Isabelle ; Fabry

Heymans

Van Bortel

e w 2 hemodynamic effects of tocolytic drugs have never been studied before. In this study, the acute effects of therapeutic doses of ritodrine and atosiban were evaluated on central and peripheral blood pressure, central-to-peripheral blood pressure amplification and pulse wave reflections in healthy non-pregnant female volunteers.

Methods

Subjects

Twenty healthy non-pregnant female volunteers, either non-smokers or smokers (≤10 cigarettes per day) with adequate non-uteral contraception were recruited from the local population. All participants gave written informed consent upon screening, which was organised within two weeks before the planned first drug administration. They were apparently healthy (no cardiovascular diseases -including arrhythmias, obstructive lung diseases, chronic kidney diseases or diabetes mellitus). Breastfeeding women or women with a severe addiction were excluded.

Subjects were asked not to eat, smoke and drink caffeine-containing beverages for at least 3 hours before and during the measurements. They also had to refrain from drinking alcohol for at least 10 hours before measurements. [16] Design

Introduction

The importance of tocolysis has been discussed extensively. [1][2][3] Beta-2 adrenoceptor agonistic drugs like ritodrine have been the reference tocolytic drugs in most countries. [3,4] Their efficacy in prolonging pregnancy compared to placebo is proven although no benefit in neonatal morbidity or mortality has been demonstrated. [5] Beta-mimetics are not highly selective and have many contraindications. Side-effects are frequent due to beta-1 and -2 adrenoceptor agonistic cardiovascular effects. Even serious complications such as pulmonary oedema and maternal death, though rare, have been reported. [5] Atosiban, a newer tocolytic drug, is a competitive antagonist of oxytocin at uterine oxytocin receptors and has less cardiovascular side effects. [6] The 13 th revision (08 July 2009) of the European Public Assessment Report (EPAR) on Tractocile® (atosiban) of the EMEA (European Medicines Agency) states that atosiban has the same effect as beta-mimetics on surrogate endpoints related to tocolysis. A better tolerability was observed, but there was no advantage in improving foetal and maternal outcome in comparison with beta-mimetics. This benefit of safety with atosiban has to be balanced against its cost. [7,8] A study of Ferriols et al. [9] revealed that the cost-effectiveness obtained with a protocol including ritodrine as firstchoice drug was three times less than when atosiban was used. In pregnant women with high risk of developing acute pulmonary oedema, or cardiovascular disease (e.g. preeclampsia, cardiomyopathies and cardiovascular syndromes), atosiban may be an appropriate alternative.

Although large studies using atosiban have been performed in both pregnant and nonpregnant groups [5,8,[10][11][12], there is mainly a subjective reporting of adverse reactions during infusion with a focus on peripheral blood pressure data.

Central blood pressure and pulse wave reflections may provide additional information regarding cardiovascular risk beyond peripheral blood pressure. [13][14][15] The central 3 (PrePar®, Eumedica, Belgium) in a single-blind way. The effects of drugs were compared after 95 minutes of infusion when kinetic steady state was reached for atosiban and ritodrine, being 15 minutes after starting the highest dose (400µg/min) of ritodrine. Hemodynamic measurements were done by one investigator with the subjects in supine position and under standardized conditions (derived from the Task Force III, clinical applications for arterial stiffness [16]) in a temperature controlled room (22 ± 1 °C).

Medication

Medication and placebo were infused for 120 minutes at the left arm. Both atosiban and ritodrine were given with glucose 5% as vehiculum. Atosiban was given at a constant dose of 300µg/min at a constant infusion rate of 0.4ml/min. Ritodrine was given in a dose escalation scheme starting with a dose of 100µg/min gradually upgraded to a dose of 400µg/min. The infusion rate varied with each dosing step (from 0.23 to 0.53ml/min). Glucose 5% was given as placebo at a constant infusion rate of 0.4ml/min. Dosing was based on previous studies using atosiban or ritodrine [3,[17][18][19][20] and on the manufacturers prescriptions. Each period, a total volume of 48ml was infused.

Stopping criteria for dosing were: a heart rate increase above 75% of the age-based maximal heart rate or blood pressure changes from baseline of more than 30 mmHg for systolic (SBP) and 15 mmHg for diastolic blood pressure (DBP) or a SBP above 180 mmHg or less than 90 mmHg and DBP more than 110 mmHg. When the subject suffered intolerable side effects, the infusion was also ended. Healthcare, Hoofddorp, The Netherlands). [21] Blood pressure and HR were recorded predose and at kinetic steady state. Mean arterial pressure (MAP) was calculated by adding 40% of the pulse pressure (PP) to the measured DBP. [22] Applanation tonometry Radial (RA) and carotid (CCA) pressure waveforms (PWFs) were recorded non-invasively with a Sphygmocor® applanation tonometry system (AtCor Medical, Sydney, Australia). [23] To obtain local blood pressure at the CCA and RA, calibration of the recorded PWFs is required. Calibration is based on the validated assumption that DBP and MAP remain constant throughout the large arteries, while SBP and PP (the difference between SBP and DBP) change. [24] SBP at an arterial site (SBP X ) is SBP X =DBP+PP X . Using our calibration method, PP at an arterial site (PP X ) can be calculated from PP at the brachial artery (PP BA ) as PP X =PP BA x FF BA /FF X [25] FF BA and FF X are the form factors (FF) at the brachial artery and the target artery, respectively, and are defined as: [26] FF X =(MAP-DBP)/PP X Brachial FF was calculated as FF BA =FF RA +0.625(FF CCA -FF RA ). This formula estimates FF BA from carotid (FF CCA ) and radial FF (FF RA ) assuming the ratio (FF BA -FF RA )/(FF CCA -FF RA ) to be fixed. This ratio was calculated in an age matched population (265 subjects (aged 19-40 years) from the Asklepios database [27] and from unpublished data).

Measurements

Sphygmomanometry

Semi

Augmentation index

Carotid augmentation index (AIx) was calculated from the carotid pressure waveform as the ratio of the amplitude of the pressure wave above its systolic shoulder to the total pulse pressure. [28] Carotid AIx is a surrogate for aortic AIx and is considered to be an index of [29] Since the AIx has an inverse, linear relationship with the HR, the AIx was corrected for HR. [30]

Data analysis

The median of 3 measurements was used for data-analysis. Statistics were done using SPSS version 15 (SPSS Inc., Chicago, United States).

A non-parametric Friedman test was run on data of the 8 subjects who also received ritodrine to compare for repeated observations on the same subject. If statistically significant, this was followed by a Wilcoxon signed rank test for 2-point comparison. Comparison between atosiban and placebo was done on all subjects using the Wilcoxon signed rank test.

Demographic differences between study groups were analysed using the non-parametric Mann-Whitney U test.

Results

Demographic data

At study entry, the subgroup of 8 subjects also receiving ritodrine did not differ statistically from the whole group (n=20). All subjects received the total amount of the planned dose, which was 300µg/min for atosiban and 400µg/min as the highest dose for ritodrine.

Central and peripheral blood pressure, and augmentation index

Data of blood pressure and augmentation index are shown in Table 2. With ritodrine, central SBP (at the CCA) increased with 13% versus placebo (p = 0.012) and peripheral SBP (at the BA) increased with 12% (p = 0.004). But SBP at the radial artery did not differ statistically from placebo. Compared to placebo, the DBP and MAP in the ritodrine group decreased with 19% (p = 0.008) and 8% (p = 0.044), respectively. With atosiban, central and peripheral pressures did not differ from placebo. With atosiban and placebo the systolic pressure rises from the central CCA towards the peripheral RA. Although not statistically significant (p = 0.180) from placebo and atosiban, with ritodrine there was a substantial loss of this systolic pressure amplification.

With ritodrine, the non-corrected AIx decreased (497%; p= 0.005) and the AIx corrected for heart rate (AIx@HR75) tended to decrease (289%; p= 0.225), while AIx did not change with atosiban.

Discussion

To the best of our knowledge this is the first placebo-controlled, randomized study investigating the effects of ritodrine and atosiban on the central blood pressure, pressure wave reflection and central-to-peripheral pressure amplification. [31][32][33][34] The results from this study show that ritodrine has important and significant effects on the cardiovascular system, whereas atosiban shows no significant effects on the parameters which we investigated.

Sphygmomanometer blood pressure

Similar effects have previously been reported. In studies with pregnant women, ritodrine caused a decrease in DBP (a drop of 4 and 7 mmHg from beginning to the end of the treatment [3,7,31]). In studies with other beta-receptor agonists, DBP and MAP were reduced because of peripheral vasodilatation. [35][36][37] For atosiban, no significant fall in DBP occurred, which is consistent with earlier work. [3,32,38,39]

Tonometry data

In a general population the best estimate of the brachial form factor is 0.4. [22] However this form factor can depend on different factors like age. [40] We also observed that medication can change substantially the form factor at different arterial sites (RA: 33.4 during placebo - present study the FF BA of each measurement was estimated using a procedure that expresses the FF BA as a (fixed) ratio of FF CCA and FF RA , circumventing the lack of brachial tonometry recordings.

The SBP CCA during ritodrine was higher and AIx was lower in comparison with those during atosiban and placebo. No other data on the effect of these tocolytic drugs on central SBP or AIx are available in the literature.

AIx has been proposed as an index of early wave reflections at the central arteries. Heart rate is a well-known determinant of AIx. But also after adjustment to a heart rate of 75 beats per minute (bpm) AIx tended to be lower during ritodrine than during atosiban or placebo, suggesting that ritodrine reduces wave reflections. As pulse wave reflections occur at changes in impedance (like at arterial branches), the vasodilation with ritodrine may account for the decrease in arterial impedance and reduction in wave reflections. Studies [35,37,41] investigating effects of salbutamol (another beta-receptor agonist) on arterial stiffness, revealed similar significant effects on AIx due to endothelial nitric oxide synthase (NOS)dependent vascular relaxation.

In the present study, systolic pressure measured by applanation tonometry at the CCA was used as a non-invasive surrogate for cSBP. [25] The central blood pressure (cBP) may have a higher predictive value for cardiovascular events compared to traditional sphygmomanometer measurements since it is an important determinant of the left ventricular workload and coronary blood flow. [14] In contrast to peripheral blood pressure, early pulse wave reflections (PWR) can boost the cSBP reflected by a positive AIx. Early PWR is being recognized as an important factor that contributes to the increase in central blood pressure. [42] As AIx was negative with ritodrine, early wave reflections cannot contribute to the increase in cSBP during ritodrine, which is due to the beta-agonistic positive inotropic effect witnessed by the increase in heart rate. During ritodrine, there was also a rise in SBP at both the levels of the BA and the RA. This is not in accordance with other studies reporting a decrease in sphygmomanometrically taken SBP. [3,7,31] This difference can be explained by the fact that the subjects in those studies were pregnant. Pregnant women in premature labour have an already higher baseline SBP in comparison with the non-pregnant group. It may not be excluded that in those studies with pregnant women, the anxiety for premature birth and the hospital setting artificially boosted SBP at the start of treatment. Finally, an altered effect of ritodrine in pregnant women cannot be fully excluded. [43,44] The effect of atosiban on the SBP, at the levels of the CCA, BA or RA in our study were not significantly different from placebo which is in accordance with other studies. [3,34] Blood pressure amplification Mean and diastolic blood pressure hardly change in the large artery tree. [24,25] In contrast, when early wave reflections are absent like in young subjects, a clear amplification of the systolic blood pressure from the heart to the periphery is present. This explains why blood pressure measured at the brachial artery may overestimate the pressure seen by the heart. [45] This pressure amplification is due to a gradual decrease in arterial compliance (C) from central to peripheral arteries [46] and can be influenced by pulse wave reflections. [47] For ritodrine, in contrast with atosiban and placebo, pressure amplification between the CCA and the upper limb is nearly absent (with a difference of only 2 mmHg between the CCA and the RA). This loss of pressure amplification with ritodrine can be explained by an increased arterial compliance, due to beta 2 mediated vasodilatation. [48]

Study limitations and clinical implications

The present study was carried out in non pregnant women. It is not clear whether the present results in non pregnant women can be fully extrapolated to pregnant women. Physiologic changes during pregnancy like an increase in cardiac output, a decrease in peripheral resistance [49,50] and modulation of oxytocin receptors during pregnancy [51,52] may alter the magnitude of the pharmacodynamic effects. On the other hand, pain and stress during premature labour may also confound the effects of tocolytic drugs. To elucidate these issues, this study would ideally be performed in late pregnant women without signs of premature labour. But this may be difficult because of ethical issues.

Ritodrine has important but contradictory effects on the cardiovascular system. In the UK, the Royal College of Obstetricians and Gynaecologists' guidelines on beta-mimetics recommend close monitoring of maternal pulse and blood pressure every 15 minutes during the infusion in every patient receiving a β-agonist as tocolyticum. [7] The fall in DBP, MAP and AIx due to the relaxation of the vascular smooth muscle cells, may appear an advantage in cases where the peripheral resistance is raised, like in gestational hypertension or pre-eclampsia. However, in hypertensive pregnancies, the circulating components of the RAAS (Renin-Angiotensin-Aldosteron system) are decreased. [53] On the contrary, beta-agonists like ritodrine may trigger the RAAS with enhanced renal sodium absorption resulting in vascular overfilling with increased risk of pulmonary oedema and acute heart failure. [54,55] In pregnant women with cardiovascular complications like gestational hypertension, preeclampsia and cardiomyopathies or in pregnant women with a history of cardiovascular complications during pregnancy, ritodrine may not be the first-line agent to control premature labour. Atosiban is then one of the alternatives.

In conclusion, the present study shows that the effects of ritodrine on blood pressure, blood pressure amplification and pulse wave reflections are significantly different from atosiban and placebo. The present study clearly demonstrates the absence of those hemodynamic effects during atosiban infusion.

Introduction

The importance of tocolysis has been discussed extensively. [1][2][3] Beta-2 adrenoceptor agonistic drugs like ritodrine have been the reference tocolytic drugs in most countries. [3,4] Their efficacy in prolonging pregnancy compared to placebo is proven although no benefit in neonatal morbidity or mortality has been demonstrated. [5] Beta-mimetics are not highly selective and have many contraindications. Side-effects are frequent due to beta-1 and -2 adrenoceptor agonistic cardiovascular effects. Even serious complications such as pulmonary oedema and maternal death, though rare, have been reported. [5] Atosiban, a newer tocolytic drug, is a competitive antagonist of oxytocin at uterine oxytocin receptors and has less cardiovascular side effects. [6] The 13 th revision (08 July 2009) of the European Public Assessment Report (EPAR) on Tractocile® (atosiban) of the EMEA (European Medicines Agency) states that atosiban has the same effect as beta-mimetics on surrogate endpoints related to tocolysis. A better tolerability was observed, but there was no advantage in improving foetal and maternal outcome in comparison with beta-mimetics. This benefit of safety with atosiban has to be balanced against its cost.[7,8] A study of Ferriols et al. [9] revealed that the cost-effectiveness obtained with a protocol including ritodrine as firstchoice drug was three times less than when atosiban was used. In pregnant women with high risk of developing acute pulmonary oedema, or cardiovascular disease (e.g. preeclampsia, cardiomyopathies and cardiovascular syndromes), atosiban may be an appropriate alternative.

Although large studies using atosiban have been performed in both pregnant and nonpregnant groups [5,8,[10][11][12], there is mainly a subjective reporting of adverse reactions during infusion with a focus on peripheral blood pressure data.

Central blood pressure and pulse wave reflections may provide additional information regarding cardiovascular risk beyond peripheral blood pressure. [13][14][15] The central

Methods

Subjects

Twenty healthy non-pregnant female volunteers, either non-smokers or smokers (≤10 cigarettes per day) with adequate non-uteral contraception were recruited from the local population. All participants gave written informed consent upon screening, which was organised within two weeks before the planned first drug administration. They were apparently healthy (no cardiovascular diseases -including arrhythmias, obstructive lung diseases, chronic kidney diseases or diabetes mellitus). Breastfeeding women or women with a severe addiction were excluded.

Subjects were asked not to eat, smoke and drink caffeine-containing beverages for at least 3 hours before and during the measurements. They also had to refrain from drinking alcohol for at least 10 hours before measurements. standardized conditions (derived from the Task Force III, clinical applications for arterial stiffness [16]) in a temperature controlled room (22 ± 1 °C).

Medication

Medication and placebo were infused for 120 minutes at the left arm. Both atosiban and ritodrine were given with glucose 5% as vehiculum. Atosiban was given at a constant dose of 300µg/min at a constant infusion rate of 0.4ml/min. Ritodrine was given in a dose escalation scheme starting with a dose of 100µg/min gradually upgraded to a dose of 400µg/min. The infusion rate varied with each dosing step (from 0.23 to 0.53ml/min). Glucose 5% was given as placebo at a constant infusion rate of 0.4ml/min. Dosing was based on previous studies using atosiban or ritodrine [3,[17][18][19][20] and on the manufacturers prescriptions. Each period, a total volume of 48ml was infused.

Stopping criteria for dosing were: a heart rate increase above 75% of the age-based maximal heart rate or blood pressure changes from baseline of more than 30 mmHg for systolic (SBP) and 15 mmHg for diastolic blood pressure (DBP) or a SBP above 180 mmHg or less than 90 mmHg and DBP more than 110 mmHg. When the subject suffered intolerable side effects, the infusion was also ended.

Measurements

Applanation tonometry

Radial (RA) and carotid (CCA) pressure waveforms (PWFs) were recorded non-invasively with a Sphygmocor® applanation tonometry system (AtCor Medical, Sydney, Australia). [23] To obtain local blood pressure at the CCA and RA, calibration of the recorded PWFs is required. Calibration is based on the validated assumption that DBP and MAP remain constant throughout the large arteries, while SBP and PP (the difference between SBP and DBP) change. [24] SBP at an arterial site (SBP X ) is SBP X =DBP+PP X . Using our calibration method, PP at an arterial site (PP X ) can be calculated from PP at the brachial artery (PP BA ) as PP X =PP BA x FF BA /FF X [25] FF BA and FF X are the form factors (FF) at the brachial artery and the target artery, respectively, and are defined as: [26] FF X =(MAP-DBP)/PP X Brachial FF was calculated as FF BA =FF RA +0.625(FF CCA -FF RA ). This formula estimates FF BA from carotid (FF CCA ) and radial FF (FF RA ) assuming the ratio (FF BA -FF RA )/(FF CCA -FF RA ) to be fixed. This ratio was calculated in an age matched population (265 subjects (aged 19-40 years) from the Asklepios database [27] and from unpublished data).

Augmentation index

Carotid augmentation index (AIx) was calculated from the carotid pressure waveform as the ratio of the amplitude of the pressure wave above its systolic shoulder to the total pulse pressure. [28] Carotid AIx is a surrogate for aortic AIx and is considered to be an index of pressure wave reflection (PWR). [29] Since the AIx has an inverse, linear relationship with the HR, the AIx was corrected for HR. [30] Data analysis

The median of 3 measurements was used for data-analysis. Statistics were done using SPSS version 15 (SPSS Inc., Chicago, United States).

A non-parametric Friedman test was run on data of the 8 subjects who also received ritodrine to compare for repeated observations on the same subject. If statistically significant, this was followed by a Wilcoxon signed rank test for 2-point comparison. Comparison between atosiban and placebo was done on all subjects using the Wilcoxon signed rank test.

Demographic differences between study groups were analysed using the non-parametric Mann-Whitney U test.

Results

Demographic data

At study entry, the subgroup of 8 subjects also receiving ritodrine did not differ statistically from the whole group (n=20). All subjects received the total amount of the planned dose, which was 300µg/min for atosiban and 400µg/min as the highest dose for ritodrine.

Central and peripheral blood pressure, and augmentation index

Data of blood pressure and augmentation index are shown in Table 2. With ritodrine, central SBP (at the CCA) increased with 13% versus placebo (p = 0.012) and peripheral SBP (at the BA) increased with 12% (p = 0.004). But SBP at the radial artery did not differ statistically from placebo. Compared to placebo, the DBP and MAP in the ritodrine group decreased with 19% (p = 0.008) and 8% (p = 0.044), respectively. With atosiban, central and peripheral pressures did not differ from placebo. With atosiban and placebo the systolic pressure rises from the central CCA towards the peripheral RA. Although not statistically significant (p = 0.180) from placebo and atosiban, with ritodrine there was a substantial loss of this systolic pressure amplification.

With ritodrine, the non-corrected AIx decreased (497%; p= 0.005) and the AIx corrected for heart rate (AIx@HR75) tended to decrease (289%; p= 0.225), while AIx did not change with atosiban.

Discussion

To the best of our knowledge this is the first placebo-controlled, randomized study investigating the effects of ritodrine and atosiban on the central blood pressure, pressure wave reflection and central-to-peripheral pressure amplification. [31][32][33][34] The results from this study

show that ritodrine has important and significant effects on the cardiovascular system, whereas atosiban shows no significant effects on the parameters which we investigated.

Sphygmomanometer blood pressure

Similar effects have previously been reported. In studies with pregnant women, ritodrine caused a decrease in DBP (a drop of 4 and 7 mmHg from beginning to the end of the treatment [3,7,31]). In studies with other beta-receptor agonists, DBP and MAP were reduced because of peripheral vasodilatation. [35][36][37] For atosiban, no significant fall in DBP occurred, which is consistent with earlier work. [3,32,38,39] Tonometry data

In a general population the best estimate of the brachial form factor is 0.4. [22] However this form factor can depend on different factors like age. [40] We also observed that medication can change substantially the form factor at different arterial sites (RA: 33. suggesting that ritodrine reduces wave reflections. As pulse wave reflections occur at changes in impedance (like at arterial branches), the vasodilation with ritodrine may account for the decrease in arterial impedance and reduction in wave reflections. Studies [35,37,41] investigating effects of salbutamol (another beta-receptor agonist) on arterial stiffness, revealed similar significant effects on AIx due to endothelial nitric oxide synthase (NOS)dependent vascular relaxation.

In the present study, systolic pressure measured by applanation tonometry at the CCA was used as a non-invasive surrogate for cSBP. [25] The central blood pressure (cBP) may have a higher predictive value for cardiovascular events compared to traditional sphygmomanometer measurements since it is an important determinant of the left ventricular workload and coronary blood flow. [14] In contrast to peripheral blood pressure, early pulse wave reflections (PWR) can boost the cSBP reflected by a positive AIx. Early PWR is being recognized as an important factor that contributes to the increase in central blood pressure. [42] As AIx was negative with ritodrine, early wave reflections cannot contribute to the increase in cSBP during ritodrine, which is due to the beta-agonistic positive inotropic effect witnessed by the increase in heart rate. During ritodrine, there was also a rise in SBP at both the levels of the BA and the RA. This is not in accordance with other studies reporting a decrease in sphygmomanometrically taken SBP. [3,7,31] This difference can be explained by the fact that the subjects in those studies were pregnant. Pregnant women in premature labour have an already higher baseline SBP in comparison with the non-pregnant group. It may not be excluded that in those studies with pregnant women, the anxiety for premature birth and the hospital setting artificially boosted SBP at the start of treatment. Finally, an altered effect of ritodrine in pregnant women cannot be fully excluded. [43,44] The effect of atosiban on the SBP, at the levels of the CCA, BA or RA in our study were not significantly different from placebo which is in accordance with other studies. [3,34] Blood pressure amplification Mean and diastolic blood pressure hardly change in the large artery tree. [24,25] In contrast, when early wave reflections are absent like in young subjects, a clear amplification of the systolic blood pressure from the heart to the periphery is present. This explains why blood pressure measured at the brachial artery may overestimate the pressure seen by the heart. [45] This pressure amplification is due to a gradual decrease in arterial compliance (C) from central to peripheral arteries [46] and can be influenced by pulse wave reflections. [47] For ritodrine, in contrast with atosiban and placebo, pressure amplification between the CCA and the upper limb is nearly absent (with a difference of only 2 mmHg between the CCA and the RA). This loss of pressure amplification with ritodrine can be explained by an increased arterial compliance, due to beta 2 mediated vasodilatation. [48]

Study limitations and clinical implications

The present study was carried out in non pregnant women. It is not clear whether the present results in non pregnant women can be fully extrapolated to pregnant women. Physiologic changes during pregnancy like an increase in cardiac output, a decrease in peripheral resistance [49,50] and modulation of oxytocin receptors during pregnancy [51,52] may alter the magnitude of the pharmacodynamic effects. On the other hand, pain and stress during premature labour may also confound the effects of tocolytic drugs. To elucidate these issues, this study would ideally be performed in late pregnant women without signs of premature labour. But this may be difficult because of ethical issues.

Ritodrine has important but contradictory effects on the cardiovascular system. In the UK, the Royal College of Obstetricians and Gynaecologists' guidelines on beta-mimetics recommend close monitoring of maternal pulse and blood pressure every 15 minutes during the infusion in every patient receiving a β-agonist as tocolyticum. [7] The fall in DBP, MAP and AIx due to the relaxation of the vascular smooth muscle cells, may appear an advantage in cases where the peripheral resistance is raised, like in gestational hypertension or pre-eclampsia. However, in hypertensive pregnancies, the circulating components of the RAAS (Renin-Angiotensin-Aldosteron system) are decreased. [53] On the contrary, beta-agonists like ritodrine may trigger the RAAS with enhanced renal sodium absorption resulting in vascular overfilling with increased risk of pulmonary oedema and acute heart failure. [54,55] In pregnant women with cardiovascular complications like gestational hypertension, preeclampsia and cardiomyopathies or in pregnant women with a history of cardiovascular complications during pregnancy, ritodrine may not be the first-line agent to control premature labour. Atosiban is then one of the alternatives.

In conclusion, the present study shows that the effects of ritodrine on blood pressure, blood pressure amplification and pulse wave reflections are significantly different from atosiban and placebo. The present study clearly demonstrates the absence of those hemodynamic effects during atosiban infusion.

F

  -recumbent brachial SBP and DBP and heart rate (HR) were recorded at the right upper arm with a validated semi-automated oscillometric device (OMRON 705IT, OMRON

  28.0 during ritodrine; CCA: 43.4 during placebo -28.7 during ritodrine). Therefore in the

  tocolytic drugs have never been studied before. In this study, the acute effects of therapeutic doses of ritodrine and atosiban were evaluated on central and peripheral blood pressure, central-to-peripheral blood pressure amplification and pulse wave reflections in healthy non-pregnant female volunteers.

[ 16 ] 3 (

 163 DesignA double-blind, randomised, crossover trial was carried out at the Drug Research Unit Ghent of the Ghent University Hospital, Belgium. The study was approved by the Ethics Committee of Ghent University and conducted according to ICH Good Clinical Practice and in compliance with the Declaration of Helsinki (last amended in 2008 in Seoul).Twenty female volunteers were given atosiban (Tractocile®, Ferring, Sweden) and placebo (Glucose 5%) intravenously. Eight of them were randomly chosen to also get ritodrine PrePar®, Eumedica, Belgium) in a single-blind way. The effects of drugs were compared after 95 minutes of infusion when kinetic steady state was reached for atosiban and ritodrine, being 15 minutes after starting the highest dose (400µg/min) of ritodrine. Hemodynamic measurements were done by one investigator with the subjects in supine position and under

Sphygmomanometry

  Semi-recumbent brachial SBP and DBP and heart rate (HR) were recorded at the right upper arm with a validated semi-automated oscillometric device (OMRON 705IT, OMRON , The Netherlands).[21] Blood pressure and HR were recorded predose and at kinetic steady state. Mean arterial pressure (MAP) was calculated by adding 40% of the pulse pressure (PP) to the measured DBP.[22]

Table 1 :

 1 Subjects' characteristics at study entry

	Parameters		Characteristics
		Double-blinded study part	Single-blinded study part	p-value*
		(n=20)	(n=8)
	Age (years)	25±7	25±11	0.862
	BMI (kg.m -2 )	21±3	22±4	0.980
	Height (m)	1.69±0.07	1.68±0.06	0.901
	Smoking n (%)	4 (20)	1 (12.5)	0.784
	SBP (mmHg)	101±7	101±9	0.826
	DBP (mmHg)	65±5	67±6	0.748
	HR (bpm)	58±8	58±9	0.901

Twenty non-pregnant, healthy female volunteers received atosiban and placebo in a double-blinded way whereas eight of them also received ritodrine in a single-blinded way; BMI (Body Mass Index); data of SBP (systolic BP), DBP (diastolic BP) and HR (heart rate) are median of tree measurements, bpm (beats per minute); *p-value is based on non-parametric Mann-Whitney U-test. All data are shown as mean ± standard deviation, except for smoking history.

Table 2 :

 2 Effects on the central and peripheral blood pressures and augmentation indices

	Parameter	Ritodrine (n=8)	Atosiban (n=20)	Placebo (n=20)	p-value $
	SBP CCA (mmHg)	115±12 *#	102±8	102±7	0.012
	SBP BA (mmHg)	116±12 *#	105±8	104±6	0.004
	SBP RA (mmHg)	117±14	110±10	110±6	0.180
	BP amplification	2.47±8.85	8.07±5.73	8.33±4.07	0.180
	DBP BA (mmHg)	55±11 *#	69±6	68±5	0.008
	MAP (mmHg)	78±10 *#	87 ±9	85±5	0.044
	HR (bpm)	111±20 *#	59±10	58 ±10	0.002
	AIx	-19.43±6.75 *#	4.89±5.04	4.89±5.80	0.005
	AIx@HR75	-8.67±12.30	5.58±16.78	4.58±13.33	0.225

SBP (systolic BP), BP-amplification (blood pressure difference from CCA to RA), DBP (diastolic BP), MAP (mean arterial pressure); HR (heart rate), bpm (beats per minute), AIx (augmentation index, not corrected for heart rate), AIx@HR75 (augmentation index at heart rate 75). $ Friedman-test on 8 subjects receiving all treatments; * p<0.005 vs. atosiban, # p<0.005 vs. placebo. All data are shown as mean ± standard deviation.

  FF BA of each measurement was estimated using a procedure that expresses the FF BA as a (fixed) ratio of FF CCA and FF RA , circumventing the lack of brachial tonometry recordings.The SBP CCA during ritodrine was higher and AIx was lower in comparison with those during atosiban and placebo. No other data on the effect of these tocolytic drugs on central SBP or AIx are available in the literature.AIx has been proposed as an index of early wave reflections at the central arteries. Heart rate is a well-known determinant of AIx. But also after adjustment to a heart rate of 75 beats per minute (bpm) AIx tended to be lower during ritodrine than during atosiban or placebo,

	present study the
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	4 during placebo -
	28.0 during ritodrine; CCA: 43.4 during placebo -28.7 during ritodrine). Therefore in the
	7

Table 1 :

 1 Subjects' characteristics at study entry Twenty non-pregnant, healthy female volunteers received atosiban and placebo in a double-blinded way whereas eight of them also received ritodrine in a single-blinded way; BMI (Body Mass Index); data of SBP (systolic BP), DBP (diastolic BP) and HR (heart rate) are median of tree measurements, bpm (beats per minute); *p-value is based on non-parametric Mann-Whitney U-test. All data are shown as mean ± standard deviation, except for smoking history.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 :

 2 Effects on the central and peripheral blood pressures and augmentation indices BP-amplification (blood pressure difference from CCA to RA), DBP (diastolic BP), MAP (mean arterial pressure); HR (heart rate), bpm (beats per minute), AIx (augmentation index, not corrected for heart rate), AIx@HR75 (augmentation index at heart rate 75). $ Friedman-test on 8 subjects receiving all treatments; * p<0.005 vs. atosiban, # p<0.005 vs. placebo. All data are shown as mean ± standard deviation.

	Parameter	Ritodrine (n=8)	Atosiban (n=20)	Placebo (n=20)	p-value $
	SBP CCA (mmHg)	115±12 *#	102±8	102±7	0.012
	SBP BA (mmHg)	116±12 *#	105±8	104±6	0.004
	SBP RA (mmHg)	117±14	110±10	110±6	0.180
	BP amplification	2.47±8.85	8.07±5.73	8.33±4.07	0.180
	DBP BA (mmHg)	55±11 *#	69±6	68±5	0.008
	MAP (mmHg)	78±10 *#	87 ±9	85±5	0.044
	HR (bpm)	111±20 *#	59±10	58 ±10	0.002
	AIx	-19.43±6.75 *#	4.89±5.04	4.89±5.80	0.005
	AIx@HR75	-8.67±12.30	5.58±16.78	4.58±13.33	0.225
	SBP (systolic BP),

European Journal of Clinical Pharmacology

Acknowledgements

The authors would like to thank all included subjects, Mr Dries Mahieu, Mrs Tine De Backer, Mrs Fabienne De Boeck, Mrs Griet Van Lancker, Mrs Charline Wehlou, Mr Marc Twagirumukiza, Mr Jan Kips for the blinded medication preparation and the staff of the Drug Research Unit Ghent for their excellent technical support and know-how.

Trial Number

EudractNr: 2008-000845-76

Acknowledgements

The authors would like to thank all included subjects, Mr Dries Mahieu, Mrs Tine De Backer, Mrs Fabienne De Boeck, Mrs Griet Van Lancker, Mrs Charline Wehlou, Mr Marc Twagirumukiza, Mr Jan Kips for the blinded medication preparation and the staff of the Drug Research Unit Ghent for their excellent technical support and know-how.

Trial Number

EudractNr: 2008-000845-76

A double-


Maria Cusato

Massimo Allegri

Tekla Niebel

Pablo Ingelmo

Monica Broglia

Antonio Braschi

Dr Mario Regazzi
email: regazzim@smatteo.pv.it

Flip-flop kinetics of ropivacaine

Introduction

Ropivacaine (RPV) is a long-acting amide-type local anesthetic with a similar structure to bupivacaine [START_REF] Mcclellan | Ropivacaine: an update of its use in regional anaesthesia[END_REF]. Many literature studies have demonstrated the clinical efficacy of ropivacaine 0.2% epidural infusion, with only some individual case reports of major side-effects. [2,3,4,5] Although ropivacaine is a safer alternative to bupivacaine, excessive plasma concentrations of ropivacaine can cause severe central nervous system and cardiac toxicity. After epidural administration, both the systemic absorption and the systemic disposition of ropivacaine play an important role in determining the clinical profile and the risk of systemic toxicity. [6,7,8,9] Ropivacaine calculated terminal half-life is significantly longer after epidural than after intravenous administration. Ropivacaine absorption after epidural administration cannot be derived directly from plasma concentration-time curves because of flip-flop kinetics. [9,10] The flip-flop model implies slow absorption of ropivacaine from the epidural space into the systemic circulation rate-limits drug elimination. Therefore, when the process of absorption is a limiting factor, the terminal half-life reflects the rate and the extent of absorption and not the elimination process. [10,11] In a system consisting of a series of processes, the decrease in concentrations after administration is discontinued and the time to reach the steady-state depends on the slowest process in the chain. This has implication for designing epidural continuous infusion schedules of ropivacaine, considering the clinical significance of accumulation reaching either toxic or ineffective concentrations.

The primary goal of this study was to evaluate the pharmacokinetics of ropivacaine accumulation in adults receiving continuous epidural analgesia during the first 48 h after major surgery.

Material and methods

After approval from the Ethics Committee of San Matteo Hospital (EudraCT number 2010-019393-32) and written informed consent 49 adult patients, ASA class I-II, scheduled for major abdominal or urological surgery and planned to receive a postoperative epidural infusion after surgery for pain control were enrolled. Exclusion criteria were emergency surgery, coagulopathy, renal, hepatic, neurological or psychiatric diseases, a history of peripheral neuropathies and chronic opioid therapy.

Before anesthesia induction epidural catheter was positioned in sitting position, using the loss resistance technique. In patients scheduled for upper abdominal surgery the epidural catheter was placed between T7-T8 or T9-T10. In patients scheduled for lower abdominal or urologic surgery between T10-T11. General anesthesia was induced with Propofol 2mg/kg, Fentanyl 2 µg/kg, Cisatracurium 0.15 mg/kg, and was maintained with Fentanyl and Sevoflurane 2%. Forty five minutes before the end of surgery patients received an epidural loading dose (RPV 0.2% 5 ml + Sufentanil 10 µg), followed by a second bolus (RPV 02% 5 ml) 15 minutes later. At the end of surgery an epidural infusion of Ropivacaine 0.2% plus Sufentanil 0.8 µg/ml was started.

Ropivacaine/Sulfentanyl epidural infusion was constantly condelivered at a rate of 5ml/h using an electronic infusion pump during the first 48 hs after surgery.

Patients were evaluated every 6 hours during the first day after surgery and every 12 hours in the second postoperative day. We recorded vital signs (arterial pressure and heart rate), pain according to the Numeric Rate Scale (at rest -NRS -and after movement -mNRS-), analgesic rescue dose (administered if NRS>4), Holmen scale (to assess if there was sensory block), sedation scale and clinical side-effects (nausea, vomiting, itching, hypotension, systemic neurological side effects)

Reagents

RPV was a gift of the Department of Anesthesia and Intensive Care. Phenacetin was purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used were of the highest commercially available quality. The HPLC system consisted of Varian Prostar Mod.430 a pump equipped with variable-wavelength UV detector (Varian Prostar Mod.330). Separation was achieved using a reverse-phase C-18 column (250×4.6 mm I.D., particle size 5µm, Hypersil BDS-CPS Analitica, Italy). The mobile phase was acetonitrile-methanol-50mM potassium dihydrogen phosphate (pH=6) (180:160:660, v/v/v) delivered at a flow rate of 1.3 mL/min. Chromatographic separation was performed at laboratory room temperature and monitored at 210nm.

The HPLC method has been validated over the range 0.1-4 µg/mL. The assay was linear over the entire concentration range (r 2 = 0.99). Intra-and inter-day precision and accuracy was less than 6%.

The limit of detection (LOD) was 0.050 µg/mL, the limit of quantification (LOQ) was 0.1 µg/mL.

Preparation of Standard solution and standard curves

A standard stock solution (1mg/mL) of drug was prepared in methanol-water (50/50), stored at -20°C and were stable for at least 6 months. Plasma standards and controls were prepared at concentrations of 0.1, 0.2, 0.4, 1.0, 2.0, 4.0 µg/mL and 0.12 (Low), 0.8 (Medium) and 1.6 (High) µg/mL, respectively, by diluting appropriate aliquots of the stock solution with drug-free serum. The calibration curve was obtained by linear regression of the peak-height ratios of ropivacaine to the internal standard and plotting these against the nominal concentration of the drug.

Blood sampling

Blood samples were obtained immediately before bolus drug administration and 3, 6, 12, 24, 48, 54 and 60 h after starting the infusion. Plasma was separated by centrifugation, transferred to clean prelabeled tubes and frozen at -20°C until analysis.

Sample preparation

250 µL of distilled deionized water,500 µL of dichloromethane and 10 µL phenacetin (10 µg/mL, I.S.) were added to 250 µL of plasma. After vortex mixing for 3 minutes, the tubes were centrifuged at 1200g for 10 min. The organic phase was transferred into a clean conical tube and evaporated to dryness under a gentle nitrogen stream. The residue was dissolved in 200 µL mobile phase and 50 µL was injected into HPLC.

Pharmacokinetic analysis

The concentration-time relationship of ropivacaine levels (corresponding to 6 data points) during an epidural infusion preceded by 2 sequential boluses was analyzed using a population pharmacokinetic method based on a mixed-effect-model approach.

This method separates explicitly the variability of drug concentrations in the population into two parts: interindividual variability characterized by the distribution of pharmacokinetic parameters in the population and residual variability (e.g., measurement errors). A parametric approach [12,13] When bioavailability (F) is not known, estimates of CL should be related to F, i.e., CL/F. Bioavailability was fixed to 1, assuming total (100%) absorption after epidural administration. Model evaluation was conducted as recommended in the P-PHARM manual and also by examination of a plot of measured/predicted concentration data versus time.

After the end of infusion, the terminal rate constant (λ ,terminal ) was determined for total ropivacaine by linear regression of the last 3 data points on the plasma concentration- µg/mL at 48 and 54 hours, respectively. Total ropivacaine concentrations up to 4.0 µg/mL were tolerated during long-term epidural ropivacaine infusion.

Figure 1 shows all the measured plasma ropivacaine concentrations and the drug concentration-time curve resulting from the average population parameters estimated with P-Pharm.

Estimated population characteristics of the kinetic parameters together for ropivacaine clearance (CL) and the apparent volume of distribution (Vd) with their coefficient of variation are presented in Table II.

The dosing period absorption/distribution/elimination rate constant of ropivacaine calculated by the average population parameters (λ DP = CL/Vd) was 0.064h -1 and the corresponding t 1/2,DP = (0.693*Vd)/CL = 10.8 hours.

Following the end of epidural infusion of ropivacaine, the terminal half-life (t 1/2,terminal ), determined for total ropivacaine by linear regression of the last 3 data points on the plasma concentration-time curve was 7.4 hours.

The pharmacokinetic population parameters were subsequently used to fit the concentration data up to 48 h using the Bayesian estimation method. This procedure enables the estimation of individual pharmacokinetic parameters for ropivacaine. The Through covariate analysis, we found that age was the only independent predictor identified.

In fact, the stepwise inclusion performed on P-PHARM revealed an age effect on CL, there are predictors that can determine a change in the pharmacokinetic profile.

which
In fact, plasma concentration profiles and the potential risk of systemic toxicity after epidural administration of ropivacaine depends on the administered dose and the interaction between the rate processes involved in drug absorption and systemic disposition. [2,16,17,18,19] Unfortunately, absorption and disposition kinetics cannot be derived directly from the plasma concentration-time profile, since local anesthetics exhibit flip-flop kinetics after epidural administration. In a system consisting of a series of processes or compartments, not only the decrease in the concentrations after stopping administration but also the time to reach steady-state depend on the slowest process in the chain.

Half-life is the most clinically relevant pharmacokinetic parameter to predict drug accumulation time in a patient upon constant rate administration. Problems arise when we have to pick the half-life upon which to calculate drug accumulation. Also, when estimating the time for the epidural administration to achieve steady-state, we have to decide if we should work with the actual elimination half-life of ropivacaine obtained from the PK of the intravenous formulation (t 1/2,IV ),consider the terminal half-life calculated after the end of the infusion (t 1/2,terminal ), or take into account the "mixed" absorption/distribution/elimination half-life estimated during the epidural infusion (t 1/2,DP ).

When the process of absorption is not a limiting factor, half-life is a hybrid parameter controlled by plasma clearance and extent of distribution. When the process of absorption is a limiting factor, the terminal half-life reflects mainly the rate and extent of absorption and not the elimination process (flip-flop pharmacokinetics). [20] Our data indicate that the true half-life of ropivacaine, as reported after IV administration (t 1/2,IV 1.8 ± 0.7 hours) [21], is irrelevant to the time to reach steady-state. of the levels that would be achieved at the steady-state.

Our data showed that prediction of ropivacaine accumulation can be improved by considering patient age. After epidural infusion, a trend towards a lower total plasma clearance with increasing age was observed (Fig. 3). Simon et al. [24] studied the influence of age on pharmacokinetics of ropivacaine after single epidural administration and reported similar results: clearance is significantly decreased in the oldest patients relative to the youngest ones. In our study we confirmed that also during continuous infusion there is an inversely correlation between age and clearance. In fact RPV clearance in a 40-year-old individual is about twice as high as in a 70-year-old subject.

As ropivacaine is extensively metabolized in the liver [8], the age dependence in clearance may presumably reflect decreased enzyme activity.

Studies during prolonged epidural infusion of ropivacaine showed that postoperative increases in plasma alpha 1 -acid glycoprotein concentrations are associated with increases in the plasma protein binding and in the total plasma concentrations of ropivacaine. Ropivacaine total clearance depends on liver enzyme activity and on the activity. [25,26] Therefore, changes in the fraction bound to proteins (alpha 1 -acid glycoprotein) may affect the drug's total clearance but might also alter the relationship between total drug concentration and possible toxic effects. [26,27,28] We did not measure alpha 1 acid glycoprotein concentrations and to some extent this is a limitation of our study. However, the postoperative course of the patients included in our trial did not exhibit any pathophysiological changes other than those expected in patients undergoing this type of surgical procedure.

In conclusion, we demonstrate that during continuous epidural infusion of ropivacaine there is a flip flop kinetic and that we have to avoid the acritical use of a drug's terminal half-life to predict the drug's accumulation rate. Furthermore, we found an equation that could correlate the ropivacaine clearance to age also during epidural continuous infusion. It will be necessary to perform other trials in order to improve ropivacaine dosage individualization on the basis of pharmacokinetic data of patients.

Abstract

Background

Ropivacaine has an optimal toxicity profile for epidural anesthesia in adults, but there are currently no studies concerning its pharmacokinetics during continuous infusion.

The primary objective of this study was to evaluate the pharmacokinetics and safety of ropivacaine in adults during a 48-hours continuous epidural infusion.

Materials and methods

We enrolled 43 adults (ASA I-II) scheduled for major abdominal or urologic surgery with postoperative continuous epidural analgesia with ropivacaine 0.2% (5mL/h) and sufentanil 0.75 µg/mL for 48 hours.

Ropivacaine blood samples were collected, during continuous epidural infusion, before the bolus and 3, 6, 12, 24, 48, 54, 60 hours after the bolus; plasma concentrations were measured on HPLC-UV. The concentration-time relationship of ropivacaine levels was analyzed using a population pharmacokinetic method based on a mixed-effect-model approach (P-PHARM software).

Results

Mean plasma concentration of ropivacaine at the end of epidural infusion (C 48h ) was 1.69 µg/mL (0.21-3.8 µg/mL). Mean (range) C max, was 1.82 µg/L (0.61-4.0 µg/mL); the area under the plasma concentration curve, AUC (0-60) , was 67.48  30.60 g.h/mL.

Total plasma ropivacaine concentrations fell mainly within (84%) or below (12%) the range reported to be safe in adults (1.0-3.0 µg/mL). Only 2 patients (5%) reached ropivacaine plasma levels higher than 3 µg/mL, namely 3.8 and 4.0 µg/mL at 48 and 54 hours, respectively. Total ropivacaine concentrations up to 4.0 g/mL were tolerated during long-term epidural ropivacaine infusion.

Mean clearance for total ropivacaine was 5.33 L/h. Ropivacaine calculated terminal half-life is significantly longer after epidural than after intravenous administration. Ropivacaine absorption after epidural administration cannot be derived directly from plasma concentration-time curves because of flip-flop kinetics.

9,10

The flip-flop model implies slow absorption of ropivacaine from the epidural space into the systemic circulation rate-limits drug elimination. Therefore, when the process of absorption is a limiting factor, the terminal half-life reflects the rate and the extent of absorption and not the elimination process. 10,11 In a system consisting of a series of processes, the decrease in concentrations after administration is discontinued and the time to reach the steady-state depends on the slowest process in the chain.

This has implication for designing epidural continuous infusion schedules of ropivacaine, considering the clinical significance of accumulation reaching either toxic or ineffective concentrations.

The primary goal of this study was to evaluate the pharmacokinetics of ropivacaine accumulation in adults receiving continuous epidural analgesia during the first 48 h after major surgery.

Material and methods

After approval from the Ethics Committee of San Matteo Hospital (EudraCT number 2010-019393-32) and written informed consent 49 adult patients, ASA class I-II, scheduled for major abdominal or urological surgery and planned to receive a postoperative epidural infusion after surgery for pain control were enrolled. Exclusion criteria were emergency surgery, coagulopathy, renal, hepatic, neurological or psychiatric diseases, a history of peripheral neuropathies and chronic opioid therapy.

Before anesthesia induction epidural catheter was positioned in sitting position, using the loss resistance technique. In patients scheduled for upper abdominal surgery the epidural catheter was placed between T7-T8 or T9-T10. In patients scheduled for lower abdominal or urologic surgery between T10-T11. General anesthesia was induced with Propofol 2mg/kg, Fentanyl 2 μg/kg, Cisatracurium 0.15 mg/kg, and was maintained with Fentanyl and Sevoflurane 2%. Forty five minutes before the end of surgery patients received an epidural loading dose (RPV 0.2% 5 ml + Sufentanil 10 µg), followed by a second bolus (RPV 02% 5 ml) 15 minutes later. At the end of surgery an epidural infusion of Ropivacaine 0.2% plus Sufentanil 0.8 µg/ml was started.

Ropivacaine/Sulfentanyl epidural infusion was constantly condelivered at a rate of 5ml/h using an electronic infusion pump during the first 48 hs after surgery.

Patients were evaluated every 6 hours during the first day after surgery and every 12 hours in the second postoperative day. We recorded vital signs (arterial pressure and heart rate), pain according to the Numeric Rate Scale (at rest -NRS -and after movement -mNRS-), analgesic rescue dose (administered if NRS>4), Holmen scale (to The HPLC method has been validated over the range 0.1-4 g/mL. The assay was linear over the entire concentration range (r 2 = 0.99). Intra-and inter-day precision and accuracy was less than 6%.

assess
The limit of detection (LOD) was 0.050 g/mL, the limit of quantification (LOQ) was 0.1 g/mL.

Preparation of Standard solution and standard curves

A standard stock solution (1mg/mL) of drug was prepared in methanol-water (50/50), stored at -20°C and were stable for at least 6 months. Plasma standards and controls were prepared at concentrations of 0.1, 0.2, 0.4, 1.0, 2.0, 4.0 g/mL and 0.12 (Low), 0.8 (Medium) and 1.6 (High) g/mL, respectively, by diluting appropriate aliquots of the stock solution with drug-free serum. The calibration curve was obtained by linear regression of the peak-height ratios of ropivacaine to the internal standard and plotting these against the nominal concentration of the drug.

Blood sampling

Blood samples were obtained immediately before bolus drug administration and 3, 6, 12, 24, 48, 54 and 60 h after starting the infusion. Plasma was separated by centrifugation, transferred to clean prelabeled tubes and frozen at -20°C until analysis.

Sample preparation

250 L of distilled deionized water,500 L of dichloromethane and 10 L phenacetin

Pharmacokinetic analysis

The concentration-time relationship of ropivacaine levels (corresponding to 6 data points) during an epidural infusion preceded by 2 sequential boluses was analyzed using a population pharmacokinetic method based on a mixed-effect-model approach.

This method separates explicitly the variability of drug concentrations in the population into two parts: interindividual variability characterized by the distribution of pharmacokinetic parameters in the population and residual variability (e.g., measurement errors). A parametric approach 12,13 was used (using the P-PHARM software, which is now incorporated into the Innaphase Kinetica suite) to estimate the When bioavailability (F) is not known, estimates of CL should be related to F, i.e., CL/F.

Bioavailability was fixed to 1, assuming total (100%) absorption after epidural administration. Model evaluation was conducted as recommended in the P-PHARM manual and also by examination of a plot of measured/predicted concentration data versus time.

After the end of infusion, the terminal rate constant ( ,terminal ) was determined for total ropivacaine by linear regression of the last 3 data points on the plasma concentrationtime curve (from the 48 th hour to the 60 th hour):

C'=C 48h . e - terminal . t' t 1/2,terminal was defined as the 0.693/ terminal ratio and represents the terminal half-life after the infusion is stopped. t' is the time since the end of the infusion.

The area under the plasma concentration time curve was calculated by numeric integration using the linear trapezoidal rule. C max was the highest concentration achieved; the time to C max and the plasma concentrations at 3, 6, 12, 24, 48, 54 and 60 h after starting ropivacaine administration (C 3h , C 6h , C 12h , C 24h , C 48h , C 54h , C 60h , respectively) were derived directly from ropivacaine levels.

Statistical analysis

Patient characteristics are summarized as median values (range). Total plasma ropivacaine concentrations and derived pharmacokinetic data are summarized as geometric mean (geometric coefficient of variation, CV%) and/or mean (SD) values.

Multiple-linear regression analysis with stepwise inclusion between maximum a posteriori estimates of all pharmacokinetic parameters and all covariates was performed with the analysis of variance to determine the relevant relationships (with the P-PHARM software). 12 A value of P<0.05 was considered significant.

Results

Forty-nine adults consecutive were enrolled and six were excluded after discontinuation of epidural continuous infusion because of clinical or hemodynamic problems not related to ropivacaine epidural infusion; data collection was incomplete in one other patient, whose data were nevertheless included in our PK analysis. Forty-three patients completed the analysis.

Thirty-five patients underwent major abdominal surgery and eight underwent urological surgery (29 males and 14 females). Patient age ranged from 41 to 83 years (median=63 years) and body weight ranged from 50 to 100 kg (median= 75.5 kg).

Analgesia was satisfactory in all cases and there were no cardiocirculatory side-effects, such as major hypotension or arrhythmia, which could require discontinuation of the epidural infusion. There were no minor (facial dysthesia, tinnitus, changes in sensitivity and strength) or major (convulsions, hallucinations) neurological complications during continuous epidural infusion of ropivacaine.

A total of 330 plasma concentrations from 43 patients were used to compute pharmacokinetic parameters including a baseline sample, four samples collected during ropivacaine infusion and three samples collected during the first 12 h after ropivacaine infusion discontinuation.

Pharmacokinetic data (geometric mean and geometric coefficient of variation, CV%) are summarized in Table I.

During the epidural infusion (ropivacaine 10mg/h) preceded by 2 sequential boluses of 10 mg each, plasma concentrations of ropivacaine leveled off, as expected. Exposure to ropivacaine during epidural infusion was highly variable.

The plasma concentration of ropivacaine at the end of epidural infusion (C 48h ) was 1.60 ± 0.89 µg/mL. After 12 hours and 24 hours, respectively, 50% and 75% of the 48hour level of ropivacaine were reached. C max, was 1.82 ± 0.89 µg/L and was observed at a mean time (t max ) of 48 hours.

Total plasma ropivacaine concentrations were mainly within (84%) or below (12%) the range reported to be safe in adults (1.0-3.00µg/mL). 5,6, 9, 14,15,16 Only 2 patients (5%) reached ropivacaine plasma levels higher than 3.0 µg/mL, namely 3.8 and 4.0 Through covariate analysis, we found that age was the only independent predictor identified.

In fact, the stepwise inclusion performed on P-PHARM revealed an age effect on CL, of the levels that would be achieved at the steady-state.

which
Our data showed that prediction of ropivacaine accumulation can be improved by considering patient age. After epidural infusion, a trend towards a lower total plasma clearance with increasing age was observed (Fig. 3). Simon et al.24 studied the influence of age on pharmacokinetics of ropivacaine after single epidural administration and reported similar results: clearance is significantly decreased in the oldest patients relative to the youngest ones. In our study we confirmed that also during continuous infusion there is an inversely correlation between age and clearance. In fact RPV clearance in a 40-year-old individual is about twice as high as in a 70-year-old subject.

As ropivacaine is extensively metabolized in the liver 8, the age dependence in clearance may presumably reflect decreased enzyme activity.

Studies during prolonged epidural infusion of ropivacaine showed that postoperative increases in plasma alpha 1 -acid glycoprotein concentrations are associated with increases in the plasma protein binding and in the total plasma concentrations of ropivacaine. Ropivacaine total clearance depends on liver enzyme activity and on the free drug fraction in plasma (CL  CL i * f u ; CL i = drug intrinsic clearance, f u : unbound drug fraction), while the clearance of the unbound drug depends solely on enzyme activity. 25,26 Therefore, changes in the fraction bound to proteins (alpha 1 -acid glycoprotein) may affect the drug's total clearance but might also alter the relationship between total drug concentration and possible toxic effects. 26,27,28 We did not measure alpha 1 acid glycoprotein concentrations and to some extent this is a limitation of our study. However, the postoperative course of the patients included in our trial did not exhibit any pathophysiological changes other than those expected in patients undergoing this type of surgical procedure.

In conclusion, we demonstrate that during continuous epidural infusion of ropivacaine there is a flip flop kinetic and that we have to avoid the acritical use of a drug's terminal half-life to predict the drug's accumulation rate. Furthermore, we found an equation that could correlate the ropivacaine clearance to age also during epidural continuous infusion. It will be necessary to perform other trials in order to improve ropivacaine dosage individualization on the basis of pharmacokinetic data of patients.

  infusion since it appears to better reflect the interplay interference between volume distribution and absorption rate during the accumulation phase. Prediction of ropivacaine accumulation can be improved by considering patient age.

Formatted

Formatted

Formatted

  was used (using the P-PHARM software, which is now incorporated into the Innaphase Kinetica suite) to estimate the mean and interindividual variance of each pharmacokinetic parameter in the population, to calculate the Bayesian maximum a posteriori estimates of individual pharmacokinetic parameters, and to test the relationships between these individual estimates and several covariates (gender, age, body weight, height, renal and hepatic function indices). During covariate modeling, P-PHARM can automatically search for a linear relationship between covariates and the population parameter estimates. Pharmacokinetic data were fitted with different compartmental models. The most appropriate model was chosen based on the Akaike information criterion [12] and the examination of residual plots. A one-compartment model with sequential bolus and infusion inputs was fitted to the total plasma ropivacaine concentration-time data. The plasma concentration versus time curve of ropivacaine during continuous epidural infusion (R 0 : rate of epidural infusion) was described by the equation: C = (R 0 /CL). (1-e -λ DP .t ) λ DP indicates the "mixed" (absorption/distribution/elimination) rate constant of ropivacaine observed during 48-hour epidural drug administration and calculated by the CL/Vd ratio. t 1/2,DP was defined as the 0.693/λ DP ratio and represents the infusion dosing half-life. Ropivacaine pharmacokinetic starting estimates for the population analysis were obtained from previous studies. A heteroscedastic error variance (1/Y) model was used to describe residual error, and a log-normal distribution was used to describe apparent clearance (CL) and apparent volume of distribution (Vd).

  individual posterior Bayesian estimates of ropivacaine pharmacokinetic parameters were generated using a maximum a posteriori probability Bayesian fitting procedure.23 ] The performance of individualized Bayesian estimation steps is shown in the plots of the observed versus estimated plasma concentrations in Figure2, and by the frequency distribution histogram of the normalized residuals which revealed a distribution very close to the expected one (normal with zero mean and unitary variance).

Formatted

  in plasma (CL ∼ CL i * f u ; CL i = drug intrinsic clearance, f u : unbound drug fraction), while the clearance of the unbound drug depends solely on enzyme

Figure 1 .Figure 2 .Fig 3 .

 123 Figure 1. Total plasma ropivacaine concentration versus time during and after an epidural continous infusion of 10mg/hour preceeded by 2 sequential epidural boluses.The solid line shows the computer-generated line of the best fit up to 48 h, for a one-compartment model with zero-order input and first-order elimination.

(

  10 g/mL, I.S.) were added to 250 L of plasma. After vortex mixing for 3 minutes, the tubes were centrifuged at 1200g for 10 min. The organic phase was transferred into a clean conical tube and evaporated to dryness under a gentle nitrogen stream. The residue was dissolved in 200 L mobile phase and 50 L was injected into HPLC.

  mean and interindividual variance of each pharmacokinetic parameter in the population, to calculate the Bayesian maximum a posteriori estimates of individual pharmacokinetic parameters, and to test the relationships between these individual estimates and several covariates (gender, age, body weight, height, renal and hepatic function indices). During covariate modeling, P-PHARM can automatically search for a linear relationship between covariates and the population parameter estimates. Pharmacokinetic data were fitted with different compartmental models. The most appropriate model was chosen based on the Akaike information criterion 12 and the examination of residual plots. A one-compartment model with sequential bolus and infusion inputs was fitted to the total plasma ropivacaine concentration-time data. The plasma concentration versus time curve of ropivacaine during continuous epidural infusion (R 0 : rate of epidural infusion) was described by the equation: C = (R 0 /CL). (1-e - DP .t )  DP indicates the "mixed" (absorption/distribution/elimination) rate constant of ropivacaine observed during 48-hour epidural drug administration and calculated by the CL/Vd ratio. t 1/2,DP was defined as the 0.693/ DP ratio and represents the infusion dosing half-life. Ropivacaine pharmacokinetic starting estimates for the population analysis were obtained from previous studies. A heteroscedastic error variance (1/Y) model was used to describe residual error, and a log-normal distribution was used to describe apparent clearance (CL) and apparent volume of distribution (Vd).

  at 48 and 54 hours, respectively. Total ropivacaine concentrations up to 4.0 µg/mL were tolerated during long-term epidural ropivacaine infusion.

Figure 1

 1 Figure 1 shows all the measured plasma ropivacaine concentrations and the drug concentration-time curve resulting from the average population parameters estimated with P-Pharm. Estimated population characteristics of the kinetic parameters together for ropivacaine clearance (CL) and the apparent volume of distribution (Vd) with their coefficient of variation are presented in TableII.The dosing period absorption/distribution/elimination rate constant of ropivacaine calculated by the average population parameters ( DP = CL/Vd) was 0.064h -1 and the corresponding t 1/2,DP = (0.693*Vd)/CL = 10.8 hours.Following the end of epidural infusion of ropivacaine, the terminal half-life (t 1/2,terminal ), determined for total ropivacaine by linear regression of the last 3 data points on the plasma concentration-time curve was 7.4 hours.The pharmacokinetic population parameters were subsequently used to fit the concentration data up to 48 h using the Bayesian estimation method. This procedure enables the estimation of individual pharmacokinetic parameters for ropivacaine. The individual posterior Bayesian estimates of ropivacaine pharmacokinetic parameters were generated using a maximum a posteriori probability Bayesian fitting procedure.23 The performance of individualized Bayesian estimation steps is shown in the plots of the observed versus estimated plasma concentrations in Figure2, and by the frequency distribution histogram of the normalized residuals which revealed a distribution very close to the expected one (normal with zero mean and unitary variance).

Figure 1 .Figure 2 .Fig 3 .

 123 Figure 1. Total plasma ropivacaine concentration versus time during and after an epidural continous infusion of 10mg/hour preceeded by 2 sequential epidural boluses.The solid line shows the computer-generated line of the best fit up to 48 h, for a one-compartment model with zero-order input and first-order elimination.

  In order to predict drug accumulation upon epidural infusion dosing, it is more appropriate to use the apparent infusion dosing half-life t 1/2,DP since this parameter appears to better reflect the interplay interference between volume distribution and absorption rate during the accumulation phase. Our data indicate that this infusion Modeling with sums of exponentials is feasible, as long as the results are reported in terms of macroscopic parameters (coefficients and exponents) and model-independent parameters, such as mean systemic or oral clearance, which is always dose over AUC (TableII).

	dosing period half-life (t 1/2DP =10.8 hours) was higher (↑ 45%) than the terminal half-life validation [20]. For a drug such as ropivacaine, the time to reach the steady-state may be influenced
	(t 1/2terminal = 7.4 hours); a more pronounced sensitivity of the t 1/2DP to the absorption rate by the absorption rate (if administered by extravascular route), by the volume of
	process probably explains the difference. This difference seems to confirm the distribution and by clearance; however, mean steady-state concentration depends only
	indication by Sahin and Benet [22] to avoid the acritical use of a drug's terminal half-life on clearance. Also, it is well recognized that clearance (divided by bioavailability)	Formatted: Not Superscript/ Subscript
	to predict the drug's accumulation rate. defines the appropriate rate of administration of a particular drug through the	Formatted: Not Superscript/ Subscript
	A one-compartment model with zero-order input and first-order elimination was optimal relationship: R 0 = CL.C ss,ave .F. For ropivacaine (10 mg/hour), a drug following linear
	F o for describing the data in the accumulation phase during epidural infusion of ropivacaine. The same model was used by Hansen et al. [16] to describe the trend of ropivacaine concentrations during the accumulation phase in pediatric patients (mean o kinetics, the predicted concentration at steady-state, C ss,ave , based on individual a F posteriori Bayesian estimates of ropivacaine pharmacokinetic clearance resulted in a	Formatted: Not Superscript/ Subscript
	r r
	P P
	e e r e e r
	R e v i e w R e v i e w	Formatted: Not Superscript/ Subscript Formatted: Not Superscript/ Subscript
		Formatted: Not Superscript/
		Subscript

age: 3.3 years, range: 0.3-7.3 years) under continuous epidural infusion. In the majority of children ropivacaine concentrations had reached the steady-state within 36 hours and the half life (t 1/2DP ) of total ropivacaine was 4.9 hours. The apparent total clearance (0.51L/h/kg) and the apparent volume of distribution (3.1L/kg) were about six and three times the values obtained in our adult patients, respectively. The pharmacokinetic parameters obtained in our study from data following continuous epidural infusion are different from the corresponding values reported after IV administration (Vd: 88.8L vs. 41.7L, CL: 5.7L/h vs. 23.2L/h) in adult patients. On the other hand, the final parameters of ropivacaine obtained by a population pharmacokinetic analysis

[23]

(NONMEN) after loco-regional administration as an anesthetic in hip or knee replacement surgery were quite similar: the apparent clearance was 3.5L/h and the volume of distribution was 65.3L. When evaluating the individual pharmacokinetic parameters, though, we must keep in mind that modeling is always dangerous if no IV data are available, because we may get a good fit of data to our equations but the parameters are hybridized and therefore incorrect. It has been underlined that when one models with sums of exponentials, one can simply assume consistency (best to assume this for the equation and not the model). Thus, good fits do not validate our model but only indicate consistency. In most cases, like this one, it is not strictly necessary to assume mean value of 2.0 (±0.62) µg/mL. At the end of infusion (48 h after the start of treatment) the ropivacaine plasma levels achieved corresponded on average to ∼ 90%

Table I .

 I Total plasma ropivacaine concentrations (geometric mean, geometric coefficient of variation (%))at different time points during epidural infusion (ropivacaine 10mg/h) preceded by 2 sequential boluses of 10 mg each.

	Trauma Patients Receiving Long-Term Continuous Peripheral Nerve Block Table II -Ropivacaine population pharmacokinetic results
	Catheters. Anesth Analg 2010; 110: 630-4.
	Parameter	Unit	Mean		Percent Coefficient of	Formatted: Not Superscript/
						Variation	Subscript
	PK parameter Apparent Clearance (CL/F)	Unit L/h	Geometric Mean 5.33	Geometric variation (%) coefficient of 21.69	Formatted: Not Superscript/ Subscript Deleted:
	C 0h Apparent Volume of	µg/mL L	0.00 92.15			0.00 16.42	Deleted:
	C 3h Distribution (Vd/F)	µg/mL	0.40			87.7
	C 6h Values are mean (percent coefficient of variation) µg/mL	0.59			56.1
	C 12h	µg/mL	0.88			31.5
	C 24h	µg/mL	1.22			20.6
	F o r F o r F o r F o r µg/mL µg/mL µg/mL F o F o C 3h /C 48h C 48h C 54h C 60h -r r	1.60 0.81 0.56 0.25			3.1 8.5 39.5 28.3
	C 12h /C 48h C 24h /C 48h C max t max AUC (0-60h)	P e e r P e e r P e e r P e e r -0.55 -0.76 P P µg/mL 1.82 hours 43 µg.h/mL 67.48 e e r e e r	10.2 6.6 2.9 2.4 2.2
	CL=(D/AUC tot )	L/h	6.96	R e v i e w R e v i e w R e v i e w R e v i e w 8.6 R e v i e w R e v i e w

  if there was sensory block), sedation scale and clinical side-effects (nausea, vomiting, itching, hypotension, systemic neurological side effects)

	The HPLC system consisted of Varian Prostar Mod.430 a pump equipped with
	variable-wavelength UV detector (Varian Prostar Mod.330). Separation was achieved
	using a reverse-phase C-18 column (2504.6 mm I.D., particle size 5m, Hypersil
	BDS-CPS Analitica, Italy). The mobile phase was acetonitrile-methanol-50mM
	potassium dihydrogen phosphate (pH=6) (180:160:660, v/v/v) delivered at a flow rate of
	1.3 mL/min. Chromatographic separation was performed at laboratory room
	temperature and monitored at 210nm.
	F o
	r
	P
	e e r
	R e v i e w
	Reagents
	RPV was a gift of the Department of Anesthesia and Intensive Care. Phenacetin was
	purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used
	were of the highest commercially available quality.
	Instrumentation

  DiscussionPharmacokinetic of epidural ropivacaine was well described after single dose, but it was important to detect what it can happen during continuous infusion. Whereby we investigated which kind of kinetic we have during epidural continuous infusion and if there are predictors that can determine a change in the pharmacokinetic profile.In order to predict drug accumulation upon epidural infusion dosing, it is more appropriate to use the apparent infusion dosing half-life t 1/2,DP since this parameter appears to better reflect the interplay interference between volume distribution and Modeling with sums of exponentials is feasible, as long as the results are reported in terms of macroscopic parameters (coefficients and exponents) and model-independent parameters, such as mean systemic or oral clearance, which is always dose over AUC (TableII).For a drug such as ropivacaine, the time to reach the steady-state may be influenced by the absorption rate (if administered by extravascular route), by the volume of distribution and by clearance; however, mean steady-state concentration depends only on clearance. Also, it is well recognized that clearance (divided by bioavailability) defines the appropriate rate of administration of a particular drug through the relationship: R 0 = CL.C ss,ave .F. For ropivacaine (10 mg/hour), a drug following linear kinetics, the predicted concentration at steady-state, C ss,ave , based on individual a posteriori Bayesian estimates of ropivacaine pharmacokinetic clearance resulted in a mean value of 2.0 (±0.62) µg/mL. At the end of infusion (48 h after the start of treatment) the ropivacaine plasma levels achieved corresponded on average to  90%

	validation 20.
	absorption rate during the accumulation phase. Our data indicate that this infusion
	dosing period half-life (t 1/2DP =10.8 hours) was higher (↑ 45%) than the terminal half-life
	In fact, plasma concentration profiles and the potential risk of systemic toxicity after (t 1/2terminal = 7.4 hours); a more pronounced sensitivity of the t 1/2DP to the absorption rate
	epidural administration of ropivacaine depends on the administered dose and the process probably explains the difference. This difference seems to confirm the
	interaction between the rate processes involved in drug absorption and systemic indication by Sahin and Benet 22 to avoid the acritical use of a drug's terminal half-life
	F o r F o r disposition. 2,16,17,18,19 Unfortunately, absorption and disposition kinetics cannot be derived directly from the plasma concentration-time profile, since local anesthetics F o to predict the drug's accumulation rate. F o exhibit flip-flop kinetics after epidural administration. In a system consisting of a series r r
	P P of processes or compartments, not only the decrease in the concentrations after stopping administration but also the time to reach steady-state depend on the slowest P P
	e e r e e r Half-life is the most clinically relevant pharmacokinetic parameter to predict drug process in the chain. accumulation time in a patient upon constant rate administration. Problems arise when e e r e e r
	R e v i e w R e v i e w we have to pick the half-life upon which to calculate drug accumulation. Also, when estimating the time for the epidural administration to achieve steady-state, we have to decide if we should work with the actual elimination half-life of ropivacaine obtained from the PK of the intravenous formulation (t 1/2,IV ),consider the terminal half-life calculated after the end of the infusion (t 1/2,terminal ), or take into account the "mixed" absorption/distribution/elimination half-life estimated during the epidural infusion (t 1/2,DP ). R e v i e w 41.7L, CL: 5.7L/h vs. 23.2L/h) in adult patients. On the other hand, the final parameters of ropivacaine obtained by a population pharmacokinetic analysis 23 (NONMEN) after loco-regional administration as an anesthetic in hip or knee replacement surgery were R e v i e w
	When the process of absorption is not a limiting factor, half-life is a hybrid parameter quite similar: the apparent clearance was 3.5L/h and the volume of distribution was
	controlled by plasma clearance and extent of distribution. When the process of 65.3L. When evaluating the individual pharmacokinetic parameters, though, we must
	absorption is a limiting factor, the terminal half-life reflects mainly the rate and extent of keep in mind that modeling is always dangerous if no IV data are available, because
	absorption and not the elimination process (flip-flop pharmacokinetics). 20 Our data we may get a good fit of data to our equations but the parameters are hybridized and
	explains 39.8% of the interindividual variability of the population pharmacokinetic indicate that the true half-life of ropivacaine, as reported after IV administration (t 1/2,IV therefore incorrect. It has been underlined that when one models with sums of
	parameters. Ropivacaine clearance correlated inversely with age. The linear correlation
	between age and ropivacaine clearance was best described by the equation (least
	square regression; r = 0.63, p<0.001):

1.8  0.7 hours) 21, is irrelevant to the time to reach steady-state.

A one-compartment model with zero-order input and first-order elimination was optimal for describing the data in the accumulation phase during epidural infusion of ropivacaine. The same model was used by Hansen et al. 16 to describe the trend of ropivacaine concentrations during the accumulation phase in pediatric patients (mean age: 3.3 years, range: 0.3-7.3 years) under continuous epidural infusion. In the majority of children ropivacaine concentrations had reached the steady-state within 36 hours and the half life (t 1/2DP ) of total ropivacaine was 4.9 hours. The apparent total clearance (0.51L/h/kg) and the apparent volume of distribution (3.1L/kg) were about six and three times the values obtained in our adult patients, respectively. The pharmacokinetic parameters obtained in our study from data following continuous epidural infusion are different from the corresponding values reported after IV administration (Vd: 88.8L vs. exponentials, one can simply assume consistency (best to assume this for the equation and not the model). Thus, good fits do not validate our model but only indicate consistency. In most cases, like this one, it is not strictly necessary to assume

Table I .

 I Total plasma ropivacaine concentrations (geometric mean, geometric coefficient of variation (%))at different time points during epidural infusion (ropivacaine 10mg/h) preceded by 2 sequential boluses of 10 mg each.

	PK parameter	Unit	Geometric Mean	Geometric coefficient of variation (%)
	C 0h			g/mL	0.00	0.00
	C 3h			g/mL	0.40	87.7
	C 6h C 12h C 24h F o	g/mL g/mL g/mL	0.59 0.88 1.22	56.1 31.5 20.6
	C 48h	r	g/mL	1.60	3.1
	C 54h C 60h C 3h /C 48h C 12h /C 48h	g/mL g/mL P --e e r 0.81 0.56 0.25 0.55	8.5 39.5 28.3 10.2
	C 24h /C 48h C max t max AUC (0-60h) CL=(D/AUC tot )	-g/mL hours g.h/mL L/h	0.76 1.82 43 67.48 6.96 R e v i e 6.6 2.9 2.4 2.2 8.6 w

Table II -

 II Ropivacaine population pharmacokinetic results

	Parameter	Unit	Mean	Percent Coefficient of
				Variation
	Apparent Clearance	L/h	5.33	21.69
	(CL/F)
	Apparent Volume of	L	92.15	16.42
	Distribution (Vd/F)
	Values are mean (percent coefficient of variation) F o
	r
	P
	e e r
			R e v i e w

European Journal of Clinical Pharmacology

Acknowledgements:

This research is supported by a grant of Scientific Direction of Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

This research is supported by a grant of Scientific Direction of Fondazione IRCCS