二氢卟吩p6-13,15-环酰亚胺类光敏剂的设计合成

目的 设计合成比紫红素-18（2）化学性质更稳定的二氢卟吩p6-13,15-环酰亚胺类光敏剂（1）。方法 以蚕沙叶绿素a粗提物（糊状叶绿素）酸水解产物脱镁叶绿酸a（3）的五元β-酮基羧酸酯环经碱性条件下空气氧化降解制得的紫红素-18（2）为原料,用各种胺包括羧基保护的氨基酸与其酸酐环发生缩合反应制得目标化合物。结果 以32.6%~65.2%的收率成功合成了目标化合物1a~1j,其结构经电喷雾质谱（ESI-MS）、氢谱（¹H NMR）、紫外谱（UV）和元素分析确证。结论 紫红素-18（2）和胺反应可以制得目标化合物1。该合成路线具有原料廉价易得、反应条件温和、操作简便等优点。     

Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer.

An improved formulation of the photosensitizer chlorin e6 (Ce6) in combination with the hydrophilic polymer polyvinylpyrrolidone (PVP) was investigated for its potential clinical applications in fluorescence diagnosis and photodynamic therapy (PDT) of cancer. This study reports the comparative preclinical biodistribution and efficacy of Ce6 delivered with or without PVP versus dimethyl sulfoxide (DMSO). The safety and fluorescence pharmacokinetics of Ce6-PVP in humans was also accessed. Biodistribution results showed that Ce6-PVP had higher tumor to normal tissue ratio compared to the other formulations. The sensitivity and specificity derived from the area under the receiver operating characteristics curves showed that the formulations were able to discriminate tumor from peritumoral muscle in the following order: Ce6-PVP>Ce6>Ce6-DMSO. In vitro PDT results showed that Ce6-PVP was found to induce selective phototoxicity in leukemic cells compared to peripheral mononuclear blood cells. In addition, in vivo light irradiation at 1h after Ce6-PVP was found to induce greater tumor necrosis without causing animal toxicity. In patients, preferential accumulation of Ce6-PVP was observed in angiosarcoma lesions compared to normal skin following intravenous administration. In conclusion, PVP significantly enhanced the Ce6 concentration in tumors compared with Ce6 alone and increased the therapeutic index of PDT without any side effects in animal model. No serious adverse events were observed in human as well.     

Isolation and identification of impurities in chlorin e6

The tetrapyrrolic compound chlorin e6 is currently used as a pharmaceutical substance for Photolon formulation, which is utilized in photodynamic therapy of various diseases. It was found that chlorin e6 could contain both process- and degradation-related impurities. In order to understand their origin, the most abundant impurities were prepared by liquid extraction, preparative chromatography or chemical synthesis. By means of HPLC-PDA-MS, 1D and 2D NMR spectroscopy, these impurities were identified as chlorin e6 17(4)-ethyl ester, chlorin e4, 15-hydroxyphyllochlorin, rhodochlorin, 15(1)-hydroxymethylrhodochlorin delta-lactone, rhodochlorin-15-oxymethyl delta-lactone, rhodochlorin-15-oxymethyl delta-lactone 17(4)-ethyl ester, 15(1)-hydroxymethylrhodoporphyrin delta-lactone, rhodoporphyrin-15-oxymethyl delta-lactone and purpurin 18. The possible routes of formation of the chlorin derivatives upon the production and storage of chlorin e6 are discussed.     

In vitro and in vivo antimicrobial effect of photodynamic therapy using a highly pure chlorin e6 against Staphylococcus aureus Xen29

Photodynamic therapy (PDT) has been recommended as an alternative therapy for various diseases including microbial infection. Recently, we developed a new method for the preparation of highly pure chlorin e(6) (Ce(6)), which has been widely used as a second-generation photosensitizer. PDT using Ce(6) was very effective for inhibition of in vitro growth of several bacterial strains. To clarify a possibility for its clinical application, in this study, we examined in vitro and in vivo antimicrobial effects of Ce(6)-mediated PDT in mice model of skin infection of Staphylococcus aureus Xen29. Inhibition zone analysis and colony forming unit (CFU) count revealed that Ce(6)-mediated PDT inhibited effectively in vitro bacterial growth. In addition, biofilm formation ability of S. aureus Xen29 was decreased by Ce(6)-mediated PDT. In vivo experiment, mice receiving Ce(6)-mediated PDT exhibited less intensity of bioluminescent signal, showing significant inhibition of bacterial growth. Furthermore, in histopathological examination, marked neutrophilic infiltration and massive bacterial colonies were seen in control mice and mice receiving laser or Ce(6) alone, but not in mice treated with PDT. These results suggest that PDT using Ce(6) extracted by our new method can be clinically useful against bacterial infectious diseases.     

Accumulation and photodynamic activity of chlorin e6 in cisplatin-resistant human lung cancer cells

Cisplatin or cis-diamminedichloroplatinum (II) (CDDP) is one of the most potent anticancer drugs, but the emergence of acquired resistance to CDDP is a major problem in clinical settings. The purpose of this study was to investigate and compare the cellular accumulation and photodynamic activity of chlorin e6, a photosensitizer, in human lung cancer A549 cell line and its CDDP-resistant subline. First, we established the CDDP-resistant (A549/CDDP) cell subline with fourfold greater half-maximal inhibitory concentration value of CDDP on cell growth than that of the parent A549 cells. The cellular accumulation of chlorin e6 was temperature sensitive, and there were no significant differences in chlorin e6 accumulation between the two cell lines. The mRNA expression levels of ABCC1, ABCC2, and ABCG2 in A549/CDDP cells were comparable to those in A549 cells, whereas ABCB1 mRNA level was significantly higher in A549/CDDP cells than in A549 cells. In consistent with chlorin e6 accumulation, chlorin e6-mediated photodynamic activity on A549/CDDP cells was similar to that on A549 cells, whereas no cytotoxicity was observed when these cell lines were kept in dark. Our observations suggest that CDDP resistance has no significant influence on accumulation and photodynamic activity of chlorin e6 in A549 cells.     

Induction of apoptosis in HaCaT cells by photodynamic therapy with chlorin e6 or pheophorbide a

The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.     

双模抗肿瘤光敏剂二氢卟吩e6-偕氟尿嘧啶的合成和生物活性

目的 文献报道氟尿嘧啶（5-Fu）与光敏剂联用具有协同抗肿瘤作用，笔者设计合成二氢卟吩e₆（化合物3）与5-Fu经酰腙键偶联的pH响应性、光化疗双模抗肿瘤光敏剂（化合物1），研究其初步体外光动力抗癌活性及作用机制。方法 首先，5-Fu与五硫化二磷于吡啶中回流反应形成4-硫代-5-氟尿嘧啶，再和水合肼于甲醇中反应制得5-氟尿嘧啶-4-腙（化合物2）；然后，将脱镁叶绿素a（化合物4）酸碱降解产物3经EDC·HCl催化缩合形成二氢卟吩e₆-13¹,15²-酸酐中间体后，直接与2发生选择性酰化反应，制得目标化合物1，并考察其体外pH响应性5-Fu释放及对黑素瘤B16-F10和肝癌HepG2细胞的光动力抗癌活性和作用机制。结果 化合物1在微酸（pH 5.0）环境中能有效释放5-Fu，24 h累积释放率可达60.3%；其在光照下对黑素瘤B16-F10和肝癌HepG2细胞的半数抑制浓度（IC₅₀）分别为0.73 μmol/L和0.90 μmol/L，均显著优于先导物3和上市药物他拉泊芬（talaporfin），且能显著提升肿瘤细胞内活性氧（ROS）水平和诱导肿瘤细胞凋亡，并阻滞肿瘤细胞周期于S期。其结构经紫外、电喷雾质谱、氢谱和元素分析确证。结论 新型双模抗肿瘤光敏剂化合物1具有光动力抗癌活性强、治疗指数（暗毒/光毒比）高，且可在微酸（pH 5.0）环境响应性释放5-Fu等优点，从而实现“单分子”光化疗双重抗肿瘤作用，值得进一步开发研究。     

二氢卟吩p6醚类光敏剂的合成及光动力抗癌活性研究

目的 基于前期研究表明二氢卟吩f的3-乙烯基成醚修饰具有更强的光敏抗肿瘤活性而设计合成二氢卟吩p₆醚类光敏剂（1）,研究其初步体外光动力抗癌活性。方法 以蚕沙叶绿素a粗提物酸水解产物脱镁叶绿酸a（5）经碱及空气氧化降解制得的紫红素-18（4）为先导物,通过碱水解和CH₂N₂甲基化制得二氢卟吩p₆三甲酯（2）,2与33% HBr加成后再和烷氧醇发生亲核取代反应生成目标化合物（1）,并评价其对黑色素瘤B16-F10细胞的光动力杀伤效应。结果 6个目标化合物1a-1f对黑色素瘤B16-F10细胞的体外光动力抗癌活性均优于同类阳性对照药他拉泊芬和维替泊芬,其结构经电喷雾质谱（ESI-MS）、氢谱（¹H NMR）、碳谱（¹³C NMR）及电喷雾高分辨质谱（ESI-HRMS）确证。结论 二氢卟吩p₆醚类光敏剂具有光动力抗癌活性强、治疗指数（暗毒/光毒比）高等优点,值得进一步开发研究。     

缓释制剂体内研究有关问题的思考

文中对缓释制剂体内研究涉及的有关问题进行了分析讨论,重点讨论了缓释制剂的生物利用度及生物等效性研究的基本要求、多规格缓释制剂体内研究的考虑、缓释制剂临床试验的基本研究思路,强调了食物对生物利用度影响研究及餐后生物等效性试验在缓释制剂体内研究中的重要性,并对体内研究与药学研究的关系以及如何结合体内外研究结果开展进一步工作进行了阐述。     

Evaluation of the in vivo genotoxicity of liposomal formulation for delivering anticancer estrogenic derivative (ESC8) in a mouse model

The genotoxic potential of glucocorticoid receptor (GR)-targeted liposomal formulations of the anticancer drug molecule ESC8 was studied in vivo. A methodical literature review discovered no previous studies on the genotoxicity of ESC8. Genotoxicity was assessed in both male and female mice by various assay systems, such as comet assay, chromosomal aberrations and micronuclei assay, which detect different abnormalities. Eleven groups of male mice and eleven groups of female mice, containing six animals per group, were used in the present study: group I served as vehicle control; group II received the positive control (cyclophosphamide 40 mg/kg; CYP); and animals in group III to XI received free drug (ESC8), DX liposome and drug-associated DX liposomal formulation (DXE), respectively, dissolved in 5% solution of glucose at a drug-dose of 1.83, 3.67 and 7.34 mg/kg, respectively. Same drug treatments were followed for the female mice groups. The obtained data revealed the safety of DXE, which did not show substantial genotoxic effects at different dose levels. In contrast, the positive control, CYP, exhibited highly substantial irregular cytogenetic variations in comparison with the control group in different assays.     

The solubility behaviour and thermodynamic relations of the three forms of Venlafaxine free base

The polymorphic and solubility behaviour of the active pharmaceutical ingredient Venlafaxine free base, which is used as an antidepressant, is studied. Using differential scanning calorimetry and slurry experiments, an enantiotropic relation between the three forms was found. Transition temperatures were determined using solubility data and compared with calculated transition temperatures based on the melting enthalpies and temperatures of the different forms. The solubility of Venlafaxine in heptane, toluene and methanol shows a large deviation from ideal behaviour. The deviations are to a large extent determined by the temperature dependence of the difference in fusion enthalpy of the undercooled melt and the solid.     

In vitro and in vivo studies with flutrimazole, a new imidazole derivative with antifungal activity.

1-[(2-Fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) is a new topical imidazole antifungal agent which displays potent broad-spectrum in vitro activity against dermatophytes, filamentous fungi and yeasts, saprophytic and pathogenic to animals and humans (MIC = 0.025-5.0 micrograms/ml). In most of these studies the activity of flutrimazole was comparable to that of clotrimazole and markedly higher than that of bifonazole (MIC differences of greater than or equal to 1 order of magnitude). Tested against Scopulariopsis brevicaulis (8 strains), both flutrimazole and clotrimazole exhibited fungistatic and fungicidal activity, and clotrimazole appeared something less active (MIC = 0.3-2.5 micrograms/ml) than flutrimazole (MIC = 0.15-0.6 micrograms/ml). In animal experiments, topical application of 1% and 2% flutrimazole, as a cream or solution, was highly effective in models of rat vaginal candidiasis and guinea-pig trichophytosis, giving a rate of cured or cured plus markedly improved animals higher than 80%. Flutrimazole shares the mode of action of other imidazole or triazole-containing antifungals, i.e. inhibition of fungal lanosterol 14 alpha-demethylase, as it strongly inhibits ergosterol biosynthesis in a cell-free homogenate of Candida albicans, with an IC50 value of 0.071 mumol/l.     

Comparative study on the in vitro and in vivo activities of heparinoids derivative investigated on the animal model.

In this study we compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulfate (Na-PPS, Ca-PPS), unfractionated heparin (UFH), and low-molecular-weight heparin (Fraxiparin). The antithrombotic effects of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules with a diameter of 20-30 microm were injured by well-defined argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities [activated partial thromboplastin time (aPTT), Heptest] of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses >10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS, an antithrombotic effect was not observed. Oral application of Ca-PPS in doses >20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose of 10 mg/kg, the aPTT increased threefold and the Heptest 2.5-fold compared with controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS, no effect on the coagulation test could be measured. Intravenous injection of UFH prolonged the Heptest after 1 min and the aPTT after 30 min. In ex vivo studies of aPTT and Heptest performed in rat plasma between 2 and 24 h after s.c. injection of 0.2 mg/kg Fraxiparin, no inhibition of any coagulation test was measured. The antithrombotic effect of 0.2 mg/kg Fraxiparin after s.c. injection was significant. Intravenous injection of 20 U/kg UFH significantly inhibited thrombus formation. The smallest antithrombotic effect was after i.v. injection of UFH.     

Improved photoinactivation of gram-negative and gram-positive methicillin-resistant bacterial strains using a new near-infrared absorbing meso-tetrahydroporphyrin: a comparative study with a chlorine e6 photosensitizer photolon

The antimicrobial activity of new tetracationic and water-soluble meso-substituted tetrahydroporphyrin tetratosylat (BL1065) and of dicationic water-soluble chlorine e6 Photolon (BLC1013) is described. The dark toxicity and photosensitizing potentials of both photosensitizers were tested on Gram-positive (Staphylococcus aureus and MRSA) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria in phosphate-buffered saline (PBS, pH 7.4), PBS + horse serum (HS) and PBS + sheep blood (SB). The results show that BLC1065 and BLC1013 did not inhibit the growth of S. aureus in the dark, but efficiently inactivated this Gram-positive bacterium after illumination. Contrary to BLC1013, BLC1065 has a photodynamic activity toward Gram-negative bacteria as well, at least in PBS. Results suggest that tetracationic BLC1065 can bind better to both Gram-positive and Gram-negative bacterial cell envelope than the dianionic chlorine BLC1013 resulting in better efficiency of photoinactivation.     

Improvement of meta-tetra(hydroxyphenyl)chlorin-like photosensitizer selectivity with folate-based targeted delivery. synthesis and in vivo delivery studies

The cell membrane folate receptor (FR) is a molecular target for tumor-selective drug delivery, including delivery of photosensitizers for anticancer photodynamic therapy (PDT). Tumor selectivity of meta-tetra(hydroxyphenyl)chlorin ( m-THPC), a photosensitizer used in PDT clinical trials, demonstrates a low tumor-to-normal epithelial uptake ratio. We report on the synthesis and on the photophysical properties of a m-THPC-like photosensitizer 1 conjugated to folic acid (compound 8). A comparative study of the accumulation of photosensitizers 1 and 8 is described. Nude mice were xenografted with FR-alpha-positive KB or HT-29 cells lacking FR-alpha as a negative control. Using optical fiber fluorimetry, we demonstrated that conjugate 8 exhibited enhanced accumulation in KB tumors compared to 1 4 h after injection. No significant difference between KB and HT-29 tumors was observed in case of compound 1. Tumor-to-normal tissue ratio exhibited a very interesting selectivity for conjugate 8 (5:1) in KB tumors 4 h postinjection.     

In vitro and in vivo studies of sustained-release floating dosage forms containing salbutamol sulfate

Peroral sustained-release floating capsules containing salbutamol sulfate were formulated using different combinations of hydrocolloids of natural and semi-synthetic origin. The floating properties and release rate characteristics were determined for the capsules in simulated gastric fluid USP XXI and HCl (0.1 mol.l-1) as dissolution media. Also, a marketed sustained-release non-floating capsule containing salbutamol sulfate was studied for its release rate characteristics. The floating capsule formulated showed a Higuchian release profile while the marketed product released only about 80% of the total dose in the stipulated 12 h in the dissolution medium. In vivo X-ray studies of the abdomen were carried out to locate the floating and non-floating (fabricated) dosage forms at various time intervals of uniform duration. The floating capsule definitely indicated a residence time (up to 8-9 h) in the stomach greater than for the non-floating capsule.     

In vitro and in vivo percutaneous absorption of topical dosage forms: case studies

This article evaluated the influence of vehicle compositions on topical drug availability. In vitro drug release and in vivo experiments were performed in case of the hydrophilic ketamine hydrochloride and the lipophilic piroxicam. Ketamine hydrochloride is a NMDA receptor antagonist that has been useful for anesthesia and analgesia. The study of transdermal ketamine delivery is a novelty, because nobody has investigated the hypnotic effects of ketamine after this administration route. In vitro measurements gave a good basis for screening among the developed products. The physiological changes after ketamine administration showed, that there were significant differences among the parameters tested (breathing rate, duration of sleep) from the developed products (hydrogel, lyotropic liquid crystal and o/w cream) compared to the reference product (Carbopol gel). The in vivo feedback for piroxicam was the measurement of the anti-inflammatory activity by edema inhibition percentage. Significant differences were measured in case of the developed systems compared to the reference.