Nicolas Azzopardi

An enzyme-linked immunosorbent assay for therapeutic drug monitoring of cetuximab.

An enzyme-linked immunosorbent assay (ELISA) measuring serum infliximab concentrations in treated patients was developed. Microtiter plates were sensitized with tumor necrosis factor α (TNF-α) and saturated with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Samples diluted 1:100 in PBS-1% BSA were added and bound infliximab was detected using peroxidase-conjugated goat anti-human immunoglobulin G specific for Fc fragment (HRP-anti hIgG). Reading was performed using an ELISA plate reader. The limit of detection, calculated by assaying 10 replicates of a drug-free serum sample or blank sample and defined as the lowest concentration distinguishable from zero at 2 standard deviations, was 0.014u2009μg/mL. Each quality control sample was tested on 7 occasions on 1 day and on 5 separate days. The intraday precision indices of the method were (percent coefficients of variation, CV%) 11.7%, 6.2%, and 6.9% for 0.04u2009μg/mL, 2u2009μg/mL, and 4.5u2009μg/mL, respectively. The corresponding bias measures (percent deviation) were −5.5%, −1.9%, and −7.9%, respectively. The between-days precision was 9.8%, 5.3%, and 5.3% for 0.04u2009μg/mL, 2u2009μg/mL, and 4.5u2009μg/mL, respectively. The corresponding bias were +0.3%, −0.3%, and −7.8%, respectively. Lower limit of quantitation and upper limit of quantitation were 0.04u2009μg/mL and 4.5u2009μg/mL, respectively. Trough serum concentrations of infliximab were measured in 6 adult patients with various diseases and in 5 pediatric patients with Crohns disease. For the latter group, samples drawn 1 hour after the end of the infusion and repeated measurements also were available. Data were described using a 1-compartment population pharmacokinetic model. Terminal elimination half-life was 10.9 days. This method is rapid, accurate, and reproducible, and may be useful in therapeutic drug monitoring of infliximab.

Cetuximab Pharmacokinetics Influences Progression-Free Survival of Metastatic Colorectal Cancer Patients

Purpose: An ancillary phase II study was conducted to study interindividual variability in cetuximab pharmacokinetics and its influence on progression-free survival (PFS) in metastatic colorectal cancer patients cotreated with irinotecan and 5-fluorouracil. Experimental Design: Ninety-six patients received cetuximab as an infusion loading dose of 400 mg/m2 followed by weekly infusions of 250 mg/m2. Doses of irinotecan and 5-fluorouracil were adjusted individually. Cetuximab concentrations were measured by ELISA. Compartmental pharmacokinetic parameters were estimated by a population approach, and PFS was analyzed using a Cox model. Results: Cetuximab pharmacokinetics was best described using a two-compartment model with both first-order and saturable (zero-order) elimination. Estimated pharmacokinetic parameters (% standard error) were as follows: central volume of distribution V1 = 2.96 L (4%), peripheral volume of distribution V2 = 4.65 L (6%), elimination clearance CL = 0.497 L/d (4%), distribution clearance Q = 0.836 L/d (8%), and zero-order elimination rate k0 = 8.71 mg/d (10%). Body surface area influenced V1, V2, and k0. Pretreatment serum albumin influenced CL. Risk of disease progression decreased with cetuximab global clearance (cumulative dose/cumulative area under the concentration versus time curve; P = 0.00016). Median PFS of patients with a cetuximab residual concentration on day 14 below median value was 3.3 months as compared with 7.8 months for the other patients (P = 0.004). Conclusions: Cetuximab pharmacokinetics in colorectal cancer patients can be described using a model combining linear and nonlinear elimination rates. PFS is influenced by global clearance of cetuximab, a parameter that can be estimated using cetuximab residual concentration on day 14. Clin Cancer Res; 17(19); 6329–37. ©2011 AACR.

The Airways, a Novel Route for Delivering Monoclonal Antibodies to Treat Lung Tumors

ABSTRACTPurposeLung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited, with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer. We have shown that cetuximab, a chimeric anticancer anti-EGFR mAb, is suitable for airway delivery as it resists the physical constraints of aerosolization, and have evaluated the aerosol delivery of cetuximab in vivo.MethodsWe developed an animal model of lung tumor sensitive to cetuximab by injecting Balb/c Nude mice intratracheally with A431 cells plus 10xa0mM EDTA and analyzed the distribution, pharmacokinetics and antitumor efficacy of cetuximab aerosolized into the respiratory tract.ResultsAerosolized IgG accumulated durably in the lungs and the tumor, but passed poorly and slowly into the systemic circulation. Aerosolized cetuximab also limited the growth of the mouse tumor. Thus, administering anticancer mAbs via the airways is effective and may limit systemic side effects.ConclusionDelivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.

Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system.

Monoclonal antibodies (mAbs) are usually delivered systemically, but only a small proportion of the drug reaches the lung after intravenous injection. The inhalation route is an attractive alternative for the local delivery of mAbs to treat lung diseases, potentially improving tissue concentration and exposure to the drug while limiting passage into the bloodstream and adverse effects. Several studies have shown that the delivery of mAbs or mAb-derived biopharmaceuticals via the airways is feasible and efficient, but little is known about the fate of inhaled mAbs after the deposition of aerosolized particles in the respiratory system. We used cetuximab, an anti-EGFR antibody, as our study model and showed that, after its delivery via the airways, this mAb accumulated rapidly in normal and cancerous tissues in the lung, at concentrations twice those achieved after intravenous delivery, for early time points. The spatial distribution of cetuximab within the tumor was heterogeneous, as reported after i.v. injection. Pharmacokinetic (PK) analyses were carried out in both mice and macaques and showed aerosolized cetuximab bioavailability to be lower and elimination times shorter in macaques than in mice. Using transgenic mice, we showed that FcRn, a key receptor involved in mAb distribution and PK, was likely to make a greater contribution to cetuximab recycling than to the transcytosis of this mAb in the airways. Our results indicate that the inhalation route is potentially useful for the treatment of both acute and chronic lung diseases, to boost and ensure the sustained accumulation of mAbs within the lungs, while limiting their passage into the bloodstream.

Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman® technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs’ distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

Dose – response relationship of bevacizumab in hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder associated with epistaxis and hepatic shunts, is responsible for high-output cardiac failure in rare cases. Bevacizumab, which targets vascular endothelial growth factor, was shown to decrease both cardiac index (CI) and epistaxis duration in HHT patients with severe liver involvement. The relationship between its serum concentration and change in both CI and epistaxis duration was investigated to design the bevacizumab maintenance dosing regimen of future therapeutic studies. Twenty-five HHT patients with dyspnea and high CI were included in a prospective non-comparative study. They received bevacizumab at a dose of 5 mg/kg per infusion every 14 days for a total of 6 injections. The relationships between bevacizumab serum concentration and both CI and epistaxis duration were described using transit compartments and direct inhibition pharmacokinetic-pharmacodynamic models. The performances of different maintenance regimens were evaluated using simulation. Infusions every 3, 2 and one months were predicted to maintain 41%, 45% and 50% of patients with CI <4 L/min/m2 at 24 months, respectively. The fraction of patients with <20 min epistaxis per month was predicted to be 34%, 43% and 60%, with infusion every 3, 2 or one months, respectively. Simulations of the effects of different maintenance dosing regimens predict that monthly 5 mg/kg infusions of bevacizumab should allow sustained control of both cardiac index and epistaxis.

Pharmacokinetics of adalimumab in Crohn’s disease

Adalimumab, an anti-TNF-α monoclonal antibody, is approved for the treatment of rheumatoid arthritis (RA), ankylosing spondylitis, psoriatic arthritis, Crohn’s disease (CD), and ulcerative colitis. The pharmacokinetics (PK) of adalimumab in RA patients was investigated using compartmental modeling [1, 2], but its characteristics in CD patients has never been reported. Patients may develop antiadalimumab antibodies (AAA) which are responsible for a decrease in adalimumab serum concentration [3] and a loss of clinical response [4, 5, 3, 6, 7]. Antibodies towards another anti-TNF-α monoclonal antibody, infliximab, were shown to increase its clearance [8-11]. The quantitative influence of anti-adalimumab antibodies (AAA) on adalimumab clearance has not been reported yet. We report the first quantitative description of adalimumab pharmacokinetics in Crohn’s disease patients. This is a post hoc analysis of a prospective cohort of 65 patients, being part of a larger cohort, who participated in an observational study in the Inflammatory Bowel Disease unit of the University Hospital of Leuven and for whom at least four adalimumab concentrations were available. [3] Patients were treated subcutaneously either with 160/80 mg at weeks 0/2, or 80/40 mg as an induction phase. Following this phase, all patients were treated with 40-mg adalimumab every 2 weeks. Median follow-up was 20 months. Median [range] age and body weight were 37 years [17–61] and 68 kg [43– 109], and 49 (75 %) were women. For 30 out of 65 patients, dosing regimen was increased to 40 mg every week. A total of 341 adalimumab serum concentrations were available. Antiadalimumab antibodies were detected in nine patients. Adalimumab concentrations were measured using an ELISA technique derived from the one developed for infliximab, [12] and AAA were measured using a double-antigen ELISA based on their capture by adalimumab-coated plates. Adalimumab concentrations and AAA detection were performed in Tours University Hospital, France. Adalimumab pharmacokinetics was studied by a population approach using MONOLIX 4.3.2 (Lixoft, Saclay, France). A one-compartment model with first-order absorption and elimination rates was used. Apparent volume of distribution (V/F), clearance (CL/F), and first-order absorption rate constant (ka) were estimated. Interindividual and residual models were exponential and mixed additive-proportional, respectively. The presence of AAAwas tested as a covariate on V/F and CL/F. All parameters were estimated with satisfactory accuracy, and no obvious model misspecification was observed. Estimated typical parameters (relative standard error) were V/F = 13.5 L (10 %), CL/F = 0.42 L/day (9 %) and ka = 0.15 day . Interindividual standard deviations for V/F and CL/F (relative standard error) were Konstantinos Karmiris is the Principal investigator.

Bevacizumab Pharmacokinetics Influence Overall and Progression-Free Survival in Metastatic Colorectal Cancer Patients.

ObjectiveClinical response to bevacizumab varies between patients treated for metastatic colorectal cancer (mCRC). The aim of this study was to quantify individual factors affecting bevacizumab pharmacokinetic variability and assess the relationship between bevacizumab concentrations and clinical outcomes.MethodsBevacizumab pharmacokinetics were assessed in 130 mCRC patients using a two-compartment pharmacokinetic population model. Overall and progression-free survival (PFS) were analyzed using Cox models.ResultsThe bevacizumab volume of distribution increased with height (pxa0=xa010−10) and was higher in patients with a 3/3 variable number tandem repeat of the FCGRT (Fc fragment of IgG receptor and transporter) gene (pxa0=xa00.039). The elimination rate constant increased with baseline carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) concentrations, and was higher in patients with extra-hepatic metastases (pxa0=xa00.00029, 0.011, and 0.014). A bevacizumab trough concentration ≤15.5xa0mg/L was associated with both shorter overallxa0survival and PFS (hazard ratio [95xa0% CI] 1.90 [1.20–2.99] and 1.76 [1.20–2.58], respectively).ConclusionHigh tumour burden is associated with low bevacizumab concentrations, and high bevacizumab concentration are associated with both decreased overall and progression-free survivals.

VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-rasLA1 model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.

Trough infliximab concentration may predict long-term maintenance of infliximab in ankylosing spondylitis

Infliximab is approved for refractory ankylosing spondylitis (AS) but 39% of patients experience no response to treatment after 24 weeks (1). de Vries et al reported that the clinical response was related to infliximab concentration, so exposure to infliximab may account for the variability in response (2). However, more recent data have not confirmed that responsiveness is associated with infliximab concentration (3). We analysed the relationship between trough infliximab concentration and long-term maintenance of infliximab treatment in patients with AS routinely treated in our centre. Patients at initiation of treatment were excluded from the study. On the day of the infusion (baseline), infliximab concentration was measured by enzyme-linked immunosorbent assay as described previously (4), as was positivity for antibodies towards infliximab (ATI). Disease activity was assessed at each infusion, for 52 weeks, by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Infliximab treatment was categorized by comparing infusion dose and dosing interval with those recorded at the baseline infusion. We considered infliximab dosage stable if both the dose (in mg/kg) and the interval (94 days) were equivalent between baseline and subsequent infusions. Dose modification and discontinuation of infliximab were based on disease activity and tolerance. Patients were divided into two groups on the basis of their staying with the same or lower dosage (group A infliximab success) or switching to another biopharmaceutical or requiring an increase in dosage (group B infliximab failure). Infliximab maintenance in group A at the last follow-up was compared between categories of infliximab concentration at baseline according to median infliximab concentration by Kaplan Meier curves and a logrank test. We analysed data for 22 patients with AS; the demographic, clinical, and biological characteristics are presented in Table 1. Patients with an infliximab concentration above the median at baseline showed a low, although not statistically significant, risk of treatment failure (p 0.14). Three patients were positive for ATI at baseline with no detectable infliximab concentration. During follow-up, one patient switched to etanercept 12 weeks after baseline measurement, one discontinued treatment 30 weeks after baseline measurement, and one remained in group A at 56 weeks after baseline measurement. Patients with high concentrations of infliximab at baseline showed sustained maintenance of infliximab treatment, although the results were not statistically significant, possibly because of better control of disease activity (Figure 1). In an open-label study (5) and a randomized controlled trial (1), patients with inadequate response to infliximab were found to benefit from dosage escalation. Systematic treatment was also found to produce a better response than an ‘on-demand’ regimen (6). Our study was based on a single measurement of serum infliximab concentration at baseline. A repeated measurement of infliximab concentration would have allowed us to confirm the status of the patients in terms of their exposure to infliximab, and to analyse the impact of exposure on treatment maintenance more precisely. Positivity for ATI was shown to influence response to infliximab. The study by de Vries et al found that