How is erythrityl tetranitrate synthesized

Pentaerithrityl tetranitrate, glyceryl trinitrate, their bioactive metabolites and other NO-based vasodilators

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1 Pentaerithrityl tetranitrate, glyceryl trinitrate, their bioactive metabolites and other NO-based vasodilators Relationship between structure and effect Dissertation to obtain the academic degree doctor rerum naturalium (Dr. rer. Nat.) Submitted to the Council of the Biological-Pharmaceutical Faculty of the Friedrich Schiller University Jena by Andreas König born on February 24, 1977 in Mühlhausen / Thür.

2 reviewers: 1. Prof. Dr. Jochen Lehmann 2. Prof. Dr. Erika Glusa 3. Prof. Dr. Hans-Jürgen Duchstein Public Defense Day:

3 Acknowledgments The present work was carried out between January 2004 and December 2007 at the Institute for Pharmacy of the Friedrich-Schiller University Jena. In a special way, I would like to thank two people equally very warmly. On the one hand, Professor Dr. Erika Glusa, who introduced me to pharmacological issues with a lot of expertise, drive and academic discipline, at the same time combined with human cordiality, and who was extremely helpful to me in all phases of the work. I would also like to thank Professor. Dr. Jochen Lehmann, who suggested the topic, supported the work with great commitment and made a pleasant working atmosphere possible with his friendly, accommodating and creative manner. I would also like to thank him for the fact that I was able to continue the work I started in Erfurt in Jena in his working group and that I was able to enjoy many wonderful and unforgettable moments such as the NO conference in Monterey or the PETN expert meeting. Professor Dr. I thank Hans-Jürgen Duchstein for kindly taking over the submission. For the great willingness to cooperate, successful cooperation and support in the joint projects, I thank PD Dr. habil. Michael Decker, Dr. Dirk Stalleicken, Dr. Dr. habil. Andreas Daiber, Dr. Jörg Konter, Ms. Kathrin Lange and Ms. Carolin Roegler. Last but not least, I would like to thank all my colleagues and the many friends I have made at the institute, with whom I was able to spend a wonderful time together in Jena in addition to the pleasant cooperation and helpfulness. My greatest and most private thanks go to my family, my parents Doris and Gerhard König and my brother Alexander König, who have supported and encouraged me in every way on my life path so far, deepest thanks also to Kathi Gülland simply for a wonderful time together.

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5 Contents I 1. Introduction to pharmacotherapy for cardiovascular diseases Coronary heart disease Significance and areas of application of organic nitrates 2 The first organic nitrate: glyceryl trinitrate 2 Development of long-acting nitrates 4 Current drug inventory of coronary drugs 7 Comparative evaluation Mechanism of action of organic nitrates Anti-ischemic principle Active principle at the molecular level Nitrate activation 13 Nitrate activation from nitrates? 13 Various bioactivation hypotheses 14 The mitochondrial aldehyde dehydrogenase 15 Highly potent metabolic pathway via ALDH-2 16 Low-potential metabolic pathway via? Nitrate tolerance Pseudotolerance Classical tolerance 20 Explanatory approaches and theories Objective Preliminary remarks Tasks Overview of publications 29

6 II Contents 4. Publications 33 Publication 1 33 Pharmacological characterization of pentaerithrityl tetranitrate, its nitrate-containing metabolites and other organic nitrates in the isolated pulmonary artery of the pig. King, Andreas; Pietig, Imke; Homann, Alexander; Glusa, Erika; Fricke, Uwe; Lehmann, Jochen. In: ERDMANN, E .; MUTSCHLER, E .; STALLEICKEN, D. (Ed.): Pentaerithrityl Tetranitrat: Evidence-Oriented Therapy Concept for Cardiac Diseases. Steinkopff Verlag, Darmstadt (2004), publication 2 43 Synthesis and vasorelaxant properties of hybrid molecules out of NO-donors and the beta-receptor blocking drug propranolol. Decker, Michael; King, Andreas; Glusa, Erika; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters (2004), 14, publication 3 46 Potency and in vitro tolerance of organic nitrates: partially denitrated metabolites contribute to the tolerance-devoid activity of pentaerythrityl tetranitrate. King, Andreas; Lange, Kathrin; Konter, Jörg; Daiber, Andreas; Stalleicken, Dirk; Glusa, Erika; Lehmann, Jochen. Journal of Cardiovascular Pharmacology (2007), 50, Publication 4 53 NO donors. Part 16: investigations on structure-activity relationships of organic mononitrates reveal 2-nitrooxyethylammonium nitrate as a high potent vasodilator. King, Andreas; Roegler, Carolin; Lange, Kathrin; Daiber, Andreas; Glusa, Erika; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters (2007), 17, publication 5 58 NO donors. Part 18: synthesis and vasorelaxant properties of the bioactive metabolites of GTN and PETN. Lange, Kathrin; King, Andreas; Roegler, Carolin; Seeling, Andreas; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters. In preparation.

7 Table of contents III 5. Unpublished results Influence of the number of nitrate groups on the vasodilatory potency Stronger vasoactivity of direct NO donors using the example of NONOates compared to organic nitrates In vitro activity profile of nitrates, nitrites and NONOates In vitro development of cross-tolerance between organic nitrates and nitrites according to EC 50 - Bolus administration Vasodilatory properties of NSAID-nitrate hybrid molecules Methods for the detection of in vitro nitrate tolerance Studies on the development of cross tolerance GTN vs. 1,2-GDN Aminoethyl nitrate: in vitro tolerance studies Aminoethyl nitrate: bioactivation via ALDH-2? Investigation of the formation of ROS species in some selected mononitrates compared to GTN Discussion Influence of the number of nitrate groups on the vasoactivity Influence of the nitrate-bearing residue on the vasoactivity Synthesis of structurally different organic mononitrates Differences in the active activities and their possible cause A highly potent mononitrate: 2-aminoethyl nitrate Nitrate Hybrid Compounds Influence of the Structure of Organic Nitrates on Tolerance Approaches to Explanations for the Clinical Effect Profile of PETN Summary 101

8 IV Table of contents 8. Conclusion Bibliography 10. Appendix Curriculum vitae List of publications Declaration of independence

9 Introduction 1 1. Introduction 1.1. Pharmacotherapy of cardiovascular diseases Cardiovascular diseases are currently the most common causes of death in women and men in Germany and other European countries. 1,2 From the annual report on health in Germany drawn up by the Robert Koch Institute on behalf of the federal government, it emerges for 2006 that in Germany almost every second deceased was caused by a disease of the cardiovascular system. Nevertheless, the number of deaths caused by cardiovascular diseases has steadily declined in Germany since 1990 due to continuously improved treatment methods. Coronary heart disease Within the group of cardiovascular diseases, coronary or ischemic heart disease (CHD) is of the greatest pathological importance. In this chronic disease of the coronary vessels, morphological or pathophysiological changes (e.g. narrowing or occlusion) of one or more vessels cause an insufficient blood supply to the heart (ischemia). This disrupts the balance between oxygen supply and consumption in the heart muscles, depending on the stress situation. The larger coronary vessels in particular can be affected by the manifestation of arteriosclerosis, known as coronary sclerosis. A narrowing (stenosis) of the coronary artery is the result and can also lead to partial or complete occlusion of coronary artery branches due to the formation of thrombi. Coronary sclerosis is one of the most important causes of CHD. In addition to this, however, heart valve defects, vegetative malfunctions, arrhythmias, anemia or high blood pressure can also cause or promote the clinical picture. The highly variable spectrum of CHD ranges, depending on the severity, from the asymptomatic form at the beginning of the disease to angina pectoris to myocardial infarction (heart attack) or death by a second (heartbeat). Confirmed risk factors promoting coronary sclerosis are smoking, hypertension, hyperlipoproteinemia, diabetes mellitus, obesity and ultimately resulting from it

10 2 Introduction to endothelial dysfunction. Avoiding these risk factors, including through preventive medical and therapeutic measures, is of great importance. 2, Significance and areas of application of organic nitrates The modern standard in the clinical treatment of CAD include bypass operations and, in particular, interventional therapy strategies such as percutaneous transluminal coronary angioplasty (PTCA) and stent implantations. 4,5 These procedures are intended to restore the blood flow to the heart muscle in the sense of a revascularization of the coronary vessels. Regardless of these established interventional procedures, there is a need for symptomatic pharmacotherapy. 6 This is based on the fact that a CHD is usually based on a multiple disease of the vascular wall and not all vascular occlusions and constrictions are operable; moreover, nothing changes in the conditions of the microcirculation in the surgical procedures. The aim of the drug treatment of CHD is to eliminate the imbalance between oxygen supply and consumption in the myocardial areas affected by the ischemia. Three drug groups with antianginal effects are available: organic nitrates, calcium channel blockers (Ca 2+ antagonists) and ß-adrenoceptor antagonists (ß-blockers). 7 The organic nitrates, also called nitric acid esters or often chemically incorrectly called nitrovasodilators, have been used for over 120 years in the prevention of attacks and angina pectoris. They are therefore among the first chemically defined active ingredients to be used in clinical medicine. The first organic nitrate: glyceryl trinitrate In order to produce potential explosives, the Turin chemist Ascanio Sobrero treated polyhydric alcohols with a mixture of sulfuric and nitric acid. The implementation was problematic, as their strongly exothermic course often led to a detonation if there was not enough cooling. But Sobrero's efforts were successful, in 1846 he received the nitric acid ester of glycerol, Glycerolbzw. Glyceryl trinitrate (GTN), also known as nitroglycerin, which decomposes when hit or suddenly heated. 8,9 In the present work the chemically correct name glyceryl trinitrate should be used.

11 Introduction 3 It was also Sobrero who described the first observations on the effect of the substance on humans. During the organoleptic test that was customary at the time, he put what appeared to be a small amount on his tongue and registered a tearing throb on his temples and severe headaches. He then named the new compound pyroglycerin. 8 Sobrero, badly marked by an explosion in his face himself, considered GTN too dangerous for any practical application. 10 The German doctor and homeopath Constantin Hering, co-founder of homeopathy in America, immediately (1849) became aware of Sobrero's publications and saw the substance as an ideal potential candidate in the range of homeopathic remedies. In terms of homeopathic therapy, the headache-inducing GTN should be used in large dilutions for headache treatment. 11 As is customary in homeopathy today, the new substance was tested in advance on healthy test subjects in increasing, non-homeopathic doses until an effect ceased. Even then, Hering described the spectrum of effects and side effects of GTN relatively comprehensively, although at that time conventional medicine showed little interest in the substance Hering referred to as glonoins. 12 For the first time, in 1858, the British doctor Alfred Field attributed the substance to have an anti-spasmic effect after successfully treating a woman with angina pectoris-like complaints and thus aroused broad interest in the medical profession. 13 Before GTN was introduced in the treatment of angina pectoris, the Scottish doctor Thomas L. Brunton described the use of amyl nitrite, an organic nitrite ester with an analogous mode of action, Brunton published a study in the Lancet in which he applied 5-10 drops of amyl nitrite to a cloth and administered it to a patient for inhalation. Within a few seconds his face blushed, the pulse became stronger and the anginal symptoms were relieved. However, the disadvantage of the application of the volatile, unpleasant-smelling compound was the inaccurate dosage and the short duration of action. He also noted that the dose has to be increased with prolonged use in order to achieve the desired effect, an allusion to tolerance phenomena (Section 1.3.). 15 Organic nitrites also lead to an undesirable formation of methaemoglobin. Their use in angina pectoris is therefore obsolete today, but they are used in cyanide poisoning and improperly as so-called poppers. The London doctor William Murrell recognized that GTN had clear therapeutic advantages over amyl nitrite, especially in the time profile, and published a study in the Lancet in 1879

12 4 Introduction to the effectiveness of the substance on 35 people, angina pectoris patients and acquaintances and praised it as the remedy par excellence for angina pectoris. 16 GTN entered the British Pharmacopoeia and established itself in clinical treatment. In addition to an inpatient treatment scheme, Murrell equipped the discharged patient with an emergency vial for seizure docking, a measure comparable to today's use of bite capsules or sprays. Murrell also devoted himself to finding the dose to avoid the nitrate headache, usually caused by overdosing, as well as the pharmaceutical processing of the explosive agent. The liquid formulation used at the time had many disadvantages and so Murrell asked the pharmacist and chemist William Martindale for a more stable and transportable preparation. He then processed one hundredth of a grain GTN (1 grain = 0.0648 grams) into chocolate. 17 GTN was available in pill form in five strengths as early as 1882 and was produced on an industrial scale by Parke Davis & Company (Detroit, USA). 12 In 1933 Sir Thomas Lewis reported an expansion of the coronary arteries and an improved supply of oxygen to the myocardium after the administration of GTN. The preload-lowering effect of nitrates was first described by Gorlin in 1959. 18 Development of long-acting nitrates Although Bradbury introduced various simple-structured alkyl nitrates as early as 1895 under the title SOME NEW VASO-DILATORS, 19 apart from amyl nitrite, only GTN was available therapeutically for all forms of angina pectoris until World War II. From the 30s of the last century, intensive research work began with the endeavor to find nitrates with fewer side effects and longer-lasting effects. John C. Krantz Jr. and his colleagues have done a lot of work that deals with the metabolism and pharmacology of sugar alcohols (polyols), corresponding anhydrides and their nitrate and nitrite esters. 20,21 Mono- or dianhydrides are created by removing water from sugar alcohols. After the discovery of insulin, it was hoped to obtain substances that the body cells use as an energy source without insulin. 22 This was not the case, however, because these anhydrides are practically not metabolized and rapidly excreted by the kidneys unchanged, so isosorbide was used as an oral, osmotically active diuretic (hydronol). 23 Thus the anhydrides were predestined as carriers of nitrate groups without their own active component.

13 Introduction 5 In 1939 Krantz published a study in which he examined seven organic nitrate anhydrides with regard to the extent and duration of the lowering of blood pressure in anesthetized dog. 24 Compared to GTN, he observed a lower but significantly longer-lasting drop in blood pressure, which he attributed to poorer hydrolysability and better water solubility of the chemically stable compounds. In addition to the favorable therapeutic profile, he also recognized advantages in the manufacture and handling of the substances due to the lower explosiveness. In further studies, the stereoisomers isomannide (IMDN) and isosorbide dinitrate (ISDN) in particular were examined. 25 Goldberg compared the two compounds experimentally and pharmacologically with regard to toxicity, blood pressure, respiration, coronary circulation in mice and clinically with regard to drop in blood pressure and headache in 24 hypertensive men. 26 Due to its higher activity and longer duration of action, he suggested the therapeutic use of ISDN for high blood pressure and angina pectoris and thus laid the foundation for the establishment of the drug. Between 1938 and 1962 numerous papers were written which dealt with the development of new nitrates as potential medicinal substances. These included, among other things, methyl and ethyl derivatives of GTN, halogenated and non-halogenated cyclic and aliphatic nitrates, alkyl nitrates of glycolic acid and its salts, choline nitrate esters and nitrates with an ethylamine partial structure. 22 Other compounds such as erythrityl tetranitrate (ETN) or mannitol hexanitrate had long been known from the explosives industry. Particularly noteworthy is the pentaerithrityl tetranitrate (PETN) synthesized by Vignon and Gerin in 1901.27 In 1937 Takeshita demonstrated that PETN had an antihypertensive effect on rabbits, 28 the first clinical studies were carried out in 1943 by Bjerlöv, who found mostly more favorable effects for PETN than ETN in 165 patients. The first approval was in the USA, followed by the Scandinavian countries, France and the GDR (1964, Pentalong). While pentalong was the only long-term nitrate available in the GDR that was widely used in therapy, the analog preparation Dilcoran was more of a stepchild in the FRG. In the USA there were over 30 formulations with the ingredient PETN in mono- or combination preparations in the 1960s. The most common therapeutic agents included combinations (two-phase tablets) of GTN with diuretics of the thiazide type, hypnotics, tranquilizers and sedatives. 30th

14 6 Introduction In addition, there were numerous other organic nitrates on the pharmaceutical market in various countries, but are currently mostly obsolete (Fig. 1) What is striking are the sometimes considerable differences in the dosage recommendations for the compounds, which even then indicate differences in pharmacokinetic behavior. ONO 2 ONO 2 ONO 2 O 2 NO ONO 2 ONO 2 Mannityl Hexanitrate Molloid (DDR), Nitromaxitate (I), Vascunitol (USA) ED: 3.3 mg TD: up to 6.6 mg O 2 NO ONO 2 O 2 NO ONO 2 Pentaerithrityl tetranitrate Pentalong (D), Peritrate (A, CDN, USA, F, I, ZA.), Nitrodex (CH), Mycardol (GB) ED: mg TD: up to 240 mg ONO 2 ONO 2 O 2 NO ONO 2 Erithrityl tetranitrate Cardilate (CDN, I, USA), Cardiwell (F) ED: 30 mg TD: up to 100mg O 2 NO O 2 NO NN Tenitramin Tenitran (I, E), ED: nn TD: n.n. ONO 2 ONO 2 O 2 NO ONO 2 ONO 2 O 2 NO ONO 2 N ONO 2 2 H 3 PO 4 O 2 NO ONO 2 ONO 2 glyceryl trinitrate Angiplex (S.), Glytrin (GB), Nitrolingual (D), Tridil ( AUS, ZA) ED: 0.2-5 mg TD: up to 15 mg trol nitrate phosphate Nitralettae (Slovakia) ED: 8 mg TD: up to 16 mg propatyl nitrate Etrynit (E), Sustrate (BR) ED: 10 mg TD: nn O 2 NO O HO ONO ONO 2 isosorbide dinitrate Cedocard (CDN, GB, NL), isoket (D., Schw.), Dilanid (ZA) ED: mg TD: up to 120 mg ONO 2 Cl ONO 2 clonitrate dylate (nn) ED : nn TD: n.n. O ONO 2 isosorbide mononitrate Elan (I), Monoket (S), Olicard (S), Uniket (E) ED: mg TD: up to 120 mg ON ONO 2 H Nicorandil Sigmart (J), Dancor (S, A) ED: 5 -20 mg TD: up to 40 mg Fig. 1: Overview of the structural formulas of therapeutically used organic nitrates and their trade names in the individual countries, single dose (ED), daily dose (TD) and approval status (= obsolete) From the mid-1960s, the metabolism Therapeutically important organonitrates deciphered by means of radioactive labeling. Interesting findings for ISDN emerged. Pharmacokinetic studies in humans and dogs have shown that ISDN has a pronounced first-pass effect after oral administration. 41,42 The main metabolite 5-isosorbide mononitrate (5-ISMN) was detected in plasma in concentrations 6-10 times higher than that of its isomer 2-isosorbide mononitrate (2-ISMN). ISDN itself was only detectable for a few minutes and only in concentrations that were not therapeutically relevant, whereas the metabolite 5-ISMN was detectable in the plasma for 6 hours. 42 This made it clear that the first nitrate group had to be split off enzymatically and stereoselectively and that the long-term effect of ISDN was solely due to 5-ISMN. These findings led to the introduction of 5-ISMN as a medicinal substance in 1981 and explain the dosage regimen analogous to the parent substance ISDN (Fig. 1).

15 Introduction 7 A structurally clearly different representative among the nitrates was developed in the late 1970s, the nicotinamide derivative nicorandil (Fig. 1). 43 Nicorandil is the first representative from the group of nitrate hybrid molecules. In addition to the nitrate effect, it has another active component in that, as a so-called potassium channel opener, it increases the probability of opening potassium channels in the cell membrane of the smooth muscle cells. Nicorandil thus simultaneously reduces cardiac preload (preload) and afterload (afterload) and is a potent coronary dilator. 44 The drug is currently said to have cardioprotective properties, after the IONA study on 5000 angina pectoris patients demonstrated a significant reduction in coronary events when nicorandil was administered, 45 in addition, no cross-tolerance to GTN was observed. 46 However, the desired use for unstable angina pectoris or as a reserve drug for patients who do not respond to conventional angina pectoris therapy has so far only caught on to a limited extent; the preparation is only on the market in a few countries. Current drug inventory of coronary drugs Drugs for the symptomatic treatment of CHD are summarized in the indication group coronary drugs. 47 The most important official representatives in Germany currently include the following organic nitrates and substances similar to substances, each with different therapeutic profiles. In addition to the antianginal properties, numerous other uses have been described for GTN in particular. 1. Glyceryl trinitrate (commercial preparations: including Aquo-Trinitrosan, Corangin Nitro, Nitrangin, Nitrolingual, Nitroderm TTS), as a spray or bite capsule (clinically also IV infusion). First choice in the treatment of seizures of stable angina pectoris that occurs under stress and unstable angina pectoris, acute myocardial infarction and catheter-induced coronary spasm (onset of action approx. 1 min); to a small extent use as nitrate plaster for the treatment of chronic congestive heart failure and for the prophylaxis of chronic angina pectoris. GTN are also said to have beneficial effects in the non-cardiovascular area, for example for the treatment of pain in diabetic neuropathy, 48 as an adjuvant to opioid therapy, 49 for pain relief in chronic anal fissures (Rectogesic), 50 for maintaining organ function in transplantations 51 and for the combination therapy of inoperable, small-cell cell cancers. 52

16 8 Introduction Numerous homeopathic combination preparations with the ingredient Glonoinum for GTN are still currently on the market in D3 - D8 potencies for a wide variety of complaints (including Hevert migraine, Ypsiloheel). 2. Pentaerithrityl tetranitrate (Pentalong, Nirason), prophylaxis and long-term treatment of angina pectoris without a so-called nitrate break. 3. Isosorbide dinitrate (isoket spray, sublingual tablets, ointment; ISDN Stada, ISDNratiopharm; nitrosorbon), mainly as interval therapy for prophylaxis and long-term treatment of angina pectoris as sustained-release capsules and tablets; sublingual or as a spray for angina attack treatment as well as preventive intake immediately before physical exertion in the case of stable angina pectoris (onset of action approx. 5 min), compared to GTN, however, slower onset of action isosorbide mononitrate (Corangin, IS 5 mono-ratiopharm, Mono Mack) than Interval therapy for the prophylaxis and long-term treatment of angina pectoris, mainly as sustained-release capsules and tablets. 5. Molsidomine (Corvaton, MolsiHexal), real-like compound that releases nitric oxide (NO) without the mediation of SH groups (Fig. 2). 53 At the same time, however, this spontaneous reaction also generates peroxynitrite (ONOO -) from radical superoxide anions (O - 2) and NO, which increases the oxidative stress status in the cell. However, since bioactivation via an intermediate reductive step is not necessary in comparison to nitrates, tolerance formation typical for organic nitrates (see Section 1.3.) Should not occur either in vitro or in vivo. However, these positive properties are offset by toxicological findings which indicate increased carcinogenicity in animal experiments when high doses are given. The indication of molsidomine is therefore very limited; it is used as a reserve drug for elderly patients and / or who do not respond to conventional prophylaxis and long-term treatment of angina pectoris. Bridging the nightly nitrate break with molsidomine is also often practiced, but it is controversial. 6. Sodium nitroprusside (nipruss), active-like complex compound that releases non-enzymatically NO or a related species and has a dilating effect on the venous and arterial blood vessels. Because of its rapid gastrointestinal inactivation, sodium nitroprusside can only be infused intravenously. The compound has clinical application as the drug of choice

17 Introduction 9 life-threatening hypertensive crises and for the control of controlled hypotension in surgery, since the blood pressure can be titrated precisely due to the short plasma half-life of 3-4 minutes. During the infusion, sodium thiosulphate must be infused at the same time in order to detoxify released cyanide ions. 7 O N O Enzymatic cleavage N N N N NH N O N O O O Linsidomine SIN-1 ring opening O O 2 N N SIN-1A N O Oxidation N Peroxynitrite formation ONOO O 2 O O N SIN-1C N NO + H N NO release N N N O N Fig. 2: Metabolism of molsidomine. Enzymatic cleavage of the carboxyethyl group to linsidomine (SIN-1), which under neutral conditions spontaneously converts to the acyclic nitrosohydrazine derivative SIN-1A. Oxygen or oxidizing agents are in turn involved in the conversion to a cation radical with the formation of superoxide (O 2 -). NO is released spontaneously from this intermediate; with deprotonation, modified according to 53, the stable iminoacetonitrile derivative SIN-1C is formed. Comparative evaluation Although 49 coronary drugs were represented among the 3,000 most frequently prescribed drugs in Germany in 2005, their position in the drug treasury has steadily declined in recent years due to a decline in medical prescriptions according to the 2006 drug prescription report. 54 This tendency is based on worrying findings that these drugs only have a purely symptomatic effect or even have arteriosclerosis-promoting properties that increase cardiovascular morbidity and mortality and thus the long-term prognosis, e.g. with nitrate administration, worsen. 55,56 ISDN and ISMN are the most frequently used nitrates, since both substances can be used to effectively prevent seizures if the dose is correct and the nitrate-free interval is adhered to. In therapy, ISMN has only theoretical advantages over ISDN from the current scientific point of view, e.g. a higher bioavailability, which, however, except for the

18 10 Introduction Dose determination, have no application-relevant meaning. The disadvantage of ISMN is the relatively slow resorption, which excludes the treatment of acute angina pectoris attacks even with sublingual application. For several years now, PETN has been used increasingly as the only long-term nitrate, since extensive research work in vitro, on animals and on healthy test persons indicates a significant superiority in terms of tolerance development and nitrate headache compared to other organic nitrates, but a final controlled, randomized, clinical long-term study is still pending . The frequency of prescribing molsidomine has remained almost constant over the past few years and is now in the same order of magnitude as the declining ISMN. This is astonishing since, according to more recent comparative studies, even with the administration of molsidomine, as with ISDN, a clear weakening of the anti-ischemic effects occurs after 1-4 days. 60 In addition to the potential carcinogenicity, there are also no controlled endpoint studies for molsidomine. Mechanism of action of organic nitrates Anti-ischemic principle Organic nitrates are endothelial-independent vasodilators with a predominant effect on the venous circulation and coronary vessels. To achieve a dilatation of the arteriole, significantly higher nitrate concentrations are necessary. This leads to a unique hemodynamic spectrum of action in the pharmacotherapy of cardiac diseases: improved blood flow through dilatation of large coronary vessels and, among other things, increased collateral flow, especially in the area of ​​partial stenoses, lowering of the preload leads to a reduced O 2 requirement of the heart, improved endocardial blood flow through a decreased intramyocardial wall tension and an increase in perfusion pressure, no coronary steal, as the tone of the arterial resistance vessels is retained. In addition, nitrates, as well as molsidomine and sodium nitroprusside, have an anti-aggregation effect and can thus prevent thrombus formation and myocardial infarction. 7th

19 Introduction Active principle at the molecular level For a long time, therapy with organic nitrates was an unusual situation. On the one hand, the antianginal properties of drugs have been used extensively since 1879, on the other hand, their mechanism of action at the cellular level was completely unknown for a long time. In the mid-1970s, Ferid Murad found out that vasodilators such as GTN, sodium nitroprusside and similar substances activate soluble guanylyl cyclase (sgc) and, as a result, the conversion of guanosine-5-triphoshate to cyclic guanosine-3,5-monophosphate (cgmp ) is stimulated in vascular smooth muscle cells; identical biological effects were observed for nitric oxide (NO). 61,62 Furchgott and Zawadzki observed in vitro, initially rather coincidentally, that in vascular specimens with damaged endothelium, after the addition of acetylcholine, relaxation did not occur. They then postulated the existence of a substance that is released from the endothelium under the influence of numerous stimuli (e.g. hormones, neurotransmitters, shear stress) and triggers relaxation in the surrounding smooth vascular muscle cells. 63 They named this substance very labile, easily diffusing and of unknown structure EDRF (Endothelium-Derived-Relaxing-Factor). It was discovered that EDRF is NO; a very short-lived, gaseous nitrogen radical that was previously only associated with air pollution, cigarette smoke or smog. 64,65 In the period that followed, it became increasingly clear which important role NO plays as a messenger substance for many biological processes; in the last five years alone, for example, papers on NO have been published, NO was voted Molecule of the year by the journal Science and the Nobel Prize in 1998 for Physiology and Medicine to the pioneers of NO research, Robert F. Furchgott, Louis J. Ignarro and Ferid Murad. 67,68 It is now accepted that the nitrate-induced relaxation in smooth vascular muscle cells is caused by the following chain-like mechanism of action (Fig. 3). Activation of the NO-sensitive soluble sgc, increasing the concentration of cgmp and subsequent activation of cgmp-dependent protein kinases (cgk-i) and ion channels with the result of a reduction in the intracellular Ca 2+ concentration. 69 An important substrate of cgk-i is VASP (VAsodilator-Stimulated-Phosphoprotein). 70 NO formed from the amino acid L-arginine by the NO synthases (NOS) also acts endogenously via this signal path; it is differentiated into: endothelial, neuronal and inducible NO synthase (enos, nnos, inos). 71

20 12 Introduction Direct NO donors: Molsidomine Nitroprusside sodium NONOate Nitrosothiols O O N N R N R Shear forces Na non-enzymatic release N O NO scavenger? sgc stimulators sgc activators reduced oxidized sgc oxidative stress sgc sgc inhibitors endothelial cells NO synthase L-arginine reductases α β α β R ONO 2 enzymatic organic nitrate bioactivation NONOX GTP PDE inhibitors PDE GMP cgmp cgk-i GTP Ca 2+ Glatte Vascular muscle cells Cellular mechanism Vasodilation Fig. 3: Mechanism of action of organic nitrates and direct NO donors. Organic nitrates are bioactivated in the smooth vascular muscles to form nitric oxide (NO) and / or related species (NO x), while direct NO donors such as diazenium diolates (NONOates) are non-enzymatic under certain chemical conditions (e.g. pH value, temperature) and direct NO release. This sets the reaction cascade described in the text in motion. Soluble guanylyl cyclase (sgc), guanosine triphosphate (GTP), cyclic guanosine monophosphate (cgmp), phosphodiesterase (PDE), cgmp-dependent modified according to protein kinase 72 (cgk-i). SGC is a heterodimeric heme protein with an α 1,2 and β 1,2 subunit and is activated by binding NO to the centrally bound iron atom (Fe 2+) of the heme group. The associated change in conformation at the catalytic center triples the affinity of the sgc for guanosine triphosphate (GTP) and increases the rate of formation of the secondary messenger substance cgmp. Numerous inhibitors of the NO-sensitive SGC have been described, the newer tricyclic quinoxaline derivatives ODQ or NS2028 are among the most potent and selective inhibitors in that they cause an oxidation and / or a change in the coordination of heme iron. 73,74 With their development, the importance of significantly less specific SGC inhibitors such as porphyrin derivatives or redox-sensitive dyes (methylene blue and LY 83583) has decreased. 75,76 An increase in the SGC enzyme function can be caused by two mechanisms and is closely related to the two redox states of SGC: a NO-sensitive, reduced and a NO-insensitive, oxidized form (Fig. 3). The sgc can be stimulated allosterically in the reduced state (reduced heme) by sgc stimulators (e.g. YC-1, BAY), a significantly increased one

21 Introduction 13 Sensitivity to NO is the consequence In the heme-oxidized or heme-free, NO-immune state of the SGC, the SGC stimulators are ineffective, while in this state so-called SGC activators (BAY, HMR-1766) activate the SGC effect without NO or heme. 72,80 In both cases, however, the result is an increase in the intracellular concentration of the second messenger cgmp. The extent to which these new and interesting classes of active ingredients, successfully applied in experimental pharmacological research, can be used therapeutically is currently open.Both classes of compounds are currently in preclinical tests for the treatment of chronic heart failure and pulmonary hypertension. 81 The degradation of the second messenger cgmp (as well as camp) to the corresponding acyclic nucleotide GMP (or AMP) is catalyzed by various phosphodiesterase enzyme families (PDE 1-11) (Fig. 3). 82 Selective inhibitors of phosphodiesterase type 5 (PDE-5) are used in the treatment of erectile dysfunction (e.g. sildenafil in Viagra, tadalafil in Cialis, vardenafil in Levitra). Recently, PDE-5 inhibitors have also been approved to improve performance in the treatment of pulmonary hypertension of WHO functional class III (Sildenafil in Revatio). 47.83 The simultaneous administration of organic nitrates or active compounds and PDE-5 inhibitors is strictly contraindicated due to a massive drop in blood pressure due to the accumulation of cgmp Bioactivation of organic nitrates In contrast to direct NO donors (e.g. diazeniumdiolates) or NO + donors ( For example nitrosothiols, nitrates (oxidation level: +5) require prior reductive bioactivation in the smooth vascular muscle cells (Fig. 3). Organic nitrates are therefore classic prodrugs. After the discovery of EDRF, the idea was obvious to regard NO (oxidation level: +2) as the actual active principle of organic nitrates, 84 however, the detection of NO in biological matrices has always been difficult due to the reactivity and short life of the molecule. NO from nitrates? Some studies confirm the formation of NO from GTN both in vivo and in vitro using the oxyhemoglobin assay and chemiluminescence detection, but only after application of therapeutically irrelevant high concentrations

22 14 Introduction Recent studies refute the thesis that GTN can increase vascular NO levels in lower therapeutic (nanomolar) doses. Kleschyov et al. determined the NO donor properties of GTN with the help of a combination of NO spintrapping (with iron (II) diethyldithiocarbamate - Fe [DECT] 2) and electron spin resonance spectroscopy and compared this with the extent of phosphorylation of the VASP at Ser239 ( P-VASP) as a marker for cgk-1 activity. 70,88 In vessels with intact endothelium there was a noticeable discrepancy (up to three powers of ten) between strong vascular activity (increase in P-VASP and observed vasorelaxation in the nanomalor range) and its weak NO donor properties (increase in NO-Fe [DECT] 2 at micromolar concentrations). In contrast, a direct correlation was observed with ISDN and calcium ionophores A23187, an endothelium-dependent vasodilator. In addition, it was shown that inhibition with the NO scavenger Carboxy-PTIO attenuates the vasodilatory activity of GTN significantly less than that of conventional NO donors. 89.90 It is conceivable that in therapeutically effective (nanomolar) concentrations a sgc-activating species (NO x) closely related to NO is formed, which cannot be detected with NO spintrapping (Fig. 3). The following explanations also exist: an indirect increase in activity or modulation by changing the position of the SGC within the cell (as also described for direct NO donors); Interaction of GTN and / or its bioactive metabolites with SGC binding proteins; as well as a partial agonism of the so-called NO is, according to the current state of knowledge, not generally the active principle of organic nitrates, rather the confirmation of the NO hypothesis depends on the selected NO analysis, the nitrate concentration used, the selected nitrate compound and the respective underlying bioactivation mechanisms . Different bioactivation hypotheses Which enzymes ultimately catalyze bioactivation has long been contradicting itself. It was known early on that compounds containing thiol groups (R-SH) are required as cofactors for the vascular relaxation of organic nitrates. 95 The depletion of R-SH pools was and is closely related to nitrate tolerance. 94 Needleman and Hunter proposed as early as 1965 the theory that GTN is metabolized by a glutathione-dependent nitrate reductase; the enzyme was later identified as glutathione-S-transferase (GST). 96 In addition to GST, the xanthine oxidoreductase and the cytochrome P450 system have often been suggested as possible candidates for the implementation of GTN. Also a thiol-dependent GTN receptor as well as the

23 Introduction 15 in vitro observed and described non-enzymatic conversion of organic nitrates with thiols, especially with cysteine, was often used to explain bioactivation. 97 However, the relevance of these mostly experimental pharmacological findings for the significance of nitrate bioactivation is questionable. For example, the GST inhibitor sulfobromophthalein has no inhibiting effect on the GTN-induced increase in the intracellular cgmp concentration. Within the GST enzyme family, the GST µ isoform has been described to have the highest GTN metabolism activity in the vessel wall. A genetic defect in GST µ, as can occur in humans, however, has no influence whatsoever on the effectiveness of GTN in the affected patients. 98 Above all, none of the alleged bioactivation enzymes catalyzes selectively the conversion of GTN to the bioactive metabolite 1,2-glyceryl dinitrate (1,2-GDN). This was detected in vascular muscle cells in concentrations 2 to 7 times higher than 1,3-glyceryl dinitrate (1,3-GDN). 99 Neither has a correlation between nitrate tolerance and change in activity been demonstrated for these enzymes. 100 Mitochondrial aldehyde dehydrogenase Chen et al. identified the mitochondrial isoform of alcohol dehydrogenase (ALDH-2) as the key enzyme for bioactivation for GTN in 2002 and were able to conclusively clarify the underlying biochemical mechanism, especially in therapeutically relevant concentrations (<1 µm) for the first time. 101 According to current findings, the bioactivating signal chain varies with low, therapeutic (<1µM) compared to high, pharmacological (> 1 µm) GTN concentration, which is why GTN is currently divided into a low-potency and a high-potency metabolic pathway. This thesis was extended to other nitrates; Vasoactive highly potent nitrates such as the tetranitrate PETN, the trinitrate GTN and pentaerythrityl trinitrate (PEtriN, bioactive PETN metabolite) are bioactivated via the ALDH-2 at low concentrations (so-called highly potent metabolic pathway). 102 The vasoactive low-potency dinitrates ISDN and pentaerithrityl dinitrate (PEdiN, bioactive PETN metabolite), the mononitrates ISMN and pentaerithrityl mononitrate (PEmoN, bioactive PETN metabolite) as well as the above-mentioned tetra- and trinitrate-2 in very high concentrations, however, are subject to ALD-2 - independent bio-activation pathway, the so-called low-potent biotransformation path (Fig. 4). It was shown for the first time that organic nitrates can be metabolized via various bioactivation pathways.

24 16 Introduction Highly Potent Nitrates Low Potent Nitrates M Low Dose High Dose Smooth ER Vasorelaxation Fig. 4: Metabolic pathways for high and low potency nitrates. At therapeutically relevant doses, PETN and GTN are metabolized via ALDH-2 to nitrite and other bioactive metabolites (PEtriN and 1,2-GDN), whereby PEtriN itself has an affinity for ALDH-2. The nitrite is converted further reductively in the respiratory chain (e.g. cytochrome c oxidase) or through acidic disproportionation to NO or a related species (NO x). Low-potency nitrates and high-potency nitrates in high doses are probably bioactivated in the endoplasmic reticulum (smooth ER) by means of cytochrome P450 enzymes, resulting in NO. 94 Highly potent metabolic pathway via ALDH-2 ALDH-2 is a key enzyme in the breakdown of ethanol. In addition to the well-known NAD + - dependent dehydrogenase function, it also has an esterase activity which is important for the bioactivation of highly potent nitrates (Fig. 5). 103 The enzymatic reaction, however, is not a simple ester hydrolysis, but involves a reduction of the nitrate group. In GTN, 1,2-GDN and nitrite were detected in a stoichiometric ratio as metabolites. 100 Although the bioactivation reaction, in contrast to the degradation of ethanol, has a reductive character, NAD + significantly accelerates the reaction rate. Redox-sensitive thiol groups are involved in the reaction in the catalytic center of the enzyme, which mediate the reductive step with the formation of disulfide bridges, resulting in inactivation of ALDH-2. The inactive thiol-oxidized enzyme can be restored by thiol donors such as dithiotreitol (DTT) or 2-mercaptoethanol; mitochondrial dihydrolipoic acid is a natural reactivator in a complex repair mechanism. 104 The conversion of the formed nitrite (oxidation level: +3) into a sgc-activating species has not yet been clarified. The redox-independent formation of

25 Introduction 17 S-nitrosothiols (oxidation state: +3) as intermediates. 105 It is known of these that they serve as a transport and stabilization form of NO (NO carrier) by low molecular weight peptides, which is why in the broader sense EDRF is also regarded as S-nitrosothiol in addition to NO. 106 Ignarro suspected early on that S-nitrosothiols would spontaneously convert to NO with disulfide formation. However, a further reduction through the respiratory chain (cytochrome bc1 complex, cytochrome c oxidase) or disproportionation in the acidic environment of the intramembrane space via nitrous acid as an intermediate is also described. 107,108 An iron-porphyrin-NO complex is also described as a NO-like species, since a nitrite reduction has been described for deoxyhemoglobin. 109 No study, however, demonstrated NO release by these enzymes at therapeutic GTN concentrations (<1µM). R S H O R O N O 1 O R S N O + R OH O R S N R S H O 2 O N O + R S S R Fig. 5: Crystal structure (left) of the bovine ALDH-2 and postulated reaction mechanism (right) in the bioactivation of highly potent, organic nitrates. The structure shows a monomer of a tetramer complex, in the active center there are three thiol groups of the amino acid cysteine ​​(Cys 301-3), one amino acid (presumably Cys 301) is located near the cofactor NAD +. In the first reaction step, a thionitrate intermediate is formed by nucleophilic attack by the thiol on the nitrate nitrogen. In the second step, a further nucleophilic attack by a thiol on the thionitrate follows, with the formation of a disulfide and nitrite as a leaving group, which in turn reacts further (see text). The enzyme function is reactivated by reducing the disulfide bond to the free thiols using mitochondrial dihydrolipoic acid. 100,102 Specific (daidzin, benomyl) and non-specific inhibitors (chloral hydrate, disulfiram, cyanamide) of ALDH-2 as well as high substrate concentrations (acetaldehyde) reduce nitrate-induced vasorelaxation and lowering of blood pressure in rats and rabbits, while acetylcholine or SNP-induced vasorelaxation remains unaffected. 101,102 vascular rings from wild-type mice, which were maximally inhibited with a selective inhibitor of ALDH-2, gave concentration-effect curves which, interestingly, agreed with those of ALDH-2 deficient animals (ALDH-2 - / -). 110 This became the

26 18 Introduction Molecular-biological evidence provided that ALDH-2 has a determining role in GTN bioactivation. In all cases (inhibition, genetic defect, tolerance), however, the inhibitory effect is limited to a maximum of two powers of ten for the highly potent nitrates. Around million people in Asia are affected by a mutation in the ALDH-2 gene (E487K). Current clinical studies have shown that these patients respond less well to GTN. 111 Accordingly, there is a contraindication for GTN with the drugs disufiram, chlorine hydrate and acetaminophenone. Low potency metabolic pathway via? At high nitrate concentrations (> 1µM), NO formation for GTN and ISDN has been described in vivo as well as in vitro, so NO is probably the active principle for this metabolic pathway. 87,112 A non-enzymatic conversion of GTN with thiols (thiosalicylic acid, cysteine, acetylcysteine) does not seem to have any physiological significance, since this reaction was only observed at high, millimolar concentrations. 113 Especially cytochrome P 450 enzymes are held responsible for the low-potency metabolic pathway (Fig. 4). Induction of hepatic CYP isoforms by glucocorticoids markedly increased cgmp and NO formation, 114 while a 3-day GTN infusion resulted in a decrease in hepatic CYP expression. 115 CYP isoforms have also been described for NO formation from GTN (10µM) in human and animal vessels, in particular CYP1A2 had the highest activity. 116, Nitrate tolerance The therapeutic effectiveness of organic nitrates, especially GTN, is weakened with chronic administration. The so-called nitrate tolerance has been described in various forms since the first clinical studies with nitrates and is currently one of the limiting factors in nitrate therapy. In medical databases (e.g. PubMed) around 1000 scientific articles and 200 reviews on nitrate tolerance are kept and are an indication of the relevance of this phenomenon and how extremely complex the underlying mechanism is. 118 Much of the work in the past was primarily devoted to the substance GTN (gold standard); the results were simply transferred to other nitrates in a relatively uncritical manner. From today's point of view, this is inadmissible because the individual nitrates are significant

27 Introduction 19 show differences in pharmacokinetic and pharmacodynamic behavior and are dosed and administered very differently. So they have no matching therapeutic profiles. In particular, organic nitrates can be subject to different bioactivation mechanisms at the molecular level, as explained under Chap. The key to clarifying the different degrees of tolerance phenomena may be found here. The in vivo nitrate tolerance is due to a nitrate-unspecific, neurohumoral counter-regulation that can also occur with other vasodilators (pseudotolerance) and a nitrate-specific vascular tolerance that is based on the mechanism of action (also classic tolerance). A reduction in the vascular effectiveness caused by a certain nitrate compared to endogenously formed (endothelium) or externally supplied by other nitrates or NO donors is referred to as cross tolerance. The in vivo tolerance caused under therapeutic conditions and chronic ingestion must be differentiated from the in vitro tolerance; this is generated in the isolated vessel at comparatively high concentrations. This phenomenon is also known as acute tolerance or, more generally, but less specifically, as tachyphylaxis and is associated with a rapid loss of effectiveness, which is, however, largely reversible. The in vitro tolerance is nevertheless of great interest in experimental pharmacology, since it enables timely assessments of structure-activity or -tolerance relationships to the exclusion of pharmacokinetic and neurohumoral influences with little study effort. All the studies on nitrate tolerance presented in this work deal with in vitro tolerance. In clinical pharmacology, the rebound effect is also common, which is associated with a significant worsening of symptoms compared to the pre-therapy phase in the event of a nitrate pause or the end of therapy. A large number of mechanisms are set in motion as a result: increase in catecholamine, vasopressin, angiotensin and aldosterone concentrations in the plasma. An intravascular increase in volume (lowering of the hematocrit) with nitrate administration is the result and can weaken the preload-lowering effect of nitrates. The nitrate headache, the second significant therapy-limiting side effect of nitrates, is probably closely related to pseudotolerance. Both phenomena are

28 20 Introduction characteristic especially at the start of therapy, very dose-dependent and most pronounced for GTN Classical tolerance Various mechanisms have been postulated as causes of classical nitrate tolerance: impairment of bioactivation, desensitization of sgc, inactivation of cgmp-dependent protein kinases, stimulation of cgmp / camp-degrading ones Phosphodiesterases, impairment of endothelial function up to endothelial dysfunction and increased sensitivity to vasoconstrictors (endothelin, angiotensin II). 94 Many of these factors are closely related and should not be viewed in isolation. Explanatory approaches and theories Basically, two main theories have become established. Early on, Needleman linked the impairment of GTN bioactivation with a depletion of free thiol groups and saw this as the cause of the decrease in sensitivity with chronic GTN administration. The other by Münzel et al. Propagated theory sees the formation of reactive oxygen species (ROS) as the main cause of nitrate tolerance, which leads to oxidative stress in the cardiovascular system and a reduced bioavailability to NO. The vascular NADPH-dependent oxidase and the decoupled eno-synthase are viewed as potential superoxide sources. 119,120 Although evidence has been provided for both theories, many phenomena such as endothelial dysfunction (cross tolerance), increased sensitivity to vasoconstrictors or nitrate-specific differences in tolerance behavior cannot be conclusively clarified and related. Only the Chen et al.The postulated key role of ALDH-2 for the bioactivation of organic nitrates enabled a better understanding of the biochemical processes that can lead to tolerance. In vitro and in vivo, GTN inhibited ALDH-2 and led to a significant decrease in the total activity (approx. 50%) of ALDH-2 in vascular homogenates and isolated mitochondria from rat aortas and hearts. It is of secondary importance whether the ALDH-2 inhibition is triggered by GTN pretreatment, ALDH-2 deficiency or specific inhibition with daidzin; the decrease in the vasodilatory response was relatively consistent in each case.

29 Introduction 21 An increase in reactive oxygen species in endothelial and vascular muscle cells was found in tolerant vessels and could be reduced by ROS radical scavengers such as vitamin C, ebselen or disufide-reducing substances such as dithiotreitol (DTT). Pathophysiologically, this oxidative imbalance leads to a large number of more or less specific reactions (enzyme inhibition, activation, decoupling, changes in expression), which are summarized in Fig. 6. Catecholamines Intravascular volume Vasopressin RAAS Pseudotolerance NOS decoupling Respiratory chain Endothelial dysfunction (cross tolerance) Disturbed bioactivation Vasoconstrictor hypersensitivity Disturbed function of the smooth vascular muscles (nitrate tolerance) Fig. 6: Mechanisms of nitrate tolerance. Neurohumoral counter-regulation (pseudotolerance) usually occurs immediately at the start of continuous, low-dose GTN therapy (RAAS: Renin-Angiotensin-Aldosterone System). 94

30 22 Introduction However, it is unclear by which chemical mechanism GTN induces ROS formation in the mitochondria, but the cause is probably the redox-dependent bioactivation step. As already mentioned in chapter, the metabolism of highly potent nitrates is mediated by cysteine ​​residues (Cys) in the active center of ALDH-2 and these are oxidized to disulfides in the course of a reduction through hydrogen transfer. The enzyme is then in an inactive, non-functional state. New studies by Daiber et al. indicate a connection between ROS formation and impaired function of manganese superoxide dismutase (Mn-SOD). 121 Mn-SOD is the mitochondrial isoform of SOD and catalyzes the breakdown of superoxide to hydrogen peroxide, which in turn is detoxified to water by glutathione peroxidase. Superoxide is permanently produced in the respiratory chain under physiological conditions by misdirected electrons (approx. 1% of the total electron flow), the Mn-SOD therefore plays a key role in protecting mitochondrial respiration from decoupling. Using a Mn-SOD +/- mouse model, it was shown that GTN clearly leads to an increase in vascular oxidative stress in chronic and acute administration. In particular, GTN-tolerant aortas from Mn-SOD +/- deficient mice showed a significant deterioration in sensitivity (EC 50) to both GTN itself (in vitro tolerance) and acetylcholine (cross tolerance). These findings were supported by a decreased P-VASP expression as a marker for the state of the NO / sGC / cGK-I signal chain and an increase in mitochondrial ROS formation by means of chemiluminescence in Mn-SOD +/- - compared to wild-type mice. As a consequence of these disturbed biochemical processes, the ALDH-2 dehydrogenase and esterase functions were also significantly reduced in Mn-SOD +/- mice. Daiber suspected that chronic or acute high-dose treatment with highly potent nitrates leads to exhaustion of the reducing capacity (probably dependent on thiol) and inactivation of ALDH-2. An accumulation of nitrates could contribute to the formation of ROS through interaction with the mitochondrial respiratory chain. It is known that superoxide traps vasodilating species such as NO with the formation of peroxynitrite, resulting in a disturbed NO signal transduction pathway. On the other hand, peroxynitrite can oxidatively modify the Mn-SOD, which triggers the uninhibited formation of superoxide. A vicious circle is set in motion, which appears as nitrate tolerance. By inducing endothelial dysfunction, the long-term prognosis in CHD patients on GTN treatment can be worsened. These biochemical insights into the intracellular process cannot generally be transferred to other therapeutically and non-therapeutically used nitrates.

31 Objective Objective 2.1. Preliminary remarks The officinal nitrates (Kap) presented in the introduction are used in dosages that differ significantly from one another (Table 1). PETN GTN ISDN ISMN structural formula O 2 NO O 2 NO ONO 2 ONO 2 O 2 NO ONO 2 ONO2 O 2 NO OO HO OO ONO 2 ONO 2 Number of nitrate groups Dosage ED: mg TD: up to 240 mg ED: 0.2-2.4 mg TD: up to 15 mg ED: mg TD: up to 120 mg ED: mg TD: up to 120 mg onset of action min 1-3 min min min plasma half-life not known 6 min min 4-6 h duration of action 6-8 h min 4-6 h 8-10 h Table 1: Data on the clinical activity profile of therapeutically relevant and approved organic nitrates. The information on onset of action, plasma half-life and duration of action refer to peroral application for PETN, ISDN and ISMN, and sublingual application for GTN. GTN was recommended for oral administration in the following dosage: ED: 2.5 mg; TD to 15 mg; however, there is no information on the action profile (nitrolingual retard, retard capsule 2.5 mg, obsolete). 36,47,122 ED: single dose; TD: daily dose. Even the dosage of the same drug as that of GTN has often been varied in clinical pharmacological studies and is currently within a wide range depending on the therapy regimen and guidelines. Obviously, the therapeutic dosage of nitrates has no relation to the number of nitrate groups in the molecule. This is remarkable because only the nitrate group causes the desired vascular relaxation. Therefore, an increase in the number of nitrate groups in the molecule should increase the vasodilatory potency, unless other physicochemical or steric influences counteract this.

32 24 Objective So far, little is known about in vitro structure-activity relationships in organic nitrates. Classical organ bath experiments on isolated vessels to record isometric relaxation curves are ideally suited for the comparative characterization of the pharmacodynamics of a large number of different nitrates. With the exclusion of pharmacokinetic parameters or neurohumoral counter-regulation mechanisms, they enable a rapid determination of the vasoactivity, in particular also in comparative parallel experiments. In addition, compared to in vivo measurements (e.g. animal experiments), the test method offers high reproducibility with relatively little equipment. The use of specific inhibitors also allows conclusions to be drawn about the underlying mechanisms of action of the compounds examined. Repeated measurements also allow tolerance phenomena to be recognized. The pulmonary artery of the pig should serve as the vessel type for the comparative qualitative and quantitative assessment of vasoactive connections. In preliminary examinations it showed a high sensitivity to nitrates, it shows an intact endothelial function with a high NO capacity and is relatively easy and quick to dissect from the lungs. In addition, findings from our working group are already available on this type of vessel. 123 Force transducer 10 volt volt 10 bridge amplifier input input arteria pulmonalis of pigs 37 C 37 C organ bath Carbogen writing system Fig. 7: Schematic structure of an organ bath apparatus, further details in publication 1 - Method.

33 Objective Tasks 1. Is there a correlation between the number of nitrate groups in the molecule and their vasoactivity in official organic nitrates in vitro? In the past, individual compounds were only unsystematically tested in preliminary pharmacological examinations for clinical tests; in most cases, neither vessel types nor test methods match in the various publications. An assessment of these results, even of nitrate compounds that have long been known, is therefore only inadequately possible. The aim of the investigations was therefore to test for the first time all therapeutically used organic nitrates (PETN, GTN, ISDN, ISMN) under standardized conditions on one type of vessel (pulmonary artery) and comparatively with regard to the vasodilatory potency in the organ bath. 2. Can a sequence of vasodilatory activity be recognized in the bioactive metabolites of the officinal organic nitrates? The special feature of oligonitrates is that, unlike mononitrates, they form bioactive metabolites. The 3 metabolites pentaerithrityl trinitrate (PEtriN), pentaerithrityl dinitrate (PEdiN) and pentaerithrityl mononitrate (PEmoN) result from the drug PETN. Together with PETN itself, this results in an ideal set of compounds on which the correlation between the number of nitrate groups and vasodilatory potency can be investigated. The pentaerithritol molecule is also symmetrical, enantiomers can also be excluded. The same tests should be carried out on the bioactive metabolites of GTN, namely with 1,2-GDN and 1,3-GDN. The results are highly relevant because it can be assumed that the entire clinical effect of the nitrates is largely determined by the bioactive metabolites. 3. What influence does the vascular endothelium have on the potency of organic nitrates? It is known that arteriosclerosis is associated with endothelial dysfunction, in which, as part of a complex pathophysiological process, not enough nitric oxide (NO) is provided for local regulation of the vasotonus due to decoupling of the endothelial NO synthase (enos). It should therefore be investigated whether

34 26 Objective Shifts in the concentration-activity curve due to damage to the endothelial cell layer or inhibition of endothelial NO synthase can be observed, and whether the vascular endothelium in general influences structure-activity relationships in organic nitrates. 4. Is vascular relaxation generally mediated by a sgc-dependent mechanism? As mentioned in the introduction, the vasodilatory effect of organic nitrates is attributed to a sgc-dependent mechanism (Chap.). In the past, however, there have been repeated reports that indicate, at least in the case of direct NO donors, an independent, as yet unidentified mechanism of action. 124 It should therefore also be checked in the in vitro relaxation studies for all organic nitrates whether their effect is directly mediated by the sgc. 5. Do all organic nitrates consistently lead to in vitro tolerance? Most of the findings with regard to nitrate tolerance were obtained in clinical studies; there are also individual comparative in vitro studies that have not been carried out systematically. In the experimental pharmacological investigations, it should now be determined whether there is a correlation between the number of nitrate groups and the extent of the weakening of the effect after repeated administration (in vitro tolerance) for the available drugs and their bioactive metabolites. The design of the study should be adapted in such a way that the in vitro tolerance is not only achieved by preincubation with matching and comparatively high micromolar amounts of nitrate (e.g. 300µM bolus) as has been the case up to now, but also by preincubation with doses of the same potency (EC 90 and EC 100 ) is induced. In contrast to earlier studies, the different vasoactivity of the organic nitrates examined could be taken into account and more relevant structure-tolerance relationships obtained. 6. Synthesis of novel mononitrate compounds. In order to identify structure-activity relationships in organic nitrates, the question should initially be clarified whether there is a correlation, and if so which, between potency and number of nitrate groups. It should go further beyond that

35 Objective 27 the structural influence of the organic nitrate-bearing residual molecules should be investigated. A larger number of organic mononitrates should therefore be systematically designed, synthesized, structurally and analytically secured. The aim was to vary the nitrate carrier with the aim of high structural divergence in terms of electronic and steric properties. 7. How do novel mononitrate compounds differ in their vasoactivity? The established in vitro methods should be used to systematically and reproducibly test the vasodilatory potency of the new mononitrates. For selected compounds, the extent to which endothelium-dependent relaxation and tolerance development can be demonstrated should be investigated. Since a connection between lipophilicity and active activity was frequently postulated in earlier studies, a logp value should be calculated as a measure of lipopilicity for each tested compound. 8. Possible bioactivation pathways and in vitro tolerance of novel mononitrate compounds? These examinations are to be carried out in cooperation with Dr. habil. Andreas Daiber, Clinic of Johannes Gutenberg University Mainz, II. Medical Clinic, Cardiology. As explained in the introduction (section), organic nitrates can be bioactivated differently from one another. It should be checked whether mononitrates, which were found to be comparatively highly potent in the relaxation studies, are bioactivated via the ALDH-2-mediated, highly potent metabolic pathway. This metabolic pathway has so far only been described for tetra- and trinitrates. The investigations were carried out comparatively on wild-type and ALDH-2 deficient (ALDH2 - / -) mice. In this context, it should also be examined whether selected mononitrates induce tolerance in vitro and, in this context, ROS species. 9. Pharmacological characterization of NO donor and nitrate ß-receptor blocker hybrid molecules. In cooperation with PD Dr. habil. Michael Decker from his own working group, NO donor and nitrate ß-receptor blocker hybrid molecules should be examined with regard to their vascular effects. The synthesized compounds are said to have vasodilating properties of nitroso or nitrate groups with the heart rate and

36 28 Objective to combine the contractility-reducing effect of ß-adrenoceptor blockers. The active activities of the individual components of the hybrid should also be examined. 10. Pharmacological characterization of NONOates, NONOate prodrugs and other NO / NO x donors. In cooperation with Dr. Jörg Konter from his own working group was to determine the vasodilatory potencies and investigate in vitro tolerance for selected NO donors and NO donor prodrugs of the diazeniumdiolate type (NONOate). This should clarify to what extent there is a connection between NO release and vascular activity in these compounds. Independently of this, other NO / NO x donors (nitrosothiols, nitrites) should also be characterized with regard to the time course and reversibility of the vasodilatory effect in comparison to the organic nitrates. 11. Pharmacological characterization of nitrate-NSAID hybrids. This project should be worked on in collaboration with Mrs. Kathrin Lange, a pharmacist. Nonsteroidal anti-inflammatory drugs (NSAIDs or NSAIDs) such as flurbiprofen and indomethacin were linked to bioactive metabolites of the drug PETN in different NSAID: nitrate ratios. These new derivatives can be viewed as gastroportective, anti-inflammatory drugs as well as potential drugs of Alzheimer's disease. To evaluate pharmacological parameters, among other things, their vascular effectiveness should be determined ..

37 Overview of the publications Overview of the publications The following lists all publications that have resulted from the experimental work in the context of the submitted dissertation. The own shares are characterized below. Publications as first author: In publications 1, 3 and 4 I carried out the experimental pharmacological investigations, as well as the evaluation of the data and the creation of the manuscripts. In publication 4 I also synthesized and purified the mononitrates described. Publications as co-author: In publications 2 and 5, the described nitrates were experimentally tested by me with regard to vasorelaxant properties, and I participated in the preparation of the manuscripts. Publication 1 Pharmacological characterization of pentaerithrityl tetranitrate, its nitrate-containing metabolites and other organic nitrates in the isolated pulmonary artery of the pig. King, Andreas; Pietig, Imke; Homann, Alexander; Glusa, Erika; Fricke, Uwe; Lehmann, Jochen. In: ERDMANN, E .; MUTSCHLER, E .; STALLEICKEN, D. (Ed.): Pentaerithrityl Tetranitrat: Evidence-Oriented Therapy Concept for Cardiac Diseases. Steinkopff Verlag, Darmstadt (2004), In this introductory, experimental-pharmacologically oriented work, all therapeutically relevant, organic nitrates are presented for the first time comparing their vasodilatory potency under identical test conditions in vitro. With these simply structured alkyl nitrates there is a strict correlation between the number of nitrate groups in the molecule and their in vitro activity. In addition, it was systematically investigated to what extent there were differences in the effects of these nitrates on vessel sections with damaged endothelial barrier and function. Finally, it was shown that all of the investigated nitrates mediated a via the soluble guanylyl cyclase

38 30 Overview of the publications Induce relaxation. This work laid the foundations for further investigations into structure-activity relationships of new organic nitrates.Publication 2 Synthesis and vasorelaxant properties of hybrid molecules out of NO-donors and the beta-receptor blocking drug propranolol. Decker, Michael; King, Andreas; Glusa, Erika; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters (2004), 14, the subject of this publication are NO donor and nitrate-ß-receptor blocker hybrid molecules, which, thanks to the two different operating principles, reduce blood pressure and reduce oxygen consumption of the myocardium and thus protect against Should lead to heart failure. In this work the synthesis and pharmacological characterization of two structurally different hybrid molecules is presented in comparison to the corresponding individual components. Both hybrid molecules are able to induce complete vasorelaxation. Compared to the respective individual components, however, they have significantly different in vitro active activities. Publication 3 Potency and in vitro tolerance of organic nitrates: partially denitrated metabolites contribute to the tolerance-devoid activity of pentaerythrityl tetranitrate. King, Andreas; Lange, Kathrin; Konter, Jörg; Daiber, Andreas; Stalleicken, Dirk; Glusa, Erika; Lehmann, Jochen. Journal of Cardiovascular Pharmacology (2007), 50, This publication includes, in addition to the therapeutically relevant organic Ni presented in publication 1, the investigation of the in vitro vascular effectiveness of SNAP (S-nitroso-nacetylpenicillamine) and the diazenium diolate PHEPIPERAZI / NO. In particular, an in vitro tolerance model is presented, which for the first time takes into account the different vasoactivity of the various organic nitrates in the implementation of the experiment. The investigations made it clear that the in vitro tolerance is closely related to the number of nitrate groups in the molecule. Particularly with the tetra (PETN) and trinitrates (GTN and PEtriN) a clear weakening was observed after repeated administration. The in vivo tolerance of

39 Overview of the publications 31 PETN is obviously decisively determined by the slow influx of the active and low-tolerance di- and mononitrate metabolites PEdiN and PEmoN. PETN itself, on the other hand, is excreted unmetabolized in large quantities and is very likely not involved in the effect. Publication 4 NO donors. Part 16: investigations on structure-activity relationships of organic mononitrates reveal 2-nitrooxyethylammonium nitrate as a high potent vasodilator. King, Andreas; Roegler, Carolin; Lange, Kathrin; Daiber, Andreas; Glusa, Erika; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters (2007), 17, publication 4 deals with the synthesis and in vitro vasoactivity of organic mononitrates. In the previous investigations it became clear that the structure-giving carbon structure carrying nitrate groups definitely has an influence on the potency. Therefore, the systematic presentation of new organic mononitrates was carried out with the aim of achieving the highest possible divergence of the substituents with regard to electronic and steric properties. Significant differences in the vasodilatory potency were observed within the group of 14 mononitrates; the active activities ranged from 32 to nm. A frequently postulated correlation between lipophilicity and in vitro potency, however, was not observed. Surprisingly, the 2-aminoethyl nitrate nitrate salt (2-nitrooxyethyl ammonium nitrate) turned out to be a strong vasodilatory compound and has practically the same effect as the highly potent trinitrate GTN. The vasoactivity can therefore be controlled by varying the nitrate-bearing molecular residue. Publication 5 NO donors. Part 18: synthesis and vasorelaxant properties of the bioactive metabolites of GTN and PETN. Lange, Kathrin; King, Andreas; Roegler, Carolin; Seeling, Andreas; Lehmann, Jochen. Bioorganic & Medicinal Chemistry Letters. In preparation. Publication 5 describes the synthesis of the bioactive metabolites of GTN and PETN. PEtriN, PEdiN, PEmoN, 1,2-GDN, 1,3-GDN, 1-GMN, 2-GMN are important reference compounds for pharmacokinetic studies of the two drugs.

40 32 Overview of the publications In addition, the vasodilatory properties of these compounds are presented. The postulated correlation between vasoactivity and number of nitrate groups could also be confirmed for the GTN metabolites. However, in this case too, metabolites with the same number of nitrate groups show different relaxation effects, which are closely related to the position of the nitrate group on the carrier molecule.

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