1 Introduction

The standard dose regimen for active pulmonary tuberculosis (TB) consists of an initial 2-month intensive treatment phase with rifampicin, isoniazid, pyrazinamide, and ethambutol, followed by a 4-month continuation phase with rifampicin, isoniazid, and ethambutol [1]. Despite the use of standard dose regimens, weight-banding, and directly observed treatment, the pharmacokinetics (PK) of rifampicin show very high interindividual variability, which may be explained by various factors, including variable oral absorption, pharmacogenetic differences in drug-metabolizing/transporter activities, nutritional status, sex differences, drug–drug interactions, comorbidities such as diabetes, and HIV co-infection [2,3,4]. The high interindividual variability in the PK of rifampicin leads to highly variable systemic exposure, with supratherapeutic plasma concentrations potentially leading to adverse reactions such as liver toxicity, and subtherapeutic plasma concentrations resulting in slow response to treatment and development of drug resistance [5, 6]. Consequently, therapeutic drug monitoring of first-line anti-TB drugs has been proposed to improve treatment outcomes in certain patient groups, such as slow responders, patients with diabetes, and those with HIV co-infection [7, 8]. This commentary focuses on the potential consequences of interpatient variability in plasma binding of rifampicin on its PK and pharmacodynamics (PD).

2 Plasma Protein Binding of Rifampicin: What is Known?

Earlier studies on the plasma binding of rifampicin reported an abnormally wide range of binding values (4–91%) when equilibrium dialysis was used, probably related to methodological problems [9,10,11,12]. In recent studies, the binding of rifampicin to plasma from TB patients receiving the four first-line anti-TB drugs, as measured by ultrafiltration and taking nonspecific binding to the ultrafiltration device into account, was very similar and ranged from approximately 72 to 92% [13,14,15]. Unlike rifampicin, the other first-line anti-TB drugs display a much lower plasma protein binding, and the reported values, although very different between studies, do not exceed 40% [14]. At such low plasma binding, interpatient variability will have only limited consequences for the drug’s PK and PD. Indeed, only for drugs with a relatively high plasma binding, such as rifampicin, can a realistic interpatient variability in binding lead to an important change in the fraction unbound (fu), which could then significantly contribute to the variability in the drug’s PK/PD (Table 1).

Table 1 The effect of interpatient variability in percentage plasma binding of isoniazid (low binding) and rifampicin (high binding) on the unbound fraction in plasma
Full size table

Surprisingly, some fundamental aspects of the plasma protein binding of rifampicin have not yet been resolved. For example, it is not clear to what extent the various protein fractions in plasma contribute to the binding of rifampicin. By using electrophoresis, a higher binding of rifampicin to the γ-globulin compared with the albumin fraction was demonstrated in plasma from TB patients [11, 16]. In another study, the degree of binding of rifampicin was similar in whole plasma compared with aqueous solutions at physiological concentrations of albumin and α1-acid glycoprotein [12]. Using fluorescence spectroscopy, a well-established technique to study drug–protein interactions, it was shown that binding of rifampicin to α1-acid glycoprotein was negligible at therapeutic rifampicin plasma concentrations, but increased at higher concentrations [17]. Therefore, it is still not clear whether only albumin or other plasma proteins significantly contribute to the binding of rifampicin. In addition, conflicting results have been published on the effect of increasing plasma concentrations of rifampicin on its plasma binding [9, 12,13,14,15, 17].

Albumin and α1-acid glycoprotein are the most important drug-binding proteins in plasma [18]. α1-Acid glycoprotein is an acute-phase plasma protein that has a high affinity for basic drugs, but it may also bind to neutral lipophilic molecules and to acidic drugs [19]. Whereas albumin plasma concentrations may be low in TB patients due to malnutrition, α1-acid glycoprotein plasma concentrations have been shown to be abnormally high in TB patients, and to decrease during treatment with first-line anti-TB drugs [20]. Depending on which protein fractions are important, the plasma binding of rifampicin may not be stable in TB patients during treatment with first-line anti-TB drugs, thus potentially contributing to the inter- and intrapatient variability of the PK and PD of rifampicin.

3 Variability in Plasma Protein Binding and Pharmacokinetics of Rifampicin

Rifampicin is primarily eliminated by metabolism, presumably in the liver [10, 11]. The main metabolite, 25-desacetyl-rifampicin, which has been shown to retain approximately 50–100% of the antimicrobial activity of rifampicin against clinical isolates of Mycobacterium tuberculosis, is predominantly eliminated via biliary excretion [21, 22]. Renal excretion of unchanged rifampicin is a relatively unimportant route of elimination; following single-dose administration, the urinary excretion of rifampicin plus 25-desacetyl-rifampicin is < 20%, and decreases following multiple daily dose administration due to enzyme induction [11, 23]. Moreover, renal clearance (CLr) of rifampicin is only approximately 12% of the glomerular filtration rate, indicating that filtration at the glomerulus, which is restricted to the unbound fraction of rifampicin in plasma, is very likely the main mechanism of renal excretion [10]. In addition, rifampicin seems to have a relatively low hepatic extraction coefficient, even after chronic administration, when the enzymes involved in its metabolism are auto-induced [24]. For all drugs that are nearly completely absorbed into the gut wall following oral administration, and are partly eliminated by the liver, the unbound plasma fraction (fu) and hepatic intrinsic clearance (CLh,int) are the driving forces for the hepatic contribution (CLh) to the drug’s plasma clearance (CL) [25, 26]:

$$CL_{\text{h}} = f_{\text{u}} \cdot CL_{\text{h,int}} .$$

Consequently, it is reasonable to assume that rifampicin’s overall plasma CL (CL = CLh + CLr), i.e. both the hepatic and renal component, will be directly related to the unbound fraction of rifampicin in plasma. The plasma CL of rifampicin will therefore be approximately three times higher in a patient having a rifampicin plasma binding of 70% (fu = 0.3) compared with another patient with a plasma binding of 90% (fu = 0.1) (Table 1). As a result, the former patient will have total plasma concentrations that will only be approximately one-third of those in the latter patient, whereas the unbound plasma concentrations will be similar in both patients [26]. In such a situation, PK/PD analysis based on total plasma concentrations of rifampicin will be clearly misleading (see below).

In a recent systematic review, it was shown that based on a total of 21 publications that assessed the effect of HIV co-infection on the PK of rifampicin, most patient groups, both HIV-negative and HIV-positive, did not achieve the generally accepted threshold maximum concentration (Cmax) for rifampicin of 8 μg/ml [3, 27]. In these studies, factors such as sex, age, severity of HIV infection (e.g. CD4 count), nutritional status and supplementation, diarrhea, antiretroviral treatment, and others were assessed for correlation with rifampicin systemic exposure measures (Cmax, area under the plasma concentration–time curve [AUC]). Plasma protein binding of rifampicin was not measured in any of the studies. Serum albumin and α1-acid glycoprotein levels were mentioned in only six and none of the 21 studies, respectively. In some studies, the serum albumin levels were normal in the patient groups investigated, whereas in others, some of the patients had hypoalbuminemia [28,29,30,31]. In two of the studies, rifampicin plasma levels (Cmax, AUC) were substantially lower in patients with low serum albumin levels [32, 33]. The authors of this systematic literature review concluded that the heterogeneity in the systemic exposure to rifampicin remains largely unexplained. In our opinion, it is likely that the variability in plasma binding of rifampicin may have been a contributing factor that deserves more attention in future studies.

In an older study in undernourished TB patients with hypoalbuminemia on rifampicin therapy, the unbound plasma fraction of rifampicin (fu = 0.57) was 2.6-fold higher and the AUC was 2.5-fold lower than in well-nourished control subjects (fu = 0.22) [34]. The urinary recovery of rifampicin was also significantly reduced in undernourished patients, but it was not mentioned by how much. The reduced plasma protein binding of rifampicin and/or the decrease in the oral absorption of rifampicin could explain the significantly reduced total plasma concentrations.

4 Variability in Plasma Protein Binding and Pharmacodynamics of Rifampicin

The antimicrobial activity of rifampicin is concentration-dependent and AUC/minimum inhibitory concentration (MIC) and Cmax/MIC are the PK/PD indices closely related to long-term clinical outcome and the suppression of resistance, respectively [35, 36]. Since only the unbound drug is pharmacologically active, these indices should ideally be expressed in terms of unbound drug exposure, i.e. AUCu/MIC and Cmax,u/MIC [37]. In three recent studies, the unbound fraction of rifampicin in plasma of TB patients receiving the four first-line anti-TB drugs was found to vary approximately two to threefold [13,14,15]. In one of the studies, patients with TB were treated with either rifampicin 10 mg/kg or rifampicin 35 mg/kg combined with standard doses of isoniazid, pyrazinamide, and ethambutol [13]. AUCu and Cmax,u of rifampicin showed a 3.6- and 6.4-fold variation, respectively, for the 10 mg/kg dose, and 2.4- and 1.9-fold variation for the 35 mg/kg dose. Because of this large interpatient variability in the systemic exposure to unbound rifampicin, it is important to express the target PD indices as AUCu/MIC and Cmax,u/MIC rather than AUC/MIC and Cmax/MIC [37]. This concept was recently proposed to select the appropriate vancomycin dose in neonates in whom the unbound fraction of vancomycin (median 0.9) is 1.5-fold higher compared with that in adults (median 0.6) [38].

Finally, from a PK/PD standpoint, it is also important to consider the possible contribution of 25-desacetyl-rifampicin to the in vivo antimicrobial activity following treatment with rifampicin. The plasma concentrations of 25-desacetyl-rifampicin are much lower than those of rifampicin in patients treated with the four first-line anti-TB drugs. For example, Ruslami et al. found mean 25-desacetyl-rifampicin/rifampicin ratios for Cmax and AUC of 0.16 and 0.13, respectively [39]. However, the microbially active part of the total plasma concentrations of 5-desacetyl-rifampicin and rifampicin is the fraction not bound to plasma proteins. No information was found in the literature regarding the plasma protein binding of 25-desacetyl-rifampicin. The plasma protein binding of a drug metabolite is usually lower than that of the parent compound, e.g. the plasma binding of the 25-desacetyl metabolite of rifapentine is 93%, compared with 98% for rifapentine [40]. Consequently, without any information on the plasma protein binding of 25-desacetyl-rifampicin, it is not possible to predict whether it is imperative to measure only rifampicin or rifampicin plus 25-desacetyl-rifampicin plasma levels for PK/PD analysis.

5 Conclusions

To unravel the contribution of plasma protein binding of rifampicin to interpatient variability in the PK and PD of this important first-line anti-TB drug, the following approach is proposed:

  1. 1.

    A standard, validated method should be used to measure the plasma protein binding of rifampicin in TB patients to make a comparison between the results obtained by different laboratories possible. Ultrafiltration may be the best choice because plasma binding of rifampicin based on equilibrium dialysis has shown extreme variability between studies.

  2. 2.

    In view of the fact that the antimicrobial activity of 25-desacetyl-rifampicin against clinical isolates of M. tuberculosis is similar to that of rifampicin, the plasma protein binding of this metabolite should be investigated to decide whether 25-desacetyl-rifampicin significantly contributes to the antimicrobial activity.

  3. 3.

    It is of utmost importance to establish the relative contribution of albumin, α1-acid glycoprotein, and other plasma protein fractions such as lipoproteins to the plasma binding of rifampicin (and possibly 25-desacetyl-rifampicin).

  4. 4.

    When studying the PK of rifampicin, its unbound fraction in plasma should be measured, together with the serum levels of the proteins important for its binding, to prove (or disprove) the effect of plasma protein binding and fluctuations in their serum concentrations on the PK of rifampicin. The association between plasma protein concentrations (albumin, α1-acid glycoprotein, etc.) and systemic rifampicin exposure (Cmax, AUC) should be systematically explored.

  5. 5.

    In view of the large interpatient variability in the fraction of unbound rifampicin, the PK/PD target should be based on unbound rifampicin plasma concentrations (or possibly the unbound plasma concentrations of rifampicin + 25-desacetyl-rifampicin).

“I do not care about protein binding because I always measure free, unbound concentrations”. Nicholas Holford (at the World Conference on Dosing of Antiinfectives: Celebrating the 150th Birthday of Paul Ehrlich, 9–11 September 2004, Nürnberg, Germany).