Search

Herpes simplex viruses-1 and -2 (HSV-1 and -2) are two of the three human alphaherpesviruses that cause infections worldwide. Since both viruses can be acquired in the absence of visible signs and symptoms, yet still result in lifelong infection, it is imperative that we provide interventions to keep them at bay, especially in immunocompromised patients. While numerous experimental vaccines are under consideration, current intervention consists solely of antiviral chemotherapeutic agents. This review explores all of the clinically approved drugs used to prevent the worst sequelae of recurrent outbreaks by these viruses.

Background: The use of oral methadone in opioid substitution treatment (OST) for the management of opioid use disorder is established clinical practice. Confounding treatment is the increased risks of contracting Mycobacterium tuberculosis, the mainstay treatment of which incorporates the potent CYP 2B6 inducer rifampicin., Methods: This study applied pharmacokinetic modelling using virtual clinical trials, to pharmacokinetically quantify the extent and impact of rifampicin-mediated drug-drug interactions (DDI) on methadone plasma concentrations. An R-methadone model was developed and validated against 11 retrospective clinical studies prior to use in all subsequent studies. The aims were to investigate: (i) the impact of the DDI on daily methadone doses of 60 mg, 90 mg and 120 mg; (ii) dose escalation during rifampicin and (iii) dose reduction following rifampicin cessation., Results: A dose increase to 160 mg daily during rifampicin treatment phases was required to maintain peak methadone plasma concentrations within a derived therapeutic window of 80-700 ng/mL. Dose escalation prior to rifampicin initiation was not required and resulted in an increase in subjects with supra-therapeutic concentrations. However, during rifampicin cessation, a dose reduction of 10 mg every 2 days commencing prior to rifampicin cessation, ensured that most patients possessed a peak methadone plasma concentration within an optimal therapeutic window., Implications: Rifampicin significantly alters methadone plasma concentrations and necessitates dose adjustments. Daily doses of almost double those used perhaps more commonly in clinical practice are required for optimal plasma concentration and careful consideration of dose reduction strategies would be required during the deinduction phase., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide- N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.

Purpose: The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients., Methods: In the trabeculectomy mouse model, intracameral injection(s) of the PlGF inhibitor (5D11D4) were compared to MMC or aflibercept and to the combination of both compounds. Treatment outcome was studied by bleb investigation and by Sirius Red staining. The effect of subconjunctival PlGF administration and topical 5D11D4 on IOP was investigated in normotensive mice and was compared to topical administration of latanoprost, the gold standard for IOP-lowering., Results: Combination of MMC and 5D11D4 was able to significantly improve surgical outcome compared to monotherapy of MMC or 5D11D4 (n = 20; P < 0.001). Compared to combined treatment of MMC with aflibercept, the simultaneous administration of MMC and 5D11D4 was equally efficacious in improving surgical outcome (n = 15; P = 0.88). In normotensive mice, 5D11D4 was able to significantly reduce the IOP-elevation induced by PlGF (n = 10; P < 0.05), whereas no effect of 5D11D4 was seen in naive mice, which was in contrast to latanoprost., Conclusions: The current data suggest that application of MMC together with PlGF inhibition may have complementary effects in the improvement of surgical outcome and is equally efficacious as the combined treatment of MMC and aflibercept. Inhibition of PlGF also might open alternative perspectives as IOP-lowering strategy for glaucoma patients with increased aqueous PlGF levels.

Aims: To extend the physiologically based pharmacokinetic (PBPK) model developed for 6-mercaptopurine to account for intracellular metabolism and to explore the role of genetic polymorphism in the TPMT enzyme on the pharmacokinetics of 6-mercaptopurine., Methods: The developed PBPK model was extended for 6-mercaptopurine to account for intracellular metabolism and genetic polymorphism in TPMT activity. System and drug specific parameters were obtained from the literature or estimated using plasma or intracellular red blood cell concentrations of 6-mercaptopurine and its metabolites. Age-dependent changes in parameters were implemented for scaling, and variability was also introduced for simulation. The model was validated using published data., Results: The model was extended successfully. Parameter estimation and model predictions were satisfactory. Prediction of intracellular red blood cell concentrations of 6-thioguanine nucleotide for different TPMT phenotypes (in a clinical study that compared conventional and individualized dosing) showed results that were consistent with observed values and reported incidence of haematopoietic toxicity. Following conventional dosing, the predicted mean concentrations for homozygous and heterozygous variants, respectively, were about 10 times and two times the levels for wild-type. However, following individualized dosing, the mean concentration was around the same level for the three phenotypes despite different doses., Conclusions: The developed PBPK model has been extended for 6-mercaptopurine and can be used to predict plasma 6-mercaptopurine and tissue concentration of 6-mercaptopurine, 6-thioguanine nucleotide and 6-methylmercaptopurine ribonucleotide in adults and children. Predictions of reported data from clinical studies showed satisfactory results. The model may help to improve 6-mercaptopurine dosing, achieve better clinical outcome and reduce toxicity., (© 2015 The British Pharmacological Society.)

Objective: To review the chemistry, pharmacology, microbiology, pharmacodynamics, pharmacokinetics, clinical efficacy, tolerability, drug interactions, dosing, and administration of fidaxomicin (FDX)., Data Sources: A search of PubMed using the terms "fidaxomicin," "OPT-80," "PAR-101," "OP-1118," "difimicin," "tiacumicin," and "lipiarmycin" was performed. All English-language articles from 1983 to November 2013 were reviewed for relevance. Bibliographies of all articles were reviewed as well as the manufacturer's Web site to further identify relevant information., Study Selection: All English-language articles from 1983 to November 2013 appearing in these searches were reviewed for relevance to this paper. In addition, their bibliographies were reviewed to identify any articles not identified in the searches., Data Synthesis: FDX is the first macrolide antibiotic with a narrow spectrum of activity targeted against Clostridium difficile. It is administered orally without regard to food. The primary route of elimination is fecal excretion. Advanced age, hepatic dysfunction, or renal impairment do not alter its disposition. Phase III clinical trials have demonstrated that FDX 200 mg twice daily for 10 days is noninferior to vancomycin 125 mg four times daily for 10 days in the treatment of adults with C. difficile infection and is associated with lower recurrence rates. FDX has a favorable side effect profile and a low potential for drug interactions., Conclusion: FDX has been shown to be safe and effective in the treatment of adults with C. difficile infection. Further research and pharmacoeconomic studies are needed to clarify and refine its role in the treatment of patients at high risk for recurrence.

A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 10⁸ RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To review the literature concerning the role of rifampin in the combination treatment of Legionella pneumophila pneumonia., Data Sources: A search of MEDLINE and Ovid databases was conducted (January 1970-May 2011) using the search terms Legionella pneumophila, pneumonia, Legionnaires' disease, rifampin or rifampicin, macrolide, fluoroquinolone, erythromycin, clarithromycin, levofloxacin, ciprofloxacin, and moxifloxacin, Study Selection and Data Extraction: In vivo studies published in English that compared antimicrobial therapies including rifampin for the treatment of Legionella pneumonia, as well as in vitro studies including an assessment of rifampin bioactivity, were included., Data Synthesis: Macrolides and fluoroquinolones have been effective as monotherapy in the treatment of L. pneumophila pneumonia. This review includes evidence summaries from 4 bioactivity evaluations, 6 clinical studies, and 6 reported cases of combination rifampin use. Combined with supporting evidence, the role of combination rifampin therapy is further delineated., Conclusions: Interpretation of the data is limited by the potential for selection bias and lack of consistent comparators. Rifampin therapy should be considered only for patients with severe disease or significant comorbid conditions (eg, uncontrolled diabetes, smoking, or obstructive lung disease) including immunocompromised hosts and those refractory to conventional monotherapy regimens. Caution for significant adverse drug events and drug-drug interactions should be taken with the addition of rifampin.

The incidence of Clostridium difficile has doubled over the past 15 years, and rising mortality rates associated with this infection have followed in its wake. C. difficile infection (CDI) has supplanted methicillin-resistant Staphylococcus aureus as the major cause of nosocomial infection. An insufficient response rate to currently available CDI therapies has prompted the search for new and alternative treatment modalities for this disease. The investigational pipeline includes evaluation of new antimicrobial agents that exhibit good activity against C. difficile without altering normal gut flora, C. difficile toxin-absorbing compounds, and preformed antibodies and vaccines against C. difficile toxin. In two robust clinical trials comparing fidaxomicin to vancomycin in the treatment of CDI, treatment with fidaxomicin demonstrated a superior global cure (cure without recurrence) rate compared with the current gold standard, vancomycin. Fidaxomicin, the first of a new class of macrocyclic antimicrobial agents, represents an advance in the management of CDI.

Filibuvir (PF-868554), being developed by Pfizer, is an orally administered, non-nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase for the potential treatment of chronic HCV infection. An estimated 180 million people worldwide are infected with HCV and at risk of developing chronic liver diseases that can lead to cirrhosis or hepatocellular carcinomas. HCV infection is the main cause of liver transplantation in industrialized nations. Filibuvir is a potent and specific inhibitor of the virally-encoded NS5B polymerase, and inhibited genotype 1 subgenomic HCV replication in the cell-based replicon system. Filibuvir demonstrated a good pharmacokinetic profile and oral bioavailability in preclinical animal studies, which is consistent with twice-daily dosing in humans. In phase I and a IIa clinical trial in treatment-naïve patients infected with genotype 1 HCV, filibuvir monotherapy or in combination with pegylated IFNα2a/ribavirin (the standard of care [SoC] for HCV infection) for up to 4 weeks significantly reduced HCV RNA levels compared with placebo or SoC alone. The incidence and severity of adverse events were similar to SoC and placebo. At the time of publication, phase I pharmacokinetic clinical trials were ongoing in healthy volunteers and a phase IIb clinical trial was assessing filibuvir in combination with SoC for up to 24 weeks in treatment-naïve patients infected with genotype 1 HCV. Results of this trial will help to characterize the potential of this drug class for the treatment of HCV infections.

Background and Purpose: Antiviral hyper-activation-limiting therapeutic agents (AV-HALTs) are a novel experimental drug class designed to both decrease viral replication and down-regulate excessive immune system activation for the treatment of chronic infections, including human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome. VS411, a first-in-class AV-HALT, is a single-dosage form combining didanosine (ddI, 400 mg), an antiviral (AV), and hydroxyurea (HU, 600 mg), a cytostatic agent, designed to provide a slow release of ddI to reduce its maximal plasma concentration (C(max)) to potentially reduce toxicity while maintaining total daily exposure (AUC) and the AV activity., Experimental Approach: This was a pilot phase I, open-label, randomized, single-dose, four-way crossover trial to investigate the fasted and non-fasted residual variance of AUC, C(max) and the oral bioavailability of ddI and HU, co-formulated as VS411, and administered as two different fixed-dose combination formulations compared to commercially available ddI (Videx EC) and HU (Hydrea) when given simultaneously., Key Results: Formulation VS411-2 had a favourable safety profile, displayed a clear trend for lower ddI C(max) (P= 0.0603) compared to Videx EC, and the 90% confidence intervals around the least square means ratio of C(max) did not include 100%. ddI AUC(∞) was not significantly decreased compared to Videx EC. HU pharmacokinetic parameters were essentially identical to Hydrea, although there was a decrease in HU exposure under fed versus fasted conditions., Conclusions and Implications: A phase IIa trial utilizing VS411-2 formulation has been fielded to identify the optimal doses of HU plus ddI as an AV-HALT for the treatment of HIV disease., (© 2010 ViroStatics srl. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Purpose: To investigate the cellular effects of mitomycin C (MMC) treatment on corneal endothelial (CE) cells at clinically relevant applications and dosages., Methods: Radial and posterior diffusion of MMC was determined by an Escherichia coli growth inhibition bioassay. A modified version of the comet assay (single cell gel electrophoresis) was used to detect DNA cross-linking. Immunostaining detected the nuclear phosphorylated histone variant H2AX (gamma-H2AX) indicating DNA double-strand breaks. Apoptosis in MMC-treated cells was detected with annexin V staining., Results: Topical application of 0.02% MMC to intact goat globes resulted in MMC in the CE at 0.37 microg/mL and produced a significant increase in CE DNA cross-linking with as little as 6 seconds of topical MMC treatment. DNA cross-linking was also demonstrated in cultured CE cells by using MMC exposures similar to those detected in CE of intact eyes. Such MMC treatment of CE produced elevated and persistent gamma-H2AX-positive cells indicative of DNA double-strand breaks. Similarly, there was an increase in the proportion of apoptotic CE cells, evidenced by positive annexin V staining., Conclusions: The results demonstrate that exposure to MMC at times and concentrations commonly used in refractive surgery produces cross-linking of corneal endothelial DNA, persistent DNA damage, and endothelial death via apoptosis. Current practices of MMC application during refractive surgeries may increase the potential for long-term and permanent deleterious effects on the health of the corneal endothelium.

The considerable interindividual differences in efficacy and side effects of commonly used medications in Crohn's disease are partly owing to genetic polymorphisms. Many genetic variants have been studied in genes possibly involved in the metabolism or mechanism of action of therapeutic agents such as glucocorticosteroids, azathioprine/6-mercaptopurine, methotrexate, calcineurin inhibitors or anti-TNF agents. However, the only test translated into clinical practice is thiopurine S-methyltransferase (TPMT) genotyping for hematological toxicity of thiopurine treatment. To date, there are no other meaningful applications for pharmacogenomics in clinical practice of Crohn's disease. In the future, designed therapeutic trials should possibly permit the development of predictive models including genotypic markers, such as that proposed for the clinical outcome after infliximab therapy, which includes an apoptotic pharmacogenetic index. The recent identification of new susceptibility genes provides additional candidate markers that have possible effects on the outcomes of therapies, and prioritizes new therapeutic targets, such as the IL-23 pathway. Further innovative approaches might be relevant for the pharmacogenetic investigation of gene variants implied in innate immune pattern recognition and autophagy.

Purpose: Iododeoxyuridine (IUdR) is a halogenated pyrimidine recognized as the thymidine substitute in DNA. When labeled with iodine 125, IUdR can be used as a carrier to incorporate the isotope into DNA and target the dividing cells. The purpose of this study was to assess the maximum uptake of IUdR by proliferating smooth muscle cells (SMCs) in vitro to determine the optimal concentration to be administered in an in vivo experiment. The long-term goal is to use radioactive IUdR to inhibit SMC proliferation and recurrent stenosis of arteries after balloon angioplasty in vivo., Materials and Methods: Porcine vascular SMCs were cultured in 5% fetal bovine serum medium and stimulated to proliferate by adding a medium containing 10% fetal bovine serum and insulin. IUdR was added to the proliferating SMCs at concentrations of 5, 10, 20, 30, and 40 micro mol/L on days 1, 3, 5, and 7 of incubation. One group of cells--the control group--did not receive IUdR. The SMCs were harvested and double-stained with an anti-IUdR antibody and propidium iodide, and fluorescence-activated cell scanning was performed to determine the ratio of IUdR-labeled cells to the total cell population for each IUdR concentration and at each time point. The data were measured three times at each time point. The doubling times, growth curve, and cell density of the proliferating SMCs were investigated by using the Coulter particle counter and digital microscopy., Results: The percentage of proliferating SMCs that showed IUdR uptake increased from 1 to 5 days incubation with all concentrations of IUdR; the incorporation rate reached a peak value at day 5 and then decreased by day 7. IUdR uptake on day 5 was higher with concentrations of 10 and 20 micro mol/L. When compared with that of the control group, the doubling times increased with an increase in IUdR concentration, whereas the proliferating cell number and density decreased significantly by days 5 (P < .05) and 7 (P < .01)., Conclusions: IUdR uptake peaked on day 5, and the optimal concentration of IUdR for in vitro uptake in proliferating SMCs was 10-20 micro mol/L. IUdR inhibited the proliferation of the SMCs, and the inhibitory effect was related to the concentration.

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

Like anybody else, pregnant women are susceptible to infections. The correct treatment of these women, however, must consider along with pathogens, the infection site and antibiotic pharmacokinetics, the fetus and possible side effects to the child. When prescribing over this special condition, the physician must remember that the prescription will affect two organism and the drug must treat the mother without affecting the fetus. Beta-lactams having a long history of use without significant deleterious effects on the fetuses still are the safest choice during pregnancy. However, considering the constant increase of multi-resistant microorganisms, the physician has been forced to use different antimicrobial agents. Usually, data regarding safety during pregnancy are very limited, which causes serious doubts during prescription. In addition, many studies regarding the safe use of antibiotics during pregnancy are inconclusive or demand more evidence. The present study is a wide revision regarding the use of antibiotics during pregnancy, considering their pharmacokinetics and the clinical experience in recent years. It also intends to assist the physician during prescription and to give information to the pharmacists to help pregnant women.

This study was designed to experimentally evaluate the optimal X-ray energy for increasing the radiation energy absorbed in tumours loaded with iodinated compounds, using the photoelectric effect. SQ20B human cells were irradiated with synchrotron monochromatic beam tuned at 32.8, 33.5, 50 and 70 keV. Two cell treatments were compared to the control: cells suspended in 10 mg ml(-1) of iodine radiological contrast agent or cells pre-exposed with 10 microM of iodo-desoxyuridine (IUdR) for 48 h. Our radiobiological end point was clonogenic cell survival. Cells irradiated with both iodine compounds exhibited a radiation sensitisation enhancement. Moreover, it was energy dependent, with a maximum at 50 keV. At this energy, the sensitisation calculated at 10% survival was equal to 2.03 for cells suspended in iodinated contrast agent and 2.60 for IUdR. Cells pretreated with IUdR had higher sensitisation factors over the energy range than for those suspended in iodine contrast agent. Also, their survival curves presented no shoulder, suggesting complex lethal damages from Auger electrons. Our results confirm the existence of the 50 keV energy optimum for a binary therapeutic irradiation based on the presence of stable iodine in tumours and an external irradiation. Monochromatic synchrotron radiotherapy concept is hence proposed for increasing the differential effect between healthy and cancerous tissue irradiation.

ViroPharma, under license from GlaxoSmithKline, is developing maribavir, a DNA synthesis inhibitor for the potential prevention and treatment of human cytomegalovirus infections related to transplants (including solid organ and hematopoietic stem cells), congenital transmission, and in patients with HIV infection. In July 2004, a phase II trial was initiated.

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.

Recently, a benzo-1,2,4-thiadiazine was shown to be a potent, specific inhibitor of the hepatitis C virus (HCV) RNA polymerase [J. Biol. Chem. 277 (2002) 32327]. Herein, we present several lines of evidence to demonstrate that thiadiazine compound 4 (C(21)H(21)N(3)O(4)S) is highly synergistic with interferon-alpha (IFN-alpha) and disrupts HCV replicon RNA synthesis with a distinct kinetic profile. A time course analysis after a single treatment with 5 microM compound 4 showed a loss of viral RNA consistent with replicon RNA half-life, suggesting inhibition of 90% of ongoing or newly initiated replicative intermediates. This finding is consistent with the mechanism of action recently reported for compound 4, an RNA synthesis initiation inhibitor [J. Biol. Chem. 278 (2003) 16602]. Further, unlike IFN-alpha, an immediate reduction of HCV replicon RNA synthesis was apparent upon addition of compound 4. Treatment with IFN-alpha showed a delay of approximately 4h prior to inhibition of viral RNA replication, consistent with its signaling kinetics.

Background: The aim of the study was to determine whether hepatic regeneration stimulates growth of tumour residing within the liver, and whether a difference in the rate of DNA synthesis in liver and tumour may be used to target cancer using the radiolabelled thymidine analogue 5-iodo-2'-deoxyuridine (IUdR)., Methods: Partial hepatectomy was performed on Buffalo rats bearing solitary nodules of syngeneic Morris hepatoma. Liver and tumour DNA synthesis was measured by incorporation of radioactive IUdR. [(125)I]IUdR was tested as an adjuvant therapy after hepatectomy in Buffalo rats bearing diffuse microscopic Morris hepatomas to simulate the clinical situation., Results: Liver regeneration enhanced liver and tumour DNA synthesis as measured by incorporation of radioactive IUdR. Liver DNA synthesis returned to baseline by 7 days, whereas tumour DNA synthesis remained above baseline level. Hepatectomy enhanced the growth of microscopic liver tumours. [(125)I]IUdR (250 micro Ci or 1 mCi/kg) administered 4 days after hepatectomy significantly reduced tumour growth without signs of systemic toxicity or liver dysfunction., Conclusion: The local environment of the regenerating liver stimulates tumour growth. The thymidine analogue [(125)I]IUdR may be used preferentially to target tumour DNA synthesis in the regenerating liver, and may prove useful as an adjuvant therapy for hepatic tumours after surgical resection., (Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Background: Episcleral application of mitomycin-C (MMC) during glaucoma filtration surgery hinders the post-operative wound healing. Diffusion through the sclera might result in a toxic effect on the ciliary body resulting in reduced aqueous humor production leading to post-operative hypotony. We developed an experimental model to investigate the influence of intraocular pressure on the diffusion of MMC through the sclera and in subscleral compartments., Methods: Scleral quadrants of 10 human donor eyes were mounted on PMMA tubes filled with saline imitating the intraocular volume. By height variation of a coupled infusion line different intraocular pressures were simulated (0, 8, 23 and 80 mmHg). Additionally the model included a subscleral sponge to mimic the compartment of the ciliary body. The episcleral sides of the scleral quadrants were exposed for 1 min to sponges soaked with 200 microg ml(-1) MMC. An 8-mm-diameter scleral disk was punched out with a trephine and horizontally dissected with a kryotome. The MMC concentrations of scleral layers, epi-and subscleral sponges and the fluid within the tubes were analysed by means of high-performance liquid chromatography., Results: The MMC concentration gradually declined from the episcleral sponge (165 microg ml(-1)) to the superficial (3.3 microg ml(-1)) and deep scleral layers (1.2 microg ml(-1)), and to the subscleral sponge (0.2 microg ml(-1)). We were able to detect very small concentrations of MMC in the fluid within the PMMA tubes (0.01 microg ml(-1))., Conclusion: We developed a new experimental in vitro model for investigating transscleral MMC diffusion. The different simulated intraocular pressures had no effect on the concentration gradient through the investigated compartments of our model.

Purpose: Episcleral application of mitomycin-C (MMC) during glaucoma surgery hinders the post-operative wound healing but may lead to side effects to the ciliary body. We present an experimental model to investigate the influence of irrigation on diffusion of MMC through the sclera and into subscleral compartments., Materials and Methods: Scleral quadrants of 10 human donor eyes were mounted on PMMA tubes filled with saline imitating the intraocular fluid. Beneath the sclera a subscleral sponge was fixed to mimic the ciliary body. The episcleral sides of the scleral quadrants were exposed to sponges soaked with 0.2 mg/ml MMC and thereafter irrigated with 0, 40, 100 and 200 ml saline. Mitomycin-C concentrations of the sclera and the subscleral compartments were analyzed by means of high-performance liquid chromatography. In a second experiment, after the application time of one minute, different times of free diffusion until analysis were allowed, to evaluate the time-course of MMC penetration., Results: MMC concentrations declined from the scleral layers (1-13 microg/ml, depending on depth and irrigation) to the subscleral sponge (0.17-0.27 microg/ml) and to the subscleral fluid (0.002-0.05 microg/ml). Irrigation significantly reduced MMC concentrations in the superficial scleral layers and in the subscleral fluid to 10-30% of the no irrigation value. No effect was noted in the deep scleral layers and subscleral sponges. In the second experiment, the subscleral compartments showed higher MMC concentrations with longer diffusion times, whereas no effect on intrascleral MMC concentrations was observed., Conclusion: Irrigation reduced MMC concentrations in the superficial scleral layers and in the subscleral fluid by the same amount. The clinical value of irrigation should be reconsidered; a lower dose MMC application without irrigation might be a more rationale approach.

Multidrug resistance conferred to cancer cells is often mediated by the expression of efflux transporter "pumps". It is also believed that many of the same transporters are involved in drug efflux from numerous normal endothelial and epithelial cell types in the intestine, brain, kidney, and liver. Etoposide transport kinetics were characterized in Caco-2 cells and in well established Madin-Darby canine kidney (MDCKII) cell lines that were stably-transfected with a human cDNA encoding P-glycoprotein (Pgp), human multidrug resistance protein (MRP1), or the canalicular multispecific organic anion (cMOAT) transporters to determine the roles of these transporters in etoposide efflux. Etoposide transport kinetics were concentration-dependent in the MDCKII-MDR1 and MDCKII-cMOAT cells. The apparent secretory Michaelis constant (Km) and carrier-mediated permeability (Pc) values for Pgp and cMOAT were 254.96 +/- 94.39 microM and 5.96 +/- 0.41 x 10(-6) cm/s and 616.54 +/- 163.15 microM and 1.87 +/- 0.10 x 10(-5) cm/s, respectively. The secretory permeability of etoposide decreased significantly in the basal to apical (B to A) (i.e., efflux) direction, whereas the permeability increased 2.3-fold in the apical to basal (A to B) direction in MDCKII-MDR1 cells in the presence of elacridar (GF120918). Moderate inhibition of etoposide efflux by leukotriene C4 (LTC4) was observed in MDCKII-cMOAT cells. Furthermore, etoposide inhibited LTC4 efflux, confirming the involvement of cMOAT. The flux of etoposide in MDCKII-MRP1 cells was similar to that in MDCKII/wt control cells. The current results demonstrate that the secretory transport mechanism of etoposide involves multiple transporters, including Pgp and cMOAT but not MRP1. These results demonstrate that Pgp and cMOAT are involved in the intestinal secretory transport of etoposide. Since the intestinal secretion of etoposide was previously reported in the literature, it also suggests that they may be involved in the in vivo intestinal secretion of etoposide; however, mechanistic in vivo studies are required to confirm this.

Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude.

Five new oligoethylene ester derivatives (9-13) of 5-iodo-2'-deoxyuridine (IDU) were synthesized and assayed to determine their lipophilicity by both experimental lipophilicity indices (log K') and calculated partition coefficients (CLOGP). In vitro experiments were carried out to evaluate the chemical and enzymatic stability and fluxes through excised human skin of these new IDU derivatives. Esters 9-13 showed increased lipophilicity compared with the parent drug (IDU), had good stability in phosphate buffer (pH 7.4), and were readily hydrolyzed by porcine esterase. No correlation between lipophilicity and skin permeation fluxes of synthesized esters 9-13 was observed. Results from in vitro percutaneous absorption studies showed that, among all esters synthesized, only esters 9 and 10 significantly increased the cumulative amount of IDU that penetrated through excised human skin compared with the parent drug (IDU)., (Copyright 2002 Wiley-Liss, Inc.)

Raltitrexed (Tomudex) is a specific inhibitor of thymidylate synthase with clinical activity in colorectal cancer. The combination of raltitrexed and 5-iodo-2'-deoxyuridine (IdUrd, a cytotoxic pyrimidine analog) resulted in increased IdUrd incorporation into DNA and exhibited in vitro synergism against colon and bladder human carcinoma cell lines. We designed a phase I trial to determine the MTD, pharmacokinetics, and biologic effects of escalating doses of the combination of IdUrd given as a 24-hour infusion after a raltitrexed 15-minute infusion every three weeks. Thirty-four patients received 95 courses of raltitrexed and IdUrd at doses ranging from raltitrexed 1 mg/m2 and IdUrd 750 mg/m2 to raltitrexed 2.5 mg/m2 and IdUrd 10,400 mg/m2. The median number of cycles administered was 2 (range 1-10). Dose limiting hematologic toxicity occurred at doses of raltitrexed 2.5 mg/m2 and IdUrd 10,400 mg/m2. In addition, we determined the mean plasma concentrations C(SS) of IdUrd, the iodouracil level at 22 hours and the IdUrd clearance. Raltitrexed did not appear to affect the pharmacokinetics of IdUrd in the dose range tested. The recommended phase II dose is raltitrexed 2 mg/m2 and IdUrd 10,400 mg/m2 repeated every three weeks. Evidence of potential antitumor activity was observed: 1 patient (with colon cancer) had a partial response while 15 others had stable disease.

We describe our preliminary studies on the development of methods to measure hypoxia in standard paraffin sections of human tumors. Three parameters were investigated. First, image analysis of tumor vascularity yielded the parameter diffusion limited fraction (DLF), which is the amount of tumor tissue greater than a fixed distance from the nearest blood vessel. Secondly, the amount of tumor tissue stained with antibodies against bound reduced products of the bioreductive marker pimonidazole was assessed. Finally, the fraction of blood vessels showing no surrounding tumor tissue labeled with lUdR, a cell kinetic marker, was measured. DLF and pimonidazole monitor primarily chronic hypoxia, while it is hypothesized that the IUdR-negative fraction monitors acute hypoxia. Feasibility was demonstrated in a series of 10 esophageal and 10 rectal tumors (no drug administration), 10 cervix tumors (pimonidazole) and 14 head and neck tumors (pimonidazole and lUdR). Significant differences between tumors were found for all parameters. DLF correlated significantly with the pimonidazole fraction when all images of all tumors were included, although mean values per tumor showed no correlation. The IUdR-negative fraction did not correlate with either of the other two parameters. We conclude that it is feasible to measure hypoxia-related, and possibly perfusion-related, parameters on paraffin sections for predictive purposes, although each method needs further validation. Each parameter will be correlated with outcome in a larger study on head and neck tumors treated with surgery with or without postoperative radiotherapy.

The purpose of this study was to investigate the impact of different diffusion times of mitomycin-C (MMC) on the intrascleral concentration vs depth profile of MMC in an experimental model. Scleral quadrants of eight human donor eyes were exposed to sponges soaked with MMC for an application time of 1 min. After irrigation with 40 ml saline, we allowed further diffusion of MMC in the sclera for 1, 5, 14 and 29 min until the specimens were further processed. A central 8 mm diameter scleral disk was horizontally dissected with a kryotome at -20 degrees C. MMC concentrations of six layers of 140 microm thickness were analysed by means of high-performance liquid chromatography. The MMC concentrations (microg g(-1)) of layer 1 were: 13.45+/- 5.9 (mean +/- S.D. at 2 min diffusion time), 7.6+/-2.5 (6 min diffusion), 5.6+/-3.1 (15 min diffusion) and 3.6+/-1.7 (30 min diffusion). The corresponding MMC concentrations of layer 6 were: 0.61+/-0.48, 1.47 +/-0.66, 1.83+/-0.42 and 2.98+/-0.97 microg g(-1). The superficial concentration of intrascleral MMC decreased with increasing diffusion time, the deep concentrations increased. After 30 min of diffusion time, equal concentrations of MMC were found in all layers. Even with current low-dose application regimens of MMC the concentrations in the inner side of the sclera rapidly increase beyond the limits of the therapeutic range. Owing to this fast diffusion of MMC, the only means of reducing ciliary body concentrations of MMC is to reduce the dose., (Copyright 2000 Academic Press.)

The purpose of this study was to investigate the impact of different concentrations and volumes of Mitomycin-C (MMC) on the intrascleral concentration vs depth profile of MMC in an experimental model. The episcleral sides of scleral quadrants of human donor eyes were exposed for 1 min to sponges (corneal light shield, Merocel Corp.) soaked with MMC. After irrigation with 40 ml saline a central 8 mm diameter scleral disk was horizontally dissected with a cryotome. MMC concentrations of six layers of 140 microns thickness were analysed by means of high-performance liquid chromatography. In Experiment 1 (11 eyes) the sponges were soaked with 50 microliters of 10, 100 and 200 micrograms ml-1 MMC solutions. In Experiment 2 (12 eyes) the sponges were soaked with 10, 30, 50 and 80 microliters of a 200 micrograms ml-1 isotonic MMC solution. In Experiment 1 the MMC concentrations (microgram g-1) of layer 1 were 0.35 (+/- 0.20; 10 micrograms ml-1 group) and 9.22 (+/- 2.92; 200 micrograms ml-1 group). In Experiment 2 the MMC concentrations were 2.57 (+/- 1.17; 10 microliters group), 7.35 (+/- 2.49; 30 microliters group) and 11.67 (+/- 3.25; 80 microliters group). The scleral MMC concentrations were significantly influenced by the applied concentrations (layers 1-5) and by the applied volumes (all layers) of MMC solution. The intrascleral MMC concentration increased linearly with increasing concentration and not linearly with increasing volume of the applied MMC solution. To achieve more predictable scleral concentrations of MMC after trabeculectomy with MMC it seems advisable to control both the concentration and the volume of the MMC solution used to soak the sponge.

We examined whether the administration of methotrexate (MTX) prior to the injection of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in rats with intrathecal (i.t.) TE-671 human rhabdomyosarcoma would enhance 125IUdR uptake by tumor cells and augment the therapeutic efficacy of this Auger-electron-emitting radiopharmaceutical. TE-671 cells were exposed in vitro to medium +/- MTX, and the percentage of cells in various phases of the cell cycle and the uptake of 125IUdR assessed. In addition, nude rats were injected i.t. with TE-671 cells and later infused i.t. with saline or MTX for 24 h prior to 125IUdR injection, and the radioactivity associated with their spinal cords was determined. Exposure of tumor cells in vitro to MTX leads to an increase in the uptake of 125IUdR as a consequence of both a rise in the absolute uptake per cell and an increase in the percentage of S-phase cells. A corresponding increase of radioactivity within the spinal cords of tumor-bearing rats also occurs in the presence of MTX. Tumor-bearing animals were infused/injected with MTX and/or 125IUdR, and the onset of paralysis was determined as a function of time. We find that: (i) MTX infusion leads to a slight increase in time to onset of paralysis (median [M] = 24 vs. 22 days, p = 0.79); (ii) 125IUdR injection results in a statistically significant delay (p < 0.01) in the onset of paralysis (M= 39 days); (iii) MTX administration prior to 122IUdR injection further increases the therapeutic efficacy (M = 45 days).

It was found recently that 17beta-estradiol (E2) could be activated by the epoxide-forming oxidant dimethyldioxirane (DMDO) resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in vitro (Carcinogenesis, 17, 1957-1961, 1996). To further study the mechanism of this inhibition, several synthetic DNAs with different base content and sequence were used to study the transcriptional effects and binding specificities of E2 after DMDO activation in vitro. The results show: (1) E2 after activation had a strong inhibitory effect on the template function of both A-T and G-C containing double-stranded DNAs, e.g. poly[d(A-T)], polydG x polydC and poly[d(I-C)], and only a weak inhibition on the single-stranded DNA template, polydC. The inhibition was dose-dependent, and only after DMDO activation. (2) 3H-labeled E2, after DMDO activation, was able to bind DNAs containing both A-T and G-C bases. The order of the binding preference was: calf thymus DNA > poly[d(A-T)] > poly[d(G-C)]. (3) The covalent binding nature of E2 to DNA after activation was further confirmed by 32P-post-labeling analysis using calf thymus DNA. (4) The absorption spectrum of E2 changed, after DMDO treatment, from a peak around 280-290 nm to 260-270 nm with a shoulder appearing around 300-320 nm. These studies have not only confirmed our earlier observation that E2, after DMDO activation, can inhibit DNA-dependent RNA synthesis, but also provided new insights into the DNA-binding properties after activation. Additionally, since epoxidation is often required for the activation of chemical carcinogens to bind DNA, these studies lend further support to our proposed hypothesis that E2 epoxidation may play an initiation role in estrogen carcinogenesis.

Hydroxyurea is used in the treatment of various forms of cancer, sickle-cell anaemia and HIV infection. Oral absorption of the drug is virtually complete, the volume of distribution is equivalent to total body water and elimination is through both renal and nonrenal mechanisms. Nonrenal elimination of hydroxyurea is characterised by Michaelis-Menten kinetics. Further studies are necessary to clarify several aspects of the pharmacokinetics and pharmacodynamics of hydroxyurea: the effect of age and disease state, concentration-effect relationship, the role of therapeutic drug monitoring, and the mechanisms of renal and nonrenal elimination. The recent development of improved assays for hydroxyurea should have benefits for future pharmacokinetic studies.

Some kinetic parameters of clastogenic activity of cyclophosphamide were inferred by means of the comparison of its kinetics of micronucleated polychromatic erythrocytes (MN-PCE) formation with the kinetics induced by radiation. The same reasoning was also applied to the kinetics obtained by treatment with mitomycin C (MMC), arabinocyl cytosine (Ara-C) and 6-mercaptopurine (6-MOP), based on previously reported data from the literature. The results indicate that the latency period (LP) and half-lives (HL) vary from one mutagen to another. For MMC, they are very similar to radiation indicating a rapid distribution and reaction. CP presents very long LP and HL which agree with the requirement of mutagen activation. Ara-C showed a very short LP which suggests a rapid activation and fast induction of damage in DNA. 6-MOP presented a very long LP which agreed with the requirement of its incorporation into DNA to cause micronucleus (MN). From the data obtained in the present work, it can be concluded that the comparison of the kinetics of MN-PCE formation induced by chemical agents with that obtained by the exposure to an acute dose of radiation permits one to estimate some parameters of the kinetics of clastogenic activity of chemical agents, like the LP and the HL. This seems to be valid for agents that act through the induction of DNA lesions; in the case of agents whose clastogenic activity is through other mechanisms, such as the inhibition or alteration of the process of duplication of the DNA, the kinetic parameters are not equivalent to the LP and HL; however, they could provide information on their possible mechanism of action.

A mathematical structure is described for determining teratogenic sensitivity or susceptibility from analysis of malformation incidence, dose-response, and pharmacokinetic data obtained during pregnancy as a result of exposure to a teratogenic agent. From the dosage or exposure of laboratory animals, embryonic and maternal concentrations of the xenobiotic are calculated using a physiologically based pharmacokinetic (PBPK) model. Malformations observed in the progeny are linked to the PBPK-derived target tissue concentrations with a model for the sensitivity calculated as a function of the embryonic age. The PBPK model for internal disposition of chemicals during pregnancy was developed previously. This report focuses on the development of the mathematical relations for the sensitivity of the embryo and effect functions on different organs. The concentrations of a xenobiotic calculated for the site of action or target tissue(s) in the embryo are weighted using both a nonlinear dose-response curve and a sensitivity distribution function that depends on the age or stage of development of the embryo. This weighted "exposure" of the target tissue is regressed with the number of observed malformations to quantify the parameters of the model. This approach lends itself to integration of diverse sources of experimental data, with hydroxyurea data taken from several sources in the literature as an example. This sensitivity function obtained from laboratory animal data serves as a vehicle for prediction and extrapolation to human pregnancy for the teratogenic potential of a substance.