Cost-minimization analysis of alemtuzumab compared to fingolimod and natalizumab for the treatment of active relapsing-remitting multiple sclerosis in the Netherlands.

AIM: In active relapsing remitting multiple sclerosis (RRMS) patients requiring second-line treatment, the Dutch National Health Care Institute (ZiN) has not stated a preference for either alemtuzumab, fingolimod, or natalizumab. The aim was to give healthcare decision-makers insight into the differences in cost accumulation over time between alemtuzumab-with a unique, non-continuous treatment schedule-and fingolimod and natalizumab for second-line treatment of active RRMS patients in the Netherlands. METHODS: In line with ZiN's assessment, a cost-minimization analysis was performed from a Dutch healthcare perspective over a 5-year time horizon. Resource use was derived from hospital protocols and summaries of product characteristics, and validated by two MS specialists. Unit costs were based on national tariffs and guidelines. Robustness of the base case results was verified with multiple sensitivity and scenario analyses. RESULTS: Alemtuzumab results in cost savings compared to fingolimod and natalizumab from, respectively, 3.3 and 2.8 years since treatment initiation onwards. At 5 years, total discounted costs per patient of alemtuzumab were €79,717, followed by fingolimod with €110,044 and natalizumab with €122,238, resulting in cost savings of €30,327 and €42,522 for alemtuzumab compared to fingolimod and natalizumab, respectively. Key drivers of the model are drug acquisition costs and the proportions of patients that do not require further alemtuzumab treatment after either two, three, or four courses. LIMITATIONS: No treatment discontinuation and associated switching between treatments were incorporated. Consequences of JC virus seropositivity while continuing natalizumab treatment (e.g. additional monitoring) were omitted from the base case. CONCLUSION: The current cost-minimization analysis demonstrates that, from the Dutch healthcare perspective, treating active RRMS patients with alemtuzumab results in cost savings compared to second-line alternatives fingolimod and natalizumab from ∼3 years since treatment initiation onwards. After 5 years, alemtuzumab's cost savings are estimated at €30k compared to fingolimod and €43k compared to natalizumab.

Prevention of anaemia by early intervention with once weekly epoetin alfa during chemotherapy.

This study compared the effects of early intervention with standard use of epoetin alfa on haemoglobin (Hb) levels and transfusion requirements in cancer patients receiving chemotherapy. Patients with Hb>10 and < or= 12 g/dL were randomised 1:1 to epoetin alfa (40,000 IU, subcutaneously, once weekly), initiated within 7d of the start of the first on-study chemotherapy cycle (defined as early intervention) versus epoetin alfa when Hb

Effects of early intervention with epoetin alfa on transfusion requirement, hemoglobin level and survival during platinum-based chemotherapy: Results of a multicenter randomised controlled trial.

This work was conducted to evaluate the effect of early intervention with epoetin alfa (EPO) on transfusion requirements, hemoglobin level (Hb), quality of life (QOL) and to explore a possible relationship between the use of EPO and survival, in patients with solid tumors receiving platinum-based chemotherapy. Three hundred and sixteen patients with Hb12.1g/dL were randomised 2:1 to EPO 10000 IU thrice weekly subcutaneously (n = 211) or best supportive care (BSC) (n = 105). The primary end point was proportion of patients transfused while secondary end points were changes in Hb and QOL. The protocol was amended before the first patient was recruited to also prospectively collect survival data. EPO therapy significantly decreased transfusion requirements (P < 0.001) and increased Hb (P < 0.005). EPO-treated patients had significantly improved QOL compared with BSC patients (P < 0.05). Kaplan-Meier estimates showed no differences in 12-month survival (P = 0.39), despite a significantly greater number of patients with metastatic disease in the EPO group (78% vs. 61%, P = 0.001). EPO was well tolerated. This study has shown that early intervention with EPO can result in a significant reduction of transfusion requirements and increases in Hb and QOL in patients with mild anemia during platinum-based chemotherapy.

A physiologically-based pharmacokinetic(PB-PK) model for ethylene dibromide: relevance of extrahepatic metabolism.

A physiologically-based pharmacokinetic (PB-PK) model was developed for ethylene dibromide (1,2-dibromoethane, EDB) for rats and humans, partly based on previously published in vitro data (Ploemen et al., 1997). In the present study, this PB-PK model has been validated for the rat. In addition, new data were used for the human class ThetaGST T1-1. Validation experiments are described in order to test the predictive value of kinetics to describe "whole-body" metabolism. For the validation experiments, groups of cannulated rats were dosed orally or intravenously with different doses of EDB. Obtained blood concentration-time curves of EDB for all dosing groups were compared to model predictions. It appeared that metabolism, which previously was assumed to be restricted to the liver, was underestimated. Therefore, we extended the PB-PK model to include all the extrahepatic organs, in which the enzymes involved in EDB metabolism have been detected and quantified. With this extended model, the blood concentrations were much more accurately described compared to the predictions of the "liver-model". Therefore, extrahepatic metabolism was also included in the human model. The present study illustrates the potential application of in vitro metabolic parameters in risk assessment, as well as the use of PB-PK modelling as a tool to understand and predict in vivo data.

Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity.

In this review, an overview is presented of the current knowledge of genetic polymorphisms of four of the most important enzyme families involved in the metabolism of xenobiotics, that is, the N-acetyltransferase (NAT), cytochrome P450 (P450), glutathione-S-transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. The emphasis is on two main topics, the molecular genetics of the polymorphisms and the consequences for xenobiotic metabolism and toxicity. Studies are described in which wild-type and mutant alleles of biotransformation enzymes have been expressed in heterologous systems to study the molecular genetics and the metabolism and pharmacological or toxicological effects of xenobiotics. Furthermore, studies are described that have investigated the effects of genetic polymorphisms of biotransformation enzymes on the metabolism of drugs in humans and on the metabolism of genotoxic compounds in vivo as well. The effects of the polymorphisms are highly dependent on the enzyme systems involved and the compounds being metabolized. Several polymorphisms are described that also clearly influence the metabolism and effects of drugs and toxic compounds, in vivo in humans. Future perspectives in studies on genetic polymorphisms of biotransformation enzymes are also discussed. It is concluded that genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity. This may lead to interindividual variability in efficacy of drugs and disease susceptibility.

Disposition of 1,2-[14C]Dibromoethane in male Wistar rats.

In this study the disposition of 1,2-[14C]dibromoethane (1, 2-[14C]DBE) was investigated in male Wistar rats. 1,2-DBE is a cytotoxic and carcinogenic compound that has been used as an additive in leaded gasoline and as a fumigant. 1,2-[14C]DBE was administered orally or iv. Radioactivity was recovered (mostly within 48 hr after administration) in urine (75-82% of the dose), feces (3.2-4% of the dose), and expired air (0.53-7.2% of the dose). One hundred-sixty-eight hours after administration of 1,2-[14C]DBE, most of the radioactivity in tissues was found in the liver, lungs, and kidneys (<1% of the dose) and the red blood cells (0.3% of the dose). Identified urinary metabolites were S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, together accounting for, on average, 78% of the total amount of radioactivity in urine. In addition to S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, several compounds were anticipated as potential urinary metabolites of 1,2-DBE, i.e. S-(carboxymethyl)mercapturic acid, S-(2-hydroxyethyl)thioacetic acid, S-(2-hydroxyethyl)thiopyruvic acid, S-(carboxymethyl)thiopyruvic acid, S-(2-hydroxyethyl)thiolactic acid, and S-(carboxymethyl)thiolactic acid. All of the postulated urinary metabolites were synthesized and searched for in urine samples. None of these metabolites could be detected in urine, however. The data obtained in the present study might be useful for risk assessment and biomonitoring studies of 1,2-DBE and will also be used to further validate a physiologically based pharmacokinetic model for 1, 2-DBE in rats and humans that was recently developed.

Toxicity of fotemustine in rat hepatocytes and mechanism-based protection against it.

Fotemustine is a relatively novel DNA-alkylating 2-chloroethyl-substituted N-nitrosourea (CENU) drug, clinically used for the treatment of disseminated malignant melanoma in different visceral and non-visceral tissues. Thrombocytopenia has been observed in patients treated with fotemustine and liver and renal toxicities as well. In this study, firstly the metabolism of fotemustine was investigated in vitro and secondly the undesired cytotoxicity of fotemustine as well as different ways of protection against it. In rat hepatocytes, chosen as a model system, fotemustine was shown to cause lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, GSSG-formation and lipid peroxidation (LPO). A reactive metabolite, DEP-isocyanate, is most likely responsible for these undesired cytotoxic effects. Based on the observed cytotoxicity mechanisms, chemoprotection with several sulfhydryl-containing nucleophiles and antioxidants was investigated. The sulfhydryl nucleophiles; GSH, N-acetyl-L-cysteine (NAC) and glutathione isopropylester (GSH-IP) protected almost completely against fotemustine-induced LDH-leakage and LPO. NAC and GSH protected partly against fotemustine-induced GSH-depletion. The antioxidant, vitamin E protected completely against fotemustine-induced LPO, but only partly against fotemustine-induced LDH-leakage and not against GSH-depletion. Ebselen, a peroxidase-mimetic organoselenium compound, did not show protective effects against the cytotoxicity of fotemustine, possibly because GSH is required for the bioactivation of ebselen. It is concluded that co-administration of sulfhydryl nucleophiles, in particular NAC and GSH-IP, possibly in combination with antioxidants, such as vitamin E, are effective against the toxicity of fotemustine in vitro. It might, therefore, be worthwhile to investigate the cytoprotective potency of these agents against undesired toxicities of fotemustine in vivo as well.

Urinary thiodiacetic acid. A selective biomarker for the cytochrome P450-catalyzed oxidation of 1,2-dibromoethane in the rat.

1,2-Dibromoethane (1,2-DBE) is a carcinogenic compound that is metabolized both by cytochrome P450 (P450) and glutathione S-transferase (GST) enzymes, and that has been used by us as a model compound to study interindividual variability in biotransformation reactions. In this study, the excretion of thiodiacetic acid (TDA) and S-(2-hydroxyethyl)-N-acetyl-l-cysteine (2-HEMA) were measured in the urine of rats dosed with 1,2-DBE, and experiments were performed to investigate to what extent P450 and GST enzymes contribute to the formation of TDA. To this end, CYP2E1, the main P450 isoenzyme catalyzing the oxidation of 1,2-DBE, was inhibited using disulfiram and diallylsulfide. Significant inhibition of CYP2E1, as confirmed by inhibition of the hydroxylation of chlorzoxazone, as well as inhibition of the formation of TDA from 1,2-DBE, was observed upon pretreatment of rats with these inhibitors, indicating that the P450-catalyzed oxidation of 1,2-DBE plays the major role in the TDA formation. No significant excretion of TDA was observed after administration of intermediate products of the GST pathway [i.e. S-(2-hydroxyethyl)glutathione and 2-HEMA], indicating that the GST-catalyzed metabolism of 1,2-DBE does not contribute to a significant extent to the formation of TDA. The results of this study show that TDA is specifically formed by P450 metabolites of 1,2-DBE, whereas the conjugation of 1,2-DBE to glutathione by GST enzymes does not contribute to the formation of TDA. TDA, excreted in urine, may thus be used as a biomarker of exposure to 1,2-DBE selectively reflecting the P450-catalyzed oxidation. In addition to 2-HEMA and S-[2-(N7-guanyl)ethyl]-N-acetyl-l-cysteine, TDA may be a valuable tool for biomonitoring and mechanistic studies into the metabolism and toxicity of 1,2-DBE.

The use of human in vitro metabolic parameters to explore the risk assessment of hazardous compounds: the case of ethylene dibromide.

Ethylene dibromide (1,2-dibromoethane, EDB) is metabolized by two routes: a conjugative route catalyzed by glutathione S-transferases (GST) and an oxidative route catalyzed by cytochrome P450 (P450). The GST route is associated with carcinogenicity. An approach is presented to use human purified GST and P450 enzymes to explore the importance of these metabolic pathways for man in vivo. This strategy basically consists of four steps: (i) identification of the most important isoenzymes in vitro, (ii) scaling to rate per milligram cytosolic and microsomal protein, (iii) scaling to rate per gram liver, and (iv) incorporation of data in a physiologically based pharmacokinetic (PBPK) model. In the first step, several GST isoenzymes were shown to be active toward EDB and displayed pseudo-first-order kinetics, while the EDB oxidation was catalyzed by CYP2E1, 2A6, and 2B6, which all displayed saturable kinetics. In the second step, the predictions were in agreement with the measured activity in a batch of 21 human liver samples. In the third step, rat liver P450 and GST metabolism of EDB was predicted to be in the same range as human metabolism (expressed per gram). Interindividual differences in GST activity were modeled to determine "extreme cases." For the most active person, an approximately 1.5-fold increase of the amount of conjugative metabolites was predicted. Lastly, it was shown that the GST route, even at low concentrations, will always contribute significantly to total metabolism. In the fourth step, a PBPK model describing liver metabolism after inhalatory exposure to EDB was used. The saturation of the P450 route was predicted to occur faster in the rat than in man. The rat was predicted to have a higher turnover of EDB from both routes. Nevertheless, when all data are combined, it is crucial to recognize that the GST remains significantly active even at low EDB concentrations. The limitations and advantages of the presented strategy are discussed.

Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes.

1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies. The apparent Km and Vmax values were 3.3 mM and 0.17 pmol/min per pmol P450 for CYP2A6, 9.7 mM and 3.18 pmol/min per pmol P450 for CYP2B6 and 42 microM and 1.3 pmol/min per pmol P450 for CYP2E1, respectively. In all of 21 human liver samples studied, 1,2-DBE was oxidized with activities ranging from 22.2 to 1027.6 pmol/min per mg protein, thus showing a 46-fold inter-individual variability. The kinetics of the oxidative metabolism of 1,2-DBE to 2-BA in human liver microsomes were linear, indicating the involvement of primarily one single P450 isoenzyme. There was a tendency towards a positive correlation between the oxidative metabolism of 1,2-DBE in the human liver microsomes and the 6-hydroxylation of chlorzoxazone, a selective substrate for CYP2E1. Furthermore, the oxidative metabolism of 1,2-DBE was inhibited by the specific CYP2E1 inhibitors disulfiram (DS) and diethyldithiocarbamate (DDC). In contrast, a poor correlation was found between the immunochemically quantified amount of CYP2E1 and the microsomal chlorzoxazone 6-hydroxylation or the 1,2-DBE oxidation. The results indicate that CYP2E1 is probably the major P450 isoenzyme involved in the oxidative hepatic metabolism of 1,2-DBE in humans. The inter-individual variability in the oxidative bioactivation of 1,2-DBE in humans, largely due to inter-individual variability in the catalytic activity of hepatic CYP2E1, may have important consequences for the risk assessment for human exposure to 1,2-DBE.

In vitro inhibition of rat and human glutathione S-transferase isoenzymes by disulfiram and diethyldithiocarbamate.

The drug disulfiram (DSF, Antabuse) has been used in the therapy of alcohol abuse. It is a potent inhibitor of aldehyde dehydrogenase. Its reduced form, diethyldithiocarbamate (DDTC), and further metabolites show similar activities. DSF and DDTC have also been widely used to inhibit mixed-function oxidases. In this study, the reversible inhibition and time-dependent inactivation of the major rat and human glutathione S-transferase (GST) isoenzymes by DSF and DDTC was investigated. Reversible inhibition, using 1-chloro-2,4-dinitrobenzene as substrate for the GST alpha-, mu-, and pi-class, expressed as I50 (in microM), ranged from 5-18 (human A1-1), 43-57 (rat 4-4) and 66-83 (rat 1-1), for both DSF and DDTC. The I50 for rat GST theta, using 1,2-epoxy-3-(p-nitrophenoxy)-propane as substrate, was 350 microM for DDTC. The other GSTs were significantly less sensitive to inhibition. The major part of reversible inhibition by DSF was shown to be due to DDTC, formed rapidly upon reduction of DSF by the glutathione (GSH) present in the assay to measure GST activity. The oxidized GSH formed upon reduction of DSF might also have made a minor contribution to reversible inhibition. The rat and human pi-class was, by far, the most sensitive class for time-dependent inactivation by DSF, but no such inactivation was observed for any of the GSTs by DDTC. Moderate susceptibility to inactivation by DSF of all the other GSTs was observed, except for human A2-2, which does not possess a cysteine residue. Consistent with the assumption that a thiol residue is involved in this inactivation, a significant part of the activity could be restored by treatment of the inactivated GST with GSH or dithiotreitol.

Molecular mechanisms of toxic effects of fotemustine in rat hepatocytes and subcellular rat liver fractions.

Fotemustine is a clinically used DNA-alkylating 2-chloro-ethyl-substituted N-nitrosourea, which sometimes shows signs of haematotoxicity and reversible liver and renal toxicity as toxic side-effects. Mechanistic data on these side-effects are scarce and incomplete. In this study, firstly the cytotoxicity of fotemustine in freshly isolated rat hepatocytes was investigated and secondly the metabolism of fotemustine and possible mechanisms involved in the observed cytotoxicity. Fotemustine caused concentration- and time-dependent cytotoxic effects in rat hepatocytes. Extensive GSH-depletion and formation of GSSG were first observed, followed by lipid peroxidation and finally by cell death measured as LDH-leakage. 2-Chloroethyl analogues of fotemustine, which in contrast to fotemustine have no carbamoylating potency, were not toxic to rat hepatocytes. The data suggest that the cytotoxicity of fotemustine is resulting from its reactive decomposition product, DEP-isocyanate. GSH-conjugation of DEP-isocyanate was shown to protect against the cytotoxicity of fotemustine, however, only temporary and not completely. Synthetical DEP-SG, the GSH-conjugate of DEP-isocyanate, was also toxic to rat hepatocytes, albeit to a significantly lesser extent than fotemustine. In rat liver microsomes, no fotemustine-induced LPO was observed, suggesting that reactive decomposition products of fotemustine do not directly cause peroxidation of cellular membranes. Fotemustine did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase, GSSG-reductase and GSH S-transferases. Thus, direct effects on these antioxidant enzymes are not likely to explain the cytotoxic effects of fotemustine in hepatocytes. In conclusion, it is proposed that the cytotoxicity of fotemustine in rat hepatocytes is caused by rapid and extensive depletion of GSH by DEP-isocyanate, a reactive decomposition product of fotemustine, consequently hampering the endogenous protection against its own toxicity. Knowledge of molecular mechanisms of the cytotoxicity of fotemustine may contribute to a more rational design of selective protection against toxic side-effects which occur upon therapy of patients with fotemustine.

Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats.

The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2-BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF-induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2-DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes.

Polymorphism in the glutathione conjugation activity of human erythrocytes towards ethylene dibromide and 1,2-epoxy-3-(p-nitrophenoxy)-propane.

In this study a polymorphism in the conjugating activity of human erythrocyte cytosol towards the dihaloethane, ethylene dibromide (EDB; 1,2-dibromoethane) was found. Two out of 12 human erythrocyte cytosols did not catalyze the formation of glutathione (GSH) conjugates of [1,2-14C]EDB. Ten cytosols formed the S,S'-ethylenebis(GSH) conjugate at a rate ranging from 0.5 to 3.2 (mean 1.76 +/- 0.95) pmol min-1 (mg protein)-1. The activity of the cytosols towards EDB was compared with the activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) and 1-chloro-2,4-dinitrobenzene (CDNB). The GSH conjugates formed from EDB, EPNP and CDNB were all quantified by HPLC. Every cytosol was active with the classical GST substrate CDNB (2.04 +/- 0.74 nmol min-1 (mg protein)-1). The two samples not showing any detectable activity towards EDB were also inactive towards EPNP: The activity towards EDB correlated significantly with EPNP (rs = 0.90, P < 0.005; Spearman's rank correlation), but not with CDNB (rs = 0.36, P > 0.10). In the incubations with EPNP, the alpha-, mu-, and pi- class glutathione S-transferase (GST) inhibitor S-hexyl(GSH) was included, indicating that the class-theta GST is the principal GST class conjugating EDB in erythrocyte cytosol. The apparent polymorphism of GST-theta which has recently been recognized to be crucial for several mono- and dihalomethanes, will thus also have considerable implications for the risk assessment of EDB.