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This study aims to develop a framework grounded in theoretical and empirical findings regarding the ways in which co-innovation processes are carried out in the context of the Indonesian music industry. This study uses a qualitative case research method via interviews. Through literature reviews, four key concepts are proposed as the basis of rival analyses to the empirical findings. From the data collection and analysis, this study establishes that co-innovation processes in the music business constitute four stages of processes—i.e., co-discovery, co-creation, co-delivery, and co-capture, in which various theoretical concepts mentioned in the literature are embedded in each stage. The framework resulting from this study is the first to integrate value chain thinking concepts within the co-innovation processes.

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 - 10,000 ng/mL and 250 - 6000 ng/mL in rat and monkey K 2 EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A hybrid immunoaffinity LC-MS/MS assay was developed and validated for the quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma. PYX-201 was proteolyzed using trypsin, and a characteristic peptide fragment PYX-201 P1 with ten amino acids IPPTFGQGTK from the complementarity-determining regions (CDRs) was used as a surrogate for the quantitation of the TAb from PYX-201. Stable isotope labelled (SIL) peptide I( 13 C 6 , 15 N)PPTFG( 13 C 9 , 15 N)QGTK was used as the internal standard (IS). We performed chromatographic analysis using a Waters Acquity BEH Phenyl column (2.1 mm × 50 mm, 1.7 µm). Quantification of PYX-201 TAb was carried out on a Sciex triple quadrupole mass spectrometer API 6500 using multiple reaction monitoring (MRM) mode with positive electrospray ionization. To validate PYX-201 TAb, a concentration range of 0.0500 µg/mL to 20.0 µg/mL was used, yielding a correlation coefficient (r) of ≥ 0.9947. For intra-assay measurements, the percent relative error (%RE) ranged from -23.2% to 1.0%, with a coefficient of variation (%CV) of ≤ 14.2%. In terms of inter-assay measurements, the %RE was between -10.5% and -5.7%, with a %CV of ≤ 12.7%. The average recovery of the analyte was determined to be 81.4%, while the average recovery of the internal standard (IS) was 97.2%. Furthermore, PYX-201 TAb demonstrated stability in human plasma and human whole blood under various tested conditions. This assay has been successfully applied to human sample analysis to support a clinical study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

PYX-201 is an investigational antibody drug conjugate (ADC) with an engineered, fully human IgG1 antibody, a cleavable chemical linker, and a toxin (Aur0101) with an average drug-antibody ratio (DAR) of ∼ 4. A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated to determine the presence in human plasma, of free payload Aur0101 from PYX-201 to assess drug safety and efficacy. Aur0101 and its deuterated internal standard (IS), Aur0101_d 8 , were extracted from 25 µL of human plasma using a solid liquid extraction (SLE) method. Chromatographic analysis was carried out on a Waters Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 µm, 130 A) column. Quantitation of free Aur0101 was conducted on a Sciex triple quadrupole mass spectrometer API 6500 + using multiple reaction monitoring (MRM) mode via positive electrospray ionization. The calibration curve was linear over the concentration range of 25.0 to 12,500 pg/mL with correlation coefficient, r 2  ≥ 0.9988. The intra-assay %RE was between -4.3% to 14.3% with % CV was ≤ 6.2%. The inter-assay %RE was between -0.2% to 9.5% with % CV was ≤ 6.1%. The average analyte recovery was 89.7% and the average IS recovery was 88.7%. Aur0101 was found to be stable in human plasma and human whole blood under various tested conditions with and without the presence of PYX-201. To our knowledge, this is the first published fully validated assay for free, unconjugated Aur0101 in any matrix, from any species. This assay has been successfully applied to clinical sample analysis to support clinical studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Aim: Aur0101 is a cytotoxic and small-molecule microtubule depolymerizing agent, and is the payload conjugated to antibody-drug conjugate PYX-201. Developing and validating a sensitive bioanalytical method to quantitate Aur0101 was novel and crucial in preclinical PYX-201 studies. Materials & methods: Reference standard Aur0101 and its stable isotope labelled internal standard Aur0101-d 8 were used in this LC-MS/MS method. Results: This sensitive assay was validated at a lower limit of quantitation of 15 pg/ml and successfully applied to support preclinical rat and monkey toxicology studies. Preclinical plasma toxicokinetic parameters were presented. Conclusion: A sensitive and robust LC-MS/MS assay was validated for Aur0101 in rat and monkey plasma.

PYX-201 is an anti-extra domain B splice variant of fibronectin (EDB + FN) antibody drug conjugate (ADC) composed of a fully human IgG1 antibody, a cleavable mcValCitPABC linker, and four Auristatin 0101 (Aur0101, PF-06380101) payload molecules. To better understand the pharmacokinetic (PK) profile of PYX-201 after it is administered to cancer patients, the development of a reliable bioanalytical assay to accurately and precisely quantitate PYX-201 in human plasma is required. In this manuscript, we present a hybrid immunoaffinity LC-MS/MS assay used to successfully analyze PYX-201 in human plasma. PYX-201 was enriched by MABSelect beads coated with protein A in human plasma samples. The bound proteins were subjected to "on-bead" proteolysis with papain to release the payload Aur0101. The stable isotope labelled internal standard (SIL-IS) Aur0101-d 8 was added and the released Aur0101 was quantified as a surrogate for the total ADC concentration. The separation was performed on a UPLC C18 column coupled with tandem mass spectrometry. The LC-MS/MS assay was validated over the range 0.0250 to 25.0 µg/mL with excellent accuracy and precision. The overall accuracy (%RE) was between -3.8% and -0.1% and the inter-assay precision (%CV) was <5.8%. PYX-201 was found to be stable in human plasma for at least 24 h on ice, 15 days after being stored at -80 °C, as well as after five freeze/thaw cycles of being frozen at -25 °C or -80 °C and thawed on ice. The assay this paper reports on, has been successfully applied to human sample analysis to support clinical studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aim: PYX-201 is a novel antibody-drug conjugate targeting the extra domain B splice variant of fibronectin in the tumor microenvironment. Accurate quantification of PYX-201 is critical for PYX-201 pharmacokinetics profiling in preclinical studies. Materials & methods: ELISA was performed using reference standard PYX-201, mouse monoclonal anti-monomethyl auristatin E antibody, mouse IgG1, mouse monoclonal anti-human IgG horseradish peroxidase and donkey anti-human IgG horseradish peroxidase. Results: This assay was validated at 50.0-10,000 ng/ml in rat dipotassium EDTA plasma and 250-10,000 ng/ml in monkey dipotassium EDTA plasma. Conclusion: This is the first time for a PYX-201 bioanalytical assay in any matrix to be reported.

Purpose: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have shown promise in treating Duchenne muscular dystrophy (DMD). We evaluated a semi-mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) model to capture the relationship between plasma and muscle tissue exposure/response in mdx mice treated by mouse surrogate PPMO., Methods: A single or repeated (every 4 weeks for 20 weeks) intravenous PPMO dose was administered to mdx mice (n = 6/timepoint). A PK/PD model was built to characterize data via sequential modeling. A 2-compartment model was used to describe plasma PK. A simultaneous tissue PK/PD model was subsequently developed: 2-compartment model to describe muscle PK; linked to an indirect response model describing stimulation of synthesis of skipped transcript, which was in turn linked to stimulation of synthesis of dystrophin protein expression., Results: Model performance assessment via goodness-of-fit plots, visual predictive checks, and accurate parameter estimation indicated robust fits of plasma PK and muscle PK/PD data. The model estimated a PPMO tissue half-life of 5 days-a useful parameter in determining the longevity of PPMOs in tissue and their limited accumulation after multiple doses. Additionally, the model successfully described dystrophin expression after single dosing and associated protein accumulation after multiple dosing (increasing ~ twofold accumulation from the first to last dose)., Conclusions: This first PK/PD model of a PPMO in a DMD disease model will help characterize and predict the time course of PK/PD biomarkers in mdx mice. Furthermore, the model framework can be used to develop clinical PK/PD models and can be extended to other exon-skipping therapies and species., (© 2021. The Author(s).)

During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC 50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC 50 : 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay., Competing Interests: The authors declare the following competing financial interest(s): S.H. and F.X.W. are or were Summit Therapeutics plc employees or consultants at the time that the work was conducted. K.E.D., S.G.D. and A.J.R. are minor shareholders of Summit Therapeutics plc., (© 2020 American Chemical Society.)

Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27 , led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[ d ]oxazole (ezutromid, 1 ) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo . No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture., (© 2019 The Authors.)

Ezutromid (SMT C1100) is a small-molecule utrophin modulator that was developed to treat Duchenne muscular dystrophy (DMD). Previous clinical trials of this agent revealed lower exposure in DMD patients compared with healthy volunteers, which may reflect differences in diet. This study evaluated the pharmacokinetics of ezutromid in patients with DMD who followed a balanced diet. This was a multicenter, double-blind, placebo-controlled, ascending single and multiple oral dose study. Twelve pediatric patients were randomly allocated to 1 of 3 treatment sequences within which were 3 treatment periods of 2 weeks each. Each patient received, in a dose-escalating fashion, 1250 mg and 2500 mg twice daily (BID) of ezutromid administered orally as a microfluidized suspension (F3) with placebo in the other treatment period. Throughout the study, patients followed a balanced diet including recommended proportions of major food groups and administration of drug accompanied with 100 mL of full-fat milk. This approach improved the absorption of ezutromid, resulting in higher systemic exposure, with considerable variability in exposure between patients at each dose level. Single and multiple oral doses of 1250 mg and 2500 mg BID were considered safe and well tolerated. No severe or serious adverse events and no study discontinuations due to adverse events were reported. This study provides assurance that, with the formulation tested (F3) and instructions regarding food (balanced diet and whole-fat milk), 2500 mg BID of ezutromid achieves plasma concentrations that, based on preclinical studies, should be able to modulate utrophin expression in future clinical trials., (© 2019, The American College of Clinical Pharmacology.)

The influence of surfactants and coreactants on Ru(bpy) 3 2+ electrogenerated chemiluminescence (ECL) was investigated comparatively. Specifically, the influence that the two tertiary amines, N-butyldiethanolamine (NBEA) and 2-(dibutylamino)ethanol (DBAE) have on the ECL reaction, alone and in the presence of the two surfactants Triton™ X-100 and Zonyl® FSN, was investigated, in comparison with that of the well-studied and established coreactant tripropylamine (TPA). Experiments were conducted on poly(methyl methacrylate) substrates coated with plasma-evaporated gold as used in many miniaturized systems. Upon optimization and study of the various combinations, the combination of NBEA/Zonyl FSN provided superior ECL signal characteristics. A limit of detection (LOD) of 2.2 nM Ru(bpy) 3 2+ was obtained. Compared with the LOD of 0.59 μM for the commonly used TPA/Triton™ X-100 system, the resulting LOD is enhanced by a factor of 250. In addition, significantly more stable signals lead to an increase in sensitivity by a factor of 50. This makes the NBEA/Zonyl® FSN system an attractive ECL strategy, especially for miniaturized analytical systems. Furthermore, it became clear that previously postulated effects of surfactants on the enhancement of coreactant-based ECL do not translate to other surfactants and coreactants. We could demonstrate that more complicated mechanisms are at play as the ECL intensity for Ru(bpy) 3 2+ /NBEA was the highest in the presence of Zonyl® FSN, whereas the ECL signal decreased significantly upon introduction of the surfactant with DBAE as coreactant.

We present studies on ruthenium-based electrochemiluminescence (ECL) focusing on conditions supporting signal enhancement and low oxidation potentials. Low oxidation potentials (LOPs) are especially attractive for miniaturized ECL biosensors, as microfabricated electrodes tend to detach from their support when used with high currents and operated at high potentials. Furthermore, high potentials or current densities can lead to damage of typical biosensor surface coatings and biological probes. The possibility of generating LOP ECL signals at a potential below 900 mV was therefore studied for Ru(bpy) 3 2+ with two typical coreactants, i.e. 2-(dibutylamino)ethanol (DBAE) and tripropylamine (TPA), as well as with the tertiary amine N-butyldiethanolamine (NBEA). Furthermore, the effect of buffer components and pH values on ECL signal generation was investigated. We could show a significant LOP ECL signal for NBEA. We found that Tris buffer, with its ability to form complexes with transition metal ions, has a positive influence on this ECL signal in terms of signal strength and LOP capabilities. Specifically, at basic pH values significant increases in ECL signals were observed at 900 mV and at 1.2 V. In fact, the ECL signal at 1.2 V was three times higher than the signal observed in phosphate buffer at a pH of 7, and it was thirty times higher than the ECL signal for TPA under these conditions. The LOP signal for NBEA in Tris buffer at pH 8.5 was similar to the signal obtained for TPA in phosphate buffer at pH 8.5 but three times higher than for TPA at pH 7.0. Interestingly, the coreactant DBAE was neither significantly influenced by the buffer system or pH nor did it present a valuable LOP ECL signal. Finally, it was found that high peak currents in cyclic voltammograms are not the indicators for high ECL signals, which should be obvious because the ECL mechanism requires more complex electron transfers. Overall, the standard TPA ECL at 1.2 V in phosphate buffer at pH 7.0 can successfully be replaced by NBEA ECL at 900 mV in Tris at pH 8.5 providing significantly higher signals accompanied by more gentle electrochemical conditions.

Mechanism-based inactivation (MBI) often involves metabolic bioactivation of the xenobiotic by cytochrome P450s (CYPs) to an electrophilic reactive intermediate and results in quasi-irreversible or irreversible inactivation. Such reactive intermediate can cause quasi-irreversible inhibition through coordination to the ferrous state, Fe(II), of the P450 enzyme forming a tight noncovalent bond leading to the formation of metabolic-intermediate complex (MIC). By contrast, irreversible inactivation is one of the most common causes for the observed drug–drug interaction (DDI) and usually implies the formation of a covalent bond between the metabolite and the enzyme via alkylation of either the heme or the P450 apoprotein. Here we illustrate the important points of the current literature understanding of the mechanisms of inhibition of CYP enzymes with emphasis on general mechanistic aspects of MBI for some drugs/moieties associated with the phenomenon. Additionally, the utility of computational and in silico approaches to address bioactivation issues will be briefly discussed.

In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.

Oxidative deamination of the GABA(A) partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL(int)) values were determined and compared to the corresponding in vivo oral clearance (CL(PO)) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A. The intrinsic clearance, CL(int), of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL(int), values were calculated as 0.008 and 0.002 ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)(PO), values in humans were calculated as 724 and 178 ml/min/kg for CP-409,092 and sumatriptan, respectively. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

Background: The purpose of this study was to evaluate the predictability of fistula closure using the ratio of C-reactive protein to prealbumin (C:P ratio)., Methods: A database of 89 patients with gastrointestinal fistulas (1994-2009) was created based on the records of our Nutrition Support Services Team. All patients had weekly blood work including C-reactive protein level, prealbumin level, and albumin level. Forty-three fistulas were managed without surgery for 6 weeks or more; of these, 29 closed., Results: The median C:P ratio for those fistulas that remained open after 6 weeks of conservative management differed significantly from those that closed (.10 vs .35; P < .001). For patients with a C:P ratio of .25 or less, fistula closure occurred in 87.0% (95% confidence interval, 74.0-94.3), whereas for patients with a ratio of greater than 1.0, no fistulas closed., Conclusion: Our results suggest that the C:P ratio is a predictor of fistula closure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

1-[4-Aminomethyl-4-(3-chlorophenyl)-cyclohexyl]-tetrahydro-pyrimidin- 2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme. Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-Carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO(2), UDP glucuronosyltransferase (UGT) reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species-mouse, rat, hamster, dog, monkey, and human. Structure elucidation was performed on a mass spectrometer with accurate mass measurement and MS(n) capabilities. (13)C-labeled carbon dioxide was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with (13)C-labeled CO(2) in rat liver microsomes revealed that CO(2) from the bicarbonate buffer (in equilibrium with exogenous CO(2)) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species, mainly rat and hamster, followed by similar formation in dog, monkey, mouse, and human. M1 could be detected in UGT1A1, UGT1A3, and UGT2B7 Supersomes in a CO(2)-rich environment. In conclusion, our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests incorporation of CO(2) from the bicarbonate buffer, in equilibrium with exogenous CO(2), into the carbamoyl moiety of the formed N-carbamoyl glucuronide.

Amplification and overexpression of erbB2 (Her-2/neu) proto-oncogene has been linked to human malignancies including tumors of the breast, ovary, and stomach. It has been implicated in tumor growth, sensitivity to standard chemotherapy, prognosis of patients, and disease-free survival. Although the clinical use of trastuzumab (Herceptin) has prolonged the survival of breast cancer patients with erbB2-overexpressing tumors, there is an urgent need for more potent and orally bioavailable small-molecule inhibitors. CP-724,714 is a potent inhibitor of erbB2 receptor autophosphorylation in intact cells and is currently undergoing phase I clinical trials. Here, we describe the effects of CP-724,714 in vitro and in vivo in human breast cancer models. CP-724,714 is selective for inhibiting growth of HER2-driven cell lines. In addition, we show that it induces G1 cell cycle block in erbB2-overexpressing BT-474 human breast carcinoma cells and inhibits erbB2 autophosphorylation in xenografts when administered p.o. to athymic mice. It induces a marked reduction of extracellular signal-regulated kinase and Akt phosphorylation, tumor cell apoptosis, and release of caspase-3. P.o. administration (q.d. or b.i.d.) of CP-724,714 inhibits the growth of erbB2-overexpressing tumors in athymic mice without overt adverse effects.

The role of single electron transfer (SET) in P450-catalyzed N-dealkylation reactions has been studied using the probe substrates N-cyclopropyl-N-methylaniline (2a) and N-(1'-methylcyclopropyl)-N-methylaniline (2b). In earlier work, we showed that SET oxidation of 2a by horseadish peroxidase leads exclusively to products arising via fragmentation of the cyclopropane ring [Shaffer, C. L.; Morton, M. D.; Hanzlik, R. P. J. Am. Chem. Soc. 2001, 123, 8502-8508]. In the present study, we found that liver microsomes from phenobarbital pretreated rats (which contain CYP2B1 as the predominant isozyme) oxidize [1'-(13)C, 1'-(14)C]-2a efficiently (80% consumption in 90 min). Disappearance of 2a follows first-order kinetics throughout, indicating a lack of P450 inactivation by 2a. HPLC examination of incubation mixtures revealed three UV-absorbing metabolites: N-methylaniline (4), N-cyclopropylaniline (6a), and a metabolite (M1) tentatively identified as p-hydroxy-2a, in a 2:5:2 mole ratio, respectively. 2,4-Dinitrophenylhydrazine trapping indicated formation of formaldehyde equimolar with 6a; 3-hydroxypropionaldehyde and acrolein were not detected. Examination of incubations of 2a by (13)C NMR revealed four (13)C-enriched signals, three of which were identified by comparison to authentic standards as N-cyclopropylaniline (6a, 33.6 ppm), cyclopropanone hydrate (11, 79.2 ppm), and propionic acid (12, 179.9 ppm); the fourth signal (42.2 ppm) was tentatively determined to be p-hydroxy-2a. Incubation of 2a with purified reconstituted CYP2B1 also afforded 4, 6a, and M1 in a 2:5:2 mole ratio (by HPLC), indicating that all metabolites are formed at a single active site. Incubation of 2b with PB microsomes resulted in p-hydroxylation and N-demethylation only; no loss or ring-opening of the cyclopropyl group occurred. These results effectively rule out the participation of a SET mechanism in the P450-catalyzed N-dealkylation of cyclopropylamines 2a and 2b, and argue strongly for the N-dealkylation of 2a via a carbinolamine intermediate formed by a conventional C-hydroxylation mechanism.

As a model for the reaction of chemically reactive quinone metabolites with cellular proteins in vivo, the reactions of benzoquinone (BQ; 1-64 mol/mol of protein) with bovine pancreatic ribonuclease A (RNase), reduced RNase, S-(amidomethylated) reduced RNase, and reduced guanidinated RNase were investigated. The reaction stoichiometry and products were characterized by means of HPLC, UV-vis spectrophotometry, electrospray mass spectrometry, amino acid analysis, alkaline permethylation analysis, and measurement of the covalent binding of [14C]BQ to protein. Native RNase and S-(amidomethylated) reduced RNase show no reaction with BQ over 60 min at pH 7.4-9.6, whereas reduced RNase, which has 8 free SH groups/mol, reacts rapidly with exactly 24 molecules of BQ, of which 8 become covalently bound to protein-SH groups while 16 are reduced to hydroquinone. Half of the latter is formed via BQ oxidation of the initially formed S-(2,5-dihydroxyphenyl)cysteine moieties. Michael addition of a second protein nucleophile to each resulting S-(2,5-quinoyl)cysteine moiety, followed by reoxidation of that addition product by BQ, generates the second group of 8 molecules of HQ and results in cross-linking. Reduced guanidinated RNase, in which most of the lysines are blocked by guanidination with O-methylisourea, also reacts rapidly with BQ, but only ca. 16 equiv are consumed; of these, 8 become covalently bound to protein-SH groups while the others are reduced to HQ. Thus, even though the lysine residues in native RNase and S-(amidomethylated) reduced RNase do not react with BQ, they may react with (2,5-quinonyl)-S-protein moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism and enzymology of metabolic ibuprofen isomerization constituted the focus of this investigation. Comparative in vivo studies revealed that this biotransformation proceeded via a proton abstraction mechanism in all tested species of mammals, which is in agreement with the previous reports. Direct evidence supporting this conclusion stemmed from the in vitro epimerization of ibuprofen-CoA thioester in rat liver homogenates. Chemically synthesized (R)-ibuprofen-CoA thioester was rapidly transformed to its (S)-counterpart by subcellular hepatic preparations. Examination of this epimerase activity in various rat tissue homogenates indicated that this enzyme was highly tissue specific. This biochemical reaction mainly took place in the liver and kidney, whereas low levels of enzyme activity were associated with other tissues. Nevertheless, the liver and kidney homogenates failed to invert (R)-ibuprofen directly even in the presence of all the necessary cofactors. Presumably, the failure to characterize this bioconversion was due to the lack of enzymatic acyl-CoA synthesis in these homogenates. It is noteworthy that the '2-arylpropionyl-CoA epimerase' catalyzed the transformation from either direction and with high turnover rates. The catalytic efficiency of (S)-ibuprofen CoA epimerization appeared to be greater than that of the (R)-counterpart. These in vitro findings suggest that the step of acyl-CoA formation assume a pivotal role in controlling the stereoselectivity and efficiency of the in vivo metabolism. As the responsible acyl-CoA synthetase(s) in different species of animals may exert the reaction with different degrees of enantiomeric preference and efficiency, the resulting stereochemical outcome and metabolic rates of this bioinversion vary accordingly. Consequently, in guinea pigs, this biotransformation proceeds in both directions with nearly equal efficiency, whereas it is virtually unidirectional and slow in humans. Currently, the purification and characterization of this novel '2-arylpropionyl-CoA epimerase' from rat livers constitute the focus of this investigation.