Abstract:
The invention provides a method of evaluating metabolism-based drug interactions. The method involves selecting time points for the determination of the inactivation rate constant of a time-dependent enzyme inhibitor based on the results of a multi-time point IC50 test. Advantageously, with the subject invention, the determination and use of the multi-time point IC50 test provides an indication of the inactivation rate of a test compound and eliminates trial and error tests associated with the selection of appropriate assay conditions for the second assay conducted to determine the inactivation rate constant of the test compound.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to U.S. Provisional Patent Application No. 60/976,653, filed Oct. 1, 2007, the entire contents of which are hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The subject invention is directed to methods of evaluating metabolism-based drug interactions, more particularly, of evaluating the ability of a test compound to inhibit catalytic activity of a target enzyme. 
       BACKGROUND OF THE INVENTION 
       [0003]    Inhibition of enzymes is a major mechanism of metabolism-based drug interactions. This enzyme inhibition is typically evaluated using in vitro models during drug discovery and development, the results of which are generally submitted in regulatory submissions. One such type of enzyme inhibition is time- and cofactor (e.g., NADPH (nicotineamide adenine dinucleotide phosphate))-dependent inhibition (TDI). In this type of inhibition, the inhibitor is generated from the test compound (which may or may not be a direct inhibitor itself) during the assay by an enzyme present in the reaction mix. 
         [0004]    Currently, the typical in vitro test for TDI is cumbersome and requires a great deal of trial and error in testing. The general test includes two separate and sequential assays: first, the IC50 shift experiment; and second, a kinetic experiment (referred to as the K l /K inact  deterrnination). The IC50 shift experiment typically includes an assay of various concentrations of the test compound in either the presence or absence of certain cofactors (e.g., NADPH or a NADPH regenerating system), with subcellular fractions containing the target enzyme of interest. At one predetermined time, typically about 30 minutes, a fraction of this initial incubation is diluted (typically at ratios of 1:5 to 1:20) into a secondary incubation, which contains a probe substrate (at a concentration of approximately the K m  of the reaction) along with the enzyme of interest and cofactors. After a second incubation time for the diluted concentration, which is dictated by the probe substrate, the reaction is stopped and the amount of metabolite formed at each condition (each concentration, with and without the cofactor) is measured and expressed as a percent of control (control being 0% concentration of the test compound). The IC50 value, that is, the test compound concentration associated with 50% decrease in metabolite formation, may be calculated for both cofactor conditions, i.e. in the presence of the cofactor and in the absence of the cofactor. A significant difference in the IC50 values between the two cofactor conditions (IC50 value without cofactor being greater than the IC50 value with cofactor, known as an “IC50 shift”) generally indicates TDI by the test compound, and a second, follow-up experiment is conducted to determine the TDI values. 
         [0005]    The second experiment is the K l /K inact  determination, which measures the kinetic parameters for TDI. This assay uses the same general design as the IC50 test, assaying multiple concentrations of the test compound and an enzyme of interest in the presence of certain cofactors. In this experiment, however, at several predetermined time points, a fraction of the primary incubation is diluted into a secondary incubation, which contains a high concentration (at a concentration above the K m  of the reaction, preferably 5-10× the K m ) of the probe substrate, and the cofactors. After a second incubation time for the diluted concentration, the reaction is stopped and the amount of metabolite formed at each concentration is determined. For each test compound concentration, the degree of metabolism (expressed as the natural log of percent of control) is plotted against the pre-incubation time. The slope associated with each test compound concentration is determined and plotted against the test compound concentration. This data set is then used to derive kinetic parameters for TDI (K l  and K inact ) using non-linear regression or other techniques. 
         [0006]    A problem with the above test procedure is in determining the appropriate time points at which to dilute the incubation in the K l /K inact  assay. For example, if the test compound acts rapidly (i.e., metabolizes quickly), the dilutions should preferably take place at quicker intervals in order to achieve better slope determinations and thus, more accurate results. By contrast, if the test compound acts slower, the dilutions should desirably take place at longer intervals to permit sufficient time to achieve accurate results. The problem lies in knowing whether the test compound will act rapidly or slowly, which is not known from a typical IC50 shift experiment. Without knowing whether the test compound is rapid or slow, the kinetic experiments may require iterative trial and error in testing various sets of incubation times to get accurate results. Such trial and error is cumbersome and adds substantial cost to the procedure. 
       SUMMARY OF THE INVENTION 
       [0007]    In one embodiment of the present invention, there is provided a method of evaluating the ability of a test compound to inhibit catalytic activity of a target enzyme, the method including the steps of performing a first assay of the test compound. The first assay includes the steps of: preparing a first set of samples of the test compound and the target enzyme, the first set including samples containing the target enzyme and varying concentrations of the test compound; preparing a second set of samples of the test compound and the target enzyme, the second set including samples containing the target enzyme and varying concentrations of the test compound; evaluating the amount of metabolite formed in the first set of samples after diluting the samples at a first time interval to obtain first results; and evaluating the amount of metabolite formed in the second set of samples after diluting the samples at a second time interval to obtain second results, the second time interval being different from the first time interval. The method further includes the steps of: comparing the first and second results, selecting time points for a secondary inhibition assay of the test compound based on the comparison, and performing a secondary inhibition assay. The secondary inhibition assay includes the steps of: preparing a secondary set of samples of the test compound and the target enzyme, the secondary set including samples containing the target enzyme and varying concentrations of the test compound; and evaluating the amount of metabolite formed in the secondary set of samples after diluting the samples at the selected time points. Advantageously with the subject invention, the determination and use of the selected time points provides an indication of the metabolizing rate of the test compound and an elimination of trial and error tests in the second assay to compare results based on the actual metabolization rate. 
         [0008]    These and other features of the invention will be better understood through a study of the following detailed description and accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0009]      FIG. 1  is a schematic representation of the first assay of the present invention. 
           [0010]      FIG. 2  is a schematic representation of the second assay of the present invention. 
           [0011]      FIG. 3  is a graphic depiction of the results of a first assay conducted in accordance with the present invention for a rapid acting inhibitor. 
           [0012]      FIG. 4  is a graphic depiction of the results of a first assay conducted in accordance with the present invention for a slow acting inhibitor. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0013]    The invention provides a method of evaluating metabolism-based drug interactions. It particularly provides a method of evaluating metabolism-based drug interactions by incorporating a multi-time period IC50 test procedure. As used herein, the IC50 test is referred to as the test to determine the concentration of a test compound wherein there is associated a 50% decrease in metabolite formation. 
         [0014]    As used herein, “NADPH” refers to nicotinamide adenine dinucleotide phosphate. Also as used herein, “+NADPH” refers to conditions where NADPH is present, while “−NADPH” refers to conditions where NADPH is not present. 
         [0015]    With reference to  FIGS. 1 and 2 , there is depicted an embodiment of the present invention, which includes two separate assays, a first assay  10  and a second assay  12 , which are described more fully below. 
         [0016]    The First Assay 
         [0017]    With reference to  FIG. 1 , the first assay  10  is useable to determine the presence of an IC50 shift of a test compound relative to a target enzyme. The first assay  10  includes a first step  14 , which includes preparing samples containing various concentrations of a test compound in the presence of a target enzyme. A set of samples are prepared, some of which include cofactors, while others do not include cofactors. Any desired cofactors suitable for the target enzyme may be used. Preferably, for studying a target enzyme of cytochrome P450, the cofactors include NADPH and/or NADPH-regenerating systems. The concentrations of the test compound may be in any amount desired, and may include any concentration from 0% to 100% (including a control concentration of 0%). 
         [0018]    The first step  14  also includes an initial incubation conducted preferably at approximately 37° C., but may be conducted at higher or lower temperatures, depending upon the target enzymes being evaluated. 
         [0019]    The target enzyme may be contained in subcellular fractions in the test samples. Preferably, the enzyme used is cytochrome P450. However, any acceptable enzyme may be used in the assay to evaluate the inhibition of catalytic activity thereof. Alternative target enzymes may include UDP-glucuronosyltransferases, A-acetyltransferases, flavine monooxygenases, or other enzymes that might be susceptible to TDI. 
         [0020]    The initial preparations are allowed to incubate for at least two predetermined periods of time. After each time interval, a fraction of the primary incubation is diluted into a secondary incubation. This secondary incubation includes a probe substrate, and additionally includes the selected cofactor. In the embodiment shown in  FIG. 1 , first and second time periods  16  and  18  are selected. After each time period  16 ,  18 , the initial concentration is diluted with the secondary incubation. Preferably, a step  20  of diluting a first fraction of the primary incubation occurs after the first time period  16 . The first time period  16  may be between about 5 minutes to about 15 minutes, more preferably about 10 minutes. Preferably, a step  22  of diluting a second fraction of the primary incubation occurs after the second time period  18 . The second time period  18  may be between about 25 minutes and about 35 minutes, more preferably about 30 minutes. The second time period  18  is to be different from, preferably greater than, the first time period  16 . In one preferred embodiment, a first fraction of the primary incubation is diluted at about 10 minutes, and a second fraction of the primary incubation is diluted at about 30 minutes. Additional fractions of the primary incubation may be diluted at different times to provide additional data points. 
         [0021]    Any level of dilution of the initial incubation may be used. Preferably, the amount of dilution is in a ratio of from about 1 to 5 to about 1 to 20. It is preferred that the concentration of the probe substrate be equal to approximately the K m  of the reaction. 
         [0022]    Any probe substrate may be used in the step  20  of the first dilution and the step  22  of the second dilution. Generally, the particular probe substrate selected will depend upon the target enzyme selected. Possible examples for probe substrates include midazolam (CYP3A4), phenacetin (CYP1A2), diclofenac (CYP2C9), dextromethorphan (CYP2D6), S-mephenytoin (CYP2CP), bupropion (CYP2B6), amodiaquine (CYP2C8), and testosterone (CYP3A4). 
         [0023]    After diluting with the respective probe substrates, the first and second dilutions are each incubated. The post-dilution incubation period is determined by the respective probe substrate. 
         [0024]    After a first incubation time  24 , which is preferably a time sufficient for the metabolism of the probe substrate after the first dilution  20 , a step  26  occurs of stopping the reaction for the first dilution. The amount of probe substrate metabolite formed at each particular condition (e.g., the various concentrations and presence or absence of cofactors) for the first dilution may then be measured. 
         [0025]    After a second incubation time  28 , a step  30  of stopping the reaction for the second dilution occurs. As with the first dilution, the amount of probe substrate metabolite formed at each particular condition for the second dilution may then be measured. The post-dilution incubation times  24  and  28  may be any time desired, depending upon the probe substrate used. Preferably, the post-dilution incubation times  24  and  28  are between about 5 to about 20 minutes. In a preferred embodiment, and to achieve best comparative results, the incubation times after the first dilution  24  and the incubation time after the second dilution  28  are the same (within reasonable error), but may vary if desired (understanding that the starting times for each may vary). By having the same post-dilution incubation times, the achieved test results are in best condition for comparative analysis. 
         [0026]    After the reactions are stopped, the various levels of probe substrate metabolite in the samples are determined by testing (steps  26  and  30 , respectively). Any known means to measure the amount of probe substrate metabolite that has been formed may be used. In a preferred embodiment, the method of measuring the amount of probe substrate metabolite formed is via mass spectrometry. 
         [0027]    In one embodiment, the amount of metabolite formed at each condition (which includes all levels of test compound concentration as well as the presence or absence of NADPH) is considered as a percentage of the control. The “control” measurement is determined as the amount of metabolite formed with a test compound concentration of 0% in the presence of the target enzyme and the probe substrate for the respective condition (with or without cofactors). As explained above, the percentages of control may then be used to calculate the IC50 values for both the +NADPH concentrations and the −NADPH concentrations. As explained above, a significant difference in the IC50 values between the +NADPH and the −NADPH groups generally indicates TDI. With an indication of TDI, the second assay  12  may be conducted to better evaluate the test compound&#39;s ability to inhibit catalytic activity of the target enzyme. 
         [0028]    The Second Assay 
         [0029]    The second assay  12  may include an experiment to determine the kinetic parameters for the test compound relative to the target enzyme. With reference to  FIG. 2 , the second assay  12  may incorporate the same general assay design as the first assay  10 , including an initial step  32  of preparing a new set of samples (not utilized in the first assay  10 ) of multiple concentrations of the test compound and target enzyme. The target enzyme may be contained in subcellular fractions in the initial preparations. Preferably, the second assay  12  is conducted in the presence of the desired cofactors, most particularly the cofactors used in the first assay  10 . The second assay  12  may be conducted at any temperature suitable for the incubation of the samples, and preferably is conducted at about 37° C. In the second assay  12 , a fraction of the primary incubation is diluted into a secondary incubation at predetermined multiple time points as discussed in more detail below. As with the first assay  10 , the secondary incubation in the second assay  12  contains the cofactors of interest. Unlike with the first assay  10 , the secondary incubation in the second assay  12  preferably contains a high concentration (preferably, 5-10× of the K m  of the reaction) of the probe substrate. 
         [0030]      FIG. 2  depicts one embodiment of the invention incorporating a multiple time point second assay  12 . By way of non-limiting example, after a first time point  34 , step  36  of diluting a first fraction of the primary incubation into a secondary incubation occurs. After a second time point  38 , step  40  of diluting a second fraction of the primary incubation into a secondary incubation occurs. Finally, after a third time point  42 , step  44  of diluting a third fraction of the primary incubation into a secondary incubation occurs. It is preferred that the first, second, and third time points  34 ,  38  and  42  be different. More or less time points may be utilized. Each of the secondary incubations includes the probe substrate and cofactor of interest. 
         [0031]    After dilution of the fractions of the primary incubation, the diluted compositions are allowed to incubate for a time sufficient for the probe substrate to be metabolized. Referring to  FIG. 2 , after a first post-dilution incubation time  46 , step  48  of stopping the first dilution occurs. After a second post-dilution incubation time  50 , step  52  of stopping the second dilution occurs. After a third post-dilution incubation time  54 , step  56  of stopping the third dilution occurs. Preferably, the post-dilution incubation times  46 ,  50  and  54  are the same (within reasonable error), but may vary if desired (even if initiated at different times). By having the same post-dilution incubation times, the achieved test results are in best condition for comparative analysis. The amount of probe substrate metabolite formed at each concentration level and after each dilution time may then be measured (steps  48 ,  52 ,  56 , respectively). 
         [0032]    The data obtained in the second assay  12  may be used to derive kinetic parameters for time- and NADPH-dependent inhibition. As explained above, the degree of metabolism, which may be plotted as the natural log of percent of control, may be plotted against the pre-incubation time. The slope associated with each test compound concentration may be used to derive kinetic parameters for TDI. Preferably, the derivation is achieved using non-linear regression or other known techniques. 
         [0033]    Any number of time points may be used in the second assay  12  (i.e., time points where a fraction of the primary incubation is diluted). In a preferred embodiment, there are about 3 to about 10 predetermined time points at which the primary incubation may be diluted into the secondary incubation. The particular time points at which the various dilutions will take place are preferably determined by comparing the results obtained in the first assay  10 , as will be explained in more detail below. 
         [0034]    Determining Time Points for the Second Assay 
         [0035]    With reference to  FIG. 1 , once the values for the amount of metabolite formed at each condition are obtained in the first assay  10 , one may then undertake the step  58  of comparing and evaluating the results. In one embodiment, the amount of metabolite formed in the concentrations that have been diluted after the first time period is compared to the amount of metabolite formed in the concentrations that have been diluted after the second time period. Preferably, the comparison of the two results is achieved by using graphic depictions of the results. In one embodiment, a linear plot for each condition is graphed and evaluated. Most preferably, the results obtained from the dilutions prepared at the first time period are graphed separately from the results obtained from the dilutions prepared at the second time period. As will be readily recognized by one skilled in the art, similar methodology is applied to any additional time points. 
         [0036]    A significant difference in the over-time formation of metabolite between the first time period dilution and the second time period dilution (e.g., as determined by comparing the graphs of the two results from the first assay  10 ) will generally indicate that the test compound is a “slow acting” inhibitor relative to the target enzyme. An insignificant difference in the over-time formation of metabolite between the first time period dilution and the second time period dilution will generally indicate that the test compound is a “rapid acting” inhibitor relative to the target enzyme. Generally, a “rapid acting” inhibitor is one that is substantially metabolized in about 10 minutes or less, while a “slow acting” inhibitor is one that is substantially metabolized in more than about 10 minutes. 
         [0037]    Once it has been determined whether the test compound is a slow acting or rapid acting inhibitor, the time points for the second assay may be selected. Preferably, where the test compound is a “rapid acting” inhibitor, the time periods for the second assay may be closer together. Additionally, for a “rapid acting” inhibitor, the first time period is preferably at a time soon after the initial incubation has begun. In one exemplary embodiment, the first time period at which a dilution may take place for a “rapid acting” inhibitor is between approximately 0.5-2 minutes, with subsequent dilutions of the initial incubation taking place between approximately every 0.5-3 minutes thereafter. 
         [0038]    Preferably, where the test compound is a “slow acting” inhibitor, the time periods for the second assay may be spaced further apart. Additionally, for a “slow acting” inhibitor, the first time period may begin at a time later after the initial incubation has begun. In one exemplary embodiment, the first time period at which a dilution may take place for a “slow acting” inhibitor is between approximately 5-10 minutes, with subsequent dilutions of the initial incubation taking place between approximately every 5-10 minutes thereafter. 
       EXAMPLES 
     Example 1 
       [0039]    A two time point IC50 shift experiment was performed for the test compound azamulin, which is known to be a rapid acting inhibitor. The target enzyme used was cytochrome P450 3A4, with midazolam as the probe substrate. Various concentrations of the test agent azamulin were prepared and allowed to incubate. Additionally, some of the initial incubations included NADPH (+NADPH), while others were void of NADPH (−NADPH). One group of concentrations was allowed to incubate for approximately 10 minutes, at which point the initial incubations were diluted and allowed to incubate for 5 minutes. The extent of midazolam metabolite formation was then tested as a percent of control. A second group of concentrations was allowed to incubate for approximately 30 minutes, at which point the initial incubations were diluted and allowed to incubate for 5 minutes. The extent of midazolam metabolite formation in the second group was then tested as a percent of control. 
         [0040]    The results are set forth below in Table 1: 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 IC50 Values (μM) 
               
             
          
           
               
                   
                 Inhibition Time 
                 +NADPH 
                 −NADPH 
                 Fold Difference 
               
               
                   
                   
               
               
                   
                 10 minutes 
                 0.0031 
                 0.095 
                 31 
               
               
                   
                 30 minutes 
                 0.0027 
                 0.117 
                 44 
               
               
                   
                   
               
             
          
         
       
     
         [0041]      FIG. 3  shows a graphical depiction of the results of this first assay at the various conditions tested. As can be seen in  FIG. 3 , there is very little difference in the graphical representations of the IC50 values for the dilution after 10 minutes (A) and the dilution after 30 minutes (B). Because there is little difference between the 10-minute dilution and the 30-minute dilution, it is surmised that the test compound has been reacted within about 10 minutes. Thus, azamulin may be considered a “rapid acting” inhibitor and the selected time points for the K inact  test should begin sooner, and be spaced closer to each other. 
       Example 2 
       [0042]    A two time point IC50 shift experiment was performed for the test compound verapamil, which is known to be a slow acting inhibitor. The target enzyme used was cytochrome P450 3A4, with midazolam as the probe substrate. Additionally, some of the initial incubations included NADPH (+NADPH), while others were void of NADPH (−NADPH). One group of concentrations was allowed to incubate for approximately 10 minutes, at which point the initial incubations were diluted and allowed to incubate for 5 minutes. The extent of midazolam metabolite formation was then tested as a percent of control. A second group of concentrations was allowed to incubate for approximately 30 minutes, at which point the initial incubations were diluted and allowed to incubate for 5 minutes. The extent of midazolam metabolite formation in the second group was then tested as a percent of control. 
         [0043]    The results are set forth below in Table 2: 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 IC50 Values (μM) 
               
             
          
           
               
                   
                 Inhibition Time 
                 +NADPH 
                 −NADPH 
                 Fold Difference 
               
               
                   
                   
               
             
          
           
               
                   
                 10 minutes 
                 4.7760 
                 22.592 
                 4.7 
               
               
                   
                 30 minutes 
                 0.4293 
                 26.399 
                 61.5 
               
               
                   
                   
               
             
          
         
       
     
         [0044]      FIG. 4  shows a graphical depiction of the results of this first assay at the various conditions tested. As can be seen in  FIG. 4 , there is a significant difference in the graphical representations of the IC50 values for the 10 minute dilution (A) and for the dilution after 30 minutes (B). Because there is a significant difference between the 10-minute dilution and the 30-minute dilution, it may be surmised that the test compound has not been significantly reacted until after about 10 minutes. Thus, verapamil may be considered a slow acting inhibitor, and the time points for the K inact  test should start later after the initial incubation, and should be more spaced apart to achieve a more accurate test.