Basic Pharmacologic Principles

Introduction

Pharmacology is a science concerned with the interaction of substances (e.g., drugs) with cells, tissues, and organisms. The in vivo efficacy of a drug is guided by two principles of pharmacology, namely pharmacokinetics and pharmacodynamics. Pharmacokinetics deals with the processes of drug concentration in the tissue compartments and therefore involves absorption, distribution, biotransformation, and excretion. Pharmacodynamics applies to the study of mechanisms of action of drugs. Application of these concepts to the newborn requires the practitioner to take into account developmental changes in pharmacokinetics and pharmacodynamics. In this chapter, we present basic pharmacologic principles and introduce how drug disposition and actions can be altered by developmental changes and disorders of the immature subject.

Principles of Drug Absorption, Bioavailability, and Distribution

Drug Absorption

The majority of drugs administered to the premature newborn are injected intravenously and therefore are not affected by factors that govern systemic absorption. Some agents are administered intramuscularly (e.g., vitamin K, vaccines ), given by an enteral route (e.g., thiazides, caffeine, ranitidine, acetaminophen, lansoprazole, sildenafil ), or applied topically or by inhalation (e.g., topical antiseptics, anesthetics, nitric oxide, bronchodilators). Regardless of the route of administration, drugs must often cross cell membranes to reach their sites of action. Therefore, the mechanisms that govern the passage of drugs across cell membranes and the physicochemical properties of molecules and membranes are important to consider in drug transfer. Among the most important physicochemical properties of drug molecules are lipid solubility, degree of ionization (p K _a ), molecular weight, and protein binding.

Although drugs with molecular weight less than 200 Da cross the cellular lipid membranes by diffusion, many require transporters. The greater the lipid solubility and the lower the degree of ionization, the more easily a drug will transfer across the cell membrane. Furthermore, the binding affinity of drugs to proteins affects their distribution, and therefore their activity. It is the unbound fraction of drugs that exhibits pharmacologic effects, because they have greater access to their site of action.

Transport Mechanisms

In addition to physicochemical properties of the drug molecules, there are a number of physiologic transport processes that influence the mechanism by which a drug traverses the cell membrane. Such processes include passive diffusion, active transport, facilitated diffusion, and pinocytosis.

1.
Passive diffusion is the principal transmembrane process for a number of small drugs. According to Fick’s laws of diffusion, drug molecules diffuse from a region of high drug concentration to a region of low drug concentration according to the equation

d Q / d t = P (C_{1} - C_{2})

$d Q / d t = P (C_{1} - C_{2})$

where d Q /d t = rate of diffusion, D = diffusion coefficient, K = partition coefficient, A = surface area of membrane, h = membrane thickness, and C ₁ − C ₂ = concentration gradient across the membrane. Because D , A , K , and h are constants under usual conditions, a combined constant P or permeability coefficient may be defined: P = DAK / h. Therefore, the previous equation can be simplified to:

d Q / d t = P (C_{1} - C_{2})

$d Q / d t = P (C_{1} - C_{2})$

This equation does not take into account the ionization state of the drug molecule, the effect of regional blood flow, or the influence of tissue affinity on drug partitioning. The ionization state is affected by the pH on both sides of the membrane, according to the Henderson-Hasselbalch equation:

pH = p K_{a} + \log (base / acid)

$pH = p K_{a} + \log (base / acid)$

Therefore, acidic compounds, such as salicylic acid and furosemide, diffuse across cell membranes more readily when the environmental pH is low, such as in the stomach, because they are less ionized at low pH. Accordingly, the opposite applies to basic compounds, such as propranolol, that tend to cross membranes more readily in basic environments, such as in the small intestine. Regardless of the acidic or basic nature of drugs, most absorption occurs in the small intestine because of its larger surface area. Regional blood flow also influences the rate of diffusion by altering the delivery and consequently, the local concentration of the drug. Finally, some drugs demonstrate increased affinity for a particular tissue component, which influences the concentration of drug on either side of the membrane. For example, tetracyclines form a complex with calcium in the bones and teeth.
2.
Active transport is a carrier-mediated transmembrane process that plays an important role in the renal, intestinal, and biliary secretion and absorption of many drugs and metabolites. Active transport is characterized by a transfer of molecules against their concentration gradient. Therefore, energy must be consumed to achieve this process. One example of this transport system is adenosine triphosphate (ATP)-dependent secretion of organic acids in the renal tubule, permitting secretion of indomethacin. Of note, this process, in contrast to diffusion, is saturable and therefore follows Michaelis-Menten kinetics. The Michaelis-Menten equation describes the rates of enzymatic reactions by relating the rate of substrate conversion or uptake by protein (e.g., enzyme, receptor, transporter) to the concentration of the substrate.
3.
Facilitated diffusion is also a carrier-mediated transport system, in which, in contrast with active transport, molecules move along their concentration gradient. This system does not require energy input but is saturable and selective. Major classes of transporters involved in facilitated diffusion include the solute carrier (SLC) superfamily and the ATP-binding cassette (ABC) superfamily ^, ^, ; examples of drugs transported by these transporters include corticosterone, ethacrynic acid, and captopril.
4.
Pinocytosis is the process of engulfing large molecules such as immunoglobins.

Membrane Transporters

Transporters are integral membrane proteins that primarily facilitate movement of nutrients and waste products such as amino acids, di- and tripeptides, sugars, nucleosides, vitamins, and bile salts into and out of the cells. Based on their physiologic role, membrane transporters can be classified as uptake transporters (transport solutes into the cell) and efflux transporters (carrying substances out of the cell). In the intestines, it has become increasingly clear that several of these transporters also play a significant role in systemic absorption of xenobiotics, including various pharmacologic agents.

To date, more than 400 genes, encoding membrane transporters, have been identified in human genome and are broadly divided into two superfamilies, SLC and ABC. Members of the SLC superfamily (such as organic anion transporters [OATs] and cation transporters) utilize either facilitated transport (along the electrochemical gradient) or cotransport (against the electrochemical gradient using electrochemical gradient of another solute) mechanisms for import or export of the substance of interest. Sodium and protons are frequently cotransported with the compound of interest in the latter case. Because the activity of such a transporter is a function of the electrochemical gradient driving force, the efficiency of the cotransport is influenced by the activity of the Na ⁺ -H ⁺ antiporter and that of the Na ⁺ -HCO ₃ ⁻ symporter. On the other hand, members of the ABC superfamily (such as multidrug resistance-associated protein [MRP] transporters) utilize energy derived from ATP hydrolysis to transport solutes against their electrochemical gradient. The tissue distribution of membrane transporters is diverse and includes liver, kidney, intestine, and blood-tissue barriers (e.g., blood-brain barrier). For description of types and tissue distributions of various membrane transporters, see the review by König et al.

A number of membrane transporters play key roles in drug import, as is the case for SLC members. These include the H ⁺ -dipeptide symporters, facilitative glucose transporter-related proteins, Na ⁺ -glucose cotransporters (SGLTs), Na ⁺ -nucleoside cotransporters (CNTs), amino acid transporters, Na ⁺ -neurotransmitter symporters, organic anion transporting polypeptides (OATPs), and the OATs and organic cation transporters (OCTs). ^, ^, ^, The H ⁺ -dipeptide symporters carry cephalosporins and angiotensin-converting enzyme (ACE) inhibitors (such as captopril and valgancyclovir ); OCTs carry antihistamines (such as cimetidine, ranitidine, famotidine, clonidine, dopamine), hormones (such as corticosterone), and β-blockers (such as propranolol) ; OATPs carry a wide variety of drugs such as antibiotics (ciprofloxacin, penicillin, cephalosporins, rifampicin), β-blockers (sotalol), vasopressin, montelukast, and digoxin. ^,

Net absorption is also affected by the activity of exporters (efflux transporters), commonly present on the brush border membrane of luminal epithelial cells (e.g., proximal tubular epithelium of kidney). The MRP and the P-glycoprotein (P-gp) families are prototypes of these transporters. These proteins are ATP-dependent transporters that significantly contribute to the excretion of potentially toxic waste products such as bile salts as well as drugs such as steroids, cyclosporins, and digoxin out of the cells.

These ports of entry across cellular membranes, along with their structural polymorphisms, may pose an additional challenge to pharmacokinetics and drug delivery. For instance, drug-drug interaction affect their activity, such as rifampin induction of P-gp activity, which increase digoxin elimination. Moreover, neonates with an OATP1B1 mutation are at higher risk of developing severe hyperbilirubinemia. Also, efflux pumps may also play an important role in etiopathology of some diseases. For example, mutations of hepatocellular bile salt export pump (BSEP), a type of ABC efflux transporter, have been associated with progressive familial intrahepatic cholestasis (PFIC) type-II.

The distribution of drugs often requires transport across multiple compartments, which affects their concentration at the site of action. Along these lines, P-glycoproteins are major efflux transporters in the blood-brain barrier and, consequently, influence the concentration of various drugs in the central nervous system. Nevertheless, transcellular transport may not be the only function served by P-glycoproteins. Interestingly, they have been localized on intracellular vesicles and more importantly at the inner membrane of the nuclear envelope, suggesting that P-glycoprotein expression at the nuclear envelope may play a role in subcellular distribution of various endogenous substrates and drugs. This intracellular localization is particularly relevant in the context of expression of cognate transmembrane receptors at subcellular organelles, notably on the nuclear envelope.

Kinetics of Absorption

Most pharmacokinetic models assume that drug absorption and elimination follow first-order kinetics (i.e., the rate of change in the concentration of a drug depends on the amount of drug present at that particular time). This process is described in the following equation:

d D_{B} / d t = F K_{a} D_{si} - K_{el} D_{B}

$d D_{B} / d t = F K_{a} D_{si} - K_{el} D_{B}$

where d D _B /d t = rate of change of drug in the body ( D _B ), F = fraction absorbed (bioavailability term), K _a and K _el = absorption and elimination constants, and D _si = drug concentration at the site of absorption. This equation applies to a single-compartment pharmacokinetic model. In a single-compartment model, the drug rapidly equilibrates with tissues of the body. Therefore, changes in the concentration of the drug in serum or plasma mirror those in the tissues. Although many drugs follow multiple-compartment kinetics, the K _a may still be calculated from a one-compartment model. The importance of the K _a lies in the design of a multiple dosage regimen. Knowledge of the K _a and K _el allows for the prediction of the peak and trough steady-state plasma drug concentrations ( C _{ss max} and C _{ss min} ) following multiple dosing concentrations (see “Clinical Pharmacokinetics” and Fig. 15.1 ) :

\begin{matrix} C s s \max = (F D_{si} / V d) / (l - e^{- Kelτ}) \\ C s s \min = C s s \max • e^{- Kelτ} \end{matrix}

$\begin{matrix} C s s \max = (F D_{si} / V d) / (l - e^{- Kelτ}) \\ C s s \min = C s s \max • e^{- Kelτ} \end{matrix}$

Fig. 15.1, Two-compartment model for serum drug disappearance curve. The α-phase represents the distribution phase, and the β-phase the elimination phase.

where τ = dosing interval. From these equations, it is evident that peak and trough concentrations depend on the absorption rate, the volume of distribution, the elimination rate constant, and the dosing interval.

Factors That Affect Absorption of Drugs

The systemic absorption of a drug from its site of administration depends on the variables discussed previously, which constitute the physicochemical properties of the drug and those of the membrane. Other factors can also influence the efficiency of drug absorption, including the disintegration, dissolution, and solubility of the compound; the blood flow to the site of absorption; the surface area available for absorption; transit time of the drug through the gastrointestinal tract; export of drugs via P-glycoproteins in enterocytes; and in situ metabolism of the agent, including the first-pass effect. A first-pass effect is defined as the rapid uptake and metabolism of an agent into inactive compounds by the liver, immediately after enteric absorption and before it reaches the systemic circulation. Drugs that exhibit a first-pass effect include morphine, isoproterenol, propranolol, and hydralazine. Each of the factors that affect absorption, taken separately or in conjunction, may have profound effects on the efficacy and toxicity of a drug.

Compounds can be administered to newborns via various routes: intravenous, intramuscular, sublingual, oral, enteral, rectal, buccal (submucosal), dermal, inhalational, intravitreal, and conjunctival. Depending on the route, the absorption may be impacted by various factors that are maturational or not. Method of administration can also impact absorption (and bioavailability of the compound of interest), such as the type of intravenous or gavage tubing used, with lipophilic drugs adhering more to the material. While evaluating the absorption of drugs through enteral route in the newborn, the developmental stages of this organ must be taken into account. Gastric acid secretion is lower in premature infants. The increased pH (>4.0) results in a reduced absorption of weak acids (such as phenobarbital) and bases, but may lead to increased absorption of acid-labile drugs (such as penicillin G). By contrast, lipid-soluble drugs (such as methylxanthines) are more easily absorbed in newborns than in older children. Furthermore, the rate of gastric emptying (slower in newborns) and the intestinal motility may be affected by gestational and postnatal age, as well as impacted by the nutritional content (increased with human milk, decreased by rising caloric density and medium-chain triglycerides). ^, Bile salt secretion is also diminished in the newborn infant, which can affect micelle formation and enterohepatic recirculation, and secondarily reduce the absorption of fats and lipid-soluble vitamins, such as vitamins D and E ^, ; on the other hand, vitamin E is adequately absorbed in premature infants, probably due to a lower intake of iron. Gestational and postnatal age, as well as pathologic processes of the newborn, may also affect intestinal surface area and permeability, impacting absorption of enterally administered compounds. In addition, permeability decreases with administration of milk (faster with breast milk compared to formula). ^,

Rectal administration, depending upon depth, may result in first-pass enteral metabolism (bypassing metabolism). Specifically, distal rectal exposure leads to absorption via the medium and inferior rectal veins, while deeper rectal absorption proceeds through the superior rectal veins to the liver. ^, ^, The submucosal buccal route is commonly used with midazolam or fentanyl administration, which may lead to increased and faster absorption compared to the enteral route.

Enzymatic development of the gastrointestinal tract may also alter drug absorption. The elevated activity of β-glucuronidase in the brush border of newborn intestine may cleave drug-glucuronide conjugates, resulting in enhanced reabsorption of free unconjugated drug into the systemic circulation; this may prolong the pharmacologic activity of certain agents, such as indomethacin. In contrast, the presence of P-glycoproteins, highly distributed in the apical brush border of the gastrointestinal tract epithelium as well as in the bile canalicular face of hepatocytes, can reduce drug absorption and bioavailability ; lower expression of P-glycoproteins in the immature subject may contribute to variable bioavailability. Although modulators of P-glycoproteins, primarily cyclosporin A and verapamil, have been used with marginal effectiveness to enhance drug action, such as in cancer treatment, ^, these drugs exert their own toxicity; selective inhibitors of P-glycoproteins and related MRPs have yet to be developed. Another factor that influences drug absorption and its access to the target organ is the presence of metabolizing enzymes in the intestinal epithelium; this is especially the case for cytochrome P450 enzymes. Depending on their activity, limited or excess bioavailability may be observed.

In comparison with adults, newborn infants also exhibit qualitative and quantitative differences in the bacterial colonization of the gastrointestinal tracts. The development of the intestinal flora has been clearly shown to affect the absorption of vitamin K. The microbiome can also impact the production of bile acids. Therefore, maturation of the gastrointestinal tract may also explain some of the characteristics of intestinal absorption of drugs in the growing child.

Finally, in addition to the physiologic changes in the gastrointestinal tract that occur during development, drug absorption can also be altered by disease processes. Diseases of genetic (e.g., cystic fibrosis), microbial, or circulatory (e.g., necrotizing enterocolitis) origin may alter the intestinal mucosa and result in a reduced absorptive surface. There are a number of developmental differences in the newborn that affect non-enteral absorption. For instance, topical skin absorption depends on the dermal thickness, permeability, skin maturation, skin perfusion, and the body surface area to weight ratio (increased in newborns). Stratum corneum maturation accelerates in the immediate weeks of postnatal life, adjusting to extrauterine life and independent of gestational age. ^, Inhalation is of particular interest in the newborn as numerous drugs are administered via this route as is the case for nitric oxide, inhaled anesthetics, steroids, and endotracheal epinephrine. Neonates have a decreased functional residual capacity but an increased alveolar surface area relative to weight, factors that can affect absorption. Also, they frequently have a bidirectional or right-to-left shunts via an inter-atrial communication or patent-ductus-arteriosus, especially in the first few days of life (or in the context of pulmonary hypertension and/or right ventricular dysfunction). Shunt direction may also impact compound absorption. In the context of pulmonary pathologies, ventilation-perfusion mismatch (such as in meconium aspiration syndrome or bronchopulmonary dysplasia) may also affect impact bioavailability of compounds administered by inhalation. Conjunctival absorption may also lead to systemic distribution of drugs; the same applies to intravitreal administration. ^, Intramuscular absorption is affected by circulation through local capillaries. Following intramuscular administration, there is an early systemic absorption, followed by a delayed release (deep compartment behavior), making it ideal for certain drugs, such as vitamin K for prevention of neonatal bleeding.

Bioavailability

Drug bioavailability is the fraction of the administered dose that reaches the systemic circulation. For the clinician, the most relevant consideration is the percentage of active drug that reaches the central compartment. Bioavailability does not take into account the rate at which the drug is absorbed but is affected by factors that influence absorption. The absolute availability of a drug may be determined by comparing the respective area under the plasma concentration curves (AUC) after oral and intravenous administration.

Absolute availability = [{AUC}_{PO} / {dose}_{PO}] / [{AUC}_{IV} / {dose}_{IV}]

$Absolute availability = [{AUC}_{PO} / {dose}_{PO}] / [{AUC}_{IV} / {dose}_{IV}]$

This measurement may be performed as long as the volume of distribution ( V _d ) and the elimination rate constant ( K _el ) are independent of the route of administration.

Distribution

The disposition of a drug refers to its passage in the body from absorption to excretion. Following absorption, a drug is distributed to various body compartments. This distribution determines its efficacy as well as its toxicity. The distribution of drugs is influenced by several factors, including the size of the body-water and lipid compartments, regional hemodynamic features, the degree of binding of drugs to plasma and tissue proteins, and the tissue expression of transporter proteins (importers and exporters). The initial phase of distribution reflects regional blood flow. Organs that are well perfused, such as the brain, the heart, and the kidneys, are first to get exposed to the drug. The second phase of distribution involves a large fraction of the body mass, including muscles and adipose tissue. Therefore, the various distribution compartments form the apparent volume of distribution ( V _d ), which is expressed by the following equation:

V_{d} = Total drug in the body / Concentration of drug in plasma

$V_{d} = Total drug in the body / Concentration of drug in plasma$

Assuming instant equilibration of the drug after administration, V _d can be determined by extrapolating the drug concentration to time zero ( C ₀ ) and dividing the dose of drug administered by the concentration of drug at time zero ( C ₀ ). This equation, however, can only be applied to a single-compartment model. V _d may also be calculated using the following equation, which is independent of the model used:

V_{d} = Dose / Kel {[AUC]}_{0}^{\infty}

$V_{d} = Dose / Kel {[AUC]}_{0}^{\infty}$

Physiologic and Pathologic Factors Affecting Distribution of Drugs

The factors that influence the distribution of drugs in the body are subject to developmental changes (weight, body composition, plasma protein concentration, permeability of compartment targeted). The amount and distribution of total body water undergo marked changes in the perinatal period. Total body water and extracellular fluid volume decrease with increasing gestational age. Consequently, the volume of distribution of many drugs has been observed to increase in preterm neonates. This results in increased volume of distribution; accordingly, lipophilic drugs (such as propofol) exhibit decreased volumes of distribution and accumulate with risks of toxicity. ^, After birth, free fat mass and total body water decrease and the volume of intracellular fluid increases relative to that of the extracellular fluid. In the term newborn, as well as in the older child, the degree of insensible water loss is linked to the metabolic rate of the infant. In contrast, in the preterm newborn, there is no fixed relationship between metabolic rate and insensible water loss, and in the very low-birth-weight infant, evaporative heat loss is substantially greater than heat produced by the basal metabolic rate. ^,

Many disorders of the newborn as well as drugs administered to critically ill newborn infants (such as diuretics ^, ) can affect total body water and, secondarily, the distribution of drugs. For instance, renal and hepatic dysfunction may have important consequences on both elimination and distribution of xenobiotics. Similarly, diseases that lead to increased total body water (e.g., congestive heart failure, syndrome of inappropriate secretion of antidiuretic hormone) can have profound effects on drug pharmacokinetics and pharmacodynamics. Therefore, any change in either total body water content or the relationship between extracellular and intracellular fluid volume may have significant effects on the distribution of drugs within the body.

The extent and the disposition of the lipid mass in the body also contribute to the distribution of drugs. The adipose tissue mass changes markedly during development. Between 28 and 40 weeks of gestation, the amount of adipose tissue (expressed as a percentage of total body mass) increases from 1% to 15%, and by 1 year of age, it represents approximately 25% of the body mass.

The nervous system contains a high proportion of lipids. Normally, the maximal increment in weight of the human brain occurs in the few weeks preceding term gestation; however, a substantial part of myelination (and lipid deposition) occurs postnatally. The entry of drugs into the central nervous system is generally restricted. In contrast with capillaries elsewhere in the body, endothelial cells of brain capillaries exhibit a predominance of tight junctions, producing nonfenestrated capillaries, which restrict the entry of hydrophilic substances into the brain. Consequently, ionized molecules, such as quaternary amines (e.g., neostigmine), exhibit limited capacity to diffuse into the central nervous system, whereas lipid-soluble compounds, such as cefotaxime and pentobarbital, traverse the blood-brain barrier more readily.

The distribution of drugs into brain and other organs is also dependent on specific transporters of nutrients and endogenous compounds, as described earlier. Furthermore, efflux carriers present in brain endothelium and glia, primarily P-glycoproteins, limit drug concentration in the brain. This is well described for a number of drugs including human immunodeficiency virus (HIV) protease inhibitors, vinca alkaloids, and anthracyclines. Of interest, P-glycoproteins are also present in placenta and function as export transporters to limit fetal exposure to potentially toxic agents. In the newborn, the blood-brain barrier is relatively more permeable than in the older subject. Newborns also display higher cerebral-to-systemic blood flow and greater volume of cerebrospinal fluid, which together increase drug concentrations in the central nervous system. ^,

A major determinant of drug distribution is the cardiac output and blood flow to various organs. Marked changes in the neonatal circulation take place during the perinatal period. ^, In addition, regional blood flow may also change acutely as a result of congestive heart failure (secondary to patent ductus arteriosus or other congenital heart diseases), as a result of sudden changes in acid-base balance (especially respiratory acidosis), or secondary to the limited ability of the stressed preterm neonate to autoregulate regional blood flow.

Plasma Proteins

The affinity of a drug for plasma proteins is another important variable that affects drug distribution. The degree of binding is inversely related to the volume of distribution, such that increased protein binding tends to maintain the drug within the vascular space. Protein binding affects renal and plasma clearance, the half-life, and the efficacy of the agent at its site of action. Table 15.1 lists the protein binding of some commonly used agents in the neonate.

Table 15.1

Plasma Protein Binding of Commonly Used Drugs in the Newborn.

Data from References 73–80.

Drug	Percent Protein-Bound
Ampicillin	∼10
Atropine	25
Caffeine	25
Cefotaxime	25–50
Dexamethasone	65
Digoxin	20
Ethacrynic acid	95
Furosemide	95
Gentamicin	45
Hydrochlorothiazide	40
Indomethacin	95
Morphine	30
Phenobarbital	∼20
Phenytoin	∼80
Theophylline	35

Several factors modify the binding of drugs to plasma proteins, notably the amount of plasma binding proteins; the number of binding sites; the affinity of the drug for the protein; and pathophysiologic conditions that alter drug-protein binding (such as blood pH, free fatty acids, bilirubin, and disease states, e.g., renal failure, liver failure). Albumin binds principally acidic drugs, whereas basic agents are bound to lipoproteins, β-globulins, and α ₁ -acid glycoproteins. Albumin contains a few high-affinity and several low-affinity binding sites. In the preterm newborn, both albumin and α ₁ -acid glycoprotein concentrations and binding affinities are deficient, resulting in an increased fraction of free drug and increased distribution of free drug outside the vascular compartment. Numerous conditions may further reduce the binding of drugs to proteins. For example, a decrease in pH may enhance the dissociation of weak acids from their albumin-binding sites. Therefore, the frequent occurrence of acidosis in premature infants may significantly change the binding of drugs to plasma proteins, especially albumin. The elevated plasma-free fatty acid content of the newborn may also alter drug binding to plasma proteins ^, ; this effect, however, may be questionable. In a similar fashion, maternal drugs that have crossed placental barrier or other agents (including other drugs) concomitantly administered to the infant may also compete for the same plasma protein-binding sites in the newborn.

The potential interference of endogenous compounds, particularly unconjugated bilirubin, on drug-protein binding has been well addressed. A displacement of bilirubin from its albumin-binding site may result in free circulating unconjugated bilirubin, which can penetrate into the brain and ultimately cause injury. Interestingly, however, bilirubin itself is tightly bound to albumin and may displace drugs from their protein-binding sites. In addition, free bilirubin is only sparingly lipophilic. Thus, the drug-induced displacement of bilirubin from albumin-binding sites possibly plays a minor role in the development of bilirubin-induced encephalopathy. Nonetheless, a few drugs can alter the binding affinity of albumin for bilirubin ( Box 15.1 ).

Box 15.1

Drugs That Cause Significant Displacement of Bilirubin From Albumin In Vitro

Sulfonamides
Ibuprofen
Moxalactam
Fusidic acid
Radiographic contrast media for cholangiography (sodium iodipamide, sodium ipodate, iopanoic acid, meglumine ioglycamate)
Aspirin
Apazone
Tolbutamide
Albumin preservatives (sodium caprylate and N -acetyl tryptophan: rapid infusions in vivo)
High concentrations of ampicillin (rapid infusions in vivo)
Long-chain free fatty acids (FFA) at high molar ratios of FFA:albumin

The volume of distribution of certain compounds is also affected by their binding to proteins outside the vascular space; for example, digoxin exhibits a higher degree of binding to myocardial and skeletal muscle proteins in the newborn than in the adult. ^, This results in an increase in the volume of distribution of digoxin. Altogether, in the immature newborn the concentration of albumin is lower and that in the fetus, resulting in decreased binding of weak acids.

In conclusion, numerous factors can influence the distribution of drugs in the body. These factors are themselves affected by development and disease conditions of the newborn infant. Major changes in the distribution of fluids and fat and their proportion relative to body mass occur at the end of gestation and during the neonatal period. Perinatal and neonatal alterations in cardiac output and regional blood flow, secondary to physiologic and disease states, also occur. Furthermore, the degree of drug binding to plasma proteins between the newborn and adult varies for several drugs. These variables should be taken into account when deciding on the appropriate drug dosage for a newborn.

Principles of Drug Elimination

The relatively high lipophilicity of many drugs does not permit their rapid elimination. After filtration through the glomerulus or passage into the bile, these agents are readily absorbed by the renal tubule or gastrointestinal mucosa. Consequently, the elimination of most drugs from the body requires a step of biotransformation prior to their excretion. This section reviews the different biotransformation processes that take place in the human body, and the mechanisms of renal drug excretion, with particular reference to developmental aspects.

Drug Biotransformation

Drug biotransformation converts drug molecules into more polar derivatives that are less able to diffuse across cell membranes. As a consequence of biotransformation, these converted molecules do not reach their receptors and in addition are not reabsorbed by the renal tubule. Therefore, biotransformation of drugs not only facilitates their excretion from the body but also may diminish their pharmacologic activity.

The metabolism of drugs does not always produce inactive compounds. Initial biotransformation of certain agents results in the formation of active metabolites. For instance, codeine is demethylated to morphine, acetylsalicylic acid is hydrolyzed to salicylic acid, theophylline is methylated to caffeine, propranolol converted to 4-hydroxypropranolol (via hydroxylation), and diazepam is converted to oxazepam. ^, Furthermore, oxidation of certain aromatic compounds produces highly reactive (and toxic) electrophiles (compounds that serve as electron acceptors). This latter reaction may be primary (aromatic hydrocarbons) or may be an increasingly active secondary reaction resulting from an inhibited or overwhelmed primary metabolic pathway (as, for example, with an excessive dosage of the agent, such as with acetaminophen overdose). Therefore, biotransformation can produce relatively innocuous metabolites or highly toxic compounds.

The mechanisms that affect the biotransformation of drugs are usually the same as those that metabolize endogenous products (e.g., hormones). Most biotransformation takes place in the liver, but some may occur at other sites, such as the kidneys, intestinal mucosa, and lungs. Biotransformation reactions are classically divided into two phases: phase I, the nonsynthetic reactions, and phase II, the synthetic or conjugation reactions ( Table 15.2 ). Each phase has reactions that can take place in the microsomes or outside of the microsomal system. The great majority of phase I reactions (oxidation, reduction, and hydrolysis) are largely catalyzed by microsomal enzymes, while phase II reactions (other than glucuronidation) are predominantly extramicrosomal.

Table 15.2

Biotransformation Reactions.

Adapted from References 85 and 87.

Reaction	Examples of Drug Substrates
Phase I (Nonsynthetic Reactions)
(a) Oxidation:
Aromatic ring hydroxylation	Phenytoin, Phenobarbital
Aliphatic hydroxylation	Ibuprofen
N-hydroxylation	Acetaminophen
N -, O -, S -dealkylation	Morphine, codeine
Deamination	Diazepam
Sulfoxidation, N -oxidation	Cimetidine
(b) Reduction:
Azoreduction	Prontosil
Nitroreduction	Chloramphenicol
Alcohol dehydrogenase	Ethanol
(c) Hydrolysis:
Ester hydrolysis	Acetylsalicylic acid
Amide hydrolysis	Indomethacin
Phase I (Microsomal Enzymes)
CYP1A2	Caffeine, theophylline
CYP2A6	Nicotine
CYP2B6	Diazepam
CYP2C9	Warfarin, ibuprofen, indomethacin, phenytoin, sildenafil
CYP2C19	Diazepam, omeprazole, lansoprazole
CYP2D6	Codeine, imipramine, propranolol, timolol, tamoxifen
CYP2E1	Acetaminophen, caffeine, ethanol
CYP3A	Erythromycin, midazolam, 6β-hydroxycortisol, sildenafil
Phase II (Synthetic Reactions: Conjugations)
(a) Glucuronide conjugation	Morphine, acetaminophen, bilirubin
(b) Glycine conjugation	Salicylic acid
(c) Sulfate conjugation	Acetaminophen, α-methyldopa
(d) Glutathione conjugation	Ethacrynic acid
(e) Methylation	Dopamine, epinephrine
(f) Acetylation	Sulfonamides, clonazepam

Phase I Reactions (Nonsynthetic Reactions)

Microsomal

The microsomal enzymes that metabolize drugs are localized in the smooth endoplasmic reticulum. Oxidative enzymes of this system, called mixed-function oxidases or monooxygenases , consist of three principal components: an electron transporter, NADPH-cytochrome P450 reductase (a flavoprotein), and multiple cytochrome P450 isoenzymes (oxidase hemoproteins). This system requires both a reducing agent (NADPH) and molecular oxygen (O ₂ ). The end result of cytochrome P450-catalyzed reactions is the incorporation of one oxygen atom into the compound being metabolized (hence the name monooxygenase ) and formation of water after reduction of the second oxygen atom.

Reactions catalyzed by monooxygenases include aromatic ring and aliphatic side chain hydroxylation, N -, O -, S -dealkylation, deamination, dehalogenation, sulfoxidation, N -oxidation, N -hydroxylation, nitroreduction, and azoreduction. Epoxides are also formed by monooxygenases, converting aromatic moieties of agents to arene and alkene oxides, which are in turn detoxified by epoxide hydrolases present in endoplasmic reticulum. These electrophilic compounds react avidly with proteins and nucleic acids, exerting potential mutagenic and carcinogenic effects; polychlorinated and polybrominated biphenyls exert their toxicity via their metabolites.

Several drugs, including fluconazole, spironolactone, amiodarone, cimetidine, erythromycin, ciprofloxacin, and metronidazole, can inhibit cytochrome P450 enzyme activity. ^, This inhibition reduces the metabolism of potential substrates and secondarily delays their elimination, as seen in fluconazole inhibition of zidovudine elimination. In contrast, other substrates can act as inducers of the cytochrome P450 system. Prototypes of the most extensively studied inducers of cytochrome P450 isozymes are phenobarbital (CYP3A), rifampin (CYP1A, CYP2C), and the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (CYP1A). Other examples of CYP450 inducers are phenytoin and carbamazepine. ^,

Approximately 1000 cytochrome P450s are known; only 50 or so are functionally active in humans. Seventeen families and many subfamilies have been sequenced. CYP1, CYP2, and CYP3 families are involved in the majority of drug metabolism reactions; members of the other families are important in the synthesis and degradation of steroids, fatty acids, vitamins, and other endogenous compounds. Individual CYP isoforms tend to have substrate specificities, but overlap is common. CYP3A4 and CYP3A5 are similar isoforms, which together are involved in metabolism of approximately 50% of drugs. CYP2C and CYP2D6 are also involved in the metabolism of many drugs. CYP1A1/2, CYP2A6, CYP2B1, and CYP2E1 are not extensively involved in drug metabolism but rather in activation of procarcinogenic agents including aromatic amines and aromatic hydrocarbons.

The cytochrome P450–dependent monooxygenase activity develops in fetal life and significantly increases during the perinatal period, often triggered by parturition. Nonetheless, its activity in the fetal and newborn liver remains considerably lower than in the adult liver. ^, The diminished enzyme activity may be clinically important because drugs that are oxygenated slowly by these enzymes (e.g., phenobarbital and phenytoin) can exhibit a prolonged half-life in the young infant ; this is especially the case for the CYP1A2, CYP2A6, CYP2B6, CYP2C, CYP2D6, CYP2E1, and CYP3A4 substrates ( Table 15.3 ). ^, However, some CYP enzymes, including CYP1A1 and CYP3A7, are expressed in higher levels in the fetal and newborn liver. ^, ^, ^, CYP3A7 expression peaks 1 week after birth and declines thereafter, while CYP3A4, a structurally related isoform, increases concomitantly in the first year of life to become the major isoform in the adult liver. Although substrate specificities overlap between CYP3A4 and CYP3A7, some drugs, such as midazolam, are mainly metabolized by CYP3A4, leading to prolonged half-life in neonates. Of interest, CYP2E1 is induced and metabolized by ethanol in the fetus and has been proposed to be implicated in the development of fetal alcohol syndrome.

Table 15.3

Ontogeny of Human Phase I and II Metabolizing Enzymes.

Data from References 89–103.

Enzyme	Fetus	Newborn	Infancy	Adult
Phase I
CYP1A1	+/−	−	−	−
CYP1A2	−	+/−	+/−	+
CYP2A6	−	NA	NA	+
CYP2B6	−	NA	NA	+
CYP2C9	−	+/−	+	+
CYP2C19	+/−	+/−	+/−	+
CYP2D6	+/−	+/−	+/−	+
CYP2E1	+/−	+/−	+	+
CYP3A4	−	+/−	+/−	+
CYP3A5 ^a	+/−	+/−	+/−	+/ −
CYP3A7	+	+	−	−
Phase II
UGT1A1	−	+	+	+
UGT2B7	+/−	+/−	+	+
GST α	+/−	+	+	+
GST µ	+/−	+	+	+
GST π	+	−	−	−
SULT1A1	+/−	+/−	+	+
SULT1A3	+	NA	NA	+/−

UGT , UDP-glucuronosyl transferase; GST , glutathione S-transferase; SULT , sulfotransferase.

a CYP3A5 expression does not vary significantly with age but has a high interindividual variability.

Extramicrosomal

A few of the oxidative and reductive reactions are mediated by enzymes in the mitochondria, and cytosol of the liver and other tissues. These enzymes include those involved in oxidation of alcohols and aldehydes; alcohol and aldehyde dehydrogenases; and enzymes that partake in the metabolism of catecholamines, tyrosine hydroxylase, and monoamine oxidase. Although the activity of some of these enzymes can be detected early in gestation, their full activity is reached only in early childhood. However, once again marked ontogenic differences between enzymes are observed. For instance, class I alcohol dehydrogenase, the major ethanol-metabolizing enzyme, tends to be well expressed in fetal liver, whereas class III alcohol dehydrogenase is relatively deficient in the fetus.

Phase II Reactions (Synthetic Reactions)

In phase II reactions, molecules that are naturally present in the body are conjugated or combined with the drug or other endogenous compounds. The drug may have first undergone a phase I reaction, or the original drug may be directly conjugated. Conjugation converts drugs into more polar compounds, which are pharmacologically less active and are more readily excreted; an exception applies to acetylation, whereby the metabolite is often less water-soluble. Although it was previously thought that conjugation reactions represented true inactivation and detoxification reactions, it is presently known that certain conjugation reactions (e.g., N -acetylation of isoniazid) may lead to the formation of reactive species responsible for hepatotoxicity. Diclofenac biotransformation produces quinoneimine, an over-oxidized metabolite responsible for idiosyncratic hepatotoxicity. The major conjugation reactions are listed in Table 15.2 .

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