An in Vitro Model for Assessing Muscle Irritation Due to Parenteral Antibiotics

An in Vitro Model for Assessing Muscle Irritation Due to ParenteralAntibiotics. WILLIAMS, P. D., MASTERS, B. G., EVANS, L. D.,LASKA, D. A., AND HOTTENDORF, G. H. (1987). Fundam. Appl. Toxicol.9, 10–17. A rat skeletal muscle cell line (L6) was evaluatedfor its potential to discriminate the muscle-irritating liabilityof several parenteral antibiotics. The cells were exposed toclinical as well as diluted concentrations of tetracycline,cefoxitin, cephalothin, carbenicillin, erythromycin, ceforanide,cefazolin, and cephaloridine for 1 hr. Control cells were similarlyexposed to culture media for 1 hr. The cells were subsequentlyassayed for their content of the muscle-associated enzyme creatinekinase (CK). Depletion of CK relative to control cultures wasutilized as the index of cellular damage. The results of theseanalyses revealed the following ranking of antibiotic toxicityto L6 muscle cells: tetracycline, erythromycin, cefoxitin >cephalothin, carbenicillin > ceforanide, cefazolin > cephaloridine.The relative order of toxicity of these antibiotics to L6 cellsis in good agreement with their reported muscle-irritating liabilityin man. The correlation between the results obtained in vitroand the irritancy data in vivo suggests that this model maybe a useful adjunct to in vivo testing of parenteral antibioticsfor muscle-irritation liability.     

Predicting Injection Site Muscle Damage I: Evaluation of Immediate Release Parenteral Formulations in Animal Models

Purpose. The current animal model generally accepted by the pharmaceutical industry and the FDA for assessment of muscle damage following intramuscular injection (IM) is the rabbit lesion volume model (RbLV). However, this model is resource intensive. The goal of this study was to find a resource sparing alternative to the rabbit lesion model for assessing injection site toleration in IM formulation screening. Methods. Short term animal model alternatives to RbLV for evaluating IM formulations were examined. In addition to RbLV, myeloperoxidase (MPO), p-nitrophenyl N-acetyl--glucosaminide (NAG) and/or plasma creatine phosphokinase (CK) activities were determined in rabbits (Rb) and rats (Rt) after injection of formulations (digoxin, azithromycin and danofloxacin). The edema from these formulations 24 hr after subcutaneous injection into the rat footpad (RFE) was also determined. Results. MPO and NAG were not considered very useful as biochemical predictors of muscle damage for these formulations. Histology generally correlated with RbLV values. Compared to saline, RbLV was marked for all formulations within 1–3 days of injection. After day 3, lesions quickly resolved, and no significant differences were found. For these formulations, all CK animal models and RFE were generally predictive of RbLV. A formulation with RtCK > 1000 U/L or RbCK > 3000 U/L, was predicted to be poorly, tolerated. Conclusions. Due to ease, number of animals, time and intrinsic mechanism, we concluded that for most formulations, 2 and 4 hr RtCK data alone should be reasonably predictive of muscle damage.     

Predicting Injection Site Muscle Damage II: Evaluation of Extended Release Parenteral Formulations in Animal Models

Purpose. The goal of this study was to find a resource sparing alternative to the rabbit lesion model (RbLV) for assessing injection site toleration in extended release (ER) intramuscular (IM) formulation screening. Methods. ER formulations (danofloxacin oily and aqueous suspensions) were evaluated in RbLV, rat and rabbit plasma creatine phospho-kinase (CK), and rat foot edema (RFE) models as described in the companion article. Results. None of the short term models could consistently predict acute and chronic effects of the. For example, RFE predicted little muscle damage from aqueous vehicle (0.03 ± 0.03 g) and 60 mg/ml (0.08 ± 0.03 g) formulation; while RbLV_{days l–3} was marked and greater (p < 0.05) for 60 mg/ml (6.0 ± 3.1) than vehicle (2.2 ± 2.9) formulations. Furthermore, RbLV_{days l–3} for vehicle (6.5 ± 7.5) and 60 mg/ml (4.9 ± 4.6) danofloxacin oily formulations were worse (p < 0.05) than oil alone (1.4 ± 2.2); an observation not predicted by CK models, since they apparently reflected only the acute muscle damage of formulation components immediately available to surrounding tissue at the time of injection. Conclusions. The CK models may be useful to screen those ER formulations with unacceptable acute damage due to immediately available components. However, to evaluate potential delayed effects from ER formulations, the long-term model RbLV was still recommended.     

Pain on Injection and Muscle Irritation: A Comparison of Animal Models for Assessing Parenteral Antibiotics

Pain on Injection and Muscle Irritation: A Comparison of AnimalModels for Assessing Parenteral Antibiotics. COMERESKI, C. R.,WILLIAMS, P. D., BREGMAN, C. L., AND HOTTENDORF, G. H. (1986).Fundam. Appl. Toxicol. 6, 335–338. Pain on injection dueto parenteral administration of cephaloridine, cephalothin,and cefoxitin with or without 1% lidocaine was examined in arat paw-lick model and the results compared with those obtainedin a rabbit intramuscular model of irritation. In both animalmodels, cephaloridine caused similar or a slightly greater responsethan sterile water. Conversely, cefoxitin and cephalothin causeda much greater reaction than water in both models. The onlymajor difference in the rankings by the two models was withformulations in which an anesthetic agent was incorporated intothe diluent. As expected, the presence of a local anestheticmasked pain on injection but not muscle damage. The rat paw-lickmodel is an alternative to the traditional rabbit muscle irritationmodel for rapidly assessing both pain on injection and muscleirritation of parenteral formulations.     

Predicting Injection Site Muscle Damage III: Evaluation of Intramuscular Formulations in the L6 Cell Model

Pharmaceutical Research -     

重组人皮肤模型用于评价外用液体药用高密度聚乙烯瓶的原发性皮肤刺激性研究

目的 探讨重组人皮肤模型用于评价外用液体药用高密度聚乙烯瓶原发性皮肤刺激的可行性。方法 采用Epikutis^®3D表皮模型和EpiSkin^TM重组人皮肤模型对6批外用液体药用高密度聚乙烯瓶的原发性皮肤刺激进行检测,并与家兔法所得的结果进行比较。结果 2种重组人皮肤模型检测结果与家兔法检测结果均为无刺激性,体内外试验的一致率为100%。结论 重组人皮肤模型可用于外用液体药用高密度聚乙烯瓶的原发性皮肤刺激性检测。     

EpiSkin™和Epikutis®模型在体外皮肤刺激性和腐蚀性检测应用中的比较

目的 对Episkin™和Epikutis®模型在体外皮肤刺激性和腐蚀性检测各方面进行对比。方法 参考体外皮肤刺激实验（OECD指导原则439）和体外皮肤腐蚀实验（OECD指导原则431）,对2种模型在质量控制、检测方法、结果判断、运输等方面进行探讨。结果 2种模型在质量控制、检测方法、结果判断等方面存在差异,在实验操作性和成本上也各有优缺点,但2种模型均能满足体外皮肤刺激性和腐蚀性实验要求。结论 2种模型均能很好地用于体外皮肤刺激性和腐蚀性检测。     

Comparison of in vitro and In vivo Models to Assess Venous Irritation of Parenteral Antibiotics

Comparison of in vitro and In vivo Models to Assess Venous Irritationof Parenteral Antibiotics. HOOVER, D. M., GARDNER, J. B., TIMMERMAN,T. L., KLEPFER, J. A., LASKA, D. A., WHITE, S. L., MCGRATH,J. P., BUENING, M. K., AND WILLIAMS, P. D. (1990). Fundam. Appl.Toxicol. 14, 589–597. The venous irritation potentialof four parenteral antibiotics, tetracycline hydrochloride (TET),erythromycin lactobionate (ERY), amphotericin B (AMP), and cephalori-dine(CEP), was evaluated in an In vivo model using the rabbit earvein. Lateral ear veins of New Zealand White rabbits were infusedfor 1 hr with test solutions containing TET (0.25, 2.5, or 10mg/ml), ERY (2.5, 5, or 25 mg/ml), AMP (0.05, 0.1, or 0.5 mg/ml),or CEP (4 or 20 mg/ml). Control rabbits received comparablevolumes of 0.9% NaCl or 5% dextrose. Approximately 24 hr postinfusion,the rabbits were evaluated for visually evident changes in thetreated ears. Pathologic evaluation of the veins was performedusing histologic sections and scanning electron microscopy.TET, ERY, and AMP caused concentration-dependent changes inveins characterized primarily by loss of endothelium with associatedinflammation and thrombus formation, consistent with the knownclinical irritancy of these antibiotics. CEP, on the other hand,was well tolerated in the rabbit ear vein, paralleling its lowirritancy potential in man. Test solutions identical to thoseused In vivo in rabbits were also evaluated in established invitro assays for hemolytic potential when mixed with whole bloodfrom monkeys and for damage to L6 muscle cells as determinedby loss of creatine phosphokinase. Results of the in vitro testsystems paralleled those of the rabbit ear model, with TET,ERY, and AMP exhibiting dose-dependent hemol-ysis and musclecell toxicity, while CEP was comparatively nontoxic. Of thethree models, the rabbit ear vein had the greatest sensitivitywhen histopathologic evaluation was employed. The in vitro musclecell toxicity test was slightly less sensitive than the In vivomodel, followed by the in vitro hemolysis test. Results indicatethat the use of these in vitro and In vivo models to evaluatevenous irritancy may assist prec.linical assessment of potentialclinical reactions to new parenteral drug formulations.     

An In Vitro Model to Evaluate Muscle Damage Following Intramuscular Injections

An isolated rat muscle preparation was developed to screen for muscle damage (myotoxic potential) following intramuscular injections. Myotoxicity is evaluated by the total cumulative efflux of the enzyme creatine kinase from the extensor digitorum longus muscle into the incubation medium over a 2-hr period or by the slope of the cumulative creatine kinase efflux curve. The system allows for rapid screening of compounds and/or formulations regarding their myotoxic potential and is not sensitive to fluctuations of in vivo creatine kinase levels caused by animal handling or patient conditions. A good rank-order correlation was obtained between this in vitro technique and the in vivo myotoxicity of a number of pharmaceutical formulations, as indicated by circulating creatine kinase levels and histological observations.     

Fuzzy Logic-Based Expert System for Evaluating Cake Quality of Freeze-Dried Formulations

Freeze-drying of peptide and protein-based pharmaceuticals is an increasingly important field of research. The diverse nature of these compounds, limited understanding of excipient functionality, and difficult-to-analyze quality attributes together with the increasing importance of the biosimilarity concept complicate the development phase of safe and cost-effective drug products. To streamline the development phase and to make high-throughput formulation screening possible, efficient solutions for analyzing critical quality attributes such as cake quality with minimal material consumption are needed. The aim of this study was to develop a fuzzy logic system based on image analysis (IA) for analyzing cake quality. Freeze-dried samples with different visual quality attributes were prepared in well plates. Imaging solutions together with image analytical routines were developed for extracting critical visual features such as the degree of cake collapse, glassiness, and color uniformity. On the basis of the IA outputs, a fuzzy logic system for analysis of these freeze-dried cakes was constructed. After this development phase, the system was tested with a new screening well plate. The developed fuzzy logic-based system was found to give comparable quality scores with visual evaluation, making high-throughput classification of cake quality possible.     

Evaluating the IVIVC by Combining Tiny-tim Outputs and Compartmental PK Model to Predict Oral Exposure for Different Formulations of Ibuprofen

Nowadays, the ever-increasing costs of research and development in the pharmaceutical industry have created a big demand for predicting the performances of drug candidates. Of those, the desire to establish an in vitro-in vivo correlation (IVIVC) to better predict the oral drug exposure for different drug products is a growing need. Once a robust IVIVC is established, the performance of different drug products can be predicted and selected for testing in clinical trials with greater confidence. This tool will significantly reduce the cost and speed of drug development and provide new therapy to the patient faster. In this study, we explore combining the outputs of Triskelion's Gastro-Intestinal Model (Tiny-TIM) and multi-compartment pharmacokinetic model for a 200 mg ibuprofen product. The Loo-Riegelman method was used to calculate the amount of ibuprofen absorbed and was combined with the Tiny-TIM data to establish the IVIVC. The IVIVC was used to predict the exposures of both fast release and liquid gel formulations in humans. In general, the predicted exposure using Tiny-TIM-based IVIVC has good agreement with the clinical findings.     

临床药师参与肠外营养相关性肝损害患者治疗的药学监护

目的:探讨临床药师在肠外营养相关性肝损害(PNALD)患者的临床营养治疗中的作用,促进临床营养药物的合理应用。方法:临床药师参与1例PNALD患者的治疗过程,建议将原有的"全合一"营养液配方中的5%葡萄糖氯化钠注射液500 m L调整为0.9%氯化钠注射液500 m L,30%长链脂肪乳剂250 m L调整为20%中/长链脂肪乳剂250 m L,8.5%复方氨基酸注射液(18AA)1 000 m L调整为8%复方氨基酸注射液(15AA)750 m L,并加用20%丙氨酰谷氨酰胺注射液100 m L和10%ω-3鱼油脂肪乳注射液100 m L,其余配方不变。另加用保肝药物还原型谷胱甘肽、S-腺苷蛋氨酸进行保肝退黄治疗,并逐渐减少肠外营养(PN)的量,过渡到肠内营养(EN)。结果:医师采纳临床药师的调整建议,该患者的肝功能逐渐恢复正常。结论:PN使用时间,PN中脂质种类、摄入量,营养物过剩等是造成PNALD的主要因素。临床药师在患者的临床营养治疗中开展药学监护可确保营养药物安全、有效地利用,减少并发症及药物不良反应的发生。     

Heptanol预处理对细胞膜及线粒体Cx43参与的大鼠心肌细胞缺氧/复氧损伤的作用研究

目的通过对细胞膜及线粒体Cx43表达变化的研究,探讨不同浓度庚醇(Heptanol)预处理对大鼠心肌细胞缺氧/复氧损伤作用。方法将SD乳鼠培养的心肌细胞随机分为5组:正常对照组(control组),缺氧/复氧组(AR组),溶酶组(DMSO组),0.1mmol/L庚醇预处理组(0.1HT组),0.5mmol/L庚醇预处理组(0.5HT组),1.0mmol/L庚醇预处理组(1.0HT组),2.0mmol/L庚醇预处理组(2.0HT组)。除Control组外,H/R组,DMSO组以及各浓度heptonal预处理组均进行缺氧/复氧处理。检测Cx43mRNA水平,并提取心肌细胞线粒体蛋白,应用Western blot检测其含量。结果 AR组、0.1HT组和0.5HT组较control组的Cx43mRNA表达水平及线粒体Cx43含量明显减少,差异有统计学意义(P<0.01)。1.0 HT组和2.0HT组与AR组相比较,Cx43mRNA表达水平及线粒体Cx43含量明显增加,差异有统计学意义(P<0.01)。结论 heptanol预处理可以逆转I/R导致的细胞膜及线粒体Cx43的减少,但作用效果与浓度有关。线粒体Cx43可能参与了heptanol预处理的心肌保护作用。     

The Living Dermal Equivalent as an In Vitro Model for Predicting Ocular Irritation

Abstract

The Living Dermal Equivalent (LDE), composed of human dermal fibroblasts in a collagen-containing matrix, was used as a model system for examining toxic responses to a broad range of test chemicals and final product formulations. The thiazolyl blue (MTT) assay for mitochondrial function and the release of proinflammatory eicosanoids (prostaglandin E₂ and prostacyclin) were used as end points to rank order the relative irritancy of test samples. In these studies a total of 65 test samples including liquids, creams, emulsions, and solids were applied topically to the air-exposed surface of the LDE. Protocols were designed to maximize the discrimination between samples with known ocular irritation potentials. For a given set of samples, altering the experimental conditions, including the dosage and time of exposure, is shown to influence the degree to which responses of the LDE agree with in vivo data. Strategies for using the LDE as an in vitro ocular irritation model are presented and recommendations for standardized protocols are discussed.     

鼠肌核心蛋白聚糖的研制

目的 :从动物组织中提取纯化核心蛋白聚糖 (Decorin) ,检测其活性。方法 :以大鼠肌肉为原料 ,采用匀浆、透析、层析等步骤分离、纯化Decorin ,经SDS PAGE检测其分子量和纯度 ,并经细胞株A5 49检测生物学活性结果 :Decorin在SDS PAGE显示分子量约为 37～ 38kD两条带 ,得率约为 2mg/g组织 ,对A5 49具有明显的抑制生长活性 ,抑制率 >5 0 %。结论 :此法获得的Decorin将有可能作为天然药物用于治疗肿瘤。     

A PBPK Model for Evaluating the Impact of Aldehyde Dehydrogenase Polymorphisms on Comparative Rat and Human Nasal Tissue Acetaldehyde Dosimetry

Acetaldehyde is an important intermediate in the chemical synthesis and normal oxidative metabolism of several industrially important compounds, including ethanol, ethyl acetate, and vinyl acetate. Chronic inhalation of acetaldehyde leads to degeneration of the olfactory and respiratory epithelium in rats at concentrations > 50 ppm (90 day exposure) and respiratory and olfactory nasal tumors at concentrations ≥ 750 ppm, the lowest concentration tested in the 2-yr chronic bioassay. Differences in the anatomy and biochemistry of the rodent and human nose, including polymorphisms in human high-affinity acetaldehyde dehydrogenase (ALDH2), are important considerations for interspecies extrapolations in the risk assessment of acetaldehyde. A physiologically based pharmacokinetic model of rat and human nasal tissues was constructed for acetaldehyde to support a dosimetry-based risk assessment for acetaldehyde (Dorman et al., 2008). The rodent model was developed using published metabolic constants and calibrated using upper-respiratory-tract acetaldehyde extraction data. The human nasal model incorporates previously published tissue volumes, blood flows, and acetaldehyde metabolic constants. ALDH2 polymorphisms were represented in the human model as reduced rates of acetaldehyde metabolism. Steady-state dorsal olfactory epithelial tissue acetaldehyde concentrations in the rat were predicted to be 409, 6287, and 12,634 μM at noncytotoxic (50 ppm), and cytotoxic/tumorigenic exposure concentrations (750 and 1500 ppm), respectively. The human equivalent concentration (HEC) of the rat no-observed-adverse-effect level (NOAEL) of 50 ppm, based on steady-state acetaldehyde concentrations from continual exposures, was 67 ppm. Respiratory and olfactory epithelial tissue acetaldehyde and H⁺ (pH) concentrations were largely linear functions of exposure in both species. The impact of presumed ALDH2 polymorphisms on human olfactory tissue concentrations was negligible; the high-affinity, low-capacity ALDH2 does not contribute significantly to acetaldehyde metabolism in the nasal tissues. The human equivalent acetaldehyde concentration for homozygous low activity was 66 ppm, 1.5% lower than for the homozygous full activity phenotype. The rat and human acetaldehyde PBPK models developed here can also be used as a bridge between acetaldehyde dose-response and mode-of-action data as well as between similar databases for other acetaldehyde-producing nasal toxicants.     

Acute Changes in Muscle Blood Flow and Concomitant Muscle Damage after an Intramuscular Administration

No HeadingPurpose. The intramuscular route (IM) is widely used but commonly induces injection site muscle damage. This study investigates the hemodynamic changes in an acute lesion induced by the IM administration of propylene glycol (PG) in rabbits.Methods. Control groups received 1, 2, or 3 ml of PG (IM). Others were pretreated with pancuronium, dantrolene, indomethacin, or SR140333 and then received 2 ml of PG. The muscle blood flow (MBF) was assessed using fluorescent microspheres before and at 15, 45, 60, 90 min, 3 and 6 h after IM administration. Different areas within the muscle damage were quantified.Results. Muscle contractions as well as a transient but major MBF increase were observed at the injection site. All treatments reduced hyperemia by up to 81% (dantrolene, 15 min) at 15, 45, and 90 min (p < 0.05). MBF had returned to basal values in all groups at 6 h. The central necrotic area was not modified, but peripheral damage (8.0 ± 1.3 g) was reduced by dantrolene, indomethacin, and SR140333 (p < 0.05), but not by pancuronium.Conclusions. Muscle contraction and hyperemia are not responsible for muscle damage at the injection site, which is the multifactorial phenomenon, involving intracellular calcium and inflammation.     

Clinical Pathology Changes Related to Cutaneous Irritation in the Fischer 344 Rat and New Zealand White Rabbit

Abstract

An evaluation of 27 repeated dose cutaneous application studies (9 applications of 6 h over an 11-day period) indicated that several hematologic and clinical chemistry parameters may be altered by chemically induced skin irritation. Irrespective of species, values that were generally decreased included hemoglobin concentration, hematocrit, erythrocyte count, and serum concentrations of calcium, potassium, inorganic phosphorus, and creatinine. Values that were increased included the neutrophil and total leukocyte counts. Some species differences were seen; for example, while the platelet count and serum globulin concentration were increased in rabbits only, the serum glucose, sodium, and chloride concentrations were increased in rats only. The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and serum albumin and total protein concentrations were variably affected. Changes were generally well associated with the degree of cutaneous irritation, but did not appear to be related to the chemical class of the test substances, decreased food consumption, loss of body weight, or systemic toxicity of the chemical. However, the relationship of the changes in the clinical pathology measurements to cutaneous irritation or secondary effects was not always clear. Some measurements were considered to be secondary to the cutaneous inflammation while others may be related to vascular and fluid balance alterations. Regardless of the cause of these changes, their frequent occurrence and consistent direction of change suggest a relationship to cutaneous irritation rather than systemic toxicity of the test substances. It is considered important that the interpreter of toxicologic studies be aware of these irritation-induced changes when evaluating the findings of repeated cutaneous application studies.