Oral therapy with proteolytic enzymes decreases excessive TGF-beta levels in human blood.

UNLABELLED: 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.

Stimulation of reactive oxygen species production and cytotoxicity in human neutrophils in vitro and after oral administration of a polyenzyme preparation.

Polymorphonuclear neutrophils (PMN) can be primed for enhanced release of reactive oxygen species (ROS) by exposure to cytokines and biological response modifiers. ROS are considered to possess tumoricidal activity. The polyenzyme preparation Wobenzym (WE) contains pancreatin, papain, bromelain trypsin and chymotrypsin and is used in adjuvant tumor therapy. We investigated killing of WE-exposed PMN against tumor cells and analyzed WE influence on ROS production in a chemiluminescence assay in PMN in vitro and in vivo. Depending on dose WE stimulates the cytotoxic capacity of PMN in vitro against tumor cells (50 micrograms/ml:p < 0.01). Exposure of PMN to Wobenzym caused a time-dependent significant (p < 0.02) increase in release of ROS. Similarly, oral administration of Wobenzym to healthy volunteers (n = 28) resulted in significant increases (p < 0.01) in ROS production, depending on dose (peak with 20 tablets) and time (peak 4 hours after Wobenzym administration). In contrast, ROS production was not elevated in the PMN of healthy volunteers receiving placebo (n = 8) or no treatment (n = 16). These findings point to an immunomodulatory capacity of WE in adjuvant tumor therapy.

Proteolytic enzymes and amylase induce cytokine production in human peripheral blood mononuclear cells in vitro.

In vitro treatment of human peripheral blood mononuclear cells (PBMNC) with proteolytic enzymes (bromelain, papain) and amylase leads to the production of large amounts of tumor necrosis factor-alpha (TNF-alpha), interleukin-1-beta (IL-1 beta), and interleukin-6 (IL-6) in a time and dose dependent manner. Increased TNF-alpha and IL-6 production was already found after 4-6 hours of incubation, and plateau levels were reached after 12-16 hours. Plateau levels up to 1500 pg TNF-alpha/ml/10(6) PBMNC, 13000 pg IL-1 beta/ml/10(6) PBMNC, and 23000 pg IL-6/ml/10(6) PBMNC were observed. Control cultures contained below 35 pg/ml/10(6) PBMNC of TNF-alpha, IL-1 beta or IL-6. In contrast to TNF-alpha which was undetectable after more than 24 hours, peak levels of IL-1 beta and IL-6 were still present at 24 hours. After incubation of the enzyme solution for some hours at 56 degrees C the cytokine inducing capacity disappeared. Neutralization experiments with inactivating antibodies, radioimmunoassay, and western blotting after electrophoretic separation showed that the TNF-like activity found in the lytic assay was due to TNF-alpha. Interferon-alpha (IFN-alpha) and Interferon-gamma (IFN-gamma), which had no effect alone, synergistically increased TNF-alpha production when applied together with the enzymes. A commercial mixture of these enzymes (Wobenzym), which was also investigated, showed a similar concentration and time dependence, as well as synergism with the interferons. A synergistic effect on TNF-alpha production was also found with the enzymes and phorbol ester (PMA).

Cytokine synthesis in human peripheral blood mononuclear cells after oral administration of polyenzyme preparations.

Pharmaceutical preparations containing mixtures of various proteolytic and nonproteolytic enzymes have been suggested for use in the treatment of malignant diseases. However, the mode of action of such preparations was not clear. We have shown before that intact bromelain, papain or amylase, which are components of a commercial polyenzyme preparation, induce cytokine production in peripheral blood mononuclear cells in vitro. IFN-alpha and IFN-gamma which had no effect alone, synergistically increased TNF production when applied together with the enzymes. Here we show that trypsin alone had only a small inducing effect. The tryptic but not the autolytic fragments of papain and bromelain have a higher (10- to 40-fold) inducing capacity for TNF production than the untreated enzyme. Additionally we demonstrate that after ingestion of milligram doses of the polyenzyme preparation (as recommended for clinical use), PBMNC of healthy donors acquire the ability to produce TNF-alpha, IL-1 beta and IL-6 when incubated ex vivo with IFN-gamma. Our results indicate that the biological effects observed after oral administration of polyenzyme preparations are related to their ability to induce cytokine production. This may explain the antitumor effects of such enzymes. Our results also suggest that polyenzyme preparations may have a stronger immunomodulary effect when used in combination with IFN-gamma.

Induction of tumor necrosis factor in human peripheral-blood mononuclear cells by proteolytic enzymes.

We could demonstrate that polyenzyme preparations as well as bromelain and papain stimulate the production of tumor necrosis factor-alpha in human peripheral-blood mononuclear cell cultures in a time-dependent manner. We give evidence that immunomodulation and especially the release of cytokines may contribute to the therapeutic effect of these preparations.