Department of Animal Medicine and Surgery. Department of Pharmacology, Toxicology and Legal and Forensic Medicine. Equine Sport Medicine Center

Faculty of Veterinary Medicine, University of Córdoba, Spain

PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES OF THREE -CONVERTING INHIBITORS IN HEALTHY NORMOTENSIVE HORSES

MANUEL GÓMEZ DÍEZ

Supervisors: Dr. Ana Muñoz Juzado Dr. Juan Manuel Serrano Rodríguez

Córdoba, 2017 TITULO: Pharmacokinetic and pharmacodynamic studies of three angiotensin-converting enzyme inhibitors in healthy normotensive horses

AUTOR: Manuel Gómez Díez

© Edita: UCOPress. 2017 Campus de Rabanales Ctra. Nacional IV, Km. 396 A 14071 Córdoba www.uco.es/publicaciones [email protected] Manuel Gómez Díez

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TÍTULO DE LA TESIS:

PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES OF THREE ANGIOTENSIN- CONVERTING ENZYME INHIBITORS IN HEALTHY NORMOTENSIVE HORSES

(Estudios farmacocinéticos y farmacodinámicos de tres inhibidores de la enzima de conversión de la angiotensina en caballos sanos normotensos)

DOCTORANDO/A: MANUEL GÓMEZ DÍEZ

INFORME RAZONADO DE LOS DIRECTOR/ES DE LA TESIS La presente tesis doctoral ha sido realizada bajo la tutela de dos directores pertenecientes a los Departamentos de Medicina y Cirugía Animal (Dr. Ana Muñoz) y de Farmacología, Toxicología y Medicina Legal y Forense (Dr. Juan Manuel Serrano- Rodríguez), de la Universidad de Córdoba. La tesis se presenta como un compendio de cuatro publicaciones, todas ellas publicadas en revistas indexadas, situadas en el primer tercil, según su índice de impacto y en inglés, al optar a mención internacional.

Durante la realización de esta tesis doctoral, el doctorando ha mostrado un gran interés y dedicación al trabajo y ha sido capaz de desarrollar una labor de investigación con rigor científico. Hemos encontrado una importante progresión en cuanto a su capacidad investigadora.

La presente tesis ha generado hasta este momento la siguiente producción científica:

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Artículos publicados

Gómez-Díez M, Muñoz A, Caballero JM, Riber C, Castejón F, Serrano-Rodríguez JM. 2014. Pharmacokinetics and pharmacodynamics of enalapril and its active metabolite, enalaprilat, at four different doses in healthy horses. Research in Veterinary Science, 97(1):105-110.

Muñoz A, Esgueva M, Gómez-Díez M, Serrano-Caballero JM, Castejón-Riber C, Serrano-Rodríguez JM. 2016. Modulation of acute transient exercise-induced hypertension after oral administration of four angiotensin-converting enzyme inhibitors in normotensive horses. The Veterinary Journal, 208:33-37.

Serrano-Rodríguez JM, Gómez-Díez M, Esgueva M, Castejón-Riber C, Mena-Bravo A, Priego-Capote F, Serrano Caballero JM, Muñoz A. 2016. Pharmacokinetics and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of ramipril in healthy horses. The Veterinary Journal, 208: 38- 43.

Serrano-Rodríguez JM, Gómez-Díez M, Esgueva M, Castejón-Riber C, Mena-Bravo A, Priego-Capote F, Ayala N, Caballero JM, Muñoz A. 2017. Pharmacokinetic/pharmacodynamic modeling of benazepril and benazeprilat after administration of intravenous and oral doses of benazepril in healthy horses. Research in Veterinary Science, 114:117-122.

Comunicaciones presentadas en congresos

Solano E, Muñoz A, Gómez-Díez M, Serrano JM, Granados MM, Ruiz I, Serrano Rodríguez JM. 2012. Concentraciones sanguíneas de enalapril y enalaprilato tras la administración intravenosa de enalaprilo en el caballo. XIII Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Gómez-Díez M, Serrano Rodríguez JM, Serrano Caballero JM, Galán A, Martínez C, Muros A, Muñoz A. 2012. Inhibición de la enzima de conversión de la angiotensina tras la administración de enalaprilo a diferentes dosis en caballos. XIII Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Solano E, Gómez-Díez M, Serrano Rodríguez JM, Serrano Caballero JM, Muñoz A. 2013. Actividad de la enzima de conversión de la angiotensina (ECA) tras la administración de ramiprilo en el caballo. XIV Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Gómez-Díez M, Serrano Rodríguez JM, Serrano Caballero JM, Riber C, Castejón F, Muñoz A. 2013. Angiotensin-converting enzyme (ACE) activity after administration of oral ramipril in horses. 11th Congress of the World Equine Veterinary Association WEVA, Budapest, Hungría.

Gómez-Díez M, Muñoz A, Esgueva M, Serrano Caballero JM, Rubio MD, Rodríguez González-Miranda R, Serrano Rodríguez JM. 2014. La administración oral de quinaprilo en el caballo reduce la actividad sérica de la enzima de conversión de la angiotensina más de un 70%. XV Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Esgueva M, Serrano Rodríguez JM, Gómez-Díez M, Serrano Caballero M, Castejón-Riber C, Granados MM, Muñoz A. 2014. El benaceprilo inhibe la hipertensión aguda temporal inducida por ejercicio, pero el enalaprilo no. XV Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

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Gómez-Díez M, Muñoz A, Serrano Caballero JM, Esgueva M, Castejón-Riber C, Serrano Rodríguez JM. 2015. Actividad comparada de la enzima de conversión de la angiotensina (ECA) tras la administración de inhibidores de la ECA por vía intravenosa y oral en el caballo. XVI Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Además, el doctorando ha colaborado en otros trabajos de investigación del grupo AGR-111 (Medicina Deportiva Equina), en el que se integró. De este modo, ha participado en las siguientes publicaciones:

Muñoz A, Riber C, Trigo P, Gómez-Díez M, Castejón FM. 2012. Bacterial endocarditis in two Spanish foals after neonatal septicemia. Journal of Equine Veterinary Science 32:760-766 (T2).

Tofé E, Muñoz A, Castejón F, Trigo P, Castejón-Riber C, Gómez-Díez M, Riber C. 2013. Behavior of renin angiotensin aldosterone axis during pulling exercises in euhydrated and dehydrated horses. Research in Veterinary Science, 95(2):616-622 (T1).

Muñoz A, Rodríguez RGM, Riber C, Trigo P, Gómez-Díez M, Castejón F. 2013. Subclinical Theileria equi and rhabdomyolysis in three endurance horses. Pakistan Veterinary Journal 33(2):256-258 (T1).

Muñoz A, Riber C, Gómez-Díez M, Castejón FM. 2013. Interpretación fisiológica y clínica de las pruebas de esfuerzo en el caballo de deporte. Utilidad de la ergoespirometría. Equinus 37:6-23 (sin índice de impacto).

Gómez-Díez M, Muñoz A, Castejón F, Santisteban R, Vivo R, Riber C. 2014. Rectal tear with hematoma after accidental breeding as a cause of peritonitis and recurrent colic in a mare. Pakistan Veterinary Journal 34(4):557-559 (T1).

Rodríguez R, Cerón JJ, Riber C, Castejón F, Gómez-Díez M, Serrano-Rodríguez JM, Muñoz A. 2014. Acute phase proteins in Andalusian horses infected with Theileria equi. The Veterinary Journal 202:182-183 (T1).

y en las siguientes comunicaciones a congresos:

Castejón-Riber C, Muñoz A, Roldán J, Trigo P, Gómez-Díez M, Riber C. 2011. Hematology, plasma proteins and fibrinogen in endurance horses completing 500 km in 6 days. 13th Conference of the European Society of Veterinary Clinical Pathology, Dublin, Irlanda.

Muñoz A, Riber C, Vivo R, Santisteban R, Tovar P, Agüera S, Rubio MD, Agüera EI, Escribano BM, Trigo P, Castejón-Riber C, Gómez-Díez M, Castejón F. 2011. Investigación en cardiología en el caballo de deporte. Curso de Investigación en Veterinaria y Ciencia y Tecnología de los Alimentos, Córdoba, España.

Satué K, Gómez-Díez M, Muñoz A. 2012. Serum triglyceride concentrations during pregnancy in Spanish mares with different body condition scoring. Congreso de la Sociedad Italiana de Veterinarios de Équidos SIVE, Bolonia, Italia.

Muñoz A, Riber C, Gómez-Díez M, Castejón-Riber C, Castejón F, Cerón J. 2012. Evolución temporal de las proteínas de fase aguda tras una inflamación inducida por vacunación en el caballo. XIII Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

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Gómez-Díez M, Tofé E, Riber C, Castejón F, Muñoz A. 2013. Effect of strength exercise on testosterone, cortisol and muscle in horses. European Veterinary Conference Voorjaarsdagen, Amsterdam, Holanda.

Gómez-Díez M, Riber C, Castejón F, Roldán J, Muñoz A. 2013. Pérdida de rendimiento en competición en un caballo de raid con eritrocitosis absoluta. XIV Congreso Internacional de Medicina y Cirugía Equina, Sevilla, España.

Muñoz A, Castejón F, Trigo P, Roldán J, Rubio MD, Gómez-Díez M, Castejón-Riber C, Riber C. 2013. Veterinary clinical examination of endurance horses in competition: correlation with laboratory data. Congreso de la Sociedad Italiana de Veterinarios de Équidos SIVE. Arrezzo, Italia.

Gómez-Díez M, Muñoz A, Trigo P, Roldán J, Castejón R, Castejón-Riber C, Riber C. 2013. Response to a 7- day endurance competition in horses with normal and raised serum muscle enzyme activities. Congreso de la Sociedad Italiana de Veterinarios de Équidos SIVE. Arrezzo, Italia.

Vega L, Gómez-Díez M, Muñoz A. 2013. Características laboratoriales diferenciales entre caballos de raid exitosos y eliminados por alteraciones metabólicas y extenuación. VIII Jornadas Complutenses, VII Congreso Nacional de Investigación en Ciencias de la Salud para Alumnos de Pregrado y XII Congreso de Ciencias Veterinarias y Biomédicas. Universidad Complutense de Madrid, España.

Castejón-Riber C, Muñoz A, Trigo P, Castejón F, Gómez-Díez M, Riber C. 2013. Oxygen uptake and ventilatory thresholds in endurance horses with different fitness. Congress of the World Equine Veterinary Association WEVA, Budapest, Hungría.

Tofé E, Muñoz A, Gómez-Díez M, Castejón F, Rubio MD, Castejón-Riber C, Riber C. 2013. Activation of the sympathetic in euhydrated and dehydrated horses during exercise. Congress of the World Equine Veterinary Association WEVA, Budapest, Hungría.

Debido al rigor científico, calidad de la tesis, formación científica y satisfactoria labor realizada por el doctorando, se autoriza la presentación de la presente tesis doctoral.

Córdoba, diciembre 2017

Fdo.: Ana Muñoz Juzado Fdo.: Juan Manuel Serrano Rodríguez

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“Ignorance more frequently begets confidence than does knowledge: it is those who know little, and not those who know much, who so positively assert that this or that problem will never be solved by science. “

“La ignorancia engendra más confianza de la que con frecuencia engendra el conocimiento: son aquellos que saben poco, y no aquellos que saben mucho, los que afirman positivamente que tal o cual problema jamás podrá ser resuelto por las ciencias.”

Charles Darwin

“Science is literally a against the charlatans”.

“La ciencia es, literalmente; una vacuna contra los charlatanes”.

Neil deGrasse Tyson

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DEDICATORIA

A mis padres Jose Ángel y Teresa; fuente de todo lo bueno que yo pueda ser hoy y espejo en el que me queda mucho por mirar. Vosotros habéis encendido en mi la mecha de la ciencia y me habéis enseñado grandes valores: el amor por el trabajo, la importancia del esfuerzo y, muy importante; el arte de dudar. Sois grandes científicos y mejores personas; os quiero enormemente y quiero poder seguir compartiendo con vosotros cada pequeño logro de mi carrera.

Manuel.

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AGRADECIMIENTOS

Quiero agradecer especialmente la labor de mis directores de tesis Ana y Juan Manuel. Que este proyecto haya llegado a término no ha sido fácil. He tenido que trabajar en el extranjero durante gran parte del tiempo que ha durado la redacción de esta tesis y sin la inestimable ayuda de ellos estoy seguro de que no habría logrado terminarla.

Además de su enorme implicación con la tesis quiero agradecerles todo lo aprendido durante mi periodo de formación en la Universidad de Córdoba y el haberme dado la oportunidad de investigar con caballos. Sois grandes profesores y os estaré siempre agradecido.

Quiero agradecer también a Francisco Castejón, director del CEMEDE; el haber tenido un papel especial en toda mi etapa como estudiante e investigador en la Universidad de Córdoba. No sólo puso a mi disposición las instalaciones y medios que dirige, sino que me ofreció la posibilidad de comenzar mi carrera profesional. Fuiste un gran mentor y consejero y recuerdo con enorme cariño el tiempo que pasamos juntos.

Por último, quiero agradecer en gran medida la labor desinteresada de todos los alumnos internos del CEMEDE con los que tuve el placer de trabajar durante los ensayos. Considero que tuve mucha suerte de contar con vuestra ayuda, supisteis trabajar de forma seria pero animosa, eficaz y muy profesional. Estoy muy orgulloso de todos y gracias a vosotros guardo grandes recuerdos de toda la fase experimental de esta tesis. Manuel

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INDEX

1. GENERAL INTRODUCTION TO THE STUDY ...... 15 2. OBJECTIVES AND HYPOTHESIS ...... 21 3. LITERATURE REVIEW ...... 25

3.1. OVERVIEW OF CARDIOVASCULAR DISEASES AND SYSTEMIC HYPERTENSION IN EQUINE MEDICINE ...... 27 3.2. DEFINITION OF FAILURE AND COMPENSATORY MECHANISMS ...... 35 3.2.1. VASOCONSTRICTOR MECHANISMS ...... 37 3.2.1.1. Sympathetic nervous system activation ...... 37 3.2.1.2. Renin angiotensin aldosterone vasopressin axis ...... 39 3.2.1.3. ...... 45 3.2.2. VASODILATOR MECHANISMS ...... 48 3.2.2.1. Natriuretic peptides...... 48 3.2.2.2. -derived releasing factor and prostacyclin ...... 52 3.3. THERAPEUTIC BLOCKAGE OF THE RENIN ANGIOTENSIN ALDOSTERONE VASOPRESSIN AXIS ...... 53 3.3.1. RENIN INHIBITORS ...... 54 3.3.2. ANGIOTENSIN CONVERTING ENZYME INHIBITORS ...... 56 3.3.3. ANGIOTENSIN I RECEPTOR ANTAGONISTS ...... 59 3.3.4. RECEPTOR ANTAGONISTS ...... 60 3.3.5. ANGIOTENSIN RECEPTOR NEPRILYSIN INHIBITORS ...... 61 3.3.6. β-BLOCKERS ...... 63 3.3.7. CHANNEL BLOCKERS ...... 64 3.3.8. BLOCKERS OF RECEPTORS OF VASOPRESSIN ...... 65 3.4. BASIC KNOWLEDGE OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS ...... 67 3.4.1. THE ANGIOTENSIN CONVERTING ENZYME ...... 67 3.4.2. ANGIOTENSIN CONVERTING ENZYME INHIBITORS: CHEMICAL STRUCTURE AND ANGIOTENSIN CONVERTING ENZYME BINDING ...... 68 3.5. PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS ...... 71 3.5.1. PHARMACOKINETIC OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS 71 3.5.2. PHYSIOLOGICAL COMPARTMENTAL PHARMACOKINETIC PHARMACODYNAMIC MODELS ...... 73 3.6. CURRENT KNOWLEDGE OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS IN THE HORSE ...... 81 4. BRIEF DESCRIPTION OF THE STUDIES ...... 93

4.1. DESCRIPTION OF THE STUDY I ...... 95 4.1.1. JUSTIFICATION ...... 95 4.1.2. OBJECTIVES ...... 96 4.1.3. MATERIAL AND METHODS ...... 96 4.1.4. BRIEF DESCRIPTION OF THE RESULTS ...... 97 4.1.5. CONCLUSIONS ...... 98 4.2. DESCRIPTION OF THE STUDY II ...... 99 4.2.1. JUSTIFICATION ...... 99

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4.2.2. OBJECTIVES ...... 100 4.2.3. MATERIAL AND METHODS ...... 100 4.2.4. BRIEF DESCRIPTION OF THE RESULTS ...... 102 4.2.5. CONCLUSIONS ...... 103 4.3. DESCRIPTION OF THE STUDY III ...... 105 4.3.1. JUSTIFICATION ...... 105 4.3.2. OBJECTIVES ...... 106 4.3.3. MATERIAL AND METHODS ...... 106 4.3.4. BRIEF DESCRIPTION OF THE RESULTS ...... 108 4.3.5. CONCLUSIONS ...... 109 4.4. DESCRIPTION OF THE STUDY IV ...... 111 4.4.1. JUSTIFICATION ...... 111 4.4.2. OBJECTIVES ...... 112 4.4.3. MATERIAL AND METHODS ...... 112 4.4.4. BRIEF DESCRIPTION OF THE RESULTS ...... 113 4.4.5. CONCLUSIONS ...... 113 5. DISCUSSION ...... 115 5.1. PHARMACOKINETICS: THE CASE OF ENALAPRIL IN HORSES...... 118 5.2. PHARMACOKINETICS: RAMIPRIL AND BENAZEPRIL, MORE LIPOPHILIC PRO- DRUGS ...... 123 5.3. PHARMACODYNAMICS: ANGIOTENSIN CONVERTING ENZYME INHIBITION AND SIMULATIONS ...... 127 5.4. EFFECTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS ON PRESSURE IN THE HORSE ...... 131 5.5. USE OF EXERCISE AS A MODEL TO STUDY RENIN ANGIOTENSIN ALDOSTERONE VASOPRESSIN AXIS ACTIVATION AND HYPERTENSION IN THE HORSE ...... 133 6. CONCLUSIONS ...... 139 7. RESUMEN ...... 143 8. SUMMARY ...... 149 9. REFERENCES ...... 155 10. LIST OF ABBREVIATIONS ...... 193 11. PUBLISHED PAPERS ...... 199

11.1. PAPER I ...... 200 11.2. PAPER II ...... 202 11.2.1 SUPPLEMENTARY MATERIALS FOR PAPER II ...... 203 11.3. PAPER III ...... 206 11.3.1 SUPPLEMENTARY MATERIALS FOR PAPER III ...... 209 11.4. PAPER IV ...... 216

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1. GENERAL INTRODUCTION TO THE STUDY

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Cardiovascular diseases are common in the horse, being valvulopathies and other structural alterations of the heart (Kriz et al., 2000; Young and Wood, 2000; Young et al., 2008; Stevens et al., 2009; Zucca et al., 2010; Ireland et al., 2012), together with disturbances of the cardiac rhythm or arrhythmias (Ryan et al., 2005; José-Cunilleras et al., 2006; Buhl et al., 2010; Trachsel et al., 2010; Reef et al., 2014; Slack et al., 2015; Flethøj et al., 2016) the most common findings in the clinical examination of these animals.

However, due to the great cardiovascular reserve of this animal species, many of these diseases are well tolerated, and might not be associated with clinical signs unless the horse is subjected to high intensity exercise, where the limits of cardiovascular function are reached (Buhl et al., 2010; Trachsel et al., 2010; Reef et al., 2014). For this reason, the study of cardiovascular diseases in horses has not traditionally received the same level of attention and development as in other domestic animal species, such as dogs and cats.

However, in the last years, there has been a plethora of scientific information concerning cardiovascular diseases as well as those diseases associated with systemic/pulmonary hypertension. This growing evidence in the diagnosis of these diseases in horses has been linked to the increasing interest for equine sports medicine, equine geriatric medicine and equine endocrinology.

Equine sports medicine is a field that has increased its relevance at the same time as equestrian disciplines have been professionalized and the weight of the sport horse industry has increased. Cardiovascular disorders are considered the third major group of causes leading to reduced sport performance in equine athletes, documented in a 21% of the animals presented for investigation of poor performance (Martin et al., 2000).

The average age of the equine population has increased, especially in those animals used for sport, leisure and company. Certain valve pathologies in the horse have been related to age, especially aortic valve regurgitation (Sage, 2002; Ireland et al., 2012; Marr, 2016). Therefore, as some of them might end in acute or chronic

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(congestive) heart insufficiency o failure (CHF), there has been an increasing interest in the pathophysiology and treatment of cardiac disease and failure in equine patients. The aortic and mitral valves are the most common sites for equine valvular pathology (Ireland et al., 2012). In horses, the aortic valve tissue is not simply passive and can contract in response to numerous mediators, including angiotensin II (ANG-II) (Bowen et al., 2014). Certain cardiovascular abnormalities, including aortic regurgitation, have been related with an increased risk of pulmonary hypertension and CHF in the horse (Reef et al., 2014). In the same way, severe mitral valve disease can lead to signs of CHF and ultimately to death (Clark et al., 1987; Reef et al., 1998; Davis et al., 2002; Ireland et al., 2012).

In the later years, there has also been a rising interest in equine endocrinopathies, with equine metabolic syndrome (EMS) as one of the most diagnosed disorders in this context (Morgan et al., 2015). EMS is a complex syndrome with multisystem involvement where the increased activity of the adipose tissue plays an important role (Frank et al., 2010). EMS might be analogous to the syndrome observed in human beings, which is characterized by insulin resistance, dyslipidemia, systemic inflammation and hypertension (Reaven, 2002; Lamounier-Zepter et al., 2006). Arterial hypertension has been recently considered a component of EMS (Bailey et al., 2008; Frank et al., 2010) and has also been related to the human metabolic syndrome (MetS) along with insulin resistance, and systemic inflammation (Lamounier-Zepter et al., 2006). A genetic predisposition to laminitis as also a part of the EMS, which has been found to happen in certain pony breeds (Bailey et al., 2008). These individuals present a pre-laminitic phenotype that included seasonal arterial hypertension as well as insulin resistance, and increased plasma concentrations of triglycerides and uric acid. This pre- laminitis phenotype could be the consequence of cardiovascular abnormalities in the equine digital vessels, because human patients affected by this syndrome also are at high risk of developing cardiovascular accidents (Paneni et al., 2014; Guarner and Rubio- Ruíz, 2015; González et al., 2017).

Because of all the above-mentioned factors, there has been a big effort to understand and to control systemic hypertension in the equine patient. Within the

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Angiotensin-converting enzyme inhibitors in the horse pathophysiological mechanisms of the CHF and systemic hypertension, the renin- angiotensin-aldosterone-vasopressin axis (RAAV) has a pivotal role, and an activated axis has been reported in small animals with different cardiovascular diseases, mainly mitral valve regurgitation (Ware et al., 1990; Pedersen et al., 1995; Pedersen, 1996; Ames et al., 2017) and dilated cardiomyopathy, DCM (Ware et al., 1990; Koch et al., 1995; Tidholm et al., 2001). Similarly, increased serum aldosterone (ALD) concentrations have been reported in a study with 40 horses with valvular regurgitation (Gehler et al., 2008), and the rise in ALD was positively correlated with the severity of the disease. More recently, the RAAV axis has been investigated in two neonatal Spanish foals with bacterial endocarditis associated with septicemia, but activation could not have been demonstrated (Rovira et al., 2009). Pharmacological control of the RAAV axis has been traditionally the cornerstone of medical management of hypertension in humans and small animals (Lefèbvre et al., 2007).

The traditional inhibition mechanism of the RAAV axis has been the administration of inhibitors of the angiotensin converting enzyme (ACEIs). This is a promising group of drugs, with a widespread use in human and veterinary medicine for small domestic animals, because of their potential to improve clinical signs and to increase survival rate and life quality in patients with CHF (Lefèbvre et al., 2007). From this group, only the pharmacokinetic (PK) and pharmacodynamic (PD) properties of enalapril had been tested in horses before we started our studies, but the investigated doses were limited and lacked effect to provide significant inhibition of the angiotensin- converting enzyme (ACE), either after individual (Gardner et al., 2004) or repeated (Sleeper et al., 2008) oral (PO) administrations. Ramipril and quinapril have been previously administered PO to horses, showing a potentially appropriate absorption. In this way, ramipril appeared to reduce the hypertensive response to angiotensin-I (ANG- I) in healthy horses after PO administration (Luna et al., 1995; Luciani et al., 2007). Because of these results, ramipril was used by these authors to treat CHF in one horse combined with the Henle loop , furosemide (Guglielmini et al., 2002). In the case of quinapril, it was used successfully to increase , and performance in horses with mitral valve regurgitation (Gehlen et al., 2003). The PO doses for both drugs in these studies were extrapolated from human medicine and their PK-

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PD profiles in the horse were not evaluated. The use of human doses in veterinary medicine can have undesirable effects ranging from the lack of effectiveness to potentially serious adverse effects (Huang et al., 2015) and the doses for each species should be specifically calculate after the generation of a PK profile.

The evidence of increased ALD concentrations (Gehlen et al., 2008), showing therefore a RAAV axis activation in equines with heart valvular disease potentially leading to CHF and hypertension was the main motivation for our group to conduct these investigations. Heart diseases in horses can have a significant clinical impact that range from mild exercise intolerance to life threatening disease. Finding effective strategies for managing hypertension and CHF in these patients could be crucial to improve clinical signs, survival rate and exercise tolerance as it has been already proven in human beings and small animals, and therefore, could have an important impact in equine medicine and equine industry.

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2. OBJECTIVES AND HYPOTHESIS

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The present investigation has been performed in order to increase our knowledge about the PK and PD characteristics of ACEIs in the horse. The following specific objectives were proposed:

First objective: To confirm whether three ACEIs that are widely used in small animals (enalapril, ramipril and benazepril) have pharmacokinetic and pharmacodynamic characteristics that could be of clinical interest in horses.

Second objective: To study the pharmacokinetic characteristics of each drug after oral and intravenous administration of different doses. Theoretical models will be used to predict and to propose possible multiple administration protocols.

Third objective: To analyze the possible effects of these drugs to control hypertension, using an experimental hypertension model based on intense exercise, and considering the pharmacokinetic and pharmacodynamic characteristics of each drug.

Fourth objective: To compare the results of the different researches to evaluate which of the ACEI drugs studied have more adequate properties in order to be clinically useful for equine practice.

Consequently, the final objective of this project was to provide scientific information to aid in the selection of an ACEI drug for the treatment of cardiovascular diseases and hypertension in horses, especially concerning the control of cardiac failure and systemic arterial hypertension.

The following hypotheses will be checked: First hypothesis: That, the pharmacokinetic characteristics of the different ACEIs in the horse might be conditioned by their and could therefore be different for each drug.

Second hypothesis: That, whether the different drugs show different bioavailability, the effects on and circulating angiotensin converting enzyme activity levels might also vary between them.

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Third hypothesis: That, the posology derived from theoretical models after analyzing pharmacokinetic-pharmacodynamic profile of each drug might be used to treat affected horses.

Fourth hypothesis: That, the modulation of hypertension when used in horses in an experimental model of induced hypertension might also be different when comparing the three different drugs and, this modulation might be associated with the degree of inhibition of the angiotensin-converting enzyme reached by each one.

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3. LITERATURE REVIEW

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3.1. OVERVIEW OF CARDIOVASCULAR DISEASES AND SYSTEMIC HYPERTENSION IN EQUINE MEDICINE

Cardiovascular diseases in the horse are less frequent than those affecting other organ systems, even though they can have a critical significance for individual patients particularly for equine athletes and geriatric horses.

The heart of an untrained horse weights normally about 0.6% of the total body weight (Hanson et al., 1994) although there is an important influence of breed type, sex and age. In thoroughbred horses, the heart weight ranges between 0.85 to 1% of the total body weight (Gunn, 1989). A high level of training will also influence heart size by physiologic hypertrophy (Young, 1999; Buhl et al., 2005; Buhl and Ersboll, 2012).

The heart acts as a physiologic pump that interacts with both the pulmonary and systemic circulations, providing oxygenated blood to the body and allowing the oxygenation of venous blood in the lungs. Specialized cardiocytes located in both atria and ventricles have also a secretory function and produce the atrial natriuretic peptide (ANP), and the brain natriuretic peptide (BNP), so the heart is also considered an endocrine organ (Trachsel et al., 2012; 2015; Van der Vekens et al., 2016). Natriuretic peptides have important roles in blood pressure (BP) regulation and electrolytic balance, acting as RAAV system antagonists, with substantial functions in the early stages of congestive or CHF. The function of the ANP has been studied in exercising horses (Kokkonen et al., 1995; 1999) and recently it has been demonstrated that there are increased circulating ANP concentrations in horses with atrial dilation (Trachsel et al., 2012; 2015; Van der Vekens et al., 2016).

Morphological heart diseases can be classified into congenital malformations, pericardial, myocardial, endocardial and, valvular diseases, diseases of the vessels and cardiac and vascular tumors (Buergelt, 2003). Early reports described that valvular lesions are responsible for about 25% of all cardiovascular changes detected in equine cardiologic examinations (Else and Holmes 1972a,b). In addition, it is very common to find heart arrhythmias without morphological alterations in horses (Reef et al., 2014). Arrhythmias are even found in healthy horses in response to exercise. In fact,

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Manuel Gómez Díez arrhythmias have been documented during exercise in endurance horses (Flethøj et al., 2016), show jumpers (Buhl et al., 2010), dressage horses (Barbesgaard et al., 2010) and Standardbred racehorses (Physick-Sheard and McGurrin, 2010). However, it should be kept in mind that arrhythmias are an important cause of loss of performance in the equine athlete, both during submaximal and maximal exercises (Martin et al., 2000; Ryan et al., 2005; José-Cunilleras et al., 2006). Further, heart arrhythmias might be involved in sudden heart disease and death during exercise (Navas de Solís, 2016).

Congenital malformations do not produce clinical signs in all affected horses but can include exercise intolerance, murmurs, hypertension, respiratory distress and cyanosis. The most common congenital malformations described in horses are the following. The defect of the ventricular septum appears to be the most commonly encountered congenital abnormality (Reef, 1985; Kraus et al., 2005; Marr, 2015). A defect can occur at different locations in the ventricular septum and a systolic murmur will be herd predominantly on the right side. Clinical signs will depend on the severity of the defect and the amount of left to right blood shunt, but small defects are usually well tolerated and can be asymptomatic (Reef, 1985; Kraus et al., 2005; Marr, 2015).

The defect of the atrial septum is much more infrequent than ventricular septum defect and blood flow from one atria into another can occur in any direction. A systolic murmur may be detected, and volume overload can affect the right heart if there is excessive left to right blood shunt at the atrial level (Wilson and Haffner, 1987).

Persistence of the patent ductus arteriosus (PDA) is another congenital heart alteration in the foal. During fetal development, the ductus arteriosus provides communication between the pulmonary trunk and the aorta. This communication closes physiologically in the first 72 h of life, giving origin to the ligamentum arteriosum. Failure to close will lead to permanent communication of these structures and usually a left to right blood shunt that might lead to pulmonary hypertension and a continuous murmur audible on auscultation. This pathology in the adult horse is rare and the clinical signs will vary depending the diameter and extension of the PDA. Most severe cases can end with cyanosis, and pulmonary aneurism (Reimer et al., 1993).

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Other congenital heart diseases in the horse are tricuspid valve atresia, common truncus arteriosus, tetralogy of Fallot, complete transposition of the greater vessels and double-outlet right (Meurs et al., 1997; Kraus et al., 2005; Sleeper and Palmer, 2005; Hall et al., 2010; Marr, 2015; Krüger et al., 2016).

Pericardial diseases including effusive non-septic pericarditis, fibrinous pericarditis and pericardial neoplasia have been reported in the horse (Buergelt, 2003). Effusive pericarditis is characterized by significant accumulation of sterile pericardial fluid of a non-determined origin with nonspecific cytological characteristics. Fibrinous pericarditis is usually associated with septic colonization of the pericardial space and subsequent accumulation of fluid and fibrin (Bolin et al., 2005; Armstrong et al., 2014). Several agents have been cultured from horses with fibrinous pericarditis and hematogenous dissemination and traumatic injury have been the most frequent infection routes (Bolin et al., 2005). Neoplasia affecting the pericardial space of horses have been previously reported, including mesothelioma and hemangiosarcoma (Buergelt, 2003). Pericardial diseases in the horse often lead to cardiac tamponade and cardiac failure.

Myocardial diseases are normally categorized into primary degenerative processes and primary inflammatory processes. Primary myocardial degenerative processes are influenced by a variety of factors including nutritional alterations and toxic insults while primary inflammatory conditions are usually related to infectious agents (either bacterial, fungal or viral). Most frequent nutritional alterations leading to cardiac pathology include hyperlipidemia and nutritional myopathy.

Hyperlipidemia is a metabolic condition that originates from a negative energy balance in ill equines, most frequently donkeys and miniature ponies, and that leads to a status of hyperlipidemia due to large mobilization of fat deposits (Platt and Whitwell, 1971; Burden et al., 2011; McKenzie, 2011; Dunkel et al., 2014). The condition is more frequent in situations of high energetic demand like feed restriction, systemic illness, pregnancy or lactation. Multiple organs can be affected, with the liver being the most frequently recognized, but the myocardium can also undergo fatty infiltration that will

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Manuel Gómez Díez significantly impact normal myocardial function (Reef, 1985; Kraus et al., 2005; Marr, 2015).

Vitamin E and selenium deficits can lead to nutritional myopathy, also known as “white muscle disease” in foals and young horses (Owen et al., 1977; Streeter et al., 2012; Delesalle et al., 2017). E and selenium are very important elements involved in the protection of cell membranes from the oxidizing action of free radicals. This disease can have either an acute presentation with sudden death, usually from (Delesalle et al., 2017) or a subacute form that will often present with cardiac muscular weakness. The disease can be present at birth in foals born from vitamin E and selenium deficient mares and affected animals can recover with adequate supplementation (Streeter et al., 2012; Delesalle et al., 2017).

Toxic insults can affect the myocardium in different ways and with different severity depending on the type and amount ingested of the toxic agent. Most frequent intoxications affecting the myocardium of horses are due to ingestion of ionophore , mycotoxins and the toxic plant Nerium oleander (Smith et al., 2002; Decloedt et al., 2012).

Ionophore antibiotics are used in animal production industry and for coccidiosis control in poultry and were used in cattle as growth promotors in the past. Horses are extremely sensible to these chemicals and can be exposed to them after contamination of the feed mixing equipment. These products have a degenerative effect on the myocardium producing myocytolysis and vacuolar degeneration and leading to cardiac arrhythmias and sudden death (Decloedt et al., 2012). Chronic cases can also occur and result in heart failure with ventral edema and pleural effusion (Muylle et al., 1981; Hughes et al., 2009). Horses have also been described as especially sensitive to aflatoxins. Ingestion of these mycotoxins can cause myocardial degeneration with lipid deposition (Angsubhakorn et al., 1981; Smith et al., 2002). Another frequent intoxication that leads to heart failure in livestock and horses occurs after the ingestion of leaves of the plant Nerium Oleander (Galey et al., 1996). Clinical signs after intoxication vary depending on the amount of leaves ingested but acute cardiac arrhythmias and arrest can occur. Cardiac damage is due to interstitial edema and myocyte necrosis (Renier et

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Angiotensin-converting enzyme inhibitors in the horse al., 2013; Butler et al., 2016). Other toxic agents that can lead to heart arrhythmias and failure in the horse with fewer incidences in Europe are the seed of the plant Cassia occidentalis and the insects of the genus Epicauta (Martin et al., 1981; Helman and Edwards, 1997).

Primary inflammatory myopathies include myocarditis, infarcts and traumatic events. The term myocarditis defines myocardial inflammation that is not typical of an infarction. Inflammation might be diffuse or focal and can be associated with myocytolysis and myocyte necrosis. causes include virus (equine influenza, equine infectious , and equine viral arteritis), bacteria (Staphylococcus aureus, Streptococcus equi and Borrelia burgdorferi or Lyme disease) and parasites (Strongylus vulgaris and Onchocerca spp.) (Jesty and Reef, 2006). Alterations of the cardiac rhythm might occur due to inflammation and scarring of myocardial areas that involve segments of the conducting system (Buergelt, 2003).

Infarcts affecting the myocardium can origin either from distant emboli that end up in the coronary arterial circulation or from narrowing of the coronary themselves. The migrating parasite Strongylus vulgaris has been proposed as a causative agent that could favor thrombi formation (Cranley and McCullagh, 1981).

Cardiomyopathy is a term to describe a well-known condition in human medicine consisting on muscle disease affecting the myocardium in the absence of preceding circulatory, pulmonary, or valvular alterations and toxic or infectious insults. In humans and in small animals, there are three different forms of the disease (dilated, DCM; hypertrophic, HCM; and restrictive, RCM) (Broschk and Distl, 2005; Lój et al., 2012; Kittleson et al., 2015; Simpson et al., 2015). Cardiomyopathy in horses has not been well documented although isolated cases of HCM or DCM leading to cardiomegaly and heart failure have been reported (Davis et al., 2002; Raftery et al., 2015). Additionally, five cases of HCM have been reported recently (Navas de Solís et al., 2013).

Endocardial diseases affect the connective tissue membrane that covers the myocardium internally, lining the atria, the ventricles and the valvular structures. Although endocardial fibrosis and calcification can be found in about 60% of horses over

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20 years of age, this does not result in CHF (Buergelt, 2003). This is due to chronic exposure to the myocardium to turbulent blood flow and subsequent irritation affects more usually the apex of the left ventricle.

Endocardial fibroelastosis has been described in horses as a cause of cardiomegaly and CHF (Belgrave et al., 2010; Cushing, 2013). In these cases, the entire left ventricle and left atria are affected by thickened endocardium that causes an insufficient contraction and cardiac output and can affect the Purkinje fibers (Cushing, 2013).

Although not common, depositions can affect the endocardium, valvular surfaces and inner surfaces of the greater vessels. This problem has been reported in cases of vitamin D3 intoxications by plants like Cestrum diurnum (Krookl et al., 1975) or hypervitaminosis D from excessive nutritional supplementation (Harrington, 1982) as well as in cases of intestinal lymphosarcoma (Mair et al., 1990).

The term endocarditis is normally reserved to describe endocardial inflammation related to either an infectious agent colonization or endocardial sensitization by predisposing factors such as toxins that lead to posterior endocardial damage. Valvular endocarditis is more frequent and carries a worse prognosis than mural affection alone with the left heart being more commonly affected (aortic and mitral valves) (Buergelt et al., 1985; Buergelt, 2003; Muñoz et al., 2012). Valvular endocarditis can present in various forms, acute, subacute or chronic. The most frequently isolated bacteria in cases of infective vegetative valvulitis in the horse have been Streptococcus sp. and Actinobacillus equuli but many other bacteria and fungi have been occasionally described (Porter et al., 2008). The parasite Strongylus vulgaris has been signaled as a causative agent for lesions in the aortic valve (Buergelt et al., 1985; Buergelt, 2003).

Valvular endocardiosis or degenerative valve disease (DVD) describes the presence of fibrous nodules caused by friction in the valves in absence of inflammatory cells (Buergelt, 2003) and fibrous thickening of the valvular matrix in the atrioventricular valves, more commonly over the mitral valve (Sage, 2002). The latest affection leads to

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Angiotensin-converting enzyme inhibitors in the horse an impaired function of the valve and blood regurgitation into the atrium and its origin is believed to be related to wear and tear of the valvular structures that could be increased during strenuous exercise (Sage, 2002).

Rupture of the chordae tendineae of the valves has been reported (Holmes and Miller, 1984; Reef, 1987; Marr et al., 1990) due to weakening of the caudal insertion of the chordae at the level of the papillary muscle due to fibrosis or because of valvular endocardiosis that extended into the chordae.

Cardiac neoplasia is very rare in horses and normally occur secondarily as a metastatic complication of other neoplasms. Multicentric lymphoma, hemangiosarcoma, infiltrative lipoma and rhabdomyosarcoma have been described in horses (Buergelt, 2003; Castleman et al., 2011; Penrose et al., 2012; Marr, 2016).

Vascular diseases, including vasculitis, verminous arteritis, purpura hemorrhagica, thrombosis and others, are not described in this review due to the low relation with systemic hypertension and CHF.

As explained in this review, atrioventricular valves dysfunctions or regurgitations are some of the most frequent cardiac problems encountered in the horse and, depending on their severity, can lead to exercise intolerance and pulmonary hypertension (mitral valve regurgitation) and signs of CHF with ventral subcutaneous edema and venous distention (tricuspid valve regurgitation). Horses with this type of pathology constituted most of the equine patients admitted into cardiology consultations and therefore there is a great interest in the different therapeutically options to for their medical management (Reef et al., 1998; Young et al., 2006; 2008; Stevens et al., 2009; Zucca et al., 2010; Trachsel et al., 2013; Ven et al., 2016).

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3.2. DEFINITION OF HEART FAILURE AND COMPENSATORY MECHANISMS

Heart failure is a term used to describe a syndrome characterized by congestion, edema and poor peripheral that can occur either at rest or just during exercise and that, in most severe cases, can ultimately lead to systemic hypotension and cardiogenic . CHF specifically refers to the clinical situation where heart failure manifests with vascular congestion. Heart failure is not always present in heart disease and can affect the left heart, the right heart or both (McIntosh Bright, 2010).

Heart failure can be caused by different mechanisms depending on the original heart disease. The mechanisms for heart failure have been traditionally classified into three main groups: volume overload, systolic failure and diastolic failure. Of the three mechanisms, volume overload is the most common mechanism implied in heart failure in horses, normally caused by valvular regurgitation or by a left-to-right blood shunt. Systolic failure might be found in horses with myocardial disease, due to reduced myocardium contractibility. Diastolic failure that implies impairment in diastolic function leading to inadequate ventricular filling has also been described as a source of heart failure in horses with restricted pericardial disease (Mann, 1999; Dixon and Spinale, 2009; Dubi and Arbel, 2010; McIntosh Bright, 2010).

Horses with heart failure will suffer two main hemodynamic abnormalities: increased ventricular filling pressures, which clinically would appear as peripheral edema, distensions and other signs of congestion, and reduced cardiac output, which would lead to signs of poor peripheral perfusion (Davis et al., 2002; Marr, 2010, 2016; Leroux et al., 2013).

Horses with left-sided heart failure will suffer from increased left ventricular filling pressures that will ultimately lead to increased pressures in the left atrium and pulmonary circulation and eventually pulmonary edema. These patients will often show respiratory clinical signs including tachypnea, cough, foamy nasal discharge and increased lung sounds at auscultation. Horses with right side heart failure will suffer from increased right ventricular filling pressures that will ultimately lead to increased right atrial and systemic venous pressures. These patients will often present peripheral

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Manuel Gómez Díez edema, jugular and/or peripheral venous distention (Davis et al., 2002; Marr and Bowen, 2010; Leroux et al., 2013; Marr, 2016). Horses with long standing left-sided heart failure will develop pulmonary and pulmonary vascular remodeling that can subsequently induce right-sided heart failure with typical signs of peripheral edema and jugular distention. This process is named “cor pulmonale” (Marr, 2010; Leroux et al., 2013; Hanka et al., 2015; Marr, 2016). When severity of heart failure increases, reduced cardiac output will lead to signs of poor peripheral perfusion that will include exercise intolerance, fatigue, weakness, cold extremities, hypothermia and increased capillary refill time (Marr, 2010).

In situations of heart failure, the organism tries to maintain circulatory homeostasis activating different neuroendocrine compensatory mechanisms which might be broadly classified into vasoconstrictor and vasodilator mechanisms. Vasoconstrictor mechanisms include activation of the sympathetic and of the RAAV systems. Other minor vasoconstrictor mechanisms are the release of other vasoconstrictive substances, such as thromboxane and endothelins (ET). The most important vasodilator mechanism is the synthesis and released of the ANP by atrial myocardiocytes, BNP from ventricular myocardiocytes, vasodilating prostaglandins, E2 and I2, dopamine, increased production of endothelin-derived releasing factors (EDFR) like endothelium-derived , NO (Dzau, 1992; De Bold and De Bold, 2005; Pandey, 2005; Elliott and Bowen, 2010).

Although the above mentioned neuroendocrine compensatory mechanisms try to compensate and to control heart failure, they also induce an increase in sodium (Na+) retention, antidiuresis and vasoconstriction that will be responsible for many of the clinical manifestations of CHF. Antidiuresis and venoconstriction will further rise ventricular filling pressures and exacerbate signs of congestion, while constriction of arterioles along with increased blood volume due to water retention will further reduce stroke volume and increase myocardial oxygen demand (Marr, 2010; Elliott and Bowen, 2010).

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3.2.1. VASOCONSTRICTOR MECHANISMS

3.2.1.1. Sympathetic nervous system activation

Activation of the sympathetic (adrenergic) nervous system (SNS) is a hallmark of heart failure. Reduced cardiac output and decreased BP leads to an activation of the adrenergic nervous system which results in an increase in , and myocardial contractibility, together with a selective redirecting of blood flow to vital centers (Sisson, 2004; Marr, 2010). The systemic effects of the sympathetic system stimulation include arteriolar constriction, which helps to maintain tissue perfusion pressures, and venous constriction, which results in increased or venous return and increased cardiac output. Despite these rises in preload and tissue perfusion pressures, the resulting increases in venous and capillary pressures are mechanisms involved in the development of symptomatic congestion (Braunwald and Bristow, 2000; Klein et al., 2003; Weiss et al., 2003; Sisson, 2004).

On the other hand, long exposure to increased concentrations of catecholamines contributes to vascular and cardiac remodeling, promotes arrhythmogenesis and induces premature myocyte death (Braunwald and Bristow, 2000; Klein et al., 2003; Weiss et al., 2003). Increased sympathetic tone also increases RAAV activation and contributes to elevated circulating concentrations of arginine vasopressin, AVP and ET (Braunwald and Bristow, 2000).

Human studies have demonstrated that plasma norepinephrine concentrations in patients with CHF positively correlate with the severity of disease and negatively correlate with the survival rate (Cohn et al., 1984; Cohn and Rector, 1988). Additionally, progressively increasing concentrations of norepinephrine in human patients when treated for CHF are associated with a decline in clinical status (Kao et al., 1989).

Limited investigation has been carried out in veterinary medicine regarding SNS activation in patients with heart failure. Cats with CHF due to HCM or RCM were found to show higher plasma concentrations of epinephrine and norepinephrine that did cats with HCM or RCM without signs of heart failure (Sisson et al., 2003).

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Dogs with heart failure caused by DCM and DVD were also found to have increased plasma concentrations of norepinephrine that correlated directly with severity of disease, being higher in those dogs with DCM (Ware el. al., 1990; Sisson, 2004). Tidholm et al. (2005) studied 15 dogs with clinical signs of DCM, 15 with preclinical DCM and 15 control dogs, and they found that urine norepinephrine related to urine creatinine were higher in dogs with clinical DCM, followed by those animals with preclinical DCM. Therefore, this study showed that preclinical heart disease also alters neuroendocrine pattern and the authors suggested that the beneficial effects of β- adrenergic receptor antagonists should be investigated in dogs with CHF and DCM. Similarly, Marcondes Santos et al. (2006) found increased norepinephrine concentrations in dog with CDM and with DVD compared to controls, but without significant differences between both groups of diseased dogs. More interestingly, a negative correlation was found between circulating norepinephrine and fractional shortening, an echocardiographic marker of myocardium contractibility. The concentrations of catecholamines appear to be useful markers of deterioration of heart function as a consequence of a heart disease in the dog. Thus, O’Sullivan et al. (2007), in normal Doberman Pinschers and in those with DCM, described a progressive rise in blood norepinephrine in those animals with worse prognosis.

Plasma concentrations of norepinephrine did not differ among healthy horses and those with aortic insufficiency with and without clinical signs of cardiovascular compromise at rest, whereas epinephrine concentrations were significant different among healthy horses and those with aortic regurgitation (Horn, 2002).

Physiologically, the organic response to increased levels of circulating catecholamines is a decrease in cardiac muscle β-adrenergic receptors (Colucci and Braunwald, 2005). Interestingly, there are some evidences that show that equine myocardium does not suffer from down regulation of β-adrenergic receptors in situations of heart failure (Horn et al., 2002).

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3.2.1.2. Renin angiotensin aldosterone vasopressin axis

3.2.1.2.1. Physiology of the renin angiotensin aldosterone vasopressin axis The RAAV axis is a complex endocrine system with a capital role in preserving hemodynamic stability and it acts in response to changes in blood volume and pressure and electrolytes and water levels in the organism (McKeever and Hinchcliff, 1995; Sisson, 2004; McKeever, 2008; Otte and Spier, 2009; Muñoz et al., 2010a,b,c). As a result of these physiological factors, pharmacological manipulation of the RAAV axis has become the mainstay of therapy for heart and kidney disease in veterinary medicine (Mochel and Danhof, 2015).

Renin (REN) is a proteolytic enzyme that is produced in the renal juxtaglomerular cells. These cells act as feedback sensors to changes in flow and BP, Na+ and chloride (Cl- ) concentrations and arterial oxygen pressure, being highly sensitive to reduced renal perfusion (McKeever and Hinchcliff, 1995; McKeever, 1998; Muñoz et al., 2010a). The release of REN from the juxtaglomerular apparatus constitutes the initial point of the axis, as REN cleaves the inactive substrate angiotensinogen to form ANG-I (McKeever and Hinchcliff, 1995; McKeever, 1998; Sisson, 2004; Otte and Spier, 2009; Muñoz et al., 2010a). The main stimuli for the release of REN are increased β-adrenergic activity, decreased renal perfusion and reduced Na+ reabsorption by the renal tubules (Griendling et al., 1993; McKeever and Hinchcliff, 1995; McKeever, 1998; Sisson, 2004; Otte and Spier, 2009; Muñoz et al., 2010a).

The produced ANG-I will then be converted to ANG-II by the action of the ACE. This enzyme is found in most tissues and also circulating in plasma, however its activity in the lungs is particularly high (McKeever, 1998; Sisson, 2004; Otte and Spier, 2009). ACE activity on ANG-I also produce angiotensin III (ANG-III), that has actions similar but less potent than ANG-II (Griendling et al., 1993). Enzymes other than ACE might contribute to the conversion of ANG-I to ANG-II. Chymase, cathepsine G, tonin and other proteases have been described as alternative pathways of ANG-II production (Weber et al., 1995; Mochel and Danhof, 2015).

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In addition to the recognized importance in the kidney RAAV system, local REN and ANG systems have been demonstrated in different organs, such as brain, heart, blood vessels and adrenal glands (Danser, 1996; Danser et al., 1997; Mochel and Danhof, 2015). These ‘local’ RAAV axes function as autocrine or paracrine systems and regulate tissue growth and repair processes. Because of these actions, the local systems play an important role in the development of most of the pathological changes associated with this axis, mainly pathological remodeling, including vascular and myocardial hypertrophy, inflammation and fibrosis (Braunwald and Bristow, 2000; Brewster et al., 2003; Brewster and Perazella, 2004; Klein et al., 2003; Sisson, 2004; Otte and Spier, 2009).

Nevertheless, ANG-II is the most important piece of this cascade, mediating in most of the clinical effects of the RAAV axis with the activation of two different receptors. The ANG type 1 receptors (AT1) are widely distributed in the vasculature, kidneys, adrenal glands, heart, liver and brain. The ANG type 2 receptors (AT2) are abundant during fetal life in the brain, kidney and other tissues, but their presence rapidly decreases after transition to the extrauterine life (Wintour et al., 1999; Sisson, 2004; Atlas, 2007; Otte and Spier, 2009; Muñoz et al., 2010a). However, despite their low expression in adult tissues, the AT2 receptors appear to mediate , and have antiproliferative and apoptotic effects in vascular smooth muscle, inhibiting growth and remodeling of the heart (Lumbers, 1999; Wintour et al., 1999; Atlas, 2007; Otte and Spier, 2009; Muñoz et al., 2010a).

Despite these data, most physiological effects mediated by ANG-II in the adult will occur after stimulation of AT1 receptors. ANG-II has very strong vasoconstrictor effects mediated both by stimulation of the sympathoadrenal system and by inhibition of the parasympathetic control (Lumbers, 1999). It also promotes renal reabsorption of Na+ (Atlas, 2007, Muñoz et al., 2010a,c). Another effect of ANG-II consists on the regulation of the secretion of ALD. ANG-II activates the AT1 receptors of the adrenal zona glomerulosa, inducing secretion of ALD. The concentration of AT1 receptors in the adrenal glands is responsive to blood Na+ concentrations, increasing in situations of Na+ depletion, and decreasing when Na+ excess occur (Atlas, 2007, Muñoz et al., 2010a,c).

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ALD is a mineralocorticoid with similar systemic functions to ANG-II, increasing potassium (K+) excretion and Na+ and water reabsorption at the renal level, as well as Na+ reabsorption at the salivary and sweat glands and colon (McKeever, 1998; Lumbers, 1999). The major stimuli for ALD production and release are increased ANG-II, elevated plasma K+ concentrations and release of adrenal corticotropic hormone (ACTH) (McKeever, 1998).

ANG-II also increases the release of cytokines and growth factors due to its mitogenic capacity, activating inflammation, formation of fibroblasts and collagen deposition (Brewster et al., 2003). ALD has also mitogenic capacity that increased production of certain growth factors leading to fibrosis and inflammation.

Both ALD and ANG-II have implications in the process by increased production, increased activity of the plasminogen activator inhibitor type 1 (PAI- 1) and increased platelet aggregation (Weber, 2001, Muñoz et al., 2010a). Hypokalemia, ANP and dopamine inhibit ALD, whereas it seems that plasma Na+ levels have little or no direct effect on ALD secretion, at least as have been confirmed in exercising horses (McKeever et al., 1991; 1992; Muñoz et al., 2010b;c). ANP, produced by atrial myocytes in response to left atrial enlargement and , inhibits REN secretion and moreover, it blocks the action of ANG-II on ALD secretion (Weber, 2001; Brewster et al., 2003; Brewster and Perazella, 2004).

The antidiuretic hormone (ADH), also named arginine vasopressin (AVP), is a hormone produced in the paraventricular and supraoptic nuclei of the hypothatalamus, being after stored within the caudal lobe of the hypophysis or neurohypophysis for subsequent release into the bloodstream (McKeever, 1998; Muñoz et al., 2010a,b). It is a highly conserved molecule in most mammals and identical in human beings, dogs, cats and horses (Sisson, 2004). Release of AVP from the neurohypophysis into the circulation is stimulated by increased plasma osmolality or hypovolemia. Thus, when hypovolemia or hypotension appears, stretch receptors in the atria and large decrease their firing rate, stimulating AVP release (Lee et al., 2003). Sympathetic stimulation and ANG- II release also promote AVP release (Schrier and Abraham, 1999; Lee et al., 2003). After that, AVP reacts with receptors in the vasculature and heart, leading to weak

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vasoconstrictive and inotropic responses (V1A receptor stimulation), as well as water reabsorption in the renal collecting tubules (V2 receptor stimulation) (Wong and Verbalis, 2001; Verbalis, 2002; Mutlu and Factor, 2004; Wong et al., 2008; Muñoz et al., 2010a,c). The response to the stimulation of the baroreceptor consists in lowering the heart rate to maintain arterial blood pressure within the normal range.

3.2.1.2.2. Evidences of altered renin angiotensin aldosterone vasopressin axis in cardiopathic patients The RAAV axis has been largely studied in with heart failure in human and small animal medicine, but little information is available regarding horses.

In human beings, RAAV axis activation has been proven in patients with DCM and is known to be partially responsible for the course of the disease (Unger and Li, 2004). An activated RAAV system has also been implied in the pathogenesis of vascular remodeling, atherosclerosis and both pulmonary and systemic hypertension (Fiebeler et al., 2007; Ayari, 2013; Lastra and Sowers, 2013). RAAV activation has also been related to the occurrence of arrhythmias in human patients (Dixen et al., 2007; Iravanian and Dudley, 2008; Dogan et al., 2009). Recently, it has been demonstrated that plasma REN activity is a strong and independent prognostic indicator in patients with acute decompensated heart failure treated with RAAV system inhibitors (Ueda et al., 2015). Elevated plasma AVP concentrations are also found in some human patients with CHF, particularly those with severe heart failure and dilutional hyponatremia (Verbrugge et al., 2015).

There is a uniform agreement that the RAAV axis is activated in dogs and cats with cardiomyopathy and signs of CHF, particularly if are administered as a part of the therapy (Knowlen et al., 1983; Koch et al., 1995; Tidholm et al., 2001; Sisson et al., 2003). However, many authors failed to detect increased concentrations of the hormones of this axis in small animal with heart disease. Häggström et al. (1997) found low plasma concentrations of ANG-II and ALD in Cavalier King Charles Spaniel dogs with valve heart disease, 1 year before, 1 to 6 months before and at the time of the onset of clinical signs of CHF. These authors suggested that fluid retention in the early stages of

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Angiotensin-converting enzyme inhibitors in the horse heart failure was not due to an activated RAAV and therefore, other pathophysiological mechanisms should be investigated in order to explain early Na+ and fluid retention in dogs with mitral regurgitation (Häggström et al., 1997). In fact, these authors suggested that increased circulating ANP concentrations suppress RAAV in dogs with early compensated DVD. Similarly, Fujii et al. (2007) in 5 beagles with experimentally induced mild mitral valve regurgitation, without clinical signs, but with eccentric hypertrophy in , did not observe significant differences in plasma REN activity and plasma ANG-II and ALD concentrations in comparison with healthy dogs of the same breed.

By contrast, Pedersen et al. (1995) y Pedersen (1996) described increased plasma REN activity and elevated concentrations of ALD in asymptomatic and mildly symptomatic dogs with DVD, even when diet was taken into account. Similarly, Pedersen et al. (1999) studied the neuroendocrine changes associated with canine mitral valve prolapse and found that the severity of the prolapse was correlated positively with plasma REN activity and tended to correlate negatively with plasma ALD. However, these correlations were not found in dogs with mitral valve regurgitation (Pedersen et al., 1999). Tidholm et al. (2001) compared 15 symptomatic dogs with DCM and 15 healthy dogs, and found increased plasma REN activity and plasma ALD concentrations in the diseased dogs. However, they concluded that, although the analysis of the neurohormonal system might aid in the identification of clinical stages of DCM for groups of dogs, the range is too great and there are too many dogs that have neurohormonal concentrations within the reference ranges to assess dogs on an individual basis (Tidholm et al., 2001). Therefore, it is commonly accepted that activation of the RAAV axis is particularly intense in dogs and cats with acquired heart disease, particularly when furosemide is used as an additional therapy (Knowlen et al., 1983; Koch et al., 1995; Tidholm et al., 2001; Sisson et al., 2003). However, most of the dogs with less severe heart disease (New York Heart Association NYHA class I and II), concentrations/activities of the hormones of the RAAV are within the normal range or variably elevated (Pedersen et al., 1995; Pedersen, 1996; Häggström et al., 1997; Rush et al., 2000; Fujii et al., 2007; Oyama, 2009).

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These apparently contradictory results have been in part associated with the difficulty in the interpretation of the results of the RAAV assays. Thus, although CHF is one of the many patients’ circumstances causing an activation of RAAV, it is not the only. Low-salt diets, dehydration, blood loss and exercise stimulate the release of REN from the juxtaglomerular apparatus as a consequence of diminished renal blood flow (Sisson, 2004; Oyama, 2009). On the other hand, as explained before, the physiological effects of the RAAV axis include volume expansion and vasoconstriction. Both of these physiological responses serve to diminish REN production and consequently, changes of the RAAV axis tends to be phasic. Probably this is the reason of the uncertainty regarding the initiation of the RAAV axis over expression in dogs with heart disease.

Only few data are available regarding circulating AVP levels in dogs or cats with spontaneously appearing heart disease, but this knowledge deficit will likely be remedied in the near future. Tidholm et al. (2005) found significantly greater plasma concentrations of AVP in dogs with clinical signs of DCM and dogs with preclinical DCM compared with control dogs. In addition, the first group of diseased dogs had higher plasma AVP concentrations than the preclinical animals. More recently, Scollan et al. (2013) compared plasma AVP concentrations in healthy dogs and dogs with CHF as a result of chronic DVD or DCM. In dogs with CHF, plasma AVP concentrations were higher than in healthy dogs.

The scientific information about the activation of the RAAV axis in horses with heart disease is very limited. Gehlen et al. (2008) performed a prospective clinical trial with 8 healthy horses as control group and 40 horses with heart valve disease (mitral, tricuspid and aortic valves). This last group was divided into groups according to the severity of the heart disease, groups I (heart valve insufficiency without dimensional changes in the heart), II (left atrial dilation), III (left ventricular dilation) and IV (left atrial and ventricular dilation). They found a significant increase in ALD concentrations in the four groups compared to controls. Thus, serum ALD concentrations achieved average values of 23.95, 45.5, 95.9, 141.6 and, 161.2 pg/ml, in control and the 4 diseased groups respectively (Gehlen et al., 2008). These results led to the authors to suggest that serum/plasma ALD concentrations might be helpful biomarkers in the evaluation of

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equine heart disease since a greater increase was observed in horses with heart dilation. In addition, in human beings, a positive correlation between left atrial diameter and serum ALD concentrations has been documented (Hayashi et al., 2001). Further, and also in human patients, the increase in circulating ALD concentrations has been proven to be proportional to the severity in functional loss of the left ventricle (Mizuno et al., 2001). These correlations were not found in the study of Gehlen et al. (2008) with horses, although a positive correlation between ALD concentrations and left ventricle internal dimensions was found in one of the diseased horse group (group III).

Another report measured RAAV axis activity in two Spanish foals with endocarditis but without clinical signs of heart failure, and a significant activation of the axis was not observed (Rovira et al., 2009). Concentrations of REN, ANG-II and ALD were measured in this study, and in both foals, they were within the reference range for animals of the same age, sex and breed (Rovira et al., 2009):

3.2.1.3. Endothelins Endothelins (ET) are peptides produced mainly in the endothelium with a very important role in vascular homeostasis and with a potent vasoconstrictor effect. Three isoforms (ET-1, ET-2 and ET-3) and two receptors (ET-A and ET-B) have been identified. The ET-A receptor has increased affinity for the ET-1 and ET-2 isoforms, while the ET-B receptor shows equal affinity for all the three endogenous ligands (Masaki and Sawamura, 2006). ET-1 is secreted by vascular endothelial cells, is the most potent vasoconstrictor known and shows inotropic, chemotactic and mitogenic properties. ET- 1 also influences Na+ and water homeostasis at the renal level and stimulates the RAAV system (Agapitov and Haynes, 2002).

As indicated before, the main site for production of ET-1 is the vascular endothelial cells, although a lesser degree of production also occurs in the heart, kidney, central nervous system and posterior hypophysis gland. ET-2 is mainly produced in the endothelial cells, heart and kidney (Howard et al., 1992) and ET-3 is expressed in the

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Manuel Gómez Díez endocrine, gastrointestinal and central nervous systems, but is not produced by vascular endothelial cells (Howard et al., 1992).

Vascular actions of ET primarily consist on a strong vasoconstriction that was first demonstrated in vitro (Yanagisawa et al., 1988). It was later confirmed that ET-1, ET-2 and ET-3 induce a transient vasodilation before inducing sustained vasoconstriction (Cocks et al., 1989; Cardillo et al., 2000). Initial vasodilation is mediated by ET-B receptors localized in the endothelial cells that will increase NO and prostacyclin secretion, whereas the final vasoconstriction is mediated by the action of ET-A and ET- B receptors localized in the vascular smooth muscle cells (Agapitov and Haynes, 2002).

ET-1 shows also in vitro and inotropic effects (Ishikawa et al., 1988) and when administered intracoronary it causes myocardial and ventricular arrhythmias (Ezra et al., 1989). Pharmacological antagonism of ET-A and ET-B receptors in humans increases plasma renal flow without affecting glomerular filtration rate, which suggests that endogenous ET-1 acts primarily over the efferent arteriole (Fleisch et al., 2000; Agapitov and Haynes, 2002). ET-1 has a natriuretic effect due to inhibition of Na+ reabsorption by tubular Na+/K+ ATPase in the proximal tubule and the collecting duct and a decrease in Cl- flux in the loop of Henle (Plato et al., 2000). ET-1 also decreases water reabsorption in the collecting due to inhibition of the effects of ADH (Oishi et al., 1991; Agapitov and Haynes, 2002).

Under in vitro conditions, ET-1 stimulates the local RAAV system in isolated rat mesenteric bed and increased ALD production from isolated cortical zona glomerulosa cells (Rakugi et al., 1990; Kawaguchi et al., 1991). ANG-II has been showed to increase tissular activity of the ET converting enzyme (ECE) and concentrations of ET-1 under in vitro conditions, suggesting a positive feedback between ANG-II and ET-1 in some diseases like CHF (Moreau et al., 1997; Agapitov and Haynes, 2002).

For clinical purposes, measurement of the precursor of ET-1 (named big ET-1) is preferred, because ET-1 has a very short half-life, whereas big ET-1 circulates longer (O’Sullivan et al., 2007). In healthy people and animals, circulating ET-1 levels are low. Increased plasma big ET-1 concentrations have been documented in human patients

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Angiotensin-converting enzyme inhibitors in the horse with heart failure, and their levels appear to correlate with heart disease (Rodeheffer et al., 1992; Wei et al., 1994). Furthermore, plasma ET concentrations have shown to correlate inversely with survival (Rubens et al., 2001). Myocardial ET-1 production is thought to substantively contribute to the rise of circulating ET levels described in patients with CHF (Rodeheffer et al., 1992; Wei et al., 1994; Rossi et al., 1999). ET-1 concentrations are also consistently elevated in patients with pulmonary hypertension and some forms of renal disease, but interestingly, not in patients with systemic hypertension (Rubens et al., 2001). More recently, it has been reported that serial evaluation of ET-1 relative to other biomarkers, such as natriuretic peptides, is very useful for prognosis of severity and mortality in (Mayyas et al., 2015), acute and CHF (Yin et al., 2011; Pérez et al., 2016), congenital heart disease in adults (Miyamoto et al., 2015), left ventricular dysfunction after acute myocardial injury and infarct (Olivier et al., 2017) and systolic CHF (Gaggin et al., 2017).

Similar results to those described before for human patients have been also found in dogs. O’Sullivan et al. (2007) found that Doberman Pinschers dogs with overt DCM had significantly greater big ET-1 concentrations than controls and dogs with occult DCM. In addition, increasing big ET-1 levels over time were associated with a shorter survival time (0’Sullivan et al., 2007). Prosek et al. (2007) studied concentrations of different hormones in dogs with dyspnea, in order to differentiate cardiac and non- cardiac processes. They found that dogs with dyspnea of cardiac origin presented significantly higher ET1 concentrations (mean values: cardiac dyspnea: 1.26 fmol/ml; non-cardiac dyspnea: 0.32 fmol/ml).

In the horse, ET-1 has been shown to induce vasoconstriction in both digital and pulmonary vasculature (Benamou et al., 2003; Katz et al., 2003; Keen et al., 2008) and its concentrations increased in endotoxemic status and in carbohydrate loading (Menzies-Gow et al., 2005; Eades et al., 2007). Additionally, ET-1 concentrations might be important in the pathogenesis of laminitis and exercise-induced pulmonary hemorrhage, EIPH (Padilla et al., 2006, Stack et al., 2013).

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3.2.2. VASODILATOR MECHANISMS

3.2.2.1. Natriuretic peptides

There is a family of structurally related peptides with natriuretic properties, which include ANP, BNP and C-type natriuretic peptide (CNP) (Forssman et al., 1998). Natriuretic peptides modulate blood volume and BP, they are functional antagonists of the RAAV axis (Forssman et al., 1998; Levin et al., 1998; Trachsel et al., 2012; 2013; 2015), alter vascular permeability (Levin et al., 1998), inhibit smooth muscle cell proliferation (Chen and Burnett, 1998) and induce bronchodilation (Angus et al., 1996), as more relevant physiological functions.

ANP is synthesized in the cardiac atrial myocytes, secreted into the vasculature, and transported to the kidney where it induces diuresis and natriuresis (Turk, 2000; Sisson, 2004, Oyama and Singletary, 2010; Trachsel et al., 2015). BNP is known to be produced in greater amounts in the heart, especially in the ventricles (Borgeson et al., 1998), whereas CNP is produced by the vascular endothelium of many organs (Borgeson et al., 1998; Levin et al., 1998; Chen and Burnett, 1998). ANP is encoded by genes as form of precursor prohormone (prepro). Stretch is the primary stimulus for the synthesis and release of ANP (Ruskoaho et al., 1997; Levin et al., 1998). The preproANP mRNA is not expressed in the contractile ventricular cardiomyocytes of clinically normal adult animals. However, it is upregulated in many forms of cardiac hypertrophy (Vikstrom et al., 1998). Secretion of ANP is stimulated by the release of vasoactive substances, including ANG-II, catecholamines (via α1-adrenoceptors), ET-1 and AVP (Ruskoaho et al., 1997; Soualmia et al., 1997; Levin et al., 1998; Thibault et al., 1999). Basic fibroblast growth factor, hypoxia, substance P, and transforming growth factor β also stimulate ANP release (Baertschi et al., 1990; Harada et al., 1997; Perhonen et al., 1997). Secretion of ANP is mediated by increasing intracellular calcium (Ca2+) concentrations and appears to be dependent on contractile activity of cardiomyocytes (Eble et al., 1998). Secretion of ANP is inhibited by NO (Thibault et al., 1999).

BNP is also produced after stretching of the heart, in the cardiac atria and ventricles of adult animals. ANG-II, hypoxia, α1-adrenoceptor agonists, ET-1,

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Angiotensin-converting enzyme inhibitors in the horse transforming growth factor-β, and AVP stimulate BNP production (Harada et al., 1997; Perhonen et al., 1997; Ruskoaho et al., 1997; Levin et al., 1998). As described before, CNP is produced within the blood vessels of many organs. The vasoactive peptides ANP, BNP, ANG-II and ET-1 upregulate the production of CNP by vascular endothelial cells (Nazario et al., 1995). Basic fibroblast growth factor, hypoxia, thrombin, transforming growth factor-β and tumor necrosis factor-α also stimulate CNP production (Turk, 2000).

There are four different groups of physiological and pathological situations in which this family of peptides are involved: plasma volume regulation, vasomotor tone and hypertension, cardiac function and pulmonary function (Turk, 2000). Volume overload increases (Stewart et al., 1992) and dehydration decreases (Vollmar et al., 1994) plasma concentrations of ANP in dogs. Plasma ANP concentrations increase during exercise in dogs (Beliveau et al., 1994) and horses (Nyman et al., 1998; Kokkonen et al., 2002), in association with the intensity of dehydration. ANP, BNP and CNP have direct tubular actions in the kidneys that include inhibition of ANG-II-stimulated Na+ and water transport in the proximal convoluted tubules (Forssman et al., 1998). In addition, these hormones inhibit the release and actions of AVP and the secretion of REN and ALD (Levin et al., 1998; Woods and Jones, 1999). ANP decreases sympathetic tone of the peripheral vasculature (Levin et al., 1998). Further, CNP induces a potent venodilation that increases venous capacitance, reduces venous return, and decreases cardiac filling pressures (Brandt et al., 1994; Chen and Burnett, 1998). Infusion of exogenous ANP, BNP or CNP reduces BP in dogs (Woods and Jones, 1999). They also inhibit smooth muscle cell proliferation (Chen and Burnett, 1998), and consequently, they might retard the vascular remodeling associated with chronic hypertension.

Plasma ANP and BNP concentrations have been shown to be increased in congenital cardiac abnormalities that cause atrial dilation (Nagaya et al., 1998). Ventricular expression of ANP and BNP increases in cardiomyopathies in human beings (Hasegawa et al., 1993), cattle (Takemura et al., 1990) and dogs (Asano et al., 1999; Oyama and Singletary, 2010; Smith et al., 2015), and recently, the same has been proved to happen in horses with atrial dilation (Trachsel et al., 2012; 2013; 2015; Leroux, 2015). Heart failure in human patients is characterized by substantial elevations of ANP and

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BNP, and several studies have demonstrated that measurement of plasma natriuretic peptide concentrations, particularly BNP, are helpful for discriminating patients with dyspnea caused by heart disease from those with pulmonary disease or other disorders (Cowie et al., 1997; McDonagh et al., 1998; Tsutamoto et al., 1997; Hammerer-Lercher et al., 2001; McCullough et al., 2002; 2003). A study with cats with myocardial disease, measures of ANP and BNP appear to have diagnostic potential (Sisson et al., 2003). Plasma BNP levels elevated more than 10-fold were an aid to distinguish between cats with heart failure from control cats, more useful than the increase in ANP levels, which was only 4 to 5-fold (Sisson et al., 2003). BNP values, however, seem to be less prognostic in dogs than in cats. Thus, plasma BNP concentrations do not increase markedly until the later stages of heart failure in dogs with DVD, and the magnitude of these changes was less evident than that observed in cats and human beings (Asano et al., 1999; Häggström et al., 2000; MacDonald et al., 2003). For that reason, some authors defend that the measurement of pro-ANP could be more useful than BNP as a marker of heart disease and heart failure in dogs (Häggström et al., 1994; 2000; Asano et al., 1999; MacDonald et al., 2003). At the present time, it is not clear whether this finding reflects a species-related difference in the physiology of the natriuretic peptides o whether it is simply an artifact or a problem with the measurement assays. In a study of dogs presented for dyspnea, it was found that plasma pro-ANP was better than plasma BNP or ET-1 for identifying the dogs with CHF (Smith et al., 2015).

In human beings, circulating BNP concentrations are increased in asymptomatic patients with systolic left ventricular dysfunction, left ventricular diastolic dysfunction, in cases of ventricular hypertrophy caused by systemic or pulmonary hypertension, and in HCM (Hasegawa et al., 1993; Yamamoto et al., 1996; Nagaya et al., 1998). On base of these data, some authors have advocated using circulating BNP levels to screen patients for early left ventricular dysfunction, and the same might be applied to cats with this type of cardiomyopathy, particularly when equivocal results have been found with other diagnostic modalities, including echocardiography (Smith et al., 2015; Parzeniecka-Jaworska et al., 2016). Studies of human patients with CHF have proven than plasma BNP levels are useful in formulating an accurate prognosis, particularly

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Angiotensin-converting enzyme inhibitors in the horse when measured before and after therapy (Yamamoto et al., 1996; Tsutamoto et al., 1997; Wallen et al., 1997; Maeda et al., 1999).

Investigations on the concentrations of plasma ANP in horses with heart disease have produced variable results with one study showing no significant differences in ANP concentrations between horses with valvular disease and controls (Gehlen et al., 2007) and some studies showing an increase in ANP concentrations in horses with left atrium enlargement due to mitral valve regurgitation when compared to controls (Trachsel et al., 2012; 2013; 2015). The different results between studies could have been related to ANP instability, and possible differences in sampling protocols and assays.

To the authors’ knowledge, pharmacological control of the activity levels of the natriuretic peptides has been scarcely investigated in veterinary medicine but has been tried in humans with successful clinical effects that vary when compared to ACE inhibition alone (Klapholz et al., 2001; Ferrario et al., 2002; Packer et al., 2002). As the heart disease progresses the beneficial effects of endogenous natriuretic peptides are progressively blunted and therefore, the antagonist effects exerted on the activated RAAV axis decrease, being this idea the main justification for administration of exogenous natriuretic peptides in human patients with CHF (Plante et al., 2014; Li et al., 2016; Díez, 2017). Furthermore, in human patients with acute CHF, intravenous (IV) infusion of BNP is an adjunctive means to decrease dyspnea, pulmonary capillary wedge pressure and and increase cardiac output, urine volume, and urine Na+ excretion (Yancy et al., 2016; 2017). Similarly, early studies in dogs with experimental CHF, exogenous BNP administration reduced left atrial and mean systolic arterial pressure, and increased urine volume and Na+ excretion (Chen et al., 2000). More recently, the feasibility, safety and tolerance of subcutaneous synthetic canine BNP have been evaluated in healthy dogs and dogs with mitral valve disease, but the clinical relevance of this treatment has not been assessed yet in dogs with naturally acquired heart disease (Oyama et al., 2017).

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3.2.2.2. Endothelin-derived releasing factor and prostacyclin Endothelial cells have an important role in the control of vascular tone by releasing powerful vasodilators like prostacyclin I2 and endothelium derived relaxing factor (EDRF). EDRF was first identified as an extremely instable substance released from the vascular endothelium that had a half-life between 3 and 50 s and that induced vascular smooth muscle relaxation and inhibited platelet aggregation and adhesion. Combination of chemical and biological investigations allowed demonstrating that the chemical composition of EDRF was, in fact, NO (Moncada et al., 1988). NO release is mostly abluminal, it does not reach significant concentrations in the circulatory bed and is easily detectable from fresh arterial tissue compared to venous tissue (Busse et al., 1985; Moncada et al., 1988).

There is a synergic relationship between NO and prostaglandins (PGs), and both substances are powerful vasodilators with a synergic activity. In vitro studies on porcine coronary arteries revealed that PGs have a direct relaxant effect on vascular smooth muscle but also stimulated secretion of EDRF from the endothelial cells (Shimokawa et al., 1988).

Concentrations of prostacyclin I2 have been found increased in people with heart failure (Dzau et al., 1984) and correlated to the levels of circulating ANG-II (Elliott and Bowen, 2010).These vasodilator mechanisms can counterbalance the negative effects of the neuroendocrine vasoconstrictor and antidiuretic mechanisms in the early stages of heart failure, but as disease progresses, renal perfusion will be severely compromised and natriuretic peptides and vasodilating PGs will no longer be effective to compensate the deleterious vasoconstriction, antidiuresis and cardiac remodeling that will enter a self-perpetuating cycle (Elliott and Bowen, 2010).

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3.3. THERAPEUTIC BLOCKAGE OF THE RENIN ANGIOTENSIN ALDOSTERONE VASOPRESSIN AXIS

The RAAV axis, as indicated before, comprises a series of physiological regulatory mechanisms of homeostasis that, under certain circumstances, can misadjust resulting in a series of cardiovascular diseases of great importance in humans, small animals, and horses (Lefèbvre et al., 2007; Muñoz et al., 2010a). This axis is of great importance in the regulation of hydroelectric balance and BP and includes both circulating and tissue elements that encompass the kidney, central nervous system, myocardium and adipose tissue (Matsusaka and Ichikawa, 1997). The discovery of each of the members of the RAAV axis, including their cardiovascular, BP, and renal actions, and the development of pharmacological drugs to target the key enzymes and receptors to treat cardiovascular, hypertensive and renal diseases have become a successful story in the cardiovascular, hypertensive, and renal research (Li et al., 2017).

Pharmacologic control of the RAAV system can help in the treatment of multiple diseases where there is an activation of this axis. These diseases include arterial hypertension, CHF and renal insufficiency. Therefore, blockage of this axis is nowadays one of the main battle fields of cardiovascular pharmacology. This blockage can be achieved with the administration of eight different drugs groups (Brewster et al., 2003; Atlas, 2007; Ferrario and Mullick, 2017). Then, several pharmacological classes of drugs are described in relation to their mechanisms of action, which are analyzed through a pharmacological approach. Different point keys as the molecular receptor targets, the various sites along the arterial system, and the extra-arterial sites of action are discussed, in order to better understand in which type of diseases a given pharmacological class of drug could be more indicated (Laurent, 2017).

The eight major pharmacological classes of drugs detailed here are: direct REN inhibitors (DRIs; Group 1), ACEIs (Group 2), ANG receptors blockers or antagonists of AT1 receptors (AARs; Group 3), mineralocorticoid receptor antagonists (MRAs; Group 4), ANG receptor neprilysin inhibitors (ARNIs; Group 5), β-blockers (Group 6), Ca2+ channel blockers (CCBs, Group 7) and blockers of receptors of AVP (Group 8). However, at this point, it is necessary to indicate that the groups 1 to 4 are the classical drugs

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considered into the RAAV system, while 5, 6, 7, and 8 groups can be considered new drugs or indirect drugs over RAAV system, according to the criteria of different authors and researchers(Ferrario and Mullick, 2017; Laurent 2017). The RAAV axis and its pharmacological blockade are shown in figure 1.

FIGURE 1. Pharmacological agents used to manipulate the RAAV axis (SNS: Sympathetic nervous system; RAAS: Renin angiotensin aldosterone system; ACEIs: Angiotensin converting enzyme inhibitors; ACE: Angiotensin converting enzyme; DRI: Direct ; AAR: Angiotensin receptor blocker; NEP: Neprilysin; ANP: Atrial natriuretic peptide; BNP: Brain natriuretic peptide; MRA: Mineralocorticoid receptor antagonist; ARNI: Angiotensin receptor neprilysin inhibitor)

3.3.1. RENIN INHIBITORS

Angiotensinogen is cleavaged by REN, considered the rate-limiting step in ANG- II generation, is a logical step to inhibit the RAAV axis from a pharmacological point of view, being the inhibition of REN one of the most important targets (Pantzaris et al., 2017).

There are three generations of REN inhibitors. The first generation includes agents that required parenteral administration and showed limited activity in healthy

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Angiotensin-converting enzyme inhibitors in the horse volunteers as REN inhibitor H142 (Webb et al., 1985). The next generation produces orally active compounds like enalkiren, remikiren and zankiren that were developed by their clinical potential but were not pursued as they had poor bioavailability, short half- life, and weak antihypertensive activity (Ferrario and Mullick, 2017). However, the last generation is clinically important, with aliskiren fumarate, the first non-peptide direct REN inhibitor with favorable pharmacokinetic properties (Pantzaris et al., 2017). Aliskiren was approved in 2007 by the US Food and Drug Administration (FDA) and the Europe Medicine Agency (EMA) regulatory bodies for the treatment of hypertension, either as a monotherapy or in combination with other antihypertensive agents (Atlas, 2007).

Aliskiren administration decreases plasmatic activity of REN by direct inhibition and has been used in monotherapy showing significant dose-related BP lowering effects. These effects are similar to those observed with some AARs as valsartan or irbesartan, or some ACEIs as lisinopril and a placebo-like tolerability profile in humans. Besides, other studies have concluded that aliskiren had superior or similar BP lowering effects than ramipril (Pantzaris et al., 2017). Nevertheless, the combination with other RAAV inhibitors or antagonists has become the most important therapeutic rationale use for aliskiren over the last decade (Ferrario and Mullick, 2017). In fact, different researches have demonstrated that incomplete RAAV suppression was achieved with a single agent, suggesting additive and synergic effects when aliskiren was combined with AARs or ACEIs (Dzau et al., 2002). These combinations demonstrated beneficial effects on surrogate endpoints, such as reductions in BP and proteinuria and adverse effects such as hyperkalemia, renal impairment or hypotension were sufficiently low as to not cause concern (McMurray et al., 2016). Despite these positive results, other trials with treatments of longer duration and worse cardiovascular or renal outcomes showed no clinical utility and unfortunately, adverse effects were detected (Gheorghiade et al., 2013). It has been suggested that the antihypertensive response produced by concomitant inhibition of both REN and ANG-II triggers significant renal injury due to loss of autoregulatory capacity (Moniwa et al., 2013).

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Up to date, aliskiren is a well-established antihypertensive agent, but its optimal use remains to be further tested. Although specific human patients seem to benefit more from their direct inhibition of REN (e.g., MetS and resistant hypertension) for others, it is just another viable option to achieve adequate BP control. As often a multidrug anti-hypertensive therapy is required, aliskiren, the only available direct REN inhibitor in the market, might play an important role as a RAAV axis blocking drug option in combination regimens (Pantzaris et al., 2017). However, it is necessary to indicate that, to our knowledge, the research in veterinary medicine of DRIs is limited to the study of aliskiren in a canine model of atrial fibrillation in order to be applied to human medicine (Satoh et al., 2017).

3.3.2. ANGIOTENSIN CONVERTING ENZYME INHIBITORS

This is the most important pharmacologic group in the inhibition of the RAAV system and the most developed, both in human and veterinary medicine. It comprises a large group of drugs that act as ACE inhibitors leading to decreased production of ANG- II. They produce a strong and selective inhibition with large specificity and affinity for both circulating and tissular (endothelial, pulmonary, renal, etc.…) ACE. The ACE enzyme acts also on other substrates, including bradykinin. Bradykinin is a potent vasodilator and its levels tend to increase after administration of an ACEI (Lefèbvre et al., 2007). Because PO absorption of these drugs is poor they are administered as more lipophilic pro-drugs with a better PO bioavailability. After absorption, they are hydrolyzed to its active form, the real ACEI than can inhibit the ACE enzyme with high affinity and efficacy (Mochel et al., 2015).

Unlike other drugs used to block the RAAV axis, ACEIs have been intensively studied in veterinary medicine, especially in dogs and cats. In these species, PK and clinical assays have been described in healthy and non-healthy animals (Lefèbvre et al., 2007; Mochel et al., 2013; Davis et al., 2014). The main pharmacologic effects and relatively benign side and adverse effect profile of ACEI described in human and veterinary medicine are summarized in the table 1 (Lefèbvre et al., 2007; Miyagawa et al., 2016; Ferrario and Mullick, 2017; Lavallee et al., 2017).

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Main pharmacologic effects Main clinical uses Main reported adverse effects Vasodilation at the venous Arterial hypertension Dry cough and headache and arteriole level because of inhibition of ANG-II synthesis. CHF Low BP and hypotension in patients that receive simultaneous Inhibition of bradykinin Cerebral reperfusion administration of diuretics. Disorders in geriatric Renal failure or deterioration of Decrease in Na+ and water animals renal function in cases of heart retention because of failure with decreased renal decreased ALD secretion. Diabetic nephropathy. perfusion.

Diarrhea and gastric alterations occasioned by their acid characteristics.

TABLE 1. Main pharmacological effects, clinical uses and adverse effects reported following ACEIs administration in human and veterinary medicine (ANG-II: Angiotensin II; CHF: Congestive heart failure; BP: blood pressure; Na+: sodium; ALD: aldosterone)

By decreasing systemic , ACEIs are known to improve cardiac and exercise capacity in human and dog patients (Lefèbvre et al., 2007). Benazepril, enalapril, and ramipril are currently pro-drugs approved for use in dogs with CHF in USA and EU. In fact, benazepril hydrochloride is a nonsulfhydryl pro-drug which is converted in vivo by hepatic esterases into its active metabolite, benazeprilat, a highly potent and selective inhibitor of ACE (Mochel et al., 2015) with well-documented effectiveness in symptomatic canine CHF (Lefèbvre et al, 2007; Häggström et al., 2008; 2013; King et al., 2017). Enalaprilat derived from PO administration of the pro-drug enalapril, has been intensely studied in dogs in recent researches suggesting that the efficacy of this ACEI in suppressing tissue RAAV, following ACE inhibition, could be caused by unknown mechanisms in relation to other pathways different but related to the known RAAV axis (Lantis et al., 2015; Ferrario and Mullick, 2017).

Although most of the preclinical investigations for dose selection of ACEIs have used ACE activity as a surrogate marker of efficacy in small animals, recent literature suggests that this may not be a sensitive approach to properly assess the modulatory effect of ACEI on the RAAV system (Mochel et al., 2015; Afonso et al., 2017b). Using a

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Manuel Gómez Díez low-Na+ diet (LSD) model of RAAV activation, benazeprilat markedly influenced the dynamic of the systemic RAAV following single and repeated PO administrations of benazepril in dogs (Mochel et al., 2013). In this study, treatment with benazepril triggered an apparent decrease in ANG-II and ALD, together with a sustained elevation of REN activity, as a consequence of benazeprilat-induced interruption of the ANG II- REN negative feedback loop. As expected, changes in ALD levels were followed by a significant reduction of K+ renal excretion. This modulatory action of benazeprilat on the functioning of the circulating RAAV was further characterized using an integrated mechanism-based PK-PD modeling approach (Mochel et al., 2015). The final model included modeling of placebo data, suggested that an overestimation of the effect of ACEI on ANG-II and ALD concentrations would have resulted, together with an underestimation on REN activity. Simulations from the model allowed quantifying the extent and duration of effects of PO benazepril on the RAAV axis. Results showed a two- to three-fold change in systemic RAAV levels at steady-state benazeprilat peak concentrations, and a more prolonged effect on REN activity compared with ANG-II and ALD levels. Such discrepancies could have been related to the production of ANG-II by up-regulation of an ACE-independent pathways in response to REN and ANG-I accumulation during acute and long-term use of ACEIs (Ferrario et al., 2017). However other studies in human medicine have demonstrated that the use of ACE as a surrogate marker might be useful because the objective of the model is the understanding of the complex biological system, such as RAAV axis, and the simplifications by the use of ACE models may be enough for scientific and clinical purposes (Levitt and Schoemaker, 2006.)

As a consequence of these pharmacologic effects and main clinical uses, abundant information regarding these drugs has been published over the last twenty years, being small animals, mainly dogs, the most studied animal species (Lantis et al., 2015; Qian et al., 2016). Moreover, new studies have appeared in horses in the last decade (Afonso et al., 2013; 2017b; Davis et al., 2014). On the other hand, while the antihypertensive effects of these drugs have been proven and many studies have confirmed their ability to acutely suppress the conversion of ANG-I into ANG-II, a consistent suppression of tissue or plasma ANG-II concentrations has not been

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demonstrated. While an ACE escape mechanism has been suggested to account for the recovery of ANG-II, insufficient attention has been paid to the multiple studies showing that chymase, rather than ACE, is the main ANG-II-forming enzyme from either ANG-I or ANG-(1-12) in humans, mice and rats in tissues like heart and vascular wall, suggesting the existence of other alternative pathways that may also contribute to ANG-II generation (Ahmad et al., 2016; Ferrario et al., 2016).

3.3.3. ANGIOTENSIN I RECEPTOR ANTAGONISTS

The ANG receptor blockers or antagonists of AT1 receptors (AARs) are other important group of drugs that interact with the RAAV axis and frequently are used as an alternative for patients intolerant to ACEIs. AARs are non-peptide, orally active, AT1 receptor antagonists, that bind competitively to and dissociate slowly from AT1 receptors. Blocking their actions causes vasodilation, reduces the secretion of AVP and reduces the production and secretion of ALD, among other actions. The combined effects all of these actions are a reduction in BP, being used as vasodilators agents (Tamargo and Tamargo, 2017). These drugs differ from ACEIs because they compete with ANG-II in its place of action, even though clinically both groups have similar effects and share many clinical indications (diabetes, hypertension, CHF, diabetic nephropathy). These drugs are considered vasodilators and anti-hypertensive and have a widespread use in human medicine. On the other hand, all AARs used are selective ligands of AT1 receptors and the pharmacological studies showed a significant potency in their ability to cause a rightward shift in the dose-response curve to ANG-II (Ferrario and Mullick, 2017). Large clinical trials in humans using this type of compounds (, valsartan, candesartan, irbesartan, telmisartan and olmesartan) have proven their ability to control BP in hypertensive patients, reduce stroke risk, and improve the prognosis of diabetes nephropathy. Nowadays their use in veterinary medicine is very limited. There are only a few PK-PD studied in dogs, as well as reports in canine models of atrial fibrillation and clinical assays for treatment of refractory proteinuria (Christ et al., 1994; Baek et al., 2013; Zhao et al., 2013; Bugbee et al., 2014).

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An important disadvantage of AARs is that they might induce compensatory pathways that increase circulating ANG-II levels as well as increased expression of metabolites like ANG (1-7) (Ferrario and Mullick, 2017). Following the results of clinical experiences in human medicine to date, and the knowledge in veterinary medicine, some disadvantages of ACEIs and AARs can be described (Ferrario and Mullick, 2017). Firstly, the generation of ANG-II by non-ACE-related enzymes limits the ability of ACEIs to block the RAAV system, whereas the existence of other non-AT1 receptors limits the ability of AARs to block this system. Secondly, both ACEIs and AARs lead to increased plasma REN levels, produce hyperkalemia and can deteriorate renal function in patients with renal artery stenosis. Thirdly, both classes of drugs only transiently decrease plasma ALD concentrations, which might limit their ability to block the RAAV system after term therapy (Laurent, 2017).

In conclusion, AARs are an important group of drugs in cardiovascular therapy. In spite of their extensive use in human medicine, their possibilities and clinical implications in veterinary medicine are unknown and further studies of these drugs should be performed for their potential into a clinical context.

3.3.4. MINERALOCORTICOID RECEPTOR ANTAGONISTS

These drugs are competitive antagonists which reduce the action of ALD by competing for the intracellular mineralocorticoid receptor of the distal convoluted tubule. This competition increases the excretion of water and Na+ while decreasing K+ excretion. Drugs as spironolactone, eplerenone or finerenone share the same mechanism of action, acting as K+-sparing diuretics. Their main use is the treatment of hyperaldosteronism and they are usually combined with stronger diuretics like furosemide when treating hypertensive patients. Moreover, they play a major role in the control of RAAV axis like ACEIs, AARs or DRIs (Ruilope and Tamargo, 2017). Spironolactone and eplerenone are frequently prescribed in combination with other antihypertensive agents for the treatment of hypertension or CHF (Ferrario and Mullick, 2017). However, the increased selectivity for mineralocorticoid receptors of the new antagonist finerenone is the current focus for the treatment of CHF, chronic kidney

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disease, and diabetic nephropathy in human patients, because it acts as a full antagonist of the mineralocorticoid receptors and shows an even accumulation of the drug in the heart and kidney, contrasting with the much larger kidney accumulation of spironolactone and eplerenone (Ruilope and Tamargo, 2017). On the other hand, only a modest natriuretic effect can be expected, since a smaller fraction of filtered load of Na+ is reabsorbed compared to the more proximal site of action of loop diuretics and thiazide diuretics. However, as a consequence of their mechanism of action, hyperkalemia is a common side effect, particularly in patients with chronic renal disease and CHF or diabetes, receiving K+-sparing diuretics or K+ supplements, or taking an ACEIs, an AARs, or a non-steroidal anti-inflammatory drug, NSAID (Laurent, 2017). Other side effects are related to the sexual effects of spironolactone, since it inhibits the binding of dihydrotestosterone to androgen receptors, which results in an increased clearance of testosterone. Treatments with eplerenone, which is a more selective ALD antagonist, are much less complicated because of these sexual side effects (Laurent, 2017).

More recently, MRAs have also been registered for use in canine patients suffering from CHF and are frequently used in dogs (Ovaert et al., 2010). In a study from Bernay et al. (2010) with dogs, spironolactone reduced by a factor of 2 the risk of cardiac-related death, euthanasia, or severe worsening of the clinical condition when used in addition to conventional therapy (ACEIs plus furosemide and digoxin, if required). These results were however discussed by Kittleson and Bonagura (2010) on the grounds of several methodological flaws (e.g., patient categorization, definition of CHF…). In addition, Schuller et al. (2011) could not find any significant effect of low-dose of spironolactone on survival when used as adjunct treatment to conventional CHF treatment in dogs. Interestingly enough, MRAs have shown a significant reduction in mortality in human patients with CHF when combined with ACEIs, whereas AABs have not (Ferrario and Mullick, 2017).

3.3.5. ANGIOTENSIN RECEPTOR NEPRILYSIN INHIBITORS

ARNIs are a new class of agents that combine two drugs, one AAR and one inhibitor of NEP. These combinations block the RAAV axis by two different pathways,

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Manuel Gómez Díez the classic antagonism of ANG-II receptor plus the inhibition of NEP enzyme. To date, the only group approved in human medicine is the combination of the NEP inhibitor sacubitril with valsartan as knowledge as LCZ696 (sacubitril/valsartan) that can be used for the treatment of heart failure. However, their use in veterinary medicine is minimal and only PK studies have been published in dogs (Ferrario and Mullick, 2017).

NEP catalyzes the degradation of natriuretic peptides that regulate cardiovascular and renal homeostasis. NEP actions in ANG metabolism include degradation of ANG-II and ANG-(1-7), and these actions would be expected to produce opposing effects on vasomotor tone and BP. However, it is the proteolysis of natriuretic peptides the most important mechanism. In fact, the expression and release of these peptides, such as ANP and BNP from cardiomyocytes is increased during heart wall stress and hypertrophy, common CHF features. Their activation promotes BP and volume reductions that act to counteract the increased cardiac wall stress. Such effects oppose the actions of ANG-II. Therefore, maintaining elevated levels of natriuretic peptides via inhibition of NEP are an attractive strategy for combating the undesirable effects of inappropriate RAAV activation (Ferrario and Mullick, 2017). For that reason, NEP inhibition represents a potential alternative strategy to prevent the breakdown of endogenous natriuretic peptides.

Candoxatril, the first NEP inhibitor available PO was associated with a dose- dependent increase in ANP and natriuresis but also increased concentrations of ANG-II because of the effect of NEP on the breakdown of ANG-II. Another NEP inhibitor, ecadotril, was tested in a dose-ranging study in human patients with CHF in which safety and efficacy were assessed. However, there were numerically more deaths in the patients receiving ecadotril and no evidence of efficacy, so development of the compound was stopped (Singh and Lang, 2015). Omapatrilat was the first representative drug acting through a dual NEP and RAAV system inhibition mechanism. As an inhibitor of both NEP and ACE, this drug was proved to be more potent than candoxatril in lowering BP and improving hemodynamics in patients with CHF. Although these initial results were promising, an outcomes trial in patients with CHF failed to show substantial benefits in comparison with the ACEI enalapril (MacDonald, 2015).

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LCZ696 is a first class of ARNI approved in human medicine (Ferrario and Mullick, 2017). LCZ696 is a novel, dual-acting complex composed of the NEP inhibitor sacubitril and the AARs valsartan. Soon after oral absorption, LCZ696 dissociates into sacubitril which is enzymatically bio transformed to the active form LBQ657 and valsartan. LCZ696 was designed to have a reduced risk for angioedema because it inhibits only one of the enzymes responsible for bradykinin breakdown (Singh and Lang, 2015). Their PK-PD relationships have been studied in rats and dogs and good properties as oral bioavailability, safety and clinical efficacy were demonstrated to the treatment of hypertension and heart failure (Gu et al., 2010).

3.3.6. β-BLOCKERS

β-blockers constitute a classic heterogeneous pharmacological group with PD properties that depend on their cardiac-selectivity, partial agonist activity and associated vasodilating properties. They all lower BP to the same extent, although they produce various amounts of reduction in cardiac output and vasodilation, according to their pharmacological properties. In fact, their effects can be related with their selectivity to the adrenergic receptor, showing mechanisms of action that are associated with the characteristics of the β-blocker (Laurent, 2017).

β-blockers bind to β-adrenergic receptors and prevent the activation of the signaling pathway protein Gs-adenilil cyclase-cyclic adenosine monophosphate- phosphokinase A by catecholamines. As a consequence, they reduce cellular levels of cyclic adenosine monophosphate (AMPc) and inhibit the activation of protein phosphokinase A and the phosphorylation of various cellular proteins (Frishman and Alwarshetty, 2002). In relation to the type of receptors they block, they are classified into non-selective (block β1 and β2 receptors), selective (at low concentrations block mainly β1 receptors) and mixed (block the α- and β-adrenergic receptors). Their selectivity is a dose-dependent phenomenon, which disappears by increasing the dose of the β-blockers. However, at these doses, these drugs are used as antihypertensive agents similar to those of the non-selective (Laurent, 2017).

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Different mechanisms of action have been suggested in order to explain the antihypertensive action of β-blockers. First, the reduction in cardiac output, in response to , is one of the most important factors for lowering mean BP. Secondly, a reduction of plasma REN activity secondary to receptor blocking of β1-adrenergic receptors in the juxtaglomerular renal cells has been suggested (Sica and Black, 2008). Third, a central effect was hypothesized because these agents accumulate in the hippocampus and interventricular septum, producing an antihypertensive response. Fourth, they induce an increased synthesis and release by endothelial cells of NO and prostaglandin I2, which have vasodilatory and antiplatelet properties. Fifth, a reduction of the sympathetic tone and peripheral vascular resistance after several days of treatment was observed. For all of these characteristics, these drugs are used to treat cardiac arrhythmias and hypertension in human and veterinary medicine (Mentz et al., 2013).

β-blockers should not be used in animals with moderate to severe asthma (since adrenergic bronchodilatation requires intact β-2 receptors), neither in unstable CHF resulting from systolic dysfunction. β-blockers may worsen glucose intolerance and mask hypoglycemic symptoms. For example, propranolol may cause bronchospasm and is contraindicated in asthmatic dogs and contraindicated in animals with heart failure or sinus bradycardia (Jacobs et al., 1997). Other important drug in this group, atenolol, is contraindicated in animals with hepatic disease. Although atenolol is selective for β1- receptors, it should be used cautiously in animals with asthma or a history of bronchospasm, because at high doses it can block β2-receptors as well (Ghaffari et al., 2011).

3.3.7. CALCIUM CHANNEL BLOCKERS

CCBs are a heterogeneous class of drugs, which include verapamil, diltiazem, and amlodipine. Although these drugs are also referred to as Ca2+ antagonists, they do not directly antagonize Ca2+. Rather, they inhibit the entry of Ca2+ into the cell or inhibit its mobilization from intracellular stores. In fact, they inhibit the voltage- dependent channels in vascular smooth muscle at significantly lower concentrations

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than that necessary to interfere with the release of intracellular Ca2+ or receptor- operated channels (Lantis et al., 2015). The PD effects of the CCBs reflect differences in potency at the various tissue receptors (Creevy et al., 2013). They are vasodilators of small resistance arteries. Acutely administered, they reduce total peripheral resistance and mean BP, and they increase cardiac output. After chronic administration, cardiac output returned toward pretreatment levels, and mean BP and systemic vascular resistance remained low (Laurent, 2017).

Amlodipine is often the drug of choice for treatment of feline hypertension (Jepson et al., 2014; Bijsmans et al., 2016; Taylor et al., 2017). Because of its actions are independent of the RAAV axis, they can be administered in combination with ACEIs (Helms, 2007). However, some side effects have been reported in dogs treated with amlopidine, as severe or diffuse edema (Creevy et al., 2013) and gingival hyperplasia (Thomason et al., 2009; Pariser and Berdolay, 2011).

3.3.8. BLOCKERS OF RECEPTORS OF ARGININE VASOPRESSIN

The neurohormonal factor AVP, as described before, produces potent systemic vasoconstriction as well as water retention in the kidneys via V1A and V2 receptors, respectively. Therefore, AVP may be considered as an aggravating factor of cardiac failure (Mao et al., 2009). AVP may play a role in a number of diseases, including CHF, hypertension, renal disease, edema, hyponatremia and the syndrome of inappropriate ADH hormone secretion. The development of AVP antagonists appears essential for assessing the pathophysiological roles of AVP and could lead to new therapeutic agents (Mao et al., 2009).

Up to date, the only drug used in this context is conivaptan, a combined blocker of V1A and V2 receptors. Conivaptan has shown efficacy in dogs with experimentally induced heart failure and in human patients with severe symptomatic CHF (Yatsu et al., 1999; Mao et al., 2009). Different studies have been made in rats, humans and dogs and the derived results suggest the potential usefulness of conivaptan as a neurohormonal antagonist for the treatment of CHF (Yatsu et al., 2002). Conivaptan hydrochloride

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Manuel Gómez Díez improved plasma Na+ concentration and plasma osmolality in hyponatremic rats, and its effectiveness was demonstrated in hyponatremic patients (Arai et al., 2009). This drug has been approved for use in the USA, which will bring relief to patients with hyponatremia (Arai et al., 2009).

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3.4. BASIC KNOWLEDGE OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS

3.4.1. THE ANGIOTENSIN CONVERTING ENZYME

The ACE, also known as kininase II, is a zinc protease that is under minimal physiological control. It is not a rate-limiting step in the generation of ANG-II and is a relatively nonspecific dipeptidyl carboxypeptidase that requires only a tripeptide sequence as a substrate which does not include proline as the second-last amino acid. Therefore, ANG-II is not further metabolized by ACE as it includes proline in the second- last position. The lack of specificity exhibited by ACE derives from its involvement in the bradykinin pathway. The degradation of bradykinin to inactive peptides occurs through the actions of ACE. Therefore, ACE not only increases the production of a potent vasoconstrictor but also inactivates a potent vasodilator (Figure 2) (Sisson, 2004; Ferrario and Mullick, 2017).

In 1965, a report concluded that the venom of the South American pit viper (Bothrops Jararaca) contained factors that potentiated the action of bradykinin (Ferreira et al., 1965). These factors were denominated bradykinin-potentiating factors (BPFs) and, when isolated, were found to form a family of peptides containing 5 to 13 amino acid residues. Their effect on bradykinin activity was linked to the inhibition of its enzymatic breakdown and the inhibition of ANG-I conversion to ANG-II. BPFs were seen as lead compounds for the development of new antihypertensive agents due to their dual activity of bradykinin potentiation and ANG-I conversion inhibition (Mentz et al., 2013). The identification of the amino acid sequences of ANG-I and bradykinin as ACE substrates and the discovery of the BFPs conditioned the synthesis and clinical development of ACEIs.

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FIGURE 2. Summary of the factors involved in REN release and the effects mediated by ANG-II and schematic representation of the bradykinin pathway and its relationship to ACE and the REN-ANG pathway

3.4.2. ANGIOTENSIN CONVERTING ENZYME INHIBITORS: CHEMICAL STRUCTURE AND ANGIOTENSIN CONVERTING ENZYME BINDING

With the knowledge from substrate-binding specificities of BPFs and the fact that ACE has properties similar to those of pancreatic carboxypeptidases, a hypothetical model of the enzyme active site was developed. The binding of a substrate to carboxypeptidase involves three major interactions (Figure 3). First, the negatively charged carboxylate terminus of the amino acid substrate binds to the positively charged arginine-145 on the enzyme. Second, a hydrophobic pocket in the enzyme provides specificity for a C-terminal aromatic or nonpolar residue. Third, the zinc atom is located close to the labile peptide bond and serves to stabilize the negatively charged tetrahedral intermediate, which results when a molecule of water attacks the carbonyl bond between the C-terminal and penultimate amino acid residues (Harold, 2008). Due to this knowledge, a new generation of substances was introduced being the first of a new class of drugs known as ACEIs.

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FIGURE 3. Structure–activity relationship of ACEIs. Left; interaction of a bradykinin- potentiating factor with ACE binding sites. Right; interaction of a captopril with ACE binding sites

ACEIs are carboxyalkyl dipeptide or tripeptide molecules whose chemistry yields three classes of drugs: the sulfhydryl-containing drugs, which are structurally related to captopril; dicarboxyl-containing drugs related to enalapril, including lisinopril, benazepril, ramipril, and quinapril; and phosphorous-containing drugs related to fosinopril (Ranadive et al., 1992; Mentz et al., 2013).

These compounds effectively block the conversion of ANG-I to ANG-II and have similar therapeutic and physiological effects. The different molecules differ primarily in their potency and PK profiles. Additionally, the sulfhydryl group in captopril is responsible for certain effects not seen with the other agents (Ranadive et al., 1992).

The present research has been developed using three dicarboxylate-containing inhibitors authorized for veterinary use, enalapril, ramipril and benazepril. For that reason, PK and PD discussion will be focused on these three drugs instead of the wide variety of ACEIs available in human medicine. The chemical structure of enalapril, ramipril, benazepril and their actives metabolites enalaprilat, ramiprilat and benazeprilat are shown in figure 4.

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FIGURE 4. Structure of drugs studied in this research. Up; dicarboxyl-containing pro-drugs enalapril, ramipril and benazepril. Down; active metabolites as ACEIs enalaprilat, ramiprilat and benazeprilat, respectively.

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3.5. PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS

3.5.1. PHARMACOKINETIC OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS

ACEIs are commonly used as vasodilator agents normally administered PO due to the prolonged length of treatment regimes. Nevertheless, the oral bioavailability of these drugs is low, and they must be administered as pro-drug components, which are lipophilic with better intestinal absorption. Food does not seem to alter absorption process for most ACEIs (Afonso et al., 2013). The peak plasma concentration of the pro-drug is observed within 30-40 min after administration for most ACEIs in dogs (Toutain and Lefèbvre, 2004).

After intestinal absorption, the pro-drug is transformed into its active metabolite by a carboxyl esterase. The bioavailability of the pro-drugs benazepril and enalapril in the dog ranges from 20 to 40%, as measured with radiolabeled products (Waldmeieir et al., 1989; Morrison et al., 1996). The overall bioavailability of benazeprilat and ramiprilat after PO administration was estimated to a value less than 7% (Toutain and Lefèbvre 2004). Lipophilic properties of the ACEIs may influence the tissue distribution, especially at the brain level, and could theoretically lead to differences in therapeutic effects, although there is no evidence that such differences are clinically relevant in veterinary medicine (Mochel et al., 2015).

Another important point to assess is the tissue distribution of ACEIs and their subsequent actions, which are among the most complex in pharmacology, being influenced by liposolubility, systemic and local binding, enzyme-affinity and, for pro- drugs, presence of local metabolizing enzymes (Toutain and Lefèvbre, 2004). Liposolubility, known as lipophilicity, determines distribution, particularly of the pro- drug, into special tissues such as the brain, where local esterases produce the active metabolite by hydrolysis. Accordingly, more lipophilic pro-drugs such as fosinopril are more likely to affect the central nervous system compared with more hydrophilic pro- drugs such as enalapril (Ferrario and Mullick, 2017). Protein binding is the most complex

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Manuel Gómez Díez property of ACEIs, and its effect and influence on PK-PD relationships is the main characteristic that has developed specific models that will be described later in the next point of this thesis (Lees et al., 1989; Mochel et al., 2014).

Elimination routes also differ between ACEIs. For example, in dogs, enalaprilat is mainly cleared (95%) by the kidney while benazeprilat is cleared by both renal (45%) and hepatic (55%) routes. These differences may result indifferent drug disposition in cases of chronic renal failure. Renal dysfunction has a limited impact on the concentration profiles of ACEIs as benazeprilat and ramiprilat, mainly cleared by hepato-renal routes, while increases the levels of ACEIs which are cleared by the kidney, such as enalaprilat (Lefèbvre et al., 1999; Toutain et al., 2000b). In cats, benazeprilat is excreted predominantly (about 85%) by the liver. Renal dysfunction does not alter benazeprilat pharmacokinetics in this species (King et al., 2002).

Despite the amount of papers published in small animals concerning ACEIs, the studies in equine patients are scarce, although in recent years some studies have been published in horses with enalapril and quinapril (Gardner et al., 2004; Davis et al., 2014), and PD researches with enalapril, quinapril, peridronpril and benazepril (Afonso et al., 2013). In main lines, the PK properties of ACEIs could differ between species, particularly the oral absorption because the of enalapril were lower than that described in dogs (Gardner et al., 2004; Sleeper et al., 2008; Davis et al., 2014). Whether these differences could be related to specific interspecies characteristic in horses is unknown and this is one of the goals of our studies. In fact, absorption and elimination could be different in horses and a consequence the ACE inhibition would also differ, because actives metabolites would not be sufficiently available in blood in comparison with other species as dogs or cats (Mochel et al., 2014). For all these reasons, it is mandatory to investigate the PK and PD properties of ACEIs in the horse.

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3.5.2. PHYSIOLOGICAL COMPARTMENTAL PHARMACOKINETIC PHARMACODYNAMIC MODELS

The PK-PD properties of a drug must be previously established in order to achieve

therapeutic concentrations. Parameters as volume of distribution at steady state (Vss),

clearance (Cl), bioavailability (F%) or half-live (t1/2), among others, must be considered. The majority of drugs used in human and veterinary medicine can be described using conventional PK-PD models. However, a few drugs need special attention because they have different characteristic which can difficult their use, as happen with ACEIs (Toutain and Lefévbre, 2004).

The most important property of ACEIs is their protein binding, which is absolutely different to other drugs, it influences the PK and also the PD and contributes to the complex responses due to the existence of different protein bindings (Toutain et al., 2000a). On one hand, binding to plasmatic proteins, as albumin, is nonspecific and similar to other drugs, yielding an inactive state. On the other hand, specific binding to ACE enzyme results in an active complex which produces pharmacological effects (Lefèvbre et al., 2007). This is very important because nonspecific binding is non- saturable, whereas specific binding is saturable, with this latter effect resulting in nonlinear PK (Levitt and Schoemaker, 2006). In addition, there are two ACE pools, located in circulation and vascular endothelium where ACEIs can bind. Consequently, PK-PD relationships of ACEIs are very complicated because they must take into account all these bindings (King et al., 2003).

From data obtained in humans, it has been proved that conventional PK analysis is inappropriate for ACEIs, because they cannot adequately describe their different bindings to ACE, which influence both PK-PD relationships and dosing regimen designs (Lees et al., 1989; Levitt and Schoemaker, 2006). However, and more importantly, this issue is not specific to humans, also exists for dogs and cats, as has been previously demonstrated (Toutain et al., 2000a; King et al., 2003; Mochel et al., 2014). However, it is unknown in horses.

Complex PK-PD models have been used to describe the time-concentration curves of ACEIs, and have been linked with surrogate markers as ACE inhibition, ANG-II

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Manuel Gómez Díez levels or REN activity, being ACE inhibition the most frequently used (Lees et al., 1989; Mochel et al., 2014). To our best knowledge, these models had not been applied in horses before our investigations, but are required in order to allow interpretation of the different phases of the plasma profiles of ACEIs (Toutain and Lefèvbre, 2004).

When ACEIs are administered orally as pro-drugs, different processes occur, as shown in figure 5. In the left panel, the ACEI disposition curve can be interpreted in terms of classical compartmental analysis, with three phases: absorption, distribution and elimination (metabolism and excretion). In this context, the last phase is only controlled by drug Cl and is known as the classic elimination terminal phase which is represented with t1/2 parameter. However, in the right panel and using a physiologically meaning which include the binding of ACEI to ACE, the understanding is completely different. Firstly, the rapid phase of increase is a combination of pro-drug absorption plus biotransformation to its active metabolite (the true ACEI). Secondly, the next phase is interpreted as a combined distribution and elimination phase controlled by the elimination rate constant k10, which can be influenced by renal and hepatic process (excretion and metabolism). Thirdly, the last phase is not a pure phase of elimination. It is a phase which mainly reflects the binding of ACEI to ACE and is influenced by binding parameters and by the elimination processes, being this last one, the limiting phase. In this third phase, the ACEI is slowly cleaved from ACE, and for that reason, the longer t1/2 observed in these drugs are not the time required to eliminate half the remaining amount of drug in serum, as opposite as what happen for the majority of drugs. In fact, this terminal phase is the time required to cleave to ACE (circulating and tissular) and next, to be eliminated. As a consequence, this phase is controlled by the total binding to

ACE or Bmax, the ACEI affinity to the enzyme or kd, and the elimination processes expressed by the constant rate of drug elimination or k10.(Lees et al., 1989; Toutain and Lefèvbre, 2004; Levitt and Schoemaker, 2006; Mochel et al., 2014).

According to the analysis derived from this type of model, circulating ACE represents approximately 5%, 10%, and 30% of the total ACE pool in dogs, cats, and humans respectively, in equilibrium with ACE from tissues (Lees et., 1989; Toutain and Lefèvbre, 2004). Endothelial ACE is present in many tissues, as central nervous system,

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Angiotensin-converting enzyme inhibitors in the horse but predominates in lungs. ACE bound to endothelium is cleaved to yield circulating ACE by hydrolysis, which is, in turn, bound to circulating plasma proteins to ensure a steady supply of ACEIs (King et al., 2003). Both ACE enzymes, circulating and endothelial, are accessible to ACEIs due to their high affinity. Therefore, the saturable nature of binding to ACE by the ACEI results in a non-linear dose response curve where detectable concentrations do not correlate well to response. Thus, unlike most drugs, the true terminal curve for ACEIs reflects slower elimination at very low, but non-detectable concentrations in many cases, which produce longer t1/2 values (Lees et al., 1989; Mochel et al., 2015).

FIGURE 5. Comparative conventional and non-conventional PK disposition of concentration-time curves of ACEIs. For further explanation, see the text (Figure modified from Toutain and Lefèvbre, 2004)

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However, because bound drug into the terminal phase is the active inhibitor, the complex PK-PD relationship of ACEIs affects the design of a dosing regimen, particularly when adjusting doses for diseases (Toutain et al., 2000a,b; Lefèvbre et al., 2006). This complexity reflects three main properties which differ from those of other drugs. Firstly, the pharmacological response correlates better with tissue concentrations that are not detectable, unlike classic drugs. Secondly, the saturation of tissue enzymes is frequently characterized by a lag time that produce a very flat slope of the elimination terminal, and maximum response may not be evident for several days (Toutain and Lefèvbre, 2004; Lefèvbre et al., 2006). Thirdly, once saturation occurs, further increases in dose are not likely to cause an increase in response, as has been demonstrated in simulations studies (Mochel et al., 2015).

In humans, dogs and cats, different physiologically PK models have been investigated using the ACE inhibition as a surrogate marker of the pharmacological effect (Toutain and Lefèvbre, 2004; Levitt and Schoemaker, 2006; Mochel et al., 2014). The most common model used for clinical purposes is described in figure 6. This is a non- conventional compartment model previously developed by Lees et al. (1989) with the approach of Picard-Hagen et al. (2001), and modified by Toutain and Lefèvbre (2004). The select model includes one compartment for the pro-drug and one compartment for the ACEI directly linked into a direct effect (Toutain and Lefèbvre, 2004). Furthermore, for PO administration, a depot compartment must be included.

ACEI-ACE . PRO-DRUG (1 - fcirc) Bmax (Noncirculating)

ka kd . fcirc Bmax k f ACEI-ACE PRO-DRUG ACEI k d (circulating) Vc Vc

k10p k10

FIGURE 6. General physiology based model for ACEI PO pro-drug administration. For intravenous administration of ACEI, the model is similar without an absorption phase of the pro-drug.

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This model consists on a single compartment with a distribution volume of Vc in which the pro-drug is absorbed with a rate constant absorption ka, and transformed to

ACEI with a biotransformation rate constant kf. Then, the transformation rate is conditioned by ka and kf, respectively. Moreover, pro-drug might be eliminated via other pathways expressed by the parent elimination rate constat k10p. Once it has been formed, the ACEI bound non-specifically to albumin, bound to ACE (circulating and endothelial) or remain free in blood (the free ACEI fraction). However, free and albumin- bound fractions are indistinguishable in these classes of models. Consequently, free ACEI corresponds to free ACEI plus ACEI bound to albumin. Thus, two ACEIs might be considered: free fraction (free and albumin-bound fractions), and bound ACE fraction which are equilibrated between them (Toutain and Lefèvbre, 2004; Mochel et al., 2014).

The binding to ACE is described by three main parameters, kd, or equilibrium dissociation constant which expresses ACEI affinity for the ACE, Bmax, the size of ACE pool, and fcirc, the fraction between circulating and noncirculating ACE pool, where circulating ACE can be calculated as Pmax = fcirc Bmax. (Lefèbvre et al., 2006; Mochel et al., 2014).

Only the free ACEI fraction can be eliminated as excretion or metabolism, and are expressed with the first order rate constant k10. Free ACEI clearance must be calculated as Cl = k10·Vc and the elimination half-live of ACEI as t½k10 = (ln2)/k10 (Toutain and Lefèvbre, 2004; Mochel et al., 2014). After PO pro-drug administration, the volume of distribution Vc/F and clearance Cl/F of ACEI can be calculated, with F indicating systemic oral bioavailability of ACEI after PO administration of pro-drug. The F value can be calculated from the ratio of Cl of ACEI after IV pro-drug and Cl/F of ACEI after PO pro- drug (Toutain and Lefèbvre, 2004).

These models can be presented as differential equations systems and are shown from equation 1 to 3, where Cpro-drugGI is the pro-drug orally administered, Cprod-drugAb is the amount of pro-drug absorbed following PO administration, and Cdrugfree is the free ACEI fraction concentration that can binding to ACE or being eliminated systemically (Lees et al., 1989; Picard-Hagen et al., 2001; Toutain and Lefévbre, 2004).

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= − 푘 ∙ 퐶

(Equation 1)

= 푘 ∙ 퐶 − 푘 ∙ 퐶 − 푘 ∙ 퐶 (Equation 2)

( ∙ ∙ ) = (/)∙ (Equation 3)

 )(C γ   drugfree  EtE 0 1)(  γ  γ   50  drugfree)(C)(IC 

(Equation 4)

For PD data, the observed effect E(t) is defined as the serum ACE activity at each time expressed as percent of control before administration of the pro-drug (E0), IC50 is the free ACEI concentration required to produce 50% of ACE inhibition and γ was the coefficient which describes the steepness of the sigmoid curve (Hill coefficient). Cdrugfree is the free ACEI concentration obtained after PK modeling (Lefèbvre et al., 2006; Mochel et al., 2014). These parameters can be fitted by the Hill equation and are shown in equation 4.

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Other parameters that can be obtained are maximum serum ACE inhibition between 0-12 h (%Imax), time to reach %Imax (T%Imax), and serum ACE inhibition at 12, 24,

48 and 72 h (%I12h, %I24h, %I48h and %I72h, respectively).

Because a long-term therapy is indicated for ACEIs, comparative simulations of PO administrations between minimum to maximum pro-drug doses should be done after single and repeated doses administered daily for several days. Predictions can be performed with parameters obtained with single PO pro-drug dose after PK-PD analysis (Lefèbvre et al., 2006; Mochel et al., 2014).

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3.6. CURRENT KNOWLEDGE OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS IN THE HORSE

The researches regarding ACEI in the horse started with the early paper of Tillman and Moore (1989), where the in vitro suppression of equine serum ACE with enalapril, captopril and enalaprilat was investigated. Further, these authors demonstrated that IV ANG-I administration to six healthy horses produced a dose proportional vasopressor response. Tillman and Moore (1989) induced a mild increase in BP with ANG-I administration and the attenuation of this response was further demonstrated in a single horse by the prior IV administration of enalaprilat at a dose of 8.71 µg/kg.

Some years later, Muir et al. (2001) based their research on the vasodilatory effect of these drugs, previously demonstrated in human beings and dogs. In fact, they initiated their study stating that ACEI drugs produce generalized vasodilation in the aforementioned species, reducing cardiac preload and and decreasing arterial and pulmonary vascular blood pressure (Colson et al., 1999). ACEIs also inhibited the degradation of bradykinin and enhanced bradykinin-mediated release of NO, decreasing peripheral and pulmonary vascular resistance (Colson et al., 1999). More importantly, it was confirmed that ACEI decrease pulmonary capillary wedge pressure (Barra et al., 1997; Colson et al., 1999) and this effect was the starting point of the study of Muir et al. (2001). An increase in this pressure is believed to be responsible for stress-induced failure in pulmonary capillaries (Birks et al., 1994) and it is involved in the pathogenesis of EIPH. Under these premises, Muir et al. (2001) hypothesized that pre-exercise administration of enalaprilat would decrease exercise-induced increases in pulmonary and systemic vascular BPs, and consequently, would limit the clinical importance of EIPH in exercising horses.

In order to test this hypothesis, Muir et al. (2001) administered 0.5 mg/kg of IV enalaprilat prior to a simulated race in 6 horses, and they compared the obtained results with the administration of the same volume of saline solution (0.9% NaCl IV), with both trials separated at least 14 days. They monitored different hemodynamic variables (systolic BP, SBP; diastolic BP, DBP, mean pulmonary arterial pressure, pulmonary

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Manuel Gómez Díez capillary wedge pressure, right ventricular systolic pressure, right ventricular end- diastolic pressure, right ventricular positive maximal rate of change, mean , cardiac output and systemic vascular resistance) and blood gas values in arterial and venous blood (pH, oxygen and carbon dioxide tension, bicarbonate, base excess, packed cell volume) at fixed times: before and immediately after the simulated races and at 35, 35, 60 and 90 min after. Muir et al. (2001) found that IV enalaprilat inhibited ACE activity to reach values lesser than 25% of the baseline activity, but it did not change cardiorespiratory or blood gas values during and after exercise in the medicated horses, compared with horses that had received saline solution. These results led the authors to conclude that, although IV enalaprilat almost completely inhibited ACE activity in horses, the lack of changes in the hemodynamic response to intense exercise made this drug unlike to be of clinical value in preventing EIHP (Muir et al., 2001).

Several years later, and taking into account the results of Muir et al. (2001), Gardner et al. (2004) thought that a potential indication for the use of ACEI in the horse would be the medical management of laminitis (Brown and Vaughn, 1998). This disorder was associated with hypertension, tachycardia, activation of the RAAV axis (high ALD concentrations, increased REN activity and hypokalemia) and vasoconstriction of the digital venules (Amoss et al., 1988; Allen et al., 1990; Moore et al., 1991). Therefore, Gardner et al. (2004) thought that ACE inhibition might improve pedal blood flow and be useful in the prevention and treatment of laminitis. More specifically, they based their research on previous studies that showed: 1) an activation of the RAAV axis in the laminitic horse; 2) a vasodilation in the post-capillary venules induced by enalapril; 3) an improvement in blood supply to the equine foot with the administration of other vasodilators (Harkema et al., 1978; Hinckley et al., 1996). In addition, Gardner et al. (2004) emphasized the importance of study the utility of PO enalapril, because of the necessity for chronic therapy.

Gardner et al. (2004) carried out three experiments. In the experiment 1, they administered a dose of IV enalapril at 0.5 mg/kg and they took blood samples at fixed times immediately after and during the following 24 h after administration, in order to

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Angiotensin-converting enzyme inhibitors in the horse measure serum enalapril and enalaprilat concentrations. In the experiment 2, performed two weeks after, Gardner et al. (2004) administered PO enalapril at the same dose (0.5 mg/kg), and took blood samples and measured SBP, DBP and mean BP (MBP) at the same times than in experiment 1. In this second experiment, they also measured serum ACE activity, serum K+, creatinine and blood ureic nitrogen (BUN) concentrations, blood gases and lactate. In the experiment 3, they administered PO enalapril at 0.5 mg/kg, and they provided IV ANG-I (dose: 0.5 µg/kg) at different times, in order to mimic an activation of the RAAV axis. These researchers wanted to know whether enalapril administration at time 0 would blunt the hemodynamic response to ANG-I in the horse.

In the experiment 1, enalapril and enalaprilat dispositions were both best described by two-compartment models, with terminal t1/2 of 0.86 and 2.01 h respectively. In the experiment 2, they found that serum concentrations of enalapril and enalaprilat were below the limit of quantification (LOQ), with the exception of one horse, in which measurable enalaprilat concentrations were found 45 min after drug administration. In humans, the t1/2ʎz of enalapril is about 2 h, and enalaprilat elimination is biphasic, with an initial phase that reflects renal Cl, with a t1/2 of about 4 h and a protracted phase that reflects saturable binding to tissue and plasma ACE with t1/2ʎz of approximately 35 h (Ulm et al., 1982; Till et al., 1984; Biollaz et al., 1985; MacFadyen et al., 1993). Because of ACE binding, enalaprilat disposition might be best described in humans by a physiologically based model (Lees et al., 1989; Donnelly et al., 1990; Meredith et al., 1990; Reid and Meredith, 1990; MacFadyen et al., 1993). This model was also used to describe enalaprilat disposition in dogs (Toutain et al., 2000a,b). However, it was not used in the study of Gardner et al. (2004) because drugs serum concentrations were below the LOQ. Therefore, they concluded that systemic availability of enalapril was poor after PO administration of 0.5 mg/kg once.

In the second experiment, they found a non-significant trend towards a reduction in serum ACE activity at 45 min and 1 and 2 h after PO enalapril administration. In fact, ACE activity was never suppressed by more than 16%. Previously, Tillman and Moore (1989) reported that ACE activity should be suppressed by 91% for substantial suppression of the pressor response in the horse. The vasopressor response was

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Manuel Gómez Díez decreased by 50% at enalaprilat concentrations of 52 ng/ml. Serum enalaprilat concentrations required for 50% ACE inhibition appear to be similar for horses, dogs, and humans (Abrams et al., 1984; Biollaz et al., 1985; Toutain et al., 2000b). Studies in dogs showed that approximately 75% ACE inhibition was associated with clinical benefits in patients with heart failure, with 0.5 mg/kg producing approximately 75% maximum ACE suppression (The COVE Study Group, 1995; The IMPROVE Study Group, 1995; Hamlin et al., 1996; The LIVE Study Group, 1998; The BENCH Study Group, 1999). In human beings, it was recommended that serum enalaprilat concentrations of 10 ng/ml at least should be reached in order to obtain the maximal blockade of ANG conversion (Biollaz et al., 1985).

Gardner et al. (2004), in their second experiment, observed also a tendency toward a decrease in MBP 6 and 8 h after PO enalapril administration. In human beings, enalapril lowers BP in normotensive individuals in a dose-related fashion without increasing heart rate (MacGregor et al., 1981; Millar et al., 1982; Shoback et al., 1983). In humans, enalapril produces small increases in serum K+ concentrations, associated with a decrease in urinary K+ excretion and no change in glomerular filtration rate (Davies et al., 1984). Gardner et al. (2004) reported that serum concentrations of K+, creatinine, BUN, venous blood gases and lactate concentrations did not change during the experiment.

In the experiment 3, Gardner et al. (2004) found a tendency toward a decrease in MBP at 4-24 h post-administration of IV ANG-I and PO enalapril, but statistical significance was not achieved. In human volunteers, enalapril inhibited the vasopressor response to exogenously administered ANG-I (Biollaz et al., 1981).

All these data led to Gardner et al. (2004) to the conclusion that systemic availability of enalapril was poor after PO administration, with minimal effects on ACE inhibition, hemodynamic properties and serum K+ concentrations. Therefore, it was hypothesized that higher doses would be necessary to achieve the effects required for clinical benefits in the horse.

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The first clinical study with a large number of horses receiving ACEI was made in the School of Veterinary Medicine of Hannover, in Germany. In 2003, Gehlen et al. evaluated the effect of quinapril in 20 horses with mitral valve insufficiency, but without signs of clinical heart failure, as well as 5 horses without signs of heart disease. They administered a PO dose rate of 120 mg/horse/day of quinapril for 8 weeks. Gehlen et al. (2003) only performed a clinical study, based on echocardiographic measures, and therefore, ACE inhibition, serum concentrations of quinaprilat and quinapril were not measured, and PK-PD studies were not performed. These authors found a statistically significant increase in stroke volume and cardiac output, together with a decrease in regurgitation velocity time integral, but the regurgitation blood velocity remained unchanged. The severity of mitral valve insufficiency underwent a mild to moderate improvement in 5 horses after therapy. Changes in cardiac dimensions and shortening fractions were not found after treatment. In the healthy group, no significant changes were observed after treatment.

Luciani et al. (2007) evaluated the hemodynamic effects of quinapril in healthy horses, administering 500 µg/kg of ramipril PO daily for 5 consecutive days. In this study, food was withheld 12 h before drug administration. The article was based on heart rate and BP changes. It was found a decrease in BP each day, during the first 6 h following administration of ramipril. Heart rate did not change during the experiment.

By this time, enalapril was very expensive and because of this reason, enalapril therapy could have been avoided in many clinical cases in horses. Considering the economic constraints, the most likely candidate for ACEI therapy would be the aged breeding stallion with valvular insufficiency that might gain additional breeding seasons with the therapy of these drugs. However, erectile dysfunction has been reported as side effect linked to enalapril therapy in men (Montvale, 1994) and this could limit the use in breeding animals. Under this premise, Sleeper et al. (2008) evaluated the effects of a 2-month course of PO enalapril treatment on erection and ejaculatory function in normal stallions and further, they also evaluated the effect of chronic PO enalapril treatment.

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Sleeper et al. (2008) studied 12 Shetland pony stallions divided into two groups: control or placebo (n=6) and enalapril group (n=6), which received 0.5 mg/kg PO daily of this ACEI for 2 months with the food. After one moth of treatment, blood samples were collected at defined times for 48 h in order to measure serum ACE activity. In addition, and because of their proposed aims, sexual function and semen output were evaluated and echocardiography was made. No significant differences in serum ACE activity over time were found. Echocardiographic parameters neither change and reproductive function was not different between placebo and enalapril group. Sleeper et al. (2008) suggested that PO enalapril was either not absorbed adequately by the equine , underwent intestinal degradation or was inadequately converted to enalaprilat, but the design of the research avoided the selection of the most probable hypothesis.

This was the state-of-art when we started our researches in 2010. From this moment, additional papers have been published concerning this subject. In the present review, we will only present the studies performed by authors different from our group. In this way, the most relevant papers regarding ACEI in horses have been published in the last 5 years, coinciding temporally with the researches carried out by our group. These investigations would be described following a chronologic order.

Afonso et al. (2013) considered that the lack of data regarding the relative potency of PO ACEI in the horse was a major problem that prevented the rational use of these agents in equine patients with CHF and/or heart disease. In their study, they made a comparison of the relative potency of benazepril, quinapril, perindorpil and ramipril in terms of their ability to inhibit serum ACE activity. The study was divided into three different experiments. In their first experiment, Afonso et al. (2013), compared different doses (low and high) of each drug (benazepril: 0.25 and 0.5 mg/kg; perindopril: 0.05 and 0.1 mg/kg; quinapril: 0.125 and 0.25 mg/kg; ramipril: 0.1 and 0.3 mg/kg). These dosages were extrapolated from the low and high ends of the dosage regimen recommended for people (perindopril; López-Sendón et al., 2004), dogs (benazepril and ramipril; Lefèbvre et al., 2007) and horses (quinapril; Gehlen et al., 2003). In all the cases, the 8 used horses were fasted overnight before administration. Afonso et al. (2013) took blood samples

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Angiotensin-converting enzyme inhibitors in the horse and measured SBP, DBP and MBP. A significant effect of the drug and the dose in ACE maximal inhibition was found. The ACE inhibition was greater for benazepril (86.9 and 72.2%), followed by ramipril (59.7 and 38.2%), quinapril (40.6 and 29.6%) and perindopril (25.2 and 19.9%), for high and low doses respectively. According to their results, only PO benazepril resulted in a maximal inhibition greater than 60%. The authors highlighted that the minimum circulating ACE inhibition that is required to improve life quality and survival in equine patients with CHF is unknown. Another interesting result of this paper was that for the four ACEIs tested, ACE inhibition was always greater after administration of the high dose compared to the low dose. This result suggested that serum inhibition might be dose-dependent. However, several studies in dogs showed that this dose-dependent effect reaches a plateau at higher doses (Desmoulins et al., 2008), finding that was associated with their saturable binding ability to ACE enzyme (King et al., 2003).

One aspect to highlight is that the different ACE inhibition of each drug could have been partially associated with the different doses administered. In this way, the lower maximal inhibition caused by PO perindopril could have been linked by the lower dose used, because the higher dose of perindopril was similar to the lower dose of quinapril and ramipril. Similarly, the greater activity of benazepril might be caused by the higher dose used, even though the ACE maximal inhibition was greater than for quinapril at equivalent doses of both drugs.

Despite the significant difference in this ACE inhibition between drugs and doses, each drug and dose combination caused a similar reduction in BP. This decrease was of minor intensity (only 5-10 mm Hg), observed between 1 and 6 h after administration. The magnitudes and times of reduction in BP showed by Afonso et al. (2013) were similar to those reported by (Lefèbvre and Toutain, 2004) in dogs. However, it should be taken into account that Afonso et al. (2013) only measured BP until 8 h after ACEI administration. Because of these results, Afonso et al. (2013) initially questioned the clinical usefulness of these drugs. However, a decrease in systemic vascular resistance is only one of the expected beneficial effects of these drugs. As a consequence, the lack

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Manuel Gómez Díez of significant differences in the reduction of BP between drugs cannot be taken as an evidence of equivalent clinical efficacy.

The second experiment carried out by Afonso et al. (2013) aimed to assess the influence of feeding on the PD properties of benazepril at a dose of 0.5 mg/kg. The experimental procedure was the same than in the first experiment, but the horses had free access to grass hay and water before and after administration of the drugs. In agreement with the results of previous studies performed in cats (Toutain and Lefèbvre, 2004), feeding did not appear to affect the PD properties of benazepril.

In their third experiment, Afonso et al. (2013) administered low (0.25 mg/kg) or high (0.5 mg/kg) dose of PO benazepril once a day for 7 days, in order to investigate PDs of this drug after repeated PO administration. According to the relative long t1/2ʎz (18.6 h) and high inhibition at 24 h (54.0%), obtained after PO benazepril, PD modeling would have predicted a considerable cumulative effect of repeated daily administration on ACE inhibition. However, Afonso et al. (2013) found a significant decrease in the inhibition at

24 h and AUC0-24 (Area under the curve from 0 to 24 h) from day 1 to day 7 of daily administration. Previous studies in people and dogs demonstrated that minimal or no accumulative effects on ACE inhibition after repeated daily administration (King et al., 1995; Harder et al., 1998; Toutain and Lefèbvre, 2004; Desmoulins et al., 2008).

The main conclusions of the study of Afonso et al. (2013) were those: 1) Administration of benazepril caused a significant greater serum ACE inhibition compared to the other three ACEI (quinapril, ramipril and perindopril) at the studied doses; 2) Feeding did not appear to influence PD properties of benazepril and 3) Administration of multiple daily doses of PO benazepril did not result in a cumulative effect on serum ACE inhibition.

In 2014, Davis et al. studied more deeply the PK and PD properties of quinapril. They considered that, from the studied ACEI in the horse, ramipril and quinapril might provide scientific evidences for their use to treat CHF, because of their promising PO bioavailability. These authors stated that incomplete or inadequate pharmacological data limit the clinical use of ACEI in the horse. Therefore, with their study, Davis et al.

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(2014) aimed to demonstrate that quinapril was converted sufficiently to its active metabolite, quinaprilat, leading to a significant ACE inhibition and as a consequence, REN activity, the first point of the RAAV axis, would increase.

Davis et al. (2014) used a similar experimental design to the previously described papers. In fact, they performed 3 trials, administering IV quinapril (120 mg/horse), PO quinapril at low doses (120 mg/horse, corresponding to doses between 0.196 and 0.244 mg/kg) and PO quinapril at high doses (240 mg/horse, corresponding to doses between 0.392 and 0.488 mg/kg). They obtained blood samples from 10 min to 24 h post- administration, measured ACE inhibition, REN activity, quinapril and quinaprilat concentrations and they also measured BP at the same times. ACE activity decreased significantly from baseline at 30 and 60 min after 120 mg IV quinapril, as expected, with a maximum inhibition of 72.51% reached at 30 min. The maximum inhibition of this enzyme was reached at 1 h after PO administration, with values of 47.11% and 53.43% for the low and high doses respectively. The ACE inhibition caused by 120 mg PO of quinapril had been previously shown to have a positive clinical effect in horses with mitral regurgitation and atrial fibrillation (Gehlen et al., 2003, Goltz et al., 2009). However, the mild increase in ACE inhibition with the high dose, compared to the low dose, might suggest that 240 mg of quinapril per horse would be more effective in horses with chronic activation of the RAAV system.

REN concentrations (or activity, depending on the used assay and authors), typically increases after ACEI therapy. This effect is due to a diminished ALD synthesis and release together with a lack of a direct negative feedback of ANG-II on REN release (Seifarth et al., 2002). Davis et al. (2014) did not find significant changes in circulating REN activity in the horse after IV or PO quinapril. Similarly, these authors did not observe a significant effect on BP. The authors attributed their results to an inadequate dosing period. In fact, according to the results of Luciani et al. (2007), a significant reduction in BP is not seen until 4 days of daily administration of quinapril, whereas Davis et al. (2014) measured BP only for 24 h after one only PO dose of quinapril. As previously other authors did (Afonso et al., 2013), Davis et al. (2014) also suggested that the decrease in BP would not occur unless the horse is hypertensive, with CHF or with a RAAV activated.

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In relation to PK studies, Davis et al. (2014) reported that IV quinapril had a short t1/2, with a maximum concentration Cmax of 144.99, occurring 10 min after quinapril administration, moderate volume of distribution and high Cl. However, is necessary to note that serum pro drug and drug concentrations were described using non- compartmental analysis instead of physiologically nonlinear PK models, as has been recommend (Toutain and Lefèbvre, 2004; Mochel et al., 2014). Despite these differences, oral bioavailability of quinapril was low (less than 5%). In man and other species, a significant amount of the drug uptake is believed to be via peptide carrier- mediated transport (PEPT) (Rubio-Aliaga and Daniel, 2002). However, the importance and influence of these proteins into the mediated ACEI absorption has been questioned, especially because both quinapril and quinaprilat show low affinities for these transporters (Knütter et al., 2008). Also, recent studies have suggested that these transporters could also be present in horses (Cehak et al., 2013), but in low quantities (Davis et al., 2014). Thus, Davis et al. (2014) speculated that the horses lack these transporters or alternatively, they might have in very low quantities. Whether or not these proteins are related to ACEI absorption in horses is unknown at this moment.

After PO quinapril, quinaprilat showed a prolonged terminal t1/2 (mean of 8.57 h), using a traditional non-compartmental modeling approach, although Davis et al. (2014) stated that it is unlikely that this model represents the terminal elimination phase of the metabolite. According to previous works with ACE inhibition in other species, the prolonged t1/2 with the traditional modeling procedures was due to the fact that the measured concentrations were the sum of the free drug concentration, drug bound non- specifically to plasma proteins (albumin mainly) and drugs specifically bound to circulating ACE (Toutain and Lefèbvre, 2004). Therefore, physiologically based models would be more interesting.

The last study concerning ACEI in horses has been published very recently (Afonso et al., 2017b). Although inhibition of circulating ACE activity is a commonly used surrogate marker to investigate the activity of ACEI (Gardner et al., 2004; Gómez-Díez et al., 2014; Afonso et al., 2013; Davis et al., 2014; Serrano-Rodríguez et al., 2016; 2017), circulating ACE concentrations only represents approximately 5-30% of the total body

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ACE pool, depending on the species (Kaplan, 2012). Consequently, measuring of circulating ACE inhibition might not be an accurate reflex of the effects of these drugs on the large tissular ACE pool (Toutain et al., 2000a, benazeprilat; Toutain and Lefèbvre, 2004). In normotensive people, measurement of the percentage of attenuation of the pressure response to a dose of exogenous ANG-I that increases SBP by 20-30 mm Hg is considered the best method for assessing the predicted efficacy on an ACEI (Biollaz et al., 1981; Brunner et al., 1983; 1994).

Because of these ideas, Afonso et al. (2017b) tested the lowest dose of benazepril that would result in a maximal attenuation in the response of SBP to exogenous ANG-I. They tested four PO benazepril doses: 0.5, 1, 2 and 4 mg/kg. ANG-I were administered IV before benazepril, in three incremental doses, in order to select the dose that would increase SBP by 20-30 mm Hg approximately. The three ANG-I concentrations (20, 60 and 200 ng/kg) resulted in significant rises in SBP (20.8, 38.8 and 52.6 mm Hg respectively).

The maximal ACE inhibition obtained in this study after administering IV ANG-I at a dose of 20 ng/kg were 19%, 47%, 43% and 56%, after 0.5, 1, 2 and 4 mg/kg of PO benazepril. It has been reported that the response of BP to exogenous ANG-I with the administration of ACEI is dose dependent until reaching a plateau (Biollaz et al., 1981; Brunner et al., 1983). As the results of Afonso et al. (2017b) appeared to indicate, PO benazepril at 2 and 4 mg/kg did not lead to greater ACE inhibition, compared with the lowest doses (0.5 and 1.0 mg/kg PO benazepril).

Despite Afonso et al. (2017b) used doses of benazepril up to 4 mg/kg, an attenuation of the rise in SBP greater than 75% was not achieved, being this the upper range of those labeled for use in the treatment of hypertension in people, dogs and cats (Buchwalder-Csajka et al., 1999). However, enalapril, benazepril and lisinopril have been shown to be clinically effective at the lowest labeled dose in people with CHF despite a maximum inhibition of only 45 to 60%. Afonso et al. (2017b) thought than this result was unlikely to be a simple consequence of the lower oral bioavailability in horses compared to other species, because the maximum inhibition of ACE exceeded 90% at a dose of 1 mg/kg, and was nearly 100% with doses of 2 and 4 mg/kg. Therefore, the maximum

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4. BRIEF DESCRIPTION OF THE STUDIES

The following studies have been financially supported by the Research Project AGL2010-17431 (Spanish Government; Research Council)

This research has been approved by the Ethical Committee for Animal Experimentation of the University of Córdoba (Reference: 55.60 PE, date of approval: 1 February 2010).

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4.1. DESCRIPTION OF THE STUDY I

TITLE: PHARMACOKINETICS AND PHARMACODYNAMICS OF ENALAPRIL AND ITS ACTIVE METABOLITE, ENALAPRILAT, AT FOUR DIFFERENT DOSES IN HEALTHY HORSES

4.1.1. JUSTIFICATION The first study was based on the research of Gardner et al. (2004), where PKs of a dose of enalapril (0.5 mg/kg IV) and PDs of a dose of enalapril (0.5 mg/kg PO) were investigated in 5 mares. These authors measured enalapril and enalaprilat concentrations and ACE at fixed times after that. In this research, if was found that elimination t1/2 of enalapril and enalaprilat were 0.59 and 1.25 h respectively after IV administration. After PO administration, enalapril and enalaprilat were not detectable in serum. Although there was a tendency toward a decrease in ACE activity 45-120 min after enalapril administration, ACE activity was never suppressed more than 16%. In healthy people, serum ACE activity must be reduced by approximately 90% from baseline values to completely abolish BP increase in response to exogenously administered ANG-I (Burnier et al., 1981; Biollaz etal, 1985). The minimum percentage of ACE inhibition to be clinically beneficial in veterinary patients needs to be elucidated. Some studies conducted in dogs have shown that approximately 75% maximum suppression of ACE after PO enalapril at 0.50 mg/kg is associated with clinical benefits in patients with heart failure (Hamlin et al., 1996; Hamlin and Nakayama, 1998; The IMPROVE Study Group, 1995; The LIVE Study Group, 1998). Lower ACE inhibitions (63%) have been linked with extended life span in dogs with CHF (The COVE Study Group, 1995; The BENCH Study Group, 1999).

Therefore, the study of Gardner et al. (2004) demonstrated that a single dose of enalapril at 0.5 mg/kg PO did not have significant availability, PD effect or cause substantial suppression of ACE activity. However, it should be taken into account that these authors only test a dose (0.5 mg/kg). The administration of higher ACE doses might lead to higher bioavailability and greater ACE inhibition, being this hypothesis the beginning of our research.

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4.1.2. OBJECTIVES

The objectives of this study were: 1) To establish and to compare the serum concentration-time profile of enalapril and its active metabolite (enalaprilat) in healthy horses after IV administration of a single dose of enalapril at 0.5 mg/Kg and PO administration of a single dose with different concentrations, 0.5, 1.0 and 2.0 mg/Kg; 2) To determine the PO bioavailability of enalapril at the three mentioned doses; 3) To investigate their effects on serum ACE activity and BP and 4) To select the most appropriate PO dose to potentially use in pathologic situations in horses.

4.1.3. MATERIAL AND METHODS Seven cross-bred adult healthy horses (2 mares and 5 geldings) were used in this study following a cross-over design. Each animal received enalapril at 0.5 mg/Kg IV and 0.0 (placebo), 0.5, 1.0 and 2.0 mg/Kg PO by a nasogastric tube with a minimal washout period of 15 days between each experiment.

SBP and DBP were measured non-invasively in the coccygeal artery. Blood samples were obtained at different intervals and serum was separated and stored frozen until analysis. Serum ACE activity, serum BUN concentration and creatinine concentration were measured using spectrophotometry. Serum concentrations of enalapril and enalaprilat were analyzed by liquid chromatography.

The serum concentration-time data of enalapril and enalaprilat and the serum ACE activity were analyzed using non-compartmental methods. Analyzed parameters included: elimination rate constant, mean residence time (MRT), serum initial

concentration of enalapril, Cmax, time to reach Cmax (Tmax), Vss, area under the response

curve (AURC), serum ACE inhibition at the different sampling times, Imax, Tmax, minimum

SBP (SBPmin), minimum DBP (DBPmin), time to reach SBPmin (TSBPmin), time to reach DBPmin

(TDBPmin), SBP at maximum serum ACE inhibition (SBPImax) and DBP at maximum serum

ACE inhibition (DBPImax).

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4.1.4. BRIEF DESCRIPTION OF THE RESULTS

In this first study, as well as happened in the other articles compiled in this thesis, no local or systemic reactions were detected during or after IV or PO administrations.

Serum enalapril and enalaprilat concentrations after IV administration of 0.5 mg/kg of enalapril reached the highest values immediately after administration and suffered a rapid decrease. Enalapril concentrations after PO administrations were below LOQ (10 ng/ml) in all horses. Enalaprilat concentrations were also below LOQ in four horses. Two horses had measurable concentrations of enalaprilat 1 h after PO administration of enalapril at 0.5 mg/kg (12.33 and 11.55 ng/ml) and another horse, a concentration of 16.14 ng/ml at 2 h after PO enalapril at 2.0 mg/kg.

The elimination t1/2 of enalapril and enalaprilat were 0.67 and 2.76 h respectively

after IV enalapril. The Cl, Vss, AUC0→∞ and AUMC0→∞ values of IV enalapril, and enalaprilat formed after IV enalapril fell in the range to the data reported by Gardner et al (2004). However oral bioavailability F% could not have been calculated due to the lower concentration detected.

Regarding ACE concentrations, the maximal inhibition (Imax) was found after IV

administration of enalapril at 0.5 mg/kg (mean values: 88.38%). Imax values of 21.69%, 26.11% and 30.19% were observed after administration of PO 0.5, 1.0 and 2.0 mg/kg. No significant differences were found in SBP and DBP at any of the recording times. Similarly, significant differences in BUN, creatinine and electrolyte concentrations were not found when comparing routes, doses and sampling times. In all the cases, moreover, concentrations were within the reference range used in the laboratory for horses. Range values were those: for serum BUN (20.00-43.00 mg/dl; mean: 34.80 mg/dl); creatinine (0.50-1.80 mg/dl; mean: 1.23 mg/dl), Na+ (139-147 mmol/l; mean: 141.6 mmol/l), K+( 3.4-5.2 mmol/l; mean: 3.99 mmol/l) and Cl- (91-99.90 mmol/l; mean: 99.90 mmol/l).

Our results confirmed the low bioavailability of PO enalapril in the horse even when administered at four times the dose that has been traditionally used in humans and small animal species.

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4.1.5. CONCLUSIONS The PO availability of enalapril at doses comprised between 0.50 and 2.00 mg/kg was low. Our study suggested that, although there was a trend towards a decrease in serum ACE activity, these values were never suppressed by more than 30% when enalapril was administered PO. These results suggest that oral absorption of enalapril was very low and dose independent. However, a marked inhibition (88.38%) was found after enalapril IV. To date, it is unknown where an ACE inhibition near 30%, as found in the current article, with a dose of enalapril PO at 2.0 mg/kg could be enough to counteract the vasoconstrictive effects of the RAAV axis in horses with hypertension and/or CHF.

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4.2. DESCRIPTION OF THE STUDY II

TITLE: PHARMACOKINETICS AND PHARMACODYNAMICS OF RAMIPRIL AND RAMIPRILAT AFTER INTRAVENOUS AND ORAL DOSIS OF RAMIPRIL IN HEALTHY HORSES

4.2.1. JUSTIFICATION

PK and PD studies on ACEIs in the horse have been limited to enalapril (Gardner et al., 2004; Gómez-Díez et al., 2014). Previously, a clinical report of a horse with pan- valvular insufficiency, atrial fibrillation and CHF, treated with PO ramipril at a dose of 50 µg/kg, was published (Guglielmini et al., 2002). The animal experienced a clinical improvement and a reduction of SBP and DBP. However, although the authors associated this decrease in BP with ramipril administration, the horse also received diuretics as a part of the treatment (furosemide). Some years later, Luciani et al. (2007) administered ramipril PO at 200 µg/kg in healthy horses for 5 days and they found mild decreases in MBP, SBP and DBP. According to these authors, ramipril might be a promising tool for the treatment of CHF in this animal species.

Some years later, Afonso et al. (2013) evaluated serum ACE inhibition and changes in BP after the administration of two PO doses of ramipril in horses (low dose, 0.1 mg/kg; high dose, 0.3 mg/kg) compared to other three ACEI (benazepril, quinapril and perindopril). In this article, only two doses of ramipril were investigated, IV ramipril was not administered and serum concentrations of ramipril and ramiprilat were not measured. The changes found in serum ACE inhibition could have derived from differences in oral bioavailability, differences in conversion from the to the active metabolite in the liver, differences in the ability of each drug to inhibit circulating ACE or a combination of these factors (Afonso et al., 2013). The most probable hypothesis could not have been confirmed because of the absence of data regarding serum concentrations of ramipril and ramiprilat. Therefore, we felt that more information would be needed about this drug in the horse and that was the main motivation for this second article.

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In addition, in human beings and small animals, PK and PD studies of ramipril were made following traditional non-compartment modeling approaches. These approaches are complicated because ACEI drugs have a non-linear binding to ACE, and because there are two ACE pools, circulating and tissular. Therefore, we suggested that physiologically based models considering binding parameters of ACEI to ACE would allow more appropriate interpretation of the different phases of disposition curves, being this one another reason for our study. Moreover, by means of the use of these physiological models, the effect of multiple dosing regimens might be predicted.

4.2.2. OBJECTIVES The objectives of the present study were: 1) To establish the serum concentration–time profile for ramipril, and its active metabolite ramiprilat after IV ramipril at 0.05 mg/kg, PO ramipril at 0 (placebo), and five different PO doses (0.05, 0.10, 0.20, 0.40 and 0.80 mg/kg), using a physiologically based PK/PD model; 2) To determine the effects of ramiprilat on serum ACE; 3) To investigate the effects on BP with these doses and routes; and 4) To predict and to simulate the most appropriate PO dose that suppresses ACE activity after single and multiple doses of ramipril.

4.2.3. MATERIAL AND METHODS

Six cross-bred adult healthy horses (2 mares and 4 geldings) were used for this study, which was divided into two trials. The first trial consisted in the administration of IV ramipril at 0.05 mg/kg. The second trial, performed after a week of washout, was made following a randomized Latin square (6x6 design), in which each animal received PO placebo or ramiprilat at 0.05, 0.1, 0.2, 0.4 or 0.8 mg/kg with at least 1 week of washout between trials. Venous blood samples were taken and SBP and DBP were measured at fixed times: before administration (time 0) and at 5, 10, 15 and 30 min and at 1, 2, 4, 8, 12, 24, 36 and 48 h after each administration. Serum ACE activity was measured by spectrophotometry, and serum concentrations of ramipril and ramiprilat were measured using a liquid chromatography-tandem mass spectrometry.

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For each animal, concentration-time data of ramipril and ramiprilat were analyzed simultaneously using a physiologically based compartment model (Lees et al., 1989; Toutain et al., 2000a; Lefèbvre et al., 2006). This model is described in the article

II and briefly, it consisted in a single compartment model, with a Vc, in which ramipril is absorbed and transformed to ramiprilat following first-order rate constants of ka and kf of absorption and formation respectively. In addition, a constant k10p was included, because ramipril might be eliminated via other pathways, as urinary excretion and biotransformation to other metabolites (Van Griensven et al., 1995). Ramipril is bound to ACE or to albumin. However, free and albumin-bound fractions cannot be distinguished by this physiological model. As a consequence, free ramiprilat corresponded to free ramiprilat plus ramiprilat bound to albumin (Lefèbvre et al., 2006). A graphical representation of this model is presented in the published article 2 (Fig. 1).

In this study, the following variables were calculated: Vc/F (volume of distribution of ramiprilat), Cl/F (clearance of ramiprilat), and F (systemic bioavailability of ramiprilat after PO administration of ramipril). In addition, these binding parameters of ramiprilat to ACE were calculated for the above described physiological model: kd (equilibrium dissociation constant which expresses ramiprilat affinity for ACE), Bmax (size of ACE pool), fcirc (fraction between circulating and non-circulating ACE) and circulating ACE (Pmax=fcirc·

Bmax (Lefèbvre et al., 2006)).

Other parameters included in this second study were these: E(t) (serum ACE activity at each time expressed as a percentage of control), IC50 (free ramiprilat concentration that induced a 50% of ACE inhibition), maximum serum ACE inhibition between 0 and 12 h (%Imax), time to reach it (T%Imax) and serum ACE inhibition at fixed times (%I12h, %I24h and %I48h).

Moreover, and because of the necessity of a chronic PO therapy of these drugs, simulations were made after oral ramipril from 0.0063 to 6.30 mg/kg with single and repeated doses every 24 h for a week. These predictions were obtained with PK/PD parameters derived from a representative horse after PO ramipril at 0.8 mg/kg, and the effect-time relationship and the influence of the dosing were analyzed (D’Argenio et al., 2009).

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4.2.4. BRIEF DESCRIPTION OF THE RESULTS

Serum concentrations of ramipril and ramiprilat after PO ramipril at 0.05 and 0.10 mg/kg were not detected in blood (concentrations were below LOQ with exception of some sampling times). However, measurable concentrations were found after 0.2 mg/kg PO ramipril but were not enough to be modulated with the PK-PD model. IV and PO concentrations from 0.40 to 0.80 mg/kg dose were analyzed and interesting

parameters were obtained. Thus, kf values close to 4.78-6.70 1/h were obtained showing a fast biotransformation from ramipril to ramiprilat and were not influenced by dose

and route. Regarding binding values, kd fell in the range described by ramiprilat in human

and small animals whereas Bmax and fcirc values were similar to dogs but lower than cats and humans suggesting that fraction between ACE pools could be different between species as has been indicated in other studies (Toutain and Lefèvbre, 2004). More importantly, the oral bioavailability was low, between 6 and 9%, lower than the values obtained in humans and small animals. However, ramipril had sufficient enteral absorption and bioconversion to ramiprilat to induce high and long-lasting serum ACE inhibitions, particularly at 0.8 mg/kg dose (Figure 3 from the paper) (Lefèvbre et al., 2006; Levitt and Schoemaker, 2006).

The PD parameters described by the IC50 and 훾 values revealed two important results: ramiprilat have high potency to inhibit ACE pool with values close to 0.88-1.07 nmol/l, and inhibition was long lasting due to the γ values, close to 0.60, indicating that a relative shallow concentration effect relationship and that some residual inhibition can be obtained for very low ramiprilat concentrations, particularly at 0.80 mg/kg of ramipril (Figure 3 from the paper).

Mean percentages of 98.88, 27.68, 39.27, 46.67, 76.13 and 84.27 for %Imax were found after 0.05 mg/kg IV and 0.05, 0.10, 0.20, 0.40 and 0.80 mg/kg PO of ramipril respectively. The time to reach these maximal inhibitions ranged between 0.21 h (for IV ramipril) and 2.50 h (for PO ramipril at 0.80 mg/kg). Maximum serum inhibitions at 12, 24 and 48 h showed values of 74.53, 63.54 and 38.01% for PO ramipril at 0.80 mg/kg. SBP, DBP did not vary during the experiments.

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After simultaneous simulations of concentration and serum ACE inhibition from PO ramipril at 0.0063 to 6.30 mg/kg for a week we observed two important effects. Firstly, values from 0.0063 to 0.20 mg/kg produced less dose-related serum ACE inhibition and the maximum effect were not reached for a week, and therefore, longer times would have been required. Secondly, results achieved from 0.80 to 6.30 mg/kg were very similar and faster to reach a steady state from the first administration predicting that an initial dose between 0.80 to 1.6 mg/kg could be administered to immediately inhibit ACE followed by the same maintenance doses every day (Figure 4 from the paper). These results are in agreement with the data obtained in humans and dogs (Lefèvbre et al., 2006; Levitt and Schoemaker, 2006).

4.2.5. CONCLUSIONS

The current study demonstrated that PO availability of ramipril, when administered at doses comprised between 0.05 and 0.80 mg/kg was low. In spite of this, serum ramiprilat concentrations achieved were able to induce high serum ACE inhibition. In fact, the maximal serum ECA inhibition after PO administration was reached with a dose of 0.80 mg/kg (84.27%), lower than the maximal inhibition obtained after IV administration (98.99%), using an IV dose 16-fold lower than the PO dose. Currently, we do not know whether this reduction in circulating ACE activity would be of enough intensity to counteract vasoconstriction in pathological situations.

In addition, the use of physiological compartmental PK/PD models allowed the study of the pharmacology of ramiprilat after IV and PO doses of ramipril in horses. Undoubtedly, these models are more complex than the traditional approaches, but they might be more accurate to interpret the disposition phases of ACEIs concentration-time curves. Besides, after simulations produced by the models, different dosage designs were investigated. The information derived could be very useful for future studies of ramipril in equine patients.

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4.3. DESCRIPTION OF THE STUDY III

TITLE: PHARMACOKINETICS AND PHARMACODYNAMICS MODELING OF BENAZEPRIL AND BENAZEPRILAT AFTER ADMINISTRATION OF INTRAVENOUS AND ORAL DOSES OF BENAZEPRIL IN HEALTHY HORSES

4.3.1. JUSTIFICATION

Although it is not completely accurate, most of the authors have used the percentage of inhibition of serum ACE as a surrogate marker of the clinical potential of the ACEI drugs (Gardner et al., 2004; Afonso et al., 2013; 2017b; Davis et al., 2014; Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016; 2017). All the five different ACEI drugs investigated up to now in the horse (i.e., enalapril, ramipril, quinapril, perindopril and benazepril), the greatest circulating ACE inhibition has been reached with the administration of benazepril. In fact, Afonso et al. (2013), compared quinapril (0.25 mg/kg), ramipril (0.3 mg/kg PO), benazepril (0.5 mg/kg) and perindopril (0.1 mg/Kg) in horses. The ACE inhibition was greater for benazepril (86.9%), followed by ramipril (59.7%), quinapril (40.6%) and perindopril (25.2%) for the studied doses. According to these results, the greatest ACE inhibition was achieved with benazepril, resulting in a maximal inhibition greater than 60%, even though the doses administered of these four ACEI drugs were different. Furthermore, Muñoz et al. (2016) compared the effects of four different ACEI drugs (enalapril, ramipril, quinapril and benazepril) administered PO 2 h before exercise and the response of BP to a brief intense hypertensive exercise was investigated. These authors found that only benazepril (at the studied doses, i.e. enalapril at 2.0 mg/kg; quinapril at 1.0 mg/kg; ramipril at 0.2 mg/kg and benazepril at 0.5 mg/kg) was able to blunt the hypertensive response of BP to exercise. More recently, Afonso et al. (2017b) evaluated the response of BP to ANG-I administration after four different benazepril PO doses (0.5, 1.0, 2.0 and 4.0 mg/kg). They based their research on benazepril because before (Afonso et al., 2013) they have concluded that administration of benazepril resulted in significantly greater serum ACE inhibition than the other ACEI drugs in the healthy horse.

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Similarly, it has been suggested that the ACEI drugs differ in their affinity for binding of tissue ACE. Such differences in ability might be related to dosage, bioavailability, half- life, tissue penetration, and tissue retention (Dzau et al., 2001; Sauer et al., 2004). Tissue affinity, from greatest to least of the commonly used ACEIs in small animal medicine is as follows: benazepril > ramipril > lisinopril > enalapril > captopril (Ames et al., 2015). Because of these reasons, benazepril has become one of the most used and studied drug to control CHF, hypertension and chronic renal disease in the dog (Häggström et al., 2008; 2013; Pouchelon et al., 2008; Tenhündfeld et al., 2009; Kittleson et al., 2009; O’Grady et al., 2009; Cortadellas et al., 2014; Mochel et al., 2015; King et al., 2017).

All these data together seem to reveal the promising clinical utility of benazepril for horses. However, PK-PD studies of this ACEI in the horse are lacking, blood benazepril and benazeprilat concentrations have not been measured and the effects of the administration of multiple doses PO are also unknown, being these the main motivations that led us to carry out this third research.

4.3.2. OBJECTIVES

The current study aimed: 1) To establish the serum concentration-time profile for benazepril, and its active metabolite benazeprilat after IV benazepril at 0.50 mg/kg, after PO placebo and after administration of three different PO benazepril doses (0.25, 0.50 and 1.0 mg/kg); 2) To determine the effects of benazeprilat on serum ACE activity; 3) To investigate the effects on BP with these doses and routes; 4) To predict and to simulate the most appropriate PO dose that suppresses ACE activity after single of multiple PO doses of benazepril.

4.3.3. MATERIAL AND METHODS

Six healthy normotensive adult crossbred horses were included in the study, which was conducted in two trials. The first trial consisted in the administration of IV benazepril at 0.50 mg/kg. The second trial was performed a week later (period of washout), and the animals received PO placebo (0.0 mg/kg), or PO benazepril at 0.25, 0.50 or 1.0 mg/kg according to a blinded and randomized Latin square, with a 4x6 design.

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The animals did not have access to food between 12 h before until 8 h later of each experiment.

Blood samples were withdrawn and SBP and DBP were measured before and at fixed times until the 72 h after administration. Serum ACE activity was measured (spectrophotometry), as well as the serum concentrations of benazepril and benazeprilat (liquid chromatography-tandem mass spectrometry).

Because ACEI drug have non-linear binding to ACE, compartment PK-PD models with binding parameters to the ACE enzyme were used (Toutain et al., 2000a,b), in order to explain disposition phases of ACEI curves. For each individual, PK- PD studies were performed following the models developed by Lees et al. (1989), with the approach of Picard-Hagen et al. (2001). The selected model included one compartment for benazepril and one compartment for benazeprilat (Toutain and Lefèbvre, 2004), and a deport compartment for PO administration. The PK parameters studied included: ka

(first order absorption rate constant for benazepril), kf (elimination rate constant for benazepril by formation of benazeprilat), Vc/F (apparent volume of distribution with respect to bioavailability and bioconversion from benazepril), systemic clearance for benazepril (Cl/F), elimination half-life (t1/2) (King et al., 2003). Binding parameters of benazeprilat to ACE studied in this research were: kd (equilibrium dissociation constant),

Bmax (size of ACE pool), fcirc (fraction between circulating and non-circulating ACE) (Toutain and Lefèbvre, 2004).

For PD data, the observed effect was defined as serum ACE activity at each sampling time presented as a percentage of control before drug administration. The following parameters were estimated: IC50 (free benazeprilat concentration needed to produce 50% ACE inhibition), γ coefficient or Hill coefficient (description of the steepness of the sigmoid curve), %Imax (maximum serum ACE inhibition between 0-12 h), T%Imax (time to reach %Imax), and %I12h, %I24h, %I48h and %I72h (serum ACE inhibition at 12, 24, 48 and 72 h respectively).

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Comparative simulations of PO doses from 0.25 to 8.0 mg/kg after single and repeated doses administered daily for seven days were made with parameters obtained PO benazepril at 1.0 mg/kg after PK-PD analysis.

4.3.4. BRIEF DESCRIPTION OF THE RESULTS

Serum benazepril and benazeprilat concentrations were low and experienced a quick reduction after IV benazepril. Benazepril concentrations were lower than

benazeprilat concentrations, and this rapid elimination had a mean kf of 7.471/h. The

decreasing phase of benazeprilat had a t1/2 of 0.60 h (approx. 36 min). High serum ACE inhibition values (close to 99.63%) were found between 0.08 and 3.0 h after IV benazepril.

The ACE-binding and PD results obtained suggested that benazeprilat has greater

affinity for ACE with a low kd value, and indicated a relatively shallow and long-lasting

concentration effect relationship due to the lower γ and IC50 values observed. However,

Bmax and fcirc values fell in the range obtained previously from ramiprilat in horses, suggesting that these parameters are dependent of the species investigated (Toutain and Lefèbvre, 2004; Serrano-Rodríguez et al., 2016).

Bioavailability of benazeprilat after PO benazepril was very low (3.28-4.30%). However, it is necessary to indicate that, the serum benazeprilat concentrations achieved were enough to induce high serum ACE inhibition despite this low bioavailability measured, as has been described with ramiprilat in horses in a previous study (Serrano-Rodríguez et al., 2016). In fact, mean maximum values of serum ACE inhibition were of 6.77, 78.91, 85.74 and 89.51% after PO doses of placebo, 0.25, 0.5 and 1.0 mg/kg respectively. In all the cases, these values were lower than those found after IV benazepril (99.63%), even though the time of inhibition were longer after PO doses compared to IV. The longer time of inhibition was found after 0.5 and 1.0 mg/kg of benazepril from 12 to 72 h (77.97%, 64.56%, 51.98% and 40.08% at 12, 24, 48 and 72 h after PO 0.5 mg/kg; 80.58%, 70.80%, 51.69%, 38.84% after PO 1.0 mg/kg at the same times). Moreover, long lasting serum ACE inhibitions were obtained, and these findings

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might suggest that benazeprilat, after PO benazepril should be investigated in horses due to these promising results compared to others ACEIs as enalaprilat or quinaprilat (Gómez-Díez et al., 2014; Davis et al., 2014). (Figure 2 from the paper).

PD parameters from PK-PD modeling as IC50 and γ values were close to 0.55-1.00 and 0.60, respectively, indicating a relatively shallow and long-lasting concentration effect relationship (Figure 2 from the paper). For that reason, higher inhibition values could be obtained for very low benazeprilat concentrations at 0.50 and 1.00 mg/kg of benazepril.

Changes in SBP and DBP were not observed during the study, even at the times when maximum serum ACE inhibitions were attained, as has been described before suggesting that in normotensive horses, BP effect of ACEIs is low (Afonso et al., 2013; 2017b). These results agree with the data obtained with ramipril in horses (Serrano- Rodríguez et al., 2016).

When single and multiple PO simulations were investigated after PK-PD modeling, an important finding was observed. Although serum ACE inhibition rose at higher doses, the effect was similar from 1.0 to 8.0 mg/kg PO doses (Figure 3 from the paper). In the case of multiple oral doses, inhibition values ranging 70-90% could be maintained after PO benazepril at 1.0 mg/kg administering every 24 h. These results might indicate that 1.0 mg/kg of oral benazepril dose could be useful in horses.

4.3.5. CONCLUSIONS

Despite the low oral bioavailability of benazeprilat in horses, a mean maximum serum ACE inhibition of 89.51% was found with the PO dose of 1.0 mg/kg, values slightly lower than those obtained after benazepril IV (99.63%). Simulations after single and multiples doses with the use of physiological compartment PK/PD model were in agreement with recent researches of benazepril in horses. Because of these data, benazepril might be a promising ACEI for the treatment of heart disease, CHF and hypertension in the horse.

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4.4. DESCRIPTION OF THE STUDY IV

TITLE: MODULATION OF ACUTE TRANSIENT EXERCISE-INDUCED HYPERTENSION AFTER ORAL ADMINISTRATION OF FOUR ANGIOTENSIN-CONVERTING ENZYME INHIBITORS IN NORMOTENSIVE HORSES

4.4.1. JUSTIFICATION

From a theoretical point of view, ACEI administration, because of the blocking of ACE activity, and as a consequence, the inhibition of conversion of ANG-I to ANG-II, releases hypertension and modulates hyperactivation of RAAV axis. In addition, ACEI administration also increases bradykinin concentrations, which in turn leads to the release of NO and other vasoactive prostaglandins (Gehlen et al., 2008). Therefore, in patients with CHF, the venous and arterial vasodilatory effects of ACEI provide relief of pulmonary congestion, decrease peripheral vascular resistance, increase cardiac output, and reduce hypertrophy (López-Sendón et al., 2004). All of these effects should result in an improvement in the quality of life and survival time in the patient with CHF. Consequently, the study of the effects of these drugs in BP in the horse is of great clinical relevance.

Until 2017, the PK-PD studies carried out with ACEI drugs in horses were tested in normotensive horses. Different authors have described a lack of significant variations in BP (SBP and DBP) after enalapril (Gardner et al., 2004; Gómez-Díez et al., 2014), quinapril (Davis et al., 2014), ramipril (Serrano-Rodríguez et al., 2016) and benazepril (Serrano-Rodríguez et al., 2017) administration in healthy normotensive horses. A mild non-significant reduction in BP was found in a comparative study of four different ACEI in the horse (quinapril, benazepril, ramipril and perindopril) (Afonso et al., 2013). It has been suggested that in the normotensive animal, BP reduction would be expected to be a relative insensitive indicator of the drug effect, as the vasodilatory potential of these drugs might be masked in the absence of a RAAV axis activation (Afonso et al., 2013; 2017b; Davis et al., 2014).

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Brief intense exercise in the horse has been shown to induce a dramatic and sudden rise in BP (Masri et al., 1990), together with an activation of the RAAV axis (McKeever et al., 1992). In fact, these last authors reported significant increases in serum REN activity (from 1.9 to 5.2 ng/ml/h), ALD (from 48 to 191 pg/ml) and AVP (from 4 to 95 pg/ml) concentrations after an intense exercise in horses. Consequently, the exercise-induced hypertension might be used as an experimental model to compare the effect of different ACEI on BP, being this idea the main reason to carry out this fourth research.

4.4.2. OBJECTIVES The current research was designed with the following purposes: 1) To elucidate whether pre-exercise PO ACEI administration results in a blunted hypertensive response to exercise compared to placebo and if this was the case, 2) To evaluate which of the four ACEI administered leads to a greater modulation of BP response to exercise, and 3) To assess whether the changes in BP after exercise are correlated with the percentage of serum ACE inhibition.

4.4.3. MATERIAL AND METHODS

Six healthy mature crossbred horses were included in a blinded and randomized Latin square design in five trials: two h before exercise, each animal received a single PO dose of placebo, 2.0 mg/kg of enalapril, 1.0 mg/kg of quinapril, 0.2 mg/kg of ramipril or 0.5 mg/kg of benazepril, with a week of washout after each trial. Two h later of administration of the placebo or the drug (according to the trial), the horses were subjected to a brief intense treadmill exercise, consisting in a warming period, followed by 1 min at 8 m/s with a treadmill slope of 6%. Blood samples were taken before the experiment (rest, R), 2 h after PO ACEI or placebo (pre-exercise, pre-E), and within the first 20 s after exercise (post-exercise, post-E), in order to measure serum ACE activity. SBP and DBP were measured at the same times.

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4.4.4. BRIEF DESCRIPTION OF THE RESULTS

Two h after ACEI administration (pre-E), serum ACE activity was reduced with the four ACEI administered, reaching the highest decrease with PO benazepril, as expected and in concordance with several studies performed before in horses (percentages of ACE inhibition of 4.75%, 39.43%, 46.40%, 54.96% and 71.68% after placebo, enalapril, quinapril, ramipril and benazepril administration, respectively). By this time, all the horses remained normotensive, without experienced significant changes in BP.

ACE activity did not vary significantly with exercise (comparison between pre-E and post-E samples in the placebo trial). Exercise resulted in a marked increase in SBP (67.49%) and a mild increase in DBP (20.64%) from resting values when ACEI drugs were not provided. Increases in SBP of 52.71%, 43.11%, 26.64%, and 4.21% were found after exercise when enalapril, quinapril, ramipril and benazepril were administered 2 h before exercise. Increases of SBP after exercise were not different after enalapril, quinapril and ramipril administration compared to the rise in SBP observed in the placebo trial. However, a significant difference in the increase in SBP was found when compared placebo with benazepril PO. In addition, the percentage of serum ACE inhibition at blood sampling time post-E compared to R, was negatively correlated with the percentage of increase in SBP.

4.4.5. CONCLUSIONS

A brief intense treadmill exercise induced systolic hypertension in healthy horses. Maximum inhibition of serum ACE activity was found 2 h after PO benazepril administration, compared with placebo, enalapril, ramipril and quinapril administration. In comparison with placebo, exercise-induced systolic hypertension was lower when ACEI drugs were administered 2 h before exercise. The exercise-induced increase in SBP was greater for enalapril and quinapril, and lower for ramipril and benazepril. Administration of PO benazepril at 0.5 mg/kg 2 h before exercise maintained normotension immediately after exercise. The use of an intense hypertensive exercise was of utility to mimic a pathological hypertensive situation in the horse, although it should be kept in mind that exercise is an acute hypertensive situation, contrasting with

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Manuel Gómez Díez the long duration or chronic hypertension of pathological conditions, with more complex interactions between vasoconstrictive and vasodilatory mechanisms.

Benazepril showed PD properties that were useful in this exercise model. However due to the low ramipril doses used, it can be suggested that higher doses of ramipril could also be useful, as described in PK-PD studies. However, further work in equine patients should be conducted to support the clinical utility of benazepril, and perhaps ramipril.

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5. DISCUSSION

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From different points of view (physiological, anatomical and pharmacological), the horse is different to other animal species, and as a consequence, the appropriate use of many drugs can be particularly challenging in equine patients. In veterinary pharmacology, the dog is the prototype species of basic clinical research while for food- producing animals, cattle or pig, are frequently used animal models (Toutain et al., 2010a). In this way, a lot of information has been published in the last decades for dogs, cats, cows or pigs, while horse has received less attention and the results and researches from dogs have been extrapolated to horses (Maxwell, 2015). However, dogs are not mini horses and horses are not super dogs. There are many physiological and anatomical differences than can make the PK and the PD properties in the horse highly unpredictable, especially the PKs. For example, drugs that are well absorbed after PO administration, safe, and efficaciously in other species may be poorly absorbed, toxic, or ineffective in horses (Davis et al., 2014; Gómez-Díez et al., 2014). In fact, some agents as β-lactam antibiotics, furosemide, tramadol or ciprofloxacin, which are adequately absorbed after PO administration in other monogastric species, are poorly absorbed in horses (Ensink et al., 1992; Johansson et al., 2004; Davis et al., 2014). Moreover, not only PO absorption can be different, the drug interactions mediated by the hepatic metabolism, specifically those originated from the cytochrome P450 enzymes, are highly variable between species and the horse is not an exception, expressing high metabolizing activity for some drugs but low activity for others in comparison with other animals (Nebbia et al., 2003).

In this context, ACEI pro-drugs are a special case, because are well known by their low PO absorption in humans, dogs or cats. However, the lower serum concentrations achieved are useful for therapeutic uses, and more importantly, for treatment for cardiovascular diseases where long-term therapies are used and only the PO route is suitable (Lefèvbre et al., 2007; Ferrario and Mullick, 2017). Over the last decades, many studies have been published in small animals with enalapril, ramipril and benazepril as principal agents (Toutain and Lefèbvre 2004, Mochel et al., 2014). However, these drugs had received little attention in the horse when we started this research, being enalapril the most studied (Muir et al. 2001; Gardner et al. 2004; Sleeper et al. 2008). However, in the last four years (from 2013) these drugs have been studied more intensely, because

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of the greater awareness in relation to the relevance of some potentially pathologic hypertensive situations in equine medicine, including cardiovascular diseases and EMS (Afonso et al., 2013; Navas de Solís et al., 2013; Davis et al., 2014; Gómez-Díez et al 2014; Muñoz et al., 2016). The studies developed by our research group, combined with other researches recently published, have permitted to confirm the conclusions of some of the previous studies on ACEIs in the horse, as well as to provide new evidences of the PK and PD behavior of these molecules when administered to horses.

5.1. PHARMACOKINETICS: THE CASE OF ENALAPRIL IN HORSES

In main lines, the most important key point to select a drug to be administered by PO dosing is their enteric absorption. If a drug is not well absorbed even at high doses, their clinical utility can be low or completely ineffective. In this context, it is necessary to indicate that PO enalapril, unlike ramipril or benazepril, did not show adequate absorption and their bioavailability was very low. From a PK point of view, it can be suggested that enalapril will not be effective in horses after PO administration, unlike the other drugs tested, because they had better PK properties. In fact, ramipril and benazepril, as has been demonstrated in the studies published by our research group and other groups, can be absorbed after PO dosing at different doses and they can inhibit serum ACE activity (Afonso et al., 2013; 2017b; Serrano-Rodríguez et al., 2016, Muñoz et al., 2016, Serrano-Rodríguez et al., 2017). These results are promising, despite the fact that PO bioavailability was low for both ramipril and benazepril pro-drugs, although they had sufficient absorption and bioconversion into their active ACEIs to induce serum ACE inhibitions near to 84-89% at doses between 0.8-1.0 mg/kg and they could be useful in a clinical equine context.

Enalapril, and its active metabolite enalaprilat, have been studied in PK-PD models and in clinical veterinary researches (The COVE Study Group, 1995; The IMPROVE Study Group, 1995; Ettinger et al., 1998; Kvart et al., 2002; Atkins et al., 2007; Toutain et al., 2000a,b; Thomason et al., 2014; Ames et al., 2015; Lantis et al., 2015; Coleman et al., 2016). PK-PD studies have shown PO bioavailabilities close to 30% and serum ACE inhibition dose dependent (Hamlin and Nakayama, 1998; Uechi et al., 2002). However,

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Angiotensin-converting enzyme inhibitors in the horse when the doses used in dogs were extrapolated to horses, the results obtained were not satisfactory because PO absorption was limited, even when multiple doses were used (Gardner et al., 2004; Sleeper et al., 2008). Unlike IV PKs, where enalapril was metabolized to its active metabolite enalaprilat and ACE was inhibited, the PK properties were not determined because only some residuals drug concentrations were detected (Gardner et al., 2004). In these researches, only PO doses of 0.5 mg/kg were used, and this could indicate that higher doses should be required to achieve serum concentrations to induce ACE inhibitions that might be of utility in an equine clinical setting. Moreover, if higher doses are not enough to obtain therapeutic drugs concentrations of enalapril and enalaprilat, other ACEIs should be investigated (Gómez- Díez et al., 2014).

The IV administration of enalapril at the usually referenced dose (0.5 mg/Kg) confirmed adequate biotransformation of the molecule to its active metabolite (enalaprilat) in the horse, evidenced by increased concentrations of enalaprilat 15 min after IV enalapril administration (Figure 7) and by a significant degree of ACE inhibition close to 88.38%. This value differed significantly from the inhibition values obtained after PO administration of 0.5, 1.0 and 2.0 mg/kg (3.24, 21.69 and 30.19% respectively). In fact, after PO administration of enalapril at three different doses, we also found that the PO absorption was low and dose independent, as long as IV pharmacokinetics was similar than that previously reported (Gardner et al., 2004), and therefore, an increase in dosage did not improve PO absorption (Gómez-Díez et al., 2014). This study evidenced the low bioavailability of enalapril in healthy horses, with serum concentrations of enalaprilat remaining below the LOQ after administration of a dose four times higher than the IV and PO doses usually referenced in the literature (Thind, 1990; Morrison et al., 1996; Toutain et al., 2000a,b).

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FIGURE 7. Semilogarithmic plot of serum concentrations (mean ± SD) of enalapril (-●-) and enalaprilat (-●-) after IV enalapril at 0.50 mg/kg in horses (n = 7).

PK data of enalapril and enalaprilat obtained after IV dosing were analyzed, unlike PO data. However, our data were not modeled using physiologically non- conventional single-compartment models as we did with ramipril or benazepril. Only non-compartmental analysis was done and there is a reason for this choice. Several physiologically based models with different equations and parameters were previously used to model IV data. However, these models could not be used in our study because IV serum concentrations data, of pro-drug and drug, were not well fitted to the models. In some cases, the model was not able to solve the equations, in other cases, the number of observations (serum data vs time) of the model was lower than the number of parameters, and the software could not resolve the equations. For that reason, a physiologically based model was refused, although it could have been more appropriate. Also, only serum data after IV dosing could be detected because serum data after PO dosing were below the LOQ (10 ng/mL) in all horses, with the exception of three cases. Therefore, an appropriate interpretation of the different phases of the disposition curve of the drug and pro-drug after PO doses were not possible. For all these reasons, the data after IV dose were analyzed used non-compartmental modeling, as has been used

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Angiotensin-converting enzyme inhibitors in the horse with enalapril and enalaprilat (Gardner et al., 2004) and others ACEIs as quinaprilat (and quinapril) in horses (Davis et al., 2014).

Non-compartmental modeling IV PK parameters obtained were compared to the data reported by Gardner et al (2004), and were very similar for Cl, Vss and AUC0→∞ for enalapril and enalaprilat, because the same dose was used and therefore our data fell in the same range. The Cl, Vss, AUC0→∞ and AUMC0→∞ values of enalapril were very close to the data reported by Gardner et al (2004) (1.65 l/h/kg, 0.64 l/kg, 320 ng/ml·h and 120

2 ng/ml·h , respectively). In the same way, the t½λz values for enalapril and enalaprilat found in our study were similar to the values reported by Gardner et al (2004) (0.86 and 2.01 h, respectively). However, it is mandatory to indicate that these values could no represent the true terminal t1/2. Serum concentrations of ACEIs are the sum of free drug in serum, drug bound nonspecifically to plasma proteins and specifically bound to circulating ACE (Toutain and Lefèbvre 2004). Therefore, the binding parameters as Bmax, kd and the partition fcirc values should be determined. However, this information is currently not available for enalapril and enalaprilat in horse, as previously indicated.

Based on these results, we can suggest that the ineffectiveness of enalapril when administered PO to horses in terms of ACE inhibition was due to an absorption deficit more than to limitations in distribution and metabolism of the drug, because IV data could be detected and analyzed. These results were in agreement with those obtained by Gardner et al. (2004), Also, the bioavailability of this agent is low for other monogastric species, 40% at 5.0-20.0 mg for humans (Thind, 1990), and 20-40% at 0.06- 6.0 mg/kg for dogs (Morrison et al., 1996). However, in these studies, oral PK data were modeled because lower serum concentrations were detected, unlike PO data in horses. Nevertheless, despite moderate bioavailability of enalapril, several experimental studies involving around 870 animals have proved their clinical efficacy in treating hypertension, especially in situations of CHF in cats and dogs, whereas in horses this drug appears to be ineffective (Gardner et al., 2004; Lefèbvre et al., 2007; Gómez-Díez et al., 2014).

Extensive research has been done regarding the PKs and PDs of ACEIs in humans and small animals (cats and dogs) (Lefèbvre et al., 2007, Mochel et al., 2014). Bioavailability of enalapril and benazepril in dogs varies from 20 to 40 % (Waldmeieir et

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Manuel Gómez Díez al., 1989; Morrison et al., 1996) while the bioavailability of their active metabolite is significantly lower (2.6 % for benazeprilat) (Lefèbvre et al., 2007; Mochel et al., 2014). Once again, this difference is related to liposolubility of the compound, which is significantly higher in the pro-drugs.

Different hypothesis can be suggested with regard to the poor PO bioavailability of enalapril in the horse. ACEIs can be absorbed either by direct passive diffusion (positively related to lipophilicity) and also actively transported by PEPT, possibly with a key role of the low-affinity H+ peptide co-transporter (PEPT1) (Knütter et al., 2008). Enalapril is considered a prototypical PEPT1 substrate (Swaan et al., 1995) and recent studies have concluded that PEPT1 transporter could be present in horses in low amounts (Cehak et al., 2013; Davis et al., 2014). However, other authors have shown that intestinal absorption of enalapril could not be predictable by the activity of PEPT1 transporter because its affinity was weak compared to others ACEIs as benazepril (Knütter et al., 2008). Moreover, it has been shown that a low affinity by PEPT1 transporter was associated with a low lipophilicity, and intestinal absorption by simple passive diffusion might be sufficient in many cases (Knütter et al., 2008). In addition to this, intestinal permeability and aqueous solubility of the administered molecules are important factors that contribute to PO absorption of drugs and are not well characterized in the horse (Davis et al., 2006). Enalapril is more hydrophilic than ramipril and benazepril (Ranadive et al., 1992) and this could affect intestinal absorption, like it has been suggested by the greater ACE inhibition rates obtained with ramipril, quinapril, perindopril and benazepril (Afonso et al., 2013; Davis et al., 2014; Serrano-Rodríguez et al., 2016; 2017).

Different factors intrinsic to the equine species could also influence PO absorption of certain molecules as the equine digestive physiology greatly differs from humans, small animals and ruminants. The horse is a hind gut fermenter, and therefore the cecum and colon are major places of microbial digestion and this factor determines the horse increased susceptibility for induced colitis (Maxwell 2015). In relation to this question, the diet plays an important role in interspecific differences of the PK properties of certain drugs. The horse diet is largely composed by fiber, which

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implicates a large quantity of cellulose. Certain drugs can suffer from large binding to cellulose from the time of ingestion until they reach the colon and cellulose is digested by the bacterial flora. This could significantly impact the availability of a drug to be absorbed in the anterior segments of the gastrointestinal tract (Toutain, 2010). Although these particularities have been proven for other drug classes like NSAIDs, they could potentially affect other medications including ACEIs, but this remains to be studied (Afonso et al., 2013).

5.2. PHARMACOKINETICS: RAMIPRIL AND BENAZEPRIL, MORE LIPOPHILIC PRO-DRUGS

Ramipril and benazepril are two pro-drugs that are metabolized to their active metabolites ramiprilat and benazeprilat. These drugs have been intensively studied in small animals using PK-PD and clinical assays, and are used for treatment of CHF associated with myocardial failure in small animals (Lefèbvre et al., 2006; 2007; Kittleson et al., 2009; O’Grady et al., 2009; Häggström et al., 2013; Mochel et al., 2014).

After IV administration, these agents are biotransformed to their active ACEIs in horses, as shown in figures 8a and 8b, respectively. In the case of ramipril, serum ramipril and ramiprilat concentrations were low and declined rapidly after IV administration of ramipril with a long terminal phase for ramiprilat until 36 h. Ramipril concentrations were lower than ramiprilat concentrations from 0.26 h suggesting a rapid elimination by

biotransformation to ramiprilat, and by other routes with kf and k10p values of 4.78 and

1.86 1/h, respectively. The t1/2 of elimination of ramiprilat was 0.16 h (9.6 min approximately). This very short value can be due to the small volume of distribution and particularly to the high Cl observed. The rapid decreasing phase of ramiprilat is

controlled by k10 and reflects the elimination process. Moreover, the terminal phase observed for ramiprilat reflects the binding to ACE and is influenced by binding

parameters and by k10 (Lefèbvre et al., 2006).

For benazepril, similar conclusions can be obtained. In fact, serum benazepril and benazeprilat concentrations were low and declined rapidly after IV administration of benazepril. Benazepril concentrations were lower than benazeprilat concentrations from 0.16 h (Fig. 8b), suggesting a rapid elimination by biotransformation to

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benazeprilat with a mean kf value of 7.471/h. The decreasing phase of benazeprilat is also controlled by k10 and reflects the elimination process with a t1/2 of 0.60 h (36 min approximately). In the same way of ramiprilat, the terminal phase observed for benazeprilat reflects the binding to ACE and is influenced by binding parameters and by k10 (Lefèbvre et al., 2006).

FIGURE 8. a) Semilogarithmic plot of serum concentrations (mean ± SD) of ramipril (-●-) and ramiprilat (-●-) after IV ramipril at 0.05 mg/kg in horses (n = 6) and b) semilogarithmic plot of serum concentrations (mean ± SD) of benazepril (-●-) and benazeprilat (-●-) after IV benazepril at 0.50 mg/kg in horses (n = 6). The results obtained after IV administration have provided important information about the metabolism from ramipril or benazepril to ramiprilat or benazeprilat respectively in horses, as described by other ACEIs (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez et al., 2014). However important differences related to the dose administered are discussed in the next paragraph.

On one hand, ramipril was administered at 0.05 mg/kg whereas benazepril was administered at 0.5 mg/Kg dose, 10-fold higher and similar to enalapril (Figure 7). However, concentrations of ramipril were lower than those of benazepril despite the fact that ramiprilat and benazeprilat were detected until 36 h post pro-drug dosing, and both ACEIs could be modeled using non-conventional physiologically PK models, unlike to enalapril and enalaprilat. On other hand, the biotransformation rate expressed as kf was higher for benazeprilat suggesting that benazepril was metabolized to benazeprilat

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Angiotensin-converting enzyme inhibitors in the horse faster compared with metabolism of ramipril to ramiprilat in horses. However, in dogs, these differences were not found (Toutain et al., 2000a; Lefèbvre et al., 2006). Moreover, both drugs, compared to enalapril, showed higher serum concentrations, particularly benazepril, that was administered at the same dose of 0.50 mg/kg. These results could indicate that enalapril and enalaprilat were metabolized faster in horses and as consequence their concentrations declined faster than ramipril and benazepril, with a rapid elimination.

In relation to PO administration, the physicochemical properties of ramipril and benazepril pro-drugs are more suitable to PO dosing, because are more lipophilic than enalapril, and are substrates that actively are transported in the jejunum by PEPT. In fact, enalapril is more hydrophilic (logP = 0.16) than ramipril, or benazepril (logP values of 1.03, and 4.54, respectively) (Knütter et al., 2008). Also, for drugs that are actively transported with high affinity, their intestinal permeability would be greater than for passive absorbed drugs (Riviere, 2011).

These properties have a good correlation with the results obtained after PO absorption in small animals and horses (Toutain et al., 2000a,b; Lefèvbre et al., 2006; Serrano-Rodríguez et al., 2016; 2017). Ramipril and benazepril are orally absorbed and metabolized to their active metabolites in horses, unlike enalapril, and oral serum concentrations could be detected and modeled using non-conventional physiologically PK models for ramipril at 0.40 and 0.80 mg/Kg and benazepril at 0.25, 0.50 and 1.00 mg/Kg. Similar results have been described in humans, dogs and cats (Van Griensven et al., 1995; Lefèvbre et al., 2006; Mochel et al., 2014). The oral concentrations-curves observed and detected for both pro-drugs and theirs ACEIs are shown in figure 9.

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FIGURE 9. (a) Semilogarithmic plot of serum concentrations (mean ± SD) of ramipril (-●- ,-■-, -♦-) and ramiprilat (--o--, --□--, --◊--) after PO ramipril at 0.20, 0.40 and 0.80 mg/kg in horses, respectively. (B) Semilogarithmic plot serum concentrations (mean ± SD) of benazepril (-●-,-■-, -♦-) and benazeprilat (--o--, --□--, --◊--) after PO benazepril at 0.25, 0.50 and 1.00 mg/kg in horses, respectively.

Ramipril showed higher bioavailability (6.5 to 9%) compared to benazepril (3.2 to 4.3%) although benazeprilat had a higher distribution volume (4.2 to 4.7 l/kg) than ramiprilat (1.9 to 2.3 l/kg). These data suggest that although benazepril has a slightly higher absorption rate after PO administration, once absorbed and metabolized, its active metabolite has a higher tissue penetration, with less amount of drug resting within systemic circulation, perhaps due to the higher liposolubility of benazepril. Ramipril and benazepril were the only ACEIs in our study that reached sufficient PO absorption to enter in a PK analysis, although bioavailability values were significantly lower to those found in small animals and humans close to 30 % (Toutain and Lefèbvre, 2004). The same characteristics particular to the equine species that have been proposed as absorption limiting factors for enalapril, could also be responsible for the absorption of ramipril and benazepril (Serrano-Rodríguez et al., 2017).

The t1/2 of both drugs were moderately different between them, with benazepril showing the longer values after PO administration (0.37 to 0.51 h) compared to ramipril (0.13 to 0.17 h). IV administration revealed similar findings, with benazepril showing

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significantly longer t1/2 (0.60 h) than ramipril (0.16 h). This parameter is correlated with long-term inhibition of the ACE (48 h post administration), which was significantly higher for the highest dose of benazepril when compared to the highest dose of ramipril, as it will be discussed later in this section.

Cl/F values for benazepril after PO administration ranged between 6.4 and 7.8 l/h/kg, while values for ramipril showed a significant variation between doses (7.7 to 12.82 l/h/kg). As this parameter correlates clearance with bioavailability, the differences found between the two doses of ramipril was attributed to the differences in bioavailability values. Cl after IV administration for benazepril was lower (0.29 l/h/kg) than for ramipril (0.77 l/h/kg). As Cl values describe the velocity at which a drug is eliminated from the circulating volume, this difference also correlates with the longer duration of the inhibitory effects found after administration of benazepril.

In relation to bindings parameters, the Bmax and fcirc values obtained after using physiologically model were similar between benazeprilat and ramiprilat because they are species dependent parameters, as has been described previously (Lefèvbre et al.,

2007). These results suggest that approximately ACE pool expressed as Bmax ranged from 70-110 nmol/l, similar to dogs but lower than cats and humans. Therefore, it could be hypothesized that the relationships or fractions between ACE pools could be different according to the species as has been indicated in other studies (Toutain and Lefèvbre,

2004). On the other hand, the affinity for ACE, described by kd values, was similar between drugs, close to 1-2 nmol/l, suggesting that both could be able to bind and inhibit ACE pool in the same way (Serrano-Rodríguez et al., 2017).

5.3. PHARMACODYNAMICS: ANGIOTENSIN CONVERTING ENZYME INHIBITION AND SIMULATIONS

In terms of PK parameters, IV administration of enalapril showed the smaller percentage of ACE inhibition (88.3%) but produced the fastest maximal inhibition (0.13 h), while ramipril and benazepril produced significant degrees of inhibition (98.8 and 99.3%) with ramipril showing a significantly shorter maximal inhibition (0.21 h) when compared to benazepril (0.75h), as has shown in figure 10. IV benazepril produced a

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Manuel Gómez Díez long-lasting serum ACE inhibition until 72 h, unlike ramipril and especially enalapril. These data suggest a rapid and effective transformation of the pro-drugs into their active metabolites, finding that has been confirmed by serum quantification of pro-drug and active metabolite for each ACEI tested (Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016; 2017).

However, IV inhibition curves were similar between enalapril at 0.50 mg/kg and ramipril at 0.05 mg/kg, but ramipril was administered 10-fold doses lower than enalapril, and higher doses of ramipril close to 0.50 mg/kg could produce similar serum inhibition curves of benazepril at 0.50 mg/kg. Despite the fact that IV 0.50 mg/kg of ramipril were not tested, it could be hypothesized that IV inhibition could be similar at the same dose between ramipril and benazepril because PO inhibition were very similar at 0.80 and 1.00 mg/kg dose (Serrano-Rodríguez et al., 2016; 2017).

FIGURE 10. Serum ACE inhibitions values after intravenous enalapril, ramipril and benazepril (-●-,-●-,--o--) at 0.50, 0.05 and 0.50 mg/kg in horses, respectively.

PO administration of enalapril at 0.5, 1.0 and 2.0 mg/kg, despite not producing quantifiable concentrations of serum enalapril or enalaprilat, led to a discrete level of ACE inhibition (3.2 to 30.1%) between 1.7 and 4 h post administration.

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The mean maximal %I24h post administration were comprised between 17.1 and 20.1 % and at 48 h, decreased to levels between 6.5 and 11.8%. There was a positive correlation between dose and Imax, although this correlation was not seen with inhibition percentages at 24 and 48 h post administration.

Ramipril and benazepril induced ACE inhibition levels significantly higher than enalapril in all doses tested and close to a complete ACE activity inhibition (98.8 and 99.6% respectively) although ramipril obtained maximal inhibition faster than benazepril (0.21 and 0.75 h respectively), as shown in figure 11. In the PO administration tests, ramipril obtained ACE inhibition levels ranging from 27.6 to 84.2% in 2.2 to 2.5 h post administration, while benazepril produced the highest inhibition levels of this study ranging from 78.9 to 89.5 % in 3.5 to 4 h post administration. Imax levels correlated with dose in all cases. Due to its PD properties after PO administration of a single dose, benazepril appeared to be the most promising ACEI of the three tested in our study, due to its highest maximal ACE inhibition and the longest duration of the inhibitory effect. This PD advantage is most likely related to its greater PO bioavailability in the horse, which could be attributed to the more intense liposolubility of benazepril compared to the other drugs used in this study.

FIGURE 11. Serum ACE inhibitions values after oral enalapril, ramipril and benazepril (- ●-,-●-,--o--) at 2.00, 0.80 and 1.00 mg/kg in horses, respectively.

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Virtual simulations of various repeated PO administration regimes were performed for ramipril and benazepril and revealed interesting findings. Although inhibition degrees were always dose related, similar values were obtained with ramipril with doses between 0.8 to 6.3 mg/kg and inhibition was always over 80%. Due to a progressive ACE saturation effect, repeated administration of a relatively low dose of ramipril (0.6 to 1.6 mg/kg once a day) could maintain ACE inhibition over 80% which could be interesting in a clinical setting (we should keep in mind that a 75% of ACE inhibition has been reported to be the minimum inhibition percentage to obtain a clinical benefit in clinically ill dogs; Hamlin et al., 1996; The IMPROVE Study Group, 1995; The COVE Study Group, 1995; The LIVE Study Group, 1998; The BENCH Study Group, 1999), although this value in horses is unknown (Afonso et al., 2013).

In a similar way, simulation of repeated PO administration of benazepril at different doses revealed a dose-related inhibition capacity but with similar values (all of them over 90%) when doses ranged between 1.0 and 8.0 mg/Kg, data that agreed with Afonso et al (2017b), who proved that increasing doses of benazepril did not produce greater attenuation in BP response to exogenous ANG-I. Therefore, daily PO administration of 1.0 mg/kg benazepril was proven to be the most interesting multiple administration regimens to be tested under clinical conditions in horses.

An important finding observed in this research was the IC50 and γ values modeled for ramiprilat and benazeprilat were close between them, unlike enalaprilat due to the use of non-conventional PK-PD models (Serrano-Rodríguez et al., 2017). The values for

IC50 indicates that ramiprilat and benazeprilat were potent agents with high affinity to inhibit the ACE pool, and γ values suggest that long lasting inhibition values could be obtained after multiple dosage, as has been demonstrated with the single PO administration obtained in our investigations (Serrano-Rodríguez et al., 2016; 2017).

More importantly, this similarity of PD properties between benazepril and ramipril can produce similar multiple PO dosing. In fact, a comparative simulation after 0.80 and 1.0 mg/kg dose of ramipril and benazepril respectively is shown in figure 12. These profiles seemed to indicate that multiple PO doses of benazepril or ramipril, with doses comprised between 0.80 and 1.0 mg/kg, administered daily, could be a useful

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treatment for horses. At this point, benazepril seems to be the most useful pro drug ACEI, but ramipril, which has close PK-PD relationships, could be another interesting option. Nevertheless, our studies are preliminary reports, carried out with healthy horses and further works in equine patients should be conducted to support the clinical utility of benazepril and ramipril in horses (Serrano-Rodríguez et al., 2016; 2017).

FIGURE 12. Comparative simulations of serum ACE inhibitions values after oral ramipril at 0.80 mg/kg (------), and oral benazepril at 1.00 mg/kg (------) in horses for a week

5.4. EFFECTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS ON BLOOD PRESSURE IN THE HORSE Several studies have revealed the beneficial effect of ACEI drugs in controlling pathological hypertension in small animals in situations like CHF and renal disease (Brown et al., 2003; Atkins et al., 2007; Lefèbvre et al., 2007; Mishina and Watanabe, 2008; Pouchelon et al., 2008; Häggström et al., 2008; 2013; Thomason et al., 2016; Vollmar and Fox, 2016). This effect is related to a reduction in circulating ANG-II and ALD, which result in peripheral vasodilation and decrease Na+ retention. Hypotension has been described as a possible side effect of ACEIs, normally of moderate to low

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Manuel Gómez Díez severity, in human beings (Lucas et al., 2006) and dogs (Lefèbvre et al., 2007), usually related to drug overdose, pre-existent hypotension or combination with diuretics. Nevertheless, it seems that there are interspecies differences regarding the potency of the hypotensive effect of ACEIs. These effects have been found to be much lower in cats (Lefèbvre et al., 2007), so this species has been signaled with a much lower risk of hypotension side effect.

The results of different investigations regarding the effects of ACEIs on BP in normotensive horses have been controversial. One study concluded that repeated administration of ramipril induced a moderate decrease in BP in healthy horses (Luciani et al., 2007). Other studies have failed to evidence a hypotensive effect of ACEIs when administered to healthy individuals (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016; 2017). As stated above, Afonso et al (2017b) evidenced a protective effect of benazepril that inhibited or limited hypertension in healthy horses that received exogenous ANG-I. This might suggest that the BP controlling effects of ACEIs could depend on increased circulating concentrations of ANG-I, like patients with CHF or healthy patients with administered exogenous ANG-I. To the authors knowledge, no clinical trials in hypertensive horses have been conducted with any ACEI drug and therefore it is unproven if an antihypertensive effect like that in humans and small animals could be reached in horses. Nevertheless, there are some case reports were ACE inhibitors were included in the treatment plan of horses with various cardiopathies with variable success (Gehlen et al., 2003; Orhan et al., 2016). Very recently, Afonso et al. (2017b) treated six horses with mitral or aortic regurgitation with benazepril at a dose of 1 mg/kg every 12 h, compared to placebo. It was found that horses that received benazepril for 28 days experienced a greater reduction in left ventricular internal diameter in and aortic sinus diameter, together with greater increases in cardiac output and fractional shortening compared to the placebo group. In addition, despite profound serum ACE inhibition, REN activity and ANG-II and ALD concentrations were not different between benazepril-treated and placebo horses. The echocardiographic changes presented in the treated horses were indicative of reduced cardiac afterload in left-sided valvular regurgitation (Afonso et al., 2017a).

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On the other hand, we have to indicate that, up to know, and to the best knowledge of the authors, the presence of hypertension in horses with CHF, cardiac disease and metabolic problems has not been confirmed in a large number of animals, and only anecdotical evidences exist. Horses appear to develop more commonly pulmonary hypertension than systemic hypertension when cardiac problems exist (Reef et al., 2014). However, in the last years, changes in BP have been associated also with metabolic diseases, with some discrepancies according to the authors. In this way, Nostell et al. (2016) documented a positive correlation between insulin sensitivity and BP in Warmblood horses. By contrast, Heliczer et al. (2007) compared cardiovascular findings in two groups of ponies, with EMS and control, and differences in BP between groups were not detected by these authors.

From our data, it is clear that more studies about diseases leading to secondary hypertension are needed in the horse and in these animals, it would be of great clinical interest to evaluate the effects of the ACEI drugs.

5.5. USE OF EXERCISE AS A MODEL TO STUDY RENIN ANGIOTENSIN ALDOSTERONE VASOPRESSIN AXIS ACTIVATION AND HYPERTENSION IN THE HORSE As we have discussed before, the effects of ACEI drugs on BP in normotensive human beings and animals are not completely elucidated, and controversial results have been reported by different authors. In human beings, administration of several PO ACEIs in Na+-repleted normotensive subjects induced a mild reduction in BP, being this decrease more evident in Na+-depleted normotensive and hypertensive subjects (Niarchos et al., 1979; Kiowski et al., 1992; Gainer et al., 1998). No changes in BP were found after PO administration of enalapril in normotensive dogs with chronic renal failure (Grodecki et al., 1997), and in normotensive healthy cats, PO enalapril produced a mild reduction in BP (Uechi et al., 2002).

If we take into account the above described data, it appears that there are two factors to consider when evaluating the effects of ACEIs administration on BP: Na+ status (i.e. depletion or repletion) and degree of activation of the RAAV axis. In fact, it has been proven in human beings that acute changes in BP are correlated with pre-treatment REN

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Manuel Gómez Díez activity and ANG levels, such that the greatest reductions in BP are seen in human patients with the highest plasma REN activity (Case et al., 1980; Waeber et al., 1980; Given et al., 1984; Brown and Vaughan, 1998). However, with long-term therapy, a greater percentage of patients achieve a decrease in BP, and the antihypertensive effects no longer correlated with pre-treatment REN activity (Case et al., 1980; Waeber et al., 1980; 1982; Given et al., 1984; Brown and Vaughan, 1998).

In our opinion, the different effects on BP associated with ACEI administration in horses might be attributed to the degree of activation of the RAAV. In order to study the influence of ACEI on BP, an experimental hypertensive situation has been provoked with administration of exogenous ANG-I, that increases SBP by 20-30 mmHg and according to some authors, it is a better method that the inhibition of ACE activity for evaluating the supposed clinical efficacy of an ACEI (Biollaz et al., 1981; Brunner et al., 1983; 1984; Afonso et al., 2017b). In these studies, doses resulting in more than 75% attenuation of the pressor response were considered likely to be clinically efficacious.

Exercise has been demonstrated to be an important activation of the RAAV axis in the horse (McKeever et al., 1991; 1992; McKeever, 1998; Muñoz et al., 2010a,b,c). As a consequence, our study used brief intense exercise as a model of physiological hypertension to evaluate BP response to the different ACEI drugs and to assess whether that response was associated with the intensity of ACE inhibition. We selected brief maximal exercise because reasons to activate RAAV axis during this type of exercise differ from those that lead to an activation of the axis during a prolonged submaximal exercise (Muñoz et al., 2010a,b,c). In fact, long term control of BP during prolonged exercise is more associated with the thermoregulatory responses.

After ACEI administration and before exercise, changes in BP were not detected, in accordance to what was reported before by Gardner et al. (2004), Davis et al., (2014) and also in our studies, already described. As discussed before, it is unclear why no evidence of BP change has been found after administration of ACEI drugs in healthy horses (with the exception of the paper of Luciani et al., 2007, where a mild reduction was described).

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In agreement with our previous and simultaneous studies (Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016; 2017), the degrees of ACE inhibition were low for enalapril and quinapril, moderate for ramipril and high for benazepril. This data is also in agreement with the results from Afonso et al (2013). It is important to remark that the dose of enalapril was relatively large compared to the doses of quinapril, ramipril and benazepril (2, 4 and 10-fold respectively). Once again, the PK properties of the different ACEIs, specially the bioavailability after PO administration, are responsible for these results.

Our study revealed that brief intense exercise induced a transient and moderate increase in BP that could be modulated by pre-exercise PO administration of an ACEI drug. Enalapril, quinapril, ramipril and benazepril were tested for this purpose, and we found that benazepril yields the most significant hypertension modulation effect. In fact, whereas the selected type of exercise induced an increase in systolic BP of 67.4%, this rise was only of 4.2% when benazepril was administered 2 h before exercise. The increase in BP (SBP and DBP) associated with exercise, was in all cases negatively correlated with the percentage of ACE inhibition.

We failed to find significant changes in serum ACE activity with exercise, as opposite as described by De Mello Costa et al. (2012), authors that reported an increase in ACE activity with sprint exercise to fatigue, returning to baseline values at 30 min after exercise. As these same authors described a lack of significant modifications in serum ACE after a submaximal prolonged exercise (Costa et al., 2011), it might be hypothesized that the duration and intensity of the exercise could influence the response of ACE.

Undoubtedly, our results are important findings, but the strongest limitation of this study relied on the differences between acute transient exercise induced hypertension and pathologic chronic hypertension, which results from activation of vasoconstrictive mechanisms rather than SNS activation. Although this model has permitted to stablish a difference between different ACEIs regarding their potential interest for clinical use, conclusions cannot be completely extrapolated and further investigations including diseased animals are necessary. Nevertheless, the procedure to test the potential clinical efficacy of these drugs consistent in administering ANG-I to

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Manuel Gómez Díez study the blunted response to the vasopressor response of this agent, in our opinion, is neither a good alternative. Up to now, the increase in ANG-I concentrations in horses with cardiac disease, CHF or renal chronic disease has not been demonstrated.

Hypertension is a physiological consequence of short intense exercise, needed for a redistribution of cardiac output, with an increase in blood flow towards the tissues implied in exercise (including muscles for movement and skin for heat dissipation) and a reduction of blow flow towards organs that do not participate actively in exercise (organs of splanchic area) (Muñoz et al., 2017). Therefore, it could be thought that modeling of this physiological process in the exercising horse might have detrimental effects for performance. On the contrary, our research demonstrated that, exercise 2 h after ACEI administration modulate BP rise, but did not provoke hypotension. A substantial decrease in BP has been evidenced more clearly in Na+ depleted dogs or when ACE inhibitors are administered in conjunction with a diuretic (Lefèbvre et al 2007). In the current study, no detrimental effects were seen, although the inter-species difference regarding BP response to ACEIs has already been discussed and one study did not find hypotension in exercising horses after IV administration of enalaprilat (Muir et al 2001). In our opinion, the possible hypotension derived from concomitant ACEI and diuretics administration deserves further investigation. Diuretics (i.e. furosemide) are allowed in some countries (USA and Japan) before speed racing in order to reduce EIPH (Hinchcliff et al., 2011; 2015; Sullivan et al., 2015). We cannot affirm that hypotension will not appear in these cases.

The results of our study suggested that, a lower increase or a lack of increase in SBP under exercise was achieved in relation with higher percentages of ACE inhibition. The different ACEIs administered could have been effective in preventing transient exercise induced hypertension, being benazepril the most effective. These data did not agree with the results of the study of Muir et al. (2001), performed with enalapril. May be, the differences between PK and PD properties of enalapril and the other ACEIs could be the responsible for these discrepancies. Nevertheless, we do not have clear evidences that the modulating effect on BP after exercise could justify clinical usage of benazepril in horses with pathological hypertension, although in our opinion, it should

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Angiotensin-converting enzyme inhibitors in the horse be the most recommended drug to be investigated in a clinical setting. Afonso et al. (2017a) shared with us the same opinion and hence their last paper, still in press, where they administered benazepril in horses with left-sided regurgitation (mitral and aortic regurgitation).

Another interesting field for future research is the possible application of ACEIs in the study and prevention of EIPH in the horse. This disease is responsible for reduced performance in a large population of equine athletes, especially those that perform high intensity exercise (mainly Thoroughbreds but also Standardbreds). The etiology of the pathology is not completely understood but an increased pressure at the capillary bed of the pulmonary circulation during maximal exercise has been hypothesized as the most probable cause (Hinchcliff et al., 2015). Diuretic therapy, especially loop diuretics like furosemide, as indicated before, has been the gold standard preventive strategy most likely due to an attenuation of the increase in and pulmonary capillary pressures. The prophylactic use of this medication in sport horses is controversial due to moderate evidence on its capacity to enhance performance (Hinchcliff et al., 2015) and in fact, it is not allowed before racing in Europe.

ACEI administration has been reported not to alter pulmonary pressure in horses (Muir et al 2001) and therefore have not been proposed for EIPH prevention. Nevertheless, this conclusion is based on studies that used IV enalapril at doses of 0.5 mg/kg, which was the drug with less antihypertensive effect from our trials. Our findings permit to hypothesize that ramipril and benazepril might potentially be adequate alternatives to diuretic therapy in exercising horses. Nevertheless, further studies to determine pulmonary pressure under exercise in pre-treated horses and studies with bleeding horses under high intensity exercise are required to confirm this hypothesis. Recently, Costa et al. (2012) and De Mello Costa (2014) found that mean ACE activity in horses treated with furosemide was lower that the values obtained in horses not treated with this diuretic. In addition, when horses with EIPH were analyzed, those treated with furosemide had significantly lower ACE activity than non-treated horses. Further, a regression analysis indicated a polynomial relationship between mean ACE activity and EIPH grade. Current legislation for professional equine sport disciplines include most

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ACEIs as either controlled or banned substances for competition. For that reason, Costa et al. (2012) and De Mello Costa (2014) suggested a potential use of ACE as a biomarker for EIPH severity, although controlled studies were recommended.

Further studies on the ability of ACEIs to alter performance would be interesting if they reveal potential for EIPH prevention. Finally, we would like to express our repulse to the administration of doping agents in sport horses. However, and because despite great efforts, the control of doping is sometimes difficult for the veterinarians involved in equine competitions, we should need to know as much as we can about the possible effects of the different drugs, even though they are banned for competition according to the FEI (Fédération Equéstre Internationalle).

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6. CONCLUSIONS

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The current research focused on the PK and PD properties of three ACEIs in the horse, enalapril, ramipril and benazepril, and assessed the changes in serum ACE activity and in BP in response to different routes of administration (IV and PO) and different doses of these compounds. In addition, the research evaluated the effects of four ACEIs (the three mentioned before plus quinapril) on BP response to exercise when administered 2 h before it, in order to provide information about the actions of these compounds in modeling acute transient hypertension.

Our research has led us to obtain the following conclusions:

First conclusion. Oral bioavailability of the enalapril (at doses from 0.05 to 2.0 mg/kg), ramipril (at doses from 0.05 to 0.8 mg/kg) and benazepril (at doses from 0.25 to 1.0 mg/kg) was low, probably attributed to a limited capacity of the equine digestive system to absorb these prodrugs and/or a low amount or absence of transporters that might act as peptide carriers.

Second conclusion. Mean maximum serum angiotensin-converting enzyme inhibitions were reached with oral ramipril at 0.8 mg/kg (84.27%) and oral benazepril at 1.0 mg/kg (89.51%), percentages significantly higher than those found after oral enalapril at 2.0 mg/kg (30.19%), findings possible due to the higher lipophilicity of ramipril and benazepril. These data might suggest that ramipril or benazepril would be reasonable choices to treat horses with and/or hypertension.

Third conclusion. Physiological compartment pharmacokinetic-pharmacodynamic models, despite their complexity, appear to be a useful method to study pharmacology of the angiotensin converting drugs in the horse after intravenous and oral administration, allowing a proper study of the disposition phases of these compounds.

Fourth conclusion. Multiple PO doses simulations showed that ramipril at 0.80 mg/kg, and benazepril at 1.00 mg/kg every 24 hours could reach sufficient serum concentrations to induce, and maintain, serum ACE inhibitions between 70 and 90 %. These data suggest that both dosages could be useful in equine patients.

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Fifth conclusion. The inhibition of the circulating angiotensin converting enzyme activity after intravenous and oral doses of the three prodrugs tested did not modify blood pressure and the horses remained normotensive during the experiments. In addition, local or systemic effects were not observed after administration of the prodrugs.

Sixth conclusion. A brief maximal treadmill exercise was a useful experimental method to induce acute transient systolic hypertension in healthy normotensive horses.

Seventh conclusion. In agreement with our previous conclusions, oral benazepril at 0.5 mg/kg, administered 2 h before treadmill exercise, inhibited systolic hypertension associated with a short bout of maximal intensity exercise.

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7. RESUMEN

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ESTUDIOS FARMACOCINÉTICOS Y FARMACODINÁMICOS DE TRES INHIBIDORES DE LA ENZIMA DE CONVERSIÓN DE LA ANGIOTENSINA EN CABALLOS SANOS NORMOTENSOS

INTRODUCCIÓN. En los últimos años, se ha incrementado el interés por patologías que conllevan a pérdida de rendimiento en el caballo, incluyendo enfermedades cardiovasculares, insuficiencia cardiaca congestiva e hipertensión, ésta última asociada a enfermedades metabólicas. Las posibles causas de esta circunstancia son la prevalencia elevada de endocrinopatías y una elevación de las poblaciones de caballos geriátricos y de deporte. En estas patologías, se supone que se produce una activación del eje renina-angiotensina-aldosterona-vasopresina. Por ello, en medicina humana y en pequeños animales, el uso de fármacos que bloquean dicho eje es esencial en el control de la progresión de la enfermedad cardiovascular e hipertensión, usando fundamentalmente los compuestos que inhiben la enzima de conversión de la angiotensina (ACEI). Esta enzima interviene en la transformación de angiotensinógeno en angiotensina, compuesto con una importante actividad vasopresora. Hasta el comienzo de nuestros estudios, solo había un estudio que evaluaba las propiedades farmacocinéticas (PK) y farmacodinámicas (PD) de un ACEI en el caballo, el enalaprilo.

OBJETIVOS. 1) Establecer un perfil concentración-tiempo para tres diferentes IECAs (enalaprilo, ramiprilo y benaceprilo), y sus metabolitos activos (enalaprilato, ramiprilato y benaceprilato), después de la administración intravenosa (IV) de enaprilo (0.5 mg/kg), ramiprilo (0.05 mg/kg) o benaceprilo (0.5 mg/kg) y oral (PO) de placebo (0.0 mg/kg), enalaprilo (0.5, 1.0 y 2.0 mg/kg), ramiprilo (0.05, 0.1, 0.2 y 0.4 mg/kg) y benaceprilo (0.25, 0.5 y 1.0 mg/kg); 2) Determinar los efectos del enalaprilato, ramiprilato y benaceprilato sobre la actividad sérica de la enzima ACE; 3) Investigar los efectos de estos fármacos sobre la presión sanguínea (BP), considerando las dosis y las vías de administración; 4) Predecir y simular la dosis PO más apropiada capaz de suprimir la actividad ACE tras una única dosis y tras dosis múltiples de los tres ACEIs, utilizando un modelo farmacocinético-farmacodinámico; 5) Analizar si, la administración PO de estos

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Manuel Gómez Díez fármacos antes del ejercicio resultaría en una modulación de la hipertensión inducida por un ejercicio intenso, evaluando si los cambios en BP estarían correlacionados con el porcentaje de inhibición de la enzima ACE.

MATERIAL Y MÉTODOS. Se han estudiado 7 caballos (enalaprilo) o 6 caballos (ramiprilo, benaceprilo y control de la hipertensión inducida por ejercicio), adultos, sanos y normotensos. En todos los casos, se monitorizó la BP y se obtuvieron muestras de sangre para la medición de la actividad enzimática ACE (espectrofotometría) y de las concentraciones de los fármacos (enalaprilato, ramiprilato y benaceprilato) y de los profármacos (enalaprilo, ramiprilo y benaceprilo), mediante cromatografía líquida con detección fluorescente y por espectrometría de masas). Estas determinaciones se hicieron antes de la administración de los profármacos y a tiempos definidos durante las primeras 48 o 72 h post-administración. Para ramiprilo (y ramiprilato), benaceprilo (y benaceprilato), y para cada animal, se analizaron los datos concentración-tiempo junto con los datos inhibición ACE-tiempo usando un modelo farmacocinético- farmacodinámico compartimental fisiológico. En relación al estudio de la hipertensión inducida por ejercicio, se administró un ACEI PO (0.0 mg/kg placebo; 2.0 mg/kg enalaprilo; 1.0 mg/kg quinaprilo; 0.2 mg/kg ramiprilo; 0.5 mg/kg benaceprilo) y a las 2 h, los caballos fueron sometidos a un ejercicio intenso y de corta duración en cinta rodante de alta velocidad (calentamiento seguido de 1 min al galope, a una velocidad de 8 m/s, con una inclinación de la cinta rodante del 6%). Se midió la BP y se tomaron muestras de sangre para cuantificar el grado de inhibición de la enzima ACE, antes de la administración del placebo o de los ACEI, 2 h después de su administración (antes del ejercicio) y dentro de los 20 s posteriores a la conclusión del ejercicio.

RESULTADOS. No se ha apreciado ningún efecto negativo de la administración de los 3 fármacos estudiados. Los valores de BP no experimentaron variaciones significativas durante los diversos estudios, permaneciendo los animales, en todos los casos, en normotensión. De los tres fármacos estudiados, el enalaprilo mostró una absorción oral inadecuada y no fue posible su modelado farmacocinético-farmacodinámico de forma precisa, a diferencia del ramiprilo y benaceprilo y sus metabolitos activos. El modelado proporcionó datos importantes como los niveles de ACE circulante en caballos (10-18

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Angiotensin-converting enzyme inhibitors in the horse nmol/l) y su fracción respecto a la ACE tisular (10-14%), el grado de fijación e inhibición de cada ACEI a la ACE (kd = 1-2 nmol/l, IC50 = 0.5-1.5 nmol/l), y su biodisponibilidad oral (2-6%). Los valores medios máximos de inhibición de la enzima ACE fueron los siguientes: para el enalaprilo (0,05 mg/kg IV: 88,38%; 0,5 mg/kg PO: 21,69%; 1,0 mg/kg PO: 26,11%; 2,0 mg/kg PO: 30,19%), ramiprilo (0,05 mg/kg IV: 98,88%; 0,05 mg/kg PO: 27,68%; 0,1 mg/kg PO: 39,27%; 0,2 mg/kg PO: 46,67%; 0,4 mg/kg PO: 76,13%; 0,8 mg/kg PO: 84,27%) y benaceprilo (0,05 mg/kg IV: 99,63%; 0,25 mg/kg PO: 78,91%; 0,5 mg/kg PO: 85,71%; 1,0 mg/kg PO: 89,51%). Las simulaciones de dosis múltiples con ramiprilo a 0,8 mg/kg y benaceprilo a 1,0 mg/kg cada 24 h sugirieron que estas posologías podrían ser útiles en pacientes equinos. El ejercicio resultó en un incremento marcado de la BP sistólica (67,59%), junto con un aumento leve de la BP diastólica (20,64%) (experimento placebo). La elevación de la BP sistólica durante el ejercicio, realizado a las 2 h de la administración de los ACEIs alcanzó valores medios de 52,71%, 43,11%, 26,64% y 4,21%, para enalaprilo, quinaprilo, ramiprilo y benaceprilo respectivamente. El aumento de la BP no fue diferente entre enalaprilo, quinaprilo y ramiprilo, si bien fue inferior en comparación con el experimento placebo. La administración de benaceprilo 2 h antes del ejercicio moduló la elevación de la BP en respuesta al ejercicio. El porcentaje de inhibición de la enzima ACE estuvo correlacionado negativamente con el porcentaje de aumento experimentado por la BP.

CONCLUSIONES. El benaceprilo, a las dosis analizadas, fue el fármaco ACEI que indujo una mayor inhibición de la enzima ACE. La biodisponibilidad obtenida del 2-4 % fue baja pero suficiente para inducir inhibiciones de la ACE sérica cercanas al 80-90%. Sin embargo, los resultados obtenidos con ramiprilo fueron de la misma magnitud, sugiriendo que ambos fármacos podrían ser utilizados en caballo, a diferencia del enalaprilo, cuyas propiedades farmacocinéticas no fueron adecuadas

RELEVANCIA CLÍNICA. En función de las características PK-PD y por el grado de inhibición de la enzima ACE, los fármacos más prometedores clínicamente parecen ser el benaceprilo y el ramiprilo. El efecto de estos fármacos en el control de la hipertensión y de la progresión de la enfermedad cardiovascular necesita ser investigado en caballos enfermos.

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PALABRAS CLAVE. Caballos. Eje renina angiotensina aldosterona vasopresina. Farmacocinética. Farmacodinamia. Hipertensión. Inhibidores de la enzima de conversión de la angiotensina. Patologías cardiovasculares.

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8. SUMMARY

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PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES OF THREE ANGIOTENSIN-CONVERTING ENZYME INHIBITORS IN HEALTHY NORMOTENSIVE HORSES

BACKGROUND. Diseases that potentially might lead to loss of performance, congestive heart failure and hypertension, the last clinical signs linked to metabolic diseases, have been growing in clinical importance in the last years in equines, in association with an enhanced prevalence of endocrinopathies, and rising of populations of geriatric and sport horses. An activation of the renin-angiotensin-aldosterone-vasopressin axis is supposed to happen in these diseases. In human and small animal medicine, the administration of blockers of this axis is of great importance to control the progression of cardiovascular disease and systemic hypertension, being the most commonly used treatment the drugs that inhibit the angiotensin-converting enzyme (ACEI). This enzyme acts in the conversion of angiotensinogen in angiotensin, a vasopressor compound. Until the onset of our studies, there was only one paper published regarding pharmacokinetic (PK) and pharmacodynamic (PD) properties in one ACEI drug, the enalapril.

PURPOSES. 1) To establish a concentration-time profile for three different ACEI drugs (enalapril, ramipril and benazepril) and for their active metabolites (enalaprilat, ramiprilat and benazeprilat), after intravenous (IV) administration of enalapril (0.5 mg/kg), ramipril (0.05 mg/kg) or benazepril (0.5 mg/kg) and oral (PO) placebo (0.0 mg/kg), enalapril (0.1, 1.0 and 2.0 mg/kg), ramipril (0.05, 0.1, 0.2 and 0.4 mg/kg) and benazepril (0.25, 0.5 and 1.0 mg/kg); 2) To determine the effects of enalaprilat, ramiprilat and benazeprilat on serum ACE activity; 3) To investigate the effects of these drugs on blood pressure (BP), taken into consideration doses and administration routes; 4) To predict and to simulate the most appropriate dose to suppress ACE activity after one and multiple doses of the three ACEIs using a PK-PD model; 5) To analyze whether the administration of these drugs before exercise would result in a modulation of exercise-induced hypertension, evaluating if these changes in BP might be associated with the percentage of inhibition of serum ACE activity.

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MATERIAL AND METHODS. A total of 7 adult, healthy and normotensive horses (enalapril experiment) or 6 horses (ramipril, benazepril and modeling of exercise- induced hypertension experiments), were recruited for this research. In all the cases, BP was monitored, and blood samples were taken in order to measure serum ACE activity (spectrophotometry) and drug (enalaprilat, ramiprilat and benazeprilat) and pro-drug (enalapril, ramipril and benazepril) concentrations (liquid chromatography-tandem mass spectrometry). These measurements and analysis were made before administration of the pro-drugs and at fixed times during the first 48 or 72 h after administration. For ramipril (and ramiprilat) and benazepril (and benazeprilat) and for each animal, concentration-time and ACE inhibition-time data were analyzed using a physiological compartmental PK-PD model. In the exercise-induced hypertension study, a PO ACE (0.0 mg/kg placebo; 2.0 mg/kg enalapril; 1.0 mg/kg quinapril; 0.2 mg/kg ramipril; 0.5 mg/kg benazepril) was provided 2 h before exercise and after that, horses were subjected to a treadmill intense brief exercise (warming-up, followed by 1 min galloping at 8 m/s with a treadmill slope of 6%). BP was measured, and blood samples were obtained to measure serum ACE activity, before placebo or ACEI administration, 2 h after administration (immediately before exercise) and within the first 20 s after exercise.

RESULTS. Negative or side effects of the administration of the drugs were not observed. BP pressure did not change significantly during the experiments, and in all the cases, the horses remained normotensive. Of the three ACEI investigated, enalapril showed an inadequate oral absorption, unlike ramipril and benazepril and their active metabolites. Modeling studies provided important data including levels of circulating ACE in horses (10-18 nmol/l) and its fraction in relation to tissular ACE (10-14%), degree of fixation and inhibition of each ACEI to ACE enzyme (kd = 1-2 nmol/l, IC50 = 0.5-1.5 nmol/l), and their oral bioavailability (2-6%). Mean maximum inhibition values for serum ACE enzyme were those: for enalapril (0.05 mg/kg IV: 88.38%; 0.5 mg/kg PO: 21.69%; 1.0 mg/kg PO: 26.11%; 2.0 mg/kg PO: 30.19%), ramipril (0.05 mg/kg IV: 98.88%; 0.05 mg/kg PO: 27.68%; 0.1 mg/kg PO: 39.27%; 0.2 mg/kg PO: 46.67%; 0.4 mg/kg PO: 76.13%; 0.8 mg/kg PO: 84.27%) and benazepril (0,05 mg/kg IV: 99.63%; 0.25 mg/kg PO: 78.91%; 0.5 mg/kg PO: 85.71%; 1.0 mg/kg PO: 89.51%). Simulations with multiple doses with ramipril at 0.8

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Angiotensin-converting enzyme inhibitors in the horse mg/kg and benazepril at 1.0 mg/kg every 24 h suggested that these posologies might be useful for equine patients. Brief intense exercise resulted in a marked and significant rise in systolic BP (67.59%) and a mild rise in diastolic BP (20.64%) (placebo experiment). The elevation of systolic BP during exercise, performed 2 h after placebo or ACEI administration, reached values of 52.71%, 43.11%, 26.64% and 4.21% for enalapril, quinapril, ramipril and benazepril respectively. This rise in BP did not differ between enalapril, quinapril and ramipril, and in the three cases, it was lower compared with the increase observed in the placebo experiment. Administration of benazepril 2 h before exercise resulted in a modulation of the rise in BP in response to exercise. The percentage of serum ACE inhibition was negatively correlated with the percentage of rise of BP.

CONCLUSIONS. Benazepril, at the tested doses, was the ACEI drug that induced a greater serum ACE inhibition. The bioavailability obtained was low, 2-4%, but of enough intensity to reach serum ACE inhibitions near 80-90%. The results with ramipril were almost of the same magnitude, suggesting that both ACEIs might be used in diseased horses, unlike enalapril, whose PK properties were not appropriate.

CLINICAL REVELANCE. As a consequence of their PK-PD properties and of the inhibition of the serum ACE activity, the clinically most promising ACEI drugs appear to be benazepril and ramipril. The effect of these drugs in the control of hypertension and progression of cardiovascular disease needs to be investigated in diseases horses in a near future.

KEY WORDS. Angiotensin-converting enzyme inhibitor. Cardiovascular diseases. Hypertension. Horses. Pharmacodynamic. Pharmacokinetic. Renin-angiotensin- aldosterone-vasopressin axis.

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SISSON D. Neuroendocrine evaluation of SERRANO-RODRÍGUEZ JM, GÓMEZ-DÍEZ M, cardiac disease. Veterinary Clinics of North ESGUEVA M, CASTEJÓN-RIBER C, MENA- America: Small Animal Practice. 2004; BRAVO A, PRIEGO-CAPOTE F, AYALA N, 34(5):1105-1126. CABALLERO JM, MUÑOZ A. Pharmacokinetic/ pharmacodynamic modeling of benazepril and benazeprilat SISSON DD, OYAMA MA, SOLTER PF. Plasma after administration of intravenous and oral levels of ANP, BNP, epinephrine, doses of benazepril in healthy horses. norepinephrine, serum aldosterone, and Research in Veterinary Science. 2017; plasma renin-activity in healthy cats and cats 114:117-122. with myocardial disease. Journal of Veterinary Internal Medicine. 2003; 17:438 (abstract). SHIMOKAWA H, FLAVAHAN NA, LORENZ RR, VANHOUTTE PM. Prostacyclin releases SLACK J, BOSTON R, SOMA L, REEF V. endothelium-derived relaxing factor and Occurrence of cardiac arrhythmias in potentiates its action in coronary arteries of Standardbred racehorses. Equine Veterinary the pig. British Journal of Pharmacology. Journal. 2014; 47(4):398-404. 1988; 95(4):1197-1203.

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SLEEPER MM, PALMER JE. Echocardiographic STEWART JM, DEAN R, BROWN M, diagnosis of transposition of the great DIASPARRA D, ZEBALLOS GA, SCHUSTEK M, arteries in a neonatal foal. Veterinary GEWITZ MH, THOMPSON CI, HINTZE TH. Radiology and Ultrasound. 2005; 46(3):259- Bilateral atrial appendectomy abolishes 262. increased plasma atrial natriuretic peptide release and blunts sodium and water SMITH GW, CONSTABLE PD, FOREMAN JH, excretion during volume loading in conscious EPPLEY RM, WAGGONER AL, TUMBLESON dogs. Circulation Research. 1992; 724-732. ME, HASCHEK WM. Cardiovascular changes STREETER R, DIVERS T, MITTEL L, KORN A, associated with intravenous administration WAKSHLAG J. Selenium deficiency of fumonisin B1 in horses. American Journal associations with gender, breed, serum of Veterinary Research. 2002; 63(4):538- vitamin E and creatine kinase, clinical signs 545. and diagnoses in horses of different age groups: a retrospective examination. Equine SMITH KF, QUINN RL, RAHILLY LJ. Biomarkers Veterinary Journal. 2012; 43:31-35. for differentiation of causes of respiratory distress in dogs and cats: part 1. Cardiac SULLIVAN SL, WHITTEM T, MORLEY PS, diseases and pulmonary hypertension. HINCHCLIFF KW. A systemic review and Journal of Veterinary Emergency and Critical meta-analysis of the efficacy of furosemide Care (San Antonio). 2015; 25(3):311-329. for exercise-induced pulmonary hemorrhage in Thoroughbred and Standardbred SOUALMIA H, BARTHELEMY C, MASSON F, racehorses. Equine Veterinary Journal. 2015; MAISTRE G, EURIN J, CARAYON A. 47(3):341-349. Angiotensin II-induced phosphoinositide production and atrial natriuretic peptide SWAAN PW, STEHOUWER MC, TUKKER JJ. release in rat atrial tissue. Journal of Molecular mechanisms for the relative Cardiovascascular Pharmacology. 1997; binding affinity to the intestinal peptide 29:605-611. carrier. Comparison of three ACE-inhibitors: STACK A, DERKSEN FJ, SORDILLO LM, enalapril, enalaprilat, and lisinopril. WILLIAMS KJ, STICK JA, BRANDENBERGER C, Biochimica et Biophysica Acta. 1995; STEIBEL JP, ROBINSON NE. Effects of exercise 1236(1):31-38. on markers of venous remodeling in lungs of TAKEMURA N, KOYAMA H, SATO T, ANDO K, horses. American Journal of Veterinary MOTOYOSHI S, MARUMO F. Bovine atrial Research. 2013; 74(9):1231-1238. natriuretic peptide in heart failure. Journal of Endocrinology. 1990; 124:463-467. STEVENS K, MARR C, HORN J, PFEIFFER D, PERKINS J, BOWEN I, ALLAN EJ, CAMPBELL J, TAMARGO M, TAMARGO J. Future drug ELLIOTT J. Effect of left-sided valvular discovery in renin angiotensin aldosterone regurgitation on mortality and causes of system intervention. Expert Opinion on Drug death among a population of middle-aged Discovery. 2017; 12(8):827-848.

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THE IMPROVE STUDY GROUP. Acute and TIDHOLM A, HAGGSTROM J, HANSSON K. short-term hemodynamic, Effects of dilated cardiomyopathy on the echocardiographic and clinical effects of renin-angiotensin-aldosterone system, atrial enalapril maleate in dogs with naturally natriuretic peptide activity, and thyroid acquired heart failure: results of invasive hormone concentrations in dogs. American multicenter prospective veterinary Journal of Veterinary Research. 2001; evaluation of enalapril study. Journal of 62(6):961-967. Veterinary Internal Medicine. 1995; 9:234- 242. TIDHOLM A, HÄGGSTRÖM J, HANSSON K. Vasopressin, cortisol, and catecholamine THE LIVE STUDY GROUP. Effects of enalapril concentrations in dogs with dilated maleate on survival in dogs with naturally cardiomyopathy. American Journal of acquired heart failure: results of the long- Veterinary Research. 2005; 66(10):1709- term investigation of preliminary enalapril 1717. (LIVE Study Group). Journal of American

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TOUTAIN PL, LEFÈBVRE HP, LAROUTE V. New TSUTAMOTO T, ATSUYUKI M, MAEDA K, insights on effect of kidney insufficiency on HISANAGA T, MAEDA Y, FUKAI D, OHNISHI disposition of angiotensin-converting M, SUGIMOTO Y, KINOSHITA M. Attenuation enzyme inhibitors: case of enalapril and of compensation of endogenous natriuretic benazepril in dogs. Journal of Pharmacology peptide system in chronic heart failure. and Experimental Therapeutics. 2000b; Prognostic role of plasma brain natriuretic 292(3):1094-1103. peptide concentration in patients with chronic symptomatic left ventricular TOUTAIN PL, LEFÈBVRE HP. Pharmacokinetic dysfunction. Circulation. 1997; 86:509-516. and pharmacokinetic/pharmacodynamic TURK JR. Physiologic and pathophysiological relationships for angiotensin-converting effects of natriuretic peptides and their enzyme inhibitors. Journal of Veterinary implications in cardiopulmonary disease. Pharmacology and Therapeutics. 2004; Journal of American Veterinary Medical 27:515-525. Association. 2000; 216(12):1970-1976.

TRACHSEL D, GRENACHER B, UECHI M, IMAMOTO S, ISHIKAWA Y. Dose- SCHWARZWALD C. Plasma atrial/A-type dependent inhibition of angiotensin- natriuretic peptide (ANP) concentration in converting enzyme by enalapril in cats. horses with various heart diseases. Journal

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VERBALIS JG. Vasopressin V2 receptor UEDA T, KAWAKAMI R, NISHIDA T, ONOUE K, antagonists. Journal of Molecular SOEDA T, OKAYAMA S, TAKEDA Y, Endocrinology. 2002; 29(1):1-9. WATANABE M, KAWATA H, UEMURA S, SAITO Y. Plasma renin activity is a strong and independent prognostic indicator in patients VERBRUGGE FH, STEELS P, GRIETEN L, NIJST with acute decompensated heart failure P, TANG WH, MULLENS W. Hyponatremia in treated with renin-angiotensin system acute decompensated heart failure: inhibitors. Circulation Journal. 2015; depletion versus dilution. Journal of the 79(6):1307-1314. American College of Cardiology. 2015; 65(5):480-492. ULM EH, HINCHENS M, GOMEZ HJ, TILL AE, HAND E, VASSIL TC, BIOLLAZ J, BRUNNER HR, VIKSTROM KL, BOHLMEYER T, FACTOR SM, SCHELLING JL. Enalapril maleate and a lysine LEINWAND LA. Hypertrophy, pathology, and analogue (MK-521): disposition in the man. molecular markers of cardiac pathogenesis. British Journal of Clinical Pharmacology. Circulation Research. 1998; 20:773-778. 1982; 14:357-362. VOLLMAR AC, FOX PR. Long-term outcome of Irish Wolfhound dogs with preclinical UNGER T, LI J. The role of the renin cardiomyopathy, atrial fibrillation, or both angiotensin aldosterone system in heart treated with pimobendan, benazepril failure. Journal of Renin Angiotensin hydrochloride, or methyldigoxin Aldosterone System. 2004; 1:S7-S10. monotherapy. Journal of Veterinary Internal Medicine. 2016; 30(2):553-559. VAN DER VEKENS N, DECLOEDT A, DE CLERCQ D, VEN S, SYS S, VAN LOON G. Atrial VOLLMAR AM, MONTAG C, PREUSSER U, natriuretic peptide vs. N-terminal-pro-atrial KRAFT W, SCHULTZ R. Atrial natriuretic natriuretic peptide for the detection of left peptide and plasma volume of dogs suffering atrial dilatation in horses. Equine Veterinary from heart failure or dehydration. Journal. 2016; 48(1):15-20. Zentralblatt Veterinarmedicine A. 1994; 41:548-557. VAN GRIENSVEN JM, SCHOEMAKER RC, WAEBER B, BRUNNER HR, BRUNNER DB, COHEN AF, LUUS HG, SEIBERT-GRAFE M, CURTET AL, TURINI CA, GAVRAS H. RÖTHIG HJ. Pharmacokinetics, Discrepancy between antihypertensive pharmacodynamics and bioavailability of the effect and angiotensin converting enzyme ACE inhibitor ramipril. European Journal of inhibition by captopril. Hypertension. 1980; Clinical Pharmacology. 1995; 47: 513-518 2:236-242.

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YATSU T, KUSAYAMA T, TOMURA Y, ARAI Y, YOUNG LE. Cardiac responses to training in AOKI M, TAHARA A, WADA K, TSUKADA J. 2-year-old thoroughbreds: an Effect of conivaptan, a combined echocardiographic study. Equine Veterinary vasopressin V(1a) and V(2) receptor Journal. 1999; 30:195-198. antagonist, on vasopressin-induced cardiac and hemodynamic changes in anaesthetized

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ZHAO J, WANG X, HANG P, YUN F, ZHAO H, ZUCCA E, FERRUCCI F, STANCARI G, SAPORITI XU W, SUN D, SUN L, LI Y. Valsartan inhibits T, FERRO E. The prevalence of cardiac transient receptor potential canonical-3 murmurs among Standardbred racehorses channel in canine atrial fibrillation. presented with poor performance. Journal International Journal of Cardiology. 2013; of Veterinary Medical Science. 2010; 168(4):4417-4418. 72(6):781-785.

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10. LIST OF ABBREVIATIONS

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ABBREVIATIONS MEANING

%Imax Maximum serum angiotensin converting enzyme inhibition

%I12h Serum angiotensin converting enzyme inhibition at 12 h

%I24h Serum angiotensin converting enzyme inhibition at 24 h

%I36h Serum angiotensin converting enzyme inhibition at 36 h

%I48h Serum angiotensin converting enzyme inhibition at 48 h

%I72h Serum angiotensin converting enzyme inhibition at 72 h

%TImax Time to reach maximum serum angiotensin converting enzyme inhibition AAR Angiotensin receptor blockers or antagonist of AT1 receptor ACE Angiotensin converting enzyme ACEI Angiotensin converting enzyme inhibitor ACTH Adrenal corticotropic hormone ADH Antidiuretic hormone ALD Aldosterone AMPc Cyclic adenosine monophosphate ANG Angiotensin ANG-I Angiotensin I ANG-II Angiotensin II ANG-III Angiotensin III ANP Atrial natriuretic peptide ARNI Angiotensin receptor neprilysin inhibitor AT1 Angiotensin receptor type 1 AT2 Angiotensin receptor type 2

AUC0-24 Area under the curve from 0 to 24 h AURC Area under the response curve AVP Arginine vasopressin

Bmax Total binding of angiotensin converting enzyme pool BNP Brain natriuretic peptide BP Blood pressure BPFs Bradykinin potentiating factors BUN Blood ureic nitrogen Ca2+ Calcium CCB

Cdrugfree Free angiotensin converting enzyme inhibitor fraction concentration that can binding to angiotensin converting enzyme or being eliminated CHF Congestive or chronic heart failure Cl Drug clearance

Cl/F Drug clearance after extravascular administration or after biotransformation of parent drugs Cl- Chloride

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Cmax Maximum metabolite or drug concentration CNP C-type natriuretic peptide

Cprod-drugAb Amount of pro-drug absorbed following oral administration

Cpro-drugGI Pro-drug orally administered DBP Diastolic blood pressure

DBPImax Diastolic blood pressure at maximum serum angiotensin converting enzyme inhibition

DBPmin Minimum diastolic blood pressure DCM Dilated cardiomyopathy DRI Direct renin inhibitor DVD Degenerative valve disease E(t) Observed effect as serum angiotensin activity at each time expressed as a percentage of control values ECE Endothelin converting enzyme EDFR Endothelin-derived releasing factors EIPH Exercise-induced pulmonary hemorrhage EMA Europe Medicine Agency EMS Equine metabolic syndrome ET Endothelin ET-1 Endothelin 1 ET-2 Endothelin 2 ET-3 Endothelin 3 ET-A Receptor A of endothelin ET-B Receptor B of endothelin F% Oral bioavailability

fcirc Fraction between circulating and noncirculating (endothelial) angiotensin converting enzyme pools FDA US Food and Drug Administration FEI Fédération Equéstre Internationelle h Hours HCM Hypertrophic cardiomyopathy

IC50 Free angiotensin converting enzyme inhibitor concentration required to produce a 50% of angiotensin converting enzyme inhibition

Imax Maximal inhibition IV Intravenous K+ Potassium

K10 Rate constant of elimination of free angiotensin converting enzyme fraction

K10p Rate constant of elimination of pro-drug by other pathways

Ka Rate constant of absorption

Kd Constant of dissociation of angiotensin converting enzyme

Kf Rate constant of biotransformation for pro-drug to angiotensin converting enzyme

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LOQ Limit of quantification LSA Low sodium diet MBP Mean blood pressure MetS Human metabolic syndrome min Minutes MRA Mineralocorticoid receptor antagonist MRT Mean residence time Na+ Sodium NEP Neprilysin NO Nitric oxide NSAID Non-steroidal anti-inflammatory drug NYHA New York Heart Association PAI-1 Plasminogen activator inhibitor type 1 PD Pharmacodynamic PDA Patent ductus arteriosus PEPT Peptide carrier mediated transport PG Prostaglandin PK Pharmacokinetic

Pmax Circulating angiotensin converting enzyme fraction PO Orally, per os RAAV Renin angiotensin aldosterone vasopressin axis RCM Restrictive cardiomyopathy REN Renin

SBPmin Minimum systolic blood pressure SBP Systolic blood pressure

SBPImax Systolic blood pressure at maximum serum angiotensin converting enzyme inhibition SNS Sympathetic nervous system t1/2 Half-life t1/2ʎz Terminal half-life t1/2k10 Terminal half-life of free angiotensin converting enzyme inhibitor fraction

TDBPmin Time to reach minimum diastolic blood pressure

Tmax Time to reach maximum metabolite concentration

TSBPmin Time to reach minimum systolic blood pressure V1A Receptor of arginine vasopressin V2 Receptor of arginine vasopressin

Vc Volume of distribution

Vss Volume of distribution at steady state

VC/F Volume of distribution after extravascular administration or after biotransformation of parent drugs γ Coefficient that describes the steepness of the sigmoid curve, Hill coefficient

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11. PUBLISHED PAPERS

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11.1. PAPER I

Pharmacokinetics and Pharmacodynamics of Enalapril and its Active Metabolite, Enalaprilat, at four Different Doses in Healthy Horses

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Research in Veterinary Science 97 (2014) 105–110

Contents lists available at ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

Pharmacokinetics and pharmacodynamics of enalapril and its active metabolite, enalaprilat, at four different doses in healthy horses Manuel Gómez-Díez a, Ana Muñoz a,b,*, Juan Manuel Serrano Caballero c, Cristina Riber a,b, Francisco Castejón a, Juan Manuel Serrano-Rodríguez c a Equine Sport Medicine Centre, CEMEDE, School of Veterinary Medicine, University of Córdoba, Spain b Department of Animal Medicine and Surgery, School of Veterinary Medicine, University of Córdoba, Spain c Department of Pharmacology, Toxicology and Legal and Forensic Medicine, School of Veterinary Medicine, University of Córdoba, Spain

ARTICLE INFO ABSTRACT

Article history: Pharmacokinetic and pharmacodynamic of IV enalapril at 0.50 mg/kg, PO placebo and PO enalapril at Received 24 December 2013 three different doses (0.50, 1.00 and 2.00 mg/kg) were analyzed in 7 healthy horses. Serum concentra- Accepted 8 June 2014 tions of enalapril and enalaprilat were determined for pharmacokinetic analysis. Angiotensin- converting enzyme (ACE) activity, serum ureic nitrogen (SUN), creatinine and electrolytes were measured, Keywords: and blood pressure was monitored for pharmacodynamic analysis. Angiotensin-converting enzyme inhibitor The elimination half-lives of enalapril and enalaprilat were 0.67 and 2.76 h respectively after IV enalapril. Blood pressure Enalapril concentrations after PO administrations were below the limit of quantification (10 ng/ml) in Enalapril Enalaprilat all horses and enalaprilat concentrations were below the limit of quantification in 4 of the 7 horses. Horses Maximum mean ACE inhibitions from baseline were 88.38, 3.24, 21.69, 26.11 and 30.19% for IV enalapril at 0.50 mg/kg, placebo and PO enalapril at 0.50, 1.00 and 2.00 mg/kg, respectively. Blood pressures, SUN, creatinine and electrolytes remained unchanged during the experiments. © 2014 Published by Elsevier Ltd.

1. Introduction with chronic heart failure (Hamlin et al., 1996; The COVE Study Group, 1995; The IMPROVE Study Group, 1995; The LIVE Study The renin-angiotensin-aldosterone system (RAAS) is pivotal for Group, 1998). preserving hemodynamic and cardiovascular function (Atlas, 2007; These drugs are commonly used as vasodilator agents and as they Brewster et al., 2003). Angiotensin II is the primary effector of RAAS- are indicated for long-term therapy, only the oral route is conve- induced physiological and pathological situations (Atlas, 2007; nient. However, their oral bioavailability is low, and they must be Brewster et al., 2003; Brown and Vaughan, 1998). used as pro-drugs. Indeed, the pro-drugs are esters that can be ab- Dysregulation of the RAAS is involved in the pathogenesis of some sorbed from the digestive tract because they are more lipophilic. hypertensive and non-hypertensive disorders, such as chronic heart After intestinal absorption, the pro-drug is transformed into its active failure and other edematous disorders (cirrhosis with ascitis and metabolite by a carboxyl esterase (Toutain and Lefèbvre, 2004). nephrotic syndrome) (Atlas, 2007; Brewster et al., 2003; Gehlen et al., The pharmacokinetic (PK) and pharmacodynamic (PD) proper- 2008; Koch et al., 1995). ties of ACEIs have been studied in dogs and cats (Hamlin and Pharmacological inhibition of the RAAS can be achieved with the Nakayama, 1998; King et al., 2003; Lefèbvre et al., 1999; Uechi et al., administration of angiotensin-converting enzyme inhibitors (ACEIs), 2002), and the pharmacokinetic/pharmacodynamic (PK/PD) rela- which blocks the synthesis of angiotensin II. The use of these drugs tionships have been established by using serum angiotensin- to treat hypertension, heart and renal failure, has revolutionized vet- converting enzyme (ACE) inhibition as a surrogate marker (Toutain erinary cardiology and nephrology. The benefits of ACEIs in chronic and Lefèbvre, 2004). However, PK/PD studies of ACEIs have re- heart failure have been largely demonstrated in human patients (Barr ceived little attention in the horse (Afonso et al., 2013; Gardner et al., et al., 1997; Eriksson et al., 1994; Funck-Brentano et al., 2011). Sim- 2004), being the most studied enalapril and enalaprilat (Gardner ilarly, ACEIs have been proven to improve exercise tolerance, quality et al., 2004; Muir et al., 2001; Sleeper et al., 2008). of life, electrolyte status, disease progression and survival in dogs The PK and PD properties of enalapril and enalaprilat after in- travenous (IV) and oral (PO) administration of enalapril at 0.50 mg/ kg was investigated by Gardner et al. (2004). They found that the * Corresponding author. Tel.: +34 957 21 10 68; fax: +34 957 21 10 93. PO dose did not suppress serum ACE activity by more than 16%, E-mail address: [email protected] (A. Muñoz). unlike IV dosing, because the oral bioavailability was poor. PO http://dx.doi.org/10.1016/j.rvsc.2014.06.006 0034-5288/© 2014 Published by Elsevier Ltd. 106 M. Gómez-Díez et al./Research in Veterinary Science 97 (2014) 105–110 enalapril was also studied by Sleeper et al. (2008), at 0.50 mg/kg, 2.4. Blood processing and analysis for three months in ponies, and these authors reached the same con- clusions than Gardner et al. (2004). Blood samples were poured into plain tubes, without antico- These previously published articles described a good suppres- agulant. After centrifugation (1500 g for 15 min), serum was ob- sion of serum ACE activity in horses after IV administration of the tained and frozen at −90 °C until analysis. pro-drug at 0.50 mg/kg. However, the necessity of investigating the Serum ACE activity, serum urea concentration (SUN) and effects of higher doses by oral route was emphasized (Afonso et al., creatinine concentrations were measured by spectrophotometry 2013; Gardner et al., 2004; Sleeper et al., 2008). (Biosystems, model A-15), using specific reagents for this instru- The objectives of the present study were: (1) to establish the ment. Serum ACE activity was determined with the method de- serum concentration–time profile and to derive PK data for enalapril, scribed by Maguire and Price (1985). This method quantifies serum and its active metabolite enalaprilat, in healthy horses after IV dosing ACE activity by the hydrolysis of the N-[3-(2-furyl)acrylolyl]-L- at 0.50 mg/kg, and PO dosing at 0.50, 1.00 and 2.00 mg/kg; (2) to phenylalanylglycylglycine) (FAPGG), forming furylacryloyl- determine the absorption rate and bioavailability of PO enalapril phenylalanine (FAP). Serum Na+,K+ and Cl– electrolytes were at the three doses tested; (3) to investigate the effects on blood pres- measured with an analyzer with selective electrodes (Vetlyte, sure, and serum ACE activity with these doses and routes; and (4) Idexx®). to select the most appropriate PO dose that suppresses ACE activ- Serum concentrations of enalapril, enalaprilat and ramipril at ity compared to IV dose. We hypothesized that PO enalapril at doses 20 μg/ml as internal standard (IS) were measured using an HPLC higher than 0.5 mg/kg would lead to greater reductions in serum method with fluorescence detection previously reported (Oliva et al., ACE activity in healthy horses. 2005; Prieto et al., 2001). The system was equipped with a model LC-10Asvp pump, an RF-10Axl Fluorescence Detector and an SIL- 10Advp autoinjector (Shimadzu). The HPLC was connected to a com- 2. Material and methods puter with a Shimadzu Class-VP Chromatography Data System TM program. The chromatographic column was a Supelcosil LC18 The experimental protocol was approved by the Ethical Com- (250 × 4.6 m, 5 μm) preceded by a guard column (20 × 4 mm) of the mittee for Animal Experimentation of the University of Córdoba. same packing material. After addition of 10 μl of the IS solution to 0.7 ml of serum, 1 ml of methanol was added. Proteins were precipitated by shaking in 2.1. Horses an ultrasonic bath for 5 min, followed by centrifugation at 28,300 g for 10 min at 4 °C (Wang et al., 2007). The supernatant was ex- Seven cross-bred horses, 2 mares and 5 geldings, aged between tracted, and 3 ml of ethyl acetate was added. The mixture was mixed 5 and 11 years (9.14 ± 2.19), and weighing between 342 and 452 kg and centrifuged for 5 min at 2000 g. The upper organic layer was

(396.70 ± 33.85) were studied. Prior to the experiment, complete evaporated to dryness under N2 at 40 °C (Yuan et al., 2008). The physical examinations, hematology, serum biochemistry, includ- residue was reconstituted and sonicated in 200 μl of methanol and ing fibrinogen and myocardiac troponin concentrations, resting elec- transferred to autosampler vials, and an aliquot of 50 μl was in- trocardiography and echocardiography, were performed. Only healthy jected. The mobile phase was a mixture of solvent A (25:75 v/v animals were included in the experiment. methanol-phosphate buffer at 10 mM at pH 2.5) and solvent B (50:50 The animals were located in individual paddocks and received v/v methanol-phosphate buffer at 10 mM at pH 2.5) with a gradi- the same food, consisting of ryegrass haylage, at a volume of 4–5% ent elution of: 0–5 min, 100% A; 5–7 min, 50% A and 50% B; body weight. The horses did not have access to salt or electrolyte 7–30 min, 100% B; 30–35 min 100% A. The flow rate was 1.0 ml/ supplements during the study. min and the column temperature was maintained at 24 °C. Under these conditions, enalaprilat, enalapril and ramipril were eluted at 13, 16 and 24 min, respectively. The detection was performed at an 2.2. Experimental protocol excitation wavelength of 215 nm, and an emission wavelength of 284 nm. A cross-over design was used in five phases. Each animal re- Linear calibrations of enalapril and enalaprilat between 10 and ceived enalapril at doses of 0.50 mg/kg IV route and 0.00 (placebo), 500 ng/ml were obtained (r > 0.99). The accuracy and precision fell 0.50, 1.00, and 2.00 mg/kg PO route, using a nasogastric tube, with within the ranges of 6.2–7.2%. The limits of quantification (LOQ) and at least a 15-day of washout period. Food was withdrawn from 12 h detection (LOD) were 10 ng/ml and 7.5 ng/ml respectively. before administration of the pro-drug to 8 h after administration. Enalapril maleate (≥98% purity, Sigma-Aldrich, Madrid, Spain) was dissolved at 25 mg/ml in sterile water. This solution was used 2.5. Pharmacokinetic and pharmacodynamic analysis both for IV and PO routes. After PO dosing, the nasogastric tube was rinsed with 250 ml of water. Blood samples were taken from a jugular The serum concentration–time data of enalapril and enalaprilat, vein-catheter at the following times: before administration (value and the serum ACE activity were analyzed by non-compartmental at time 0), and at 5, 10, 15 and 30 min and at 1, 2, 4, 8, 12, 24, 36 methods, using a WinNonlin program (Version 5.3, Pharsight Cor- and 48 h after administration. poration, Mountain View, CA, USA). The elimination rate constant

(λZ) was estimated by linear regression of time vs. log concentra-

tion, and the elimination half-life as t½λz = (ln2)/λZ. The area under

2.3. Blood pressure monitoring the curve (AUC0→∞) and the first moment–time curve (AUMC0→∞) were calculated using the trapezoidal rule. Mean residence time was

Systolic and diastolic arterial pressures (SAP, DAP) were mea- calculated as MRT = AUMC0→∞/AUC0→∞. The serum initial concen- sured non-invasively in each blood sampling time in the coccy- tration of IV enalapril C0 was estimated by log-linear regression of geal artery (S/5 Datex-Ohmeda Compact®, multiparametric monitor). initial time points. The maximum metabolite concentration Cmax and

Three measurements of each blood pressure were made in each sam- the time to reach Cmax (Tmax) were taken directly from the data. Clear- pling time and the results are presented as the means of the three ance was calculated as Cl = Dose/AUC0→∞ and volume of distribu- measurements. tion at steady state as Vss = Cl·MRT (Gibaldi and Perrier, 1982). M. Gómez-Díez et al./Research in Veterinary Science 97 (2014) 105–110 107

Table 1 Pharmacokinetic parameters (mean ± SD) for enalapril and its metabolite enalaprilat in horses (n = 7) after IV enalapril at 0.50 mg/kg.

Parameters Units Enalapril Enalaprilat

λz 1/h 1.03 ± 0.31 0.25 ± 0.04 a t1/2λz h 0.67 2.76 C0 ng/ml 3814.48 ± 1975.06 – Cmax ng/ml – 584.78 ± 234.43 Tmax h – 0.09 ± 0.03 AUC0→∞ ng/ml·h 339.77 ± 117.87 866.16 ± 102.27 2 AUMC0→∞ ng/ml·h 117.50 ± 113.22 3077.23 ± 529.67 MRT h 0.50 ± 0.22 3.54 ± 0.38 Cl l/h/kg 1.66 ± 0.67 – Vss l/kg 0.83 ± 0.55 –

λz: elimination rate constant; t1/2λz: terminal half-life; C0: serum initial concentra- tion of intravenous pro-drug; Cmax: peak or maximum metabolite serum concen- tration following pro-drug administration; Tmax: time to reach peak or maximum metabolite serum concentration following pro-drug administration; AUC0→∞:area under the plasma concentration–time curve from zero to infinity; AUMC0→∞:area under the first moment curve from zero to infinity; MRT: mean residence time; Vss: apparent volume of distribution at steady state; Cl: total body clearance of drug from the plasma. a Harmonic mean.

Fig. 1. Semilogarithmic plot of serum concentrations (mean ± SD) of enalapril (-●-) and enalaprilat (-○-) after IV enalapril at 0.50 mg/kg in horses (n = 7).

after IV administration have been analyzed by PK methods and the obtained parameters are shown in Table 1. For the pharmacodynamic data, the area under the response curve Mean serum ACE activity values, expressed as percent of control (AURC) was calculated using the trapezoidal rule. Other param- (value at time 0) from 0 to 48 h are plotted in Fig. 2, for each ad- eters included maximum serum ACE inhibition between 0 and 12 h ministration route and dose. Table 2 shows the mean (±SD) AURC

(Imax), and the time to reach Imax (TImax), serum ACE inhibition at 24 values, as well as the inhibition values (Imax,I24% and I48%), the blood and 48 h, I24% and I48%, minimum systolic arterial pressure SAPmin, pressures values (SAPmin,DAPmin,SAPImax and DAPImax) and the time and the time to reach SAPmin (TSAPmin), minimum diastolic arterial to reach these values. pressure DAPmin,andthetimetoreachDAPmin (TDAPmin), SAPImax:sys- Significant differences in SUN, creatinine and electrolytes con- tolic arterial pressure at maximum serum ACE inhibition and DAPImax: centrations were not found when comparing routes, doses and sam- diastolic arterial pressure at maximum serum ACE inhibition, pling times. In all the cases, the concentrations were within the respectively. reference range used in our laboratory for horses (Muñoz et al., 2010). SUN concentrations ranged between 20.00 and 43.00 mg/dl ± 2.6. Statistical analysis (34.80 5.55 mg/dl) and creatinine concentrations were between 0.50 and 1.80 mg/dl (1.23 ± 0.25 mg/dl). Na+ concentrations between ± + Arithmetic mean and standard deviation were calculated. Har- 139 and 147 (141.6 3.20 mmol/l), K concentrations between 3.4 ± − monic means were calculated for the half-lives of elimination (Lam and 5.2 (3.99 0.47 mmol/l) and Cl concentrations between 91 and ± et al., 1985). The Wilcoxon rank sum test was used to test param- 107 (99.90 4.79 mmol/l) were found. eters for significant differences between IV and PO routes at 0.50 mg/ kg. The Kruskal–Wallis test was used for significant differences between PO doses including placebo. When differences were found, a Wilcoxon rank sum test was used (Powers, 1990). The statistical software used was Statgraphics Centurion XVI.I for windows (StatPoint Technologies). The minimum significant difference level was established at P < 0.05. Figures were computed using Sigmaplot software (SigmaPlot for Windows version 11.0, 2008, Systat Soft- ware Inc).

3. Results

No local or systemic adverse reactions were detected during or after IV and PO administrations. The mean (±SD) of serum concentrations of enalapril and enalaprilat after IV enalapril at 0.50 mg/kg are plotted in Fig. 1. Serum enalapril and enalaprilat concentrations were low and rapidly declined after IV administration. However, enalapril concentra- tions after PO administrations were below the LOQ (10 ng/ml) in all horses. Enalaprilat concentrations were also below the LOQ with the exception of three cases. Two horses had measurable concen- Fig. 2. Mean serum ACE activity expressed as percent of control after IV enalapril trations (12.33 and 11.15 ng/ml) at 1 h after PO enalapril at 0.50 mg/ at 0.50 mg/kg (black solid line; -○-), after PO enalapril at 0.00 mg/kg (placebo, black kg, and another horse, a concentration of 16.14 ng/ml at 2 h after dash line; --○--), at 0.50 mg/kg (grey solid line; -●-), at 1.00 mg/kg (grey dash line; PO enalapril at 2.00 mg/kg. Therefore, only serum concentrations --■--) and at 2.00 mg/kg (black dash line; -●-) in horses (n = 7). 108 M. Gómez-Díez et al./Research in Veterinary Science 97 (2014) 105–110

Table 2 Mean (±SD) of pharmacodynamic parameters after IV enalapril at 0.50 mg/kg, and PO enalapril at 0.00, 0.50, 1.00 and 2.00 mg/kg.

Route IV PO PO PO PO

Dose (mg/kg) 0.50 0.00 0.50 1.00 2.00 a,b c Imax (%) 88.38 ± 3.83 3.24 ± 1.84 21.69 ± 3.61 26.11 ± 6.47 30.19 ± 4.20 b b b TImax (h) 0.13 ± 0.06 1.17 ± 0.76 3.00 ± 1.41 4.00 ± 2.19 2.33 ± 1.53 b b b,d I24h (%) 16.78 ± 2.79 3.75 ± 1.42 17.13 ± 8.43 20.12 ± 6.40 14.06 ± 4.70 I48h (%) 16.35 ± 9.40 6.54 ± 2.82 11.89 ± 5.97 8.92 ± 5.57 6.52 ± 3.50 AURC (%·h) 1226.44 ± 422.95 88.20 ± 45.70 711.30 ± 313.22a,b 701.16 ± 257.05b 676.00 ± 150.25b SAPmin (mmHg) 125.57 ± 11.34 93.10 ± 19.89 110.00 ± 12.43 116.83 ± 13.54 107.80 ± 6.06 b TSAPmin (h) 4.67 ± 2.83 4.67 ± 2.83 4.33 ± 2.83 2.33 ± 1.53 4.00 ± 3.06 DAPmin (mmHg) 64.00 ± 8.51 62.50 ± 5.93 61.67 ± 13.97 61.00 ± 7.21 72.40 ± 8.24 b b TDAPmin (h) 3.00 ± 1.41 4.67 ± 3.06 2.33 ± 1.53 1.81 ± 0.77 5.00 ± 2.00 b SAPImax (mmHg) 148.60 ± 18.97 109.30 ± 8.17 130.67 ± 18.61 123.40 ± 15.57 114.20 ± 6.02 b DAPImax (mmHg) 83.20 ± 7.76 69.00 ± 12.91 76.33 ± 15.06 70.40 ± 12.14 83.53 ± 4.04

Imax: maximum serum ACE inhibition expressed as percent of control from 0 to 12 h; TImax: time to reach the maximum serum ACE inhibition; I24%: maximum serum ACE inhibition at 24 h expressed as percent of control; I48%: maximum serum ACE inhibition at 48 h expressed as percent of control; AURC: area under the response curve from 0to48h;SAPmin: minimum systolic arterial pressure; TSAPmin: time to reach the minimum systolic arterial pressure; DAPmin: minimum diastolic arterial pressure; TDAPmin: time to reach the minimum diastolic arterial pressure; SAPImax: systolic arterial pressure at maximum serum ACE inhibition; DAPImax: diastolic arterial pressure at maximum serum ACE inhibition. a Significantly different between IV dose at 0.50 mg/kg and PO at dose at 0.50 mg/kg (P < 0.05). b Significantly different from PO dose at 0.00 mg/kg (placebo) (P < 0.05). c Significantly different from PO dose at 0.50 mg/kg (P < 0.05). d Significantly different from PO dose at 1.00 mg/kg (P < 0.05).

4. Discussion were higher than those previously presented (0.38 h and 0.38 h, re- spectively, Gardner et al., 2004).

ACEIs have represented a great advance in the treatment and The t½λz values for enalapril and enalaprilat found in the current control of progression of cardiac disease and failure, chronic kidney study were similar to the values reported by Gardner et al. (2004) disease and systemic hypertension in small animals (Atkins et al., (0.86 and 2.01 h, respectively). However, it is necessary to note that 2007; Miller et al., 1999). Of these conditions, heart disease is by these values could not represent the true terminal half life. Serum far the most common in horses. Compared to small animals and concentrations of ACEIs are the sum of free drug in serum, drug human beings, vascular and myocardial diseases are uncommon bound nonspecifically to plasma proteins and specifically bound to in horses. By contrast, valvular disorders are quite common. A recent circulating ACE (Toutain and Lefèbvre, 2004). Therefore, the binding research performed on a hospital-based population of 3434 horses, parameters as the total binding capacity of the circulating ACE revealed that mitral regurgitation (4.4%), atrial fibrillation (2.3%), (Bmax), the binding affinity (Kd) and the partition of ACEI between atrial regurgitation (2.1%) and tricuspid regurgitation (1.7%) were circulating and tissular ACE(fcirc) should be determined. However, the most commonly diagnosed cardiac diseases (Leroux et al., 2013). this information is currently not available for enalaprilat in horse, Despite this prevalence, it is accepted that most of the cardiac and further studies are necessary in order to determine these diseases are not sufficiently severe to result in congestive or parameters. chronic heart failure (Davis et al., 2002). Few data are available on In the present study, it has been found that systemic availabil- the prevalence of congestive heart failure in horses, and only one ity of PO enalapril was low because the concentrations of enalapril study reported 7.9% of death attributable to cardiac diseases and enalaprilat were below the LOQ (10 ng/ml) in most of the horses. among a population of middle-aged and older horses (Stevens et al., These results agree with those obtained by Gardner et al. (2004), 2009). despite their analytical technique being more sensitive (LOQ of 5 ng/ ACEI drugs have received little attention in horses, despite the ml). These results indicate that, perhaps, the availability of enalapril suggestion that their use could improve life quality and increase life and enalaprilat in horses after PO administration of enalapril from span in horses with congestive heart failure (Gardner et al., 2004). 0.50 to 2.00 mg/kg is very low. The bioavailability of enalapril and More importantly, these drugs could limit the progression of a cardiac enalaprilat after PO enalapril is also low for other monogastric disease to a congestive heart failure. species, 40% at 5.00–20.00 mg for humans (Thind, 1990), and 20– PK/PD studies of enalapril were performed in healthy horses at 40% at 0.06–6.00 mg/kg for dogs (Morrison et al., 1996).

0.50 mg/kg dose by Gardner et al. (2004) by IV and PO routes. After The Imax value after IV enalapril at 0.50 mg/kg (88.38%) was similar PO dose, serum ACE inhibition was lower than 16%. It could be to that obtained by Muir et al. (2001) (>75%). This value was higher thought that higher doses could lead to higher ACE inhibition. For than obtained after PO dosing of enalapril (3.24, 21.69, 26.11 and that reason, the present study describes PK and PD properties of 30.19% at 0.00, 0.50, 1.00 and 2.00 mg/kg respectively). However, enalapril and enalaprilat, after administration of enalapril IV at the Imax value achieved after PO enalapril at 0.5 mg/kg was slightly 0.50 mg/kg compared to placebo PO and three different doses of PO higher that the value reported by Gardner et al. (2004) (16%). The enalapril 0.50, 1.00 and 2.00 mg/kg, in healthy horses. reduced inhibition of serum ACE activity after PO enalapril in horses A rapid biotransformation of enalapril to its active metabolite contrasts with the results obtained in humans (96.37% at 20 mg) was found, as serum enalaprilat concentrations were higher than (Rouini et al., 2002) and dogs (75%) (Hamlin and Nakayama, 1998). serum enalapril concentrations from 0.15 h post-administration of However, in cats, a single dose of enalapril PO at 0.50 mg/kg induced

IV enalapril. The Cl, Vss, AUC0→∞ and AUMC0→∞ values of enalapril an inhibition near 40%, lower than that described in dogs, but higher were very similar to the data reported by Gardner et al. (2004) (1.65 l/ than that found in horses (Uechi et al., 2002). h/kg, 0.64 l/kg, 320 ng/ml·h and 120 ng/ml·h2, respectively). However, The present study showed that inhibition of serum ACE activi-

AUC0→∞ and AUMC0→∞ values of enalaprilat were 816.16 ng/ml·h and ty by enalapril was low after PO doses, from 0.50 to 2.00 mg/kg, 3077.23 120 ng/ml·h2, respectively. Therefore, these parameters were because PO availability was low. Three main hypotheses can be pro- higher in our research (Gardner et al., 2004; 320 ng/ml·h and 120 ng/ posed to explain the low availability of PO enalapril and the limited ml·h2, respectively). The MRT values for enalapril and enalaprilat serum ACE inhibition in horses. M. Gómez-Díez et al./Research in Veterinary Science 97 (2014) 105–110 109

Firstly, after PO administration, ACEI pro-drugs can be ab- ing vasodilation and fluid loss, both desirable in congestive heart sorbed by passive diffusion and the higher intestinal uptake is as- failure (Brown and Vaughan, 1998). sociated with lipophilicity. However, it has been hypothesized that Some concerns about renal safety of ACEIs have arisen, because they can be actively transported by peptide carrier-mediated trans- angiotensin II maintains glomerular filtration rate, and therefore, port (PEPT). The low-affinity H+/peptide co-transporter, or PEPT1, ACEIs therapy could induce azotemia (Atkins et al., 2002; Brown and could play an important role in intestinal absorption of ACEIs (Sadée Vaughan, 1998). There are, however, some data suggesting that renal et al., 1999). In this regard, enalapril is considered as a prototypi- function improves after ACEIs in some glomerular diseases (Maschio cal PEPT substrate (Swaan et al., 1995). Recent studies have sug- et al., 1996). In the current research, none of the horses developed gested that these transporters could be present in horses (Cehak azotemia during the experiments in agreement with the results of et al., 2013), but in low quantities (Davis et al., 2014). However, other Gardner et al. (2004). Similarly, administration of IV and PO enalapril authors have shown that intestinal absorption of enalapril could not in our research did not result in significant changes in serum elec- be predictable by the activity of the PEPT1 transporter because its trolyte concentrations. In normotensive humans, dogs and cats, a affinity for enalapril is weak compared with other ACEIs as benazepril mild hyperkalemia has been described, attributable to a decrease (Knütter et al., 2008; Moore et al., 2000). Moreover, it has been shown in urinary K excretion (Davies et al., 1984; The COVE Study Group, that a low affinity by the PEPT1 transporter was associated with a 1995; Häggström et al., 1996). The lack of changes in serum elec- low lipophilicity, and intestinal absorption by simple passive dif- trolytes in our research is consistent with a normal renal function. fusion might be sufficient in many cases (Knütter et al., 2008). Secondly, the factors that contribute to PO absorption of drugs 5. Conclusions (intestinal permeability and aqueous solubility) have not been well characterized for many drugs in the horse (Davis et al., 2006). The current study shows that PO availability of enalapril at doses = Enalapril is more hydrophilic (logP 0.16) than other ACEIs such as from 0.50 to 2.00 mg/kg in horses is low. Our results suggest that, peridonpril, ramipril, quinapril or benazepril (logP values of 0.55, although there was a tendency toward a decrease in serum ACE ac- 1.03, 1.44 and 4.54, respectively) (ADC Labs Software, 2013; Ranadive tivity, these values were never suppressed by more than 30% unlike et al., 1992; Thind, 1990), and this property might affect intestinal IV data of 88.38% (3-fold to PO inhibition). Up to now, it is unknown absorption. Also, for drugs that are actively transported with high whether an ACE inhibition near 30%, as found in our study with affinity, their intestinal permeability would be greater than for enalapril PO at 2.0 mg/kg, could be enough to counteract the va- passive absorbed drugs (Riviere, 2011). It has been demonstrated soconstrictive effects of angiotensin II in horses with congestive heart that other more lipophilic pro-drugs could be absorbed and con- failure. verted to the active ACEI metabolites in the horse after PO admin- istration. I values of 47 and 53% after PO quinapril at 120 and max Acknowledgement 240 mg respectively have been reported (Davis et al., 2014; Kruger et al., 2011). In other study, I values of 19.9–25.2%, 38.2–59.7%, max This research has been generously supported by the Science and 29.6–40.6%, and 72.2–86.9% were obtained after PO peridonpril at Innovation Council of Spain (Research Project AGL2010-17431). 0.05–0.10 mg/kg, ramipril at 0.10–0.30 mg/kg, quinapril at 0.125– 0.250 mg/kg and benazepril at 0.25–0.50 mg/kg, respectively (Afonso et al., 2013). In addition, results from our laboratory shows that References serum ACE activity can be suppressed by more than 50% and 58% after PO ramipril at 0.20 mg/kg and quinapril at 0.48 mg/kg (un- ADC Labs Software, 2013. ACD/Structure Elucidator, version 12.01, Advanced Chemistry Development, Inc. (ACD/Labs), Toronto, ON. Available from: http:// published observations). www.acdlabs.com accessed 13 October 2013. Thirdly, the interspecies differences in the gastrointestinal Afonso, T., Giguère, S., Rapoport, G., Berghaus, L.J., Barton, M.H., Coleman, A.E., 2013. anatomy and physiology, and the influence of the diet in the oral Pharmacodynamic evaluation of 4 angiotensin-converting enzyme inhibitors in absorption of drugs in horses compared to other monogastric species healthy adult horses. Journal of Veterinary Internal Medicine 27, 1185–1192. Atkins, C.E., Brown, W.A., Coats, J.R., Crawford, M.A., DeFrancesco, T.C., Edwards, J., should be considered (Riviere, 2011; Toutain et al., 2010). et al., 2002. Effects of long-term administration of enalapril on clinical indicators SAP and DAP values did not change during the experiment, in of renal function in dogs with compensated mitral regurgitation. Journal of agreement with the results of Gardner et al. (2004), also in horses. American Veterinary Medical Association 221, 654–658. Atkins, C.E., Keene, B.W., Brown, W.A., Coats, J.R., Crawford, M.A., DeFrancesco, T.C., This may be due to, in normotensive subjects, ACEIs having little et al., 2007. Results of the veterinary enalapril trial to prove reduction in onset effect on blood pressure (Brown and Vaughan, 1998). However, a of heart failure in dogs with chronically treated enalapril alone for compensated, decrease in angiotensin II through inhibition of ACE includes dose- naturally occurring mitral valve insufficiency. Journal of American Veterinary Medical Association 231, 1061–1069. dependent generalized vasodilation and reductions in SAP and DAP Atlas, S.A., 2007. The renin-angiotensin-aldosterone system: pathophysiological role in hypertensive subjects (Atlas, 2007; Brown and Vaughan, 1998; and pharmacologic inhibition. Journal of Management and Care Pharmacy 13, Lefèbvre et al., 2007). S9–S20. Barr, C.S., Naas, A.A., Fenwick, M., Struthers, A.D., 1997. Enalapril reduces QTc In healthy humans, serum ACE activity must be reduced by ap- dispersion in mild congestive heart failure secondary to coronary artery disease. proximately 90% from baseline values to completely abolish blood The American Journal of Cardiology 79, 328–333. pressure increase in response to exogenously administered angio- Biollaz, J., Schelling, J.L., Jacob Des Combes, B., Brunner, D.B., Despong, G., Brunner, tensin I (Biollaz et al., 1985; Burnier et al., 1981). The minimum per- H.R., et al., 1985. Enalapril maleate and a lysine analogue (MK-521) in normal volunteers: relationship between plasma drug level and the renin angiotensin centage of ACE inhibition to be clinically beneficial in veterinary system. British Journal of Clinical Pharmacology 14, 363–368. patients needs to be established. Studies conducted in dogs have Brewster, U.C., Setaro, J.F., Perazella, M.A., 2003. The renin-angiotensin-aldosterone shown that approximately 75% maximum suppression of ACE after system: cardiorenal effects and implications for renal and cardiovascular disease states. American Journal of Medical Science 32, 15–24. PO enalapril at 0.50 mg/kg is associated with clinical benefits in pa- Brown, N.J., Vaughan, D.E., 1998. Angiotensin-converting enzyme inhibitors. tients with heart failure (Hamlin et al., 1996; Hamlin and Nakayama, Circulation 97, 1411–1420. 1998; The IMPROVE Study Group, 1995; The LIVE Study Group, 1998). Burnier, M., Turini, G.A., Brunner, H.R., Porchet, M., Kruithof, D., Vukovich, R.A., et al., 1981. RHC 3659: a new orally active angiotensin converting enzyme inhibitor Lower ACE inhibitions (63%) have been associated with extended in normal volunteers. British Journal of Clinical Pharmacology 12, 893–899. life span in dogs with congestive heart failure (The BENCH Study Cehak, A., Schröder, B., Feige, K., Breves, G., 2013. 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Manuel Gómez Díez

11.2. PAPER II

Pharmacokinetics and Pharmacodynamics of Ramipril and Ramiprilat after Intravenous and Oral Doses of Ramipril in Healthy Horses

Page 202

The Veterinary Journal 208 (2016) 38–43

Contents lists available at ScienceDirect

The Veterinary Journal

journal homepage: www.elsevier.com/locate/tvjl

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. Mena-Bravo 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, Córdoba, Spain b Equine Sport Medicine Centre, CEMEDE, University of Cordoba, Córdoba, Spain c Department of Analytical Chemistry, Faculty of Sciences, University of Cordoba, Córdoba, 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, Córdoba, Spain

ARTICLE INFO ABSTRACT

Article history: The pharmacokinetics and pharmacodynamics (PK/PD) of the angiotensin-converting enzyme inhibitor Accepted 8 October 2015 (ACEI) ramiprilat after intravenous (IV) and oral (PO) administration of ramipril have not been evalu- ated in horses. This study was designed to establish PK profiles for ramipril and ramiprilat as well as to Keywords: determine the effects of ramiprilat on serum angiotensin converting enzyme (ACE) and to select the most Angiotensin-converting enzyme inhibitors appropriate ramipril dose that suppresses ACE activity. Six healthy horses in a cross-over design re- Ramiprilat ceived IV ramipril 0.050 mg/kg, PO at a dose of 0 (placebo), and 0.050, 0.10, 0.20, 0.40 and 0.80 mg/kg Ramipril ramipril. Blood pressures were measured and blood samples obtained at different times. Serum ramipril Pharmacokinetic–pharmacodynamic Horses and ramiprilat concentrations and serum ACE activity were measured by liquid chromatography- tandem mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. Systemic bioavailability of ramiprilat after PO ramipril was 6–9%. Percentages of maximum ACE in- hibitions from baseline were 98.88 (IV ramipril), 5.31 (placebo) and 27.68, 39.27, 46.67, 76.13 and 84.27 (the five doses of PO ramipril). Blood pressure did not change during the experiments. Although oral avail- ability of ramiprilat was low, ramipril has sufficient enteral absorption and bioconversion to ramiprilat to induce serum ACE inhibitions of almost 85% after a dose of 0.80 mg/kg ramipril. Additional research on ramipril administration in equine patients is indicated. © 2015 Elsevier Ltd. All rights reserved.

Introduction mainly at the surface of vascular endothelium. Circulating ACE origi- nates mainly from the vascular endothelium, representing about Ramipril is an angiotensin converting enzyme inhibitor (ACEI) 5–30% of serum ACE in different species including humans, dogs and administered orally (PO) for long-term therapy of cardiovascular cats (Toutain and Lefebvre, 2004; Afonso et al., 2013). Therefore, disease, hypertension and renal failure in humans and small animals physiologically based models with binding parameters of ACEI to (Brewster et al., 2003; Lefebvre et al., 2007). Ramipril is a pro- ACE are required to allow appropriate interpretation of the differ- drug, and after PO absorption, is hydrolyzed in the liver to the active ent phases of disposition curves (Toutain and Lefebvre, 2004). To drug (ramiprilat). Ramiprilat prevents conversion of angiotensin I the best of our knowledge, these models have not been applied in to II by inhibiting angiotensin converting enzyme, ACE (Toutain and horses. Lefebvre, 2004; Lefebvre et al., 2007). Because angiotensin II causes PK/PD studies of ACEIs in horses are limited to enalaprilat and vasoconstriction, ACEIs are used as vasodilator agents (Toutain and quinaprilat after intravenous (IV) and PO enalapril and quinapril Lefebvre, 2004). (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez et al., 2014). Pharmacokinetic and pharmacodynamic (PK/PD) relationships Recently, Afonso et al. (2013) administered two PO doses of ramipril of ACEIs are complicated because they have a nonlinear binding to in horses (low, 0.1 mg/kg; high, 0.3 mg/kg) and evaluated serum ACE ACE, the target enzyme, and because there are two ACE pools, cir- inhibition and changes in blood pressure (BP). In this study, however, culating and in tissues. ACE is distributed throughout the body but IV ramipril was not administered and serum concentrations of ramipril and ramiprilat were not measured. The objectives of the present study were: (1) to establish the serum concentration–time profile for ramipril, and its active me- * Corresponding author. Tel.: +34 957 218702. tabolite ramiprilat after IV ramipril at 0.050 mg/kg, PO ramipril at E-mail address: [email protected] (J.M. Serrano-Rodríguez). 0 (placebo), and five different PO doses (0.050, 0.10, 0.20, 0.40 and http://dx.doi.org/10.1016/j.tvjl.2015.10.024 1090-0233/© 2015 Elsevier Ltd. All rights reserved. J.M. Serrano-Rodríguez et al./The Veterinary Journal 208 (2016) 38–43 39

0.80 mg/kg), using a physiologically based PK/PD model; (2) to de- termine the effects of ramiprilat on serum ACE; (3) to investigate the effects on BP with these doses and routes; and (4) to predict and to simulate the most appropriate PO dose that suppresses ACE activity after single and multiple doses of ramipril.

Material and methods

Horses

Six crossbred horses, two mares and four geldings, aged between 5 and 11 years (10.8 ± 2.35), and weighing between 378 and 530 kg (458.2 ± 59) were studied. Prior to the experiment, physical examinations, hematology, serum biochemistry, includ- ing fibrinogen and myocardial troponin concentrations, resting , echocardiography and blood pressure (BP) measurements, were performed. Only healthy animals were studied. The animals were located in individual paddocks and received the same food, consisting of rye-grass haylage, at 1% bodyweight. The horses did not have access Fig. 1. Physiologically based model for ramiprilat disposition after PO ramipril. For to salt or electrolyte supplements during the study. IV disposition of ramiprilat the model was similar without absorption phase of ramipril. For further explanation, see the text. Experimental protocol

The research was approved by the Ethical Committee for Animal Experimenta- tion of the University of Córdoba (Reference: 55.60 PE, date of approval: 1 February described (Lees et al., 1989; Toutain et al., 2000; Lefebvre et al., 2006).The model is 2010). presented in Fig. 1 and consists of a single compartment with a volume of distri- The study was conducted in two trials. In the first trial, each animal received bution of Vc in which ramipril is absorbed and transformed to ramiprilat according IV ramipril at 0.050 mg/kg. After a week of washout, the second trial was under- to absorption and formation first-order rate constants of ka and kf, respectively. taken, using a blinded and randomized Latin square (6 × 6) design. Each animal Ramipril might be eliminated via other pathways, including urinary excretion and received PO placebo or ramipril at 0.050, 0.10, 0.20, 0.40 or 0.80 mg/kg, with at least biotransformation to other metabolites (Van Griensven et al., 1995). For that reason, 1 week of washout between trials. Food was withdrawn from 12 h before admin- a first order elimination rate constant k10pwas included. Ramiprilat is bound to ACE istration of the pro-drug to 8 h after. or free or non-specifically bound to albumin. However, free and albumin-bound frac- For IV dosing, ramipril (Sigma-Aldrich) was dissolved in 0.7% saline and 1% NaHCO 3 tions are indistinguishable in the model. Consequently free ramiprilat corresponds (King et al., 2003). For PO doses, ramipril tablets (Vasotop P 5 mg, Merck Sharp and to free ramiprilat plus ramiprilat bound to albumin (Lefebvre et al., 2006). Free Dohme Animal Health) were dissolved and suspended in 150 mL of water, soni- ramiprilat fraction was eliminated according to a first order rate constant k10.Free cated in an ultrasonic bath for 15 min and stored at 4 °C before trials. For placebo, ramiprilat clearance was calculated as Cl = k10·Vc and the elimination half-live as t1/2 the equivalent amount of water without the pro-drug was used. Pro-drugs and placebo k10 = (ln2)/k10. were administered by nasogastric intubation and after, the nasogastric tube was rinsed After PO ramipril, the volume of distribution Vc/F and clearance Cl/F of ramiprilat with 250 mL of water. were calculated, with F indicating systemic bioavailability of ramiprilat after PO ad- In each assay, venous blood samples were taken before administration (time 0), ministration of ramipril. The F value was calculated from the ratio of Cl of ramiprilat and at 5, 10, 15 and 30 min and at 1, 2, 4, 8, 12, 24, 36 and 48 h after. after IV ramipril and Cl/F of ramiprilat after PO ramipril (Toutain and Lefebvre, 2004). Ideally, IV ramiprilat would have been administered to determine Vc, Cl and F values. Blood pressure monitoring However, the necessary amount of ramiprilat for IV formulation was not available. Administration of IV ramipril does, however, provide important information about Systolic (SBP) and diastolic (DBP) blood pressures were measured non-invasively the metabolism from ramipril to ramiprilat in horses as described by other ACEIs with a multiparametric monitor (S/5 Datex-Ohmeda Compact) from the coccygeal (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez et al., 2014). artery at each sampling time. Three BP measurements were made at each sam- Binding parameters of ramiprilat to ACE calculated by the model were: Kd, equi- pling point and the results are presented as the means of the three measurements. librium dissociation constant which expresses ramiprilat affinity for the ACE; Bmax, size of ACE pool; fcirc, fraction between circulating and non-circulating ACE and cir- Blood processing and analysis culating ACE as Pmax = fcirc ·Bmax. Data were weighted with the inverse of the square of the observation (Lefebvre et al., 2006). After extraction, blood samples were transferred to tubes without anticoagu- For PD data, the observed effect E(t) was defined as the serum ACE activity at lant and left to coagulate in a refrigerator for 30 min. They were then centrifuged, each time expressed as percent of control before administration of the pro-drug (E0), serum was obtained, and samples stored at −90 °C until analysis. IC50 was the free ramiprilat concentration required to produce 50% of ACE inhibi- Serum ACE activity was measured by spectrophotometry (Afonso et al., 2013; tion and γ was the coefficient which describes the steepness of the sigmoid curve. Gómez-Díez et al., 2014). This method quantifies serum ACE activity by hydrolysis C was the free ramiprilat concentration obtained after PK modeling (Lefebvre et al., of the N-(3-[2-furyl]acrylolyl)-Lphenylalanyl-glycyl-glycine(FAPGG), forming 2006). These parameters were fitted by the Hill equation: furylacryloyl-phenylalanine (FAP), that results in a decrease in absorbance at 340 nm. Linear calibration curves from 3 to 150 IU/L were obtained, and the correlations co- ⎛ ()γ ⎞ ()=⋅− C efficients (r) were >0.99. Precision of the technique was between 2.86% and 6.21%. Et E0 ⎜1 γγ⎟ ⎝ ()IC+ () C ⎠ The limits of quantification (LOQ) and detection (LOD) were 5 and 3 IU/L, respectively. 50 Serum concentrations of ramiprilat (Santa Cruz Biotechnology), ramipril and Other parameters obtained were the maximum serum ACE inhibition between enalapril as internal standard (Sigma-Aldrich) were measured using a liquid 0 and 12 h (%I ), the time to reach %I (T ), and serum ACE inhibition at 12, chromatography-tandem mass spectrometry (LC-MS/MS) assay (Yuan et al., 2008). max max %Imax 24 and 48 h (%I ,%I and %I ), respectively. A volume of 700 μL of horse serum was spiked with internal standard solution and 12h 24h 48h Because a long term therapy is indicated for ACEIs, simulations of PO doses of then 1 mL of methanol was added. After shaking in an ultrasonic bath for 5 min, and ramipril from 0.0063 to 6.30 mg/kg were made after single and repeated doses every centrifugation at 28,300 g for 10 min at 4 °C, supernatant was extracted, and 3 mL 24 h for a week. Predictions were performed with PK/PD parameters obtained from of ethyl acetate were added. The mixture was mixed and centrifuged for 5 min at a representative horse after PO ramipril at 0.80 mg/kg to describe the effect-time 2000 g. The upper organic layer was evaporated to dryness under N2 at 40 °C relationship and the influence of the dosing. All PK/PD modeling and simulations (Gómez-Díez et al., 2014). The residue was reconstituted in 400 μL of methanol and included in this work were developed using ADAPT5 software (D’Argenio et al., 2009). aliquot of 5 μL were injected into the LC-MS/MS system. Linear calibrations of ramipril and ramiprilat between 0.3 and 1000 ng/mL were obtained (r > 0.99). The accuracy and precision were within 1.2–4.0%. Values of LOQ and LOD for both analytes were Statistical analysis 0.5 ng/mL and 0.15 ng/mL. See Appendix: Supplementary material for more information. Arithmetic mean, standard deviation and harmonic mean were calculated. The Wilcoxon rank sum test was used to assess significant differences between IV and Pharmacokinetic and pharmacodynamic analysis PO routes at 0.050 mg/kg. The Kruskal–Wallis test was used to evaluate significant differences between PO doses including placebo. When differences were found, a For each animal, concentration–time data of ramipril and ramiprilat were ana- Wilcoxon rank sum test was used. The significance level was fixed at P < 0.05 using lyzed simultaneously by a physiologically based compartmental model previously Statgraphics Centurion XVI.I as statistic software (StatPoint Technologies). 40 J.M. Serrano-Rodríguez et al./The Veterinary Journal 208 (2016) 38–43

Results Table 1 Pharmacokinetic-pharmacodynamic parameters (mean ± SD) for ramipril and ramiprilat after different IV and PO ramipril doses. No local or systemic adverse reactions were detected during or after IV and PO administrations. Parameters Routes and doses Mean (±SD) serum concentrations of ramipril and ramiprilat after IV (0.05 mg/kg) PO (0.40 mg/kg) PO (0.80 mg/kg)

IV and PO ramipril are plotted in Fig. 2. Ramipril and ramiprilat con- Ramipril centrations after PO ramipril at 0.050 and 0.10 mg/kg were below ka (1/h) 0.23 ± 0.06 0.12 ± 0.06 LOQ in all horses with exception of some sampling times. However, k10p (1/h) 1.86 ± 0.66 1.94 ± 0.61 1.60 ± 0.90 ± ± ± both ramipril and ramiprilat had measurable concentrations after kf (1/h) 4.78 1.20 6.70 0.77 5.87 2.26 0.20 mg/kg of ramipril, although this was not enough to be fitted Ramiprilat k10 (1/h) 5.09 ± 2.46 5.49 ± 0.91 4.10 ± 1.70 by the model. Therefore, only concentrations of ramipril and a t1/2 k10 (h) 0.16 (0.09–0.31) 0.13 (0.10–0.16) 0.17 (0.10–0.27) ramiprilat after IV ramipril at 0.050 mg/kg and PO ramipril at 0.40 Vc (L/kg) 0.15 ± 0.04 and 0.80 mg/kg were analyzed by the PK/PD models (Table 1). Vc/F (L/kg) 2.35 ± 0.84 1.97 ± 0.74 Mean (±SD) serum ACE activities, expressed as percent of control Cl (L/kg/h) 0.77 ± 0.34 Cl/F (L/kg/h) 12.82 ± 4.46 7.79 ± 3.37 (value at time 0) from 0 to 48 h are plotted in Fig. 3. Mean values F (%) 6.52 ± 3.51 9.08 ± 4.01 of %Imax,T%Imax, and %I12h,%I24h, and %I48h for each administration route Kd (nmol/L) 1.35 ± 0.31 1.47 ± 0.29 1.19 ± 0.30 and dose are presented in Table 2. Differences in SBP and DBP values fcirc (%) 5.01 ± 1.21 5.80 ± 0.58 6.08 ± 1.43 ± ± ± after IV and PO doses of ramipril or placebo were not found between Bmax (nmol/L) 109.46 27.14 79.31 21.54 91.72 47.05 ± ± ± trials, even at maximum serum time ACE inhibitions. Pmax (nmol/L) 5.27 0.85 4.60 1.38 5.08 1.08 IC50 (nmol/L) 1.05 ± 0.21 1.07 ± 0.24 0.88 ± 0.36 For the simulations, the predicted effects on serum ACE activi- γ 0.63 ± 0.08 0.61 ± 0.12 0.60 ± 0.11 ty after single and multiple PO doses of ramipril from 0.0063 to k , absorption rate constant of ramipril; k , elimination rate constant of ramipril; 6.30 mg/kg every 24 h for a week were calculated (Fig. 4). a 10p kf, formation rate constant for ramiprilat from ramipril; k10, elimination rate con- stant of free ramiprilat; t1/2 k10, half-life of elimination of free ramiprilat; Vc, apparent Discussion 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; In the present study, serum ramipril and ramiprilat concentra- Kd, equilibrium dissociation constant of free ramiprilat producing saturation of 50% tions were low and declined rapidly after IV administration of of ACE; fcirc, fraction between circulating and non-circulating ACE; Bmax, total binding ramipril with a long terminal phase for ramiprilat until 36 h. Ramipril capacity of the ACE; Pmax, total binding capacity of the circulating ACE; IC50,free concentrations were lower than ramiprilat concentrations from ramiprilat concentration required to produce 50% of ACE inhibition; γ, coefficient which describes the steepness of the concentration effect curve. 0.26 h (Fig. 2a), suggesting a rapid elimination by biotransforma- a Harmonic mean and range. tion to ramiprilat, and by other routes with a kf and k10p values of 4.78 and 1.86 1/h, respectively (Table 1). The half-life of elimina- tion of ramiprilat was 0.16 h (9.6 min approximately). This very short value can be due to the small volume of distribution and particu- larly to the high clearance observed (Table 1). The rapid decreasing to 98.88% at 0.17 and 0.25 h (Toutain and Lefebvre, 2004). More- phase of ramiprilat is controlled by k10 and reflects the elimina- over, the terminal phase observed for ramiprilat reflects the binding tion process (Fig. 2a). It is likely that during this phase serum ACE to ACE and is influenced by binding parameters and by k10 (Lefebvre is saturated, explaining the high inhibition values (Fig. 3a) and close et al., 2006).

Fig. 2. (a) Semilogarithmic plot of serum concentrations (mean ± SD) of ramipril (-●-) and ramiprilat (-)-) after IV ramipril at 0.05 mg/kg in horses. (b) Semilogarithmic plot of serum concentrations (mean ± SD) of ramipril (-il at 0.05 mg/kg iramiprilat (-●-, -■-, -▲-) after PO ramipril at 0.20, 0.40 and 0.80 mg/kg in horses, respectively. J.M. Serrano-Rodríguez et al./The Veterinary Journal 208 (2016) 38–43 41

Fig. 3. (a) Mean serum ACE activity expressed as percent of control after IV ramipril at 0.05 mg/kg (-♦-) and PO ramipril at 0.05 mg/kg (-●-). (b) Mean serum ACE activity expressed as percent of control after PO doses of placebo (0.00 mg/kg, -●-), of ramipril at 0.05 (-●-), at 0.10 (-▲-), at 0.20 (-▼-), at 0.40 (-■-) and at 0.80 mg/kg (-□-), respectively.

Following PO ramipril administration, the bioavailability of ramipril (Fig. 2b). Visually, the next phase of the curve is more dif- ramiprilat was low (6.62–9.08%), with values similar to those re- ficult to interpret, because it is an elimination phase influenced by ported in dogs but lower than those in humans (Van Griensven et al., k10, but also a binding phase to ACE controlled by Kd,Bmax and fcirc 1995; Lefebvre et al., 2006). However, in the current research, higher (Lefebvre et al., 2006). The elimination of ramiprilat described by

PO doses were used compared to IV dose (8- and 16-fold), unlike k10 was different between PO doses (P < 0.05), and also t1/2 k10 the studies in dogs and humans. These findings might indicate that (Table 1). The elimination was slightly lower at 0.80 mg/kg of most of the pro-drug administered was not absorbed and higher ramipril, possibly because ACE was more saturated than at 0.40 mg/ PO doses should be used compared to the IV dose in order to achieve kg. Moreover, owing to the low concentrations of ramiprilat observed, the same ACE inhibition (Fig. 3a) unlike other species (Van Griensven it is unlikely that a maximum saturation of ACE occurred at the doses et al., 1995; Lefebvre et al., 2006). tested, since %Imax only reached 84.27% at times close to 2 h, unlike

Different studies have shown that PO availability of ACEIs in IV ramipril, with a %Imax value of 98.88% between 0.75 and 0.25 h horses is low (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez (Table 2). However, the inhibition after PO doses lasted longer than et al., 2014). Because of the limited knowledge of the factors that IV administration (Fig. 3), particularly with 0.40 and 0.80 mg/kg of contribute to PO absorption of drugs in the horse, various hypoth- ramipril at 24 and 48 h (Table 2). eses have been proposed, including interspecific differences in Binding parameters derived from the model indicated that the ACE gastrointestinal anatomy and physiology relative to enteral absorp- pool binding capacity was close to 80–100 nmol/L, and approximate- tion, and a possible minor influence of intestinal uptake by peptide ly 5% of the ACE pool was circulating (Table 1). These results differ transporter 1 (PepT 1) transport compared to passive diffusion (Davis from those previously obtained in dogs and cats (Toutain and Lefebvre, et al., 2014; Gómez-Díez et al., 2014; Maxwell, 2015). 2004). This is because Bmax and fcirc are species-related properties and

Values for ka of ramipril were low and differed between both might be lower in horses. On the other hand, Kd is a drug-related prop- doses, because the ascending phase of ramipril is PO absorption and erty, and the value determined, close to 1.35 nmol/L, was slightly bioconversion to ramiprilat, and the decreasing phase is elimina- higher than the value obtained in dogs, but fell into the nanomolar tion by biotransformation to ramiprilat and other pathways. For range suggesting that ramiprilat has a greater affinity for ACE (Toutain ramiprilat, the ascending phase is mainly a bioconversion phase from and Lefebvre, 2004; Lefebvre et al., 2006).

Table 2 Serum ACE inhibitions (mean ± SD) after different IV and PO ramipril doses.

Dose (mg/kg)/route %Imax T%Imax (h) %I12h %I24h %I48h 0.05/IV 98.88 ± 0.80 0.21 ± 0.04 32.61 ± 10.43 21.97 ± 6.63 8.02 ± 4.05 0.00/PO 5.31 ± 2.24 0.68 ± 0.67 0.48 ± 3.15 1.77 ± 3.39 −5.40 ± 4.58 0.05/PO 27.68 ± 10.88 2.20 ± 1.10 12.57 ± 6.67 13.47 ± 6.29 5.30 ± 4.29 0.10/PO 39.27 ± 5.72 1.80 ± 0.45 26.60 ± 11.10 22.79 ± 7.93 3.74 ± 3.48 0.20/PO 46.47 ± 14.39 2.40 ± 1.52 27.36 ± 8.88 23.37 ± 13.23 12.72 ± 9.11 0.40/PO 76.13 ± 3.99 1.67 ± 0.52 55.44 ± 8.63 35.76 ± 9.94 8.92 ± 6.69 0.80/PO 84.27 ± 4.00 2.50 ± 1.22 74.53 ± 4.24 63.54 ± 8.16 38.01 ± 9.08

%Imax, maximum serum ACE inhibition from 0 to 12 h; T%Imax, time to reach the %Imax value; %I12h, maximum serum ACE inhibition at 12 h; %I24h, maximum serum ACE in- hibition at 24 h; %I48h, maximum serum ACE inhibition at 48 h. 42 J.M. Serrano-Rodríguez et al./The Veterinary Journal 208 (2016) 38–43

Fig. 4. (a) Plot of predicted effects on serum ACE activity in horses after single PO doses of ramipril from 0.0063 to 6.30 mg/kg. (b) Plot of predicted effects on serum ACE activity in horses after single PO doses of ramipril from 0.0063 to 6.30 mg/kg every 24 h for a week.

The values of IC50 of ramiprilat, 1.05 nmol/L after IV ramipril and Conclusions 0.88 and 1.07 nmol/L after PO ramipril 0.40 and 0.80 mg/kg, ob- served between 7, 20 and 36 h, were consistent with the inhibition The use of physiological compartmental PK/PD models permit- values observed in those times (Fig. 3). Moreover, γ values were close ted us to study the pharmacology of ramiprilat after IV and PO doses to 0.60, indicating that a relative shallow concentration effect re- of ramipril in horses. Despite its complexity, these models can in- lationship and that some residual inhibition can be obtained for very terpret more appropriately the disposition phases of ACEIs low ramiprilat concentrations, especially at 0.80 mg/kg of ramipril concentration–time curves. The current study shows that PO avail- (Fig. 3). These data are in agreement with the results obtained with ability of ramipril at doses from 0.050 to 0.80 mg/kg in horses is ramiprilat and other ACEIs in different species (King et al., 2003; low. Although there was a tendency toward a decrease in serum ACE Lefebvre et al., 2006). activity, these values were never suppressed by more than 84.27% Our model was also used to predict PK/PD profiles with differ- unlike the IV data of 98.88%, using a dose 16-fold lower than the ent dosing regimens of ramipril. After single doses, visual inspection PO dose. It is unknown whether an ACE inhibition near 80%, as found of the curves showed that although ACE inhibition increased at in our study with PO ramipril, would be sufficient to counteract va- higher doses, the effect was quite similar from 0.80 to 6.30 mg/kg soconstriction in pathological conditions. This is a preliminary study (Fig. 4a). Moreover, after multiple ramipril administration, inhibi- with healthy horses and further work with different doses or for- tion values from 0.0063 to 0.20 mg/kg produced less dose-related mulations in equine patients should be conducted. inhibition of serum ACE activity and it took longer to reach steady state. In fact, the maximum effect was not reached for a week with Conflict of interest statement these doses and longer times would be required. However, PO doses from 0.80 to 6.30 mg/kg were very similar and faster to reach a steady None of the authors of this paper has a financial or personal re- state from the first administration. These findings are in agree- lationship with other people or organizations that could ment with the simulations described with PO benazepril in cats and inappropriately influence or bias the content of the paper. dogs (Toutain et al., 2000; King et al., 2003) and indicate that it is possible to inhibit ACE at very low doses due to the progressive sat- uration by ramiprilat. In addition, the model predicts that an initial Acknowledgments dose between 0.80 and 1.6 mg/kg could be administered to imme- diately inhibit ACE followed by the same maintenance doses every This research has been generously supported by the Science and day (Fig. 4a). Innovation Council of Spain (Research Project AGL2010-17431). The lack of variations in BP during the experiments was in agree- ment with other authors studying other ACEIs in horses (Gardner Appendix: Supplementary material et al., 2004; Davis et al., 2014; Gómez-Díez et al., 2014). This may indicate that ACEIs have little effect on BP in normotensive sub- Supplementary data to this article can be found online at jects (Lefebvre et al., 2007; Gómez-Díez et al., 2014). doi:10.1016/j.tvjl.2015.10.024. J.M. Serrano-Rodríguez et al./The Veterinary Journal 208 (2016) 38–43 43

References 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 Afonso, T., Giguère, S., Rappaport, G., Berghaus, L.J., Barton, M.H., Coleman, A.E., 2013. inhibitor, S-9780, in man: Evidence of tissue binding. Journal of Pharmacokinetics Pharmacodynamic evaluation of 4 angiotensin-converting enzyme inhibitors in and Biopharmaceutics 17, 529–550. healthy adult horses. Journal of Veterinary Internal Medicine 27, 1185–1192. Lefebvre, H.P., Jeunesse, E., Laroute, V., Toutain, P.L., 2006. Pharmacokinetic and Brewster, U.C., Setaro, J.F., Pezarella, M.A., 2003. The renin-angiotensin-aldosterone pharmacodynamic parameters of ramipril and ramiprilat in healthy dogs and system. Cardiorenal effects and implications for renal and cardiovascular disease dogs with reduced glomerular filtration rate. Journal of Veterinary Internal states. American Journal of Medical Sciences 32, 15–24. Medicine 20, 499–507. Davis, J.L., Kruger, K., Lafevers, D.H., Barlow, B.M., Schirmer, J.M., Breuhaus, B.A., 2014. Lefebvre, H.P., Brown, S.A., Chetboul, V., King, J.N., Puchelon, J.L., Toutain, P.L., 2007. Effects of quinapril on angiotensin-converting enzyme and plasma renin activity Angiotensin-converting enzyme inhibitors in veterinary medicine. Current as well as pharmacokinetic parameters of quinapril and its active metabolite, Pharmaceutical Design 13, 1347–1361. quinaprilat, after intravenous and oral administration to mature horses. Equine Maxwell, L., 2015. Horse of a different color: Peculiarities of equine pharmacology. Veterinary Journal 46, 729–733. In: Equine Pharmacology, First Ed. John Wiley and Sons, Ames, IA, pp. 3–15. D’Argenio, D.Z., Schumitzky, A., Wang, X., 2009. ADAPT 5 User’s Guide: Toutain, P.L., Lefebvre, H.P., 2004. Pharmacokinetic and pharmacokinetic/ Pharmacokinetic/Pharmacodynamic Systems Analysis Software. Biomedical pharmacodynamic relationships for angiotensin-converting enzyme inhibitors. Simulations Resource, Los Angeles, CA. Journal of Veterinary Pharmacology and Therapeutics 27, 515–525. Gardner, S.Y., Atkins, C.E., Rams, R.A., Schwabenton, A.B., Papich, M.G., 2004. Toutain, P.L., Lefebvre, H.P., King, J.N., 2000. Benazeprilat disposition and effect in Characterization of the pharmacokinetic and pharmacodynamic properties of dogs revisited with a pharmacokinetic/pharmacodynamics modeling approach. the angiotensin-converting enzyme inhibitor, enalapril, in horses. Journal of Journal of Pharmacology and Experimental Therapeutics 292, 1087–1093. Veterinary Internal Medicine 18, 231–237. Van Griensven, J.M., Schoemaker, R.C., Cohen, A.F., Luus, H.G., Seibert-Grafe, M., Röthig, Gómez-Díez, M., Muñoz, A., Caballero, J.M., Riber, C., Castejón, F., Serrano-Rodríguez, H.J., 1995. Pharmacokinetics, pharmacodynamics and bioavailability of the ACE J.M., 2014. Pharmacokinetics and pharmacodynamics of enalapril and its active inhibitor ramipril. European Journal of Clinical Pharmacology 47, 513– metabolite, enalaprilat, at four different doses in healthy horses. Research in 518. Veterinary Science 97, 105–110. Yuan, B., Wang, X., Zhang, F., Jia, J., Tang, F., 2008. Simultaneous determination of King, J.N., Maurer, M., Toutain, P.L., 2003. Pharmacokinetic/pharmacodynamic ramipril and its active metabolite ramiprilat in human plasma by LC–MS-MS. modelling of the disposition and effect of benazepril and benazeprilat in Chromatographia 68, 533–539. Angiotensin-converting enzyme inhibitors in the horse

11.2.1 SUPPLEMENTARY MATERIALS FOR PAPER II

Analytical method for ACEIs determination.

Reagents

Chromatographic grade acetonitrile and water were used for LC–MS/MS analyses. Mass spectrometry grade formic acid was used as ionization agent.

Chromatographic grade methanol and ethyl acetate were used.

Sample treatment

A volume of 700 μL of serum sample was spiked with IS solution and then, 1 mL of methanol was added. Proteins were precipitated by shaking in an ultrasonic bath for 5 min, followed by centrifugation at 28300 g for 10 min at 4º C. Supernatant was extracted, and 3 mL of ethyl acetate were added. The mixture was mixed and centrifuged for 5 min at 2000 g. The upper organic layer was evaporated to dryness under N2 at 40ºC. The residue was reconstituted and sonicated in 400 μL of methanol and transferred to auto sampler vials, and an aliquot of 5 μL was injected [reference 7 in paper].

Instruments and apparatus

Chromatographic separation was carried out with an Agilent (Palo Alto, CA, USA) 1200 Series LC system coupled to an Agilent 6460 triple quadrupole mass spectrometer equipped with a

JetStream® Technology electrospray (ESI) ion source. The data were processed using

MassHunter Workstation Software (V-B.05) for qualitative and quantitative analyses. The analytical column was a Mediterranea Sea C18 analytical column (3 μm particle size, 50 mm ×

4.6 mm, Teknokroma, Barcelona, Spain) that was thermostated at 25°C.

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Manuel Gómez Díez

Chromatographic and mass spectrometry conditions

An isocratic method was adopted for chromatographic separation of the target analytes. The mobile phase consisted of 0.1% formic acid in acetonitrile/water 90/10 (v/v) (mobile phase A) and in acetonitrile (mobile phase B) at 0.3 mL/min flow rate. The injection volume was 5 μL and the total run time was 4 min. The ESI conditions were as follows: gas heater (N2) 325 ºC, gas flow

(N2) 12 L/min, nebulizer pressure 45 psi, sheath gas heater (N2) 350 ºC, sheath gas flow (N2) 11 psi, voltage capillary +3500 V. Determination of the target compounds was carried out by selected reaction monitoring (SRM) mode by using the parameters reported by Yuan et al.

[reference 10 in paper], which are listed in Table 1. Figure 1 shows MS/MS spectra obtained for each precursor ion to justify the product ions used for quantitative and qualitative transitions.

The representative chromatograms are shown in Figure 2.

Sensitivity and linear calibration range

Calibration plots were run using the peak area ratio between the target analyte and that of the

IS (32 ng/ml of Enalapril) as a function of the standard concentration of Ramipril and Ramiprilat.

Regression coefficients were 0.9996 for both analytes. The analytical features of the calibration models are listed in Table 2. The limits of detection (LOD) and quantitation (LOQ) for both analytes were 0.15 ng/mL and 0.5 ng/mL respectively. The LOQ values were below the concentrations detected in horse serum.

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Tables and Figures

Analyte Precursor ion Fragmentor Collision energy Quantitative ion Qualitative ion

[m/z] [eV] [m/z] [m/z]

Ramipril 417.1 120 20 234.1 129.9 343.1

Ramiprilat 389.1 105 20 206.1 156 343.1

Enalapril 377.1 135 20 234.1 160 303.1

Table 1. Selected reaction monitoring (SRM) parameters for quantitative analysis of

ramipril and ramiprilat.

Calibration range LOQ LOD Analyte Equation R2 (ng/mL) (ng/mL) (ng/mL)

Ramipril 0.3 – 1000 0.3 0.09 y = 0.0737x + 0.494 0,99967

Ramiprilat 0.5 – 1000 0.5 0.15 y = 0.0027x + 0.0044 0,99965

Table 2. Analytical features of the method.

A B

C

Fig. 1. MS/MS spectra of analytes and IS: (A) Ramipril, (B) Ramiprilate, (C) Enalapril.

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Fig. 2. Total Ion Chromatogram (TIC) and individual chromatograms for analytes and IS:

(A) Ramipril, (B) Ramiprilate, (C) Enalapril.

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11.3. PAPER III

Pharmacokinetic/Pharmacodynamic Modeling of Benazepril and Benazeprilat after Administration of Intravenous and Oral Doses of Benazepril in Healthy Horses

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Research in Veterinary Science 114 (2017) 117–122

Contents lists available at ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

Pharmacokinetic/pharmacodynamic modeling of benazepril and MARK benazeprilat after administration of intravenous and oral doses of benazepril in healthy horses

⁎ Juan Manuel Serrano-Rodrígueza, , Manuel Gómez-Díezb, María Esguevab, Cristina Castejón-Riberb, Antonio Mena-Bravoc,d, Feliciano Priego-Capotec,d, Nahúm Ayalaa, Juan Manuel Serrano Caballeroa, Ana Muñozb,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

ARTICLE INFO ABSTRACT

Keywords: Pharmacokinetic and pharmacodynamic (PK/PD) properties of the angiotensin-converting enzyme inhibitor Angiotensin-converting enzyme inhibitors (ACEI) benazeprilat have not been evaluated in horses. This study was designed to establish PK profiles for Benazeprilat benazepril and benazeprilat after intravenous (IV) and oral (PO) administration of benazepril using a PK/PD Benazepril model. This study also aims to determine the effects of benazeprilat on serum angiotensin converting enzyme Pharmacokinetic-pharmacodynamic (ACE), selecting the most appropriate dose that suppresses ACE activity. Six healthy horses in a crossover design Horses received IV benazepril at 0.50 mg/kg and PO at doses 0 (placebo), 0.25, 0.50 and 1.00 mg/kg. Blood pressures (BP) were measured and blood samples were obtained at different times in order to measure serum drug concentrations and serum ACE activity, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. Systemic bioavailability of benazeprilat after PO benazepril was 3–4%. Maximum ACE inhibitions from baseline were 99.63% (IV benazepril), 6.77% (placebo) and 78.91%, 85.74% and 89.51% (for the three PO benazepril doses). Significant differences in BP were not found. Although oral availability was low, benazeprilat 1.00 mg/kg, reached sufficient serum concentrations to induce long lasting serum ACE inhibitions (between 88 and 50%) for the first 48 h. Additional research on benazepril administration in equine patients is indicated.

1. Introduction and cats, there is a huge amount of information derived from clinical studies (reviewed by Lefèbvre et al., 2007). However, studies of ACEIs Benazepril is a pro-drug, which after per os (PO) absorption under- in horses are scarce, with a progressively increasing number of reports goes hydrolization to its active metabolite, benazeprilat. It is an being published in recent years (Afonso et al., 2013; Davis et al., 2014; angiotensin converting enzyme inhibitor (ACEI), as it prevents conver- Gómez-Díez et al., 2014; Muñoz et al., 2016; Serrano-Rodríguez et al., sion of angiotensin I into angiotensin II (Toutain and Lefèbvre, 2004). 2016). The interest in these pro-drugs has risen recently in horses Angiotensin II causes vasoconstriction, and therefore, benazeprilat and because there is an increased awareness of cardiovascular abnormalities other ACEIs such as ramiprilat or enalaprilat are used to modulate and their consequences in sport horses. In addition, new researchers blood pressure (BP) in human and veterinary medicine in pathological have highlighted the clinical relevance of some hypertensive situations processes in which hypertension is a concern (Lefèbvre et al., 2007). in equids. Five cases of hypertensive cardiomyopathy have been Considering this, the main clinical indications for ACEI administration reported (Navas de Solís et al., 2013), and there are also indications are chronic heart failure, essential hypertension and chronic renal that systemic hypertension might be a feature of equine metabolic failure. Because of the clinical importance of these disorders in dogs disease, as ponies with this syndrome and previous laminitis have been

⁎ Corresponding author at: Faculty of Veterinary Medicine, University of Cordoba, Pharmacology, Toxicology and Legal and Forensic Medicine, Campus de Rabanales, Carretera N-IV km 396-a, Córdoba, Andalucía, Spain. E-mail address: [email protected] (J.M. Serrano-Rodríguez). http://dx.doi.org/10.1016/j.rvsc.2017.03.016 Received 20 June 2016; Received in revised form 10 February 2017; Accepted 27 March 2017 0034-5288/ © 2017 Elsevier Ltd. All rights reserved. J.M. Serrano-Rodríguez et al. Research in Veterinary Science 114 (2017) 117–122 reported to have elevated systemic BPs in summer (Bailey et al., 2008). benazeprilat was not detected. For placebo, the equivalent amount of Pharmacokinetic and pharmacodynamic (PK/PD) studies of ACEIs water without the pro-drug was used. Pro-drugs and placebo were in equine patients are limited to enalaprilat, quinaprilat and ramiprilat administered by nasogastric intubation at ambient temperature (be- after intravenous (IV) and PO enalapril, quinapril and ramipril, tween 20 and 24 °C approximately). respectively (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez In each assay, venous blood samples were taken before administra- et al., 2014; Serrano-Rodríguez et al., 2016). To the authors' knowl- tion (time 0), and at 5, 10, 15, 30 and 45 min and at 1, 1.5, 2, 3, 4, 6, 8, edge, there are three papers regarding benazepril, another ACEI. In one 12, 24, 36, 48 and 72 h after. paper, Afonso et al. (2013) administered two different PO doses of benazepril, considered as low (0.25 mg/kg) and high (0.50 mg/kg), 2.3. Blood pressure monitoring extrapolated from the dosage recommended for dogs. These authors measured ACE inhibition and BP, but because they did not administer Systolic (SBP) and diastolic (DBP) BP measurements were made IV benazepril, only a pharmacodynamic analysis was made. Later, non-invasively with a multiparametric monitor (S/5 Datex-Ohmeda Muñoz et al. (2016) compared the effect of different ACEIs adminis- Compact) in the coccygeal artery. Three BP measurements were made tered 2 h before exercise and the response of BP after an intense at each sampling point and the results are presented as the means of the exercise. It was found that only benazepril was able to avoid the three measurements. hypertensive response to exercise. More recently, Afonso et al. (2016) evaluated BP in response to angiotensin I administration after four 2.4. Blood processing and analysis different benazepril PO doses (0.5, 1, 2 and 4 mg/kg). They found that the attenuation of BP was modest despite achieving an adequate serum After extraction, blood samples were poured into tubes without ACE inhibition. Although in these recent years the number of studies , left to coagulate in a refrigerator for 30 min and regarding benazepril has increased, pharmacokinetic analyses are still centrifuged. Harvested serum was stored at −90 °C until analysis. lacking. Moreover, the previously mentioned articles did not measure Serum ACE activity was measured by spectrophotometry (Afonso blood benazepril-benazeprilat concentrations. Additionally, the effects et al., 2013; Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016). of multiple PO doses are also unknown. Linear calibration curves from 3 to 150 IU/l were obtained, and the Therefore, the current study aims to: (1) establish serum concen- correlations coefficients (r) were > 0.99. Precision of the technique tration–time profile for benazepril, and its active metabolite benaze- expressed as mean, standard deviation and coefficient of variation were prilat after IV benazepril at 0.50 mg/kg, PO (placebo), and three 25.10 ± 0.80 IU/l (3.18%) and 75.04 ± 4.29 IU/l (5.72%) for intra- different PO benazepril doses (0.25, 0.50 and 1.00 mg/kg); (2) deter- assay validation, and 25.63 ± 1.04 (4.04%) and 75.89 ± 4.90 mine the effects of benazeprilat on serum ACE activity; (3) investigate (6.45%) for inter-assay validation, respectively. The limits of quantifi- the effects on BP with these doses and routes; and (4) predict and cation (LOQ) and detection (LOD) were 5 and 3 IU/l, respectively. simulate the most appropriate PO dose that suppresses ACE activity Serum concentrations of benazeprilat (CymitQuímica, Barcelona, after single and multiple PO doses of benazepril. Spain), benazepril and enalapril as internal standard (Sigma- AldrichQuímica, Madrid, Spain) were measured using a liquid chroma- 2. Material and methods tography-tandem mass spectrometry (LC-MS/MS) assay (Yuan et al., 2008). A volume of 700 μl of horse serum was spiked with internal 2.1. Horses standard solution at 50 ng/ml, and then 1 ml of methanol was added. After shaking in an ultrasonic bath for 5 min, and centrifugation at Six crossbred horses, two mares and four geldings, between 5 and 28300 g for 10 min at 4 °C, supernatant was extracted, and 3 ml of ethyl 11 years (10.8 ± 2.35), and weighing between 391 and 594 kg acetate were added. The mixture was blended and centrifuged for 5 min

(477.44 ± 67.58) were studied. Prior to the experiment, physical at 2000 g. The upper organic layer was evaporated to dryness under N2 examinations, hematology, serum biochemistry, myocardial troponin at 40 °C for about 15 min. The residue was reconstituted in 100 μlof concentrations, electrocardiography, echocardiography and BP mea- methanol and an aliquot of 5 μl was injected into the LC-MS/MS system. surements, were performed. Only healthy animals were studied, and Linear calibrations of benazepril and benazeprilat between 0.1 and they did not receive any other medication during the period of study. 500 ng/ml were obtained (r > 0.99). Precision of the technique The animals were located in individual paddocks and were fed with expressed as mean, standard deviation and coefficient of variation rye-grass haylage. The horses did not have access to salt or electrolyte were 19.69 ± 1.04 ng/ml (5.27%) and 39.81 ± 1.44 ng/ml (3.62%) supplements during the study. for benazepril and 4.04 ± 0.10 ng/ml (2.52%) and 8.13 ± 0.47 ng/ ml (5.77%) for benazeprilat after intra-assay validation, respectively. 2.2. Experimental protocol For both analytes these values were 18.97 ± 1.06 ng/ml (5.57%) and 41.24 ± 2.56 ng/ml (6.22%), and 4.24 ± 0.32 ng/ml (7.55%) and The research was approved by the Ethical Committee for Animal 8.33 ± 0.47 ng/ml (5.63%) after inter-assay validation, respectively. Experimentation of the University of Córdoba (Reference: 55.60 PE, The limits of detection (LOD) and quantitation (LOQ) for both drugs date of approval February 1st 2010). were 0.03 and 0.1 ng/ml, respectively (see chromatographic methodol- The study was conducted in two trials. In the first trial, each animal ogy in Supplement data). received IV benazepril at 0.50 mg/kg. After a week of washout, the second trial was undertaken, using a blinded and randomized Latin 2.5. Pharmacokinetic and pharmacodynamic analysis square (6 × 4) design. Each animal received PO placebo or benazepril at 0.25, 0.50, or 1.00 mg/kg, with at least 1 week of washout between ACEI drugs have nonconventional PKs due to nonlinear binding to trials. Food was withdrawn from 12 h before administration of the pro- ACE, the target enzyme, and because there are two ACE pools, drug to 8 h after. circulating and non-circulating. Circulating ACE originates mainly from For IV study, benazepril (Sigma-Aldrich) was dissolved in 0.7% vascular endothelium, representing about 5–30% of ACE pool in saline and 1% NaHCO3 (King et al., 2003). For PO doses, benazepril different species (Toutain and Lefèbvre, 2004). Therefore, compart- hydrochloride tablets (Fortekor 20 mg, Novartis) were dissolved and mental PK/PD models with binding parameters of ACEI to ACE are suspended in 150 ml of water, sonicated in an ultrasonic bath for required to allow an appropriate interpretation of the different disposi- 15 min and stored at 4 °C before trials. After this dissolution process, tion phases of ACEI curves (Toutain et al., 2000). benazepril concentration was tested by the analytical technique and For each animal, PK-PD data were analyzed using the models

118 J.M. Serrano-Rodríguez et al. Research in Veterinary Science 114 (2017) 117–122 developed by Lees et al. (1989) with the approach of Picard-Hagen et al. (2001). The final selected model included one compartment for benazepril and one compartment for benazeprilat directly linked into a direct effect (Toutain and Lefèbvre, 2004). Furthermore, for PO administration, a depot compartment was included (see figures and equations in PK/PD modeling in Supplement data).

The parameters estimated for PK modeling were: ka, the first order absorption rate constant for benazepril; kf, the elimination rate constant for benazepril by formation of benazeprilat; and Vc/F, the apparent volume of distribution with respect to bioavailability and bioconversion from benazepril. The systemic clearance for benazeprilat with respect to the bioavailability and bioconversion from benazepril was determined as Cl/F = k10·Vc/F, with k10 indicating the elimination rate constant of benazeprilat. The elimination half-life was obtained as t½k10 = (ln2)/ k10. The F value was calculated from the ratio of Cl of benazeprilat after IV benazepril and Cl/F of benazeprilat after PO benazepril (King et al., 2003). Ideally, IV benazeprilat would have been administered to determine Vc, Cl and F values. However, the necessary amount for IV formulation was not available. Administration of IV benazepril, how- ever, provides important information about the biotransformation to benazeprilat, as described for other ACEIs in horses (Davis et al., 2014; Serrano-Rodríguez et al., 2016).

Binding parameters of benazeprilat to ACE were: Kd, equilibrium dissociation constant that expresses the affinity for ACE; Bmax, size of ACE pool and fcirc, fraction between circulating and non-circulating ACE. Circulating ACE was obtained as Pmax =Bmax·fcirc.(Toutain and Lefèbvre, 2004). However, it is necessary to note that benazeprilat is bound to ACE, or free, or non-specifically bound to albumin. Free and albumin-bound fractions are indistinguishable in these models, and free benazeprilat concentration used for PD modeling corresponded to free benazeprilat plus benazeprilat bound to albumin (Toutain et al., 2000). For PD data, the observed effect was defined as the serum ACE activity at each time expressed as a percentage of the control before administration of the pro-drug. The estimated parameters were IC50, free benazeprilat concentration required to produce 50% ACE inhibi- tion, and γ coefficient, also known as Hill coefficient, which describes the steepness of the sigmoid curve. Other parameters obtained were maximum serum ACE inhibition between 0 and 12 h (%Imax), time to reach %Imax (T%Imax), and serum ACE inhibition at 12, 24, 48 and 72 h (%I12h,%I24h, %I48h and %I72h, respectively). Because a long-term therapy is indicated for ACEIs, comparative simulations of PO doses of benazepril from 0.25 to 8.00 mg/kg were made after single and repeated doses administered daily for seven days. Predictions were performed with parameters obtained with PO bena- Fig. 1. (a) Semilogarithmic plot of serum concentrations (mean ± SD) of benazepril zepril at 1.00 mg/kg after PK-PD analysis. The software used for (−■-) and benazeprilat (−□-) after IV benazepril at 0.50 mg/kg, and benazepril (−■-) modeling was ADAPT 5 (D'Argenio et al., 2009), and the simulations and benazeprilat (−□-) after PO benazepril at 0.50 mg/kg in horses. (b) Semilogarithmic were developed using Berkeley Madonna software (Krause and Lowe, plot of serum concentrations (mean ± SD) of benazepril (−●-,-■-, −♦-) and benaze- 2014). prilat (−○-,-□-,-◊-) after PO benazepril at 0.25, 0.50 and 1.00 mg/kg in horses, respectively. 2.6. Statistical analysis after IV and PO benazepril and mean ( ± SD) serum ACE activities, Because the data did not follow a Gaussian distribution, non- expressed as a percentage of controls (values at time 0) from 0 to 72 h parametric tests were used. Data are presented as median with are outlined in Fig. 1 and 2, respectively. PK-PD parameters obtained interquartile range. A Wilcoxon rank sum test was used to assess and mean values of %Imax,T%Imax, and %I12h,%I24h,%I48h and %I72h significant differences between IV and PO routes at 0.50 mg/kg. A for each administration route and dose are presented in Tables 1 and 2, Kruskal-Wallis test was used to evaluate significant differences between respectively. Neither IV nor PO benazepril administration resulted in PO doses including placebo. When differences were found, a Wilcoxon significant changes in SBP and DBP, even at the times when maximum rank sum test was used. The significance level was fixed at p<0.05 serum ACE inhibitions were attained. (Statgraphics Centurion XVI·I, StatPoint Technologies). Serum benazepril and benazeprilat concentrations were low and declined rapidly after IV administration of benazepril. Benazepril 3. Results concentrations were lower than benazeprilat concentrations from 0.16 h (Fig. 1a), suggesting a rapid elimination by biotransformation No local or systemic adverse reactions were detected during or after to benazeprilat with a mean kf value of 7.471/h (Table 1). The fl IV and PO administrations. SBP and DBP remained statistically un- decreasing phase of benazeprilat is controlled by k10 and re ects the changed during the whole experiment. elimination process with a half-life of 0.60 h (36 min approximately). It Mean ( ± SD) serum concentrations of benazepril and benazeprilat is likely that during this phase, serum ACE would be saturated,

119 J.M. Serrano-Rodríguez et al. Research in Veterinary Science 114 (2017) 117–122

Table 1 Pharmacokinetic-pharmacodynamic parameters (median with interquartile ranges) for benazepril and benazeprilat after different PO and IV benazepril doses.

Doses and routes

Parameters 0.50 mg/kg 0.25 mg/kg 0.50 mg/kg 1.00 mg/kg (IV) (PO) (PO) (PO) Benazepril

ka (1/h) 0.09 (0.03) 0.07 (0.05) 0.08 (0.03)

kf (1/h) 7.47 (0.93) 6.53 (0.51) 6.86 (0.38) 5.80 (0.47) Benazeprilat

Vc (L/kg) 0.25 (0.05)

Vc/F (L/kg) 4.45 (0.37) 4.26 (0.62) 4.72 (1.00) Cl (L/h/kg) 0.29 (0.06) Cl/F (L/h/kg) 6.47 (1.56) 7.87 (1.65) 6.57 (1.55) F (%) 4.29 (1.19) 3.28 (1.74) 4.30 (1.39)

k10 (1/h) 1.17 (0.30) 1.50 (0.49) 1.88 (0.53) 1.36 (0.52)

t1/2k10 (h) 0.60 (0.16) 0.46 (0.13) 0.37 (0.12) 0.51 (0.20) a a Kd (nmol/l) 1.91 (0.21) 4.22 (4.40) 1.22 (1.75) 1.22 (0.84)

Bmax (nmol/l) 107.74 96.98 (59.60) 84.18 (12.73) 144.04 (68.98) (101.30)b b fcirc (%) 16.55 (11.15) 18.68 (3.55) 14.48 (3.73) 10.72 (8.26) b Pmax (nmol/l) 13.31 (23.63) 18.56 (14.57) 12.16 (4.86) 15.74 (27.40)

IC50 (nmol/l) 0.55 (0.31) 0.81 (0.09) 0.80 (0.07) 1.12 (0.17) γ 0.60 (0.03) 0.65 (0.08) 0.58 (0.04) 0.61 (0.15)

ka, absorption rate constant of benazepril; kf, elimination rate constant of benazepril by

formation of benazeprilat; Vc, apparent volume of distribution for benazepril and

benazeprilat; Vc/F, apparent volume of distribution for benazepril and benazeprilat with respect to the bioavailability; Cl, clearance of free benazeprilatafter IV benazepril; Cl/F, clearance of free benazeprilat with respect to the bioavailability; F, PO bioavailability of

benazeprilat after IV benazepril; k10, elimination rate constant of free benazeprilat; t1/

2k10, half-life of elimination of free benazeprilat; Kd,equilibrium dissociation constant of

free benazeprilat producing saturation of 50% of ACE; Bmax, total binding capacity of the

ACE; fcirc, fraction between circulating and noncirculating ACE; Pmax, total binding

capacity of the circulating ACE; IC50, free benazeprilat concentration required to produce 50% of ACE inhibition; γ, Hill coefficient which describes the steepness of the concentration effect curve. a Significantly different from 0.50 mg/kg PO dose (p<0.05). b Significantly different from 0.25 and 0.50 mg/kg PO doses (p<0.05).

Table 2 Serum ACE inhibitions (median with interquartile ranges) after different IV and PO benazepril doses.

Doses and routes

Dose 0.50 mg/kg 0.00 mg/kg 0.25 mg/ 0.50 mg/ 1.00 mg/kg kg kg Route IV PO (placebo) PO PO PO %I 99.63 (0.12) 6.77 (8.67) 78.91 85.74 89.51 (6.70)a Fig. 2. (a) Mean serum ACE activity expressed as percent of control after IV benazepril at max (2.64) (8.31)a,b 0.50 mg/kg (−■-) and PO benazepril at 0.50 mg/kg (−●-). (b) Mean serum ACE activity T (h) 0.75 (0.63) 2.50 (3.25) 3.50 3.00 4.00 (2.25)c expressed as percent of control after PO doses of placebo (0.00 mg/kg, −□-), of %Imax (1.00)c (1.00)b,c benazepril at 0.25 (−●-), at 0.50 (−●-) and at 1.00 mg/kg (−○-), respectively. a %I12h 73.93 (13.03) 4.74 (3.20) 69.99 77.97 80.58 (8.70) (10.41)c (5.32)a,b a explaining the high inhibition values, close to 99.63% between 0.08 %I24h 46.54 (22.69) 3.60 (9.04) 52.52 64.56 70.80 (8.58) c a,b and 3.00 h (Fig. 2a). Moreover, the terminal phase observed for (20.03) (6.11) a benazeprilat reflects the binding to ACE and is influenced by binding %I48h 34.66 (22.59) 9.14 (4.54) 24.50 51.98 51.69 (9.15) (29.95)c (10.40)a,b parameters and by k (Lefèbvre et al., 2006). a 10 %I72h 16.61 (19.19) 4.27 (3.02) 2.11 40.08 38.84 (8.64) Following PO benazepril administration, the bioavailability of (6.21) (14.13)a,b benazeprilat was low (3.28–4.30%). Maximum inhibition values of 78.91, 85.74 and 89.51% after PO benazepril at 0.25, 0.50 and 1.00 mg/kg were obtained, respectively, and these data were lower %Imax, maximum serum ACE inhibition from 0 to 12 h; T%Imax, time to reach the %Imax value; %I12h, maximum serum ACE inhibition at 12 h;%I24h, maximum serum ACE than IV benazepril values at 0.50 mg/kg (Fig. 2). However, the inhibition at 24 h; %I48h, maximum serum ACE inhibition at 48 h; %I72h, maximum serum inhibition after PO doses lasted longer than IV administration ACE inhibition at 72 h. (Fig. 2), particularly with 0.50 and 1.00 mg/kg of benazepril from 12 a Significantly different from 0.00 mg/kg (placebo) and 0.25 mg/kg PO dose to 72 h, without significant differences between them (Table 2). (p<0.05). b Significantly different from 0.50 mg/kg IV dose (p<0.05). Values for k of benazepril were low. The elimination of benaze- c a Significantly different from 0.00 mg/kg (placebo) (p<0.05). prilat described by k10 and t1/2k10 was not different between PO doses (p>0.05), unlike the IV values. Binding parameters derived from the circulating (Table 1).The Kd value was in the range of 1.22 to model indicated that the ACE pool binding capacity Bmax was between 4.22 nmol/l. 80 and 160 nmol/l, and approximately 10–18% of the ACE pool was For benazeprilat, IC50 values of 0.55 nmol/l were found at 24 h after

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activity in horses with exercise-induced pulmonary hemorrhage; there- fore, it might be a promising marker of this condition. However, this together with the vasodilatory effects might lead to fraudulent admin- istration of these pro-drugs despite the fact that they are a prohibited (benazepril) or controlled (enalapril) medication according to the FEI (Fédération Equestre Internationalle). Nevertheless, we have to con- sider that these types of drugs could be the cornerstone to treat and even to delay clinical progression in cardiopathic patients or in geriatric horses at risk of heart disease and failure. The current study aims to define PK/PD properties of benazepril and compare these with other ACEIs previously studied in the horse. After PK/PD analysis following a physiologically based interpretation of the serum curves, PO absorption of benazepril was found to be low. This finding might indicate that most of the pro-drug administered is not absorbed and higher PO doses should be used compared to the IV dose in order to achieve the same ACE inhibition. Consequently, the oral bioavailability of benazepril in horses was low, near 4%, and similar to the values described by previous researchers with other ACEIs. In fact, values close to 2–6% for quinaprilat and ramiprilat, and even lower for enalapril have been published suggesting that oral absorption of ACEIs in horses is low (Davis et al., 2014; Gómez-Díez et al., 2014; Serrano- Rodríguez et al., 2016). Several hypotheses related to the enteral absorption of these kinds of drugs, and the influence of intestinal uptake by peptide transporter 1 (PepT1) compared to passive diffusion have been proposed to explain these findings (Davis et al., 2014; Gómez-Díez et al., 2014; Maxwell, 2015). Despite the low oral bioavailability, benazepril had sufficient enteral absorption and bioconversion to benazeprilat to induce max- imum serum ACE inhibitions close to 78–89% at doses from 0.25 to 1.00 mg/kg of benazepril at times between 2 and 4 h post-administra- tion. Similar values have been reported with ramiprilat after PO ramipril at 0.4 and 0.8 mg/kg (76.13–84.27%) but lower with quina- prilat after PO quinapril at 0.25 and 0.5 mg/kg (47.11–53.43%) and enalaprilat after PO enalapril at 1.00 and 2.00 mg/kg (26.11–30.19%). These findings suggest that benazepril and ramipril, at the doses tested, produce a higher ACE inhibition than quinapril and especially enalapril (Davis et al., 2014; Gómez-Díez et al., 2014; Serrano-Rodríguez et al., 2016).

In relation to ACE-binding and PD results, the low Kd data obtained suggest that benazeprilat has a greater affinity for ACE and indicates a relatively shallow and long-lasting concentration effect relationship due

to the low γ and IC50 values observed (Fig. 2). For that reason, inhibition values up to 50% between 48 and 72 h can be obtained for very low benazeprilat concentrations at 0.50 and 1.00 mg/kg of Fig. 3. (a) Plot of predicted effects on serum ACE activity in horses after single PO doses benazepril unlike other ACEIs such as enalaprilat, ramiprilat or of benazepril from 0.25 to 8.00 mg/kg. (b) Plot of predicted effects on serum ACE activity quinaprilat (Gómez-Díez et al., 2014, Davis et al., 2014; Serrano- in horses after multiple PO benazepril from 0.25 to 8.00 mg/kg for a week. Rodríguez et al., 2016). At this point, it is necessary to indicate that Afonso et al. (2013) found that PO benazepril resulted in a greater IV benazepril at 0.5 mg/kg. For the doses of PO benazepril at 0.5 and serum ACE inhibition compared with quinapril, ramipril and peridon- 1.0 mg/kg, IC50 values ranged between 0.80 and 1.12 nmol/l and were pril in horses, and no feeding effect was observed in any PD parameters, obtained at times comprised between 48 and 60 h (Table 1). These data suggesting that benazepril could be more useful in equine patients than were consistent with the inhibition values observed in those times other inhibitors. (Fig. 2). Moreover, γ values close to 0.65 were found. After simulation with single and multiple doses, a close examination For the simulations, the predicted effects on serum ACE activity of the curves showed that although serum ACE inhibition rose at higher after single and multiple PO doses of benazepril from 0.25 to 8.00 mg/ doses, the effect was similar from 1.00 to 8.00 mg/kg and PO doses kg once a day, for a week were calculated and shown in Fig.3. from 1.00 to 8.00 mg/kg were not statistically different between them (Fig.3a, 3b). The data derived from these simulations are consistent 4. Discussion with the experimental results reported in the recent paper of Afonso et al. (2016). This group described the effect on BP in response to ACEI pro-drugs have been studied more intensely in horses in recent exogenous angiotensin I administration after single PO benazepril years, because of the greater awareness in relation to the relevance of administrations from 0.50 to 4.00 mg/kg. It was observed that the some potentially pathologic hypertensive situations in equine medicine, greater ACE inhibition found with increasing doses of PO benazepril did including cardiovascular diseases and equine metabolic syndrome. not lead to a greater BP attenuation after angiotensin I administration. Furthermore, heart valve regurgitation is associated with an activation All of these results taken into consideration together might indicate that of the renin angiotensin aldosterone axis (Gehlen et al., 2002). In a PO benazepril at 1.00 mg/kg would be useful in horses with hyperten- preliminary report, Costa et al. (2012) also found greater serum ACE sion. In another study, horses received four different PO ACEIs or

121 J.M. Serrano-Rodríguez et al. Research in Veterinary Science 114 (2017) 117–122 placebo 2 h before being exposed to an intense exercise that resulted in References systemic hypertension. It was found that the increase in BP immediately after exercise reached values over above baseline of 67.6% (placebo Afonso, T., Giguère, S., Rappaport, G., Berghaus, L.J., Barton, M.H., Coleman, A.E., 2013. experiment), 52.7% (enalapril 2 mg/kg), 43.1% (quinapril 1 mg/kg), Pharmacodynamic evaluation of 4 angiotensin-converting enzyme inhibitors in healthy adult horses. J. Vet. Intern. Med. 27, 1185–1192. 26.6% (ramipril 0.2 mg/kg) and 4.2% (benazepril 0.5 mg/kg) (Muñoz Afonso, T., Gigüere, S., Rapoport, G., Barton, M.H., Brown, S.A., Coleman, A.E., 2016. et al., 2016). These findings appear to indicate that benazepril might be Attenuation of the blood pressure response to exogenous angiotensin I after oral more effective than other ACEIs in the management of hypertension in administration of benazepril to healthy adult horses. J. Vet. Intern. Med [Epub ahead of print] http://dx.doi.org/10.1111/evj.12593. horses. In most of the studies, as with the present research, normoten- Bailey, S., Habershon-Butcher, J.L., Ransom, K.J., Elliot, J., Menzies-Gow, N.J., 2008. sive individuals do not experience evident changes in BP with the Hypertension and insulin resistance in a mixed-breed population of ponies administration of these types of drugs (Davis et al., 2014; Gómez-Díez predisposed to laminitis. Am. J. Vet. Res. 69, 122–129. et al., 2014; Muñoz et al., 2016; Serrano-Rodríguez et al., 2016). This is Costa, M.F., Ronchi, F.A., Ivanow, A., Carmona, A.K., Casarini, D., Slocombe, R.E., 2012. Association between circulating angiotensin-converting enzyme and exercise-induced an important concern because in humans, hypotension could appear pulmonary haemorrhage in thoroughbred racehorses. Res. Vet. Sci. 93, 993–994. with the use of these drugs, until response to treatment is evaluated and D'Argenio, D.Z., Schumitzky, A., Wang, X., 2009. ADAPT 5 user's Guide: an individual dosage is established for the patient. This hypotension can Pharmacokinetic/Pharmacodynamic Systems Analysis Software. Biomedical Simulations Resources, Los Angeles, CA. even be accentuated with a simultaneous inclusion of diuretics in the Davis, J.L., Kruger, K., Lafevers, D.H., Barlow, B.M., Schirmer, J.M., Breuhaus, B.A., 2014. therapy (Yusuf et al., 2000). It should be kept in mind that these drugs Effects of quinapril on angiotensin-converting enzyme and plasma renin activity as might also be administered in cardiopathic normotensive horses. well as pharmacokinetic parameters of quinapril and its active metabolite, quinaprilat, after intravenous and oral administration to mature horses. J. Equine Despite the complexity of the PK/PD model used in this research, Vet. 46, 729–733. the results and simulations of the present research suggest that Gardner, S.Y., Atkins, C.E., Rams, R.A., Schwabenton, A.B., Papich, M.G., 2004. benazeprilat, after PO benazepril administration, could be the most Characterization of the pharmacokinetic and pharmacodynamic properties of the angiotensin-converting enzyme inhibitor, enalapril, in horses. J. Vet. Intern. Med. 18, promising ACEI in the horse in comparison to other drugs from the 231–237. same class. However it is necessary to note that there are too few Gehlen, H., Hundermann, T., Rohn, K., Stadler, F., 2002. Aldosterone plasma studies on the effects of ACEIs in equine patients unlike in small animals concentration in horses with heart valve insufficiencies. Res. Vet. Sci. 85, 340–344. Gómez-Díez, M., Muñoz, A., Caballero, J.M., Riber, C., Castejón, F., Serrano-Rodríguez, or humans. At this point, benazepril seems to be the most useful ACEI, J.M., 2014. Pharmacokinetics and pharmacodynamics of enalapril and its active but another such as ramipril, which has closer PK/PD relationships in metabolite, enalaprilat, at four different doses in healthy horses. Res. Vet. Sci. 97, horses with benazepril, could be another interesting option. 105–110. Nevertheless, those are preliminary studies with healthy horses and Serrano-Rodríguez, J.M., Gómez-Díez, M., Esgueva, M., Castejón-Riber, C., Mena-Bravo, A., Priego-Capote, F., Serrano-Caballero, J.M., Muñoz, A., 2016. Pharmacokinetics further work in equine patients should be conducted to support the and pharmacodynamics of ramipril and ramiprilat after intravenous and oral doses of clinical utility of benazepril in horses. ramipril in healthy horses. Vet. J. 208, 38–43. King, J.N., Maurer, M., Toutain, P.L., 2003. Pharmacokinetic⁄pharmacodynamic modelling of the disposition and effect of benazepril and benazeprilat in cats. J. Vet. 5. Conclusions Pharmacol. Ther. 26, 213–224. Krause, A., Lowe, P.J., 2014. Visualization and communication of pharmacometric models with Berkeley Madonna. CPT Pharmacometrics & Systems Pharmacology 3, The use of physiological compartmental PK/PD models allowed the 1–20. pharmacological study of benazeprilat after IV and PO doses of Lees, K.R., Kelman, A.W., Reid, J.L., Whiting, B., 1989. Pharmacokinetics of an ACE benazepril in horses. This study shows that PO availability of benazepril inhibitor, S-9780, in man: evidence of tissue binding. J. Pharmacokinet. Biopharm. 17, 529–550. in horses was low. Serum ACE activity was never suppressed by > Lefèbvre, H.P., Jeunesse, E., Laroute, V., Toutain, P.L., 2006. Pharmacokinetic and 89.51% after PO benazepril, unlike the IV data of 99.63%, using half pharmacodynamic parameters of ramipril and ramiprilat in healthy dogs and dogs the dose of PO administration. It would be interesting to test whether a with reduced glomerular filtration rate. J. Vet. Intern. Med. 20, 499–507. Lefèbvre, H.P., Brown, S.A., Chetboul, V., King, J.N., Puchelon, J.L., Toutain, P.L., 2007. serum ACE inhibition near 90%, as found in our study with PO Angiotensin-converting enzyme inhibitors in veterinary medicine. Curr. Pharm. benazepril at 1.00 mg/kg, would be sufficient to counteract vasocon- Design 13, 1347–1361. striction in pathological conditions. However, data presented in this Maxwell, L., 2015. Horse of a different color: Peculiarities of equine pharmacology. In: Equine Pharmacology, first ed. John Wiley and Sons, pp. 3–15. research together with those reported by other groups indicate that Muñoz, A., Esgueva, M., Gómez-Díez, M., Serrano-Caballero, J.M., Castejón-Riber, C., clinical efficacy of benazepril might have promising results in equine Serrano-Rodríguez, J.M., 2016. Modulation of acute transient exercise-induced patients. hypertension after oral administration of four angiotensin-converting enzyme inhibitor in normotensive horses. Vet. J. 208, 33–37. Navas de Solís, C., Slack, J.A., Boston, R.C., Reef, V.B., 2013. Hypertensive Conflict of interest statement cardiomyopathy in horses: 5 cases (1995–2011). J. Am. Vet. Med. Assoc. 243, 126–130. Picard-Hagen, N., Gayrard, V., Alvinerie, M., Smeyers, H., Ricou, R., Bousquet-Melou, A., None of the authors of this paper has a financial or personal Toutain, P.L., 2001. A nonlabeled method to evaluate cortisol production rate by relationship with other people or organizations that could inappropri- modeling plasma CBG-free cortisol disposition. Am. J. Physiol. Endocrinol. Metab. – ately influence or bias the content of the paper. 281, E946 E956. Toutain, P.L., Lefèbvre, H.P., 2004. Pharmacokinetic and pharmacokinetic/ pharmacodynamic relationships for angiotensin-converting enzyme inhibitors. J. Vet. – Acknowledgments Pharmacol. Ther. 27, 515 525. Toutain, P.L., Lefèbvre, H.P., King, J.N., 2000. Benazeprilat disposition and effect in dogs revisited with a pharmacokinetic/pharmacodynamics modeling approach. J. This research has been generously supported by the Science and Pharmacol. Exp. Ther. 292, 1087–1093. Innovation Council of Spain (Research Project AGL2010-17431). 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. Chromatographia 68, 533–539. ff Appendix A. Supplementary data Yusuf, S., Sleight, P., Poque, L., Bosch, J., Davies, R., Dagenais, G., 2000. E ects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high- risk patients The heart outcomes prevention evaluation study investigators. N. Engl. Supplementary material. J. Med. 342, 145–153.

122 Angiotensin-converting enzyme inhibitors in the horse

11.3.1 SUPPLEMENTARY MATERIALS FOR PAPER III

Chromatographic methodology

Analytical method for ACEIs determination.

Reagents

Chromatographic grade acetonitrile and water were used for LC–MS/MS analyses. Mass spectrometry grade formic acid was used as ionization agent.

Chromatographic grade methanol and ethyl acetate were used.

Sample treatment

A volume of 700 μL of serum sample was spiked with IS solution and then, 1 mL of methanol was added. Proteins were precipitated by shaking in an ultrasonic bath for 5 min, followed by centrifugation at 28300 g for 10 min at 4º C. Supernatant was extracted, and 3 mL of ethyl acetate were added. After, it was mixed and centrifuged for 5 min at 2000 g. The upper organic layer was evaporated to dryness under N2 at 40ºC. The residue was reconstituted and sonicated in 100 μL of methanol and transferred to auto sampler vials, and an aliquot of 5 μL was injected.

Instruments and apparatus

Chromatographic separation was carried out with an Agilent (Palo Alto, CA, USA) 1200

Series LC system coupled to an Agilent 6460 triple quadrupole mass spectrometer equipped with a JetStream® Technology electrospray (ESI) ion source. The data were processed using

MassHunter Workstation Software (V-B.05) from Agilent for qualitative and quantitative analyses.

The analytical column was a Mediterranea Sea C18 analytical column (3 μm particle size, 50 mm

× 4.6 mm, Teknokroma, Barcelona, Spain) that was thermostated at 25°C.

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Manuel Gómez Díez

Chromatographic and mass spectrometry conditions

An isocratic method was adopted for chromatographic separation of the target analytes.

The mobile phase consisted of 0.1% formic acid in 85/15 (v/v) acetonitrile/water at 0.3 mL/min flow rate. The injection volume was 5 μL and the total time for each run was 4 min. The ESI conditions were as follows: gas heater (N2) 325 ºC, gas flow (N2) 12 L/min, nebulizer pressure 45 psi, sheath gas heater (N2) 350 ºC, sheath gas flow (N2) 11 psi, voltage capillary +3500 V.

Determination of the target compounds was carried out by selected reaction monitoring (SRM) mode using the parameters listed in Table 1. Figure 1 show MS/MS spectra obtained for each precursor ion to justify the product ions used for quantitative and qualitative transitions. The representative chromatograms of serum samples are shown in Figure 2

Sensitivity, linear calibration range and recovery

Calibration plots were run using the peak area ratio between the target analyte and that of the IS (50 ng/ml of enalapril) as a function of the standard concentration of benazepril and benazeprilat. The absence of enalapril in the target samples was previously confirmed by LC–

MS/MS analysis. Regression coefficients were 0.994 for benazepril and 0.993 for benazeprilat.

The analytical features of the calibration models are listed in Table 2. The limits of detection (LOD) and quantitation (LOQ) for both analytes were 0.03 ng/mL and 0.1 ng/mL respectively. The accuracy and precision were within 1.97 – 5.27 %. The mean percentage recoveries of benazepril, benazeprilat and enalapril as internal standard were 91.17 ± 11.81, 95.26 ±13.01 and 93.11 ±

8.33%, respectively.

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Angiotensin-converting enzyme inhibitors in the horse

Tables and Figures

Precursor ion Q1 voltage Collision energy Quantitative ion Qualitative ions Analyte [m/z] [V] [eV] [m/z] [m/z]

Benazepril 425.3 135 20 351.1 190.0 234.8 Benazeprilat 397.2 120 20 351.1 190.0 313.9 Enalapril 377.1 135 20 234.1 160 303.1 Table 1. Selected reaction monitoring (SRM) parameters for quantitative analysis of benazepril

and benazeprilat.

Calibration range Analyte LOQ LOD Calibration equation R2 (ng/mL) (ng/mL) (ng/mL) Benazepril 0.1 – 500 0.1 0.03 y = 0.0392x – 0.1698 0.994

Benazeprilat 0.1 – 500 0.1 0.03 y = 0.0331x – 0.1393 0.993

Table 2. Analytical features of the method.

A B

C

Fig. 1. MS/MS spectra of analytes and IS: (A) Benazepril, (B) Benazeprilat, (C) Enalapril.

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Manuel Gómez Díez

A A

BB

C C

Fig. 2. SRM chromatograms obtained for analytes and IS: (A) Benazepril, (B) Benazeprilat, (C)

Enalapril.

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Angiotensin-converting enzyme inhibitors in the horse

PK/PD modeling

benazepril kf benazeprilat benazeprilat absorbed free kd bound to Vc Vc Serum ACE

k10 f . B ka circ max

benazepril GI tract

Model 1: PK/PD disposition model and differential equations for benazeprilat and benazepril after PO benazepril administration.

푑퐶 = − 푘 ∙ 퐶 푑푡

푑퐶 = 푘 ∙ 퐶 − 푘 ∙ 퐶 푑푡

푑퐶 (푘 ∙ 퐶 − 푘 ∙ 퐶 ) = 푑푡 1 + 푓 ∙ 퐵 ∙

퐶 퐸(푡) = 퐸 ∙ 1 − 퐶 + 퐼퐶

benazepril kf benazeprilat benazeprilat intravenous free kd bound to Vc Vc Serum ACE f . B k10 circ max

Model 2: PK/PD disposition model and differential equations for benazeprilat and benazepril after IV benazepril administration.

푑퐶 = −푘 ∙ 퐶 푑푡

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Manuel Gómez Díez

푑퐶 (푘 ∙ 퐶 − 푘 ∙ 퐶 ) = 푑푡 1 + 푓 ∙ 퐵 ∙

퐶 퐸(푡) = 퐸 ∙ 1 − 퐶 + 퐼퐶

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11.4. PAPER IV

Modulation of Acute Transient Exercise-Induced Hypertension after Oral Administration of four Angiotensin-Converting Enzyme Inhibitors in Normotensive Horses

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The Veterinary Journal 208 (2016) 33–37

Contents lists available at ScienceDirect

The Veterinary Journal

journal homepage: www.elsevier.com/locate/tvjl

Modulation of acute transient exercise-induced hypertension after oral administration of four angiotensin-converting enzyme inhibitors in normotensive horses Ana Muñoz a,b,*, María Esgueva b, Manuel Gómez-Díez b, Juan Manuel Serrano-Caballero c, Cristina Castejón-Riber b, Juan Manuel Serrano-Rodríguez c a Department of Animal Medicine and Surgery, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain b Equine Sport Medicine Centre, CEMEDE, School of Veterinary Medicine, University of Córdoba, Córdoba 14004, Spain c Department of Pharmacology, Toxicology, and Legal and Forensic Medicine, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain

ARTICLE INFO ABSTRACT

Article history: Changes in blood pressure (BP) during acute hypertension in response to angiotensin-converting enzyme Accepted 10 October 2015 inhibitors (ACEIs) have not been investigated in normotensive horses. In this study, six healthy horses were subjected to five trials, consisting in a treadmill exercise workload of 8 m/s for 1 min, 2 h after oral Keywords: administration (PO) of placebo (0 mg/kg), enalapril (2.0 mg/kg), quinapril (1.0 mg/kg), ramipril (0.2 mg/ Equine kg) or benazepril (0.5 mg/kg). Serum angiotensin converting enzyme (ACE) activity was measured and Exercise systolic (SBP) and diastolic (DBP) blood pressures were recorded at rest (R), 2 h after placebo or ACEI Angiotensin-converting enzyme inhibitors administration (pre-E) and within the first 20 s after exercise (post-E). Blood pressure Hypertension Mean maximum serum ACE inhibition 2 h after PO administration was 4.8% (placebo), 39.4% (enalapril), 46.4% (quinapril), 55.0% (ramipril) and 71.68% (benazepril). There were no significant differences in serum ACE inhibition between enalapril and quinapril. SBP and DBP at times R and pre-E were not different in any of the five trials. In response to exercise, SBP increased by 67.6% (placebo), 52.7% (enalapril), 43.1% (quinapril), 26.6% (ramipril) and 4.2% (benazepril). In response to exercise, DBP increased by 20.6, 13.2, 11.7, 16.6 and 3.7% after placebo, enalapril, quinapril, ramipril and benazepril administration, respec- tively. Serum ACE activity changed during exercise, but statistical significance was not achieved. In conclusion, administration of PO benazepril at a dose of 0.5 mg/kg modulated physiological hyperten- sion induced by exercise in horses that were otherwise normotensive. © 2015 Elsevier Ltd. All rights reserved.

Introduction and inhibit angiotensin converting enzyme (ACE) (Toutain and Lefebvre, 2004). Angiotensin-converting enzyme inhibitors (ACEIs) prevent con- The effects of ACEIs on blood pressure (BP) in normotensive version of angiotensin I to II and attenuate the actions of angiotensin human beings or animals have not been completely elucidated, with II (Brewster et al., 2003; Toutain and Lefebvre, 2004; Atlas, 2007). discrepant results between studies. In normotensive human beings, Angiotensin II activates AT1 receptors on vascular smooth muscle Gainer et al. (1998) did not find significant changes in systolic (SBP), of pre-capillary arterioles and post-capillary venules, causing va- diastolic (DBP) and mean (MBP) blood pressures after ACEI admin- soconstriction (Brewster et al., 2003; Atlas, 2007). ACEIs cause mixed istration (captopril). In other studies, BP decreased in normotensive vasodilatation, by decreasing available angiotensin II and sympa- humans when they were sodium-depleted (Vidt et al., 1982; Todd thetic release of adrenaline, and by enhancing available bradykinin and Heel, 1986). Several studies have shown that acute changes in and bradykinin-mediated release of nitric oxide (Muir et al., 2001). BP are correlated with pre-treatment plasma renin activity and an- ACEIs are administered per os (PO) as pro-drugs (i.e., benazepril), giotensin concentrations, with the greatest reductions in BP in which are more lipophilic and better absorbed from the gastroin- patients with the highest plasma renin activity (Waeber et al., 1982; testinal tract than the active drugs (i.e. benazaprilat). Pro-drugs are Given et al., 1984). Conversely, Shoback et al. (1983) reported that subsequently transformed to their active metabolites by esterases administration of an ACEI (enalapril) lowered BP in normotensive humans in a dose-related fashion. These differences between studies in the effects of ACEIs on BP have been also observed in normotensive horses; PO administra-

* Corresponding author. Tel.: +34 957 211068. tion of 0.5–2.0 mg/kg enalapril did not influence BP (Gardner et al., E-mail address: [email protected] (A. Muñoz). 2004; Gómez-Díez et al., 2014). Davis et al. (2014) did not find http://dx.doi.org/10.1016/j.tvjl.2015.10.036 1090-0233/© 2015 Elsevier Ltd. All rights reserved. 34 A. Muñoz et al./The Veterinary Journal 208 (2016) 33–37 significant changes in BP after PO administration of 0.25–0.5 mg/ intense exercise (post exercise, post-E). Blood samples allowed to coagulate in plain kg quinapril. The lack of vasodilatory effects of ACEIs was attributed tubes in a refrigerator for 30 min, then centrifuged and the serum was stored at −90 °C until analysis. to normotension and to a non-activated renin–angiotensin– aldosterone axis. In contrast, Afonso et al. (2013) observed a mild Measurement of angiotensin-converting enzyme inhibitor activity reduction in BP (5–10 mm Hg) after PO administration of low and high doses of ramipril, quinapril, benazepril and peridonpril. Serum ACE activity was quantified by the hydrolysis of N-[3-(2-furyl) acrylolyl]- The effects of ACEIs on BP in normotensive horses during an acute L-phenylalanyl-glycyl-glycine (FAPGG), forming furylacryloyl-phenylalanine (FAP), which results in a decrease in absorbance at 340 nm (Maguire and Price, 1985). This hypertensive physiological situation have not been investigated. We method has been used to measure serum ACE activity in horses previously (De Mello used an experimental model of temporal hypertension based on ex- Costa et al., 2010, 2012; Costa et al., 2011; Afonso et al., 2013; Gómez-Díez et al., ercise; brief, strenuous exercise in horses induces a dramatic and 2014). FAPGG (500 μL) was mixed with 50 μL serum and incubated for 5 min at 37 °C. sudden rise in BP (Masri et al., 1990). In the current study, four dif- The absorbance was measured at 340 nm with a spectrophotometer (Biosystems, ferent ACEIs and placebo were administered PO before exercise; BP model A-15). Quality control was performed using specific reagents for this assay. Linear calibration curves from 3 to 150 IU/L were obtained; the correlation coeffi- and inhibition of serum ACE activity were measured before and after cients (r) were >0.99, the precision was 2.86–6.21% and the limit of quantification exercise. The main aims of the study were: (1) to elucidate whether (LOQ) and limit of detection (LOD) were 5 and 3 IU/L, respectively. pre-exercise PO administration of an ACEI reduces the hyperten- sive response to exercise, and (2) to assess whether the changes in Measurement of blood pressure BP after exercise are associated with inhibition of circulating/ Systolic and diastolic blood pressures (SBP and DBP, respectively) were mea- serum ACE activity. It was hypothesised that the acute transient sured non-invasively from the coccygeal artery (S/5 Datex-Ohmeda CompactR). Three hypertensive response to exercise would be lower after PO ACEI ad- measurements were made during R and pre-E; results are presented as the mean ministration and that this effect would be proportional to the degree of each of the three recordings. At post-E, only one measurement was made, because of inhibition of serum ACE activity. of the rapid decrease in BP after exercise.

Statistical analysis Materials and methods

Descriptive statistical parameters including arithmetic mean, maximum and Horses minimum were calculated. The Kruskal–Wallis test was used to compare differ- ences for each parameter measured at the three time points (R, pre-E, post-E). When Six healthy mature unfit crossbred horses (three mares and three geldings), aged significant differences were found, a Wilcoxon rank sum test was used as a second 8–12 years and with body weights of 382–525 kg, were included in the study. The test (pairwise comparison). Correlations between the different parameters were cal- horses were found to be healthy on physical examination, haematology and serum culated using Spearman’s rank correlation. The significance level was set at P < 0.05. biochemical profile, BP monitoring and echocardiography. During the experi- The statistical software was Statgraphics Centurion XVI.I for Windows (StatPoint ments, the horses were housed outdoors, in paddocks, with free access to water, Technologies). but not to salt blocks. They were fasted for 12 h prior to and 8 h after PO adminis- tration of ACEIs/placebo, even though it has been demonstrated that feeding does not affect serum ACE inhibition (Afonso et al., 2013). On days outside the experi- Results ment, horses were maintained on their normal ration of ryegrass haylage. The six horses tolerated PO administration of the four ACEIs Experimental design without any noticeable adverse effects. Serum ACE activity and SBP A blinded and randomised Latin square design in five trials was used. Two hours and DBP values for the five trials and for the three sampling times before exercise, each animal received a single PO dose of placebo or ACEI (2.0 mg/ are presented in Figs. 1–3, respectively. The baseline ACE activity kg enalapril, 1.0 mg/kg quinapril, 0.2 mg/kg ramipril or 0.5 mg/kg benazepril). These before administration of ACEI or placebo did not change over the doses and the time between dosage and exercise were selected according to the results time of study between trials (P = 0.53) or between animals (P = 0.58). of previous ACEI pharmacokinetics–pharmacodynamics (PK-PD) studies, which found that maximum serum ACE inhibition occurs at ~2 h (Afonso et al., 2013; Davis et al., Differences in SBP and DBP at time R were not found between trials 2014; Gómez-Díez et al., 2014, unpublished observations from our group). There and the horses were normotensive. Two hours after PO adminis- was a washout period of 1 week between each experiment. The study was ap- tration (time pre-E), serum ACE activity did not change in the placebo proved by the Ethical Committee for Animal Experimentation of the University of trial, but decreased in the four ACEI trials, achieving the greatest Córdoba (approval number 55.60 PE; date of approval 1 February 2010). reduction after PO benazepril (Fig. 1). At this time, all the horses were normotensive and a significant effect on BP was not detected. Preparation of angiotensin-converting enzyme inhibitors There was no significant increase in serum ACE activity with ex- Enalapril hydrochloride (Enacard 20 mg, Merial), ramipril (Vasotop P 5 mg, Merck ercise in any of the five trials. Exercise after placebo administration Sharp and Dohme Animal Health), quinapril hydrochloride (Acuprel 40 mg, Pfizer) induced a marked increase in SBP and a mild increase in DBP and benazepril hydrochloride (Fortekor 20 mg, Novartis) were used in the study. For (Figs. 1–3). The exercise-induced rise in SBP was reduced when ACEIs each pro-drug, tablets were dissolved and suspended in 150 mL of water, soni- cated in an ultrasonic bath for 15 min and stored at 4 °C before trials. For placebo were administered PO, and the maximal reduction was achieved in dosing, the equivalent amount of water without pro-drug was used. Pro-drug and the benazepril trial. placebo administration was made by nasogastric intubation, then the nasogastric The percentages of serum ACE inhibition and increases in SBP tube was rinsed with 250 mL of water. and DBP, comparing post-E and R values for each trial, are pre- sented in Table 1. The percentage of serum ACE inhibition at time Treadmill exercise post-E compared to R time was negatively correlated with the per- Horses were subjected to a brief intense treadmill exercise (Mustang 2000, Kagra) centage of increase in SBP (r =−0.861), but correlations with the to induce hypertension. The exercise was preceded by 5 min at walk (1.3–1.5 m/s) percentage of increase presented by DBP were not significant and 5 min at trot (3.5–3.7 m/s), on the treadmill without inclination. Intense exer- (r =−0.270). cise consisted of a workload of 8 m/s for 1 min, with the treadmill inclined by 6%. The horses were unfit and untrained and, therefore, this exercise protocol was of sufficient intensity to induce hypertension. After exercise, horses performed a cooling- Discussion down exercise, with 5 min at trot and 5 min at walk on the treadmill uninclined. The aims of this study were to elucidate whether PO adminis- Collection of blood samples tration of different ACEI pro-drugs, prior to the exercise, could modulate the physiological hypertensive response to exercise and Venous blood samples were collected and BP was measured three times: at rest, before starting the experiments, with horses in the paddock (R), 2 h after PO ad- whether the reduced response of the BP to intense exercise was as- ministration of placebo or ACEIs (pre-exercise, pre-E) and within the first 20 s after sociated with the degree of inhibition of serum ACE activity. The A. Muñoz et al./The Veterinary Journal 208 (2016) 33–37 35

Fig. 1. Serum ACE activity at rest, at 2 h after PO administration of placebo or ACEIs (pre-E) and immediately after intense exercise (post-E). Different letters indicate sig- nificant differences between trials at each of the sampling times. * Significant differences from resting values. P < 0.05.

main findings were: (1) the maximum inhibition of serum ACE ac- ity is caused by the release of the enzyme from the lungs. The lack tivity was found with PO benazepril at 0.5 mg/kg; (2) benazepril of a significant effect of exercise suggests that a longer exercise time modulated the increase in BP associated with a short intense hy- is needed to release local or pulmonary ACE. pertensive exercise; and (3) the increase in SBP in response to The percentage of serum ACE inhibition after 2.0 mg/kg enalapril exercise after administration of ACEIs was negatively correlated with PO (39.4%) was slightly higher than the values previously pre- the percentage of inhibition of serum ACE activity. sented at the same dose (30.2%; Gómez-Díez et al., 2014). After PO After brief maximal exercise, there were no significant changes quinapril, a maximum ACE inhibition of 46.4% was found, in agree- in serum ACE activity in the present study. These results do not agree ment with the mean values of 40.6% reported by Afonso et al. (2013) with those reported by De Mello Costa et al. (2012), who de- in response to a dose of 0.25 mg/kg and 53.4% reported by Davis scribed an increase from 99.5 ± 4.7 to 122.5 ± 7.5 IU/L after a treadmill et al. (2014) in response to a dose of 0.5 mg/kg. Our data for Ramipril exercise, consisting of a warming-up period (duration not indi- were consistent with those reported by Afonso et al. (2013), who cated), followed by seven workloads of 2 min each; the duration of reported maximal serum ACE inhibition of 38.2 and 59.7% with doses exercise, excluding warming-up, was 14 min. In the present study, of 0.1 and 0.3 mg/kg, respectively. Results from our laboratory show the workload was only 1 min in duration, since it was found that a maximum ACE inhibition of 54.0% with a dose of 0.2 mg/kg of this exercise load induces the largest increase in BP. Therefore, dif- ramipril (unpublished observations). After PO administration of ferences in serum ACE concentrations between studies may have benazepril at 0.5 mg/kg, Afonso et al. (2013) found a maximum been associated with their different durations of exercise. It has been serum ACE inhibition of 86.9%, which is slightly higher than the speculated that an exercise-induced increase in serum ACE activ- values (71.7%) observed in our study using the same dose.

Fig. 2. Systolic blood pressure at rest, at 2 h after PO administration of placebo or ACEIs (pre-E) and immediately after intense exercise (post-E). Different letters indicate significant differences between trials at each of the sampling times. * Significant differences from resting values. P < 0.05. 36 A. Muñoz et al./The Veterinary Journal 208 (2016) 33–37

Fig. 3. Diastolic blood pressure at rest, at 2 h after PO administration of placebo or ACEIs (pre-E) and immediately after intense exercise (post-E). Different letters indicate significant differences between trials at each of the sampling times. * Significant differences from resting values. P < 0.05.

The inhibition of serum ACE activity at 2 h after PO ACEI ad- Brief intense exercise requires the release of vasopressin ministration was lowest for enalapril and quinapril, intermediate and angiotensin II to cause vasoconstriction in non-obligated for ramipril and greatest for benazepril. These data are in agree- tissues (splanchnic vascular beds) to facilitate the increase in ment with other PK-PD studies in horses (Afonso et al., 2013; Davis BP for optimal cardiovascular function (McKeever and Hinchcliff, et al., 2014; Gómez-Díez et al., 2014). However, it should be noted 1995). As a consequence, this type of exercise in unfit untrained that the dose of enalapril was 2-fold, 4-fold and 10-fold higher than horses was used to trigger an acute transient hypertensive quinapril, benazepril and ramipril, respectively. The PK-PD prop- situation. erties of enalapril compared to other ACEIs could have been There are three main considerations in relation to the results ob- responsible for these findings (Knütter et al., 2008; Gómez-Díez et al., tained in the current study. Firstly, a reduction in BP during exercise 2014). These data might indicate that the systemic availability after could have detrimental consequences for the equine athlete, since PO ACEI administration could be different at the doses tested, and hypertension results in a redistribution of blood flow to the tissues therefore, the degree of serum ACE inhibition. involved in exercise. Marked hypotension would promote In this study, administration of ACEIs did not modify SBP and hypoperfusion and collapse. The vasodilatory properties of ACEIs DBP in a normotensive situation (during R and pre-E), but it reduced are mild, even though a larger reduction in BP can be found in the exercise-induced increase in SBP. Furthermore, this effect ap- sodium-depleted individuals or when ACEIs are administered si- peared to be associated with the capacity of the ACEIs to inhibit multaneously with diuretics (Lefebvre et al., 2007). In the current serum ACE activity. study, caution was taken with hypotension, although none of the The effects of ACEIs on BP in the normotensive state are not clear. horses experienced negative effects during exercise. Similarly, Muir PO administration of ACEIs in sodium-repleted normotensive human et al. (2001) did not find hypotension in exercising horses after IV subjects induces a mild decrease in BP. This reduction was greater administration of enalaprilat. in hypertensive and sodium-depleted normotensive human beings Secondly, pathological hypertension results from activation of (Niarchos et al., 1979; Kiowski et al., 1992; Gainer et al., 1998). Ad- vasoconstrictive mechanisms which interact with counteractive ministration of ACEIs elicits a more intense SBP reduction in mechanisms. These mechanisms (both vasoconstrictive and hypertensive than in normotensive dogs (Grauer et al., 2000). In nor- vasodilatory) and their interactions probably are different in acute motensive healthy cats, PO enalapril leads to a slight but significant transient physiological exercise-induced hypertension, and acute (or decrease in SBP (Uechi et al., 2002). In agreement with other studies, chronic) pathological hypertension. As a consequence, the modu- BP was not influenced by PO administration of ACEIs in normoten- lation of BP during exercise achieved in the present study after sive horses (Gardner et al., 2004; Davis et al., 2014; Gómez-Díez et al., administration of ACEIs should not be extrapolated to pathologi- 2014). cal hypertension.

Table 1 Percentages of serum ACE inhibition and increase of systolic and diastolic blood pressures after intense exercise compared to rest in horses after placebo or oral adminis- tration of four different ACEIs.

PO doses (mg/kg) % Serum ACE inhibition % Increase of SBP % Increase of DBP

Placebo 0 4.75 (1.77–8.33) 67.49 (61.68–72.82) 20.64 (2.70–40.98) Enalapril 2 39.43 (30.39–48.60) 52.71 (38.53–73.27) 13.17 (−13.04–23.88) Quinapril 1 46.40 (39.25–56.31) 43.11 (31.07–55.00) 11.68 (1.37–25.00) Ramipril 0.2 54.96 (33.62–64.22) 26.64 (16.67–38.61) 16.62 (0.00–38.98) Benazepril 0.5 71.68 (64.08–78.81) 4.21 (1.69–14.56) 3.74 (−8.82–10.77)

ACE, angiotensin-converting enzyme; SBP, systolic blood pressure; DBP, diastolic blood pressure. Data are presented as means with minimum and maximum values. A. Muñoz et al./The Veterinary Journal 208 (2016) 33–37 37

Thirdly, the results of our study suggest that there was a lower as well as pharmacokinetic parameters of quinapril and its active metabolite, increase or a lack of increase in SBP in response to exercise when quinaprilat, after intravenous and oral administration to mature horses. Equine Veterinary Journal 46, 729–733. higher percentages of serum ACE inhibitions were achieved. This De Mello Costa, M.F., Anderson, G.A., Davies, H.M., El-Hage, C.M., Slocombe, R.F., 2010. corroborates the hypothesis that serum ACE inhibition induced by Circulating angiotensin converting enzyme in endurance horses: Effects of the ACEIs might have been effective. However, further studies are exercise on blood levels and its value in predicting performance. Equine Veterinary Journal 38, 152–154. necessary to investigate the effects of differences in doses for each De Mello Costa, M.F., Anderson, G.A., Davies, H.M., Slocombe, R.F., 2012. Effects of individual drug on BP in horses. acute exercise on angiotensin I-converting enzyme (ACE) activity in horses. Equine The data derived from the current study are in disagreement with Veterinary Journal 44, 487–489. Gainer, J.V., Morrow, J.D., Loveland, A., King, D.J., Brown, N.J., 1998. Effect of those described by Muir et al. (2001), who exercised six horses at bradykinin-receptor blockade on the response to angiotensin converting enzyme supramaximal velocities immediately after IV treatment with 0.5 mg/ inhibitor in normotensive and hypertensive subjects. New England Journal of kg enalaprilat. However the PK-PD relationships of IV enalaprilat Medicine 339, 1285–1292. are likely to differ from PO enalapril (Gómez-Díez et al., 2014). Muir Gardner, S.Y., Atkins, C.E., Rams, R.A., Schwabenton, A.B., Papich, M.G., 2004. Characterization of the pharmacokinetic and pharmacodynamic properties of et al. (2001) reported values of SBP and DBP similar to those found the angiotensin-converting enzyme inhibitor, enalapril, in horses. Journal of after placebo administration. Despite our data, there is currently no Veterinary Internal Medicine 18, 231–237. evidence that this modulation of BP could limit acute or chronic Given, B.D., Taylor, T., Hollenberg, N.K., Williams, G.H., 1984. Duration of action and short-term hormonal responses to enalapril (MK 421) in normal subjects. Journal pathological hypertensive situations. of Cardiovascular Pharmacology 6, 436–441. A limitation of the present study was that the cuff width-to- Gómez-Díez, M., Muñoz, A., Caballero, J.M., Riber, C., Castejón, F., Serrano-Rodríguez, tail circumference ratio was not measured, and BP recordings were J.M., 2014. Pharmacokinetics and pharmacodynamics of enalapril and its active metabolite, enalaprilat, at four different doses in healthy horses. Research in not corrected for vertical distances from the tail to the heart base, Veterinary Science 97, 105–110. as recommended by Parry et al. (1984) and Navas de Solís et al. Grauer, G.F., Greco, D.S., Getzy, D.M., Cowgill, L.D., Vaden, S.L., Chew, D.J., Polzin, D.J., (2013). These influences were probably of minor importance, because Barsanti, J.A., 2000. Effects of enalapril versus placebo as a treatment for canine idiopathic glomerulo-nephritis. Journal of Veterinary Internal Medicine 14, the experimental conditions and the animals used for the five trials 526–533. were the same. Kiowski, W., Linden, L., Kleinbloesem, C., Van Brummelen, P., Bühler, F.R., 1992. Blood pressure control by renin-angiotensin system in normotensive subjects. Assessment of angiotensin converting enzyme and renin inhibition. Circulation Conclusions 85, 1–8. 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