Randomized Controlled trial on 21 puppies using Sildenafil suggests the drug reduces the LES tone and facilitating the emptying of the esophagus, could represent a useful drug for the clinical management of Congenital Idiopathic Megaesophagus in Dogs. Veterinary Record (2017) F. Quintavalla, A. Menozzi, C. Pozzoli, E. Poli, P. Donati, D.K. Wyler, P. Serventi, S. Bertini

Sildenafil improves clinical signs and radiographic features in dogs with congenital idiopathic megaoesophagus: a randomised controlled trial
F. Quintavalla, A. Menozzi, C. Pozzoli, E. Poli, P. Donati, D. K. Wyler, P. Serventi, S. Bertini

We evaluated the efficacy of oral sildenafil citrate in dogs with congenital idiopathic megaoesophagus (CIM). Twenty-one puppies were randomly assigned to two groups (treatment and control). The dogs were given sildenafil oral suspension 1 mg/kg every 12 hours for 14 days or placebo in a masked fashion. Clinical signs (frequency of regurgitation and weight gain) and oesophagrams (relative oesophageal diameter, ROD) were evaluated in order to assess the efficacy of drug treatment, by examiners who were unaware of the study protocol. In addition, a set of in vitro experiments on isolated samples of canine lower oesophageal sphincter (LOS) was performed, and the effects of increasing concentrations of sildenafil on basal tone and electrically-stimulated motility were assessed. Sildenafil administration significantly reduced the number of regurgitation episodes (0.88 ±1.40 v 2.65±1.56, P<0.0001) and significantly increased weight gain in the treated dogs compared to controls (79.76±28.30 per cent v 53.40±19.30 per cent, P=0.034). ROD values, at the end of the treatment period, were significantly decreased in the sildenafil group, compared to pre-treatment values (0.97±0.19 v 0.24±0.14, P<0.0001), in contrast to control subjects (0.98±0.17 v 1.10±0.25, P=0.480). In accordance with the in vivo findings, sildenafil dose-dependently reduced basal tone and increased electrically-induced relaxation of dog LOS samples. These results suggest that sildenafil citrate helps ameliorate clinical and radiographic signs in dogs with CIM by reducing LOS tone, and could represent a novel therapeutic tool for the treatment of this disease.

The term megaoesophagus is used to describe a disease characterised by reduced or absent oesophageal motility which causes the accumulation of ingesta, dilatation of the oesophageal lumen, food regurgitation (which is often mistaken for vomit by the dog owner),and weight loss as the main clinicalsigns. Megaoesophagus may be idiopathic, congenital or acquired, or secondary to different aetiologies, such as myasthenia gravis, hypothyroidism or Addison’s disease. Congenital idiopathic megaoesophagus (CIM) is often observed at or before 10 weeks of life, and the condition
frequently affects more than one animal in the same litter (Harveyand others 1974, Glidewell 1983). CIM causes poor weight gain (WG) in puppies shortly after weaning, and, even though most animals tend to show spontaneous improvement over time, they require long-lasting physical and nutritional support, and the risk of fatal complications such as aspiration pneumonia is high. The pathogenesis of CIM is currently unclear. A predisposition for the disease has been reported in large and giant-breed dogs such as the German shepherd, Great Dane, Irish setter, Labrador retriever, Irish wolfhound and Newfoundland (Knowles and others 1990), and genetics might play a role in the aetiology of CIM because autosomal dominant inheritance has been demonstrated in miniature Schnauzers and fox terriers (Washabau 2003). A suspected hereditary form has also been reported in Bouvier des Flandres dogs (Peeters and others 1991). It has been hypothesised that the congenital form of the disease is linked to a reduced or delayed development of the oesophageal neuromuscular system, in particular of the afferent vagal innervation, which fails to respond to the mechanical stimulus induced by food, thus resulting in ineffective peristalsis (Holland and others 1994, 1996, 2002). Manometric studies have found a normal tone and functioning of the lower oesophageal sphincter (LOS) in dogs with idiopathic megaoesophagus (Diamant and others 1973), unlike in other oesophageal motility
Veterinary Record (2017) doi: 10.1136/vr.103832 F. Quintavalla, DVM, A. Menozzi, DVM, PhD, P. Serventi, BSc, S. Bertini, DVM, PhD, Department of Veterinary Science, Universityof Parma, Parma, Italy C. Pozzoli, PhD, E.Poli, PhD, Department of Neuroscience, Universityof Parma, Parma, Italy P. Donati, DVM, Cerro Maggiore, Milano, Italy D. K.Wyler, DVM, The Animal Medical Hospital and Whitestone Veterinary Care, New York, NY, USA E-mail for correspondence: alessandro. menozzi@unipr.it Provenance: Not commissioned; externally peer reviewed Accepted January 23, 2017
April 22, 2017 | Veterinary Record

group.bmj.com on May 15, 2017 – Published by http://veterinaryrecord.bmj.com/Downloaded from disorders in humans, such as achalasia or diffuse oesophageal spasm, where a hypertonicity of sphincter muscle is present (Pohl and Tutuian 2007, Roman and Kahrilas 2012). However, a failure by the LOS to relax in response to intraluminal balloon distension has been observed (Tan and Diamant 1987), further supporting the hypothesis of a functional defect of oesophageal sensory innervation. CIM treatment is frustrating, resulting in high mortality from directly related causes such as malnutrition and aspiration pneumonia or because euthanasia is required due to the continuing clinical signs (Harvey and others 1974, McBrearty and others 2011). In the majority of cases, drugs are not adequately effective, and the treatment is based mostly on nutritional support and alterations in body position (Chandra and others 1989). Several pharmacological approaches, especially with prokinetic drugs such as metoclopramide, domperidone or cisapride, have been proposed, with modest or varying results (Washabau 2003). However, recent studies with high-resolution manometry showed that cisapride significantly increased LOS pressure in healthy dogs, and this could represent a serious concern in dogs with megaoesophagus (Kempf and others 2014, Ullal and others 2016). Swallowing and oesophageal motility are complex processes involving a multifaceted interplay between excitatory innervation, mostly vagal cholinergic fibres, and inhibitory innervation, which releases nitric oxide (NO) as the main neurotransmitter. Endogenous NO induces smooth muscle relaxation through the synthesis of the second messenger cyclic guanosine monophosphate (cGMP). Sildenafil, a selective phosphodiesterase-type 5 (PDE-5) inhibitor, indirectly potentiates the action of endogenous NO by reducing cGMP degradation due to PDE-5 (Zhu and others 2007). Sildenafil is an effective vasodilator and is widely prescribed for the treatment of erectile disorders in man; however, it is also used to treat pulmonary hypertension, and it relaxes the smooth muscle of other organs such as the uterus (Méhats and others 2006) and the gallbladder (Degirmenci and others 2006). The vasorelaxant properties of sildenafil have also been observed in dogs (Souza-Silva and others 2005, Bach and others 2006), and this drug represents a valid option for the treatment of pulmonary hypertension in this species. In humans and cats sildenafil has already been shown to induce the relaxation of the LOS (Zhang and others 2001, Fox and others 2007). Therefore, in the current study we evaluated the therapeutic efficacy of sildenafil in dogs affected by CIM, on the premise that a decreased LOS tone would facilitate the entry of the ingesta into the stomach, thus reducing the pressure inside the oesophageal lumen. The effects of sildenafil were assessed by evaluating the clinical signs of the disease, and by means of oesophagrams. Moreover, in order to understand better the effects of sildenafil observed in vivo, a set of in vitro experiments on smooth muscle samples of canine LOS was also performed.

Materials and methods
Animals Twenty-one puppies of both sexes with clinical and radiographic signs of CIM, sourced from six breeding kennels, were enrolled in the study (Table 1). The sample size was determined on the basis of a previous study (Lee and others 2003). CIM was initially suspected on the basis of patient age (≤50 days), history and clinical findings, such as post-prandial regurgitation of undigested food, palpable enlargement of the oesophagus and poor body condition; the diagnosis was then confirmed by plain radiography and oesophagrams. Pre-admission exclusion criteria included the presence of one or more of the following conditions: diarrhoea, cardiovascular abnormalities, distension of the oesophagus limited to the cervical region, and clinical signs (fever, nasal discharge, cough) or radiographic evidence of aspiration pneumonia. None of the puppies had received any medication within 48 hours from the
beginning of the study, and none had been previously treated with prokinetic drugs. The present study was conducted as a randomised controlled trial; therefore the dogs eligible for enrolment were randomly assigned to two parallel groups, treatment and control (placebo), with an allocation ratio of 1:1. Informed consent about the nature of the diagnostic and experimental procedures to be performed was obtained from the dog owners (breeders), before enrolling their puppies. The trial was conducted in compliance with institutional guidelines for research on animals, and it was approved by the Ethics Committee of the University of Parma (O.P.B.A.), Prot. N. 136/ OPBA/2016. Each dog was given the daily amount of food, according to the caloric requirements of each breed, divided into six equal small meals. All the dogs were fed with the same homogenised commercial canned puppy food from an elevated position, and none was managed with a percutaneous endoscopy gastrostomy tube. Moreover, all the dogs were kept in an elevated position for 10 minutes after each meal, and carefully observed for the following 30 minutes to detect possible regurgitation episodes. For each dog, the breeder was asked to randomly choose one of two identical bottles (labelled ‘A’ and ‘B’), and therefore was masked to the nature of the content. Bottles labelled ‘A’ contained sildenafil citrate (Revatio 10 mg/ml oral suspension, Pfizer Italia), whereas the bottles labelled ‘B’ contained placebo, that is, a suspension prepared with only water and the excipients present in Revatio commercial formulation, and with the same physical aspect. A dose of 0.1 ml/kg of suspension ‘A’ or ‘B’ was administered directly into the oral cavity of each dog, using a syringe, every 12 hours for two weeks, by a member of our research group who was unaware of the treatment protocol. The dogs allocated in the treatment group therefore received 1 mg/kg sildenafil every 12 hours; this dose was chosen on the basis of previous studies in humans and cats (Bortolotti and others 2000, Zhang and others 2004), in which the same dose was effective in relaxing the LOS. In order to obtain a prolonged effect on LOS tone, the drug was administered twice daily, since the half-life of sildenafil in dogs is about five hours (Walker and others 1999). The dogs’ owners were asked to record (on an appropriate data sheet) the frequency of regurgitation occurring within a 24 hour period at different times: D0 (the day before the beginning of the treatment), D1 (first day of treatment), D2 (second day of treatment), D3 (third day of treatment), D4 (fourth day of treatment), D5 (fifth day of treatment), D7 (seventh day of treatment), D10 (10th day of treatment), D14 (14th day of treatment), D21 (seven days after the end of treatment), and D45 (30 days after the end of treatment) (Fig 1). Dogs in both groups were weighed daily for the precise dose calculation, and the weight measured at D0, and the day after the last sildenafil or placebo administration (D15), (named W1 and W2, respectively) were used to calculate the WG for each dog, expressed as percentage (Fig 1). Dog owners were also asked to report immediately to the investigators any adverse event observed during or after the drug administration period. Apart from the regurgitation count, which took place in the breeding kennels, all the other evaluations were performed in the veterinary hospital facilities.
Radiographic evaluation Lateral radiographs of each dog were taken, without any pharmacological restraint, before and immediately after the administration of 4ml/kg of a barium suspension (Prontobario 60 per cent, Bracco Imaging Italia, Milan), mixed with 3–4 boluses of canned food, without keeping the dog in lateral recumbency. Radiographic evaluation was performed at D0, and the day after the last sildenafil or placebo administration (D15). The oesophageal diameter (OD) was measured in each radiograph at its widest point, perpendicularly to the oesophageal longitudinal axis, at the level of its luminal surface. The thoracic inlet (TI) was also measured in the same radiograph, from the Veterinary Record | April 22, 2017

group.bmj.com on May 15, 2017 – Published by http://veterinaryrecord.bmj.com/Downloaded from
ventral aspect of the vertebral column at the mid-point of the first rib, to the inner aspect of the manubrium at the point of the narrowest diameter of the TI. In order to minimise the differences in weight and size of the dogs in the two groups, the relative OD (ROD) was adopted instead of OD, using the function OD/TI, as proposed by Wray and Sparkes (2006) (Fig 2). All measures were performed with an image analysis software (Image J, version 1.49 NIH), by an examiner who was unaware of the study protocol.
In vitro experiments Following laparotomy, the gastroesophageal junction was excised from six dogs of different breeds, euthanased at the Animal Hospital of the Department of Veterinary Science for reasons unrelated to pathologies of the digestive system. Each segment of oesophagus was put in cooled (4°C) modified Krebs-Henseleit Solution (KHS) of the following composition: sodium chloride (NaCl) 113.0 mM, potassium chloride (KCl) 4.7 mM, magnesium sulphate heptahydrate (MgSO4·7H2O) 1.2 mM, calcium chloride dehydrate (CaCl2·2H2O) 1.8 mM, monopotassium phosphate (KH2PO4) 1.2 mM, sodium bicarbonate (NaHCO3) 25.0 mM and dextrose 11.2 mM, and immediately carried to the laboratory. The tissuewas cut longitudinally, pinned flat and the mucosa removed. Eight strips of circular smooth muscle (0.3–0.4 by 1.0– 1.5 cm) were obtained from the LOS region. The strips were tied at each end with silk thread and set up in organ baths (10 ml) filled with KHS, maintained at 37°C and continuously bubbled with 95 per cent oxygen (O2) and 5 per cent carbon dioxide (CO2). After a period of stabilisation (45–60 minutes), the mechanical activity was measured by means of an isotonic transducer developing a passive load of 2–3 g to the preparation throughout the entire experiment. In a separate set of experiments, electrical field stimulation (EFS) was applied with a pair of coaxial platinum electrodes positioned 10 mm from the longitudinal axis of the preparation and used to deliver trains of square wave pulses (0.4 ms duration, 50 Vamplitude) every 120 seconds to the tissue at a frequency of 20 Hz. For each experiment, the intensity was adjusted to a level giving 70–80 per cent of the maximum tissue response (usually 250–300 mA). Under these conditions, depolarisation of intrinsic nerve endings and neurotransmitter release were induced, as described previously (Poli and others 1994, Rakestraw and others 1996). All experiments were performed in the presence of atropine (10−6 M), guanethidine (10−5 M) and indomethacin (10−5 M), to prevent the contractile effect evoked byendogenousacetylcholine, catecholaminesand prostaglandins, respectively. When concentration-response curves were needed, drugs were added cumulatively to the bath solution in 1 log unit increments of concentration. For in vitro experiments, sildenafil citrate 0.8 mg/ml (Revatio intravenous solution, Pfizer Italia) was used. The effect of drugs on basal tone was measured as the modification of the muscle length (Δ cm) with respect to the pre-drug level (baseline); the drug-induced variations of EFS-evoked responses of the preparation were expressed as a percentage of the pre-drug amplitude, assumed as 100 per cent. All recordings were performed by means of a pen-writing polygraph (Basile, Milan, Italy).
Drugs Sildenafil citrate pharmacological forms (Revatio oral suspension, Revatio intravenous solution) were purchased from Pfizer Italia; atropine, guanethidine, indomethacin, tetrodotoxin (TTX), L-NG-nitroarginine methyl ester (L-NAME), and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) were purchased from Sigma (Sigma-Aldrich, St Louis, Missouri, USA).

Statistical analysis Data are expressed as mean±sd. Unpaired t tests were used for the comparison of data between the treatment and control groups, while paired t tests were employed to compare pre- and post-treatment data in the same group. All analyses were performed using a commercial statistical software (GraphPad Prism for Mac V.6.0f, GraphPad Software Inc, USA).

Results The trial was conducted between November 2013 and January 2016. The treatment group (n=12) consisted of seven Great Danes (four of which were littermates), three German shepherd
TABLE 1: Weight values of dogs in control and treatment group at day 0 (W1), day 15 (W2), and WG. Regurgitation frequency (D=day number)*
Weight assessment Number of regurgitation episodes in 24 hours W1 (kg) W2 (kg) WG (%) D0 D1 D2 D3 D4 D5 D7 D10 D14 D21 D45
Control group 3.63 ±0.92
5.45 ±0.86
53.40 ±19.30
5.00 ±0.86
4.56 ±1.33
3.44 ±0.88
3.33 ±0.87
3.00 ±1.00
2.11 ±0.93
2.00 ±1.00
2.33 ±0.71
1.44 ±0.73
1.11 ±0.78
0.78 ±0.67
Treatment group
3.23 ±0.92
5.60 ±0.84
79.76 ±28.30*
4.58 ±1.24
1.58 ±0.90
0.83 ±0.72
0.33 ±0.49
0.67 ±0.49
0.42 ±0.90
0.50 ±0.52
0.25 ±0.45
0.17 ±0.39
0.17 ±0.39
0.17 ±0.39
*All values are expressed as mean±sd. p=0.034 treatment v control group. WG, weight gain.
D0 D1
beginning of treatment
D2 D3 D4 D5 D6 D7
sildenafil or placebo
regurgitation frequency evaluation
D9 D10 D11 D12 D13
end of treatment
D15 D21 D45
FIG 1: Study design scheme indicating: the duration of the study (from day 0 to day 45, D0–D45); sildenafil (1 mg/kg twice daily) or placebo administration protocol; times of regurgitation frequency evaluation; weight recordings (W1 and W2), and radiographic evaluation times (RX)
April 22, 2017 | Veterinary Record

group.bmj.com on May 15, 2017 – Published by http://veterinaryrecord.bmj.com/Downloaded from
dogs and two Labrador retrievers. The control group (n=9) consisted of five Great Danes (two of which were littermates) and four German shepherd dogs. The mean ages of the dogs were 28.17±6.07 days (range 22–45 days) and 28.44±3.00 days (range 25–35 days) in the treatment and control groups, respectively (P=0.389). Mean weight at the start of the study (W1) for the treatment group was 3.23±0.92 kg (range 2–5.1 kg) whereas for the control group it was 3.63±0.92 kg (range 2.8–5.2 kg) (P=0.453). The mean WG at D15 in the treatment group (79.76 ±28.30 per cent) was significantly higher (P=0.034) than the control group (53.40±19.30 per cent). The values of mean ages and weights in the two groups are shown in Table 1. The owners did not observe regurgitation episodes within three hours after the administration of either ‘A’ or ‘B’ suspension. There was no significant difference in frequency of regurgitation between the treatment and control groups at D0 (P=0.540). The number of regurgitation episodes were notably decreased in puppies of the treatment group after the first sildenafil dose (Table 1). Regurgitation episodes ceased almost completely after 10 days of sildenafil administration (D10), and no relapses were observed up to one month after the end of the treatment (D45). Conversely, regurgitation persisted in the control group, although a gradual reduction in frequency was noted with nutritional management alone (Table 1). Overall, puppies in the control group had more than two-fold total regurgitation episodes throughout the study period, compared to the treatment group (262 v 116). The mean number of regurgitation episodes in 24 hours was significantly lower in the dogs receiving sildenafil compared to the placebo-treated dogs (0.88±1.40 v 2.65±1.56, P<0.0001). No adverse effects were reported by the dog owners during the entire trial period. Lateral thoracic radiograph measurements are shown in Fig 3. The mean ROD at D0 in the control group was 0.98±0.17 (range 0.67–1.21) and in the treatment group it was 0.97±0.19 (range 0.69–1.44) (P=0.663). The values of ROD at D15 in the treatment group dogs were significantly lower (0.24±0.14) (range 0.02–0.44) compared to the control group dogs, in which the mean ROD value was 1.10±0.25 (range 0.82–1.47) (P<0.0001). Sildenafil administration was also able to reduce mean OD in a significant fashion, as observed by comparing ROD values at D0 v D15 (P<0.0001). By contrast, no significant difference was recorded in the control group between the ROD values at D0, with respect to D15 (P=0.480). In the experiments performed in vitro, sildenafil (10−9 to 10−5 M) induced a concentration-dependent decrease of basal tone of LOS preparations, as shown by the fall of the baseline with respect to pre-drug level (Fig 4). In the presence of atropine, guanethidine and indomethacin, EFS evoked non-cholinergic non-adrenergic phasic relaxations of the LOS muscle (Fig 4), which were abolished by neuronal sodium channel blocker (TTX), by NO-synthase inhibitor (L-NAME), and by guanylyl cyclase inhibitor (ODQ). Sildenafil (10−10 to 3×10−6 M) enhanced the amplitude of these relaxations in a concentrationdependent fashion (Fig 4).

To date, there is no specific and effective pharmacological treatment for idiopathic megaoesophagus. In dogs, the oesophagus possesses a striated muscle layer throughout its entire length, excluding the LOS; therefore prokinetic agents which act on smooth muscle, such as metoclopramide and cisapride, are ineffective and could be contraindicated (Washabau 2003). In accordance with this, 5-HT4 serotonin receptors were not detected in the oesophageal muscle of dogs (Cohen and others 1994). Moreover, metoclopramide and cisapride tend to increase LOS tone, further hindering the emptying of oesophageal content, and thus worsening the clinical signs (Washabau and Hall 1997). On the other hand, bethanechol, a muscarinic agonist, was instead shown to increase the amplitude of contractions in dogs with idiopathic megaoesophagus (Diamant and others 1974). Due to the scarce results obtained with drugs aiming to enhance the contractions of the oesophageal body, a possible therapeutic strategy could be to relax LOS smooth muscle, in order to promote the emptying of the oesophagus. Indeed, calcium channel blockers were shown to be able to decrease LOS pressure in humans with oesophageal motor dysfunctions (Baunack and others 1991), and nifedipine administration resulted in a temporary clinical improvement in dogs with idiopathic megaoesophagus (Chandra and others 1989). A possible detrimental effect exerted by calcium antagonists on overall oesophagus peristalsis cannot be excluded, though, and it may represent a serious concern. The importance of NO in basal and swallowing-induced LOS relaxation, as well as the ability of sildenafil to modify LOS tone, have been demonstrated several times in different species over the past two decades. For example, seminal work in the opossum demonstrated that the inhibition of NO synthesis antagonised swallowing-induced LOS relaxation, and caused an increase in basal LOS pressure (Tøttrup and others 1991, Yamato and others 1992). Indeed, sildenafil was shown to decrease LOS tone in healthy humans or in patients with achalasia or other oesophageal motility disorders (Bortolotti and others 2000, 2002, Rhee and others 2001, Eherer and others 2002, Lee and others 2003, Fox and others 2007). An average basal LOS relaxation of
FIG 2: Radiographic measurement technique as proposed by Wray and Sparkes (2006). The oesophageal diameter (OD) was measured in each radiograph at its widest point, perpendicularly to the oesophageal longitudinal axis, at the level of its luminal surface. The thoracic inlet (TI) was also measured in the same radiograph, from the ventral aspect of the vertebral column at the mid-point of the first rib, to the inner aspect of the manubrium at the point of narrowest diameter of the TI. The relative OD (ROD) was calculated, using the OD/TI ratio
Co D0 Co D15 Sil D0
Sil D15
FIG 3: Mean±sd of relative oesophageal diameter (ROD) (OD/ thoracic inlet (TI)) values measured at day 0 (D0) and day 15 (D15) for control group (Co) and treatment group (Sil). **P<0.0001 Sil D15 v Co D15; ##P<0.0001 Sil D15 v Sil D0
Veterinary Record | April 22, 2017

group.bmj.com on May 15, 2017 – Published by http://veterinaryrecord.bmj.com/Downloaded from
50 per cent was also observed in sildenafil-treated cats (Zhang and others 2004). The present study provides the first evidence documenting the benefits of sildenafil citrate in relieving the clinical signs associated with CIM in dogs. Although a decrease in the number of regurgitations from D0 to D14 was observed in both groups, it was notably higher in the sildenafil group. Moreover, in the puppies treated with sildenafil, the mean frequency of regurgitation episodes in 24 hours was significantly lower, compared to non-treated subjects, and the clinical improvement was supported by a significant increase of WG in the treatment group with respect to controls. The beneficial effects of the drug were also observed radiographically: in the treatment group dogs, a marked reduction of the OD was measured at the end of the treatment period, as indicated by the significantly lower mean ROD values, compared to control group. By contrast, in all the dogs enrolled in the control group the OD was wider at D15, with respect to the beginning of the study. In placebo-treated dogs a gradual decrease of regurgitation episodes was observed despite a worsening of oesophageal enlargement; although this discrepancy might seem surprising, there is usually poor correlation between the severity of clinical signs and the degree of oesophageal distension in dogs with megaoesophagus (Guilford 1990), and spontaneous improvement with time may be due to feeding from the upright position (Sokolovsky 1972). NO is the principal inhibitory neurotransmitter released from myenteric neurons which induces relaxation of the LOS, through activation of cGMPsynthesis (Mittal and Bhalla 2004). The importance of the NO/cGMP pathway for the relaxation of LOS muscle in dogs was corroborated by the results of in vitro experiments, which indicated that NO-synthase inhibitor L-NAME inhibited EFS-evoked relaxation spikes of LOS preparations, in accordance with what was observed previously (Yamato and others 1992). Moreover, ODQ, a guanylyl cyclase inhibitor, abolished such relaxations, confirming that they were mediated by cGMP, and thus could be susceptible to sildenafil action. In fact, sildenafil enhanced EFS-evoked relaxation spikes and reduced basal tone in a concentration-dependent manner, showing that this PDE-5 inhibitor is able to induce the relaxation of isolated LOS in the dog. These results strongly support the hypothesis that the clinical and radiographic improvement observed in dogs treated with sildenafil are indeed due to a reduced LOS tone, with subsequent easier transit of food from the oesophagus into the stomach. The ability of sildenafil to relax smooth muscle could also represent a concern, though, as it might hinder oesophageal peristalsis further. As a matter of fact, in previous studies in humans and in cats, sildenafil significantly reduced oesophageal contractile pressures (Bortolotti and others 2000, 2002, Eherer and others 2002, Zhang and others 2004). Unlike in humans and cats, however, oesophageal muscle in dogs is almost entirely of the striated type, and thus is not affected by sildenafil. Indeed, the work by Zhang and others showed that the contractile amplitude in oesophageal portions with striated muscle was unaffected. Another concern of the reduced tone of LOS induced by sildenafil could be represented by a potential increased risk of gastro-oesophageal reflux (GOR); a previous study, though, found that sildenafil altered LOS function without causing GOR in human patients (Kim and others 2006). Since peristalsis of the oesophagus is unchanged, the dogs affected by CIM treated with sildenafil would still require to be fed from an elevated position; however, they could benefit greatly from the easier oesophageal emptying and the decrease in oesophageal dilatation, resulting in an improvement in clinical signs and general health status. Moreover, serious complications such as aspiration pneumonia are less likely to occur. Interestingly, sildenafil seemed to achieve results that go beyond mere symptomatic treatment, since puppies in the sildenafil group had only occasional episodes of regurgitation up to 30 days after the drug administration was discontinued, whereas the clinical signs, though improved, were considerably worse in the control subjects. CIM is a chronic disease, so it would be very important in future studies to expand the knowledge about sildenafil effects over time. Further experiments with different doses of sildenafil and with similar drugs, like tadalafil, will be necessary for a better understanding of the efficacy of PDE-5 inhibitors against idiopathic megaoesophagus in dogs. Moreover, gastro-oesophageal manometric studies should be performed to determine the effect of sildenafil activity on oesophageal and LOS tone and contractility. The current dosage was well tolerated in all treated puppies. Apart from the possible decrease in blood pressure, several adverse reactions following sildenafil administration have been reported in the literature. Abbott and others (2004), for example, described species-specific effects in dogs (Beagle pain syndrome), mice and rats. For this reason, additional clinical studies in dogs would benefit from arterial pressure measurement, urinalysis, haematological and serum biochemical analyses in sildenafiltreated patients. In conclusion, this preliminary study suggests, for the first time, that sildenafil citrate, by reducing LOS tone and facilitating the emptying of the oesophagus, could represent a useful drug for the clinical management of CIM in dogs.

Acknowledgements The authors would like to thank Professor Ezio Bottarelli, University of Parma, for the precious assistance with the statistical analysis. The present study was supported by a local grant (FIL 2012-2013).
0.1 a b c 140
–10 –9 –8 Log [sildenafil] –7 –6 –5
EFS-evoked relaxation (% of pre-drug level)
EFS-evoked relaxation (% of pre-drug level)
–7 –6
–5 Log [M]
–4 –3
–0.2 baseline (cm)
–9 –8 –7 Log [sildenafil] –6 –5 –4
FIG 4: Effect of sildenafil (10−9 to 10−5 M) on basal tone (baseline) of dog lower oesophageal sphincter (LOS) (A). Effects of L-NG-nitroarginine methyl ester (L-NAME) (10−6 to 10−3 M), tetrodotoxin (TTX) (10−6 M), and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) (10−6 to 10−4 M) on the amplitude of electrical field stimulation (EFS)-evoked relaxation spikes of dog LOS (B). Effect of sildenafil (10−10 to 10−5 M) on the amplitude of EFS-evoked relaxation spikes of dog LOS (C). All the experiments were performed in the presence of atropine (10−6 M), guanethidine (10−5 M), and indomethacin (10−5 M) (not shown). Data represent mean±sd of eight experiments
April 22, 2017 | Veterinary Record

group.bmj.com on May 15, 2017 – Published by http://veterinaryrecord.bmj.com/Downloaded from
References ABBOTT, D., COMBY, P., CHARUEL, C., GRAEPEL, P., HANTON, G., LEBLANC, B., LODOLA, A., LONGEART, L., PAULUS, G., PETERS, C. & STADLER, J. (2004) Preclinical safety profile of sildenafil. International Journal of Impotence Research 16, 498–504 BACH, J. F., ROZANSKI, E. A., MACGREGOR, J., BETKOWSKI, J. M., RUSH, J. M. & RUSH, J. E. (2006) Retrospective evaluation of sildenafil citrate as a therapy for pulmonary hypertension in dogs. Journal of Veterinary Internal Medicine 20, 1132–1135 BAUNACK, A. R. & WEIHRAUCH, T. R. (1991) Clinical efficacy of nifedipine and other calcium antagonists in patients with primary oesophageal motor dysfunctions. Arzneimittelforschung 41, 595–602 BORTOLOTTI, M., MARI, C., LOPILATO, C., PORRAZZO, G. & MIGLIOLI, M. (2000) Effects of sildenafil on oesophageal motility of patients with idiopathic achalasia. Gastroenterology 118, 253–257 BORTOLOTTI, M., PANDOLFO, N., GIOVANNINI, M., MARI, C. & MIGLIOLI, M. (2002) Effects of sildenafil on hypertensive lower oesophageal sphincter. European Journal of Clinical Investigation 32, 682–685 CHANDRA, N. C., MCLEOD, C. G. Jr & HESS, J. L. (1989) Nifedipine: atemporizing therapeutic option for the treatment of megaesophagus in adult dogs. Journal of the American Animal Hospital Association 25, 175–179 COHEN, M. L., SUSEMICHEL, A. D., BLOOMQUIST, W. & ROBERTSON, D. W. (1994) 5-HT4 receptors in rat but not guinea pig, rabbit or dog oesophageal smooth muscle. General Pharmacology 25, 1143–1148 DEGIRMENCI, B., ACAR, M., ALBAYRAK, R., YUCEL, A., HAKTANIR, A., DEMIREL, R. & ELLIDOKUZ, E. (2006) Effect of sildenafil citrate on postprandial gallbladder motility. Southern Medical Journal 99, 208–211 DIAMANT, N., SZCZEPANSKI, M. & MUI, H. (1973) Manometric characteristics of idiopathic megaesophagus in the dog: an unsuitable animal model for achalasia in man. Gastroenterology 65, 216–223 DIAMANT, N., SZCZEPANSKI, M. & MUI, H. (1974) Idiopathic megaesophagus in the dog: reasons for spontaneous improvement and a possible method of medical therapy. The Canadian Veterinary Journal 15, 66 –71 EHERER, A. J., SCHWETZ, I., HAMMER, H. F., PETNEHAZY, T., SCHEIDL, S. J., WEBER, K. & KREJS, G. J.(2002) Effect of sildenafil on oesophageal motor function inhealthysubjectsandpatientswithoesophagealmotordisorders.Gut50,758 –764 FOX, M., SWEIS, R., WONG, T. & ANGGIANSAH, A. (2007) Sildenafil relieves symptoms and normalizes motility in patients with oesophageal spasm: a report of two cases. Neurogastroenterologyand Motility 19, 798–803 GLIDEWELL, H. S. (1983) Clinical signs of idiopathic megaoesophagus in Great Dane puppies. Veterinary Medicine and Small Animal Clinician 78, 202–205 GUILFORD, W. G. (1990) Megaesophagus in the dog and cat. Seminars in Veterinary Medicine (Small Animals) 5, 37 –45 HARVEY, C. E., O’BRIEN, J. A., DURIE, V. R., MILLER, D. J. & VEENEMA, R. (1974) Megaoesophagus in the dog: a clinical survey of 79 cases. Journal of the American Veterinary Medical Association 165, 443–446 HOLLAND, C. T., SATCHELL, P. M. & FARROW, B. R. (1994) Vagal afferent dysfunction in naturally occurring canine esophageal motility disorder. Digestive Diseases and Sciences 39, 2090–2098 HOLLAND, C. T., SATCHELL, P. M. & FARROW, B. R. (1996) Vagal esophagomotor nerve function and esophageal motor performance in dogs with congenital idiopathic megaesophagus. American Journal of Veterinary Research 57, 906–913 HOLLAND, C. T., SATCHELL, P. M. & FARROW, B. R. H. (2002) Selective vagal afferent dysfunction in dogs with congenital idiopathic megaoesophagus. Autonomic Neuroscience 99, 18 –23 KEMPF, J., LEWIS, F., REUSCH, C. E. & KOOK, P. H. (2014) High-resolution manometric evaluation of the effects of cisapride and metoclopramide hydrochloride administered orally on lower esophageal sphincter pressure in awake dogs. American Journal of Veterinary Research 75, 361–366 KIM, H. S., CONKLIN, J. L. & PARK, H. (2006) The effect of sildenafil on segmental oesophageal motility and gastro-oesophageal reflux. Alimentary Pharmacology and Therapeutics 24, 1029–1036 KNOWLES, K. E., O’BRIEN, D. P. & AMANN, J. F. (1990) Congenital idiopathic megaoesophagus in a litter of Chinese Shar-Pei: clinical, electrodiagnostic, and pathological findings. Journal of the American Hospital Animal Association 26, 313–318 LEE, J. I., PARK, H., KIM, J. H., LEE, S. I. & CONKLIN, J. L. (2003) The effect of sildenafil on oesophageal motor function in healthy subjects and patients with nutcracker oesophagus. Neurogastroenterologyand Motility 15, 617–623 MCBREARTY, A. R., RAMSEY, I. K., COURCIER, E. A., MELLOR, D. J. & BELL, R. (2011) Clinical factors associated with death before discharge and overall survival time in dogs with generalized megaoesophagus. Journal of the American Veterinary Association 12, 1622–1628 MÉHATS, C., SCHMITZ, T., BREUILLER-FOUCHÉ, M., LEROY, M. J. & CABROL, D. (2006) Should phosphodiesterase 5 selective inhibitors be used for uterine relaxation? American Journal of Obstetrics and Gynecology 195, 184–185 MITTAL, R. K. & BHALLA, V. (2004) Oesophageal motor functions and its disorders. Gut 53, 1536–1542 PEETERS, M. E., VENKER-VAN HAAGEN, A. J., GOEDEGEBUURE, S. A. & WOLVEKAMP W. T. (1991) Dysphagia in Bouviers associated with muscular dystrophy; evaluation of 24 cases. Veterinary Quarterly 13, 65 –73 POHL, D., TUTUIAN, R. (2007) Achalasia: an overview of diagnosis and treatment. Journal of Gastrointestinal and Liver Diseases 16, 297–305 POLI, E., POZZOLI, C., CORUZZI, G. & BERTACCINI, G. (1994) Signal transducing mechanisms coupled to histamine H3 receptors and alpha-2 adrenoceptors in the guinea pig duodenum: possible involvement of N-type Ca++ channels. Journal of Pharmacologyand Experimental Therapeutics 270, 788–794 RAKESTRAW, P. C., SNYDER, J. R., WOLINER, M. J., SANDERS, K. M. & SHUTTLEWORTH, C. W. (1996) Involvement of nitric oxide in inhibitory neuromuscular transmission in equine jejunum. American Journal of Veterinary Research 57, 1206–1213 RHEE, P. L., HYUN, J. G., LEE, J. H., KIM, Y. H., SON, H. J., KIM, J. J., PAIK, S. W., RHEE, J. C. & CHOI, K. W. (2001) The effect of sildenafil on lower esophageal sphincter and body motility in normal male adults. The American Journal of Gastroenterology 96, 3251–3257 ROMAN, S. & KAHRILAS, P. J. (2012) Distal esophageal spasm. Dysphagia 27, 115–123 SOKOLOVSKY, V. (1972) Achalasia and paralysis of the canine esophagus. Journal of American Veterinary Medical Association 160, 943–955 SOUZA-SILVA, A. R., DIAS-JUNIOR, C. A., UZUELLI, J. A., MORENO, H. Jr, EVORA, P. R. & TANUS-SANTOS, J. E. (2005) Hemodynamic effects of combined sildenafil and L-arginine during acute pulmonary embolism-induced pulmonary hypertension. European Journal of Pharmacology 524, 126–131 TAN, B. J. & DIAMANT, N. E. (1987) Assessment of the neural defect in a dog with idiopathic megaesophagus. Digestive Diseases and Sciences 32, 76 –85 TØTTRUP, A., KNUDSEN, M. A. & GREGERSEN, H. (1991) The role of the L-arginine-nitric oxide pathway in relaxation of the opossum lower oesophageal sphincter. British Journal of Pharmacology 104, 113–116 ULLAL, T. V., KASS, P. H., CONKLIN, J. L., BELAFSKY, P. C. & MARKS, S. L. (2016) High-resolution manometric evaluation of the effects of cisapride on the esophagus during administration of solid and liquid boluses in awake healthy dogs. American Journal of Veterinary Research 77, 818–827 WALKER, D. K., ACKLAND, M. J., JAMES, G. C., MUIRHEADS, G. J., RANCE, D. J., WASTALL, P. & WRIGHT, P. A. (1999) Pharmacokinetics and metabolism of sildenafil in mouse, rat, rabbit, dog and man. Xenobiotica 29, 297–310 WASHABAU, R. J. (2003) Gastrointestinal motility disorders and gastrointestinal prokinetic therapy. Veterinary Clinics of North America: Small Animal Practice 33, 1007–1028 WASHABAU, R. J. & HALL, J. A. (1997) Gastrointestinal prokinetic therapy: serotoninergic drugs. Compendium: Continuing Education for the Practicing Veterinarian 19, 473–480 WRAY, J. D. & SPARKES, A. H. (2006) Use of radiographic measurements in distinguishing myasthenia gravis from other causes of canine megaoesophagus. Journal of Small Animal Practice 47, 256–263 YAMATO, S., SAHA, J. K. & GOYAL, R. K. (1992) Role of nitric oxide in lower oesophageal sphincter relaxation to swallowing. Life Sciences 50, 1263–1272 ZHANG, X., TACK, J., JANSSENS, J. & SIFRIM, D. A. (2001) Effect of sildenafil, a phosphodiesterase-5 inhibitor, on oesophageal peristalsis and lower oesophageal sphincter function in cats. Neurogastroenterology and Motility 13, 325–331 ZHANG, X., TACK, J., JANSSENS, J. & SIFRIM, D. A. (2004) Neural regulation of tone in the oesophageal body: in vivo barostat assessment of volume-pressure relationships in the feline oesophagus. Neurogastroenterology and Motility 16, 13–21 ZHU, H., XU, X. & CHEN, J. D. Z. (2007) Inhibitory effects of sildenafil on gastric motility and gastric slow waves in dogs. Neurogastroenterology and Motility 19, 218–224