The issue of clarithromycin and increased cardiac deaths #4 - Where are the potential sources of variability?

1. Bioavailability
Regardless of its touted lipophilicity, clarithromycin has a reported average bioavailability of only about 50%. Generally, as a guiding principle, the lower the bioavailability, the greater the potential for variability in systemic availability.

2. Uncertain target site concentrations
There are two associated problems here.

Firstly, clarithromycin has an elimination half life of about 3-5 hours at low doses and 5-7 at higher doses. At a 12 hourly dosing intervals, there will be significant fluctuations in the plasma concentration profile. Even if it is administered at 8 hourly intervals, and if half-life is assumed to be at the high end of the range, say 8 hours, there will be at least a 2 fold fluctuation between peaks and trough. While this may meet the needs of anti-bacterial efficacy (assuming we keep trough levels above MIC), the levels of the peaks may predispose to cardiac toxicity if it is able to inhibit HERG potassium channels. To some extent, we can mitigate the fluctuations by using extended release formulations, but this may be at the expense of even more variability in bioavailability.

[source:http://medlibrary.org/lib/images-rx/clarithromycin-24/img-0238.jpg]

Comparison between normal formulation and extended release formulations

Secondly, since we do not routinely measure either plasma or tissue concentrations, we have little idea if adequate concentrations are being achieved at the target site. Here, there is some more uncertainty. Tissue and cellular concentrations tend to be higher than plasma unbound concentrations, but concentrations in the extra-cellular fluid (where the bugs are) are variable and may be lower than unbound concentrations of clarithromycin. These are functions of variable protein binding and the variable net activities of specific influx and efflux membrane transporters.

Consequent upon the previous two points, the differential effects of clarithromycin on the bacteria and on HERG channels may be variable between individuals not only because they relate to different effect compartments but the latter may relate to heights of the peak while the former to trough concentrations being above the MIC.

[Source:http://www.fda.gov/ohrms/dockets/ac/03/briefing/Image66.gif]

Relationship between QT prolongation ad clarithromycin concentrations

Although the IC50 for clarithromycin on the HERG channel is about a 100 times higher than the MIC, arrhythmic risk is associated with lower extent of inhibition. Hence cardiac risk is seen at much lower IC10 or IC20 concentrations

Added to all these, is the uncertainty contributed by an active 14-OH metabolite of clarithromycin.

3. Inter-individual variability in pharmacokinetics
Clarithromycin is both a substrate and inhibitor of CYP3A4. This metabolic pathway is also responsible to generating the active 14-OH metabolite. Variable CYP3A4 activity therefore results in a variable mix of clarithromycin and its active 14-OH metabolite.

There is a very high extent of variabilty in CYP3A4 activity in any population studied. There are also significant differences in activity between men and women (women generally higher). While there are genetic polymorphisms associated with CYP3A4, no single genetic variant has been able to account for the variability within a population. On the other hand, CYP3A4 is also vulnerable to many food and drug interactions.

To make matters more complicated, clarithromycin inhibits its own metabolism by CYP3A4, and exhibits a non-linear pharmacokinetic profile.

4. Inter-individual variability in susceptibility to QT prolongation
The HERG potassium channel gene is genetically polymorphic and variants may predispose to variable susceptibility to QT prolongation. Added to this is the uncertainty about appropriate dosing regiments between different ethnic populations, who may have different body weights and distributional volumes, as well as different exposures to CYP3A4 food and drug interactions.

5. Variability in microbial susceptibility
Apart from differences in anti-microbial efficacy due to variability in drug permeation to target sites, bacteria do differ in how susceptible they are to concentrations of clarithromycin. While sensitive bacteria generally have MICs in easily achievable range, resistance genes have become more prevalent and differences in bacterial sensitivity has become more common.

6. Compliance issues
One must never forget the variability that may be caused by failure of the patient to medicate according to instructions, leading to highly irregular dosing intervals and therefore variable degree of fluctuations in circulating drug concentrations.


Taking all these uncertainties into consideration, the question is how to ensure the patient gets optimal dosing? Think about it.

[To be continued]

The issue of clarithromycin and increased cardiac deaths #2 - Pharmacology

Clarithromycin is a macrolide bacteriostatic antimicrobial that came onto the market in 1991. It enjoyed considerable success as an orally administrable macrolide, being relatively lipophilic and having a slightly longer elimination half-life. Came off patent about 10 years ago.

It acts by inhibiting bacterial protein synthesis by blocking the ribosomal RNA. Resistance develops as bacteria acquire various resistance genes, such as the plasmid erm (A) gene that confers an ability to methylate the adenine in the binding site.

Clarithromycin can be administered orally with a bioavailability of about 50%. Its permeability across biological membranes is only due in part to its lipophilicity. A significant part of the process depends on a complex interplay between influx and efflux transporters expressed on various membranes. Consequently intra-cellular, and tissue concentrations do not correlate with circulating unbound drug concentrations. Interestingly, tissue interstitial fluid concentrations are lower than free drug concentrations in plasma, but intra-cellular concentrations are to a variably extent much higher than plasma free concentrations.

The protein binding of clarithromycin is about 60-70%. The Volume of Distribution is about 10 L/kg, which is consistent with significant permeability into tissues. Again this increased permeability results not only from lipophilicity but from the complex interplay of influx and efflux transporters, in this case clearly favouring influx.

Clarithromycin is eliminated by both hepatic metabolism and renal elimination. It is extensively metabolized by CYP3A4 (which it also inhibits), to a principal metabolite 14-(R) hydroxyclarithromycin, which is also pharmacologically (less) active. The pharmacokinetics is not linear, and the elimination half-life increases from 3-5 hours at lower doses, to 5-7 hours at higher doses. Tissue concentrations persist for much longer.

Clarithromycin produces a range of adverse reactions, but the one that concerns us for this discussion is with respect to cardiac death. Like many of the macrolides, clarithromycin has an effect on the myocardial delayed potassium rectifier current, leading a prolongation of the QT interval of the ECG. This prolongation of the QT interval is associated with risk of torsades de pointe and a fatal ventricular arrhythmia.

The usual adult dosage is 250-500 mg 12 hourly for 7-14 days.

Clarithromycin is a drug with very interesting pharmacological properties. Give a thought as to how these properties contribute to variability in the clinical response and the risk-benefit ratio particularly with respect to the problem of cardiac death.

(To be continued)

Phytochemicals, pharmacogenetics and pharmacokinetics

When one appreciates the extensive range on interactions that exist between phytochemicals essentially entering our bodies through our diet, and the various processes involved in host protection, it becomes very clear that dietary modifications of pharmacokinetic process should be a relative given in our understanding of drug ADME. In fact it should be a central theme in our understanding of interindividual and interpopulational variability in drug behaviour, rather than just being accorded the occasional consideration as a determinant of drug behaviour.

As a corollary, pharmacogenetics cannot hope to fully explain the pharmacokinetic behaviour of any drug in an individual. Environmental chemicals, largely phytochemicals, modify pharmacokinetic processes according to the genetic constitution of the individual, while the genetic makeup of the individual can only be fully expressed in response to environmental chemical effects.

Which leads us to automatically consider what the epigenetic mechanisms might be, in shaping the PK environment of the individual. Perhaps these might be more important than the occasional loss/gain of function variants that we find in the population.

Polyphenols - another type of phytochemical

Another probably more significant group of phytochemicals are the polyphenols. These are molecules of various sizes but which have more than one phenolic unit in their structure. Examples of polyphenols are flavonoids, lignins and tannins.

One of the oldest functions of polyphenols might be protection against UV damage. But polyphenols tend to have varied and complex biological roles. Some of these roles include antioxidation, cell signalling and insect/herbivore signalling.

The non-flavonoid polyphenol, curcumin, for example, is principle member of a family of co

ngeners found in turmeric.

Turmeric itself is a rhizome and a relative of the ginger. The Chinese call it the 'yellow ginger', and the Malays call it 'kunyit'. It's biological activity is recognized in in many cultures and is listed in many traditional pharmacopoieas. It is best known however for the yellow flavouring used in many curries.

Curcumin is however, actually, poorly absorbed when taken orally. The reason has been attributed to poor absorption and rapid metabolism and elimination. It has been shown to induce apoptosis of cancer cells, and thus thought to have anti-cancer properties in the colon. Some clinical studies have also shown that curcumin taken in gram amounts over a period of time can inhibit CYP1A2 while enhancing CYP2A6. Apparently, when given in these large amounts, significant absorption occurs to enable enzyme inhibition. In vitro work suggest inhibition also of CYP2B6 and CYP3A4. Curcumin is otherwise quite harmless even in large doses and therefore appear to serve only the purpose of discouraging eating of the raw rhizome. Although quite flavourful when cooked with other spices, the raw turmeric root is quite unpalatable due to the bitter and pungent taste of curcuminoids.

Similar to the alkaloids, the polyphenols interact extensively with drug metabolizing enzymes and membrane transporters. These interactions are not limited to direct interactions with the proteins, but are also mediated through interaction with regulatory processes of the various enzyme and transporter genes.

Plant alkaloids and chemodefense

Although many alkaloids have toxicity which is immediate and topical so as to discourage predation, many alkaloids do get absorbed into the predator's body and can therefore produce pharmacological and toxicological effects beyond the point of exposure. Some of these effects are extreme and may cause severe reactions in the predator, again discouraging predation.

Animals learn to stay away from such plants. Alternatively, they develop protective mechanisms against the toxicological effects of the alkaloids. Apart from the biological membranes which provide an initial protective barrier against insoluble and hydrophilic chemicals, many organisms also have evolved protective mechanisms such as the cytochrome P450 enzyme systems and the efflux transport proteins to detoxify and repel the more permeable alkaloids which may be able to escape past the biological membranes. In response, plants, over time, evolve even more complex chemicals to overcome animal defense mechanisms. This plant-animal arms-race create the complex environment which now can be seen to determine pharmacokinetic behaviour of the the drugs we use.

Drug metabolism and drug transport must therefore be seen as component parts of an integrated process to protect animals from the toxicity of plant alkaloids.

Read this interesting account of the mustard oil bomb.

A well known groups of alkaloids are the methylxanthines.

Caffeine: R₁ = R₂ = R₃ = CH₃

Theobromine: R₁ = H, R₂ = R₃ = CH₃

Theophylline: R₁ = R₂ = CH₃, R₃ = H

The three main members are caffeine (1,3,7-trimethyxanthine), theobromine (3,7-dimethylxanthine) and theophylline (1,3-diethyxanthine). Caffeine is found in tea and coffee, while theobromine is the main methylxanthine found chocolate. The methylxanthines are phospohodiesterase inhibitors.

The metabolism of caffeine is shown below. Caffeine has been use as a probe substrate to develop metabolic ratios for CYP1A2 and N-acetytransferase 2.

Clopidogrel - variability in response

Indian Heart Journal. 2008 Nov-Dec; 60(6): 543-7

The use of clopidogrel presents another interesting challenge with respect to the variability in drug response.

Clopidogrel is a a platelet inhibitor, acting through irreversible binding to the P2Y12 purinergic receptor on the platelet membrane; though it is not clopidogrel itself that binds, but the active metabolite. The PK of clopidogrel itself is quite complex. Upon oral administration about 90% of clopidogrel is removed through the action of circulating and hepatic esterases to inactive metabolites. Only about 10-15% gets activated by CYP2C19 and CYP3A4 to the final metabolite that binds to the P2Y12 receptor. As the receptor inactivation is irreversible, the recovery of function is dependent on fresh platelet regeneration from megakaryocytes.

The way clopidogrel produces its action is therefore fraught with all kinds of problems which clearly contributes to the observed variability in therapeutic response. These are potential sources of variability:

a) high first pass and low active metabolite bioavailability

b) variability of CYP3A4 and CYP2C19 activities due to pharmacogenetics and food/drug interactions

c) irreversible binding to receptor

d) temporal delay in onset, as well as in recovery of platelet function

e] variability in rate of platelet recovery.

This extent of variability really points to a crying need for dosages of clopidogrel to be optimized according to some clinical measure of drug response. Unlike the situation with warfarin however, there isn't a universally accepted way of monitoring plate function. Nevertheless, platelet function test is shaping up to become a standard bedside test for this very reason. A recent review by Williams et al (Thromb Haemost 2010; 103: 29–33) is worth a read.

Drug level testing would clearly not be useful as it is not clopidogrel itself but the metabolite that is active. Furthermore the irreversible binding to the platelet purinergic receptor would not allow concentrations of the active metabolite to be useful in predicting the level of platelet inhibition.

Warfarin - variability in response

Frequency distribution of warfarin daily dose requirement

Pharmacogenomics. 2009, 10 (12) :1955-1965

Warfarin presents a very good case study with respect to drug response variability, and the management of the uncertainty that surrounds the therapeutic use of warfarin.

Warfarin inhibits the reductase that recycles warfarin epoxide (Vit K epoxide reductase C1) so that it can be used again in the production of the Vit K dependent clotting factors. Conceptually very simple, but a number of things complicate this schematic. Firstly warfarin is optically active, and the two isomers, R and S warfarin, have different potencies and PK characteristics. The S warfarin has 5 times the potency of R warfarin and so often has been taken to represent the active ingredient of racemic warfarin. This is a convenient over-simplification, and it is by no means true that all warfarin activity is accounted for by only the S isomer. This is further

complicated by the fact that the isomers are metabolized preferentially by different CYP450 enzymes and have different elimination halflives.

S warfarin has quite a long halflife - an average of 40 hours. In some individuals it may be up to or longer than 60 hours. This means that after initiation of dosing, S warfarin doesn't achieve steady-state concentrations until about a week of dosing. Using a loading dose will get you closer to the steady-state concentrations, but will still need 5 halflives to settle into 'steady-state'. To make it worse, this does not even mean that warfarin's anticoagulant effects stabilize after one week. In fact the anticoagulant effects are not just dependent on warfarin kinetics but also on the kinetics of the clotting factors, which have their own halflives of elimination. This means that after warfarin steady-state is reached, some more time is required for the clotting factors, and consequently the fully anticoagulant effect, to settle into 'steady-state'. Simulations suggest that the whole process of anticoagulation may take up to about 2 weeks to reach steady-state.

Practically this means that the sooner you can settle into the correct maintenance dose, the sooner the patient will be at a stable level of anticoagulation. Every time you tweak the dose, it will require another 2 weeks to settle down. This makes dosage optimization particularly problematic.

The main sources of variability for warfarin response may be anticipated to relate to the following:

a] body weight

b] diet (Vit K supply, inhibitors.inducers of CYP enzymes),

c] smoking

d] genetics of CYP enzymes

-particularly CYP2C9 for S-warfarin, but cannot ignore other CYP enzymes involved with R warfarin.

e] genetics of CYP4F2 involved in breakdown of Vit K

f] genetics of Vit epoxide reductase complex 1 (VKORC1)

g] drug interaction with CYP enzymes

What saves the situation for warfarin is that it has an excellent direct measurement of drug response, - the INR (International Normalized Ratio) which directly measures the state of anticoagulation produced by warfarin. The INR allows a very convenient way to adjust warfarin dosages according to a 'target' level of response. This is called a target response strategy. For warfarin, drug level monitoring is of little use because of i) the delay in response because of the clotting factors halflives, ii) the presence of 2 active warfarin isomers, and iii) because there are different sensitivities to warfarin effects because of genetic variants affecting VKORC1.

Though the INR is a useful 'direct' measure of warfarin response, it is in reality only a 'surrogate' measure of the true warfarin efficacy, which is the eventual effect warfarin has in reducing morbidity and mortality associated with thromboembolism, strokes etc. These can only be assessed through monitoring therapeutic outcomes. However, these outcome measures, do not help us in the day to day optimization of the patient's warfarin dose.

Olanzapine and CYP1A2 genotypes

Shirley et al, Neuropsychopharmacology (2003) 28, 961–966

Olanzapine, an atypical antipsychotic agent used in the treatment of schizophrenia, is metabolized to 10- and 4'-N-glucuronide, 4'-N-desmethylolanzapine via CYP1A2. Here is report demonstrating the relationship between (orally administered) olanzapine clearance/F and the metabolic ratio (PMR = 17X/137X) measured using caffeine.

Laika et al, The Pharmacogenomics Journal (2010) 10, 20–29

Notwithstanding the lack of predictive value of CYP1A2 genetics on CYP1A2 activity, here is a study looking at the effect of the presence of CYP1A2*1F allele on olanzapine steady state plasma concentrations, in the presence/absence of inducers such as smoking and carbamazepine.

While both inducers and *1F/*1F genotype showed significant effects on average olanzapine concentrations, the scatter within each group remains very large, and is clearly not explained by either effects.

The Cytochrome p450 enzymes belong to probably the largest gene superfamilies known. Comprising more than 6500 genes, there are 57 enzymes alone in humans, involved with the metabolism of endogenous and exogenous compounds. The original CYP450 gene is a very ancient one, tracing its origins back perhaps 2 billion years. All the known members of the gene superfamily probably arose from this ancient precursor through gene duplications etc.

This vast numbers of members is required at least in part for the metabolism of endogenous substrates, but it is likely that many have developed for the purpose of dealing with environmental toxicants which enter the body through mucosal barriers of the gut, and lungs, but also the skin. As the main route for the entry of environmental chemicals is via oral ingestion, the largest amount of p450 is found in the liver, as well as the intestinal linings.

The largest amount present in the liver belong to the 3A family. However, the abundance of the enzymes does not translate directly to the relatively importance of the enzyme with respect to the metabolism of pharmaceuticals. This is likely because the enzymic isoforms have evolved primarily for environmental chemicals, while pharmaceuticals as a subset of environmental chemicals, are chemicals with a very short and recent history.

The 3 most important families for pharmaceutical detoxification are CYP3A, CYP2D6 and CYP2C.

The recognition of CYP450's role in dealing with environmental chemicals allows us to anticipate that interactions between environmental chemicals (including dietary phytochemicals) and pharmaceuticals at the level of the CYP450 enzymes may be more prevalent than we have previously anticipated.

Drug metabolism

Our current ideas of drug metabolism have been shaped to a large extent by the landmark review by Richard Techwyn Williams in 1947 (see Detoxification Reactions, by RT Williams 1947). Prior to this, detoxification reactions were seen to be applicable mainly to poisons, and molecules which were structurally related to endogenous chemicals. Prof Williams himself wrote that "detoxification reactions exist for natural and structurally related foreign compounds; metabolism unlikely for totally foreign structures".

By 1959 however, the ideas had expanded to include drug metabolism.

Prof Williams went on to propose a schematic for drug metabolism that still applies today. Drug molecules were metabolized via a Phase I set of reactions which included oxidations, reductions and hydrolyses; and then a phase II synthetic reactions. These reactions were not necessarily detoxification reactions but could actually result in the formation of a toxic metabolite.

These series of reactions did however, convert molecules which were lipophilic to metabolites that were hydrophilic and more water soluble. It was also thought at that time, and for a long time after that, that these lipophilic drug molecules were freely permeable across membranes.

These ideas are however, currently being challenged as we increasingly recognize that drug molecules are not free permeable across membranes and do require the involvement of drug transporters. But this is a story for a later telling.