Method for use of microdialysis

Abstract

It has been surprisingly found that very accurate measurements of mass transfer can be made rapidly by permitting diffusion of an agent desired to be measured into a small, known volume of receiver or out of a known volume of donor, then rapidly pumping or flushing (“pulsing”) the receiver with a known volume of fluid. More specifically, a novel method of transferring small quantities of a contained material (either dissolved or suspended) between two media, based on such pulsing, hereinafter called pulsatile microdialysis (PMD), is disclosed. In a preferred embodiment, one medium (the dialysate) is inside a small, permeable tube (microdialysis probe window) and the other (external medium) is outside. The transfer of material between the two media can be utilized, for example, to sample drug concentrations in the external medium, or the release of drugs from systems within the dialysate, or for other measurements as disclosed herein. In PMD, a dialysate fluid is pumped into a microdialysis probe window, allowed to occupy the probe window while at rest for some resting time, and then flushed at a high rate out as a single pulse. A model that is based on a Fick's Laws was solved, and equations were derived to calculate the effects of various experimental parameters. The models were verified against experimental data using methazolamide, warfarin and benzocaine as test drugs. The data followed the mathematical models. For cases in which the concentration of free drug in the medium outside the probe was constant or changed very slowly, the concentration calibration plots were linear. In simulated first order uptake studies, the PMD and direct donor sampling data were in nearly exact agreement with the theoretical values of k=0.09 min−1. In another experiment, the free concentration of warfarin sodium in the medium outside the probe was made to decline rapidly in a known first order manner, with rate constants as high as 0.077 sec−1. The concentration in the external medium calculated from the PMD data was in nearly exact agreement with the known concentration at various times, and the experimental rate constants were in nearly exact agreement with the theoretical rate constants. For binding of methazolamide to activated charcoal, and for the binding of sodium warfarin to bovine serum albumin, PMD was able to generate sufficient data points to accurately characterize the rapid initial binding. This invention demonstrates that PMD is an accurate method of sampling drug concentrations and measuring rates and extents of a number of processes, including protein binding, adsorption to binding agents such as activated charcoal, release from microemulsion drug delivery systems, and the determination of drug diffusion coefficients, and for various other purposes which will occur to those skilled in the art. Compared to known methods such as traditional (continuous) microdialysis, the present invention offers the ability to sample more frequently, and over much shorter time intervals, thereby accurately obtaining data not heretofore available.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The invention relates to an improved method of effecting and measuring mass transfer. In particular, the invention relates to the use of an improved method of microdialysis for measuring the transfer of relatively small quantities of dissolved, suspended or otherwise dispersed material between two media (one inside and one outside the microdialysis probe). The transfer can be characterized by the loss of material from the medium in which it is contained and / or the collection by the other medium, and in particular can be used to sample drug concentrations and / or characterize the rates at which various processes occur and the extent of transfer. Examples include determination of drug solubility, and processes such as binding of drugs to proteins, chelation and complexation of drugs, adsorption of drugs in solution onto charcoal and other adsorbing agents, and release of drugs from emulsions and microemulsion systems. ...

AI Technical Summary

Benefits of technology

[0032] Pulsatile microdialysis has surprisingly been found to provide not only accurate but very rapid results, thus greatly facilitating, for instance, the ability to correlate in vitro with in vivo results in the case of drugs. Similarly, the methods of this invention have surprisingly been found applicable to determining a free drug concentration, as well as the concentration of the free form in the presence of other forms, including bound, precipitated, adsorbed, or complexed forms, or the presence of proteins, enzymes, and other large molecules, structures or particles. In addition, the present invention has surprisingly been found to be very useful for accurately determining the permeability of a porous device such as a microdialysis probe, and for accurately determining other properties of such a probe such as its window volume. Moreover, the instant invention provides surprisingly accurate measurement of diffusion coefficients, the rate of release of an agent dissolved in emulsion and / or microemulsion droplets, the rate of binding of a material under conditions of adsorption to particles, complexation, and chemical reaction.

[0033] For all the above uses, the present invention has been found to be extremely valuable for the accurate determination of changes in quickly changing systems.

[0096] If a long resting time is used in a PMD experiment, then the concentrations of the dialysate resting in the probe window and in the medium outside the probe will equilibrate, so FRP→1 long tR   (44) and Equation (24) becomes FR=FRQ+VW⁡(1-FRQ)⁢1VS(45) A plot of FR vs. 1 / VS will give an intercept of FRQ and a slope of VW (1−FRQ), which will allow the calculation of VW. (FRQ can also be measured independently from CFMD data.) A variation of this method that will not require long resting times is to obtain a matrix of FR vs. 1 / VS for a range of resting times, and perform a nonlinear regression on the matrix. However, the preferred method of using Equation (45) is preferred because it avoids the potential numerical problems associated with nonlinear regressions.

[0154] Measuring the value of γ1 in this way is one of the preferred ways because it does not require a knowledge of VW. However, knowledge of VW is still desirable because it can be used to improve the accuracy of the method.

[0165] Measuring the value of γ1 in this way is one of the preferred ways because it does not require a knowledge of FRQ. It also does not require knowledge of VW, but knowing of VW is desirable because it can be used to improve the accuracy of the method.

Problems solved by technology

Microdialysis performed in a continuous manner is a known method for sampling drug concentrations from media in biological tissues or in vitro systems; however, certain deficiencies, as will be discussed more fully below, have prevented its optimum application.

At these flow rates, however, the time required for sampling is relatively long, and the time resolution of the samples (i.e., the ability to associate a specific concentration with a specific time or a short time interval) is poor.

In addition, there are problems associated with generating sufficient sample volumes (5-20μL) in short time intervals (less than 30 seconds, perhaps less than 5-10 seconds).

For instance, the sample concentrations become very dilute and may fall below the detection limit of the assay being utilized.

Consequently, CFMD is poorly suited for studies in which concentrations change relatively rapidly.

For setups like these, the inability of CFMD to sample every 10-15 seconds is a great disadvantage.

In addition, for sampling methods such as spiking, which requires separating the cells from the buffer, large errors can potentially occur because the uptake process continues during the sample preparation.

Another problem that can be associated with CFMD is that, at typical perfusion flow rates, the recovery of drug and the resulting sampling efficiency can be poor.

For situations in which the concentration of the external medium changes appreciably during the time a microdialysis sample is taken, the FR defined above is not applicable because CD is changing with time.

As discussed above, this is further complicated by the fact that taking samples rapidly is often difficult because processes can be ongoing during separation or other cleanup steps prior to sample assay.

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