Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses

Accepted Manuscript Title: Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses Author: J.M. Serrano-Rodríguez, M. Gómez-Díez, M. Esgueva, C. CastejónRiber, A. Mena-Bravo, F. Priego-Capote, J.M. Serrano Caballero, A. Muñoz PII: DOI: Reference:

S1090-0233(15)00433-5 http://dx.doi.org/doi: 10.1016/j.tvjl.2015.10.024 YTVJL 4662

To appear in:

The Veterinary Journal

Accepted date:

8-10-2015

Please cite this article as: J.M. Serrano-Rodríguez, M. Gómez-Díez, M. Esgueva, C. CastejónRiber, A. Mena-Bravo, F. Priego-Capote, J.M. Serrano Caballero, A. Muñoz, Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses, The Veterinary Journal (2015), http://dx.doi.org/doi: 10.1016/j.tvjl.2015.10.024. 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.

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Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses J.M. Serrano-Rodríguez a, M. Gómez-Díez b, M. Esgueva b, C. Castejón-Riber b, A. MenaBravo c,d, F. Priego-Capote c,d, J.M. Serrano Caballero a, A. Muñoz b,e,* a

Department of Pharmacology, Toxicology and Legal and Forensic Medicine, Faculty of Veterinary Medicine, University of Cordoba, Spain b Equine Sport Medicine Centre, CEMEDE, University of Cordoba, Spain c Department of Analytical Chemistry, Faculty of Sciences, University of Cordoba, Spain d Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía University Hospital, University of Córdoba, Córdoba, Spain e Department of Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, Spain

* Corresponding author. Tel.: +34 957 218702. E-mail address: [email protected] (J.M. Serrano-Rodríguez).

23 24 Highlights 25 26 27  The angiotensin converting enzyme inhibitor ramiprilat was evaluated in horses after 28 different IV and PO doses of ramipril. 29 30  Pharmacokinetic and pharmacodynamic relationships were obtained and investigated. 31 32  The oral bioavailability of ramiprilat after PO ramipril was low. 33 34  Higher oral doses of ramipril had sufficient absorption and bioconversion to 35 ramiprilat to induce serum ACE inhibitions near to 85%. 36 37  Further work with different doses or formulations in equine patients should be 38 undertaken

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39 Abstract 40

The pharmacokinetics and pharmacodynamics (PK/PD) of the angiotensin-converting

41 enzyme inhibitor (ACEI) ramiprilat after intravenous (IV) and oral (PO) administration of 42 ramipril have not been evaluated in horses. This study was designed to establish PK profiles 43 for ramipril and ramiprilat as well as to determine the effects of ramiprilat on serum 44 angiotensin converting enzyme (ACE) and to select the most appropriate ramipril dose that 45 suppresses ACE activity. Six healthy horses in a cross-over design received IV ramipril 0.050 46 mg/kg, PO at a dose of 0 (placebo), and 0.050, 0.10, 0.20, 0.40 and 0.80 mg/kg ramipril. 47 Blood pressures were measured and blood samples obtained at different times. Serum 48 ramipril and ramiprilat concentrations and serum ACE activity were measured by liquid 49 chromatography-tandem

mass

spectrometry

(LC-MS/MS)

and

spectrophotometry,

50 respectively. 51 52

Systemic bioavailability of ramiprilat after PO ramipril was 6-9%. Percentages of

53 maximum ACE inhibitions from baseline were 98.88 (IV ramipril), 5.31 (placebo) and 27.68, 54 39.27, 46.67, 76.13 and 84.27 (the five doses of PO ramipril). Blood pressure did not change 55 during the experiments. Although oral availability of ramiprilat was low, ramipril has 56 sufficient enteral absorption and bioconversion to ramiprilat to induce serum ACE inhibitions 57 of almost 85% after a dose of 0.80 mg/kg ramipril. Additional research on ramipril 58 administration in equine patients is indicated. 59 60 Keywords: Angiotensin-converting enzyme inhibitors; Ramiprilat; Ramipril; 61 Pharmacokinetic-pharmacodynamic; Horses 62

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63 Introduction 64

Ramipril is an angiotensin converting enzyme inhibitor (ACEI) administered orally

65 (PO) for long-term therapy of cardiovascular disease, hypertension and renal failure in 66 humans and small animals (Brewster et al., 2003; Lefevbre et al., 2007). Ramipril is a pro67 drug, and after PO absorption, is hydrolysed in the liver to the active drug (ramiprilat). 68 Ramiprilat prevents conversion of angiotensin I to II by inhibiting angiotensin converting 69 enzyme, ACE (Lefevbre et al., 2007; Toutain and Lefebvre, 2004). Because angiotensin II 70 causes vasoconstriction, ACEIs are used as vasodilator agents (Toutain and Lefebvre, 2004). 71 72

Pharmacokinetic and pharmacodynamic (PK/PD) relationships of ACEIs are

73 complicated because they have a nonlinear binding to ACE, the target enzyme, and because 74 there are two ACE pools, circulating and in tissues. ACE is distributed throughout the body 75 but mainly at the surface of vascular endothelium. Circulating ACE originates mainly from 76 the vascular endothelium, representing about 5-30% of serum ACE in different species 77 including humans, dogs and cats (Toutain and Lefebvre, 2004; Afonso et al., 2013). 78 Therefore, physiologically based models with binding parameters of ACEI to ACE are 79 required to allow appropriate interpretation of the different phases of disposition curves 80 (Toutain and Lefebvre, 2004). To the best of our knowledge, these models have not been 81 applied in horses. 82 83

PK/PD studies of ACEIs in horses are limited to enalaprilat and quinaprilat after

84 intravenous (IV) and PO enalapril and quinapril (Davis et al., 2014; Gadner et al., 2004; 85 Gómez-Díez et al., 2014). Recently, Afonso et al. (2014) administered two PO doses of 86 ramipril in horses (low, 0.1 mg/kg; high, 0.3 mg/kg) and evaluated serum ACE inhibition and

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87 changes in blood pressure (BP). In this study, however, IV ramipril was not administered and 88 serum concentrations of ramipril and ramiprilat were not measured. 89 90

The objectives of the present study were: (1) to establish the serum concentration–

91 time profile for ramipril, and its active metabolite ramiprilat after IV ramipril at 0.050 mg/kg, 92 PO ramipril at 0 (placebo), and five different PO doses (0.050, 0.10, 0.20, 0.40 and 0.80 93 mg/kg), using a physiologically based PK/PD model; (2) to determine the effects of 94 ramiprilat on serum ACE; (3) to investigate the effects on BP with these doses and routes; 95 and (4) to predict and to simulate the most appropriate PO dose that suppresses ACE activity 96 after single and multiple doses of ramipril. 97 98 Material and methods 99 Horses 100

Six crossbred horses, two mares and four geldings, aged between 5 and 11 years (10.8

101 ± 2.35), and weighing between 378 and 530 kg (458.2 ± 59) were studied. Prior to the 102 experiment, physical examinations, haematology, serum biochemistry, including fibrinogen 103 and myocardial troponin concentrations, resting electrocardiography, echocardiography and 104 blood pressure (BP) measurements, were performed. Only healthy animals were studied. 105 106

The animals were located in individual paddocks and received the same food,

107 consisting of rye-grass haylage, at 1% bodyweight. The horses did not have access to salt or 108 electrolyte supplements during the study. 109 110 Experimental protocol

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111

The research was approved by the Ethical Committee for Animal Experimentation of

112 the University of Córdoba (Reference: 55.60 PE, date of approval1 February 2010). 113 114

The study was conducted in two trials. In the first trial, each animal received IV

115 ramipril at 0.050 mg/kg. After a week of washout, the second trial was undertaken, using a 116 blinded and randomized Latin square (6  6) design. Each animal received PO placebo or 117 ramipril at 0.050, 0.10, 0.20, 0.40 or 0.80 mg/kg, with at least 1 week of washout between 118 trials. Food was withdrawn from 12 h before administration of the pro-drug to 8 h after. 119 120

For IV dosing, ramipril (Sigma-Aldrich) was dissolved in 0.7% saline and 1%

121 NaHCO3 (King et al., 2003). For PO doses, ramipril tablets (Vasotop P 5 mg, Merck Sharp 122 and Dohme Animal Health) were dissolved and suspended in 150 mL of water, sonicated in 123 an ultrasonic bath for 15 min and stored at 4 ºC before trials. For placebo, the equivalent 124 amount of water without the pro-drug was used. Pro-drugs and placebo were administered by 125 nasogastric intubation and after, the nasogastric tube was rinsed with 250 mL of water. 126 127

In each assay, venous blood samples were taken before administration (time 0), and at

128 5, 10, 15 and 30 min and at 1, 2, 4, 8, 12, 24, 36 and 48 h after. 129 130 Blood pressure monitoring 131

Systolic (SBP) and diastolic (DBP) blood pressures were measured non-invasively

132 with a multiparametric monitor (S/5 Datex-Ohmeda Compact) in the coccygeal artery at each 133 sampling time. Three BP measurements were made at each sampling point and the results are 134 presented as the means of the three measurements.

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135 136 Blood processing and analysis 137

After extraction, blood samples were poured in tubes without anticoagulant and left to

138 coagulate in a refrigerator for 30 min. They were then centrifuged, serum was obtained, and 139 samples stored at -90 ºC until analysis. 140 141

Serum ACE activity was measured by spectrophotometry (Afonso et al., 2013;

142 Gómez-Díez et al., 2014). This method quantifies serum ACE activity by hydrolysis of the N143 (3-[2-furyl]acrylolyl)-Lphenylalanyl-glycyl-glycine(FAPGG),

forming

furylacryloyl-

144 phenylalanine (FAP), that results in a decrease in absorbance at 340 nm. Linear calibration 145 curves from 3 to 150 IU/L were obtained, and the correlations coefficients (r) were >0.99. 146 Precision of the technique was between 2.86–6.21%. The limits of quantification (LOQ) and 147 detection (LOD) were 5 and 3 IU/L, respectively. 148 149

Serum concentrations of ramiprilat (Santa Cruz Biotechnology), ramipril and enalapril

150 as internal standard (Sigma-Aldrich) were measured using a liquid chromatography-tandem 151 mass spectrometry (LC-MS/MS) assay (Yuang et al., 2008). A volume of 700 μL of horse 152 serum was spiked with internal standard solution and then 1 mL of methanol was added. 153 After shaking in an ultrasonic bath for 5 min, and centrifugation at 28300 g for 10 min at 4º 154 C, supernatant was extracted, and 3 mL of ethyl acetate were added. The mixture was mixed 155 and centrifuged for 5 min at 2000 g. The upper organic layer was evaporated to dryness under 156 N2 at 40 ºC (Gómez-Díez et al., 2014). The residue was reconstituted in 400 μL of methanol 157 and aliquot of 5 μL were injected into the LC-MS/MS system. Linear calibrations of ramipril 158 and ramiprilat between 0.3-1000 ng/mL were obtained (r>0.99). The accuracy and precision

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159 were within 1.2 – 4.0%. Values of LOQ and LOD for both analytes were 0.5 ng/mL and 0.15 160 ng/mL. See Appendix (Supplementary material) for more information. 161 162 Pharmacokinetic and pharmacodynamic analysis 163

For each animal, concentration-time data of ramipril and ramiprilat were analyzed

164 simultaneously by a physiologically based compartmental model previously described 165 (Lefebvre et al., 2006; Lees et al., 1989; Toutain et al., 2000).The model is presented in Fig. 1 166 and consists of a single compartment with a volume of distribution of Vc in which ramipril is 167 absorbed and transformed to ramiprilat according to absorption and formation first-order rate 168 constants of ka and kf, respectively. Ramipril might be eliminated via other pathways, 169 including urinary excretion and biotransformation to other metabolites (Van Griensven et al., 170 1995). For that reason, a first order elimination rate constant k10pwas included. Ramiprilat is 171 bound to ACE or free or non-specifically bound to albumin. However, free and albumin172 bound fractions are indistinguishable in the model. Consequently free ramiprilat corresponds 173 to free ramiprilat plus ramiprilat bound to albumin (Lefebvre et al., 2006). Free ramiprilat 174 fraction was eliminated according to a first order rate constant k10. Free ramiprilat clearance 175 was calculated as Cl = k10· Vc and the elimination half-live as t½k10 = (ln2)/k10. 176 177

After PO ramipril, the volume of distribution Vc/F and clearance Cl/F of ramiprilat

178 were calculated, with F indicating systemic bioavailability of ramiprilat after PO 179 administration of ramipril. The F value was calculated from the ratio of Cl of ramiprilat after 180 IV ramipril and Cl/F of ramiprilat after PO ramipril (Toutain and Lefebvre, 2004). Ideally, IV 181 ramiprilat would have been administered to determine Vc, Cl and F values. However, the 182 necessary amount of ramiprilat for IV formulation was not available. Administration of IV

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183 ramipril does, however, provide important information about the metabolism from ramipril to 184 ramiprilat in horses as described by other ACEIs (Davis et al., 2014; Gadner et al., 2004; 185 Gómez-Díez et al., 2014). 186 187

Binding parameters of ramiprilat to ACE calculated by the model were: Kd,

188 equilibrium dissociation constant which expresses ramiprilat affinity for the ACE; Bmax, size 189 of ACE pool; fcirc, fraction between circulating and non-circulating ACE and circulating ACE 190 as Pmax = fcirc ∙ Bmax. Data were weighted with the inverse of the square of the observation 191 (Lefebvre et al., 2006). 192 193

For PD data, the observed effect E(t) was defined as the serum ACE activity at each

194 time expressed as percent of control before administration of the pro-drug (E0), IC50 was the 195 free ramiprilat concentration required to produce 50% of ACE inhibition and γ was the 196 coefficient which describes the steepness of the sigmoid curve. C was the free ramiprilat 197 concentration obtained after PK modelling (Lefebvre et al., 2006). These parameters were 198 fitted by the Hill equation: 199 200

γ  (C) E (t )  E 0   1  γ γ  (IC 50 )  (C) 

   

201 202

Other parameters obtained were the maximum serum ACE inhibition between 0-12 h

203 (%Imax), the time to reach %Imax (T%Imax), and serum ACE inhibition at 12, 24 and 48 h 204 (%I12h, %I24h and %I48h), respectively. 205

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206

Because a long term therapy is indicated for ACEIs, simulations of PO doses of

207 ramipril from 0.0063 to 6.30 mg/kg were made after single and repeated doses every 24 h for 208 a week. Predictions were performed with PK/PD parameters obtained from a representative 209 horse after PO ramipril at 0.80 mg/kg to describe the effect-time relationship and the 210 influence of the dosing. All PK/PD modelling and simulations included in this work were 211 developed using ADAPT5 software (D’Argenio et al., 2009). 212 213 Statistical analysis 214

Arithmetic mean, standard deviation and harmonic mean were calculated. The

215 Wilcoxon rank sum test was used to assess significant differences between IV and PO routes 216 at 0.050 mg/kg. The Kruskal-Wallis test was used to evaluate significant differences between 217 PO doses including placebo. When differences were found, a Wilcoxon rank sum test was 218 used. The significance level was fixed at P< 0.05 using Statgraphics Centurion XVI.I as 219 statistic software (StatPoint Technologies). 220 221 Results 222

No local or systemic adverse reactions were detected during or after IV and PO

223 administrations. 224 225

Mean (±SD) serum concentrations of ramipril and ramiprilat after IV and PO ramipril

226 are plotted in Fig. 2. Ramipril and ramiprilat concentrations after PO ramipril at 0.050 and 227 0.10 mg/kg were below LOQ in all horses with exception of some sampling times. However, 228 both ramipril and ramiprilat had measurable concentrations after 0.20 mg/kg of ramipril, 229 although this was not enough to be fitted by the model. Therefore, only concentrations of

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230 ramipril and ramiprilat after IV ramipril at 0.050 mg/kg and PO ramipril at 0.40 and 0.80 231 mg/kg were analyzed by the PK/PD models (Table 1). 232 233

Mean (±SD) serum ACE activities, expressed as percent of control (value at time 0)

234 from 0 to 48 h are plotted in Fig. 3. Mean values of %Imax, T%Imax, and %I12h, %I24h, and %I48h 235 for each administration route and dose are presented in Table 2. Differences in SBP and DBP 236 values after IV and PO doses of ramipril or placebo were not found between trials, even at 237 maximum serum time ACE inhibitions. 238 239

For the simulations, the predicted effects on serum ACE activity after single and

240 multiple PO doses of ramipril from 0.0063 to 6.30 mg/kg every 24 h for a week were 241 calculated (Fig. 4). 242 243 Discussion 244

In the present study, serum ramipril and ramiprilat concentrations were low and

245 declined rapidly after IV administration of ramipril with a long terminal phase for ramiprilat 246 until 36 h. Ramipril concentrations were lower than ramiprilat concentrations from 0.26 h 247 (Fig. 2a), suggesting a rapid elimination by biotransformation to ramiprilat, and by other 248 routes with a kf and k10p values of 4.78 and 1.86 1/h, respectively (Table 1). The half-life of 249 elimination of ramiprilat was 0.16 h (9.6 min approximately). This very short value can be 250 due to the small volume of distribution and particularly to the high clearance observed (table 251 1). The rapid decreasing phase of ramiprilat is controlled by k10 and reflects the elimination 252 process (Fig. 2a). It is likely that during this phase serum ACE is saturated, explaining the 253 high inhibition values (Fig. 3a) and close to 98.88% at 0.17 and 0.25 h (Toutain and

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254 Lefevbre, 2004). Moreover, the terminal phase observed for ramiprilat reflects the binding to 255 ACE and is influenced by binding parameters and by k10 (Lefebvre et al., 2006). 256 257

Following PO ramipril administration, the bioavailability of ramiprilat was low (6.62

258 – 9.08%), with values similar to those reported in dogs but lower than those in humans 259 (Lefebvre et al., 2006; Van Griensven et al; 1995). However, in the current research, higher 260 PO doses were used compared to IV dose (8 and 16-fold), unlike the studies in dogs and 261 humans. These findings might indicate that most of the pro-drug administered was not 262 absorbed and higher PO doses should be used compared to the IV dose in order to achieve the 263 same ACE inhibition (Fig. 3a) unlike other species (Lefebvre et al., 2006; Van Griensven et 264 al; 1995). 265 266

Different studies have shown that PO availability of ACEIs in horses is low (Davis et

267 al., 2014; Gadner et al., 2004; Gómez-Díez et al., 2014). Because of the limited knowledge of 268 the factors that contribute to PO absorption of drugs in the horse, various hypotheses have 269 been proposed, including interspecific differences in gastrointestinal anatomy and physiology 270 relative to enteral absorption, and a possible minor influence of intestinal uptake by peptide 271 transporter 1 (PepT 1) transport compared to passive diffusion (Davis et al., 2014; Gómez272 Díez et al., 2014; Maxwell, 2015). 273 274

Values for ka of ramipril were low and differed between both doses, because the

275 ascending phase of ramipril is PO absorption and bioconversion to ramiprilat, and the 276 decreasing phase is elimination by biotransformation to ramiprilat and other pathways. For 277 ramiprilat, the ascending phase is mainly a bioconversion phase from ramipril (Fig. 2b).

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278 Visually, the next phase of the curve is more difficult to interpret, because it is an elimination 279 phase influenced by k10, but also a binding phase to ACE controlled by Kd, Bmax and fcirc 280 (Lefebvre et al., 2006). The elimination of ramiprilat described by k10 was different between 281 PO doses (P<0.05), and also t1/2

k10

(Table1). The elimination was slightly lower at 0.80

282 mg/kg of ramipril, possibly because ACE was more saturated than at 0.40 mg/kg. Moreover, 283 owing to the low concentrations of ramiprilat observed, it is unlikely that a maximum 284 saturation of ACE occurred at the doses tested, since %Imax only reached 84.27% at times 285 close to 2 h, unlike IV ramipril, with a %Imax value of 98.88% between 0.75 and 0.25 h (Table 286 2). However, the inhibition after PO doses lasted longer than IV administration (Fig. 3), 287 particularly with 0.40 and 0.80 mg/kg of ramipril at 24 and 48 h (Table 2). 288 289

Binding parameters derived from the model indicated that the ACE pool binding

290 capacity was close to 80-100 nmol/L, and approximately 5% of the ACE pool was circulating 291 (Table 1). These results differ from those previously obtained in dogs and cats (Toutain and 292 Lefebvre, 2004). This is because Bmax and fcirc are species-related properties and might be 293 lower in horses. On the other hand, Kd is a drug-related property, and the value determined, 294 close to 1.35 nmol/L, was slightly higher than the value obtained in dogs, but fell into the 295 nanomolar range suggesting that ramiprilat has a greater affinity for ACE (Toutain and 296 Lefebvre, 2004; Lefevbre et al., 2006). 297 298

The values of IC50 of ramiprilat, 1.05 nmol/L after IV ramipril and 0.88 and 1.07

299 nmol/L after PO ramipril 0.40 and 0.80 mg/kg, observed between 7, 20 and 36 h, were 300 consistent with the inhibition values observed in those times (Fig. 3). Moreover, γ values 301 were close to 0.60, indicating that a relative shallow concentration effect relationship and that

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302 some residual inhibition can be obtained for very low ramiprilat concentrations, especially at 303 0.80 mg/kg of ramipril (Fig. 3). These data are in agreement with the results obtained with 304 ramiprilat and other ACEIs in different species (King et al., 2003, Lefevbre et al., 2006). 305 306

Our model was also used to predict PK/PD profiles with different dosing regimens of

307 ramipril. After single doses, visual inspection of the curves showed that although ACE 308 inhibition increased at higher doses, the effect was quite similar from 0.80 to 6.30 mg/kg 309 (Fig. 4a). Moreover, after multiple ramipril administration, inhibition values from 0.0063 to 310 0.20 mg/kg produced less dose-related inhibition of serum ACE activity and it took longer to 311 reach steady state. In fact, the maximum effect was not reached for a week with these doses 312 and longer times would be required. However, PO doses from 0.80 to 6.30 mg/kg were very 313 similar and faster to reach a steady state from the first administration. These findings are in 314 agreement with the simulations described with PO benazepril in cats and dogs (King et al., 315 2003; Toutain et al., 2000) and indicate that it is possible to inhibit ACE at very low doses 316 due to the progressive saturation by ramiprilat. In addition, the model predicts that an initial 317 dose between 0.80 to 1.6 mg/kg could be administered to immediately inhibit ACE followed 318 by the same maintenance doses every day (Fig. 4a). 319 320

The lack of variations in BP during the experiments was in agreement with other

321 authors studying other ACEIs in horses (Davis et al., 2014; Gadner et al., 2004; Gómez-Díez 322 et al., 2014). This may indicate that ACEIs have little effect on BP in normotensive subjects 323 (Lefevbre et al., 2007, Gómez-Díez et al., 2014). 324 325 Conclusions

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326

The use of physiological compartmental PK/PD models permitted us to study the

327 pharmacology of ramiprilat after IV and PO doses of ramipril in horses. Despite its 328 complexity, these models can interpret more appropriately the disposition phases of ACEIs 329 concentration-time curves. The current study shows that PO availability of ramipril at doses 330 from 0.050 to 0.80 mg/kg in horses is low. Although there was a tendency toward a decrease 331 in serum ACE activity, these values were never suppressed by more than 84.27% unlike the 332 IV data of 98.88%, using a dose 16-fold lower than the PO dose. It is unknown whether an 333 ACE inhibition near 80%, as found in our study with PO ramipril, would be sufficient to 334 counteract vasoconstriction in pathological conditions. This is a preliminary study with 335 healthy horses and further work with different doses or formulations in equine patients should 336 be conducted. 337 338 Conflict of interest statement 339

None of the authors of this paper has a financial or personal relationship with other

340 people or organizations that could inappropriately influence or bias the content of the paper. 341 342 Acknowledgments 343

This research has been generously supported by the Science and Innovation Council

344 of Spain (Research Project AGL2010-17431). 345 346 Appendix: Supplementary material 347

Supplementary data associated with this article can be found in the online version at

348 doi: setters please insert doi number 349

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350 References 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395

Afonso, T., Giguère, S., Rappaport, G., Berghaus, L.J., Barton, M.H., Coleman, A.E., 2013. Pharmacodynamic evaluation of 4 angiotensin-converting enzyme inhibitors in healthy adult horses. Journal of Veterinary Internal Medicine 27, 1185-1192. Brewster, U.C., Setaro, J.F., Pezarella, M.A., 2003. The renin-angiotensin-aldosterone system. Cardiorenal effects and implications for renal and cardiovascular disease states. American Journal of Medical Sciences 32, 15-24. D’Argenio, D.Z., Schumitzky, A., Wang, X.,2009. ADAPT 5 user’s guide: pharmacokinetic/pharmacodynamic systems analysis software. Biomedical Simulations Resource, Los Angeles, CA. Davis, J.L., Kruger, K., Lafevers, D.H., Barlow, B.M., Schirmer, J.M, Breuhaus, B.A., 2014. Effects of quinapril on angiotensin-converting enzyme and plasma renin activity as well as pharmacokinetic parameters of quinapril and its active metabolite, quinaprilat, after intravenous and oral administration to mature horses. Equine Veterinary Journal 46, 729-733. Gardner, S.Y., Atkins, C.E., Rams, R.A., Schwabenton, A.B., Papich, M.G., 2004. Characterization of the pharmacokinetic and pharmacodynamic properties of the angiotensinconverting enzyme inhibitor, enalapril, in horses. Journal of Veterinary Internal Medicine 18, 231-237. Gómez-Díez, M., Muñoz, A., Caballero, J.M., Riber, C., Castejón, F., Serrano-Rodríguez, J.M., 2014. Pharmacokinetics and pharmacodynamics of enalapril and its active metabolite, enalaprilat, at four different doses in healthy horses. Research in Veterinary Science 97, 105110. King, J.N., Maurer, M., Toutain, P.L., 2003. Pharmacokinetic⁄pharmacodynamic modelling of the disposition and effect of benazepril and benazeprilat in cats. Journal of Veterinary Pharmacology and Therapeutics 26, 213-224. Lees, K.R., Kelman, A.W., Reid, J.L., Whiting, B., 1989. Pharmacokinetics of an ACE inhibitor, S-9780, in man: evidence of tissue binding. Journal of Pharmacokinetics and Biopharmaceutics17, 529-550. Lefebvre, H.P., Brown, S.A., Chetboul, V., King, J.N., Puchelon, J.L., Toutain, P.L., 2007. Angiotensin-converting enzyme inhibitors in veterinary medicine. Current Pharmaceutical Design 13, 1347-1361. Lefebvre, H.P., Jeunesse, E., Laroute, V., Toutain, P.L., 2006. Pharmacokinetic and pharmacodynamic parameters of ramipril and ramiprilat in healthy dogs and dogs with reduced glomerular filtration rate. Journal of Veterinary Internal Medicine 20, 499-507. Maxwell, L., 2015. Horse of a Different Color: Peculiarities of Equine Pharmacology. In: Equine Pharmacology, First Ed. John Wiley and Sons, pp. 3-15.

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Toutain, P.L., Lefebvre, H.P., 2004. Pharmacokinetic and pharmacokinetic/ pharmacodynamic relationships for angiotensin-converting enzyme inhibitors. Journal of Veterinary Pharmacology and Therapeutics 27, 515-525. Toutain, P.L., Lefebvre, H.P., King, J.N., 2000. Benazeprilat disposition and effect in dogs revisited with a pharmacokinetic/pharmacodynamics modeling approach. Journal of Pharmacology and Experimental Therapeutics 292, 1087-1093. Van Griensven, J.M., Schoemaker, R.C., Cohen, A.F., Luus, H.G., Seibert-Grafe, M., Röthig, H.J., 1995. Pharmacokinetics, pharmacodynamics and bioavailability of the ACE inhibitor ramipril. European Journal of Clinical Pharmacology47, 513-518. Yuan, B., Wang, X., Zhang, F., Jia, J., Tang, F., 2008. Simultaneous determination of ramipril and its active metabolite ramiprilat in human plasma by LC–MS-MS. Chromatographia68, 533-539.

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413

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414 Figure legends 415 Fig. 1. Physiologically based model for ramiprilat disposition after PO ramipril. For IV 416 disposition of ramiprilat the model was similar without absorption phase of ramipril. For 417 further explanation, see the text. 418 419 Fig. 2. (a) Semilogarithmic plot of serum concentrations (mean ± SD) of ramipril (-●-) and 420 ramiprilat (-●-) after IV ramipril at 0.05 mg/kg in horses. (b) Semilogarithmic plot of serum 421 concentrations (mean ± SD) of ramipril (-●-, -■-, -▲-) and ramiprilat (-●-, -■-, -▲-) after PO 422 ramipril at 0.20, 0.40 and 0.80 mg/kg in horses, respectively. 423 424 Fig. 3. (a) Mean serum ACE activity expressed as percent of control after IV ramipril at 0.05 425 mg/kg (-♦-) and PO ramipril at 0.05 mg/kg (-●-). (b) Mean serum ACE activity expressed as 426 percent of control after PO doses of placebo (0.00 mg/kg, -●-), of ramipril at 0.05 (-●-), at 427 0.10 (-▲-), at 0.20 (-▼-), at 0.40 (-■-) and at 0.80 mg/kg (-□-), respectively. 428 429 Fig. 4. (a) Plot of predicted effects on serum ACE activity in horses after single PO doses of 430 ramipril from 0.0063 to 6.30 mg/kg. (b) Plot of predicted effects on serum ACE activity in 431 horses after single PO doses of ramipril from 0.0063 to 6.30 mg/kg every 24 h for a week. 432 433 434 435

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436 Table 1 437 Pharmacokinetic-pharmacodynamic parameters (mean ± SD) for ramipril and ramiprilat after 438 different IV and PO ramipril doses.

Parameters Ramipril ka (1/h) k10p (1/h) kf (1/h) Ramiprilat k10 (1/h) t1/2 k10 (h)1 Vc (L/kg) Vc/F (L/kg) Cl (L/kg/h) Cl/F (L/kg/h) F (%) Kd (nmol/L) fcirc (%) Bmax (nmol/L) Pmax (nmol/L) IC50 (nmol/L) γ 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

Routes and doses IV (0.05 mg/kg)

PO (0.40 mg/kg)

PO (0.80 mg/kg)

1.86 ± 0.66 4.78 ± 1.20

0.23 ± 0.06 1.94 ± 0.61 6.70 ± 0.77

0.12 ± 0.06 1.60 ± 0.90 5.87 ± 2.26

5.49 ± 0.91 0.13 (0.10 – 0.16)

4.10 ± 1.70 0.17 (0.10 – 0.27)

2.35 ± 0.84

1.97 ± 0.74

12.82 ± 4.46 6.52 ± 3.51 1.47 ± 0.29 5.80 ± 0.58 79.31 ± 21.54 4.60 ± 1.38 1.07 ± 0.24 0.61 ± 0.12

7.79 ± 3.37 9.08 ± 4.01 1.19 ± 0.30 6.08 ± 1.43 91.72 ± 47.05 5.08 ± 1.08 0.88 ± 0.36 0.60 ± 0.11

5.09 ± 2.46 0.16 (0.09 – 0.31) 0.15 ± 0.04 0.77 ± 0.34

1.35 ± 0.31 5.01 ± 1.21 109.46 ± 27.14 5.27 ± 0.85 1.05 ± 0.21 0.63 ± 0.08

ka, absorption rate constant of ramipril; k10p, elimination rate constant of ramipril; kf, formation rate constant for ramiprilat from ramipril; k10, elimination rate constant of free ramiprilat; t1/2k10, half-life of elimination of free ramiprilat; Vc, apparent volume of distribution; Vc/F, apparent volume of distribution with respect to the bioavailability; Cl, clearance of the free ramiprilat; Cl/F, clearance of the free ramiprilat with respect to the bioavailability; F, bioavailability of ramiprilat after oral ramipril; Kd, equilibrium dissociation constant of free ramiprilat producing saturation of 50% of ACE; fcirc, fraction between circulating and non-circulating ACE; Bmax, total binding capacity of the ACE; Pmax, total binding capacity of the circulating ACE; IC50, free ramiprilat concentration required to produce 50% of ACE inhibition; γ, coefficient which describes the steepness of the concentration effect curve. 1 Harmonic mean and range.

456

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457 Table 2. 458 Serum ACE inhibitions (mean ± SD) after different IV and PO ramipril doses. Dose (mg/kg)/route 0.05/IV 0.00/PO 0.05/PO 0.10/PO 0.20/PO 0.40/PO 0.80/PO

%Imax 98.88 ± 0.80 5.31 ± 2.24 27.68 ± 10.88 39.27 ± 5.72 46.47 ± 14.39 76.13 ± 3.99 84.27 ± 4.00

T%Imax (h) 0.21 ± 0.04 0.68 ± 0.67 2.20 ± 1.10 1.80 ± 0.45 2.40 ± 1.52 1.67 ± 0.52 2.50 ± 1.22

%I12h 32.61 ± 10.43 0.48 ± 3.15 12.57 ± 6.67 26.60 ± 11.10 27.36 ± 8.88 55.44 ± 8.63 74.53 ± 4.24

%I24h 21.97 ± 6.63 1.77 ± 3.39 13.47 ± 6.29 22.79 ± 7.93 23.37 ± 13.23 35.76 ± 9.94 63.54 ± 8.16

%I48h 8.02 ± 4.05 -5.40 ± 4.58 5.30 ± 4.29 3.74 ± 3.48 12.72 ± 9.11 8.92 ± 6.69 38.01 ± 9.08

459 460 %Imax, maximum serum ACE inhibition from 0 to 12 h; T%Imax, time to reach the %Imax value; 461 %I12h, maximum serum ACE inhibition at 12 h; %I24h, maximum serum ACE inhibition at 24 462 h; %I48h, maximum serum ACE inhibition at 48 h. 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482

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