Targeted delivery of HSP90 inhibitors for efficient therapy of CD44-positive acute myeloid leukemia and solid tumor-colon cancer.

Heat shock protein 90 (HSP90) is overexpressed in numerous cancers, promotes the maturation of numerous oncoproteins and facilitates cancer cell growth. Certain HSP90 inhibitors have entered clinical trials. Although less than satisfactory clinical effects or insurmountable toxicity have compelled these trials to be terminated or postponed, these results of preclinical and clinical studies demonstrated that the prospects of targeting therapeutic strategies involving HSP90 inhibitors deserve enough attention. Nanoparticulate-based drug delivery systems have been generally supposed as one of the most promising formulations especially for targeting strategies. However, so far, no active targeting nano-formulations have succeeded in clinical translation, mainly due to complicated preparation, complex formulations leading to difficult industrialization, incomplete biocompatibility or nontoxicity. In this study, HSP90 and CD44-targeted A6 peptide functionalized biomimetic nanoparticles (A6-NP) was designed and various degrees of A6-modification on nanoparticles were fabricated to evaluate targeting ability and anticancer efficiency. With no excipients, the hydrophobic HSP90 inhibitor G2111 and A6-conjugated human serum albumin could self-assemble into nanoparticles with a uniform particle size of approximately 200 nm, easy fabrication, well biocompatibility and avoidance of hepatotoxicity. Besides, G2111 encapsulated in A6-NP was only released less than 5% in 12 h, which may avoid off-target cell toxicity before entering into cancer cells. A6 peptide modification could significantly enhance uptake within a short time. Moreover, A6-NP continues to exert the broad anticancer spectrum of Hsp90 inhibitors and displays remarkable targeting ability and anticancer efficacy both in hematological malignancies and solid tumors (with colon tumors as the model cancer) both in vitro and in vivo. Overall, A6-NP, as a simple, biomimetic and active dual-targeting (CD44 and HSP90) nanomedicine, displays high potential for clinical translation.

LZY3016, a novel geldanamycin derivative, inhibits tumor growth in an MDA-MB-231 xenograft model.

A novel geldanamycin derivative LZY3016 was synthesized as an antitumor agent. Compound LZY3016 exhibited potent anti-proliferation activity toward MDA-MB-231 (IC50 = 0.06 µM), which was more effective than positive drug 17-AAG. In vivo hepatotoxicity assay displayed that serum AST/ALT levels in LZY3016-treated mice were both significantly less than those in the geldanamycin (GA) group. LZY3016 showed potent antitumor activity in an MDA-MB-231 xenograft mouse model, suggesting LZY3016 is an up-and-coming antitumor candidate. The theoretical binding mode between LZY3016 and Hsp90 was obtained by molecular dynamics simulation.

Smart and Generalizable Cytarabine Derivative-Triggered Nanoparticles for Synergistic Therapy of Relapsed/Refractory Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is rapidly progressed hematologic malignancy with relapsed and refractory characteristics. Cytarabine combined with the BCL2 inhibitor venetoclax showed impressive response rates in the treatment of relapsed/refractory acute myeloid leukemia (R/R AML), while it requires complicated administration regimens and brings added toxicity. In this work, we synthesized a mercaptopropionic acid-substituted derivative of Ara-C (Ara-SH) and used it as the trigger to fabricate a smart cytarabine and venetoclax-coloaded nanoparticle (AV-NP) through self-assembly. The AV-NP characterized with redox-responsive drug release, rapid uptake by leukemia cells, and long retention in circulation had the potential to accumulate in leukemia-enriched sites. It generated a remarkable synergistic effect with higher antileukemia activity in vitro and better safety in the hematologic system compared with free drugs and significantly improved the therapeutic effect on orthotopic AML mice in vivo. These similar results were also confirmed in primary cells from R/R-AML patients. Besides, the AV-NP has the superiority of facile fabrication and generalizability, rendering it easy for clinical translation.

DT7 peptide-modified lecithin nanoparticles co-loaded with γ-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemia and reduce gastrointestinal toxicity.

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematologic malignancy and glucocorticoid resistance is the main recurrent cause for a high relapsed and death rate. Here, we proposed an effective therapeutic regimen of combining gamma-secretase inhibitors (GSIs) with dexamethasone (DEX) to overcome glucocorticoid resistance. Moreover, the bone marrow targeting DT7 peptide-modified lecithin nanoparticles co-loaded with DEX and GSI (TLnp/D&G) were developed to enhance T-ALL cells recognition and endocytosis. In vitro cytotoxicity studies showed that TLnp/D&G significantly inhibited cell survival and promoted apoptosis of T-ALL cells. Mechanically, we found that GSIs promoted DEX-induced cell apoptosis by two main synergetic mechanisms: 1) GSIs significantly upregulated glucocorticoid receptor (GR) expression in T-ALL and restored the glucocorticoid-induced pro-apoptotic response. 2) Both DEX and GSI synergistically inhibited BCL2 and suppressed the survival of T-ALL cells. Furthermore, in vivo studies demonstrated that TLnp/D&G showed high bone marrow accumulation and better antileukemic efficacy both in leukemia bearing models and in systemic Notch1-induced T-ALL models, with excellent biosafety and reduced gastrointestinal toxicity. Overall, our study provides new strategies for the treatment of T-ALL and promising bone marrow targeting systems with high transformation potential.

A targeted and redox/pH-responsive chitosan oligosaccharide derivatives based nanohybrids for overcoming multidrug resistance of breast cancer cells.

A novel folic acid mediated chitosan oligosaccharide-grafted disulfide-containing polyethylenimine copolymer-based silica nanohybrids were fabricated for co-delivering paclitaxel and P-shRNA. These nanoparticles could efficiently protect P-shRNA against degradation, and exhibited well redox-responsive P-shRNA release and pH-responsive drug release behaviors. Folic acid as the targeting head, could improve cellular uptake of nanoparticles by multidrug-resistant breast cancer cells. Moreover, these nanoparticles showed excellent delivery P-shRNA into cells and displayed high gene silencing efficiency at the targeted mRNAs to downregulate the expression of P-gp which induced up to 63% decrease. Finally, nanoparticles could completely reverse the resistance of breast cancer cells to paclitaxel and the resistance reversion index was 50.59. These results suggested that our nanoparticles could efficiently co-deliver paclitaxel and P-shRNA into cancer cells to exert its synergistic antitumor effect, and opened up a new avenue for overcoming multidrug resistance.

Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors.

A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated for their inhibitory activity against IDH2 R140Q mutant. The preliminary results indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest inhibitory activity, with the IC50 value of 0.26 µM, which was more active than positive control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.

Synthesis and biological evaluation of geldanamycin-ferulic acid conjugate as a potent Hsp90 inhibitor.

A novel geldanamycin-ferulic acid conjugate LZY228 was prepared and evaluated for anti-proliferation activity on human cancer cell line MDA-MB-231. Compound LZY228 exhibited potent cytotoxicity with IC50 value of 0.27 µM, which was more potent than 17-AAG. Hepatotoxicity test in mice demonstrated that the levels of both AST and ALT of LZY228-treated group were lower than that of GA-treated group, indicating that LZY228 was a promising antitumor candidate. In addition, excellent in vivo antitumor potency of LZY228 was observed in MDA-MB-231 xenograft model, which was superior to reference drug 17-AAG. Docking and MD refinement of the Hsp90-LZY228 complex give us an explanation of theoretical binding model of 17-ferulamido-17-demethoxygeldanamycins at molecular level.

Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance.

The objective of the study is to fabricate multifunctional mesoporous silica nanoparticles for achieving co-delivery of conventional antitumor drug paclitaxel (PTX) and the multidrug resistance reversal agent tetrandrine (TET) expecting to overcome multidrug resistance of MCF-7/ADR cells. The nanoparticles were facile to prepare by self-assemble in situ drug loading approach. Namely, PTX and TET were solubilized in the cetyltrimethylammonium bromide (CTAB) micelles and simultaneously silica resources hydrolyze and condense to form nanoparticles. The obtained nanoparticles, denoted as PTX/TET-CTAB@MSN, exhibited pH-responsive release property with more easily released in the weak acidic environment. Studies on cellular uptake of nanoparticles demonstrated TET could markedly increase intracellular accumulation of nanoparticles. Furthermore, the PTX/TET-CTAB@MSN suppressed tumor cells growth more efficiently than only delivery of PTX (PTX-CTAB@MSN) or the free PTX. Moreover, the nanoparticle loading drugs with a PTX/TET molar ratio of 4.4:1 completely reversed the resistance of MCF-7/ADR cells to PTX and the resistance reversion index was 72.3. Mechanism research showed that both TET and CTAB could arrest MCF-7/ADR cells at G1 phase; and besides PTX arrested cells at G2 phase. This nanocarrier might have important potential in clinical implications for co-delivery of multiple drugs to overcome MDR.

In vitro and in vivo Evaluation of 17-phenylpropylamine/phenoxyethylamine- 17-demethoxygeldanamycins as Potent Hsp90 Inhibitors.

A series of 17-phenylpropylamine/phenoxyethylamine-substituted derivatives of geldanamycin (GA) was synthesized and evaluated for the anti-proliferation activity on human cancer cell line MDA-MB-231. All the derivatives exhibited potent cytotoxicity with IC50 values range from 0.35 to 1.03 µM. Among them, 17-(2-phenoxyethylamino)-17-demethoxygeldanamycin (3) was identified as the most potent compound. Hepatotoxicity test in mice demonstrated that the levels of both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of 3-treated group were lower than that of GA-treated group, indicating that compound 3 was a promising antitumor candidate. Additionally, the Hsp90 inhibitory activity of compound 3 was more active than 17-AAG. Docking and molecular dynamics (MD) refinements of this new series of GA derivatives were also investigated, suggesting a theoretical model between 17- phenylpropylamine/phenoxyethyl-amines and Hsp90.

Discovery of novel 17-phenylethylaminegeldanamycin derivatives as potent Hsp90 inhibitors.

Twenty-six 17-phenylethylamine-modified geldanamycin derivatives were synthesized and evaluated for antiproliferation activity in human cancer cell lines, LNCaP and MDA-MB-231. Five derivatives (2j, 2q, 2v, 2x, and 2 y) showed excellent in vitro antitumor activities. Among them, compound 2 y was the most potent lead, with IC50 values of 0.27 ± 0.11 and 0.86 ± 0.23 µm for LNCaP and MDA-MB-231, respectively. In particular, compound 2 y was more active than its precursor geldanamycin against LNCap cells. Liver injury test in mice demonstrated that 2 y group showed no significant difference for serum alanine aminotransferase (ALT) activity versus vehicle control, indicating that 2 y was a promising antitumor candidate. Preliminary structure-activity relationship (SAR) and molecular dynamics (MD) simulations of this new series of geldanamycin derivatives were also investigated, suggesting a theoretical model of 17-phenylethylaminegeldanamycins binding to Hsp90.

Discovery of diamine-linked 17-aroylamido-17-demethoxygeldanamycins as potent Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is an attractive target for the development of antitumor agents. Geldanamycin (GA), the first Hsp90 inhibitor, has potent antitumor activity, but showed significant hepatotoxicity. To get rid of the hepatotoxicity of GA, in this study we incorporated aroyl groups via three types of linkers (4-aminomethylpiperidine, 1,4-butanediamine, and 1,6-hexanediamine) to the 17-position of GA and synthesized fifty-three 17-diamine-linked 17-aroylamido-17-demethoxygeldanamycins. All the derivatives were evaluated by MTT assay for their inhibitory activities against human breast cancer cell line MDA-MB-231. Among these compounds, 17-(6-(3,4,5-trimethoxycinnamamido)hexylamino)-17-demethoxygeldanamycin (7h29) showed the most potent cytotoxicity against MDA-MB-231 (IC50 = 0.19 ± 0.02 µM) with the lowest hepatotoxicity (AST = 181.0 ± 23.6 U/L, ALT = 40.4 ± 11.8 U/L). Compared to tanespimycin (17-AAG), 7h29 exhibited lower hepatotoxicity in mice, higher Hsp90 inhibitory activity in vitro and antitumor activity in human breast carcinoma (MDA-MB-231) xenograft nude mice.

Design, synthesis and biological evaluation of 17-arylmethylamine-17-demethoxygeldanamycin derivatives as potent Hsp90 inhibitors.

Thirty-three 17-arylmethylamine-substituted derivatives of geldanamycin (GA) were designed, synthesized and evaluated for the anti-proliferation activity on human cancer cell lines, LNCaP and MDA-MB-231. Three derivatives (22, 33 and 34) exhibited potent cytotoxicity with IC50 values range from 0.05 to 0.51 µM against both cell lines. Hepatotoxicity test in mice demonstrated that the levels of both AST and ALT of 34-treated group were lower than that of 17-AAG group. Western blot assay indicated that 34 was more potent than 17-AAG in the down-regulation of Hsp90 client proteins CDK4, Her2, EGFR and Raf. Moreover, 34 showed excellent in vivo antitumor activity in the MDA-MB-231 xenograft nude mice, which is superior to 22 and 33, and 17-AAG, indicating that 34 was a promising antitumor candidate. Additionally, preliminary structure-activity relationship (SAR) and molecular dynamics (MD) simulations of this new series of GA derivatives were also investigated, suggesting a theoretical model of 17-arylmethylamine geldanamycins binding to Hsp90.

Self-assembled nanoparticles based on galactosylated O-carboxymethyl chitosan-graft-stearic acid conjugates for delivery of doxorubicin.

A novel polymer, i.e. galactosylated O-carboxymethyl chitosan-graft-stearic acid (Gal-OCMC-g-SA) was synthesized for liver targeting delivery of doxorubicin. The chemical structure was characterized by FT-IR, (1)H NMR and elemental analysis. Gal-OCMC-g-SA could self-assemble into nanoparticles with diameter of 160 nm by probe sonication in aqueous medium and exhibited a low critical aggregation concentration of 0.047 mg/mL. The DOX-loaded Gal-OCMC-g-SA (Gal-OCMC-g-SA/DOX) self-assembled nanoparticles were almost spherical in shape with an average diameter of less than 200 nm and zeta potential of around -10 mV. In vitro release revealed that the Gal-OCMC-g-SA/DOX nanoparticles exhibited a sustained and pH-dependent drug release manner. Furthermore, the hemolysis test demonstrated the good safety of Gal-OCMC-g-SA in blood-contacting applications. These results indicated that Gal-OCMC-g-SA/DOX nanoparticles were highly potential to be applied in cancer therapy.

Synthesis, characterization, in vitro and in vivo evaluation of PEGylated oridonin conjugates.

Oridonin (ORI), a diterpenoid compound with promising antitumor activity, was proved to possess potent antileukemia efficacies in vitro and in vivo recently. However, the development and application of ORI was limited by its poor solubility and rapid plasma clearance. The purpose of this study was to solve these problems. PEGylated oridonin linked with succinic acid (SA) as spacer moiety (PEG-SA-ORI conjugate) was synthesized. mPEG amines with four specifications of molecular weight (MW) were utilized. All polymeric conjugates showed satisfactory aqueous solubility and in vitro studies implied that the drug solubility and release features of conjugates were relevant to PEGs. The drug solubility increased more when the MW of PEG was lower, while more significant sustained-release effect was shown with higher PEG MW. Moreover, the release behaviors of conjugates showed a pH-sensitive property. In vivo pharmacokinetic studies demonstrated that the elimination half-life was prolonged in comparison with ORI solution. PEGylation could be a promising method to obtain better efficacy in the field of drug delivery system.

Nanosuspension for parenteral delivery of a p-terphenyl derivative: preparation, characteristics and pharmacokinetic studies.

Recently, nanosuspension technology has evolved into a mature drug delivery system, which can enhance the saturation solubility and dissolution velocity of poorly soluble drugs. In this study, nanosuspensions of a p-terphenyl derivative (H2) were prepared by combining microfluidization and precipitation method and transformed into dry powder by lyophilization. The resultant nanosuspensions had a mean particle size of 201.7±5.87nm and a zeta potential of -21.07±0.57mV. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis verified that the crystalline state of H2 was not transformed when it was prepared to nanosuspensions. An increased saturation solubility (1.46ug/ml) and accelerated dissolution velocity were achieved. The percent drug release of bulk H2 and H2 nanosuspension dried powder was 7.16% and 93.5% at 120min point, respectively. The pharmacokinetic test in rats indicated that the area under plasma concentration-time curve (AUC0~∞) value of H2 nanosuspension (5.183mg/Lh) was about 5-fold higher than that of H2 solution (1.094mg/Lh). Additionally, the mean retention time (MRT) value of H2 nanosuspension (2.832h) was significantly longer than that of H2 solution (0.997h).

In vitro and in vivo evaluation of paclitaxel-loaded mesoporous silica nanoparticles with three pore sizes.

In the present study, mesoporous silica nanoparticles (MSNs) with three pore size were manufactured by the etch method. A typical chemotherapeutic agent, paclitaxel (PTX) was loaded into these MSNs. The in vitro drug release behavior, the in vitro anti-tumor activity, the morphological apoptosis cell changes, cell apoptosis rate and pharmacokinetics were extensively evaluated to clarify the biomedical roles of these MSNs in the application of drug delivery. The results showed that paclitaxel-loaded MSNs not only demonstrated effective drug loading but also exhibited pore-size-dependent drug release performance in vitro. In addition, MSNs exhibited pore-size-dependent anti-tumor activity against breast cancer MCF-7 cells. The apoptosis mechanism study demonstrated that the percentage of early and late apoptosis of all PTX-loaded MSNs treated MCF-7 cells were significantly higher than that of free PTX, and additionally the percentage of apoptosis for PTX-loaded MSNs increased as the pore size of carriers enlarged. The pharmacokinetics results showed that PTX-loaded MSNs with the largest pore size exhibited the pharmacokinetic property similar to the PTX solution and the other drug loaded MSNs displayed sustained release behavior. These results demonstrate that MSNs could be a very promising drug delivery system for pore-size controllable drug release and enhancing the anti-tumor activity.

In vitro and in vivo evaluation of riccardin D nanosuspensions with different particle size.

Riccardin D (RD) is a novel compound extracted from Chinese liverwort Marchantia polymorpha L. It exhibits various anticancer activities and can be used during lung cancer treatment. However, the compound's low solubility hinders its development. Recently nanosuspension has been developed as one of the most promising formulations for poorly water-soluble drugs. In order to understand the dissolution behavior of riccardin D in vitro and in vivo, two nanosuspensions of riccardin D with markedly different sizes were prepared. The particle size of nanosuspension A prepared by bottom-up method was 184.1±3.15 nm, while that of nanosuspension B prepared by top-down method was 815.4±9.65 nm. The main purpose of this study was to investigate the effects of particle size on pharmacokinetics and tissue distribution after intravenous administration. Riccardin D dissolving in organic solution was studied as control group. In pharmacokinetics study in Wistar rats, nanosuspension A showed properties similar to the control group, while nanosuspension B exhibited rather different properties. In tissue distribution research on Kunming strain mice, nanosuspension A had a multi-peak phenomenon because of reticulate endothelial system (RES) while nanosuspension B showed a high uptake in RES organs that passively target to the lungs. In conclusion, particle size of riccardin D nanosuspensions had obvious effects on pharmacokinetics and tissue distribution.

Successfully tailoring the pore size of mesoporous silica nanoparticles: exploitation of delivery systems for poorly water-soluble drugs.

A novel approach was applied to fabricate mesoporous silica nanoparticles (MSNs) with different pore size in this study. The pore size of MSNs can be modulated conveniently from 3 nm to 10nm by controlling the etching time of MSNs with the NaBH(4) solution. The as-synthesized MSNs were investigated as carriers for loading and delivery of the model drug paclitaxel (PTX). The characteristics, drug loading capacity, in vitro drug release behavior, anti-tumor activity and the mechanism of cell uptake were systematically studies. The resultant MSNs showed uniform and mono-dispersed sphere with high drug loading capacity (12-21%). The in vitro drug release exhibited that the released rate of PTX from MSNs could be controlled by the pore size and the larger the pore size, the faster the release rate of PTX. The in vitro anti-tumor studies demonstrated that PTX-loaded MSNs produced higher cytotoxicity than free PTX. Besides, the PTX-loaded MSNs with largest pore size showed the highest anti-tumor activity. These results indicated that these MSNs could provide a promising platform for delivering water-insoluble drugs, controlling the release rate of drugs and increasing the anti-tumor activity.

In vivo studies on the oridonin-loaded nanostructured lipid carriers.

Oridonin (ORI)-loaded Nanostructured lipid carriers (NLC) were prepared by emulsion-evaporation and low temperature-solidification technique, and evaluated for morphological observation, particle size, zeta potential and in vitro drug release. Next, the characteristics of biodistribution and pharmacokinetics in vivo were examined. The average particle size of resultant NLC was 245.2 nm and the zeta potential was found to be -38.77 mV. The in vivo characteristics of ORI-loaded NLC were studied after intravenous administration using Kunming strain mice as experimental animals. An ORI control solution was studied parallelly. In tested organs, the distribution of ORI-loaded NLC to liver was higher than that of free drug. ORI-loaded NLC showed higher AUC (area under tissue concentration-time curve) values and circulated in the blood stream for a longer time compared with ORI solution. These results support the potential applications of NLC for the delivery of ORI.

Galactosylated chitosan nanoparticles for hepatocyte-targeted delivery of oridonin.

In this study, oridonin-loaded nanoparticles coated with galactosylated chitosan (ORI-GC-NP) were prepared for tumor targeting and their characteristics were evaluated for the morphologies, particle size and zeta potential. Oridonin-loaded nanoparticles (ORI-NP) without galactosylated chitosan were prepared as a control. The entrapment efficiency of ORI-GC-NP and ORI-NP were 72.15% and 85.31%, respectively. The in vitro drug release behavior from nanoparticles displayed biphasic drug release pattern with initial burst release and consequently sustained release. Next, the pharmacokinetics and tissue distribution of ORI-GC-NP, ORI-NP and ORI solution were carried out. Pharmacokinetic analysis showed that ORI-GC-NP and ORI-NP could prolong the drug plasma levels compared with ORI solution. Meanwhile, the distribution of ORI-GC-NP to liver was higher than that of ORI-NP and free drug. In conclusion, ORI-GC-NP, as a promising intravenous drug delivery system for ORI, could be developed as an alternative to the conventional ORI preparations.