Paola Brusa

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drugs antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel.

The taxanes, paclitaxel and docetaxel, are anticancer agents used in clinical trials against ovarian carcinoma, breast, lung and head/neck cancer. Paclitaxel, very insoluble in water, is generally formulated using Cremophor EL. Docetaxel, more soluble in water, is formulated using Tween 80 and ethanol. Tween 80, albeit less toxic than Cremophor EL, may be responsible of some toxic effects. To eliminate these vehicles and improve the drugs antitumor efficacy, taxanes have been incorporated in liposomes. We compared formulation, stability, biodistribution and pharmacokinetics of docetaxel in conventional and PEGylated liposomes. Of the several formulations examined, docetaxel-liposomes composed of ePC/PG/CHOL 9:1:2 and ePC/PG/DSPE-PEG2000/CHOL 9:1:2:0.7 were the most effective. Both conventional and PEGylated docetaxel-liposomes were stable at 4 degrees C after 15 days, whereas in the presence of serum at 37 degrees C they were less stable. The IC50 values of docetaxel-liposomes, evaluated on HT-29 and Igrov1 cell lines, remained very high. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of [14C]docetaxel, formulated in Tween 80 or in 3H-labeled conventional or PEGylated liposomes. The t(1/2)beta, which was low for docetaxel (52.3 min), rose to 260 min for conventional docetaxel-liposomes and to 665 min for PEGylated docetaxel liposomes. Biodistribution studies confirmed the pharmacokinetics.

Preparation, characterization and properties in vitro and in vivo of a paclitaxel-albumin conjugate

Paclitaxel (taxol) is in routine clinical use for treatment of a variety of cancers. Because of its low aqueous solubility, it requires Cremophor EL (polyethoxylated castor oil) and ethanol as a vehicle (Diluent 12). These agents cause severe allergic reactions upon intravenous administration. In this study paclitaxel was covalently attached to human serum albumin. The 2′-hydroxyl group of the drug was esterefied with succinic anhydride and then derivatized to give the N-hydroxy-3-sulfo-succinimide active ester, highly reactive to lysyl amino groups of the protein. Two different conjugate populations (with 6 or 30 average molecules of drug linked to each albumin molecule) were prepared, purified and characterized. The conjugates were stable in physiological solution and in serum whereas the presence of proteases or liver extract released the drug in a linear fashion. The antitumor activity of free drug and conjugates was tested on three different tumor cell lines. The conjugates maintained high cytotoxicity with efficient cell binding and internalization followed by release of the drug inside the cell. The pharmacokinetics of the conjugate (after iv administration) was evaluated and compared to that of the free drug. Both followed a bicompartmental model but elimination of the conjugate from the plasma was much slower than the free drug, giving a relevant rise in AUC and MRT values. The conjugate also released of parent drug continuously to the plasma over prolonged periods, thus providing a depot effect. The acute toxicity noted with the standard formulation of taxol was strongly reduced in our albumin-conjugated preparation.

Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components

Immunotoxins and antibody-drug conjugates are protein-based drugs combining a target-specific binding domain with a cytotoxic domain. Such compounds are potentially therapeutic against diseases including cancer, and several clinical trials have shown encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit conjugates’ therapeutic use, including inefficient cellular uptake, low cytotoxicity, and off-target effects. During the preparation of immunoconjugates by chemical synthesis, the choice of the hinge component joining the two building blocks is of paramount importance: the conjugate must remain stable in vivo but must afford efficient release of the toxic moiety when the target is reached. Vast efforts have been made, and the present article reviews strategies employed in developing immunoconjugates, focusing on the evolution of chemical linkers.

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.

Folate-mediated targeting of albumin conjugates of paclitaxel obtained through a heterogeneous phase system

The study developed cytotoxic macromolecular conjugates that specifically target the folate receptor and deliver the drug into cell cytoplasm. The anticancer agent paclitaxel was conjugated to human serum albumin (HSA) and this drug-albumin conjugate was further equipped with folic acid, linked via an extended poly(ethylene glycol) spacer. Preparation was carried out in a heterogeneous phase system exploiting the binding ability of Cibacron Blue dye to HSA. Unreacted reagents were easily removed and, after purification by gel filtration, the conjugate was fully characterized. Binding and in vitro cytotoxicity studies on human nasopharyngeal epidermal carcinoma KB and colorectal carcinoma HT-29 cells (as negative control) demonstrated increased selectivity and anti-tumoral activity.

A randomized double-blind placebo controlled phase I-II study on clinical and molecular effects of dietary supplements in men with precancerous prostatic lesions. Chemoprevention or "chemopromotion"?

Antioxidants effectiveness in prostate cancer (PCa) chemoprevention has been severely questioned, especially after the recent results of the Selenium and Vitamin E Cancer Prevention Trial. We present the results of a double‐blind randomized controlled trial (dbRCT) on the pharmacokinetic, clinical, and molecular activity of dietary supplements containing lycopene, selenium, and green tea catechins (GTCs) in men with multifocal high grade prostatic intraepithelial neoplasia (mHGPIN) and/or atypical small acinar proliferation (ASAP).

Inhibition of 2,3-oxidosqualene cyclase and sterol biosynthesis by 10- and 19-azasqualene derivatives

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [14C] acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

Comparison of blocked and non-blocked ricin-antibody immunotoxins against human gastric carcinoma and colorectal adenocarcinoma cell lines

SummaryTo avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linkedricin-antibody IT, in which the galactosebinding site of ricin had lost the ability to bind to galactosidic residues of Sepharose 6B gel. As carrier agent, the monoclonal antibody AR-3, which defines the CAR-3 tumour-associated antigenic determinant expressed selectively on different human carcinoma cell lines, was used. Purification of the new conjugate was performed in three sequential steps: (1) by HPLC gel filtration on TSK G3000SW to remove the unconjugated ricin: (2) by affinity chromatography on Affi-Gel Blue to separate the free antibody from the conjugate and (3) by affinity chromatography on Sepharose 6B to separate the galactose-binding IT from the non-binding moiety. The cytotoxicity of the blocked and non-blocked thioether-linked IT was compared with that of classical ricin-antibody IT conjugated via SPDP and that of ricin A chain IT. The comparison was made on two different target cell lines (KATO III human gastric carcinoma and HT-29 human colorectal carcinoma) versus two control cell lines (HL-60 promyelocytic pre-leukaemic and COLO38 melanoma). The results showed that the blocked thioether IT displayed a more selective toxicity to target cells than the non-blocked IT and was much more potent than the ricin A chain conjugate.

Diffusion of counterfeit drugs in developing countries and stability of galenics stored for months under different conditions of temperature and relative humidity

Aim To investigate the diffusion of counterfeit medicines in developing countries and to verify the stability of galenic dosage forms to determine the stability of galenics prepared and stored in developing countries. Methods We purchased 221 pharmaceutical samples belonging to different therapeutic classes both in authorized and illegal pharmacies and subjected them to European Pharmacopoeia, 7th ed. quality tests. An UV-visible spectrophotometric assay was used to determine the galenics stability under different conditions of temperature (T) and relative humidity (RH). Results A substantial percentage of samples was substandard (52%) and thus had to be considered as counterfeit. Stability tests for galenics showed that the tested dosage forms were stable for 24 months under “standard” (t = 25 ± 2°C, RH = 50 ± 5%) conditions. Under “accelerated” (t = 40 ± 2°C, RH = 50 ± 5%) conditions, samples were stable for 3 months provided that they were stored in glass containers. Stability results of samples stored in “accelerated” conditions were similar to those obtained by on site in tropical countries and could so supply precious information on the expected stability of galenics in tropical countries. Conclusion This study gives useful information about the presence of counterfeit medicinal products in the pharmacies of many developing countries. This should serve as an alarm bell and an input for the production of galenics. We recommend setting up of galenic laboratories in developing countries around the globe.