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... Currently, in silico tools have been widely used to predict the toxicity endpoints of DPs (Pinheiro et al., 2017). Besides the advantage of reducing animal testing, the risk assessment of the DPs through in silico approaches is useful to establish the limits of potentially toxic compounds (ICH, 2006a;Pinheiro et al., 2017). ...
... Currently, in silico tools have been widely used to predict the toxicity endpoints of DPs (Pinheiro et al., 2017). Besides the advantage of reducing animal testing, the risk assessment of the DPs through in silico approaches is useful to establish the limits of potentially toxic compounds (ICH, 2006a;Pinheiro et al., 2017). ...
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Injectable solutions containing Epinephrine (EPI) and Norepinephrine (NE) are not stable and their degradation is favored mainly by the oxidation of catechol moiety. Since studies of these drugs under forced degradation conditions are scarce, herein we report the identification of their degradation products (DP) in anesthetic formulations by the development of stability‐indicating HPLC method. Finally, the risk assessment of the major degradation products was evaluated using in silico toxicity approach. HPLC method was developed to obtain a higher selectivity allowing adequate elution for both drugs and their DPs. The optimized conditions were developed by using a C18 HPLC column, sodium 1‐octanesulfonate and methanol (80:20 v/v) as mobile phase, with a flow rate of 1.5 mL/min, UV detection at 199 nm. The analysis of standard solutions with these modifications resulted in greater retention time for EPI and NE, which allow the separation of these drugs from their respective DPs. Then, Five DPs were identified and analyzed by in silico studies. Most of the DPs showed important alerts as hepatotoxicity and mutagenicity. To the best of our acknowledgment, this is the first report of a stability‐indicating HPLC method which can be used with formulations containing catecholamines.
... 3.5. Prediction of toxicity of impurity-I in comparison to drug by ADMET Predictor ® software ADMET Predictor ® software has been widely applied as a tool for evaluation of potential toxicity of impurities or metabolites in drug discovery and development [21,22]. The results of in silico toxicological profile of sugammadex sodium and impurity-I were presented in Table S2. ...
Article
Sugammadex sodium is the first selective relaxant binding agent (SRBA) indicated for reversal of neuromuscular blockade induced by rocuronium or vecuronium during surgery. The chemical synthesis of sugammadex involved the nucleophilic substitution reaction between 6-per-deoxy-6-per-halo-γ-cyclodextrin and 3-mercaptopropionic acid under basic conditions. During the manufacture of sugammadex sodium, an unknown process-related impurity was observed in pilot batches in the range of 0.21–1.9 % based upon HPLC analysis. The same impurity was also detected in commercial Bridion® samples at the levels of more than 0.1 %. Thus this unknown impurity was enriched from the mother liquor of reaction by preparative HPLC and characterized by LC–MS/QTOF, 1D-NMR (1H, 13C, DEPTQ) and 2D-NMR (1H-1H COSY, TOCSY, HSQC, HMBC, NOESY) techniques. Based on spectroscopic analysis and the synthetic route of sugammadex sodium, this new impurity was identified as monocyanoethyl sugammadex (impurity-I). The prospects to the formation mechanism and control strategy of impurity-I were discussed in detail. Moreover, the toxicological properties of impurity-I were evaluated using ADMET Predictor® software.
... In addition, the metabolic profile of the most active chalcone derivatives (LC32, LC39, and LC41) was evaluated by predicting if the derivatives could act as substrates or inhibitors of nine cytochrome P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). The toxicological endpoints evaluated were hepatotoxicity, mutagenicity, carcinogenicity, acute toxicity, and cardiotoxicity (Pinheiro et al., 2017;Tiwari et al., 2018). Hepatotoxicity parameters were specifically studied using five relevant biomarkers, ALP, SGOT, SGPT, GGT, and LDH enzymes. ...
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Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea, acting as a key enzyme in the biosynthesis of polyamines. Leishmania growth and survival is dependent on polyamine biosynthesis; therefore, inhibition of Leishmania arginase may be a promising therapeutic strategy. Here, we evaluated a series of thirty-six chalcone derivatives as potential inhibitors of Leishmania infantum arginase (LiARG). In addition, the activity of selected inhibitors against L. infantum parasites was assessed in vitro. Seven compounds exhibited LiARG inhibition above 50% at 100 µM. Among them, compounds LC41, LC39, and LC32 displayed the greatest inhibition values (72.3 ± 0.3%, 71.9 ± 11.6%, and 69.5 ± 7.9%, respectively). Molecular docking studies predicted hydrogen bonds and hydrophobic interactions between the most active chalcones (LC32, LC39, and LC41) and specific residues from LiARG's active site, such as His140, Asn153, His155, and Ala193. Compound LC32 showed the highest activity against L. infantum promastigotes (IC 50 of 74.1 ± 10.0 µM), whereas compounds LC39 and LC41 displayed the best results against intracellular amastigotes (IC 50 of 55.2 ± 3.8 and 70.4 ± 9.6 µM, respectively). Moreover, compound LC39 showed more selectivity against parasites than host cells (macrophages), with a selectivity index (SI) of 107.1, even greater than that of the reference drug Fungizone ®. Computational pharmacokinetic and toxicological evaluations showed high oral bioavailability and low toxicity for the most active compounds. The results presented here support the use of substituted chalcone skeletons as promising LiARG inhibitors and antileishmanial drug candidates.
... The software predicted that most of the formed DPs were potentially hepatotoxic. [65] Degradation behavior of Tedizolid phosphate was investigated by UFLC and structural characterization of its four DPs was performed by using UPLC-QTRAP MS n , LC-HRMS, and NMR techniques. Acute toxicity, carcinogenicity, and mutagenicity of DPs were anticipated with help of web-based CISOC-WEBPAT prediction system. ...
Article
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Degradation studies of drug give insightful information about the intrinsic stability of the molecule, the possible degradents formed during shelf life and thus, aid in the subsequent development of its stable formulation. For identification and characterization of degradents and impurities formed during stress studies, various analytical techniques are required including hyphenated techniques. Chromatographic methods play a crucial role in the field of degradation and impurity profiling. Many of these methods have been introduced for the qualitative and quantitative analysis of drugs and their formulations. This article encompasses current trends in analytical methods used for degradents, foreign matter, genotoxic impurity, and impurity profiling studies of the last five years (2013–2017). It further provides an insight into the development of various analytical methods such as hyphenated and non-hyphenated techniques those used in the analysis of pharmaceuticals. Various components used in chromatographic development such as mobile phase, buffer solutions, elution modes, columns, column temperature, and detectors are emphasized. Techniques developed by systematic Analytical Quality by Design (AQbD) approach are also discussed. Additionally, the in silico toxicity prediction studies and the methods developed for genotoxic impurity analysis are briefly explained.
... En plus de ces études, dans lesquelles une toxicité des produits de dégradation a été établie, d'autres cas rapportés démontrent par des approches informatiques que certains produits de dégradation présentent une toxicité potentiellement supérieure à celle du principe actif (Patel et al., 2015 ;Devrukhakar et al., n.d. ;Pinheiro et al., 2017). ...
Thesis
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Au cours de son cycle de vie, le principe actif se retrouve en solution dans différentes situations : dans desformes pharmaceutiques liquides, dans l’organisme et dans les eaux usées. Or, par rapport à l’état solide, lamise en solution du principe actif l’expose davantage à des facteurs susceptibles de conduire à sa dégradation.Les transformations modifient sa structure chimique et donc potentiellement ses activités pharmacologiques ettoxicologiques.L’objectif de ce travail de thèse est de présenter une méthodologie et des études visant à prédire le devenir ensolution de principes actifs et les impacts potentiels consécutifs à leur dégradation.Trois principes actifs ont été sélectionnés pour la réalisation de ce travail. Ils ont en commun de présenter,d’une part, une activité pharmacologique élevée corrélée à une toxicité potentielle de leurs produits dedégradation et, d’autre part, l'absence de données sur leurs comportements en solution. Dans tous les cas,bien que le contexte soit singulier pour chaque molécule, l’approche méthodologique suivie intègre aussi biendes travaux expérimentaux que des études ab initio et in silico.La première étude porte sur le devenir de l’apixaban, principe actif actuellement commercialisé sous formeorale solide, en solution aqueuse. Les données expérimentales ont mis en évidence des groupementschimiques du principe actif pouvant contribuer à son instabilité. L’approche ab initio a permis d’expliquer larégio-spécificité de la réaction d’hydrolyse dépendamment du pH. À partir de la structure des produits dedégradation caractérisés, l’étude de leur potentiel toxique a été réalisée par approche in silico. Ces donnéesconcourent à la démarche d'analyse et évaluation des risques déployée lors de développements de formespharmaceutiques liquides ou des situations particulières impliquant la mise en solution de l'apixaban aumoment de l'administration.De telles approches ont également été employées pour caractériser les mécanismes de photodégradation del’argatroban et évaluer le potentiel toxique des produits de dégradation. Les processus initiant laphotodégradation ont fait l’objet d’études complémentaires reposant sur des calculs d’énergies. Cesconnaissances pourront apporter le rationnel nécessaire au choix de procédés capables de réduire laphotodégradation de l’argatroban et son impact sur les patients. Elles pourront également servir à anticiper lessituations d’écarts pouvant mettre en jeu le rapport bénéfice risque du médicament telles que le mésusage oula modification de la forme pharmaceutique administrée.Enfin, dans un contexte autre que le contexte pharmaceutique, une étude de dégradation du pémétrexed parphotocatalyse via un procédé d'oxydation avancée a été réalisée. Il s'agit d'un procédé particulièrement étudiépour sa capacité à réduire l’empreinte environnementale de composés organiques en accélérant leurdégradation. Le choix de ce principe actif utilisé comme anticancéreux a été justifié par son caractère toxiqueet rémanent dans les eaux de surface, ce qui en fait un produit à haut risque environnemental. Ce travail amontré que des produits de plus faible masse résultant de la transformation photocatalytique du pémétrexedsont malheureusement plus toxiques et encore plus rémanents que la molécule mère elle-même. Ces résultatscontribuent donc à souligner que les procédés d'oxydation avancée, bien qu'efficaces pour l'élimination despolluants médicamenteux, sont à évaluer au regard de l'existence d'un risque accru pour l'environnementavant toute perspective d'utilisation à grande échelle.Les approches et les résultats présentés dans cette thèse pourront être employés pour d’autres études visant àprédire, prévenir et réduire l’impact de la dégradation du principe actif sur le patient et l’environnement.
... It was the first drug approved by the FDA for the treatment of COPD and is commercially available as Daliresp? oral tablets containing 500 ?g of roflumilast ( Pinheiro et al., 2017;Sun et al., 2015). However, it has been reported that the full clinical impact of orally active PDE4 in- hibitors has been limited by their mechanism-based gastrointestinal side effects and unexplained weight loss (Tashkin, 2014). ...
... Therefore, the data can either be procured directly from the manufacturers, or as mentioned in section 6.5, where evidence was provided of enhanced cooperation between USFDA and USP, the regulatory can share the available qualification data with the pharmacopoeial agency before fixing of specifications for IMPs/DPs in the respective monographs. Incidentally, many publications have appeared in the literature [149][150][151][152][153][154] that carry in silico toxicity data for IMPs and DPs, and a very recent trend is the use of software package like ADMET Predictor TM [155][156][157], which can calculate not only the toxicity properties, but even the physicochemical, biopharmaceutical and metabolism properties for each known structure. Even several publications exist that report in vitro cytotoxicity data for IMPs/DPs [158][159][160][161][162][163][164][165][166][167][168][169]. ...
Article
The reference standards of impurities (IMPs) and degradation products (DPs) are required by pharmaceutical industry the world over to comply with stringent thresholds prescribed in the compendial tests. In all the major pharmacopoeias, more and more monographs are currently being modified to include tests for IMPs and DPs. This review does a critical analysis of the whole situation with respect to development, supply, costs, etc. of reference standards of IMPs and DPs. It is found that significant differences exist not only regarding the above-mentioned issues, but even definitions, nomenclatures, types, instructions for use, storage, handling, etc. Typical suggestions have been put forth, which may prove handy to pharmacopoeial agencies during harmonization exercise. The write-up also discusses difficulties faced by the users, recent developments and the trends.
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Macitentan is a dual endothelin receptor antagonist indicated for the treatment of pulmonary arterial hypertension, a chronic and complex disease. Under different stress conditions, such as changes in pH and temperature, the drug can generate a large number of degradation products, while many process-related impurities can occur during the four main synthetic routes. The assessment of the potential toxicity of these impurities is an essential regulatory requirement for the quality and safety of drugs. The goal of this study was to identify all metabolites and potential impurities for macitentan and evaluate their in silico toxicity. Thirty-five compounds related to macitentan were found reported in the literature, two of which were described simultaneously as metabolites, degradation products, and process-related impurities. In the present study, the main degradation products and the conditions under which they could be formed, and the major impurities according to the synthetic route, are discussed. The types and amounts of process-related impurities were dependent on the synthesis route and process controls, while macitentan was found to be more susceptible to degradation in acidic media resulting in the most different types of degradation products. The structure of each compound was generated and the potential risk for mutagenicity and carcinogenicity were determined using three different in silico platforms, in addition the metabolic substrate/inhibition profile for each compound was assessed. Overall, five compounds were considered critical as they had a possible toxicity risk in terms of mutagenicity, tumorigenicity, irritation, and reproductive effects. These data support the current legislation for raw materials and pharmaceutical products containing macitentan as to prevent any adverse effects from this drug.
Article
Pharmaceutical drug analysis (PDA), besides quantifying drugs and related substances (RS), can enrich drug discovery (DD) by suggesting new leads. PDA may be extended towards comparative in-silico predictions for drugs and RS. This may lead to the assessment of drug likeliness of nontoxic RS as an incentive to study them further. This review overviews the in-silico profiling of drugs and RS as an innovative scope. It may help extending classical PDA with in-silico determinations to widen horizon from regulatory toxicology evaluation towards drug discovery. The virtual screening of selected RS may indicate them as potential DD leads. The extension of impurity profiling, after toxicity evaluation, to ADME estimation, QSAR studies, molecular docking, and bioactivity prediction, has widened the purposes of drug analysis. The RS which are predicted to have low toxicity may be in-silico exploited for their therapeutic potential prior to entering into the DD path.
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Human immunodeficiency virus type 1 (HIV-1) is a public health problem that affects over 38 million people worldwide. Although there are highly active antiretroviral therapies, emergence of antiviral resistant strains is a problem which leads to almost a million death annually. Thus, the development of new drugs is necessary. The viral enzyme reverse transcriptase (RT) represents a validated therapeutic target. Because the oxoquinolinic scaffold has substantial biological activities, including antiretroviral, a new series of 4-oxoquinoline ribonucleoside derivatives obtained by molecular hybridization were studied here. All synthesized compounds were tested against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and 9a and 9d displayed the highest antiviral activities, with IC50 values of 1.4 and 1.6 μM, respectively. These compounds were less cytotoxic than AZT and showed CC50 values of 1486 and 1394 μM, respectively. Molecular docking studies showed that the most active compounds bound to the allosteric site of the enzyme, suggesting a low susceptibility to the development of antiviral resistance. In silico pharmacokinetic and toxicological evaluations reinforced the potential of the active compounds as anti-HIV candidates for further exploration. Overall, this work showed that compounds 9a and 9d are promising scaffold for future anti-HIV-1 RT drug design.
Article
Roflumilast is a phosphodiesterase type 4 inhibitor that is administered orally as a long-term, in the clinical treatment of chronic obstructive pulmonary disease and asthma. Launched in 2010 for the European market, it currently does not have an official monograph. Here, a reproducible gradient RP-HPLC method was developed and validated for the separation and determination of Roflumilast in the presence of its six major degradation products. Separation was performed on a C18 analytical column (250 × 4.6 mm, 5 µm) with a mobile phase-A of ACN and a phase-B of ammonium acetate buffer (5 mM, pH 4.2) containing triethylamine (0.5% v/v). The most effective RP-HPLC gradient program was determined to be 0/80, 35/10, 36/80, 40/80 (time in minutes/% mobile phase-B). The flow rate was 1.0 ml/min and the column temperature was 25°C. The success of separation of the degradation products with different chemical characteristics was obtained by extending the time of the gradient, changing the proportion of the mobile phases and increasing the velocity of the flow. Two detectors were evaluated for the identification of degradation products and Roflumilast: a diode-arrary detector and a charged aerosol detector. The inability of the charged aerosol detector to dectect one of the six degradation products indicated that the method developed with RP-HPLC and the diode-array detector was more suitable for Roflumilast analysis. The method was validated according to specificity, linearity, LOD, LOQ, accuracy, precision and robustness.
Article
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A comprehensive stability indicating HPLC with diode array detection method was developed for determination of the recently approved phosphodiesterase type 4 (PDE-4) inhibitor roflumilast (RFL). Effective chromatographic separation was achieved using Durashell C18 column (4.6 × 250 mm, 5 μm particle size) with isocratic elution of the mobile phase composed of 0.0065 M ammonium acetate pH 6.3, methanol and acetonitrile in the ratio of 30:35:35 (by volume). The mobile phase was pumped at a flow rate of 1.3 mL/min, and quantification of RFL was based on measuring its peak areas at 251 nm. RFL eluted at retention time 6.2 min. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, range, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range was 2.5–200 μg/mL with correlation coefficient >0.9998. The drug was subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drug from its forced degradation products. Moreover, specificity of the method was verified by resolution of drug from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was successfully applied to the analysis of the cited drug in its tablet dosage form. The proposed method made use of DAD as a tool for peak identity and purity confirmation.
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A forced degradation study on roflumilast drug substance was conducted under the conditions of hydrolysis, oxidation, thermal and photolysis. The method was developed and optimized by analyzing forcefully degraded samples. The best separation was achieved on a Zorbax SB C18 1.8 µm column with 0.005 M ammonium formate buffer pH 3.5 and acetonitrile as mobile phase in a 13 min run time. The proposed method was able to resolve all the possible degradation products formed during stress study. The drug was stable to neutral, thermal and photolytic conditions but unstable to acidic, alkaline and oxidative conditions at 80° for 24 h. The degradation products resulting from stress study did not interfere in assay and related substances of roflumilast and thus the method can be regarded as stability indicating. An alternate method was also developed on a conventional 250×4.6 mm, 5 µm column wherein runtime was 38 min. Thus rapid resolution high throughput column was able to reduce the run time from 38 min to 13 min.
Article
Roflumilast analogs are a group of drugs which act as selective photodiesterase (PDE-4) inhibitor for the treatment severe chronic pulmonary disease associated with chronic brochnonities. Structural identification of degradation products using high resolution mass spectrometry and theoretical investigation by density functional theory have been successfully carried out on roflumilast to identify four degradation products namely, 3,5-dichloropyridin-4-amine, N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)-3-hydroxy benzamide, N-(3,5-dichloropyridin-4-yl)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzamide and 3-(cyclopropylmethoxy)-N-(3,5-dichloro-1-oxidopyridin-4-yl)-4-(difluoro methoxy) benzamide, generated in alkali, acidic and oxidative conditions. © 2015, Korean Society for Mass Spectrometry. All rights reserved.
Article
A reversed phase HPLC method was developed and validated for analysis of roflumilast, its related substances and degradation products, using Ecosil C18 column (250×4.6 mm, 5 μm) with a flow rate of 1.0 ml/min and detection wavelength of 215nm. The mobile phase was a mixture of acetonitrile and 0.005mol·L-1 ammonium dihydrogen phosphate buffer pH 3.5 in the ratio of 48:52 (v/v). The samples were analyzed using 20 μl injection volume and the column temperature was maintained at 30°C. The limit of detection and limit of quantitation were found to be 2.6 ng/ml and 8ng/ml, respectively. The stability-indicating capability of method was established by forced degradation studies and method demonstrated successful separation of drug, its related substances and degradation products. The method is sensitive, specific, accurate, precise and stability indicating for the quantitation of drug, its related substances and other degradation compounds.
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This review describes an epigrammatic impression of the recent trends in analytical perspectives of degradation and impurities profiling of pharmaceuticals including active pharmaceutical ingredient (API) as well as drug products during 2008-2012. These recent trends in forced degradation and impurity profiling were discussed on the head of year of publication; columns, matrix (API and dosage forms) and type of elution in chromatography (isocratic and gradient); therapeutic categories of the drug which were used for analysis. It focuses distinctly on comprehensive update of various analytical methods including hyphenated techniques for the identification and quantification of thresholds of impurities and degradants in different pharmaceutical matrices.
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Liquid chromatography-mass spectrometry (LC-MS) is considered today as a mainstay tool for the structure characterization of minor components like impurities (IMPs) and degradation products (DPs) in drug substances and products. A multi-step systematic strategy for the purpose involves high resolution mass and multi-stage mass studies on both the drug and IMPs/DPs, followed by comparison of their fragmentation profiles. Its successful application requires consideration of many practical aspects at each step. The same are critically discussed in this review.
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Different pharmaceutical preparations against the common cold containing phenylephrine (PHE) and saccharose were studied. New impurities were discovered in these preparations after exposure using isocratic ion-pair chromatography separation on a C18 column. LC-MS and NMR techniques were employed to identify and to fully characterize these new compounds. The products were identified as 1-[5-(hydroxymethyl)-2-furyl]-2-methyl-1,2,3,4-tetrahydroisochinolin-4,8-diol and 1-[5-(hydroxymethyl)-2-furyl]-2-methyl-1,2,3,4-tetrahydroisochinolin-4,6-diol. Identification of these degradation products allowed to understand and to confirm their formation mechanism. The developed HPLC method separates of all known impurities and impurities originated from PHE as well.
Article
High-resolution mass spectrometry has become ever more accessible with improvements in instrumentation, such as modern FT-ICR and Orbitrap mass spectrometers. This has resulted in an increase in the number of articles submitted for publication quoting accurate mass data. There is a plethora of terms related to accurate mass analysis that are in current usage, many employed incorrectly or inconsistently. This article is based on a set of notes prepared by the authors for research students and staff in our laboratories as a guide to the correct terminology and basic statistical procedures to apply in relation to mass measurement, particularly for accurate mass measurement. It elaborates on the editorial by Gross in 1994 regarding the use of accurate masses for structure confirmation. We have presented and defined the main terms in use with reference to the International Union of Pure and Applied Chemistry (IUPAC) recommendations for nomenclature and symbolism for mass spectrometry. The correct use of statistics and treatment of data is illustrated as a guide to new and existing mass spectrometry users with a series of examples as well as statistical methods to compare different experimental methods and datasets.
Article
The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats. The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the 32P-postlabeling assay. For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4'-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF). In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines. 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP. Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis. Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent. N-hydroxy-ADCP was essentially inactive in this assay. For the 32P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days. Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled. The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P1 and butanol enhancement methods. Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies. No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs. In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.
Article
This write-up provides a review on the development of validated stability-indicating assay methods (SIAMs) for drug substances and products. The shortcomings of reported methods with respect to regulatory requirements are highlighted. A systematic approach for the development of stability-indicating methods is discussed. Critical issues related to development of SIAMs, such as separation of all degradation products, establishment of mass balance, stress testing of formulations, development of SIAMs for combination products, etc. are also addressed. The applicability of pharmacopoeial methods for the analysis of stability samples is discussed. The requirements of SIAMs for stability study of biotechnological substances and products are also touched upon.
Article
Roflumilast is an orally administered phosphodiesterase 4 inhibitor that has potential for use in pediatric patients with asthma. The pharmacokinetics of roflumilast and roflumilast N-oxide were examined in adolescents and children with stable mild to moderate asthma in an open-label crossover study with age-stratification and 2 treatment periods (100-microg dose in period 1, 250-microg dose in period 2) separated by a washout period. Plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined using standard noncompartmental methods and compared between study groups and within the entire cohort. Roflumilast was well tolerated. Linear relationships were evident for dose and area under the plasma drug concentration-time curve extrapolated to infinity for both roflumilast (r(2) = 0.36, P < .01) and roflumilast N-oxide (r(2) = 0.39, P < .01). With the exception of dose-normalized maximum plasma concentration (mean 1.1 and 0.8 microg/L per 1 microg/kg dose for adolescents and children, respectively), pharmacokinetic parameters for roflumilast and roflumilast N-oxide were not different between age groups and were similar to adults.
Implementation of QbD approach to the RP-HPLC method development and validation of roflumilast in bulk and tablet dosage form: an application in degradation study
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Analytical method development and validation of RP-HPLC for estimation of roflumilast in bulk drug and tablet dosage form
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Identification and quantitative analysis of related substances in roflumilast
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Critical practical aspects in the application of liquid chromatography-mass spectrometric studies for the characterization of impurities and degradation products
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Identification and characterization of degradation products of phenylephrine in several pharmaceutical formulations against the common cold
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Lack of DNA binding in the rat nasal mucosa and other tissues of the nasal toxicants Roflumilast
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