Ingeborg Walter-Sack

Jens Rengelshausen

Heike Oberwittler

Juergen Burhenne

Olaf Mueller

Peter Meissner

Gerd Mikus
email: gerd.mikus@med.uni-heidelberg.de

: H Oberwittler

PHARMACOKINETICS AND DISPOSITION

Keywords: Bioavailability, Chloroquine, Kinetics, Methylene blue

Purpose Methylene blue (MB) has recently been reevaluated for malaria treatment. With the aim of excluding treatment failures due to low bioavailability, we have investigated the absolute bioavailability of MB given as an aqueous oral formulation and its interaction with chloroquine (CQ). Methods A phase I study in 16 healthy individuals was performed as a monocenter prospective open randomized intra-individual cross-over comparison of MB single doses [50 mg intravenous (i.v.), 500 mg orally, separated by a 1week wash-out]. After a second week, the group was split for a randomized parallel group comparison of CQ 750 mg administered orally alone or combined with 500 mg MB orally.

Results Mean MB plasma area under the substrate concentration-time curve (AUC 0À1 ) was 7,639±3,384 ng/ mL*h and 51,171±17,147 ng/mL*h after i.v. and oral administration, respectively (dosage 1:10), and 76,897± 46,037 ng/mL*h after MB combined with CQ. The absolute bioavailability was 72.3±23.9%. Co-administration with CQ significantly increased MB plasma concentrations (p≤0.016); CQ kinetics remained unaffected. Conclusion The absolute bioavailability of MB is high. Co-administration of MB and CQ increases plasma, but not whole blood MB concentrations.

Introduction

Methylene blue (MB, methylthioninium chloride) has recently been reinvestigated for various aspects of its antimalarial activity, such as its selective inhibition of glutathione reductase of Plasmodium falciparum [START_REF] Akoachere | In vitro assessment of methylene blue on chloroquine-sensitive and -resistant Plasmodium falciparum strains reveals synergistic action with artemisinins[END_REF][START_REF] Schirmer | Methylene blue as an antimalarial agent[END_REF], and MB-based combination therapy is currently under development for the treatment of uncomplicated malaria in Sub-Saharan Africa [START_REF] Mandi | Safety of the combination of chloroquine and methylene blue in healthy adult men with G6PD deficiency from rural Burkina Faso[END_REF][START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF][START_REF] Meissner | High-performance chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples[END_REF][START_REF] Zoungrana | falciparum malaria: a randomized controlled trial from Burkina Faso[END_REF]. Methylene blue was used more than a 100 years ago against malaria in children and adults in South America and Europe [START_REF] Ehrlich | Chemotherapeutics: scientific principles, methods, and results[END_REF][START_REF] Ferreira | Sur l`emploi du bleu de méthylène dans la malaria infantile[END_REF][START_REF] Guttmann | Ueber die Wirkung des Methylenblau bei Malaria[END_REF], but it was abandoned when other treatments became available. Apart from malaria, MB is still used for a number of other relevant indications, mainly for the treatment of methemoglobinemia and for the prevention and treatment of ifosfamide-induced encephalopathy in cancer chemotherapy [START_REF] Aeschlimann | Comparative pharmacokinetics of oral and intravenous ifosfamide/mesna/ methylene blue therapy[END_REF][START_REF] Kuepfer | Prophylaxis and reversal of ifosfamide encephalopathy with methylene blue[END_REF][START_REF] Pelgrims | Methylene blue in the treatment and prevention of ifosfamide-induced encephalopathy: report of 12 cases and review of the literature[END_REF][START_REF] Zulian | Methylene blue for ifosfamide-associated encephalopathy[END_REF]. Other applications of MB include visualization of target tissues during surgery [START_REF] Khan | Prolonged postoperative altered mental status after methylene blue infusion during parathyroidectomy: a case report and review of the literature[END_REF], supportive treatment of anaphylactic reactions [START_REF] Evora | Role of nitric oxide production in anaphylaxis and its relevance for the treatment of anaphylactic hypotension with methylene blue[END_REF][START_REF] Neto | Methylene blue: an effective treatment for contrast medium-induced anaphylaxis[END_REF], and pathogen inactivation in fresh frozen plasma [START_REF] Politis | Quality and safety of fresh-frozen plasma inactivated and leuco-reduced with the Theraflex methylene blue system including the Blueflex filter: 5 years' experience[END_REF][START_REF] Simonsen | Clinical tolerance of methylene blue virus-inactivated plasma[END_REF][START_REF] Williamson | Methylene bluetreated fresh-frozen plasma: what is its contribution to blood safety[END_REF]. It has even been reported to be beneficial against sepsis [START_REF] Donati | Does methylene blue administration to septic shock patients affect vascular permeability and blood volume[END_REF].

For systemic treatment regimens, MB can be administered orally and intravenously (i.v.). Following oral intake, in humans the drug is absorbed from the gastrointestinal tract, reaching maximum plasma concentrations after 1-2 h [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF][START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF]. Maximum whole blood MB concentrations after oral or i.v. administration of 100 mg were observed to differ in the order of 1:100 [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF]; a difference in the same order of magnitude was reported for AUC 0-4h (9 vs. 137 nmol/mL*min). The plasma half-life was found to be about 5-6.5 h [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF][START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF]. Renal excretion of MB was, on average, 74% (range 53-97%) after an oral dose of 10 mg [START_REF] Disanto | Pharmacokinetics of highly ionized drugs II: methylene blue-absorption, metabolism, and excretion in man and dog after oral administration[END_REF], and about 20-30% after of an oral dose of 100 mg [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF]. Data from animal studies showed that MB crosses the blood-brain barrier, with i.v. administration leading to much higher concentrations in the central nervous system than oral intake, indicating differences in organ distribution after oral and i.v. MB administration [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF]. Such observations may at least in part explain why MB taken orally is reported to be safe in much higher doses than when it is given intravenously.

For safe oral drug administration in very young pediatric patients, especially those under the age of 2 years, it is essential to use liquid medicines [START_REF]European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use[END_REF]. This means that the recent development of MB for effective malaria treatment in African children has to include a liquid MB formulation. In order to exclude a low bioavailability of MB as a reason for potentially insufficient therapeutic effects, an assessment of oral bioavailability was required. The primary aim of this study was to determine the absolute bioavailability of MB when given as an aqueous oral formulation. A supplemental part of the study was linked to a bioavailability assessment evaluating chloroquine (CQ) and MB concentrations when using higher MB doses than those tested in our previous phase I study, which had indicated a slight decrease of CQ concentrations during the initial 24 h after co-administration of MB and CQ [START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF].

Materials and methods

Study design

This phase I study in 16 healthy individuals was performed as a monocenter prospective open randomized intraindividual cross-over comparison of single doses of MB (50 mg i.v., 500 mg orally, study days 1 and 8, separated by a washout phase of 1 week; Fig. 1,part A). After a further washout phase of 1 week, the group was split for a supplemental randomized parallel group comparison of CQ (single oral dose of 750 mg) given alone or combined with 500 mg MB orally (study day 15; Fig. 1,part B). Because of the long elimination half-life of CQ, it was not feasible to include the second part into the cross-over comparison. Venous blood samples were drawn up to 24 h (Fig. 1, part A) and 72 h (Fig. 1, part B). Since malaria parasites are mainly found in erythrocytes, whole blood MB concentrations in addition to plasma concentrations were analyzed in order to estimate MB within erythrocytes as the intended target tissue.

The study was carried out in accordance with the Declaration of Helsinki adopted by the World Medical Association in 1964, Version of Somerset West 1996 (according to physicians' professional law at the time of study conduction), further specific legal regulations in Germany (as required prior to implementation of the EU Directive 2001/20/EC into national law), and the Note of Guidance on Good Clinical Practice (CPMP/ICH/135/95). Written approval by the Ethics Committee of the Medical Faculty of the University of Heidelberg was obtained prior to beginning the study. Voluntarily signed informed consent after a full explanation of the study verbally and in writing was obtained from each participant prior to inclusion.

Study drugs

The aqueous liquid oral preparation of MB containing 25 mg/mL (2.5 %) was supplied by Mayrhofer Pharmazeutika GmbH, Linz, Austria, (manufacturer) under contract of DSM Fine Chemicals Austria. It was part of the investigational study lot which was produced for bioavailability testing and a phase II study in African children [START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF]. For intravenous application, a commercially available MB formulation was used, purchased by the Hospital Pharmacy (Methylenblau VITIS®). The CQ tablets were also commercially available (CQ phosphate, Resochin® tablets 250 mg) and purchased by the hospital pharmacy.

Study population and procedures

Sixteen healthy individuals (nine males and seven females) were included in the study. Their mean age was 26.3± 4.9 years, mean weight was 67.1±10.4 kg, mean height was 173±7 cm, and mean body mass index was 22.2±2. Eligibility was based on an uneventful medical history, normal pre-study findings of a comprehensive physical examination, 12-lead electrocardiograph (ECG), safety clinical chemistry and hematology screening, determination of glucose-6-phosphate dehydrogenase (G6PD), urine analysis, and a qualitative pregnancy test in female participants performed within 3 weeks prior the first scheduled dosing of the study medication. Important exclusion criteria were renal insufficiency, G6PD deficiency, retinopathy, blood disorders, myasthenia gravis, epilepsy, psoriasis, allergies (except for mild forms of hay fever) or history of hypersensitivity reactions, pregnancy or lactation, as well as any other acute or chronic disorder that could interfere with the safety of the participants or the study objectives. Administration of any other investigational drug during the 2 months preceding study onset or other drug treatments necessary during the 4-week period prior to study onset also led to exclusion. This especially referred to treatments with a known inhibitor or inducer of drug-metabolizing enzymes or transport proteins within a period of less than ten times the respective elimination half-life. Co-administration of oral contraceptive drugs was permitted, but changes in usage were not allowed during the study. If intermittent drug treatment was necessary, the investigator could decide whether this was acceptable for participation in the study or whether the individual needed to be excluded for aspects of his or her own safety or for aspects of reliability of the data. In addition, smoking, excessive drinking of alcoholic beverages (more than approximately 30 g alcohol/day), drug addiction or a positive result in urinary drug screening, excessive coffee drinking (more than six cups per day), excessive physical activities during the days prior to the study, and blood donation within the last 2 months were reasons for exclusion.

Upon admission on the first study day, the individual was not included if there was any doubt about the status of unimpaired health. In female participants, a qualitative pregnancy test was performed and analyzed before drug administration on study days 1, 8, and 15 and, additionally, a plasma sample was analyzed for β-human chorionic gonadotropin. A post-study follow-up examination was scheduled within 8-14 days after the last pharmacokinetic blood sampling to verify the health of the participant.

The randomization list was prepared by computer generation (http://www.randomization.com). Participants were allocated to the numbers in chronological order. An individual drug treatment regimen had been predefined for each participant number (bioavailability assessment: randomized order of i.v. and oral preparation of MB; subsequent study part B: randomized assignment of participants to CQ alone or combined MB-CQ administration). After an overnight fast, the study drugs were administered at approximately 8:00 a.m. The time of swallowing the liquid MB formulation and/or the CQ tablets and the end of the i.v. injection were defined as 0 h and 0 min. All previous and following study times refer relatively to this time point. During the initial 10 h after the administration of the study drugs the participants were asked to stay in a semi-recumbent position, and they were only allowed to leave the Clinical Research Unit after the 10 h-blood sample had been drawn. Alcoholic and caffeinated beverages were not allowed from the time the study drug was administered until the last blood sample for the pharmacokinetics analysis had been drawn (24 h in the bioavailability study and 72 h in the supplemental study). Caffeine-free beverages were supplied during all study days. For lunch and supper, standardized meals were provided by the hospital.

On study day 1 or 8, respectively, 50 mg MB, dissolved in 10 mL 0.9% NaCl, was injected i.v. over 9.5 min, followed by a bolus of 10 mL 0.9% NaCl within 30 s. Alternatively, 20 mL of the liquid oral preparation containing 25 mg MB/mL, corresponding to 500 mg MB orally, was taken together with 180 mL water. On study day 15, a single oral dose of 750 mg CQ (three tablets, equivalent to an initial therapeutic dose in normal weight adults), either alone or together with 500 mg MB (20 mL of the liquid oral preparation), was taken together with 180 mL water. When MB had to be injected i.v., blood samples [7.5-mL blood samples into a heparinized tube (for plasma drug concentrations) and 2.7 mL into an EDTA tube (for whole blood drug concentrations] were drawn prior to medication (at the end of the injection= 0 min) and at 5, 10, 15, 25, 35, 45 and 60 min and 2, 3, 4, 6, 8, 10, 23.5, and 24 h after drug administration. When MB was taken orally, equivalent samples were drawn prior to medication(=0 min), and at 30, 60, 90, and 120 min and at 3, 4, 5, 6, 7, 8, 9, 10, 23.5, and 24 h after drug administration. On study day 15, the timing of blood sampling was the same as after oral MB intake; however, the 48-h and 72-h samples were added because of the long half life of CQ. Heparinized blood samples were centrifuged immediately at +4°C; plasma and whole blood samples were kept frozen at -20°C until analysis.

For safety reasons a 12-lead ECG was recorded continuously for the first 4 h following the administration of CQ. Blood pressure was measured simultaneously at constant intervals. Both were registered and documented using the Surveyor II Monitoring System (Mortara Instruments, Essen, Germany). Female participants had to ensure effective contraceptive measures (co-application of two independent measures recommended) during the entire period of study participation and for 6 months after the administration of CQ. Any adverse event had to be adequately documented. The outcome was followed until recovery or stabilization of the participant's state.

Analytical methods

Plasma and whole blood concentrations of MB were determined by selective and sensitive methods based on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS); these method had been validated according to the Food and Drug Administration's (FDA) standards [START_REF] Burhenne | Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry[END_REF]. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for whole blood and plasma. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as the internal standard. Depending on the sample volume (whole blood and plasma: 250 μL), the method was linear at least within the range of 75 ng/mL and 10,000 ng/mL, respectively, and the limit of quantification (LOQ) in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood and plasma methods varied between -4.5 and +6.6% and -3.7 and +7.5%, respectively, with the corresponding precision ranging from 3.8 to 11.8% CV.

The concentrations of CQ in the plasma and whole blood were determined using high performance liquid chromatography (HPLC) with ultra-violet (UV) detection after liquid/ liquid extraction. These methods were based on published methods [20], but were modified in terms of chromatographic and sample extraction details as described in [START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF]. The LOQ in whole blood and plasma was 24 ng/mL.

Pharmacokinetic calculations and biometrical evaluation

No published data were available allowing sample size calculation for determination of the absolute bioavailability of MB. The calculation of pharmacokinetic parameters and biometrical evaluation of MB and CQ data were carried out using WinNonlin ver. 5.2 software (Pharsight Corporation, Mountain View, CA). The area under the substrate concentration time curves (AUC) was calculated by the trapezoidal rule with linear interpolation. The absolute bioavailability of MB (total AUC oral / total AUC iv, corrected for the difference in dosage) was determined intra-individually. The maximum concentration C max and the time to reach this concentration T max were obtained directly from the raw data. The terminal elimination halflife was determined according to T 1/2 =ln 2/λ z . A maximum of seven data points was used to calculate T 1/2 ; in three participants, only three data points were used in study part A after oral MB administration. Mean values ± standard deviation (SD) are given for all measured variables. A Wilcoxon matched-pairs signed-rank test was calculated for the intra-individual comparison of the total AUC (AUC 0À1 ) after oral MB alone and combined MB-CQ treatment, assessed separately for plasma and whole blood respectively. The AUC 0À1 of CQ was compared between plasma and whole blood, separately for CQ alone and for the combined MB-CQ treatment. The level of significance for a difference between two variables was set at the 5% level one-sided, corresponding to a two-sided 90% confidence interval (CI).

Results

MB plasma concentrations

MB plasma concentration time curves are shown in Figs. 2 and3 (note the different time and concentration scales). Concentrations rose promptly after oral application and were well above the detection limit after 24 h or even When MB was combined with CQ and both were given orally (part B, n=8), the AUC 0À1 of MB was 76,897± 46,037 ng/mL*h. The mean plasma MB AUC 0À1 in participants who had received MB alone in part A and the combined MB-CQ treatment in part B was 45,449±22,039 and 76,897 ±46,037 ng/mL*h, respectively (n=8); an intra-individual comparison showed a significant difference (p ≤ 0.016). In participant No. 11, who had vomited within 30 min after dosing, the AUC 0-72 and AUC 0À1 were in the lower range of the data. The residual AUC not covered by MB measurements (AUC 0À1 -AUC 0-24 , study part A, 24-h drug determination) corresponded to 31.3±16.5 and 33.6±11.8% of the total AUC after i.v. and oral MB, respectively, and to 11.7±8.7% after combined MB-CQ treatment (AUC 0À1 -AUC 0-72 , study part B, 72-h drug determination). The mean plasma elimination half-lives, T 1/2 , of MB were 18.5±11.8, 18.3±7.2, and 24.7 ± 9.4 h after i.v., oral, and combined MB-CQ administration, respectively. In participants No. 1 and 11, who had vomited in study part A or B, respectively, seven and five data points, respectively, were available for calculating T 1/2 . Therefore, the results are assumed to be sufficiently reliable despite vomiting.

MB whole blood concentrations

Individual pharmacokinetic characteristics are given in Table 2. The mean AUC 0À1 was 6,467±3,311 ng/mL*h when given i.v., and 50,578±30,125 ng/mL*h when given orally (dosage ratio i.v./ oral=1:10). From these data, the fraction absorbed (bioavailability) amounts to 84.0±16.7% (n=10). Based on the AUC covered by MB measurements (AUC 0-24 ), the bioavailability amounted to 81.7±16.9. When MB was combined with CQ, both given orally, (n= 8), the AUC 0À1 of MB was 45,075±21,989 ng/mL*h. The mean whole blood MB AUC 0À1 in participants who had received MB alone in part A and the combined MB-CQ treatment in part B was 50,387±35,138 and 45,075± 21,989 ng/mL*h, respectively (n=8); the difference is not significant. The mean T 1/2 of MB were 13.6±3.7, 14.7± 4.4, and 19.4±5.6 h after i.v., oral, and combined MB-CQ administration, respectively. In participant No. 11, who had vomited within 30 min after dosing, the results were not remarkably different from the other data.

Ratio of whole blood to plasma MB concentrations (study part A)

In participants who had received MB i.v. and in whom a complete set of the corresponding whole blood and plasma AUC 0À1 had been obtained (n=10), the mean ratio was 0.98±0.39. In participants who had received MB orally and in whom a complete set of the corresponding whole blood and plasma AUC 0À1 had been obtained (n=12), the mean ratio was 1.07±0.44.

CQ plasma and whole blood concentrations (study part B)

Mean CQ plasma and whole blood concentrations are shown in Fig. 4 (n = 14), individual pharmacokinetic characteristics are given in Table 3. The mean CQ total area under the plasma concentration time curve (AUC 0À1 ) was 8,385±3,018 ng/mL*h when given alone (n=6), and 8,80±4126 ng/mL*h when given with MB (n=8). The mean CQ whole blood AUC 0À1 was 35,029±12,448 ng/mL*h when CQ was given alone (n =6), and 34,999± 16,794 ng/mL*h when given with MB (n =8).

An intra-individual comparison of CQ whole blood and plasma AUC 0À1 after CQ intake alone (n=6; mean values see above) showed a significant difference (p<0.05); a comparison of CQ whole blood and plasma AUC 0À1 after combined MB-CQ intake (n=8; mean values see above) gave a highly significant difference (p ≤0.001). There was no influence of MB on the pharmacokinetics of CQ. Therefore, the mean ratio of CQ whole blood to plasma concentrations was assessed for the total group (n=14); the ratio remained close to 4 during the entire period of observation.

Safety and tolerability of the study medication

No relevant safety concerns arose during the study; no serious adverse event occurred. However, the tolerability of MB was limited, mainly due to an intensively bitter and metallic taste of the liquid oral formulation which resulted in immediate nausea after 14 of 23 drug administrations and provoked vomiting in three occasions-once 7 min after MB alone and twice after the combination of MB and CQ (after 25 min and about 1 h respectively). One male participant vomited 20 min after i.v. administration of MB, but not after oral intake. Mild diarrhea was reported five times after MB administration, and mild and self-limiting dysuria was reported twice after MB. On three occasions, a mild to moderate headache was reported, twice in association with CQ intake and once after i.v. MB. Completely reversible blue staining of the oral mucosa and teeth (including foreign material) for several hours and of the urine for several days was noticed by all participants, but this was no problem.

Discussion

The development of MB for the treatment of malaria was initially primarily intended for countries where other treatments are too expensive to be sufficiently available for the large populations affected by this disease [START_REF] Mandi | Safety of the combination of chloroquine and methylene blue in healthy adult men with G6PD deficiency from rural Burkina Faso[END_REF][START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF][START_REF] Meissner | High-performance chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples[END_REF][START_REF] Zoungrana | falciparum malaria: a randomized controlled trial from Burkina Faso[END_REF]. The MB-based treatment of uncomplicated malaria in Sub-Saharan Africa was first investigated in combination with CQ [START_REF] Mandi | Safety of the combination of chloroquine and methylene blue in healthy adult men with G6PD deficiency from rural Burkina Faso[END_REF][START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF][START_REF] Meissner | High-performance chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples[END_REF]. When MB was given at a dose of 2 mg/kg twice daily over 3 days, treatment was safe, even in G6PDdeficient adults and children [START_REF] Mandi | Safety of the combination of chloroquine and methylene blue in healthy adult men with G6PD deficiency from rural Burkina Faso[END_REF][START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF], but it was not sufficiently effective [START_REF] Meissner | Safety of the combination of chloroquine and methylene blue in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF]. In a subsequent dose-finding study in a comparable population of young children, a significantly better efficacy was demonstrated when threeto sixfold higher MB doses were given over 3 days. Such MB doses again were safe, even in G6PD deficient children. However, despite a significant reduction, especially in early treatment failure rates, an unacceptably high rate of recrudescence, most likely due to the high background resistance of the local parasites against CQ, was observed [START_REF] Meissner | High-performance chromatographic method for determination of amodiaquine, chloroquine and their monodesethyl metabolites in biological samples[END_REF]. This has led to the search for other MB combinations [START_REF] Zoungrana | falciparum malaria: a randomized controlled trial from Burkina Faso[END_REF]. However, given the most recent problem-that of parasite resistance against artemisinin derivatives [START_REF] Noedl | Artemisinin resistance in Cambodia. Oral presentation[END_REF]-MB may even gain importance in populations who do have sufficient access to other effective therapies. A reevaluation of MB as an antimalarial agent is indeed reasonable based on earlier reports of its beneficial therapeutic effects [START_REF] Ehrlich | Chemotherapeutics: scientific principles, methods, and results[END_REF][START_REF] Ferreira | Sur l`emploi du bleu de méthylène dans la malaria infantile[END_REF][START_REF] Guttmann | Ueber die Wirkung des Methylenblau bei Malaria[END_REF] and more recent knowledge on its mechanisms of action [START_REF] Schirmer | Methylene blue as an antimalarial agent[END_REF]. However, the therapeutic efficacy of oral treatment regimens is not only related to the properties of the active drug, but also to its bioavailability. Very little information on systemic MB concentrations in humans was available at the onset of this investigation, and what was available suggested a very low bioavailability.

Our own data on the interaction of MB and CQ from a previous study [START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF] did not allow us to estimate bioavailability. In a study comparing 100 mg MB orally and i.v., the AUC 0-4h of whole blood MB concentrations was only about 1% of the corresponding value after i.v. injection [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF]. Therefore, in the investigation reported here, the oral dose was relatively high, whereas the i.v. dose was less than that in the earlier bioavailability study [START_REF] Peter | Pharmacokinetics and organ distribution of intravenous and oral methylene blue[END_REF] and less than used for some diagnostic procedures [START_REF] Khan | Prolonged postoperative altered mental status after methylene blue infusion during parathyroidectomy: a case report and review of the literature[END_REF]. However, in contrast to our expectation, bioavailability turned out to be high: about two thirds of the oral dose of MB taken as an aqueous solution reached the systemic circulation. One important explanation for this finding is probably the fact that the sensitivity of analytical procedures has improved remarkably in the time between our study and previous ones [START_REF] Burhenne | Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry[END_REF]; this has enabled us to detect total MB concentrations in plasma and whole blood much better than previously, with a difference in the order of 100:1. In our interaction study with CQ [START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF], we probably quantified only the unbound fraction, and it is possible that other published reports also relate to free rather than to total MB plasma and whole blood concentrations.

Our intention was to clarify the order of magnitude of MB bioavailability. This information is essential to the decision-making process on whether or not the development of MB as an antimalarial drug should be pursued. We found that the bioavailability of MB is certainly high enough to justify continuation. The fact that there was appreciable inter-individual variation and that the extrapolated AUC in study part A on average exceeded the usual upper limit of 20% is not a relevant issue because the bioavailability values based on the total AUC and on the AUC 0-24 are similar. From the data generated in study part B, with an observation period of 72 h, which resulted in a low residual AUC of only about 12%, we can conclude that investigations of a new taste-masked pharmaceutical formulation of MB should be based on 72-h or 96-h observation periods to also meet the strict criteria set for bioavailability studies requested by regulatory agencies for pre-marketing authorization. In participants No. 1 and No. 11, who vomited within 30 min after dosing in study part A or B, respectively, the results did not differ remarkably from the data of the other study participants; we therefore assume that the data on these two participants are sufficiently reliable.

Plasma and whole blood MB concentrations did not differ when only MB was administered alone, indicating that MB does not accumulate within a cellular compartment, such as the erythrocytes. Interestingly, MB plasma concentrations rose significantly when MB was given concurrently with CQ in part B of our study, whereas whole blood concentrations remained unaffected. This study part was conducted because our former data had suggested a decrease in CQ whole blood concentrations during the combined treatment. The present data do not confirm this type of interaction as CQ kinetics were not influenced at all. Rather, a different interaction became apparent: higher concentrations of MB in plasma while whole blood concentrations are unchanged suggest a shift of MB from an intracellular compartment into the plasma. Further studies are needed to clarify this aspect, which could be relevant for the choice of future partner drugs in MB-based combination therapy.

The administration of oral MB was safe in this study, but the tolerability of oral MB was insufficient due to its bad taste. Developing MB as an antimalarial drug therefore has to include a further improvement of the pharmaceutical formulation. Since mainly liquid medicines are acceptable to very young children (only dispersible, very small granules to be mixed with food might be an alternative [START_REF]European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use[END_REF]), optimal taste masking is essential. A new type of formulation based on other principles than merely an aqueous solution [START_REF] Gut | Cation exchange resins as pharmaceutical carriers for methylene blue: Binding and release[END_REF] is a promising new approach for this goal. A taste-masked pediatric MB formulation was consequently developed and field-tested in the year 2007 (unpublished data). Other adverse events were mainly limited to temporary blue staining of the oral mucosa and teeth, including inserted dental material, mild and selflimiting dysuria, and blue discoloration of the urine. In contrast to a published report on permanent discoloration of a silicone intraocular lens by MB [START_REF] Stevens | Corneal edema and permanent blue discoloration of a silicone intraocular lens by methylene blue[END_REF], all discolorations had been reversed within a few days among our participants.

The concentrations of CQ were also determined in our study. The whole blood maximum concentrations and the ratio of whole blood to plasma concentrations correspond to our previous observations, whereas the AUC values are not comparable due to the different dosage regimens. In this latest study, the participants received a single dose of CQ, whereas in our previous investigation [START_REF] Rengelshausen | Pharmacokinetic interaction of chloroquine and methylene blue combination against malaria[END_REF] repetitive doses with a loading regimen were used. The present data are not sufficiently comparable with published data [START_REF] Ducharme | Clinical pharmacokinetics and metabolism of chloroquine. Focus on recent advancements[END_REF][START_REF] Frisk-Holmberg | The single dose kinetics of chloroquine and its major metabolite desethylchloroquine in healthy subjects[END_REF][START_REF] Gustafsson | Disposition of chloroquine in man after single intravenous and oral doses[END_REF], mainly because we measured the drug levels only up to 72 hours after dosing.

Conclusion

The absolute bioavailability of MB given as an aqueous oral formulation is high. Therefore, bioavailability should not be considered to be a problem for the development of

Fig. 1

 1 Fig. 1 Study design. Intra-individual cross-over of intravenous (i.v.) and oral methylene blue (MB) administration (part A), followed by a parallel group comparison of MB alone and MB combined with chloroquine (CQ) (part B)

  Fig.2Mean ± standard error of the mean (SEM) for MB plasma concentrations after i.v. injection of 50 mg MB

Fig. 4

 4 Fig. 4 Mean (± SEM) chloroquine (CQ) whole blood (filled triangles) and plasma concentrations (filled squares)

Table 1

 1 Individual pharmacokinetic characteristics and mean values ± SD of methylene blue (MB) in plasma after a single intravenous dose (50 mg) and a single oral dose (500 mg) of MB, and after the combination of a single oral dose of MB (500 mg) and a single oral dose of chloroquine (750 mg)

		7000
		6000
		5000
	MB (ng/mL)	3000 4000
		2000
		1000
		0
		0	10	20	30	40	50	60	70	80
						time (h)
	Fig. 3 Mean (± SEM) for MB plasma concentrations after oral intake
	of 500 mg MB either alone (filled diamond) or combined with 750 mg
	chloroquine (filled circles). Note the different plasma concentration
	and time scales in Figs. 2 and 3 as a consequence of the different
	dosing regimens

SD, Standard deviation; CV, coefficient of variation; AUC, area under the substrate concentration time curve; C max , maximum concentration; T max , time to reach maximum concentration; T 1/2 , terminal elimination half-life a Subject vomited within the initial 30 min after drug administration.

Table 1

 1 Individual pharmacokinetic characteristics and mean values ± SD of methylene blue (MB) in plasma after a single intravenous dose (50 mg) and a single oral dose (500 mg) of MB, and after the combination of a single oral dose of MB (500 mg) and a single oral dose of chloroquine (750 mg)

Table 2

 2 Individual pharmacokinetic characteristics and mean values ± SD of MB in whole blood after a single intravenous dose (50 mg) and a single oral dose (500 mg) of MB, and after the combination of a single oral dose of MB (500 mg) and a single oral dose of CQ (750 mg) Mode of administration/subject (nr.) Part AUC 0À1 (ng/mL*h) AUC 0-24 (ng/mL*h C max (ng/mL) T max (h) T 1/2 (h) F 0À1 % ð Þ

	Intravenous
	3	A	8,404.4	7,200.8	1,084.8	0.58	8.6
	4	A	12,848.4	11,282.3	2,063.2	0.17	9.2
	5	A	8,653.1	4,968.6	1,386.7	0.17	20.5
	6	A	7,500.4	5,835.0	1,624.5	0.17	12.5
	11	A	3,701.3	2,534.5	1,695.7	0.17	16.0
	12	A	4,810.4	3,457.3	1,241.3	0.17	13.0
	13	A	8,944.1	6,214.3	1,506.4	0.33	16.0
	14	A	2,407.0	1,581.9	503.2	0.17	15.9
	15	A	3,796.1	2,912.4	2,599.7	0.17	14.0
	16	A	3,604.6	2,948.5	478.8	0.17	10.5
	Mean		6,467	4,894	1,418	0.22	13.6
	SD		3,311	2,888	650	0.14	3.7
	CV (%)		51.2	59.0	45.8	59.8	26.9
	Oral
	2	A	33,977.4	24,299.7	2,189.8	3.0	13.1	n.d.
	3	A	76,626.4	54,998.7	4,951.6	4.0	15.5	91.2
	4	A	124,521.4	84,253.6	7,920.8	2.0	15.4	96.9
	5	A	54,920.9	31,897.4	3,372.4	2.0	18.2	63.5
	6	A	50,276.9	37,794.3	4,177.5	1.5	14.9	67.0
	7	A	29,940.8	21,777.7	2,757.0	1.5	13.9	n.d.*
	11	A	29,902.0	24,461.3	2,708.0	2.0	9.7	80.8
	12	A	46,756.6	26,429.0	3,586.2	1.0	25.9	97.2
	13	A	80,177.0	63,012.9	7,798.6	2.0	12.9	89.6
	14	A	25,531.2	17,808.6	2,448.4	1.0	16.6	106.1
	15	A	21,118.0	18,877.7	2,607.8	3.0	8.7	55.6
	16	A	33,184.2	25,215.2	2,971.4	3.0	12.2	92.1
	Mean		50,578	35,902	3,957	2.2	14.7	84.0
	SD		30,125	20,766	1,981	0.9	4.4	16.7
	CV (%)		59.6	57.8	50.1	42.1	30.0	19.9
	Oral MB-CQ combination			AUC 0-72
	2	B	33,329.9	30,904.4	2,477.9	1.5	22.6
	4	B	68,683.4	56,449.1	4,011.3	2.0	19.1
	6	B	31,068.8	30,470.5	2,862.1	2.0	14.5
	7	B	41,616.6	36,963.5	2,388.1	1.5	26.0
	11 a	B	35,930.1	32,175.2	4,865.0	1.5	24.5
	13	B	89,357.7	79,738.4	5,765.3	3.0	23.1
	15	B	30,651.7	29,532.6	2,650.3	3.0	10.4
	16	B	29,960.2	28,961.8	2,180.2	4.0	15.0
	Mean		45,075	40,649	3,400	2.3	19.4
	SD		21,989	18,212	1,327	0.9	5.6
	CV (%)		48.8	44.8	39.0	39.9	28.8
	n.d., Not determined

a Subject vomited within the initial 30 min after drug administration.

Table 3

 3 Individual pharmacokinetic characteristics and mean values ± SD of CQ in plasma and whole blood after a single oral dose of CQ (750 mg), and after the combination of a single oral dose of MB (500 mg) and a single oral dose of CQ (750 mg) MB as an antimalarial drug. Plasma and whole blood MB concentrations do not differ, suggesting that MB is taken up into cellular blood components, but it does not accumulate intracellularly. The finding that the co-administration of MB and CQ increases MB plasma concentrations while whole blood concentrations are unchanged has potential consequences for the selection of partner drugs in MB-based combination therapies and deserves further investigation.

	Drug therapy/subject (nr.)	AUC 0À1 (ng/mL*h)	C max (ng/mL)	T max (h)
		CQ plasma
	CQ alone/subject (nr.)
	1	10,161.1	256.2	2.0
	3	4,330.2	186.3	2.0
	5	12,928.6	334.3	3.0
	8	6,905.8	180.5	7.0
	12	9,180.0	285.8	3.0
	14	6,805.3	150.3	5.0
	Mean	8,385	232	3.7
	SD	3,018	71	2.0
	CV (%)	36.0	30.7	53.6
	CQ-MB combination
	2	13,224.5	353.8	3.0
	4	5,498.7	343.3	1.5
	6	13,750.5	534.8	3.0
	7	7,932.0	390.9	1.0
	11	1,784.8	79.2	5.0
	13	9,287.9	307.1	1.0
	15	9,262.3	216.0	4.0
	16	4,696.8	117.5	6.0
	Mean	8,180	293	3.1
	SD	4,126	150	1.9
	CV (%)	50.4	51.1	60.7
		CQ whole blood
	CQ alone
	1	28,285.6	630.0	1.5
	3	17,660.4	631.6	2.0
	5	54,300.3	1,214.5	3.0
	8	38,794.6	699.2	7.0
	12	40,122.7	978.9	3.0
	14	31,012.7	610.9	5.0
	Mean	35,029	794	3.6
	SD	12,448	248	2.1
	CV (%)	35.5	31.2	57.5
	CQ-MB combination
	2	47,927.0	1,324.9	3.0
	4	19,631.4	1,013.7	1.5
	6	64,346.1	1,634.9	3.0
	7	38,085.0	1,111.4	1.0
	11	17,046.2	311.8	7.0
	13	44,774.3	1,211.0	2.0
	15	28,797.1	689.8	2.0
	16	19,383.3	483.3	2.0
	Mean	34,999	973	2.7
	SD	16,794	447	1.9
	CV (%)	48.0	45.9	69.6

Eur J Clin Pharmacol (2009) 65:179-189

Acknowledgements

The study was funded by an award from DSM Fine Chemicals Austria, Linz, Austria and by the Deutsche Forschungsgemeinschaft (SFB 544 "Control of Tropical Infectious Diseases" at the Ruprecht-Karls-University Heidelberg). We thank Mrs. Monika Maurer and Mrs. Brigitte Tayrouz for their excellent technical assistance.

Financial disclosure None of the authors has financial or other relationships that are relevant to the study.


Christian Queckenberg
email: christian.queckenberg@uk-koeln.de

Uwe Fuhr

Influence of posture on pharmacokinetics

Keywords: Absorption, Gastric emptying, Pharmacokinetics, Posture

Introduction Body position may influence physiological characteristics, such as perfusion, gastrointestinal function and plasma volume. These characteristics may interact with key factors determining the pharmacokinetics of drugs (dissolution, absorption, distribution, metabolism, excretion). Objectives Based on a systematic literature search, current data on the effect of posture on physiological characteristics and/or pharmacokinetics are summarized, and the relevance of possible effects, such as those presenting in clinical practice and clinical pharmacokinetic studies, is assessed. Results Postures which favour rapid gastric emptying (sitting, standing, recumbent right) accelerate the absorption of orally administered drugs. Consequently, these postures favour a shorter time to reach peak plasma drug concentration (t max ) and a higher maximum plasma drug concentration (C max ) and-in the case of transient saturation of first-pass metabolism-total exposure (area under the concentration-time curve, AUC) in comparison to recumbent left and supine positions (e.g. nifedipine: AUC 30 and 38% higher in standing and right lateral position vs. left lateral position; C max 149 and 80% higher, respectively). The magnitude of these postural effects depends strongly on the nature and amount of liquids and food ingested before drug administration and is most pronounced in the fasting state and after administration with a nonnutrient liquid. Changes in splanchnic-hepatic blood flow (e.g. reduction of estimated hepatic perfusion by 37% in standing vs. supine position) may substantially affect the metabolism of orally administered drugs, especially of those with a high/saturable first-pass metabolism. For highly protein-bound drugs (e.g. phenytoin, imipramine), the total plasma concentration has been found to be approximately 10% higher in standing than lying subjects due to changes in plasma volume. Conclusions Positioning of a patient may be an effective method of enhancing or retarding absorption of some drugs in appropriate clinical situations (e.g. toxic ingestions, bedridden patients). In clinical pharmacokinetic trials, such as bioequivalence studies, defining and maintaining posture precisely is a useful approach for reducing within-and between-subject variability.

Introduction

The influence of body position on physiological characteristics and pharmacokinetics has been investigated for several decades. The first study on this topic (effects of posture on gastric emptying) was reported to have been performed as early as 1918 [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF] when it was observed that barium was emptied faster when subjects lay on the right side compared to other positions. Since then, a multitude of studies has addressed various aspects of postural effects, especially those on gastrointestinal motility, haematological parameters/perfusion and the pharmacokinetics of orally administered drugs.

However, due to the variety of different postures, characteristics, and study procedures that have been chosen, a general consensus on the extent and relevance of postural effects has not yet been reached. Furthermore, the number of potential mechanisms of these effects is quite large, thus complicating a general assessment. In particular, the importance and magnitude of possible postural effects on orally administered drugs has not yet been completely elucidated.

In this review, we assess by which means posture can influence physiological characteristics and identify the links between posture, physiology and pharmacokinetics. We also examine the clinical relevance of postural effects and evaluate whether posture may affect the outcome of clinical pharmacokinetic trials by, for example, influencing the pharmacokinetic key characteristics [area under the concentration-time curve (AUC) and time to reach maximum plasma concentration (C max )] and/or affecting the within-subject and betweensubject variability of these characteristics.

Data

In this review, we have summarized the results of clinical studies that have investigated the influence of posture on gastrointestinal function/motility, haematological parameters, plasma volume, splanchnic-hepatic blood flow and the pharmacokinetics of drugs.

To this end, a comprehensive Medline/PubMed search for reports on areas relating to this topic was carried out, using the key words "posture", "body position", "pharmacokinetics", "gastric emptying" and "absorption". In addition, we manually explored the references of original or review articles and found publications not captured by our systematic keyword research strategy. We only included peer-reviewed articles published up to April, 2008. Conference abstracts, data only given in the product information and information available only on the webpage of the U.S. drug registration agency (FDA) were not included.

Effects of posture on the gastric residence time of fluids and meals

For nonnutrient liquids, the mechanisms by which posture affects gastric motility and gastric emptying were clarified by Anvari et al. [START_REF] Anvari | Effects of posture on gastric emptying of nonnutrient liquids and antropyloroduodenal motility[END_REF]. These researchers examined the effects of posture (sitting vs. left lateral) on gastric emptying, intragastric distribution and antropyloroduodenal motility following ingestion of 150 ml saline, both with and without coadministration of intravenous atropine (see Table 1). As expected, atropine slowed gastric emptying in both postures (time required for 50% total stomach emptying in sitting position: 18 vs. 14 min; left lateral: 35 vs. 28 min). However, both with and without atropine, emptying of the saline solution was faster in sitting subjects. The authors concluded that posture influences the intragastric distribution of liquids due to the effect of gravity such that, for anatomical reasons, in the left lateral (= "pylorus up") and supine posture, gravity cannot accelerate gastric emptying. Normally, the liquid is emptied according to a first-order process (exponentially): the larger the volume, the faster the emptying [START_REF] Golub | Physiologic considerations in drug absorption from the gastrointestinal tract[END_REF].

Moreover, the trial showed that the effects of gravity on gastric emptying of nonnutrient liquids are also likely to reflect changes in antropyloric motility. The sitting position was associated with increases in (antro-) pyloric pressure waves and in basal pyloric pressure and, hence, with more "active pumping".

The importance of gravity for gastric emptying was further demonstrated by a trial of Horowitz et al., who coadministered 60 ml olive oil and 290 ml aqueous soup. They also compared sitting with left lateral position. Gastric emptying of the oil was slower in the sitting position; the aqueous phase, however, was emptied much slower in left lateral position (see Table 1), indicating that both phases were emptied according to their specific density due to intragastric "layering" of the nonemulsified oil and aqueous phase according to gravity [START_REF] Horowitz | The effect of posture on gastric emptying and intragastric distribution of oil and aqueous meal components and appetite[END_REF]. Similar results were obtained by Burn-Murdoch et al., who studied the emptying of 750 ml of a saline drink in the sitting, left lateral and right lateral position. They found the emptying to be most rapid in the right lateral position ("pylorus down"), with the difference being significant compared to emptying in the left lateral position [START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF].

For high nutrient/high calorie liquids (e.g. glucosecontaining drinks or olive oil), the effects of changes in posture are much less pronounced than those for non-or low-nutrient liquids (e.g. saline or low-nutrient beef consommé soup) [START_REF] Horowitz | The effect of posture on gastric emptying and intragastric distribution of oil and aqueous meal components and appetite[END_REF][START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF][START_REF] Jones | Effects of posture on gastric emptying, transpyloric flow, and hunger after a glucose drink in healthy humans[END_REF]. In the trial cited above, Burn-Murdoch et al. did not find any significant influence of posture on the emptying rate of 750 ml of a glucose drink. Despite the force of gravity being the same for both kinds of liquids, corresponding studies confirmed this difference [START_REF] Hunt | The effect of gravity on gastric emptying with various test meals[END_REF][START_REF] Jones | Effects of posture on gastric emptying, transpyloric flow, and hunger after a glucose drink in healthy humans[END_REF]. Whereas intragastric volume and gravity appear to be the driving forces controlling gastric emptying of nonnutrient liquids [START_REF] Anvari | Effects of posture on gastric emptying of nonnutrient liquids and antropyloroduodenal motility[END_REF][START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF][START_REF] Moore | Effect of body posture on radionuclide measurements of gastric emptying[END_REF], for nutrient liquids, feedback from small intestinal "nutrient" receptors is the decisive factor. These receptors, which are sensitive to, for example, carbohydrates and digestion products of fat, mediate the slowing of gastric emptying [START_REF] Anvari | Effects of posture on gastric emptying of nonnutrient liquids and antropyloroduodenal motility[END_REF][START_REF] Horowitz | The effect of posture on gastric emptying and intragastric distribution of oil and aqueous meal components and appetite[END_REF][START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF][START_REF] Moore | Effect of body posture on radionuclide measurements of gastric emptying[END_REF]. This is also reflected in the emptying pattern, which is predominantly linear for nutrient liquids and exponential for nonnutrient ones [START_REF] Anvari | Effects of posture on gastric emptying of nonnutrient liquids and antropyloroduodenal motility[END_REF]. Consequently, nutrient liquids are emptied slower, and the process is largely independent of posture [START_REF] Anvari | Effects of posture on gastric emptying of nonnutrient liquids and antropyloroduodenal motility[END_REF][START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF] (see results of Burn-Murdoch et al., Table 1). In terms of the effects of posture on the gastric emptying of solid food, conflicting results have been published. Whereas Asada et al. [START_REF] Asada | Effect of body position on gastric emptying of solid food-a study using a sulfamethizole capsule food method[END_REF] found gastric emptying to be more delayed in the supine position versus the sitting position, Doran et al. did not see any relevant influence of posture (left lateral vs. sitting) following the administration of both a small and a large meal [START_REF] Doran | Effects of meal volume and posture on gastric emptying of solids and appetite[END_REF]. Doran et al. concluded that posture has only a minor influence on intragastric distribution and no effect at all on gastric emptying of solid food. Similar results were obtained by Mannell et al. [START_REF] Mannell | Gastric emptying of a solid meal[END_REF]. Two trials investigating gastric emptying of a meal containing a nutritive liquid and solid food found the food to be emptied slower in the supine position compared to the sitting [START_REF] Moore | Effect of body posture on radionuclide measurements of gastric emptying[END_REF], [START_REF] Spiegel | Effects of posture on gastric emptying and satiety ratings after a nutritive liquid and solid meal[END_REF] and standing position [START_REF] Moore | Effect of body posture on radionuclide measurements of gastric emptying[END_REF] (see Table 1). Both authors concluded that the effects of posture and gravity on the rate of gastric emptying are more pronounced after coadministration of solid food and nutrient liquid than after the administration of either component alone. After coadministration, propulsive motor activity apparently does not increase sufficiently to overcome the effects of gravity [START_REF] Spiegel | Effects of posture on gastric emptying and satiety ratings after a nutritive liquid and solid meal[END_REF].

Posture and appetite

Somehow, surprisingly, appetite may also be influenced by posture. After ingestion of a 600-ml glucose test drink, hunger and prospective consumption were significantly less in the sitting position than in the supine position, continuously from 45 min until the end of the observation period 180 min after ingestion [START_REF] Jones | Effects of posture on gastric emptying, transpyloric flow, and hunger after a glucose drink in healthy humans[END_REF]. Horowitz et al. found a reduction of hunger in the left lateral decubitus position compared to sitting. After the administration of olive oil and soup, hunger was less in the subjects lying on their left side at both at 120 and at 180 min post-ingestion [START_REF] Horowitz | The effect of posture on gastric emptying and intragastric distribution of oil and aqueous meal components and appetite[END_REF]. Corresponding results were obtained by Carney et al. for the same postures and a similar meal [START_REF] Carney | Gastric emptying of oil and aqueous meal components in pancreatic insufficiency: effects of posture and on appetite[END_REF]. In contrast, Spiegel et al. found no posture-dependent differences in postprandial hunger after the ingestion of soup and a sandwich (seated vs. supine), which they explained by a different meal composition and a shorter observation period (0-120 min post-ingestion) [START_REF] Spiegel | Effects of posture on gastric emptying and satiety ratings after a nutritive liquid and solid meal[END_REF]. After the ingestion of a solid meal (without liquid component), no statistical significant difference in satiety ratings for sitting and left lateral position could be observed [START_REF] Doran | Effects of meal volume and posture on gastric emptying of solids and appetite[END_REF].

Effects of posture on the absorption of orally administered drugs

The extent of absorption from the stomach is negligible for the vast majority of drugs [START_REF] Davis | Transit of pharmaceutical dosage forms through the small intestine[END_REF][START_REF] Heading | The dependence of paracetamol absorption on the rate of gastric emptying[END_REF]. Thus, the rate of gastric emptying is the principal factor and one of the most important rate-limiting steps affecting the absorption of orally administered drugs [START_REF] Backon | The lateral decubitus position may affect gastric emptying through an autonomic mechanism: the skin pressure-vegetative reflex[END_REF]. In the case of saturable absorption kinetics (e.g. amoxycillin [START_REF] Sanchez | New formulations of amoxicillin/clavulanic acid: a pharmacokinetic and pharmacodynamic review[END_REF]) and if there is a small "absorption window", accelerated gastric emptying may decrease overall systemic bioavailability [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF][START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF]. Usually, as has been shown for a solution of acetaminophen (20 mg/kg), the first-order rate constant for drug transfer from the intestinal lumen into the systemic circulation is greater than the first-order rate constant for gastric emptying [START_REF] Clements | Kinetics of acetaminophen absorption and gastric emptying in man[END_REF]. The mean half-time for drug transfer was 6.8 ± 0.9 min in this trial, whereas the mean gastric emptying half-time was 12 min. Consequently, gastric emptying, which is predominantly pulsatile, is the ratelimiting step in the early phase of absorption of orally administered drugs (provided the drug is well absorbed/ highly soluble according to the Biopharmaceutics Classification System classification and given as a solution or rapidly dissolving dosage form; if dissolution is slow, it is rate-limiting). As posture is known to influence gastric emptying [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF][START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF][START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF], in many cases the rate-limiting step for absorption can be influenced by posture. Moreover, in addition to the factors discussed above, it may also depend on posture as to whether the rate-limiting step is dissolution or gastric emptying, with respective consequences for the pharmacokinetic profile.

The resulting differences in the rate (and possibly extent) of absorption may affect various pharmacokinetic characteristics. As shown in Table 2, several studies, for example, with paracetamol [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF][START_REF] Vance | Optimal patient position for transport and initial management of toxic ingestions[END_REF][START_REF] Wallden | The delay of gastric emptying induced by remifentanil is not influenced by posture[END_REF], nifedipine [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF], amoxycillin [START_REF] Roberts | Effect of posture and sleep on pharmacokinetics. I. Amoxycillin[END_REF] and theophylline [START_REF] Warren | Posture and theophylline kinetics[END_REF], have found an increase in the time to reach the maximum serum drug concentration (t max )/mean residence time (MRT) and/or a decrease in maximum plasma drug concentration (C max )/ (early) AUC for left lateral and supine position compared to other positions (right lateral, prone, sitting, standing). The effects of the respective postures on pharmacokinetics correspond to their effects on gastric emptying, as discussed above (left lateral and supine positions tend to decrease the rate of gastric emptying). These differences were reported consistently by the studies; however, the extent of these postural effects on pharmacokinetics differs widely, ranging from "no effect" to an increase of 149% (C max of nifedipine in standing vs. left lateral position; see Table 2 [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF]). For nifedipine, however, the additive postural effect on metabolism also has to be taken into consideration (see below). The extent of postural effects on absorption depends on several factors, especially on the dissolution and absorption characteristics of the drug/dosage form and on the quality/quantity of food and liquids ingested before or shortly after intake of the drug. Based on the considerations stated above, it can be expected that slow dissolution/absorption kinetics of the dosage form/agent and coadministration of solid food or nutrient liquids will diminish or abolish the effect of posture on the absorption and pharmacokinetics of orally administered drugs.

Influence of posture on haematological parameters, plasma volume and distribution

Another way by which body position may affect pharmacokinetics is the postural influence on plasma volume and haematological parameters. Due to gravity, the standing position results in a high hydrostatic pressure in lower body parts, leading to an intravascular fluid loss caused by filtration of plasma into the interstitium [START_REF] Maw | Redistribution of body fluids during postural manipulations[END_REF]. This fluid loss accounts for approximately 9.5% of the blood volume and 16.2% of plasma volume, respectively (compared to the supine position; see Table 1) [START_REF] Hagan | Plasma volume changes with movement to supine and standing positions[END_REF]. Thus, as the plasma concentrations of drugs can depend on plasma volume and its changes (especially in the case of high-molecular and highly protein-bound drugs), changes in pharmacokinetic characteristics (e.g. C max ) up to a similar magnitude may be expected for some drugs.

The effects of posture in this context have been studied extensively over the past decades, with the results being largely consistent [START_REF] Maw | Redistribution of body fluids during postural manipulations[END_REF][START_REF] Hagan | Plasma volume changes with movement to supine and standing positions[END_REF][START_REF] Dukova-Peneva | Effect of body position on substrates, enzymes and electrolytes in the serum of healthy subjects[END_REF][START_REF] Penev | Effect of body position on laboratory hematological indices[END_REF]. A change in body position from recumbent to sitting and to erect results in a significant increase in haematological laboratory values, such as haemoglobin, haematocrit, erythrocytes, leucocytes and platelets, due to reversible changes in the concentration and redistribution of the blood [START_REF] Hagan | Plasma volume changes with movement to supine and standing positions[END_REF][START_REF] Penev | Effect of body position on laboratory hematological indices[END_REF]. Relative to the sitting position, blood volume increases by 89 ml (mean) after 30 min in supine posture and decreases by 406 ml while standing, with these differences being primarily the result of plasma shifts between the interstitium and circulation [START_REF] Maw | Redistribution of body fluids during postural manipulations[END_REF] (see Table 1). The haemo-concentration while standing is supposed to involve all blood compounds-and drugsthat cannot permeate the capillary membrane. Equivalent changes have been found for total protein, albumin and the activity of several enzymes [START_REF] Dukova-Peneva | Effect of body position on substrates, enzymes and electrolytes in the serum of healthy subjects[END_REF].

Not surprisingly, the same mechanism applies to highly protein-bound drugs, such as phenytoin, imipramine and desipramine, whose total plasma concentrations increase significantly after standing for 40 min compared to lying (mean increase: 10.5, 9.8 and 11.5%, respectively) [START_REF] Abalan | Effect of posture on imipramine and desipramine plasma concentrations[END_REF][START_REF] Abalan | Effect of posture on total phenytoin plasma concentration[END_REF]. In addition, the plasma level of endogenous hormones (thyroxine, cortisol) can be influenced to a similar extent (mean increase: 6.8 and 32.6%, respectively) [START_REF] Abalan | Effect of posture on total thyroxine plasma concentration[END_REF][START_REF] Abalan | Effect of posture on total cortisol plasma concentration[END_REF]. The large increase in cortisol is explained by a second effect of posture in this case: standing induces cortisol secretion as a physiological response to the resulting changes in plasma volume, circulation and blood pressure.

Apart from these effects on blood composition and plasma volume, it is likely that there is no further relevant influence of posture on the distribution of drugs. The results of Rumble et al. on paracetamol suggest that previously reported changes in the volume of distribution do not exist [START_REF] Rumble | Effects of posture and sleep on the pharmacokinetics of paracetamol (acetaminophen) and its metabolites[END_REF]. As a general rule, because distribution is mainly determined by the physico-chemical properties of the administered drug, significant changes due to posture seem unlikely. The free (unbound) concentration of a drug in plasma is not supposed to be influenced by posture, provided the drug is able to permeate the capillary membranes. For high-molecular drugs unable to permeate the membrane (e.g. heparin), from a theoretical point of view the free concentration may also depend on posture.

Influence of posture on splanchnic-hepatic blood flow and metabolism

There is ample evidence that posture, as well as food and exercise, may influence splanchnic-hepatic blood flow [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF][START_REF] Modi | Influence of posture on hepatic perfusion and the presystemic biotransformation of propranolol: simulation of the food effect[END_REF][START_REF] Stromberg | Exercise alters the pharmacokinetics of midazolam[END_REF]. In subjects standing upright for 15 min, estimated hepatic blood flow (clearance of indocyanine green) was reduced by 37% compared to supine position [START_REF] Daneshmend | Physiological and pharmacological variability in estimated hepatic blood flow in man[END_REF]. The authors of this study concluded that the influence of posture should be considered during pharmacokinetic assessment of high-clearance drugs. Similarly, Culbertson et al. also reported a mean 37% reduction of estimated hepatic blood flow in the standing compared to supine position [START_REF] Culbertson | The effect of the upright posture upon hepatic blood flow in normotensive and hypertensive subjects[END_REF].

Changes in splanchnic-hepatic blood flow may substantially affect the absorption and metabolism of orally administered drugs, especially of those with a high firstpass metabolism [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF][START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF]. In cases of partial saturation of first-pass metabolism, overall systemic bioavailability can depend on the rate of absorption [START_REF] Ludden | Nonlinear pharmacokinetics: clinical Implications[END_REF] and, therefore, on posture as well.

As shown above, gastric emptying and intestinal motility affect the rate of absorption of drugs; this may also influence the extent of first-pass metabolism [START_REF] Pond | First-pass elimination. Basic concepts and clinical consequences[END_REF]. Postures which favour rapid gastric emptying (i.e. standing, sitting and recumbent right) may cause a transient saturation of first-pass metabolism due to the more rapid delivery of the agent to the metabolizing enzymes in the gut wall and liver. This is especially true if the administered drugs are absorbed rapidly after gastric emptying and undergo extensive presystemic elimination, as faster gastric emptying may increase the overall bioavailability of drugs with saturable first-pass metabolism, such as propranolol [START_REF] Keller | Saturable first-pass kinetics of propranolol[END_REF], nifedipine [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF] and ethanol [START_REF] Holford | Clinical pharmacokinetics of ethanol[END_REF]. On the other hand, slower absorption may decrease overall systemic availability [START_REF] Ludden | Nonlinear pharmacokinetics: clinical Implications[END_REF].

The possible effects of posture in this context can be illustrated by the results of the study by Renwick et al. [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF] (see Table 2). These researchers co-administered nifedipine and paracetamol and compared the left lateral, right lateral and standing positions, with the respective posture being subsequently maintained for 4 h. In left lateral position, t max was prolonged for both drugs, indicating slower gastric emptying; C max and AUC were lower, but with this difference being significant for nifedipine only. When the left lateral (= "pylorus up") position was compared to the right lateral (= "pylorus down") position, even one pharmacodynamic parameter (increase in heart rate following nifedipine administration) was significantly greater when the subject was lying on the right side.

The significantly higher AUC of nifedipine (but not of paracetamol) in the two postures that favour rapid gastric emptying (right lateral 38% higher, standing position 30%) suggests that there was an enhanced bioavailability of nifedipine due to a reduced first-pass extraction during transient saturation of first-pass metabolism [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF].

In contrast to these findings, several other studies have found no significant effect of posture on the first-pass metabolism of highly extracted drugs [START_REF] Modi | Influence of posture on hepatic perfusion and the presystemic biotransformation of propranolol: simulation of the food effect[END_REF][START_REF] Gupta | The effect of food, time of dosing, and body position on the pharmacokinetics and pharmacodynamics of verapamil and norverapamil[END_REF][START_REF] Challenor | The effects of food and posture on the pharmacokinetics of a biphasic release preparation of nifedipine[END_REF]. These contradictory results can be explained by various differences between the studies, such as different elimination characteristics of the drugs used: if elimination is a saturable (mixed order) process, a significant effect of posture is possible; on the other hand, if elimination is a first-order process, posture will have no impact on bioavailability even though extraction may be high. Furthermore, differences between the postures (including their definition/duration of maintaining) and dosage forms may have contributed to the differing results. For example, in the case of osmotically controlled release (Gupta et al. [START_REF] Gupta | The effect of food, time of dosing, and body position on the pharmacokinetics and pharmacodynamics of verapamil and norverapamil[END_REF]), the rate of drug release from the formulation is the limiting factor for the rate of absorption; hence, posture cannot influence the rate-limiting step within this setting.

Effects on renal elimination/clearance

Posture may also influence the renal elimination of drugs. After oral administration of lithium to volunteers who adopted either a supine or sitting position or walked around, lithium and sodium clearance tended fell in the sitting position and fell significantly while the subjects were walking around (30, 29 and 26 ml/min, respectively) [START_REF] Kamper | The influence of body posture on lithium clearance[END_REF]. The decreased lithium clearance was not associated with any significant change in glomerular filtration rate, indicating an increase in both the absolute and the fractional proximal tubular reabsorptive rate (APR: walking 92, supine 83 ml/min; p<0.005. FPR: walking 0.78, supine 0.73 ml/min; p<0.025).

Effects on the skin pressure-vegetative reflex

In terms of the right and left lateral decubitus position, pharmacokinetic characteristics may be additionally affected by posture through an autonomic mechanism [START_REF] Backon | The lateral decubitus position may affect gastric emptying through an autonomic mechanism: the skin pressure-vegetative reflex[END_REF]. There are indications that pressure on the right side increases vagal tone due to the skin pressure-vegetative reflex, whereas lying on the left side increases sympathetic tone [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF][START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF][START_REF] Backon | Right lateral decubitus position via skin pressure vegetative reflex may prevent anxiety, adverse autonomic reactions, and syncope in blood donors[END_REF]. As vagal and sympathetic tone affect gastrointestinal motility, this mechanism may have contributed to the fact that several studies [START_REF] Burn-Murdoch | Does lying on the right side increase the rate of gastric emptying?[END_REF][START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF][START_REF] Vance | Optimal patient position for transport and initial management of toxic ingestions[END_REF] have shown faster gastric emptying/absorption in subjects lying on the right side compared to the left.

Posture and route of administration

The influence of posture on the pharmacokinetics of drugs depends also on the route of administration. Both the mechanism and extent of postural effects on pharmacokinetic characteristics differ between drugs administered orally, intravenously or intramuscularly.

Several studies failed to show any relevant postural effect on metabolism or elimination of the drugs administered intravenously [START_REF] Hinderling | The pharmacokinetics of furazlocillin in healthy humans[END_REF][START_REF] Breiby | Effect of posture on ampicillin pharmacokinetics, glomerular filtration rate and renal plasma flow in resting subjects[END_REF][START_REF] Rumble | The effect of posture on the pharmacokinetics of intravenous benzylpenicillin[END_REF][START_REF] Kates | Influence of prolonged recumbency on drug disposition[END_REF]. As there is no absorption phase after intravenous application, the effects of posture on gastric emptying are not relevant. Effects on splanchnichepatic blood flow may occur, but in absence of a (potentially posture-sensitive) first-pass effect, these effects are less significant. The effects on C max , such as higher peak plasma concentrations in the upright (18.3 mg/l) position versus the supine position (12.4 mg/l), which were seen initially after intravenous administration of theophylline [START_REF] Warren | Theophylline concentrations and posture[END_REF][START_REF] Warren | The effect of posture on the sympathoadrenal response to theophylline infusion[END_REF], are supposed to be based principally on the lower volume of the central compartment due to reversible haemo-concentration and, possibly, to vasoconstriction while standing [START_REF] Warren | The effect of posture on the sympathoadrenal response to theophylline infusion[END_REF]. Hence, these effects are usually short-time effects prior to the final distribution of the drug (except for drugs that are not distributed outside the systemic circulation). With the same exception, total exposure (AUC) is not supposed to be affected by posture after intravenous administration.

Rumble et al. studied the effects of posture on the pharmacokinetics of benzylpenicillin after intramuscular administration. Their results suggest that no significant changes occur (ambulation vs. supine: t ½ 52.5 and 50.9 min; AUC: 1175 and 1032 min mg/l, respectively). The authors concluded that these small differences are unlikely to be clinically relevant. Absorption seemed to be independent of posture; the major source of posturally determined (but not significant) pharmacokinetic differences after intramuscular application appeared to be clearance [START_REF] Rumble | The effects of posture on the pharmacokinetics of intramuscular benzylpenicillin[END_REF].

In summary, posture may influence the pharmacokinetics of orally administered drugs by affecting:

1. gastrointestinal motility/rate of gastric emptying 2. absorption 3. distribution/plasma volume 4. splanchnic-hepatic blood flow 5. metabolism/presystemic elimination 6. renal elimination/clearance 7. the skin pressure-vegetative reflex It therefore appears that all pharmacokinetically relevant steps after oral ingestion of a drug can be affected by posture-with the exception of the dissolution of the agent from the pharmaceutical preparation (from a purely theoretical point of view, dissolution may be influenced indirectly by posture via effects on gastric motility and the rate of gastric emptying).

However, the magnitude of postural effects on pharmacokinetics of an orally administered drug depends on various factors, including [START_REF] Oosterhuis | Pharmacokinetic studies in healthy volunteers in the context of in vitro/in vivo correlations[END_REF]

Implications for clinical practice

Compared to individual key factors that influence the pharmacokinetics of orally administered drugs and its variability (e.g. concomitant medication, phenotype of metabolizing enzymes/elimination, food intake), the effect of posture appears to be of minor importance. Generally, the occurrence of a clinically relevant effect of posture on pharmacokinetics might be expected if the drug has a steep concentration-response curve, is absorbed rapidly, undergoes saturable presystemic elimination, has a narrow therapeutic range, is administered orally in fasted state with a non-nutrient liquid, and if a rapid onset of the pharmacological effect is important. In these cases, in combination with prolonged maintenance of the same posture after intake, pharmacokinetic differences due to body position may be clinically relevant. For example, the positioning of a patient may be an effective method of enhancing or retarding drug absorption in appropriate clinical situations [START_REF] Backon | The lateral decubitus position may affect gastric emptying through an autonomic mechanism: the skin pressure-vegetative reflex[END_REF]. After the administration of acetaminophen (paracetamol) in an oral overdose model (80 mg/kg), Vance et al. found the AUC 0-2h to be significantly lower for both the left lateral (6.0) and supine posture (6.6) compared to the prone (7.4), sitting (8.6) and right lateral positions (9.0 min mg/l; see Table 2) [START_REF] Vance | Optimal patient position for transport and initial management of toxic ingestions[END_REF]. Hence, mean absorption and total systemic exposure were (at least) during the first 2 hours significantly lower in these two former postures (up to 33% lower for left compared to right lateral position). Based on these results, the authors recommended the use of the left lateral decubitus position for initial management of patients with toxic ingestions. On the other hand, for rapid onset of therapeutic effects, it would be advisable to avoid the left lateral and supine positions after the oral intake of a drug.

In normal clinical-especially ambulant-practice, however, daily activities of the patients are likely to cause a multitude of alterations in posture after drug administration. Therefore, and because for most of the drugs total AUC is not affected by posture, the clinical impact for mobile patients would seem to be quite limited. In terms of bedridden patients, particularly those with severe illness and/or those taking drugs with a narrow therapeutic range, the situation may be different: to position a patient in right lateral posture may accelerate the onset of therapeutic effects; on the other hand, avoiding the right lateral posture after intake may help to prevent toxic peak concentrations. Thus, in some cases, the adoption of a certain posture or adjustment of the dose regimen will be advisable for pharmacokinetic reasons. Additionally, in some cases (bisphosphonates), adoption/avoiding of certain postures after intake is required not for pharmacokinetic reasons, but to prevent side effects (oropharyngeal ulcer).

Implications for clinical pharmacokinetic studies

Pharmacokinetic data are commonly derived from subjects staying in bed and/or maintaining a standardized posture during the initial assessment of an orally administered drug. However, posture is not always defined strictly in these trials. As stated above, posture can influence the ratelimiting step in absorption (both whether it is gastric emptying or dissolution and, if it is gastric emptying, its rate), with respective consequences for the pharmacokinetic profile of orally administered drugs. The possible influences of posture on decisive pharmacokinetic characteristics, such as AUC and C max , clearly indicate that the posture of participating volunteers should be defined and maintained precisely, especially in the case of drugs which are absorbed rapidly and are subject to presystemic elimination [START_REF] Renwick | The influence of posture on the pharmacokinetics of orally administered nifedipine[END_REF]. At least throughout the phase of absorption, any change of posture should be avoided. This is especially relevant for bioequivalence studies, where the subjects should be carefully instructed and supervised. An inadequate definition and control of posture may result in an increase in both between-subject and within-subject variability of the various pharmacokinetic characteristics.

In bioequivalence trials, possible alterations in the key characteristics AUC and C max may cause a broader confidence interval, thus increasing the risk of failing to show bioequivalence. C max in particular, which is considerably sensitive to changes in the rate of absorption, can be notably influenced by posture. Hence, the precise definition of posture may help to reduce costs, for example, in the number of subjects required, and/or improve the outcome of bioequivalence studies.

However, as shown in Table 2, the data available to date is limited, and the sample sizes in most of the trials are quite small. Experimental clinical trials, such as large bioequivalence studies comparing tight posture control versus no control, would be needed to confirm and assess quantitatively the impact of posture on pharmacokinetics and its variability.

Table 1

 1 Influence of posture on gastric emptying and on haematological parameters of healthy volunteers

	Outcome/conclusion				Gastric contents recovered at 10 min (saline),	20 min (glucose):	Saline: right side 215 ml, left 431 ml (p<0.005);	sitting: 308 ml	Glucose: no significant differences (555, 589	and 564 ml)	Gastric emptying half time: 15.8 min (sitting)	vs. 18.3 min (UPD) (p=0.07)	Emptying pattern: linear for UPD, exponential for sitting	Gastric emptying delayed in supine position	Sitting: emptying of oil slower than of aqueous phase	Lag phase (oil) 44.3 min vs. 7.6 min	(aqueous phase); p<0.01	Left side: emptying of oil faster than of aqueous phase	Lag phase (oil) 16.7 min vs. 160.9 min	(aqueous phase); p<0.01	No effect on gastric emptying	No effect on gastric emptying	Supine position slowed GE of beef, compared	to both sitting (t ½ 116.9 vs. 75.8 min;	p=0.012) and standing (70.4; p=0.015)	GE slower in supine position for both soup	(p<0.05) and sandwich (p<0.025)	Atropine slows gastric emptying (both postures; p<0.05).	GE on left side slower, both with and without	atropine (p<0.05)	Values of haemoglobin, haematocrit, erythrocytes,	leucocytes and platelets increase significantly	with the change from recumbent to sitting and to erect	Significant increase of total protein, albumin,	calcium and of activity of creatine kinase,	alkaline phosphatase, gamma GTP
		Sitting Standing Upside	down		x						x x			x	x						x	x	x x			x		x			x x			x x
			Supine Prone											x								x	x			x					x			x
	Body position	Lying on:	Right Left	side side	x x										x						x							x
	Characteristic/	parameter			Rate of gastric	emptying					GE			GE	GE						GE	GE	GE			GE		GE, motility,	intragastric	distribution	Haematological	lab values		Total protein,	substrates,	enzymes and	electrolytes	in serum
	Author Intervention; number	of volunteers			Burn-Murdoch [5] 750 ml saline drink 750 ml	glucose drink (n=5)					Steingoetter [50] 300 ml water (n=12)			Asada [9] Solid food (n =30)	Horowitz [4] 60 ml olive oil plus 290 ml	soup (aqueous) (n=11)					Doran [10] Large and small meal (n=8)	Jones [7] 600 ml glucose drink (n=8)	Moore [8] Standard beef meal, orange	juice (n=8)		Spiegel [12] Tomato soup, egg sandwich	(n=9)	Anvari [2] 150 ml saline drink, with and	without atropine i.v. (n=7)		Penev [27] Change of body position	(15 min per posture; n=32)		Dukova-Peneva [26] Change of body position	(15 min per posture; n=32)

Table 1 (

 1

		Outcome/conclusion			Relative to seated: increase of blood volume	in supine posture (mean: 89 ml), decrease while	standing (406 ml), primarily due to plasma shifts	between circulation and interstitium.	In standing position vs. supine: increase of mean hct	(10.3%), hb (10.8%), plasma proteins (20.8%);	decrease of mean blood and plasma volume	(9.5 and 16.2%, respectively)
			Sitting Standing Upside	down	x x				x
				Supine Prone	x				x
		Body position	Lying on:	Left Right	side side
		Characteristic/	parameter		Distribution of	intra-and	extravascular	fluid volumes	Haematological	lab values,	blood and	plasma volume
		Intervention; number	of volunteers		Change of body	position (30 min	per posture; n=8)		Change of body	position (35 min	per posture; n=7)
	continued)	Author			Maw [24]				Hagan [25]

GE, Gastric emptying; i.v. intravenous; hct, haematocrit; hb, haemoglobin; GTP, glutamyltranspeptidase

Table 2

 2 Influence of posture on the pharmacokinetic characteristics of orally administered drugs and endogenous hormones

	Body position
	Characteristic
	Study drug,	number of	volunteers
	Author

max , time to reach maximum serum drug concentration