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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), which caused novel corona virus disease-2019 (COVID-19) pandemic, necessitated a global demand for studies related to genes and enzymes of SARS-CoV2. SARS-CoV2 infection depends on the host cell Angiotensin-Converting Enzyme-2 (ACE2) and Transmembrane Serine Protease-2 (TMPRSS2), where the virus uses ACE2 for entry and TMPRSS2 for S protein priming. The TMPRSS2 gene encodes a Transmembrane Protease Serine-2 protein (TMPS2) that belongs to the serine protease family. There is no crystal structure available for TMPS2, therefore, a homology model was required to establish a putative 3D structure for the enzyme. A homology model was constructed using SWISS-MODEL and evaluations were performed through Ramachandran plots, Verify 3D and Protein Statistical Analysis (ProSA). Molecular dynamics simulations were employed to investigate the stability of the constructed model. Docking of TMPS2 inhibitors, camostat, nafamostat, gabexate, and sivelestat, using Molecular Operating Environment (MOE) software, into the constructed model was performed and the protein-ligand complexes were subjected to MD simulations and computational binding affinity calculations. These in silico studies determined the tertiary structure of TMPS2 amino acid sequence and predicted how ligands bind to the model, which is important for drug development for the prevention and treatment of COVID-19.

The major impact produced by the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) focused many researchers attention to find treatments that can suppress transmission or ameliorate the disease. Despite the very fast and large flow of scientific data on possible treatment solutions, none have yet demonstrated unequivocal clinical utility against coronavirus disease 2019 (COVID‑19). This work represents an exhaustive and critical review of all available data on potential treatments for COVID‑19, highlighting their mechanistic characteristics and the strategy development rationale. Drug repurposing, also known as drug repositioning, and target based methods are the most used strategies to advance therapeutic solutions into clinical practice. Current in silico, in vitro and in vivo evidence regarding proposed treatments are summarized providing strong support for future research efforts.

As of April 9, 2020, a novel coronavirus (SARS-CoV-2) had caused 89,931 deaths and 1,503,900 confirmed cases worldwide, which indicates an increasingly severe and uncontrollable situation. Initially, little was known about the virus. As research continues, we now know the genome structure, epidemiological and clinical characteristics, and pathogenic mechanisms of SARS-CoV-2. Based on this knowledge, potential targets involved in the processes of virus pathogenesis need to be identified, and the discovery or development of drugs based on these potential targets is the most pressing need. Here, we have summarized the potential therapeutic targets involved in virus pathogenesis and discuss the advances, possibilities, and significance of drugs based on these targets for treating SARS-CoV-2. This review will facilitate the identification of potential targets and provide clues for drug development that can be translated into clinical applications for combating SARS-CoV-2., (© 2020 The British Pharmacological Society.)

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC) 50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC 50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of -14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.

The number of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly increased, although the WHO declared a pandemic. However, drugs that function against SARS-CoV-2 have not been established. SARS-CoV-2 has been suggested to bind angiotensin-converting enzyme 2, the receptor of the SARS coronavirus. SARS coronavirus and coronavirus 229E, the cause of the common cold, replicate through cell-surface and endosomal pathways using a protease, the type II transmembrane protease. To examine the effects of protease inhibitors on the replication of coronavirus 229E, we pretreated primary cultures of human nasal epithelial (HNE) cells with camostat or nafamostat, each of which has been used for the treatment of pancreatitis and/or disseminated intravascular coagulation. HNE cells were then infected with coronavirus 229E, and viral titers in the airway surface liquid of the cells were examined. Pretreatment with camostat (0.1-10 μg/mL) or nafamostat (0.01-1 μg/mL) reduced the titers of coronavirus 229E. Furthermore, a significant amount of type II transmembrane protease protein was detected in the airway surface liquid of HNE cells. Additionally, interferons have been reported to have antiviral effects against SARS coronavirus. The additive effects of interferons on the inhibitory effects of other candidate drugs to treat SARS-CoV-2 infection, such as lopinavir, ritonavir and favipiravir, have also been studied. These findings suggest that protease inhibitors of this type may inhibit coronavirus 229E replication in human airway epithelial cells at clinical concentrations. Protease inhibitors, interferons or the combination of these drugs may become candidate drugs to inhibit the replication of SARS-CoV-2.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Chronic pancreatitis (CP) is a progressive, fibro-inflammatory disease characterized by enzymatic autoactivation and subsequent fibrotic replacement of acinar cells. A significant proportion of patients develop pain, which may be due to many causes, including perineural inflammation, altered central processing of pain signals, parenchymal structural changes, and ductal obstruction. Currently there are no approved medical treatment options for CP-associated pain. NI-03 (camostat mesilate) is an orally administered serine protease inhibitor that reduces pancreatic enzyme activity and has been widely used for the treatment of CP-associated pain in Japan. The current study will assess the safety and efficacy of NI-03 for reduction of CP-associated pain in the USA., Methods: The current study consists of two phases. First, a phase I study will be performed to establish the pharmacokinetics and safety profile over a 1-week period following a single dose (100, 200, or 300 mg). Subsequently, a phase II study will be performed consisting of a double-blind, randomized, controlled trial (RCT). This RCT will evaluate the efficacy of each of the three doses of NI-03 given three times daily compared to placebo over 28 days. A 7-day, single-blind, run-in period will precede the double-blind phase to assess baseline pain characteristics. The primary efficacy outcome is the average of worst daily pain scores (numeric rating scale of 0-10) over the terminal 7 days of the study period compared to baseline. Secondary efficacy outcomes include change in opioid dose and quality of life measures, and time to first rescue intravenous analgesic. Adverse events will be recorded., Discussion: NI-03 has been used successfully and safely in Japan to treat CP-associated pain. The aim of the current study is to assess the safety and efficacy of NI-03 using a rigorous RCT in a population in the USA. This study may fill an important clinical gap to provide an effective medical treatment option for CP-associated pain., Trial Registration: ClinicalTrials.gov, NCT02693093 . Registered through the National Institutes of Health on 26 February 2016.

Trypsin is the major serine protease responsible for intestinal protein digestion. An inhibitor, camostat (CS), reduced weight gain, hyperglycemia, and dyslipidemia in obese rats; however, the mechanisms for these are largely unknown. We reasoned that CS creates an apparent dietary protein restriction, which is known to increase hepatic fibroblast growth factor 21 (FGF21). Therefore, metabolic responses to CS and a gut-restricted CS metabolite, FOY-251, were measured in mice. Food intake, body weight, blood glucose, branched-chain amino acids (LC/MS), hormone levels (ELISA), liver pathology (histology), and transcriptional changes (qRT-PCR) were measured in ob/ob , lean and diet-induced obese (DIO) C57BL/6 mice. In ob/ob mice, CS in chow (9-69 mg/kg) or FOY-251 (46 mg/kg) reduced food intake and body weight gain to a similar extent as pair-fed mice. CS decreased blood glucose, liver weight, and lipidosis and increased FGF21 gene transcription and plasma levels. In lean mice, CS increased liver FGF21 mRNA and plasma levels. Relative to pair feeding, FOY-251 also increased plasma FGF21 and induced liver FGF21 and integrated stress response (ISR) transcription. In DIO mice, FOY-251 (100 mg/kg po) did not alter peak glucose levels but reduced the AUC of the glucose excursion in response to an oral glucose challenge. FOY-251 increased plasma FGF21 levels. In addition to previously reported satiety-dependent (cholecystokinin-mediated) actions, intestinal trypsin inhibition engages non-satiety-related pathways in both leptin-deficient and DIO mice. This novel mechanism improves metabolism by a liver-integrated stress response and increased FGF21 expression levels in mice. NEW & NOTEWORTHY Trypsin inhibitors, including plant-based consumer products, have long been associated with metabolic improvements. Studies in the 1980s and 1990s suggested this was due to satiety hormones and caloric wasting by loss of protein and fatty acids in feces. This work suggests an entirely new mechanism based on the lower amounts of digested protein available in the gut. This apparent protein reduction may cause beneficial metabolic adaptation by the intestinal-liver axis to perceived nutrient stress.

We previously reported that camostat mesilate (CM) had renoprotective and antihypertensive effects in rat CKD models. In this study, we examined if CM has a distinct renoprotective effect from telmisartan (TE), a renin-angiotensin-aldosterone system (RAS) inhibitor, on the progression of CKD. We evaluated the effect of CM (400 mg/kg/day) and/or TE (10 mg/kg/day) on renal function, oxidative stress, renal fibrosis, and RAS components in the adenine-induced rat CKD model following 5-weeks treatment period. The combination therapy with CM and TE significantly decreased the adenine-induced increase in serum creatinine levels compared with each monotherapy, although all treatment groups showed similar reduction in blood pressure. Similarly, adenine-induced elevation in oxidative stress markers and renal fibrosis markers were significantly reduced by the combination therapy relative to each monotherapy. Furthermore, the effect of the combination therapy on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was similar to that of TE monotherapy, and CM had no effect on both PRA and PAC, suggesting that CM has a distinct pharmacological property from RAS inhibition. Our findings indicate that CM could be a candidate drug for an add-on therapy for CKD patients who had been treated with RAS inhibitors., (Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

Aim: To examine the effects of pancreatic rest, stimulation and rest/stimulation on the natural course of recovery after acute pancreatitis., Methods: Acute hemorrhagic pancreatitis (AP) was induced in male rats by intraductal infusion of 40 μL/100 g body weight of 3% sodium taurocholate. All rats took food ad libitum. At 24 h after induction of AP, rats were divided into four groups: control (AP-C), pancreas rest (AP-R), stimulation (AP-S), and rest/stimulation (AP-R/S). Rats in the AP-C, AP-R and AP-S groups received oral administration of 2 mL/kg body weight saline, cholecystokinin (CCK)-1 receptor antagonist, and endogenous CCK release stimulant, respectively, twice daily for 10 d, while those in the AP-R/S group received twice daily CCK-1 receptor antagonist for the first 5 d followed by twice daily CCK release stimulant for 5 d. Rats without any treatment were used as control group (Control). Biochemical and histological changes in the pancreas, and secretory function were evaluated on day 12 at 24 h after the last treatment., Results: Feeding ad libitum (AP-C) delayed biochemical, histological and functional recovery from AP. In AP-C rats, bombesin-stimulated pancreatic secretory function and HOMA-β-cell score were significantly lower than those in other groups of rats. In AP-R rats, protein per DNA ratio and pancreatic exocrine secretory function were significantly low compared with those in Control rats. In AP-S and AP-R/S rats, the above parameters recovered to the Control levels. Bombesin-stimulated pancreatic exocrine response in AP-R/S rats was higher than in AP-S rats and almost returned to control levels. In the pancreas of AP-C rats, destruction of pancreatic acini, marked infiltration of inflammatory cells, and strong expression of α-smooth muscle actin, tumor necrosis factor-α and interleukin-1β were seen. Pancreatic rest reversed these histological alterations, but not atrophy of pancreatic acini and mild infiltration of inflammatory cells. In AP-S and AP-R/S rats, the pancreas showed almost normal architecture., Conclusion: The favorable treatment strategy for AP is to keep the pancreas at rest during an early stage followed by pancreatic stimulation by promoting endogenous CCK release.

Objective: To observe the effect of gut protease activity on visceral hypersensitivity in rats with acute restraint stress., Methods: Sprague-Dawley rats were given 30, 100 or 300 mg/kg camostat mesilate (CM), a protease inhibitor, or saline intragastrically 30 min before acute restraint stress induced by wrapping the fore shoulders, upper forelimbs and thoracic trunk for 2 h. Visceral perception of the rats was quantified as the visceral motor response with an electromyography, and the rectal mucosa and feces protease activity and spinal c-fos expression were measured., Results: CM dose-dependently reduced visceral sensitization elicited by rectal distension, but these doses did not completely inhibit stress-induced visceral sensitization. In normal rats, c-fos expression was found mainly in the superal spinal cord dorsal horn, and after the administration the CM, c-fos-positive cells decreased significantly in all dose groups (P<0.05). In 30 mg/kg CM group, fecal and rectal mucosal protease activity significantly decreased as compared with that in the stress group (P<0.05), and as CM dose increased to 100 and 300 mg/kg, the protease activity decreased even further (P<0.01)., Conclusion: The gut protease is involved in acute stress-induced visceral hypersensitivity, and CM can lower the visceral sensitivity and spinal c-fos expression in rats.

Background/aims: Ciprofloxacin is considered to be a safe and effective treatment for acute infectious colitis. However, this drug may cause drug-induced pancreatitis, albeit rarely., Methods: From March 2007 to February 2012, we studied 227 patients who were hospitalized for infectious colitis at St. Mary's Hospital. All of the patients received ciprofloxacin therapy for the treatment of infectious colitis. We observed a few cases of rare adverse events, including ciprofloxacin-induced acute pancreatitis diagnosed based on the Naranjo algorithm., Results: During ciprofloxacin therapy, seven of 227 patients (3.1%) developed rare pancreatitis as defined by the Naranjo algorithm; pancreatic enzyme activity was sporadically elevated with ciprofloxacin use. After ciprofloxacin administration, the average interval until the development of pancreatitis was 5.5 days (range, 4 to 7 days). On abdominal computed tomography, pancreatic swelling and homogenous enhancement was noted in three of seven patients. Complicating acute pancreatitis was gradually but completely resolved after cessation of ciprofloxacin administration. The mean recovery time was 11.3 days (range, 8 to 15 days)., Conclusions: We observed that ciprofloxacin-induced pancreatitis may occur with an incidence of approximately 3%. Ciprofloxacin-induced pancreatitis presents a short latency, suggesting an idiosyncratic hypersensitivity reaction. Practitioners should be aware that drug-induced pancreatitis can occur during ciprofloxacin therapy.

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

Interstitial fibrosis is a final common pathway for the progression of chronic kidney diseases. Activated fibroblasts have an extremely important role in the progression of renal fibrosis, and transforming growth factor (TGF)-β₁ is a major activator of fibroblasts. Since previous reports have indicated that serine protease inhibitors have a potential to inhibit TGF-β₁ signaling in vitro, we hypothesized that a synthetic serine protease inhibitor, camostat mesilate (CM), could slow the progression of renal fibrosis. TGF-β₁ markedly increased the phosphorylation of TGF-β type I receptor, ERK 1/2, and Smad2/3 and the levels of profibrotic markers, such as α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1, in renal fibroblasts (NRK-49F cells), and they were all significantly reduced by CM. In protocol 1, 8-wk-old male Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO) and were concurrently treated with a slow-release pellet of CM or vehicle for 14 days. Protocol 2 was similar to protocol 1 except that CM was administered 7 days after UUO. CM substantially improved renal fibrosis as determined by sirius red staining, collagen expression, and hydroxyproline levels. The phosphorylation of ERK1/2 and Smad2/3 and the levels of α-SMA, CTGF, promatrix metalloproteinase-2, and matrix metalloproteinase-2 were substantially increased by UUO, and they were all significantly attenuated by CM. These antifibrotic effects of CM were also observed in protocol 2. Our present results suggest the possibility that CM might represent a new class of therapeutic drugs for the treatment of renal fibrosis through the suppression of TGF-β₁ signaling.

The number of the chronic renal failure (CRF) patients is increasing explosively. Hypertension, proteinuria, inflammation, fibrosis, and oxidative stress are intertwined in a complicated manner that leads to the progression of CRF. However, the therapeutic strategies to delay its progression are limited. Since serine proteases are involved in many processes that contribute to these risk factors, we investigated the effects of a synthetic serine protease inhibitor, camostat mesilate (CM), on the progression of CRF in 5/6 nephrectomized (Nx) rats. Eighteen male Sprague-Dawley rats were divided into three groups: a sham-operated group (n = 6), a vehicle-treated Nx group (n = 6), and a CM-treated Nx group (n = 6). Following the 9-wk study period, both proteinuria and serum creatinine levels were substantially increased in the vehicle-treated Nx group, and treatment with CM significantly reduced proteinuria and serum creatinine levels. The levels of podocyte-associated proteins in glomeruli, such as nephrin and synaptopodin, were markedly decreased by 5/6 nephrectomy, and this was significantly ameliorated by CM. CM also suppressed the levels of inflammatory and fibrotic marker mRNAs including transforming growth factor-β1, TNF-α, collagen types I, III, and IV, and reduced glomerulosclerosis, glomerular hypertrophy, and interstitial fibrosis in histological studies. Furthermore, CM decreased the expression of NADPH oxidase component mRNAs, as well as reactive oxygen species generation and advanced oxidative protein product levels. Our present results strongly suggest the possibility that CM could be a useful therapeutic agent against the progression of CRF.

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

Background & Aims: Growth of exocrine pancreas is regulated by gastrointestinal hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates Nuclear Factor of Activated T cells (NFATs), but the genetic underpinnings and feedback mechanisms that regulate this response are not known., Methods: Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI stimulation was performed on Affymetrix 430A chips, and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with "flox-on" Rcan1 mice., Results: CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1-8 hours course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice., Conclusions: We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator, and firmly establish that CN-NFAT signaling is required for this response., (Copyright (c) 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The activities of cell surface serine proteases are markedly enhanced in malignant tumours. Proteolytic degradation of the extracellular matrix and basal membrane of normal cells is an important event for tumour cell growth and invasion. Two well-known broad-spectrum inhibitors of serine protease, Foy-305 and Ono-3403, were evaluated for their ability to affect the growth rate and survival of MCF7 breast cancer cells co-cultured with MRC5 lung fibroblasts as feeder cells in the absence of serum. Flow cytometry and differential staining demonstrated that in the mixed culture, the rate of tumor growth was dependent upon the presence of the feeder MRC5 lung fibroblasts and could be obviated by the additional presence of the inhibitors of serine proteases.

Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1-24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1-2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.

The papilla of Vater emptying into the duodenal bulb site is extremely rare and considered an aberrant condition. We report here a case with recurrent duodenal ulcer bleeding associated with this anomaly. A 42-year-old man was admitted to St. Mary Hospital because of tarry stool for three days. Despite no documented etiology to explain recurrent ulceration, the patient had about ten episodes of ulcer bleeding since 1995. On duodenoscopy, 1.0 x 0.6 cm sized active stage duodenal ulcer with oozing was observed at the posterior wall side below the pylorus. The papilla of Vater was bulging just below the pylorus. Bile juice was excreted from its opening. Pancreatic duct and common bile duct, which drained into the bulb site, were observed on ERCP. In this report, we show that recurrent duodenal ulcer can be associated with the papilla of Vater just below the pylorus.

Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.

Gabexate mesilate (GM) and camostat mesilate (CM) are protease inhibitors used for the treatment of pancreatitis, and have been reported to show anticancer effects in vivo. Lipid emulsions (20% fractionated soybean oil) were investigated in terms of physicochemical interaction between the drugs and lipid emulsions as a possible drug carrier. The result showed that the drugs did not distribute in the oil phase but were adsorbed at the phospholipid interface of oil droplets. With increasing concentration of the drugs, the adsorption amount at the interface rose steeply to around 2.2x10(-11) mol/cm2 for GM and 1.2x10(-11) mol/cm2 for CM, respectively, followed by further adsorption deviated from the Langmuir adsorption manner after the inflection. To interpret this two-stage adsorption of the drugs, surface potential and fluorescence changes were examined in addition to thermodynamics for their interaction with the interfacial lipid layer. The primary adsorption was exothermic and was due to electrostatic interaction and van der Waals interaction between drug molecules and phospholipid molecules. Both acidic and neutral phospholipids in the lipid were involved in the adsorption of GM, while acidic phospholipids were mainly involved in the adsorption of CM. On the other hand, the secondary adsorption was endothermic and was entropy-driven most probably due to hydrophobic interaction for GM and CM in common, including peripheral penetration of drug molecules into the interfacial lipid layer.

Pancreatic secretion of protein, water, chloride, and bicarbonate under basal conditions and in response to intravenous and intraduodenal stimuli were studied in awake rats fully recovered from surgery. During the basal phase of pancreatic secretion, protein output and water output were weakly correlated or uncorrelated, consistent with separate regulation and distinct cellular origin of enzyme (acinar cells) and water (duct cells), referred to as the two-component paradigm of pancreatic secretion. When pancreatic secretion was stimulated physiologically, water and protein output abruptly became strongly and significantly correlated, suggesting that protein secretion and water secretion are tightly coupled or that protein secretion is dependent on water secretion. The apparent function of this coupling is to resist or prevent increases in protein concentration as protein output increases. This pattern of secretion was reproduced by intravenous infusion of the CCK-58 form of cholecystokinin, which strongly stimulates pancreatic water and chloride secretion, but not by CCK-8, which only weakly stimulates water and chloride secretion in a non-dose-dependent manner. The remarkably tight association of water and protein secretion in food-stimulated and CCK-58-stimulated pancreatic secretion is consistent with a single cell type as the origin of both water and enzyme secretion, i.e., the acinar cell, and is not consistent with the two-component paradigm of pancreatic secretion. Because CCK-58 is the only detectable endocrine form of cholecystokinin in the rat and its bioactivity pattern is markedly and qualitatively different from CCK-8, actions previously recorded for CCK-8 should be reexamined.

Camostat mesilate (CM), an oral protease inhibitor, has been used clinically for the treatment of chronic pancreatitis in Japan. However, the mechanism by which it operates has not been fully understood. Our aim was to evaluate the therapeutic efficacy of CM in the experimental pancreatic fibrosis model induced by dibutyltin dichloride (DBTC), and we also determined the effect of CM on isolated monocytes and panceatic stellate cells (PSCs). In vivo, chronic pancreatitis was induced in male Lewis rats by single administration of 7 mg/kg DBTC and a special diet containing 1 mg/g CM was fed to the DBTC+CM-treated group from day 7, while the DBTC-treated group rats were fed a standard diet. At days 0, 7, 14 and 28, the severity of pancreatitis and fibrosis was examined histologically and enzymologically in both groups. In vitro, monocytes were isolated from the spleen of a Lewis rat, and activated with lipopolysaccharide stimulation. Thereafter, the effect of CM on monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) production from monocytes was examined. Subsequently, cultured rat PSCs were exposed to CM and tested to see whether their proliferation, MCP-1 production and procollagen alpha1 messenger RNA expression was influenced by CM. In vivo, the oral administration of CM inhibited inflammation, cytokines expression and fibrosis in the pancreas. The in vitro study revealed that CM inhibited both MCP-1 and TNF-alpha production from monocytes, and proliferation and MCP-1 production from PSCs. However, procollagen alpha1 expression in PSCs was not influenced by CM. These results suggest that CM attenuated DBTC-induced rat pancreatic fibrosis via inhibition of monocytes and PSCs activity.

Metastasis is one of the major causes of mortality in cancer. It is well known that the activities of cell surface serine proteases are especially enhanced in malignant tumors. Proteolytic degradation of the extracellular matrix and basal membrane is a crucial event for tumor cell invasion and metastasis formation. FOY-305 (Foypan), a remedy for tumor pancreatitis, is a broad spectrum synthetic serine protease inhibitor which inhibits enzymatic activities including trypsin, thrombin, kallikrein and plasmin. Using Lewis lung carcinoma cell, we found that FOY-305 inhibited both spontaneous and experimental pulmonary metastasis. Furthermore, the combined treatment of FOY-305 and a traditional anti-cancer drug, 5-FU or bleomycin, resulted in marked enhancement of anti-pulmonary metastatic activity.

Context: The tumor protein p53-induced nuclear protein 1 (TP53INP1) gene was found using DNA microarray technology as an overexpressed gene in acute pancreatitis. However, expression of TP53INP1 in chronic pancreatitis has not been previously reported., Objective: This study investigated TP53INP1 gene expression and its relationship with p53 and apoptosis in spontaneous chronic pancreatitis in the Wistar-Bonn/Kobori rat., Methods: Ninety four-week-old male Wistar-Bonn/Kobori rats were fed a special breeding diet until sacrifice. Camostat mesilate (n=30) or a herbal medicine (Saiko-keishi-to; n=30) were mixed with the diet, while the other 30 rats were untreated. The rats were sacrificed every 4 weeks for 20 weeks, and the pancreas was examined. In addition, 6 four-week-old male Wistar-Bonn/Kobori rats were sacrificed and studied as starting reference. Finally, Wistar rats (n=36) were studied as controls., Main Outcome Measure: TP53INP1 mRNA expression was determined by reverse transcription-polymerase chain reaction using semi-quantitative analysis, direct sequencing and in situ hybridization., Results: TP53INP1 mRNA was strongly expressed at 12 weeks when chronic pancreatitis developed, with a second peak at 20 weeks. The expression kinetics of TP53INP1 mRNA paralleled acinar cell apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The p53 mRNA expression showed a single peak at 12 weeks. In situ hybridization revealed that TP53INP1 mRNA was expressed mainly in acinar cells. Therapeutic drugs such as camostat mesilate and a herbal medicine Saiko-keishi-to suppressed the TP53INP1 mRNA expression. TP53INP1 mRNA induction in acinar cells was confirmed with in vitro experiments using an arginine-induced rat pancreatic acinar AR4-2J cell injury model., Conclusions: TP53INP1 expression may reflect the acute-phase response and apoptosis of acinar cells in the course of chronic pancreatitis.

CCK acts on pancreatic acinar cells to increase intracellular Ca(2+) leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca(2+) and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.

Duchenne muscular dystrophy is known to be caused by a defective gene of dystrophin, a 427-kDa cytoskeletal protein, but the effective therapeutic drug is presently unavailable. We previously reported that a trypsin-like protease designated as dystrypsin is markedly activated in the muscle microsomal fraction immediately before onset of the clinical signs in mdx mice, a dystrophin-deficient hereditary animal model for human Duchenne muscular dystrophy. In order to examine the possible participation of dystrypsin in the occurrence of the disease, we investigated the therapeutic effects of dystrypsin inhibitors on the occurrence and progress of muscular dystrophy. Here, we show that camostat mesilate, a low-molecular-weight inhibitor of trypsin-like proteases, including dystrypsin, is a candidate drug for Duchenne muscular dystrophy.

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in rats.

Our recent evidence suggests that pancreatic digestive enzymes in the lumen of the intestine may play a major role in the production of cardiovascular stimulatory factors during splachnic artery occlusion and reperfusion. These stimulators are detected in plasma, but their origin and mechanism of production has remained uncertain. We examine here in the rat the role of pancreatic enzymes with and without administration of a serine protease inhibitor (FOY) into the lumen of the small intestine during splanchnic artery occlusion (90 min) and reperfusion (120 min). In the presence of pancreatic enzyme inhibition in the lumen of the intestine, there is significantly enhanced survival rate, lower levels of inflammatory mediator production, the femoral artery blood pressure is maintained close to control levels, and there are significantly lower levels of cell activators in plasma. These results support the hypothesis that pancreatic enzymes may escape across the brush border barrier during intestinal ischemia and thereby initiate the production of a powerful set of cytotoxic mediators. Blockade of pancreatic enzymes in the lumen of the intestine may be a tool to interfere with inflammation and early indicators of multiorgan failure.

Background & Aims: Proteolytic release and activation of latent transforming growth factor beta (TGF-beta) by the hepatic stellate cells (HSCs) are key events for pathogenesis of hepatic fibrosis, and protease inhibitors suppress TGF-beta generation by cultured HSCs, suggesting their potential use as antifibrogenic agents. We explored this idea using camostat mesilate, a serine protease inhibitor, to determine its effects and mechanisms of action in vivo., Methods: Camostat mesilate was either added to cultured rat HSCs or administered orally to rats during porcine serum treatment, followed by overexpression of urokinase. We measured cellular and hepatic levels of plasmin, TGF-beta, TGF-beta activity, activated HSC markers (increased cell number, morphologic change, and expression of both alpha-smooth muscle actin and collagen(alpha2)[I]), and fibrosis (Azan-staining and quantification of hydroxyproline content)., Results: Camostat mesilate (500 micromol/L) inhibited generation of TGF-beta by suppressing plasmin activity and reduced the activity of TGF-beta, which blocked in vitro activation of HSCs. In the in vivo model, camostat mesilate (1-2 mg/g of diet) markedly attenuated an increase in hepatic plasmin and TGF-beta levels, HSC activation, and hepatic fibrosis without apparent systemic or local side effects, all of which were reverted by restoration of hepatic plasmin activity., Conclusions: Camostat mesilate prevents porcine serum-induced rat hepatic fibrosis via a profound reduction in TGF-beta generation.

Background & Aims: Cholecystokinin (CCK) is known to stimulate the synthesis of digestive enzymes in the pancreas at the translational level. We investigated in vivo the biochemical regulation of initiation factors important for the stimulation of translation of digestive enzyme protein in rat pancreas by CCK., Methods: Intraperitoneal injection of CCK or intragastric administration of a trypsin inhibitor to elicit endogenous CCK release was followed by removal and preparation of pancreas for protein evaluation. Isoelectric focusing was used to evaluate the phosphorylation of the initiation factor eIF4E, and Western blotting and immunoprecipitation followed by Western blotting were used to study the phosphorylation state and amount of other interacting factors., Results: CCK treatment induced a time- and dose-dependent phosphorylation of pancreatic eIF4E and its binding protein (PHAS-I). Because the release of eIF4E from its binding protein as a result of phosphorylation is followed by formation of a messenger RNA cap-binding complex that includes the initiation factor eIF4G, we evaluated the association of eIF4G with released eIF4E and showed that it was increased by CCK. These events occurred over a range of CCK doses from 0.2 to 5 microg/kg. We also evaluated the effect of endogenous CCK by administering a synthetic trypsin inhibitor, camostat (100 mg/kg). Camostat treatment markedly increased the phosphorylation of both PHAS-I and eIF4E and the formation of eIF4E-eIF4G complex. Thus, both exogenous and endogenous CCK activate translational initiation factors in vivo., Conclusions: Activation of translational machinery necessary for initiation of protein synthesis likely contributes to the normal postprandial synthesis of pancreatic digestive enzymes.

The effects of oral administration of two synthetic trypsin inhibitors (camostate and ONO-3403) and soybean trypsin inhibitor (SBTI) on cholecystokinin (CCK), secretin gene expression and pancreatic secretion were examined in CCK-A-receptor-deficient (OLETF) rats. The rats were fed chow containing 0.1% trypsin inhibitors for 7 days. To examine pancreatic secretion, the rats were prepared with cannulae to drain the bile and pancreatic juice separately, a duodenal cannula and an external jugular vein cannula. The animals were maintained in Bollman cages and the experiments were conducted 4 days after surgery. The levels of CCK mRNA were significantly increased by each treatment. The levels of secretin mRNA were significantly increased by camostate and SBTI, but not by ONO-3403. Bicarbonate secretion was significantly increased in rats treated with camostate and ONO-3403, but not SBTI, while protein secretion was not affected by any treatment. These observations suggest that increased bicarbonate secretion produced by synthetic trypsin inhibitors in CCK-A-receptor-deficient rats may not be due to secretin but due to ONO-3403 in the circulation.

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 micrograms/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 micrograms/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.

Background & Aims: Feedback regulation of pancreatic enzyme secretion is well established in animals, and their pancreases are able to adapt to intraduodenal inhibition of pancreatic enzymes by proteinase inhibitors such as Camostate (FOY-305; Schwarz GmbH, Monheim, Germany). In this study, we addressed whether similar adaptive changes occur in the human pancreas after 4 weeks of 2 g/day Camostate application., Methods: Before, at the end of, and 2 weeks after 4-week Camostate treatment (four times 500 mg daily), pancreatic changes were analyzed with the use of a secretin-cerulein test, a test-meal stimulation, cholecystokinin plasma measurement, and standardized ultrasonographic investigations of the pancreas., Results: Duodenal trypsin output after secretion stimulation was significantly increased (+44%; P < 0.01) and duodenal bicarbonate output decreased 22% (P < 0.05) after 4 weeks of Camostate application. The size of the pancreatic head (vertical) increased 8% (P < 0.05) at week 4 and decreased to pretreatment values 2 weeks after treatment (week 6). The other three diameters measured (head oblique, body, and tail) showed a similar pattern. Stimulated cholecystokinin plasma levels 15 minutes after application of a standard test meal increased 62% (P < 0.05)., Conclusions: The human pancreas adapts to oral application of the proteinase inhibitor Camostate. These findings support the theory that feedback control of the exocrine pancreas operates in humans.

1. It is now well established that cholecystokinin (CCK) has a major physiological role in the regulation of pancreatic secretion and gastro-intestinal (GI) motility. Both these actions are mediated by stimulation of CCK(A)-receptors located on pancreatic acini and GI smooth muscle cells. While chronic administration of CCK-like peptides invariably causes pancreatic hypertrophy and hyperplasia, their action on gastric growth remains controversial. 2. In the present investigation the action of exogenous and endogenous CCK on both pancreatic and gastric growth was studied in the same animal. In addition, the ability of dexloxiglumide, a new potent and selective CCK(A)-receptor antagonist, to counteract CCK-mediated effects was evaluated. 3. The amphibian peptide caerulein (1 microg kg(-1) intraperitoneally three times daily) was used as a CCK agonist, while camostate (200 mg kg(-1) intragastrically once daily), a synthetic protease inhibitor, was used to release endogenous CCK. They were administered to rats for seven days with or without dexloxiglumide (25 mg kg(-1) subcutaneously 15 min before the stimulus). On the eighth day, animals were killed, the pancreas and stomach excised, weighed, homogenized and their protein and DNA content measured. 4. Both exogenous and endogenous CCK increased the weight of the pancreas as well as the total pancreatic protein and DNA content. Dexloxiglumide, which alone did not affect pancreatic size and composition, was able to counteract both caerulein- and camostate-induced pancreatic changes. Neither stimuli affected gastric growth in respect of weight and composition of the oxyntic gland area and the antrum. 5. These results show different effects of CCK on pancreatic and gastric growth. The CCK-induced pancreatic hypertrophy and hyperplasia are blocked by the potent and specific CCK(A)-receptor antagonist, dexloxiglumide. This compound therefore represents a useful tool to investigate CCK-receptor interactions in peripheral organs.

The effect of methylcarbonylmethyl 2(S)-14-(4-guanidino-benzoyloxy) phenyl] propionate methanesulfonate (TT-S24) on experimental pancreatitis in rats was examined in comparison with that of camostat. TT-S24 showed a preventive effect on increases in plasma amylase activity and pancreatic weight induced by cerulein injection. TT-S24 also reduced an increase in plasma amylase activity induced by taurocholate. TT-S24 effectively prevented the mortality induced by an injection of a mixture of trypsin and taurocholate. TT-S24 showed no effect on an increase in amylase activity 6 h after duodenum ligation (closed duodenal loop pancreatitis), indicating that the drug had no effect on the initiation and propagation step of closed duodenal loop pancreatitis. On the other hand, TT-S24 reduced an increase in amylase activity 6 h after release of the duodenum ligation. TT-S24 showed anti-trypsin, anti-kallikrein, anti-thrombin and anti-plasmin activities. The effect of TT-S24 on some experimental pancreatitis models was nearly equal to or somewhat more potent in most instances to that of camostat. Therefore, TT-S24 should be useful in the clinical treatment of pancreatitis.

Patients with early non-insulin-dependent diabetes mellitus (NIDDM) empty glucose solutions from their stomachs more rapidly than non-diabetic control subjects, and this exacerbates postprandial hyperglycaemia. To determine if accelerated gastric emptying occurred in a rat model of NIDDM and influenced postprandial hyperglycaemia, gastric emptying of glucose was measured, and the effect of slowing the gastric emptying rate on postprandial hyperglycaemia was observed. We tested eight male obese Zucker diabetic rats and eight age-matched lean Zucker controls at 10-13 weeks of age to measure gastric emptying of glucose (by gamma scintigraphy). Rats fasted overnight were gavaged with 30% glucose at 1 ml/100 g body weight. Separately, six Zucker diabetic rats and six lean controls were tested for sensitivity to the inhibitory effects of cholecystokinin and secretin on gastric emptying. The diabetic rats emptied glucose significantly faster than controls (t1/2 = 37.3 +/- 1.5 vs 58.8 +/- 2.3 min in controls), and aging exaggerated this differential. Camostat, a stimulant of cholecystokinin and secretin release, added to the glucose meal significantly slowed gastric emptying (t1/2 = 123 +/- 23 and 166 +/- 19 min, diabetic vs lean, respectively), and significantly reduced postprandial hyperglycaemia in diabetic rats. Compared to Zucker lean controls, Zucker diabetic rats were as sensitive (cholecystokinin) or more sensitive (secretin) to gastrointestinal hormones that inhibit gastric emptying. The results demonstrate accelerated gastric emptying in a rat model of NIDDM, consistant with similar observations in humans with early NIDDM. These results also support the proposal that interventions to slow gastric emptying improve glucose control in this disease.

Effects of a new cholecystokinin (CCK)A-receptor antagonist, T-0632 [sodium (S)-1-(2-fluorophenyl)-2, 3-dihydro-3-[(3-isoquinolinylcarbonyl) amino]-6-methoxy-2-oxo-1H-indole-3-propanoate], on caerulein-induced and pancreatic duct ligation-induced pancreatitis models were studied and compared with the CCKA-receptor antagonist loxiglumide and the orally active protease inhibitor camostate, respectively. In rats, orally administered T-0632 potently prevented the caerulein-induced increases in pancreatic digestive enzymes in plasma and suppressed the histological changes in the pancreas. The estimated ED50 values of T-0632 and loxiglumide were 0.0092 and 8.9 mg/kg, respectively. In dogs, T-0632 (0.1, 1 mg/kg, i.d.) prevented the caerulein-induced increase in plasma amylase activity in a dose-dependent manner. Loxiglumide (100 mg/kg, i.d.) did not show any preventive effects. In pancreatic duct ligation (6 hr)-induced pancreatitis of the rat, T-0632 (0.001-0.1 mg/kg, p.o.) partially prevented both the increase in plasma amylase activity and the histological changes in the pancreas, whereas camostate (10, 100 mg/kg, p.o.) did not show any preventive effects. In pancreatic duct ligation (3 hr)-induced pancreatitis, caerulein injection (1 microgram/kg, s.c.) caused a further increase in plasma amylase activity, and T-0632 (0.01, 0.1 mg/kg, p.o.) dose-dependently decreased the aggravation by caerulein. We conclude that T-0632 showed preventive effects on all of these pancreatitis models by oral or intraduodenal administration. These results suggest that CCK plays an important role in progression and aggravation of acute pancreatitis, and T-0632 may have a therapeutic value in these disease states.