Monday, February 16, 2009
The Kidney as Excretory Organ
Most drugs are eliminated in urine ei-ther chemically unchanged or as metab-olites. The kidney permits eliminationbecause the vascular wall structure inthe region of the glomerular capillaries allows unimpeded passage of bloodsolutes having molecular weights (MW)5000. Filtration diminishes progres-sively as MW increases from 5000 to70000 and ceases at MW 70000. Withfew exceptions, therapeutically useddrugs and their metabolites have muchsmaller molecular weights and can,therefore, undergo glomerular filtra-tion, i.e., pass from blood into primaryurine. Separating the capillary endothe-lium from the tubular epithelium, thebasal membrane consists of chargedglycoproteins and acts as a filtrationbarrier for high-molecular-weight sub-stances. The relative density of this bar-rier depends on the electrical charge ofmolecules that attempt to permeate it.Apart from glomerular filtration, drugs present in blood may passinto urine by active secretion. Certaincations and anions are secreted by theepithelium of the proximal tubules intothe tubular fluid via special, energy-consuming transport systems. Thesetransport systems have a limited capac-ity. When several substrates are presentsimultaneously, competition for thecarrier may occur.During passage down the renal tu-bule, urinary volume shrinks more than100-fold; accordingly, there is a corre-sponding concentration of filtered drugor drug metabolites. The resultingconcentration gradient between urineand interstitial fluid is preserved in thecase of drugs incapable of permeatingthe tubular epithelium. However, withlipophilic drugs the concentration gra-dient will favor reabsorption of the fil-tered molecules. In this case, reabsorp-tion is not based on an active processbut results instead from passive diffu-sion. Accordingly, for protonated sub-stances, the extent of reabsorption isdependent upon urinary pH or the degree of dissociation. The degree of disso-ciation varies as a function of the uri-nary pH and the pKa, which representsthe pH value at which half of the sub-stance exists in protonated (or unproto-nated) form. This relationship is graphi-cally illustrated with the example ofa protonated amine having a pKa of 7.0.In this case, at urinary pH 7.0, 50 % of theamine will be present in the protonated,hydrophilic,membrane-impermeantform (blue dots), whereas the other half,representing the uncharged amine(orange dots), can leave the tubular lu-men in accordance with the resultingconcentration gradient. If the pKa of anamine is higher (pKa = 7.5) or lower (pKa= 6.5), a correspondingly smaller orlarger proportion of the amine will bepresent in the uncharged, reabsorbableform. Lowering or raising urinary pH byhalf a pH unit would result in analogouschanges for an amine having a pKa of7.0.The same considerations hold foracidic molecules, with the importantdifference that alkalinization of theurine (increased pH) will promote thedeprotonization of -COOH groups andthus impede reabsorption. Intentionalalteration in urinary pH can be used inintoxications with proton-acceptor sub-stances in order to hasten elimination ofthe toxin (alkalinizationphenobarbi-tal; acidification amphetamine).
Enterohepatic Cycle
After an orally ingested drug has beenabsorbed from the gut, it is transportedvia the portal blood to the liver, where itcan be conjugated to glucuronic or sul-furic acid (shown in B for salicylic acidand deacetylated bisacodyl, respective-ly) or to other organic acids. At the pH ofbody fluids, these acids are predomi-nantly ionized; the negative charge con-fers high polarity upon the conjugateddrug molecule and, hence, low mem-brane penetrability. The conjugatedproducts may pass from hepatocyte intobiliary fluid and from there back intothe intestine. O-glucuronides can becleaved by bacterial inthe colon, enabling the liberated drugmolecule to be reabsorbed. The entero-hepatic cycle acts to trap drugs in thebody. However, conjugated productsenter not only the bile but also theblood. Glucuronides with a molecularweight (MW) > 300 preferentially passinto the blood, while those with MW >300 enter the bile to a larger extent.Glucuronides circulating in the bloodundergo glomerular filtration in the kid-ney and are excreted in urine becausetheir decreased lipophilicity preventstubular reabsorption.Drugs that are subject to enterohe-patic cycling are, therefore, excretedslowly. Pertinent examples include digi-toxin and acidic nonsteroidal anti-in-flammatory agents.Conjugations (B)The most important of phase II conjuga-tion reactions is glucuronidation. Thisreaction does not proceed spontaneous-ly, but requires the activated form ofglucuronic acid, namely glucuronic aciduridine diphosphate. Microsomal glucu-ronyl transferases link the activatedglucuronic acid with an acceptor mole-cule. When the latter is a phenol or alco-hol, an ether glucuronide will beformed. In the case of carboxyl-bearingmolecules, an ester glucuronide is theresult. All of these are O-glucuronides.
Amines may form N-glucuronides that,unlike O-glucuronides, are resistant tobacterial Soluble cytoplasmic sulfotrans-ferases conjugate activated sulfate (3’-phosphoadenine-5’-phosphosulfate)with alcohols and phenols. The conju-gates are acids, as in the case of glucuro-nides. In this respect, they differ fromconjugates formed by acetyltransfe-rases from activated acetate (acetyl-coenzyme A) and an alcohol or a phenol.Acyltransferases are involved in theconjugation of the amino acids glycineor glutamine with carboxylic acids. Inthese cases, an amide bond is formedbetween the carboxyl groups of the ac-ceptor and the amino group of the do-nor molecule (e.g., formation of salicyl-uric acid from salicylic acid and glycine).The acidic group of glycine or glutamineremains free.
Amines may form N-glucuronides that,unlike O-glucuronides, are resistant tobacterial Soluble cytoplasmic sulfotrans-ferases conjugate activated sulfate (3’-phosphoadenine-5’-phosphosulfate)with alcohols and phenols. The conju-gates are acids, as in the case of glucuro-nides. In this respect, they differ fromconjugates formed by acetyltransfe-rases from activated acetate (acetyl-coenzyme A) and an alcohol or a phenol.Acyltransferases are involved in theconjugation of the amino acids glycineor glutamine with carboxylic acids. Inthese cases, an amide bond is formedbetween the carboxyl groups of the ac-ceptor and the amino group of the do-nor molecule (e.g., formation of salicyl-uric acid from salicylic acid and glycine).The acidic group of glycine or glutamineremains free.
Biotransformation of Drugs
Many drugs undergo chemical modifi-cation in the body (biotransformation).Most frequently, this process entails aloss of biological activity and an in-crease in hydrophilicity (water solubil-ity), thereby promoting elimination viathe renal route. Since rapid drugelimination improves accuracy in titrat-ing the therapeutic concentration, drugsare often designed with built-in weaklinks. Ester bonds are such links, beingsubject to hydrolysis by the ubiquitousesterases. Hydrolytic cleavages, alongwith oxidations, reductions, alkylations,and dealkylations, constitute Phase I re-actions of drug metabolism. These reac-tions subsume all metabolic processesapt to alter drug molecules chemicallyand take place chiefly in the liver. InPhase II (synthetic) reactions, conju-gation products of either the drug itselfor its Phase I metabolites are formed, forinstance, with glucuronic or sulfuric ac-id.The special case of the endogenoustransmitter acetylcholine illustrateswell the high velocity of ester hydroly-sis. Acetylcholine is broken down at itssites of release and action by acetylchol-inesterase so rapidly as tonegate its therapeutic use. Hydrolysis ofother esters catalyzed by various este-rases is slower, though relatively fast incomparison with other biotransforma-tions. The local anesthetic, procaine, is acase in point; it exerts its action at thesite of application while being largelydevoid of undesirable effects at other lo-cations because it is inactivated by hy-drolysis during absorption from its siteof application.Ester hydrolysis does not invariablylead to inactive metabolites, as exempli-fied by acetylsalicylic acid. The cleavageproduct, salicylic acid, retains phar-macological activity. In certain cases,drugs are administered in the form ofesters in order to facilitate absorption(enalaprilenalaprilate; testosteroneundecanoatetestosterone) or to re-duce irritation of the gastrointestinal mucosa (erythromycin succinateerythromycin). In these cases, the esteritself is not active, but the cleavageproduct is. Thus, an inactive precursoror prodrug is applied, formation of theactive molecule occurring only after hy-drolysis in the blood.Some drugs possessing amidebonds, such as prilocaine, and of course,peptides, can be hydrolyzed by pepti-dases and inactivated in this manner.Peptidases are also of pharmacologicalinterest because they are responsiblefor the formation of highly reactivecleavage products andpotent mediators frombiologically inactive peptides.Peptidases exhibit some substrateselectivity and can be selectively inhib-ited, as exemplified by the formation ofangiotensin II, whose actions inter aliainclude vasoconstriction. Angiotensin IIis formed from angiotensin I by cleavageof the C-terminal dipeptide histidylleu-cine. Hydrolysis is catalyzed by “angio-tensin-converting enzyme”. Pep-tide analogues such as captopril block this enzyme. Angiotensin II is de-graded by angiotensinase A, which clipsoff the N-terminal asparagine residue.The product, angiotensin III, lacks vaso-constrictor activity.
The Liver as an Excretory Organ
As the chief organ of drug biotransfor-mation, the liver is richly supplied withblood, of which 1100 mL is receivedeach minute from the intestinesthrough the portal vein and 350 mLthrough the hepatic artery, comprisingnearly 1/3 of cardiac output. The bloodcontent of hepatic vessels and sinusoidsamounts to 500 mL. Due to the widen-ing of the portal lumen, intrahepaticblood flow decelerates. Moreover,the endothelial lining of hepatic sinu-soids contains pores largeenough to permit rapid exit of plasmaproteins. Thus, blood and hepatic paren-chyma are able to maintain intimatecontact and intensive exchange of sub-stances, which is further facilitated bymicrovilli covering the hepatocyte sur-faces abutting Disse’s spaces.The hepatocyte secretes biliaryfluid into the bile canaliculi (darkgreen), tubular intercellular clefts thatare sealed off from the blood spaces bytight junctions. Secretory activity in thehepatocytes results in movement offluid towards the canalicular space.The hepatocyte has an abundance of en-zymes carrying out metabolic functions.These are localized in part in mitochon-dria, in part on the membranes of therough (rER) or smooth (sER) endoplas-mic reticulum.Enzymes of the sER play a most im-portant role in drug biotransformation.At this site, molecular oxygen is used inoxidative reactions. Because these en-zymes can catalyze either hydroxylationor oxidative cleavage of -N-C- or -O-C-bonds, they are referred to as “mixed-function” oxidases or hydroxylases. Theessential component of this enzymesystem is cytochrome P450, which in itsoxidized state binds drug substrates (R-H). The FeIII-P450-RH binary complex isfirst reduced by NADPH, then forms theternarycomplex,O2-FeII-P450-RH,which accepts a second electron and fi-nally disintegrates into FeIII-P450, oneequivalent of H2O, and hydroxylateddrug (R-OH).
Compared with hydrophilic drugsnot undergoing transport, lipophilicdrugs are more rapidly taken up fromthe blood into hepatocytes and morereadily gain access to mixed-functionoxidases embedded in sER membranes.For instance, a drug having lipophilicityby virtue of an aromatic substituent(phenyl ring) can be hydroxylatedand, thus, become more hydrophilic. Besides oxi-dases, sER also contains reductases andglucuronyl transferases. The latter con-jugate glucuronic acid with hydroxyl,carboxyl, amine, and amide groups; hence, also phenolic products ofphase I metabolism (Phase II conjuga-tion). Phase I and Phase II metabolitescan be transported back into the blood— probably via a gradient-dependentcarrier — or actively secreted into bile.Prolonged exposure to certain sub-strates, such as phenobarbital, carbama-zepine, rifampicin results in a prolifera-tion of sER membranes.This enzyme induction, a load-depen-dent hypertrophy, affects equally all en-zymes localized on sER membranes. En-zyme induction leads to acceleratedbiotransformation, not only of the in-ducing agent but also of other drugs (aform of drug interaction). With contin-ued exposure, induction develops in afew days, resulting in an increase in re-action velocity, maximally 2–3 fold, thatdisappears after removal of the induc-ing agent.
Compared with hydrophilic drugsnot undergoing transport, lipophilicdrugs are more rapidly taken up fromthe blood into hepatocytes and morereadily gain access to mixed-functionoxidases embedded in sER membranes.For instance, a drug having lipophilicityby virtue of an aromatic substituent(phenyl ring) can be hydroxylatedand, thus, become more hydrophilic. Besides oxi-dases, sER also contains reductases andglucuronyl transferases. The latter con-jugate glucuronic acid with hydroxyl,carboxyl, amine, and amide groups; hence, also phenolic products ofphase I metabolism (Phase II conjuga-tion). Phase I and Phase II metabolitescan be transported back into the blood— probably via a gradient-dependentcarrier — or actively secreted into bile.Prolonged exposure to certain sub-strates, such as phenobarbital, carbama-zepine, rifampicin results in a prolifera-tion of sER membranes.This enzyme induction, a load-depen-dent hypertrophy, affects equally all en-zymes localized on sER membranes. En-zyme induction leads to acceleratedbiotransformation, not only of the in-ducing agent but also of other drugs (aform of drug interaction). With contin-ued exposure, induction develops in afew days, resulting in an increase in re-action velocity, maximally 2–3 fold, thatdisappears after removal of the induc-ing agent.
Possible Modes of Drug Distribution
Following its uptake into the body, thedrug is distributed in the blood andthrough it to the various tissues of thebody. Distribution may be restricted tothe extracellular space (plasma volumeplus interstitial space) or may alsoextend into the intracellular space.Certain drugs may bind strongly to tis-sue structures, so that plasma concen-trations fall significantly even beforeelimination has begun.After being distributed in blood,macromolecular substances remainlargely confined to the vascular space,because their permeation through theblood-tissue barrier, or endothelium, isimpeded, even where capillaries arefenestrated. This property is exploitedtherapeutically when loss of blood ne-cessitates refilling of the vascular bed,e.g., by infusion of dextran solutions. The vascular space is, moreover,predominantly occupied by substancesbound with high affinity to plasma pro-teins; determination of the plas-ma volume with protein-bound dyes).Unbound, free drug may leave thebloodstream, albeit with varying ease,because the blood-tissue barrier is differently developed in different seg-ments of the vascular tree. These re-gional differences are not illustrated inthe accompanying figures.Distribution in the body is deter-mined by the ability to penetrate mem-branous barriers. Hydrophilicsubstances (e.g., inulin) are neither tak-en up into cells nor bound to cell surfacestructures and can, thus, be used to de-termine the extracellular fluid volume. Some lipophilic substances diffusethrough the cell membrane and, as a re-sult, achieve a uniform distribution.Body weight may be broken downas follows:
Further subdivisions are shown inthe table.The volume ratio interstitial: intra-cellular water varies with age and bodyweight. On a percentage basis, intersti-tial fluid volume is large in premature ornormal neonates (up to 50 % of bodywater), and smaller in the obese and theaged.The concentration of a solutioncorresponds to the amount of sub-stance dissolved in a volume; thus, c= D/V. If the dose of drug and itsplasma concentrationare known, avolume of distributioncan be calcu-lated from V = D/c. However, this repre-sents an apparent volume of distribu-tion (Vapp), because an even distributionin the body is assumed in its calculation.Homogeneous distribution will not oc-cur if drugs are bound to cell mem-branes or to membranes of intracel-lular organelles or are stored withinthe latter. In these cases, Vapp can ex-ceed the actual size of the available fluidvolume. The significance of Vapp as apharmacokinetic parameter is dis-cussed.
Having entered the blood, drugs maybind to the protein molecules that arepresent in abundance, resulting in theformation of drug-protein complexes.Protein binding involves primarily al-bumin and, to a lesser extent, lins and acidic glycoproteins. Otherplasma proteins (e.g., transcortin, trans-ferrin, thyroxin-binding globulin) servespecialized functions in connectionwith specific substances. The degree ofbinding is governed by the concentra-tion of the reactants and the affinity of adrug for a given protein. Albumin con-centration in plasma amounts to4.6 g/100 mL or O.6 mM, and thus pro-vides a very high binding capacity (twosites per molecule). As a rule, drugs ex-hibit much lower affinity (KD approx.10–5 –10–3 M) for plasma proteins thanfor their specific binding sites (recep-tors). In the range of therapeutically rel-evant concentrations, protein binding ofmost drugs increases linearly with con-centration (exceptions: salicylate andcertain sulfonamides).The albumin molecule has differentbinding sites for anionic and cationic li-gands, but van der Waals’ forces alsocontribute. The extent of bindingcorrelates with drug hydrophobicity(repulsion of drug by water).Binding to plasma proteins is in-stantaneous and reversible, i.e., anychange in the concentration of unbounddrug is immediately followed by a cor-responding change in the concentrationof bound drug. Protein binding is ofgreat importance, because it is the con-centration of free drug that determinesthe intensity of the effect. At an identi-cal total plasma concentration (say, 100ng/mL) the effective concentration willbe 90 ng/mL for a drug 10 % bound toprotein, but 1 ng/mL for a drug 99 %bound to protein. The reduction in con-centration of free drug resulting fromprotein binding affects not only the in-tensity of the effect but also biotransfor-mation (e.g., in the liver) and elimina-tion in the kidney, because only free drug will enter hepatic sites of metab-olism or undergo glomerular filtration.When concentrations of free drug fall,drug is resupplied from binding sites onplasma proteins. Binding to plasma pro-tein is equivalent to a depot in prolong-ing the duration of the effect by retard-ing elimination, whereas the intensityof the effect is reduced. If two substanc-es have affinity for the same binding siteon the albumin molecule, they maycompete for that site. One drug may dis-place another from its binding site andthereby elevate the free (effective) con-centration of the displaced drug (a formof drug interaction). Elevation of thefree concentration of the displaced drugmeans increased effectiveness and ac-celerated elimination.A decrease in the concentration ofalbumin (liver disease, nephrotic syn-drome, poor general condition) leads toaltered pharmacokinetics of drugs thatare highly bound to albumin.Plasma protein-bound drugs thatare substrates for transport carriers canbe cleared from blood at great velocity,e.g., p-aminohippurate by the renal tu-bule and sulfobromophthalein by theliver. Clearance rates of these substanc-es can be used to determine renal or he-patic blood flow.
Further subdivisions are shown inthe table.The volume ratio interstitial: intra-cellular water varies with age and bodyweight. On a percentage basis, intersti-tial fluid volume is large in premature ornormal neonates (up to 50 % of bodywater), and smaller in the obese and theaged.The concentration of a solutioncorresponds to the amount of sub-stance dissolved in a volume; thus, c= D/V. If the dose of drug and itsplasma concentrationare known, avolume of distributioncan be calcu-lated from V = D/c. However, this repre-sents an apparent volume of distribu-tion (Vapp), because an even distributionin the body is assumed in its calculation.Homogeneous distribution will not oc-cur if drugs are bound to cell mem-branes or to membranes of intracel-lular organelles or are stored withinthe latter. In these cases, Vapp can ex-ceed the actual size of the available fluidvolume. The significance of Vapp as apharmacokinetic parameter is dis-cussed.
Having entered the blood, drugs maybind to the protein molecules that arepresent in abundance, resulting in theformation of drug-protein complexes.Protein binding involves primarily al-bumin and, to a lesser extent, lins and acidic glycoproteins. Otherplasma proteins (e.g., transcortin, trans-ferrin, thyroxin-binding globulin) servespecialized functions in connectionwith specific substances. The degree ofbinding is governed by the concentra-tion of the reactants and the affinity of adrug for a given protein. Albumin con-centration in plasma amounts to4.6 g/100 mL or O.6 mM, and thus pro-vides a very high binding capacity (twosites per molecule). As a rule, drugs ex-hibit much lower affinity (KD approx.10–5 –10–3 M) for plasma proteins thanfor their specific binding sites (recep-tors). In the range of therapeutically rel-evant concentrations, protein binding ofmost drugs increases linearly with con-centration (exceptions: salicylate andcertain sulfonamides).The albumin molecule has differentbinding sites for anionic and cationic li-gands, but van der Waals’ forces alsocontribute. The extent of bindingcorrelates with drug hydrophobicity(repulsion of drug by water).Binding to plasma proteins is in-stantaneous and reversible, i.e., anychange in the concentration of unbounddrug is immediately followed by a cor-responding change in the concentrationof bound drug. Protein binding is ofgreat importance, because it is the con-centration of free drug that determinesthe intensity of the effect. At an identi-cal total plasma concentration (say, 100ng/mL) the effective concentration willbe 90 ng/mL for a drug 10 % bound toprotein, but 1 ng/mL for a drug 99 %bound to protein. The reduction in con-centration of free drug resulting fromprotein binding affects not only the in-tensity of the effect but also biotransfor-mation (e.g., in the liver) and elimina-tion in the kidney, because only free drug will enter hepatic sites of metab-olism or undergo glomerular filtration.When concentrations of free drug fall,drug is resupplied from binding sites onplasma proteins. Binding to plasma pro-tein is equivalent to a depot in prolong-ing the duration of the effect by retard-ing elimination, whereas the intensityof the effect is reduced. If two substanc-es have affinity for the same binding siteon the albumin molecule, they maycompete for that site. One drug may dis-place another from its binding site andthereby elevate the free (effective) con-centration of the displaced drug (a formof drug interaction). Elevation of thefree concentration of the displaced drugmeans increased effectiveness and ac-celerated elimination.A decrease in the concentration ofalbumin (liver disease, nephrotic syn-drome, poor general condition) leads toaltered pharmacokinetics of drugs thatare highly bound to albumin.Plasma protein-bound drugs thatare substrates for transport carriers canbe cleared from blood at great velocity,e.g., p-aminohippurate by the renal tu-bule and sulfobromophthalein by theliver. Clearance rates of these substanc-es can be used to determine renal or he-patic blood flow.
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