Alessandra Semenzato

Stability of vitamin C derivatives in solution and topical formulations.

The stability of ascorbic acid, ascorbyl palmitate and magnesium ascorbyl phosphate (VC-PMG) in both standard solutions and topical formulations was investigated by direct RP-HPLC analysis after sample dilution with a suitable aqueous-organic solvent mixture. The results showed that, whereas the two vitamin C derivatives were more stable than ascorbic acid, the ascorbyl esters showed significant differences. Esterification with palmitic acid in 6 position did not prevent hydrolysis of the molecule, either in solution or in emulsion; only the special preparation of products with high viscoelastic properties was able to reduce the typical behaviour of this compound. Conversely, the introduction of the phosphoric group in 2 position protected the molecule from break-up of the enediol system, confirming VC-PMG as a very stable derivative of vitamin C that may be easily used in various types of cosmetic products.

New cyclodextrin bioconjugates for active tumour targeting

A new cyclodextrin-based carrier for active targeting of low soluble and degradable drugs has been synthesized and characterized. β-Cyclodextrins were first reacted with excess hexamethylene diisocyanate and the resulting CD–(C6–NCO)5 derivative was reacted with 700 Da diamino-PEG to yield CD–(C6–PEG–NH2)5. About one out of five free amino groups of PEG were functionalised with folic acid (FA) as a tumour targeting moiety. The chemical structures of the intermediates as well as the final product, CD–(C6–PEG)5–FA, were characterized by 1H and 13C NMR, reverse phase and gel permeation chromatography, and UV-Vis spectroscopy. After modification, the haemolytic activity of β-cyclodextrins decreased by about 70%. In the presence of the new carrier, the β-estradiol solubility increased by more than 300 fold and the chlorambucil degradation rate decreased by 50–60%. CD–(C6–PEG)5–FA formed an inclusion complex with curcumin displaying an association constant of 954,732 M− 1. The new carrier increased the curcumin solubility by about 3200 fold as compared to native β-cyclodextrins and reduced its degradation rate at pH 6.5 and 7.2 by 10 and 45 fold, respectively. FA receptor-overexpressing human nasopharyngeal tumour KB cell lines and non-folic acid receptor-expressing human breast cancer MCF7 cells were used to evaluate the targeting properties of the new drug delivery system. The in vitro studies demonstrate that the new carrier possesses potential selectivity for the folate receptor-overexpressing tumour cells as ED50 values of 52 μM, 58 μM and 21 μM were obtained with curcumin-loaded CD–(C6–PEG–NH2)5, curcumin in foetal serum medium and CD–(C6-PEG)5–FA, respectively.

High-performance liquid chromatographic determination of free formaldehyde in cosmetics

An improved, sensitive method for the determination of formaldehyde in cosmetics and other commercial products is reported. The procedure is based on dilution of the sample with tetrahydrofuran-water (9:1), followed by precolumn derivatization with 2,4-dinitrophenylhydrazine and direct reversed-phase high-performance liquid chromatography. The formaldehyde derivative is stabilized in the reaction medium by addition of phosphate buffer and neutralization and detected in less than 10 min by the standard additions methods. The method also appears to be suitable for the direct evaluation of the formaldehyde donors used in cosmetics as preservatives.

Poly(ethylene glycol)–avidin bioconjugates: suitable candidates for tumor pretargeting

Avidin-poly(ethylene glycol) (PEG) conjugates were obtained by derivatization of about 10% of the protein amino groups (four amino groups per protein molecule) with linear 5 kDa PEG or branched 10 or 20 kDa PEGs. Circular dichroism analysis showed that the polymer conjugation neither altered the protein structure nor the environment of the aromatic amino acids which are present at the level of the biotin binding site. Spectroscopic studies were carried out to evaluate the biotin recognition activity of the conjugates either in terms of number of biotin binding sites or avidin/biotin affinity. Avidin-PEG 5 kDa and avidin-PEG 10 kDa displayed over 90% of the native protein biological activity while a reduction in the recognition of biotinylated antibodies of about 25% was found with PEG 20 kDa. In vivo studies demonstrated that the protein immunogenicity was in the order: wild type avidin>avidin-PEG 5 kDa>avidin-PEG 10 kDa>avidin-PEG 20 kDa. By intravenous injection into mice bearing a solid tumor, all conjugates displayed prolonged permanence in the circulation with respect to the native protein. The area under the curve values of avidin-PEG 5 kDa, avidin-PEG 10 kDa and avidin-PEG 20 kDa were about 3-, 7- and 30-times higher than the wild type avidin with reduced accumulation in kidneys and liver. Interestingly, all conjugates accumulated significantly in the tumor mass. In particular, in the case of avidin-PEG 20 kDa, 8% of the injected dose (ID)/g of tissue accumulated in the tumor after 5 h from the administration and over 6% of the ID/g was maintained throughout 72 h.

Poly(hydroxyethylaspartamide) derivatives as colloidal drug carrier systems.

Poly(hydroxyethylaspartamide) (PHEA) derivatives bearing at the polyaminoacidic backbone poly(ethyleneglycol) (2000 or 5000 Da) or both poly(ethyleneglycol) and hexadecylalkylamine as pendant moieties were investigated as polymeric colloidal drug carriers. The ability of the PHEA derivatives to solubilize hydrophobic drugs was investigated using paclitaxel, amphotericin B and methotrexate. The results demonstrated that the drug solubility depends on both macromolecule composition and drug physicochemical properties. In particular, PEG/hexadecylalkylamine co-grafting increased significantly the solubilization properties of PHEA for the considered drugs while the conjugation of PEG only did not endow PHEA with drug carrier properties. A stability study carried out with paclitaxel/PHEA-PEG(5000)-hexadecylalkylamine demonstrated that the drug/carrier system is characterized by physicochemical instability, which is strictly related to the incubation pH. However, the carrier was found to partially prevent drug degradation. Investigations performed using murine myeloid leukaemia NFS-60 cell line showed that paclitaxel loaded PHEA-PEG(5000)-hexadecylalkylamine possesses high pharmacological activity with IC(50) value of 22.3 ng/ml. Pharmacokinetic studies carried out by intravenous administration of paclitaxel loaded PHEA-PEG(5000)-hexadecylalkylamine to Balb/c mice demonstrated that the carrier modifies the in vivo paclitaxel fate. In particular, PHEA-PEG(5000)-hexadecylalkylamine prolonged the drug distribution and elimination phase of 6 and 17 times, respectively; in addition, it increased the systemic availability (AUC) by about 30 times.

Allergic contact sensitivity in elderly patients

Background and aims: Aging has been shown to be correlated with the rate and type of contact sensitization, but only a few studies have evaluated patch test reactivity in elderly subjects with an adequately large population. Methods: The response patterns to patch testing in 1444 elderly subjects (>65 years) with suspected allergic contact dermatitis were studied, and the results compared with a control group of individuals with suspected allergic contact dermatitis, aged between 20 and 40 years. Results: The prevalence of the positive patch test to at least one hapten was significantly lower in the group of elderly patients compared with adult patients (40.7 vs 47.8%, p<0.0001). However, some allergens, i.e., primin, diaminodiphenylmethane, neomycin, lanolin alcohols, paraben mix, Euxyl K400 and quinoline mix, showed an increased sensitization rate in elderly patients compared with adult patients. These allergens are now less frequently employed in the workplace, or are substances particularly used in the formulation of topical treatment of age-related diseases, i.e., leg ulcer and xerosis. It was also found that the intensity of positive patch test reactions was significantly lower in elderly patients compared with younger subjects, with higher proportions of weak (+) positive reactions. Moreover, elderly patients showed a dynamic pattern of increasing intensity of patch test reactions at the second reading after 3 days compared with the first reading after 2 days more frequently than younger patients (60 vs 53%, p<0.0001 ). Conclusions: These findings suggest an age-dependent decline of overall positive patch test reactions, but a higher sensitization rate to some allergens frequently used in the composition of topical treatments. The development of an allergic response in elderly patients was found to be delayed, and this may require an additional reading after 7 days and the interpretation of even weak reactions as valid positive patch test reactions.

Glycosilated Macromolecular Conjugates of Antiviral Drugs with a Polyaspartamide

Two new polymeric conjugates for specific liver targeting were prepared by conjugation of sugar moieties and antiviral drugs to α, β-poly[N-2-(hydroxyethyl)-dl-aspartamide] (PHEA). PHEA-galactopyranosylphenylthiocarbamide-mono-O-succinylganciclovir (conjugate 7) and PHEA-mannopyranosylphenylthiocarbamide-O-succinylacyclovir (conjugate 8) were synthesized according to a multi-step procedure which allowed for obtaining high product yield and process standardization. Conjugate 7 contained 7.5 and 8.5% of galactose and ganciclovir (substituent/repeating unit, mol/mol), respectively, and conjugate 8 contained 14.2 and 10.8% of mannose and acyclovir, respectively. In vitro studies demonstrated that both acyclovir and ganciclovir are released from the polymeric adducts at a release rate, which depended on the incubation medium. Though a detailed study evidenced that the two bioconjugates undergo different hydrolysis pathways, in both cases high drug release rate was found in plasma, while the glycosidic moiety was not released. Pharmacokinetic studies carried out by intravenous administration of the bioconjugates to Balb/c mice demonstrated that the conjugation of glycosidic moieties promotes the disappearance of the polymer from the bloodstream. The two derivatives displayed a different pharmacokinetic profile. In particular, the mannosyl conjugation promoted the rapid disposition of the macromolecule in the kidneys and in the liver, while prevented the accumulation in the spleen. On the contrary, the galactosyl derivative was found to dispose in the liver at the same extent of the naked polymer. Few considerations on the different behavior of the conjugates were reported.

High-performance liquid chromatographic determination of ionic compounds in cosmetic emulsions: application to magnesium ascorbyl phosphate

Abstract A rapid, reliable method based on reversed-phase high performance liquid chromatography for the qualitative and quantitative determination of magnesium ascorbyl phosphate (VC-PMG) in both standard solutions and cosmetic products is reported. The procedure, based on an aminic stationary phase and acetonitrile-0.3 M phosphate buffer (pH 4) (40:60) as the mobile phase, allows the determination of VC-PMG in cosmetic samples after dilution with a tetrahydrofuran-0.3 M phosphate buffer (pH 4) (3:7) solvent mixture.

High-performance liquid chromatographic determination of free formaldehyde in cosmetics preserved with Dowicil 200

We recently reported a rapid and reliable method for the determination of free formaldehyde in cosmetics; the procedure is based on sample dilution with a tetrahydrofuran (THF) -water solvent mixture, followed by precolumn derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) and direct high-performance liquid chromatographic (HPLC) analysis. Our studies showed the applicability of this method when preservative donors are present in the cosmetic formula: only Dowicil 200 [cis-1-(chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride] is not compatible with the procedure, because of its instability in the acidic media

Reversed-phase high-performance liquid chromatography applied to the direct analsis of untreated heterophasic systems

Abstract A simple and rapid approach for the direct analysis of multi-component heterophasic matrices is reported. The procedure is based on sample dilution with tetrahydrofuran—water (9:1) followed by direct reversed-phase high-performance liquid chromatography. The method is suitable for quality control and stability studies of drugs, food and cosmetic products.