Sandeep ppt

kinetics of protein binding

1. KINETICS OF PROTEIN BINDING, BIOEQUIVALANCE, NON COMPARTMENTAL ANALYSIS Presented by Grandhi Sandeep Ganesh

2. PROTEIN BINDING The macromolecules such as protein, DNA or adipose are particularly responsible for complex formation with drug. Such interaction of drug with protein is called protein binding of drug. The proteins commonly involved in binding with drugs are albumin, lipoproteins and al-acid-glycoprotein (AGP). The bound drug is kept in the blood stream while the unbound component may be metabolized or excreted. The unbound drug shows the pharmacological action while the bound drug is neither metabolized nor excreted due to its pharmacokinetic and pharmacodynamic inertness. protein + drug = protien -drug complex

3. KINETICS OF PROTEIN BINDING If P represents proteins and D the drug ,applying law of mass action. P+D PD At equilibrium, K a = [PD]= K a [P][D] …………(1) where, [P]=concentration of free protein [D]=concentration of free drug [PD]=concentration of protein-drug complex K a = association rate constant K d =dissociation constant

4. K a > K d indicates forward reaction i.e. protein-drug binding is favored. If P t is the total concentration of protein present , bound and unbound, then : P t =[PD]+[P]………….(2) If r is the number of moles of drug bound to total moles of protein, then, r= ……….(3) Substituting the value of [PD] from equation (1) in equation (3) r = = ………(4) Equation (4) holds when there is only one binding site on the protein drug complex is 1:1 complex

5. if N is number of binding sites available per mole of protein , then: r = …….(5) the value of association constant k a and number of binding sites then N can be obtained by plotting equation four in different ways. Direct plot Scatchard plot Klotz plot/ lineweaver -burke plot (double reciprocal plot) Hitchcock plot

6. DIRECT PLOT It is made by plotting r vs [D]. Note that all binding sites are occupied by the drug. The protein is saturated and plateau is reached r=N where r=N/2 , [D]=1/k a

7. SCATCHARD PLOT It is made by transforming equation (5) into linear form r= r+r k a [D] r=N k a [D]-r k a [D] therefore, =N k a -r k a A plot of r/[D] vs r yields a straight line slope line=- k a y-intercept=N k a and x-intercept=N

8. KLOTZ PLOT/LINEWEAVER-BURKE PLOT(DOUBLE RECIPROCAL PLOT The reciprocal of equation = A plot of 1/r vs 1/[D] yields a straight line with slope 1/N k a and y-intercept 1/N

9. HITCHCOCK PLOT It is made by re-writing equation (5) as =1+ k a [D] Dividing both sides with N k a gives =

10. BIOEQUIVALENCE STUDIES Equivalence : It is a relative term that compares drug products with respect to a specific characteristic or function or to a defined set of standards. There are several types of equivalences. Chemical Equivalence:- It indicates that two or more drug products contain the same labeled chemical substance as an active ingredient in the same amount.   Pharmaceutical Equivalence:- This term implies that two or more drug products are identical in strength, quality, purity, content uniformity and disintegration and dissolution characteristics. They may, however, differ in containing different excipients .

11. Bioequivalence :- It is a relative term which denotes that the drug substance in two or more identical dosage forms, reaches the systemic circulation at the same relative rate and to the same relative extent i.e. their plasma concentration-time profiles will be identical without significant statistical differences. When statistically significant differences are observed in the bioavailability of two or more drug products, bio­inequivalence is indicated.  Therapeutic Equivalence :- This term indicates that two or more drug products that contain the same therapeutically active ingredient elicit identical pharmacological effects and can control the disease to the same extent.

12. TYPES OF BIOEQUIVALENCE STUDIES Bioequivalence can be demonstrated either- In vivo, or In vitro OBJECTIVE If a new product is intended to be a substitute for an approved medicinal product as a pharmaceutical equivalent or alternative, the equivalence with this product should be shown or justified. In order to ensure clinical performance of such drug products, bioequivalence studies should be performed

13. In vivo Bioequivalence Studies:- The following sequence of criteria is useful in assessing the need for invivo studies 1.Oral immediate-release products with systemic action- Indicated for serious conditions requiring assured response. Narrow therapeutic margin. Pharmacokinetics complicated by absorption <70 o/o or absorption window, nonlinear kinetics, presystemic limination >70%.  Unfavorable physiochemical properties, e.g. low solubility, metastable modification, Instability, etc. Documented evidence for bioavailability problems. No relevant data available, unless justification by applicant that invivo study is not necessary.  Non-oral immediate-release products. Modified-release products with systemic action

14. INVITRO BIOEQUIVALENCE STUDIES The drug product differs only in strength of the active substance It contains, provided all the following conditions hold- Pharmacokinetics are linear The qualitative composition is the same. The ratio between active substance and the excipients is the same, or (in the case of small strengths) the ratio between the excipients is the same. Both products are produced by the same manufacturer at the same production site. A bioavailabilty or bioequivalence study has been performed with a original product. Under the same test conditions, the in vitro Dissolution rate is the same.

15. 2.The drug product has been slightly reformulated or the manufacturing method has been slightly modified by the original manufacturer in ways that can convincingly be argued to be irrelevant for the bioavailability. 3.The drug product meets all of the following requirements - The product is in the form of solution or solubilised form (elixir, syrup, tincture, etc). The product contains active ingredient in the same concentration as the approved drug product. The product contains no excipients known to significantly affect absorption of the active ingredient. 4.An acceptable IVIVC and the invitro dissolution rate of the new product is equivalent with that of the already approved medicinal product, Moreover, The product is intended for topical administration (crean1,ointment,gel,etc.) for local effect.

16. BIOEQUIVALENCE STUDY DESIGNS A. STANDARD BIOEQUIVALENCE STUDY DESIGNS (NON-REPLICATE): This is the most common design where in two formulations are compared in a randomized two-period, to-sequence single dose crossover design. The order of treatment administration in a crossover experiment is called a sequence and the time of treatment administration is called a period. The treatment periods should be separated by a wash out period sufficient to ensure that drug concentration are below the lower limit of bioanalytical quantification in all subjects at the beginning of the second period normally 5 elimination half lives are necessary to achieve this.

17. 2. ALTERNATIVE BIOEQUIVALENCE STUDY DESIGNS 1. Parallel design: It is used for drug substances with very long half-life, e.g.Fingolimod . 2. Replicate designs: For substances with highly variable pharmacokinetic characteristics,e.g.lansoprazole . The advantages of replicate study designs may be that they allow comparisons of within-subject variances for the test and reference products. Partial replicate: where in one treatment (test or reference) is administered to the same subject on to separate occasions. Full replicate : where in both treatments of test and reference product are administered to the same subject on to separate occasions.

18. BIOEQUIVALENCE PROTOCOL Title Principle investigator Project number and date Study objective Study design Design Drug products Test products Reference product Dosage regimen Sample collection schedule Housing 4. Study population Subjects Subject selection 5. Clinical procedures Dosage and drug administration Biological sampling schedule 6. Ethical considerations 7. Facilities 8. Data analysis 9. Drug accountability 10. appendix

19. NON COMPARTMENTAL ANALYSIS The non compartment analysis, also called as model-independent method, does not require the assumption of specific compartment model. This method is however, based on the assumption that the drugs or metabolites follow linear kinetics, and on this basis, this technique can be applied to any compartment model. The compartmental approach, based on the statistical moments theory, involves collection of experimental data following a single dose of drug. If one considers the time course of drug concentration in plasma as a statistical distribution curve, then

20. MRT = where, MRT = mean residence time AUMC = area under the first-moment curve AUC = area under the zero-moment curve AUMC is obtained from a plot of product of plasma drug concentration and time t from zero to infinity. mathematically it is expressed by equation; AUMC =

21. AUC AND AUMC PLOTS

22. AUC is obtained from a plot of plasma drug concentration versus time from zero to infinity. mathematically it is expressed by equation:- AUC = Practically, the AUMC and AUC can be calculated from the respective graphs by the trapezoidal rule ( the method involves dividing the curve by a series of vertical lines into a number of trapezoids, calculating separately the area of each trapezoid and adding them together). MRT is defined as the average amount of time spent by the drug in the body before being eliminated. In this sense, it is the statistical moment analogy of half-life t 1/2 . In effect MRT represents bolus dose to be eliminated. The values will always be greater when the drug is administered in a fashion other than I.v bolus

23. APPLICATIONS It is widely used to estimate the important pharmacokinetic parameters like bioavailability, clearance and apparent volume of distribution. The method is also useful in determining half-life, rate of absorption and first-order absorption rate constant of the drug

24. ADVANTAGES Ease of derivation of pharmacokinetic parameters by simple algebraic equations. The same mathematical treatment can be applied to almost any drug or metabolite provided they follow first-order kinetics. DISADVANTAGES It provided limited information regarding the plasma drug concentration-time profile more often, it deals with averages. The method does not adequately treat non-linear cases.

25. REFERENCES Biopharmaceutics and pharmacokinetics by-treatise by D.M.Brahmankar , Sunil B.jaiswal https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2880414/ https://www.sciencedirect.com/topics/neuroscience/drug-binding