Natalie D. Eddington

The rate of absorption and relative bioavailability of caffeine administered in chewing gum versus capsules to normal healthy volunteers

OBJECTIVE The purpose of this study was to evaluate the rate of absorption and relative bioavailability of caffeine from a Stay Alert chewing gum and capsule formulation. METHODS This was a double blind, parallel, randomized, seven treatment study. The treatment groups were: 50, 100, and 200 mg gum, 50, 100, and 200 mg capsule, and a placebo. Subjects consisted of 84 (n=12 per group); healthy, non-smoking, males who had abstained from caffeine ingestion for at least 20 h prior to dosing and were randomly assigned to the treatment groups. Blood samples were collected pre-dose and at 5, 15, 25, 35, 45, 55, 65, 90 min and 2, 3, 4, 6, 8, 12, 16 and 29 h post administration. Plasma caffeine levels were analyzed by a validated UV-HPLC method. RESULTS Mean Tmax for the gum groups ranged from 44.2 to 80.4 min as compared with 84.0-120.0 min for the capsule groups. The Tmax, for the pooled data was significantly lower (P<0.05) for the gum groups as compared with the capsule groups. Differences in Tmax were significant for the 200 mg capsule versus 200 mg gum (P<0.05). The mean ka values for the gum group ranged from 3.21 to 3.96 h-1 and for the capsule groups ranged from 1.29 to 2.36 h-1. Relative bioavailability of the gum formulation after the 50, 100 and 200 mg dose was 64, 74 and 77%, respectively. When normalized to the total drug released from the gum (85%), the relative bioavailability of the 50, 100 and 200 mg dose were 75, 87, and 90%, respectively. No statistical differences were found for Cmax and AUCinf for comparisons of the gum and capsule formulations at each dose. Within each dose level, there were no significant formulation related differences in Cmax. No significant differences were observed in the elimination of caffeine after the gum or capsule. CONCLUSIONS The results suggest that the rate of drug absorption from the gum formulation was significantly faster and may indicate absorption via the buccal mucosa. In addition, for the 100 and 200 mg groups, the gum and capsule formulations provide near comparable amounts of caffeine to the systemic circulation. These findings suggest that there may be an earlier onset of pharmacological effects of caffeine delivered as the gum formulation, which is advantageous in situations where the rapid reversal of alertness and performance deficits resulting from sleep loss is desirable.

Transport of Poly(Amidoamine) Dendrimers across Caco-2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling

PurposeTo investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. MethodsCationic PAMAM-NH2 (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5–G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. 14C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity.ResultsThe overall rank order of PAMAM permeability was G3.5COOH > G2NH2 > G2.5COOH > G1.5COOH > G2OH. 14C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns.ConclusionsThese studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.

Plasma pharmacokinetics and tissue distribution of paclitaxel in CD2F1 mice.

We defined the pharmacokinetics of paclitaxel after i. v., i. p., p. o., and s. c. administration of 22.5 mg/kg to CD2F1 mice. Additional mice were studied after i. v. bolus dosing at 11.25 mg/kg or 3-h continuous i.v. infusions delivered at 43.24 μg kg−1 min−1. Plasma was sampled between 5 min and 40 h after dosing. Brains, hearts, lungs, livers, kidneys, skeletal muscles, and, where applicable, testicles were sampled after i.v. dosing at 22.5 mg/kg. Liquid-liquid extraction followed by isocratic high-performance liquid chromatography (HPLC) with UV detection was used to determine paclitaxel concentrations in plasma and tissues. After i.v. administration to male mice, paclitaxel clearance (CLtb) was 3.25 ml min−1 kg−1 and the terminal half-life (t1/2) was 69 min. After i.v. administration to female mice, paclitaxel CLtb was 4.54 ml min−1 kg−1 and the terminalt1/2 was 43 min. The bioavailability of paclitaxel was ∼10%, 0, and 0 after i.p., p.o., and s.c. administration, respectively. Paclitaxel bioavailability after i.p. administration was the same when the drug was delivered in a small volume to mimic the delivery method used to evaluate in vivo antitumor efficacy or when it was delivered in a large volume to simulate clinical protocols using i.p. regional therapy. Paclitaxel was not detected in the plasma of mice after i.p. delivery of the drug as a suspension in Klucel: Tween 80. Pharmacokinetic parameters were similar after i.v. delivery of paclitaxel at 22.5 and 11.25 mg/kg; however, the CLtb calculated in these studies was much lower than that associated with 3-h continuous i.v. infusions. After i.v. administration, paclitaxel was distributed extensively to all tissues but the brain and testicle. These data are useful in interpreting preclinical efficacy studies of paclitaxel and predicting human pharmacokinetics through scaling techniques.

Characterization of hepatic cytochrome P4503A activity in patients with end‐stage renal disease

The cytochrome P450 (CYP) oxidative enzyme system, located primarily in the liver and small intestine, is responsible for metabolism and detoxification of numerous endogenous and exogenous substances. The most abundant CYP enzyme, CYP3A, is known to be involved in the metabolism of more than 200 commonly used medications. In experimental models of renal failure, both hepatic function and CYP enzyme content are reduced; however, direct evidence in humans is lacking. Evaluation of drug metabolism in patients with end‐stage renal disease is important because these patients use a large number of medications and are at risk of adverse reactions and drug‐drug interactions.

Enaminones-versatile therapeutic pharmacophores. Further advances.

Enaminones, enamines of ss-dicarbonyl compounds, have been know for many years. In our initial account (Current Med. Chem. 1994, 1, 159-175), we reported on the anticonvulsant activity of a series of enaminones, notably methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxo-cyclohex-3-en- 1-oate, 9a (R=CH3, R1=4-Cl), which, in animal tests, compared favorably to phenytoin and carbamazepine. Since that time, further research in our laboratory and other laboratories have expanded the therapeutic potential of these compounds. In addition to new anticonvulsant derivatives, we have uncovered a novel brain transport mechanism for the enaminones and developed a preliminary regression model for further synthetic direction. These topics will each be presented and elaborated.

Synthesis and anticonvulsant activity of enaminones. 4. Investigations on isoxazole derivatives

Due to the exceptional anticonvulsant activity displayed by substituted aniline enaminones, related pyridine derivatives and phenothiazines synthesised in our laboratories, the further investigation of various aromatic heterocycles was undertaken. Condensation of cyclic 1,3-diketo esters with 3-, and 5-aminoisoxazole derivatives led to a series of potent anti-maximal electroshock (MES) analogues, three of which occurred in the 3-amino series: ethyl ester (10), orally (po) active in rats [ED(50) 68.9 mg kg(-1), TD(50) > 500 mg kg(-1), protective index (PI = TD(50)/ED(50)) > 49.6]; methyl ester (9), ED(50) 68.9 mg kg(-1) intraperitoneally (ip) in mice, TD(50) > 500 mg kg(-1), PI > 7.3, and tert-butyl ester (8), ED(50) 28.1 mg kg(-1) po in rats, TD(50) > 500 mg kg(-1), PI > 17.8. Sodium channel binding studies, as well as evaluations against pentylenetetrazol, bicuculline, and picrotoxin on isoxazole 10 were all negative, leading to an unknown mechanism of action. X-ray diffraction patterns of a representative of the 3-amino series (isoxazoles 6-11) unequivocally display the existence of intramolecular hydrogen bonding of the nitrogen to the vinylic proton in the cyclohexene ring, providing a pseudo three ring structure which was also shown previously with the vinylic benzamides. Physicochemical-permeability across the BBB suggested an efflux mechanism for the previously synthesised aniline enaminones, but not with isoxazole 10.

Determination of the nutraceutical, glucosamine hydrochloride, in raw materials, dosage forms and plasma using pre-column derivatization with ultraviolet HPLC.

A selective and specific high performance liquid chromatography method was developed to quantitate glucosamine hydrochloride in raw materials, dosage forms and plasma. Reverse phase chromatography using pre-column derivatization with phenylisothiocyanate, and ultraviolet detection (lambda = 254 nm) was used to quantify the eluate. The mobile phase consisted of MeOH/H2O/CH3COOH (10:89.6:0.04) and was pumped at a flow rate of 1.2 ml/min. The standard curves for glucosamine hydrochloride showed linearity (r > or = 0.99) over the selected concentration range from 6.65 to 16.63 microg/ml for raw materials and dosage forms. The precision of the dosage form assay, expressed as the % relative standard deviation (R.S.D.), was < 5% at all concentrations. The intra-day and inter-day accuracy, as indicated by the relative error (R.E.), ranged from - 2.54 to 2.70% for glucosamine hydrochloride. For the plasma assay, beagle dog plasma was used to prepare standard curves in the concentration range of 1.25-20 microg/ml. Precipitation of plasma proteins was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. The supernatant was derivatized using phenylisocyanate in phosphate buffer (pH = 8.3) and subsequently evaporated to dryness under a nitrogen stream at 42 degrees C. The residue was dissolved in 250 microl mobile phase and injected onto the chromatographic system. The assay was linear in concentration ranges of 1.25-20 microg/ml (r > or = 0.999). Intra- and inter-day precision was < or = 5.23 and 5.65%, respectively and the intra- and inter-day accuracy, indicated by R.E., ranged from - 8.6 to 10.35%. The method was found to be specific and with excellent linearity, accuracy and precision and is well suited for the quantitation of glucosamine hydrochloride in raw materials, dosage forms, and pharmacokinetic studies.

Development and internal validation of an in vitro-in vivo correlation for a hydrophilic metoprolol tartrate extended release tablet formulation

AbstractPurpose. To develop and validate internally an in vitro-in vivo correlation (IVIVC) for a hydrophilic matrix extended release metoprolol tablet. Methods. In vitro dissolution of the metoprolol tablets was examined using the following methods: Apparatus II, pH 1.2 & 6.8 at 50 rpm and Apparatus I, pH 6.8, at 100 and 150 rpm. Seven healthy subjects received three metoprolol formulations (100 mg): slow, moderate, fast releasing and an oral solution (50 mg). Serial blood samples were collected over 48 hours and analyzed by a validated HPLC assay using fluorescence detection. The f2 metric (similarity factor) was used to analyze the dissolution data. Correlation models were developed using pooled fraction dissolved (FRD) and fraction absorbed (FRA) data from various combinations of the formulations. Predicted metoprolol concentrations were obtained by convolution of the in vivo dissolution rates. Prediction errors were estimated for Cmax and AUC to determine the validity of the correlation. Results. Apparatus I operated at 150 rpm, and pH of 6.8 was found to be the most discriminating dissolution method. There was a significant linear relationship between FRD and FRA when using either two or three of the formulations. An average percent prediction error for Cmax and AUC for all formulations of less than 10% was found for all IVIVC models. Conclusions. The relatively low prediction errors for Cmax and AUC observed strongly suggest that the metoprolol IVIVC models are valid. The average percent prediction error of less than 10% indicates that the correlation is predictive and allows the associated dissolution data to be used as a surrogate for bioavailability studies.

Synthesis and anticonvulsant activity of enaminones. Part 7. Synthesis and anticonvulsant evaluation of ethyl 4-[(substituted phenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylates and their corresponding 5-methylcyclohex-2-enone derivatives.

Further investigation of the potential anticonvulsant activity of the enaminones was attempted to discern the possible role of metabolites as the active/co-active entities of the esters of the enaminones. A series of 5-methyl-2-cyclohexene enaminones, the hypothesised metabolites corresponding to a sequence of active and inactive esters were synthesised and evaluated for anticonvulsant activity. With two exceptions, ethyl 4-[(4-cyanophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1k), and 3-[N-(4-cyanophenyl)amino]-5-methyl-2-cyclohexenone (3g), and ethyl 4-(phenylamino)-6-methyl-2-cyclohexenone (1n), and 3-N-(phenylamino)-5-methyl-2-cyclohexenone (3j), anticonvulsant screening data were parallel, with the ester and their putative decarboxylated analogue displaying similar activity. The most active analogue evaluated in this series, ethyl 4-[(4-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate (1e), which displayed an ED(50) of 16.7 mg kg(-1) and a TD(50) of 110.7 mg kg(-1) (protective index, PI = TD(50)/ED(50) = 6.6) in the maximal electroshock seizure (MES) test in mice and an ED(50) of 3.0 mg kg(-1) and a TD(50) >250 mg kg(-1) (PI > 83.3) in rats in the same evaluation, making this compound the most potent enaminone emanating from our laboratories. Pharmacokinetic evaluation of compound 1e in rats using LC/MS analysis unequivocally provides evidence that this compound is converted into the decarboxylated analogue 3a in the brain and the urine.

Enhanced permeability of molecular weight markers and poorly bioavailable compounds across Caco-2 cell monolayers using the absorption enhancer, zonula occludens toxin

AbstractPurpose. Zonula occludens toxin (Zot), a protein elaborated from Vibrio cholerae, has been shown to be capable of reversibly opening tight junctions. The objective of this work was to determine the stability of Zot and to examine the permeability of a series of molecular weight hydrophilic markers and therapeutic agents in the presence of Zot. Methods. The transport of molecular weight markers (i.e., PEG 4000, FITC-dextran 10,000 and inulin) and therapeutic agents (i.e., acyclovir, cyclopsorin, paclitaxel, doxorubicin) was evaluated with Zot (0, 1, 2, and 4 μg/mL) using Caco-2 cell monolayers. Results. Zot was found to be stable over a 10-day period. Significantly higher (p < 0.05) permeability of the molecular weight markers, inulin, and PEG4000 were observed with Zot (4 μg/mL). The transport of each therapeutic marker was significantly increased with paclitaxel displaying a >3-fold enhancement in Papp values with Zot (4 μg/mL). A 30% decrease in transepithelial electrical resistance values was observed, which returned to baseline 30 min after Zot was removed. Conclusions. Considering the problems of poor oral bioavailability, it is concluded that Zot is a promising drug delivery technology to be used to enhance drug transport across the intestinal mucosa. Future applications are targeted at assessing its usefulness in oral drug delivery using in vivo systems.