Dionyssis S. Ithakissios

PLGA-MPEG NANOPARTICLES OF CISPLATIN: IN VITRO NANOPARTICLE DEGRADATION, IN VITRO DRUG RELEASE AND IN VIVO DRUG RESIDENCE IN BLOOD PROPERTIES

The in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties of PLGA-mPEG nanoparticles of cisplatin were investigated. The nanoparticles were prepared by a double emulsion method and characterized with regard to their morphology, size, zeta potential and drug loading. The rate of in vitro degradation of the PLGA-mPEG nanoparticles in PBS (pH 7.4) depended on their composition, increasing when the mPEG content (mPEG:PLGA ratio) of the nanoparticles increased. Sustained cisplatin release over several hours from the PLGA-mPEG nanoparticles in vitro (PBS) was observed. The composition of the nanoparticles affected drug release: the rate of release increased when the mPEG content of the nanoparticles increased. Within the range of drug loadings investigated, the drug loading of the nanoparticles did not have any significant effect on drug release. The loading efficiency was low and needs improvement in order to obtain PLGA-mPEG nanoparticles with a satisfactory cisplatin content for therapeutic application. The i.v. administration of PLGA-mPEG nanoparticles of cisplatin in BALB/c mice resulted in prolonged cisplatin residence in systemic blood circulation. The results appear to justify further investigation of the suitability of the PLGA-mPEG nanoparticles for the controlled i.v. delivery and/or targeting of cisplatin.

Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA-mPEG nanoparticles.

The effect of nanoparticle dose on the biodistribution and pharmacokinetics of conventional PLGA and stealth poly(Lactide-co-glycolide)-monomethoxypoly(ethyleneglycol) (PLGA-mPEG) nanoparticles was investigated. The precipitation-solvent diffusion method was used to prepare PLGA and PLGA-mPEG nanoparticles labeled with 125I-cholesterylaniline. These were administered intravenously (i.v.) in mice and at predetermined time intervals the animals were sacrificed and their tissues were excised and assayed for radioactivity. Within the dose range applied in this study, blood clearance and mononuclear phagocyte system (MPS) uptake of the PLGA nanoparticles depended on dose whereas they were independent of dose in the case of the PLGA-mPEG nanoparticles. Increasing the dose, decreased the rates of blood clearance and MPS uptake of the PLGA nanoparticles, indicating a certain degree of MPS saturation at higher doses of PLGA nanoparticles. The dose affected the distribution of PLGA nanoparticles between blood and MPS (liver) but it did not affect the nanoparticle levels in the other tissues. Within the range of doses applied here, the PLGA nanoparticles followed non-linear and dose-dependent pharmacokinetics whereas the PLGA-mPEG nanoparticles followed linear and dose-independent pharmacokinetics. In addition to the prolonged blood residence, the dosage-independence of the pharmacokinetics of the PLGA-mPEG nanoparticles would provide further advantages for their application in controlled drug delivery and in drug targeting.

Effect of copolymer composition on the physicochemical characteristics, in vitro stability, and biodistribution of PLGA–mPEG nanoparticles

The physicochemical properties, the colloidal stability in vitro and the biodistribution properties in mice of different PLGA-mPEG nanoparticle compositions were investigated. The nanoparticles were prepared by a precipitation-solvent evaporation technique. The physical characteristics and the colloidal stability of the PLGA-mPEG nanoparticles were significantly influenced by the composition of the PLGA-mPEG copolymer used to prepare the nanoparticles. PLGA-mPEG nanoparticles prepared from copolymers having relatively high mPEG/PLGA ratios were smaller and less stable than those prepared from copolymers having relatively low mPEG/PLGA ratios. All PLGA-mPEG nanoparticle compositions exhibited prolonged residence in blood, compared to the conventional PLGA nanoparticles. The composition of the PLGA-mPEG copolymer affected significantly the blood residence time and the biodistribution of the PLGA-mPEG nanoparticles in liver, spleen and bones. The in vivo behavior of the different PLGA-mPEG nanoparticle compositions did not appear to correlate with their in vitro stability. Optimum mPEG/PLGA ratios appeared to exist leading to long blood circulation times of the PLGA-mPEG nanoparticles. This may be associated with the effects of the mPEG/PLGA ratio on the density of PEG on the surface of the nanoparticles and on the size of the nanoparticles.

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.

Effect of preparative variables on the properties of poly(dl-lactide-co-glycolide)-methoxypoly(ethyleneglycol) copolymers related to their application in controlled drug delivery.

The effect of certain preparative variables, such as the composition of the feed, the reaction time and the reaction temperature, on the properties of prepared poly(dl-lactide-co-glycolide)-methoxypoly(ethyleneg lycol) (PLGA-mPEG) copolymers and on the yield of the reaction was investigated. The results with regard the molecular weight and yield were discussed in relation to a polymerization mechanism proposed recently (Du et al., 1995. Macromolecules 28, 2124-2132). The higher the PEG content of the feed the lower the molecular weight of the copolymer and the yield of the reaction. The breadth of the molecular weight distribution decreased initially with time, but appeared to stabilize later at low values. Both the ethylene oxide content and the lactide to glycolide molar ratio in the copolymer depended on the reaction temperature and varied with the reaction time. PLGA and mPEG appeared to be partially miscible, and copolymers containing approximately 40% mol or higher ethylene oxide exhibited crystallinity.

Analytical techniques for determining biotin

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Synthesis and angiogenetic activity in the chick chorioallantoic membrane model of thymosin beta-15.

Thymosin beta-15 (Tbeta15), a 44 amino acid peptide (MW = 5173) localized in human prostate and breast cancer tissues was successfully synthesized in multigram quantities using Fmoc solid-phase peptide synthesis. The synthesized product was shown to have the right structure by ESI and MALDI mass spectral techniques and amino acid analysis. Relatively high yield was achieved, which might be due to enhanced acid stability of the p-cyanotrityl resin used. The effect of the synthesized Tbeta15 on the angiogenesis process was investigated using the chick chorioallantoic membrane (CAM) in vivo model. At concentrations above 1 microg/10 microl per disc, Tbeta15 exhibited a positive effect on angiogenesis, comparable to the effect of the intense angiogenetic factor phorbol 12-myristate 13-acetate at a standard concentration of 0.1 microg/10 microl per disc. The results of this study contribute to the further elucidation of the biological regulatory role of thymosin peptides and provide helpful information in the investigation of their possible therapeutic potential.

Colorimetric determination of reactive primary amino groups of macro- and microsolid supports

The several factors that could affect the sensitivity and the accuracy of the determination of solid-supported amino groups using 2-iminothiolane (Trauts reagent) and 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellmans reagent) are described. The authors found that by using 0.1M phosphate buffer, pH 8.0, instead of ethanol as solvent for the reaction of the solid supports with the 2-iminothiolane, using 0.1M phosphate buffer adjusted at pH 7.27 instead of 8.0 as diluent of 5,5′-dithiobis-(2-nitrobenzoic acid), and selecting carefully the concentration of the latter reagent, it was possible to produce a very sensitive assay capable of quantitatively determining the surface amino groups of very different types of samples. The assay is well adapted for quantitative determination of amino-carrying plastic beads, permitting the determination of nanomolar quantities. In addition, the assay is well suited for microparticulated solid supports (e.g., AH-Sepharose).

Direct ELISA Method for the Specific Determination of Prothymosin Alpha in Human Specimens

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

Serum Biotin Levels in Patients Undergoing Chronic Hemodialysis

Dionyssis S. Ithakissios, Ph. D, Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Patras 261 10 (Greece) Dear Sir, A number of clinical problems have recently been related to biotin deficiency, and this has rekindled the interest in the role of this vitamin in nutrition and its place in biochemistry [1,2]. Since the levels of biotin in biological fluids is an important tool for clinical investigation, we developed a biotin-avidin-double-antibody radioli-gand method freed from the problems of existing assays [3–5]. Details of the method will be reported elsewhere. The main improvements are the ability for easy simultaneous handling of more than 100 samples in less than 4 h and excellent assay characteristics with sensitivity less than 10 pg/ml. We used this assay to measure biotin serum levels in 88 patients undergoing hemodialysis treatment for at least 3 years, since an earlier report [6] suggested biotin treatment in these patients due to an acquired biotin deficiency status. It was found (fig. 1) that the serum biotin levels were in the range of 0.5–2.4 ng/ml (the value of one sample was 3.0 ng/ml) while those in 51 serum samples, obtained from apparently healthy men and women, measured under the same conditions, were in the range of 0.1–0.8 ng/ml (the value of one sample was 0.9 ng/ml). Thus, the concentration of biotin in 75 serum samples (85%) of patients undergoing hemodialysis treatment was found to be higher than the upper limits of the normal range of the assay, whereas 13 serum samples (15%) were in the higher levels of the normal range. The same patients also showed significantly higher concentrations, above the normal range, for endogenous small molecules such as urea, creatinine, uric acid, which did not interfere with the accuracy of the assay. Fifteen patients under biotin treatment (10 mg/day for several months) had serum biotin values between 12.7 and 108.9 ng/ml. We have no explanation yet for the high biotin values determined in patients undergoing maintenance hemodi-