Purpose. 5-HMF is a potential antisickling agent forming a Schiff-adduct with hemoglobin (Hb). In-vitro studies were designed to identify the metabolic pathways of 5-HMF in human hepatic cytosol, to assess inter-species differences in its hepatic metabolism, and to predict in-vivo PK properties. Moreover, metabolism of 5-HMF in human RBCs was investigated. Finally, in-vitro studies were done to characterize 5-HMF binding kinetics with human Hb and albumin (HSA). Methods. NAD+ reduction was monitored at 340 nm in human hepatic cytosol for 5-HMF (26 mM) and prototypical ADH and ALDH substrates in the presence or absence of their inhibitors. Furthermore, concentration-dependency studies were performed for 5-HMF (1.5-96 mM) in mouse, rat, dog, and human hepatic cytosol and fitted by Michaelis Menten (MM) model. In-vitro-in-vivo-extrapolation (IVIVE) was performed using the well-stirred model. Moreover, metabolic studies of 5-HMF (12-142 mM) in human RBCs were done under similar conditions. Time- and concentration-dependent binding studies were conducted for 5-HMF (5 µM-5 mM), in Hb (217 μM) and HSA (63 and 202 μM) solutions. Ultrafiltered 5-HMF concentrations were measured by a validated HPLC-UV assay. After correction for nonspecific binding, rate constants, binding affinity, and capacity were estimated by nonlinear regression. Results. In human hepatic cytosol, 5-HMF followed MM kinetics with Km: 218(±74) mM and was mainly inhibited by the ALDH inhibitor. In all animal species, 5-HMF exhibited millimolar Km values and is expected to have low hepatic extraction, high oral bioavailability, and first-order PK for relevant blood concentrations. The IVIVE-predicted in-vivo half-lives for 5-HMF were adequate for the mouse and dog but overestimated for humans. In RBCs, 5-HMF had Clintin-vitro of 0.34(± 0.02) ml/min/ml RBCs scaled-up to 9.9 ml/min/kg. Time-dependent binding of 5-HMF was demonstrated for both Hb and HSA. Steady-state studies revealed saturable Hb binding and non-saturable HSA binding. Conclusions. 5-HMF is an ALDH/ADH substrate in hepatic cytosol. Across animal species, 5-HMF is expected to be a low-hepatic-extraction-ratio drug with high oral bioavailability. 5-HMF is subject to RBCs metabolism in human. 5-HMF is expected to show fast association with, but slow dissociation from its drug target, Hb, which may lead to a prolonged in-vivo PD effect.
","abstract_html":"<p>Purpose. 5-HMF is a potential antisickling agent forming a Schiff-adduct with hemoglobin (Hb). In-vitro studies were designed to identify the metabolic pathways of 5-HMF in human hepatic cytosol, to assess inter-species differences in its hepatic metabolism, and to predict in-vivo PK properties. Moreover, metabolism of 5-HMF in human RBCs was investigated. Finally, in-vitro studies were done to characterize 5-HMF binding kinetics with human Hb and albumin (HSA). Methods. NAD+ reduction was monitored at 340 nm in human hepatic cytosol for 5-HMF (26 mM) and prototypical ADH and ALDH substrates in the presence or absence of their inhibitors. Furthermore, concentration-dependency studies were performed for 5-HMF (1.5-96 mM) in mouse, rat, dog, and human hepatic cytosol and fitted by Michaelis Menten (MM) model. In-vitro-in-vivo-extrapolation (IVIVE) was performed using the well-stirred model. Moreover, metabolic studies of 5-HMF (12-142 mM) in human RBCs were done under similar conditions. Time- and concentration-dependent binding studies were conducted for 5-HMF (5 µM-5 mM), in Hb (217 μM) and HSA (63 and 202 μM) solutions. Ultrafiltered 5-HMF concentrations were measured by a validated HPLC-UV assay. After correction for nonspecific binding, rate constants, binding affinity, and capacity were estimated by nonlinear regression. Results. In human hepatic cytosol, 5-HMF followed MM kinetics with Km: 218(±74) mM and was mainly inhibited by the ALDH inhibitor. In all animal species, 5-HMF exhibited millimolar Km values and is expected to have low hepatic extraction, high oral bioavailability, and first-order PK for relevant blood concentrations. The IVIVE-predicted in-vivo half-lives for 5-HMF were adequate for the mouse and dog but overestimated for humans. In RBCs, 5-HMF had Clintin-vitro of 0.34(± 0.02) ml/min/ml RBCs scaled-up to 9.9 ml/min/kg. Time-dependent binding of 5-HMF was demonstrated for both Hb and HSA. Steady-state studies revealed saturable Hb binding and non-saturable HSA binding. Conclusions. 5-HMF is an ALDH/ADH substrate in hepatic cytosol. Across animal species, 5-HMF is expected to be a low-hepatic-extraction-ratio drug with high oral bioavailability. 5-HMF is subject to RBCs metabolism in human. 5-HMF is expected to show fast association with, but slow dissociation from its drug target, Hb, which may lead to a prolonged in-vivo PD effect.</p>","abstract_has_math":false,"creators":["Obied, Taghrid"],"institution":null,"degree_name":"Doctor of Philosophy","degree_level":"Dissertation","degree_discipline":"Pharmaceutics","degree_department":null,"school":null,"contributors":["Jürgen Venitz"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010-01-01T08:00:00Z","date_published":"2010-01-01T08:00:00Z","updated_at":"2026-07-24T05:53:26Z","subjects":["5-HMF","metabolism","protein binding","antisickling","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":["© The Author"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarscompass.vcu.edu/etd/120"],"render_values":[{"text":"https://scholarscompass.vcu.edu/etd/120","href":"https://scholarscompass.vcu.edu/etd/120","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.25772/5M2S-1C59","outbound_label":"DOI","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Jürgen Venitz"]},{"key":"dc:creator","label":"Author","values":["Obied, Taghrid"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2015-08-09T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutics"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["5-HMF","metabolism","protein binding","antisickling","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["© The Author"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://doi.org/10.25772/5M2S-1C59","https://scholarscompass.vcu.edu/etd/120"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Purpose. 5-HMF is a potential antisickling agent forming a Schiff-adduct with hemoglobin (Hb). In-vitro studies were designed to identify the metabolic pathways of 5-HMF in human hepatic cytosol, to assess inter-species differences in its hepatic metabolism, and to predict in-vivo PK properties. Moreover, metabolism of 5-HMF in human RBCs was investigated. Finally, in-vitro studies were done to characterize 5-HMF binding kinetics with human Hb and albumin (HSA). Methods. NAD+ reduction was monitored at 340 nm in human hepatic cytosol for 5-HMF (26 mM) and prototypical ADH and ALDH substrates in the presence or absence of their inhibitors. Furthermore, concentration-dependency studies were performed for 5-HMF (1.5-96 mM) in mouse, rat, dog, and human hepatic cytosol and fitted by Michaelis Menten (MM) model. In-vitro-in-vivo-extrapolation (IVIVE) was performed using the well-stirred model. Moreover, metabolic studies of 5-HMF (12-142 mM) in human RBCs were done under similar conditions. Time- and concentration-dependent binding studies were conducted for 5-HMF (5 µM-5 mM), in Hb (217 μM) and HSA (63 and 202 μM) solutions. Ultrafiltered 5-HMF concentrations were measured by a validated HPLC-UV assay. After correction for nonspecific binding, rate constants, binding affinity, and capacity were estimated by nonlinear regression. Results. In human hepatic cytosol, 5-HMF followed MM kinetics with Km: 218(±74) mM and was mainly inhibited by the ALDH inhibitor. In all animal species, 5-HMF exhibited millimolar Km values and is expected to have low hepatic extraction, high oral bioavailability, and first-order PK for relevant blood concentrations. The IVIVE-predicted in-vivo half-lives for 5-HMF were adequate for the mouse and dog but overestimated for humans. In RBCs, 5-HMF had Clintin-vitro of 0.34(± 0.02) ml/min/ml RBCs scaled-up to 9.9 ml/min/kg. Time-dependent binding of 5-HMF was demonstrated for both Hb and HSA. Steady-state studies revealed saturable Hb binding and non-saturable HSA binding. Conclusions. 5-HMF is an ALDH/ADH substrate in hepatic cytosol. Across animal species, 5-HMF is expected to be a low-hepatic-extraction-ratio drug with high oral bioavailability. 5-HMF is subject to RBCs metabolism in human. 5-HMF is expected to show fast association with, but slow dissociation from its drug target, Hb, which may lead to a prolonged in-vivo PD effect.
"]},{"key":"dc:title","label":"Title","values":["IN-VITRO METABOLISM AND PROTEIN BINDING OF 5-HMF, A POTENTIAL ANTISICKLING AGENT"]}]}],"canonical_facts":{"dc:contributor":["Jürgen Venitz"],"dc:creator":["Obied, Taghrid"],"dc:date.available":["2015-08-09T07:00:00Z"],"dc:description.abstract":["Purpose. 5-HMF is a potential antisickling agent forming a Schiff-adduct with hemoglobin (Hb). In-vitro studies were designed to identify the metabolic pathways of 5-HMF in human hepatic cytosol, to assess inter-species differences in its hepatic metabolism, and to predict in-vivo PK properties. Moreover, metabolism of 5-HMF in human RBCs was investigated. Finally, in-vitro studies were done to characterize 5-HMF binding kinetics with human Hb and albumin (HSA). Methods. NAD+ reduction was monitored at 340 nm in human hepatic cytosol for 5-HMF (26 mM) and prototypical ADH and ALDH substrates in the presence or absence of their inhibitors. Furthermore, concentration-dependency studies were performed for 5-HMF (1.5-96 mM) in mouse, rat, dog, and human hepatic cytosol and fitted by Michaelis Menten (MM) model. In-vitro-in-vivo-extrapolation (IVIVE) was performed using the well-stirred model. Moreover, metabolic studies of 5-HMF (12-142 mM) in human RBCs were done under similar conditions. Time- and concentration-dependent binding studies were conducted for 5-HMF (5 µM-5 mM), in Hb (217 μM) and HSA (63 and 202 μM) solutions. Ultrafiltered 5-HMF concentrations were measured by a validated HPLC-UV assay. After correction for nonspecific binding, rate constants, binding affinity, and capacity were estimated by nonlinear regression. Results. In human hepatic cytosol, 5-HMF followed MM kinetics with Km: 218(±74) mM and was mainly inhibited by the ALDH inhibitor. In all animal species, 5-HMF exhibited millimolar Km values and is expected to have low hepatic extraction, high oral bioavailability, and first-order PK for relevant blood concentrations. The IVIVE-predicted in-vivo half-lives for 5-HMF were adequate for the mouse and dog but overestimated for humans. In RBCs, 5-HMF had Clintin-vitro of 0.34(± 0.02) ml/min/ml RBCs scaled-up to 9.9 ml/min/kg. Time-dependent binding of 5-HMF was demonstrated for both Hb and HSA. Steady-state studies revealed saturable Hb binding and non-saturable HSA binding. Conclusions. 5-HMF is an ALDH/ADH substrate in hepatic cytosol. Across animal species, 5-HMF is expected to be a low-hepatic-extraction-ratio drug with high oral bioavailability. 5-HMF is subject to RBCs metabolism in human. 5-HMF is expected to show fast association with, but slow dissociation from its drug target, Hb, which may lead to a prolonged in-vivo PD effect.
"],"dc:identifier":["https://doi.org/10.25772/5M2S-1C59","https://scholarscompass.vcu.edu/etd/120"],"dc:rights":["© The Author"],"dc:subject":["5-HMF","metabolism","protein binding","antisickling","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"],"dc:title":["IN-VITRO METABOLISM AND PROTEIN BINDING OF 5-HMF, A POTENTIAL ANTISICKLING AGENT"],"thesis:degree_discipline":["Pharmaceutics"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy"]},"updated_at":"2026-07-24T05:53:26Z"}