Hematologic Toxicity of AZT and ddC Administered as Single Agents and in Combination to Rats and Mice

Hematologic Toxicity of AZT and ddC Administered as Single Agentsand in Combination to Rats and Mice. THOMPSON, M. B., DUNNICK,J. K., SUTPHIN, M. E., GILES, H. D., IRWIN, R. D., AND PREJEAN,J. D. (1991). Fundam. Appl. Toxicol. 17, 159-176. Toxicity studiesof 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine(ddC) were conducted in F344/N rats and B6C3F, mice. The drugswere administered as single agents and in combination. In allstudies, animals were treated by oral gavage twice a day, 7days a week. In studies of the individual compounds, each wasadministered for 13 weeks at the following concentrations; AZTin rats, 0, 125, 250, 500, 1000 mg/kg and in mice, 0, 25, 50,100, 400, 1000 mg/kg; ddC in rats and mice, 0, 250, 1000, 2000mg/kg. Additional male rats and female mice that were treatedwith 0, 250, 1000, or 2000 mg/kg ddC and male and female micetreated with 0, 50, 400, 1000 mg/kg AZT were maintained for30 days after treatment was stopped (at 94 days) to evaluatethe reversibility of toxic effects. Hematologic variables weremeasured on Days 5, 23, and 94 (last day of dosing), and onDay 123 (after a 30-day period without treatment). AZT and ddCproduced dose-related, poorly regenerative, macrocytic anemiasas evidenced by decreases in erythrocyte counts, he-matocrits,and hemoglobin concentrations and increases in mean corpuscularhemoglobin and mean corpuscular volume. Bone marrow samplesin rats treated with AZT were hyperplastic whereas those inmice treated with AZT and rats and mice treated with ddC werehypoplastic. The hematologic toxicity of AZT was more severethan that of ddC. Generally, toxic effects of either chemicalwere greater in mice than in rats and more pronounced in femalethan in male animals. After 30 days without dosing, hematologiceffects either resolved or dramatically improved. In studiesin which ddC and AZT were administered in combination for 4weeks at concentrations of 0/0, 0/500, 500/0, 10/500, 100/500,500/500, and 500/1000 mg/kg ddC/AZT, there was macrocytic anemiain animals in the lower doses and marked microcytic anemia insurviving male mice in higher dose groups. Most female micedied in the 500/500 and 500/1000 mg/kg ddC/ AZT dose groups.At lower concentrations (100/500, 500/1000 mg/kg ddC/AZT), effectsof the two drugs were similar to those in the single drug studies.At higher concentrations (500/500 and 500/1000 mg/kg ddC/AZT),the combination treatment produced enhanced hematopoietic toxicity.These studies demonstrated the early and progressive time courseof toxicity of AZT and ddC, species differences in sensitivitiesand responses, and reversibility of effects after terminationof treatment. Based on these findings, a chronic toxicity studyis being conducted with AZT in mice.     

Differential Effect of Imatinib and Synergism of Combination Treatment with Chemotherapeutic Agents in Malignant Glioma Cells

Abstract: Imatinib mesylate (STI571, Gleevec^®) is a signal transduction inhibitor and novel anti‐cancer agent. It selectively inhibits aberrantly activated tyrosine kinases in malignant cells, for example, bcr‐abl in leukaemia, platelet‐derived growth factor receptor and stem cell factor receptor (c‐Kit) in solid cancers including malignant glioma. However, recently published clinical studies with imatinib monotherapy in patients with malignant glioma demonstrated only very modest anti‐tumour activity. The aim of this study was to investigate the biological activity of imatinib, its cellular mechanisms of action and its synergism with other chemotherapeutic agents in human malignant glioma cells in culture. Expression of PDGF/R and c‐Kit was analyzed by RT‐PCR. Proliferation was measured by MTT assays and drug synergy was assessed by the Chou–Talalay method. Cell cycle and apoptosis were analyzed by flow cytometry and migration by monolayer migration assays. Multi‐immunoblot was performed on imatinib‐treated and control malignant glioma cells. Results indicate that imatinib is more effective in inhibiting cell colony formation and migration rather than proliferation. Imatinib treatment caused cell cycle arrest of glioma cells in G0–G1 or G2/M, with significant elevation of a few cyclin‐dependent kinases. Furthermore, imatinib acted synergistically with chemotherapy agents, such as the DNA alkylating agent, temozolomide, and riboneucleotide reductase inhibitors, for example, hydroxyurea at varied effective dose levels. In conclusion, imatinib exerts varied biological effects on malignant glioma cells in culture. Synergistic interaction of imatinib with chemotherapy agents may be related to cell cycle control mechanisms and could be potentially important in a clinical setting.     

氯萘磺酰胺类化合物的合成与蛋白激酶C免疫调节作用

为了研究蛋白激酶Ｃ抑制剂及激活剂对巨噬细胞释放肿瘤坏死因子的影响，作者设计并合成了十八个５－氯－１－萘磺酰胺类化合物，经初步药理试验表明，本类化合物中的抑制剂均能显著抑制ＴＮＦ释放，而激活剂单独应用时不能引起ＴＮＦ释放，但可增强内毒素引起的ＴＮＦ释放。     

蛋白激酶C的研究进展

阐述蛋白激酶C的生化性质,在肿瘤多药耐药中的作用及近年来研究发现的蛋白激酶C抑制剂。     

蛋白激酶C抑制剂星形孢菌素对肺腺癌A549细胞侵袭力的影响研究

目的:探讨蛋白激酶C(PKC)抑制剂星形孢菌素(STS)对肺腺癌A549细胞侵袭移动的影响及作用机制。方法:取A549细胞加入不同浓度STS(1、10、100 nmol.L-1)处理后,应用Transwell小室检测A549细胞移动抑制率和侵袭抑制率;免疫荧光染色法检测细胞内蛋白水解酶基质金属蛋白水解酶-9(MMP-9)和尿激酶型纤溶酶原激活剂(uPA)蛋白表达;激光共聚焦法观测细胞内骨架蛋白微管蛋白α-tubulin的分布,Western blot法检测胞膜和胞浆内PKC-α活性。同时设立不加STS处理的对照组进行比较。结果:与对照组比较,A549细胞的移动抑制率、侵袭抑制率、MMP-9和uPA蛋白表达量均明显下降(P<0.05或P<0.01);胞浆内α-tubulin蛋白分布不均;胞膜内PKC-α蛋白含量减少,而胞浆内PKC-α蛋白含量无明显变化。结论:PKC抑制剂STS能抑制A549细胞移动和侵袭;其机制可能与抑制PKC-α的活性,减弱肿瘤细胞中MMP-9、uPA表达,诱导肿瘤细胞骨架变化相关。     

Effects of Sustained Administration of Quetiapine Alone and in Combination with a Serotonin Reuptake Inhibitor on Norepinephrine and Serotonin Transmission

Quetiapine is now used in the treatment of unipolar and bipolar disorders, both alone and in combination with other medications. In the current study, the sustained administration of quetiapine and N-Desalkyl quetiapine (NQuet) in rats in a 3 : 1 mixture (hQuetiapine (hQuet)) was used to mimic quetiapine exposure in patients because rats do not produce the latter important metabolite of quetiapine. Sustained administration of hQuet for 2 and 14 days, respectively, significantly enhanced the firing rate of norepinephrine (NE) neurons by blocking the cell body α₂-adrenergic autoreceptors on NE neurons, whether it was given alone or with a serotonin (5-HT) reuptake inhibitor. The 14-day regimen of hQuet enhanced the tonic activation of postsynaptic α₂- but not α₁-adrenergic receptors in the hippocampus. This increase in NE transmission was attributable to increased firing of NE neurons, the inhibition of NE reuptake by NQuet, and the attenuated function of terminal α₂-adrenergic receptors on NE terminals. Sustained administration of hQuet for 2 and 14 days, respectively, significantly inhibited the firing rate of 5-HT, whether it was given alone or with a 5-HT reuptake inhibitor, because of the blockade of excitatory α₁-adrenergic receptors on 5-HT neurons. Nevertheless, the 14-day regimen of hQuet enhanced the tonic activation of postsynaptic 5-HT_1A receptors in the hippocampus. This increase in 5-HT transmission was attributable to the attenuated inhibitory function of the α₂-adrenergic receptors on 5-HT terminals and possibly to direct 5-HT_1A receptor agonism by NQuet. The enhancement of NE and 5-HT transmission by hQuet may contribute to its antidepressant action in mood disorders.     

丝裂原活化蛋白激酶抑制剂对急性肺损伤影响

目的探讨p38MAPK抑制剂SB203580对大鼠急性肺损伤影响。方法采用LPS建立SD大鼠急性肺损伤模型，随机分为对照组、LPS组、SB203580组。造模后注射p38MAPK抑制剂SB203580，在1h、3h、6h及12h时，剖杀大鼠，观察肺组织病理改变，ELISA法测血清中的TNF—α及IL-6；免疫组化检测肺组织中的p38MAPK及其磷酸化-p38MAPK。结果注射LPS后，SB203580治疗组较LPS组血清中的TNF—α及IL-6显著减少（P〈0．05 or 0.01）；肺组织中的磷酸化-p38MAPK的表达显著减轻，而p38丝裂原活化蛋白激酶未明显减轻。结论p38MAPK抑制剂SB203580可减轻血清中的TNF-α及IL-6和肺组织中的磷酸化-p38MAPK的表达，从而减轻LPS诱导的急性肺损伤。     

Protein Kinase C Targeting in Antineoplastic Treatment Strategies

Neoplastic cell survival is governed by a balance between pro-apoptotic and anti-apoptotic signals. Noteworthy among several anti-apoptotic signaling elements is the protein kinase C (PKC) isoenzyme family, which mediates a central cytoprotective effect in the regulation of cell survival. Activation of PKC, and subsequent recruitment of numerous downstream elements such as the mitogen-activated protein kinase (MAPK) cascade, opposes initiation of the apoptotic cell death program by diverse cytotoxic stimuli. The understanding that the lethal actions of numerous antineoplastic agents are, in many instances, antagonized by cytoprotective signaling systems has been an important stimulus for the development of novel antineoplastic strategies. In this regard, inhibition of PKC, which has been shown to initiate apoptosis in a variety of malignant cell types, has recently been the focus of intense interest. Furthermore, there is accumulating evidence that selective targeting of PKC may prove useful in improving the therapeutic efficacy of established antineoplastic agents. Such chemosensitizing strategies can involve either (a) direct inhibition of PKC (e.g., following acute treatment with relatively specific inhibitors such as the synthetic sphingoid base analog safingol, or the novel staurosporine derivatives UCN-01 and CGP-41251) or (b) down-regulation (e.g., following chronic treatment with the non-tumor-promoting PKC activator bryostatin 1). In preclinical model systems, suppression of the cytoprotective function(s) of PKC potentiates the activity of cytotoxic agents (e.g., cytarabine) as well as ionizing radiation, and efforts to translate these findings into the clinical arena in humans are currently underway. Although the PKC-driven cytoprotective signaling systems affected by these treatments have not been definitively characterized, interference with PKC activity has been associated with loss of the mitogen-activated protein kinase (MAPK) response. Accordingly, recent pre-clinical studies have demonstrated that pharmacological disruption of the primary MEK-ERK module can mimic the chemopotentiating and radiopotentiating actions of PKC inhibition and/or down-regulation.     

Isoarenarol,A New Protein Kinase Inhibitor from the Marine Sponge Dysidea arenaria

The novel sesquiterpene hydroquinone isoarenarol (1) and the known compound arenarol (2) were isolated from extracts of the marine sponge Dysidea arenaria Bergquist as part of a search for new protein kinase inhibitors. Both 1 and 2 showed potent and selective protein kinase inhibition in vitro.     

Protein Kinase C Activators as Synaptogenic and Memory Therapeutics

The last decade has witnessed a rapid progress in understanding of the molecular cascades that may underlie memory and memory disorders. Among the critical players, activity of protein kinase C (PKC) isoforms is essential for many types of learning and memory and their dysfunction, and is critical in memory disorders. PKC inhibition and functional deficits lead to an impairment of various types of learning and memory, consistent with the observations that neurotoxic amyloid inhibits PKC activity and that transgenic animal models with PKCβ deficit exhibit impaired capacity in cognition. In addition, PKC isozymes play a regulatory role in amyloid production and accumulation. Restoration of the impaired PKC signal pathway pharmacologically results in an enhanced memory capacity and synaptic remodeling / repair and synaptogenesis, and, therefore, represents a potentially important strategy for the treatment of memory disorders, including Alzheimer's dementia. The PKC activators, especially those that are isozyme‐specific, are a new class of drug candidates that may be developed as future memory therapeutics.     

Overview: Recent Advances in Protein Kinase C Inhibitors

Glycolipids are important components of mammalian cell membranes and play an important role as signal mediators in cellular biology. Recently, the glycolipid mediated biological pathways have been drawing researchers’ attention. There has been a resurgence of interest in the signalling roles of glycolipid derivatives in the biochemical and biomedical research field. The design of glycolipid derivatives that block cell adhesion and regulate the immune response has highlighted potential therapeutic uses in the treatment of cancer, infection and other diseases originating in cell regulation disorder. This review summarises recent developments in the design of the four kinds of glycolipid related compounds (steroidal glycoside derivatives, glycosyl ceramide derivatives, glucosamine derivatives and glycero glycolipid derivatives) for therapeutic use.     

蛋白激酶C抑制药enzastaurin对吉非替尼耐药的人非小细胞肺癌细胞株生长的影响

目的：观察新型靶向制剂enzastaurin单独或联合吉非替尼对耐药人非小细胞肺癌细胞株的影响,设计合理的治疗药物组合。方法：CCK8法检测细胞增殖,Chou-Talalay联用指数法判定联合用药效果,流式细胞仪检测细胞周期变化。结果：单药吉非替尼及enzastaurin作用NCI-H460耐药肺癌细胞72 h的半数抑制浓度（IC50）分别为6.99μmol·L-1（95%CI 3.55~13.79μmol·L-1）,7.25μmol·L-1（95%CI 4.77~1.02μmol·L-1）。两药联合应用对肺癌细胞的抑制作用增强（P 〈0.05）,同时给药组抑制效果更显著（P〈0.01）。同时给药组、序贯给药组（先用吉非替尼）和序贯给药组（先用enzastaurin）吉非替尼对H460细胞的IC50值分别为0.006μmol·L-1（95%CI 0.002~0.020μmol·L-1）,0.02μmol·L-1（95%CI 0.011~0.037μmol·L-1）,0.085μmol·L-1（95%CI 0.042~0.170μmol·L-1）。联合指数法显示在吉非替尼浓度0.05μmol·L-1以上时,同时给药组联合指数均〈1。细胞周期分布实验结果显示同时给药组可显著提高G0/G1期细胞比例（P〈0.05）,阻滞细胞于G1期。结论：蛋白激酶C抑制药enzastaurin与EGFR抑制药吉非替尼联用具有较好的协同作用,两药联合应用可能是出现吉非替尼耐药的非小细胞肺癌后续治疗的一个新选择。     

Stimulation of Enterocyte Protein Kinase C by Laxatives In-vitro

Abstract— To elucidate the role of protein kinase C in the mechanism of action of stimulatory laxatives, experiments were performed with preparations of rat lysed enterocytes. The phorbol ester 4-β-phorbol 12-myristate 13-acetate (PMA) concentration-dependently (2–200 μg mL^?1) stimulated the activity of protein kinase C in this preparation. Ricinoleic acid, the active principle of castor oil, deacetylbisacodyl, the active principle of bisacodyl, and deoxycholic acid exerted the same effect, although less efficiently. This reflects their potency for inducing intestinal fluid secretion and prostaglandin release, effects that are also induced more potently by PMA. Accordingly, the potency of the three C₁₈ fatty acids, ricinoleic acid, stearic acid and oleic acid on protein kinase C activity in-vitro, on prostaglandin E₂ release and on net fluid secretion in-vivo runs in parallel. It is therefore concluded that stimulatory laxatives activate protein kinase C., leading to prostaglandin E₂ release, thus resulting in net fluid secretion.     

Toxicity Sudies of Acetone Administered in the Drinking Water of Rodents

Toxicity Studies of Acetone Administered in the Drinking Waterof Rodents. DIETZ, D. D., LEININGER, J. R., RAUCKMAN, E. J.,THOMPSON, M. B., CHAPIN, R. E., MORRISSEY, R. L., AND LEVINE,B. S. (1991). Fundam Appl. Toxicol 17, 347–360. Two- andthirteen-week toxicity studies were conducted using male andfemale F344/N rats and B6C3F1 mice. Animals were exposed tothe following concentrations of acetone in their drinking water:two-week studm 0; 5000; 10,000; 20,000; 50,000; or 100,000 ppmacetone. Thirteen-week rat and female mouse studies 0; 2500;5000; 10,000; 20,000; or 50,000 ppm acetone. Thirteen week malemice were exposed to 0; 1250; 2500; 5000; 10,000; or 20,000ppm acetone. Depressed body weight gain was restricted to the50,000 and 100,000 ppm exposure groups. Male and female miceexposed respectively to 20,000 or 50,000 ppm acetone for 2 weeksdeveloped hepatocellular hypertrophy. This change was not apparentafter 13 weeks of exposure although relative and alsolute liverweight was increased in high dose female mice. Bone marrow hypoplasiawas observed In 5/5 high dose (100,000 ppm) male rats duringthe 2-week studies. Treatment of male rats for 13 weeks resultedin a variety of mild and subtle hematological changes that oftenoccurred at relatively low levels of exposure (5000 ppm) andresembled those seen during the clinical condition of megaloblasticanemia Changes characteristic of hypogonadism (depressed spermmotility and cauda epididymal and epididymal weight and elevatedincidence of abnormal sperm) were observed in male rats raceiving50,000 ppm acetone for 13 weeks. The incidence and severityof a kidney laion that is morphologically similar to the spontaneouslyoccurring nephropathy among aging F-344 rats were increasedat 20,000 and 50,000 ppm acetone, respectively, in 13-week malerats. In summary, the effects of acetone were either subtlein nature or occurred during very high levels of exposure confirmingacetone's low level of toxicity. The daily levels of acetoneexposure were often several-fold greater than possibly encounteredby humans during the accidental consumption of contaminatedgroundwater (250 ppm; 5 mg/day) and frequently exceeded maximumlevels reported following acute toxic exposures (2,500 mg/kg).     

The Protein Kinase C Agonist PEP005 (Ingenol 3-Angelate) in the Treatment of Human Cancer: A Balance between Efficacy and Toxicity

The diterpene ester ingenol-3-angelate (referred to as PEP005) is derived from the plant Euphorbia peplus. Crude euphorbia extract causes local toxicity and transient inflammation when applied topically and has been used in the treatment of warts, skin keratoses and skin cancer. PEP005 is a broad range activator of the classical (α, β, γ) and novel (δ, ε, η, θ) protein kinase C isoenzymes. Direct pro-apoptotic effects of this drug have been demonstrated in several malignant cells, including melanoma cell lines and primary human acute myelogenous leukemia cells. At micromolar concentrations required to kill melanoma cells this agent causes PKC-independent secondary necrosis. In contrast, the killing of leukemic cells occurs in the nanomolar range, requires activation of protein kinase C δ (PKCδ) and is specifically associated with translocation of PKCδ from the cytoplasm to the nuclear membrane. However, in addition to this pro-apoptotic effect the agent seems to have immunostimulatory effects, including: (i) increased chemokine release by malignant cells; (ii) a general increase in proliferation and cytokine release by activated T cells, including T cells derived from patients with chemotherapy-induced lymphopenia; (iii) local infiltration of neutrophils after topical application with increased antibody-dependent cytotoxicity; and (iv) development of specific anti-cancer immune responses by CD8(+) T cells in animal models. Published studies mainly describe effects from in vitro investigations or after topical application of the agent, and careful evaluation of the toxicity after systemic administration is required before the possible use of this agent in the treatment of malignancies other than skin cancers.     

The Developmental Toxicity of Orally Administered Oxytetracycline in Rats and Mice

The Developmental Toxicity of Orally Administered Oxytetracyclinein Rats and Mice. MORRISSEY, R.E., TYL, R.W., PRICE, C.J., LEDOUX,T.A., REEL, J.R., PASCHKE, L.L., MARR, M.C, AND KJMMEL, C.A.(1986). Fundam. Appl. Toxicol. 7, 434-443. Timed-pregnant CDrats and CD-1 mice were dosed by gavage with oxytetracyclinehydrochloride (OXT) in corn oil on gestational days (gd) 6-15(0, 1200, 1350, or 1500 mg/kg/day for rats; 0, 1325, 1670, or2100 mg/kg/day for mice). Deaths among treated females occurredin a dose-related manner in all OXT dose groups (2-7%, mice;5-24%, rats), but no maternal deaths occurred in the vehiclecontrol groups. Significant dose-related decreases in maternalweight gain during treatment, as well as for corrected gestationalweight gain (i.e., maternal gestational weight gain minus graviduterine weight), were observed at all doses in rats but notin mice. Gravid uterine weight was reduced in a dose-relatedmanner only in mice, with the high-dose group significantlyreduced compared to the control group. At termination (gd 20,rats; gd 17, mice), the status of uterine implantation siteswas recorded and live fetuses were weighed. Fetuses were examinedfor external, visceral, and skeletal abnormalities. There wereno significant effects of OXT in either species on the incidenceof postimplantation loss (resorptions plus dead fetuses) ormalformations. In both species, there was a significant trendtoward reduced fetal body weight, and each group of rats receivingOXT was significantly reduced compared to the control group.Administration of OXT during organogenesis at doses exceedingthe therapeutic range for humans produced maternal and fetaltoxicity, but did not produce any treatment-related increasein malformations.     

The Developmental Toxicity of Orally Administered Theophylline in Rats and Mice

The Developmental Toxicity of Orally Administered Theophyllinein Rats and Mice. LIND-STRÖM, P., MORRISSEY, R. E., GEORGE,J. D., PRICE, C. J., MARR, M. C, KJMMEL, C. A., AND SCHWETZ,B. A. (1990). Fundam. Appl. Toxicol. 14, 167–178. Theophylline(THEO), a widely prescribed anti-asthmatic, was evaluated fordevelopmental toxicity. It was administered continuously onGestational Days 6 through 15 to pregnant Sprague-Dawley (CD)rats in the feed (0,0.15,0.30, or 0.40%) and to pregnant Swiss(CD-1) mice in the drinking water (0,0.075, 0.15, or 0.20%).Estimated intake of THEO for rats was 0, 124, 218, or 259 mg/kg/day,while for mice it was 0, 282, 372, or 396 mg/kg/day. In rats,maternal weight gain parameters (weight gain during gestationand treatment, as well as corrected weight gain) decreased at0.40%. While food consumption was lower only in the 0.40% treatmentgroup, water consumption was higher in all treated groups. Therewas a dose-related decreasing trend in gravid uterine weight.The number of live fetuses per litter decreased at 0.40% andthe average male and female fetal weight per litter decreasedat 0.30 and 0.40%. There was no increase in malformations. Inmice, maternal corrected body weight and weight gain duringgestation decreased at 0.15 and 0.20%, and weight gain duringtreatment and gravid uterine weight decreased at 0.20%. Waterconsumption was reduced by as much as 30-45% of controls at0.15 and 0.20%, respectively, while food consumption did notchange with THEO treatment There was an increase in percentageresorp-tions per litter and a decrease in the average male andfemale fetal weight per litter at 0.15 and 0.20%. An increasingtrend was noted for percentage malformed fetuses per litter,and percentage litters with externally malformed fetuses wereslightly increased in the mid- and high-dose groups. However,these increases were not statistically significant. In summary,there were developmental effects seen in rats at a dose (0.30%)that did not produce overt maternal toxicity, but the adversedevelopmental effects in mice were observed at doses that causedreduced maternal water consumption and body weight gain. Itis possible that water deprivation contributed to the effectsseen in mice after THEO treatment. For maternal toxicity, noobservable adverse effect levels (NOAELs) were 218 mg/kg forrats and 282 mg/kg for mice. NOAELs for developmental toxicitywere 124 mg/kg for rats and 282 mg/kg for mice. These NOAELsare approximately 10-to 30-fold greater than doses requiredto maintain humans on serum THEO concentrations that are clinicallyuseful.