Antibacterial efficacy and drug-induced tooth discolouration of antibiotic combinations for endodontic regenerative procedures.

Elimination of microbial contamination from the root canal system is a precondition for successful root canal treatment. Teeth with immature root development, necrotic pulps and apical periodontitis present multiple challenges for successful treatment. Disinfection is achieved by irrigation followed by the placement of an intracanal medicament. A mixture of ciprofloxacin, metronidazole and minocycline (3-MIX S) has been shown to be very effective in eliminating endodontic pathogens in vitro and in vivo. Among the components of the mixture, minocycline can induce tooth discolouration after long-term oral use. Therefore, the elimination of minocycline from the above-mentioned combination has been suggested to prevent the occasion of this undesirable effect. The aim of this study was to investigate the potential antimicrobial efficacy of alternative antibiotic combinations [3-MIX C (clarithromycin); 3-MIX F (fosfomycin)] against bacteria from infected root canals. An additional objective was to evaluate their discolouration potential as possible alternatives to minocycline-based intracanal medicaments. Our in vitro results clearly demonstrated that 3-MIX C and 3-MIX F had a greater antimicrobial activity than 3-MIX S, underlying that clarithromycin still had a higher capacity to kill endodontic pathogens in vitro compared to fosfomycin. Both 3-MIX C and 3-MIX F were able to avoid the permanent staining effect of the crown.

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 drug's 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 water-soluble prodrugs of paclitaxel.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 2'-succinyl, 2'-methylpyridinium acetate and 2'-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 2'-mPEG (5000)-paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mgml(-1)). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t(1/2)beta, plasma lifetime, which was longer in liposomes containing 2'-mPEG (5000)-paclitaxel.

Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs.

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

In vitro and in vivo antitumor activity of immunoconjugates prepared by linking 5-fluorouridine to antiadenocarcinoma monoclonal antibody.

5-Fluorouridine (5-FUr), a cytotoxic antitumoral agent not in clinical use because of its systemic toxicity, and AR-3, a monoclonal antibody specific to a human colorectal adenocarcinoma, were covalently linked via two different strategies. 5-FUr was 5' succinilated after protection of the secondary hydroxyl groups and the carboxylate derivative was then activated as N-hydroxysuccinimidyl ester in order to react with the amino groups present in the monoclonal antibody, giving an amide linkage. Alternatively, a 5-FUr immunoconjugate containing an acid-cleavable hydrazone bond was formed from the reaction between an acyl hydrazide derivative of 5-FUr and a periodate oxydized antibody with approximately 12 aldehyde groups in its carbohydrate region. An average of 9 to 12 drug molecules were attached to the antibody. In a cytotoxic assay on the human colorectal carcinoma cell line HT-29, the hydrazone containing drug conjugate was equally active as the succinylamido conjugate and the free drug. However, ELISA showed that while in the case of the succinylamido conjugate the Mab immunoreactivity was not affected after conjugation, there was a significant loss of reactivity in the acid cleavable conjugate. In a model of a disseminated intraabdominal carcinomatosis by HT-29 intraperitoneal graft in nude mice, the 5-FUr immunoconjugate selected was more effective than the unconjugated drug in medium-term therapy (21 days after the graft and 16 days after drug treatment), albeit in the longer period the efficacy of the two formulations was similar. The toxic effect of the drug-conjugate in vivo was much weaker, demonstrating its clear advantage over the drug, in terms of pharmacological efficacy.

Synthesis of different immunotoxins composed by ribosome inactivating proteins non-covalently bound to monoclonal antibody.

This study describes the synthesis of a panel of immunotoxins made by a non-covalent interaction between a monoclonal antibody derivatized with a dichlorotriazinic dye and six different ribosomal inhibitor proteins: colocin 1, momorcochin, momordin, bryodin, saporin 6 and PAP-S. The scheme of preparation showed several advantages respect to commercially available heterobifunctional cross-linkers, such as an higher overall yield of production and the homogeneity of the obtained conjugates. Nevertheless this procedure allowed the synthesis of immunoconjugates only for the four glycosilated RIPs since the not glycosilated ones saporin 6 and PAP-S precipitated in the presence of the dye. The non covalent linkage did not significantly affect the toxic activity of the glycosilated RIPs, as shown by their antitumoral activity on three different cell lines: HT-29 and A431 as target and MeWo as control. Furthermore, the monoclonal antibody cell-antigen recognition was preserved if a maximum derivatization ratio of 4 between the antibody and the dye was applied. The described original procedure may be of general application to prepare panels of immunotoxin for clinical use avoiding the expected RIP-related immune reaction.

New coupling reagents for the preparation of disulfide cross-linked conjugates with increased stability.

To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.