Improved Cellular Delivery of Antisense Oligonucleotides Using Transferrin Receptor Antibody-Oligonucleotide Conjugates

This work is dedicated to the memory of the late Dr Ian Walker.     

Short-Term Distribution, Metabolism, and Excretion of 2,2′,5-Tri-, 2,2′,4,4′-Tetra-, and 3,3′,4,4′-Tetrachlorobiphenyls in Prepubertal Rats

The excretion and tissue retention of three ¹⁴C-labeled lower chlorinated biphenyls were examined in prepubertal male and female Sprague-Dawley rats following IV administration. Urine and feces were collected individually at different time intervals up to 72 h for pharmacokinetic analyses. After 72 h, different organs were removed and extracted in acetone:hexane (1:1, v/v) to determine radioactivity. Within the first 10 h after dosing, 2,2′,5-trichlorobiphenyl (PCB 18) was rapidly excreted in urine (8–18% of the administered dose), whereas only 0.6–0.8% of 2,2′,4,4′-tetrachlorobiphenyl (PCB 47) and 0.3–0.8% 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) were found in urine during this time period. The half-life of elimination was shortest for PCB 18 (37.5 to 49.2 h). The half-lives for PCB 47 and PCB 77 were 351 to 672 h and 152 to 186 h, respectively. The cumulative total excretion (urinary + fecal) of PCB 18 within 72 h was 51–62%, of PCB 77 was 22–25%, and of PCB 47 was 7–10%. No parent PCBs were detected in urine. PCB 47 accumulated preferentially in adipose tissues (subcutaneous fat > mesenteric fat); relatively high levels of PCB 47 were also found in adrenals, ovaries, lungs, liver, and skin. The highest concentration of PCB 77 was found in serum, followed by adipose tissues. Very low concentrations of PCB 18 were found in most tissues; the highest being found in serum, followed by ovaries and adrenal glands. This study suggests that prepubertal rats retain higher short-term serum levels and have lower excretion rates than adult rats. Received: 3 February 1998/Accepted: 16 August 1998     

Toxicity and tissue distribution of 2,2',4,4'- and 3,3',4, 4'-tetrachlorobiphenyls in houseflies

Insects selectively retain different polychlorinated biphenyls (PCBs) which are then contributed to the food chain. To quantitate specific differences, adult female houseflies (Musca domestica L.) were topically dosed with 0.5 microgram of two structurally distinct PCB congeners (14C-2,2',4,4'-tetraCB or 14C-3,3',4,4'-tetraCB). Total radioactivity in the acetone rinse of intact flies, several tissues, and excrement was determined at 11 time points over a 48-h period. Ninety-seven percent of the applied 2,2',4,4'-tetraCB disappeared from the surface following an initial rapid absorption of 79% within 3 h. The absorbed 2,2',4,4'-tetraCB was immediately found within the thoracic cuticle, then spreading laterally to the abdominal cuticle and head, through the wax layers, or penetrating to the alimentary canal and ovaries. Penetration of 3,3',4, 4'-tetraCB was markedly slower; even though 87% of the applied dose was absorbed within 48 h, only 19% of the dose penetrated into the fly body within the first 3 h. This PCB very slowly distributed into the tissues. Toxicities of the sublethal doses were determined by monitoring changes in activity of houseflies following dosing with 0. 5 microg of radioactive PCBs. Flies treated with 2,2',5-triCB and 3, 3',4,4'-tetraCB remained very active, whereas 2,2',4, 4'-tetraCB-treated flies were less active, consistent with the previously reported toxicity of this congener.     

Association between vitamin D deficiency and serum Homocysteine levels and its relationship with coronary artery disease

Journal of Thrombosis and Thrombolysis - Homocysteine (Hcy) elevation and vitamin D deficiency have emerged as potential markers of coronary artery disease (CAD). However, even tough...     

Diabetes-induced renal vascular dysfunction is normalized by inhibition of epidermal growth factor receptor tyrosine kinase

Contribution of receptor tyrosine kinase activation to development of diabetes-induced renal artery dysfunction is not known. We investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of tyrosine kinases (TKs), and AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) TK activity, to modulate the altered vasoreactivity of isolated renal artery ring segments to common vasoconstrictors in streptozotocin-induced diabetes. In diabetic renal artery, the vasoconstrictor responses induced by norepinephrine, endothelin-1 and angiotensin II were significantly increased. Inhibition of TKs or the EGFR pathway did not affect the agonist-induced vasoconstrictor responses in the non-diabetic control animals. However, inhibition of TKs by genistein or EGFR TK by AG1478 treatment produced a significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. Treatment with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor responses in the diabetic animals. Western blotting showed that phosphorylated EGFR protein levels were increased in vehicle-treated diabetic animals. In renal arteries from AG1478-treated diabetic animals, EGFR protein levels were similar to non-diabetic control animals. These data suggest that activation of TK-mediated pathways, including the EGFR TK signalling pathway, are involved in the development of diabetic vascular dysfunction in the renal artery.     

Toxicity of fungicides to Pisum sativum: a study of oxidative damage,growth suppression,cellular death and morpho-anatomical changes

Considering the fungicidal threat to the sustainable agro-environment, the toxicological impacts of three fungicides, namely kitazin, hexaconazole and carbendazim, on the biological, chemical and morpho-anatomical changes of peas were assessed. Fungicide applications in general caused a slow but gradual reduction in growth, symbiosis and yields of peas, which, however, varied appreciably among species and concentrations of the three fungicides. Of the three fungicides, carbendazim had the most lethal effect, in which it delayed seed germination and also diminished the overall pea growth. Carbendazim at 3000 μg kg⁻¹ maximally reduced the germination, SVI, size of roots and shoots and total dry matter accumulation in roots, shoots and whole plants distinctly by 40%, 84%, 72%, 73%, 68%, 75% and 73% (p ≤ 0.05), respectively. Hexaconazole at 120 μg kg⁻¹ significantly (p ≤ 0.05) declined total chlorophyll, carotenoids, grain yields, grain protein, root P and shoot N by 19%, 28%, 46%, 69%, 48% and 51%, respectively, over the control. The synthesis of stress biomarkers and oxidative stress were increased with increasing dosage rates of fungicides. Proline content in roots, shoots, leaves and grains, MDA, electrolyte leakage and H₂O₂ of plants grown in soil treated with 288 μg kg⁻¹ kitazin were increased significantly (p ≤ 0.05) by 73%, 52%, 41%, 24%, 59%, 40% and 27%, respectively, relative to the control. Antioxidant defence enzymes were greater in pea foliage. The SEM and CLSM images revealed an obvious alteration in root tips, enhanced cellular damage and cell death when plants were raised under fungicide stress. Also, morpho-anatomical variations in fungicide-treated foliage were visible in the SEM images. Overall, the present study suggests that a careful and secure strategy should be adopted before fungicides are chosen for enhancing pulse production in different agro-climatic regions.

Considering the fungicidal threat to the sustainable agro-environment, the toxicological impacts of three fungicides, namely kitazin, hexaconazole and carbendazim, on the biological, chemical and morpho-anatomical changes of peas were assessed.     

Results of ACDF using Alexandria Modular Cage-Plate Construct in cervical canal stenosis

Background

The purpose of this study was to assess prospectively operative results and complications of treatment of cervical spinal canal stenosis (CCS) by anterior cervical discectomy and fusion (ACDF) using a newly introduced Alexandria Modular Cage-Plate Construct (AMCPC).

Methods

Fifteen patients (eight males, seven females) with symptomatic CCS were treated by ACDF, with a mean age of 51.2 years. Post-operative assessment depended upon clinical and radiological results.

Results

Total number of operated levels was 25 levels, with a mean 1.67 levels/patient. Mean operative time was 69.6 min/level and 116 min/patient. Average blood loss was 78 mL/level and 130 mL/patient. Mean hospital stay was 2.8 days. Post-operative dysphagia/dysphonia persisted in one patient for 1 year. One patient developed C5 radiculopathy with grade 2 deltoid weakness that recovered after 3 months. According to Odom’s criteria, results were excellent in 13 patients (86.67 %), good in 1 (6.67 %), and fair in 1 patient. In 13 patients (23 cage-plates, 92 %), the implant showed to be completely contained until the end of follow-up (24 months). One patient had a broken screw and one had screw backing-out, both did not necessitate revision. The achieved sagittal profile was maintained without sinking-in of any cage.

Conclusion

AMCPC can be used safely for a variety of disorders requiring instrumentation and fusion. It is advantageous to stand-alone cage and to rigid cage-plates when intraoperative flexibility is needed. It overcomes the disadvantages of stand-alone cage, sinking-in, cage dislodgement and post-operative rekyphosis. In addition, it obviates the need for wearing post-operative neck collar.     

Ocular findings in aplastic anemia

OBJECTIVE: To analyze the ocular findings in aplastic anemia. Design: Eighteen patients with aplastic anemia were examined. Results: Ocular findings included cotton wool spots (38%), nerve fiber layer or preretinal hemorrhages (67%), vitreous hemorrhages (13%), a picture resembling central retinal vein occlusion (13%) and optic disk edema (6%). Preretinal hemorrhages were the presenting sign of aplastic anemia in 2 patients (13%). CONCLUSIONS: A blood profile is needed in patients with unexplained retinal hemorrhages. Patients with aplastic anemia need to avoid ocular massage and Valsalva maneuvers to decrease ocular morbidity.     

Co-delivery of an antisense oligonucleotide and 5-fluorouracil using sustained release poly (lactide-co-glycolide) microsphere formulations for potential combination therapy in cancer

Antisense oligonucleotides (AODNs) can selectively inhibit oncogene expression by Watson-Crick hybridisation to target mRNA and are being increasingly considered for use in combination with conventional drugs for potential anticancer therapy. Combination therapy of AODNs and cytotoxic agents using biodegradable polymeric delivery systems potentially offers several advantages including site-specific or organ-directed targeting, protection from digesting enzymes, and improved pharmacokinetics/pharmacodynamics resulting from sustained delivery of the entrapped drugs. Using a model AODN targeting the epidermal growth factor receptor (that is over-expressed in several cancers including breast and brain cancer) and the commonly used cytotoxic agent, 5-fluorouracil (5-FU), we have examined the use of poly (lactide-co-glycolide) (P(LA-GA)) microsphere formulations for co-delivery of these agents. Both agents were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations and release profiles determined in vitro. Using a double emulsion method for preparing the P(LA-GA) microspheres suitable entrapment and sustained release over 35 days was observed in both types of formulation. Release of AODN and 5-FU from all formulations appeared to be biphasic. However, the release rates of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Electrophoretic mobility shift assays suggested that this might be, in part, due to an interaction of 5-FU with the oligodeoxynucleotide (ODN). Further, our data suggest that by mixing individual formulations of 5-FU and ODNs at different mass ratios allowed greater flexibility in achieving the desired release profile as well as avoiding potential drug-drug interactions. Thus, co-administration of individual P(LA-GA) microsphere formulations of AODNs and 5-FU, at appropriate mass ratios, appears worthy of further investigation for the potential co-delivery of these anti-cancer agents in vivo.     

Toxicogenomics of cationic lipid-based vectors for gene therapy: impact of microarray technology

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.