Antibody production and pharmacokinetics of METH in rats following vaccination with the METH vaccine, IXT-v100, adjuvanted with GLA-SE.

BACKGROUND: Methamphetamine use disorder continues to be inadequately treated, but improvements are being made in the field of immunotherapeutics, including vaccines, which could provide new options for treatment. Cocaine and nicotine vaccines have been tested clinically, but have yet to elicit the necessary antibody concentrations required to be effective. Methamphetamine vaccines have been tested in multiple nonclinical models and appear promising. Improved adjuvants have the potential to further stimulate the immune system to reach effective levels of antibodies. Previously, the methamphetamine vaccine IXT-v100 was administered with GLA-SE, a toll-like receptor 4 agonist, in mice to produce higher levels of antibodies than when it was administered with two other widely used adjuvants, Alhydrogel and Sigma Adjuvant System. METHODS: The purpose of this research was to evaluate IXT-v100, given in combination with the adjuvant GLA-SE, to determine its efficacy in antagonizing methamphetamine disposition in a rat pharmacokinetic study. Additional rat studies were conducted to compare the ability of IXT-v100 manufactured with greater hapten densities to elicit higher antibody levels. RESULTS: As expected based on prior studies with anti-methamphetamine monoclonal antibodies, the antibodies resulting from vaccination with IXT-v100 altered methamphetamine pharmacokinetics by increasing serum concentrations and extending the half-life. Furthermore, intentional variations in the ratio of components during manufacturing led to production of vaccines with higher hapten densities. The higher hapten densities resulted in production of antibodies that maintained the ability to bind methamphetamine with high affinity. CONCLUSIONS: The results support continued development of IXT-v100 for the treatment of methamphetamine use disorder.

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use.

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.

Vaccination protects rats from methamphetamine-induced impairment of behavioral responding for food.

(+)-Methamphetamine (METH) addiction is a chronic disease that interferes with fundamental brain-mediated behaviors and biological functions like eating. These studies present preclinical efficacy and safety profiles for a METH conjugate vaccine (IC(KLH)-SMO9) designed to treat METH abuse. ICKLH-SMO9 efficacy and safety were assessed over a 16-week period by monitoring general health and stability of responding in a food maintained behavioral paradigm. Male Sprague-Dawley rats were trained to lever press for food reinforcers until stable behavior was established. Rats (n=9/group) were then immunized with 100 µg of a control antigenic carrier protein (IC(KLH)-Cys) or IC(KLH)-SMO9 in Alhydrogel adjuvant, with booster immunizations at 4, 8 and 12 weeks. Health, immunization site and behavior were assessed daily. No adverse effects were found. During weeks 14-16, when antibody titers and METH affinity (K(d)=13.9 ± 1.7 nM) were maximal, all rats received progressively higher METH doses (0.3-3.0 mg/kg) every 3-4 days, followed by behavioral testing. Even though the lower METH doses from 0.3 to 1.0 mg/kg produced no impairment in food maintained behavior, 3.0-mg/kg in control rats showed significantly (p<0.05) reduced response rates and number of reinforcers earned, as well as reduced food intake. In sharp contrast, the IC(KLH)-SMO9 group showed no changes in food maintained behavior at any METH dose, even though METH serum concentrations showed profound increases due to anti-METH antibody binding. These findings suggest the IC(KLH)-SMO9 vaccine is effective and safe at reducing adverse METH-induced effects, even at high METH doses.

Soy immunotherapy for peanut-allergic mice: modulation of the peanut-allergic response.

BACKGROUND: Allergen-specific immunotherapy (IT) is an effective therapeutic modality to prevent further anaphylactic episodes in patients with insect sting hypersensitivity and is being investigated for peanut allergy. So far, peanut-specific IT has been unsuccessful because of the side effects of therapy. Soybean seed storage proteins share significant homology with the respective peanut allergens. OBJECTIVE: This study was undertaken in mice to investigate whether specific doses of soybean would desensitize peanut-allergic mice. METHODS: C3H/HeJ mice were sensitized to peanut with 3 intraperitoneal (IP) injections of crude peanut extract. The mice were desensitized by IP injections with either crude peanut or soybean extract for 4 weeks, 3 times a week. Controls included placebo desensitization with PBS and naive mice. After 2 weeks of rest, mice were challenged IP with crude peanut extract. Thirty minutes later, symptom scores and body temperatures were recorded. Serum immunoglobulins, peanut-induced splenocyte proliferation, and secreted cytokines were measured before and after desensitization. RESULTS: The clinical symptoms in the soybean- and peanut-desensitized animals were markedly reduced compared with the placebo-treated mice. Specific IgG1 levels to crude peanut were significantly lower in the soy IT group than in the peanut IT group. The cellular response to crude peanut was also downregulated in the soy IT group, as shown by decreased peanut-specific stimulation indices and a cytokine profile skewed toward a T H 1 response. CONCLUSIONS: Soy IT can be used to desensitize/downregulate peanut-specific response in peanut-allergic mice and could provide a new therapeutic intervention for peanut allergy.

Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation.

BACKGROUND: Peanut allergy is a major health concern due to the increased prevalence, potential severity, and chronicity of the reaction. The cDNA encoding a third peanut allergen, Ara h 3, has been previously cloned and characterized. Mutational analysis of the Ara h 3 IgE-binding epitopes with synthetic peptides revealed that single amino acid changes at critical residues could diminish IgE binding. METHODS: Specific oligonucleotides were used in polymerase chain reactions to modify the cDNA encoding Ara h 3 at critical IgE binding sites. Four point mutations were introduced into the Ara h 3 cDNA at codons encoding critical amino acids in epitopes 1, 2, 3 and 4. Recombinant modified proteins were used in SDS-PAGE/Western IgE immunoblot, SDS-PAGE/Western IgE immunoblot inhibition and T cell proliferation assays to determine the effects of these changes on in vitro clinical indicators of peanut hypersensitivity. RESULTS: Higher amounts of modified Ara h 3 were required to compete with the wild-type allergen for peanut-specific serum IgE. Immunoblot analysis with individual serum IgE from Ara-h-3-allergic patients showed that IgE binding to the modified protein decreased approximately 35-85% in comparison to IgE binding to wild-type Ara h 3. Also, the modified Ara h 3 retained the ability to stimulate T cell activation in PBMCs donated by Ara-h-3-allergic patients. CONCLUSIONS: The engineered hypoallergenic Ara h 3 variant displays two characteristics essential for recombinant allergen immunotherapy; it has a reduced binding capacity for serum IgE from peanut-hypersensitive patients and it can stimulate T-cell proliferation and activation.