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Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers
Accepted Manuscript Title: Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers Author: Song Guo Jens P. Goetze Jørgen L. Jeppesen John C Burnett Jes Olesen Inger Jansen-Olesen Messoud Ashina PII: DOI: Reference:
S0196-9781(15)00268-5 http://dx.doi.org/doi:10.1016/j.peptides.2015.09.008 PEP 69549
To appear in:
Peptides
Received date: Revised date: Accepted date:
25-8-2015 21-9-2015 23-9-2015
Please cite this article as: Guo Song, Goetze Jens P, Jeppesen Jorgen L, Burnett John C, Olesen Jes, Jansen-Olesen Inger, Ashina Messoud.Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers.Peptides http://dx.doi.org/10.1016/j.peptides.2015.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers Song Guo1, Jens P. Goetze2, Jørgen L. Jeppesen3, John C Burnett4, Jes Olesen1, Inger JansenOlesen1, Messoud Ashina1* MD PhD DMSc [email protected] 1
Danish Headache Center and Department of Neurology, Rigshospitalet Glostrup, Faculty of
Health Sciences, University of Copenhagen, 2600 Glostrup, Copenhagen, Denmark 2
Department of Clinical Biochemistry, Rigshospitalet Blegdamsvej, Faculty of Health and
Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark 3
Department of Medicine, Hvidovre Hospital Glostrup, Faculty of Health and Medical
Sciences, University of Copenhagen, 2600 Glostrup, Copenhagen, Denmark 4
Departments of Internal Medicine and Physiology, Division of Cardiovascular Disease,
Cardiorenal Research Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55906, USA *Corresponding author at: Danish Headache Center and Department of Neurology, Rigshospitalet Glostrup, Faculty of Health and Medical Sciences, University of Copenhagen, Nordre Ringvej 57, 2600 Glostrup, Copenhagen, Denmark, Tel.: (+45) 38 63 33 85.
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Highlights
Natriuretic peptides in physiological and pharmacological doses have no effects on cerebral arteries in healthy volunteers.
Natriuretic peptides showed dose-dependent relaxation of cerebral arteries in guinea pigs.
This is the first study investigating the effects of natriuretic peptides on cerebral arteries in humans.
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Abstract The natriuretic peptides (NPs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), have vasoactive functions that concern humans and most animals, but their specific effects on cerebral circulation are poorly understood. We therefore examined the responsiveness of cerebral arteries to different doses of the natriuretic peptides in animals and humans. We conducted a dose-response experiment in guinea pigs (in vitro) and a double-blind, three-way cross-over study in healthy volunteers (in vivo). In the animal experiment, we administered cumulative doses of NPs to pre-contracted segments of cerebral arteries. In the main study, six healthy volunteers were randomly allocated to receive two intravenous doses of ANP, BNP or CNP, respectively, over 20 min on three separate study days. We recorded blood flow velocity in the middle cerebral artery (VMCA) by transcranial Doppler. In addition, we measured temporal and radial artery diameters, headache response and plasma concentrations of the NPs. In guinea pigs, ANP and BNP but not CNP showed significant dose-dependent relaxation of cerebral arteries. In healthy humans, NP infusion had no effect on mean VMCA, and we found no difference in hemodynamic responses between the NPs. Furthermore, natriuretic peptides did not affect temporal and radial artery diameters or induce headache. In conclusion, natriuretic peptides in physiological and pharmacological doses do not affect blood flow velocity in the middle cerebral artery or dilate extracerebral arteries in healthy volunteers.
Keywords: Natriuretic peptides; atrial; brain; C-type; cerebral arteries; cerebral blood flow; headache.
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1. Introduction Natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and Ctype natriuretic peptide (CNP), are structurally related hormones [1]. The biological effect of these peptides in humans and most animals is to maintain cardiovascular homeostasis through their vasoactive and diuretic properties [2–6]. ANP and BNP are predominantly produced in the heart [7, 8], but all three natriuretic peptides are expressed in the brain and blood vessels [9–14]. Since their discovery, the diagnostic and therapeutic importance of natriuretic peptides has increased tremendously, especially in the field of clinical cardiology [15–18]. However, the biological effects of natriuretic peptides on cerebral circulation are still poorly understood [19]. To our knowledge, no in vivo or in vitro studies in humans of the effect of natriuretic peptides on cerebral or extracranial arteries have been conducted. Four animal in vitro studies have examined the vasodilatory response of natriuretic peptides on cerebral arteries. Two studies in guinea pigs and rabbits showed vasodilation [20, 21] while the other two in rats showed no response [22, 23]. Possible interspecies differences between humans and animals have never been investigated, but potential clinical implications of the peptides may, however, be relevant in neurovascular disorders such as stroke and migraine [19]. We hypothesized that natriuretic peptides dilate the cerebral arteries in a dosedependent manner. Therefore, we examined the responsiveness of cerebral arteries to natriuretic peptides with different doses in animals (in vitro) and in healthy volunteers (in vivo). In addition, we measured the diameters of the superficial temporal artery and radial artery, blood flow in face microvasculature, the headache inducing properties of the natriuretic peptides and their plasma concentrations before, during and after infusion.
2. Materials and Methods
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We conducted three separate experiments (Fig. 1). The first experiment was an in vitro animal experiment where we tested the effect of the three natriuretic peptides on isolated middle cerebral arteries (MCA) and basilar arteries (BA) from guinea pigs. The second was a pilot experiment with healthy volunteers to determine the optimal dose of the natriuretic peptides for the main experiment. In the third (and main) experiment, we examined the vasodilatory effects of all three natriuretic peptides. All participants gave written informed consent before inclusion. The study was approved by the ethics committee of Copenhagen (H-3-2011-065) and was conducted according to the Helsinki II declaration. The study was also registered at ClinicalTrials.gov (NCT01637662). The natriuretic peptides were purchased from CASLO ApS (Lyngby, Denmark) and prepared for infusion by central pharmacy (Marielundvej, Herlev). We performed identification test of all three peptides by monoisotopical mass spectroscopy and molecule weight determination, which showed the same results as the certificate analysis from CASLO. All stock solutions were stored at −80 °C before usage.
2.1 Design of the animal experiment Four male guinea pigs, 350–450 g, were sacrificed in the morning by decapitation after an overdose of barbiturate. The brain was removed and the basilar artery and the middle cerebral artery were dissected free. The arteries were placed in an ice-cold buffer solution (NaCl 119, NaHCO3 15, KCl 4.6, CaCl2 1.5, NaHPO4 1.2, MgCl2 1.2, glucose 5.5, mM, pH 7.4) aerated with 95% O2 and 5% CO2 and transported to the laboratory for performance of the experiment. The retrieval of animal tissue followed the ethical guidelines for animal studies in Denmark.
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To determine vessel tension, each segment was mounted on two metal wires 40 μm in diameter in a myograph (Model 610M, Danish Myo Technology, Denmark). The buffer solution was continuously aerated with 5% CO2 in O2 to maintain a stable pH of 7.4. The artery segments were allowed to equilibrate for approximately 30 min. The vessels were stretched to the internal circumference the vessel would have if relaxed and exposed to a passive transmural pressure of 100 mmHg (13.3 kPa). This was in order to achieve maximal active force development [24]. Following a second 30-min equilibration period, the vessels were constricted twice with 60 mM KCl in a modified buffer solution in which NaCl was substituted for KCl on an equimolar basis. The contraction amounted to 4.65±0.15 mN/mm (n=16) in basilar and 1.98±0.16 mN/mm (n=16) in middle cerebral arteries. In order to study the relaxant effect of ANP, BNP and CNP the arteries were pre-contracted with 3×10−6 M prostaglandin F2α (PGF2α). In our preparations, it resulted in a stable tension of 3.63±0.40 mN (n=16) in basilar and 1.87±0.17 mN (n=16) in middle cerebral arteries to which the agonist was added in cumulative concentrations. The tension lasted for at least 20–30 min without a significant fall in tone. Stock solutions of ANP, BNP and CNP (10−4 M) and PGF2α (Sigma, USA) were prepared by dissolving the peptides in distilled water and further diluted in buffer immediately prior to the experiment.
2.2 Design of the pilot experiment In a double-blind, three-way cross-over design, four healthy volunteers were randomly allocated to receive three high pharmacological doses (Fig. 1) of ANP, BNP or CNP over 20 min on 3 study days, separated by at least a week. The natriuretic peptides were administered intravenously and technicians, who were blinded in respect to the natriuretic peptides and their doses, performed all measurements. The purpose was to determine the optimal dose of
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the natriuretic peptides for the main study. We wanted to find a dose that would cause detectable changes in mean blood flow velocity of the middle cerebral artery (VMCA) by transcranial Doppler (TCD) without intolerable adverse effects. Inclusion criteria for the pilot experiment were: healthy individuals 18–35 years old and weighting 50-90 kg. Fertile women were included only if using either oral contraceptives or intrauterine devices. Exclusion criteria were: a history of migraine, first degree relatives with migraine, episodic tension-type headache more than once a week, history or clinical findings suggesting neurological or cardiovascular disorders, asthma or bronchospasm and daily intake of any kind of medicine except oral contraceptives. The subjects arrived at the clinic at 8:00 a.m. on each study day fasting and received intravenous natriuretic peptides in step-wise increasing doses. Subjects had to be free from headache for at least the last 48 h. Each infusion lasted 20 min separated by a 100min washout period. VMCA, diameter of superficial temporal artery and radial artery, blood flow in face microvasculature, end-tidal partial pressure of CO2 (PetCO2), vital signs, ECG, headache intensity and adverse events were recorded at baseline and then every 10 min until 60 min after start of each infusion.
2.3 Design of the main experiment In a double-blind, three-way cross-over design, six healthy volunteers were randomly allocated to receive two doses (a high physiological and a pharmacological dose) of ANP, BNP or CNP over 20 min on 3 study days, separated by at least a week (Fig. 1). The doses used in the main experiment were based on the results from the pilot experiment. The natriuretic peptides were administered intravenously and skilled technicians, who were blinded in respect to the natriuretic peptides and their doses, performed all measurements.
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Inclusion criteria for the main experiment were the same as in the pilot experiment. Full medical history and examination was performed prior inclusion. Each study day consisted of two administration rounds separated by a washout
period. The participants arrived at the clinic at 8.30 a.m. fasting for the last 8 h and were offered a glass of water (200 ml) to avoid dehydration. Coffee, tea, cocoa or other methylxanthine-containing foods or beverages was not allowed for at least 8 h before start of the study. The participants had to be free from headache for at least 48 h. They were informed that the natriuretic peptides might induce headache in some individuals, but the timing or the type of headache was not discussed. None of the participants had previously participated in similar studies. All procedures were performed in a quiet room and the room temperature was kept between 23–26 ºC. The participants were placed in the supine position and venous catheters (Venflon®) were inserted into both antecubital veins for infusion and blood sampling. The subjects then rested for 30 min before baseline recordings. Infusion was given for 20 min using a time and volume controlled infusion pump (Braun Perfusor, Melsungen, Germany). VMCA, diameter of the superficial temporal artery and radial artery, end-tidal partial pressure of CO2 (PetCO2), blood flow in face microvasculature, vital signs, ECG, headache intensity and adverse events were recorded at baseline and then every 10 min until 60 min after start of infusion (Table 1). Blood samples were drawn only in the first administration round at time points 0, 5, 10, 30, 45 and 60 min. The first administration round was followed by a 2 h washout period where 500 ml isotonic water was given intravenously. After the wash out period, a second administration round with the second dose was initiated following the same protocol as the first administration round (Fig. 2) except that no blood samples were drawn. Subsequently,
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the subjects were instructed to register adverse events and headache score using a questionnaire [25] every hour until 12 h after discharge from the hospital.
2.4 Mean blood flow velocity of middle cerebral artery (VMCA) Mean VMCA was recorded bilaterally using hand-held 2-MHz probes and transcranial Doppler (TCD; Multidop X, DWL, Sipplingen, Germany). Marks were drawn on the skin to ensure accurate repositioning in each acquisition. Four recordings were taken and averaged at each time point. One recording was a time-averaged mean over 4 s or approximately four cardiac cycles. We avoided fixed probes because they may cause discomfort and even headache [26]. Identification of the middle cerebral artery (MCA) and marking reproducible fixed points were done as previously described [27, 28]. Every TCD recording of the same participant was performed by the same examiner (LE or NKR). Partial end-tidal carbon dioxide (PetCO2) was recorded simultaneously with the TCD measurements using an open mask that caused no respiratory resistance (ProPaq Encore®; Welch Allyn Protocol, Beaverton, OR, USA).
2.5 Diameter of superficial arteries The diameter of the frontal branch of the superficial temporal artery (STA) and the radial artery (RA) was measured using high-resolution ultra-sonography, 20 MHz, bandwidth 15 MHz (Dermascan C, Hadsund, Denmark) [29, 30]. Marks were drawn on the skin and the coordinates of these recorded to ensure reproducibility of the measuring points from day to day. The same skilled examiner (WG) performed all the measurements.
2.6 Blood flow in face microvasculature For blood flow measurement in the microvasculature of the face we used laser speckle contrast imager, moorFLPI (Moor Instruments, Devon, UK); a commercially available Page 9 of 34
system. The instrument head containing the camera was positioned 30 cm straight above the face of the participants and focus was optimized. The imager was calibrated using a reference flux signal which was generated by the laser light scattered from a suspension of polystyrene microspheres in water undergoing thermal or Brownian motion. We used previously published [31, 32] and made measurements of the entire face, while the participants were lying completely still on the bed with eyes closed. The recorded contrast images were processed to produce a scaled color-coded live flux image (red equaled high perfusion, blue equaled low perfusion), which correlated with the blood flow velocity in the face skin. At each time point, based on the flux images the mean perfusion of the entire face was calculated.
2.7 Plasma concentration measurements An intravenous cannula was placed in the ante-cubital vein of the subjects. Blood was withdrawn into a plastic syringe after we aspirated and discarded the first three ml of blood. The catheter was flushed with isotonic salt water after each sample taking. Blood samples for ANP, BNP and CNP measurements were collected in 10 ml EDTA tubes. The tubes were inverted several times and put in an ice-chilled box. Within 30 min they were centrifuged at 5 °C at 3,000 rpm for 10 min. Plasma was then transferred to a polypropylene tube and stored at -20° C or colder temperature. Measurement of ANP was performed as earlier reported [15]; BNP was measured on an Advia Centaur platform (Siemens, Germany); and CNP was measured using a commercial ELISA (Phoenix Peptides, Germany). All blood samples were measured in the same run. The intra- and interassay imprecision (coefficient of variations, CV) for all methods were below 10%.
2.8 Adverse events
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Adverse events and headache score were recorded every 10 min and every 1 h after discharge using a questionnaire. Headache intensity was recorded on a verbal rating scale (VRS) from 0 to 10. 0 is no headache, 1 represented a very mild headache (including a sensation of pressing or throbbing or otherwise altered sensation in the head not associated with pain), 5 is headache of moderate intensity and 10 is the worst headache imaginable [33].
2.9 Vital signs Heart rate (HR) and mean arterial blood pressure (MAP) were measured every 10 min using an auto-inflatable cuff (Omega 1400, Orlando, FL, USA). ECG (Cardiofax V, NihonCohden, Shinjuku-ku, Tokyo, Japan) was monitored and recorded on paper every 10 min as well.
2.10 Statistics Data are presented as mean ± SD, except headache score, which is listed as medians (range). Concentration curves are plotted graphically with values given as means±SEM. Only descriptive analysis of data was performed in the animal and pilot experiment. Baseline was defined as mean of T-10 and T0 before the start of infusion of each dose. We used GraphPad Prism 5 for the descriptive analysis of data. Sample size was calculated based on detection of at least 15% change of mean VMCA with a 5% level of significance (2-tailed) and 90% power. With an anticipated 10.7% [34] variation and a correction for correlation (paired comparison), we calculated that six patients should be sufficient for comparison in a two-way crossover study based on the following formula: 2 x f(α,1-β) x (σ/Δ)2 [35]. In the animal experiment, Imax (maximum relaxant effect obtained with the peptides) and pIC50 (negative logarithm of the concentration of peptide that elicited half-
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maximum relaxation) were calculated arithmetically from each individual concentration– response curve. Maximum two segments from each guinea pig. In the main experiment, primary end-points were: 1) changes in mean VMCA between baseline and 20 min after start of infusion analyzed with Wilcoxon signed rank test, 2) differences in response for mean VMCA (AUCVMCA) between dose 1 and 2 calculated as area under the curve (AUC) according to the trapezium rule [36] and 3) differences in dose 1 and 2 for mean VMCA between ANP, BNP and CNP analyzed with Friedman test. Secondary endpoints were differences in diameter of STA (AUCSTA), diameter of RA (AUCRA), blood flow in face microvasculature, mean arterial blood pressure (MAP; AUCMAP), heart rate (HR; AUCHR), response in PetCO2 (AUCPetCO2) and incidence of any headache between dose 1 and 2 for ANP, BNP and CNP. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. Due to multiple testing, p-values of secondary end-points except for vital parameters (HR and MAP) were adjusted with Holm-Bonferroni correction to avoid type 1 errors. Primary end-points were not adjusted with Holm-Bonferroni correction. The half-life of ANP, BNP and CNP were calculated according to the following formula: C60 = C30e-kt where C30 and C60 are plasma concentrations at 30 min and 60 min after start of start of infusion. Plasma concentrations were plotted on semi logarithmic graph paper after subtraction of basal values. Linear regression of the logarithm of plasma concentration vs. time was computed to yield the slope (k), from which the half-life was calculated by dividing into 0.693. All analyses were performed with SPSS for Mac v11.0 (Chicago, IL, USA). Five percent (P<0.05) was accepted as the level of significance.
3. Results
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3.1 Effect of the natriuretic peptides on isolated guinea pig cerebral arteries In the animal experiment, cumulative application of ANP, BNP and CNP to PGF2α precontracted vessel segments resulted in dilation of guinea pig basilar and middle cerebral arteries with the following order of potency in the basilar artery: ANP = BNP >> CNP (Fig 3A) and in the middle cerebral arteries ANP > BNP >> CNP (Fig 3B). The response to ANP was significantly (P=0.02, Mann-Whitney test) more potent in the middle cerebral arteries than in the basilar arteries. There was no difference in relaxant response to BNP and CNP between the two arteries. CNP hardly induced any relaxation with a maximum response at 4 ± 4 % (n=2) in basilar arteries and 14 ± 1 % (n=2) in middle cerebral arteries.
3.2 Pilot experiment None of the four healthy volunteers completed the pilot experiment or even the whole study day due to adverse events. Two male subjects experienced a vasovagal syncope incident approximately 70 min after infusion of respectively ANP (0.5 µg/kg/min) and BNP (0.2 µg/kg/min) during urination in the toilet. They both had no clear affection of the blood pressure before the incident. One female subject experienced decreased mean arterial blood pressure (mean arterial pressure, MAP = 60) after administration of ANP (2.0 µg/kg/min). We lowered the doses of BNP for the last female subject to 0.04 µg/kg/min and 0.06 µg/kg/min, but she experienced decreased mean arterial blood pressure (MAP = 62) as well during the second dose (0.06 µg/kg/min). The pilot experiment was therefore terminated. The incomplete results of the pilot experiment are shown in Table 2. ANP and BNP caused decrease in VMCA (12% to 15%), dilatation of the STA (16% to 29%) and increase of HR (11% to 40 %) after 20 min of infusion. One male subject developed a delayed headache 2 h after discharge with moderate intensity (VRS=4) after ANP. Furthermore, a female subject developed delayed mild headache (VRS=2) 1 hour after
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ANP and CNP. Both subjects took paracetamol 1g as rescue medication for the delayed headache, which was bilateral, pressing and localized in the frontal or temporal regions without migraine characteristics.
3.3 Main experiment Six subjects (3F/3M) completed the main study. The mean age was 25 (range 21-28 years) and mean weight was 69 kg (range 62-80 kg). Plasma concentrations of all three natriuretic peptides increased significantly after 10 min of infusion (P=0.028 for all three peptides); ANP increased by 40-fold, BNP by 22-fold, while CNP only increased by 3-fold. The increase of the natriuretic peptides quickly declined after the cessation of infusion (Fig. 4A). After ANP infusion plasma concentrations for cGMP increased around 3-fold after 10 min and 5-fold after 30 min (P=0.028 for both time points) (Fig. 4B). Plasma half-lives of ANP and BNP were calculated to be respectively 13.9 min and 18.7 min. Half-life for CNP could not be calculated due to the low concentrations. Mean VMCA for dose 1 and 2 changed less than 10% from baseline during the periods of measurements for ANP, BNP and CNP (Fig. 5). VMCA did not change after 20 min of infusion and we found no difference between dose 1 and 2 of the three peptides. In addition, there was no difference in changes of VMCA between ANP, BNP or CNP for either dose 1 (P=1.000) or dose 2 (P=1.000). Diameter of STA, diameter of RA, blood flow in face microvasculature, response in PetCO2, MAP and HR did not change after 20 min of ANP, BNP or CNP infusion, and there were also no difference between dose 1 and 2 (Table 3). Additionally, there was no difference between ANP, BNP and CNP for the recorded variables (data not
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shown). However, there was a clear trend of increase in HR after ANP for both dose 1 (P=0.063) and dose 2 (P=0.067). Adverse events were few and not statistically different between ANP, BNP and CNP (data not shown). Single subjects reported warm sensation, hunger, dizziness and muscle tension. Urination urge was also reported (n=1 after ANP, n=4 after BNP, and n=2 after CNP). In addition, few subjects (n=2 after ANP, n=3 after BNP, and n=3 after CNP) developed a mild headache (VRS 1-3) after infusion of ANP, BNP or CNP, but the median headache score intensity for the entire duration, both in-hospital and out-hospital, was 0 for all three peptides.
3.4 MAP & HR Descriptive data of HR and MAP are shown in Fig. 6. As earlier mentioned, we found no change of HR and MAP over time and no difference between dose 1 and dose 2.
4. Discussion We found that ANP, BNP and CNP in physiological and pharmacological doses (≤0.15 μg/kg/min for ANP and ≤0.06 μg/kg/min for BNP and CNP) did not affect mean blood flow velocity of the middle cerebral artery (VMCA), which is an established surrogate measurement for vasodilatation of deep cerebral arteries [27]. Moreover, the natriuretic peptides caused no changes in mean VMCA after 20 min of infusion, showed no difference between the two doses we used and demonstrated no difference between ANP, BNP and CNP. Furthermore, natriuretic peptides did not affect diameter of superficial temporal artery (STA), radial artery and blood flow in face microvasculature despite showing increase in plasma concentrations during infusion.
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In contrast, ANP and BNP but not CNP showed significant dose-dependent relaxation of both MCA and BA in vitro, which is in line with a previous study [20]. Using very high pharmacological doses in the pilot study, ANP and BNP but not CNP seemingly caused a decrease in VMCA and an increase of STA diameter. These findings indicate that natriuretic peptides may exert dilatory effects on cerebral arteries at least when infused in very high doses. Additionally, ANP caused some changes of HR in the main study (up to 18%). Previous studies using similar doses and long continuous infusion of ANP and BNP showed increase of HR (up to 20%) and decrease of MAP (up to 6%), as well as being diuretic [37– 40]. In the pilot study using very high pharmacological doses, we also saw an effect on STA but not on radial artery. This is in contrast to previous studies showing that infusions of all three peptides into brachial arteries of healthy men induce a dose-dependent increase of forearm blood flow by dilating forearm resistance vessels [3, 41–45]. However, the doses of ANP and BNP used in these studies were different from those we used in the present study. To our knowledge, no previous studies have directly measured the diameter of radial artery with ultrasound technique. The natriuretic peptides did not induce headache despite the fact that they activate the cGMP-dependent pathway via pGC and NO in vascular cells [46, 47], an important pathway in the induction of headache and migraine [48, 49]. However, in the pilot study two participants reported delayed headache after infusion of very high pharmacological doses of ANP and CNP, respectively. This could indicate that the headache inducing effects of natriuretic peptides are dose-dependent, but very high doses are not possible due to severe side effects. Moreover, the delayed headache suggests induction by secondary processes rather than by the systemically circulating natriuretic peptides from infusion. Consequently, we cannot rule out that natriuretic peptides may induce headache due to endogenous release
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of ANP, BNP, CNP or other neuropeptides from perivascular nerve endings of cerebral arteries.
4.1 Interpretation of results Transcranial Doppler measurements of velocity alone must also be interpreted with some caution because velocity is dependent on both the diameter of the MCA and the regional cerebral blood flow (CBF). It is uncertain whether natriuretic peptides may cause changes in CBF [19]. To date, results from animal studies have been contradictory and inconsistent regarding what response natriuretic peptides elicit on CBF [21, 50, 51]. These experiments were performed in different animals albeit with similar doses of the natriuretic peptides. However, TCD is still useful to give an idea of possible effects on arteries and a decrease in velocity is regarded as an expression for vasodilatation [27]. Measurements are the most precise in the MCA with a CV of 26% between participants and 11% between days [27, 34]. To reduce the impact of intersubject and interobserver variability of TCD measurements, the present study was designed as a crossover study and the same trained investigator performed all measurements. There is also a correlation between PetCO2 and VMCA, which has previously been established [52], but in the present study PetCO2 was more or less constant for all natriuretic peptides and can therefore not influence our VMCA measurements. Highfrequency ultrasound determination of the diameter of the STA and radial artery is highly accurate and reproducible as documented in many previous studies [29, 53]. The lack of response on mean VMCA in the main study may have several explanations. One explanation could be that the doses we used in the main study were too low. However, the doses we used were as high as those used in previous drug trials of ANP (Carperitide) and BNP (Nesiritide) in healthy volunteers and heart failure patients [16, 54– 57]. Using higher doses would hold the risk of unwanted side effects as we experienced in
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our pilot study. Another possibility is that interspecies difference may play a role and that human arteries are less sensitive so much higher doses were needed in our healthy subjects to see the same response as in animals. The effects of the natriuretic peptides may also have some latency due to autoregulatory mechanisms. There was a clear trend of an increase in HR after ANP and BNP, which could indicate sympathetic activation. It is possible that the vasodilatory effects of the natriuretic peptides were counter regulated by the sympathetic nervous system. Furthermore, in contrast to previous studies, we infused the natriuretic peptides in a high dose over only 20 min. ANP and BNP given as therapy for heart failure patients is administered as continuous infusion for 24–48 hours [16]. The half-lives of ANP and BNP are biphasic consisting, of a fast phase (1.7–2.1 min) and also a slow phase (13–37 min) [58, 59], i.e. long infusion duration may contribute to achieving a higher plasma concentration. Previous studies with continuous infusion of ANP and BNP reported a progressive response in HR and MAP developing over two hours [60, 61]. This may also explain why two participants in our pilot study experienced a vasovagal syncope incident over one hour after the cessation of ANP and BNP infusion. In addition, the lack of effect on MCA could also be because natriuretic peptides pass the blood–brain barrier (BBB) poorly [62] and may therefore not reach the vascular smooth muscle cells of the cerebral arteries unless given in very high doses. In our in vitro experiment which showed dilatation of MCA, natriuretic peptides were applied both intra- and extraluminally. Therefore, a model that allows selective intra- and extraluminal application of the natriuretic peptides would have been interesting. The distribution of natriuretic peptide receptors (NPR) on cerebral arteries may also be different from the peripheral vascular system. To our knowledge, no studies on the distribution of NPRs on cerebral arteries have been done.
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5. Conclusion This is the first study investigating the effects of natriuretic peptides on cerebral arteries in humans. We showed that natriuretic peptides in physiological and pharmacological doses (≤0.15 μg/kg/min for ANP and ≤0.06 μg/kg/min for BNP and CNP) had no significant effects on cerebral arteries in healthy volunteers. However, ANP and BNP showed significant dosedependent relaxation of cerebral arteries in guinea pigs. In addition, we cannot exclude that ANP and BNP may exert vasodilatory properties on the middle cerebral artery and the superficial temporal artery in very high doses (≥0.5 μg/kg/min for ANP and ≥0.2 μg/kg/min for BNP), whether these being pharmacological or caused by severe cardiac disease. It is still unclear whether natriuretic peptides may have potential implications in diseases where alterations of the cerebral hemodynamic play a role and more studies are warranted.
Acknowledgements The authors thank all the volunteers who participated in this study. Special thanks to Denise M Hublein and Dijana Terzic for performing the biochemical analyses, and Niklas Kahr Rasmussen, Lene Elkjær and Winnie Grønning for technical assistance. Further thanks to funding from Novo Nordisk Foundation (NNF11OC1014333) and Independent ResearchMedical Sciences (FSS) (DFF-1331-00210A), Lundbeck Foundation (R155-2014-171) and the FP7-EUROHEADPAIN (602633).
Disclosures Song Guo has received a travel grant from Autonomic Technologies Inc. Jes Olesen has received grants/research support from and/or has been a consultant/scientific adviser for, and/or has been in the speakers’ bureau of Allergan Inc, AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim, Eli Lilly, GlaxoSmithKline, Janssen Pharmaceutical Products,
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Lundbeck, Merck, Amgen, Alder and Pfizer. Messoud Ashina has received consulting fees, speaking/teaching fees, and/or honoraria – Alder, Allergan, Amgen, ATI and Eli Lilly. Jens P. Goetze has no conflicts of interest to report.
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Page 25 of 34
Figure Captions Figure 1: Flow chart of the experiments in animal and humans investigating effects of the natriuretic peptides on cerebral arteries. Pilot and main experiments were conducted in a double-blind, randomized three-ways crossover design. Participants were allocated to receive intravenous infusion of different doses of ANP, BNP or CNP on each study day.
Page 26 of 34
Figure 2: Experimental protocol for each study day in the main experiment.
Page 27 of 34
Figure 3: ANP and BNP showed clear relaxation of both BA and MCA, whereas generally no response was seen for CNP. Concentration response curves are plotted graphically with values given as means±SEM.
Page 28 of 34
Figure 4: (A) Plasma ANP, BNP and CNP concentrations up to one hour after the start of infusion in healthy subjects (mean±SEM). Infusion was given from 0-20 min. (B) Plasma concentrations of cGMP up to one hour after the start of ANP infusion (0.05 µg/kg/min). Data are shown as individual and mean±SEM.
Page 29 of 34
Figure 5: Median of mean flow velocities in the middle cerebral artery (VMCA) for dose 1 and 2, and changes (%) from baseline of mean VMCA with 95% CI in six healthy subjects after ANP, BNP or CNP. There was no significant change in VMCA after 20 min of infusion for dose 1 and dose 2 compared to baseline. We found no difference in area under the curve (AUC) between dose 1 and dose 2. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. *P-value: Wilcoxon signed rank test.
Page 30 of 34
Figure 6: Median changes with 95% CI for HR and MAP after infusion (20 min) of ANP, BNP and CNP in six healthy subjects. There was no difference in the AUC for HR or MAP between dose 1 and dose 2.
Page 31 of 34
Tables
Table 1: Time points and measurements () for the first administration round. The second administration round was exactly the same except no blood samples were taken. Time:
-10
0
5
10
20
30
40
45
50
60
──────►
Infusion Headache questions
Blood flow velocity in MCA
Radial and temporal arteries
BP, HR and pCO2
ECG Blood flow in face microvasculature Blood samples
Page 32 of 34
Table 2: Recorded variables in pilot experiment for dose 1, 2 and 3 of ANP, BNP and CNP in one to three subjects. Mean change (± %) after 20 min of infusion compared to baseline is shown in the table. Due to adverse events BNP dose 1 and 2 were reduced to 0.04 and 0.06 µg/kg/min. ANP
BNP
CNP
Dose 1 0.5 (n=3)
Dose 2 1.0 (n=2)
Dose 3 2.0 (n=2)
Dose 1 0.04 (n=1)
Dose 2 0.06 (n=1)
Dose 3 0.2 (n=1)
Dose 1 0.2 (n=2)
Dose 2 0.4 (n=2)
Dose 3 0.8 (n=1)
Mean VMCA
-15%
-12%
-14%
-9%
-17%
-16%
-3%
0%
+6%
Temporal artery (STA)
+16%
+29%
+28%
-8%
+20%
+39%
-7%
+9%
+4%
Radial artery (RA)
+1%
-3%
+1%
-18%
-2%
+4%
-4%
+5%
+5%
Blood flow of face
+1%
+18%
+9%
-9%
-11%
+3%
-7%
-7%
-16%
MAP
-7%
-9%
-5%
-9%
-8%
-9%
+2%
-4%
-9%
+11%
+40%
+30%
+26%
+18%
+21%
-2
+4%
4%
1
2
0
1
0
1
0
1
0
µg/kg/min: number of subjects:
Heart rate Subjects who felt urge to urinate (n) Immediate headache (during in-hospital) Delayed headache (after discharge from hospital)
None
None
None
Yes (n=1)
Not reported due to AE
Yes (n=1)
Page 33 of 34
Table 3: Recorded variables in the main study for dose 1 and dose 2 of ANP, BNP and CNP in six healthy subjects. Table shows mean change (± %) after 20 min of infusion compared to baseline with calculated P-value (Wilcoxon signed rank test). Difference between dose 1 and dose 2 of ANP, BNP or CNP are calculated as area under the curve (AUC) and shown as PAUC-value. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. *P-values for secondary end-points were adjusted with HolmBonferroni correction.
ANP (n=6) µg/kg/min: Temporal artery* Radial artery * Blood flow of face * PetCO2* MAP Heart rate
Dose 1
Dose 2
0.05
BNP (n=6) Dose 1
Dose 2
0.15
PAUCvalue
0.02
+2%
+4%
1.000
P=1.000
P=1.000
0%
-2%
P=1.000
P=1.000
-6%
-3%
P=0.624
P=1.000
+1%
-2%
P=1.000
P=1.000
-4%
+7%
P=0.496
P=0.095
+18%
+12%
P=0.063
P=0.067
0.252 1.000 0.468 0.094 1.000
CNP (n=6) Dose 1
Dose 2
0.06
PAUCvalue
0.02
0.06
PAUCvalue
+8%
+3%
0.375
+10%
+4%
0.124
P=0.124
P=0.876
P=0.124
P=0.345
-6%
+1%
+1%
0%
P=0.124
P=1.000
P=1.000
P=1.000
0%
-5%
+2%
-7%
P=1.000
P=0.939
P=1.000
P=0.124
-1%
0%
-1%
-1%
P=1.000
P=1.000
P=1.000
P=0.980
+5%
-2%
-2%
-1%
P=0.163
P=0.581
P=0.570
P=0.834
-2%
+8%
-3%
-3%
P=0.752
P=0.203
P=0.343
P=0.399
0.124 0.438 0.375 0.156 0.063
Page 34 of 34
1.000 1.000 1.000 0.313 0.688
S0196-9781(15)00268-5 http://dx.doi.org/doi:10.1016/j.peptides.2015.09.008 PEP 69549
To appear in:
Peptides
Received date: Revised date: Accepted date:
25-8-2015 21-9-2015 23-9-2015
Please cite this article as: Guo Song, Goetze Jens P, Jeppesen Jorgen L, Burnett John C, Olesen Jes, Jansen-Olesen Inger, Ashina Messoud.Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers.Peptides http://dx.doi.org/10.1016/j.peptides.2015.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers Song Guo1, Jens P. Goetze2, Jørgen L. Jeppesen3, John C Burnett4, Jes Olesen1, Inger JansenOlesen1, Messoud Ashina1* MD PhD DMSc [email protected] 1
Danish Headache Center and Department of Neurology, Rigshospitalet Glostrup, Faculty of
Health Sciences, University of Copenhagen, 2600 Glostrup, Copenhagen, Denmark 2
Department of Clinical Biochemistry, Rigshospitalet Blegdamsvej, Faculty of Health and
Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark 3
Department of Medicine, Hvidovre Hospital Glostrup, Faculty of Health and Medical
Sciences, University of Copenhagen, 2600 Glostrup, Copenhagen, Denmark 4
Departments of Internal Medicine and Physiology, Division of Cardiovascular Disease,
Cardiorenal Research Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55906, USA *Corresponding author at: Danish Headache Center and Department of Neurology, Rigshospitalet Glostrup, Faculty of Health and Medical Sciences, University of Copenhagen, Nordre Ringvej 57, 2600 Glostrup, Copenhagen, Denmark, Tel.: (+45) 38 63 33 85.
Page 1 of 34
Highlights
Natriuretic peptides in physiological and pharmacological doses have no effects on cerebral arteries in healthy volunteers.
Natriuretic peptides showed dose-dependent relaxation of cerebral arteries in guinea pigs.
This is the first study investigating the effects of natriuretic peptides on cerebral arteries in humans.
Page 2 of 34
Abstract The natriuretic peptides (NPs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), have vasoactive functions that concern humans and most animals, but their specific effects on cerebral circulation are poorly understood. We therefore examined the responsiveness of cerebral arteries to different doses of the natriuretic peptides in animals and humans. We conducted a dose-response experiment in guinea pigs (in vitro) and a double-blind, three-way cross-over study in healthy volunteers (in vivo). In the animal experiment, we administered cumulative doses of NPs to pre-contracted segments of cerebral arteries. In the main study, six healthy volunteers were randomly allocated to receive two intravenous doses of ANP, BNP or CNP, respectively, over 20 min on three separate study days. We recorded blood flow velocity in the middle cerebral artery (VMCA) by transcranial Doppler. In addition, we measured temporal and radial artery diameters, headache response and plasma concentrations of the NPs. In guinea pigs, ANP and BNP but not CNP showed significant dose-dependent relaxation of cerebral arteries. In healthy humans, NP infusion had no effect on mean VMCA, and we found no difference in hemodynamic responses between the NPs. Furthermore, natriuretic peptides did not affect temporal and radial artery diameters or induce headache. In conclusion, natriuretic peptides in physiological and pharmacological doses do not affect blood flow velocity in the middle cerebral artery or dilate extracerebral arteries in healthy volunteers.
Keywords: Natriuretic peptides; atrial; brain; C-type; cerebral arteries; cerebral blood flow; headache.
Page 3 of 34
1. Introduction Natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and Ctype natriuretic peptide (CNP), are structurally related hormones [1]. The biological effect of these peptides in humans and most animals is to maintain cardiovascular homeostasis through their vasoactive and diuretic properties [2–6]. ANP and BNP are predominantly produced in the heart [7, 8], but all three natriuretic peptides are expressed in the brain and blood vessels [9–14]. Since their discovery, the diagnostic and therapeutic importance of natriuretic peptides has increased tremendously, especially in the field of clinical cardiology [15–18]. However, the biological effects of natriuretic peptides on cerebral circulation are still poorly understood [19]. To our knowledge, no in vivo or in vitro studies in humans of the effect of natriuretic peptides on cerebral or extracranial arteries have been conducted. Four animal in vitro studies have examined the vasodilatory response of natriuretic peptides on cerebral arteries. Two studies in guinea pigs and rabbits showed vasodilation [20, 21] while the other two in rats showed no response [22, 23]. Possible interspecies differences between humans and animals have never been investigated, but potential clinical implications of the peptides may, however, be relevant in neurovascular disorders such as stroke and migraine [19]. We hypothesized that natriuretic peptides dilate the cerebral arteries in a dosedependent manner. Therefore, we examined the responsiveness of cerebral arteries to natriuretic peptides with different doses in animals (in vitro) and in healthy volunteers (in vivo). In addition, we measured the diameters of the superficial temporal artery and radial artery, blood flow in face microvasculature, the headache inducing properties of the natriuretic peptides and their plasma concentrations before, during and after infusion.
2. Materials and Methods
Page 4 of 34
We conducted three separate experiments (Fig. 1). The first experiment was an in vitro animal experiment where we tested the effect of the three natriuretic peptides on isolated middle cerebral arteries (MCA) and basilar arteries (BA) from guinea pigs. The second was a pilot experiment with healthy volunteers to determine the optimal dose of the natriuretic peptides for the main experiment. In the third (and main) experiment, we examined the vasodilatory effects of all three natriuretic peptides. All participants gave written informed consent before inclusion. The study was approved by the ethics committee of Copenhagen (H-3-2011-065) and was conducted according to the Helsinki II declaration. The study was also registered at ClinicalTrials.gov (NCT01637662). The natriuretic peptides were purchased from CASLO ApS (Lyngby, Denmark) and prepared for infusion by central pharmacy (Marielundvej, Herlev). We performed identification test of all three peptides by monoisotopical mass spectroscopy and molecule weight determination, which showed the same results as the certificate analysis from CASLO. All stock solutions were stored at −80 °C before usage.
2.1 Design of the animal experiment Four male guinea pigs, 350–450 g, were sacrificed in the morning by decapitation after an overdose of barbiturate. The brain was removed and the basilar artery and the middle cerebral artery were dissected free. The arteries were placed in an ice-cold buffer solution (NaCl 119, NaHCO3 15, KCl 4.6, CaCl2 1.5, NaHPO4 1.2, MgCl2 1.2, glucose 5.5, mM, pH 7.4) aerated with 95% O2 and 5% CO2 and transported to the laboratory for performance of the experiment. The retrieval of animal tissue followed the ethical guidelines for animal studies in Denmark.
Page 5 of 34
To determine vessel tension, each segment was mounted on two metal wires 40 μm in diameter in a myograph (Model 610M, Danish Myo Technology, Denmark). The buffer solution was continuously aerated with 5% CO2 in O2 to maintain a stable pH of 7.4. The artery segments were allowed to equilibrate for approximately 30 min. The vessels were stretched to the internal circumference the vessel would have if relaxed and exposed to a passive transmural pressure of 100 mmHg (13.3 kPa). This was in order to achieve maximal active force development [24]. Following a second 30-min equilibration period, the vessels were constricted twice with 60 mM KCl in a modified buffer solution in which NaCl was substituted for KCl on an equimolar basis. The contraction amounted to 4.65±0.15 mN/mm (n=16) in basilar and 1.98±0.16 mN/mm (n=16) in middle cerebral arteries. In order to study the relaxant effect of ANP, BNP and CNP the arteries were pre-contracted with 3×10−6 M prostaglandin F2α (PGF2α). In our preparations, it resulted in a stable tension of 3.63±0.40 mN (n=16) in basilar and 1.87±0.17 mN (n=16) in middle cerebral arteries to which the agonist was added in cumulative concentrations. The tension lasted for at least 20–30 min without a significant fall in tone. Stock solutions of ANP, BNP and CNP (10−4 M) and PGF2α (Sigma, USA) were prepared by dissolving the peptides in distilled water and further diluted in buffer immediately prior to the experiment.
2.2 Design of the pilot experiment In a double-blind, three-way cross-over design, four healthy volunteers were randomly allocated to receive three high pharmacological doses (Fig. 1) of ANP, BNP or CNP over 20 min on 3 study days, separated by at least a week. The natriuretic peptides were administered intravenously and technicians, who were blinded in respect to the natriuretic peptides and their doses, performed all measurements. The purpose was to determine the optimal dose of
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the natriuretic peptides for the main study. We wanted to find a dose that would cause detectable changes in mean blood flow velocity of the middle cerebral artery (VMCA) by transcranial Doppler (TCD) without intolerable adverse effects. Inclusion criteria for the pilot experiment were: healthy individuals 18–35 years old and weighting 50-90 kg. Fertile women were included only if using either oral contraceptives or intrauterine devices. Exclusion criteria were: a history of migraine, first degree relatives with migraine, episodic tension-type headache more than once a week, history or clinical findings suggesting neurological or cardiovascular disorders, asthma or bronchospasm and daily intake of any kind of medicine except oral contraceptives. The subjects arrived at the clinic at 8:00 a.m. on each study day fasting and received intravenous natriuretic peptides in step-wise increasing doses. Subjects had to be free from headache for at least the last 48 h. Each infusion lasted 20 min separated by a 100min washout period. VMCA, diameter of superficial temporal artery and radial artery, blood flow in face microvasculature, end-tidal partial pressure of CO2 (PetCO2), vital signs, ECG, headache intensity and adverse events were recorded at baseline and then every 10 min until 60 min after start of each infusion.
2.3 Design of the main experiment In a double-blind, three-way cross-over design, six healthy volunteers were randomly allocated to receive two doses (a high physiological and a pharmacological dose) of ANP, BNP or CNP over 20 min on 3 study days, separated by at least a week (Fig. 1). The doses used in the main experiment were based on the results from the pilot experiment. The natriuretic peptides were administered intravenously and skilled technicians, who were blinded in respect to the natriuretic peptides and their doses, performed all measurements.
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Inclusion criteria for the main experiment were the same as in the pilot experiment. Full medical history and examination was performed prior inclusion. Each study day consisted of two administration rounds separated by a washout
period. The participants arrived at the clinic at 8.30 a.m. fasting for the last 8 h and were offered a glass of water (200 ml) to avoid dehydration. Coffee, tea, cocoa or other methylxanthine-containing foods or beverages was not allowed for at least 8 h before start of the study. The participants had to be free from headache for at least 48 h. They were informed that the natriuretic peptides might induce headache in some individuals, but the timing or the type of headache was not discussed. None of the participants had previously participated in similar studies. All procedures were performed in a quiet room and the room temperature was kept between 23–26 ºC. The participants were placed in the supine position and venous catheters (Venflon®) were inserted into both antecubital veins for infusion and blood sampling. The subjects then rested for 30 min before baseline recordings. Infusion was given for 20 min using a time and volume controlled infusion pump (Braun Perfusor, Melsungen, Germany). VMCA, diameter of the superficial temporal artery and radial artery, end-tidal partial pressure of CO2 (PetCO2), blood flow in face microvasculature, vital signs, ECG, headache intensity and adverse events were recorded at baseline and then every 10 min until 60 min after start of infusion (Table 1). Blood samples were drawn only in the first administration round at time points 0, 5, 10, 30, 45 and 60 min. The first administration round was followed by a 2 h washout period where 500 ml isotonic water was given intravenously. After the wash out period, a second administration round with the second dose was initiated following the same protocol as the first administration round (Fig. 2) except that no blood samples were drawn. Subsequently,
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the subjects were instructed to register adverse events and headache score using a questionnaire [25] every hour until 12 h after discharge from the hospital.
2.4 Mean blood flow velocity of middle cerebral artery (VMCA) Mean VMCA was recorded bilaterally using hand-held 2-MHz probes and transcranial Doppler (TCD; Multidop X, DWL, Sipplingen, Germany). Marks were drawn on the skin to ensure accurate repositioning in each acquisition. Four recordings were taken and averaged at each time point. One recording was a time-averaged mean over 4 s or approximately four cardiac cycles. We avoided fixed probes because they may cause discomfort and even headache [26]. Identification of the middle cerebral artery (MCA) and marking reproducible fixed points were done as previously described [27, 28]. Every TCD recording of the same participant was performed by the same examiner (LE or NKR). Partial end-tidal carbon dioxide (PetCO2) was recorded simultaneously with the TCD measurements using an open mask that caused no respiratory resistance (ProPaq Encore®; Welch Allyn Protocol, Beaverton, OR, USA).
2.5 Diameter of superficial arteries The diameter of the frontal branch of the superficial temporal artery (STA) and the radial artery (RA) was measured using high-resolution ultra-sonography, 20 MHz, bandwidth 15 MHz (Dermascan C, Hadsund, Denmark) [29, 30]. Marks were drawn on the skin and the coordinates of these recorded to ensure reproducibility of the measuring points from day to day. The same skilled examiner (WG) performed all the measurements.
2.6 Blood flow in face microvasculature For blood flow measurement in the microvasculature of the face we used laser speckle contrast imager, moorFLPI (Moor Instruments, Devon, UK); a commercially available Page 9 of 34
system. The instrument head containing the camera was positioned 30 cm straight above the face of the participants and focus was optimized. The imager was calibrated using a reference flux signal which was generated by the laser light scattered from a suspension of polystyrene microspheres in water undergoing thermal or Brownian motion. We used previously published [31, 32] and made measurements of the entire face, while the participants were lying completely still on the bed with eyes closed. The recorded contrast images were processed to produce a scaled color-coded live flux image (red equaled high perfusion, blue equaled low perfusion), which correlated with the blood flow velocity in the face skin. At each time point, based on the flux images the mean perfusion of the entire face was calculated.
2.7 Plasma concentration measurements An intravenous cannula was placed in the ante-cubital vein of the subjects. Blood was withdrawn into a plastic syringe after we aspirated and discarded the first three ml of blood. The catheter was flushed with isotonic salt water after each sample taking. Blood samples for ANP, BNP and CNP measurements were collected in 10 ml EDTA tubes. The tubes were inverted several times and put in an ice-chilled box. Within 30 min they were centrifuged at 5 °C at 3,000 rpm for 10 min. Plasma was then transferred to a polypropylene tube and stored at -20° C or colder temperature. Measurement of ANP was performed as earlier reported [15]; BNP was measured on an Advia Centaur platform (Siemens, Germany); and CNP was measured using a commercial ELISA (Phoenix Peptides, Germany). All blood samples were measured in the same run. The intra- and interassay imprecision (coefficient of variations, CV) for all methods were below 10%.
2.8 Adverse events
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Adverse events and headache score were recorded every 10 min and every 1 h after discharge using a questionnaire. Headache intensity was recorded on a verbal rating scale (VRS) from 0 to 10. 0 is no headache, 1 represented a very mild headache (including a sensation of pressing or throbbing or otherwise altered sensation in the head not associated with pain), 5 is headache of moderate intensity and 10 is the worst headache imaginable [33].
2.9 Vital signs Heart rate (HR) and mean arterial blood pressure (MAP) were measured every 10 min using an auto-inflatable cuff (Omega 1400, Orlando, FL, USA). ECG (Cardiofax V, NihonCohden, Shinjuku-ku, Tokyo, Japan) was monitored and recorded on paper every 10 min as well.
2.10 Statistics Data are presented as mean ± SD, except headache score, which is listed as medians (range). Concentration curves are plotted graphically with values given as means±SEM. Only descriptive analysis of data was performed in the animal and pilot experiment. Baseline was defined as mean of T-10 and T0 before the start of infusion of each dose. We used GraphPad Prism 5 for the descriptive analysis of data. Sample size was calculated based on detection of at least 15% change of mean VMCA with a 5% level of significance (2-tailed) and 90% power. With an anticipated 10.7% [34] variation and a correction for correlation (paired comparison), we calculated that six patients should be sufficient for comparison in a two-way crossover study based on the following formula: 2 x f(α,1-β) x (σ/Δ)2 [35]. In the animal experiment, Imax (maximum relaxant effect obtained with the peptides) and pIC50 (negative logarithm of the concentration of peptide that elicited half-
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maximum relaxation) were calculated arithmetically from each individual concentration– response curve. Maximum two segments from each guinea pig. In the main experiment, primary end-points were: 1) changes in mean VMCA between baseline and 20 min after start of infusion analyzed with Wilcoxon signed rank test, 2) differences in response for mean VMCA (AUCVMCA) between dose 1 and 2 calculated as area under the curve (AUC) according to the trapezium rule [36] and 3) differences in dose 1 and 2 for mean VMCA between ANP, BNP and CNP analyzed with Friedman test. Secondary endpoints were differences in diameter of STA (AUCSTA), diameter of RA (AUCRA), blood flow in face microvasculature, mean arterial blood pressure (MAP; AUCMAP), heart rate (HR; AUCHR), response in PetCO2 (AUCPetCO2) and incidence of any headache between dose 1 and 2 for ANP, BNP and CNP. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. Due to multiple testing, p-values of secondary end-points except for vital parameters (HR and MAP) were adjusted with Holm-Bonferroni correction to avoid type 1 errors. Primary end-points were not adjusted with Holm-Bonferroni correction. The half-life of ANP, BNP and CNP were calculated according to the following formula: C60 = C30e-kt where C30 and C60 are plasma concentrations at 30 min and 60 min after start of start of infusion. Plasma concentrations were plotted on semi logarithmic graph paper after subtraction of basal values. Linear regression of the logarithm of plasma concentration vs. time was computed to yield the slope (k), from which the half-life was calculated by dividing into 0.693. All analyses were performed with SPSS for Mac v11.0 (Chicago, IL, USA). Five percent (P<0.05) was accepted as the level of significance.
3. Results
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3.1 Effect of the natriuretic peptides on isolated guinea pig cerebral arteries In the animal experiment, cumulative application of ANP, BNP and CNP to PGF2α precontracted vessel segments resulted in dilation of guinea pig basilar and middle cerebral arteries with the following order of potency in the basilar artery: ANP = BNP >> CNP (Fig 3A) and in the middle cerebral arteries ANP > BNP >> CNP (Fig 3B). The response to ANP was significantly (P=0.02, Mann-Whitney test) more potent in the middle cerebral arteries than in the basilar arteries. There was no difference in relaxant response to BNP and CNP between the two arteries. CNP hardly induced any relaxation with a maximum response at 4 ± 4 % (n=2) in basilar arteries and 14 ± 1 % (n=2) in middle cerebral arteries.
3.2 Pilot experiment None of the four healthy volunteers completed the pilot experiment or even the whole study day due to adverse events. Two male subjects experienced a vasovagal syncope incident approximately 70 min after infusion of respectively ANP (0.5 µg/kg/min) and BNP (0.2 µg/kg/min) during urination in the toilet. They both had no clear affection of the blood pressure before the incident. One female subject experienced decreased mean arterial blood pressure (mean arterial pressure, MAP = 60) after administration of ANP (2.0 µg/kg/min). We lowered the doses of BNP for the last female subject to 0.04 µg/kg/min and 0.06 µg/kg/min, but she experienced decreased mean arterial blood pressure (MAP = 62) as well during the second dose (0.06 µg/kg/min). The pilot experiment was therefore terminated. The incomplete results of the pilot experiment are shown in Table 2. ANP and BNP caused decrease in VMCA (12% to 15%), dilatation of the STA (16% to 29%) and increase of HR (11% to 40 %) after 20 min of infusion. One male subject developed a delayed headache 2 h after discharge with moderate intensity (VRS=4) after ANP. Furthermore, a female subject developed delayed mild headache (VRS=2) 1 hour after
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ANP and CNP. Both subjects took paracetamol 1g as rescue medication for the delayed headache, which was bilateral, pressing and localized in the frontal or temporal regions without migraine characteristics.
3.3 Main experiment Six subjects (3F/3M) completed the main study. The mean age was 25 (range 21-28 years) and mean weight was 69 kg (range 62-80 kg). Plasma concentrations of all three natriuretic peptides increased significantly after 10 min of infusion (P=0.028 for all three peptides); ANP increased by 40-fold, BNP by 22-fold, while CNP only increased by 3-fold. The increase of the natriuretic peptides quickly declined after the cessation of infusion (Fig. 4A). After ANP infusion plasma concentrations for cGMP increased around 3-fold after 10 min and 5-fold after 30 min (P=0.028 for both time points) (Fig. 4B). Plasma half-lives of ANP and BNP were calculated to be respectively 13.9 min and 18.7 min. Half-life for CNP could not be calculated due to the low concentrations. Mean VMCA for dose 1 and 2 changed less than 10% from baseline during the periods of measurements for ANP, BNP and CNP (Fig. 5). VMCA did not change after 20 min of infusion and we found no difference between dose 1 and 2 of the three peptides. In addition, there was no difference in changes of VMCA between ANP, BNP or CNP for either dose 1 (P=1.000) or dose 2 (P=1.000). Diameter of STA, diameter of RA, blood flow in face microvasculature, response in PetCO2, MAP and HR did not change after 20 min of ANP, BNP or CNP infusion, and there were also no difference between dose 1 and 2 (Table 3). Additionally, there was no difference between ANP, BNP and CNP for the recorded variables (data not
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shown). However, there was a clear trend of increase in HR after ANP for both dose 1 (P=0.063) and dose 2 (P=0.067). Adverse events were few and not statistically different between ANP, BNP and CNP (data not shown). Single subjects reported warm sensation, hunger, dizziness and muscle tension. Urination urge was also reported (n=1 after ANP, n=4 after BNP, and n=2 after CNP). In addition, few subjects (n=2 after ANP, n=3 after BNP, and n=3 after CNP) developed a mild headache (VRS 1-3) after infusion of ANP, BNP or CNP, but the median headache score intensity for the entire duration, both in-hospital and out-hospital, was 0 for all three peptides.
3.4 MAP & HR Descriptive data of HR and MAP are shown in Fig. 6. As earlier mentioned, we found no change of HR and MAP over time and no difference between dose 1 and dose 2.
4. Discussion We found that ANP, BNP and CNP in physiological and pharmacological doses (≤0.15 μg/kg/min for ANP and ≤0.06 μg/kg/min for BNP and CNP) did not affect mean blood flow velocity of the middle cerebral artery (VMCA), which is an established surrogate measurement for vasodilatation of deep cerebral arteries [27]. Moreover, the natriuretic peptides caused no changes in mean VMCA after 20 min of infusion, showed no difference between the two doses we used and demonstrated no difference between ANP, BNP and CNP. Furthermore, natriuretic peptides did not affect diameter of superficial temporal artery (STA), radial artery and blood flow in face microvasculature despite showing increase in plasma concentrations during infusion.
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In contrast, ANP and BNP but not CNP showed significant dose-dependent relaxation of both MCA and BA in vitro, which is in line with a previous study [20]. Using very high pharmacological doses in the pilot study, ANP and BNP but not CNP seemingly caused a decrease in VMCA and an increase of STA diameter. These findings indicate that natriuretic peptides may exert dilatory effects on cerebral arteries at least when infused in very high doses. Additionally, ANP caused some changes of HR in the main study (up to 18%). Previous studies using similar doses and long continuous infusion of ANP and BNP showed increase of HR (up to 20%) and decrease of MAP (up to 6%), as well as being diuretic [37– 40]. In the pilot study using very high pharmacological doses, we also saw an effect on STA but not on radial artery. This is in contrast to previous studies showing that infusions of all three peptides into brachial arteries of healthy men induce a dose-dependent increase of forearm blood flow by dilating forearm resistance vessels [3, 41–45]. However, the doses of ANP and BNP used in these studies were different from those we used in the present study. To our knowledge, no previous studies have directly measured the diameter of radial artery with ultrasound technique. The natriuretic peptides did not induce headache despite the fact that they activate the cGMP-dependent pathway via pGC and NO in vascular cells [46, 47], an important pathway in the induction of headache and migraine [48, 49]. However, in the pilot study two participants reported delayed headache after infusion of very high pharmacological doses of ANP and CNP, respectively. This could indicate that the headache inducing effects of natriuretic peptides are dose-dependent, but very high doses are not possible due to severe side effects. Moreover, the delayed headache suggests induction by secondary processes rather than by the systemically circulating natriuretic peptides from infusion. Consequently, we cannot rule out that natriuretic peptides may induce headache due to endogenous release
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of ANP, BNP, CNP or other neuropeptides from perivascular nerve endings of cerebral arteries.
4.1 Interpretation of results Transcranial Doppler measurements of velocity alone must also be interpreted with some caution because velocity is dependent on both the diameter of the MCA and the regional cerebral blood flow (CBF). It is uncertain whether natriuretic peptides may cause changes in CBF [19]. To date, results from animal studies have been contradictory and inconsistent regarding what response natriuretic peptides elicit on CBF [21, 50, 51]. These experiments were performed in different animals albeit with similar doses of the natriuretic peptides. However, TCD is still useful to give an idea of possible effects on arteries and a decrease in velocity is regarded as an expression for vasodilatation [27]. Measurements are the most precise in the MCA with a CV of 26% between participants and 11% between days [27, 34]. To reduce the impact of intersubject and interobserver variability of TCD measurements, the present study was designed as a crossover study and the same trained investigator performed all measurements. There is also a correlation between PetCO2 and VMCA, which has previously been established [52], but in the present study PetCO2 was more or less constant for all natriuretic peptides and can therefore not influence our VMCA measurements. Highfrequency ultrasound determination of the diameter of the STA and radial artery is highly accurate and reproducible as documented in many previous studies [29, 53]. The lack of response on mean VMCA in the main study may have several explanations. One explanation could be that the doses we used in the main study were too low. However, the doses we used were as high as those used in previous drug trials of ANP (Carperitide) and BNP (Nesiritide) in healthy volunteers and heart failure patients [16, 54– 57]. Using higher doses would hold the risk of unwanted side effects as we experienced in
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our pilot study. Another possibility is that interspecies difference may play a role and that human arteries are less sensitive so much higher doses were needed in our healthy subjects to see the same response as in animals. The effects of the natriuretic peptides may also have some latency due to autoregulatory mechanisms. There was a clear trend of an increase in HR after ANP and BNP, which could indicate sympathetic activation. It is possible that the vasodilatory effects of the natriuretic peptides were counter regulated by the sympathetic nervous system. Furthermore, in contrast to previous studies, we infused the natriuretic peptides in a high dose over only 20 min. ANP and BNP given as therapy for heart failure patients is administered as continuous infusion for 24–48 hours [16]. The half-lives of ANP and BNP are biphasic consisting, of a fast phase (1.7–2.1 min) and also a slow phase (13–37 min) [58, 59], i.e. long infusion duration may contribute to achieving a higher plasma concentration. Previous studies with continuous infusion of ANP and BNP reported a progressive response in HR and MAP developing over two hours [60, 61]. This may also explain why two participants in our pilot study experienced a vasovagal syncope incident over one hour after the cessation of ANP and BNP infusion. In addition, the lack of effect on MCA could also be because natriuretic peptides pass the blood–brain barrier (BBB) poorly [62] and may therefore not reach the vascular smooth muscle cells of the cerebral arteries unless given in very high doses. In our in vitro experiment which showed dilatation of MCA, natriuretic peptides were applied both intra- and extraluminally. Therefore, a model that allows selective intra- and extraluminal application of the natriuretic peptides would have been interesting. The distribution of natriuretic peptide receptors (NPR) on cerebral arteries may also be different from the peripheral vascular system. To our knowledge, no studies on the distribution of NPRs on cerebral arteries have been done.
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5. Conclusion This is the first study investigating the effects of natriuretic peptides on cerebral arteries in humans. We showed that natriuretic peptides in physiological and pharmacological doses (≤0.15 μg/kg/min for ANP and ≤0.06 μg/kg/min for BNP and CNP) had no significant effects on cerebral arteries in healthy volunteers. However, ANP and BNP showed significant dosedependent relaxation of cerebral arteries in guinea pigs. In addition, we cannot exclude that ANP and BNP may exert vasodilatory properties on the middle cerebral artery and the superficial temporal artery in very high doses (≥0.5 μg/kg/min for ANP and ≥0.2 μg/kg/min for BNP), whether these being pharmacological or caused by severe cardiac disease. It is still unclear whether natriuretic peptides may have potential implications in diseases where alterations of the cerebral hemodynamic play a role and more studies are warranted.
Acknowledgements The authors thank all the volunteers who participated in this study. Special thanks to Denise M Hublein and Dijana Terzic for performing the biochemical analyses, and Niklas Kahr Rasmussen, Lene Elkjær and Winnie Grønning for technical assistance. Further thanks to funding from Novo Nordisk Foundation (NNF11OC1014333) and Independent ResearchMedical Sciences (FSS) (DFF-1331-00210A), Lundbeck Foundation (R155-2014-171) and the FP7-EUROHEADPAIN (602633).
Disclosures Song Guo has received a travel grant from Autonomic Technologies Inc. Jes Olesen has received grants/research support from and/or has been a consultant/scientific adviser for, and/or has been in the speakers’ bureau of Allergan Inc, AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim, Eli Lilly, GlaxoSmithKline, Janssen Pharmaceutical Products,
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Lundbeck, Merck, Amgen, Alder and Pfizer. Messoud Ashina has received consulting fees, speaking/teaching fees, and/or honoraria – Alder, Allergan, Amgen, ATI and Eli Lilly. Jens P. Goetze has no conflicts of interest to report.
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Figure Captions Figure 1: Flow chart of the experiments in animal and humans investigating effects of the natriuretic peptides on cerebral arteries. Pilot and main experiments were conducted in a double-blind, randomized three-ways crossover design. Participants were allocated to receive intravenous infusion of different doses of ANP, BNP or CNP on each study day.
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Figure 2: Experimental protocol for each study day in the main experiment.
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Figure 3: ANP and BNP showed clear relaxation of both BA and MCA, whereas generally no response was seen for CNP. Concentration response curves are plotted graphically with values given as means±SEM.
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Figure 4: (A) Plasma ANP, BNP and CNP concentrations up to one hour after the start of infusion in healthy subjects (mean±SEM). Infusion was given from 0-20 min. (B) Plasma concentrations of cGMP up to one hour after the start of ANP infusion (0.05 µg/kg/min). Data are shown as individual and mean±SEM.
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Figure 5: Median of mean flow velocities in the middle cerebral artery (VMCA) for dose 1 and 2, and changes (%) from baseline of mean VMCA with 95% CI in six healthy subjects after ANP, BNP or CNP. There was no significant change in VMCA after 20 min of infusion for dose 1 and dose 2 compared to baseline. We found no difference in area under the curve (AUC) between dose 1 and dose 2. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. *P-value: Wilcoxon signed rank test.
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Figure 6: Median changes with 95% CI for HR and MAP after infusion (20 min) of ANP, BNP and CNP in six healthy subjects. There was no difference in the AUC for HR or MAP between dose 1 and dose 2.
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Tables
Table 1: Time points and measurements () for the first administration round. The second administration round was exactly the same except no blood samples were taken. Time:
-10
0
5
10
20
30
40
45
50
60
──────►
Infusion Headache questions
Blood flow velocity in MCA
Radial and temporal arteries
BP, HR and pCO2
ECG Blood flow in face microvasculature Blood samples
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Table 2: Recorded variables in pilot experiment for dose 1, 2 and 3 of ANP, BNP and CNP in one to three subjects. Mean change (± %) after 20 min of infusion compared to baseline is shown in the table. Due to adverse events BNP dose 1 and 2 were reduced to 0.04 and 0.06 µg/kg/min. ANP
BNP
CNP
Dose 1 0.5 (n=3)
Dose 2 1.0 (n=2)
Dose 3 2.0 (n=2)
Dose 1 0.04 (n=1)
Dose 2 0.06 (n=1)
Dose 3 0.2 (n=1)
Dose 1 0.2 (n=2)
Dose 2 0.4 (n=2)
Dose 3 0.8 (n=1)
Mean VMCA
-15%
-12%
-14%
-9%
-17%
-16%
-3%
0%
+6%
Temporal artery (STA)
+16%
+29%
+28%
-8%
+20%
+39%
-7%
+9%
+4%
Radial artery (RA)
+1%
-3%
+1%
-18%
-2%
+4%
-4%
+5%
+5%
Blood flow of face
+1%
+18%
+9%
-9%
-11%
+3%
-7%
-7%
-16%
MAP
-7%
-9%
-5%
-9%
-8%
-9%
+2%
-4%
-9%
+11%
+40%
+30%
+26%
+18%
+21%
-2
+4%
4%
1
2
0
1
0
1
0
1
0
µg/kg/min: number of subjects:
Heart rate Subjects who felt urge to urinate (n) Immediate headache (during in-hospital) Delayed headache (after discharge from hospital)
None
None
None
Yes (n=1)
Not reported due to AE
Yes (n=1)
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Table 3: Recorded variables in the main study for dose 1 and dose 2 of ANP, BNP and CNP in six healthy subjects. Table shows mean change (± %) after 20 min of infusion compared to baseline with calculated P-value (Wilcoxon signed rank test). Difference between dose 1 and dose 2 of ANP, BNP or CNP are calculated as area under the curve (AUC) and shown as PAUC-value. Baseline was subtracted before calculating the AUC to reduce variation between sessions within subject. *P-values for secondary end-points were adjusted with HolmBonferroni correction.
ANP (n=6) µg/kg/min: Temporal artery* Radial artery * Blood flow of face * PetCO2* MAP Heart rate
Dose 1
Dose 2
0.05
BNP (n=6) Dose 1
Dose 2
0.15
PAUCvalue
0.02
+2%
+4%
1.000
P=1.000
P=1.000
0%
-2%
P=1.000
P=1.000
-6%
-3%
P=0.624
P=1.000
+1%
-2%
P=1.000
P=1.000
-4%
+7%
P=0.496
P=0.095
+18%
+12%
P=0.063
P=0.067
0.252 1.000 0.468 0.094 1.000
CNP (n=6) Dose 1
Dose 2
0.06
PAUCvalue
0.02
0.06
PAUCvalue
+8%
+3%
0.375
+10%
+4%
0.124
P=0.124
P=0.876
P=0.124
P=0.345
-6%
+1%
+1%
0%
P=0.124
P=1.000
P=1.000
P=1.000
0%
-5%
+2%
-7%
P=1.000
P=0.939
P=1.000
P=0.124
-1%
0%
-1%
-1%
P=1.000
P=1.000
P=1.000
P=0.980
+5%
-2%
-2%
-1%
P=0.163
P=0.581
P=0.570
P=0.834
-2%
+8%
-3%
-3%
P=0.752
P=0.203
P=0.343
P=0.399
0.124 0.438 0.375 0.156 0.063
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1.000 1.000 1.000 0.313 0.688