Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. TRPV1 is pivotal in initiating symptoms of rhinitis. characterization of SB-705498 in guinea pig isolated trigeminal ganglion cell bodies from cells innervating nasal mucosa and the pharmacology of SB-705498 on capsaicin and hypertonic saline-induced intranasal fluid secretion in the guinea pig, as measured by MRI. Materials and methods All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. SB-705498 was manufactured at GlaxoSmithKline. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny an Axoxlamp 200B amplifier. 10 mM stock aliquots of capsaicin in ethanol and a 10 mM stock solution of SB-705498 in DMSO were prepared and stored at ?20C. Drug applications were controlled by a fast application system (Biologic RSC200, time for solution exchange 30 ms) using a triple-barrel pipette assembly. DiI Cpositive cells bodies were identified punctuate fluorescence. Preliminary experiments demonstrated that repeated application of capsaicin to trigeminal cell bodies underwent significant tachyphylaxis. Macroscopic currents to 1 M capsaicin, however, were stable over a 60 s period (Figure 2) enabling the effect of SB-705498 to be examined. Until stable, 1 M capsaicin was applied (20 s) and then SB-705498 and capsaicin solutions were co applied for 20 s before switching back to 1 M capsaicin. A single concentration of SB-705498 was tested on each trigeminal cell body and the data pooled. Open in a separate window Figure 2 SB-705498 inhibits the capsaicin-induced current in nasally innervated guinea pig trigeminal cell bodies. (A) 1 M capsaicin (black bar) produced an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response (red bar) produced almost complete inhibition that was rapidly reversed upon washout of SB-705498. (B) Pooled data generated from similar experiments to those described in A. Data were fitted with the Hill equation and gave an pIC50 estimate of 7.2 and a Hill coefficient of 1.47. Data was graphically represented using Graphpad Prism (Version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and a non-linear regression fitted using the built in equation log (inhibitor) versus normalized response C variable slope (also known as the Hill equation: = 100{1 + 10[(logIC50 ? = normalized response and = log [SB-705498]) to generate a pIC50 value and Hill coefficient. Study details In vivo guinea pig studies Female Dunkin-Hartley guinea pigs were obtained from Harlan, UK at 180C200 g upon arrival. Animals were housed in groups of six in a temperature and humidity controlled environment, with a 12-h light : dark cycle. Food and water were available allowed subsequent identification of the isolated ganglionic cell bodies with afferents terminating in the nasal mucosa. These fluorescently labelled cells were used in the patch clamp experiments. Figure 2A shows that 1 M capsaicin generated an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. The potency of SB-705498 was evaluated in trigeminal ganglion cell bodies; a single concentration of SB-705498 was evaluated per cell and the data pooled for concentrationCresponse analysis (multiple concentrations per cell were not possible because of run-down). The concentration response curve generated a pIC50 of 7.2 and Hill coefficient of 1.47. < 0.05 compared with capsaicin vehicle challenge; = 6) was achieved at 0.3 and 1 mM capsaicin (50 L). No significant increases were seen with the vehicle challenge group. The 0.3 mM capsaicin response was shown to be completely inhibited by 10 mgmL?1 atropine (Figure 4). All further studies characterizing the effect of SB-705498 used a capsaicin challenge concentration/volume of.Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post 0.3 mM capsaicin ipsilateral challenge. were examined. Key Results SB-705498 concentration-dependently inhibited capsaicin-induced currents in isolated trigeminal ganglion cells (pIC50 7.2). oral and intranasal routes; efficacy was optimized using particle-reduced SB-705498. We propose that TRPV1 is pivotal in initiating symptoms of rhinitis. characterization of SB-705498 in guinea pig isolated trigeminal ganglion cell bodies from cells innervating nasal mucosa and the pharmacology of SB-705498 on capsaicin and hypertonic saline-induced intranasal fluid secretion in the guinea pig, as measured by MRI. Materials and methods All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. SB-705498 was manufactured at GlaxoSmithKline. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny an Axoxlamp 200B amplifier. 10 mM stock aliquots of capsaicin in ethanol and a 10 mM stock solution of SB-705498 in DMSO were prepared and stored at ?20C. Drug applications were controlled by a fast application system (Biologic RSC200, time for HTH-01-015 solution exchange 30 ms) using a triple-barrel pipette assembly. DiI Cpositive cells bodies were identified punctuate fluorescence. Preliminary experiments demonstrated that repeated application of capsaicin to trigeminal cell bodies underwent significant tachyphylaxis. Macroscopic currents to 1 M capsaicin, however, were stable over a 60 s period (Figure 2) enabling the effect of SB-705498 to be examined. Until stable, 1 M capsaicin was applied (20 s) and then SB-705498 and capsaicin solutions were co applied for 20 s before switching back to 1 M capsaicin. A single concentration of SB-705498 was tested on each trigeminal cell body and the data pooled. Open in a separate window Figure 2 SB-705498 inhibits the capsaicin-induced current in nasally innervated guinea pig trigeminal cell bodies. (A) 1 M capsaicin (black bar) produced an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response (red bar) produced almost complete inhibition that was rapidly reversed upon washout of SB-705498. (B) Pooled data generated from similar experiments to those described in A. Data were fitted with the Hill equation and gave an pIC50 estimate of 7.2 and a Hill coefficient of 1.47. Data was graphically represented using Graphpad Prism (Version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and a non-linear regression fitted using the built in equation log (inhibitor) versus normalized response C variable slope (also known as the Hill equation: = 100{1 + 10[(logIC50 ? = normalized response and = log [SB-705498]) to generate a pIC50 value and Hill coefficient. Study details In vivo guinea pig studies Female Dunkin-Hartley guinea pigs were obtained from Harlan, UK at 180C200 g upon arrival. Animals were housed in groups of six in a temperature and humidity controlled environment, with a 12-h light : dark cycle. Food and water were available allowed subsequent identification of the isolated ganglionic cell bodies with afferents terminating in the nasal mucosa. These fluorescently labelled cells were used in the patch clamp experiments. Figure 2A shows that 1 M capsaicin generated an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. The potency of SB-705498 was evaluated in trigeminal ganglion cell bodies; a single concentration of SB-705498 was evaluated per cell and the data pooled for concentrationCresponse analysis (multiple concentrations per cell were not possible because of run-down). The concentration response curve generated a pIC50 of 7.2 and Hill coefficient of 1.47. < 0.05 compared with capsaicin vehicle challenge; = 6) was achieved at 0.3 and 1 mM capsaicin (50 L). No significant increases were seen with the vehicle challenge group. The 0.3 mM capsaicin response was shown to be completely inhibited by 10 mgmL?1 atropine (Figure 4). All further studies characterizing the effect of SB-705498 used a capsaicin challenge concentration/volume of 0.3 mM/50 L. Open in a separate window Figure 3 Effect of capsaicin on contralateral fluid secretion. Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post-capsaicin ipsilateral challenge. *< 0.05; compared with vehicle pretreatment group, = 7/8 per group. anova, with Dunnett's follow-up analysis. Open in a separate window Figure 4 Effect of intranasal atropine on capsaicin and hypertonic saline-induced nasal secretion. Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post 0.3 mM capsaicin ipsilateral challenge. **< 0.001; compared with vehicle pretreatment group, = 5/8 per group. anova, with Dunnett's follow-up analysis. The stimulatory effect of hypertonic saline is also shown in Figure 4. Whereas 0.9% saline was ineffective in.At the lowest concentration of 1 mgmL?1 (which given as a 50 L bilateral dose equates to a 50 g total dose per animal) a 50% inhibition of the capsaicin response was observed. and hypertonic saline-induced intranasal fluid secretion in the guinea pig, as measured by MRI. Materials and methods All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. SB-705498 was manufactured at GlaxoSmithKline. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny an Axoxlamp 200B amplifier. 10 mM stock aliquots of capsaicin in ethanol and a 10 mM stock solution of SB-705498 in DMSO were prepared and stored at ?20C. Drug applications were controlled by a fast application system (Biologic RSC200, time for solution exchange 30 ms) using a triple-barrel pipette assembly. DiI Cpositive cells bodies were identified punctuate fluorescence. Preliminary experiments demonstrated that repeated application of capsaicin to trigeminal cell bodies underwent significant tachyphylaxis. Macroscopic currents to 1 M capsaicin, however, were stable over a 60 s period (Figure 2) enabling the effect HTH-01-015 of SB-705498 to be examined. Until stable, 1 M capsaicin was applied (20 s) and then SB-705498 and capsaicin solutions were co applied for 20 s before switching back to 1 M capsaicin. A single concentration of SB-705498 was tested on each trigeminal cell body and the data pooled. Open in a separate window Figure 2 SB-705498 inhibits the capsaicin-induced current in nasally innervated guinea pig trigeminal cell bodies. (A) 1 M capsaicin (black bar) produced an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response (red bar) produced almost complete inhibition that was rapidly reversed upon washout of SB-705498. (B) Pooled data generated from similar experiments to those described in A. Data were fitted with the Hill equation and gave an pIC50 estimate of 7.2 and a Hill coefficient of 1.47. Data was graphically represented using Graphpad Prism (Version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and a non-linear regression fitted using the built in equation log (inhibitor) versus normalized response C variable slope (also known as the Hill equation: = 100{1 + 10[(logIC50 ? = normalized response and = log [SB-705498]) to generate a pIC50 value and Hill coefficient. Study details In vivo guinea pig studies Female Dunkin-Hartley guinea pigs were obtained from Harlan, UK at 180C200 g upon arrival. Animals were housed in groups of six in a temperature and humidity controlled environment, with a 12-h light : dark cycle. Food and water were available allowed subsequent identification of the isolated ganglionic cell bodies with afferents terminating in the nasal mucosa. These fluorescently labelled cells were used in the patch clamp experiments. Figure 2A shows that 1 M capsaicin generated an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. The potency of SB-705498 was evaluated in trigeminal ganglion cell bodies; a single concentration of SB-705498 was evaluated per cell and the data pooled for concentrationCresponse analysis (multiple concentrations per cell were not possible because of run-down). The concentration response curve generated a pIC50 of 7.2 and Hill coefficient of 1.47. < 0.05 compared.This long duration of action of SB-705498 seen in the nose of the guinea pig may therefore be in part associated with the clearance mechanisms required for a suspension that is mucocilliary and or absorption through the mucosal epithelium. In addition to its inhibitory action on capsaicin-induced neuronal responses, SB-705498 was also effective in inhibiting fluid secretory responses to 10% hypertonic saline, demonstrating that TRPV1 plays a major role in hypertonic saline responses in vivo. and the pharmacology of SB-705498 on capsaicin and hypertonic saline-induced intranasal fluid secretion in the guinea pig, as measured by MRI. Materials and methods All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. SB-705498 was manufactured at GlaxoSmithKline. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny an Axoxlamp 200B amplifier. 10 mM stock aliquots of capsaicin in ethanol and a 10 mM stock solution of SB-705498 in DMSO were prepared and stored at ?20C. Drug applications were controlled by a fast application system (Biologic RSC200, time for solution exchange 30 ms) using a triple-barrel pipette assembly. DiI Cpositive cells bodies were identified punctuate fluorescence. Preliminary experiments demonstrated that repeated application of capsaicin to trigeminal cell bodies underwent significant tachyphylaxis. Macroscopic currents to 1 M capsaicin, however, were stable over a 60 s period (Figure 2) enabling the effect of SB-705498 to be examined. Until stable, 1 M capsaicin was applied (20 s) and then SB-705498 and capsaicin solutions were co applied for 20 s before switching back to 1 M capsaicin. A single concentration of SB-705498 was tested on each trigeminal cell body and the data pooled. Open in a separate window Figure 2 SB-705498 inhibits the capsaicin-induced current in nasally innervated guinea pig trigeminal cell bodies. (A) 1 M capsaicin (black bar) produced an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response (red bar) produced almost complete inhibition that was rapidly reversed upon washout of SB-705498. (B) Pooled data generated from similar experiments to those described in A. Data were fitted with the Hill equation and gave an pIC50 estimate of 7.2 and a Hill coefficient of 1.47. Data was graphically represented using Graphpad Prism (Version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and a non-linear regression fitted using TRADD the built in equation log (inhibitor) versus normalized response C variable slope (also known as the Hill equation: = 100{1 + 10[(logIC50 ? = normalized response and = log [SB-705498]) to generate a pIC50 value and Hill coefficient. Study details In vivo guinea pig studies Female Dunkin-Hartley guinea pigs were obtained from Harlan, UK at 180C200 g upon arrival. Animals were housed in groups of six in a temperature and humidity controlled environment, with a 12-h light : dark cycle. Food and water were available allowed subsequent identification of the isolated ganglionic cell bodies with afferents terminating in the nasal mucosa. These fluorescently labelled cells were used in the patch clamp experiments. Figure 2A shows that 1 M capsaicin generated an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. The potency of SB-705498 was evaluated in trigeminal ganglion cell bodies; a single concentration of SB-705498 was evaluated per cell and the data pooled for concentrationCresponse analysis (multiple concentrations per cell were not possible because of run-down). The concentration response curve generated a pIC50 of 7.2 and Hill coefficient of 1.47. < 0.05 compared with capsaicin vehicle challenge; = 6) was achieved at 0.3 and 1 mM capsaicin (50 L). No significant increases were seen with the vehicle challenge group. The 0.3 mM capsaicin response was shown to be completely inhibited by 10 mgmL?1 atropine (Figure 4). All further studies characterizing the effect of SB-705498 used a capsaicin challenge concentration/volume of 0.3 mM/50 L. Open in a separate window Figure 3 Effect of capsaicin on contralateral fluid secretion. Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post-capsaicin ipsilateral challenge. *< 0.05; compared with vehicle pretreatment group, = 7/8 per group. anova, with Dunnett's follow-up analysis. Open in a separate window Figure 4 Effect of intranasal atropine on capsaicin and hypertonic saline-induced nasal secretion. Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post 0.3 mM capsaicin ipsilateral challenge. **< 0.001; compared with vehicle pretreatment group, = 5/8 per group. anova, with Dunnett's follow-up analysis. The stimulatory effect of hypertonic saline is also shown in Figure 4. Whereas 0.9% saline was ineffective in producing a contralateral fluid response (Figure 3), 10% saline induced a significant increase of approximately 26 mm3: this response.Pretreatment = micronized suspension of SB-705498 at 10 mgmL?1. trigeminal ganglion cell bodies from cells innervating nasal mucosa and the pharmacology of SB-705498 on capsaicin and hypertonic saline-induced intranasal fluid secretion in the guinea pig, as measured by MRI. Materials and methods All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. SB-705498 was manufactured at GlaxoSmithKline. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny an Axoxlamp 200B amplifier. 10 mM stock aliquots of capsaicin in ethanol and a 10 mM stock solution of SB-705498 in DMSO were prepared and stored at ?20C. Drug applications were controlled by a fast application system (Biologic RSC200, time for solution exchange 30 ms) using a triple-barrel pipette assembly. DiI Cpositive cells bodies were identified punctuate fluorescence. Preliminary experiments demonstrated that repeated application of capsaicin to trigeminal cell bodies underwent significant tachyphylaxis. Macroscopic currents to 1 M capsaicin, however, were stable over a 60 s period (Figure 2) enabling the effect of SB-705498 to be examined. Until HTH-01-015 stable, 1 M capsaicin was applied (20 s) and then SB-705498 and capsaicin solutions were co applied for 20 s before switching back to 1 M capsaicin. A single concentration of SB-705498 was tested on each trigeminal cell body and the data pooled. Open in a separate window Figure 2 SB-705498 inhibits the capsaicin-induced current in nasally innervated guinea pig trigeminal cell bodies. (A) 1 M capsaicin (black bar) produced an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response (red bar) produced almost complete inhibition that was rapidly reversed upon washout of SB-705498. (B) Pooled data generated from similar experiments to those described in A. Data were fitted with the Hill equation and gave an pIC50 estimate of 7.2 and a Hill coefficient of 1.47. Data was graphically represented using Graphpad Prism (Version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and a non-linear regression fitted using the built in equation log (inhibitor) versus normalized response C variable slope (also known as the Hill equation: = 100{1 + 10[(logIC50 ? = normalized response and = log [SB-705498]) to generate a pIC50 value and Hill coefficient. Study details In vivo guinea pig studies Female Dunkin-Hartley guinea pigs were obtained from Harlan, UK at 180C200 g upon arrival. Animals were housed in groups of six in a temperature and humidity controlled environment, with a 12-h light : dark cycle. Food and water were available allowed subsequent identification of the isolated ganglionic cell bodies with afferents terminating in the nasal mucosa. These fluorescently labelled cells were used in the patch clamp experiments. Figure 2A shows that 1 M capsaicin generated an inward current that was stable over the 60 s application period. Application of 300 nM SB-705498 during the capsaicin response produced almost complete inhibition that was rapidly reversed upon washout. The potency of SB-705498 was evaluated in trigeminal ganglion cell bodies; a single concentration of SB-705498 was evaluated per cell and the data pooled for concentrationCresponse analysis (multiple concentrations per cell were not possible because of run-down). The concentration response curve generated a pIC50 of 7.2 and Hill coefficient of 1.47. < 0.05 compared with capsaicin vehicle challenge; = 6) was achieved at 0.3 and 1 mM capsaicin (50 L). No significant increases were seen with the vehicle challenge group. The 0.3 mM capsaicin response was shown to be completely inhibited by 10 mgmL?1 atropine (Figure 4). All further studies characterizing the effect of SB-705498 used a capsaicin challenge concentration/volume of 0.3 mM/50 L. Open in a separate window Figure 3 Effect of capsaicin on contralateral fluid secretion. Baseline measurements = pre capsaicin challenge, 10 min data point = 10 min post-capsaicin ipsilateral challenge. *< 0.05; compared with vehicle pretreatment group, = 7/8 per group. anova, with Dunnett's follow-up analysis. Open in a separate window Figure.