Discussion In the case example, acetaminophen was implicated as a cause of hepatic necrosis in the child. Diagnosis was delayed because CT abdominal fi ndings, performed 3 days aft e r the presenting injury, were misinterpreted as hepatic trauma. Th e pattern of necrosis de scribed in this child \200\231s liver is consistent with damage related to acetaminophen wh ere zone 3 is the main area of concentration of the enzyme system responsible for the conve rsion of acetami- nophen into hepatotoxic metabolites. None of the other drugs used in his management are commonly implicated in hepatoxicity, although co-administration of anticonvu lsants may induce hepatic microsomal enzymes resulting in increased NAPQI aft er subse- que nt acetaminophen administration. While some anticonvulsant drugs may induce CYP2E1, phenyto in and phenobarbital are not metabolized through CYP2E1 and have not been considered to be CYP2E1 inducers, although there are reports that suggest pheno- barbital induces a modest i ncrease (1.7-fold) in CYP2E1 (Madan et al., 2003 ). hepatic transaminase concentrations with peak concentrations occurring within 24 h of the i schaemic episode. It is likely that the combination of therapeutic acetaminophen doses in t he presence of a liver compromised by ischaemic hepatitis, contributed to this boy \200\231 s death. Consideration should be given to both the indication for acetaminophen and the dose in chil dren with hepatic pathol- ogy. Acetaminophen has poor eff ect on temperature changes due to cerebral pathology. Temperature reduction in this child would have been better achieved by external cooling and neuromuscu- lar blockade to prevent shivering. Acetaminophen contribu tes lit- tle additional analgesia and does not reduce opioid adverse eff ects when used to supplement morphine (Maund et al., 2011 ). Th e use of acetaminophen to control fever conti nues to be debated. It has not been established conclusively that the benefi ts of antipyre tic therapy outweigh its risks (Plaisance and Mackowiak, 2000 ). References Allegaert , K. , Cossey , V. , Debeer , A. , Langhendries , J. P. , Van Overmeir e , B. , De Hoon , J. , et al . ( 2005 ). Th e impact of ibuprofen on renal clearance in pr eterm infants is independent of the gestational age . Pediatr Nephrol , 20 , 740 \200\2237 43. Acetaminophen has recognized hepatotoxicity aft er doses above 150 mg/kg in children (Penna and Buchanan, 1991 ), although data subsequent to this report has implicated lower thresho ld doses (75 \200\22390 mg/kg/day) used for 3 days or more (Kearns et al., 1998 ). Children may have raised serum concentrations aft er 2 \200\2233 days of repeated therapeutic doses , resulting in hepatic damage. Liver func- tion is further reduced in the presence of ischa emic hepatitis, which occurs in association with severe shock and is due to a reduction in liver blood fl ow. Th e condition is manifested by elevation of plasma Allegaert , K. , Murat , I. , and Anderson , B. J. ( 2007 ). Not all intravenous paracetamo l formulations are created equal . Paediatr Anaesth , 17 , 811 \200\223812. Allegaert , K. , Palmer , G. M. , and Anderson , B. J. ( 2011 ). Th e pharmacokinetics of i ntravenous paracetamol in neonates: size matters most . Arch Dis Child , 96 , 575 \200\223 580. CHAPTER 43 the non-steroidal anti-inflammatory drugs and acetaminophen 447 Demiraran , Y. , Ozturk , O. , Guclu , E. , Iskender , A. , Ergin , M. H. , and Tokmak , A. ( 2008 ). Vasoconstriction and analgesic effi cacy of locally infi ltrated levobupivacaine for nasal surgery . 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Litalien , C. and Jacqz-Aigrain , E. ( 2001 ). Risks and benefi ts of nonsteroidal anti-inf l ammatory drugs in children: a comparison with paracetamol . Paediatr Drugs , 3 , 817 \200\223858. Rumack , B. H. , and Matthew , H. ( 1975 ). Acetaminophen poisoning and toxicity . Pediatrics , 55 , 871 \200\223876. Madan , A. , Graham , R. A. , Carroll , K. M. , Mudra , D. R. , Burton , L. A. , Krueger , L. A. , et al . ( 2003 ). Eff ects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes . Drug Metab Dispos , 31 , 421 \200\223431. Scott , C. S. , Retsch-Bogart , G. Z. , Kustra , R. P. , Graham , K. M. , Glasscock , B. J. , and Smith , P. C. ( 1999 ). Th e pharmacokinetics of ibuprofen suspension, chewable tabl ets, and tablets in children with cystic fi brosis . J Pediatr , 134 , 58 \200\22363. Mandema , J. W. , and Stanski , D. R. ( 1996 ). Population pharmacodynamic Standing , J. F. , Tibboel , D. , Korpela , R. , and Olkkola , K. T. 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A single-tablet fi xed-dose combination of racemic ibuprofen/ paracetamol in the management of moderate to severe postoperative dental pain in adult and adolescent pat ients: a multicenter, two-stage, randomized, double-blind, parallel-group, placebo-controll ed, factorial study . Clin Th er , 32 , 1033 \200\2231049. Van Der Marel , C. D. , Anderson , B. J. , Romsing , J. , Jacqz-Aigrain , E. , and Tibboel , D. ( 2004 ). Diclofenac and metabolite pharmacokinetics in children . Paediatr Anaesth , 14 , 443 \200\223451. Van Overmeire , B. , Touw , D. , Schepens , P. J. , Kearns , G. L. , and Van Den Anker , J. N. ( 2001 ). Ibuprofen pharmacokinetics in preterm infants with patent ductus arteriosus . Clin Pharmacol Th er , 70 , 336 \200\223343. Miyagi , S. J. , and Collier , A. C. ( 2011 ). The development of UDP- glucuronosyltransfer ases 1A1 and 1A6 in the pediatric liver . Drug Metab Dispos , 39 , 912 \200\223919. Walls , L. , Baker , C. F. , and Sarkar , S. ( 2007 ). 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Phase I trial of high dose paracetamol and carmustine in patients wi th metastatic melanoma . Melanoma Res , 13 , 189 \200\223196. Palmer , G. M. ( 2005 ). A teenager with severe asthma exacerbation following ibuprofen . Anaesth Intensive Care , 33 , 261 \200\223265. Wong , D. L. , and Baker , C. M. ( 1988 ). Pain in children: comparison of Penna , A. , and Buchanan , N. ( 1991 ). Paracetamol poisoning in children and assessment scales . Pediatr Nurs , 14 , 9 \200\22317. hepatotoxicity . Br J Clin Pharmacol , 32 , 143 \200\223149. CHAPTER 44 Developmental pharmacology of opioids Gareth J. Hathway Summary Recognition of the need for alternative analgesic regimens for managing neonatal an d childhood pain has led to a rich literature concerning the ways in which early life pain diff ers from that at older ages. As in adults, opiates are oft en considered the gold stan d- ard analgesic class of drugs, of which morphine is the prototypi- cal agent. Th ere is a wealth of data detailing clinical observations, measurements, and interventions with regar d to the use of opioids in treating pain in children. Studies in the early part of this cen - tury have highlighted that, in humans, age is an important factor that infl uences the mo rphine requirement of neonates following surgery; and dose requirements are infl uenced by both pharma- cokinetic and pharmacodynamic factors. Laboratory studies have extended our un derstanding of changes within the peripheral and central nervous systems that underlie alte rations in nociception in early life. Th is chapter will review what is currently known abo ut the actions of opioids upon nociceptive and nociresponsive ele- ments of the nervous sys tem in early life, how they diff er from adult responses, and ask whether manipulating endo genous opioid sys- tems in early life may have consequences on neurodevelopment. cord (Baccei and Fitzgerald, 2004 , 2006 ; Baccei et al., 2003 ; see also Walker and Baccei , Chapter 6, this volume). Further sources of inhibitory drive to the adult spinal cord are descending projections fro m the ventral brainstem. In the adult, these projections that emanate from the rostroventra l medulla (RVM) are able to powerfully facilitate and inhibit spinal excitabil- ity, increa sing and decreasing spinal refl exes (Fields and Anderson, 1978 ; Fields et al., 1983 ). Fu rthermore, there is considerable evi- dence that these brainstem centers are important for the mainte- nance of chronic pain states in adults (Porreca et al., 2002 ; Zhang et al., 20 09 ). Th e RVM receives a dense aff erent input from the peri- aqueductal gray (PAG) which in turn acts as a common target for numerous more rostral structures such as the amygdala, hypotha- lamus, hippocampus, and anterior cingulate cortex (Fields et al., 2006 ; see Fitzg erald, Chapter 8, this volume). As this brief summary demonstrates, pain is a complex pheno menon involving numerous parts of the peripheral and central nervous systems. When con- sid ering the analgesic action of opioids, it is therefore necessary to consider the expression and actions of both endogenous and exog- enous ligands, at multiple sites within the nervo us system. Opioid peptides History Opium has been used as a recreational and analgesic drug for centur ies, yet it was Stert rner \200\231s isolation of the active ingredient morphine in 1806 that began the pharmacological evaluation of how this drug and other similar drugs act. So me considerable time passed before the fi rst description of specifi c opiate receptors (se e later) (Pert and Snyder, 1973 ; Simon et al., 1973 ; Terenius, 1973 ). Th e ability of op iates to mediate analgesia in a dose-dependent manner when administered into the midbrain o f rats and humans, and the observation that electrical stimulation of the same brain region s resulted in similar inhibition of pain, led to the hypothesis that an endogenous opiate-l ike neurotransmission system existed in the mammalian CNS (Mayer and Liebeskind, 1974 ; Rey nolds, 1969 ; Tsou and Jang, 1964 ). Introduction Th e response of neonatal and young animals to pain can be gener- ally conside red to be hyperexcitable; and characterized by poorly coordinated, inappropriately organize d refl exes and reduced ther- mal and mechanical pain thresholds (Fitzgerald, 2005 ). Th e dorsal horn (DH) of the spinal cord is important for the primary integra- tion of somatosen sory and nociceptive information in the central nervous system (CNS). Delayed maturation of inhibitory neuro- transmission in the spinal cord contributes to the alterations in nocice ptive responses exhibited in early life. At a cellular level this is manifested as an incre ased cutaneous receptive fi eld of nocire- sponsive neurons in the spinal DH (Fitzgerald an d Jennings, 1999 ). Th ese immature physiological responses are underpinned by a lack of sy naptic inhibition in the DH through the fi rst 2 to 3 postna- tal weeks in the rat (a time period which correlates with infancy through to late childhood in human development). Intri nsic spinal neurotransmitters such as GABA and glycine fail to evoke measur- able mIPSCs (i nhibitory currents) in the neonatal and infant spinal Th e pioneering work of many groups in the late 1960s and 1970s led to the fi nal identifi cation of a family of endogenous opioid- like peptides in the forebrain that were intrinsic ally linked with SECTION 6 pharmacological interventions 450 Impact of developmental interventions on opioid systems Stress and maternal separation Subs equent research has focussed on pre- or perinatal manipula- tions to either the rat pups or their mothers, and the impact this has upon the development of opioidergic systems, partic ularly in the forebrain (Belcheva et al., 1998 ; Lin et al., 2009 ; McLaughlin et al., 2002 ). Most of these manipulations take the form of stress such as maternal separation in the early postnatal period, or of pharmacological manipulations such as exposure to opiates whi lst in utero . Th e overriding hypotheses of these studies are that the ontogeny of the opi oidergic system in the CNS will be altered, and there is considerable supporting data. In o ne example (Gustafsson et al., 2008 ), rats were either collectively or individually separa ted from their mothers for 15 or 360 min over the fi rst three postna- tal weeks. Th is res ulted in altered met-enkephalin and dynorphin levels in a number of forebrain regions in 3- week and 10-week old off spring, and this was particularly pronounced if pups were indi- vi dually separated. Whilst many areas of the brain were assayed, focusing on the PAG which is directly involved in pain processing, it can be seen that the normal increase in met-enkep halin levels seen in control rats is blunted in rats that underwent separation, with inconc lusive changes in dynorphin levels. Th is study, which is one of many, demonstrates that ea rly life interventions can radically change the expression and therefore the functional rol e of endog- enous opioid systems through to adulthood. nociception (Goldstein et al., 1979 ; Hughes et al., 1975 ; Li et al., 1976 ; Loh et al., 1 976 ). Th e endogenous opioids include -endorphin, the endomorphins 1 and 2, the enkepha lins and dynorphins. Each of these are synthesized from precursor peptides: pre-opiomelano- cortin (POMC), pro-enkephalin, and pro-dynorphin respectively. Work on the ontogeny of the se systems was largely carried out in the 1980s and is expertly reviewed by McDowell and Ki tchen ( 1987 ). Although predating developments within the fi eld of molecular neurobiology , this is a valuable review of the subject. Speci \201 c peptides -endorphin can be detected prenatally in the rat from embryonic d ay 13 (E13), levels increase within several brain areas with age (Ng et al., 1984 ) until E 18 (just prior to birth at E20 \200\22321 in the rat), when adult levels have been reached (Bayon et al., 1979 ; Bloom et al., 1980 ). Analysis of the literature from this time shows general agree- ment that -endorphin levels change postnatally, including in the pons, w hich has direct relevance to the descending control of pain processing. However, there is s ignifi cant disagreement as to the tem- poral profi le of these changes and the absolute le vels of -endorphin that can be measured in specifi c brain regions (McDowell and Kitchen , 1987 ), but marked regional diff erences in the development of -endorphin expression i n the CNS are apparent. Th e mecha- nisms for -endorphin release in the brain are not fu lly developed at birth; both the spontaneous and potassium-stimulated release of the peptid e from hypothalamic slices appear to be immature until at least postnatal day 20 (P20) in t he rat (Hompes et al., 1982 ). Enkephalin (both Met and Leu) can also be detected prenatally. Within the brainstem levels are thought to peak around the second postnatal week in the rat (McDowell and Kitchen, 1987 ; Ng et al., 1984 ) with levels then diminishing to those found in the adult. It has been proposed that the development of the enkephalinergic system proceeds in a rostrocaudal mann er (Tsang et al., 1982 ). Opioid exposure Clinically it is important to determine whether exposure to opi- ate analge sics early in life has any eff ect on subsequent response to the same compounds later in li fe. In rats, administration of opioids either prenatally to the mother, or in the fi rst 10 postnatal days, can signifi cantly shift the morphine dose \200\223response relationship l ater in life. Single morphine administration on postnatal days 1 or 2 (1 mg/kg) produced \200\230tolerance \200\231 to subsequent morphine analgesia at postnatal day 29 (Bardo and Hughes, 1981 ). Repeated administra- tion of morphine over the fi rst 9 postnatal days (3 m g/kg subcutane- ously) resulted in signifi cantly lower basal thermal and mechanical thresh olds in the juvenile period (P29). Additionally, there was a signifi cant rightward-shift i n the cumulative dose \200\223response curves at both postnatal days 20 and 49 (Zhang and S weitzer, 2008 ). Prenatal exposure (twice daily from E5 to E12) altered morphine dose \200 \223response in off spring at postnatal day 35 (O \200\231Callaghan and Holtzman, 1976 ). T h ese data suggest that opiate exposure early in life can alter the development of componen ts of the nervous system necessary for mediating the analgesic eff ects of exogenous opioid s; and also that early life opiate exposure can alter other elements of the \200\230pain p athway \200\231 development as indexed by alterations in basal sensory thresholds in some s tudies (Zhang and Sweitzer, 2008 ). Pro-dynoprhin is the pro-peptide responsible for the synthesis of dynorphin. Studies detail ing the developmental expression profi le of either the pro-peptide or the actual peptide i n pain related areas of the CNS are very sparse and no clear consensus or opinion has been reached (McDowell and Kitchen, 1987 ). Developmental expression It is apparent from the current literature that investigations int o the normal development of the endogenous opioid system have slowed considerably from the late 1980s and early 1990s. Th ere is little data regarding the presence of endogenous opio id peptides in the pri- mary aff erent fi bres through postnatal development. Th ere is lim - ited evidence for the presence of opioid peptides in some aff erent fi bres in the prenat al environment (Haynes et al., 1982 ), but this weak immunoreactivity disappeared postnatal ly and was restricted to white matter tracts. Fiber staining within the spinal DH was absen t. Th is study pre-dated a full description of the composition of the family of opioid pept ides and concentrated only on the expres- sion of -endorphin. More recent studies regard ing the distribution of these peptides in the peripheral nervous system has focussed on the adult rat. In these studies (Marviz n et al., 2009 ), although enkephalin was never obs erved to be co-localized with primary aff erent terminals in the spinal DH, there was some co-localiza- tion of dynorphin with calcitonin gene-related peptide (CGRP), a marker of pep tidergic C fi bres. A thorough study through postnatal development has not yet been perform ed. Role of opioid peptides in normal development Do opioid peptides have a role in normal deve lopment? Reviewing the available literature suggests that opioid peptides act as inhibi- to ry growth factors at a cellular level, with antagonism of opioid receptors in vivo resultin g in increased dendritic length and spine CHAPTER 44 developmental pharmacology of opioids 451 formation in neurons in the cerebellum (Hauser et al., 1987 , 1989 ). However, alterations in the anatomical development of the nerv- ous system by opioids are not restricted to neur ons. Astrocyte numbers have decreased in mouse cortical cultures exposed to met-enkephalin by a receptor-dependent mechanism (Stiene- Martin and Hauser, 1990 ). More recently this ef f ect of opioids on the development of non-neuronal cells in the nervous system has been co nfi rmed. To model eff ects of prolonged opiate exposure of off spring of opiate-dependent mothers, Sanchez et al. ( 2008 ) implanted pregnant rat dams with osmotic mini-pumps that c on- tinuously delivered buprenorphine from day 7 of gestation until postnatal day 21, ensur ing that pups were exposed prenatally to the drug via the placenta and postnatally via mate rnal milk. In these pups it was shown that lower doses of buprenorphine increased the expre ssion of myelin basic protein whilst higher doses signifi cantly decreased levels of this p rotein, particularly when animals were examined at postnatal day 12. Anatomical examination of the cor- pus callosum showed that in rats exposed to buprenorphine there were signifi c antly more large caliber myelinated fi bres but the same distributions of non-myelinated fi bres compared to controls. Th is increase in caliber was also associated with a thinner my elin sheath surrounding these axons suggesting an altered balance between axon growth and m yelin thickness. Th us, exposure to high levels of opioid for relatively prolonged periods during prenatal develop- ment can alter CNS structure. Further studies are required to eval u- ate the impact of diff erent doses and durations of exposure during postnatal developmen t. ontogeny of adult endogenous opioid networks in the CNS, how- ever the application of quant itative genomic and post-genomic techniques would signifi cantly add to this work. Equally, utilizing immunohistochemical techniques to uncover the distribution of these peptides and the co-transmitters utilized in diff erent neuro- nal populations through the postnatal pe riod would be valuable. Many of the supraspinal structures involved in pain modulation are anatomically small and the analyzes detailed earlier were too crude to detect subtle variat ions in expression levels within refi ned brain nuclei. Even with today \200\231s state-of- the art RNA amplifi cation meth- ods accurately and repeatedly dissecting these areas from postmor- tem tissue is technically challenging. Opioid receptors Opioid peptides mediate their eff ects via interactions with a family of o pioid receptors, which are metabotropic, membrane-expressed receptors that act through seco nd messengers (i.e. in contrast to ionotropic receptors that form an ion channel pore). Th ere are three main subclasses of opioid receptor named mu ( ), delta ( ), and kappa ( ), as well as a less well recognized and investigated receptor, ORL-1 (opioid-like rece ptor). Receptors are composed of seven hydrophobic membrane-spanning domains, with extra- c ellular components that are involved in specifying interactions with ligands, and intracell ular domains that interact with intracel- lular signaling mediators. Opioid receptors media te their actions via second messenger G-proteins (see later). In the adult nervous systems activation of opioid receptors engage intracellular signal- ling systems that are in large part inhibitory, and act to decrease neuronal excitability by increasing inwardly rectifyin g potassium (K + ) conductances, decreasing voltage-dependent calcium (Ca 2+ ) channels, an d by decreasing the production of cyclic adenosine monophosphate (cAMP). More recently data from our laboratory has demonstrated a specifi c trophic role for endoge nous opioids in the maturation of supraspinal centers responsible for the descending contro l of spinal pain processing (Hathway et al., 2012 ). In this study we implanted subcutaneou s mini-pumps containing naloxone HCl at P14, P21, or P28, and continuously delivered the dr ug for 7-day periods. Animals were then allowed to mature to P40. In the group that had dru g delivered between P21 to P28, the RVM failed to mature properly, and descending inhibitio n of spinal pain processing could not be evoked in adulthood. Th ese data showed that the a ction of endogenous opioids are central to the normal maturation of supraspinal pain contro l systems, but even more interestingly that this occurs relatively late in postnatal develo pment, at a period when rats would be judged to be adolescent. Th is is a true \200\230cri tical period \200\231 as the same manipulation one week before or aft er this does not prev ent or eff ect normal maturation. In an extension of this study we implanted neonatal, 7-da y-old, rats with mini-pumps that delivered morphine for 7 days and showed that at developme ntal stages where the RVM is normally immature and unable to provide descending inhibitory control of spinal processing, that neonatal morphine treatment had accelerated the maturati on of the RVM. In these animals adult like stimulus response profi les from the RVM were ev oked. Th is work demonstrates the importance of the opioidergic system for the normal postn atal development of pain processing in the nervous system. Peripheral nervous system Th e peripheral nervous system detects stimuli and conveys sensor y information to the CNS. Peripheral sensory aff erent neurons are subdivided into three br oad categories according to their physi- ological and anatomical properties: \231 A fi bres are large, myelinated fi bres that usually convey non- noxious information to the CNS. \231 A fi bres are small myelinated fi bres and convey both noxious and non-noxious information.