RESUMEN
AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8 degrees C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.
Asunto(s)
Acrilamidas/química , Antineoplásicos/química , Drogas en Investigación/química , Compuestos Organoplatinos/química , Tecnología Farmacéutica/métodos , Acrilamidas/análisis , Antineoplásicos/análisis , Química Farmacéutica , Drogas en Investigación/análisis , Liofilización/métodos , Infusiones Parenterales , Compuestos Organoplatinos/análisis , Compuestos de Platino/análisis , Compuestos de Platino/químicaRESUMEN
This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.
Asunto(s)
Antineoplásicos/química , Dimetilsulfóxido/análogos & derivados , Dimetilsulfóxido/química , Metástasis de la Neoplasia/prevención & control , Compuestos Organometálicos/química , Tecnología Farmacéutica/métodos , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Química Farmacéutica , Dimetilsulfóxido/administración & dosificación , Dimetilsulfóxido/farmacocinética , Liofilización , Infusiones Parenterales , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/farmacocinética , Rutenio/administración & dosificación , Rutenio/química , Rutenio/farmacocinética , Compuestos de RutenioRESUMEN
AP 5280 is a novel polymer-conjugated platinum anticancer agent currently undergoing phase I clinical trials. It is pharmaceutically formulated as a lyophilized product containing 200 mg platinum per dosage unit. The aim of this study was to determine the reconstitution and dilution fluid of choice, and to investigate the stability and compatibility of AP 5280 in solution under different storage conditions and with several container materials. Furthermore, the hemolytic potential of AP 5280 infusion solution was investigated. AP 5280 slowly released small platinum species in all solutions, although this release was enhanced in normal saline. Accordingly, 5% dextrose in water (D W) was selected for reconstitution and dilution of AP 5280. Container material [glass or polyvinylchloride (PVC)] did not influence the stability of AP 5280 in solution. Storage at refrigerated temperature (2-8 degrees C) marginally decreased the release rate of liberated platinum. The infusion solutions are compatible with the PVC infusion system and do not cause hemolysis. In conclusion, AP 5280 lyophilized product should be reconstituted and diluted to infusion concentration with D W, and administered within 8 h after preparation to ensure that less than 1.0% of the total platinum concentration is present as liberated platinum.
Asunto(s)
Acrilamidas/química , Antineoplásicos/química , Drogas en Investigación/química , Hemólisis/efectos de los fármacos , Compuestos Organoplatinos/química , Platino (Metal)/análisis , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada/química , Dietilhexil Ftalato/análisis , Embalaje de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Técnicas In Vitro , Bombas de Infusión , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Polímeros/química , SolucionesRESUMEN
The clinical pharmacology of itraconazole is presented in relation to its use in the treatment of fluconazole-resistant oropharyngeal candidosis. The oral solution is a new formulation of itraconazole in which itraconazole is solubilised with the use of cyclodextrin. This formulation has a higher bioavailability and leads to higher local concentrations in the oral cavity which are advantages over the solid capsule formulation. Literature, in which the use of itraconazole oral solution was described to treat fluconazole-resistant oral candidosis, is reviewed. In about 55% of the patients signs and symptoms of oral candidosis were resolved after treatment with itraconazole oral solution. Although all the reviewed studies lack data to objectively qualify all the included patients as having a fluconazole-resistant candidosis, the authors conclude, that based on the available information itraconazole oral solution 100 or 200mg twice daily can be effective for fluconazole-resistant oropharyngeal candidosis (OPC) and should be considered prior to salvage therapy with intravenous amphotericin B. The use of itraconazole, however, requires careful monitoring of the patients co-medication for potential serious drug-drug interactions.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Antifúngicos/farmacología , Candidiasis Bucal/tratamiento farmacológico , Fluconazol/farmacología , Infecciones por VIH/complicaciones , Itraconazol/farmacología , Infecciones Oportunistas Relacionadas con el SIDA/complicaciones , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Administración Oral , Antifúngicos/administración & dosificación , Candida/efectos de los fármacos , Candida/aislamiento & purificación , Candidiasis Bucal/complicaciones , Farmacorresistencia Fúngica , Humanos , Itraconazol/administración & dosificación , Pruebas de Sensibilidad Microbiana , SolucionesRESUMEN
Kahalalide F is a novel antitumor agent isolated from the marine mollusk Elysia rufescens; it has shown highly selective in vitro activity against androgen-independent prostate tumors. The purpose of this study was to develop a stable parenteral formulation of kahalalide F to be used in early clinical trials. Solubility and stability of kahalalide F were studied as a function of polysorbate 80 (0.1%-0.5% w/v) and citric acid monohydrate (15-15 mM) concentrations using an experimental design approach. Stabilities of kahalalide F lyophilized products containing crystalline (mannitol) or amorphous (sucrose) bulking agents were studied at +5 degrees C and +30 degrees C +/- 60% relative humidity (RH) in the dark. Lyophilized products were characterized by infrared (IR) spectroscopy and differential scanning calorimetry (DSC). Recovery studies after reconstitution of kahalalide F lyophilized product and further dilution in infusion fluid were carried out to select an optimal reconstitution vehicle. It was found that a combination of polysorbate 80 and citric acid monohydrate is necessary to solubilize kahalalide F. Lyophilized products were considerably less stable with increasing polysorbate 80 and citric acid monohydrate concentrations, with polysorbate 80 being the major effector. A combination of 0.1% w/v polysorbate 80 and 5 mM citric acid monohydrate was selected for further investigation. Lyophilized products containing sucrose as a hulking agent were more stable compared to the products containing mannitol. The glass transition temperature of the sucrose-based product was determined to be + 46 degrees C. The amorphous state of the product was confirmed by IR analysis. A solution composed of Cremophor EL, ethanol, and water for injection (5%/5%/90% v/v/v CEW, kept kahalalide F in solution after reconstitution andfurther dilution with 0.9% w/v sodium chloride (normal saline) to 1.5 microg/m. A stable lyophilized formulation was presented containing 100 microg of kahalalide F, 100 mg sucrose, 2.1 mg citric acid monohydrate, and 2mg polysorbate 80 to be reconstituted with a vehicle composed of 5%/5%/90% v/v/v CEW and to be diluted further using normal saline.
Asunto(s)
Antineoplásicos/administración & dosificación , Depsipéptidos , Péptidos/administración & dosificación , Algoritmos , Antineoplásicos/química , Rastreo Diferencial de Calorimetría , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Excipientes , Liofilización , Humedad , Infusiones Parenterales , Péptidos/química , Soluciones Farmacéuticas , Vehículos Farmacéuticos , Solubilidad , Espectrofotometría Infrarroja , SacarosaRESUMEN
The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca(2+)-dependent PKC isoforms (alpha, betaI, betaII) within the SCN of selected house mouse lines that differ in behavioral circadian rhythm parameters. Optical density measurements revealed that the adult mice selected for low levels of nest-building behavior (small nest-builders) had more than threefold higher PKCbetaI immunostaining in the SCN than the mice selected for high levels of nest-building behavior (big nest-builders). A similar twofold difference between the adult small and big nest-builders was observed for the number of PKCbetaI-containing cells in the SCN. The non-selected control lines were intermediate. Ten-day-old pups revealed similar differences in PKCbetaI immunostaining in the SCN between the small and big nest-builders. PKCalpha and PKCbetaII immunostaining in the SCN was not different among the lines. PKCbetaI immunostaining was not different among the selected lines in the lateroanterior hypothalamic nucleus (LA) and the cornu ammonis field 1 (CA1) of the dorsal hippocampus and confirms the specificity of the difference in PKCbetaI immunostaining in the SCN among the selected lines. The significance of these findings is discussed in the context of differences among the lines in arginine-vasopressin (AVP) and light-induced Fos expression in the SCN, behavioral phase-delay responses to 15-min light pulses in constant darkness, and measures of the strength of the circadian activity rhythm expressed.
Asunto(s)
Arginina Vasopresina/metabolismo , Isoenzimas/metabolismo , Ratones Endogámicos/metabolismo , Proteína Quinasa C/metabolismo , Núcleo Supraquiasmático/enzimología , Envejecimiento/fisiología , Animales , Recuento de Células , Ritmo Circadiano/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos/anatomía & histología , Actividad Motora/fisiología , Comportamiento de Nidificación/fisiología , Estimulación Luminosa , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Isoformas de Proteínas/metabolismo , Proteína Quinasa C beta , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas c-fos/metabolismo , Tiempo de Reacción/fisiología , Transducción de Señal/fisiología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismoRESUMEN
Kahalalide F is a novel marine-derived antitumor agent isolated from the marine mollusk Elysia rufescens, an organism living in the seas near Hawaii. The compound has shown highly selective in vitro activity against prostate tumors and phase I trials in patients with androgen independent prostate tumors incorporating a daily times five and weekly schedule have been initiated. Kahalalide F is pharmaceutically formulated as a lyophilized product containing 150 microg active substance per dosage unit. Prior to i.v. administration it is reconstituted with a solution composed of Cremophor EL, ethanol absolute and Water for Injection (CEW, 5/5/90% v/v/v) with further dilution in 0.9% w/v sodium chloride for infusion. The aim of this study was to investigate the compatibility and stability of kahalalide F with different infusion systems prior to the start of clinical trials with the compound. Due to the presence of Cremophor EL in the infusion solution, leaching of diethylhexyl phthalate (DEHP) from polyvinyl chloride infusion containers (PVC, Add-a-Flex) was found. Loss of kahalalide F as a consequence of sorption to contact surfaces was shown with an infusion container composed of low density polyethylene (LD-PE, Miniflac). We conclude that kahalalide F must be administered in a 3-h infusion in concentrations of 0.5 microg/mL to 14.7 microg/mL using an administration set consisting of a glass container and a low-extrables, DEHP-free extension set. Kahalalide F 150 microg/vial powder for infusion reconstituted with 5/5/90% v/v/v CEW is stable in the original container for at least 24 h at room temperature (+20-25 degrees C) and ambient light conditions. Infusion solutions stored in glass infusion containers at either room temperature (+20-25 degrees C, in the dark) or refrigerated conditions (+2-8 degrees C, in the dark) are stable for at least 5 days after preparation.
Asunto(s)
Antineoplásicos/química , Depsipéptidos , Glicerol/análogos & derivados , Péptidos/química , Absorción , Animales , Área Bajo la Curva , Materiales Biocompatibles , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Estabilidad de Medicamentos , Glicerol/química , Bombas de Infusión , Infusiones Intravenosas , Venenos de Moluscos , Factores de TiempoRESUMEN
This paper describes several successful cost-effective attempts to couple capillary electrophoresis (CE) and mass spectrometry (MS) without make-up flow or nebulizing gas. An in-depth analysis of several interfaces using conductive spray tips was performed as well as an easy-to-prepare T-junction with direct electrode contact, the latter being the most robust interface. No coating is necessary and the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries. The T-junction interface is made by puncturing a small piece of transparent rubber. The on-line preconcentration CE-MS system allows immunoassay sensitivity, as is demonstrated by a calibration plot in the picomolar range for angiotensin II and gonadorelin. It also shows good reproducibility and has the ability of excellent automation. The secure electrical contact gives a constant spray quality, even with 100% aqueous separation buffers. The described setup has a wide applicability as is demonstrated by the analysis of larger peptides, such as insulin and cytochrome c. Detailed information is given on critical factors in the preparation of the described interfaces.
Asunto(s)
Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Angiotensina II/análisis , Animales , Automatización , Análisis Costo-Beneficio , Grupo Citocromo c/análisis , Electroforesis Capilar/economía , Electroforesis Capilar/instrumentación , Electroforesis Capilar/normas , Hormona Liberadora de Gonadotropina/análisis , Humanos , Insulina/análisis , Espectrometría de Masas/economía , Espectrometría de Masas/instrumentación , Espectrometría de Masas/normasRESUMEN
An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.
Asunto(s)
Angiotensina II/sangre , Electroforesis Capilar/métodos , Hormona Liberadora de Gonadotropina/sangre , Péptidos/análisis , Humanos , Espectrometría de Masas/métodos , Péptidos/uso terapéutico , Propilaminas , Silanos , Espectrofotometría Ultravioleta/métodos , Rayos UltravioletaRESUMEN
This overview highlights the possibilities of on- or in-line preconcentration procedures in combination with a CZE separation, focused on the determination of peptides and proteins. The discussed methods, including sample stacking, field-amplified injection, isotachophoresis, solid phase extraction, membrane preconcentration, electroextraction, supported liquid membranes, hollow fibers, immunoaffinity, and molecularly imprinted polymers technology preconcentration are categorized in electrophoresis-based and chromatography-based preconcentration. The chromatography-based preconcentration is subdivided in low-specificity and high-specificity methods. A number of preconcentration methods are available, however, this paper demonstrates that various compounds in different media (aqueous solutions, urine, and plasma) require different preconcentration systems. The preconcentration techniques of first choice in general seem to be solid-phase extraction and membrane preconcentration, because of their high concentration ability, multiapplicability, relative simplicity and clean-up capability. For the future, hollow fibers seem to hold a great potential as preconcentration technique, yielding high concentration factors, using simple designs. New techniques, such as hollow fibers, molecularly imprinted polymers technology and supported liquid membranes may have the potential to supersede the conventional preconcentration techniques in some cases. The larger the arsenal of preconcentration techniques becomes, the more efficiently peptides and proteins may be analyzed in the future. These techniques, in some cases, require pre-cleanup procedures, to ensure the purity of the samples to concentrate.
Asunto(s)
Electroforesis Capilar/métodos , Proteínas/análisis , Adsorción , Biopolímeros , Cromatografía/métodos , Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Técnicas de Inmunoadsorción , Concentración Osmolar , Péptidos/análisis , Unión Proteica , Sensibilidad y Especificidad , Manejo de Especímenes , Moldes GenéticosRESUMEN
An in vitro biocompatibility study was performed with the pharmaceutical formulation of the investigational, marine-derived anticancer agent kahalalide F developed for early clinical studies. The pharmaceutical formulation consists of a lyophilized product containing 150 micrograms kahalalide F, 3 mg citric acid, 3 mg polysorbate 80, and 150 mg of sucrose per dosage unit, to be reconstituted with 3 mL of a mixture composed of Cremophor EL, ethanol, and water (5/5/90% v/v/v), resulting in a solution of pH 3 and to be further diluted in normal saline for infusion. The reconstituted product, infusion solutions, and Cremophor/ethanol (CE) vehicle were tested for hemolytic potential and buffer capacity. No significant hemolysis due to the kahalalide F formulation as well as the CE vehicle was found using both a static and dynamic test model. FB-ratio's (ratio of formulation solution (F) and volume of blood simulant (B) necessary to maintain physiological pH) as a measure of the buffer capacity of the kahalalide F infusion solutions examined indicated that no vascular irritation due to pH effects is expected in the intended administration schedule in the forthcoming Phase I study.
Asunto(s)
Antineoplásicos/farmacología , Química Farmacéutica/métodos , Depsipéptidos , Hemólisis/efectos de los fármacos , Péptidos/farmacología , Animales , Antineoplásicos/química , Materiales Biocompatibles , Tampones (Química) , Etanol , Glicerol/análogos & derivados , Concentración de Iones de Hidrógeno , Péptidos/química , Vehículos Farmacéuticos , RatasRESUMEN
The degradation kinetics of aplidine were investigated using reversed-phase high-performance liquid chromatography combined with UV detection. Aplidine consists of at least two isomers that undergo interconversion at a low rate. Influences of pH, temperature, buffer ions and ionic strength on the degradation kinetics were studied. The log kobs) -pH profile can be divided into three parts, a proton, a solvent and a hydroxyl-catalysed section. The stability-indicating properties of the used analysis technique as well as the identities of the main degradation products were checked using gradient liquid chromatography and mass spectrometric detection. The overall degradation rate constant as a function of the temperature under acidic and alkaline conditions obeys the Arrhenius equation. No catalytic influences were observed with phosphate and carbonate buffers and, in addition, the ionic strength showed no substantial effect on the stability, as expected. Results from gradient LC-MS indicated that hydrolysis of the ester groups present in the ring structure was the main degradation route. There is no difference in degradation rate constants for the individual isomers.
Asunto(s)
Antineoplásicos/química , Depsipéptidos , Oligopéptidos/química , Péptidos Cíclicos/química , Tampones (Química) , Cromatografía Liquida/métodos , Concentración de Iones de Hidrógeno , Isomerismo , Cinética , Espectrometría de Masas , Conformación Molecular , Estructura Molecular , Concentración Osmolar , Soluciones , TemperaturaRESUMEN
This article describes the phase response curve (PRC), the effect of light on Fos immunoreactivity (Fos-IR) in the suprachiasmatic nucleus (SCN), and the effect of SCN lesions on circadian rhythms in the murid rodent, Arvicanthis niloticus. In this species, all individuals are diurnal when housed without a running wheel, but running in a wheel induces a nocturnal pattern in some individuals. First, the authors characterized the PRC in animals with either the nocturnal or diurnal pattern. Both groups of animals were less affected by light during the middle of the subjective day than during the night and were phase delayed and phase advanced by pulses in the early and late subjective night, respectively. Second, the authors characterized the Fos response to light at circadian times 5, 14, or 22. Light induced an increase in Fos-IR within the SCN during the subjective night but not subjective day; this effect was especially pronounced in the ventral SCN, where retinal inputs are most concentrated, but was also evident in other regions. Both light and time influenced Fos-IR within the lower subparaventricular area. Third, SCN lesions caused animals to become arrhythmic when housed in a light-dark cycle as well as constant darkness. In summary, Arvicanthis appear to be very similar to nocturnal rodents with respect to their PRC, temporal patterns of light-induced Fos expression in the SCN, and the effects of SCN lesions on activity rhythms.
Asunto(s)
Hipotálamo/metabolismo , Muridae/fisiología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Núcleo Supraquiasmático/metabolismo , Animales , Temperatura Corporal/fisiología , Ritmo Circadiano/fisiología , Luz , Masculino , Actividad Motora/fisiología , Estimulación Luminosa , Núcleo Supraquiasmático/fisiologíaRESUMEN
Previous divergent selection for nest-building behavior at 22 +/- 1 degrees C resulted in a 40-fold difference between the high and the low lines in amount of cotton used to build a nest. Correlated responses to selection indicated positive genetic correlations with body weight, nest-building at 4 +/- 1 degrees C, and litter size and negative genetic correlations with food consumption. At generation 46, the replicate high-selected (High 1 x High 2), randomly bred control (Control 1 x Control 2), and low-selected (Low 1 x Low 2) lines were crossed and the F1 showed significant heterosis for nest-building behavior. Regression of the F3 on the F2 generation gave heritability estimates of 0.16 +/- 0.10 for the high and 0.07 +/- 0.10 for the low cross, revealing a potential to break the selection limit (at least in the high direction), which had been reached at about 20 generations of selection. Indeed, renewed selection resulted in responses in both the high and the low directions of nesting, yielding realized heritabilities of 0.29 +/- 0.02 and 0.30 +/- 0.004, respectively. Replicated renewed selection, using the F3 generation as the base population, in the high direction of nesting resulted in correlated increases in nest-building at 4 +/- 1 degrees C, litter size, and food consumption. Body weight did not change. The positive correlation with food consumption is opposite in sign compared to the original selection experiment. This indicates that the evolutionary potential of a population to adapt to a changing environment not only depends on its current genetic variability in one adaptive trait, but may be constrained by genetic correlations changing over the course of selection.
Asunto(s)
Regulación de la Temperatura Corporal/genética , Comportamiento de Nidificación/fisiología , Selección Genética , Animales , Peso Corporal/genética , Cruzamientos Genéticos , Conducta Alimentaria/fisiología , Femenino , Masculino , Ratones , TemperaturaRESUMEN
An on-capillary adsorptive phase in combination with capillary electrophoresis (CE), frequently referred to as preconcentration CE, for quantitative analysis of low peptide concentrations was developed. The capillary containing the on-line analyte preconcentrator can be constructed within 5 min from commercially available extraction disks. These disks contain poly(styrenedivinylbenzene) adsorbent particles incorporated in a matrix of inert Teflon, creating a mechanically stable sorbent. Therefore, no frits are needed in the capillary to hold the stationary phase in place. Several parameters, such as the required minimal elution volume, required elution strength, sample application speed or ionic strength, and the capacity were investigated and special interest was given to the quantitative properties of the method. Instead of nL injections, volumes up to a least 25 microL are possible, yielding improvements in detection limits of 3-4 orders of magnitude. The observed limit of detection for both model peptides was 20 pg, corresponding to a 20 microL injection of a 1 ng/mL solution of both model peptides. Using low-wavelength UV detection, reproducibility and linearity in the low nanogram range were satisfactory. No influence of matrix salt concentrations was observed, extending the use of CE to all kinds of samples.
Asunto(s)
Electroforesis Capilar/métodos , Péptidos/análisis , Péptidos/farmacología , Sensibilidad y EspecificidadRESUMEN
Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was characterized by using several chromatographic and spectrometric techniques. These experiments showed that aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-drying was carried out with a 500 micrograms/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection (WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to determine the optimal freeze-drying cycle parameters. The prototype, containing 500 micrograms aplidine and 25 mg D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both reconstituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formulation is stable for at least 1 year when stored at +2-8 degrees C in the dark.
Asunto(s)
Antineoplásicos/administración & dosificación , Depsipéptidos , Oligopéptidos/administración & dosificación , Péptidos Cíclicos , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Liofilización , Espectroscopía de Resonancia Magnética , Oligopéptidos/química , Control de Calidad , SolubilidadRESUMEN
In this study, criteria for the acceptability of comparative in vitro equivalence testing are proposed. Furthermore, the following equivalence limits for in vitro impaction methods are postulated: the 90% confidence interval (CI) of the in vitro deposition ratio of the test product and the reference product should lie within 0.80-1.20. The aim of this study was to challenge these limits by applying them to in vitro impaction results of several groups of pressurized metered-dose inhalers and dry powder inhalers containing salbutamol and beclomethasone dipropionate. The deposition results were obtained with the Twin Impinger. All products had a marketing authorization in The Netherlands and were considered therapeutically equivalent within each group. The postulated equivalence limits/group were challenged by fictitiously assigning a preparation as a test product or reference product and calculating the 90% CI of the deposition ratio of the test and reference products. All possible combinations of products within a group were tested. The products were considered equivalent if the 90% CI of the quotient lay within 0.80-1.20. In most cases, the quotient of the test product and reference product remains within 0.80-1.20, but due to a high variability in the deposition results of several products, the 90% CI of the quotient sometimes falls outside the proposed equivalence limits. It is concluded that the equivalence limits postulated are rather conservative, with respect to accepting equivalence. The limits can therefore serve as a prudent predictor of equivalence within the acceptability criteria proposed, but have to be further validated.
Asunto(s)
Nebulizadores y Vaporizadores/estadística & datos numéricos , Polvos/administración & dosificación , Albuterol/administración & dosificación , Albuterol/análisis , Antiasmáticos/administración & dosificación , Antiasmáticos/análisis , Beclometasona/administración & dosificación , Beclometasona/análisis , Broncodilatadores/administración & dosificación , Broncodilatadores/análisis , Cromatografía Líquida de Alta Presión , Equivalencia TerapéuticaRESUMEN
Increased light intensity of a 5-min light pulse is positively correlated with Fos mRNA and Fos protein levels in the suprachiasmatic nucleus (SCN) of hamsters. These findings suggest that the level of Fos activation is proportional to the light intensity and that the magnitude of the phase-shift response depends on the level of Fos activation. However, to what extent different phase-delay responses to the same light pulse are mediated by differential Fos activation is unknown. To elucidate this, the authors used selected house mouse lines that reveal an almost threefold difference in phase-delay responses in constant darkness (DD) between circadian time (CT) 16 and CT 20 to the same light pulse. The authors measured wheel-running activity and subjected male mice of these lines to a 15-min light pulse at CT 16 after 2 weeks in DD. The behavioral response was measured and 10 to 12 days later the animals were again subjected to the same light pulse at CT 16. One hour after the start of the second light pulse, the animals were sacrificed for Fos immunocytochemistry. Results indicate a significant difference between the lines in the phase-delay response (F2,26 = 5.112, p < 0.017) and the level of Fos activation (F2,26 = 27.15, p < 0.0001) after a 15-min light pulse at CT 16. These findings support the hypothesis that the magnitude of the phase-delay response is proportional to the number of cells in the SCN that exhibit Fos induction after the same 15-min light pulse at CT 16 in DD. It also indicates a possible difference in the input pathways among the lines.
Asunto(s)
Luz , Ratones/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efectos de la radiación , Animales , Ritmo Circadiano/efectos de la radiación , Oscuridad , Inmunohistoquímica , Masculino , Ratones/metabolismo , Actividad Motora/efectos de la radiaciónRESUMEN
In this study, we report the immunocytochemical localization of the calcium-binding protein calretinin (CAL) in the suprachiasmatic nuclei (SCN) of male and female rodents including rats, mice, golden hamsters, and Arvicanthis niloticus. The results revealed that CAL is present in different subdivisions of the SCN in the different species studied and CAL can, therefore, not be considered a marker for particular subdivisions within the SCN. No differences were found between males and females.
Asunto(s)
Proteína G de Unión al Calcio S100/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Biomarcadores , Calbindina 2 , Cricetinae , Femenino , Inmunohistoquímica , Masculino , Mesocricetus , Ratones , Ratas , Ratas Endogámicas Lew , Ratas Long-Evans , Roedores , Distribución TisularRESUMEN
Three model tripeptides, N-acetyl-Tyr-Pro-azaGly-NH(2) (NYPaG), Tyr-Pro-azaGly-NH(2) (YPaG), and Tyr-Pro-Gly-NH(2)(YPG), were subjected to a systematic degradation study to get information about the degradation of the azaglycinamido residue. The degradation products were characterized with LC-MS. Main degradation products of NYPaG possess partially or totally eliminated azaglycinamido residues, while YPaG and YPG are exhibit cyclo(Tyr-Pro) formation, a diketopiperazine. The influence of the pH on the degradation rate constant k(obs) was investigated for NYPaG and YPaG in the pH range 0.4-11. An U-shaped profile with an inflexion around pH 9 was found for NYPaG while the degradation rate of YPaG was independent of the pH. NYPaG apparently was subject to proton-, solvent-, and hydroxyl-catalyzed degradation reactions whereas YPaG only underwent solvent-catalyzed reactions. Some influence of acetate and phosphate ions on k(obs) was found for YPaG. Arrhenius plots of NYPaG and YPaG were found to be linear.