Farzana Alam

Oral drug delivery systems using chemical conjugates or physical complexes

Oral delivery of therapeutics is extremely challenging. The digestive system is designed in a way that naturally allows the degradation of proteins or peptides into small molecules prior to absorption. For systemic absorption, the intact drug molecules must traverse the impending harsh gastrointestinal environment. Technologies, such as enteric coating, with oral dosage formulation strategies have successfully provided the protection of non-peptide based therapeutics against the harsh, acidic condition of the stomach. However, these technologies showed limited success on the protection of therapeutic proteins and peptides. Importantly, inherent permeability coefficient of the therapeutics is still a major problem that has remained unresolved for decades. Addressing this issue in the context, we summarize the strategies that are developed in enhancing the intestinal permeability of a drug molecule either by modifying the intestinal epithelium or by modifying the drug itself. These modifications have been pursued by using a group of molecules that can be conjugated to the drug molecule to alter the cell permeability of the drug or mixed with the drug molecule to alter the epithelial barrier function, in order to achieve the effective drug permeation. This article will address the current trends and future perspectives of the oral delivery strategies.

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

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 (KD) were measured using surface plasmon resonance. The KD 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.

Functional transformations of bile acid transporters induced by high-affinity macromolecules.

Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery.

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.

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.

Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

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.

Preliminary safety evaluation of a taurocholate-conjugated low-molecular-weight heparin derivative (LHT7): a potent angiogenesis inhibitor

In our previous studies, taurocholic acid (TA)‐conjugated low‐molecular‐weight heparin derivative (LHT7) has been proven to be a potent anti‐angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism‐related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg–1 doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg–1 doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism‐related studies showed that blood pressure at 50 mg kg–1 did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg–1 repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material. Copyright

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

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 5 mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62±0.05 IU/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 10 days) 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.

Multi-Purposable Filaments of HPMC for 3D Printing of Medications with Tailored Drug Release and Timed-Absorption

Graphical abstract Figure. No Caption available. Abstract Three‐dimensional printing (3DP), though developed for nonmedical applications and once regarded as futuristic only, has recently been deployed for the fabrication of pharmaceutical products. However, the existing feeding materials (inks and filaments) that are used for printing drug products have various shortcomings, including the lack of biocompatibility, inadequate extrudability and printability, poor drug loading, and instability. Here, we have sought to develop a filament using a single pharmaceutical polymer, with no additives, which can be multi‐purposed and manipulated by computational design for the preparation of tablets with desired release and absorption patterns. As such, we have used hydroxypropyl‐methylcellulose (HPMC) and diltiazem, a model drug, to prepare both drug‐free and drug‐impregnated filaments, and investigated their thermal and crystalline properties, studied the cytotoxicity of the filaments, designed and printed tablets with various infill densities and patterns. By alternating the drug‐free and drug‐impregnated filaments, we fabricated various types of tablets, studied the drug release profiles, and assessed oral absorption in rats. Both diltiazem and HPMC were stable at extrusion and printing temperatures, and the drug loading was 10% (w/w). The infill density, as well as infill patterns, influenced the drug release profile, and thus, when the infill density was increased to 100%, the percentage of drug released dramatically declined. Tablets with alternating drug‐free and drug‐loaded layers showed delayed and intermittent drug release, depending on when the drug‐loaded layers encountered the dissolution media. Importantly, the oral absorption patterns accurately reproduced the drug release profiles and showed immediate, extended, delayed and episodic absorption of the drug from the rat gastrointestinal tract (GIT). Overall, we have demonstrated here that filaments for 3D printers can be prepared from a pharmaceutical polymer with no additives, and the novel computational design allows for fabricating tablets with the capability of producing distinct absorption patterns after oral administration.

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

&NA; 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 50 mg/kg oral dose was administered. Further findings of oral bioavailability during a low‐dose daily administration of the complex (10 mg/kg) for 3 weeks 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 10 mg/kg (74.09% vs. control, P < 0.01) and 20 mg/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 10 mg/kg and 20 mg/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 10 mg/kg daily oral dose. Our study thus describes an effective and safe oral formulation of carboplatin as a metronomic chemotherapy. Graphical abstract Figure. No caption available.