Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Summary of Bound Residue Chemistry Session Nicholas E. Weber To cite this article: Nicholas E. Weber (1990) Summary of Bound Residue Chemistry Session, Drug Metabolism Reviews, 22:6-8, 677-680, DOI: 10.3109/03602539008991461 To link to this article: http://dx.doi.org/10.3109/03602539008991461
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 10
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of California Santa Barbara]
Date: 05 November 2015, At: 17:43
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
DRUG METABOLISM REVIEWS, 22(6-8). 677-680 (1990)
SUMMARY OF BOUND RESIDUE CHEMISTRY SESSION* NICHOLAS E. WEBER Food and Drug Administration, HFV-144 Centerfor Veterinary Medicine 5600 Fishers Lane Rockville. Maryland 20857
At the beginning of this session, I indicated that we would be hearing about state-of-the-art approaches to identifying and characterizing xenobiotic adducts, i.e., bound residues. Furthermore, I indicated that not only would we review the various approaches to understanding binding and mechanisms but also listen to what each of these speakers had to say about validating their approaches comparing their in vitro systems with in vivo tests for biological significance, A tiered examination of their studies permits judgments about the biological significance of their findings and in some cases a potential safety evaluation. 1 will now outline the highlights from my perspective as to the scientific and regulatory significance of these talks. Our first speaker, Dr. Stevens, discussed three major areas, all of which are important to us in evaluating bound residues. First, he addressed the likelihood of cysteine being a target for binding of electrophilic metabolites
*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 677 Copyright 0 1991 by Marcel Dekker. Inc.
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
678
WEBER
of veterinary drugs. His assessment included the probability of binding to cysteine in glutathione as well as cysteine in protein polypeptide chains. He then discussed the likely bioavailability of cysteine adducts if they are released in the gut by proteolysis. Furthermore, he reviewed the uptake of cysteine conjugates via the portal vein to the liver where the mercapturate is synthesized. The latter is usually eliminated from the kidneys or further metabolized back to the cysteine adduct (conjugate). Finally, Dr. Stevens reviewed the mechanisms by which cysteine adducts can yield toxic manifestations with the emphasis that both toxic and nontoxic reactions of these adducts with cells can and apparently do occur. However, the range of toxic interactions has thus far been limited to a narrow group of compounds. The mechanism that he extensively discussed involved a group of enzymes called cysteine conjugate P-lyase, which cleave the adduct and yield a sulfur-containing xenobiotic residue. Dr. Steven’s paper gave us an excellent review of several areas where specific information is now available from which we can see both the utility of a particular approach as well as what is known about the mechanism and the potential for toxicity of certain adduct residues. Further, his council that adduct residues have a potential to be absorbed and yield a toxic response in the consumer is prudent at this time. While his suggestion that the threat of danger to consumers is minimal, it is the responsibility of the regulatory agency to develop ways to quantitate the “threat.” The second speaker in the session was Dr. Bakke from the USDA Laboratory in Fargo. Dr. Bakke indicated that it was his intent “to create an awareness of the possibilities that exist in the catabolism of GSH conjugates for the formation of reactive intermediates.” He first showed how the complexity of adduct residues can arise from the various cogeners of the xenobiotic-GSH adduct if they each have a reactive intermediate or second reactive site with which to bind to DNA, RNA, or protein. He also explained that adduct residues can also be partially hydrolyzed by the action of peptidase to yield cysteinyl- and cysteinylglycine-adducts. Dr. Bakke then discussed some of the reversible reactions of glutathione. Finally, he discussed at length the reactions of the P-lyase enzyme and its various specificities depending on tissue source with the G.I. tract enzyme having the broadest substrate specificity. In addition he reviewed the metabolic reactions that the adducts may undergo as well as the reactions that occur to the P-lyase cleaved, thiol-containing metabolites. Dr. Bakke’s contribution at this session gave us an in-depth look at the myriad of potential results of xenobiotic conjugation to glutathione. While he did not speak at length about cysteine adducts of residues in protein, it is clear that those portions of the metabolic information dealing with cysteinyl residues are directly applicable to the metabolism of bound residues. Fur-
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
SUMMARY BOUND RESIDUE CHEMISTRY
679
thermore, since glutathione will also react with the activated drug metabolites to form conjugates, this paper also gives us some insight into the metabolism of soluble drug-glutathione adducts. Dr. Wislocki was our third speaker, and he outlined an approach to assess the toxicological potential of bound residues of ronidazole. He reviewed the studies that Merck scientists had undertaken, which included in vitro metabolism work using rat liver microsomes and in vivo studies in rats and swine to confirm the in vitro model. After comparing the similarity of the two models, their strategy was to determine the mutagenic potency of the bound adducts in an Ames test since the parent compound, ronidazole, as well as aromatic amines which are potential reduced metabolites are both positive. The speaker’s review outlined a number of studies that he and his colleagues at Merck had done initially in vitro and subsequently in vivo to establish the metabolism and mechanism of covalent binding of ronidazole to cellular components such as proteins. This work found that cysteine and cysteine-containing compounds such as glutathione were primary targets for activated ronidazole metabolites. The ronidazole workers then proceeded to validate the in virro studies by confirming that the residue behaved chemically in a similar manner when comparing the labeling patterns and the extracted portions to in vivo generated residues under various conditions. Finally, Dr. Wislocki described studies to evaluate the toxicological significance of the drug adducts and bound residues. Three types of residue were examined using the Ames test. Those tested first were compounds believed to be structurally related to residue identifications supported by the in vitro results. Second, the microsomally produced free and bound residues were evaluated. Lastly, the in vitro produced bound residue was tested afer exposing it to digestive enzymes found in the G.I. tract. All residue fractions tested were found devoid of mutagenic activity under the conditions of the test except where it could be attributed to unmetabolized ronidazole. While this group of experiments are not completely unambiguous, they represent the largest effort to date to evaluate a veterinary drug bound residue by using the in vitro chemical biological strategy. Such an approach will undoubtedly be used in the future for regulatory decision making. Our last speaker in this session, Dr. Vroomen, also gave us a look at an actual veterinary drug (furazolidone) and his efforts to understand the nature of its bound residue. He outlined how he confirmed that the covalent residues were not endogenous compounds that resulted from fragmentation of the parent drug by showing that the residue adducts had different chemical properties from endogenous compounds. His study then turned to testing the bioavailability of the bound residue in the rat. After orally administering the bound residue to rats, not only were the bound residues bioavailable
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
680
WEBER
in this instance but they also had the capability of binding in the tissues of the rat surrogate consumer. Dr. Vroomen also studied the metabolism of furazolidone with rat and swine liver microsomes. While the qualitative profile of metabolites was similar for the most part, he found significant differences in the quantitative proportions of the metabolites. Furthermore, he found that a reactive metabolite binds reversibly to sulfhydryl-containing compounds including protein. One such compound was the mercaptoethanol adduct of the openchain acrylonitrile metabolite of furazolidone. This adduct was found to bind to protein and was also found to be mutagenic when tested in the classic Ames salmonella test. This last speaker emphasized that much of his work had been done with in v i m techniques and that the results should be confirmed with in vivo studies. However, he was quick to point out that his in vivo bioavailability study gave strong support to a similar mechanism and continued toxicological concern for tissue residues of furazolidone. His overall use of a mixture of in vivo bioavailability along with in v i m mechanistic and mutagenicity tests has also given us another example of an integrated chemical and biological approach that should be useful to regulatory decision making. This session can be concluded by stating that we have been shown through four excellent papers the state of the art and where the frontier is in the chemicaVbiologica1 strategy approach to the evaluation of bound residues. We have learned that drug adducts can be isolated and in some cases identified and that they can be tested and in some cases found to exhibit biological activity. While it is clear that one must be diligent to design unambiguous experiments, it is also clear that great strides have been made at focusing our scientific attention upon a developing area of drug metabolism and toxicological testing that has gone unaddressed too long. Our speakers have shown us that a scientific evaluation of bound residues can be made and that prudent regulatory decisions using the information developed is a reasonable expectation. Furthermore, the production of adducts by employing the simple substrate competition experiments used by the speakers may be a shortcut to generating significant amounts of adducts to be used in toxicoligical evaluation studies. I believe that this session has brought us a step closer to understanding a significant portion of the bound residue issue and some of the tools and strategies available to address it. Armed with this understanding of the chemical and some of the biological capabilities, the next session will build on that base and examine the multitude of biological testing strategies that are available to evaluate the toxicological significance of bound residues.
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Summary of Bound Residue Chemistry Session Nicholas E. Weber To cite this article: Nicholas E. Weber (1990) Summary of Bound Residue Chemistry Session, Drug Metabolism Reviews, 22:6-8, 677-680, DOI: 10.3109/03602539008991461 To link to this article: http://dx.doi.org/10.3109/03602539008991461
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 10
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of California Santa Barbara]
Date: 05 November 2015, At: 17:43
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
DRUG METABOLISM REVIEWS, 22(6-8). 677-680 (1990)
SUMMARY OF BOUND RESIDUE CHEMISTRY SESSION* NICHOLAS E. WEBER Food and Drug Administration, HFV-144 Centerfor Veterinary Medicine 5600 Fishers Lane Rockville. Maryland 20857
At the beginning of this session, I indicated that we would be hearing about state-of-the-art approaches to identifying and characterizing xenobiotic adducts, i.e., bound residues. Furthermore, I indicated that not only would we review the various approaches to understanding binding and mechanisms but also listen to what each of these speakers had to say about validating their approaches comparing their in vitro systems with in vivo tests for biological significance, A tiered examination of their studies permits judgments about the biological significance of their findings and in some cases a potential safety evaluation. 1 will now outline the highlights from my perspective as to the scientific and regulatory significance of these talks. Our first speaker, Dr. Stevens, discussed three major areas, all of which are important to us in evaluating bound residues. First, he addressed the likelihood of cysteine being a target for binding of electrophilic metabolites
*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 677 Copyright 0 1991 by Marcel Dekker. Inc.
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
678
WEBER
of veterinary drugs. His assessment included the probability of binding to cysteine in glutathione as well as cysteine in protein polypeptide chains. He then discussed the likely bioavailability of cysteine adducts if they are released in the gut by proteolysis. Furthermore, he reviewed the uptake of cysteine conjugates via the portal vein to the liver where the mercapturate is synthesized. The latter is usually eliminated from the kidneys or further metabolized back to the cysteine adduct (conjugate). Finally, Dr. Stevens reviewed the mechanisms by which cysteine adducts can yield toxic manifestations with the emphasis that both toxic and nontoxic reactions of these adducts with cells can and apparently do occur. However, the range of toxic interactions has thus far been limited to a narrow group of compounds. The mechanism that he extensively discussed involved a group of enzymes called cysteine conjugate P-lyase, which cleave the adduct and yield a sulfur-containing xenobiotic residue. Dr. Steven’s paper gave us an excellent review of several areas where specific information is now available from which we can see both the utility of a particular approach as well as what is known about the mechanism and the potential for toxicity of certain adduct residues. Further, his council that adduct residues have a potential to be absorbed and yield a toxic response in the consumer is prudent at this time. While his suggestion that the threat of danger to consumers is minimal, it is the responsibility of the regulatory agency to develop ways to quantitate the “threat.” The second speaker in the session was Dr. Bakke from the USDA Laboratory in Fargo. Dr. Bakke indicated that it was his intent “to create an awareness of the possibilities that exist in the catabolism of GSH conjugates for the formation of reactive intermediates.” He first showed how the complexity of adduct residues can arise from the various cogeners of the xenobiotic-GSH adduct if they each have a reactive intermediate or second reactive site with which to bind to DNA, RNA, or protein. He also explained that adduct residues can also be partially hydrolyzed by the action of peptidase to yield cysteinyl- and cysteinylglycine-adducts. Dr. Bakke then discussed some of the reversible reactions of glutathione. Finally, he discussed at length the reactions of the P-lyase enzyme and its various specificities depending on tissue source with the G.I. tract enzyme having the broadest substrate specificity. In addition he reviewed the metabolic reactions that the adducts may undergo as well as the reactions that occur to the P-lyase cleaved, thiol-containing metabolites. Dr. Bakke’s contribution at this session gave us an in-depth look at the myriad of potential results of xenobiotic conjugation to glutathione. While he did not speak at length about cysteine adducts of residues in protein, it is clear that those portions of the metabolic information dealing with cysteinyl residues are directly applicable to the metabolism of bound residues. Fur-
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
SUMMARY BOUND RESIDUE CHEMISTRY
679
thermore, since glutathione will also react with the activated drug metabolites to form conjugates, this paper also gives us some insight into the metabolism of soluble drug-glutathione adducts. Dr. Wislocki was our third speaker, and he outlined an approach to assess the toxicological potential of bound residues of ronidazole. He reviewed the studies that Merck scientists had undertaken, which included in vitro metabolism work using rat liver microsomes and in vivo studies in rats and swine to confirm the in vitro model. After comparing the similarity of the two models, their strategy was to determine the mutagenic potency of the bound adducts in an Ames test since the parent compound, ronidazole, as well as aromatic amines which are potential reduced metabolites are both positive. The speaker’s review outlined a number of studies that he and his colleagues at Merck had done initially in vitro and subsequently in vivo to establish the metabolism and mechanism of covalent binding of ronidazole to cellular components such as proteins. This work found that cysteine and cysteine-containing compounds such as glutathione were primary targets for activated ronidazole metabolites. The ronidazole workers then proceeded to validate the in virro studies by confirming that the residue behaved chemically in a similar manner when comparing the labeling patterns and the extracted portions to in vivo generated residues under various conditions. Finally, Dr. Wislocki described studies to evaluate the toxicological significance of the drug adducts and bound residues. Three types of residue were examined using the Ames test. Those tested first were compounds believed to be structurally related to residue identifications supported by the in vitro results. Second, the microsomally produced free and bound residues were evaluated. Lastly, the in vitro produced bound residue was tested afer exposing it to digestive enzymes found in the G.I. tract. All residue fractions tested were found devoid of mutagenic activity under the conditions of the test except where it could be attributed to unmetabolized ronidazole. While this group of experiments are not completely unambiguous, they represent the largest effort to date to evaluate a veterinary drug bound residue by using the in vitro chemical biological strategy. Such an approach will undoubtedly be used in the future for regulatory decision making. Our last speaker in this session, Dr. Vroomen, also gave us a look at an actual veterinary drug (furazolidone) and his efforts to understand the nature of its bound residue. He outlined how he confirmed that the covalent residues were not endogenous compounds that resulted from fragmentation of the parent drug by showing that the residue adducts had different chemical properties from endogenous compounds. His study then turned to testing the bioavailability of the bound residue in the rat. After orally administering the bound residue to rats, not only were the bound residues bioavailable
Downloaded by [University of California Santa Barbara] at 17:43 05 November 2015
680
WEBER
in this instance but they also had the capability of binding in the tissues of the rat surrogate consumer. Dr. Vroomen also studied the metabolism of furazolidone with rat and swine liver microsomes. While the qualitative profile of metabolites was similar for the most part, he found significant differences in the quantitative proportions of the metabolites. Furthermore, he found that a reactive metabolite binds reversibly to sulfhydryl-containing compounds including protein. One such compound was the mercaptoethanol adduct of the openchain acrylonitrile metabolite of furazolidone. This adduct was found to bind to protein and was also found to be mutagenic when tested in the classic Ames salmonella test. This last speaker emphasized that much of his work had been done with in v i m techniques and that the results should be confirmed with in vivo studies. However, he was quick to point out that his in vivo bioavailability study gave strong support to a similar mechanism and continued toxicological concern for tissue residues of furazolidone. His overall use of a mixture of in vivo bioavailability along with in v i m mechanistic and mutagenicity tests has also given us another example of an integrated chemical and biological approach that should be useful to regulatory decision making. This session can be concluded by stating that we have been shown through four excellent papers the state of the art and where the frontier is in the chemicaVbiologica1 strategy approach to the evaluation of bound residues. We have learned that drug adducts can be isolated and in some cases identified and that they can be tested and in some cases found to exhibit biological activity. While it is clear that one must be diligent to design unambiguous experiments, it is also clear that great strides have been made at focusing our scientific attention upon a developing area of drug metabolism and toxicological testing that has gone unaddressed too long. Our speakers have shown us that a scientific evaluation of bound residues can be made and that prudent regulatory decisions using the information developed is a reasonable expectation. Furthermore, the production of adducts by employing the simple substrate competition experiments used by the speakers may be a shortcut to generating significant amounts of adducts to be used in toxicoligical evaluation studies. I believe that this session has brought us a step closer to understanding a significant portion of the bound residue issue and some of the tools and strategies available to address it. Armed with this understanding of the chemical and some of the biological capabilities, the next session will build on that base and examine the multitude of biological testing strategies that are available to evaluate the toxicological significance of bound residues.
