Non-serotonergic 3H-ketanserin binding sites in human platelets: characteristics and interaction with calcium antagonists. Here we report the identification of binding sites for 3H-ketanserin in human platelet membranes. At 4 degrees C, 3H-ketanserin binding is saturable (Bmax = 0.58 pmol/mg protein), rapid (equilibrium being attained within 20 min) and reversible. The kinetics of the association and dissociation curves are consistent with the existence of a single class of binding sites, as confirmed also by computer-assisted analysis of the saturation curve. Specific binding is increased by Ca2+ and Mg2+. 3H-ketanserin binding is inhibited by serotonin (Ki = 48.5 microM), unlabeled ketanserin (Ki = 3-15 nM), as well as by another antiserotonergic drug, methysergide (Ki = 32.6 microM). However, other selective 5-HT2 ligands, such as ritanserin, spiperone and cyproheptadine fail to interact with 3H-ketanserin binding. On the contrary, tetrabenzine, a monoamine depleting agent, when preincubated at 30 degrees C, did inhibit the specific binding completely. 3H-ketanserin specific binding is inhibited in a dose-dependent fashion by some calcium blocking agents, with different potencies: verapamil (Ki = 2.25 microM), diltiazem (Ki = 139 microM) and SIM6080, a new Ca(2+)-antagonist related to the phenylalkylamines (Ki = 5.22 microM). Flunarizine inhibited 3H-ketanserin specific binding only at relatively high concentrations (IC50 greater than 100 microM), while nitrendipine did not show any inhibitory effect up to 20 microM. The present evidence indicates that all the sites labeled by 3H-ketanserin at 4 degrees C might be coincident with the monoamino transporter identified in other systems, and that they might play a role in the modulation of platelet aggregation exerted by some calcium blocking agents.


Vigabatrin does not affect the intestinal absorption of phenytoin in rat duodeno-jejunal loops in situ. The antiepileptic drug vigabatrin (GVG) is known to decrease significantly the serum concentration of concurrently administered phenytoin (PHT) in epileptic patients. To assess a possible mechanism for this interaction, the effect of GVG on the intestinal absorption of PHT was investigated by means of circulation experiments in an in situ rat duodeno-jejunal loop. GVG did not affect the rate of disappearance of PHT from the loop perfusing medium, providing evidence against occurrence of GVG-induced impairment of PHT absorption.


UN-1, a murine monoclonal antibody recognizing a human thymocyte undescribed antigen. A monoclonal antibody (MoAb), UN-1, specific for a human thymocyte cell surface antigen was produced. The same MoAb recognized in immunofluorescence also cells of the human T line HPB-ALL, while did not bind human peripheral blood cells and cells of some T, B and myeloid lines. Western blotting analysis of thymocyte membranes with MoAb UN-1 revealed a structure with a molecular weight of 150 kDa. Therefore both the pattern of expression and the size of the antigen recognized by MoAb UN-1 did not correspond to any previously described cell surface molecule.


Glucocorticoid-induced DNA fragmentation: role of protein-kinase-C activity. Glucocorticoid hormones (GCH) and IL-2 induce apoptotic cell death by a PKC-dependent mechanism. IL-4 counteracts apoptosis by inhibiting PKC activity. GCH and IL-2 show antagonistic effects on apoptosis when administered together. These data indicate that PKC activation in response to different stimuli can both enhance or reduce thymocyte survival.


The relationship of ideology to developing community health nursing theory. Ideology plays a major role in developing theory, guiding education, and directing practice in nursing. This study investigated the opinions of community health nurse educators regarding specific elements of community health nursing ideology and its relationship to the educators' opinions about the appropriateness of existing theory for professional use. The majority of participants agreed that a specific set of beliefs can be called a community health nursing ideology. Covariant factor analysis indicated that it is not a undimensional, internally consistent single set of beliefs or values, but rather is a set of two types of values, one focusing on the individual and the other on society. Individually oriented beliefs were positively related to satisfaction with existing theory. Socially oriented beliefs were negatively related to satisfaction with existing theory, but the relationship was not statistically significant. Anyone engaged in developing community health nursing theory would do well to consider which ideologic model is undergirding the process.


Toward redefining public health nursing in Canada: challenges for education. Faced with a recent shift from a hospital-based to a community-based health care system, Canadian public health nurses are being challenged to redefine their roles. To empower these nurses to respond effectively, it is imperative that the profession be reclarified as a specialty with a distinct philosophy and mission. Nursing education is key to facilitate this process, and public health nursing educators must lead the way. Certain barriers hinder the response of public health nursing education, and four areas are suggested in which efforts to advance public health nursing may be best directed.


Health maintenance, health promotion: is there a difference? Nurses in community settings are seeing more health care consumers who are medically stable and who are asking for assistance in areas such as exercise, stress reduction, nutrition, and illness and accident prevention. These requests can be characterized as concerns of health maintenance or health promotion. Two nursing diagnoses, altered health maintenance and health-seeking behaviors (specify), enable the practitioner to address these concerns in clinically useful ways. Through case studies and comparative tables, the differences between these two diagnoses is clarified to enable nurses to identify, treat, and evaluate efforts made to enhance health-oriented behaviors.


Secondary data on health-related subjects: major sources, uses, and limitations. Although secondary data are generally neglected by nurses, they are useful for nurse researchers, practitioners, educators, administrators, and policy makers. Major sources of data on health-related subjects are medical records, administrative records, the census, vital statistics, surveys of the National Center for Health Statistics, social science archives, and nursing archives, each with its specific uses and limitations.


Adaptation themes for prenatal care delivered by public health nurses. An adaptation process involving three cognitive themes provides the framework for community nursing model for prenatal care on the island of Hawaii. The themes are based on the cognitive adaptation model developed by Taylor (1983) and include search for meaning, sense of mastery, and self-esteem. Each theme is used to guide public health nurses and is identified during designated encounters between the nurse and woman throughout the duration of prenatal care. A focus on adaptation themes for each trimester of pregnancy expands on the medical model of prenatal care to highlight developmental processes of childbearing and broaden the scope of practice for public health nurses responsible for assessment and intervention in the community.


Maternal time and the care of disabled children. Caring for disabled children has become increasingly the responsibility of parents, even when the medical care is complex. To assess the time commitment required, 133 mothers of disabled children were asked to estimate by specific task categories the extra time required to care for the children. Total average daily care time was reported at 12 hours and 6 minutes, with 6 hours and 30 minutes consumed in "vigilant" tasks (i.e., watching a child who cannot be left alone and/or providing emotional support). Multiple regression analysis showed an increase in total caregiving hours associated with a younger child who was more physically and mentally impaired, and who required more medical treatments. The amount of time required by parents to care for their chronically ill children of necessity results in lost opportunities. Because this burden is experienced primarily by women and because professional nurses could alleviate some of the burden, this issue deserves further study.


Homelessness as a determinant of health. The homeless population in the United States is variously estimated at between 350,000 and 2.5 million. Families and children are among the fastest-growing segments of this population. A combination of social and economic changes occurring nationally have contributed to this phenomenon. Conditions associated with homelessness have a profound effect on the individual's ability to maintain health, to have access to treatment, and to recuperate. Several health problems are specific to the homeless, and have implications for public health practitioners.


Registered nurses' perceptions of their communication with Spanish-speaking migrant farmworkers in North Carolina: an exploratory study. Communication forms the foundation for all that nurses do. Recently, nurses working with migrant farmworkers have become challenged as the language of this population has become predominantly Spanish. To explore how nurses in North Carolina are managing communication issues, 55 nurses at 12 state health care agencies that serve migrant farmworkers was surveyed. Data were collected to determine what nurses believed about various communication issues involving these Spanish-speaking clients, including how they were presently communicating with them, in what areas of nursing functions they believed communication barriers were problematic, what they were doing to improve their communication, and what they envisioned the potential solutions were concerning communication barriers. Language differences proved to be substantial barriers to adequate nursing care for these individuals. Nurses cited many problems and said that they were receiving little assistance from their agencies to improve direct communication with the clients. Responses indicated a need for and a willingness by nurses to focus on improving communication with Spanish-speaking clients. Classes held at their agencies were seen as the most effective way of learning Spanish.


How elderly women cope: concerns and strategies. Forty-two elderly women were asked to describe the problems they faced with aging, and how they coped with both real-life and hypothetical problems in seven areas. Content analysis indicated that their spontaneously reported ongoing strategies were consistent with current theoretical formulations, the strategies differed by area of concern and for real-life versus hypothetical problems, and direct action strategies were related to lower levels of psychologic distress. These results have implications for nursing research and practice.


Approaching the media with confidence.

Approaching the media with confidence. The media are useful in communicating health information to the public. They are also a powerful way to promote and maintain positive nursing images. This paper examines the past portrayals of nurses in the media, explores the benefits of communicating nursing knowledge to the profession and to the community, and suggests strategies to access the media.


Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations. Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (alpha-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8-A9 was observed. Formation of this split product is species dependent: bovine greater than porcine greater than human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.


Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations. Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4-45 degrees C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures greater than or equal to 25 degrees C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.


Derivatives of 4-(2-N,N-di-n-propylaminoethyl)-5-hydroxyindole: synthesis and pharmacological effects. 5-Methoxy-1-methyl-4-(2-N,N-di-n-propylaminoethyl)indole (12) was synthesized from 5-hydroxyindole by a multistep synthesis. This target compound was designed as a bioisostere of "p-dimethoxy" catechol congeners of dopaminergic agonists derived from a variety of ring systems, in some of which p-dimethoxy-substituted systems are potent, active dopaminergic agonists. To complete the indole series, all possible combinations of N- and O-demethylated derivatives of 12 were prepared and were also evaluated pharmacologically. All members of this indole-derived series showed a low order of cardiovascular activity, which appeared to be independent of dopamine receptors. The lack of dopaminergic activity of 12 is cited as yet another example of the unpredictable effect of replacement of the catechol moiety of a dopaminergic agonist with a p-dimethoxy moiety.


Structural modification study of anthracyclinones: synthesis and biological activity of several derivatives of eta-pyrromycinone. On the basis of the N-O-O triangular pharmacophore hypothesis postulated earlier in our laboratory, selected side chains with or without the nitrogen atom at the strategic position were incorporated to eta-pyrromycinone, one of the anthracyclinones derived from the antibiotic cinerubins. Since none of the anthracyclinones (the aglycones of anthracyclines) were reported to have antineoplastic activity, the validity of the proposed hypothesis could be tested. Results indicated that a compound designed in this manner, 1,4-bis[2-(2,2-dimethyloxazolidin-3-yl)ethylamino]-1,4-didehydr oxy-eta- pyrromycinone (9c) possessed both in vitro and in vivo antineoplastic activity comparable to that of mitoxantrone. The structure-activity relationship of this class of compounds is discussed.


Improved delivery through biological membranes. XLV. Synthesis, physical-chemical evaluation, and brain uptake studies of 2-chloroethyl nitrosourea delivery systems. The dihydropyridine in equilibrium with pyridinium redox chemical delivery system (CDS) was supplied to two 2-chloroethylnitrosoureas, i.e., HECNU and CCNUOH, and the physicochemical properties of the delivery systems were studied to assess their potential as improved delivery forms to the CNS. Detailed physicochemical evaluation and brain uptake studies were performed on one of the delivery systems (CCNUOH-CDS) derived from trans-4-hydroxy-CCNU, an active metabolite of CCNU. Two aqueous-based formulations derived from hydroxypropyl-beta-cyclodextrin (HP beta CD) and Tween 80:ethanol:water system were developed for CCNUOH-CDS to overcome the poor aqueous solubility conferred upon it by its high lipophilicity. The formulations enabled a 200- to 400-fold improvement in the water solubility of CCNUOH-CDS. Dose- and vehicle-dependent comparative tissue distribution studies in rats indicated improved brain-to-organ ratios of the delivery system at lower doses.


Parenteral formulation of the kappa agonist analgesic, DuP 747, via micellar solubilization. The nonopioid kappa agonist analgesic amine, DuP 747, as a hydrochloride salt exhibited an aqueous solubility of 3 mg/ml. This solubility was insufficient to provide the desired dose in a solution formulation for intramuscular administration. Aqueous solutions of the hydrochloride salt exerted surface activity behavior; however, the critical micellar concentration (CMC) was not reached at the saturation solubility. Enhanced aqueous solubility required to reach the CMC could lead to micellization of the compound and a possible i.m. solution formula. The methanesulfonate salt was more water soluble than the hydrochloride salt and yielded a micellar solution with a concentration of 60 mg/ml.


Acceleration of heat transfer in vial freeze-drying of pharmaceuticals. II. A fluid cushion device. A simple device for the improvement of freeze-drying efficiency is described. The device is an aluminum foil bag which contains a small amount of glycerin. The device can be either reusable or disposable. When placed on a freeze-drying tray the liquid is about 1 mm thick. When vials are placed on the device it conforms to the shape of the vial bottoms. Since both the aluminum foil and the glycerin are better heat conductors than a vacuum, the device improves heat transfer from the shelf to the vial. Drying times obtained with and without the device are compared for different sizes as well as different types of vials. In most cases the use of the device reduces the drying time by nearly a factor of two. The use of the device also increases vial-to-vial uniformity and minimizes the effect of spillage.


Microencapsulation of solid dispersions: release of griseofulvin from griseofulvin:phospholipid coprecipitates in microspheres. The preparation, characteristics, and behavior of microspheres of poly(L-lactic acid) (PLA) containing griseofulvin (Gris) or Gris:phospholipid coprecipitates are described. Microspheres were spherical and increased in size from 17 microns (empty) to 30 microns, containing 22% Gris. The release of coprecipitated Gris after 60 min from 146,000 MW PLA microspheres in pH 2.0 buffer at 37 degrees C was twofold greater than that from microspheres containing pure Gris. Also, the release profile from pure Gris microspheres was 25% lower than its dissolution profile, whereas the dissolution and microspheres of Gris coprecipitate suspended in PEG 600 in hard gelatin capsules for 1 week released Gris at levels comparable to the dissolution of coprecipitate. Decreasing the MW of PLA substantially increased the release of Gris from microspheres of coprecipitate after 20 min but insignificantly from microspheres of pure Gris. These findings suggest that microsphere formulation offers some new opportunities in the development of solid dispersions which normally encounter processing difficulties.


The pharmacokinetics of pulmonary-delivered insulin: a comparison of intratracheal and aerosol administration to the rabbit. The pulmonary deposition and pharmacokinetics of insulin, administered via an endotracheal tube as an aerosol and instillate, in formulations containing either 113mIn-DTPA or 99mTc-DTPA (for gamma scintigraphic imaging) have been studied in four male New Zealand White rabbits. Using a randomized crossover design, the pharmacokinetics of intravenous insulin were also characterized. Recovery of immunoreactive insulin after nebulization was greater than 90%, indicating that the aerosolisation procedure did not cause appreciable insulin degradation. Gamma scintigraphy demonstrated that the penetration index (peripheral:central deposition) for the aerosolized formulation (1.52) was much greater than that for the instillate (0.32). Gamma scintigraphy also allowed exact quantification of the dose deposited after aerosol administration and thus permitted accurate determination of bioavailabilities. The bioavailable fraction for aerosolized insulin was 10-fold greater than for instilled insulin (57.2 vs 5.6%). Mucociliary clearance was likely to be greater for the instillate since it showed a preferential central deposition; this may account for the lower bioavailability. Insulin pharmacokinetics from both pulmonary formulations were absorption rate limited, resulting in postpeak half-lives which were approximately 20-fold greater than the intravenous elimination half-life (3 min). The apparent absorption rate constants resulting from instillation and aerosolisation were statistically equivalent (0.015 and 0.011 min-1, respectively). Mucociliary clearance of insulin would result in an overestimation of the true absorption rate constant; hence if mucociliary transport were greater for the instillate, then the true airways to blood transfer rate constant will be higher for the aerosolized formulation.


Immobilization of active urokinase on albumin microspheres: use of a chemical dehydrant and process monitoring. A method of immobilizing urokinase on albumin microspheres has been developed. Laser scattering, which was used to follow particle size from the initial emulsification stage to the final aqueous resuspension of the microsphere stage, showed that particle coalescence and crosslinking were critical parameters in manufacturing the microspheres. Chemical dehydration with 2,2-dimethoxypropane was used to convert an albumin emulsion into an albumin suspension and to reduce coalescence. An optimal amount of dehydrant produced 0.3-micron particles which resisted a 50 degrees C temperature challenge. Since oil/glutaraldehyde emulsion resulted in large particles with no urokinase activity, the cross-linking concentration of glutaraldehyde was reduced by solubilizing 25% (w/v) glutaraldehyde in the oil phase with n-propanol. A concentration of 0.015% (v/v) glutaraldehyde effectively immobilized urokinase and stabilized albumin microspheres. Amidolytic activity using the specific chromogenic substrate for urokinase, S-2444, showed that enzyme activity could be retained during this glutaraldehyde cross-linking.


Macrophage activation by polymeric nanoparticles of polyalkylcyanoacrylates: activity against intracellular Leishmania donovani associated with hydrogen peroxide production. Nanoparticles of polyalkylcyanoacrylates (PACA) can be useful carrier for the targeting of antileishmanial drugs into macrophages and also possess significant antileishmanial activity by themselves. No significant difference in antileishmanial activity could be detected between nanoparticles of five PACAs with differing alkyl side chains, suggesting that the main degradation products of PACA are not involved in their antileishmanial action. The effect of polyisohexylcyanoacrylate (PIHCA) on the induction of the respiratory burst in a macrophage-like cell line (J774G8) was assessed in non-infected macrophages and in macrophages infected with amastigotes of Leishmania donovani infantum, by measuring nitroblue tetrazolium (NBT) reduction and hydrogen peroxide production. Phagocytosis of PIHCA nanoparticles led to a respiratory burst, which was more pronounced in infected than in uninfected macrophages. The production of reactive oxygen intermediates associated with the respiratory burst was inhibited by addition of superoxide dismutase and catalase to the cell suspensions. The addition of catalase to the culture medium together with PIHCA nanoparticles significantly reduced the antileishmanial activity of PIHCA. Moreover PIHCA nanoparticles did not induce interleukin-1 release by macrophages. It is suggested that the antileishmanial action of PIHCA and other PACA nanoparticles results from the activation of respiratory burst in macrophages.


The mobile order theory versus UNIFAC and regular solution theory-derived models for predicting the solubility of solid substances. The theory of mobile order of Huyskens is tested against the UNIFAC model, the regular solution model, and the extended Hildebrand or Hansen solubility approaches in predicting the solubility of naphthalene in both polar and nonpolar solvents at 40 degrees C. While all models correctly predict the solubility in nonpolar and moderately polar solvents, a substantial improvement is achieved by Huyskens' model, particularly in alcohols. This improvement originates from the correct description of the entropy effects as well as of the hydrophobic effects in the particular case of the alcohols. The model necessitates the knowledge of only one parameter not known a priori, i.e., the naphthalene modified nonspecific solubility parameter, the value of which is deduced from its solubility in hexane.


Cholesterol (thermodynamic) activity determinations in bile salt-lecithin-cholesterol systems and cholesterol-rich liquid crystalline mesophase formation. Previous in vitro studies have shown that tauroursodeoxycholate (TUDC)-lecithin (L) micellar solutions solubilize cholesterol (Ch) poorly compared to its 7 alpha-epimer, taurochenodeoxycholate (TCDC). However, in clinical studies ursodeoxycholic acid (UDC) has been found to be as effective as chenodeoxycholic acid (CDC) in Ch gallstone dissolution, and it has been suggested that, during UDC therapy, liquid crystalline mesophase formation may be involved in enhancing micellar Ch dissolution and dispersion. The purpose of the present study was to investigate whether measurements of the Ch thermodynamic activity (A(T) would provide new insights into the problem of Ch solubilization and mesophase formation in bile salt-lecithin-Ch systems. Using the silicone polymer uptake method developed in this laboratory, A(T) was measured as a function of Ch concentration in the TUDC-L-Ch and TCDC-L-Ch model bile systems. In the TCDC systems Henry's law was obeyed almost up to unit activity (i.e., A(T) was proportional to Ch concentration almost up to A(T) = 1.0). However, in many of the TUDC-containing systems negative deviations from Henry's law were observed well below unit activity and these systems became visibly turbid before saturation with respect to cholesterol monohydrate (ChM) was reached. The effects of varying the TCDC/TUDC ratio upon the A(T) behavior were also studied. With increasing TCDC/TUDC ratio, the onset of mesophase formation was shifted to higher A(T) values. A(T) measurements were also conducted in BS-L-Ch mixtures simulating biles of patients undergoing UDC therapy. The results obtained suggest that mesophase formation may not always occur in biles of patients undergoing UDC therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


Plasma concentration clamping in the rat using a computer-controlled infusion pump. We have developed a computer-controlled infusion pump to achieve rapidly and then maintain stable plasma thiopental concentrations in rats. Initially we derived the parameters of a triexponential pharmacokinetic model for thiopental, administered as a brief infusion to 10 rats, using nonlinear regression and standard pharmacokinetic equations. These parameters were incorporated into the pharmacokinetic model of a computer-controlled infusion pump. In a second group of animals this device was used to maintain three consecutive target thiopental concentrations ranging from 5 to 100 micrograms/ml in a stepwise fashion. Arterial blood gases were kept normal through controlled ventilation when necessary. The plasma thiopental concentrations in this second group of animals were generally higher than the target concentrations. The bias in pump performance (median prediction error) was +25%, and the inaccuracy (median absolute prediction error) was 26%. We fit the parameters of a three-compartment model to the plasma thiopental concentrations observed in the second group of animals. This produced a second set of thiopental pharmacokinetic parameters with the unique characteristic of having been derived from a computer controlled infusion study. These parameters were tested prospectively with a computer-controlled infusion pump in a third group of animals. This second set of thiopental pharmacokinetic parameters performed better, with a median prediction error of 0% and a median absolute prediction error of 15%. This study shows that it is possible to achieve rapidly and maintain steady plasma thiopental concentrations in the rat. Our results suggest that it is feasible to derive robust pharmacokinetic parameters from unusual drug dosing approaches, such as employed by a computer-controlled infusion pump.(ABSTRACT TRUNCATED AT 250 WORDS)


Stability of the new anticancer platinum analogue 1,2-diaminomethyl-cyclobutane-platinum(II)-lactate (lobaplatin; D19466) in intravenous solutions. The chemical stability of the new anticancer platinum analogue 1,2-diaminomethyl-cyclobutane-platinum(II)-lactate (D19466) in infusion media was studied in an accelerated stability testing experiment with a selective HPLC-UV method. Variables were time, temperature, light, concentration, and infusion mixture. Mean reaction rate constants of decomposition were, respectively, 0.9555 *10(-2), 2.127 *10(-2), and 4.221 *10(-2) hr-1 at 37, 56, and 66 degrees C at a concentration of 200 mg/L in normal saline. From the Arrhenius equation, shelf lives (5% loss) at 4, 22, 37, and 121 degrees C were, respectively, calculated to be 41.6, 13.2, 5.7, and 0.15 hr. Mean reaction rate constant in 5% dextrose was 3.106 *10(-2) hr-1 (200 mg/L; 56 degrees C) and differed from that in normal saline (P less than 0.005). Mean reaction rate constant in Ringer lactate was 2.084 *10(-2) hr-1 (200 mg/L; 56 degrees C) (P greater than 0.05). There was no influence of normal daylight on the rate of decomposition. It is recommended to prepare D19466 infusions in normal saline. Chemical stability is then maximal 12 hr at room temperature or 24 hr at 4 degrees C. No protection against normal daylight is required. Sterilization by heat is not possible.


Simultaneous microdetermination of rifampin, deacetylrifampin, isoniazid, and acetylisoniazid in plasma by liquid chromatography with dual electrochemical and spectrophotometric detection. A rapid liquid chromatographic method for simultaneous determination of isoniazid (INH), acetylisoniazid (AINH), rifampin (RIF), and deacetylrifampin (DARIF) in microsamples of deproteinized plasma is described. The compounds and internal standard (IS) (diphenylcarbazide) were separated on a 10-microns, 8 mm x 10-cm phenyl Radial Pak cartridge in conjunction with a binary linear gradient system at a flow rate of 3 ml/min. A dual electrochemical (+800 mV) and spectrophotometric (334 nm) detection system with a computerized data station was employed to measure the above compounds in the effluent. Prior to injection, the plasma sample was diluted (2:1) with a pH 3, 0.075 M phosphate buffer after adding the internal standard (6.67 micrograms/ml of plasma) and passed through an Amicon Centrifree-MS filter at 2000g. Under these conditions, no interference in the analysis was observed, and the retention times of AINH, INH, IS, DARIF, and RIF were 3.95, 4.89, 15.82, 17.25, and 19.34 min, respectively. The linearity of the assay for all four compounds was excellent (r greater than 0.9925), and the between- and within-day CV was not greater than 8% at any concentration. This method is currently being used for therapeutic monitoring and pharmacokinetic studies of INH, RIF, and their major metabolites in patients with tuberculosis.


Pharmacodynamics of insulin following intravenous and enteral administrations of porcine-zinc insulin to rats. Previous work from this laboratory showed site-dependent variations in the apparent permeability of insulin as measured using the everted rat gut sac technique, with the greatest permeability in the distal jejunum and the lowest in the duodenum (5). To quantify better the rate and extent of insulin absorption from the small intestine, closed-loop in situ experiments were performed in nondiabetic rats. Results correlated with the everted gut sac technique in that the absolute bioavailability determined in situ was higher for insulin solution administered to the more distal region of the intestine (0.133%) than that absorbed from an earlier portion of the intestine (0.059%). While the difference in regional bioavailabilities was not significant (P = 0.08), the blood glucose response showed highly significant differences (P = 0.0015), with severe and prolonged hypoglycemia resulting from insulin delivered to the distal jejunum/proximal ileum. Insulin administered iv followed a two-compartment pharmacokinetic model. Whole-body elimination rate constants were similar for both iv and enteral insulin. Although therapeutic quantities of insulin were absorbed from the distal small intestine, absorption enhancers would be necessary to decrease the dose of insulin required.


Measurement of serum [3H]tetracycline kinetics and indices of kidney function facilitate study of the activity and toxic effects of bisphosphonates in bone resorption. The [3H]tetracycline ([3H]TC) model is based on the observation that TC is released from the bones of rats prelabeled with [3H]TC via first-order kinetics, a factor directly reflecting the kinetics of bone resorption. In the present paper we applied the [3H]TC elimination model to rats treated with antiresorptive drugs. The validity of this model was evaluated by examining the effect of the bisphosphonate, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (ABP), and a novel bisphosphonate, dihydrogen disodium adipoylbisphosphonate (AdBP), on serum TC levels and the elimination rate constant. ABP and AdBP significantly inhibited the TC elimination rate. However, ABP treatment caused impairment of bone mineralization, renal dysfunction, and inhibition of somatic growth. It is concluded that antiresorptive effects of bisphosphonates could be evaluated by the [3H]TC model, but this model is limited to animals with normal kidney function. The experimental conditions provide a technically simple method which is sensitive enough to examine antiresorptive properties in a healthy animal and to detect adverse effects on the kidney. The activity of the novel bisacylphosphonate, AdBP, and lack of its adverse effects indicate the potential of this drug for clinical applications.


Enhancement of nasal delivery of a renin inhibitor in the rat using emulsion formulations. Nasal absorption of O-(N-morpholino-carbonyl-3-L-phenylaspartyl-L-leucinamide of (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane (I), a renin inhibitor, was evaluated in two rat nasal models, one involving surgery and the other requiring no surgical intervention. Oleic acid/monoolein emulsion formulations were tested along with a control PEG 400 solution. The percent absolute bioavailability of the compound was enhanced from 3-6% (PEG 400 solution) to 15-27% when the emulsion formulations were used. The different nasal model techniques (with and without surgery) did not produce any statistical difference in the absolute bioavailability values for I. Emulsion formulations did not produce appreciable damage as assessed morphologically. It is suggested that emulsion formulations containing membrane adjuvants such as oleic acid and monoolein can be used to enhanced the nasal delivery of low-bioavailable, lipid-soluble drugs.


The transport barrier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit. The transport barrier of the epithelial presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal approximately nasal greater than or equal to bronchial greater than or equal to tracheal greater than vaginal greater than or equal to rectal greater than corneal greater than buccal greater than skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)


Quantitative evaluation of aqueous isopropanol enhancement of skin flux of terbutaline (sulfate). I. Ion associations and species equilibria in the formulation. It has been reported previously that saturated terbutaline sulfate in aqueous isopropanol significantly enhances the terbutaline flux through human skin in vitro. This paper demonstrates that the effect of isopropanol on the permeant species in the formulation contributes to the flux enhancement. This demonstration is based on studies involving measurements of conductivity and pKa as well as NMR spectroscopy in isopropanol-water mixtures. Increasing isopropanol concentration inhibits the proton dissociation of terbutaline and results in the ion associations between the protonated terbutaline and its counterion, sulfate anion. The species present in the formulation include protonated terbutaline, the negatively charged terbutaline-sulfate (1:1) ion pair, and the neutral terbutaline-sulfate (2:1) ion triplet. The results of the studies provide the basis for a quantitative evaluation of the species equilibria in solutions of terbutaline sulfate. The saturated terbutaline sulfate in 60% isopropanol produces the maximum concentration of the neutral ion triplet. This result is almost parallel to the terbutaline skin flux, which maximized at 60-80% isopropanol.


Sonophoresis. II. Examination of the mechanism(s) of ultrasound-enhanced transdermal drug delivery. We have shown previously that high-frequency ultrasound (sonophoresis) can significantly enhance the transdermal delivery of a topically applied drug in vivo and that the augmentation of transport was caused by the action of the ultrasound on the skin. However, these earlier experiments did not reveal (i) the mechanism of sonophoresis, (ii) the pathway of drug permeation under the influence of ultrasound, and (iii) any potentially detrimental effects of the enhancement procedure on skin structure and morphology. In the study reported here, these three key issues have been addressed using electron microscopy to follow the penetration of an electron-dense, colloidal tracer (lanthanum hydroxide; LH). Experiments have again been performed using the hairless guinea pig animal model. Colloidal LH suspensions were applied to skin sites, which were then immediately exposed to ultrasound (at 10 or 16 MHz) for 5 or 20 min. Passive transport of LH under identical conditions (but without ultrasound) provided the control measurements. Tissue processing after the treatment periods utilized standard electron microscopy staining procedures. We found the following: (1) LH does not permeate the skin by passive diffusion; under the influence of ultrasound, on the other hand, it penetrates through the stratum corneum (SC) and the underlying viable epidermal cell layers via an apparently intercellular route. (2) LH transports through the epidermis to the upper dermis, even after only 5 min of ultrasound treatment, a remarkable and unexpected finding.(ABSTRACT TRUNCATED AT 250 WORDS)


Brain parenchymal metabolism of 5-iodo-2'-deoxyuridine and 5'-ester prodrugs. In an attempt to generate derivatives of 5-iodo-2'-deoxyuridine (IDU) with enhanced blood-brain barrier (BBB) permeability, a series of 5' ester prodrugs of IDU was synthesized and their metabolism studied in rat brain homogenate and its different subcellular fractions. The rate of hydrolysis was dependent on the steric and polar nature of the ester substituent. Ester hydrolyzing activities were associated primarily with the cytosolic fraction and were due mainly to the presence of cholinesterases as confirmed by inhibition experiments performed with different esterase inhibitors. The metabolism of IDU to 5-iodouracil (5-IU) by the cytosolic fraction, in the presence and absence of specific pyrimidine nucleoside phosphorylase inhibitors, also suggests that there are two specific enzyme systems catalyzing two different metabolic processes. IDU 5'-esters competitively inhibit the metabolism of IDU and the inhibitory effect depends on the affinity of a particular ester toward the enzyme and also on the rate by which the ester itself undergoes hydrolysis. In the absence of any 5'-ester, 95% IDU was metabolized within 6 hr. However, in the presence of an eightfold molar excess of butyryl-IDU, the hydrolysis of IDU was completely inhibited over a 6-hr time period.


Disposition, bioavailability, and serum protein binding of pentachlorophenol in the B6C3F1 mouse. The toxicokinetics of pentachlorophenol (PCP) were studied in B6C3F1 mice, a strain in which PCP was previously found to be carcinogenic. In a crossover design, doses of 15 mg/kg were given intravenously (bolus) and orally (gastric intubation) to six animals. Concentrations of PCP in blood, urine, and feces were measured by capillary gas chromatography with electron-capture detection. After intravenous administration, the values of clearance and volume of distribution were 0.057 +/- 0.007 L/hr/kg and 0.43 +/- 0.06 L/kg, respectively. These two parameters exhibited low intermouse variability (coefficients of variation less than 14%). The elimination half-life was 5.2 +/- 0.6 hr. After oral administration, the PCP peak plasma concentration (28 +/- 7 micrograms/ml) occurred at 1.5 +/- 0.5 hr and absorption was complete (bioavailability = 1.06 +/- 0.09). The elimination half-life was 5.8 +/- 0.6 hr. Only 8% of the PCP dose was excreted unchanged by the kidney. PCP was primarily recovered in urine as conjugates. A portion of the dose was recovered in urine as the mutagen, tetrachlorohydroquinone (5%) (TCHQ), and its conjugates (15%). For both PCP and TCHQ, sulfates accounted for 90% or more of the total conjugates (glucuronides and sulfates).


Pharmacokinetic analysis of the structural requirements for forming "stable" analogues of valpromide. The following valpromide (VPD) analogues were synthesized and their structure-pharmacokinetic relationships explored: 3-ethyl pentanamide (EPD), methylneopentylacetamide (MND), 1-methyl cyclohexanecarboxamide (MCD), cycloheptanecarboxamide (CHD), and t-butylacetamide (TBD). Two aliphatic (EPD and MND) and two cyclic amides (MCD and CHD) underwent complete or partial conversion to their corresponding acids. The only amide found in this study to be "stable" to the amide-acid biotransformation was TBD. It also had the lowest clearance and the longest half-life and mean residence time. Unlike the other investigated amides, TBD contained two substitutions of two methyl moieties at the beta position of its chemical structure. A "stable" valpromide analogue must have either two substitutions at the beta position, such as in the case of TBD, or a substitution in the alpha and beta positions, such as in the case of the VPD isomer valnoctamide (VCD). This paper discusses the antiepileptic potential of stable VPD analogues which may be more potent and less teratogenic than their biotransformed isomers.


Pilocarpine disposition and salivary flow responses following intravenous administration to dogs. Oral doses of pilocarpine increase salivary flow rates in patients afflicted with xerostomia (dry mouth). This study examined the pharmacokinetics of and a pharmacodynamic response (salivation) to intravenous pilocarpine nitrate administration in dogs. Disposition was linear over a dose range of 225-600 micrograms/kg; plasma concentrations were 10-120 micrograms/L. Elimination was rapid and generally biphasic, with a terminal elimination half-life of approximately 1.3 hr. The systemic clearance of pilocarpine was high (2.22 +/- 0.49 L/kg/hr) and its steady-state volume of distribution (2.30 +/- 0.64 L/kg) was comparable to that of many other basic drugs. All doses of pilocarpine induced measurable submaxillary and parotid salivary flow rates which could be maintained constant over time. Cumulative submaxillary saliva flow was linearly related to total pilocarpine dose. Plasma pilocarpine concentration was linearly related to both steady-state and postinfusion submaxillary salivary flow rates.


Nonlinear disposition of intravenous 2',3'-dideoxyinosine in rats. The pharmacokinetics of 2',3'-dideoxyinosine (ddI) were examined in rats given intravenous doses of 8, 40, or 200 mg/kg. The concentrations of ddI in whole blood and plasma were identical. The concentration decline was multiexponential, with mean half-lives of 2 and 20 min for the first and second phases, respectively. At the highest dose, a slower third phase with a half-life of 56 min was observed. The total-body clearances were 99, 77, and 37 ml/min-kg for the 8, 40, and 200 mg/kg doses. The steady-state volume of distribution showed a trend for a decrease with increasing doses, but the difference was not statistically significant. Twenty-four-hour urinary recovery of unchanged drug for the three doses was similar at about 20%, suggesting that a major fraction of the dose was metabolized. Urinary excretion of ddI metabolite, hypoxanthine, accounted for less than 5% of the dose. Renal and metabolic clearances decreased with increased doses. ddI was metabolized in blood; the addition of inorganic phosphate, a cosubstrate in phosphorylase-mediated nucleoside catabolism, enhanced the degradation by about fourfold. In summary, these data indicate equal distribution of ddI in the extracellular and intracellular spaces in blood, its enzymatic degradation in blood, and nonlinear elimination kinetics.


Is ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] unusually reactive for an imide? The hydrolysis of ICRF-187 and two model compounds, 4-methylpiperazine-2,6-dione (4-MP) and 3-methylglutarimide (3-MG), was investigated over the neutral to alkaline pH range at 25 degrees C and an ionic strength of 0.5 (sodium chloride). The purpose of the study was to correlate the influence of molecular changes to the reactivity of these imides. Additionally, an improved chromatographic resolution of all the components of the degradation and NMR confirmation of the identity of the degradation products are presented. Based on the study of 4-MP, which is essentially half of an ICRF-187 molecule, and 3-MG, which has a carbon in place of the piperazine nitrogen, several conclusions can be drawn with regard to the stability of ICRF-187. The tertiary piperazine nitrogen/s of 4-MP and ICRF-187 contributed to the base-catalyzed hydrolysis of these compounds above pH 7 and caused a significant decrease in the pKa values of the imide moiety of ICRF-187 and 4-MP compared with 3-MG. One 2,6-piperazinedione ring of ICRF-187 was shown to affect only minimally the rate of hydrolysis of the second ring. ICRF-187 hydrolyzes by parallel consecutive pathways forming two monoacids with one ring opened and, subsequently, the diacid with both rings hydrolyzed.


Formation of the racemic compound of ephedrine base from a physical mixture of its enantiomers in the solid, liquid, solution, or vapor state. Physical mixtures (conglomerates) of the two enantiomers of ephedrine base, each containing 0.5% (w/w) of water, were observed to be converted to the 1:1 racemic compound in the solid, liquid, solution, or vapor state. From a geometrically mixed racemic conglomerate of particle size 250-300 microns (50-60 mesh), the formation of the racemic compound follows second-order kinetics (first order with respect to each enantiomer), with a rate constant of 392 mol-1 hr-1 at 22 degrees C. The reaction appears to proceed via the vapor phase as indicated by the growth of the crystals of the racemic compound between diametrically separated crystals of the two enantiomers in a glass petri dish. The observed kinetics of conversion in the solid state are explained by a homogeneous reaction model via the vapor and/or liquid states. Formation of the racemic compound from the crystals of ephedrine enantiomers in the solution state may explain why Schmidt et al. (Pharm. Res. 5:391-395, 1988) observed a consistently lower aqueous solubility of the mixture than of the pure enantiomers. The solid phase in equilibrium with the solution at the end of the experiment was found to be the racemic compound, whose melting point and heat of fusion are higher than those of the enantiomers. An association reaction, of measurable rate, between the opposite enantiomers in a binary mixture in the solid, liquid, solution, or vapor state to form the racemic compound may be more common than is generally realized.


Aqueous stability and solubility of CI-988, a novel "dipeptoid" cholecystokinin-B receptor antagonist. The aqueous solubility and solution stability of the N-methylglucamine and sodium salts of CI-988 (CI-988 NMG and CI-988 Na) were evaluated to aid in the development of a parenteral formulation for preclinical and clinical testing. CI-988 ([R-(R*,R*)]-4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)- carbonyl]amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) is a selective "dipeptoid" cholecystokinin-B receptor antagonist. The shape of the pH-solubility profile, generated at 30 degrees C, is consistent with the ionization of the terminal carboxyl group (pKa of 4.34). The pH-rate profile is independent of the salt form and is well described by two reaction pathways: spontaneous or water-catalyzed degradation of the nonionized form and specific base-catalyzed catalyzed degradation of the ionized form. The primary mechanism of degradation from the former pathway is consistent with intramolecular, carboxyl-assisted, amide-bond cleavage, whereas the primary mechanism of degradation from the latter pathway appears to be intramolecular cyclization to a hydantoin product with expulsion of 2-adamantanol. The pH dependencies of the solubility and stability show that a simple aqueous buffered solution of CI-988 has a predicted t90 of 2.1 years and a solubility of 0.94 mg/ml at pH 6.5, the theoretical pH of maximum stability, and 30 degrees C.


Protein-peptide interactions as probed by tryptophan fluorescence: serum albumins and enkephalin metabolites. Binding of Leu-enkephalin and the enkephalin metabolite, tyrosine-glycine-glycine (TGG), to bovine serum albumin (BSA) was studied as a model to investigate protein peptide interactions. TGG and Leu-enkephalin quench the tryptophyl fluorescence of BSA. Stern-Volmer quenching constants were typically in the range of 40 to 300 M-1, depending on the experimental conditions. The addition of Cu(II) or Ni(II) did not change the quenching constant, indicating that TGG does not compete for the metal binding sites on BSA. From fluorescence quenching studies with TGG, tyrosyl-glycine, tyrosine and glycyl-glycine, it was concluded that the presence of the tyrosine residue is required for the observed quenching. The phenolic group in tyrosine accounted for the quenching mechanism because phenol was efficient in quenching BSA fluorescence, whereas phenylalanine had no detectable effect. A large solvent isotope effect on the quenching constant of phenol and TGG with BSA strongly suggests an active role of the -OH functionality in the quenching mechanism.


Structural specificity of mucosal-cell transport and metabolism of peptide drugs: implication for oral peptide drug delivery. The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/tripeptide)-type drugs with or without an N-terminal alpha-amino group, including beta-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Polypeptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.


An implantable dosage form for the treatment of bone infections. The object of this investigation was the development of an implantable sustained-release dosage form, for the treatment of bone infections. Cross-linked polydimethylsiloxane (PDMS) was used as the matrix material. The drug delivery system was prepared by incorporating tobramycin, as a free base (C18H37N5O9.H2O) or as a sulfate salt [(C18H37N5O9)2.5H2SO4], into the matrix and molding into spherical beads. Following in vitro studies, the cumulative amount of drug released when plotted as a function of the square root of time was linear for both the base and the salt. The addition of glycerol to the matrix substantially accelerated the rate of drug release and the plots of cumulative amount of drug released continued to be linear as a function of the square root of time. The glycerol-incorporated beads swelled in contact with the aqueous medium but a negligible amount of glycerol was released even after exposure to the medium for 20 days. 13C solid-state and high-resolution NMR studies indicated that a fraction of the added glycerol participated in the cross-linking reaction of the polymer. The effect of the initial molecular weight of PDMS and the effect of the concentration of the cross-linker on the kinetics of drug release were investigated.


Transport of thyrotropin releasing hormone in rabbit buccal mucosa in vitro. The transport of thyrotropin releasing hormone (TRH) in rabbit buccal mucosa in vitro has been investigated with respect to (a) rate and type of metabolism of TRH on mucosal and serosal sides of buccal mucosa, (b) mechanism of TRH transport including charge effect on its permeability, and (c) pathway and rate-limiting regions of TRH movement. In addition, the integrity of excised buccal mucosa has been evaluated for purposes of in vitro solute diffusion experiments using tissue ATP level data, transmission electron microscopy, and TRH transport kinetic data. The results indicate that excised rabbit buccal mucosa can be used for TRH diffusion studies for approximately 6 hr. In addition, TRH apparently traverses buccal mucosa by simple diffusion with a steady-state permeability of about 10(-7) cm/sec, and this permeability is independent of pH. Moreover, the primary pathway appears to be via the intercellular space in the rate-limiting barrier, i.e., the upper 50 microns of the epithelium. Finally, TRH is degraded predominantly by deamidase activity, which is followed by, to a lesser degree, carboxypeptidase metabolism.


Assessment of the twin impinger for size measurement of metered-dose inhaler sprays. The calibration of the first stage of the twin-stage impinger, an instrument proposed for use in measuring the spray size from metered-dose inhalers, was performed with monodisperse aerosols by a standard technique for cascade impactors. The mean cut point was found to be not particularly sensitive to operating variables which may be expected to occur in practice. The cut point was close to that reported previously, although the collection efficiency curve was found to be slightly sharper. Calculations are reported on the expected results of measurements on aerosols in a two-stage instrument with an idealized perfect collection efficiency curve as well as the curve measured for the twin impinger. These results indicate that important characteristics of spray size distribution cannot be distinguished with an ideal two-stage instrument; the twin impinger is less capable than an ideal instrument.


Calcium alginate beads as core carriers of 5-aminosalicylic acid. The utilization of calcium alginate beads as core carriers for delayed dissolution followed by burst release as a potential method of intestinal site specific drug delivery was investigated. 5-Aminosalicylic acid was spray-coated on dried calcium alginate beads and then coated with different percentages of enteric coating polymer and/or sustained-release polymer. Beads coated with more than 6% (w/w) methacrylic copolymer plastisized with dibutyl sebacate and triethyl citrate resisted release in 2-hr acid fluid challenge and allowed immediate dissolution upon transfer to simulated intestinal fluid. With 6% (w/w) methacrylic copolymer on top of 4% (w/w) ethylcellulose polymer, the major portion of drug did not release in 2 hr of acid treatment or the next 3 hr of simulated intestinal fluid treatment. This dosage form provides the possibility to deliver drug to the lower intestinal tract with minimal early release, followed by sustained release in the colon.


Indomethacin-loaded microspheres: design and preparation by a multiple-emulsification technique and their in vitro evaluation. A new oral controlled-release drug delivery system was developed with two polymers using a multiple-emulsification technique. Powdered drug was dispersed in methyl cellulose sol, which was emulsified in ethyl cellulose solution in ethyl acetate. The primary emulsion thus formed was reemulsified in aqueous medium. During this phase, discrete microspheres were formed under optimized conditions. The size distribution of the microspheres was investigated, and scanning electron microscopy revealed the surface topography of the microspheres. The in vitro drug release followed first-order diffusion-controlled dissolution. More than 85% of the drug was released over 6 hr at pH 6.2 for all dissolution batches.


Evaluation of mucoadhesive polymers in ocular drug delivery. II. Polymer-coated vesicles. Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P less than 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.


Percutaneous absorption and metabolism of lonapalene in psoriatic skin. The percutaneous absorption and metabolism of lonapalene (6-chloro-2,3-dimethoxynaphthalene-1,4-diol-diacetate; RS-43179), a topically effective 5-lipoxygenase inhibitor, has been measured in six subjects with stable plaque-type psoriasis of the lower extremities. Lonapalene readily penetrates psoriatic skin, is rapidly and completely metabolized, and is almost entirely excreted in the urine. Unexpectedly we observed a trend for thigh (T) plaque skin to be more permeable than lower leg (LL) plaque skin as measured by total absorption (T, 44.8 +/- 13.4%; LL, 24.9 +/- 12.6% applied dose excreted), peak plasma levels (T, 209 +/- 107; LL, 146 +/- 81 ng Eq/ml), and peak rate of urinary excretion (T, 591.7 +/- 112.2; LL, 318.4 +/- 143.9 micrograms Eq/hr). There were also differences in the metabolic profiles between the two sites as measured by the quantity and proportion of dealkylated and conjugated products excreted in the urine.


Correlation between the inhibitory effects of basic drugs on the uptake of cardiac glycosides and taurocholate by isolated rat hepatocytes. The role of the multispecific bile acid transporter for cardiac glycoside uptake is still controversial. This study was designed to examine the inhibitory effects of basic drugs (verapamil, dipyridamole, nifedipine, chlorpromazine, disopyramide, quinidine, propranolol, and lidocaine) on taurocholate uptake by isolated rat hepatocytes and to compare these effects with inhibition of ouabain uptake. Sodium-dependent taurocholate uptake was significantly reduced, to 50-70% of the control value, by 50 microM verapamil, dipyridamole, and nifedipine. Sodium-independent taurocholate uptake was more extensively inhibited, to 20-40%, by these basic drugs. The inhibition of ouabain uptake correlated better with sodium-independent taurocholate uptake (gamma = 0.918) than with sodium-dependent taurocholate uptake (gamma = 0.714). Taurocholate competitively inhibited ouabain uptake in the absence of sodium. These results indicate that the cardiac glycoside transport system is similar to the sodium-independent taurocholate transport system.


Cyclodextrins as nasal absorption promoters of insulin: mechanistic evaluations. The safety and effectiveness of cyclodextrins (CD) as nasal absorption promoters of peptide-like macromolecules have been investigated. The relative effectiveness of the cyclodextrins in enhancing insulin nasal absorption was found to be in the descending order of dimethyl-beta-cyclodextrin (DM beta CD) greater than alpha-cyclodextrin (alpha-CD) greater than beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) greater than gamma-cyclodextrin (gamma-CD). A direct relationship linking absorption promotion to nasal membrane protein release is evident, which in turn correlates well with nasal membrane phospholipid release. The magnitude of the membrane damaging effects determined by the membrane protein or phospholipid release may provide an accurate, simple, and useful marker for predicting safety of the absorption enhancers. In order to estimate further the magnitude of damage and specificity of cyclodextrin derivatives in solubilizing nasal membrane components, the enzymatic activities of membrane-bound 5'-nucleotidase (5'-ND) and intracellular lactate dehydrogenase (LDH) in the perfusates were also measured. HP beta CD at a 5% concentration was found to result in only minimal removal of epithelial membrane proteins as evidenced by a slight increase in 5'-ND and total absence of LDH activity. On the other hand, 5% DM beta CD caused extensive removal of the membrane-bound 5'-ND. Moreover, intracellular LDH activity in the perfusate increased almost linearly with time. The cyclodextrins are also capable of dissociating insulin hexamers into smaller aggregates, and this dissociation depends on cyclodextrin structure and concentration. Enhancement of insulin diffusivity across nasal membrane through dissociation may provide an additional mechanism for cyclodextrin promotion of nasal insulin absorption.


In vivo percutaneous absorption of hydrocortisone: multiple-application dosing in man. Percutaneous absorption of hydrocortisone was measured in six healthy adult men from whom informed consent had been obtained. The study compared a single topical dose to multiple-topical dose treatments (one vs three applications) on the same day. 14C-Labeled hydrocortisone in acetone was applied to 2.5 cm2 of ventral forearm skin and protected with a nonocclusive polypropylene chamber. The amount of 14C measured in urine collected over 7 days was used to determine hydrocortisone absorption. The treatments, performed 2 to 3 weeks apart, each utilized adjacent sites on the same individuals. A single dose of 13.33 micrograms/cm2 delivered 0.056 microgram/cm2 of hydrocortisone through the skin. When the single dose was tripled to 40 micrograms/cm2, the amount delivered through the skin increased by nearly three times, from 0.056 to 0.140 micrograms/cm2; the expected delivery was 3 x 0.056 micrograms/cm2 = 0.168 microgram/cm2. Three serial doses of 13.33 micrograms/cm2 (total, 40 micrograms/cm2) were also expected to deliver 0.168 micrograms/cm2 with or without soap and water washing between doses, but the observed amount of hydrocortisone delivered through the skin significantly exceeded our expectations. This indicates that multiple-dosing treatments resulted in a significant increase in bioavailability. It is postulated that increased vehicle application and washing dissolved and mobilized previously dosed hydrocortisone and increased bioavailability.


Effects of transdermal penetration enhancers on the permeability of shed snakeskin. The effects of Azone and lauryl alcohol on the permeability of shed snakeskin were examined. Permeability of a variety of compounds through shed snakeskin was increased after Azone or lauryl alcohol pretreatment but the magnitude of the enhancement varied depending on the lipophilicity and the molecular size of the permeant. It was found that the shed snakeskin became more permeable after Azone or lauryl alcohol pretreatment, with a greater permeability increase for more hydrophilic and larger-molecular size permeants. As has been shown for untreated shed snakeskins, both the lipophilicity and the molecular size of the permeants are important in skin penetration and in determining the effects of transdermal penetration enhancers.


Enhanced delivery of 5-iodo-2'-deoxyuridine to the brain parenchyma. 5'-Ester derivatives of 5-iodo-2'-deoxyuridine (IDU) with varying degrees of lipophilicity were examined to evaluate the effectiveness of lipophilic ester prodrugs for enhanced and sustained delivery of IDU to the brain parenchyma. Approximately 1.0% (1.0 +/- 0.19; n = 4) of the total radioactivity was found in the brain at 30 min following intravenous administration of the lipophilic benzoyl-5'-ester of 125I-labeled IDU, whereas IDU per se yielded only 0.01% (0.01 +/- 0.06; n = 4). Since the IDU 5'-esters generated significantly higher levels of IDU in the brain, an HPLC analysis of IDU in the presence of 5'-esters and the metabolite 5-iodouracil was developed to characterize IDU uptake in the brain. The drug was detected at levels of 6.6 and 9.5 micrograms/g of brain tissue at 3 hr following intravenous administration of valeryl and benzoyl IDU, respectively, at a dose level of 40 mg/kg IDU equivalent each. IDU, on the other hand, when injected at a similar dose level, produced concentration levels below 0.01 micrograms/g of brain tissue, which was too low to be detected accurately by the HPLC assay. These results suggest that the 5'-ester derivatives cross the blood-brain barrier effectively and generate significantly higher brain levels of the parent drug in the brain parenchyma. The regenerated hydrophilic drug because of its polarity is "locked in" the brain and is subsequently metabolized by pyrimidine phosphorylase to 5-iodouracil.(ABSTRACT TRUNCATED AT 250 WORDS)


Direct suppression of phagocytosis by amphipathic polymeric surfactants. Recent studies have demonstrated that phagocytosis of colloidal particles by the mononuclear phagocytes of the liver and spleen can be controlled by either coating or stabilizing particulate carriers with the amphipathic polymeric surfactants, F108 and T908. These surfactants consist of copolymers of polypropylene oxide (PPO) and polyethylene oxide (PEO) and, when adsorbed to particulate surfaces, significantly decrease sequestration of particulates by the mononuclear phagocytes (MPS) of the liver. To evaluate these observations further, murine peritoneal macrophages were incubated for varying periods with surfactant-coated and noncoated polystyrene particles (PSPs). Phagocytosis was monitored using gamma counting and quantitative fluorescence microscopy. The data show that phagocytosis is decreased when PSPs are coated with F108 and T908. In addition, suppression of phagocytic activity was observed when cells were pretreated with the surfactant and then challenged with noncoated particles. The data confirm previous observations that polymeric surfactants consisting of PEO and PPO protect particulate carriers from rapid uptake by the MPS of the liver. Further, F108 and T908 suppress phagocytosis directly without affecting the integrity, viability, or functional state of the cell.


Nasal membrane and intracellular protein and enzyme release by bile salts and bile salt-fatty acid mixed micelles: correlation with facilitated drug transport. The effects of four bile salts, one fusidate derivative, and one mixed micellar formulation of bile salt-fatty acid combination on the nasal mucosal protein and enzyme release have been investigated in rats using an in situ nasal perfusion technique. Deoxycholate (NaDC) was found to possess the maximum protein solubilizing activity, followed by taurodihydrofusidate (STDHF), cholate, glycocholate (NaGC), and taurocholate (NaTC) in a descending order. The difference in protein solubilization of NaDC and NaGC was further characterized by the release of 5'-nucleotidase (5'-ND), a membrane-bound enzyme, and lactate dehydrogenase (LDH), an intracellular enzyme, in the perfusate. While both NaDC and NaGC caused comparable 5'-ND release from nasal membrane, intracellular LDH release was significantly higher with NaDC. The greater protein and LDH solubilizing effects of NaDC corresponded well with its faster rate of disappearance from the nasal perfusate. Therefore, the dihydroxy bile salt NaDC tends to cause intracellular damage and cell lysis, whereas the trihydroxy bile salt NaGC appears to produce primarily mucosal membrane perturbations. Linoleic acid in the form of soluble mixed micelles with glycocholate caused a further increase in nasal protein release. However, the rate and extent of nasal membrane protein release by the mixed micelles composed of 15 mM glycocholate and 5 mM linoleic acid were significantly lower than those caused by either deoxyholate or STDHF at the same concentrations. Nasal absorption of acyclovir, a non-absorbable hydrophilic model antiviral agent, was found to be enhanced in the presence of conjugated trihydroxy bile salts and bile salt-fatty acid mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of gastrointestinal site of drug delivery on the absorption characteristics of ranitidine. The absorption characteristics of ranitidine after delivery to three locations in the gastrointestinal tract were compared in an open-label study of eight healthy males. Subjects received ranitidine HCl (150 mg) for injection via a nasoenteric tube directly into their stomach, jejunum, or cecum sequentially in three separate periods (24 hr apart). Plasma samples were collected at periodic time intervals for 12 hr following each dosing and analyzed for ranitidine concentration by high-pressure liquid chromatography. Mean concentrations following cecal dosing were lower (P less than 0.05) than concentrations following gastric or jejunal dosing at each sampling time except baseline. Mean concentrations following gastric and jejunal dosing were similar except at 2 hr (gastric greater than jejunal). Mean pharmacokinetic parameters for cecal administration were different (P less than 0.05) from either the gastric or the jejunal periods with the exception of Tmax. There was no difference in any pharmacokinetic parameter after gastric or jejunal dosing. The relative bioavailability after cecal administration was less than 15% of that observed after administration into the stomach or jejunum. Additionally, Wagner-Nelson analysis indicated that the rate of ranitidine absorption was much slower following cecal administration than after gastric or jejunal dosing. Two plasma concentration peaks were observed in three of eight subjects after gastric dosing, in eight of eight subjects after jejunal dosing, and in zero of eight subjects after cecal dosing. These data demonstrate that the absorption profile of ranitidine is equivalent, in extent and duration, after delivery to the stomach or jejunum, while absorption from the cecum is significantly less.(ABSTRACT TRUNCATED AT 250 WORDS)


Quantitative analysis of mercaptoundecahydrododecaborate by Fourier transform infrared spectroscopy. Mercaptoundecahydrododecaborate (BSH) is an important agent in boron neutron capture therapy (BNCT) of various cancers. A simple and rapid analytical method for the measurement of mercaptoundecahydrododecaborate in aqueous solution and in urine by Fourier transform infrared spectroscopy has been developed. A thin-pathlength sampling apparatus was used to minimize the strong absorption of water. The subtraction of water absorbance from sample spectra resolved a B-H band at 2493 cm-1. The quantitative measurement of BSH concentration was carried out by integrating the B-H band above baseline in the range of 2534-2440 cm-1. The lower limit of measuring the concentration of sodium BSH (Na2B12H11SH) in our experiment was 10 micrograms/ml (about 5 ppm of boron). This method measures the hydroborate (B-H) concentration instead of total boron and, thus, may be utilized to measure the BSH concentration in in vivo samples for metabolic studies.


Solid-state stability studies of 13-cis-retinoic acid and all-trans-retinoic acid using microcalorimetry and HPLC analysis. The solid-state stabilities of 13-cis-retinoic acid and all-trans-retinoic acid in the presence and absence of oxygen were investigated. The samples were first evaluated using microcalorimetry. The rate laws of different samples under different conditions were deduced from the shapes of the heat flow curves, and the activation energies of the reactions were determined from Arrhenius plots. Under an air atmosphere, the decomposition of 13-cis-retinoic acid is an autocatalytic reaction, while all-trans-retinoic acid undergoes a zero-order process. The degradation of the two compounds at a selected elevated temperature was also determined utilizing HPLC analysis. This technique confirmed the decomposition kinetics. Hence, their half-lives and shelf lives at room temperature could be calculated. Under a nitrogen atmosphere, the microcalorimetric experiment showed a first-order phenomenon for both samples, but HPLC analysis showed no degradation, suggesting that the two samples, in the absence of oxygen, undergo only a physical change.


An unexpected hydrolysis pH-rate profile, at pH values less than 7, of the labile imide, ICRF-187: (+)-1,2-bis-(3,5-dioxopiperazin-1-yl)propane. The hydrolysis of the cardioprotectant and anticancer agent, ICRF-187 (or ADR-529) and the structurally similar model compound, 4-methylpiperazine-2,6-dione (4-MP), was investigated in the acid to neutral pH range at 25 degrees C and an ionic strength of 0.5 (sodium chloride). Their solution stability was shown to be compromised compared to 3-methylglutarimide (3-MG) and other imides. It appears that the tertiary piperazine nitrogens of ICRF-187 and 4-MP significantly contributed to the instability of these compounds over this pH range. Unexpectedly, bell-shaped curves were observed in the pH-rate profiles. A change in the rate-determining step from tetrahedral intermediate formation in the weakly acidic pH region to breakdown of the tetrahedral intermediate in the more acidic pH regions was proposed as an explanation for the bell-shaped curves. The piperazine nitrogen was implicated in the hydrolytic pathways that occur within these pH regions; the mechanism of involvement was dependent on the state of ionization of the parent molecule and the tetrahedral intermediate.


Molecular basis of type IA (tyrosinase negative) oculocutaneous albinism. Type IA (Tyrosinase negative) oculocutaneous albinism (OCA) is produced by mutations of the tyrosinase gene. We have found a total of 13 different mutations associated with type IA OCA. Analysis of the distribution of the 9 missense mutations shows that most of these mutations cluster in three areas of the gene. All but one of these mutations involve amino acids that are conserved between the mouse and human. Two clusters involve the copper A and copper B binding sites, and could disrupt the metal ion-protein interaction necessary for enzyme function. The third cluster is in exon I and could represent an important functional domain of the enzyme such as the tyrosine binding site. The deletion or insertion frameshift mutations are distributed throughout the coding region and do not appear to cluster. We conclude that a diverse number of mutations are responsible for type IA OCA and many individuals are compound heterozygotes for mutations responsible for this genetic disease (Table 3).


Epidemiology of malignant melanoma in central Europe: risk factors and prognostic predictors. Results of the Central Malignant Melanoma Registry of the German Dermatological Society. The Central Malignant Melanoma Registry (CMMR) of the German Dermatological Society was established in 1983, and 7789 cutaneous malignant melanomas (CMM) were registered by 35 dermatological departments in Germany, Austria and Switzerland until the end of 1989. Population-based incidence rates, risk factors for developing CMM and prognostic parameters for predicting the final outcome were investigated in separate multicenter studies performed by the CMMR. Among the 7789 CMM registered, there was a preponderance of females (57.7%) versus males (42.3%). The age distribution peaked in the 5th and 6th decade of life for both sexes with a mean age of 52 years. The mean detection age was 50 years for superficial spreading melanoma, 53 for nodular melanoma, and 65 for lentigo maligna melanoma. Mean tumor thickness decreased from 2 mm in 1983 to 1.5 mm in 1989, indicating better CMM-awareness of the population and the medical community in this area. 90% of the patients presented with clinical stage I CMM without detectable metastases at first diagnosis. The incidence of CMM in Berlin (West) was assessed based on 960 cases diagnosed between 1980 and 1986. The incidence increased by 49% between 1980-81 and 1985-86, and the age standardized-incidence rate (European standard population) was 9.8 for males and 7.8 for females per 100,000 inhabitants and year in 1985-86. Mortality rates decreased in this period from 3.5 to 2.6 for males and slightly increased for females from 1.2 to 1.6 per 100,000 inhabitants and year. A case control study on the relative risk (RR) for developing CMM revealed the total number of melanocytic nevi (MCN) to be the strongest risk predictor (15x -50x increased RR), followed by the presence of dysplastic MCN (7x increased RR) and the skin type I (2x increased RR). Interestingly, no differences between CMM-cases and controls were found with respect to the history of sunburns or other parameters of sun exposure in this study. Multivariate analysis of 5093 stage I CMM-patients from four departments with long-term follow-up revealed that tumor thickness is the strongest predictor of survival with an almost linear correlation to the risk of death for tumor thickness up to 6 mm with no further increase in mortality for higher tumor thickness. The best classification of tumor thickness for survival prediction was less than or equal to 1 mm, 1.01-2 mm, 2.01-4 mm and greater than 4 mm in our data set on 5093 patients.(ABSTRACT TRUNCATED AT 400 WORDS)


The stability of 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid in human urine. 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid are important intermediate metabolites in the formation of cutaneous melanin pigment. Since they both are serious candidates as markers of melanoma progression, their stability in urine has been investigated during storage at various conditions. The results show that storage at -20 degrees C is necessary. Both compounds are nonstable at room temperature, particularly if the urine was not acidified to pH 4-5. Reference levels were obtained from analysis of urine from 31 men and 40 women. The mean (SD) excretion of 5-S-cysteinyldopa was 32 (12.5) mumol/mol creatinine (women). Corresponding figures for 6H5MI2C were 23 (10.3) and 24 (8.1) mumol/mol creatinine for men and women respectively.


Regulatory factors of pheo- and eumelanogenesis in melanogenic compartments. Melanogenesis, i.e., synthesis of melanin and melanosomes, is a "cascade" of event which is channelled by internal and external regulatory factors. The recognition and selection of this information and subsequent differentiation of melanogenesis (melanin type and melanosomal development) would be regulated significantly by melanosomal membrane. The melanogenesis type could be switched relatively easily by UV light, hormone, and availability of tyrosinase substrate. The role of sulphydryl compounds as a regulatory factor in melanogenesis type (in particular for pheomelanogenesis) may not be tied to its absolute presence or absence, but rather, to the effective concentration within the melanocyte at a given time. It is, therefore, probable that the morphogenesis of melanosomes may not follow immediately in response to melanogenesis-type changes, hence the melanocyte revealing more often mosaic forms of melanosomes in nature after exposure to non-genetic factors. The switch of melanogenesis would be significantly controlled by structural and functional availability of vesiculoglobular bodies which are encoded or associated with HMSA-5 (69 kDa) glycoprotein. This HMSA-5 protein shares a significant homology with gp75 "b-locus" protein. However, because of our hypothesis that vesiculoglobular bodies carry post- (and pre-) tyrosinase regulatory factors involving in both pheo- and eumelanogenesis, the term "b-protein" which focuses only on eumelanogenesis may not be applied to HMSA-5.


Identification of mutations in the pigment cell-specific gene located at the brown locus in mouse. The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.


Molecular bases of tyrosinase-negative oculocutaneous albinism: a single base insertion or a missense point mutation in the tyrosinase gene. We have identified two different mutations in the tyrosinase genes of Japanese patients with tyrosinase-negative oculocutaneous albinism (OCA). One is a single base insertion in the exon 2 of the tyrosinase gene that shifts the reading frame and introduces a premature termination codon (TGA) after the amino acid residue 298 (codon 316). The other is a G to A transition at residue 312, leading to a single amino acid substitution, arginine at position 59 (codon 77) to glutamine. The promoter activity of the patients' tyrosinase genes was evaluated in the cell-free transcription system prepared from pigmented melanoma cells, indicating that the patients' genes were accurately transcribed in vitro. It is therefore conceivable that the tyrosinase gene is expressed in their melanocytes. Furthermore, transient expression of the mutated genes indicates that the truncated tyrosinase or the tyrosinase containing glutamine 59 is unable to form melanin in melanocytes. We therefore propose that these mutations in the tyrosinase genes lead to a phenotype of tyrosinase-negative OCA.


Materia melanica: further dark thoughts. The view is advanced that melanogenesis arose evolutionarily as a detoxification pathway for intrinsically-generated orthoquinones. The primary impetus for the production of orthoquinones may have been their general antibiotic properties and the utility of these chemical species in forming covalent cross-links between proteins, as illustrated by cuticular sclerotization in insects. It is argued that polymerization to give rise to visible pigments may have originated as a pathway for the inactivation of orthoquinones. The possible evolutionary advantages accruing from the generation of melanin are discussed with special reference to acuity of photoreceptors and the physico-chemical properties of melanin, as well as the contribution of melanin to protective colouration or display.


B700 antigen as a component of an antimelanoma vaccine: formalinized extracellular antigens. Formalin fixation has enjoyed widespread use in the preparation of antibacterial and other vaccines, but rather less use in antitumor vaccines. Previous studies from our laboratories have demonstrated the efficacy of antimelanoma vaccines in mice, produced from formalinized antigens shed by cultured melanoma cells. In this study, we provide evidence that the immunodominant component of that vaccine is the well-characterized B700 melanoma antigen.


Synthesis and characterization of melanins from dihydroxyindole-2-carboxylic acid and dihydroxyindole. Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 micron filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.).(ABSTRACT TRUNCATED AT 250 WORDS)


Melanin biosynthesis from dopamine. II. A mass spectrometric and collisional spectroscopic investigation. Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry together with collisional activation (CA) experiments were applied to the study of the oxidation pathway of dopamine by tyrosinase. In order to prevent attachment of the protein to the highly reactive intermediates, ultrafiltration was employed to remove the enzyme at different reaction times. FAB, privileging molecular species formation, was successfully used for identification of transient intermediates and their relative concentrations with respect to time, directly in the reaction mixture. The presence of isobaric molecular species made chromatographic separation necessary. Further EI mass spectrometry and collision spectroscopy led to structural identification of pure components. Of these, dopamine-o-quinone, leucoaminochrome, and aminochrome semiquinone were characterized for the first time as real intermediates in dopamine melanogenesis, in agreement with previous hypotheses. This approach elucidated the pathway of dopamine melanogenesis.


Melanin standard method: particle description. Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.


Melanin standard method: empirical formula. Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.


[Mortality from cancer in France. Changes between 1950 and 1989 in the population aged 35-64]. In the French population aged 35-64, cancer mortality increased yearly by 1 percent in males and decreased yearly by 0.6 percent in females between 1950 and 1989. In males, the increase is mostly due to the sites associated with tobacco and alcohol. The important increase of lung cancer is related to the increase in tobacco consumption from 4.7 g per adult per day in 1950 to 6.3 g in 1976. The increase observed in head and neck cancers is related to tobacco and alcohol. In females, the moderate decrease is due to the decrease observed in uterine cervix and stomach cancer mortality, uncompensated by the increase in breast cancer. Contrary to what is observed in the USA, there is no marked increase in lung cancer mortality for women.


[Isolated dissecting aneurysm of the superior mesenteric artery. Dysplasia, a rare cause of mesenteric ischemia. Report of a case]. We report a case of successful surgical revascularization of the superior mesenteric artery in a patient presenting with intestinal ischaemia due to fibrodysplasia. This is a rare pathology, with 5 cases operated in our institution and only 16 cases reported in the literature. Angiography may show other sites of arterial dysplasia. In such cases, success depends on early surgical revascularization.


[Value of corticosteroids in bacterial meningitis]. Bacterial meningitis mortality largely results from the intense host inflammation response to infection. Lysis of bacteria releases bacterial components that stimulate production of cytokines. Cerebrospinal fluid concentrations of these cytokines have been shown to correlate with the severity of the meningitis in both experimental and clinical situations. Dexamethasone has an antiinflammatory effect superior to methylprednisolone. It is the only agent with which significant clinical studies have yet been performed. A recent study recommends the routine use of dexamethasone with Haemophilus influenzae meningitis. The immediate and long-term clinical profiles indicate significantly better outcomes for the dexamethasone group. It is prudent to administer dexamethasone before the first parenteral dose of antibiotic to prevent the release of bacterial components induced by bacterial lysis. The search for agents that down modulate inflammation more strongly continues. Some monoclonal antibodies are superior to dexamethasone in experimental meningitis without modification of antibiotic pharmacokinetics.


[Effect of the captopril-hydrochlorothiazide combination on blood lipids. Study in a population of hypertensive hyperlipidemic patients]. In this placebo-controlled, randomized double-blind, parallel study the effects of the fixed captopril 50 mg + hydrochlorothiazide (HCTZ) 25 mg combination on plasma lipids were assessed in 42 hypertensive, type IIa or IIb hyperlipidaemic patients on diets. Some patients received oral hypolipidaemic treatment and some did not. Blood pressure and plasma lipids levels were measured before and after 1, 2 and 3 months of treatment. From the first month onward blood pressure decreased more in the treated group than in the placebo group (P < 0.05). Neither the combination nor the placebo altered the following parameters of lipid metabolism: total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein A1 and apolipoprotein B. The combination was well tolerated; 2 patients in each group had one or several adverse events. The results of this study show that treatment with the captopril-HCTZ combination in hypertensive, hyperlipidaemic patients has no influence on the normolipidaemic effects of diet and lipid-lowering treatment.


[Structural approach of vascular aging]. The cardiovascular system is one of those target-organ systems of senescence where the effects of physiological ageing meet the consequences, accumulated with time, of pathological disorders. In man, these two processes are not easily disentangled, and despite the advances achieved in ultrasonic techniques the approach of structural parameters remains difficult. On the other hand, the morphological and functional unicity of the vascular wall in different species is such that observations made in animals are relevant. In rats, the structure-function relationship can be determined by histomorphometric analysis of the myocardium and vascular wall under standardized conditions of treatment. As the animals get older, the cardiac mass, related or not to body-weight, increases while the cardiac efficacy decreases. Hypertrophy of the heart is accompanied by a change in the enzymatic property of myosin. Simultaneously, the walls of the greater arteries become thicker, more rigid and less compliant, hypertrophy of the smooth muscle cells being an essential component of vascular wall thickening. At the same time, the collagen fraction and the amount of collagen-bound calcium increase. The elastic component decreases, at least relatively, and the elastin-collagen ratio clearly diminishes with age. Altogether, these alterations are not different from those observed in human arterial hypertension. They result in a lesser permeability of the tunica media, facilitate the accumulation in the subendothelium of lipidic and/or proteinic compounds originating in plasma and constitute a link between ageing and atheromatous processes.