Effect of the synthetic protease inhibitor [N,N-dimethylcarbamoyl-methyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate on carcinogenesis by 3-methylcholanthrene in mouse skin.

The effect of the potent synthetic protease inhibitor [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate (FOY-305) on skin carcinogenesis in ddY mice was examined over a total observation period of 105 days. Administration of 0.1% FOY-305 in the diet suppressed the incidence of carcinomas induced by repeated local application of the carcinogen 3-methylcholanthrene (MCA) (P less than .05) in mouse skin and delayed the time of appearance of the skin tumors (P less than .001). There was no significant difference in the number of tumors per tumor-bearing mouse and the size of the tumors between mice treated with MCA and mice treated with MCA plus FOY-305.

Potent growth-suppressive activity of a serine protease inhibitor, ONO-3403, toward malignant human neuroblastoma cell lines.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 and FO-349 are its derivatives. The effects of these compounds on the proliferation of 13 human neuroblastoma cell lines were investigated in vitro by MTT colorimetric assay. The half maximum inhibition concentrations of ONO-3403 varied between 22 and 90 microg/ml while those of FOY-305 and FO-349 were higher than 100 microg/ml. ONO-3403 showed higher growth-inhibitory activity for N-myc-amplified neuroblastomas as compared with that for non-amplified cells. Since N-myc amplification in neuroblastomas is well correlated with a poor prognosis, ONO-3403 could be an effective anticancer drug for malignant neuroblastomas.

Enhancement of anti-proliferative activities of 5-FU, HCFU, SN-38, THP and ACNU by a serine protease inhibitor, FOY-305.

The effects of a synthetic serine protease inhibitor, FOY-305, on the proliferation of normal and transformed mouse fibroblasts were investigated in vitro by MTT colorimetric assay. FOY-305 inhibited the growth of normal NIH3T3 cells and their src- and erbB2-transformed cells with a half maximum inhibitory concentration (IC50) of 1-1.2 mg/ml whereas it suppressed the growth of ras-transformed cells more effectively (IC50 was 0.5-0.6 mg/ml). Flow cytometric analysis using synchronized NIH3T3 cells has shown that the growth-inhibitory activity of FOY-305 was due to the suppression of G(1)/S transition. The synergistic effects between FOY-305 and traditional anticancer drugs were also investigated by the MTT assay and the results showed that FOY-305 significantly enhanced the antiproliferative activities of 5-fluorouracil (5-FU), 1-hexylcarbamoyl-5-fluorouracil (HCFU), 7-ethyl-1-hydroxy-7-ethyl-10-[4-(1-piperidino)-1-piperidino] camptothecin (SN-38), pirarubicin (THP) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU).

Growth-suppressive activities of serine protease inhibitors, FOY-305, ONO-3403 and FO-349 toward human carcinoma cells.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 and FO-349 are its derivatives. The effects of these compounds on the proliferation of several human carcinoma cell lines were investigated in vitro by MTT colorimetric assay. ONO-3403 showed the most potent growth-inhibitory activity among these protease inhibitors. The half maximum inhibition concentrations of ONO-3403 toward BxPC-3 pancreatic carcinoma, T24 bladder carcinoma and A431 epidermoid carcinoma cells were 20-30 mu g/ml whereas those toward pancreatic carcinomas, PANC-1 and Mia PaCa-2, were 60-80 mu g/ml. Since FOY-305 has been shown to be effective in chemotherapy for human oral cancer, ONO-3403 is expected to be a more effective anticancer drug.

Growth-suppressive activities of serine protease inhibitors, ONO-3403 and ONO-5046, toward normal and transformed murine fibroblasts.

ONO-3403 and ONO-5046 are potent synthetic protease inhibitors of trypsin and elastase, respectively. These compounds suppressed the proliferation of polyoma virus- and Kirsten sarcoma virus-transformed BALB/c 3T3 cells more effectively than their normal counterparts. SV40-transformed 3Y1 and v-Ha-ras-transformed NIH3T3 cells were also more sensitive to ONO-3403 and ONO-5046 than the parent normal cells. These results suggest that ONO-3403 and ONO-5046 are useful for selective suppression of the proliferation of rapidly growing transformed cells.

Growth inhibition of mouse skin tumor by serine protease inhibitor ONO-3403.

The new serine protease inhibitor, ONO-3403 is an analog of FOY-305 (Foypan). The IC50s values of ONO-3403 toward serine proteases, such as trypsin, plasmin, kallikrein and thrombin are much lower than that of FOY-305. To investigate the growth-suppressing effect of ONO-3403 on 3-methylcholanthrene-induced autochthonous solid tumors in mice, ONO-3403 was intraperitoneally administered to mice at a dose of 4 mg/kg twice a day for 5 weeks. All seven mice receiving the drug had a solitary tumor and showed potent growth suppression (p<0.001) without any apparent side effects such as hair loss and body weight loss. The results suggest that ONO-3403 may be useful for the treatment of squamous cell carcinoma.

Effects of serine protease inhibitor FOY-305 and heparin on the growth of squamous cell carcinoma.

Effects of serine proteinase inhibitor FOY-305 and heparin on autochthonous skin carcinoma were examined using ddY female mice with single tumor induced by methylcholanthrene. FOY-305 given intraperitoneally for 9 consecutive weeks inhibited the growth of the pre-existing dermal tumors. Heparin alone was equally therapeutically effective. However, heparin induced severe mucocutaneous bleeding of the gastrointestinal mucosa. In contrast, the combined administration of FOY-305 and heparin suppressed tumor growth without any visible side effects. There was a significant increase in well differentiated cancer cells in mice treated with FOY-305 and heparin in combination, suggesting that co-administration of FOY-305 and heparin may be useful for the treatment of squamous cell carcinoma.

Anti-invasive activity of synthetic serine protease inhibitors and its combined effect with a matrix metalloproteinase inhibitor.

Tumor invasion into the extracellular matrix (ECM) and basement membrane (BM) is a crucial step of tumor metastasis. In order to investigate the possible therapeutic procedure for the tumor invasion, we investigated the anti-invasive activities of several synthetic serine protease inhibitors. FOY-305, a serine protease inhibitor, showed no cytotoxic activity against human HT-1080 fibrosarcoma cells at concentrations ranging from 0.1 to 100 micrograms/ml, while its analogs ONO-3403 and FO-349 showed slight cytotoxic activities at the concentration of 100 micrograms/ml. These compounds inhibited the activity of urokinase-type plasminogen activator (u-PA) which is one of serine proteases and considered to be associated with tumor invasion and metastasis in fibrin zymography. FOY-305 more potently inhibited the invasion of HT-1080 cells into the reconstituted BM Matrigel, as well inhibited u-PA activity, compared with ONO-3403 and FO-349. These results suggest that the anti-invasive activity of these compounds is consistent with their anti-fibrinolytic activities. In addition, the combined treatment of FOY-305 with FC-336 processing anti-invasive and anti-MMP properties resulted in marked enhancement of anti-invasive activity. In conclusion, FOY-305 inhibited the invasion of tumor cells through interference with the u-PA activity of tumor cells, and this inhibitory activity was augmented by the combination with a MMP inhibitor.

The effect of cryotherapy on intraarticular temperature and postoperative care after anterior cruciate ligament reconstruction.

The objective of this study was to elucidate how cryotherapy after anterior cruciate ligament reconstruction affects intraarticular temperature and clinical results. A prospective and randomized study was performed on 21 knees of 21 patients. The ligament reconstruction was performed by single-incision arthroscopy using autogenous hamstring tendon. On completion of the surgery, thermosensors were implanted in the suprapatellar pouch and the intracondylar notch, and the intraarticular temperature was monitored while the joint was cooled. Cooling was performed in one group at 5 degrees C (N = 7) and in another at 10 degrees C (N = 7), for 48 hours. A control group (N = 7) did not undergo cryotherapy. The cooled groups showed three temperature phases: a low-temperature phase immediately after the ligament reconstruction, followed by a temperature-rising phase and a thermostatic phase. The control group had no low-temperature phase and immediately entered a thermostatic phase. During the low-temperature phase in the treated groups, the temperature of the suprapatellar pouch and of the intercondylar notch were significantly lower than the body temperature. The pain score and the number of times an analgesic had to be administered were both significantly lower in the 10 degrees C group than in the control group. Blood loss was significantly less in the 5 degrees C group than in the control group.

Uveitopathogenic sites in recoverin.

PURPOSE: This study was carried out in order to determine the most potent and novel uveitopathogenic sites of recoverin using synthetic peptides. METHODS: Several synthetic peptides containing the recoverin sequence plus adjuvants were injected into Lewis rats, and the uveitopathogenic sequence was defined, clinically, histologically, and immunologically. RESULTS: Peptides containing of amino acids 57-85 and 136-167 induced severe EAU, and the lowest doses to induce EAU were 20 microg and 10 microg, respectively. Lymphocyte proliferative reactions were also positive for peptides 57-85 and 136-167. The core sequences within the uveitopathogenic site were 65-79 and 153-164. Peptides of amino acids 65-79 within 57-85 and 149-167 within 136-167 were the smallest in the recoverin sequence, respectively, that could induce severe EAU. CONCLUSION: We found recoverin has some novel potent uveitopathogenic sites, 149-167. These findings of the uveitopathogenic sites in recoverin may lead to improved understanding of the pathogenesis of uveitis and the means to design specific treatment.

Analysis of uveitogenic sites in phosducin molecule.

PURPOSE: Phosducin, a retinal photoreceptor protein, induces experimental autoimmune uveitis (EAU). In this study, we attempted to determine the numbers of uveitogenic sites in phosducin using synthetic peptides. METHODS: Antigen peptides were synthesized according to the amino acid sequence of the rat-derived phosducin with a peptide-synthesizer and purified by reversed-phase HPLC. First, 13 peptides covering the entire sequence of phosducin were synthesized, and each was injected into the hind footpad of Lewis rats for immunization, and induction of EAU was examined clinically and histologically. Next, peptides that appeared to contain sequences of a uveitogenic site were newly synthesized and examined clinically and immunologically. RESULTS: Of the 13 peptides used in the first immunization, 7 induced inflammation. Similar to other EAU antigens, clinical changes began with fibrin deposition in the anterior segment and posterior synechia, followed by posterior chamber hypopyon. Histologically, inflammation was observed mainly in the outer segment of photoreceptor cells and outer nuclear layer, and serous retinal detachment was found in cases of severe inflammation. Infiltration of inflammatory cells in the pineal gland was also observed. In experiments designed to further specify the uveitogenic sites, the presence of inflammation-inducing sequences was inferred for amino acid sequences 1-20, 23-37, 79-91, 127-142 and 198-212. The rats immunized with these peptides also exhibited high value on lymphocyte proliferation assay. CONCLUSION: Phosducin has 5 uveitogenic sites. Among others, one of them has potent and others weak uveitogenicity.

Suppression of experimental autoimmune uveoretinitis by dietary calorie restriction.

To investigate the inhibitory effect of dietary calorie restriction on experimental autoimmune uveoretinitis (EAU) in rats, and its mechanism. Lewis rats were maintained on a 50% calorie-restricted diet for 2 months or 6 months. The control group was maintained on a 90% ad libitum intake for the same length of time. Experimental autoimmune uveoretinitis was elicited in both groups by immunization with an inter-photoreceptor retinoid-binding protein or its peptide. Rats in both groups were examined clinically, histopathologically, and immunologically. The severity of EAU was milder in the restricted diet group than in the control group. In EAU rats, production of interferon-gamma (IFN-gamma) in eyes and of IFN-gamma and tumor necrosis factor-alpha in draining lymph node cells was significantly lower in the restricted diet group than in the control group. Our results indicate that a calorie-restricted diet suppresses the development of EAU. The suppressed Th1-dependent immunological response is one of the reasons for the mildness of EAU in the calorie-restricted diet group of rats.

Uveitopathogenic site of the gamma-subunit of cyclic guanosine monophosphate phosphodiesterase in Lewis rats.

PURPOSE: The gamma-subunit of cyclic guanosine monophosphate phosphodiesterase (PDEgamma) plays an important role in the phototransduction process of rod photoreceptors. A previous report indicated that experimental autoimmune uveoretinitis (EAU) could be induced in Lewis rats by immunization with PDEgamma. In this study, we identified the uveitopathogenic site of PDEgamma synthetic peptides and identified pivotal amino acid residues using analogue peptides. METHODS: Several synthetic peptides derived from PDEgamma plus adjuvants were injected in Lewis rats. The induction of EAU was examined clinically and histologically. In addition, humoral and cellular immunity against peptides was investigated. RESULTS: The smallest uveitopathogenic peptide was identified as PDEgamma 64-76 (ITVICPWEAFNHL), which consists of 13 amino acid residues, and the core sequence was identified as PDEgamma 70-76 (WEAFNHL), which consists of 7 amino acid residues. The lowest dose of peptide to induce EAU was 0.03 nmol. The pivotal amino acid residues for eliciting EAU are at 70(W), 71(E), 73(F), and 75(H). CONCLUSION: Our findings demonstrated the presence of a potent uveitopathogenic site in PDEgamma whose potency in Lewis rats was comparable to that of interphotoreceptor retinoid-binding protein.

[Review [new antibiotics series III]: micronomicin (author's transl)].

Micronomicin is a new aminoglycosidic antibiotic discovered and developed by Kyowa Hakko Kogyo Co., Ltd. It is produced by Micromonospora sagamiensis var. nonreducans. Investigation of micronomicin performed in 134 research facilities in Japan led to the following results. 1) Micronomicin showed a broad antibacterial spectrum against Gram positive and Gram negative bacteria. 2) In susceptibility tests of clinical isolates, micronomicin was almost similarly active to GM. 3) Bactericidal activity of micronomicin against Pseudomonas aeruginosa and E. coli was higher than those of TOB and DKB. 4) Micronomicin showed a synergistic antibacterial activity against Pseudomonas aeruginosa and E. coli with CBPC and SBPC. 5) The therapeutic activity of micronomicin in mice infected with Pseudomonas aeruginosa and Serratia sp. was in high correlation with in vitro antibacterial activity similarly to that of GM. 6) Micronomicin was confirmed to be stable against aminoglycoside 6'-acetyltransferase of Pseudomonas aeruginosa and to be not inactivated. 7) Pharmacokinetics of micronomicin was almost similar to those of GM with respect to the concentrations in the serum, urine and tissues. 8) Ototoxicity of micronomicin in guinea pigs was found to be approximately four times less than that of GM. 9) Nephrotoxicity of micronomicin in rabbits was estimated to be less than those of GM and DKB. In rats, nephrotoxicity of micronomicin was approximately 4 times less than that of GM. 10) Micronomicin was effective on 964 cases out of 1,469 cases from 127 research facilities in Japan (65.6%), suggesting its favorable activity against respiratory tract infections and against urinary tract infections. 11) Side effects with the drug were observed in 43 cases out of 1,532 cases (2.81%). Abnormalities in laboratory findings were also recognized, but transient without severe cases. 12) In conclusion, micronomicin is a favorable drug having lesser ototoxicity and nephrotoxicity as well as antibacterial and bactericidal activity of aminoglycosidic antibiotics usually used.

[The problem in the incubation of injective solutions. The difference between ampule and vial].

Variations in the actual doses with vials and ampules due to causes of dosage forms and human operations have been discussed. The differences between the labeled doses and actually administered doses with ampules and vials have been studied. The comparison of resulting blood levels revealed peaks of 4.88 +/- 1.08 micrograms/ml and 3.85 +/- 0.71 micrograms/ml actually determined with ampule preparations and vial preparations, respectively, showing some appreciable differences. These values were analyzed with compartment models. Cmax values were 5.13 micrograms/ml and 3.94 micrograms/ml, respectively, showing significant differences (P less than 0.05) between the ampule and vial preparations. However, AUC and Tmax values were equal to each other, so that it was assumed that there would be no problem about the similarity of the 2 types of dosage forms. As to the differences due to human operations, the nurse A did normally collect only 83.0% (75.2 mg) volume of the labeled doses of vials, and, even when she did it with greater care, she collected still 90.0% (81.5 mg) of the labeled doses. On the other hand, the nurse B normally collected 89.8% (81.4 mg) of the labeled dose of vials, and when she used greater care, she collected 92.4% (83.7 mg) of the labeled doses. In the group of ampule preparations, the nurses A and B collected 93.1% (94.8 mg) and 98.1% (99.9 mg), respectively. It was beyond the amount expected in advance for the actually collected dose from ampules. The differences in the collected doses between ampules and vials were within the expected range because ampule preparations usually contain approximate 10% overage, but as to the differences added to this difference due to the human operations, the nearly twice as much speed for collecting the filled preparation by the nurse A would not have been denied for the smaller doses collected on the basis of the above-mentioned results. It was noted therefore that care should be taken in collecting the filled doses from containers into injection syringes.

[Antibacterial activities of new cephems against clinically isolated organisms (author's transl)].

The antibacterial activities of cefotaxime (CTX), cefoperazone (CPZ), ceftizoxime (CZX), cefmenoxime (CMX), latamoxef (LMOX), cefotiam (CTM), cefazolin (CEZ), gentamicin (GM) and cefsulodin (CFS) were investigated. All causative organisms were isolated from patients with urinary tract infections treated in Tokai University Hospital. The results were as follows. 1) The MICs of CMX, CTX, and CZX against most of clinically isolated strains of E. coli, K. pneumoniae, Indole (-) Proteus sp. were 0.1 microgram/ml and lower. And then CTM, LMOX and CPZ showed similar antibacterial activities. 2) LMOX and GM showed potent antibacterial activities against C. freundii which was considered to be causative organisms of infections in rare cases. 3) Against S. marcescens, CMX, CTX, CZX, and LMOX showed very potent antibacterial activities. 4) Against P. aeruginosa, CFS, GM and CPZ showed moderate antibacterial activities. 5) Against Enterobacter sp., GM and CMX showed potent antibacterial activities.

[Clinical experiment of gentamicin in patients with urological diseases (author's transl)].

1. Gentamicin was effective in 20 patients out of 25 with urological diseases. 2. Gentamicin was more effective on acute symptoms than on chronicones. 3. No marked side effects were noted. 4. No conclusion was drawn on difference in efficacy of gentamicin by dosage, duration of administration and kinds of organisms in this clinical trial.

[Clinical study on intravenous infusion of micronomicin].

In recent years, aminoglycoside agents as well as beta-lactam antibiotics have been increasingly used with increased incidence of opportunistic infection caused mainly by Gram-negative bacteria. Therefore, we administered micronomicin sulfate (MCR), reportedly lower in nephrotoxicity, at doses of 60 and 120 mg by intravenous drip infusion for 1 and 2 hours to healthy male volunteers and determined the blood level and the urinary recovery rate. The peak of blood level after 1 hour infusion of MCR was 7.3 micrograms/ml in the 60 mg group and 9.5 micrograms/ml in the 120 mg group. T 1/2 (beta) was 3.34 and 2.48 hours respectively. The peak of blood level after 2 hours infusion of MCR was 5.7 micrograms/ml in the 60 mg group and 8.7 micrograms/ml in the 120 mg group. T 1/2 (beta) was 3.36 and 3.71 hours respectively. In the 120 mg group, the urinary recovery rate for the first 24 hours was 53.5% after 1 hour infusion and 60.9% after 2 hours infusion. In the 60 mg group, the rate was higher, 90.1 and 98.6% respectively. It was suggested that intravenous drip infusion of 120 mg of MCR for 1 hour is comparable to intramuscular injection of the same dose. Further, safety and effectiveness of this drug were studied in 7 clinical cases of urological infection. Good results were obtained in 7 clinical cases given 60 or 120 mg of MCR by intravenous drip infusion. Neither side effects nor abnormal laboratory findings were observed in clinical cases.

[Studies on tobramycin by the intravenous drip infusion method].

Fundamental and clinical efficacy evaluation has been made in the study on the therapy with intravenous drip infusion of tobramycin (TOB) which is one of the aminoglycoside antibiotics and which has been recently approved of intravenous administration. The fundamental evaluations were made with intravenous drip infusion of 60 mg of TOB for 1 hour and that of 120 mg of TOB for 2 hours to 4 healthy adult volunteers on crossover method. Follow-up determinations were made for comparison on its concentrations in blood and urine samples which were collected both at intervals thereafter, using 2 assay methods of the bioassay and the EMIT methods. The maximum blood level of TOB based on the intravenous drip infusion of 60 mg for 1 hour was 5.9 micrograms/ml and 4.6 micrograms/ml by the bioassay and the EMIT methods at the end of intravenous drip infusion, respectively, and 0.3 micrograms/ml and 0.4 micrograms/ml, respectively, at 6 hours after the drip infusion. The T 1/2 value was 1.81 hours with bioassay and 2.41 hours with the EMIT method. The maximum blood level of TOB through drip infusion method of 120 mg of TOB for 2 hours was 7.5 micrograms/ml by bioassay and 5.8 micrograms/ml by EMIT method. The T 1/2 value was 1.87 hours and 1.99 hours, respectively. The recovery rate of TOB from urine until 24 hours after the administration was 83.0% and 94.4% with the doses of 60 mg and 120 mg, respectively, which were determined only with the bioassay. The correlation coefficient between the agar well method and the EMIT method was 0.963 and 0.972 in the groups of intravenous drip infusion of 60 mg for 1 hour and 120 mg for 2 hours, respectively, showing proximity to each other. TOB was kept administered with a daily dose of 120 mg at a time to 11 cases and at 2 times to 1 case with chronic complicated infections of urinary tracts (comprising 9 cases with chronic complicated cystitis, 2 with chronic complicated pyelonephritis, and 1 with acute epididymitis). Of the 12 patients, 8 could meet the criteria for evaluation of drug efficacy and 6 of them proved to be effective, while 2 were ineffective. The efficacy rate was so high as 75%. The rate of disappearance of bacteria was 80.0%, which was very high. No side effect was specifically found out, except 1 case showing slight rise in S-GOT.

[Well-controlled comparative study on aztreonam and cefoperazone in the treatment of complicated urinary tract infections].

A well-controlled comparative study was conducted in order to evaluate the efficacy, safety and usefulness of the monobactam antibiotic aztreonam (AZT) in complicated urinary tract infections (UTI) using cefoperazone (CPZ) as the control drug. Patients with complicated UTI due to Gram-negative bacteria were examined. However, in polymicrobial infections, the cases caused by Gram-negative and Gram-positive bacteria were also examined. Both drugs were administered 1 g, twice a day by intravenous drip infusion for 5 days. Overall clinical efficacy was evaluated by the criteria proposed by the UTI committee in Japan. Of the total 394 cases, the clinical efficacy was evaluated for 152 cases in the AZT group and 143 cases in the CPZ group excluding 99 cases of exclusion or dropout. There was no statistically significant difference in the back ground characteristics between the 2 groups. The overall clinical efficacy rate was 55.3% for AZT and 55.2% for CPZ with difference not significant. As for clinical efficacy, in the monomicrobial infection after postprostatectomy (G-2), AZT was superior to CPZ (P less than 0.05), whereas in the polymicrobial infection without indwelling catheter (G-6), CPZ was superior to AZT (P less than 0.1). The overall bacteriological eradication rate was 77.2% for AZT and 74.5% for CPZ. For Gram-negative bacteria only the eradication rate for AZT (83.2%) was superior to that for CPZ (74.6%). This was probably due to the stability of AZT to beta-lactamase. Side effects were observed in 3 cases out of 201 in the AZT group and 5 cases out of 189 in the CPZ group. No severe abnormal laboratory finding was observed in any group; there was no significant difference between the 2 groups. Consequently, AZT was judged to be an antibiotic with clinical usefulness equal to, or even superior to that of CPZ.