RESUMEN
Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and it is also present on the neovasculature within many non-prostate solid tumors. Herein, we report on the construction and biological testing of novel tubulysin B-containing therapeutic agents for the treatment of PSMA-expressing cancer. One of these compounds, EC1169, emerged as a lead candidate for preclinical development and phase 1 clinical testing. This water-soluble conjugate was shown to have high affinity for PSMA-positive cells. When tested in vitro, EC1169 was found to inhibit the growth of PSMA-positive cells, but it displayed no activity against PSMA-negative cells. Brief treatment of nude mice bearing PSMA-positive LNCaP human xenografts with EC1169 led to complete remissions and cures. Furthermore, this activity occurred in the absence of weight loss. In contrast, the nontargeted tubulysin B drug proved to be inactive against the LNCaP tumor model when administered at doses near to or greater than the maximum tolerated level. PSMA-negative KB tumors did not appreciably respond to EC1169 therapy, thereby confirming this compound's targeted specificity for PSMA-positive cells. Finally, treatment of LNCaP-bearing mice with docetaxel (the most active chemotherapeutic agent approved for late stage prostate cancer therapy) was found to produce only modest anti-tumor activity, and this outcome was also associated with severe weight loss. Taken together, these results strongly indicate that PSMA-positive tumors may be effectively treated using highly potent, PSMA-targeted small-molecule drug conjugates using regimens that do not cause undesirable side effects.
Asunto(s)
Antígenos de Superficie/análisis , Antineoplásicos/uso terapéutico , Glutamato Carboxipeptidasa II/análisis , Oligopéptidos/uso terapéutico , Ácidos Pipecólicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Masculino , Ratones Desnudos , Oligopéptidos/química , Ácidos Pipecólicos/química , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of a reactive intermediate possessing an aldehyde and aromatic amine. An intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine and, as a result, the diazepine ring. In our pro-PBDs, we mask the aldehyde as a hydrolytically sensitive oxazolidine moiety which in turn is a part of a reductively labile self-immolative linker system. To prove the range of applications for this new class of latent DNA-alkylators, we designed and synthesized several novel latent warheads: pro-PBD dimers and hybrids of pro-PBD with other sequence-selective DNA minor groove binders. Preliminary preclinical pharmacology studies showed excellent biological activity and specificity.
Asunto(s)
Benzodiazepinas/química , Benzodiazepinas/metabolismo , Diseño de Fármacos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Profármacos/síntesis química , Profármacos/metabolismo , Pirroles/química , Pirroles/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Técnicas de Química Sintética , Humanos , Células KB , Neoplasias/patología , Profármacos/química , Pirroles/farmacología , Pirroles/uso terapéuticoRESUMEN
Vintafolide is a potent folate-targeted vinca alkaloid small molecule drug conjugate (SMDC) that has shown promising results in multiple clinical oncology studies. Structurally, vintafolide consists of 4 essential modules: (1) folic acid, (2) a hydrophilic peptide spacer, (3) a disulfide-containing, self-immolative linker, and (4) the cytotoxic drug, desacetylvinblastine hydrazide (DAVLBH). Here, we report a structure-activity study evaluating the biological impact of (i) substituting DAVLBH within the vintafolide molecule with other vinca alkaloid analogues such as vincristine, vindesine, vinflunine, or vinorelbine; (ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide with alternative hydrophilic spacers of varied composition; and (iii) varying the composition of the linker module. A series of vinca alkaloid-containing SMDCs were synthesized and purified by HPLC and LCMS. The SMDCs were screened in vitro against folate receptor (FR)-positive cells, and anti-tumor activity was tested against well-established subcutaneous FR-positive tumor xenografts. The cytotoxic and anti-tumor activity was directly compared to that produced by vintafolide. Among all the folate vinca alkaloid SMDCs tested, DAVLBH-containing SMDCs were active, while those constructed with vincristine, vindesine, or vinorelbine analogues failed to produce meaningful biological activity. Within the DAVLBH series, having a bioreleasable, self-immolative linker system was found to be critical for activity since multiple analogues constructed with thioether-based linkers all failed to produce meaningful activity both in vitro and in vivo. Substitutions of some or all of the natural amino acids within vintafolide's hydrophilic spacer module did not significantly change the in vitro or in vivo potency of the SMDCs. Vintafolide remains one of the most potent folate-vinca alkaloid SMDCs produced to date, and continued clinical development is warranted.
Asunto(s)
Antineoplásicos/farmacología , Ácido Fólico/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Alcaloides de la Vinca/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ácido Fólico/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Conformación Molecular , Neoplasias Experimentales/patología , Estereoisomerismo , Relación Estructura-Actividad , Alcaloides de la Vinca/químicaRESUMEN
During a phase I trial of EC145 (a folate-targeted vinca alkaloid conjugate), constipation was identified as the dose-limiting toxicity, probably from a nonfolate receptor-related liver clearance process capable of releasing unconjugated vinca alkaloid from EC145 and shuttling it to the bile. Here, we report on the selective placement of novel carbohydrate segments (1-amino-1-deoxy-glucitolyl-γ-glutamate) spaced in-between the folate and vinca alkaloid moieties of EC145, which yielded a new agent (EC0489) that is equipotent but less toxic than EC145. Whereas both compounds could cure tumor-bearing mice reproducibly, EC0489 differed from EC145 with i) a shorter elimination half-life, ii) approximately 70% decrease in bile clearance, iii) a 4-fold increase in urinary excretion, and iv) improved tolerability in rodents. This combination of improvements justified the clinical evaluation of EC0489 where currently administered dose levels have exceeded the maximal tolerated dose of EC145 by approximately 70%, thereby reflecting the translational benefits to this new approach.
Asunto(s)
Antineoplásicos/farmacocinética , Ácido Fólico/análogos & derivados , Hígado/metabolismo , Alcaloides de la Vinca/farmacocinética , Animales , Antineoplásicos/toxicidad , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Femenino , Receptores de Folato Anclados a GPI/fisiología , Ácido Fólico/farmacocinética , Ácido Fólico/toxicidad , Riñón/metabolismo , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Alcaloides de la Vinca/toxicidadRESUMEN
Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been chemically synthesized; however, these efforts have been hampered by poor yields and arduous purifications. In contrast, treatment of a mixture of natural tubulysins A, B, C, G, and I, obtained from a fermentation batch with trifluoroacetic acid results in the formation of a single N-acyliminium ion. Subsequent addition of butyric, isopentyl, or acetic acid results in the formation of tubulysin B, A, or I, respectively, as a single species. New tubulysin analogs can be formed upon treatment of the acyliminium ion with other nucleophiles such as alcohols, thiols, and nitriles, resulting in corresponding N-acyl-N,O-acetals, N-acyl-N,S-thioacetals, and N,N'-diacyl-aminals. Carbon-carbon bond formation is also possible with a modification of this protocol. The cytotoxicies of the natural tubulysins and tubulysin analogs synthesized by this method were evaluated on KB cells.
Asunto(s)
Productos Biológicos/síntesis química , Técnicas de Química Sintética/métodos , Myxococcales/química , Oligopéptidos/síntesis química , Ácidos Pipecólicos/síntesis química , Moduladores de Tubulina/síntesis química , Ácido Acético/química , Productos Biológicos/química , Técnicas de Química Sintética/economía , Iminas/química , Iones/química , Oligopéptidos/química , Ácidos Pipecólicos/química , Moduladores de Tubulina/químicaRESUMEN
Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors. EC2629 treatment of nude mice bearing FR-positive KB human xenografts led to cures in 100% of the test animals with very low dose levels (300 nmol/kg) following a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing). Complete responses were also observed against FR-positive paclitaxel (KB-PR) and cisplatin (KB-CR) resistant models. When evaluated against FR-positive patient derived xenograft (PDX) models of ovarian (ST070), endometrial (ST040) and triple negative breast cancers (ST502, ST738), EC2629 showed significantly greater anti-tumor activity compared to their corresponding standard of care treatments. Taken together, these studies thus demonstrated that EC2629, with its distinct DNA reacting mechanism, may be useful in treating FR-positive tumors, including those that are classified as drug resistant.
Asunto(s)
Antineoplásicos/farmacología , Reactivos de Enlaces Cruzados/farmacología , ADN/química , Neoplasias Endometriales/tratamiento farmacológico , Receptores de Folato Anclados a GPI/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Alquilantes/química , Animales , Bovinos , Cisplatino/administración & dosificación , Perros , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Femenino , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Humanos , Concentración 50 Inhibidora , Células KB , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Paclitaxel/administración & dosificación , Ratas , Alcaloides de la Vinca/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We recently developed a new group of folate-conjugated Vinca alkaloids, one of which, EC145, emerged as a candidate for clinical development. Brief treatment of nude mice bearing approximately 100 mm(3) folate receptor-positive human xenografts led to complete response (CR) in 5/5 mice and cures (i.e., remission without a relapse for >90 days post-tumor implantation) in 4/5 mice. Multiple CRs and cures were also noted when EC145 was used to treat mice initially bearing tumors as large as 750 mm(3). Likewise, complete cures (5/5) resulted following the treatment of an aggressive folate receptor-positive J6456 lymphoma model. The activity of EC145 was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, no significant antitumor activity (0/5 CR) was observed in EC145-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating the target-specific activity of EC145. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the unconjugated drug (desacetylvinblastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose levels. Subsequent dose regimen studies confirmed that EC145 given on a more frequent, qdx5 schedule resulted in the most effective antitumor response as compared with an equivalent total dose given on thrice- or single-injection-per-week schedule. Taken together, these studies show that EC145 has significant antiproliferative activity and tolerability, thus lending support to an ongoing phase 1 trial for the treatment of advanced malignancies.
Asunto(s)
Antineoplásicos/farmacología , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Alcaloides de la Vinca/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Esquema de Medicación , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ácido Fólico/administración & dosificación , Humanos , Células KB , Linfoma/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Alcaloides de la Vinca/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
EC131, a new folate receptor (FR)-targeted drug conjugate, was prepared by covalently attaching the vitamin folic acid (FA) to a potent microtubule-inhibiting agent, maytansinoid DM1, via an intramolecular disulfide bond. When tested on cells in culture, EC131 was found to retain high affinity for FR-positive cells and to provide FR-specific cytotoxicity with an IC(50) in the low nanomolar range. The activity of EC131 was completely blocked in the presence of an excess of free FA, and no activity was detected against FR-negative cells. When evaluated against s.c. FR-positive M109 tumors in BALB/c mice, EC131 showed marked antitumor efficacy. Furthermore, this therapeutic effect occurred in the apparent absence of weight loss or noticeable organ tissue degeneration. In contrast, no significant antitumor activity was observed in EC131-treated animals that were codosed with an excess of FA, thus demonstrating the targeted specificity of the in vivo activity. EC131 also showed marked antitumor activity against FR-positive human KB tumors, but not against FR-negative A549 tumors, in nude mice with no evidence of systemic toxicity during or after the therapy. In contrast, therapy with the free maytansinoid drug (in the form of DM1-S-Me) proved not to be effective against the KB model when administered at its maximum tolerated dose (MTD). Taken together, these results indicate that EC131 is a highly potent agent capable of producing therapeutic benefit in murine tumor models at sub-MTD levels.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Ácido Fólico/farmacología , Maitansina/farmacología , Extractos Vegetales/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Receptores de Folato Anclados a GPI , Ácido Fólico/química , Ácido Fólico/metabolismo , Humanos , Células KB , Dosis Máxima Tolerada , Maitansina/química , Maytenus/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors' natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.
RESUMEN
EC1456 is a folate-tubulysin conjugate constructed with an all-D enantiomeric spacer/linker configuration. When tested against folate receptor (FR)-positive cells, EC1456 demonstrated dose-responsive activity with an approximate 1000-fold level of specificity. Treatment of nude mice bearing FR-positive human xenografts (as large as 800 mm3) with non-toxic doses of EC1456 led to cures in 100% of the mice. Combinations of low dose EC1456 with standard of care agents such as platins, taxanes, topotecan and bevacizumab, safely and significantly augmented the growth inhibitory effects of these commonly used agents. When tested against FR-positive human tumor xenograft models having confirmed resistance to a folate-vinca alkaloid (vintafolide), cisplatin or paclitaxel, EC1456 was found to generate partial to curative responses. Taken together, these studies demonstrate that EC1456 has significant anti-proliferative activity against FR-positive tumors, including models which were anticancer drug resistant, thereby justifying a Phase 1 trial of this agent for the treatment of advanced human cancers.
Asunto(s)
Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácido Fólico/química , Neoplasias/tratamiento farmacológico , Oligopéptidos/química , Ácidos Pipecólicos/química , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Bevacizumab/administración & dosificación , Carboplatino/administración & dosificación , Línea Celular Tumoral , Cisplatino/administración & dosificación , Docetaxel/administración & dosificación , Evaluación Preclínica de Medicamentos/métodos , Humanos , Ratones Desnudos , Neoplasias/patología , Paclitaxel/administración & dosificación , Topotecan/administración & dosificación , Carga Tumoral/efectos de los fármacosRESUMEN
Folic acid (pteroylglutamic acid) represents a useful ligand for targeted cancer therapies because it binds to a common epithelial tumor antigen known as the folate receptor. We previously devised an immunotherapy strategy that uses a bispecific ligand, a folate-hapten (FITC) conjugate, to redirect endogenously induced anti-FITC antibodies to folate receptor-positive tumor cells following parenteral administration. Here, we present results from preclinical pharmacokinetic and tissue biodistribution studies using a radioactive folate-FITC conjugate and results from dose optimization studies done in tumor-bearing animals. Folate-FITC was found to be rapidly eliminated in non-immunized mice; however, in immunized hosts, folate-FITC was shown to form immune complexes with FITC-specific antibodies, the consequence of which was a approximately 173-fold increase in drug exposure (i.e., area under the curve). Using a newly developed ELISA assay, the extent of circulating anti-FITC antibodies occupied by parenterally given folate-FITC was determined to be proportional to the given dose. Furthermore, high doses of folate-FITC were found to promote the cosaturation of tumor cell surface folate receptors and circulating FITC-specific antibodies, blocking the immune recognition of tumor cells and thereby reducing antitumor activity. Nonetheless, by extending the duration of treatment and administering subsaturating doses of folate-FITC, enhanced antitumor response was observed in mice bearing established folate receptor-positive M109 tumors. Overall, results from the present study may help to guide clinicians through on-going clinical investigations of folate-targeted immunotherapy.
Asunto(s)
Citocinas/farmacocinética , Ácido Fólico/farmacocinética , Inmunoconjugados/farmacocinética , Inmunoterapia/métodos , Animales , Citocinas/farmacología , Femenino , Fluoresceína-5-Isotiocianato/farmacocinética , Ácido Fólico/farmacología , Haptenos/farmacología , Inmunoconjugados/farmacología , Ratones , Ratones Endogámicos BALB C , Distribución Tisular , TritioRESUMEN
PURPOSE: EC0305 represents a folate-tubulysin B construct capable of specifically eradicating folate receptor (FR)-positive subcutaneous tumors from mice (Leamon et al., Cancer Res 68:9839-9844, 8). Herein we report on the use of multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl-γ-glutamate) placed in-between the folate and tubulysin B moieties of EC0305 creating a new conjugate, herein referred to as EC0531, with more desirable biological properties. METHODS: The synthesis of EC0531 and its tritium-labeled counterpart are described. EC0531's affinity for FR binding and specific cytotoxic activity was assessed using standard in vitro assays. Human tumor xenografts were used to directly compare EC0305 and EC0531's antitumor activity. Finally, bile duct cannulated, female Sprague-Dawley rats were used to compare hepatobiliary clearance of these two targeted chemotherapeutic agents. RESULTS: EC0531 tightly binds to the FR with an affinity about half that of folic acid. It was found to specifically inhibit the growth of FR+ cells (IC50 of ~2 nM) in a dose-dependent manner. Using 3H-labeled compounds, more than a 12-fold higher amount of tubulysin was measured in a FR + human tumor xenograft compared to the unconjugated drug, a finding that explains, in part, why EC0531 displays curative activity, whereas the unconjugated tubulysin agent is essentially inactive. EC0531 was found to produce greater FR-specific anti-tumor activity at lower dose levels than EC0305; furthermore, EC0531's maximum tolerated dose level was significantly higher than that of EC0305, likely because EC0531's saccharopeptidic-based spacer allows for ~sixfold reduction in hepatic clearance. CONCLUSIONS: These data provide additional evidence that the therapeutic range of targeted small-molecule drug conjugates can be favorably increased using molecular spacers constructed with 1-amino-1-deoxy-glucitolyl-γ-glutamate residues.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Receptor 1 de Folato/efectos de los fármacos , Ácido Fólico/análogos & derivados , Oligopéptidos/química , Oligopéptidos/farmacología , Ácidos Pipecólicos/química , Animales , Antineoplásicos/farmacocinética , Bilis/metabolismo , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Femenino , Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Humanos , Marcaje Isotópico , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratas , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Ácido Fólico/análogos & derivados , Expresión Génica , Alcaloides de la Vinca/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Análisis por Conglomerados , Biología Computacional/métodos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Receptores de Folato Anclados a GPI/antagonistas & inhibidores , Ácido Fólico/farmacología , Perfilación de la Expresión Génica , Humanos , Ratones , Platino (Metal)/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Our group previously reported on the synthesis and characterization of a novel (99m)Tc-based folate-peptide chelator called EC20. This agent was found to bind folate receptor (FR)-positive cells and tissues with high affinity and was deemed useful for radiodiagnostic applications. In this study, we investigated the effect of D-amino acid substitution within EC20 on its tissue biodistribution. Expanded in vivo studies were also performed with unmodified (99m)Tc-EC20 to determine the effect of tumor FR expression, tumor size, tumor location, route of dose administration, and rodent diet on the agent's tissue biodistribution pattern. METHODS: EC20 and EC53, the all-D-isomer of EC20, were synthesized and radiolabeled with (99m)Tc. The relative affinity of EC53 to the FR with respect to EC20 was then determined in cultured tumor cells. The ability of (99m)Tc-EC20 and (99m)Tc-EC53 to target tumors in vivo was examined using BALB/c mice with subcutaneously inoculated M109 or 4T1 cells, yielding 0.1- to 0.5-g tumors in 20 d. RESULTS: The D-amino acid substitutions of EC20 were found to reduce the uptake of the agent into tumor and major organs. Subsequent studies using the original (99m)Tc-EC20 agent confirmed that its net tumor uptake was specific and proportional to FR expression levels in tumor cells as well as linear with respect to the overall tumor size. Further, (99m)Tc-EC20 uptake was found to be independent of both solid tumor location (intraperitoneal vs. subcutaneous) and the route of administration (intraperitoneal vs. intravenous). Interestingly, leucovorin supplementation of a commonly used folate-deficient laboratory chow had no effect on the agent's overall tissue biodistribution pattern. But, tumor-to-nontumor ratios could be increased up to 2.7-fold when 1 equivalent of free folic acid was coinjected with (99m)Tc-EC20. CONCLUSION: Taken together, these results confirm that (99m)Tc-EC20 has the potential to be a clinically useful noninvasive radiodiagnostic agent for detecting the locus of FR-positive cancers.
Asunto(s)
Proteínas Portadoras/metabolismo , Cisteína/análogos & derivados , Ácido Fólico/metabolismo , Neoplasias Experimentales/diagnóstico por imagen , Compuestos de Organotecnecio , Receptores de Superficie Celular , Animales , Cisteína/farmacocinética , Femenino , Receptores de Folato Anclados a GPI , Ratones , Ratones Endogámicos BALB C , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
PURPOSE: When evaluated in patients with ovarian and other cancer, vintafolide (EC145), a potent folate-targeted vinca alkaloid conjugate, displayed a toxicity profile that seemed to be nonoverlapping with many standard-of-care cancer therapeutics. It was, therefore, hypothesized that combining vintafolide with certain approved anticancer drugs may afford greater therapeutic efficacy compared with single-agent therapy. To explore this concept, vintafolide was evaluated in combination with pegylated liposomal doxorubicin (PLD; DOXIL), cisplatin, carboplatin, paclitaxel, docetaxel, topotecan, and irinotecan against folate receptor (FR)-positive models. EXPERIMENTAL DESIGN: FR-expressing KB, M109, IGROV, and L1210 cells were first exposed to graded concentrations of vintafolide, either alone or in combination with doxorubicin (active ingredient in PLD), and isobologram plots and combination index values generated. The vintafolide combinations were also studied in mice bearing various FR-expressing tumors. RESULTS: Vintafolide displayed strong synergistic activity against KB cells when combined with doxorubicin, and no less-than-additive effects resulted when tested against M109, IGROV, and L1210 cells. In contrast, when either desacetylvinblastine hydrazide (DAVLBH; the vinca alkaloid moiety in vintafolide) or vindesine (the vinca alkaloid most structurally similar to DAVLBH) were tested in combination with doxorubicin, less-than-additive antitumor effects were observed. In vivo, all vintafolide drug combinations produced far greater antitumor effect (complete responses and cures) compared with the single agents alone, without significant increase in overall toxicity. Importantly, these benefits were not observed with combinations of PLD and DAVLBH or vindesine. CONCLUSIONS: On the basis of these encouraging preclinical results, clinical studies to evaluate vintafolide drug combination therapies are now under way.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Camptotecina/farmacología , Carboplatino/administración & dosificación , Carboplatino/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/farmacología , Docetaxel , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Sinergismo Farmacológico , Ácido Fólico/administración & dosificación , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Células HeLa , Humanos , Irinotecán , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Desnudos , Neoplasias/patología , Neoplasias Experimentales/patología , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Taxoides/administración & dosificación , Taxoides/farmacología , Topotecan/administración & dosificación , Topotecan/farmacología , Resultado del Tratamiento , Alcaloides de la Vinca/administración & dosificación , Alcaloides de la Vinca/farmacologíaRESUMEN
Herein we report on the potencies of 4 related folate-conjugated tubulysins constructed with either tubulysin B hydrazide (EC0305), tubulysin A hydrazide (EC0510), the N,O-acetal derivative of natural tubulysins (EC0317) or a tubulysin B ester (EC0302). Our results confirmed that EC0305 is the most favorable conjugate of the group due to its potent antitumor activity [100% cures at 1 micromol/kg, three times a week (TIW) for 2 weeks] and its favorably low toxicity profile. In contrast, the natural tubulysin B drug proved to be inactive against a human nasopharyngeal tumor model when administered at doses near to or greater than the maximum tolerated dose (MTD). When tested against more chemoresistant folate receptor expressing M109 and 4T1-cl2 tumors, EC0305 displayed superior antitumor activity over a previously disclosed folate conjugate of desacetylvinblastine monohydrazide (EC145). These studies demonstrate that EC0305 has significant antiproliferative activity against FR expressing tumors, including those which are generally more chemoresistant, and that EC0305 should be considered for development as a candidate for the treatment of advanced FR-expressing human cancers.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Ácido Fólico/análogos & derivados , Oligopéptidos/química , Oligopéptidos/farmacología , Animales , Antineoplásicos/toxicidad , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Bovinos , Línea Celular Tumoral , Perros , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/química , Ácido Fólico/farmacología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Oligopéptidos/toxicidad , Unión Proteica , Conejos , Ratas , Receptores de Superficie Celular/metabolismo , Relación Estructura-ActividadRESUMEN
The membrane-bound high-affinity folate receptor (FR) is highly expressed on a wide range of primary and metastatic human cancers, such as those originating in ovary, lung, breast, endometrium, kidney, and brain. Because folate-linked conjugates bind to and become internalized within FR-expressing cells (similar to that of free folic acid), we explored the possibility of using the folate ligand to target a potent, semisynthetic analogue of the microtubule inhibitor tubulysin B to FR-enriched tumors. When tested in vitro, a novel folate conjugate, herein referred to as EC0305, was found to specifically inhibit the growth of a panel of FR-positive cell lines (IC50 range, 1-10 nmol/L) in a dose-dependent manner, whereas cells lacking FR expression were unaffected. The potency of EC0305 was also confirmed against a human KB xenograft-nu/nu mouse cancer model. Here, a brief three times per week, 2-week regimen yielded remarkable antitumor activity (100% tumor-free animals) without causing significant weight loss or major organ tissue degeneration. In contrast, antitumor activity was completely abolished in EC0305-treated animals that were co-dosed with an excess of a nontoxic folate-containing analogue, thereby confirming that the antitumor effect of this agent was mediated by FRs. The advantage provided by folate conjugation was further proved by the untargeted free drug, which was found to be completely inactive at both tolerable and highly toxic dose levels. Collectively, these results show that this potent antiproliferative tubulysin compound can be specifically delivered to FR-positive tumors to provide substantial therapeutic benefit using well-tolerable dosing regimens.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Oligopéptidos/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Antineoplásicos/metabolismo , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos/métodos , Etilenodiaminas/farmacología , Femenino , Fluoresceína/farmacología , Receptores de Folato Anclados a GPI , Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Humanos , Células KB , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Oligopéptidos/metabolismo , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have designed a new type of tumor-targeted agent by tethering two different drug molecules, with distinct biological mechanisms of action, to the same ligand. This compound, named EC0225, represents the "first in class" multidrug, folate receptor (FR)-targeted agent to be disclosed. It was constructed with a single folate molecule, extended by a hydrophilic peptide-based spacer, which was in turn attached to mitomycin and Vinca alkaloid units via two separate disulfide-containing linkers. EC0225 produced potent, dose-responsive activity in vitro, and curative activity was observed against FR-positive syngeneic and xenograft tumors following the administration of well-tolerated dosing regimens. Multiple complete responses and cures were also noted when EC0225 was used to treat mice initially bearing tumors as large as 750 mm (3) in volume. Overall, EC0225's impressive preclinical activity allowed for its selection as a development candidate and for the start of Phase 1 clinical trials, which began in March of 2007, for the treatment of advanced malignancies.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos , Ácido Fólico/metabolismo , Neoplasias/metabolismo , Animales , Antineoplásicos/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Ácido Fólico/química , Ácido Fólico/farmacología , Ácido Fólico/toxicidad , Humanos , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias/patología , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
EC140 is a water soluble folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, subcutaneous folate receptor (FR)-positive tumors in multiple animal models. Recent structure-activity and optimization studies have yielded a disulfide bond-containing counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently observed in EC145-treated tumor-bearing animals. Furthermore, EC145 was not found to be active against a FR-negative tumor model. Additional preclinical studies are therefore warranted to better understand EC145's breadth of activity against FR-positive tumors.
Asunto(s)
Ácido Fólico/análogos & derivados , Neoplasias Experimentales/prevención & control , Pterinas/farmacología , Alcaloides de la Vinca/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/prevención & control , Animales , Unión Competitiva , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Receptores de Folato Anclados a GPI , Ácido Fólico/química , Ácido Fólico/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Unión Proteica , Pterinas/química , Pterinas/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Tiempo , Carga Tumoral , Alcaloides de la Vinca/química , Alcaloides de la Vinca/metabolismoRESUMEN
A novel folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), herein referred to as EC140, was designed and evaluated for biological activity against folate receptor (FR)-positive cells and tumors. EC140 was produced by coupling a peptidic analogue of the vitamin folic acid to DAVLBH via an acylhydrazone bond. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Initial in vivo tests confirmed EC140's activity in both syngeneic and xenograft models. Hence, enduring complete responses were observed in animals bearing established, subcutaneous tumors prior to therapy using regimens that produced minor toxicity. In contrast, treatment with the unconjugated DAVLBH drug produced nominal efficacy when dosed at its MTD. Overall, EC140's performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.