Development of Extraction and Detection Method for a Chemotherapeutic Drug with Phenytoin in Biological Samples

Dexamethasone is classified as a corticosteroid and is commonly used among cancer patients to decrease the amount of swelling around the tumor. Among patients with cancer, in particular brain tumors, seizures can become a daily routine in their everyday lives. To counteract the seizures, an antiepileptic drug such as phenytoin is administered to act as an anticonvulsant. Phenytoin and dexamethasone are frequently administrated concurrently to brain cancer patients. A previous study has shown that phenytoin serum concentration decreases when administrated concurrently with dexamethasone. Thus, it is important to monitor the concentration of these two drugs in biological samples to ensure that the proper dosages are administrated to the patients. This study aims to develop an effective extraction and detection method for dexamethasone and phenytoin. A reverse-phase high-performance liquid chromatography (HPLC) method with UV/Vis detection has been developed to separate phenytoin and dexamethasone at 219 nm and 241 nm respectively from urine samples. The mobile phase consists of a mixture of 0.01 M KH2PO4, acetonitrile, and methanol adjusted to pH 5.6 (48:32:20) and is pumped at a flow rate of 1.0 mL/min. Calibration curves were prepared for phenytoin and dexamethasone (r2 > 0.99). An efficient solid-phase extraction (SPE) method for the extraction of dexamethasone and phenytoin from urine samples was developed with the use of C-18 cartridges. The percent recovery for phenytoin and dexamethasone is 95.4% (RSD = 1.15%) and 81.1% (RSD = 3.56%) respectively.

Pregnenolone 16α-carbonitrile negatively regulates hippocampal cytochrome P450 enzymes and ameliorates phenytoin-induced hippocampal neurotoxicity

The central nervous system is susceptible to the modulation of various neurophysiological processes by the cytochrome P450 enzyme(CYP),which plays a crucial role in the metabolism of neurosteroids.The antiepileptic drug phenytoin(PHT)has been observed to induce neuronal side effects in patients,which could be attributed to its induction of CYP expression and testosterone(TES)metabolism in the hippocampus.While pregnane X receptor(PXR)is widely known for its regulatory function of CYPs in the liver,we have discovered that the treatment of mice with pregnenolone 16α-carbonitrile(PCN),a PXR agonist,has differential effects on CYP expression in the liver and hippocampus.Specifically,the PCN treatment resulted in the induction of cytochrome P450,family 3,subfamily a,polypeptide 11(CYP3A11),and CYP2B10 expression in the liver,while suppressing their expression in the hippocampus.Functionally,the PCN treatment protected mice from PHT-induced hippocampal nerve injury,which was accompanied by the inhibition of TES metabolism in the hippocampus.Mechanistically,we found that the inhibition of hippocampal CYP expression and attenuation of PHT-induced neurotoxicity by PCN were glucocorticoid receptor dependent,rather than PXR independent,as demonstrated by genetic and pharmacological models.In conclusion,our study provides evidence that PCN can negatively regulate hippocampal CYP expression and attenuate PHT-induced hippocampal neurotoxicity independently of PXR.Our findings suggest that glucocorticoids may be a potential therapeutic strategy for managing the neuronal side effects of PHT.

Effect of Cyclooxygenase Inhibition on P-Glycoprotein Expression and Phenytoin Level in Brain Tissue of Pilocarpine Induced Epilepsy in Rats

Background: Increased brain P-glycoprotein (P-gp) expression may play important role in resistance to antiseizure drugs. The present work aimed to overcome the drug resistance that develop due to overexpression of P-gp with subsequent increase in brain phenytoin level in epileptic rats, using either non-selective (indomethacin) or selective (celecoxib) cyclooxygenase inhibitors. Methods: Fifty-six adult male albino rats were randomly divided into seven groups. Epilepsy was induced using the lithium pilocarpine model. Rats received indomethacin (2.5 mg/kg) or celecoxib (20 mg/kg), either alone or combined with phenytoin (50 mg/kg). Seizures were evaluated using Racine score. Motor coordination was assessed using open field and rotarod tests. Phenytoin brain level was measured using High Performance Liquid Chromatography (HPLC), glutamate expression was measured using Enzyme Linked Immunosorbent Assay (ELISA), ATP Binding Cassette Subfamily B Member 1 (ABCB1) gene expression was assessed using Real Time-Polymerase Chain Reaction (RT-PCR), and immunohistochemical analysis was done for P-gp expression. Results: Phenytoin combination with either indomethacin or celecoxib had improved the Racine score, motor coordination on rotarod apparatus, and open field test results. Also, phenytoin combination with either indomethacin or celecoxib decreased brain glutamate level, ABCB1 gene and P-gp expression, and increased brain phenytoin level compared to treatment with phenytoin alone. This indicated that both P-gp inhibitors indomethacin and celecoxib, increased the level of phenytoin that reached the brain of rats. However, brain uptake of phenytoin was significantly enhanced using celecoxib rather than indomethacin (CI 95%, 17.092: 32.808, P-value Conclusion: Cyclooxygenase inhibition using either celecoxib or indomethacin resulted in downregulation of P-gp expression, with subsequent increase in brain phenytoin level in epileptic rats.

Formulation of a new phenytoin-containing mucoadhesive and evaluation of its healing effects on oral biopsy ulcers

Background and Objective: Several studies have shown the wound healing effect of topical phenytoin, which is applied by its effect on connective tissue intracellular matrix. However, there are still some controversies about its effect on various kinds of wounds, especially in the experimental models. This study is aimed at evaluating the effect of mucoadhesive paste compared to phenytoin mucoadhesive paste on wound healing after oral biopsy. Material and Methods: In this double blind randomized clinical trial, 20 patients who were eligible for oral biopsy were allocated into the case and control groups. After the biopsy, patients having ulcers ranging between one and two centimeters were treated by simple or 1% phenytoin mucoadhesive paste. All patients were instructed to apply their paste at least three times a day for five days after the biopsy. Patients in both groups were evaluated every other day for size of the ulcer, degree of pain and diameter of the inflammatory halo. Statistical analysis was done using SPSS software and Mann-Whitney test. Results: After the second and third appointments, it was observed that the rate of wound healing and decrease in the size of the ulcers were significantly quicker in the treatment group (p = 0.001 and p = 0.003 respectively) and the patients in the phenytoin group reported less pain. Diameter of the inflammatory halo was not significantly different between two groups. Conclusion: Applying 1% phenytoin mucoadhesive paste on biopsy ulcers resulted in accelerated wound healing and decrease in pain, but had no effect on the diameter of the inflammatory halo.

Liposome Immunoassay for Determination of Phenytoin

Homogeneous liposome immunoassay for the determination of phenytoin was studied.Liposomes were prepared from cholesterol (CH) and phospholipids including dipalmitoyl phosphatidyl ethanolamine (DPPE) tbr conjugation with thiol-containing antibodies. Hemin chloride was entrapped in the liposome and antibody was modified by reaction with 3' (2-pyndyl-dithio) propionyl N-hydroxysuccinimide ester (SPDP) to introduce thiol groups for efficient coupling. Antibody-coupled liposomes (immunoliposomes) were incubated with phenytoin and complement, and then with hemin substrate. The amount of hemin released from immunoliposomes, which increases with concentration increase of phenytoin, can be detected rapidly by determining the fluorescence with its substrate p-hydroxyphenyl propionic acid (HPPA)and hydrogen peroxide.

Effects of duration of phenytoin administration on mRNA expression of cytochrome P450 and P-glycoprotein in the liver and small intestine of rats

Phenytoin(5,5-diphenylhydantoin;DPH) induces expression of cytochromes P450(CYPs). Interactions between DPH and tacrolimus suggested that the persistence of CYP induction after discontinuation of DPH is dependent on the history of administration and dosing period of DPH. However, the relationship between the duration of DPH administration and expression of CYPs in the liver and small intestine of rats is not known. Alterations in levels of P-glycoprotein(P-gp;MDR1;ABCB1) as well as CYPs cause drug interactions in the small intestine. We examined the effects of the duration of DPH administration on expression of CYPs and P-gp in the liver and small intestine of rats. Rats were treated with DPH(100 mg/kg,peroral(p.o.) twice a day(b.d.)) for 2, 4, 8, and 16 d. mRNA levels of CYPs and P-gp were examined using the total RNA extracted from the liver and duodenum 2 h and 24 h after the final administration of DPH. CYP3 A activities were determined using microsomes. DPH administration for 2 d and 4 d markedly increased m RNA levels of CYPs such as CYP3 A1, CYP3 A2,CYP2 B1, and CYP2 B2 in the liver. A relatively long duration of DPH administration(8 d and16 d) resulted in abolition of the induction of hepatic CYP but increased CYP3 A activities were maintained. These results suggest that the duration of DPH administration could be an important determinant of hepatic CYP induction.

Jiedu Tongbi Tang Plus Phenytoin Sodium for Severe Acute Sciatica

Case 1: Mr. Li, a 55-year farmer from Hebeiprovince, paid his first visit on August 11, 1995. Thepatient complained of a sudden pain in the area fromhis left buttock down to the posterolateral aspect ofthe leg.

在恒河猴苯妥英(Phenytoin)与炔诺酮的相互作用

已知,抗癫痫药物能诱导肝内的甾体代谢酶系,从而可改变外源甾体的作用,有报道,服口服避孕药的癫痫妇女出现经间出血及意外妊娠并归之于抗癫痫药苯妥英的诱导代谢酶作用。本文研究了给恒河猴用苯妥英对于炔诺酮代谢的影响。

Phenytoin-Induced Elevation of the Intracellular Calcium Concentration by Stimulation of Calcium-Sensing Receptors in Gingival Fibroblasts

Background:The mechanism concerning gingival overgrowth as a side effect of phenytoin, a therapeutic drug for epilepsy has been still unclear. As one of mechanisms, by measuring the intracellular calcium concentration ([Ca2+]i) of the gingival fibroblasts, it has been advocated that there is relationship between gingival overgrowth and phenytoin-induced alterations in the [Ca2+]i in gingival fibroblasts. To confirm that phenytoin elevates the [Ca2+]i, and if so, to find out its mode of action. Methods: The [Ca2+]i was measured with the Ca2+-sensitive fluorescent dye fura-2/AM. Cells were soaked in a flexiperm chamber and perfused by a saline. Drugs at appropriate concentrations were added to the perfusate. Results: Phenytoin concentration-dependently elevated the [Ca2+]i. NPS2390, a calcium-sensing receptor (CaSR) blocker, significantly suppressed the phenytoin-induced [Ca2+]i elevation. U73122, a phospholipase C (PLC) inhibitor, inihibited the phenytoin-induced [Ca2+]i elevation. TMB-8, a blocker of inositol triphophate (IP3) receptors in ER, significantly depressed the phenytoin-induced [Ca2+]i elevation. m-3M3FBS, a PLC activator, enhanced the phenytoin-induced [Ca2+]i elevation. From the findings obtained, it is discussed as follows: The Ca2+-free saline and NPS2390, a CaSR antagonist, inhibited the phenytoin-induced [Ca2+]i rise;These results indicate that CaSRs exist in gingival fibroblasts and that CaSRs are involved in the phenytoin-induced [Ca2+]i rise;U73122 and TMB-8 depressed the phenytoin-induced [Ca2+]i elevation and furthermore, m-3M3FBS enhanced the phenytoin-induced [Ca2+]i elevation, showing that the Ca2+ release from the ER is involved in the phenytoin-induced [Ca2+]i elevation. Conclusion: We have concluded that phenytoin elevates the [Ca2+]i by activating CaSRs and enhancing the Ca2+ release from the Ca2+ stores in gingival fibroblasts.

Phenytoin Effects on Proliferation and Induction of IL1<i>β</i>and PGE2 in Pediatric and Adults’ Gingival Fibroblasts

Background: Gingival Overgrowth (GO) is a well documented and unwanted side effect that occurs mainly as a result of certain antiseizure, phenytoin. The aim of this study was to compare the effect of phenytoin on proliferation and production of IL1β and PGE2 in cultured human gingival fibroblasts (HGF) of children and adults. Materials and Methods: Normal HGFs were obtained from 4 healthy children and 4 adult and then were cultured with phenytoin (20 mg/ml). MTT test was used to evaluate the proliferation and ELISA to determine the level of IL1β and PGE2 production by HGFs. Analysis of proliferation were assessed by Independent T-Test and ANOVA analysis was used to assess the level of IL1β and PGE2 production with an a error level less than 0.05. Results: The proliferation of HGF was not affected significantly by phenytoin in both cultured fibroblast sources (P > 0.05). Phenytoin induced a significantly higher formation of IL1β and PGE2 in child’s HGFs as compared to adult’s HGFs (P < 0.05). Conclusion: The results suggest that different inflammatory responses and cytokine formation by child’s and adult’s HGFs are the probable key elements that cause different reactions of phenytoin therapy. More advanced and systematic studies are needed to verify these findings.

Physico-Chemical and Microbiological Study for the Stability of Phenytoin Sodium Extemporaneously Compounded Suspension in Saudi Arabia Hospitals

Epilepsy is a chronic and the fourth most common neurological disorder which affects people of all age groups. Recently research and awareness on epilepsy-related deaths have rapidly grown over the past two decades. Many previous studies are attributed to the guidelines that apprise health care professionals in handling these deaths, but there is a relative scarcity of information accessible for clinicians and pharmacists who are responsible for manufacturing or preparing the extemporaneous anti-epileptic suspensions in the hospitals. Mostly in partial seizures, phenytoin is one of the first-choice drugs. In Saudi Arabian hospitals, the extemporaneous preparation of phenytoin suspension is common, but the hot climatic weather in Saudi Arabia possesses stability problems that should be tackled as the prepared suspension should pass all the stability tests to ensure uniform dosage of the extemporaneous formulation. In the current study, the commercial capsules were used to prepare the oral phenytoin sodium extemporaneous suspension. The physical, chemical and microbiological stability of phenytoin sodium suspension is analyzed at various temperatures.

高原低氧环境下HDAC5在大鼠体内P-gp表达中的作用及对苯妥英钠药代影响

目的探究高原低氧环境下HDAC5在大鼠体内P-gp表达中的关键作用及对苯妥英钠药代的影响。方法Wistar大鼠运至海拔4010 m的青海玉树巴塘,每组6只。不同组分别给予苯妥英钠、苯妥英钠联用金丝桃素、苯妥英钠联用维拉帕米。在高原地区收集服药后不同时间的血浆和肝组织。采用UFLC-MS法测定血浆中苯妥英钠的浓度,计算药代动力学参数。Western blot检测肝组织中HDAC5、P-gp蛋白表达的变化。结果UFLC-MS结果表明,给予P-gp激动剂后,AUC(0-t)、AUC(0-∞)、MRT(0-t)、MRT(0-∞)、T_(1/2z)、CL_(z)/F、V_(z)/F增加;给予P-gp抑制剂后,AUC(0-t)、AUC(0-∞)、MRT(0-t)、MRT(0-∞)、T_(1/2z)降低。同时,高原低氧环境下,HDAC5参与P-gp表达的调控。当给予P-gp激动剂和抑制剂时,HDAC5与P-gp表达呈现相同变化趋势。结论高原低氧环境下,肝中转运体P-gp的表达影响了P-gp底物苯妥英钠的药代动力学,P-gp的变化水平与HDAC5有关。

拉莫三嗪结合苯妥英钠片治疗癫痫患者的效果及对临床症状、认知功能的影响

目的探讨拉莫三嗪结合苯妥英钠片治疗癫痫患者的效果。方法选取2019年1月至2020年12月收治的108例癫痫患者为研究对象,按照随机法将其分为单药组(54例,苯妥英钠片治疗)和联合组(54例,拉莫三嗪结合苯妥英钠片治疗)。比较两组的治疗效果。结果联合组的治疗总有效率高于单药组(P<0.05)。两组的药副反应总发生率无显著差异(P>0.05)。治疗后,联合组的简易精神状态评价量表(MMSE)评分高于单药组(P<0.05)。治疗后,联合组的α脑电频率低于单药组,θ、δ脑电频率高于单药组(P<0.05)。治疗后,联合组的同型半胱氨酸(Hcy)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)水平低于单药组(P<0.05)。结论拉莫三嗪结合苯妥英钠片治疗癫痫患者可取得显著疗效,能明显改善临床症状及认知功能,且用药安全性较高。

强直性肌营养不良一家系5例报告并文献复习

目的探讨强直性肌营养不良1型(DM1)一家系的临床表现和遗传学特点。方法对郑州大学附属郑州中心医院2019年4月收治的该家系部分成员进行体格检查、血生化、肌电图和基因检测等检查,绘制家族系谱图,分析该家系的临床特点。结果该家系共12人,其中5人为强直性肌营养不良(myotonic dystrophy,DM)病人,存在明显的遗传早现现象。5例DM病人均为慢性病程,以肌无力、肌强直为主要表现,伴心脏、内分泌、呼吸等多系统受累,肌电图可见特征性肌强直放电和肌源性损害,血清肌酶轻度增高或正常。结论DM主要以肌强直、肌无力、肌萎缩为主要表现的多系统受累疾病,临床表现复杂多样,肌电图、病理活检和基因检测有助于诊断和分型。

四氯苯醌荷移分光光度法测定苯妥英钠

通过研究电子供体苯妥英钠和电子受体四氯苯醌间的荷移反应,建立了四氯苯醌荷移分光光度法测定片剂中苯妥英钠含量的实验方法。实验结果表明,苯妥英钠和四氯苯醌在乙醇介质中,50℃水浴反应10 min,可以生成稳定的荷移络合物。该络合物的络合比为1∶1,在430 nm处有最大吸收,表观物质的量吸光系数为3.47×10^(2) L·mol^(-1)·cm^(-1)。在苯妥英钠的质量浓度为10~250 mg·L^(-1)范围内,生成的络合物浓度与吸光度遵守比尔定律,相关系数是0.9993。药物苯妥英钠的质量浓度为250 mg·L^(-1)时,实验结果的相对标准偏差(n=11)是1.49%。应用本实验方法测定了片剂中的苯妥英钠含量,平均回收率处于98.8%~101.9%范围内。

苯妥英钠血药浓度与CYP2C19基因多态性关系的研究

目的:探讨苯妥英钠药物浓度与CYP2C19基因多态性的关系,以指导临床个体化用药。方法:运用血药浓度监测仪测定患者血药浓度和变性高效液相色谱法检测癫痫患者的CYP2C19基因多态性位点,对二者结果进行相关性分析。结果:21例中国汉族癲痫患者中有12例含突变型CYP2C19基因。在7例(58.33％)苯妥英钠血药浓度的比值高于剂量比值的患者中,6例(85.7％)为突变型基因携带者(慢代谢者)。结论:CYP2C19是苯妥英钠的主要代谢酶。CYP2C19基因突变等位基因携带者苯妥英钠代谢减慢,应给予小剂量苯妥英钠,以减少药物不良反应的发生和药物资源的浪费。

苯妥英钠和丙戊酸钠预防术后癫痫的对照研究

目的 对比苯妥英钠和丙戊酸钠预防术后癫痫的作用、毒副反应以及和血药浓度的关系。方法 采用随机前瞻对照性研究 ,选择幕上开颅手术病人 ,苯妥英纳组 72例 ,丙戊酸钠组 80例 ,术前术后分别规则服用苯妥英钠和丙戊酸钠 ,监测抗癫痫药物的血药浓度和术后癫痫及毒副反应发生情况。结果 两组共 15例术后发生早期癫痫 ,10例 (10 /15 )达到有效血浓度 ;其中 ,苯妥英钠组 6例 (8 3 % )发生早期癫痫 ,2例达到有效血浓度 ,丙戊酸钠组 9例 (11 3 % )发生早期癫痫 ,8例达到有效血浓度 ;远期癫痫发作 7例中丙戊酸钠组 5例 ,苯妥英钠组 2例。苯妥英钠组 11例 (15 3 % )及丙戊酸钠组2例 (2 5 % )出现了毒副反应。经χ2 检验 ,两组术后癫痫发生率无显著性差异 ,术后未发癫痫组和癫痫发作组间药物血浓度无显著性差异 (P >0 0 5 ) ,苯妥英钠组毒副反应发生率高于丙戊酸钠组。结论 苯妥英钠和丙戊酸钠预防控制术后癫痫无差别 ,苯妥英钠毒副反应较丙戊酸钠严重。

HPLC同时测定苯巴比妥、苯妥英、卡马西平和氯硝西泮血药浓度

目的建立同时测定血清中摹巴比妥（PB）、苯妥荚（PT）、卡马西平（CBZ）和氯硝西泮浓度的HPLC.方法分析柱为Agilent C18柱（4．6mm×250mm,5μm），流动相为水-甲醇-乙腈（45：45：10），流速为1．0mL·min^-1，检测波长为254nm，柱温30℃、阿普唑仑为内标物。结果PB，PT，CBZ和氯硝西泮的线性范围分别为2．5～80，1．25～40，0.75～24，0.02～0.64μg·mL^-1，最低检测浓度分别为0．2,0．2,0．1，0．005μg·mL^-1，方法回收率分别为99．1％，100.8％，100.1％，98.1％，日内、日间RSD均〈3．5％，绝对回收率分别为91．7％，90．2％，89．9％和89．3％，RSD均〈2％。结论该方法快速、灵敏、买用，适用于治疗药物监测。