No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Immunophilin-loaded erythrocytes as a new delivery strategy for immunosuppressive drugs
Abstract
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.
... To obtain RBC-based multimodal theranostic probes, the RBCs were first obtained from healthy female nude mice. Second, the complexes of ICG with bovine serum albumin (BSA) were loaded into RBCs through the strategy of hypotonic dialysis (RBC@ICG) to avoid destructing the RBC membranes [47,51]. Third, to link the avidin-modified nanoprobes, the membrane proteins of RBC@ICG were biotinylated by biotin-modified phospholipids (DSPE-PEG 2000 -biotin) (RBC@ICG@biotin) [47]. ...
... Second, the complexes of ICG with bovine serum albumin (BSA) were loaded into RBCs through the strategy of hypotonic dialysis (RBC@ICG) to avoid destructing the RBC membranes [47,51]. Third, to link the avidin-modified nanoprobes, the membrane proteins of RBC@ICG were biotinylated by biotin-modified phospholipids (DSPE-PEG 2000 -biotin) (RBC@ICG@biotin) [47]. Finally, according to the bio-interaction of avidin and biotin, the UCNPs@RB@RGD@avidin were then crosslinked with RBC@ICG@biotin (Figure 1). ...
... Surface modification of RBC membranes was conducted as follows. RBC surface functionalization is obtained by following a previously reported method [47]. To prepare biotin-modified membranes of RBCs, 400 μL concentrated RBCs were redispersed in PBS (4 mL, 1 X, pH 7.4). ...
The therapeutic efficacy of fluorescence image-guided tumor surgery and photodynamic therapy (PDT) is impaired by the penetration depth limitation, low signal-to-noise ratio of traditional first near-infrared window (NIR I) fluorescence and the hypoxic tumor microenvironment. Here, a “red blood cell-based multimodal probe” was proposed to achieve enhanced tumor targeting and retention of fluorescent probes after an intravenous injection, so that second near-infrared window (NIR II) fluorescence bioimaging-guided complete tumor resection and high-efficiency photodynamic therapy could then be realized.
Methods: The hexanoic acid ester-modified rose bengal (RB-HA), RGD (Arginine-Glycine-Aspartic) peptide and avidin were covalently coupled onto amine-modified upconversion nanoparticles (UCNPs) via EDC/NHS reaction (UCNPs@RB@RGD@avidin). Afterwards, the complex of ICG with bovine serum albumin (BSA) was loaded into RBCs through hypotonic dialysis (RBC@ICG). Then, the membrane proteins of RBC@ICG were biotinylated by biotin-modified phospholipids (RBC@ICG@biotin). Finally, the RBCp (Red Blood Cell based probe) was obtained by crosslinking UCNPs@RB@RGD@avidin to RBC@ICG@biotin through the interaction of avidin and biotin. The obtained multimodal RBCp was extensively characterized, both in vitro and in vivo, including analysis of chemical, physical and fluorescent features, O2 delivery ability, tumor accumulation, NIR II fluorescence bioimaging ability, photodynamic therapeutic efficiency, and biosafety.
Results: The RBCp experienced efficient tumor targeting and long tumor retention for almost 4 h after intravenous injection, and the superior signal-to-noise ratio at the optimal time window can be used for guiding precise tumor resection under an 808-nm laser irradiation to facilitate lymph popliteal metastasis surgical delineation. Meanwhile, the RBCp can provide laser-responsive O2 release to enhance the PDT efficiency of popliteal lymph node metastasis under NIR II fluorescence bioimaging guidance. These excellent performances obviously lead to remarkably enhanced synergistic therapeutic effects of tumor surgery and metastatic inhibition.
Conclusion: The proposed strategy will develop a new platform to increase surgical resection completeness and improve PDT efficiency, resulting in the successful and complete inhibition of tumor and metastasis, which could offer a promising approach for the clinical translation of malignant tumor treatment.
... The incorporation of an antifolate and antimetabolite agent methotrexate (used for the treatment of blood-related diseases and malignancy) into red blood cells can be used to enhance the lifetime of hepatoma cells of rats which is comparatively less when the cells are treated with the free form of MTX. It was demonstrated by Biagiotti et al. that erythrocytes-based carriers play an important role in the delivery of immunosuppressive agents (Biagiotti et al., 2011). It has been observed that red blood cells can be loaded with different drugs to treat different diseases, such as δ-aminolevulinate is used to treat lead poisoning, β-glucocerebrosidase is used to treat enzyme replacement therapy in Gaucher's disease and gentamicin is used to inhibit bacterial infection. ...
... The incorporation of inhibitors of insulin in the erythrocytes directly stabilizes the amount of insulin in the cells by preventing its degradation. But still, further research is required to carry out this system in vivo (Bird et al., 1983). ...
The use of drug delivery systems (DDSs) using biological carriers, especially blood cells is a rapidly emerging field. The blood cells have many exceptional advantages, for example, longer circulation time such as erythrocytes, targeted drug release capacity (platelets), and adhesive properties in the case of leukocytes and platelets. The blood cells particularly the erythrocytes are well studied for their role as carriers for drugs owing to the slow release of the drug or for targeted drug delivery to certain organs such as the reticuloendothelial system, spleen, and liver. The erythrocytes have extensively been considered to carry a wide range of drugs including antibiotics, antiviral agents, antiinflammatory, and therapeutic enzymes. In this chapter, the recent advances in the blood cell-based drug delivery systems for the therapeutic management of various diseases have been discussed. Further, we have discussed the advantages and drawbacks of blood cell-based DDSs.
... Moran et al. [81] found that when thymidine phosphorylase (TP) was carried by red blood cells, the levels of deoxythymidine and deoxyuridine in mitochondrial of neuro gastro intestinal encephalomyopathy (MNGIE) patients were significantly reduced. Biagiottiet et al. [82] confirmed that immune suppressants can be encapsulated into red blood cells in the presence of corresponding target proteins, and red blood cells can serve as a promising delivery system for immunosuppressive agents [82]. Hubbell demonstrated that albumin conjugated with peptides binding to red blood cells leads to T-cell deletion and immune tolerance to the antigen [83], this study demonstrated a tolerogenic effect of red blood cells coupling that has significant implications on their use as delivery vehicles for biologic agents. ...
... Moran et al. [81] found that when thymidine phosphorylase (TP) was carried by red blood cells, the levels of deoxythymidine and deoxyuridine in mitochondrial of neuro gastro intestinal encephalomyopathy (MNGIE) patients were significantly reduced. Biagiottiet et al. [82] confirmed that immune suppressants can be encapsulated into red blood cells in the presence of corresponding target proteins, and red blood cells can serve as a promising delivery system for immunosuppressive agents [82]. Hubbell demonstrated that albumin conjugated with peptides binding to red blood cells leads to T-cell deletion and immune tolerance to the antigen [83], this study demonstrated a tolerogenic effect of red blood cells coupling that has significant implications on their use as delivery vehicles for biologic agents. ...
Most conventional drugs accumulate in non-target areas in the body of patients and are responsible for adverse side effects due to poor solubility and poor selectivity after injection, with only a tiny portion of drugs reaching their targets (cells or tissues). Drug delivery is believed to overcome this challenge by allowing control over drug distribution, metabolism, and excretion, which also improves drug safety and efficacy. Nano-drugs (nanoscale particles and vehicles) have attracted great interest because of the enhanced permeation and retention (EPR) effect for tumors. In this review, we summarized the types of nano-drug carriers, described the advantages and adverse effects of the carriers, and discussed the mechanism of single nano-drug transport in living cells. Better understanding the nano-drug delivery mechanism is necessary to ensure the potential chemotherapy of nano-drugs.
... The co-loading of drugs with protein binding partners was originally developed to increase the amount of drug that can be retained by RBCs. For example, loading the immunosuppressive agents, cyclosporine and FK506, in the presence of cyclophilin A and FKBP12, respectively, increases drug-loading capacity by 10-fold (Biagiotti et al., 2011). An analogous observation was reported for phenytoin, a hydrophobic antiepileptic agent, which was introduced into RBCs in the presence of BSA (Hamidi et al., 2011). ...
... An analogous observation was reported for phenytoin, a hydrophobic antiepileptic agent, which was introduced into RBCs in the presence of BSA (Hamidi et al., 2011). Although the rate of drug release from drug/protein-RBCs relative to drug-only RBCs was either not reported (Biagiotti et al., 2011) or showed only modest improvements (Hamidi et al., 2011), it is possible to imagine that intracellular drug-protein complexes could dramatically alter the in vivo pharmacokinetics of drug release. ...
Cell-based drug delivery systems offer the prospect of biocompatibility, large-loading capacity, long in vivo lifespan, and active targeting of diseased sites. However, these opportunities are offset by an array of challenges, including safeguarding the integrity of the drug cargo and the cellular host, as well as ensuring that drug release occurs at the appropriate time and place. Emerging strategies that address these, and related, issues, are described herein.
... So far, the cell-mediated drug delivery concept has been evaluated in the context of cancer, infectious diseases, and inflammatory and neurological disorders. Subsequent studies have demonstrated positive therapeutic effects by using cellular drug vehicles such as erythrocytes [45][46][47][48][49][50][51][52][53][54] , fibroblasts [55,56] , encapsulated or inactivated cancer cells [57][58][59] , mesenchymal [60][61][62] and neural [63][64][65][66][67][68] progenitor/stem cells, myoblasts [69] , Sertoli cells [70] and leukocytes such as dendritic cells [71] , monocytes/macrophages [72][73][74][75][76][77][78][79] , lymphocytes [80] , lymphokine-activated killer (LAK) cells [6,81,82] , NK cells [83] , and T lymphocytes [11,[84][85][86][87][88][89][90][91] . Members of following therapeutic classes were used as payloads for the aforementioned cellular carriers: derivatives of alkaloids [11] , anthracyclines [ , antimetabolites [45,47,72] , antimicrobials [48,51,79] , cytokines [90] , enzymes [52][53][54]77] , immunomodulators [91] , immunosuppressors [49,50,70,71] , nanoparticulate gold [75] and iron/iron oxide [73] , photosensitizers [47] , plant toxins [82,84] , radioactive isotopes [80] , and viruses [62,68,89] . ...
... Subsequent studies have demonstrated positive therapeutic effects by using cellular drug vehicles such as erythrocytes [45][46][47][48][49][50][51][52][53][54] , fibroblasts [55,56] , encapsulated or inactivated cancer cells [57][58][59] , mesenchymal [60][61][62] and neural [63][64][65][66][67][68] progenitor/stem cells, myoblasts [69] , Sertoli cells [70] and leukocytes such as dendritic cells [71] , monocytes/macrophages [72][73][74][75][76][77][78][79] , lymphocytes [80] , lymphokine-activated killer (LAK) cells [6,81,82] , NK cells [83] , and T lymphocytes [11,[84][85][86][87][88][89][90][91] . Members of following therapeutic classes were used as payloads for the aforementioned cellular carriers: derivatives of alkaloids [11] , anthracyclines [ , antimetabolites [45,47,72] , antimicrobials [48,51,79] , cytokines [90] , enzymes [52][53][54]77] , immunomodulators [91] , immunosuppressors [49,50,70,71] , nanoparticulate gold [75] and iron/iron oxide [73] , photosensitizers [47] , plant toxins [82,84] , radioactive isotopes [80] , and viruses [62,68,89] . ...
... Methotrexate (MTX), an antimetabolite and antifolate agent used in solid tumors and hematological diseases, can be encapsulated by erythrocytes, and the average survival time of rat hepatoma cells is enhanced with MTXloaded erythrocyte treatment compared with that of cells treated with native MTX. 55 Biagiotti et al 56 confirmed that immunosuppressants can be encapsulated into erythrocytes in the presence of corresponding target proteins, and RBCs can serve as a promising delivery system for immunosuppressive agents. 56 The use of RBCs as a drug delivery system for chemotherapeutic agents, especially in vitro and in vivo use of antineoplastic agents, has been widely investigated. ...
... 55 Biagiotti et al 56 confirmed that immunosuppressants can be encapsulated into erythrocytes in the presence of corresponding target proteins, and RBCs can serve as a promising delivery system for immunosuppressive agents. 56 The use of RBCs as a drug delivery system for chemotherapeutic agents, especially in vitro and in vivo use of antineoplastic agents, has been widely investigated. Other therapeutic drugs delivered by RBCs include gentamicin for bacterial infection, 57 δ-aminolevulinate dehydratase for lead poisoning, 58 β-glucocerebrosidase for enzyme replacement therapy in Gaucher's disease, 59 adenosine deaminase for adenosine deaminase deficiencies, 60 enalaprilat for hypertension management and congestive heart failure, 61 and heparin for thromboembolism 62 and carrier for thrombolytic agents. ...
In the past few years, nanomaterial-based drug delivery systems have been applied to enhance the efficacy of therapeutics and to alleviate negative effects through the controlled delivery of targeting and releasing agents. However, few drug carriers can achieve high targeting efficacy, even when targeting modalities and surface markers are introduced. Immunological problems have also limited their wide applications. Biological drug delivery systems, such as erythrocytes, platelets, and albumin, have been extensively investigated because of their unique properties. In this review, erythrocytes, platelets, and albumin are described as efficient drug delivery systems. Their properties, applications, advantages, and limitations in disease treatment are explained. This review confirms that these systems can be used to facilitate a specific, biocompatible, and smart drug delivery.
... Erythrocytes have been proposed for the delivery of a chemotherapeutic agent, and the process of its encapsulation in erythrocytes has been studied [17]. Erythrocytes have also been considered as a container for immunosuppressive agents, the systemic administration of which can significantly improve the outcome of organ transplantation [18]. Moreover, thrombolytic agents have been obtained for the treatment of a wide range of diseases [19]. ...
Citation: Mayorova, O.A.; Gusliakova, O.I.; Prikhozhdenko, E.S.; Verkhovskii, R.A.; Bratashov, D.N. Magnetic Platelets as a Platform for Drug Delivery and Cell Trapping. Pharmaceutics 2023, 15, 214. Abstract: The possibility of using magnetically labeled blood cells as carriers is a novel approach in targeted drug-delivery systems, potentially allowing for improved bloodstream delivery strategies. Blood cells already meet the requirements of biocompatibility, safety from clotting and blockage of small vessels. It would solve the important problem of the patient's immune response to embedded foreign carriers. The high efficiency of platelet loading makes them promising research objects for the development of personalized drug-delivery systems. We are developing a new approach to use platelets decorated with magnetic nanoparticles as a targeted drug-delivery system, with a focus on bloodstream delivery. Platelets are non-nuclear blood cells and are of great importance in the pathogenesis of blood-clotting disorders. In addition, platelets are able to attach to circulating tumor cells. In this article, we studied the effect of platelets labeled with BSA-modified magnetic nanoparticles on healthy and cancer cells. This opens up broad prospects for future research based on the delivery of specific active substances by this method.
... Haematocrit was positively correlated with the WB:IC tacrolimus concentration ratio. This relationship can be explained by the high binding capacity of tacrolimus to erythrocytes [30]. As haematocrit reflects the erythrocyte concentration, a higher haematocrit might result in a higher binding capacity, which in turn decreases the shift to the intracellular compartment. ...
Background and objective:
The tacrolimus concentration within peripheral blood mononuclear cells may correlate better with clinical outcomes after transplantation compared to concentrations measured in whole blood. However, intracellular tacrolimus measurements are not easily implemented in clinical practice. The prediction of intracellular concentrations based on whole-blood concentrations would be a solution for this. Therefore, the aim of this study was to describe the relationship between intracellular and whole-blood tacrolimus concentrations in a population pharmacokinetic (popPK) model.
Methods:
Pharmacokinetic analysis was performed using non-linear mixed effects modelling software (NONMEM). The final model was evaluated using goodness-of-fit plots, visual predictive checks, and a bootstrap analysis.
Results:
A total of 590 tacrolimus concentrations from 184 kidney transplant recipients were included in the study. All tacrolimus concentrations were measured in the first three months after transplantation. The intracellular tacrolimus concentrations (n = 184) were best described with an effect compartment. The distribution into the effect compartment was described by the steady-state whole-blood to intracellular ratio (RWB:IC) and the intracellular distribution rate constant between the whole-blood and intracellular compartments. Lean body weight was negatively correlated [delta objective function value (ΔOFV) -8.395] and haematocrit was positively correlated (ΔOFV = - 6.752) with RWB:IC, and both lean body weight and haematocrit were included in the final model.
Conclusion:
We were able to accurately describe intracellular tacrolimus concentrations using whole-blood concentrations, lean body weight, and haematocrit values in a popPK model. This model may be used in the future to more accurately predict clinical outcomes after transplantation and to identify patients at risk for under- and overexposure. Dutch National Trial Registry number NTR2226.
... The RBC-based DDSs also exhibit new applications in immunological and host defense therapies. For exam-ple, steroids and inhibitors can bind to RBCs to mitigate the inflammatory response and antigens can bind and deliver to defense cells to modulate the immune response [49,[78][79][80][81]. Additionally, it was found that delivering antigens on the RBC surface to different cells could lead to diverse or even opposite immunological responses, and the determinant factors involved in these processes require further investigation [82][83][84][85]. ...
In recent years, drug delivery has emerged as a more and more popular drug administration means for the improvement of pharmacokinetics, minimization of side effects, and enhancement of clinical outcomes. However, rapid clearance from the blood flow by immune systems limits the performance of drugs injected into the blood circulation. Thus, a new avenue for drug delivery systems by using the blood cells has drawn increasing attention. Blood cells can protect the drugs from macrophage uptake, prolong their circulation time, and improve their biocompatibility and stability. Here, we review recent advances in the developments and applications of numerous blood cell-based drug delivery systems and those inspired by blood cells. We highlight examples of vascular drug delivery using carriers red blood cells, white blood cells as well as platelets. We expect that this review will provide a jumping-off point or an inspiration for further investigation in this area.
... While not specifically investigated for IBD treatment, further studies point out the certain benefits of erythrocyte-based carriers that highlight their further potential applications within the context of IBD therapy. For instance, RBCs have been introduced as efficient carriers for modifying the pharmacokinetic of immunosuppressive drugs, whereby the adverse effects of the latter can be significantly reduced [246]. Furthermore, the applicability of the intraperitoneal injection of carrier erythrocytes for the targeting of peritoneal macrophages in mice has been established [247]. ...
Current treatment strategies for inflammatory bowel disease (IBD) seek to alleviate the undesirable symptoms of the disorder. Despite the higher specificity of newer generation therapeutics, e.g. monoclonal antibodies, adverse effects still arise from their interference with non-specific systemic immune cascades. To circumvent such undesirable effects, both conventional and newer therapeutic options can benefit from various targeting strategies. Of course, both the development and the assessment of the efficiency of such targeted delivery systems necessitate the use of suitable in vivo and in vitro models representing relevant pathophysiological manifestations of the disorder. Accordingly, the current review seeks to provide a comprehensive discussion of the available preclinical models with emphasis on human in vitro models of IBD, along with their potentials and limitations. This is followed by an elaboration on the advancements in the field of biology- and nanotechnology-based targeted drug delivery systems and the potential rooms for improvement to facilitate their clinical translation.
... For example, FKBP-loaded RBCs have been developed as a DDS for tacrolimus delivery via the binding between FKBP and tacrolimus. (7) In this study, we first showed that intracellular binding of tacrolimus to FKBP was critical for the distribution of tacrolimus in RBCs. Considering the change in the amount of tacrolimus remaining in the buffer, as shown in Fig. 1, we concluded that treatment with rapamycin, which competitively inhibits the Fig. 5 Effect of ATP on the distribution of tacrolimus from buffer to RBCs. ...
PurposeTacrolimus is distributed mainly in red blood cells (RBCs) after transfer into blood. This study aimed to evaluate the effect of FK506-binding proteins (FKBPs) on RBC distribution of tacrolimus in a physiological environment.Methods
Human RBCs were isolated from fresh blood samples from healthy volunteers. The effect of FKBPs on each process of the RBC distribution of tacrolimus was evaluated in vitro. Effect of intracellular FKBPs was assessed by inhibition experiment with rapamycin, which competitively inhibits the binding of tacrolimus to FKBPs. Effect of extracellular FKBPs was examined by pre-exposure of RBCs to FKBP and preincubation of tacrolimus with FKBP.ResultsPretreatment with rapamycin significantly reduced the rate of tacrolimus distribution in RBCs in a concentration-dependent manner. Pre-exposure of RBCs to FKBP12 followed by exposure to tacrolimus significantly decreased tacrolimus distribution in RBCs in a concentration-dependent manner. In addition, preincubation of tacrolimus with FKBP12 significantly reduced the rate of tacrolimus distribution in RBCs.ConclusionsFKBP played an important role in the distribution of tacrolimus in RBCs. The effect of intracellular and extracellular FKBPs on RBC distribution of tacrolimus in circulating blood was substantial. FKBP was shown as a potential biomarker for predicting the pharmacokinetics and pharmacodynamics of tacrolimus.
... As a natural carrier of oxygen in the blood, red blood cells (RBCs) have aroused wide interest due to the stability, biocompatibility, flexibility, and other characteristics. 27 Braḧler et al. confirmed that the RBC-encapsulated superparamagnetic iron oxide nanoparticles still have good magnetic resonance imaging effects. 28 Zhang et al. further proposed and developed RBC membrane encapsulation technology, which proved that the encapsulation structure can effectively reduce the phagocytosis of nanoparticles by macrophages, increasing the blood circulation time and stability in vivo. ...
Here, ferric oxide loaded metal-organic frameworks (FeTCPP/Fe2O3 MOFs) nanorice was designed and constructed by liquid diffusion method. The introduction of iron metal nodes and the loading of Fe2O3 can effectively catalyze fenton reaction to produce hydroxyl radicals (•OH), and overcome the hypoxic environment of tumor tissue by generating oxygen. The monodispersity and porosity of the porphyrin photosensitizers in the MOFs structure exposed more active sites, which promoted energy exchange between porphyrin molecules and oxygen molecules for photodynamic therapy (PDT) treatment. Therefore, the generated hydroxyl radicals and singlet oxygen (1O2) can synergistically act on tumor cells to achieve the purpose of improving the tumor therapy. Then the erythrocyte membrane was camouflaged to enhance blood circulation and tissue residence time in the body, and finally the targeted molecule AS1411 aptamer was modified to achieve the high enrichment of MOFs photosensitizers on tumor domain. As a result, the MOFs nanorice camouflaged by erythrocyte membrane could effectively enhance the efficacy of PDT and chemo-dynamic therapy (CDT) and reduce the side effect. This study not only improved the efficacy of PDT and CDT in essence from the MOFs nanorice, but also used the method of camouflage to further concentrate the FeTCPP/Fe2O3 on the tumor sites, achieving the goal of multiple gains. These results will provide theoretical and practical directions for the development of tumor-targeted MOFs nanomaterials
... Tacrolimus is particularly distributed into erythrocytes, next to being associated with (lipo)proteins (see Fig. 2) [38,39]. Within the erythrocytes, tacrolimus is known to be highly associated with the FK-binding protein [40][41][42][43]. In the clinically unstable transplant patient, erythrocyte counts may highly fluctuate due to bleeding, red blood cell transfusions, dilution, bone marrow depression, and hemolysis due to extracorporeal equipment. ...
The calcineurin inhibitor tacrolimus is an effective immunosuppressant and is extensively used in solid organ transplantation. In the first week after heart and lung transplantation, tacrolimus dosing is difficult due to considerable physiological changes because of clinical instability, and toxicity often occurs, even when tacrolimus concentrations are within the therapeutic range. The physiological and pharmacokinetic changes are outlined. Excessive variability in bioavailability may lead to higher interoccasion (dose-to-dose) variability than interindividual variability of pharmacokinetic parameters. Intravenous tacrolimus dosing may circumvent this high variability in bioavailability. Moreover, the interpretation of whole-blood concentrations is discussed. The unbound concentration is related to hematocrit, and changes in hematocrit may increase toxicity, even within the therapeutic range of whole-blood concentrations. Therefore, in clinically unstable patients with varying hematocrit, aiming at the lower therapeutic level is recommended and tacrolimus personalized dosing based on hematocrit-corrected whole-blood concentrations may be used to control the unbound tacrolimus plasma concentrations and subsequently reduce toxicity.
... Different types of cells, such as erythrocytes, leukocytes, platelets and stem cells, have been used to explore for the treatment of various diseases or disorders, including cancer [24,[35][36][37], chronic inflammatory diseases [38], ataxia telangiectasia [39], Gaucher disease [40], diabetes [41], autoimmune diseases [42], etc. (Table 1). ...
Functional biomaterials that are capable of effectively carrying therapeutic agents and specifically delivering therapeutics to pathological sites have been widely investigated over decades. Recently, cellular carriers and cell derivative-based bio-hybrid delivery systems have drawn extensive attention as a promising branch of therapeutic delivery systems, owing to their low immunogenicity and intriguing biomimetic capabilities. Various approaches for the fabrication of these biomimetic carriers have been developed, and some products have already been commercialized as well. In this review, we summarized various processing methods for engineering cell-derived biomimetic drug delivery systems, and discussed their future outlooks.
... The tacrolimus-FKBP12 complex in turn binds to calcineurin and blocks the activation of this calcium/calmodulin-activated phosphatase within the T-lymphocyte (Griffith et al., 1995. However, erythrocytes also have a high concentration of FKBP12 and tacrolimus is extensively distributed within the red blood cell compartment (Biagiotti et al., 2011). Approximately 80% (range 70-95%) of tacrolimus measured in whole blood is distributed in erythrocytes, where it has no immunosuppressive effect. ...
Background
After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds for approximately 80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate an UPLC‐MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs).
Methods
PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDMPREP. To each sample we added: 30 μL lysis buffer, 20 μL reconstitution buffer containing ¹³C²H4‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDMPREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. 10 μL of the supernatant was injected into the UPLC‐MS/MS system.
Results
The method was validated resulting in high sensitivity and specificity. The method was linear (r²=0.997) over the range of 5.0–1250 pg/1*10⁶ PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results.
Conclusions
A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.
... Another group proposed the usage of erythrocytes engineered for the TAC DDS for modifying its pharmacokinetics as well as restraining adverse symptoms successfully. Results suggested that TAC after diffusion gets captured up inside red blood cells because of the respective target protein loading consequently, paving a way for the immunophilin-loaded erythrocytes in subjects accidently receiving toxic dose (Biagiotti et al., 2011). Therefore, various drug delivery systems have been discussed which minimizes toxic effects of TAC and adequately reaches the target site. ...
From the current trends, tacrolimus (TAC) has become an important therapeutic option for the optimal individualization of immunosuppressive therapy especially in case of transplant recipients. TAC is used most frequently in comparison to other immunosuppressants because it offers better safety profile with increased long-term survival in patients especially in children and adolescents. This drug has developed an immense interest in the research field owing to its potential pharmacological scope but due to its poor water solubility, need of concomitant steroids and higher incidences of nephrotoxicity, there comes a need for future research to minimize such limitations and decipher maximum use of the drug. In addition, there are number of formulations attempted to enhance its erratic bioavailability through various techniques namely solid dispersions, inclusion complexes, prodrug approach, SMEDDS etc. The present review aims to acknowledge the TAC pharmacokinetic profile and novel drug delivery systems in multiple diseased conditions by particularly enhancing its poor biopharmaceutical issues as well as dose related toxicity. Collectively, we have updated the data pertaining to the drug delivery prospects of TAC for the period of last 8–10 years.
... DOX@RBC was produced based on the "hypotonic dialysis encapsulation" method reported previously. 24,26,34,35 The loading efficiency of DOX into RBCs was determined to be ~17% based on the fluorescence of DOX after lysis of DOX@RBC and extraction of free DOX by HCl / isopropanol. Subsequently, DOX@RBC was treated with Ce6 as preparations of RBC-Ce6 to get DOX@RBC-Ce6. ...
Red blood cells (RBCs), the most abundant type of cells in our blood, have shown promises as a natural drug delivery system (DDS) with inherent biocompatibility. Herein, we uncover that a photosensitizer, chlorin e6 (Ce6), could be decorated into the membrane of RBCs upon simple mixing, without affecting the membrane integrity and stability in dark. Upon light irradiation with a rather low power density, the generated singlet oxygen by Ce6 as the result of photodynamic effect would lead to rather efficient disruption of RBC membrane. With doxorubicin (DOX), a typical chemotherapy drug, as the model, we engineer a unique type of light-responsive RBC-based DDS by decorating Ce6 on the cell membrane and loading DOX inside cells. The light triggered cell membrane break down would thus trigger instant release of DOX, enabling light-controlled chemotherapy with great specificity. Beyond that, our RBC system could also be utilized for loading of larger biomolecules such as enzymes, whose release as well as catalytic function is also controlled by light. Our work thus presents a unique type of biocompatible cell-based DDS that can be precisely controlled by mild external stimuli, promising not only for cancer therapy but also for other potential applications in biotechnologies.
... 46 Immunophilin proteins that noncovalently bind to immunosuppressive drugs are entrapped within erythrocytes to increase drug retention. 47 Although clever and suitable for industrial application, this method is restricted to molecules that are ligands of proteins. Another possibility is to encapsulate a prodrug that will be ...
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.
... Mica was purchased from Novascan Technologies, Inc. (Ames, Iowa, USA). The recombinant protein FKBP12 was already available in our lab where it had been characterized [11]. All other reagents and chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). ...
In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose-response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.
Copyright © 2015 Elsevier B.V. All rights reserved.
... Cyclosporine A (CsA) is a natural compound produced by the fungus Tolypocladium inflatum (Lallemand et al., 2003). CsA belongs to the class of ''calcineurin inhibitor'' in immunosuppressant drugs and immunosuppressive activity depends mostly on its ability to bind and inhibit the enzyme calcineurin (Biagiotti et al., 2011). Due to its cyclic structure and lipophilic character, CsA is a very stable molecule (Kumar et al., 2001). ...
Abstract In the present study, cyclosporine A (CsA) was successfully incorporated into cationic chitosan nanoparticles by spray-drying method aiming ocular application. Physicochemical characterisation of particles was performed in detail. Among the particles prepared using three types of chitosan with different molecular weights, particles containing chitosan with medium molecular weight was selected for in vivo studies. Selection was dependent on higher incorporation and encapsulation efficiencies of CsA and also better release characteristic in simulated tear fluid. Sheep were used in in vivo studies. Biological samples were collected at predetermined time intervals and were analysed by enzyme immune assay. CsA could be detected in both aqueous and vitreous humour samples for the duration of 72 h. In vivo release profiles indicated prolonged release of active agent from positively charged chitosan formulations. This may be attributed to enhanced residence time at the corneal and conjunctival surfaces.
This review focuses on the role of human red blood cells (RBCs) as drug carriers. First, a general introduction about RBC physiology is provided, followed by the presentation of several cases in which RBCs act as natural carriers of drugs. This is due to the presence of several binding sites within the same RBCs and is regulated by the diffusion of selected compounds through the RBC membrane and by the presence of influx and efflux transporters. The balance between the influx/efflux and the affinity for these binding sites will finally affect drug partitioning. Thereafter, a brief mention of the pharmacokinetic profile of drugs with such a partitioning is given. Finally, some examples in which these natural features of human RBCs can be further exploited to engineer RBCs by the encapsulation of drugs, metabolites, or target proteins are reported. For instance, metabolic pathways can be powered by increasing key metabolites (i.e., 2,3-bisphosphoglycerate) that affect oxygen release potentially useful in transfusion medicine. On the other hand, the RBC pre-loading of recombinant immunophilins permits increasing the binding and transport of immunosuppressive drugs. In conclusion, RBCs are natural carriers for different kinds of metabolites and several drugs. However, they can be opportunely further modified to optimize and improve their ability to perform as drug vehicles.
In the context of bone regeneration, nanoparticles harboring osteogenic factors have emerged as pivotal agents for modulating the differentiation fate of stem cells. However, persistent challenges surrounding biocompatibility, loading efficiency, and precise targeting ability warrant innovative solution. In this study, we introduce a novel nanoparticle platform founded upon the zeolitic imidazolate framework‐8 (ZIF‐8). This new design, CDC20@ZIF‐8@eM‐Apt, involves the envelopment of ZIF‐8 within an erythrocyte membrane (eM) cloak, and coupled with a targeting aptamer. ZIF‐8, distinguished by its porosity, biocompatibility, and robust cargo transport capabilities, constitutes the core framework. We illuminate that cell division cycle protein 20 homolog (CDC20) as a new target in bone regeneration. The erythrocyte membrane plays a dual role in maintaining nanoparticle stability and facilitating fusion with target cell membranes, while the aptamer orchestrates the specific recruitment of bone marrow mesenchymal stem cells (BMSCs) within bone defect sites. Significantly, CDC20@ZIF‐8@eM‐Apt amplifies osteogenic differentiation of BMSCs via the inhibition of NF‐κB p65, and concurrently catalyzes bone regeneration in two bone defect models. Consequently, CDC20@ZIF‐8@eM‐Apt introduces a pioneering strategy for tackling bone defects and associated maladies, opening novel avenues in therapeutic intervention.
This article is protected by copyright. All rights reserved
Red Blood Cells (RBCs)-derived particles are an emerging group of novel drug delivery systems. The natural attributes of RBCs make them potential candidates for use as a drug carrier or nanoparticle camouflaging material as they are innately biocompatible. RBCs have been studied for multiple decades in drug delivery applications but their evolution in the clinical arena are considerably slower. They have been garnering attention for the unique capability of conserving their membrane proteins post fabrication that help them to stay non-immunogenic in the biological environment prolonging their circulation time and improving therapeutic efficiency. In this review, we discuss about the synthesis, significance, and various biomedical applications of the above-mentioned classes of engineered RBCs. This article is focused on the current state of clinical translation and the analysis of the hindrances associated with the transition from lab to clinic applications.
Drug delivery research pursues many types of carriers including proteins and other macromolecules, natural and synthetic polymeric structures, nanocarriers of diverse compositions and cells. In particular, liposomes and lipid nanoparticles represent arguably the most advanced and popular human-made nanocarriers, already in multiple clinical applications. On the other hand, red blood cells (RBCs) represent attractive natural carriers for the vascular route, featuring at least two distinct compartments for loading pharmacological cargoes, namely inner space enclosed by the plasma membrane and the outer surface of this membrane. Historically, studies of liposomal drug delivery systems (DDS) astronomically outnumbered and surpassed the RBC-based DDS. Nevertheless, these two types of carriers have different profile of advantages and disadvantages. Recent studies showed that RBC-based drug carriers indeed may feature unique pharmacokinetic and biodistribution characteristics favorably changing benefit/risk ratio of some cargo agents. Furthermore, RBC carriage cardinally alters behavior and effect of nanocarriers in the bloodstream, so called RBC hitchhiking (RBC-HH). This article represents an attempt for the comparative analysis of liposomal vs RBC drug delivery, culminating with design of hybrid DDSs enabling mutual collaborative advantages such as RBC-HH and camouflaging nanoparticles by RBC membrane. Finally, we discuss the key current challenges faced by these and other RBC-based DDSs including the issue of potential unintended and adverse effect and contingency measures to ameliorate this and other concerns.
Nanoscale porphyrinic metal−organic frameworks have emerged as promising therapeutic system for photodynamic therapy (PDT) of cancer in recent years. Nevertheless, their low selectivity towards malignant tissues is a hindrance for highly efficient cancer photodynamic therapy. In this study, a nano metal-organic framework ([CuL-[AlOH]2]n (Cu MOF), with Cu (II) core, as a PDT active center, was coated with red blood cell (RBC) membrane after loaded with doxorubicin (DOX), to avoid immediate clearance from the bloodstream and persist for longer periods of time in circulation. Then, a carboxylic acid-end aptamer (MUC1) was decorated on the RBC-coated DOX-loaded MOF (Apt-RBC-MOF@DOX) in order to provide targeted chemo and photodynamic therapy. The MTT and flow cytometry experiments exhibited that Apt-RBC-MOF@DOX provided highly efficient DOX transportation and toxicity against 4T1 and MCF7 breast cancer cells in vitro in comparison to non-targeted RBC-MOF@DOX. On the other hand, the single dose intravenous injection of Apt-RBC-MOF@DOX considerably reduced the tumor growth in vivo. The desirable biodistribution, faster liver clearance, and higher tumor accumulation were confirmed by ex vivo fluorescent imaging. Moreover, under irradiation at 630 nm, the fabricated nanostructures exhibited effective synergistic chemo and photodynamic therapy, proved via in vitro MTT assay and tumor ablation in vivo. The developed Apt-RBC-MOF@DOX could serve as a potent platform in clinic for targeted chemo-photodynamic dual therapy.
Tumor vasculature has long been considered as an extremely valuable therapeutic target for cancer therapy, but how to realize controlled and site-specific drug release in tumor blood vessels remains a huge challenge. Despite the widespread use of nanomaterials in constructing drug delivery systems, they are suboptimal in principle for meeting this demand due to their easy blood cell adsorption/internalization and short lifetime in the systemic circulation. Here, natural red blood cells (RBCs) are repurposed as a remote-controllable drug vehicle, which retains RBC's morphology and vessel-specific biodistribution pattern, by installing photoactivatable molecular triggers on the RBC membrane via covalent conjugation with a finely tuned modification density. The molecular triggers can burst the RBC vehicle under short and mild laser irradiation, leading to a complete and site-specific release of its payloads. This cell-based vehicle is generalized by loading different therapeutic agents including macromolecular thrombin, a blood clotting-inducing enzyme, and a small-molecule hypoxia-activatable chemodrug, tirapazamine. In vivo results demonstrate that the repurposed “anticancer RBCs” exhibit long-term stability in systemic circulation but, when tumors receive laser irradiation, precisely releases their cargoes in tumor vessels for thrombosis-induced starvation therapy and local deoxygenation-enhanced chemotherapy. This study proposes a general strategy for blood vessel-specific drug delivery.
In this study, a novel tumor-targeting drug delivery system (DDS) based on red blood cells (RBCs) were fabricated for combinational chemo-phototherapy against cancer. Cyclic peptide (cRGD) and indocyanine green (ICG) were applied to the surface of RBCs to increase the targeting and photothermal effect, respectively. Doxorubicin (DOX) as a model drug was loaded into RBCs by the hypotonic dialysis method. A series of tests have been carried out to evaluate the RBCs-based DDS and these tasks include physicochemical properties, cellular uptake, targeting ability, and combination therapeutic efficiency. As a result, the DOX was successfully loaded into RBCs and the drug loading amount was 0.84 ± 0.09 mg/mL. There was no significant change of particle size after surface modification of RBCs. The RBCs-based DDS could target to the surface of cancer cells, which delivery DOX to the lesions efficiently and accurately. Meanwhile, due to the combined treatment effect, the RBCs-based DDS can effectively inhibit tumor growth. The RBCs-based DDS constructed in this research may have promising applications in cancer therapy due to their highly synergistic efficient therapy and to investigate its possibility for tumor therapy.
Red blood cell (RBC) hitchhiking is a method of drug delivery that can increase drug concentration in target organs by orders of magnitude. In RBC hitchhiking, drug-loaded nanoparticles (NPs) are adsorbed onto red blood cells and then injected intravascularly, which causes the NPs to transfer to cells of the capillaries in the downstream organ. RBC hitchhiking has been demonstrated in multiple species and multiple organs. For example, RBC-hitchhiking NPs localized at unprecedented levels in the brain when using intra-arterial catheters, such as those in place immediately after mechanical thrombectomy for acute ischemic stroke. RBC hitchhiking has been successfully employed in numerous preclinical models of disease, ranging from pulmonary embolism to cancer metastasis. In addition to summarizing the versatility of RBC hitchhiking, we also describe studies into the surprisingly complex mechanisms of RBC hitchhiking as well as outline future studies to further improve RBC hitchhiking's clinical utility.
Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Modifying living cells using in-situ synthesized nanomaterials to endow them with new functions is highly desirable. Herein we report intra- and extra-cellular dual-modified red blood cells (RBCs) with intracellular CaCO3 nanoparticles (NPs) and extracellular polypyrrole-folic acid (PPy-FA) coating, which are exploited as a bifunctional drug carrier. The functionalized living cells (CaCO3@[email protected]) are fabricated through first the intracellular in situ reaction of exogenous Ca²⁺ and CO3²⁻ ions to generate CaCO3 NPs, then polymerization of pyrrole and finally modification of folic acid (FA) on the membrane of individual cells, forming a CaCO3@[email protected] structure. As a result, such dual-modified RBCs not only preserve the original performances of the cells but also possess the desirable properties as a drug carrier, such as high loading capacity due to the action of CaCO3 NPs, targeting and light-controlled drug release due to the action of PPy-FA. Under NIR laser stimulation, these bifunctional RBCs (DOX-CaCO3@[email protected]) present an instant release profile of doxorubicin (DOX) and have high targeting-ability toward cancer cells, achieving a marked synergistic combined photothermal–chemotherapy effect.
Biofluids act as a repository for disease biomarkers and are excellent diagnostic tools applied in establishing a disease profile based on clinical testing, evaluation and monitoring the progression of patients suffering from various conditions. Furthermore, biofluids and their derived components such proteins, pigments, enzymes, hormones and cells carry a potential in the development of therapeutic drug delivery systems or as cargo materials for targeting the drug to the site of action. The presence of biofluids with respect to their specific location reveals the information of disease progression and mechanism, delivery aspects such as routes of administration as well as pharmacological factors such as binding affinity, rate of kinetics, efficacy, bioavailability and patient compliance. This review focuses on the properties and functional benefits of some biofluids, namely blood, saliva, bile, urine, amniotic fluid, synovial fluid and cerebrospinal fluid. It also covers the therapeutic and targeting action of fluid-derived substances in various micro- or nano-systems like nanohybrids, nanoparticles, self-assembled micelles, microparticles, cell-based systems, etc. The formulation of such biologically-oriented systems demonstrate the advantages of natural origin, biocompatibility and biodegradability and offer new techniques for overcoming the challenges experienced in conventional therapies.
Background and objective:
Therapeutic drug monitoring of tacrolimus whole-blood concentrations is standard care in thoracic organ transplantation. Nevertheless, toxicity may appear with alleged therapeutic concentrations possibly related to variability in unbound concentrations. However, pharmacokinetic data on unbound concentrations are not available. The objective of this study was to quantify the pharmacokinetics of whole-blood, total, and unbound plasma tacrolimus in patients early after heart and lung transplantation.
Methods:
Twelve-hour tacrolimus whole-blood, total, and unbound plasma concentrations of 30 thoracic organ recipients were analyzed with high-performance liquid chromatography-tandem mass spectrometry directly after transplantation. Pharmacokinetic modeling was performed using non-linear mixed-effects modeling.
Results:
Plasma concentration was < 1% of the whole-blood concentration. Maximum binding capacity of erythrocytes was directly proportional to hematocrit and estimated at 2700 pg/mL (95% confidence interval 1750-3835) with a dissociation constant of 0.142 pg/mL (95% confidence interval 0.087-0.195). The inter-individual variability in the binding constants was considerable (27% maximum binding capacity, and 29% for the linear binding constant of plasma).
Conclusions:
Tacrolimus association with erythrocytes was high and suggested a non-linear distribution at high concentrations. Monitoring hematocrit-corrected whole-blood tacrolimus concentrations might improve clinical outcomes in clinically unstable thoracic organ transplants.
Clinical trial registration:
NTR 3912/EudraCT 2012-001909-24.
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
This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies
Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Herein, we fabricated efficient MR imaging probes by incorporating gadolinium oxide nanoparticles (Gd2O3) and gadolinium hybrid nanoparticles (GH) within RBCs. The Gd2O3 and GH encapsulated in the RBCs exhibited high relaxation rates and revealed high sensitivity for T1 MR imaging.
Blood cells, including erythrocytes, leukocytes and platelets are used as drug carriers in a wide range of applications. They have many unique advantages such as long life-span in circulation (especially erythrocytes), target release capacities (especially platelets), and natural adhesive properties (leukocytes and platelets). These properties make blood cell based delivery systems, as well as their membrane-derived carriers, far superior to other drug delivery systems. Despite the advantages, the further development of blood cell-based delivery systems was hindered by limitations in the source, storage, and mass production. To overcome these problems, synthetic biomaterials that mimic blood cell and nanocrystallization of blood cells have been developed and may represent the future direction for blood cell membrane-based delivery systems. In this paper, we review recent progress of the rising blood cell-based drug delivery systems, and also discuss their challenges and future tendency of development.
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.
The field of drug delivery has grown tremendously in the past few decades by developing a wide range of advanced drug delivery systems. An interesting category is cell-based drug delivery, which includes encapsulation of drugs inside cells or attached to the surface and subsequent transportation through the body. Another approach involves genetic engineering of cells to secrete therapeutic molecules in a controlled way. The next-generation systems integrate expertise from synthetic biology to generate therapeutic gene networks for highly advanced sensory and output devices. These developments are very exciting for the drug delivery field and could radically change the way we administer biological medicines to chronically ill patients. This review is covering the use of living cells, either as transport system or production-unit, to deliver therapeutic molecules and bioactive proteins inside the body. It describes a wide range of approaches in cell-based drug delivery and highlights exceptional examples.
Red blood cells (RBCs), the “innate carriers” in blood vessels, are gifted with many unique advantages in drug transportation over synthetic drug delivery systems (DDSs). Herein, a tumor angiogenesis targeting, light stimulus-responsive, RBC-based DDS is developed by incorporating various functional components within the RBC platform. An albumin bound near-infrared (NIR) dye, together with a chemotherapy drug doxorubicin, is encapsulated inside RBCs, the surfaces of which are modified with a targeting peptide to allow cancer targeting. Under stimulation by an external NIR laser, the membrane of the RBCs would be destroyed by the light-induced photothermal heating, resulting in effective drug release. As a proof of principle, RBC-based cancer cell targeted drug delivery and light-controlled drug release is demonstrated in vitro, achieving a marked synergistic therapeutic effect through the combined photothermal–chemotherapy. This work presents a novel design of smart RBC carriers, which are inherently biocompatible, promising for targeted combination therapy of cancer.
Cell-based therapy is a promising modality to address many unmet medical needs. In addition to genetic engineering, material-based, biochemical, and physical science-based approaches have emerged as novel approaches to modify cells. Non-genetic engineering of cells has been applied in delivering therapeutics to tissues, homing of cells to the bone marrow or inflammatory tissues, cancer imaging, immunotherapy, and remotely controlling cellular functions. This new strategy has unique advantages in disease therapy and is complementary to existing gene-based cell engineering approaches. A better understanding of cellular systems and different engineering methods will allow us to better exploit engineered cells in biomedicine. Here, we review non-genetic cell engineering techniques and applications of engineered cells, discuss the pros and cons of different methods, and provide our perspectives on future research directions.
Introduction:
Drug delivery by means of erythrocytes is becoming a growing field with applications that in recent years have moved from the laboratory to the clinic. Several reviews have been published in recent years and have well illustrated the developments and the different fields of application. In this paper we have summarized the techniques that make the bases for these developments and provided illustrative examples on how the erythrocytes can be used in drug delivery.
Areas covered:
We have illustrated the possibility of using erythrocytes for the slow delivery in circulation of active pharmaceutical ingredients (APIs), for the encapsulation of those ingredients that must remain confined within the erythrocyte, the encapsulation of drug-binding proteins (or protein domain) able to reversibly bind drugs of interest and for coupling of APIs on the surface of carrier erythrocytes.
Expert opinion:
Since many years erythrocytes are recognized as powerful cellular carriers for drugs. In this review, we briefly summarize different approaches that can be used to take advantage of these natural carriers. It is shown that several different APIs can be delivered by way of erythrocytes but the selection of the technique to be employed is crucial for a successful development. The examples illustrated can guide in selecting the most appropriate way of using erythrocytes as delivery systems.
Despite continued achievements in anti-thrombotic pharmacotherapy, difficulties remain in managing patients at high risk for both thrombosis and hemorrhage. Utility of anti-thrombotic agents (ATAs) in these settings is restricted by inadequate pharmacokinetics and narrow therapeutic indices. Use of advanced drug delivery systems (ADDS) may help to circumvent these problems. Various nanocarriers, affinity ligands, and polymer coatings provide ADDS that have the potential to help optimize ATA pharmacokinetics, target drug delivery to sites of thrombosis, and sense pathologic changes in the vascular microenvironment, such as altered hemodynamic forces, expression of inflammatory markers, and structural differences between mature hemostatic and growing pathological clots. Delivery of ATA using biomimetic synthetic carriers, host blood cells, and recombinant fusion proteins that are activated preferentially at sites of thrombus development has shown promising outcomes in preclinical models. Further development and translation of ADDS that spare hemostatic fibrin clots hold promise for extending the utility of ATAs in the management of acute thrombotic disorders through rapid, transient, and targeted thromboprophylaxis. If the potential benefit of this technology is to be realized, a systematic and concerted effort is required to develop clinical trials and translate the use of ADDS to the clinical arena.
Cell systems have recently emerged as biological drug carriers, as an interesting alternative to other systems such as micro- and nano-particles. Different cells, such as carrier erythrocytes, bacterial ghosts and genetically engineered stem and dendritic cells have been used. They provide sustained release and specific delivery of drugs, enzymatic systems and genetic material to certain organs and tissues. Cell systems have potential applications for the treatment of cancer, HIV, intracellular infections, cardiovascular diseases, Parkinson's disease or in gene therapy. Carrier erythrocytes containing enzymes such us L-asparaginase, or drugs such as corticosteroids have been successfully used in humans. Bacterial ghosts have been widely used in the field of vaccines and also with drugs such as doxorubicin. Genetically engineered stem cells have been tested for cancer treatment and dendritic cells for immunotherapeutic vaccines. Although further research and more clinical trials are necessary, cell-based platforms are a promising strategy for drug delivery.
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 focuses 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.
Efforts to improve the oral bioavailability of cyclosporine A (CyA) remains a challenge in the field of drug delivery. In this study, glyceryl monooleate (GMO)/poloxamer 407 cubic nanoparticles were evaluated as potential vehicles to improve the oral bioavailability of CyA. Cubic nanoparticles were prepared via the fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by sonication and homogenization. The cubic inner structure formed was verified using Cryo-TEM. The mean diameters of the nanoparticles were about 180 nm, and the entrapment efficiency of these particles for CyA was over 85%. The in vitro release of CyA from these nanoparticles was less than 5% at 12 h. The results of a pharmacokinetic study in beagle dogs showed improved absorption of CyA from cubic nanoparticles as compared to microemulsion-based Neoral®; higher Cmax (1371.18 ± 37.34 vs 969.68 ± 176.3 ng mL−1), higher AUC0–t (7757.21 ± 1093.64 vs 4739.52 ± 806.30 ng h mL−1) and AUC0–∞ (9004.77 ± 1090.38 vs 5462.31 ± 930.76 ng h mL−1). The relative oral bioavailability of CyA cubic nanoparticles calculated on the basis of AUC0–∞ was about 178% as compared to Neoral®. The enhanced bioavailability of CyA is likely due to facilitated absorption by cubic nanoparticles rather than improved release.
A new procedure for the encapsulation of non-diffusible drugs into human erythrocytes was developed. With as little as 50 ml of blood and by using a new apparatus, it was possible to encapsulate a variety of biologically active compounds into erythrocytes in 2 h at room temperature and under blood-banking conditions. The process, which is based on two sequential hypotonic dilutions of washed red cells followed by concentration with a haemofilter and resealing of red cells, allows a 35–50% cell recovery and approx. 30% encapsulation of added drugs. The resulting processed erythrocytes have a normal survival in vivo and can be modified further, with the same apparatus, to increase their recognition by tissue macrophages to perform as a drug-targeting system. The new equipment designed and built for this procedure was named ‘Red Cell Loader’.
We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.
Sirolimus and tacrolimus are potent immunosuppressants that are delivered by drug-eluting stents (DES) for the prevention of in-stent restenosis. Balloon angioplasty with stent implantation has emerged as a successful treatment for coronary stenoses; angioplasty dilates the vessel lumen and the stent prevents elastic recoil of the vessel walls. However, angioplasty and stent placement both produce vascular injuries that potently stimulate the proliferation of smooth muscle cells, resulting in a thickening of the vascular wall. The purpose of DES is to deliver pharmacological agents that counteract neointimal hyperplasia. The sirolimus-eluting-stent reduces the incidence of in-stent restenosis significantly, whereas the tacrolimus-eluting-stent demonstrates no improvement in clinical benefit compared with a bare stent. Although sirolimus and tacrolimus have similar molecular structures, these drugs regulate immune activation via different mechanisms of action. The effects of this class of drugs are mediated by binding to the FK-506-binding proteins (FKBPs), which are highly evolutionarily conserved across species. This review highlights the structure and function of sirolimus, tacrolimus and FKBPs, with particular focus on recent observations that the two drugs target signaling pathways involved in the control of vascular smooth muscle apoptosis and proliferation directly.
Efforts to improve the oral bioavailability of cyclosporine A (CyA) remains a challenge in the field of drug delivery. In this study, glyceryl monooleate (GMO)/poloxamer 407 cubic nanoparticles were evaluated as potential vehicles to improve the oral bioavailability of CyA. Cubic nanoparticles were prepared via the fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by sonication and homogenization. The cubic inner structure formed was verified using Cryo-TEM. The mean diameters of the nanoparticles were about 180 nm, and the entrapment efficiency of these particles for CyA was over 85%. The in vitro release of CyA from these nanoparticles was less than 5% at 12 h. The results of a pharmacokinetic study in beagle dogs showed improved absorption of CyA from cubic nanoparticles as compared to microemulsion-based Neoral((R)); higher C(max) (1371.18 +/- 37.34 vs 969.68 +/- 176.3 ng mL(-1)), higher AUC(0-t) (7757.21 +/- 1093.64 vs 4739.52 +/- 806.30 ng h mL(-1)) and AUC(0-infinity) (9004.77 +/- 1090.38 vs 5462.31 +/- 930.76 ng h mL(-1)). The relative oral bioavailability of CyA cubic nanoparticles calculated on the basis of AUC(0-infinity) was about 178% as compared to Neoral((R)). The enhanced bioavailability of CyA is likely due to facilitated absorption by cubic nanoparticles rather than improved release.
The aim of this study was to investigate the ability of liquid loadable tablets (LLT) to be loaded with a self-microemulsifying drug delivery system (SMEDDS) containing cyclosporine (CyA). LLT were prepared by direct compression of the porous carrier magnesium aluminometasilicate and subsequently loaded with SMEDDS by a simple absorption method. SMEDDS was evaluated regarding visual appearance and droplet size distribution after dispersion in aqueous media. The developed SMEDDS was found to be similar to Neoral. LLT were characterized before and after loading regarding weight variation, tablet hardness, disintegration time, and in vitro drug release. It was found that LLT with high porosities suitable for liquid loading and further processing could be prepared. Adding a tablet disintegrant was found to improve in vitro drug release. Additionally, the volume-based loading capacity of LLT was evaluated and found to be comparable to soft gelatin and hard two-piece capsules. Furthermore, the pharmacokinetic performance of CyA from loaded LLT was tested in two PK-studies in dogs. Absorption of CyA from SMEDDS loaded into LLT was found in the first study to be significantly lower than the absorption of CyA from SMEDDS filled into a capsule. However, addition of a superdisintegrant improved the absorption markedly. The bioavailability of CyA from SMEDDS loaded into disintegrating LLT was found in the second study to be at the same level as from capsule formulation. In conclusion, the LLT technology is therefore seen as a promising alternative way of achieving a solid dosage form from liquid drug delivery systems.
The use of the calcineurin inhibitors cyclosporine and tacrolimus led to major advances in the field of transplantation, with excellent short-term outcome. However, the chronic nephrotoxicity of these drugs is the Achilles' heel of current immunosuppressive regimens. In this review, the authors summarize the clinical features and histologic appearance of both acute and chronic calcineurin inhibitor nephrotoxicity in renal and nonrenal transplantation, together with the pitfalls in its diagnosis. The authors also review the available literature on the physiologic and molecular mechanisms underlying acute and chronic calcineurin inhibitor nephrotoxicity, and demonstrate that its development is related to both reversible alterations and irreversible damage to all compartments of the kidneys, including glomeruli, arterioles, and tubulo-interstitium. The main question--whether nephrotoxicity is secondary to the actions of cyclosporine and tacrolimus on the calcineurin-NFAT pathway--remains largely unanswered. The authors critically review the current evidence relating systemic blood levels of cyclosporine and tacrolimus to calcineurin inhibitor nephrotoxicity, and summarize the data suggesting that local exposure to cyclosporine or tacrolimus could be more important than systemic exposure. Finally, other local susceptibility factors for calcineurin inhibitor nephrotoxicity are reviewed, including variability in P-glycoprotein and CYP3A4/5 expression or activity, older kidney age, salt depletion, the use of nonsteroidal anti-inflammatory drugs, and genetic polymorphisms in genes like TGF-beta and ACE. Better insight into the mechanisms underlying calcineurin inhibitor nephrotoxicity might pave the way toward more targeted therapy or prevention of calcineurin inhibitor nephrotoxicity.
The discovery of cyclosporine A was a milestone in organ transplantation and the treatment of autoimmune diseases. However, developing an efficient oral delivery system for this drug is complicated by its poor biopharmaceutical characteristics (low solubility and permeability) and the need to carefully monitor its levels in the blood. Current research is exploring various approaches, including those based on emulsions, microspheres, nanoparticles, and liposomes. Although progress has been made, none of the formulations is flawless. This review is a brief description of the main pharmaceutical systems and devices that have been described for the oral delivery of cyclosporine A in the context of the physicochemical properties of the drug and the character of its interactions with lipid membranes.
Cremophor EL (CrEL) is a formulation vehicle used for various poorly-water soluble drugs, including the anticancer agent paclitaxel (Taxol). In contrast to earlier reports, CrEL is not an inert vehicle, but exerts a range of biological effects, some of which have important clinical implications. Its use has been associated with severe anaphylactoid hypersensitivity reactions, hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral neuropathy. The pharmacokinetic behaviour of CrEL is dose-independent, although its clearance is highly influenced by duration of the infusion. This is particularly important since CrEL can affect the disposition of various drugs by changing the unbound drug concentration through micellar encapsulation. In addition, it has been shown that CrEL, as an integral component of paclitaxel chemotherapy, modifies the toxicity profile of certain anticancer agents given concomitantly, by mechanisms other than kinetic interference. A clear understanding of the biological and pharmacological role of CrEL is essential to help oncologists avoid side-effects associated with the use of paclitaxel or other agents using this vehicle. With the present development of various new anticancer agents, it is recommended that alternative formulation approaches should be pursued to allow a better control of the toxicity of the treatment and the pharmacological interactions related to the use of CrEL.
Cyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is a peptidylprolyl cis-trans-isomerase and the major target of the potent immunosuppressive drug cyclosporin A. Although expressed predominantly as an intracellular molecule, CyPA is secreted by cells in response to inflammatory stimuli and is a potent neutrophil and eosinophil chemoattractant in vitro and in vivo. The mechanisms underlying CyPA-mediated signaling and chemotaxis are unknown. Here, we identified CD147 as a cell surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation. Both signaling and chemotactic activities of CyPA depended also on the presence of heparans, which served as primary binding sites for CyPA on target cells. The proline 180 and glycine 181 residues in the extracellular domain of CD147 were critical for signaling and chemotactic activities mediated by CD147. Also crucial were active site residues of CyPA, because rotamase-defective CyPA mutants failed to initiate signaling events. These results establish cyclophilins as natural ligands for CD147 and suggest an unusual, rotamase-dependent mechanism of signaling.
One of the main tasks in the management of organ transplantation is the optimization of immunosuppressive therapy, in order to provide therapeutic efficacy limiting drug-related toxicity. In the past years major efforts have been carried out to define therapeutic windows based on blood/plasma levels of each immunosuppressant relating those concentrations to drug dosing and clinical events. Although this traditional approach is able to identify environmental and nongenetic factors that can influence drug exposure during the course of treatment, it presents limitations. Therefore, complementary strategies are advocated. The advent of the genomic era gives birth to pharmacogenomics, a science that studies how the genome as a whole, including single genes as well as gene-to-gene interactions, may affect the action of a drug. This science is of particular importance for drugs characterized by a narrow therapeutic index, such as the immunosuppressants. Preliminary studies focused on polymorphisms of genes encoding for enzymes actively involved in drug metabolism, drug transport and pharmacological target. Pharmacogenomics holds promise for improvement in the ability to individualize immunosuppressive therapy based on the patient's genetic profile, and can be viewed as a support to traditional therapeutic drug monitoring. However, the clinical applicability of this approach is still to be proven.
Several new immunosuppressive agents have recently been approved for use in solid organ transplantation. Many of these agents have narrow therapeutic ranges, which necessitates drug concentration monitoring in order to optimise efficacy, minimise toxicity and individualise dosages. Some of the lessons learned with the clinical use of the revolutionary agent cyclosporin have been applied to the newer agents tacrolimus and sirolimus. The agent mycophenolate mofetil has been in clinical use without widespread drug concentration monitoring; however, recent data suggest that therapeutic monitoring may improve clinical outcomes, especially in certain high risk subsets of patients. This review focuses on the literature published to date on drug concentration monitoring of the newer immunosuppressive agents — tacrolimus, mycophenolate mofetil and sirolimus. In addition, pertinent aspects of the clinical pharmacokinetics and metabolism of each agent are reviewed.
Cyclosporin is a lipophilic cyclic polypeptide immunosuppressant that interferes with the activity of T cells chiefly via calcineurin inhibition. The original oil-based oral formulation of this drug (Sandimmun®)1 was characterised by high intra- and interpatient pharmacokinetic variability, with poor bioavailability in many patients; a novel microemulsion formulation (Neoral®)1 was therefore developed to circumvent these problems. Studies show increases, attributable chiefly to improved absorption in patients who absorb the drug only poorly from the original formulation, in mean systemic exposure to cyclosporin with the microemulsion, with no clinically significant differences in tolerability or drug interaction profiles.
Cyclosporin microemulsion is at least as effective as the oil-based formulation in renal, liver and heart transplant recipients, with trends towards decreased incidence of acute rejection with the microemulsion formulation in some (statistically significant in a few) trials. Cyclosporin microemulsion and tacrolimus appear to have similar efficacy in preventing acute rejection episodes in most renal, pancreas-kidney, liver and heart transplant recipients. However, there are indications of superior efficacy for tacrolimus in some trials, particularly in the prevention of severe acute rejection and in Black transplant recipients. Current 12-month data also indicate equivalent efficacy of sirolimus in renal transplantation.
Conversion from the oil-based to microemulsion formulation in stable renal, liver and heart transplant recipients is achievable with no change in acute rejection rates. The addition of an anti-interleukin-2 receptor monoclonal antibody and/or mycophenolate mofetil to cyclosporin microemulsion plus corticosteroids decreases rates of acute rejection; corticosteroid withdrawal without increased acute rejection rates was also achieved on the addition of these agents in some trials.
Pharmacoeconomic analyses have shown savings in direct healthcare costs in kidney or liver transplantation when cyclosporin microemulsion is used in preference to the oil-based formulation, although studies incorporating indirect costs or expressing costs in terms of therapeutic outcomes are currently unavailable.
Conclusions: The introduction of cyclosporin microemulsion has consolidated the place of the drug as a mainstay of therapy in all types of solid organ transplantation; research into optimisation of outcomes through more effective therapeutic monitoring in patients receiving this formulation is ongoing. Several novel immunosuppressants have been introduced in recent years: further clinical and pharmacoeconomic research will be needed to clarify the relative positioning of these agents, particularly with respect to specific patient groups. Other new drugs (basiliximab/daclizumab and mycophenolate mofetil) offer particular advantages when used in combination with cyclosporin.
Overview of Pharmacodynamic Properties
Cyclosporin inhibits the activation of the calcium/calmodulin-activated phospha-tase calcineurin via complex formation with cyclophilin, and thereby prevents the translocation of the transcription factor nuclear factor of activated T cells (NF-AT). The drug also inhibits activation of the transcription factor NF-κB, and T cell activation is suppressed by inhibition of interleukin-2 gene expression. In vitro study of porcine aortic endothelial cells has shown complete suppression by cyclosporin of tumour necrosis factor-a-mediated induction of class II major histocompatibility complex expression.
Cyclosporin also has hypertensive effects; potential underlying mechanismsinclude effects on the sympathetic nervous system, upregulation of angiotensin II receptors in vascular smooth muscle cells, increased plasma levels of en-dothelin-1, and effects on whole blood viscosity and plasma fibrinogen levels.
Pharmacokinetic Properties and Monitoring of Therapy
The original oil-based oral formulation of cyclosporin is characterised by widely varying bioavailability. The microemulsion, however, has self-emulsifying properties that enhance bioavailability and reduce pharmacokinetic variability between and within patients.
Assay Methods, Pharmacokinetic Monitoring and Clinical Outcomes. Measurement of cyclosporin concentrations in whole blood by immunoassay is currently used most commonly for monitoring therapy in patients receiving the drug for immunosuppression. Trough cyclosporin concentrations have been most frequently used to direct dosage adjustment, although they have little predictive value with respect to actual systemic exposure in patients receiving the original oil-based formulation.
The introduction of the microemulsion has led to new research into the therapeutic monitoring of cyclosporin therapy, with increasing emphasis on the importance of the 4-hour absorption phase that follows oral administration. Correlation has been shown between areas under curves of cyclosporin concentrations in whole blood versus time (AUCs) taken over 4 hours (abbreviated AUC) and a full 12-hour administration interval in patients undergoing renal transplantation. Other data suggest utility of 2-point sampling (at 2 and 6 hours), and a strong correlation has been shown between freedom from liver graft rejection during the first month after surgery and 6-hour AUCs (AUC6) or peak drug concentrations in blood (Cmax) in patients receiving cyclosporin microemulsion. Close correlations have been reported between drug concentrations measured in blood 2 hours post-dose and 4- or 6-hour AUCs, and there is evidence of improved overall clinical outcome with 2-hour over trough concentration monitoring.
General Pharmacokinetic Properties of Cyclosporin. Cyclosporin undergoes extensive extravascular distribution, with a volume of distribution at steady state of 3 to 5 L/kg after intravenous administration. The drug is 90 to 98% bound to plasma proteins, crosses the placenta, and is distributed into human milk.
Blood concentrations of cyclosporin generally decline in a biphasic manner. The initial elimination half-life is reported to average 1.2 hours, whereas the average terminal elimination half-life is reported to be 8.4 to 27 hours. The drug is metabolised extensively to at least 30 metabolites, chiefly by the hepatic cyto-chrome P450 3A enzyme system. Elimination is primarily biliary; around 6% of each dose is excreted in the urine, with 0.1 % eliminated in the urine as unchanged drug. Clearance is not affected to any significant extent by haemodialysis or renal failure.
Pharmacokinetic Properties of Cyclosporin Microemulsion. In general, significant increases in mean systemic exposure of patients to cyclosporin, with attendant reductions in time to Cmax (tmax), are seen when the microemulsion is used in place of the original oil-based formulation. These overall increases are attributable predominantly to improved absorption in patients who absorb cyclosporin only poorly from the oil-based formulation, with little or no change in good absorbers.
In renal transplantation, increases in AUC of up to 64% have been reported in randomised comparisons and in studies in which patients were converted from the older formulation to the microemulsion, with marked increases in drug exposure in patients previously classified as poor absorbers. Comparisons of variance data in several studies indicate significant reductions in intra- and interpatient pharmacokinetic variability relative to the oil-based formulation in patients receiving the microemulsion. Substantially increased AUCs (median 71% increase in one 6-month study in 25 patients) relative to the oil-based formulation have been reported with the microemulsion in children undergoing renal transplantation.
Significant increases in AUC and Cmax, and decreases in tmax, with cyclosporin microemulsion relative to the oil-based formulation have also been reported in patients undergoing liver transplantation. In one study, no clinically relevant effect of food intake was reported in microemulsion recipients. Most notably, the Canadian NOF-11 trial in 32 children showed exposure to cyclosporin (mean 8-hour AUC) to be increased by over 200% relative to the oil-based formulation in the early post-transplant period in patients treated with the microemulsion.
In general, systemic exposure to cyclosporin given as microemulsion appears greater than with the oil-based formulation when T tubes are open during the first few days after liver transplantation, although data are available to indicate that absorption of cyclosporin from the microemulsion is not fully independent of bile flow.
Enhancement of absorption of cyclosporin from the microemulsion relative to the oil-based formulation has also been reported in patients receiving heart and/or lung allografts. Increases were particularly marked in patients receiving lung transplants, with an increase in mean AUC6 of just over 70% relative to the oil-based formulation after 12 months in a comparative study in 50 recipients of new allografts.
Relative to adults, absorption of and systemic exposure to cyclosporin are substantially reduced in children undergoing bone marrow transplantation who receive the microemulsion. AUCs were increased significantly by GI inflammation in one study. Increased systemic exposure to cyclosporin with the microemulsion has also been reported in renal transplant recipients with diabetes mellitus.
The pharmacokinetic characteristics of cyclosporin are not altered to any clinically significant extent by advanced age.
Effect of Formulation on Cyclosporin Dosage. Cyclosporin dosage reductions were required to maintain required drug concentrations in whole blood after conversion from the oil-based formulation to microemulsion in 12.3 to 87.2% of patients in case series of stable renal transplant recipients. Overall reductions in mean dosage (after initial conversion on a 1: 1 basis) ranged from 4.7 to 14.7% over 8 weeks to 12 months in studies in a total of 1381 patients, and were predominantly statistically significant. Dosage reductions with the microemulsion relative to the original formulation have also been shown in randomised comparative studies, although statistical significance was not attained consistently. Reduced dosage requirements with the microemulsion have also been reported in liver and heart/lung transplant recipients.
Drug Interactions. A wide variety of agents increase (e.g. erythromycin, ketoconazole) or decrease (e.g. phenytoin, phenobarbital) plasma or whole blood concentrations of cyclosporin by competitive hepatic enzyme inhibition or induction, or by other mechanisms (e.g. absorption or binding to P-glycoprotein). Some drugs (e.g. aminoglycosides) are also associated with enhancement of nephrotoxicity of cyclosporin when coadministration takes place.
Recent data indicate possible enhancement by cyclosporin of the potential of HMG-CoA reductase inhibitors to induce rhabdomyolysis. Mycophenolate mofetil may increase systemic exposure to cyclosporin, but the proton pump inhibitor pantoprazole has no apparent pharmacokinetic effect when coad-ministered with the drug.
Therapeutic Efficacy
Comparisons with Cyclosporin Oil-Based Formulation (Sandimmun®). The overall ranges of incidence of biopsy-confirmed acute rejection episodes in the various trials in adult de novo transplant recipients receiving cyclosporin microemulsion or the original oil-based cyclosporin formulation at trough blood concentration-controlled dosages were 25 to 44.2% versus 22 to 60.5% at 3 to 24 months for renal transplantation, 45.9 to 62.7% versus 49.2 to 59.1% at 4 to 24 months for liver transplantation, and (in a single study) 86.2 versus 84.9% at 6 months for heart transplantation. Azathioprine and corticosteroids were given concomitantly in most trials. A trend for improved efficacy in this respect, and for the incidence of more than one acute rejection episode, with the microemulsion formulation was seen in most renal transplantation trials, with a statistically significant difference at 3 months in one for both parameters. There were no significant differences in the end-point incidence of acute rejection between the formulations in adult recipients of liver transplants, but the microemulsion formulation appeared significantly superior in a small trial in children (35 vs 80%; p = 0.01) at 12 months.
The incidence of severe (corticosteroid-resistant in most studies) acute rejection tended to be lower in adult patients receiving the microemulsion formulation than in those receiving the oil-based formulation (0 to 18.5% vs 10.8 to 20.0% at 4 to 24 months) in liver transplantation but not in heart transplantation (46.3 vs 45.8% at 2 years). The difference between the formulations was more apparent in children receiving liver transplants in this respect (6 vs 53% at 12 months; p = 0.004).
There was a trend for fewer recipients of the microemulsion than the oil-based formulation to require antilymphocyte antibody treatment for acute rejection over the first 3 months after renal transplantation. This difference was more marked in heart transplant recipients (6.9 vs 17.7%; p = 0.002 at 24 months).
Graft survival rates for the microemulsion and oil-based formulations were 91 to 96% versus 89 to 98% at 3 to 24 months in renal transplant recipients and 90 to 94.1% versus 86 to 93.8% at 4 to 24 months in liver transplant recipients. Patient survival rates for the microemulsion and oil-based formulations were 98 to 100% versus 99 to 100% in renal transplant recipients and 84.2 to 100% versus 85.9 to 94% in liver transplant recipients. Graft/patient survival rates were 88.3 versus 85.4% at 2 years in heart transplant recipients.
Comparisons with Other Modified Formulations. There are preliminary indications of clinical equivalence between cyclosporin Neoral® and cyclosporins SangCya®, Consupren® and Neoplanta® in de novo renal transplant recipients. Equivalence has also been demonstrated between Neoral® and Consupren® or SangCya® in two small studies in patients with stable existing transplants who were transferred from therapy with the original oil-based formulation of cyclosporin. Available comparisons, however, are predominantly nonblind and are based on small numbers of patients only.
Comparisons with Tacrolimus. The incidence of acute rejection in de novo cyclosporin microemulsion (initially 8 to 15 mg/kg/day) and tacrolimus (initially 0.1 to 0.2 mg/kg/day; both dosages concentration-controlled) recipients was 10 to 39% versus 9 to 40% at 3 to 24 months in renal transplantation, 11 versus 11% at 3 months in simultaneous pancreas-kidney transplantation, 23 to 82.5% versus 17 to 66% at 1 to 30 months in liver transplantation (p < 0.01 favouring tacrolimus in one of seven trials) and 30 versus 24% at 12 months in heart transplantation (in one trial).
The incidence of severe acute rejection in cyclosporin microemulsion and tacrolimus recipients was 0 to 14% versus 0 to 7% at 3 to 24 months in most trials of renal transplantation, 6 to 25% versus 0 to 19% at 1 to 30 months in liver transplantation (p < 0.01 favouring tacrolimus in one of seven trials) and 30 versus 21% at 12 months in heart transplantation. Tacrolimus 0.3 mg/kg/day was associated with significantly lower incidences of acute (20 vs 37%; p < 0.001) and severe acute rejection (9 vs 21%; p < 0.001) than cyclosporin microemulsion 8 to 10 mg/kg/day in the largest trial in renal transplant recipients, a nonblind comparative 6-month study in 577 patients in 50 European centres.
Graft survival rates in cyclosporin and tacrolimus recipients were 78 to 97% versus 83 to 100% at 3 to 24 months in renal transplantation (p < 0.05 favouring tacrolimus in one of nine trials) and 62 to 92% versus 68 to 95% at 1 to 30 months in liver transplantation (p < 0.05 favouring tacrolimus in one of seven trials). Patient survival rates in cyclosporin and tacrolimus recipients were 86 to 100% versus 90 to 100% at 3 to 24 months in renal transplantation, 67 to 98% versus 72 to 98% at 1 to 30 months in liver transplantation and 85 versus 85% at 12 months in heart transplantation.
Interim 6-month data from 425 of 606 liver transplant recipients taking part in a randomised, nonblind study in the UK and Ireland indicate a lower incidence of death, retransplantation or treatment failure for immunological reasons with tacrolimus than with cyclosporin microemulsion (17 vs 28%; p = 0.01).
Black recipients of renal transplants tended to do better on tacrolimus than on cyclosporin microemulsion (acute rejection 14 vs 38%, respectively; severe acute rejection 7 vs 14%, respectively). Similarly, Black recipients of heart transplants did significantly better on tacrolimus at 12 months (acute rejection episodes requiring treatment, p = 0.01; patient/graft survival, p = 0.04).
Comparisons with Sirolimus. The efficacy of cyclosporin microemulsion appears similar to that of sirolimus on the basis of results from two 12-month, randomised, nonblind studies in a total of 161 patients undergoing de novo renal transplantation. Graft and patient survival rates were similar between treatments in both trials; rates of biopsy-confirmed acute rejection were also not statistically significantly different, although there was a trend in favour of cyclosporin in one study (18 vs 27.5%).
Conversion to Cyclosporin Microemulsion. Conversion of stable renal, liver and heart transplantation patients from the oil-based cyclosporin formulation (Sandimmun®) to the microemulsion formulation, at an initial 1: 1 dosage ratio, appears not to affect the rate of acute rejection.
Preliminary evidence suggests that conversion from tacrolimus to cyclosporin microemulsion because of adverse effects or lack of efficacy is comparatively successful in renal and liver transplant recipients.
Use of Other Agents with Cyclosporin Microemulsion-Based Immuno-suppression. Incidences of presumed or biopsy-proven acute rejection were significantly decreased on the addition of mycophenolate mofetil 2 g/day to cyclosporin microemulsion plus corticosteroids in nonblind studies in 173 renal transplant recipients. The addition of mycophenolate mofetil 2 or 3 g/day to cyclosporin microemulsion (initial daily dosage 5 to 15 mg/kg/day) plus corticosteroid-based immunosuppression significantly reduced the incidence of biopsy-proven rejection or treatment failure over 1 year in a randomised, multicentre, double-blind, placebo-controlled study in 491 recipients of first or second renal allografts. A significantly lower incidence of acute rejection was reported with the addition of mycophenolate mofetil to cyclosporin microemulsion and corticosteroids than with the addition of azathioprine in a nonblind study in 57 liver transplant recipients (21.4 vs 44.8%; p < 0.05).
In a double-blind study (n = 376), rates of acute and severe acute rejection at 6 months were significantly reduced in patients receiving concomitant basiliximab 20mg on days 0 and 4 of renal transplantation compared with those receiving cyclosporin microemulsion and corticosteroids alone. A similar study in 346 renal transplant recipients showed statistically significant reductions in 12-month incidences of first acute rejection, second rejection, biopsy-confirmed rejection, and rejection episodes requiring treatment with augmented immuno-suppression (other than corticosteroids) with the addition of basiliximab to cyclosporin microemulsion plus corticosteroid-based immunosuppression. Significantly reduced incidence relative to placebo of biopsy-proven acute rejection has also been noted with addition of daclizumab to cyclosporin microemulsion and corticosteroid therapy.
Two multicentre placebo-controlled, double-blind trials in a total of 1295 patients undergoing renal transplantation showed statistically significant reductions relative to placebo or azathioprine in a composite end-point of acute rejection, graft loss and death when sirolimus 2 or 5 mg/day was added to immunosuppression with cyclosporin microemulsion and corticosteroids.
The addition of basiliximab and/or mycophenolate mofetil also allowed the elimination of corticosteroids from the immunosuppressive regimen without affecting the rate of acute rejection in a number of small studies in patients undergoing renal transplantation. However, a larger (n = 266), placebo-controlled, double-blind study has indicated an increase in risk of acute rejection (particularly among Black patients) upon withdrawal of corticosteroids from renal transplant recipients also receiving cyclosporin microemulsion and mycophenolate mofetil. Similar findings were reported in a further double-blind study in 500 renal transplant recipients, 447 whom received cyclosporin microemulsion in addition to mycophenolate mofetil, although the authors stated that the increase in frequency of serious rejection episodes when corticosteroids were withdrawn was acceptable. Results of corticosteroid withdrawal studies in liver allograft recipients receiving cyclosporin microemulsion or tacrolimus, either as monotherapy or in combination with mycophenolate mofetil, are inconclusive.
Pharmacoeconomic Considerations
Various cost analyses have been carried out from a healthcare provider’s or third party payer’s perspective to assess potential pharmacoeconomic advantages of the use of cyclosporin microemulsion in place of the older oil-based formulation.
Details from a study reported as an abstract have suggested a monthly cost saving of $US52 per patient after conversion from the oil-based formulation to microemulsion in 181 French individuals with stable renal allografts. Costs accounted for and year of costing were not given, however, for this 6-month analysis, which appeared to have been carried out from a healthcare provider’s perspective.
Prospectively gathered resource utilisation data from the MILTON study in 390 de novo liver transplant recipients showed savings (relative to treatment with the oil-based formulation) from a healthcare system perspective of 8 to 10% over the 4-month post-transplant period in patients receiving cyclosporin microemulsion. This was attributed partly to a more rapid discontinuation of intravenous cyclosporin therapy in patients receiving the microemulsion.
Examination of healthcare utilisation based on time in hospital and treatment of acute rejection indicated a cost saving of 2162 Canadian dollars per patient (year of costing and statistical significance not stated) relative to the oil-based formulation in a 3-month retrospective case-control study in 20 de novo liver transplant recipients. Three other analyses in patients undergoing liver transplantation have indicated reductions in direct healthcare costs when patients receive cyclosporin microemulsion rather than the original formulation.
Data from studies in patients receiving de novo kidney or liver transplants have suggested that the direct cost of using cyclosporin microemulsion is similar to or lower than that with tacrolimus. In one study, 6-month direct healthcare costs in 89 renal transplant recipients were £13 216 with cyclosporin microemulsion and £12 982 with tacrolimus (year of costing not stated). In 86 patients receiving liver transplants, the mean cost of cyclosporin microemulsion was 22% lower than that of tacrolimus (on the basis of dosages used over 1 year), although few details were available for this analysis (abstract published only).
Tolerability
The tolerability profile of cyclosporin is characterised by a number of potentially serious adverse effects that are related to exposure, including acute or chronic nephrotoxicity, hypertension and neurotoxicity. The main dose-limiting adverse effect of cyclosporin is nephrotoxicity, which usually presents as a reversible decrease in glomerular filtration rate. Nephrotoxicity is reported to affect 25 to 37% of kidney, heart or liver transplant recipients being treated with cyclosporin and may progress to permanent renal dysfunction in up to 15% of patients. Glomerular capillary thrombosis, progressing to graft failure in some patients, may also occur in transplant patients receiving cyclosporin.
In comparative trials conducted in recipients of renal transplants, hypertension was reported in fewer than 25% of patients treated with either cyclosporin microemulsion or the oil-based formulation. Hypertension was also reported in recipients of liver or heart transplants treated with either cyclosporin formulation.
Neurological symptoms, such as headaches, tremor, paraesthesia and convulsions, are also common adverse effects of cyclosporin in patients who have received transplants (1 source notes tremor in 12 to 21, 31 and 55% of patients receiving kidney, heart or liver transplants, respectively). Factors contributing to the development of convulsions in patients receiving cyclosporin therapy include hypomagnesaemia, hypertension, high-dose methylprednisolone therapy, nephrotoxicity and hypocholesterolaemia.
Numerous comparative double-blind or nonblind clinical trials have shown that the increased bioavailability of cyclosporin and greater systemic exposure achieved with the microemulsion formulation does not result in an increase in incidence or severity of adverse events compared with the original oil-based formulation in stable renal, liver or heart transplant recipients (provided that the dose of the microemulsion formulation is adjusted on the basis of target trough cyclosporin concentrations in whole blood).
Muscle weakness, oedema, epigastric pain, headache and hypertension were the most common events in stable renal transplant patients receiving treatment with cyclosporin microemulsion in a large comparative trial. About 40% of patients treated with either the cyclosporin microemulsion or the oil-based formulation experienced adverse events that were described as ‘serious’ in this study.
In patients who had received primary orthotopic liver transplants, the most common adverse events reported during therapy with cyclosporin microemulsion or the oil-based formulation were infections, cardiovascular effects, hypertension, nervous system effects and renal failure. Clinical diabetes mellitus, hir-sutism and gum hyperplasia developed in small numbers of patients in each treatment group.
Overall, both formulations of cyclosporin were equally well tolerated in a randomised double-blind trial in 380 de novo heart transplant recipients. However, relative to the oil-based formulation, patients treated with the microemul-sion had a lower (not statistically significant) incidence of candidiasis (5.9 vs 10.9%), cytomegalovirus infections (10.1 vs 15.1%) and de novo diabetes mellitus (3.9 vs 8.5%), whereas incidences of gingival hyperplasia and GI symptoms were higher in the microemulsion treatment group than in the comparator group(3.2 vs 2.6% and 81.9 vs 76.5%); these adverse events were transient and mild to moderate in severity.
The tolerability profile of cyclosporin microemulsion was broadly similar to that of tacrolimus (both drugs were given in combination with a corticosteroid and azathioprine) in cadaveric renal transplant recipients in a nonblind randomised study. In contrast, significant differences in the biochemical profiles of patients treated with either cyclosporin microemulsion or tacrolimus were reported in another study in renal transplant recipients. In a study in 577 renal transplant recipients, the incidences of new-onset diabetes mellitus after 6 months’ treatment were 4.5% with tacrolimus group and 2% with cyclosporin microemulsion (statistical significance not stated). Mean serum creatinine levels were similar for both drugs from the end of month 1 to study completion.
Thrombocytopenia and diarrhoea were reported significantly more frequently with sirolimus than with cyclosporin microemulsion in a randomised, nonblind comparison in 78 renal transplant recipients. Increased serum creatinine levels, hyperuricaemia, cytomegalovirus infection and tremor were more frequent with cyclosporin.
At present, there are no published well designed and controlled studies of the efficacy and tolerability of cyclosporin microemulsion in pregnant transplant recipients and their offspring. However, in a retrospective analysis, no notable malformation trends were evident among the 175 children (mean age 4.4 years) of renal transplant recipients who had been treated with cyclosporin during pregnancy.
Dosage and Administration
Cyclosporin microemulsion is available variously in different countries as 10,25, 50 and l00 mg soft gelatin capsules and as an oral solution containing 100 mg/ml. The oral solution may be made more palatable by diluting with orange or apple juice. Blood cyclosporin concentrations increase when cyclosporin microemulsionis taken with grapefruit/grapefruit juice, which should therefore be avoided by patients taking the drug.
Cyclosporin microemulsion is indicated for the prophylaxis of organ rejection in patients who have undergone allogeneic renal, liver or heart transplantation. Cyclosporin microemulsion should be taken twice daily (in two equal doses). An optimal dosage of the drug will produce trough whole blood concentrations sufficient to achieve immunosuppression while preventing high peak blood concentrations and drug-related toxicity. Importantly, because cyclosporin is more bioavailable from the oral microemulsion than from the oil-based oral formulation, the two formulations cannot be interchanged without careful monitoring of the patient by a physician.
Whole blood concentrations of cyclosporin should be measured frequently (three to four times weekly or daily in the early post-transplantation period) in patients receiving treatment with cyclosporin microemulsion, as lower than recommended therapeutic concentrations may result in rejection of the transplanted organ and higher concentrations are likely to produce drug-related toxicity. Renal function, liver function and blood pressure should be monitored closely in patients receiving treatment with cyclosporin microemulsion. In addition, levels of serum lipids, potassium and magnesium should be checked regularly during treatment with the drug. In randomised controlled trials in transplant recipients, most patients received an initial dosage of 10 mg/kg/day. Dosages were adjusted thereafter to achieve target therapeutic trough concentrations in whole blood and then further titrated according to assessments of transplant rejection and tolerability.
Stable transplant recipients receiving the original oil-based formulation of cyclosporin may have their therapy changed to the microemulsion formulation with careful monitoring. In these patients, it is recommended that the initial dosage of cyclosporin microemulsion is the same as that of the previously administered cyclosporin formulation. Thereafter, the dose of cyclosporin microemulsion should be adjusted to obtain a whole blood trough cyclosporin concentration the same as that achieved previously with the original formulation.
A meeting of 14 transplant and pharmacokinetic specialists from Europe and North America was convened in November 2001 to evaluate scientific and clinical data regarding the use of different formulations of cyclosporin A (CsA). The following consensus was achieved. (1) CsA is a critical-dose drug with a narrow therapeutic window. Clinical outcomes after transplantation are affected by the pharmacokinetic properties of CsA, particularly by its bioavailability, and by intrapatient variability in CsA exposure. (2) Standard bioequivalence criteria do not address differences in CsA pharmacokinetics between transplant recipients and healthy volunteers, or between subpopulations of transplant recipients. (3) In some circumstances, currently available formulations of CsA that meet standard bioequivalence criteria are likely to be nonequivalent with respect to pharmacokinetic characteristics. (4) The choice of CsA formulation can affect the short- and long-term clinical outcome. Currently, there is a lack of clinical comparisons between generic CsA formulations and the Neoral® formulation (Novartis Pharmaceuticals Corporation, East Hanover, New Jersey). Initial retrospective data from the Collaborative Transplant Study suggest that use of generic CsA formulations may result in reduced graft survival at 1 year. (5) Management of transplant recipients by monitoring Neoral concentrations 2 hours after dosing (C2) reduces the incidence and severity of acute rejection compared with monitoring of trough concentrations with no increase in toxicity. C2 monitoring has been developed based on the pharmacokinetics of Neoral only and has not been evaluated or validated for generic formulations of CsA. (6) The major costs of care after transplantation relate to the management of poor clinical outcomes and toxicity. CsA formulations with different pharmacokinetic properties may be associated with varying clinical outcomes, which would be expected to affect total health care costs. (7) The transplant physician is responsible for selecting immunosuppressive agents and formulations for his or her patients. Any switch between CsA formulations in a particular patient should take place only in a controlled setting with adequate pharmacokinetic monitoring.
The objective of this work was to suggest the biowaiver potential of biopharmaceutical classification system (BCS) Class II drugs in self-microemulsifying drug delivery systems (SMEDDS) which are known to increase the solubility, dissolution and oral absorption of water-insoluble drugs. Cyclosporine was selected as a representative BCS Class II drug. New generic candidate of cyclosporine SMEDDS (test) was applied for the study with brand SMEDDS (reference I) and cyclosporine self-emulsifying drug delivery systems (SEDDS, reference II). Solubility and dissolution of cyclosporine from SMEDDS were critically enhanced, which were the similar behaviors with BCS class I drug. The test showed the identical dissolution rate and the equivalent bioavailability (0.34, 0.42 and 0.68 of p values for AUC₀(→)₂₄(h), C(max) and T(max), respectively) with the reference I. Based on the results, level A in vitro-in vivo correlation (IVIVC) was established from these two SMEDDS formulations. This study serves as a good example for speculating the biowaiver extension potential of BCS Class II drugs specifically in solubilizing formulation such as SMEDDS.
In the therapy of chronic inflammatory skin diseases, the epicutaneous application of anti-inflammatory drugs in combination with maintenance therapy leads to ideal therapeutic long term effects. In this work, the development of well-tolerated colloidal carrier systems (ME) containing tacrolimus is described. A comprehensive physico-chemical characterization of the novel systems was performed using different techniques. The potential of three ME compared to an ointment as suitable carrier for dermal delivery of tacrolimus was determined. The penetration studies demonstrated that in comparison to the standard vehicle ointment, all three ME resulted in higher concentrations of tacrolimus in the deeper skin layers independent of the time of incubation. Particularly, the percentage of the bioavailable amount of tacrolimus (sum of the amount found in the dermis and acceptor compartment) from the ME with concentrations up to 20.95 ± 12.03% after 1000 min incubation time differed significantly (p<0.01), when compared to the ointment which yielded a concentration of 6.41 ± 0.57%. As a result of these experiments, using colloidal carrier systems, the penetration profile of tacrolimus was enhanced significantly (p<0.01). High drug amounts penetrated the target site in a short period of time after applying the ME.
Jie Lai1,2, Yi Lu1, Zongning Yin2, Fuqiang Hu3, Wei Wu11School of Pharmacy, Fudan University, Shanghai, China, 2West China School of Pharmacy, Sichuan University, Chengdu, China, 3School of Pharmacy, Zhejiang University, Hangzhou, ChinaAbstract: Efforts to improve the oral bioavailability of cyclosporine A (CyA) remains a challenge in the field of drug delivery. In this study, glyceryl monooleate (GMO)/poloxamer 407 cubic nanoparticles were evaluated as potential vehicles to improve the oral bioavailability of CyA. Cubic nanoparticles were prepared via the fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by sonication and homogenization. The cubic inner structure formed was verified using Cryo-TEM. The mean diameters of the nanoparticles were about 180 nm, and the entrapment efficiency of these particles for CyA was over 85%. The in vitro release of CyA from these nanoparticles was less than 5% at 12 h. The results of a pharmacokinetic study in beagle dogs showed improved absorption of CyA from cubic nanoparticles as compared to microemulsion-based Neoral®; higher Cmax (1371.18 ± 37.34 vs 969.68 ± 176.3 ng mL-1), higher AUC0–t (7757.21 ± 1093.64 vs 4739.52 ± 806.30 ng h mL-1) and AUC0–∞ (9004.77 ± 1090.38 vs 5462.31 ± 930.76 ng h mL-1). The relative oral bioavailability of CyA cubic nanoparticles calculated on the basis of AUC0–∞ was about 178% as compared to Neoral®. The enhanced bioavailability of CyA is likely due to facilitated absorption by cubic nanoparticles rather than improved release.Keywords: nanoparticles, cubosomes, cyclosporine A, glyceryl monooleate, oral drug delivery, bioavailability, beagle dogs
Ophthalmic drug delivery with long pre-corneal retention time and high penetration into aqueous humor and intraocular tissues is the key-limiting factor for the treatment of ocular diseases and disorders. Within this study, the conjugate of cysteine-polyethylene glycol monostearate (Cys-PEG-SA) was synthesized and was used to compose the thiolated nanostructured lipid carrier (Cys-NLC) as a potential nanocarrier for the topical ocular administration of cyclosporine A (CyA). The rapid cross-linking process of Cys-PEG-SA in vitro was found in simulated physiological environment. The in vitro CyA release from Cys-NLC was slower than that of non-thiolated nanostructured lipid carriers (NLC) due to the cross-linking of thiomers on the surface of nanocarriers. After topical ocular administration in rabbits, the in vivo ocular distribution of CyA was investigated in comparison of Cys-NLC with non-thiolated NLCs and oil solution. The results showed that CyA concentration in systemic blood was very low and close to the detection limit. The area-under-the-curve (AUC(0-24h)) and mean retention time (MRT(0-24h)) of Cys-NLC group in aqueous humor, tear and eye tissues were significantly higher than that of oil solution, non-thiolated NLCs (p<0.05). These results demonstrated that the thiolated NLC could deliver high level of CyA into intraocular tissues due to its bioadhesive property and sustained release characteristics.
Cyclosporine A (CyA) is a useful immunosuppressive agent for steroid-dependent or steroid-refractory ulcerative colitis. However, side effects have been reported in clinical trials of ulcerative colitis treated with CyA. Biodegradable microspheres (MS) have been investigated as drug delivery system. We evaluated the effect of a drug delivery system with poly(d,l-lactic acid)-MS containing CyA. Colitis was induced in C57BL/6 mice by 3% dextran sulfate sodium (DSS). Mice with DSS-induced colitis were treated with oral administration of CyA or CyA-MS: CyA (0.2 mg/kg/day)-MS; CyA (2 mg/kg/kg)-MS). Serum levels of CyA were significantly less elevated after oral administration of CyA (2 mg/kg/day)-MS compared with CyA (2 mg/kg/day) (CyA (2 mg/kg/day), 44.7 ± 0.8 ng/ml; CyA (2 mg/kg/day)-MS, 7.7 ± 1.3 ng/ml). The body weight at day 10 was significantly recovered in the mice treated with CyA (0.2 mg/kg/day)-MS and CyA (2 mg/kg/day)-MS compared with CyA (0). The histological score and myeloperoxidase activity in the mice treated with CyA-MS was significantly lower than CyA (0). Gene expressions of interleukin-1β (IL-1β), IL-6, and CXCL1 in the mice treated with CyA (0.2 mg/kg/day)-MS and CyA (2 mg/kg/day)-MS were downregulated compared with CyA (0)-MS. CyA-MS might be possible to treat ulcerative colitis effectively by decreasing the total dosage without the elevation of the serum level or the side effects of CyA.
Three solid dispersions containing poorly water-soluble tacrolimus were prepared with hydroxypropyl-β-cyclodextrin (HP-β-CD) and dioctyl sulfosuccinate (DOSS) using a spray-drying technique via the solvent-evaporation method with a methylene chloride/ethanol mixture, the solvent-wetting method with ethanol and the surface-attached method with water, respectively. The solubility and dissolution of the drug in the three solid dispersions were evaluated compared to drug powder. Furthermore, their physicochemical properties were investigated using SEM, DSC and powder X-ray diffraction. The solubility and dissolution of the drug were significantly improved in the order of the tacrolimus-loaded solid dispersion prepared by: solvent-evaporation method > solvent-wetting method > surface-attached method. The solid dispersions prepared by solvent evaporation appeared as an aggregated form with the amorphous form. In particular, the solid dispersion prepared by the solvent-evaporation method improved solubility about 900-fold and dissolution of tacrolimus 15-fold because of its reduced particle size, increased surface area and close contact between the hydrophilic carrier and the drug. In the solvent-wetting method, the drug, which was changed to an amorphous form, was attached onto the surface of undissolved carriers. However, the solid dispersion prepared by the surface-attached method gave an unchanged crystalline form. In this solid dispersion, the carriers were attached to the surface of the undissolved drug, resulting in changing the drug from being hydrophobic to hydrophilic. As the crystal form of drug in this solid dispersion was not converted to the amorphous form unlike other solid dispersions, it gave relatively less solubility and dissolution of the drug than did the others. Thus, in the development of a solid-dispersion system containing poorly water-soluble drugs, the method of preparation plays an important role in the solubility and crystallinity of the drugs.
Immunophilin ligand FK506 has been treated as adjunct therapy for nerve repair due to its potent neurotrophic and neuroprotective actions. It was hypothesized that FK506 releasing from biodegradable chitosan guide provided better nerve regenerative response than the guide with no FK506. The drug was entrapped in the semi-permeable wall of chitosan guide with the drug-loading of 647 microg/g. Rat sciatic nerve defect model treated with FK506-releasing chitosan guide showed more mature appearance of myelinated fibers 8 weeks after surgery; furthermore, the motor functional reinnervation occurred, the amplitude and velocity of compound muscle action potentials reached 60% and 73% with respect to the normal. Thus, FK506-releasing chitosan guide should be acted as a long-lasting delivery device of immunosuppressive and neuroregenerative agent for peripheral nerve repair.
Combination therapy will soon become a reality, particularly for those patients requiring poly-therapy to treat co-existing disease states. This becomes all the more important with the increasing cost, time and complexity of the drug discovery process prompting one to look at new delivery systems to increase the efficacy, safety and patient compliance of existing drugs. Along this line, we attempted to design nano-scale systems for simultaneous encapsulation of cyclosporine A (CsA) and coenzyme Q10 (CoQ10) and model their encapsulation and release kinetics. The in vitro characterization of the co-encapsulated nanoparticles revealed that the surfactant nature, concentration, external phase volume, droplet size reduction method and drug loading concentration can all influence the overall performance of the nanoparticles. The semi-quantitative solubility study indicates the strong influence of CoQ10 on CsA entrapment which was thought to be due to an increase in the lipophilicity of the overall system. The in vitro dissolution profile indicates the influence of CoQ10 on CsA release (64%) to that of individual particles of CsA, where the release is faster and higher (86%) on 18th day. The attempts to model the encapsulation and release kinetics were successful, offering a possibility to use such models leading to high throughput screening of drugs and their nature, alone or in combination for a particular polymer, if chi-parameters are understood.
In an effort to improve lymphatic targeting efficiency and reduce the toxicity of tacrolimus, the emulsification-diffusion method was used to load the drug into nanoparticles (NP). Poly(lactide-co-glycolide) (PLGA) and PLGA surface-modified with poly(ethylene glycol) (PEG) were used as polymers. Mean particle size and drug encapsulation efficiency of PLGA were 218+/-51nm and 60.0+/-1.2% and for PEG-PLGA NP were 220+/-33nm and 60.3+/-2.0%. NP were characterized by thermal analyzer and X-ray diffractometry (XRD), and their shapes were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In vitro release profiles were affected by the pH of dissolution media. The prepared NP and commercial product of tacrolimus (Prograf) inj.) were intravenously administered to rats to compare their pharmacokinetic characteristics and lymphatic targeting efficiency. The area under the whole blood concentration-time curve (AUC), mean residence time (MRT), and total clearance (CL(t)) of PEG-PLGA NP were significantly different (P<0.05) compared with those of Prograf inj., and lymphatic targeting efficiencies of both NP formulations at the mesenteric lymph node significantly increased (P<0.05). These results showed that the prepared tacrolimus-loaded NP are good possible candidates as a lymphatic delivery system of tacrolimus.
When liposomes are used as drug delivery systems, it is important that the therapeutic agent is efficiently and adequately encapsulated. In this study, cyclosporine A (CyA) was chosen as a model drug for two reasons. First, liposomes are a potential delivery system for CyA, as it has been shown that CyA has decreased side effects when encapsulated in liposomes. Secondly, if the aqueous solubility of a lipophilic drug can be increased, then it is possible to increase liposomal encapsulation by additionally loading the drug into the liposomes' aqueous compartments. Therefore, we investigated the use cyclodextrins (CDs) for complexing CyA to increase aqueous solubility as a strategy to increase liposomal loading. The effect of CyA loading on the liposomes' characteristics, stability and rigidity of the bilayer, and also the drug release profile were evaluated. Liposomes encapsulating CyA, liposomes containing CyA-CD complexes, and liposomes loaded with both plain drug and complex (double-loaded liposomes) were prepared. For evaluation of the effect of CD on bilayer rigidity and integrity, the permeability of the liposomal membrane in terms of carboxyfluorescein (CF) leakage was studied. Among liposomal formulations containing only CyA, 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (CHOL) (1:1) and hydrogenated soybean phosphatidylcholine (HSPC):CHOL (1:1) formulations demonstrated maximum drug entrapments of 65.94 +/- 4.68% and 75.03 +/- 4.87%, respectively. There was no significant difference in encapsulation efficiencies between different liposomal formulations for those containing CyA-CD complexes (P > 0.05). Measurement of encapsulation efficiency showed that the amount of drug entrapped in the lipid bilayers was identical when prepared in the form of CyA inclusion complexes. Drug entrapment in double-loaded liposomes was increased by approximately 2-fold. The release profile of all liposomal formulations was biphasic, with an initial rapid phase during the first 5 h followed by a continuous and slower release thereafter. During the first 5 h, CyA used as the complex was released to a greater extent than free CyA. Leakage of CF from liposomes was affected by the inclusion of CD. The leakage rate was minimum for CyA liposomes and maximum for double-loaded (CyA and CyA-CD) liposomes. In conclusion, it is possible to encapsulate CyA both in the aqueous and lipidbilayers of liposomes if the aqueous solubility of CyA is increased by complexation with CD. Although entrapment of a higher amount of drug was achieved, the stability of the liposomes was compromised and should therefore be considered.
To develop a novel tacrolimus-loaded solid dispersion with improved solubility, various solid dispersions were prepared with various ratios of water, sodium lauryl sulfate, citric acid and carboxylmethylcellulose-Na using spray drying technique. The physicochemical properties of solid dispersions were investigated using scanning electron microscopy, differential scanning calorimetery and powder X-ray diffraction. Furthermore, their solubility and dissolution were evaluated compared to drug powder. The solid dispersion at the tacrolimus/CMC-Na/sodium lauryl sulfate/citric acid ratio of 3/24/3/0.2 significantly improved the drug solubility and dissolution compared to powder. The scanning electron microscopy result suggested that carriers might be attached to the surface of drug in this solid dispersion. Unlike traditional solid dispersion systems, the crystal form of drug in this solid dispersion could not be converted to amorphous form, which was confirmed by the analysis of DSC and powder X-ray diffraction. Thus, the solid dispersion system with water, sodium lauryl sulfate, citric acid and CMC-Na should be a potential candidate for delivering a poorly water-soluble tacrolimus with enhanced solubility and no convertible crystalline.
Inhaled drug delivery after lung transplantation provides a unique opportunity for direct treatment of a solid organ transplant. At present, no inhaled therapies are approved for this population though several have received some development. Primary potential applications include inhaled immunosuppressive and anti-infective drugs.
The objective of this article is to review potential applications of inhaled medications for lung transplant recipients, the techniques used to develop inhaled drugs and the challenges of aerosol delivery in this specific population.
The results of relevant studies are reviewed and two developmental examples are presented.
Inhaled medications may provide significant advantages for lung transplant recipients. Past studies with inhaled cyclosporine and amphotericin-B provide useful guidance for clinical development of new preparations.
Tacrolimus (FK 506), a poorly soluble immunosuppressant is currently formulated in nonaqueous vehicle containing hydrogenated castor oil derivative for intravenous administration. Hydrogenated castor oil derivatives are associated with acute anaphylactic reactions. This proposes to overcome the problems of poor aqueous solubility of the drug and the toxicity associated with currently used excipients by the development of a new parenterally acceptable formulation using self-microemulsifying drug delivery system (SMEDDS). Solubility of FK 506 in various oils, surfactants, and cosurfactants was determined to identify SMEDDS components. Phase diagrams were constructed at different ratios of surfactants:cosurfactant (K(m)) to determine microemulsion existence area. Influence of oily phase content, K(m), aqueous phase composition, dilution, and incorporation of drug on mean globule size of microemulsions was studied. SMEDDSs were developed using ethyl oleate as oily phase and Solutol HS 15 as surfactant. Glycofurol was used successfully as a cosurfactant. Developed SMEDDS could solubilize 0.8% (wt/wt) FK 506 and on addition to aqueous phase could form spontaneous microemulsion with mean globule size < 30 nm. The resulting microemulsion was iso-osmotic, did not show any phase separation or drug precipitation even after 24 h, and exhibited negligible hemolytic potential to red blood cells.
Biodistribution and lymphoscintigraphy of cyclosporine A (CyA) and technetium-99m (99mTc) were studied using 99mTc-labeled dextran acetate (DxA) including CyA. DxA particles were prepared from dextran with acetic anhydride, and CyA was loaded into them. Lymphatic delivery of 99mTc-labeled DxA particles containing CyA was evaluated after subcutaneous injection into the foot pad of rats and compared with those of 99mTc-labeled human serum albumin (HSA). The labeling efficiency of CyA-loaded 99mTc-DxA particles was about 95% at 30 min. The labeling efficiency maintained stably above 80% for 12 h. The percent injected dose (%ID) of CyA-loaded 99mTc-DxA was similar to that of 99mTc-HSA at the inguinal lymph node after 40 min. The CyA-loaded 99mTc-DxA could be as well distributed as 99mTc-HSA through the lymph node. The DxA particles could steadily distribute the CyA as well as the 99mTc radiolabeling through the lymph node.
This study proposes a new concept of double coated nanocapsules to improve the oral bioavailability of a P-glycoprotein (P-gp) substrate drug, tacrolimus, without modulating the physiological activity of the P-gp pump. Tacrolimus was incorporated in nanocapsules with different ratios of two polymethacrylate polymers followed by microencapsulation of these nanocapsules within hydroxypropylmethylcellulose using a spray drying technique. The influence of different formulations of tacrolimus administered orally to rats and pigs on the drug's absorption was investigated. Histopathological studies were performed on rats to follow the nanocapsule path in enterocytes. The novel formulations that released mostly drug loaded nanocapsules in the intestine were shown to enhance markedly the oral absorption of tacrolimus. The relative oral bioavailability of tacrolimus was 4.9 and 2.45 fold compared to the commercial product in rats and pigs respectively. Although there is no direct evidence that intact nanocapsules internalized in the enterocytes, numerous small oil cores were detected within the enterocytes showing the potential of P-gp substrates incorporated in such nanocarriers to escape the efflux pump.
Nearly 25% of patients with ulcerative colitis (UC) requiring steroids therapy become steroid-dependent after 1 yr, and virtually all develop steroid-related adverse events. We planned a controlled study to investigate the efficacy and safety of dexamethasone 21-P (Dex 21-P) encapsulated into erythrocytes (DEE).
Forty patients with mild-to-moderate UC, refractory to mesalamine, were randomly assigned to one of the following three treatments: two DEE infusions 14 days apart (group A, N = 20), oral prednisolone (0.5 mg/kg for 14 days followed by a 6 mg/weekly tapering (group B, N = 10), and sham infusions (group C, N = 10). The clinical, biochemical, and endoscopic parameters were monitored at inclusion and after 8 wk.
In group A, a mean dose of 9.9 +/- 4.1 mg Dex 21-P was loaded into autologous erythrocytes at each infusion. At 8 wk, 15 patients in group A (75%), 8 in group B (80%), and 1 in group C (10%, P < 0.001 vs A and B) were in clinical and endoscopic remission. When compared with the baseline values, C-reactive protein (CRP) dropped in groups A (1.6 mg/dL vs 0.4 mg/dL, P= 0.006) and B (1.0 vs 0.5, P= 0.02), but not in group C. No steroid-related adverse events were apparent in the patient treated with DEE, compared with 8 out of 10 patients on oral steroids (P< or = 0.01).
Low doses of Dex (mean total dose +/- 20 mg) loaded into autologous erythrocytes were significantly more effective than sham infusions in terms of symptoms relief, endoscopic, and biochemical improvements in UC patients refractory to mesalamine. In addition, in contrast to oral prednisolone (mean total dose +/- 1 g), no steroid-related adverse events were induced.
Nanamolar concentrations of the immunosuppressive drug FK-506 inhibit the induction of T-lymphocyte proliferation by the lectins concanavalin A (Con A) and phytohaemagglutinin (PHA). Activation by Con A is more sensitive to inhibition than the response to PHA. FK-506 inhibits an early Ca2+-dependent step in the activation process, and its effects are not reversible by the addition of recombinant interleukin-2 (IL-2) or lymphokine-rich culture supernatant. While the effects of suboptimal concentrations of FK-506 and cyclosporin A (CsA) are additive, FK-506 does not enhance the effects of optimal concentrations of CsA. Both drugs also have similar effects on the expression of specific mRNA in Con A-activated lymphocytes. A brief preincubation of unstimulated cells with FK-506 irreversibly inhibits their subsequent responsiveness to Con A. The mechanism of action of FK-506 thus resembles that of CsA, except that it is effective at two to three orders of magnitude lower concentrations and its effects are much less readily reversible.
Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.
Previous studies on the distribution of circulating ciclosporin have shown that the majority of the drug is associated with erythrocytes. In order to investigate the nature of ciclosporin-erythrocyte binding, binding studies were performed on isolated erythrocytes. At therapeutic concentrations (approx. 0.5 microgram/ml in whole blood) greater than 90% of the erythrocyte associated ciclosporin was found in the cytosol. The cytosolic binding capacity was approximately (2-2.5).10(5) molecules of ciclosporin per cell. A lower affinity binding of the drug to the plasma membrane occurred only at higher ciclosporin concentrations. The ciclosporin-binding species was purified from erythrocyte cytosol using ciclosporin-Affigel affinity chromatography. This revealed a 16 kDa protein, similar in size to the ciclosporin-binding protein, cyclophilin, previously identified in lymphocyte cytosol. Immunochemical analysis using rabbit anti-bovine spleen cyclophilin antisera revealed that the erythrocyte ciclosporin-binding protein was either cyclophilin or a closely related protein. It is concluded that intracellular ciclosporin-binding within erythrocytes is mostly attributable to the presence of a single protein or protein family represented by cyclophilin. The presence of (2-2.5).10(5) copies of this binding protein within each erythrocyte is responsible for the ciclosporin found associated with erythrocytes.
This chapter describes the radioiodination of proteins by the use of the chloramine-T method. The incorporation of radioiodine into protein molecules provides increased dimensions to the study of their interactions and subsequent fates. This incorporation involves the covalent linkage of radioiodine in the tyrosyl and to a lesser degree histidine residue of the protein molecule. Information gained through the use of radioiodinated proteins can be used with confidence if it is established that minimal, if any, alteration occurs in the reactivity of the radioiodinated protein versus that observed in its native form. Chloramine-T radioiodination of proteins involves both the oxidation of radioiodide to its reactive state, H2OI+, and the oxidation of sulfhydryl groups of the protein molecules. There must be sufficient chloramine-T available to complete both the reactions. Therefore, proteins, with high reducing capacity, may be poorly radioiodinated under the standard conditions. For example, keyhole limpet hemocyanin under standard conditions is minimally radioiodinated. However, by increasing the chloramine-T to 100/μg per milligram of protein, an average 50% incorporation of radioiodine is achieved with no apparent denaturation. Proteins that cannot be radioiodinated successfully with standard conditions should be repeated at increased chloramine-T to the protein ratios.
The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.
Tacrolimus is a macrolide immunosuppressive drug undergoing clinical trials for organ transplantation. Whole animal studies were undertaken to assess the rabbit as an animal model for tacrolimus pharmacokinetics. The disposition of tacrolimus in rabbits following 0.5 mg/kg iv and 2.0 mg/kg po doses is similar to man. The apparent plasma clearance 1.67 liters/hr/kg is more than 5-fold higher than blood clearance 0.31 liters/hr/kg. The steady-state volume of distribution is 30.7 liters/kg for plasma and 6.3 liters/kg for whole blood. The bioavailability after oral administration calculated from plasma and whole blood is low with a mean value of 9.7%. The in vitro studies and fitting using a nonlinear red blood cell (RBC)-plasma binding/diffusion model showed slow diffusion of drug from RBC to plasma (t1/2 = 7 min, CLD = 0.085 ml/min). In perfused liver studies, the extraction ratio of tacrolimus from blood with hematocrit of 0.1 is low (0.20). However, extraction of drug from plasma is moderate (0.42), and plasma concentrations are elevated by an average of 28% because of redistribution of tacrolimus from RBC. This creates a lower apparent plasma clearance (DO/AUC) for equilibrated (30 min at 37 degrees C) samples (15.4 ml/min) compared with samples centrifuged within 5 min (22.1 ml/min). RBC efflux was accounted for using a comprehensive perfusion/diffusion model. The intrinsic metabolic clearance averaged 29.2 ml/min. Simulations showed that the apparent plasma clearance of tacrolimus is closely correlated with RBC binding capacity (whole blood:plasma ratio). Higher ratios caused greater apparent plasma clearance (lower concentration of tacrolimus in reservoir plasma) because strong binding of drug by erythrocytes prevents tacrolimus from diffusion into plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
Cyclosporin is a lipophilic cyclic polypeptide which produces calcium-dependent, specific, reversible inhibition of transcription of interleukin-2 and several other cytokines, most notably in T helper lymphocytes. This reduces the production of a range of cytokines, inhibiting the activation and/or maturation of various cell types, including those involved in cell-mediated immunity. Thus, cyclosporin has immunosuppressive properties, and has a proven place as first line therapy in the prophylaxis and treatment of transplant rejection. Cyclosporin has also been evaluated in a large range of disorders where immunoregulatory dysfunction is a suspected or proven aetiological factor, and this is the focus of the present review. In patients with severe disease refractory to standard treatment, oral cyclosporin is an effective therapy in acute ocular Behçet's syndrome, endogenous uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, active Crohn's disease and nephrotic syndrome. Concomitant low dose corticosteroid therapy may improve response rates in some disorders. The drug can be considered as a first line therapy in patients with moderate or severe aplastic anaemia who are ineligible for bone marrow transplantation, with the additional benefit of reducing platelet alloantibody titres. It may also be of considerable therapeutic benefit in patients with primary biliary cirrhosis, particularly those with less advanced disease. Limited evidence suggests cyclosporin is effective in patients with intractable pyoderma gangrenosum, polymyositis/dermatomyositis or severe, corticosteroid-dependent asthma. Indeed, the steroid-sparing effect of cyclosporin is a significant advantage in a number of indications. Furthermore, the drug has shown some efficacy in a wide range of other, generally uncommon disorders in which controlled clinical trials are lacking and/or are unlikely to be performed. Cyclosporin does not appear to be effective in patients with allergic contact dermatitis, multiple sclerosis or amyotrophic lateral sclerosis. It is only temporarily effective in patients with type I (insulin-dependent) diabetes mellitus and should not be used in this indication. To avoid relapse after control of active disease, patients should receive cyclosporin maintenance therapy at the lowest effective dosage. However, maintenance therapy appears to be of no benefit in patients with Crohn's disease and cyclosporin should be discontinued in these patients once active disease is controlled. Hypertrichosis, gingival hyperplasia, and neurological and gastrointestinal effects are the most common adverse events in cyclosporin recipients, but are usually mild to moderate and resolve on dosage reduction. Changes in laboratory variables indicating renal dysfunction are relatively common, although serious irreversible damage is rare.(ABSTRACT TRUNCATED AT 400 WORDS)
More than 70% of cyclosporine A (CsA) is bound to erythrocytes at whole blood concentrations of 50–1000 ng·ml−1. Cytosolic CsA is bound to the erythrocyte peptidyl-prolyl cis-trans isomerase cyclophilin. Measurements of serum CsA levels under clinical conditions are hampered by a temperature-dependent translocation of CsA into erythrocytes during cooling of the probes to room temperature. In order to characterize the kinetics of CsA uptake and to find a specific uptake inhibitor, we developed a method to measure the velocity of uptake based on rapid cooling of the erythrocyte suspension.
The total erythrocyte-binding capacity for CsA amounted to 43·10−5 nmol per 106 erythrocytes or 2.6·105 molecules per erythrocyte. Whereas the erythrocyte-binding capacity of CsA was temperature-independent between 10°C and 42°C, uptake kinetics of CsA were temperature-dependent. The Arrhenius plot for CsA uptake in human erythrocytes was linear and no transition temperature between 0°C and 42°C could be detected. Therefore the CsA uptake process in human erythrocytes did not fulfil the criteria of carrier-mediated transport.
This indicates that CsA diffuses passively into human erythrocytes. Hence, erythrocyte CsA uptake cannot be specifically inhibited.
FK506 (tacrolimus) is a safe and effective immunosuppressant for the prevention of organ rejection after organ transplantation. FK506 has a relatively narrow therapeutic index and the correlation of dose to blood concentration is poor as a result of moderate variability in pharmacokinetic parameters between patients. Therapeutic monitoring of whole blood FK506 drug concentrations has been used in an effort to determine whether a relationship exists between concentrations of FK506 in the blood and the development of toxicity or the risk for organ rejection. An analysis of the relationship between FK506 blood levels and the occurrence of toxicity and rejection was carried out using data from four recent clinical trials. Trough FK506 levels within a 7-day window before the onset of rejection or toxicity were analyzed using logistic regression models. In kidney transplant patients (n=92), a significant correlation between FK506 levels and the incidence of both toxicity (P=0.01) and rejection (P=0.02) was seen. In liver transplant patients from three clinical trials, FK506 levels correlated well with the incidence of toxicity (P < or = 0.01); however, there was no significant relationship between FK506 levels and the incidence of rejection. It is concluded that therapeutic monitoring of whole blood FK506 levels may be useful for minimizing the risks of both toxicity and rejection in kidney transplant patients and for minimizing the risk of toxicity in liver transplant recipients.
Tacrolimus is an immunosuppressive agent used for organ transplantation. Studies were performed to examine the influence of different perfusate hematocrits and albumin concentrations on hepatic extraction of tacrolimus. In vitro binding, efflux and influx between red blood cells (RBCs) and buffer or plasma, and rabbit liver perfusion with use of human erythrocytes were studied. In the range of hematocrits from 0.05 to 0.4, plasma concentrations of tacrolimus were not affected by increased albumin content. Increased hematocrit caused decreases in whole blood:plasma (buffer) concentration ratios. The binding capacity of drug with RBCs was independent of hematocrit, with a value of 440 ng/ml of RBCs; the binding affinity was 0.876 ng/ml using plasma or buffer. Diffusion of tacrolimus from RBCs to buffer was rapid with a clearance of 0.940 ml/min, and equilibration was achieved within 2 min. Diffusion in the opposite direction (buffer-RBCs) was slower with a clearance of 0.576 ml/min. In such diffusion studies, plasma produced a greater difference between efflux (1.70 ml/min) and influx (0.276 ml/min) clearances. During liver perfusion, the major factor regulating elimination of tacrolimus was hematocrit. Both well-stirred and parallel-tube models reflected a low extraction ratio drug with values of 0.15 and 0.17 for the 0.05 and 0.2 hematocrits. Intrinsic clearances were 8.43 and 17.44 ml/min for the well-stirred and parallel-tube models. Albumin had a negligible influence on liver extraction of drug. A model-building process of characterizing nonlinear RBC binding, RBC diffusion rates, and liver perfusion parameters allows the complexities of tacrolimus hepatic clearance to be dissected and shows that strong RBC binding can be artificially perceived as causing a high clearance of the drug.
A new procedure for the encapsulation of non-diffusible drugs into human erythrocytes was developed. With as little as 50 ml of blood and by using a new apparatus, it was possible to encapsulate a variety of biologically active compounds into erythrocytes in 2 h at room temperature and under blood-banking conditions. The process, which is based on two sequential hypotonic dilutions of washed red cells followed by concentration with a haemofilter and resealing of red cells, allows a 35-50% cell recovery and approx. 30% encapsulation of added drugs. The resulting processed erythrocytes have a normal survival in vivo and can be modified further, with the same apparatus, to increase their recognition by tissue macrophages to perform as a drug-targeting system. The new equipment designed and built for this procedure was named 'Red Cell Loader'.
Successful pregnancy outcomes are possible after solid organ transplantation. While there are risks to mother and fetus, there has not been an increased incidence of malformations noted in the newborn of the transplant recipient. It is essential that there is closely coordinated care that involves the transplant team and an obstetrician in order to obtain a favourable outcome.
Current data from the literature, as well as from reports from the National Transplantation Pregnancy Registry (NTPR), support the concept that immuno suppression be maintained at appropriate levels during pregnancy. At present, most immunosuppressive maintenance regimens include combination therapy, usually cyclosporin or tacrolimus based. Most female transplant recipients will be receiving maintenance therapy prior to and during pregnancy. For some agents, including monoclonal antibodies and mycophenolate mofetil, there is either no animal reproductive information or there are concerns about reproductive safety.
The optimal (lowest risk) transplant recipient can be defined by pre-conception criteria which include good transplant graft function, no evidence of rejection, minimum 1 to 2 years post-transplant and no or well controlled hypertension. For these women pregnancy generally proceeds without significant adverse effects on mother and child.
It is of note that the epidemiological data available to date on azathioprine-based regimens are favourable in the setting of a category D agent (i.e. one that can cause fetal harm). Thus, there is still much to learn regarding potential toxicities of immunosuppressive agents. The effect of improved immunosuppressive regimens which use newer or more potent (and potentially more toxic) agents will require further study.
A novel macromolecular prodrug of Tacrolimus (FK506), FK506-dextran conjugate, was developed and its physico-chemical, biological and pharmacokinetic characteristics were studied. The conjugate was estimated to contain 0.45% of FK506 and the coupling molar ratio was approximately 1:1 (dextran-FK-506). Adsorption experiments using ion exchangers indicated that FK506-dextran conjugate acted as a weakly negatively charged macromolecule. Low molecular weight radioactive compound(s), which was eluted in the same fractions as [(3)H]FK506, was released from [(3)H]FK506-dextran conjugate by chemical hydrolysis with a half-life of 150 h in phosphate buffer. In vitro immunosuppressive activity of the conjugate, as assessed by the rat lymphocyte stimulation test, was almost comparable to that of free FK506, suggesting that biologically active FK506 could be liberated from the conjugate. In vitro biodistribution studies demonstrated that conjugation with the dextran derivative dramatically changed the pharmacokinetic properties of FK506 after intravenous injection in rats. AUC of the FK506-dextran conjugate was almost 2000 times higher than that of free FK506 and organ uptake clearances of the conjugate were significantly smaller than those of the free drug. Thus, the present study has demonstrated that the FK506-dextran conjugate behaves as a prodrug of FK506 with an extended blood circulating time and can be expected to have an improved therapeutic potency.
Cyclosporine A therapy for prophylaxis against graft rejection revolutionized human organ transplantation. The immunosuppressant drugs cyclosporin A (CsA), FK506 and rapamycin block T-cell activation by interfering with the signal transduction pathway. The target proteins for CsA and FK506 were found to be cyclophilins and FK506-binding proteins, (FKBPs), respectively. They are unrelated in primary sequence, although both are peptidyl-prolyl cis-trans isomerases catalyzing the interconversion of peptidylprolyl imide bonds in peptide and protein substrates. However, the prolyl isomerase activity of these proteins is not essential for their immunosuppressive effects. Instead, the specific surfaces of the cyclophilin-CsA and FKBP-FK506 complexes mediate the immunosuppressive action. Moreover, the natural cellular functions of all but a few remain elusive. In some cases it could be demonstrated that prolyl isomerization is the rate-limiting step in protein folding in vitro, but many knockout mutants of single and multiple prolyl isomerases were viable with no detectable phenotype. Even though a direct requirement for in vivo protein folding could not be demonstrated, some important natural substrates of the prolyl isomerases are now known, and they demonstrate the great variety of prolyl isomerization functions in the living cell: (i) A human cyclophilin binds to the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1) virion and was found to be essential for infection with HIV to occur, probably by removal of the virion coat. (ii) Together with heat shock protein (HSP) 90, a member of the chaperone family, high molecular weight cyclophilins and FKBPs bind and activate steroid receptors. This example also demonstrates that prolyl isomerases act together with other folding enzymes, for example the chaperones, and protein disulfide isomerases. (iii) An FKBP was found to act as a modulator of an intracellular calcium release channel. (iv) Along with the cyclophilins and FKBPs, a third class of prolyl isomerases exist, the parvulins. The human parvulin homologue Pin1 is a mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. These findings place proline isomerases at the intersection of protein folding, signal transduction, trafficking, assembly and cell cycle regulation.
Human erythrocytes from ten patients with chronic obstructive pulmonary disease (COPD) were loaded with increasing amounts of dexamethasone 21-phosphate and were re-infused into the original donors. Drug-loaded erythrocytes acted as circulating bioreactors, converting the non-diffusible dexamethasone 21-phosphate into the diffusible dexamethasone. Pharmacokinetic analyses on these patients showed that a single administration of drug-loaded erythrocytes was able to maintain detectable dexamethasone concentrations in blood for up to seven days. This continuous release of dexamethasone was paralleled by the suspension of beta2-agonist and oral corticosteroid treatments by all of the patients. Thus dexamethasone 21-phosphate-loaded erythrocytes are safe carriers for corticosteroid analogues and are a useful alternative to frequent oral or inhaled drugs in elderly patients with COPD.
Unlabelled:
Cyclosporin is a lipophilic cyclic polypeptide immunosuppressant that interferes with the activity of T cells chiefly via calcineurin inhibition. The original oil-based oral formulation of this drug (Sandimmun)l was characterised by high intra- and interpatient pharmacokinetic variability, with poor bioavailability in many patients; a novel microemulsion formulation (Neoral)1 was therefore developed to circumvent these problems. Studies show increases, attributable chiefly to improved absorption in patients who absorb the drug only poorly from the original formulation, in mean systemic exposure to cyclosporin with the microemulsion, with no clinically significant differences in tolerability or drug interaction profiles. Cyclosporin microemulsion is at least as effective as the oil-based formulation in renal, liver and heart transplant recipients, with trends towards decreased incidence of acute rejection with the microemulsion formulation in some (statistically significant in a few) trials. Cyclosporin microemulsion and tacrolimus appear to have similar efficacy in preventing acute rejection episodes in most renal, pancreas-kidney, liver and heart transplant recipients. However, there are indications of superior efficacy for tacrolimus in some trials, particularly in the prevention of severe acute rejection and in Black transplant recipients. Current 12-month data also indicate equivalent efficacy of sirolimus in renal transplantation. Conversion from the oil-based to microemulsion formulation in stable renal, liver and heart transplant recipients is achievable with no change in acute rejection rates. The addition of an anti-interleukin-2 receptor monoclonal antibody and/or mycophenolate mofetil to cyclosporin microemulsion plus corticosteroids decreases rates of acute rejection; corticosteroid withdrawal without increased acute rejection rates was also achieved on the addition of these agents in some trials. Pharmacoeconomic analyses have shown savings in direct healthcare costs in kidney or liver transplantation when cyclosporin microemulsion is used in preference to the oil-based formulation, although studies incorporating indirect costs or expressing costs in terms of therapeutic outcomes are currently unavailable.
Conclusions:
The introduction of cyclosporin microemulsion has consolidated the place of the drug as a mainstay of therapy in all types of solid organ transplantation; research into optimisation of outcomes through more effective therapeutic monitoring in patients receiving this formulation is ongoing. Several novel immunosuppressants have been introduced in recent years: further clinical and pharmacoeconomic research will be needed to clarify the relative positioning of these agents, particularly with respect to specific patient groups. Other new drugs (basiliximab/daclizumab and mycophenolate mofetil) offer particular advantages when used in combination with cyclosporin.
This paper reviews the pharmacokinetics of tacrolimus, with special reference to its combination with adjunctive immunosuppressants. Oral bioavailability of tacrolimus, which is variable between patients, averages approximately 25%. This is largely due to extrahepatic metabolism of tacrolimus in the gastrointestinal epithelium. Nevertheless, intra-patient variability is low, as evidenced by the small number of dose changes required to maintain patients within the recommended tacrolimus target levels. Tacrolimus is distributed extensively in the body with most partitioned outside the blood compartment. Concentrations of tacrolimus in blood are used as a surrogate marker of clinically relevant concentration of the drug at the site(s) of action. Convenient whole-blood sampling within a +/-2-h window around 12 h post-dose (C(min)) is highly predictive of systemic exposure to tacrolimus and is thus used to optimise therapy. Sampling at other time-points offers no advantage over C(min) monitoring. The interactions of tacrolimus with other immunosuppressive agents are well characterized. After cessation of concomitant corticosteroid treatment, exposure to tacrolimus increases by approximately 25%. In contrast, there is no pharmacokinetic interaction between mycophenolate mofetil (MMF) and tacrolimus. Therefore, systemic exposure to the active metabolite of MMF, mycophenolic acid, is higher with MMF-tacrolimus combination than with MMF-cyclosporin combination. Therefore, 1 g/day MMF may be an adequate maintenance dose in tacrolimus-based regimens. Co-administration of tacrolimus and sirolimus, while having no effect on exposure to sirolimus, results in reduced exposure to tacrolimus at sirolimus doses of 2 mg/day and above. In conclusion, tacrolimus levels should be monitored when sirolimus is co-administered at doses >2 mg/day and after cessation of corticosteroid treatment.