Pharmacology Journal

Antilymphocyte globulins (ALGs) are polyclonal antibodies used in organ transplantation to prevent or treat acute allograft rejection. ALGs are obtained by immunizing animals (rabbits or horses) with human lymphoid cells (lymphoblasts, lymphocytes or thymocytes). Although ALGs have been in use for several decades, the interindividual variability in patient response remains poorly understood. Monitoring of drug effect is based on lymphocyte numeration, because ALG doses may be decreased when lymphopenia is obtained or the drug readministered to maintain it. Although lymphocyte count is an established biomarker of ALG therapeutic effect, the study of its quantitative relationship with ALG concentrations has not been reported.

The mechanism of action of ALG is probably complex, but it eventually results in target cell destruction. Several mechanisms have been observed in vitro, including apoptosis, complement-dependent cytotoxicity (CDC) and also antibody-dependent cellular cytotoxicity (ADCC) in the presence of cytotoxic cells expressing receptors for the Fc portion of IgG (FcγR). The FCGR3A gene, which encodes FcγRIIIa, a FcγR expressed by macrophages and natural killer cells, displays a single nucleotide polymorphism at position 559 of the cDNA (G or T, rs number 17857127), which generates two FcγRIIIa allotypes, with a valine (V) or a phenylalanine (F) at position 158, respectively. Non-Hodgkin’s lymphoma patients with the 158V allotype have a better response to rituximab, an anti-CD20 cytolytic therapeutic antibody, than patients with the 158F allotype.

FCGR3A polymorphism may therefore be a genetic factor influencing the effect of cytolytic therapeutic antibodies, although it remains to be demonstrated that this is also true for antibodies of animal origin (with an animal Fc portion). The aim of this study was to analyse the concentration–effect relationship of horse ALG in renal transplant patients, with the hypothesis that FCGR3A polymorphism may influence the ALG effect, as measured by lymphocyte depletion, and to explain part of the interindividual variability of its effect.

Patients

This study was based on both a prospective and a retrospective group of patients. The prospective group consisted of 14 kidney transplant patients included in a noncomparative study of horse ALG pharmacokinetics (PK) and pharmacodynamics (PD), recruited in four renal transplant centres (Caen, Poitiers, Reims and Tours, France). They had received a first cadaveric renal transplantation with a cold ischaemia time <48 h. The patients were not hyperimmunized (panel-reactive IgG antibodies <75%) and had never been treated with horse ALG. This prospective study was approved by the local ethics committee (CCPPRB of Tours) and all patients gave written informed consent, both for the clinical study and for FCGR3A genotyping.

The retrospective group was also studied to increase the number of patients and to validate our models. It consisted of six renal transplant recipients treated with the same horse ALG. They had been transplanted in the centre of Tours and gave written informed consent for FCGR3A genotyping. Patients’ characteristics are shown in.

Treatment

Antilymphocyte globulin dosing regimen In the prospective group, ALG (Lymphoglobuline^®; Genzyme Imtix-SangStat, Lyon, France) was administered through a central venous catheter or an arteriovenous fistula. The first infusion was started within the first hour after patient entry in the recovery room and lasted for 24 h. The second infusion was started at least 12 h after the end of the first infusion and subsequent infusions over a period of 8–12 h, separated by a 24-h period. The initial dose was 10 mg kg^−1 day^−1 without exceeding 600 mg per infusion. After the first two infusions, the dose was halved if T-cell lymphopenia was achieved, with a minimum of 100 mg per infusion. T-cell lymphopenia was defined by one of the two following criteria: a CD3+ lymphocyte count of <20 cells mm^−3 (measured using flow cytometry) or a total lymphocyte count of <200 cells mm^−3. The conditions leading to ALG discontinuation were a platelet count of <50 000 cells mm^−3 or a leucocyte count of <1500 mm^−3. When severe adverse effects possibly related to the studied ALG were observed, they were replaced by Thymoglobuline^® (Genzyme Imtix-SangStat), an ALG of rabbit origin. Patients in the retrospective group had been administered an initial dose of 10 mg kg^−1. Subsequent doses had been adjusted to maintain a CD3+ count of <20 cells mm^−3.

Concomitant immunosuppressive treatment Concomitant immunosuppressive treatment consisted of ciclosporin (Neoral^®; Novartis Pharmaceuticals, Basel, Switzerland), mycophenolate mofetil (Cellcept^®; Roche, Neuilly sur-Seine, France) and corticosteroids. Ciclosporin was given at an initial dose of 8 mg kg^−1 day^−1, starting after the sixth infusion of ALG, if serum creatinine was <250 µmol l^−1. A 48-h overlap between ciclosporin introduction and ALG discontinuation was maintained, in order to ensure adequate immunosuppression. The dose of ciclosporin was individually adjusted to reach trough concentrations of 150–250 ng ml^−1. Mycophenolate mofetil was administered orally prior to transplantation, at an initial dose of 2 g day^−1, subsequently adjusted according to clinical and haematological parameters. On the day of transplantation, 250 mg of methylprednisolone were infused before and after surgery, in the latter case before the start of ALG infusion. During the first week, 1 mg kg^−1 day^−1 of methylprednisolone or prednisolone were given before each ALG infusion. After day 7, the dose was progressively reduced. Before each ALG infusion, 5 mg of dexchlorpheniramine was administered.

Biological measurements

Antilymphocyte globulin serum concentrations At the time of the first infusion, ALG serum concentrations were measured before and 12 h and 18 h after the start of the first infusion, on days 4, 6, 8, 10 and 14 before the beginning of each ALG infusion and on days 30, 60 and 90 after the end of the first infusion. The number of available serum samples was reduced if horse ALG had to be discontinued because of side-effects. The day 30 sample was missing for patients P1, P4 and P7. Days 60 and 90 samples were missing for patients P1, P4, P6 and P7. Serum concentrations of horse ALG were measured by Imtix-SangStat (Marcy l’Etoile, France) using an enzyme-linked immunosorbant assay (ELISA) method. Briefly, the ELISA plates were coated with a goat antibody directed against equine IgG and incubated with diluted patient’s serum (1 : 500, 1 : 1500 and 1 : 4500). The presence of ALG was revealed by a goat antihorse IgG labelled with peroxidase. The lower limit of quantification was 0.3 ng ml^−1.

To detect possible immunization against horse ALG, blood samples were drawn before the first infusion and on days 10, 14 and 30. Measurements of human IgG and IgM directed against ALG were done by an ELISA using goat antihuman IgG or IgM labelled with peroxidase.

Lymphocyte count In both the prospective and retrospective groups, the total lymphocyte count was measured before transplantation, at the end of the first infusion and every day from day 2 to day 14. For patients P1, P4, P7 and P14, who were switched to another ALG during the course of the study, lymphocyte data were truncated, the first data point to be removed being the one measured after the first infusion of the other ALG.

FCGR3A genotyping FCGR3A genotyping was performed using a real time allele-specific polymerase chain reaction assay based on SYBR Green fluorescence.

Pharmacokinetic and pharmacokinetic–pharmacodynamic modelling

Pharmacokinetic modelling Models with one, two or three compartments and first-order elimination from the central compartment were tested. Compartmental PK parameters, i.e. volumes and clearances of central (V_c and CL_c) and peripheral compartments (V_p and CL_p), and half-lives (distribution and elimination, T_1/2-α and T_1/2-β) were estimated. Both individual and population approaches were used. Separate modelling of the infusion period (i.e. from the beginning of the first to the end of the last infusion) and the postinfusion period showed that some estimated PK parameters were time dependent: notably, V_c was smaller during the infusion period than after. A population PK model was therefore developed to take into account the decrease of V_c with time.

Filed under: Pharmacogenetics