RESUMO
VL-2397 is an antifungal drug with a novel mechanism of action, rapid fungicidal in vitro activity, and potent in vivo activity against Aspergillus fumigatus, including azole-resistant strains. VL2397-101, a phase 1 first-in-human, randomized, double-blind, placebo-controlled dose-escalation study, was conducted in healthy adults to determine the safety, tolerability, and pharmacokinetics (PK) of single and multiple ascending intravenous (i.v.) doses of VL-2397. All dosing cohorts were fully enrolled; all subjects completed the safety follow-up. A safety committee reviewed the safety data for each dosing cohort prior to recommending the initiation of each subsequent cohort. No serious adverse events (SAEs) occurred; the majority of treatment-emergent adverse events (TEAEs) were mild and self-limited. The most common drug-related TEAEs were infusion site reactions. No clinically concerning trends were noted in vital signs, electrocardiograms, physical examinations, or safety laboratory results. Following single infusions of VL-2397, the overall and maximum exposures rose less than proportionally with increasing doses from 3 mg to 1,200 mg as indicated by area under the concentration-time curve over 24 h (AUC24) and maximum concentration (Cmax). No signs of VL-2397 accumulation were observed following i.v. infusions of 300, 600, and 1,200 mg every 24 h (q24h) for 7 days. Renal elimination played a major role in total body clearance, with up to 47% of unmetabolized drug in urine 24 h after administration at single doses of >30 mg. Overall, VL-2397 dosing in the study appeared to be safe and well tolerated in the healthy subjects. The safety profile, consistent PK, and lack of drug accumulation support further development of VL-2397 in patients with invasive aspergillosis.
Assuntos
Antifúngicos/farmacocinética , Antifúngicos/uso terapêutico , Complexos de Coordenação/farmacocinética , Complexos de Coordenação/uso terapêutico , Peptídeos Cíclicos/farmacocinética , Peptídeos Cíclicos/uso terapêutico , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Eletrocardiografia/métodos , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Human cytomegalovirus (CMV) establishes a lifelong persistent infection characterized by periods of latency and sporadic viral replication and is a major infectious cause of birth defects following congenital infection. Currently, no licensed vaccine is available that would prevent CMV infection. In an effort to develop a prophylactic CMV vaccine, the effects of different formulations, immunization routes and delivery devices on the immunogenicity of plasmid DNA (pDNA)-based vaccines were evaluated in rabbits and mice. Compared with PBS- and poloxamer-based formulations, significantly higher antibody responses were obtained with pDNA formulated with Vaxfectin (®) , a cationic lipid-based adjuvant. With low vaccine doses, the intradermal (ID) route resulted in higher antibody responses than obtained when the same dose was administered intramuscularly (IM). Since the IM route allowed injection of larger volumes and higher doses than could be administered at a single ID site, better antibody responses were obtained using the IM route. The needle-free injection system Biojector (®) 2000 and electroporation devices enhanced antibody responses only marginally compared with responses obtained with Vaxfectin (®) -formulated pDNA injected IM with a needle. A single-vial Vaxfectin (®) formulation was developed in a dosage form ready for use after thawing at room temperature. Finally, in a GLP-compliant repeat-dose toxicology study conducted in rabbits, single-vial Vaxfectin (®) -formulated vaccines, containing pDNA and Vaxfectin (®) up to 4.5 mg and 2 mg/injection, respectively, showed a favorable safety profile and were judged as well-tolerated. The results support further development of a Vaxfectin (®) -formulated pDNA vaccine to target congenital CMV infection.
Assuntos
Vacinas contra Citomegalovirus/imunologia , Plasmídeos/genética , Vacinas de DNA/uso terapêutico , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Feminino , Injeções Intradérmicas , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Vacinas de DNA/imunologiaRESUMO
Vaxfectin(®) is a lipid-based adjuvant initially developed for use with plasmid DNA (pDNA) vaccines. Here we present detailed nonclinical assessments performed prior to Vaxfectin(®)'s first-in-man use, as an adjuvant in the H5N1 influenza vaccine VCL-IPT1. Following IM delivery to rabbits, VCL-IPT1 pDNA localized primarily to injection sites, where levels steadily declined over the 2 months examined. Risk of pDNA integration into genomic DNA was negligible. Toxicology studies in rabbits revealed mild inflammatory/immune responses at injection sites characteristic of IM vaccine delivery; Vaxfectin(®) directly contributed to these responses. These data support clinical development of H5N1 pDNA vaccines, and also present an encouraging profile for further development of Vaxfectin(®) as an adjuvant for vaccines in general.
Assuntos
Adjuvantes Imunológicos/farmacocinética , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/farmacocinética , Fosfatidiletanolaminas/farmacocinética , Vacinas de DNA/farmacocinética , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Animais , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/genética , Injeções Intramusculares , Masculino , Fosfatidiletanolaminas/administração & dosagem , Fosfatidiletanolaminas/efeitos adversos , Coelhos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/genéticaRESUMO
BACKGROUND: Development of vaccines against highly pathogenic avian influenza virus H5N1 subtypes posing a pandemic threat remains a priority. Limitations in manufacturing capacity and production time of conventional inactivated vaccines highlight the need for additional approaches. METHODS: We conducted two double-blind, placebo-controlled phase 1 studies involving a total of 103 healthy adults who received two intramuscular injections of Vaxfectin-adjuvanted plasmid DNA vaccine or placebo 21 days apart. Vaccine cohorts received either a monovalent vaccine containing an A/Vietnam/1203/04 H5 hemagglutinin-encoding plasmid or a trivalent vaccine with plasmids encoding H5, NP, and M2 proteins in doses from 0.1 to 1mg of DNA/injection. RESULTS: All doses were well tolerated without vaccine-related serious adverse events or discontinuations. In the monovalent cohorts, hemagglutination inhibition (HI) titers of > or =40 and 4-fold rises from baseline were achieved in 47-67% of subjects and H5-specific T-cell responses in 75-100%. Trivalent cohorts had lower HI response rates (< or = 20%), but 72% of subjects achieved T-cell and/or antibody responses to one or more antigens. CONCLUSIONS: Vaxfectin-adjuvanted monovalent H5 DNA vaccines were well tolerated and induced HI response rates and titers in the reported range of inactivated protein-based H5 vaccines, suggesting that adjuvanted DNA vaccines with rapid vaccine production could be useful for pandemic control.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/efeitos adversos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adulto , Anticorpos Antivirais/sangue , Método Duplo-Cego , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunização Secundária/métodos , Vírus da Influenza A/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Intramusculares , Masculino , Proteínas do Nucleocapsídeo , Fosfatidiletanolaminas/administração & dosagem , Fosfatidiletanolaminas/efeitos adversos , Placebos/administração & dosagem , Plasmídeos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
We previously reported the capacity of the cationic lipid-based formulation, Vaxfectin, to enhance the immunogenicity and protective efficacy of a low dose plasmid DNA vaccine against Plasmodium yoelii malaria in mice. Here, we have extended this finding to human Plasmodium falciparum genes, evaluating the immune enhancing effect of Vaxfectin formulation on a mixture, designated CSLAM, of five plasmid DNA vaccines encoding antigens from the sporozoite (PfCSP, PfSSP2/TRAP), intrahepatic (PfLSA1), and erythrocytic (PfAMA1, PfMSP1) life cycle stages of P. falciparum administered at 2, 10 or 50microg doses. Vaxfectin formulation enhanced both antibody and cellular immune responses to each component of the multi-antigen vaccine mixture, as assessed by ELISA, IFAT, and IFN-gamma ELIspot, respectively. There was no apparent antigenic competition, as indicated by comparison of responses induced in mice immunized with PfCSP vs. CSLAM. These data showing that Vaxfectin can enhance the immunogenicity of plasmid DNA vaccines administered at low doses per body weight, and in combinations, has important clinical implications for the development of a vaccine against malaria, as well as against other public health threats.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antiprotozoários/sangue , Vacinas Antimaláricas/imunologia , Fosfatidiletanolaminas/administração & dosagem , Proteínas de Protozoários/imunologia , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Animais , Feminino , Imunoensaio/métodos , Vacinas Antimaláricas/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/genética , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Vacinas de DNA/administração & dosagemRESUMO
Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV formulated with Vaxfectin, a cationic lipid-based adjuvant. Increasing doses of Vaxfectin resulted in increased hemagglutination-inhibition or anti-TIV ELISA antibody titers, with up to a 200-fold increase obtained with 900 microg of Vaxfectin. A >or=10-fold dose-sparing effect was demonstrated with Vaxfectin formulations. Vaxfectin preferentially increased IgG2 titers compared to IgG1 titers, resulting in a balanced IgG isotype distribution. Lower doses of Vaxfectin (30 microg) did not enhance antibody responses, but increased the number of IFN-gamma secreting T-cells by up to 18-fold. The data demonstrate that Vaxfectin enhances Th1 responses with protein-based seasonal influenza vaccine, and suggest that cellular or humoral immune responses may be preferentially induced by modifying the Vaxfectin:antigen ratio in the vaccine formulation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Fosfatidiletanolaminas/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Antígenos Virais/imunologia , Cátions/imunologia , Cátions/farmacologia , Células Cultivadas , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Inibição da Hemaglutinação , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Vacinas contra Influenza/administração & dosagem , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Fosfatidiletanolaminas/administração & dosagem , Fosfatidiletanolaminas/imunologia , Baço/citologia , Baço/imunologia , Células Th1/imunologiaRESUMO
Plasmid DNA (pDNA) vaccines represent an alternative to conventional inactivated influenza vaccines that are likely to experience supply constraints during a pandemic. Several Vaxfectin-formulated pDNA vaccines were tested in mice and ferrets for efficacy against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) influenza virus strain; the vaccines encoded influenza A virus hemagglutinin (HA), and/or nucleoprotein (NP), and M2 protein. Complete protection from death and disease was achieved in mice and ferrets with 2 doses of a Vaxfectin-formulated vaccine containing H5 HA, NP, and M2 plasmids and in ferrets with only 1 dose. A Vaxfectin-formulated vaccine containing NP and M2 pDNA provided significant protection against death in mice and provided some benefit in ferrets (i.e., 17% survival, delayed time to illness and death, and significant reduction in viral load compared with that in negative control animals). These experiments support the clinical testing of pDNA vaccine candidates that may ultimately increase global vaccine supply options during pandemics.
Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Plasmídeos , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/sangue , Peso Corporal , Química Farmacêutica , Furões , Camundongos , Camundongos Endogâmicos BALB C , Líquido da Lavagem Nasal/virologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Fosfatidiletanolaminas , Fatores de TempoRESUMO
BACKGROUND: Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or treatment of infectious diseases. Broader applications may benefit from the identification of safe and potent vaccine adjuvants. This report describes the development of a novel polymer-based formulation to enhance the immunogenicity of pDNA-based vaccines. METHODS: Plasmid DNA was formulated with a nonionic block copolymer, poloxamer CRL1005, and the cationic surfactant benzalkonium chloride (BAK) to produce a thermodynamically stable, self-assembling system. The influence of parameters such as polymer concentration and BAK composition on the immune responses was evaluated in mice vaccinated with pDNA encoding influenza nucleoprotein. RESULTS: At concentrations of 7.5 mg/ml CRL1005, 0.3 mM BAK and 5 mg/ml pDNA, CRL1005/BAK/pDNA particles had a mean diameter of 261 +/- 0.2 nm and a surface charge of - 11.6 +/- 0.9 mV. The negative surface charge and atomic force microscopy images suggested that pDNA binds to BAK adsorbed to the surface of poloxamer particles. The CRL1005/BAK/pDNA formulation significantly enhanced antigen-specific cellular and humoral immune responses, and increased transgene levels in muscle and serum. The complexity of the formulation was reduced by replacing the commercial BAK, which is a mixture of four alkyl chains, with a C14 BAK homolog. The substitution yielded an analytically preferable formulation with equivalent physical characteristics and immunogenicity. CONCLUSIONS: The results suggest that the CRL1005/BAK/pDNA formulation may enhance immunogenicity by improving the delivery of pDNA-based vaccines. This formulation is currently being evaluated for the prevention of CMV-associated disease in a phase 2 clinical trial.
Assuntos
Adjuvantes Imunológicos/metabolismo , Compostos de Benzalcônio/metabolismo , DNA/metabolismo , Poloxâmero/metabolismo , Vacinas de DNA/imunologia , Análise de Variância , Ensaio de Imunoadsorção Enzimática , Microscopia de Força Atômica , Microscopia de Fluorescência , Tamanho da Partícula , Polímeros/metabolismo , Vacinas de DNA/metabolismoRESUMO
The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point. Survival and weight loss data showed that protection can be achieved in mice with 50 microg of phosphate-buffered saline-formulated LEC DNA or 2 microg of Vaxfectin-formulated LEC DNA. Survival correlated with neutralizing antibody titers (hemagglutination inhibition, HAI); titers obtained after vaccination with LEC were equivalent to those obtained with HA (H3N2) plasmid DNA control. Vaccination with heterotypic H1 HA-LEC DNA provided no protection against viral challenge.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Reação em Cadeia da Polimerase , Vacinas de DNA/imunologia , Animais , Linhagem Celular , Cães , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Coelhos , PerusRESUMO
Next generation influenza vaccines containing conserved antigens may enhance immunity against seasonal or pandemic influenza virus strains. Using a plasmid DNA (pDNA)-based vaccine approach, we systematically tested combinations of NP, M1, and M2 antigens derived from consensus sequences for protection against lethal influenza challenge and compared formulations for adjuvanting low pDNA vaccine doses. The highest level of protection at the lowest pDNA doses was provided by Vaxfectin-formulated NP + M2. Vaxfectin adjuvanticity was confirmed with a low dose of HA pDNA. These promising proof-of-concept data support the clinical development of Vaxfectin-formulated pDNA encoding NP + M2 consensus proteins.
Assuntos
Vacinas contra Influenza/imunologia , Nucleoproteínas/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Fosfatidiletanolaminas/farmacologia , Proteínas de Ligação a RNA/imunologia , Vacinas de DNA/imunologia , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/sangue , Vacinas contra Influenza/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/imunologia , Fosfatidiletanolaminas/administração & dosagem , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Vacinação , Vacinas de DNA/genética , Proteínas do Core Viral/genética , Proteínas da Matriz Viral/genética , Proteínas ViraisRESUMO
Experiments were conducted with a cationic lipid-formulated pDNA vaccine (VCL-AB01) to evaluate the models used to determine biodistribution, persistence and the potential for integration (into genomic DNA) of plasmid DNA-based vaccines. Mice were injected with a high-dose volume of 50 microL unilaterally containing approximately 1.33 x 10(13) plasmid copy numbers (PCN) or a low-dose volume of 20 microL bilaterally ( approximately 5.3 x 10(12) PCN). Rabbits were injected bilaterally with a 0.5 mL ( approximately 1.33 x 10(14) PCN) volume. Injection site muscle tissue was harvested two days, one month, and two months postinjection for the low-dose murine and rabbit models and two days and two months postinjection for the high-dose murine model. Total DNA was extracted and analyzed by real-time quantitative PCR for sequences specific to the injected pDNA. The geometric mean PCN/microg of total DNA from the high and low dose models were compared to determine if injection volume impacts clearance and/or persistence. Results from these studies showed that PCN clearance over two months was similar in mice injected with 20 microL and rabbits injected with 0.5 mL, but PCN clearance was slower in mice injected with similar PCN in 50 microL (1.33 x 10(13) PCN) compared to 20 microL (5.3 x 10(12) PCN). Persistence at two months in the rabbit and low-dose murine models was comparable, with geometric mean of 5.22 x 10(3) PCN/microg of total DNA for the low-dose volume murine model and 2.81 x 10(3)/microg DNA for the rabbit model. Interanimal variability in persistence was not impacted by dose volume.
Assuntos
Plasmídeos , Vacinas de DNA/farmacocinética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , Coelhos , Distribuição TecidualRESUMO
Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.
Assuntos
Vacinas contra Citomegalovirus/farmacocinética , Citomegalovirus , Poloxâmero/farmacocinética , Vacinas de DNA/farmacocinética , Proteínas Virais/imunologia , Animais , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/administração & dosagem , Vacinas contra Citomegalovirus/genética , Vacinas contra Citomegalovirus/imunologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Injeções Intramusculares , Camundongos , Poloxâmero/química , Coelhos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas Virais/genéticaRESUMO
Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.
Assuntos
Vacinas contra Antraz/farmacocinética , Bacillus anthracis , Lipídeos/farmacocinética , Fosfatidiletanolaminas/farmacocinética , Plasmídeos/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Vacinas de DNA/farmacocinética , Animais , Antraz/genética , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/imunologia , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Injeções Intramusculares , Lipídeos/imunologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fosfatidiletanolaminas/imunologia , Plasmídeos/genética , Plasmídeos/imunologia , Compostos de Amônio Quaternário/imunologia , Coelhos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
The large number of cytofectin and co-lipid combinations currently used for lipoplex-mediated gene delivery reflects the fact that the optimal cytofectin/co-lipid combination varies with the application. The effects of structural changes in both cytofectin and co-lipid were systematically examined to identify structure-activity relationships. Specifically, alkyl chain length, degree of unsaturation and the head group to which the alkyl side chain was attached were examined to determine their effect on lipoplex structure and biological activity. The macroscopic lipoplex structure was assessed using a dye-binding assay and the biological activity was examined using in vitro transfection in three diverse cell lines. Lipoplexes were formulated in three different vehicles currently in use for in vivo delivery of naked plasmid DNA (pDNA) and lipoplex formulations. The changes in dye accessibility were consistent with structural changes in the lipoplex, which correlated with alterations in the formulation. In contrast, transfection activity of different lipoplexes was cell type and vehicle dependent and did not correlate with dye accessibility. Overall, the results show a correlation between transfection and enhanced membrane fluidity in both the lipoplex and cellular membranes.