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.

1 . A microdialysis process comprising pumping the dialysate in a pulsed manner and analyzing at least some of such pulsed dialysate for its content of a desired material.  
     
     
         2 . In a microdialysis process comprising pumping a dialysate through a probe, the improvement comprising pumping the dialysate in a pulsed manner and analyzing at least some of such pulsed dialysate for its content of a desired material.  
     
     
         3 . A method of performing microdialysis, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium into which or from which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate, which may or may not contain said agent, into the relatively highly permeable section of the probe;    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate from the external medium (acting as a donor medium), or to diffuse from the dialysate into the external medium (acting as a receiver medium);    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at a known flow rate.    
     
     
         4 . A method of  claim 1  for determining the concentration of a free agent, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium containing said diffusible agent, into which or from which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate, which may or may not contain said agent, into the relatively highly permeable section of the probe;    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate from the external medium (acting as a donor medium), or to diffuse from the dialysate into the external medium (acting as a receiver medium);    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at a known flow rate;    f) assaying said dialysate to determine the amount of said diffusible agent that was transferred into or out of said dialysate.    
     
     
         5 . A method of  claim 1  for determining the concentration of a free form of dissolved diffusible agent in the presence of other forms, including bound, precipitated or complexed forms, proteins, enzymes, and other large molecules, structures or particles, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium outside the probe containing said diffusible agent in its free form, along with bound, complexed or precipitated forms, proteins, enzymes, microemulsions, or other large molecules or particles, from which said agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate, which may or may not contain said agent, into the relatively highly permeable section of the probe;    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate from the external medium (acting as a donor medium), or to diffuse from the dialysate into the external medium (acting as a receiver medium);    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at a known flow rate;    f) assaying said dialysate to determine the amount of said diffusible agent that was transferred into or out of said dialysate.    
     
     
         6 . A method of  claim 1  for accurately determining the concentration of drugs or other diffusible agents comprising small molecules, and the exchange of said molecules between two media, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium into which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate containing said agent into the relatively highly permeable section of the probe, for which the radius and volume are known, at a known flow rate (Q);    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse out of the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at the same flow rate used in step (c), above;    f) determining the concentration of said agent in said medium.    
     
     
         7 . A method  claim 1  for accurately determining the concentration of drugs or other diffusible agents comprising other small molecules, and the exchange of said molecules between two media, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium from which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate containing said agent into the relatively highly permeable section of the probe, for which the radius and volume are known, at a known flow rate (Q);    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse out of the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at the same flow rate used in step (c), above;    f) determining the concentration of said agent in said medium.    
     
     
         8 . A method for accurately determining the permeability of a wall of a microdialysis probe, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium into which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate containing said agent in a known concentration (C 0 ) into the relatively highly permeable section of the probe at a known flow rate (Q) and known volume (V W ) and surface area (A);    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse out of the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at the same flow rate used in step (c), above;    f) determining the concentration of said agent in said medium;    g) repeating steps (c) through (f) with the same flow rate and sample volume, and at least one different resting time;    h) plotting the results of steps (f) and (g) to determine how said amount of agent changes with t R ;    i) taking the slope (dM/dt) of the plot of step (h);    j) using said slope to calculate probe wall permeability (P).    
     
     
         9 . A method for accurately determining the window volume (V w ) of a microdialysis probe, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium outside the probe in which is contained a known concentration C D  of the agent to be transferred by diffusion;    c) perfusing a known quantity of a dialysate into the relatively highly permeable section of the probe;    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at a known flow rate;    f) determining the concentration of said agent in said dialysate;    g) calculating the fractional recovery (F R ) of the dialysate sample from Equation (1);    h) repeating steps (c) through (f) with at least one different sample volume V S ;    i) plotting the results of steps (f) and (g) to determine how said concentration changes with 1/V S ;    j) taking the slope of the plot of step (h);    k) using said slope to calculate probe window volume V W .    
     
     
         10 . A method for accurately determining the permeability of a microdialysis probe wall, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium from which an agent in known concentration (C D ) is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate not containing said agent into the relatively highly permeable section of the probe at a known flow rate (Q);    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at the same flow rate used in step (c), above;    f) determining the concentration of said agent in said dialysate;    g) repeating steps (c) through (f) with the same flow rate and sample volume, and at least one different resting time;    h) plotting the results of steps (f) and (g) to determine how said amount of agent changes with t R ;    i) taking the slope (dM/dt) of the plot of step (h);    j) using said slope to calculate the probe permeability.    
     
     
         11 . A method of  claim 1  for obtaining the diffusion coefficient of an agent contained in a medium comprising: 
 a) obtaining the window volume V W  of a microdialysis probe of known length L to at least the accuracy provided by Equation (24);    b) calculating the radius of the probe a and its surface area A=2πaL;    c) measuring F R  for a range of resting times with a constant flow rate, then obtaining F RP  from Equation (24) using the known value of V W  for the said probe;    d) constructing a plot of ln(1−F RP ) vs. t P  and taking γ 1  as the negative of its slope;    e) calculating the diffusion coefficient from V W , A, a, and γ 1 .    
     
     
         12 . A method  claim 1  for accurately determining the rate of release of an agent dissolved in emulsion and/or microemulsion droplets, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of emulsion and an outlet to a receptacle, and through which membrane particles are to be transferred;    b) putting said probe in contact with a medium into which the particles are to be transferred by diffusion;    c) perfusing a known quantity (V W ) of dialysate containing said emulsion into the relatively highly permeable section of the probe;    d) allowing said known quantity of emulsion to remain stationary for a resting time (t R ) sufficient to permit at least some of said emulsion particles to diffuse into the said medium;    e) flushing out said known quantity of emulsion with a single pulse of a known volume (V S ) of emulsion into said receptacle at a flow rate sufficiently high that less than about 10% of said agent transfers out of the dialysate into said medium;    f) determining the concentration of said agent remaining in said known volume (V S );    g) repeating steps (c) through (f) with at least one different (t R );    h) plotting the results of steps (f) and (g) to determine how said concentration changes with (t R );    i) taking the slope of the plot of step (h), which is the rate of release of said agent from the emulsion.    
     
     
         13 . A method of  claim 1  for accurately determining the concentration of a diffusible agent in a medium when the concentration of said agent is changing rapidly, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane said agent is to be transferred;    b) determining the value of γ 1  for the probe using a chosen sample volume, flow rate and resting time;    c) putting said probe in contact with said medium in which is contained the agent to be transferred by diffusion;    d) perfusing a known quantity of a dialysate into the relatively highly permeable section of the probe at the same flow rate Q used to calculate γ 1 ;    e) allowing said known quantity of dialysate to remain stationary for the same resting time t R  used to calculate γ 1 ;    f) flushing out said known quantity of dialysate with a single pulse to collect a sample of dialysate, of the same volume as that chosen in step (b), of a known volume V S , into said receptacle at the same flow rate used in step (d), above;    g) determining the concentration of said agent in said dialysate;    h) repeating steps (d) through (g) with the same flow rate, sample volume, and resting time;    i) determining the fractional recovery at the beginning of a sample interval by calculating F R   0  or F R  and taking the plotting time as the midpoint of the sampling interval according to Equation (78);    j) calculating the concentration in the medium at the beginning of the sample interval as C D =C S /F R   0 , or at the midpoint of the sample interval as C D =C S /F R .    
     
     
         14 . A method  claim 1  for accurately determining the rate at which a diffusible agent interacts with an interactive agent, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) determining the value of γ 1  for the probe using a chosen sample volume, flow rate and resting time;    c) putting said probe in contact with said medium in which is contained the agent to be transferred by diffusion;    d) to said medium containing the agent that will transfer by diffusion, adding a second agent that will interact with the diffusible agent;    e) perfusing a known quantity of a dialysate into the relatively highly permeable section of the probe at the same flow rate Q used to calculate γ 1 ;    f) allowing said known quantity of dialysate to remain stationary for the same resting time t R  used to calculate γ 1 ;    g) flushing out said known quantity of dialysate with a single pulse to collect a sample of dialysate, of the same volume as that chosen in step (b), of a known volume V S , dialysate into said receptacle at the same flow rate used in step (d), above;    h) determining the concentration of said diffusible agent in said dialysate;    i) repeating steps (e) through (h) with the same flow rate, sample volume, and resting time;    j) determining the fractional recovery at the beginning of a sample interval by calculating F R   0 ;    k) calculating the concentration in the medium at the beginning of the sample interval as C D =C S /F R   0 .    
     
     
         15 . A method of  claim 3  wherein said probe comprises a tubular section of highly permeable membrane connected at each end to a section of relatively impermeable membrane.  
     
     
         16 . A method of  claim 3  wherein said probe comprises a needle-type probe comprising a highly permeable tubular membrane concentrically positioned within a relatively impermeable tube.  
     
     
         17 . A method of  claim 3  wherein the agent is contained in the dialysate and the dialysate acts as the donor medium.  
     
     
         18 . A method of  claim 14  in which the interactive agent at least partially binds with the diffusible agent.  
     
     
         19 . A method of  claim 14  in which the interactive agent at least partially adsorbs the diffusible agent.  
     
     
         20 . A method of  claim 14  in which the interactive agent at least partially forms a complex with the diffusible agent.  
     
     
         21 . A method of  claim 14  in which the interactive agent causes the diffusible agent to at least partially precipitate.  
     
     
         22 . A method of  claim 14  in which the interactive agent causes the diffusible agent to at least partially form an emulsion or suspension.  
     
     
         23 . A method of  claim 14  in which the interactive agent is an enzyme or catalyst, which will cause the diffusible agent to chemically degrade.  
     
     
         24 . A method of  claim 14  in which the interactive agent is a chemical that reacts with the diffusible agent to create a different chemical entity.  
     
     
         25 . A method of  claim 14  in which the interactive agent is the diffusible agent itself and causes itself to at least partially precipitate due to supersaturation.  
     
     
         26 . A method of  claim 1  for determining the concentration of a free form of dissolved diffusible agent in the presence of other forms, including bound, precipitated or complexed forms, proteins, enzymes, and other large molecules, structures or particles, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium outside the probe which may or may not contain said agent;    c) perfusing a known quantity of a dialysate, containing said diffusible agent in its free form, along with bound, complexed or precipitated forms, proteins, enzymes, microemulsions, or other large molecules or particles, from which said agent is to be transferred by diffusion, into the relatively highly permeable section of the probe;    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse into the dialysate from the external medium (acting as a donor medium), or to diffuse from the dialysate into the external medium (acting as a receiver medium);    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at a known flow rate;    f) assaying said dialysate to determine the amount of said diffusible agent that was transferred into or out of said dialysate.    
     
     
         27 . A method of  claim 1  for obtaining the diffusion coefficient of an agent contained in a medium comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) putting said probe in contact with a medium from which an agent is to be transferred by diffusion;    c) perfusing a known quantity of a dialysate containing said agent into the relatively highly permeable section of the probe, for which the radius and volume are known, at a known flow rate (Q);    d) allowing said known quantity of dialysate to remain stationary for a resting time (t R ) sufficient to permit at least some of said contained agent to diffuse out of the dialysate;    e) flushing out said known quantity of dialysate with a single pulse of a known volume (V S ) of dialysate into said receptacle at the same flow rate used in step (c), above;    f) determining the concentration of said agent in said medium;    g) calculating the radius of the probe a and its surface area A=2πaL;    h) measuring F R  for a range of resting times with a constant flow rate, then obtaining F RP  using the known value of V W  for the said probe;    i) constructing a plot of ln(1−F RP ) vs. t P  and taking γ 1  as the negative of its slope;    j) calculating the diffusion coefficient from V W , A, a, and γ 1 .    
     
     
         28 . A method of  claim 1  for accurately determining the rate at which a diffusible agent is dissolved or released from an agent containing said diffusible agent, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) determining the value of γ 1  for the probe using a chosen sample volume, flow rate and resting time;    c) putting said probe in contact with said medium which may or may not contain the agent to be transferred by diffusion;    d) to said medium, adding the agent containing said diffusible agent;    e) perfusing a known quantity of a dialysate into the relatively highly permeable section of the probe at the same flow rate Q used to calculate γ 1 ;    f) allowing said known quantity of dialysate to remain stationary for the same resting time t R  used to calculate γ 1 ;    g) flushing out said known quantity of dialysate with a single pulse to collect a sample of dialysate, of the same volume as that chosen in step (b), of a known volume V S , dialysate into said receptacle at the same flow rate used in step (d), above;    h) determining the concentration of said diffusible agent in said dialysate;    i) repeating steps (e) through (h) with the same flow rate, sample volume, and resting time;    j) determining the fractional recovery at the beginning of a sample interval by calculating F R   0 ;    k) calculating the concentration in the medium at the beginning of the sample interval as C D =C S /F R   0 .    
     
     
         29 . A method of  claim 1  for accurately determining the apparent supersaturated solubility and rate at which a diffusible agent is precipitated from a solution or supersaturated solution due to change in temperature, pressure or addition of other agents containing the diffusible agent in a form that can bring about supersaturation of said agent, comprising: 
 a) providing a probe comprising a section of relatively highly permeable membrane relative to any materials to which the membrane is attached for support and positioned between an inlet to a source of dialysate and an outlet to a receptacle, and through which membrane a diffusible agent is to be transferred;    b) determining the value of γ 1  for the probe using a chosen sample volume, flow rate and resting time;    c) putting said probe in contact with said medium in which is contained the agent to be transferred by diffusion;    d) to said medium, creating a supersaturated solution by decreasing the temperature or pressure, or adding other agents that can create a supersaturation of said diffusible agent;    e) perfusing a known quantity of a dialysate into the relatively highly permeable section of the probe at the same flow rate Q used to calculate γ 1 ;    f) allowing said known quantity of dialysate to remain stationary for the same resting time t R  used to calculate γ 1 ;    g) flushing out said known quantity of dialysate with a single pulse to collect a sample of dialysate, of the same volume as that chosen in step (b), of a known volume V S , dialysate into said receptacle at the same flow rate used in step (d), above;    h) determining the concentration of said diffusible agent in said dialysate;    i) repeating steps (e) through (h) with the same flow rate, sample volume, and resting time;    j) determining the fractional recovery at the beginning of a sample interval by calculating F R   0 ;    k) calculating the concentration in the medium at the beginning of the sample interval as C D =C S /F R   0 .    
     
     
         30 . A method of  claim 29  in which the supersaturation of the diffusible agent is brought about by the addition of solids or solid mixtures of nanometer to micron size containing the diffusible agent.  
     
     
         31 . A method of  claim 29  in which the supersaturation of the diffusible agent is brought about by the addition of a microemulsion containing the diffusible agent.  
     
     
         32 . A method of  claim 29  in which the supersaturation of the diffusible agent is brought about by the addition of a solids or solid mixtures containing said diffusible agent in amorphous forms or crystalline forms of higher energy than the most stable crystalline form.  
     
     
         33 . A method of  claim 28  in which said agent is a solid or mixture of solids.  
     
     
         34 . A method of  claim 28  in which said agent is a liquid.