Boris Vaisman

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron — providing protection to the ischemic heart via preconditioning (PC).

Paclitaxel tumor biodistribution and efficacy after intratumoral injection of a biodegradable extended release implant.

PURPOSE The aim of this study was to investigate the effectiveness of paclitaxel controlled release from intratumorally injected polymer. METHODS The effectiveness of paclitaxel-polymer formulation injected intratumorally was tested in mouse bladder tumor model. To determine paclitaxel biodistribution in tumor at predetermined time periods the tumor was excised, frozen and sectioned, and the paclitaxel concentrations were determined in the tumor tissue and in plasma by HPLC. Histopathological evaluation of the necrosis and inflammation was performed on tumor sections. RESULTS In the paclitaxel/polymer group mice were injected intratumorally with 0.2 ml of the 10% (w/w) paclitaxel formulation, the tumor disappeared completely 5 days after injection, and mice survived till the end of the study (50 days post-tumor cells inoculation). In biodistribution studies, the highest paclitaxel concentration in the tumor tissue was 40 microg/mg 1 day after the intratumoral injection and decreased gradually during 10 days to 5 microg/mg that is still high enough to induce cytotoxic effect, and the necrotic effect of paclitaxel on the tumors was confirmed by histopathology. CONCLUSIONS Treatment with local injection of polymer-paclitaxel formulation inhibited the growth of solid tumors. Distribution studies of paclitaxel after intratumoral injection showed high and effective drug concentrations in tumor.

Ferritin expression in maturing normal human erythroid precursors

We studied the expression of H‐ and L‐ferritin subunits at sequential stages of maturation of normal human erythroid precursors. The erythroid cells developed in liquid culture and were purified immunomagnetically before analysis. It was found that the content of both ferritin subunits decreased exponentially with maturation: the decrease was rapid when cellular haemoglobin was low, and it slowed down when the haemoglobin was increased. This mode of decline was especially pronounced for the L‐subunits. The H‐/L‐subunit ratio did not change significantly during the investigated period. The synthesis of both subunits was equal at each given developmental stage, and declined significantly with maturation. However, this decline was just slightly faster than that of total protein synthesis. The data indicated that the degradation of H‐ and L‐ferritin also declined as maturation proceeded. No decrease was observed in mRNA levels of either ferritin subunit. Thus, the ferritin content and turnover were maximal at the beginning of haemoglobin accumulation and diminished later. As the rate of ferritin turnover determines the rate of incorporation and release of its iron, the results presented suggest that ferritin mediates cellular iron transport and donates iron for haem synthesis, mainly at the beginning of haemoglobin accumulation. The synthesis of both ferritin subunits is regulated during erythroid maturation at the post‐transcriptional level.

Biocompatibility and safety evaluation of a ricinoleic acid-based poly(ester-anhydride) copolymer after implantation in rats

The aim of this study was to evaluate the safety and tissue compatibility of an injectable biodegradable poly(ester-anhydride) copolymer of ricinoleic acid (RA) and sebacic acid (SA) in rats. The absorbable biomaterial containing 70% w/w of RA and 30% w/w of SA [P(SA-RA) 3:7] was implanted in rats in two separate studies: (1) at high doses subcutaneously (SC) and intramuscularly (IM) simultaneously into the same animal and (2) intracranially (IC). The safety was established in the high-dose administration experiment. No systemic tissue damage, polymer-related lesions, or abnormalities could be detected in the animals. The histopathological evaluation of the SC and IM P(SA-RA) 3:7 implanted sites suggested a typical foreign body reaction (FBR) to biomaterials, and was characterized by excellent tissue repair and good tissue tolerance. In the second experiment, no neurological deficits or behavior changes suggestive of systemic or localized toxicity were observed in the animals implanted IC with the polymer. Only minimal, well-demarcated inflammatory response was observed on days 14 and 21 and consisted of glia cells. No abnormalities were noted in the brain tissue parenchyma located further from the edges of the implant. These results demonstrated that the P(SA-RA) 3:7 copolymer was tolerated well by the animals and compatible with rat subcutaneous, muscle and brain tissues. The biodegradable polymeric system described here could be used as a scaffold for varied applications in localized and sustained delivery of therapeutic agents.

Intravenous and Regional Paclitaxel Formulations

Paclitaxel has been proven to be effective against different types of cancer. A delivery system loaded with paclitaxel at tumor site should provide a high local concentration of the drug detrimental to malignant cells, which prevents the re-growth and metastasis of tumor. In this review, paclitaxel formulations for systemic and for intratumoral administration are discussed.

Cisplatin Tumor Biodistribution and Efficacy after Intratumoral Injection of a Biodegradable Extended Release Implant

Local delivery of chemotherapeutic drugs has long been recognized as a potential method for reaching high drug doses at the target site while minimizing systemic exposure. Cisplatin is one of the most effective chemotherapeutic agents for the treatment of various tumors; however, its systemic toxicity remains the primary dose-limiting factor. Here we report that incorporation of cisplatin into a fatty acid-based polymer carrier followed by a local injection into the solid tumor resulted in a successful tumor growth inhibition in heterotopic mouse bladder tumor model in mice. Platinum concentration in the tumor tissue surrounding the injected implant remained above the therapeutic level up to 14 days after the injection, while the plasma levels were several orders of magnitude lower comparing to systemic delivery. The reported delivery system increased the maximum tolerated dose of cisplatin 5 times compared to systemic delivery, thus potentially improving antitumor efficacy of cisplatin in solid tumor model.

In Vivo Degradation and Elimination of Injectable Ricinoleic Acid- Based Poly(ester-anhydride)

The in vivo degradation and elimination after subcutaneous implantation of injectable p(SA-RA) 3:7 copolymer in rats, followed by characterization of the polymer matrix composition during hydrolysis and erosion, is reported. Major chemical changes were observed during the first few days post implantation, the anhydride bonds hydrolyzed along with about 45% weight loss and a significant decrease in the molecular weight. 1H NMR spectral analysis was used to determine the structures and content of ricinoleic acid containing oligomeric chains present in the degraded polymer. The polymer degrades into ester oligomers of 2-4 ricinoleic acid units which further degrade to ricinoleic acid, a natural fatty acid. The polymer hydrolytic degradation process fit the in vitro degradation process.

Efficacy of poly(sebacic acid-co-ricinoleic acid) biodegradable delivery system for intratumoral delivery of paclitaxel

The effectiveness of an injectable polymeric formulation, based on poly(sebacic acid-co-ricinoleic acid) and paclitaxel against a heterotopic tumor model was studied. An injectable pasty polymer that releases an incorporated drug over a period of weeks was used. The degradation rate of formulations with paclitaxel was examined in vitro and in vivo. The effectiveness of the polymeric carrier of paclitaxel was investigated using a melanoma heterotopic model in C57BL/6 mice. Tumor bearing animals were injected intratumorally with 0.1 ml of formulations containing 5%, 10%, 15%, and 20% paclitaxel. Formulations with 5% and 10% paclitaxel content degraded faster in vivo then in vitro. Changes in tumor progression, survival time, and body weight were observed over a period of 77 days. The highest tumor size was reported for the control groups that did not receive paclitaxel in their treatment regiment: 3.6 g on day 20, while in all groups treated with polymer loaded with paclitaxel the tumor size was much smaller than that in the blank polymer or non treatment groups and ranged from 1.3 g to 0.3 g. Intratumoral injection of paclitaxel loaded in the polymer was found to be an effective treatment for localized tumors.

Isolation of Large and Pure Samples of Human Erythroid Precursors at Different Stages of Maturation Using Immunomagnetic Separation of Cells from Liquid Cultures

The two-phase liquid culture, which supports the development of human erythroid progenitors into hemoglobin-containing orthochromatic normoblasts, provides high yield of erythroblasts with synchronized development. However, other cell types are also present in the culture. In the present report, we used immunomagnetic separation for the purification of the developing erythroid precursors. At different stages of the culture, aliquots of the cells were incubated with anti-glycophorin A, B monoclonal antibodies, followed by anti-mouse IgG-coated magnetic beads, and separated by a magnet. In mature cultures, all isolated glycophorin-positive cells contained hemoglobin, as indicated by their staining with benzidine. All glycophorin-negative cells were benzidine negative. In earlier cultures, morphological examination and measurements of hemoglobin content showed that the erythroid precursors could be isolated at different stages of maturation. Thus, the combination of the liquid culture procedure with the immunomagnetic separation thechnique permitted to obtain large samples of pure erythroid cells, which were synchronized at subsequent stages of maturation. The method enables comprehensive studies of developing erythroid cells from normal donors as well as from patients with various disorders of erythropoiesis.

An ELISA for the H-subunit of human ferritin which employs a combination of rabbit poly- and mice monoclonal antibodies and an enzyme labeled anti-mouse-IgG.

Abstract We describe a sensitive ELISA for measuring the H-type subunit of human ferritin. A high detection sensitivity was attained by the use of antibodies from different species and an enzyme-conjugated secondary antibody. It consisted of a sandwich assay using a solid phase coated with a rabbit polyclonal antibody for human ferritin from term placenta and a soluble monoclonal antibody for human H-ferritin, followed by a secondary anti-mouse immunoglobulin (Ig)G conjugated to β-galactosidase. The assay was calibrated with purified recombinant human H-ferritin from E. coli. The colorigenic chlorophenol red β-D-galactopyranoside and the fluorogenic 4-methyl-umbelliferyl-β-D-galactopyranoside substrates were used with similar outcome. The described method permits the measurement of human H-ferritin at a concentration ranging from 0.1 to 100 μg/l (or 20–20000 pg per 200 μl sample) and is accurate at a concentration as low as 0.3 μg/l. The coefficient of variation of the assay was 6.05–10.3 % and the recovery of H-ferritin added to cell lysates was 105.8 ± 7.52 %. Depending on the H-ferritin content of the cell line tested, only 600 to 60 000 cells of different human cell lines were needed to measure their H-territin content.