Studies of the aggregation behavior of cyclic gemini surfactants.

The specific conductance, surface tension, mean aggregation number, and apparent molar volume properties of aqueous solutions of a novel series of N,N'-bis(cyclododecyldimethyl)-alpha,omega-alkanediammonium dibromide (c12-s-c12) surfactants, where s is the spacer chain length, are reported. Surfactants with s = 3, 4, and 6 have been prepared and characterized in terms of their Krafft temperature (T(Kr)), critical micelle concentration (cmc), surfactant head group area (a) at the air-water interface, mean aggregation number (N(agg)), and the volume change upon micelle formation (deltaV(phi,M)). The c12-3-c12 shows little evidence of aggregate formation, while the results obtained for the c12-4-c12 and c12-6-c12 homologues suggest the formation of small, poorly defined micellar aggregates in aqueous solution.

Evidence that a New Antibiotic Flavone Glycoside Chemically Defends the Sea Grass Thalassia testudinum against Zoosporic Fungi.

Significantly fewer thraustochytrid protists (zoosporic fungi) were observed in association with healthy leaf tissue of the marine angiosperm Thalassia testudinum than in association with sterilized samples that were returned to the collection site for 48 h. In support of the hypothesis that sea grass secondary metabolites were responsible for these differences, extracts of healthy T. testudinum leaf tissues inhibited the growth of the co-occurring thraustochytrid Schizochytrium aggregatum and deterred the attachment of S. aggregatum motile zoospores to an extract-impregnated substrate. By using S. aggregatum for bioassay-guided chemical fractionation, a new flavone glycoside was isolated and structurally characterized as luteolin 7-O-beta-d-glucopyranosyl-2"-sulfate. Whole-leaf tissue concentrations of this metabolite (4 mg/ml of wet leaf tissue) inhibited S. aggregatum attachment, and a significantly lower concentration (270 mug/ml) reduced thraustochytrid growth by 50%, suggesting that natural concentrations are at least 15 times greater than that needed for significant microbiological effects. These results offer the first complete chemical characterization of a sea grass sulfated flavone glycoside and provide evidence that a secondary metabolite chemically defends T. testudinum against fouling microorganisms.

Evidence that a New Antibiotic Flavone Glycoside Chemically Defends the Sea Grass Thalassia testudinum against Zoosporic Fungi.

[This corrects the article on p. 1491 in vol. 64.].

Application of total organic carbon analysis to cleaning validation.

Cleaning validation is the process of assuring that cleaning procedures effectively remove residue from manufacturing equipment/facilities below a predetermined level. This is necessary to assure the quality of future products using the equipment, to prevent cross-contamination and as a GMP requirement. Currently, cleaning validation samples are measured using HPLC or spectrophotometric methods of analysis which are often time consuming and subject to a number of interferences. Total Organic Carbon (TOC) analysis is a new method which has previously only been applied to measurement of carbon residues on production surfaces for biopharmaceuticals. We have applied the TOC analysis method to a number of traditional pharmaceutical products including antibiotics, steroids, and antinauseants in addition to biopharmaceuticals. The method offers extremely low detection capability (ppm and ppb), rapid sample analysis time and therefore quick turn-around of production equipment and facilities. TOC analysis is also applicable to on-line analysis. The method allows the measurement of extraneous materials such as process intermediates, cleaning agents, and protein materials not possible by other methods.

In vitro evaluation of the plasma and blood compatibility of a parenteral formulation for ditekiren, a novel renin inhibitor pseudopeptide.

Ditekiren (U-71038; Boc-Pro-Phe-N-MeHis-Leu-psi[CHOHCH2]-Val-Ile-(aminomethyl)pyridine ) is a potent renin inhibitor peptide and was formulated for clinical intravenous administration in acidified dextrose. This formulation of ditekiren was evaluated in vitro with human and monkey plasma as to its potential for forming a precipitate either of drug or of plasma proteins. Analysis by centrifugation showed that no drug precipitation occurred in plasma from either species at concentrations 25 times higher than anticipated in clinical studies. Results obtained by turbidimetry indicated that formulated ditekiren did not cause aggregation of human platelets or flocculation of proteins at concentrations approaching the solubility limit of the drug in plasma. Ditekiren or vehicle also caused no detectable lysis of red cells at concentrations representing 10 times the maximum clinical level. Therefore, ditekiren solutions as formulated are judged completely compatible with blood and plasma upon clinical intravenous administration.

Derivatization of primary amines by 2-naphthalenesulfonyl chloride for high-performance liquid chromatographic assay of neomycin sulfate.

A normal phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of neomycin sulfate. The method involves pre-column derivatization with 2-naphthalenesulfonyl chloride (NSCl) followed by extraction in chloroform and chromatography using a normal phase silica column with detection at 254 nm. The standard curve for the HPLC assay of neomycin sulfate is linear with a correlation coefficient of 0.9996 over the range of 0.02 to 0.4 mg/ml. Neomycins B, and C, and neamine can be separated and quantified isocratically with relative standard deviations of 0.92% and 1.4% for neomycin (B + 1/2C) and neamine, respectively. Prednisolone is used as an internal standard to aid in quantification. Mass spectrometric data confirms that neomycin is derivatized at all the six primary amines on the neomycin molecule. Eight lots of neomycin sulfate were used to compare the HPLC [NSCl and 1-fluoro-2,4-dinitrobenzene (DNFB)], gas-liquid chromatographic and microbiological assay methods. The average results of the NSCl-HPLC method fell between those of the microbiological and DNFB-HPLC methods. Also, good correlation of the neomycin C contents in neomycin were obtained between the NSCl-HPLC and DNFB-HPLC methods.

Derivatization of secondary amines with 2-naphthalene-sulfonyl chloride for high-performance liquid chromatographic analysis of spectinomycin.

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of spectinomycin hydrochloride and spectinomycin sulfate for detection at 254 nm. The method involves pre-column derivatization of secondary amines of spectinomycin with 2-naphthalenesulfonyl chloride (NSCl) using a catalyst. Lincomycin, 1-methylpyrrole, 2-acetyl-1-methylpyrrole, and 2-acetyl-pyrrole act as catalysts for sulfonylation of spectinomycin. Without a catalyst, the derivatization reaction forms a considerable amount of actinospectinoic acid, a degradation compound of spectinomycin, and peak area:weight ratio of the derivative is approximately 15% lower than those with the catalyst. Following derivatization the sample is extracted and chromatographed on a normal-phase silica column with detection at 254 nm. The method is applicable for the analysis of both the hydrochloride and sulfate salt forms of spectinomycin. All the known degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and the biosynthesis intermediates, dihydrospectinomycin diastereoisomers, are completely separated with this method. Mass spectrometric data confirms that spectinomycin is derivatized with NSCl at the secondary amines located at positions 6 and 8 of the ring structure. The standard curves for the HPLC assay of spectinomycin hydrochloride and sulfate are linear with correlation coefficients of 0.9997 and 0.9999, respectively over the range of 0.05 mg/ml to 0.3 mg/ml. The relative standard deviations (R.S.D.) of the HPLC assay methods for spectinomycin hydrochloride and sulfate are 0.67% and 0.86%, respectively. Spectinomycin hydrochloride and sulfate bulk drugs were assayed by the HPLC method and compared to gas-liquid chromatography and microbiological assay results. The HPLC method was used to assay spectinomycin in a veterinary formulation, Linco-Spectin soluble powder. The sensitivity of the HPLC assay was determined to be approximately 4 ng sample load on the column, which suggests applicability in serum and residue level studies.

Human intestinal mononuclear cells. II. Demonstration of a naturally occurring subclass of T cells which respond in the allogeneic mixed leukocyte reaction but do not effect cell-mediated lympholysis.

We have examined the T-cell functional capabilities of human intestinal mononuclear cells isolated from surgically obtained normal and inflammatory bowel disease intestinal specimens. Intestinal mononuclear cells have T cells present which respond to the mitogenic lectins E-PHA, Con A, and PWM and to foreign cell-surface antigens in the allogeneic-mixed leukocyte reaction. Intestinal mononuclear cells from ulcerative colitis patients exhibit a significantly decreased responsiveness in comparison to normal intestinal mononuclear cells with regard to both mitogenesis and the allogeneic-mixed leukocyte reaction; however, these defects may be secondary to the severity of the disease process that led to intestinal resection or the therapy which patients had received. Although both normal and inflammatory bowel disease intestinal mononuclear cells exhibit responsiveness in the allogeneic-mixed leukocyte reaction, thus indicating recognition of and sensitization by foreign cell-surface determinants, intestinal mononuclear cells do not subsequently kill the sensitizing cells in cell-mediated lympholysis. Therefore, the subclass of T cells which mediates cell-mediated lympholysis may either be absent from intestinal mononuclear cells or nonfunctional, while the subclass of T cells which responds in the allogeneic-mixed leukocyte reaction is both present and functional. This observation adds to the evidence of major functional differences between intestinal and peripheral blood mononuclear cells. Therefore, it will be necessary to better understand the factors regulating the effector capabilities of intestinal mononuclear cells before delineation of immunopathologic events in these tissues.