Can We Panelize Seizure?

Seizure liability remains a significant cause of attrition in drug discovery and development, leading to loss of competitiveness, delays, and increased costs. Current detection methods rely on observations made in in vivo studies intended to support clinical trials, such as tremors or other abnormal movements. These signs could be missed or misinterpreted; thus, definitive confirmation of drug-induced seizure requires a follow-up electroencephalogram study. There has been progress in in vivo detection of seizure using automated video systems that record and analyze animal movements. Nonetheless, it would be preferable to have earlier prediction of seizurogenic risk that could be used to eliminate liabilities early in discovery while there are options for medicinal chemists making potential new drugs. Attrition due to cardiac adverse events has benefited from routine early screening; could we reduce attrition due to seizure using a similar approach? Specifically, microelectrode arrays could be used to detect potential seizurogenic signals in stem-cell-derived neurons. In addition, there is clear evidence implicating neuronal voltage-gated and ligand-gated ion channels, GPCRs and transporters in seizure. Interactions with surrounding glial cells during states of stress or inflammation can also modulate ion channel function in neurons, adding to the challenge of seizure prediction. It is timely to evaluate the opportunity to develop an in vitro assessment of seizure linked to a panel of ion channel assays that predict seizure, with the aim of influencing structure-activity relationship at the design stage and eliminating compounds predicted to be associated with pro-seizurogenic state.

Toxicology Evaluation of Drugs Administered via Uncommon Routes: Intranasal, Intraocular, Intrathecal/Intraspinal, and Intra-Articular.

As the need for nasal, ocular, spinal, and articular therapeutic compounds increases, toxicology assessments of drugs administered via these routes play an important role in human safety. This symposium outlined the local and systemic evaluation to support safety during the development of these drugs in nonclinical models with some case studies. Discussions included selection of appropriate species for the intended route; conducting nonclinical studies that closely mimic the intended use with adequate duration; functional assessment, if deemed necessary; evaluation of local tissues with special histological staining procedure; and evaluations of safety margins based on local and systemic toxicity.

HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Immunomodulation and lymphoma in humans.

Observational and clinical studies have associated increased cancer risks with primary or acquired immunodeficiencies, autoimmunity, and use of immunotherapies to treat chronic inflammation (e.g. autoimmunity) or support organ engraftment. Understanding of the relationship between immune status and cancer risk is generally grounded in two juxtaposing paradigms: that the immune system protects the host via surveillance of tumors and oncogenic viruses (e.g. immunosurveillance model) and that chronic inflammation can augment tumor growth and metastasis (inflammation model). Whereas these models support a role of immune status in many cancers, they are insufficient to explain the disproportionate increase in B-cell lymphoma risk observed across patient populations with either chronic immunosuppression or inflammation. Evaluation for the presence of Epstein-Barr virus (EBV) in lymphomas obtained from various populations demonstrates a variable role for the virus in lymphomagenesis across patient populations. An evaluation of the DNA alterations found in lymphomas and an understanding of B-cell ontogeny help to provide insight into the unique susceptibility of lymphocytes, primarily B-cells, to oncogenic transformation. EBV-independent B-cell oncogenic transformation is driven by chronic antigenic stimulation due to either inflammation (as seen in patients with autoimmune disease or a tissue allograft) or to unresolved infection (as seen in immunosuppressed patients), and the transformation arises as a result of DNA damage from genomic recombination and mutation during class switching and somatic hypermutation. This model explains the increased background rate of lymphoma in some patients with autoimmunity, and highlights the challenge of resolving the confounding that occurs between disease severity and use of targeted immunotherapies to treat chronic inflammation. The ability to distinguish between disease- and treatment-related risk of lymphoma and an appreciation of the etiology of B-cell transformation is central to an improved risk assessment by scientists, clinicians and regulators, including the approval, labeling, and chronic use of immunotherapies.

Uniform assessment and ranking of opioid µ receptor binding constants for selected opioid drugs.

The safe disposal of unused opioid drugs is an area of regulatory concern. While toilet flushing is recommended for some drugs to prevent accidental exposure, there is a need for data that can support a more consistent disposal policy based on an assessment of relative risk. For drugs acting at the Mu-opioid receptor (MOR), published measurements of binding affinity (K(i)) are incomplete and inconsistent due to differences in methodology and assay system, leading to a wide range of values for the same drug thus precluding a simple and meaningful relative ranking of drug potency. Experiments were conducted to obtain K(i)'s for 19 approved opioid drugs using a single binding assay in a cell membrane preparation expressing recombinant human MOR. The K(i) values obtained ranged from 0.1380 nM (sufentanil) to 12.486 µM (tramadol). The drugs were separated into three categories based upon their K(i) values: K(i) > 100 nM (tramadol, codeine, meperidine, propoxyphene and pentazocine), K(i)=1-100 nM (hydrocodone, oxycodone, diphenoxylate, alfentanil, methadone, nalbuphine, fentanyl and morphine) and K(i) < 1 nM (butorphanol, levorphanol, oxymorphone, hydromorphone, buprenorphine and sufentanil). These data add to the understanding of the pharmacology of opioid drugs and support the development of a more consistent labeling policies regarding safe disposal.

Continuing education course #3: current practices and future trends in neuropathology assessment for developmental neurotoxicity testing.

The continuing education course on Developmental Neurotoxicity Testing (DNT) was designed to communicate current practices for DNT neuropathology, describe promising innovations in quantitative analysis and noninvasive imaging, and facilitate a discussion among experienced neuropathologists and regulatory scientists regarding suitable DNT practices. Conventional DNT neuropathology endpoints are qualitative histopathology and morphometric endpoints of particularly vulnerable sites (e.g., cerebral, cerebellar, or hippocampal thickness). Novel imaging and stereology measurements hold promise for automated analysis of factors that cannot be effectively examined in routinely processed specimens (e.g., cell numbers, fiber tract integrity). The panel recommended that dedicated DNT neuropathology data sets be acquired on a minimum of 8 sections (for qualitative assessment) or 3 sections (for quantitative linear and stereological analyses) using a small battery of stains to examine neurons and myelin. Where guidelines permit discretion, immersion fixation is acceptable for younger animals (postnatal day 22 or earlier), and peripheral nerves may be embedded in paraffin. Frequent concerns regarding DNT data sets include false-negative outcomes due to processing difficulties (e.g., lack of concordance among sections from different animals) and insensitive analytical endpoints (e.g., qualitative evaluation) as well as false-positive results arising from overinterpretation or misreading by inexperienced pathologists.

Compilation of international regulatory guidance documents for neuropathology assessment during nonclinical general toxicity and specialized neurotoxicity studies.

Neuropathology analyses as end points during nonclinical efficacy and toxicity studies are challenging and require trained personnel and particular equipment to achieve optimal results. Accordingly, many regulatory agencies have produced explicit guidelines for designing and performing neuropathology assessments for nonclinical studies. This compilation of international regulatory guidance for toxicologic neuropathology end points represents a set of criteria recommended for general toxicity studies and specialized neurotoxicity studies that should facilitate the efforts of individuals who plan, perform, analyze, and report neuropathology evaluations in nonclinical toxicity studies.

Use of anesthetic agents in neonates and young children.

BACKGROUND: Some drugs used for sedation and anesthesia produce histopathologic central nervous system changes in juvenile animal models. These observations have raised concerns regarding the use of these drugs in pediatric patients. We summarized the findings in developing animals and describe the steps that the Food and Drug Administration (FDA) and others are taking to assess potential risks in pediatric patients. The FDA views this communication as opening a dialog with the anesthesia community to address this issue. METHODS: We reviewed the available animal studies literature examining the potential neurotoxic effects of commonly used anesthetic drugs on the developing brain. The search strategy involved crossing the keywords neurotoxic and neuroapoptosis with the following general and specific terms: anesthetic, N-methyl-d-aspartate (NMDA), ketamine, midazolam, lorazepam, fentanyl, methadone, morphine, meperidine, isoflurane, nitrous oxide, sevoflurane, halothane, enflurane, desflurane, propofol, etomidate, barbiturate, methoxyflurane, and chloral hydrate. We summarized several studies sponsored by the FDA in rats and monkeys, initially examining the potential for ketamine, as a prototypical agent, to induce neurodegeneration in the developing brain. RESULTS: Numerous animal studies in rodents indicate that NMDA receptor antagonists, including ketamine, induce neurodegeneration in the developing brain. The effects of ketamine are dose dependent. The data suggest that limiting exposure limits the potential for neurodegeneration. There is also evidence that other general anesthetics, such as isoflurane, can induce neurodegeneration in rodent models, which may be exacerbated by concurrent administration of midazolam or nitrous oxide. There are very few studies that have examined the potential functional consequences of the neurodegeneration noted in the animal models. However, the studies that have been reported suggest subtle, but prolonged, behavioral changes in rodents. Although the doses and durations of ketamine exposure that resulted in neurodegeneration were slightly larger than those used in the clinical setting, those associated with isoflurane were not. There are insufficient human data to either support or refute the clinical applicability of these findings. CONCLUSIONS: Animal studies suggest that neurodegeneration, with possible cognitive sequelae, is a potential long-term risk of anesthetics in neonatal and young pediatric patients. The existing nonclinical data implicate not only NMDA-receptor antagonists, but also drugs that potentiate gamma-aminobutyric acid signal transduction, as potentially neurotoxic to the developing brain. The potential for the combination of drugs that have activity at both receptor systems or that can induce more or less neurotoxicity is not clear; however, recent nonclinical data suggest that some combinations may be more neurotoxic than the individual components. The lack of information to date precludes the ability to designate any one anesthetic agent or regimen as safer than any other. Ongoing studies in juvenile animals should provide additional information regarding the risks. The FDA anticipates working with the anesthesia community and pharmaceutical industry to develop strategies for further assessing the safety of anesthetics in neonates and young children, and for providing data to guide clinicians in making the most informed decisions possible when choosing anesthetic regimens for their pediatric patients.

Reversal of acute effects of high dose morphine on lymphocyte activity by chlorisondamine.

To explore the mechanisms mediating the effects of acute morphine on the immune system, effects of ganglionic blockade with chlorisondamine on acute high dose morphine-induced alterations in blood lymphocyte proliferation, white blood cell counts, spleen lymphocyte proliferation and splenic natural killer (NK) cell cytolytic activity were examined in male Sprague--Dawley rats. Two hours after morphine (30 mg/kg, s.c.) administration, blood lymphocyte proliferation (ConA) was decreased 85%; this effect was antagonized by chlorisondamine (5 mg/kg, i.p.). Notably, however, such morphine exposure did not significantly decrease splenic lymphocyte proliferation, although depression of NK cell activity was also evident and appeared to be chlorisondamine-sensitive. Immune effects of morphine 1 h after treatment were somewhat different. In this case, blood lymphocyte proliferation decreased and plasma levels of corticosterone increased, with ED(50) values of 2.2 and 7.8 mg/kg, respectively. Splenic lymphocyte proliferation and NK activity were also significantly depressed in the 1-h exposure paradigm, but only after administration of 30 mg/kg morphine. These results indicate that chlorisondamine blocks the effects of relatively high doses of morphine on blood lymphocyte activity and indicate that blood lymphocyte proliferation is more sensitive to effects of acute morphine exposure than splenic lymphocyte proliferation, NK cell cytolytic activity and activation of the HPA axis.

Discovery of radio emission from the brown dwarf LP944-20.

Brown dwarfs are not massive enough to sustain thermonuclear fusion of hydrogen at their centres, but are distinguished from gas-giant planets by their ability to burn deuterium. Brown dwarfs older than approximately 10 Myr are expected to possess short-lived magnetic fields and to emit radio and X-rays only very weakly from their coronae. An X-ray flare was recently detected on the brown dwarf LP944-20, whereas previous searches for optical activity (and one X-ray search) yielded negative results. Here we report the discovery of quiescent and flaring radio emission from LP944-20, with luminosities several orders of magnitude larger than predicted by the empirical relation between the X-ray and radio luminosities that has been found for many types of stars. Interpreting the radio data within the context of synchrotron emission, we show that LP944-20 has an unusually weak magnetic field in comparison to active M-dwarf stars, which might explain the previous null optical and X-ray results, as well as the strength of the radio emissions compared to those at X-ray wavelengths.

Acute effects of morphine on blood lymphocyte proliferation and plasma IL-6 levels.

Administration of morphine (10 mg/kg) to rats was found to decrease the proliferative potential of blood lymphocytes by 60-80% and concurrently elevate circulating levels of the cytokine, interleukin-6 (IL-6), 2- to 4-fold. Both parameters were similarly altered upon the central administration of morphine and were blocked upon pretreatment of animals with the opioid receptor antagonist, naltrexone. These results suggest that the activation of central opioid receptors is involved in morphine-induced inhibition of lymphocyte proliferation as well as increases in circulating levels of IL-6. Studies addressing the potential peripheral mechanisms demonstrated that intact ganglionic transmission was required for both effects of morphine. Although the suppression by morphine of lymphocyte proliferation appeared to be largely independent of stimulation of the hypothalamic-pituitary-adrenal axis, the elevation of IL-6 was completely abolished in adrenalectomized animals. Collectively, these results suggest that central opioid receptor activation results in changes in different immune parameters that can be mediated through distinct peripheral mechanisms.

The effects of morphine, nicotine and epibatidine on lymphocyte activity and hypothalamic-pituitary-adrenal axis responses.

Acute administration of morphine alters various neuroendocrine and immune parameters via opioid receptors located within the central nervous system. Similar effects have been reported after systemic nicotine treatment. To examine the possible relationship between opioid and nicotinic receptor activation on the immune system, we compared the effects of morphine with both nicotine and the highly selective nicotinic agonist, epibatidine. Male Sprague-Dawley rats were treated with either morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. = 1 mg/kg freebase), or epibatidine (5 microg/kg, s.c.) and sacrificed 2 hours later. Each drug increased plasma corticosterone levels and decreased the magnitude of the peripheral blood lymphocyte proliferation response to the T cell mitogen concanavalin A. None of the treatments had a significant effect on splenic or thymic lymphocyte responses. The effects of nicotine treatment were dose-dependent. Pretreatment with the quaternary ganglionic antagonist chlorisondamine (0.5 mg/kg, i.p.), completely blocked the effect of epibatidine on blood lymphocytes without altering the elevation of corticosterone levels. Although naltrexone (10 mg/kg, s.c.) blocked all effects of morphine, the effects of epibatidine were not blocked by the opioid receptor antagonist. Furthermore, in contrast to morphine (), central injection of neither nicotine (30 or 240 nmol) nor epibatidine (5, 50, or 500 ng) altered blood lymphocyte responses. These results suggest that, like morphine, nicotinic agonists decrease blood lymphocyte proliferation responses, apparently independent of elevated corticosterone. However, unlike morphine, nicotinic agonists appear to act predominantly at peripheral receptors, suggesting that nicotinic receptors are downstream of opioid receptors in a centrally mediated opioid-induced immunomodulatory pathway.

Relationship between endocervical canal length between 15-24 weeks gestation and obstetric history.

The object was to determine whether there is a correlation between the obstetric history and the ultrasonographically determined endocervical canal length between 15 and 24 weeks gestation. A retrospective cohort study was performed in singleton pregnancies of multigravidas with normal and abnormal obstetric histories. They underwent sonographic evaluation for the determination of the endocervical canal length between 15 and 24 weeks gestation. The shortest endocervical canal length measurements between 15 and 20 weeks and also between 21 and 24 weeks of gestation were recorded. An ultrasound diagnosis of cervical incompetence was defined as progressive shortening of the endocervical canal length to <2 cm or a single endocervical canal length measurement <2 cm. A multivariable general linear regression model was used to correlate the relationship between endocervical canal lengths at 15-20 weeks and 21-24 weeks gestation in the current pregnancy with the earliest gestational age at delivery of prior pregnancies. Chi-square test was used to determine the relationship between the development of an ultrasound diagnosis of cervical incompetence and the earliest gestational age at delivery of prior pregnancies. A total of 155 pregnancies were studied. The number of women according to the obstetric history categories were: 57 had delivered <24 weeks, 12 between 24 and 26 weeks, 16 between 27 and 32 weeks, 16 between 33 and 36 weeks, and 54 delivered > or =37 weeks. There was a significant correlation between the endocervical canal length measurements between 15-20 (P < 0.0001) weeks and 21-24 weeks (P < 0.0001) in the studied pregnancy and the earliest gestational age at delivery of prior pregnancies. A significant relationship between the ultrasound diagnosis of cervical incompetence and the obstetric history category (P = 0.0026) was observed. There were 36 cases of ultrasound diagnosed cervical incompetence with 91.7% (33/36) occurring in women who had a prior <27 weeks' gestation delivery. These data provide further evidence that cervical incompetence is a relative condition and not an "all or none" phenomenon. In addition, women with a prior delivery <30 weeks gestation should be followed with second trimester serial cervical sonography to rule out cervical incompetence.

Evidence for central opioid receptors in the immunomodulatory effects of morphine: review of potential mechanism(s) of action.

This review will discuss studies demonstrating that activation of opioid receptors within the central nervous system alters various immune system parameters. Specifically, natural killer cell cytolytic activity and lymphocyte proliferative responses to mitogen appear to be modulated predominantly, if not exclusively, through central opioid receptors. The potential mechanisms by which central opioid receptors appear to modulate these peripheral immune functions will be examined by evaluating the role of both the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system. The studies discussed below indicate that acute administration of morphine or related compounds appears to primarily alter peripheral immune function through the sympathetic nervous system, while more prolonged exposure to opioids alter the immune system predominantly by activation of the HPA axis. Finally, the potential clinical relevance of these observations are discussed in relationship to both the therapeutic use, as well as the abuse of opioid compounds.

Role of central opioid receptor subtypes in morphine-induced alterations in peripheral lymphocyte activity.

Morphine has been shown to decrease proliferative responses of rat T-lymphocytes via central opioid receptors, however, the specific receptor subtype(s) mediating this effect have not been established. To determine the potential role of central mu opioid receptors in morphine-mediated suppression of T-lymphocyte proliferation, 20 nmol/2 microliters of either morphine sulfate or DAMGO (mu-selective agonist) were administered into the lateral ventricle of freely moving Sprague-Dawley rats. Lymphocyte proliferative response to the polyclonal T cell mitogen concanavalin A (ConA), changes in splenic natural killer cell (NK) cytolytic activity, activation of the hypothalamic-pituitary-adrenal (HPA) axis and antinociception (tail-flick latency) were examined. Results indicated that like morphine, DAMGO decreased blood lymphocyte proliferative responses by 80% and elevated both tail-flick latency and plasma corticosterone when compared to saline-treated animals. The proliferation response of lymphocytes from the spleen or thymus and splenic NK cell activity were not significantly altered by either morphine or DAMGO treatment. The effects of DAMGO were determined to be dose-dependent and completely antagonized by naltrexone pretreatment. Central administration of DPDPE (delta-selective agonist) and U-50488 (kappa-selective agonist) produced between 40-50% suppression of blood lymphocyte proliferation responses only at a dose five times greater (100 nmol) than DAMGO treatment, without altering antinociception or activation of the HPA axis. To determine the central opioid receptor subtype(s) involved in the effects of morphine, selective opioid antagonists were microinjected into the lateral ventricle prior to morphine treatment (6 mg/kg, s.c.). CTOP (mu-selective antagonist, 5 micrograms/2 microliters) completely blocked the effects of morphine on all parameters measured, however, naltrindole (delta-selective antagonist, 2 micrograms/2 microliters) or nor-binaltorphimine (kappa-selective antagonist, 73.5 micrograms/2 microliters) failed to block the effects of morphine. Collectively, these results provide evidence that morphine acts primarily through central mu receptors to modulate peripheral blood lymphocyte proliferation responses. Further, the antinociception and blood lymphocyte effects show greater sensitivity to opioids than either natural killer cell cytolytic activity or activation of the HPA axis.

Tracer technique to measure in vivo chemical transport rates within an implantable cell transplantation device.

An in vivo tracer technique that uses radiolabeled insulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the host's circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device.