Application of HILIC stationary phase to determination of dimethindene maleate in topical gel.

A novel high performance liquid chromatography method for the determination of dimethindene maleate in pharmaceutical gel using hydrophilic interaction liquid chromatography (HILIC) with UV detection was developed and validated. Following optimal conditions for the analysis of dimethindene maleate were used: analytical column SeQuant ZIC-HILIC (50mmx2.1mm, 5microm), and mobile phase consisted of a mixture of acetonitrile and aqueous solution of acetic acid (25mM) and ammonium acetate (2.5mM) (87.5:12.5, v:v). The analysis time was less than 3min at a flow rate of 0.3mlmin(-1). UV detection was performed at 258nm. The method was validated and system suitability parameters were evaluated. The method is suitable for application for routine determination of dimethindene maleate in topical pharmaceutical preparation.

HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment.

A novel fast isocratic reversed-phase HPLC method for simultaneous determination of chlorhexidine and its degradation product p-chloroaniline was developed. Zorbax SB Phenyl column (75 mm x 4.6 mm, 3.5 microm) was used for the separation. Mobile phase composed of acetonitrile and buffer solution of 0.08 M sodium phosphate monobasic containing 5 ml of triethylamine (0.5%) and adjust with 85% phosphoric acid to pH 3.0 in ratio 35:65 (v/v) pumped isocratically at flow rate 0.6 ml min(-1) was used. UV detection was performed at 239 nm, the total analysis time was about 10 min. The method is suitable for practical routine analysis of topical ointment in the quality control laboratory.

Simultaneous HPLC determination of ketoprofen and its degradation products in the presence of preservatives in pharmaceuticals.

A novel and quick high-performance liquid chromatography (HPLC) method with UV spectrophotometric detection was developed and validated for the determination of five compounds in topical gel. The described method is suitable for simultaneous determination of active component ketoprofen, two preservatives methylparaben and propylparaben and two degradation products of ketoprofen--3-acetylbenzophenone and 2-(3-carboxyphenyl) propionic acid--in a topical cream after long-term stability tests using ethylparaben as an internal standard. The chromatographic separation was performed on a 5microm Supelco Discovery C18 column (125mm x 4mm i.d., Sigma-Aldrich); the optimal mobile phase for separation of ketoprofen, methylparaben, propylparaben, degradation products 3-acetylbenzophenone and 2-(3-carboxyphenyl) propionic acid and ethylparaben as internal standard consists of a mixture of acetonitril, water and phosphate buffer pH 3.5 (40:58:2, v/v/v). At a flow rate of 1.0ml min(-1) and detection at 233nm, the total time of analysis was less than 10min. The method was applied for routine analysis (batch analysis and stability tests) of these compounds in topical pharmaceutical product.

Simultaneous determination of methylparaben, propylparaben, hydrocortisone acetate and its degradation products in a topical cream by RP-HPLC.

A novel reversed-phase high-performance liquid chromatographic method with UV spectrophotometric detection was developed and validated for the determination of compounds in topical cream. The method describes determination of active component hydrocortisone acetate (HCA), its degradation products hydrocortisone (HC) and cortisone acetate (occurring in formulation after long-term stability tests) and two preservatives presented in the cream-methylparaben and propylparaben, using dexamethasone as an internal standard. The chromatographic separation was performed on a 5 microm SUPELCO Discovery C18 125 x 4-mm ID column. The optimised mobile phase for separation of all the compounds consists of methanol, acetonitrile and water (15:27:58, v/v/v), with the analysis time less than 13 min. The method was applicable for routine analysis (assays and stability tests) of active compound HCA, preservatives and degradation products in pharmaceutical product--topical cream Hydrocortizone cream 1%.

Determination of methylparaben, propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC.

A novel reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of active component triamcinolone acetonide, its degradation product triamcinolone (occurring in formulation after long-term stability tests) and two preservatives presented in the cream-methylparaben and propylparaben, using hydrocortisone as an internal standard. The chromatographic separation was performed on a Supelco Discovery C18 column; the mobile phase for separation of all compounds consists of a mixture of acetonitrile and water (40:60 v/v). The analysis time was less than 9 min, at a flow rate of 0.6 mL min(-1) and detection at 240 nm. The method was found to be applicable for routine analysis (stability tests, homogeneity) in the pharmaceutical product topical cream Triamcinolon cream 0.1%.

Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Peritumoral administration of antigen-unstimulated bone marrow-derived dendritic cells inhibits tumour growth.

Murine BM cells from B6 mice were grown in vitro in medium supplemented with GM-CSF and IL-4 to differentiate DC from DC precursors. After 10 days of culture, approximately 20% of the cell population exhibited the characteristic morphology of BMDC. In cytofluorometric analysis the morphological changes of cells were accompanied by upregulation of the expression of the MHC class II, CD11c, CD80, and CD86 molecules. The BMDC were pulsed with a lysate of syngeneic MK16 carcinoma cells and used for in vitro activation of SC. Co-cultivation of the carcinoma lysate-pulsed BMDC with SC induced a proliferative response of the syngeneic SC. Priming of the proliferative responses was more efficient when the BMDC were grown in the presence of GM-CSF and IL-4 for 10 days than for 7 days. The in vivo effect of mature, tumour lysate-unstimulated BMDC was examined in mice carrying syngeneic MK16 carcinoma transplants. It has been found that local pretreatment with BMDC inhibits growth of a subsequent challenge inoculum of the MK16 cells. Similarly, treatment of mice carrying small MK16 tumours and of those with MK16 surgical minimal residual disease performed with BMDC significantly inhibited tumour growth. It can be concluded from these results that local concentration of mature BMDC at the tumour site can control the development and growth of the transplanted tumour inoculum.

Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16.

Experiments were designed to examine the efficacy of IL-2 gene therapy in a surgical minimal residual tumour disease, using moderately immunogenic MK16/1/IIIABC murine cells transformed by activated ras and HPV 16 E6/E7 oncogenes (MK16 cells). Previously we demonstrated that surgical minimal residual tumour disease (SMRTD) could be effectively cured when murine Mc12 sarcoma had been resected and the operated mice were treated with irradiated Mc12 sarcoma cells engineered to secrete IL-2. In this study we performed IL-2 gene therapy of MK16 carcinoma with two types of irradiated MK16-unrelated tumour cell vaccines. One type of vaccine was derived from MHC class I-matched Mc12 sarcoma cells engineered to secrete IL-2 and the other from MHC class I-discordant IL-2 producing plasmacytoma X63-m-IL-2. The vaccines did not share any tumour rejection antigen with the MK16 cells and served exclusively as a local source of IL-2 production. Both vaccines were capable of inhibiting MK16 tumours when administered peritumorally up to 15 days after MK16 tumour challenge. The irradiated MHC class I-matched and IL-2-producing Mc12 sarcoma vaccine was then selected for therapy of MK16 SMRTD. Whereas the recurrence rate in the operated MK16 carcinoma bearers was 80%, so that only 20% of mice were cured by surgery, approximately 65% of the MK16 carcinoma bearers were permanently protected when the surgery was followed by local administration of the IL-2-producing Mc12 sarcoma vaccine.

Granulocyte-macrophage colony-stimulating factor-producing tumour vaccines.

Murine sarcoma MC12 cells were transfected with the gene coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumorigenicity of a variety of cell clones with different expression of the inserted gene was assessed. All of the genetically manipulated MC12 cell clones examined were found to be less tumorigenic than the parental MC12 cell population. No correlation was observed between the production of GM-CSF by the clones and their tumorigenicity. It has been found that irradiation of the GM-CSF-producing cells with the dose of 150 Gy did not significantly inhibit the GM-CSF production during the period of 5 days after irradiation. These findings provided us with the rationale for using the irradiated GM-CSF-producing MC12 sarcoma vaccine for therapy. It has further been found than immunosensitivity of the genetically manipulated, GM-CSF-producing tumour targets to the IL-2-activated killer (LAK) cell-mediated cytolysis was significantly increased, as compared to the parental target cell population. Irradiated, GM-CSF-producing tumour vaccines were used for therapy of 3-day-old MC12 sarcoma transplants in syngeneic mice and for therapy of surgically induced minimal residual tumour disease. Neither small tumour transplants, nor tumour residua after surgery were significantly sensitive to the therapy with GM-CSF-producing tumour vaccines.

Interleukin 2 gene therapy of surgical minimal residual tumour disease: characterization of cytolytic effector cells from tumour progressors and regressors.

Experiments were designed to characterize cytolytic effector cells from mice with SMRTD treated with IL-2 gene therapy. Mice were inoculated with syngeneic murine MK16 carcinoma cells. When the tumours reached 8-12 mm in diameter, they were excised and the operated mice were randomized into two groups. The first group without any further treatment was designated as operated-only; the second group, vaccinated 3 days after the operation with IL-2-producing tumour vaccine, is referred to as operated-vaccinated. Tumour recurrence rate in the operated-only mice was 90 percent; in the operated-vaccinated group the recurrence rate was 38.5 percent (progressors). The remaining 61.5 percent of mice were permanently protected (regressors). On day 53, the tumour progressors, regressors and healthy controls were sacrificed, and their spleen cells were used for 51Cr microcytotoxicity assay. Splenocytes from any group of mice were not cytolytic when allowed to react with MK16, YAC-1 (NK sensitive) and C1498 (NK resistant) targets. However, when grown for 3 days in IL-2-containing medium, the splenocytes from all groups of mice could develop cytolytic activity. The cytolytic activity of splenocytes from tumour progressors and regressors was substantially lower then that of splenocytes from healthy controls. In addition, significantly lower cytolytic activity was observed with IL-2-activated splenocytes from tumour progressors as compared to that of tumour regressors. Depletion of NK1.1+ cells or CD4+ plus CD8+ cells prevented the induction of significant IL-2-stimulated cytotoxicity directed against MK16 and C1498 targets in spleen cell cultures from tumour progressors, regressors, and healthy control mice, indicating that both, NK1.1+ and CD4+ plus CD8+, cells participate in the antitumour effect of IL-2 gene therapy. This was further supported by the finding that after depletion of CD4+ plus CD8+ cells, a residual cytolytic activity directed exclusively against NK-sensitive YAC-1 cells was observed.