Chronic intratracheal application of the soluble guanylyl cyclase stimulator BAY 41-8543 ameliorates experimental pulmonary hypertension

Dysfunction of the NO/sGC/cGMP signaling pathway has been implicated in the pathogenesis of pulmonary hypertension (PH). Therefore, agents stimulating cGMP synthesis via sGC are important therapeutic options for treatment of PH patients. An unwanted effect of this novel class of drugs is their systemic hypotensive effect. We tested the hypothesis that aerosolized intra-tracheal delivery of the sGC stimulator BAY41-8543 could diminish its systemic vasodilating effect. Pharmacodynamics and -kinetics of BAY41-8543 after single intra-tracheal delivery was tested in healthy rats. Four weeks after a single injection of monocrotaline (MCT, 60 mg/kg s.c.), rats were randomized to a two-week treatment with either placebo, BAY 41-8543 (10 mg/kg per os (PO)) or intra-tracheal (IT) instillation (3 mg/kg or 1 mg/kg). Circulating concentrations of the drug 10 mg/kg PO and 3 mg/kg IT were comparable. BAY 41-8543 was detected in the lung tissue and broncho-alveolar fluid after IT delivery at higher concentrations than after PO administration. Systemic arterial pressure transiently decreased after oral BAY 41-8543 and was unaffected by intratracheal instillation of the drug. PO 10 mg/kg and IT 3 mg/kg regimens partially reversed pulmonary hypertension and improved heart function in MCT-injected rats. Minor efficacy was noted in rats treated IT with 1 mg/kg. The degree of pulmonary vascular remodeling was largely reversed in all treatment groups. Intratracheal administration of BAY 41-8543 reverses PAH and vascular structural remodeling in MCT-treated rats. Local lung delivery is not associated with systemic blood pressure lowering and represents thus a further development of PH treatment with sGC stimulators.

oral placebo (400 µl of 2 % methylcellulose solution) by gavage (n = 5); b) intratracheal placebo (400 µl of 0.2 % surfactant solution) by intratracheal instillation 12 (n = 5); c) BAY 41-8543 (10 mg/kg) orally by gavage (n = 5); d) BAY 41-8543 (3 mg/kg) by intratracheal instillation (n = 5); e) BAY 41-8543 (1 mg/kg) by intratracheal 14 instillation (n = 5). One, three, and six hours after treatment, blood samples were collected from vena saphenous under anaesthesia with 3% isoflurane. Plasma was 16 obtained from the blood samples by centrifugation at 4°C 10 min 1000g. Afterwards, rats were sacrificed and bronchoalveolar lavage (BAL) was performed with 10 ml of 18 isotonic saline solution. BAL fluid (BALF) was immediately frozen and stored at -80 °C. After misternal thoracotomy pulmonary artery was canulated and flushed with 15 20 ml of isotonic saline. Afterwards, the lung tissue was harvested, immediately frozen, and stored at -80 °C. 22 Pressure transmitter implantation. Under anesthesia with isoflurane 3-5%, rats 24 were intubated and ventilated using small animal ventilator with frequency 60 min -1 and 150 ml minute ventilation. Skin of the antero-medial surface of the rat's right leg 26 was shaved and wet with Braunoderm. After cutting skin, subcutane tissue was prepared and femoral artery was prepared free from surrounding tissue. PhysioTel ® 28 PA-C40 small animal pressure transmitter (DSI International, Tilburg, Netherlands) was implanted into femoral artery and fixed with ligatures. The body of the transmitter 30 was placed in the cavity generated in the lateral abdominal subcutaneous connective tissue. Subcutaneous tissue and skin was closed with two series of sutures. To 32 release the post-operative pain, Buprenorphine 50 µg/kg and Indometacine 20 mg/kg was injected subcutaneously. Animals were allowed to recover under heating lamp 34 breathing 50 % oxygen and were housed individually in standard rat cages. The pressure signal was transferred to a remote receiver (model RPC-1) and a data-36 exchange matrix connected to a computer. After surgery, rats were allowed to recover for 3 days. The SAP stabilized in the first 24 hours. None of the animals 38 manifested signs of inflammation or infection. Pressure measurements were done 30 min before drug administration and continued for 24 hours thereafter. 40

MCT treatment
Monocrotaline (Sigma, Deishofen, Germany) was dissolved in 1 M HCl, adjusted to 42 pH 7.4 with 1 M NaOH, sterilized by passing through Minisart sterile filter (Sartorius, Göttingen, Germany) 0.2 µm, and administered as a single subcutaneous injection at 44 the dose of 60 mg/kg body weight, as described 31 . Control rats received an equal volume of isotonic saline. 46

Hemodynamic and right ventricular hypertrophy measurements
For measurement of hemodynamic parameters, rats were anaesthetised with intra-48 peritoneal injection of ketamine (9 mg/kg body weight) and medetomidine (100 µg/kg body weight). Afterwards, rats received intramuscular injection of atropine (250 µg/kg 50 body weight) to minimise vasovagal side effects during the preparation. The rats were tracheotomised and artificially ventilated at a constant frequence of 60 breaths 52 per minute with inspiratory flow rate of 500-600 cc/min. Positive end-expiratory pressure was set at 1 cmH 2 O. The left carotid artery was isolated and cannulated 54 with a polyethylene tubing connected to a fluid-filled force transducer and the systemic arterial pressure (SAP) was measured. A catheter was inserted through the 56 right jugular vein into the right ventricle for measurement of right ventricular systolic pressure (RVSP). Cardiac output was calculated using the Fick's principle, by 58 employing the mixed venous oxygen and the arterial oxygen content as previously described (Schermuly Am J Respir Crit Care Med 160:1500-1506. Arterial and 60 mixed venous samples were collected (150 μL) and analyzed for partial pressure of oxygen, pH and carbon dioxide tension, haemoglobin and oxygen saturation 62 (Rapidlab 348, Bayer Diagnostics, Leverkusen, Germany). Pulmonary vascular resistance indexed to the body weight (PVRI) was determined as described 64 previously (Schermuly Circulation 115:2331-2339. The animals were ex-sanguinated and the lungs were flushed with sterile saline to get rid of blood. The left lung was 66 fixed for histology in 3.5 % neutral buffered formalin and the right lung was snap frozen in liquid nitrogen. The ventricles were dissected free of the great vessels and 68 atria. The right ventricle (RV) was separated from the left ventricle and ventricular septum (LV + S). The RV and (LV + S) were patted dry and weighed. We evaluated 70 right ventricular hypertrophy using the ratio of RV to (LV + S).

High-resolution echocardiography
Anesthesia was induced with 3% isoflurane gas and maintained with 1.0 -1.5% 74 isoflurane in 100% O 2 . Rats were laid supine on a heating platform with all legs taped to ECG electrodes for heart rate (HR) monitoring. Body temperature was monitored 76 via a rectal thermometer (Indus Instruments, Houston, TX) and maintained at 36.5-37.5°C using a heating pad and an infrared lamp. The chest of the rats was shaved 78 and treated with a chemical hair remover to reduce ultrasound attenuation. To provide a coupling medium for the transducer, a pre-warmed ultrasound gel was 80 spread over the chest wall. in the parasternal short-axis view and defined as the interval from the onset to the maximal velocity of forward flow. All echocardiographic parameters were calculated 92 off-line using tool section of the Visual Sonics Vevo770 system. All the studies were performed by an experienced sonographer who was blinded to results of invasive 94 and morphometric studies.

Histology and pulmonary vascular morphometry 96
The formalin-fixed lungs were subjected to paraffin embedding. The paraffinembedded tissues were subject to sectioning to yield 3 μm thick sections. Elastica 98 staining was performed according to common histopathological procedures. The degree of muscularization of small peripheral pulmonary arteries was assessed by 100 double-staining the 3 μm sections with an anti-α-smooth muscle actin antibody (dilution 1:900, clone 1A4, Sigma, Saint Louis, Missouri) and antihuman von 102 Willebrand factor antibody (vWF, dilution 1:900, Dako, Hamburg, Germany) followed by analysis of the vessels using a computerized morphometric analysis system 104 (QWin; Leica, Wetzlar, Germany). In each rat, 80 to 100 intra-acinar arteries (25 to 50 μm diameter) were categorized as muscular, partially muscular, or non-muscular. 106 Arteries of the same size were additionally analyzed for the medial wall thickness as previously described (Schermuly J Clin.Invest 115:2811-2821. All analyses were 108 done in a blinded fashion.
Photomicrographs were quantified to determine the mean cross-sectional area of 120 cardiomyocytes and interstitial collagen fraction using Leica Qwin V3 computerassisted image analysis software (Leica Microsystem, Wetzlar, Germany). Average 122 data reflect results from at least four or five different hearts in each group (more than 90 cells for each heart).