Microdetermination of procainamide in human serum. An electron-capture GLC method to measure procainamide (0.1-1 microgram/sample) in human serum was developed. An internal standard, p-amino-N-[2-(dipropylamino)ethyl]benzamide, is added to the serum before the sample is alkalinized with pH 10.5 phosphate buffer and extracted with ethyl acetate. The ethyl acetate phase is evaporated to dryness, and the residue is reacted with pentafluoropropionic anhydride. N-Pentafluoropropionyl derivatives of the drug and the internal standard had retention times of 5 and 8 min, respectively, when chromatographed at 235 degrees on a 1-m (4-mm i.d.) glass column packed with 5% OV-17 (carrier gas flow of 40 ml/min). The coefficient of variation was less than 5% for spiked standards. Furthermore, N-acetylprocainamide added to samples did not interfere. One hundred and eighty-six samples from 16 patients receiving procainamide intravenously were assayed by this GLC procedure and by a standard colorimetric method. Linear regression analysis yielded a correlation coefficient of 0.985 (slope, 1.040; intercept, 0.015).


Sheabutter V: effect of particle size on release of medicament from ointment. An ointment prepared from fat (sheabutter) obtainable from an indigenous (African) fruit released medicaments faster than either of the two ointment bases recommended by the British Pharmacopoeia. Reduction in particle size of crystalline medicaments incorporated in ointments increased surface area and transfer from the ointment to the substrate, possibly due to greater dissolution in the base, and, therefore, increased the availability of medicament at the ointment-substrate interface.


Influence of cobalt (dietary), cobalamins, and inorganic cobalt salts on phenytoin- and cortisone-induced teratogenesis in mice. Various cobalt-containing agents (cyanocobalamin, sodium cobaltinitrite, and cobaltous chloride), which formerly had been shown to prevent the onset of cleft palate in CF-1 mice injected with cortisone, were studied to determine whether they would afford similar protection against phenytoin. Phenytoin, however, failed to cause cleft palate in the mouse fetus when given to pregnant animals alone; and cortisone, on the contrary, induced this anomaly in the presence of the so-called cobalt antagonists as well as when administered in their absence. It is suggested from these results that high dietary intake of cobalt prevents cleft palate caused by phenytoin challenge and also negates the protective effects associated with the acute administration of cobalt compounds. Therefore, it is concluded that these well-known teratogens inhibit palatal closure in mice by different mechanisms.


Relationship between osmolality and osmolarity. Since the compendia require the osmolarity of certain parenterals to be labeled and since experimentally only the osmolality can be measured, it is necessary to obtain the relationship between these two quantities. This relationship was determined by considering fundamental physical-chemical definitions. The osmolality of a solution was found to be simply related to the osmotic coefficient. The conversion to osmolarity requires the use of the partial molal volume(s) of the solute(s). A single conversion factor is required for a particular solute system; i.e., the conversion factor is independent of the solution concentration.


Solid-state dispersions employing urethan. The dissolution rates of a number of drug-urethan solid-state dispersion systems were studied. A marked enhancement of the initial dissolution rates of several poorly water-soluble drugs was found when they were incorporated into a urethan matrix by heat fusion. These differences were considerable when pure substances such as griseofulvin, hydrocortisone, chloramphenicol, and acetaminophen were compared to the urethan-drug solid dispersion. Physical mixtures of the medicinal agents with urethan also gave a marked increase in the amount of drug in solution, with the value in most cases being over one-half that of the solid-state dispersion. Data are given, comparing ultrafiltration with samples filtered through cotton, regarding drug content remaining in solution.


Synthesis and evaluation of N-deacetyl-N-glycosylalkylthiocolchicines as antileukemic agents. A series of N-deacetyl-N-glycosylalkylthiocolchicines (glucosyl, galactosyl, mannosyl, ribosyl, and arabinosyl) was prepared by heating N-deacetylalkylthiocolchicines with the appropriate monosaccharides in methanol. Some compounds (glucosyl-, mannosyl-, and ribosylalkylthiocolchicines) were per-O-acetylated in dry pyridine with acetic anhydride. The compounds were tested against leukemia L-1210 and P-388 systems. Preliminary results showed that the antileukemic activity of the glycosyl compounds in vitro is similar to that of the N-deacetylalkylthiocolchicines used for their preparation. However, the presence of a glycosyl moiety in the molecule gives the advantage of greater solubility in water. Of the results obtained to date in lymphoid leukemia screening in vivo, five glycosyl compounds showed promising activity levels and have now reached confirmed active status.


Ambiguities in IR and X-ray characterization of amphotericin B. Two distinct IR spectra of amphotericin B have been reported. These differences can be obtained from the same sample by surprisingly small changes in the method of sample preparation. Type I spectra (hand-ground samples) are characterized by a sharp C = O stretching band at 1692 cm-1, and Type II spectra (vibrator-ground samples) are characterized by a broad C = O stretching band near 1710 cm-1. X-ray powder diffraction demonstrates that vibrator grinding promotes a transition from a crystalline to an amorphous phase. The two phases are not bioequivalent. Differential thermal analysis reveals a transition near 157 degrees and samples heated to 158 degrees give only Type II IR spectra. However, a marked color change accompanies such heating (i.e., structural changes affecting the chromophore have been thermally induced), while X-ray spectra show an increase of only about 30% in amorphous content. Furthermore, hand-ground samples heated to 120 degrees still display only Type I IR spectra. Thus, the vibrator-induced transition is not solely a static thermal effect. Many observed spectral lines can be assigned to specific functional groups.


Synthesis of diastereoisomeric 4-dimethylamino-3-phenyl-2-butanols and related esters for antimicrobial evaluation. 4-Dimethylamino-3-phenyl-2-butanone was reduced to the corresponding diastereoisomeric alcohols, which were separated by fractional crystallization of the corresponding hydrochloride salts. The configuration of the diastereoisomeric alcohols was determined by PMR spectroscopy. The assignments were confirmed by a consideration of the mass spectral data obtained for the two alcohols. Acylation of the alcohols gave the corresponding esters. Antimicrobial evaluation of the compounds prepared showed that 4-dimethylamino-3-phenyl-2-butanone had a promising level of antifungal activity while the other derivatives showed either a low level of potency or were inactive.


Simple high-pressure liquid chromatographic determination of trisulfapyrimidines in human serum. A simple and rapid high-pressure liquid chromatographic method was developed for the determination of sulfadiazine, sulfamerazine, and sulfamethazine in human serum. After the trichloroacetic acid precipitation of the serum proteins, an aliquot of the supernate is injected into a high-pressure liquid chromatograph equipped with a reversed-phase microparticulate column and a fixed wavelength UV detector. For each of the three components of trisulfapyrimidines, a linear calibration curve was observed in the 1-30-microgram/ml range, with the precision of the assay estimated to be +/- 2% (RSD). Preliminary pharmacokinetic data are also presented.


New method of preparing gelatin microcapsules of soluble pharmaceuticals. A new method of preparing gelatin microcapsules of soluble pharmaceuticals is described. Spherical droplets of a gelatin dispersion prepared in the drug solution were produced by the capillary method, and the droplets were congealed rapidly to yield discrete units in the form of a free-flowing powder. The microcapsules obtained were spherical in shape and showed no tendency to form agglomerates. Hardening of the microcapsules resulted in a significant reduction of the release rate without altering the reproducibility. The results indicated that the process of microencapsulation described is simple, reproducible, economical, and amenable to industrial application.


Synthesis of methaqualone and its diphasic titration in pure and tablet forms. A one-step synthesis of methaqualone from N-acetylanthranilic acid and o-toluidine in the absence of a catalyst is described. A rapid diphasic titration procedure for its microestimation in pure and tablet forms, using dioctyl sodium sulfosuccinate and dimethyl yellow screened with oracet blue B, is proposed. The data were compared with those obtained from nonaqueous titration methods.


S-(4-Chlorophenyl) 3-aryl-3-hydroxypropanethioates as antibacterial agents. A series of S-(4-chlorophenyl) 3-aryl-3-hydroxypropanethioates was prepared and shown to have in vitro activity against several selected bacterial species.


Assessment of enterohepatic circulation of 3H-digoxin with a minimal interruption technique. The biliary excretion of 3H-digoxin in rats prepared for bile sampling with minimal interruption of the enterohepatic circulation was compared with that in rats with complete interruption after intraduodenal or intravenous administration. Following dosage by either route, significantly more radioactivity was recovered from animals with nearly intact enterohepatic circulation. The method described allows direct measurement of this cycle in unanesthetized animals without the consequences of bile depletion.


GLC determination of pemoline in biological fluids. A specific GLC method for the determination of microgram quantities of the central stimulant pemoline in biological fluids is described. Extraction problems due to the low solubility of pemoline are avoided by acid hydrolysis of the drug to 5-phenyl-2,4-oxazolidinedione, which then can be easily isolated by two-phase extraction with dichloromethane, ether, or chloroform. Amide-imide tautomerism enables a cleanup of the extract. Quantitative determination at the microgram level is done on a methylated fraction of the dichloromethane extract by GLC using a suitable internal standard. For supporting evidence of the GLC method's specificity, the compound is also identified by examining an aliquot of the final dichloromethane extract by TLC.


Synthesis of 4,5-dimethoxykynuramine and its in vivo conversion to 6,7-dimethoxy-4-quinolinol. 4,5-Dimethoxykynuramine was synthesized in a three-step sequence originating with veratrole. Indirect evidence indicates that the drug was converted in vivo to the hypotensive agent 6,7-dimethoxy-4-quinolinol by the action of monoamine oxidase.


Separation and quantitation of possible degradation products of procarbazine hydrochloride in its dosage form. A stability-indicating assay for the degradation products of procarbazine hydrochloride was developed using high-pressure liquid chromatography. The method uses a buffered methanol-water mobile phase on a reversed-phase column. Concentrations of degradation products as low as 0.04 mg/ml, 0.02% degradation, can be quantitated using an internal standard of cinnamyl alcohol. The typical range for degradation products in procarbazine capsules is 0.1-0.5% after as long as 4.5 years.


Alkaloids from Lupinus argenteus var. stenophyllus. TLC and GLC of an alkaloid extract of the aboveground portions of Lupinus argenteus Pursh. var. stenophyllus (Rydb.) Davis (Leguminosae) suggested the presence of sparteine, beta-isosparteine, delta5-dehydrolupanine, alpha-isolupanine, lupanine, thermopsine, and anagyrine. GLC-mass spectrometry confirmed these preliminary findings. Preparative TLC was used to isolate sparteine, and this alkaloid was further characterized by IR spectral analysis and derivatization.


Simultaneous salt and ethanol removal from human serum albumin. Diafiltration of salt and ethanol from human serum albumin was shown to be a competitive alternative to freeze drying. At least 99% of the original alcohol content could be removed in approximately five volume changes. Data on changes in ionic strength, bacteriological buildup, permeate flux, and dimer contents are presented.


Constituents of West African medicinal plants XX: quindoline from Cryptolepis sanguinolenta. An ethanol extract of the roots of Cryptolepis sanguinolenta (Asclepidaceae) afforded, after partitioning and chromatography, the alkaloids quindoline and CSA-3. The structural elucidation of CSA-3 is currently in progress. Cryptolepine was also isolated from this species.


New spectrophotofluorometric assay for probenecid. A new spectrophotofluorometric assay for probenecid is presented based on conversion of the drug to a fluorescent anthranilic acid derivative. The assay is especially applicable with "clean" biological fluids such as cerebrospinal fluid and offers severalfold greater sensitivity than the commonly used UV method.


Determination of fluoxymesterone, norethandrolone, prednisolone, and prednisone in tablets by differential pulse polarography. A differential pulse polarographic method for the determination of fluoxymesterone, norethandrolone, prednisolone, and prednisone in tablets is described. This method is more sensitive than dc polarography, and the measurement of diffusion current is greatly simplified. Sørensen phosphate buffer, pH 5.6, was used as the supporting electrolyte. No apparent interference was observed from tablet excipients; the method is rapid, simple, and relatively precise.


Griseofulvin---phenobarbital interaction: a formulation-dependent phenomenon. The reported interaction of griseofulvin with phenobarbital was studied in the rat following oral administration of different dosage forms. A single oral dose of 15 mg of phenobarbital/kg 24 hr prior to the oral administration of a suspension of 100 mg og griseofulvin/kg in 0.5% polysorbate 80 significantly reduced plasma griseofulvin levels. An increase in the concentration of polysorbate 80 to 2% reduced the extent of the interaction from 50 to 32%. Phenobarbital did not influence plasma griseofulvin levels when griseofulvin was given in either 70% polyethylene glycol 300 (suspensions of 20 or 100 mg/kg) or 100% polyethylene glycol 600 (solution of 50 mg/kg). It is concluded that the observed interaction is formulation dependent and is a result of diminished dissolution and, consequently, reduced absorption of griseofulvin.


Micro and macro GLC determination of ethambutol in biological fluids. Previously available GLC methods for ethambutol may only be used to measure quantities of drugs much greater than those found in biological fluids such as plasma and dialysate. A previously published GLC method for plasma samples is extended to measure ethambutol in dialysate. A second GLC method, involving derivatization with bis(trimethylsilyl)trifluoroacetamide and subsequent quantitation using a flame-ionization detector, is described for urine samples. With a dual-column and dual-detector gas--liquid chromatograph, simultaneous micro (plasma and dialysate) and macro (urine) determinations of ethambutol are possible.


Hofmann elimination with diazomethane on curare bases and selected quaternary tetrahydroisoquinoline alkaloids. Use of a large excess of alkali-free diazomethane resulted in a Hofmann elimination with selected curare bases and some other quaternary tetrahydroisoquinoline alkaloids. (+)-Tubocurarine chloride provided a monostilbene methine, O,O-dimethyltubocurinemethine, and a monostilbene--monostyrene compound, O,O-dimethyltubocurinedimethine. The major elimination products of (+)-isotubocurarine chloride and (+)-carnegine methiodide were monostyrene methines, O,O-dimethyltubocurineisomethine and carneginemethine, respectively. Treatment of (+)-laudanosine methiodide with potassium hydroxide, under the conditions of Hofmann degradation, or alkali-free diazomethane solution provided the same stilbene compound, laudanosinemethine. The structures of the elimination compounds were further confirmed by catalytic reduction and quaternization with methyl iodide.


Determination of urinary indolic metabolites. A rapid mass spectral assay for tryptophol, 5-hydroxytryptophol, and 3-indoleacetic acid, employing stable-isotope labeled internal standards, is described. The compounds were extracted from urine or buffer with ethyl acetate and quantitatively measured by chemical-ionization mass spectrometry. The calibration curves were linear over a range of 0.06--2.8 microgram/ml. The technique was applied to the analysis of urine from patients with carcinoid tumors. In addition, the first synthesis of 5-methoxy-3-indoleacrylic acid is reported.


Inhibition of rat liver mitochondrial monoamine oxidase by chloramphenicol and 2-amino-1-p-nitrophenylpropane-1,3-diol. Inhibition of rat liver mitochondrial monoamine oxidase by chloramphenicol and its hydrolytic product, 2-amino-1-p-nitrophenylpropane-1,3-diol, was studied. The enzyme activity and its inhibition by these two compounds were optimum at pH 7.0 after preincubation for 60 min, the time needed for maximum enzyme--inhibitor complex formation. Enzyme activity could be restored after prolonged dialysis. Monamine oxidase inhibition by chloramphenicol and its hydrolytic product was noncompetitive and reversible. Deamination of various monamines was not to the same degree by these compounds. Of the different antimicrobials studied, only chloramphenicol and its hydrolytic produced has a strong inhibitory effect on monoamine oxidase.


New GLC analysis of urushiol congeners in different plant parts of poison ivy, Toxicodendron radicans. Methods are presented for the direct GLC analysis of the catechol C15 alkenyl side-chain congeners contained in the urushiol fraction of poison ivy (Toxicodendron radicans) and the C17 homologs of poison oak (Toxicodendron diversilobum). A number of liquid phases were investigated and demonstrated varying degrees of separation. The methods developed were applied to the analysis of the urushiol fractions obtained from different plant parts of poison ivy. The effects of extraction before and after drying demonstrated tht a larger percentage of urushiol was obtained when the fresh plant material was extracted with ethanol.


Thermodynamics of mercaptopurine dehydration. The hydrate form of mercaptopurine was shown to undergo peritectic decomposition of its water molecule, localized dissolution, and dehydration around 125 degrees. The anhydrate form was prepared by a thermal method, whose effectiveness was confirmed by X-ray diffraction, NMR spectroscopy, and differential scanning calorimetry. The activation energy for mercaptopurine dehydration calculated by various methods ranged from 45.74 to 63.04 kcal/mole. The dehydration enthalpy was calculated to be 8.27 kcal/mole by differential scanning calorimetry. The solution enthalpy for the hydrate was calculated to be 4.85 kcal/mole from its saturation solubility and differential scanning calorimetry. Anhydrate solubility in water was calculated based on initial dissolution rate data since the anhydrate converts to hydrate in aqueous media. The high degree of stability against interconversion of the hydrate and anhydrate forms and the higher solubility of the anhydrate suggest that use of the anhydrate might improve mercaptopurine bioavailability.


Ultrasonic method for direct and simultaneous determination of alcohol and soluble solids content of mouthwash. A technique for the determination of certain soluble species in solution by the changes they produce in the speed of propagation of ultrasonic waves in the solution was applied to measure the alcohol and soluble solids levels of mouthwashes. The simultaneous determination of these two quantities is made possible by measuring the wave velocity at two different temperatures. The method gives accurate, precise results for the general range of mouthwash compositions in use with appropriate calibration. The advantages of this method over other current methods are precision, speed, and convenience. It is not a suitable regulatory method, however, because calibration must be done with known variations of the particular mouthwash composition to be analyzed.


Influence of wax coatings on release rate of anions from ion-exchange resin beads. Ion-exchange resin beads were coated with various waxes to improve and control their release. The in vitro release rates of benzoate ions from the coated-resin beads were then investigated using a rotating sieve basket technique. The dramatic differences in release rates observed with the different waxes can be discussed in terms of the wax to resin ration and the solubility characteristics of the waxes. The initial release rates can be expressed in terms of a mathematical expression previously reported for the diffusion of ions in ion-exchange resins, thereby aiding in the elucidation of the effect of the waxes on release.


Correlation of log P with molecular connectivity in hydroxyureas: influence of conformational system on log P. The correlation of log P (in octanol--water) with the nonempirical, topologically dependent, calculated molecular connectivity index (1chiv) delineates substituted hydroxyureas into two families of linearly related groups of compounds. The first group, composed of the 3-substituted ethyl, n-propyl, and n-butyl analogs, is more hydrophilic than the 1-substituted methyl and ethyl and the 3-substituted isopropyl and tert-butyl analogs. The unsubstituted model compound hydroxyurea appears between the two groups in equal volumes of octanol. In octanol--water ratios of 5:1, log P approaches the range of the more hydrophilic group in high concentrations and becomes more lipophilic (similar to the other group) in lower concentrations. The differences in the relative hydrophilicities--lipophilicities of the two groups are rationalized in terms of the equilibri of internally hydrogen-bonded, conformers to those that allow optimal interactions with solvent, water, or other hydroxyurea molecules. The concentration dependency observed with hydroxyurea appears to be due to the ease of interconversion of intermolecularly bonded conformers to those interacting with water, whereas the involvement of internally bonded conformers, which are apparently present to a greater degree in lower concentrations, increases the relative lipophilicity.


Benzocaine diffusion from polyethylene glycol through human stratum corneum. The diffusion, penetration, and surface effects of benzocaine incorporated in various polyethylene glycol ointment bases through human stratum corneum were studied. Benzocaine diffusion was measured by following the benzocaine concentration in the receiving compartment of a diffusion cell. The ointment was placed in the other cell compartment and was separated from the receiving compartment by sheets of human stratum corneum. Surface effects were monitored by scanning electron micrographs of the stratum corneum. Results showed a decrease in drug diffusion in the presence of relatively high amounts of the lower molecular weight portions of polyethylene glycol. Scanning electron microscope studies showed that both benzocaine and polyethylene glycol affect the surface structure of the stratum corneum. Thermal analysis indicated that benzocaine dissolves in polyethylene glycol.


Thermal and photolytic decomposition of methotrexate in aqueous solutions. The chemical kinetics of thermal and photolytic degradation of methotrexate in aqueous solutions were studied. At above pH 7 and 85 degrees, methotrexate hydrolyzed to yield mainly N10-Methylpteroylglutamic acid. The hydrolysis rate followed first-order kinetics with respect to methotrexate concentration and increases rapidly above pH 9. When methotrexate solutions were kept under laboratory fluorescent light, the major degradation products were 2,4-diamino-6-pteridinecarbaldehyde, 2,4-diamino-6-pteridinecarboxylic acid, and p-aminobenzoylglutamic acid. The photolytic reaction followed zero-order kinetics with respect to methotrexate concentration and was catalyzed by bicarbonate ion. Evidence is presented for a proposed sequential cleavage of methotrexate by a free radical mechanism for the photolysis. Commercial parenteral methotrexate was found to be quite stable as marked when stored in the original vial at room temperature.


Effects of compression force, particle size, and lubricants on dissolution rate. The effects of compression force, particle size, and lubricant concentration on the dissolution rates of compressed disks of salicylic acid, aspirin, and an equimolar mixture of aspirin and salicylic acid were investigated. Compression forces from 450 to 9100 kg had no effect on dissolution rates. With 5% starch incorporated into an equimolar mixture of aspirin and salicylic acid, the dissolution rates were independent of compression forces from 910 to 9100 kg. A 10-fold change of particle size of the materials being compressed did not affect the dissolution rates. An increase in the concentration from 0.1 to 5% of calcium stearate, glyceryl monostearate, magnesium stearate, and stearic acid progressively slowed the dissolution rate. An increase in the concentration from 0.1 to 5% talc and polyethylene glycol 4000 did not affect the dissolution rates. An increase in the concentration of starch from 0.1 to 5% progressively increased the dissolution rates.


Recommendations to eliminate subjective olfactory methods from compendial identification tests. Substitution of IR and UV absorption spectroscopy and use of the various test papers listed in the compendia provide satisfactory means of identifying drug and other substances that presently require subjective odor identification.


Quantitative determination of nalbuphine in plasma using electron-capture detection. A procedure for the determination of nalbuphine in plasma is presented. The compound and an internal standard are extracted into benzene-2-propanol from plasma at pH 10.4, followed by back-extraction into 0.1 N HCl. After the acid phase is washed with benzene and the compound is reextracted into benzene-2-propanol, the heptafluorobutyryl derivatives are formed and determined using electron-capture GLC. The lower limit of sensitivity is approximately 0.5 ng/ml, and the upper limit of the linear dynamic range is greater than 50 ng/ml. Peak plasma levels of 50 and 150 ng were observed in two dogs 10 and 20 min, respectively, after subcutaneous administration of 1 mg of nalbuphine/kg. Nalbuphine was detectable in plasma 5 hr after administration.


Anti-inflammatory 1-substituted 2-imidazolidinones. The synthesis and anti-inflammatory activity of a series of 1-substituted 2-imidazolidinones are reported. These compounds exerted marked anti-inflammatory activity in the carrageenan-induced hindpaw edema assay in the rat in doses that failed to elicit side effects. Their ED50 values were near the value of aspirin but markedly higher than the value of indomethacin.


Potential source of error in official diazepam assays. A possible source of interference by a benzophenone hydrolysis product with the USP XIX spectrophotometric determination of diazepam in dosages forms is reported. A minor adaptation of the official assay procedures is briefly proposed as one method to correct this error.


Antitumor agents XXVII: Effects of helenalin on anaerobic and aerobic metabolism of Ehrlich ascites cells. Evidence is presented that the antitumor agent helenalin, a sesquiterpene lactone, suppresses anaerobic glycolytic enzymes of tumor cells at a number of sites and not exclusively at glycogen synthetase and phosphofructokinase, previously proposed sites for inhibition by alpha-methylene-gamma-lactones. Of the enzymes tested, the sulfhydryl-containing enzyme hexokinase was inhibited the maximum, i.e., 83%, by helenalin treatment, whereas phosphofructokinase and glycogen synthetase were suppressed approximately 45%. Another sulfhydryl-bearing enzyme, aldolase, was decreased approximately 43%. Phosphorylase a was inhibited 65%, glucose-6-phosphatase was inhibited 46%, and succinic dehydrogenase was inhibited 59% by helenalin treatment. Mitochondrial oxidative phosphorylation processes were also significantly depressed in the presence of helenalin in vitro with either succinate or alpha-ketoglutarate as substrates. Thus, a number of enzymes of anaerobic and aerobic carbohydrate metabolism of Ehrlich ascites cells appear to be inhibited by helenalin, which supposedly can alkylate functional groups, e.g., sulfhydryl groups of these enzymes, by a rapid Michael-type addition.


Intrapatient variability of serial steady-state plasma tricyclic antidepressant concentrations. Nine or 10 serial steady-state plasma measurements of amitryptyline, desipramine, desmethyldoxepin, doxepin, imipramine, nortriptyline, or protriptyline were made in 23 depressed patients.  Each patient was monitored for compliance by pill counts, and sampling time was controlled carefully to determine intrapatient variability of steady-state tricyclic levels on a day-to-day basis. The coefficients of variation during serial sampling of the various ingested drugs were: amitriptyline, 21%; desipramine, 26%; doxepin, 21%; imipramine, 14%; nortriptyline, 13%; and protriptyline, 17%. The therapeutic ranges for the tricyclic antidepressants are relatively wide, so coefficients of variation of these magnitudes indicate that the position of an individual patient in relation to the optimal therapeutic range can be reliably determined on a clinical basis.


Paper chromatographic determination of chlortetracycline. Chlortetracycline was successfully separated from its degradation products and from other tetracyclines on a paper chromatogram previously impregnated with a mixture of edetate disodium and urea solution in pH 5.0 McIlvaine buffer. Chlortetracycline separation from its degradation products and from both tetracycline and oxytetracycline was attributed primarily to the complexation of these antibiotics with urea and edetate on the chromatogram. The separated spots were located under UV light, and their Rf values were calculated. Chlortetracycline recovery from the paper chromatogram was satisfactory (98.3 +/- 0.002%). The developed method was successfully applied to the determination of chlortetracycline in various dosage forms. In addition, the method was adequate for monitoring chlortetracycline hydrochyloride stability in aqueous solutions.


tert-Butoxycarbanyl as a convenient protecting group in synthesis of potential centrally active dopamine derivatives. Several pivaloyl and pivaloyloxy esters and amides of dopamine were synthesized for possible antiparkinson activity. The compounds were synthesized by select O- and N-acylation and N-methylation procedures. The tert-butoxycarbonyl function is an effective and easily removed nitrogen-protecting group for dopamine. Preliminary biological testing results showed that all compounds tested elicited a hypothermic response in mice, while only O,O-dipivaloyl-N,N-dimethyldopamine reversed reserpine-induced motor depression in mice. However, it is difficult to conclude from the preliminary data that the observed biological effects were due to central dopaminergic receptor stimulation.


Drug absorption VIII: Kinetics of GI absorption of methotrexate. The absorption of methotrexate from the lumen of the rat small intestine, in situ, was found to obey Michaelis--Menten kinetics. The values of Vmax and Km for the absorption process were 4.78 X 10(-7) M/min and 1.49 X 10(-5) M, respectively.


Effects of quinone derivatives on an experimental tumor. In continuation of studies on the activity of known solid tumor inhibitors, four acetylated glycosyl derivatives of 1,4-quinones were prepared and tested against Ehrlich ascitic tumor. All four compounds significantly inhibited growth of this neoplasm. UV, IR, and mass spectra are given for the three new synthetic quinone derivatives.


Structure--activity relationships of anesthetic ethers using molecular connectivity. The structure--activity relationships in the anesthetic and toxic actions of 28 aliphatic ethers were simple functions of molecular connectivity indexes. These quantitative relationships are discussed briefly in the light of theories of the mechanism of action of general anesthetics.


Molecular connectivity in quantitative structure--activity relationship study of anesthetic and toxic activity of aliphatic hydrocarbons, ethers, and ketones. The nonempirical molecular connectivity indexes of 27 aliphatic hydrocarbons, ethers, and ketones were calculated. Very good correlations were obtained between anesthetic (AD100) and toxic (LD100 and LD50) activities of these compounds and their molecular connectivity indexes. These quantitative structure--activity relationships are discussed briefly in the light of general anesthesia theories.


Correlation of quinidine absorption with disintegration and dissolution rates. The dissolution profiles of four commercial quinidine sulfate tablets were determined using the USP rotating-basked dissolution apparatus. Substantial differences in dissolution half-times were noted and compared to previously reported disintegration times, absorption rate constants, and times of appearance of peak serum concentrations. Rank-order correlations were observed among all combinations of in vivo and in vitro parameters, indicating that the absorption rates of these tablets are controlled by both disintegration and dissolution.


Separation and spectrophotometric determination of theophylline and hydroxyethyltheophylline in a pharmaceutical syrup. A simple chromatographic procedure was developed for the separation and determination of theophylline and hydroxyethyltheophylline. An aliquot of diluted syrup was pipetted into a chromatographic column containing a strong anion-exchange resin, which retains theophylline and allows the hydroxyethyl derivative to pass through it. Theophylline was subsequently eluted with 1 N HCl and determined by difference spectrophotometry at 284 nm; its derivative was determined at 272 nm.


Use of MULTDOS for pharmacokinetic analysis of ethosuximide data during repetitive administration of single or divided daily doses. MULTDOS, a computer method to curve fit data obtained on multiple dosing, was used with either the 1969 or 1974 version of the NONLIN program to compare the pharmacokinetic parameters of ethosuximide during repetitive administration of single or divided daily doses. Elimination rate constants, excretion rate constants, and apparent volumes of distribution were similar between the two dosing regimens and essentially identical between the two nonlinear regression programs.


Synthesis and antimicrobial activity of ethyl N-aryl-S-(triphenylstannyl)isothiocarbamates. Five ethyl N-aryl-S-(triphenylstannyl)isothiocarbamates were synthesized by the reaction of triphenyltin iodide with the appropriate ethyl N-arylthiocarbamate in the presence of triethylamine. The IR spectrum of each compound was obtained over the 4000--200-cm--1 range, and some bands were assigned. These new compounds were found to be generally better antifungal agents than the previously tested N-substituted N'-cyano-S-(triphenylstannyl)isothioureas. The new compounds were also investigated for antibacterial activity and were especially inhibitory toward Gram-positive species. Except for their lower activity toward Bacillus subtilis, their antibacterial activity was identical to the previously tested N-phenyl-N'-cyano-S-(triphenylstannyl)isothiourea.


Antifungal properties of halofumarate esters. Alkyl esters (C1--C4) of the four halofumaric acids were tested for antifungal activity against Candida albicans, Aspergillus niger, Mucor mucedo, and Trichophyton mentagrophytes at pH 5.6 and 7.0 in the absence and presence of 10% beef serum in Sabouraud dextrose agar. The most toxic compound to each organism was: C. albicans, ethyl iodofumarate (0.054 mmole/liter); A. niger, methyl bromofumarate (0.090 mmole/liter); M. mucedo, methyl fluorofumarate (0.037 mmole/liter); and T. mentagrophytes, ethyl iodofumarate (0.020 mmole/liter). The order of overall activity of the six most toxic compounds was: ethyl iodofumarate greater than ethyl chlorofumarate greater than methyl iodofumarate = methyl bromofumarate greater than methyl chlorofumarate greater than bromofumarate.


Quantitative determination of acetaminophen in plasma. A simple method is described for the rapid quantitative analysis of acetaminophen in plasma. The acetaminophen and its conjugates present in the plasma following drug administration are hydrolyzed with 4 N HCl to p-aminophenol. This compound is coupled with 5% vanillin reagent to form a stable yellow color whose concentration is determined spectrophotometrically at 395 nm. Application of this method to a study of three dogs treated with 650 mg of acetaminophen is described.


Synthesis of 2-(substituted anilino) 4-(substituted phenyl)thiazoles. 2-(Substituted anilino) 4-(substituted phenyl)thiazoles were synthesized by condensing 2-haloketones with substituted thioureas. The biological screening of some compounds indicated hypoglycemic and hyperglycemic activity.


Synthesis and local anesthetic properties of secondary alkoxyalkylaminoacylanilides. Nineteen secondary alkoxyalkylaminoacylanilides were prepared. They were weaker bases and were more hydrophilic than the corresponding analogs lacking the ether function. In 2% solution, these compounds blocked the rat sciatic nerve in vivo after relatively short onset times with good frequency of anesthesia. The duration of block was 0.4--1.5 times that of lidocaine. Their systemic toxicity was low, and their irritation liability in most instances was acceptable.


Syntheses and pharmacological activity of N-acyl-substituted imidazolidinethiones and thioimidazolines. A series of imidazolidinethiones and thioimidazolines was synthesized and tested for their effect on both forced and spontaneous motor activities as well as their ability to raise the convulsion threshold. The N-acyl-substituted 2-p fluorobenzylthioimidazolines were the most active compounds, producing protection against maximal electroshock seizures and tonic convulsions induced by pentylenetetrazol. Both compounds had high LD50 values and safety indexes.


Preliminary pharmacokinetic model of pilocarpine uptake and distribution in the eye. A pharmacokinetic model that permits prediction of aqueous humor pilocarpine levels following topical application to rabbit eyes was developed. The model is able to account for changes in both instilled solution volume and drug concentration. The model, although simplified, relies mainly on experimentally verifiable and independently measured parameters. Its utility lies in its ability to account quantitatively for the large drainage loss of instilled drug solutions and its predictive ability regardless of the instilled volume or concentration. The framework established by this model will allow further sophistication as more experimental data become available and should be adaptable to other ophthalmic drugs.


Kinetics of drug transport and concurrent metabolism in human cell cultures I: theory. A method for evaluating the passive permeability and single-step metabolism of drugs in suspension cultures of mammalian cells was formulated assuming linear kinetics. It was assumed that the metabolizing enzymes are driven by endogenous substrates present in steady-state quantities. The presence of the drug in radiolabeled tracer quantities was assumed to cause only a small perturbation from the endogenous steady-state operating point. The time course solutions for the drug and its metabolite are given in terms of macroscopic constants, and their physical interpretations are given in terms of metabolic and transport parameters.


Dissolution and absorption of the antineoplastic agent ellipticine. Ellipticine, a poorly water-soluble alkaloid, is active in several experimental tumor systems. Marked solubility increases were produced by polyvinylpyrrolidone of varying molecular weights (10,000--160,000) and were optimal (approximately 13 mg/ml at 25 degrees) with polyvinylpyrrolidone mol. wt 10,000. Dissolution of ellipticine--polyvinylpyrrlidone (1:22 w/w) tablets in simulated gastric juice was superior to that of ellipticine hydrochloride polymers without affecting maximum dissolution at 37 degrees. Physiological disposition of ellipticine--polyvinylpyrrolidone was compared with that of the hydrochloride salt and ellipticine in suspension following oral administration at 250 mg/kg in fasted mice. In comparison to the suspension, ellipticine tissue levels were about threefold higher with polyvinylpyrrolidone or hydrochloride preparations. Antitumor activity of the three preparations was evaluated intraperitonneally and orally versus L-1210 leukemia. The optimal dose of ellipticine--polyvinylpyrrolidone and ellipticine hydrochloride was lower than that of the suspension and suggested improved absorption.


Spectrophotometric determinations of 3-dimethylaminomethylkhellin hydrochloride and khellin. Spectrophotometric assays are proposed for the determination of 3-dimethylaminomethylkhellin hydrochloride and khellin in bulk chemical and dosage forms. The acid dye method, using methyl orange at pH5, is applied to assay the amine in the form of an ion-pair extractable in chloroform with maximum abosrbance at 420 nm. The perchloric acid method, depending on formation and extraction of the oxonium salts of both compounds, is used to assay the amine and khellin at 333 or 430 nm and at 325 or 410 nm, respectively. The reineckate method can be used to assay the amine as the reineckate derivative in acetone with maximum absorbance at 530 nm. However, small amounts of the amine (1.5--3 mg) can be determined as the reineckate in methanol with maximum absorbance at 245 nm. Stability determination of the two compounds can be done by the acid dye and perchloric acid methods. The three methods are sufficiently accurate, sensitive, and precise.


Identification and quantitation of impurities in methotrexate. The impurities in bulk, commercial, and investigational methotrexate samples were chromatographically separated by gradient elution from a diethylaminoethylcellulose column. Besides the already known impurities in the bulk and commercial samples, two other impurities, 4-amino-N10-methylpteroyl-N-methylglutamine and 2,4-di-amino-N10-methylpteramide, were identified. A new batch of methotrexate, synthesized for the National Cancer Institute for investigational use only, showed a different spectrum of impurities. The purity of all methotrexate samples studied was about 80% calculated on an anhydrous basis.


Thermodynamics of aqueous solutions of parabens. The solubility of a related series of parabens was determined in water at four temperatures. The parabens chosen were the methyl through n-butyl p-hydroxybenzoates, and the temperature variations were 5 degrees increments from 25 to 40 degrees. These solutes are useful preservatives, especially combinations of the methyl and propyl ester derivatives. The chemical relationship of these compounds varied by successive linear methylene additions on the ester portion of the molecules. The thermodynamic values obtained for these aqueous systems could be related to these molecular variants since the remainder of the molecule was constant. For the overall thermodynamics, the free energy functions such as the ideal, actual, and excess were found to be smooth, nonlinear functions of the number of carbon atoms in the alkyl portion of the paraben esters. A linear relationship with the number of carbon atoms in the ester portion of these esters was found with the partial excess free energy of the solute.


Electron-capture GLC determination of ibuprofen in serum. To evaluate drug-protein binding, a sensitive method for the determination of ibuprofen in submilliliter amounts of serum was required. A specific and highly sensitive procedure, based on benzene extraction of the acidified specimen. TLC of the benzene extract residue, fomation of the pentafluorobenzyl esters of the materials eluted from the thin-layer chromatogram, and quantification of the pentafluorobenzyl esters by GLC, was developed. Utilizing electron-capture detection, the method is sensitive to 0.1 microgram of ibuprofen/0.1 ml of serum. Statistical analyses indicated an average recovery of 97.7% with a standard deviation of +/- 7.3%. Mass spectrometric analysis, in conjunction with GLC, confirmed the specificity of the method for the intact drug. The procedure was applied successfully to drug absorption and drug-protein binding studies in humans.


Effect of enantiomeric purity on solubility determination of dexclamol hydrochloride. The solubility of (+/-)-4a,13b-trans)-[3(OH),13b(H)-trans]-3-isopropyl - 2,3,4,4a,8,9,13b,14- octahydro-1H - benzo[6,7]cycloheptal[1,2,3-de]pyrido[2,1-a]isoquinolin-3-ol hydrochloride [(+/-)-I] was 3.3 mg/ml in pure water. Its resolved enantiomers exhibited about a fivefold increase in solubility. Dexclamol hydrochloride, the (+)-I enantiomer, is bioactive as a neuroleptic and is soluble to the extent of 16.4 mg/ml. However, raw material purity in relation to the amounts of (+/-)-I racemate present significantly influences solubility and optimum conditions for dissolution. The effect of trace amounts of (-)-I or (+/-)-I forms of the solubility of (+)-I enantiomer raw materials was explained through phase solubility studies. Conditions required for preparation of clear solutions using  raw materials of varying purity can be determined with derived expressions. The application and limitations of using an enantiomer as a tool to improve solubility are discussed.


Quantitative determination of chlordiazepoxide and its metabolites in serum by fluorescence TLC--densitometry. A sensitive fluorescence TLC--densitometric procedure was developed for the specific determination of chlordiazepoxide (I) and its two metabolites, demoxepam (II) and desmethylchlordiazepoxide (III), in serum. After extraction from serum with ether, I, II, and II were separated by TLC and converted with a sulfuric acid spray to pale blue (Rf 0.63), green (Rf 0.54), and blue (Rf 0.45) fluorescence spots, respectively. Quantitation was accomplished by scanning the plate with a densitometer at 390 (I), 430 (II), and 390 (III) nm. The sensitivities were 0.05 (I), 0.01 (II), and 0.01 (III) microgram/ml of serum. The procedure was successfully applied to measurement of I-III in human serum after oral administration of 20 mg of chlordiazepoxide hydrochloride.


Alanine derivatives with reactive groups. The synthesis of diazoketone analogs of amino acids and associated problems were investigated with N-phthaloyl-DL-alanine serving as a model. The carboxyl was activated by conversion to the acid chloride or, under mild conditions, to the mixed anhydride obtained with ethyl chloroformate or dicyclohexylcarbodiimide; the product was reacted with diazomethane. Deblocking the phthaloyl group with hydrazine gave 3-amino-1-diazo-2-butanone as a well-characterized solid salt and as a less stable oil. Further reactions of the blocked diazoketone of DL-alanine, such as conversion to alpha-haloketones, Wolff rearrangement, and deuterium exchange on C-1 were investigated. 3-A-mino-1-diazo-2-butanone had moderate inhibitory activity against mouse mammary adenocarcinoma in cell culture.


Simultaneous determination of reserpine and hydrochlorothiazide in two-component tablet formulations by high-performance liquid chromatography. A high-performance liquid chromatographic procedure is presented for the simultaneous determination of reserpine and hydrochlorothiazide in two-component tablet formulations. An aliquot of a tetrahydrofuran extract of the tablet, containing polylythiazide as an internal standard, is chromatographed on a microparticulate silica gel column using a mobile phase of 0.01% (v/v) diethylamine, 5% (v/v) chloroform, and 18% (v/v) 2-propanol in n-hexane. The relative standard deviations are 1.2 and 0.6% for the simultaneous determination of reserpine and hydrochlorothiazide, respectively. Seven commericial tablet formulations were found to contain 92.7--101.0% and 98.3--101.4% of the labeled amounts of reserpine and hydrochlorothiazide, respectively.


Relationships between chemical structure and inhibition of human placental choline acetyltransferase by keto analogs of acetylcholine. Seven keto analogs of acetylcholine were synthesized and evaluated as inhibitors of human placental choline placental choline acetyltransferase. Their potencies for inhibition of horse serum cholinesterase and stimulation of cholinergic receptors in the longitudinal ileal muscle of the guinea pig were investigated. The most potent and selective inhibitor of choline acetyltransferase was (2-benzoylethyl)trimethylammonium chloride with an I50 of 3 X 10(-6) M. It exhibited considerably low activities at muscarinic and nicotinic receptors and cholinesterases. Its high potency for inhibiting choline acetyltransferase was atrributed to: (a) its cationic terminal, a site for an electron acceptor interaction; (b) an aryl moiety for hydrophobic and electron donor contributions; and (c) a positive charge on the carbon atom adjacent to the benzene ring due to the presence of the carbonyl group, which interacts with the nucleophilic residue on the enzyme.


Spectrophotometric determination of isoniazid in presence of its hydrazones. A spectrophotometric determination of isoniazid in the presence of its hydrazones was developed. The method involves the reaction between isoniazid and 2,3-dichloro-1,4-naphthoquinone in the presence of ammonia in an ethanolic medium. The colored product has an absorbance maxium at 640 nm. The Lambert-Beer law is obeyed in the 1--14-microgram/ml range. The proposed method was applied to the analysis of isoniazid tablets. In commercial tablets, hydrazone formation due to the reaction between isoniazid and lactose was detected by TLC. The analysis of lactose-containing isoniazid tablets showed 10--22% lower recovery than that obtained by the official method. Hydrazone formation in tablets probably interferes with isoniazid bioavailability.


Mathematical basis of point-area deconvolution method for determining in vivo input functions. The point-area method for deconvolution derives a "staircase" input function which, when convolved onto the characteristic function, gives an output function coincidental with the given output data points. The area--area method for deconvolution is shown to be erroneous.


High-pressure liquid chromatographic determination of cycloheximide in ointment and suspension formulations. A high-pressure liquid chromatographic procedure is described for the assay of the antibiotic cycloheximidein bulk drug and two experimental formulations. The method utilizes a reversed-phase C18 chromatographic column and refractive index detection. Possible impurities or degradation products, isocycloheximide, anhydrocycloheximide, and dimethylcyclohexone, are well separated from cycloheximide by this procedure. Complete extraction of cycloheximide from the formulations was obtained. The assay has a relative standard deviation of approximately 1%.


Separation of multisized drug suspensions into narrow distributions by centrifugal elutriation. A centrifugal elutriator rotor was used to separate suspensions of micronized hydrocortisone acetate and prednisolone acetate. Up to five distinct particle fractions could beisolated from a single parent suspension, and reasonable reproducibility was exhibited between various elutriator runs. Arithmetic means and associated standard deivations, based on volume, were calculated for each isolated fraction. These data demonstrate the narrowness and uniqueness of each fraction. The particle-size data show an apparent log-normal distribution for each isolate. The uniqueness of each fraction also was demonstrated by dissolution experiments where Fraction 1, the smallest fraction of prednisolone acetate, dissolved much more rapidly than Fraction 4, the largest fraction. The substantial difference in dissolution behavior of these two fractions of prednisolone acetate powder was biopharmaceutically significant.


Liquid chromatography in pharmaceutical analysis IX: Determination of muscle relaxant--analgesic mixtures using normal phase chromatography. High-pressure liquid chromatography was used to optimize the resolution of eight widely prescribed therapeutic agents commonly found in muscle relaxant--analgesic mixtures. The compounds were chromatographed on normal phase porpous silica or cyanopropylsilane columns, using various solvent systems paired on the basis of Synder's solvent selectivity scheme to give a polarity index for each system of 3.3. A carisoprodol, phenacetin, and caffeine mixture was selected to demonstrate the utility of the separation and quantification method. The mixture was chromatographed on a porous silica column, using tetrahydrofuran--toluene (50:50) at a flow rate of 2.0 ml/min. Each determination can be achieved in approximately 8 min with an accuracy of 3--5%.


High-pressure liquid chromatographic analysis of melphalan in plasma. A new sensitive and rapid high-pressure liquid chromatographic determination of melphalan in plasma was developed. Recovery of 1 microgram added to 1 ml of plasma at 23 degrees was 94% but was greatly reduced at higher temperature. The method has been applied to plasma determinations of melphalan in rats and humans and is currently being utilized for human pharmacokinetic studies.


Hydrolysis and protein binding of melphalan. Melphalan (30 microgram/ml) is completely hydrolyzed in water at 37 degrees after 8 hr. At lower temperatures, hydrolysis proceeds at slower rates. The presence of bovine serum albumin retards hydrolysis of melphalan (30 microgram/ml) in water. The melphalan hydrolysis rate is directely releated to the bovine serum albumin concentration. At 37 degrees, 8 g of bovine serum albumin/100 ml of water gives a recovery rate of melphalan similar to that of human plasma. In vitro alkylation of melphalan at 37 degrees with human plasma containing 30 microgram/ml, calculated by equilibrium dialysis, methanol extraction, and high-pressure liquid chromatographic analysis, is 30% after 8 hr.


Elimination of a quaternary pyridinium salt delivered as its dihydropyridine derivative from brain of mice. 1-Methylpyridine-2-carbaldehyde oxime, a quaternary pyridinium salt, can be delivered efficiently through the blood-brain barrier in its dihydropyridine prodrug form. This redox system was used to study the elimination rate from the brain of a small quaternary salt. It was found that the oxime is eliminated relatively fast from the brain, which supports a hypothesis for the existence of an active transport mechanism for eliminating organic ions from the brain. The possibilities of using the pyridinium salt in equilibrium dihydropyridine redox system for specific delivery of drugs to the brain are discussed.


Permeability of interstitial space of muscle (rat diaphragm) to solutes of different molecular weights. The transport characteristics of muscle interstitial space were determined using an isolated rat diaphragm preparation. Permeability of the interstitial space for extracellular solutes is one-thirtieth to one-fiftieth that of an equivalent thickness of water. However, most of this low permeability can be accounted for by correcting for the tortuosity and relative volume of interstitial space. The estimated diffusivity of solutes (mol. wt. 100-70,000) in the interstitial space of muscle is only about one-half to one-fifth less than in water alone.


Synthesis of anticholinergic agents: N-methyl-4-piperidinyl alpha-benzoyloxy-alpha-cyclopentylphenylacetate salts. The synthesis of a new anticholinergic agent, N-methyl-4-piperidinyl alpha-benzoyloxy-alpha-cyclopentylphenylacetate, obtained by reacting N-methyl-4-piperidinyl alpha-cyclopentylmandelate with benzoyl chloride in the presence of methyllithium, is reported. This material may be useful as an antiperspirant.


Theoretical relationships between area under the curve and route of administration of drugs and their precursors for evaluating sites and pathways of metabolism. The bioavailability of a drug administered extrasystemically is a measure of the initial extraction of a compound by a series of eliminating events involving the intestinal mucosal enzymes, the gut bacterial microflora, the liver, and the lung. A theoretical analysis is presented to differentiate the process of gut wall elimination and hepatic removal of a drug during this first-pass effect. The area under the blood concentration--time curve (AUC) for a drug and its metabolite is shown to be useful in determining the presence of these processes when a drug and its metabolite are administered concomitantly by different routes of administration. Furthermore, the fraction of a precursor transformed to its metabolite also can be determined by pharmacokinetic analysis of the AUC of a drug and its metabolite after administration of both substances.


Electronic structure--activity relationships of antibacterial acridines. The antibacterial activity of a series of amino- and fluorinated acridines was studied in the framework of their electronic structures. To calculate the electronic structure, a simple Hückel molecular orbital theory was used. A statistical regression analysis revealed linear correlations between the activity and the electronic indexes, particularly the electron density at the ring nitrogen.


Synthesis and antitumor evaluation of 4-ethoxycarbonyl cyclophosphamide analogs. 4-Ethoxycarbonyl analogs of cyclophosphamide and its five-membered ring homolog were synthesized utilizing the cyclization method previously described. N,N-Bis(2-chloroethyl)-4-ethoxycarbonyl-1,3,2-oxazaphospholidin-2-amine 2-oxide demonstrated activity against L-1210 lymphoid leukemia whereas N,N-bis(2-chloroethyl)-4-(ethoxycarbonyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide did not. The oxazaphosphorin-2-amine was evaluated against human epidermoid carcinoma of the nasopharynx (cell culture). The results again were negative: ED50 = 2.8 X 10.


Simple and rapid high-pressure liquid chromatographic simultaneous determination of aspirin, salicylic acid, and salicyluric acid in plasma. A rapid and sensitive high-pressure liquid chromatographic assay was developed for aspirin, salicylic acid, and salicyluric acid in plasma. The procedure involves the solvent extraction of these compounds from plasma and separation using a reversed-phase column eluted by acidified aqueous acetonitrile. Small quantitites of aspirin can be assayed directly in the presence of a large quantity of salicylic acid. The assay is also free from blank interference.


Comparison of stationary phases in reversed-phase TLC for correlation between structure and biological response of probiotics. A series of C16,C18 and C20 fatty acids and their ethyl esters and alcohols were investigated as possible stationary phases in reversed-phase TLC for the correlation between structure and biological response (antistaphylococcal activity). Ten probiotics (omego-amino acids and their L-histidine dipeptides) were used as the biologically active compounds. The mobile phase was 70% acetone in water. The best correlations were obtained with hexadecanoic acid (palmitic acid) or cis9,cis-12,cis-15-octadecatrienoic acid, 1-hexadecanol or cis-9-octadeconol, and ethyl hexadecanoate for the fatty acids, their alcohols, and their ethyl esters, respectively. Among all compounds, the following relation was obtained: fatty acids = alcohols greater than ethyl esters greater than white paraffin oil.


Modified NF method for quantitative determination of pentaerythritol tetranitrate. A modified NF method for the quantitative determination of pentaerythritol tetranitrate is reported. A solution of powder is made directly from the dosage form in glacial acetic acid and is then reacted with phenoldisulfonic acid TS. The proposed method saves approximately 75% of the time required with the NF method. The results on six differnet commercial dosage forms with four different colors and three other active ingredients are reported.


Synthesis of an anti-inflammatory 10,10a-dihydro-1H,5H-imidazo[1,5-b)isoquinoline-1,3(2H)-dione. A new synthesis of imidazo[1,5-b]isoquinolines is reported. 2-[2-(Piperidino)ethyl]-10,10a-dihydro-1H,5H-imidazo[1,5-b]isoquinoline-1,3(2H)-dione hydrochloride was found to posses anti-inflammatory activity.


dc Polarographic determination of hydroxylaminoeverninomicin D. An analytical procedure for a tertiary alkylhydroxylamine, hydroxylaminoeverninomicin D, was developed. It involved the autoxidation of the compound in the presence ofCu(II) and subsequent polarographic reduction. Conditions are described for the quantitative determination of the possible impurities (everninomicin D and nitrosoeverninomicin D) in hydroxylaminoeverninomicin D.


Characterization of products derived from aprindine hydrochloride photolysis. An investigation of the products resulting from the photolysis of apridine hydrochloride is described. The compounds were characterized by GLC spiking experiments and combined GLC--mass spectrometry. In some cases, Rf values derived from TLC and/or preparative TLC and subsequent high-resolution mass spectral measurements also were employed for identification.


Determination of plasma hydrochlorothiazide levels in humans. A method for determining plasma hydrochlorothiazide levels was developed with a sensitivity of 5 ng/ml. Accuracy and precision were demonstrated over the 5--648-ng/ml range by an overall recovery of 95 +/- 8%. The detector response was linear for the 5--250-ng/ml range. The method was sufficiently sensitive for hydrochlorothiazide bioavailability studies and also was applicable for the determination of whole blood drug levels. Plasma levels in two subjects reached peak levels of 428 and 450 ng/ml at 2.5 and 2 h, respectively, after a 50-mg dose. Whole blood levels at 3 hr after the same dose were 547 and 851 ng/ml and were approximately 2.5 times the 3-hr plasma levels.


Determination of clonazepam in tablets by dc polarography. A simple and convenient method for the routine determination of clonazepam in tablets by dc polarography is described. Clonazepam is extracted from the sample by ethanol and diluted with pH 4.15 acetate buffer in a volumetric flask. The filtered solution is then polarographed at the dropping mercury electrode versus the saturated calomel electrode. The polarographic wave is well developed; the determination is quantitative, precise, and accurate.


Solubility of calcium oxalate in 1-alkanols and ethanol--water mixtures. Solubility profiles for calcium oxalate were determined in pure 1-alkanols and ethanol--water mixtures at 20 degrees. The magnitude of solubility in the aliphatic alcohols was highest in methanol and decreased in a nonlinear fashion as the dielectric constant decreased, going to n-decanol. In ethanol--water mixtures, the solubility increased nonlinearly with water content or increasing dielectric constants. The work factor for these systems was also calculated and, as expected, a "mirror image" to the solubility profiles was evidenced. In the mixed solvent system, a dramatic change in the magnitude of solubility occurred after a dielectric constant value of about 60, indicating ionic association or ion-pairs below this value. The Born relationship of solubility and ionic size was tested in the ethanol--water system, and the ionic size of the divalent species of about 2.27 A agreed with the theoretical ionic size of about 2.4 A. These results indicate that the water number of calcium is 8 or that a monolayer of dipoles surrounds this divalent cationic species.


Pyrrole, furan, and thiophene oxamates as potential antiallergy agents. Nine heterocyclic oxamic acid derivatives were synthesized and tested in the rat passive cutaneous anaphylactic assay as potential antiallergy agents. Some compounds also were tested for their effects on cholesterol-lipoprotein levels and for diuretic, antidiabetic, and antifertility activities in rats.


New compounds: isoquinoline derivatives as simple emetine models. Several new isoquinolines were prepared using Reissert compounds. A few compounds were screened, and they exhibited no antineoplastic activity.


Effects of organophosphate insecticides on the cholinergic receptors of frog skeletal muscle. The organophosphates octamethyl pyrophosphoramide, Bidrin, and phosphoric acid 2,2-dichlorovinyl dimethyl ester inhibit the membrane voltage response of frog sartorius muscles to carbamylcholine in a manner expected of either a slowly reversing competitive inhibitor or a noncompetitive inhibitor. The inhibition reverses with a time course of minutes, depending upon the temperature. The inhibition by these compounds is reduced by d-tubocurarine but is unaffected by hexamethonium. This may indicate that the organophosphate binding sites are near the cholinergic binding site since both d-tubocurarine and hexamethonium are competitive inhibitors of cholinergic agonists and d-tubocurarine is the larger compound.


Neostigmine-induced alterations at the mammalian neuromuscular junction. II. Ultrastructure. Brief and chronic exposure of rats to neostigmine methylsulfate produced marked morphological alterations of the fine structure at the end-plate region of the extensor digitorum longus muscles. These changes were dose and time dependent and were restricted primarily to the subjunctional myofibrillar apparatus and membrane-bound organelles. In addition, significant presynaptic alterations were observed including synaptic vesicle depletion and the appearance of numerous coated vesicles and membrane cisternae, which indicated continuing nerve terminal hyperactivity. With chronic treatment, degeneration and partial recovery of the nerve axon also were observed. The morphological changes of the end-plate region induced by neostigmine did not occur in most fibers after brief denervation and were eliminated entirely by chronic nerve section. Thus, the postsynaptic degenerative changes caused by neostigmine treatment observed in nondenervated animals appear to result primarily from greatly increased synaptic activity and not primarily from a direct neostigmine reaction with the pre- or postsynaptic membranes. Since the myopathic changes observed in this study were produced by neostigmine, a drug which is commonly employed in the routine treatment of human patients with myasthenia gravis, continued use of neostigmine for long-term therapy in noncrisis situations may not be accepted as being free from risk.


Adrenergic storage vesicles in rat heart: quantitation of membrane-bound and membrane-enclosed dopamine beta-hydroxylase. Norepinephrine-containing storage particles in rat heart have been disrupted to determine their stability and to quantitate the amount of dopamine beta-hydroxylase (DBH) that exists in a soluble, membrane enclosed pool. Vesicles were relatively resistant to lysis by hypotonic shock or by up to five cycles of freezing and thawing. These procedures have been shown to release catecholamines from adrenal chromaffin granules readily. However, rat heart vesicles were sensitive to temperature and they could be lysed by mechanical disruption using a sonicator or a Polytron homogenizer. Increasing the force or duration of the disruption resulted in an increased release of norepinephrine and DBH into a supernatant fraction after high-speed centrifugation. Extrapolation of the data from such experiments gave an estimate for membrane-enclosed DBH of 37% of the total amount of enzyme activity. This estimate of the proportion of membrane-enclosed enzyme is considerably higher than those previously reported for membrane-enclosed DBH in sympathetic neurons.


Effects of aprindine HCl on slow channel action potentials and transient depolarizations in canine Purkinje fibers. The electrophysiologic effects of aprindine hydrochloride were studied on normal Purkinje fibers, on Purkinje fibers superfused with Tyrode's solution containing 22 mM KCl and isoproterenol (1 X 10(-5) M) and on transient depolarizations induced by exposure to acetylstrophanthidin (1.7--2 X 10(-7) M). Aprindine (3 X 10(-6) M) significantly reduces the action potential amplitude and dV/dtmax and shortens the action potential duration but does not alter the resting membrane potential. Transient depolarizations were suppressed by aprindine at a dose of 2 X 10(-6) M. Isoproterenol (1 X 10(-5) M) failed to restore the transient depolarizations after suppression with aprindine. Slow responses induced in K-depolarized, isoproterenol-treated fibers were unchanged by aprindine (3 X 10(-6)-1 X 10(-5) M) in the presence as well as in the absence of acetylstrophanthidin. These experiments suggest that aprindine does not have slow channel blocking properties and that an inward current through the slow channel cannot be considered as the sole basis of the digitalis-induced transient depolarization.


Interaction of hydralazine and hydrazone derivatives with contractile mechanisms in rabbit aortic smooth muscle. The mechanism of action and relative potency of hydralazine (H) and tow hydrazone derivatives were investigated using isolated rabbit aortic strips. H, hydralazine acetone hydrazone (HA) and hydralazine butanone hydrazone (HBH) relaxed established K+ and norepinephrine (NE) contractures, and inhibited the development of contractures to these two agents on preincubation. H, HA and HBH increased the threshold to Ca++ and decreased the maximum tension responses during K+-Ca++-contractures (HA greater than H, P less than .05; HBH greater than H P less than .01). The Ca++-dependent and Ca++-independent components of NE contractures were both inhibited by H, HA and HBH. NE contractures were more sensitive to the effects of H than K+ contractures. These results are consistent with the conclusion that H and hydrazone derivatives produce effects on vascular muscle both by interactions with the fluxes of Ca++ from the extracellular space and effects on release from cell stores. However, other possibilities need to be assessed experimentally.


Enhanced exocytotic release of norepinephrine consequent to nerve stimulation by low concentrations of cyclic nucleotides in the presence of phenoxy-benzamine. Stimulation at 5 Hz of postganglionic nerves to the isolated guinea-pig heart which had been labeled with [3H]norepinephrine (NE) resulted in a proportional release of NE, total 3H, [3H]NE, and dopamine beta-hydroxylase. Phenoxybenzamine (3 micrometer) caused a significant increase in the release of all these indices of neurotransmitter release throughout a series of four consecutive stimulations. Stimulation in the presence of dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (1 X 10(-9) M) and 8-bromo cyclic guanosine 3':5'-monophosphate (8-bromo cyclic GMP) (1 X 10(-8) M) failed to alter any of the measured indices of release when compared with control. However, when perfused in combination with phenoxybenzamine, both cyclic nucleotide analogs significantly increased total 3H, [3H]NE, NE and dopamine beta-hydroxylase outflow with stimulation, as compared with phenoxybenzamine alone. Lower concentrations of both agents (1 X 10(-10) M dibutyryl cyclic AMP and 1 X 10(-9) M 8-bromo cyclic GMP) were less effective in augmenting release. The increased release of NE with nerve stimulation in the presence of phenoxybenzamine alone and with both phenoxybenzamine and the cyclic nucleotide analogs was associated with a significant increase in intraventricular pressure. In contrast, only the combination of 1 X 10(-9) M dibutyryl cyclic AMP plus phenoxybenzamine resulted in an increase in heart rate. The results suggest that phenoxybenzamine and the cyclic nucleotides, probably operating by two distinct mechanisms to enhance neurally mediated release, can act in concert to enhance neurotransmitter release when subeffective concentrations of cyclic nucleotides are used in conjunction with an effective concentration of phenoxybenzamine.


Calcium-mediated effects of carbachol on cation pumping and Na uptake in rat parotid gland. Carbachol (10(-4) M), a cholinergic secretagogue, significantly increased the ouabain-sensitive uptake of 86Rb by rat parotid gland slices. This effect was blocked by the omission of Ca from the bathing medium. When extracellular Na was decreased from 125 to 5 mM, the effect of carbachol was reversed (86Rb uptake was significantly decreased). Increasing intracellular Na (by incubation in medium lacking K) produced a significant stimulation of 86Rb influx. A role for Na in the response was suspected, and so the metabolism, of 22Na by the slices was characterized. The 60-minute distribution of 22Na could be described by three kinetic components; one extracellular (0.315 ml/g, tau = 1.94 minutes) and two intracellular (0.075 ml/g, tau = 7.63 minutes and 0.017 ml/g, tau = 58.4 minutes). Carbachol enhanced the uptake of 22Na into the intracellular components (primarily the 7.63-minute component). The increased uptake of 22Na required Ca in the bathing medium. These observations could be explained by assuming that cholinergic receptor activation stimulates Ca influx which in turn enhances Na uptake. The resulting elevated intracellular Na acts to stimulate activity of the Na,K pump.


Digoxin disposition kinetics in dogs before and during azotemia. The purpose of this study was to evaluate the disposition kinetics of digoxin after the administration of a single intravenous dose to the same dogs before and during azotemia. The digoxin plasma concentration-time data were fitted to a multicompartment model using nonlinear regression analysis. During azotemia, the biological half-life of digoxin was prolonged in six of seven dogs, while digoxin renal clearance, body clearance and apparent volume of distribution were significantly decreased. There was a corresponding increase in the apparent volume of the "central" compartment of digoxin. Approximately 45% of a digoxin dose was excreted by the kidney in these animals indicating a substantial nonrenal component to digoxin elimination in the dog. This nonrenal elimination did not change during azotemia, despite a decrease in renal clearance by 61%.


A radioimmunoassay for the determination of combined amitriptyline and nortriptyline concentrations in microliter samples of plasma. A sensitive radioimmunoassay for amitriptyline and nortriptyline in blood has been developed. The antibodies used in the radioimmunoassay were raised in a sheep against a conjugate of nortriptyline and bovine serum albumin. Using tritiated amitriptyline as the label, the assay is capable of detecting concentrations as low as 2.0 ng/ml in a 50 microliter sample of plasma. Cross-reactivity studies have demonstrated the specificity of the radioimmunoassay for both amitriptyline and nortriptyline, and comparison with gas-liquid chromatography assay has indicated the applicability of the assay to a routine situation. The radioimmunoassay has been used to study the plasma drug levels after single oral administration of amitriptyline to four volunteers. A wide variation in maximum drug concentrations, ranging from 18 to 62 ng/ml, was seen, with the time taken to reach the maxima ranging between 1.5 and 3 hours. A second concentration peak was seen in three of the volunteers, at 4 to 5 hours after ingestion of the drug.


Strategies for sentence perception.

Strategies for sentence perception. Three experiments are reported in which rapid sequential visual presentation of sentences--RSVP--was used to determine some perceptual processes in sentence perception. It was found that adjectives, when they are actually or potentially modifiers of a noun, are far less likely to be reported than any other part of the sentence. On the other hand, adjectives which are attribute adjectives are as likely to be reported as any other part of the sentence. Conclusions are drawn concerning sentence perception in general.


[Risks in modern diagnosis].

[Risks in modern diagnosis]. The first principle in diagnosis, as in treatment, is: nil nocere. Modern methods of diagnosis are becoming more and more technical. They often create physical and mental stress to the patient, and have undesirable side-effects of varying kinds and degrees. To be aware of contra-indications is important not only for the doctor but also for the patient, who must be informed of the risk of a diagnostic procedure. The data presented gives statistical information from which the doctor can see the number likely to be at risk from his diagnostic methods. The most common methods and their adverse effects are mentioned: intravenous use of X-ray contrast media, invasive tests (peripheral, cerebral, coronary, renal angiographs), laparoscopies with and without liver biopsies, kidney biopsies, endoscopies (oesophago-gastro-duodenoscopy, colonoscopy, retrograde cholangio-pancreatography), percutaneous lung biopsy and mammography. Although the mortality rates of all methods of diagnosis, even the most hazardous ones, are relatively low, all techniques create a stress to which not every patient is accustomed; they all demand considerable co-operation for which not every patient is prepared. There is now a trend towards non-invasive diagnostic techniques, creating little or no stress, but which nevertheless give at least a comparable level of diagnostic efficiency (e.g. ultrasonics, isotopes and computer tomography).


[The use of simultaneous angiography to determine the signification of vascular images in renal scintiscans with technetium (author's transl)]. Combined angiography and renal scintiscans were used by the authors in an attempt to determine the signification of the images and vascular tracings obtained after intravenous injection of technetium (99mTc). They conclude that the scintiscan images are essentially the reflection of cortical vascularization and that its duration, as determined by the scintiscan, is roughly equal to the mean transit time of the indicator employed (Tc). Arterial stenosis cannot be demonstrated by scintiscans using technetium.


[Angiographic abnormalities after punch-biopsy of the liver (author's transl)]. The authors report on 10 cases of complications during punch-biopsy of the liver, an arterio-portal fistula being the one most frequently encountered (8 out of 10 cases). The clinical condition improved in 9 cases out of 10. The authors stress the frequency of arterioportal fistula after punch-biopsy of the liver (60% it arteriography is carried out during the first week). They recall the benign character of these fistulae and their tendency towards spontaneous healing.


[Interest of a combination of film and screen reinforcer in mammography (author's transl)]. The authors present the results of their comparative experimental study on phantoms, of three mammographies. Their study clearly demonstrates the advantages gained by the use of combined film and screen reinforcer compared to classical radiographic procedures using fine-grain film without screen reinforcers. The quality of the images obtained appeared to them to be equal, if not superior, and this seemed to be confirmed by use in clinical practice. Irradiation doses are about one third of the usual dose. They conclude that it would be of value to use mammographs powered by strong generators, as the irradiation dose, under these conditions, would be reduced even further.


[Abnormal partial pulmonary venous return of the left lung in an adult (author's transl)]. The authors report a case of left-sided abnormal partial pulmonary venous return. This abnormality was discovered following weight loss with a strongly positive tuberculin skin test. A plain chest X-ray showed an externally convex opacity filling the aortico-pulmonary space, suggestive of a lymphadenopathy in that area. Pulmonary angiography and selective opacification of the different afferent branches of this abnormal vein defined the territory of drainage of this A.P.V.R.


[Gastroduodenal complications after the ingestion of hydrochloric acid. One case (author's transl)]. The ingestion of a caustic acid resulted in retractile fibrosis of 2/3 of the stomach with a secondary duodenal fistula. The antro-pyloro-duodenal circuit was re-established at around the 3rd after ingestion. However, the duodenal fistula caused the formation of encysted pelvic abscesses which were difficult to drain and satisfactorily treat. The clinical course stretched over a period of 4 years.


[An arterial catheter for multiple use in pediatrics (author's transl)]. The authors describe a pediatric catheter for use mainly in highly selective abdominal angiography (splenic, gastrica sinistra, and intestinal arteries). The catheter is made of polyurethane, is 65 cm long and is curved. It has a bore of 5 F and is introduced with a wo 32 guide, allowing a flow-rate of 10 ml/sec. The catheter point is polished by chemical means allowing penetration of the arterial wall without preliminary dilatation. This avoids vascular spasm and damage to the vascular wall at the site of puncture.


[Oesophageal perforations from instrumentation during high fibroscopy. Radiological signs (author's transl)]. The authors report on a survey of perforation following fibroscopy in the Hôpital Saint-Antoine. A total of 10,600 diagnostic examinations were reviewed and 5 perforations were recorded, to which must be added 2 perforations following oesophageal dilatation. A chesy X-ray, taken within 24 hours of the incident, showed, in most cases, the presence of air in the mediastinum with early signs of subcutaneous emphysema in the cervical or cervicothoracic regions. A hydro-pneumothorax in the right side of the chest was also observed in the cases studied, without air in the mediastinum. Oesophageal transit was studied by means of a water-soluble compound, and showed oesophageal effraction in only 4 cases, and in 3 of these cases it was possible to see the location of the orifice with precision.


[Cicatricial digitiform pseudopolyposis of the colon (author's transl)]. A report on a patient aged 30 years with cicatricial digitiform pseudopolyposis discovered ten years after a severe hemorrhagic rectocolitis in which almost complete functional healing had occurred. Cicatricial digitiform pseudopolyposis is a rare condition. We have found reports on only 12 cases in the literature from 1952. Recently, ten cases in 190 patients with Crohn's disease have also been reported. This particular type of pseudopolyposis appears in two different forms: diffuse or localized ("giant"). Diagnosis can be difficult if there is no history of colitis which is nearly always from hemorrhagic rectocolitis or Crohn's disease. The diffuse form can be confused with polyposis of the colon from any cause, while the localized "giant" form can evoke a tumour especially of the villous type. An essential diagnostic procedure is the anatomical and pathological examination of the polypoid mass by surgical biopsy. Cicatricial pseudopolyposis does not require surgical intervention except when rare complications such as invagination caused by the pseudopolypoid mass occurs. Radiologists should bear in mind this particular form of pseudopolyposis, as correct identification can avoid unjustified surgical intervention.


[X-ray examination of the sterno-manubrial joint in ankylosing spondylitis and Reiter's disease (author's transl)]. In 19 patients with ankylosing spondylitis, 11 presented some lesions of the manubrio-sternal joint. In 3 cases these lesions were more advanced than the sacro-iliac alterations which were very slight or even doubtful. In 8 patients with severe Reiter's disease, 7 developed an ankylosing spondylitis and 2 presented involvement of the manubrio-sternal joint 8 weeks, respectively 10 weeks after the appearance of the first symptoms.


[A new case of macrodactyly of the hand reported in France. An attempt at a statistical study (author's transl)]. A boy of 4 years presented with hypertrophy of the last three fingers of the right hand associated with syndactyly of the middle and ring fingers. The fingers were of adult length and wider. Arteriography did not show any evidence of an aneurism but demonstrated the existence of an extensive collateral circulation. Thermography confirmed the hypervascularization with the presence of hot points. The authors found reports on 118 cases in the literature published during the last century which could be analyzed statistically. There is a slight predominance in boys (55%) and on the right side. Two fingers are usually involved, the index and middle finger being those affected. Discussions as to aetiology (GOURNET) usually vary between a neurological cause with the possibility of a fibrolipoma of the middle finger being present, and it being due to a phacomatosis. Effective therapy in adults is amputation. In the young there are several possibilities; ablation of the neurovascular masses at the base of the fingers (even though the metacarpals are often involved), destruction of proliferating cartilage, etc.


Steroid hormones in the postovulatory follicle of the domestic fowl (Gallus domesticus). Progesterone, androstenedione, testosterone, oestrone and oestradiol were measured in the postovulatory follicle (POF) at various times up to 52 h after ovulation. The 3-fold decrease in progesterone, the major constituent, which occurred over the first 15--20 h resembled the changes previously described for the enzyme, 3beta-hydroxy-steroid dehydrogenase. At 1 h before ovulation the granulosa cells of the anteovulatory follicle (AOF) contained 50 times more progesterone than the POF granulosa fraction collected 2--3 h later. The thecal portion of the AOF had progesterone concentrations 5 times those of the POF theca, but the latter contained higher concentrations of androstenedione and oestrone.


The role of placental gonadotrophins (PMSG and hCG) in pregnancy in the rat. Treatment of rats with anti-PMSG and anti-hCG for 5 days from Days 6, 11 or 16 of pregnancy caused variable necrosis of fetal tissue, the extent of which increased from the chorionic cells to the inner trophoblastic layers with the increase in dose from 4 to 10 mg anti-PMSG. At 10 mg/day, fetal death and resorption were invariable in early to mid-pregnancy, whilst in late pregnancy some litters were carried to term. The main histological change in the corpora lutea was hyperaemia in the anti-PMSG-treated rats. Immunofluorescence studies with conjugates of anti-hCG and anti-PMSG showed sharp localization in the giant and chorionic cell layer at the feto-maternal junction. We conclude that an hCG/PMSG-like substance is produced by rat chorionic cells and that it plays an immunosuppressive role during pregnancy.


Branched-chain amino acid aminotransferase in mouse testicular tissue. Branched-chain amino acid aminotransferase (L-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest Km were obtained with L-leucine. Activity with L-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.


Uterine DNA synthesis and cell proliferation during early decidualization induced by oil in mice. [3H]Thymidine autoradiography was used to study cell proliferation during decidualization induced by intraluminal oil in ovariectomized mice treated with oestrogen and progesterone. Development of the decidual reaction involves two distinct populations of stromal cells. Periluminal cells start to synthesize DNA 11--15 h after instillation and by 17--20 h, without dividing, differentiate into epithelioid decidual cells which continue to incorporate [3H]thymidine, presumably becoming polyploid. Cells peripheral to this zone also start to synthesize DNA between 11 and 15 h, but at 18.5 h many have divided before differentiating. None of these dividing cells had been arrested in G2. The periluminal and peripheral cells do not appear to differ in their proliferative antecedents.


Cryogenic preservation of fish and mammalian spermatozoa. Various combinations of sucrose, reduced glutathione and potassium bicarbonate were tested for the cryogenic preservation of salmon spermatozoa. When a fast freezing procedure was followed, the extender that gave the best results was composed of 1 part of dimethyl sulphoxide (DMSO), as a protective agent, and 7 parts of a medium containing 125 mM-sucrose, 6.50 mM-reduced glutathione and 100 mM-potassium bicarbonate. Salmon and cod spermatozoa were kept frozen in this extender for 1 year. Freezing resulted in a reduction in the number of motile spermatozoa but had no significant effect on the degree of progression of motile spermatozoa or on their fertilizing capacity. When glycerol replaced DMSO in the extender and a slow freezing procedure was followed, similar results were obtained for the spermatozoa of bull or man; although the number of motile spermatozoa was reduced, there was no effect on the progressive motility score.


Effect of abdominal vagotomy of the pregnant rat on LH and progesterone concentrations and fetal resorption. Abdominal vagotomy on Day 8 of pregnancy in rats decreased the number of live fetuses at Day 16 and increased the number of resorbing fetuses. The activity of delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the corpus luteum and interstitial gland, LH and progesterone values in plasma and progesterone values in ovarian tissue were all lower in vagotomized rats than in sham-operated controls. Ovarian PGF levels were not affected. We suggest that these effects were caused by a direct effect of vagotomy on LH secretion which in turn lowers 3beta-HSD activity and progesterone levels in ovarian tissue and plasma, leading to fetal resorption.


Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro. Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.


The influence of the testis on the fertilizing life of spermatozoa in the ligated vas deferens of the golden hamster. Spermatozoa in the vas deferens of the hamster lose their fertilizing capacity 3 days after ligation of the initial part of the duct and after 2 days if the testes are removed at the time of ligation. Sham-ligation had no effect on the fertilizing life of vasal spermatozoa on the contralateral side even 3 days after bilateral castration. Unilateral castration for 3 days had no effect on the fertilizing capacity of spermatozoa from the ipsilateral unobstructed duct, whereas no eggs were fertilized by spermatozoa from the contralateral ligated duct associated with the remaining testis. Unlike testosterone, 5alpha-dihydrotestosterone injected daily or implanted subcutaneously in Silastic tubes maintained normal fertilizing capacity for 2 days in castrates and for 3 days in intact males. Within each group, ligation had no effect on the level of fructose in the seminal vesicle on that side compared with the level in the gland on the other side.


An ultrastructural study of the human micropolycystic ovary. The basement membrane of follicles in the micropolycystic ovaries of infertile women was thickened compared to that in the ovaries of normal women or those with typical polycystic ovaries.


Inhibition of testosterone 5alpha-reductase activity and growth of accessory sex tissues in castrated male mice. The steroid 4-androsten-3-one-17beta-carboxylic acid (17betaC) reduced the growth-promoting actions of testosterone, but not those of DHT in accessory sex tissues of castrated mice. The 5alpha-reduction of testosterone to DHT in these tissues was also reduced by 17betaC treatment, suggesting that DHT formation is a required step in the mechanism of action of testosterone.


Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during pregnancy in the guinea-pig. During unilateral pregnancy in the guinea-pig there is loss of formaldehyde-induced fluorescence from the adrenergic nerves supplying the uterus and its vasculature. This loss occurs initially near the site of implantation at about Day 20 of gestation and spreads progressively. Implantation of wax pellets containing progesterone into the uterine lumen or the gastrocnemius muscle of virgin guinea-pigs for 7 days produced loss of fluorescence from all local adrenergic nerves. No diminution of fluorescence was seen when pellets containing oestradiol were substituted. Chronic denervation studies showed that the adrenergic axons supplying the uterus and its arteries originated from both the ovarian artery and the pelvic region. Our results suggest that loss of adrenergic fluorescence within the uterus during pregnancy is due to an effect of placental progesterone which is localized to the uterus because the high concentration of progesterone necessary to cause fluorescence loss is not attained in the systemic circulation.


Inhibitors of adenylate cyclase from ejaculated human spermatozoa. Adenylate cyclase from ejaculated human spermatozoa was inhibited by fluoride, Cu2+, Zn2+, Ni2+ and several carboxylic acids.


Effect of the odour of female rat urine on plasma testosterone concentrations in male rats. Testosterone levels were significantly higher in males, regardless of sexual experience, exposed to the odour of urine from females than in those exposed to saline or kept in ordinary groups.


Effects of sexual stimulation on plasma levels of LH and testosterone in rams from high- and low-fertility flocks. Plasma LH and testosterone levels did not differ significantly between high and low fertility rams before or after sexual stimulation (ejaculation or teasing). Repeated stimulation caused significant elevation of mean plasma LH and an almost significant rise in testosterone concentration. Plasma testosterone peaks followed those of LH after 30-60 min. A single sexual stimulation did not always cause an LH peak or increase the mean LH level.


Induction of implantation in the mouse by intrauterine injection of adenosine monophosphate. Like cyclic AMP or dibutyryl cyclic AMP, intrauterine administration of AMP (non-cyclic) induced implantation in ovariectomized, progesterone-treated mice.


Evidence for a physiological role of prostaglandins in oviposition by the hen. A single oral dose of indomethacin (25 mg) given 2--4 h before expected oviposition delayed oviposition by about 12 h and suppressed plasma PGE levels by about 90%. Intrauterine injection of PGE-1 (0.2--0.4 microgram/hen) to 12 hens pretreated with 25 mg indomethacin induced oviposition in all birds in about 7 1/2 min. Passive immunization with goat antiserum to PGE-1 delayed oviposition (69%) in 4 hens. It is concluded that prostaglandins play a functional role in oviposition.


Demonstration of acrosin in mouse spermatozoa. Gelatinolytic activity of whole mouse spermatozoa was demonstrated by the gelatin film test. The presence of a trypsin-like protease (acrosin) in acidic extracts of mouse spermatozoa was shown by an electrophoretic method to separate the enzyme from a putative inhibitor.


Absence of effect of maternal immunization to paternal antigens on placental weight, fetal weight and litter size in the mouse. No evidence was obtained that immunization to paternal antigens affected placental weight, fetal weight or litter size on Day 18 of pregnancy in female mice.


Preovulatory secretion of gonadotrophins and TSH in cyclic rats with small lesions in the preoptic area. Animals with unilateral or bilateral lesions, with respectively 85 and 70% of intact preoptic area tissue remaining, returned to regular oestrous cycles. By comparison with sham-operated controls, rats with lesions had more days with a leucocytic smear in each of the 1st, 2nd or 3rd post-operative cycle, before returning to a 4-day cycle. Hormone measurements of blood collected during the afternoon of pro-oestrus showed that, in rats with bilateral lesions, the duration of the preovulatory LH secretion was significantly shortened, although peak levels were unchanged; the initial phase of the preovulatory FSH secretion was lost; and much of the prolactin surge was also inhibited. The TSH surge was unaffected.


A scanning electron microscope study of the hatching of bovine blastocysts in vitro. The blastocysts were recovered from cows 7--10 days after oestrus and cultured. The zona pellucida has a spongy fibrous structure. Hatching begins, about 24 h after culture, through a relatively small hole out of which the blastocyst appears to escape by its own activity. Later the zona becomes split and the two edges of the slit surround part of the blastocyst. The emerging cells were large or small blastomeres which were generally covered by a dense mass of microvilli.


Effects of progesterone and oestradiol-17beta on the spontaneous meiotic maturation of mouse oocytes. The spontaneous meiotic maturation of cumulus-free oocytes was not affected by progesterone or oestradiol-17beta at 2 microgram/ml, but both steroids decreased polar body production at 10 microgram/ml and progesterone decreased the number undergoing germinal vesicle breakdown.