Recruitment or activation of mast cells in the liver aggravates the accumulation of fibrosis in carbon tetrachloride-induced liver injury.

Liver diseases caused by viral infections, alcoholism, drugs, or chemical poisons are a significant health problem: Liver diseases are a leading contributor to mortality, with approximately 2 million deaths per year worldwide. Liver fibrosis, as a common liver disease characterized by excessive collagen deposition, is associated with high morbidity and mortality, and there is no effective treatment. Numerous studies have shown that the accumulation of mast cells (MCs) in the liver is closely associated with liver injury caused by a variety of factors. This study investigated the relationship between MCs and carbon tetrachloride (CCl4)-induced liver fibrosis in rats and the effects of the MC stabilizers sodium cromoglycate (SGC) and ketotifen (KET) on CCl4-induced liver fibrosis. The results showed that MCs were recruited or activated during CCl4-induced liver fibrosis. Coadministration of SCG or KET alleviated the liver fibrosis by decreasing SCF/c-kit expression, inhibiting the TGF-ß1/Smad2/3 pathway, depressing the HIF-1a/VEGF pathway, activating Nrf2/HO-1 pathway, and increasing the hepatic levels of GSH, GSH-Px, and GR, thereby reducing hepatic oxidative stress. Collectively, recruitment or activation of MCs is linked to liver fibrosis and the stabilization of MCs may provide a new approach to the prevention of liver fibrosis.

Sustained Delivery of Olopatadine from Vitamin-E Loaded Contact Lenses.

Purpose: Topical antihistamines, such as olopatadine hydrochloride, an H1 receptor antagonist, are commonly prescribed for treating allergic conjunctivitis. Drug delivery via eye drops has many deficiencies including a short residence time due to tear drainage via the nasolacrimal duct, which results in a low bioavailability and potential for side effects. These deficiencies could be mitigated by a drug-eluting contact lens such as the recently approved ACUVUE® THERAVISION™ WITH KETOTIFEN which is a daily disposable etafilcon, a drug-eluting contact lens with ketotifen (19 µg per lens). Here, we investigate the feasibility of designing a drug-eluting lens with sustained release of olopatadine for treating allergies using an extended wear lens. Methods: Nanobarrier depots composed of vitamin-E (VE) are formed through direct entrapment by ethanol-driven swelling. The drug-loaded lenses are characterized for transparency and water content. In vitro release is measured under sink conditions and fitted to a diffusion control release model to determine diffusivity and partition coefficient. Results: In vitro studies indicate that ACUVUE OASYS® and ACUVUE TruEye™ lenses loaded with ∼0.3 g of VE/g of hydrogel effectively prolong olopatadine dynamics by 7-fold and 375-fold, respectively. Incorporation of VE into the lenses retains visible light transmission and other properties. Conclusion: The VE incorporation in commercial lenses significantly increases the release duration offering the possibility of antiallergy extended wear lenses.

Ketotifen directly modifies the fibrotic response of human skin fibroblasts.

Fibrosis is a destructive, end-stage disease process. In the skin, it is associated with systemic sclerosis and scarring with considerable health burden. Ketotifen is a clinical antihistamine and mast cell stabilizer. Studies have demonstrated mast cell-dependent anti-fibrotic effects of ketotifen but direct effects on fibroblasts have not been determined. Human dermal fibroblasts were treated with pro-fibrotic transforming growth factor-ß1 (TGFß) followed by ketotifen or control treatments to determine direct effects on fibrotic fibroblasts. Ketotifen impaired TGFß-induced α-smooth muscle actin gene and protein responses and decreased cytoskeletal- and contractility-associated gene responses associated with fibrosis. Ketotifen reduced Yes-associated protein phosphorylation, transcriptional coactivator with PDZ binding motif transcript and protein levels, and phosphorylation of protein kinase B. In a fibroblast-populated collagen gel contraction assay, ketotifen reduced the contractile activity of TGFß-activated fibroblasts. In a murine model of bleomycin-induced skin fibrosis, collagen density and dermal thickness were significantly decreased in ketotifen-treated mice supporting in vitro findings. These results support a novel, direct anti-fibrotic activity of ketotifen, reducing pro-fibrotic phenotypic changes in fibroblasts and reducing collagen fibres in fibrotic mouse skin. Together, these findings suggest novel therapeutic potential and a novel mechanism of action for ketotifen in the context of fibrosis.

Comparative evaluation of single and combined efficacy of dipyridamole, ketotifen and quercetin on cyclosporine induced hepatorenal toxicity.

Cyclosporine is an immunosuppressive drug that is used to prevent tissue rejection in organ transplants and to treat autoimmune diseases such as psoriasis and rheumatoid arthritis. It has important toxic effects in many organs such as the liver and kidney. The aim of this study was to determine and compare the effectiveness of the single and combined treatment of dipyridamole, which is a vasodilator and has an antioxidant effect, ketotifen which is toll-like receptor-4 inhibitory and has an antioxidant effect, quercetin which is an antioxidant and has an anti-inflammatory effect in cyclosporine-induced hepatorenal toxicity. Forty-eight Wistar Albino rats were divided into 7 groups. The research period was 21 days. The cyclosporine increased serum ALT and AST levels, in contrast to their increased levels prevented by all the treatments. The serum creatinine level decreased significantly with ketotifen and combined treatment, while cyclosporine partially increased serum creatinine and urea levels. The urine microalbumin and protein levels were increased significantly by cyclosporine, whereas they decreased with dipyridamole treatment. The protein levels decreased by quercetin and combined treatments. The kidney injury molecule- 1 and retinol-binding protein levels were increased by the cyclosporine, while ketotifen treatment partially decreased them. In conclusion, ketotifen and dipyridamole can prevent cyclosporine- induced hepatorenal toxicity and quercetin can increase the effectiveness of this treatment.

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

Abdominal Massage Improves the Symptoms of Irritable Bowel Syndrome by Regulating Mast Cells via the Trypase-PAR2-PKCε Pathway in Rats.

Background: Irritable bowel syndrome (IBS) is a clinical disease mainly characterized as a syndrome of abdominal pain and discomfort, which frequently occurs in humans aged 20-50. Abdomen massage is of great medical significance for the health of the human body, including promoting intestinal peristalsis, relieving constipation, and facilitating weight loss. However, its potential benefits in alleviating IBS and the underlying mechanisms remain elusive. Methods: In this study, we established an IBS model in rats to evaluate the effects of abdomen massage. Forty male Sprague Dawley (SD) rats were randomly assigned into 4 groups: the normal (control) group, IBS group, abdominal massage group, and abdominal massage + ketotifen treatment group (n = 10 rats in each group). Abdominal massage was performed once a day for 5 minutes for 14 days. On day 14, the rats were euthanized and the tissues were analyzed by transmission electron microscopy (TEM), immunohistochemistry or immunofluorescence staining, and laser confocal focus to visualize the micromorphology of the intestinal mucosa. The expression of TRPV1 and the release of trypase were determined by RT-qPCR and western blot. Results: We found that compared with the control group, the mast cells in the IBS group were significantly increased and the increased MC was partially decreased by an abdominal massage with or without ketotifen treatment. We also found that TRPV1 was upregulated in the IBS group. Abdominal massage with or without ketotifen treatment could attenuate the upregulation of TRPV1 in IBS. Mechanically, results of IHC and western Blot suggested that abdominal massage reduces the sensitivity of IBS by regulating the trypase-PAR2-PKCε pathway. Conclusion: Overall, our results suggested that abdominal massage produces a beneficial effect in improving the symptoms of IBS through reducing mast cell recruitment and attenuating the trypase-PAR2-PKCε pathway. Ketotifen could promote the effect of abdominal massage on IBS treatment, which can serve as a potential therapeutic strategy for IBS.

Mechanism of ketotifen fumarate inhibiting renal calcium oxalate stone formation in SD rats.

OBJECTIVES: To investigate the inhibitory effect of ketotifen fumarate (KFA), a mast cell membrane stabilizer, on renal calcium oxalate stone (CaOx) formation and its possible molecular mechanism. METHODS: We used the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for functional and pathway enrichment analyses of osteopontin (OPN), CD44 and fibronectin (FN). Blood biochemistry, reactive oxygen species ratio (ROS), mast cells, proteins (CD44, OPN and FN) and OPN receptor integrin family genes were detected by ELISA, flow cytometry, immunohistochemistry and RT-QPCR, respectively. RESULTS: The crystal area of CaOx in the KFA and Control group was significantly smaller than that in the Model group. The number of activated mast cells, the expression levels of OPN and CD44 in the Control and KFA groups were significantly lower than those in the Model group, and the percentage of ROS in the KFA group was also significantly lower than that in the Model group. The mRNA expression levels of ITGB1, ITGA9, ITGAV and ITGA4 genes in the prominent OPN receptor integrin family increased significantly in the Model group. CONCLUSIONS: Ketotifen can effectively inhibit the crystal formation of CaOx and reduce the inflammatory response of tissue in SD rats. The mechanism may be to reduce the infiltration and activation of mast cells in renal tissue and down-regulate the expression of OPN, CD44 and FN in renal tubules and renal interstitium. And affect the synthesis of integrins (ITGA9, ITGA4, ITGAV, ITGB1, ITGB3 and ITGB5) and ROS.

Ketotifen is a Prodrug. Norketotifen is the active metabolite.

Evaluation of the in vitro human liver microsome and hepatocyte metabolism of ketotifen demonstrated that norketotifen (NK) is the major demethylated hepatic metabolite of ketotifen. It is here reported that NK is completely devoid of the severe and dose-limiting sedative effects of ketotifen. Thus, while ketotifen is clinically dose-limited to 1 mg, bid, there are no dose-limiting sedative effects elicited by NK, even after the highest single-dose (16 mg) or after repeat-doses (8 mg × 7 days) in humans or after the highest doses given to dogs in repeat-dose toxicological studies (40 mg/kg × 14 days). In addition, NK-but not ketotifen-was found to express potent and dose-dependent inhibition of the release of the pro-inflammatory cytokine TNFα from activated human buffy coat preparations. Thus, when used as an anti-inflammatory drug, ketotifen is the sedating prodrug which is converted to NK a nonsedating metabolite with anti-inflammatory activity.

Topical Ketotifen Fumarate Inhibits Choroidal Mast Cell Degranulation and Loss of Retinal Pigment Epithelial Cells in Rat Model for Geographic Atrophy.

Purpose: This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model. Methods: Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function. Results: PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF. Conclusions: Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA. Translational Relevance: The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Mast Cell Mediated Regulation of Small Intestinal Chloride Malabsorption in SAMP1/YitFc Mouse Model of Spontaneous Chronic Ileitis.

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn's disease, DRA (Downregulated in Adenoma) mediated Cl:HCO3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (ß-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the ß-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

In Vitro Assessment of the Antiviral Activity of Ketotifen, Indomethacin and Naproxen, Alone and in Combination, against SARS-CoV-2.

The 2019 coronavirus infectious disease (COVID-19) is caused by infection with the new severe acute respiratory syndrome coronavirus (SARS-CoV-2). Currently, the treatment options for COVID-19 are limited. The purpose of the experiments presented here was to investigate the effectiveness of ketotifen, naproxen and indomethacin, alone or in combination, in reducing SARS-CoV-2 replication. In addition, the cytotoxicity of the drugs was evaluated. The findings showed that the combination of ketotifen with indomethacin (SJP-002C) or naproxen both reduce viral yield. Compared to ketotifen alone (60% inhibition at EC50), an increase in percentage inhibition of SARS-CoV-2 to 79%, 83% and 93% was found when co-administered with 25, 50 and 100 µM indomethacin, respectively. Compared to ketotifen alone, an increase in percentage inhibition of SARS-CoV-2 to 68%, 68% and 92% was found when co-administered with 25, 50 and 100 µM naproxen, respectively. For both drug combinations the observations suggest an additive or synergistic effect, compared to administering the drugs alone. No cytotoxic effects were observed for the administered dosages of ketotifen, naproxen, and indomethacin. Further research is warranted to investigate the efficacy of the combination of ketotifen with indomethacin (SJP-002C) or naproxen in the treatment of SARS-CoV-2 infection in humans.

Mechanistic Studies of the Antiallergic Activity of Phyllanthus amarus Schum. & Thonn. and Its Compounds.

Phyllanthus amarus Schum. & Thonn. (Phyllanthaceae) is a medicinal plant that is commonly used to treat diseases such as asthma, diabetes, and anemia. This study aimed to examine the antiallergic activity of P. amarus extract and its compounds. The antiallergic activity was determined by measuring the concentration of allergy markers release from rat basophilic leukemia (RBL-2H3) cells with ketotifen fumarate as the positive control. As a result, P. amarus did not stabilize mast cell degranulation but exhibited antihistamine activity. The antihistamine activity was evaluated by conducting a competition radioligand binding assay on the histamine 1 receptor (H1R). Four compounds were identified from the high performance liquid chromatography (HPLC) analysis which were phyllanthin (1), hypophyllanthin (2), niranthin (3), and corilagin (4). To gain insights into the binding interactions of the most active compound hypophyllanthin (2), molecular docking was conducted and found that hypophyllanthin (2) exhibited favorable binding in the H1R binding site. In conclusion, P. amarus and hypophyllanthin (2) could potentially exhibit antiallergic activity by preventing the activation of the H1 receptor.

Mast cell stabilizer modulates Sirt1/Nrf2/TNF pathway and inhibits oxidative stress, inflammation, and apoptosis in rat model of cyclophosphamide hepatotoxicity.

Objectives: Cyclophosphamide (CYC) is the most common cytotoxic alkylating agent which considered as chemotherapy but its clinical usefulness is challenged with different forms of organ damage including hepatotoxicity. Hepatic mast cells (MC) have an important role in the pathophysiology of liver toxicity. We aimed to evaluate the possible protective effect of mast cell stabilizer, ketotifen in CYC induced-hepatotoxicity.Materials and methods: Twenty-four adult male albino Wistar rats were divided into four groups: control group, ketotifen group (received ketotifen 10 mg/kg/day, p.o.) for 14 days, CYC group (received CYC 200 mg/kg i.p.) as a single dose at the ninth day and ketotifen plus CYC group (received ketotifen and CYC). We measured serum enzyme biomarkers [alanine transaminase (ALT) and aspartate transaminase (AST)], total antioxidant capacity (TAC), interluken-1ß (IL-1ß), tissue malondialdehyde (MDA), nitric oxide (NOx), reduced glutathione (GSH), P-glycoprotein (P-gp), Sirtuin type 1 (Sirt1) and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Furthermore; histological changes, tumor necrosis factor (TNF) and caspase-3 immuno-expressions were evaluated.Results: CYC group showed hepatotoxic effect in the form of a significant increase in ALT, AST, MDA, NOx, IL-1ß levels; TNF and caspase-3 immuno-expression. Moreover; it showed toxic histological changes of marked liver injury meanwhile, there is a significant decrease in TAC, GSH, P-gp, Sirt1, and Nrf2 levels. Ketotifen showed a significant improvement in all parameters.Conclusion: Mast cell stabilizer, ketotifen possesses potent ameliorative effects against the hepatotoxic effect of CYC by reducing oxidative stress, inflammatory process, and apoptosis through regulation of Sirt1/Nrf2/TNF pathway.

Cell-based, animal and H1 receptor binding studies relative to the sedative effects of ketotifen and norketotifen atropisomers.

OBJECTIVES: Ketotifen (K) and its active metabolite norketotifen (N) exist as optically active atropisomers. They both have antihistaminic and anti-inflammatory properties but the S-atropisomer of N (SN) causes less sedation than K and RN in rodents. This study investigated whether this could be related to a lower concentration of SN in brain or a lower affinity of SN for rat brain H1 receptors. METHODS: Ketotifen and norketotifen atropisomers were quantified using a validated chiral HPLC assay. RBE4 and Caco-2 cell monolayers were used in uptake and permeability studies, respectively. Free and total brain-to-plasma (B/P) ratios were determined after injecting racemic K and N into rat tail veins. Affinity for rat brain H1 receptors (KI ) was determined using the [3 H]mepyramine binding assay. KEY FINDINGS: Uptake and permeation studies indicate no stereoselective transport for K or N. B/P ratios reveal the brain concentration of N is lower than K with no stereoselective transport into brain. Finally, the [3 H]mepyramine binding assay shows SN has the lowest affinity for rat brain H1 receptors. CONCLUSION: The lower sedative effect of SN in rodents is probably due to a combination of a lower uptake of N than K into the brain and less affinity of SN for CNS H1 receptors.

In vitro safety of ketotifen as a topical nasal rinse.

BACKGROUND: Ketotifen is a second-generation noncompetitive H1-antihistamine and mast-cell stabilizer. It is commonly used to treat or prevent allergic conjunctivitis, asthma, chronic urticaria, anaphylaxis, mast-cell, and other allergic-type disorders. However, it has never been studied in aspirin-exacerbated respiratory disease (AERD), an aggressive phenotype of chronic rhinosinusitis with nasal polyps, where the mast cell plays a prominent role its pathogenesis. METHODS: Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ketotifen powder was dissolved in saline to make 4 test solutions at 1.04, 2.08, 10.4, and 20.8 µg/mL. Control (saline) or ketotifen solution was added apically to ALI cultures from tissue of 5 unique patients, and ciliary beat frequency (CBF) changes were recorded. Lactate dehydrogenase was measured at 24 and 48 hours to estimate long-term cellular toxicity. RESULTS: Apical application of ketotifen at all concentrations was neither ciliotoxic nor ciliostimulatory, with no change in CBF over a period of 15 minutes after application. Cellular toxicity for all concentrations at 24 and 48 hours after application was <3% and <7%, respectively, that of lysed cultures. CONCLUSION: Topical application of ketotifen to an in vitro model of sinonasal epithelium is safe, as evaluated by CBF and lactate dehydrogenase. Ketotifen is neither ciliotoxic nor ciliostimulatory, and no long-term cellular toxicity was observed. Ketotifen may have promise as a topical nasal rinse in the treatment of AERD.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord.

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

A new zinc chelator, IPZ-010 ameliorates postoperative ileus.

Zinc was discovered to be a novel second messenger in immunoreactive cells. We synthesized a novel free zinc chelator, IPZ-010. Here, we investigated the effects of IPZ-010 in a mouse postoperative ileus model and determined the effects of zinc signal inhibition as a new therapeutic strategy against postoperative ileus. Zinc waves were measured in bone marrow-derived mast cells (BMMCs) loaded with a zinc indicator, Newport green. Degranulation and cytokine expression were measured in BMMCs and bone marrow-derived macrophages (BMDMs). Postoperative ileus model mice were established with intestinal manipulation. Mice were treated with IPZ-010 (30 mg/kg, s.c. or p.o.) 1 h before and 2 h and 4 h after intestinal manipulation. Gastrointestinal transit, inflammatory cell infiltration, and expression of inflammatory mediators were measured. Free zinc waves occurred following antigen stimulation in BMMCs and were blocked by IPZ-010. IPZ-010 inhibited interleukin-6 secretion and degranulation in BMMCs. IPZ-010 inhibited tumor necrosis factor-α mRNA expression in BMMCs stimulated with lipopolysaccharide or adenosine triphosphate, whereas IPZ-010 had no effects on tumor necrosis factor-α mRNA expression in BMDMs stimulated with lipopolysaccharide or adenosine triphosphate. In postoperative ileus model mice, IPZ-010 inhibited leukocyte infiltration and cytokine expression, which ameliorated gastrointestinal transit. Furthermore, ketotifen (1 mg/kg) induced similar effects as IPZ-010. These effects were not amplified by co-administration of IPZ-010 and ketotifen. IPZ-010 inhibited zinc waves, resulting in inhibition of inflammatory responses in activated BMMCs in vitro. Targeting zinc waves in inflammatory cells may be a novel therapeutic strategy for treating postoperative ileus.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord / 亚洲男科学杂志(英文版)

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

Ketotifen Modulates Mast Cell Chemotaxis to Kit-Ligand, but Does Not Impact Mast Cell Numbers, Degranulation, or Tumor Behavior in Neurofibromas of Nf1-Deficient Mice.

Neurofibromatosis Type 1 (NF1) is one of the most common genetic tumor predisposition syndromes in humans. Mutant NF1 results in dysregulated RAS allowing neoplasms throughout the neuroaxis. Plexiform neurofibromas (pNF) afflict up to 50% of patients with NF1. They are complex tumors of the peripheral nerve that cause major morbidity via nerve dysregulation and mortality via conversion to malignant sarcoma. Genetically engineered mouse models (GEMM) of NF1 provide valuable insights for the identification of therapies that have utility in people with pNF. Preclinical studies in GEMMs implicate mast cells and the c-Kit/Kit ligand pathway in pNF tumorigenesis. Kit ligand is a potent chemokine secreted by tumorigenic, Nf1-deficient Schwann cells. Ketotifen is an FDA-approved drug for the treatment of allergic conjunctivitis and asthma that promotes mast cell stabilization and has been used in prior case studies to treat or prevent pNFs. This study investigated the effect of ketotifen on mast cell infiltration and degranulation in the presence and absence of Kit ligand provocation and the effect of ketotifen on shrinking or preventing pNF formation in the Nf1flox/flox ;PostnCre + GEMM. Ketotifen decreased mast cell infiltration in response to exogenous Kit ligand administration, but did not affect mast cell degranulation. Importantly, ketotifen did not reduce mast cells numbers or activity in pNF and did not prevent pNF formation or decrease the volume of established pNF despite administration of pharmacologically active doses. These findings suggest that ketotifen has limited use as monotherapy to prevent or reduce pNF burden in the setting of Nf1 mutations.

Inhibition of Mast Cell Degranulation Relieves Visceral Hypersensitivity Induced by Pancreatic Carcinoma in Mice.

Cancer pain induced by pancreatic carcinoma is one of the most common symptoms and is difficult to endure, especially in the advanced stage. Evidence suggests that mast cells are recruited and degranulate in enteric disease-related visceral hypersensitivity. However, whether mast cells promote the visceral pain induced by pancreatic carcinoma remains unclear. Here, using toluidine blue staining and western blotting, we observed that mast cells were dramatically recruited to tissues surrounding pancreatic carcinoma, but not inside the carcinoma in patients with severe visceral pain. The levels of mast cell degranulation products, including tryptase, histamine, and nerve growth factor, were significantly increased in pericarcinoma tissues relative to their levels in normal controls, as evidenced by enzyme-linked immunosorbent assay. We determined that systemic administration of mast cell secretagogue compound 48/80 exacerbated pancreatic carcinoma-induced visceral hypersensitivity in a male BALB/c nude mouse model as assessed by measuring the hunching behavior scores and mechanical withdrawal response frequency evoked by von Frey stimulation. In contrast, the mast cell stabilizer ketotifen dose-dependently alleviated pancreatic cancer pain. In addition, we observed incomplete development of abdominal mechanical hyperalgesia and hunching behavior in mast cell-deficient mice with pancreatic carcinoma. However, ketotifen did not further attenuate visceral hypersensitivity in mast cell-deficient mice with carcinoma. Finally, we confirmed that intraplantar injection of pericarcinoma supernatants from BALB/c nude mice but not mast cell-deficient mice caused acute somatic nociception. In conclusion, our findings suggest that mast cells contribute to pancreatic carcinoma-induced visceral hypersensitivity through enrichment and degranulation in pericarcinoma tissues. The inhibition of mast cell degranulation may be a potential strategy for the therapeutic treatment of pancreatic carcinoma-induced chronic visceral pain.