New neplanocin analogues. 1. Synthesis of 6'-modified neplanocin A derivatives as broad-spectrum antiviral agents.

Novel neplanocin A analogues modified at the 6'-position, i.e., 6'-deoxy analogues (2, 3, 6, 9, 20), 6'-O-methylneplanocin A (15), and 6'-C-methylneplanocin A's (22a and 22b) have been synthesized and evaluated for their antiviral activity in a wide variety of DNA and RNA virus systems. These compounds showed an activity spectrum that conforms to that of S-adenosylhomocysteine hydrolase inhibitors. They were particularly active against pox- (vaccinia), paramyxo-(parainfluenza, measles, respiratory syncytial), arena- (Junin, Tacaribe), rhabdo- (vesicular stomatitis), reo-, and cytomegalovirus. In order of (increasing) antiviral activity, the compounds ranked as follows: 3 less than 15 approximately 20 less than 6 less than 9 approximately 2 less than 22a. Of the two diastereomeric forms of 22, only 22a was active; 22a surpassed neplanocin A both in antiviral potency and selectivity. Compound 22a appears to be a promising candidate drug for the treatment of pox-, paramyxo-, arena-, rhabdo-, reo-, and cytomegalovirus infections.

AS-924, a novel, orally active, bifunctional prodrug of ceftizoxime: physicochemical properties, oral absorption in animals, and antibacterial activity.

AS-924 is an oral prodrug of the antibiotic ceftizoxime (CTIZ), a parenteral use cephalosporin. This novel prodrug, produced by esterifying CTIZ with a lipophilic pivaloyloxymethyl (POM) group and introducing a water soluble L-alanyl group, is expected to increase the bioavailability and thereby, augment the antibacterial activity of CTIZ in vivo compared with existing prodrugs. To study the effect of the L-alanyl group in AS-924 on its bioavailability, the plasma concentration profiles of CTIZ in dogs were examined following the dosing of AS-924 and CTIZ-POM, in powder form, after pretreatment with the antacid ranitidine, and following the dosing of AS-924 after pretreatment with a gastrointestinal motility stimulant metoclopramide or suppressant scopolamine butylbromide. The absorption rate of AS-924 was constant under these different conditions due to its unique balance of lipophilicity and water solubility. CTIZ is as antibacterially active as pre-existing oral cephalosporins against Gram-positive clinical isolates, while being more active against all Gram-negative isolates-particularly Enterobacteriaceae and Haemophilus influenzae. A simulation model for the eradication profile of bacteria in computer programmed pharmacokinetic (PK) system was carried out to study the antibacterial action of CTIZ in human. CTIZ was proven to eradicate Streptococcus pneumoniae and H. influenzae effectively, while cefpodoxime (CPOD), the active moiety of CPOD proxetil, eradicated S. pneumoniae, but not H. influenzae. These results confirm that, AS-924 is a potent oral antibiotic and would be expected to be clinically effective and efficient.

The acetylation of 6'-amino group of amikacin by a new enzyme prepared from serratia sp.

It was found that Serratia marcescens 43, Serratia proteamaculans 48 and Serratia sp. 45, all of which were clinically isolated, produced a new type of aminoglycoside acetyltransferase which acetylated amikacin at the 6'-amino group. 1-N-[(S)-3-Amino-2-hydroxypropionyl]-gentamicin B (HAPA-B, SCH 21420) and gentamicin C2 were hardly inactivated by the enzymes and had effective antimicrobial activities against these strains both in vitro and in vivo. This kind of aminoglycoside acetyltransferase should be classified into a new group other than previously reported AAC(6') enzymes.

[Effects of isepamicin and beta-lactam antibiotics on the release of endotoxin from Pseudomonas aeruginosa].

Antibiotic-induced release of endotoxin (lipopolysaccharide, LPS) from Pseudomonas aeruginosa was investigated in in vitro with different antibiotic concentrations and in the pharmacokinetic autosimulation system. We compared the effect of isepamicin (ISP) with those of 3 beta-lactam antibiotics, piperacillin (PIPC), ceftazidime (CAZ) and imipenem (IPM). ISP showed a strong bactericidal activity, but the amount of free LPS did not increase by 6 hrs (28 +/- 2 ng/ml at 1MIC). PIPC and CAZ caused a gradual killing and a large amount of LPS release at 4 hrs (515 ng/ml and 493 ng/ml, respectively, at 1 MIC). At 1/4 x MIC, PIPC and CAZ did not reduce colony forming counts and induced more release of free LPS. The organism treated with IPM released less LPS, while it was killed rapidly. The viable cell counts decreased dramatically after administration of ISP in the pharmacokinetic autosimulation system. ISP inhibited the bacterial regrowth and the following release of free LPS by 8 hrs. Great amounts of free LPS were released 4 hrs after the administration of PIPC and CAZ in the simulation system, associated with morphological changes; elongation, cell lysis or regrowth. IPM showed a strong bactericidal activity and less liberation of free LPS, but the free LPS level increased at 8 hrs, accompanied by the regrowth of the organism. The total amounts of LPS released by P. aeruginosa PAO1 in 8 hours of the pharmacokinetic simulation system were as follows; ISP < IPM < CAZ < PIPC.

Long-term infusion of brain-derived neurotrophic factor reduces food intake and body weight via a corticotrophin-releasing hormone pathway in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) has been implicated in learning, depression and energy metabolism. However, the neuronal mechanisms underlying the effects of BDNF on energy metabolism remain unclear. The present study aimed to elucidate the neuronal pathways by which BDNF controls feeding behaviour and energy balance. Using an osmotic mini-pump, BDNF or control artificial cerebrospinal fluid was infused i.c.v. at the lateral ventricle or into the paraventricular nucleus of the hypothalamus (PVN) for 12 days. Intracerebroventricular BDNF up-regulated mRNA expression of corticotrophin-releasing hormone (CRH) and urocortin in the PVN. TrkB, the receptor for BDNF, was expressed in the PVN neurones, including those containing CRH. Both i.c.v. and intra-PVN-administered BDNF decreased food intake and body weight. These effects of BDNF on food intake and body weight were counteracted by the co-administration of alpha-helical-CRH, an antagonist for the CRH and urocortin receptors CRH-R1/R2, and partly attenuated by a selective antagonist for CRH-R2 but not CRH-R1. Intracerebroventricular BDNF also decreased the subcutaneous and visceral fat mass, adipocyte size and serum triglyceride levels, which were all attenuated by alpha-helical-CRH. Furthermore, BDNF decreased the respiratory quotient and raised rectal temperature, which were counteracted by alpha-helical-CRH. These results indicate that the CRH-urocortin-CRH-R2 pathway in the PVN and connected areas mediates the long-term effects of BDNF to depress feeding and promote lipolysis.

Nucleotide sequence of aminoglycoside 6'-N-acetyltransferase [AAC(6')] determinant from Serratia sp. 45.

Gene for aminoglycoside 6'-N-acetyltransferase [AAC(6')] from Serratia sp. 45 was cloned into E. coli. The enzyme produced in E. coli carrying the recombinant plasmid was compared to the Serratia enzyme. Both enzymes acetylated the 6'-C position of amikacin, dibekacin, tobramycin, sisomicin, gentamicin C1a and kanamycin but effected gentamicin C1, gentamicin C2 and micronomycin minimally. No significant difference in optimal pH, isoelectric point or molecular weight was detected. The nucleotide sequence of the gene was determined. Initiating with a GTG codon for methionine, it was composed of 552 base pair coding for 184 amino acids. The molecular weight of the enzyme was about 20418. Comparison of the amino acid sequence of this AAC(6') with the amino acid sequence of aacA4 gene from Serratia marcescens (G. Tran Van Nhieu and E. Collatz, J. Bacteriol., 169, 5708(1987)) showed 98.3% homology.

Distribution of mefE and ermB genes in macrolide-resistant strains of Streptococcus pneumoniae and their variable susceptibility to various antibiotics.

From February to October 1995, 62 erythromycin-resistant strains of Streptococcus pneumoniae isolated at Yamanashi Red Cross Hospital were tested to determine their susceptibility to various macrolides, subjected to resistance induction tests by the disc diffusion method and analysed for genes encoding resistance to macrolides (ermB and mefE). On the basis of resistance induction testing, the isolates were classified as having either inducible (59.7%) or non-inducible (40.3%) macrolide resistance. The ermB gene was always detected in resistance-inducible type isolates, either alone or in combination with mefE. The mefE gene alone was found only in non-inducible type isolates. Isolates with non-inducible resistance (those with only the mefE gene) had an intermediate level of resistance to 14-membered macrolides, and were susceptible to rokitamycin, a 16-membered macrolide. According to NCCLS guidelines, 9.6% of S. pneumoniae strains were judged to be susceptible to penicillin, 62.9% of reduced susceptibility and 27.4% penicillin resistant. No correlation was detected between the presence of particular macrolide-resistance genes (ermB, ermB + mefE, or mefE) and resistance to penicillin G.

Antitumor effect of photodynamic therapy with zincphyrin, zinc-coproporphyrin III, in mice.

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.

New neplanocin analogues. IV. 2-Fluoroneplanocin A: an adenosine deaminase-resistant equivalent of neplanocin A.

2-Fluoro- and 2-chloroneplanocin A's (2 and 3) were synthesized as an adenosine deaminase resistant-equivalent of neplanocin A, and evaluated for their antitumor and antiviral activities. Of these, 2 was completely resistant to adenosine deaminase and showed more significant antitumor and antiviral activities than neplanocin A.