Evaluation of Patients with Heart Failure to Determine Eligibility for Treatment with Sacubitril/Valsartan: Insights from a Veterans Administration Healthcare System.

STUDY OBJECTIVE: Despite evidence that supports the use of sacubitril/valsartan - the first angiotensin II receptor blocker-neprilysin inhibitor - for mortality reduction in patients with heart failure (HF), it remains underprescribed. The objective of this study was to evaluate eligibility for initiation of sacubitril/valsartan treatment in patients with HF within the largest Veterans Administration healthcare system in the United States. DESIGN: Cross-sectional study. SETTING: Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS). PATIENTS: A total of 2985 patients with a HF diagnosis who were alive as of November 1, 2017. MEASUREMENTS AND MAIN RESULTS: Eligibility for sacubitril/valsartan initiation was based on inclusion and exclusion criteria from the Prospective Comparison of Angiotensin Receptor-Neprilysin Inhibitor with Angiotensin-Converting-Enzyme Inhibitor to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial and the VA Criteria for Use. The proportion of eligible patients was estimated, and characteristics of eligible patients were compared with those in the PARADIGM-HF trial. Of the 2985 patients with HF who were alive as of November 1, 2017, 965 (32.3%) had HF with reduced ejection fraction (HFrEF). Of these patients with HFrEF, 263 (27.3%) fulfilled eligibility criteria and were considered candidates for sacubitril/valsartan initiation. Of the 702 patients who did not fulfil eligibility criteria, the most common reasons were New York Heart Association functional class I (35.3%) and B-type natriuretic peptide level of 100 pg/ml or lower (22.2%). Compared with patients in the PARADIGM-HF trial, VAGLAHS patients were older (70.4 vs 63.8 yrs) and more likely to be male (98.5% vs 79.0%), and a higher proportion had New York Heart Association functional class III symptoms (35.4% vs 23.1%). Of the 965 patients with HFrEF, 34 (3.5%) had an active sacubitril/valsartan prescription as of November 1, 2017, of whom 27 (79.4%) did not meet criteria. CONCLUSION: Whereas 27% of patients with HFrEF were eligible to initiate sacubitril/valsartan, only 3.5% of these patients were prescribed the medication. Although sacubitril/valsartan reduced morbidity and mortality in clinical trials, it remains underused within this VA healthcare system. This analysis provides important insights into the VA and other healthcare systems regarding the opportunity for optimizing guideline-directed HF therapy.

Disrupting P-glycoprotein function in clinical settings: what can we learn from the fundamental aspects of this transporter?

P-glycoprotein is one of the most well-studied drug transporters, significant for its role in cancer multiple drug resistance. However, using P-gp inhibitors with the aim of enhancing the therapeutic efficacy of anti-cancer drugs has led to disappointing outcomes. Furthermore, several lead compounds suggested by in vitro and pre-clinical studies have shown variable pharmacokinetics and therapeutic efficacies when applied in the clinical setting. This review will highlight the need to revisit a sound approach to better design and apply P-gp inhibitors in light of safety and efficacy. Challenges confronting the issue hinge upon myriad studies that do not necessarily represent the heterogeneous target population of this therapeutic approach. The application of P-gp modulators has also been complicated by the promiscuous substrate-binding behaviour of P-gp, as well as toxicities related to its intrinsic presence in healthy tissue. This review capitalizes on information spanning genetics, energetics, and pharmacology, bringing to light some fundamental aspects that ought to be reconsidered in order to improve upon and design the next generation of P-gp inhibitors.

Autologous Adipose-Derived Mesenchymal Stromal Cells for the Treatment of Psoriasis Vulgaris and Psoriatic Arthritis: A Case Report.

Psoriasis is a dermatologic disease of immune origins with no definitive cure. We report the Makati Medical Center experience of utilizing autologous mesenchymal stromal cells (MSCs) for one patient with psoriasis vulgaris (PV) and another with psoriatic arthritis (PA). Patients were educated and gave informed consent, according to the principles of the Declaration of Helsinki. The protocol was approved by the Cellular Transplantation Ethics Committee of the Makati Medical Center. Autologous MSCs were cultured from lipoaspirate and expanded in a clean room class 100 facility (Cellular Therapeutics Center, Makati Medical Center). MSCs were infused intravenously at a dose of 0.5-3.1 million cells/kg after complying with quality control parameters. Psoriasis area and severity index (PASI) evaluations were conducted by third-party dermatologists. The PA patient, who was previously unresponsive to standard treatment modalities, demonstrated a decrease in PASI (from 21.6 to 9.0, mild state after two infusions). No improvements were noted in joint pain until further treatment with etanercept and infliximab. The PV patient, who was previously dependent on methotrexate, showed a decrease in PASI from 24.0 to 8.3 after three infusions; this clinical improvement was sustained for 292 days (9.7 months) without methotrexate. The PV patient illustrated a marginal reduction in serum tumor necrosis factor-α (TNF-α), while significant (3.5- to 5-fold) decreases in reactive oxygen species (ROS) activity were noted. The ROS levels correlated with the clinical improvement of the PV patient. No serious adverse events were noted for either patient as a result of MSC infusions. This report demonstrates safe and tolerable transplantation of autologous MSCs for the treatment of psoriasis and warrants large clinical studies to investigate the long-term safety and efficacy of this approach.

Modeling cyclosporine A inhibition of the distribution of a P-glycoprotein PET ligand, 11C-verapamil, into the maternal brain and fetal liver of the pregnant nonhuman primate: impact of tissue blood flow and site of inhibition.

UNLABELLED: Through PET imaging, our laboratory has studied the dynamic biodistribution of (11)C-verapamil, a P-gp substrate, in the nonhuman primate Macaca nemestrina. To gain detailed insight into the kinetics of verapamil transport across the blood-brain barrier (BBB) and the blood-placental barrier (BPB), we analyzed these dynamic biodistribution data by compartmental modeling. METHODS: Thirteen pregnant macaques (gestational age, 71-159 d; term, ∼172 d) underwent PET imaging with (11)C-verapamil before and during infusion (6, 12, or 24 mg/kg/h) of cyclosporine A (CsA, a P-glycoprotein [P-gp] inhibitor). Dynamic (11)C-verapamil brain or fetal liver (reporter of placental P-gp function) activity was assessed by a 1- or 2-tissue-compartment model. RESULTS: The 1-tissue-compartment model best explained the observed brain and fetal liver distribution of (11)C-radioactivity. When P-gp was completely inhibited, the brain and fetal liver distribution clearance (K1) approximated tissue blood flow (Q); that is, extraction ratio (K1/Q) was approximately 1, indicating that in the absence of P-gp function, the distribution of (11)C-verapamil radioactivity into these compartments is limited by blood flow. The potency of CsA to inhibit P-gp was tissue-independent (maternal BBB half-maximal inhibitory concentration [IC50], 5.67 ± 1.07 µM, vs. BPB IC50, 7.63 ± 3.16 µM). CONCLUSION: We propose that on deliberate or inadvertent P-gp inhibition, the upper boundary of increase in human brain (or fetal) distribution of lipophilic drugs such as verapamil will be limited by tissue blood flow. This finding provides a means to predict the magnitude of P-gp-based drug interactions at the BBB and BPB when only the baseline distribution of the drug (i.e., in the absence of P-gp inhibition) across these barriers is available through PET. Our data suggest that P-gp-based drug interactions at the human BBB and BPB can be clinically significant, particularly for those P-gp substrate drugs for which P-gp plays a significant role in excluding the drug from these privileged compartments.

Autologous dendritic cell transplantation as an adjuvant treatment in pleomorphic lung carcinoma

We embarked on autologous dendric cells (DC) transplantation as an adjuvant therapy with chemotherapy in a pleomorphic lung carcinoma patient. DC were isolated from PBMC and primed with the autologous tumor lysate. No adverse event was noted in DC transplantation. DC administration also correlated with immunomodulation, as evidenced by an approximately 5-fold increase in serum interferon gamma after 2 months. The utility of autologous DC transplantation may offer a clinical benefit with virtually no adverse event.

Simultaneous PET imaging of P-glycoprotein inhibition in multiple tissues in the pregnant nonhuman primate.

UNLABELLED: Studies in rodents indicate that the disruption of P-glycoprotein (P-gp) function increases drug distribution into the developing fetus and organs such as the brain. To simultaneously and serially evaluate the effect of P-gp activity and inhibition on the tissue distribution of drugs in a more representative animal model, we tested the feasibility of conducting whole-body PET of the pregnant nonhuman primate (Macaca nemestrina). We used (11)C-verapamil as the prototypic P-gp substrate and cyclosporine A (CsA) as the prototypic inhibitor. METHODS: Four pregnant macaques (gestational age, 145-159 d; gestational term, 172 d) were imaged after the intravenous administration of (11)C-verapamil (30-72 MBq/kg) before and during intravenous infusion of CsA (12 or 24 mg/kg/h, n = 2 each). The content of verapamil and its metabolites in plasma samples was determined using a rapid solid-phase extraction method. The plasma and tissue time-radioactivity concentration curves of (11)C were integrated over 0-9 min after each verapamil injection. The tissue or arterial plasma area under the time-concentration curve (AUC(tissue)/AUC(plasma)) served as a measure of the tissue distribution of (11)C radioactivity. CsA effect on (11)C radioactivity distribution was interpreted as P-gp inhibition. The change in the fetal liver AUC ratio served as a reporter of placental P-gp inhibition. RESULTS: CsA effect on tissue distribution of (11)C radioactivity (AUC ratios) did not increase with the mean blood concentration of CsA, indicating a near-maximal P-gp inhibition. CsA increased maternal brain and fetal liver distribution of (11)C radioactivity by 276% +/- 88% (P < 0.05) and 122% +/- 75% (P < 0.05), respectively. Changes in other measured tissues were not statistically significant. CONCLUSION: These data demonstrate for the first time, to our knowledge, the feasibility of simultaneous, serial, noninvasive imaging of P-gp activity and inhibition in multiple maternal organs and the placenta in the nonhuman primate. Our findings, consistent with previous data in rodents, indicate that the activity of P-gp in the placenta and the blood-brain barrier is high and that the inhibition of P-gp facilitates drug distribution across these barriers.

Pregnancy does not increase CYP3A or P-glycoprotein activity in the non-human primate, Macaca nemestrina.

Plasma concentrations of protease inhibitors are lower in pregnant women than in nonpregnant women or men. Using nelfinavir as a model protease inhibitor, we have shown that this phenomenon can be reproduced in a representative non-human primate model, Macaca nemestrina (J Pharmacol Exp Ther 329:1016-1022, 2009). Nelfinavir is cleared from the body predominantly by CYP3A metabolism and P-glycoprotein (P-gp) efflux. Therefore, using midazolam (MDZ) as a CYP3A probe and digoxin (DIG) as a P-gp probe, we determined the antepartum (73-118 days) and postpartum (61-130 days) in vivo intestinal and hepatic CYP3A or P-gp activity in the macaque. Although the systemic clearance of MDZ was significantly increased ( approximately 70%) during pregnancy after intra-arterial (IA) administration of the drug ((15)N-labeled MDZ; 40 microg/kg), pregnancy did not affect the oral clearance of the drug administered simultaneously (1 mg/kg p.o.) with the IA dose. In vitro studies in hepatic and intestinal S-9 fractions indicated no effect of pregnancy on CYP3A activity or protein expression in the small intestine or liver. In contrast, neither the oral (100 microg/kg) nor the IA (10 microg/kg) clearance of DIG was significantly altered by pregnancy, indicating no effect of pregnancy on P-gp activity. Assuming that MDZ and DIG are selective substrates of the macaque CYP3A enzymes and P-gp, respectively, these results suggest that factors other than increased CYP3A or P-gp activity contribute to the increased clearance of protease inhibitors during M. nemestrina pregnancy.

As in humans, pregnancy increases the clearance of the protease inhibitor nelfinavir in the nonhuman primate Macaca nemestrina.

The apparent oral clearance of protease inhibitors (PIs) is increased in pregnant women. Although this phenomenon is reproduced in the mouse, because of the multiplicity of mouse cytochrome P450 isoforms, lack of information on their substrate and inhibitor selectivity, and lack of reagents (e.g., antibodies, purified protein), it is difficult to study the mechanistic basis of this phenomenon in this animal model. To investigate the mechanistic basis of this phenomenon in a more representative model, the nonhuman primate, we first determined whether this phenomenon could be reproduced in Macaca nemestrina, using nelfinavir as a model PI. Consistent with the human and mouse studies, we found that the apparent oral clearance of nelfinavir (NFV) in the macaques was significantly increased (3.14-fold) antepartum (n = 3) versus postpartum (n = 4). This increased apparent oral clearance was a result of an increased systemic clearance (1.9-fold) and a decreased bioavailability (approximately 45%) during pregnancy. In vitro, pregnancy significantly enhanced the rate of NFV depletion in hepatic, but not intestinal S-9 fractions. Human CYP3A inhibitors erythromycin (0.5 mM), ketoconazole (0.5 microM), and troleandomycin (0.01-1 mM), but not the CYP2C inhibitor, sulfaphenazole (3 microM), significantly inhibited the depletion of NFV in hepatic S-9 fractions and expressed rhesus CYP3A64 enzyme. Based on these data, we conclude that increased hepatic activity of NFV-metabolizing enzymes (perhaps CYP3A enzymes) results in increased clearance of PIs during pregnancy in the macaques. The M. nemestrina should be further investigated as a model to study the mechanisms by which the clearance of PIs is increased during pregnancy.

Rapid solid-phase extraction method to quantify [(11)C]-verapamil, and its [(11)C]-metabolites, in human and macaque plasma.

INTRODUCTION: P-glycoprotein (P-gp), an efflux transporter, is a significant barrier to drug entry into the brain and the fetus. The positron emission tomography (PET) ligand, [(11)C]-verapamil, has been used to measure in vivo P-gp activity at various tissue-blood barriers of humans and animals. Since verapamil is extensively metabolized in vivo, it is important to quantify the extent of verapamil metabolism in order to interpret such P-gp activity. Therefore, we developed a rapid solid-phase extraction (SPE) method to separate, and then quantify, verapamil and its radiolabeled metabolites in plasma. METHODS: Using high-performance liquid chromatography (HPLC), we established that the major identifiable circulating radioactive metabolite of [(11)C]-verapamil in plasma of humans and the nonhuman primate, Macaca nemestrina, was [(11)C]-D-617/717. Using sequential and differential pH elution on C(8) SPE cartridges, we developed a rapid method to separate [(11)C]-verapamil and [(11)C]-D-617/717. Recovery was measured by spiking the samples with the corresponding nonradioactive compounds and assaying these compounds by HPLC. RESULTS: Verapamil and D-617/717 recovery with the SPE method was >85%. When the method was applied to PET studies in humans and nonhuman primates, significant plasma concentration of D-617/717 and unknown polar metabolite(s) were observed. The SPE and the HPLC methods were not significantly different in the quantification of verapamil and D-617/717. CONCLUSIONS: The SPE method simultaneously processes multiple samples in less than 5 min. Given the short half-life of [(11)C], this method provides a valuable tool to rapidly determine the concentration of [(11)C]-verapamil and its [(11)C]-metabolites in human and nonhuman primate plasma.

Consequences of increased vascular permeability induced by treatment of mice with 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and thalidomide.

PURPOSE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) (AS1404), a small-molecule vascular disrupting agent currently in clinical trial, increases vascular permeability and decreases blood flow in both murine and human tumours. DMXAA induces tumour necrosis factor (TNF) in mice and the effects on vascular permeability are hypothesised to result from both direct (DMXAA) and indirect (TNF) effects. Skin temperature decreases in mice treated with high doses of DMXAA, raising the question of whether host toxicity is mediated by the induction of increased vascular permeability in normal tissue. Thalidomide is an anti-inflammatory agent that potentiates the anti-tumour activity of DMXAA but decreases induction of TNF in plasma. We wished to determine how it potentiated the effects of DMXAA. METHODS: Vascular permeability was measured in Colon 38 tumour and liver tissue by uptake of Evans Blue dye. Blood haematocrit and body temperature were also measured. RESULTS: Tumour vascular permeability was increased following administration of DMXAA (25 mg/kg i.p.), minimally affected following thalidomide (100 mg/kg i.p.) but strongly increased following co-administration of both drugs. In contrast, dye uptake into liver tissue was decreased following administration of DMXAA, thalidomide or both drugs. Administration of DMXAA at a potentially toxic dose (35 mg/kg i.p. or 50 mg/kg orally) was found to decrease body temperature and to increase the blood haematocrit, while administration of thalidomide alone (100 mg/kg i.p.) had no effect. Co-administration of thalidomide potentiated the effects of DMXAA on both body temperature and haematocrit but surprisingly did not increase toxicity. CONCLUSIONS: The results are consistent with the hypothesis that the host toxicity of high-dose DMXAA is mediated by effects on host vasculature. Co-administration of thalidomide increases the effective dose of DMXAA by reducing clearance but also, by inhibiting production of circulating TNF, reduces the host toxicity of DMXAA.

The role of transporters in drug interactions.

Transport proteins play an important role in the adsorption, distribution and elimination of a wide variety of drugs. Therefore, it is not surprising that transporter-based drug interactions can occur in the clinic. These interactions can lead to changes in toxicity and/or efficacy of the affected drug. Here, we review such interactions and ask if these interactions could have been predicted from in vitro data. Conducting such in vitro-in vivo correlation is important for predicting future transporter-based drug interactions.

Dose-response effects of Lepidium meyenii (Maca) aqueous extract on testicular function and weight of different organs in adult rats.

Lepidium meyenii (Brassicaceae) known as Maca grows exclusively between 4000 and 4500 m over the sea level in the Peruvian central Andes. The dried hypocotyls of Maca are traditionally used as food and for its supposed fertility-enhancing properties. A dose-response study was performed to determine the effect of 7 days oral administration of an aqueous lyophilized extract of Maca at 0.01-5 g/kg (corresponding to 0.022-11 g dry hypocotyls of Maca/kg) on body and different organ weights, stages of the seminiferous tubules, epididymal sperm count and motility, and serum testosterone and estradiol levels in rats. In doses up to 5 g extract/kg, no toxicity was observed. Almost all organ weights were similar in controls and in the Maca extract-treated groups. Seminal vesicles weight was significantly reduced at 0.01 and 0.10 g extract/kg. Maca increased in length of stages VII-VIII of the seminiferous tubules in a dose-response fashion, with highest response at 1.0 g/kg, while caput/corpus epididymal sperm count increased at the 1.0 g dose. Cauda epididymal sperm count, sperm motility, and serum estradiol level were not affected at any of the doses studied. Serum testosterone was lower at 0.10 g extract/kg. Low-seminal vesicle weights correlated with low-serum testosterone levels (R2=0.33; P<0.0001) and low-testosterone/estradiol ratio (R2=0.35; P<0.0001). Increase in epididymal sperm count was related to lengths of stages VII-VIII. Highest effect on stages VII-VIII of the seminiferous tubules was observed at 1.0 g Maca aqueous extract/kg. The present study demonstrated that Maca extract in doses up to 5 g/kg (equivalent to the intake of 770 g hypocotyls in a man of 70 kg) was safe and that higher effect on reproductive parameters was elicited with a dose of 1 g extract/kg corresponding to 2.2 g dry Maca hypocotyls/kg.

Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m).

Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO.min(-1).g heart(-1) (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.

Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients.

PURPOSE: Thalidomide has a variety of biological effects that vary considerably according to the species tested. We sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide. EXPERIMENTAL DESIGN: Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concentrations of thalidomide were measured after drug administration using high performance liquid chromotography. Plasma samples from five multiple myeloma patients over 24 hours after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liquid chromatography-mass spectrometry. RESULTS: Plasma concentration-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concentration curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 micromol/L. hour, respectively, and corresponding elimination half-lives were 0.5, 2.2, and 7.3 hours, respectively. Large differences were also observed between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients. CONCLUSIONS: Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metabolism. We suggest that the interspecies differences in biological effects of thalidomide may be attributable, at least in part, to the differences in its metabolism and hence pharmacokinetics.

Modulation of thalidomide pharmacokinetics by cyclophosphamide or 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice: the role of tumour necrosis factor.

PURPOSE: There is considerable current interest in the use of thalidomide as a single agent or in combination with drugs such as cyclophosphamide in the treatment of multiple myeloma and other cancers. Our previous work has shown that thalidomide potentiates the antitumour activity of both cyclophosphamide and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) against murine Colon 38 tumours. In both of these cases, thalidomide extends the half-life (t(1/2)) of the other drug. We wished to determine whether cyclophosphamide and DMXAA altered the t(1/2) of thalidomide. Since both thalidomide and DMXAA modulate tumour necrosis factor (TNF), we also wished to determine the role of TNF in this interaction. METHODS: Mice with Colon 38 tumours were treated with cyclophosphamide (220 mg/kg) and/or thalidomide (20 mg/kg) or DMXAA (25 mg/kg) and thalidomide (100 mg/kg), combinations that have previously demonstrated synergistic activity. Plasma and tumour tissue drug concentrations were analysed by high-performance liquid chromatography. To determine the role of TNF, similar experiments were performed using mice defective in the TNF gene (TNF(-/-)) or the TNF receptor-1 gene (TNFR1(-/-)). RESULTS: Coadministration of cyclophosphamide increased the thalidomide t(1/2) by 3.9- and 3.6-fold, respectively, in plasma and tumour tissue, with a corresponding increase in the concentration-time curve (AUC). The corresponding values following coadministration of DMXAA were 3.0- and 4.6-fold, respectively. Coadministration of cyclophosphamide had similar effects on thalidomide t(1/2) in C57Bl/6, TNF(-/-) and TNFR1(-/-) mice, while coadministration of DMXAA did not alter the t(1/2) or AUC in TNF(-/-) and TNFR1(-/-) mice. CONCLUSIONS: Both cyclophosphamide and DMXAA have a pharmacokinetic interaction with thalidomide, increasing t(1/2) and AUC. TNF mediates the effect of DMXAA on thalidomide pharmacokinetics but not that of cyclophosphamide.

Effect of 3-fluorothalidomide and 3-methylthalidomide enantiomers on tumor necrosis factor production and antitumor responses to the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is an antivascular drug that induces tumor necrosis factor (TNF) in mice. Thalidomide inhibits TNF induction by DMXAA and also potentiates its antitumor activity. We investigated whether these effects were enantiomer specific, using the R- or S-enantiomers of two nonracemizable thalidomide analogues. Racemic 3-fluorothalidomide (3FThal) and racemic 3-methylthalidomide (3MeThal) were separated into enantiomers of greater than 98% optical purity using preparative chiral column chromatography. C57Bl/6 mice implanted with subcutaneous Colon 38 tumors were treated with DMXAA (25 mg/kg) alone or together with the pure R- or S-enantiomers by a single i.p. injection. TNF levels in the serum or tumor tissues 3 h after treatment were measured using ELISAs and tumor growth was also measured. 3FThal and 3MeThal, at their respective single maximum tolerated doses (MTD) of 15 and 50 mg/kg, were more toxic in mice than thalidomide (100 mg/kg). The R- and S-enantiomers of either 3FThal or 3MeThal, at their respective MTD, inhibited DMXAA-induced TNF activity in serum and tumor tissue, but no significant differences were observed between the enantiomers. Coadministration of racemic or enantiomers of 3FThal or 3MeThal at their respective MTD did not potentiate the antitumor responses above that obtained with DMXAA alone, and no enantioselectivity was apparent. We conclude that there is no advantage in using the nonracemizable thalidomide analogues to improve the antitumor activity of DMXAA.