Reversing diet-induced metabolic dysregulation by diet switching leads to altered hepatic de novo lipogenesis and glycerolipid synthesis

In humans, low-energy diets rapidly reduce hepatic fat and improve/normalise glycemic control. Due to difficulties in obtaining human liver, little is known about changes to the lipid species and pathway fluxes that occur under these conditions. Using a combination of stable isotope, and targeted metabolomic approaches we investigated the acute (7–9 days) hepatic effects of switching high-fat high-sucrose diet (HFD) fed obese mice back to a chow diet. Upon the switch, energy intake was reduced, resulting in reductions of fat mass and hepatic triacyl- and diacylglycerol. However, these parameters were still elevated compared to chow fed mice, thus representing an intermediate phenotype. Nonetheless, glucose intolerance and hyperinsulinemia were completely normalized. The diet reversal resulted in marked reductions in hepatic de novo lipogenesis when compared to the chow and HFD groups. Compared with HFD, glycerolipid synthesis was reduced in the reversal animals, however it remained elevated above that of chow controls, indicating that despite experiencing a net loss in lipid stores, the liver was still actively esterifying available fatty acids at rates higher than that in chow control mice. This effect likely promotes the re-esterification of excess free fatty acids released from the breakdown of adipose depots during the weight loss period.

The influence of dietary fatty acid and fasting on the hepatic lipid metabolism of barramundi (Lates calcarifer)

For many fish species, dietary fish oil (FO) has been substituted with other oils such as poultry oil (PO) without affecting growth performance. However, in barramundi, the mechanisms by which fatty acid metabolism is regulated are poorly understood, and the effects of FO substitution are unknown. This study defined changes in the expression of genes controlling the metabolism of fatty acids in barramundi over a 24-h time period after a single meal. From one to 12 h after a single feeding event, the expression of fatty acid synthesis genes in the liver was upregulated, while genes involved in the β-oxidation showed minimal alteration. However, the expression of β-oxidation genes was significantly correlated with the expression of genes regulating fatty acid synthesis. In a second experiment, the changes in liver fatty acid composition and gene expression were defined after FO was substituted with PO. Liver fatty acid profile reflected the diet composition, with some subtle exceptions supporting the enrichment of certain long-chain polyunsaturated fatty acids in the liver. The fish from all experimental groups preferentially retained more docosahexaenoic acid than eicosapentaenoic acid in the liver, suggesting a bioconversion of this fatty acid to intermediate fatty acids. Replacement of FO with PO significantly regulated genes controlling both fatty acid synthesis and catabolism pathways, potentially related to a higher percentage of monounsaturated fatty acids, in the livers of fish fed these diets. The results demonstrated that diet composition significantly altered the lipid metabolism in barramundi and that there was a balance between direct dietary effects and endogenous synthetic capacity.

Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multi-vesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of tx mice

Tanis: a link between type 2 diabetes and inflammation?

Here we describe a novel protein, which we have named Tanis, that is implicated in type 2 diabetes and inflammation. In Psammomys obesus, a unique polygenic animal model of type 2 diabetes and the metabolic syndrome, Tanis is expressed in the liver in inverse proportion to circulating glucose (P = 0.010) and insulin levels (P = 0.004) and in direct proportion with plasma triglyceride concentrations (P = 0.007). Hepatic Tanis gene expression was markedly increased (3.1-fold) after a 24-h fast in diabetic but not in nondiabetic P. obesus. In addition, glucose inhibited Tanis gene expression in cultured hepatocytes (P = 0.006) as well as in several other cell types (P = 0.001–0.011). Thus, Tanis seems to be regulated by glucose and is dysregulated in the diabetic state. Yeast-2 hybrid screening identified serum amyloid A (SAA), an acute-phase inflammatory response protein, as an interacting protein of Tanis, and this was confirmed by Biacore experiments. SAA and other acute-phase proteins have been the focus of recent attention as risk factors for cardiovascular disease, and we contend that Tanis and its interaction with SAA may provide a mechanistic link among type 2 diabetes, inflammation, and cardiovascular disease.

Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Elevation in tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells

Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.

Effects of alternative dietary lipid sources on performance, tissues chemical composition, mitochondrial fatty acid oxidation capabilities, and sensory characteristics in brown trout (Salmo trutta L.)

The efficiency of five dietary lipid sources (fish oil as control—C; canola oil—CO; poultry fat—PF; pork lard—PL; and oleine oil—OO) were evaluated in juvenile brown trout (58.4±0.7 g) in an experiment conducted over 70 days at 14.6±0.4 °C. The best growth was observed in fish fed the C diet whereas the PL diet fed fish had the best feed utilization. Significant differences in carcass and muscle proximate composition, but not in liver, were noted among fish fed the different dietary treatments. The fatty acid composition of muscle largely reflected that of the diets, while total cholesterol was not affected. The atherogenicity and the thrombogenicity qualities of the trout flesh were modified by the lipid sources. Sensory analysis did not show any significant differences among the cooked fillets with respect to dietary treatments, while in uncooked products, some significant differences were observed. The carnitine palmitoyltransferase I and II (CPT-I and CPT-II) activities of liver and white muscle were assayed for a better understanding of the potential β-oxidation capability of the different dietary lipid sources. The hepatic, but not white muscle CPT-I and CPT-II activities were affected by dietary treatments. This study showed that alternative lipid sources could be used effectively for oil coating extruded diets for brown trout.

A mechanistic study on altered pharmacokinetics of irinotecan by St. Johns wort

Irinotecan (CPT-11) is an important anticancer drug in management of advanced colon cancer. A marked protective effect on CPT-11-induced blood and gastrointestinal toxicity is obtained by combination of St. John's wort (SJW) in recent clinical and rat studies. However, the mechanism is unclear. This study aimed to explore the effects of SJW on the pharmacokinetics of CPT-11 and its major metabolites (SN-38 and SN-38 glucuronide) in rats and the underlying mechanisms using several in vitro models. Short-term (3 days) and long-term (14 days) pretreatment with SJW were conducted in rats to examine the effects of co-administered SJW on the plasma pharmacokinetics of CPT-11, SN-38 and SN- 38 glucuronide. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were utilized to study the effects of aqueous and ethanolic extracts (AE and EE) and major active components (hyperforin, hypericin and quercetin) of SJW on CPT-11 and SN-38 metabolism and intracellular accumulation. Co-administered SJW for consecutive 14 days significantly decreased the initial plasma concentration (C0) of CPT-11, the area under the concentration-time curve (AUC0-10hr) and maximum plasma concentration (Cmax) of SN-38. The ethanolic extracts (EE) of SJW at 5 μ g/ml significantly decreased SN-38 glucuronidation by 45% (P < 0.05) in rat hepatic microsomes. Pre-incubation of aqueous SJW extracts (AE) at 10 g/ml, SJW EE at 5 μg/ml, and quercetin at 10μ M significantly increased the glucuronidation of SN-38 in H4- II-E cells. A 2-hr pre-incubation of quercetin (100μ M) significantly increased the intracellular accumulation of CPT-11 (P < 0.05). However, pre-incubation of hypericin (20 nM and 200 nM) and hyperforin (1μ M) significantly decreased the intracellular accumulation of CPT-11. In addition, pre-incubation of hypericin, SJW EE and quercetin significantly increased the intracellular accumulation of SN-38. Aqueous and ethanolic SJW extracts and its major active components did not alter the plasma protein binding of CPT-11 and SN-38. These results indicated that the aqueous and ethanolic extracts of SJW and its major active components could markedly alter glucuronidation of SN-38 and intracellular accumulation of CPT-11 and SN-38, which probably provides partial explanation for the altered plasma pharmacokinetics of CPT-11 and SN-38 and the antagonizing effects on the toxicities of CPT-11. Further studies are needed to explore the role of both pharmacokinetic and pharmacodynamic components in the protective effect of SJW against the toxicities of CPT-11.

Effect of carbohydrate ingestion on glucose kinetics during exercise in the heat

Six endurance-trained men [peak oxygen uptake (VO₂) = 4.58 ± 0.50 (SE) l/min] completed 60 min of exercise at a workload requiring 68 ± 2% peak VO₂ in an environmental chamber maintained at 35°C (<50% relative humidity) on two occasions, separated by at least 1 wk. Subjects ingested either a 6% glucose solution containing 1 µCi [3-³H]glucose/g glucose (CHO trial) or a sweet placebo (Con trial) during the trials. Rates of hepatic glucose production [HGP = glucose rate of appearance (R_a) in Con trial] and glucose disappearance (R_d), were measured using a primed, continuous infusion of [6,6-^₂H]glucose, corrected for gut-derived glucose (gut R_a) in the CHO trial. No differences in heart rate, VO₂, respiratory exchange ratio, or rectal temperature were observed between trials. Plasma glucose concentrations were similar at rest but increased (P < 0.05) to a greater extent in the CHO trial compared with the Con trial. This was due to the absorption of ingested glucose in the CHO trial, because gut R_a after 30 and 50 min (16 ± 5 µmol · kg^-1 · min^-1) was higher (P < 0.05) compared with rest, whereas HGP during exercise was not different between trials. Glucose R_d was higher (P < 0.05) in the CHO trial after 30 and 50 min (48.0 ± 6.3 vs 34.6 ± 3.8 µmol · kg^-1 · min^-1, CHO vs. Con, respectively). These results indicate that ingestion of carbohydrate, at a rate of ~1.0 g/min, increases glucose Rd but does not blunt the rise in HGP during exercise in the heat.

Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson's disease

Background and Aim: The toxic milk (tx) mouse is a non-fatal animal model for the metabolic liver disorder, Wilson's disease. The tx mouse has a mutated gene for a copper-transporting protein, causing early copper accumulation in the liver and late accumulation in other tissues. The present study investigated the efficacy of liver cell transplantation (LCT) to correct the tx mouse phenotype.

Methods: Congenic hepatocytes were isolated and intrasplenically transplanted into 3–4-month-old tx mice, which were then placed on various copper-loaded diets to examine its influence on repopulation by transplanted cells. The control animals were age-matched untransplanted tx mice. Liver repopulation was determined by comparisons of restriction fragment length polymorphism ratios (DNA and mRNA), and copper levels were measured by atomic absorption spectroscopy.

Results: Repopulation in recipient tx mice was detected in 11 of 25 animals (44%) at 4 months after LCT. Dietary copper loading (whether given before or after LCT, or both) provided no growth advantage for donor cells, with similar repopulation incidences in all copper treatment groups. Overall, liver copper levels were significantly lower in repopulated animals (538 ± 68 µg/g, n = 11) compared to non-repopulated animals (866 ± 62 µg/g, n = 14) and untreated controls (910 ± 103 µg/g, n = 6; P < 0.05). This effect was also seen in the kidney and spleen. Brain copper levels remained unchanged.

Conclusion: Transplanted liver cells can proliferate and correct a non-fatal metabolic liver disease, with some restoration of hepatic copper homeostasis after 4 months leading to reduced copper levels in the liver and extrahepatic tissues, but not in the brain.

Impact of in vivo fatty acid oxidation blockade on glucose turnover and muscle glucose metabolism during low-dose AICAR infusion

AMPK plays a central role in influencing fuel usage and selection. The aim of this study was to analyze the impact of low-dose AMP analog 5-aminoimidazole-4-carboxamide-1-ß-D-ribosyl monophosphate (ZMP) on whole body glucose turnover and skeletal muscle (SkM) glucose metabolism. Dogs were restudied after prior 48-h fatty acid oxidation (FAOX) blockade by methylpalmoxirate (MP; 5 x 12 hourly 10 mg/kg doses). During the basal equilibrium period (0–150 min), fasting dogs (n = 8) were infused with [3-³H]glucose followed by either 2-h saline or AICAR (1.5–2.0 mg·kg^–1·min^–1) infusions. SkM was biopsied at completion of each study. On a separate day, the same protocol was undertaken after 48-h in vivo FAOX blockade. The AICAR and AICAR + MP studies were repeated in three chronic alloxan-diabetic dogs. AICAR produced a transient fall in plasma glucose and increase in insulin and a small decline in free fatty acid (FFA). Parallel increases in hepatic glucose production (HGP), glucose disappearance (Rd tissue), and glycolytic flux (GF) occurred, whereas metabolic clearance rate of glucose (MCR_g) did not change significantly. Intracellular SkM glucose, glucose 6-phosphate, and glycogen were unchanged. Acetyl-CoA carboxylase (ACC~pSer²²¹) increased by 50%. In the AICAR + MP studies, the metabolic responses were modified: the glucose was lower over 120 min, only minor changes occurred with insulin and FFA, and HGP and Rd tissue responses were markedly attenuated, but MCR_g and GF increased significantly. SkM substrates were unchanged, but ACC~pSer²²¹ rose by 80%. Thus low-dose AICAR leads to increases in HGP and SkM glucose uptake, which are modified by prior FA_ox blockade.

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.

Drug-herb interactions: eliminating toxicity with hard drug design.

By searching the literatures, it was found that a total of 32 drugs interacting with herbal medicines in humans. These drugs mainly include anticoagulants (warfarin, aspirin and phenprocoumon), sedatives and antidepressants (midazolam, alprazolam and amitriptyline), oral contraceptives, anti-HIV agents (indinavir, ritonavir and saquinavir), cardiovascular drug (digoxin), immunosuppressants (cyclosporine and tacrolimus) and anticancer drugs (imatinib and irinotecan). Most of them are substrates for cytochrome P450s (CYPs) and/or P-glycoprotein (PgP) and many of which have narrow therapeutic indices. However, several drugs including acetaminophen, carbamazepine, mycophenolic acid, and pravastatin did not interact with herbs. Both pharmacokinetic (e.g. induction of hepatic CYPs and intestinal PgP) and/or pharmacodynamic mechanisms (e.g. synergistic or antagonistic interaction on the same drug target) may be involved in drug-herb interactions, leading of altered drug clearance, response and toxicity. Toxicity arising from drug-herb interactions may be minor, moderate, or even fatal, depending on a number of factors associated with the patients, herbs and drugs. Predicting drug-herb interactions, timely identification of drugs that interact with herbs, and therapeutic drug monitoring may minimize toxic drug-herb interactions. It is likely to predict pharmacokinetic herb-drug interactions by following the pharmacokinetic principles and using proper models that are used for predicting drug-drug interactions. Identification of drugs that interact with herbs can be incorporated into the early stages of drug development. A fourth approach for circumventing toxicity arising from drug-herb interactions is proper design of drugs with minimal potential for herbal interaction. So-called ”hard drugs” that are not metabolized by CYPs and not transported by PgP are believed not to interact with herbs due to their unique pharmacokinetic properties. More studies are needed and new approached are required to minimize toxicity arising from drug-herb interactions.

Metabolism and transport of oxazphosphorines and the clinical implications

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity. CPA is widely used as an anticancer drug, an immunosuppressant, and for the mobilization of hematopoetic progenitor cells from the bone marrow into peripheral blood prior to bone marrow transplantation for aplastic anemia, leukemia, and other malignancies. New oxazaphosphorines derivatives have been developed in an attempt to improve selectivity and response with reduced toxicity. These derivatives include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). This review highlights the metabolism and transport of these oxazaphosphorines (mainly CPA and IFO, as these two oxazaphosphorine drugs are the most widely used alkylating agents) and the clinical implications. Both CPA and IFO are prodrugs that require activation by hepatic cytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxic nitrogen mustards capable of reacting with DNA molecules to form crosslinks and lead to cell apoptosis and/or necrosis. Such prodrug activation can be enhanced within tumor cells by the CYP-based gene directed-enzyme prodrug therapy (GDEPT) approach. However, those newly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC 612567 and NSC 613060, do not need hepatic activation. They are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by plain phosphodiesterase (PDEs) in plasma and other tissues or by the high-affinity nuclear 3'-5' exonucleases associated with DNA polymerases, such as DNA polymerases and ε. The alternative CYP-catalyzed inactivation pathway by N-dechloroethylation generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Various aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs) are involved in the detoxification of oxazaphosphorine metabolites. The metabolism of oxazaphosphorines is auto-inducible, with the activation of the orphan nuclear receptor pregnane X receptor (PXR) being the major mechanism. Oxazaphosphorine metabolism is affected by a number of factors associated with the drugs (e.g., dosage, route of administration, chirality, and drug combination) and patients (e.g., age, gender, renal and hepatic function). Several drug transporters, such as breast cancer resistance protein (BCRP), multidrug resistance associated proteins (MRP1, MRP2, and MRP4) are involved in the active uptake and efflux of parental oxazaphosphorines, their cytotoxic mustards and conjugates in hepatocytes and tumor cells. Oxazaphosphorine metabolism and transport have a major impact on pharmacokinetic variability, pharmacokinetic-pharmacodynamic relationship, toxicity, resistance, and drug interactions since the drug-metabolizing enzymes and drug transporters involved are key determinants of the pharmacokinetics and pharmacodynamics of oxazaphosphorines. A better understanding of the factors that affect the metabolism and transport of oxazaphosphorines is important for their optional use in cancer chemotherapy.

Pharmacokinetic mechanisms for reduced toxicity of irinotecan by coadministered Thalidomide

The clinical use of irinotecan (CPT-11) is hindered by dose-limiting diarrhea and myelosuppression. Recent clinical studies indicate that thalidomide, a known tumor necrosis factor-alpha inhibitor, ameliorated the toxicities induced by CPT-11. However, the mechanisms for this are unknown. This study aimed to investigate whether combination of thalidomide modulated the toxicities of CPT-11 using a rat model and the possible role of the altered pharmacokinetic component in the toxicity modulation using in vitro models. The toxicity model was constructed by treatment of healthy rats with CPT-11 at 60 mg/kg per day by intravenous (i.v.) injection. Body weight, acute and delayed-onset diarrhea, blood cell counts, and macroscopic and microscopic intestinal damages were monitored in rats treated with CPT-11 alone or combined therapy with thalidomide at 100 mg/kg administered by intraperitoneal (i.p.) injection. Single dose and 5-day multiple-dose studies were conducted in rats to examine the effects of concomitant thalidomide on the plasma pharmacokinetics of CPT-11 and its major metabolites SN-38 and SN-38 glucuronide (SN-38G). The effect of CPT-11 on thalidomide's pharmacokinetics was also checked. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were used to study the in vitro metabolic interactions between these two drugs. H4-II-E cells were also used to investigate the effect of thalidomide and its hydrolytic products on the transport of CPT-11 and SN-38. In addition, the effect of thalidomide and its hydrolytic products on rat plasma protein binding of CPT-11 and SN-38 was examined. Administration of CPT-11 by i.v. for 4 consecutive days to rats induced significant body weight loss, decrease in neutrophil and lymphocyte counts, severe acute- and delayed-onset diarrhea, and intestinal damages. These toxicities were alleviated when CPT-11 was combined with thalidomide. In both single-dose and 5-day multiple-dose pharmacokinetic study, coadministered thalidomide significantly increased the area under the plasma concentration-time curve (AUC) of CPT-11, but the AUC and elimination half-life (t(1/2)) of SN-38 were significantly decreased. However, CPT-11 did not significantly alter the pharmacokinetics of thalidomide. Thalidomide at 25 and 250 microM and its hydrolytic products at a total concentration of 10 microM had no significant effect on the plasma protein binding of CPT-11 and SN-38, except for that thalidomide at 250 microM caused a significant increase in the unbound fraction (f(u)) of CPT-11 by 6.7% (P < 0.05). The hydrolytic products of thalidomide (total concentration of 10 microM), but not thalidomide, significantly decreased CPT-11 hydrolysis by 16% in rat liver microsomes (P < 0.01). The formation of both SN-38 and SN-38G from CPT-11, SN-38 glucuronidation, or intracellular accumulation of both CPT-11 and SN-38 in H4-II-E cells followed Michaelis-Menten kinetics with the one-binding site model being the best fit for the kinetic data. Coincubation or 2-hr preincubation of thalidomide at 25 microM and 250 microM and its hydrolytic products at 10 microM did not show any significant effects on CPT-11 hydrolysis and SN-38 glucuronidation. However, preincubation of H4-II-E cells with thalidomide (250 microM), its hydrolytic products (total concentration of 10 microM), or phthaloyl glutamic acid (one major thalidomide hydrolytic product, 10 microM) significantly increased the intracellular accumulation of SN-38, but not CPT-11 (P < 0.01). The dose-limiting toxicities of CPT-11 were alleviated by combination with thalidomide in rats and the pharmacokinetic modulation by thalidomide may partially explain its antagonizing effects on the toxicities of CPT-11. The hydrolytic products of thalidomide, instead of the parental drug, modulated the hepatic hydrolysis of CPT-11 and intracellular accumulation of SN-38, probably contributing to the altered plasma pharmacokinetics of CPT-11 and SN-38. Further studies are needed to explore the role of both pharmacokinetics and pharmacodynamic components in the protective effect of thalidomide against the toxicities of CPT-11.