RESUMO
Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.
Assuntos
Antivirais , Proteínas do Capsídeo , Capsídeo , Animais , Camundongos , Antivirais/farmacologia , Modelos Animais de Doenças , Relação Estrutura-Atividade , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/metabolismoRESUMO
AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 µM; EC90 = 0.33 to 1.32 µM) with no significant cytotoxicity (50% cytotoxic concentration > 10 µM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.
Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Montagem de Vírus/efeitos dos fármacos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , DNA Circular/metabolismo , DNA Viral/sangue , DNA Viral/metabolismo , Feminino , Guanina/análogos & derivados , Guanina/farmacologia , Vírus da Hepatite B/crescimento & desenvolvimento , Humanos , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , RNA Viral/genéticaRESUMO
BACKGROUND: CPX-351, a liposomal formulation of cytarabine and daunorubicin co-encapsulated at an optimized synergistic 5:1 molar ratio, has demonstrated improved clinical outcomes over conventional cytarabine/daunorubicin treatment in a randomized phase 2 trial in patients with AML as well as superior efficacy against preclinical leukemia models when compared to the free drugs in combination. PROCEDURES: Given the promising phase 2 data, limited toxicities observed, and the known clinical activities of cytarabine/daunorubicin, we assessed the efficacy of CPX-351 against a panel of childhood ALL xenograft models. Plasma pharmacokinetics of cytarabine and daunorubicin following CPX-351 treatment were determined by HPLC in order to correlate efficacy with drug exposure. RESULTS: CPX-351, at a dose of 5 units/kg (corresponding to 5 mg/kg cytarabine and 2.2 mg/kg daunorubicin), was highly efficacious against all xenografts tested, inducing complete responses in four B-lineage xenografts and partial response in one T-lineage xenograft. These therapeutic responses were achieved with CPX-351 doses that provided drug exposures (based on Cmax and AUC) comparable to those observed in patients with AML. CONCLUSIONS: These results suggest that CPX-351 may be a promising chemotherapeutic to be utilized in the treatment of ALL and support its testing in pediatric patients with leukemia.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Criança , Pré-Escolar , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Feminino , Humanos , Lipossomos , Masculino , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pediatria , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Approved therapies for hepatitis B virus (HBV) treatment include nucleos(t)ides and interferon alpha (IFN-α) which effectively suppress viral replication, but they rarely lead to cure. Expression of viral proteins, especially surface antigen of the hepatitis B virus (HBsAg) from covalently closed circular DNA (cccDNA) and the integrated genome, is believed to contribute to the persistence of HBV. This work focuses on therapies that target the expression of HBV proteins, in particular HBsAg, which differs from current treatments. Here we describe the identification of AB-452, a dihydroquinolizinone (DHQ) analogue. AB-452 is a potent HBV RNA destabilizer by inhibiting PAPD5/7 proteins in vitro with good in vivo efficacy in a chronic HBV mouse model. AB-452 showed acceptable tolerability in 28-day rat and dog toxicity studies, and a high degree of oral exposure in multiple species. Based on its in vitro and in vivo profiles, AB-452 was identified as a clinical development candidate.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Camundongos , Ratos , Animais , Cães , Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B , Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , RNA Viral/genética , Relação Estrutura-Atividade , Naftiridinas/farmacologia , Naftiridinas/uso terapêutico , DNA Viral/genética , Replicação ViralRESUMO
HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC90), compared to concentrations in plasma which were substantially below its EC90, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161's clinical development.
Assuntos
Complexos de Coordenação , Vírus da Hepatite B , Hepatite B Crônica , Naftalenossulfonatos , Masculino , Camundongos , Ratos , Animais , Cães , Vírus da Hepatite B/fisiologia , Antígenos de Superfície da Hepatite B/genética , RNA Viral , RNA Mensageiro , Antivirais/farmacologia , Antivirais/uso terapêutico , DNA Viral/genética , Hepatite B Crônica/tratamento farmacológico , DNA CircularRESUMO
The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Nucleosídeos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , SARS-CoV-2/efeitos dos fármacos , Humanos , Nucleosídeos/farmacologia , Nucleosídeos/química , Animais , Descoberta de Drogas , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Chlorocebus aethiops , Células Vero , COVID-19/virologia , RNA-Polimerase RNA-Dependente de CoronavírusRESUMO
Disruption of the HBV viral life cycle with small molecules that prevent the encapsidation of pregenomic RNA and viral polymerase through binding to HBV core protein is a clinically validated approach to inhibiting HBV viral replication. Herein we report the further optimisation of clinical candidate AB-506 through core modification with a focus on increasing oral exposure and oral half-life. Maintenance of high levels of anti-HBV cellular potency in conjunction with improvements in pharmacokinetic properties led to multi-log10 reductions in serum HBV DNA following low, once-daily oral dosing for key analogues in a preclinical animal model of HBV replication.
RESUMO
AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: â¼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Proteínas do Core Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacocinética , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Feminino , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Camundongos , Ratos , Montagem de Vírus/efeitos dos fármacosRESUMO
Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections.
Assuntos
Antígeno B7-H1/metabolismo , Endocitose , Inibidores de Checkpoint Imunológico/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/farmacologia , Antivirais/farmacologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Cricetulus , Modelos Animais de Doenças , Feminino , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/metabolismo , Multimerização Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
We demonstrate here that cytarabine and daunorubicin, a standard drug combination used in the treatment of leukaemia, exhibits drug ratio-dependent synergistic antitumor activity in vitro and in vivo. A cytarabine:daunorubicin molar ratio of 5:1 displayed the greatest degree of synergy and minimum antagonism in a panel of 15 tumor cell lines in vitro. Co-encapsulating cytarabine and daunorubicin inside liposomes maintained the synergistic drug ratio in plasma for 24h post-injection. Liposome-encapsulated cytarabine:daunorubicin combinations exhibited drug ratio-dependent in vivo efficacy with the 5:1 molar drug ratio (designated CPX-351) having the greatest therapeutic index, despite using sub-MTD daunorubicin doses. CPX-351 exhibited superior therapeutic activity compared to free-drug cocktails, with high proportions of long-term survivors, consistent with in vivo synergy. The therapeutic advantage of CPX-351 was associated with prolonged maintenance of synergistic drug ratios in bone marrow. These results indicate that in vitro informatics on cytarabine:daunorubicin cytotoxicity can be translated in vivo to optimize the efficacy of anticancer drug combinations by controlling the exposure of drug ratios with drug delivery vehicles.
Assuntos
Antineoplásicos/farmacologia , Citarabina/farmacologia , Daunorrubicina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células , Citarabina/administração & dosagem , Citarabina/farmacocinética , Daunorrubicina/administração & dosagem , Daunorrubicina/farmacocinética , Citometria de Fluxo , Humanos , Lipossomos , CamundongosRESUMO
A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles. These nanoparticles were shown to have an elimination half-life of approximately 24 h in vivo. The rate at which the prodrug was released from the nanoparticles could be controlled by adjusting the hydrophobicity of the lipid anchor, resulting in release half-lives ranging from 1 to 24 h. The diglycolate and succinate cross-linked prodrugs were 1-2 orders of magnitude less potent than paclitaxel in vitro. Nanoparticle formulations of the succinate prodrugs showed no evidence of efficacy in HT29 human colorectal tumor xenograph models. Efficacy of diglycolate prodrug nanoparticles increased as the anchor hydrophobicity increased. Long circulating diglycolate prodrug nanoparticles provided significantly enhanced therapeutic activity over commercially formulated paclitaxel at the maximum tolerated dose.
Assuntos
Paclitaxel/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Camundongos , Camundongos Nus , Micelas , Nanopartículas , Transplante de Neoplasias , Paclitaxel/química , Paclitaxel/farmacologia , Polietilenoglicóis , Poliestirenos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Transplante HeterólogoRESUMO
In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.
Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Viral/genética , Elementos de Resposta , Sítios de Ligação , Genótipo , Humanos , Ligação Proteica , Estabilidade de RNA/efeitos dos fármacos , Transfecção , Replicação ViralRESUMO
Whether anticancer drug combinations act synergistically or antagonistically often depends on the ratio of the agents being combined. We show here that combinations of irinotecan and floxuridine exhibit drug ratio-dependent cytotoxicity in a broad panel of tumor cell lines in vitro where a 1:1 molar ratio consistently provided synergy and avoided antagonism. In vivo delivery of irinotecan and floxuridine coencapsulated inside liposomes at the synergistic 1:1 molar ratio (referred to as CPX-1) lead to greatly enhanced efficacy compared to the two drugs administered as a saline-based cocktail in a number of human xenograft and murine tumor models. When compared to liposomal irinotecan or liposomal floxuridine, the therapeutic activity of CPX-1 in vivo was not only superior to the individual liposomal agents, but the extent of tumor growth inhibition was greater than that predicted for combining the activities of the individual agents. In contrast, liposome delivery of irinotecan:floxuridine ratios shown to be antagonistic in vitro provided antitumor activity that was actually less than that achieved with liposomal irinotecan alone, indicative of in vivo antagonism. Synergistic antitumor activity observed for CPX-1 was associated with maintenance of the 1:1 irinotecan:floxuridine molar ratio in plasma and tumor tissue over 16-24 h. In contrast, injection of the drugs combined in saline resulted in irinotecan:floxuridine ratios that changed 10-fold within 1 h in plasma and sevenfold within 4 h in tumor tissue. These results indicate that substantial improvements in the efficacy of drug combinations may be achieved by maintaining in vitro-identified synergistic drug ratios after systemic administration using drug delivery vehicles.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Floxuridina/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Camptotecina/administração & dosagem , Camptotecina/sangue , Camptotecina/farmacocinética , Sobrevivência Celular , Relação Dose-Resposta a Droga , Antagonismo de Drogas , Combinação de Medicamentos , Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Floxuridina/sangue , Floxuridina/farmacocinética , Humanos , Injeções Intravenosas , Irinotecano , Lipossomos , Camundongos , Neoplasias/metabolismo , Veículos Farmacêuticos , Taxa de Sobrevida , Distribuição Tecidual , Carga Tumoral , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The purpose of these studies was to design an intravenous drug formulation consisting of two active agents having synergistic in vitro activity. Specifically, we describe a novel drug combination consisting of a cytotoxic agent (vinorelbine) with an apoptosis-inducing lipid (phosphatidylserine, PS). In vitro cytotoxicity screening of PS and vinorelbine, alone and in combination, against human MDA435/LCC6 breast cancer and H460 lung cancer cells was used to identify the molar ratio of these two agents required for synergistic activity. PS and vinorelbine were co-formulated in a lipid-based system at the synergistic molar ratio and the pharmacokinetic and antitumor characteristics of the combination assessed in mice bearing H460 tumors. The cytotoxicity of the lipid, and the synergy between the lipid and vinorelbine, were specific to PS; these effects were not observed using control lipids. A novel formulation of PS, incorporated as a membrane component in liposomes, and encapsulating vinorelbine using a pH gradient based loading method was developed. The PS to vinorelbine ratio in this formulation was 1/1, a ratio that produced synergistic in vitro cytotoxicity over a broad concentration range. The vinorelbine and PS dual-agent treatment significantly delayed the growth of subcutaneous human H460 xenograft tumors in Rag2M mice compared to the same dose of free vinorelbine given alone or given as a cocktail of the free vinorelbine simultaneously with empty PS-containing liposomes. These studies demonstrate the potential to develop clinically relevant drug combinations identified using in vitro drug-drug interactions combined with lipid-based delivery systems to co-formulate drugs at their synergistic ratios.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Fosfatidilserinas/administração & dosagem , Vimblastina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Relação Dose-Resposta a Droga , Composição de Medicamentos , Desenho de Fármacos , Sinergismo Farmacológico , Feminino , Humanos , Injeções Intravenosas , Lipossomos , Neoplasias Pulmonares/patologia , Camundongos , Fosfatidilserinas/química , Fosfatidilserinas/farmacocinética , Tecnologia Farmacêutica , Vimblastina/administração & dosagem , Vimblastina/química , Vimblastina/farmacocinética , Vinorelbina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio-dependent synergy. In each case, synergistic interactions were observed in vitro at certain drug/drug molar ratio ranges (1:1, 5:1, and 10:1, respectively), whereas other ratios were additive or antagonistic. We were able to maintain fixed drug ratios in plasma of mice for 24 hours after i.v. injection for all three combinations by controlling and overcoming the inherent dissimilar pharmacokinetics of individual drugs through encapsulation in liposomal carrier systems. The liposomes not only maintained drug ratios in the plasma after injection, but also delivered the formulated drug ratio directly to tumor tissue. In vivo maintenance of drug ratios shown to be synergistic in vitro provided increased efficacy in preclinical tumor models, whereas attenuated antitumor activity was observed when antagonistic drug ratios were maintained. Fixing synergistic drug ratios in pharmaceutical carriers provides an avenue by which anticancer drug combinations can be optimized prospectively for maximum therapeutic activity during preclinical development and differs from current practice in which dosing regimens are developed empirically in late-stage clinical trials based on tolerability.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Camptotecina/sangue , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/sangue , Citarabina/administração & dosagem , Citarabina/sangue , Daunorrubicina/administração & dosagem , Daunorrubicina/sangue , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Floxuridina/administração & dosagem , Floxuridina/sangue , Humanos , Irinotecano , Lipossomos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations. EXPERIMENTAL DESIGN: Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation. RESULTS: Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism. CONCLUSION: These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.
Assuntos
Doxorrubicina/farmacologia , Lipossomos/metabolismo , Vincristina/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/patologia , Calcimicina/farmacologia , Linhagem Celular Tumoral , Corantes/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Ionóforos/farmacologia , Íons , Metabolismo dos Lipídeos , Manganês/farmacologia , Compostos de Manganês/farmacologia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Prótons , Sulfatos/farmacologia , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de TempoRESUMO
Pharmacokinetic modeling and simulation is a powerful tool for the prediction of drug concentrations in the absence of analytical techniques that allow for direct quantification. The present study applied this modeling approach to determine active drug release from a nanoparticle prodrug formulation. A comparative pharmacokinetic study of a nanoscale micellar docetaxel (DTX) prodrug, Procet 8, and commercial DTX formulation, Taxotere, was conducted in bile duct cannulated rats. The nanoscale (~40nm) size of the Procet 8 formulation resulted in confinement within the plasma space and high prodrug plasma concentrations. Ex vivo prodrug hydrolysis during plasma sample preparation resulted in unacceptable error that precluded direct measurement of DTX concentrations. Pharmacokinetic modeling of Taxotere and Procet 8 plasma concentrations, and their associated biliary metabolites, allowed for prediction of the DTX concentration profile and DTX bioavailability, and thereby evaluation of Procet 8 metabolism. Procet 8 plasma decay and in vitro plasma hydrolytic rates were identical, suggesting that systemic clearance of the prodrug was primarily metabolic. The Procet 8 and Taxotere plasma profiles, and associated docetaxel hydroxy-tert-butyl carbamate (HDTX) metabolite biliary excretion, were best fit by a two compartment model, with both linear and non-linear DTX clearance, and first order Procet 8 hydrolysis. The model estimated HDTX clearance rate agreed with in vitro literature values, supporting the predictability of the proposed model. Model simulation at the 10mg DTX equivalent/kg dose level predicted DTX formation rate-limited kinetics and a peak plasma DTX concentration of 39ng/mL at 4h for Procet 8, in comparison to 2826ng/mL for Taxotere. As a result of nonlinear DTX clearance, the DTX AUCinf for the Procet 8 formulation was predicted to be 2.6 times lower than Taxotere (775 vs. 2017h×ng/mL, respectively), resulting in an absolute bioavailability estimate of 38%. As DTX clearance in man is considered linear, this low bioavailability is likely species-dependent. These data support the use of pharmacokinetic modeling and simulation in cases of complex formulations, where analytical methods for direct measurement of free (released) drug concentrations are unavailable. Uses of such models may include interpretation of preclinical toxicology studies, selection of first in man dosing regimens, and PK/PD model development.
Assuntos
Antineoplásicos/metabolismo , Bile/metabolismo , Nanopartículas/metabolismo , Pró-Fármacos/metabolismo , Taxoides/metabolismo , Animais , Antineoplásicos/farmacocinética , Simulação por Computador , Docetaxel , Feminino , Hidrólise , Cinética , Masculino , Micelas , Modelos Biológicos , Pró-Fármacos/farmacocinética , Ratos , Ratos Sprague-Dawley , Taxoides/farmacocinéticaRESUMO
OBJECTIVE: To evaluate the possibility of improved selective killing of acute myeloid leukemia (AML) cells with CPX-351 (a liposomal formulation of cytarabine and daunorubicin). CPX-351 and the same molar ratio of free drugs were compared for cytotoxicity against colony-forming cells (CFCs) and subpopulations of cells enriched for primitive progenitors from AML patients and normal granulocyte colony-stimulating factor-mobilized peripheral blood (PB) and bone marrow (BM) donors. MATERIALS AND METHODS: AML blasts (n = 13) and normal PB and BM cells (n = 7) were incubated for 24 hours in various concentrations of CPX-351 or free drugs before plating in CFC assay or staining with anti-CD34 and anti-CD38 antibodies, Annexin-V, and propidium iodide followed by fluorescence-activated cell sorting analysis. High performance liquid chromatography was used to measure intracellular daunorubicin accumulation. RESULTS: AML blasts and progenitors from patients who achieved complete remission were more sensitive to both CPX-351 and free drugs than the same cells from patients with chemotherapy refractory leukemia. However, AML CFCs and CD34(+)CD38(-) AML blasts (enriched for candidate leukemia stem cells) from the same patient showed similar sensitivity to the liposomal or free drug formulations. In contrast, CFCs and CD34(+)CD38(-) cells from normal PB and BM were fivefold more sensitive to the free drugs than to CPX-351. Consistent with these observations, preferential intracellular accumulation of CPX-351 in AML over normal cells was observed, while there was little difference in drug uptake between AML and normal cells with the free drug cocktail. CONCLUSIONS: CPX-351, as compared to free cytarabine:daunorubicin, shows enhanced selective in vitro cytotoxicity for AML rather than normal progenitors.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Citarabina/farmacologia , Daunorrubicina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Doença Aguda , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Composição de Medicamentos , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Leucócitos Mononucleares/metabolismo , Lipossomos , Células-Tronco/metabolismo , Células Tumorais CultivadasRESUMO
A newly identified form of multidrug resistance (MDR) in tumor cells is presented, pertaining to the commonly encountered resistance of cancer cells to anticancer drug combinations at discrete drug:drug ratios. In vitro studies have revealed that whether anticancer drug combinations interact synergistically or antagonistically can depend on the ratio of the combined agents. Failure to control drug ratios in vivo due to uncoordinated pharmacokinetics could therefore lead to drug resistance if tumor cells are exposed to antagonistic drug ratios. Consequently, the most efficacious drug combination may not occur at the typically employed maximum tolerated doses of the combined drugs if this leads to antagonistic ratios in vivo after administration and resistance to therapeutic effects of the drug combination. Our approach to systematically screen a wide range of drug ratios and concentrations and encapsulate the drug combination in a liposomal delivery vehicle at identified synergistic ratios represents a means to mitigate this drug ratio-dependent MDR mechanism. The in vivo efficacy of the improved agents (CombiPlex formulations) is demonstrated and contrasted with the decreased efficacy when drug combinations are exposed to tumor cells in vivo at antagonistic ratios.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antagonismo de Drogas , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Humanos , Lipossomos/química , Lipossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
CPX-351, a liposomal formulation co-encapsulating cytarabine (Cyt) and daunorubicin (Daun), has been developed, which delivers synergistic Cyt:Daun molar ratios to bone marrow. CPX-351 has demonstrated markedly superior anti-leukemic activity over free Cyt:Daun drug cocktails in preclinical models. Given the prolonged plasma lifetime of CPX-351, we examined the relationship between therapeutic efficacy and the frequency of treatment in the consolidation setting using a bone marrow-engrafting human leukemia xenograft model. Adding a day 1,3,5 consolidation treatment course for CPX-351 therapy improved the increase in lifespan (ILS) from 116% and no cures for a single induction course, to 268% plus a 33% cure rate for an induction plus consolidation course. In contrast, free Cyt:Daun cocktail treatment provided much lower ILS values with no cures. Administering CPX-351 as consolidation therapy starting on day 42 using a day 1,3, day 1,5, or day 1,7 schedule yielded ILS values of 154%, 185%, and 108%, respectively. The increased efficacy observed for the day 1,3 and day 1,5 consolidation schedules was associated with elevated bone marrow drug accumulation for the second doses. The enhanced efficacy obtained for intermediate dosing frequency in the consolidation setting suggests that the anti-leukemic activity of synergistic drug ratios is dependent on both duration of exposure and maintenance above a therapeutic threshold.