An Microsomal Stability Assay Biology Essay

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Drugs are most often eliminated by biotransformation and/or excretion into urine or bile. The liver is the major organ for xenobiotic biotransformation and is thereby important in characterizing the metabolism stability, toxicology, and drug-drug interaction properties of drugs. Drug metabolism is achieved via two major enzyme reactions within the liver, Phase I and Phase II reactions. Phase I enzymes include the cytochrome-P450 (CYP) family of enzymes which are located in the smooth endoplasmic reticulum. The basic processes in phase I reactions are oxidation, reduction and/or hydrolysis many of which are catalyzed by the CYP system and require NADPH as a cofactor. Phase II enzymes are located in the cytoplasm and endoplasmic reticulum and are characteristic of conjugation reactions including glucuronic acid, glutathione, sulfate, and glutamine conjugations. Phase II reactions generally inactivate the drug if it is not already therapeutically inactive following Phase I metabolism, and make the drug more water soluble to facilitate its elimination. Some drugs are metabolized by Phase I or Phase II enzymes alone whereas others are metabolized by both Phase I and Phase II enzymes. (Rodrigues, A.D 1994)

Microsomal stability assay

Metabolic stability is defined as the susceptibility of a chemical compound to biotransformation, and is expressed as in vitro half-life (t1/2) and intrinsic clearance (CLin). By using these values, in vivo pharmacokinetic parameters like bioavailability and in vivo half-life can be calculated. The drug metabolic enzymes possess broad substrate specificity and can metabolize multiple compounds. So the risk for metabolism-based drug-drug interactions is always a potential problem during the drug development process. For this reason, inhibition and induction in vitro screens are used early, before selection of a candidate drug, to estimate the risk for clinically significant drug-drug interactions. (Baranczewski paweÅ‚ 2006)

Liver Microsomes

Microsomes are defined operationally as the particulate fraction obtained from a tissue homogenate by performing ultra centrifugation after the nuclear and mitochondrial fractions have been removed at low rpm. Electron microscopy has shown that microsomes are composed primarily of closed sacs of membrane called vesicles. The vesicles are copied from rough and smooth endoplasmic reticulum (ER). Membrane vesicles derived from the Golgi apparatus, peroxisomes, endosomes and other intermediate compartments consist of a minor component of microsomes. Liver microsomes contain rough and smooth ER vesicles (2:1) ratio, and, components held over in protein secretory pathway are multitude of proteins which involves in lipid/lipoprotein biosynthesis, and drug metabolism. The Endoplasmic reticulum is the richest membrane in the active cells. About 2-3 mg of microsomal protein is obtained from liver per gram of wet tissue. As such, microsomal preparation is used to study the relationships between lipid -protein interaction, enzyme structure, protein and protein binding and the functional properties of membrane attached enzymes. Many of the most microsomal proteins have been studied extensively; many are hanging about to be isolated and considered.

Preparation of microsomes

After the selection of tissue for study, the composition of the homogeneous buffer, the homogenization method, and the low centrifugation speed and time are the important variables in the preparation of microsomes. The homogeneous buffer is isotonic and contains (usually 10-100 mM Tris, HEPES, or Triethanolamine, pH 7.5-8.1), a chelating agent, and a reducing agent such as 1 mM dithiothreitol (DTT). Depending on the tissue been selected and the protein or activity of interest, it may be beneficial to add magnesium (1-5 mM), and/or protease inhibitor(s) to the homogenizing buffer. The nature and volume of tissue to be processed determines the choice of technique for homogenization. Delicate tissues such as brain and liver are readily homogenized with Potter-Elvehjem tissue grinders. A large volume of tissue is homogenized in a Warring blender. Microsomes are pelleted by centrifugation from the post-mitochondrial supernatant at approximately 200,000 g (45,000 RPM) for 30-60 minutes in the Ultracentrifuge. Rabbit liver microsomes can be prepared by homogenizing minced tissue from two animals (combiningly the liver weight shoukd be approximately 160 g) in 800 ml 100 mM Tris acetate, pH 7.5, 100 mM KCl, 1 mM EDTA, and 0.1 mM DTT in a Warring blender. The livers are perfused with buffer prior to homogenization and to limit contamination with haemoglobin and serum proteins. It is important not to over-homogenize the suspension so as to avoid the formation of nuclear and mitochondrial fragments. Differential centrifugation processes are then applied in series to remove unbroken cells, nuclei, and mitochondria. Sequential centrifugation at 600 x g and 10,000 x g gives pellets designated the "nuclear" and "mitochondrial" fractions. Centrifugation of the post mitochondrial supernatant at 105,000 g for ninety minutes yields the microsomes in a pelleted form. The isolated microsomal fraction consists of smooth and rough microsomes, the latter having ribosomes attached on their outer surface. A density gradient sedimentation process can be used at this point to separate the rough and smooth microsomes. The w/w density concentrations can be made as follows 20%, 30%. 40% and 50% in 5 ml steps for each concentration and 5 ml of the microsome pellet suspension can be layered on each four-step gradient. It was found that after centrifugation at 200,000 x g for about 90 min the density of smooth microsomes and rough microsomes were 30% and 45% range. Slow acceleration and slow deceleration programs for the ultra centrifuge should be used for the fixed angle rotor to prevent sample/gradient mixing before or after the centrifugation run. A hypodermic syringe and long needle should be used to remove the visible microsome zones in the gradient (F.S Heinemann and Juris Ozols, 1998).

CYP: cytochrome P450, NQ01: NADPH:quinine oxidoreductase (DT diaphorase); DPD: dihydropyrimidine dehydrogenase; ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase

HMT: histamine methyltransferase; TPMT: thiopurine methyltransferase; COMT: catechol O-methyltransferase; UGT: Uridine Glucuronosyl-S-Transferases; ST: Sulfotransferase; GST: Glutathione-S-Transferases.

Hepatic Clearance :

For certain drugs, the normal clearances can be assumed as equal to hepatic clearance ClH . It is given as :

ClH = ClT - ClR

An equation parallel to the above equation can also be written for hepatic clearance :


Where QH = Hepatic blood flow (about 1.5 litres/min) and ERH = hepatic extraction ratio. The hepatic clearance of drugs can be divided into two groups: drugs with hepatic blood flow rate-limited clearance, and with intrinsic capacity-limited clearance.

1.Hepatic Blood Flow :

When ERH is one ClH approaches its maximum value i.e. hepatic blood flow. In such a situation, hepatic clearance is said to be perfusion rate-limited or flow -dependent. Alteration in hepatic blood flew significantly affects the elimination of drugs with high ERH e.g. propranolol, lidocaine, etc. Such drugs are removed from the blood as rapidly as they are presented to the liver (high first-pass hepatic metabolism). Indocyanine green is so rapidly eliminated by the human liver that its clearance is often used as an, indicator of hepatic blood flow rate. First-pass hepatic extraction is suspected when there is lack of unchanged drug in systemic circulation alter oral administration. An extension of the same equation is the non-compartmental method of estimating F:

F = I - ERH = AUC

On the contrary, hepatic blood flow has very little or no effect on drugs with low ERH eg. theophylline. For such drugs, whatever concentration of drug present in the blood perfuses liver, is more than what the liver can eliminate (low first-pass hepatic metabolism). Similar discussion can be extended to the influence of blood flow on renal clearance of drugs. This is illustrated in Table. Hepatic clearance of a drag with high ER is independent of protein binding.

2. Intrinsic Capacity Clearance:

Denoted as ClintH is defined as the inherent ability of an organ to irreversibly remove a drug in the absence of any flow limitation, it depends, in this case, upon the hepatic enzyme activity. Drugs with low ERH and with elimination primarily by metabolism are greatly affected by changed in enzyme activity. Hepatic clearance of such drugs is said to be capacity limited. Eg, Theophylline the such drugs show great inter subject variability. Hepatic clearance of drugs with low ER is independent of blood flow rate but sensitive to changes in protein binding. (Brahmankar and jaiswal 1995)

In vitro measurement of intrinsic clearance (Clin)

The in vitro measurement of intrinsic clearance (CLin) using hepatic microsomes and/or hepatocytes is frequently used to estimate the in vivo metabolic stability of new drug entities in both rat and human (Houston, 1994; Obach, 1999; McGinnity and Riley, 2001),for long time metabolite formation method has been used for measurement of in vitro CLin, where the initial rate of metabolite production using hepatic microsomes or hepatocytes is measured over a range of substrate concentrations under linear conditions with respect to protein concentration/cell density and time (Madan et al 2002; Houston et al 2003). Short incubation times and low enzyme (protein) concentrations are used in these studies, in ordert to be in compliance with the Michaelis-Menten equation assumes less than 10% substrate consumption. Therefore, issues such as the stability of the enzyme preparation (resulting from long incubation times), nonspecific binding (resulting from high enzyme concentrations), and end product inhibition (resulting from the accumulation of the phase-I hydroxylated metabolite in the microsomal incubation) are not generally limitations. However, the main disadvantage requirement of prior knowledge of the particular metabolic pathway under study and its importance to the overall metabolic fate of the drug to ensure a true and accurate prediction of clearance. This may be problematic if multiple metabolic pathways are involved. For many drugs, especially new drug candidates, this information is not known; therefore, the use of this approach is limited. More recently, the substrate depletion approach has been used, where the consumption of parent drug is monitored over time. This method is particularly popular in the pharmaceutical industry because formal kinetic characterization and metabolite quantification are not required, allowing the rapid screening of compounds (Lave´ et al. 1997; Obach, 2001). However, compared with the metabolite formation approach, this method has been poorly defined. Although CLint is expressed per unit time and per unit enzyme (protein), initial linearity studies are usually not performed and arbitrary values for enzyme concentration and incubation time are used. Linearity is a necessary requirement when scaling CLin from in vitro incubations to the whole liver (Houston, 1994). In contrast to the metabolite formation method, for analytical/sensitivity reasons, normally at least 20% of the substrate must be metabolized within the incubation period, so that any substrate depletion can be distinguished from baseline variability. Consequently, longer incubation times and higher protein concentrations are used than for metabolite formation studies (e.g., substrate depletion incubation conditions of up to 90 min and 10 mg/ml have been reported by Austin et al., 2002, and Obach, 1999, respectively). For these reasons, the stability of the enzyme preparation, nonspecific binding, and end-product inhibition (resulting from the more extensive metabolism) must be considered. . CLin was calculated according to

Where dose is the initial amount of drug in the incubation mixture (unit of mol/mg microsomal protein) and AUC∞ is the area under the concentration versus time curve, extrapolated to infinity. The unit for CLin is l/h/mg protein. (Claudio Giulino, 2005).


Formation of a protein drug complex is termed as drug-protein binding. Drug protein binding may be a reversible or an irreversible process. Irreversible drug protein binding is usually as a result of chemical activation of drug, when then attaches strongly to the protein or macromolecule by covalent chemical bonding. Reversible drug protein binding implies that the drug binds the protein with weaker chemical bonds such as hydrogen bonds or vanderwaals forces. The amino acids that compose the protein chain have hydroxyl, carboxyl or other sites available for reversible drug interaction.

Drugs may bind to various macromolecular components in the blood including albumin, α1 acid glycoprotein, lipoproteins, immunoglobulins (IgG) and erythrocytes (RBC).Albumin is a protein synthesised by the liver with a molecular weight of 65,000 to 69,000d.Albumin is the major component of plasma proteins responsible for reversible drug binding. In the body, albumin is distributed in the plasma and in the extra cellular fluids of skin, muscle and various other tissues.

Albumin is responsible for maintaining osmotic pressure of the blood and for the transport of endogenous and exogenous substances. Lipoproteins are macromolecular complexes of lipids and proteins. Lipoproteins are responsible for the transport of plasma lipids and may be responsible for binding of the drugs if the albumin sites become saturated.

Reversible drug-protein binding is of major interest in pharmacokinetics. The protein bound drug is a large complex that cannot easily cross cell membranes and therefore has a restricted distribution. The protein bound drug is usually pharmacologically inactive. In contrast, the free or unbound drug crosses cell membranes and is therapeutically active.

Drug protein binding is influenced by a number of factors like:-

The drug


Affinity between drug and protein

Drug interactions

The pathophysiologic condition of patient. ( Brahmankar and jaiswal 1995)


The kinetics of reversible drug-protein binding site can be described by law of mass action as follows.

Protein + drug → Drug-Protein complex

[P] + [D] → [PD] --------------1

From this equation and law of mass action, an association constant, Ka can be expressed as the ratio of molar concentration of the products and molar concentrations of the reactants. It assumes only one binding site per protein molecule.

Ka = --------------2

To study the binding behaviour of drugs, a determinable ratio, r is defined as follows.

r =

r = -----------------3

According to eqn 2 [PD] =Ka [P] [D] by substitution into eqn 3.

r =

r = --------------------4

The eqn describes 1mole of drug binds to 1 mole of protein in a 1:1 complex. Therefore this assumes only one independent binding site for each molecule of drug.

If there are "n" independent identical binding sites the equation 4 becomes

r = ---------------------5

In terms of Kd =1/Ka eqn 5 reduces to

r =

Since protein molecules are large in size, more than one type of binding site is present and drugs bind independently on each binding site with its own association constant and hence eqn 6 becomes;-

r =

Where, 1, 2:- different binding sites

K: - binding constants

n :- number of binding sites

As per the equations we assume that each drug binds to the protein at an independent binding site and the affinity of a drug for one binding site does not influence binding to other sites.

In reality a drug protein binding sometimes exhibits a phenomenon called cooperativity. For these drugs the binding of first drug molecule at one site on the protein molecule influences successive binding of other drug molecules.

Ex:-Binding of oxygen to haemoglobin ( Brahmankar and jaiswal 1995)



r = --------------1

As per the equation quoted above, as free drug concentration increases number of moles of drug bound per mole of protein becomes saturated and plateaus. Thus drug protein binding resembles a Langmuir adsorption isotherm which is also similar to adsorption of a drug to an adsorbent absorbent becoming saturated as drug concentration increases.

Reciprocal of equation written above gives

1 = ---------------------------2

1 = ---------------------------3

A graph of 1/r versus 1/[D] is called as double reciprocal plot. The y intercept is 1/n and slope is 1/nKa.From this graph, the number of binding sites may be determined from y intercept and the association constant may be determined from the slope if the value for n is known.

If the graph of 1/r versus 1/[D] doesn't yield a straight line then the drug protein binding process is probably more complex.eqn 1 assumes one type of binding site and no interaction among the binding sites.


Reciprocal plots cannot be used if the exact amount and nature of protein in the experimental system is unknown the percent of drug bound is often to used to describe the extent of drug protein binding in the plasma the fraction of drug bound, β, can be determined experimentally and is equal to the ratio of the concentration of bound drug, D β,and the total drug concentration of, Dr, in the plasma as follows.

β =

The value of the association constants can be determined even though the nature of the plasma proteins binding the drug is unknown by rearranging the equation above as

r = =


D β:-bound drug concentration

D : - free drug concentration

PT :- total protein concentration

Rearrangement of the above equation yields the expression

D β = nKaPT-KaD β

Concentrations of both free and bound drug may be found experimentally and a graph obtained by plotting D β/D versus D β will yield a straight line from which the slope is the association constant Ka.

The above equation shows that the ratio of bound Cp to free Cp is influenced by the affinity constant, the protein concentration Pt which may change during disease states and with the drug concentration in the body.

The values for n and Ka give a general estimate of the affinity and binding capacity of the drug as plasma contains a complex mixture of proteins. The drug protein binding in plasma may be influenced by competing substances such as ions, free fatty acids, drug metabolites and other drugs. Measurements of drug protein binding should be obtained over a wide range of concentration range, because at low drug concentrations a high affinity low capacity binding site might be missed or at a higher drug concentration saturation of the protein binding sites may occur

Clinical significance of drug-protein binding.

Most drugs bind to plasma proteins to some extent. When the clinical significance of the fraction of drug bound is considered it is important to know whether the study was performed using pharmacological or therapeutic plasma drug concentrations. The fraction of drug bound can change with plasma drug concentration and dose of drug administered.

When a highly protein bound drug is displaced from binding by a second drug or agent, a sharp increase in the free drug concentration in the plasma may occur leading to toxicity. Displacement occurs when a second drug is taken that competes for the same binding site in the protein as initial drug. This will lead to increased apparent volume of distribution and an increased half life, but clearance will remain unaffected. If administered by multiple doses, the mean steady state remain unaffected; however the mean steady state free drug level will be increased due to displacement. The therapeutic effect will therefore increase. (Leon shargel, Susanna wu-pong, 2005).


Drug-drug interaction refers to an alteration of the effect of one drug caused by the presence of a second drug. Drug-nutrient interactions similarly refer to the alteration of the effect of a drug or nutrient caused by the presence of a second agent. Drug interactions can be beneficial or detrimental. One example would be administering a drug product like carbidopa/levodopa (Sinemet®). Levodopa is converted to dopamine in the central nervous system (CNS), thereby exerting an effect against symptoms of Parkinson's disease. Carbidopa acts as a chemical decoy, which binds to the enzyme that converts levodopa to dopamine outside the CNS. This increases dopamine levels in the CNS while limiting side effects of increased dopamine in peripheral tissues.

In combination, the paired drugs produce additive effects. Patients with numerous disease states may require treatment with interacting drugs. Where these interactions cannot be avoided, the fact is taken into account when planning therapy. Many times dosing is not altered at all, but usual monitoring is increased.

The risk of having drug interactions will be increased as the number of medications taken by an individual increases. This also implies a greater risk for the elderly and the chronically ill as they will be using more medications than the general population. Risks also increase when a patient's regimen originates from multiple prescribers. Filling all prescriptions in a single pharmacy may decrease the risk of undetected interactions. (Beverly J. McCabe, Eric H. Frankel,2004)

Types of Drug Interactions

Drug interactions are often classified as


pharmacokinetic interactions.


This can be defined depending only on the pharmacology of the given drug.


Interactions Resulting from Alterations in Gastrointestinal Absorption.

The rate and extent of drug absorption after oral administration may be grossly altered by other agents. Absorption of a drug is a function of the drug's ability to diffuse from the lumen of the gastrointestinal tract into the systemic circulation .Changes in intestinal pH may profoundly affect drug diffusion as well as dissolution of the dosage form. For example, the absorption of ketoconazole is reduced by the co-administration of antacids or H2-blockers.

Interactions Resulting from Alterations in Metabolizing Enzymes

The liver is the major, though not exclusive, site for drug metabolism. Other sites include the kidney and the lining of the gastrointestinal tract. The two main types of hepatic drug metabolism are phase I and phase II reactions. Phase I oxidative reactions are the initial step in drug biotransformation, and are mediated by the cytochrome P-450 (CYP) system. This complex super family of enzymes has been subclassified into numerous enzymatic subfamilies. The most common CYP subfamilies include CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. These enzymes may be induced or inhibited by other agents, thereby leading to an increase or decrease in the metabolism of the primary drug. Phase II reactions occur following Phase I reactions. In this process, drug metabolites are converted into more water-soluble compounds that can be more easily eliminated by the kidneys.

Enzyme induction may result in increased CYP enzyme synthesis, faster drug metabolism, subtherapeutic drug concentrations and the risk for ineffective drug therapy. The rapidity of the enzyme induction is dependent upon the half-life of the inducing drug as well as the rate of synthesis of new enzymes. Examples of drugs that cause enzyme induction are the barbiturates, some anticonvulsants.

Enzyme inhibition may result from noncompetitive or competitive inhibition of CYP enzymes by a second drug, an effect that may occur rapidly. Examples of hepatic enzyme inhibitors include cimetidine, fluconazole and erythromycin. The result of noncompetitive enzyme inhibition by addition of a second agent is slower metabolism of the first drug, higher plasma drug concentrations, and a risk for toxicity. In the case of competitive inhibition, the metabolism of both drugs can be reduced, resulting in higher than expected concentrations of each drug.

Interactions Resulting from Alterations in Protein Binding

Drugs may exist in plasma either reversibly bound to plasma proteins or in the free (unbound) state. The primary drug-binding plasma proteins are albumin and α1-acid glycoprotein. It is free drug that exerts the pharmacological effect. Drugs may compete with each other for plasma protein binding sites, and when this occurs, one drug may displace another that was previously bound to the protein. Displacement of a drug from its binding sites will therefore increase that agent's unbound concentrations, perhaps resulting in toxicity.

Interactions Resulting from Changes in Renal Excretion

The majority of renally eliminated drugs are excreted via passive glomerular filtration. Some drugs are eliminated via active tubular secretion, such as penicillins, cephalosporins, and most diuretics. The active secretion may be inhibited by secondary agents, such as cimetidine, nonsteroidal anti-inflammatory agents and probenecid, with resulting elevations in the serum drug concentrations and reduced urinary drug concentrations. In some cases, the interaction is desirable, while others may lead to adverse therapeutic outcomes.( Beverly J. McCabe ,Eric H. Frankel,2004).