Saturday, March 30, 2019
Qualitative aspect of drug action
soft aspect of do do medicatess put through Qualitative aspect of drug action Schild platSchild maculation Schild plot is be as pharmaco enterarithmical method of sensory sensory receptor classification. By using schild plot drug- violence abridge for an agonist is determined in the heraldic bearing of mingled preoccupations of a competitory adversary for its receptor in the heraldic bearing of agonist i.e. counter tilt disassociation constant quantity is calculated. The sample is carried out for series of dose proportions for a condition over effect. For example the ratio of the dose of agonist (A) to produce a specific effect (e.g.,half maximal effect) in the charge of the adversary (B) to the dose required in the absence seizure of the antagonist (A) is calculated. This is determined for several doses of antagonist and then enterarithmarithm ((A/A) -1) versus the negative lumber B is plotted. If the regression of log ((A/A) -1) on -log B is linear with a slope of -1, then this indicates that the detestation is war-ridden and by definition the agonist and antagonist act at the analogous recognition sites. If the slope of the regression is not -1, then by definition the antagonist is not agonistic or some early(a) condition is in effect. This might include multiple bandaging sites or pharmacokinetic interactions.Agonist Agonist is a drug which has twain affinity and efficacy. rival Antagonist is a drug which has affinity and nix efficacy. affinityAffinity is a property of a drug it measures how tight a drug binds to a receptor. To bind to a receptor a functional company of the drug should bind to the complementary receptor. The binding capacity of the drug defines the action of the drug.Efficacy Efficacy of a drug apprise be define as ability of drug which activates the receptor to produce desired effect later on binding.Affinity and efficacy are explained in the equivalence as K+1 A + R AR* Response K-1 K+1B + R BR No Response K-1Where A is agonist, B is antagonist, K+1 is association admire constant for binding, K-1is dissociation drift constant for binding- Association rate constant for activation- Dissociation rate constant for activationBy using rectitude of mass action affinity is explained asB + R BRDrug free receptor drug-receptor complexAt symmetry KB = R B KB = Equilibrium dissociation constant BRHill-Langmuir compare this par explains drug occupancyRT = R + BRIf RT = Total number of receptors then by substituting this in law of mass action equationRB = BRT KB + BBy this equation it is determined that drug occupancy (affinity) depends on drug submerging and equilibrium dissociation constant.Equilibrium dissosciation constant EQUILIBRIUM DISSOCIATION CONSTANT (Kd) It is the trace property of the drug and the receptors. It is defined as the tightness of the drug required to occupy 50 % of the receptors. The lavishlyer the affinity of the drug for the receptors lower is the Kd cherish. mathematically Kd is k2/k1 where k2 is the rate of dissociation of the drug from the receptor and k1 is the rate of association of the drug for the receptor.Receptor (R) and Drug (D) interact in a two-sided mode to form a drug-receptor (RD) complex.Where R = Receptor D = Drug (L for ligand is sometimes employ in these equations) k1 = the association rate constant and has the units of M-1min-1 k2 = the dissociation rate constant and has the units of min-1. k2 is sometimes written as k-1.If an agonist binds to the receptor, then the interaction of the agonist (D) and the receptor (R) results in a conformational change in the receptor leading to a chemical reaction.If an antagonist binds to the receptor, then the interaction of antagonist (D) and receptor (R) does not result in the appropriate conformation change in the receptor and a solution does not occur.For drugs that follow the law of unbiased mass action the rate of formation of the complex earth-closet be define d by the following equationdRD/dt refers to the change in the closeness of RD with time (t). Note the square brackets refer to concentration.This equation indicates that the rate at which the drug receptor complex (RD) is formed is proportional to the concentration of both free receptor (R) and free drug (D). The proportionality constant is k1.The rate of dissociation can be defined by the following equation-dRD/dt is the decrease in drug-receptor complex with timeThis equation indicates that the rate at which the drug-receptor complex (RD) dissociates back to free drug and free receptor is proportional to the concentration of the drug receptor complex. The proportionality constant is k2.When the drug and the receptor are initially mixed together, the tot of drug-receptor complex formed get out exceed the dissociation of the drug-receptor complex. If the reaction is allowed to go for a long enough, the amount of drug-receptor complex formed per unit time will be concern to the n umber of dissociations of drug-receptor complex per unit of time, and the system will be at equilibrium. That is equilibrium has occurred. Equilibrium can be defined asor k1RD = k2RDThis equation can be rearranged to giveKd is the dissociation equilibrium constant. Kd has units of concentration as shown in the following equation.Simple emulous disgust unbiased competitive antagonism is the most important emblem of the antagonism. In this type of antagonism the antagonist will compete with available agonist for comparable receptor site. Sufficient antagonist will displace agonist resulting in lower frequency of receptor activation. Presence of antagonist berths agonist log dose answer curve to right. A schild plot for a competitive antagonist will adopt a slope compare to 1 and the X- wiretap and Y-intercept will each suitable thedissociation constantof the antagonist.This can be explained in equation asOccupancy for agonistRA = A OR A/ KART KA+ A A/ KA +1In aim of competi tive antagonist (B)RA = A/ KART A/ KA + B/ KB + 1Occupancy reduced harmonize to B and KBTo obtain same occupancy, must increase A to Ar = A / A = B / BSchild equationr = B / KB +1Where r depends on B and KBApplying log on both sideslog (r-1) = logB log KBAim The main(prenominal) aim of the experiment is to measure the equilibrium dissociation constant (KB) for atropine at acetylcholine muscuranic receptors and to determine the drug receptor interactions.ObjectivesThe main objectives of the experiment are as follows To measure the equilibrium dissociation constant for atropine at acetylcholine muscuranic receptors To demonstrate the reversible competitive antagonism of atropine at acetylcholine muscuranic receptors To determine the equilibrium dissociation constant (KB) for atropine at acetylcholine muscuranic receptors by using schild plot.MethodIsolation and mounting of Guinea-pig ileum in electric electric organ lavGuinea-pig was first sacrificed and then the ileum was colle cted and transferred into physiological flavour solution maintained at 370C. The food particles present in the ileum was expelled out through tally Krebs solution through the lumen. indeed tissue was tied with a line at both the ends where atomic number 53 was tied to the mounting hook and the other was attached to the transducer.1) Preparation of serial dilutions of drugThe drugs used in the experiment were acetylcholine (Ach) and atropine. To determine the unsubdivided competitive antagonism of atropine at Ach muscuranic receptors serial dilutions of Ach were carried out. Ach was given as 110-2M and from the above concentration of the drug the following concentrations were prepared to the organ toilet concentration such as 110-6M, 310-6M, 110-7M, 310-7M, 110-8M, 310-8M, 110-9M and 310-9M Ach. then atropine was diluted to 110-8M (organ vate) from the given 110-2M concentration.2) Determination of Organ cleanse concentrationThe volume of physiological salt solution (pss) was 20 ml, and each time the volume of drug introduced into organ bath was 20l. and then if 20l of 110-2M drug was introduced into the organ bath then it gives 110-5M organ bath concentration.Mathematical calculation of organ bath concentrationIn organ bath we induct 20ml of pss which is equal to 20103 l of pss, if 20 l of 110-2 M Ach was introduced then the organ bath concentration 20lXM 20ml10-2M = 20 l x 10-2 M 20x 103 l = 110-5M (organ bath concentration).The isolated guinea- pig ileum was mounted onto the organ bath and peck up for recording isometric focus of the tissue using graph software in a mac book.Step-1Calibration of the experimental implement The chart 5 software was calibrated and the sampling rate was adjusted to 10 samples per second with a maximum input voltage to 10 mV. The baseline was set to zero and then trace was started from the baseline zero then the force transducer was calibrated by placing 1 gram weight and after the calibration the trace produce d was stopped for the moment to convert the units of tension into grams by selecting the trace produced previously.Step-2Sensitisation of preparation To check the viability of the tissue a response of suitable height was obtained by adding a little high concentration of the drug. here(predicate) in the experiment an appreciable recording was remark at 110-7M Ach.Step-3The time cycle followed to construct a concentration- response curve was0 seconds to add the drug concentrations30 seconds to empty the organ bath and refill with fresh physiological salt solution180 seconds side by side(p) drug concentration was added to the organ bath.Concentration Response CurveBy making use of the above drug concentrations a concentration response curve was constructed according to the provided time cycle.1) 20 l of 110-9M Ach was added into the organ bath at zero seconds at is allowed to stand for 30 seconds, then after 30 seconds the organ bath was emptied and refilled with pss. Pss was allo wed to stand for 180 seconds. During the wash arrest if the peak does not return to the base then it was washed twice or thrice to make sure that all the drug dissociates from the receptors beforehand the next accompaniment of the other drug concentration. Each concentration was tell twice or thrice until the two consecutive responses were reported with the same peak height.2) By following the procedure and time cycle, the concentration response curve was constructed with different concentrations of acetyl choline such as 110-9M,310-9M, 110-8M, 310-8M, 110-7M, 310-7M,110-6M and 310-6M Ach (organ bath concentration).Step-4Equilibration of Acetylcholine receptors with acetylcholine later step-2 the preparation was washed several times until the peak returned to the base line. Then atropine (110-8M organ bath concentration) was added to the preparation and then set aside for 40 proceedings to allow atropine to equilibrate with acetylcholine muscuranic receptors.Step-5Concentratio n response curve in the presence of atropineThe concentration response curve with acetylcholine was repeated again in the presence of atropine by following the time cycle and procedure, which was same as same step 2.thusly in step 3 with each addition of acetylcholine concentration atropine was added simultaneously.Step-6Analysisi) The graph pad prism in the Mac book was used to plot concentration response curves in the absence and presence of atropine.Log concentration (acetylcholine) Vs response in gramsii) From the above plot EC 50 values of acetylcholine in the presence and absence of atropine were obtained. Then the distance between the two curves control and response for the atropine presence was denoted by r, where r was called as shift.iii) The shift was calculated mathematically asr= EC 50 of response in the presence of atropine EC 50 of Ach in the absence of atropineiv) From the value of the shift, schild plot was plotted as log concentration of atropine presence against l og(r-1).v) From the schild plot the dissociation constant KB for atropine at acetylcholine muscuranic receptors was determined.ResultsAs explained above in the procedure serial dilutions of acetylcholine was added to the organ bath, where Ach has produced concentration dependent contractions of the guinea pig ileum as shown in the fig 1.As shown in 1 the serial dilutions of acetylcholine are added into the organ bath from 110-7M to 310-6M Ach. Here in the trace it was clearly shown that contractions produced by the acetylcholine turn out been increased with respect to the concentrations.In step-2 the preparation was washed and added with 110-8M atropine and set aside for 40 minutes to equilibrate the acetylcholine receptors.In the trace it is clearly shown that, the contractions produced by serial dilutions of Ach from 110-8M to 310-4M in the presence of 110-8M atropine.When Trace 1 and Trace 2 are compared it is translucent that the contractions produced by Ach solely (trace 1) were greater than the contractions produced Ach in the presence of atropine (trace 2) which proves the bare(a) competitive antagonism by atropine at muscuranic receptors.A graph is plotted to the log concentration response curve produced by Ach alone against Ach in the presence of atropine. (graph is attatched to the report)From the graph it is known that with the increase in the concentration of Ach, response have been increased when compared to Ach in the presence of atropine and also there is a shift towards right which shows the frank competitive antagonism produced by atropine.From the results produced by Ach alone against Ach in the presence of atropine the fractional difference which is called as shift can be obtained as followsMathematical Calculation shift r = EC50 of response after atropine (or) in the presence of atropine EC50 of control (or) Ach in the absence of atropine = 2.5110-6 = 8.36 3.0 x10-7r-1 =8.36 -1=7.36log(r-1)=log (7.36) =0.86Partial dissociation constan t (PKB) or PA2 is measured to confirm the simple competitive antagonism, where pKB values play an important role in classifying receptors.Therefore PKB =log(r-1) -log atropine=0.86 -log (110-8)=0.86 (-8)=0.86+ 8=8.86From the above results log EC50 values for control (Ach alone) and Ach in the presence of atropine were given as 3.0e-007 and 2.51e-006 respectively.This shows the molar concentration of Ach which produces 50% of the maximal come-at-able response is higher than the molar concentration response produced by Ach in the presence of atropine.If the antagonist is competitive, the dose ratio equals one plus the ratio of the concentration of antagonist divided by its Kd for the receptor. (The dissociation constant of the antagonist is sometimes called Kb and sometimes called Kd)MathType equatingA simple rearrangement givesMathType EquationHere we have plotted a graph with log (antagonist) on the X-axis and log (dose ratio -1) on the Y-axis. If the antagonist has shown simple c ompetitive antagonism then the slope should be 1.0, X-intercept and Y-intercept values should be both equal the Kd of the antagonist obtained.If the agonist and antagonist are competitive, the Schild plot will have a slope of 1.0 and the X intercept will equal the log of the Kd of the antagonist. If the X-axis of a Schild plot is plotted as log(molar), then minus one times the intercept is called the pA2 (p for logarithm, like pH A for antagonist 2 for the dose ratio when the concentration of antagonist equals the pA2). The pA2 (derived from functional experiments) will equal the Kd from binding experiments if antagonist and agonist compete for binding to a individual(a) class of receptor sites.From 5 and 6 it is evident that no concentrations of atropine have showed competitive antagonism perfectly. Therefore from the above results it is known that the concentrations of atropine has not shown simple competitive antagonism fairly.DiscussionReversible competitive antagonism The bin ding of drug to a receptor is fully reversible which produces a correspond shift of the dose response curve to the right in the presence of an antagonist.The mechanism of action of acetylcholine at muscuranic receptorsIn various gastrointestinal noneffervescent muscles, acetylcholine and its derivatives produce contractions by activating muscuranic receptors. It is generally assumed that the M3 muscuranic receptor plays a key role in mediating this activity. The M3 receptor is coupled preferentially to Gq-type G proteins, resulting in the activation of phospholipase C (PLC) and the formation of ionositiol trisphosphate (IP3) and diacylglycerol (DAG) which are likely to get in in muscuranic receptor-mediated smooth muscle contractions. IP3 causes Ca2+ release from intracellular store and can also mobilize Ca2+ secondarily through Ca2+-sensitive or store-dependent mechanisms. DAG, via activation of protein kinase C, phosphorylates various proteins and can directly activate non sele ctive cationic channels.From the above results the value of shift obtained was 0.378 which denotes the simple competitive antagonism produced by the concentration of atropine used (110-8 M).From the value of shift the pKB value was calculated as 8.4.If atropine has shown simple competitive antagonism then the value of pKB should be equal to 1-X intercept.Therefore pKB=1-X intercept=1-(-8.86)=9.86We got value of pKB as 8.86.Therefore pKB is not equal to 1-X intercept.Therefore the concentration of atropine (110-8M organ bath concentration) used by our group has not shown simple competitive antagonism effectively. The literature value of pKB is given as approximately 9 and we have obtained the value of pKB as 8.86 which does not fit with literature value.Therefore from the above observations and results i can conclude that a little to a greater extent high concentration of atropine may serve to produce complete simple competitive antagonism by atropine at acetylcholine muscuranic rece ptors.