MONO- AND BIS-NITROSYLATED PROPANEDIOLS FOR THERAPEUTIC USE

Abstract

The present invention relates to methods of treating a condition wherein NO has a beneficial effect, wherein such treatment comprises administering certain mono- and/or bis nitrosylated propanediols, including compositions and formulations thereof, wherein administration of said compounds, compositions or formulations is indirect to the pulmonary circulation and/or the systemic circulation of a patient in need thereof.

Claims

1. A compound of formula (I): ##STR00018## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; wherein when n is 0, R.sup.1 is H; and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO, for use in the treatment of a condition wherein NO has a beneficial effect, wherein the compound of formula (I) is administered indirectly to the pulmonary circulation and/or systemic circulation of a patient.

2. A substantially non-aqueous composition comprising: (a) one or more compounds of formula (I): ##STR00019## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; and wherein when n is 0, R.sup.1 is H and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO and (b) a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H, for use in the treatment of a condition wherein NO has a beneficial effect, wherein the compound of formula (I) is administered indirectly to the pulmonary circulation and/or systemic circulation of a patient.

3. The substantially non-aqueous composition for use according to claim 2, wherein the substantially non-aqueous composition comprises from about 0.01% to about 9% by weight of the one or more compounds of formula (I).

4. The substantially non-aqueous composition for use according to claim 2 or claim 3, wherein the substantially non-aqueous composition is substantially free of dissolved nitric oxide.

5. The substantially non-aqueous composition for use according to any one of claims 2 to 4, wherein the substantially non-aqueous composition consists essentially of the one or more compounds of formula I and a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.

6. The substantially non-aqueous composition for use according to any one of claims 2 to 5, wherein the substantially non-aqueous composition is comprised in a pharmaceutical formulation, optionally comprising one or more pharmaceutically acceptable excipients.

7. The substantially non-aqueous composition for use according to claim 6, wherein the one or more pharmaceutically acceptable excipients are non-aqueous.

8. The compound for use according to claim 1, or the substantially non-aqueous composition for use of any one of claims 2 to 7, wherein the compound of formula (I) is administered dermally, gastrointestinally, subcutaneously, intramuscularly, sublingually, intranasaly, intravesically or via inhalation.

9. The compound for use according to claim 1, or the substantially non-aqueous composition for use of any one of claims 2 to 7, wherein the compound of formula (I) is administered to an epithelial layer of a patient.

10. The compound for use according to claim 9 or the non-aqueous composition for use as claimed in claim 9, wherein the epithelial layer that the compound of formula (I) is administered to is a serous membrane, a cutaneous membrane, a synovial membrane, uroepithelial membrane, or a mucous membrane, preferably wherein the epithelial layer is a mucous membrane.

11. The compound for use according to any one of claims 1, or 8 to 10, or the substantially non-aqueous composition for use of any one of claims 2 to 10, wherein compound of formula (I) is administered dermally, gastrointestinally, sublingually, intranasally, intravesically or via inhalation.

12. The compound for use according to any one of claims 1, or 8 to 11 or the substantially non-aqueous composition for use of any one of claims 2 to 11, wherein the compound of formula (I) is administered across an epithelial layer, preferably a mucous membrane, in the mouth, nose, eyelids, trachea, lungs, stomach, intestines, rectum, renal pelvis, ureters, urethra or urinary bladder of the patient.

13. The compound for use according to any one of claims 1, or 8 to 11 or the substantially non-aqueous composition for use of any one of claims 2 to 11, wherein the compound of formula (I) is administered across an epithelial layer in the skin.

14. The compound for use according to claim 1 or claim 8, or the substantially non-aqueous composition for use of any one of claims 2 to 8, wherein the compound of formula (I) is administered subcutaneously.

15. The compound for use according to claim 1 or claim 8, or the substantially non-aqueous composition for use of any one of claims 2 to 8, wherein the compound of formula (I) is administered intramuscularly.

16. The compound for use according to claim 1, or any one of claims 8 to 15, or the substantially non-aqueous composition for use of any one of claims 2 to 15, wherein the condition is selected from the group consisting of: acute pulmonary vasoconstriction of different genesis; pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; preclampsia; eclampsia; conditions of different genesis in need of vasodilation; erectile dysfunction; systemic hypertension of different genesis; regional vasoconstriction of different genesis; local vasoconstriction of different genesis; acute heart failure (with or without preserved ejection fraction (HFpEF)); coronary heart disease; myocardial infarction; ischemic heart disease; angina pectoris; instable angina; cardiac arrhythmia; acute pulmonary hypertension in cardiac surgery patients; acidosis; inflammation of the airways; cystic fibrosis; COPD; immotile cilia syndrome; inflammation of the lung; pulmonary fibrosis; acute lung injury (ALI); adult respiratory distress syndrome; acute pulmonary oedema; acute mountain sickness; asthma; bronchitis; hypoxia of different genesis; ischemic disease of different genesis; stroke; cerebral vasoconstriction; inflammation of the gastrointestinal tract; gastrointestinal dysfunction; gastrointestinal complication; IBD; Crohn's disease; ulcerous colitis; liver disease; pancreas disease; inflammation of the bladder of the urethral tract; inflammation of the urinary bladder and ureters of the urethral tract; inflammation of the skin; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of different genesis; wound healing; organ protection in ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterus relaxation; cervix relaxation; and conditions where smooth muscle relaxation is needed.

17. The compound for use, or the substantially non-aqueous composition for use, of claim 16, wherein the condition is selected from the group consisting of pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; and acute heart failure (with or without preserved ejection fraction (HFpEF)).

18. A method of treating a condition wherein NO has a beneficial effect, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) indirectly to the pulmonary circulation and/or the systemic circulation of the patient: ##STR00020## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; wherein when n is 0, R.sup.1 is H; and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO.

19. A method of treating a condition wherein NO has a beneficial effect, comprising administering to a patient in need thereof a therapeutically effective amount of a substantially non-aqueous composition indirectly to the pulmonary circulation and/or systemic circulation of the patient, wherein the substantially non-aqueous composition comprises: (a) one or more compounds of formula (I): ##STR00021## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; and wherein when n is 0, R.sup.1 is H and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO and (b) a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H,

20. The method of treatment according to claim 18 or claim 19, wherein the compound of formula (I) is administered to an epithelial layer of a patient.

21. The method of treatment according to any one of claims 18 to 20, wherein the administration is to a serous membrane a synovial membrane, an uroepithelial membrane, or a mucous membrane, preferably wherein the administration is to a mucous membrane.

22. The method of treatment according to any one of claims 18 to 21, wherein the administration is carried out dermally, gastrointestinally, sublingually, intranasally, intravesically or via inhalation.

23. The method of treatment according to any one of claims 18 to 22, wherein the administration is to an epithelial layer, preferably a mucous membrane, in the mouth, nose, eyelids, trachea, lungs, stomach, intestines, rectum, ureters, urethra or urinary bladder of the patient.

24. The method of treatment according to claim 18 or claim 19, wherein the compound of formula (I) is administered subcutaneously.

25. The method of treatment according to claim 18 or claim 19, wherein the compound of formula (I) is administered intramuscularly.

26. The method of treatment according to any one of claims 18 to 25, wherein the condition is selected from the group consisting of: acute pulmonary vasoconstriction of different genesis; pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; preclampsia; eclampsia; conditions of different genesis in need of vasodilation; erectile dysfunction; systemic hypertension of different genesis; regional vasoconstriction of different genesis; local vasoconstriction of different genesis; acute heart failure (with or without preserved ejection fraction (HFpEF)); coronary heart disease; myocardial infarction; ischemic heart disease; angina pectoris; instable angina; cardiac arrhythmia; acute pulmonary hypertension in cardiac surgery patients; acidosis; inflammation of the airways; cystic fibrosis; COPD; immotile cilia syndrome; inflammation of the lung; pulmonary fibrosis; acute lung injury (ALI); adult respiratory distress syndrome; acute pulmonary oedema; acute mountain sickness; asthma; bronchitis; hypoxia of different genesis; ischemic disease of different genesis; stroke; cerebral vasoconstriction; inflammation of the gastrointestinal tract; gastrointestinal dysfunction; gastrointestinal complication; IBD; Crohn's disease; ulcerous colitis; liver disease; pancreas disease; inflammation of the bladder of the urethral tract; inflammation of the urinary bladder and ureters of the urethral tract; inflammation of the skin; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of different genesis; wound healing; organ protection in ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterus relaxation; cervix relaxation; and conditions where smooth muscle relaxation is needed.

27. The method of treatment according to claim 26, wherein the condition is selected from the group consisting of pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; and acute heart failure (with or without preserved ejection fraction (HFpEF)).

28. A device for administering a substantially non-aqueous composition comprising: (a) one or more compounds of formula (I): ##STR00022## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; and wherein when n is 0, R.sup.1 is H and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO and (b) a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H, wherein the administration is via inhalation.

29. The device according to claim 28, wherein the device comprises a vaporiser or atomiser for vaporising or atomising the substantially non-aqueous composition.

30. The device according to claim 28 or claim 29, wherein the device comprises a reservoir for containing the substantially non-aqueous composition.

31. The device according to any one of claims 28 to 30, wherein the device is an electronic cigarette comprising: a. a reservoir for containing the substantially non-aqueous composition; b. a vaporiser for vaporising the substantially non-aqueous composition; c. a mouthpiece; d. a battery; e. a microprocessor; and f. a sensor for detecting when a user inhales on the mouthpiece.

32. A cartridge for use with the device of any one of claims 28 to 31, wherein the cartridge comprises a substantially non-aqueous composition comprising: (a) one or more compounds of formula (I): ##STR00023## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; and wherein when n is 0, R.sup.1 is H and wherein when n is 1, R.sup.2 is H, provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO and (b) a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.

33. The cartridge according to claim 32, wherein the cartridge is removable from the device of any one of claims 28 to 31.

Description

DESCRIPTION OF THE FIGURES

[0325] FIG. 1 details the effects of inhaled PDNO administration (by means of an electrical cigarette (eGo AIO, Joyetech), n=1) systemic arterial pressure (SAP, panel A), mean pulmonary arterial pressure (mPAP, panel B) and end-tidal carbon dioxide (ETCO2, panel C) in an anesthetised pig subjected to hypercapnoic aPH. The first and third arrow indicate administration of PDNO, whereas the middle arrow indicates administration of room air.

[0326] FIG. 2 details the effects of intranasal PDNO administration (n=6) on exhaled nitric oxide (ETNO, panel A), mean pulmonary arterial pressure (MPAP, panel B) and mean arterial pressure (MAP, panel C) in anesthetised naïve pigs. Data are means with standard error of the mean.

[0327] FIG. 3 details the effects of PDNO on end-tidal nitric oxide (ETNO, panel A) and mean arterial pressure (MAP, panel B) administered intravenously (5-80 nmol kg-1 min-1 for 30 min each, n=6), subcutaneously (100-1600 nmol kg-1 min-1 for 5 min each, n=6) and intramuscularly (50-800 nmol kg-1 min-1 for 5 min each, n=4-6) in anesthetised naïve pigs having normal pulmonary vascular resistance. Data are means with standard error of the mean. The intravenous data is included herein as a reference example.

[0328] FIG. 4 details the effects of PDNO on end-tidal nitric oxide (ETNO, panel A), mean pulmonary arterial pressure (MPAP, panel B) and mean arterial pressure (MAP, panel C) administered either intravenously (5, 15 and 45 nmol kg-.sub.1 min-.sub.1 for 15 min each, n=4) or subcutaneously (200, 600 and 1800 nmol kg-.sub.1 min-.sub.1 for 5 min each, n=5) in anesthetised pigs with aPH (induced by a continuous intravenous infusion of U46619). Data are means with standard error of the mean. The intravenous data is included herein as a reference example.

[0329] FIG. 5 depicts a typical device for use in the third aspect of the invention for administering the substantially non-aqueous composition to a patient via inhalation. The device (100) comprises a battery (102), a microprocessor (104), a heating element (106), a wick (108), a mouthpiece (112) and a removable cartridge reservoir (110) for containing the substantially non-aqueous composition.

[0330] FIG. 6 depicts the mean systemic and pulmonary arterial pressure (MAP and MPAP, respectively) in an anesthetised and mechanically ventilated pig where pulmonary arterial pressure was increased by permissive hypercapnia (end-tidal carbon dioxide fraction of approximately 8-9%). One-part PDNO (203 mM) was dissolved in four parts of sodium bicarbonate (50 mg-1) and nebulised with an ordinary intensive care unit nebuliser for 5-20 min.

[0331] FIG. 7 depicts mean systemic and pulmonary arterial pressure (MAP and MPAP, respectively) in an anesthetised and mechanically ventilated pig where pulmonary arterial pressure was increased by permissive hypercapnia (end-tidal carbon dioxide fraction of approximately 8-9%). A few ml of PDNO (203 mM) were applied in a commercially available electronic cigarette (eGo AIO, Joyetech). Gas from the electronic cigarette was sampled in a 50 ml syringe and injected into the inspiratory limp of the ventilator circuit. This procedure was repeated for approximately 10 times with very short interval.

[0332] FIG. 8 depicts mean systemic arterial pressure (MAP, panel A), mean pulmonary arterial pressure (MPAP, panel B) and end-tidal concentration of nitric oxide (ETNO, panel C) in an anesthetised pig subjected to increasing doses of PDNO applied sublingually by a small compress soaked with 2 ml PDNO in increasing concentrations (1 mM-203 mM) for 15 min.

[0333] FIG. 9 depicts systemic and pulmonary arterial pressure (AP, panel A and B), and end-tidal concentration of nitric oxide (FENNO, panel C) in an anesthetised pig subjected air pulmonary embolization by a fast injection of 300 microl/kg air intravenously and sublingual PDNO by a small compress soaked with 2 ml PDNO in (100 mM).

[0334] FIG. 10 depicts mean systemic arterial pressure (MAP, panel A), mean pulmonary arterial pressure (MPAP, panel B) and end-tidal concentration of nitric oxide (ETNO, panel C) in an anesthetised pig (25 kg) subjected to dermal application of 3 small compresses soaked with 2 ml PDNO (203 mM) or 3 compresses without PDNO (control). The skin was pretreated with transdermal catalyser menthone.

[0335] FIG. 11 depicts change in mean systemic arterial pressure (Delta MAP, panel A) and mean pulmonary arterial pressure (MPAP, panel B) in an anesthetised pig subjected to injection of PDNO (2-4 ml of 1 mM, 10 mM, 100 mM and 200 mM) via catheter in various body cavities.

EXAMPLES

[0336] The invention is illustrated by way of the following examples, which are not intended to be limiting on the general scope of the invention.

[0337] Abbreviations

[0338] aq aqueous

[0339] conc concentration

[0340] GC gas chromatography

[0341] NMR nuclear magnetic resonance

[0342] equiv. equivalent(s)

[0343] rel. vol. relative volume(s)

[0344] For the avoidance of doubt, compounds of formula (I) may also be referred to herein as compounds of the invention and may be referred to by the acronym PDNO, which will indicate that such compounds, including all embodiments and particular features thereof, are used in the methods and uses as described in relation to the present invention. Furthermore, when compositions of PDNO are described that also contain PD, the PD refers to the corresponding propanediol to the compound of formula (I), that is to say the PD is the same compound according to formula (I), but wherein but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.

[0345] However, in the context of the examples below, the term “PDNO” specifically refers to compounds according to Formula (II). In conjunction with this, the term “PD” refers specifically to 1,2-propanediol, being the starting material from which PDNO is prepared.

[0346] General Procedures

[0347] Starting materials and chemical reagents specified in the preparations described below are commercially available from a number of suppliers, such as Sigma Aldrich.

[0348] All NMR experiments were performed at 298K on a Bruker 500 MHz AVI instrument equipped with a QNP probe-head with Z-gradients using the Bruker Topspin 2.1 software. Signals were referenced to residual CHCl.sub.3 at 7.27 ppm, unless stated otherwise.

[0349] Stability Assays

[0350] Assays of the stability samples were performed by GC/FID, under the following conditions. 1,4-Dioxane was used as the Internal Standard (IS; approximately 0.50 mg/ml in CH.sub.3CN).

[0351] GC column: Rxi-5Sil MS, 20 m×0.18 mm, 0.72 μm

[0352] Carrier gas: Helium

[0353] Inlet: 200° C., split ratio 30:1

[0354] Constant flow: 1.0 ml/min

[0355] Oven temperature profile: 40° C. (3 min), 10° C./min, 250° C. (3 min)

[0356] FID: temp 300° C.; H.sub.2 flow 30 ml/min, Air flow 400 ml/min, make-up flow (N.sub.2) 25 ml/min

Example 1—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite

[0357] 1,2-propanediol (15 mL, 205 mmol), water (100 mL), dichloromethane (200 mL) and sodium nitrite (57 g, 826 mmol) were added to a 500 mL three-necked round bottom flask. The mixture was cooled down to 0° C. with an ice bath. Concentrated sulphuric acid (30 mL, 546 mmol) and water (30 mL) were added to a dropping funnel and cooled to 5° C. in a refrigerator. The funnel was adapted to the round bottom flask and the acid added to the nitrite mixture during two hours. The mixture was stirred with a magnet for 20 minutes and then poured into a separation funnel together with more dichloromethane (100 mL) and water (100 mL). The organic phase was separated and dried with sodium sulphate, and reduced on a rotavapor to yield a mixture of 1,2-propanediol (3 wt. %), 1-(nitrosooxy)-propan-2-ol (23 wt. %) 2-(nitrosooxy)-propan-1-ol (13 wt. %) and 1,2-bis(nitrosooxy)propane (57 wt. %).

Example 2—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite

[0358] 1,2-propandiol (20 mL, 273.4 mmol), water (60 mL), dichloromethane (120 ml) and sodium nitrite (37.72 g, 546.7 mmol) were added to a 0.5 reactor fitted with a stirrer and flushed with nitrogen and kept during the course of the following reaction under nitrogen. The mixture was cooled down to below 5° C. by cooling the mantle to 0° C. Concentrated sulphuric acid (26.3 g, 260.1 mmol) and water were added to a dropping funnel. The funnel was attached (to the reactor and the acid was added to the nitrite mixture during 33 minutes. The mixture was stirred for 54 minutes and then poured into a flask containing an aqueous saturated sodium bicarbonate solution (100 mL). The mixture was transferred to a separation funnel and the organic phase was washed. The aqueous phase was discarded, and the organic phase was washed with additional aqueous saturated sodium bicarbonate solution (100 mL). The organic phase was dried with magnesium sulphate and then transferred to a 1 L round bottom flask together with 1,2-propandiol (120 ml, 1640 mmol). The solution was reduced on a rotavapor under reduced pressure until the dichloromethane was removed. The removal of dichloromethane was monitored by NMR. A clear solution (134 g) containing 1,2-propandiol (82.8 wt. %), 1-(nitrosooxy)-propan-2-ol (10.4 wt. %), 2-nitrosooxy)-propan-1-ol (6 wt. %) and 1,2-bis(nitrosooxy)propane (0.8 wt. %) was obtained.

[0359] .sup.1H-NMR, δ ppm: 5.61 (br s 1H), 4.75-5.58 (m, 2H), 4.11 (br s, 1H), 3.90-3.87 (m, 1H), 3.83-3.69 (m, 2H), 3.60 (dd, J=3.0, 11.2 Hz, 1H), 3.38 (dd, J=7.9, 11.2 Hz, 1H),1. 47 (d, J=6.6 Hz, 3H), 1. 39 (d, J=6.4 Hz, 3H), 1.26 (d, J=6.4 Hz, 3H), 1.15 (d, J=6.3 Hz, 3H), Signals for CH and CH.sub.2 of the 1,2-bis(nitrosooxy)propane were below the detection limit.

Example 3—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with tert-butyl nitrite

[0360] Tert-butyl nitrite (2 mL, 15.1 mmol) was added to a round bottom flask with 1,2-propanediol (11 mL, 150.3 mmol) and the obtained solution was stirred at ambient temperature. 1 mL of the reaction solution was then mixed with 7.5 mL 1,2-propanediol.

Example 4—Stability of non-aqueous mixtures of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-propanediol

[0361] Three different concentrations of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol in 1,2-propanediol were prepared and stored in both a refrigerator (5° C.) and freezer (−20° C.). Aliquots of each solution were taken periodically and analysed by GC to determine the concentration of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol.

[0362] The results of the GC analysis are shown in the table below (column: Rxi-5Sil MS, 20 m×0.18 mm, 0.36 film thickness; carrier: He; Inlet: 250° C., split ratio 100:1; constant flow: 1.0 mL/min; oven temperature profile: 40° C. (3 min), 10° C./min, 80° C. (0 min), 30° C./min, 250° C. (3 min); FID: 300° C., H.sub.2 flow 30 mL/min, air flow 400 mL/min, make-up flow (N.sub.2) 25 mL/min; internal standard: 1,1,1,3,5,5,5-heptamethyl trisiloxane):

TABLE-US-00001 Refrigerator (5° C.) Freezer (−20° C.) Concentration (% w/w) Concentration (% w/w) 1- 2- 1- 2- Stability Sample Nitrite Nitrite Total Nitrite Nitrite Total Start High conc. 3.75 2.94 6.69 3.75 2.94 6.69 Start Medium conc. 0.81 0.61 1.42 0.81 0.61 1.42 Start Low conc. 0.08 0.06 0.14 0.08 0.06 0.14 14 days High conc. 3.72 2.91 6.63 3.76 2.89 6.65 10 days Medium 0.86 0.67 1.53 0.81 0.63 1.44 conc. 10 days Low conc. 0.08 0.06 0.14 0.08 0.06 0.14 28 days High conc. 3.67 2.90 6.57 3.72 2.93 6.65 27 days Medium 0.81 0.63 1.44 0.74 0.57 1.31 conc. 27 days Low conc. 0.09 0.07 0.16 0.07 0.06 0.13 56 days High conc. 3.47 2.69 6.16 3.55 2.74 6.29 64 days Medium 0.73 0.57 1.30 0.74 0.58 1.32 conc. 64 days Low conc. 0.07 0.06 0.13 0.07 0.06 0.13 84 days High conc. 3.33 2.59 5.92 3.50 2.71 6.21 84 days Medium 0.77 0.60 1.37 0.78 0.62 1.40 conc. 84 days Low conc. 0.07 0.06 0.13 0.08 0.06 0.14 Note: no build-up of pressure was observed for any of the samples.

Example 6—Solvent free preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol, and 1,2-bis(nitrosooxy)propane with sodium nitrite

[0363] Water (30 mL) and sodium nitrite (19.01 g, 272.8 mmol) were added to a 100 mL three-necked round bottom flask, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. 1,2-Propanediol (10 mL, 136.7 mmol) was added. Concentrated sulphuric acid (7 mL, 127.4 mmol) and water (20 mL) were pre-cooled to room temperature and added dropwise during one hour via a dropping funnel. During the addition, the water layer formed a thick slurry and a green second layer was formed. Before completion of acid addition (5 mL remaining) the flask was removed from the cooling bath and the green layer was decanted into a separation funnel and washed with 2×saturated aqueous NaHCO.sub.3 solution. The green layer faded to yellow and after separation was dried over Na.sub.2SO.sub.4 and filtered through a syringe filter (Acrodisc® 13 mm, 0.45 μM SUPOR®) to yield 1.1g mixture of approximately 0.25/0.1/1 of 1-(nitrosooxy)-propan-2-ol/2-(nitrosooxy)-propan-1-ol/1,2-bis(nitrosooxy)propane. No starting-material 1,2-propanediol could be detected within the limits of NMR sensitivity.

[0364] .sup.1H-NMR, δ ppm: 5.81-5.76 (m, br, 1.0 H), 5.63 (br, 0.1 H), 4.93 (br, 2.08 H), 4.73-4.65 (br, m, 0.47 H), 4.14 (br, 0.19 H), 3.84-3.77 (br, m, 0.22 H), 1.49 — 1.48 (br, m, 3.21 H), 1.43 (br, 0.51 H), 1.28 (br, 0.72 H).

Example 7—Preparation of (2S)-1-(nitrosooxy)-propan-2-ol, (25)-2-(nitrosooxy)-propan-1-ol and (2S)-1,2-bis(nitrosooxy)propane

[0365] (S)-1,2-propanediol (5 mL, 66.97 mmol), water (15 mL), dichloromethane (30 mL) and sodium nitrite (9.34 g, 134 mmol) were added to a 100 mL three-necked round bottom flask, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. Concentrated sulphuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre-cooled to room temperature and added dropwise via a syringe-pump during 1 h. After addition the mixture was stirred for additional 60 minutes. After separation of the two layers, the DCM layer was diluted with additional DCM (15 mL) and washed with sat. aq. NaHCO.sub.3 (15 mL), followed by brine (15 mL), then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo. The residue was taken up again in 30 mL DCM, washed with 1.4% w/w aq. bicarbonate solution, then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo to yield 1 g of product mixture. The mixture of consisted of (2S)-1,2-propanediol (3%), (2S)-1-(nitrosooxy)-propan-2-ol (23%), (2S)-2-(nitrosooxy)-propan-1-ol (14%) and (2S)-1,2-bis(nitrosooxy)propane (60%) based on NMR.

[0366] .sup.1H-NMR, δ ppm: 5.83-5.74 (m, 1.0 H), 5.66-5.57 (br, 0.22 H), 4.99-4.85 (br, 1.98 H), 4.76-4.59 (br, 0.77 H), 4.17-4.07 (br, 0.38 H), 3.86-3.73 (br, 0.40 H), 1.8-1.6 (br, 0.97 H), 1.48 (d, J=6.7 Hz, 3.12 H), 1.40 (d, J=6.6Hz, 0.63 H), 1.28 (d, J=6.5 Hz, 1.15 H).

Example 8—Preparation of (2R)-1-(nitrosooxy)-propan-2-ol, (2R)-2-(nitrosooxy)-propan-1-ol and (2R)-1,2-bis(nitrosooxy)propane

[0367] (R)-1,2-propanediol (5 mL, 66.97 mmol), water (15 mL), dichloromethane (30 mL) and sodium nitrite (9.34 g, 134 mmol) were added to a 100 mL three-necked round bottom flask, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. Concentrated sulphuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre-cooled to room temperature and added dropwise via a syringe-pump during 1 h. After addition the mixture was stirred for additional 55 minutes. After separation of the two layers, the DCM layer was diluted with additional DCM (10 mL) and washed with saturated aqueous NaHCO.sub.3 (20 mL), then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo. The mixture of consisted of (2R)-1,2-propanediol (17%), (2R)-1-(nitrosooxy)-propan-2-ol (16%), (2R)-2-(nitrosooxy)-propan-1-ol (7%) and (2R)-1,2-bis(nitrosooxy)propane (59%) based on NMR.

[0368] .sup.1H-NMR, δ ppm: 5.83-5.74 (m, 1.0 H), 5.66-5.57 (br, 0.12 H), 4.99-4.85 (br, 2.10 H), 4.76-4.59 (br, 0.53 H), 4.17-4.07 (br, 0.24 H), 3.86-3.73 (br, 0.28 H), 2.4-2.1 (br, 0.38 H), 1.48 (d, J=6.8 Hz, 3.20 H), 1.40 (br, 0.56 H), 1.28 (br(d), 0.88 H).

Example 9—Preparation of 1-(nitrosom)propan-3-ol and 1,3-bis(nitrosooxy)propane

[0369] 1,3-propanediol (2.5 g, 32.86 mmol), water (7 mL), dichloromethane (15 mL) and sodium nitrite (4.53 g, 65.7 mmol) were added to a 100 mL round bottom flask, flushed with nitrogen and cooled down to 0° C. for 15 min on a water bath cooled with an external cooler. Concentrated sulphuric acid (1.7 mL, 31.2 mmol) and water (5 mL) were pre-cooled to room temperature and added dropwise for 5 minutes. After addition the mixture was stirred for additional 60 minutes at 0° C. The two layers was then separated, and the organic phase was diluted with additional DCM (10 mL), washed with saturated aqueous NaHCO.sub.3 (2×25 mL), dried over MgSO.sub.4, filtered over a sintered glass filter. Finally, 1,3-propanediol (16.4 g 216 mmol) was added to the organic phase followed by removal of DCM in vacuo.

[0370] Based on NMR the mixture (18.1 g) contained 1,3-propandiol (86.9 wt. %), 1-(nitrosooxy)-propan-3-ol (11.8 wt. %), and 1,3-bis(nitrosooxy)propane (1.3 wt. %).

[0371] 1H-NMR, δ 4.76-4.88 (m, 2H), 3.83 (t, J=5.7 Hz, 2H), 3.73 (t, J=6.1 Hz, 2H), 2.79 (s, 1H), 2.18 (quintet, J=6.3 Hz, 2H), 1.99 (quintet, J=6.2 Hz, 2H), 1.80 (quintet, J=5.7 Hz, 2H).

Example 10—Scaled up Process for the Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite

[0372] 10.1 Chemicals Used

[0373] Starting materials were purchased from the list of suppliers in the table below. Unless otherwise noted the chemicals were used as received without further purification.

TABLE-US-00002 List of used chemicals and solvents Chemical/Solvent Grade Supplier 1,2-Propanediol EMPROVE ® ESSENTIAL Merck Ph. Eur. or BP or USP, ≥99% Sodium nitrite Conforms to current ACS, VWR, Acros USP or Ph. Eur., ≥97% Sulfuric acid ≥95.0, Conforms to current VWR, Acros ACS, USP or Ph. Eur. TBME Conforms to current ACS, VWR, Acros USP or Ph. Eur., ≥99% Sodium bicarbonate Conforms to current ACS, VWR, Acros USP or Ph. Eur. Magnesium sulfate USP, dried VWR, Acros Argon 4.8 or higher Linde AG, Westfalen AG

[0374] 10.2 General Procedure for the Synthesis of PDNO Using DCM as Solvent (Origin Process)

[0375] A round bottom flask was equipped with a stirrer and dropping funnel. Water (3.0 veq.) was added and sodium nitrite (2.0 equiv.) was charged to the flask. The solution was cooled (0° C.) and PD (1.0 equiv.) and DCM (6 rel. vol.) were also added. During further cooling, a sulfuric acid solution (1.0 eq. H.sub.2SO.sub.4, 2.0 rel. vol. water) was prepared. The sulfuric acid solution was further added dropwise to the reaction mixture while keeping the reaction mixture between 0° C. and 5° C. After complete addition of the acid, the solution was further stirred for 1 h to complete reaction.

[0376] Then, the reaction was quenched with saturated NaHCO.sub.3 solution (6.0 rel. vol.). The phases were separated, and the organic layer was further washed with NaHCO.sub.3 solution (6.0 rel. vol.). The organic phase was dried over MgSO.sub.4, filtered, diluted with PD, and concentrated under reduced pressure using a rotary evaporator (water bath temperature 40° C.).

[0377] The product was obtained as a slightly yellowish liquid.

[0378] 10.3 General Synthesis of PDNO Using TBME as Solvent

[0379] A round bottom flask was equipped with stirrer and dropping funnel. Argon was flushed through for several minutes. A diluted sulfuric acid solution (1.0 eq. H.sub.2SO.sub.4, 2.0 rel. vol. water) was prepared in advanced and precooled (−30° C.). Water was added to the flask (3.0 rel. vol.). Sodium nitrite (2.0 equiv.) was added into the water. TBME (7.5 rel. vol.) was added. Propanediol (1.0 equiv.) was added and the reaction mixture was cooled (−20° C.) flushing constantly with argon. The reaction mixture was stirred well while adding dropwise the precooled sulfuric acid. The reaction temperature was monitored during the entire addition of the acid. After addition, the reaction mixture was further stirred (30-60 min) at cold temperature (−20° C.). Afterwards, the reaction mixture was allowed to warm up (−5° C.). The reaction was stopped by quenching with saturated NaHCO.sub.3solution (6.0 rel. vol.). The phases were separated. The organic layer was further washed with saturated NaHCO.sub.3 solution until a pH value of 7-8 was obtained. The organic phase was then dried over MgSO.sub.4. The crude PDNO solution was diluted with PD (3 rel. vol.) and further concentrated under reduced pressure at ambient temperature (25° C.).

[0380] The crude PDNO solution was further purified using a vertical tube evaporation apparatus.

[0381] PDNO was obtained as a slightly yellowish liquid.

[0382] 10.4 Detailed Synthesis of PDNO Using TBME as Solvent

[0383] The process was designed to produce approx. 7.5 L of 7% PDNO solution with one synthesis (one “run”). The synthesis was performed several times, to give the desired batch size. GC analysis was used each single run for purity determination. The runs which are within the specifications for the organic related compounds can be blended together to yield one batch. The entire crude PDNO batch was then purified. After purification, the strong PDNO solution was then further diluted with PD to yield the desired concentration (usually 7% PDNO solution).

[0384] A suitable double wall reactor (60 L) was equipped with specific “cup-stirrer”, dropping funnel and attachment for argon. The reactor was flushed for 5 min to 10 min with a constant argon stream. Water (3.0 L) was added to the reactor. Sodium nitrite (2.0 equiv., 1886 g) was added through the reactor. The reaction was further stirred until all of the salt was dissolved. 1,2-propanediol (1.0 equiv., 1040 g, 1L) was added, followed by tert-butylmethyl ether (7.5 rel. vol., 7.5 L). The reaction mixture was then cooled by continuous stirring and argon flow at an inner reaction temperature of —20° C. Meanwhile sulfuric acid (1.0 equiv., 1340 g, 728 mL) was diluted with water (2.0 L) and cooled at —30° C. After reaching an inner reaction temperature of −20° C., the diluted acid was added dropwise to the reaction mixture while vigorous stirring.

[0385] The stirring speed was varied during the addition of the acid. Starting with approx. 350 rpm to a slower stirring speed by the end of the reaction (approx. 180 rpm.). This variation of the stirring speed is due the two-phase reaction system and the slowly precipitation of sodium sulfate by further progress of the reaction (due to the addition of more and more sulfuric acid).

[0386] During the entire addition of the sulfuric acid, the reaction temperature was monitored. The temperature should ideally be in range of (−20±3) ° C. In addition, the reaction was stirred for 30-60 min at (−20±3) ° C.

[0387] The reaction was allowed to warm up to —5° C. to 0° C. The reaction was stopped by the addition of saturated NaHCO.sub.3 solution (6.0 rel. vol 6.0 L) followed by the addition of water (10 L). The phases were separated and the organic layer was transferred into a separate double wall reactor and chilled at 0° C. to −5° C. The organic layer was washed several times (approx. 2-3 times) with saturated NaHCO3 solution (4.0 rel. vol., 4.0 L). The pH value of the water phase was monitored after each washing step. The pH value was about 7-8. The water phases were discarded. The organic layer was dried over MgSO.sub.4 and filtered over a Whatman filter paper.

[0388] The crude PDNO (solution in TBME) was diluted by the addition of further PD (3.0 rel. vol., 3.0 L). This crude PDNO was transferred to a rotary evaporator and concentrated under reduced pressure. The water bath temperature during the evaporation was maintained at a maximum temperature of 25° C. The evaporation of the main amount of TBME was removed in a time range between 1.5 h and 2.0 h.

[0389] The evaporation of the organic solvents could then be continued at a water bath temperature at (0±2) ° C. for several hours using a high vacuum pump (during the development the PDNO purity was monitored at these conditions, and over a period of 6 h the product purity was not affected).

[0390] 10.5 Further Purification of the Crude PDNO Solution

[0391] The final purification of the PDNO solution was done by vertical tube evaporation. The PDNO solution was distilled under high vacuum with a continuous thin steam of PDNO at 0° C. The storing tank for the “crude” PDNO solution was chilled at 0° C. The entire distillation was performed at 0° C. The storage tank for the “purified” PDNO was also chilled at −10° C. to 0° C. After each run of the evaporation of the entire batch PDNO, the residual organic solvent (TBME) can be checked via GC. This evaporation was continued until the desired limit for the residual solvents was achieved. In the case of PDNO the limit for the residual solvent is 1000 ppm.

[0392] 10.6 Preparation of the Final Dilution

[0393] After purification, PDNO was further diluted to reach the favoured concentration. The first step was to filter the PDNO solution into a clean glass bottle via Whatman filter. In addition, the assay of the PDNO solution was determined via q-NMR. The amount of PD for dilution can be calculated. The PD was filtered first over a Whatman filter. The final dilution can be done at ambient temperatures. The calculated amount of PD was added to the PDNO solution (or the other way around). The resulting mixture was shaken for several minutes to obtain a homogeneous solution. The final PDNO solution was filled into the product bottles.

[0394] PDNO (7.5 kg; 7 wt. % solution) was yielded as a slightly yellowish liquid.

Example 11—First set of In Vivo Studies 11.1 Material and Methods

[0395] Prior to experimentation, ethical approval was received from Linkoping's regional animal ethics committee (Linköping, Sweden; approval number 953). Anaesthetic management, surgical instrumentation and methods for measurements were recently described (Dogan et al. 2018, Sadeghi et al. 2018, Stene Hurtsén 2020). In brief, 13 male and female pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; 3-4 months old; 24-26 kg) were premedicated with azaperone at the farm and transported to the laboratory.

[0396] At the laboratory, anaesthesia was induced with a mixture of tiletamine, zolazepam and azaperone (intramuscular injection). Propofol was given in a peripheral venous catheter in an ear vein, if needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were endotracheally intubated and mechanically ventilated (5 cm H.sub.2O in positive end-expiratory pressure, minute ventilation was adjusted to normoventilation). General anaesthesia was maintained with propofol and remifentanil via continuous intravenous infusions, and additional bolus doses were given if needed. Ringer's acetate and glucose solutions were continuously administered intravenously to substitute for fluid loss. Heparin was given as an intravenous bolus dose after the surgical instrumentation. After the experiments, the animals were killed in general anaesthesia with a propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and asystolia was confirmed.

[0397] The animals were instrumented with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introduction of a pulmonary-arterial catheter. This catheter was used for continuous measurement of pulmonary arterial blood pressure, semi-continuous cardiac output and intermittent pulmonary wedge pressure. A central venous catheter was inserted in the left external jugular vein for drug and fluid administration. All fluid and drug administrations were done by motorised syringe or drip pumps. The urinary bladder was catheterized. Respiratory gases including the fraction of nitric oxide, pressures and volumes were measured at the endotracheal tube. Respiratory and hemodynamic variables were measured by a Datex AS/3 (Helsinki, Finland) and data were collected by a computerised system (MP100 or MP150/Acknowledge 3.9.1, BIOPAC systems, Goleta, Calif., USA). After surgical instrumentation, a 1 h intervention-free period followed.

[0398] Data were presented as means and standard error of the means where applicable.

[0399] 11.2 Experimental Protocol

[0400] After collecting baseline data, several administration routes of PDNO (i.e. the product that comprises one of, or a mixture of, 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane prepared as outlined above) were investigated in the same animal with stabilisation in between.

[0401] 11.2.1 Experiments at Normal Pulmonary Vascular Resistance

[0402] Intravenous infusions of PDNO into a carrier flow of a solution of sodium bicarbonate (14 mg ml-1; pH approximately 8; infusion rate 9 times of the PDNO infusion rate) in increasing doses (5, 10, 20, 40 and 80 nmol kg.sup.−1 min.sup.−1) for 30 min at each dose were done. Subcutaneous (in the neck) and intramuscular (gluteal muscles) infusion in increasing dose (subcutaneous: 100, 200, 400, 800 and 1600 nmol kg.sup.−1 min.sup.−1; intramuscular: 50, 100, 200, 400 and 800 nmol kg.sup.−1 min.sup.−1) for 5 min followed by 25 min observation for each dose were done. Intranasal bolus application of PDNO in two doses (500 and 2500 nmol kg.sup.−1) were done in one nostril.

[0403] 11.2.2 Experiments at Increased Pulmonary Vascular Resistance

[0404] Pulmonary arterial pressure was increased to approximately 35 mmHg by a continuous intravenous infusion of U46619 (Cayman Chemical, MI, USA). Thereafter, intravenous and subcutaneous infusions of PDNO in increasing doses (intravenous: 5, 15 and 45 nmol kg.sup.−1 min.sup.−1 for 15 min at each dose; subcutaneous: 200, 600 and 1800 nmol kg.sup.−1 min.sup.−1 for 5 min followed by 10 min observation at each dose).

[0405] In an additional experiment, pulmonary arterial pressure was increased by permissive hypercapnia (end-tidal carbon dioxide fraction of approximately 8%). Thereafter, a few ml of PDNO (203 mM) were applied in a commercially available electronic cigarette (eGo AIO, Joyetech). Gas from the electronic cigarette was sampled in a 50 ml syringe and injected into the inspiratory limp of the ventilator circuit in one single breath, thus administering PDNO via inhalation. It was repeated after stabilisation and control inhalations were made with room air.

[0406] 11.3 Results

[0407] At normal vascular resistance, intravenous, subcutaneous, intramuscular and intranasal administration of PDNO caused dose-dependent increments of end-tidal fraction of nitric oxide and lowering of systemic mean arterial pressure (FIG. 2-3). At increased pulmonary vascular resistance, intravenous and subcutaneous infusions of PDNO caused dose-dependent increments of end-tidal fraction of nitric oxide and lowering of systemic and pulmonary mean arterial pressure (FIG. 4). Inhalation of PDNO caused small decrements of mean pulmonary arterial pressure whereas mean systemic arterial pressure was unchanged (FIG. 4).

Example 12—Second set of In Vivo Studies

[0408] 12.1 Material and Methods

[0409] Prior to experimentation, ethical approval was received from Linkoping's regional animal ethics committee (Linkoping, Sweden; approval number 953). Anaesthetic management, surgical instrumentation and methods for measurements were recently described (Dogan et al. 2018, Sadeghi et al. 2018, Stene Hurtsén 2020). In brief, 11 male and female pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; 3-4 months old; 20-35 kg) were premedicated with azaperone at the farm and transported to the laboratory. At the laboratory, anaesthesia was induced with a mixture of tiletamine, zolazepam and azaperone (intramuscular injection). Propofol was given in a peripheral venous catheter in an ear vein, if needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were endotracheally intubated and mechanically ventilated (5 cm H.sub.2O in positive end-expiratory pressure, minute ventilation was adjusted to normoventilation). General anaesthesia was maintained with propofol and remifentanil via continuous intravenous infusions, and additional bolus doses were given if needed. Ringer's acetate and glucose solutions were continuously administered intravenously to substitute for fluid loss. Heparin was given as an intravenous bolus dose after the surgical instrumentation. After the experiments, the animals were killed in general anaesthesia with a propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and asystolia was confirmed.

[0410] The animals were instrumented with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introduction of a pulmonary-arterial catheter. This catheter was used for continuous measurement of pulmonary arterial blood pressure, semi-continuous cardiac output and intermittent pulmonary wedge pressure. A central venous catheter was inserted in the left external jugular vein for drug and fluid administration. All fluid and drug administrations were done by motorised syringe or drip pumps. The urinary bladder was catheterized. Respiratory gases including the fraction of nitric oxide, pressures and volumes were measured at the endotracheal tube. Respiratory and hemodynamic variables were measured by a Datex AS/3 (Helsinki, Finland) and data were collected by a computerised system (MP100 or MP150/Acknowledge 3.9.1, BIOPAC systems, Goleta, Calif., USA). After surgical instrumentation, a 1 h intervention-free period followed.

[0411] 12.2 Experimental Protocol

[0412] After collecting baseline data, several administration routes of PDNO (i.e. the product that comprises one of, or a mixture of, 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane prepared as outlined above) were investigated in the same animal with stabilisation in between.

[0413] 12.2.1 Nebulisation of PDNO

[0414] Pulmonary arterial pressure was increased by permissive hypercapnia (end-tidal carbon dioxide fraction of approximately 8-9%). One-part PDNO (203 mM) was dissolved in four parts of sodium bicarbonate (50 mg-1) and nebulised with an ordinary intensive care unit nebuliser for 5-20 min (n=3).

[0415] 12.2.2 Inhalation of PDNO Using an E-cigarette

[0416] A few ml of PDNO (203 mM) were applied in a commercially available electronic cigarette (eGo AIO, Joyetech). Gas from the electronic cigarette was sampled in a 50 ml syringe and injected into the inspiratory limp of the ventilator circuit. This procedure was repeated for approximately 10 times with very short interval, thus administering PDNO via inhalation (n=1).

[0417] 12.2.3 Sublingual Administration of PDNO

[0418] PDNO was applied sublingually by a small compress soaked with 2 ml PDNO (1 mM-203 mM) for 10-20 min each (one or several doses in three animals). In two experiments acute pulmonary hypertension was induced by a fast injection of air (300 microl/kg) intravenously (air pulmonary embolization).

[0419] 12.2.4 Dermal Application of PDNO

[0420] PDNO was applied on the skin of the abdomen by three small compresses soaked with 2 ml PDNO (203 mM). Three compresses without PDNO (control) were used for comparison. The skin was pretreated with menthone.

[0421] 12.2.5 Gastrointestinal and Urinary Bladder Administration of PDNO

[0422] PDNO (2-4 ml of 1 mM, 10 mM, 100 mM and 200 mM) was injected via catheters in the urinary bladder, stomach, small and large intestine.

[0423] 12.3 Results

[0424] Using pulmonary and systemic arterial pressure measurements and measurements of end-tidal concentration, it was found that PDNO administered via inhalation, nebulisation, sublingual application, dermal application, gastrointestinal application and urinary bladder administration elicited biological responses (decreases in blood pressures) via NO donation (increases in end-tidal NO concentration, not measured in all experiments (FIGS. 6 to 11). Furthermore, it was found that such administration was efficient to counteract acute pulmonary hypertension (induced either by air pulmonary embolization and hypercapnia).