RESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing lamivudine as the only active pharmaceutical ingredient were reviewed. The solubility and permeability data of lamivudine as well as its therapeutic index, its pharmacokinetic properties, data indicating excipient interactions, and reported BE/bioavailability (BA) studies were taken into consideration. Lamivudine is highly soluble, but its permeability characteristics are not well-defined. Reported BA values in adults ranged from 82% to 88%. Therefore, lamivudine is assigned to the biopharmaceutics classification system (BCS) class III, noting that its permeability characteristics are near the border of BCS class I. Lamivudine is not a narrow therapeutic index drug. Provided that (a) the test product contains only excipients present in lamivudine IR solid oral drug products approved in the International Conference on Harmonization or associated countries in usual amounts and (b) the test product as well as the comparator product fulfills the BCS dissolution criteria for very rapidly dissolving; a biowaiver can be recommended for new lamivudine multisource IR products and major post-approval changes of marketed drug products.
Asunto(s)
Lamivudine/administración & dosificación , Lamivudine/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Fenómenos Químicos , Excipientes , Humanos , Lamivudine/química , Lamivudine/toxicidad , Solubilidad , Equivalencia Terapéutica , Distribución TisularRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release solid oral dosage forms containing mefloquine hydrochloride as the only active pharmaceutical ingredient (API) are reviewed. The solubility and permeability data of mefloquine hydrochloride as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability studies were taken into consideration. Mefloquine hydrochloride is not a highly soluble API. Since no data on permeability are available, it cannot be classified according to the Biopharmaceutics Classification System with certainty. Additionally, several studies in the literature failed to demonstrate BE of existing products. For these reasons, the biowaiver cannot be justified for the approval of new multisource drug products containing mefloquine hydrochloride. However, scale-up and postapproval changes (HHS-FDA SUPAC) levels 1 and 2 and most EU type I variations may be approvable without in vivo BE, using the dissolution tests described in these regulatory documents.
Asunto(s)
Antimaláricos/administración & dosificación , Antimaláricos/farmacocinética , Mefloquina/administración & dosificación , Mefloquina/farmacocinética , Administración Oral , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Disponibilidad Biológica , Formas de Dosificación , Aprobación de Drogas , Excipientes , Humanos , Mefloquina/química , Mefloquina/uso terapéutico , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of new multisource and reformulated immediate release (IR) solid oral dosage forms containing ciprofloxacin hydrochloride as the only active pharmaceutical ingredient (API) are reviewed. Ciprofloxacin hydrochloride's solubility and permeability, its therapeutic use and index, pharmacokinetics, excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. Solubility and BA data indicate that ciprofloxacin hydrochloride is a BCS Class IV drug. Therefore, a biowaiver based approval of ciprofloxacin hydrochloride containing IR solid oral dosage forms cannot be recommended for either new multisource drug products or for major scale-up and postapproval changes (variations) to existing drug products.
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Ciprofloxacina/administración & dosificación , Ciprofloxacina/farmacocinética , Administración Oral , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Disponibilidad Biológica , Ciprofloxacina/química , Ciprofloxacina/uso terapéutico , Formas de Dosificación , Aprobación de Drogas , Excipientes , Humanos , Absorción Intestinal , Permeabilidad , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature and new experimental data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing furosemide are reviewed. The available data on solubility, oral absorption, and permeability are sufficiently conclusive to classify furosemide into Class IV of the Biopharmaceutics Classification System (BCS). Furosemide's therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems are also taken into consideration. In view of the data available, it is concluded that the biowaiver procedure cannot be justified for either the registration of new multisource drug products or major postapproval changes (variations) to existing drug products.
Asunto(s)
Furosemida/farmacocinética , Disponibilidad Biológica , Biofarmacia , Formas de Dosificación , Excipientes , Humanos , Permeabilidad , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing doxycycline hyclate are reviewed. According to the Biopharmaceutics Classification System (BCS), doxycycline hyclate can be assigned to BCS Class I. No problems with BE of IR doxycycline formulations containing different excipients and produced by different manufacturing methods have been reported and hence the risk of bioinequivalence caused by these factors appears to be low. Doxycycline has a wide therapeutic index. Further, BCS-based dissolution methods have been shown to be capable of identifying formulations which may dissolve too slowly to generate therapeutic levels. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing doxycycline hyclate as the single Active Pharmaceutical Ingredient (API) provided that (a) the test product contains only excipients present in doxycycline hyclate IR solid oral drug products approved in the International Conference on Harmonization (ICH) or associated countries; and (b) the comparator and the test products comply with the BCS criteria for "very rapidly dissolving" or, alternatively, when similarity of the dissolution profiles can be demonstrated and the two products are "rapidly dissolving.".
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Doxiciclina/análogos & derivados , Antibacterianos/química , Antibacterianos/uso terapéutico , Formas de Dosificación , Doxiciclina/administración & dosificación , Doxiciclina/química , Doxiciclina/farmacocinética , Doxiciclina/uso terapéutico , Aprobación de Drogas , Humanos , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of new multisource and reformulated immediate release (IR) solid oral dosage forms containing rifampicin as the only Active Pharmaceutical Ingredient (API) are reviewed. Rifampicin's solubility and permeability, its therapeutic use and index, pharmacokinetics, excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. Solubility and absolute BA data indicate that rifampicin is a BCS Class II drug. Of special concern for biowaiving is that many reports of failure of IR solid oral dosage forms of rifampicin to meet BE have been published and the reasons for these failures are yet insufficiently understood. Moreover, no reports were identified in which in vitro dissolution was shown to be predictive of nonequivalence among products. Therefore, a biowaiver based approval of rifampicin containing IR solid oral dosage forms cannot be recommended for either new multisource drug products or for major scale-up and postapproval changes (variations) to existing drug products.
Asunto(s)
Antibióticos Antituberculosos/administración & dosificación , Antibióticos Antituberculosos/farmacocinética , Leprostáticos/administración & dosificación , Leprostáticos/farmacocinética , Rifampin/administración & dosificación , Rifampin/farmacocinética , Administración Oral , Antibióticos Antituberculosos/química , Antibióticos Antituberculosos/uso terapéutico , Disponibilidad Biológica , Formas de Dosificación , Aprobación de Drogas , Estabilidad de Medicamentos , Excipientes , Interacciones Alimento-Droga , Humanos , Leprostáticos/química , Leprostáticos/uso terapéutico , Permeabilidad , Rifampin/química , Rifampin/uso terapéutico , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data are reviewed regarding the scientific advisability of allowing a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing either diclofenac potassium and diclofenac sodium. Within the biopharmaceutics classification system (BCS), diclofenac potassium and diclofenac sodium are each BCS class II active pharmaceutical ingredients (APIs). However, a biowaiver can be recommended for IR drug products of each salt form, due to their therapeutic use, therapeutic index, pharmacokinetic properties, potential for excipient interactions, and performance in reported BE/bioavailability (BA) studies, provided: (a) test and comparator contain the same diclofenac salt; (b) the dosage form of the test and comparator is identical; (c) the test product contains only excipients present in diclofenac drug products approved in ICH or associated countries in the same dosage form, for instance as presented in this paper; (d) test drug product and comparator dissolve 85% in 30 min or less in 900 mL buffer pH 6.8, using the paddle apparatus at 75 rpm or the basket apparatus at 100 rpm; and (e) test product and comparator show dissolution profile similarity in pH 1.2, 4.5, and 6.8.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacocinética , Diclofenaco/administración & dosificación , Diclofenaco/farmacocinética , Administración Oral , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/química , Disponibilidad Biológica , Fenómenos Químicos , Diclofenaco/efectos adversos , Diclofenaco/química , Excipientes/química , Humanos , Solubilidad , Comprimidos , Equivalencia TerapéuticaRESUMEN
Literature data are reviewed relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of new multisource and reformulated immediate release (IR) solid oral dosage forms containing quinidine sulfate. Quinidine sulfate's solubility and permeability, its therapeutic use and index, pharmacokinetics, excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. The available data are not fully conclusive, but do suggest that quinidine sulfate is highly soluble and moderately to highly permeable and would likely be assigned to BCS Class I (or at worst BCS III). In view of the inconclusiveness of the data and, more important, quinidine's narrow therapeutic window and critical indication, a biowaiver based approval of quinidine containing dosage forms cannot be recommended for either new multisource drug products or for major postapproval changes (variations) to existing drug products.
Asunto(s)
Antiarrítmicos/administración & dosificación , Antiarrítmicos/farmacocinética , Antimaláricos/administración & dosificación , Antimaláricos/farmacocinética , Quinidina/administración & dosificación , Quinidina/farmacocinética , Administración Oral , Antiarrítmicos/química , Antiarrítmicos/uso terapéutico , Antimaláricos/química , Antimaláricos/uso terapéutico , Disponibilidad Biológica , Formas de Dosificación , Aprobación de Drogas , Excipientes , Humanos , Permeabilidad , Quinidina/química , Quinidina/uso terapéutico , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing (biowaiver) for the approval of immediate release (IR) solid oral dosage forms containing aciclovir are reviewed. Aciclovir therapeutic use and therapeutic index, pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) studies were also taken into consideration in order to ascertain whether a biowaiver can be recommended. According to the Biopharmaceutics Classification System (BCS) and considering tablet strengths up to 400 mg, aciclovir would be BCS Class III. However, in some countries also 800 mg tablets are available which fall just within BCS Class IV. Aciclovir seems not to be critical with respect to a risk for bioinequivalence, as no examples of bioinequivalence have been identified. It has a wide therapeutic index and is not used for critical indications. Hence, if: (a) the test product contains only excipients present in aciclovir solid oral IR drug products approved in ICH or associated countries, for instance as presented in this article; and (b) the comparator and the test product both are very rapidly dissolving, a biowaiver for IR aciclovir solid oral drug products is considered justified for all tablet strengths.
Asunto(s)
Aciclovir/administración & dosificación , Antivirales/administración & dosificación , Aciclovir/farmacocinética , Administración Oral , Antivirales/farmacocinética , Disponibilidad Biológica , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing pyrazinamide as the only active pharmaceutical ingredient (API) are reviewed. Pyrazinamide is BCS Class III, with linear absorption over a wide dosing range. The risk of bioinequivalence is estimated to be low. Depending on the definition used, pyrazinamide can be classified as a narrow therapeutic index (NTI) drug, which is usually a caveat to biowaiving but may be deemed acceptable if the Summary of Product Characteristics (SmPCs) of the test product stipulates the need for regular monitoring of liver function. It is concluded that a biowaiver can be recommended for IR solid oral dosage only when the test product (a) contains only excipients present in pyrazinamide IR solid oral drug products approved in ICH or associated countries, (b) these excipients are present in amounts normally used in IR solid oral dosage forms, (c) the test product is very rapidly dissolving, (d) the SmPC of the test product indicates the need for monitoring of the patient's liver function.
Asunto(s)
Antituberculosos , Pirazinamida , Comprimidos , Administración Oral , Antituberculosos/administración & dosificación , Antituberculosos/efectos adversos , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Disponibilidad Biológica , Excipientes , Humanos , Pirazinamida/administración & dosificación , Pirazinamida/efectos adversos , Pirazinamida/farmacocinética , Pirazinamida/farmacología , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data are reviewed relevant to the decision for a biowaiver of immediate release (IR) solid oral dosage forms containing metoclopramide hydrochloride. In addition, new solubility data, obtained under Biopharmaceutics Classification System (BCS) conditions are presented. Metoclopramide HCl is conservatively assigned to BCS Class III. Taken also into consideration excipient interactions reported in metoclopramide drug products, its pharmacokinetic properties and therapeutic use and therapeutic index, a biowaiver can be recommended when: (a) the test product contains only excipients present also in metoclopramide HCl containing IR solid oral drug products approved in ICH or associated countries, for instance as presented in this paper, (b) in amounts in normal use in IR solid oral dosage forms, and (c) the test product and the comparator both comply with the criteria for very rapidly dissolving.
Asunto(s)
Antieméticos , Antagonistas de Dopamina , Metoclopramida , Comprimidos , Administración Oral , Antieméticos/administración & dosificación , Antieméticos/farmacocinética , Antieméticos/farmacología , Disponibilidad Biológica , Antagonistas de Dopamina/administración & dosificación , Antagonistas de Dopamina/farmacocinética , Antagonistas de Dopamina/farmacología , Excipientes , Humanos , Metoclopramida/administración & dosificación , Metoclopramida/farmacocinética , Metoclopramida/farmacología , SolubilidadRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing acetazolamide are reviewed. Acetazolamide's solubility and permeability characteristics according to the Biopharmaceutics Classification System (BCS), as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems are taken into consideration. The available data on solubility, on oral absorption and permeability are not sufficiently conclusive to classify acetazolamide with certainty. Taking a conservative approach, no biowaiver is considered justified for the registration of new multisource drug products. However, SUPAC level 1 and level 2 postapproval changes and most EU Type I variations can be approved waiving in vivo BE studies.
Asunto(s)
Acetazolamida/administración & dosificación , Inhibidores de Anhidrasa Carbónica/administración & dosificación , Acetazolamida/farmacocinética , Administración Oral , Inhibidores de Anhidrasa Carbónica/farmacocinética , Formas de Dosificación , Excipientes , Humanos , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing ethambutol dihydrochloride as the only active pharmaceutical ingredient (API) are reviewed. Ethambutol dihydrochloride is a Biopharmaceutics Classification System (BCS) Class III drug with permeability properties approaching the border between BCS Class I and III. BE problems of ethambutol formulations containing different excipients and different dosages forms have not been reported and hence the risk of bioinequivalence caused by excipients is low. Ethambutol has a narrow therapeutic index related to ocular toxicity. However, as long as the prescribers' information of the test product stipulates the need for regular monitoring of ocular toxicity, the additional patient risk is deemed acceptable. It is concluded that a biowaiver can be recommended for IR solid oral dosage forms provided that the test product (a) contains only excipients present in ethambutol IR solid oral drug products approved in ICH or associated countries, for instance as presented in this paper, (b) complies with the criteria for "very rapidly dissolving" and (c) has a prescribers' information indicating the need for testing the patient's vision prior to initiating ethambutol therapy and regularly during therapy.
Asunto(s)
Etambutol/administración & dosificación , Absorción , Administración Oral , Células CACO-2 , Etambutol/química , Etambutol/farmacocinética , Excipientes , Humanos , Permeabilidad , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing prednisone are reviewed. Due to insufficient data prednisone cannot be definitively classified according to the current Biopharmaceutics Classification System (BCS) criteria as both the solubility and the permeability of prednisone are on the borderline of the present criteria of BCS Class I. Prednisone's therapeutic indications and therapeutic index, pharmacokinetics and the possibility of excipient interactions were also taken into consideration. Available evidence indicates that a biowaiver for IR solid oral dosage forms formulated with the excipients tabulated in this article would be unlikely to expose patients to undue risks.
Asunto(s)
Prednisona/farmacocinética , Administración Oral , Excipientes/administración & dosificación , Humanos , Permeabilidad , Prednisona/administración & dosificación , Prednisona/química , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing prednisolone are reviewed. Data on its solubility, oral absorption, and permeability are not totally conclusive, but strongly suggest a BCS Class 1 classification. Prednisolone's therapeutic indications and therapeutic index, pharmacokinetics, and the possibility of excipient interactions were also taken into consideration. Available evidence indicates that a biowaiver for IR solid oral dosage forms formulated with the excipients tabulated in this article would be unlikely to expose patients to undue risks.
Asunto(s)
Antiinflamatorios/farmacocinética , Prednisolona/farmacocinética , Administración Oral , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/efectos adversos , Antiinflamatorios/química , Disponibilidad Biológica , Biofarmacia , Química Farmacéutica , Ensayos Clínicos como Asunto , Formas de Dosificación , Aprobación de Drogas , Excipientes/química , Humanos , Absorción Intestinal , Permeabilidad , Prednisolona/administración & dosificación , Prednisolona/efectos adversos , Prednisolona/química , Medición de Riesgo , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing isoniazid as the only active pharmaceutical ingredient (API) are reviewed. Isoniazid's solubility and permeability characteristics according to the Biopharmaceutics Classification System (BCS), as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. Isoniazid is "highly soluble" but data on its oral absorption and permeability are inconclusive, suggesting this API to be on the borderline of BCS Class I and III. For a number of excipients, an interaction with the permeability is extreme unlikely, but lactose and other deoxidizing saccharides can form condensation products with isoniazid, which may be less permeable than the free API. A biowaiver is recommended for IR solid oral drug products containing isoniazid as the sole API, provided that the test product meets the WHO requirements for "very rapidly dissolving" and contains only the excipients commonly used in isoniazid products, as listed in this article. Lactose and/or other deoxidizing saccharides containing formulations should be subjected to an in vivo BE study.
Asunto(s)
Isoniazida/administración & dosificación , Disponibilidad Biológica , Excipientes , Humanos , Absorción Intestinal , Isoniazida/química , Isoniazida/farmacocinética , Permeabilidad , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data are reviewed on the properties of acetaminophen (paracetamol) related to the biopharmaceutics classification system (BCS). According to the current BCS criteria, acetaminophen is BCS Class III compound. Differences in composition seldom, if ever, have an effect on the extent of absorption. However, some studies show differences in rate of absorption between brands and formulations. In particular, sodium bicarbonate, present in some drug products, was reported to give an increase in the rate of absorption, probably caused by an effect on gastric emptying. In view of Marketing Authorizations (MAs) given in a number of countries to acetaminophen drug products with rapid onset of action, it is concluded that differences in rate of absorption were considered therapeutically not relevant by the Health Authorities. Moreover, in view of its therapeutic use, its wide therapeutic index and its uncomplicated pharmacokinetic properties, in vitro dissolution data collected according to the relevant Guidances can be safely used for declaring bioequivalence (BE) of two acetaminophen formulations. Therefore, accepting a biowaiver for immediate release (IR) acetaminophen solid oral drug products is considered scientifically justified, if the test product contains only those excipients reported in this paper in their usual amounts and the test product is rapidly dissolving, as well as the test product fulfils the criterion of similarity of dissolution profiles to the reference product.
Asunto(s)
Acetaminofén/farmacocinética , Analgésicos no Narcóticos/farmacocinética , Acetaminofén/administración & dosificación , Acetaminofén/química , Administración Oral , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/química , Disponibilidad Biológica , Química Farmacéutica , Formas de Dosificación , Excipientes , Solubilidad , Equivalencia TerapéuticaRESUMEN
Herbimycin A (HA), a known inducer of the heat shock response, was investigated for its ability to increase survival of a human cell line following thermal injury. Its effect on transcriptional activity was also assessed with cDNA arrays to provide new targets for cytoprotection. Pretreatment with at least 0.75 microg/ml HA significantly increased the fraction of cells surviving thermal injury by up to 50% (based on 8s exposure) compared to untreated controls. HA also significantly induced transcription of mRNA for HSP90 and HSP70, and protein production for HSP40 and HSP70. Gene expression profiling demonstrated that the most highly elevated genes included growth factors and transcription factors, while prominently suppressed genes included transcription factors and kinases. These results suggest that cytoprotection may be due to the contribution of the products of a significant number of genes in addition to the classic stress response genes, suggesting that modulation of these genes might induce thermotolerance and amelioration of thermal injury.
Asunto(s)
Citoprotección/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Calor/efectos adversos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Quinonas/farmacología , Benzoquinonas , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citoprotección/fisiología , ADN Complementario/biosíntesis , Evaluación Preclínica de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Lactamas Macrocíclicas , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Quinonas/uso terapéutico , Rifabutina/análogos & derivadosRESUMEN
To assess the feasibility of using cDNA microarrays to understand the response of endothelial cells to lipopolysaccharide (LPS) and to evaluate potentially beneficial agents in treatment of septic shock, human umbilical vein endothelial cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h. Total RNA was isolated and reverse-transcribed into (33)P-labeled cDNA probes that were hybridized to human GeneFilter microarrays containing approximately 4,000 genes. The mRNA levels of several genes known to respond to LPS changed after stimulation. In addition, a number of genes not previously implicated in the response of endothelial cells to LPS also appeared to be altered in expression. Nuclear factor-kappaB (NF-kappaB) was shown to play an important role in regulating genes identified from the microarray studies. Pretreatment of endothelial cells with a specific NF-kappaB translocation inhibitor eliminated most of the alterations in gene expression. Quantitative RT-PCR results independently confirmed the microarray results for monocyte chemotactic protein-1 and interleukin-8, and enzyme-linked immunosorbent assays demonstrated that augmented transcription was followed by translation and secretion.
Asunto(s)
ADN Complementario/biosíntesis , Endotelio Vascular/efectos de los fármacos , Endotoxinas/farmacología , Lipopolisacáridos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Cultivadas , Quimiocina CCL2/biosíntesis , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Humanos , Procesamiento de Imagen Asistido por Computador , Interleucina-8/metabolismo , FN-kappa B/metabolismoRESUMEN
PURPOSE: The degradation kinetics of thymopentin (RKDVY) and its analogs (RKDVW and RPDVY) in aqueous solution was studied by isothermal and nonisothermal methods. METHODS: The isothermal decomposition of thymopentin and its analogs was investigated as a function of pH (2-10), temperature (37, 57, and 80 degrees C) and ionic strength (micro = 0.02 to 1). Nonisothermal decomposition studies were performed using a linear temperature programmer. The temperature increasing rate was set to 0.25 degrees C per hour and the temperature interval varied from 40 to 88 degrees C. RESULTS: The decomposition of thymopentin and its analogs followed first order kinetics. The dependence of the rate constant on temperature followed a linear Arrhenius plot. This indicated that the degradation mechanism of thymopentin and its analogs might be the same within the temperature range studied. The energies of activation were found to be in close agreement for the isothermal and nonisothermal studies, suggesting that the nonisothermal studies may save considerable amount of time in the early stages of drug development. The logK-pH profile of thymopentin suggests that maximum stability is achieved in the pH range of 6-8. CONCLUSIONS: These results indicate that the nonisothermal methodology provides an attractive alternative to isothermal methods, as it requires a much lower amount of both material and time, to determine the peptide stability and to estimate the shelf-life for peptide pharmaceutical preparations.