1、CalcuSynfor WindowsSoftware for Dose Effect AnalysisPublished and Distributed by Copyright 1996 -2005PO Box 10938, FergusonMO 63135, USA2D Dolphin Way, StaplefordCambridge, CB2 5DW, UKDISCLAIMERThe author and publisher make no representation or warranties with respect to the programs or manual and s
2、pecifically disclaim any implied warranties of merchantability or fitness for purpose.COPY RESTRICTIONSThe purchaser may make a single backup copy of CalcuSyn for his or her own use. Production of other copies is not permitted. Storage of this program in any other computer format or retrieval system
3、 or transmittal in any form without prior permission of the publisher is strictly forbidden.Prined in the U.K.CalcuSyn 2Contents 3ContentsCONTENTS.3INTRODUCTION.7System Requirements7Installing CalcuSyn on your Computer 7Registration.7Technical Support 7Contacting Biosoft8THEORY OF DOSE-EFFECT ANALYS
4、IS 9The Mass-Action Law .9Why a Physical-chemical Approach:.9A Derived Unified Theory:.9The Median-Effect Principle 10The Median-effect Equation of Chou 10The Median-effect Plot and Parameters .10The Combination Index and Isobologram11The Combination Index Equation and Plot of Chou-Talalay.11The Iso
5、bolograms and their Equations .12The Dose-reduction Index of Chou.12What is Synergism: Complexity and Pitfalls .12Experimental Design for Drug-combination Studies .13APPLICATION OF THE CHOU-TALALAY METHOD 15Scope of Application 15By Type of Drugs or Modality of Treatment Agents15By Experimental Desi
6、gn15Example of Previously Published Applications 16CalcuSyn 4How much Synergism is Synergy?.17CI and Isobol Equations for n Drugs. .18CALCUSYN: USING THE PROGRAM .19Creating a New Experiment .19Using the Contents Pane .19Entering a Single Drug.20Using the Add Drug Wizard 20Adding a Drug Manually23En
7、tering a Combination Drug27Using the Add Drug Wizard 27Adding a Combination Manually .29Entering a Non-Constant-Ratio Drug32View Reports .34Report Contents34View Graphs 36Importing Raw Data37Raw Data Grid Import .37Files.38ODBC39Drug Import .40Command Line Import 40Exporting Results .44Raw Data 44Re
8、ports44Graphs 44Monte Carlo Simulation45Reports45Graphs 45CalcuSyn 5Worked examples .45GLOSSARY, SYMBOLS AND DEFINITIONS 57STATISTICAL CONSIDERATIONS61 REFERENCES65For Single Drugs:65For Drug Combinations: 65Statistical Considerations67CalcuSyn 6Introduction 7IntroductionSystem RequirementsIn order
9、to use CalcuSyn you will need the following:(i) CPU a computer with at least a Pentium processor running Windows 98 or later(ii) 16 MB RAM(iii) 4MB of available hard disk space for the program filesInstalling CalcuSyn on your ComputerIf you have an older version of CalcuSyn or the demo version of th
10、is release (V2) installed on your computer then deinstall them. Run the installation program, called setup.exe if the program was supplied on CD, or calcusyn.exe if the program was received by online fulfillment.Note that the CD version has Autorun and, if your computer is suitable, that will presen
11、t a menu on-screen from which you can select the Install option.RegistrationThe first time you run CalcuSyn, you will be presented with a dialog containing a serial number and requesting a password. Contact BIOSOFT at one of the addresses given on page 8 with:(iv) Your name and address.(v) The seria
12、l number generated by the program.(vi) Your order reference code/license number.BIOSOFT will then issue you with the password that you must enter into the dialog. Once you have entered the correct password, you will be able to run CalcuSyn repeatedly.Note that until you have entered the correct pass
13、word, you will not be able to run CalcuSyn at all.Technical SupportBefore contacting BIOSOFT for Technical Support please ensure that you have sought answers in the documentation supplied with the program.CalcuSyn 8If you do need to contact BIOSOFT Technical Support, please make sure that you have t
14、he following information available:(i) The version number of the program you are using. You can get this from the Help | About menu option.(ii) Your order reference code or license number.(iii) The version of Windows you are using.(iv) The exact text of any error or warning messages produced by Calc
15、uSyn or Windows when the problem occurred.(v) Whether or not you have been able to replicate the problem and, if so, the steps necessary to do so.The preferred method for contacting BIOSOFT for technical support is by sending an email to .Contacting BiosoftBIOSOFT2D Dolphin Way, StaplefordCambridge,
16、 CB2 5DW, UKBIOSOFTPO Box 10938Ferguson, MO 63135, USATel: +44 (0)1223 841700 Tel: 314-524-8029Fax: +44 (0)1223 841802 Fax: 314-524-8129email - Theory of Dose-Effect Analysis 9Theory of Dose-Effect Analysis The Mass-Action LawWhy a Physical-chemical Approach: The mass-action law is the basic law in
17、Nature that forms the basis for the chemical equilibrium dynamics, physical absorption isotherm of Langmuir, biological receptor theory and enzyme kinetics. It is a rigorous and well developed discipline. Unlike empirical or statistical approaches in dose-effect analysis in biology, the mass-action
18、law provides well defined models for mathematical derivations. A Derived Unified Theory: By using the enzyme kinetic models of the mass-action law, Chou derived and reported several hundred equations for different numbers of substrates, products, reaction sequences, and different types of inhibition
19、s (1-4). By dividing the equations in the presence and absence of inhibitor and by using the method of mathematical induction and deduction, the “median-effect equation” was derived by Chou (2,5). It is further shown that Chous median-effect equation can be readily used for deriving the four major e
20、quations in biochemistry: The Michaelis-Menten equation, the Hill equation, the Hendersen-Hasselbalch equation and the Scatchard equation (see Refs. 3,10,13).By deriving equations for more than one inhibitor for inhibiting different enzyme reaction mechanisms, the median-effect equation and the medi
21、an-effect plot of Chou has been extended from first order to higher order, and the introduction of the Ki/I50equation (1,2), the distribution equation (1,2) and the general multiple drug-effect equation (6). In 1981, the concept of “Combination Index” was introduced which includes the combination in
22、dex (CI) equation and Fa-CI plot (6-9). In 1988, the concept of Dose-Reduction Index (DRI) was introduced by Chou and Chou (12).According to the Science Citation Index published by the Institute for Scientific Information, Philadelphia, PA. the median-effect equation and the combination-index equati
23、on and its computer software (18,19) have been used in scientific papers published in over 140 different biomedical journals world wide during 1985-1996.There are ten review articles that have been published, including three in encyclopedias (11,13,17).CalcuSyn 10The Median-Effect PrincipleThe Media
24、n-effect Equation of ChouA general equation for dose-effect relationship was derived by Chou (1,2) through mathematical induction using hundreds of enzyme kinetic models. It correlates the “Dose” and the “Effect” in the simplest possible form:fa/fu= (D/Dm)mEq. 1where D: the dose of drugDm: the media
25、n-effect dose signifying the potency. It is determined from the x-intercept of the median-effect plot. fa: the fraction affected by the dosefu: the fraction unaffected, fu=1-fam: an exponent signifying the sigmoidicity (shape) of the dose effect curve. It is determined by the slope of the median-eff
26、ect plot.The alternative forms of the median-effect equation are:fa= 1/1+ (Dm/D)m Eq. 2D = Dmfa/(1-fa)1/mEq. 3From Eq. 2, if Dmand m are known, the effect (fa) can be determined for any dose (D).From Eq. 3, if Dmand m are known, the dose (D) or (Dx) can be determined for any effect (fa). Thus, Dmand
27、 m parameters representing the potency and shape, respectively, determine the entire dose-effect curve.The Median-effect Plot and ParametersThe median-effect plot is a plot of x = log (D) vs y = log (fa/fu) introduced by Chou in 1976 (2,14). It is based on the logarithmic form of Chous median-effect
28、 equation Eq. 1: log (fa/fu) = m log (D) - m log (Dm) Eq. 4Eq. 4 has the form of a straight line, y = mx+b.Thus, the slope yields m value and the x-intercept yields log (Dm) value, and thus, the Dmvalue. The Dmcan also be determined by: Dm= 10-(y-intercept)/m)Theory of Dose-Effect Analysis 11Note th
29、at fa/fu= fa/(1-fa) = (fu)-1-1 = (fa)-1-1-1. The goodness of fit for the data to the median-effect equation is represented by the linear correlation coefficient r of the median-effect plot. Usually, the experimental data from enzyme or receptor systems have r 0.96, from tissue culture r 0.90; and fr
30、om animal systems, r 0.85.The Combination Index and IsobologramThe Combination Index Equation and Plot of Chou-TalalayThe combination index (CI) equation is based on the multiple drug-effect equation of Chou-Talalay derived from enzyme kinetic models (5,7). An equation determines only the additive e
31、ffect rather than synergism or antagonism. However, we define synergism as a more than expected additive effect, and antagonism as a less than expected additive effect. Chou and Talalay in 1983 (7) proposed the designation of CI = 1 as the additive effect, thus from the multiple drug effect equation
32、 of two drugs, we obtain:CI =(D )( D )+(D )( D )1x12x2Eq. 5for mutually exclusive drugs that have the same or similar modes of action, and it is further proposed that CI =(D )( D )+(D )( D )+(D ) (D )( D ) ( D )1x12x212x1x2Eq. 6for mutually non-exclusive drugs that have totally independent modes of
33、action. CI 1 indicates synergism, additive effect, and antagonism, respectively. Eq. 5 or Eq. 6 dictates that drug 1, (D)1, and drug 2, (D)2, (in the numerators) in combination inhibit x% in the actual experiment. Thus, the experimentally observed x% inhibition may not be a round number but most fre
34、quently has a decimal fraction. (Dx)1and (Dx)2(in the denominators) of Eq. 5 or 6 are the doses of drug 1 and drug 2 alone, respectively, inhibiting x%. Dxcan be readily calculated from Eq. 3.For simplicity, mutual exclusivity is usually assumed when more than two drugs are involved in combinations
35、(8,9,14).CalcuSyn 12The Isobolograms and their EquationsFor Eq. 5 and Eq. 6, when CI = 1, they represent the classical isobologram and conservation isobologram equations, respectively. One can generate isobolograms at different effect levels, e.g., ED50, ED70and ED90. However, if too many effect lev
36、els are selected, the isobolograms frequently become messy and difficult to read. For this reason, the Fa-CI table or the Fa-CI plot are recommended over the isobolograms, although they are based on the same CI equation (8,14). The general isobologram equation for the n drug combination has been der
37、ived by Chou and Talalay (8,9).The Dose-reduction Index of ChouThe dose-reduction index (DRI) is a measure of how much the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses for each drug alone. This terminology was formerly introduced by J.
38、 Chou and T.C. Chou in 1988 (12), and has since been used in many publications (14-17). For two drug combinations, we may define from Eq. 5 to obtain (13-17): (DRI)1 = (Dx)1/(D)1 and (DRI)2 = (Dx)2/(D)2. Therefore:CI = (D)1/(Dx)1+ (D)2/(Dx)2= 1/(DRI)1+ 1/(DRI)2Eq. 7The DRI is important in clinical s
39、ituations, where dose-reduction leads to reduced toxicity toward the host while retaining the therapeutic efficacy. Although DRI 1 is beneficial, it not necessarily indicates synergism since, from the above equation, additive effect or even slight antagonism may also lead to DRI 1. If drug A and dru
40、g B each inhibits 50%, and if (0.5A + 0.5B) also inhibits 50% (A and B can be the same drug, or different drugs), then (DRI)1= 2 and (DRI)2= 2. There will be an additive effect if A = B, and this will have no real benefit. However, when A = B (A and B are different drugs) and both drugs have no over
41、lapping toxicity toward the host, then DRI 1 is beneficial indeed.What is Synergism: Complexity and PitfallsThere are ambiguous definitions of synergism and numerous unsubstantiated claims of synergy in biomedical literature. For example, in one review alone (Goldin, A. 1 : 4, 4 : 1, etc.) to form a
42、 Latin square or a checker board design.An Idealized Experimental Design Showing the Layout of Dose Range and Dose Density of Two Drugs for Drug Combination AnalysisDrug 100.25x(ED50)10.5x(ED50)11x(ED50)12x(ED50)14x(ED50)10 Control(fa)0(fa)1(fa)1(fa)1(fa)1(fa)10.25x(ED50)2(fa)2(fa)1,20.5x(ED50)2(fa)
43、2(fa)1,2Drug 2 1x(ED50)2(fa)2(fa)1,22x(ED50)2(fa)2(fa)1,24x(ED50)2(fa)2(fa)1,2Application of the Chou-Talalay Method 17How much Synergism is Synergy?A frequently asked question is “How much synergism is synergy?“ Is CI = 0.95 synergistic? The errors of estimate can be from several sources, e.g., acc
44、uracy of assays, experimental conditions, biological variations and the conformity of the data to the mass-action law.The r value of the median-effect plot provides a first line of statistics. For those who require it, a thorough statistical treatment is given in the section on “Statistical Consider
45、ations“ in the latter part of this manual which provides derivation of CI and its 95% confidence intervals in terms of an algebraic estimate or Monte Carlo analysis.There is no easy way to answer the synergy question. Several relevant points need to be considered:1. Accuracy of measurement and biolo
46、gical variability.2. Synergism at what dose level? or at what effect level?3. Experimental conditions, e.g., temperature, oxygen tension, pH, etc. may affect the data and conclusions.4. Whether synergy is treatment schedule dependent or combination ratio dependent?The following table shows the recom
47、mended symbols and descriptions for presenting the degrees of synergism or antagonism.Recommended Symbols for Describing Synergism or Antagonism in Drug Combination Studies Analyzed with the Combination Index (CI) MethodaRange of CI Symbol Description10 Very strong antagonismaThe combination index m
48、ethod is based on that described by Chou and Talalay (8,14) and the computer software of Chou and Chou (18,19) and CalcuSyn. The ranges of CI and the symbols are refined from those described earlier by Chou (14). CI 1 indicate synergism, additive effect and antagonism, respectively (8,14). When CI v
49、alues change with Fa, the averaged CI at EC50, EC75, EC90and EC95(or at EC75, EC90, and EC95) are used. If any CI value(s) are greater than 2, it is recommended that the antilog of the averaged log (CI) value is used to avoidsignificant biases of overestimating antagonism. Overall results may also be represented by the averaged number of + or symbols where + and may be canceled out.CI and Isobol Equations for n Drugs.For mutually exclusive drugs, the combination index equation for the combination of n inhibitors (6, 8, 1
