The utility of endogenous glycochenodeoxycholate-3-sulfate and 4β-hydroxycholesterol to evaluate the hepatic disposition of atorvastatin in rats

The liver is an important organ for drugs disposition, and thus how to accurately evaluate hepatic clearance is essential for proper drug dosing. However, there are many limitations in drug dosage adjustment based on liver function and pharmacogenomic testing. In this study, we evaluated the ability of endogenous glycochenodeoxycholate-3-sulfate (GCDCA-S) and 4β-hydroxycholesterol (4β-HC) plasma levels to evaluate organic anion-transporting polypeptide (Oatps)-mediated hepatic uptake and Cyp3a-meidated metabolism of atorvastatin (ATV) in rats. The concentration of ATV and its metabolites, 2-OH ATV and 4-OH ATV, was markedly increased after a single injection of rifampicin (RIF), an inhibitor of Oatps. Concurrently, plasma GCDCA-S levels were also elevated. After a single injection of the Cyp3a inhibitor ketoconazole (KTZ), plasma ATV concentrations were significantly increased and 2-OH ATV concentrations were decreased, consistent with the metabolism of ATV by Cyp3a. However, plasma 4β-HC was not affected by KTZ treatment despite it being a Cyp3a metabolite of cholesterol. After repeated oral administration of RIF, plasma concentrations of ATV, 2-OH ATV and 4-OH ATV were markedly increased and the hepatic uptake ratio of ATV and GCDCA-S was decreased. KTZ did not affect plasma concentrations of ATV, 2-OH ATV and 4-OH ATV, but significantly decreased the metabolic ratio of total and 4-OH ATV. However, the plasma level and hepatic metabolism of 4β-HC were not changed by KTZ. The inhibition of hepatic uptake of GCDCA-S by RIF was fully reversed after a 7-d washout of RIF. Plasma concentration and hepatic uptake ratio of GCDCA-S were correlated with the plasma level and hepatic uptake of ATV in rats with ANIT-induced liver injury, respectively. These results demonstrate that plasma GCDCA-S is a sensitive probe for the assessment of Oatps-mediated hepatic uptake of ATV. However, Cyp3a-mediated metabolism of ATV was not predicted by plasma 4β-HC levels in rats.     

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.     

Acute nociception mediated by hindpaw P2X receptor activation in the rat

1. The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5′-triphosphate (ATP), α,β-methylene ATP, adenosine 5′-diphosphate (ADP) and adenosine.
2. Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, α,β-methylene ATP. Nociceptive behaviours continued for 15 min following administration of α,β-methylene ATP (200 nmol) and were dose-related (0–5 min hindpaw lifting times after injection of α,β-methylene ATP 100 nmol and 1000 nmol were 89±26 s and 232±11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0–5 min hindpaw lifting time 66±19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity.
3. Morphine (3 mg kg⁻¹, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either α,β-methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88±5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34±11%) in morphine-pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar α,β-methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished α,β-methylene ATP-evoked nociceptive behaviours.
4. Hindpaw lifting and licking mediated by α,β-methylene ATP (600 nmol, subplantar) were inhibited (72±15% and 95±5%, respectively) by 30 min local pretreatment with 600 nmol α,β-methylene ATP. Subplantar α,β-methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw.
5. Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 μg) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with α,β-methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml).
6. We conclude that activation of P2X receptors present on small diameter (capsaicin-sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.

     

Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes.     

Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity

Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug–food or drug–herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver–Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver–Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC₅₀ values of 0.07 and 0.03 μM, respectively. The K_i value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.     

In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes

ContextPeucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy.ObjectiveThe interaction between peucedanol and CYP450s was investigated.Materials and methodsPeucedanol (0, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro.ResultsPeucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC₅₀ values of 6.03, 13.57, and 7.58 μM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a K_i value of 4.07 μM and a competitive inhibitor of CYP1A2 and 2D6 with a K_i values of 3.39 and 6.77 μM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the K_i/K_inact value of 5.44/0.046 min/μM.Discussion and conclusionsIn vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.     

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

1. The γ-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA_A receptors are favoured by β₂- or β₃- versus β₁-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β_{2 N290}, β_{3 N289}, β_{1 S290}). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster.
2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at −60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC₅₀ for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl_M314N) or a serine (Rdl_M314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC₅₀ of 4.8±0.2 μM.
3. Utilizing the appropriate GABA EC₁₀, etomidate (300 μM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl_M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC₅₀ of 64±3 μM and an E_max of 68±2% (of the maximum response to GABA).
4. The actions of etomidate at Rdl_M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μM R-(+)-etomidate and S-(−)-etomidate enhancing the current induced by GABA (EC₁₀) to 52±6% and 12±1% of the GABA maximum respectively.
5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl_M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (Rdl_M314N=18±3% of the GABA maximum).
6. Etomidate acting at β₁ (S290)-containing mammalian GABA_A receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at Rdl_M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl_M314N receptors.
7. Etomidate acting at human α₆β₃γ_2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA_A receptor complex. Mutation of the human β₃ subunit asparagine to methionine (β_{3 N289M} found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10–300 μM) acting at α₆β_{3 N289M}γ_2L receptors.
8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

     

Metabolism of bilirubin by human cytochrome P450 2A6

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K_i of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme.     

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL_int) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL_int rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. The electrophysiological actions of several agonists which may differentiate between P2X₁- and P2X₃-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
2. β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P¹,P⁵-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
3. The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
5. This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X₃-subtype to produce these effects.

     

Effects of cytochrome P450 3A4 and non-genetic factors on initial voriconazole serum trough concentrations in hematological patients with different cytochrome P450 2C19 genotypes

1.?Polymorphisms of cytochrome P450 2C19 (CYP2C19) is an important factor contributing to variability of voriconazole pharmacokinetics. Polymorphisms of CYP3A4, CYP3A5, CYP2C9 and non-genetic factors such as age, gender, body mass index (BMI), transaminase levels, concomitant medications might also affect voriconazole initial steady serum trough concentration (VIC_min) in haematological patients, but the effects were not clear.

2.?Eighteen single-nucleotide polymorphisms in CYP2C19, CYP3A4, CYP3A5, CYP2C9 were genotyped. Patients were stratified into two groups according to CYP2C19 genotype. Group 1 were patients with CYP2C19*2 or CYP2C19*3, and Group 2 were homozygous extensive metabolizers. The effects were studied in different groups. VIC_min was adjusted on daily dose (VIC_min/D) for overcoming effect of dose.

3.?A total of 106 blood samples from 86 patients were included. In final optimal scaling regression models, polymorphisms of rs4646437 (CYP3A4), age, BMI was identified to be factors of VIC_min/D in Group 1 (R^2?=^?.255, p?<?.001). Only age was confirmed as a factor of VIC_min/D in Group 2 (R^2?=^?0.144, p?=?.021).

4.?Besides polymorphisms of CYP2C19, in individualized medication of voriconazole in haematological patients, polymorphisms of CYP3A4, and non-genetic factors as BMI, age should also be taken into account, especially for individuals with CYP2C19*2 or CYP2C19*3.     

Isoform-specific regulation of cytochrome P450 expression and activity by estradiol in female rats

Estradiol (E2) is the major endogenous estrogen, and its plasma concentration increases up to 100-fold during pregnancy in humans. Accumulating evidence suggests that an elevated level of E2 may influence hepatic drug metabolism, potentially being responsible for altered drug metabolism during pregnancy. We characterized effects of E2 on expression and activities of cytochrome P450 enzymes (CYPs) in an in vivo system using rats. To this end, female rats were treated with estradiol benzoate (EB) or known CYP inducers. Liver tissues were obtained after 5 days of treatment, and mRNA and protein expression levels as well as activities of major hepatic CYPs were determined by qRT-PCR, immunoblot, and microsomal assay. E2 increased CYP1A2 expression and activity to a smaller extent than β-naphthoflavone did. E2 also enhanced CYP2C expression (CYP2C6, CYP2C7, and CYP2C12) to levels comparable to those observed by phenobarbital. E2 upregulated CYP3A9 expression, while expression of CYP3A1 was downregulated. Expression of hepatic nuclear receptors (PXR and CAR) and the obligate redox partner of CYPs (POR) was downregulated in EB-treated rats, suggesting their potential involvement in regulation of CYP expression and activity by E2. In summary, in female rats E2 regulates expression of hepatic CYPs in a CYP isoform-specific manner although the directional changes are different from those clinically observed during human pregnancy. Further study is warranted to determine whether the changes in drug metabolism during human pregnancy are attributable to involvement of hormones other than E2.     

己烯雌酚对人肝微粒体内CYP3A4和CYP2C9酶的抑制作用

目的:体外实验考察己烯雌酚(DES)对细胞色素P450 3A4(CYP3A4)和细胞色素P450 2C9(CYP2C9)活性的抑制作用,以评佑DES通过抑制这两个重要的细胞色素P450(CYP)亚型而引发药物-药物相互作用的可能性.方法:混合人肝微粒体与不同浓度的DES(或阳性抑制剂),CYP3A4或CYP2C9的探针...     

CYP3A5 polymorphism affects the increase in CYP3A activity after living kidney transplantation in patients with end stage renal disease

AimsIt has been reported that cytochrome P450 (CYP)3A activity increases significantly in patients with end stage renal disease (ESRD) after kidney transplantation, with wide interindividual variability in the degree of increase. The aim of this study was to evaluate the influence of CYP3A5 polymorphism on the increase in CYP3A activity after living kidney transplantation, by measuring the plasma concentration of 4β‐hydroxycholesterol.MethodsThis prospective study recruited 22 patients with ESRD who underwent a first living kidney allograft transplantation, comprising 12 patients with CYP3A5*1 allele (CYP3A5*1/*1 or *1/*3) and 10 patients without CYP3A5*1 allele (CYP3A5*3/*3).ResultsNo significant difference in estimated glomerular filtration rate over time was observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele, suggesting that the degrees of recovery in renal function after living kidney transplantation were similar in the two groups. However, plasma concentrations of 4β‐hydroxycholesterol on days 90 (57.1 ± 13.4 vs. 39.5 ± 10.8 ng ml⁻¹) and 180 (55.0 ± 14.5 vs. 42.4 ± 12.6 ng ml⁻¹) after living kidney transplantation were significantly higher in the presence of the CYP3A5*1 allele than in the absence of the CYP3A5*1 allele [P = 0.0034 (95% confidence interval of difference 6.55, 28.6) and P = 0.043 (95% confidence interval of difference 0.47, 24.8), respectively], suggesting that CYP3A activity may increase markedly associated with recovery of renal function in patients with the CYP3A5*1 allele.ConclusionsThese findings suggest that the presence of the CYP3A5*1 allele contributes to marked elevation of CYP3A activity associated with recovery of renal function after kidney transplantation.     

Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567

BACKGROUND AND PURPOSE

An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist.

EXPERIMENTAL APPROACH

We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain.

KEY RESULTS

JNJ-47965567 is potent high affinity (pK_i 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC₅₀ of 78 ± 19 ng·mL⁻¹ (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg⁻¹) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test.

Conclusion and Implications

JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology.     

Toll-like receptor 3 agonist Poly I:C protects against simulated cerebral ischemia in vitro and in vivo

Aim:

To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo.

Methods:

Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNFα, IL-6 and interferon-β (IFNβ) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNFα and IL-6 in ischemic brain were measured using ELISA.

Results:

Pretreatment with Poly I:C (10 and 20 μg/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFNβ production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNFα and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNFα and IL-6 in the ischemic striatum and cortex.

Conclusion:

Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.     

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.
2. α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED₅₀ values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.
3. Although the contraction caused by α,β-mATP was strongly diminished by Ca²⁺-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.
4. UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.
5. The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
6. In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.
7. These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.