Application of modified in vitro screening procedure for identifying herbals possessing sulfonylurea-like activity.

We describe here the application of a modified in vitro procedure for identifying herbs potentially possessing sulfonylurea-like activity. The procedure consists of the combination of an SUR1 receptor binding assay and an insulin secretion assay in cultures of HIT-T15 cells. This procedure could be used as an initial step in identifying new safe and efficacious agents for the management of Type II diabetes. The application of this screening procedure to a set of selected herbs produced results that were consistent with the previously reported properties of those herbs. The collected data suggest that the hypoglycemic properties of bitter melon (Momordica charantia, Linn. Family, Cucurbitacea), cerasse (Momordica charantia, Linn. wild variety, Family, Cucurbitacea) and American ginseng (Panax quinquefolius, Linn., Family Araliacea) are at least partially due to their sulfonylurea-like activity.

Liquid chromatographic determination of nystatin in pharmaceutical preparations.

A rapid, reversed-phase liquid chromatographic method was developed for the assay of nystatin in the bulk drug and a variety of dosage forms. Analysis was performed on a Symmetry C18 reversed-phase column using a mobile phase of methanol-water-dimethylformamide (DMF; 55 + 30 + 15, v/v/v), with detection by UV at 305 nm. Quantitation is based on the sum of the peak areas of the 2 major isomers of nystatin. The linearity of the assay was determined for a concentration range of 0.05 to 0.2 mg/mL (correlation coefficient > 0.999). Accuracies and precision showed good reproducibility.

Isolation and identification of process impurities in trimethoprim drug substance by high-performance liquid chromatography, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy.

Twenty-two lots of recently synthesized trimethoprim drug substance, from five different manufacturers, in three different countries of origin, China, Israel and the United States, were investigated for the presence of impurities. A liquid chromatographic system, using gradient elution, and a mobile phase consisting of 0.25% TEA/0.1% formic acid (pH 5.8)--acetonitrile, was used to separate and detect two significant, recurring impurities in trimethoprim drug substance. The two impurities were isolated by preparative liquid chromatography and identified, using a combination of liquid chromatography/mass spectroscopy and nuclear magnetic resonance, as 2,4-diamino-5-(4-ethoxy-3,5-dimethoxybenzyl) pyrimidine and 2,4-diamino-5-(3-bromo-4,5-dimethoxybenzyl) pyrimidine. These impurities were not detected by the compendial method and were present at significant levels in 17 of the lots tested. Total impurity concentrations were in the range of 0.1-2.1%.

Synthesis and antinociceptive activity of [D-Met2, Pro5] enkephalin [N1,5-beta-D-2,3,4,6-O-tetraacetylglycosyl]--amide and [D-Met2, Pro5] enkephalinamide.

Tetra-O-acetylgalactopyranosylamine and tetra-O-acetylglucopyranosylamine of D-Met2, Pro5 enkephalin were designed and synthesized to enhance their membrane penetration, biological activity and resistance to proteolytic hydrolysis. Three approaches to the synthesis were attempted, which lead to a new synthetic scheme with a higher yield and enhanced ease of purification. The improved procedure involved attaching the tetra-O-acetylglycopyranosylamine to a t-Boc-Gly-Phe-Pro-OH peptide, removing the t-Boc, and condensing it with t-Boc-Tyr-D-Met-OH. Biological evaluation in vivo showed that these acetylglycopyranosylamine derivatives bind to mu and delta opioid receptors in homogenate binding assays and possess analgesic activity. The analgesic potency was less than that of the parent compound D-Met2, Pro5 enkephalin. These acetylglycopyranosylamine derivatives showed enhanced lipophilicity compared to their parent compound by a partition coefficient study and they also showed greater membrane permeability, using the rabbit cornea as a model system. These derivatives also are resistant to hydrolytic enzymes as compared to the endogenous met-enkephalin when evaluated in homogenized iris-ciliary body and aqueous humor from rabbit eyes.

GC/MS comparison of the West Indian aphrodisiac "Love Stone" to the Chinese medication "chan su": bufotenine and related bufadienolides.

The death of a 23-year-old man resulting from digoxin-like toxicity and heart failure was attributed to ingestion of a West Indian aphrodisiac known as "Love Stone." GC/MS analyses identified bufotenine, a controlled substance under both US and New York State statutes. In addition, a series of bufadienolides, namely resibufogenin, bufalin, and cinobufagin, were also identified. Bufadienolides, which are derived from toad venom or secretions, are cardiotonic steroids that cause symptoms similar to digoxin. GC/MS analyses of the Chinese medication "Chan Su," a product derived from toads, produced a highly similar elution profile and contained the same compounds as "Love Stone." The data demonstrate that the aphrodisiac was also derived from toads.

The combined use of DSC and TGA for the thermal analysis of atenolol tablets.

The New York Regional Laboratory's forensic pharmaceutical group used thermal analysis (TA) to ensure adherence to batch formulations by drug manufacturers. TA in our laboratory consisted of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). DSC and TGA were used to analyse finished dosage forms and their components. In this study the components of atenolol formulations were qualitatively identified by analysing individual components and comparing with the finished product. DSC and TGA were used together to develop a profile of batch formulations, with each analytical technique giving complementary types of information. For example, the excipient sodium starch glycolate was identified by DSC and confirmed by TGA. These experimental results demonstrated the use of TA for the characterization of finished dosage forms and the qualitative identification of some of the individual components in batch formulations.

Solubility enhancement of nucleosides and structurally related compounds by complex formation.

Water-soluble vitamins, amino acids, and nontoxic pharmaceutical excipients were studied as solubilizing agents for poorly water-soluble adenine (nucleic acid base), guanosine (nucleoside), and structurally related drugs (acyclovir and triamterene). The apparent solubility of the substrates (adenine, guanosine, acyclovir, or triamterene) was appreciably increased by forming complexes with the ligands (vitamins, amino acids, or other ligand). Apparent association constants (Ka) values were measured at 25 degrees C in pH 7 phosphate buffer using phase solubility analysis. The effect of combination ligands on substrate solubility was also studied. Additive solubility enhancement was obtained for several ligand pairs.

Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.

Incorporation of [1-14C]-Isopentenyl Pyrophosphate into Monoterpenes by a Cell-Free Homogenate Prepared from Callus Cultures of Chrysanthemum cinerariaefolium.

Callus culture of CHRYSANTHEMUM CINERARIAEFOLIUM (Trev.) Bocc. (Asteraceae), initiated with axillary buds from the plant, have been shown to produce chrysanthemyl alcohol and chrysanthemic acid. Incubation of a cell-free homogenate from the callus with [1- (14)C]-isopentenyl pyrophosphate demonstrated incorporation of radioactivity into chrysanthemyl alcohol and chrysanthemic acid as well as products from isopentenyl pyrophosphate isomerase (EC 5.3.3.2; isopentenyl diphosphate Delta (3)-Delta (2)-isomerase) and prenyltransferase activities (EC 2.5.1.11; allylic-terpene-diphosphate:isopentenyl diphosphate terpenoid-allyltransferase).

Selection of high pyrethrin producing tissue cultures.

Axenic callus and shoot cultures of pyrethrum have been shown to be capable of pyrethrin biosynthesis. The influence of explant source on the biosynthetic capacity in cultured tissue was examined. Explants of various plant organs were taken from high and low yielding plant selections. The conditions necessary for the establishment of these explants in culture are described. Analytical screening of the tissue lines enabled the selection of a few "high yielding" strains which were derived from high yielding plant selections. Differentiated cultures tended to produce more pyrethrins than did callus cultures.

Distribution of pyrethrins in oil glands and leaf tissue of Chrysanthemum cinerariaefolium.

Leaves of Chrysanthemum cinerariaefolium at various stages of development were found to contain differing amounts of pyrethrins. The variation corresponds to the number of oil glands per unit area of leaf surface. Leaf tissue was mechanically and enzymatically separated into lower and upper epidermis and mesophyll cells. Pyrethrins were found to be present in each layer, more pyrethrins were present in the epidermal layers of young leaves while the mesophyll cells were found to have higher pyrethrins in older, more mature cells.

Thebaine from root cultures of Papaver bracteatum.

Root cultures derived from cell suspension cultures of Papaver bracteatum were shown to produce thebaine (yield 0.03%).

Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase.

Sodium boro[3H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates epsilon-N-[3H]pyridoxyl lysine which is degraded to [3H]pyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium boro[3H]hydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme x substrate complex through the reduction of the Schiff's base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme x substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff's base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme.