The History of Glyceryl Trinitrate As a Prescription Drug

Home , Amyl nitrite

patches for transdermal delivery and in tablet form. It ARTICLE may also be injected. Th e history of glyceryl trinitrate as a prescription drug

Anthony Butler

Abstract A number of prescribed drugs possessing a nitro group

(-NO2) are vasodilators and are used to relieve the symptoms of angina pectoris. Th e most widely prescribed drug in this group is glyceryl trinitrate (GTN). It was fi rst synthesized by an Italian chemist, Ascanio Sobrero, in 1847. Its clinical use was pioneered by phy- sicians including William Murrell and Sir Th omas Lauder Brunton, and it is now an established remedy for the condition. Its mode of action in vivo was a com- plete mystery until the discovery in the 1980s of nitric oxide (NO) as the naturally occurring messenger molecule that triggers the process of vasodilation. However, exactly how the nitro group in GTN is converted into NO is still uncertain, although the general belief is that an enzyme is responsible.

Introduction Th e nineteenth and twentieth centuries witnessed a Figure 1. Ascanio Sobrero. (Source: University of Torino surge in the number of new compounds made in the Archive) laboratory that were available for testing as possible drugs. Not only were the physical properties (colour, GTN was fi rst synthesised by an Italian, Ascanio So- crystal form, melting point and so on) of new com- brero (1812-1888) around 1847. (Figure 1). He was born pounds recorded, but the discoverer might do a few in the small town of Casale Monferrato in the Pied- simple tests to see whether the compound had any in- mont region of Italy and studied medicine at the near- teresting biological properties. It was quite common for by University of Torino (Turin). He found chemistry a chemist to taste the compound he had made, some- the most interesting part of the course and discontin- thing that would be unthinkable now. Normally, tast- ued the study of medicine. On making this decision ing did not lead anywhere, but in one instance at least he moved, in 1840, to Paris to study with the distin- it led to the development of one of the most frequently guished French chemist Th éophile-Jules Pelouze. Pe- prescribed drugs of all time: glyceryl trinitrate (GTN), louze appears not to have discovered anything of sig- also known as nitroglycerine. It is used to alleviate the nifi cance but is famous for being distinguished. Sobrero symptoms of angina pectoris, and having been used stayed there until the early part of 1843 when, to en- for over 130 years is unlikely to be replaced in the near hance further his chemical understanding, he moved future. How it was discovered and how its use was de- to Giessen to study with the great German chemist Jus- veloped is a tale that spans several countries in Europe. tus von Liebig. Th ere he studied the distillation and de- Angina pectoris is one of the commonest cardio- composition of guajaco (guiacum) resin, a component vascular diseases. Its most conspicuous symptoms are of many a masculine fragrance. However, he was there an agonising chest pain and breathlessness on exertion for only a few months when the opportunity to return or stress, but both disappear on resting. Th e risk fac- to his home city of Turin arose. He was appointed to the tors for stable angina are having an unhealthy lifestyle, post of assistant at the Arsenale and later became lec- including not exercising, smoking, being overweight, turer in applied chemistry at the Technical School and and having a history of heart disease. Its use is predict- started his work on the nitration of glycerine.1 ed to increase as the population ages and as the inci- Th ere are errors in some published accounts of So- dence of diabetes, hypertension and obesity increases. brero’s invention of GTN. For example, Sneader2 refers GTN can be used as a spray for sublingual dosing, in to him as ‘Ascagne Sobrero, a French chemist’, both

6 PHARMACEUTICAL HISTORIAN · 2021 · Volume 51/1 Marsh and Marsh^{3 and Fant4 appear to suggest that credit to Sobrero for the invention of GTN, the latter he made GTN while working with Pelouze in Paris, felt he deserved more recognition. Nobel reappears in while a Wikipedia entry5 states that Pelouze supervised our story later in a somewhat humbler role. this work at the University of Turin. Th e truth emerged during the centennial celebrations for Sobrero’s birth in Biological action of GTN 1912. In Paris he had witnessed the production of gun- Although a full account7 was given in Italian in 1849, cotton by the action of fuming nitric acid on cotton. the fi rst announcement of the invention and proper- Pelouze had tried the same reaction using glycerine in ties of GTN was given in an article in the French sci- place of cotton, but the result was oxidation to give ox- entifi c journal Comptes rendus8 in February 1847. It is alic acid and carbon dioxide. described as an extract from a letter to Pelouze. As was Th ere are easier ways to make oxalic acid. It was at the custom of the time, Sobrero tasted the material he the Technical School in Turin, without any supervi- had made. He placed a ‘very small quantity’ (ce qu’on sion by Pelouze, that Sobrero attempted the nitration peut en prendre en y mouillant) on his tongue, and the of glycerine using – not concentrated nitric acid – but result was a severe headache that lasted several hours. It a mixture of concentrated nitric and sulphuric acids, had the same eff ect on other members of the laboratory which is a nitrating rather than an oxidising agent. He staff . Th is observation was not without signifi cance, al- obtained a compound that reminded him of olive oil though there were many twists and turns on its journey and he called it pyroglycerine, but today we know it as to becoming a treatment for angina pectoris. glyceryl trinitrate. Its systematic name is 1,2,3-trini- Th e fact that GTN caused headaches when ingested troxypropane. He soon came to realise that it was a haz- was of great interest to homeopaths. During the Victo- ardous material, exploding with considerable violence, rian era homeopathy was a popular form of medicine, and he was injured during one experiment. He delayed and when one considers the nauseating and ineff ective making any announcement of his discovery because of medicines prescribed at that time, giving a patient a the hazards involved in the manipulation of GTN. De- solution so dilute that it contained nothing was an at- termining its chemical structure was not without diffi - tractive option. One essential principle of homeopathy culties, but it is now known to be as shown (Figure 2). was similia similibus curantur (like cures like). If GTN caused headaches then it must also be a cure for them. Th e full story of GTN in homoeopathic medicine is re- counted in detail by Fye.9 As a preliminary to its homeopathic use as a treatment for headaches, it underwent the process of Prüfung or ‘proving’ by the prominent homeopath Constantine Hering, under the guidance of Samuel Hahnemann himself, the founder of homeopathic medicine. It was renamed Glonoine and given to a number of believers who reported on its eff ect. Hering Figure 2. Chemical structure of glyceryl trinitrate reported the results10 and concluded that it ‘will cure headaches and other complaints’, but there are no re- Dynamite ports if it having been used in this way. Although ho- Some years later, when information about GTN was meopathy is now largely discredited, the case of GTN generally available, it was taken up by the Swedish ex- represents a benefi cial crossover from homeopathic to plosives entrepreneur, Alfred Nobel. He incorporated allopathic prescribing, as its ‘proving’ provided useful it into a diatomaceous earth (kieselguhr) to give dyna- information on its biological activity and toxicity. mite. Th is proved far safer to handle than GTN itself, although Nobel’s younger brother, Emil, and a student (C.E. Hertzman) were killed by an explosion during the early testing of dynamite in 1864. According to his bi- ographer, this event preyed on Nobel’s mind all his life, although he rarely spoke of it.6 Dynamite is used extensively in civil engineering, and Nobel deeply regretted Figure 3. Chemical structure of isoamyl nitrite its use in warfare. However, it is also one of the products that contributed to Nobel’s fortune and allowed him Amyl nitrite to establish the Nobel Prizes, the highest honour that At about the same time the French chemist Antoine Jé- any scientist can receive. Although Nobel always gave rôme Balard (1802-1876) produced what is known as}

PHARMACEUTICAL HISTORIAN · 2021 · Volume 51/1 7 amyl nitrite but is, in fact, iso-amyl nitrite (Figure 3). showed that inhaling amyl nitrite greatly lowered arte- He began life as an apothecary in Montpellier and end- rial tension in both animals and humans. Among the ed up as professor of chemistry at the Collège de France. young housemen in Edinburgh at that time was Th om- He is most famous for the discovery of bromine (also as Lauder Brunton (1844-1916) (Figure 4). discovered by Carl Jacob Löwig at around the same Brunton was a gifted physician as well as physiolo- time) but, in fact, the invention of amyl nitrite is just gist and noted that the severe pain of angina in his pa- as signifi cant. Th e biological eff ects of inhaling amyl tients could be alleviated by venesection. He reasoned nitrite were studied extensively by the British chemist that it worked because of the lowering of arterial ten- Frederick Guthrie (1833-1886) who noted that there sion. Venesection did not cure the condition but alle- was acceleration in the action of the heart.11 Benjamin viated some of its most conspicuous symptoms. It was Richardson (1828-1896), a London physician, worked inconvenient, and he looked around for an easier way on possible medical applications, noting that it caused to bring about the same end. After consultation with dilation of the capillaries in a frog’s foot. Th is line of his colleague Gamgee he chose the inhalation of amyl research was taken up by a number of medical men in nitrite. He tried it on a number of in-patients, and it Edinburgh, and it was their persistence and skill that was gratifyingly successful in temporarily relieving the gave the world a treatment for the symptoms of angina. discomfort of angina, but the reason for this relief was not clear. Close observation of patients led him to understand angina better.12 During an angina attack the patient’s pulse became small, rapid and thready, but, after the administration of amyl nitrite, it was slower and fuller. He suggested, therefore, that angina was due to excessive contraction of arteries and arterioles and this was why administration of a vasodilator like amyl nitrite had a benefi cial eff ect. He continued to study its clinical uses when – after time spent studying in continental Europe – he was appointed to St Bartholomew’s Hospital in London. Later, the French physiologist and physician Charles-Émile François-Frank (1849-1921) concluded that the action of amyl nitrite was to dilate spe- cifi cally the coronary arteries.13 In his textbook on the circulation Brunton writes: ‘Such a dilating action of amyl nitrite…upon the coronary vessels would easily explain the relief they off er in angina pectoris, even in cases where the blood pressure is normal’.14 Th is is an example of Brunton’s aim of putting drug prescription on a fi rm scientifi c basis rather than relying on empirical reputation. However, amyl nitrite is not the ideal drug, as its eff ect quickly wears off . Th e search Figure 4. Sir Th omas Lauder Brunton. (Source: Wellcome began for another vasodilator, possibly another nitro- Image) compound, but for many years amyl nitrite was regu- larly prescribed. Rather curiously for a man with strong Th e Edinburgh story Scottish antecedents, Brunton was one of a small num- Although a member of a notable Edinburgh family, Ar- ber of Victorians that converted to Islam.15, 16 thur Gamgee (1833-1886) was born in Florence, Italy, Such was the extensive use of amyl nitrite in Vic- where his father had gone in order to learn more about torian medicine that it is mentioned in a number of the care of horses. He studied medicine in Edinburgh novels written at that time. For example, in a Sherlock while other members of the family established a veteri- Holmes story entitled Th e Resident Patient, the detective nary college there. After further study in continental interviews a doctor who specialises in the treatment of Europe in 1870 he was appointed to the post of lec- cataleptic fi ts. In some editions of the story the doctor turer in physiology at Surgeon’s Hall and physician to states that he ‘had obtained good results in such cases the Hospital for Sick Children in Edinburgh. While he by inhalation of nitrate of amyl.’ However, nitrate of was there, by the use of a primitive sphygmograph, he amyl (amyl nitrate) is a very diff erent compound from

8 PHARMACEUTICAL HISTORIAN · 2021 · Volume 51/1 amyl nitrite, having none of its relaxant properties. It large, avoided by keeping the dosage down, but for a is more likely to explode than heal, and so the error is a fair number of patients, they are an unpleasant side ef- signifi cant one. It is noteworthy that the author, Arthur fect in the use of the drug. Conan Doyle, was a qualifi ed doctor.17 Amyl nitrite has By 1892 GTN had become the treatment of choice been used in recent times under the street name ‘pop- for angina relief and was included in the British Phar- pers’. When inhaled it enlarges arteries to the brain macopoeia as an established remedy. As a liquid it is and makes the user feel elated and light-headed. It was not entirely convenient, and Marsh and Marsh3 recount popular amongst participants in the disco scene of the that Murrell asked his friend William Martindale to 1970s and the rave scene of the 1980s. Online pharma- prepare a more portable preparation. He proposed to cies sold amyl nitrite as ‘Liquid Gold’. Th e use of pop- put it into chocolate which could be eaten all at once pers was common among homosexuals, and early in the or nibbled until the pain subsided. Alfred Nobel reap- AIDS pandemic of the 1980s it was suggested that amyl pears in the story at this point, as towards the end of his nitrite was the cause of that condition.18 life he suff ered from angina and was prescribed GTN. A compound that had contributed to his wealth now Th e coming of GTN played an important part in saving his life.20 In the search for a better vasodilator for the treatment of angina, GTN was considered, although why nitro- Why is GTN a vasodilator? compounds had a vasodilating eff ect was still a com- GTN is a remarkable pharmaceutical material for two plete mystery. Th at GTN was bioactive was not in main reasons. Firstly, such has been its record of safe- doubt – Hering and the homeopaths had shown that ty and effi cacy over many years that patients, relatives – but the eff ects observed by investigators when GTN and doctors no longer have concerns about taking what was inhaled varied greatly, and the occurrence of severe is also a highly explosive material. Secondly, for a very headaches was a barrier to its use. A doctor at Westmin- long time no-one knew why it acts as a vasodilator and, ster Hospital in London, William Murrell (1853-1912), in so doing, relieves the symptoms of angina. Th e sec- decided to investigate the matter fully in the hope of ond matter was resolved, partially at least, by a totally clarifying the situation. His fi rst experience is well de- unexpected discovery that was made after GTN had scribed in his own words: been in use for over 130 years. In 1987 two research groups, one in the USA and the other in Britain, report- I determined to try its action on myself. Accordingly, ed that the messenger molecule bringing about vasodi- I obtained some 1 per cent solution. One afternoon, lation by activating the enzyme guanylate cyclase was

whilst seeing outpatients, I remembered I had the the gas nitric oxide NO (not nitrous oxide N2O which bottle in my pocket. Wishing to taste it, I applied is an anaesthetic). the moistened cork to my tongue, and a moment Th is simple, diatomic molecule had never been sug- after, a patient coming in, I had forgotten all about gested as having any physiological role, and this is why it. Not for long … I experienced a violent pulsation the discovery was so unexpected. It is produced in the in my head …. (that) rapidly increased and soon endothelial cells lining the inside of an artery from the became so severe that each beat of the heart seemed amino acid arginine by an enzymatic process. Th e role to shake my whole body…I was afraid the patient of NO in human physiology has been reviewed many would notice my distress, and think I was ill or in- times.21, 22 It was the American physiologist Robert toxicated.19 Furchgot23 who fi rst showed that a messenger molecule was produced in endothelial cells, and it was iden- In spite of the rather alarming nature of the experi- tifi ed as nitric oxide by Sir Salvador Moncada24 at the ment, he persevered with the use of GTN in the treat- Wellcome Laboratories in London and by Lou Ignarro ment of angina. He used a primitive sphygmomanom- in the USA.25 Furchgott and Ignarro received a share of eter to compare the eff ect of amyl nitrite and GTN on the Nobel Prize in 1998, but Moncada was overlooked, a patient’s pulse. With GTN the onset of the pulse in- a decision that is diffi cult to understand. crease is slow but the eff ect is more sustained. Eventu- Th e essential role of nitric oxide in vasodilation ap- ally he showed that GTN was an eff ective treatment for pears to make it easier to understand how GTN might angina relief and was better than amyl nitrite for this be a vasodilator because of the presence of the nitrate purpose. Th is was soon admitted by Brunton and his group, but exactly how that transformation occurs is colleagues when they heard of Murrell’s success. People still far from clearly understood. Th e matter has been varied in their response to GTN and this may explain reviewed at some length by Mayer and Beretta 26 and by some of the earlier confusion. Headaches were, by and Th atcher et al 27 but no fi rm conclusion has emerged.

PHARMACEUTICAL HISTORIAN · 2021 · Volume 51/1 9 Th e conversion of nitrate to nitric oxide involves Endnotes and References a 3-electron reduction and these are diffi cult to bring 1. Guareschi, I. Ascanio Sobrero nei centenario della sua about. Some in vitro experiments suggest28 that GTN nascita. Isis. 1913: 1(3); 351-358. 2. Sneader, W. Drug Discovery: Th e Evolution of Modern reacts with a thiol to give a nitrosothiol (R-S-N=O) Medicines. Chichester, UK: John Wiley & Sons, 1986: 141. but this result has been questioned. Th e intermediacy 3. Marsh, N. and Marsh, A. A short history of nitroglyc- of a nitrosothiol is a sound idea as these rather obscure erine and nitric oxide in pharmacology and physiology. Clinical compounds occur naturally and readily yield nitric ox- and Experimental Pharmacology and Physiology. 2000: 27; 313- ide29 but their formation from GTN in vivo has not 319. been demonstrated. A more likely explanation of the 4. Fant, K. Alfred Nobel. A Biography. New York, Arcade Publishing. 1991: 56-57. transformation is an enzymatic process. Chen, Zhang 5. Ascanio Sobrero – Wikipedia. Accessed 10 February 30 and Stamler suggest that the enzyme involved is mi- 2020. tochondrial aldehyde dehydrogenase which converts 6. Fant, K. Alfred Nobel. (Note 4) 1991: 60-63. GTN into 1,2-glyceryl dinitrate and nitrite. 7. Sobrero, A. Sopra alcuni nuovi composti fulminanti ot- However, nitrite is not a powerful vasodilator and teranti con mezzo dell’azione dell’acido nitrico sulle sostante or- ganiche vegetali. Memorie della Reale Accademia delle Scienze di further enzymatic action would be required. Th is Torino 2nd Series. 1849: 10; 195-201. 31 mechanism has been questioned by Feelisch et al 8. Sobrero, A. Sur plusieurs composés détonants produits who, using a metabonomic approach, examined the avec l’acide nitrique et le sucre, la dextrine, la lactine la mannite global metabolism when GTN was introduced into a et la glycérine. Comptes rendu. 1847: 24; 247-248. rat in vivo. Th ey found extensive S and N-nitrosation 9. Fye, WB. Vasodilator therapy for angina pectoris: Th e in both tissue and red blood cells. Th e extent of nitro- intersection of homeopathy and scientifi c medicine. Journal of the History of Medicine and Allied Sciences. 1990: 45(3); 317-340. sation varied with the dose given. Th ese results paint 10. Hering, C. Glonoine, a new medicine for headache etc. a more complex picture and still leave us in doubt on American Journal of Homeopathy. 1849: 4; 3-9. the mechanism of GTN therapy. Mayer and Beret- 11. Guthrie, F. Contributions to the knowledge of the amyl ta26 also focus on mitochondrial dehydrogenase and group. Journal of the Chemical Society. 1859: 11; 245-252. suggest that is does bring about the 3-electron reduc- 12. Richardson, BW. Report of the physiological action of nitrite of amyl. Report British Association for the Advancement of tion of GTN to nitric oxide without the intermediacy Science. 1864: 34; 120-129. 32 of nitrous acid, a view confi rmed by others. Clearly, 13. Mosso, A., Raichle, ME., and Shepherd, GM. Angelo further research is required. Mosso’s Circulation of blood in the human brain. Oxford: New York: Oxford University Press; 2014. Th e future of GTN 14. Brunton, TL. A Textbook of Pharmacology, Th erapeutics It might be argued that, as long as the patient responds and Materia Medica. Philadelphia, USA: Macmillan, 1885. 15. Geaves, R. Islam in Victorian Britain. Markfi eld: Kube positively, it does not matter that we do not know how Publishing Ltd, 2016: 269. the drug works. Th at might be seen as satisfactory if 16. Gümüş, MS. Why did they become Muslims? Hakȋkat it were not for one factor: GTN has one great failing. Kitâbevi Publications, 2008: Number 17. Within hours of ingestion tolerance emerges, and a rest 17. Doyle, AC. Th e Memoirs of Sherlock Holmes. London: period is necessary before a further dose can be given.33, John Murray and Jonathan Cape, 1974 edition. 18. Duesberg, P., Koehnlein, C., and Rasnick, D. Th e 34 Th ose receiving GTN transdermally from a patch chemical bases of the various AIDS epidemics: recreational usually arrange that the non-responsive period is while drugs, antiviral chemotherapy and malnutrition. Journal of Bio- they are asleep. Once that period is over, a new patch sciences 2003, 28(4); 383-412. works perfectly well. If we knew exactly why GTN is 19. Murrell, W. Nitro-glycerine as a remedy for angina pec- such an eff ective vasodilator, bringing relief to thou- toris. Th e Lancet. 1879: 113(2890); 80-81. sands, we might be able to modify it chemically so that 20. Fant, K. Alfred Nobel. (Note 4) 1991: 302. 21. Moncada, S., Palmer, RMJ., and Higgs, EA. Nitric ox- tolerance does not occur. Th at is the hope. In the mean- ide. Physiology, pathophysiology and pharmacology. Pharmaco- time, to prevent the need for GTN therapy in the fi rst logical Reviews. 1991: 43; 109-142. place, one can use another version of nitrate therapy: 22. Butler, AR. and Williams, DLH. Th e physiological eating plenty of green vegetables.35 role of nitric oxide. Chemical Society Reviews. 1993: 233-237. 23. Furchgott, RF. and Zawadzki., JV. Th e obligatory role of endothelial cells in the relaxation of arterial smooth muscle Acknowledgments by acetylcholine. Nature. 1980: 288; 373-376. I would like to thank Charlotte Barlow for assistance 24. Palmer, RMJ., Ferrige, AG., and Moncada, S. Nitric with the references. oxide release accounts for the biological activity of the endothelium-derived relaxing factor. Nature. 1987: 327(6122); 524-526. Author’s address: Professor Anthony Butler, Flat 1, 80 25. Ignarro, LJ., Byrnes, RE., Buga, GM., and Wood, KS. Argyle Street, St Andrews, KY16 9BU. Email: arb3@ Endothelium-derived relaxing factor from pulmonary artery and st-andrews.ac.uk. vein possesses pharmacologic and chemical properties identical

10 PHARMACEUTICAL HISTORIAN · 2021 · Volume 51/1 to those of nitric oxide radical. Circulation Research. 1987: 61(6); 31. Janero DR., Bryan, NS., Saijo, F. et al. Diff erential 866-879. nitros(yl)ation of blood and tissue constituents during glyceryl 26. Mayer, B. and Beretta, M. Th e enigma of nitroglycer- trinitrate biotransformation in vivo. Proceedings of the National ine bioactivation and nitrate tolerance: news, views and troubles. Academy of Sciences of the USA. 2004: 101(48); 16958-16963. British Journal of Pharmacology. 2008: 155(2); 170-184. 32. Kowaluk, EA., Chung, SJ., and Fung, HL. Nitrite ion is 27. Th atcher, GRJ., Nicolescu, AC., Bennett, BM., and not an active intermediate in the vascular metabolism of organic Toader, V. Nitrates and NO release: Contemporary aspects in nitrates and organic nitrites to nitric oxide. Drug Metabolism and biological and medicinal chemistry, Free Radical Biology and Disposition. 1993: 21(5); 967-969. Medicine. 2004: 37(8); 1122-1143. 33. Fung, HL. and Poliszczuk, R. Nitrosothiol and nitrate 28. Ignarro, LJ. and Gruetter, CS. Requirement of thiols for tolerance. Zeitschrift für Kardiologie. 1986: 75; 25-27. activation of coronary arterial guanylate cyclase by glyceryl trini- 34. Gori, T. and Parker, JD. Th e puzzle of nitrate tolerance: trate and sodium nitrite possible involvement of S-nitrosothiols. pieces smaller than we thought? Circulation. 2002: 106(18); Biochimica et Biophysica Acta. 1980: 631(2); 221-231. 2404-2408. 29. Butler, AR. and Rhodes, P. Chemistry, analysis and bi- 35. Wink, DA. and Paolocci, N. Mother was right: Eat your ological roles of S-nitrosothiols. Analytical Biochemistry. 1997: vegetables and do not spit. Hypertension. 2008: 51(3); 1-3. 249(1); 1-9. 30. Chen, Z., Zhang, J., and Stamler, JS. Identifi cation of the enzymatic mechanism of nitroglycerin bioactivation. Pro- ceedings of the National Academy of Sciences of the USA. 2002: 99(12); 8306-8311.

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