In vitro metabolism of etoposide (VP-16-213) by liver microsomes and irreversible binding of reactive intermediates to microsomal proteins

doi:10.1016/0006-2952(87)90362-5

Biochemical Pharmacology

Volume 36, Issue 4, 15 February 1987, Pages 527-536

https://doi.org/10.1016/0006-2952(87)90362-5 Get rights and content

Abstract

We have studied the metabolism of VP-16-213 (etoposide, VP-16), an antitumor agent, by mouse liver microsomes to reactive intermediates and the subsequent covalent binding to microsomal proteins. This metabolism was shown to involve the O-demethylation of VP-16 and resulted in the formation of a 3', 4'-dihydroxy derivative (DHVP-16) which was identified by both HPLC and mass spectrometry. The formation of DHVP-16 was cytochrome P-450-mediated as indicated by its dependence on NADPH, its increased production following treatment of mice with phenobarbital, and its marked inhibition by SKF-525A and piperonyl butoride. Furthermore, DHVP-16 formation required oxygen. Microsomal incubation of VP-16 resulted in an irreversible binding of the drug to the proteins, which was also shown to be cytochrome P-450 dependent. The covalent binding of the VP-16 metabolite(s) was inhibited by DHVP-16 in a dose-dependent fashion, suggesting that the reactive intermediates that bound to proteins were derived from DHVP-16. Electron spin resonance studies indicated that the same semiquinone radical was formed during enzymatic (oxidation or reduction) metabolism of DHVP-16 and the o-quinone derivative of VP-16 (VP-16-Q). VP-16-Q and its semiquinone radical are suggested to be the bioalkylating species.

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Particularly, it is a semi-synthetic derivative of podophyllotoxin, which is isolated from the dried roots and rhizomes of species of the genus Podophyllin. Ιn human body, ETO undergoes hepatic metabolism phase I converting into 3′-demethyletoposide [7–9]. It also undergoes metabolism phase II glutathione and glucuronide conjugation.
This study presents the photolytic and photocatalytic degradation of antineoplastic drug etoposide (ETO) in aqueous solutions under UV irradiation. Photolytic degradation of ETO was slow with insufficient mineralization. The quantum yield of ETO’s photolytic degradation was calculated and ranged from 0.00029 to 0.00273 mol Einstein⁻¹. Photocatalytic degradation was proven effective, especially at pH 4, where k_obs reached 0.963 min⁻¹. Overall, 29 TPs of photocatalysis were detected at pH 4 and 7. Structures were proposed for 23 of them, based on complementary use of low-/high- resolution MS and Quantitative Structure-Retention Relationship (QSRR) prediction models. The main proposed transformations were: addition and/or demethylation of hydroxyl groups, cyclization, dehydrogenation, full or partial deprotection of diols and the loss of sugar or 2,6-dimethoxyphenol moiety. Mineralization did not follow ETO’s degradation. Toxicity assessment using Vibrio fischeri bioassay and in silico prediction model revealed the formation of partially recalcitrant and possibly toxic TPs.
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Like TPT•, the VP-16• is stable at physiological pH; however, it is still extremely reactive with glutathione, depleting glutathione in cells and in vivo [35,55]. VP-16•, however, also undergoes significant metabolism forming various reactive products that bind to DNA and proteins [30,56], including topo II, inhibiting its function [57]. At present, we do not know whether TPT• also undergoes metabolism to generate other species that contribute to TPT cytotoxicity.
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Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells
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In the present study, we examined the effects of NO/NO-related species generated via PPNO, an NO-donor, on topo II and certain topo II-poisons used clinically (i.e., VP-16 and DOX) to treat human tumors. While VP-16 undergoes extensive metabolism, catalyzed by cytochrome P450 and peroxidases, to form reactive o-quinone [9–11], the mechanism of cytotoxicity of VP-16 is considered to be due exclusively to its interactions with topo II. The mechanism of resistance of VP-16 involves both overexpression of p-glycoprotein [12,13] and decreases in the cellular target protein and/or its decreased activity as low levels of topo II protein are correlated with VP-16 resistance [14,15].
Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions.
Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin.
Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines.
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As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.
Involvement of P-glycoprotein and CYP 3A4 in the enhancement of etoposide bioavailability by a piperine analogue
2011, Chemico-Biological Interactions
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Etoposide is known to undergo biotransformation predominantly via O-demethylation in rat and humans, catalyzed by CYP 3A4 [43,44]. O-demethylation of etoposide has also been demonstrated in isolated mouse liver microsomes [45–47]. Many experimental and clinical studies are documented wherein CYP 3A4 has prominently been implicated to play a major role in the etoposide pharmacokinetics [5–7,11,14,17], and several CYP 3A4 inhibitors have been found to enhance etoposide bioavailability, such as cyclosporine A [6,10,12], ketoconazole [8,9,14], grapefruit juice [11].
Etoposide, a semi-synthetic derivative of podophyllotoxin, is widely used anticancer agent. Etoposide presents low bioavailability with wide inter-, and intra-patient variability after oral dosing. In an earlier study a piperine analogue, namely, 4-ethyl 5-(3, 4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1), was shown to cause 2.32-fold enhancement of the absolute bioavailability of co-dosed etoposide in mice. In the present investigation a mechanistic evaluation was undertaken using various in vitro and animal-derived models. In everted rat gut sac studies PA-1 enhanced mucosal uptake of the drug while it inhibited efflux of Rh-123, a P-glycoprotein substrate from serosal-to-mucosal direction. In a single pass in situ perfusion experiment PA-1 significantly reduced the intestinal exsorption rate, exsorption clearance and the total plasma clearance of etoposide. On the other hand PA-1 did not alter the passive diffusion pattern of the drug in PAMPA assay. PA-1 was inhibitory to NADPH-assisted deethylation and demethylation reactions catalyzed by erythromycin N-demethylase, 7-methoxycoumarin-O-demethylase (MOCD) and ethoxyresorufin-O-deethylase (EROD). PA-1 was not cytotoxic to mucosal membrane and showed no adverse effect in acute toxicity determination. The results suggested that PA-1-mediated enhancement in the oral bioavailability of etoposide could possibly be due to its ability to modify P-gp/CYP 3A4 mediated drug disposition mechanisms.
Effects of prednisone and genetic polymorphisms on etoposide disposition in children with acute lymphoblastic leukemia
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Etoposide is a substrate for P-glycoprotein, CYP3A4, CYP3A5, and UGT1A1. Glucocorticoids modulate CYP3A and P-glycoprotein in preclinical models, but their effect on clinical etoposide disposition is unknown. We studied the pharmacokinetics of etoposide and its catechol metabolite in children with acute lymphoblastic leukemia, along with polymorphisms in CYP3A4, CYP3A5, MDR1, GSTP1, UGT1A1, and VDR. Plasma pharmacokinetics were assessed at day 29, after 1 month of prednisone (n = 102), and at week 54, without prednisone (n = 44). On day 29, etoposide clearance was higher (47.4 versus 29.2 mL/min/m², P < .0001) than at week 54. The day 29 etoposide or catechol area under the curve (AUC) was correlated with neutropenia (P = .027 and P = .0008, respectively). The relationship between genotype and etoposide disposition differed by race and by prednisone use. The MDR1 exon 26 CC genotype predicted higher day 29 etoposide clearance (P = .002) for all patients, and the CYP3A5 AA and GSTP1 AA genotypes predicted lower clearance in blacks (P = .02 and .03, respectively). The UGT1A1 6/6, VDR intron 8 GG, and VDR Fok 1 CC genotypes predicted higher week 54 clearance in blacks (P = .039, .036, and .052, respectively). The UGT1A1 6/6 genotype predicted lower catechol AUC. Prednisone strongly induces etoposide clearance, genetic polymorphisms may predict the constitutive and induced clearance of etoposide, and the relationship between genotype and phenotype differs by race.
Alteration of nuclear factor-κB (NF-κB) expression in bone marrow stromal cells treated with etoposide
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One direct means by which VP-16 may alter nuclear translocation of p65 is through physical interaction that either masks the nuclear localization signal, or alters nuclear pore permeability. However, while VP-16 has been shown to directly bind various proteins [48,49], no binding of [3H]VP-16 to immunoprecipitated stromal cell p65 was observed (data not shown). To address the possibility that VP-16 masked the protein sites necessary for antibody interaction, immunoprecipitations were completed with nuclear protein, unlabeled VP-16, and α-p65 antibody.
Bone marrow stromal cells are an essential regulatory component in the hematopoietic microenvironment. Regulation of hematopoietic cell development is mediated, in part, through interaction of progenitor cells with stromal cell vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 expression has been shown to be driven primarily by binding of nuclear factor-κB (NF-κB) to two consensus binding sites in the promoter region. In this study, we show that down-regulation of VCAM-1 by the chemotherapeutic agent etoposide (VP-16) is associated with altered cellular localization of NF-κB. We demonstrated that VCAM-1 was diminished at the transcriptional level following treatment of stromal cells with VP-16, without alteration of VCAM-1 stability. Culture of bone marrow stromal cells in VP-16 resulted in reduced nuclear RelA (p65), a modest increase in nuclear NF-κB1 (p50), and reduced NF-κB binding to its DNA consensus sequence. Total levels of the NF-κB inhibitor Iκ-Bα were reduced during exposure to VP-16. Following removal of VP-16 from the culture, p65 and p50 nuclear profiles approximated those of untreated stromal cells, and VCAM-1 protein expression was restored. The current study indicates that NF-κB is a target molecule that is responsive to VP-16-induced damage in bone marrow stromal cells. As the primary transcription factor that promotes VCAM-1 expression, the observed changes in p65 and p50 cellular localization during treatment have a direct consequence for stromal cell function. The myriad of genes regulated by NF-κB, including both adhesion molecules and cytokines that contribute to stromal cell function, make chemotherapy-induced disruption of NF-κB biologically significant. Alterations in NF-κB activity may provide one measure by which the effects of aggressive treatment strategies on the bone marrow microenvironment can be evaluated.

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^☆: Presented in part at the meeting of the American Society for Pharmacology and Experimental Therapeutics, Boston, MA, August 18–22, 1985.

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In vitro metabolism of etoposide (VP-16-213) by liver microsomes and irreversible binding of reactive intermediates to microsomal proteins☆

Abstract

Biochem. Pharmac.

Biochem. Pharmac.

J. biol. Chem.

Biochem. Pharmac.

Biochem. biophys. Res. Commun.

Biochem. Pharmac.

J. Chromat.

Eur. J. Cancer clin. Oncol.

Biochem. biophys. Res. Commun.

Biochem. Pharmac.

Eur. J. Cancer clin. Oncol.

Cancer, N.Y.

New Engl. J. Med.

Biochemistry

Cancer Res.

Cancer Res.

Biochemistry