Microfluidic nanoprecipitation of PEGylated PLGA nanoparticles with rapamycin and performance evaluation.

Rapamycin (RAP) is currently being developed as potential antibreast cancer drug. However, its poor solubility completely limits its use. The aim of this study was to develop polyethylene glycol-poly(lactide-co-glycolide) (PEG-PLGA)-based nanoparticles (NPs) to load RAP via microfluidics with an appropriate polyethylene glycol (PEG) content to enhance the bioavailability of RAP. Polydimethylsiloxane (PDMS) chips with a Y-shaped channel were designed to obtain RAP-loaded PEG-PLGA NPs (RAP-PEG-PLGA). The entrapment efficiency (EE) and drug loading (DL) as well as release profile of RAP-PEG-PLGA were evaluated, and their resistance to plasma albumin adsorption of NPs with different PEG contents was evaluated and compared. RAW264.7 and 4T1 cells were used to assess the antiphagocytic and anticancer cells effect of NPs, respectively. RAP-PEG-PLGA of around 124 nm in size were successfully prepared with the EE of 82.0% and DL of 12.3%, and sustained release for around 40 d. A PEG relative content of 10% within the PEG-PLGA molecule was shown superior in resisting protein adsorption. RAP-PEG-PLGA inhibited the growth of breast cancer cells when the concentration was over 10 µg/mL, and the inhibition efficiency was significantly higher than free RAP. Hence, the current RAP-PEG-PLGA could be a potential therapeutic system for breast cancer treatment.

Predictors of sirolimus pharmacokinetic variability identified using a nonlinear mixed effects approach: a systematic review.

Several sirolimus (SRL) population pharmacokinetics (PopPK) were conducted to explain its pharmacokinetic variability, and the results varied across studies. Thus, we conducted a systematic review to summarize significant predictors influencing SRL pharmacokinetic variability. Moreover, discrepancies in model methodologies across studies were also reviewed and discussed. Four databases (PubMed, CINAHL Complete, Science Direct, and Scopus) were systematically searched. The PICO framework was used to identify eligible studies conducted in humans and employ a nonlinear-mixed effects strategy. Based on the inclusion and exclusion criteria, 20 studies were included. SRL pharmacokinetics were explained using 1- or 2-compartment models. Only one study assessed the model using an external approach, while the rest employed basic or advanced internal approaches. Significant covariates influencing SRL pharmacokinetics were bodyweight, age, CYP3A5 polymorphism, gender, BSA, height, cyclosporine dose or trough concentration, triglyceride, total cholesterol, hematocrit, albumin, aspartate aminotransferase, alanine aminotransferase, and total bilirubin. Of these, bodyweight, age, and CYP3A5 polymorphism were the three most identified significant predictors for SRL clearance. This review summarizes significant predictors to predict SRL clearance, which can subsequently be used to individualize SRL maintenance dose. However, the PopPK model selected for such prediction should be based on the resemblance of population characteristics between the target population and those used to conduct the model. Moreover, the predictability of the models in the target population should be assessed before implementation in clinical practice.

Population Pharmacokinetic Analysis and Dosing Optimization of Sirolimus in Children With Tuberous Sclerosis Complex.

Sirolimus is confirmed to be effective in the treatment of tuberous sclerosis complex (TSC) and related disorders. The study aims to establish a population pharmacokinetic model of oral sirolimus for children with TSC and provide an evidence-based approach for individualization of sirolimus dosing in the pediatric population. A total of 64 children were recruited in this multicenter, retrospective pharmacokinetic study. Whole-blood concentrations of sirolimus, demographic, and clinical information were collected and analyzed using a nonlinear mixed-effects population modeling method. The final model was internally and externally validated. Then Monte Carlo simulations were performed to evaluate and optimize the dosing regimens. In addition, the efficacy and safety of sirolimus therapy was assessed retrospectively in patients with epilepsy or cardiac rhabdomyomas associated with TSC. Finally, the sirolimus pharmacokinetic profile was described by a 1-compartment model with first-order absorption and elimination along with body weight and total daily dose as significant covariates. The typical population parameter estimates of apparent volume of distribution and apparent clearance were 69.48 L and 2.79 L/h, respectively. Simulations demonstrated that dosage regimens stratified by body surface area may be more appropriate for children with TSC. These findings could be used to inform individualized dosing strategies of sirolimus for pediatric patients with TSC.

Macrophage membrane camouflaged reactive oxygen species responsive nanomedicine for efficiently inhibiting the vascular intimal hyperplasia.

BACKGROUND: Intimal hyperplasia caused by vascular injury is an important pathological process of many vascular diseases, especially occlusive vascular disease. In recent years, Nano-drug delivery system has attracted a wide attention as a novel treatment strategy, but there are still some challenges such as high clearance rate and insufficient targeting. RESULTS: In this study, we report a biomimetic ROS-responsive MM@PCM/RAP nanoparticle coated with macrophage membrane. The macrophage membrane with the innate "homing" capacity can superiorly regulate the recruitment of MM@PCM/RAP to inflammatory lesion to enhance target efficacy, and can also disguise MM@PCM/RAP nanoparticle as the autologous cell to avoid clearance by the immune system. In addition, MM@PCM/RAP can effectively improve the solubility of rapamycin and respond to the high concentration level of ROS accumulated in pathological lesion for controlling local cargo release, thereby increasing drug availability and reducing toxic side effects. CONCLUSIONS: Our findings validate that the rational design, biomimetic nanoparticles MM@PCM/RAP, can effectively inhibit the pathological process of intimal injury with excellent biocompatibility.

Functionalized nanoparticles with monocyte membranes and rapamycin achieve synergistic chemoimmunotherapy for reperfusion-induced injury in ischemic stroke.

BACKGROUND: Ischemic stroke is an acute and severe neurological disease, and reperfusion is an effective way to reverse brain damage after stroke. However, reperfusion causes secondary tissue damage induced by inflammatory responses, called ischemia/reperfusion (I/R) injury. Current therapeutic strategies that control inflammation to treat I/R are less than satisfactory. RESULTS: We report a kind of shield and sword nano-soldier functionalized nanoparticles (monocyte membranes-coated rapamycin nanoparticles, McM/RNPs) that can reduce inflammation and relieve I/R injury by blocking monocyte infiltration and inhibiting microglia proliferation. The fabricated McM/RNPs can actively target and bind to inflammatory endothelial cells, which inhibit the adhesion of monocytes to the endothelium, thus acting as a shield. Subsequently, McM/RNPs can penetrate the endothelium to reach the injury site, similar to a sword, and release the RAP drug to inhibit the proliferation of inflammatory cells. In a rat I/R injury model, McM/RNPs exhibited improved active homing to I/R injury areas and greatly ameliorated neuroscores and infarct volume. Importantly, in vivo animal studies revealed good safety for McM/RNPs treatment. CONCLUSION: The results demonstrated that the developed McM/RNPs may serve as an effective and safe nanovehicles for I/R injury therapy.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5-50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

Challenging the CNS Targeting Potential of Systemically Administered Nanoemulsion Delivery Systems: a Case Study with Rapamycin-Containing Fish Oil Nanoemulsions in Mice.

PURPOSE: Despite extensive preclinical investigations, in-vivo properties and formulation characteristics that improve CNS drug delivery following systemic dosing of nanoemulsions remain incompletely understood. METHODS: The CNS targeting potential of systemically administered nanoemulsions was evaluated by formulating rapamycin containing fish oil nanoemulsions, and testing the combined effect of formulation characteristics such as the circulation half-life and particle size distribution, on CNS delivery of rapamycin containing fish oil nanoemulsions in mice. RESULTS: Results generated with rapamycin nanoemulsions suggested that circulation half-life and particle size distribution did not impact the brain targeting efficiency of rapamycin containing fish oil nanoemulsions. Further, in the absence of any improvement in the systemic exposures of rapamycin, nanoemulsions did not outperform their aqueous counterpart with respect to the extent of CNS drug delivery. CONCLUSIONS: Our findings confirm that BBB penetration, which primarily depends on intrinsic drug-related properties, may not be significantly improved following encapsulation of drugs in nanoemulsions. Graphical Abstract The CNS targeting potential of systemically administered nanoemulsions was investigated by formulating various rapamycin containing fish oil nanoemulsions associated with different formulation characteristics such as the circulation half-life and particle size distribution. The targeting efficiency (TE) defined as the ratio of the brain exposures to the accompanying systemic exposures of rapamycin was estimated for each formulation following IV dosing in mice.

Dose Escalation Study to Assess the Pharmacokinetic Parameters of a Nano-amorphous Oral Sirolimus Formulation in Healthy Volunteers.

BACKGROUND AND OBJECTIVES: Sirolimus (Rapamune®) exhibits low bioavailability, high variability and moderate food effect following oral administration. This makes therapeutic blood monitoring of sirolimus concentrations necessary for kidney transplant patients. Furthermore, reaching therapeutic blood sirolimus concentrations in renal cancer patients was found to be challenging when the marketed drug was administered alone. A novel, nano-amorphous formulation of the compound was developed and its pharmacokinetic properties were investigated in a dose escalation study in a first-in-human clinical trial. The effect of food at the highest dose on the pharmacokinetic parameters was also assessed. METHODS: Each group received one of the escalating doses (0.5-2-10-40 mg) of sirolimus as the novel formulation in the fasted state. Following a 2- to 3-week washout period, the 40-mg group then also received another 40 mg dose in the fed state. Sirolimus whole blood concentrations were determined for up to 48 h. To avoid degradation of sirolimus in the acidic environment in the stomach, 40 mg famotidine was administered 3 h pre-dose in all regimens. The main pharmacokinetic parameters were calculated and data were compared with pharmacokinetic data reported for dose escalation studies for Rapamune®. RESULTS: Thirty-two healthy volunteers were divided into 4 cohorts of 8 volunteers. Dose increments resulted in approximately dose-proportional increases of maximal plasma concentrations (Cmax) and area under the concentration-time curve (AUC)0-48 h up to 10 mg, while less than dose-proportional increases were observed when the dose was increased from 10 to 40 mg. Mean AUCinf at the 40 mg dose in the fasted state was 4,300 ± 1,083 ng·h/ml, which is 28% higher than the AUC reported following the administration of 90 (2 × 45) mg Rapamune® and 11% higher than the exposure reported for 25 mg intravenous pro-drug temsirolimus (3,810 ng·h/ml). At the 40 mg dose, food reduced Cmax by 35.5%, but it had no statistically significant effect on AUC. Inter-individual variability of the pharmacokinetic parameters mostly fell in the 20-30% (CV) range showing that sirolimus administered as the nano-amorphous formulation is a low-to-moderate variability drug. CONCLUSION: Based on the pharmacokinetic profiles observed, the nano-amorphous formulation could be a better alternative to Rapamune® for the treatment of mammalian target of rapamycin-responsive malignancies. Therapeutically relevant plasma concentrations and exposures can be achieved by a single 40 mg oral dose. Furthermore, the low variability observed might make therapeutic blood monitoring unnecessary for transplant patients taking sirolimus as an immunosuppressant.

Preincubation With Everolimus and Sirolimus Reduces Organic Anion-Transporting Polypeptide (OATP)1B1- and 1B3-Mediated Transport Independently of mTOR Kinase Inhibition: Implication in Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 mediate hepatic uptake of many drugs including lipid-lowering statins. Current studies determined the OATP1B1/1B3-mediated drug-drug interaction (DDI) potential of mammalian target of rapamycin (mTOR) inhibitors, everolimus and sirolimus, using R-value and physiologically based pharmacokinetic models. Preincubation with everolimus and sirolimus significantly decreased OATP1B1/1B3-mediated transport even after washing and decreased inhibition constant values up to 8.3- and 2.9-fold for OATP1B1 and both 2.7-fold for OATP1B3, respectively. R-values of everolimus, but not sirolimus, were greater than the FDA-recommended cutoff value of 1.1. Physiologically based pharmacokinetic models predict that everolimus and sirolimus have low OATP1B1/1B3-mediated DDI potential against pravastatin. OATP1B1/1B3-mediated transport was not affected by preincubation with INK-128 (10 µM, 1 h), which does however abolish mTOR kinase activity. The preincubation effects of everolimus and sirolimus on OATP1B1/1B3-mediated transport were similar in cells before preincubation with vehicle control or INK-128, suggesting that inhibition of mTOR activity is not a prerequisite for the preincubation effects observed for everolimus and sirolimus. Nine potential phosphorylation sites of OATP1B1 were identified by phosphoproteomics; none of these are the predicted mTOR phosphorylation sites. We report the everolimus/sirolimus-preincubation-induced inhibitory effects on OATP1B1/1B3 and relatively low OATP1B1/1B3-mediated DDI potential of everolimus and sirolimus.

Pharmacokinetic Drug-Drug Interactions Between Letermovir and the Immunosuppressants Cyclosporine, Tacrolimus, Sirolimus, and Mycophenolate Mofetil.

Letermovir (AIC246, MK-8228) is a human cytomegalovirus terminase inhibitor indicated for the prophylaxis of cytomegalovirus infection and disease in allogeneic hematopoietic stem cell transplant recipients that is also being investigated for use in other transplant settings. Many transplant patients receive immunosuppressant drugs, of which several have narrow therapeutic ranges. There is a potential for the coadministration of letermovir with these agents, and any potential effect on their pharmacokinetics (PK) must be understood. Five phase 1 trials were conducted in 73 healthy female participants to evaluate the effect of letermovir on the PK of cyclosporine, tacrolimus, sirolimus, and mycophenolic acid (active metabolite of mycophenolate mofetil [MMF]), as well as the effect of cyclosporine and MMF on letermovir PK. Safety and tolerability were also assessed. Coadministration of letermovir with cyclosporine, tacrolimus, and sirolimus resulted in 1.7-, 2.4-, and 3.4-fold increases in area under the plasma concentration-time curve and 1.1-, 1.6-, and 2.8-fold increases in maximum plasma concentration, respectively, of the immunosuppressants. Coadministration of letermovir and MMF had no meaningful effect on the PK of mycophenolic acid. Coadministration with cyclosporine increased letermovir area under the plasma concentration-time curve by 2.1-fold and maximum plasma concentration by 1.5-fold, while coadministration with MMF did not meaningfully affect the PK of letermovir. Given the increased exposures of cyclosporine, tacrolimus, and sirolimus, frequent monitoring of concentrations should be performed during administration and at discontinuation of letermovir, with dose adjustment as needed. These data support the reduction in clinical dosage of letermovir (to 240 mg) upon coadministration with cyclosporine.

Significant effect of infection and food intake on sirolimus pharmacokinetics and exposure in pediatric patients with acute lymphoblastic leukemia.

BACKGROUND: Sirolimus is increasingly investigated as a new targeted therapy in pediatric populations. To date, population pharmacokinetic (PK) studies have identified several factors that explain in part the large between-patient variability in sirolimus PK. However, within-patient variability in sirolimus PK is not well documented. This study presents examples of model-based PK-guided dosing of sirolimus in children with acute lymphoblastic leukemia (ALL), where patients experienced significant changes in sirolimus blood concentrations due to infection and food intake during the treatment period. METHODS: Clinical PK data were obtained as part of a prospective pilot study of sirolimus combined with multiagent chemotherapy in pediatric patients with ALL (ClinicalTrial.gov identifier: NCT01658007). A PK model-informed loading dose of 1.8 mg/m2 every 8 h was started on the first day of sirolimus treatment. Subsequent doses were adjusted based on concentration measurements the first blood draw scheduled 24 h into the regimen on the morning of day 2. Sirolimus blood concentrations were determined by a validated LC-MS/MS assay. All dosing recommendations were generated in real time using the PK model with Bayesian estimation. RESULTS: Three patients were enrolled in this study. Two patients achieved target concentration attainment with the PK model-informed loading dose on day 1 of sirolimus treatment. Subsequent unexpected high sirolimus concentrations were observed in two patients, where patients had flulike symptoms such as fever and cough. A sudden decrease in sirolimus concentrations was observed in one patient after switching sirolimus administration from the fed to the fasting state. CONCLUSIONS: This study highlights within-patient fluctuations in sirolimus concentrations associated with intercurrent infection and with changes in diet. These findings highlight the challenge of maintaining a target sirolimus concentration as a patient's clinical status changes, and the benefit of intensive monitoring of therapeutic drug levels in children treated with sirolimus. Intra-patient alternations in sirolimus PK due to similar disease/food interactions may be relevant in pediatric patients treated with sirolimus for other disease indications.

Rapamycin Prevents Surgery-Induced Immune Dysfunction in Patients with Bladder Cancer.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction.

Efficacy and absorption of topical sirolimus for the treatment of vascular anomalies in children: A case series.

BACKGROUND/OBJECTIVES: Efficacy of topical sirolimus has recently been described in lymphatic anomalies but not in other types of vascular anomalies. To our knowledge, systemic absorption of topical sirolimus in these lesions has not yet been reported. The objective was to evaluate the efficacy, tolerance, and absorption of topical sirolimus 0.1% with different types of vascular anomalies in children. METHODS: Sirolimus 0.1% was applied on cutaneous vascular anomalies in six children aged 2-17. These anomalies consisted of three extratruncular micro- and macrocystic lymphatic malformations and one each verrucous venous malformation, truncular lymphatic malformation with angiokeratomas, and infantile hemangioma. Sirolimus blood levels were measured after 1 week, 1 month, and 3 months. RESULTS: A rapid decrease in the size of superficial lymphatic malformations in three of six patients and a significant decrease in discharge from oozing lesions were observed. Response occurred in less than 3 months. The truncular lymphatic malformation, verrucous venous malformation, and infantile hemangioma did not respond to topical sirolimus. Sirolimus levels were undetectable. Adverse effects were limited to local irritation. CONCLUSIONS: Topical sirolimus 0.1% is a useful treatment for cutaneous manifestations of extratruncular lymphatic malformations. The only adverse effect is local irritation. No systemic effects are expected, because blood levels are clinically insignificant.

The role of mTOR in ovarian Neoplasms, polycystic ovary syndrome, and ovarian aging.

The mammalian target of rapamycin, mTOR, is a serine-threonine protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-AKT axis. The pathway can regulate cell growth, proliferation, and survival by activating ribosomal kinases. Recent studies have implicated the mTOR signaling pathway in ovarian neoplasms, polycystic ovary syndrome (PCOS) and premature ovarian failure (POF). Preclinical investigations have demonstrated that the PI3K/AKT/mTOR pathway is frequently activated in the control of various ovarian functions. mTOR allows cancer cells to escape the normal biochemical system and regulates the balance between apoptosis and survival. Some recent studies have suggested that involvement of the mTOR signaling system is an important pathophysiological basis of PCOS. Overexpression of the mTOR pathway can impair the interaction of cumulus cells, lead to insulin resistance, and affect the growth of follicles directly. The roles of mTOR signaling in follicular development have been extensively studied in recent years; abnormalities in this process lead to a series of pathologies such as POF and infertility. To improve understanding of the role of the mTOR signaling pathway in the pathogenesis and development of ovarian diseases, here we review the roles of mTOR signaling in such diseases and discuss the corresponding therapeutic strategies that target this pathway. Clin. Anat. 31:891-898, 2018. © 2018 Wiley Periodicals, Inc.

Enhanced rapamycin delivery to hemangiomas by lipid polymer nanoparticles coupled with anti-VEGFR antibody.

The most common tumors in children are infantile hemangiomas which could cause morbidity and severe complications. The development of novel alternative drugs to treat infantile hemangiomas is necessary, since Hemangeol is the only US Food and Drug Administration-approved drug for infantile hemangiomas. However, Hemangeol has several disadvantages, including a high frequency of administration and adverse effects. Rapamycin is a well­established antiangiogenic drug, and we have previously developed rapamycin lipid polymer nanoparticles (R­PLNPs) as a local sustained­release drug delivery system to achieve controlled rapamycin release and to decrease the frequency of administration and side effects of rapamycin. To improve the targeting of R­PLNPs to infantile hemangiomas in the present study, R­PLNPs were modified to include an antibody against vascular endothelial growth factor receptor (VEGF). The characteristics, and the anti­hemangioma activity of the resulting R­PLNPs coupled with the anti­VEGFR2 antibody (named R­PLNPs­V) were examined in vitro and in vivo. R­PLNPs­V possessed a small size (115 nm) and sustained drug release for 6 days. The anti­VEGFR2 antibody promoted the targeting and cytotoxic effect of R­PLNPs­V to human hemangioma endothelial cells and human umbilical vein endothelial cells. Using a subcutaneous infantile hemangioma xenograft in mice, the in vivo therapeutic effect (evaluated with hemangioma weight, volume, and microvessel density) of R­PLNPs­V was demonstrated to be superior compared with rapamycin alone and other non­targeted nanoparticles, without any total body weight loss. In summary, R­PLNPs­V could facilitate targeted delivery and sustained release of rapamycin to infantile hemangiomas, and thus may represent a promising candidate treatment for infantile hemangiomas.

Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo.

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

Induced prodrug activation by conditional protein degradation.

Enzyme prodrug therapies hold potential as a targeted treatment option for cancer patients. However, off-target effects can be detrimental to patient health and represent a safety concern. This concern can be alleviated by including a failsafe mechanism that can abort the therapy in healthy cells. This feature can be included in enzyme prodrug therapies by use of conditional degradation tags, which degrade the protein unless stabilized. We call this process Degradation-Directed Enzyme Prodrug Therapy (DDEPT). Herein, we use traceless shielding (TShld), a mechanism that degrades a protein of interest unless it is rescued by the addition of rapamycin, to test this concept. We demonstrated that TShld rapidly yielded only native protein products within 1h after rapamycin addition. The rapid protection phenotype of TShld was further adapted to rescue yeast cytosine deaminase, a prodrug converting enzyme. As expected, cell viability was adversely affected only in the presence of both 5-fluorocytosine (5-FC) and rapamycin. We believe that the DDEPT system can be easily combined with other targeting strategies to further increase the safety of prodrug therapies.

Association of NR1I2, CYP3A5 and ABCB1 genetic polymorphisms with variability of temsirolimus pharmacokinetics and toxicity in patients with metastatic bladder cancer.

PURPOSE: Temsirolimus is a mammalian target of rapamycin (mTOR) inhibitor that exhibits antitumor activity in renal cell carcinoma and mantle cell lymphoma. The metabolism of temsirolimus and its active metabolite sirolimus mainly depends on cytochrome P450 3A4/5 (CYP3A4/A5) and the ABCB1 transporter. Differently from sirolimus, no pharmacogenetic study on temsirolimus has been conducted. Therefore, the aim of this pilot study was to identify genetic determinants of the inter-individual variability in temsirolimus pharmacokinetics and toxicity. METHODS: Pharmacokinetic profiles were obtained for 16 patients with bladder cancer after intravenous infusion of 25 mg temsirolimus. Non-compartmental analysis was performed to calculate the pharmacokinetic parameters of temsirolimus and sirolimus, its main metabolite. The presence of single nucleotide polymorphisms (SNPs) in CYP3A5, ABCB1 and in their transcriptional regulator NR1I2 (PXR) was assessed by genotyping. Non-parametric statistical tests were used to assess associations between candidate SNPs and temsirolimus pharmacokinetics and toxicity. RESULTS: The ratio between sirolimus AUC and temsirolimus AUC was 1.6-fold higher in patients who experienced serious toxic events (p = 0.034). The frequency of adverse events was significantly higher in patients homozygous for the NR1I2-rs6785049 A allele (OR = 0.065, p = 0.04) or NR1I2-rs3814055 C allele (OR = 0.032, p = 0.006). These NR1I2 SNPs were also predictive of temsirolimus half-life and global exposure to temsirolimus and sirolimus. Finally, the effect of the ABCB1-rs1128503, ABCB1-rs2032582 and CYP3A5*3 SNPs on sirolimus pharmacokinetics was confirmed. CONCLUSIONS: Our findings suggest that SNPs of NR1I2 and its target genes CYP3A5 and ABCB1 are genetic determinants of temsirolimus pharmacokinetics and toxicity in patients with bladder cancer.