Pharmacodynamic Investigations Research Articles

Pharmacokinetic and pharmacodynamic investigation of ELND006, a novel APP-selective gamma-secretase inhibitor, on amyloid-β concentrations in the brain, CSF and plasma of multiple nonclinical species following oral administration

Sequential processing of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase produces Aβ, a peptide that is central to the pathogenesis of Alzheimer's disease. We have identified novel gamma-secretase inhibitors which demonstrate in vitro substrate selectivity towards inhibiting APP cleavage compared to Notch. ELND006 is one of these potent inhibitors and is currently being evaluated in Phase 1 human clinical trials. We present here the in vivo characterization of ELND006 after oral administration in multiple preclinical models. The potency of ELND006 for inhibiting Aβ production resulting from gamma-secretase-mediated cleavage of APP and the selectivity of ELND006 for inhibition of APP cleavage compared to Notch cleavage was determined in enzymatic and cellular assays. Aβ and compound levels were analyzed in the brain, CSF and/or plasma of FVB wildtype mice, PDAPP mice, Sprague Dawley rats and Hartley Guinea pigs after single or repeat dose oral administration of ELND006 or vehicle. Compound levels were determined by an LC-MS/MS assay. Aβ levels (1-x, x-40 or x-42) were determined by an ELISA assay. ELND006 inhibited APP and Notch cleavage with an IC50 = 0.34 nM and 5.3 nM respectively. ELND006 inhibited Aβ production and Notch signaling in cells with an IC50 = 1.1 nM and 81.6 nM respectively. After oral administration of ELND006 in multiple species, ELND006 penetrated the brain (Kp > 1) and significantly reduced brain and CSF Aβ at doses between 0.3 to 30 mg/kg. In all species tested, plasma ELND006 concentrations needed to reduce plasma Aβ were higher than those needed to reduce brain Aβ. Elevated plasma Aβ was observed under some ELND006 treatment conditions, consistent with results achieved with other gamma-secretase inhibitors. Plasma ELND006 concentrations of 8-35 ng/mL were associated with lowering of Aβ in the brain of approximately 25%. ELND006 is a potent, APP-selective gamma-secretase inhibitor and is approximately 15- to 70-fold more selective towards inhibiting gamma-secretase mediated cleavage of APP than Notch cleavage. ELND006 plasma concentrations required to reduce brain Aβ were highly consistent across species, offering an opportunity to translate these nonclinical data into a clinical setting. Phase 1 clinical trials with ELND006 are in progress.

Phase I, pharmacokinetic study of temsirolimus in combination with nelfinavir in patients with solid tumors.

2572 Background: Temsirolimus (CCI-779, TEM) is a selective inhibitor of mTOR with anticancer activity in a limited number of cancer types. A potential mechanism of drug resistance of TEM is feedback activation of the PI3k signaling pathway. Dual inhibition of PI3 kinase and mTOR may overcome this resistance. Nelfinavir (NFV), a human immunodeficiency protease inhibitor and a strong CYP3A4 inhibitor, has PI3kinase-inhibiting activity. In this phase I dose-escalation study TEM and NFV were combined (start dose of weekly 10 mg IV TEM infusion and daily 1,500 mg oral NFV in a 3-week cycle). Methods: Standard dose escalation was used in cohorts of 3-6 patients (pts). PK profiles of TEM, its active metabolite sirolimus (SIR), and NFV were obtained in each pt after the first administration and after combination of TEM and NFV (D1, D4, and D11of Cycle 1). Peripheral blood mononuclear cells and, where possible, tumor material of pts were obtained to determine the PD effects. Results: Pretreated pts received NFV at dose level 1 (1,500 mg, 7 pts) and dosel evel 0 (1,000 mg, 3 pts) in combination with 10mg TEM. Pts (6 females, 4 males, median age: 49 years [24-66], WHO 0-2, main tumor types: pancreas, glioblastoma, sarcoma, and galbladder) received up to 6 cycles. The most common reason for treatment discontinuation was disease progression. DLT was seen in 2 pts at dose level 1 (1,500 mg NFV and 10 mg TEM): grade 3 mucositis and nausea. Ulcerative mucositis was the most common toxicity (all grades) (7/10pts), then nausea (5/10 pts) and diarrhea (5/10pts). Neither CRs nor PRs were observed. TEM plasma exposure alone and in combination with NFV did not change (mean AUC0-24 1,435 vs. 1,250 nghr/mL, respectively), while SIR plasma exposure increased when TEM was combined with NFV (mean AUC0-24 678 vs. 1,050 respectively, p<0.05). NFV showed a high interpatient variability alone as well as in combination with TEM (CV= 55 and 71%, respectively). Pharmacodynamic investigations are ongoing. Conclusions: Nelfinavir 1,000 mg/day and temsirolimus 10 mg/wk can be safely combined. DLT was mucositis and nausea. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Wyeth

Pharmacokinetic and pharmacodynamic investigation of irinotecan hydrochloride in pediatric patients with recurrent or progressive solid tumors

A multicenter Phase I/II study of Irinotecan hydrochloride (CPT-11; 40-45 mg/m(2)/dose) was conducted for the treatment of refractory pediatric solid tumors. The pharmacokinetics of CPT-11 and its metabolites were characterized using both traditional noncompartmental analysis and population pharmacokinetics using NONMEM VI; pharmacokinetic pharmacodynamic relationships of SN-38 with indices of toxicity were also evaluated. 11 patients between 3 and 18 years were enrolled. Pharmacokinetic parameters and consideration of relevant covariates (performance status (PS), BSA, corrected body weight (CBW), exponent of 3/4 on weight, etc.) were evaluated. Relationships between pharmacokinetic parameters of SN-38 and percentage change from baseline in patient biochemical response data were investigated via regression analysis. CPT-11 exhibited a mean clearance (CL) of 15.31 +/- 5.95 (l/h) (13.06 +/- 3.58 (l/hr/m(2))) and AUC(0-inf) of 3547.0 +/- 1406.5 (ng x h/ml); the AUC ratio of parent CPT-11 to SN-38 was 5.0%. Based on the population pharmacokinetic analysis, decreasing PS was significantly dependent on reduction in CL of CPT-11 (p < 0.001). The final model for CPT-11 are as follows: CL (l/h) = 1.31 x CBW(0.75) (omegaCL = 21.7%), Vss (l) = 2.66 x CBW (omegaVss = 21.2%), Vc (l) = 1.13 x CBW, inter-compartment CL (l/h) = 0.257 x CBW(0.75). Percentage changes of leucocyte and neutrophil count within a first month treatment were significantly correlated with Cmax of SN-38 (r = 0.78 and r = 0.74) and AUC0-2 of SN-38 (r = 0.73 and r = 0.73). Pharmacokinetic parameters were similar to results published in several past reports. An allometric scaling of CBW(0.75) would seem to provide a good index of dosage requirement of CPT-11 in pediatric patients.

Spectroscopic measurements with dispersion encoded full range frequency domain optical coherence tomography in single- and multilayered non- scattering phantoms

In this study, depth resolved measurements of absorption profiles in the wavelength range of 800 nm with a bandwidth of 140 nm are demonstrated using high speed spectroscopic frequency domain OCT(SOCT) and a full range reconstruction algorithm (dispersion encoded full range, DEFR). The feasibility of the algorithm for SOCT is tested in simulation and experiment. With proper calibration, SOCT with DEFR is able to extract absolute, depth resolved absorption profiles over the whole wavelength range at once without the need of tuning and performing measurements at single wavelengths sequentially. The superior acquisition speed and better phase stability in frequency domain as compared to time domain results in a better reproducibility and practicability for spectroscopic measurements. In addition, high acquisition speed in excess of 20 kHz allows to measure absorption dynamics with 50 micros time resolution, which might be useful for the investigation of pharmacokinetics or pharmacodynamics. SOCT of approximately 600 microm thick single- and multilayered, weakly scattering phantoms with varying absorption in the range of 5-80 cm(-1), equivalent to blood absorption in capillaries, is presented. SOCT measurements are compared with those using a spectrometer in transmission mode. For Indocyanine Green (ICG), a dynamic absorption measurements are demonstrated.

Morphine Glucuronidation in Preterm Neonates, Infants and Children Younger than 3 Years

A considerable amount of drug use in children is still unlicensed or off-label. In order to derive rational dosing schemes, the influence of aging on glucuronidation capacity in newborns, including preterms, infants and children under the age of 3 years was studied using morphine and its major metabolites as a model drug. A population pharmacokinetic model was developed with the nonlinear mixed-effects modelling software NONMEM V, on the basis of 2159 concentrations of morphine and its glucuronides from 248 infants receiving intravenous morphine ranging in bodyweight from 500 g to 18 kg (median 2.8 kg). The model was internally validated using normalized prediction distribution errors. Formation clearances of morphine to its glucuronides and elimination clearances of the glucuronides were found to be primarily influenced by bodyweight, which was parameterized using an allometric equation with an estimated exponential scaling factor of 1.44. Additionally, a postnatal age of less than 10 days was identified as a covariate for formation clearance to the glucuronides, independent of birthweight or postmenstrual age. Distribution volumes scaled linearly with bodyweight. Model-based simulations show that in newborns, including preterms, infants and children under the age of 3 years, a loading dose in microg/kg and a maintenance dose expressed in microg/kg1.5/h, with a 50% reduction of the maintenance dose in newborns younger than 10 days, results in a narrow range of morphine and metabolite serum concentrations throughout the studied age range. Future pharmacodynamic investigations are needed to reveal target concentrations in this population, after which final dosing recommendations can be made.

Physiologically Based Pharmacokinetic (PBPK) Models for Ethanol

Physiologically based pharmacokinetic models have been used to describe the distribution and elimination of ethanol after intravenous administration. These models have been used to estimate the ethanol infusion profile that is sufficient for achieving a prescribed breath ethanol concentration time course in individuals, providing a useful platform for several pharmacokinetic and pharmacodynamic investigations. Mathematical foundations of these models are examined, including the derivation of an explicit set of governing equations in the form of a system of nonlinear ordinary differential equations. These equations can then be used to formulate and refine parameter identification and control strategies. Finally, a framework in which models related to this model can be constructed and analyzed is described.

Polyclonal antibody production against bovine serum albumin conjugated artemisinin in rabbit

Abstract: The aim of the present study was to produce a polyclonal antibody against bovine serum albumin (BSA) conjugated with artemisinin. To gain an immunogenic character of artemisinin, a carboxyl group was added to it using mixed anhydride method. Then, the reactive compound of artemisinin was conjugated with BSA. The BSA+artemisinin were injected to white female New Zealand rabbits for two times. In the first injection, the Fraud's complete adjuvant was used, and two weeks later, a booster injection was carried out with a Fraud's incomplete adjuvant. Two weeks later, blood samples were collected; their serum were separated and frozen until assessment. The production of antibody against BSA+artemisinin was assayed by Immunoblot technique. Antibody was separated and concentrated by saturated ammonium sulfate. The assay confirmed the successful production of an antibody against BSA+artemisinin as a fundamental step in investigation of pharmacodynamics and pharmacokinetics of artemisinin. Key words: Artemisia sieberi, artemisinin, polyclonal antibody, BSA-conjugation

A safety, pharmacokinetic and pharmacodynamic investigation of deferasirox (Exjade®, ICL670) in patients with transfusion-dependent anemias and iron-overload: a Phase I study in Japan

The pharmacokinetics (PK) and pharmacodynamics (PD) of the once-daily, oral ironchelating agent, deferasirox (Exjade, ICL670), have been evaluated further in a Phase I, openlabel, multicenter, dose-escalation study in Japanese patients with myelodysplastic syndromes, aplastic anemia, and other anemias. Deferasirox was initially administered as a single dose of 5 (n = 6), 10 (n = 7), 20 (n = 6) or 30 (n = 7) mg/(kg day) and then after 7 days seven daily doses were administered. Linear PK (C (max) and AUC) were observed at all doses after a single dose and at steady state, and dose-dependent iron excretion was observed. Pharmacokinetic/pharmacodynamic parameters were similar to those reported in a Caucasian beta-thalassemia cohort. Following the single- and multiple-dose phases, 21 of 26 patients progressed to a 3-year extension phase of the study, where dose reductions and increases [5-30 mg/(kg day)] were allowed following safety and efficacy assessments. In the interim, 1-year data show that deferasirox was well tolerated, with generally infrequent and mild adverse events. Reductions in serum ferritin levels were observed and a negative iron balance achieved at doses of 20-30 mg/(kg day). These data suggest that deferasirox has a stable and predictable PK/PD profile, irrespective of underlying disease or race, and a predictable and manageable safety profile suitable for chronic administration.

Leveraging Exploratory Investigational New Drug Studies to Accelerate Drug Development

In 2006, the U.S. Food and Drug Administration published its guide on exploratory investigational new drug (IND) studies with the goal of making the approach to early-stage, pilot clinical trials more flexible within the context of current regulations. The exploratory IND allows sponsors to initiate clinical trials of limited scale with reduced preclinical requirements. These studies may be important vehicles for the conduct of proof-of-principle pharmacodynamic investigations of highly potent molecules, for bioavailability studies that require only a single drug dose to be administered, and for imaging trials that permit critical dosimetry and biodistribution investigations of new molecules. These trials were done with no therapeutic intent and must be followed by traditional dose-escalation investigations that are supported by standard preclinical toxicologic and pharmacologic studies. To the extent that they allow early evaluations of essential drug characteristics that can only be obtained in humans, exploratory IND trials have the potential to limit the cost and improve the development times of new agents.

Mechanistic model for the acute effect of fluvoxamine on 5-HT and 5-HIAA concentrations in rat frontal cortex

A mechanistic model is proposed to predict the time course of the concentrations of 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in rat frontal cortex following acute administration of SSRIs. In the model, SSRIs increase synaptic 5-HT concentrations by reversible blockade of the SERT in a direct concentration-dependent manner, while the 5-HT response is attenuated by negative feedback via 5-HT autoreceptors. In principle, the model allows for the description of oscillatory patterns in the time course of 5-HT and 5-HIAA concentrations in brain extracellular fluid. The model was applied in a pharmacokinetic–pharmacodynamic (PK/PD) investigation on the time course of the microdialysate 5-HT and 5-HIAA response in rat frontal cortex following a 30-min intravenous infusion of 3.7 and 7.3 mg/kg fluvoxamine. Directly after administration of fluvoxamine, concentrations of 5-HT were increased to approximately 450–600% of baseline values while 5-HIAA concentrations were decreased. Thereafter 5-HT and 5-HIAA concentrations gradually returned to baseline values in 6–10 h, respectively. The PK/PD analysis revealed that inhibition of 5-HT reuptake was directly related to the fluvoxamine concentration in plasma, with 50% inhibition of 5-HT reuptake occurring at a plasma concentration of 1.1 ng/ml (EC 50). The levels of 5-HT at which 50% of the inhibition of the 5-HT response was reached (IC 50) amounted to 272% of baseline. The model was unable to capture the oscillatory patterns in the individual concentration time curves, which appeared to occur randomly. The proposed mechanistic model is the first step in modeling of complex neurotransmission processes. The model constitutes a useful basis for prediction of the time course of median 5-HT and 5-HIAA concentrations in the frontal cortex in behavioral pharmacology studies in vivo.

Near-Infrared Spectroscopy (NIRS): A Non-Invasive In Vivo Methodology for Analysis of Brain Vascular and Metabolic Activities in Real Time in Rodents

Near infrared spectroscopy (NIRS) was first used as a tool for the in vivo monitoring of tissue oxygenation in the late seventies. Today, NIRS instruments are more and more used in clinical environments since they are now easy to use, sensitive, robust, provide rapid analysis and could be complementary to other non invasive methodologies such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET). The feasibility of non-invasive analysis of brain activities is studied in the attempt to overcome the major limitation of invasive in vivo methodologies. In the present work, optic fibre probes were used as optical head of a novel, highly sensitive near infrared continuous wave spectroscopy (CW-NIR) instrument adapted for in vivo NIRS measurements in the brain of rodents. This prototype was designed for non-invasive analysis of the 2 main forms of haemoglobin: oxy-haemoglobin (HbO(2)) and deoxy-haemoglobin (Hb), chromophores present in biological tissues as these are markers of the degree of tissue oxygenation, thus providing an index of blood level and therefore of tissue metabolism. It was first tested in peripheral tissue (human gastrocnemius muscle) and then reset to perform measurement on rat brain. In animal studies, the optical head was firmly placed using stereotaxic apparatus upon the sagittal line of anaesthetised adult rat's head, without any surgery. 'Physiologic' (i.e. with exogenous oxygen (O(2)) or carbon dioxide (CO(2)) supplied orally) or pharmacologic (i.e. with drugs of abuse such as cocaine) experiments have been performed to support the effectiveness of the methodology in preclinical studies. In addition, the possibility that changes in brain metabolism as measured by NIRS might be a useful index of brain penetration of new chemical entities has been investigated using different compounds from different chemical classes that were selected on the basis of their known brain penetration and overall pharmacokinetic profile. Finally, the feasibility of coupling NIRS with another although invasive in vivo method such as electrophysiology for concomitant analysis of cerebral cell firing in discrete brain areas was tested in the attempt to study in real time the putative correlation between blood levels, brain metabolism and neuronal activities in rat CNS, i.e. apply NIRS to pharmacodynamic investigations. The data gathered in rat treated with exogenous O(2), indicate an original relationship between NIRS analysis of brain metabolism and electrical changes in this major nucleus of CNS involved in neurophysiologic and pathologic activities.

Ixabepilone and the Narrow Path to Developing New Cytotoxic Drugs

During the last decade, the focus of attention in the field of new drug development has drifted away from the uninterrupted quest of new and better cytotoxic drugs to the newer and more glamorous grounds of targeted therapy, proof of principle and proof of concept, and sophisticated pharmacodynamic investigations. After years of relentless work behind the scenes, ixabepilone is emerging from the somewhat neglected list of cytotoxic compounds to stand on stage as something more than an apprentice, as reported in this issue of the Journal of Clinical Oncology (JCO). There are reports of five phase II studies of the drug, four in metastatic breast cancer (MBC) and one in non–small-cell lung cancer (NSCLC). Ixabepilone (BMS-247550) is an analog of epothilone B and a reference compound of a novel class of tubulin-active drugs that blocks tubulin polymerization in a way similar to that of the taxanes. Ixabepilone binds to a different site of the tubulin molecule than the taxanes; such a feature may be relevant to the reported lack of complete cross resistance with paclitaxel and docetaxel in preclinical models. The data presented in this issue deserve attention for multiple reasons, forcing us to reconsider the role of the development of old-fashioned chemotherapy to complement the current armamentarium of drugs for cancer treatment in the era of targeted therapies. Ixabepilone enters with the almost mandatory ambition of challenging the still undisputed role of the taxanes. The data presented in this issue of JCO, as summarized in Table 1, support such ambition: ixabepilone worked well as first-line therapy in women with MBC, and had clear antitumor activity in tumors resistant to or even refractory to multiple other classes of cytotoxic drugs and—most important—to paclitaxel or docetaxel. For NSCLC, the activity was observed despite progression within 4 months from treatment with cisplatin or carboplatin. Such therapeutic activity is an encouraging start to widening the range of therapeutic options after failure of the taxanes, prompting the testing of ixabepilone in early stages of disease. But ixabepilone treatment comes at a price: neurotoxicity. Tubulin-active drugs almost invariably cause neurologic adverse effects. Ixabepilone, which was well tolerated regarding bone marrow and other organ toxicities, is not an exception, causing a paclitaxel-like neuropathy that was primarily sensory in nature, cumulative, and slowly reversible in the majority of patients. The neuropathy associated with ixabepilone had much to do with the uneven and slower than expected clinical development of the drug. Table 1 reveals a situation that points to the differences of dose and schedule of ixabepilone in the different trials reported in this issue of JCO. The difference is indeed larger than it appears from the table, due to the extent and severity of the neuropathy initially observed in four of the studies that forced a lowering of the dose from 50 to 40 or even to 32 mg/m per cycle to make treatment feasible with the schedule of administration once every 3 weeks. The only exception to the remedy of amending the dose was the study in women who never received taxanes before ixabepilone, in which daily administration for 5 consecutive days was adopted. One may be tempted to conclude that the daily schedule was less neurotoxic. However, after dose titration to feasibility of the once-per-cycle dosing, there was no obvious pattern of different

Assessment of in vitro and in vivo recovery of sinomenine using microdialysis

For microdialysis studies in the skin, laboratory-made linear probes are often used. The application of the microdialysis technique to the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of the linear probes' performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of sinomenine. Using different lengths of the dialysis membrance and different perfusion flow rates, a flow rate of 2 microL/min with 20-min sampling intervals was selected for the subsequent studies, based on the analysis of sinomenine from the microdialysis probe. In vitro recovery of sinomenine from the microdialysis probe was independent of concentration, stable over an 8-h period. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of sinomenine from rat subcutaneous tissue. The performance of the microdialysis system was stable over an 8-h study, resulting in a mean in vitro recovery of 51.91+/-1.29%. Recovery obtained using the ZNF plot was 52.43%. In vivo recovery of sinomenine was 34.46+/-0.76% and was stable over the 7-h study period. The in vitro and in vivo performance of the microdialysis technique was established for the study of sinomenine. It would prove to be a useful and reliable tool to study the pharmacokinetics and pharmacodynamics of sinomenine. The data obtained in our study highlight the importance of a systematic examination of microdialysis linear probe validation.

Direct and rapid inhibition of factor Xa by otamixaban: A pharmacokinetic and pharmacodynamic investigation in patients with coronary artery disease

New anticoagulants that combine effective anticoagulation with low bleeding rates are still sought after. We investigated the safety, pharmacokinetics, and pharmacodynamics of otamixaban, a direct factor Xa inhibitor, in patients with stable coronary artery disease. This was a randomized, placebo-controlled, double-blind, multicenter study in 119 patients with stable coronary artery disease taking maintenance doses of their comedication. Of these patients, 50% had mild renal impairment (creatinine clearance >45 mL/min but <80 mL/min). Patients were randomized in a 4:1 ratio to receive either otamixaban or placebo as a 1-minute bolus followed by a 24-hour continuous infusion. Anti-factor Xa activity, clotting times (activated partial thromboplastin time, dilute prothrombin time, Russell's viper venom test), and international normalized ratio were measured. All patients completed the study according to the protocol. No major or minor bleeding occurred according to Thrombosis in Myocardial Infarction criteria. Anti-factor Xa activity and anticoagulant effect were measurable early after the start of the infusion and remained during the infusion. Upon cessation, these effects declined rapidly and returned to baseline within 6 hours after the end of infusion. Anti-factor Xa activity coincided with the otamixaban plasma concentrations. The fold changes from baseline at the end of infusion with regard to the clotting times ranged from 1.7 to 4.4 (1.15 for placebo), 1.29 to 3.15 (0.98 for placebo), and 1.19 to 2.11 (0.94 for placebo) for Russell's viper venom test, dilute prothrombin time, and activated partial thromboplastin time, respectively, and ranged from 0.94 to 1.70 (0.94 for placebo) for the international normalized ratio. In patients with stable coronary artery disease taking maintenance doses of their usual concomitant medication, otamixaban exerts a rapid onset of anticoagulation and anti-factor Xa activity. Our data provide evidence that further studies are warranted to investigate the safety and efficacy of otamixaban in the target population.

The hollow fibre model - facilitating anti-cancer pre-clinical pharmacodynamics and improving animal welfare

We describe a modified hollow fibre assay (HFA) for investigating the potential of novel molecules as pharmaceutical agents. In particular the assay provides drug/target interaction data that can facilitate the selection of lead compounds for further evaluation in more sophisticated solid tumour models, whilst successfully implementing the 3Rs - the 'replacement' 'refinement' and 'reduction' of animals. This more ethical and rapid approach to early drug development does not compromise on the validity, sensitivity, predictivity or efficacy of preclinical evaluation. We present novel data using the standard cross-linker mitomycin C (MMC) as a positive control, and two investigational DNA interactive molecules (C1311/ SJG-136). Tumour cells were seeded in fibres and implanted into mice. Following treatment with an intraperitoneal injection, fibres were excised and cells retrieved for pharmacodynamic analysis using the comet assay/fluorescence microscopy. Microscopy results revealed nuclear uptake and localisation within cytoplasmic organelles of HT29 colorectal adenocarcinoma cells following treatment with C1311 (150 mg/kg). Following treatment with SJG-136 (0.3 mg/kg) a 27.3% (p<0.001) DNA cross-linking (s.c.) effect was observed in the HL60 acute promyelocytic leukaemia cell line. DNA cross-linking effects of 55% (i.p) and 50% (s.c.) (p<0.005) were observed in the A549 lung carcinoma cell line following administration of MMC (6 mg/kg). These data are consistent with previous activity defined using solid tumour models, and support the use of the HFA for in vivo pharmacodynamic investigation whilst significantly reducing animal numbers and the influence of tumour growth on the welfare of mice.

Clofarabine and nelarabine: two new purine nucleoside analogs

Both clofarabine and nelarabine recently received an accelerated approval by the US Food and Drug Administration for use in refractory or relapsed pediatric acute lymphoblastic leukemia and in refractory-relapsed T-cell acute lymphoblastic leukemia or T-cell lymphoblastic lymphoma. Both drugs have been studied for their metabolism and mechanisms of action in preclinical investigations and for their efficacy in clinical trials. This review will summarize these investigations and will suggest future plans with these nucleoside analogs. Clofarabine and nelarabine were designed based on preclinical and clinical findings with other nucleoside analogs or normal deoxynucleotides such as dGTP. Studies in cell lines have demonstrated that triphosphate is the active metabolite for both these purine nucleoside analogs. Pharmacokinetic and pharmacodynamic investigations during clinical trials have verified the importance of triphosphate levels in achieving clinical responses. Several phase I and II clinical explorations have suggested the utility of clofarabine in acute leukemias and nelarabine in T-cell diseases. Dose-limiting toxicities were nonhematologic: hepatotoxicity for clofarabine and neurotoxicity for nelarabine. Clofarabine is the first deoxyadenosine analog that shows promise in adult and pediatric acute leukemias without untoward toxicity. Nelarabine, as expected from its design, is a drug that may be directed to T-cell diseases.

Challenges of marijuana research

The use of any drug ideally represents a decision based on objective, scientifically based cost–benefit analyses that factor in both the short and long-term effects of that exposure. Pharmacological, toxicological, pharmacokinetic and pharmacodynamic investigations that are deemed to be essential for the rational use of any therapeutic agent are therefore part of the usual drug approval process. With regard to marijuana, sociopolitical factors have intervened in this scientific process. Three major lay perspectives appear to dominate the societal view of marijuana—the ‘reefer madness’ camp holding the view that there are no redeeming attributes to the ‘evil weed’, the ‘innocuous’ camp who consider it to be a harmless recreational substance and the ‘medical marijuana’ camp that believes marijuana to be a panacea for a multitude of aches, pains and chronic diseases with, of course, every shade of opinion in-between. On the scientific front, three trends preface nearly every recent journal article concerning marijuana—the significant prevalence of marijuana use, the changes in age of first use (Kohn et al ., 2005; Monshouwer et al ., 2005) and the increasing strength of the drug, leading to higher acute and chronic exposures (National Institute on Drug Abuse, 2005; Pijlman et al ., 2005). For example, in 2004 ∼96.8 (40.2%), 25.5 (10.6%) and 14.6 (6.1%) million Americans aged 12 and older were considered to have used marijuana within their lifetime, the last year, and the last month, respectively (Office of National Drug Control Policy, 2006). However, in the 3 of the 11 states in which medical use of marijuana is legal, and for which statistics are available, 0.05% of the population are registered, i.e. legal, users (General Accounting Office, 2002). Even though marijuana is available from pharmacies in the Netherlands, 80% of users secure their drug via illicit channels (Erkens et al ., …

Pharmacokinetics and Pharmacodynamics of Meropenem in Febrile Neutropenic Patients with Bacteremia

Pharmacodynamic investigations with antimicrobials define the relationship between the infecting organism and achievable drug concentrations with clinical outcome. To examine this relationship for meropenem in a population of patients who are at high risk of infection-related morbidity and mortality. The study was a retrospective analysis of a multicenter, randomized, blinded clinical trial. A population-based predictive model was created using data from adults with febrile neutropenia and the nonparametric modeling program, NPEM. Patient age, body weight, and serum creatinine level were covariates in the model used to predict unbound concentrations for each patient. Pathogen susceptibility was estimated using product literature minimum inhibitory concentrations for effectiveness against 50% of microorganisms (MIC50) for specific organisms. The pharmacodynamic index of percent time above MIC (% T>MIC) was analyzed for its association with clinical outcome. A 2-compartment pharmacokinetic model using patient covariates of body weight and renal function best described the pharmacokinetics of meropenem in febrile neutropenic patients. Sixty patients with confirmed gram-positive or -negative bacteremia were studied. An average of 83% T>MIC was identified for the 42 clinical responders compared with 59% T>MIC for the 18 nonresponders (p = 0.04). An 80% clinical response rate was evident when the % T>MIC for meropenem exceeded 75% of the dosing interval (p = 0.01). To our knowledge, this is the first published report of a relationship between a pharmacodynamic index and clinical outcome in a febrile neutropenic population. Based on this relationship, dosing with intravenous meropenem 500 mg every 6 hours is predicted to be comparable to the currently recommended 1 g every 8 hours for serious infections. Our model provides further justification for a prospective clinical trial to evaluate a pharmacodynamically targeted meropenem dosing schedule as to its ability to improve clinical outcome in these patients.

Transcriptional signature of flavopiridol-induced tumor cell death

Flavopiridol has been shown to inhibit the proliferation of a variety of human tumor cells and is currently undergoing clinical evaluation in cancer treatment. Although the antiproliferative effect of flavopiridol has been attributed to the inhibition of cyclin-dependent kinases 2 and 4, recent reports indicate that the mechanism responsible for the cell death induced by this agent is more complex. To provide insight into the molecular processes mediating flavopiridol-induced cytotoxicity and to investigate the availability of markers indicative of its activity, we have applied cDNA microarray technology. Gene expression profiles were determined for four human tumor cell lines (prostate carcinomas PC3 and DU145 and gliomas SF359 and U251) following exposure to selected concentrations of flavopiridol. Treatment of these cell lines with a concentration of flavopiridol sufficient to reduce survival to 10% resulted in the identification of a set of 209 genes, the expression of which were altered in each of the cell lines. This common set of 209 gene expression changes suggested that flavopiridol-induced cell death can be defined in terms of a specific transcriptome. The flavopiridol death transcriptome consisted primarily of down-regulated genes; however, there were also a significant number of genes with increased expression. Whereas causal relationships were not established, these data suggest molecular events/processes that may be associated with flavopiridol-induced tumor cell death. Moreover, the identification of a set of gene expression changes in four human tumor cell lines suggests that such a transcriptome may be applicable to investigations of flavopiridol pharmacodynamics.

A pharmacodynamic investigation of tacrolimus in pediatric liver transplantation

Although monitoring of tacrolimus blood concentrations is standard clinical practice following liver transplantation, a greater understanding of the relationship between trough concentrations and clinical outcome is required. The aim of this study was to perform a pharmacodynamic investigation of tacrolimus in pediatric liver transplant recipients. A retrospective analysis was performed on 35 pediatric liver recipients who received oral tacrolimus as the primary immunosuppressant. Outcome data were recorded corresponding to the times that tacrolimus trough concentrations had been determined (using a validated high-performance liquid chromatography-tandem mass spectrometry assay). A Mann-Whitney rank sum test was used to investigate differences between median concentrations at which drug toxicity, infection, and organ rejection did and did not occur. Outcome data and trough concentrations were recorded from the immediate post-transplant time to over 3 years (656 concentration-effect measures). Seventy one percent of data was obtained after the first 90 days post-transplant. Patients were identified as having experienced hypomagnesemia (20), hypertension (14), hyperkalemia (12), infection (11), nephrotoxicity (8), diarrhea (6), hyperglycemia (3), neurotoxicity(3), and rejection (3) during retrospective follow-up. Median trough concentrations were significantly higher (P <.05) at times patients experienced tacrolimus toxicity compared to no toxicity for nephrotoxicity (11.8 vs. 6.1 ng/mL) and neurotoxicity (15.1 vs. 6.2 ng/mL) and at times when patients suffered from diarrhea (8.9 vs. 6.0 ng/mL). No significant difference could be found between median trough concentrations at which hypertension, hyperkalemia, hyperglycemia, infection and organ rejection did and did not occur. A statistically significant relationship exists between some tacrolimus toxicities and tacrolimus trough concentrations. To minimize toxicity in the later post-transplant period, we propose a target trough tacrolimus concentration of 6 ng/mL.