Can preclinical drug development help to predict adverse events in clinical trials?

The development of novel therapeutics is associated with high rates of attrition, with unexpected adverse events being a major cause of failure. Serious adverse events have led to organ failure, cancer development and deaths that were not expected outcomes in clinical trials. These life-threatening events were not identified during therapeutic development due to the lack of preclinical safety tests that faithfully represented human physiology. We highlight the successful application of several novel technologies, including high-throughput screening, organs-on-chips, microbiome-containing drug-testing platforms and humanised mouse models, for mechanistic studies and prediction of toxicity. We propose the incorporation of similar preclinical tests into future drug development to reduce the likelihood of hazardous therapeutics entering later-stage clinical trials.

Genotoxicity test battery - An assessment of its utility in early drug development.

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

Pre-clinical study of IRDye800CW-nimotuzumab formulation, stability, pharmacokinetics, and safety.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a target for cancer therapy as it is overexpressed in a wide variety of cancers. Therapeutic antibodies that bind EGFR are being evaluated in clinical trials as imaging agents for positron emission tomography and image-guided surgery. However, some of these antibodies have safety concerns such as infusion reactions, limiting their use in imaging applications. Nimotuzumab is a therapeutic monoclonal antibody that is specific for EGFR and has been used as a therapy in a number of countries. METHODS: Formulation of IRDye800CW-nimotuzumab for a clinical trial application was prepared. The physical, chemical, and pharmaceutical properties were tested to develop the specifications to determine stability of the product. The acute and delayed toxicities were tested and IRDye800CW-nimotuzumab was determined to be non-toxic. Non-compartmental pharmacokinetics analysis was used to determine the half-life of IRDye800CW-nimotuzumab. RESULTS: IRDye800CW-nimotuzumab was determined to be non-toxic from the acute and delayed toxicity study. The half-life of IRDye800CW-nimotuzumab was determined to be 38 ± 1.5 h. A bi-exponential analysis was also used which gave a t1/2 alpha of 1.5 h and t1/2 beta of 40.8 h. CONCLUSIONS: Here, we show preclinical studies demonstrating that nimotuzumab conjugated to IRDye800CW is safe and does not exhibit toxicities commonly associated with EGFR targeting antibodies.

Pre-clinical and cellular toxicity evaluation of 7-methylxanthine: an investigational drug for the treatment of myopia.

The present study entails the toxicity evaluation of 7-methyl xanthine (7-MX), first of its kind molecule found effective in phase II clinical trials for the treatment of myopia, in comparison to other clinically used xanthines i.e., caffeine and theobromine. For acute toxicity evaluation, 7-MX was administered orally in two rodent species (rat and mice) at the doses of 300 mg/kg and 2000 mg/kg and for repeated dose 28-d oral toxicity, at 250, 500, and 1000 mg/kg in rats. Further, cellular toxicity was evaluated in normal breast epithelial (fR2), rat brain C6 glioma (C6 glioma) and human colorectal (Caco-2) cell lines. Also, the cell uptake assay to determine the intestinal permeability of drug was performed in Caco-2 cells. In acute toxicity, 7-MX treatment showed no mortality and toxicity, whereas 66.6% (mice) and 33.3% (rat) mortality was observed in both caffeine and theobromine treatment groups. In repeated dose 28-d oral toxicity, 7-MX treatment was found to have no-observed-adverse-effect level up to the dose of 1000 mg/kg in the present study conducted as per OECD guidelines 407. Also, very high IC50 value of 305.5 and 721 µg/mL was observed for 7-MX in fR2 and C6 glioma cells, respectively. In Caco-2 cells, linear bioavailability and high % cell viability was observed. Thus, 7-MX may be classified as Globally Harmonized System (GHS) category 5 drug with LD50 >2000-5000 mg/kg. Also, the repeated dose 28-d oral toxicity study demonstrated 7-MX to be nontoxic in nature, with cell line toxicity results further endorsing its nontoxic nature.

New phenotypic cytotoxicity assay for ROS-inducing compounds using rat renal epithelial cells.

An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15 % of compounds not cytotoxic to HepG2 cells (below 50 µM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.

Phase IA/IB study of single-agent tislelizumab, an investigational anti-PD-1 antibody, in solid tumors.

BACKGROUND: The programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in suppressing antitumor immunity; axis dysregulation can be used by cancer cells to evade the immune system. Tislelizumab, an investigational monoclonal antibody with high affinity and binding specificity for PD-1, was engineered to minimize binding to FcγR on macrophages to limit antibody-dependent phagocytosis, a potential mechanism of resistance to anti-PD-1 therapy. The aim of this phase IA/IB study was to investigate the safety/tolerability, antitumor effects and optimal dose and schedule of tislelizumab in patients with advanced solid tumors. METHODS: Patients (aged ≥18 years) enrolled in phase IA received intravenous tislelizumab 0.5, 2, 5 or 10 mg/kg every 2 weeks; 2 or 5 mg/kg administered every 2 weeks or every 3 weeks; or 200 mg every 3 weeks; patients in phase IB received 5 mg/kg every 3 weeks. Primary objectives were to assess tislelizumab's safety/tolerability profile by adverse event (AE) monitoring and antitumor activity using RECIST V.1.1. PD-L1 expression was assessed retrospectively with the VENTANA PD-L1 (SP263) Assay. RESULTS: Between May 2015 and October 2017, 451 patients (n=116, IA; n=335, IB) were enrolled. Fatigue (28%), nausea (25%) and decreased appetite (20%) were the most commonly reported AEs. Most AEs were grade 1-2 severity; anemia (4.9%) was the most common grade 3-4 AE. Treatment-related AEs led to discontinuation in 5.3% of patients. Grade 5 AEs were reported in 14 patients; 2 were considered related to tislelizumab. Pneumonitis (2%) and colitis (1%) were the most common serious tislelizumab-related AEs. As of May 2019, 18% of patients achieved a confirmed objective response in phase IA and 12% in phase IB; median follow-up duration was 13.6 and 7.6 months, respectively. Pharmacokinetics, safety and antitumor activity obtained from both phase IA and IB determined the tislelizumab recommended dose; ultimately, tislelizumab 200 mg intravenous every 3 weeks was the dose and schedule recommended to be taken into subsequent clinical trials. CONCLUSIONS: Tislelizumab monotherapy demonstrated an acceptable safety/tolerability profile. Durable responses were observed in heavily pretreated patients with advanced solid tumors, supporting the evaluation of tislelizumab 200 mg every 3 weeks, as monotherapy and in combination therapy, for the treatment of solid tumors and hematological malignancies. TRIAL REGISTRATION NUMBER: NCT02407990.

Cardiotoxicity Assessment of Drugs Using Human iPS Cell-Derived Cardiomyocytes: Toward Proarrhythmic Risk and Cardio-Oncology.

Growing evidence suggests that Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) can be used as a new human cell-based platform to assess cardiac toxicity/safety during drug development. Cardiotoxicity assessment is highly challenging due to species differences and various toxicities, such as electrophysiological and contractile toxicities, which can result in proarrhythmia and heart failure. To explore proarrhythmic risk, the Multi-Electrode Array (MEA) platform is widely used to assess QT-interval prolongation and the proarrhythmic potential of drug candidates using hiPSC-CMs. Several consortiums, including the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japanese iPS Cardiac Safety Assessment (JiCSA), have demonstrated the applicability of hiPSC-CMs/MEA for assessing the torsadogenic potential of drug candidates. Additionally, contractility is a key safety issue in drug development, and efforts have been undertaken to measure contractility by a variety of imaging-based methods using iPS-CMs. Therefore, hiPSC-CMs might represent a standard testing tool for evaluating the proarrhythmic and contractile potentials. This review provides new insights into the practical application of hiPSC-CMs in early or late-stage nonclinical testing during drug development.

Comparisons of false negative rates from a trend test alone and from a trend test jointly with a control-high groups pairwise test in the determination of the carcinogenicity of new drugs.

Interest has been expressed in using a joint test procedure that requires that the results of both a trend test and a pairwise comparison test between the control and the high groups be statistically significant simultaneously at the levels of significance recommended in the FDA 2001 draft guidance for industry document for the separate tests in order for the drug effect on the development of an individual tumor type to be considered as statistically significant. Results of our simulation studies show that there is a serious consequence of large inflations of the false negative rate through large decreases of false positive rate in the use of the above joint test procedure in the final interpretation of the carcinogenicity potential of a new drug if the levels of significance recommended for separate tests are used. The inflation can be as high as 204.5% of the false negative rate when the trend test alone is required to test if the effect is statistically significant. To correct the problem, new sets of levels of significance have also been developed for those who want to use the joint test in reviews of carcinogenicity studies.

A 3D Human Airway Model Enables Prediction of Respiratory Toxicity of Inhaled Drugs In Vitro.

Respiratory tract toxicity represents a significant cause of attrition of inhaled drug candidates targeting respiratory diseases. One of the key issues to allow early detection of respiratory toxicities is the lack of reliable and predictive in vitro systems. Here, the relevance and value of a physiologically relevant 3D human airway in vitro model (MucilAir) were explored by repeated administration of a set of compounds with (n = 8) or without (n = 7) respiratory toxicity following inhalation in vivo. Predictability for respiratory toxicity was evaluated by readout of cytotoxicity, barrier integrity, viability, morphology, ciliary beating frequency, mucociliary clearance and cytokine release. Interestingly, the data show that in vivo toxicity can be predicted in vitro by studying cell barrier integrity by transepithelial electrical resistance (TEER), and cell viability determined by the Resazurin method. Both read-outs had 88% sensitivity and 100% specificity, respectively, while the former was more accurate with receiver operating characteristic (ROC) AUC of 0.98 (p = .0018) compared with ROC AUC of 0.90 (p = .0092). The loss of cell barrier integrity could mainly, but not fully, be attributed to a loss of cell coverage in 6 out of 7 compounds with reduced TEER. Notably, these effects occurred only at 400 µM, at concentration levels significantly above primary target cell potency, suggesting that greater attention to high local lung concentrations should be taken into account in safety assessment of inhaled drugs. Thus, prediction of respiratory toxicity in 3D human airway in vitro models may result in improved animal welfare and reduced attrition in inhaled drug discovery projects.

The Identification of Pivotal Transcriptional Factors Mediating Cell Responses to Drugs With Drug-Induced Liver Injury Liabilities.

Drug-induced liver injury (DILI) is a leading cause of drug attrition during drug development and a common reason for drug withdrawal from the market. The poor predictability of conventional animal-based approaches necessitates the development of alternative testing approaches. A body of evidence associates DILI with the induction of stress-response genes in liver cells. Here, we set out to identify signal transduction pathways predominantly involved in the regulation of gene transcription by DILI drugs. To this end, we employed ATTAGENE's cell-based multiplexed reporter assay, the FACTORIAL transcription factor (TF), that enables quantitative assessment of the activity of multiple stress-responsive TFs in a single well of cells. Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nat. Methods 5, 253-260). Using this assay, we assessed TF responses of the human hepatoma cell line HepG2 to a panel of 64 drug candidates, including 23 preclinical DILI and 11 clinical DILI compounds and 30 nonhepatotoxic compounds from a diverse physicochemical property space. We have identified 16 TF families that specifically responded to DILI drugs, including nuclear factor (erythroid-derived 2)-like 2 antioxidant response element, octamer, hypoxia inducible factor 1 alpha, farnesoid-X receptor, TCF/beta-catenin, aryl hydrocarbon receptor, activator protein-1, E2F, early growth response-1, metal-response transcription factor 1, sterol regulatory element-binding protein, paired box protein, peroxisome proliferator-activated receptor, liver X receptor, interferone regulating factor, and P53, and 2 promoters that responded to multiple TFs (cytomegalovirus and direct repeat 3/vitamin D receptor). Some of TFs identified here also have previously defined role in pathogenesis of liver diseases. These data demonstrate the utility of cost-effective, animal-free, TF profiling assay for detecting DILI potential of drug candidates at early stages of drug development.

Designing More Efficient Preclinical Experiments: A Simulation Study in Chemotherapy-Induced Myelosupression.

A new more efficient preclinical study design (referred to as a compact design) is proposed that removes the need for satellite animals for the collection of toxicokinetic (TK) data by sampling from the main study animals, taking no more than one sample in 24 h to build up a full profile over the course of the study. The compact design's performance was tested with a simulation study, using an example of chemotherapy-induced myelosupression in rats. Data sets were simulated from a model based on available data, following both the compact design and a traditional design using satellite animals, with 100 studies being simulated for each. The effect of the compact design on parameter and variance estimates for the TK and neutrophil models were investigated, as well as the potential effect of interoccasion variability (IOV). The compact design performed equally as well as the traditional design, and had little impact on parameter or variation estimates, indicating that it would be a suitable alternative to traditional satellite designs while reducing the number of animals required. When IOV was present but not accounted for during the TK analysis some parameter estimates were biased and interindividual variation and residual errors inflated; this was reduced by allowing for IOV in the analysis. Using the compact design removes the need for a satellite group, reducing the number of animals required, without affecting the ability to model the data. If large IOV is suspected, caution should be exercised to avoid parameter estimation bias, and inflation of variability and residual error.

Multiparametric assay using HepaRG cells for predicting drug-induced liver injury.

The utility of HepaRG cells as an in vitro cell-based assay system for assessing drug-induced liver injury (DILI) risk was investigated. Seventeen DILI-positive and 15 DILI-negative drugs were selected for the assay. HepaRG cells were treated with each drug for 24h at concentrations that were 1.6-, 6.3-, 25-, and 100-fold the therapeutic maximum plasma concentration (Cmax). After treatment, the cell viability, glutathione content, caspase 3/7 activity, lipid accumulation, leakage of lactate dehydrogenase, and albumin secretion were measured. The sensitivity and specificity were calculated to assess the ability of the assay to predict DILI. Our multiparametric assay using HepaRG cells exhibited a 67% sensitivity and 73% specificity at a 100-fold concentration of Cmax and a 41% sensitivity and 87% specificity at a 25-fold concentration of Cmax. When a 25-fold Cmax cut-off was applied, approximately 70% of drugs exhibiting positive responses were classified into the high DILI risk category. HepaRG cells distinguished relatively safe drugs from their high-risk analogs. Our study indicates that HepaRG cells may be of use to (1) prioritize drug analogs, (2) analyze the mechanism of DILI, and (3) assess the risk for DILI in the early drug discovery stage.

Genomic models of short-term exposure accurately predict long-term chemical carcinogenicity and identify putative mechanisms of action.

BACKGROUND: Despite an overall decrease in incidence of and mortality from cancer, about 40% of Americans will be diagnosed with the disease in their lifetime, and around 20% will die of it. Current approaches to test carcinogenic chemicals adopt the 2-year rodent bioassay, which is costly and time-consuming. As a result, fewer than 2% of the chemicals on the market have actually been tested. However, evidence accumulated to date suggests that gene expression profiles from model organisms exposed to chemical compounds reflect underlying mechanisms of action, and that these toxicogenomic models could be used in the prediction of chemical carcinogenicity. RESULTS: In this study, we used a rat-based microarray dataset from the NTP DrugMatrix Database to test the ability of toxicogenomics to model carcinogenicity. We analyzed 1,221 gene-expression profiles obtained from rats treated with 127 well-characterized compounds, including genotoxic and non-genotoxic carcinogens. We built a classifier that predicts a chemical's carcinogenic potential with an AUC of 0.78, and validated it on an independent dataset from the Japanese Toxicogenomics Project consisting of 2,065 profiles from 72 compounds. Finally, we identified differentially expressed genes associated with chemical carcinogenesis, and developed novel data-driven approaches for the molecular characterization of the response to chemical stressors. CONCLUSION: Here, we validate a toxicogenomic approach to predict carcinogenicity and provide strong evidence that, with a larger set of compounds, we should be able to improve the sensitivity and specificity of the predictions. We found that the prediction of carcinogenicity is tissue-dependent and that the results also confirm and expand upon previous studies implicating DNA damage, the peroxisome proliferator-activated receptor, the aryl hydrocarbon receptor, and regenerative pathology in the response to carcinogen exposure.

Automated volumetric growth plate measurement using magnetic resonance imaging for monitoring skeletal toxicity in children treated on investigational drug trials.

PURPOSE: Targeted anticancer agents have been reported to have side effects on the skeletal system such as thickening of the epiphyseal growth plate in preclinical models of juvenile, but not mature, animals. Careful evaluation of skeletal toxicity in the clinical development of targeted therapies for children is required. We validated a novel method to measure the growth plate volume using MRI. EXPERIMENTAL DESIGN: A semiautomated method of volumetric growth plate measurement was developed on the basis of the differences of pixel intensity of the growth plate from surrounding bone on T(1) sagittal MRI. Two observers measured the femoral growth plate volume and thickness on three different days using 20 pediatric knee MRIs obtained at the NIH. Five subjects had two knee MRIs obtained on the same day to evaluate intrasubject reproducibility. RESULTS: Volumetric analysis showed low intraobserver variability, with the coefficient of variation for the two observers ranging from 0.2% to 6.1%. Interobserver correlation was 0.99, and good concordance was shown with a mean volume difference of -1.8 mm(3). One-dimensional measurements had poorer intra and interobserver consistency. No statistically significant differences in volumetric measurements were observed between the two scans done on the same day in five subjects (P = 0.5). CONCLUSIONS: MRI volumetric growth plate measurement is a reproducible and sensitive method to evaluate meaningful growth plate volume changes over time. This tool, along with close monitoring of height and laboratory evaluations for bone metabolism, may be used to evaluate potential bone and growth toxicities of children enrolled in trials of investigational drugs.

Investigational new drug-directed toxicology and pharmacokinetic study of 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257) in Beagle dogs.

4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a 2-nitroimidazole-based hypoxia-selective cytotoxin has been shown to target hypoxic regions of solid tumours. The present study is one of several pre-clinical toxicology studies conducted in support of an 'investigational new drug' (IND) application to test this agent as an adjuvant to radio/chemotherapy for the treatment of cancer in humans. Twenty-four dogs were each assigned to one vehicle control group or to one of three test article-treated groups (three dogs/sex/treatment group). Intravenous (i.v.) doses of 0, 2.74, 5.48 and 10.95 mg/kg/day (54.8, 109.6 or 219 mg/m(2)/day) were administered on a per day x 5 days (qd x 5) schedule. NLCQ-1 was formulated as a solution in sterile saline at 1.5 mg/ml. None of the dogs died during this 33-day study. With few exceptions, most of the clinical signs of toxicity were noted within 2 hr following dosing in the 10.95 mg/kg/day dose group. These observations included aggressive behaviour, ataxia, tachypnea, emesis, hypoactivity, excessive salivation, tremors, and involuntary urination and defecation. Aggressive behaviour was judged to be dose-limiting. No clinical signs of toxicity were noted during the 28-day observation period that followed the 5-day dose period. Findings in a functional observation battery examination were consistent with the clinical observations. No drug-related effects were noted on the body weight or food consumption values, and no drug-related changes were noted during ocular examinations made on these animals prior to scheduled necropsy or during examination of electrocardiogram recordings made at 15 min. and 2 hr after dosing on days 1 and 5. No definitive changes in haematology, clinical chemistry or coagulation values were noted in dogs treated with NLCQ-1. NLCQ-1 was detected in the plasma of treated dogs on days 1 and 5, up to 60 min. after dosing (2.74 and 5.48 mg/kg/day) and up to 8 hr after dosing (10.95 mg/kg/day). There was a dose-related increase in maximum plasma concentration of NLCQ-1 at 5 min. after dosing; comparable concentrations were noted on days 1 and 5. No definitive test article-related lesions were noted during microscopic evaluation of tissues from dogs in this study, although lesions noted at the injection site and in the vascular tissue, lungs, thymus, prostate gland, muscle, adrenal cortex and tongue may have resulted from treatment with this drug. Any drug-related toxicity noted was readily reversible and not cumulative. No sex difference was detected in the susceptibility to NLCQ-1-induced toxicity.

Investigational new drug-directed, 5-day repeat dose toxicity study of 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257) administered with or without Taxol in Sprague-Dawley rats.

In pre-clinical studies, 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257) is a weak DNA-intercalating, hypoxia-selective cytotoxin with a promising profile as an adjuvant to radio/chemotherapy and it is about to enter phase I clinical trials. The present investigation was undertaken to further evaluate potential systemic toxicity induced by i.v. doses of NLCQ-1 alone or in combination with Taxol in Sprague-Dawley rats, in support of an investigational new drug application. Doses of NLCQ-1 were based on previous range-finding studies. In the present study, NLCQ-1 was administered either alone, at 0, 6, 9 or 12 mg/kg/dose to male rats and 8, 12 or 16 mg/kg/dose to female rats or, at 9 (male rats) and 12 (female rats) mg/kg/dose, in combination with Taxol, on a qd x 5 schedule. Taxol was administered i.v. at 3.5 mg/kg/dose 1 hr before NLCQ-1. Observations were recorded for mortality/moribundity, clinical signs of toxicity, body weights, food consumption, haematology, clinical chemistry, gross lesions at necropsy and histopathology. Blood samples were taken from 10 animals from each dose group on each of 2 days (days 8 and prior to scheduled necropsy on day 33). Administration of i.v. doses of NLCQ-1 alone, on a qdx5 schedule, resulted in no signs of toxicity over the 33-day study. Taxol-induced toxicity included minimal decreases in the group mean RBC, haemoglobin and haematocrit values, minimal increases in group mean reticulocyte counts (females), marked decreases in group mean neutrophil counts and minimal decreases in group mean monocyte and eosinophil counts. Lymphoid atrophy of thymus, atrophy of bone marrow and atrophy of the germinal epithelium of the testis were also associated with the administration of Taxol. There was no additional toxicity associated with the co-administration of NLCQ-1 and Taxol. In the present study, the 'no observable adverse effect level' for NLCQ-1, when administered on a qdx5 schedule, was >12 and >16 mg/kg/dose in male and female rats respectively. Daily administration of 9 (male rats) or 12 (female rats) mg/kg of NLCQ-1 1 hr after i.v. administration of Taxol (3.5 mg/kg) had no effect on Taxol-induced toxicity.

Current regulatory perspectives on genotoxicity testing for botanical drug product development in the U.S.A.

Genotoxicity testing is an important part of preclinical safety assessment of new drugs and is required prior to Phase I/II clinical trials. It is designed to detect genetic damage such as gene mutations and chromosomal aberration, which may be reflected in tumorigenic or heritable mutation potential of the drug. Botanical new drugs in the U.S. are entitled to a waiver for preclinical pharmacology/toxicology studies, including genotoxicity testing, in support of an initial clinical trial under IND, contingent on previous human experience. Recently, ethical concerns have been raised over conducting Phase I/II clinical trials of new drugs with positive genotoxicity findings in healthy volunteers. Although the relevance of this issue to patients, as opposed to healthy volunteers, depends on the drug's indication, duration of treatment, and specific findings related to the assays, the regulatory view is to avoid exposing patients to genotoxic compounds unnecessarily in clinical trials. This philosophy may impact on herbal supplement marketing and botanical drug development, in that genotoxicity data are often lacking while consumers are exposed to the herbal supplement, or healthy volunteers are tested in an initial Phase I/II clinical trial on the botanical drug. This paper presents results of a survey conducted on genotoxicity data in botanical INDs submitted to the Agency and discusses the significance of this information. The information presented indicates that the sponsors of botanical INDs have increasingly recognized the importance of genotoxicity information and may have prioritized its acquisition in their strategic drug development programs.

A two-stage algorithm for designing phase I cancer clinical trials for two new molecular entities.

The continual reassessment method (CRM) and subsequent developments of the Bayesian approach provide important tools for the design of Phase I cancer clinical trials for a new molecular entity. In recent years the idea of developing a treatment composed of two molecular entities has been proposed. For example, for some tumor types there may be two signaling pathways, both of which need to be blocked simultaneously using two molecules to achieve therapeutic benefit. A two-stage Bayesian and likelihood based algorithm is introduced herein for designing Phase I cancer clinical trials for two new molecular entities. It starts with a modified CRM approach in the first stage and makes use of the accumulated data from the first stage to provide likelihood estimates of model parameters for use in the second stage.

Alternative strategies for toxicity testing of species-specific biopharmaceuticals.

Although toxicology studies should always be conducted in pharmacologically relevant species, the specificity of many biopharmaceuticals can present challenges in identification of a relevant species. In certain cases, that is, when the clinical product is active only in humans or chimpanzees, or if the clinical candidate is active in other species but immunogenicity limits the ability to conduct a thorough safety assessment, alternative approaches to evaluating the safety of a biopharmaceutical must be considered. Alternative approaches, including animal models of disease, genetically modified mice, or use of surrogate molecules, may improve the predictive value of preclinical safety assessments of species-specific biopharmaceuticals, although many caveats associated with these models must be considered. Because of the many caveats that are discussed in this article, alternative approaches should only be used to evaluate safety when the clinical candidate cannot be readily tested in at least one relevant species to identify potential hazards.

[Experimental treatment of malignant melanoma and its rationale]. / Experimentelle Tumortherapie beim malignen Melanom und ihre Rationale.

To treat malignant melanoma successfully currently means to recognize the tumor at an early stage and to remove it immediately. Aside from individual cases, available treatment modalities are not able to increase survival, especially in the palliative situation. Thus innovative experimental approaches are urgently needed to strongly improve the palliative and adjuvant treatment of melanoma. Anti-tumor effects are expected from targeted therapies, which are directed against defined molecules decisive for tumor pathogenesis. Crucial points of attack are signaling pathways, angiogenesis and apoptosis resistance. New diagnostic and therapeutic developments have enhanced the efficacy of chemotherapies. Increasing insights into tumor immunology provide new treatment approaches of vaccination, cell transfer and especially of blocking immune tolerance mechanisms. It will be challenging for the future to identify and characterize more precisely those patients who might most benefit from a certain treatment approach.