Acetylspiramycin (ASPM) was fractionated using high performance liquid chromatography (HPLC). The peak fractions were named F1 to F7 successively in order of increasing retention times (Rt), i.e., increasing hydrophobicity, and studied for 1) antibacterial activities (MIC), 2) antibacterial potency against Bacillus subtilis ATCC 6633, 3) therapeutic effect on mice infected with Streptococcus pneumoniae III, Staphylococcus aureus SMITH, 4) acute toxicity by i.p. administration to mice (LD₅₀) and 5) cytotoxicities to fibroblasts derived from Chinese-hamster lung (CHL), cow pulmonary artery endothelial cells (CPAE) and rat hepatic cells.
The results obtained are summarized below.
1. Components F1 and 4″-acetylspiramycin F2 had significantly different biological activities from those of other components: F1 showed the lowest antibacterial potency of 492 μg (potency) /mg, F2 showed the highest antibacterial potency of 2,040 μg (potency) /mg and correspondingly the lowest LD₅₀ value of 692 mg/kg (the highest toxicity). The therapeutic effect of F2 on infections in mice was found to be the second smallest and was superior only to that of F1. The LD₅₀ value of Fl was 1,200 mg/kg and similar to that of ASPM.
2. Antibacterial potencies of F3, F4, F5 and F6 were 1,165, 1,266, 1,374 and 1,530 itg (potency) /mg, respectively; fraction with the higher antibacterial activities corresponded to the longer retention times, i.e., the greater hydrophobicities. The most hydrophobic component, F7, 3-propiony1-3″, 4″-diacetylspiramycin, however, showed a low antibacterial potency of 1,085 μg (potency) /mg, next to the lowest one, F1, a fact which was in contradiction to with the sequential relation between hydrophobicities and potencies from F3 to F6. The structure of F7 was also distinctively different from the compound with the highest antibacterial potency, F2, in which OH at position 3 was unsubstituted and only OH at position 4 was acetylated. The LD₅₀ values of F3, F4 and F5 were within a narrow range from 943 to 949 mg/kg, and those of F6 and F7 were higher than 1,430 mg/kg, indicating a low toxicity. 3. A sequential relationship existed between the cytotoxicities and hydrophobicities of there components: The in vitro cytotoxicity increased with the increase of hydrophobicity but the in vivo toxicity (LD₅₀) tended to decrease with increase of hydrophobicity. Further details of the different tox cities observed in vivo and in vitro remain to be elucidated by future pharmacodynamic studies.

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We analyzed antibiotic susceptibilities of urinary isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Indole (+) Proteus group to ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ) and gentamicin (GM) in 69 laboratories in 1988 and also studied changing patterns of susceptibilities from 1980 to 1988. Minimal inhibitory concentrations were determined using the agar dilution method (MUELLER-HINTON agar, BBL) with inoculation of 10⁶ cfu/ml of bacteria.
Ninety to 99% of the strains of E. coli, Klebsiella spp. and P. mirabilis were inhibited at a concentration of 6.25 μg/ml of CEZ and CMZ and of 1.56 μg/ml of GM. Approximately 80% of the strains of Indole (+) Proteus group were inhibited at concentrations of 6.25 μg/ml of CMZ and of 1.56 μg/ml of GM. However, resistance to ABPC and CEZ was high, with 83% and 81% of the strains being not inhibited at a concentration of 50 μg/ml of ABPC and CEZ, respectively.
No significant changes in susceptibilities of the 4 bacteria to the above 4 antibiotics were observed over the 9 year period. No increase was found in the incidence of the resistant strains of the 4 bacteria to CMZ and GM, nor of E. coli and Klebsiella spp. to CEZ.

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With a new simple sample pretreatment, microbiological assay methods for quantitative determination of BMY-28100 and some other cephem antibiotics in serum and tissues have been developed.
Presented sample pretreatment was founded on the extraction with EVANS' blue, a high serumprotein binding dye, and the ultrafiltration using cellulose membrane (Millipore; MW cutoff is 10,000) to which EVANS' blue was specifically absorbed. Regardless of sample media, antibiotic contents in filtrates were ditermined accurately using calibration curves based on standard solutions prepared with 1/15 Mphosphate buffer, pH 7.4.
Four cephems, ceftriaxone (CTRX), cefazolin (CEZ), BMY-28100 and cefadroxil (CDX) were examined in human serum and rat liver tissues. Corrections were made for antibiotic recoveries into 1/15 M phosphate buffer, pH 7.4. CTRX, CEZ, BMY-28100 and CDX in serum and tissues were recovered into buffer at rates of 98.4-101.6%, 97.6-102.0%, 99.8-101.4% and 97.5-98.2% of spiked (theoretical) values, respectively regardless of antibiotic concentrations. Furthermore, these concentrations obtained using the present bioassay method were well in agreement with those obtained by the HPLC method.
When BMY-28100 was administered to volunteers, concentrations of BMY-28100 in serum obtained by the present bioassay method were well in agreement with those obtained by the usual bioassay method which required the fresh human serum to prepare the standard solutions.

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Antimicrobial activities of minocycline (MINO) against various clinical isolates, 270 strains obtained in 1988, were determined and the reliability of the MINO disc susceptibility test in estimating approximate values of MICs was studied. Clinical significance of a 4 category system for the interpretation of the disc tests, which is widely used in Japan, and that of a 3 category system used in the USA and Europe, were also evaluated to determine which system would be more suitable for the evaluation of proper dose levels of administration.
In this study, MICs were determined using the agar dilution method at an inoculum level of 10⁶ CFU/ml. MIC₈₀ values of MINO against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae were all S 0.78 gg/ml. Those against Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Proteus vulgaris were 0.39, 6.25, 3.13, 25, 50 μg/ml, respectively. MIC₈₀ values against Pseudomonas aeruginosa, Serratia marcescens, Enterobacter spp., and Citrobacter spp. were 50,100, 50 and 12.5 μg/ml, respectively. The inhibition zones obtained with the discmethod were compared with MICs.The results of MINO disc susceptibility test either with 200μg disc (Showa) or30μg disc (prepared in this laboratory) were well correlated with MICs, showing the reliability of the disc method in estimating approximate values of MICs.
In the 4 category classification system currently used, break points in MIC values proposed are (+++) MIC<2μg/ml,(++) MIC>2-10μg/ml,(+) >10-50μg/ml,(-) MIC>50μg/ml.The results obtained with 200μg and 30μg discs showed false positive in26.6%and20.5% of the samples, and false negative in5.8%and23.6% of the samples, respectively.The disc results of s.aureus, S.epidermidis, S.pneumoniae, etc.were relatively well classified, but those of E.coli K.pneunoniae, Proteus spp.were not, showing more false positive results.
Changing the lower 2MIC break points in the 4category system to: (+++) MIC<μg/ml and (++) MIC>3-15μg/ml, the false positive results with both200μg and 30μg discs were reduced tol2%and 6.2%, respectively.The false negative results were5.8%and23.6%, respectively.
Since British Society for Antimicrobial Chemotherapy has recommended in vitro MIC break point of tetracycline to be1μg/ml, a study was carried out to evaluate whether or not MIC break point of1μg/ml could be identified with inhibitory zone diameters in 200μg and30μg disc tests.It was possible to identify the break point of MIC1μg/ml on inhibitory zone diameters with both200μg and 30μg discs with an accuracy over90%.
In the 3 category classification system, the MIC break points are defined sensitive (S) for MIC<4 μg/ml and resistant (R) for MIC>16μg/ml.In this study, 200μg and 30μg disc test results showed false positive in9.7%and8.5% of the samples tested and false negative in5.0%and6.9% of the samples, respectively.
A pharmacokinetic examination with the recommended dose schedule for MINO (200mg loading dose, there after 100mg a day or 100 mg twice a day orally) showed that plasma levels of MINO reach2-4μg/ml.MINO levels in sputum and tear averaged0.83-0.93μg/mland 0.16-0.2μg/ml, respectively.Thesse levels are far above reported MIC values against Chlamnydia spp.and Mycoplasma spp.The concentration of MINO in spinal fluid was 20 to 30% of plasma levels.MINO was excreted into the bile at a concentration 20-30times higher than that in plasma.The excretion into urine was about8-12% of the administered amounts.
Antimicrobial activities of antibiotic agents, particularlyβ-lactams, have been reported to be reduced by serum protein binding.

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The reliability of the piperacillin (PIPC) disc susceptibility test in estimating approximate values of MICs was studied with various clinical isolates totaling 284 strains using Showa discs and the discs prepared in this laboratory (both 8 mm diameter containing 30 μg of PIPC).
Clinical significance of a 4 category system for the interpretation of the PIPC disc tests, which iswidely used in Japan, was reevaluated to determine whether this system would be suitable or not for the evaluation of proper dose levels of administration. Break points in MIC values proposed for the classification of bacteria into 4 categories of susceptibility were: (+++) MIC< 3, μg/ml,(++) MIC > 3-15 μg/ml,(+) MIC>15-60μg/ml,(-) MIC>60μg/ml. A 3 category system for interpreting of disctest, which is generally used in the USA and Europe, was also evaluated. MIC break points in the 3 category system proposed for the classification of the PIPC test are sensitive (S) MIC < 64 μg/ml and resistant (R) MIC >256, μg/ml. In addition, British Society for Antimicrobial Chemotherpy recently proposed in vitro MIC break points for PIPC in therapeutic use, recommending MICs 16 and 64 μg/ml.MIC 16 μg/ml can be used for general infections treated with usual administrative doses for such infections and MIC 64 μg/ml may apply to increased dosage or to normal dosage when PIPC is locally concentrated (mainly urinary or biliary tract infections).
The results obtained with the disc method were compared with MICs determined using the agar dilution method at an inoculum level of 10⁶ CFU/ml. The results of the PIPC disc susceptibility test either with Showa discs or discs prepared in this laboratory were well correlated with MICs, showing the reliability of the disc method in estimating approximate values of MICs.
In the 4 category classification system of the Showa disc test, 33 out of 284 strains (11.6%) tested showed false positive results and 5 strains (1.8%) false negative results. Similarly, in the test of 30 μg discs prepared in this laboratory, of 284 strains 26 (9.2%) showed false positive results and 8 (2.8%) false negative results.
In the tests of Showa discs and discs prepared in this laboratory, both containing 30 μg of PIPC, no inhibitory zones were observed against the strains with MIC > 100, ug/ml. Therefore, using these 30 μg discs it is impossible to classify bacteria into 3 categories of (S) MIC < 64 ug/ml,(I) MIC > 64-<256μg/ml, and (R) MIC > 256μg/ml.
However, it is possible to classify bacteria into 3 categories using the criteria proposed by the British Society for Antimicrobial Chemotherapy. In this system, false positive and false negative results in the Showa disc test were 21 out of 284 strains (7.4%) and 3 strains (1.1%), respectively. Those in the tests with discs prepared in this laboratory were 18 out of 284 strains (6.3%) and 4 strains (1.4%), respectively.
Antimicrobial activities of PIPC against various clinical isolates obtained in 1988 were as follows: Staphylococcus aureus and Staphylococcus epidermidis inhibited by PIPC at 12.5 μg/ml were 43.3 and 72.4% of the strains tested, respectively and Enterococcus faecalis thus inhibited was 93.3%. Similarly, Escherichia coli, Klebsiella pneumoniae, indole negative and indole positive Proteus spp. inhibited at 12.5μg/ml were 71, 90,100 and 100% of the strains, respectively. Those of Pseudomonas aeruginosa, Serratia spp., Enterobacter spp. and Citrobacter spp. were 70, 81.3, 65.5 and 68.8% of the strains, respectively. At a dose level of 50 μg/ml of PIPC, 73.3-100% of various clinical isolates were inhibited except S. aureus of which only 43.3% was inhibited.

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A preliminary study was carried out to see whether or not the synergistic enhancement of antimicrobial activity of piperacillin (PIPC) and amikacin (AMK) in combinations against Pseudomonasaeruginosa can be evaluated using combination discs of these antibiotics. A synergistic antimicrobial effect was observed between these antibiotics, when evaluated using FIC index, against piperacillinsensitive strains with MIC <12.5 μg/ml under the conditions of the present investigation using the agar dilution method. However, no synergy was found between these antibiotics against piperacillin-resistant strains with MIC <25 μg/ml, even when a higher concentrations of the drugs were used.
Using strains of P. aeruginosa against which the synergistic effect between these antibiotics was observed, it was found that inhibitory diameters obtained with combination discs of PIPC/AMK at concentrations of 30 μg/30 μg or 30 μg/15 μg were more than 3 mm larger than those obtained with discs containing either PIPC or AMK alone. Using strains against which no synergistic effects were observed, however, the above mentioned enlargement of inhibitory zone diameters with combination discs were not clearly found. Further studies are deemed necessary to determine whether or not the combination discs of PIPC and AMK are useful in routine clinical work.

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A study was done on the combined actions of an aminoglycoside, isepamicin (ISP), and 3 β-lactam antibiotics (cefoperazone (CPZ), latamoxef (LMOX) and imipenem/cilastatin sodium (IPM/CS)) against clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae.Minimal inhibitory concentrations of individual antibiotics were compared first. ISP and IPM/CS had strong antibacterial activities against all 3 bacterial species while the antibacterial activities of CPZ againstP. aeruginosa and S. marcescens, and that of LMOX against P. aeruginosa were much weaker than those of IPM/CS or ISP. Fractional inhibitory concentration indices determined by the checker-board dilution method were compared next. ISP, when used in combination with β-lactam antibiotics (CPZ, LMOX, or IPM/CS), showed synergistic or additive effect on most strains of the all 3 species, the combination of ISP and CPZ being most effective. Although less effective, synergistic or additive effects were also observed with the combinations of 2 β-lactam antibiotics (CPZ and IPM/CS, LMOX and IPM/CS). Time course experiments demonstrated that ISP combined with CPZ had bactericidal activities against all 3 bacterial species at concentrations at which the respective drug alone showed only bacteriostatic activity.

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Effects of cefminox (CMNX) on hemostasis and blood coagulation system were studied. Adult in-patients admitted to 237 centers (310 clinics) nationwide in Japan during the period from April 1988 to March 1989 were followed up using a newly designed uniformed protocol. Case cards recovered were inspected by an evaluation committee and patients to be included in analysis were determined according to the protocol. Presence or absence of abnormalities in the hemostasis and blood coagulation system was examined objectively using criteria for evaluation prepared by the committee.
Out of 1,374 patients included in analysis, 10 patients were judged as having abnormalities which were suspected to have causal relationships with CMNX. Prolongation in prothrombin time was observed in 4 cases (0.29%), prolongation in activated partial thromboplastin time in 4 cases (0.29%), and decrease in fibrinogen in 2 cases (0.15%). Decrease in platelet count was not detected in any of the cases. Cross-sectional analysis according to background factors in these 10 cases revealed that abnormalities of the hemostasis and blood coagulation systems were significantly higher (P < 0.01) for the group positive for underlying disease or complications (“positive” group) than the “negative” group. Five out of 9 patients of the positive group had malignant neoplasm. Other than this factor, no items showed statistically significant differences.
From these results it is considered that the administration of CMNX is nearly free of effects on the hemostasis and blood coagulation system and development of labolatory abnomalities is chiefly due to the patients' condition.

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We have carried our laboratory and clinical studies on cefodizime (CDZM, THR-221).
The results were summarized as follows.
CDZM was given by 30-minute drip infusion to 2 children at a single dose of 10 mg/kg and to 2 children at a single dose of 20 mg/kg and to 3 children at a single dose of 40 mg/kg. After the 30-minute drip infusion, mean serum levels of CDZM obtained for the 3 dose levels were 76.16 ± 5.52 μg/ml, 170.49 ± 16.70 μg/ml, 270.01 ±50.44 μg/ml at the end of injection, respectivery, and serum half-lives were 2.03 ± 0.78 hours, 2.03 ± 0.38 hours, 2.28 ±0.30 hours, respectively.
The mean urinary excretion rate of CDZM were 83.3 ±22.3%, 73.1 ±13.9%, 51.1±8.5% in the first 8 hours after the 30-minute drip infusion of 10 mg/kg, 20 mg/kg, 40 mg/kg, respectively.
Treatment with CDZM was made in 28 cases of pediatric bacterial infections; 5 cases of tonsillitis, 2 cases of bronchitis, 10 cases of pneumonia, 6 cases of enteritis, 3 cases of urinary tract infection and 1 case each of maxillary sinusitis and laryngitis. Results obtained were excellent in 13 cases, good in 7 cases, fair in 2 cases, poor in 6 cases.
No significant side effect due to the drug was observed except one case of thrombocytosis and 2 cases each of elevated GOT and elevated GOT and GPT.

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Clinical effects of a combinaion therapy using aspoxicillin (ASPC) and ceftazidime (CAZ) were investigated in 88 patients with severe infections which were complicating hematological disorders. ASPC and CAZ were administered intravenously at daily doses of 8 g and 4 to 6 g, respectively, in 2 to 4 divided doses for at least 3 days.
The treatment was markedly effective in 20 cases; effective in 31; fairly effective in 4; and ineffective in 33 cases. Seventy-seven patients with whom detailed data were obtained showed an efficacy rate of 63.6%.
Bacteria were detected in 9 patients, from whom 10 strains were isolated. The results of bacteriological effects were: 3 strains disappeared, 1 decreased, 3 unchanged, and 3 unclear. The bacteriological eradication rate was 42.9%. Of the detected 10 strains, 3 were identified as Pseudomonas aeruginosa, with 2 of the 3 strains eradicated and 1 decreased.
An evaluation of the relationship between clinical efficacies and neutrophil counts before and after the ASPC-CAZ combination therapy showed that the patients with 500/mm³ or higher neutrophil counts before the therapy or those with increased neutrophils after the therapy tended to be more responsive to the therapy.
Side effects were observed in 4 patients, but all of them disappeared upon discontinuation of the therapy.
The combination therapy with ASPC and CAZ appears to be useful for the treatment of severe
infections complicating hematological disorders.

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We conducted a clinical investigation on the use of S6472, an sustained release preparation of cefaclor, in the dermatological area.
Thirty-two patients serving as subjects were divided into group I (14 cases, including folliculitis and acne pustulora), group II (16 cases, including furuncle, furunculosis and carbuncle) and others (2 cases).One or 2 wrappers (375 or 750 mg) of S6472 were orally administered to patients twice daily after meals.
In group I, the efficacy rate was 100%. Clinical results were as follows: Excellent (6 cases), good (8 cases), fair (0), and poor (0). In group II, the efficacy rate was 93.8%: Excellent (10 cases), good (5cases), fair (1 case), and poor (0).
A decrease of the bacteriological effect was observed in 1 case with Staphylococcus epidermidis. A superinfection was noted in 1 case Staphylococcus aureus. The causative bacteria were all eradicated in the remaining 24 cases. No side effects were detected.
From the above results, S6472 appears to be a useful preparation, twice daily oral administration has excellent effects against dermatological infections.

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Pharmacokinetical, bacteriological and clinical studies on norfloxacin (NFLX), a quinolonecarboxylic acid antibacterial agent, were conducted in the pediatric field.
1. Serum concentrations and urinary excretion of NFLX after single dose of 2.2-5.6 mg/kg (mean 4.4±1.2 mg/kg) were determined in 13 children with ages between 6 and 11 years.
The mean peak serum concentration of the drug was 0.37 ±0.20 itg/ml at 2 hours after administration. The mean half-life of the drug in serum was 2.8 ±0.4 hours and the serum concentration at 8 hours was 0.11 ± 0.06 μg/ml.
The mean urinary concentration reached a maximum of 125.2 ± 166.2 μg/ml in pooled urine from 0to 2 hours and the mean urinary recovery rate in the first 8 hours after administration was 22.1±6.0%.
A dose-response relationship was observed between doses/body weight and peak serum concentrations.
2. The clinical efficacy, bacteriological efficacy and the safety of NFLX were evaluated in 65pediatric patients with ages between 2 years 10 months and 15 years 7 months with infections. In 62assessable cases (acute purulent tonsillitis 9 cases, acute pneumonia 3 cases, cronic rhinitis 1 case, urinary tract infections 15 cases, and acute colitis 34 cases), clinical efficacies were excellent in 48 cases, good in 13 casses. and fair in 1 case with an overall efficacy rate of 98.4%.
Staphylococcus aureus 1 strain, Staphylococcus epidermidis 1 strain, Escherichia coli 10 strains, Salmonella sp. 5 strains, Morganella morganii 1 strain, Pseudomonas aeruginosa 3 strains, Haemophilus parainfluenzae 1 strain and Campylobacter jejuni 12 strains were isolated from the patients as pathogens.Bacteriologically, all of these strains were eradicated except that 3 strains of C. jejuni only decreased.
With regard to side effects, dizziness and nausea were observed in 1 case each but they were slight and the continuation of the treatment was possible. No abnormal laboratory test data were observed.
From the above results, NFLX was considered to be a useful drug for the treatment of pediatric infections.

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To assess the usefulness of single dose treatment with isepamicin (ISP) against chronic complicated urinary tract infection (CC-UTI), laboratory and clinical studies were carried out. The results are summarised as follows.
1. Serum concentrations
Serum concentrations of the drug were assayed for patients after the administration of ISP at a doseof 400 mg by intravenous drip infusion for 30 or 60 minutes. Patients tested consisted of 3 groups with different degrees of renal functions (Ccr: ml/min); 1) normal, 130.8, 2) slightly impaired, 70.8, 3) moderately impaired, 45.9. When the peak/trough concentrations in the 3 groups were compared on 1st - 2nd day/5th-6th day of the administration, no significantly different values were recognized among the 3 groups in the peak/trough concentrations.
2. Clinical efficacy
Twenty three patients including 18 (78%) patients over 61 years of age were treated at a dose of 400 mg once a day for 4-10 days. Of 21 evaluable cases, 15 (71%) were evaluated as excellent or moderate according to the Japanese UTI criteria. Against the clinical isolates, 21 (68%) out of 31 strains were eradicated after the treatment.
3. Safety
Neither subjective side reactions nor clinical abnormal values were encountered throughout the treatment.
4. Conclusions
The clinical effectiveness of daily single dose treatment with ISP (400 mg once a day, i.v.d.) was considered to be almost comparable to the treatment with 200 mg twice a day. Furthermore, since no problems on safety were noted even in treating aged patients with slight to moderate renal impairments, the drug appears to be evaluated as highly useful.

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