Imatinib-N-oxide through chemical modification of imatinib and investigation of in-vitro potential toxic metabolite formation.

An own experience of effective treatment of a patient with a disseminated form of gastrointestinal stromal tumor (GIST) with a preparation from the group of tyrosine kinase inhibitors (imatinib) is presented.Relevance. Therapy of gastrointestinal stromal tumors is still a complex problem of modern oncology. Since 2001, a breakthrough has occurred in the treatment of patients with GISTO due to the successful use of a targeted drug from the group of tyrosine kinase inhibitors — imatinib, which is effective in the first line of inoperable and / or metastatic GISTs, and is also used for the neoadjuvant, adjuvant therapy of localized GISTs. The lack of response to therapy and, consequently, the progression of the disease, may be associated with a decrease in the therapeutic concentration of imatinib in the blood plasma. Determining the concentration of active metabolites of imatinib in the serum allows timely identification of potential causes of insufficient response to therapy and individual correction of the dose of the drug.Materials and methods. In order to determine the significance of the correlation between increasing / decreasing the dose of imatinib and achieving a therapeutic response, we used a laboratory method of high performance liquid chromatography to determine the concentration of imatinib in serum.Conclusion. Determination of the reduced concentration of active metabolites of imatinib in the blood plasma by high performance liquid chromatography with the detection of tandem mass spectrometry in a patient with disseminated form of GIST allowed to correct the dose of the drug and achieve a positive effect.

Methylisoeugenol (1,2-dimethoxy-4-propenylbenzene, 1) is a minor constituent of essential oils, naturally occurring as a mixture of cis/trans isomers. 1 is a U.S. Food and Drug Administration-approved food additive and has been given "Generally Recognized as Safe" status. Previously, metabolism of 1 has been studied in the rat, revealing mainly nontoxic cinnamoyl derivatives as major metabolites. However, data concerning the possible formation of reactive intermediary metabolites are not available to date. In this study, the oxidative metabolism of 1 was studied using liver microsomes of rat [not induced, rat liver microsomes (RLM); Aroclor1254 induced RLM (ARLM)], bovine, and human (pooled from 150 donors) origin. Incubations of these microsomes with 1 provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by NMR and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Identity was confirmed by comparison with (1)H NMR spectra of synthesized reference compounds. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol (10) synthesized as the internal standard. From incubations of ARLM with 1, seven metabolites could be detected, with 3'-hydroxymethylisoeugenol (2), isoeugenol and isochavibetol (3 + 4), and 6-hydroxymethylisoeugenol (5) being the main metabolites. Secondary metabolites derived from 1 were identified as the α,β-unsaturated aldehyde 3'-oxomethylisoeugenol (6) and 1',2'-dihydroxy-dihydromethylisoeugenol (7). We were surprised to find that formation of allylic 6-hydroxymethyleugenol (8) was observed starting at approximately 30 min after the beginning of incubations with ARLM. HLM did not form ring-hydroxylated metabolites but were most active in the formation of 6 and 7. ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes. In conclusion, we present a virtually complete pattern of nonconjugated microsomal metabolites of 1 comprising reactive metabolites and suggest the formation of reactive intermediates that need more investigation with respect to their possible adverse properties.

Olmutinib (Olita™) is an orally bioavailable third generation epidermal growth factor receptor tyrosine kinase inhibitor. Olmutinib was approved in South Korea in May 2016 for the treatment of patients suffering from locally advanced or metastatic epidermal growth factor receptor T790M mutation-positive non-small cell lung cancer. Reactive olmutinib intermediates may be responsible for the severe side effects associated with the treatment. However, literature review revealed no previous reports on the structural identification of reactive olmutinib metabolites. In this work, the formation of reactive olmutinib metabolites in rat liver microsomes was investigated. Methoxylamine, glutathione, and potassium cyanide were used as capturing agents for aldehyde, iminoquinones, and iminium intermediates, respectively. The stable complexes formed were identified using liquid chromatography-tandem mass spectrometry. The major phase I metabolic pathway observed in vitro was hydroxylation of the piperazine ring. Seven potential reactive intermediates were characterized, including three iminium ions, three iminoquinones, and one aldehyde. Based on the findings, various bioactivation pathways were postulated. Hence, identifying the reactive intermediates of olmutinib that may be the cause of severe side effects can provide new insights, leading to improved treatments for patients.

Reactive metabolites are believed to be one of the main reasons for unexpected drug-induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione-trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide-trapped metabolites were found for eight and semicarbazide-trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup.

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes.

Tyrosine kinase inhibitors (TKI) are small heterocyclic molecules targeting transmembrane and cytoplasmic tyrosine kinases that have met with considerable success in clinical oncology. TKI are associated with toxicities including liver injury that may be serious and even life-threatening. Many of them require warnings in drug labeling against liver injury, and five of them have Black Box Warning (BBW) labels. Although drug-induced liver injury is a matter of clinical and industrial concern, little is known about the underlying mechanisms that likely involve reactive metabolites (RM). RM are electrophiles or radicals originating from the metabolic activation of particular functional groups, known as structural alerts or toxicophores. RM are able to covalently bind to proteins and macromolecules, causing cellular damage and even cell death. If the adducted protein is the enzyme involved in RM formation, time-dependent inhibition of the enzyme-also called mechanism-based inhibition (MBI) or inactivation-can occur and lead to pharmacokinetic drug-drug interactions. To mitigate RM liabilities, common practice in drug development includes avoiding structural alerts and assessing RM formation via RM trapping screens with soft and hard nucleophiles (glutathione, potassium cyanide, and methoxylamine) in liver microsomes. RM-positive derivatives are further optimized to afford drug candidates with blocked or minimized bioactivation potential. However, different structural alerts are still commonly used scaffolds in drug design, including in TKI structures. This review focuses on the current state of knowledge of the relations among TKI structures, bioactivation pathways, RM characterization, and hepatotoxicity and cytochrome P450 MBI in vitro.

Intra- and extra-cellular formation of metabolites from chemically reactive species.

Drug-induced toxicity has, in many cases, been linked to oxidative metabolism resulting in the formation of reactive metabolites and subsequent covalent binding to biomolecules. Two structurally related antipsychotic drugs, clozapine (CLZ) and olanzapine (OLZ), are known to form similar nitrenium ion reactive metabolites. CLZ-derived reactive metabolites have been linked to agranulocytosis and hepatotoxicity. We have studied the oxidative metabolism of CLZ and OLZ as well as two known metabolites of CLZ, desmethyl-CLZ (DCLZ), and CLZ-N-oxide (CLZ-NO), using in vitro rat liver microsomal (RLM) incubations with glutathione (GSH) trapping of reactive metabolites and liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Reactive metabolite binding to selected standard peptides and recombinant purified human proteins was also evaluated. Bottom-up proteomics was performed using two complementary proteases, prefractionation of peptides followed by LC-HRMS/MS for elucidating modifications of target proteins. Induced RLM was selected to form reactive metabolites enzymatically to assess the complex profile of reactive metabolite structures and their binding potential to standard human proteins. Multiple oxidative metabolites and several different GSH adducts were found for CLZ and OLZ. Modification sites were characterized on human glutathione S-transferase (hGST) alpha 1 (OLZ-modified at Cys112), hGST mu 2 (OLZ at Cys115), and hGST pi (CLZ, DCLZ, CLZ-NO and OLZ at Cys170), human microsomal GST 1 (hMGST1, CLZ and OLZ at Cys50), and human serum albumin (hSA, CLZ at Cys34). Furthermore, two modified rat proteins, microsomal GST 1 (CLZ and OLZ at Cys50) and one CYP (OLZ-modified, multiple possible isoforms), from RLM background were also characterized. In addition, direct effects of the reactive metabolite modifications on proteins were observed, including differences in protease cleavage specificity, chromatographic behavior, and charge-state distributions.

Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib

Oncological efficiency of wide local excision in dermatofibrosarcoma protuberans

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.

In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported.

Reactive metabolites are estimated to be one of the main reasons behind unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. Due to their high reactivity and short lifespan, reactive metabolites are analyzed after chemical trapping with nucleophilic agents such as glutathione or cyanide. Recently, unexplained and uncharacterized methylated reaction products were reported in a human liver microsome based reactive metabolite trapping assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was utilized to produce mono- or dimethylated and further cyanide-trapped reaction products from propranolol, amlodipine and ciprofloxacin, followed by ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) experiments for their more detailed structural elucidation. Formation of all observed cyanide-trapped products was clearly NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways included N-methylation leading to iminium formation in primary and/or secondary amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation reaction was suggested to be involved in formation of the actual reactive iminium ion, the observed cyanide-trapped products were experimental artifacts rather than trapped reactive metabolites. The results stress that to avoid overestimating the formation of reactive metabolites in vitro, this methylation phenomenon should be taken into account when interpreting the results of cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes the importance of identification of the observed cyano conjugates during such studies. Yet, metabolite identification has a high importance to avoid overestimation of in vitro metabolic clearance in the cases where this kind of metabonate formation has a high impact in the disappearance rate of the compound.

Methylisoeugenol (1,2-dimethoxy-4-propenylbenzene, 1) is a minor constituent of essential oils, naturally occurring as a mixture of <i>cis/trans</i> isomers. 1 is a U.S. Food and Drug Administration-approved food additive and has been given “Generally Recognized as Safe” status. Previously, metabolism of 1 has been studied in the rat, revealing mainly nontoxic cinnamoyl derivatives as major metabolites. However, data concerning the possible formation of reactive intermediary metabolites are not available to date. In this study, the oxidative metabolism of 1 was studied using liver microsomes of rat [not induced, rat liver microsomes (RLM); Aroclor1254 induced RLM (ARLM)], bovine, and human (pooled from 150 donors) origin. Incubations of these microsomes with 1 provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by NMR and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Identity was confirmed by comparison with ¹H NMR spectra of synthesized reference compounds. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol (10) synthesized as the internal standard. From incubations of ARLM with 1, seven metabolites could be detected, with 3′-hydroxymethylisoeugenol (2), isoeugenol and isochavibetol (3 + 4), and 6-hydroxymethylisoeugenol (5) being the main metabolites. Secondary metabolites derived from 1 were identified as the α,β-unsaturated aldehyde 3′-oxomethylisoeugenol (6) and 1′,2′-dihydroxy-dihydromethylisoeugenol (7). We were surprised to find that formation of allylic 6-hydroxymethyleugenol (8) was observed starting at approximately 30 min after the beginning of incubations with ARLM. HLM did not form ring-hydroxylated metabolites but were most active in the formation of 6 and 7. ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes. In conclusion, we present a virtually complete pattern of nonconjugated microsomal metabolites of 1 comprising reactive metabolites and suggest the formation of reactive intermediates that need more investigation with respect to their possible adverse properties.

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.