Nanomedicines of Hedgehog inhibitor and PPAR-γ agonist for treating liver fibrosis.

PURPOSE: Hedgehog (Hh) and peroxisome proliferator-activated receptor gamma (PPAR-γ) are major signaling pathways involved in the pathogenesis of liver fibrosis. Since Hh inhibitor, vismodegib (GDC) and PPAR-γ agonist, rosiglitazone (RSG) have poor water solubility, our objective was to formulate biodegradable polymeric nanoparticles encapsulating GDC and RSG for treating liver fibrosis. METHODS: Methoxy-polyethylene-glycol-b-poly(carbonate-co-lactide) [mPEG-b-p(CB-co-LA)] was synthesized and characterized using (1)H NMR. Nanoparticles were prepared using this polymer by emulsification/solvent evaporation method to encapsulate GDC and RSG either alone or in combination. Nanoparticles were characterized for particle size, drug loading, drug release, and anti-fibrotic efficacy after tail vein injection into common bile duct ligated (CBDL) fibrotic rats. RESULTS: mPEG-b-p(CB-co-LA) copolymer has molecular weight of 30,000 Da as determined by (1)H NMR. Nanoparticles were monodisperse with a mean particle size of 120-130 nm. Drug loading was 5% and 2% w/w for GDC and RSG, respectively. Nanoparticles carrying both GDC and RSG were formulated at half of their individual drug loading. Systemic administration of drug loaded nanoparticles protected liver injury in CBDL rats by suppressing the activation of hepatic stellate cells, and decreasing inflammatory cytokines. CONCLUSION: Polymeric nanoparticles for co-delivery of Hh inhibitor and PPAR-γ agonist have the potential to treat liver fibrosis by intervening complex fibrotic cascade.

Mesenchymal stem cell-based therapy.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells which have self-renewal capacity and differentiation potential into several mesenchymal lineages including bones, cartilages, adipose tissues and tendons. MSCs may repair tissue injuries and prevent immune cell activation and proliferation. Immunomodulation and secretion of growth factors by MSCs have led to realizing the true potential of MSC-based cell therapy. The use of MSCs as immunomodulators has been explored in cell/organ transplant, tissue repair, autoimmune diseases, and prevention of graft vs host disease (GVHD). This review focuses on the clinical applications of MSC-based cell therapy, with particular emphasis on islet transplantation for treating type I diabetes.

A phase 1, open-label, randomized drug-drug interaction study of zanubrutinib with moderate or strong CYP3A inhibitors in patients with B-cell malignancies.

BTK inhibitor exposure increases significantly when coadministered with CYP3A inhibitors, which may lead to dose-related toxicities. This study explored the pharmacokinetics, efficacy, and safety of zanubrutinib when coadministered with moderate or strong CYP3A inhibitors in 26 patients with relapsed or refractory B-cell malignancies. Coadministration of zanubrutinib (80 mg BID) with moderate CYP3A inhibitors fluconazole and diltiazem or zanubrutinib (80 mg QD) with strong CYP3A inhibitor voriconazole resulted in comparable exposures to zanubrutinib (320 mg QD) with AUC0-24h geometric least squares mean ratios approaching 1 (0.94, 0.81, and 0.83, for fluconazole, diltiazem, and voriconazole, respectively). The most common treatment-emergent adverse events were contusion (26.9%), back pain (19.2%), constipation and neutropenia (15.4% each), and rash, diarrhea, and fall (11.5% each). This study supports current United States Prescribing Information dose recommendations for the coadministration of reduced-dose zanubrutinib with moderate or strong CYP3A inhibitors and confirms the favorable efficacy and safety profile of zanubrutinib.

Formulation and characterization of polyester/polycarbonate nanoparticles for delivery of a novel microtubule destabilizing agent.

PURPOSE: Since our newly synthesized potent 5-indolyl derivative, (2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293), to treat resistant melanoma was hydrophobic, our objective was to synthesize a biodegradable copolymer for formulating this drug into nanoparticles and to determine its anticancer activity and mechanism of action. METHODS: Methoxy poly (ethylene glycol)-b-poly (carbonate-co-lactide) [mPEG-b-P (CB-co-LA)] was synthesized for formulating LY293 into nanoparticles by o/w emulsification and stabilization by solvent evaporation. Particle size, drug release profile, in vitro efficacy in multiple melanoma cells, and mechanism of action of drug-loaded nanoparticles were determined. RESULTS: LY293-loaded nanoparticles with 170 nm mean size and 2.2 and 4.16% drug loading efficiently inhibited proliferation of A375 and B16F10 cells with IC(50) of 12.5 nM and 25 nM, respectively. LY293 circumvented multidrug resistance and inhibited proliferation of Pgp overexpressing MDA-MB435/LCC6 MDR1 melanoma cells. Upon treatment with LY293-loaded nanoparticles, A375 cells underwent cell cycle arrest in G2/M phase and apoptotic cell death. Immunofluorescence images showed inhibition of tubulin polymerization after treatment with LY293. CONCLUSION: LY293-loaded mPEG-b-P (CB-co-LA) nanoparticles showed excellent efficacy and induced apoptosis in melanoma cells. These polyester/polycarbonate-based nanoparticles provided an excellent platform to deliver different poorly soluble drugs to melanoma.

Pharmacokinetics and biodistribution of polymeric micelles containing miRNA and small-molecule drug in orthotopic pancreatic tumor-bearing mice.

Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation. Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment. Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC 0-∞ ) by 7-fold, while t1/2,e and AUC 0-∞ of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation. Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.

Polymer conjugate of a microtubule destabilizer inhibits lung metastatic melanoma.

Melanoma is the most aggressive type of skin cancer. It is highly metastatic, migrating through lymph nodes to distant sites of the body, especially to lungs, liver and brain. Systemic chemotherapy remains the mainstay of treatment; however, the development of multidrug resistance (MDR) restricts the efficacy of current chemotherapeutic drugs. We synthesized a series of microtubule destabilizers, substituted methoxybenzoyl-ary-thiazole (SMART) compounds, which inhibited tubulin polymerization and effectively circumvented MDR. Due to poor water solubility of SMART compounds, co-solvent delivery is required for their systemic administration, which is usually associated with hepatotoxicity, nephrotoxicity and hemolysis. To solve this problem and also to increase circulation time, we synthesized a new SMART analogue, SMART-OH, and its polymer-drug conjugate, methoxy-poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate-graft-SMART-graft-dodecanol) (abbreviated as P-SMART), with 14.3±2.8% drug payload of SMART-OH. Similar to its parent drug, P-SMART showed significant anticancer activity against melanoma cells in cytotoxicity, colony formation, and cell invasion studies. In addition, P-SMART treatment led to cell cycle arrest at G2/M phase and cell accumulation in sub-G1 phase. We established a model of metastatic melanoma to the lung in C57/BL6 albino mice to determine in vivo efficacy of P-SMART and SMART-OH at the dose of 20mg/kg. P-SMART treatment resulted in significant inhibition of tumor growth and prolonged mouse median survival. In conclusion, P-SMART, a novel polymer-microtubule destabilizer conjugate, has the potential to treat metastatic melanoma.

Nanoparticle-mediated drug delivery for treating melanoma.

Melanoma originated from melanocytes is the most aggressive type of skin cancer with limited treatment options. New targeted therapeutic options with the discovery of BRAF and MEK inhibitors have shown significant survival benefits. Despite the recent progress, development of chemoresistance and systemic toxicity remains a challenge for treating metastatic melanoma. While the response from the first line of treatment against melanoma using dacarbazine remains only 5-10%, the prolonged use of targeted therapy against mutated oncogene BRAF develops chemoresistance. In this review, we will discuss the nanoparticle-based strategies for encapsulation and conjugation of drugs to the polymer for maximizing their tumor distribution through enhanced permeability and retention effect. We will also highlight photodynamic therapy and design of melanoma-targeted nanoparticles.

Micellar formulation of indocyanine green for phototherapy of melanoma.

Phototherapy (PT), a light activated treatment modality, is a potential therapeutic option for the treatment of melanoma. In spite of the excellent safety profile and absorption in the near infrared (NIR) range, clinical potential of indocyanine green (ICG) as PT is limited by its short half-life and inefficient tumor accumulation. In this study, we have covalently conjugated ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling, which self-assembled into micelles with a particle size of 30-50 nm and high ICG loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiate on at 4 °C revealed photodynamic and photothermal as mechanism of cytotoxicity of ICG solution and ICG conjugated micelles, respectively. In vivo NIR imaging demonstrated that ICG conjugated micelles prolonged its circulation and increased tumor accumulation through enhanced permeability and retention (EPR) effect. Enhanced tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to ICG solution and control in a A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for PT and imaging of melanoma.

Systemic delivery of nanoparticle formulation of novel tubulin inhibitor for treating metastatic melanoma.

Clinical translation of tubulin inhibitors for treating melanoma is limited by multidrug efflux transporters, poor aqueous solubility, and dose-limiting peripheral toxicities. Tubulin inhibitors with efficacy in taxane-resistant cancers are promising drug candidates and can be used as single agent or in conjunction with other chemotherapy. Systemic therapy of such a novel tubulin inhibitor, 2-(1H-indol-5-yl)thiazol-4-yl)3,4,5-trimethoxyphenyl methanone (abbreviated as LY293), is limited by its poor aqueous solubility. The objective of this study was to design a polymeric nanocarrier for systemic administration of LY293 to improve tumor accumulation and reduce side effects of tubulin inhibitor in a lung metastasis melanoma mouse model. Methoxy polyethylene glycol-b-poly(carbonate-co-lactide) (mPEG-b-P(CB-co-LA)) random copolymer was synthesized and characterized by ¹H NMR and gel permeation chromatography (GPC). Polymeric nanoparticles were formulated using oil/water (o/w) emulsification method with a mean particle size of 150 nm and loading efficiency of 7.40%. Treatment with LY293-loaded nanoparticles effectively inhibited the proliferation of melanoma cells in vitro and exhibited concentration-dependent cell cycle arrest in G2/M phase. Mitotic arrest activated the intrinsic apoptotic machinery by increasing the cellular levels of cleaved poly ADP ribose polymerase (PARP) and fraction of sub-G1 cells. In vivo, LY293-loaded nanoparticles significantly inhibited the proliferation of highly aggressive metastasized melanoma in a syngeneic lung metastasis melanoma mouse model without toxicity to vital organs. In conclusion, we have designed a promising polymeric nanocarrier for systemic delivery of LY293 for treating metastatic melanoma while minimizing the toxicity associated with the administration of cosolvents.

Genetically modified human bone marrow derived mesenchymal stem cells for improving the outcome of human islet transplantation.

The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl) /SzJ (NSG) diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF). hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet transplantation.

Attenuation of early liver fibrosis by pharmacological inhibition of smoothened receptor signaling.

Hedgehog (Hh) signaling is involved in the pathogenesis of liver fibrosis. It has been previously shown that Hh-inhibitor cyclopamine (CYA) can reduce liver fibrosis in rats. However, CYA is not stable in vivo, which limits its clinical application. This study compares the antifibrotic potential of two known Hh antagonists, vismodegib (GDC-0449, abbreviated to GDC) and CYA. GDC is a synthetic molecule presently in clinical cancer trials and has been reported to be safe and efficacious. These drugs attenuated early liver fibrosis in common bile duct ligated rats, improved liver function, and prevented hepatic stellate cell (HSC) activation, thereby suppressing epithelial to mesenchymal transition (EMT). While both CYA and GDC increased the number of proliferating cell nuclear antigen positive liver cells in vivo, only CYA increased Caspase-3 expression in HSCs in rat livers, suggesting that while GDC and CYA effectively attenuate early liver fibrosis, their hepatoprotective effects may be mediated through different modes of action. Thus, GDC has the potential to serve as a new therapeutic agent for treating early liver fibrosis.