Equivalence in test assay method comparisons for the repeated-measure, matched-pair design in medical device studies: statistical considerations

In medical device clinical studies, including therapeutic device and in vitro test assay method studies, the investigator is frequently interested in demonstrating the equivalence of clinical response, generally in continuous measurements, between a standard assay method and a new assay method over various occasions or times. The new assay method may be less invasive or more convenient or cheaper to use than the standard assay method. In this paper, several statistical approaches are discussed, including various repeated-measure regression models, the simultaneous 95% confidence interval for paired mean differences derived from Hotelling's multivariate T2 analysis for repeated-measure, paired data, repeatability and reproducibility studies, and concordance correlation coefficient.     

Development of the SoloSTAR® insulin pen device: design verification and validation

Background: SoloSTAR^® (SOL; sanofi-aventis, Deutschland, GmbH) is a new, disposable insulin injection pen device for use by people with type 1 or type 2 diabetes to administer long- or short-acting insulin. Objectives: To discuss factors that have underlined the design process of the SOL device. In addition, to highlight the studies that shaped the direction of its development, such as addressing the unmet needs of people with diabetes, which included a need for better differentiation features and a lower injection force compared with existing prefilled disposable pen devices. Results: The development of the SOL pen device was an iterative process involving both patients and the design team, which has lead to a manufacturable, tailor-made pen device. Patients' needs have been taken into account in the pen design; there are numerous differentiators on the device, which avoids confusion between insulin types. Furthermore, the SOL device has a lower injection force compared with other marketed pen devices. Finally, studies have shown that the SOL device is more accurate, easier to use and is preferred by patients over other pens on the market. Conclusions: The SOL device has undergone rigorous user and laboratory testing, which has captured evolving improvements to better meet the needs of people with diabetes.     

Pharmacokinetic study of a tripeptide HIV-1 protease inhibitor,KNI-174, in rats after intravenous and intraduodenal administrations

Recently, as a new type of anti-AIDS drug, an HIV-1 protease inhibitor, KNI-174, has been synthesized; it shows a potent and selective HIV-1 protease inhibitory activity in vitro. In this study, we developed an HPLC assay system for KNI-174 in rat plasma and examined the pharmacokinetics of KNI-174 in rats using this assay method after both intravenous (i.v.) and intraduodenal (i.d.) administrations to obtain the disposition characteristics and bioavailability of this new anti-AIDS drug. This HPLC assay method is specific to KNI-174 and the standard curve was linear from 0.02 to 30 μg ml^?1 plasma. After i.v. administration, 10.0 mg kg^?1, KNI-174 disappeared from the rats' plasma in a three-exponential decay. The mean terminal elimination half-life, t_1/2ÀZ, was 3.97 ± 0.19 (S.E.)h, the total body clearance, CL_tot, was 9.53 ± 1.08 ml min^?1 and the distribution volume at steady state, V_{d, ss}′ was 7070 ± 960 ml kg^?1. In the case of the i.d. administration, 10.0 mg kg^?1, the mean peak plasma concentration, C_max, and the peak time, t_max, were 0.196 ± 0.076 μg ml^?1 and 0.444 ± 0.193 h, respectively. The bioavailability of KNI-174 till infinity, BA^(0-infinity), was 5.37 per cent. Because the IC₅₀ of KNI-174 against HIV-1 in PHA-PBM was 138 ng ml^?1, the time needed for maintaining the concentrations above IC₅₀ after a single i.d. administration of KNI-174 is estimated to be 0.350 ± 0.184 h.     

Safety assessment of aditoprim acute,subchronic toxicity and mutagenicity studies

Aditoprim (ADP), a new developed dihydrofolate reductase (DHFR) inhibitor, has great potential in clinical veterinary medicine because of its greater pharmacokinetic properties than structural analogs. Preclinical toxicology studies were performed to assess the safety of ADP including an acute oral toxicity test, a subchronic toxicity test and five mutagenicity tests. In the acute oral toxicity test, ADP was administered singly by oral gavage to Wistar rats and Kunming mice. The LD₅₀ calculated was 1400 mg kg^–1 body weight (BW) day^–1 in rats and 1130 mg kg^–1 BW day^–1 in mice. In a subchronic study, Wistar rats were administered ADP at dose levels of 0, 20, 100 and 1000 mg kg^–1 diet for 90 days. Significant decreases were observed on body weight and food efficiency in the high‐dose group. Treatment‐related changes in clinical serum biochemistry were found in the medium‐ and high‐dose groups. Significant increases in the relative weights of livers and kidneys in females and testis in males in the 1000 mg kg^–1 diet, and significant decrease in relative weights of livers in males in the 100 mg kg^–1 diet were noted. Histopathological observations revealed that the 1000 mg kg^–1 ADP diet could induce lymphocytic infiltration and hepatocytic necrosis near the hepatic portal area. The genotoxicity of ADP was negative in tests, such as the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay, in vitro chromosomal aberration test, in vitro cho/hgprt mammalian cell mutagenesis assay and mice testicle cells chromosome aberration. Based on the subchronic study, the no‐observed‐adverse‐effect level for ADP was a 20 mg kg^–1 diet, which is about 1.44‐1.53 mg kg^–1 BW day^–1 in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

用紫外导数分光光度法测定复方替米沙坦氨氯地平片的含量

目的 测定复方片中替米沙坦和氨氯地平的含量。方法 采用紫外导数分光光度法,替米沙坦测定波长为236 nm,氨氯地平测定波长为390 nm。结果 替米沙坦浓度在（4~20）×10^-3mg/ml范围内与一阶导数值有良好的线性关系,其标准曲线方程为Y=0.0043X-0.0005,R²=0.9993;氨氯地平浓度在（10~90）×10^-3 mg/ml范围内与一阶导数值有良好的线性关系,其标准曲线方程为Y=0.0003X+0.0002,R²=0.9995。结论 该方法具有较好的灵敏度、准确度和精密度;样品检测结果与HPLC法基本一致;是测定复方替米沙坦氨氯地平片含量的一种较好的分析方法。     

A new phenanthrenequinone from Dendrobium draconis

A number of Dendrobium species (Orchidaceae) have been used as health foods. In Thailand, the tea prepared from the stems of Dendrobium draconis Rchb.f. (Orchidaceae) has been used as a blood tonic. Our phytochemical investigation of this plant led to the isolation of a new compound namely 5-methoxy-7-hydroxy-9,10-dihydro-1,4-phenanthrenequinone (1), along with four known stilbenoids including hircinol (2), gigantol (3), batatasin III (4), and 7-methoxy-9,10-dihydrophenanthrene-2,4,5-triol (5). The structures of these compounds were determined through extensive spectroscopic studies, including ¹H and ¹³C NMR, DEPT, COSY, NOESY, HMQC, HMBC, ESI-MS, and HR-ESI-MS experiments. In the DPPH-free radical assay, these stilbene derivatives showed appreciable antioxidant activity.     

Evaluation of a tiered in vitro testing strategy for assessing the ocular and dermal irritation/corrosion potential of pharmaceutical compounds for worker safety

Introduction: Irritation reactions are a frequently reported occupational illness. The potential adverse effects of pharmaceutical compounds (PCs) on eye and skin can now be assessed using validated in vitro methods.

Objectives: Our overall aim is to reduce animal testing by replacing the historically utilized in vivo test methods with validated in vitro test methods which accurately determine the ocular and dermal irritation/corrosion potential of PCs to inform worker safety within the pharmaceutical space. Bristol–Myers Squibb (BMS) and the Institute for In Vitro Sciences (IIVS) have therefore conceptualized and internally qualified a tiered in vitro testing strategy to inform occupational hazards regarding eye and skin irritation and corrosivity of PCs. For the small scale pre-qualification phase, we paired historical in vivo and newly generated in vitro data for 15 PCs to determine the predictive capacity of in vitro assays already validated for the eye and skin irritation/corrosion endpoints and accepted for certain regulatory submissions. During the post-qualification phase, a group of 24 PCs were subjected exclusively to the developed tiered testing strategy, which is based on three Organisation for Economic Co-operation and Development (OECD) in vitro methods.

Materials and methods: The qualified in vitro testing strategy utilizes the Corrositex^® assay for the corrosivity (OECD TG 435), the Bovine Corneal Opacity and Permeability (BCOP) assay for ocular irritation (OECD TG 437), and the EpiDerm? tissue model-based Skin Irritation Test (SIT) for dermal irritation (OECD TG 439). In the first step, the pH of each PC was determined. For compounds with pH extremes ≥11 or ≤2, the Corrositex^® assay was generally conducted first. For compound(s) that were incompatible with or were negative in the Corrositex^® assay or had pH values between 2 and 11, the BCOP assay and SIT were performed first.

Results: The results of the tiered testing strategy’s qualification phase demonstrated that the BCOP assay is sensitive enough to identify a wide range of eye irritation/corrosion potentials and its over-prediction rate was considered acceptable to inform occupational hazards and ensure the proper handling practices of PCs. The SIT correctly predicted the skin irritation potential of 14 out of the 15 PCs included in the qualification phase, only over-predicting one PC. In the post-qualification phase, four PCs out of four tested were predicted corrosive by the Corrositex^® assay and thus no further testing was needed or conducted. The rest of the PCs were evaluated in the BCOP assay (both neat and as a 20% dilution), with the higher response being used for hazard classification. Four PCs were determined to be severe eye irritants, 1 a moderate irritant, 8 were mild irritants, and 8 were non-irritants. The same set of PCs was evaluated using the SIT and were classified as non-irritants to skin. These results are consistent with the BMS historical in vivo results showing a very low number of PCs as skin irritants.

Conclusions: This tiered in vitro testing strategy, which replaces the use of animal studies, was found to be reasonably accurate in its predictive capacity when compared to historical in vivo results and represents a conservative and reliable platform that can be utilized for the prediction of ocular and dermal irritation/corrosion potential of PCs and for subsequent GHS classification and worker safety hazard communications.     

Evaluation of the vascular components of the chorioallantoic membrane assay as a model for eye irritation potential: II

Abstract

Previous research has shown that the chorioallantoic membrane (CAM) of a fertile hen's egg can be a useful model for predicting eye irritation potential. However, the specific test procedure utilized will affect the degree of correlation between the CAM response and the results from the standard Draize rabbit eye irritation test. We studied how various components of a standard CAM assay, such as age of CAM and the endpoint evaluated, have an impact on the relationship of the CAM response to the in vivo eye response. These studies suggested the basis for a new CAM assay procedure that improves the correlation with the in vivo test. Herein is described an assay method that involves treating a 14-day-old CAM and evaluating the vascular changes that occur 30 min following treatment. This method is referred to as the CAM vascular assay (CAMVA). The CAMVA is shown to produce fewer false-positive responses than previously described CAM procedures and is especially effective at differentiating irritants from non-irritants as classified for in vivo test results by U.S. regulatory guidelines.     

A critical assessment of edaravone acute ischemic stroke efficacy trials: is edaravone an effective neuroprotective therapy?

Importance of the field: Edaravone (Radicut^®) is a free radical scavenger marketed in Japan by Mitsubishi Tanabe Pharma Corp. to treat acute ischemic stroke (AIS) patients presenting within 24 h of the attack. Injectable edaravone ampoules (30 mg b.i.d., i.v., 14 days) were first approved on 23 May 2001. On 19 January 2010, as a new innovation, the Radicut BAG (Intravenous BAG) was approved by the Japanese Ministry of Health and Welfare. Efficacy of edaravone ranges from large significant clinical improvements to only modest improvements in clinical function measured using standard stroke scales when administered 6 – 72 h following an ischemic stroke. With almost 17 years of edaravone clinical experience, a few adverse events – including acute renal failure – have been noted.

What the reader will gain: This is the only article to date to critically review available clinical efficacy and toxicology data published in the literature to ascertain whether edaravone should be further pursued as a candidate for development worldwide.

Areas covered in this review: This review covers clinical studies carried out over the period 1993 – 2008.

Take home message: Edaravone may be a useful neuroprotective agent to treat the > 15 million victims worldwide who are devastated by stroke annually. Additional clinical studies are necessary to verify the efficacy of edaravone.     

Usability of a pre-filled insulin injection device in a 3-month observational survey of everyday clinical practice in Australia*

ABSTRACT

Objective: SoloSTAR^{* ?} (SR) is a new pre-filled insulin pen device for administration of insulin glargine and insulin glulisine. This article reports on the usability of SR, as reported by healthcare professionals (HCPs) and participants, in clinical practice in Australia.

Methods: Individuals with Type 1 or Type 2 diabetes were eligible for this 3-month observational survey. Participants were supplied with the insulin glargine SR pens, the instruction leaflet and a toll-free helpline number. Training was offered to all participants. Independent telephone interviews were conducted with participants and HCPs after 6–10 weeks of use of SR.

Results: Overall, 150 HCPs across 93 sites supported this survey. Of these, 65 HCPs (14 doctors; 51 diabetes educators, covering 1669 patients) provided feedback, with the remaining 85 HCPs not responding. All HCPs rated participant training as ‘very easy’ or ‘easy’, and most reported that SR had, in their opinion, made training easier (85%) and quicker (98%). Most of the 536 participants reported that ease of learning to use (98%), ease of using (98%) and features (≥?89%) of SR were ‘excellent’ or ‘good’. SR had positive impacts on various psychological aspects for people with diabetes, including helping overcome reluctance to use insulin, and increasing confidence in their ability to manage their diabetes using insulin.

Conclusions: In this non-randomized, non-interventional, open-label, observational survey of clinical practice, HCPs reported that SR was easy to teach and easy to use for people with diabetes. People with diabetes reported that SR was easy to use and rated specific features of SR highly. Further follow-up surveys and comparative studies in clinical practice are needed to confirm these findings and to determine the impact of SR in diabetes management.     

A Quantitative Assay of Telomerase Activity

Purpose. Telomerase is a ribonucleoprotein that extends telomeres at the ends of chromosome. Increased telomerase activity is associated with cellular immortality. The currently available assay for telomerase, i.e., telomeric repeat amplification protocol (TRAP), consists of 2 steps: (a) telomerase-mediated extension of an oligonucleotide primer by the enzyme-containing extracts of cells and tissues, and (b) amplification of the telomerase-extended primer products by polymerase chain reaction (PCR) and detection of the PCR products. It is generally accepted that the current TRAP assay lacks quantitative precision. The present study was to develop a quantitative telomerase assay with greater precision and sensitivity. Methods. This new method used the primer extension method as in TRAP, plus the following modifications: (a) used a lysis buffer that yielded complete lysis of nuclei; (b) removal of PCR inhibitors by phenol/chloroform extraction after primer extension; and (c) used primers for the internal standard that were designed to reduce their competition with the telomerase products for PCR. Results. The modified method showed a good correlation (r² = 0.99, P < 0.001) between telomerase amount (expressed as total protein in cell lysate) and its activity (expressed as telomerase products). Compared to the conventional TRAP, the new method (a) was more sensitive (average of 5.5-fold in cultured cancer cells and >5.9-fold in patient tumors), (b) had a lower inter- and intra-day variability (>3-fold), and (c) showed a 2 to 4-fold broader range of linearity in the standard curve. The higher assay sensitivity further enabled the use of a nonradioactive method, i.e., ethidium bromide staining of DNA, to detect the TRAP products, as opposed to the use of radioactive nucleotide and the more labor-intensive autoradiography mandated by the conventional TRAP. Conclusion. We report here a quantitative assay for telomerase activity in cultured human cancer cells and patient tumors.     

A highly sensitive assay for xanthine oxidoreductase activity using a combination of [13C2,15N2]xanthine and liquid chromatography/triple quadrupole mass spectrometry

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [¹³C₂,¹⁵N₂]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [¹³C₂,¹⁵N₂]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [¹³C₂,¹⁵N₂]xanthine as a substrate, [¹³C₂,¹⁵N₂]UA as an analytical standard, and [¹³C₃,¹⁵N₃]UA as an internal standard. The [¹³C₂,¹⁵N₂]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R² = 0.998, weighting of 1/x²) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial‐diluted mouse plasma was measured. Thereby, the XOR activity of the 1024‐fold‐diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high‐sensitivity measurements required for XOR activity analysis on various organs or human plasma.     

Observations on conducting whole‐cell patch clamping of the hERG cardiac K+ channel in pure human serum

Inhibition of the human ether‐a‐go‐go‐related gene (hERG) K⁺ channel by drugs leads to QT prolongation on the electrocardiogram and can result in serious cardiac arrhythmia. For this reason, screening of drugs on hERG is mandatory during the drug development process. Patch clamp electrophysiology in a defined physiological saline solution (PSS) represents the standard method for assaying drug effects on the channel. To make the assay more translatable to clinical studies, we have conducted whole‐cell patch clamping of hERG using pure human serum as the extracellular medium. Pure human serum had little effect on the hERG channel waveform or the current–voltage relationship when compared to PSS. hERG current recordings were highly stable in serum at room temperature, but prolonged recordings at the physiological temperature required prior heat inactivation of the serum. Compared to PSS, the IC₅₀ values, conducted at room temperature, of the classic hERG blocking drugs cisapride, moxifloxacin, and terfenadine were shifted to the right by an extent predicted by their known plasma protein binding, but we did not detect any differences in IC₅₀s between male and female serum. Total plasma levels of these drugs associated with clinical QT prolongation corresponded to small (<15%) inhibition of hERG current in pure serum suggesting that minor inhibition of the channel leads to observable pharmacodynamic effects. Conducting whole‐cell patch clamping of hERG in human serum has the potential to make the assay more translatable to clinical studies and improve its predictive value for safety testing. Copyright © 2016 John Wiley & Sons, Ltd.     

Evolution of the percutaneous penetration and distribution of uranyl nitrate as a function of skin-barrier integrity: an in vitro assessment

As recommended by OECD Guidelines, percutaneous penetration studies consider intact skin, but rarely injured skin. Recent years have witnessed a growing concern for these two types of dermal exposure in the industry, particularly in the nuclear industry. The aim of this study was to show that a method based on an in vitro device can be used to realistically assess how skin-barrier alterations caused by occupational accidents can modify the percutaneous penetration and distribution of radionuclides, particularly uranium. Wounds encountered in the nuclear industry (i.e., nitric acid burns and abrasion) were simulated on hairless rat skin. Skin-barrier alterations were characterized by means of a histological study and by measuring transepidermal water loss (TEWL) and skin thickness. The percutaneous penetration of uranyl nitrate through intact or injured skin biopsies was then measured in vitro. The maximum uranium flux values obtained for intact skin, skin abrasion with stratum corneum removal, and skin exposed to 2 N HNO₃, 5 N HNO₃, and 14 N HNO₃ were, respectively, 0.6?±?0.02, 1.2?±?0.03, 1.2?±?0.04, 42.0?±?1.0, and 174.0?±?8.7?ng.cm^?2.h^?1. These results demonstrated that the percutaneous absorption of uranium increased with the increased impairment of the stratum corneum. TEWL, combined with maximum uranium flux values measured in vitro, yielded a good prediction of the percutaneous penetration of uranium through injured skin, previously observed in vivo. To conclude, this in vitro assay provides a conservative estimate of the percutaneous diffusion of uranium through intact or injured skin, making it a good alternative method for toxicological studies and risk assessments.     

In vitro stability and metabolism of O2′, O3′, O5′-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat,dog and human plasma: Chemical hydrolysis and role of plasma esterases

1. O2′, O3′, O5′-tri-acetyl-N⁶-(3-hydroxylaniline)adenosine (WS070117), a new structure-type lipid regulator, is being developed in pre-clinical study. In order to monitor drug kinetics it is essential to understand pre-analytical factors that may affect drug assay.

2. In vitro stability and metabolism were investigated using high-performance liquid chromatography (HPLC) method in this study. The hydrolysis products were identified by HPLC–mass spectrometry (MS)/MS method. The esterases involved in WS070117 hydrolysis was assigned via inhibition rate assay.

3. It was found that WS070117 was chemically unstable in alkaline solutions compared to acidic and near neutral solutions. Enzymatic hydrolysis was even more rapid. Hydrolytic rate constants differ between species, being 4.24, 5.96?×?10^?3 and 6.85?×?10^?2 min^?1 in rat, dog and human plasma at 37°C, respectively. The hydrolysis was catalyzed by plasma esterase because NaF (sodium fluoride: a general esterase inhibitor) inhibited WS070117 hydrolysis and metabolite production. Hydrolysis was fast in rat plasma and was catalysed by carboxylesterase and butyrylcholinesterase. In dog plasma, carboxylesterase, butyrylcholinesterase and paraoxonase were mainly responsible. Butyrylcholinesterase was the major esterase involved in WS070117 hydrolysis in human plasma. The WS070117 hydrolysis in plasma proceeded by gradual loss of acetyl groups.

4. The knowledge of in vitro drug stability and metabolic pathways identified in this study will be essential for future pre-clinical and clinical pharmacokinetics studies.

     

Bazedoxifene when paired with conjugated estrogens is a new paradigm for treatment of postmenopausal women

Importance of the field: The concept of the tissue selective estrogen complex (TSEC) combining a selective estrogen receptor modulator (SERM) with one or more estrogens, aims to provide comparable efficacy to combination estrogen and progestin therapy for symptomatic menopausal women with a uterus without the need for a progestin.

Areas covered in this review: Published multi-center randomized blinded clinical trials with bazedoxifene alone and paired in combination with conjugated estrogens show an effect in hot flashes, vaginal atrophy, quality of life measures, sleep, bone density, and breast and uterine safety.

What the reader will gain: A new concept for menopausal women, bazedoxifene with conjugated estrogens (BZA-CE) TSEC, appears to provide the selective benefits of a SERM with additional benefits of estrogen without the need for a progestin. Preclinical studies with bazedoxifene alone showed that it was antagonistic in the uterine and breast tissue while an agonist in the bone. Phase II and III clinical studies of BZA-CE reveal relief from hot flashes and vaginal atrophic changes, and improvement in bone density, quality of life and sleep without breast or uterine stimulation.

Take home message: Bazedoxifene paired with conjugated estrogens in postmenopausal women relieves vasomotor symptoms and vulvovaginal atrophic changes with prevention of bone loss. Adverse events include a twofold increase risk of venous thrombosis. No evidence of stimulation of the breast, uterus or ovary was seen.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

高效液相色谱法分离测定大鼠肝微粒体中普萘洛尔葡醛酸化代谢产物

目的　建立分离测定外消旋普萘洛尔两种对映体的葡醛酸化代谢产物的反相高效液相色谱法。方法用生物合成法合成R-(+)-普萘洛尔葡醛酸苷,并经C₁₈固液萃取柱纯化、浓集,得到标准液储备液,以此标准液进行普萘洛尔葡醛酸化代谢物的分析测定。结果　R-(+)-普萘洛尔葡醛酸苷在0.24-73.17μmol·L^-1 浓度范围内线性良好,方法专属性高,平均回收率为99.4%±1.0% ,日内精密度RSD小于3.3% ,日间精密度小于4.5%。结论　此法可用于外消旋普萘洛尔两种对映体体外葡醛酸化代谢研究     

Performance of a miniaturized algal bioassay in phytotoxicity screening

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC₅₀ results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r ² = 0.975, p < 0.0017) or environmental samples toxicity (r ² = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r ² = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.     

Quantification of CKD-501, lobeglitazone, in rat plasma using a liquid-chromatography/tandem mass spectrometry method and its applications to pharmacokinetic studies

CKD-501 (i.e., lobeglitazone), a potent agonist for both PPARα/γ, is a new drug that has potential clinical applications in the management of type-2 diabetes. The objective of this study was to develop a rapid and sensitive method for the determination of CKD-501 in rat plasma and to assess the applicability of the assay to pharmacokinetic studies. Rat plasma samples were processed using a fast flow protein precipitation (FF-PPT) method and then introduced onto an LC–MS/MS system for quantification. The analyte and rosiglitazone, an internal standard, were analyzed by multiple reactions monitoring (MRM) at m/z transitions of 482.0 → 258.0 for CKD-501 and 358.0 → 135.0 for the internal standard. The lower limit of quantification (LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10,000 ng/mL (R > 0.999). Validation parameters such as accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the acceptance criteria of the assay validation guidelines, indicating that the assay is applicable to estimating the concentration in the range studied. The concentration of CKD-501 was readily quantifiable in plasma samples up to 24 h post-dose in rats that had received an oral dose of 1 mg/kg. These observations suggest, therefore, that the validated assay can be used in pharmacokinetic studies of CKD-501 in small animals such as the rat.