RESUMEN
A liquid chromatography-tandem mass spectrometry assay was developed and qualified for the multiplexed quantitation of a small molecule stimulator of soluble guanylate cyclase (sGC) and its target engagement biomarker, 3',5'-cyclic guanosine monophosphate (cGMP), in ocular tissues and plasma from a single surrogate matrix calibration curve. A surrogate matrix approach was used in this assay due to the limited quantities of blank ocular matrices in a discovery research setting. After optimization, the assay showed high accuracy, precision, and recovery as well as parallelism between the surrogate matrix and the biological matrices (rabbit plasma, vitreous, and retina-choroid). This assay provided pharmacokinetic and target engagement data after intravitreal administration of the sGC stimulator. The nitric oxide-sGC-cGMP pathway is a potential target to address glaucoma. Increasing sGC-mediated production of cGMP could improve aqueous humor outflow and ocular blood flow. The sGC stimulator showed dose-dependent exposure in rabbit vitreous, retina-choroid, and plasma. The cGMP exhibited a delayed yet sustained increase in vitreous humor but not retina-choroid. Multiplexed measurement of both pharmacokinetic and target engagement analytes reduced animal usage and provided improved context for interpreting PK and PD relationships.
Asunto(s)
GMP Cíclico , Guanilato Ciclasa , Animales , Guanilato Ciclasa/metabolismo , Óxido Nítrico , Conejos , Transducción de Señal , Guanilil Ciclasa Soluble/metabolismoRESUMEN
PURPOSE: To prepare an oligo(lactic acid)8-rapamycin prodrug (o(LA)8-RAP)-loaded poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) micelle for injection and characterize its compatibility and performance versus a RAP-loaded PEG-b-PLA micelle for injection in vitro and in vivo. METHODS: Monodisperse o(LA)8 was coupled on RAP at the C-40 via DCC/DMAP chemistry, and conversion of o(LA)8-RAP prodrug into RAP was characterized in vitro. Physicochemical properties of o(LA)8-RAP- and RAP-loaded PEG-b-PLA micelles and their antitumor efficacies in a syngeneic 4 T1 breast tumor model were compared. RESULTS: Synthesis of o(LA)8-RAP prodrug was confirmed by 1H NMR and mass spectroscopy. The o(LA)8-RAP prodrug underwent conversion in PBS and rat plasma by backbiting and esterase-mediated cleavage, respectively. O(LA)8-RAP-loaded PEG-b-PLA micelles increased water solubility of RAP equivalent to 3.3 mg/ml with no signs of precipitation. Further, o(LA)8-RAP was released more slowly than RAP from PEG-b-PLA micelles. With added physical stability, o(LA)8-RAP-loaded PEG-b-PLA micelles significantly inhibited tumor growth relative to RAP-loaded PEG-b-PLA micelles in 4 T1 breast tumor-bearing mice without signs of acute toxicity. CONCLUSIONS: An o(LA)8-RAP-loaded PEG-b-PLA micelle for injection is more stable than a RAP-loaded PEG-b-PLA micelle for injection, and o(LA)8-RAP converts into RAP rapidly in rat plasma (t1/2 = 1 h), resulting in antitumor efficacy in a syngeneic 4 T1 breast tumor model.
Asunto(s)
Antineoplásicos/administración & dosificación , Portadores de Fármacos/química , Lactatos/química , Polietilenglicoles/química , Profármacos/administración & dosificación , Sirolimus/administración & dosificación , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Femenino , Lactatos/toxicidad , Ácido Láctico/química , Ratones , Micelas , Polietilenglicoles/toxicidad , Profármacos/química , Profármacos/farmacología , Profármacos/toxicidad , Ratas , Transducción de Señal , Sirolimus/química , Sirolimus/farmacología , Sirolimus/toxicidad , Solubilidad , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles are nanocarriers for poorly water-soluble anticancer agents and have advanced paclitaxel (PTX) to humans due to drug solubilization, biocompatibility, and dose escalation. However, PEG-b-PLA micelles rapidly release PTX, resulting in widespread biodistribution and low tumor exposure. To improve delivery of PTX by PEG-b-PLA micelles, monodisperse oligo(l-lactic acid), o(LA)8 or o(LA)16, has been coupled onto PTX at the 7-OH position, forming ester prodrugs: o(LA)8-PTX and o(LA)16-PTX, respectively. As expected, o(LA)n-PTX was more compatible with PEG-b-PLA micelles than PTX, increasing drug loading from 11 to 54%. While in vitro release of PTX was rapid, resulting in precipitation, o(LA)n-PTX release was more gradual: t1/2 = 14 and 26 h for o(LA)8-PTX and o(LA)16-PTX, respectively. Notably, o(LA)8-PTX and o(LA)16-PTX in PEG-b-PLA micelles resisted backbiting chain end scission, based on reverse-phase HPLC analysis. By contrast, o(LA)8-PTX and o(LA)16-PTX degraded substantially in 1:1 acetonitrile:10 mM PBS, pH 7.4, at 37 °C, generating primarily o(LA)2-PTX. The IC50 value of o(LA)2-PTX was â¼2.3 nM for A549 human lung cancer cells, equipotent with PTX in vitro. After weekly IV injections at 20 mg/kg as PEG-b-PLA micelles, o(LA)8-PTX induced tumor regression in A549 tumor-bearing mice, whereas PTX delayed tumor growth. Surprisingly, o(LA)8-PTX caused less toxicity than PTX in terms of change in body weight. In conclusion, o(LA)n acts as a novel promoiety, undergoing backbiting conversion without a reliance on metabolizing enzymes, and o(LA)n-PTX improves PTX delivery by PEG-b-PLA micelles, providing a strong justification for clinical evaluation.
Asunto(s)
Portadores de Fármacos/química , Liberación de Fármacos , Lactatos/química , Micelas , Paclitaxel/química , Poliésteres/química , Polietilenglicoles/química , Profármacos/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Portadores de Fármacos/farmacocinética , Humanos , Lactatos/farmacocinética , Paclitaxel/metabolismo , Paclitaxel/farmacología , Polietilenglicoles/farmacocinética , Profármacos/metabolismo , Distribución TisularRESUMEN
The subcutaneous administration of therapeutic peptides would provide significant benefits to patients. However, subcutaneous injections are limited in dosing volume, potentially resulting in high peptide concentrations that can incur significant challenges with solubility limitations, high viscosity, and stability liabilities. Herein, we report on the discovery that low-shear resonant acoustic mixing can be used as a general method to prepare stable nanoparticles of a number of peptides of diverse molecular weights and structures in water without the need for extensive amounts of organic solvents or lipid excipients. This approach avoids the stability issues observed with typical high-shear, high-intensity milling methods. The resultant peptide nanosuspensions exhibit low viscosity even at high concentrations of >100 mg/mL while remaining chemically and physically stable. An example nanosuspension of cyclosporine nanoparticles was dosed in rats via a subcutaneous injection and exhibited sustained release behavior. This suggests that peptide nanosuspension formulations can be one approach to overcome the challenges with high-concentration peptide formulations.
RESUMEN
Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles affect drug solubilization, and a paclitaxel (PTX) loaded-PEG-b-PLA micelle (PTX-PM) is approved for cancer treatment due to injection safety and dose escalation (Genexol-PM®) compared to Taxol®. However, PTX-PM is unstable in blood, has rapid clearance, and causes dose-limiting toxicity. We have synthesized a prodrug for PTX (7-OH), using oligo(lactic acid) as a novel pro-moiety (o(LA)8-PTX) specifically for PEG-b-PLA micelles, gaining higher loading and slower release of o(LA)8-PTX over PTX. Notably, o(LA)8-PTX prodrug converts into PTX by a backbiting reaction in vitro, without requiring esterases. We hypothesize that o(LA)8-PTX-loaded PEG-b-PLA micelles (o(LA)8-PTX-PM) has a lower Cmax and higher plasma AUC than PTX-PM for improved therapeutic effectiveness. In Sprague-Dawley rats at 10â¯mg/kg, compared to o(LA)8-PTX-PM (10% w/w loading) and PTX-PM (10%), o(LA)8-PTX-PM (50% w/w loading) produces a 2- and 3-fold higher plasma AUC0-24 of PTX, lactic acid-PTX, and o(LA)2-PTX (o(LA)0-2-PTX), respectively. For o(LA)8-PTX-PM at 10 and 50% w/w loading, PTX and lactic acid-PTX are major bioactive metabolites, respectively. Fast prodrug conversion of o(LA)8-PTX in vivo versus in vitro (by backbiting) suggests that o(LA)8 is a good substrate for esterases. At 60â¯mg/kg (qwx3), o(LA)8-PTX-PM (50%) has higher antitumor activity than o(LA)8-PTX-PM (10%) and PTX-PM (10%) in a syngeneic 4T1-luc breast tumor model based on measurements of tumor volume, 4T1-luc breast tumor bioluminescence, and survival. Importantly, intravenous administration of o(LA)8-PTX-PM is well tolerated by BALB/c mice. In summary, oligo(lactic acid)8-PTX is more compatible than PTX with PEG-b-PLA micelles, more stable, and may expand the role of PEG-b-PLA micelles from "solubilizer" into "nanocarrier" for PTX as a next-generation taxane for cancer.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/química , Paclitaxel/administración & dosificación , Animales , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/farmacología , Área Bajo la Curva , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Lactatos/química , Ácido Láctico/química , Masculino , Ratones , Ratones Endogámicos BALB C , Micelas , Paclitaxel/farmacocinética , Paclitaxel/farmacología , Polietilenglicoles/química , Profármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Herein we demonstrate the formation of stereocomplex prodrugs of oligo(l-lactic acid) n-gemcitabine (o(LLA) n-GEM) and oligo(d-lactic acid) n-gemcitabine (o(DLA) n-GEM) for stable incorporation in poly(ethylene glycol)- block-poly(d,l-lactic acid) (PEG- b-PLA) micelles. O(LLA) n or o(DLA) n was attached at the amino group (4-( N)) of GEM via an amide linkage. When n = 10, a 1:1 mixture of o(LLA)10-GEM and o(DLA)10-GEM (o(L+DLA)10-GEM) was able to form a stereocomplex with a distinctive crystalline pattern. Degradation of o(L+DLA)10-GEM was driven by both backbiting conversion and esterase contribution, generating primarily o(L+DLA)1-GEM and GEM. O(L+DLA)10-GEM stably loaded in PEG- b-PLA micelles in the size range of 140-200 nm with an unexpected elongated morphology. The resulting micelles showed improved physical stability in aqueous media and inhibited backbiting conversion of o(L+DLA)10-GEM within micelles. Release of o(L+DLA)10-GEM from micelles was relatively slow, with a t1/2 at ca. 60 h. Furthermore, weekly administration of o(L+DLA)10-GEM micelles i.v. displayed potent antitumor activity in an A549 human non-small-cell lung carcinoma xenograft model. Thus, stereocomplexation of isotactic o(LLA) n and o(DLA) n acts as a potential prodrug strategy for improved stability and sustained drug release in PEG- b-PLA micelles.
Asunto(s)
Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Lactatos/farmacología , Ácido Láctico/farmacología , Micelas , Polietilenglicoles/farmacología , Profármacos/farmacología , Células A549 , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/química , Desoxicitidina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Lactatos/química , Ácido Láctico/química , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Polietilenglicoles/química , Profármacos/química , Estereoisomerismo , GemcitabinaRESUMEN
Triolimus is a multi-drug loaded polymeric micelle containing paclitaxel (PTX), 17-allylamino-17-demethoxygeldanamycin (17-AAG), and rapamycin (RAP). This study examines the radiosensitizing effect of Triolimus in vitro and in vivo. Radiosensitizing effects of Triolimus on A549 cells are dose dependent and at 2 × 10-9 m, Triolimus shows significant radiosensitization even at low radiation doses (2 Gy). By sensitivity enhancement ratio, PTX alone, dual drug combinations, and Triolimus treatment at 2 × 10-9 m have radiosensitizing effects with potency as follows: PTX alone (PTX) > PTX and RAP (P/R) > Triolimus (TRIO) > PTX and 17-AAG (P/17) >17-AAG and RAP (17/R). In vivo, fractionated radiation of 15 Gy preceded by infusion of PTX alone, dual drug combinations, or an intermediate dose of Triolimus (Int. TRIO: PTX/17-AAG/RAP at 15/15/7.5 mg kg-1 ) strongly inhibits A549 tumor growth. Notably, pretreatment with high dose of Triolimus (High TRIO: PTX/17-AAG/RAP at 60/60/30 mg kg-1 ) before the fractionated radiation leads to tumor control for up to 24 weeks. An enhanced radiosensitizing effect is observed without an increase in acute toxicity compared to PTX alone or radiation alone. These results suggest that further investigations of Triolimus in combination with radiation therapy are merited.
Asunto(s)
Benzoquinonas/uso terapéutico , Lactamas Macrocíclicas/uso terapéutico , Micelas , Paclitaxel/uso terapéutico , Polímeros/química , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Sirolimus/uso terapéutico , Células A549 , Antineoplásicos/farmacología , Benzoquinonas/farmacología , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Fenómenos Químicos , Células Clonales , Combinación de Medicamentos , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Lactamas Macrocíclicas/farmacología , Necrosis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Paclitaxel/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Sirolimus/farmacologíaRESUMEN
BACKGROUND: Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. METHODS: This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. RESULTS: In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. CONCLUSION: These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Micelas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Benzoquinonas/administración & dosificación , Benzoquinonas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Proteínas HSP90 de Choque Térmico/metabolismo , Immunoblotting , Lactamas Macrocíclicas/administración & dosificación , Lactamas Macrocíclicas/farmacología , Masculino , Ratones Noqueados , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/administración & dosificación , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Taxoides/administración & dosificación , Taxoides/farmacologíaRESUMEN
Over-expression of ATP-binding cassette transporters is one of the most important mechanisms responsible for multidrug resistance. Here, we aimed to develop a stable polymeric nanoparticle system by flash nanoprecipitation (FNP) for enhanced anticancer drug delivery into drug resistant cancer cells. As an antisolvent precipitation process, FNP works best for highly lipophilic solutes (logP>6). Thus we also aimed to evaluate the applicability of FNP to drugs with relatively low lipophilicity (logP=1-2). To this end, doxorubicin (DOX), an anthracycline anticancer agent and a P-gp substrate with a logP of 1.3, was selected as a model drug for the assessment. DOX was successfully incorporated into the amphiphilic diblock copolymer, polyethylene glycol-b-polylactic acid (PEG-b-PLA), by FNP using a four-stream multi-inlet vortex mixer. Optimization of key processing parameters and co-formulation with the co-stabilizer, polyvinylpyrrolidone, yielded highly stable, roughly spherical DOX-loaded PEG-b-PLA nanoparticles (DOX.NP) with mean particle size below 100nm, drug loading up to 14%, and drug encapsulation efficiency up to 49%. DOX.NP exhibited a pH-dependent drug release profile with higher cumulative release rate at acidic pHs. Surface analysis of DOX.NP by XPS revealed an absence of DOX on the particle surface, indicative of complete drug encapsulation. While there were no significant differences in cytotoxic effect on P-gp over-expressing LCC6/MDR cell line between DOX.NP and free DOX in buffered aqueous media, DOX.NP exhibited a considerably higher cellular uptake and intracellular retention after efflux. The apparent lack of cytotoxicity enhancement with DOX.NP may be attributable to its slow DOX release inside the cells.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Precipitación Química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Nanopartículas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Composición de Medicamentos , Liberación de Fármacos , Resistencia a Antineoplásicos/genética , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Lactatos/química , Nanopartículas/metabolismo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/química , Povidona/químicaRESUMEN
Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αß structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αß peptide scaffolds for the design of novel peptide-based antibiotics.