Anti-TNF Monoclonal Antibodies in Inflammatory Bowel Disease: Pharmacokinetics-Based Dosing Paradigms

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1 nature publishing group state Anti-TNF Monoclonal Antibodies in Inflammatory Bowel Disease: Pharmacokinetics-Based Dosing Paradigms Ingrid Ordás 1,2, Diane R. Mould 3, Brian G. Feagan 4 and William J. Sandborn 1 Crohn s disease and ulcerative colitis are chronic inflammatory disorders resulting from immune dysregulation. Patients who fail conventional medical therapy require biological treatment with monoclonal antibodies (mabs). Although mabs are highly effective for induction and maintenance of clinical remission, not all patients respond, and a high proportion of patients lose response over time. One factor associated with loss of response is immunogenicity, whereby the production of antidrug antibodies accelerates mab clearance. However, other factors related to patient and disease characteristics also influence the pharmacokinetics of mabs. These factors include gender, body size, concomitant use of immunosuppressive agents, disease type, serum albumin concentration, and the degree of systemic inflammation. Because it is important to maintain clinically effective concentrations to provide optimal clinical response and drug exposure is affected by patient factors, a better understanding of the pharmacology of mabs will ultimately result in better patient care. In the past decade, therapy with monoclonal antibodies (mabs) has revolutionized the therapeutic paradigms of immune diseases such as rheumatoid hritis (RA), psoriasis, and the inflammatory bowel diseases (IBDs) Crohn s disease (CD) and ulcerative colitis (UC). The process for generating hybridomas, discovered by Köhler and Milstein in 1975, allowed for the production and isolation of mabs as therapeutic agents. 1 Early therapeutic mabs were murine, and their use in clinical practice was limited because of immunogenicity. Subsequently, modern genetic engineering techniques led to the development of humanized and fully human mabs. Although, in general, humanized and fully human mabs are less immunogenic as compared with murine antibodies, they also can induce antidrug antibody (ADA) formation. Infliximab, the first mab used for treatment of patients with CD, 2 was approved by the US Food and Drug Administration for that indication in The rapid adoption of infliximab into clinical practice, and its high level of efficacy in patients with CD unresponsive to conventional therapies, led to great interest in the development of additional mabs to block tumor necrosis factor (TNF) activity and other proinflammatory cytokines that play key roles in the activation and perpetuation of the inflammatory response in patients with IBD. Even though mabs have been used in clinical practice for more than a decade, little is known about their exposure response relationship and the factors that may affect their disposition. Understanding these factors is essential to further improving the therapeutic efficacy of these drugs. This review evaluates the factors known to influence the pharmacokinetics (PK) of the currently approved mabs for the treatment of IBD infliximab, adalimumab, and certolizumab and speculates on the future role of therapeutic drug monitoring and the role of individualized dosing for these agents. Monoclonal Antibodies Structure mabs are engineered immunoglobulin G (IgG) therapeutic proteins. IgG molecules are constructed by a basic unit of four polypeptide chains including two identical heavy chains (C H ) and two identical light chains (C L ). Each antibody comprises two domains: (i) the variable region or Fab (antigen-binding region), which is specific for the antigen target (each antibody has two Fabs), and (ii) the constant region or the Fc (Figure 1). 3 Depending on their structure or isotype, mabs can be classified as murine antibodies (suffix nomenclature: -omab; 1 Division of Gastroenterology, University of California San Diego, La Jolla, California, USA; 2 Gastroenterology Depment, Hospital Clinic of Barcelona, CIBER-EHD, IDIBAPS, University of Barcelona, Barcelona, Spain; 3 Projections Research Inc., Phoenixville, Pennsylvania, USA; 4 Robs Research Institute, University of Western Ontario, London, Ontario, Canada. Correspondence: WJ Sandborn (wsandborn@ucsd.edu) Received 4 October 2011; accepted 16 November 2011; advance online publication 22 February doi: /clpt Clinical pharmacology & Therapeutics 1

2 Fab S S C H S S S S C H1 C H2 C H3 V H S S C L V L Fc of macrophages inhibits proliferation of activated T cells and produces anti-inflammatory cytokines. 9 TNF-α can be detected in serum in its soluble form or expressed as a cell-surface polypeptide on activated macrophages, monocytes, and T cells in its transmembrane form. Overall, infliximab, adalimumab, and certolizumab have similar intrinsic binding properties and affinities for both soluble and transmembrane forms of TNF-α. 10 Infliximab and adalimumab also have similar ability to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. By contrast, certolizumab, because of the absence of the IgG1 Fc portion, exhibits neither complement-dependent cytotoxicity nor antibody-dependent cell-mediated cytotoxicity. 10 The C H2 and C H3 domains of the IgG1 Fc portion are involved in the binding to Fc receptors of natural killer cells, which leads to the lysis of target cells. Therefore, unlike infliximab and adalimumab, certolizumab does not induce apoptosis of activated immune cells. Figure 1 General structure of IgG antibodies. Each antibody is composed of two identical heavy chains (C H ) and two identical light chains (C L ). The light chain contains two domains: one variable (V L ) and one constant (C L ). The heavy chain is composed of a variable domain (V H ) and three constant domains (C H1, C H2, and C H3 ). The antigen-binding region (Fab) includes V H, V L, C L, and C H1, and the constant region (Fc) includes C H2 and C H3. -S-S-, disulfide bond. e.g., tositumomab), chimeric (-ximab; e.g., infliximab), humanized (-zumab; e.g., certolizumab), or fully human (-umab; e.g., adalimumab) (Supplementary Figure S1 online). Infliximab is a chimeric IgG1 mab composed of a variable murine Fab region linked by disulfide bonds to a human IgG1 κ constant region. 4 Infliximab is produced by cell culture using Chinese hamster ovary cells. Adalimumab is also a recombinant IgG1 mab, but, unlike infliximab, it is fully human. It is composed of human-derived variable regions (Fabs) and a human IgG1 κ constant region. 5 Adalimumab is produced by cell culture using Chinese hamster ovary cells. Certolizumab pegol, a pegylated humanized mab, is composed of an IgG4 isotype Fab fragment chemically linked to a polyethylene glycol moiety, which slows clearance, thereby increasing the half-life of the drug. 6 Certolizumab is produced by cell culture using Escherichia coli. Mechanism of action and therapeutic target Improved understanding of the pathogenesis of RA, psoriasis, and IBD at the cellular and molecular level led to the development of biological agents targeting TNF-α, a potent proinflammatory cytokine that plays a key role in orchestrating the inflammatory process in multiple autoimmune diseases. IBD is characterized by a dysregulated mucosal immune response toward the commensal enteric flora in genetically susceptible individuals. 7 This immune dysregulation results in an overproduction of TNF-α by monocytes, macrophages, and T cells. 8 Interestingly, mabs targeting TNF-α (infliximab, adalimumab, and certolizumab) induce the formation of regulatory macrophages with immunosuppressive properties. This population Mechanisms of absorption, distribution, degradation, and elimination Absorption. The majority of approved mabs are administered intravenously; however, some of these agents are designed for extravascular administration, either subcutaneous (s.c.) or intramuscular. Infliximab is administered intravenously, whereas adalimumab and certolizumab are administered by s.c. injection. In general, the route of administration of mabs affects their pharmacokinetic behavior. Intravenous therapy allows administration of large volumes of drug, achieves immediate central distribution, results in less variability in drug exposure between subjects, and is usually less immunogenic. The mechanism of absorption after s.c. administration is not fully understood but is likely to occur by means of lymphatic drainage. The main drawbacks of s.c. administration are that a smaller volume must be administered (generally no more than 1 ml), in comparison with the intravenous route, and the fraction of dose absorbed is variable, which leads to higher pharmacokinetic variability between patients and doses. Reported bioavailability of mabs administered subcutaneously is highly variable among individual patients, ranging from 50 to 100%. 11 In addition, the s.c. route is often more immunogenic than the intravenous route because the skin is highly specialized for processing foreign antigens. After s.c. injection, mabs undergo slow absorption, with maximum concentrations being achieved 8 to 10 days after administration. Distribution. After administration, mabs distribute mainly within the central compment (extracellular fluid), whereas penetration inside cells is limited because of their high molecular weight and hydrophilicity. mabs seem to have a volume of distribution on the order of 0.1 l/kg, approximately equal to the extracellular fluid volume. 12 For example, the volume of distribution of infliximab at steady state ranges from 4.5 to 6 l. 13 Because of the relatively large loading doses and the intravenous route of administration, infliximab yields acute concentration time profiles with very high peak concentrations 2

3 a Infliximab concentration (µg/ml) b Infliximab concentration (µg/ml) 1, , Time (days) Time (days) Model predicted infliximab concentration Observed infliximab concentration Figure 2 Concentration vs. time curves of infliximab in (a) ulcerative colitis (UC) and (b) Crohn s disease (CD). Observed (dots) and simulated (lines) median concentration time profiles of patients with UC (data from ACT trials) and CD (data from ACCENT I trial). Patients received treatment with infliximab 5 mg/kg at weeks 0, 2, and 6 and every 8 weeks thereafter. The serum infliximab concentrations are higher at early time points in the graph, corresponding with the induction phase of treatment (loading doses). After 14 weeks (100 days), infliximab serum concentrations tend to stabilize. ACCENT I, A Crohn s Disease Clinical Trial Evaluating Infliximab in a New, Longterm Treatment Regimen; ACT, Active Ulcerative Colitis Trial. Adapted with permission from refs. 14 and 15. and low trough levels and, hence, high peak-to-trough ratios (Figure 2). 14,15 By contrast, adalimumab and certolizumab, given their slow absorption and elimination rates, exhibit more uniform concentration time profiles at steady state. 16 The pharmacokinetic properties of infliximab, adalimumab, and certolizumab are shown in Table 1. Degradation and elimination. Although the exact mechanisms by which mabs are cleared from the circulation is not well understood, the primary route of antibody clearance is via proteolytic catabolism after receptor-mediated endocytosis in the cells of the reticuloendothelial system (RES). 17 Antibody salvage and recirculation is mediated by the Brambell receptor (FcRn), which is essential for maintaining immunoglobulin and albumin homeostasis. 18 In adults, FcRn is primarily expressed in the vascular endothelial cells or the RES and at lower levels on monocyte cell surfaces, tissue macrophages, and dendritic cells. 19 This Fc receptor plays a critical role in protecting IgG antibodies and albumin from the ongoing catabolic activities, thus prolonging their half-lives. 20 However, this system is saturable at high IgG concentrations, resulting in an inverse relationship between concentration and halflife (the higher the concentration of the antibody, the lower its half-life). 21 Thus, one would anticipate that high levels of endogenous IgG, as is seen in chronic inflammatory diseases, could potentially shorten the half-life of exogenously administered mabs. In addition to FcRn, three other classes of Fc receptors (FcγRI, FcγII, and FcγIII) for IgG binding have been identified in humans. 22 These receptors are expressed by macrophages, natural killer cells, B and T cells, and platelets. Fc gamma receptor (FcγR) polymorphisms have been associated with clinical response to TNF antagonists in patients with IBD. 23 Whether FcγRs polymorphisms may contribute to clearance of TNF antagonists needs further investigation. FcRn binds to IgG with ph-dependent affinity. 24 Antibodies bind tightly to FcRn inside endosomes, which have an acidic environment. Inside endolysosomes, the IgG FcRn complexes do not undergo catabolism, whereas the antibody bound to FcγR is degraded. Eventually, the antibody bound to FcRn is returned Table 1 Pharmacokinetic properties of infliximab, adalimumab, and certolizumab Infliximab Adalimumab Certolizumab CD UC CD a 70 UC CD 71 UC Cmax 118 µg/ml 4.7 ± 1.6 µg/ml N/A µg/ml N/A T 1/ days days N/A 14 days N/A Tmax Within an hour 5.46 ± 2.3 days N/A days N/A Vd liters liters liters N/A N/A V ml/kg liters 14 V ml/kg liters 14 Cl 5.42 ml/kg/d 15 (15.8 ml/h) b 0.4 liters/d 14 (16.7 ml/h) 12 ml/h N/A 17 ml/h N/A CD, Crohn s disease; Cl, clearance; Cmax, maximum concentration; N/A, not available; T 1/2, half-life; Tmax, time to reach maximum plasma concentration; UC, ulcerative colitis; V1, volume of distribution in the central compment; V2, volume of distribution in the peripheral compment. a Following a single 40-mg s.c. administration to healthy adult subjects. b Assuming a mean body weight of 70 kg. Clinical pharmacology & Therapeutics 3

4 Antibody 5. Cell surface (ph 7.4) Antibody is released from FcRn back into circulation 1. mab uptake via FcRn and FcγR interaction FcRn 2. Endosome formation (ph 6) FcγR 4. Antibody bound to FcRn is returned to cell surface 3. Antibody bound to FcγR is degraded and antibody bound to FcRn protected Figure 3 Mechanism of degradation of monoclonal antibodies. FcgR, Fc gamma receptor; FcRn, Brambell receptor. Adapted with permission from ref. 54. to the cell surface where, at physiologic ph, it dissociates from the receptor and is released again back into the circulation (Figure 3). It has been shown that in mice genetically lacking expression of FcRn, IgG follows hypercatabolism and thus accelerated clearance. 25 These results support the importance of FcRn in regulating the catabolism of antibodies and therefore their pharmacokinetic behavior. In addition, IgG affinity for FcRn is species-specific. Human FcRn shows high affinity for human IgG, whereas the human receptor shows little affinity for IgG derived from most other species, including mice. This observation helps to explain the higher clearance that murine mabs experience in humans. 26 Thus, the structure of mabs influences their pharmacokinetic behavior; the Fc region is responsible for the prolonged half-life of IgG antibodies through its binding to FcRn. Fc conjugation is a common method used to extend the half-life of mabs by decreasing their clearance through improvement of FcRn binding. As expected, the half-life of mabs generally increases with their level of humanization. Murine antibodies display a short half-life (1 2 days); chimeric antibodies have a half-life of approximately 10 to 14 days, and humanized and fully human antibodies exhibit longer half-lives of approximately 10 to 20 days. 27 PEGylation is another modification that has been used to increase the half-life of mabs that do not have a functional Fc region. The addition of polyethylene glycol to mabs structurally protects them from proteolytic breakdown and immunologic recognition, thus decreasing the likelihood of neutralizing antibody formation. Clearance of mabs can be either linear or nonlinear. Generally, mabs targeting cell-surface receptors tend to exhibit nonlinear clearance that is dependent on antigen expression, whereas mabs directed against soluble antigens (e.g., cytokines) typically exhibit dose-proportional behavior with linear clearance, which is often affected by body weight. 28 However, it should be noted that the relationship between mab clearance and body weight is generally less than linear, suggesting that mg/kg dosing may lead to patient exposure that is below the target range in patients with low body weight. There is some suggestion, however, that the clearance of mabs targeting soluble receptors may be influenced by receptor expression as well. 28 The contribution of receptor-mediated clearance to overall clearance depends on several factors such as mab concentration and distribution together with target receptor expression, internalization, and turnover rates. In some cases, cell-surface receptors are released into the serum, circulating as free antigens. mabs can bind to these shed receptors, resulting in the formation of antibody antigen complexes that may result in an accelerated mab clearance. 12 Overview of Ibd Treatment with Mabs CD and UC, the two main forms of IBD, are chronic diseases that result from immune dysregulation in genetically susceptible individuals. 7 Because the specific cause of CD and UC is 4

5 unknown, they have conventionally been treated using a broad spectrum of anti-inflammatory agents, including aminosalicylates, corticosteroids, and standard immunosuppressive agents such as purine analogs (azathioprine and 6-mercaptopurine), methotrexate, and cyclosporine. 29 However, a high proportion of patients fail to respond to these therapies and require biological treatment with TNF antagonists (mabs). TNF antagonists have shown clear benefits in randomized controlled trials for inducing and maintaining clinical remission in both CD and UC However, despite their therapeutic efficacy, more than one-third of patients show no response to induction therapy (primary nonresponders), and in up to 50% of responders, TNF antagonist therapy becomes ineffective over time (secondary nonresponders). 36 Thus, a critical need exists to develop new approaches and strategies that further optimize the efficacy of these drugs. In recent years, interest in the mechanistic causes of TNF antagonist treatment failure has intensified. It is now well established that patients lacking objective evidence of inflammation, such as presence of ulcers at the endoscopic examination or increased serum C-reactive protein concentrations, do not benefit from treatment with TNF antagonists. 37,38 Furthermore, symptoms usually do not correlate well with the presence of mucosal lesions or with elevated biomarkers of inflammation. 39,40 Hence, when loss of response to TNF antagonists occurs, dose intensification or switching between mabs based exclusively on symptoms will frequently lead to incorrect therapeutic decisions and should be avoided. Instead, before making any decision, patients with suspected active inflammation based on symptoms should undergo endoscopic or radiologic evaluation to ensure that they exhibit objective evidence of inflammation that could potentially benefit from dose intensification. A therapeutic algorithm for the treatment of patients with moderate and severe IBD is shown in Supplementary Figure S2 online. The structural characteristics and dosage of mabs for the treatment of IBD are detailed in Table 2. The development of immunogenicity due to inappropriate administration strategies (episodic administration rather than scheduled administration and monotherapy instead of combined therapy with an immunosuppressive) is a common cause of treatment failure. The optimal strategies to minimize the risk of immunogenicity and the potential benefits of tailoring therapy based on the determination of serum drug concentrations and the presence or absence of neutralizing ADAs are examined below. Strategies to minimize the risk of immunogenicity The development of immunogenicity is an important determinant of both the efficacy and safety of TNF antagonists. As discussed above, a high proportion of patients who initially respond to mabs lose response over time, owing, in p, to development of ADAs Two therapeutic strategies have been associated with a reduction in ADA formation: (i) use of TNF antagonists in a scheduled maintenance regimen rather than episodic administration and (ii) concomitant use of immunosuppressive agents (azathioprine, mercaptopurine, or methotrexate) with a TNF antagonist. Scheduled vs. episodic treatment. Episodic infliximab treatment strategy in patients with CD has been associated with a higher rate of antibody formation and a higher rate of infusion reactions as compared with scheduled maintenance therapy. 44 Infusion reactions to infliximab are strongly associated with the presence of ADAs. Intravenous hydrocortisone premedication reduces the formation of ADAs but does not eliminate the risk of infusion reactions. 45 The best strategy to minimize this risk is to administer infliximab on a scheduled basis and to use concomitant immunosuppressive therapy. In addition, detectable trough infliximab concentrations, which are associated with better outcomes, are higher in patients undergoing a regularly scheduled treatment strategy. 44 Therefore, the optimal strategy to reduce the risk of ADAs to infliximab is to use an induction dosing regimen (5 mg/kg intravenously at weeks 0, 2, and 6) followed by a maintenance strategy every 8 weeks rather than episodic therapy. With adalimumab, episodic administration has been associated with inferior clinical outcomes, but data regarding immunogenicity have not been obtained. 33 With Table 2 Characteristics and dosage of mabs for inflammatory bowel disease Infliximab Adalimumab Certolizumab Natalizumab 73 Ustekinumab 74 Golimumab 75 Approved indications CD UC RA PsA AS CD UC RA PsA AS JRA Psoriasis CD RA CD MS Psoriasis Phase II/III studies for CD RA PsA AS Phase II/III studies for UC Structure Chimeric IgG1 κ Human IgG1 κ Humanized Humanized IgG4 Human IgG1 κ Human IgG1 κ pegylated Fab IgG4 Therapeutic target TNF-α TNF-α TNF-α α4 integrin IL-12/23 (p40 subunit) TNF-α Dosage in IBD 5 mg/kg and every 8 wk mg/2 wk and 40 mg eow 400 mg wk and every 4 wk 300 mg every 4 wk 45 mg at baseline, 4 wk after, and every 12 wk thereafter (90 mg if weight >100 kg) 50 mg monthly Administration route i.v. s.c. s.c. i.v. s.c. s.c. Brand name Remicade Humira Cimzia Tysabri Stelara Simponi AS, ankylosing spondylitis; CD, Crohn s disease; eow, every other week; Fab, antigen-binding region; IBD, inflammatory bowel disease; IgG, immunoglobulin G; IL, interleukin; i.v., intravenous; JRA, juvenile rheumatoid hritis; mabs, monoclonal antibodies; MS, multiple sclerosis; PsA, psoriatic hritis; RA, rheumatoid hritis; s.c., subcutaneous; TNF-α, tumor necrosis factor α; UC, ulcerative colitis; wk, weeks. Clinical pharmacology & Therapeutics 5

6 certolizumab, episodic treatment strategy in patients with CD has been associated with a higher rate of antibody formation as compared with scheduled maintenance therapy. 32 Combined therapy vs. monotherapy. Combination therapy with TNF antagonists and immunosuppressive agents has been shown to reduce the risk of ADA formation in patients with CD using episodic or scheduled treatment strategy; the magnitude of reduction is amplified when the TNF antagonist is administered following a scheduled strategy. 37,41,46,47 Baert et al. showed that the rate of ADA formation in patients with refractory CD treated with infliximab episodically was significantly lower in those receiving concomitant immunosuppressive therapy as compared with those who were not taking these agents (43% vs.75%, respectively; P < 0.01). 41 Vermeire et al. reproduced those results in a prospective cohort study evaluating the effectiveness of concomitant immunosuppressive therapy in suppressing the formation of ADAs in patients with CD treated with infliximab in an on-demand schedule. Concomitant use of azathioprine or methotrexate was associated with a lower incidence of ADAs as compared with patients not taking immunosuppressive agents (46% vs. 73%, respectively; P < 0.001). No difference was found between azathioprine (48%) and methotrexate (44%) in reducing this risk. 46 Hanauer et al. demonstrated that concomitant use of immunosuppressives in patients receiving infliximab in a scheduled strategy significantly reduces the risk of immunogenicity as compared with patients receiving infliximab monotherapy (10% vs. 18%, respectively; P = 0.02). 47 The SONIC trial in which patients with CD who were naive to immunosuppressive and TNF antagonist therapy were randomized to receive azathioprine or infliximab or a combination of both demonstrated that the rate of ADA formation was significantly lower in the subgroup of patients receiving combination therapy (0.9%) as compared with patients receiving infliximab monotherapy (14.6%). 37 Feagan et al. also demonstrated that the use of concomitant methotrexate with infliximab in patients with CD significantly reduces the rate of ADA formation (4% in patients receiving combined therapy vs. 20.4% in patients treated with infliximab monotherapy following a scheduled strategy). 48 Adalimumab and certolizumab can also induce the formation of neutralizing antibodies. 32,42,49 Similarly to infliximab, this risk is also decreased when these mabs are given concomitantly with immunosuppressives and administered as a scheduled maintenance regimen. 32,49 In patients with UC, combined immunosuppression with azathioprine and infliximab also reduces ADA formation and increases infliximab trough concentrations. 31,50,51 It is therefore clear that concomitant use of immunosuppressive agents with a TNF antagonist reduces the risk of ADA development in patients with CD and UC. In terms of efficacy, it has recently been shown that combined therapy with infliximab and azathioprine is more efficacious than either drug alone for induction of clinical remission and mucosal healing in both CD and UC. 37,51 It is likely that, at least in p, this increased efficacy is due to lower rates of ADA formation and higher infliximab drug concentrations among patients receiving combined therapy. Therapeutic monitoring: determination of trough concentrations and ADAs Loss of response to TNF antagonists, mainly due to development of neutralizing ADAs and subtherapeutic drug concentrations, is a challenging problem in the management of patients with IBD. Emerging data indicate that a strong relationship exists between serum drug concentrations (PK) and efficacy (pharmacodynamics, PD). Studies conducted in both RA and IBD have shown that patients with higher trough drug concentrations achieve superior outcomes. 43,44,52 This observation holds out the possibility that therapeutic drug monitoring may direct dose adjustment and clinical decision making. Infliximab concentrations 12 µg/ml 4 weeks after infusion or >1.4 µg/ml at dosing trough are considered to be predictive of therapeutic response. 41 These cutoff values are based on a study in which patients were treated using infliximab episodically rather than on a scheduled basis and have not been prospectively validated. In a retrospective study, Afif et al. evaluated the clinical utility of measuring ADAs and trough drug concentrations in patients with loss of response to infliximab. 53 In patients with antibodies against infliximab, switching to another TNF antagonist was associated with a complete or pial response in a very high proportion of patients (92%), whereas increasing infliximab dose had a response in only 17% of patients. Conversely, dose escalation in patients with subtherapeutic infliximab concentrations was associated with clinical response in 86% of patients, whereas the rate of clinical response in patients changing to another TNF antagonist agent was 33%. Therefore, increasing the dose of infliximab in patients who have developed ADAs is ineffective. Accordingly, measurement of ADAs and trough drug concentrations in patients with loss of response is potentially a clinically useful strategy (Supplementary Figure S2 online). Nevertheless, the added value of tailoring TNF antagonist maintenance therapy in individual patients based on trough drug concentrations and the presence or absence of ADAs deserves further evaluation by prospective studies. Factors Affecting the Pk and Pd of Mabs The pharmacology of therapeutic mabs is complex and depends not only on the structure of the antibody but also on the properties of the target antigen and on patient- and disease-related factors. To date, only limited information exists regarding the factors, other than the formation of neutralizing antibodies, that influence the PK of mabs. Identification of factors that influence disposition and elimination of mabs is essential to understanding their PK PD relationship. PK is a branch of pharmacology dedicated to study the mechanisms of absorption, distribution, metabolism, and elimination of an administered drug (i.e., what the body does to the drug), whereas PD studies the relationship between drug exposure and therapeutic effect (i.e., what the drug does to the body). PK and PD are interrelated. PK PD analyses play an important role during drug development and are critical to select the appropriate 6

7 dose regimens. 54 Furthermore, PK PD modeling analyses are essential to understanding the relationship between drug concentration (PK) and therapeutic response (PD). PK PD modeling is a valuable tool in drug research and development for several reasons: (i) it may help to reduce the number of unnecessary and unproductive studies, (ii) it may help generate pivotal decision-making algorithms (e.g., dose optimization), (iii) it may help to improve the overall drug safety and efficacy, and (iv) it may lead to savings in time and money. 55 PK PD modeling has evolved rapidly over the past decade, but unfortunately no formal prospective studies evaluating the PK PD relationship of mabs in patients with IBD have been conducted. Serum mab concentrations have been shown to be highly variable between individuals and differ over time even within an individual patient. The differences in the observed concentration time profiles and the exposure characteristics among mabs can be explained by different molecular properties (such as structure, physiology of the therapeutic target, and clearance mechanisms), differences in the dosage or administration regimens (e.g., route of administration and administration frequency), and patient and disease characteristics. These factors are discussed below. Population PK studies seek to identify the factors that influence changes in the relationship between the administered dose and the achieved serum concentrations. Hence, if therapeutic concentrations are not reached, dosage can be appropriately modified according to these factors. Unfortunately, several uncertainties regarding the pharmacokinetic profile of mabs persist. Specifically, the covariates that may influence drug clearance are not well defined. Considering trough serum drug concentrations as a surrogate for pharmacokinetic analysis may be misleading and related to errors. Estimates from nontrough PK observations are more robust, providing more information on the disposition of the drug, and are thus preferred for PK PD modeling studies. Population PK models, using random variables with mean and variance parameters instead of an individual data analysis, help to identify and quantify the sources of variability through the identification of covariates on each pharmacokinetic parameter. In addition, population PK analyses supported by large data sets with sparse sampling yield good-quality pharmacokinetic parameter estimates and can be better extrapolated to the target patient population as compared with the values obtained from studies involving single-dose administrations and a small number of subjects because the results obtained from population PK analyses reflect information from a wide range of patients undergoing treatment at multiple clinical centers. Therefore, PK modeling studies using intensive blood sampling instead of measuring only trough drug concentrations, together with evaluation of several patient and disease covariates, are more accurate and yield more information on the sources of betweenpatient variability in mab exposure. Although therapeutic mabs have been commercially available for two decades, little is known about their PK PD relationship. Conventional wisdom implicates neutralizing ADAs as a primary cause of therapeutic failure. However, although ADAs can Table 3 Factors affecting the pharmacokinetics of monoclonal antibodies Presence of ADAs Concomitant use of IS High baseline (TNF-α) Low albumin High baseline CRP Body size Gender Impact on pharmacokinetics Decreases serum (mabs) Threefold-increased clearance Worse clinical outcomes Reduces ADA formation Increases serum (mabs) Decreases mabs clearance Better clinical outcomes May decrease (mabs) by increasing clearance Increases clearance Worse clinical outcomes Increases clearance High body mass index may increase clearance Males have higher clearance ADA, antidrug antibody; CRP, C-reactive protein; IS, immunosuppressive agent; mab, monoclonal antibody; TNF-α, tumor necrosis factor-α. Terms in parentheses refer to serum concentration. profoundly affect drug clearance, resulting in low or nonmeasurable trough drug concentrations and loss of response, other factors that affect the PK of TNF antagonists exist, including concomitant use of immunosuppressives, serum albumin concentration, body weight, the degree of systemic inflammation (e.g., serum albumin concentration and TNF burden), and disease type (e.g., CD vs. UC). Collectively, these factors probably account for the large interindividual differences in PK and clinical efficacy observed after standard dosing of mabs (Table 3). Determinants of the PK PD of mabs: challenges in interpreting the literature It should be noted that, to date, the majority of publications evaluating the relationship between the PK and PD of mabs have been compromised by the following problems: (i) retrospective study designs that are not optimally designed to identify relevant PK PD relationships; (ii) failure to accurately sample serum at the time of treatment failure/success; (iii) failure to perform pharmacokinetic sampling at informative times, thus limiting analytical power (peak and concentrations measured during the beta decline phase are more informative of drug clearance than trough samples); (iv) use of ADA assays that cannot detect ADAs in the presence of circulating drug; (v) use of inappropriate statistical methods ( as observed analyses do not adequately account for patients who withdraw from treatment prematurely and who are therefore more likely to have measurable ADAs and low serum drug concentrations; intent-to-treat evaluations suffer from other issues); 56 (vi) failure to account analytically for the effects of confounders; (vii) inclusion of patients without evidence of active inflammation, thus reducing statistical power; and (viii) failure to use objective PD end points (the majority of studies have used symptoms instead of objective findings of inflammation). Factors that may influence the PK and hence the PD of mabs in patients with IBD are reviewed below. Clinical pharmacology & Therapeutics 7

8 Role of ADAs. Immunogenicity is a major issue related to therapeutic efficacy of mabs. Development of ADAs can affect the safety profile of these drugs because of hypersensitivity reactions. mabs are exogenous proteins and can therefore induce an immune response leading to the production of endogenous ADAs, which in turn leads to a reduced therapeutic efficacy of the drug. Depending on the structure of mabs, ADAs can be classified as human antimouse antibodies, human antichimeric antibodies, or human antihuman antibodies. Theoretically, immunogenicity decreases with the degree of humanization. Although infliximab, because of its chimeric structure, is theoretically more immunogenic than adalimumab and certolizumab, data from clinical trials evaluating the efficacy of these mabs in patients with IBD confirm that both adalimumab and certolizumab can also induce immunogenicity and that the degree of immunogenicity seems to be relatively similar to that seen with infliximab. 32,42,49,57 Development of ADAs results in the formation of immune complexes that accelerate drug clearance by the RES and/or impaired binding to target. If the drug antibody complex is inactive ( neutralizing antibody ) and if the antibody binding capacity is similar to the total concentration of the therapeutic protein, decreased efficacy may ensue as a result of a decline in free concentrations of the active agent. Alternatively, if the drug antibody complex is active, enhanced bioactivity may result. 55 In some cases, the drug antibody complex will be cleared ( clearing antibody ), which provides an alternative clearance pathway for the therapeutic protein, decreasing total and free concentrations and leading to decreased bioactivity. Therefore, immunogenicity usually impacts clinical response to therapy in a negative manner by affecting bioavailability, PK, and PD. 58 However, this topic has been controversial because of methodological challenges. The majority of published data evaluating the influence of ADAs on the pharmacokinetic properties of mabs are based on solid-phase enzyme-linked immunosorbent assays in which the presence of circulating drug renders the test insensitive in detecting ADAs. In addition, solid-phase enzyme-linked immunosorbent assay are associated with false-positive results due to nonspecific binding to immunoglobulins other than infliximab. However, highly sensitive liquid-phase mobility-shift assays and liquid-phase radioimmunoassays that measure ADAs in the presence of circulating drug are emerging, which should provide more accurate evaluation of the rate and intensity of sensitization early in the course of treatment with mabs. This advance highlights the potential of therapeutic drug monitoring to direct interventions, such as dose intensification or immunosuppression, that may prevent primary and secondary loss of response. Several studies have consistently linked the presence of ADAs to inferior outcomes. 37,41,42 Baert et al. showed that the presence of a high titer of ADAs in patients with CD was associated with a reduced duration of response in comparison with nonsensitized patients (35 days vs. 71 days, respectively; P < 0.001). 41 Similarly, the formation of ADAs in patients with CD has been associated with lower rates of prednisone-free clinical remission (57.1% (prednisone-free clinical remission in patients with positive ADAs) vs. 70.6% (prednisone-free clinical remission in patients with negative ADAs), respectively). 37 Role of concomitant immunosuppressive therapy. Although the mechanism by which immunosuppressives (azathioprine, mercaptopurine, and methotrexate) increase the concentration of serum mabs is not well established, they are likely to exert this function by reducing the formation of ADAs and/or downregulating RES-mediated drug clearance. In some cases, immunosuppressives downregulate receptors for mabs, which also slows the clearance of mabs. Because of the methodological problems previously described, the role of concomitant immunosuppressive therapy as a determinant of the PK of mabs is poorly understood. Post hoc analysis of four randomized controlled trials showed that concomitant use of immunosuppressives with infliximab was associated with higher serum infliximab concentrations. 50 In the SONIC trial, patients with active CD who received combination therapy (infliximab plus azathioprine) had higher trough infliximab concentrations than those who received infliximab monotherapy (3.5 μg/ml vs. 1.6 μg/ ml, respectively; P < 0.001). These findings correlated with better outcomes in terms of higher corticosteroid-free remission rates in the combination therapy arm. 37 Although it is apparent that coadministration of azathioprine decreased drug clearance in the SONIC trial, the mechanisms responsible are unclear. One likely mechanism is reduction of ADA formation (0.9% in patients receiving combination therapy vs. 14.6% in patients receiving infliximab monotherapy). As mentioned above, coadministration of immunosuppressives with TNF antagonists increases serum drug concentrations and decreases the formation of ADAs. 41,46,47,50 In patients with UC, however, factors other than immunosuppressive-mediated clearance may have, quantitatively, the most critical effect in determining the PK of infliximab, at least during the acute phase of therapy. Role of the RES and disease severity. Because of their high molecular weight, mabs do not undergo renal elimination or metabolism by hepatic enzymes; rather, as previously described, proteolytic catabolism within the cells of the RES is the primary route of elimination. 12 Disease severity may influence elimination of mabs through RES-mediated mechanisms. In regard to this observation, it has been shown that patients with elevated C-reactive protein and serum albumin concentrations below the normal range have accelerated drug clearance. 14,15,59 The presence of systemic inflammation may therefore increase mabs catabolism in the RES. Unfortunately, there is no practical way to measure this phenomenon. However, generating data for each mab and disease using a range of covariates related to the inflammatory burden of the disease could be an indirect way to determine the degree of systemic inflammation required before the PK of mabs is substantially affected. This assumption holds out the possibility that patients with more severe inflammation may require higher than average drug exposure for optimal results. This hypothesis may account for the suboptimal infliximab concentrations that have been observed 8

9 in patients with severe UC undergoing infliximab induction therapy. 43 As an additional route of mab clearance, the intestinal clearance of IgG was investigated in patients with IBD (inactive and active CD and UC) as well as in a control group. The authors reported increases in intestinal clearance of monomeric IgG that were closely related to the severity of the intestinal lesions. 60 Role of the antigen sink. The antigen-dependent clearance pathway is often referred as an antigen sink. It has been shown that receptor density (i.e., antigen targeted by mabs) clearly influences the PK of mabs. Therefore, differences in PK do exist with different indications, and, indeed, changes in PK with patient response have been reported. In the case of gemtuzumab, an antibody targeting CD33-positive blast cells with chemotherapeutic properties in patients with acute myeloid leukemia, serum concentrations and half-life increased after a second dose as compared with the first dose of the drug. This observation was attributed to a decreased clearance by CD33-positive blast cells due to a reduced tumor burden after the first dose. 61 One potential explanation for lack of response to TNF antagonists is incomplete suppression of TNF-α activity because of insufficient serum drug concentrations to block the excess of TNF. A high inflammatory burden at baseline is associated with higher concentrations of TNF-α in both tissue and serum. Patients with a higher degree of systemic inflammation may therefore require, in a stoichiometric fashion, greater amounts of drug to neutralize this excess of TNF-α. In turn, this could result in lower mab serum concentrations and less functional available drug. In this paradigm, the higher the baseline TNF-α concentration, the higher the dose of drug required to achieve a pharmacodynamic response. In addition, baseline TNF-α serum concentrations may predict the need of dose escalation in cases of loss of response. 62 Ainsworth et al. evaluated 33 patients with CD treated with infliximab classified by response status (primary nonresponse, secondary loss of response, and sustained response) and found that patients who were primary nonresponders to infliximab had higher affinity to bind TNF-α in serum than patients with secondary loss of response and had no antibodies against infliximab (Supplementary Figure S3 online). The authors concluded that measurement of serum TNF-α binding capacity in conjunction with ADAs may provide new insights into the causes of treatment failure (sensitization vs. non-tnf inflammatory pathway vs. inadequate drug concentration in the absence of sensitization). 63 Interestingly, not only can the measurement of serum TNF-α concentration be used as a surrogate marker of the PK of mabs but its measurement in colonic tissue seems to also be useful for this purpose. Olsen et al. demonstrated that the likelihood of inducing clinical or endoscopic remission after induction therapy with infliximab in patients with UC was inversely associated with pretreatment concentration of TNF-α in colorectal mucosa. 64 The clinical implication of this observation is that pretreatment values of colorectal TNF-α may be used, together with other factors, as a surrogate marker to individualize infliximab dosing regimen. Patients with higher pretreatment TNF-α colonic concentration may require higher doses. Role of disease type: CD vs. UC. Potential pharmacokinetic differences between CD and UC exist that may or may not result from differences between the previously discussed factors. Infliximab clearance seems to be similar for CD, RA, and psoriasis. In distinction, potentially important pharmacokinetic differences exist between CD and UC. 43,44 Seow et al. demonstrated that patients with moderate UC had higher rates of clinical response (70% vs. 41%; P = 0.004), clinical remission (41% vs. 17%; P = 0.015), and endoscopic remission (26% vs. 4%; P = 0.046) than those with severe disease after infliximab induction treatment. Undetectable trough infliximab concentrations were associated with less favorable outcomes. 43 In the study by Seow et al., the proportion of patients with nonmeasurable infliximab trough concentrations was higher than that in a previous study performed by the same investigators in patients with CD. 44 One potential explanation of these findings is that patients with UC have a more rapid clearance of infliximab than patients with CD. It is noteworthy that two large-scale randomized con trolled trials have shown relatively low rates of remission with s.c. adalimumab induction therapy in UC 35,65 at doses that result in relatively high rates of remission in CD. In distinction, both intravenously administered infliximab and subcutaneously administered adalimumab are effective in CD. 30,49 These findings raise the possibility that fundamental pharmacokinetic differences with important clinical consequences may exist between the two diseases. One hypothesis that may account for these differences is that the overall inflammatory burden in patients with severe and extensive UC is higher than that in patients with CD owing to a greater inflamed intestinal surface. This higher inflammatory burden may result in higher production of TNF-α. Therefore, higher doses of mabs may be required to neutralize the excessive production of the target antigen. Alternatively, patients with UC may have a greater area of the mucosal surface affected than is observed in patients with CD, resulting in greater loss of drug in the intestinal lumen. Although the concept of a drug-losing enteropathy is entirely hypothetical, future pharmacokinetic studies should explore this hypothesis. Role of patient factors. It is worth noting that the influence of weight on the clearance, and therefore area under the curve, of mabs is not linear. Hence, dosing based on weight does not always produce drug exposure that is efficacious. Monitoring of serum drug concentrations is consequently more important in patients with both low weight and high inflammatory burden than in patients with higher weight and/or less inflammation. Gender has also been shown to independently influence the disposition of mabs, with clearance being higher in men. 14,66 However, because weight and gender are generally somewhat correlated (men weighing more than women), this finding may be related to weight. The clearance of mabs is not affected by either renal or hepatic dysfunction. However, an interesting association between baseline serum albumin concentrations and serum infliximab concentrations in both UC and CD has recently been reported. 15,67 Patients with a baseline serum albumin concentration below the Clinical pharmacology & Therapeutics 9

10 a CL (ml/kg/d) b CL (ml/kg/d) Base model SAC (g/dl) Base model (the lower the albumin concentration, the higher the infliximab clearance) (Figure 4). Recently, a study of patients with RA treated with infliximab has shown that a high body mass index negatively influences clinical response. 68 Research into the role of mesenteric fat in chronic inflammatory diseases has intersected with investigation into the importance of adipose tissue as a metabolically active source of proinflammatory cytokines (e.g., TNF) in patients with insulin resistance. It would be expected, therefore, that obese patients with CD would inherently have higher TNF production than patients with normal weight, suggesting also that the mg/ kg dose paradigm might be inappropriate for obese patients; these patients may require higher drug doses than those currently recommended. This observation requires confirmation in additional studies. Accordingly, measurement of body mass index (and potentially quantitative assessment of mesenteric fat) should be incorporated into future pharmacokinetic studies, especially in patients with CD, in whom adipose tissue is involved in the inflammatory process. 69 In summary, preliminary evidence suggests that multiple factors influence the PK of mabs. Understanding the determinants of the PK of mabs has great potential to improve and optimize the therapeutic management of patients with IBD. c % Responders Albumin level (g/dl) 2 3 P = < Placebo SAC (g/dl) 4 5 P = P = < IFX-Treated All albumin concentrations Placebo IFX-Treated Figure 4 Albumin as a predictive factor of infliximab clearance. Serum albumin concentration (SAC) is inversely related to infliximab clearance (CL) in both (a) ulcerative colitis (UC) and (b) Crohn s disease. (c) Relationship between serum album concentrations and clinical response rates in patients with UC treated with infliximab (IFX) and placebo. Patients with serum albumin concentration below the normal range achieved lower response rates. Adapted with permission from refs. 15 and 67. normal range (a common finding associated with severe inflammation) have lower remission rates after treatment with infliximab. This observation suggests that an inverse relationship exists between serum albumin concentration and infliximab clearance Conclusions and Future Directions The use of anti-tnf mabs in the treatment of IBD has led to improved disease outcomes. However, further optimization is needed because a high proportion of patients fail to respond to these therapies. Monitoring serum drug concentrations and ADAs (immunogenicity) may lead to more appropriate therapeutic management of patients with loss of response. The PK of mabs seems to be strongly influenced by several factors related to patient and disease characteristics. Evaluation of the covariates that influence the disposition of mabs may help in identifying patients who are more likely to benefit from receiving higher doses as a result of accelerated drug clearance. Unfortunately, studies integrating these variables into a single PK model in targeted populations have not been performed yet. The number of approved mabs for the treatment of IBD is expected to increase. Therefore, a better understanding of the factors that impact the PK and PD of mabs is crucial to ensure more efficient dosing regimens, which in turn may enhance the therapeutic success of these therapies. Combined clinical, imaging, and PK studies should lead to further advances in customizing drug dosage and monitoring therapeutic response. Finally, individualized and tailored dosing approaches guided by PK algorithms may be safer, more effective, and even cost-effective. SUPPLEMENTARY MATERIAL is linked to the online version of the paper at Conflict of Interest The authors declare no financial or other conflict of interest in relation to the content of this icle. I.O. does not have any stocks, equity, a contract of employment, or a named position on a company board in companies 10

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