Intracellular signaling by phospholipase D as a therapeutic target.

The pharmaceutical industry has recently focused on intracellular signaling as a means to integrate the multiple facets of complex disease states, such as inflammation, because these pathways respond to numerous extracellular signals and coordinate a collection of cell responses contributing to pathology. One critical aspect of intracellular signaling is regulation of key cell functions by lipid mediators, in particular the generation of a key mediator, phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine by phospholipase D (PLD). Research in this field has intensified, due in part to the recent cloning and partial characterization of the two PLD isoforms in mammalian cells, and this work has contributed significantly to our understanding of events downstream of PA generation. It is these effector functions of PLD activity that make this pathway attractive as a therapeutic target while the biochemical properties of the PLD isozymes make them amenable to small molecule intervention. Recent studies indicate that PA, and its immediate metabolites diacylglycerol and lyso-PA, affect numerous cellular pathways including ligand-mediated secretion, cytoskeletal reorganisations, respiratory burst, prostaglandin release, cell migration, cytokine release, and mitogenesis. This review summarises the data implicating signaling via PLD in these cell functions, obtained from: (i) molecular analyses of PLD/effector interactions, (ii) correlation between PA production and cell responses, (iii) experimental manipulation of PA levels, (iv) inhibition of PLD regulators, and (v) direct inhibition of PA production. The utility of targeting PLD signaling for the treatment of acute/chronic inflammation and other indications is discussed in light of these data.

Characterization of two polymorphs of salmeterol xinafoate crystallized from supercritical fluids.

PURPOSE: To characterize two polymorphs of salmeterol xinafoate (SX-I and SX-II) produced by supercritical fluid crystallization. METHODS: SX-I and SX-II were crystallized as fine powders using Solution Enhanced Dispersion by Supercritical Fluids (SEDS). The two polymorphs and a reference micronized SX sample (MSX) were characterized using powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), aqueous solubility (and dissolution) determination at 5-40 degrees C, BET adsorption analysis, and inverse gas chromatography (IGC). RESULTS: Compared with SX-I, SX-II exhibited a lower enthalpy of fusion, a higher equilibrium solubility, a higher intrinsic dissolution rate, a lower enthalpy of solution (based on van't Hoff solubility plots), and a different FTIR spectrum (reflecting differences in intermolecular hydrogen bonding). Solubility ratio plot yielded a transition temperature (-99 degrees C) below the melting points of both polymorphs. MSX showed essentially the same crystal form as SX-I (confirmed by PXRD and FTIR), but a distinctly different thermal behaviour. Mild trituration of SX-I afforded a similar DSC profile to MSX while prolonged grinding of SX-I gave rise to an endotherm at -109 degrees C, corresponding to solid-solid transition of SX-I to SX-II. Surface analysis of MSX, SX-I, and SX-II by IGC revealed significant differences in surface free energy in terms of both dispersive (nonpolar) interactions and specific (polar) acid-base properties. CONCLUSIONS: The SEDS-processed SX-I and SX-II display high polymorphic purity and distinctly different physical and surface properties. The polymorphs are related enantiotropically with SX-I being the thermodynamically stable form at room temperature.

Pharmacokinetics of acetaminophen in Hong Kong Chinese subjects.

The pharmacokinetics of acetaminophen have been well studied in different populations, especially in Caucasians. However, limited studies on acetaminophen pharmacokinetics have been conducted in the native Chinese and few such data have been reported in the English language literature. Previous published studies suggested that environmental and genetic factors may cause inter-individual difference in acetaminophen disposition, thus we investigated the pharmacokinetics of acetaminophen in Hong Kong Chinese subjects. A single 500 mg oral dose of acetaminophen was administered to 12 healthy male Chinese subjects under fasting conditions. Multiple blood samples were obtained after drug administration. Plasma acetaminophen concentrations were determined using HPLC, and its main pharmacokinetic parameters were generated. In comparison to other published data, acetaminophen half-life was considerably longer (15-62%), and oral clearance was lower (16-56%) in Hong Kong Chinese as compared to Australian Chinese, Caucasians (USA, UK, Australia), and subjects from Pakistan, Denmark, Spain and South Africa. Similarities however were found in the pharmacokinetic parameters between Hong Kong Chinese and Mainland Chinese subjects. The observed pharmacokinetic parameters of acetaminophen in Hong Kong Chinese subjects may be different from other ethnic populations. Further studies are needed to verify this hypothesis.

Study of the hygroscopic properties of selected pharmaceutical aerosols using single particle levitation.

PURPOSE: To use a single particle levitation technique to investigate the equilibrium water sorption characteristics in both the evaporation and growth of four respiratory drugs at 37 degrees C: atropine sulfate (AS), isoproterenol hydrochloride (IPHC) and isoproterenol hemisulfate (IPHS) and disodium cromoglycate (DSCG). METHODS: The equilibrium water content was measured as a function of relative humidity (RH) by a single particle levitation technique using an electrodynamic balance (EDB). The change of water content was determined by the voltage required to balance the weight of the levitated particle electrostatically. The water activities of bulk samples were also measured. Growth ratios were determined and compared with values in the literature. RESULTS: Crystallization or deliquescence was not observed for AS, IPHC and IPHS. The hysteresis in the water cycle was not observed for any of the drugs. At RH approximately 0%, AS particles still contain about 5% water but IPHC and IPHS particles do not contain any residual water. The aerodynamic growth ratio from RH 0% to 99.5% is 2.60, 2.86, 2.42 and 1.26 for AS, IPHC, IPHS and DSCG, respectively. Supersaturated droplets of IPHC and IPHS are expected to exist in the ambient conditions. DSCG is in a solid state in the RH range of 10-90%. CONCLUSIONS: It is expected that some aerosolized drugs of low solubility may experience supersaturation before they enter the human body and this could exert a significant influence both on particle loss before inhalation and on the deposition of the drugs in the lungs. The EDB is a convenient and reliable tool for studying the hygroscopic properties of pharmaceutical aerosols, especially for supersaturated solutions.

High performance liquid chromatographic analysis of a novel aminoalkylpyridine anticonvulsant 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane.

A simple, rapid and specific high performance liquid chromatographic (HPLC) method for the quantitation of 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane (AAP-Cl) and identification of its putative metabolite, 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethanol (beta-AA) in rat blood and urine has been developed. AAP-Cl, beta-AA and an appropriate internal standard were extracted from rat biofluids by a solid phase extraction technique using C18 cartridges prior to the HPLC analysis. The extractibility was 92% for AAP-Cl and 98% for beta-AA. The HPLC analysis employed a symmetrical or standard reversed-phase HPLC column (Apex ODS, 5 microm, 25 cm x 0.46 cm) for blood or urine analysis, a mobile phase of water methanol acetonitrile (40:30:30) containing 20 microl 100 ml(-1) diethylamine at a flow rate of 1 ml min(-1), and UV detection at 254 nm. The limit of detection was 100 ng ml(-1) for both analytes in both blood and urine. The calibration curves for AAP-Cl in rat biofluids were shown to be linear in both low and high concentration ranges (blood: 0-1 and 1-10 microg ml(-1); urine: 0-10 and 10-100 microg ml(-1)) with intra- and inter-day coefficients of variation of no more than 18% for blood and 14% for urine. The method developed was successfully applied to a preliminary analysis of intact AAP-Cl in both blood and urine obtained from rats dosed with AAP-Cl.

Study of hygroscopic properties of aqueous mixtures of disodium fluorescein and sodium chloride using an electrodynamic balance.

PURPOSE: The purposes of this study are: a) to demonstrate the use of an Electrodynamic Balance (EDB) to investigate the hygroscopic properties of pharmaceutical aerosols; and b) to evaluate the applicability of the Zdanovskii-Stokes-Robinson model (ZSR) in the associated data analysis with multicomponent pharmaceutical aerosols. METHODS: The compositional dependence of the water activity of two model materials commonly employed in the study of pharmaceutical aerosols, namely, NaCl and Disodium Fluorescein (DF), was investigated using an EDB. The water contents of single levitated droplets of NaCl and DF and their mixtures at mass ratios of 1:3, 1:1, 3:1, and 6:1 from dilute concentration to high supersaturation were determined as a function of relative humidity (RH). RESULTS: At decreasing ambient RH, supersaturated aqueous NaCl droplets lose water and crystallize to form dry solid particles at an RH of approximately 50%. Aqueous DF droplet continues to lose water until it reaches a final state containing about 20% by mass of residual water. Mixed solutions of DF and NaCl crystallize at an RH of approximately 50% and then continue to lose water at lower RHs. The resulting "dried" particle still contains water whose amount depends on the mass ratios of DF and NaCl in the mixture. Good prediction of water activity of the DF-NaCl mixture can be achieved with the ZSR model. Collection of a full set of water activity-composition data at each mass ratio of DF-NaCl requires only a few hours. CONCLUSIONS: The EDB, together with the application of the ZSR model in data treatment, appears to be a valuable tool for studying the hygroscopic properties of pharmaceutical aerosols.

Mathematical evaluation of the influence of adverse effect on therapeutic efficacy in a direct response system: a case of pharmacodynamic variability of adverse effect.

Two major concerns in therapeutics are the efficacy (E) and adverse effects (Ae) exhibited by a pharmacological agent. Although these elements are studied routinely in both animals and humans during the drug development processes, the data are usually considered independently and in different contexts. Since E and Ae are different yet inseparable components in drug therapy, E should not be used as the sole descriptor for drug responses. To accommodate the influence of Ae on E, the present approach requires the observed Ae data to be transformed to the equivalencies of E data (Ae'). Using appropriate pharmacokinetic modeling techniques, this approach permits the prediction of the adjusted therapeutic effect (ATE) (i.e. , E minus Ae') as a function of time. Effects of pharmacodynamic variability of Ae due to variations in Hill's parameters (i.e., Ae (max), AeC (50), and n (Ae) ) on ATE were studied by computer simulations for a hypothetical one-compartment model drug that displays simple first-order absorption and elimination with central sites for E and Ae. An increase in Ae' (max) and a decrease in AeC (50) and n (Ae) cause a downward shift and peak inversion on the ATE versus time curves coupling with a longer duration of influence of Ae on E. Results also showed that the downward shift of these curves was more apparent with decreasing n (Ae) values and that peak inversion became less noticeable for n (Ae) values <1.5. Subsequent analyses established the optimal dose for the hypothetical drug studied. This approach allows a more comprehensive description of the time course of the overall drug responses and is potentially useful for therapeutic drug monitoring and dose selection during the drug development process.