International seminar on the red blood cells as vehicles for drugs.

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications. A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic ‘The Red Blood Cells as Vehicles for Drugs’. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of the applications, particularly for clinical use, of this innovative formulation. Indeed, due to the intrinsic properties of erythrocytes, their use as a drug carrier is one of the most promising drug delivery systems investigated in recent decades. Of the different methods developed to encapsulate therapeutic agents into RBCs [1,2,] the most widely used method is the lysis of the RBCs under tightly controlled hypotonic conditions in the presence of the drug to be encapsulated, followed by resealing and annealing under normotonic conditions (Figure 1). This results in uniform encapsulation of the material into the cells and a final product with good stability, reproducibility and viability. This process, which has now been developed to an industrial scale, is the technique chosen by the majority of the experts presenting their work in this seminar (by R Franco). Figure 1 The process of reversible hypotonic lysis of RBCs to entrap molecules Keywords: carriers, drug delivery, erythrocytes, red blood cells, targeting 1. Therapeutic enzyme-loaded RBCs Therapy using RBC encapsulated enzymes has the advantage of prolonging the half-life of the enzyme and maintaining therapeutic blood levels, reducing the dosage and frequency of therapeutic interventions, and preventing the need for expensive chemical modification [3]. The therapeutic index can be strongly improved, especially by reducing immunogenic reactions, which are often observed in enzyme replacement (Figure 2). Figure 2 The red blood cell as a bioreactor: the substrate (yellow) contained in the plasma permeates the erythrocyte membrane, with the entrapped enzyme (green) catalyzing the metabolism of the substrate to its normal product inside the red cell 1.1 L-asparaginase-loaded RBCs for ASNS-deficient tumor (by E Dufour) L-asparaginase has been used in the treatment of acute lymphoblastic leukemia for > 40 years. This enzyme converts plasmatic L-asparagine (L-Asn) into L-aspartate plus ammonia. Its use is motivated by the fact that malignant cells (especially leukemic) are deficient in asparagine synthetase (ASNS). Because these cells are unable to synthesize L-Asn to meet metabolic demands, L-Asn deprivation, due to L-asparaginase activity, kills the cancerous cells. However, L-asparaginase can also be responsible for adverse events such as hypersensitivity reactions or blood coagulations disorders, in addition to L-Asn depletion. An approach to decreasing side effects of free L-asparaginase in vivo is to entrap the enzyme in RBCs. Reversible hypotonic dialysis remains the most controlled and reproducible method. Indeed, with this process, human RBCs can be loaded with 116 ± 15 IU of L-asparaginase per milliliter of red cells. The resulting product acts as a bioreactor allowing transport of L-Asn through the RBC membrane where L-asparaginase hydrolyzes it. Due to the RBC membrane, the enzyme is protected from rapid catabolism as well as from potential neutralizing antibodies, resulting in an increased half-life and a reduction in hypersensitivity reactions. A Phase I–II trial testing GRASPA® (ERYTECH Pharma, France) on 24 patients in relapsed acute lymphoblastic leukemia showed a strong reduction in hypersensitive reactions, coagulation disorders and hepatic dysfunctions [4]. The L-asparaginase half-life is enhanced (40 days vs 1 day with the free form) and the mean duration of L-Asn depletion is 18.57 days at a dose of 150 IU/kg in a single injection that corresponds to eight injections of Escherichia coli native L-asparaginase. This improvement in tolerance allows the introduction of L-asparaginase treatment to other hematological malignancies, such as acute myeloid leukemia, and also in solid tumors. Indeed, the level of expression of L-ASNS, the enzyme responsible for the synthesis of L-Asn in mammalian cells, provides a rationale for testing L-asparaginase in several cancers. For example, about 30 and 40% of pancreatic ductal adenocarcinoma patients (85 – 90% of all pancreatic cancer subjects) have no or low level of expression of ASNS, respectively. A Phase I clinical study is ongoing with pancreatic adenocarcinoma patients.