Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs

Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while diminishing its action against other cellular targets. Thus, a set of NDGA analogs (7a-7h) was prepared and examined for inhibitory potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs was based upon a cursory Topliss approach in which one of NDGA's aromatic rings was modified with various substituents. Structural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may serve as a new lead in developing novel therapeutic agents for breast cancer.     

A novel drug delivery system for type 1 diabetes: insulin-mimetic vanadyl-poly(gamma-glutamic acid) complex

Insulin-mimetic vanadyl-poly(gamma-glutamic acid) complex, VO-gamma-PGA, is proposed as a novel drug delivery system for treating type 1 diabetic animals. The structure of VO-gamma-PGA in solution as well as in solid state was analyzed by electronic absorption, infra-red, and electron spin resonance spectra, and proposed that the equatorial coordination mode of VO(2+) is in either carboxylate(O)-VO-(OH(2))(3) or 2 carboxylate(O(2))-VO-(OH(2))(2). In vitro insulin-mimetic activity, metallokinetic feature in the blood of healthy rats, and in vivo normoglycemic effect of the complex prepared in solution were evaluated in streptozotocin(STZ)-induced type 1 diabetic mice, and these effects were compared with those of a solution containing only VOSO(4) as a positive control. The in vitro insulin-mimetic activity of VO-gamma-PGA was examined by determining both inhibition of free fatty acid (FFA) release and glucose uptake in isolated rat adipocytes, in which the concentration of VO-gamma-PGA for 50% inhibition of FFA release was significantly lower than that of VOSO(4). Metallokinetic study suggested that the bioavailability of VO-gamma-PGA complex was much higher than that of VOSO(4). The complex showed a significant hypoglycemic activity within at least 4h after a single oral administration, the effect being sustained for at least 24h. Furthermore, VO-gamma-PGA normalized the hyperglycemia in STZ-mice within 3 days when it was given orally at doses of 5-10mgVkg(-1) body mass for 16 days. The improvement in diabetes was also supported by the results on oral glucose tolerance test, HbA(1c) levels, and blood pressure.     

Modification of sterculia gum with methacrylic acid to prepare a novel drug delivery system

The present paper deals with the modification of the sterculia gum with methacrylic acid (MAAc) to hydrogels for use in drug delivery. The hydrogels were characterized by SEMs, FTIR and swelling studies. The release dynamics of model anti-ulcer drug (ranitidine hydrochloride) from the hydrogels has been studied for the evaluation of the release mechanism. The values of the diffusion exponent 'n' (0.55, 0.54 and 0.59) and gel characteristic constant 'k' (2.109 x 10(-2), 3.698 x 10(-2) and 2.427 x 10(-2)) have been obtained, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer. The release of the drug from the hydrogels occurred through non-Fickian diffusion mechanism.     

Development and in vitro evaluation of thiolated chitosan--Poly(methacrylic acid) nanoparticles as a local mucoadhesive delivery system

The main objective of this study was to develop a local, oral mucoadhesive metronidazole benzoate (MET) delivery system that can be applied and removed by the patient for the treatment of periodontal diseases. The results of present study revealed that the retention time of MET at its absorption site could be increased by formulating it into nanoparticles using thiolated chitosan (TCS)-poly(methacrylic acid) (PMAA). The nanoparticles of MET prepared from TCS-PMAA may represent a useful approach for targeting its release at its site of absorption, sustaining its release and improving its oral availability.     

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.     

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery

N_ω-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N_ω-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N_ω-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8–40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.Revisions requested 8 April 2005; Revisions received 6 May 2005     

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.     

A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.     

Specific antitumor targetable beta-cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconjugate

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to beta-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA specifically interacts with immobilized folate binding protein (FBP) while the naked beta-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD-PEG-FA was devoid of cell toxicity. [(3)H]-folic acid/CD-PEG-FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD-PEG-FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M(-)(1) inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD-PEG-FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded beta-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD-PEG-FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.     

Mutagenic evaluation of tromaril (N-beta-phenylethylanthranilic acid), a novel anti-inflammatory drug, using mammalian test systems

The mutagenic effect of tromaril, a new anti-inflammatory drug, was assessed in Swiss male mice employing bone-marrow micronucleus induction, abnormal sperm formation, and the induction of meiotic chromosome anomalies in spermatocytes as the test parameters. Administration of tromaril induced significantly higher percentages of MN and abnormal sperm at 600 and 900 mg/kg body wt., whereas chromosomal anomalies in spermatocytes were significantly higher at all the 3 doses of 300, 600 and 900 mg/kg body wt. at time intervals of 7, 15 and 30 days as compared to parallel controls.     

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeobacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

Nanotechnology based drug delivery system(s) for the management of tuberculosis

The era of nanotechnology has allowed new research strategies to flourish in the field of drug delivery. Nanoparticle-based drug delivery systems are suitable for targeting chronic intracellular infections such as tuberculosis. Polymeric nanoparticles employing poly lactide-co-glycolide have shown promise as far as intermittent chemotherapy in experimental tuberculosis is concerned. It has distinct advantages over the more traditional drug carriers, i.e. liposomes and microparticles. Although the experience with natural carriers, e.g. solid lipid nanoparticles and alginate nanoparticles is in its infancy, future research may rely heavily on these carrier systems. Given the options for oral as well as parenteral therapy, the very nature of the disease and its complex treatment urges one to emphasize on the oral route for controlled drug delivery. Pending the discovery of more potent antitubercular drugs, nanotechnology-based intermittent chemotherapy provides a novel and sound platform for an onslaught against tuberculosis.     

Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.     

Acid- and salt-triggered multifunctional poly(propylene imine) dendrimer as a prospective drug delivery system

A novel dendrimeric compound is designed with the objective of simultaneously addressing issues commonly encountered in drug delivery, i.e., stability in biological milieu as well as targeting. For this purpose, a multifunctional dendrimeric system derived from diaminobutane poly(propylene imine) dendrimers (DAB) is prepared bearing at its external surface poly(ethylene glycol) chains and guanidinium moieties. For these moieties, it has been established that they exhibit protective and targeting properties, respectively. The release of encapsulated compounds is triggered by titration with acids followed by the addition of sodium chloride solution. Specifically for pyrene, the solubilization site of which can be clearly traced, protonation leads to a distribution between the core and the poly(ethylene glycol) chains in the periphery of the dendrimer while it is released to the aqueous bulk solution by the addition of sodium chloride. The release of betamethasone valerate is also triggered by the addition of sodium chloride solution.     

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Transport characteristics of a novel peptide platform for CNS therapeutics

New and effective therapeutics that cross the blood‐brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep‐2 peptide, a sequence derived from ligands that bind to low‐density lipoprotein receptor‐related protein‐1 (LRP‐1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep‐2 conjugate and immunohistochemical studies of injected Angiopep‐2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co‐localization with the neuronal nuclei‐selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [¹²⁵I]‐Angiopep‐2 into brain endothelial cells occurred by a saturable mechanism involving LRP‐1. The primary sequence and charge of Angiopep‐2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.