Belinda W.Y. Cheung

The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease

Vascular diseases such as atherosclerosis, stroke or myocardial infarction are a significant public health problem worldwide. Attempts to prevent vascular diseases often imply modifications and improvement of causative risk factors such as high blood pressure, obesity, an unfavorable profile of blood lipids or insulin resistance. In addition to numerous preventive and therapeutic drug regimens, there has been increased focus on identifying dietary compounds that may contribute to cardiovascular health in recent years. Food-derived bioactive peptides represent one such source of health-enhancing components. They can be released during gastrointestinal digestion or food processing from a multitude of plant and animal proteins, especially milk, soy or fish proteins. Biologically active peptides are considered to promote diverse activities, including opiate-like, mineral binding, immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic and antihypertensive actions. By modulating and improving physiological functions, bioactive peptides may provide new therapeutic applications for the prevention or treatment of chronic diseases. As components of functional foods or nutraceuticals with certain health claims, bioactive peptides are of commercial interest as well. The current review centers on bioactive peptides with properties relevant to cardiovascular health.

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (μD) is the only tool available that explicitly provides data on the extracellular space. Although μD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of μD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of μD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on μD as a tool in drug research and development.

The application of sample pooling methods for determining AUC, AUMC and mean residence times in pharmacokinetic studies

For high‐throughput screening in drug development, methods that can reduce analytical work are desirable. Pooling of plasma samples from an individual subject in the time domain to yield a single sample for analysis has been used to estimate the area under the concentration–time curve (AUC). We describe a pooling procedure for the estimation of the area under the first moment curve (AUMC). The mean residence time (MRT), and where intravenous dosing has been used, the steady‐state volume of distribution can then be determined. Plasma samples from pharmacokinetic studies in dogs and humans analyzed in our laboratory were used to validate the pooling approach. Each plasma sample containing a prokinetic macrolide and three of its metabolites was first analyzed separately, and AUCs and AUMCs were calculated using the linear trapezoidal rule. The procedures for the estimation of AUC by sample pooling have been reported by Riad et al. [Pharm. Res. (1991) vol. 8, pp. 541–543]. For the estimation of AUMC, the volume taken from each of n samples to form a pooled sample is proportional to tn(tn+1 − tn−1), except at t0 where the aliquot volume is 0 and at tlast where the aliquot volume is proportional to tlast(tlast − tlast−1). AUMC to tlast is equal to Cpooled × T2/2, where T is the overall experimental time (tlast − t0). The ratio between AUMC and AUC yields the mean residence time (MRT). Bivariate (orthogonal) regression analysis was used to assess agreement between the pooling method and the linear trapezoidal rule. Bias and root mean square error were used to validate the pooling method. Orthogonal regression analysis of the AUMC values determined by pooling (y‐axis) and those estimated by the linear trapezoidal rule (x‐axis) yielded a slope of 1.08 and r2 of 0.994 for the dog samples; slope values ranged from 0.862 to 0.928 and r2 values from 0.838 to 0.988 for the human samples. Bias, expressed as percentage, ranged from −25.1% to 14.8% with an overall average of 1.40%. The results support the use of a pooled‐sample technique in quantitating the average plasma concentration to estimate areas under the curve and areas under the first moment curve over the sampling time period. Mean residence times can then be calculated.

Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method

Lack of reliable methods to accurately measure hydrogen sulfide (H(2)S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H(2)S in cell culture media following incubation with H(2)S-releasing compounds. However, this method is inaccurate because H(2)S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H(2)S, allowing measurement of H(2)S accumulated with time. The amount of H(2)S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H(2)S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 μM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 μM NaHS, we were able to recover twice more H(2)S than conventional methods. When H(2)S-releasing compounds L-cysteine and N-acetylcysteine were added to the cell culture, the amount of H(2)S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H(2)S generated in vitro. This method could be used to screen compounds to identify potential H(2)S donors and inhibitors for therapeutic use.

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives report from the Joint AAPS-FDA Workshop, November 4–5, 2005, Nashville, TN.

AAPS-FDA workshop white paper : Microdialysis principles, application, and regulatory perspectives report from the joint AAPS-FDA workshop, November 4-5, 2005, Nashville, TN

Heme oxygenase-1 is a novel target and antioxidant mediator of S-adenosylmethionine.

The sulfur compound and dietary supplement S-adenosylmethionine (SAM) has been reported to have cytoprotective and antioxidant properties. However, the underlying mechanisms remain unresolved. The present study investigates the effect of SAM on the expression of the antioxidant stress proteins heme oxygenase-1 (HO-1) and ferritin in endothelial cells. Induction of the HO-1/ferritin-system leads to protection of tissues against several inflammatory stimuli. SAM increased the protein and mRNA levels of HO-1 in cultured endothelial cells. Induction of HO-1 gene expression was associated with elevated ferritin protein levels and regulated at the transcriptional level via increased promoter activity. HO-1 upregulation by SAM was causally related to a decrease in NADPH-mediated production of oxygen radicals. Our results demonstrate that the HO-1/ferritin-system is a novel target of the antioxidant compound SAM.

Microdialysis Studies of the Distribution of Antibiotics into Chinchilla Middle Ear Fluid

For conditions such as acute otitis media, in which antibiotic penetration into middle ear fluid (MEF) may be slow or limited, antibiotic plasma levels may not reflect the concentrations at the site of infection that are relevant to clinical outcome. In such cases, a model is needed that will enable prediction of the time course of unbound, pharmacologically active antibiotic levels in MEF. We describe the use of microdialysis as a sampling tool for measurement of unbound antibiotic concentrations in the MEF of the awake, freely moving chinchilla. Results of studies of MEF penetration of the β‐lactam antibiotic, cefdinir, with use of this technique are also described. Preliminary results of studies of the penetration of antibiotics into MEF of the chinchilla appear consistent with clinical findings and suggest that the chinchilla microdialysis model may prove to be a useful tool for predicting antibiotic efficacy in patients.

Estimating Amoxicillin Influx/Efflux in Chinchilla Middle Ear Fluid and Simultaneous Measurement of Antibacterial Effect

ABSTRACT Understanding the transport process and the factors that control the influx/efflux of antibiotics between plasma and middle ear fluid is essential in optimizing the antimicrobial efficacy in the treatment of acute otitis media. In this study, an experimental chinchilla model with the application of a microdialysis technique was utilized to evaluate amoxicillin middle ear distribution kinetics. Amoxicillin solutions at various doses were instilled into the middle ear with a simultaneous intravenous bolus dose. Unbound amoxicillin levels were monitored by microdialysis in both ears. Serial phlebotomy provided samples for the measurement of unbound amoxicillin concentration in plasma ultrafiltrates. In infected chinchillas, discrete middle ear fluid samples were plated and cultured to characterize Streptococcus pneumoniae growth-kill kinetics. Noncompartmental analysis was used to estimate distributional and elimination clearances assuming linear pharmacokinetics. A nonlinear Michaelis-Menten equation was also used to determine the efflux clearance (from middle ear fluid to plasma) in a mammillary compartment model. No difference was observed in amoxicillin pharmacokinetics between control and infected chinchillas. Influx clearance was (4.6 ± 2.4) × 10−3 ml/min-kg and significantly lower than the efflux clearance estimated as (19.2 ± 9.7) × 10−3 ml/min-kg (P < 0.002). Nonlinear kinetics was observed in the locally dosed ear. The microdialysis procedure did not interfere with the bacterial growth-kill profile, thereby enabling pharmacokinetic and pharmacodynamic evaluation concurrently. In conclusion, the results suggested that the distribution equilibrium of amoxicillin in the middle ear favors efflux to plasma over influx. An active transport mechanism across middle ear mucosal epithelium may be involved in amoxicillin distribution.

The chinchilla microdialysis model for the study of antibiotic distribution to middle ear fluid

In cases of slow or limited penetration of an antibiotic to the site of infection such as in acute otitis media (the middle ear), plasma levels of the agent may not reflect the concentrations that are relevant in determining clinical outcome. There is a need for a model that allows prediction of the time-course of unbound, pharmacologically active drug levels in middle ear fluid (MEF). This article introduces microdialysis as a sampling tool to measure unbound antibiotic concentrations in the MEF of the chinchilla, and briefly summarizes the results of studies of MEF penetration of a cephalosporin, a macrolide, and a ketolide antibiotic using this technique. The general concurrence of preliminary results of the chinchilla studies with clinical findings suggests that the chinchilla microdialysis model may be useful in predicting efficacy in patients.

Chapter 6.7 Application of microdialysis in pharmacokinetic studies

Abstract This chapter focuses on the recent advances and applications of microdialysis in pharmacokinetic studies. The first section addresses the technical and theoretical issues regarding microdialysis experiments with an emphasis on obtaining pharmacokinetic data. The principles and methodologies for microdialysis probe calibration are examined. Various setups for microdialysis sampling and subsequent data analysis are also illustrated. The second section includes research articles since the year 2000, chosen from an extensive literature review to report and discuss the utilization and implications of microdialysis sampling in pharmacokinetics. These articles are discussed in three subsections dealing with the kinetics of drug delivery to specific targets, the kinetics of drug transport and metabolism, and the use of microdialysis to study the effects of pathophysiology or surgical intervention on pharmacokinetics. A concluding section briefly underscores the critical need to characterize microdialysis recovery in pharmacokinetic investigations.