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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., Biochem Pharm, 48(12): 2147 (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 (CLint). Based on these values, in vivo pharmacokinetic parameters such as bioavailability and in vivo half-life can be calculated. The drug metabolic enzymes possess broad substrate specificity and can metabolize multiple compounds. Therefore, 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.
Microsomes are defined operationally as the particulate fraction obtained from a tissue homogenate by ultra centrifugation after the nuclear and mitochondrial fractions have been removed by low speed centrifugation. Electron microscopy has shown that microsomes are composed primarily of closed sacs of membrane called vesicles. Most of the vesicles are derived from rough and smooth endoplasmic reticulum (ER). Membrane vesicles derived from the Golgi apparatus, peroxisomes, endosomes, the trans Golgi network, and other intermediate compartments comprise a minor component of microsomes. Liver microsomes contain rough and smooth ER vesicles in a roughly 2:1 ratio, and, in addition to components of the protein secretory pathway, contain a multitude of proteins involved in lipid/lipoprotein biosynthesis, and drug metabolism. The ER is by far the most abundant membrane in metabolically active cells. Some 2-3 mg of microsomal protein is obtained from liver per gram of wet tissue. As such, microsomes are an ideal preparation in which to study the relationships between enzyme structure, protein-protein and lipid-protein interactions, and the functional properties of membrane bound enzymes. Although many of the most abundant microsomal proteins have been studied extensively, many more remain to be isolated and characterized.
Preparation of microsomes
After a tissue has been selected for study, the composition of the homogenizing buffer, the method of homogenization, and the time and force of the low speed centrifugation step are the primary variables in the preparation of microsomes. The homogenizing buffer is usually isotonic and contains buffer (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 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 choice of technique for homogenization is determined by the nature and volume of tissue to be processed. Delicate tissues such as brain and liver are readily homogenized with Potter-Elvehjem tissue grinders. A large volume of tissue is most conveniently 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 (combined liver weight approximately 160 g) in 800 ml 100 mM Tris acetate, pH 7.5, 100 mM KCl, 1 mM EDTA, 0.1 mM DTT in a Warring blender. The livers are perfused with buffer prior to homogenization to limit contamination with hemoglobin 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 steps are then applied in sequence 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 step 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. After centrifugation at 200,000 x g for 90 min the smoothe microsomes can be found at the 30% density range and the rough microsomes can be found 45% density 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, Frontiers in Science 3:483, 1998.)
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 :
ClH = QH.ERH
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.
In vitro measurement of intrinsic clearance (Clin)
The in vitro measurement of intrinsic clearance (CLin) using hepatic microsomes and/or hepatocytes is frequently used in both academia and the pharmaceutical industry to estimate the in vivo metabolic stability of new drug entities in both rat and human (Houston, 1994; Obach, 1999; McGinnity and Riley, 2001)Traditionally, the 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). In general, short incubation times and low enzyme (protein) concentrations are used in these studies, since 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 to this approach is that it requires 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 particularly 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 adopted, 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 (LaveÂÂ´ et al.,1997; Obach, 2001; Obach and Reed-Hagen, 2002; Austin et al.,2002). 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). What remains unresolved is the impact of this lack of formality on the predictive utility of this method. 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, in general, 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 AUCâË†Å¾ is the area under the concentration versus time curve, extrapolated to infinity. The unit for CLin is l/h/mg protein. (Claudio Giulino et.al, 2005).