Preclinical and clinical developments in enzyme-loaded red blood cells: an update.

INTRODUCTION: We have previously described the preclinical developments in enzyme-loaded red blood cells to be used in the treatment of several rare diseases, as well as in chronic conditions. AREA COVERED: Since our previous publication we have seen further progress in the previously discussed approaches and, interestingly enough, in additional new studies that further strengthen the idea that red blood cell-based therapeutics may have unique advantages over conventional enzyme replacement therapies in terms of efficacy and safety. Here we highlight these investigations and compare, when possible, the reported results versus the current therapeutic approaches. EXPERT OPINION: The continuous increase in the number of new potential applications and the progress from the encapsulation of a single enzyme to the engineering of an entire metabolic pathway open the field to unexpected developments and confirm the role of red blood cells as cellular bioreactors that can be conveniently manipulated to acquire useful therapeutic metabolic abilities. Positioning of these new approaches versus newly approved drugs is essential for the successful transition of this technology from the preclinical to the clinical stage and hopefully to final approval.

Myelin basic protein recovery during PKU mice lifespan and the potential role of microRNAs on its regulation.

Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages, which are accompanied by myelin basic protein (MBP) impairment. Despite many assumptions, the primary explanation of the mentioned cerebral outcomes remains elusive. In this study, MBP protein and mRNA expression on brains of wild type (WT) and phenylketonuric (ENU2) mice were analyzed throughout mice lifespan (14-60-180-270-360-540 post-natal days, PND). The results confirmed the low MBP expression at first PND times, while revealed an unprecedented progressive MBP protein expression recovery in aged ENU2 mice. Unexpectedly, unaltered MBP mRNA expression between WT and ENU2 was always observed. Additionally, for the same time intervals, a significant decrease of the phenylalanine concentration in the peripheral blood and brain of ENU2 mice was detected, to date, for the first time. In this scenario, a translational hindrance of MBP during initial and late cerebral development in ENU2 mice was hypothesized, leading to the execution of a microRNA microarray analysis on 60 PND brains, which was followed by a proteomic assay on 60 and 360 PND brains in order to validate in silico miRNA-target predictions. Taken together, miR-218-1-3p, miR-1231-3p and miR-217-5p were considered as the most impactful microRNAs, since a downregulation of their potential targets (MAG, CNTNAP2 and ANLN, respectively) can indirectly lead to a low MBP protein expression. These miRNAs, in addition, follow an opposite expression trend compared to MBP during adulthood, and their target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and on a possible treatment, emphasizing the potential modulating role of differentially expressed microRNAs in MBP expression on PKU brains during PKU mouse lifespan.

Engineering new metabolic pathways in isolated cells for the degradation of guanidinoacetic acid and simultaneous production of creatine.

Here we report, for the first time, the engineering of human red blood cells (RBCs) with an entire metabolic pathway as a potential strategy to treat patients with guanidinoacetate methyltransferase (GAMT) deficiency, capable of reducing the high toxic levels of guanidinoacetate acid (GAA) and restoring proper creatine levels in blood and tissues. We first produced a recombinant form of native human GAMT without any tags to encapsulate into RBCs. Due to the poor solubility and stability features of the recombinant enzyme, both bioinformatics studies and extensive optimization work were performed to select a mutant GAMT enzyme, where only four critical residues were replaced, as a lead candidate. However, GAMT-loaded RBCs were ineffective in GAA consumption and creatine production because of the limiting intra-erythrocytic S-adenosyl methionine (SAM) content unable to support GAMT activity. Therefore, a recombinant form of human methionine adenosyl transferase (MAT) was developed. RBCs co-entrapped with both GAMT and MAT enzymes performed, in vitro, as a competent cellular bioreactor to remove GAA and produce creatine, fueled by physiological concentrations of methionine and the ATP generated by glycolysis. Our results highlight that metabolic engineering of RBCs is possible and represents proof of concept for the design of novel therapeutic approaches.

Intellectual Disability and Brain Creatine Deficit: Phenotyping of the Genetic Mouse Model for GAMT Deficiency.

Guanidinoacetate methyltransferase deficiency (GAMT-D) is one of three cerebral creatine (Cr) deficiency syndromes due to pathogenic variants in the GAMT gene (19p13.3). GAMT-D is characterized by the accumulation of guanidinoacetic acid (GAA) and the depletion of Cr, which result in severe global developmental delay (and intellectual disability), movement disorder, and epilepsy. The GAMT knockout (KO) mouse model presents biochemical alterations in bodily fluids, the brain, and muscles, including increased GAA and decreased Cr and creatinine (Crn) levels, which are similar to those observed in humans. At the behavioral level, only limited and mild alterations have been reported, with a large part of analyzed behaviors being unaffected in GAMT KO as compared with wild-type mice. At the cerebral level, decreased Cr and Crn and increased GAA and other guanidine compound levels have been observed. Nevertheless, the effects of Cr deficiency and GAA accumulation on many neurochemical, morphological, and molecular processes have not yet been explored. In this review, we summarize data regarding behavioral and cerebral GAMT KO phenotypes, and focus on uncharted behavioral alterations that are comparable with the clinical symptoms reported in GAMT-D patients, including intellectual disability, poor speech, and autistic-like behaviors, as well as unexplored Cr-induced cerebral alterations.

Preclinical developments of enzyme-loaded red blood cells.

INTRODUCTION: Therapeutic enzymes are currently used in the treatment of several diseases. In most cases, the benefits are limited due to poor in vivo stability, immunogenicity, and drug-induced inactivating antibodies. A partial solution to the problem is obtained by masking the therapeutic protein by chemical modifications. Unfortunately, this is not a satisfactory solution because frequent adverse events, including anaphylaxis, can arise. AREA COVERED: Among the delivery systems, we focused on red blood cells for the delivery of therapeutic enzymes. Erythrocytes possess a long circulation time, a reduced immunogenicity, there is no need of chemical modifications and the encapsulated enzyme remains active because it is protected by the cell membrane. Here we discuss some representative applications of the preclinical developments of the field. Some of these are currently in clinic, others are approaching the clinic and others are illustrative of the development process. The selected examples are not always the most recent, but they are the most useful for a comparative approach. EXPERT OPINION: The results discussed confirm the central role that red blood cells can play in the treatment of several conditions and suggest the benefit in using a natural cellular carrier in terms of pharmacokinetic, biodistribution, safety, and efficacy.

Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells.

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Redox homeostasis as a target for new antimycobacterial agents.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and ß-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1ß), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response.

Ongoing Developments and Clinical Progress in Drug-Loaded Red Blood Cell Technologies.

Engineered red blood cells (RBCs) appear to be a promising method for therapeutic drug and protein delivery. With a number of agents in clinical trials (e.g., dexamethasone 21-phosphate in ataxia telangiectasia, asparaginase in pancreatic cancer/acute lymphoblastic leukemia, thymidine phosphorylase in mitochondrial neurogastrointestinal encephalomyopathy, RTX-134 in phenylketonuria, etc.), this leading article summarizes the ongoing efforts in developing these agents, focuses on the clinical progress, and provides a brief background into engineered RBCs and the different ways in which they can be exploited for therapeutic/diagnostic purposes. References to available data on safety, efficacy, and tolerability are reported. Due to the continuous progress in this field, the information is updated as of January 2020 from databases, websites, and press releases of the involved companies and information that is in the public domain.

Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination.

Methionine deprivation induces growth arrest and death of cancer cells. To eliminate l-methionine we produced, purified, and characterized the recombinant pyridoxal 5'-phosphate (PLP)-dependent l-methionine γ-lyase (MGL)- BL929 from the cheese-ripening Brevibacterium aurantiacum Transformation of an Escherichia coli strain with the gene BL929 from B. aurantiacum optimized for E. coli expression led to production of the MGL-BL929. Elimination of l-methionine and cytotoxicity in vitro were assessed, and methylation-sensitive epigenetics was explored for changes resulting from exposure of cancer cells to the enzyme. A bioreactor was built by encapsulation of the protein in human erythrocytes to achieve sustained elimination of l-methionine in extracellular fluids. Catalysis was limited to α,γ-elimination of l-methionine and l-homocysteine. The enzyme had no activity on other sulfur-containing amino acids. Enzyme activity decreased in presence of serum albumin or plasma resulting from reduction of PLP availability. Elimination of l-methionine induced cytotoxicity on a vast panel of human cancer cell lines and spared normal cells. Exposure of colorectal carcinoma cells to the MGL-BL929 reduced methyl-CpG levels of hypermethylated gene promoters including that of CDKN2A, whose mRNA expression was increased, together with a decrease in global histone H3 dimethyl lysine 9. The MGL-erythrocyte bioreactor durably preserves enzyme activity in vitro and strongly eliminates l-methionine from medium.

A new therapy prevents intellectual disability in mouse with phenylketonuria.

Untreated phenylketonuria (PKU) results in severe neurodevelopmental disorders, which can be partially prevented by an early and rigorous limitation of phenylalanine (Phe) intake. Enzyme substitution therapy with recombinant Anabaena variabilis Phe Ammonia Lyase (rAvPAL) proved to be effective in reducing blood Phe levels in preclinical and clinical studies of adults with PKU. Aims of present study were: a) to gather proofs of clinical efficacy of rAvPAL treatment in preventing neurological impairment in an early treated murine model of PKU; b) to test the advantages of an alternative delivering system for rAvPAL such as autologous erythrocytes. BTBR-Pahenu2-/- mice were treated from 15 to 64 post-natal days with weekly infusions of erythrocytes loaded with rAvPAL. Behavioral, neurochemical, and brain histological markers denoting untreated PKU were examined in early treated adult mice in comparison with untreated and wild type animals. rAvPAL therapy normalized blood and brain Phe; prevented cognitive developmental failure, brain depletion of serotonin, dendritic spine abnormalities, and myelin basic protein reduction. No adverse events or inactivating immune reaction were observed. In conclusion present study testifies the clinical efficacy of rAvPAL treatment in a preclinical model of PKU and the advantages of erythrocytes as carrier of the enzyme in term of frequency of the administrations and prevention of immunological reactions.

Reengineering red blood cells for cellular therapeutics and diagnostics.

Recently optimized technologies that permit the reversible opening of nanopores across the red blood cell membrane, give the extraordinary opportunity for reengineering human erythrocytes to be used in different biomedical applications, both for therapeutic and diagnostic purposes. Engineered erythrocytes have been exploited as a system for the controlled release of drugs in circulation upon encapsulation of prodrugs or small molecules; as bioreactors when they are endowed of recombinant enzymes able to catalyze the conversion of toxic metabolite into inert products; as drug targeting system for the delivery of compounds to the reticuloendothelial system inducing proper senescent signals on the drug-loaded erythrocyte membrane; as carrier of contrasting agents for diagnostic procedures. Preclinical development of these different applications has taken advantage from the use of proper animal models whose erythrocytes can be reengineered as the human ones or the encapsulation procedures can be adapted on the basis of their specific erythrocyte biological features. Successful results, obtained both in vitro and in preclinical studies, have prompted several clinicians to start pilot clinical investigations in different conditions and some new companies to start the industrialization of selected loading technologies and to initiate clinical development programs. This short review summarizes the key features that, to the best of our knowledge, have been crucial to advance the products toward regulatory clinical approval making reengineering of erythrocytes a modality to treat patients with limited or absent therapeutic options. WIREs Nanomed Nanobiotechnol 2017, 9:e1454. doi: 10.1002/wnan.1454 For further resources related to this article, please visit the WIREs website.

Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment.

Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

Engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution.

Pharmacokinetics, biodistribution, and biological activity are key parameters that determine the success or failure of therapeutics. Many developments intended to improve their in vivo performance, aim at modulating concentration, biodistribution, and targeting to tissues, cells or subcellular compartments. Erythrocyte-based drug delivery systems are especially efficient in maintaining active drugs in circulation, in releasing them for several weeks or in targeting drugs to selected cells. Erythrocytes can also be easily processed to entrap the desired pharmaceutical ingredients before re-infusion into the same or matched donors. These carriers are totally biocompatible, have a large capacity and could accommodate traditional chemical entities (glucocorticoids, immunossuppresants, etc.), biologics (proteins) and/or contrasting agents (dyes, nanoparticles). Carrier erythrocytes have been evaluated in thousands of infusions in humans proving treatment safety and efficacy, hence gaining interest in the management of complex pathologies (particularly in chronic treatments and when side-effects become serious issues) and in new diagnostic approaches.

Erythrocyte-mediated delivery of phenylalanine ammonia lyase for the treatment of phenylketonuria in BTBR-Pah(enu2) mice.

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by defects in the phenylalanine hydroxylase gene. Preclinical and clinical investigations suggest that phenylalanine ammonia lyase (PAL) could be an effective alternative for the treatment of PKU. The aim of this study is to investigate if erythrocytes loaded with PAL may act as a safe delivery system able to overcome bioavailability issues and to provide, in vivo, a therapeutically relevant concentration of enzyme. Murine erythrocytes were loaded with recombinant PAL from Anabaena variabilis (rAvPAL) and their ability to perform as bioreactors was assessed in vivo in adult BTBR-Pah(enu2) mice, the genetic murine model of PKU. Three groups of mice were treated with a single i.v. injection of rAvPAL-RBCs at three different doses to select the most appropriate one for assessment of efficacy. Repeated administrations at 9-10 day-intervals of the selected dose for 10 weeks showed that the therapeutic effect was persistent and not affected by the generation of antibodies induced by the recombinant enzyme. This therapeutic approach deserves further in vivo evaluation either as a potential option for the treatment of PKU patients or as a possible model for the substitutive enzymatic treatment of other inherited metabolic disorders.

Erythrocytes as a novel delivery vehicle for biologics: from enzymes to nucleic acid-based therapeutics.

Biological drugs are among the most exciting drugs of the future, offering better treatment options for patients than ever before but they need an appropriate delivery vehicle. Carrier erythrocytes are one of the most promising drug-delivery systems. Application of erythrocytes as containers for various drugs minimizes toxicity, decreasing the risk of side effects and pathologic immune reactions against encapsulated agents as well as improving their efficacy, leading to better patient compliance. This review discusses the rationale for the use of erythrocytes as a vehicle for biopharmaceuticals and summarizes the categories of these new encapsulable compounds that are currently under investigation. The authors' intent is to describe the development of this delivery system to give the reader an overview of the remarkable potential of erythrocytes as naturally designed carriers and their versatility in the field of biologics for the treatment of various pathological conditions.

Immunophilin-loaded erythrocytes as a new delivery strategy for immunosuppressive drugs.

Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5±1.3, 7.5±3.1 and 15.5±0.4nmol FKBP12 were able to bind 3.5±1.5, 6.0±1.9 and 11.4±2.9µg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0±0.6, 5.0±0.8 and 15.9±2.4nmol of cyclophilin A could bind 8.9±3.4, 12.2±3.5 and 17.0±3.2µg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0±0.3µg FK506 and 3.2±0.3µg CsA). Moreover, FK506 released from FKBP12-RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.

Cytotoxic activity of 2-Fluoro-ara-AMP and 2-Fluoro-ara-AMP-loaded erythrocytes against human breast carcinoma cell lines.

Fludarabine phosphate (2-Fluoro-ara-AMP) is a purine analogue approved for the clinical treatment of haematological malignancies. This antimetabolite has also shown 'in vitro' antiproliferative activity against experimental models of solid mammary tumor. In this perspective, we have determined the cytotoxic effects of 2-Fluoro-ara-AMP against two human breast cancer cell lines (the ER-positive MCF-7 and the ER-negative MDA-MB-435), by adding the drug both in its free form and encapsulated into erythrocytes, as a strategy to modify the pharmacokinetic profile of the compound in order to increase its efficacy and decrease its toxicity. Similar antiproliferative activity of 2-Fluoro-ara-AMP in the two cell lines was obtained, reaching an almost complete abrogation of growth already after just 24 h of free drug exposure at all the tested doses. Meanwhile, encapsulated 2-Fluoro-ara-AMP was successfully released from erythrocytes into the culture media in a time-dependent manner with an efficacy comparable to that of the free drug treatment. This result suggests the possibility of administering 2-Fluoro-ara-AMP in patients with breast cancer using autologous erythrocytes as a system to slowly and constantly deliver 2-Fluoro-ara-A into circulation. In addition, possible mechanisms involved in the antiproliferative activity of 2-Fluoro-ara-AMP, such as the effects on cell cycle progression, p53 expression and STAT1 pathway activation in ER+ and ER- cancer cell lines, are proposed.

Prolonged islet allograft survival in diabetic mice upon macrophage depletion by clodronate-loaded erythrocytes.

Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4+/-0.89 and 20+/-2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25+/-1.9 days in the group treated with free-clodronate and 35+/-6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.

Inhibition of HIV-1 replication in macrophages by a heterodinucleotide of lamivudine and tenofovir.

OBJECTIVES: (i) To generate a new heterodinucleotide (3TCpPMPA) comprising the drugs lamivudine and tenofovir which have been shown to act synergistically and (ii) to protect macrophages from 'de novo' HIV-1-infection through its administration. METHODS: 3TCpPMPA was obtained by coupling the morpholidate derivative of tenofovir with the mono n-tri-butylammonium salt of lamivudine 5'-monophosphate. Stability and metabolism were evaluated in vitro and in vivo in mice. 3TCpPMPA was encapsulated into autologous erythrocytes by a procedure of hypotonic dialysis, isotonic resealing and reannealing. 3TCpPMPA-loaded erythrocytes were modified to increase their phagocytosis by human macrophages. Macrophages were infected by HIV-1(Ba-L) and inhibition of HIV-1 replication was assessed by HIV p24(gag) quantification. RESULTS: Pharmacokinetic studies in mice revealed a rapid disappearance of the heterodinucleotide from circulation (t(1/2)=15 min) without any advantage compared with the administration of single drugs. Adding free 3TCpPMPA to macrophages (18 h), a 90% inhibition of viral replication up to 35 days post-treatment was achieved, while only a 60% inhibition was obtained by the combined treatment 3TC and (R)PMPA. When 3TCpPMPA was selectively targeted to the macrophage compartment by a single addition of loaded erythrocytes, the protection of macrophages from 'de novo' infection (99% protection 3 weeks post-treatment) was nearly complete. CONCLUSIONS: Erythrocytes loaded with 3TCpPMPA and modified to increase their phagocytosis are able to protect macrophages from 'de novo' HIV-1 infection. 3TCpPMPA acts as an efficient antiviral pro-drug that, once inside macrophages, can be slowly converted into 3TCMP and (R)PMPA protecting these cells for a longer period of time.

Erythrocyte-based drug delivery.

The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimising inevitable adverse side effects, is an extremely fascinating perspective. The behaviour of erythrocytes as a delivery system for several classes of molecules (i.e., proteins, including enzymes and peptides, therapeutic agents in the form of nucleotide analogues, glucocorticoid analogues) has been studied extensively as they possess several properties, which make them unique and useful carriers. Furthermore, the possibility of using carrier erythrocytes for selective drug targeting to differentiated macrophages increases the opportunities to treat intracellular pathogens and to develop new drugs. Finally, the availability of an apparatus that permits the encapsulation of drugs into autologous erythrocytes has made this technology available in many clinical settings and competitive with other drug delivery systems.