Rev-erbα agonists suppresses TGFß1-induced fibroblast-to-myofibroblast transition and pro-fibrotic phenotype in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFß1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFß-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFß1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFß1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFß1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFß1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders.

Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer.

Chemotherapy is limited by the low availability of drug at the tumor site and drug resistance by the tumor. In this report we describe a combination therapy for codelivery of two anticancer drugs with spatiotemporal control by ultrasound pulses. We developed curcumin and topotecan-coencapsulated nanoconjugates Cur_Tpt_NC.MB to have an ultrasound contrast property. MDA MB 231 and B16F10 cells were incubated with Cur_Tpt_NC.MB and exposed to ultrasound. Ultrasound exposure reduced the IC50 concentration of topotecan and curcumin significantly (P < 0.05) compared with free drug. Antitumor efficacy study of the Cur_Tpt_NC.MB in B16F10 melanoma tumor-bearing C57BL/6 mice showed that ultrasound exposure of right tumor reduced growth by 3.5 times compared with the unexposed left tumor of same mice and 14.8 times compared with a group treated with a physical mixture of drugs. These results suggest that the nanotherapeutic system we developed induces site-specific inhibition of tumor growth at a high rate and has the potential to be used as a therapeutic regimen for spatiotemporal delivery of dual drugs for treatment of cancer.

Lung mechanics showing sex-based differences and circadian time-of-day response to bleomycin-induced lung injury in mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that impairs lung mechanical properties due to dysregulated extracellular matrix remodeling. Lung function assessment is an important physiological endpoint in the mouse model of pulmonary fibrosis (PF) that has gained a broader scientific acceptance in the field. IPF pathophysiology shows sex-based differences, disproportionately affecting more men compared to women. Prior reports suggest that the circadian clock is perturbed during the pathogenesis of PF. We have comprehensively assessed the sex-based differences and time-of-day response (at Zeitgeber time: ZT0/6:00 a.m. or ZT12/6 p.m.) in lung mechanics among sham (control) versus bleomycin (BLM)-challenged female and male (C57BL/6: WT) mice using Flexi-vent. BLM challenge altered lung function significantly in males in both total lung (reduced dynamic compliance, and increased resistance and elastance) as well as lung tissue-specific parameters (increased tissue elastance and tissue damping) but less pronounced in females. BLM-challenged mice showed a time-of-day response in lung function at ZT0 versus ZT12, which was pronounced in the ZT0 BLM group. Overall, these findings provide a comprehensive analysis of altered lung function in female and male mice and the time-of-day difference in lung function parameters following BLM-induced lung fibrosis.

Synthesis and biological evaluation of theranostic Trastuzumab-SN38 conjugate for Near-IR fluorescence imaging and targeted therapy of HER2+ breast cancer.

Here, we report on the design, synthesis, and biological evaluation of a new theranostic antibody drug conjugate (ADC), Cy5-Ab-SS-SN38, that consists of the HER2-specific antibody trastuzumab (Ab) connected to the near infrared (NIR) pentamethine cyanine dye Cy5 and SN38, which is a bioactive metabolite of the anticancer drug irinotecan. SN38 is bound to an antibody through a glutathione-responsive self-immolative disulfide carbamate linker. For the first time, we explored this linker in ADC and found that it to reduce the drug release rate, which is important for safe drug delivery. The developed ADC exhibited specific accumulation and nanomolar anti-breast cancer activity on HER2-positive (HER2+) cell lines but no effect on HER2-. Animals treated with this ADC exhibited good tolerance. In vivo studies have shown that the ADC had good targeting ability for HER2+ tumors with much higher anticancer potency than trastuzumab itself or a mixture of trastuzumab with SN38. Side-by-side HER2+/HER2-xenograft at the 10 mg/kg dose exhibited specific accumulation and reduction of HER2+ tumor but not accumulation or growth inhibition of HER2-counterpart. The self-immolative disulfide linker implemented in this study was proven to be successful, broadening its utilization with other antibodies for targeted anticancer therapy in general. We believe that the theranostic ADCs comprising the glutathione-responsive self-immolative disulfide carbamate linker are applicable for the treatment and fluorescent monitoring of malignancies and anticancer drug delivery.

A novel, dual action chimera comprising DNA methylating agent and near-IR xanthene-cyanine photosensitizer for combined anticancer therapy.

A facile synthesis, biological evaluation and photodynamic properties of novel activatable anticancer molecular hybrids (chimeras) Ch and I-Ch are described. The chimeras consist of DNA methylating methyl triazene moiety and fluorogenic xanthene-cyanine (XCy) or iodinated xanthene-cyanine (I-XCy) photosensitizer. These two anticancer core structures are bound by means of a self-immolative 4-aminobenzyl alcohol linker. The hydrolytic cleavage of the carbamate protecting group promotes activation of both DNA methylating monomethyl triazene and phototoxic xanthene-cyanine dye providing, in addition, a near-IR emission signal for detection of the drug activation events. Preliminary antiproliferative assay demonstrates that the developed chimeras exhibit higher antitumor activity in the breast cancer cell line upon near-IR light irradiation compared to their structural constituents, xanthene-cyanine photosensitizer and monomethyl triazene substance.

Discovery of Dolastatinol: A Synthetic Analog of Dolastatin 10 and Low Nanomolar Inhibitor of Tubulin Polymerization.

We developed a highly potent anticancer agent, dolastatinol, which is a methylene hydroxyl derivative of dolastatin 10. Dolastatinol is a synthetic analog of dolastatin 10, synthesized by a solid-phase peptide Fmoc chemistry protocol on 2-chlorotrityl chloride resin utilizing a pH-triggering self-immolative monosuccinate linker. The introduction of the C-terminus hydroxyl methylene functionality preserves the anticancer properties of the parent dolastatin 10, including strong suppression of the cell proliferation, migration, high cytotoxicity. Our research establishes a new facile route toward the further development of C-terminus-modified dolastatin-10-based microtubule inhibitors for anticancer treatment.

Targeted methylation facilitates DNA double strand breaks and enhances cancer suppression: A DNA intercalating/methylating dual-action chimera Amonafidazene.

A DNA intercalating agent Amonafide interferes with topoisomerase 2 (Topo II) activity and prevents re-ligation of DNA strands, leading to double strand breaks (DSB). If DSB repair fails, cells stop dividing and eventually die. In a search of approaches to enhance anti-cancer activities of Topo II inhibitors, we hypothesized that introduction of additional damage in proximity to the DSB may suppress DNA repair and enhance cancer cell killing. Accordingly, chimeric molecules were created that target a DNA alkylating component to the proximity of Topo II-induced DSBs. These chimeras consist of Amonafide or its 4-amino isomer, and DNA methylating methyl triazene moiety Azene protected with a carbamate group, connected via linker. Treatment of cancer cells with the chimeric molecules leads to significantly higher number of DSBs, which were repaired slower compared to Amonafide or monomethyl triazene-treated cells. On the other hand, methyl triazene linked to non-intercalating Amonafide analogs was ineffective. Together, these data strongly support our hypothesis. In line with increased DSBs, the chimeric molecules exhibited significantly higher antiproliferative activity in cancer cell lines compared to Amonafide or monomethyl triazene constituent Azene. We utilized the fluorescent properties of chimera Amonafidazene to develop ''photo-switchable'' reporting system to monitor the prodrug activation. Using this approach, we found that the chimera accumulated and was activated at the tumor sites specifically and demonstrated significantly stronger tumor suppressing activities compared to Amonafide in a xenograft model. Therefore, targeting alkylating groups to the proximity of DSB sites may become an effective approach towards enhancing anti-cancer activities of inhibitors of topoisomerases.

Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor.

Systemic toxicity caused by conventional chemotherapy is often regarded as one of the major challenges in the treatment of cancer. Over years, the trigger-based modality has gained much attention as it holds the spatiotemporal control over release and internalization of the drug. In this article, we are reporting an increase in the anti-tumor efficacy of curcumin due to ultrasound pulses. MDA MB 231 breast cancer and B16F10 melanoma cells were incubated with lecithin-based curcumin encapsulated nanoemulsions and exposed to ultrasound in the presence and absence of microbubble. Ultrasound induced sonoporation enhanced the cytotoxicity of curcumin in MDA MB 231 and B16F10 cancer cells in the presence of microbubble by 100- and 64-fold, respectively. To study the spatiotemporal delivery of curcumin, we developed B16F10 melanoma subcutaneous tumor on both the flanks of C57BL/6 mice but only the right tumor was exposed to ultrasound. Insonation of the right tumor spatially enhanced the cytotoxicity and enabled the substantial regression of the right tumor compared to the unexposed left tumor which grew continuously in size. This study showed that the ultrasound has the potential to target and increase the drug's throughput to the tumor and enable effective treatment.

Trigger responsive polymeric nanocarriers for cancer therapy.

Conventional chemotherapy for the treatment of cancer has limited specificity when administered systemically and is often associated with toxicity issues. Enhanced accumulation of polymeric nanocarriers at a tumor site may be achieved by passive and active targeting. Incorporation of trigger responsiveness into these polymeric nanocarriers improves the anticancer efficacy of such systems by modulating the release of the drug according to the tumor environment. Triggers used for tumor targeting include internal triggers such as pH, redox and enzymes and external triggers such as temperature, magnetic field, ultrasound and light. While internal triggers are specific cues of the tumor microenvironment, external triggers are those which are applied externally to control the release. This review highlights the various strategies employed for the preparation of such trigger responsive polymeric nanocarriers for cancer therapy and provides an overview of the state of the art in this field.