Designing functional formulations of a taurocholate conjugated low molecular weight heparin derivative for targeting tumor angiogenesis and metastasis

Abstract

Heparin is a biomimetic molecule, which is commercially used as an anti-coagulant drug. But years of research have shown its efficacy in many other biological systems, such as, in immunology, angiogenesis and metastasis. Among all of the above mentioned effects, the anti-cancer effect of heparin showed promising results in many studies. However, unfractionated heparin cannot be used as anti-cancer drug because of its side effects, such as- heparin induced thrombocytopenia (HIT). So, low molecular weight heparin (LMWH) and its chemically modified heparin derivatives are under extensive studies as anti-cancer drugs in many cancer related fields. Previously, we synthesized several low molecular weight heparin moieties, which can be used as anticancer drugs. Among them seven taurocholate conjugated low molecular weight heparin derivative (LHT7) showed significant effect as an angiogenesis inhibitor. In our previous studies, LHT7 proved as highly effective in the inhibition of tumor angiogenesis by targeting multiple growth factors, such as, VEGF, bFGF, PDGF, HGF, etc. However, during safety evaluation in preclinical study, LHT7 showed unwanted accumulation in liver and kidney and thus showed functional abnormalities of these organs. Moreover, the half-life of LHT7 is short (2 h) and thus the modification in the delivery through either the change in administration or the development of effective formulation is necessary to increase the half-life with lowering the toxicological effect of this noble molecule. Finally, intravenous (i.v) administration of LHT7 could be less desirable to some patients for the maintenance anti-cancer therapy. So, developing patient compatible oral formulation is also a necessary part of the LHT7s development. As the LHT7 showed toxic effects upon daily administration to the animals in preclinical studies, we at first change the administration condition from i.v blous form to i.v infusion. At first, for the evaluation of safety, the method of intravenous infusion was compared with that of i.v bolus (at rate 400 μl/min/kg of body weight for 30 min). Then, for pharmacokinetic analysis, organ accumulation and plasma concentration profiles of LHT7 were measured. Furthermore, the anticancer effects of LHT7 were measured in murine (SCC7) and human xenograft (MDAMB-231) model, and the general safety studies were performed in beagle dogs. The result of the pharmacokinetic studies showed that the reduced organ accumulation in mice and the AUC(0-96h) was increased up to 1257.38 ± 0.11 h*μg/m. The efficacy, at dose 1mg/kg/2 days was higher for i.v infusion than for i.v bolus administration in both murine and human cancer model. The preliminary safety analysis of SD rats showed that there were no organ specific side effects in higher doses. So, from our study, we concluded that, LHT7 showed sustained effects with minimized adverse events when it is administered through i.v infusion. It was also shown that a maximum dose of 12 mg/kg (through i.v infusion) could be safely used for further clinical development of LHT7 as a multi-targeting anti-angiogenic agent. On the other hand, 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, with a protamine to form a self-assembling nanocomplex with a mean diameter of 200 nm with zeta potential of -14.2 ± 1.2 mV, which is designed to produce effective anti-angiogenic effect. At first, LHT7 was modified with poly-ethylene-glycol (PEG) and form nanocomplex with positively charged protamine (in buffer). We observed that PEG-LHT7/protamine nanocomplex was stable in buffer and slowly dissociated in plasma (9% dissociation after 24 h). Compared to the free form of PEG-LHT7, the mean residence time of PEG-LHT7/protamine nanocomplex was found higher (15.9 h) with its increased accumulation in tumor that was found in biodistribution study. Most importantly, PEG-LHT7/protamine nanocomplex was diffused and extravasated through the dense collagen matrix of tumor. Although the nanocomplex showed accumulation in liver, beside tumor in the biodistribution study, the serological evaluation of this nanocomplex did not show any functional variation of liver. 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 with minimized side effect. 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. Moreover, in biodistribution study, LHT7 mainly showed increased accumulationin tumor after 30 min of administration. 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. Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. As metastasis is a longer process, so the therapy should be more compatible to the patients. Considering these theories, we used an orally active polymeric bile acetylated taurocholate conjugated low molecular weight heparin derivative (LHTD4) with a formulation containing synthetic bile acid enhancer (DCK) to target human metastasis breast cancer. In case of breast cancer, TGFβ1 and CXCL12 possess enhanced metastatic activity during the initiation and the progression (seeding in other organs). So, in our study, we focus the binding effect of LHTD4 with TGFβ1 and CXCL12. We carried out computer simulation study and SPR analysis for the binding confirmation of LHTD4 with TGFβ1 and CXCL12. Here, we found that the KD values of TGFβ1 and CXCL12 with LHTD4 were 0.85 and 0.019 µM respectively. Moreover, the simulation showed stable binding affinities of the dp4 moieties of LHTD4 with through the strong electrostatic interaction. After confirming the binding affinity, we carried out in vitro phosphorylation assays of the consecutive receptors of TGFβ1 and CXCL12 (TGFβ1R1 and CXCR4 receptor respectively)

where the successful inhibition of the phosphorylation of those receptors was observed with the treatment of LHTD4. The expression changes of EMT marker proteins, such as, E-cadherin (degradation), Vimentin (increased expression) and SNAIL (increased expression), were prevented by the LTHD4 treatment in our in vitro studies with TGFβ1 treated MDAMB231 cells. Moreover, cell migration (induced by TGFβ1) and chemotaxis (mediated by CXCL12) of MDAMB-231 cells were inhibited by LHTD4 treatment. Finally, through accelerated lung metastasis model and by orthotopic MDAMB-231 breast cancer model, the metastasis inhibitory effect was evaluated in the mice. Finally, through metastasis inhibition analysis of the breast cancer cells in in vivo confirmed the anti-metastatic effect of LHTD4/DCK by inhibiting TGFβ1 and CXCL12. In conclusion, LHT7 is a noble molecule, which can be applied clinically to target tumor angiogenesis as a second line cancer therapy. With i.v infusion administration, LHT7 can be effective and thus increased tumor accumulation can showed less organ accumulation and reduced toxic events. More over, nanocomplex of PEGylated LHT7 with protamine showed increased the circulation time and tumor accumulation

thus proved to be reasonable to apply to the patients to avoid daily administration. Finally, for patient compliance and maintenance therapy oral formulation showed more applicability in the outdoor therapy with similar efficacy. Moreover, besides targeting angiogenesis, oral tetra-deoxycholate conjugated LHT7, LHTD4 can effectively use as an anti-metastasis drug by targeting TGFβ1 and CXCL12, which play key roles in the initiation and development of breast cancer metastasis.