Ex vivo evaluation of adhesive strength and barrier effect of a novel treatment for esophagitis.

AIM: To investigate the mucoadhesive strength and barrier effect of Esophacare® (Atika Pharma SL, Las Palmas de Gran Canaria) in an ex vivo model of gastro-oesophageal reflux. METHODS: An ex vivo evaluation through the Falling Liquide Film Technique with porcine esophagi was performed, compared to a positive control (Ziverel®; Norgine, Amsterdam), after different washing periods with saline, acidified saline (pH 1.2) and acidified saline with pepsin (2000U/mL). RESULTS: The adhesive mean strength on the oesophageal mucosa of Esophacare was 94.7 (6.0)%, compared to 27.6 (19.1)% of the positive control (p<0.05). These results were homogeneous across the different washes and throughout the tissue. The area covered by 1mL of Esophacare, and its respective persistence after washing was also assessed, yielding a mean global persistence of 74.29 (19.7)% vs. 18.9 (12.3)% for the control (p<0.05). In addition, after 30min exposure to acidified saline with pepsin, Esophacare shows a protective effect on the oesophageal mucosa, detectable histologically: preserved integrity and structure of the apical layers was observed, as well as reduced permeability to the washing solution. CONCLUSIONS: Esophacare shows an adhesive strength close to 100%, irrespective of the washing solution applied or the oesophageal region studied. Histologically, it reduces the abrasive effects of the acidic solution on the oesophageal epithelium, reducing permeability to the washing solution. The results in this ex vivo model of gastro-oesophageal reflux disease (GERD) support its therapeutic potential.

Upper Esophageal Sphincter Compression Device as an Adjunct to Proton Pump Inhibition for Laryngopharyngeal Reflux.

BACKGROUND: The Reflux Band, an external upper esophageal sphincter (UES) compression device, reduces esophago-pharyngeal reflux events. This study aimed to assess device efficacy as an adjunct to proton pump inhibitor (PPI) therapy in patients with laryngopharyngeal reflux (LPR). METHODS: This two-phase prospective clinical trial enrolled adults with at least 8 weeks of laryngeal symptoms (sore throat, throat clearing, dysphonia) not using PPI therapy at two tertiary care centers over 26 months. Participants used double dose PPI for 4 weeks in Phase 1 and the external UES compression device nightly along with PPI for 4 weeks in Phase 2. Questionnaire scores and salivary pepsin concentration were measured throughout the study. The primary endpoint of symptom response was defined as reflux symptom index (RSI) score ≤ 13 and/or > 50% reduction in RSI. RESULTS: Thirty-one participants completed the study: 52% male, mean age 47.9 years (SD 14.0), and mean body mass index (BMI) 26.2 kg/m2 (5.1). Primary endpoint was met in 11 (35%) participants after Phase 1 (PPI alone) and 17 (55%) after Phase 2 (Device + PPI). Compared to baseline, mean RSI score (24.1 (10.9)) decreased at end of Phase 1 (PPI alone) (21.9 (9.7); p = 0.06) and significantly decreased at end of Phase 2 (Device + PPI) (15.5 (10.3); p < 0.01). Compared to non-responders, responders to Device + PPI had a significantly lower BMI (p = 0.02) and higher salivary pepsin concentration (p = 0.01). CONCLUSION: This clinical trial highlights the potential efficacy of the external UES compression device (Reflux Band) as an adjunct to PPI for patients with LPR (ClinicalTrials.Gov NCT03619811).

A non-interventional, observational study of a fixed combination of pepsin and amino acid hydrochloride in patients with functional dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a gastrointestinal disorder characterized by recurrent and diverse symptoms and pathophysiology that remains unexplained following routine clinical investigation. Enzynorm®f is a pharmaceutical preparation comprising fixed amounts of pepsin of biological origin and organically bound acid in the form of amino acid hydrochloride. It is traditionally used as a mild agent to support gastric function and to stimulate the stomach's proteolytic activities in FD. METHODS: In a non-interventional, observational, post-marketing surveillance study, patients with an established diagnosis of FD were treated with a fixed combination of pepsin and amino acid hydrochloride taken as tablets three times daily for 6 weeks. The primary objective of this study was to assess the change in symptoms using the validated Gastrointestinal Symptom Score (GIS©). Secondary objectives included patients' assessment of their gastrointestinal symptoms as well as treatment safety and tolerability. RESULTS: A total of 97 patients (mean age 58.4 ± 13.9 years; 63.2% females) were included in the study, with 72 data having GIS© score data at baseline and at 6 weeks, and 34 also at 3 weeks. The overall GIS© sum score decreased by 4.1 (p < 0.0001) from 11.6 (±4.8) at baseline to 7.4 (± 4.6) reflecting an improvement of clinical symptomatology after 6 weeks of treatment. In a subgroup of 70 patients who had FD meeting the Rome III criteria a GIS© score reduction of ≥50% was observed after 3 weeks treatment in 24% and in 30.8% after 6 weeks. Adverse events were mostly gastrointestinal in nature and consistent with the underlying disease; no unexpected adverse reactions were reported. Twenty-seven patients discontinued the study, mostly because of gastrointestinal symptoms. CONCLUSION: The results of this study support the efficacy of a fixed combination of pepsin and amino acid hydrochloride for the treatment of patients with FD and also suggest good to moderate treatment tolerability. These findings should be further explored in a randomised, placebo-controlled clinical trial. CLINICAL TRIAL REGISTRATION: This study has been retrospectively registered in the ClinicalTrials.gov registry, trial identifier NCT03076411 .

[Peptic enzymes for irritable stomache syndrome: a new, old therapeutic option? Results of a survey among family doctors]. / Verdauungsenzyme beim Reizmagensyndrom: eine neue alte Therapieoption? : Ergebnisse einer Hausarztbefragung.

OBJECTIVE AND METHOD: The treatment of patients with functional dyspepsia (irritable stomache syndrome) was recorded in a nationwide survey among family doctors. On a questionnaire, the physicians should provide information on the use of a combination preparation of gastric enzyme (pepsin) and amino acids, duration of use, therapy success and patients' contentment. RESULTS: A total of 874 family doctors were contacted, 201 questionnaires were completed and evaluated. The physicians administered the combination preparation predominantly for feeling of fullness and pressure (82%), flatulence (78%) and upper abdominal pain (57%), usually over 3-6 weeks. They confirmed, that patients who suffered from these symptoms were very content with the combination preparation. 85% of the doctors rated the therapeutic success as "good" to "very good". CONCLUSION: The combination of gastric enzyme (pepsin) and amino acids can be an effective therapeutic option for functional dyspepsia (irritable stomache syndrome) in medical practice.

The role of pepsin in the laryngopharyngeal reflux.

Laryngopharyngeal reflux (LPR) is a common defect among laryngological and phoniatric patients. Although LPR is categorized as a superficial gastroesophageal reflux disease (GERD), differential diagnosis should treat these two diseases separately. LPR symptoms can be assessed in the interview using as a tool the reflux symptom index (RSI). In addition, changes in the larynx that occur during LPR might be seen during laryngoscopy and classified according to the reflux finding score (RFS). One of the main mucosal irritants in LPR is pepsin which digests proteins and impairs the functions of the upper respiratory tract cells by affecting carbonate anhydrase (CAIII) and the Sep 70 protein. Pepsin initiates inflammatory changes within the larynx, nasopharynx and nasal cavity. The use of pepsin detection in upper and lower throat secretions is a new direction in LPR diagnostics.

Reconditioning of an injured lung graft with intrabronchial surfactant instillation in an ex vivo lung perfusion system followed by transplantation.

BACKGROUND: We tested whether an injured lung graft from category-3 donation after cardiac death donor could be reconditioned with an ex vivo lung perfusion (EVLP) system by intrabronchial diluted surfactant lavage before transplantation. METHODS: In a pig model, cardiac arrest was induced by deconnecting from the ventilator. Left lung injury was done by intrabronchial instillation of 1 mL/kg pepsin + HCl. After retrieval, the heart-lung block was stored at 4°C for 2 h. In the treated group, transplantation was performed after reconditioning with intrabronchial diluted surfactant lavage in EVLP system. RESULTS: During EVLP, surfactant group showed better oxygenation and lower pulmonary vascular resistance. After transplantation, better oxygenation, lower mean pulmonary artery pressure, and lower lung edema were observed in surfactant group. Lower blood IL-1 beta and IL-6 cytokine levels were measured in the surfactant group. In bronchoalveolar lavage, the percentage of neutrophils, IL-1 beta and IL-6 cytokine levels, amount of protein, and neutrophil infiltration in the lung tissue at the end of the experiment were significantly lower in the surfactant group. CONCLUSIONS: Our data demonstrate the feasibility of reconditioning and transplantation of an acutely damaged lung graft due to aspiration from a category-3 DCD donor. Implementation of an EVLP system is an efficacious tool to recondition and assess a questionable graft before transplantation.

Review article: reflux and its consequences--the laryngeal, pulmonary and oesophageal manifestations. Conference held in conjunction with the 9th International Symposium on Human Pepsin (ISHP) Kingston-upon-Hull, UK, 21-23 April 2010.

BACKGROUND: Gastro-oesophageal reflux disease (GERD) is one of the commonest diseases of Western populations, affecting 20 to 30% of adults. GERD is multifaceted and the classical oesophageal symptoms such as heartburn and regurgitation often overlap with atypical symptoms that impact upon the respiratory system and airways. This is referred to as extra-oesophageal reflux disease (EERD), or laryngopharyngeal reflux (LPR), which manifests as chronic cough, laryngitis, hoarseness, voice disorders and asthma. AIM: The 'Reflux and its consequences' conference was held in Hull in 2010 and brought together a multidisciplinary group of experts all with a common interest in the many manifestations of reflux disease to present recent research and clinical progress in GERD and EERD. In particular new techniques for diagnosing reflux were showcased at the conference. METHODS: Both clinical and non-clinical key opinion leaders were invited to write a review on key areas presented at the `Reflux and its consequences' conference for inclusion in this supplement. RESULTS AND CONCLUSION: Eleven chapters contained in this supplement reflected the sessions of the conference and included discussion of the nature of the refluxate (acid, pepsin, bile acids and non-acid reflux); mechanisms of tissue damage and protection in the oesophagus, laryngopharynx and airways. Clinical conditions with a reflux aetiology including asthma, chronic cough, airway disease, LPR, and paediatric EERD were reviewed. In addition methods for diagnosis of reflux disease and treatment strategies, especially with reference to non-acid reflux, were considered.

Does DIAGNOdent provide a reliable caries-removal endpoint?

OBJECTIVES: To compare mineral density of residual dentine after excavation with different caries-removal techniques and to evaluate the diagnostic potential of laser-induced fluorescence (LIF), measured by DIAGNOdent, as a tool to determine the caries-removal endpoint. METHODS: Carious teeth were excavated by tungsten-carbide round burs (Komet), ceramic burs (CeraBurs, Komet), sono-abrasion (Cariex TC tips, Kavo), and by chemo-mechanical excavation using two enzyme-based solutions (exp. SFC-V and SFC-VIII, 3M-ESPE) or a sodium hypochlorite-based solution (Carisolv, MediTeam). The caries-excavated teeth were scanned by micro-CT (1172, Skyscan), after which the mineral density at the bottom dentine was correlated to LIF measurements at the same region. A micro-CT threshold for dentine caries was defined by comparison with surface-hardness measurements. The intensity of dentine staining was evaluated by analysing the component 'L*' in CIE-L*a*b-converted images from the excavated teeth. RESULTS: No statistically significant difference in mineral density was found at the bottom of the cavities prepared with the different caries-excavation techniques, except for exp. SFC-V that left residual dentine with a significantly higher mineral density than when CeraBurs were used (Tukey-Kramer, p<0.05). Absence of residual caries was associated with darker staining of dentine. No significant correlation was found between the distance from the deepest cavity point to the pulp-chamber roof and LIF measurements. A strong negative correlation (R=-0.86, p<0.01) was however found between L* values and LIF measurements, indicating that staining in residual dentine leads to higher LIF measurements. CONCLUSIONS: LIF measured by DIAGNOdent is influenced by staining in residual dentine. Therefore, its use to determine the caries-removal endpoint is doubtful.

An in vitro evaluation of microtensile bond strengths of two adhesive bonding agents to residual dentine after caries removal using three excavation techniques.

OBJECTIVES: To assess amounts of residual dentine retained after using three excavation techniques; the microtensile bond strengths (microTBS) to residual dentine, comparing etch-rinse vs. self-etching adhesives. METHODS: 42 carious molars were subdivided (N=21) dependent upon adhesive/composite system (Adper Scotchbond 1XT and Filtek Supreme vs. Filtek Silorane adhesive and composite). Dividing into three (N=7), dependent upon caries excavation technique employed (hand vs. chemo-mechanical: Carisolv gel vs. experimental enzymatic gel (SFC-V)), caries removal was assessed using visual/tactile criteria and in situ autofluorescence (AF) confocal fibre-optic micro-endoscopy (CFOME). Post-restoration/four-week hydrated storage, four 0.9 mm(2) beams per tooth underwent microTBS testing/microscopic analysis of fractured surfaces. Three cavities from each excavation group were analysed using SEM. RESULTS: SEM revealed surface roughness with smear layer occluding tubule orifices in hand-excavated samples and a reduced, variable smear layer for both chemo-mechanical systems. CFOME AF assessment indicated hand excavation left sound dentine, Carisolv left affected dentine and SFC-V slightly under-prepared clinically. Mean microTBS values from etch-rinse samples (27 MPa (SD 3.9), hand; 22 MPa (SD 5.1), Carisolv; 26 MPa (SD 4.4), SFC-V) showed statistical differences between hand and Carisolv groups. Mean microTBS data for self-etch samples (22 MPa (SD 3.3), hand; 27 MPa (SD 6.1), Carisolv; 25 MPa (SD 4.7), SFC-V) showed significant differences between hand and Carisolv, and hand vs. SFC-V. Failure loci distribution in etch-rinse samples was between dentine-adhesive, within adhesive and within composite whereas self-etch samples exhibited failure predominantly between adhesive and composite. CONCLUSIONS: Data indicated that all null hypotheses were disproved.

Self-limiting caries therapy with proteolytic agents.

PURPOSE: To determine the extent to which artificial carious dentin can be removed by agents that do not seem to attack sound dentin such as pepsin, trypsin, collagenase and NaOCl, and to evaluate the effect of the enzyme pepsin and a new enzymatic solution SFC-V (pepsin in mild acidic buffer) as a self-limiting caries therapy in deep dentin carious lesions using our new model for artificial dentin caries. METHODS: Artificial dentin caries was used to investigate different proteolytic agents which have the potential to remove carious tissue. 408 slices of coronal dentin were subjected to a demineralization regime which produces dentin caries very similar to natural lesions: acetic acid (pH 5) or lactic acid (pH 4) were used (7 days). Subsequently, sodium hypochlorite, collagenase, trypsin and pepsin were dissolved each in a suitable buffer and the demineralized dentin was treated for 10 minutes or 24 hours with these solutions. To differentiate the influence of the acidic buffer in case of pepsin, a second experiment was performed. 192 slices were exposed to lactic acid for 1 week. Subsequently the demineralized dentin surfaces were treated with either the enzyme pepsin in its acidic buffer, the acidic buffer alone, and in addition a neutral buffer as a control. In addition a fourth group was added where a new enzyme-based solution SFC-V was used. This second experiment differentiated further the influence of "diffusion enhanced by agitation" versus "diffusion" alone. The application time of the solutions was 3 minutes with and without agitation using a stiff nylon brush. To obtain information on the morphology of the pre- and post-treatment dentin surfaces, high resolution FE-SEM was used. Descriptive statistics were used based on cross tabulation of the morphological criteria. RESULTS: Lactic acid produced demineralized dentin covered with a surface layer removable by proteolytic enzymes while acetic acid produced only demineralized dentin. The amount of tissue removed with the current proteolytic agents ranked as follows: trypsin < pepsin < collagenase < NaOCl. The neutral and the acidic buffers did not affect the surface precipitates while the enzyme pepsin and the solution SFC-V were effective in removing the degraded organic matrix.

[Enzymatic activity and therapeutic action of pepsin K in chronic gastritis with secretory failure]. / Fermentativnaia aktivnost' i lechebnoe deistvie pepsina K pri khronicheskom gastrite s sekretornoi nedostatochnost'iu.

Pepsin K (chicken pepsin) in the test for hemoglobin proteolysis is 1.7 times superior to abomin (porcine pepsin) in the medium pH 2 and 44 times more potent in the medium pH 3. Even in pH 4 chicken pepsin retains its enzymatic activity while porcine pepsin in pH 4 looses its activity. These biochemical findings show that pepsin K is more effective in the treatment of enzymatic insufficiency of the stomach. Intake of 1-3 pepsin K tablets after meal for 4 weeks by 82 patients having chronic gastritis with secretory insufficiency was effective in 75% of patients.

[Comparative experimental enzymatic activity and clinical efficacy of pepsin K and abomin]. / Fermentativnaia aktivnost' pepsina K v sravnenii s abominom v éksperimente i ego lechebnoe deistvie v klinike.

Hen pepsin (pepsin K) in the experiment on proteolytic activity of hemoglobin surpasses porcine pepsin (abomin) with pH of medium 2 in 1.7 times, with pH of medium 3 in 44 times, that makes it more acceptable for treatment of enzymatic insufficiency of stomach. In the clinic reception of pepsin K in tablets for 4 weeks had a positive effect for all 10 patients after the surgical intervention on the stomach. The results were good for seven of them, and satisfactory for three patients.

Spasmocanulase in irritable bowel syndrome.

OBJECTIVES: Irritable bowel syndrome is a functional gastro intestinal disorder, with various symptomatology and difficult to treat using several medications. Spasmocanulase, which has an anti-spasmodic and anti-flatulence effect and contains several ingredients, was tried in patients with irritable bowel syndrome, who had been on other medications previously without improvement. METHODS: At the gastroenterology out-patient clinic, Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia, 21 patients who were diagnosed with irritable bowel syndrome for more than 2 years on treatment and did not benefit from these medications, received spasmocanulase one tablet 3 times a day and followed in the gastroenterology out-patient clinic, every 6 weeks for 6 months. Their previous medications were discontinued which, had been used for different durations and in different combinations included Mebeverine, Colpermin, Normacol, Importal, Librax. The main symptoms were different types of abdominal pain, bloating, flatulence, diarrhea, constipation or both. RESULTS: There was improvement or disappearance of the symptoms in more than 50% of the patients who were not improved on previous medication used for more than 2 years. The overall improvement in symptoms ranged between 43-75% in these patients, when followed up in the clinic at 6 weeks and 3 months. Spasmocanulase caused improvement in abdominal pain, flatulence and bloating compared to their previous medications. Only 43% of patients with diarrhea, showed improvement on spasmocanulase. CONCLUSION: Although the number of patients, we studied is small, this study has shown that spasmocanulase is beneficial in improving symptoms of irritable bowel syndrome.

Effects of neutral pepsin on the deposition of dietary antigens in glomeruli from IgA nephropathy.

Recently we reported on the participation of dietary antigens in the pathogenesis of IgA nephropathy. Particularly, the glomerular deposition of dietary antigens (casein, soybean protein, rice protein) was observed in some cases with IgA nephropathy. It is known that human urinary pepsin (HUP) prepared from the urine of a healthy adult acts as an immune complexase at neutral pH. In this study, the effects of incubation with HUP on the glomerular deposition of dietary antigens was investigated by an immunofluorescence technique in 48 patients with IgA nephropathy, four patients with Henoch-Schönlein purpura nephritis and 69 with other glomerulonephritides. In 33 cases (68.8%) soybean protein and in 12 (25.0%) casein was found in biopsies of patients with IgA nephropathy. After a 3-day incubation with HUP there was no change in the deposition of IgA in 12 cases, a decrease in 22 and complete elimination in 14 cases. The deposits of soybean protein increased in 12 cases, did not change in 20, decreased or was abolished in five and was positively converted in 11. Exceptionally, in five patients treated with HUP, the deposition of soybean protein appeared concomitantly with the disappearance of IgA deposits. Such a concomitance did not occur either in other glomerulonephritides or in studies of other dietary antigens. Thus it appears that soybean protein may be deposited in the glomerular mesangium as an antigen in some patients with IgA nephropathy.

Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization.

A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.

[Substantiation of the use of pepsidil in secretory insufficiency of the digestive glands caused by antitubercular drugs]. / Obosnovanie ispol'zovaniia pepsidila pri sekretornoi nedostatochnosti pishchevaritel'nykh zhelez, vyzvannoi protivotuberkuleznymi preparatami.

The experiments on rats were undertaken to study the action of pepsidil to make up for the biosynthesis deficiency of digestive enzymes in the stomach and pancreas caused by antituberculous drugs (methazide + PAS, isoniazid + PAS, isoniazid + rifampicin + ethambutol). It was proved that pepsidil reduced an inhibiting action of antituberculous drugs on the activity of gastroenteric tract proteinases. The given condition should be taken into account when antituberculous treatment is conducted.

[The effects of human and mouse pepsins on Masugi nephritis].

The effects of intravenous administration of human or mouse pepsin were investigated on Masugi nephritis in rats and mice, which is an experimental model of glomerulonephritis in humans. Injection of anti-kidney serum to rats significantly increased the urinary protein excretion, serum levels of fibrinogen, cholesterol and immune complex, significantly decreased serum immunoglobulin G and complement levels, and caused histopathological changes such as detachment of endothelial cells from basement membrane, thickening of basement membrane, fusion of foot process and increase in hyaline cast in renal tubuli. Deposit of anti-rat IgG on glomerular capillary wall was also observed. All of these changes were ameliorated or showed a tendency to be ameliorated by intravenous administration of human pepsin, and the mechanism responsible for these effects was suggested to be selective decomposition of immune complex. Marked increase in urinary protein excretion was also observed in mice that intravenously received anti-kidney serum. This increase was suppressed by intravenous administration of human or mouse serum, the latter showing a stronger effect.

Pepsin and autoimmune disease mouse.

The effects of pepsin on autoimmune glomerulonephritis of MRL/1 mice were investigated. Intravenous administration of pepsin significantly improved survival rate and suppressed progressive increase in urinary excretion of protein and various histopathological changes in kidney. Increased serum levels of immune complex and anti-DNA antibody in MRL/1 mice were decreased by pepsin. Pepsin ameliorated abnormalities in lymphocyte subsets and lymphocyte functions. The fact that pepsin ameliorated abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progress of immune complex nephritis.