Yann Godfrin

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. n nA 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). n n n nFigure 1 n nThe process of reversible hypotonic lysis of RBCs to entrap molecules n n n n nKeywords: carriers, drug delivery, erythrocytes, red blood cells, targeting n n n1. Therapeutic enzyme-loaded RBCs nTherapy 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). n n n nFigure 2 n nThe 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 n n n n1.1 L-asparaginase-loaded RBCs for ASNS-deficient tumor (by E Dufour) nL-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. n nThis 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.

In situ targeting of dendritic cells by antigen-loaded red blood cells: A novel approach to cancer immunotherapy

Red blood cells (RBCs) were shown to be efficient antigen carriers to target dendritic cells (DCs) and induce cytotoxic T-cell responses. Mouse RBCs were loaded with ovalbumin (RBC-OVA) and injected with Poly (I:C) into mice. Phagocytosis of RBC-OVA by macrophages and DCs was demonstrated to induce OVA-specific CD4(+) and CD8(+) T cell activation. Moreover, these CD8(+) T cells produced IFN-gamma and were able to induce OVA-specific cell lysis. Finally, T-cell response was demonstrated to be dependent on the dose-amount of antigen entrapped and this response could be maintained for up to 30 days.

Drug-loaded erythrocytes: on the road toward marketing approval

Erythrocyte drug encapsulation is one of the most promising therapeutic alternative approaches for the administration of toxic or rapidly cleared drugs. Drug-loaded erythrocytes can operate through one of the three main mechanisms of action: extension of circulation half-life (bioreactor), slow drug release, or specific organ targeting. Although the clinical development of erythrocyte carriers is confronted with regulatory and development process challenges, industrial development is expanding. The manufacture of this type of product can be either centralized or bedside based, and different procedures are employed for the encapsulation of therapeutic agents. The major challenges for successful industrialization include production scalability, process validation, and quality control of the released therapeutic agents. Advantages and drawbacks of the different manufacturing processes as well as success key points of clinical development are discussed. Several entrapment technologies based on osmotic methods have been industrialized. Companies have already achieved many of the critical clinical stages, thus providing the opportunity in the future to cover a wide range of diseases for which effective therapies are not currently available.

Methionine tumor starvation by erythrocyte‐encapsulated methionine gamma‐lyase activity controlled with per os vitamin B6

Erymet is a new therapy resulting from the encapsulation of a methionine gamma‐lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)‐dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U‐87 MG‐luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event‐free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half‐life was increased from <24 h to 9–12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation‐sensitive tumors.