Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library.

Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The K(m) and V(max) of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with IC(50) values of 100 and 200 microM, respectively. As these compounds competitively inhibited the catalysis of PGH(2), their binding sites appeared to be located near the PGH2 binding pocket.

Modeling of drug release from partially coated matrices made of a high viscosity HPMC.

A mathematical model able to describe the release kinetics of two model drugs (Diprophylline and Theophylline) from partially coated hydroxypropylmethylcellulose (HPMC, Methocel) K4M) matrices is presented. As solvent interaction with the system and drug release can only take place in one direction, the physical frame to be modeled turns out simpler. The model was developed starting from the established equation describing drug dissolution and taking into account the resistance to drug release given by the presence of a growing gel barrier around a matrix system. The model fits the release data obtained from both series of hydrophilic matrices containing increasing amounts (from 0.2 to 0.8 mass ratio) of the two xanthine derivatives. Differences were found in drug release rate according to the different solubility of the actives. Interestingly, however, there is no further reduction in the outer gel layer permeability when the polymer mass fraction exceeds a certain value, with both Theophylline and Diprophylline systems. Results confirm the importance of the fraction of the glassy/rubbery interface held by the active substance in defining the release rate from hydrophilic systems.

Pharmacokinetics and the effect of probenecid on the renal excretion mechanism of diprophylline.

The mechanism of renal excretion of diprophylline (DPP) and the effect of probenecid on the active transport of DPP in renal tubules were investigated in rats. The concentration of DPP in plasma increased in proportion to the doses of 10, 30, and 60 mg/kg. The pharmacokinetic parameters and the urinary excretion of DPP did not change significantly with the dose. These findings indicate that DPP possesses dose-independent pharmacokinetics. Pharmacokinetic parameters for tubular secretion of DPP, as determined by a single-injection renal clearance method, were 21.25 micrograms/mL for the Michaelis-Menten constant and 102.38 micrograms/min for maximum velocity. Coadministration of probenecid decreased the total body clearance of DPP but did not change in the steady-state volume of distribution of DPP. The effect of probenecid concentration on the steady-state renal clearance of DPP was evaluated by continuously infusing probenecid at various rates. The renal clearance of DPP decreased as the probenecid concentration increased, a result indicating that probenecid inhibits the tubular secretion of DPP. However, probenecid did not inhibit the renal secretion of DPP completely, probably because of the existence of probenecid-insensitive transport systems for DPP in the renal proximal tubule. The Michaelis-Menten constant, maximum velocity, and glomerular filtration rate, as calculated with the competitive inhibition model for renal clearance of DPP, correlated well with estimated values after a single intravenous administration, as described earlier. The competitive inhibition constant of probenecid was 15.86 micrograms/mL.

Absorption of intra-bronchial diprophylline-methodology and preliminary results.

With the objective of measuring alveolo-capillary permeability, a diprophylline solution (molecular weight 254 D) was instilled as a bolus. Instillation was performed through distallized impacted fiber bronchoscope and 3 normal volunteers and 3 patients with pulmonary fibrosis were evaluated. Blood samples were collected at regular intervals with subsequent pharmacokinetic study, mainly aimed at determining absorption kinetics. This technique enabled us to distinguish the two groups of individuals which had different absorption rates. In controls, kinetics apparently followed a simple, one compartment model for absorption, which was slower and occurred to a lesser extent. Patients with a pulmonary fibrosis had a faster and higher degree of absorption, with a larger plasma Cmax. In the latter group two-compartment kinetics for absorption was found. We conclude that absorption kinetic parameters can disclose data on structural integrity of epithelia and possible lesions of the lung interstitium.

Dyphylline liposomes for delivery to the skin.

Delivery of dyphylline to the skin using liposomes was investigated. Xanthines are inhibitors of cAMP phosphodiesterase and have been considered for treatment of psoriasis. Dyphylline was chosen because of its solubility in water, which should allow for incorporation of higher concentrations within the liposomes. Liposomes containing dyphylline were prepared by a method using sonication. Transmission electron micrography (TEM) visualization showed small particles ranging from 40 to 100 nm, and particle size distribution determined by light scattering showed the vesicles to have an average diameter of 360 nm. The transdermal delivery of free dyphylline and dyphylline incorporated in unilamellar liposomes was measured from polyethylene glycol (PEG), Carbopol gel, a PEG enhancer base, and water. For comparison, similar experiments were carried out with theophylline as well. When the drugs were incorporated in Carbopol gel, a large difference was seen between their fluxes, with free dyphylline having the highest permeation, followed by liposomal dyphylline, and then theophylline. With the PEG enhancer base, a very high permeation of theophylline was observed relative to dyphylline and liposomal dyphylline. From the PEG base, liposomal dyphylline exhibited the lowest skin permeation flux relative to other bases. Using the PEG base for dyphylline incorporated in liposomes, a high skin partitioning of the drug, along with low transdermal permeation, was measured. These results may indicate that the drug is localized in the skin.

Pharmacokinetic characteristics of N7-substituted theophylline derivatives and their interaction with quinolone in rats.

Disposition of diprophylline (DPP) and proxyphylline (PXP) and the effect of enoxacin on their disposition were investigated in rats. Concentrations of the two drugs in plasma and urine were measured by HPLC. The pharmacokinetic parameters of the two drugs were estimated by model-independent methods. Although the chemical structures of the two drugs are very similar, remarkable differences in the disposition of the two drugs were observed. Total body clearance (CLT) of DPP was 1.77 L/h/kg, which was sevenfold greater than that of PXP (0.26 L/h/kg). Diprophylline was excreted in an almost completely unchanged form in the urine, but only 50% of PXP was excreted. However, no binding of either drug to proteins in rat plasma was observed. The DPP renal clearance (CLR) was 1.75 L/h/kg, approximately 13-fold the CLR for PXP (0.13 L/h/kg) and sevenfold the rat glomerular filtration rate. This study indicates that in rats, DPP is mainly excreted by active tubular secretion and that renal tubular reabsorption contributes to renal excretion of PXP with glomerular filtration. No significant changes in any pharmacokinetic parameters of the two drugs were observed when they were coadministered with enoxacin, compared with the drug administered alone, suggesting that enoxacin had no effect on the pharmacokinetics of either drug.

Protective effect of methylxanthines against carbachol-induced bronchoconstriction in normal subjects.

The bronchoprotective effect of bioequivalent doses of theophylline (TH; 234 mg) and a combination of TH, proxyphylline (PPH) and diprophylline (DPH) in the proportion 2:3:3 (Neo-Biphyllin, NB; 600 mg) against carbachol-induced bronchoconstriction was studied in 10 healthy non-smokers in a randomised controlled double-blind cross-over trial. The subjects were on a methylxanthine-free diet 4 days prior to and during each study day. Bronchial provocation tests were conducted in the morning and afternoon of the three separate study days--control and two medication days--using a standardised technique. On the control day, the dose of carbachol required to reduce the partial expiratory flow volume at 25% of vital capacity (V25p) by at least 25% was established. A significant protective effect was achieved with both TH (p less than 0.05) and NB (p less than 0.001) as measured by V25p. Bronchoprotection was achieved with low maintenance serum levels of TH.