Case study 2. Practical analytical considerations for conducting in vitro enzyme kinetic studies.

Characterization of enzyme kinetics in an experiment is dependent on measurement of a change in concentration of either the substrate (loss of parent) or the product (formation of metabolite). Modern analytical techniques such as ultrahigh pressure liquid chromatography and high-resolution mass spectrometry have allowed accurate characterization of minute changes in concentration. Therefore, complex kinetic data such as a sigmoidal phase at low substrate concentrations or terminal half-life in a PK curve can be evaluated by stretching the limits of analytical quantification.

Enzyme kinetics in drug metabolism: fundamentals and applications.

Enzymes are protein catalysts that lower the energy barrier for a reaction and speed the rate of a chemical change. The kinetics of reactions catalyzed by enzymes, as well as several mechanisms underlying the kinetics, have been comprehensively studied and written in textbooks (1, 2). The importance of quantitative evaluation of enzymatic processes has been recognized in many fields of study, including biochemistry, molecular biology, and pharmaceutical sciences to name a few.

Modulation of xylanase production from alkaliphilic Bacillus pumilus VLK-1 through process optimization and temperature shift operation.

This study was aimed at enhancing the production of xylanase from an alkaliphilic Bacillus pumilus VLK-1 in submerged fermentation using wheat bran, a cheap and abundantly available agro-residue, through process optimization and to monitor the effect of temperature shift operation on it. The potential of xylanase in saccharification of wheat straw was also investigated. The results showed that optimization of the fermentation process by one variable approach increased the enzyme yield from 402 to 4,986 IU/ml.

Herb-drug interactions: challenges and opportunities for improved predictions.

Supported by a usage history that predates written records and the perception that "natural" ensures safety, herbal products have increasingly been incorporated into Western health care. Consumers often self-administer these products concomitantly with conventional medications without informing their health care provider(s). Such herb-drug combinations can produce untoward effects when the herbal product perturbs the activity of drug metabolizing enzymes and/or transporters.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs).

Studies on Tinospora cordifolia monosugars and correlation analysis of uronic acids by spectrophotometric methods and GLC.

Cold water-soluble (CWSP) and hot water soluble polysaccharides (HWSP) from Tinospora cordifolia stems were isolated and purified in 2.99% and 1.99% yield respectively.

A multi-institutional international study of risk factors for hematoma after thyroidectomy.

Background: Cervical hematoma can be a potentially fatal complication after thyroidectomy, but its risk factors and timing remain poorly understood.

Methods: We conducted a retrospective, case-control study identifying 207 patients from 15 institutions in 3 countries who developed a hematoma requiring return to the operating room (OR) after thyroidectomy.

Results: Forty-seven percent of hematoma patients returned to the OR within 6 hours and 79% within 24 hours of their thyroidectomy.

Source-dependent intracellular distribution of iron in lens epithelial cells cultured under normoxic and hypoxic conditions.

Purpose: Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs).

Compartmental models for apical efflux by P-glycoprotein--part 1: evaluation of model complexity.

Purpose: With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data.

Methods: Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated.

Hurthle cell carcinoma: an update on survival over the last 35 years.

Background: Hurthle cell carcinoma (HCC) of the thyroid is a variant of follicular cell carcinoma (FCC). A low incidence and lack of long-term follow-up data have caused controversy regarding the survival characteristics of HCC. We aimed to clarify this controversy by analyzing HCC survival over a 35-year period using the Surveillance, Epidemiology, and End Results (SEER) database.

Compartmental models for apical efflux by P-glycoprotein: part 2--a theoretical study on transporter kinetic parameters.

Purpose: The impact of efflux transporters in intracellular concentrations of a drug can be predicted with modeling techniques. In Part 1, several compartmental models were developed and evaluated. The goal of Part 2 was to apply these models to the characterization and interpretation of saturation kinetic data.

Identification of domperidone metabolites in plasma and urine of gastroparesis patients with LC-ESI-MS/MS.

Domperidone is a prokinetic agent used to treat gastroparesis. Previous studies reported oxidative metabolites of domperidone, detected by radiometric high-performance liquid chromatography or single quadrupole mass spectrometric techniques. Our aim was to identify domperidone Phase I and Phase II metabolites using liquid chromatography combined with electrospray ionization-enabled tandem mass spectrometry.

Intracellular drug concentrations and transporters: measurement, modeling, and implications for the liver.

Intracellular concentrations of drugs and metabolites are often important determinants of efficacy, toxicity, and drug interactions. Hepatic drug distribution can be affected by many factors, including physicochemical properties, uptake/efflux transporters, protein binding, organelle sequestration, and metabolism. This white paper highlights determinants of hepatocyte drug/metabolite concentrations and provides an update on model systems, methods, and modeling/simulation approaches used to quantitatively assess hepatocellular concentrations of molecules.

ITC recommendations for transporter kinetic parameter estimation and translational modeling of transport-mediated PK and DDIs in humans.

This white paper provides a critical analysis of methods for estimating transporter kinetics and recommendations on proper parameter calculation in various experimental systems. Rational interpretation of transporter-knockout animal findings and application of static and dynamic physiologically based modeling approaches for prediction of human transporter-mediated pharmacokinetics and drug-drug interactions (DDIs) are presented. The objective is to provide appropriate guidance for the use of in vitro, in vivo, and modeling tools in translational transporter science.

Covalent immobilization of xylanase produced from Bacillus pumilus SV-85S on electrospun polymethyl methacrylate nanofiber membrane.

Polymethyl methacrylate (PMMA) nanofiber membrane (NFM) was synthesized by an electrospinning technique. These membranes were utilized as a support for immobilization of xylanase enzyme to study its pH stability, thermal stability, and reusability. The morphology of aligned NFM was studied by optical microscopy and scanning electron microscopy.

Pharmacokinetic application of a bio-analytical LC-MS method developed for 5-fluorouracil and methotrexate in mouse plasma, brain and urine.

In the past we have reported significant cognitive deficits in mice receiving 5-fluorouracil in combination with low-dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio-analytical method was therefore developed and validated for 5-fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer.

Pulmonary metabolism of resveratrol: in vitro and in vivo evidence.

The role of pulmonary metabolism in trans-resveratrol (RES) pharmacokinetics was studied in a mouse model. Plasma concentrations of RES and its major metabolites trans-resveratrol-3-sulfate (R3S) and trans-resveratrol-3-glucuronide (R3G) were compared after administration of RES by intravenous (IV) and intra-arterial (IA) routes. Total area under the curve (AUC) of RES decreased by approximately 50% when RES was administered by the IV route compared with the IA route.

Biobleaching application of cellulase poor and alkali stable xylanase from Bacillus pumilus SV-85S.

The potential of extracellular alkali stable and thermo tolerant xylanase produced by Bacilluspumilus SV-85S through solid state fermentation was investigated in pulp bleaching in association with conventional bleaching using chlorine and chlorine dioxide. The biobleaching of kraft pulp with xylanase was the most effective at an enzyme dose of 10 IU/g oven dried pulp, pH 9.0 and 120 min incubation at 55 °C.

An open-label pharmacokinetic study of oxymorphone extended release in the presence of naltrexone in the older adult.

Objective: The aging population generally has greater need for analgesics and is best served by having as many good therapeutic options as possible. Geriatric analgesia requires special consideration of age-associated physiologic changes that can affect drug dosing. The study of extended-release (ER) oxymorphone in older (≥ 65 years of age) versus younger (18-40 years of age) male and female volunteers was described.

In vivo-formed versus preformed metabolite kinetics of trans-resveratrol-3-sulfate and trans-resveratrol-3-glucuronide.

Metabolites in safety testing have gained a lot of attention recently. Regulatory agencies have suggested that the kinetics of preformed and in vivo-formed metabolites are comparable. This subject has been a topic of debate.

Commentary: nonspecific protein binding versus membrane partitioning: it is not just semantics.

Nonspecific binding or sequestration results in differences between free and total drug concentrations, both in vitro and in vivo. Membrane partitioning and not protein binding is the primary mechanism of drug sequestration. Therefore, physicochemical properties, e.

Observational study of the outcomes and costs of initiating maintenance therapies in patients with moderate exacerbations of COPD.

Background: There are limited data describing patients with moderate COPD exacerbations and evaluating comparative effectiveness of maintenance treatments in this patient population. The study examined COPD patients with moderate COPD exacerbations. COPD-related outcomes were compared between patients initiating fluticasone propionate-salmeterol 250/50 mcg (FSC) vs anticholinergics (ACs) following a moderate COPD exacerbation.

An analysis of fine needle aspiration versus core needle biopsy in clinically palpable breast lesions: a report on the predictive values and a cost comparison.

Background: Although fine-needle aspiration (FNA) is an established tool in the biopsy of breast masses, there has been a trend toward using core-needle biopsy (CNB). The aim of this study was to determine whether FNA has comparable predictive value with CNB and whether FNA is more cost effective.

Methods: A retrospective review was conducted on 162 patients who underwent either FNA or CNB of palpable breast lesions and had histologic confirmation with surgical biopsy in calendar year 2005.

Ceruloplasmin alters intracellular iron regulated proteins and pathways: ferritin, transferrin receptor, glutamate and hypoxia-inducible factor-1α.

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp.