Basic and translational research in neonatal pharmacology - Nature

Journal of Perinatology (2006) 26, S8–S12 r 2006 Nature Publishing Group All rights reserved. 0743-8346/06 $30 www.nature.com/jp

ORIGINAL ARTICLE

Basic and translational research in neonatal pharmacology RM Ward1,2, RH Lane2 and KH Albertine2 1

Pediatric Pharmacology Program, University of Utah Department of Pediatrics, Salt Lake City, UT, USA and 2Division of Neonatology, University of Utah Department of Pediatrics, Salt Lake City, UT, USA

Pharmacologic study is needed in the extremely immature newborns who currently survive. Study is needed of both the drug treatment previously established in more mature neonates and of novel drug therapy. Carefully controlled studies are needed to identify accurately both beneficial and harmful drug therapy and the mechanisms of that toxicity. Careful pharmacologic study of drug disposition and its mechanisms might lead to dosing paradigms or patient selection that minimize toxicity and maximize efficacy. In vivo, translational models of neonatal diseases are limited, but can be used to identify novel treatments and study mechanisms of established, successful therapy. Findings from such studies can generate hypotheses for study in humans leading to a continuing scientific interchange from bedside to bench to bedside. Similarly, clinical observations can generate hypotheses for study in translational models where more invasive analyses are possible. Specific areas of drug treatment should focus on neonatal disorders with long-term, adverse outcomes, such as chronic lung disease, that is amenable to translational study with animal models. National data show a progressive decrease in the clinician-scientist pool entering biomedical research. The future of neonatal pharmacology studies requires an increase in training programs for the physician-scientist whose clinical education in neonatology can be complemented by rigorous basic-science training. Success as a clinician-scientist will require collaboration with full-time basic scientists who can continue studies during periods of clinical work and provide critical study methodology to the overall study design. Such a work environment must be supported by academic institutions and may require more flexibility in the promotion and tenure schedule and process, such as the nature of what it rewards. To complement this, the NIH could modify its grant reporting process to identify co-investigators in studies who may provide unique input to the study concepts and design, such as clinical correlations or clinical investigations. Journal of Perinatology (2006) 26, S8–S12. doi:10.1038/sj.jp.7211423

Keywords: translational research; developmental pharmacology; neonatal chronic lung disease; vitamin A; retinoic acid receptor alpha; VEGF; Flk-1

Correspondence: Dr RM Ward, Pediatric Pharmacology Program, 417 Wakara Way, Suite 3510, Salt Lake City, UT 84108, USA. E-mail: [email protected] Received 13 September 2005; accepted 6 October 2005

Pharmacologic investigations have not kept pace with the increasing survival of the most immature newborns during the last two decades. Drug therapy for these tiny infants has often been based on extrapolation from more mature newborns or even adults, because few studies of pharmacokinetics and efficacy have been repeated in this population. More importantly, few translational studies have been conducted to investigate mechanisms of drug actions and toxicity using immature animal models or in vitro systems modeled after immature humans. Neonatal conditions associated with long-term morbidity and developmental delay in the extremely low birth weight (ELBW) population identify important areas to focus study of the mechanisms of disease and treatment.1 Conditions identified by the NICHD Neonatal Network members to be associated with adverse long-term outcome include severe intracranial hemorrhage or periventricular leukomalacia, postnatal steroid treatment, male gender, maternal education confined to high school or less, and chronic lung disease (CLD).1 To investigate one of these disorders, an animal model of neonatal CLD has been developed in mechanically ventilated preterm sheep and used to investigate mechanisms of the disease and its treatment.2 This paper will discuss several aspects of ELBW neonates: increasing survival, unique aspects of pharmacology, toxicities from inadequate pharmacologic study, and translational studies of pathophysiology and treatment of CLD. The increased survival of premature newborns during the last 20 years is illustrated in data from the University of Utah in 5-year epochs from 1981 to 2000 (Figure 1). These statistics show that survival of the most immature newborns of 22–24 weeks gestation has increased from 0 to 50–75% by 1996–2000.3 In this extremely premature population, improvements in survival have been accompanied by improvements in outcome, so the proportion of premature neonates with developmental problems has actually decreased.4,5 Innovations in drug therapy during these 20 years with surfactant,6 nitric oxide,7 and newer antimicrobials have contributed to these improvements. At the same time, drug therapy has contributed to developmental disorders, as with dexamethasone treatment for BPD.1 Developmental disorders are not equally distributed among the most immature newborns. Data from the Vermont Oxford Neonatal Network show that the more immature

Neonatal pharmacology research RM Ward et al

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Figure 1 Percentage survival of newborns admitted to the University of Utah Newborn ICU from 1981–2000 in 5-year epochs. Every gestational age group/epoch includes 8–80 patients.

the infant, the greater the risk of developmental disorders, emphasizing the need to increase study in this population.8 During growth and development of these infants, drug treatment without adequate pharmacologic study may produce either excessive or inadequate drug concentrations at the sites of action to achieve the treatment goals. Careful and extensive study is needed to determine whether drug treatment, especially of the ELBW infants, contributes to these developmental disorders and if so, how. Drug therapy of the ELBW newborn is often extrapolated from older populations of newborns, children, or even adults. Yet, drug therapy in this extremely immature group may not produce similar effects to those in the older population for a variety of reasons.9 The concentrations of unbound drug at the site of action may be quite different in the immature newborn because of differences in distribution volume, protein binding, metabolism and excretion that are unlikely to be accurately predicted from studies in more mature infants. Beyond achieving similar concentrations at the site of action, the effects of those concentrations may also differ. Receptor number, binding, affinity, and coupling of those receptors to intracellular effects, such as protein kinases, may each be unique in the ELBW infant. Other aspects of prematurity may influence pharmacology through penetration of drug into sites that are usually shielded, such as the blood–brain barrier and wellkeratinized skin. Drug dosages for extremely premature newborns estimated from dosages for older populations have caused crippling toxicity and even death.10 One of the earliest examples was chloramphenicol, which caused lethal cardiovascular collapse, using dosages estimated from adults.11 More recent examples include tolazoline12 and possibly dexamethasone (see below). Study of developmental changes in drug metabolism has extended slowly into the population of extremely premature newborns leaving the clinicians to treat empirically without guidance from adequately-powered, randomized controlled trials. One of the few studies of developmental changes in drug metabolism in premature newborns was conducted by Lacroix

et al. who studied the cytochrome P450 3A family of enzymes that metabolizes more drugs than any other in the body.13 They studied the developmental changes in these enzymes in human liver samples as they switch from the fetal form, CYP3A7 (testosterone 16-a hydroxylation), to the adult form, CYP3A4 (testosterone 6-b hydroxylation). Infants were divided into those born before and after 30 weeks gestation at birth and the enzymes were also analyzed according to age after birth. CYP3A7 activity was higher in the group born before 30 weeks gestation and then increased for a week before it started to decrease. CYP3A4 increased progressively until it reached adult activities and largely replaced CYP3A7 after 12 months of age. Many other pathways of drug disposition need study, both phase I reactions that usually increase the polarity of the parent drug and the phase II reactions of conjugation that increase drug elimination. Systematic study of the pharmacokinetics of many more drugs in the extremely premature newborn population will begin to describe patterns of change in various pathways of drug disposition during development that can guide dosing decisions. Drug therapy of the most immature newborns who are severely ill with complex combinations of diseases often evolves into patterns of treatment reflecting regional beliefs and styles of practice because of the lack of guidance from a careful study in this population. Unanticipated toxicity from such patterns of treatment has been reported for a variety of drugs over the years, such as chloramphenicol, tolazoline, propylene glycol as a solvent, and dexamethasone.10 Dexamethasone, a potent halogenated synthetic corticosteroid, has been used to treat bronchopulmonary dysplasia (BPD) and to facilitate weaning neonates from mechanical ventilation since the1980s,14,15 although its kinetics in the very premature newborn were not reported until the mid-1990s.16,17 Those studies show dexamethasone clearance to be inversely related to gestational age and to be quite low at 24–27 weeks gestation. Some recent reports have correlated dexamethasone treatment of immature newborns with reduced brain growth18 and long-term neurological impairment.19,20 Other controlled trials have not observed developmental impairment.21 As with chloramphenicol, dexamethasone’s toxicity may be related to extrapolation of dose and dosing intervals from older children and adults to the ELBW newborn, leading to excessive concentrations, or dexamethasone may cause a unique form of toxicity, possibly in concert with other factors or other drugs. Some authors have generalized the toxicity to corticosteroids, as a group, without acknowledging that the pharmacology of a halogenated corticosteroid, such as dexamethasone, may differ significantly from that of corticosteroids that resemble cortisol and serve as substrates for the same endogenous enzymes. In our zeal to protect newborns and prevent unnecessary neurologic injury, we must be careful not to impugn a specific, potentially useful drug, such as dexamethasone, without understanding whether it is simply a matter of inadequate Journal of Perinatology


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pharmacokinetic study, a toxicity unique to the drug, or a type 1 statistical error related to confounding factors in an immature and quite ill population. The mechanism of dexamethasone toxicity to the immature newborn needs further translational study using in vitro cell cultures and animal models, that are underway, to complement existing studies.22,23 The correlation of dexamethasone treatment of ELBW infants with adverse neurologic outcomes illustrates the broad spectrum of possible mechanisms that might be involved. Dexamethasone dosing has not been guided by pharmacokinetic study, so the concentrations may have accumulated to ‘toxic levels.’ Toxic levels may point to a direct neurotoxic effect within the CNS or secondary effects such as vasoconstriction producing ischemic damage. Direct neurotoxic effects may point to a specific effect of this synthetic, halogenated corticosteroid on the neuron, increased apoptosis, arrest of neuronal maturation or growth, or even a currently unrecognized mechanism of neurotoxicity. Many of these possibilities can be tested in vitro or in immature animals. Unfortunately, few extrauterine animal models exist with a similar degree of neural immaturity as that exhibited in the 23/40 weekgestation human newborn. Mechanisms of adverse outcomes in care of ELBW infants are not easy to study in this population. Small numbers of subjects cared for at a single institution and variations in care among institutions challenge identification of factor(s) that cause adverse outcomes. This increases the need for translational studies to identify how medications may cause adverse effects in this immature population as well as the mechanisms through which medications may reduce adverse outcomes. Studies must integrate clinical findings from the bedside with translational study in animals and with molecular approaches to explain the findings from the other two. Surfactant therapy has been a significant therapeutic advance during the last two decades, first with calf-lung surfactant extracts and with those supplemented with exogenous chemicals. Surfactant replacement for extremely premature newborns acutely improves RDS. Surfactant treatment, however, has not improved the frequency of CLD. The pathological definition of CLD includes the disruption of air-sac partitioning by secondary crests and distancing of capillaries from air sacs. These pathological changes reduce the surface of the air–blood barrier, thereby impairing gas exchange. Initiation of mechanical ventilation appears to be the single greatest predictor of CLD.24,25 Because models of prolonged ventilation of the premature rodent are not feasible secondary to size, our group utilizes a ventilated, premature sheep model of CLD to study interventions that moderate the pulmonary morbidity associated with prematurity.2,26,27 We have demonstrated that conventional ventilation in preterm lambs disrupts air-sac partitioning, decreases pulmonary compliance, alters mesenchymal apoptotic homeostasis, and decreases pulmonary angiogenesis likely through reduction in vascular endothelial growth factor (VEGF) and its functional receptor in the lung, fetal liver kinase Journal of Perinatology

1 (Flk-1). These pathological changes reduce the surface of the air–blood barrier, thereby impairing gas exchange. In contrast, the use of nasal continuous positive airway pressure (nCPAP) in preterm lambs produces findings that are similar to the normal term lung, and thereby becomes the ‘gold standard’ similar to what has been observed after nCPAP treatment of preterm baboons.28 Vitamin A treatment of premature newborns significantly reduces CLD. A recent Cochrane Database System Review analyzed the results of seven trials involving vitamin A supplementation in infants weighing less than 1500 g.29 The meta-analysis of these trials suggested that vitamin A supplementation reduces oxygen requirement at 4 weeks postnatal age and 36 weeks postmenstrual age, traditional clinical definitions of CLD. To identify the mechanisms through which vitamin A affects the development of CLD, our group has developed an approach that utilizes tools that include physiological assessments, pharmacological interventions, and molecular approaches. We have proposed the following hypothesis: treatment of conventionally ventilated lambs with vitamin A will permit normal lung development similar to that observed in the preterm lamb treated with nCPAP alone, and these effects occur through the retinoic acid receptor (RAR). To prove this hypothesis, we have treated preterm lambs with vitamin A and have found pressure volume curves, histology, P53 (a pro-apoptotic transcription factor) expression, and VEGF/Flk-1 expression profiles that are similar to the lambs treated with nCPAP. Moreover, when conventionally ventilated lambs are treated with AM-580, a specific RAR-alpha agonist, the lambs respond as if they are on nCPAP. Therefore, by using this multifaceted translational approach, not only can we demonstrate specific molecular pathways through which vitamin A permits normal lung development in preterm lambs, but we can attribute to our molecular findings the appropriate physiological significance (Figure 2).30,31

Figure 2 Histopathology of terminal respiratory units (TRU) in lung parenchyma from preterm lambs managed for 3 days by conventional ventilation (CV). (a) CV alone. The TRU has wide airspaces that are devoid of secondary septa. The walls of the airspaces are thick and cellular.(b) CV þ vitamin A (Aquasol; 5000 U/kg/day, i.m.). The TRU has areas that are wide and other areas that are narrow and have secondary septa. The walls of the airspaces are thinner and less cellular compared to CV alone. (c) CV þ AM580 (Retinoic acid receptor-a agonist; 0.01 mg/kg/day, i.m.). The TRU has uniformly small airspaces that are penetrated by secondary septa. The walls of the airspaces are thin and the least cellular. The three panels are of the same magnification.


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Advances in drug therapy of the ELBW newborn require a combination of straightforward, simple clinical pharmacology studies of pharmacokinetics, safety and efficacy coupled with more complex translational study in animal models or in vitro molecular approaches to explain molecular mechanisms. The extremely immature newborns who now survive represent a new frontier of pharmacology, requiring repetition of older studies in these infants whose extreme immaturity may create a unique physiology. The increasing survival of the extremely immature newborn is accompanied by a high frequency of developmental delay. Risk factors associated with such delay include intracranial hemorrhage (ICH) or periventricular leukemalacia (PVL), male gender, postnatal steroid treatment, reduced maternal education and CLD.1 In the care of sick preterm newborns, it is often unclear whether addition of new drug therapy increases or decreases survival and how to separate adverse outcomes from the disease from those caused by treatment. These factors identify important areas of study for neonates to identify effective and safe treatments. Pediatric clinical pharmacologists have always needed to know the principles of pharmacokinetics and drug actions within the context of developmental changes that characterize pediatric medicine. The next generation of neonatal clinical pharmacologists, however, will also require knowledge of the regulatory background for clinical study of drugs32,33 and of pharmacogenetics that explains variations in both diseases and drug metabolism.34 To succeed as a clinician-scientist, the next generation will also need to work in a group collaborating with the laboratory-based scientists who can sustain a research effort while the clinician returns to the clinical setting. To achieve recognition of important research contributions in such a setting, the NIH needs to recognize and acknowledge the co-investigators on grants and institutions must do the same in the promotion and tenure process.

Acknowledgments This study was supported in part by Grant NICHD PPRU 1 U10 HD045986-01 to RM Ward Grant R01 HD41075 to RH Lane; and Children’s Health Research Center Grants R01 HL62875, P50 HL5640, and T35 HL07744 to KH Albertine

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