S-1 and oxaliplatin versus tegafur-uracil and leucovorin as post-operative adjuvant chemotherapy in patients with high-risk stage III colon cancer: updated 5-year survival of the phase III ACTS-CC 02 trial.

BACKGROUND: The ACTS-CC 02 trial demonstrated that S-1 plus oxaliplatin (SOX) was not superior to tegafur-uracil and leucovorin (UFT/LV) in terms of disease-free survival (DFS) as adjuvant chemotherapy for high-risk stage III colon cancer (any T, N2, or positive nodes around the origin of the feeding arteries). We now report the final overall survival (OS) and subgroup analysis according to the pathological stage (TNM 7th edition) for treatment efficacy. PATIENTS AND METHODS: Patients who underwent curative resection for pathologically confirmed high-risk stage III colon cancer were randomly assigned to receive either UFT/LV (300 mg/m2 of UFT and 75 mg/day of LV on days 1-28, every 35 days, five cycles) or SOX (100 mg/m2 of oxaliplatin on day 1 and 80 mg/m2/day of S-1 on days 1-14, every 21 days, eight cycles). The primary endpoint was DFS and the patients' data were updated in February 2020. RESULTS: A total of 478 patients in the UFT/LV group and 477 patients in the SOX group were included in the final analysis. With a median follow-up time of 74.3 months, the 5-year DFS rate was 55.2% in the UFT/LV group and 58.1% in the SOX group [stratified hazard ratio (HR) 0.92; 95% confidence interval (CI) 0.76-1.11; P = 0.3973], and the 5-year OS rates were 78.3% and 79.1%, respectively (stratified HR 0.97; 95% CI 0.76-1.24; P = 0.8175). In the subgroup analysis, the 5-year OS rates in patients with T4N2b disease were 51.0% and 64.1% in the UFT/LV and SOX groups, respectively (HR 0.72; 95% CI 0.40-1.31). CONCLUSION: Our final analysis reconfirmed that SOX as adjuvant chemotherapy is not superior to UFT/LV in terms of DFS in patients with high-risk stage III colon cancer. The 5-year OS rate was similar in the UFT/LV and SOX groups.

Quality evaluation of health foods containing licorice in the Japanese Market.

Focusing on licorice, a highly used raw material in health foods, quantitative analysis of functional/medicinal components and a safety and functional evaluation was carried out for herbal medicines, health food ingredients, and so-called health foods. A functional component, glabridin, was detected in herbal medicines from Glycyrrhiza glabra and G. inflata, health food ingredients, and in commercially available health foods that contain licorice. Likewise, glycyrrhizin, a medicinal component, was detected in these sources, except in licorice oil extract. Estrogen activity in vitro was detected in some of the herbal medicines, health food ingredients, and in health foods containing licorice. In the in vivo study, liver weight in ovariectomized (OVX) mice treated with licorice oil extract was significantly higher than that in OVX and sham mice in a dose dependent manner. These results suggest that excessive intake of licorice oil extract from health foods should be avoided, even though these ingredients might be beneficial for medical use in order to maintain bone health in postmenopausal women. Measurement of hepatic cytochrome P-450 (CYP) activity, reproductive organ weight, and fat and bone mass in OVX mice was considered useful for evaluating the safety and efficacy of estrogenic health food ingredients derived from herbal medicines.

Influence of Ankle Braces on the Maximum Strength of Plantar and Toe Flexor Muscles.

This study sought to investigate the influence of ankle braces on maximum strength of plantar and toe flexor muscles. 21 healthy participants volunteered, and their maximum isometric toe flexor muscle strength (TFS), plantar flexor muscle strength (PFS) and passive range of motion of the ankle joint (ROM) were measured. TFS, PFS and ROM were compared among barefoot as a control (BAR), ankle support (SUP) and ankle splint (SPL) conditions. TFS was significantly lower in SPL (90.7±32.5 N, p<0.0001) compared to BAR (117.3±39.5 N) and it was significantly lower in SPL than in SUP (110.5±36.7 N, p=0.0001), whereas it was not significantly different between SUP and BAR (p=0.2587). On the contrary, PFS and ROM were significantly lower in SUP (p=0.0087 for PFS; p=0.0095 for ROM) and SPL (p<0.0001 for PFS; p<0.0001 for ROM) compared to BAR, and they were significantly lower in SPL than in SUP (p=0.0073 for PFS; p<0.0001 for ROM). The ankle support could provide ankle joint stabilization without a large decrease in the muscle strength of the foot. The proper use of an ankle brace is required to prevent major impairment of the muscle functions of the ankle-foot complex.

Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

OBJECTIVES: Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). MATERIALS AND METHODS: We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. RESULTS: We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. CONCLUSIONS: Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks.

(-)-Epigallocatechin gallate inhibits basic fibroblast growth factor-stimulated interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (FGF-2) activates the mitogen-activated protein (MAP) kinase superfamily in osteoblast-like MC3T3-E1 cells and that p38 MAP kinase functions as a positive regulator in the FGF-2-stimulated synthesis of interleukin-6 (IL-6), a potent bone-resorptive agent, in these cells. In the present study, we investigated the exact mechanism of IL-6 and the effects of (-)-epi-gallocatechin gallate (EGCG), one of the major green tea flavonoids, on the synthesis of IL-6. PD98059, an inhibitor of MEK, but not SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase, suppressed FGF-2-stimulated IL-6 synthesis. EGCG significantly reduced the IL-6 synthesis stimulated by FGF-2 in a dose-dependent manner. EGCG attenuated the FGF-2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. These results strongly suggest that EGCG inhibits the FGF-2-stimulated synthesis of IL-6 at least partly via suppression of the p44/p42 MAP kinase pathway and the p38 MAP kinase pathway in osteoblasts.

Alpha1-adrenoceptors are required for normal male sexual function.

BACKGROUND AND PURPOSE: Alpha(1)-adrenoceptor antagonists are extensively used in the treatment of hypertension and lower urinary tract symptoms associated with benign prostatic hyperplasia. Among the side effects, ejaculatory dysfunction occurs more frequently with drugs that are relatively selective for alpha(1A)-adrenoceptors compared with other drugs of this class. This suggests that alpha(1A)-adrenoceptors may contribute to ejaculation. However, this has not been studied at the molecular level. EXPERIMENTAL APPROACH: The physiological contribution of each alpha(1)-adrenoceptor subtype was characterized using alpha(1)-adrenoceptor subtype-selective knockout (KO) mice (alpha(1A)-, alpha(1B)- and alpha(1D)-AR KO mice) since the subtype-specific drugs available are only moderately selective. We analysed the role of alpha(1)-adrenoceptors in the blood pressure and vascular response as well as ejaculation by determining these variables in alpha(1)-adrenoceptor subtype-selective KO mice and in mice with all their alpha(1)-adrenoceptor subtypes deleted (alpha(1)-AR triple-KO mice). KEY RESULTS: The pregnancy rate was reduced by 50% in alpha(1A)-adrenoceptor KO mice, and this reduction was dramatically enhanced in alpha(1)-adrenoceptor triple-KO mice. Contractile tension of the vas deferens in response to noradrenaline was markedly decreased in alpha(1A)-adrenoceptor KO mice, and this contraction was completely abolished in alpha(1)-adrenoceptor triple-KO mice. This attenuation of contractility was also observed in the electrically stimulated vas deferens. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that alpha(1)-adrenoceptors, particularly alpha(1A)-adrenoceptors, are required for normal contractility of the vas deferens and consequent sperm ejaculation as well as having a function in fertility.

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.

Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription.

BACKGROUND: TFIIH is one of the general transcription factors required for accurate transcription of protein-coding genes by RNA polymerase II. TFIIH has helicase and kinase activities, plays a role in promoter opening and promoter escape, and is also implicated in efficient activator-dependent transcription. RESULTS: We have established a reconstitution system of recombinant TFIIH using a three-virus baculovirus expression system. The recombinant TFIIH was active in CTD kinase and DNA helicase assays, and showed both basal and activator-dependent transcriptional activities that were indistinguishable from those of HeLa cell-derived TFIIH. Further analyses using recombinant TFIIH confirmed a critical role of TFIIH in activator-dependent transcription. The dose response of TFIIH in activator-dependent transcription suggested that mere recruitment of TFIIH is not sufficient for transcriptional activation. The sensitivity of activator-dependent transcription to nonhydrolysable ATP analogues indicated the importance of the enzymatic activities of TFIIH in transcriptional activation. CONCLUSIONS: Our results raise a possibility that transcriptional activation by GAL4-VP16 requires enzymatic activities. Recombinant TFIIH reconstituted from this baculovirus system should be useful for analysis of the mechanisms of activation by GAL4-VP16.

Early microcirculatory derangement in mild and severe pancreatitis models in mice.

An in vivo microscopic technique was used to clarify the increase in microvascular permeability and enhanced leukocyte-endothelium interaction of pancreatic microcirculation in experimental pancreatitis of differing severity. Using bovine albumin fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDASE) as tracers, the change in permeability and the behavior of leukocytes in the acinar microcirculation were quantified during the initial 1, 2, 6, and 12h after the induction of caerulein pancreatitis in mice. Cold stress was added to produce the severe model. It was revealed that the early microcirculatory changes in the pancreas of caerulein pancreatitis included the increased permeability of endothelial lining and an accumulation of extravasated fluid in the perilobular space, which were more severe if cold stress was added. A decrease in flow velocity was also noted 2h after the onset of severe pancreatitis. Leukocyte adherence to the endothelial cells was not observed during the first 12h in either model of severity. In contrast, observation of the hepatic microcirculation revealed a significant number of adherent leukocytes 2h after the induction of severe pancreatitis. These results suggest that during the early course of acute pancreatitis, leukocyte adherence in the pancreatic microcirculation is a secondary event following the increase in pancreatic vascular permeability.

In vivo evaluation of the early events associated with liver metastasis of circulating cancer cells.

The mechanism of metastasis formation remains still largely unknown. Many studies underline the importance and complexity of the initial arrest of the circulating tumour cells in the target organ, a key stage in metastasis occurrence. In our study, we evaluated by visual means the metastasis formation using an in vivo microscopy system in a murine model. Moreover, we investigated the involvement of P-selectin in these processes using immunohistochemistry and P-selectin knockout mice. The present study offers direct evidence of distinct pathways for tumour metastasis formation by a lymphoma cell - EL-4 and a solid tumour cell - C26. Off-line analysis of the images and histological data confirmed that mechanical entrapment of the solid tumour cell, which had a bigger diameter than that of the liver sinusoids, promoted metastasis without any detectable involvement of adhesion molecules. On the other hand, we observed that lymphoma cells, in spite of their smaller diameter as compared to the sinusoids, promoted liver metastasis as well, but with the essential participation in their arrest of P-selectin, indicating an adhesion molecule-mediated pathway.

Tocopherol-associated protein is a ligand-dependent transcriptional activator.

Vitamin E is a term that encompasses a group of potent, lipid-soluble, chain-breaking antioxidants. Structural analysis reveals that molecules having vitamin E activity include four isomers (alpha, beta, gamma, and delta) of both tocopherols and tocotrienols. Alpha-tocopherol has been shown to have the highest biological vitamin E activity in mammalian tissues based on fetal resorption assays, and it reverses vitamin E deficiency symptoms. Although the molecular functions fulfilled specifically by alpha-tocopherol have yet to be fully described, it is unlikely that they are limited to general antioxidant functions. Here we show the functional characterization of alpha-tocopherol associated protein, TAP, which displays significant sequence similarity to the alpha-tocopherol transfer protein. Ligand competition analysis showed that recombinant TAP binds to alpha-tocopherol but not to other isomers of tocopherols. Using GFP fusion protein expression system, we observed that TAP translocates from cytosol to nuclei in alpha-tocopherol-dependent fashion. Transient transfection experiment showed that TAP activates transcription of the reporter gene in alpha-tocopherol-dependent manner. These results suggest that the biological function of alpha-tocopherol is not only as an antioxidant but also as a transcriptional regulator of gene expression via association with a transcription factor TAP.

Beta2-adrenergic receptor/cyclic adenosine monophosphate (cAMP) leads to JNK activation through Rho family small GTPases.

Gi- and Gq-coupled G protein-coupled receptors (GPCRs) have been shown to activate c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (MAPKs), through Rho family small GTPases in mammalian cells. We investigated the signaling pathway linking the Gs-coupled beta2-adrenergic receptor with JNK, using smooth muscle DDT1 MF-2 cells, which natively express the beta2-adrenergic receptor. Stimulation of the beta2-adrenergic receptor activated JNK in a time-dependent manner, and a cell-permeable cyclic adenosine monophosphate analogue (8-Br-cAMP) activated JNK. The beta2-adrenergic receptor- or 8-Br-cAMP-induced activation of JNK required Rho family small GTPases. Also, the beta2-adrenergic receptor or 8-Br-cAMP induced activation of Rho family small GTPases. These results demonstrate that the beta2-adrenergic receptor/cAMP leads to JNK activation through Rho family small GTPases in DDT1 MF-2 cells. Activation of Rho family small GTPases may provide a common step in GPCR-mediated JNK activation.

Revascularization and microcirculation of freely grafted islets of Langerhans.

A considerable number of experimental studies have demonstrated that the reestablishment of an appropriate microvascular supply is an essential prerequisite for successful pancreatic islet transplantation. Freely transplanted islets show the first signs of angiogenesis (i.e., capillary sprout formation and protrusion) as early as 2 days after transplantation, and the entire vascularization process is completed after 10 to 14 days. Cryopreservation and culture of the isolated islets before transplantation and hyperglycemia of the transplant recipient seem not to affect the vascularization process essentially. In addition, immunosuppressive drugs, such as cyclosporin A and 15-deoxyspergualin, do not or only slightly inhibit revascularization of syngeneic islets; however, they are not able to prevent completely xenograft-induced microvascular perfusion failure. In contrast, novel immunosuppressants (e.g., RS-61443) or dietary supplementation of the antioxidant vitamin E were shown to prevent microvascular graft rejection almost completely, including leukocyte recruitment and capillary perfusion failure. Thus the development of novel strategies to improve posttransplant islet function should include concepts that accelerate the vascularization process and protect the newly formed microvasculature from rejection-mediated injury. The improvement of islet graft vascularization and the maintenance of adequate microvascular perfusion will contribute to the increased success of pancreatic islet transplantation.

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis.

TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases. Although it has been intensively analyzed in vitro and in Saccharomyces cerevisiae, in vivo studies of vertebrate TBP have been limited. We applied gene-targeting techniques using chicken DT40 cells to generate heterozygous cells with one copy of the TBP gene disrupted. Such TBP-heterozygous (TBP-Het) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G2/-M- than G1-phase cells, and a high proportion of sub-G1, presumably apoptotic, cells. Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs. To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G2/M transition, and unexpectedly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells. Providing an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly and specifically reduced. These properties were all due to decreased TBP levels, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant form lacking the species-specific N-terminal domain. Our results indicate that small changes in TBP concentration can have profound effects on cell growth in vertebrate cells.

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation.

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.

Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading.

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.

[The false positive reaction of the Triage panel drug-of-abuse by herbal drugs ma-huang (Ephedra sinica (Ephedraceae))].

We investigated false-positive reactions obtained from a drug screening test using a Triage panel. We detected 2 cases giving false-positive reaction for AMP (amphetamine, methamphetamine) during the screening of 187 normal subjects. Subsequent follow up testing by high-performance liquid chromatography (HPLC), showed both to be false-positive reactions. As both cases have a history of ingesting the herbal drug, Ma-huang (Ephedra sinica (Ephedraceae)), containing ephedrine, we examined the relationship between false-positive reactions on Triage and Ma-huang. All urine samples collected from 7 healthy volunteers following administration of Ma-huang indicated AMP positive on Triage. Also a high ratio of AMP positives was observed in the patients who were administered Ma-huang-containing drugs at the hospital. However, none of them were identified as true-positives by HPLC or gas chromatography mass spectrometry (GC/MS) analysis. The extract of Ma-huang contained in herbal drugs, which otherwise contain neither amphetamine nor its derivatives, gives (AMP) positive indications on Triage. We speculate that unidentified components of Ma-huang cause the false-positive reactions. We suggest that follow-up tests by GC/MS or HPLC are needed wherever a positive result is obtained from a screening test by Triage. Furthermore, it will be established to continue collecting information on prescribed and non-prescribed drugs.

Continuous regional application of protease inhibitor in the treatment of acute pancreatitis. An experimental study using closed duodenal obstruction model in dogs.

In clinical settings, the effectiveness of protease inhibitors in the treatment of acute pancreatitis has been still controversial. With the concept that sufficient tissue concentration of protease inhibitor in the pancreas has to be included to achieve its potent inhibitory effect, we applied a continuous regional intra-arterial (CRI) application of low-molecular-weight protease inhibitor, nafamostat mesilate (FUT-175), for closed duodenal loop obstruction model in mongrel dogs. The use of CRI application led to a higher concentration of FUT-175 in the pancreatic tissue (4,453 +/- 758 ng/g) when compared with that applied intravenously (905 +/- 48 ng/g). Consequently, pancreatic parenchyma in CRI application animals was remarkably preserved, as assessed by the lower extent of pancreatic necrosis (12.4 +/- 2.6% in CRI vs. 25.6 +/- 1.9% in intravenous). Additionally, the elevation of trypsin-like activity in the pancreas was significantly inhibited in CRI animals. Based on these findings, the dose as well as the route of protease inhibitors should be carefully considered to achieve its beneficial effect.