Clinical pharmacology of tocilizumab for the treatment of systemic juvenile idiopathic arthritis

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1 For reprint orders, please contact Clinical pharmacology of tocilizumab for the treatment of systemic juvenile idiopathic arthritis Expert Rev. Clin. Pharmacol. 6(2), (213) Xiaoping Zhang* 1, Peter N Morcos 1, Tomohisa Saito 2 and Kimio Terao 2 1 Hoffmann-La Roche Inc., Nutley, NJ, USA 2 Chugai Pharmaceutical Co. Ltd, Tokyo, Japan *Author for correspondence: Tel.: zhangxiaoping@aol.com Tocilizumab is a humanized anti-il-6 receptor monoclonal antibody approved for the treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis. Biweekly doses of 8 mg/kg for patients who weigh 3 kg and 12 mg/kg for patients who weigh <3 kg produce adequate blockade of IL-6 receptors and normalization of C-reactive protein levels. The mean area under the curve during a 2-week dosing interval, maximum and minimum serum concentrations for both doses were 1341 ± 415 µg day/ml, 245 ± 57.2 and 57.5 ± 23.3 µg/ml, respectively. Tocilizumab pharmacokinetic exposure parameters and clinical end points were comparable between these two dose groups. Proportions of patients achieving clinical end points were comparable across exposure quartiles, suggesting that pharmacokinetic exposures are within the plateau of the exposure response curve. Keywords: Actemra clinical pharmacology C-reactive protein exposure response relationship IL-6 IL-6 receptor pharmacokinetics systemic juvenile idiopathic arthritis tocilizumab Systemic juvenile idiopathic arthritis (sjia) is a type of arthritis that can occur in adolescents and children 16 years of age and younger; it is accompanied by a daily (quotidian) acute fever of 39 C or higher that persists for longer than 2 weeks plus one of the following clinical features: characteristic evanescent erythematous skin rash, lymphadenopathy, pericarditis, pleuritis or hepatosplenomegaly [1]. This disorder in sjia patients is one of the most common physically disabling conditions of their childhood [1], and the associated long-lasting inflammation causes anemia, impairment of growth and development and amyloidosis. Moreover, the acute complication known as macrophage activating syndrome is associated with serious morbidity and sometimes death [2]. Patient age at presentation has a broad peak between and 5 years of age, with 2 years of age the most common [3,4]. However, sjia can occur in children of any age and, rarely, in young adults. Unlike the other subtypes of juvenile idiopathic arthritis (JIA), which have a female preponderance in younger age groups, sjia incidence is similar in both sexes in the Caucasian populations of Europe [5]. The prevalence of JIA is approximately cases per, children; of that, sjia accounts for 7% of the cases in Europe and North America [5 7]. Retrospective reviews and surveys suggest, however, that sjia accounts for a higher proportion of JIA cases in Japan and India (54 and 24%, respectively) [8,9]. This severe inflammatory disease is often refractory to various cytotoxic and immunosuppressive medications. High doses and long durations of corticosteroid therapy are commonly used for suppressing disease activity, often resulting in iatrogenic Cushing-like syndrome, osteoporosis and compression fractures, growth impairment, cataracts and increased susceptibility to infection [1]. Treatment remains challenging because of the limited efficacy of metho trexate [1] and TNF inhibition [11,12]. Efficacy of the IL-1 inhibitor anakinra has been reported in a subset of sjia patients [13 15]. Clearly, unmet needs in sjia therapy have led to efforts to find alternative treatments. Tocilizumab was approved in the EU [16] and in the USA [17] in April and August 211, respectively, for the treatment of patients with sjia. Approved doses in the EU and in the USA are 12 mg/kg for patients weighing <3 kg and /ECP Expert Reviews Ltd ISSN

2 Zhang, Morcos, Saito & Terao 8 mg/kg for patients weighing 3 kg, administered every 2 weeks by intravenous infusion. It should be noted that the approved tocilizumab dose in Japan is 8 mg/kg for all bodyweights every 2 weeks. However, the dosing interval can be shortened if the amelioration of symptoms and the suppression of IL-6 effects, as indicated by CRP level, are insufficient [18]. The objective of the present review is to summarize the available clinical pharmacology data for tocilizumab from four clinical studies conducted in patients with sjia (Table 1) [19]. The first study was an open-label, Phase II, single ascending dose trial of 2, 4 and 8 mg/kg tocilizumab intravenously in the UK and France. This trial was conducted in 18 children with active sjia for at least 3 months while receiving >.2 mg/kg/day prednisolone or its equivalent [2]. The second study was an individual escalating dose trial conducted in Japan in 11 children with active sjia despite treatment with NSAIDs, corticosteroids, cyclosporin or methotrexate [21]. Tocilizumab doses studied were 2, 4 and 8 mg/kg administered intravenously every 2 weeks. The third study was a randomized, double-blind, placebo-controlled, withdrawal Phase III trial conducted in Japan in 56 children undergoing conventional treatment whose disease was refractory to treatment [22]. The tocilizumab dose studied was 8 mg/kg administered intravenously every 2 weeks for all patients. The fourth study is a pivotal global, multicenter, 5-year, Phase III trial (TENDER) conducted in 112 patients with severe sjia with inadequate response to NSAIDs and corticosteroids because of toxicity or lack of efficacy [23,24]. Tocilizumab doses under evaluation were 12 mg/kg for patients weighing <3 kg and 8 mg/kg for patients weighing 3 kg. This trial consisted of three parts: a randomized, doubleblind, placebo-controlled, parallel, two-group, 12-week phase; a single-arm, open-label, 92-week extension; and an open-label, 3-year continuation [23,24]. Part 3 of the study is still ongoing. Efficacy statistics are summarized for the four studies in Table 1. IL-6 & tocilizumab Tocilizumab is a humanized monoclonal antibody that acts as an IL-6 receptor inhibitor [17]. This agent was originally developed by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan) in collaboration with the researchers at Osaka University (Osaka, Japan) [25,26] and is now marketed by Roche Pharmaceuticals (Basel, Switzerland), Genentech Inc. (CA, USA) and Chugai Pharmaceutical Co. Ltd (Tokyo, Japan). The rationale for IL-6 blockade as a potential sjia treatment stems from the pathogenic role of the cytokine in this disease. IL-6 is a monomeric protein that binds to soluble and membrane-bound IL-6 receptors (IL-6Rs) [27]. The soluble receptor (sil-6r) is contained in many body fluids, including synovial fluid and serum, whereas the transmembrane IL-6R (mil-6r) is expressed in a limited number of cell types including hepatocytes, monocytes, macrophages and some lymphocytes. When stimulated by IL-6, the mil-6r binds to gp13, triggering receptor dimerization and intracellular signal transduction. Similarly, when bound to IL-6, sil-6r assembles with gp13 on the cell membrane to transmit intracellular signals [27 29]. Signaling through the IL-6R activates JAK, thus activating either JAK/STAT or mitogen-activated protein kinase pathways. The JAK/STAT pathway has a key role in immune response control, and the acute-phase reactant CRP is a target of this pathway [28 3]. IL-6 is markedly elevated in the blood and synovial fluid in patients with sjia [31], and abnormal IL-6 regulation is responsible for the thrombocytosis and anemia seen in this disease [32]. In vitro studies have documented increased IL-6 production by peripheral blood mononuclear cells from patients with sjia [33]. Signs and symptoms of disease seen in patients with sjia are also attributable to an imbalance in IL-6 homeostasis [34,35]. Chronic overexpression of IL-6 in transgenic mice induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development [36]. In contrast to IL-6, TNF-α levels are not increased in sjia. Thus, IL-6 and IL-6R may play a central role in the induction and progression of sjia and its complications [32]. Tocilizumab binds to both mil-6r and sil-6r, blocking signal transduction pathways through competitive inhibition of IL-6 binding [27]. This has pleiotropic effects on the immune system (including preventing signal transduction by inflammatory mediators that summon B and T cells), inflammation (reducing production of acute-phase reactants) and potentially at other systemic sites where effects of IL-6 have been found (including bone and blood vessels) [29]. Chemistry Tocilizumab was created by selecting a mouse antihuman IL-6R monoclonal antibody with the most potent inhibitory activity in a mouse myeloma cell xenograft system. Humanization was performed by grafting the complementarity-determining region of the mouse antihuman IL-6R monoclonal antibody onto a human IgG 1 κ antibody framework, followed by transfection of both light and heavy chain genes into Chinese hamster ovary cells [11]. The chemical name for tocilizumab is recombinant humanized antihuman IL-6R monoclonal antibody. The H2L2 polypeptide structure consists of two light chains and two heavy chains held together by disulfide bonds. Each light chain and each heavy chain consist of 214 and 448 amino acids, respectively. The molecular weight of tocilizumab is approximately 148 kda. Pharmacodynamics IL-6 plays a role in a number of systemic effects that are relevant to the systemic features of chronic inflammatory diseases. IL-6 is the principal stimulator of liver production of acute-phase proteins, including CRP, serum amyloid A and fibrinogen [37]. In patients with rheumatoid arthritis (RA) treated with tocilizumab, rapid (1 week from first infusion) and sustained normalization of CRP has been observed [38], showing that in RA, CRP production is dependent on IL-6. IL-6 may also be involved in neutrophil recruitment into peripheral blood [39], modulation of neutrophil apoptosis [4] and suppressed neutrophil infiltration [41]. IL-6 is also a pleiotropic cytokine implicated in the immune response, inflammation, hematopoiesis and bone metabolism [39]. Levels of pharmacodynamic markers (IL-6, sil-6r, CRP and erythrocyte sedimentation rate [ESR]) in patients with sjia were studied in the four clinical 124 Expert Rev. Clin. Pharmacol. 6(2), (213)

3 Clinical pharmacology of tocilizumab Table 1. Clinical trial study design and efficacy results for tocilizumab in the treatment of systemic juvenile idiopathic arthritis patients. Study (year) Design Patients and dose ACR3 ACR5 ACR7 ACR9 Ref. Woo et al., 11 of 15 8 of 15 NR NR [2] Europe (5) Yokota et al., Japan (5) Yokota et al., Japan (8) De Benedetti et al. (worldwide including Europe and USA), TENDER (212) Multicenter, OL, single ascending dose, Phase II study Single-center, OL, intrapatient dose escalation, Phase II study from 6 to 14 weeks with 1-year extension phase Multicenter Phase III study consisting of three parts: 6-week OL lead-in phase, 12-week DB, randomized, placebo-controlled phase, OLE phase of 48 weeks 12-week randomized, DB, placebocontrolled, parallel two-arm study; 92-week single-arm OLE; 3-year singlearm, OL continuation TCZ 2, 4 and 8 mg/kg iv.; n = 18; 6 at each dose level. n = 15 included in analysis of efficacy (patients were split into two age groups: 2 5 and 6 18 years) TCZ 2, 4 and 8 mg/kg iv. biweekly for three doses escalating to next dose level; n = 11 Three doses of TCZ 8 mg/kg, every 2 weeks in OL phase: n = 56. Patients aged 2 19 years. Three doses of TCZ 8 mg/kg, every 2 weeks or placebo in DB phase: n = 43; 2 on TCZ (three doses of TCZ 8 mg/kg, every 2 weeks) and 23 on placebo. Patients aged 2 19 years. OLE: n = 5 Week 12: n = 112 (37 placebo, 75 TCZ [bodyweight <3 kg, 12 mg/kg; bodyweight 3 kg, 8 mg/kg]) 2 weeks after third fixed dose: 9.9% OL phase: 51/56 (91%) DB phase: 4/23 (17%) maintained ACR3 and CRP <15 mg/l in placebo group compared with 16/2 (8%) in TCZ group*, OLE phase: 47/48 (98%) At week 12, 85.3% achieved ACR3 and absence of fever compared with 24.3% for placebo* 2 weeks after third fixed dose: 9.9% OL phase: 48/56 (86%); OLE phase: 45/48 (94%) At week 12, 85.3% for TCZ compared with 1.8% for placebo* 2 weeks after third fixed dose: 63.6% OL phase: 38/56 (68%); OLE phase: 43/48 (9%) At week 12, 7.7% for TCZ and 8.1% for placebo* NR [21] NR [22] At week 12, 37.3% for TCZ compared with 5.4% for placebo* *p <.1. ACR3: American College of Rheumatology 3 response; ACR5: American College of Rheumatology 5 response; ACR7: American College of Rheumatology 7 response; ACR9: American College of Rheumatology 9 response; DB: Double blind; iv.: Intravenous; NR: Not reported; OL: Open label; OLE: Open-label extension phase; TCZ: Tocilizumab. [17,23] trials mentioned [2 22,24,42], and the effects of tocilizumab on these pharmacodynamic markers are described here. IL-6 In tocilizumab clinical trials, serum IL-6 levels were determined using a method based on ELISA of human IL-6 (Quantikine, R&D Systems, Inc., MN, USA). In the open-label, single ascending dose study in patients with sjia [2], the mean IL-6 concentration increased dramatically (Figure 1A) and thereafter gradually decreased following single-dose tocilizumab infusions of 2, 4 and 8 mg/kg (six patients in each group). Dose-related increases in mean IL-6 levels of 423, 623 and 752 pg/ml relative to baseline were observed at the first postdose assessment (48 h) in the 2, 4 and 8 mg/kg dose groups, respectively [Data on File]. Larger mean increases were seen in older patients than in younger patients in each dose group. Furthermore, IL-6 increases were transient, with mean values returning to near baseline levels by week 2 for the 2 mg/kg dose and by week 3 for the 4 and 8 mg/kg doses. In the TENDER study, mean ± SD IL-6 concentrations were 129 ± 177 pg/ml (8 mg/kg dose in sjia patients 3 kg) and 133 ± 135 pg/ml (12 mg/kg dose in sjia patients <3 kg) at baseline (Table 2) [19,42] [Data on File]. After multiple-dose tocilizumab infusions of the weight-based 8 and 12 mg/kg doses [43], mean IL-6 concentrations increased rapidly by week 2 (53 ± 657 and 655 ± 882 pg/ml, respectively) and then decreased over time through 52 weeks of treatment (Figure 1B); by week 52, IL-6 levels approached baseline (132 ± 188 and 128 ± 143 pg/ml, respectively) (Table 2)

4 Zhang, Morcos, Saito & Terao A IL-6 (pg/ml) C sil-6r (ng/ml) mg/kg (n = 2 4) 4 mg/kg (n = 5 6) 8 mg/kg (n = 3 5) mg/kg (n = 2 4) 4 mg/kg (n = 5 6) 8 mg/kg (n = 3 5) Figure 1. Serum concentrations of IL-6 and soluble IL-6 receptor after singleand multiple-dose administration for two bodyweight groups through week 52. Error bars indicate standard error of mean. (A & C): Single-dose administration. (B & D): Multiple-dose administration. sil-6r: Soluble IL-6 receptor. (A & C) [Data on File]; (B & D) data taken from [43]. B IL-6 (pg/ml) sil-6r (ng/ml) D 8 mg/kg (n = 29 47) 12 mg/kg (n = 24 41) 8 mg/kg (n = 3 52) 12 mg/kg (n = 26 48) At baseline in the TENDER study [43], mean IL-6 levels in sjia patients were significantly higher than those in healthy subjects (5 ± 3 pg/ml) and in patients with RA (53 ± 53 pg/ml) [42], suggesting that sjia pathogenesis may more heavily involve IL-6 than the RA disease state. In animal studies, it was noted that increases in IL-6 levels induced by anti- IL-6R antibody (e.g., tocilizumab) were mediated by the blockade of IL-6 binding to IL-6R (hence, inhibition of the receptormediated IL-6 clearance pathway) rather than through the induction of IL-6 production [44]. An evaluation of IL-6 kinetics after the administration of tocilizumab to healthy subjects, patients with RA and patients with Castleman s disease demonstrated that the observed increase in free serum IL-6 was due to the inhibition of IL-6R-mediated clearance of IL-6 because of the unavailability of tocilizumab-free IL-6R [45]. The same study demonstrated that the serum levels of free IL-6 during IL-6R inhibition by tocilizumab represents the actual endogenous IL-6 production and the true disease activity of patients with different diseases much better than do serum levels of IL-6 before tocilizumab treatment [45]. These findings suggest that the efficacy of tocilizumab in sjia is due to its binding to IL-6R, thereby blocking receptor binding to IL-6 and hence IL-6 signaling, and is not due to the reduction of IL-6 production. Furthermore, these results support Table 2. Soluble IL-6 receptor, IL-6, erythrocyte sedimentation rate and CRP levels at baseline and at weeks 2, 12 and 52 in the TENDER study. Treatment and visit sil-6r (ng/ml) IL-6 (pg/ml) ESR (mm/h) CRP (mg/l) TCZ 8 mg/kg (BW 3 kg) Baseline 43. ± 12.9 (52) ± (47) 54. ± 36.9 (52) ± (52) Week ± (44) 53.2 ± (4) 11. ± 12.8 (51) 3.2 ± 41.3 (51) Week ± (38) ± (37) 4. ± 3.8 (52).7 ± 1.3 (5) Week ± (34) ± (31) 4. ± 3.1 (41) 2.36 ± (37) TCZ 12 mg/kg (BW <3 kg) Baseline 42. ± 11. (48) ± (41) 6 ± 31.3 (5) ± (5) Week ± (43) ± (35) 9. ± 11.9 (5) 19.9 ± 26.1 (49) Week ± 247. (33) ± (28) 3. ± 2.5 (49) 2.9 ± 18.1 (49) Week ± (26) ± (24) 3. ± 2.7 (35).18 ±.14 (35) Mean ± SD (n) is displayed. BW: Bodyweight; ESR: Erythrocyte sedimentation rate; sil-6r: Soluble IL-6 receptor; TCZ: Tocilizumab. Data taken from [19,42]; [Data on File]. 126 Expert Rev. Clin. Pharmacol. 6(2), (213)

5 Clinical pharmacology of tocilizumab the prominently exaggerated production of IL-6 in sjia and the mechanism of action of tocilizumab. sil-6r Serum sil-6r, as determined by ELISA with the use of the human IL-6sR Quantikine (R&D Systems) kit, is detected as four forms of IL-6R: free from IL-6 or tocilizumab; complexed with IL-6; complexed with IL-6 and sgp13; in an immune complex (IC) with tocilizumab [45,46]. sil-6r levels were studied and increased after tocilizumab administration in three studies conducted in sjia patients [2,21,42]. On single-dose administration of 2, 4 and 8 mg/kg tocilizumab in the Phase II study, sil-6r concentrations increased, reaching 45 ng/ml [Data on File]. Dose-related increases in mean sil-6r (free and bound to tocilizumab) levels were observed at the first assessment (48 h) postdose, with maximal mean increases of 137.5, and 277. ng/ml at week 1 in the 2, 4 and 8 mg/kg groups, respectively (Figure 1C). In the 2 and 4 mg/kg groups, values returned to approximately predose levels by week 2 and 3, respectively, whereas at the highest dose level, sil-6r levels were still elevated at week 3. After multiple-dose administration of tocilizumab every 2 weeks at 8 mg/kg for patients weighing 3 kg and 12 mg/kg for patients weighing <3 kg in the TENDER trial [43], mean sil-6r levels increased rapidly to 515 ng/ml by week 2 and continued to increase until they reached a plateau through week 52 ( ng/ml; Figure 1D & Table 2). The observed changes in sil-6r levels were similar between the two bodyweight-based dosing groups, and the sustained increase in sil-6r levels observed for both groups suggested persistent binding of tocilizumab to sil-6r. sil-6r serum accumulation with increasing numbers of tocilizumab infusions is explained by the prolonged half-life for tocilizumab/sil-6r IC compared with sil-6r alone [45]. Mean sil-6r levels in patients with sjia (42 43 ng/ml; Table 2), patients with RA (~43 ng/ml [47]) and healthy subjects (~35 ng/ml [46, Data on File]) are similar at baseline. Serum sil-6r can be free or bound to IL-6 before tocilizumab administration; after tocilizumab administration, tocilizumab binds to sil-6r to form sil-6r/tocilizumab IC. In healthy subjects after tocilizumab administration, more than 95% of the sil-6r molecules are bound to tocilizumab as long as free tocilizumab concentrations remain 1 µg/ml [45]. When free tocilizumab concentrations fall below 1 µg/ml, levels of sil-6r/tocilizumab IC decrease and those of free sil-6r increase [45]. A CRP (mg/l) C CRP (mg/l) CRP & ESR CRP is synthesized by hepatocytes as a direct effect of IL-6 stimulation [3], and elevated CRP levels are an indication of inflammation [22,42]. As a result of its blockade of IL-6R, tocilizumab is expected to lower CRP levels. ESR is the rate at which red blood cells sediment and is a nonspecific measure of inflammation. In patients with sjia, the ESR is increased [22]; tocilizumab treatment is expected to decrease ESR in this patient population. After single-dose administration of 2, 4 and 8 mg/kg tocilizumab in the Phase II study [2], decreased mean CRP levels were observed at the first assessment time point of 48 h and remained low until week 1 [Data on File]. In addition, mean CRP levels increased between weeks 1 and 2 (Figure 2A). A similar profile was observed for ESR but showed a short time delay in response; the maximum decrease was observed at week 1 (Figure 2B; [Data on File]). These findings support the hypothesis that IL-6 is a key cytokine in the upregulation of genes crucial to the inflammatory process associated with sjia [48]. In the dose escalation study, elevated baseline CRP levels and baseline ESR returned to normal ranges after tocilizumab treatment within 1 week of the first administration of 2 mg/kg tocilizumab [21]. However, eight of the 11 patients mg/kg (n = 2 4) 4 mg/kg (n = 5 6) 8 mg/kg (n = 3 5) 8 mg/kg (n = 37 52) 8 mg/kg (n = 35 5) B Figure 2. Serum concentrations of CRP and erythrocyte sedimentation rate after single-dose (A & B) and multiple-dose (C & D) administration for two bodyweight groups through week 52. (C & D) Error bars indicate standard error of mean. ESR: Erythrocyte sedimentation rate. (A & B) [Data on File]; (C & D) data taken from [43]. ESR (mm/h) D ESR (mm/h) mg/kg (n = 2 4) 4 mg/kg (n = 5 6) 8 mg/kg (n = 3 5) 8 mg/kg (n = 41 52) 12 mg/kg (n = 35 5)

6 Zhang, Morcos, Saito & Terao showed increases in CRP levels and needed additional, increasing doses of tocilizumab (4 and 8 mg/kg) [21]. The same investigators noted, based on their experience with the compassionate use of tocilizumab, that CRP levels increased before manifestations of sjia could be detected clinically [21]. In the Phase III study conducted in Japan, both median CRP concentration and ESR decreased rapidly within 2 weeks and remained low after patients received 8 mg/kg tocilizumab during the open-label, lead-in phase (dosed every 2 weeks for 6 weeks) but increased after patients receiving placebo entered the 12-week, double-blind phase [22]. In the Phase III TENDER study, rapid decreases in mean CRP concentration and mean ESR occurred after the first tocilizumab dose; mean CRP concentration and mean ESR remained low through week 52 (Figures 2C & D, Table 2). Observed changes in CRP and ESR were similar between the two bodyweight-based regimens [43]. Blockade of IL-6 signaling by tocilizumab effectively reduced levels of inflammatory markers to the normal range for long-term treatment. Neutrophil counts In a pooled analysis of neutropenia in RA patients by percentile of tocilizumab exposure during 6 months of treatment, percentages of patients experiencing common toxicity criteria (CTC) grade 3 neutropenia (neutrophils < /l) increased steadily with increasing exposure level, with the highest rates (7%) occurring at >95% of steady state area under the curve (AUC) 28days [38]. In this 6-month analysis, no patients experienced grade 4 neutropenia Neutrophils (1 9 /l) years (n = 2 3) 6 11 years (n = 26 29) years (n = 12 13) years (n = 29 3) ULN: 2 5 years ULN: 6 17 years LLN: years LLN: 2 11 years 1 12 Figure 3. Mean plot for neutrophils from baseline to week 12 by age group. Error bars indicate standard error of the mean. LLN: ( /l for patients 2 12 years of age and /l for patients years of age); ULN: ( /l for patients 2 5 years of age and /l for patients 6 17 years of age). LLN: Lower limit of normal; ULN: Upper limit of normal. Source: [Data on File]. (neutrophils < /l) [38]. It was therefore expected that neutrophil counts would also decrease in sjia patients treated with tocilizumab. Mean neutrophil counts over time from baseline to week 12 in the TENDER study are shown in Figure 3. At baseline, neutrophil counts were high in all age groups and close to the upper limit of normal (ULN) because of the presence of inflammatory disease. After tocilizumab treatment, mean neutrophil levels returned to normal ranges (except at week 6 in the 2-year-old age category) and remained normal until week 12 [Data on File]. During the 12-week controlled phase of the study, a decrease in neutrophils below /l occurred in 7% of the sjia patients [17]. To investigate whether neutrophil count reduction was dependent on tocilizumab pharmacokinetic exposure, mean AUC 2 weeks was summarized by the worst neutrophil CTC grade (grade : lower limit of normal (LLN); grade 1: /l to <LLN; grade 2: 1. to < /l; grade 3:.5 to < /l; and grade 4: < /l). For patients randomly assigned to tocilizumab treatment for 12 weeks in the TENDER study, no patients with available pharmacokinetic data reported neutrophil CTC grade 4. Based on worst neutrophil CTC grade up to week 12, AUC 2 weeks at week 12 was similar between patients with grade (1313 ± 435 µg day/ml; n = 59) and grade 2 (1357 ± 34 µg day/ml; n = 1). Mean AUC 2 weeks for the five patients with CTC grade 3 were higher (1672 ± 272 µg day/ml; n = 5) than for those with CTC grade [43]. In the extension phase of the TENDER study, no patients with pharmacokinetic data reported neutrophil CTC grade 4 up to week 52. Mean observed values were not different among patients with grade (65. ± 29.5 µg/ml; n = 26), grade 1 (71.3 ± 33.9 µg/ml; n = 2), grade 2 (83.8 ± 31 µg/ml; n = 16) and grade 3 neutropenia (62.2 ± 28.1 µg/ml; n = 14) [43]. Pharmacokinetics Pharmacokinetics of tocilizumab was investigated in all four clinical trials identified in Table 1. In the Phase II single-dose study [2], tocilizumab serum concentrations in the 2, 4 and 8 mg/kg groups decreased gradually within 1 week after infusion, with most serum concentrations decreasing to below 1 µg/ml (assay detection limit) 2 weeks after infusion (four of the five patients in the 4 mg/kg group; four of the six patients in the 8 mg/kg group). Tocilizumab serum concentrations were undetectable by 3 weeks after infusion in all patients. C max and AUC inf (mean ± SD) after singledose administration of 8 mg/kg tocilizumab were 96.3 ± 21.2 µg/ ml and 433 ± 137 µg day/ml (Table 3). It was noted that tocilizumab AUC inf for the 8 mg/kg group (11.6 ± 4.5 mg h/ml for patients 2 5 years of age and 9.2 ± 1.7 mg h/ml for patients 6 18 years of age) was two-fold to three-fold lower than AUC inf after single-dose 8 mg/kg administration to adult patients with RA (31.2 ± 7.7 mg h/ml [12]) [Data on File], and total clearance for sjia patients (.78 ±.34 ml/kg for patients 2 5 years of age and.89 ±.18 ml/kg for patients 6 18 years of age) was higher than for adult RA patients (.16 ±.12 ml/kg) for the 8 mg/kg dose [12, Data on File]. As such, patients with sjia may require higher doses or greater dosing frequency, or both, of tocilizumab for successful treatment. Differences in total clearance between sjia and RA may occur because bodyweight-based dosing overcorrects 128 Expert Rev. Clin. Pharmacol. 6(2), (213)

7 Clinical pharmacology of tocilizumab Table 3. Pharmacokinetic parameters from systemic juvenile idiopathic arthritis patients across various studies and from rheumatoid arthritis Phase III studies. PK parameters TENDER study 8 mg/kg Q2W TENDER 12 mg/kg Q2W Open-label Phase II study 8 mg/kg single dose Phase III Japanese study 8 mg/kg Q2W Adult RA patients (n = 1793) # 4 mg/kg Q4W 8 mg/kg Q4W C max (µg/ml) 226 ± 54.5 (37) 263 ± 54.1(38) 96 ± (6) 29 ± 68.3 (5) 88 ± ± 86 AUC (µg day/ml) 1337 ± ± ± 137 (6) 127 ± ± ± 667 Apparent t ½ (days) NA 5.1 ± 1.3 (23) Up to 11 Up to 13 at week 12 (µg/ml) 54.5 ± 2.7 (34) 6.5 ± 25.5(3) NA 56.7 ± 18.8 (18) 1.5 ± ± 11 R obs 3.16 ± 1.37 (32) 3.17 ± 1.16 (28) NA 2.43 ± 1.8 (14) Mean ± SD (n) is displayed. WA18221 (international Phase III study): Pharmacokinetic parameters after sixth dose at week 12; AUC: AUC 2 weeks ; R obs : ratio of the observed predose concentration at week 12 to the predose concentration before the second infusion (week 2). Data taken from [24,42]. LRO32 (Phase II study conducted in UK/France [2]). AUC is AUC inf after a single dose of tocilizumab administration [Data on File]. MRA316JP (Phase III study conducted in Japan) [22]. C max is concentration at the end of infusion after three doses of 8 mg/kg tocilizumab every 2 weeks; AUC is AUC last after the third infusion. t ½ estimated from patients receiving placebo in the withdrawal phase of the study. R obs is the accumulation ratio for -ss (steady state)/-1st (first dose). The reported was at week 18 [Data on File]. # Population PK model simulated PK parameters at steady state based on the PK model developed from RA patients in Phase III studies [49]. AUC: Area under the curve; NA: Not applicable; PK: Pharmacokinetic; Q2W: Every 2 weeks; Q4W: Every 4 weeks; RA: Rheumatoid arthritis. Data taken from [17,19,2,22,24,39,42,51]. bodyweight (scaling factor for body surface area on clearance was.7 instead of 1) [49]. Differences in total clearance between sjia and RA may also be associated with differences between diseases and disease-associated pharmacodynamic markers, including baseline IL-6 levels (129 ± 177 pg/ml in sjia patients with bodyweight 3 kg and 133 ± 135 pg/ml in sjia patients with bodyweight <3 kg [43] vs 53 ± 53 pg/ml in RA [42] at baseline). In the dose escalation trial [21], patients were administered tocilizumab at a dose of 2 mg/kg, with intrapatient dose escalations at 2-week intervals (based on the study objective to normalize CRP response). The final tocilizumab dose was 2 mg/kg in three patients; five patients required dose increases to 4 mg/kg, and three patients required dose increases to 8 mg/kg. In the three patients in the 2 mg/kg group, serum tocilizumab concentrations before the second and third infusions and at the time of the last observation were undetectable. In the 4 and 8 mg/kg groups, three of the eight patients treated with 4 mg/kg for three doses had low tocilizumab concentrations (two undetectable, one at 1.24 µg/ml) at 2 weeks after the last infusion [Data on File]. It appeared therefore that the dose had to be increased to 8 mg/kg in patients in whom detectable tocilizumab concentrations (>1 µg/ml) could not be maintained at 4 mg/kg. As a result, a dose of 8 mg/kg every 2 weeks was assessed in the subsequent Phase III trial conducted in patients with sjia in Japan [22]. In the same Phase III study in Japan, pharmacokinetic and pharmacodynamic data were not included in the published paper [22]. However, mean ± SD predose serum tocilizumab concentrations increased with the number of infusions, from 2.2 ± 12.4 µg/ml at 2 weeks after the start of treatment to 34.3 ± 17.2 µg/ml at 4 weeks and 43. ± 2.5 µg/ml at 6 weeks. The mean predose serum tocilizumab concentration in the tocilizumab group continued to increase until approximately 1 weeks after the start of the treatment and thereafter remained at a level of approximately 5 µg/ml. Pharmacokinetic parameters calculated by noncompartmental analyses are shown in Table 3 for C max (29 ± 68.3 µg/ml, end of infusion after third tocilizumab dose), AUC last (127 ± 496 µg day/ml after the third infusion) and (56.7 ± 18.8 µg/ml, week 18) [Data on File]. In the TENDER study [43], mean predose serum tocilizumab concentrations rose with increasing numbers of tocilizumab doses until they became stable at week 12 (Figure 4). Serum tocilizumab concentrations were similar between tocilizumab treatment groups (8 mg/kg for bodyweight 3 kg and 12 mg/kg for bodyweight TCZ (µg/ml) mg/kg 12 mg/kg Figure 4. Serum concentrations of tocilizumab for two bodyweight groups through week 52. Patients weighing <3 kg were treated with 12 mg/kg TCZ, and patients weighing 3 kg were treated with 8 mg/kg TCZ. Error bars indicate standard deviation. TCZ: Tocilizumab. Data taken from [43]

8 Zhang, Morcos, Saito & Terao <3 kg) at all sampling time points (Figure 4). Mean (SD) trough serum tocilizumab concentrations increased approximately threefold (R obs ) from week 2 to week 12 (Table 3). Tocilizumab concentrations at the end of infusion followed a pattern similar to that for preinfusion concentrations but were stable by week 4; mean ± SD concentrations ranged from 237 ± 76.9 µg/ml at week 4 to 268 ± 77.4 µg/ml at week 1 [24]. The tocilizumab pharmacokinetic profile has been described by a two-compartment model with combined first-order and concentration-dependent saturable elimination. Post-hoc estimations of pharmacokinetic exposures (C max, and AUC 2 weeks ) based on the final population pharmacokinetic modeling at week 12 were similar between the two treatment groups in the TENDER trial (Tables 3 & 4) [12]. Pharmacokinetic exposure parameters were also comparable between Japanese [2] and Caucasian patients with sjia (Table 3) [24,42]. The accumulation ratio for the observed was 2.43 (week 6/week 2) following 8 mg/kg administration in the Japanese Phase III trial and 3.17-fold following 8 mg/kg (bodyweight 3 kg), and 12 mg/kg (bodyweight <3 kg) (week 12/ week 2) in the TENDER trial (Table 3). The observed at week 52 in TENDER was not significantly different from at week 12 for either tocilizumab dose (p >.5), indicating that steady state was reached by week 12, at which time patients had received six doses of tocilizumab [43]. Furthermore, no clear effects on estimated AUC 2 weeks, C max or based on bodyweight (range: kg) or body surface area (range: m 2 ) were observed in TENDER. Mean pharmacokinetic exposures were similar among age categories (2 5, 6 12 and years), indicating the lack of age effect on tocilizumab pharmacokinetics across the entire pediatric age range studied in TENDER when two bodyweight-based dosing regimens were used [12]. At low tocilizumab serum concentrations, the nonlinear clearance pathway was the dominant component of total clearance, reflecting target-mediated clearance due to tocilizumab binding to IL-6R. Linear clearance was the dominant pathway for tocilizumab elimination at high serum concentrations as a result of the saturation of the target-mediated clearance [49]. As such, the half-life (t ½ ) of tocilizumab, which is dependent on clearance, is also concentration dependent [17]. The estimated terminal elimination t ½ of tocilizumab in patients with sjia was 5.1 ± 1.3 days after three infusions using a noncompartmental analysis method (Table 3), whereas the estimated t ½ of tocilizumab in patients with sjia ranged from 18 to 23 days for the two bodyweight categories at week 12 in TENDER. Post-hoc estimated pharmacokinetic exposure parameters using population modeling in patients with sjia (TENDER) [12] and in adult patients with RA (four pooled Phase III studies) [49] are summarized in Table 3. After 8 mg/kg tocilizumab infusion, C max was slightly higher in patients with sjia than in patients with RA (226 vs 187 µg/ml), was sixfold higher in patients with sjia than in adult patients with RA (54.5 vs 8.6 µg/ml at 8 mg/kg) and AUC 2 weeks in patients with sjia was similar to AUC 4 weeks in adult patients with RA (1337 vs 1417 µg day/ml). When adjusted to the same treatment duration of 4 weeks, AUC 4 weeks for the patients with sjia (2674 ± 818 for 8 µg day/ml every 2 weeks [Q2W] and 2692 ± 852 µg day/ml for 12 mg/kg Q2W) was approximately double that for the patients with RA. Table 4. Tocilizumab pharmacokinetic exposures at week 12 by American College of Rheumatology response status for all patients in the TENDER study. Responders Parameter JIA ACR3 (n = 68) JIA ACR5 (n = 64) JIA ACR7 (n = 53) JIA ACR9 (n = 28) C max (µg/ml) Mean ± SD 249 ± ± ± ± 43.1 CV% (µg/ml) Mean ± SD 59. ± ± ± ± 2. CV% AUC 2 weeks (µg day/ml) Mean ± SD 1363 ± ± ± ± 345 CV% Nonresponders Parameter JIA ACR3 (n = 7) JIA ACR5 (n = 11) JIA ACR7 (n = 22) JIA ACR9 (n = 47) C max (µg/ml) Mean ± SD 25 ± ± ± ± 64. CV% (µg/ml) Mean ± SD 44.2 ± ± ± ± 25.3 CV% AUC 2 weeks (µg day/ml) Mean ± SD 1133 ± ± ± ± 452 CV% ACR3: American College of Rheumatology 3 response; ACR5: American College of Rheumatology 5 response; ACR7: American College of Rheumatology 7 response; ACR9: American College of Rheumatology 9 response; AUC: Area under the curve; CV%: Coefficient of variation (=SD/mean); JIA: Juvenile idiopathic arthritis; SD: Standard deviation. Data taken from [5]. 13 Expert Rev. Clin. Pharmacol. 6(2), (213)

9 Clinical pharmacology of tocilizumab Relationship between tocilizumab exposure & pharmacodynamic markers in serum Results from two Phase III studies in patients with sjia show the relationships between tocilizumab concentration and several pharmacodynamic markers (IL-6, sil-6r, CRP and ESR). Scatter plots of the pharmacodynamic markers versus tocilizumab in the open-label period of the Japanese Phase III study [22] are shown in Figure 5. CRP and ESR levels were low when tocilizumab concentrations were above the assay detection limit (1 µg/ml). In addition, there was no obvious relationship between IL-6 and tocilizumab concentration, and the level of sil-6r reached a plateau when tocilizumab concentration was greater than approximately 1 µg/ml [19, Data on File]. Scatter plots of the pharmacodynamic markers IL-6, sil-6r, CRP and ESR versus tocilizumab at week 12 in TENDER are shown in Figure 6. There was no appreciable relationship between tocilizumab and IL-6 levels. In addition, sil-6r levels were high when tocilizumab was >1 µg/ml, whereas CRP and ESR levels were low when tocilizumab was >1 µg/ml [24]. Exposure efficacy relationship As indicated, four clinical studies conducted in patients with sjia are summarized in Table 1 [19]. The first study was an open-label Phase II trial of single ascending tocilizumab doses in Caucasian children with sjia. This proof-of-principle study demonstrated the efficacy of IL-6R blockade [2]. Eighteen patients were divided into three groups receiving 2, 4 or 8 mg/kg tocilizumab by intravenous infusion. Of 15 patients included in the efficacy analysis, 11 achieved JIA ACR3, ACR5 or greater. A dose-dependent increase in the duration of JIA ACR3, ACR5 and ACR7 was observed; after single doses of 2, 4 and 8 mg/kg tocilizumab, patients achieved JIA ACR3, ACR5 and ACR7 up to 3, 6 and 8 weeks, respectively [2]. The second study was an intrapatient dose escalation study conducted in 11 sjia patients [21]. First, each patient was administered an intravenous dose of 2 mg/kg tocilizumab, and then each child without active inflammation was given identical second and third doses at 2-week intervals after the first dose. By contrast, each child with disease flare according to laboratory marker assessment received a 4 mg/kg dose. Those without disease flares at the higher dose received second and third 4 mg/kg doses at 2-week intervals, whereas those with active inflammation received three additional doses of 8 mg/kg tocilizumab. Of the 11 children requiring 2 mg/kg tocilizumab, seven (63.6%) achieved both JIA ACR3 and ACR5 response and one (9.1%) CRP (mg/dl) IL-6 (pg/ml) TCZ (µg/ml) CRP IL TCZ (µg/ml) ESR Figure 5. Relationships between concentration of tocilizumab at the end of a dosing interval ( ) and pharmacodynamic parameters after 12 weeks of treatment. (A) CRP, (B) ESR, (C) IL-6 and (D) sil-6r in the open-label period of a Phase III study conducted in Japan. ESR: Erythrocyte sedimentation rate; sil-6r: Soluble IL-6 receptor; TCZ: Tocilizumab. Data taken from [19] and [Data on File]. ESR (mm/h) sil-6r (ng/ml) TCZ (µg/ml) sil-6r TCZ (µg/ml) achieved JIA ACR7 response at the last observation. Of the eight children infused three times with 4 mg/kg tocilizumab, seven (87.5%) achieved both JIA ACR3 and ACR5 response and four (5.%) achieved JIA ACR7 response at the last observation. All three children who received three doses of 8 mg/kg tocilizumab achieved JIA ACR3/5/7 response 2 weeks after the last infusion. Overall clinical efficacy results indicated that although 2 4 mg/kg tocilizumab could suppress disease activity, 8 mg/kg was the more appropriate dose for controlling disease activity in patients with sjia. In addition, accelerated accumulation of serum tocilizumab was determined to be key in stabilizing the disease [21]. The third study was a Phase III study conducted in Japanese patients with sjia [22]. Fifty-six children (aged 2 19 years) with disease refractory to conventional treatment were each given three doses of tocilizumab 8 mg/kg every 2 weeks during a 6-week, open-label lead-in phase. Patients achieving a JIA ACR3 response and a CRP <5 mg/l were randomly assigned to receive placebo or to continue tocilizumab treatment for 12 weeks or until withdrawal for rescue medication in a double-blind phase. The primary end point of the double-blind phase was JIA ACR3 response and CRP concentration <15 mg/l. Patients responding to tocilizumab and needing further treatment were enrolled in an open-label extension phase for at least 48 weeks [22]. At the end of the open-label lead-in phase, ACR3, ACR5 and ACR7 response was achieved by 51 (91%), 48 (86%) and 38 (68%) 131

10 Zhang, Morcos, Saito & Terao A B CRP (mg/l) ESR (mm/h) TCZ (µg/ml) TCZ (µg/ml) C IL-6 (pg/ml) D sil-6r (ng/ml) TCZ (µg/ml) TCZ (µg/ml) Figure 6. Relationships between concentration of tocilizumab at the end of a dosing interval ( ) and pharmacodynamic parameters after 12 weeks of treatment in the TENDER study. (A) CRP, (B) ESR, (C) IL-6 and (D) sil-6r at week 12. ESR: Erythrocyte sedimentation rate; sil-6r: Soluble IL-6 receptor; TCZ: Tocilizumab. Data taken from [24]. patients, respectively. Forty-three patients entered the doubleblind phase and were included in the efficacy analysis. Four (17%) of 23 placebo patients maintained JIA ACR3 response and a CRP concentration <15 mg/l compared with 16 (8%) of 2 tocilizumab patients (p <.1). By week 48 of the open-label extension phase, ACR3, ACR5 and ACR7 response was achieved by 47 (98%), 45 (94%) and 43 (9%) of 48 patients, respectively [22]. After 6 weeks of treatment, the proportion of patients reaching ACR5 response was lower for those weighing <3 kg (83%) than for those weighing 3 kg (%) [12]. Similarly, the proportion of patients who reached ACR7 was lower for those weighing <3 kg (63%) than for those weighing 3 kg (85%) [12]. This difference was explained by the visible trend toward lower systemic exposure to tocilizumab in patients with lower bodyweight (Figure 7). Population pharmacokinetic modeling and simulation were performed using pooled pharmacokinetic data from the two early studies [2,21]; the goal was to explore an alternative dosing regimen to achieve uniform exposure across the entire bodyweight range. Post-hoc estimates of systemic tocilizumab exposures using the final population pharmacokinetic model predicted that a 12 mg/kg tocilizumab dose in patients <3 kg would yield systemic exposure similar to that observed in patients 3 kg. With the knowledge gained from the modeling and simulation exercise, the subsequent pivotal Phase III TENDER study evaluated tocilizumab 8 mg/kg (patients weighing 3 kg) and 12 mg/kg (patients weighing <3 kg) administered every 2 weeks [43]. Indeed, the dosing regimens in the TENDER study resulted in similar serum concentrations at sampling time points over time (Figure 4) and similar systemic exposure (Table 3) between the chosen bodyweight categories [Data on File,42,12]. Additionally, 94.7 and 75.7% of the patients achieved the primary efficacy end point of JIA ACR3 response and absence of fever at week 12 for 132 Expert Rev. Clin. Pharmacol. 6(2), (213)

11 Clinical pharmacology of tocilizumab the 12 (bodyweight <3 kg) and 8 mg/kg (bodyweight 3 kg) doses, respectively, compared with only 24.3% in the placebo group [12]. Uniform pharmacokinetic exposure achieved with the two bodyweight-based dosing regimens resulted in comparable efficacy across a wide bodyweight range. A summary of pharmacokinetic exposures (AUC 2 weeks, and C max ) by JIA ACR3/5/7/9 response status at week 12 is provided in Table 4 for the TENDER trial [12]. No clear trend toward higher mean pharmacokinetic exposures (AUC 2 weeks, and C max ) was evident in responders compared with nonresponders. In addition, no clear difference in mean pharmacokinetic exposures was evident across JIA ACR3/5/7/9 responders. A summary of pharmacokinetic exposures (C max, and AUC 2 weeks ) by primary end point (JIA ACR3 and absence of fever) is provided in Table 5 [12]. Despite the approximate twofold increase in AUC 2 weeks and C max and the approximate threefold increase in from quartile 1 to quartile 4, the percentages of patients achieving JIA ACR3 response and absence of fever at week 12 were similar across exposure quartiles [12]. Proportions of patients who achieved secondary end points (JIA ACR3/5/7/9 response) were comparable across pharmacokinetic exposure quartiles (AUC 2 weeks or ) [43]. Results support the idea that even the lowest systemic exposures occurring within the range of exposures (Table 5) was sufficient to achieve the desired efficacy in this patient population. Therefore, tocilizumab pharmacokinetic exposure reached a plateau on the exposure response curve in the TENDER trial. Exposure safety relationship In study MRA11JP, at the time of enrollment, sjia patients had active disease despite treatment with nonsteroidal anti-inflammatory drugs, corticosteroids, cyclosporin or methotrexate, or any combination of those for at least 4 weeks [21]. Treatment with tocilizumab in patients with sjia was safe and well tolerated and provides greater clinical benefit than treatment with conventional corticosteroids [21]. Common adverse events are gastrointestinal, nasopharyngeal and upper respiratory tract infections and are generally mild [22]. For the TENDER study, the most frequently reported adverse events (AEs) in placebo patients in TENDER were disease flare (juvenile arthritis; 13.5%) and pyrexia (16.2%), and most of these events led to escape therapy [12]. AEs at an incidence of 5% in either treatment group to week 12 was comparable for tocilizumab dose group versus placebo dose group (upper respiratory tract infection: 13.3 vs 1.8%, headache: 9.3 vs 8.1%, nasopharyngitis: 1.7 vs 2.7%, diarrhea: 6.7 vs 2.7% and pharyngitis: 2.7 vs 5.4%), further supporting the use of the 12 mg/kg dose in patients with sjia weighing <3 kg. Common laboratory abnormalities (worst CTC grade at any time point) reported at up to the 52-week follow-up included increased alanine aminotransferase levels (grade 3: >5 2 ULN in 6.3% of Tocilizumab AUC (µg/ml d) mg/kg for bodyweight <3 kg 8 mg/kg for bodyweight 3 kg Bodyweight (kg) Figure 7. Area under the curve versus bodyweight in Japanese pediatric systemic juvenile idiopathic arthritis patients. Dosing with 8 mg/kg tocilizumab results in lower exposure in Japanese systemic juvenile idiopathic arthritis patients with lower bodyweights. AUC: Area under the curve. Adapted with permission from [12]. the patients; grade 4 [>2 ULN] in.9% of the patients) and neutropenia (grade 3 in 15.2% of the patients; grade 4 in 1.8% of the patients) [23]. In addition, there was a slight trend toward an association of grade 3 neutropenia with higher tocilizumab AUC 2 weeks (1672 ± 272 µg day/ml) than grade neutropenia (1313 ± 435 µg day/ml) during first 12 weeks of treatment [43]. Percentages of patients reporting AEs by body system and preferred term were analyzed by AUC 2 weeks, C max and quartiles in TENDER; results are shown in Table 6 [24,12]. When combining tocilizumab treatment groups and comparing AEs across pharmacokinetic exposure quartiles, no trend was observed toward an increased incidence in AEs or in the percentage of patients reporting at least one AE with increasing tocilizumab exposure. For AEs reported by >11% of the patients, the analysis was also performed in each system organ class. As a result, no trend toward increased percentages of patients with at least one AE was observed with increasing tocilizumab exposure in infection and infestation; GI disorder; skin and subcutaneous Table 5. Percentages of patients achieving juvenile idiopathic arthritis American College of Rheumatology 3 response and absence of fever status at week 12 by exposure quartiles in the TENDER study. Parameter Responders, n (%) Quartile 1 (n = 19) Quartile 2 (n = 19) Quartile 3 (n = 19) Quartile 4 (n = 18) n (%) C max 5 (78.9) 17 (89.5) 16 (84.2) 16 (88.9) n (%) 16 (84.2) 17 (89.5) 15 (78.9) 16 (88.9) n (%) AUC 2 weeks 16 (84.2) 17 (89.5) 16 (84.2) 15 (83.3) Quartile 1: to 25; quartile 2: >25 to 5%; quartile 3: >5 to 75%; quartile 4: >75 to %. Mean AUC 2 weeks (µg day/ml) are as follows: quartile 1: 849 ± 147; quartile 2: 1178 ± 68.4; quartile 3: 1445 ± 15; quartile 4: 1925 ± 187 µg day/ml. AUC: Area under the curve; CV%: Coefficient of variation (=SD/mean). Data taken from [12]