Prolonged Intracellular Retention Research Articles

Targeting HIF-1α with Specific DNA Yokes for Effective Anticancer Therapy.

Hypoxia, a ubiquitous hallmark in cancer, underscores the significance of targeting HIF-1α, the principal transcriptional factor of hypoxic responses, for effective cancer therapy. Herein, DNA yokes, a novel class of DNA nanomaterials harboring specific HIF-1α binding sequences (hypoxia response elements, HREs), are introduced as nanopharmaceuticals for cancer treatment. Comprising a basal tetrahedral DNA nanostructure and four HRE-bearing overhanging chains, DNA yokes exhibit exceptional stability and prolonged intracellular retention. The investigation reveals their capacity to bind HIF-1α, thereby disrupting its interaction with the downstream genomic DNAs and impeding transcriptional activity. Moreover, DNA yokes facilitate HIF-1α degradation via the ubiquitination pathway, thereby sequestering it from downstream targets and ultimately promoting its degradation. In addition, DNA yokes attenuate cancer cell proliferation, migration, and invasion under hypoxic conditions, while also displaying preferential accumulation within tumors, thereby inhibiting tumor growth and metastasis in vivo. This study pioneers a novel approach to cancer therapy through the development of DNA-based drugs characterized by high stability and low toxicity to normal cells, positioning DNA yokes as promising candidates for cancer treatment.

Conidial Melanin of the Human-Pathogenic Fungus Aspergillus fumigatus Disrupts Cell Autonomous Defenses in Amoebae.

The human-pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal lung infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How this virulence trait could have been selected for in natural environments is unknown. Here, we found that surface exposure of the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin can protect conidia from phagocytic uptake and intracellular killing by the fungivorous amoeba Protostelium aurantium and delays its exocytosis from the nonfungivorous species Dictyostelium discoideum To elucidate the antiphagocytic properties of the surface pigment, we followed the antagonistic interactions of A. fumigatus conidia with the amoebae in real time. For both amoebae, conidia covered with DHN-melanin were internalized at far lower rates than were seen with conidia lacking the pigment, despite high rates of initial attachment to nonkilling D. discoideum When ingested by D. discoideum, the formation of nascent phagosomes was followed by transient acidification of phagolysosomes, their subsequent neutralization, and, finally, exocytosis of the conidia. While the cycle was completed in less than 1 h for unpigmented conidia, the process was significantly prolonged for conidia covered with DHN-melanin, leading to an extended intracellular residence time. At later stages of this cellular infection, pigmented conidia induced enhanced damage to phagolysosomes and infected amoebae failed to recruit the ESCRT (endosomal sorting complex required for transport) membrane repair machinery or the canonical autophagy pathway to defend against the pathogen, thus promoting prolonged intracellular persistence in the host cell and the establishment of a germination niche in this environmental phagocyte.IMPORTANCE Infections with Aspergillus fumigatus are usually acquired by an inhalation of spores from environmental sources. How spores of a saprophytic fungus have acquired abilities to withstand and escape the phagocytic attacks of innate immune cells is not understood. The fungal surface pigment dihydroxynaphtalene-melanin has been shown to be a crucial factor for the delay in phagosome maturation. Here, we show that this pigment also has a protective function against environmental phagocytes. Pigmented conidia escaped uptake and killing by the fungus-eating amoeba Protostelium aurantium When ingested by the nonfungivorous phagocyte Dictyostelium discoideum, the pigment attenuated the launch of cell autonomous defenses against the fungal invader, such as membrane repair and autophagy, leading to prolonged intracellular retention. Membrane damage and cytoplasmic leakage may result in an influx of nutrients and thus may further promote intracellular germination of the fungus, indicating that A. fumigatus has acquired some of the basic properties of intracellular pathogens.

Enhanced anticancer effect of doxorubicin by TPGS-coated liposomes with Bcl-2 siRNA-corona for dual suppression of drug resistance

Multiple drug resistance (MDR) is a tough problem in developing hepatocellular carcinoma (HCC) therapy. Here, we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin (Dox) i.e., Bcl-2 siRNA/Dox-TPGS-LPs, to enhance anticancer effect of Dox in HCC-MDR. TPGS i.e., d-α-tocopheryl polyethylene glycol 1000 succinate, inhibited P-glycoprotein (P-gp) efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein. The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy. The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGS-LPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum. In drug-resistant cells, TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes. In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model. MTT assay confirmed the IC50 value of Dox was 20–50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells. Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo. Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS- (or siRNA-) linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells, and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice. In conclusion, TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.

Uptake of Ranibizumab but Not Bevacizumab into Uveal Melanoma Cells Correlates with a Sustained Decline in VEGF-A Levels and Metastatic Activities.

Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. Here, we evaluated whether bevacizumab and ranibizumab, a humanized Fab-fragment against VEGF-A, can enter UM cells and induce a sustained physiological response. The primary and metastatic UM cell lines Mel-270 and OMM-2.5 were exposed to bevacizumab or ranibizumab for one day and were maintained further in untreated medium for a total of three days. Both antibodies significantly reduced the levels of extracellular VEGF-A and the angiogenic potential of the conditioned medium after one day. These inhibitory effects of bevacizumab diminished by day three. Ranibizumab suppressed the metabolic activity, proliferation, and intracellular VEGF-A levels in a cell-type and concentration-dependent manner, whereas bevacizumab exerted no effect. Both drugs were detected inside early endosomes within the UM cells, with the stronger and sustained colocalization of ranibizumab. Our results therefore demonstrated the more potent and persistent suppressive activity of ranibizumab on the UM cells, possibly due to its higher level of uptake and prolonged intracellular retention. Further research on the endosome dynamics in UM cells might provide valuable insight into the response of these heterogenous tumors to therapeutic antibodies.

A potential long-acting bictegravir loaded nano-drug delivery system for HIV-1 infection: A proof-of-concept study

Bictegravir (BIC), a newly FDA-approved integrase strand transfer inhibitor (INSTI), as a single tablet regimen has proven efficacious in treating HIV-1 and SIV viruses, with reduced resistance. BIC clinical trials have not investigated its prophylaxis potency. This study investigates the HIV prevention potency of a novel long-acting BIC nano-formulation aimed to improve adherence. Poly (lactic-co-glycolic acid) loaded BIC nanoparticles (BIC NPs) were formulated using an oil-in-water emulsion methodology. BIC NPs were <200 nm in size, with 47.9 ± 6.9% encapsulation efficiency. A novel, sensitive and high throughput LC-MS/MS method was used to estimate intracellular pharmacokinetics (PK) of BIC NPs and compared to BIC solution demonstrated prolonged intracellular BIC retention. BIC NPs safety was assessed based on cytotoxicity. Further, in-vitro prevention study of BIC NPs vs BIC solution was assessed against HIV-1NLX and HIV-1ADA on TZM-bl cell line and PBMCs, respectively. BIC nanoencapsulation demonstrated elevated cellular cytotoxicity concentration (CC50: 2.25 μM (BIC solution) to 820.4 μM (BIC NPs)] and lowers HIV-1 inhibitory concentration [EC50: 0.604 μM (BIC solution) to 0.0038 μM (BIC NPs)) thereby improving selectivity index (SI) from 3.7 (BIC solution) to 215,789 (BIC NP) for TZM-bl cells. Comparable results in PBMCs were obtained where BIC NPs improved SI from 0.29 (BIC solution) to 523.33 (BIC NPs). This demonstrates long-acting BIC nano-formulation with sustained drug-release potency, improved BIC cytotoxicity and enhanced HIV-1 protection compared to BIC in solution.

The LXR-623-induced long non-coding RNA LINC01125 suppresses the proliferation of breast cancer cells via PTEN/AKT/p53 signaling pathway

LXR-623 (WAY-252623), a liver X receptor agonist, reduces atherosclerotic plaque progression and remarkably inhibits the proliferation of glioblastoma cells, owing to its brain-penetrant ability. However, the role of LXR-623 against the proliferation of other cancer cells and the underlying mechanism remain unknown. Long non-coding RNAs (lncRNAs) serve as novel and crucial regulators that participate in cancer tumorigenesis and diverse biological processes. Here, we report a previously uncharacterized mechanism underlying lncRNA-mediated exocytosis of LXR-623 via the phosphatase and tensin homolog (PTEN)/protein kinase B (AKT)/p53 axis to suppress the proliferation of cancer cells in vitro. We found that LXR-623 significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at S phase in breast cancer cells in a concentration- and time-dependent manner. Experiments using a xenograft mouse model revealed the inhibitory effects of LXR-623 on tumor growth. We used lncRNA microarray to investigate the potential genes regulated by LXR-623. As a result, LINC01125 was found to be significantly upregulated in the cells treated with LXR-623. Gain- and loss-of-function assays were conducted to investigate the anti-proliferation role of LINC01125. LINC01125 knockdown resulted in the inhibition of the cytotoxic effect of LXR-623; in contrast, LINC01125 overexpression significantly enhanced the effect of LXR-623. LXR-623 and LINC01125-mediated anti-growth regulation is, at least in part, associated with the participation of the PTEN/AKT/mouse double minute 2 homolog (MDM2)/p53 pathway. In addition, SF1670, a specific PTEN inhibitor with prolonged intracellular retention, may strongly block the anti-proliferation effect induced by LXR-623 and LINC01125 overexpression. Chromatin immunoprecipitation (ChIP) assay results suggest that p53 binds to the promoter of LINC01125 to strengthen the expression of the PTEN/AKT pathway. Taken together, our findings suggest that LXR-623 possesses significant antitumor activity in breast cancer cells that is partly mediated through the upregulation in LINC01125 expression and enhancement in apoptosis via the PTEN/AKT/MDM2/p53 pathway.

P2.03a-011 Population Pharmacokinetic/Pharmacodynamic Monitoring of Pemetrexed to Predict Survival in Patients with Advanced NSCLC

A major challenge in advanced non-small cell lung cancer (NSCLC) remains the identification of predictors of clinical benefit from pemetrexed. Total systemic exposure to pemetrexed and its metabolites could be predictive for progression-free and overall survival (PFS/OS). However, sampling times in population pharmacokinetic (PK) analyzes of pemetrexed are limited. We performed population PK modeling of pemetrexed during total treatment period and evaluated total systemic exposure as a predictor. In a prospective observational multi-center study, treatment-naive patients with stage IIIB or IV NSCLC receiving pemetrexed/platinum treatment were enrolled. Pemetrexed, dosed based on body surface area (500mg/m2), was administered as a 10-minute intravenous infusion every 21 days. Prior to and weekly after each pemetrexed administration, plasma sampling was performed (cycle-PK). Simultaneously, glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboriation (CKD-EPI) formula. In a subgroup, blood samples were collected at 10, 30 minutes and 1,2,4, 8, 24 hours after start of pemetrexed infusion (24-hour-PK). We used a recently validated assay to quantify plasma pemetrexed concentrations (Stoop et al, J Pharm Biomed Anal 2016;128:1-8). Population PK analyzes were performed using nonlinear mixed effects modeling (NONMEM version 7.2). The final model, based on both cycle-PK and 24-hour-PK, was used to estimate the area under the plasma concentration versus time curve during cycle 1 (AUCcycle). With a Cox-regression analysis we examined the relation between AUCcycle and OS/PFS, adjusted for known prognostic factors (sex, disease stage, WHO performance-score). For 97 of the 151 patients, concentrations of pemetrexed were quantified (24-hour-PK, n=15; cycle-PK, n=82). A two-compartment model parametrized (population estimate (%standard error of the estimate) in terms of clearance (CL; 5.44L/h (14.9%)), central distribution volume (V1; 19.6L (11.3%)), peripheral distribution volume (V2; 173L (32.7%)), intercompartimental clearance (Q; 0.19L/h (31.6%)) and incorporated between-patient variability of CL (10.7%), estimated cycle-PK appropriately. GFR and other covariates did not improve the estimation of the parameters. The AUCcylce was significantly different between males (190.8±64.9mg·h/L) and females (165.4±47.2mg·h/L). When we stratified for sex, the highest quartile of AUC independently predicted worse OS (HR=3.06, 95% CI: 1.43-6.57) and PFS (HR=2.79 95% CI: 1.36-5.70), adjusted for the remaining prognostic factors, GFR and pemetrexed dosage. Paradoxically worse OS and PFS in patients with high plasma pemetrexed AUC may suggest lower intracellular levels of pemetrexed. Another possibility is that these patients poorly metabolize pemetrexed into its more effective metabolites, which inhibit tumor growth more strongly by prolonged intracellular retention and higher affinity to target enzymes.

Tumor-Triggered Drug Release with Tumor-Targeted Accumulation and Elevated Drug Retention To Overcome Multidrug Resistance

Multidrug resistance (MDR) is one of the main causes of the failure in cancer chemotherapy. To address this challenge, this work develops a tumor-triggered nanomedicine (HA-MSDOX-KLA; comprised of a tumor active targeting segment (hyaluronic acid, HA) and two programmed therapeutic agents, MMP-substrate peptide (CPLGLAGG) conjugated doxorubicin (designated as MSDOX) and proapoptotic peptide (KLAKLAK)2 (designated as KLA)) with switchable morphology transformation that significantly enhances tumor-specific accumulation and retention for overcoming MDR. Morphology changes could be triggered by tumor overexpressed matrix metalloproteinases (MMPs), achieving elevated drug accumulation and prolonged intracellular drug retention. Additionally, the adjunctive release of proapoptotic peptide KLA would targetedly induce the dysfunction of mitochondria with decreased adenosine 5′-triphosphate (ATP) generation to further inhibit the drug efflux. In vitro and in vivo studies demonstrate that, compared with free doxor...

Reconstituted high density lipoprotein mediated targeted co-delivery of HZ08 and paclitaxel enhances the efficacy of paclitaxel in multidrug-resistant MCF-7 breast cancer cells

In the past decades, reconstituted high density lipoprotein (rHDL) has been successfully developed as a drug carrier since the enhanced HDL-lipids uptake is demonstrated in several human cancers. In this paper, rHDL, for the first time, was utilized to co-encapsulate two hydrophobic drugs: an anticancer drug, paclitaxel (PTX), and a new reversal agent for P-gp (P-glycoprotein)-mediated multidrug resistance (MDR) of cancer, N-cyano-1-[(3,4-dimethoxyphenyl)methyl]-3,4-dihydro-6,7-dimethoxy-N′-octyl-2(1H)-isoquinoline-carboximidamide (HZ08). We proposed this drug co-delivery strategy to reverse PTX resistance. The study aimed to develop a biomimetic nanovector, reconstituted high density lipoprotein (rHDL), mediating targeted PTX-HZ08 delivery for cancer therapy. Using sodium cholate dialysis method, we successfully formulated dual-agent co-delivering rHDL nanoparticles (PTX-HZ08-rHDL NPs) with a typical spherical morphology, well-distributed size (~100nm), high drug encapsulation efficiency (approximately 90%), sustained drug release properties and exceptional stability even after storage for 1month or incubation in 10% fetal bovine serum (FBS) DMEM for up to 2days. Results demonstrated that PTX-HZ08-rHDL NPs significantly enhanced anticancer efficacy in vitro, including higher cytotoxicity and better ability to induce cell apoptosis against both PTX-sensitive and -resistant MCF-7 human breast cancer cell lines (MCF-7 and MCF-7/PTX cells). Mechanism studies demonstrated that these improvements could be correlated with increased cellular uptake of PTX mediated by scavenger receptor class B type I (SR-BI) as well as prolonged intracellular retention of PTX due to the HZ08 mediated drug-efflux inhibition. In addition, in vivo investigation showed that the PTX-HZ08-rHDL NPs were substantially safer, have higher tumor-targeted capacity and have stronger antitumor activity than the corresponding dosage of paclitaxel injection. These findings suggested that rHDL NPs could be an ideal tumor-targeted nanovector for simultaneous transfer of insoluble anticancer drug and drug resistance reversal agents. The PTX-HZ08-rHDL NPs co-delivery system might be a new promising strategy to overcome tumor drug resistance.

Nanoparticle tumor localization, disruption of autophagosomal trafficking, and prolonged drug delivery improve survival in peritoneal mesothelioma.

The treatment outcomes for malignant peritoneal mesothelioma are poor and associated with high co-morbidities due to suboptimal drug delivery. Thus, there is an unmet need for new approaches that concentrate drug at the tumor for a prolonged period of time yielding enhanced antitumor efficacy and improved metrics of treatment success. A paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) system is described that addresses two unique challenges to improve the outcomes for peritoneal mesothelioma. First, following intraperitoneal administration, eNPs rapidly and specifically localize to tumors. The rate of eNP uptake by tumors is an order of magnitude faster than the rate of uptake in non-malignant cells; and, subsequent accumulation in autophagosomes and disruption of autophagosomal trafficking leads to prolonged intracellular retention of eNPs. The net effect of these combined mechanisms manifests as rapid localization to intraperitoneal tumors within 4h of injection and persistent intratumoral retention for >14 days. Second, the high tumor-specificity of PTX-eNPs leads to delivery of greater than 100 times higher concentrations of drug in tumors compared to PTX alone and this is maintained for at least seven days following administration. As a result, overall survival of animals with established mesothelioma more than doubled when animals were treated with multiple doses of PTX-eNPs compared to equivalent dosing with PTX or non-responsive PTX-loaded nanoparticles.

Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

Overcoming Multidrug Resistance with Mesoporous Silica Nanorods as Nanocarrier of Doxorubicin

Multidrug resistance (MDR) is a major obstacle to the effective chemotherapy in many human malignancies. Nanoparticulate drug delivery systems (NDDSs) have been reported to be able to bypass MDR, but the cancer therapeutic efficacy is still limited. In this study, we firstly designed the nonspherical mesoporous silica nanorods (MSNRs) with aspect ratio (AR) of 1.5 and 5 as drug delivery systems of doxorubicin to overcome multidrug resistance. For drug loading, the long-rod MSNRs (NLR, AR = 5) showed higher drug loading capacity of doxorubicin (DOX) than the short-rod MSNRs (NSR, AR = 1.5). NLR encapsulated DOX had increased intracellular DOX accumulation in drug-resistant Chinese hamster ovary (CHO) cells compared with free DOX by observablly increased cellular uptake and significantly prolonged intracellular drug retention. It further exhibited increased cytotoxicity compared with free DOX under different drug concentrations. These findings may provide a new perspective for designing high-performance nanoparticulate drug delivery systems for bypassing multidrug resistance of cancer therapy.

Elacytarabine — A New Agent in the Treatment of Relapsed/Refractory Acute Myeloid Leukaemia

The prognosis for patients with relapsed acute myeloid leukaemia (AML) is poor, and effective treatments for this patient group remain a substantially unmet clinical need. Elacytarabine is a promising new cytotoxic nucleoside agent made by an esterification reaction between cytarabine and elaidic acid, currently in development for use in the treatment of relapsed/refractory AML. Unlike cytarabine, cellular uptake of elacytarabine is independent of human equilibrative nucleoside transporter-1 (hENT-1) and results in prolonged intracellular retention of the active nucleoside. In addition, elacytarabine inhibits DNA synthesis for twice the duration seen with cytarabine and exhibits a different intracellular distribution. A Phase I trial in patients with AML identified a recommended dose of 2,000 mg/m²/day for five days and showed limited non-haematological side effects, liver toxicity being dose-limiting. Elacytarabine can be safely combined with idarubicin. A recent Phase II trial demonstrated an improved complete remission rate and overall survival with elacytarabine as a single agent in patients with advanced AML, as compared with a historical control group treated with second salvage therapy. Following these encouraging results, the results of an ongoing Phase III clinical trial comparing elacytarabine with the investigator’s choice of standard of care are awaited with interest.

A Folate Receptor-Targeting Nanoparticle Minimizes Drug Resistance in a Human Cancer Model

Resistance to chemotherapy is a major obstacle in cancer therapy. The main purpose of this study is to evaluate the potential of a folate receptor-targeting nanoparticle to overcome/minimize drug resistance and to explore the underlying mechanisms. This is accomplished with enhanced cellular accumulation and retention of paclitaxel (one of the most effective anticancer drugs in use today and a well-known P-glycoprotein (P-gp) substrate) in a P-gp-overexpressing cancer model. The folate receptor-targeted nanoparticle, HFT-T, consists of a heparin-folate-paclitaxel (HFT) backbone with an additional paclitaxel (T) loaded in its hydrophobic core. In vitro analyses demonstrated that the HFT-T nanoparticle was superior to free paclitaxel or nontargeted nanoparticle (HT-T) in inhibiting proliferation of P-gp-overexpressing cancer cells (KB-8-5), partially due to its enhanced uptake and prolonged intracellular retention. In a subcutaneous KB-8-5 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably prolonged retention within tumor tissues. Importantly, HFT-T treatment markedly retarded tumor growth in a xenograft model of resistant human squamous cancer. Immunohistochemical analysis further indicated that increased in vivo efficacy of HFT-T nanoparticles was associated with a higher degree of microtubule stabilization, mitotic arrest, antiangiogenic activity, and inhibition of cell proliferation. These findings suggest that when the paclitaxel was delivered as an HFT-T nanoparticle, the drug is better retained within the P-gp-overexpressing cells than the free form of paclitaxel. These results indicated that the targeted HFT-T nanoparticle may be promising in minimizing P-gp related drug resistance and enhancing therapeutic efficacy compared with the free form of paclitaxel.

Enhanced antiproliferative activity of Herceptin (HER2)-conjugated gemcitabine-loaded chitosan nanoparticle in pancreatic cancer therapy

Currently, effective drug delivery in pancreatic cancer treatment is a major challenge. Nanomedicine plays an essential role by delivering anticancer drugs in a targeted manner to the malignant tumor cells, leading to increased efficacy by reducing the toxicity of anticancer drugs to normal, sensitive sites. This study investigated the preparation and characterization of a targeted system represented by Herceptin-conjugated gemcitabine-loaded chitosan nanoparticles (HER2-Gem-CS-NPs) for pancreatic cancer therapy. The targeted NPs displayed superior antiproliferative activity along with an enhanced S-phase arrest, leading to apoptosis in comparison with unconjugated gemcitabine-loaded nanoparticles and free gemcitabine due to higher cellular binding with eventual uptake and prolonged intracellular retention. Thus, HER2-Gem-CS-NPs are able to provide an efficient and targeted delivery of gemcitabine for pancreatic cancer treatment. From the Clinical EditorThis study investigated the preparation and characterization of a targeted drug delivery system consisting of Herceptin-conjugated gemcitabine-loaded chitosan nanoparticles for pancreatic cancer therapy.

Cellular processing in the SW1222 cell line of mAb A33 directly and indirectly radiohalogenated

Investigations into the cellular processing of radiolabeled monoclonal antibodies (mAbs) for their further use in radioimmunodiagnosis and cancer therapy are needed in order to understand the fate of internalized and catabolized mAbs. The anti-colorectal cancer mAb, A33, was labelled with 76Br and 125I using the direct Chloramine-T method, or by labelling N-succinimidyl para-(tri-methylstannyl) benzoate and its further conjugation to the mAb. The cellular processing of the four conjugates was investigated in SW1222 cells in vitro. Uptake of mAb was rapid, peaking after 14-16 h. Intracellular degradation was slow and the early loss of radioactivity was due to dissociation of cell-surface bound mAb. The indirect labelling resulted in stronger binding of the mAb as well as prolonged intracellular retention of the radiolabel. Direct and indirect halogen radiolabelling results in different cell-processing patterns of radiolabels, and radioactive catabolic products follow different routes of cellular excretion. The results of this cellular study indicate that indirect labelling is preferable to the direct Chloramine-T method.

From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates

• Are multiple intracellular targets better than one? Does inhibition of dihydrofolate reductase (DHFR), thymidylate synthase (TS) or enzymes of the purine biosynthetic pathway confer an advantage? Alternatively, is inhibition of purine biosynthesis a safety liability rather than an advantage? • Can a lipophilic molecule rather than one that uses the folate carrier mechanisms, i.e. the folate binding protein (FBP) and or the reduced folate carrier (RFC), circumvent intrinsic or acquired resistance? • Can tumor selectivity be increased by exploiting knowledge of different patterns of RFC versus FBP expression or of variations in FBP isoform expression? • How does a patient’s functional folate status or supplemental folic acid modulate efficacy and or toxicity? • Does an antifolate that is a good substrate for the intracellular enzyme folylpolyglutamate synthase (FPGS) have a selective advantage? Does polyglutamation of the antifolate confer a therapeutic advantage because it results in increased potency against the target enzyme, and increased polarity leading to increased cellular retention? Alternatively, is it a disadvantage with respect to safety of the molecule because of prolonged intracellular retention times or the potential for resistance? • Does an increase in the potency of binding to the target enzyme confer a therapeutic advantage? • Resistance mechanisms include decreased cellular accumulation due to impaired transport, decreased retention inside the cell as a consequence of a lack of polyglutamate formation, or an increase in gamma glutamyl hydrolase (GGH) activity, an increase in expression of the target enzyme, or a mutation in the target enzyme. Which of these is clinically relevant, and how does knowledge of these resistance mechanisms aid selection of the appropriate antifolate for treatment of an individual patient? • How do pharmacokinetic variables contribute to safety and efficacy? Do the newer antifolates have fewer propensities for drug-drug interactions? • Is there an intrinsic limit to the clinical activity of an antifolate as a single agent? Can antitumor activity be increased, or can an increase in activity only be achieved by combining antifolates with other anticancer drugs (either other antifolates which target different folate requiring enzymes or compounds with different mechanisms of action)? Similarly, can the spectrum of clinical activity be increased? • Can patients whose tumors have a specific set of molecular properties be identified prospectively, and matched with the pharmacological properties of an agent, i.e. can therapy of individual patients be tailored? • Can toxicity be decreased and quality of life improved? • Can patients at risk of serious toxicity to a given agent be identified before or during therapy and managed in the light of that knowledge? • Are there mechanisms for rescuing patients from toxicity, and not from antitumor activity? • What improvements can be made in the administration schedule and therefore convenience to patients?

Pemetrexed: An active new agent for breast cancer

Pemetrexed is a novel antifolate that inhibits three enzymes in the de novo purine and pyrimidine pathways including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. It becomes highly polyglutamated once inside cancer cells, resulting in prolonged intracellular retention and 60-fold greater inhibition of thymidylate synthase than the monoglutamated form. Several phase II and III studies have shown that pemetrexed has significant antitumor activity against a variety of tumor types including mesothelioma, non–small cell lung cancer, and colon, pancreatic, and breast cancers. Pemetrexed appears to provide non–cross-resistant cytotoxic activity against breast cancers that have been treated with anthracyclines, taxanes, and capecitabine. Preclinical studies have suggested that pemetrexed enhances the cytotoxicity of several other important chemotherapeutic agents active against breast cancer including doxorubicin, paclitaxel, cisplatin, and gemcitabine. In vitro studies have shown that pemetrexed treatment synchronizes cancer cells at the G 2/S interphase, leading to synchronous entry into S phase. Ongoing phase II studies of pemetrexed combinations in breast cancer hope to exploit the cell cycle modulatory effects of this drug as well as its excellent tolerability. Semin Oncol 29 (suppl 18):57-62. Copyright 2002, Elsevier Science (USA). All rights reserved.

Pemetrexed: an active new agent for breast cancer.

Pemetrexed is a novel antifolate that inhibits three enzymes in the de novo purine and pyrimidine pathways including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. It becomes highly polyglutamated once inside cancer cells, resulting in prolonged intracellular retention and 60-fold greater inhibition of thymidylate synthase than the monoglutamated form. Several phase II and III studies have shown that pemetrexed has significant antitumor activity against a variety of tumor types including mesothelioma, non-small cell lung cancer, and colon, pancreatic, and breast cancers. Pemetrexed appears to provide non-cross-resistant cytotoxic activity against breast cancers that have been treated with anthracyclines, taxanes, and capecitabine. Preclinical studies have suggested that pemetrexed enhances the cytotoxicity of several other important chemotherapeutic agents active against breast cancer including doxorubicin, paclitaxel, cisplatin, and gemcitabine. In vitro studies have shown that pemetrexed treatment synchronizes cancer cells at the G(2)/S interphase, leading to synchronous entry into S phase. Ongoing phase II studies of pemetrexed combinations in breast cancer hope to exploit the cell cycle modulatory effects of this drug as well as its excellent tolerability.

A Naturally Occurring Nonpolymerogenic Mutant of α1-Antitrypsin Characterized by Prolonged Retention in the Endoplasmic Reticulum

The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.