Ultra-fast chromatographic micro-assay for quantification of diphenidol in plasma: application in an oral multi-dose switchability trial

Pharmacokinetics of diphenidol (DPN) is limited due to the lack of analytical methodology. Here, a micro-assay for DPN quantification was developed, by coupling ultra-performance liquid chromatography with tandem mass spectrometry. The procedure involved plasma precipitation and injection of supernatant into UPLC with an Acquitytrade mark C18 column. Detection was in positive electrospray, following transitions of m/z 310.3 --> 292.3 and m/z 275.3 --> 230.2 for DPN and chlorphenamine (internal standard), respectively. The method was linear with a range of 4-400 ng/mL, and a 2 min run time. This method was applied in a switchability trial, where both formulations of DPN were bioequivalent.     

High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

Determination of chemically reduced pyrrolobenzodiazepine SJG-136 in human plasma by HPLC-MS/MS: application to an anticancer phase I dose escalation study

SJG-136 1,1'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyr- rolo[2,1-c][1,4]benzodiazepin-5-one]] (NSC 694501), is a bifunctional pyrrolobenzodiazepine (PBD) dimer that forms selective, irreversible, interstrand DNA cross-links via exocyclic N2 atoms of two guanine bases, with a preference for 5'PuGATCPy binding sites. SJG-136 is highly cytotoxic in human tumor cells in vitro and in human tumor xenograft models in vivo at subnanomolar concentrations and is currently in anticancer phase I clinical trials in the United Kingdom and United States. To support correlative pharmacokinetics studies, a highly sensitive HPLC-MS/MS assay was developed and validated for the reliable quantitation of SJG-136 in human plasma, using the structurally similar PBD dimer DSB-120 as an internal standard. Chemical reduction of SJG-136 to its corresponding amine (SJG-136-H(4), [M + H](+)m/z 561) improved HPLC peak resolution and sensitivity by minimizing complications that arose from the reactivity of the labile imine moieties. Plasma samples were processed by protein precipitation and centrifugal membrane dialysis; components were separated by HPLC using an Agilent Rapid Resolution HT 1.8 mm (2.1 mm x 50 mm) analytical column. The total analysis time from injection to injection was 11 min. Electrospray MS/MS detection of SJG-136-H(4) was based on the selected reaction monitoring (SRM) transition [M + H](+)m/z 561 --> 301. The analytical response ratio was linearly proportional to the plasma concentration of SJG-136 over the nominal concentration range of 25 pg/ml to 250 ng/ml, with a coefficient of determination of r > or = 0.999. The intrarun absolute %RE was < or =19.6, 14.2, and 14.0% at 0.056, 2.83, and 56.3 ng/ml, respectively. The corresponding %RSD was < or =14.9%, 9.01, and 4.59%. The interday %RSD was < or =2.72, 3.46, and 5.20%. The lower and upper limits of quantitation were 0.056 and 56 ng/ml, respectively; recovery of SJG-136 from plasma was > or = 62% across the validated concentration range. The sensitivity of the validated assay was sufficient to detect SJG-136 in human subjects for up to 6 h after intravenous administration of 6 microg/m(2), the starting dose of an NCI-sponsored dose escalation study.     

A Modular Method for the High‐Yield Synthesis of Site‐Specific Protein–Polymer Therapeutics

A versatile method is described to engineer precisely defined protein/peptide–polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin‐like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme‐mediated ligation; and 3) attachment of a polymer by a click reaction with near‐quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water‐soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide–polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide–polymer conjugates for therapeutic use and other applications.     

A liquid chromatography-mass spectrometry assay method for simultaneous determination of amiodarone and desethylamiodarone in rat specimens

A liquid chromatographic-mass spectrometry (LC/MS) assay method was developed for the determination of amiodarone and desethylamiodarone in rat specimens. Analytes were extracted using liquid-liquid extraction in hexane. The LC/MS system consisted of a Waters Micromass ZQtrade mark 4000 spectrometer with an autosampler and pump. A C(18) 3.5 microm (2.1 x 50 mm) column heated to 45 degrees C was used for separation. The mobile phase consisted of methanol and 0.2% aqueous formic acid pumped at 0.2 mL/min as a linear gradient. Components eluted within 12 min. The concentrations of ethopropazine (internal standard), desethylamiodarone and amiodarone were monitored for m/z of 313.10, combination of 546.9 and 617.73, and 645.83, respectively. In plasma (0.1 mL), linearity was achieved between the peak area ratios and concentrations over the range of 2.5-1000 ng/mL for both amiodarone and desethylamiodarone (r(2) > 0.999). The intraday and interday CV were equal or less than 18%, and mean error was <12%. Similarly, in homogenates containing 0.1 g of rat tissue, linearity was observed in standards ranging from 5 to 5000 ng/g. The method was successfully used to measure tissue and plasma concentrations of drug. The validated lower limit of quantitation was 2.5 ng/mL for drug and metabolite, based on 0.1 mL of plasma.     

Pharmacokinetic study of salvianolic acid A in rat after intravenous administration of Danshen injection

In order to research the pharmacokinetics of salvianolic acid A (SalA), a herbal ingredient isolated from Salvia miltiorrhiza Bunge, after intravenous administration to rats, a specific and accurate high-performance liquid chromatography (HPLC) was developed. The assay procedure involved simple liquid-liquid extraction of SalA and internal standard (IS, ethyl-p-hydroxybenzoate) from plasma into ethyl acetate. The organic layer was separated and evaporated under reduced pressure at 40 degrees C. The residue was reconstituted in the mobile phase and analyzed on an Inertsil C8 column, monitored at 285 nm. The mobile phase, which consisted of methanol-acetonitrile-water-formic acid (10:20:70:0.4, by vol), was used at a flow rate of 1.0 mL/min. The ratio of the peak area of the analyte to IS was applied to quantify the plasma samples. The standard curve for SalA was linear (r2 = 0.9999) in the concentration range of 0.75-150 microg/mL. The limit of quantitation (LOQ) of SalA was 0.75 microg/mL. The intra- and inter-day precisions (RSD) of the quality control (QC) samples were in the ranges of 2.17-3.29 and 1.24-5.28%, respectively. Accuracy in the measurement of QC samples ranged from 94.7 to 101.1%. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of SalA in rat plasma after intravenous administration of Danshen injection.     

High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of curcumin in rat plasma. Plasma was precipitated with acetonitrile after addition of the internal standard (IS), 4-hydroxybenzophenone. Separation was achieved on a Waters muBondapak C(18) column (3.9 x 300 mm, 5 microm) using acetonitrile (55%) and citric buffer, pH 3.0 (45%) as the mobile phase (flow rate = 1.0 mL/min). The UV detection wavelength was 300 and 428 nm for IS and curcumin, respectively. The extraction efficiencies were 97.08, 95.69 and 94.90% for 50, 200 and 1000 ng/mL of curcumin in rat plasma, respectively. The calibration curve was linear over the range 0.02-1 microg/mL with a correlation coefficient of r(2) > 0.999. The intra- and inter-day coefficients of variation were less than 13%, and mean intra- and inter-day errors were less than +/-6% at 50, 200 and 1000 ng/mL of curcumin. This assay was successfully applied to the pharmacokinetic studies of both solubilized curcumin and its polymeric micellar formulation in rats. It was found that polymeric micelles increased the half-life of curcumin 162-fold that of solubilized curcumin and increased the volume of distribution (Vd(ss)) by 70-fold.     

Drug Design: Where We Are and Future Prospects

Medicinal chemistry is facing new challenges in approaching precision medicine. Several powerful new tools or improvements of already used tools are now available to medicinal chemists to help in the process of drug discovery, from a hit molecule to a clinically used drug. Among the new tools, the possibility of considering folding intermediates or the catalytic process of a protein as a target for discovering new hits has emerged. In addition, machine learning is a new valuable approach helping medicinal chemists to discover new hits. Other abilities, ranging from the better understanding of the time evolution of biochemical processes to the comprehension of the biological meaning of the data originated from genetic analyses, are on their way to progress further in the drug discovery field toward improved patient care. In this sense, the new approaches to the delivery of drugs targeted to the central nervous system, together with the advancements in understanding the metabolic pathways for a growing number of drugs and relating them to the genetic characteristics of patients, constitute important progress in the field.     

Synthesis,Antibacterial and Pharmacokinetic Evaluation of Novel Derivatives of Harmine N9-Cinnamic Acid

A series of 16 new derivatives of harmine N⁹-Cinnamic acid were synthesized and fully characterized using NMR and MS. The in vitro antibacterial evaluation revealed that most of the synthesized harmine derivatives displayed better antibacterial activities against Gram-positive strains (S. aureus, S. albus and MRSA) than Gram-negative strains (E. coli and PA). In particular, compound 3c showed the strongest bactericidal activity with a minimum inhibitory concentration of 13.67 μg/mL. MTT assay showed that compound 3c displayed weaker cytotoxicity than harmine with IC₅₀ of 340.30, 94.86 and 161.67 μmol/L against WI-38, MCF-7 and HepG2 cell lines, respectively. The pharmacokinetic study revealed that the distribution and elimination of 3c in vivo were rapid in rats with an oral bioavailability of 6.9%.