Colchicine-Binding Site Agent CH-2-77 as a Potent Tubulin Inhibitor Suppressing Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2-M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.

Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics.

X-ray Crystallography-Guided Design, Antitumor Efficacy, and QSAR Analysis of Metabolically Stable Cyclopenta-Pyrimidinyl Dihydroquinoxalinone as a Potent Tubulin Polymerization Inhibitor.

Small molecules that interact with the colchicine binding site in tubulin have demonstrated therapeutic efficacy in treating cancers. We report the design, syntheses, and antitumor efficacies of new analogues of pyridopyrimidine and hydroquinoxalinone compounds with improved drug-like characteristics. Eight analogues, 5j, 5k, 5l, 5m, 5n, 5r, 5t, and 5u, showed significant improvement in metabolic stability and demonstrated strong antiproliferative potency in a panel of human cancer cell lines, including melanoma, lung cancer, and breast cancer. We report crystal structures of tubulin in complex with five representative compounds, 5j, 5k, 5l, 5m, and 5t, providing direct confirmation for their binding to the colchicine site in tubulin. A quantitative structure-activity relationship analysis of the synthesized analogues showed strong ability to predict potency. In vivo, 5m (4 mg/kg) and 5t (5 mg/kg) significantly inhibited tumor growth as well as melanoma spontaneous metastasis into the lung and liver against a highly paclitaxel-resistant A375/TxR xenograft model.

Orally available tubulin inhibitor VERU-111 enhances antitumor efficacy in paclitaxel-resistant lung cancer.

Lung cancer is the most common cause of cancer associated mortality. Chemotherapeutic agents, such as paclitaxel, are important treatment options but drug resistance often develops upon prolonged use. We report here the preclinical evaluation of a new orally available tubulin inhibitor, VERU-111, which can overcome several ABC-transporters mediated multi-drug resistance associated with taxane treatment. In vitro, VERU-111 prevents cell proliferation, invasion, migration and colony formation in both paclitaxel-sensitive and paclitaxel-resistant A549 lung cancer cells. VERU-111 effectively inhibits tubulin polymerization, arrests cells in G2/M phase, and induces cancer cell apoptosis. Further evaluation of various apoptotic proteins revealed that treatment of VERU-111 increases the expression of cleaved-PARP, cleaved-caspase-3 and p-histone H3 proteins. In vivo, orally administered VERU-111 in a paclitaxel-sensitive A549 xenograft model strongly inhibits tumor growth in a dose-dependent manner and is equally potent with paclitaxel. When tested in a highly paclitaxel-resistant A549/TxR tumor model, VERU-111 is as effective as the parental A549 model in significantly reducing the tumor volume, whereas paclitaxel is essentially ineffective. Collectively, this study showed that VERU-111 is a promising new generation of anti-tubulin agent for the treatment of taxane-resistant lung cancer.

Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53-deficient breast cancer treatment.

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

Oral pemetrexed facilitates low-dose metronomic therapy and enhances antitumor efficacy in lung cancer.

There is a growing interest in preclinical research to consider low-dose metronomic chemotherapy as antiangiogenic cancer treatment. Oral metronomic therapy, in particular, has shown much promise with its ease of daily administration and higher therapeutics window. In that regard, we developed oral pemetrexed using the physical complex with the bile acid enhancers (DCK). In a caco-2 permeability study, the oral pemetrexed/DCK complex had significantly higher drug uptake, and inhibited efflux transporter activity as well. We further observed that the mechanism of oral drug uptake was related to transcellular along with bile acid transporter mediated pathways. The oral administration of drug complex in rats revealed high bioavailability (22.37%) and extended mean residence time. Using SCC7 and A549 xenograft models, we demonstrated that antitumor effects from daily oral metronomic pemetrexed significantly reduced tumor in a dose-dependent manner. The antitumor activity of oral pemetrexed/DCK complex plus cisplatin was superior to both monotherapies. The xenograft study also revealed that oral metronomic therapy markedly reduced microvessel density, proliferation and increased apoptosis in the tumor tissues. Oral metronomic doses were significantly correlated with the elevation of plasma deoxyuridine level, an essential biomarker for pemetrexed therapy. One-month toxicity study confirmed that daily dosing of oral pemetrexed is safe by investigating apoptosis in the gut tissues from mice. Moreover, we analyzed different biochemical parameters and enzymes from the blood to prove that our newly developed oral pemetrexed complex is well tolerated.

Radiotherapy-assisted tumor selective metronomic oral chemotherapy.

Chemotherapy have commonly been used in maximum tolerated dose to completely eradicate the cancer. However, such treatments often failed due to the complex and dynamic nature of cancer. Therefore, it has been suggested that cancer should be treated as a chronic disease, controlling its growth by providing continuous therapeutic pressure for long-term. Such an approach, however, requires a therapy that is non-toxic and orally available with sufficient potency. Herein, we propose a radiotherapy-assisted orally available metronomic apoptosis-targeted chemotherapy, which delivers doxorubicin continuously to the irradiated tumor with high selectivity while causing minimal toxicities to the normal tissues. DEVD-S-DOX/DCK complex is the anticancer prodrug for our strategy that could selectively release doxorubicin in the irradiated tumor tissue with sufficient oral bioavailability. The prodrug was completely inactive by itself, but displayed potent anticancer activity when coupled with radiotherapy. Consequently, the daily oral administration of DEVD-S-DOX/DCK in combination with the low-dose radiotherapy effectively suppressed the growth of tumor in vivo with no significant systemic toxicities despite that the accumulated dose of doxorubicin exceeded 150 mg/kg. Therefore, the our novel therapy using DEVD-S-DOX/DCK complex is considered as an outstanding treatment option for treating cancer for long-term attributed to its oral availability and low-toxicity profile as well as the potent anticancer effect.

A heparin conjugate, LHbisD4, inhibits lymphangiogenesis and attenuates lymph node metastasis by blocking VEGF-C signaling pathway.

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.

Metronomic chemotherapy using orally active carboplatin/deoxycholate complex to maintain drug concentration within a tolerable range for effective cancer management.

Metronomic chemotherapy has translated into favorable toxicity profile and capable of delaying tumor progression. Despite its promise, conventional injectable chemotherapeutics are not meaningful to use as metronomic due to the necessity of frequent administration for personalized therapy in long-term cancer treatments. This study aims to exploit the benefits of the oral application of carboplatin as metronomic therapy for non-small cell lung cancer (NSCLC). We developed an orally active carboplatin by physical complexation with a deoxycholic acid (DOCA). The X-ray diffraction (XRD) patterns showed the disappearance of crystalline peaks from carboplatin by forming the complex with DOCA. In vivo pharmacokinetic (PK) study confirmed the oral absorption of carboplatin/DOCA complex. The oral bioavailability of carboplatin/DOCA complex and native carboplatin were calculated as 24.33% and 1.16%, respectively, when a single 50mg/kg oral dose was administered. Further findings of oral bioavailability during a low-dose daily administration of the complex (10mg/kg) for 3weeks were showed 19.17% at day-0, 30.27% at day-7, 26.77% at day-14, and 22.48% at day-21, demonstrating its potential for metronomic chemotherapy. The dose dependent antitumor effects of oral carboplatin were evaluated in SCC7 and A549 tumor xenograft mice. It was found that the oral carboplatin complex exhibited potent anti-tumor activity at 10mg/kg (74.09% vs. control, P<0.01) and 20mg/kg dose (86.22% vs. control, P<0.01) in A549 tumor. The number of TUNEL positive cells in the tumor sections was also significantly increased during oral therapy (3.95% in control, whereas 21.37% and 32.39% in 10mg/kg and 20mg/kg dose, respectively; P<0.001). The enhanced anti-tumor efficacy of oral metronomic therapy was attributed with its antiangiogenic mechanism where new blood vessel formation was notably decreased. Finally, the safety of oral complex was confirmed by three weeks toxicity studies; there were no significant systemic or local abnormalities found in mice at 10mg/kg daily oral dose. Our study thus describes an effective and safe oral formulation of carboplatin as a metronomic chemotherapy.

Multi-stage inhibition in breast cancer metastasis by orally active triple conjugate, LHTD4 (low molecular weight heparin-taurocholate-tetrameric deoxycholate).

Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-ß1 (TGF-ß1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-ß1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-ß1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-ß1 and CXCL12 (TGF-ß1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-ß1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-ß1 and CXCL12 with LHTD4 were 0.85 and 0.019 µM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-ß1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-ß1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-ß1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-ß1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-ß1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity.

Chemical Conjugate of Low Molecular Weight Heparin and Suramin Fragment Inhibits Tumor Growth Possibly by Blocking VEGF165.

Low molecular weight heparin (LMWH) and its derivatives have been reported to possess antiangiogenic effect via electrostatic interaction with various angiogenic growth factors such as VEGF165. However, clinical applications of LMWH for anticancer therapy have been restricted due to its anticoagulant effect and insufficient therapeutic efficacy. To overcome these limitations and enhance the antiangiogenic efficacy, LMWH was conjugated with suramin fragments that have a binding affinity to the heparin-binding domain (HBD) of proteins. The conjugation of suramin fragments to LMWH enhanced the antiangiogenic effect of LMWH by increasing the binding affinity to VEGF165, while decreasing its anticoagulant activity. The chemical conjugate of LMWH and suramin fragments (LHsura) showed a substantial inhibitory effect on VEGF165-mediated cell proliferation, migration, and tube formation of HUVECs without significant cytotoxicity in vitro. Finally, we confirmed the anticancer effect of LHsura (61.4% vs control) in a SCC7-bearing mouse model.

Functionalized heparin-protamine based self-assembled nanocomplex for efficient anti-angiogenic therapy.

Angiogenesis is a key feature of cancer development, thus it is a good target for cancer therapy. However, drugs that have been designed to block angiogenesis mainly capture growth factors in circulation, resulting not only in the transient inhibition of tumor progression but also in producing undesirable side effects. Nanoparticular drug delivery systems, on the other hand, may help overcome such drawbacks and improve the efficacy of anti-angiogenic therapies by altering the biodistribution and pharmacokinetics, improving tumor targeting ability, and reducing side effects. In this light, we propose a new approach of anti-angiogenic therapy that combines strategies of long circulating, passive tumor targeting, and anti-angiogenesis efficacy using a new polyelectrolyte complex system that combines LHT7, a previously developed heparin-based angiogenesis inhibitor, with a protamine to form a self-assembling nanocomplex with a mean diameter of 200nm, which is effective for anti-angiogenesis therapy. At first, LHT7 was modified with polyethylene glycol (PEG). We observed that PEG-LHT7/protamine nanocomplex was stable in buffer and slowly dissociated in plasma (9% dissociation for 24h). Compared to the free form of PEG-LHT7, the mean residence time of PEG-LHT7/protamine nanocomplex was found higher (15.9h) with its increased accumulation in tumor. Most importantly, PEG-LHT7/protamine nanocomplex was diffused and extravasated through the dense collagen matrix of the tumor. Thus, the study describes a successful application of functionalized PEG-LHT/protamine nanocomplex that can inhibit angiogenesis with long circulating, passive targeting, and tumor extravasating ability.

Oral delivery of a potent anti-angiogenic heparin conjugate by chemical conjugation and physical complexation using deoxycholic acid.

Angiogenesis, the formation of new blood vessels, plays a pivotal role in tumor progression and for this reason angiogenesis inhibitors are an important class of therapeutics for cancer treatment. Heparin-based angiogenesis inhibitors have been newly developed as one of such classes of therapeutics and possess a great promise in the clinical context. Taurocholate conjugated low molecular weight heparin derivative (LHT7) has been proven to be a potent, multi-targeting angiogenesis inhibitor against broad-spectrum angiogenic tumors. However, major limitations of LHT7 are its poor oral bioavailability, short half-life, and frequent parenteral dosing schedule. Addressing these issues, we have developed an oral formulation of LHT7 by chemically conjugating LHT7 with a tetrameric deoxycholic acid named LHTD4, and then physically complexing it with deoxycholylethylamine (DCK). The resulting LHTD4/DCK complex showed significantly enhanced oral bioavailability (34.3 ± 2.89%) and prolonged the mean residence time (7.5 ± 0.5 h). The LHTD4/DCK complex was mostly absorbed in the intestine by transcellular pathway via its interaction with apical sodium bile acid transporter. In vitro, the VEGF-induced sprouting of endothelial spheroids was significantly blocked by LHTD4. LHTD4/DCK complex significantly regressed the total vessel fractions of tumor (77.2 ± 3.9%), as analyzed by X-ray microCT angiography, thereby inhibiting tumor growth in vivo. Using the oral route of administration, we showed that LHTD4/DCK complex could be effective and chronically administered as angiogenesis inhibitor.