RESUMEN

Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10(12) vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH in vivo. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.

Asunto(s)

Trastornos Relacionados con Anfetaminas/genética , Trastornos Relacionados con Anfetaminas/terapia , Dependovirus , Vectores Genéticos , Metanfetamina/efectos adversos , Anticuerpos de Cadena Única/biosíntesis , Trastornos Relacionados con Anfetaminas/metabolismo , Animales , Masculino , Metanfetamina/farmacología , Ratones , Ratones Endogámicos BALB C , Anticuerpos de Cadena Única/genética

RESUMEN

PURPOSE: Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv's have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life. METHODS: The affinity of scFv7F9Cys and PEG conjugates to METH was determined in vitro via equilibrium dialysis saturation binding. Pharmacokinetic and parameters of scFv7F9Cys and scFv7F9Cys-PEG20K (30 mg/kg i.v. each) and their ability to bind METH in vivo were determined in male Sprague-Dawley rats receiving a subcutaneous infusion of METH (3.2 mg/kg/day). RESULTS: Of three PEGylated conjugates, scFv7F9Cys-PEG20K was determined the most viable therapeutic candidate. PEGylation of scFv7F9Cys did not alter METH binding functionality in vitro, and produced a 27-fold increase in the in vivo half-life of the antibody fragment. Furthermore, total METH serum concentrations increased following scFv7F9Cys or scFv7F9Cys-PEG20K administration, with scFv7F9Cys-PEG20K producing significantly longer changes in METH distribution than scFv7F9Cys. CONCLUSIONS: PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH abuse.

Asunto(s)

Estimulantes del Sistema Nervioso Central/inmunología , Metanfetamina/inmunología , Polietilenglicoles/química , Anticuerpos de Cadena Única/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Semivida , Masculino , Ratas Sprague-Dawley , Anticuerpos de Cadena Única/química , Distribución Tisular

RESUMEN

To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (± 0.3) to 26 (± 2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles.

Asunto(s)

Metanfetamina/inmunología , Nanopartículas/química , Anticuerpos de Cadena Única/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Dendrímeros/química , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Semivida , Hemólisis/efectos de los fármacos , Masculino , Metanfetamina/sangre , Metanfetamina/metabolismo , Polietilenglicoles/química , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacocinética , Espectrometría de Masas en Tándem , Distribución Tisular

RESUMEN

Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

Asunto(s)

Anfetamina/química , Anticuerpos Monoclonales/química , Afinidad de Anticuerpos , Metanfetamina/química , Secuencia de Aminoácidos , Anfetamina/antagonistas & inhibidores , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Metanfetamina/antagonistas & inhibidores , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutación , Unión Proteica , Ingeniería de Proteínas , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/uso terapéutico , Relación Estructura-Actividad

RESUMEN

Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Toward this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG cross-linker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting the PEG cross-linker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH binding function. The dendribodies were found to have affinity for METH identical to that of the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.

Asunto(s)

Fragmentos de Inmunoglobulinas/inmunología , Metanfetamina/inmunología , Nanopartículas , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción