CNS & Neurological Disorders - Drug Targets (Formerly Current Drug Targets - CNS & Neurological Disorders) - Volume 10, Issue 8, 2011

A leading hypothesis for the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS) is excitotoxicity mediated by Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The AMPA receptors are assembled from 4 subunits and their permeability for Ca2+ is determined by the absence of the glutamate receptor subunit type 2 (GluR2), associated with an increased portion of unedited GluR2 mRNA at the glutamine/arginine (Q/R) site. RNA editing at this location is predominantly catalyzed by adenosine deaminase acting on RNA 2 (ADAR2). Thus, the observation that a substantial portion of Q/R site unedited GluR2 mRNA is present in postmortem spinal cord tissue of sporadic ALS patients suggests inefficiency of ADAR2 to be a disease-specific molecular dysfunction in ALS. To decide whether this malfunction could be a cause of motor neuron death in vivo, Hideyama and colleagues developed a conditional ADAR2 knock-out mouse line (designated as AR2), which carries gene-targeted floxed ADAR2 alleles that become functionally ablated by Cre recombinase expressed from a transgene (VAChT-Cre.Fast) in approximately 50% of motor neurons. They demonstrated that only the ADAR2-lacking spinal motor neurons degenerated in these animals, and this could be prevented by generating Q/R site edited GluR2 in the absence of ADAR2 by a point mutation in the endogenous GluR2 alleles. These results convincingly demonstrate that the inefficient GluR2 Q/R site editing found in the spinal cords of patients with sporadic ALS proposes the decreased level of ADAR2 mRNA to be a direct cause of death of motor neurons in mice, which accentuates the detrimental role of elevated Ca2+ influx in motor neurons as a consequence of increased Ca2+-permeability of AMPA receptors. From therapeutic point of view, an interesting aspect of the Ca2+-mediated cell death in ALS is connected with certain emotional reactions of the patients such as depression and anxiety, which has received particular attention in ALS. Although many researchers with experience with ALS challenge the lay opinion of an increased risk of developing major depressive disorder in ALS, studies using standardized methodology assessing nonphysical symptoms reported an occurrence exceeding the prevalence of that in the general population. There is growing evidence that besides the monoaminergic system the glutamatergic system, particularly N-methyl-D-aspartate receptor mediated Ca2+-entry plays an important role in the pathobiology of major depressive disorder. It is thus intriguing that conventional tricyclic antidepressants, specific serotonin reuptake inhibitors and serotonin/norepinephrine reuptake inhibitors not only down-regulate N-methyl-D-aspartate receptors but - at least in an in vitro model system - have the potency to enhance GluR2 Q/R site-editing either by up-regulating ADAR2 mRNA expression level, or through other unidentified mechanisms. It will now be important to determine whether these drugs exert a similar protective effect on motor neurons in in vivo models of ALS, and thus constitute a new therapeutic approach for this disease.

As for other psychiatric disorders, pharmacological approaches to treating substance use disorders (SUDs) have traditionally focused on small molecules. This approach has not yielded highly effective medications. Perhaps the most glaring example is the absence of medications approved to treat stimulant (e.g. cocaine and methamphetamine) abuse, with no approvals on either the short- or mid-term horizon. Moreover, medication options to treat other SUDs, when available, are far from ideal. Using nicotine addiction as an example, the rate of sustained (1 year) abstinence in individuals treated with available smoking cessation medications (nicotine replacement therapies, bupropion, and varenicline) is below 20%. This special issue for CNS & Neurological Disorders-Drug Targets is devoted to biological approaches to treating SUDs. The approaches described here include a bioengineered esterase capable of rapidly inactivating cocaine as well as immunization strategies directed against methamphetamine, nicotine, cocaine, and heroin. The principle underlying these biological approaches is pharmacokinetic - preventing or slowing the entry of abused substance into the central nervous system. While not comprehensive, this collection of reviews provides the reader with insights about the feasibility and potential of biological approaches to treating SUDs. In this volume, Stephen Brimijoin overviews the development of a genetically engineered cocaine hydrolase as a means of “intercepting” cocaine before it reaches the CNS. As for both vaccines and monoclonal antibodies, this enzyme is highly specific, and will only reduce the entrance of cocaine (but not other stimulants or abused drugs) into the CNS. This article also highlights the potential for viral gene transfer as a novel strategy for relapse prevention. Shen and Kosten review the concept of immunotherapy for drug abuse, and describe a cocaine vaccine made by conjugating a cocaine containing hapten to inactivated cholera toxin B that is currently being tested in the clinic. Koob and coworkers describe preclinical studies using an anti-cocaine vaccine developed by coupling a cocaine analog to a disrupted adenovirus. The approach is based on studies demonstrating that adenovirus capsid proteins are highly immunogenic in humans, and if preclinical results can be successfully translated, would result in circulating levels of high-affinity, high-titer antibodies against cocaine. The contribution by Fahim et al. describes the development of a nicotine vaccine. While the initial proof of concept study [1] provided a significant efficacy signal, this vaccine failed to achieve its primary endpoint in Phase III clinical trials (NABI Pharmaceutical press release, www.nabi.com). The contribution by Stowe, et al. describes the challenges in developing a vaccine against heroin, which has several psychoactive metabolites. While the preceding chapters on vaccines rely on the ability of the immune system to raise antibodies targeted at the abused substance, the article by Owens et al. describes the development of monoclonal antibodies, a passive immunization technique, for the treatment of methamphetamine abuse. There are both advantages and disadvantages to these biological approaches. Perhaps the most compelling reason to develop biologics is the promise of a long-lived protective effect, which requires the patient to make “one good decision” to receive a biologic, compared to one (or multiple) good daily decisions to remain compliant with a traditional medication. The specificity of an effective vaccine developed against (e.g.) heroin should dramatically reduce the use of this opiate, but would neither preclude an individual from receiving maintenance therapy nor prevent the patient from abusing a structurally unrelated opiate. The ability of a patient to circumvent the therapy by taking drugs other than the one targeted by the biologic (e.g., an effective heroin vaccine would not preclude abuse of cocaine) is a limitation of these approaches. However, this is also an issue for current medications, exemplified by a heroin abuser using cocaine despite receiving methadone maintenance therapy. Given the high incidence of polysubstance abuse, even a highly effective biologic will require parallel interventions (including behavioral therapy) to modify lifestyles in order to either remove or mitigate precipitants of relapse. Immunization therapies currently in clinical trials can take weeks or months before antibody titers reach pharmacologically relevant levels [1, 2]. This “therapeutic lag”, taken together with the need for multiple immunizations and high patient expectations of a cure, could be problematic for the ultimate commercial success of such a product. Nonetheless, given the limited success of traditional pharmacological approaches to treating SUDs coupled with the rapid technological advances described in these reviews, biological approaches represent appealing alternatives that, when coupled with behavioral therapy, have the potential to dramatically change the practice of addiction medicine.

Heroin addiction is a wide-reaching problem with a spectrum of damaging social consequences. Currently approved heroin addiction medications include drugs that bind at the same receptors (e.g. opioid receptors) occupied by heroin and/or its metabolites in the brain, but undesired side effects of these treatments, maintenance dependence and relapse to drug taking remains problematic. A vaccine capable of blocking heroin's effects could provide an economical, long-lasting and sustainable adjunct to heroin addiction therapy without the side effects associated with available treatment options. Heroin, however, presents a particularly challenging vaccine target as it is metabolized to multiple psychoactive molecules of differing lipophilicity, with differing abilities to cross the blood brain barrier. In this review, we discuss the opiate scaffolding and hapten design considerations to confer immunogenicity as well as the specificity of the immune response towards structurally similar opiates. In addition, we detail different strategies employed in the design of immunoconjugates for a vaccine-based therapy for heroin addiction treatment.

Substance use disorders continue to be major medical and social problems worldwide. Current medications for substance use disorders have many limitations such as cost, availability, medication compliance, dependence, diversion of some to illicit use and relapse to addiction after discontinuing their use. Immunotherapies using either passive monoclonal antibodies or active vaccines have distinctly different mechanisms and therapeutic utility from small molecule approaches to treatment. They have great potential to help the patient achieve and sustain abstinence and have fewer of the above limitations. This review covers the cocaine vaccine development in detail and provides an overview of directions for developing anti-addiction vaccines against the abuse of other substances. The notable success of the first placebo-controlled clinical trial of a cocaine vaccine, TA-CD, has led to an ongoing multi-site, Phase IIb clinical trial in 300 subjects. The results from these trials are encouarging further development of the cocaine vacine as one of the first anti-addiction vaccines to go forward to the U.S. Food and Drug Administration for review and approval for human use.

Recent progress in enzyme engineering has led to versions of human butyrylcholinesterase (BChE) that hydrolyze cocaine efficiently in plasma, reduce concentrations reaching reward neurocircuity in the brain, and weaken behavioral responses to this drug. Along with enzyme advances, increasingly avid anti-cocaine antibodies and potent anti-cocaine vaccines have also been developed. Here we review these developments and consider the potential advantages along with the risks of delivering drug-intercepting proteins via gene transfer approaches to treat cocaine addiction.

Developing specific medications to treat (+)-methamphetamine (METH) addiction is a difficult challenge because METH has multiple sites of action that are intertwined with normal neurological function. As a result, no small molecule medication for the treatment of METH addiction has made it through the FDA clinical trials process. With the invention of a new generation of proteinbased therapies, it is now possible to consider treating drug addiction by an entirely different approach. This new approach is based on the discovery of very high affinity anti-METH monoclonal antibodies (mAbs), which are non-addictive and antagonize METH effects from the blood stream without entering the brain. Due to a very long biological half-life, anti-METH mAbs would only need to be administered once every 2-4 weeks, aiding in patient compliance. As a relapse prevention medication, anti-METH mAbs could reduce or prevent the rewarding effects of a relapse to METH use and thereby improve a patient's probability of remaining in therapy and recovering from their addiction. In this review, we discuss the discovery process of anti-METH mAbs, with a focus on the preclinical development leading to high affinity anti-METH mAb antagonists.

The challenge in developing an anti-cocaine vaccine is that cocaine is a small molecule, invisible to the immune system. Leveraging the knowledge that adenovirus (Ad) capsid proteins are highly immunogenic in humans, we hypothesized that linking a cocaine hapten to Ad capsid proteins would elicit high-affinity, high-titer antibodies against cocaine, sufficient to sequester systemically administered cocaine and prevent access to the brain, thus suppressing cocaine-induced behaviors. Based on these concepts, we developed dAd5GNE, a disrupted E1-E3- serotype 5 Ad with GNE, a stable cocaine analog, covalently linked to the Ad capsid proteins. In pre-clinical studies, dAd5GNE evoked persistent, high titer, high affinity IgG anti-cocaine antibodies, and was highly effective in blocking cocaine-induced hyperactivity and cocaine self-administration behavior in rats. Future studies will be designed to expand the efficacy studies, carry out relevant toxicology studies, and test dAd5GNE in human cocaine addicts.

Smoking is a global healthcare problem. Current smoking cessation rates using behavioral counseling and pharmacotherapeutic interventions have had modest success, with ∼1:5 smokers remaining abstinent long-term. Nicotine vaccines are a new class of immunotherapeutics under development. It is believed that anti-nicotine antibodies arising from vaccination capture nicotine and prevent or reduce its entry into the brain, as the antibody-bound nicotine is too large to cross the blood-brain barrier. This in turn decreases the pleasurable effects of smoking, reducing or eliminating positive reinforcement, thereby making it easier for a smoker to quit smoking. Four vaccine candidates have advanced into clinical testing with mixed success. Proof-of-concept has been established in that individuals with higher levels of anti-nicotine antibodies were observed to have higher smoking cessation and abstinence rates. Recently, the most advanced candidate vaccine, NicVAX, failed to meet the primary endpoint in two large phase III studies, although the correlation of higher abstinence rates in subjects with higher immunity to nicotine was observed. Although the field has had setbacks, the magnitude of the tobacco epidemic and the positive pre-clinical research and observed clinical trends indicate continued research is warranted. Several avenues are being actively pursued: a) improving vaccine potency by introducing novel carriers and/or adjuvants to stimulate higher immune response b) targeting subjects who have a robust response (e.g. personalized medicine) c) combining vaccines with pharmacotherapy for maintenance of abstinence/relapse prevention.

We assessed the effect of palmitoylethanolamide (PEA) on pain and nerve function in patients with chemotherapy-induced painful neuropathy, in 20 patients undergoing thalidomide and bortezomib treatment for multiple myeloma. All patients were evaluated before and after a two-month treatment with PEA 300 mg BID using pain and warmth thresholds; blinded examiners measured motor and sensory nerve fibre function and laser-evoked potentials. Although no variables returned to normal values, pain and all neurophysiological measures—assessing Aα, Aβ, and Aδ fibres— significantly improved (P < 0.05). In contrast, warmth thresholds, assessing unmyelinated afferents, remained unchanged (P > 0.50). Although a placebo effect might play a role in the reported pain relief, the changes in neurophysiological measures indicate that PEA exerted a positive action on myelinated fibre groups. PEA, possibly by moderating mast cell hyperactivity, relieved conduction blocks secondary to endoneural edema. In a severe condition such as painful neuropathy associated with multiple myeloma and chemotherapy, a safe substance such as PEA provides significant restoration of nerve function.

The progress of metabolic syndrome (MetS) continues with the onset of type-2 diabetes mellitus (Type-2 DM) along with linkage to other disorders such as neurodegenerative, especially Alzheimer's disease (AD), via oxidative stress and low grade systemic inflammatory process. Type-2 DM and AD are health disorders of priority research. The treatment for an individual suffering with Type- 2 DM and/or AD requires monitoring by clinicians. The aim of this study was to investigate the role of C-peptide and its correlation to insulin resistance, body mass index (BMI), β cell function, insulin sensitivity, lipid profile and hemoglobin A1c (HbA1c). The study was designed to include 96 Type-2 DM individuals from India. 58.3% males and 41.7% females were selected and fasting blood samples were collected for estimation of fasting C-peptide, fasting blood sugar (FBS), postprandial blood sugar (PPBS), HbA1c and lipid profile. Analysis was done on Hitachi912 and Elecsys 2010 using Roche reagents and standard controls. Anthropometries to calculate BMI and β cell function, insulin sensitivity, and insulin resistance were obtained. The statistical tool ANOVA, followed by calculation of p-value and r-value were applied for investigating correlation of C-peptide levels to those of high density lipoprotein-C (HDL-C), low density lipoprotein-C (LDL-C), triglycerides (TGL), HbA1c, β cell function, insulin sensitivity and insulin resistance. Highly significant positive correlations were observed in different quantiles of C-peptide levels to the studied parameters of MetS, BMI and % β cell function. Lower HDL-C level was found to be significantly related to higher C-peptide levels. Similarly, TGL and C-peptide levels displayed a significant positive correlation. A significant negative correlation was observed between C-peptide quantiles and % sensitivity. Thus, insulin resistance showed a positive correlation until the fourth quantile. No significant correlation was observed between C-peptide and HbA1c levels. This study demonstrates that assessment of C-peptide levels is a useful tool to monitor the progress of MetS among patients suffering from Type-2 DM and AD, as these disorders are intertwined to each other by common metabolic pathways. Assessment of C-peptide levels, along with HDL-C levels, in patients can be used to monitor insulin resistance.

Acetylcholinesterase (AChE)-inhibition is an area of priority research as various roles have been attributed to AChE in neurodegenerative disorders and cancer as well. In the present study, a comparative multiple 4 dimensional (4D)-approach was applied to analyze human platelet AChE-inhibition by cyclophosphamide (CP). AChE activity was assessed by measuring the hydrolysis of acetylthiocholine iodide (ASChI). The different perspective of analysis was based on two classical (Lineweaver-Burk as well as Dixon) plots, built-in equations of GOSA and a recently introduced graphical approach. Thus, various kinetic constants such as KI, Ks, Km, ksl, Vmao, Ki, ksli, Slmax, °Ks, K1/2, kcat and ksp were estimated. Previous findings of AChE (from different sources) inhibition by CP were also compared. This study extends the elucidation of the kinetic approach of analysis and quantifying enzyme-substrate and enzyme-inhibitor interactions, which is crucial to bringing any drug from bench to bedside. The acyl pocket of human AChE was found to interact with CP through the amino acid residues Y70, Y121, W233, F288, F290, Y334, F408 and Y442, while the anionic sub-site of catalytic site (CAS) interacted with the ligand through residues W84, N85, G116, G117, Y121, S122, G123, L127, Y130, E198, Y334, H443 and G444. CP displayed variable docking poses with the peripheral anionic site (PAS) of human AChE. The findings of kinetic analysis were reinforced by the results of docking experiments. Both the applied approaches strongly indicate partial mixed type of inhibition pattern for the study enzyme (AChE) and its inhibitor (CP).

Extremely low frequency electromagnetic fields (ELF-EMF) have been found to produce a variety of biological effects. These effects of ELF-EMF depend upon frequency, amplitude, and length of exposure, and are also related to intrinsic susceptibility and responsiveness of different cell types. Although the mechanism of this interaction is still obscure, ELF-EMF can influence cell proliferation, differentiation, cell cycle, apoptosis, DNA replication and protein expression. The aim of this study was to estimate various kinetic constants of catalase, cytochrome P450 and inducible nitric oxide synthase in response to ELF-EMF exposure in human HaCaT and THP-1 cell lines. In order to evaluate the effect of ELF-EMF on the modulation of cellular responses to an inflammatory stimulus, both cell lines were treated with lipopolysaccharide. To the best of our knowledge there is no available report on such type of kinetic study of selected enzymes in response to ELF-EMF in these cell lines. Therefore, the current study may reveal novel mechanism of ELFEMF biological interaction with the enzymological and hormonal systems of living organisms. These new insights may be important for ELF-EMF application particularly for wound healing, tissue regeneration, Parkinson's and Alzheimer's diseases.

This study aimed to investigate the acute modulatory effect of bromazepam, a benzodiazepine derivative drug, on alpha and beta bands (8-35Hz) in primary motor areas (M1) through event-related spectral perturbation (ERSP). Ten healthy subjects were submitted to a cross-over double-blind design. Subjects performed a visuomotor task where they had to identify rapidly the ball launched horizontally and catch it quickly, while electroencephalographic activity was acquired. We found a statistically significant difference on the time windows of 2920 ms for 13Hz in the electrodes C3 and Cz, and on the time window of 2000 ms for 18Hz in the electrodes C3, when compared the bromazepam and placebo conditions. We concluded that the acute effects of bromazepam provoked changes in information process in the left M1 represented by electrode C3 in both 13 Hz and 18 Hz. Our paradigm is relevant for a better understanding of the brain dynamics due to the information related to bromazepam effects on sensorimotor processes. We consider this report an invitation to conduct more studies in order to associate electro-cortical activity and psychometric tests.