Acute and Direct Effects of Sodium-Glucose Cotransporter 2 Inhibition on Glomerular Filtration Rate in Spontaneously Diabetic Torii Fatty Rats.

Recent clinical studies indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors exhibit a renoprotective effect. While studies at the single nephron level suggest that direct effects of SGLT2 inhibitors on renal hemodynamics may be a possible mechanism underlying their renoprotective effect, few studies have focused on such direct effects at the whole-kidney level. In the present study, we investigated the acute and direct effect of SGLT2 inhibition on creatinine clearance, an index of whole-kidney glomerular filtration rate (GFR), in a rat model of type 2 diabetes. Twelve to fifteen-week-old male Spontaneously Diabetic Torii (SDT) fatty rats and Sprague-Dawley rats were used as diabetic animals and non-diabetic controls, respectively. Under general anesthesia, baseline urine samples were collected from the left and right ureters for 1 h. The selective SGLT2 inhibitor ipragliflozin or vehicle was subsequently administered intravenously and post-drug urine was collected for 1 h. Baseline and post-drug blood samples were collected immediately before baseline urine collection and immediately after post-drug urine collection, respectively. Plasma glucose, urine volume, urinary glucose and albumin excretion were measured, and creatinine clearance was calculated. Blood pressure and heart rate were monitored continuously throughout the experiment. A single intravenous injection of ipragliflozin increased both urine output and glucose excretion, but reduced creatinine clearance without affecting systemic blood pressure. These results suggest that SGLT2 inhibition directly reduced whole-kidney GFR, most likely due to a reduction in intraglomerular pressure, by altering local renal hemodynamics, which may contribute to the renoprotective effects demonstrated in clinical studies.

Effect of ipragliflozin, an SGLT2 inhibitor, on cardiac histopathological changes in a non-diabetic rat model of cardiomyopathy.

AIMS: We investigated the effect of the selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin on cardiac dysfunction and histopathology in a non-diabetic rat model of cardiomyopathy. MAIN METHODS: Ipragliflozin was mixed with chow (0.01%, w/w) and administered to male DahlS.Z-Leprfa/Leprfa (DS/obese) rats for 8 weeks. Male DahlS.Z-Lepr+/Lepr+ (DS/lean) rats of the same age were used as controls. Systolic blood pressure (SBP) and heart rate (HR) were measured every 4 weeks. After 8 weeks of treatment, echocardiography and histopathological examinations were performed. Further, the effect of ipragliflozin on blood and urine parameters were investigated. KEY FINDINGS: In the DS/obese rats, ipragliflozin delayed the age-related increase in SBP without affecting HR, reduced left ventricular (LV) mass and intraventricular septal thickness in echocardiography, and ameliorated hypertrophy of cardiomyocytes and LV fibrosis in histopathological examination. Although ipragliflozin significantly increased both urine volume and urinary glucose excretion in DS/obese rats, it did not alter plasma glucose levels. SIGNIFICANCE: Ipragliflozin prevented LV hypertrophy and fibrosis in non-diabetic DS/obese rats without affecting plasma glucose levels. These findings suggest that SGLT2 inhibitors have a cardio-protective effect in non-diabetic patients with cardiomyopathy.

In Vitro Pharmacological Profile of Ipragliflozin, a Sodium Glucose Co-transporter 2 Inhibitor.

Ipragliflozin, a selective sodium glucose cotransporter 2 (SGLT2) inhibitor, is used for the treatment of type 2 diabetes mellitus. To date, the only known in vitro pharmacological characteristic of ipragliflozin is its selectivity for SGLT2 over SGLT1, which was previously reported by our group. Therefore, in this study, we investigated other in vitro pharmacological characteristics of ipragliflozin and compared them with those of phlorizin, a naturally occurring SGLT inhibitor. Selectivity of ipragliflozin and phlorizin for human (h) SGLT2 over hSGLT3, hSGLT4, hSGLT5, hSGLT6 and hSodium/myo-inositol (MI) cotransporter 1 (hSMIT1) was examined in Chinese hamster ovary (CHO) cells overexpressing each transporter using specific radio-ligands. Ipragliflozin had higher selectivity for hSGLT2 than other hSGLTs. Phlorizin showed lower selectivity for hSGLT2 compared to ipragliflozin. Studies using CHO cells overexpressing hSGLT2 demonstrated that both ipragliflozin and phlorizin competitively inhibited SGLT2-mediated methyl-α-D-glucopyranoside (AMG) uptake with an inhibitory constant (Ki) of 2.28 and 20.2 nM, respectively. Ipragliflozin, but not phlorizin, inhibited hSGLT2 in a wash-resistant manner, suggesting that binding of ipragliflozin to hSGLT2 was persistent. These data demonstrate that ipragliflozin is a competitive inhibitor of SGLT2, has high selectivity for SGLT2 over not only SGLT1 but also other SGLT family members, and binds persistently to hSGLT2.

First-dose effect of the SGLT2 inhibitor ipragliflozin on cardiovascular activity in spontaneously diabetic Torii fatty rats.

The first dose of a sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor induces osmotic diuresis and can thereby affect cardiovascular activity in hyperglycemic patients. We aimed to determine whether the first dose of the selective SGLT2 inhibitor ipragliflozin affects cardiovascular activity in non-diabetic Sprague-Dawley (SD) rats and Spontaneously Diabetic Torii (SDT) fatty rats in two studies, a urine collection study and a telemetry study. In the former study, urine was collected for 24 hours after a single oral dose of ipragliflozin. In the latter study, systolic blood pressure (SBP) and heart rate (HR) were continuously monitored for 24 hours under conscious and unrestrained conditions from immediately before the administration of ipragliflozin. The telemetry study was conducted in a crossover design at successive 1 week intervals. Cardiovascular autonomic nerve activity was calculated from the SBP and HR. SDT fatty rats exhibited polyuria, glucosuria and hyperglycemia. In addition, the mean and standard deviation of SBP were higher, while the coefficient of variance of HR was lower than the respective parameters in SD rats. Ipragliflozin increased both urine output and urinary glucose excretion, and the increases were more pronounced in SDT fatty rats than in SD rats. In contrast, ipragliflozin had no effect on SBP, the standard deviation of SBP, HR, and the coefficient of variance of HR, or on autonomic nerve activity in either rat strain. These results suggest that the first dose of the SGLT2 inhibitor ipragliflozin has little impact on cardiovascular activity despite causing glucosuria with osmotic diuresis.

Protective Effect of Ipragliflozin on Pancreatic Islet Cells in Obese Type 2 Diabetic db/db Mice.

Ipragliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that increases urinary glucose excretion and subsequently improves hyperglycemia in patients with type 2 diabetes mellitus (T2DM). To assess the beneficial effect of ipragliflozin on the mass and function of pancreatic ß-cells under diabetic conditions, obese T2DM db/db mice were treated with ipragliflozin for 5 weeks. Glucose and lipid metabolism parameters, pathological changes in pancreatic islet cells and insulin content were evaluated. Pathological examination of pancreatic islet cells comprised measuring the ratios of insulin- and glucagon-positive cells and levels of oxidative stress markers. Hemoglobin A1c, plasma glucose, non-esterified fatty acid and triglyceride levels in ipragliflozin-treated groups were reduced compared to the diabetic control (DM-control) group. Histopathological examination of pancreatic islet cells revealed strong insulin staining and reduced glucagon staining in the ipragliflozin 10 mg/kg-treated group compared with the DM-control group. The ratio of α- to ß-cell mass was lower in the ipragliflozin 10 mg/kg-treated group than the DM-control group and was similar to that of the non-diabetic control group. The density of immunostaining for 4-hydroxy-2-nonenal, an oxidative stress marker, in pancreatic islets was significantly lower in the ipragliflozin 10 mg/kg-treated group than the DM-control group. Pancreatic insulin content tended to be higher in the ipragliflozin-treated groups than the DM-control group. Our findings demonstrate the benefit of ipragliflozin treatment in improving glucolipotoxicity and reducing oxidative stress in pancreatic islet cells. Treatment with ipragliflozin may protect against the progressive loss of islet ß-cells in patients with T2DM.

The Sodium Glucose Cotransporter 2 Inhibitor Ipragliflozin Promotes Preferential Loss of Fat Mass in Non-obese Diabetic Goto-Kakizaki Rats.

Sodium glucose cotransporter 2 (SGLT2) inhibitors improve hyperglycemia in patients with type 2 diabetes mellitus (T2DM) by increasing urinary glucose excretion. In addition to their antihyperglycemic effect, SGLT2 inhibitors also reduce body weight and fat mass in obese and overweight patients with T2DM. However, whether or not SGLT2 inhibitors similarly affect body composition of non-obese patients with T2DM remains unclear. In this study, we investigated the effect of the SGLT2 inhibitor ipragliflozin on body composition in a Goto-Kakizaki (GK) rat model of non-obese T2DM. GK rats were treated with ipragliflozin once daily for 9 weeks, starting at 23 weeks of age. Body composition was then analyzed using dual-energy X-ray absorptiometry. Treatment with ipragliflozin increased urinary glucose excretion, reduced hemoglobin A1c (HbA1c) levels and suppressed body weight gain as the dose increased. Body composition analysis revealed that body fat mass was lower in the ipragliflozin-treated groups than in the control group, while lean body mass and bone mineral contents were comparable between groups. Thus, an SGLT2 inhibitor ipragliflozin was found to promote preferential loss of fat mass in a rat model of non-obese T2DM. Ipragliflozin might also promote preferential loss of fat in non-obese patients with T2DM.

A chronic renal rejection model with a fully MHC-mismatched rat strain combination under immunosuppressive therapy.

BACKGROUND: The Fischer-to-Lewis (LEW) rat model of kidney transplantation is a widely accepted and well-characterized model of chronic rejection. In contrast to transplantation in a clinical setting, however, the absence of treatment with immunosuppressants and only minor mismatch of major histocompatibility complexes (MHCs) are critical discrepancies. Here, we established a rat model of chronic rejection using fully MHC-mismatched strains in which kidney disease progresses even under immunosuppressive therapy. METHODS: LEW (RT1(l)) rats were used as donors and Brown Norway (BN, RT1(n)) rats as recipients. Intramuscular administration of 0.1mg/kg of tacrolimus was initiated on the day of transplantation. Post-transplantation, this dose was maintained until Day 9, suspended until Day 28 and then resumed from Day 29. Renal function, histopathology, and levels of donor-specific antibody (DSA) and several biomarkers of renal injury were assessed. RESULTS: On Day 91 post-transplantation, recipients received tacrolimus treatment with short-term suspension exhibited reduced renal function and changes in histology. Those were characteristics of chronic rejection including glomerulosclerosis, interstitial fibrosis, and tubular atrophy in human transplantation recipients. Urinary protein excretion increased in a linear fashion, and elevated levels of several biomarkers of renal injury and DSA were observed even under administration of an immunosuppressant. CONCLUSIONS: We established an allograft rejection model with impaired renal function and typical histopathological changes of chronic rejection in fully MHC-mismatched rats by controlling administration of an immunosuppressant. These findings suggest that this model more accurately reflects transplantation in a clinical setting than existing models and enables the evaluation of therapeutic agents.

Effect of ipragliflozin, an SGLT2 inhibitor, on progression of diabetic microvascular complications in spontaneously diabetic Torii fatty rats.

AIMS: We investigated the effect of the selective sodium-dependent glucose cotransporter 2 inhibitor ipragliflozin on the simultaneous progression of diabetic microvascular complications of retinopathy, nephropathy and neuropathy in individual Spontaneously Diabetic Torii (SDT) fatty rats. MAIN METHODS: Ipragliflozin was administered to male SDT fatty rats for 12weeks. Male Sprague-Dawley rats of the same age were used as non-diabetic controls. Non-fasting plasma glucose and glycated hemoglobin levels were measured every 4weeks. Cataract formation was monitored once a week, and the electroretinogram was measured after 6weeks of treatment. After the treatment period, motor nerve conduction velocity was measured and urinalysis was conducted. Tissue samples were then dissected for histopathological examination. KEY FINDINGS: Treatment with ipragliflozin reduced glycated hemoglobin levels, inhibited the progression of cataract formation, prevented the prolongation of oscillatory potential peaks in the electroretinogram, ameliorated the slowing of motor nerve conduction velocity, and reduced the severity of glomerulosclerosis in SDT fatty rats. SIGNIFICANCE: These results suggest that the control of hyperglycemia with ipragliflozin slows the progression of the diabetic complications of retinopathy, nephropathy, and neuropathy.

Functional imaging of pharmacological action of SGLT2 inhibitor ipragliflozin via PET imaging using 11C-MDG.

Sodium-dependent glucose cotransporter 2 (SGLT2) is a pharmacological target of type 2 diabetes mellitus. The aim of this study was to noninvasively visualize the pharmacological action of a selective SGLT2 inhibitor ipragliflozin in the kidney using positron emission tomography (PET) imaging with 11C-methyl-d-glucoside (11C-MDG), an SGLT-specific radio-labeled substrate. PET imaging with 11C-MDG in vehicle-treated rats demonstrated that intravenously injected 11C-MDG substantially accumulated in the renal cortex, reflecting that the compound was reabsorbed by SGLTs. In contrast, ipragliflozin-treated rats showed significantly lower uptake of 11C-MDG in renal cortex in a dose-related manner, suggesting that ipragliflozin inhibited the renal reabsorption of 11C-MDG. This method of visualizing the mode of action of an SGLT2 inhibitor in vivo has demonstrated the drug's mechanism in reducing renal glucose reabsorption in kidney in living animals.

Ipragliflozin, an SGLT2 inhibitor, exhibits a prophylactic effect on hepatic steatosis and fibrosis induced by choline-deficient l-amino acid-defined diet in rats.

Ipragliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that increases urinary glucose excretion by inhibiting renal glucose reabsorption and thereby causes a subsequent antihyperglycemic effect. As nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), is closely linked to metabolic diseases such as obesity and diabetes, we investigated the effect of ipragliflozin on NAFLD in rats fed a choline-deficient l-amino acid-defined (CDAA) diet. Five weeks after starting the CDAA diet, rats exhibited hepatic triglyceride (TG) accumulation, fibrosis, and mild inflammation. Repeated oral administration of ipragliflozin (3mg/g, once daily for 5 weeks) prevented both hepatic TG accumulation (188 vs.290 mg/g tissue vehicle-treated group; P<0.001) and large lipid droplet formation. Further, ipragliflozin exerted a prophylactic effect on liver fibrosis, as indicated by a marked decrease in hydroxyproline content and fibrosis score. Pioglitazone, which is known to be effective on hepatic fibrosis in CDAA diet-fed rats as well as NASH patients with type 2 diabetes mellitus (T2DM), also exerted a mild prophylactic effect on fibrosis, but not on hepatic TG accumulation or inflammation. In conclusion, ipragliflozin prevented hepatic TG accumulation and fibrosis in CDAA-diet rats. These findings suggest the therapeutic potential of ipragliflozin for patients with NAFLD.

Antihyperglycemic effect of ipragliflozin, a sodium-glucose co-transporter 2 inhibitor, in combination with oral antidiabetic drugs in mice.

Inhibition of sodium-glucose cotransporter 2 is a novel strategy for glycemic control in type 2 diabetes mellitus patients. As the mechanism of action of sodium-glucose cotransporter 2 inhibitors on plasma glucose levels is distinct from that of existing oral antidiabetic drugs, a combination of the two might provide a therapeutic benefit. Here, we investigated the antihyperglycemic effect of ipragliflozin, a selective sodium-glucose cotransporter 2 inhibitor, alone or in combination with oral antidiabetic drugs in a range of relevant mouse models to analyse the blood glucose-lowering properties of different drug types based on their mechanism of action. Oral glucose tolerance tests in ICR mice were used to evaluate the effect of ipragliflozin in combination with the insulin secretagogues, glibenclamide or nateglinide. Liquid meal tests in ICR mice and diabetic KK-A(y) mice were used to investigate the combined effect of ipragliflozin with the dipeptidyl peptidase-4 inhibitor, sitagliptin, and α-glucosidase inhibitor, voglibose, respectively. Four-week repeated administration tests in KK-A(y) mice were used to examine the combined effect of ipragliflozin with the insulin sensitizers, pioglitazone and metformin. In all mouse models tested, the combination of ipragliflozin and existing oral antidiabetic drugs lowered blood glucose or glycated hemoglobin levels more than either monotherapy. In conclusion, inhibition of sodium-glucose cotransporter 2 by ipragliflozin, alone or in combination with existing oral antidiabetic drugs, has a robust effect on blood glucose levels in a range of mouse models of hyperglycemia.

Pharmacokinetics and pharmacodynamics of ASP2151, a helicase-primase inhibitor, in a murine model of herpes simplex virus infection.

ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus. Here, to determine and analyze the correlation between the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of ASP2151, we examined the PD profile of ASP2151 using in vitro plaque reduction assay and a murine model of HSV-1 infection. ASP2151 inhibited the in vitro replication of HSV-1 with a mean 50% effective concentration (EC(50)) of 14 ng/ml. In the cutaneously HSV-1-infected mouse model, ASP2151 dose dependently suppressed intradermal HSV-1 growth, with the effect reaching a plateau at a dose of 30 mg/kg of body weight/day. The dose fractionation study showed that intradermal HSV-1 titers were below the detection limit in mice treated with ASP2151 at 100 mg/kg/day divided into two daily doses and at 30 or 100 mg/kg/day divided into three daily doses. The intradermal HSV-1 titer correlated with the maximum concentration of drug in serum (C(max)), the area under the concentration-time curve over 24 h (AUC(24h)), and the time during which the concentration of ASP2151 in plasma was above 100 ng/ml (T(>100)). The continuous infusion of ASP2151 effectively decreased intradermal HSV-1 titers below the limit of detection in mice in which the ASP2151 concentration in plasma reached 79 to 145 ng/ml. Our findings suggest that the antiviral efficacy of ASP2151 is most closely associated with the PK parameter T(>100) in HSV-1-infected mice. Based on these results, we propose that a plasma ASP2151 concentration exceeding 100 ng/ml for 21 to 24 h per day provides the maximum efficacy in HSV-1-infected mice.

Susceptibility of herpes simplex virus isolated from genital herpes lesions to ASP2151, a novel helicase-primase inhibitor.

ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. To evaluate the anti-HSV activity of ASP2151, susceptibility testing was performed on viruses isolated from patients participating in a placebo- and valacyclovir-controlled proof-of-concept phase II study for recurrent genital herpes. A total of 156 HSV strains were isolated prior to the dosing of patients, and no preexisting variants with less susceptibility to ASP2151 or acyclovir (ACV) were detected. ASP2151 inhibited HSV-1 and HSV-2 replication with mean 50% effective concentrations (EC(50)s) of 0.043 and 0.069 µM, whereas ACV exhibited mean EC(50)s of 2.1 and 3.2 µM, respectively. Notably, the susceptibilities of HSV isolates to ASP2151 and ACV were not altered after dosing with the antiviral agents. Taken together, these results demonstrate that ASP2151 inhibits the replication of HSV clinical isolates more potently than ACV, and HSV resistant to this novel helicase-primase inhibitor as well as ACV may not easily emerge in short-term treatment for recurrent genital herpes patients.

Tacrolimus ameliorates dextran sulfate sodium-induced colitis in mice: implication of interferon-γ and interleukin-1ß suppression.

We investigated the effect of tacrolimus, a calcineurin inhibitor, on dextran sulfate sodium (DSS)-induced colitis. After inducing colitis in C57BL/6 mice by administering DSS solution as drinking water for 7 d, the animals were treated with tacrolimus. Severity of colonic inflammation was evaluated based on colon weight per unit length. Levels of cytokines (interferon (IFN)-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-12, and tumor necrosis factor (TNF)-α) released from isolated inflamed colons of mice treated with tacrolimus or vehicle were also measured. Treatment with tacrolimus for 14 d reduced the colon weight per unit length and suppressed the release of IFN-γ and IL-1ß, but not other cytokines, in inflamed colons of colitic mice compared with vehicle-treated mice. A positive correlation was noted between colon weight per unit length and released level of IFN-γ or IL-1ß. The release of IFN-γ and IL-1ß was also suppressed after single dosing with tacrolimus to colitic mice. Taken together, these results suggested that tacrolimus ameliorated DSS-induced colitis by suppressing release of IFN-γ and IL-1ß from inflamed colon.

Preventive and therapeutic effects of tacrolimus in an interleukin-10-deficient mouse model of colitis.

OBJECTIVE: To investigate the preventive and therapeutic effects of tacrolimus on colonic inflammation in interleukin-10-deficient (IL-10(-/-)) mice, which spontaneously develop T-cell-mediated colitis. METHODS: Tacrolimus or prednisolone, an anti-inflammatory glucocorticoid, was administered to IL-10(-/-) mice with pre- or post-symptomatic colitis. Effects on colonic inflammation were examined by measuring indices of colitis such as colonic weight/length ratio, cell infiltration, and goblet cell depletion. Effects on cytokine production in colonic lamina propria mononuclear cells (LPMCs) isolated from IL-10(-/-) mice were also examined. RESULTS: Tacrolimus prevented development of colitis and improved already-developed colitis. Prednisolone prevented the development of colitis, but had no effect on already-developed colitis. Tacrolimus completely inhibited IFN-γ and TNF-α production of activated T-cells in LPMCs, but only partially inhibited IFN-γ, TNF-α, and IL-12 production of activated monocytes/macrophages in LPMCs. Prednisolone inhibited cytokine production in both cell types but exhibited greater potency on monocytes/macrophages than on T-cells. CONCLUSION: These results suggest that the preventive and therapeutic effect of tacrolimus in IL-10(-/-) mice colitis might be attributed to the inhibition of colonic T-cell activation rather than monocyte/macrophage activation. T-cell immunosuppression may thus be a promising strategy for treating colonic inflammation.

Unique "delta lock" structure of telmisartan is involved in its strongest binding affinity to angiotensin II type 1 receptor.

Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique "delta lock" structure. Our present study suggests that due to its "delta lock" structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.

Repeated topical application of glucocorticoids augments irritant chemical-triggered scratching in mice.

Topical glucocorticoids, widely used for the treatment of a variety of dermatitises, are known to exacerbate atopic dermatitis after long-term or inappropriate use. In some animal models, topical glucocorticoids augment the allergic cutaneous inflammation after repeated application, suggesting a relationship between these and clinical observations. We investigated whether topical glucocorticoids augment itching, rather than inflammation, resulting in the exacerbation of atopic dermatitis. Mice receiving repeated topical application of glucocorticoids, betamethasone valerate or dexamethasone, to the ear for 1 week showed significantly higher scratching frequency after application of an irritant chemical, 2,4-dinitrofluorobenzene (DNFB) or 12-O-tetradecanoilphorbol 13-acetate (TPA) than those receiving either a glucocorticoid or irritant chemical alone. In contrast, the increase in ear thickness induced by application of TPA was significantly suppressed by dexamethasone. Substance P (SP) and nerve growth factor (NGF) levels were higher in the ear receiving betamethasone valerate followed by DNFB application than in that receiving DNFB alone. In addition, histopathological studies revealed an increased density of nerve fibers in the ear receiving betamethasone valerate or dexamethasone followed by DNFB application. Oral administration of betamethasone valerate was not associated with an increase in either scratching frequency or SP or NGF level in the ear. These results suggest that repeated topical application of glucocorticoids may augment irritant chemical-triggered scratching through an increase in SP and NGF levels and nerve fiber density at the application site. These findings might explain the etiology of the exacerbation of atopic dermatitis and other dermatitises, occurring after long-term or inappropriate use of topical glucocorticoids.