Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to directly analyze and image pharmaceutical compounds in intact tissue. The anti-tumor drug SCH 226374 was unambiguously determined in mouse tumor tissue using MALDI-QqTOFMS (QSTAR) by monitoring the dissociation of the protonated drug at m/z 695.4 to its predominant fragment at m/z 228.1. A second drug, compound A, was detected in slices of rat brain tissue following oral administration with doses ranging from 1-25 mg/kg. Quantitation of compound A from whole brain homogenates using routine high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedures revealed that concentrations of the drug in whole brain varied from a low of 24 ng/g to a high of 1790 ng/g. The drug candidate was successfully detected by MALDI-QqTOF in samples from each dose, covering a range of approximately two orders of magnitude. In addition, good correlation was observed between the MALDI-QqTOFMS intensities at each dose with the HPLC/MS/MS results. Thus the MALDI-MS response is proportional to the amount of drug in tissue. Custom software was developed to facilitate the imaging of small molecules in tissue using the MALDI-QqTOF mass spectrometer. Images revealing the spatial localization of SCH 226374 in tumor tissue and compound A in brain tissue were acquired.     

Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry

A method is described for the evaluation of drug concentrations in plasma and brain from treated rats. The analyte is recovered from plasma or brain homogenate by liquid-liquid extraction and subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A simple experimental protocol renders the procedure valuable for obtaining information rapidly on brain penetration and plasma exposure of specific classes of compounds. This methodology has been applied to evaluate brain penetration with 30 different compounds from the same discovery program. In an attempt to increase throughput in our screening efforts, mixture dosing was evaluated. Results from single compound administration were compared with results following administration of a mixture of four compounds. Preliminary results, with specific classes of compounds, show no major differences (ranking order) in brain or plasma concentrations between mixture dosing and single compound administration, suggesting that mixture dosing could be applicable to brain penetration studies in the drug discovery phase.     

Structure-activity relationships of 6-nitroquinazolines: dual-acting compounds with inhibitory activities toward both TNF-alpha production and T cell proliferation

We synthesized various 6-nitroquinazolines by modifying the structure of compound 1 and evaluated their inhibitory activities toward both TNF-alpha production and T cell proliferation responses. The presence of the unsubstituted piperazine ring at the C(7)-position was required for both inhibitory activities. In this series of compounds, 5d and 5f, containing the 4-fluorophenyl and 3,4-difluorophenyl moiety, respectively, at the C(4)-position, showed the suppressing effects toward both responses with low cell growth inhibition. Furthermore, the oral administration of these compounds mentioned above at doses of 30 and 100 mg/kg also resulted in significant inhibition of TNF-alpha production induced by LPS in vivo.     

Prodrugs of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine: potent interferon inducing agents in monkeys

In order to improve the oral bioavailability of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine (SM-295072), a potent interferon (IFN) inducing agent, we synthesized prodrugs of it by utilizing the hydroxy groups at the C(2)-side chain and/or the C(8)-position. The carbonate prodrug at the C(8)-position was more effective than that at the C(2)-side chain for oral absorption in rats. Among the compounds prepared, compound 6 demonstrated the most preferable prodrug properties, and the maximum plasma concentration of 6 was approximately 4-fold higher than that of SM-295072. Furthermore, compound 6 was dose-dependently absorbed in monkeys by oral administration, and exhibited a potent IFN-inducting activity that correlated well with its plasma drug concentration.     

Radiation-Induced Copolymerization of Styrene and Cellulose at Low Dose Rates

A study has been made of the radiation-induced grafting of styrene to cellulose in the presence of cobalt 40 gamma irradiation at low total doses and dose rates. For copolymerization, What mans 41 filter paper was immersed in solutions of styrene in methanol at various monomer concentrations. The dose rates were varied from 140 to 3100 rads/hr while the total dose varied from lo³ to 2 × 10⁵ rads.

In the presence of oxygen the grafting results showed considerable scatter, but a statistical analysis revealed that at all concentrations a linear relationship existed between total dose and graft %. Three factors contributing to the variance about the regression line were examined. Residual oxygen in the solution, while not important at high dose rates, was shown markedly to contribute to the variance in the range examined. A linear dose-rate effect involving a decrease in graft with increasing dose rate was demonstrated. A LET effect also contributed to the over-all variance. Preliminary results indicate the presence of a maximum which may be a Trommsdorff effect.     

Systematic approach to optimize a pretreatment method for ultrasensitive liquid chromatography with tandem mass spectrometry analysis of multiple target compounds in biological samples

In current approaches for new drug development, highly sensitive and robust analytical methods for the determination of test compounds in biological samples are essential. These analytical methods should be optimized for every target compound. However, for biological samples that contain multiple compounds as new drug candidates obtained by cassette dosing tests, it would be preferable to develop a single method that allows the determination of all compounds at once. This study aims to establish a systematic approach that enables a selection of the most appropriate pretreatment method for multiple target compounds without the use of their chemical information. We investigated the retention times of 27 known compounds under different mobile phase conditions and determined the required pretreatment of human plasma samples using several solid‐phase and liquid–liquid extractions. From the relationship between retention time and recovery in a principal component analysis, appropriate pretreatments were categorized into several types. Based on the category, we have optimized a pretreatment method for the identification of three calcium channel blockers in human plasma. Plasma concentrations of these drugs in a cassette‐dose clinical study at microdose level were successfully determined with a lower limit of quantitation of 0.2 pg/mL for diltiazem, 1 pg/mL for nicardipine, and 2 pg/mL for nifedipine.     

Evaluation of the photosensitizer Tookad for photodynamic therapy on the Syrian golden hamster cheek pouch model: light dose, drug dose and drug-light interval effects

We have evaluated the efficacy of the new photosensitizer (PS) Tookad in photodynamic therapy (PDT) in vivo. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 762 and 538 nm. The light dose, drug dose and drug injection-light irradiation interval (DLI), ranging between 100 and 300 J/cm2, 1 and 5 mg/kg and from 10 to 240 min, respectively, were varied, and the response to PDT was analyzed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. The level of PDT response, macroscopically and histologically, shows a strong dependence on the DLI, light dose and drug dose at the applied conditions in the normal hamster cheek pouch. A decay of the tissular response with increasing DLI is observed corresponding to a time of half-maximum response ranging from 10 to 120 min, depending on drug dose and light dose. The tissues affected at the lowest doses are predominantly the vascularized diffuse connective tissue situated between the inner and outer striated muscle (SM) layers as well as these muscle layers themselves. The highest response at the shortest DLI and the absence of a measurable response at DLI longer than 240 min at 300 J/cm2 and drug dose of 5 mg/kg are characteristics of a predominantly vascular effect of this PS. This observation suggests that Tookad could be effective in PDT of vascularized lesions or pathologies associated with the proliferation of neovessels.     

Colon targeted drug delivery of branch-chained disulphide cross-linked polymers: design,synthesis, and characterisation studies

Drug delivery directly to the colon is a very useful approach for treating localised colonic diseases such as inflammatory bowel disease, ulcerative colitis, and Crohn’s disease. The use of disulphide cross-linked polymers in colon targeted drug delivery systems has received much attention because these polymers are redox sensitive, and the disulphide bonds are only cleaved by the low redox potential environment in the colon. The goal of this study was to synthesise tricarballylic acid-based trithiol monomers for polymerisation into branch-chained disulphide polymers. The monomer was synthesised via the amide coupling reaction between tricarballylic acid and (triphenylmethyl) thioethylamine using two synthesis steps. The disulphide cross-linked polymers which were synthesised using the air oxidation method were completely reduced after 1 h of reduction with different thiol concentrations detected for the different disulphide polymers. In simulated gastric and intestinal conditions, all polymers had low thiol concentrations compared to the thiol concentrations in the simulated colon condition with Bacteroides fragilis present. Degradation was more pronounced in polymers with loose polymeric networks, as biodegradability relies on the swelling ability of polymers in an aqueous environment. Polymer P15 which has the loosest polymeric networks showed highest degradation.     

Determination of estrogen presence in water by SPR using estrogen receptor dimerization

Estrogenic compounds are a class of pharmaceutical products harmful to animals and a cause of environmental damage. The biological activity of these compounds is high since they have been designed to act at low concentrations. Thus, even at the low concentrations found in the environment, they may produce deleterious effects on aquatic organisms as well as on humans, who might be contaminated in a number of ways (via drinking water or contaminated food, for example). We used the property of these compounds to bind a specific protein (estrogen receptor, ER) to develop a quantification method of these chemical entities. Estrogenic compound detection was performed using ER dimerization properties monitored by surface plasmon resonance (SPR). The ligand-activated ER dimer was detected by its interaction with a specific DNA consensus sequence estrogen response element. The concentration and the nature of the estrogenic compounds modified the SPR signal and were characteristic of the ligand-dependent homodimerization of ER. For 17β-estradiol, dimerization of ER was experimentally determined at an ER to 17β-estradiol ratio near 1:1. Estrogenic compounds (17β-estradiol, estriol, estrone, ethynyl estradiol) activated the dimerization process at different concentration levels, while some others (tamoxiphen, resveratrol, genistein, bisphenol A) did not seem to have any effects on it. We demonstrated that this method allows the direct detection of 17β-estradiol at concentrations above 1.4 μg/L (5 nM).     

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH-sensitive drug release property

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity were developed for oral delivery of protein drugs, using bovine serum albumin (BSA) as a model drug. The composite drug-loaded microparticles with a mean particle size less than 200 μm were prepared by a convenient shredding method. Since the microparticles were formed by tripolyphosphate cross-linking, electrostatic complexation by alginate and/or pectin, as well as ionotropic gelation with calcium ions, the microparticles exhibited an improved pH-sensitive drug release property. The in vitro drug release behaviors of the microparticles were studied in simulated gastric (pH 1.2 and pH 5.0), intestinal (pH 7.4) and colonic (pH 6.0 and pH 6.8 with enzyme) media. For the composite microparticles with suitable compositions, the releases of BSA at pH 1.2 and pH 5.0 could be effectively sustained, while the releases at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for site-specific protein drug delivery through oral administration.     

New antidepressant drug candidate: Hypericum montbretti extract

This study was designed to investigate possible antidepressant-like effects of the extract prepared from the flowers of Hypericum montbretti Spach. (Guttiferae, Clusiaceae). Phytochemical constituents of the methanolic extract were analysed by HPLC method. The main flavonoid component was detected as rutin, and another highly concentrated phenolic compound was quercitrin. Antidepressant activity of the extract was examined by tail suspension and modified forced swimming tests, whereas the motor coordination of the animals was tested by the Rota-Rod apparatus. Reboxetine at a dose of 20 mg?kg?1 was used as a reference drug. Dose-dependent antidepressant activity was observed in both tests following the administration of extract at 100 and 250 mg?kg?1 doses. To the best of our knowledge, this is the first study reporting the antidepressant activity of H. montbretti extract. Additionally, the results of this work support previous papers reporting the antidepressant activity of rutin.     

Development,Validation, and Application of the LC-MS/MS Method for Determination of 4-Acetamidobenzoic Acid in Pharmacokinetic Pilot Studies in Pigs

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid–liquid extraction with only one step—protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r² ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased.     

Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.     

Gastro-intestinal absorption of ethyl 2-chloro-3-[4-(2-methyl-2-phenylpropyloxy)phenyl]propionate from different dosage forms in rats and dogs

To obtain information as to a suitable formulation of ethyl 2-chloro-3-[4-(2-methyl-2-phenylpropyloxy)-phenyl]propionate (AL-294), an antihyperlipidemic drug of low water solubility, the bioavailability after its oral administration in various dosage forms was evaluated in rats and dogs. After AL-294 was administered orally, AL-294 acid (2-chloro-3-[4-(2-methyl-2-phenylpropyloxy)phenyl]propionic acid), which is a metabolite of AL-294, was detected in the plasma. Therefore, absorbability of AL-294 was evaluated using plasma AL-294 acid levels. AL-294 in an oil solution or in a gelatin capsule showed poor absorption, whereas it's absorption was greatly enhanced in the form of an emulsion. The postprandial administration also showed better absorption. The elimination rate of AL-294 acid from the plasma after oral administration of the emulsion was similar to that after intravenous administration of a sodium salt of AL-294 acid.     

Determination of plasma protein binding of propafenone in rats, dogs and humans by highly sensitive gas chromatography-mass spectrometry

A highly sensitive method for the determination of propafenone in plasma has been established using gas chromatography-mass spectrometry with the deuterium-labelled compound as an internal standard. Plasma levels as low as 1 ng/ml were measured using 0.5-ml plasma samples. Plasma protein binding of the drug in rats, dogs and humans in vitro and in vivo was determined by the proposed method. About 90% of the drug was bound to the plasma protein in all species in vitro (20-500 ng/ml) and 69-88% in rats, 90-95% in dogs and 77-89% in humans after oral administration of the drug at doses of 50 mg/kg, 5 mg/kg and 200 mg per person, respectively.     

Interaction of carbamazepine and phenobarbital in rabbits

The interaction of carbamazepine and phenobarbital in rabbits was investigated. The drugs were administered to the rabbit orally as a single dose. By simultaneous administration the sequence of drugs was varied, with an interval between doses of 15 min. The doses of carbamazepine and phenobarbital were 100 and 25 mg, respectively. It was established that phenobarbital appears to be an inductor for carbamazepine independently sequence of administration of the drugs. Carbamazepine reveals inductive properties for phenobarbital in the case where phenobarbital enteres in the organism first. It was ascertained also that, by simultaneous administration, these drugs reduce absorption of each other in plasma.     

High-performance liquid chromatographic determination of the nitroimidazole azanidazole in human plasma and urine

Azanidazole can be measured in plasma and urine by reversed-phase high-performance liquid chromatography employing UV detection. Peak mean plasma concentrations of azanidazole, of 267 ng/ml, occurred at 1.5 h after single oral doses to human subjects, and declined with a half-life of 0.8 h. Less than 0.5% of the dose was excreted in the urine as unchanged drug. Metabolites of azanidazole were also detected by the procedures used.     

Determination of piceid in rat plasma and tissues by high-performance liquid chromatographic method with UV detection

A rapid, sensitive and selective HPLC method was developed and validated for determination of piceid in rat plasma and tissues. The drug was isolated from plasma and tissues by a simple protein precipitation procedure. Chromatographic separation was performed on a C(18) column with acetonitrile-water (26:74, v/v) as mobile phase. The method was successfully applied to the pharmacokinetics and tissue distribution research after oral administration of a 50 mg/kg dose of piceid to healthy male Wistar rats. The pharmacokinetic parameters showed that piceid was quickly absorbed, distributed and eliminated within 4 h after oral administration. The tissue distribution results showed that, at 10 min, the concentrations of piceid in most tissues reached peak level except in heart and testis. The highest level of piceid was found in stomach, then in small intestine, spleen, lung, brain, testis, liver, kidney and heart. The amount of piceid in testis and heart reached the peak level at 30 min. At 120 min, the amount of piceid in all tissues decreased to a low percentage of the initial concentration. Piceid was absorbed throughout the gastrointestinal tract with considerable absorption taking place in the stomach and small intestine. There was no long-term accumulation of piceid in rat tissues.