Souzan B Yanni

Philip B Smith

Danny K Benjamin

Patrick F Augustijns

Dhiren R Thakker

PhD Pieter P Annaert
email: pieter.annaert@pharm.kuleuven.

Biopharmacy Thakker

Daniel K Benjamin

Higher clearance of micafungin in neonates

Keywords: micafungin, neonate, clearance prediction, serum protein binding, BCRP, Breast Cancer Resistance Protein, BSEP, Bile Salt Export Pump, Cl, Clearance, f u , fraction of drug unbound, HPLC, High Performance Liquid Chromatography, MRP2 and MRP3, Multidrug Resistance-associated Protein 2 micafungin, pediatric drug disposition, serum binding, clearance prediction, antifungal

Introduction

Micafungin is a new semi-synthetic echinocandin-type antifungal drug that has been widely used for the treatment of various fungal infections in human populations.

Pharmacokinetic studies with 14 C-micafungin determined that approximately 90% of the plasma clearance occurs via biliary elimination of the parent drug [START_REF] Hebert | Concomitant cyclosporine and micafungin pharmacokinetics in healthy volunteers[END_REF], whereas urinary excretion is a minor elimination route in human and rat [START_REF] Kaneko | Metabolites of micafungin in rats and dogs[END_REF][START_REF] Yamato | Pharmacokinetics of the antifungal drug micafungin in mice, rats and dogs, and its in vitro protein binding and distribution to blood cells[END_REF][4]. Like caspofungin and anidulafungin, micafungin is a peptide-like compound. Due to the presence of a sulfate group, micafungin is negatively charged at physiological pH (see Figure 1). Micafungin displays high plasma protein binding (>99%) in humans and animals [4].

Pharmacokinetic parameters of micafungin were determined in several human age groups: neonates < 1 month old, children <8 years of age, adolescents, and adults [START_REF] Heresi | The pharmacokinetics and safety of micafungin, a novel echinocandin, in premature infants[END_REF][START_REF] Wiederhold | The echinocandin micafungin: a review of the pharmacology, spectrum of activity, clinical efficacy and safety[END_REF]. Micafungin was found to have a higher body weight-adjusted clearance in neonates than older groups.

The necessity to use higher body-weight adjusted doses in neonates in order to achieve therapeutic plasma concentrations was recently confirmed by a population pharmacokinetic analysis [START_REF] Hope | Population pharmacokinetics of micafungin in neonates and young infants[END_REF]. As of today, no studies have been conducted to elucidate the cause of this agedifference in the disposition of micafungin in humans.

In order to obtain better insight in the age-dependent disposition of micafungin, it was crucial to first understand the clearance mechanisms in the adult population. In this context, we have recently conducted in vitro transport studies with micafungin in sandwich-cultured human hepatocytes (SCHH) [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF], which revealed the following: a) the sinusoidal uptake transporter Na + -taurocholate co-transporting peptide (NTCP, SLC10A1) and the canalicular efflux transporter Bile Salt Export Pump (BSEP, ABCB11) were found to mediate the hepatobiliary disposition of micafungin; b) Breast Cancer Resistance Protein (BCRP, ABCG2), Multidrug Resistance Associated Protein 3 (MRP3, ABCC3), and Organic Anion Transporting Polypeptide (OATP, SLCO) were found to modestly associate with the disposition of micafungin in SCHH; c) by using B-CLEAR  technology (Qualyst Inc., Research Triangle Park, NC) [START_REF] Hoffmaster | Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems[END_REF][START_REF] Liu | Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats[END_REF][START_REF] Ghibellini | In vitro-in vivo correlation of hepatobiliary drug clearance in humans[END_REF], in vivo plasma and biliary clearance of micafungin were calculated based on the in vitro data obtained in SCHH and found to be in agreement with the clinical data.

Because of ethical considerations, it is almost impossible to obtain fresh liver tissue from neonates suitable for establishing SCHH, which would support investigating transportermediated disposition of micafungin and predicting in vivo hepatobiliary clearance like in adults. However, it was possible to obtain frozen liver tissue samples from neonates (and adults) to support determining the protein expression of hepatic drug transporters. In addition, because protein binding is known to influence drug distribution and drug clearance, and because it is variable as a function of age [START_REF] Mcnamara | Protein binding predictions in infants[END_REF][START_REF] Lombardo | Prediction of volume of distribution values in humans for neutral and basic drugs using physicochemical measurements and plasma protein binding data[END_REF][START_REF] Fukuda | Effect of plasma protein binding on in vitro-in vivo correlation of biliary excretion of drugs evaluated by sandwich-cultured rat hepatocytes[END_REF][START_REF] Benedetti | Differences in absorption, distribution, metabolism and excretion of xenobiotics between the paediatric and adult Populations[END_REF], the extent of micafungin serum binding in neonates was determined in vitro.

In this study, we calculated in vivo hepatobiliary clearance of micafungin in neonates based on: a) the in vitro intrinsic clearance in adults as determined previously [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF]; b) scaling factors applicable for human neonatal liver; and c) micafungin fraction unbound in serum of neonates. We showed that the calculated in vivo plasma and biliary clearance in neonates are in agreement to those observed in clinic. Our data provide evidence for the mechanism underlying higher clearance of micafungin in neonates compared to adults. GAPDH were purchased from Alexis (San Diego, CA). Horseradish peroxidase-conjugated secondary antibody suitable for each primary antibody and the chemi-luminescent substrate SuperSignal ® West Dura were purchased from Pierce Chemical (Rockford, IL).

Protein Expression of Drug Transporters in Liver Tissues from Neonates and Adults.

Frozen liver tissues from neonates (postnatal age 0-1 month), and from adults (older than 18 years) were homogenized in 1.15% KCL buffer, pH 7.4 (1:3, w/v) and crude homogenates were prepared after centrifugation at 800 rpm for 3 min. Protein concentration in crude liver homogenates was determined by the bicinchoninic acid method against bovine serum albumin standard, and homogenates at 1mg/ml were prepared in sample buffer containing a protease inhibitors cocktail (Sigma-Aldrich, St. Louis, MO). Western blot analysis for the expression of transport proteins was performed by separating the transport proteins in liver homogenate samples loaded on SDS page gel (20 µl/well) with gel electrophoresis and the proteins were then transferred to nitrocellulose membranes according to the vendor's Horseradish peroxidase-conjugated secondary antibody suitable for each primary antibody was cross-reacted with the transporter protein-primary antibody complex; immunoreactive protein bands were detected using a Bio-Rad VersaDoc imaging system (Bio-Rad, Hercules, CA). For quantitative determination of protein expression, three density measurements of each band corresponding to BSEP, OATP1B1/3, MRP3, BCRP, GAPDH (molecular weight 190 kDa, 90 kDa, 170 kDa, 70 kDa, 35 kDa, respectively) were determined and the mean density was normalized to the mean density of the GAPDH band (used to adjust expression based on actual loaded protein samples). Relative expression of the four transporters to GAPDH in each liver homogenate sample in the neonatal or adult group was determined for each of the five liver homogenate samples per group. The mean was expressed per kg body weight based on the following scaling parameters: 35 and 70 mg protein/gram liver in neonates and adult (experimentally determined), respectively; 120 g and 1500 g average liver weight in neonates or adult (data listed by Björkman [START_REF] Björkman | Prediction of drug disposition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs[END_REF]), respectively; 3 kg and 70 kg average body weight in neonates and adult (data listed by Björkman [START_REF] Björkman | Prediction of drug disposition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs[END_REF]), respectively. The relative protein expression in neonates was expressed as percent of the mean expression in adult samples. (n=6) were spiked with micafungin (10 µM) and 70 µl aliquots of each sample were loaded into a 96-well equilibrium dialysis apparatus (HTDialysis, Gales Ferry, CT) and dialyzed against an equal volume of sodium phosphate buffer (100 mM, pH 7.4). The 96-well equilibrium dialysis apparatus was incubated for 4.5 hr with orbital shaking (100 rpm) at 37°C. These equilibrium dialysis conditions were found to be sufficient for micafungin to reach equilibrium. Aliquots from dialyzed samples (serum side and buffer side) were processed and the concentrations of extracted micafungin in both serum and buffer matrices were determined based on calibration curves of micafungin prepared in the corresponding matrix. Micafungin f u was determined according to Equation 1.

Micafungin Serum Fraction

Analytical Method. An HPLC method was developed to separate micafungin and diclofenac (internal standard) using a C8 XTerra® column (3 x 100 mm, 3.5µ Waters); a linear gradient of mobile phase A:B (v/v) was used starting from 70:30 at 0 min to 5:95 in 9 minutes at a flow rate of 0.5 ml/min, where (A) was 5 mM ammonium acetate with 0.1% formic acid, pH 4.0, and (B) was acetonitrile with 0.1% formic acid. Micafungin was detected by fluorescence emission at 464 nm (excitation at 273 nm), while diclofenac was detected by UV at 280 nm; the retention times were 5.5 min for micafungin and 6.3 min for diclofenac.

The method was linear for a micafungin concentration range of 0.1-5 µM. Accuracy and precision of the method were 12% and 3.3% for 0.3 µM and 0.2% and 2.8% for 2.5 µM, respectively. Data Analysis. Micafungin ƒ u was determined from the following equation:

f u = [micafungin buffer ] [micafungin plasma ] ( Equation 1)
The f u values in six neonates were plotted against the age of gestation at birth (in weeks) or against postnatal age at birth (in days) of each subject. In vivo micafungin plasma and biliary clearance values were predicted based on previously obtained in vitro data [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF], and the serum binding data obtained in the present study. The in vitro intrinsic biliary clearance (ml/min/mg protein) and in vitro intrinsic accumulation clearance were first scaled to kg body weight using the parameters listed in Table 1 as published by Björkman et al. [START_REF] Björkman | Prediction of drug disposition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs[END_REF]. In the absence of albumin during in vitro incubations [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF],

unbound intrinsic biliary clearance (intrinsic Cl' biliary ) and unbound intrinsic plasma clearance (intrinsic Cl' plasma ) were assumed to correspond to in vitro intrinsic biliary clearance and in vitro intrinsic accumulation clearance, respectively. The calculated in vivo biliary clearance (Cl biliary ) and in vivo plasma (accumulation) clearance (Cl plasma ) values were calculated according to the well-stirred model of hepatic disposition, assuming a micafungin blood/plasma concentration ratio of 1: 2)

Calculated in vivo Cl biliary = Q H × f u × intrinsic Cl biliary ' Q H + f u × intrinsic Cl biliary ' ( Equation
Calculated in vivo Cl plasma = Q H × f u × intrinsic Cl plasma ' Q H + f u × intrinsic Cl plasma ' ( Equation 3)
where Q H represents the hepatic plasma flow rate. The Q H values in neonate and adult are listed in Table 1.

Statistical

Analysis. An unpaired t-test (assuming unequal variance) was performed to determine the significance of the difference in micafungin f u between neonates and adults. shown). In neonates, the mean f u from six different neonatal samples conducted at three different days was found to be 0.033 + 0.017 (96.7% bound) compared to a mean f u value of 0.004 + 0.003 (99.6% bound) in adult (Figure 3). The difference of f u between neonates and adults is statistically significant (P <0.001). Figure 4 illustrates the individual f u values in neonates along with the postgestational age at birth as well as the postnatal age. The highest f u was observed for the full term subjects 1 and 3 (40 weeks postgestation), while lower f u values were found for premature subjects 2 and 4 (24 and 31 weeks of gestation, respectively). The lowest micafungin f u was observed for subject 2, which is not only the most premature, but also the oldest (52 days after birth) baby.

These results indicate that micafungin f u in neonate serum is higher than in adult serum.

These results also indicated that, while there appears to be a weak relationship between postgestational age at birth and f u , there is no evidence for a relationship between postnatal age in neonates and f u .

Discussion

Micafungin, which is used to treat life-threatening fungal CNS infection in neonates and young infants, is mainly cleared into the bile [START_REF] Hebert | Concomitant cyclosporine and micafungin pharmacokinetics in healthy volunteers[END_REF]. Surprisingly, the clearance of micafungin in neonates, the human population generally displaying immature drug metabolizing enzymes and drug transporter function, is reported to be three times that of adults. Because it undergoes biliary clearance, we hypothesized that the higher clearance of micafungin in neonates might be due to the higher drug uptake and/or secretion by sinusoidal and canalicular liver transporters. The transporters that mediate the uptake and secretion of micafungin were identified in our previous in vitro mechanistic study using human sandwichcultured hepatocytes prepared from fresh liver of adult donors [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF]. These in vitro data confirmed the unique utility of this in vitro model system in exploring the mechanisms of hepatic uptake and biliary excretion of endogenous and exogenous compounds and in predicting in vivo biliary clearance of drugs [START_REF] Hoffmaster | Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems[END_REF][START_REF] Liu | Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats[END_REF][START_REF] Ghibellini | In vitro-in vivo correlation of hepatobiliary drug clearance in humans[END_REF][START_REF] Chandra | Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes[END_REF][START_REF] Annaert | Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes[END_REF][START_REF] Kemp | Xenobiotics inhibit hepatic uptake and biliary excretion of taurocholate in rat hepatocytes[END_REF][START_REF] Mcrae | Ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibit bile acid transport in human and rat hepatocytes[END_REF]. In our previous study, we have indeed reported that in in vitro hepatic accumulation of micafungin in adult human hepatocytes occurred mainly by transporter-mediated mechanisms. The uptake of micafungin into the hepatocytes was mediated by NTCP >> OATP, whereas the biliary excretion involved the canalicular transporters BSEP > BCRP. The results also suggested a role for the sinusoidal efflux transporter MRP3, in the hepatic disposition of micafungin.

Unfortunately, because of ethical considerations, it is nearly impossible to obtain human neonatal liver tissue suitable for establishing hepatocyte cultures. Therefore, we considered another approach to obtain better insight into the age-dependent clearance of micafungin. By measuring the protein expression of NTCP, BSEP, BCRP, MRP3 and both hepatic OATP1B isoforms in neonatal versus adult liver homogenates, we pursued to make a semi-quantitative assessment regarding the possible age-dependent activity of these Formatted: Highlight Deleted: The biliary excretion of micafungin, rather than its hepatic accumulation, appeared to be ratelimiting for its hepatic elimination in adult liver. indeed demonstrated that transporter protein expression levels correlate with function. For example, in a developmental mouse model, Pinto et al. [START_REF] Pinto | Ontogeny of renal P-glycoprotein expression in mice: correlation with digoxin renal clearance[END_REF] showed that digoxin accumulation in brain, kidney, and liver agreed with the expression level of the Mdr1a gene encoding for the P-gp efflux transporter in an age-dependent manner. Goralski et al. [START_REF] Goralski | Brain cyclosporin A levels are determined by ontogenic regulation of mdr1a expression[END_REF] have shown an agreement between the expression and function of transporters by demonstrating that deficient Mdr1a/b expression in neonatal mouse brain resulted in enhanced accumulation of digoxin and cyclosporin in neonatal mice compared to adult mice.

As mentioned earlier, we have previously shown that micafungin is predominantly relying on the bile salt transporters NTCP and BSEP for its hepatobiliary elimination. At the uptake level, our data support comparable expression of the main micafungin transporter NTCP between neonates and adults. Previous work by Hardikar et al. [START_REF] Hardikar | Differential ontogenic regulation of basolateral and canalicular bile acid transport proteins in rat liver[END_REF] in rats, indicated that Ntcp protein was expressed as early as postnatal day 1, and the expression levels on day 1 and day 7 (equivalent to 1 month human neonate) postnatal liver were also comparable to those in adults. Furthermore, as shown in this study (see Figure 2), also BSEP exhibits comparable expression in neonates and in adults. This result was consistent with previous data on quantitative expression of BSEP and other canalicular transport proteins in human fetal, pediatric and adult livers [START_REF] Chen | Developmental expression of canalicular transporter genes in human liver[END_REF][START_REF] Chen | Expression of hepatocyte transporters and nuclear receptors in children with early and late-stage biliary atresia[END_REF]. In these studies, the authors reported that BSEP protein was expressed in fetus and pediatric human liver at the same level as adult, however, in fetus, the localization of BSEP was found to be at both the canalicular and cytosolic locations. In general, very limited information on the ontogeny of drug transporters in human, not only at the functional level, but also at the expression level has been obtained. Studies on the age-dependent expression and localization of MDR1/P-gp in human brain [START_REF] Daood | ABC transporter (P-gp/ABCB1, RP1/ABCC1, BCRP/ABCG2) expression in the developing human CNS[END_REF] and small intestine [START_REF] Fakhoury | Impact of inflammation on the duodenal mRNA expression of CYP3A and P-glycoprotein in children with Crohn's disease[END_REF] Because protein binding is known to affect drug clearance, especially of those drugs that possess high protein binding like micafungin, we measured f u of micafungin in vitro in serum from neonates and adults; as mentioned in the results section, we observed a significantly higher unbound fraction in neonates. McNamara and Alcorn [START_REF] Mcnamara | Protein binding predictions in infants[END_REF] have shown that for lipophilic drugs, ~ 2-fold higher f u values can be expected in pediatric plasma compared to adults based on lower plasma protein concentrations in neonates than in adults. However, the model that was developed by McNamara and Alcorn [START_REF] Mcnamara | Protein binding predictions in infants[END_REF] under-predicted the f u value of micafungin in neonates compared to the value we have experimentally measured in the present study using in vitro equilibrium dialysis in neonate serum. One explanation for the 8fold higher f u in neonates compared to adult could be that micafungin binds not only to plasma proteins [more likely to albumin than gamma-globulin [START_REF] Abe | Role of plasma proteins in pharmacokinetics of micafungin, an antifungal antibiotic, in analbuminemic rats[END_REF]], but also to lipoproteins, cholesterol, or other serum components that were found to be higher in adult serum than in neonate serum [START_REF] Mcnamara | Protein binding predictions in infants[END_REF][START_REF] Benedetti | Differences in absorption, distribution, metabolism and excretion of xenobiotics between the paediatric and adult Populations[END_REF]. Furthermore, the physicochemical properties of a peptide like (higher free fraction in the blood) in neonates versus adults is a more common reason for enhanced drug clearance in neonates. In addition, in contrast to what we have presently observed for micafungin, the enzyme or transporter-mediated elimination pathways of many drugs show pronounced age-dependency [START_REF] Anderson | The pharmacology of anaesthetics in the neonate[END_REF][START_REF] Anderson | Optimizing pediatric dosing: a developmental pharmacologic approach[END_REF][START_REF] Yanni | In vitro hepatic metabolism explains higher clearance of voriconazole in children versus adults: role of CYP2C19 and flavin-containing monooxygenase[END_REF], thus complicating the interpretation of possible effects of altered protein binding on overall in vivo drug elimination. Caspofungin, which is another echinocandin antimycotic, also shows a small unbound fraction in blood (about 3.5%) [START_REF] Stone | Disposition of caspofungin: role of distribution in determining pharmacokinetics in plasma[END_REF]. However, in contrast to micafungin, caspofungin clearance in neonates (< 3 months) is significantly lower as compared to older children (2-11y) and, to a lesser extent, as compared to adults [START_REF] Sáez-Llorens | Pharmacokinetics and safety of caspofungin in neonates and infants less than 3 months of age[END_REF]. For this reason, the caspofungin recommended dose (normalized for body surface area) in neonates is about half the dose used in adults. While the caspofungin free fraction in neonate serum has not been measured, there is clearly no evidence that reduced caspofungin serum binding in neonates plays an important role in altered caspofungin disposition in this age group. The immunosuppressant cyclosporin A is another cyclic peptide exhibiting low unbound fraction in blood as well as age-dependent pharmacokinetics, with 2-fold lower oral clearance in neonates compared to older children and adults [START_REF] Regazzi | Two-year follow-up of the pharmacokinetics of immunosuppressive drugs in a neonate who underwent heart transplantation[END_REF]. However, cyclosporin A shows a complex binding profile in plasma (mainly to lipoproteins) as well as in blood (distribution to erythrocytes) [START_REF] Akhlaghi | Distribution of cyclosporin in organ transplant recipients[END_REF], while the ontogeny of this behavior has not been investigated. From a more general point of view, the clinical relevance of plasma protein binding alterations (most often considered from a binding competition perspective) as a mechanism underlying drug clearance differences should not be overestimated [START_REF] Rolan | Plasma protein binding displacement interactions--why are they still regarded as clinically important?[END_REF][40]. Nevertheless, the 8-fold increase in unbound micafungin in neonates as compared to adults is substantial, especially in light of the less pronounced effects reported previously for a list of more than 25 drugs [START_REF] Mcnamara | Protein binding predictions in infants[END_REF]. In this list, the largest differences were reported for propranolol, quinidine and verapamil, which all exhibited 3-4 fold increased unbound fractions in neonates compared to adults. Unfortunately, clinical PK data in children, especially neonates, are very limited or lacking. In the case of verapamil, Piovan et al.

[41] reported plasma levels of verapamil and its metabolite norverapamil in children between 15 days and 17 years. Their data demonstrated gradually increasing norverapamil/verapamil levels from birth onwards to reach a plateau by the age of 1 year, which then remained constant until adulthood. There was no evidence for any effect of an increased free verapamil fraction on verapamil clearance in neonates as compared to older children. This further suggests that for many drugs, altered plasma protein binding in neonates does not have a substantial impact on clearance. In addition, even when some effect would exist, it may not be easily observed due to concomitant changes in activity of drug metabolizing enzymes and transporters [START_REF] Anderson | The pharmacology of anaesthetics in the neonate[END_REF][START_REF] Anderson | Optimizing pediatric dosing: a developmental pharmacologic approach[END_REF][START_REF] Yanni | In vitro hepatic metabolism explains higher clearance of voriconazole in children versus adults: role of CYP2C19 and flavin-containing monooxygenase[END_REF].

As we have indicated in the results section (Table 1), the in vivo plasma clearance and biliary clearance values we calculated for micafungin in neonates were found to be three times those in adults, thus mirroring the difference in clearance between neonates and adults observed in the clinic. Since the calculated in vivo clearance was based on the adult intrinsic clearance values, the higher f u value of micafungin in neonates, is the factor that explains the higher in vivo clearance of micafungin in neonates. According to the well-stirred model applied here, neither the different relative liver protein content (mg/kg body weight) nor the higher Q H in neonates, contributed to the higher in vivo clearance of micafungin in neonates.

To summarize, similar protein expression of liver drug transporters, but higher f u of micafungin in neonates compared to adults have been determined by using in vitro methods 18 that f u may be the key contributor to the higher clearance of micafungin observed in neonates, however a definitive conclusion could not be made with the results that have been presented in this study. For instance, a possible role of one or more transporters that differ from those identified in adults may still be involved in micafungin disposition in neonatal liver. In addition, we assumed that neonates display no additional enzymatic clearance mechanisms for micafungin as compared to adults. However, Hope et al. [START_REF] Hope | Population pharmacokinetics of micafungin in neonates and young infants[END_REF] recently reported that substantially higher levels of the metabolite M5 were measured in neonates as compared to adults. It is clear that the existence of a neonate-specific metabolic pathway for micafungin could also contribute to its higher clearance, but further research is required in this respect.

The extent of renal excretion of micafungin (and/or) metabolites in neonates has not been measured. However, based on mass balance data obtained in adults, and based on the fact that no dose adjustments are needed in patients with renal impairment [START_REF] Hope | Population pharmacokinetics of micafungin in neonates and young infants[END_REF], renal micafungin elimination was demonstrated to play a very minor role as opposed to fecal elimination. This suggests that the several fold higher clearance in neonates compared to adults is unlikely to be due to (much) enhanced renal clearance in neonates compared to adults. Future nonclinical work in sandwich-cultured hepatocytes from juvenile animals or using apical and basolateral membrane vesicles prepared from human neonatal frozen liver should be conducted in order to ultimately understand all mechanisms underlying altered micafungin disposition in neonates. Intrinsic clearance values were generated as described in the methods section. Intrinsic biliary clearance was determined using B-CLEAR ® technology Qualyst, Inc.). *, Yanni et al. [START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF]; **, Barter et al. [START_REF] Barter | Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: reaching a consensus on values of human microsomal protein and hepatocellularity per gram of liver[END_REF]; ***, Björkman [START_REF] Björkman | Prediction of drug disposition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs[END_REF]; #, Heresi et al. [START_REF] Heresi | The pharmacokinetics and safety of micafungin, a novel echinocandin, in premature infants[END_REF].

  . Micafungin-sodium was a gift from Dr. John Perfect at Duke University. Serum samples from neonates or adult (n=6/group) were provided by Dr. Daniel Benjamin at Duke Center Research Institute. Frozen liver from neonates and adults (5 samples per group) were obtained from the Comparative Human Tissue Network (CHTN) (Columbus, OH) under an approved UNC-Chapel Hill Institutional Review Board protocol. NuPAGE 4-20% Bis-Tris gel and all other gel electrophoresis and Western blot reagents and supplies were purchased from Invitrogen (Carlsbad, CA). Primary antibodies against human BSEP [ABCB11] (F-6), NTCP [SLC10A1] (M-130) and OATP1B1 [SLCO1B1] (and OATP1B3 [SLCO1B3] to a lesser extent) (N-16) were purchased from Santa Cruz (Santa Cruz, CA), while those against MRP3 [ABCC3] (M3II-9), BCRP [ABCG2] (BXP-53), and

Formatted:

  Font: Italic Formatted: Font: Italic Formatted: Font: Italic Formatted: Font: Italic Formatted: Font: Italic Formatted: Font: Italic Deleted: , Carlsbad, CA). Primary antibodies against human NTCP, BSEP, OATP1B1/3, MRP3, BCRP, GAPDH were appropriately diluted (1:200, 1:200, 1:200, 1:200, 1:500, and 1:4000, respectively) and incubated with the membrane for 2 hr at 37 o C.

Figure 2 .

 2 Figure 2. As illustrated in the lower panel of Figure 2, the protein expression levels of these transporters in neonates, relative to GAPDH and expressed per kg body weight, were between 84 and 115% of adult levels. No statistically significant differences were observed in transporter expression levels between neonates and adults. Serum Fraction Unbound of Micafungin in Neonates and Adults. Using equilibrium dialysis, micafungin f u was determined as function of time and drug concentration (data not

  few studies on the ontogeny of transporter expression and function have

  from those of the model drugs used to establish the model obtained by McNamara and Alcorn [12]. Due to limited availability of clinical pharmacokinetic data in neonates, it is presently unclear to what extent lower protein binding

Formatted

  and biological samples from both age groups. The in vivo plasma and biliary clearance in neonates calculated based on in vitro transport data of adults generated by SCHH indicated Deleted: same Deleted: as those of adult Deleted: which we asssumed

Figure 1 .Figure 2 .

 12 Figure 1. Micafungin chemical structure Figure 2. Expression of Transporter Proteins in Liver from Human Neonates and Adults.Western blot analysis on liver homogenates from neonates and adults was used to determine the relative expression of hepatic transporters involved in micafungin uptake and biliary excretion. The top parts of panel A and panel B depict the protein expression bands in five neonate samples (N1-N5) and in five adult samples (A1-A5) for uptake and efflux transport proteins, respectively. GAPDH protein expression was measured for normalization. The bottom parts of both panels represent the mean (± SD) of GAPDH-normalized expression of the transporters in neonates (per kg body weight) relative to protein expression (per kg body weight) in adults where adult expression is 100%. Samples N3 and A5 were excluded from densitometric analysis for NTCP due to irregular band shape.

Figure 3 .

 3 Figure 3. Micafungin Fraction Unbound in Serum from Human Neonates Compared to Adults. Fraction unbound in serum from neonates and adults was determined by equilibrium dialysis as discussed in the methods section. Bars represent the mean (±SD) of values from 6 subjects /group. Statistical significance indicated by ***, P <0.001 based on the unpaired ttest (unequal variance).

Figure 4 .

 4 Figure 4. Micafungin Fraction Unbound in Serum from Neonates. Grey triangles (▲) represent the f u (mean ± SD for three determinations) of micafungin in each subject. Each subject's postnatal age (♦) or postgestational age at birth (■) are also plotted. The mean value of f u for the six subjects is represented by a dashed line (--).

Unbound in Neonates and Adults. Micafungin fraction unbound in serum from human neonates or adults was determined by equilibrium dialysis according to a modified method described by Kalvass and Maurer [17]. Briefly,

	23-40 weeks of gestation; postnatal age 1-52 days; body weight 0.6-3.5 kg) (n=6) or adults
	F o r
	P
	e
	e r
	R
	e
	v i e
	Spectra/Por ® 2 dialysis membranes (Spectrum Laboratories Inc., Rancho Dominguez, CA) w
	were conditioned in water for 15 min, followed by 30% ethanol for 15 min, and sodium
	phosphate buffer (100 mM, pH 7.4) for 15 min. Serum samples from neonates (prenatal age
	9

12 Prediction of in vivo Plasma and Biliary Clearance of Micafungin in Neonates.

  In vitro intrinsic accumulation Cl and in vitro intrinsic biliary Cl of micafungin have recently been determined using sandwich-cultured human hepatocytes from adult liver donors[START_REF] Yanni | In vitro investigation of the hepatobiliary disposition mechanisms of the antifungal agent micafungin in humans and rats[END_REF], and were 23.5 ± 1.6 and 14.0 ± 9.0 µl/min/mg, respectively. To estimate in vitro intrinsic clearance values for micafungin in neonates, in vitro intrinsic clearance values of adults were used, given that expressions of drug transporters associated with the clearance of micafungin in neonates and adults were comparable (Figure2). As illustrated in Table1, intrinsic clearance values were scaled to in vivo intrinsic clearance using neonate-based scaling factors as reported[START_REF] Björkman | Prediction of drug disposition in infants and children by means of physiologically based pharmacokinetic (PBPK) modelling: theophylline and midazolam as model drugs[END_REF][START_REF] Barter | Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: reaching a consensus on values of human microsomal protein and hepatocellularity per gram of liver[END_REF]. In vivo intrinsic clearance values were then used to calculate in vivo clearance values based on the well-stirred model (Equation 2 and Equation3), taking into account micafungin f u (as determined in the present study) as well as adult/neonatal values for Q H . The results shown in Table1illustrate a good agreement between the observed and calculated plasma clearance values. In addition, the calculated clearance values in neonates were 3-fold higher than in adults as it has been observed in clinic.

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  are exceptions. In the rat, Gao et al. observed that the canalicular transporter Bsep was detected at the canalicular membrane before birth but reached adult expression day 12 (day 7 in neonatal rat is equivalent to neonate in human)[START_REF] Gao | Differential expression of bile salt and organic anion transporters in developing rat liver[END_REF]. The authors indicated that by day 12, hepatocytes were fully polarized and localization of transporters (at either canalicular or sinusoidal membranes) was comparable to what was observed in adult hepatocytes. They also reported that the development of polarized hepatocyte membranes and expression of major bile salt transporters Bsep and Ntcp occurs shortly before birth and that correlated with the development of the enterohepatic circulation of bile salts. Therefore, full development of this excretion route early in neonatal life indicates that age-dependent expression (and thus function) of these transporters may not explain the age-dependent clearance of micafungin. It follows that the similarity in the expression of these transporters between neonates and adults can confidently be used to quantitatively estimate micafungin hepatic clearance in neonates, based on the in vitro intrinsic uptake and biliary clearance that we previously measured for adults.

	levels by postnatal
		Deleted: Because of the unavailability
		of a monoclonal or polyclonal antibody
	F o r	against human NTCP, our investigation of the protein expression of transporters relevant for micafungin disposition is not fully comprehensive. Deleted: However, Deleted: p Deleted: in rat liver
	P
	e e r	Deleted: Nevertheless, it is also noteworthy that determination of NTCP expression might not be as critical as the determination of BSEP levels, given the fact that biliary excretion was shown to be the rate-limiting step in micafungin hepatobiliary elimination in adults.
		Deleted: A
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	15

  Kalvass JC and Maurer TS. Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 2002; 23: Benet LZ, Hoener BA. Changes in plasma protein binding have little clinical relevance.

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	41. Piovan D, Padrini R, Svalato Moreolo G, Magnolfi G, Milanesi O, Zordan R, Pellegrino 17. 327-338. 40. Clin Pharmacol Ther 2002; 71: 115-21.	Formatted: Adjust space between Latin and Asian text, Adjust space between Asian text and numbers Formatted: Font: Bold Formatted: Font: Italic Formatted: Font: Bold
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	PA, Ferrari M. Verapamil and norverapamil plasma levels in infants and children	Formatted: Font: Bold
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	during chronic oral treatment. Ther Drug Monit 1995; 17: 60-7.	Formatted: Font: Bold
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Table 1 .

 1 Calculation of in vivo Plasma and Biliary Clearance of Micafungin in Adults and Neonates

	Physiological Parameters &
		Adult*	Neonate
	Scaling Factors
	f u	0.004	0.033
	mg protein/g liver	71	24**
	g liver/kg body weight	26	43***
	QH, ml/min/kg	21	73***
	Observed Plasma Cl (ml/min/kg)	0.229	0.622 #
	Intrinsic Accumulation Cl (ml/min/mg)	0.024 ± 0.002	0.024 ± 0.002
	Intrinsic Plasma Cl
	(ml/min/kg)	43.4 ± 3.0	24.3 ± 2.6
	Calculated Plasma Cl (ml/min/kg)	0.175 ± 0.012	0.793 ± 0.085
	Observed Biliary Cl (ml/min/kg)	0.160	Unknown
	Intrinsic Biliary Cl (ml/min/mg)	0.014 ± 0.009	0.014 ± 0.012
	Intrinsic Biliary Cl (ml/min/kg)	25.5 ± 15.7	14.3 ± 8.8
	Calculated Biliary Cl (ml/min/kg)	0.101 ± 0.062	0.467 ± 0.287

http://mc.manuscriptcentral.com/bdd Biopharmaceutics & Drug Disposition

5U10 HD045962-04), FWO .U. Leuven is acknowledged. Dr. Benjamin receives support from the United States Government for his work in pediatric and neonatal clinical and Government Contract HHSN267200700051C), the nonprofit organization Thrasher Research Foundation for his work in neonatal candidiasis (http://www.thrasherresearch.org), and from industry for neonatal and pediatric drug development (http://www.dcri.duke.edu/research/coi.jsp). http://mc.manuscriptcentral.com/bdd


Hana Slachtova
email: hana.slachtova@zuova.cz

Ulrike Gehring

Gerard Hoek

Hana Tomaskova

Heike Luttmann- Gibson

Hanns Moshammer

Anna Paldy

Sam Pattenden

Katarina Slotova

Frank Speizer

Renata Zlotkowska

Joachim Heinrich

Parental education and lung function of children in the PATY study

Keywords: lung function, parental education, combined analysis, East-West differences, PATY study, children

Studies of the relationships between low socio-economic status and impaired lung function were conducted mainly in Western European countries and North America. East-West differences remain unexplored. Associations between parental education and lung function were explored using data on 24,010 school-children from 8 cross-sectional studies conducted in North America, Western and Eastern Europe. Parental education was defined as low and high using country-specific classifications. Country-specific estimates of effects of low parental education on volume and flow parameters were obtained using linear and logistic regression, controlling for early life and other individual risk factors. Meta-regressions were used for assessment of heterogeneity between country-specific estimates. The association between low parental education and lung function was not consistent across the countries, but showed a more pronounced inverse gradient in the Western countries. The most consistent decrease associated with low parental education was found for peak expiratory flow (PEF), ranging from -2.80 % to -1.14 %, with statistically significant associations in 5 out of 8 countries. The mean odds ratio for low PEF (<75 % of predicted) was 1.34 (95% CI 1.06-1.70) after all adjustments. Although social gradients were attenuated after adjusting for known risk factors, these risk factors could not completely explain the social gradient in lung function.

ABBREVIATIONS

INTRODUCTION

Socioeconomic status (SES) has been found an important predictor of health within developed countries. Publication of the Whitehall study results [START_REF] Marmot | Inequalities in death--specific explanations of a general pattern?[END_REF], reporting differences in all-cause mortality between social classes, was followed by an increased interest in the issue of social health inequalities in epidemiological research [START_REF] Strand | Can cardiovascular risk factors and lifestyle explain the educational inequalities in mortality from ischaemic heart disease and from other heart diseases? 26 year follow up of 50,000 Norwegian men and women[END_REF][START_REF] Van Lenthe | Neighbourhood unemployment and all cause mortality: a comparison of six countries[END_REF].

Only a limited number of studies have investigated the SES effect on impaired lung function or chronic obstructive pulmonary disease. Studies on adults found evidence that the level of education was associated with airways disorders independently of smoking [START_REF] Bakke | Educational level and obstructive lung disease given smoking habits and occupational airborne exposure: a Norwegian community study[END_REF][START_REF] Prescott | Socioeconomic status, lung function and admission to hospital for COPD: results from the Copenhagen City Heart Study[END_REF]. A review by Hegewald and Crapo [START_REF] Hegewald | Socioeconomic status and lung function[END_REF] found significantly negative correlations between lung function and SES both in children and adults.

The CARDIA study [START_REF] Jackson | CARDIA Study. A matter of life and breath: childhood socioeconomic status is related to young adult pulmonary function in the CARDIA study[END_REF] on young adults found a decline of lung function over time due to lower childhood SES. A British study [START_REF] Lawlor | Association between self-reported childhood socioeconomic position and adult lung function: findings from the British Women's Heart and Health Study[END_REF] confirmed that childhood poverty was associated with poorer lung function in women aged 60-79.

American studies with large sample size [START_REF] Vedal | Risk factors for childhood respiratory disease. Analysis of pulmonary function[END_REF][START_REF] Demissie | Socioeconomic status and lung function among primary school children in Canada[END_REF] also brought evidence that low SES was associated with lower lung function in children.

An Austrian study of school age children [START_REF] Lercher | Birth weight, education, environment, and lung function at school age: a community study in an alpine area[END_REF] found that most of lung function measures were negatively associated with a lower birth weight (LBW), lower maternal education, smaller gestational age, and larger family size.

Associations between low SES and impaired lung function both in children and adults were detected mainly in Western European countries and North America. East-West differences

have not yet been explored.

The PATY project (Pollution and the Young) offers a unique opportunity to investigate the relationship between lung function in school-age children and their socioeconomic status in a large international dataset.

MATERIAL AND METHODS

Study design and study population

The present study is a combined analysis of questionnaire and lung function data from 8 cross-sectional studies in which the lung function measurements were part of the original studies. The study sample contained 24,010 school-children aged 6-12 years from the North American 24-cities study [START_REF] Raizenne | Health effects of acid aerosols on North American children: pulmonary function[END_REF], the East European CESAR study countries [START_REF] Leonardi | Respiratory symptoms, bronchitis and asthma in children of Central and Eastern Europe[END_REF], and data from Austria (Linz study [START_REF] Neuberger | Declining ambient air pollution and lung function improvement in Austrian children[END_REF]), Germany (Bitterfeldt, Hettstedt and Zerbst study [START_REF] Heinrich | Respiratory diseases and allergies in two polluted areas in East Germany[END_REF]), and the Netherlands (24-schools study [START_REF] Janssen | The relationship between air pollution from heavy traffic and allergic sensitization, bronchial hyperresponsiveness, and respiratory symptoms in Dutch schoolchildren[END_REF]). The number of children derived from the original studies into a current analysis is slightly smaller due to missing dataonly those with a full set of data were taken into the analysis. The studies were conducted across the 1990s. Some studies were conducted in several areas of one country, whereas others (CESAR and the 24-cities study) were conducted in more than one country.

The combined analyses used original data including measures of lung function, and questionnaire information on a wide range of potential risk factors including sources of indoor air pollution, socio-economic status, family medical history etc.

Definition of parental education status

Parental education was defined as the maximum of the mother"s and father"s educational level and classified into three categories (low, medium, and high) as described previously [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF].

This classification was done at country level, taking frequency distributions into account to make sure that every country had a sufficiently large number of subjects with low and high parental education, respectively [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF]. Nevertheless, for the present analyses the frequency of "low lung function" (see below) was found to be too small in low parental education category (as defined by Gehring et al. [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF]) in some countries and therefore, the low and medium parental education categories were joined and defined as "low". Only subjects with a defined parental education were included in the analysis.

Lung function outcomes

Lung function testing was performed according to the protocol of the American Thoracic Society [START_REF] Thoracic | Standardization of spirometry -1987 update[END_REF], except for the minimum exhalation time of 6 s (not feasible for children) and except for the Dutch study, which followed the protocol of the European Respiratory Society [START_REF]Standardized lung function testing. Official statement of the European Respiratory Society[END_REF].

We analyzed the following volume and flow parameters: forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), peak expiratory flow (PEF) and maximal mean expiratory flow (MMEF). MMEF data were not available from Germany.

In addition, "low lung function" was analyzed, specified by low FVC (defined as less than 85 % of predicted by age, gender, height, and weight from all study participants from the same country), low FEV1 (less than 85 % of predicted), low PEF (less than 75 % of predicted) and low MMEF (less than 75 % of predicted) [START_REF] Moshammer | Parental smoking and lung function in children: an international study[END_REF]. These cut-off levels were based on a priori clinical experience. Effect on poor lung function was analyzed by logistic regression.

Potential confounders

A set of a-priori confounders was prepared based on the literature. Confounders were divided into two groups: early life factors such as maternal smoking during pregnancy, premature birth (< 36 weeks), low birth weight (less than 2.3 kg), breastfeeding (less/more than 3 months), attendance of kindergarten, low respiratory tract infection (LRI) in first 2 years of life, and parental history of asthma; and other potential confounders like body mass index (BMI) [START_REF] Vidmar | Standardizing anthropometric measures in children and adolescents with new functions for egen[END_REF], the presence of any smoker in the household, pet ownership ever, overcrowding, presence of mould/moisture in the child"s home, use of gas for cooking or gas/kerosene/oil heater and infection prior to test.

Statistical analysis

Since lung growth during childhood is not linear, the lung function variables were logtransformed for calculation of predicted values, i.e. the linear (for continuous outcomes) regression models used the natural logarithm of each lung function variable as the dependent variable and the relationship with body parameters were described using the natural logarithm of age and weight including an interaction between sex and natural logarithm of height.

Data were analyzed in two stages. In stage one, country-specific estimates of effects of low parental education were estimated using linear regression for the continuous outcomes and logistic regression for the binary outcomes. The models included random effects at area level allowing for geographic clustering of outcomes.

In stage two, these country specific effect-estimates and their standard errors were pooled using meta-analytic methods, obtaining a mean estimate and a measure and Cochran 2 test of heterogeneity. Country-specific effects are assumed to follow a random distribution about a mean, and the estimation of this mean (and confidence interval) takes into account variations both within and between studies [START_REF] Dersimonian | Meta-analysis in clinical trials[END_REF].

All country-specific and pooled estimates were produced in three models -Model 1 controlled for age, sex, weight, height, and technician (technician who performed the lung function testing). In Model 2 early life factors were added; and in the third model the other potential confounders were considered (Table 1).

The magnitude of the effect of each confounder was tested separately. The final set of confounders was selected for all potential confounders that change odds ratios (OR) for any of the low lung function indicators by more than at least 10 % in one country (Table 1).

As indicated, not all potential confounding variables were available for all studies -sensitivity analyses were conducted to assess the impact of unavailability of covariates, by comparing results between studies with full set and restricted sets of confounders. As the US data represents the largest group further sensitivity analysis was performed on the restricted data set without American data.

Meta-regressions were used to assess whether the following study characteristics could explain heterogeneity between country-specific estimates: East/West differences based upon a hypothesis of different health status in Western countries and former Eastern block (Germany was coded Eastern country as the study was conducted in the Eastern part of Germany), response rate lower than 80 %; earlier studies (arbitrary stated cut-point 1995); proportion of young children (6-8 years versus 9-12 years); proportion of high parental education, and study design: whether the study was conducted within-towns, between-towns or a mixture.

All analyses were performed in STATA version 8.

RESULTS

The distribution of parental education, confounders and potential sources of heterogeneity across countries are summarized in Table 1. The prevalence of higher education differed between countries, ranging from 13.7 % in Germany to 67.1 % in North America.

Final set of confounders

The

Continuous lung function analyses

The association between continuous lung function parameters and low parental education was not consistent across the countries (Fig. 1). The results of the fully adjusted linear regression model varied between countries, ranging for FEV1 and FVC from changes of -1.09 % and -1.08 % respectively in Germany to changes of +1.43 % and +1.33 % respectively in Poland;

and MMEF changes ranged from -1.66 % in Slovakia to +1.70 % in Poland. The most consistent decrease was found for PEF, ranging from -2.80 % in the USA to -1.14 % in Hungary and the results were statistically significant in 5 out of 8 countries.

In the basic model low parental education was associated with a significant overall mean decrease of PEF (-2.2 %) and MMEF (-1.4 %) with no indication of heterogeneity between studies (Table 2). No association with FVC and a borderline significant association with FEV1 were found in Model I with evidence of heterogeneity (p<0.10) for both these outcomes -Table 2. After controlling for early life factors the effect of low parental education on lung function parameters slightly decreased except for PEF. Further adjustment for all other potential risk factors confirmed the only significant association between low education and PEF (-2.4 %) and still borderline significance with MMEF (-0.8 %).

Low lung function analyses

The overall distribution of low lung function varied modestly between countries. The mean distribution of low FEV1 reached 6.5 %, low FVC 6.1 %, low PEF 5.2 % and low MMEF 10.6 % across the countries (Table 3).

The mean odds ratios describing the association between parental education and prevalence of low lung function were statistically significant for FVC, PEF and MMEF and ranged from 1.18 for MMEF to 1.38 for PEF in the baseline Model I (Fig. 2). The associations with parental education persisted after additional adjustment for early life factors and other potential confounders, but were statistically significant only for PEF in Model III (Fig. 2).

Mean odds ratios for FEV1 were not statistically significant, with considerable betweencountry heterogeneity. The fully adjusted model gave slightly lower mean odds ratios for FVC and MMEF compared with the basic model. For PEF, where the largest adverse effects were seen, the mean OR changed only slightly after adjustment from 1.38 (95% CI 1.08-1.76), to 1.34 (95% CI 1.06-1.70). The results indicate that the association with parental education could not be fully explained by the additional studied risk factors.

Analysis of heterogeneity of results

Country-specific results tended to be heterogeneous. We explored this heterogeneity, focussing on the low lung function measures. The significant source of heterogeneity of the results was the differences between the Western and Eastern countries for FEV1, and differences between countries with higher (more than 80 %) and lower response-rate for PEF (Table 4).

The association between low FEV1 and low parental education appears to be confined to Western countries (OR=1.28; 95% CI 1.10-1.49), where the ORs varied from 1.20 (95% CI 1.01-1.43) in North America to 1.56 (95% CI 0.96-2.53) in the Netherlands (Table 4, Fig. 2).

The same East-West difference was not apparent for low FVC, MMEF or PEF.

All other study characteristics (proportion of young children, proportion of high parental education, and study design) could not explain heterogeneity between country-specific estimates.

Sensitivity analysis

The impact of missing confounding variables in some studies on the meta-analysis results was explored in sensitivity analyses. Study results for each of the health outcome were compared between groups of studies with the full availability of covariates, studies with 5 items missing (early life factors and overcrowding in Austria) and 3 items missing (breastfeeding, attendance of kindergarten and presence of pets in a household in North America). The sensitivity analyses indicated that the missing confounders in some studies did not significantly change the results.

Excluding the US data lead to broader confidence intervals, decreased % change of lung function parameters (FEV1, FVC, MMEF) and loss of heterogeneity of results for FEV1.

Indication of heterogeneity of results in the restricted data set has still been confirmed for FVC. Other results were found out regarding PEFthe point estimates for the percentage difference in fully adjusted models decreased from -2.4 % in the full dataset to -1.9 % in the restricted data set and heterogeneity decreased practically to zero. All the models for PEF (full data set, restricted data set and US data set itself) provided statistically significant results with narrow confidence intervals.

DISCUSSION

This study found positive associations between low lung function and low parental education.

Significant risk increases of low lung function parameters (PEF OR=1.38, FVC OR=1.28, and MMEF OR=1.18) were associated with low parental education before inclusion of potential risk factors. Odds ratios for low FVC and MMEF were reduced and became statistically nonsignificant after adjustment for other risk factors, but the risk of low PEF remained persistent after adjustment, decreasing only slightly to OR=1.34. The overall effect of SES over LF parameters was found. That only PEF remained significant is mostly due to the fact that PEF shows the smallest chance variation. So here the power to detect small effects is greatest.

Our study results support findings from other studies on socio-economic differences of lung function [START_REF] Prescott | Socioeconomic status, lung function and admission to hospital for COPD: results from the Copenhagen City Heart Study[END_REF][START_REF] Vedal | Risk factors for childhood respiratory disease. Analysis of pulmonary function[END_REF][START_REF] Demissie | Socioeconomic status and lung function among primary school children in Canada[END_REF] mostly being carried out in the Western countries and showed that the social gradient is less pronounced in Eastern European countries.

In a previous analysis of the PATY data [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF], adverse effects of low parental education were observed on prevalences of wheeze and nocturnal dry cough. The association with wheeze was mainly restricted to children from Eastern Europe, and the authors commented that different distributions of atopy and different patterns of link between wheeze, atopy, and SES in Eastern and Western countries might have played a role [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF]. The East-West effect modification for lung function, reported in the current paper, is in the opposite direction, with higher effects of low education occurring in the Western countries. We speculate that this may be partly due to a more pronounced link in Western countries between individual-level socioeconomic status and area-level deprivation, with the latteracting perhaps via factors such as housing quality, ambient air pollution, etceterahaving its own adverse effects on lung function [START_REF] Shohaimi | Area deprivation predicts lung function independently of education and social class[END_REF].

Explanation of associations with education by other risk factors

The study results might be conservative. Inclusion of other risk factors may result in overadjustment as part of the behaviours, e.g. smoking, or living conditions, e.g. mould in the home, may be in the causal chain from socio-economic conditions and health.

Low respiratory tract infection in early childhood had been hypothesized to be one of the risk factors [START_REF] Lercher | Birth weight, education, environment, and lung function at school age: a community study in an alpine area[END_REF], but in our multi-country study controlling for this risk factor changed odds ratios only slightly. The impact of early life factors on lung function data in our study confirmed the results of previous studiesmaternal smoking during pregnancy [START_REF] Moshammer | Parental smoking and lung function in children: an international study[END_REF], day-care attendance [START_REF] Demissie | The role of domestic factors and day-care attendance on lung function of primary school children[END_REF], and low-birth weight and premature birth [START_REF] Lercher | Birth weight, education, environment, and lung function at school age: a community study in an alpine area[END_REF].

Passive smoking is one of the most important confirmed risk factor of impaired lung function [START_REF] Moshammer | Parental smoking and lung function in children: an international study[END_REF][START_REF] Forastiere | Passive smoking as a determinant of bronchial responsiveness in children[END_REF][START_REF] Ware | Passive smoking, gas cooking, and respiratory health of children living in six cities[END_REF]. This was again confirmed in this study.

Brunekreef et al. [START_REF] Brunekreef | Home dampness and respiratory morbidity in children[END_REF] reported dampness and moulds to be a strong predictor of impaired lung function. But in our study mould/moisture did not relevantly affect the relationship between education and health outcomes.

Some studies reported SES differences in outdoor/indoor air pollution [START_REF] Heinrich | Social inequality and environmentally-related diseases in Germany: review of empirical results[END_REF][START_REF] Eroshina | Environmental and social factors as determinants of respiratory dysfunction in junior schoolchildren in Moscow[END_REF][START_REF] Prescott | Socioeconomic status and chronic obstructive pulmonary disease[END_REF][START_REF] Zeka | The effects of socioeconomic status and indices of physical environment on reduced birth weight and preterm births in Eastern Massachusetts[END_REF]. However adverse effects on lung function are reversible with the improvement of air quality [START_REF] Neuberger | Declining ambient air pollution and lung function improvement in Austrian children[END_REF][START_REF] Frye | Association of lung function with declining ambient air pollution[END_REF]. In spite of these reported class-specific differences of indoor exposure, the impact of gas use for cooking or use of unvented gas/kerosene/oil (water) heater had only a weak impact on relationships being investigated in our study.

Some studies reported racial differences to be a cause of different lung function outcomes [START_REF] Cunningham | Racial differences in the association between maternal smoking during pregnancy and lung function in children[END_REF][START_REF] Harik-Khan | Racial difference in lung function in African-American and White children: effect of anthropometric, socioeconomic, nutritional, and environmental factors[END_REF]. The information on race of children was not available in most of component studies.

For the Austrian, the Netherlands and the US datasets the information on nationality/race was available, but it concerned only minorities representing around 3.5 % of the study sample in the US and the Netherlands. In the Austrian data nationality was based on "child"s language" and minorities varied from 2.3 -8.4 %. To avoid confounding of effect estimates caused by genetic aspects relating with the different race (in the US data) and ethnic differences probably interacting both with SES and culturally determined life-style only data on major population was analysed.

No comparative data on physical activity was available. Instead BMI was included into a set of potential confounders as the association between lung function parameters and overweight in children was confirmed by several studies [START_REF] Chu | Extreme BMI predicts higher asthma prevalence and is associated with lung function impairment in school-aged children[END_REF][START_REF] Spathopoulos | The effect of obesity on pulmonary lung function of school aged children in Greece[END_REF][START_REF] Lazarus | Effects of body fat on ventilatory function in children and adolescents: cross-sectional findings from a random population sample of school children[END_REF].

We have to acknowledge that our variable "low education" may not be fully equivalent across all countries, and that quantitative results from pooling country-specific effect estimates may be seen as indicative rather than as numeric absolutes. Gehring et al., reporting on parental education and respiratory symptoms in the PATY study [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF], expressed a confidence that parental education really reflected the children's SES, based on consistency of the association between parental education and crowding. Furthermore, our analyses of heterogeneity did not find differences of results between countries with different proportions of high parental education. However, the country-specific effect estimates themselves and the patterns of association seen across them, remain important results, besides the mean estimates presented.

It is worthy to notice that education as previously defined (Gehring et al.) ( 17) is a proxi of SES for a broader range of possible risk factors for low lung function that can be modified, including quality of housing, poor sanitation, air pollution in a living area, ETS exposure, diet, physical activities, access to health care etc. Even when a range of these risk factors that are linked to SES (mother smoking in pregnancy, premature birth/low birth weight, breastfeeding, attending kindergarten, presence of current smoker in a household, pet ownership ever and overcrowding) are included in the model there remains an effect of low parental education that is not explained by these specific risk factors. That proxy SES measures, such as low education, can explain more variation in ill-health than the sum of the known individual risk factors, illustrates precisely the strength of SES as a concept of health relevance.

East-West differences

Analysis of heterogeneity confirmed East-West differences in study results for low FEV1 and low FVC and differences between groups of countries according the response-rate (below/above 80 %) for low PEF. Considering that higher response rates were seen in the US, Austria, and German studies, and East-West and response-rate differences greatly overlapped, it can be assumed that the differences are caused more likely by East-West than response-rate differences.

East-West differences were also found in analyses of wheeze and low parental education in the earlier paper [START_REF] Gehring | Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study[END_REF]. The opposite East-West pattern was seen in the present analysishigher risks of low FEV1 and low FVC were seen in children from less educated families in the Western countries. The observed East-West differences in relationship between low education and lung function might be ascribed to differences in factual status [START_REF] Hauser | Socioeconomic indexes for occupations: A review, Update, and Critique[END_REF][START_REF] Ganzeboom | Three Internationally Standardised Measures for Comparative Research on Occupational Status[END_REF] regarding occupational class, material resources, life-style and behavior patterns, with education in Eastern countries not necessarily a good indicator of affluence due to low consistency of socio-economic status. This speculation is supported by Davey-Smith et al.

[40] who argued that material resources related to occupational social class rather than culturally based sources related to education should be key determinants of socio-economic differentials. However, information on occupational social class of parents was not available for most studies.

Although the data were acquired in the period from 1988 to 1998, the authors are confident of its relevance. Such large datasets, especially for the US population have not been collected since that period. The East-West differences in social health inequalities might have changed since that time. Education is having a much more important role in socio-economic status in the Eastern countries. But the argument of material conditions as key determinant of socioeconomic health differences remains, and this still persists between Western and Eastern countries.

CONCLUSIONS

The results of the study confirmed a higher risk of low lung function in children from less educated families. The associations with parental education were more pronounced in the Western countries, while in the Eastern countries the direction of the association was not consistent, which might be explained by the low consistency of socio-economic status in the former socialist countries. Although social gradients were attenuated after adjusting for several known risk factors, these risk factors could not completely explain the social gradient in lung function.

Figure 1 2

 12 FIGURES

Table 2 .

 2 The mean percentage change of lung function parameters in association with low parental education for each outcome from the basic (adjusted for age, sex, height, weight and technician), early life factors and all confounder adjusted models. P-values for evidence of heterogeneity between study-specific results.

		Model I	Model II	Model III
		Basic model	Early life factors adjusted	Confounder adjusted
		change %	change % (95% CI)	change % (95% CI)
		(95% CI)
	FEV1
	Estimate (Random)	-0.4 (-1.0; 0.1)	-0.3 (-1.0; 0.3)	-0.4 (-0.9; 0.2)
	p-Heterogeneity	0.061	0.054	0.112
	FVC
	Estimate (Random)	-0.1 (-0.7; 0.5)	-0.1 (-0.9; 0.6)	-0.2 (-0.8; 0.4)
	p-Heterogeneity	0.013	0.017	0.028
	PEF
	Estimate (Random)	-2.2 (-2.7; -1.7)	-2.2 (-2.7; -1.6)	-2.4 (-2.9; -1.8)
	p-Heterogeneity	0.600	0.915	0.688
	MMEF
	Estimate (Random)	-1.4 (-2.2; -0.6)	-1.1 (-2.0; -0.2)	-0.8 (-1.6; 0.04)
	p-Heterogeneity	0.862	0.835	0.604

Table 3 .

 3 Description of distribution of low lung function accross the countries

	Abbreviation	POL HUN SLO CZE NL GER A	US All
	low FEV1* (N)	42	66	50	30 102 103 152 802 1 347
	% of study sample	6.8 5.3 5.2 3.7 6.3 6.5 6.4 7.0 6.5
	low FEV1 in high parental edu (%)	11.0 5.9 6.0 2.9 5.0 4.6 4.9 6.7 6.3
	low FEV in low parental edu (%)	5.8 5.1 4.6 4.0 7.3 6.8 7.3 7.8 6.8
	low FVC* (N)	38	77	46	29 101 150 140 681 1 262
	% of study sample	6.4 6.4 4.7 3.6 6.2 8.6 5.9 6.0 6.1
	low FVC in high parental edu (%)	7.2 6.3 5.0 4.4 4.7 4.8 5.0 5.4 5.3
	low FVC in low parental edu (%)	6.2 6.4 4.6 3.3 7.4 9.3 6.4 7.2 6.9
	low PEF* (N)	20	28	59	32	75	74 127 669 1 084
	% of study sample	3.4 2.3 6.1 4.0 4.6 4.3 4.0 5.9 5.2
	low PEF in high parental edu (%)	1.7 2.7 6.5 2.9 4.4 1.7 3.8 4.8 4.6
	low PEF in low parental edu (%)	3.8 2.2 5.8 4.3 4.8 4.7 6.3 8.1 5.9
	low MMEF* (N)	60 104	92	81 166	-276 1240 2 019
	% of study sample	10.1 8.6 9.5 10.0 10.2	-11.6 10.9 10.6
	low MMEF in high parental edu (%)	11.7 6.0 9.0 9.3 8.8	-10.4 10.4 10.1
	low MMEF in low parental edu (%)	9.8 9.6 9.8 10.3 11.3	-12.3 11.9 11.3

* Low lung function is measured lung function less than 85% (FVC, FEV1) or 75% (PEF, MMEF) of predicted lung function based upon age, sex, height and weight.

Table 4 .

 4 Sources of heterogeneity of results -comparison of mean odds ratios for the association between low lung function and low parental education across subgroups of countries. Only factors significantly associated with the size of the effect estimate are presented. * East=Eastern countries /Poland, Hungary, Slovak Republic, Czech Republic, Germany/ West=Western countries /Netherlands, Austria, US/ ** Response rate <80% /Poland, Hungary, Slovak Republic, Czech Republic, Netherlands/ Response rate ≥80% /Germany, Austria, US/

		No. of studies	Mean odds ratio (95% CI)	p-value
	FEV1 low
	East*	5	0.86 (0.56 -1.31)
	West	3	1.28 (1.10 -1.49)	0.040
	PEF low
	Response rate ≥80%**	3	1.56 (1.32 -1.84)
	Response rate <80%	5	1.01 (0.73 -1.40)	0.020

ACKNOWLEDGEMENTS

The PATY study was funded by the EU 5 th Framework Quality of Life Programme, contract Nr. QLRT-2001-02544. The authors would like to thank the scientists and technical staff involved in the original studies.

ETHICS COMMITTEE APPROVAL

The paper combines the data from the 8 previous cross-sectional studies -all of them were approved by the Ethics Committees.