Sample matrix

Sample matrix

Serum or plasma represent the matrix of choice for therapeutic drug monitoring (TDM), and although they can be used inter-changeably it is preferable to use one or the other. Saliva is a matrix of increasing utility, but only for some AEDs.

In most clinical settings the measurement of total serum concentrations will suffice and indeed most routine methods for measuring AEDs in sera do not discriminate between the component of drug that is free (unbound) and that that is bound to serum proteins. However, because only the free drug is available to move across the endothelium and to equilibrate with the concentration in the interstitial space in the brain where the pharmacological effect is to occur, in certain clinical settings when protein binding is altered, patient management would be best guided by monitoring free serum concentrations.

Settings in which protein binding impairment occurs and therefore monitoring free serum concentrations is appropriate include:

• hypoalbuminemia - eg during pregnancy, old age, liver disease, renal disease, after surgery and many other pathological conditions.
• conditions associated with accumulation of endogenous displacing agents - eg uraemia
• when drugs are administered which compete for serum protein binding sites. If the free fraction increases, the measurement of the total serum concentration will underestimate the amount of free, pharmacologically active, drug and under these circumstances therapeutic and toxic effects will be observed at total drug concentrations which are lower than usual. Because phenytoin and valproic acid are highly protein bound and consequently susceptible to variable binding, free concentrations are most commonly monitored for these drugs.

Saliva has been advocated as an alternative matrix to serum since the 1980s and the increasing interest in free drug concentration monitoring has provided a renewed impetus in saliva monitoring of AEDs.

Sampling time

Knowledge of sampling time and a meticulous dosage history is imperative if TDM is to be used to maximum utility. Sampling should be done at steady-state, which occurs at 4-5 half-lives after starting treatment or a dose change. The table below shows the minimum time one needs to wait to achieve steady-state conditions.

For AEDs with long half-lives such as phenobarbital, zonisamide and ethosuximide, the fluctuation in serum drug concentration during a dosing interval is negligible, and samples can be collected at any time. For most AEDs, however, particularly those with short or relatively short half-lives (eg carbamazepine, valproic acid, gabapentin, levetiracetam, pregabalin, tiagabine, vigabatrin, lamotrigine, and topiramate), it is important to standardize sampling time in relation to dose. Patient non-compliance within a period of 3-4 half-lives before the blood sample is drawn can significantly affect the serum concentration and cause misinterpretation of the result. If blood sampling is undertaken before reaching steady-state following a dose increment, the steady-state serum concentration at that dose will be underestimated. Consequently, if a further dose increase is undertaken, this may eventually result in toxicity for the patient. In the case of carbamazepine, sampling before autoinduction is complete will result in overestimation of the steady-state concentration. This can result in a dose that is sub-therapeutic and patients may continue to have unnecessary seizures.

The ideal blood sampling time for all AEDs is immediately before the next oral dose (trough), but if this is not possible, particularly when attending an out-patient clinic, patients should not be told to delay their morning dose for longer than 2 or 3 hours in the case of AEDs with short (<8 hours) half-lives, and it is then desirable to note the sampling time and the time medication was last ingested. In some cases, two blood samples, for example one taken at the time of trough and a second taken at the expected time of peak (or in conjunction with the appearance of symptoms suggestive of transient concentration-related toxicity) could be valuable to optimize the dosing schedule. During overdose, sampling should be undertaken as soon as the patient presents at casualty but repeated sampling might be necessary, depending on the timing of the overdose.

Saliva samples

There is substantial interest in the use of saliva in therapeutic drug monitoring and indeed saliva can be used as an alternative to blood for the monitoring of some AEDs.

The advantages of saliva

The relative value of saliva sampling, as opposed to blood sampling, may vary in different settings.

Advantages:

• collection of saliva is simple and non-invasive
• procedure may be cheaper than blood drawing
• does not require expertise of drawing blood
• preferred by children and their parents (many children fear blood sampling)
• less stress, fear and discomfort
• avoids complications of infection and thrombosis.
• easier to obtain multiple samples.
• measured concentrations reflect the free (pharmacologically relevant) concentration in blood
• saliva can be collected with minimal patient discomfort and can easily be obtained on multiple occasions
  

Saliva sampling can be particularly useful in paediatric patients and those with disabilities, as its sampling is convenient, painless and non-invasive.

Indications for monitoring antiepileptic drugs in saliva

The indications for monitoring saliva antiepileptic drug concentrations are the same as for blood monitoring namely:

1. When drug therapy is initiated and after dosage adjustments (sampling at trough concentration at steady-state).
2. Suspected therapeutic failure (identify inappropriate dosage, mal-absorption or unusually rapid metabolism [sampling at trough concentration at steady-state]).
3. Signs of clinical intoxication (sampling at peak concentration or when symptoms are present).
4. Suspected drug interaction (sampling at trough concentration).
5. Special risk groups where drug pharmacokinetics is altered (elderly patients, pregnant women and patients with compromised elimination eg renal, hepatic disease [sampling at trough concentration at steady-state]).
6. Special risk groups where assessment of efficacy or toxicity is difficult and where the patient is unable to communicate the effectiveness/toxicity of a particular drug regimen (physically and/or mentally disabled patients [sampling at trough concentration at steady-state]).
7. Suspected non-compliance, which may be responsible for unnecessary and avoidable therapeutic failure (sampling at trough concentration).
8. Suspected drug overdose (sampling as soon as possible).
   

The ideal saliva sampling time for all AEDs is immediately before the next oral dose (trough), but if this is not possible, it is desirable to note the sampling time and the time medication was last ingested.  However, in some situations sampling at trough may be contraindicated (eg overdose, toxicity).  It is generally good practice to undertake saliva sampling at steady-state.

Characteristics of some selected antiepileptic drugs

Carbamazepine

Carbamazepine is a major AED with rather unique characteristics, making it particularly amenable to monitoring.  These characteristics include the formation of a pharmacologically active metabolite (carbamazepine-epoxide), exhibits significant diurnal oscillation in blood concentrations, is associated with both autoinduction and hetero-induction and is commonly associated with pharmacokinetic drug interactions.  Therefore there is a substantial inter-patient variability in the relationship between dose and serum concentrations making monitoring essential.

Many studies on the relationship between saliva and serum carbamazepine and carbamazepine-epoxide have been published.  Saliva concentrations have been observed to be very close to that of free serum concentrations.

Phenytoin

The effective management of patients with epilepsy treated with phenytoin is crucially dependent on monitoring.  This can be attributed to the fact that phenytoin has a narrow therapeutic index, is highly protein bound and exhibits saturable metabolism resulting in disproportionate increases in blood concentration upon dosage escalation.  Furthermore, phenytoin is very loosely bound to plasma proteins and hepatic enzymes so that it is readily displaced from these sites resulting in clinically significant drug interactions that are invariably associated with patient toxicity.

Saliva phenytoin concentrations are significantly correlated with serum  free phenytoin concentrations.  Indeed, the clear benefits of monitoring salivary rather than serum total phenytoin have been shown in patients with uraemia and those receiving concomitant valproic acid therapy.

Phenobarbitone

The usefulness of monitoring phenobarbitone has been limited by the fact that inter-patient variability in the serum phenobarbitone-efficacy/toxicity inter-relationship is rather large.  Nevertheless, monitoring of phenobarbitone is undertaken and many studies have reported excellent correlation between saliva and serum free phenobarbitone concentrations.

Primidone

Because the metabolism of primidone results in the production of phenobarbitone, it is common practice to monitor phenobarbitone during primidone treatment.  The monitoring of primidone along with its pharmacologically active metabolite phenobarbitone is usually useful for ascertaining short-term drug non-compliance.  Primidone concentrations in serum and saliva correlate well and therefore monitoring of primidone in saliva can be useful.

Valproic acid (sodium valproate)

Valproic acid is substantially bound to serum albumin.  Binding is saturable and consequently dependent on it’s free fraction.  Therefore, valproic acid can be considered a drug for which the monitoring of free drug concentrations (salivary concentrations) can be helpful in patient management.  However, saliva valproic acid concentrations do not generally correlate with blood concentrations.

Ethosuximide

Results from numerous studies suggest that saliva can be used instead of serum for monitoring therapy with ethosuximide.

The new antiepileptic drugs

Since 1989 nine new AEDs (vigabatrin, lamotrigine, gabapentin, topiramate, tiagabine, oxcarbazepine, levetiracetam, pregabalin and zonisamide) have been licensed in the UK for the management of epilepsy.  The usefulness of saliva monitoring has been investigated for lamotrigine, gabapentin, levetiracetam, oxcarbazepine (10-OH-metabolite) topiramate and vigabatrin and the evidence suggests that there is good correlation between serum and saliva concentrations.  Tiagabine, pregabalin and zonisamide are measurable in saliva but the relationship between serum and saliva concentrations has not been established to date.

Table of AEDs (pdf), including ideal sampling times, time to steady-state and reference ranges.

Further reading

Liu H, Delgado MR. Therapeutic drug concentration monitoring using saliva samples. Focus on anticonvulsants. Clinical Pharmacokinetics 1999;36:453-470.

Patsalos PN, Froscher W, Pisani F, van Rijn CM. The importance of drug interactions in epilepsy therapy. Epilepsia, 2002;43: 365-385.

Patsalos PN, Berry DJ, Bourgeois BFD, Cloyd JC, Glauser TA,  Johannessen SI, Leppik IE, Tomson T, Perucca E. Antiepileptic drugs – Best practice guidelines for therapeutic drug monitoring: A position paper by the Subcommission on Therapeutic Drug Monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008;49: 1239-1276.