Dossier: Autoimmunity and Biotherapy
Multidrug resistance-1 (MDR-1) in autoimmune disorders IV. P-glycoprotein overfunction in lymphocytes from myasthenia gravis patients

https://doi.org/10.1016/j.biopha.2004.04.008Get rights and content

Abstract

Multidrug resistance (MDR) mechanisms have been widely studied in cancer. Among them, P-glycoprotein (P-gp) overfunction has been associated with resistance to several antineoplastic agents. The physiological role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space, thus inhibiting their toxic or therapeutic effects. The study of MDR-1 in diseases of autoimmune origin has just recently emerged. Corticosteroids remain the mainstay therapy for autoimmune diseases. As prednisone (PDN) is transported by P-gp, the aim of this study was to evaluate the P-gp function in lymphocytes from myasthenia gravis (MG) patients. Thirty MG patients and 25 healthy controls were studied. Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (DNR) (a fluorescent drug extruded by P-gp). Functional activity of P-gp was analyzed by flow cytometry. Results were expressed as percentage of gated lymphocytes able to efflux DNR. Overall, MG patients showed increased numbers of lymphocytes with functional P-gp activity when compared with controls (x = 4.92 ± 5.26% vs. x = 0.7 ± 0.48%, respectively) (P < 0.0001). When patients were classified as responders (n = 21) or refractory (n = 9) to treatment, the latter group exhibited higher values of functional P-gp (x = 10.18 ± 6.39%) when compared to the responder group (x = 2.66 ± 2.45%) (P = 0.0076). These data suggest, on the one hand, that drug resistance may be induced by long-term treatment or by high PDN doses and, on the other, emphasize the need for the study of P-gp antagonists in order to improve the current therapeutical schemes for the treatment of MG.

Introduction

Myasthenia gravis (MG) is an acquired, neuromuscular autoimmune disorder characterized by skeletal muscle weakness and fatigue. It is generally caused by antibodies against nicotinic acetylcholine receptor (AChR) [1]. The disease is heterogeneous with respect to age at onset, thymic pathology and distribution of muscle weakness, all of which, together with its genetics, need to be kept in mind when considering treatment [2]. Most patients benefit from pyridostigmine. In nonthymomatous ocular MG, prednisone (PDN) is often effective. Thymectomy is indicated for thymoma and is an option for AChR antibody-positive patients with generalized weakness developing under the age of 45 years. Therapeutic failures are often seen among MG patients and plasma exchange, the use of cyclosporine A, metothrexate, cyclophosphamide, or intravenous immunoglobulin G administration may provide improvement of symptoms. Notwithstanding, all of these therapeutic schemes have a high risk of side effects and their cost is often considerable. In older patients or in those failing to respond to thymectomy, PDN alone or combined with azathioprine (AZA) remains the mainstay therapy [2].

Acquired unresponsiveness to corticosteroids in MG may be related to drug resistance mechanisms. Among these, one of the most extensively studied, mainly in cancer, is the so-called multidrug resistance-1 (MDR-1) which is characterized by the overfunction of a 170 kDa P-glycoprotein (P-gp) [3]. This molecule belongs to a superfamily of the ATP-binding cassette (ABC) transporters [4]. The MDR-1 phenotype involves a decreased sensitivity to several agents including vinca alkaloids, anthracyclines and glucocorticoids among others [5]. This phenomenon is the consequence of the drug extrusion by P-gp, which diminishes the intracellular drug concentration, thus reducing its therapeutic action [6]. Although the physiological role of P-gp is not yet completely understood, it has been implicated in bacterial product detoxification, and in hormone as well as metabolite secretion [4].

Little is known about the possible role of P-gp in autoimmune diseases that require therapy with drugs actively effluxed by this energy-dependent pump, e.g. PDN. The aim of this study was to explore whether P-gp function could be related to the clinical response to treatment in MG patients.

Section snippets

Patients

The study included 30 MG patients, 20 women and 10 men (age range from 20 to 62 years, mean 36 years). The diagnostic was made on clinical grounds and confirmed by positive edrophonium chloride test and by a diminished electrophysiologic muscule response to repetitive supramaximal stimulation [7]. Individual features at the time of the study are shown in Table 1. Patients were classified into two groups according to their response to treatment as follows: (i) responders (those patients with no

Results

The whole group of MG patients showed higher percentages (x = 4.92 ± 5.26%, range 0.33–18.6%) of lymphocytes able to extrude DNR when compared with healthy subjects (x = 0.7 ± 0.48%, range 0.05–1.98%) (P < 0.0001). When patients were compared according to their clinical response to treatment a clear-cut difference was observed. Thus, the refractory patients exhibited increased P-gp values than the responder MG group (x = 10.18 ± 6.39% vs. x = 2.66 ± 2.45%, respectively; P = 0.0076) (Fig. 1A).

Discussion

The study of drug resistance mechanisms, particularly MDR-1, is no longer circumscribed exclusively to neoplastic diseases. Recent information concerning the possible role of P-gp function in other disorders like HIV infection, epilepsy, and inflammatory bowel disease as well as in autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus and immune thrombocytopenic purpura, has opened an exciting area of research, for therapeutic failures are often seen among these

Acknowledgements

We thank Dr. Juanita Romero for her collaboration with statistical analysis. Dr. Antonio Vidaller was visiting professor. This work was supported in part by a research grant from the Fundación Miguel Alemán, A.C., Mexico City, Mexico.

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