted in slower absorption but higher maximum plasma concentration and higher AUC compared to rectal administration. CLINICAL RELEVANCE: African elephants administered famciclovir via oral and rectal routes resulted in measurable serum penciclovir, and these findings may be utilized by clinicians treating viral infections in this species.

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-ß-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Brain efflux index to investigate the influence of active efflux on brain distribution of pemetrexed and methotrexate.

Antifolates, in particular methotrexate (MTX), have been widely used in the treatment of primary and secondary tumors of the central nervous system (CNS). Pemetrexed (PMX) is a novel antifolate that also exhibits potent antitumor activity against CNS malignancies. Studies have shown that brain distribution of both antifolates is significantly restricted, possible due to active efflux transport at the blood-brain barrier (BBB). This study characterizes the brain-to-blood transport of PMX and MTX and examines the role of several efflux transporters in brain distribution of the antifolates by use of the intracerebral microinjection technique (brain efflux index). The results from this study show that both PMX and MTX undergo saturable efflux transport across the BBB, with elimination half-lives of approximately 39 minutes and 29 minutes, respectively. Of the various efflux transporters this study investigated, multidrug resistance-associated protein 2 (Mrp2) does not play an important role in the brain distribution of the two antifolate drugs. Interestingly, breast-cancer resistance protein (Bcrp) makes a significant contribution to the brain elimination of MTX but not PMX. In addition, the brain-to-blood transport of both antifolates was inhibited by probenecid and benzylpenicillin, suggesting the involvement of organic anion transporters in the efflux of these compounds from the brain, with organic anion transporter 3 (Oat3) being a possibility. Our results suggest that one of the underlying mechanisms behind the limited brain distribution of PMX and MTX is active efflux transport processes at the BBB, including a benzylpenicillin-sensitive transport system and/or the active transporter Bcrp.

Phase Ib trial of the oral angiogenesis inhibitor pazopanib administered concurrently with pemetrexed in patients with advanced solid tumors.

INTRODUCTION: We sought to define the maximum tolerated dose (MTD) and evaluate the safety, pharmacokinetics, and preliminary clinical activity of pazopanib plus pemetrexed in patients with solid tumors. METHODS: This dose-escalation study used a standard 3 + 3 design to evaluate once daily pazopanib (400-800 mg) plus pemetrexed (400-500 mg/m(2) on Day 1 of each 21-day cycle). Eight additional patients were enrolled into an expansion cohort. RESULTS: Twenty-five patients were enrolled. Pazopanib 800 mg plus pemetrexed 500 mg/m(2) was the MTD. The most common adverse events at all dose levels included fatigue, neutropenia, diarrhea, and thrombocytopenia. The frequencies of non-hematologic adverse events were consistent with those of the individual agents. The rates of all-grade and Grade 4 hematologic toxicities (reversible neutropenia with median duration of 4 days) were higher with the combination regimen than with either monotherapy. Exploratory analyses revealed no association between the plasma levels of 3 biomarkers of vitamin B12 metabolism (cystathionine, homocysteine, and methylmalonic acid) and the risk of Grade 4 neutropenia and Grade 3 febrile neutropenia. Of 20 patients evaluated for efficacy, 2 (10 %) had a partial response. Pazopanib did not affect pemetrexed clearance, but increased pemetrexed maximal concentration by 22 %. In exploratory pharmacogenetic analyses, allelic variants of the VEGFA gene demonstrated weak correlation with development of severe neutropenia. CONCLUSIONS: Concomitant administration of pazopanib 800 mg once daily plus pemetrexed 500 mg/m(2) once every 21 days is feasible, albeit associated with a high frequency of brief, reversible neutropenia. Preliminary activity was observed in non-small-cell lung cancer.

Pharmacokinetics and efficacy of pemetrexed in patients with brain or leptomeningeal metastases.

Brain metastases (BM) and leptomeningeal metastases (LM) are devastating neurologic complications. Pemetrexed is a multi-targeted anti-folate agent approved for treatment of nonsquamous non-small cell lung cancer but has anti-tumor activity in other solid tumors. We performed two trials using pemetrexed in patients with BM and LM to assess CSF penetration and anti-tumor activity. Patients were treated with intravenous pemetrexed at doses of 500 (n = 3), 750 (n = 3), 900 (n = 12) or 1,050 mg/m(2) (n = 3) every 3 weeks. Neuro-imaging was done every 6 weeks. Matched CSF and plasma samples were obtained serially from three patients with Ommaya reservoirs; the remaining patients had a single paired collection. Twenty-one patients (15 women and six men) with median age of 50 years and median KPS of 90 were treated. Primary tumors included breast (13), lung (4), colorectal (1), endometrial (1), esophageal (1) and pinealoblastoma (1). Nine patients had prior whole brain RT and median number of prior chemotherapies was two including prior methotrexate in four patients. Median pemetrexed doses administered was three (range 1-14). Responses included one partial response, ten stable disease and ten progressive disease. Median time to progression and survival was 2.7 and 7.3 months; PFS six was 22 %. No major toxicities were seen. Pemetrexed distributed from the plasma to the CSF within 1-4 h with the resulting CSF concentrations < 5 % of plasma. Pemetrexed was tolerated in solid tumor patients with CNS metastases. Limited anti-tumor activity was seen, which might have been due to low CSF concentrations, although some patients displayed prolonged benefit.