Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

Short-term effects of atropine combined with orthokeratology (ACO) on choroidal thickness.

PURPOSE: To analyse the one-month change in subfoveal choroidal thickness (SFChT) of myopic children treated with 0.01 % atropine, orthokeratology (OK), or their combination. METHODS: This is a prospective, randomized controlled trial. One hundred fifty-four children aged between 8 and 12 years with a spherical equivalent (SE) of -1.00 to -6.00 diopters were enrolled. Subjects were randomly assigned to receive 0.01 % atropine and orthokeratology (ACO, n = 39), 0.01 % atropine and single vision glasses (atropine, n = 42), orthokeratology and placebo (OK, n = 36), or placebo and single vision glasses (control, n = 37). SFChT was assessed using optical coherence tomography (OCT). Ocular parameters, including axial length (AL), were measured using a Lenstar LS 900. RESULTS: SFChT significantly increased in the ACO (14.12 ±â€¯12.88 µm, p < 0.001), OK (9.43 ±â€¯9.14 µm, p < 0.001) and atropine (5.49 ±â€¯9.38 µm, p < 0.001) groups, while it significantly decreased in the control group (-4.81 ±â€¯9.93 µm, p = 0.006). The one-month change in SFChT was significantly different between the control and treatment groups (p < 0.001). The results of pairwise comparisons among the treatment groups showed that the magnitude of the SFChT change was larger in the ACO group than in the atropine group (p = 0.002). The changes in the ACO and OK groups were not significantly different (p = 0.326). CONCLUSION: The combination of OK and atropine induced a greater increase in SFChT than monotherapy with atropine, which might indicate a better treatment effect for childhood myopia control.

Axl receptor tyrosine kinase is a regulator of apolipoprotein E.

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.

Pregnancy Alters CYP- and UGT-Mediated Metabolism of Buprenorphine.

BACKGROUND: In the United States, drug addiction has become a nationwide health crisis. Recently, buprenorphine (BUP), a maintenance therapy approved by the Food and Drug Administration, has been increasingly used in pregnant women for the treatment of opioid use disorder. Pregnancy is associated with various anatomic and physiological changes, which may result in altered drug pharmacokinetics (PKs). Previously, we reported that dose-adjusted plasma concentrations of BUP are lower during pregnancy than after pregnancy. The mechanism(s) responsible for this difference has not yet been defined. Our study aimed to evaluate alterations in cytochromes P450 (CYP)- and uridine diphosphate glucunosyltransferases (UGT)-mediated metabolism of BUP during pregnancy to determine the mechanism(s) responsible for this observation. METHODS: Data from 2 clinical studies were included in the current analysis. Study 1 was a prospective, open-labeled, nonrandomized longitudinal BUP PK study in pregnant women with a singleton gestation, stabilized on twice-daily sublingual BUP opioid substitution therapy. Each subject participated in up to 3 studies during and after pregnancy (the second, third trimester, and postpartum). The design of study 2 was similar to study 1, with patients evaluated at different time points during the pregnancy (first, second-half of pregnancy), as well as during the postpartum period. In addition, the dosing frequency of BUP study 2 participants was not restricted to twice-daily dosing. At each study visit, blood samples were collected before a BUP dose, followed by multiple collection times (10-12) after the dose, for up to 12 hours or till the end of the dosing interval. Plasma concentrations of BUP and 3 metabolites were quantified using validated ultraperformance liquid chromatography-tandem mass spectrometric assays. RESULTS: In total, 19, 18, and 14 subjects completed the PK study during 1/2 trimester, third trimester, and postpartum, respectively. The AUC ratios of norbuprenorphine and norbuprenorphine glucuronide to buprenorphine, a measure of CYP3A mediated N-demethylation, were 1.89, 1.84, and 1.33 during the first and second, third trimesters, and postpartum, respectively. The AUC ratios of buprenorphine glucuronide to BUP, indicative of UGT activity, were 0.71, 2.07, and 0.3 at first/second trimesters, third trimester, and postpartum, respectively. Linear mixed-effect modeling analysis indicated that the AUC ratios of CYP- and UGT-mediated metabolism of BUP were significantly higher during pregnancy compared with postpartum. CONCLUSIONS: The CYP and UGT activities were significantly increased as determined by the metabolic ratios of BUP during pregnancy compared with the postpartum period. The increased UGT activity appeared to account for a substantial part of the observed change in metabolic activity during pregnancy. This is in agreement with the need for BUP dose increment in pregnant women to reach similar BUP exposure and therapeutic effect as in nonpregnant subjects.

Creatine based polymer for codelivery of bioengineered MicroRNA and chemodrugs against breast cancer lung metastasis.

Metastasis is the major cause for breast cancer related mortality. The combination of miRNA-based therapy and chemotherapy represents a promising approach against breast cancer lung metastasis. The goal of this study is to develop an improved therapy that co-delivers a novel bioengineered miRNA prodrug (tRNA-mir-34a) and doxorubicin (DOX) via a multifunctional nanomicellar carrier that is based on a conjugate of amphiphilic copolymer POEG-VBC backbone with creatine, a naturally occurring cationic molecule. Co-delivery of DOX leads to more effective processing of tRNA-mir-34a into mature miR-34a and down-regulation of target genes. DOX + tRNA-mir-34a/POEG-PCre exhibits potent synergistic anti-tumor and anti-metastasis activity in vitro and in vivo. Interestingly, the enhanced immune response contributes to the overall antitumor efficacy. POEG-PCre may represent a safe and effective delivery system for an optimal chemo-gene combination therapy.

Pharmacokinetics of Intravenous Isavuconazole in Solid-Organ Transplant Recipients.

Isavuconazole may be useful in treating and preventing fungal infections in solid-organ transplant (SOT) recipients due to its safety profile and activity against Aspergillus and some Mucorales Isavuconazole has favorable pharmacokinetics based on clinical trials in various patient populations, but data are limited in SOT recipients. We evaluated the steady-state pharmacokinetics of isavuconazole in 26 SOT recipients receiving the drug intravenously for prophylaxis. There was moderate interpatient variability in isavuconazole pharmacokinetic parameters (coefficients of variation of 51% for the area under the plasma concentration-versus-time curve [AUC] and 59% for the trough plasma concentration [Ctrough]). AUC and steady-state Ctrough were significantly lower in women, patients with a body mass index of ≥18.5 kg/m2, and those receiving hemodialysis. Trough plasma concentrations were highly correlated with AUCs (R2 = 0.94) and can serve as a suitable measure of isavuconazole exposure in patients. In conclusion, moderate interpatient variability in isavuconazole exposure, the identification of factors associated with lower exposure, the recognition that Ctrough is a surrogate marker for AUC, and the availability of a simple analytical method suggest that therapeutic drug monitoring (TDM) may be useful for guiding treatment in at least some SOT recipients. Future studies are needed to correlate isavuconazole exposure with patients' clinical outcomes and to determine the clinical role of TDM.

Mycophenolic Acid for Topical Immunosuppression in Vascularized Composite Allotransplantation: Optimizing Formulation and Preliminary Evaluation of Bioavailability and Pharmacokinetics.

Mycophenolic acid (MPA), is the active form of the ester prodrug mycophenolate mofetil (MMF). MMF is an FDA approved immunosuppressive drug that has been successfully used in systemic therapy in combination with other agents for the prevention of acute rejection (AR) following solid organ transplantation (SOT) as well as in vascularized composite allotransplantation (VCA). Systemic use of MMF is associated with gastrointestinal adverse effects. Topical delivery of the prodrug could thus provide graft-targeted immunosuppression while minimizing systemic drug exposure. Our goal was to develop a topical formulation of MPA with optimal in vitro/in vivo characteristics such as release, permeation, and tissue bioavailability to enable safety and efficacy evaluation in clinical VCA. Permeation studies were performed with a solution of MPA (10 mg/ml). In vitro release and permeation studies were performed for different semisolid formulations (Aladerm, Lipoderm, emollient, and VersaBase) of MPA (1% w/w) using a Franz Diffusion Cell System (FDCS). In vivo pharmacokinetic characterization of MPA release from Lipoderm was performed in rats. MPA in solution exhibited a steady state flux (3.8 ± 0.1 µg/cm2/h) and permeability (1.1 × 10-7 ± 3.2 × 10-9 cm/s). MPA in Lipoderm exhibited a steady state flux of 1.12 ± 0.24 µg/cm2/h, and permeability of 6.2 × 10-09 ± 1.3 × 10-9 cm/s across the biomimetic membrane. The cumulative release of MPA from Lipoderm, showed a linear single-phase profile with a R2 of 0.969. In vivo studies with MPA in Lipoderm showed markedly higher local tissue MPA levels and lower systemic MPA exposure as compared to values obtained after intravenous delivery of the same dose of drug (p < 0.05). We successfully developed for the first time, a topical formulation of MPA in Lipoderm with optimal in vitro/in vivo permeability characteristics and no undesirable local or systemic adverse effects in vivo. Our study provides key preliminary groundwork for translational efficacy studies of topical MPA in pre-clinical large animal VCA models and for effectiveness evaluation in patients receiving VCA.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway.

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

Doxorubicin delivered by a redox-responsive dasatinib-containing polymeric prodrug carrier for combination therapy.

Two novel prodrug polymers POEG-b-PSSDas (redox-sensitive) and POEG-b-PCCDas (redox-insensitive), which consist of poly(oligo(ethylene glycol) methacrylate) (POEG) hydrophilic blocks and dasatinib (DAS, an oncogenic tyrosine kinases inhibitor) conjugated hydrophobic blocks, were designed as dual-functional carriers for codelivery with doxorubicin (DOX). Both carriers retained antitumor activity of DAS and could form mixed micelles with DOX. Compared to POEG-b-PCCDas micelles, incorporation of disulfide linkage into POEG-b-PSSDas micelles facilitated efficient cleavage of DAS from prodrug micelles in tumor cells/tissues, leading to a higher level of anti-tumor activity in vitro and in vivo. In addition, DOX-loaded POEG-b-PSSDas micelles exhibited triggered DOX release under a redox environment (10mM glutathione, GSH), and demonstrated enhanced cytotoxicity against 4T1.2 and PC3 cell lines compared to DOX and DOX-loaded POEG-b-PCCDas micelles. More importantly, DOX-loaded POEG-b-PSSDas micelles were more effective in inhibiting the tumor growth and prolonging the survival rate in an aggressive murine breast cancer model (4T1.2) compared to DOX-loaded POEG-b-PCCDas micelles and a micellar formulation co-loaded with DOX and DAS. This redox-responsive prodrug micellar system provides an attractive strategy for effective combination of tumor targeted therapy and traditional chemotherapy, which warrants further investigation.

Programmable co-delivery of the immune checkpoint inhibitor NLG919 and chemotherapeutic doxorubicin via a redox-responsive immunostimulatory polymeric prodrug carrier.

To achieve synergistic therapeutic efficacy and prevent cancer relapse, chemotherapy and immunotherapy have been combined as a new modality for tumor treatment. In this work, we designed a redox-responsive immunostimulatory polymeric prodrug carrier, PSSN10, for programmable co-delivery of an immune checkpoint inhibitor NLG919 (NLG) and a chemotherapeutic doxorubicin (DOX). NLG-containing PSSN10 prodrug polymers were self-assembled into nano-sized micelles that served as a carrier to load DOX (DOX/PSSN10 micelles). DOX/PSSN10 micelles displayed spherical morphology with a size of ∼170 nm. DOX was effectively loaded into PSSN10 micelles with a loading efficiency of 84.0%. In vitro DOX release studies showed that rapid drug release could be achieved in the highly redox environment after intracellular uptake by tumor cells. In 4T1.2 tumor-bearing mice, DOX/PSSN10 micelles exhibited greater accumulation of DOX and NLG in the tumor tissues compared with other organs. The PSSN10 carrier dose-dependently enhanced T-cell immune responses in the lymphocyte-Panc02 co-culture experiments, and significantly inhibited tumor growth in vivo. DOX/PSSN10 micelles showed potent cytotoxicity in vitro against 4T1.2 mouse breast cancer cells and PC-3 human prostate cancer cells comparable to that of DOX. In 4T1.2 tumor-bearing mice, DOX/PSSN10 mixed micelles (5 mg DOX/kg, iv) was more effective than DOXIL (a clinical formulation of liposomal DOX) or free DOX in inhibiting the tumor growth and prolonging the survival of the treated mice. In addition, a more immunoactive tumor microenvironment was observed in the mice treated with PSSN10 or DOX/PSSN10 micelles compared with the other treatment groups. In conclusion, systemic delivery of DOX via PSSN10 nanocarrier results in synergistic anti-tumor activity.

An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy.

Immunochemotherapy combines a chemotherapeutic agent with an immune-modulating agent and represents an attractive approach to improve cancer therapy. However, the success of immunochemotherapy is hampered by the lack of a strategy to effectively co-deliver the two therapeutics to the tumours. Here we report the development of a dual-functional, immunostimulatory nanomicellar carrier that is based on a prodrug conjugate of PEG with NLG919, an indoleamine 2,3-dioxygenase (IDO) inhibitor currently used for reversing tumour immune suppression. An Fmoc group, an effective drug-interactive motif, is also introduced into the carrier to improve the drug loading capacity and formulation stability. We show that PEG2k-Fmoc-NLG alone is effective in enhancing T-cell immune responses and exhibits significant antitumour activity in vivo. More importantly, systemic delivery of paclitaxel (PTX) using the PEG2k-Fmoc-NLG nanocarrier leads to a significantly improved antitumour response in both breast cancer and melanoma mouse models.

Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes.

The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer's Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 µM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRß, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 µM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists.

A prodrug micellar carrier assembled from polymers with pendant farnesyl thiosalicylic acid moieties for improved delivery of paclitaxel.

UNLABELLED: In order to achieve enhanced and synergistic delivery of paclitaxel (PTX), a hydrophobic anticancer agent, two novel prodrug copolymers, POEG15-b-PFTS6 and POEG15-b-PFTS16 composed of hydrophilic poly(oligo(ethylene glycol) methacrylate) (POEG) and hydrophobic farnesylthiosalicylate (FTS, a nontoxic Ras antagonist) blocks, were synthesized. Both POEG-b-PFTS polymers were able to form micelles with intrinsic antitumor activity in vitro and in vivo. Employing these micelles as a carrier to load PTX, their drug loading capacity, stability, in vivo biodistribution and tumor inhibition effect were evaluated. PTX/POEG15-b-PFTS16 mixed micelles exhibited an excellent stability of 9days at 4°C with a PTX loading capacity of 8.2%, which was more effective than PTX/POEG15-b-PFTS6 mixed micelles. In vivo biodistribution data showed that DiR-loaded POEG-b-PFTS micelles were more effectively localized in the tumor than in other organs. Moreover, both PTX/POEG-b-PFTS micelles showed significantly higher antitumor activity than Taxol in a 4T1.2 murine breast tumor model, and the tumor inhibition and animal survival followed the order of PTX/POEG15-b-PFTS16>PTX/POEG15-b-PFTS6>POEG15-b-PFTS16>Taxol≈POEG15-b-PFTS6. Our data suggest that POEG-b-PFTS micelles are a promising anticancer drug carrier that warrants more studies in the future. STATEMENT OF SIGNIFICANCE: Polymerization of drug-based monomer represents a facile and precise method to obtain well-defined polymeric prodrug amphiphiles. Currently, most reports largely focus on the synthesis methods and the biophysical properties. There is limited information about their anti-tumor activity and delivery function as prodrug carriers in vitro and in vivo. In this manuscript, we report the development of two novel prodrug copolymers, POEG15-b-PFTS6 and POEG15-b-PFTS16 composed of hydrophilic poly(oligo(ethylene glycol) methacrylate) (POEG) and hydrophobic farnesylthiosalicylate (FTS, a nontoxic Ras antagonist) blocks. Both POEG-b-PFTS polymers were able to self-assemble into nano-sized micelles with intrinsic antitumor activity in vitro and in vivo. More importantly, POEG-b-PFTS polymers were effective in forming stable mixed micelles with various anticancer agents including PTX, DOX, docetaxel, gefitinib, and imatinib. Delivery of PTX via our new carrier led to significantly improved antitumor activity, suggesting effective PTX/FTS combination therapy. We believe that our study shall be of broad interest to the readers in the fields of biomaterials and drug delivery.

Pharmacokinetics of Posaconazole Suspension in Lung Transplant Patients with and without Cystic Fibrosis.

Invasive fungal infections (IFIs) are common among lung transplant recipients (LTRs). Posaconazole is an important antifungal agent for both prophylaxis and treatment of IFIs; however, detailed pharmacokinetic data are limited among LTRs, particularly those with cystic fibrosis (CF). Our objective was to conduct a pharmacokinetic study of posaconazole oral suspension among LTRs, with particular attention to patients with CF. We enrolled 20 LTRs, 7 with CF and 13 with other underlying lung diseases. Average daily doses in CF and non-CF patients were 829 and 862 mg, respectively. After ≥5 days of treatment, only 4 patients had average plasma concentrations of >0.7 µg/ml. Average steady-state plasma concentrations were 61% lower in CF patients (0.233 µg/ml) than in non-CF LTRs (0.594 µg/ml; P = 0.03). The average dose-normalized plasma area-under-the-curve (AUC) values were also lower in CF (0.007 h·µg/ml) than in non-CF LTRs (0.02 h·µg/ml; P = 0.02). The weight-normalized apparent oral clearance values were 2.51 and 0.74 liters/h/kg among CF and non-CF LTRs, respectively (P = 0.005). Despite significant interpatient variability, plasma trough concentrations were strongly correlated with posaconazole AUC across all LTRs (r(2) = 0.95, P < 0.0001). Taken together, our study highlights a critical need to incorporate new formulations of posaconazole into prophylaxis and treatment strategies for LTRs, particularly those with CF. Future pharmacokinetic studies of both tablet and intravenous formulations must consider LTR-specific factors and incorporate a therapeutic drug monitoring plan in this patient population.

Effective co-delivery of doxorubicin and dasatinib using a PEG-Fmoc nanocarrier for combination cancer chemotherapy.

A simple PEGylated peptidic nanocarrier, PEG5000-lysyl-(α-Fmoc-ε-Cbz-lysine)2 (PLFCL), was developed for effective co-delivery of doxorubicin (DOX) and dasatinib (DAS) for combination chemotherapy. Significant synergy of DOX and DAS in inhibition of cancer cell proliferation was demonstrated in various types of cancer cells, including breast, prostate, and colon cancers. Co-encapsulation of the two agents was facilitated by incorporation of 9-Fluorenylmethoxycarbonyl (Fmoc) and carboxybenzyl (Cbz) groups into a nanocarrier for effective carrier-drug interactions. Spherical nanomicelles with a small size of ∼30 nm were self-assembled by PLFCL. Strong carrier/drug intermolecular π-π stacking was demonstrated in fluorescence quenching and UV absorption. Fluorescence study showed more effective accumulation of DOX in nuclei of cancer cells following treatment with DOX&DAS/PLFCL in comparison with cells treated with DOX/PLFCL. DOX&DAS/PLFCL micelles were also more effective than other treatments in inhibiting the proliferation and migration of cultured cancer cells. Finally, a superior anti-tumor activity was demonstrated with DOX&DAS/PLFCL. A tumor growth inhibition rate of 95% was achieved at a respective dose of 5 mg/kg for DOX and DAS in a murine breast cancer model. Our nanocarrier may represent a simple and effective system that could facilitate clinical translation of this promising multi-agent regimen in combination chemotherapy.

An improved D-α-tocopherol-based nanocarrier for targeted delivery of doxorubicin with reversal of multidrug resistance.

Nanocarriers have recently emerged as an attractive platform for the delivery of various types of therapeutics including anticancer agents. Previously, we developed an improved TPGS delivery system (PEG5K-VE2) which demonstrated improved colloidal stability and greater in vivo antitumor activity. Nevertheless, the application of this system is still limited by a relatively low drug loading capacity (DLC). In this study we report that incorporation of a fluorenylmethyloxycarbonyl (Fmoc) motif at the interfacial region of PEG5K-VE2 led to significant improvement of the system through the introduction of an additional mechanism of drug/carrier interaction. Doxorubicin (DOX) could be effectively loaded into PEG5K-Fmoc-VE2 micelles at a DLC of 39.9%, which compares favorably to most reported DOX nanoformulations. In addition, PEG5K-Fmoc-VE2/DOX mixed micelles showed more sustained release of DOX in comparison to the counterpart without Fmoc motif. MTT assay showed that PEG5K-Fmoc-VE2/DOX exerted significantly higher levels of cytotoxicity over DOX, Doxil as well as PEG5K-VE2/DOX in PC-3 and 4T1.2 cells. A cytotoxicity assay with NCI/ADR-RES, a drug resistant cell line, suggested that PEG5K-Fmoc-VE2 may have the potential to reverse multidrug resistance, which was supported by its inhibition of P-gp ATPase. Pharmacokinetic (PK) and biodistribution studies showed an increased half-life in blood circulation and more effective tumor accuulation for DOX formulated in PEG5K-Fmoc-VE2 micelles. More importantly, DOX-loaded PEG5K-Fmoc-VE2 micelles showed an excellent safety profile with a MTD (~30 mgDOX/kg) that is about 3 times as much as that for free DOX. Finally, superior antitumor activity was demonstrated by PEG5K-Fmoc-VE2/DOX in both drug-sensitive (4T1.2 and PC-3) and drug-resistant (KB 8-5) tumor models compared to DOX, Doxil, and PEG5K-VE2/DOX.

Targeted delivery of anticancer agents via a dual function nanocarrier with an interfacial drug-interactive motif.

We have developed a dual-function drug carrier, polyethylene glycol (PEG)-derivatized farnesylthiosalicylate (FTS). Here we report that incorporation of a drug-interactive motif (Fmoc) into PEG5k-FTS2 led to further improvement in both drug loading capacity and formulation stability. Doxorubicin (DOX) formulated in PEG5k-Fmoc-FTS2 showed sustained release kinetics slower than those of DOX loaded in PEG5k-FTS2. The maximum tolerated dose of DOX- or paclitaxel (PTX)-loaded PEG5k-Fmoc-FTS2 was significantly higher than that of the free drug. Pharmacokinetics and biodistribution studies showed that DOX/PEG5k-Fmoc-FTS2 mixed micelles were able to retain DOX in the bloodstream for a significant amount of time and efficiently deliver the drug to tumor sites. More importantly, drug (DOX or PTX)-loaded PEG5k-Fmoc-FTS2 led to superior antitumor activity over other treatments including drugs formulated in PEG5k-FTS2 in breast cancer and prostate cancer models. Our improved dual function carrier with a built-in drug-interactive motif represents a simple and effective system for targeted delivery of anticancer agents.

Targeted delivery of Doxorubicin by folic acid-decorated dual functional nanocarrier.

Doxorubicin (DOX) is one of the most commonly used antineoplastic agents, but its clinical application is oftentimes coupled with severe side effects. Selective delivery of DOX to tumors via nanosized drug carrier represents an attractive approach to this problem. Previously, we developed a dual functional nanomicellar carrier, PEG5K-embelin2 (PEG5K-EB2), which was able to deliver paclitaxel (PTX) selectively to tumors and to achieve an enhanced therapeutic effect. In the present study, we examined the utility of PEG5K-EB2 to deliver DOX to tumors. In addition, folic acid (FA) was coupled to the surface of the PEG5K-EB2 micelles (FA-PEG5K-EB2) to further improve the selective targetability of the system. DOX-loaded PEG5K-EB2 micelles were uniformly spherical particles with a diameter of approximately 20 nm. Incorporation of FA had minimal effect on the size of the particles. The DOX loading efficiency was as high as 91.7% and 93.5% for PEG5K-EB2 and FA-PEG5K-EB2, respectively. DOX formulated in PEG5K-EB2 micelles (with or without FA decoration) demonstrated sustained kinetics of DOX release compared to free DOX. FA-PEG5K-EB2 significantly facilitated the intracellular uptake of DOX over free DOX and PEGylated liposomal DOX (Doxil) in breast cancer cells, 4T1.2, and drug resistant cells, NCI/ADR-RES. P-gp ATPase assay showed that PEG5K-EB2 significantly inhibited the function of the P-gp efflux pump. The maximum tolerated dose of DOX-loaded PEG5K-EB2 micelles was 15 mg/kg in mice, which was 1.5-fold greater than that for free DOX. Pharmacokinetics (PK) and biodistribution studies showed that both types of DOX-loaded micelles, especially FA-PEG5K-EB2, were able to significantly prolong the blood circulation time of DOX and facilitate its preferential accumulation at the tumor tissue. Finally, DOX/PEG5K-EB2 mixed micelles demonstrated significantly enhanced tumor growth inhibitory effect with minimal toxicity in comparison to free DOX and Doxil and the antitumor activity was further enhanced after the decoration by folic acid. Our data suggest that FA-PEG5K-EB2 micelles represent a promising DOX delivery system that warrants more study in the future.

Fmoc-conjugated PEG-vitamin E2 micelles for tumor-targeted delivery of paclitaxel: enhanced drug-carrier interaction and loading capacity.

The purpose of this study is to develop an improved drug delivery system for enhanced paclitaxel (PTX) loading capacity and formulation stability based on PEG5K-(vitamin E)2 (PEG5K-VE2) system. PEG5K-(fluorenylmethoxycarbonyl)-(vitamin E)2 (PEG5K-FVE2) was synthesized using lysine as the scaffold. PTX-loaded PEG5K-FVE2 micelles were prepared and characterized. Fluorescence intensity of Fmoc in the micelles was measured as an indicator of drug-carrier interaction. Cytotoxicity of the micelle formulations was tested on various tumor cell lines. The therapeutic efficacy and toxicity of PTX-loaded micelles were investigated using a syngeneic mouse model of breast cancer (4T1.2). Our data suggest that the PEG5K-FVE2 micelles have a low CMC value of 4 µg/mL and small sizes (~60 nm). The PTX loading capacity of PEG5K-FVE2 micelles was much higher than that of PEG5K-VE2 micelles. The Fmoc/PTX physical interaction was clearly demonstrated by a fluorescence quenching assay. PTX-loaded PEG5K-FVE2 micelles exerted more potent cytotoxicity than free PTX or Taxol formulation in vitro. Finally, intravenous injection of PTX-loaded PEG5K-FVE2 micelles showed superior anticancer activity compared with PEG5K-VE2 formulation with minimal toxicity in a mouse model of breast cancer. In summary, incorporation of a drug-interactive motif (Fmoc) into PEG5K-VE2 micelles represents an effective strategy to improve the micelle formulation for the delivery of PTX.

PEG-farnesyl thiosalicylic acid telodendrimer micelles as an improved formulation for targeted delivery of paclitaxel.

We have recently designed and developed a dual-functional drug carrier that is based on poly(ethylene glycol) (PEG)-derivatized farnesylthiosalicylate (FTS, a nontoxic Ras antagonist). PEG5K-FTS2 readily form micelles (20-30 nm) and hydrophobic drugs such as paclitaxel (PTX) could be effectively loaded into these micelles. PTX formulated in PEG5K-FTS2 micelles showed an antitumor activity that was more efficacious than Taxol in a syngeneic mouse model of breast cancer (4T1.2). In order to further improve our PEG-FTS micellar system, four PEG-FTS conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/FTS (1/2 vs 1/4) in the conjugates. These conjugates were characterized including CMC, drug loading capacity, stability, and their efficacy in delivery of anticancer drug PTX to tumor cells in vitro and in vivo. Our data showed that the conjugates with four FTS molecules were more effective than the conjugates with two molecules of FTS and that FTS conjugates with PEG5K were more effective than the counterparts with PEG2K in forming stable mixed micelles. PTX formulated in PEG5K-FTS4 micelles was the most effective formulation in inhibiting the tumor growth in vivo.