This chapter is probably relevant to Section G8(iii)  of the 2017 CICM Primary Syllabus, which asks the exam candidate to "understand the pharmacology of anti-hypertensive drugs".  It also has some vague relevance to Section G8(v), "understand the pharmacology of anti-anginal drugs", considering that most of the substances discussed here have been used to treat angina. In fact the entire class was developed for this purpose, and their antihypertensive properties only became important in the early 20th century, when physicians finally agreed that hypertension was bad for you. 

Nitric oxide donors like sodium nitroprusside and all the nitrates and nitrites (classically represented glyceryl trinitrate) are the topic of this chapter, with some necessary digressions on the molecular details of their mechanism of action. Fortunately, a disciplined exam-focused discussion would only need to focus on GTN and sodium nitroprusside, as these are the only drugs ever used in the college papers. Unfortunately, "disciplined" and "exam-focused" doesn't really describe the approach which was ultimately taken. Those experiencing time pressure, or otherwise experiencing the natural impatience of the sane, would be advised to limit their review to the grey summary box below and the condensed answers in these past paper questions.

  • Question 2 from the first paper of 2020 (GTN pharmacology)
  • Question 2 from the second paper of 2016 (sodium nitroprusside and GTN)
  • Question 11 from the first paper of 2015 (sodium nitroprusside toxicity)
  • Question 4(p.2) from the first paper of 2010 (metoprolol and GTN)
  • Question 8(p.2) from the second paper of 2008  (sodium nitroprusside and GTN)
  • Question 3(p.2) from the first paper of 2008 (sodium nitroprusside and GTN)

It is somewhat difficult to discuss this class, because the representative members are so different. All attempts to present nitrates as "class rules and their exceptions" has failed, as the drugs in question have so little in common apart from their mechanism of action. As such, it is perhaps better to just look at this good honest comparison table of GTN and nitroprusside:

A Comparison of Glyceryl Trinitrate and Sodium Nitroprusside
  Glyceryl trinitrate (GTN) Sodium nitroprusside
Class Nitrate vasodilator Nitrate vasodilator
Chemistry Organic nitrate Cyanide
Routes of administration Oral, sublingual, intravenous, transdermal (as patch or cream) IV only
Absorption 40% sublingual biavailability (but only 1% orally) 0% oral availability, degraded into cyanide almost immediately on contact with mucosal surfaces
Solubility pKa -5.6; very poor water solubility (and excellent fat solubility). pKa -3.3; minimally fat soluble; highly water soluble
Distribution 3.3L/kg VOD; 60% protein-bound. Virtually no protein binding.
Target receptor Soluble guanylyl cyclase (which is induced by NO) Soluble guanylyl cyclase (which is induced by NO)
Metabolism Metabolised in the liver (by reductase enzymes) but also has extrahepatic sites of metabolism, including vascular cell walls and RBC cell walls. Two main mechanisms of metabolism: spontaneous decomposition into ferrous nitrosyl (FeNO) and five cyanide molecules, or reaction with haemoglobin to form cyanmethemoglobin and four cyanide molecules.
It is also degraded by direct light, which requires opaque storage vessels and special handling techniques.
Elimination Elimination half-life is about 30 minutes Immediate onset of effect (seconds); elimination half-life of 2 minutes
Time course of action Onset of the vasodilatory effect occurs approximately 1 to 3 minutes after sublingual nitroglycerin administration and reaches a maximum by 5 minutes postdose. Effects persist for at least 25 minutes Rapid onset and offset of effect; vasodilation is seen within seconds of beginning the infusion
Mechanism of action Acts as a donor of nitric oxide (NO) which activates guanylate cyclase, resulting in an increase of guanosine 3'5' monophosphate (cyclic GMP) in vascular smooth muscle. This hyperpolarises the membrane by increasing potassium channel conductivity and decreases the availability of inracellular calcium, thereby decreasing the resting tone and contractility of vascular smooth muscle.
Clinical effects Systemic vasodilation - preferentially venodilation and cornary arterial dilation; reduced preload, reduced afterload. Increased intracranial pressure, headache, reflex tachycardia, methaemoglobinaemia (rare). Tolerance develops over sustained use (tachyphylaxis). Systemic vasodilation - balanced venodilation and arterial dilation; reduced preload as well as reduced afterload. Increased intracranial pressure, headache, reflex tachycardia, methaemoglobinaemia. Tolerance develops over sustained use (tachyphylaxis). Significant cyanide toxicity can occur with doses in excess of 2mcg/kg/min
Single best reference for further information FDA PI pamphlet for Nitrostat tablets DBL PI document

In terms of published peer-reviewed material, Münzel & Daiber (2017) has the magic combination of being recent, comprehensive, and free. Unlike many book chapters it is also well-referenced, leaving the reader with the option to ferret out even more information if they choose to. Realistically, the exam candidate could pass all conceivable nitrate questions with their reading confined to this single resource. 

Available nitrate vasodilators

Nitrate vasodilators, or nitrovasodilators/nitrodilators if you prefer unnecessary portmanteaus, all should be called "nitric oxide donors" if we really wanted our nomenclature to have some scientific accuracy. These are some of the oldest drugs in the oldest book. We are talking about the sort of book you might find being thrown out by the medical school library because of extensive mold damage. An excellent paper by Neville and Alexander Marsh (2001) details some of their early history in pharmacology, and is offered here with the expectation that reader who has made it this far down has anything better to do with their time. 

Nitrate Vasodilators
Drug Dates of availability
Amyl nitrite 1864-2018
Glyceryl trinitrate 1870s
Potassium nitrite 1880s-1950s
Sodium nitrite 1906-1950s
Isosorbide dinitrate 1940s
Sodium nitroprusside 1974
Isosorbide mononitrate 1981

In summary, there have been numerous nitrates available to medicine, and many have subsequently become less available because better alternatives were developed. For example, you really can't get amyl nitrite anywhere in Australia these days, except as a solvent. In 2018, the Australian TGA had moved this drug from Schedule 4 (where one might be able to prescribe it to patients for some reason, if one still wanted) to Schedule 9 (prohibited substances), alongside heroin and mescaline. In case you're interested, prior to 1990 it was under Schedule 3, where you could basically buy it over the counter from any morally flexible pharmacist. All the other early 20th century nitrates have either fallen into disuse or had become reclassified. For example, sodium nitrite infusion is still available for the management of cyanide toxicity, but potassium nitrite has completely disappeared from human use by the 1950s,  and even its use as a food additive has fallen into question.

Chemical structure and chemical relatives of nitrate vasodilators

If a well-prepared CICM primary candidate were pushed to the wall and asked to classify this group of vasodilators at gunpoint, they would smoothly respond by organising them into organic and inorganic groups, like so:

  • Organic nitrItes:
    • Alkyl nitrites, eg. amyl nitrite
  • Organic nitrAtes:
    • Glyceryl trinitrate
    • Isosorbide dinitrate
    • Isosorbide mononitrate
  • Inorganic nitrItes:
    • Potassium nitrite
    • Sodium nitrite
    • Sodium nitroprusside
  • Nitric oxide itself, which is cool enough to get its own separate page.

It goes without saying that nitrite (NO2-) and nitrate (NO3-) are rather distinct, and bungling these chemical names in a written exam answer could easily cost you marks if your error is discovered by the sensory filaments of a pedantic examiner. Nitrites and nitrates are ubiquitous in nature, and nitrates are reduced into nitrites (for example, bacterial reduction of organic nitrates is what produces urinary nitrite, forming the basis of that urine dipstick test).

Chronologically, the first nitrite vasodilator discovered by chemists was amyl nitrite, discovered in 1844 by Antoine Balard. However, Balard did nothing medically useful with this substance, and it had spent many years being regarded as "a physiological curiosity". In the 1860s, at a  meeting of the British Association for the Advancement of Science, Benjamin Ward Richardson engaged in probably the first incidence of its recreational use by passing around an ampoule of the substance so that his colleagues could take a hit during his lecture. Its medicinal use was not firmly established until Thomas Lauder Brunton started experimenting with it on the patients of the Edinburgh Royal Infirmary. It clearly helped some of them, which is remarkable because from the records it appears that many of them did not actually have ischaemic coronary disease. Regardless, though completely unscientific (i.e. without a physiological foundation), this technique was a marked improvement in the management of a condition for which there was no existing treatment apart from the medical recommendation to "adopt a tranquil lifestyle".

alkyl nitriite vasodilator chemical structuresAmyl nitrite, the chemical, is an alkyl nitrite. It sounds like, and is chemically related to, amyl nitrate, which is basically a diesel additive without any interesting pharmacological functions. Alkyl nitrites, in contrast, all have some biological effect, which is mainly because of their nitrite group (as the alkyl group is basically some alkane or other, i.e. pharmacologically inert unless you drink a litre of it). This group is made up of highly volatile hydrocarbons (methyl nitrite and ethyl nitrite are gases at room temperature) and all of them have some degree of vasodilator effect when inhaled or absorbed. They also feature a delightful fruity odour. Carr (1975) covers this group in more detail than anybody could ever need, and without revisiting his work, it is only necessary to say that out of all of them, only amyl nitrite has gained any sort of medicinal popularity, likely owing to the risk of methaemoglobinaemia (Titov & Petrenko, 2005) and the difficulty of handling a volatile flammable toxin.

organic nitrate vasodilator chemical structuresMedicinal nitroglycerine falls into the group, as it is basically a mutant three-headed version of the same molecule. Unlike the aforementioned alkyl molecules, this one is a nitrate. It has several chemical relatives, some of which are also vasodilators (eg. erythritol tetranitrate), and others which are also high explosives (toluene trinitrate). The latter does not appear to have any intrinsic vasodilator effects, but is hideously toxic to all the other organs instead.

Chronologically, GTN became available shortly after amyl nitrite was made popular in the mid-to-late 19th century.  We owe its discovery not to Alfred Nobel (who exploited it commercially to revive his family's armaments business which was at risk of going bankrupt after the end of the Crimean War), but to Ascanio Sobrero, an industrial chemist from Turin. This was apparently not a completely random experimental result but a part of a long-term and carefully guided effort to explore the chemistry of organic nitrates. In 1846, he made nitrocellulose, and then nitromannose (from the nitration of mannitol).  To move on to glycerol was the next logical step. Nitroglycerine (more properly, glyceryl trinitrate) was discovered in 1847, and detonated spectacularly in front of members of the Accademia delle Scienze di Torino.  

Even at this early stage we saw hints of a pharmacological effect. Sobrero reported on the intense headache which he had developed when he tasted his violently explosive creation ("for reasons which are not recorded", snicker the Marshes). By 1878, these effects of nitroglycerine were sufficiently well known that Dr. William Murrell was able to recognise them as being similar to those of amyl nitrite, surmising that both could be used to achieve the same clinical endpoints. The advantage of nitroglycerine over amyl nitrite was mainly pharmacokinetic: the duration of the angina-relieving effect was measured in hours rather than minutes. Within a couple of years, it achieved such high acceptance that physicians who were reluctant to use it were reprimanded for being too timid. By 1890, Alfred Nobel's own physicians started feeding him nitroglycerine as a means of ameliorating his angina. 

As far as the medical establishment saw it in the early 20th century, the main problem with these drugs was their short duration of effect. Well, that wasn't the only problem. They were also socially disastrous. To quote a lovingly detailed retrospective by Carr (1985)

"When an ampule or perle of amyl nitrite is crushed, as done by the patient, not only does it produce a mild explosion, but the escaping vapors and disagreeable odor fill the room. The whole procedure is somewhat dramatic, to say the least. "

Unfortunately, that also means that, wherever nitrites were handled by researchers, similar unpleasant environmental disturbances occurred. John C. Krantz, a chemist involved in nitrite research during the 1930s, faced "such violent objections by working colleagues in the department" that he was forced to look for a less obnoxious substance. A search for substances which might have daily dosing and which did not reek abominably first turned up octyl nitrite, which had a much lower vapour pressure and therefore did not tend to explode quite as much with routine use. In pursuit of something even more stable, Kranz and colleagues then borrowed reagents from the lab next door, where sugar alcohol anhydrides were being developed (intended as carbohydrate substitutes for diabetics).  The nitrated anhydride of sorbitol, isosorbide dinitrate, was, therefore, the product of convenience.  There does not appear to have been anything specific about these anhydrides to make them attractive for antihypertensive research - they were simply lying around the lab. 

Isosorbide dinitrate was only the most commercially offspring from this line of investigation. Krantz, Carr and their team produced dozens of molecules (dinitrates and tetranitrates of mannitol, sorbitol, styracitol, polygalitol, erythritol, isomannide, and so on). None of these gained very much popularity, for a variety of largely market-related reasons, but also because some of these molecules had various pharmacological effects, whereas isosorbide passed through the body unchanged.  It was at this stage unknown that the main metabolite of isosorbide dinitrate was isosorbide mononitrate, which was the main active agent responsible for the long duration of action. This daughter molecule would ultimately be the most marketable member of this drug family, made popular by its favourable pharmacokinetics (Irvine et al, 1987). 

organic nitrates - the long acting nitrates of isosorbide

The pursuit of longer-acting vasodilators also led people to consider inorganic nitrites such as sodium nitrite and potassium nitrite. Both were cheap and easily manufactured from commonly available precursors. The advantage of their prolonged action made them attractive, and the lax pharmaceutic standards of the day made them available. By 1906, you could get a 400ml bottle of sodium nitrite from Squibb for one dollar (Butler & Feelisch, 2008). These substances were listed alongside other vasodilator drugs as management options for vasospasm and hypertension. Unfortunately, various unpleasant side effects (hyperkalemia and uncontrollable belching) made these substances unpopular, and they disappeared from the pharmacological armamentarium. 

inorganic nitrates - sodium nitroprussideIt is therefore all the weirder that an inorganic nitrate with much more inconvenient side effects would become so popular, and remain popular for such a long time. Sodium nitroprusside does not cause anything as socially inconvenient as belching, but it certainly does disrupt your mitochondrial electron transport chain. It is not strictly speaking a nitrite or nitrate, but a cyanide - as it contains six C≡N groups.  It only ends up grouped with nitrates and nitrites here because of a common mechanism of effect, as well as because CICM constantly ask us to compare it to GTN. Its real relatives are other members of the cyanide coven, including nightmarishly toxic sodium and potassium salts of cyanic acid, as well as the surprisingly benign iron hexacyanide and the positively pleasant dye Prussian Blue from which cyanide gets its name, and of which you can safely ingest tens of grams to save yourself from radioactive heavy metal poisoning.

For many years nitroprusside has been available as a diagnostic reagent, for example in the detection of urinary ketones. However, nobody had the guts to test it in vivo until 1928, when Chas C. Johnson used it in "definitely effective but not fatal doses" on animals, who developed "intense respiratory stimulation, followed sometimes by tremors and convulsions in all species, and accompanied by nausea and emesis in mammals and birds". In spite of these unpromising effects, the author suggested that it may have some therapeutic use, because its circulatory effects resembled those of sodium nitrite. This was not confirmed until 1955, when Page et al infused it into hypertensive humans and established that it was reasonably safe to do so. They also infused it into rats, administered it orally to sixteen hypertensive patients. The dose, in case you are wondering, was about 30-60mg, four times a day. 

Unfortunately, nitroprusside did not become widely available in medicine until 1974, when a convenient method of mass manufacture had presented itself. It had rapidly become very popular, particularly in critical care environments, and proponents found various ways of protecting the patients from its most fearsome side effects. When they were writing in 1995, Friedrich & Butterworth reported that its popularity remains strong ("although beset with controversy") and that personal communication with an Abbott executive had revealed solid sales figures. The introduction from Hottinger et al (2014) suggests that there is still plenty of enthusiasm for this drug, even though alternatives have become available which are cheaper and which do not produce cyanide as a metabolic byproduct.  

Anyway: all these nitrates nitrites and cyanides are not the only nitric oxide donors around. All sorts of other molecules are available for this purpose, though they are less known for their vasodilatory effects and more for their potential as anticancer agents (Park et a, 2019). N-diazeniumdiolates, S-nitrosothiols and non-nitroprusside metal-nitroxyl complexes are all members of this group. 


Routes of administration and GI absorption

Though it seems absurd to issue this statement following a massive digression on the history of nitrate chemistry, it seems important not to waste any more of the reader's time, and particularly essential to concentrate the attention of the revising exam candidate on only those drugs which they are likely to encounter in exams and in real clinical life. There's really only three. One could, conceivably, include nicorandil in this group, but for various arbitrary reasons this was exiled into a chapter which deals with random miscellaneous antihypertensives.  

Bioavailability of Nitrate Vasodilators
Drug Available routes Absorption Bioavailability
Glyceryl trinitrate Oral, sublingual, transdermal, IV Rapidly and completely absorbed in the GIT less than 1% (massive first pass metabolism)
Isosorbide mononitrate Oral Rapidly and completely absorbed in the GIT 100% bioavailability (no first pass effect)
Sodium nitroprusside IV only 0% absorption - degrades into cyanide immediately on contact with mucosa 0% oral bioavailability

Yes, GTN can be given orally. These old organic nitrates enjoy excellent absorption through every body surface they seem to contact, so much so that munitions factory workers would absorb vast amounts of nitroglycerine transcutaneously, develop massive tolerance to it over their workweek, and would have angina-like withdrawal symptoms on weekends. This is mainly because it has excellent lipid solubility. In the GI tract, glyceryl trinitrate would have no trouble crossing into the circulation, in the same way it easily negotiates the skin and the oral mucosa. Unfortunately, after that it is rapidly degraded by first pass hepatic metabolism, leaving behind less than 1% of the original drug, making it rather unsuitable for oral administration.

What's more remarkable is that oral organic nitrates do have some therapeutic activity. In a trial of oral GTN delivery by a modified-release preparation,  Windsor & Berger (1975) ended up having to give their patients 2.6mg tds (which is 7800 mcg per day - compare that to a 300 mcg sublingual dose). However, it had a clinically measurable effect - incidence and severity of angina were decreased in the treatment group. This, it turns out, was because of some long-acting metabolites (dinitrates and mononitrates). Though these daughter molecules have much lower potency, their concentrations would end up being high enough with large enough oral doses of GTN that it would appear to be having a clinical antihypertensive effect. Noonan & Benet (1986) confirmed this by administering 6500 mcg of oral GTN to their healthy volunteers, and observing that dinitrate persisted for hours in their bloodstream.

Isosorbide mononitrate, by contrast, has both excellent oral absorption and impeccable bioavailability, as might be expected from the culmination of a century-long process of looking for good oral nitrate options. It will absorb anywhere in the small intestine (Kramer et al, 1994) and it undergoes minimal first pass metabolism, being metabolised mainly by renal glucouronidation (it's one of the few drugs where the kidneys act as the dominant organ of biotransformation). In fact, its bioavailability is so predictably good that a concentration-time curve of a 1-hour IV infusion can be superimposed over the pharmacokinetic curve of the same dose administered orally, as shown here in Abshagen (1992):

isosorbide mononitrate oral bioavailability

Sodium nitroprusside, on the other hand, has zero oral bioavailability. It is a vivid red powder which dissolves into a ruddy-brown liquid, and yes - one could conceivably eat or drink raw nitroprusside, but this would not have the desired antihypertensive effect. There's no record of its flavour in chemical literature, but one might surmise that it would probably taste like burning, as its effect on mucosal surfaces is quite corrosive. Indeed, in cases of large scale nitroprusside ingestion, the autopsy demonstrated significant mucosal damage, as if from drinking bleach or acid (eg. Walls, 1948; a case of intentional self-poisoning by a male nurse who had access to laboratory reagents).  The fatal effect was probably due to the elaboration of free cyanide, which represents 44% of the molecular mass of nitroprusside, and which is liberated readily when it reacts with pretty much any sulfhydryl groups on the surface of living cells. In short, immediately upon oral ingestion, most of the nitroprusside would degenerate into hydrogen cyanide, combining with local body water to form Prussic acid.  Even if it didn't do this, the molecule itself is quite polar, and would penetrate lipid bilayers only very reluctantly (Schulz, 1984), which means it is also ill-suited to inhaled or transdermal administration. 

Solubility and protein binding

The general rule is that none of these drugs are particularly protein-bound, except for GTN which is occasionally reported as being transported 60% protein bound. It's hard to study in vivo because GTN and nitroprusside are rather short-lived molecules, and so each variable had to be tracked down to some ancient monograph or chemical manual; the author is lazy and references were not kept fastidiously, but wherever possible links to the sources are offered.

Solubility and Protein Binding of Nitrate Vasodilators
Drug Solubility Protein binding pKa
Glyceryl trinitrate Poorly soluble in water (~ 1mg/ml); highly lipid soluble 11-60%  -5.6
Isosorbide mononitrate Amphoteric: water solubility and lipid solubility are both excellent Minimally protein bound, <5% -3.9
Sodium nitroprusside Minimally lipid-soluble Minimally protein bound -3.3

None of this is particularly important from a clinical perspective, as GTN is not used in a way which might be affected by its protein binding (i.e there would never be a scenario where one would be surprised by the excess of free GTN effect in a hypoalbuminaemic patient, where one could not immediately react by turning down the infusion rate). 

Half-life and duration of effect

Of the available nitrate vasodilators, the majority have short half lives, which makes them excellent immediate relievers of angina, and conveniently titratable antihypertensives. Isosorbide dinitrate and mononitrate are the only exceptions, as they were designed to be taken orally as long-acting antianginal agents.

Solubility and Protein Binding of Nitrate Vasodilators
Drug Time to peak effect Elimination half-life
Glyceryl trinitrate Seconds 2-6 minutes
Isosorbide mononitrate 30-60 minutes 5 hours
Sodium nitroprusside seconds 2 minutes

The actual half-lives here are reasonably close representatives of the duration of action. The only exception may be GTN. The trinitrate of toluene, upon the first step of its biotransformation, turns into a dinitrate and then a mononitrate, both of which have some feeble vasodilatory properties. This means that GTN has some variable persisting effect which might continue for many minutes following the end of the infusion (and perhaps for longer). This is not the case for nitroprusside (off means off, when you stop the infusion), which is one of its major advantages. 

Distribution, metabolism and elimination

Volume of distribution is easy to calculate, but there is a surprising amount of uncertainty in the scholarly literature about the metabolic fate of these substances, which is unexpected for a group of drugs we have been using for over a century.

Solubility and Protein Binding of Nitrate Vasodilators
Drug Volume of distribution Metabolism
Glyceryl trinitrate 3.3L (2.1-4.5) L/kg Multiple sites:
- hepatic (by CYP450 enzymes and glutathione S-transferases)
- peripheral (non-enzymatic conversion by cellular thiol compounds)
Isosorbide mononitrate 2-4 L/kg Renal metabolism, where is is converted to a glucuronide metabolite (which is still active)
Sodium nitroprusside 0.25 L/kg Two main mechanisms of metabolism: spontaneous decomposition into ferrous nitrosyl (FeNO) and five cyanide molecules, or combination if haemoglobin to form cyanmethemoglobin and four cyanide molecules

nit

The metabolism of GTN is probably the most unclear. It seems to be metabolised in the liver, and the metabolic process appears to be one of gradual denitration, whereby nitrate groups are stripped from the molecule until it turns into naked glycerol and gets sucked into the jet engine of glycolysis. What enzyme system performs these denitration steps, remains unclear; but clearly the liver is not the only site of GNT metabolism. Potential enzymatic suspects have been identified, including glutathione S-transferases (GSTs) and CYP450 enzymes. Additionally, some (currently unknown?) metabolic system degrades GTN at the level of the vascular endothelium - "possibly esterases", theorised Torfgård & Ahlner in 1994, but in fact it could be something non-enzymatic, mediated by cellular thiol (sulfhydryl) compounds or directly by haemoglobin and myoglobin. All of these steps ultimately lead to the liberation of nitrite ions (NO2-) which are reduced into NO (nitric oxide).

Organic nitrates - metabolism of GTN

As mentioned above, the metabolism of isosorbide mononitrate mainly seems to happen in the kidneys. There, an enzyme system which remains to be discovered converts some of it into the relatively active isosorbide-5-mononitrate-2-glucuronide (Abshagen et al, 1985, and Abshagen, 1992). Weirdly, some manufacturer propaganda contradicts this directly, claiming without any references that some of the glucouronidation occurs in the liver. Then why no first-pass metabolism, Arrowpharma? Anyway, additionally, as with GTN, the tissues have some capacity to liberate nitrite, leaving behind isosorbide which is presumably excreted in a largely unchanged form because it was marketed quite successfully as an osmotic diuretic in the 1960s. In other words, when you feed somebody an oral dose of isosorbide, that person will return most of the drug to you in the form of urine. Conceivably, some of this anhydride may also be converted to sorbitol and then metabolised to produce energy, but this is not a major mechanism of elimination.

metabolism of isosorbide mononitrate

Now, to nitroprusside. It would probably be fairer to refer to the processes of its biotransformation as decomposition rather than metabolism. This molecule undergoes spontaneous breakdown upon contact with sulfhydryl groups which are present in all cell wall everywhere. They are hanging off most protein molecules, as well as being found in several low molecular weight substances such as glutathione, coenzyme A, thioglycate and the amino acid cysteine (Rothstein, 1971). The products of this breakdown is a free iron nitrosyl (FeNO), which does all the useful work of donating NO2-, and five cyanide molecules. This is what seems to happen in acellular plasma, or any body fluid for that matter. Wherever there are erythrocytes present, nitroprusside will also enter them and react with haemoglobin, producing cyanmethaemoglobin and four cyanide molecules, which then diffuse back out into the bloodstream. This is probably the dominant mechanism, as Vesey et al (1979) report that the in vitro decomposition of nitroprusside was greatly hastened by the addition of red cells.

sodium nitroprusside metabolism

The cyanide then reacts with a variety of substrates. Specifically, Vesey et al (1979) remark that "there are at least six known ways by which HCN may be removed from the blood", including being exhaled as almond-scented breath.  Most of the cyanide ends up reacting with available thiosulfate to form harmless renally excreted thiocyanate, some reacts with methaemoglobin to create cyanmethaemoglobin, and some ends up binding to cytochrome enzymes and other proteins which contain ferrous (Fe3+) iron. As there is a finite supply of thiosulfate in the body, this ends up being the rate-liming step of safe nitroprusside use; most people only have enough of it to sustain a maximum nitroprusside infusion rate of 2mcg/kg/min before their clearance mechanisms are overwhelmed. 

Sodium nitroprusside is also degraded by light, which should probably be mentioned (even though that's not really a clearance mechanism). Blue light causes sodium nitroprusside to degrade into cyanide and nitrosyl groups. For this reason, it usually presents as opaque ampoules and hangs next to the patient in a sinister black bag.

The molecular mechanism of nitrate vasodilator activity

Unfortunately, though nitrate vasodilators became rather popular in medical research and practice in the late 19th century, its pharmacology remained poorly understood, as was the pathophysiology of angina. Horatio Wood, describing a series of nitrate experiments in 1871, commented dourly that:

"The truth is, we have no positive knowledge of the real nature of the disease alluded to. How futile then to attempt to explain the physiological action of a medicine by its effect upon it. This attempting to study physiologically a not understood medicine by its influence upon a not understood pathological condition ... resembles the youthful gambols of a kitten in pursuit of its tail -a circle of useless labour."

Fortunately, in this enlightened era, we have whole book chapters like Van de Voorde & Bogart (2000) or Münzel & Daiber (2017)  dedicated to patiently explaining this subject, and usually there is some mention of it in almost every textbook, which makes this topic interesting to the exam candidate. The mechanism of action of nitrate donors is likely to come up not only as a pharmacodynamic "hows it work" question, but also as a discussion of tachyphylaxis, of which nitrates are a good example.

Ultimately, all of the metabolic pathways of these drugs lead to one final common endpoint, which is the liberation of NO2-. In fact you could just view sodium nitroprusside and GTN as vehicles that deliver NO2- to the vascular endothelium and smooth muscle. NO2- is the substrate of nitric oxide synthase and the precursor for nitric oxide (NO), which directly stimulates soluble guanylate cyclase (this enzyme has a heme molecule which appears to be specifically there to bind NO). 

The rest  is self-explanatory:

  •  Guanylate cyclase catalyses the conversion of GTP to cGMP, which is a potent secondary messenger.
  • cGMP in turn activates several cGMP-dependent protein kinases, of which the most important is protein kinase I (cGK-I).
  • These kinases mediate downstream effects, such as:
    • Inhibition of IP3-dependent calcium release
    • Inactivation of voltage-gated calcium channels
    • Increase in the activity of the sarcolemma calcum pump by phosphorylation of phospholamban
    • Increase in the conductivity of potassium channels, which hyperpolarises the smooth muscle membrane

The net result of these changes is a vascular smooth muscle cell which is hyperpolarised, with decreased intracellular calcium concentration, and many contractile elements rendered quiescent. This cell will not be inclined to contract normally in response to vasoconstrictive stimuli, or to maintain its normal tone. The outcome is vasodilation.

Mechanisms of diminishing effect with continued nitrate use

This is probably not the most interesting topic here, but needs to be mentioned because it is a rich substrate for examiners and question-writers. "Explain the mechanisms responsible for the decreased vasodilator effect of nitrates seen with continued use", they might ask. In fact, multiple mechanisms are involved:

Pharmacodynamic tolerance: the sensitivity of vascular smooth muscle to nitrates decreases with time. This is due to multiple mechanisms, of which the most important one is probably the desensitization of the soluble guanylyl cyclase by S-nitrosylation, which happens due to exposure to S-nitrosocysteine. The nitrosylation of the heme molecule prevents the binding of NO, and disables the entire downstream cascade of effects. This can be reversed by the addition of other sulfhydryl donors, eg. N-acetylcysteine; or at least that seems to work in vitro.  In the living organism, there does not seem to be any such effect (Münzel et al, 1989).

Pharmacokinetic tolerance: the effects of nitrates (well, specifically, GTN) can diminish because of decreased tissue-level biotransformation. One must recall that GTN and its metabolites are nitrate donors who must be separated from their nitrate somehow, usually by some local metabolic process at the vascular endothelium. This metabolism can break down due to oxidative stress. This does not appear to affect other nitrates, nitrites, or nitroprusside.

Physiological tolerance: the effects of nitrate vasodilators diminish over time due to physiological homeostatic mechanisms which are designed to defend blood pressure and regional blood flow. In academic works, this is often referred to as "pseudotolerance" , which is weird because it implies that the tolerance seen here is somehow illegitimate or mistaken. That's not true - the definition of tolerance is "the requirement of higher doses of a drug to produce a given response", which is definitely what happens here. "Physiological tolerance" would be a more accurate term, as this represents a tolerance to the effects of the drug rather than to the drug itself. Physiological tolerance to nitrate vasodilators is mediated by normal cardiac reflex and humoral volume defence mechanisms, which, in summary, are:

  • Activation of the renin-angiotensin-aldosterone system
  • Increase in circulating catecholamine levels
  • Increased sympathetic nervous system tone
  • Increase in vasopressin levels

And thus,

  • Volume expansion (fluid retention)
  • Increased sodium retention
  • Increased heart rate and stroke volume

All of these compensatory mechanisms are clearly counterproductive as they will lead to oedema and increased myocardial oxygen demand, defeating the purpose of vasodilator therapy. As such, these substances are never used as solo agents for the management of anything other than angina. Chronic therapy for congestive cardiac failure or hypertension incorporates nitrates at the fourth and fifth line therapy level, added only where diuretics, beta-blockers and ACE-inhibitors are already in use.

Clinical effects of nitrate vasodilators

"Venoselective" and "arterioselective" is how people sometimes describe nitrates and nitroprusside, alluding to the common perception that GTN and its relatives are largely venodilatory molecules. Occasionally, one might overhear a snippet of conversation regarding the main antianginal effects of GTN being those of preload reduction rather than coronary vasodilation. This is not completely wrong.

Nitrate donors all have a vasodilator effect on all vessels, but the degree of effect differs between different vascular beds, and appears to be somewhat drug-specific. Well-referenced discussions of this appear in Torfgård & Ahlner (1994).  The main points are presented below, though the discussion is more interesting than exam-valuable, and so the readers are invited to just skim the bolded headings. 

GTN appears to be highly venoselectiveRösen et al (1987) found that GTN was about ten times as potent in rabbit femoral veins than it was in rabbit femoral arteries, i.e. one-tenth the dose was required to achieve the threshold of vasorelaxation. The upshot of this is a relative preservation of afterload with preload reduction, provided you stick to sane doses.  Miller (1976) found that peripheral vascular resistance was essentially unchanged following the administration of sublingual nitroglycerine, whereas the LV end-diastolic pressure dropped significantly (from 19 to 8 mmHg).  Stiefel & Kreye (1984) confirmed that this selectivity for venous capacitance vessels is also seen with isosorbide dinitrate and mononitrate.

GTN appears to be highly selective for the coronary and internal mammary arteries. The magnitude of this effect difference was rather striking in a study by Gharabeih & Gross (1984), who found that there was a 10,00-fold difference in the GTN dose which was required to dilate coronary arteries, as compared to femoral arteries. For those starting work in the cardiothoracic ICU and puzzling over why all the patients seem to come back from theatre with some laughable (1ml/hr) GTN infusion - well, this is why. 

Nitroprusside is not especially arterioselective. It just happens to be slighly less venoselective than GTN. If one had to give an honest report on its effects, one would have to describe it as "balanced". The same abovementioned study by Miller (1976) found that nitroprusside both dropped the preload (measured as LVEDP) and afterload (measured as peripheral vascular resistance).  This highlights the main difference between clevidipine and nitroprusside; as nitroprusside vasodilates veins and arteries both, it decreases both the preload and the afterload, which is optimal for a failing dilated ventricle. In contrast, clevidipine is a pure arteriodilator, and preserves preload, which is probably going to be better for the thick meaty ventricle with chronic diastolic dysfunction.

Pulmonary vascular resistance is also affected, though intravenous nitrates are not usually listed as pulmonary vasodilators. However, GTN demonstrably causes pulmonary vasodilation. Pinkerson & Kot (1963) demonstrated that, while the pulmonary vascular resistance definitely decreases, the effect is offset by the increased pulmonary blood flow. The result is that one never really sees a therapeutic effect from this. However, one can often see a substantial negative effect, when GNT vasodilates pulmonary vascular territories which ought to have remained vasoconstricted due to their poor ventilation. The result is a reversal of constructive VQ matching, and an increase in shunt. This "nitroglycerin-induced hypoxia" is a well-known phenomenon (Weygandt et al, 1980)

Other effects of nitrate donors need to be mentioned for full marks, though in all honesty if you've managed to get this far in your short answer question, you are clearly the Antichrist and do not need the extra credit. These effects are:

  • Platelet inhibition
  • Uterine relaxation
  • Headache, mediated by cerebrovascular vasodilation
  • Methaemoglobinaemia

Uses of nitrates in myocardial ischaemia

This little aside is mainly here to answer Question 4(p.2) from the first paper of 2010, which wanted a comparison of GTN and metoprolol in the specific field of managing coronary ischaemia.

Drug properties Desirable effect Adverse effect
Metoprolol decreases heart rate This decreases myocardial oxygen consumption and increases diastolic filling time, improving coronary perfusion The decreased heart rate leads to decreased cardiac output, which in turn may decrease coronary perfusion
GTN causes a compensatory increase in heart rate This ameliorates the negative inotropic effects of decreased preload Myocardial oxygen consumption is increased because of this compensatory effect

GTN decreases preload

This decreases the stroke volume and therefore reduces myocardial oxygen demand. 
Additionally, pulmonary venous congestion is decreased, which decreases the work of breathing, decreasing the demand on cardiac output
The decrease in preload can also lead to decreased cardiac output and hypotension
GTN has some minimal afterload-reducing effect GTN decreases peripheral vascular resistance, decreasing LV afterload and therefore decreasing the myocardial oxygen demand This effect is only seen at high doses, where the benefit may be outweighed by the hypotension and reflex tachycardia
GTN is a selective coronary arteriodilator Even in small doses, GTN can selectively decrease coronary vascular resistance, improving blood flow through narrowed arteries  
Metoprolol decreases contractility This decreases cardiac oxygen demand and improves supply/demand matching during periods of ischaemia. Decreased contractility can lead to decreased cardiac output and hypotension
Metoprolol is  long-acting This allows the effect to be sustained with intermittent dosing,  Adverse effects are difficult to reverse and therapy is harder to titrate
GTN is short-acting This allows rapid control of acute angina, and careful titration of therapy There is little susteined effect. Also, tolerance develops.
Other effects of metoprolol

Metoprolol preserves sinus rhythm. This improves cardiac output by preserving the diastolic "atrial kick".

Metoprolol also suppresses ventricular arrhythmogenicity.

Peripheral β2-mediated vasoconstriction may counterproductively increase afterload

Other effects of GTN   Vasodilation in other vascular beds can give rise to adverse effects (eg. headache).

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