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Keywords:

  • drug delivery;
  • carrier red blood cells;
  • immunosuppressive drugs

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Drug delivery is a growing field of interdisciplinary activities that combine the use of new materials with the biochemical properties of selected drugs, with the aim of improving their therapeutic action and reducing their toxicity. In few cases, proper medical devices have been also realized to implement new drug delivery modalities. In this article, we have summarized available information and our experience on the use of autologous Red Blood Cells as carriers for drugs to be released within the vascular system. This is not a comprehensive review, but it focusses on the mechanisms that are available to distribute drugs in circulation by carrier red blood cells and provide illustrative examples on how this is currently obtained. We have not included a summary of clinical data collected in recent years using this technology but simply provided proper references for the interested readers. Finally, a special attention is devoted to the possibility of entrapping, into autologous red blood cells, recombinant drug-binding proteins. This new strategy is opening the way at a new modality to influence the vascular distribution of drugs by realizing a dynamic circulating container (the engineered red cell) capable of reversible binding and transportation of one or more drugs of interest selected on the bases of the red cell entrapped target proteins. This new modality is not yet fully developed and explored but will certainly provide a technical solution to the problem of stabilizing drug concentration in circulation improving drug efficacy and reducing drug toxicity. © 2011 IUBMB IUBMB Life, 2011


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

There are 1,564 approved drugs, 4,367 identified targets, and 70 unique drug transporters available (http://www.drugbank.ca/stats), but this apparently large armamentarium, only partially covers the medical needs we experience every day. As the identification of a new chemical entity, and its approval for human use is a very long and expensive process, it is possible to improve the therapeutic efficacy of several lead compounds simply by improving their pharmacokinetic and/or pharmacodynamic properties. In other words, the effectiveness of drugs is often limited by side effects, possible and undesirable immune responses, premature degradation, inactivation or elimination from the body, limited bioavailability and/or interaction with plasma proteins and several of these events could be affected by the selection and use of an appropriate delivery system. The delivery systems available include carriers such as antibodies, soluble and bio-degradable polymers, polysaccharides, microcapsules, microparticles, cells, cell ghosts, lipoproteins, liposomes, and erythrocytes. Among all, red blood cells (RBC) possess unique features that make them potentially biocompatible carriers for a number of bioactive substances (1–6). In detail: 1) they are completely biodegradable without generation of toxic products and show high biocompatibility especially when autologous RBC are employed; 2) they can be easily handled by means of several techniques for the encapsulation of different molecules, without affecting their morphological, immunological and biochemical properties; 3) they have a large volume available for the encapsulation of drugs; 4) they protect the encapsulated substance from premature inactivation and degradation and protect the organism against the toxic effects of the drug; 5) they have a longer life-span in circulation, when compared with other synthetic carriers; 6) they could act as active transporters (bioreactors) due to the presence of several enzymatic activities that could convert selected pro-drugs into diffusible, active, drugs.

Because of the properties listed above, RBC could be employed as drug carriers for different purposes: (a) they could be loaded by an active payload (i.e., an enzyme) and thus be used as circulating bioreactors to remove undesired molecules from the blood stream; (b) they could be used as a drug delivery system providing a sustained release of the drug into the body, allowing therapeutic levels to be maintained in the blood for long periods of time; (c) they could be used as drug targeting systems for the selective delivery of pharmacological agents to cells responsible for or capable of erythrophagocytosis (i.e., cells of the monocyte-macrophage system), which are often involved in various pathological conditions; (d) they could be loaded with magnetic-responsive particles and targeted to a specific site of the body using external magnetic fields. Thus, the use of RBC-based drug delivery systems represents an attractive and versatile technology, suitable for several clinical applications, and immediately available for the introduction in clinical practice. Here, we summarize some examples of RBC-based vascular delivery of drugs.

RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

RBC-based drugs delivery in circulation can be achieved by either direct encapsulation of membrane-diffusible drug molecules or by internalization of impermeant pro-drugs susceptible to be metabolically converted into diffusible active drugs. In this last case, it is necessary to consider the specificity and kinetic properties of the intra-erythrocytic enzymes involved in the bioconversion of the pro-drug into the drug. The rate of release of the active drug depends also on the diffusion or transport rate of the drug through the RBC membrane. Several pro-drugs, including anti-inflammatory, antiviral, and anticancer molecules, were encapsulated in RBC to obtain a slow release in circulation.

Pro-Drug-Loaded RBC to Deliver Anti-inflammatory Molecules

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressant agents; particularly, dexamethasone (Dexa) shows the highest anti-inflammatory activity and is used to treat acute and severe inflammatory, immunological, and allergic disorders. However, GCs benefits are countered by the potential for important adverse events such as osteoporosis, skin atrophy, cushingoid appearance, diabetes, and glaucoma (7) that limit their clinical use so that, in principle, they need to be formulated in a way to be released in low and effective doses for prolonged periods of time. Dexamethasone 21-phosphate (Dex 21-P) once loaded into RBC, is slowly dephosphorylated by resident enzymes to its active diffusible metabolite Dexa that is then slowly released into the blood stream by simple passive diffusion through the red cell membrane (Fig. 1). Administration of Dex 21-P-loaded RBC could provide concentrations of the diffusible drug within therapeutic values for several days, while to maintain similar Dexa concentration the free drug should be repeatedly administered at intervals of a few hours (8). It is noteworthy that the use of RBC as a glucocorticoid analogue delivery system has recently found clinical applications in the treatment of inflammatory diseases (Table 1). This was possible because of a new apparatus that is approved in Europe as a medical device (Fig. 2) and performs the drug loading procedure under blood banking conditions. The preparation of Dex 21-P-loaded RBC guarantees a high percentage of drug encapsulation (30 ± 3%) and a good cell recovery (30–50%). It is easy to perform, reproducible and can be completed within 2 h. The procedure is safe, this being confirmed by more than 3,000 infusions of drug-loaded RBC in patients with inflammatory diseases in which clinically significant adverse effects have been never observed. Furthermore, a single dose of 10 mg of Dex 21-P loaded into autologous RBC (from just 50 mL of blood) was sufficient to maintain in vivo a plasma drug concentration between 15 and 50 nM for at least 30 days. It is worthy of note that these low amounts are capable of saturating 80 to 85% of the glucocorticoid receptors, ensuing a high efficacy of the formulation (16). In fact, the low doses of Dexa released from the autologous drug-loaded RBC received monthly by patients give the clinical benefits proper of corticosteroid treatment without the toxic side effects.

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Figure 1. Encapsulation of Dexamethasone 21-P into RBC and release of Dexamethasone. The glucocorticoid analogue is loaded into RBC as the nondiffusible prodrug Dex 21-P. According to the erythrocytic phosphatase kinetic, Dex 21-P is then dephosphorylated to its active diffusible metabolite dexamethasone (Dexa) and slowly released into the blood stream. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 2. Current medical devices by EryDel S.p.A. The Red Cell Loader (a) and the Disposable sterile and pirogen-free kit (b) are CE marked medical devices specifically designed by EryDel S.p.A. to realize the encapsulation of drugs and other active agents into RBC in a reliable and reproducible way. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1. Clinical applications of Dex 21-P-loaded erythrocytes in inflammatory diseases
Clinical applicationsReferences
Chronic obstructive pulmonary disease (COPD)(9)
Cystic fibrosis (CF)(10)
Inflammatory bowel diseases (IBDs)(11-15)

Pro-Drug-Loaded RBC to Deliver Antiviral Molecules

Besides anti-inflammatory drugs, also antiviral drugs were encapsulated into RBC. Nucleoside analogue reverse transcriptase inhibitors, such as 3′-azido-3′-deoxythymidine and 2′, 3′-dideoxycitidine (ddCyd) were experimentally loaded into human RBC under newly synthesized pro drug forms (i.e., as 5′-phosphorylated derivatives, AZT-MP and ddCMP, respectively). These are dephosphorylated by endogenous nucleotidases and subsequently released as 3′-azido-3′-deoxythymidine (AZT) and ddCyd, respectively (17, 18). The results reported suggest that RBC can be used as slow delivery system ensuring long-lasting nucleoside analogues release in circulation.

Pro-Drug-Loaded RBC to Deliver Anticancer Molecules

RBC were also investigated for the release of 2-Fluoro-ara-A by encapsulation of Fludarabine phosphate (9-b-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate, 2-Fluoro-ara-AMP, Fludara). This is a fluorinate purine analogue commonly used in the treatment of haematological malignancies (19). 2-Fluoro-ara-AMP was encapsulated in human RBC from which it was in part released as 2-fluoro-ara-A, while most of the drug was converted to the di- and triphosphate derivatives. The triphosphate derivative was in turn dephosphorylated for the supply of 2-Fluoro-ara-A over a period of days (20). The ability of 2-Fluoro-ara-AMP-loaded RBC to release 2-Fluoro-ara-A was tested on breast cancer cell lines (MCF-7 and MDA-MB-435 cells), where the drug significantly inhibited cell growth (21). The results suggest that RBC can be used as a delivery system to maintain a critical plasma concentration of 2-Fluoro-ara-A providing potential advantages in terms of both efficacy and reduction of toxicity.

Pre-Pro-Drug-Loaded RBC

The enzymatic activities of the RBC that can be exploited to convert an active pro-drug into an active drug may prove to be too high for a slow release thus, in some cases, it could be advantageous to synthesize metabolically more remote precursors of the final drug. In this case, the erythrocyte enzyme apparatus produces the final active drug through a multistep pathway starting from a pre-pro-drug. Examples of this strategy have been reported. One is represented by the possibility to use the RBC to convert the encapsulated pro-drug 5-fluoro-2′-deoxyuridine-monophosphate (FdUMP) to the membrane diffusible drug 5-fluoro-2′-deoxyuridine (FdUR), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas (22). Although such bioconversion was demonstrated to take place, the rate of intraerythrocytic conversion of FdUMP to FdUR was too high to be compatible with an efficient pharmacokinetic model of anticancer therapy (23). Accordingly, an alternative approach was developed to down-regulate FdUR release; a fluoropyrimidine dimer able to generate FdUMP and finally FdUR within RBC was synthesized. The main products deriving from this compound were FdUMP and the corresponding diphosphate and triphospahte forms, FdUDP and FdUTP, respectively, that represent transient fluoropyrimidine reservoirs for further formation of FdUR. The encapsulation of this pre-prodrug resulted in the slow production and release of FdUR according to patterns that are compatible with pharmacokinetic requirements of human therapy (24). Another dimer encapsulated into erythrocytes was represented by azydothymidine derivative, di-(thymidine-3′-azido-2′,3′-dideoxy-D-riboside)-5′-5′-p1-p2-pyrophosphate (AZTp2AZT), which was converted to AZT that in turn was released by simple diffusion (Fig. 3) (25). Because of its intra-erythrocytic metabolism, AZT-dimer can provide AZT release for few days, and its encapsulation is more advantageous with respect to that of AZT-MP described above.

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Figure 3. Encapsulation of AZT-dimer into RBC and release of AZT. AZT-dimer is loaded into RBC, where it is metabolized to AZT-TP, AZT-DP, AZT-MP and AZT which is finally released into circulation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Most drugs currently used in the clinical practice, can cross the red cell membrane by simple diffusion or by transport-mediated mechanisms. It is immediately evident that the encapsulation of such a kind of molecules would be of no added value because, even in case of a successful entrapment, they would immediately escape from the red cell container without any practical pharmacokinetic advantage over conventional delivery. To overcome these limitations, it is possible to modify the drug substance by producing a nondiffusible prodrug to be converted into a diffusible drug by red cell resident enzymes (see examples above) or develop completely new strategies aimed at retaining the diffusible drug into the red cell compartment by some forms of drug-binding substance. Of course, the binding should not be irreversible and the affinity not extremely high. Each of these alternatives has advantages and disadvantages. The chemical modification of an existing drug, already approved for human use, results in the generation of a new chemical entity that must pass again all typical approval processes for new drugs. The second approach could take advantage from the availability of drugs already on the market, and thus immediately available, but must qualify the drug-binding substance. Based on these considerations, we have designed a new drug delivery system, where the diffusible drug of interest is retained in the cell compartment by noncovalent binding to a specific drug-binding recombinant human protein (Fig. 4).

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Figure 4. Loading of drug-binding proteins for the delivery of diffusible drugs. A recombinant protein, able to selectively bind a selected drug, is entrapped into red blood cells. The protein-loaded red blood cells can reversibly bind the drug added ex-vivo (i.e., before infusion) or in vivo (i.e., after transfusion of the protein-loaded red cells and the binding of the drug present in circulation). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Proteins able to selectively bind drugs with high affinity are already known in literature, and a selected example is represented by human serum albumin (26, 27). Apart from albumin, human “druggable proteins” (i.e., human genome encoded proteins able to bind drug-like molecules) have been estimated to be about 3,000 representing 10-14% of human product genes (28), thus the challenge is not in the discovery of new leads but in the correct selection of the most useful drug-binding proteins.

Several drugs on the market already have an identified cellular counterpart that could be explored as a drug-binding protein to enhance the red blood cell abilities to perform as drug delivery systems in circulation. When such information is not available, it is in principle possible, by bioinformatic tools, to predict drug binding to selected protein motif (29, 30) or to experimentally identify the drug-protein interactions (31) or, in case a drug-binding protein is not available, it could be produced by recombinant technology.

The Immunophilin Family of Proteins

Immunophilins are a highly conserved family of proteins sharing the ability of binding immunosuppressive drugs (32). Cyclophilin was originally discovered as a specific Cyclosporine A (CsA) binding protein and FKBP12 as a FK506 (Tacrolimus) binding protein with a cis-trans peptidyl-prolyl isomerase activity (33, 34).

It is worth noting that some immunophilins are already present in native RBC. In fact, FK506 in blood is mainly associated with erythrocytes (about 85 %) followed by plasma (14 %) and lymphocytes (0.46 %) (35). This high RBC fraction is due to the presence in erythrocytes of at least two types of immunophilins that bind the drug with very high affinity: FKBP12 (cited above), a 12-kDa cytosolic protein with peptidyl-prolyl cis-trans isomerase activity, and FKBP-13, a 13-kDa membrane-associated protein with 43 % amino acid identity with FKBP12 (36). The binding capacity of RBC is estimated to be around 440 ng/mL of blood (35). This peculiar blood distribution seen for FK506 is remarkable for Cyclosporine as well. In fact, in whole blood at concentration of 50-1000 ng/mL, more than 70% of CsA is associated to erythrocytes; cytosolic CsA is bound to the erythrocyte peptidyl-prolyl cis-trans isomerase cyclophilin A (CypA) (37). The total RBC binding capacity for CsA amounted to 43x10−5 nmol per 106 RBC (37, 38) that corresponds to 2 μg CsA/mL suspension of RBC at 40% haematocrit.

CsA is characterized by high intra- and inter-patient pharmacokinetic variability and poor bioavailability. The tolerability profile of Cyclosporine is characterized by a number of potentially serious adverse effects, including acute or chronic nephrotoxicity, hypertension, and neurotoxicity. The main dose-limiting adverse effect of Cyclosporine is nephrotoxicity, which usually presents as a reversible decrease in glomerular filtration rate (39). Compared with CsA, Tacrolimus provides a better side effect profile and increased long-term survival in patients; however, despite its therapeutic efficacy, demonstrated by an in vitro potency 100 times greater than Cyclosporine and an in vivo reduction of tissue rejection incidence (40), it also possesses a very narrow therapeutic window (5–20 ng/mL whole blood 10–12 h postdose) and frequently exhibits episodes of toxicity, including nephrotoxicity, neurotoxicity, glucose intolerance, and so forth (41, 42). Moreover, as well as CsA, FK506 presents a high variability in pharmacokinetic profile among patients. Thus, the pharmacokinetic of immunosuppressive drugs is strongly dependent on their binding to red cell immunophilins, and their tolerability is also dependent on the fraction that is free in plasma because the dose transported by the red cells does not cause toxic side effects.

The idea that RBC can be used to entrap and transport diffusible drugs by binding to selected proteins was experimentally evaluated in our laboratories (43). Human FKBP12 and CypA were cloned and expressed as recombinant proteins in bacteria. The purified proteins were then entrapped into human RBC by a procedure of hypotonic dialysis, isotonic resealing, and “reannealing” as described in (44). Starting from different protein concentrations, it has been estimated that human red cells could be loaded with 3 to 15 nanomoles of FKBP12/mL of red cells and 4 to 16 nanomoles of CypA/mL red cells, in a dose dependent manner. The protein-loaded red cells are indistinguishable from unloaded red cells (i.e., red cells processed as per the encapsulation procedure without the addition of the recombinant proteins) in terms of morphology, mean cell volume (78 ± 2.9 fl), mean cell hemoglobin (24.8 ± 0.7 pg), and mean cell hemoglobin concentration (31.4 ± 1 g/dL). Furthermore, they also show the same in vitro stability at 37 °C under sterile conditions. Cell recovery at 1, 2, 3, and 6 days resulted generally equal in all loading conditions compared with unloaded erythrocytes. The drug binding capacity of the immunophilin-loaded red cells has been next investigated. Erythrocytes dialyzed in the presence of 20, 40, and 80 μM FKBP12 were able to bind 3.5 ± 1.5, 6.0 ± 1.9, and 11.4 ± 2.9 μg of FK506 per mL RBC, respectively; these values were exactly 3.5, 6, and 11 times greater than those obtained for native RBC (1.0 ± 0.3 μg/mL RBC 100% Ht). RBC dialyzed with 20, 40, and 80 μM CypA were demonstrated to be able to bind a drug amount equivalent to 8.9 ± 3.4, 12.2 ± 3.5, and 17.0 ± 3.2 μg/mL RBC, respectively, while unloaded RBC were able to carry 3.2 ± 0.3 μg CsA per mL of packed RBC. In fact, by comparing the results showed above with those reported in literature for native erythrocytes (37, 38), CypA-loaded RBC possess a clearly higher binding capacity for CsA in all loading conditions tested.

Thus, immunophilin-loaded RBC are able to bind the immunosuppressive drugs FK506 and CsA. It is worth noting that these engineered red cells are also able to release their drug cargo (but not the entrapped recombinant proteins) once resuspended in whole blood. One volume of FK506-loaded erythrocytes (at 10% haematocrit) diluted in 100 volumes of blood release free FK506 and 30% of the drug is recovered free in plasma after 1 h incubation at 37 °C.

The Potential Use of Immunophilin-Loaded Erythrocytes

We have envisaged two different uses of immunophilin-loaded RBC: (a) the engineered cells could be ex vivo incubated with the drug of interest and reinfused into a patient in need of immunosuppressive drugs; (b) the engineered cells could be administered to a patient that will later assume the drug (the modality for drug administration is not important and could be orally or intravenously or intramuscularly or in any other way that allows the drug to reach the circulatory system).

In the first case, the drug is administered associated with the carrier red cells which release the drug in circulation. Thus, when the drug is administered, it is not free. The drug released from the drug-loaded red cells could eventually bind endogenous immunophilins physiologically present into native erythrocytes circulating within the same vascular system. According to the second modality, the engineered red cells have a capacity for binding the immunosuppressive drug that is several times higher than that of the native cells. Thus, the patient will receive first the immunophilin-loaded erythrocytes which usually circulate for few months, then the drug. A significant proportion of free drugs will bind the engineered cells which preferentially will transport the drug (and release the same) allowing a slow diffusion into the circulatory system and into lymphocytes. Which of the two modalities is able to realize the most favorable pharmacokinetic is actually not known, and only in vivo experiments will provide sufficient data to guide a conclusive decision. Both modalities are used in treating a patient in need of an immunosuppressive drug. In fact, the first administration of immunophilin-loaded erythrocytes could be given upon saturation of the red cells binding capacity with the selected drug. Once in circulation, the loaded red cells will release the entrapped drug but, because the immunophilin-loaded red cells remain in circulation for months, could also bind and transport immunosuppressive drugs that will be assumed by the patient orally or by any known modality that permits the drug to enter into circulation. In a further embodiment of this application, the patient's erythrocytes could be loaded with more than one immunophilin at a time (i.e., FKBP12 and CypA), separately or together in the same erythrocyte, thus providing a new modality to increase the amount of immunosuppressive drugs that are usually administered to a patient in need of the same without drug side effects.

Red blood Cells Loaded with Druggable Proteins

The example described above could potentially be extended to all human druggable proteins present in the human genome. The main limiting step appears to be the availability of the selected proteins into recombinant forms, produced under GMP conditions and at an affordable cost. Based on the evidence available in the literature, it is possible that single protein domains or combination of the same could be employed to reach the same aim (i.e., realize RBC engineered to bind and transport all drugs a patient is in need). Furthermore, because the dissociation rate of the drug from the protein-binding partner depends on the protein affinity for the selected drug, it could be possible in future to engineer the protein by site-specific mutagenesis in a way that the drug could be released with a predefined kinetic to maintain a proper free drug concentration in circulation. If the evidences so far collected in our laboratories will be confirmed to be of practical utility, we then have a new modality to potentially administer almost all drugs a patient is in need by taking advantage of the possibility of loading autologous erythrocytes with selected drug binding proteins.

RED BLOOD CELLS AS ENZYME CARRIERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Carrier erythrocytes can be employed also for the encapsulation and the consequent transport of drug-acting proteins. The idea of entrapping proteins, particularly enzymes, into RBC arises from two different approaches: enzyme replacement therapy (EPR) to treat congenital enzyme-deficiencies or detoxification to clear toxic metabolites from the blood stream (45, 46).

Obviously, in the treatment of metabolic disorders, deficient or missing enzymes could be replaced administering them by intravenous injection, but the systemic use of proteins generates some problems such as too short circulation half-life, onset of ipersensitivity reactions, immunogenicity and tissue toxicity. The same inconveniences occur when enzyme administration is required for the detoxification from noxious metabolites in consequence of accidental poisoning. The encapsulation of enzymes into RBC can overcome all these limitations. According to the different enzymes loaded and the related condition to be treated, red cells filled with endogenous or exogenous enzymes could act as long circulating bioreactors in which substrates can diffuse, interact with the loaded enzyme and generate products (Fig. 5). Otherwise, enzyme-loaded RBC can target the proteins to the RES, where their action is required, especially in lysosomal storage disorders (LSD) (47).

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Figure 5. Enzyme-loaded RBC acting as long circulating bioreactors. RBC are first loaded with selected enzymes which confer new or improved metabolic activities. Once in circulation, the engineered red cells are able to catabolise selected substrates into nontoxic metabolic products. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Several detoxifying enzymes have been successfully encapsulated into RBC acting as long circulating bioreactors. Erythrocytes loaded with aldehyde dehydrogenase were shown to favor the depletion of acetaldehyde and ethanol in normal or alcoholic mice (48), while alcohol oxidase-loaded RBC were efficiently used to deplete methanol and formaldehyde during methanol poisoning (49), as well the co-entrapment of glucose oxidase and hexokinase was found to be effective in the glycaemia regulation (50). To concern the targeting of enzymes to phagocyic cells, galactosidase (51) and glucuronidase-loaded RBC (52) were efficaciously targeted to macrophages for the treatment of LSD.

Numerous are the examples of enzymes loaded to treat congenital diseases. Alglucerase, a modified form of the β-glucocerebrosidase, is the main enzyme used in replacement therapy in Gaucer's patients, a rare hereditary disorder which results in accumulation of the lipid glucocerebroside within macrophages owing to the deficiency of the lysosomal β-glucocerebrosidase (53). Adenosine deaminase (ADA) deficiency is an inherited disorder which leads to elevated cellular levels of deoxyadenosine triphosphate and systemic accumulation of its precursor 2-deoxyadenosine which impair lymphocyte function leading to severe immunodeficiency. Carrier erythrocyte encapsulated with native ADA has been administered in an adult-type ADA deficient patient demonstrating that encapsulated enzyme is protected from antigenic responses and therapeutic activities are sustained (54). Recently, EPR through carrier RBC has been attempted for the treatment of Mitochondrial Neuro GastroIntestinal Encephalopathy (MNGIE), an inherited disease caused by deficiency of thymidine phosphorylase (TP) resulting in severe loss of TP function and prominent plasma accumulations of the TP substrates thymidine and deoxyuridine. A clinical pilot investigated the efficacy and safety of the administration of TP-loaded RBC in an adult patient affected by MNGIE revealing the improvement of biochemical parameters but few ameliorations of patient's clinical conditions (55).

Finally, RBC as enzyme carriers could be applied to thrombolytic therapy by means of the internalization of fibrinolytic agents, because RBC could assure protection against rapid inactivation by circulating antibodies, proteolytic degradation in the blood flow and lower dosages. Furthermore, loaded erythrocytes could act only when anticoagulation is needed, that is during a clotting process, for the active agent could be released in situ when the red blood cell spontaneously lyses within the clot fragment. Among fibrinolytic agents, urokinase and streptokinase have been both encapsulated in RBC (56–58) or coupled onto cell membrane (59–62) and administered in this form to prevent thrombosis. Examples of enzymes loaded into RBC are listed in Table 2.

Table 2. Examples of enzymes loaded into RBC
Loaded enzymeModality of actionIn vitroPreclinicalClinicalProposed applicationRefs.
L-AsparaginaseLong circulating bioreactors, RES targetingXXXAcute lymphoblastic leukemia(63, 64–67)
Alcohol dehydrogenase, Aldehyde dehydrogenaseLong circulating bioreactorsXX Alcohol detoxification(48, 68)
Alcohol oxidaseLong circulating bioreactorsXX Alcohol detoxification(49)
Formate dehydrogenaseLong circulating bioreactorsXX Alcohol detoxification(69)
Hexokinase and glucose oxidaseLong circulating bioreactorsXX Iperglycemia(50)
PhosphotriesteraseLong circulating bioreactorsXX Intoxication by organophosphates(70)
Lactate–catabolizing enzymesLong circulating bioreactors    (71)
RhodanaseLong circulating bioreactorsXX Cyanide intoxication(72–74)
Delta-aminolevulinate dehydrataseRES targetingXXXLead intoxication(75, 76)
Glutamate dehydrogenaseLong circulating bioreactorsXX Hyperammoniemia(77, 78)
UricaseLong circulating bioreactorsXX Hyperuricemia(79, 80)
Urease, PEG-UreaseLong circulating bioreactorsX  Hyperuricemia(81)
Catalase, PEG-CatalaseLong circulating bioreactorsX  Against reactive species in deficiency cases(58)
Galactosidase, GlucuronidaseRES targetingXX Lysosomal storage diseases(51, 52)
Alglucerase, beta-GlucocerebrosidaseLong circulating bioreactors, RES targetingX XEPR in Gaucer's disease(53, 82)
PEG-Ademase, Adenosine deaminaseLong circulating bioreactors  XEPR in adenosine deaminase deficiences(54, 83, 84)
ArginaseLong circulating bioreactorsXX EPR in familiar hyperargininemia(85)
Thymidine phosphorylaseLong circulating bioreactors  XEPR in MNGIE(55)
UrokinaseLong circulating bioreactors X Thrombolytic therapy(56, 57)
Tissue plasminogen activatorLong circulating bioreactors X Thrombolytic therapy(59)
BrinaseLong circulating bioreactors X Thrombolytic therapy(56)

Surely, the most successful therapeutic application of enzyme-loaded erythrocytes is represented by the encapsulation of L-asparaginase for the treatment of Acute Lymphoblastic Leukemia (ALL). Asparaginase is an exogenous enzyme which converts amino acid L-asparagine to L-aspartic acid and ammonia. As ALL cells are unable to produce the amino acid Asn, its systemic depletion by asparaginase would impair protein biosynthesis leading to ALL cell death. Asparaginase from E. coli has been in clinical use since 1967; unfortunately, these available protein preparations present to date some undesirable disadvantages such as short plasma half-life and high rate of anaphylactic reactions observed in patients. These inconveniences have been partially overcome by coating the enzyme with polyethylene glycol but other important adverse effects are frequently observed. Thus to increase the outcome of the treatment, improving both pharmacokinetic parameters and enzyme efficacy, L-asparaginase loaded-erythrocytes have been proposed. Asparaginase has been first encapsulated in vitro into RBC revealing that Asn rapidly entered the red cells and was metabolized by the loaded enzyme in situ (63, 86, 87). This was confirmed in several animal models (63, 64–67). These findings led to the clinical exploitation of asparaginase-loaded RBC through human clinical trials in which both pharmacodynamic parameters and enzyme efficacy improved (65). Currently, phase II/III clinical trials, for the treatment of ALL patients with L-Asparaginase entrapped into human RBC, are ongoing in Europe. Recently, L-Asparaginase has been encapsulated into RBC by means of a novel method using the membrane-traslocating low molecular weight protamine (88). This method was demonstrated to give intact and fully functional RBC and prolong enzyme half-life, resulting very effective in the treatment of LLA in mouse models.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

It is very difficult to envisage a vascular drug delivery system better suited for the scope than the red blood cell. These “carriers” naturally circulate for 120 days and are removed from circulation not randomly but by selected recognition mechanisms. Furthermore, the technological advances in the field and the availability of a specific medical device to realize the entrapment of drugs into autologous blood, now permits the diffusion of the method in all clinical setting. The modalities described in this article for administering a wide range of new and established chemical entities permit to cover a large number of treatment applications finally responding to unsolved medical needs.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RED BLOOD CELLS AS BIOREACTORS FOR THE SLOW DELIVERY OF DRUGS
  5. NEW MODALITIES FOR THE USE OF RED BLOOD CELLS AS CARRIERS FOR DIFFUSIBLE DRUGS
  6. RED BLOOD CELLS AS ENZYME CARRIERS
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES
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