Oligomeric bile acid-mediated oral delivery of low molecular weight heparin

Al-Hilal, T.A.,Park, J.,Alam, F.,Chung, S.W.,Park, J.W.,Kim, K.,Kwon, I.C.,Kim, I.S.,Kim, S.Y.,Byun, Y. Elsevier Science Publishers 2014 Journal of controlled release Vol.175 No.-

Intestinal transporters are limited to the transport of small molecular substrates. Here, we describe the development of apical sodium-dependent bile acid transporter (ASBT)-targeted high-affinity oligomeric bile acid substrates that mediate the transmembrane transport of low molecular weight heparin (LMWH). Several oligomers of deoxycholic acid (oligoDOCA) were synthesized to investigate the substrate specificity of ASBT. To see the binding of oligoDOCA on the substrate-binding pocket of ASBT, molecular docking was used and the dissociation rate constants (K<SUB>D</SUB>) were measured using surface plasmon resonance. The K<SUB>D</SUB> for tetrameric DOCA (tetraDOCA) was 50-fold lower than that for monomeric DOCA, because tetraDOCA interacted with several hydrophobic grooves in the substrate-binding pocket of ASBT. The synthesized oligoDOCA compounds were subsequently chemically conjugated to macromolecular LMWH. In vitro, tetraDOCA-conjugated LMWH (LHe-tetraD) had highest selectivity for ASBT during its transport. Orally administered LHe-tetraD showed remarkable systemic anticoagulation activity and high oral bioavailability of 33.5+/-3.2% and 19.9+/-2.5% in rats and monkeys, respectively. Notably, LHe-tetraD successfully prevented thrombosis in a rat model of deep vein thrombosis. These results represent a major advancement in ASBT-mediated LMWH delivery and may facilitate administration of many important therapeutic macromolecules through a non-invasive oral route.

Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

Al-Hilal, Taslim A.,Chung, Seung Woo,Choi, Jeong Uk,Alam, Farzana,Park, Jooho,Kim, Seong Who,Kim, Sang Yoon,Ahsan, Fakhrul,Kim, In-San,Byun, Youngro American Society for Clinical Investigation 2016 The Journal of clinical investigation Vol.126 No.4

<P>Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis.</P>

Prevention effect of orally active heparin conjugate on cancer-associated thrombosis

Al-Hilal, T.A.,Alam, F.,Park, J.W.,Kim, K.,Kwon, I.C.,Ryu, G.H.,Byun, Y. Elsevier Science Publishers 2014 Journal of controlled release Vol.195 No.-

Thrombogenesis is a major cause of morbidity and mortality in cancer patients. Prophylaxis with low-molecular-weight heparin (LMWH) is recommended for cancer patients, but requires non-parenteral delivery methods for long-term treatments. In this study, we sought to generate a new oligomeric-bile acid conjugate of LMWH that can be used for oral delivery. We first synthesized a tetramer of deoxycholic acid (tetraDOCA), which was site-specifically conjugated at the end saccharide of LMWH. When LMWH-tetraDOCA conjugate (LHe-tetraD) was orally administered at a dose of 5mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62+/-0.05IU/mL without any evidence of liver toxicity, gastrointestinal damage, or thrombocytopenia. The cancer-associated thrombosis was induced in tumor-bearing mice by local heat application, and the fibrin deposition in tumors was evaluated. The oral administration of LHe-tetraD (either a single dose or multiple daily doses for up to 10days) in mice substantially abolished the coagulation-dependent tropism of fibrinogen in the heated tumors and significantly decreased hemorrhage, compared to the mice treated with saline or subcutaneous injection of LMWH. Thus, the anticoagulation effect of oral LHe-tetraD invokes the benefits of oral delivery and promises to provide an effective and convenient treatment for cancer patients at risk of thrombosis.

Phylogenetic Relationships of the Mutualistic Fungi Associated with Macrotermes subhyalinus in Oman

Hilal S. AlShamakhi,Abdullah M. Al-Sadi,Lyn G. Cook 한국균학회 2023 Mycobiology Vol.51 No.5

The symbiotic association between fungus-gardening termites Macrotermes and its fungalsymbiont has a moderate degree of specificity—although the symbiotic fungi (Termitomyces)form a monophyletic clade, there is not a one-to-one association between termite speciesand their fungus-garden associates. Here, we aim to determine the origin and phylogeneticrelationships of Termitomyces in Oman. We used sequences of the internal transcribed spacerregion (ITS) and the nuclear large subunit ribosomal RNA (LSU rRNA, 25S) gene and analyzedthese with sequences of Termitomyces from other geographic areas. We find no evidence formore than a single colonization of Oman by Termitomyces. Unexpectedly, we findTermitomyces in Oman is most closely related to the symbiont of M. subhyalinus in WestAfrica rather than to those of geographically closer populations in East Africa.

Design, Synthesis, and Therapeutic Evaluation of Poly(acrylic acid)–tetraDOCA Conjugate as a Bile Acid Transporter Inhibitor

Park, Jooho,Al-Hilal, Taslim A.,Jeong, Jee-Heon,Choi, Jeong uk,Byun, Youngro American Chemical Society 2015 Bioconjugate chemistry Vol.26 No.8

<P>Regulation of cholesterol and bile acid homeostasis has been attracting attention as a pharmaceutical target for the treatment of diseases, such as hypercholesterolaemia and type 2 diabetes. In recent years, small bile acid analogues have been developed for the purpose of apical sodium-dependent bile acid transporter (ASBT) inhibition. Here, we designed a novel hydrophilic ASBT inhibitor using oligomeric bile acid with a high affinity with ASBT. Polyacrylic acid-tetraDOCA conjugates (PATD) have the ability to bind to ASBT in order to induce hypocholesterolemic effects. Both the viability and the functionality of PATD were evaluated in vitro, showing that PATDs were effective in inhibiting the increases of cholesterol in the blood and oil in the liver induced by high fat diet (HFD). The results indicated that the newly developed biomaterials with oligomeric bile acids and a hydrophilic polymer are potent therapeutic agents for hyperlipidemia.</P>

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

Alam, F.,Al-Hilal, T.A.,Park, J.,Choi, J.U.,Mahmud, F.,Jeong, J.H.,Kim, I.S.,Kim, S.Y.,Hwang, S.R.,Byun, Y. IPC Science and Technology Press 2016 Biomaterials Vol.86 No.-

<P>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-beta 1 (TGF-beta 1) 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-beta 1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-beta 1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-beta 1 and CXCL12 (TGF-beta 1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-beta 1) 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 K-D values of TGF-beta 1 and CXCL12 with LHTD4 were 0.85 and 0.019 mu M respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-beta 1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-beta 1 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-beta 1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-beta 1 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-beta 1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Antiangiogenic and anticancer effect of an orally active low molecular weight heparin conjugates and its application to lung cancer chemoprevention

Kim, J.y.,Al-Hilal, T.A.,Chung, S.W.,Kim, S.Y.,Ryu, G.H.,Son, W.C.,Byun, Y. Elsevier Science Publishers 2015 Journal of controlled release Vol.199 No.-

Angiogenesis is well recognized as a pivotal process in tumor progression from the very early premalignant stages, thus making it an important target in cancer chemoprevention as well as in cancer treatment. In the present study, we introduce a recently developed oral heparin conjugate (LHTD4) for use as an inhibitor of angiogenesis and evaluate its therapeutic and preventive effects in two different animal models of lung cancer. The antiangiogenic activities of LHTD4 were evaluated using tube formation and Matrigel plug assays. VEGF- and bFGF-induced tube formations were reduced up to 77.2 and 67.3%, respectively, by LHTD4. Hemoglobin content was also significantly decreased by LHTD4 in the Matrigel plugs that were transplanted into mice. We observed that the VEGF- and bFGF-mediated phosphorylations of the receptors, VEGFR-2 and FGFR-1, were also inhibited by LHTD4. The in vivo antiangiogenic and anticancer effects of LHTD4 that developed following oral administration were verified in a tumor xenograft model of human A549 lung cancer cells: tumor volume was found to have decreased by 60.2% and the expressions of CD34 and Ki-67 in the LHTD4-treated group were affected. Finally, in a chemically induced lung carcinogenesis model, the number and area of each nodule were significantly reduced in the LHTD4-treated groups by 49.2% and 30.1%, respectively. In addition, the degree of angiogenesis in the lung tissue itself was decreased in the drug treatment group by 52.9%. Taken together, these results suggest that LHTD4 could be a promising candidate for angiogenesis inhibitor for the treatment and prevention of cancer.

Natural dye-sensitized ZnO nano-particles as photo-catalysts in complete degradation of E. coli bacteria and their organic content

Zyoud, A.,Dwikat, M.,Al-Shakhshir, S.,Ateeq, S.,Shteiwi, J.,Zu'bi, A.,Helal, M.H.S.,Campet, G.,Park, D.,Kwon, H.,Kim, T.W.,Kharoof, M.,Shawahna, R.,Hilal, H.S. Elsevier Sequoia 2016 Journal of photochemistry and photobiology Chemist Vol.328 No.-

<P>This communication describes for the first time how nano-size particles, sensitized with natural dye molecules of anthocyanin, can be used as catalysts in photo-degradation of gram negative Escherichia coli bacteria in water. The naked ZnO nano-particles degraded up to 83% of the bacteria under solar simulator light, while the dye-sensitized particles increased the bacterial loss by similar to 10%. Solar simulator light includes about 5% of UV tail (shorter than 400 nm) which means that both UV and visible light (longer than 400 nm) radiations could be involved. When a cut-off filter was used, the naked ZnO caused only 40% bacterial loss, in accordance with earlier literature that described killing of bacteria with ZnO particles both in the dark and under light. With the cut-off filter, the sensitized ZnO particles caused higher than 90% bacterial loss, which confirms sensitization of the ZnO particles to visible light. Moreover, the results show that the catalyzed photo-degradation process causes mineralization of the bacteria and their organic internal components which leach out by killing. The catalyst can be recovered and reused losing similar to 10% of its activity each time due to mineralization of the dye molecules. However, catalyst activity can be totally regained by re-sensitizing it with the anthocyanin dye. The effects of different experimental conditions, such as reaction temperature, pH, bacterial concentration and catalyst amount together with nutrient broth and saline media, will be discussed together with the role of the sensitizer. (C) 2016 Elsevier B.V. All rights reserved.</P>

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

( Farzana Alam ),( Taslim A. Al Hilal ),( Jooho Park ),( Jeong Uk Choi ),( Foyez Mahmud ),( Jee Heon Jeong ),( In San Kim ),( Sang Yoon Kim ),( Seung Rim Hwang ),( Youngro Byun ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

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 tetra-meric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1(TGF-β1) 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- βl and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF- βl and CXCL12. We carried out in vitro phosphorylation assays of the consecutive re-ceptors of TGF-βl and CXCL12 (TGF-βl R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF- βl) 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 ofTGF- β1 and CXCL12 with LHTD4 were 0.85 and 0.019 ,βM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-βl or CXCL 12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-βl R1 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-~l treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-βl and CXCL 12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models. the treatment with LHTD4 (5 mgjkg daily. p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-βl and CXCL12. and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity.

Absorption Mechanism of a Physical Complex of Monomeric Insulin and Deoxycholyl-<small>l</small>-lysyl-methylester in the Small Intestine

Mahmud, Foyez,Jeon, Ok-Cheol,Al-Hilal, Taslim A.,Kweon, Seho,Yang, Victor C.,Lee, Dong Soo,Byun, Youngro American Chemical Society 2015 MOLECULAR PHARMACEUTICS Vol.12 No.6

<P>Currently, oral administration of insulin still remains the best option to avoid the burden of repeated subcutaneous injections and to improve its pharmacokinetics. The objective of the present investigation was to demonstrate the absorption mechanism of insulin in the physical complexation of deoxycholyl-<SMALL>l</SMALL>-lysyl-methylester (DCK) for oral delivery. The oral insulin/DCK complex was prepared by making a physical complex of insulin aspart with DCK through ion-pair interaction in water. For the cellular uptake study, fluorescein-labeled insulin or DCK were prepared according to a standard protocol and applied to Caco-2 or MDCK cell lines. For the PK/PD studies, we performed intrajejunal administration of different formulation of insulin/DCK complex to diabetic rats. The resulting insulin and DCK complex demonstrated greatly enhanced lipophilicity as well as increased permeation across Caco-2 monolayers. The immunofluorescence study revealed the distribution of the complex in the cytoplasm of Caco-2 cells. Moreover, in the apical sodium bile acid transporter (ASBT) transfected MDCK, the insulin/DCK complex showed interaction with ASBT, and also demonstrated absorption through passive diffusion. We could not find that any evidence of endocytosis in relation to the uptake of insulin complex in vitro. In the rat intestine model, the highest absorption of insulin complex was observed in the jejunum at 1 h and then in the ileum at 2–4 h. In PK/PD study, the complex showed a similar PK profile to that of SC insulin. Overall, the study showed that the effect of DCK on enhancing the absorption of insulin resulted from transcellular processes as well as bile acid transporter activity.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/mpohbp/2015/mpohbp.2015.12.issue-6/mp500626a/production/images/medium/mp-2014-00626a_0009.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/mp500626a'>ACS Electronic Supporting Info</A></P>