When generating monoclonal antibodies (mAb) against small molecules, the chemical composition and molecular orientation of the drug-like hapten on the antigen is a crucial determinant. This is especially important when attempting to discover therapeutic mAb against the drugs of abuse (ϩ)-methamphetamine, and the related compound (ϩ)-3,4-methylenedioxymethamphetamine [(ϩ)-MDMA, the plus isomer in the racemic mixture known as MDMA or ecstasy]. The goal of these studies was to design and synthesize (ϩ)-METH-like haptens with structural attributes that could make them effective for generating monoclonal antibodies for treating medical problems associated with these stimulant drugs of abuse. Five prototype (ϩ)-METH-like haptens, which mimic structural aspects of these drugs, were synthesized and used to generate mAb. After screening for anti-(ϩ)-METH IgG antibodies in more than 25,000 potential mouse hybridoma cell lines, one prototype mAb from each of the five haptens was selected and studied in detail for molecular properties and preclinical efficacy. The amino acid sequences of the IgGvariable regions, structural models, affinity, and ligand specificity of each mAb were then used to help elucidate important therapeutic characteristics. Four of these antibodies exhibited high affinity and specificity to (ϩ)-METH and (ϩ)-MDMA; whereas one antibody (designated mAb4G9) exhibited high affinity and specificity to (ϩ)-METH, (ϩ)-MDMA, and (ϩ)-AMP, without significant cross-reactivity against other METH-like ligands, over-the-counter medications, or endogenous neurotransmitters. Considered together, discovery of mAb4G9 and the other antibodies in this report represent an important step in understanding the process for custom design of drug class-specific therapeutic antibodies for the treatment of drug addiction. (ϩ)-Methamphetamine [(ϩ)-METH] abuse has becomeAmerica's number one drug threat (NACo, 2005), and effective treatment strategies for abuse of (ϩ)-METH and related stimulants are greatly needed. Current pharmacotherapies for managing the acute cardiovascular system, central nervous system, and toxic effects are mostly supportive (Sato, 1992;Albertson et al., 1999;Richards et al., 1999); they do nothing to remove the drug from its sites of action in the brain. Also lacking are medications that can reduce or treat the medically crippling effects of (ϩ)-METH addiction. Monoclonal antibodies (mAb) provide an attractive potential medication that can target the drug instead of the site of action (Kosten and Owens, 2005). These high-affinity protein-based medications act as so-called pharmacokinetic antagonists, sequestering the drug in the bloodstream away from medically vulnerable tissues, such as the brain and heart. Unlike nicotine and cocaine where the effects are caused by a single, specific compound, drugs such as opiates (e.g., morphine), arylcyclohexylamines (e.g., phencyclidine) and amphetamines [e.g., (ϩ)-METH] are starting structures from which many pharmacologically similar compounds can be synthesize...

In addition to addiction, the repeated use of (+)-methamphetamine [(+)-METH], (+)amphetamine [(+)-AMP], or (±)-methylenedioxymethamphetamine [(±)-MDMA, commonly called ecstasy] canlead to life-threatening medical problems including cardiovascular injury, severe depression, and psychosis. Currently, there are no specific pharmacotherapies to treat these medical problems. In this study, we report the design and synthesis of two haptens, (S)-(+)-3-(9-carboxynonyloxy) methamphetamine [3a, (+)-METHMO10] and (S)-(+)-3-(5-carboxypentyloxy)methamphetamine [3b, (+)-METH MO6], and their use in generating high affinity (low K D value) monoclonal antibodies (mAbs) against (+)-METH, (+)-AMP, and/or (+)-MDMA. Based on results from the determination of mAb K D values and ligand specificity, the mAbs generated from hapten 3a showed the greatest promise for generating active and passive immunotherapies for treating overdose or addiction from (+)-METH-like stimulants.The abuse of (S)-(+)-methamphetamine [1a-(+)-METH] a and other amphetamine-like stimulants continues to be a major health problem worldwide. 1-3 Indeed, a study by RAND Corporation estimates the economic cost of (+)-METH use in the United States in 2005 was $23.4 billion. 4 This comprehensive estimate includes the economic burden of addiction, premature death, drug treatment, and many other aspects of the drugs impact on Americans. The 2008 National Survey on Drug Use and Health estimates that over 12 million individuals, aged 12 and older, had used (+)-METH in their lifetime, that 850,000 million had used (+)-METH during the past year, and that 314,000 individuals had used (+)-METH during the last month, which defines them as current users. 5 At present there are no specific pharmacotherapies for managing adverse (+)-METH-induced effects like acute overdose and chronic addiction. Preclinical studies in rats show that systemic administration of anti-(+)-METH monoclonal antibodies (mAbs) can rapidly remove the drug from its sites of action in critical tissues like the brain and heart suggesting that immunotherapy could provide an important new medical strategy for addressing (+)-METH-induced adverse health effects in humans, while others suggest antibody catalyzed inactivation of METH could be a possible therapeutic approach. 7 (S)-(+)-Amphetamine [1b, (+)-AMP], which is a major metabolite of (+)-METH and a drug of abuse, and (+)-methylenedioxymethamphetamine, the stimulant-inducing chemical in the racemic mixture of (±)-methylenedioxymethamphetamine [2, (±)-MDMA, commonly referred to as ecstasy] are two other widely abused and dangerous stimulants. The potency and stimulant effects of these (+)-METH-like compounds are influenced by the drug's stereochemistry, with the (+)-or (S)-isomers producing significantly more psychomimetic effects, stereotyped behavior and locomotor activity, 8 and increased production of reactive oxygen species in mice. 9 Given the medical importance of all three of these structurally related (+)-or (S)-isomers, it could be medicall...

IntroductionMonoclonal antibody (mAb) medications offer promising treatment for autoimmune disease, cancer, and a host of other diseases including substance abuse [1][2][3]. These medications have several advantages over traditional small molecule medicines. For example, mAbs have high affinity and precise selectivity for their disease targets, act as pharmacokinetic antagonists to block or blunt the action of their target antigen, and have an extremely long biological halflife (e.g., ~21 days for human IgG 1 ) [4]. The long half-life is a particularly valuable pharmacological property since it allows for very long intervals between dosing. This long mAb half-life is usually assumed to result in an equally prolonged binding function, but direct evidence for prolonged function is often difficult to obtain. The use of antibodies for treatment of drug abuse and addiction provides a novel therapeutic opportunity that would greatly benefit from a prolonged pharmacological action and less need for daily patient compliance. Toward this goal, our group has produced a significant number of mouse mAbs for use in preclinical studies of the acute and chronic treatments of adverse health effects associated with (+)-methamphetamine (METH) abuse [5][6][7]. The METHspecific mAb were produced using several METH-like haptens, coupled to a carrier protein. These METH antigens are useful for active immunizations, in addition to generating mAb for passive immunization. Our METH-specific mAbs reverse or reduce the pharmacological actions of METH by pharmacokinetic antagonism, which includes high affinity mAb binding, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptVaccine. Author manuscript; available in PMC 2010 November 23. METH's primary sites of action are in the brain [8], and the drug has extensive extravascular distribution with an apparent volume of distribution (Vd) = 9 L/kg in rats [9]. In contrast, mAb medications are confined mostly to the plasma and extracellular fluid space, which has a Vd = 0.141 L/kg for mouse mAbs in rats [10]. This suggests the maximum theoretical reduction in the METH Vd in the presence of an anti-METH mAb would be about 64-fold. MAb high affinity binding is considered a critical mechanism for therapeutic action, and it is generally assumed that in vivo efficacy (and binding function) can be predicted by the in vitro mAb affinity (or K D value) for the target ligand. However, comparison of pharmacological data from acute and chronic in vivo studies with METH and various moderate-to-high affinity anti-METH mAbs has now lead ...

In a recent randomized, double-blind, placebo-controlled trial, we were able to demonstrate the superiority of a dietary supplement composed of essential amino acids (EAAs) over whey protein, in older adults with low physical function. In this paper, we describe the comparative plasma protein expression in the same subject groups of EAAs vs whey. The plasma proteomics data was generated using SOMA scan assay. A total of twenty proteins were found to be differentially expressed in both groups with a 1.5-fold change. Notably, five proteins showed a significantly higher fold change expression in the EAA group which included adenylate kinase isoenzyme 1, casein kinase II 2-alpha, Nascent polypeptide-associated complex subunit alpha, peroxiredoxin-1, and peroxiredoxin-6. These five proteins might have played a significant role in providing energy for the improved cardiac and muscle strength of older adults with LPF. On the other hand, fifteen proteins showed slightly lower fold change expression in the EAA group. Some of these 15 proteins regulate metabolism and were found to be associated with inflammation or other comorbidities. Gene Ontology (GO) enrichment analysis showed the association of these proteins with several biological processes. Furthermore, protein–protein interaction network analysis also showed distinct networks between upregulated and downregulated proteins. In conclusion, the important biological roles of the upregulated proteins plus better physical function of participants in the EAAs vs whey group demonstrated that EAAs have the potential to improve muscle strength and physical function in older adults. This study was registered with ClinicalTrials.gov: NCT03424265 “Nutritional interventions in heart failure.”

The anti-diabetic drug, metformin, has been reported to be beneficial for the cardiovascular system and may facilitate the extension of a healthier lifespan. Doxorubicin is a leading chemotherapeutic drug used to treat a variety of cancers, yet it can cause significant adverse effects with cardiac toxicity and longterm damage. To test the hypothesis that the hypoglycaemic agent, metformin, may protect normal cells during chemotherapy treatment with liposomal doxorubicin, C2C12 myoblast cells were used to study cellular bioenergetics, variations in gene expressions and biochemical alterations induced by metformin and pegylated liposomal doxorubicin (L-Doxo) under low glucose conditions (2.7 mM or 50 mg/dL). Using confocal microscopy, we noted that treatment of C2C12 cells with 30 µg/mL L-Doxo under low glucose conditions induced a number of cellular defects. L-Doxo treatment dysregulated the expression of mitochondrial fission and fusion genes, which may influence transformation of the network’s connectivity. L-Doxo significantly reduced mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). However, pre-treatment of cells with 100 nM metformin provided protection against L-Doxo-induced damage and increased cell viability and ATP levels in cells even under low glucose conditions. In addition, metformin increased and restored the decreased OCR and ECAR. Our data provide a mechanism by which low dose metformin exerts protective effects against L-Doxo via involvement of AMPKα under low glucose conditions. Taken together, our results demonstrate that metformin protects normal cells from L-Doxo damage even under low glucose conditions.