Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation.

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

No evidence for the benefit of PPIs in the treatment of acute pancreatitis: a systematic review and meta-analysis.

Although current guidelines do not recommend the use of proton pump inhibitors (PPIs) in the standard of care of acute pancreatitis (AP), they are often prescribed in clinical practice, mainly for ulcer stress prophylaxis. In this systematic review and meta-analysis we evaluated the association between the use of PPIs in the management of AP and various clinical outcomes. We conducted the systematic research in six databases without restrictions on January 24th, 2022. We investigated adult patient with AP, who were treated with PPI compared to conventional therapy. The pooled odds ratios, mean differences, and corresponding 95% confidence intervals were calculated with random effect model. We included six RCTs and three cohort studies, consisting of 28,834 patients. We found a significant decrease in the rate of pancreatic pseudocyst formation in patients who received PPI treatment. PPI use was associated with a higher risk of GI bleeding, however this finding could be due to the patients' comorbid conditions. We found no significant difference in the rates of 7-day mortality, length of hospital stay, and acute respiratory distress syndrome between the groups. The available data on this topic are limited; therefore, further well designed RCTs are needed to evaluate the potential benefits and adverse effects of PPIs in AP.

Novel p.G250A Mutation Associated with Chronic Pancreatitis Highlights Misfolding-Prone Region in Carboxypeptidase A1 (CPA1).

Inborn mutations in the digestive protease carboxypeptidase A1 (CPA1) gene may be associated with hereditary and idiopathic chronic pancreatitis (CP). Pathogenic mutations, such as p.N256K, cause intracellular retention and reduced secretion of CPA1, accompanied by endoplasmic reticulum (ER) stress, suggesting that mutation-induced misfolding underlies the phenotype. Here, we report the novel p.G250A CPA1 mutation found in a young patient with CP. Functional properties of the p.G250A mutation were identical to those of the p.N256K mutation, confirming its pathogenic nature. We noted that both mutations are in a catalytically important loop of CPA1 that is stabilized by the Cys248-Cys271 disulfide bond. Mutation of either or both Cys residues to Ala resulted in misfolding, as judged by the loss of CPA1 secretion and intracellular retention. We re-analyzed seven previously reported CPA1 mutations that affect this loop and found that all exhibited reduced secretion and caused ER stress of varying degrees. The magnitude of ER stress was proportional to the secretion defect. Replacing the naturally occurring mutations with Ala (e.g., p.V251A for p.V251M) restored secretion, with the notable exception of p.N256A. We conclude that the disulfide-stabilized loop of CPA1 is prone to mutation-induced misfolding, in most cases due to the disruptive nature of the newly introduced side chain. We propose that disease-causing CPA1 mutations exhibit abolished or markedly reduced secretion with pronounced ER stress, whereas CPA1 mutations with milder misfolding phenotypes may be associated with lower disease risk or may not be pathogenic at all.

Preclinical testing of dabigatran in trypsin-dependent pancreatitis.

Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.

Arg236 in human chymotrypsin B2 (CTRB2) is a key determinant of high enzyme activity, trypsinogen degradation capacity, and protection against pancreatitis.

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.

Loss-of-function variant in chymotrypsin like elastase 3B (CELA3B) is associated with non-alcoholic chronic pancreatitis.

BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.

Misfolding-induced chronic pancreatitis in CPA1 N256K mutant mice is unaffected by global deletion of Ddit3/Chop.

Genetic mutations in pancreatic digestive enzymes may cause protein misfolding, endoplasmic reticulum (ER) stress and chronic pancreatitis. The CPA1 N256K mouse model carries the human p.N256K carboxypeptidase A1 (CPA1) mutation, a classic example of a pancreatitis-associated misfolding variant. CPA1 N256K mice develop spontaneous, progressive chronic pancreatitis with moderate acinar atrophy, acinar-to-ductal metaplasia, fibrosis, and macrophage infiltration. Upregulation of the ER-stress associated pro-apoptotic transcription factor Ddit3/Chop mRNA was observed in the pancreas of CPA1 N256K mice suggesting that acinar cell death might be mediated through this mechanism. Here, we crossed the CPA1 N256K strain with mice containing a global deletion of the Ddit3/Chop gene (Ddit3-KO mice) and evaluated the effect of DDIT3/CHOP deficiency on the course of chronic pancreatitis. Surprisingly, CPA1 N256K x Ddit3-KO mice developed chronic pancreatitis with a similar time course and features as the CPA1 N256K parent strain. In contrast, Ddit3-KO mice showed no pancreas pathology. The observations indicate that DDIT3/CHOP plays no significant role in the development of misfolding-induced chronic pancreatitis in CPA1 N256K mice and this transcription factor is not a viable target for therapeutic intervention in this disease.

Evolutionary expansion of polyaspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis.

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH2-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.NEW & NOTEWORTHY Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.

Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis.

The serine protease inhibitor Kazal type 1 (SPINK1) protects the pancreas from intrapancreatic trypsin activation that can lead to pancreatitis. Loss-of-function genetic variants of SPINK1 increase the risk for chronic pancreatitis, often by diminishing inhibitor expression or secretion. Variants that are secreted normally have been presumed to be pathogenic because of defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wildtype SPINK1 and seven secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 because of altered interactions with Tyr43 in the SPINK1 reactive loop. Using authentic sulfated human trypsins, we provide conclusive evidence that SPINK1 variants N34S, N37S, R65Q, and Q68R have unimpaired inhibitory activity, whereas variant P55S exhibits a small and clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1 decrease inhibitor binding by 20,000- to 30,000-fold and three- to sevenfold, respectively. Taken together, the observations indicate that defective trypsin inhibition by SPINK1 variants is an uncommon mechanism in chronic pancreatitis. The results also strengthen the notion that a decline in inhibitor levels explains pancreatitis risk associated with the large majority of SPINK1 variants.

Acid suppression therapy, gastrointestinal bleeding and infection in acute pancreatitis - An international cohort study.

BACKGROUND: Acid suppressing drugs (ASD) are generally used in acute pancreatitis (AP); however, large cohorts are not available to understand their efficiency and safety. Therefore, our aims were to evaluate the association between the administration of ASDs, the outcome of AP, the frequency of gastrointestinal (GI) bleeding and GI infection in patients with AP. METHODS: We initiated an international survey and performed retrospective data analysis on AP patients hospitalized between January 2013 and December 2018. RESULTS: Data of 17,422 adult patients with AP were collected from 59 centers of 23 countries. We found that 23.3% of patients received ASDs before and 86.6% during the course of AP. ASDs were prescribed to 57.6% of patients at discharge. ASD administration was associated with more severe AP and higher mortality. GI bleeding was reported in 4.7% of patients, and it was associated with pancreatitis severity, mortality and ASD therapy. Stool culture test was performed in 6.3% of the patients with 28.4% positive results. Clostridium difficile was the cause of GI infection in 60.5% of cases. Among the patients with GI infections, 28.9% received ASDs, whereas 24.1% were without any acid suppression treatment. GI infection was associated with more severe pancreatitis and higher mortality. CONCLUSIONS: Although ASD therapy is widely used, it is unlikely to have beneficial effects either on the outcome of AP or on the prevention of GI bleeding during AP. Therefore, ASD therapy should be substantially decreased in the therapeutic management of AP.

Measuring digestive protease activation in the mouse pancreas.

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

Engineering mouse cationic trypsinogen for rapid and selective activation by cathepsin B.

Intra-pancreatic activation of trypsin is an early event in pancreatitis. Trypsinogen can be activated to trypsin either through autoactivation (trypsin-mediated trypsinogen activation) or by the lysosomal protease cathepsin B (CTSB). Experimental separation of CTSB-mediated activation from autoactivation in mice is possible through knocking in mutations that render trypsinogen sensitive to CTSB but resistant to trypsin. Here we present biochemical studies on novel mouse cationic trypsinogen (isoform T7) mutants engineered for selective CTSB activation. First, we demonstrated that mutation K24G, which alters the activation site Lys in T7 trypsinogen, abolished autoactivation while activation by CTSB was stimulated 4-fold at pH 4.0. Interestingly, CTSB-mediated activation of the K24G mutant became more sensitive to inhibition by increasing pH. Next, Ala-scanning of the five Asp residues preceding the activation site Lys revealed that mutation D22A accelerated CTSB-mediated activation by 2-fold. Finally, combination of mutations D22A and K24G resulted in a trypsinogen mutant that exhibited 14-fold increased activation by CTSB and normal pH sensitivity. We conclude that we successfully engineered a mouse T7 trypsinogen mutant (D22A,K24G), which is robustly activated by CTSB but cannot undergo autoactivation. These studies set the stage for the generation of a preclinical mouse model of CTSB-dependent pancreatitis.

PPIs Are Not Responsible for Elevating Cardiovascular Risk in Patients on Clopidogrel-A Systematic Review and Meta-Analysis.

Background: Clopidogrel and proton pump inhibitors (PPIs) are metabolized by cytochrome P450 enzymes. Contradictory results have been reported on possible complications of simultaneous PPI and clopidogrel use. Our aim was to investigate the clinical relevance of this debate with a systematic review and meta-analysis. Methods: The PubMed, Embase, and Cochrane Central Register of Controlled Trials electronic databases were searched for human studies [randomized controlled trials (RCTs) and observational studies] using the PICO format (P: patients on clopidogrel; I: patients treated with PPI; C: patients without PPI treatment; O: cardiovascular risk). We screened eligible studies from 2009 to 2016. After study exclusions, we extracted data from 27 articles for three outcomes: major adverse cardiac event (MACE), myocardial infarction (MI) and cardiovascular (CV) death. The meta-analysis was registered on PROSPERO (CRD42017054316). Results: Data were extracted on 156,823 patients from the 27 trials included (MACE: 23, CV death: 10, MI: 14). The risks of MACE (RR = 1.22, 95% CI = 1.06-1.396, p = 0.004) and MI (RR = 1.43, 95% CI = 1.24-1.66, p < 0.001) were significantly higher in the PPI plus clopidogrel group. However, subgroup analysis demonstrated that this significance disappeared in RCTs (RR = 0.99, 95% CI = 0.76-1.28, p = 0.93) in the MACE outcome group. There was no effect of combined PPI and clopidogrel therapy on CV death outcome (RR = 1.21, 95% CI = 0.97-1.50, p = 0.09). Conclusion: Concomitant use of PPIs and clopidogrel has been proved not to be associated with elevated cardiovascular risks according to RCTs. Based on our results, no restrictions should be applied whenever PPIs and clopidogrel are administered simultaneously.

Younger age at diagnosis predisposes to mucosal recovery in celiac disease on a gluten-free diet: A meta-analysis.

BACKGROUND AND AIMS: Persistent intestinal damage is associated with higher complication rates in celiac disease. We aimed to assess the potential modifiers of mucosal recovery. MATERIALS AND METHODS: We screened databases (PubMed, Embase, Cochrane Trials, and Web of Science) for papers on celiac disease. Papers discussing (1) celiac patients (2) follow-up biopsy and (3) mucosal recovery after commencement of a gluten-free diet were included. The primary outcome was to produce a comprehensive analysis of complete mucosal recovery (i.e., Marsh 0 on follow-up). We compared children's recovery ratios to those of adults. Patients following a strict gluten-free dietary regimen were included in a subgroup. Summary point estimates, 95% confidence intervals (CIs), and 95% predictive intervals (PIs) were calculated. Heterogeneity was tested with I2-statistic. The PROSPERO registration number is CRD42016053482. RESULTS: The overall complete mucosal recovery ratio, calculated from 37 observational studies, was 0.36 (CI: 0.28-0.44, PI: -0.12-0.84; I2: 98.4%, p<0.01). Children showed higher complete mucosal recovery ratio than adults (p<0.01): 0.65 (CI: 0.44-0.85, PI: -0.10-1.39; I2: 96.5%, p<0.01) as opposed to 0.24 (CI: 0.15-0.33, PI: -0.19-1.08; I2: 96.3%, p<0.01). In the strict dietary adherence subgroup, complete mucosal recovery ratio was 0.47 (CI: 0.24-0.70, PI: -0.47-1.41; I2: 98.8%, p<0.001). On meta-regression, diagnostic villous atrophy (Marsh 3) ratio (-8.97, p<0.01) and male ratio (+6.04, p<0.01) proved to be a significant determinant of complete mucosal recovery, unlike duration of gluten-free diet (+0.01, p = 0.62). The correlation between complete mucosal recovery ratio and age on diagnosis is of borderline significance (-0.03, p = 0.05). CONCLUSIONS: There is considerable heterogeneity across studies concerning complete mucosal recovery ratios achieved by a gluten-free diet in celiac disease. Several celiac patients fail to achieve complete mucosal recovery even if a strict dietary regimen is followed. Younger age on diagnosis, less severe initial histologic damage and male gender predispose for achieving mucosal recovery.

Analysis of Research Activity in Gastroenterology: Pancreatitis Is in Real Danger.

OBJECTIVE: Biomedical investment trends in 2015 show a huge decrease of investment in gastroenterology. Since academic research usually provides the basis for industrial research and development (R&D), our aim was to understand research trends in the field of gastroenterology over the last 50 years and identify the most endangered areas. METHODS: We searched for PubMed hits for gastrointestinal (GI) diseases for the 1965-2015 period. Overall, 1,554,325 articles were analyzed. Since pancreatology was identified as the most endangered field of research within gastroenterology, we carried out a detailed evaluation of research activity in pancreatology. RESULTS: In 1965, among the major benign GI disorders, 51.9% of the research was performed on hepatitis, 25.7% on pancreatitis, 21.7% on upper GI diseases and only 0.7% on the lower GI disorders. Half a century later, in 2015, research on hepatitis and upper GI diseases had not changed significantly; however, studies on pancreatitis had dropped to 10.7%, while work on the lower GI disorders had risen to 23.4%. With regard to the malignant disorders (including liver, gastric, colon, pancreatic and oesophageal cancer), no such large-scale changes were observed in the last 50 years. Detailed analyses revealed that besides the drop in research activity in pancreatitis, there are serious problems with the quality of the studies as well. Only 6.8% of clinical trials on pancreatitis were registered and only 5.5% of these registered trials were multicentre and multinational (more than five centres and nations), i.e., the kind that provides the highest level of impact and evidence level. CONCLUSIONS: There has been a clear drop in research activity in pancreatitis. New international networks and far more academic R&D activities should be established in order to find the first therapy specifically for acute pancreatitis.