Serum free ropivacaine concentrations among patients receiving continuous peripheral nerve block catheters: is it safe for long-term infusions?

BACKGROUND: Ropivacaine is a long-acting local anesthetic used for continuous peripheral nerve catheter infusions. Catheters may remain in situ for prolonged time periods. In the present study, patients were enrolled to receive continuous peripheral nerve catheters with measurement of free serum ropivacaine concentrations. METHODS: Peripheral nerve catheters were placed for postoperative pain management in trauma patients and infused with ropivacaine 0.2% or bolused with 0.5%. Blood samples were obtained from each subject on days 0 (preinfusion), 3, 5, 7, 10, and every third day until catheter removal. Serum free ropivacaine concentrations were measured via high-performance liquid chromatography and were compared using the Wilcoxon signed rank test. RESULTS: One hundred thirty-three blood samples were analyzed in 35 patients; all serum free ropivacaine concentrations after infusion initiation (99 samples from 35 subjects) were below 0.34 mg/L (previously determined toxic threshold). The highest concentration achieved in a blood sample was 0.19 mg/L; all other values were <0.09 mg/L. The total amount of drug received during the study ranged from 1146 to 22,320 mg (median of 3722 mg). Catheters remained in situ for a median of 7 days (range: 3-23). From day 0 to 3 (preinfusion), 77% of the study participants had an increase in the serum free-fraction ropivacaine concentrations. The median concentration on day 3 was 0.025 mg/L (95% upper confidence limit for mean: 0.05, range: <0.01-0.19); P < 0.001 compared with preinfusion levels). From day 3 to 5, 68% of the participants had a decrease in the serum free ropivacaine concentrations (median level 0.016 mg/L [95% upper confidence limit for mean: 0.021] P = 0.007 for day 5 compared with day 3). CONCLUSIONS: In this study, free serum ropivacaine concentrations remained well below toxic values despite large amounts of drug administration in combat-wounded patients. The administration of continuous ropivacaine infusions over prolonged time periods, coupled with multiple drug boluses, did not produce toxic or near-toxic serum concentrations.

Kinevac stability after reconstitution with sodium chloride injection USP, 0.9%.

UNLABELLED: Our objective was to compare the stability of Kinevac when reconstituted with sodium chloride injection, USP, 0.9%, versus the manufacturer's recommended sterile water for injection, USP, and to determine the effects on stability of deviating from the manufacturer's recommended methods of product preparation. METHODS: Kinevac was reconstituted with either sterile water or 0.9% sodium chloride. Triplicate high-performance liquid chromatography was performed on each vial of reconstituted sample at time zero and at time zero plus 8 h. The concentration of each sample, as measured by the peak area, was recorded at each time point. The process was repeated over 4 consecutive days. RESULTS: Kinevac reconstituted with sterile water resulted in the recovery of 89.73% of the time zero concentration after 8 h. Kinevac reconstituted with 0.9% sodium chloride resulted in chemical stability of the injection, with 80.05% recovery of the time zero value after 8 h. CONCLUSION: Kinevac is more stable when reconstituted with sterile water than when reconstituted with 0.9% sodium chloride. Kinevac should be reconstituted with sterile water for injection as per the manufacturer's instructions.

Production of the subdomains of the Plasmodium falciparum apical membrane antigen 1 ectodomain and analysis of the immune response.

The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.

Purification, characterization, and immunogenicity of the refolded ectodomain of the Plasmodium falciparum apical membrane antigen 1 expressed in Escherichia coli.

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83(Gly) to 531(Glu)) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.