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The effects of chronic oral treatment of rats with 400 mg/kg body weight camostat, a synthetic protease inhibitor, on weight gain and both pancreatic exocrine and endocrine secretion were studied and compared with pair-fed controls. Administration of camostat was found to result in a lower weight gain than in untreated rats fed ad libitum because of reduced food intake. The pancreas of treated rats showed hypertrophy and hyperplasia with significantly higher total contents of amylase, lipase, trypsinogen and chymotrypsinogen than that of pair-fed controls. The specific activity of lipase, trypsinogen and chymotrypsinogen remained unchanged but the specific activity of amylase was significantly lower than in pair-fed controls. Stimulation of pancreatic enzymes with CCK 8 in treated rats resulted in a greater output of proteases, no difference in secretion of lipase and a lower secretion of amylase than in pair-fed rats. Glucose-dependent insulin secretion was also significantly lower in camostat-treated rats than in controls. This effect could be reversed by gastric inhibitory polypeptide. After termination of treatment with camostat all enzymes were normalized within 14 d. Our results on the hypertrophied pancreas of rats suggest preferential synthesis and secretion of protease at the expense of amylase and diminished sensitivity of insulin to stimulation by glucose. All effects of camostat on the pancreas were reversible.

In the present study the effect of atropine on feedback regulation of pancreatic function was investigated in male rats. Orogastric tube feeding of a single dose of the protease inhibitor camostate (100 mg) caused a 6-fold increase in plasma CCK. This was accompanied by a 50% decrease in pancreatic enzyme content for up to 6 h. The effect of i.v. infusion of atropine (100 micrograms.kg-1.h-1) was studied 60 and 180 min after camostate feeding. Atropine completely abolished the decrease in enzyme content 180 min after camostate feeding. The stimulated CCK plasma levels were significantly lowered by the infusion of atropine 60 min, but not 180 min, after feeding of camostate. It is discussed that a cholinergic pathway mediates CCK release in the early phase of feedback regulation of pancreatic function.

The serine proteinase inhibitor camostate was fed to rats in single, daily doses of 100 mg/kg, 200 mg/kg, and 400 mg/kg for 5, 10, or 15 days. Within 5 days, pancreatic size and protein, DNA, and enzyme content increased significantly. After prolonged administration, this trophic effect was more pronounced, and anticoordinate regulation of the synthetic rate of individual secretory proteins was observed. While enzyme content and protein synthesis of trypsinogen and chymotrypsinogen were increased, respective values for amylase were drastically reduced. Plasma levels of cholecystokinin (CCK) did not differ from controls when measured 24 h after administration of camostate. Immediately after oral feeding of camostate, CCK levels increased 10-fold above controls, reached a maximum after 90 min, and remained elevated for more than 6 h. Proglumide, a CCK-receptor antagonist, only slightly reduced the trophic action of the proteinase inhibitor. The data indicate that endogenous CCK release by a proteinase inhibitor is as potent in the modulation of pancreatic growth and individual enzyme synthesis as exogenous hormone application.

Oral administration of a synthetic protease inhibitor. [N,N-dimethylcarbamoyl-methyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate, was used to challenge 3-methylcholanthrene-induced carcinoma. This drug was administered 3 times daily via a stomach tube at doses of 12.5, 25 and 50 mg/kg in 33 mice harboring solid tumors. Oral administration of more than 50.0 mg/kg of this protease inhibitor significantly inhibited tumor growth and prolonged the survival time of cancer-harboring animals (P les than 0.05). These results indicate that oral administration of an inhibitor of kinin-forming protease has an antitumor effect on malignant tumors.

The effect of long-term oral synthetic protease inhibitor (FOY 305) administration on fasting blood sugar (FBS), body weight, glucose tolerance, plasma insulin and glucagon levels, pancreatic insulin and glucagon contents, hepatic enzyme activities, and plasma lipids in normal and streptozotocin (STZ)-induced diabetic rats was studied. Normal rats treated with oral FOY 305 for 9 weeks were found to have pancreatic hypertrophy and decreased body weight gain as compared with the untreated normal controls. FBS, glucose tolerance, plasma insulin and glucagon levels, pancreatic insulin and glucagon contents, and plasma lipids were uninfluenced in FOY 305 treated normal rats. STZ-induced diabetic rats treated with oral FOY 305 were found to have decreased FBS for 5 weeks after the beginning of FOY 305 administration as compared with the untreated diabetic controls, whereas at the 7th and 9th week after treatment there was no difference in FBS between FOY 305 treated and untreated diabetic rats. In the metabolic balance observed at the 4th week after treatment, a slight improvement of the diabetic state was found in FOY 305 treated diabetic rats. There was no apparent difference in the blood sugar curve and insulin response following oral glucose load between diabetic rats treated for 7 weeks and untreated diabetic rats. All the rats were sacrificed after 9 weeks of treatment. Diabetic rats treated with oral FOY 305 for 9 weeks showed pancreatic hypertrophy and decreased plasma glucagon level and decreased pancreatic glucagon content as compared with the untreated diabetic controls, whereas there was no difference in body weight, plasma insulin level and pancreatic insulin content between FOY 305 treated and untreated diabetic rats. Furthermore, oral FOY 305 treatment improved hyperlipidemia in STZ-induced diabetic rats and also significantly improved the hepatic pyruvate kinase and phosphoenlpyruvate carboxykinase activities of diabetic rats. These improvements might partly be due to a decreased pancreatic content and secretion of glucagon and/or a direct action of the synthetic PI, FOY 305 to tissues.

Two models of severe acute pancreatitis were chosen and pancreatitis induced by sodium taurocholate and by a choline-deficient ethionine-supplemented diet, to evaluate the effectiveness of FOY-305 (camostate), a new synthetic trypsin inhibitor. Prophylactic administration of FOY-305 had a significantly favorable effect on the course of the sodium taurocholate-induced disease and on the survival rate of the treated group. A beneficial effect on the amylase and lipase content in serum and ascites was found, but no effect was observed on enzyme concentration in pancreatic tissue or on the degree of histologically detectable organ destruction. Therapeutic administration of FOY-305 had a significantly positive influence when infused directly, 5 and 30 min after the operation, whereas enzyme increase and organ destruction remained unaffected. FOY-305 showed a beneficial effect when given prophylactically or therapeutically at the beginning of the pancreatitis induced by a CDE diet, with no significant change in enzyme increase and degree of organ destruction. The favorable effect on survival time and rate in the early phase of these two severe experimental forms of pancreatitis may justify an evaluation of FOY-305 in a clinically controlled study.

It was examined what mechanism involved in an increase of met-enkephalin (met-EK)-like peptide content in the pulp induced by noxious stimuli. The increased content of the peptides by cavity formation as noxious stimulation was not influenced by cycloheximide, but attenuated by FOY-305 [N,N-dimethyl carbamoyl-methyl 4-(4-guanidinobenzoyloxy) phenyl acetate methanesulfonate], a trypsin-like enzyme inhibitor, and enhanced by captopril, and attenuated by infusion of saline in the pulp cavity. From these results, it was suggested that noxious stimuli on the pulp led to activation of trypsin-like enzymes followed by an increased content of met-EK-like peptides, and thereafter, the peptides, such as met-EK, might be degraded by angiotensin converting enzyme (ACE). Furthermore, an immunohistochemical study demonstrated that met-EK-like immunoreactivity (met-EK-IR) of cells in the rat incisor pulp was clearly increased following tryptic digestion of the pulp section, supporting a suggestion mentioned above.

In conscious rats with cannulated pancreatic ducts, the synthetic trypsin inhibitor camostat (400 mg/kg) was instilled into the stomach once daily on 5 consecutive days. Each camostat administration stimulated the secretion of juice, bicarbonate, and protein. From day 2 to day 5, basal protein secretion measured before the daily instillation of camostat was increased, whereas basal secretion of volume and bicarbonate remained unchanged. The secretory responses to camostat increased up to day 3 of treatment without further change on days 4 and 5. In response to acute camostat instillation, parallel secretion of enzymes was found. Within 1 day, however, a change in the composition of secreted enzymes was observed. After 3 days, the percentage of amylase in pancreatic juice decreased by approximately 50%, whereas the relative amount of proteinases and the specific activity of phospholipase A2 doubled. The alteration of protein patterns in pancreatic juice parallels the effect on enzymes stored in the gland. The results demonstrate that adaptation of enzyme secretion in the exocrine pancreas occurs after a short period of treatment with camostat. Because this compound stimulated protein, volume, and bicarbonate secretion, it is suggested that other factors in addition to cholecystokinin may be involved in this adaptation process.

Neutral protease activity was significantly elevated in the cerebro-spinal fluid of patients with multiple sclerosis (MS) in exacerbation and in the acute phase of acute viral meningoencephalitis (AME) compared with that of MS in remission, amyotrophic lateral sclerosis or psychosomatic disease. Since in each relapse of MS, protease activity was higher in exacerbation than in remission, this activity may be one good marker of disease activity in MS. One hundred micro molar of FOY305, synthetic protease inhibitor, inhibited in vitro increased neutral protease activity in MS in exacerbation, which suggests the possibility of a clinical application of this protease inhibitor for MS.

The effects on pancreatic growth and plasma CCK concentration of chronic feeding of camostate (400 mg/kg day for 10 days), a potent inhibitor of serine proteases including trypsin, were assessed in the mouse. For comparison, the trophic effects of chronic exogenous administration of CCK octapeptide (sc injection of 1 microgram/kg day every eight hours for 10 days) were also studied. In addition, the effects of a proglumide-analogue CCK-receptor antagonist (CR1409) on the stimulatory actions of camostate feeding and chronic administration of exogenous CCK were studied. The effects of the combination of chronic camostate feeding and sc injections of CCK, the effects of acute camostate feeding, and the effects of the CCK-receptor antagonist given without camostate or CCK were also studied. The results show that chronic camostate feeding markedly increased CCK plasma concentrations eight-fold over control values, and that acute camostate feeding increased plasma concentration to four fold of control values. Correspondingly, chronic camostate feeding markedly increased pancreatic weight, protein and DNA content. Exogenous CCK-8 also had qualitatively similar, but quantitatively less potent stimulatory effects. The combination of camostate and CCK-8 resulted in an additive stimulatory effect. The trophic actions of exogenous and endogenous CCK grossly increased chymotrypsinogen content, but left amylase content unaffected. The CCK-receptor antagonist CR 1409 completely abolished the trophic effects of exogenous CCK and greatly inhibited the effects of chronic camostate feeding. The CCK antagonist decreased pancreatic weight, DNA and protein content compared to control values when given without any CCK or camostate. We conclude that the protease inhibitor camostate is a very strong release effector of CCK and exerts a powerful trophic effect on mouse pancreas which is probably mediated by CCK. Furthermore, physiological increases of CCK during feeding of regular chow appear to exert trophic effects on the exocrine pancreas.

This study was designed to investigate the role of ornithine decarboxylase (ODC) and polyamines in pancreatic adaptation. Cholecystokinin (CCK) is well-known to be a potent trophic stimulus on the pancreas. On the other hand, the oral application of the synthetic trypsin inhibitor camostate results in an extensive release of endogenous CCK in rats. alpha-difluoromethylornithine (DFMO), an irreversible and specific inhibitor of ODC, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. Camostate feeding (200 mg/kg b.wt. orally twice a day) resulted in a rapid elevation of ODC activity already after 2 hours, reaching a maximum after 6 hours (about 200fold above controls) followed by a significant increase in putrescine after 4 hours and spermidine after 24 hours while spermine remained unchanged. The trophic parameters increased as expected in following time-course: thymidine kinase (12 hours), DNA polymerase (12 hours), protein (24 hours), pancreatic weight (24 hours) and DNA (5 days). DFMO (2% in drinking water + 3 x 300 mg/kg b.wt. i.p. during daytime) was not able to prevent but significantly delayed and reduced the camostate-induced increase in ODC and polyamines as well as the trophic parameters. These data indicate an essential role for ODC and polyamines in camostate-induced pancreatic growth and hormonal mediated pancreatic adaptation.

Chronic oral administration of camostate, a specific serine protease inhibitor, is known to induce pancreas hypertrophy in rats. A possible influence of the protease inhibitor on the endocrine rat pancreas was studied using isolated perfused pancreas and islet incubations. The presence of camostate had no direct effect on the glucose-induced insulin release in vitro in concentrations from 1 microM to 1 mM, but enhanced the basal insulin release from islets cultured over 24 h in media containing the protease inhibitor (100 microM). Administration of camostate over 14 days to rats induced a remarkable hypertrophy of the pancreas without influencing plasma insulin or gastric inhibitory polypeptide levels and insulin concentration of the pancreas. Glucose-stimulated insulin release from the perfused pancreas was not increased despite significantly higher total insulin content. It is concluded that camostate exerts no direct effect on the glucose-stimulated insulin release and that chronic administration of the compound induces pancreas hypertrophy in vivo without influencing insulin release.

Oral application of a single dose of a new synthetic proteinase inhibitor Camostate (Foy-305) in male Wistar rats was carried out together with studies of in vitro amino acid incorporation followed by separation of proteins by two-dimensional gel electrophoresis. The aim of this experiment was to analyze changes produced by the inhibitor in total protein and individual enzyme biosynthesis. Administration of 100 mg/kg Foy-305 resulted in significant inhibition of total pancreatic protein synthesis, without changes in fractional rates for individual enzymes. 50 mg/kg Foy-305 induced a 10-fold elevation of cholecystokinin (CCK) levels in serum; this persisted for 3 h and led to a significant increase in the total rate of protein synthesis with peak values at 6 and 9 h (78% and 84% above control levels, respectively), returning to control by 15 h. Changes in fractional rates of synthesis occurred with a latency of 6 h and were restricted to amylase and the anionic form of trypsinogen and chymotrypsinogen. Amylase biosynthesis decreased by about 40% from control levels at 9 h to return to control levels by 15 h. Increased synthesis of trypsinogen and chymotrypsinogen was observed; this was also phasic. The results show similar enzyme-specific regulation as previously described for exogenous CCK stimulation and for the adaptation of the pancreas to diets enriched in protein. They demonstrate the effectiveness of pulsatory endogenous hormone release in the regulation of protein synthesis.

Pancreatic secretion and plasma cholecystokinin (CCK) and secretin levels were measured in 10 healthy volunteers after application of a serine protease inhibitor (camostate) to study the mechanism of feedback regulation. Camostate produced a strong inhibition of trypsin and chymotrypsin activity in duodenal juice for 1 h. This was accompanied by an increase in duodenal aspirate volume and pancreatic enzyme secretion under both basal and secretin-stimulated conditions. Due to inhibition of tryptic activity, survival of lipase activity in duodenal juice was prolonged. In control experiments we ruled out that the volume and the pH of the solution were responsible for stimulation of pancreatic secretion. The protease inhibitor did not alter pancreatic secretion, which was stimulated by a test meal. Plasma levels of CCK and secretin were not changed after duodenal perfusion of camostate. These observations suggest that trypsin and chymotrypsin are involved in feedback regulation of pancreatic secretion in man which is, however, not mediated by CCK or secretin.

Proglumide was shown to possess a low affinity for cholecystokinin (CCK) receptors and to inhibit the synergistic action of CCK8 on glucose-mediated insulin release in vitro. Proglumide (400 mg/kg i.p., given 15 min before an i.v. combination of CCK8 and glucose) reversed the CCK8 (0.5 nmol/kg)-induced increase of plasma insulin levels and decrease of glucose levels. It had no effect on plasma insulin and glucose levels when the glucose bolus was administered alone. Camostate (400 mg/kg p.o.; Foy-305; a trypsin inhibitor acting via endogenously released CCK) increased plasma insulin levels by 10 microU/ml during an oral glucose (500 mg/kg) tolerance test. Pretreatment with proglumide (400 mg/kg i.p.) antagonized this effect. The data indicate that proglumide has an antagonistic effect on either exogenously added or endogenously released CCK with respect to plasma insulin and glucose levels and that it has no effect on plasma insulin and glucose levels when glucose is given alone. Therefore, proglumide and the newly developed, more potent CCK receptor antagonists are able to disturb insulin and glucose homoeostasis.

Differences in pancreatic responses and CCK release to intragastric administration of a synthetic protease inhibitor (camostate) were examined in young (6-mo) and old (26-mo) female rats. When rats were sacrificed 1 hour after camostate administration (100 mg/kg), plasma CCK concentration significantly increased in both age groups and was significantly higher in young rats than in the old. Acute pancreatic responses to camostate were attenuated in old rats, compared with the young. On the other hand, 5-day administration of camostate (100 mg/kg, twice a day) increased pancreatic wet weight, chymotrypsin concentration and content in pancreas in both age groups, whereas lipase content did not increase in old rats although it significantly increased in young rats. Plasma CCK concentrations were not significantly different between young and old rats. It is concluded that the acute response of CCK release to camostate is attenuated in old rats, but the capability of chronic pancreatic adaptation of chymotrypsin content to camostate is well maintained in old rats.

Pancreatic injury was induced to rats with intraperitoneal injection of ethionine 60 mg per 100 g BW twice or three times weekly for 6 weeks. These rats were given 100 mg/kg of Camostate mesilate (CM) via a gastric tube daily for 14 days. CM administration resulted in an increase of pancreatic wet weight, hypertrophy and hyperplasia of acinar cells, and an increase of exocrine pancreatic function. Acini prepared from CM and ethionine-treated rats exhibited increased response to caerulein, but decreased sensitivity to caerulein. The plasma CCK level in rats with CM administration 24 hours later was higher than that without CM administration. However, there were no significant changes in plasma CCK and secretin level thereafter. We concluded that CM had a trophic effect on the pancrease with ethionine-induced pancreatic injury, and CCK was considered playing the same role in injured pancreas as the normal rat pancreas. Studies using CCK receptor antagonist are needed for further clarification.

The effect of the potent synthetic protease inhibitor [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate (FOY-305) on skin carcinogenesis in ddY mice was examined over a total observation period of 105 days. Administration of 0.1% FOY-305 in the diet suppressed the incidence of carcinomas induced by repeated local application of the carcinogen 3-methylcholanthrene (MCA) (P less than .05) in mouse skin and delayed the time of appearance of the skin tumors (P less than .001). There was no significant difference in the number of tumors per tumor-bearing mouse and the size of the tumors between mice treated with MCA and mice treated with MCA plus FOY-305.

A dose of FOY-305 [N,N-dimethylcarbamoylmethyl-4-(4-guanidinobenzoyloxy)phenylacetate methanesulfonate; 100 mg/kg body weight] was administered orally once a day for 30 days to a group of rats (FOY group). To the control group of rats, 10 ml/kg body weight of distilled water (a solvent of FOY-305) was administered in the same way. The FOY-305 administration induced hypertrophy of the pancreas: the increase in wet weight of the pancreas was significantly higher than the increase in DNA content. The administration also induced hyperpyknia in the FOY group as shown by the increased concentration of constituents (amylase, trypsinogen, and chymotrypsinogen) in the pancreas. The secretory responses of the exocrine pancreas to stimulation with cholecystokinin (CCK) were examined in vivo. The secretory responses induced by lowered doses of CCK in the FOY group were slightly greater than those in the control group, but the differences were not statistically significant. On the other hand, the responses induced by higher doses of CCK were significantly greater than those in the control group. The secretory responses to stimulation with CCK or acetylcholine (ACh) were examined further in vitro using an isolated perfused pancreas. Protein output induced by lower doses of the secretagogues (146-730 pM CCK or 10 nM ACh) was similar in the control and the FOY groups, but the output induced by higher doses of the secretagogues (1,460 pM CCK or 30-100 nM ACh) in the FOY group was significantly larger than that in the control group. The present results provided evidence that the FOY-305-induced hypertrophy and hyperpyknia of the rat pancreas coincided with potentiation of secretory responses to higher doses of the secretagogues.

Exocrine pancreatic amino acid transport was studied following oral treatment of rats with the synthetic protease inhibitor camostat mesilate. Camostat (200 mg/kg/day) was administered via an orogastric tube for 10 days, and kinetics of amino acid transport were then examined in the isolated perfused pancreas using a dual-isotope dilution technique. Administration of camostat doubled the rate constant (Vmax/Kt) for L-phenylalanine transport via System L by increasing the Vmax from 12.3 +/- 0.4 (n = 12) to 19.6 +/- 2.2 mumol/min/g (n = 4). Although the Kt (35 +/- 2 mM, n = 4) and Vmax (47.7 +/- 1.6 mumol/min/g) for L-serine transport via System asc increased, the Vmax/Kt ratio decreased from a control value of 1.85 to 1.36. Increases in Kt (5 +/- 2 mM, n = 4) and Vmax (5.18 +/- 1.01 mumol/min/g) for L-lysine transport via System y+ were also detected but Vmax/Kt remained unchanged. In the presence of a low Na+ perfusate, influx of L-serine, L-phenylalanine, and L-lysine were inhibited between 36 and 50%. Treatment with camostat results in an increase in the rate and Na+-dependence of large neutral amino acid transport in the rat exocrine pancreas. Pancreatic growth induced by repeated oral administration of camostat appears to be accompanied by an enhanced uptake of precursor amino acids.

Activities of plasminogen activators (PA) and their inhibitors were studied in the bullous lesions of 2 siblings with recessive dystrophic epidermolysis bullosa (RDEB). The hematological findings of the patients revealed hyperfibrinogenemia, hyper-gamma-globulinemia and marked thrombocytosis. The immunofluorescent studies showed strong deposits of plasminogen, fibrin-degenerative products and alpha 1-antitrypsin around the blister lesions. On the other hand, alpha 2-macroglobulin (alpha 2-M) was sparsely deposited. The levels of alpha 2-M in the blister fluid of the patients were also decreased in comparison with those from patients with other bullous dermatoses. The topical application of antibiotic ointment with strong PA inhibitor was clinically effective when applied to the blister and eroded lesions of the patients. These findings suggest that the increased activity of PA may play an important role in the development of blister formation in patients with RDEB.

Pancreatic exocrine function in rats given synthetic protease inhibitor camostate (200 mg/kg body weight) perorally once daily for 10 days was investigated. Pancreatic wet weight was significantly increased in the camostate-treated rats. The increase in pancreatic weight was associated with pronounced hypertrophy and moderate hyperplasia. Total amylase, trypsin, and lipase contents in the pancreas were also increased in the camostate-treated group compared with the control rats. Secretory patterns of pancreatic juice and amylase in response to caerulein were similar in both groups, whereas the dose-response curve for pancreatic juice secretion in the camostate-treated rats was shifted tenfold toward higher concentrations of caerulein. Basal and caerulein-stimulated flow rates of pancreatic juice were significantly greater in the camostate-treated rats than the control rats, although both groups showed a threefold increase over basal secretion in response to maximal stimulation. Amylase outputs in basal state and in response to submaximal doses of caerulein were significantly lower, whereas those to maximal and supramaximal doses were significantly greater in the camostate-treated animals than that in the control rats. These results indicate that treatment with camostate induces pancreatic hypertrophy and hyperplasia, and that the secretory function of the hypertrophied pancreas is quantitatively but not qualitatively altered.

Feeding of raw soya flour or other trypsin inhibitors such as camostate is a well-established method for promoting growth of (pre)neoplastic foci induced in the exocrine pancreas of rats by azaserine. The effect of trypsin inhibitors is thought to be mediated through an increased release of cholecystokinin. Using the specific cholecystokinin receptor antagonist lorglumide (CR-1409), we performed a 16-wk study to investigate the potential of this drug in inhibiting growth of putative preneoplastic foci and to determine whether and to what extent cholecystokinin is responsible for the effect of trypsin inhibitors on pancreatic growth. After initiation with 30 mg/kg of azaserine at 19 days of age, six groups of 15 rats each received one of the following treatments: camostate, cholecystokinin-8, or gelatin control, either or not in combination with CR-1409, once daily, 3 days wk for 16 wk. Plasma cholecystokinin levels, measured 30 min after the stimulus, were similar after camostate and cholecystokinin octapeptide administration. After 16 wk the pancreata were removed, weighted, and quantitatively analyzed for the number and size of putative preneoplastic foci by light microscopy. Both camostate and cholecystokinin octapeptide stimulated pancreatic growth and development of acidophilic putative preneoplastic foci, whereas growth of basophilic putative preneoplastic foci was inhibited by camostate but stimulated by cholecystokinin. CR-1409 almost completely abolished the effect of cholecystokinin and was found to cause a significant decrease in the effects of camostate. It is concluded that (a) cholecystokinin plays a significant role in camostate-stimulated growth of acidophilic putative preneoplastic foci in rat pancreas and (b) CR-1409 inhibits growth of putative preneoplastic foci induced in rat pancreas by azaserine and hence may be of potential value for the treatment of pancreatic cancer in humans.

In the present study pancreatic secretion and plasma cholecystokinin (CCK) levels were analyzed in eight volunteers after daily ingestion of the serine protease inhibitor camostate for 5 days. This was compared with the effect of a single intraduodenal dose of camostate. Prolonged administration of camostate for 5 days had no effect on basal and stimulated pancreatic secretion and plasma CCK. A single dose of camostate completely inhibited enzymatic activity of trypsin and chymotrypsin and stimulated volume, amylase, and lipase secretion but induced an only slight and insignificant increase in plasma CCK. After the ingestion of a test meal, camostate did not influence stimulated enzyme secretion and increased plasma CCK. We concluded that the intraduodenal perfusion of camostate stimulated pancreatic secretion by a feedback mechanism that is not mediated by CCK. The repeated oral administration of camostate did not induce adaptive changes in pancreatic secretion.

Major resection of the pancreas leads to disorders of the endocrine and exocrine pancreas. The effect of a trypsin inhibitor on the remnant pancreas was studied in rats after 85 percent pancreatectomy. Impairments of the glucose elimination rate and the integrated insulin response after 85 percent pancreatectomy were improved by means of oral administration of a synthetic trypsin inhibitor for 4 and 12 weeks. The pancreatic insulin content in the animals treated with trypsin inhibitor for 13 weeks increased to about 1.3 times than that obtained in control animals. The exocrine pancreatic function in 85 percent pancreatectomized rats treated with trypsin inhibitor for 4 weeks and 12 weeks showed substantial improvement as shown by the test with N-benzoyl-L-tyrosil-p-aminobenzoic acid (BT-PABA). The pancreatic amylase, lipase, and protein contents in the animals treated with trypsin inhibitor were increased to 1.9, 1.7 and 2.1-fold, respectively, as compared to control animals for 13 weeks. Histologic examination showed a decrease in abnormal islets of Langerhans, and a tendency toward hypertrophy of the acinar cells. These results suggest that oral administration of a trypsin inhibitor to rats is effective in improving pancreatic endocrine and exocrine functions after 85 percent pancreatectomy.

The present study was aimed to examine whether BANA-degrading enzyme activities could be enhanced by bradykinin(BK) in dental pulp of the rat in vitro. The results showed that BK(0.1-10 microM) dose-dependently enhanced BANA-degrading enzyme activity at pH 7.4. The effects of BK(1 microM) were found to be most effective at both pH 7 and 8, with enhancement of the enzyme activities at a wide range of pH. The BK effects at both the pH were not inhibited by FOY-305(0.1 microM), an inhibitor of trypsin-like enzymes, differing from that at pH 6 in adrenal medulla of the rat. On the other hand, the effects of BK at both the pH were remarkably inhibited by EGTA (2 mM), followed by reversal with calcium ion (2.42 mM). These results suggested as follows: 1) there might be two kinds of BANA-degrading enzymes activated by BK in the pulp. 2) it was conceivable that BANA-degrading enzymes activated by BK were quite different from serine proteinases and were interfered with them in the pulp. 3) calcium ion might play a role in BK-induced enhancement of BANA-degrading enzyme activities which were regarded as met-enkephalin (ME) processing enzyme activities in the pulp.

Since endogenous cholecystokinin (CCK) is released after oral administration of camostate, it has been suggested that camostate-induced pancreatic growth is mediated via circulating CCK. To test this concept, we investigated the effects of three potentially inhibitory substances on rat pancreatic hypertrophy caused by feeding of camostate over 2 weeks: (1) L-364,718, the novel specific highly potent nonpeptide CCK receptor antagonist, (2) octreotide (SMS 201-995), a potent long-lasting somatostatin analogue and (3) pancreastatin (33-49), the biologically active C-terminal fragment of the novel gastrointestinal peptide pancreastatin. Camostate feeding (200 mg/kg) once daily for 14 days induced a significant increase in pancreatic weight, total protein, trypsinogen and polyamine levels, whereas total amylase content was substantially diminished. Simultaneous oral or subcutaneous treatment with L-364,718 (0.3 mg/kg twice daily) completely suppressed all trophic effects of camostate. Octreotide (25 micrograms/kg twice daily s.c.) and pancreastatin (33-49) (10 micrograms/kg twice daily s.c.) did not change any trophic parameter. In case of octreotide it could be shown that two daily injections only partially suppressed elevated CCK levels. Pancreatic DNA and putrescine levels were slightly reduced in rats receiving the CCK antagonist alone. These results demonstrate that camostate-induced pancreatic hypertrophy in rats is caused by the release of endogenous CCK which may contribute to the maintenance of normal pancreatic DNA and putrescine concentrations.

The effects of peroral treatment with synthetic trypsin inhibitor on endocrine secretion in response to four different stimuli were investigated in the isolated perfused rat pancreas. After 10 days' treatment pancreatic wet weights and protein and amylase contents in the pancreas were significantly increased. On the other hand, total contents of IRI and IRG in the T.I.-treated and control pancreas were nearly the same. In spite of the lack of alteration in hormone contents, a lower initial phase of IRI response to 8.3 mM glucose, glucose plus 0.1 ng/ml caerulein or 20 mM arginine was observed in the T.I.-treated pancreas. Furthermore, the IRI response to 10 mM theophylline was both slower and the most reduced in the T.I.-treated pancreas. From these observations, it is suggested that short-term peroral administration of synthetic trypsin inhibitor not only has no trophic effect on pancreatic B-cell and A-cell in the rat but also may affect some pathway of insulin secretion from the B-cell to reduce IRI responses to secretagogues.

In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations of CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per microgram DNA basis (19.6 +/- 5.1 vs. 47.4 +/- 11.4, p less than 0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10(-10) M), less amylase was secreted in obese rats across the dose range tested (p less than 0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p less than 0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p less than 0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p less than 0.001) and by carbachol was only 20% of control (p less than 0.001). Thus, increased size by increased cell number was associated with decreased response per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Recently we reported that a kind of serine protease, SH protease, and collagenase might be involved in blister formation and, furthermore, that the cooperative action of these three proteases was essential for blister formation in recessive dystrophic epidermolysis bullosa. In this study we examined the inhibitory effect of clinically usable serine protease inhibitors for blister formation in organ culture and in clinical trials of recessive dystrophic epidermolysis bullosa patients. Camostat mesylate, a synthetic serine protease inhibitor that is available for the treatment of chronic pancreatitis, demonstrated a striking effect of inhibiting blistering in organ culture of normal human skin with recessive dystrophic epidermolysis bullosa blister fluids. Subsequently we administered camostat mesylate by topical application to four patients with recessive dystrophic epidermolysis bullosa to assess its ability to reduce blistering. Therapeutic response was favorable; a significant effect in decreasing the number of blisters was observed in three of four patients. These findings actually supported the hypothesis that a kind of serine protease had a close relationship with blistering in recessive dystrophic epidermolysis bullosa and that therapy with a clinically usable protease inhibitor was useful for the treatment of this disease.

The effect of the epsilon-guanidino acid derivate camostate (FOY 305) on lipolysis of isolated adipocytes was evaluated to test its supposed influence on the reactions of lipid metabolism in experimental shock. Isoprenaline (10(-6) M) and theophylline (10(-3) M) significantly stimulated lipolysis in vitro. The basal submaximal (isoprenaline 10(-6)-10(-9) M) and maximal (isoprenaline 10(-6) M) catecholamine-stimulated lipolysis were not affected by camostate (10(-6) M). Furthermore, camostate (0.1-100 X 10(-6) M) did not influence half-maximal theophylline-induced lipolysis of the adipocytes. Our findings do not support the suggestion that epsilon-guanidino acid derivatives diminish in shock the increase of free fatty acids in blood via an influence on lipolysis in the peripheral adipose tissue.

Cholecystokinin (CCK) is a potent inhibitor of gastric emptying. We have examined the effects of a novel potent peripheral CCK receptor antagonist (L364,718) on the action of endogenous and exogenous CCK octapeptide (CCK-8) on gastric emptying in the rat. In conscious gastric fistula rats, the recovery of liquid test meals of 3.0 ml (containing phenol red as a dilution marker) was determined over periods up to 8 min. Compared with saline, gastric emptying of solutions of peptone (4.5%), 50 mM HCl, and hyperosmolal saline was significantly delayed. The emptying of saline was also delayed by intravenous infusion of CCK-8 (400 pmol.kg-1.h-1). The CCK antagonist L364,718 reversed the effect of peptone in a dose-dependent manner and inhibited the response to exogenous CCK, but the emptying of physiological saline, 50 mM HCl, or hyperosmolal saline remained unchanged. A protease inhibitor (FOY-305) that is thought to release endogenous CCK by inhibiting negative feedback control by luminal proteases also delayed emptying, and this response was inhibited by L364,718. We conclude that CCK has a physiological role in the mediation of the effect of proteins on gastric emptying in the rat.

The influences of truncal vagotomy or surgical sympathectomy on the pancreatic trophic effect of oral administration of synthetic trypsin inhibitor (FOY-305) were examined upon normal rats and 85% major pancreatectomized rats. On normal rats, oral administration of trypsin inhibitor increased pancreatic weight, DNA content RNA content, protein content, pancreatic weight/DNA, RNA/DNA and protein/DNA. This pancreatic trophic effect seemed to be consisted of hyperplasia and hypertrophy of pancreatic acinar cell. Under truncal vagotomy or surgical sympathectomy, this trophic effect was not diminished. On major pancreatectomized rats, oral administration of trypsin inhibitor also caused pancreatic trophic action, consisted of hyperplasia mainly. And truncal vagotomy or surgical sympathectomy did not decrease this action. These results suggested that oral administration of trypsin inhibitor might be a beneficial method for functional recovery of remnant pancreas after major pancreatectomy even under the denervated state.

It has been reported that camostat (Foy-305), a synthetic proteinase inhibitor, is excreted into the biliary-pancreatic juice in rats. To confirm whether camostat is excreted into bile or pancreatic juice, camostat (5 mg/kg/h) was given intravenously to dogs with pancreatic or biliary fistulae. Camostat and its active metabolite (Foy-251) in bile and pancreatic juice were measured by high-performance liquid chromatography (HPLC) and by their trypsin-inhibitory activity. Camostat did not alter the exocrine pancreatic secretion of fluid, bicarbonate, protein or trypsin stimulated by secretin (1 Crick-Harper-Raper unit/kg/h) and cerulein (50 ng/kg/h). Camostat and its active metabolite were detected in bile by HPLC and trypsin-inhibitory activity (the mean excretion rate was 1.3% of the intravenous dose of camostat), but they were not detectable in pancreatic juice. In conclusion, camostat and its active metabolite were excreted into bile but not into pancreatic juice.

The present study evaluates the effect of atropine and of the cholecystokinin receptor antagonist loxiglumide on feedback regulation of basal pancreatic secretion in 6 healthy volunteers. The intraduodenal instillation of the protease inhibitor camostate reduced enzymatic activities of trypsin and chymotrypsin by 80%. This was accompanied by a strong increase in amylase and lipase output. The intravenous infusion of atropine (5 micrograms/kg.h) completely abolished the stimulatory effect of camostate on enzyme output. The infusion of loxiglumide (10 mg/kg.h) caused no changes in camostate-induced stimulation of enzyme output. Plasma levels of cholecystokinin were not altered after intraduodenal instillation of camostate whether atropine, loxiglumide, or saline were infused. We suggest that the protease inhibitor camostate, by inhibition of the enzymatic activity of trypsin and chymotrypsin, interferes with feedback regulation of basal pancreatic secretion in humans, and this mechanism is predominantly mediated by the cholinergic system.

A sensitive and specific radioimmunoassay system for rat plasma cholecystokinin (CCK) was employed to study the effect of trypsin inhibitor on plasma CCK levels. Feeding the trypsin inhibitor, FOY-305 200 mg/kg, to rats for 10 days stimulated the pancreatic growth. However, there was no significant difference in fasting plasma concentrations and duodenal contents of CCK and secretin. On the other hand, acute ingestion of same dosage of FOY-305 caused a marked (20-fold) and sustained elevation of plasma CCK levels. The principal form of circulating CCK was CCK-22 like on Sephadex G-50, in contrast to that of the cerebrum and duodenum where CCK-8 like form, or CCK-8 and CCK-22 like forms were main components, respectively. A significant rise in plasma levels of immunoreactive secretin was also found. These results suggest that the pancreatic growth observed by feeding the trypsin inhibitor to rats is caused by an excessive amount of CCK in conjunction with secretin.

This study was designed to investigate changes of ornithine decarboxylase and polyamines during pancreatic adaptation in response to feeding of the synthetic protease inhibitor camostate. alpha-Difluoromethylornithine, an irreversible and specific inhibitor of ornithine decarboxylase, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. Cholecystokinin (CCK) plasma levels in camostate-fed rats increased from basal values of 3-4 pmol/l to a maximal level of 27.4 pmol/l after 2h; they then decreased up to 12 h but remained elevated above controls throughout the 30-day experiments. In the camostate group pancreatic ornithine decarboxylase activity was elevated after 2 h, reaching a maximum after 6 h (1,858.5 pmol 14CO2/h/mg DNA, about 200-fold above controls) followed by a significant increase in putrescine after 4 h and spermidine after 24 h while spermine remained unchanged. The trophic parameters increased in the following time sequence: thymidine kinase (12 h), DNA polymerase (12 h), protein (24 h), pancreatic weight (24 h) and DNA (5 days). alpha-Difluoromethylornithine significantly delayed and reduced the camostate-induced increase in ornithine decarboxylase activity and polyamine concentrations as well as the trophic parameters. Application of the CCK receptor antagonist L-364,718 resulted in complete inhibition of the increases in ornithine decarboxylase, polyamines and all trophic parameters. These data indicate an important role for ornithine decarboxylase and polyamines in camostate-induced pancreatic growth and hormonal mediated pancreatic adaptation in rats.

3-Methylcholanthrene-induced tumor was challenged with a low molecular synthetic protease inhibitor, [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate. This drug at a dosage of 10 mg/kg or 20 mg/kg was administered by ip injection to 20 mice each harboring a solitary tumor twice daily for 10 weeks. This protease inhibitor significantly inhibited tumor growth and prolonged the survival time of the tumor-bearing mice (P less than 0.001). The results suggest the involvement of kinin-forming proteases, such as trypsin, plasmin and kallikrein, in the tumor growth.

Application of a single dose of a new type of proteinase inhibitor camostate (FOY-305) via orogastric tube was used in rats to study the dose-response relationship of resulting pancreatic stimulation. Doses up to 10 mg/kg failed to elicit any response, while significant decrease in enzyme content and increase in serum CCK-levels were observed with doses ranging from 25 to 400 mg/kg. A single dose of 100 mg/kg was selected for a time-sequence analysis, which revealed a 60 to 70% depletion of enzyme stores persisting over 6 h and reverting to control levels by 12 h. Peak increases in serum CCK-levels (15-fold above the elevation observed after regular food intake) were found after 30 min and persisted as an 8- to 10-fold elevation for at least 3 h, then declined to control levels by 9 h. This prolonged endogenous hormone release and resulting pancreatic stimulation were also verified in a separate group of animals in which volume, protein, and enzyme output were measured after cannulation of the pancreatic duct. While volume secretion was not altered by feeding a single dose of 100 mg/kg FOY-305, protein and enzyme output increased 2- to 3-fold over a period of 7 h. Fine-structural analysis of the pancreas demonstrated efficient depletion of zymogen granules from acinar cells with all doses between 50 and 400 mg/kg, accompanied by the appearance of membrane material in the acinar lumina at 3 and 6 h. The same transient increase in the number of lysosomal bodies predominantly containing mitochondria with all doses above 50 mg/kg was interpreted as increased organelle turnover due to persisting hormonal stimulation.