Bioavailability & Method of determination

METHODS  FOR  ASSESSING  BIOAVAILABILITY:

• Direct and indirect methods may be used to assess drug bioavailability.
• The in-vivo bioavailability of a drug product is demonstrated by the rate and extent of drug absorption, as determined by comparison of measured parameters, eg, concentration of the active drug ingredient in the blood, cumulative urinary excretion rates, or pharmacological effects. 
• For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action.
• The design of the bioavailability study depends on the objectives of the study, the ability to analyze the drug (and metabolites) in biological fluids, the pharmacodynamics of the drug substance, the route of drug administration, and the nature of the drug product. 
• Pharmacokinetic and/or pharmacodynamic parameters as well as clinical observations and in-vitro studies may be used to determine drug bioavailability from a drug product. 

1. Pharmacokinetic methods 

• These are indirect methods 
• Assumption that –pharmacokinetic profile reflects the therapeutic effectiveness of a drug.
• Advantages: - Accurate, Reliable,Reproducible
• Plasma / blood level time profile. 
• Time for peak plasma (blood) concentration (t max) 
• Peak plasma drug concentration (C max) 
• Area under the plasma drug concentration–time curve (AUC) 
• Urinary excretion studies
• Cumulative amount of drug excreted in the urine (D u) 
• Rate of drug excretion in the urine (dD u/dt) 
• Time for maximum urinary excretion (t)
• Other biological fluids 

2. Pharmacodynamic methods 

• Involves direct measurement.(measurement of  pharmacologic or therapeutic end point)
• Disadvantages:- 
• High variability
• Difficult to measure
• Limited choices
• Less reliable
• More subjective
• Drug response influenced by several  physiological & environmental factors
• Maximum pharmacodynamic effect (E max) 
• Time for maximum pharmacodynamic effect 
• Area under the pharmacodynamic effect–time curve 
• Onset time for pharmacodynamic effect 
• They involve determination of bioavailability from: 
• Acute pharmacological response. 
• Therapeutic response.

3. In-vitro dissolution studies 

• Closed compartment apparatus
• Open compartment apparatus
• Dialysis systems. 

4. Clinical observations 

• Well-controlled clinical trials 

METHODS:

1. PLASMA  DRUG  CONCENTRATION 

Measurement of drug concentrations in blood, plasma, or serum after drug administration is the most direct and objective way to determine systemic drug bioavailability. By appropriate blood sampling, an accurate description of the plasma drug concentration–time profile of the therapeutically active drug substance(s) can be obtained using a validated drug assay. 

T-max

• The time of peak plasma concentration, t max, corresponds to the time required to reach maximum drug concentration after drug  administration. 
• At t max, peak drug absorption occurs and the rate of drug absorption exactly equals the rate of drug elimination. Drug absorption still continues after t max is reached, but at a slower rate. 
• When comparing drug products, t max can be used as an approximate indication of drug absorption rate. The value for tmax will become smaller (indicating less time required to reach peak plasma concentration) as the absorption rate for the drug becomes more rapid. 
• Units for t max are units of time (eg, hours, minutes).  

C max

• The peak plasma drug concentration, C max, represents the maximum plasma drug concentration obtained after oral administration of drug. For many drugs, a relationship is found between the pharmacodynamic drug effect and the plasma drug concentration. 
• C max provides indications that the drug is sufficiently systemically absorbed to provide a therapeutic response. In addition, C max provides warning of possibly toxic levels of drug. 
• The units of C max are concentration units (eg, mg/mL, ng/mL). 
• Although not a unit for rate, C max is often used in bioequivalence studies as a surrogate measure for the rate of drug bioavailability. 

AUC

• The area under the plasma level–time curve, AUC, is a measurement of the extent of drug bioavailability. 
• The AUC reflects the total amount of active drug that reaches the systemic circulation. 
• The AUC is the area under the drug plasma level–time curve from t = 0 to t = ∞, and is equal to the amount of unchanged drug reaching the general circulation divided by the clearance.  

• where F = fraction of dose absorbed, Do = dose, k = elimination rate constant and Vd = volume of distribution. 
• The AUC is independent of the route of administration and processes of drug elimination as long as the elimination processes do not change. 
• The AUC can be determined by a numerical integration procedure, such as the trapezoidal rule method. 
• The units for AUC are concentration time (eg, µg hr/mL).
• For many drugs, the AUC is directly proportional to dose. 
• For example, if a single dose of a drug is increased from 250 to 1000 mg, the AUC will also show a fourfold increase ( and ).
• In some cases, the AUC is not directly proportional to the administered dose for all dosage levels. For example, as the dosage of drug is increased, one of the pathways for drug elimination may become saturated. 
• Drug elimination includes the processes of metabolism and excretion. Drug metabolism is an enzyme-dependent process.
• For drugs such as salicylate and phenytoin, continued increase of the dose causes saturation of one of the enzyme pathways for drug metabolism and consequent prolongation of the elimination half-life. 
• The AUC thus increases disproportionally to the increase in dose, because a smaller amount of drug is being eliminated (ie, more drug is retained). When the AUC is not directly proportional to the dose, bioavailability of the drug is difficult to evaluate because drug kinetics may be dose dependent.

2. URINARY  DRUG  EXCRETION  DATA 

Urinary drug excretion data is an indirect method for estimating bioavailability. The drug must be excreted in significant quantities as unchanged drug in the urine. In addition, timely urine samples must be collected and the total amount of urinary drug excretion must be obtained.

D∞u:

• The cumulative amount of drug excreted in the urine, D∞u, is related directly to the total amount of drug absorbed. Experimentally, urine samples are collected periodically after administration of a drug product. Each urine specimen is analyzed for free drug using a specific assay.
• The relationship between the cumulative amount of drug excreted in the urine and the plasma level–time curve shows when the drug is almost completely eliminated , the plasma concentration approaches zero and the maximum amount of drug excreted in the urine, D ∞ u, is obtained.

dDu/dt.

• The rate of drug excretion. Because most drugs are eliminated by a firstorder rate process, the rate of drug excretion is dependent on the first-order elimination rate constant k and the concentration of drug in the plasma C p. In , the maximum rate of drug excretion, (dD u/dt)max, is at point B, whereas the minimum rate of drug excretion is at points A and C. Thus, a graph comparing the rate of drug excretion with respect to time should be similar in shape as the plasma level–time curve for that drug.

t∞:

• The total time for the drug to be excreted. In and , the slope of the curve segment A–B is related to the rate of drug absorption, whereas point C is related to the total time required after drug administration for the drug to be absorbed and completely excreted t = ∞. The t ∞ is a useful parameter in bioequivalence studies that compare several drug products.

3. OTHER  BIOLOGICAL  FLUIDS

• Bioavailability can also be determined using other biological fluids:
• Eg: theophylline → salivary fluid
• Cephalosporin → CSF and bile fluids, etc.

4. ACUTE  PHARMACOLOGICAL  RESPONSE

• In some cases, the quantitative measurement of a drug in plasma or urine lacks an assay with sufficient accuracy and/or reproducibility. For locally acting, nonsystemically absorbed drug products, such as topical corticosteroids, plasma drug concentrations may not reflect the bioavailability of the drug at the site of action.
• An acute pharmacodynamic effect, such as an effect on forced expiratory volume, FEV1 (inhaled bronchodilators) or skin blanching (topical corticosteroids) can be used as an index of drug bioavailability. In this case, the acute pharmacodynamic effect is measured over a period of time after administration of the drug product.  
• Measurements of the pharmacodynamic effect should be made with sufficient frequency to permit a reasonable estimate for a time period at least three times the half-life of the drug (). This approach may be particularly applicable to dosage forms that are not intended to deliver the active moiety to the bloodstream for systemic distribution.
• The use of an acute pharmacodynamic effect to determine bioavailability generally requires demonstration of a dose–response curve . 
• Bioavailability is determined by characterization of the dose–response curve. For bioequivalence determination, pharmacodynamic parameters including the total area under the acute pharmacodynamic effect–time curve, peak pharmacodynamic effect, and time for peak pharmacodynamic effect are obtained from the pharmacodynamic effect–time curve. The onset time and duration of the pharmacokinetic effect may also be included in the analysis of the data.
• The use of pharmacodynamic endpoints for the determination of bioavailability and bioequivalence is much more variable than the measurement of plasma or urine drug concentrations.
• Effects such as change in ECG or EEG readings, pupil diameter, etc are related to the time course of a given drugs.
• Bioavailability can be determined by construction of pharmacologic effect time curve as well as dose response graph. 
• The drawback of this method is that, the response tends to more variable. Moreover, the observed response may be due to an active metabolite whose concentration is not proportional to concentration of parent drug responsible for the pharmacological effect. 

5. THERAPEUTIC  RESPONSE

• Theoretically, this method is most definite among all.  
• It‘s based on observing clinical response to a drug formulation given to a patient suffering from disease for which the drug is intended to be used. 
• A major drawback is that quantification of observed response is unreliable for assessment of bioavailability. 

6. IN VITRO  DISSOLUTION  STUDY 

• Drug dissolution studies may under certain conditions give an indication of drug bioavailability. Ideally, the in-vitro drug dissolution rate should correlate with invivo drug bioavailability (see and on in-vivo–in-vitro correlation, IVIVC). Dissolution studies are often performed on several test formulations of the same drug. The test formulation that demonstrates the most rapid rate of drug dissolution in vitro will generally have the most rapid rate of drug bioavailability in vivo. 
• The best available tool today which can at least quantitatively assure about the biological availability of a drug from its formulation. 
• The aim of these tests are mimicking the environment offered by the biological system as they must predict in vivo behavior to such an extent that in vivo bioavailability test need not be performed. 
• Closed compartment apparatus : Non sink condition 
• Open compartment apparatus : perfect sink condition 
• Dialysis system 
• This method is useful for very poorly aqueous soluble drugs for which maintenance of sink condition would require large volume of dissolution fluid. 

7. CLINICAL OBSERVATIONS

• Well-controlled clinical trials in humans establish the safety and effectiveness of drug products and may be used to determine bioavailability. 
• However, the clinical trials approach is the least accurate, least sensitive, and least reproducible of the general approaches for determining in-vivo bioavailability. 
• The FDA considers this approach only when analytical methods and pharmacodynamic methods are not available to permit use of one of the approaches described above. 
• Comparative clinical studies have been used to establish bioequivalence for topical antifungal drug products (eg, ketoconazole) and for topical acne preparations. 
• For dosage forms intended to deliver the active moiety to the bloodstream for systemic distribution, this approach may be considered acceptable only when analytical methods cannot be developed to permit use of one of the other approaches. 
• Analytical methodology 
• The selected analytical method should be  
• Sufficiently sensitive to permit detection of low concentration of drug. 
• Reproducible 
• Must be specific for unmetabolized drug as well as capable of determining concentration of drug in presence of metabolites, constituents of blood/ urine. 
• Stable Isotope Studies 
• This approach involves the simultaneous administration of test product and the reference product, using each subject as his own control. 
• The reference contains the drug, which has been synthesized to contain a stable isotope such as such as 2H, 15N, 13C or 18O in a position in a drug molecule that is not susceptible to metabolism and does not result in kinetic differences due to presence of isotope. 
• The sample is collected and the comparisons are made of quantity of labeled and unlabeled drug in each sample, using sophisticated detection systems involving mass spectroscopy. 
• Application of Stable Isotope Method in Study Bioavailability and Bioequivalence of Highly Variable Drugs and Formulations  
• The stable isotope method has been used successfully in bioavailability studies for highly variable drugs that have extensive first-pass metabolism and exhibit large intra-subject variation in clearance.  
• Recently this technique has been extended to bioequivalence studies where labeled solution is intravenously infused in both occasions while test and reference formulation are administered.  
• This report discusses the advantages and disadvantages of the stable isotope method and its application in bioavailability and bioequivalence studies of highly variable drugs and drug delivery systems.
• Bioavailability studies for a Controlled release drug product
• They are tested in a four way cross over with following treatment: 
• Administration under fasting condition 
• Administered 1 hr before a high fat content meal 
• Administered immediately after a high fat content meal. 
• Administered 2 hrs after  a high fat content meal 
• A method for the calculation of bioavailability in slow release formulations in the presence of within-individual variability  
• In the present study they propose a model-independent method based on the combination of the area under the curve of serum drug levels and the mean residence time for evaluating the amount of bioavailability when within-individual variability is present in the serum clearance of the drug, administered as a slow release formulation (SRF), and this follows linear pharmacokinetic behaviour. -The method assumes that the modifications in the area under the curve of the serum levels induced by the within-individual variability in the kinetic behaviour of the drug lead to a variation of the same proportions in the mean residence time of the serum levels curve and that this parameter can be used as a correction factor in the ratio of the areas under the curve of serum levels in bioavailability studies. -The method allows one to calculate the fraction of dose absorbed from the SRF without having to measure the disposition clearance of the drug either when using the reference formulation or when the drug is administered as a SRF. The method is easy to apply and has a minimum mathematical complexity. The validity of the method was evaluated using simulated data with either no error or containing a random error of 10%.