Strategies for the early detection of drug-induced hepatic steatosis in preclinical drug safety evaluation studies

Hepatic steatosis is characterized by the accumulation of lipid droplets in the liver. Although relatively benign, simple steatosis can eventually lead to the development of steatohepatitis, a more serious condition characterized by fibrosis, cirrhosis, and eventual liver failure if the underlying cause is not eliminated. According to the "two hit" theory of steatohepatitis, the initial hit involves fat accumulation in the liver, and a second hit leads to inflammation and subsequent tissue injury. Because some xenobiotics target liver fatty acid metabolism, especially mitochondrial β-oxidation, it is important to avoid potential drug candidates that can contribute to either the initiation of liver steatosis or progression to the more injurious steatohepatitis. The gold standard for the detection of these types of hepatic effects is histopathological examination of liver tissue. In animal studies, these examinations are slow, restricted to a single sampling time, and limited tissue sections. Recent literature suggests that rapid in vitro screening methods can be used early in the drug R&D process to identify compounds with steatotic potential. Further, progress in the identification of potential serum or plasma protein biomarkers for these liver changes may provide additional in vivo tools to the preclinical study toxicologist. This review summarizes recent developments for in vitro screening and in vivo biomarker detection for steatotic drug candidates.     

A recombinant protein LHRH-PE40 for tumour therapy: preclinical safety studies

LHRH-PE40, a recombinant DNA-derived protein composed of LHRH and Pseudomonas aeruginosa exotoxin A, is being developed for the treatment of malignant tumours. This experiment was designed to assess its preclinical safety. Reproductive toxicity studies, pharmacokinetic studies, single- and repeat-dose intraperitoneal or intravenous toxicity studies in mice, rats and monkeys were conducted to assess the toxicity of LHRH-PE40. In intravenous single-dose studies in mice, the LD50 was 731.26 microg/kg and 676.03 microg/kg in male and female mice respectively. In single-dose studies and repeat-dose range-finding studies in rats, dose-limited severe vascular leakage syndromes occurred. In repeat-dose long-term studies, except drug-related vascular leakage syndromes, other drug-related changes included decreased testis weight and testis atrophy. In single-dose and repeat-dose studies in monkeys, dose-limited acute tubular necrosis of the kidneys was the chief finding. In reproductive studies, drug-related changes were decreased food intakes, decreased testis weight and uterus weight, decreased foetus weight and increased foetus mortality, increased maternal and F1 offspring mortality and decreased maternal and F1 offspring body weight. Pharmacokinetic studies showed a similar half-time of distribution and clearance in mice and monkeys. Tissue distribution showed a high concentration in the kidneys and a low concentration in the brain. LHRH-PE40 induced vascular leak syndromes in rats and acute tubular necrosis in monkeys. It also led to testicle atrophy in rats and overt productive toxicity to parents and F1 generations in mice. Because of these findings, it should be monitored carefully in human clinical trials for things such as respiratory, urinary and reproductive toxicities.     

Novel technology to prepare oral formulations for preclinical safety studies

A novel method to prepare oral formulations, normally suspended dosage form, for preclinical safety studies in animals has been developed using a rotation/revolution mixer. Small hard balls made of zirconia were added to the mixing process to evaluate effectiveness in making a high quality suspension. The driving with balls loaded in the cylindrical container (vessel) of the mixer was quite efficient in dispersing and milling the particles of the active pharmaceutical ingredient (API) in an aqueous medium. The API powder and a small amount of oral aqueous medium (vehicle) were successfully mixed by the spinning motion of the balls in the vessel as though the paste-like suspension was kneaded with a mortar and pestle. It was found that the milled suspension with the mean size of 10–20 μm could be prepared, in addition finer milling of less than 10 μm could be achieved by selecting the material of vessel. Optimum driving conditions including mixing time, size and quantity of balls, and the standard operational procedure was established using compounds varying in physicochemical properties. The particle size and quantitative analysis by HPLC showed that the resultant suspension was well-milled and highly homogeneous with the nearly intended concentration of API. The proposed method established by this experiment could be applied to the actual safety studies in the real preparation scale of oral suspension.     

An integrated process for measuring the physicochemical properties of drug candidates in a preclinical discovery environment

Automated log P, pK(a), solubility, and chemical stability systems comprise an integrated process that provides early stage physicochemical property data to the discovery research organization. Capillary electrophoresis (CE) techniques are used to experimentally determine pK(a) and log P. Solubility is determined using a quasi-equilibrium approach employing sample quantitation by flow injection analysis with ultraviolet (UV) detection at 256 nm. Chemical stability is assessed by challenging compounds with pH 2, pH 7, pH 12, and 3% hydrogen peroxide solutions overnight, and comparing the chromatographic profiles of the stability challenged solutions to that of a freshly prepared control. Validation of the log P method using a set of drug-like compounds demonstrates that the method yields log P values within +/-0.5 units of literature values. The log P method is valid over the range -0.5-5.0, and the technique is compatible with acidic, neutral, and basic compounds. The pK(a) technique yields results within +/-0.2 units of corresponding values obtained by potentiometric titration over a pK(a) range of 2 to 12. Solubility is reported in a 3-60 microg/mL range, and the results are generally within 20% of values measured by equilibrium solubility techniques. The current level of automation supports the measurement of the physicochemical properties of 100 compounds per week. Physicochemical property data for approximately 2000 compounds have been generated to date.     

A preclinical study on the safety of a new antialcoholic drug inmecarb

A preclinical study on safety of a new antialcoholic drug inmecarb was carried out on three species of experimental animals (rats, guinea pigs, dogs) receiving the drug at different doses including a subtoxic one (1/5 of LD50) for 6 months. The authors concluded that on the basis of a relatively low toxicity and the absence of specific toxicity and long-term side effects the drug may be recommended for clinical use as an antialcoholic agent.     

The impact of preclinical drug safety on R&D productivity

The Fifth International World Pharmaceutical Congress was held in the Philadelphia Convention Centre during May 23 – 25 2006. In addition to exhibits and three plenary presentations, there were six concurrent sessions. In this report the author summarises some of the highlights of the conference entitled ‘The impact of preclinical drug safety on R&D productivity’. This focused session gave a broad overview of the present state and future directions of drug safety assessment, both from a technical and organisational perspective.     

Methodological characteristics of the preclinical study of the safety of drug preparations for children

Some aspects of the preclinical study on safety of drugs used in children during experiments on immature animals are presented. Problems of the selection of animal species and the experimental schemes are discussed.     

Improved preclinical safety assessment using micro-BAL devices: the potential impact on human discovery and drug attrition

Hepatotoxicity is often unpredictable in the early phase of drug discovery and leads to drug attrition in preclinical and clinical development. Here, we discuss the conventional preclinical liver models that do not mimic in vivo livers. We focus on key components such as new sources of hepatocyte-derived human stem cells, enhanced direct oxygenation, defined biocompatibility nanoscaffolds, organotypical cellular models, dynamic culture, and metabolite status inside and outside the cell for effective configuration for the development of a bioartificial liver (BAL) device to mimic the in vivo liver microenvironment. The potential for development of BAL devices could open up new avenues in: (i) hepatotoxicity assessment for selecting drug candidates during preclinical screening; and (ii) therapeutic approaches for liver cell therapy at the clinical stage.     

Early preclinical studies of discriminable sedative and hallucinogenic drug effects

RATIONALE: One important technique in behavioral pharmacology is to train laboratory animals to discriminate between a psychoactive drug effect and a nondrug condition. Tests with different drugs have identified several categories of drugs that have different discriminable effects. OBJECTIVES: The two authors describe and discuss the early research on discriminable effects of sedative and hallucinogenic drugs and their acquaintance with each other at Yale University prior to their early and frequent publications on discriminable drug effects. Herb Barry studied sedative drugs primarily and Jim Appel studied hallucinogenic drugs. RESULTS: Sedative drugs include ethyl alcohol, barbiturates, and benzodiazepines. Their discriminable effects are largely attributable to the activation of an inhibitory neurotransmitter, gamma-amino butyric acid. Alcohol has the most pervasive effect in accordance with the high dose required to alter behavior. Hallucinogenic drugs include lysergic acid diethylamide and mescaline. They increase the activity of the neurotransmitter 5-hydroxytryptamine and, perhaps, dopamine in the central nervous system (CNS). In spite of their relatively low concentrations in the brain, both of these neurotransmitters have many important behavioral effects. CONCLUSIONS: Various sedative drugs cause a discriminable decrease in the function of the CNS. Different types of sedatives can be discriminated from each other. Indole and phenylethylamine hallucinogens have potent discriminative stimulus properties, which are related to the actions of biogenic amine neurotransmitters in the CNS.     

Androgenetic alopecia; drug safety and therapeutic strategies

Introduction: Androgenetic alopecia (AGA) is a benign condition with variable psychosocial impact, with some individuals adapting well while others needing therapeutic support. Although 5α-reductase inhibitors like finasteride and dutasteride have proven effective in ameliorating AGA, their use/selection is currently a subject of debate.

Areas covered: Treatment of AGA with 5α-reductase inhibitors lead to variable adverse effects and relatively unstable results (therapeutic efficacy ending with treatment cessation), so the choice of optimal therapy is not straightforward. This paper presents a general perspective regarding AGA based on studies listed in PubMed, to better understand/appreciate the opportunity for long term use of medication for a biological condition having non-life threatening implications. Studies focussed on adverse effects suggest that finasteride should be used with caution in AGA, due to considerable and persistent side effects induced in some men. In contrast, efficacy data indicate that dutasteride (a stronger inhibitor) presents superior therapeutic results compared to finasteride.

Expert opinion: This paper argues that finasteride should be preferred to dutasteride in the treatment of AGA. Thus, finasteride preserves important physiological roles of dihydrotestosterone (unrelated to AGA) and, in addition, its adverse effects seem to be (at least in part) predictable.     

From nonsteroidal aromatase inhibitors to multifunctional drug candidates: classic and innovative strategies for the treatment of breast cancer

Aromatase is the enzyme responsible for the conversion of androgens to estrogens and represents the main source of local estrogens in post-menopausal breast cancer tissue. Nonsteroidal aromatase inhibitors (NSAIs) are able to reduce growth-stimulatory effects of estrogens in hormone-dependent breast cancer, and third generation NSAIs are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic advanced breast cancer. Nevertheless, some issues in this area still need to be addressed and research efforts are aimed both at identifying new molecules of therapeutic interest and at exploring different options for the modulation of this enzyme. In this review, an update of the latest developments in the field of NSAIs is presented, to provide a broad view on the recent progress in this area. Beside classical structure-activity relationships studies and development of natural product derivatives, rational approaches for both ligand- and structure-based design are described. Moreover, novel strategies for the development of multitarget-directed molecules are also presented. Finally, some possible future developments in this research area are briefly considered.     

Biological basis of sex differences in drug abuse: preclinical and clinical studies

The recent focus on drug abuse in women has brought attention to numerous differences between women and men. In this review, we discuss both preclinical and clinical findings of sex differences in drug abuse as well as mechanisms that may underlie these differences. Recent evidence suggests that the progression to dependence and abuse may differ between women and men; thus, different prevention and treatment strategies may be required. Similar sex differences in drug sensitivity and self-administration have been reported in laboratory animal studies. Females appear to be more vulnerable than males to the reinforcing effects of psychostimulants, opiates, and nicotine during many phases of the addiction process (e.g. acquisition, maintenance, dysregulation-escalation, relapse). Male and female animals differ in their behavioral, neurological, and pharmacological responses to drugs. Although the role of sex in the mechanisms of drug action remains unclear, preclinical and clinical studies indicate that ovarian hormones, particularly estrogen, play a role in producing sex differences in drug abuse. Future research is necessary to provide information on how to design more effective drug abuse treatment programs and resources that are sex specific. Electronic Publication     

Psoriasis drug discovery: methods for evaluation of potential drug candidates

INTRODUCTION: Psoriasis is a complex disease with several clinical subtypes, as well as variations in body location and severity. Many patients suffering from psoriasis now benefit from the increased understanding of the pathogenesis of the disease, which in turn drives translational efforts to test new therapeutic concepts in the clinic. However, a multitude of treatment options is currently needed to satisfy patient needs. AREAS COVERED: This review describes the drug discovery platform in relation to psoriasis with special emphasis on how the major disease mechanisms of psoriasis can be studied in experimental in vitro and in vivo settings. The value of using humanized models and experimental clinical studies is highlighted. EXPERT OPINION: The successful development of novel therapies requires a translational approach to develop and implement the best preclinical and experimental clinical models and analytical tools that capture the various biological aspects of the disease. There is a need for more advanced in vitro skin models that contain the relevant cellular constituents as well as a need for careful validation of relevant in vivo models for psoriasis.