An Attempt to Clarify the Mechanism of the Penetration Enhancing Effects of Lipophilic Vehicles with Differential Scanning Calorimetry (DSC)*

In a previous in-vivo skin penetration study, it was observed that certain lipophilic liquid vehicles enhanced drug penetration, whilst others did not. To clarify the mechanism of skin penetration enhancement, isolated sheets of human stratum corneum were measured by differential scanning calorimetry (DSC), either untreated or after pretreatment with various lipophilic liquids (highly purified light mineral oil, isopropyl myristate, caprylic/capric acid triglycerides containing 5% phospholipids, dibutyl adipate, dimethicone 100, cetearyl iso-octanoate, caprylic/capric acid triglycerides), commonly used in ointment bases. All samples were analysed over a heating range of at least — 10–130°C. All DSC curves were evaluated with regard to the phase-transition enthalpies (peak areas) and peak maximum temperatures of the lipid-phase transitions at ca 75 and 85°C. With the exception of dimethicone 100, cetearyl iso-octanoate and caprylic/capric acid triglycerides, all vehicles showed characteristic alterations of the phase-transition temperatures and enthalpies of the stratum corneum lipids. Mineral oil and isopropyl myristate caused a reduction of the enthalpy and a decrease of the phase-transition temperatures. These two vehicles are thought to fluidize the lamellar-gel phase of the stratum corneum lipids, and possibly partially dissolve the lipids. Dibutyl adipate and caprylic/capric acid triglycerides containing 5% phospholipids decreased the phase-transition enthalpy only, probably due to dissolution or extraction of the stratum corneum lipids. These DSC results provide an explanation for the in-vivo penetration-enhancing effects observed previously.     

The Reversibility of Absorption Promoter Interaction with Red Blood Cell Membranes Studied with Differential Scanning Calorimetry

Absorption promoters, or adjuvants, are used to enhance the gastrointestinal absorption of poorly absorbed drugs such as macromolecules. In the present work, adjuvant–membrane interactions have been studied by differential scanning calorimetry (DSC) using red blood cell (RBC) membranes as model membrane. These interactions caused temperature shifts, amplitude changes, and broadening of the RBC transitions. Because more than one transition may be simultaneously affected by a given adjuvant, complex overlappings occur. Gaussian modeling and nonlinear regression analysis, therefore, were used to resolve these transitions. A correlation, which may serve as an indicator of adjuvant potency, was found between adjuvant concentration and induced transition temperature shifts. Further, these shifts recovered to baseline after successive washings with buffer (for most adjuvants). Sodium lauryl sulfate induced transition alterations, however, never recovered. Thus the DSC might be useful in monitoring reversible adjuvant–membrane interactions.     

The Cryopreservation of Liposomes. 1. A Differential Scanning Calorimetry Study of the Thermal Behavior of a Liposome Dispersion Containing Mannitol During Freezing/Thawing

The thermal behavior of water in liposome dispersions and in liposome dispersions containing mannitol at subzero temperatures was investigated with differential scanning calorimetry (DSC). The cooling curves from 20 down to - 60°C for a liposome dispersion (bilayer composition PL100H/DCP), monitored at cooling rates of 5 and 10°C/min, showed several heat flows related to water crystallization. All lipid-containing dispersions showed water crystallization at temperatures below –40°C. The magnitude of this heat flow strongly depended on the experimental variables. Cooling rate, particle size, lipid concentration, and location and nature of the cryoprotectant all influenced the water crystallization behavior as shown in the DSC cooling curve. Different fractions of water–presumably related to their location in the dispersion–could be distinguished. It is concluded that DSC provides a valuable tool for the detection of changes in the physical state of water in liposome dispersions during freezing/thawing. The insights gained from these DSC studies may make it possible to select–on the basis of rational considerations rather than by trial and error–optimum conditions for the cryopreservation of liposomes containing water-soluble drugs.     

Zaltoprofen/4,4′-Bipyridine: A Case Study to Demonstrate the Potential of Differential Scanning Calorimetry (DSC) in the Pharmaceutical Field

The Zaltoprofen/4,4′-Bipyridine system gives rise to two co-crystals of different compositions both endowed - in water and in buffer solution at pH 4.5 - with considerably higher solubility and dissolution rate than the pure drug.The qualitative and quantitative analysis of the DSC measurements, carried out on samples made up of mixtures prepared according to different methodologies, allows us to elaborate and propose an accurate thermodynamic model that fully takes into account the qualitative aspects of the complex experimental framework and which provides quantitative predictions (reaction enthalpies and compositions of the co-crystals) in excellent agreement with the experimental results. Co-crystal formation and cocrystal compositions were confirmed by X-ray diffraction measurements as well as by FT-IR and NMR spectroscopy measurements.The quantitative processing of DSC measurements rationalizes and deepens the scientific aspects underlying the so-called Tammann's triangle and constitutes a model of general validity. The work shows that DSC has enormous potential, which however can be fully exploited only by paying adequate attention to the experimental aspects and the quantitative processing of the measurements.     

丙泊酚复合亚麻醉剂量氯胺酮清醒镇静应用于颈丛麻醉

目的 观察微泵静注丙泊酚复合亚麻醉剂量氯胺酮清醒镇静用于甲状腺手术中的临床效果.方法 选择ASA Ⅰ～Ⅱ级的甲状腺手术患者60例,随机分为4组,每组15例.分别在丙泊酚20ml溶液中加入氯胺酮0mg(Ⅰ组,对照组),50mg(Ⅱ组),100 mg(Ⅲ组),150mg(Ⅳ组).以首次静脉推注丙泊酚剂量50 ug/kg后,继之以微泵输注35ug/kg/min维持,监测血压、心率、SpO2、ECG、警觉/镇静(0从/S)评分及药物不良反应.结果 试验组病人术中镇静效果较对照组满意(P＜0.05),Ⅲ组病人随访满意度最高(P＜0.05),而Ⅳ组不良反应发生率明显增加(P＜0.05).结论 颈丛麻醉下行甲状腺手术时,丙泊酚复合亚麻醉剂量氯胺酮清醒镇静效果优于单纯应用丙泊酚,100 mg组氯胺酮复合丙泊酚是较为合适的配伍剂量.     

High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

Purpose

The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO? Orange) interact with surfactants, complicating DSF measurements.

Methods

CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO? Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®.

Results

Agreement is observed between SYPRO? Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening.

Conclusions

DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO? Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.

     

腹膜透析联合血液透析与腹膜透析疗效比较

目的比较腹膜透析联合血液透析与腹膜透析的疗效。方法 15例慢性肾功能衰竭患者接受腹膜透析联合血液透析与腹膜透析治疗,分析治疗前后血液及临床指标变化。结果与单腹膜透析相比较,联合治疗可明显降低血肌酐,提高血清白蛋白。结论腹膜透析联合血液透析治疗能增加透析充分性,提高患者生活质量。     

糖尿病合并高脂血症患者血脂、糖化血红蛋白及血液流变学指标的检测分析

目的:了解糖化血红蛋白、血脂水平及血液流变学与糖尿病并发动脉粥样硬化的关系。方法:取健康组100例,单纯糖尿病组65例,糖尿病伴高脂血症组47例,对各组血中糖化血红蛋白、空腹血糖、血脂各指标和血液流变学等指标进行检测,并进行统计学分析。结果:糖尿病伴高脂血症患者组糖化血红蛋白、血脂水平及血液流变学等指标较单纯糖尿病组以及健康组存有显著性差异(P<0.01或P<0.05)。结论:慢性高血糖、高糖化血红蛋白、高血脂、以及全血粘度、血浆粘度增高均是糖尿病并发动脉粥样硬化独立的危险因素。定期监控有关指标对预防、治疗和减少糖尿病患者并发症的发生具有重要的临床意义。     

纳洛酮联合呼吸机治疗COPD合并Ⅱ型呼吸衰竭疗效观察

目的观察纳洛酮联合呼吸机治疗慢性阻塞性肺疾病(COPD)合并Ⅱ型呼吸衰竭的临床疗效。方法选择2008年6月至2010年2月在河北省张家口市第一医院呼吸科诊治的慢性阻塞性肺疾病合并Ⅱ型呼吸衰竭患者104例,随机分为观察组和对照组各52例。两组患者均予以吸氧、抗感染、止咳化痰平喘等常规治疗。对照组在常规治疗基础上加用双水平气道正压通气的无创正压呼吸机治疗,选用S/T模式;观察组在对照组治疗基础上予以纳洛酮2mg,加入50mL生理盐水中,用微量泵以5mL/h的速度静脉泵入,1次/12h。全部患者均在治疗前及治疗后3d比较动脉血气分析。结果观察组插管率为11.54%,病死率6.25%,对照组插管率28.85%,病死率21.15%,两组比较差异均有统计学意义(P<0.05);治疗后72h观察组动脉血气比较差异均有统计学意义(P<0.05或P<0.01)。结论纳洛酮辅助呼吸机治疗慢性阻塞性肺疾病合并Ⅱ型呼吸衰竭能够明显改善呼吸循环功能,解除呼吸抑制,提高插管率,降低病死率。     

The use of effect sizes to characterize the nature of cognitive change in psychopharmacological studies: an example with scopolamine

Drug induced cognitive change is generally investigated using small sample sizes. In terms of null hypothesis significance testing (NHST) this can render a meaningful change non-significant, as a result of insufficient power in the statistical model. NHST leads to 'all or none' thinking, where a non-significant result is interpreted as an absence of change. An effect size calculation indicates the magnitude of change which has occurred post-intervention, and therefore whether a significant result is meaningful. We used a scopolamine challenge to demonstrate the usefulness of effect sizes. The aim of the study was to determine how effect sizes could describe the cognitive changes that occur following administration of subcutaneous scopolamine (s.c. scopolamine). Twenty four healthy young males (M = 32.6, sd = 4.5 years) were administered placebo and 0.2 mg, 0.4 mg & 0.6 mg of s.c. scopolamine using a 4-way crossover design. Memory, learning, psychomotor function, attention and executive function were assessed. Scopolamine significantly impaired performance on all tasks in a dose and time related manner. These results demonstrate the functionality of change scores to draw comparisons between different times and doses. This methodology overcomes the limitations of comparisons between studies using different tasks, doses and time at which cognitive functions are measured.     

The use of optical imaging to assess the potential for embryo-fetal exposure to an exogenous material after intravaginal administration

A β-actin-luc transgenic mouse model was used to evaluate whether embryo-fetal exposure could occur after intravaginal administration of a compound. A bioluminescent substrate, d-luciferin, was delivered intravaginally to mimic compound exposure to the female reproductive track and the embryo-fetus. Bioluminescence was observed throughout the reproductive tract during diestrus, but not during estrus, 2–5 min after intravaginal d-luciferin administration to female β-actin-luc mice. Intravaginal administration of d-luciferin to wild-type females mated with male β-actin-luc mice indicated that the substrate reached the developing embryo-fetus, with bioluminescence corresponding to transgene expression in the embryo-fetus. d-Luciferin substrate rapidly reached the embryo-fetus regardless of the administration route (intravaginal, intraperitoneal, subcutaneous, or intravenous). Vaginal ligation appeared to block at least some direct exposure to the embryo-fetus, but did not prevent d-luciferin from eventually reaching the embryo-fetus. Additional work will be necessary to form the basis for a reliable assessment of the human risk for male-mediated teratogenicity.     

Some factors related to the use of psychoactive drugs,in an institution for adults with mental handicap

A 1-day audit (on 11 March 1985) of the psychotropic drugs prescribed for 1084 institutionalized adults with mental handicap in a long-stay hospital is reported. The relevance of staffing levels, chronological age, sex and mental level is considered. Of the 1084 patients, 35 per cent were 17–44 years old and 65 per cent were 45 years or older; 51 per cent were severely and 40 per cent were profoundly mentally retarded; 23 per cent received antiepileptic drugs; 22 per cent received antipsychotic drugs. The hospital's nurse:patient ratio was nationally recognized as among the worst in England. There was no evidence that a low staffing level was associated with a high level of antipsychotic drug use, but there was an inverse relationship to age, and to mental level. Significantly more women than men received antipsychotics/anxiolytics, antidepressants or hypnotics/sedatives.     

The use of an automated hematology analyzer to observe cell growth in the chromosome aberration test using human lymphocytes

Human lymphocytes have been frequently used for in vitro chromosome aberration or micronucleus tests on whole-blood culture. However, it is difficult to observe or confirm the cell growth of lymphocytes just before chemical treatment compared with cultured cell lines, such as CHL or CHO cells. In order to overcome this drawback of using whole-blood culture, we investigated a possibility of using an automated hematology analyzer (AHA) (Sysmex XT-2000i, SYSMEX Corp. (Hyogo, Japan)) to measure the growth of lymphocytes applying a manual function of this apparatus. In this study, whole-blood samples were cultured for 4 days, and the growth of lymphocytes was measured once a day using a standard flow cytometer (FCM) with antibody CD3 and DNA staining solution, and by the AHA simultaneously. The results showed that growth curves produced employing the two methods coincided fairly well. Therefore, it can be concluded that the growth of lymphocytes in whole-blood culture can be measured using AHA in a straightforward and rapid way in in vitro chromosome aberration or micronucleus tests.     

The use of xanthan gum in an ophthalmic liquid dosage form: rheological characterization of the interaction with mucin

The development of an ocular dosage form containing xanthan gum and capable of interacting with mucin in the precorneal area is a challenge. The polymer concentration that can be applied is restricted because of the limited patient acceptability of highly viscous preparations. The precorneal mucin concentration is low and the high ionic strength of the lachrymal fluid forces xanthan gum in an ordered structure, less capable of interacting through heterotypic junctions. Intrinsic viscosity measurements and shear rheometry are used to investigate the effect of several factors (polymer concentration, additional boiling or sonication step to the preparation procedure) on the physicochemical properties of xanthan gum and the degree of interaction with a low (8%, w/v) and high (16.0%, w/v) concentrated mucin dispersion. Independent of the preparation procedure applied, a xanthan gum concentration of 1.0% (w/v) is required to obtain a measurable interaction with mucin. If an extra boiling or sonication step is added to the standard preparation procedure, the minimum mucin concentration necessary to achieve formation of heterotypic junctions is decreased. Only by sonication of the highly concentrated xanthan gum dispersion is the viscosity decreased to a level that is tolerable and comfortable to the patient. The findings of the present study clearly demonstrate that a significant interaction between a tolerable and comfortable ocular dosage form containing xanthan gum, and mucin 8% (w/v), is feasible after sonication of a highly concentrated polymer dispersion.     

The use of small interfering RNA to elucidate the activity and function of ion channel genes in an intact tissue

Small interfering RNA (siRNA) directs the targeted destruction of mRNA encoding a specific protein, in a process known as RNA interference (RNAi). This stops translation of the targeted mRNA into protein, effectively silencing the gene. RNAi is a recent discovery, identified in mammalian cells in 2001, but it has rapidly advanced into a practical technique and is being used increasingly to investigate mammalian gene function. Tools are available to induce RNAi in cell lines, intact tissue preparations and even in vivo. Depending on the method used, loss of gene expression may be transient or sustained, enabling a wide range of functions to be investigated. RNAi therefore offers a powerful technique that can be used to produce targeted knockout of ion channel genes in mammalian cells. Its applications potentially include identification of ion channel function in health and disease, identification of novel channel genes and drug target validation. This paper outlines our current understanding of siRNA and the experimental requirements for producing efficient RNAi and gene silencing. Effective RNAi requires an appropriate siRNA sequence to be designed and an efficient method for delivering the siRNA to the cells of interest. Since not all potential siRNA sequences are effective, it is also important to verify the loss of gene expression by measuring the level of channel protein remaining. Limitations of the methods available for delivering siRNA are one of the main obstacles to producing efficient RNAi, especially in intact tissue preparations. Here we describe an in vitro method for targeted RNAi against the TASK-1 potassium channel gene in an isolated vascular preparation, using a DNA construct to direct the expression of siRNA, along with a non-viral method for transfecting cells within the vessel. Successful silencing of the TASK-1 gene is verified by immunostaining with an antibody directed against the TASK-1 protein.