Fourth-generation fluoroquinolone penetration into the aqueous humor in humans.

PURPOSE: To compare the penetration and levels of the fourth-generation fluoroquinolones moxifloxacin 0.5% ophthalmic solution and gatifloxacin 0.3% solution in the aqueous humor (AH) in humans after topical application with published levels of other available fluoroquinolones under similar dosing conditions. DESIGN: Prospective, randomized, double-masked clinical trial. PARTICIPANTS: Forty-six patients undergoing cataract extraction. METHODS: Patients scheduled for routine phacoemulsification and intraocular lens implantation were provided either moxifloxacin 0.5% ophthalmic solution (n = 22) or gatifloxacin 0.3% ophthalmic solution (n = 24) to use 4 times daily the day before surgery plus 1 drop 1 hour before the surgical entry into the anterior chamber on the day of surgery. This regimen simulated a realistic postoperative dosing schedule. Aqueous humor samples were obtained and analyzed by high-pressure liquid chromatography. Aqueous humor fluoroquinolone concentrations were calculated by peak comparison with a known concentration peak for ciprofloxacin that was used as an internal standard. These values were compared with published concentrations of other available fluoroquinolones under similar dosing conditions. RESULTS: The mean age of the moxifloxacin 0.5% group was 67.8+/-9.7 years, whereas that of the gatifloxacin 0.3% group was 69.9+/-8.7 years. The moxifloxacin AH concentration was 1.86+/-1.06 microg/ml, and that of gatifloxacin was 0.94+/-0.72 microg/ml. This 2-fold difference was statistically significant (P = 0.001). CONCLUSIONS: Aqueous humor antibiotic concentrations achieved at the time of cataract surgery after topical application can serve as an effective surrogate for what can be achieved with typical postoperative topical dosing (e.g., 4 times daily). Both fourth-generation fluoroquinolones achieved a greater AH concentration after 4 times daily dosing relative to prior-generation fluoroquinolones. Moxifloxacin 0.5% ophthalmic solution achieved a 2-fold higher aqueous humor concentration than gatifloxacin 0.3% ophthalmic solution. The superior penetration of moxifloxacin into the AH may be attributed partially to its high degree of lipophilicity, greater solubility at neutral pH, and higher concentration in the commercial formulation. The enhanced penetration of moxifloxacin 0.5% ophthalmic solution may provide better protection against ocular infections.

Molecular cloning of the mature NAD(+)-dependent succinic semialdehyde dehydrogenase from rat and human. cDNA isolation, evolutionary homology, and tissue expression.

Three rat brain cDNA clones approximately 3500, 1465, and 1135 base pairs in length encoding succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24) were isolated from two cDNA libraries using a polymerase chain reaction derived probe. Restriction mapping and DNA sequencing revealed that the 3.5-kilobase clone contained an 84-base pair (28 amino acid) insert in the coding region. Composite clones encoding mature SSADH predicted proteins with 488 amino acids (M(r) = 52,188) when including the insert and 460 amino acids (M(r) = 48,854) without the insert. The cDNA clones were confirmed by expression of enzyme activity in bacteria and protein sequence data obtained from sequencing purified rat brain SSADH. Two human liver SSADH cDNA clones of 1091 and 899 base pairs were also isolated. Human and rat SSADH share 83 and 91% identity in nucleotide and protein sequence, respectively. Northern blot analysis revealed two differentially expressed SSADH transcripts of approximately 2.0 and 6.0 kilobases in both rat and human tissues. Human genomic Southern blots indicate that the two SSADH transcripts are encoded by a greater than 20-kilobase single copy gene. Mammalian SSADH contains significant homology to bacterial NADP(+)-succinic semialdehyde dehydrogenase (EC 1.2.1.16) and conserved regions of general aldehyde dehydrogenases (EC 1.2.1.3), suggesting it is a member of the aldehyde dehydrogenase superfamily of proteins.

High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.

This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with the introduction of a Ser-Thr linker to give a protein we have named rF450[mBov17A/mRatOR]L1. Similarly, the cDNA for the omega-hydroxylase of rat liver (P450 4A1) was linked with the cDNA for rat liver NADPH-P450 reductase to give rF450[mRat4A1/mRatOR]L1. A procedure involving disruption of transformed E. coli by sonication, isolation of membranes by differential centrifugation, solubilization with detergent, and affinity chromatography provided significant amounts of purified fusion proteins of approximately 118 kDa. The purified fusion proteins had turnover numbers for the metabolism of steroids (rF450[mBov17A/mRatOR]L1) or fatty acids (rF450[mRat4A1/mRatOR]L1) ranging from 10/min to 30/min in the absence of added phospholipid. Addition of purified rat liver cytochrome b5 stimulated the 17,20-lyase reaction for the conversion of 17-hydroxypregnenolone to dehydroepiandrosterone, and addition of purified rat NADPH-cytochrome P450 reductase enhanced the formation of omega--1 metabolites from lauric and arachidonic acids. NADPH oxidation was tightly coupled to substrate hydroxylation with the purified fusion proteins.

High-level expression of functional human cytochrome P450 1A2 in Escherichia coli.

Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.