Inhibitory effect of naphthoquine phosphate on Babesia gibsoni in vitro and Babesia rodhaini in vivo.

BACKGROUND: Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. METHODS: An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice. RESULTS: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 µM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. CONCLUSION: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis.

UPLC-MS/MS assay for the simultaneous determination of pyrotinib and its oxidative metabolite in rat plasma: Application to a pharmacokinetic study.

Pyrotinib is an irreversible EGFR/HER2 inhibitor that has been approved for the treatment of breast cancer. The aim of this work was to establish a quantification method for the simultaneous determination of pyrotinib and its metabolite pyrotinib-lactam in rat plasma using UPLC-MS/MS. After simple protein precipitation with acetonitrile, the analytes and internal standard (neratinib) were separated on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 µm) using a mobile phase of water containing 0.1% formic acid and acetonitrile. The detection was performed using selected reaction monitoring mode with precursor-to-product ion transitions at m/z 583.2 > 138.1 for pyrotinib, m/z 597.2 > 152.1 for pyrotinib-lactam, and m/z 557.2 > 112.1 for internal standard. The assay exhibited excellent linearity in the concentration range of 0.5-1000 ng/mL for pyrotinib and pyrotinib-lactam. The assay met the criteria of the United States Food and Drug Administration-validated bioanalytical methods and was successfully applied to a pharmacokinetic study of pyrotinib and its metabolite for the first time. Our results demonstrated that pyrotinib rapidly converted into pyrotinib-lactam, whose in vivo exposure was 21% that of pyrotinib.

The Effect of Gastric pH on the Pharmacokinetics-pharmacodynamics of Naphthoquine in Rodents, as well as in Human Predicted Using a PBPK Model.

BACKGROUND: Fixed-dose combination of artemisinin and naphthoquine (NQ) is a new artemisinin- based combination therapy for the treatment of uncomplicated Plasmodium falciparum. NQ absorption has been reported to be affected by food in humans. OBJECTIVES: The effect of gastric pH on NQ pharmacokinetics and antiplasmodial activity was investigated. METHODS: The pharmacokinetic profiles of NQ were studied in healthy rodents after an oral dose of NQ with or without gastric pH modulators, i.e., pentagastrin (stimulator) and famotidine (suppressant). The effect of gastric pH on NQ exposures in humans was predicted using a physiologically-based pharmacokinetic (PBPK) model. The effect of gastric pH on the antiplasmodial activity of NQ was evaluated in mice infected with Plasmodium yoelii. RESULTS: Neither pentagastrin nor famotidine affected NQ absorption (AUC0-t and Cmax) significantly (P > 0.05) in rodents. The predicted PK profiles of NQ in humans did not show an effect of gastric pH. Compared to pure NQ (ED90, 1.2 mg/kg), the combination with pentagastrin showed non-significantly (< 1.5-fold) higher antimalarial potency (ED90, 1.1 mg/kg). Correspondingly, the elevation of gastric pH (up to pH 5) by famotidine treatment resulted in a relatively weaker antimalarial potency for NQ (ED90, 1.4 mg/kg). Such a difference is within the acceptable range of variability in NQ pharmacokinetics and antiplasmodial activity. CONCLUSIONS: Although the food was found to significantly impact NQ pharmacokinetics, other factors except for gastric pH should account for the result, and the warning of careful use of NQ in patients with the acid-related disease is not expected to be clinically meaningful.

The gender-related variability in the pharmacokinetics and antiplasmodial activity of naphthoquine in rodents.

BACKGROUND: Naphthoquine (NQ) is a suitable partner anti-malarial for the artemisinin-based combination therapy (ACT), which is recommended to be taken orally as a single-dose regimen. The metabolism of NQ was mainly mediated by CYP2D6, which is well-known to show gender-specific differences in its expression. In spite of its clinical use, there is limited information on the pharmacokinetics of NQ, and no data are available for females. In this study, the effect of gender on the pharmacokinetics and antiplasmodial efficacy of NQ in rodents was evaluated. The underlying factors leading to the potential gender difference, i.e., plasma protein binding and metabolic clearance, were also evaluated. METHODS: The pharmacokinetic profiles of NQ were investigated in healthy male or female rats after a single oral administration of NQ. The antiplasmodial efficacy of NQ was studied in male or female mice infected with Plasmodium yoelii. The recrudescence and survival time of infected mice were also recorded after drug treatment. Plasma protein binding of NQ was determined in pooled plasma collected from male or female mice, rat or human. In vitro metabolism experiments were performed in the liver microsomes of male or female mice, rat or human. RESULTS: The results showed that the gender of rats did not affect NQ exposure (AUC0-t and Cmax) significantly (P > 0.05). However, a significant (P < 0.05) longer t1/2 was found for NQ in male rats (192.1 ± 47.7), compared with female rats (143.9 ± 27.1). Slightly higher but not significant (P > 0.05) antiplasmodial activity was found for NQ in male mice (ED90, 1.10 mg/kg) infected with P. yoelii, compared with female mice (ED90, 1.67 mg/kg). The binding rates of NQ to plasma protein were similar in males and females. There was no metabolic difference for NQ in male and female mice, rat or human liver microsomes. CONCLUSIONS: These results indicated that the pharmacokinetic profiles of NQ were similar between male and female rats, except for a longer t1/2 in male rats. The difference was not associated with plasma protein binding or hepatic metabolic clearance. Equivalent antiplasmodial activity was found for NQ in male and female mice infected with P. yoelii. This study will be helpful for the rational design of clinical trials for NQ.

Determination of Puquitinib in Human Plasma by HPLC-ESI MS/MS: Application to Pharmacokinetic Study.

BACKGROUND AND OBJECTIVE: Puquitinib mesylate (XC-302) is a new multiple-target anticancer inhibitor, which directly suppresses the activity of phosphatidylinositol 3-kinase (PI3K). This study was aimed to develop a sensitive and specific liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) method for the quantification and pharmacokinetic investigation of plasma puquitinib in cancer patients. METHODS: The analytes of human plasma were prepared by liquid-liquid extraction using methyl-t-butyl ether (MTBE). The plasma analytes were separated by HPLC on Thermo ODS Hypersil column (2.1 × 150 mm; 3 µm) at 25 °C with 5 mmol/L ammonium acetate (A)-acetonitrile (B) (30:70, v/v) as the mobile phase. RESULTS: The total run time was 3.5 min and the elution of puquitinib was at 1.38 min. The detection were analyzed by multiple reaction monitoring (MRM) mode with positive-ion electrospray ionization (ESI) interface using the respective [M + H]+ ions: m/z 318.2 → 261.1 for puquitinib and m/z 258.2 → 121.0 for the internal standard (etofesalamide). The optimized method provided a good linear relation over the concentration range of 1.00-500.00 ng/mL (r = 0.9944) for puquitinib. The intra-day and inter-day precision (relative standard deviation [RSD%]) were within 9.83%, and the intra-day and inter-day accuracy ranged from 91.05 to 103.26%. The lower limit of quantitation (LLOQ) was 1.00 ng/mL. The absolute extraction recovery was on an average of 50.43% for puquitinib and 49.3% for internal standard. In addition, the maximum plasma concentration (Cmax) of puquitinib in dosage from 50 to 800 mg/m2 in the human study showed an increased linearly (57.1-1289.2 ng/mL), which displayed that the concentrations had reached effective levels. CONCLUSIONS: The optimized method was successfully applied to the pharmacokinetic profile study in human cancer patient plasma after the oral administration of puquitinib.

A randomized, double-blind, active-control trial to evaluate the efficacy and safety of a three day course of tafenoquine monotherapy for the treatment of Plasmodium vivax malaria.

BACKGROUND: Tafenoquine is an investigational 8-aminoquinoline for the prevention of Plasmodium vivax relapse. Tafenoquine has a long half-life and the potential for more convenient dosing, compared with the currently recommended 14-day primaquine regimen. METHODS: This randomized, active-control, double-blind trial was conducted in Bangkok, Thailand. Seventy patients with microscopically confirmed P. vivax were randomized (2:1) to tafenoquine 400 mg once daily for 3 days or 2500 mg total dose chloroquine phosphate (1500 mg chloroquine base) given over 3 days plus primaquine 15 mg daily for 14 days. Patients were followed to day 120. RESULTS: Day 28 adequate clinical response rate in the per-protocol population was 93% (40/43) (90%CI 83-98%) with tafenoquine, and 100% (22/22) (90%CI 87-100%) with chloroquine/primaquine. Day 120 relapse prevention was 100% (35/35) with tafenoquine (90%CI 92-100%), and 95% (19/20) (90%CI 78-100%) with chloroquine/primaquine. Mean (SD) parasite, gametocyte and fever clearance times with tafenoquine were 82.5 h (32.3), 49.1 h (33.0), and 41.1 h (31.4) versus 40.0 h (15.7), 22.7 h (16.4), and 24.7 h (17.7) with chloroquine/primaquine, respectively. Peak methemoglobin was 1.4-25.6% (median 7.4%, mean 9.1%) in the tafenoquine arm, and 0.5-5.9% (median 1.5%, mean 1.9%) in the chloroquine/primaquine arm. There were no clinical symptoms of methemoglobinemia in any patient. DISCUSSION: Although there was no difference in efficacy in this study, the slow rate of parasite, gametocyte and fever clearance indicates that tafenoquine should not be used as monotherapy for radical cure of P. vivax malaria. Also, monotherapy increases the potential risk of resistance developing to this long-acting agent. Clinical trials of single-dose tafenoquine 300 mg combined with standard 3-day chloroquine or artemisinin-based combination therapy are ongoing. TRIAL REGISTRATION: Clinicaltrials.gov NCT01290601.

Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors, neratinib and pelitinib, with apigenin in rat plasma by UPLC-MS/MS.

Neratinib (NER) and pelitinib (PEL) are irreversible tyrosine kinase inhibitors (TKIs) that have been recently employed in cancer treatment. Apigenin (API), among other flavonoids, is known to have antioxidant, anti-proliferative, and carcinogenic effect. API can potentiate the antitumor effect of chemotherapeutic agents and/or alleviate the side effects of many anticancer agents. Since TKIs are mostly metabolized by CYP3A4 enzymes and that API could alter the enzymatic activity, potential drug interactions could be expected following their co-aministration. In the present study, a bioanalytical UPLC-MS/MS method has been developed and validated for the quantification of NER and PEL in rat plasma, using domperidone (DOM) as an internal standard. Sample preparation was carried out using solid phase extraction (SPE) with C18 cartridges with good extraction recovery of not less than 92.42% (NER) and 89.73% (PEL). Chromatographic analysis was performed on a Waters BEH C18 column with a mobile phase composed of acetonitrile and water, (70:30, v/v), each with 0.1% formic acid. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions from protonated precursor ions [M+H]+, at m/z 557.30 (NER), m/z 468.21 (PEL), and at m/z 426.27 (DOM), to selected product ions at m/z 112.05 (NER), m/z 395.22 (PEL), and at m/z 175.18 (DOM). The method was fully validated as per the FDA guidelines over the concentration range of 0.5-200ng/mL with very low lower limit of quantification (LLOQ) of 0.5ng/mL for both NER and PEL. The intra- and inter-day assay precision and accuracy were evaluated for both drugs and the calculated values of percentage relative standards deviations (%RSD) and relative errors (%Er) were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ. The applicability of the method was extended to study the possibility of drug interactions following the oral co-administration of NER/PEL with API. Thus, this study could be readily applied in therapeutic drug monitoring (TDM) of cancerous patients receiving such drug combinations.

Naphthoquine-induced Central Nervous System and Hepatic Vasculocentric Toxicity in the Beagle Dog.

Naphthoquine phosphate (NP) was considered as a partner drug with a promising antimalarial drug candidate. Here we report unexpected adverse clinical signs and microscopic findings in a canine pilot toxicology study with NP. Male and female dogs were dosed daily by oral gavage with NP at 2, 10, or 50 mg/kg/day for a maximum of 14 days. NP was not tolerated at ≥10 mg/kg/day; several animals were sacrificed in moribund condition and marked neurological clinical signs were noted at 50 mg/kg/day. The main microscopic observation was central nervous system vasculocentric inflammation (mainly lymphocytes and macrophages) in the white and gray matter of various regions of the brain at ≥2 mg/kg/day and at lower incidence in the spinal cord at ≥10 mg/kg/day. Vasculocentric microscopic changes predominantly centered on the centrilobular vein were also observed in the liver at ≥2 mg/kg/day. Females were more sensitive than males with comparable NP plasma exposure. In conclusion, under the conditions of this study, the administration of NP to dogs via daily oral gavage for up to 2 weeks was not tolerated causing moribundity, marked neurological clinical signs, and vasculocentric microscopic changes in the central nervous system and the liver.

Metabolic characterization of pyrotinib in humans by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Pyrotinib is a novel irreversible tyrosine kinase inhibitor developed for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The results of phase I clinical trial demonstrated that pyrotinib was well tolerated and exhibited potent antitumor activity. As a promising therapeutic agent for HER2-positive breast cancer, it is of great importance to investigate the biotransformation of pyrotinib in humans and identify the major enzymes involved in its metabolism during its early stage of development for safety consideration. For this purpose, a robust analytical method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was established to characterize the metabolites of pyrotinib in human plasma, feces, and urine, and identify the primary enzymes responsible for its metabolism. As a result, a total of 24 metabolites were identified, including 16 phase I metabolites resulting from dealkylation, oxidation, dehydrogenation, and carbonylation, and 8 phase II metabolites originating from cysteine and N-acetylcysteine conjugation. Pyrotinib was absorbed into blood by 1h, reached its peak level at 4h, and afterwards underwent slow elimination. The principal metabolites detected in humans (M1, M2, and M5) were products resulting from O-depicoline and pyrrolidine lactam formation, whose structures have been confirmed by the synthetic references. In addition, fecal clearance was the major route of excretion for pyrotinib. Further phenotyping experiment proved that CYP3A4 was the most active enzyme responsible for the biotransformation of pyrotinib, implying the vital necessity of the assessment of the potential CYP3A-mediated drug-drug interactions in humans. Taken together, this study provided valuable metabolic data to explicate the dynamic process of pyrotinib in humans, and important reference basis for its safety evaluation and rational clinical application. The results will also benefit the assessment of the contributions to the overall activity or toxicity from the key metabolites.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase.

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Population Pharmacokinetic and Pharmacodynamic Modeling of AZD4901 and Simulation to Support Dose Selection for the Phase 2a Study.

Significant and reversible reductions in testosterone levels were observed with AZD4901 in both preclinical and clinical testing. A comprehensive population pharmacokinetic/pharmacodynamic (PK/PD) modeling of AZD4901 concentration and testosterone relationship from 3 phase 1 studies was performed using NONMEM to support dose selection for phase 2a development. A 2-compartment model with first-order absorption and first-order elimination best described AZD4901 PK. Circadian rhythm of baseline testosterone concentrations was well described by a cosine function. An indirect response model with inhibition of testosterone production was used to link the AZD4901 concentration to testosterone response. The AZD4901 concentration to yield 50% maximum testosterone suppression (IC50) was estimated to be 230 ng/mL. Based on simulations, following 40 mg twice daily (BID) treatment, the AZD4901 steady-state trough concentration will be much higher compared to 80 mg once daily (QD). The AZD4901 concentration time above IC50 after 40 mg BID is 84% of the time of the dosing interval compared to only 49% after 80 mg QD. The mean predicted testosterone concentrations at steady state are lower and overall less variable over 24 hours for 40 mg BID dosing compared to 80 mg QD dosing. Population PK and PK/PD analyses demonstrated that AZD4901 40 mg BID is a better dosing strategy to more consistently suppress testosterone during the entire dosing interval. Consequently, 40 mg BID dosing was suggested in a phase 2a trial in females with polycystic ovary syndrome, and the trial resulted in a positive outcome as shown by significant testosterone decrease compared to placebo.

Differential cytochrome P450 2D metabolism alters tafenoquine pharmacokinetics.

Cytochrome P450 (CYP) 2D metabolism is required for the liver-stage antimalarial efficacy of the 8-aminoquinoline molecule tafenoquine in mice. This could be problematic for Plasmodium vivax radical cure, as the human CYP 2D ortholog (2D6) is highly polymorphic. Diminished CYP 2D6 enzyme activity, as in the poor-metabolizer phenotype, could compromise radical curative efficacy in humans. Despite the importance of CYP 2D metabolism for tafenoquine liver-stage efficacy, the exact role that CYP 2D metabolism plays in the metabolism and pharmacokinetics of tafenoquine and other 8-aminoquinoline molecules has not been extensively studied. In this study, a series of tafenoquine pharmacokinetic experiments were conducted in mice with different CYP 2D metabolism statuses, including wild-type (WT) (reflecting extensive metabolizers for CYP 2D6 substrates) and CYPmouse 2D knockout (KO) (reflecting poor metabolizers for CYP 2D6 substrates) mice. Plasma and liver pharmacokinetic profiles from a single 20-mg/kg of body weight dose of tafenoquine differed between the strains; however, the differences were less striking than previous results obtained for primaquine in the same model. Additionally, the presence of a 5,6-ortho-quinone tafenoquine metabolite was examined in both mouse strains. The 5,6-ortho-quinone species of tafenoquine was observed, and concentrations of the metabolite were highest in the WT extensive-metabolizer phenotype. Altogether, this study indicates that CYP 2D metabolism in mice affects tafenoquine pharmacokinetics and could have implications for human tafenoquine pharmacokinetics in polymorphic CYP 2D6 human populations.

Tafenoquine at therapeutic concentrations does not prolong Fridericia-corrected QT interval in healthy subjects.

Tafenoquine is being developed for relapse prevention in Plasmodium vivax malaria. This Phase I, single-blind, randomized, placebo- and active-controlled parallel group study investigated whether tafenoquine at supratherapeutic and therapeutic concentrations prolonged cardiac repolarization in healthy volunteers. Subjects aged 18-65 years were randomized to one of five treatment groups (n = 52 per group) to receive placebo, tafenoquine 300, 600, or 1200 mg, or moxifloxacin 400 mg (positive control). Lack of effect was demonstrated if the upper 90% CI of the change from baseline in QTcF following supratherapeutic tafenoquine 1200 mg versus placebo (ΔΔQTcF) was <10 milliseconds for all pre-defined time points. The maximum ΔΔQTcF with tafenoquine 1200 mg (n = 50) was 6.39 milliseconds (90% CI 2.85, 9.94) at 72 hours post-final dose; that is, lack of effect for prolongation of cardiac depolarization was demonstrated. Tafenoquine 300 mg (n = 48) or 600 mg (n = 52) had no effect on ΔΔQTcF. Pharmacokinetic/pharmacodynamic modeling of the tafenoquine-QTcF concentration-effect relationship demonstrated a shallow slope (0.5 ms/µg mL(-1) ) over a wide concentration range. For moxifloxacin (n = 51), maximum ΔΔQTcF was 8.52 milliseconds (90% CI 5.00, 12.04), demonstrating assay sensitivity. In this thorough QT/QTc study, tafenoquine did not have a clinically meaningful effect on cardiac repolarization.

Assessment of the prophylactic activity and pharmacokinetic profile of oral tafenoquine compared to primaquine for inhibition of liver stage malaria infections.

BACKGROUND: As anti-malarial drug resistance escalates, new safe and effective medications are necessary to prevent and treat malaria infections. The US Army is developing tafenoquine (TQ), an analogue of primaquine (PQ), which is expected to be more effective in preventing malaria in deployed military personnel. METHODS: To compare the prophylactic efficacy of TQ and PQ, a transgenic Plasmodium berghei parasite expressing the bioluminescent reporter protein luciferase was utilized to visualize and quantify parasite development in C57BL/6 albino mice treated with PQ and TQ in single or multiple regimens using a real-time in vivo imaging system (IVIS). As an additional endpoint, blood stage parasitaemia was monitored by flow cytometry. Comparative pharmacokinetic (PK) and liver distribution studies of oral and intravenous PQ and TQ were also performed. RESULTS: Mice treated orally with three doses of TQ at 5 mg/kg three doses of PQ at 25 mg/kg demonstrated no bioluminescence liver signal and no blood stage parasitaemia was observed suggesting both drugs showed 100% causal activity at the doses tested. Single dose oral treatment with 5 mg TQ or 25 mg of PQ, however, yielded different results as only TQ treatment resulted in causal prophylaxis in P. berghei sporozoite-infected mice. TQ is highly effective for causal prophylaxis in mice at a minimal curative single oral dose of 5 mg/kg, which is a five-fold improvement in potency versus PQ. PK studies of the two drugs administered orally to mice showed that the absolute bioavailability of oral TQ was 3.5-fold higher than PQ, and the AUC of oral TQ was 94-fold higher than oral PQ. The elimination half-life of oral TQ in mice was 28 times longer than PQ, and the liver tissue distribution of TQ revealed an AUC that was 188-fold higher than PQ. CONCLUSIONS: The increased drug exposure levels and longer exposure time of oral TQ in the plasma and livers of mice highlight the lead quality attributes that explain the much improved efficacy of TQ when compared to PQ.

Inhibition of keratinocyte proliferation by phospholipid-conjugates of a TLR7 ligand in a Myc-induced hyperplastic actinic keratosis model in the absence of systemic side effects.

BACKGROUND: The Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions. OBJECTIVES: To evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation. MATERIAL AND METHODS: The effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88(-/-) and Tlr7(-/-)). RESULTS: TMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases. CONCLUSION: TMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions.

Liquid chromatography-tandem mass spectrometry assay for the EGFR inhibitor pelitinib in plasma.

A bioanalytical liquid chromatography-tandem mass spectrometry assay for the tyrosine kinase inhibitor pelitinib was developed and validated in plasma. Acetonitrile containing the internal standard erlotinib was used to precipitate proteins. The extract was diluted with water and then directly injected onto the sub-2µm particle, bridged ethylsilica hybrid trifunctional bonded C18 column. A gradient consisting of 0.02% (v/v) formic acid in a methanol-water mixture was used. The ionization mode of the electrospray interface was positive and the analyte was detected by a triple quadrupole mass spectrometer in the selected reaction monitoring mode. The calibration range of the assay was 1-200ng/ml. The within day precision, the between day precision, and the accuracy were 3.5-7.4%, 4.5-8.6%, and 94.0-104.8%, respectively. The stability of the drug was sufficient under all relevant conditions. The validated assay was used to measure drug levels in wild-type FVB mice and pharmacokinetic parameters were assessed.

Therapy with TLR7 agonists induces lymphopenia: correlating pharmacology to mechanism in a mouse model.

BACKGROUND AND OBJECTIVE: Synthetic TLR7 agonists have been proposed as oral replacements for interferonα (IFNα) therapy in the treatment of hepatitis C virus infection. However, adverse effects, such as lymphopenia and cardiovascular irregularities, have been observed in the clinical following treatment with TLR7 agonists. We wished to understand and characterise the relationship between TLR7 agonism and adverse effects. METHODS: We compared responses to two prototypic TLR7 agonists (Resiquimod: R-848; and PF-04878691) in a mouse model and compared the responses to treatment with IFNα. We measured clinically relevant adverse effects such as lymphopenia and cardiovascular irregularities and related them to plasma drug levels and clinically relevant efficacy biomarkers such as the pro-inflammatory cytokine IP-10, 2'5'OAS and TLR7 receptor expression. RESULTS: By 2 h post dose all agents had induced a dose-dependent transient lymphopenia. IFNα increased heart rate immediately following dosing, persisting for 5 h, whilst PF-04878691 induced significant reductions in blood pressure. Lymphopenia co-incided with maximum plasma drug levels, raised levels of IP-10 and the auto-induction of TLR7 expression in the blood and lymph nodes. Peak levels of 2'5'OAS occurred at 24 h post-dose and only at doses which also induced lymphopenia. CONCLUSIONS: We conclude that systemic delivery of TLR7 agonists or IFNα induces similar exaggerated pharmacology, consistent with there being a narrow therapeutic window between efficacy and safety. This clinically validated mouse model will help to investigate whether more potent agonists or optimised dosing schedules, will be successful strategies for targeting TLR7 in patients.

Dried matrix spot direct analysis: evaluating the robustness of a direct elution technique for use in quantitative bioanalysis.

BACKGROUND: The surge in interest in switching from traditionally used wet plasma to dried matrix spot (DMS) sampling and analysis to support pharmaceutical drug development is due to the significant ethical, financial and data quality advantages on offer. Unfortunately these advantages do not extend to sample bioanalysis, as DMS extraction is more complex than the protein precipitation method typically used for wet plasma analysis. Direct elution techniques coupled to HPLC-MS/MS have been identified as a potential means to counter this additional complexity. RESULTS: The robustness and reproducibility of DMS HPLC-MS/MS data generated using a CAMAG DBS-MS 16 prototype automated direct elution instrument has been demonstrated to meet or exceed results obtained using a conventional manual extraction methodology. CONCLUSION: The data generated suggest that a simple and fast direct elution method of DMS samples that does not require additional sample or extract clean-up, offers sufficiently robust performance to be compatible with high-sample-throughput quantitative analysis. Further evaluation of the technique and the development of more advanced fully automated direct elution instrumentation is fully warranted.

Method of applying internal standard to dried matrix spot samples for use in quantitative bioanalysis.

A novel technique is presented that addresses the issue of how to apply internal standard (IS) to dried matrix spot (DMS) samples that allows the IS to integrate with the sample prior to extraction. The TouchSpray, a piezo electric spray system, from The Technology Partnership (TTP), was used to apply methanol containing IS to dried blood spot (DBS) samples. It is demonstrated that this method of IS application has the potential to work in practice, for use in quantitative determination of circulating exposures of pharmaceuticals in toxicokinetic and pharmacokinetic studies. Three different methods of IS application were compared: addition of IS to control blood prior to DBS sample preparation (control 1), incorporation into extraction solvent (control 2), and the novel use of TouchSpray technology (test). It is demonstrated that there was no significant difference in accuracy and precision data using these three techniques obtained using both manual extraction and direct elution.

Determination of tafenoquine in dried blood spots and plasma using LC and fluorescence detection.

BACKGROUND: The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment. RESULTS: A bioanalytical method for the determination of tafenoquine in 100 µl of capillary blood applied onto sampling paper and in 100 µl of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l. CONCLUSION: The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.