DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

Tofacitinib and Baricitinib Are Taken up by Different Uptake Mechanisms Determining the Efficacy of Both Drugs in RA.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SF) play a key role. Baricitinib and Tofacitinib both act intracellularly, blocking the ATP-binding side of JAK proteins and thereby the downstream signalling pathway via STAT-3. Therefore, we investigated the role of organic cation transporters (OCTs) in Baricitinib and Tofacitinib cellular transport. METHODS: OCT expression was analysed in SF isolated from RA and osteoarthritis (OA) patients, as well as peripheral blood mononuclear cells. The interaction of Baricitinib and Tofacitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of both drugs was quantified using LC/MS. Target inhibition for both drugs was tested using Western blot for phosphorylated JAK1 and STAT3 upon stimulation with IL-6. RESULTS: MATE-1 expression increased in OASF compared to RASF. The other OCTs were not differentially expressed. The transport of Baricitinib was not OCT dependent. Tofacitinib; however, was exported from RASF in a MATE-1 dependent way. Tofacitinib and Baricitinib showed comparable inhibition of downstream signalling pathways. CONCLUSION: We observed different cellular uptake strategies for Baricitinib and Tofacitinib. Tofacitinib was exported out of healthy cells due to the increased expression of MATE1. This might make Tofacitinib the favourable drug.

Duvelisib for the treatment of chronic lymphocytic leukemia.

INTRODUCTION: Duvelisib, a first in class, oral, dual PI3 k-delta/gamma inhibitor recently received FDA approval for previously treated CLL (chronic lymphocytic leukemia)/SLL (small lymphocytic lymphoma) and follicular lymphoma. Data coming from the phase III 'DUO' trial, in fact, showed a superior progression-free survival (PFS) in CLL patients treated with duvelisib compared to ofatumumab. AREAS COVERED: This review provides analysis of the mechanism of action of duvelisib and includes the rationale for the use of double inhibition. The authors also give their clinical experience with duvelisib. Overall, despite the high efficacy of the drug, some concern remains on duvelisib-related adverse events leading to treatment interruption in a significant proportion of patients. EXPERT OPINION: Considering the unmet need of salvage therapies in patients failing BTK and/or Bcl2 inhibitors, treatment with duvelisib represents a new valid option in the CLL therapeutic armamentarium. Therefore, the correct management of adverse events with early treatment suspension, dose reductions and prompt supportive treatment could help to manage treatment, thus improving patient outcome. Finally, the association of duvelisib with other targeted therapies, such as ibrutinib or venetoclax, could allow clinicians to capitalize on the synergistic activity of these agents.

Design, Synthesis, Anticonvulsant Activity, Preclinical Study and Pharmacokinetic Performance of N-{[3-(4-chlorophenyl)-4-oxo-3, 4-dihydroquinazolin- 2-yl] methyl}, 2-[(2-isopropyl-5-methyl) 1-cyclo Hexylidene] Hydrazinecarboxamide.

BACKGROUND: N-{[3-(4-chlorophenyl)-4-oxo-3, 4-dihydroquinazolin-2-yl] methyl}, 2-[(2- isopropyl-5-methyl) 1-cyclohexylidene] hydrazinecarboxamide QS11 was designed by computational study. It possessed essential pharmacophoric features for anticonvulsant activity and showed good docking with iGluRs (Kainate) glutamate receptor. METHODS: QSAR and ADMET screening results suggested that QS11 would possess good potency for anticonvulsant activity. QS11 was synthesised and evaluated for its anticonvulsant activity and neurotoxicity. QS11 showed protection in strychnine, thiosemicarbazide, 4-aminopyridine and scPTZ induced seizure models and MES seizure model. QS11 showed higher ED50, TD50 and PI values as compared to the standard drugs in both MES and scPTZ screen. A high safety profile (HD50/ED50 values) was noted and hypnosis, analgesia, and anaesthesia were only observed at higher doses. No considerable increase or decrease in the concentration of liver enzymes was observed. Optimized QS11 was subjected to preclinical (in-vivo) studies and the pharmacokinetic performance of the sample was investigated. The result revealed that the pharmacokinetic performance of QS11 achieved maximum plasma concentrations (Cmax) of 0.315 ± 0.011 µg/mL at Tmax of 2.0 ± 0.13 h, area under the curve (AUC0-∞) value 4.591 ± 0.163 µg/ml x h, elimination half-life (T1/2) 6.28 ± 0.71 h and elimination rate constant was found 0.110 ± 0.013 h-1 . RESULTS AND CONCLUSION: Above evidences indicate that QS11 could serve as a lead for development of new antiepileptic drugs.

Pharmacokinetic interaction between Kaempferia parviflora extract and sildenafil in rats.

Kaempferia parviflora (KP) is a plant widely used in Southeast Asia. Its major compounds are 3,5,7,3',4'-pentamethoxyflavone (PMF), 5,7,4'-trimethoxylflavone (TMF), and 5,7-dimethoxyflavone (DMF). This study investigated the effect of KP extract on the blood levels and pharmacokinetics of sildenafil co-administration in rats. Rats were randomly assigned to four groups. Groups 1, 2, and 3 were given sildenafil 20 mg/kg daily for 9 days. On days 4-9 of each treatment period, the treated rats received KP extract (250 mg/kg) and vehicle (groups 2 and 3, respectively). Group 4 received KP extract only (250 mg/kg daily for 9 days). Daily blood concentrations of sildenafil, PMF, TMF, and DMF were determined by HPLC to evaluate the daily blood level interactions. Additional blood samples were collected at various times on the last day of treatment to evaluate the pharmacokinetic interactions. The KP extract decreased blood levels of sildenafil on the first day of co-administration by 95 % but the percentage reduction was insignificant on subsequent days. When co-administered with KP extract, the area under the curve (AUC), maximum concentration (C max), and half-life (T 1/2) of sildenafil were decreased by 60-65, 40-52, and 32-54 %, respectively, with the elimination rate constant (K e) increased by 37-77 %. In addition, PMF, TMF, and DMF concentrations and their AUC, C max, T max, K e, and T 1/2 values were changed after co-administration of KP extract and sildenafil.

Effects of Nigella sativa, Lepidium sativum and Trigonella foenum-graecum on sildenafil disposition in beagle dogs.

The present study was conducted to investigate the effects of some commonly used herbs namely Nigella sativa, Lepidium sativum and Trigonella foenum-graecum on the pharmacokinetics of sildenafil in beagle dogs. The study design involved four treatments in a non-balanced crossover design. Sildenafil was given one tablet 100 mg orally to each dog and blood samples were obtained. After a suitable washout period, animals were commenced on a specific herb treatment for 1 week. Blood samples were withdrawn at different time intervals and sildenafil was analyzed by HPLC method. Oral administration of Nigella sativa resulted in reduction of AUC0-∞, C max and t 1/2 as compared to the control. Treatment of Lepidium sativum resulted in a significant reduction in the C max and AUC. There were no significant differences between the rests of the pharmacokinetic parameters relative to those of the control. For Trigonella foenum-graecum, the effects were similar to those obtained in case of Lepidium sativum. It was concluded that concurrent use of investigated herbs alters the pharmacokinetics of sildenafil. Co-administration of investigated herbs should be cautious since their concomitant use might result in decrease in sildenafil bioavailability.

Two new adenosine receptor antagonists for the treatment of Parkinson's disease: istradefylline versus tozadenant.

INTRODUCTION: Adenosine A2A receptors are localized in the brain, mainly within the caudate and putamen nuclei of the basal ganglia. Their activation leads to stimulation of the 'indirect' pathway. Conversely, administration of A2A receptor antagonists leads to inhibition of this pathway, which was translated into reduced hypomotility in several animal models of parkinsonism. AREAS COVERED: In this review, the effects of two A2A receptor antagonists, istradefylline and tozadenant, on parkinsonian symptoms in animal and humans will be discussed. EXPERT OPINION: Animal studies have shown potent antiparkinsonian effects for several A2A receptor antagonists, including istradefylline. In clinical trials, istradefylline reduced OFF time when administered with levodopa, but results are inconclusive. Results with tozadenant are scarce. Modification of thalamic blood flow compatible with reduced inhibition was noted in one small trial, followed by a significant reduction in OFF time in a larger one. Therefore, both drugs show promising efficacy for the reduction of OFF time in levodopa-treated Parkinson's disease patients, but further research is needed in order to obtain definitive conclusions.

Pharmacokinetic evaluation of sildenafil as a pulmonary hypertension treatment.

INTRODUCTION: Sildenafil citrate is a potent, selective phosphodiesterase type 5 inhibitor approved for the treatment of pulmonary arterial hypertension (PAH) and plays an important role in the management of the disease. AREAS COVERED: In this review, we focus on the current available information on the pharmacokinetics, pharmacodynamics, clinical efficacy and safety of sildenafil citrate in PAH through a MEDLINE literature search. Comparison of sildenafil citrate with tadalafil, another phosphodiesterase type 5 inhibitor was also performed. EXPERT OPINION: In the last few years, considerable progress has been made in the understanding and treatment of PAH. Sildenafil citrate has multiple advantages and whether it is first-line treatment alone or in combination for the mild form of the disease, it is one of the treatments of choice. In terms of its future use, more studies are still needed to better evaluate the benefit/risk balance of sildenafil citrate in pediatric populations.

Herb-drug interaction of Epimedium sagittatum (Sieb. et Zucc.) maxim extract on the pharmacokinetics of sildenafil in rats.

Epimedium sagittatum (Sieb. et Zucc.) Maxim is one of the herbs used to treat erectile dysfunction in Traditional Chinese Medicine. Sildenafil is a phosphodiesterase 5 inhibitor used to treat erectile dysfunction in Western Medicine. This study evaluates the herbal-drug interaction of Epimedium sagittatum extract on the pharmacokinetics of sildenafil in rats by ultra-performance liquid chromatography. The rat plasma was sampled from each anesthetized rat after pretreatment with 3-days Epimedium sagittatum extract (1/2 g/kg/day) and intravenous injection with sildenafil (10/30 mg/kg). The pharmacokinetic data demonstrate that the area under the concentration-time curve (AUC) of sildenafil (10 mg/kg) was significantly decreased in groups that received a high dose of Epimedium sagittatum extract. In conclusion, the study demonstrates that there was significant herb-drug interaction of Epimedium sagittatum extract on the pharmacokinetics of sildenafil at low and high daily doses, suggesting co-administration use of Epimedium sagittatum extract and sildenafil in clinical practice should be prevented due to possible herb-drug interactions.

Evaluation and prediction of potential drug-drug interactions of linagliptin using in vitro cell culture methods.

Linagliptin is a highly potent dipeptidyl peptidase-4 (DPP-4) inhibitor approved for the treatment of type 2 diabetes. Unlike other DPP-4 inhibitors, linagliptin is cleared primarily via the bile and gut. We used a panel of stably and transiently transfected cell lines to elucidate the carrier-mediated transport processes that are involved in linagliptin disposition in vivo and to assess the potential for drug-drug interactions (DDIs). Our results demonstrate that linagliptin is a substrate of organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp) but not of organic anion-transporting polypeptide 1B1 and 1B3; organic anion transporter 1, 3, and 4; OCT1; or organic cation/carnitine transporter 1 and 2, suggesting that OCT2 and P-gp play a role in the disposition of linagliptin in vivo. Linagliptin inhibits transcellular transport of digoxin by P-gp with an apparent IC(50) of 66.1 µM, but it did not inhibit activity of multidrug resistance-associated protein 2 and breast cancer resistance protein as represented by transport of probe substrate into membrane vesicles from respective transporter-expressing cells. In addition, the inhibitory effect of linagliptin on major solute carrier transporter isoforms was investigated. Linagliptin showed inhibitory potency against only OCT1 and OCT2 out of all major solute carrier transporter isoforms examined, and those inhibition potencies, evaluated using three different in vitro probe substrates, were substrate-specific. Considering the low therapeutic plasma concentration of linagliptin, our data clearly suggest a very low risk for transporter-mediated DDIs with comedications in clinical practice.

Discovery and optimization of novel purines as potent and selective CB2 agonists.

A focused screening strategy identified thienopyrimidine 1 as a hCB2 cannabinoid receptor agonist with moderate selectivity over the hCB1 receptor. This initial hit suffered from poor in vitro metabolic stability and high in vivo clearance. Structure-activity relationships describe the optimization and modification to a less lipophilic purine core. Examples from this novel series were found to be highly potent and fully efficacious agonists of the human CB2 receptor with excellent selectivity against CB1. Compound 10 possesses good biopharmaceutical properties, is highly water soluble and demonstrates robust oral activity in rodent models of joint pain.

The anti-proliferative side effects of AEE788, a tyrosine kinase inhibitor blocking both EGF- and VEGF-receptor, are liver-size-dependent after partial hepatectomy in rats.

BACKGROUND: Blocking of EGFR signaling by the tyrosine kinase inhibitor AEE788 was well tolerated and did not inhibit liver regeneration after standard 70% partial hepatectomy (PH) in a rat model, as demonstrated previously. However, serum levels of AEE788 at POW1 were 3-fold higher than in the non-resected control group. Therefore, we expanded theses studies to a model of extended 90%PH to investigate the role of liver size for the metabolism of AEE788 and its potential influence on side effects, liver regeneration and liver remodeling. METHOD: Rats treated with 50 mg/kg AEE788 or solvent every other day orally were subjected to 90%PH. Animals were sacrificed at 1, 2, 7 and 28 days after PH. We measured plasma and liver levels of AEE788 and assessed anti-proliferative side effects, liver regeneration, and liver architecture. RESULT: Liver regeneration and liver architecture were not impaired by AEE788 treatment after 90%PH. 90%PH caused a clinically relevant drug accumulation within 1 week of treatment (AEE788 serum and tissue levels: 90%PH*>70%PH*>normal control, *p < 0.05), suggesting a liver-size-dependent metabolism of the drug. Drug accumulation after 90%PH was associated with severe side effects (delayed body weight recovery, diarrhea, impaired hair growth) within 1 week of treatment. CONCLUSION: Treatment with AEE788 could be a potential strategy for adjuvant treatment after oncological liver resection, as liver regeneration was not impaired. Our results suggest a liver-size-dependent metabolism of AEE788 leading to drug accumulation and subsequently to severe side effects. It calls for therapeutic drug monitoring in the early postoperative phase after extended resection.

Linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor with an unusual profile for the treatment of type 2 diabetes.

IMPORTANCE OF THE FIELD: Type 2 diabetes is a progressive disease for which current treatments are often unsatisfactory with respect to achieving therapeutic goals and unwanted side effects. AREAS COVERED: Preclinical and clinical studies of linagliptin, a new oral antidiabetic agent, including data presented at Scientific Meetings and peer-reviewed studies published since 2007. WHAT THE READER WILL GAIN: This article reviews pharmacokinetic and pharmacodynamic characteristics of linagliptin. Linagliptin belongs to a new chemical class of dipeptidyl pepidase-4 (DPP-4) inhibitors, which comprise xanthine-based compounds. It is a potent, long-acting inhibitor with high selectivity for DPP-4 versus the related enzymes DPP-8 and DPP-9. The drug has modest oral availability in humans, but is absorbed rapidly to inhibit plasma DPP-4 activity by > 80% over 24 h. It is not metabolized appreciably in vivo, but binds extensively to plasma proteins, with elimination occurring primarily in the liver. Linagliptin reduces degradation of the incretin hormone glucagon-like peptide-1 and is associated with reduced fasting and postprandial glucose in preclinical and clinical studies. Limited data from longer duration clinical trials show it improves glycemic control in patients with type 2 diabetes. TAKE HOME MESSAGE: Linagliptin is a new oral antidiabetic agent associated with minimal risk of hypoglycemia, which holds promise for treatment of type 2 diabetes.

Population pharmacokinetic model of the pregabalin-sildenafil interaction in rats: application of simulation to preclinical PK-PD study design.

PURPOSE: Preliminary evidence has suggested a synergistic interaction between pregabalin and sildenafil for the treatment of neuropathic pain. The focus of this study was to determine the influence of sildenafil on the pharmacokinetics (PK) of pregabalin with the objective of informing the design of a quantitative pharmacodynamic (PD) study. METHODS: The pharmacokinetics were determined in rats following 2-hr intravenous infusions of pregabalin at doses of 4 mg/kg/hr and 10 mg/kg/hr with and without a sildenafil bolus (2.2 mg) and steady state infusion (12 mg/kg/hr for 6 h). This PK model was utilized in a preclinical trial simulation with the aim of selecting the optimal sampling strategy to characterize the PK-PD profile in a future study. Eight logistically feasible PK sampling strategies were simulated in NONMEM and examined through trial simulation techniques. RESULTS: A two-compartment population PK model best described pregabalin pharmacokinetics. Significant model covariates included either a binary effect of sildenafil administration (30.2% decrease in clearance) or a concentration-dependent effect due to sildenafil's active metabolite. CONCLUSIONS: Analysis of simulations indicated that three post-PD samples had the best cost/benefit ratio by providing a significant increase in the precision (and minor improvement in bias) of both PK and PD parameters compared with no PK sampling.

Biotransformation of sildenafil in the male rat: evaluation of drug interactions with testosterone and carbamazepine.

The biotransformation of sildenafil to its major circulating metabolite, UK-103,320, was studied in male rat liver microsomes. The conversion of sildenafil to UK-103,320 by rat microsomes followed Michaelis-Menten kinetics, for which the parameters were V(max) = 1.96 microM/minand K(m) = 27.31 microM. Using substrates of CYP3A4 of testosterone and carbamazepine, the active sites on CYP3A4 responsible for metabolizing sildenafil were also evaluated. Sildenafil biotransformation was inhibited in the individual presence of testosterone and carbamazepine. The results showed drug interaction was observed in the sildenafil-testosterone and sildenafil-carbamazepine. Although testosterone and carbamazepine can inhibit sildenafil demethylation in concentration- and incubation time-dependent manners, sildenafil did not inhibit testosterone hydroxylation or carbamazepine epoxidation. These results may be explained by a model in which multiple substrates or ligands can concurrently bind to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 cannot be ruled out in this study.

Cassette dosing pharmacokinetics of a library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 inhibitors prepared by parallel synthesis.

Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.

Pharmacokinetic studies of 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyl-oxybut-1-yl)purine, an oral prodrug for the antiviral agent penciclovir.

2-Amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)- purine (SK1899) was tested as an oral prodrug for penciclovir. SK1899 was administered orally to rats and dogs at doses up to 2 and 0.68 mmol/kg, respectively. SK1899 was well absorbed, and the major metabolites detected in plasma and urine were penciclovir, the active antiviral compound, and 6-deoxypenciclovir (M4) in both species. In rats, SK1899 was rapidly and extensively metabolized to penciclovir, which reached the peak plasma concentration (C(max)) of 39.5 microM at 0.5 h after 0.2-mmol/kg dosing. The area under the plasma concentration-time curve (AUC) for penciclovir was 57.5 microM x h. After an oral dose of 0.034 mmol/kg to dogs, extensive conversion of SK1899 to penciclovir also occurred with slower rate of formation of penciclovir from M4 than in rats. The mean C(max) and AUC for penciclovir were 4.5 microM at 2.7 h and 28.2 microM x h, respectively. The 0- to 24-h urinary recovery of penciclovir represented 36.1 and 36.3% of dose to rats and dogs, respectively. Radioactivity was found in fetuses following an oral administration of [(14)C]SK1899 to pregnant rats, but no significant accumulation was observed. Although substantial milk transfer of [(14)C]SK1899 occurred in rats, the radioactivity in milk was rapidly cleared. The values of C(max), AUC, and urinary recovery of penciclovir after dosing with SK1899 to rats and dogs were similar or slightly higher than those from famciclovir. These data indicate that introduction of an isopropoxy carbonate group into one of the two hydroxyl groups of M4 did not significantly alter the oral bioavailability of penciclovir compared with famciclovir.

Omega-3 polyunsaturated fatty acids increase purine but not pyrimidine transport in L1210 leukaemia cells.

Here we show that in vitro supplementation of L1210 murine lymphoblastic leukaemia cells with n-3 polyunsaturated fatty acids results in considerable changes in the fatty acid composition of membrane phospholipids. Incubations for 48 h with 30 microM eicosapentaenoic acid (20:5, n-3; EPA) or docosahexaenoic acid (22:6, n-3; DHA) results primarily in substitution of long chain n-6 fatty acids with long-chain n-3 fatty acids. This results in a decrease in the n-6/n-3 ratio from 6.9 in unsupplemented cultures to 1.2 or 1.6 for EPA and DHA supplemented cultures, respectively. Coincident with these changes in membrane fatty acid composition, we observed a 5-fold increase in the rate of adenosine (5 microM) uptake via a nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter in EPA- and DHA-supplemented L1210 cells, relative to unsupplemented cells. This seemed to result from a decrease in the Km for adenosine from 12.5 microM in unsupplemented cultures to 5.1 microM in DHA-treated cultures. Guanosine (50 microM) transport was similarly affected by DHA with a 3.5-fold increase in the initial rate of uptake. In contrast, pyrimidine transport, as measured by uptake of thymidine and cytidine, was not similarly affected, suggesting that substrate recognition had been altered by fatty acid supplementation. Studies using [(3)H]NBMPR showed that there was no effect of EPA or DHA on either the number of NBMPR-binding sites or the affinity of these sites for NBMPR. This observation suggests that the increases in adenosine and guanosine transport were not due to increases in the number of transported sites but rather that EPA and DHA directly or indirectly modulate transporter function.