RESUMEN
Highly cytotoxic maytansine derivatives are widely used in targeted tumor delivery. Structure-activity studies published earlier suggested the C9 carbinol to be a key element necessary to retain the potency. However, in 1984 a patent was published by Takeda in which the synthesis of 9-thioansamitocyn (AP3SH) was described and its activity in xenograft models was shown. In this article we summarize the results of an extended study of the anti-tumor properties of AP3SH. Like other maytansinoids, it induces apoptosis and arrests the cell cycle in the G2/M phase. It is metabolized in liver microsomes predominately by C3A4 isoform and doesn't inhibit any CYP isoforms except CYP3A4 (midazolam, IC50 7.84 µM). No hERG inhibition, CYP induction or mutagenicity in Ames tests were observed. AP3SH demonstrates high antiproliferative activity against 25 tumor cell lines and tumor growth inhibition in U937 xenograft model. Application of AP3SH as a cytotoxic payload in drug delivery system was demonstrated by us earlier.
Asunto(s)
Antineoplásicos , Maitansina , Humanos , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Línea Celular Tumoral , Ciclo Celular , División CelularRESUMEN
BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Vesículas Extracelulares/química , Streptococcus mutans/fisiología , Membrana Externa Bacteriana/química , Gentamicinas/farmacología , Pruebas de Sensibilidad Microbiana , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/patogenicidadRESUMEN
Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.
Asunto(s)
Antibacterianos/metabolismo , Antibiosis/fisiología , Burkholderia/metabolismo , Vesículas Extracelulares/metabolismo , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Antibacterianos/farmacología , Membrana Externa Bacteriana/metabolismo , Biopelículas/crecimiento & desarrollo , Glucolípidos/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Quinolinas/metabolismoRESUMEN
BACKGROUND: Human pancreatic carcinoids, a type of neuroendocrine tumors, are asymptomatic and difficult to diagnose, with the effects of traditional anti-cancer therapies being limited. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was evaluated for its effects alone and in combination with receptor-targeting peptide-drug conjugate via increasing drug internalization. MATERIALS AND METHODS: The in vitro and in vivo assays were used to evaluate the effects of VPA and somatostatin receptor-targeting camptothecin-somatostatin conjugate (CPT-SST). RESULTS: VPA induced proliferation suppression, cell apoptosis and cell cycle arrest. VPA acts as a HDAC inhibitor to induce a decrease of HDAC4 and an increase of acetylated histone 4 (AcH4). Meanwhile, most importantly, besides activating Notch signaling, VPA was observed to stimulate the expression of somatostatin receptor type 2 (SSTR2) that has been applied for receptor-targeting therapies. This characteristic was used for a combination therapy of VPA and CPT-SST. The combination displayed much more potent anti-tumor effects on carcinoid tumor growth by increasing SSTR2 density and drug internalization in target tumor cells. CONCLUSION: The combination of VPA and a SSTR2-targeting agent provides us a promising approach in treatment of carcinoid tumors.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Péptidos/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Somatostatina/metabolismo , Ácido Valproico/farmacología , Animales , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: We investigated the effects of the anti-epilepsy drug valproic acid (VPA) alone and in combination in treating cervical cancer. METHODS: VPA was investigated for its effects on cervical cancer Hela cell proliferation and tumor growth via in vitro and in vivo assays. RESULTS: VPA induce cell growth suppression and cell cycle arrest, with an increase of Notch1 that acts as a tumor suppressor and the change of other tumor-associated genes such as p21, p63 and PCNA. VPA was also found to induce cell morphological change, with an increase of certain cell transformation markers such as snail1, snail2 and N-cadherin. Moreover, VPA could significantly up-regulate somatostatin receptor type II (SSTR2). Our in vivo study further demonstrated that VPA via inducing SSTR2 up-regulation extremely enhanced the anti-tumor ability of the SSTR2-preferential cytotoxic COL-SST conjugate in xenografts. CONCLUSIONS: VPA could not only suppress tumor progression but also provide a novel promising therapeutic choice in combination with a receptor-targeted cytotoxic conjugate via activating the specific receptor.
Asunto(s)
Anticonvulsivantes/farmacología , Carcinoma/tratamiento farmacológico , Receptor Notch1/metabolismo , Receptores de Somatostatina/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Valproico/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colchicina/uso terapéutico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclooxigenasa 2/metabolismo , Combinación de Medicamentos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Expresión Génica , Genes p53/efectos de los fármacos , Células HeLa , Hormonas/uso terapéutico , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/metabolismo , Receptor Notch1/genética , Somatostatina/uso terapéutico , Moduladores de Tubulina/uso terapéuticoRESUMEN
The role of Notch signaling in cervical cancer is seemingly controversial. To confirm the function of Notch signaling in this type of cancer, we established a stable Notch1-activated cervical cancer HeLa cell line. We found that Notch1 activation resulted in apoptosis, cell cycle arrest, and tumor suppression. At the molecular level, we found that a variety of genes associated with cyclic AMP, G protein-coupled receptor, and cancer signaling pathways contributed to Notch1-mediated tumor suppression. We observed that the expression of somatostatin (SST) was dramatically induced by Notch1 signaling activation, which was accompanied by enhanced expression of the cognate SST receptor subtype 1 (SSTR1) and SSTR2. Certain genes, such as tumor protein 63 (TP63, p63), were upregulated, whereas others, such as B-cell lymphoma 2 (BCL-2), Myc, Akt, and STAT3, were downregulated. Subsequently, knockdown of Notch1-induced SST reversed Notch1-induced decrease of BCL-2 and increase of p63, indicating that Notch1-induced tumor suppression may be partly through upregulating SST signaling. Our findings support a possible crosstalk between Notch signaling and SST signaling. Moreover, Notch-induced SSTR activation could enhance SSTR-targeted cancer chemotherapy. Valproic acid (VPA), a histone deacetylase inhibitor, suppressed cell growth and upregulated the expression of Notch1 and SSTR2. A combination therapy with VPA and the SSTR2-targeting cytotoxic conjugate CPT-SST strongly led to greater suppression, as compared to each alone. Our findings thus provide us with a promising clinical opportunity for enhanced cancer therapy using combinations of Notch1-activating agents and SSTR2-targeting agents.
Asunto(s)
Receptor Notch1/fisiología , Receptores de Somatostatina/fisiología , Transducción de Señal/fisiología , Somatostatina/fisiología , Neoplasias del Cuello Uterino/prevención & control , Animales , Puntos de Control del Ciclo Celular , Proliferación Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Femenino , Células HeLa , Humanos , Ratones , Receptores de Somatostatina/antagonistas & inhibidores , Neoplasias del Cuello Uterino/patologíaRESUMEN
A more convenient and facile approach for the synthesis and production of camptothecin-amino acids carbamate linkers, that can be used in the synthesis of bioconjugate peptides JF-10-81, JF-10-71, and other peptide analogs designed to target somatostatin receptors has been described.
Asunto(s)
Aminoácidos/química , Camptotecina/química , Carbamatos/química , Péptidos/síntesis química , Profármacos/síntesis química , Técnicas de Síntesis en Fase SólidaRESUMEN
In our previous study, we found that several tumor cell lines displayed high receptor-specific binding affinity, one of which, the human pancreatic carcinoid BON cell line, demonstrates high affinity binding of the bombesin (BN) and somatostatin (SST) receptor-specific ligands. In the present study, BON cells, as a representative model, were further applied to evaluate various peptide analogs and cytotoxic receptor-targeted peptide conjugates. We observed quick ligand-receptor internalization in BON cells as well as high binding affinity. Furthermore, BON cells have high expression of multidrug resistance-associated genes (MDR1) and show camptothecin (CPT) resistance. Various receptor-specific cytotoxic conjugates were synthesized and evaluated in the BON cell model via in vitro and in vivo studies. We found that all the tested conjugates displayed potent antitumor ability in xenografts. Especially, the CPT conjugates, CPT-SST, and CPT-BN, are most likely to increase sensitivity to CPT-resistant BON cells. Our findings suggest that appropriately defined tumor cell lines may provide physiologically relevant cell-based evaluations of novel peptide analogs and receptor-targeted chemotherapeutics.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Bombesina/química , Portadores de Fármacos/química , Diseño de Fármacos , Neoplasias Pancreáticas/metabolismo , Receptores de Superficie Celular/metabolismo , Somatostatina/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/uso terapéutico , Bibencilos/administración & dosificación , Bibencilos/química , Bibencilos/uso terapéutico , Camptotecina/administración & dosificación , Camptotecina/química , Camptotecina/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Ligandos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Bombesina/metabolismo , Receptores de Somatostatina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The major problems of traditional chemotherapy are non-selectivity and non-specificity, resulting in severe toxic side effects. Peptides are a new-generation of drug-delivery vector to increase efficacy of this therapy and avoid the resulting damage. The cytotoxic somatostatin (SST) conjugate JF-10-81 was developed by coupling camptothecin (CPT) to the N-terminus of a SST analog (JF-07-69) using an activated carbamate linker. This conjugate selectively targets somatostatin receptor subtype 2 (SSTR2) and also retains high binding affinity and rapid internalization as well as anti-proliferative activity towards various tumor cells. JF-10-81 was tested for its inhibitory activity against the growth of human tumors which included neuroblastoma (IMR32), pancreatic cancer (CFPAC-1), leukemia (MOLT-4), pancreatic carcinoid (BON) and prostate cancer (PC-3). Both SSTR2 mRNAs and proteins were detected in all these tumor cell lines. The conjugate displayed potent in vivo inhibitory activity, although some of the potency measured in in vitro experiments was lost. JF-10-81 was found to significantly inhibit growth of these SSTR-positive tumors, resulting in 87% tumor reduction in neuroblastoma IMR32 and 97% in leukemia MOLT-4 bearing animals, even inducing regression of CFPAC-1 tumors. SSTR-overexpressing BON tumors were unfortunately relatively CPT-insensitive in vitro, however, JF-10-81 again exhibited in vivo potency presumably by specifically increasing CPT concentrations inside the tumor cells so that the inhibition rate for JF-10-81 was 85%. Also, JF-10-81 was used to treat highly invasive PC-3 tumors where s.c. injections inhibited both tumor growth (almost 60% reduction) and tumor metastasis (over 70%). This conjugate demonstrated its broad and excellent anti-tumor activity by targeting SSTR2-specific tumor tissues, supporting that short peptides and their analogs may be applied as ideal drug-delivery carriers to improve the traditional chemotherapy.
RESUMEN
The effects of vasoactive intestinal peptide-camptothecin (VIP-CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala(2,8,9,19,24.25.27), Nle(17), Lys(28))VIP, (A-NL-K)VIP, was synthesized and Lys(28) was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC(1)-R, with IC(50) values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-VIP to 3T3 cells transfected with VPAC(1)-R with IC(50) values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-Ro25-1553 to 3T3 cells transfected with VPAC(2)-R with IC(50) values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. (125)I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 degrees C but not 4 degrees C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC(1)-R agonist which is cytotoxic for breast cancer cells.
Asunto(s)
Camptotecina/farmacología , Proliferación Celular/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Células 3T3 , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Camptotecina/síntesis química , Camptotecina/química , Línea Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
The camptothecin-bombesin conjugate termed DC-51-43, as a novel targeted drug delivery system, was examined in over 10 human tumor cell lines and shows a potent antiproliferative activity. This conjugate has also demonstrated its antitumor activity in our previous experiments. In our present study, we evaluate this conjugate for its antiangiogenic activity by in-vitro and in-vivo experiments. The camptothecin-bombesin conjugate and free camptothecin show potent in-vitro inhibitory activities of cell adhesion to various extracellular matrix components and integrins alphaVbeta3 and alphaVbeta5, not beta1/alphabeta1. This conjugate displays inhibitory activity to cell migration and invasion at concentrations of 10 micromol/l or above. This conjugate is also effective against in-vitro capillary-like tube formation of endothelial cells (at 40 micromol/l), and in-vivo angiogenesis as seen by blocking the spread of host mice endothelial cells into matrigel plugs. These experimental results support the fact that the camptothecin-bombesin conjugate has therapeutic activities against angiogenesis. By binding to bombesin receptor-expressing sites, this bombesin analog, consisting of 11 amino acids, is potentially a novel delivery vector for nonspecific cytotoxic agents.
Asunto(s)
Inhibidores de la Angiogénesis , Antineoplásicos Fitogénicos/farmacología , Bombesina/análogos & derivados , Bombesina/farmacología , Camptotecina/farmacología , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Fase G1/efectos de los fármacos , Humanos , Integrinas/metabolismo , Invasividad Neoplásica/patología , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/farmacología , Movimiento Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Western Blotting , Camptotecina/química , Camptotecina/uso terapéutico , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Vitronectina/metabolismo , Somatostatina/química , Somatostatina/uso terapéuticoRESUMEN
Most human tumors overexpress or ectopically express peptide hormone/neurotransmitter receptors, which are being increasingly studied as a means to selectively deliver cytotoxic agents. Although a number of peptide ligand-constructs demonstrate tumor cytotoxicity, the role of specific tumoral receptor interaction in its mediation is unclear. To address this question, we synthesized camptothecin (CPT) bombesin (Bn) analogs, in which CPT was coupled via a novel carbamate linker, L2 [N-(N-methyl-amino-ethyl)-glycine carbamate], that were chemically similar but differed markedly in their potency/affinity for human Bn receptors. We then examined their ability to interact with Bn receptors and cause in vitro and in vivo tumor cytotoxicity. CPT-L2-[D-Tyr(6),beta-Ala(11),D-Phe(13),Nle(14)] Bn (6-14) (BA3) bound with high affinity and had high potency for all three human Bn receptor subtypes, whereas CPT-L2-[D-Tyr(6),beta-Ala(11), D-Phe(13),Nle(14)] Bn (6-14) [D-Phe-CPT-L2-BA3] had >1400-fold lower affinity/potency. (125)I-CPT-L2-BA3 but not (125)I-D-Phe-CPT-L2-BA3 was internalized by Bn receptor subtype-containing cells. CPT-L2-BA3 displayed significantly more cytotoxicity than D-Phe-CPT-L2-BA3 toward NCI-H1299 lung cancer cells in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and clonogenic assays and more potently inhibited H1299 xenograft growth in nude mice. CPT-L2-BA3 was also metabolically more stable than its parent peptide and inhibited growth of a number of other tumor cell lines in vitro and in vivo. These results demonstrate that specific tumoral receptor interaction is important in mediating the ability of peptide ligand-cytotoxic constructs to cause cytotoxicity. Because many tumors overexpress Bn receptors, these results also demonstrate that CPT-L2-BA3 will be a useful agent for delivering receptor-mediated cytotoxicity to many different human tumors.
Asunto(s)
Antineoplásicos/farmacología , Bombesina/farmacología , Camptotecina/farmacología , Receptores de Bombesina/efectos de los fármacos , Animales , Células 3T3 BALB , Bombesina/metabolismo , Camptotecina/metabolismo , Proliferación Celular/efectos de los fármacos , Estabilidad de Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológicoRESUMEN
PURPOSE: To evaluate the ocular toxicity and efficacy of intravitreal camptothecin-somatostatin conjugate (JF-10-81), a somatostatin type 2 receptor-directed, antiangiogenic compound. METHODS: Part 1: New Zealand albino rabbits (except for controls) were injected intravitreally with conjugate at various concentrations. Preoperative and postoperative ocular examinations and electroretinography were performed until animals were killed for histology. Part 2: Long-Evans pigmented rats had choroidal neovascularization (CNV) induced by argon laser. One eye per animal was injected with concentration 10M (safe dose), whereas the other eyes were controls and received 30 microL of sterile water at different time intervals after laser application. Fluorescein angiography was performed at various time points to evaluate the lesions and confirm presence of CNV. Animals were euthanized. The eyes were immediately enucleated and prepared for histologic examination. RESULTS: Part 1: No clinical changes were seen in groups receiving 10(-8)M, 10(-7)M, 10(-6)M, and 10(-5) M of conjugate. Electroretinography showed decreasing b-wave amplitude in groups receiving 10(-4) M and 10(-3) M; cataracts also developed in these eyes. Part 2: Fluorescein angiography revealed that intravitreal injection of somatostatin conjugate JF-10-81 favorably affected the development of CNV when the treatment was performed at least 1 week after the laser application. These results were statistically significant. Histologic analysis results of eyes treated 2 weeks after laser application also showed significant benefit. CONCLUSIONS: Part 1: Camptothecin-somatostatin conjugate injected intravitreally appeared safe at concentrations of 10(-5)M or less. Part 2: Conjugate JF-10-81 at a concentration of 10(-5)M administered intravitreally 1 to 3 weeks after laser demonstrated statistically significant efficacy in the treatment of choroidal neovascularization.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Camptotecina/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Somatostatina/uso terapéutico , Animales , Camptotecina/toxicidad , Neovascularización Coroidal/fisiopatología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Electrorretinografía , Angiografía con Fluoresceína , Inyecciones , Terapia por Láser , Masculino , Conejos , Ratas , Ratas Long-Evans , Retina/efectos de los fármacos , Retina/fisiopatología , Somatostatina/toxicidad , Cuerpo VítreoRESUMEN
Two CPT-SSA conjugates, JF-10-71 and JF-10-81, containing a chemically adjustable release-rate carbamate linker, have been reported previously by us to potently inhibit growth of human neuroblastoma IMR32 cells overexpressing somatostatin receptor type II (SSTR2) but are stable under buffer incubation conditions or in rat plasma. Further experiments now reveal that the conjugates performed well against many additional cell lines, particularly somatostatin receptor containing rat pancreatic CA20948 cells that were actually more sensitive to the conjugates than free camptothecin itself. JF-10-71 and JF-10-81 also were examined for their inhibitory effects on the growth of this and several other tumors transplanted into rats (CA20948) or nude mice. CA20948 tumors, known to overexpress SSTR2 and grown in Lewis rats, were treated, respectively, with nontoxic 400 nmol/kg intraperitoneal (i.p.) doses of JF-10-71 or JF-10-81. Also, SSTR2-positive human SCLC NCI-H69 tumors transplanted in nude mice were treated in a similar fashion. Human prostate PC-3 tumors, which do not contain high concentrations of SSTR2, also were grown in nude mice and treated with a 400 nmol/kg ip dose of JF-10-71. Both cytotoxic conjugates significantly inhibited growth of SSTR2-specific pancreatic and SCLC tumors, but JF-10-81 did not significantly affect PC-3 tumor growth. These experimental results suggested that CPT-SSA conjugates can effectively target and kill tumor cells growing in vivo and that the effect is mediated by somatostatin receptors resulting in either release of camptothecin at the cell surface or, more likely, after receptor-mediated cellular internalization.
Asunto(s)
Antineoplásicos/administración & dosificación , Camptotecina/administración & dosificación , Inhibidores de Crecimiento/administración & dosificación , Somatostatina/administración & dosificación , Animales , Antineoplásicos/química , Camptotecina/química , Línea Celular Tumoral , Inhibidores de Crecimiento/química , Humanos , Ratones , Ratones Desnudos , Ratas , Ratas Endogámicas Lew , Somatostatina/química , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Creation of protease-resistant somatostatin analogs has allowed development of these peptides as clinically useful drugs. Widespread diagnostic use of radiolabeled somatostatin analogs has enhanced interest in the binding and intracellular distribution of these peptides. The degree of drug internalization and length of drug retention may be critical for drug-induced cytotoxicity. We hypothesized that the ability of a radiolabeled peptide to bind to a cell, be internalized, and induce cytotoxicity is proportional to both the radioligand concentration and the exposure time. MATERIALS AND METHODS: To test this hypothesis, somatostatin receptor-expressing cells (IMR-32) were incubated with (111)In-pentetreotide, a sst 2 preferring somatostatin analogue. Radioligand exposure time and/or concentration were varied. RESULTS: Prolonged exposure to a fixed concentration of radioligand resulted in progressive increases in whole cell binding and internalization over time. Cells exposed to a relatively fixed number of microCi-Hr yielded constant whole cell binding and internalization. Increasing the microCi-Hr resulted in a proportionate increase in binding. Cytotoxicity was also proportional to the dose of radiation regardless of whether the exposure was internalized radiation (microCi-Hr from (111)In-pentetreotide) or from external beam radiation (cGy). CONCLUSION: Both drug exposure time and drug concentration contribute to cell binding and cytotoxicity in this model and their relative contributions are inversely related.
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Somatostatina/análogos & derivados , Somatostatina/administración & dosificación , Somatostatina/metabolismo , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Radioisótopos de Indio , Concentración Osmolar , Ensayo de Unión Radioligante , Somatostatina/farmacocinética , Factores de TiempoRESUMEN
A series of novel 1,4-diaryl-2-azetidinones was prepared by stereospecific Staudinger reaction as conformationally restricted analogues of combretastatin A-4 because molecular modeling studies suggested close geometric similarities. They were evaluated for cytotoxicity against a number of human tumor and normal cell lines. Strong potencies were observed, with the best compounds exhibiting IC(50)'s of 25-74 nM against human neuroblastoma IMR 32 cell growth and a variety of other cell lines. Compounds inhibited tubulin polymerization with potencies commensurate with their cytotoxic activity and a more soluble anilino-containing analogue was very effective in inhibiting the growth of AR42J rat pancreatic tumors transplanted into in nude mice. Further studies on this interesting group of compounds as anti-cancer agents appear warranted.
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Antineoplásicos/química , Azetidinas/química , Estilbenos/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Ratas , Estilbenos/síntesis química , Estilbenos/farmacologíaRESUMEN
BACKGROUND: Combretastatin A-4 (CA-4) and its analogs are potent inhibitors of tubulin polymerization and display strong inhibitory activity on both solid tumor and tumor cell growth. Since natural CA-4 is difficult to synthesize and also isomerizes to an inactive form quite readily, a recently reported new 3,4-diarylfuran-2-one-based series of CA-4 analogs was investigated, in the hope of bypassing some of these difficulties. These analogs appear to offer a valuable tool for CA-4 research because of their extremely facile synthesis from readily available starting materials. MATERIALS AND METHODS: The CA-4 analogs were evaluated by MTT assay, cell cycle analysis, tubulin polymerization and tumor-inhibiting experiments. RESULTS: Various benzene ring substitutions on the furan-2-one skeleton (analogs to the two aromatic rings on the CA-4 styrene skeleton) quickly demonstrated that the structure-activity relationships are quite similar to previously synthesized CA-4 analogs. The most interesting analog appears to be an anilino compound (NV-5-9) which was also quite soluble. Analog NV-5-9 was remarkably potent in all tested tumor cell lines and could strongly inhibit tubulin polymerization at doses as low as 1 mM. Further experiments with tumor-bearing mice indicated that NV-5-9 and other potent analogs (NV-4-82 and NV-4-86) were effective in treating human prostate PC-3 and SCLC NCI-H69 tumors at well below an oral MTD dose of around 200 mg/kg body weight. This suggests some bioavailability by this route. CONCLUSION: These data strongly support that NV-5-9 is extremely potent, readily synthesizable and apparently suitable for in vivo studies employing transplanted tumors.
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Antineoplásicos Fitogénicos/farmacología , Furanos/farmacología , Estilbenos/farmacología , Animales , Antineoplásicos Fitogénicos/toxicidad , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Furanos/toxicidad , Inhibidores de Crecimiento/farmacología , Inhibidores de Crecimiento/toxicidad , Humanos , Masculino , Ratones , Ratones Desnudos , Estilbenos/toxicidad , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Peptide hormones are often rapidly internalized after binding to and activation of their receptors which are sometimes over-expressed on tumor cells. Thus, peptide ligands are increasingly being utilized for specific tumor cell targeting and internalization of radioactive isotopes for tumor imaging and for specifically delivering and internalizing cytotoxic moieties. Here, we describe a new carbamate linker system containing a series of built-in nucleophile assisted releasing (BINAR) groups which enable the 'fine-tuning' of intracellular cleavage rates of free cytotoxic agents containing reactive OH groups. Release rates were found to fit well with the chemical model and several conjugates of camptothecin and one of combretastatin were shown to have potent cytotoxic effects on cultures of human neuroblastoma IMR-32 cells which over-express somatostatin receptors.
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Antineoplásicos/química , Carbamatos/química , Reactivos de Enlaces Cruzados/química , Péptidos/química , Estilbenos , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Bibencilos/química , Bibencilos/farmacocinética , Bibencilos/farmacología , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/farmacología , Carbamatos/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Péptidos/administración & dosificación , Ratas , Somatostatina/análogos & derivados , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Peptide nucleic acid (PNA) sequences are synthetic versions of naturally occurring oligonucleotides which display improved binding properties to DNA and RNA, but are still poorly internalized across cell membranes. In an effort to employ the rapid binding/internalization properties of somatostatin agonist analogs and the over-expression of somatostatin receptors on many types of tumor cells, PNAs complementary to target sites throughout 5'-UTR, translation start site and coding region of the n-myc oncogene were conjugated to a somatostatin analog (SSA) with retention of high somatostatin biological potency. IMR32 cells, which over-express somatostatin receptor type 2 (SSTR2) and contain the n-myc oncogene, were treated with these PNA-SSA conjugates. The results show that PNA conjugates targeted to the 5'-UTR terminus and to regions at or close to the translation start site could effectively inhibit n-myc gene expression and cell growth, whereas the non-conjugate PNAs were without effect at similar doses. The most potent inhibition of cell growth was achieved with PNAs binding to the translation start site, but those complementary to the middle coding region or middle upstream site between 5'-UTR and translation start site displayed no inhibition of gene expression. These observations were extended to four other cell lines: GH3 cells which express SSTRs with the n-myc gene, SKNSH cells containing a silent n-myc gene without SSTR2, HT-29 cells carrying the c-myc but no n-myc gene, and CHO-K1 cells lacking SSTR2 with n-myc gene. The results show that there was almost no effect on these four cell lines. Our study indicates that PNAs conjugated to SSA exhibited improved inhibition of gene expression possibly due to facilitated cellular uptake of the PNAs. These conjugates were mRNA sequence- and SSTR2-specific suggesting that many other genes associated with tumor growth could be targeted using this approach and that SSA could be a novel and effective transportation vector for the PNA antisense strategy.