S. Michael Owens

Sex differences in (+)-amphetamine- and (+)-methamphetamine-induced behavioral response in male and female Sprague-Dawley rats.

(+)-Methamphetamine (METH) and (+)-amphetamine (AMP) are structurally similar drugs that are reported to induce similar pharmacological effects in rats of the same sex. Because pharmacokinetic data suggest female rats should be more affected than males, the current studies sought to test the hypothesis that the behavioral and temporal actions of METH and AMP should be greater in female Sprague-Dawley rats than in males. Using a dosing regimen designed to reduce the possibility of tolerance and sensitization, rats were administered 1.0 and 3.0 mg/kg intravenous drug doses. Distance traveled, rearing events and focal stereotypies (e.g., head weaving, sniffing) were measured. Female rats traveled significantly greater distances and displayed a greater number of rearing events than males after both doses. Analysis of stereotypy ratings after 3.0 mg/kg revealed that focal stereotypies were more pronounced and lasted longer in females. The second study compared the potencies of METH and AMP in inducing locomotor activity and focal stereotypies in each sex. No differences in potency were found when METH and AMP effects were compared within males or females. In summary, these studies showed female rats displayed greater and longer-lasting locomotor activity and more stereotypic behaviors, supporting earlier evidence of significant sexual dimorphism in pharmacokinetics.

Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse.

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 (+)-methamphetamine [(+)-METH], (+)-amphetamine [(+)-AMP], and the related compound (+)-3,4-methylenedioxymethamphetamine [(+)-MDMA, the plus isomer in the racemic mixture known as MDMA or ecstasy]. The goal of these studies was to design and synthesize (+)-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 (+)-METH-like haptens, which mimic structural aspects of these drugs, were synthesized and used to generate mAb. After screening for anti-(+)-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 IgG-variable 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 (+)-METH and (+)-MDMA; whereas one antibody (designated mAb4G9) exhibited high affinity and specificity to (+)-METH, (+)-MDMA, and (+)-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.

Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats

Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution.

Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats.

The goal of these studies was to determine if chronic (+)methamphetamine ((+)METH) administration affects the production of anti-(+)METH antibodies during active immunization of rats. Active immunization for the treatment of chronic drug abuse has been proposed for drugs such as cocaine and nicotine. However, studies have not adequately addressed whether continual drug use during treatment would affect the development of an immune response. For the current studies, male Sprague-Dawley rats were immunized with either keyhole limpet hemocyanin (KLH; control group) or a (+)METH hapten ((+)METH with a six carbon spacer group at the para position of the ring structure)-KLH conjugate. The (+)METH-KLH animals were further divided into two groups. One group was immunized with no subsequent administration of (+)METH, while the other group was immunized and repeatedly challenged (twice a week throughout the study) with an i.p. dose of 3 mg/kg (+)METH. The results showed that the two groups of (+)METH-KLH immunized rats developed and maintained anti-(+)METH antibody titers. The anti-(+)METH immune responses of the two groups were not statistically different (P < 0.05) as measured by serum titers and the relative antibody affinities. These data suggest that repeated administration of (+)METH does not affect the generation of an anti-(+)METH antibody response in actively immunized rats.

(+)-Methamphetamine-induced spontaneous behavior in rats depends on route of (+)METH administration

These studies examined the role of (+)-methamphetamine ((+)METH) administration route on spontaneous behavioral activity vs. time relationships, and pharmacokinetic mechanisms for differences in effects. Male Sprague-Dawley rats (n=6 per administration route) received saline and three doses (0.3, 1.0 and 3.0 mg/kg) of (+)METH in a mixed-sequence design by intravenous (iv), subcutaneous (sc) or intraperitoneal (ip) administration. Locomotion and stereotypy were quantified by video-tracking analysis. The effects of (+)METH on spontaneous behavior were dose- and route-dependent. In particular, total locomotor activity was greatest following 3.0 mg/kg intraperitoneally (P<0.05) and stereotypy ratings were greatest following 3.0 mg/kg subcutaneously (P<0.05). In addition, the duration of locomotor effects was greatest after 3.0 mg/kg subcutaneously (P<0.05). Serum pharmacokinetic parameters were determined in separate rats given 3.0 mg/kg by subcutaneous and intraperitoneal administration (n=4 per administration route). The (+)METH elimination half-life was not different between the routes, but the (+)METH AUC was greater (P<0.05), and the (+)METH and (+)-amphetamine (AMP) maximum concentrations occurred later following subcutaneous than after intraperitoneal dosing (P<0.05), increasing and prolonging drug exposure. In conclusion, the overall pattern of (+)METH effects on locomotor activity depend on dose and the route of administration, which affects serum concentration and the time course of behavioral effects.

Monoclonal Antibody Form and Function: Manufacturing the Right Antibodies for Treating Drug Abuse

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

Development of active and passive human vaccines to treat methamphetamine addiction.

Methamphetamine (METH) abuse is a major worldwide epidemic, with no specific medications for treatment of chronic or acute effects. Anti-METH antibodies have the potential to save lives and reduce the crippling effects of METH abuse. While they are not expected to be the magic bullet to immediately cure addiction, immunotherapy could provide a breakthrough medication to continuously block or attenuate METH effects during a comprehensive addiction recovery plan. A unique challenge for METH antibody antagonists is the need to protect the brain from the complex direct and indirect adverse effects of long-term METH use. To meet this challenge, a new generation of passive monoclonal antibodies and active immunization therapies are at an advanced stage of preclinical development. Both of these vaccines could play an essential role in a well planned recovery program from human METH addiction by providing long-lasting protection from the rewarding and reinforcing effect of METH.

Differential and Region-Specific Activation of Mitogen-Activated Protein Kinases Following Chronic Administration of Phencyclidine in Rat Brain

We have previously demonstrated elevation of the extracellular signal-regulated kinase (ERK) pathway in the cerebellum from patients with schizophrenia, an illness that may involve dysfunction of the N-methyl-D-aspartate (NMDA) receptor. Since the NMDA antagonist, phencyclidine (PCP), produces schizophrenic-like symptoms in humans, and abnormal behavior in animals, we examined the effects of chronic PCP administration in time- and dose-dependent manner on ERK and two other members of mitogen-activated protein kinase family, c-Jun N-terminal protein kinase (JNK) and p38, in rat brain. Osmotic pumps for PCP (18 mg/kg/day) and saline (controls) were implanted subcutaneously in rats for three, 10, and 20 days. Using Western blot analysis, we found no change at three days, but a significant increase in the phosphorylation of ERK1, ERK2 and MEK in the cerebellum at 10- and 20-days of continuous PCP infusion. For the experiments involving various doses of PCP, rats were infused with PCP at concentrations of 2.5, 10, 18, or 25 mg/kg/day, or saline for 10 days. We observed a dose-dependent elevation in the phosphorylation of ERK1 and ERK2 only in the cerebellum but not in brainstem, frontal cortex or hippocampus. The activities of JNK and p38 were unchanged in all investigated brain regions including cerebellum. These results demonstrate that chronic infusion of PCP in rats produces a differential and brain region-specific activation of MAP kinases, suggesting a role for the ERK signaling pathway in PCP abuse and perhaps in schizophrenia.

Monoclonal antibodies as pharmacokinetic antagonists for the treatment of (+)-methamphetamine 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 patients 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.

Functional and biological determinants affecting the duration of action and efficacy of anti-(+)-methamphetamine monoclonal antibodies in rats

These studies examined the in vivo pharmacokinetics and efficacy of five anti-methamphetamine monoclonal antibodies (mAbs, K(D) values from 11 to 250 nM) in rats. While no substantive differences in mAb systemic clearance (t(1/2)=6.1-6.9 days) were found, in vivo function was significantly reduced within 1-3 days for four of the five mAbs. Only mAb4G9 was capable of prolonged efficacy, as judged by prolonged high methamphetamine serum concentrations. MAb4G9 also maintained high amphetamine serum concentrations, along with reductions in methamphetamine and amphetamine brain concentrations, indicating neuroprotection. The combination of broad specificity for methamphetamine-like drugs, high affinity, and prolonged action in vivo suggests mAb4G9 is a potentially efficacious medication for treating human methamphetamine-related medical diseases.