Enzyme Safety

last updated 7.22.06


Digestive enzymes are produced regularly by the pancreas in all animals including humans. They occur in raw food and by some microorganisms. Thus they are everywhere in nature and are in our bodies all the time. They are necessary for life. Taking enzymes only adds to what should be there anyway. What about the general question of safety taking enzymes? What about long-term effects? Are there upper or toxic limits to how much one can take?

In a sense, trying to show that enzymes are harmful even over a lifetime is like trying to show that drinking water is harmful even over a lifetime. Everyone gets sick at some point and eventually dies and everyone drinks water. . . so can we prove that drinking water causes death? The long-term safety of enzyme supplements has been explored in detail for a hundred years. The result is that enzymes are generally found to be highly safe with no toxic limit no matter how much you take or for how long you take them. There is research showing that it is the lack of enzymes that causes problems, not taking too many.

Enzymes have very specific mechanisms. Many are well known and characterized. Each enzymes has a very particular job. Enzymes work according to ordinary biochemical properties and are not living organisms complete with free will and instincts. Because of this, enzymes have far fewer side-effects and unknown possible reactions than other compounds, supplements, or medications. This feature makes them extremely safe.

Also, healthy tissue and cells have natural mechanisms protecting them from enzyme action. The body is full of checks and balances, including lots of enzyme inhibitors, which allow enzymes in your body to function properly without digesting you away. Enzymes are used for wound healing because they selectively degrade infection and dead tissue away while leaving healing tissue growing. Enzymes are used to remove tumors because they attack the cancerous tissue and remove it, while faciliating the growth of healthy tissue. This built-in natural selective property of enzymes can be seen on surface wounds. It has also be seen and measured in cell cultures and by monitoring internal wounds and tumors (Enzyme therapy, Wolf)

No known toxicity has been demonstrated at any level of enzyme dosing in animal studies or in humans. Animals survived outrageously large quantities of enzymes without damage so it has been impossible to find a lethal dose (no LD50). In 'Enzymes – The Fountain of Life,' Lopez relates how researchers fed guinea pigs and rats a daily enzyme dose for six months which would have corresponded to around 250 tablets per day for a 60 kilogram (134 pound) person. No ill effects. In another study, rats were fed enzymes equivalent to a human dose of 2500 tablets daily for a short period and the rats only seemed a little fatigued. Research has also looked at cell changes and mutations. They found no negative affects at all.

Recorded information on the safety of using digestive enzymes goes back to the early 1900s. This would be both reasonable and understandable to expect because, after all, our digestive system is swimming with digestive enzymes all the time anyway. Every time you eat fresh fruits, vegetables, or other raw food you are consuming digestive enzymes.

As far as long-term safety is concerned, keep in mind that you are full of enzymes throughout your lifetime. Those with certain medical conditions like malfunctioning pancreases need to take enzymes their entire lives to survive.

Digestive enzymes are classified as a safe food in the United States and have GRAS status (Generally Regarded As Safe). Since they are not in the realm of the Food and Drug Administration (FDA), specific digestive enzyme blends are neither FDA approved or unapproved, just as oatmeal is not specifically FDA approved or unapproved for you to eat. However, enzyme therapy is currently FDA approved in the treatment of certain health conditions (Lopez 1994). These include:

• cardiovascular disorders
• gastrointestinal conditions, particularly pancreatic insufficiency and related disorders
• replacement therapy for specific genetic disorders and deficiencies
• cancer treatment
• debridement of wounds (degradation or cleaning out of dying or dead/necrotic tissue)
• removal of toxin substances from the blood

Not Taking Enzymes Lead to Health Deterioration

Thinking of our water analogy: Everyone dies and everyone drinks water. . . so can we prove that drinking water causes death? The same applies to breathing oxygen: can we prove that since everyone breathes oxygen and everyone eventually dies, is a long-term effect of breathing oxygen a cause of illness and death? Maybe not since water and oxygen are necessary for life. But we can turn this around and look at: If we take away water or oxygen, will a person live the same length of time or even longer? What usually happens is that if you take away air and water, the person dies pretty rapidly. So the lack of oxygen and water is a cause of death far, far more and quicker than taking in too much.

Digestive enzymes are similar because they are a necessary part of sustaining life in animals. So far, taking too many enzymes hasn't been a cause of death or even shortening one's life span. However, not taking enzymes has been a cause of death and sharply reducing health.

examples and research coming.

Taking Enzymes Maintains Health

Here is one of many links telling about health benefits of enzymes. It contains a series of articles of enzymes and various health issues, and how enzymes may be beneficial in each one.

examples and research coming.


http://europa.eu.int/comm/environment/dansub/enzymerepcomplete.pdf (396 pages)

In the Enzyme Therapy by Dr Max Wolf, he summarizes the research to date from the early 1900s through 1972. Dr Wolf is regarded as a leader in enzyme therapy. Under General Directions for oral enzymes, he writes, "Proteolytic enzymes are non-toxic to healthy tissues, even is highest concentration."

Dr Wolf spent many years working with oral enzymes and cancers.

Comparative evaluation of alpha-galactosidase A infusions for treatment of Fabry disease.
Hopkin RJ, Bissler J, Grabowski AG. Genet Med. 2003 May-Jun;5(3):144-53. PMID: 12792421

"Enzyme therapy has proven safe and effective in preventing and reversing many manifestations in patients with Gaucher disease. On the basis of this success, enzyme therapy is now becoming a reality for Fabry disease, alpha-galactosidase A deficiency. ..."

Safety and efficacy of recombinant human alpha-galactosidase A--replacement therapy in Fabry's disease.
Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S, Caplan L, Linthorst GE, Desnick RJ; International Collaborative Fabry Disease Study Group. N Engl J Med. 2001 Jul 5;345(1):9-16. PMID: 11439963

Fabry's disease, lysosomal alpha-galactosidase A deficiency, results from the progressive accumulation of globotriaosylceramide and related glycosphingolipids. Affected patients have microvascular disease of the kidneys, heart, and brain. We evaluated the safety and effectiveness of recombinant alpha-galactosidase A in a multicenter, randomized, placebo-controlled, double-blind study of 58 patients who were treated every 2 weeks for 20 weeks. ...We also evaluated the histologic clearance of microvascular endothelial deposits of globotriaosylceramide in the endomyocardium and skin, as well as changes in the level of pain and the quality of life.

Recombinant alpha-galactosidase A replacement therapy cleared microvascular endothelial deposits of globotriaosylceramide from the kidneys, heart, and skin in patients with Fabry's disease, reversing the pathogenesis of the chief clinical manifestations of this disease.

Enzyme replacement therapy in Fabry disease: a randomized controlled trial.
Schiffmann R, Kopp JB, Austin HA 3rd, Sabnis S, Moore DF, Weibel T, Balow JE, Brady RO. JAMA. 2001 Jun 6;285(21):2743-9. PMID: 11386930

CONTEXT: Fabry disease is a metabolic disorder without a specific treatment, caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). Most patients experience debilitating neuropathic pain and premature mortality because of renal failure, cardiovascular disease, or cerebrovascular disease.

OBJECTIVE: To evaluate the safety and efficacy of intravenous alpha-gal A for Fabry disease.

DESIGN AND SETTING: Double-blind placebo-controlled trial conducted from December 1998 to August 1999 at the Clinical Research Center of the National Institutes of Health.

PATIENTS: Twenty-six hemizygous male patients, aged 18 years or older, with Fabry disease that was confirmed by alpha-gal A assay.

INTERVENTION: A dosage of 0.2 mg/kg of alpha-gal A, administered intravenously every other week (12 doses total).

MAIN OUTCOME MEASURE: Effect of therapy on neuropathic pain while without neuropathic pain medications measured by question 3 of the Brief Pain Inventory (BPI).

RESULTS: Mean (SE) BPI neuropathic pain severity score declined from 6.2 (0.46) to 4.3 (0.73) in patients treated with alpha-gal A vs no significant change in the placebo group (P =.02). Pain-related quality of life declined from 3.2 (0.55) to 2.1 (0.56) for patients receiving alpha-gal A vs 4.8 (0.59) to 4.2 (0.74) for placebo (P =.05). In the kidney, glomeruli with mesangial widening decreased by a mean of 12.5% for patients receiving alpha-gal vs a 16.5% increase for placebo (P =.01). Mean inulin clearance decreased by 6.2 mL/min for patients receiving alpha-gal A vs 19.5 mL/min for placebo (P =.19). Mean creatinine clearance increased by 2.1 mL/min (0.4 mL/s) for patients receiving alpha-gal A vs a decrease of 16.1 mL/min (0.3 mL/s) for placebo (P =.02). In patients treated with alpha-gal A, there was an approximately 50% reduction in plasma glycosphingolipid levels, a significant improvement in cardiac conduction, and a significant increase in body weight.

CONCLUSION: Intravenous infusions of alpha-gal A are safe and have widespread therapeutic efficacy in Fabry disease.

Systemic enzyme therapy in oncology: effect and mode of action.
Leipner J, Saller R. Drugs. 2000 Apr;59(4):769-80. PMID: 10804034

Plant extracts with a high content of proteolytic enzymes have been used for a long time in traditional medicine. Besides proteolytic enzymes from plants, 'modern' enzyme therapy additionally includes pancreatic enzymes. The therapeutic use of proteolytic enzymes is partly based on scientific studies and is partly empirical. The aim of the current review is to provide an overview of clinical trials of systemic enzyme therapy in oncology, and to discuss the evidence for their possible mechanisms of action. Clinical studies of the use of proteolytic enzymes in oncology have mostly been carried out on an enzyme preparation consisting of a combination of papain, trypsin and chymotrypsin. This review of these studies showed that enzyme therapy can reduce the adverse effects caused by radiotherapy and chemotherapy. There is also evidence that, in some types of tumours, survival may be prolonged. The beneficial effect of systemic enzyme therapy seems to be based on its anti-inflammatory potential. However, the precise mechanism of action of systemic enzyme therapy remains unsolved. The ratio of proteinases to antiproteinases, which is increasingly being used as a prognostic marker in oncology, appears to be influenced by the oral administration of proteolytic enzymes, probably via an induction of the synthesis of antiproteinases. Furthermore, there are numerous alterations of cytokine composition during therapy with orally administered enzymes, which might be an indication of the efficacy of enzyme therapy. Effects on adhesion molecules and on antioxidative metabolism are also reviewed.

Systemic enzyme therapy in diseases of the vascular system
Nouza K. Bratisl Lek Listy. 1995 Oct;96(10):566-9. PMID: 8620329

The treatment of autoimmune and immune complex diseases of the vascular bed consists--similarly as of immunopathologic processes of other systems--in the use of risky immunosuppressive agents and antiinflammatory as well as symptomatic therapy. In the article the author informs about the possibility to use in these indications (and in addition also in other angiologic diseases) the systemic enzyme therapy, residing in the oral application of high-dosed combinations of several animal and plant proteolytic enzymes. About four tens years of positive medical empirical experience have been supported by a concentrated sophisticated research and approximately 150 clinical studies according to GCP. These revealed in most autoimmune and immune complex diseases a surprisingly high effectiveness and complete harmlessness of the enzyme therapy. . . Strong evidence indicates that enzymotherapy ameliorates the disturbed composition and properties of blood and vessel walls, acts preventively as well as therapeutically in thromboses, thromboflebitides and consequences of venous insufficiency; it seems be prospective in afflictions of arterial bed, including vasculitides and glomerulonephritides, also. The key feature of enzymotherapy is the immunomodulatory activity. There exists a strong evidence for the favourable modulation of pathogenic autoantibodies, inhibition of the neogenesis of immune complexes and cleavage of their deposits, normalization of the T cell system, network of cytokines, adhesion molecules and inflammatory cascades. Besides the direct peptidolytic and proteolytic effects of hydrolases, the indirect effects realized in the course of interaction between the resorbed enzymes and their natural "partners"--antiproteases (mainly alfa-2-macroglobulin)--have become a topic of intensive research. The author feels, that systemic enzyme therapy should become a regular component of the treatment of immunopathologic processes in general and of angiologic diseases specially. (Ref. 42.).

The favorable effect of hydrolytic enzymes in the treatment of immunocytomas and plasmacytomas
Sakalova A, Mikulecky M, Holomanova D, Langner D, Ransberger K, Stauder G, Mistrik M, Gazova S, Chabronova I, Benzova M, et al. Vnitr Lek. 1992 Sep;38(9):921-9. PMID: 1481392

At present attention is focused on research of biomodulating influences on tumorous processes, in particular inhibition of metastatic spread of tumors. In the aetiopathogenesis an important part is played by immune complexes, interaction of cytokines. The authors tested the supporting effect of hydrolytic enzymes in plasmocytoma and immunocytoma. The enzymes were administered along with cytostatic preparations according to the MOCCA pattern. They recorded a more rapid onset and longer persistence of remissions, a marked decline of total proteins, paraproteins, beta-2-microglobulin. Complications associated with paraprotein (hyperviscosity syndrome, nephrotic syndrome, peripheral angiopathy) improved. A combination of chemotherapy and enzymatic treatment proved effective and suitable, in particular for patients with interferon intolerance.

Long-term multicomponent enzyme therapy--a developing trend in the complex treatment of suppurative wounds
Adamian AA, Gliantsev SP. Khirurgiia (Mosk). 1992 Jul-Aug;(7-8):105-14. PMID: 1469859

Prolonged local enzyme therapy of suppurative wounds
Efendiev AI, Tolstykh PI, Dadashev AI, Marshava OM. Khirurgiia (Mosk). 1991 Jul;(7):48-50. PMID: 1921198

The article analyses the results of treatment of 150 patients with purulent diseases of the soft tissues of various localization, in which after surgical treatment the purulent focus immobilized trypsin (50 patients), trypsin with lysozyme immobilized on gauze (50 patients), and antiseptics (50 patients) were used for topical treatment of the formed wounds. The results of the treatment showed that topical prolonged enzyme therapy of purulent wounds with the use of trypsin immobilized on gauze and that with lysozyme promotes cleansing of the wounds, reduces microbial dissemination, intensifies reparative processes, and reduces the terms of treatment.

Theory and practice in the individualization of oral pancreatic enzyme administration for chronic pancreatitis.
Andren-Sandberg A. Int J Pancreatol. 1989;5 Suppl:51-62. PMID: 2702251

"Pancreatic enzyme therapy may be beneficial to all patients with chronic pancreatitis, even those in whom the condition is very mild. The goal of enzyme therapy should be to restore normal gastrointestinal physiology as completely as possible. Monitoring of body weight is recommended as the main measure of treatment efficacy. Most pancreatic enzyme preparations presently employed are porcine in origin and must meet certain standards of quality for human consumption. The amount of active lipase in the duodenum determines the quantity of enzymes to be given. An appropriate diet is also important for relieving symptoms of pancreatic insufficiency and improving nutritional status. . . "

Effectiveness of enteric-coated preparations on nutritional parameters in cystic fibrosis. A long-term study.
Carroccio A, Pardo F, Montalto G, Japichino L, Iacono G, Collura M, Notarbartolo A.Digestion. 1988;41(4):201-6. PMID: 3243379

To evaluate the effectiveness of enteric-coated pancreatic enzyme supplements in comparison to conventional preparations of ingested enzyme on growth and nutritional parameters of patients with cystic fibrosis, we conducted a long-term study involving 40 patients. The data reproduced here were recorded after 6 months of therapy with powder-containing capsules or with enteric-coated products. Fat absorption was estimated by measurement of steatorrhoea with the steatocrit method. All parameters studied improved after enteric-coated pancreatic enzyme therapy, with a statistically significant increase in weight, cholesterol and haemoglobin values. Furthermore, the number of patients with positive steatocrit test was lower after therapy with enteric-coated enzyme supplementation. These findings suggest that the enteric-coated product not only reduces steatorrhoea, but above all improves the nutritional parameters and growth of patients affected by cystic fibrosis.

Efficacy of pancreatic enzyme supplementation in children with cystic fibrosis: comparison of two preparations by random crossover study and a retrospective study of the same patients at two different ages.
Ansaldi-Balocco N, Santini B, Sarchi C. J Pediatr Gastroenterol Nutr. 1988;7 Suppl 1:S40-5. PMID: 3042938

Two studies were performed in children with cystic fibrosis (CF) to compare the efficacy and tolerability of pancreatic enzymes prepared as enteric-coated microspheres with that of a conventional enzyme preparation. The parameters evaluated included the following: fecal fat excretion, coefficient of fat absorption, daily caloric intake, percent of diet as fats, proteins and carbohydrates, increase in height and weight, frequency and consistency of stools, palatability of the preparation, and patient compliance. The first study was an open randomized crossover trial of an enteric-coated microsphere preparation (Pancrease) versus conventional pancreatin given alone or with cimetidine. With Pancrease compared to conventional pancreatin, a significant improvement was observed in all the digestive parameters in addition to better patient compliance. In the second study, the response of a group of CF patients given Pancrease for at least 3 months (3-67 months) was retrospectively compared with the response to conventional pancreatin, which had been given to the same patients from 10 months to 8 years earlier. In comparison to conventional pancreatin, Pancrease provided better digestive efficacy and greater increases in the growth rate of teenage patients. With Pancrease, the number of daily dosage units is decreased even when fat intake is increased. No adverse reactions were seen with either of the enzyme preparations used in these studies.

Natural treatment of perennial allergic rhinitis.
Thornhill SM, Kelly AM. Altern Med Rev. 2000 Oct;5(5):448-54. PMID: 11056414

Perennial allergic rhinitis is an IgE-mediated inflammatory disorder of the nasal mucosa characterized by paroxysms of sneezing, nasal congestion, pruritis, and rhinorrhea. The condition may be caused by certain environmental agents, food sensitivities, structural abnormalities, metabolic conditions, or synthetic drugs. Recent health impairment outcome studies on allergic rhinitis sufferers reveal a measurable decline in physical and mental health status and the inability to perform daily activities. Antihistamines, decongestants, anticholinergic agents, and corticosteroid drug therapy, alone or in combination, are typically used in the treatment of allergic rhinitis. Reported adverse side effects include sedation, impaired learning/memory, and cardiac arrhythmias. Therapeutic strategies should seek to decrease the morbidity already associated with this condition. Urtica dioica, bromelain, quercetin, N-acetylcysteine, and vitamin C are safe, natural therapies that may be used as primary therapy or in conjunction with conventional methods.

Efficacy of enzyme supplementation after surgery for chronic pancreatitis.
Van Hoozen CM, Peeke PG, Taubeneck M, Frey CF, Halsted CH. Pancreas. 1997 Mar;14(2):174-80. PMID: 9057190

Although surgical procedures that improve pancreatic drainage alleviate abdominal pain in the vast majority of patients with chronic pancreatitis, postoperative absorption and nutritional status are less predictable. The present study was designed to determine the efficacy of pancreatic enzyme supplementation in maintaining postoperative digestion and nutrition in patients who had received the local resection-longitudinal pancreaticojejunostomy (LR-LPJ) procedure for chronic pancreatitis. We evaluated nutritional status and intestinal absorption in 11 patients who had undergone LR-LPJ. The efficacy of postoperative pancreatic enzyme supplementation was studied by measurements of intestinal absorption and nutritional status at baseline, after 4 weeks of individualized daily dosage of pancreatin (Creon), and after an additional 4 weeks of randomization to receive another 4 weeks of pancreatin or placebo. All patients demonstrated abnormal digestion of fat, protein, and total energy at baseline 3 weeks after surgery. Pancreatin supplementation significantly improved the coefficients of absorption of dietary fat and total energy over the next 4 weeks. Between 4 and 8 weeks, pancreatin significantly improved protein absorption and nitrogen balance, whereas placebo substitution worsened the absorption of dietary fat and total energy. Nutritional status was not significantly altered over the 8-week study period, although four patients receiving pancreatin gained more than 3.6 kg body weight. The data suggest that long-term postoperative pancreatic enzyme supplementation is both efficacious and necessary in chronic pancreatitis patients after LR-LPJ.

Oral pancreatic enzyme therapy in the control of diabetes mellitus in tropical calculous pancreatitis.
Mohan V, Poongothai S, Pitchumoni CS. Int J Pancreatol. 1998 Aug;24(1):19-22. PMID: 9746885

CONCLUSION: Pancreatic enzyme supplementation therapy conducted during a 6 mo period produces better control of diabetes, improvement in nutrition, and overall improvement in quality of life in patients with tropical calculous pancreatitis.

BACKGROUND: Tropical calculous pancreatitis is characterized by abdominal pain, pancreatic calculi, and diabetes. The diabetes in insulin resistant. The brittle diabetes may be owing to defective glucagon secretion and erratic absorption of nutrients.

METHODS: Patients with tropical calculous pancreatitis with diabetes and chronic pancreatitis were studied for fasting and postprandial plasma glucose, glycosylated hemoglobin, serum cholesterol, triglycerides, and calcium, liver function, and plasma C-peptide. All patients were given Creon and evaluated for quality of life.

RESULTS: Clinical parameters of the patients showed considerable improvement at the end of the trial. Abdominal pain, steatorrhea, and sense of well being improved. There was a significant reduction in postprandial plasma glucose and glycosylated hemoglobin.

Assimilation of wheat starch in patients with chronic pancreatitis. Positive effect of enzyme replacement.
Nordgaard I, Rumessen JJ, Gudmand-Hoyer E. Scand J Gastroenterol. 1992 May;27(5):412-6. PMID: 1529277

Pancreatic insufficiency due to chronic pancreatitis may lead to symptomatic malabsorption of both starch and fat. The absorption capacity of wheat starch has not been studied previously in patients with chronic pancreatitis, although this carbohydrate is a quantitatively important component of the Western diet. We studied the absorption of wheat starch and the effect of pancreatic enzyme substitution in seven patients with chronic pancreatitis and steathorrea. The malabsorption was determined from hydrogen breath tests with lactulose standards as reference. Without enzyme substitution, wheat starch (50 g) was absorbed to a lesser extent than in healthy controls (p less than 0.05). The mouth-to-cecum transit time was prolonged and correlated positively to the fat excretion before substitution with pancreatic enzymes (sigma = 1). The enzyme substitution increased the absorption of wheat starch to values seen in healthy controls (p less than 0.05) and reduced the mouth-to-cecum transit time by 19.8%.

Efficacy and tolerability of oral enzyme therapy as compared to diclofenac in active osteoarthrosis of knee joint: an open randomized controlled clinical trial.
Tilwe GH, Beria S, Turakhia NH, Daftary GV, Schiess W. J Assoc Physicians India. 2001 Jun;49:617-21. PMID: 11584936

OBJECTIVE: To compare the efficacy and tolerability of an oral enzyme preparation (Phlogenzym) with that of an NSAID (diclofenac) in the treatment of active osteoarthrosis.

METHODS: Prospective, randomized, controlled, single-blind study of seven weeks duration at a tertiary care centre wherein 50 patients aged 40-75 years, with activated osteoarthrosis of knee joint were randomized to receive phlogenzym tablets (2-3 tablets, bid) or diclofenac sodium 50 mg bid for three weeks.

RESULTS: At the end of therapy (three weeks) and at follow-up visit at seven weeks there was reduction in pain and joint tenderness and swelling in both groups, and slight improvement in the range of movement in the study group. The reduction in joint tenderness was greater (p < 0.05) in the study group receiving phlogenzym.

CONCLUSION: Phlogenzym is as efficacious and well tolerated as diclofenac sodium in the management of active osteoarthrosis over three weeks of treatment.

Efficacy and safety of phlogenzym--a protease formulation, in sepsis in children.
Shahid SK, Turakhia NH, Kundra M, Shanbag P, Daftary GV, Schiess W. J Assoc Physicians India. 2002 Apr;50:527-31. PMID: 12164403

BACKGROUND: Infections are a major cause of hospitalisation wherein the host mounts an inflammatory response against the infecting agent. Administration of proteolytic enzymes could regulate the host's immune system and help early recovery from sepsis.

OBJECTIVE: To test the efficacy and safety of an oral enzyme formulation, Phlogenzym (Mucos Pharma GmbH, Geretsried, Germany; constituents of each enteric-coated tablet were bromelain 90 mg, trypsin 48 mg, rutin 100 mg) as adjuvant therapy in treatment of sepsis in children.

SUBJECTS AND METHODS: Double-blind, randomised, controlled phase III study at a tertiary care centre wherein 60 eligible children aged one month to 12 years with sepsis were randomised to receive either phlogenzym (n=30; 17 boys) or placebo (n=30; 22 boys) tablets (1 tablet/10 kg body weight up to maximum six tablets a day in two or three divided doses for 14-21 days) along with appropriate antibiotics and supportive treatment.

RESULTS: Median time taken for fever to subside was three days (range 1-12; 95% CI--1.14 to 7.14) in the phlogenzym group vs four days (range 1-18; 95% CI--3.52 to 11.52) in the placebo group (p < 0.05); haemodynamic support was needed for two days (range 1-3; 95% CI--0.84 to 3.16) in the phlogenzym group but three days (range 1-8; 95% CI--0.76 to 5.24) in the placebo group (p < 0.05). The modified Glasgow coma scale score normalized in three days (range 1-14; 95% CI--4.62 to 9.62) in the phlogenzym group vs 5.5 days (range 1-18; 95% CI--2.52 to 13.52) in the placebo group (p > 0.05). Oral feeds could be started in four days (range 1-15; 95% CI--1.74 to 9.74) in the phlogenzym group vs five days (range 1-11; 95% CI--1.26 to 11.26) in the placebo group (p > 0.05). Two patients died in the placebo group.

CONCLUSION: Phlogenzym is effective as an adjuvant with antibiotics and supportive treatment for early improvement of pediatric patients with sepsis.

Rofecoxib, a specific inhibitor of cyclooxygenase 2, with clinical efficacy comparable with that of diclofenac sodium: results of a one-year, randomized, clinical trial in patients with osteoarthritis of the knee and hip. Rofecoxib Phase III Protocol 035 Study Group.
Cannon GW, Caldwell JR, Holt P, McLean B, Seidenberg B, Bolognese J, Ehrich E, Mukhopadhyay S, Daniels B. Arthritis Rheum. 2000 May;43(5):978-87. PMID: 10817549

OBJECTIVE: To compare the clinical efficacy of rofecoxib, a specific inhibitor of cyclooxygenase 2 (COX-2), with that of diclofenac in patients with osteoarthritis (OA) and to evaluate the safety and tolerability of rofecoxib.

METHODS: We performed a randomized, double-blind, active comparator-controlled trial in 784 adults with OA of the knee or hip. Patients were randomized to 1 of 3 treatment groups: 12.5 mg of rofecoxib once daily, 25 mg of rofecoxib once daily, and 50 mg of diclofenac 3 times daily. Clinical efficacy and safety were evaluated over a 1-year continuous treatment period.

RESULTS: Rofecoxib at dosages of 12.5 and 25 mg demonstrated efficacy that was clinically comparable to that of diclofenac, as assessed by all 3 primary end points according to predefined comparability criteria. Results from secondary end points were consistent with those of the primary end points. There were small statistical differences favoring diclofenac for 2 of the end points. All treatments were well tolerated.

CONCLUSION: Rofecoxib was well tolerated and provided efficacy that was clinically comparable, according to predefined statistical criteria, to that of 150 mg of diclofenac per day in this 1-year study. Specific inhibition of COX-2 provided therapeutic efficacy in OA.

Oral therapy with proteolytic enzymes decreases excessive TGF-beta levels in human blood.
Desser L, Holomanova D, Zavadova E, Pavelka K, Mohr T, Herbacek I. Cancer Chemother Pharmacol. 2001 Jul;47 Suppl:S10-5. PMID: 11561866

Therapy with oral proteolytic enzymes (OET) with combination drug products containing papain, bromelain, trypsin, and chymotrypsin has been shown to be beneficial in clinical settings such as radiotherapy-induced fibrosis, bleomycin pneumotoxicity and immunosuppression in cancer, all of which are nowadays known to be accompanied by excessive transforming growth factor-beta (TGF-beta) production. It has been demonstrated that proteolytic enzymes reduce TGF-beta levels in serum by converting the protease inhibitor alpha2 macroglobulin (alpha2M) from the "slow" form into the "fast" form, whereby the "fast" form binds and inactivates TGF-beta irreversibly. In this study we have investigated the effect of OET on the concentration of TGF-beta1 in serum of patients with rheumatoid arthritis (RA) (n = 38), osteomyelofibrosis (OMF) (n = 7) and herpes zoster (HZ) (n = 7). Seventy-eight healthy volunteers served as controls. TGF-beta1 levels in serum were assessed by enzyme-linked immunosorbent assay (ELISA). We have demonstrated that in healthy volunteers and in patients there exists a correlation between active and latent TGF-beta1 in serum (r=0.8021; P<0.0001). Treatment with OET had no significant effect on TGF-beta1 concentration in healthy volunteers or patients with a normal level of TGF-beta1. In patients with elevated TGF-beta1 concentration (> 50 ng/ml serum), OET reduced TGF-beta1 in RA (P < 0.005), in OMF (P < 0.05) and in HZ (P < 0.05). Conclusion: These results support the concept that OET is beneficial in diseases characterized in part by TGF-beta1 overproduction.

Modulation of growth factor binding properties of alpha2-macroglobulin by enzyme therapy.
Lauer D, Muller R, Cott C, Otto A, Naumann M, Birkenmeier G. Cancer Chemother Pharmacol. 2001 Jul;47 Suppl:S4-9. PMID: 11561872

PURPOSE: To investigate the binding of transforming growth factor-beta (TGF-beta) to human alpha2-macroglobulin upon oral treatment of patients with proteases.

METHODS: Volunteers were given a cocktail of active proteinases (Phlogenzym) composed of trypsin, bromelain and the additive rutoside orally over a period of 7 days at low dose followed by a bolus application. Before and after medication plasma was immediately withdrawn and binding of 125I-TGF-beta to the proteinase inhibitor alpha2-macroglobulin was determined by electrophoresis and gamma-counting. Cell culture experiments were performed to study the effect of transformed alpha2-macroglobulin on TGF-beta-stimulated proliferation of skin fibroblasts.

RESULTS: Ingestion of proteinases was found to trigger the formation of intermediate forms of alpha2-macroglobulin displaying high affinity to TGF-beta. Maximum binding of TGF-beta was observed 1-2 h after bolus ingestion, and steadily levelled off with time. In vitro experiments demonstrated that complex formation of diverse proteinases (trypsin, alpha-chymotrypsin, bromelain and plasmin) with alpha2-macroglobulin conferred binding of 125I-TGF-beta, alpha2-Macroglobulin transformed by methylamine or proteinases was found to abolish the TGF-beta effect on fibroblasts in cell culture.

CONCLUSIONS: Intestinal absorption of proteinases triggers the formation of TGF-beta binding species of alpha2-macroglobulin in blood. Mediated by this process high concentrations of TGF-beta might be reduced via enhanced clearance of alpha2-macroglobulin-TGF-beta complexes. Thus, proteinase therapy may have beneficial effects in treatment of fibrosis and certain cancers accompanied by excessively high TGF-beta concentrations.

Prevention of murine EAE by oral hydrolytic enzyme treatment.
Targoni OS, Tary-Lehmann M, Lehmann PV. J Autoimmun. 1999 May;12(3):191-8. PMID: 10222028

Clinical trials that test the efficacy of Phlogenzym (consisting of the hydrolytic enzymes bromelain and trypsin and the anti-oxidant rutosid) as a treatment for T cell-mediated autoimmune diseases including multiple sclerosis (MS), type 1 diabetes and rheumatoid arthritis are presently ongoing. We tested the effects of Phlogenzym treatment in the murine model for MS, experimental allergic encephalomyelitis (EAE), a disease induced in SJL mice by immunization with proteolipid protein (PLP) peptide 139-151. Oral administration of Phlogenzym resulted in complete protection from EAE. In Phlogenzym-treated mice, the dose response curve of the PLP:139-151-specific T cell response was shifted to the right, that is, the primed T cells required higher peptide concentrations to become activated. Additionally, the T cell response to this peptide was shifted towards the T helper 2 cytokine profile. Both effects are consistent with an increased T cell activation threshold. In support of this interpretation, we found that the accessory molecules CD4, CD44, and B7-1 (all of which are involved in T cell co-stimulation) were cleaved by Phlogenzym, while CD3 and MHC class II molecules (which are involved in the recognition of antigens by T cells) and LFA-1 were unaffected. These data show the efficacy of oral Phlogenzym treatment in an animal model of T cell-mediated autoimmune disease and suggest that the protective effect might be the result of an increase in the activation threshold of the autoreactive T lymphocytes brought about by the cleavage of accessory molecules involved in the interaction of T cells and antigen presenting cells. Copyright 1999 Academic Press.

Transport of proteolytic enzymes across Caco-2 cell monolayers.
Bock U, Kolac C, Borchard G, Koch K, Fuchs R, Streichhan P, Lehr CM. Pharm Res. 1998 Sep;15(9):1393-400. PMID: 9755891

PURPOSE: To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man.

METHODS: Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin.

RESULTS: Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules.

CONCLUSIONS: Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.

Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.
Mynott TL, Luke RK, Chandler DS. Gut. 1996 Jan;38(1):28-32. PMID: 8566855

The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea.

Polyenzyme preparation Wobe-Mugos inhibits growth of solid tumors and development of experimental metastases in mice.
Wald M, Zavadova E, Pouckova P, Zadinova M, Boubelik M. Life Sci. 1998;62(3):PL43-8. PMID: 9488106

Long-term rectal administration of enzyme mixture containing papain, trypsin and chymotrypsin in the same ratio as the preparation Wobe-Mugos E (Mucos Pharma, Germany) was evaluated for their antitumor effects in C57Bl6 inbred mice inoculated with Bl6 melanoma cells. 30% of animals in the test group (3 pcs) have been cured of cancer. In the rest of animals (70%) the survival time was prolonged by 58.3% compared to the control group (from average survival time of 24 days in control group to 38 days in the test group). Based on histological and immunohistochemical evaluation a faster process of metastasizing was found in control group than in the group treated with the polyenzyme preparation. In the case of melanoma Bl6 an antimetastatic effect of the preparation was thus proved.

Mixture of trypsin, chymotrypsin and papain reduces formation of metastases and extends survival time of C57Bl6 mice with syngeneic melanoma B16.
Wald M, Olejar T, Sebkova V, Zadinova M, Boubelik M, Pouckova P. Cancer Chemother Pharmacol. 2001 Jul;47 Suppl:S16-22. PMID: 11561867

PURPOSE: The aim of the present study was to investigate the effect of a mixture of proteolytic enzymes (comprising trypsin, chymotrypsin and papain) on the metastatic model of syngeneic melanoma B16.

METHODS: 140 C57B16 mice were divided into two control and two "treated" groups. Control groups received saline rectally, twice a day starting 24 h after intracutaneous transplantation (C1) or from the time point of the primary B16 melanoma extirpation (C2), respectively. "Treated" groups were rectally administered a mixture of 0.2 mg trypsin, 0.5 mg papain, and 0.2 mg chymotrypsin twice daily starting 24 h after transplantation (E1) or after extirpation of the tumor (E2), respectively. Survival of mice and B16 melanoma generalization were observed for a period of 100 days. Immunological evaluation of B16 melanoma cells in the ascites was accomplished. CD44, CD54 and CD106 cells were measured by flow cytometry.

RESULTS: Administration of proteolytic enzymes to mice inhibited the growth of primary tumors, and tumor recurrences were less numerous. Importantly, metastasis was considerably curtailed both in the vicinity of the primary tumor and at distant locales. These findings correlated with a decreased expression of CD44 and CD54 molecules in tumors exposed to proteolytic enzymes in vivo.

CONCLUSIONS: Our data suggest that serine and cysteine proteinases suppress B16 melanoma, and restrict its metastatic dissemination in C57B16 mice.

Double-blind pilot-study on the efficacy of enzyme therapy in advanced colorectal cancer.
Popiela T, Kulig J, Klek S, Wachol D, Bock PR, Hanisch J. Przegl Lek. 2000;57 Suppl 5:142. PMID: 11202281

All tested variables showed a tendency in favor for Wobe-Mugos E therapy as addition to standard therapy. Enzymes improve the quality of life by reducing cancer disease typical symptoms, they reduce side effects of chemo-/radiotherapy and they have a potential of prolonging life (preliminary data only).

The efficacy of the multienzyme preparation Wobe-Mugos E in lymphoproliferative diseases.
Kolomoiets' MIu, Antofiichuk MP. Lik Sprava. 1999 Jun;(4):150-4. PMID: 10476670

The drug Wobe-Mugos E was prescribed to patients with chronic lymphoid leukosis and multiple myeloma during and following the administration of chemotherapy treatment, 3 tablets on a three-times daily schedule 30 to 45 minutes before meals for 2 to 4 weeks. A positive effect was manifested by improvement in morphofunctional properties of erythrocytes, detoxicating and antioxidant effects. There were no cases of intolerability or side effects of the drug in the examined patients.

Enzyme therapy--an alternative in treatment of herpes zoster. A controlled study of 192 patients.
Billigmann P. Fortschr Med. 1995 Feb 10;113(4):43-8. PMID: 7713467

PROBLEM: Herpes Zoster requires an effective, inexpensive form of treatment not only because it impairs quality of life, but also on account of its relatively high incidence and the resulting costs incurred. Given the present situation in the health care sector, the high costs of treatment with the standard drug, acyclovir, often mean that herpes zoster patients do not receive medicinal therapy. AIM: The aim of the present study was to establish whether the positive results of a prior investigation involving treatment with an enzyme combination preparation could be confirmed.

METHOD: Over a period of 14 days, two groups of 96 patients each were given acyclovir or an enzyme combination preparation. During the course of the study, the intensity (score) of segmental pain and various skin lesions were investigated.

RESULTS: In terms of the first end point, "segmental pain", the test groups showed no significant difference either on day 7 or on day 14. Although the second end point "segmental reddening" did reveal a significant difference (p = 0.015) in favor of the acyclovir group on day 14, no significant difference was found for any of the other examination endpoints. Nor did any of the other skin lesions evaluated differ significantly by the end of the study.

CONCLUSIONS: Overall, the enzyme combination preparation showed identical efficacy with acyclovir. The results of the prior study were thus confirmed. Further investigations on the immunomodulatory potency, dosage and effects on postherpetic herpes neuralgia are, however, still required.

Results of a double-blind, randomized comparative study of Wobenzym-placebo in patients with cervical syndrome
Tilscher H, Keusch R, Neumann K. Wien Med Wochenschr. 1996;146(5):91-5. PMID: 8686328

The results of this study are based on 30 patients per treatment group. The primary objective of this trial was evidence of a superior analgesic effect of the verum group under concomitant treatment with physicotherapeutic measures. This objective was achieved and showed a small, but statistically significant and beneficial effect of Wobenzym in the pain self ratings of the patients. The results of erythrocyte sedimentation rates (Westergreen), leukocyte counts and lymphocyte counts as secondary study parameters indicate, that the antiphlogistic effects of Wobenzym provide an additional beneficial effect for the patients suffering from cervical syndrome compared to placebo. In combination with the available safety data of this study, the final interpretation of the authors of this study leads to a superior benefit/risk ratio judgement for Wobenzym compared to placebo.

Enzyme breakdown of immune complexes
Steffen C, Menzel J. Z Rheumatol. 1983 Sep-Oct;42(5):249-55. PMID: 6606278

4 types of immune complexes according to the Heidelberger curve were prepared as an in vitro model for immune complexes in rheumatoid arthritis and collagen diseases. Immune complexes of high and moderate antigen excess and immune complexes of high and moderate antibody excess were incubated with increasing concentrations of a mixture of enzymes or papain or pancreatin. The concentration of immune complexes was determined by a solid phase C1q radioimmunoassay before and after incubation. Up to 90% of the complexes of antigen excess were disintegrated even by low doses of enzymes (5-10 mg%); total cleavage appeared at 80 mg% enzyme concentration. Complexes of the antibody excess zone were gradually disintegrated by enzyme concentrations gradually increasing from 5 to 80 mg%, where total cleavage appeared. Single enzymes showed less cleavage activity than enzyme mixtures. Although enzymes were administered to supernatants of the Heidelberger precipitation containing soluble immune complexes as well as enzyme inhibitors, enzymatic activity was not impaired and immune complexes were disintegrated. The results of these investigations are discussed in regard to treatment with enzymes.

Safety and Tolerance of Phlogenzym®
Study No.: MU-91418/1 and MU-91418/2
Dr. med. A. Doenicke. Open clinical trial according to the GCP guidelines Biometric and medical final report

CONCLUSIONS: This examination has proven that the study treatment is a safe and good tolerated drug, which did not have an adverse effect on any of the organ systems controlled within this trial. The adverse drug reactions in particular focus on a functional impairment of the gastro-intestinal tract. A very low rate of adverse drug reactions has to be expected, if the medication is taken as commonly used i.e. in a dosage of 3 x 2 tablets. Especially the increase in leukocyte values as well as the gastrointestinal complaints observed in this drug trial have to be investigated in detail in a further examination. This examination is suggested to be performed as a double-blind study with either parallel group or crossover design. Principally there are no objection to use this preparation in clinical trials with greater population of patients and a longer duration of treatment.

Conclusions - Study No.: MU-91418/2 This second examination has also proven that the study treatment, which did not have an adverse impact on any of the organ systems controlled within this trial, is safe and well tolerated. The adverse drug reactions in particular focus on a functional impairment of the gastro-intestinal tract. These adverse drug reactions are obviously not caused by the active ingredients of the test medication, but by the supporting ingredient lactose. Since many years lactose is used in medicine as a supporting and active ingredient, the latter because of its relaxant activity. The symptoms occurring during intake of higher doses are not relevant with regard to the pharmaceutical safety. A very low rate of adverse drug reactions has to be expected if the medication is taken as commonly prescribed i.e. in a dosage of 3 x 2 tablets . The increases of leukocyte and platelet numbers, observed in this drug trial, represent no risk. Because of a possible therapeutic benefit, it should be tried to elucidate the principal mechanism of its pharmacological action. Principally there are no objections to use this preparation in clinical trials with a greater population of patients and a longer duration of treatment.

An overview of the safety evaluation of the Rhizomucor miehei lipase enzyme.
Broadmeadow A, Clare C, De Boer AS. Food Addit Contam 1994 Jan-Feb;11(1):105-19

"The Rhizomucor miehei lipase enzyme expressed in Aspergillus oryzae, is used in the production of specialty fats, the production of existing fats from new raw materials, or new fats with improved nutritional or functional qualities. It is produced by A. oryzae containing the structural gene for the precursor of R. miehei triglyceride lipase. It was subjected to a series of toxicological tests to document the safety in use. The enzyme preparation was not found to be mutagenic either in bacterial cultures (Ames test) or in the mammalian cell cultures (mouse lymphoma assay), nor did it cause chromosomal damage (human lymphocyte assay). Dietary concentrations up to 1600 mg/kg diet for up to 13 weeks caused no adverse effect in rats...."

Safety evaluation of lipase derived from Rhizopus oryzae: summary of toxicological data.
Coenen TM, Aughton P, Verhagen H. Food Chem Toxicol 1997 Mar-Apr;35(3-4):315-22

A lipase enzyme, obtained from Rhizopus oryzae produced by a fermentation process was subjected to a series of toxicological tests to document the safety for use as a food additive. The enzyme product was examined for acute, subacute and subchronic oral toxicity, and mutagenic potential. An extensive literature search on the production organism has also been conducted. No evidence of (sub)acute oral toxicity or mutagenic potential was found. Administration of the lipase at dosages of 50, 200 and 1000 mg/kg body weight/day for 90 days did not induce noticeable signs of toxicity. A few minor changes in the chemical composition of the blood in the highest dose group were of no toxicological significance. The no-observed-adverse-effect level of the tox-batch in the subchronic toxicity study was 1000 mg/kg body weight/day. It can be concluded that no safety concerns were identified in the studies conducted with this lipase preparation derived from R. oryzae and produced under controlled fermentation conditions.

Safety evaluation of lipase produced from Rhizopus oryzae: summary of toxicological data.
Flood MT, Kondo M. Regul Toxicol Pharmacol. 2003 Apr;37(2):293-304. PMID: 12726758

The toxicity of Lipase D, an enzyme preparation, was evaluated in a series of studies. Lipase D selectively hydrolyzes triglycerides of fatty acids. It also catalyzes the interesterification of edible fats and oils. In a 13-week gavage study, Sprague-Dawley rats received Lipase D at levels of 0, 500, 1000, or 2000mg/kg body wt./day. A dose dependent decrease in urinary pH was observed, but there were no effects on electrolyte balance, kidney weight, or histology of the kidney. The no-observed-adverse-effect level in rats was 1000mg/kg body wt./day. In common with other enzyme preparations, Lipase D was not genotoxic. Lipase D was tested in the Ames assay, the mouse lymphoma forward mutation assay, and the chromosome aberration assay. Finally, the particular strain of Rhizopus oryzae used to prepare Lipase D was shown to have low to moderate pathogenicity when injected into the tail vein of mice at doses up to 1.3x10(6) colony-forming units (CFU) per animal. No effects were observed when mice received up to 2.2x10(5) CFU by gavage or in their diets daily for 28 days. The results indicate that this particular strain can be handled using ordinary safety practices current in the fermentation industry. These studies support a conclusion that Lipase D is safe when used as described in the processing of dietary fatty acids and glycerides of fatty acids.

An overview of the safety evaluation of the Thermomyces lanuginosus xylanase enzyme (SP 628) and the Aspergillus aculeatus xylanase enzyme (SP 578).
Bergman A, Broadmeadow A. Food Addit Contam. 1997 May-Jun;14(4):389-98. PMID: 9205568

Xylanases SP 628 and SP 578 were produced by submerged fermentation of Aspergillus oryzae, containing a gene code originating from Thermomyces lanuginosus and Aspergillus aculeatus, respectively. Both enzymes were subject to the same series of toxicological tests to document their safety in use. The enzymes are to be applied as processing aids in the baking industry and in wheat starch separation. Neither enzyme was found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did they cause chromosomal aberrations in cultured human peripheral lymphocytes. No evidence of inhalation toxicity or skin and eye irritation was found. The enzymes are not regarded as skin-sensitizers, although the Buehler test with guinea-pigs revealed a minor potential. Oral administration up to 10.0 ml/kg bw/day (equivalent to a Total Organic Solids amount of 13.3% for SP 628 and of 11.3% for SP 578) in 13-week rat studies did not show any adverse effect.

Safety evaluation of beta-glucanase derived from Trichoderma reesei: summary of toxicological data.
Coenen TM, Schoenmakers AC, Verhagen H. Food Chem Toxicol. 1995 Oct;33(10):859-66. PMID: 7590530

Barlican, a beta-glucanase enzyme obtained from Trichoderma reesei, was produced by a fermentation process and subjected to a series of toxicological tests to document its safety for use as a feed additive. The enzyme product was examined for general oral toxicity, inhalation toxicity, irritation to eye and skin, skin sensitization and mutagenic potential. An extensive literature search on the production organism was also conducted. Furthermore, safety for target species was assessed in a 28-day oral toxicity study with broilers. A strong skin-sensitizing potential of the beta-glucanase enzyme was detected, but no other evidence of oral or inhalation toxicity, mutagenic potential, eye or skin irritancy was found. Feeding of the beta-glucanase enzyme at dietary levels up to 10,000 ppm in the 90-day subchronic toxicity study in rats did not induce noticeable signs of toxicity. In addition, no adverse effects were observed when broiler chicks were fed dietary concentrations of the beta-glucanase enzyme up to eight times the daily recommended dose. It is therefore concluded that this beta-glucanase preparation is safe for use in feed of the intended target species. However, some occupational health precautions should be taken to avoid skin contact and inhalation, as is the case for almost all enzyme proteins.

Toxicological studies on Thermomyces lanuginosus xylanase expressed by Fusarium venenatum, intended for use in food.
Pedersen PB, Broadmeadow A. Food Addit Contam. 2000 Sep;17(9):739-47. PMID: 11091786

The xylanase used in this study was produced by a submerged fermentation of Fusarium venenatum and contained a gene code originating from Thermomyces lanuginosus. The enzyme was subject to a 13-week toxicological test in rats and in vitro tests to document its safety in use. The enzyme is to be applied as a processing aid in the baking industry to improve handling and stability of dough. The enzyme was not found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes. Oral administration to rats of up to 10.0 ml/kg bw/day (equivalent to a Total Organic Solids dosage of 1.12 g/kg bw/day or a xylanase dosage of 89422 FXU (W)/kg bw/day) for 13 weeks did not cause any adverse effect.

Safety evaluation of amino peptidase enzyme preparation derived from Aspergillus niger.
Coenen TM, Aughton P. Food Chem Toxicol. 1998 Sep-Oct;36(9-10):781-9. PMID: 9737425

An amino peptidase enzyme preparation obtained from Aspergillus niger was subjected to a series of toxicological tests to document the safety for use as a processing aid for food. The enzyme preparation was examined for subacute and subchronic oral toxicity, and mutagenic potential. No evidence of oral toxicity or mutagenicity was found. Administration of the amino peptidase enzyme preparation at doses of 500, 1000 and 2000 mg/kg body weight/day for 90 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of the enzyme preparation in the subchronic toxicity study was 2000mg/kg body weight/day (equivalent to 1152 PHEA units/kg body weight/day). It can be concluded that no safety concerns were identified in the studies conducted with this amino peptidase enzyme preparation derived from Aspergillus niger and produced under controlled fermentation conditions.

Safety evaluation of a glucanase preparation intended for use in food including a subchronic study in rats and mutagenicity studies.
Elvig SG, Pedersen PB.Regul Toxicol Pharmacol. 2003 Feb;37(1):11-9. PMID: 12662905

An enzyme preparation containing glucanase produced by the fungus Trichoderma harzianum is intended to be used to improve the clarification and filtration of wines. The safety of a glucanase preparation was assessed in a series of toxicological tests to document its safety in use. Oral administration to rats of up to 10mL/kgbw/day (equivalent to a total organic solids dosage of 1258mg/kgbw/day or a glucanase dosage of 1882 BGXU/kgbw/day) for 13 weeks did not cause any adverse effect. The test substance was not found to be mutagenic in the bacterial reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes. The safety margin for exposure is estimated to be in the range of 2100-72,000 depending on the estimate for daily wine consumption. The results of these studies demonstrate that an enzyme preparation containing glucanase may be considered safe when employed in wine processing.

Safety evaluation of a lactase enzyme preparation derived from Kluyveromyces lactis.
Coenen TM, Bertens AM, de Hoog SC, Verspeek-Rip CM. Food Chem Toxicol. 2000 Aug;38(8):671-7. PMID: 10908814

Neutralact(R), the DSM brand name of a lactase enzyme preparation, obtained from a homologous rDNA strain of Kluyveromyces lactis, was subjected to a series of toxicological tests to document the safety for use as a processing aid in the dairy industry. The enzyme preparation was examined for subacute oral toxicity and mutagenic potential. As a result of these tests, no evidence of oral toxicity, mutagenicity or clastogenicity was found. Administration of the lactase enzyme preparation at doses of 500, 3000 and 10,000 mg/kg body weight/day for 28 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of the enzyme preparation in the acute toxicity study was 10,000 mg/kg body weight/day (equivalent to 114,000 NL units/kg body weight/day). It can be concluded that no safety concerns were identified in the studies conducted with this lactase enzyme preparation derived from Kluyveromyces lactis under controlled fermentation conditions.

Comparative evaluation of a high lipase pancreatic enzyme preparation and a standard pancreatic supplement for treating exocrine pancreatic insufficiency in chronic pancreatitis.

Delhaye M, Meuris S,Gohimont AC, Buedts K,Cremer M. Eur J Gastroenterol Hepatol. 1996 Jul;8(7):699-703.

Medicosurgical Department of Gastroenterology, Erasme Hospital, Free University of Brussels, Belgium.

OBJECTIVE: To compare the efficacy and safety of two enzyme-containing preparations, Pancrease HL (Cilag) containing 25,000 units of lipase per capsule and Creon (Triosol) with 8000 units of lipase per capsule, in patients with chronic pancreatitis and exocrine insufficiency. DESIGN: The study is a monocentric open crossover prospective study including 25 patients entered from March 1993 to May 1994. PATIENTS: Chronic pancreatitis was alcohol-related in 23 patients, previous surgery was performed in 9, 16 had diabetes and all had steatorrhoea (fat balance > 10g/24h). METHODS: Patients were investigated during four periods of 2 weeks, each one corresponding to a new treatment regimen: Pancrease HL, 3 capsules/day or Creon, 9 capsules/day, with or without omeprazole 20 mg/day. Stools were collected on the last 3 days at the end of each period when the patients were on a standard diet with a fixed daily intake of 100 g fat/day. RESULTS: Faecal fat, protein and energy excretion did not differ when both preparations were compared at roughly pharmaceutically equivalent doses. No significant improvement in fat and protein absorption was observed when omeprazole was taken with the pancreatic enzymes. However, omeprazole treatment was associated with a marked decrease in the fat-protein content ratio, suggesting an improvement in the fat digestive process but a decrease in the efficiency of protein digestion. Drug safety was comparable in the four groups of treatment. CONCLUSION: Pancrease HL with high lipase activity provides effective pancreatic enzyme replacement therapy in patients with chronic pancreatitis at an appreciably lower number of capsules per day than with standard preparations.

PMID: 8853261 [PubMed - indexed for MEDLINE]

Safety evaluation of a xylanase expressed in Bacillus subtilis.

Harbak L, Thygesen HV. Food Chem Toxicol. 2002 Jan;40(1):1-8. PMID: 11731030

A programme of studies was conducted to establish the safety of a xylanase expressed in a self-cloned strain of Bacillus subtilis to be used as a processing aid in the baking industry. To assess acute and subchronic oral toxicity, rat feeding studies were conducted. In addition, the potential of the enzyme to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. Acute and subchronic oral toxicity was not detected at the highest dose recommended by OECD guidelines. There was no evidence of mutagenic potential or chromosomal aberrations. Furthermore, the organism used for production of the xylanase is already accepted as safe by several major national regulatory agencies.

Safety evaluation of lipase G from Penicillium camembertii.

Kondo M, Ogawa T, Matsubara Y, Mizutani A, Murata S, Kitagawa M. Food Chem Toxicol. 1994 Aug;32(8):685-96. PMID: 8070733

Lipase G, a partial glycerides eliminating enzyme produced by Penicillium camembertii, was subjected to safety evaluation studies to establish its safety when used as a processing aid in the food industry. The toxicological studies on the enzyme included a 90-day gavage study with rats, a mutagenicity study using bacteria, and a pathogenicity study using mice. The no-adverse-effect level from the 90-day gavage toxicity study was 2000 mg/kg body weight/day for rats. There was no evidence of mutagenic potential. The micro-organism was evaluated for pathogenicity using mice and classified as a non-pathogen. Results indicate that the production and use of lipase G may be regarded as safe for the enzyme production worker and the consumer.

Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I.

Landry TD, Chew L, Davis JW, Frawley N, Foley HH, Stelman SJ, Thomas J, Wolt J, Hanselman DS. Regul Toxicol Pharmacol. 2003 Feb;37(1):149-68. PMID: 12662916 

BD5088 alpha-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications. The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans. Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids. Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain. A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion. There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests. There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database. The enzyme was labile to pepsin digestion. Based on these data, BD5088 alpha-amylase preparation may be considered safe for use in food production such as corn wet milling. Copyright 2003 Elsevier Science (USA)

Safety evaluation of tannase enzyme preparation derived from Aspergillus oryzae

Lane RW, Yamakoshi J, Kikuchi M, Mizusawa K, Henderson L, Smith M. Food Chem Toxicol. 1997 Feb;35(2):207-12. PMID: 9146733.

Tannase is an acylhydrolase enzyme preparation from Aspergillus oryzae that can be used as a processing aid for the manufacture of cold water-soluble tea beverages. A 91-day oral toxicity study in the rat and a gene mutation study in Salmonella typhimurium were performed to establish the safety of the enzyme preparation for the consumer. General toxicity was low, with no adverse effects observed at the highest dose tested, 1% in the diet. There was no evidence of mutagenic potential with or without metabolic activation. These results, together with knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, indicate that tannase can be regarded as safe for its intended use in processing tea.

Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil
Ciofalo V, Barton N, Kreps J, Coats I, Shanahan D. Regul Toxicol Pharmacol. 2006 Jun;45(1):1-8. Epub 2006 Mar 24. PMID: 16563586
Safety evaluation of lipase produced from Rhizopus oryzae: summary of toxicological data.
Flood MT, Kondo M. Regul Toxicol Pharmacol. 2003 Apr;37(2):293-304.
PMID: 12726758

Safety evaluation of lipase produced from Candida rugosa: summary of toxicological data.

Flood MT, Kondo M. Regul Toxicol Pharmacol. 2001 Apr;33(2):157-64.
PMID: 11350198

Safety evaluation of a phytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed.

Ciofalo V, Barton N, Kretz K, Baird J, Cook M, Shanahan D. Regul Toxicol Pharmacol. 2003 Apr;37(2):286-92. PMID: 12726757

BD006 phytase is the product of the Escherichia coli B app A gene expressed in Schizosaccharomyces pombe strain ASP595-1. This enzyme preparation is intended for use in animal feed applications where it improves the bioavailability of phosphate for monogastric animals. BD006 phytase was tested as an unrefined (DV006U) and a refined (DV006R) preparation for its effects on genotoxicity and in acute, inhalation and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to a high of 2000X for oral in vivo toxicity studies. The highest oral dose tested (2000X) is 2000 times the highest expected consumption of product by poultry or swine (X=50 units of phytase per kg bwt/day). There was no genotoxicity or any in vivo toxicity reported which could be directly related to systemic effects of the product. Other additional safety studies conducted were devoid of any relevant toxicity. The historic safety profile of the production organism S. pombe, and the results of the toxicity studies presented herein, attest to the safety of BD006 phytase for use in animal feed applications.

Safety evaluation of a glucanase preparation intended for use in food including a subchronic study in rats and mutagenicity studies.

Elvig SG, Pedersen PB. Regul Toxicol Pharmacol. 2003 Feb;37(1):11-9. PMID: 12662905

An enzyme preparation containing glucanase produced by the fungus Trichoderma harzianum is intended to be used to improve the clarification and filtration of wines. The safety of a glucanase preparation was assessed in a series of toxicological tests to document its safety in use. Oral administration to rats of up to 10 mL/kg bw/day (equivalent to a total organic solids dosage of 1258 mg/kg bw/day or a glucanase dosage of 1882 BGXU/kg bw/day) for 13 weeks did not cause any adverse effect. The test substance was not found to be mutagenic in the bacterial reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes. The safety margin for exposure is estimated to be in the range of 2100-72,000 depending on the estimate for daily wine consumption. The results of these studies demonstrate that an enzyme preparation containing glucanase may be considered safe when employed in wine processing. Copyright 2003 Elsevier Science (USA)

Safety evaluation of a hexose oxidase expressed in Hansenula polymorpha.

Cook MW, Thygesen HV. Food Chem Toxicol. 2003 Apr;41(4):523-9. PMID: 12615123

A programme of studies was conducted to establish the safety of hexose oxidase (HOX) from Chondrus crispus expressed in the yeast Hansenula polymorpha to be used as a processing aid in the food industry. Rat feeding studies were conducted to assess acute and subchronic oral toxicity. In addition, the potential of the enzyme to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. Acute and subchronic oral toxicity was not detected at the highest dosage recommended by OECD guidelines. There was no evidence of mutagenic potential or chromosomal aberrations. The no-observed-adverse-effect level (NOAEL) derived from the 13-week study was 5000 units/kg body weight/day. In conclusion it can be considered a safe processing aid for use in the food industry. Copyright 2003 Elsevier Science Ltd.

Safety evaluation of a lipase expressed in Aspergillus oryzae

Greenough RJ, Perry CJ, Stavnsbjerg M. Food Chem Toxicol. 1996 Feb;34(2):161-6. PMID: 8606032

A programme of studies was conducted to establish the safety of a lipase artificially expressed in Aspergillus oryzae to be used in the detergent industry and as a processing aid in the baking industry. Laboratory animal studies were used to assess general and inhalation toxicity, skin sensitization, and skin and eye irritation. Its potential to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. The pathogenicity of A. oryzae, the organism used to produce the lipase, was also assessed in laboratory animals. Basic ecotoxicity in a variety of test species was studied. General and inhalation toxicity was low. There was evidence of mild skin irritation. There was no evidence of eye irritation, skin sensitization, mutagenic potential, chromosomal aberrations, exotoxicity or notable pathogenicity. Comparison of these results with human exposure levels and previously published data indicates that the lipase appears safe for consumers in the given applications, requires no special occupational health precautions in manufacture and is of low environmental impact. Furthermore, the organism used in production of the lipase hs no notable pathogenicity.




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