Nevirapine uptake into the central nervous system of the Guinea pig: an in situ brain perfusion study.

The presence of human immunodeficiency virus (HIV) in the central nervous system (CNS) is associated with the development of HIV-1-associated dementia (HAD), a major cause of HIV-related mortality. To eradicate HIV in the CNS, anti-HIV drugs need to reach the brain and cerebrospinal fluid (CSF) in therapeutic concentrations. This involves passage through the blood-brain and blood-CSF barriers. Using a well established guinea pig in situ brain perfusion model, this study investigated whether nevirapine [6H-dipyrido(3,2-b:2',3'-e)(1,4)diazepin-6-one,11-cyclopropyl-5,11-dihydro-4-methyl], a non-nucleoside reverse transcriptase inhibitor (NNRTI), could effectively accumulate in the CNS. [(3)H]Nevirapine was coperfused with [(14)C]mannitol (a vascular/paracellular permeability marker) through the carotid arteries for up to 30 min, and accumulation in the brain, CSF, and choroid plexus was measured. [(3)H]Nevirapine uptake into the cerebrum was greater than uptake of [(14)C]mannitol, indicating significant passage across the blood-brain barrier and accumulation into the brain (this was further confirmed with capillary depletion and high-performance liquid chromatography analyses). Likewise, [(3)H]nevirapine showed a great ability to cross the blood-CSF barrier and accumulate in the CSF, compared with [(14)C]mannitol. The CNS accumulation of [(3)H]nevirapine was unaffected by 100 muM nevirapine, suggesting that passage across the blood-brain barrier can occur by diffusion. Furthermore, coperfusion with 100 muM efavirenz [2H-3,1-benzoxazin-2-one, 6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-, (4S)-; another NNRTI] did not significantly alter CNS accumulation of [(3)H]nevirapine, indicating that the efficacy of nevirapine in the CNS would not be altered by the addition of this drug to a combination therapy. Together, these data indicate that this anti-HIV drug should be beneficial in the eradication of HIV within the CNS and the subsequent treatment of HAD.

The involvement of the blood-brain and the blood-cerebrospinal fluid barriers in the distribution of leptin into and out of the rat brain.

Leptin is a 16 kDa hormone that is produced by adipose tissue and has a central effect on food intake and energy homeostasis. The ability of leptin to cross the blood-brain and blood-cerebrospinal fluid (CSF) barriers and reach or leave the CNS was studied by the bilateral in situ brain perfusion and isolated incubated choroid plexus techniques in the rat. Brain perfusion results indicated that [(125)I]leptin reached the CNS at higher concentrations than the vascular marker, confirming that [(125)I]leptin crossed the brain barriers. Leptin distribution varied between CNS regions and indicated that the blood-brain barrier, in contrast to the blood-CSF route, was the key pathway for [(125)I]leptin to reach the brain. Further perfusion studies revealed that [(125)I]leptin movement into the arcuate nucleus, thalamus, frontal cortex, choroid plexuses and CSF was unaffected by unlabelled human or murine leptin at a concentration that reflects the upper human and rat plasma leptin concentration (2.5 nM). In contrast, the cerebellum uptake of [(125)I]leptin was decreased by 73% with 2.5 nM human leptin. Thus, this site of dense leptin receptor expression would be sensitive to physiological changes in leptin plasma concentrations. The highest rate (K(in)) of [(125)I]leptin uptake was into the choroid plexuses (307.7+/-68.0 microl/min/g); however, this was not reflected in the CSF (8.9+/-4.1 microl/min/g) and indicates that this tissue tightly regulates leptin distribution. The multiple-time brain uptake of [(125)I]leptin was non-linear and suggested leptin could also be removed from the CNS. Studies using the incubated rat choroid plexus model found that [(125)I]leptin could cross the apical membrane of the choroid plexus to leave the CSF. However, this movement was not sensitive to unlabelled human leptin or specific transport inhibitors/modulators (including probenecid, digoxin, deltorphin II, progesterone and indomethacin).This study supports the concept of brain-barrier regulation of leptin distribution to the CNS, and highlights an important link between leptin and the cerebellum.

Inhibition of nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects.

1. The ability of a range of substituted imidazole compounds to inhibit mouse cerebellar neuronal nitric oxide synthase (nNOS), bovine aortic endothelial NOS (eNOS) and inducible NOS (iNOS) from lungs of endotoxin-pretreated rats was investigated. In each case the substrate (L-arginine) concentration employed was 120 nM. 2. 1-(2-Trifluoromethylphenyl) imidazole (TRIM) was a relatively potent inhibitor of nNOS and iNOS (IC50S of 28.2 microM and 27.0 microM respectively) but was a relatively weak inhibitor of eNOS (IC50, 1057.5 microM). The parent compound, imidazole, was a weak inhibitor of all three NOS isoforms (IC50S: nNOS, 290.6 microM; eNOS, 101.3 microM; iNOS, 616.0 microM). Substitution of imidazole with a phenyl group to yield I-phenylimidazole (PI) resulted in an isoform non-selective increase in inhibitory potency (IC50S: nNOS, 72.1 microM; eNOS, 86.9 microM; iNOS, 53.9 microM). Further substitution of the attached phenyl group resulted in an increase in nNOS and a decrease in eNOS inhibitory potency as in TRIM, 1-chlorophenylimidazole (CPI; IC50S: nNOS, 43.4 microM; eNOS, 392.3 microM; iNOS, 786.5 microM) and 1-(2,3,5,6-tetrafluorophenyl) imidazole (TETRA-FPI; IC50S; nNOS, 56.3 microM; eNOS, 559.6 microM; iNOS, 202.4 microM). 3. The ability of TRIM to inhibit mouse cerebellar nNOS activity in vitro was influenced by the concentration of L-arginine (0.12-10.0 microM) in the incubation medium. When mouse cerebellar nNOS was used as enzyme source a double reciprocal (Lineweaver-Burk) plot in the presence/absence of TRIM (50 microM) revealed a competitive inhibitory profile. The K(m) for L-arginine and the Ki for TRIM calculated from these data were 2.4 microM and 21.7 microM, respectively. The ability of TRIM to inhibit mouse cerebellar nNOS activity in vitro was unaffected by varying the time of exposure of the enzyme to TRIM from 0-60 min at 0 degree C. 4. TRIM exhibits potent antinociceptive activity in the mouse as evidenced by inhibition of acetic acid induced abdominal constrictions. The ED50 for TRIM following i.p. administration was 20 mg kg-1 (94.5 mumol kg-1). The antinociceptive effect of TRIM was reversed by pretreatment of animals with L-arginine (50 mg kg-1, i.p.) and was not accompanied by sedation, motor ataxia or behavioural changes (rearing, crossing, circling, dipping) as assessed by use of a box maze procedure. 5. L-NG nitro arginine methyl ester (L-NAME, 20 mg kg-1, i.v.) but not TRIM (0.5-20 mg kg-1, i.v.) increased mean arterial blood pressure (MAP) in the urethane-anaesthetized rat. 6. L-NAME (100 microM) potentiated the contractile response of the rabbit isolated aorta to phenylephrine (ED50; 0.084 +/- 0.01 microM in the presence and 0.25 +/- 0.05 microM in the absence of L-NAME; maximum response, 7.7 +/- 0.4 g in the presence and 5.6 +/- 0.5 g in the absence of L-NAME, n = 6, (P < 0.05) whilst TRIM (1-100 microM) was without effect. L-NAME (100 microM) but not TRIM (1-100 microM) also reduced carbachol-induced relaxation of the phenylephrine-precontracted rabbit aorta preparation. 7. L-NAME (50 microM) potentiated the vasoconstrictor effect of bolus-injected noradrenaline (10-1000 nmol) and reduced the vasodilator effect of carbachol (10 microM) added to the Krebs reservoir in the rat perfused mesentery preparation. L-NAME (50 microM) also reduced nitric oxide (NO) release (measured by chemiluminescence of nitrite in the Krebs perfusate) in response to noradrenaline (100 nmol; 53.8 +/- 4.0 pmol ml-1 in the presence and 84.8 +/- 8.0 pmol ml-1 in the absence of L-NAME, n = 15, P < 0.05) and carbachol (10 microM; 63.9 +/- 5.0 pmol ml-1 in the presence and 154.0 +/- 9.0 pmol ml-1 in the absence of L-NAME, n = 15, P < 0.05). TRIM (50 microM) did not affect either the vasoconstrictor response to noradrenaline or the vasodilator response to carbachol or the accompanying release of NO from the perfused rat mesentery.

The antinociceptive effect of 1-(2-trifluoromethylphenyl) imidazole (TRIM), a potent inhibitor of neuronal nitric oxide synthase in vitro, in the mouse.

1-(2-trifluoromethylphenyl)imidazole (TRIM) is a potent inhibitor of neuronal (mouse cerebellar) and inducible (lung from endotoxin-pretreated rats) isoforms of nitric oxide synthase (NOS) with IC50 values of 28.2 microM and 27.0 microM, respectively. In contrast, TRIM is a poor inhibitor of bovine aortic endothelial NOS with an IC50 of 1057.5 microM. TRIM (10-50 mg kg-1) administered i.p. exhibits dose-related antinociceptive activity in the mouse (assessed as inhibition of late phase formalin-induced hindpaw licking behaviour) with an ED50 of 85.8 mumol kg-1. In contrast, TRIM (50 mg kg-1, i.p.) failed to influence mean arterial blood pressure in the urethane-anaesthetized mouse. Thus, TRIM may be of use as an experimental tool with which to investigate the biological roles of nitric oxide (NO) within the central nervous system.

Augmented antinociception following 7-nitro indazole and flurbiprofen in the conscious mouse.

Co-administration of the nitric oxide synthase inhibitor, 7-nitro indazole (1 mg/kg i.p.), with the cyclooxygenase inhibitor, flurbiprofen (5-75 mg/kg i.p.), resulted in significantly enhanced antinociceptive activity in mice (formalin-induced hindpaw licking assay) without affecting hindpaw inflammation. No antinociception was observed in animals pretreated with 7-nitro indazole (1 mg/kg i.p.) and flurbiprofen (100 micrograms subplantar). Flurbiprofen (50 mg/kg i.p.) pretreatment did not influence the inhibition of cerebellar or spinal cord nitric oxide synthase activity observed after 7-nitro indazole (1 or 25 mg/kg i.p.) administration and did not alter blood pressure in anaesthetised animals. Thus, flurbiprofen acts by a mechanism unrelated to a local anti-inflammatory effect in the hindpaw. Since nitric oxide synthase inhibitors are antinociceptive by an effect in the spinal cord (dorsal horn) this would appear to be a likely location for the nitric oxide synthase and cyclooxygenase enzymes targetted by 7-nitro indazole and flurbiprofen respectively.

Effect of 7-nitro indazole on neurotransmission in the rat vas deferens: mechanisms unrelated to inhibition of nitric oxide synthase.

1. The effect of the nitric oxide synthase (NOS) inhibitor, 7-nitro indazole (7-NI), on sympathetic and purinergic neurotransmission in the rat isolated vas deferens preparation has been studied. 2. 7-NI (50-200 microM) caused a dose- and frequency-dependent inhibition of the phasic (predominantly purinergic) contractile response of the rat vas deferens to electrical (field) stimulation (100 V, 0.5 ms). Greatest inhibition occurred at lower frequencies of stimulation (0.1-10 Hz). The sustained tonic contractile response (predominantly noradrenergic) was inhibited only at a high frequency of stimulation (60 Hz) and only at the highest concentration of 7-NI studies (200 microM). 3. 7-NI (100 microM) significantly reduced the contractile response of the vas deferens to exogenous ATP (20 microM-5 mM) and the stable P2X purinoceptor agonist, alpha, beta-methylene ATP (2.5 and 25.0 microM) but was without effect on contractions due to noradrenaline (0.1-50 microM) indicating a lack of antagonist effect on post-junctional alpha 1 adrenoceptors. 4. The effect of 7-NI (100 microM) on the phasic contractile response to field stimulation (0.1 and 2.0 Hz) was unaffected by preincubation of preparations with yohimbine (1.0 microM) or propranolol (0.01-10.0 microM) indicating the absence of involvement of alpha 2- or beta-adrenoceptors in this response. 5. 7-NI (50-600 microM) caused dose-related inhibition of contractions elicited by addition of a depolarizing concentration of KCl (64 mM). 6. The effect of 7-NI (100 microM) on the phasic contractile response to field stimulation (0.1 and 2.0 Hz) was unaffected by preincubation of preparations with L-arginine (1 mM). Neither L-arginine (1 mM) nor NC nitro L-arginine methyl ester (L-NAME, 100 LM) affected the response of the vas deferens to field stimulation at 0.1 or 2.0 Hz. Nitric oxide synthase (NOS) enzyme activity, measured as the conversion of[3H]-L-arginine to [3H]-citrulline, was not detectable in rat vas deferens homogenates.7. 7-Nr preferentially inhibits the purinergic component of the response of the rat vas deferens to field stimulation. The mechanism of action of 7-NI is not known but is not related to NOS inhibition. It seems likely that 7-NI combines an antagonist action at smooth muscle cell P2X-purinoceptors with the ability to inhibit the cellular influx of calcium ions. Although these hitherto unrecorded effects of 7-NI occur at relatively high concentrations, the effects described may contribute to the pharmacological effects of this NOS inhibitor.

Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects.

1. 7-Nitro indazole (7-NI, 10-50 mg kg-1), 6-nitro indazole and indazole (25-100 mg kg-1) administered i.p. in the mouse produce dose-related antinociception in the late phase of the formalin-induced hindpaw licking and acetic acid-induced abdominal constriction assays. The ED50 values (mg kg-1) were as follows: 7-NI (27.5 and 22.5), 6-nitro indazole (62.5 and 44.0) and indazole (41.0 and 48.5) in the two assays respectively. 3-Indazolinone, 6 amino indazole and 6-sulphanilimido indazole (all 50 mg kg-1) were without effect. With the exception of 5-nitro indazole (50 mg kg-1) which produced sedation, none of the other indazole derivates examined caused overt behavioural changes. 2. The antinociceptive effect of 7-NI (25 mg kg-1, i.p.) in the late phase of the formalin-induced hindpaw licking assay was partially (46.7 +/- 16.2%, n = 18) reversed by pretreatment with L- but not D-arginine (both 50 mg kg-1, i.p.). 3. The time course of 7-NI induced antinociception in the mouse was correlated with inhibition of brain (cerebellum) nitric oxide synthase (NOS) activity. Maximum antinociceptive activity and NOS inhibition was detected 18-30 min following i.p. administration. In contrast, no antinociceptive effect or inhibition of cerebellar NOS was detected 75 min post-injection. 4. 7-NI, 6-nitro indazole, indazole, 3-indazolinone and 6-amino indazole (all 50 mg kg-1) failed to influence mean arterial pressure (MAP) over the 45 min after i.p. administration in the anaesthetized mouse. Similarly, 7-NI (25 mg kg-1) administered i.v. in the anaesthetized rat did not increase MAP or influence the vasodepressor effect of i.v. injected acetylcholine (ACh) over the same period.5. 7-NI (100 microM) did not influence the vasorelaxant effect of ACh (IC50, 0.2 +/- 0.04 microM, cf. 0.16+/-0.06 microM, n = 6) in phenylephrine-precontracted rabbit aortic rings.6. These data provide further evidence that antinociception following administration of 7-NI in the mouse results from inhibition of central NOS activity and is not associated with inhibition of in vivo vascular endothelial cells NOS. Accordingly, 7-NI (or a derivative thereof) may provide an alternative approach to the development of novel antinociceptive drugs.

The modulation by nedocromil sodium of proteases released from rat peritoneal mast cells capable of degrading vasoactive intestinal peptide and calcitonin gene-related peptide.

Tryptase and chymase released from activated mast cells degrade the neuropeptides calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) to peptide fragments. We have examined whether nedocromil sodium can modulate the ability of rat activated peritoneal mast cells to degrade 125I-CGRP and 125I-VIP. Mast cell-dependent degradation of both 125I-CGRP and 125I-VIP was observed with compound 48/80 (0.03-1 microgram/ml) and in the case of 125I-VIP with anti-IgE (1-20 micrograms/ml). Nedocromil sodium (10(-6)-10(-4) M) caused significant inhibition of neuropeptide degradation, with the most effective inhibition observed against anti-IgE-induced degradation of 125I-VIP. Nedocromil sodium had no inhibitory effect on the ability of lysed mast cells, bovine trypsin or chymotrypsin to breakdown 125I-VIP. These results suggest that nedocromil sodium inhibits mast cell-dependent degradation of neuropeptides, such as VIP, as a secondary consequence of inhibiting the release of mast cell proteases.

L-NG-nitro arginine p-nitroanilide (L-NAPNA) is anti-nociceptive in the mouse.

L-NG-nitro arginine p-nitroanilide (L-NAPNA), L-NG nitro arginine methyl ester (L-NAME) and L-NG-monomethyl arginine (L-NMMA) inhibit rat cerebellar nitric oxide synthase (NOS) with IC50s of 1.4 +/- 0.1 microM, 0.81 +/- 0.16 microM and 5.1 +/- 0.07 microM respectively. L-NAPNA inhibits the late phase of formalin-induced hindpaw licking (ED50, 57.2 mg kg-1) and acetic acid induced abdominal constrictions (ED50, 25 mg kg-1) in the mouse. L-NAPNA is approximately 65 times less active than L-NAME as an inhibitor of endothelium-dependent relaxation in the rabbit aorta and about 10 fold less potent as a vasopressor in the anaesthetized mouse. LNAPNA shows some degree of selectivity for the central NOS isoform and may be of clinical interest for the treatment of pain.

7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure.

7-Nitro indazole (7-NI) inhibits mouse cerebellar nitric oxide synthase (NOS) in vitro with an IC50 of 0.47 microM. Following i.p. administration in mice, 7-NI (10-50 mg kg-1) produces dose-related anti-nociception as evidenced by an inhibition of late phase (15-30 min) but not early phase (0-5 min) hindpaw licking time following subplantar injection of formalin (10 microliters, 5% v/v). The ED50 for this effect was 26 mg kg-1 (equivalent to 159.5 mumol kg-1). Similar i.p. administration of 7-NI (20 and 80 mg kg-1) in urethane-anaesthetized mice failed to increase MAP. Thus, 7-NI is a novel inhibitor of NOS which exhibits selectivity for the brain enzyme. Accordingly, 7-NI may be a useful starting point for the development of selective, centrally acting NOS inhibitors devoid of cardiovascular side effects and as a tool to study the central pharmacological effects of nitric oxide (NO).

Synergistic anti-nociceptive effect of L-NG-nitro arginine methyl ester (L-NAME) and flurbiprofen in the mouse.

1. L-NG-nitro arginine methyl ester (L-NAME) administered i.p. produces anti-nociception in the mouse assessed by the formalin-induced paw licking and acetic acid-induced abdominal constriction models. The non-steroidal anti-inflammatory drug (NSAID), flurbiprofen, was similarly anti-nociceptive in both models. 2. Combination of a sub-threshold dose of L-NAME (10 mg kg-1) with increasing doses of flurbiprofen (25- 75 mg kg-1) or a sub-threshold dose of flurbiprofen (50 mg kg-1) with increasing doses of L-NAME (10- 100 mg kg-1) resulted in potentiated anti-nociception in the formalin model. Combined therapy with sub-threshold doses of L-NAME (10 mg kg-1) and indomethacin (10 mg kg-1) also resulted in significant anti-nociception. In addition, combining sub-threshold doses of L-NAME (12.5 mg kg-1) and flurbiprofen (2 mg kg-1) significantly reduced acetic acid-induced abdominal constriction. 3. L-NAME (10 mg kg-1) administered i.p. caused a significant (approximately 35%) increase in MAP in the urethane-anaesthetized mouse. Flurbiprofen (50 mg kg-1) was inactive. Combination treatment with L-NAME (10 mg kg-1) and flurbiprofen (50 mg kg-1) failed to elevate MAP above that observed with L-NAME alone. Neither L-NAME (10 mg kg-1) nor flurbiprofen (50 mg kg-1) either alone or in combination significantly altered mouse locomotor activity. 4. These results suggest that L-NAME and flurbiprofen/indomethacin act synergistically in their anti-nociceptive action in the mouse. Combination therapy with L-NAME and flurbiprofen and a similar NSAID may provide an alternative to the clinical control of pain in man.

Catabolism of 6 keto PGE1: biological activation by the rat kidney in vitro.

An increase in anti-aggregatory but not spasmogenic activity was observed when 6 keto prostaglandin E1 (but not PGE1) was incubated at 37 degrees C with rat kidney 100 000 X g supernatant. No such biological activation was observed in boiled rat kidney supernatant. After high-pressure liquid chromatography two absorbance peaks with anti-aggregatory activity were detected. One peak had a retention time identical to authentic 6 keto prostaglandin E1 whilst the second peak did not coincide with known anti-aggregatory prostaglandins.