The effect of coencapsulation of bovine insulin with cyclodextrins in ethylcellulose microcapsules.

Polymeric microcapsules have been widely investigated for protein delivery. Common problems include: low stability, low encapsulation efficiency, lack of uniformity, and burst release. Cyclodextrins (CDs) are known to enhance stability and solubility of proteins in solution. This research examines the effect of alpha-, beta-, and gamma-CDs on: (1) stability, (2) encapsulation, and (3) release of insulin from ethylcellulose microcapsules. All CDs improved thermal stability of insulin by lowering the enthalpy of unfolding by 16-52%. alpha- and gamma-CDs also increased the encapsulation efficiency of insulin and improved uniformity of the microcapsule formulations. Two mathematical models were proposed to account for insulin release and consisted of multiple zero order and first order input processes, and a single first order output process. All CDs decreased the initial burst release of insulin by up to 30%. This research demonstrates the potential for CDs to improve stability, uniformity, and encapsulation of proteins in microcapsule formulations.

Identification of muscarinic receptor subtypes in mouse parotid gland.

Immunoprecipitation of muscarinic receptors from mouse parotid membranes by specific subtype antisera showed that M3 and M1 receptors represented 75 and 15% of the total number of precipitable receptors, respectively. [N-methyl-3H]methylscopolamine (NMS) labeled a single class of high-affinity binding sites in membranes from parotid glands with a dissociation constant of 0.67 +/- 0.02 nM and a maximum binding capacity of 176 +/- 15 fmol/mg protein. Competition curves for NMS, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydro-sila-difenidol fit best to a one-site binding model, whereas pirenzepine and methoctramine fit best to a two-site binding model, indicating 76-90% M3 receptors. Results from the use of pirenzepine indicated that the second mouse parotid receptor subtype, unlike that of the submandibular gland, has atypical characteristics for an M1 receptor. The rank order of potency of muscarinic antagonists in inhibiting phosphoinositide turnover and biphasic effects of carbachol on isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was atropine > or = 4-DAMP >> pirenzepine > AF-DX 116. A specific M1 antagonist, m1-toxin, had no effect on carbachol augmentation or inhibition of isoproterenol responses. Results suggest that M3 receptors couple to both augmentation and inhibition of stimulated cAMP levels.

The routine analysis of breast milk for drugs of abuse in a clinical toxicology laboratory.

Drug screening of breast milk in a clinical toxicology laboratory is reported. Findings from three cases include cocaine, ethylbenzoylecgonine (cocaethylene), ethanol, oxycodone, codeine, and nicotine. We believe this to be the first report of ethylbenzoylecgonine in human breast milk. One other specimen submitted for analysis was screened with negative results. Screening and confirmation procedures adapted for use with breast milk are described. Finally, the potential for cocaine intoxication from mother to baby is discussed. Estimates of infant blood cocaine concentration are given which may increase awareness of the need to monitor milk and blood cocaine concentrations in the infant when the situation warrants.

Teratogenic effects of ethanol during hyperplastic growth in cardiac myocyte cultures.

Ethanol alters cellular growth and maturation, teratologic factors that are recognized as contributing to abnormal phenotypic expression. Cultured neonatal Sprague-Dawley rat cardiac myocytes were utilized to determine how ethanol alters growth and development. Two ethanol exposure paradigms were studied: (1) constant, to cultures in closed chambers for 7 days at low (10 mM) and high (50 mM) concentrations; and (2) periodic (24-hr) to cells during hyperplastic growth. In constantly exposed cultures, 10 and 50 mM ethanol concentrations depressed the rate of leucine incorporation and the rate of thymidine uptake during early hyperplastic growth (log phase growth). A resultant slower expansion of cell populations was noted. Although the period of maximum vulnerability appeared to be the hyperplastic growth phase, a second set of experiments using 10 and 50 mM ethanol were performed to assess the effects of short (24-hr) exposures. DNA synthesis was depressed during early hyperplastic growth compared with controls (days 2-4), reflected as a decrease in thymidine incorporation and smaller cell population. This study demonstrates that ethanol depresses both DNA and protein synthesis during hyperplastic growth resulting in an insufficient, protein-deficient cell mass, incapable of participating in normal embryogenesis.

Ethanol-induced teratogenic alterations in developing cardiomyocytes in culture.

The development of an in vitro cardiogenesis model was designed to enhance our understanding of the mechanism(s) of ethanol teratogenicity. Growth and development events in the model are similar to in vivo events. Time-specific and event-specific windows of biokinetic activities during both hyperplastic and hypertrophic growth are easily controlled and independently investigated. Systematic study of the effects of ethanol on the embryogenesis model, from committed blast cells to mature, functioning cells was accomplished to determine at what stage or stages of the growth and development paradigm ethanol exerts its teratogenic potential. Using the model and comparing the data to in vivo ethanol exposures, it appears that ethanol impairs the capability of the cell to properly propagate and mature.

Characterization of beta-adrenergic receptors in DiFi and HT-29 cells.

The beta-adrenergic receptors present in two human colorectal adenocarcinoma cell lines were characterized by measuring specific binding of [125I]-cyanopindolol (CYP). Whole, cultured, DiFi (derived from a familial adenomatous polyposis [FAP] patient) and HT-29 cells were used in radioligand binding assays. Scatchard analysis of specific 125I-CYP binding gave KDS of 38.6 +/- 5.7 pM in DiFi cells and 54 +/- 9.1 pM in HT-29 cells. However, binding site density (Bmax) in the DiFi cells was greater than that in HT-29 cells. In DiFi cells, the kinetically determined KD was similar to that calculated from Scatchard analysis. Studies in DiFi cells of the displacement of specific 125I-CYP binding by nonselective (propranolol), beta 1-selective (metoprolol and atenolol), and beta 2-selective (ICI 118-551) antagonists revealed only a single class of beta 2-adrenergic receptors. This provides the first evidence that colorectal adenocarcinoma cell lines contain beta-adrenergic receptors and shows that only beta 2-adrenergic receptors are present in DiFi cells. Mechanisms possibly affecting beta-adrenergic-receptor expression in such cells are discussed in relation to colon carcinogenesis.

The effect of HCO3- on anion-stimulated ATPase from rat parotid granules.

ATPase from isolated secretory granules was stimulated in a concentration-dependent manner by HCO3- above 0.9 mM. Maximal stimulation was found at about 16 mM HCO3- and was about half of that with sulphite (SO3(2-)). The activation site(s) appeared to be similar to at least one class of SO3(2-) sites, HCO3(-)-stimulate ATPase was inhibited by SITS. Furthermore, maximal stimulation with SO3(2-) was not enhanced with HCO3-. At low Mg2+ concentrations, Ca2+ stimulated granule ATPase. At higher concentrations of Mg2+ (0.5 mM and above), Ca2+ at 0.1 mM or less had little effect on HCO3(-)-ATPase, and Ca2+ at 4 mM inhibited HCO3(-)-ATPase. At concentrations of Ca2+ above 0.44 mM, the enzyme was partially stimulated in the absence of Mg2+ and presence of HCO3-. Mitochondrial contamination did not account for the presence of ATPase in the isolated granule fraction. The granule ATPase may be regulated by HCO3- and calcium and this could be related to changes in the granule environment during exocytosis.

Stability of cephalexin monohydrate suspension in polypropylene oral syringes.

The stability of cephalexin monohydrate suspension in plastic oral syringes was studied. Commercially available cephalexin monohydrate powder for oral administration was reconstituted according to the manufacturer's instructions and stored in the original containers or drawn into 5-mL clear polypropylene oral syringes. The original containers and syringes were divided into groups and stored at -20, 4, 25, 40, 60, or 80 degrees C. Powder from two additional lots was similarly reconstituted and packaged; these original containers and syringes were stored at 80 degrees C only to assess interlot variability. Immediately after reconstitution and at specified times during storage, three syringes and the corresponding three original containers stored at each temperature were removed, and their contents were analyzed for cephalexin concentration using the standard USP iodometric assay for antibiotics. The stability-indicating nature of the assay was documented. Cephalexin monohydrate followed a first-order rate of degradation at temperatures of 40, 60, and 80 degrees C. At temperatures of -20, 4, and 25 degrees C, cephalexin monohydrate exhibited no appreciable degradation during the 90-day study period. Cephalexin monohydrate suspension reconstituted from powder as a suspension and repackaged in clear polypropylene oral syringes was stable for 90 days when stored under ambient, refrigerated, and frozen conditions.

Stability of dicloxacillin sodium oral suspension stored in polypropylene syringes.

The stability of dicloxacillin sodium in oral suspension stored in clear polypropylene oral syringes was studied. Commercially available dicloxacillin sodium powder for oral suspension from a single lot was reconstituted according to the manufacturer's instructions and drawn into 5-mL clear polypropylene oral syringes. The syringes were divided into groups and stored at -20, 4, 25, 40, 60 or 80 degrees C. Two additional lots were similarly reconstituted, repackaged, and stored at 80 degrees C only to assess interlot variability. Powder in the original containers was similarly reconstituted according to the manufacturer's instructions, and the containers were divided into groups and stored with the syringes. Immediately after reconstitution and at specified times during storage, three syringes and the original containers at each storage temperature were removed, and their contents were analyzed for dicloxacillin sodium concentration using the Standard USP Iodometric Assay. Dicloxacillin sodium follows a first-order rate of degradation at temperatures of 40, 60, and 80 degrees C. The rate of degradation changes to a zero-order process at temperatures of 25, 4, and -20 degrees C. At all temperatures, degradation occurred more rapidly when the drug was repackaged into unit dose polypropylene oral syringes than in the manufacturer's original container. Dicloxacillin sodium reconstituted from powder as oral suspension and repackaged in clear polypropylene syringes was stable for no longer than 7, 10, and 21 days when stored under ambient, refrigerated, and frozen conditions, respectively.

Relationship between planar and all-surface rate constants for drugs formulated in nondisintegrating cylindrical slow-release tablets.

The release of drug through the planar surface of a nondisintegrating tablet with an insoluble matrix has been described mathematically using the Higuchi release rate constant (kH). The release of drug through a similar all-surface tablet has been described by using a cubic equation and the all-surface rate constant (kr). Using sodium salicylate and quinidine sulfate as model drugs, the relationship between kH and kr was verified for cylindrical slow-release tablets. Accordingly, the rate constant obtained from a single exposed planar surface can be used to predict the rate constant (kr) when all surfaces of the tablet are exposed to dissolution fluid.

Stability of ampicillin trihydrate suspension in amber plastic oral syringes.

The stability of ampicillin trihydrate oral suspension stored in amber plastic oral syringes was studied. Commercially available ampicillin trihydrate powder for oral suspension was reconstituted according to manufacturer's instructions and drawn into 5-mL amber polypropylene plastic oral syringes. The syringes were divided into groups and stored at -20, 4, 25, 60, or 80 degrees C. Powder from two additional lots was similarly reconstituted and packaged and stored at 80 degrees C only to assess interlot variability. Immediately after reconstitution and at specified times during storage, three syringes at each storage temperature were removed and their contents analyzed for ampicillin trihydrate concentration by a spectrophotometric assay. Samples stored at frozen (-20 degrees C) or refrigerated (4 degrees C) temperature retained at least 90% of the initial ampicillin concentration throughout the 47-day study period. Samples stored at room temperature retained at least 90% of the initial ampicillin concentration for 30 days and exhibited an apparent zero-order degradation rate. Samples stored at heated temperatures (60 and 80 degrees C) exhibited an apparent first-order degradation process, with the concentration of ampicillin decreasing to less than 90% of initial concentration within two hours. Reconstituted ampicillin trihydrate powder for oral suspension is stable for at least 30 days when stored at room, refrigerated, or frozen temperature in the amber plastic oral syringes studied. The expiration dates recommended by the manufacturer for ampicillin trihydrate suspension stored in its original container can also be used for reconstituted suspension stored in these amber plastic syringes.

Percutaneous absorption and disposition of iodochlorhydroxyquin in dogs.

The percutaneous absorption and disposition of iodochlorhydroxyquin (5-chloro-7-iodo-8-quinolinol; I) from a 3% cream were studied in five dogs over a 28-d topical treatment period. Plasma levels, determined by HPLC, were 0.275-0.525 microgram/mL. The steady-state elimination rate of total I in urine was 2.4-3.0 mg/d. The apparent elimination rate constant and half-life were 0.25 +/- 0.05 d-1 and 3.1 +/- 0.5 d, respectively. Greater than 50% of topically applied I was absorbed over 16 h. Occlusion of the skin without the drug indicated that the skin acted as a reservoir for the drug. Feces analysis for iodochlorhydroxyquin from one dog showed that 27.1 +/- 8.5 mg/d was eliminated via this route. Tissue levels of I 15 d after the 28-d topical treatment were 0.7 microgram/g of liver, 0.2 microgram/g of kidney, and 0.8 microgram/g of mesenteric fat. The apparent rate constants of plasma level decline after a 100-mg iv bolus dose of I were alpha = 3.9 h-1 and beta = 0.6 h-1. The urinary elimination after intravenous administration was biphasic. The rate constant for the slow elimination phase was 0.4 +/- 0.1 d-1, and the half-life was 2.0 +/- 0.5 d. The primary neurological symptoms observed during topical treatment were ataxia and hind limb paralysis. Microscopic examination revealed liver necrosis. A weight loss of 15.3 +/- 2.7% was also observed over the 28-d topical treatment period. The results indicate that significant percutaneous absorption of I occurs, and that chronic high-dose topical treatment may lead to toxicity.

Ocular delivery of pilocarpine from erodible matrices.

The present study examined the feasibility of sustaining the release of a water-soluble drug, pilocarpine, to the tear film. Both gels and dried films were utilized as drug delivery systems. In vitro studies demonstrated significant prolongation of drug release from these systems as compared with simple aqueous or viscous solutions. The in vitro results were supported by in vivo miosis studies in albino rabbits.

Percutaneous absorption of iodochlorhydroxyquin in humans.

Iodochlorhydroxyquin (I) is used in the treatment of diaper rash and other skin disorders, and is presumed to undergo little or no percutaneous absorption. The absorption of (I) from a 3% cream was studied in 5 normal male subjects after a single application of the cream for 12 h. Plasma levels of the drug were followed for 24 h after initial application while urinary excretion was measured for 54 h. (I) was extracted from plasma and urine and assayed by high-performance liquid chromatography. The drug in the range of 0.37-0.56 micrograms/ml was detected in plasma 2 h after application and persisted throughout the treatment period. The mean excretion rate after 12 h of application was 58.4 micrograms/h and the excretion rate was 8.8 micrograms/h at 42 h posttreatment. The elimination rate constant was calculated to be 0.15 h-1. Approximately 40% of the drug was absorbed over the 12-h application period. From the above results it is apparent that significant percutaneous absorption of (I) occurs.

Stability of methacholine chloride in bronchial provocation test solutions.

The stability of methacholine chloride (5 mg/ml) in 0.9% sodium chloride solution was measured. A reliable colorimetric assay (530 nm) based on the formation of a hydroxamic acid-iron complex was used. At appropriate time intervals, samples were removed from solutions stored at 4, 20, 37, 60, or 80 degrees C and assayed. The degradation of methacholine chloride followed apparent first-order kinetics of methacholine chloride followed apparent first-order kinetics at all temperatures, with observed half-lives ranging from 29.3 days at 80 degrees C to 693 days 4 degrees C. Methacholine chloride in 0.9% sodium chloride solution does not degrade as rapidly as previously suggested. According to an Arrhenius plot, storage of such solutions at 30 or 4 degrees C would result in not more than 10% degradation over a period of approximately two or five months, respectively. Thus, it should be possible to prepare stock solutions of this deliquescent drug.

The use of marker drugs to measure organ function: a theoretical interpretation.

An equation is presented which describes the changes in drug clearance through an isolated organ due to alterations in the blood flow to the organ or the intrinsic clearance of the drug by the organ. In the case of hepatic clearance, example calculations of alterations in both parameters are presented from literature data for marker drugs having high and low extraction ratios. The predicted hepatic clearance of d-propranolol based on these calculations compares with observed values. For the particular example of the kidney, the blood flow and the intrinsic clearance change in proportion and, therefore, yield a simple equation relating renal clearance of any drug to endogenous creatinine clearance. This equation, though used clinically, has previously not been derived theoretically.

Pharmacokinetics of topically applied pilocarpine in the albino rabbit eye.

The temporal and spatial pattern of [3H]-pilocarpine nitrate distribution in the albino rabbit eye following topical administration was determined. A four-compartment caternary chain model describing this disposition corresponds to the precorneal area, the cornea, the aqueous humor, and the lens and vitreous. Simultaneous computer fitting of data from tissue corresponding to some compartments in the model supported the proposed model. Additional support was provided by the excellent correlation between predicted and observed values in multiple-dosing studies. Several important aspects of ocular drug disposition are evident from the model. The extensive parallel elimination at the absorption site gives rise to an apparent absorption rate constant that is one to two orders of magnitude larger than the true absorption rate constant. In addition, aqueous flow accounts for most of the drug removal. Thus, major effects on absorption and elimination, independent of the drug structure, suggest the possibility of similar pharmacokinetics for vastly different drugs.

Quinidine and dihydroquinidine interactions in human plasma.

The protein-binding characteristics of dihydroquinidine, a known impurity in drug grade quinidine, in human plasma and the effects of dihydroquinidine on quinidine interactions with these plasma constituents were studied by equilibrium dialysis. In the plasma concentration range of 1.75-23.0 mg/liter, dihydroquinidine binding was similar to the binding observed with quinidine. The data suggested the presence of a single class of binding sites for both compounds in the plasma drug concentration range and samples studied. The mean values for the association constant, K, and the total concentration of binding sites, nPt, for dihydroquinidine were 4.75 +/- 0.67 X 10(4) M-1 and 5.78 +/- 0.17 x 10(-5) M, respectively. The corresponding values for quinidine were 4.78 +/- 1.00 x 10(4) M-1 and 5.65 +/- 0.48 x 10(-5) M. In the presence of 5 and 10% (of total alkaloid content) dihydroquinidine, the plasma concentration of unbound quinidine did not change significantly. At a 20% level of dihydroquinidine, however, an increase in unbound quinidine was observed (p less than 0.05). The elevations in free quinidine concentrations were directly related to the level of dihydroquinidine present. The results of this study indicate that the interactions between dihydroquinidine and quinidine for binding sites on human plasma proteins are competitive.