Effects of hydroxypropyl-beta-cyclodextrin on the chemical stability and the aqueous solubility of thalidomide enantiomers

The aim of this work was to study the effect of hydroxypropyl-beta-cyclodextrin on the solubility and stability of thalidomide enantiomers in aqueous solutions for clinical oral administration to be used in HIV-infected children. For this reason racemic thalidomide was added to solutions containing different concentrations of hydroxypropyl-beta-cyclodextrin. True complexes were obtained by using hydroxypropyl-beta-cyclodextrin and the solubility of both thalidomide enantiomers was increased directly depending on the amount of hydroxylpropyl-beta-cyclodextrin in the medium although no enantioselective differences were observed at 37 degrees C. The chemical stability of thalidomide enantiomers is clearly improved by hydroxypropyl-beta-cyclodextrin. No enantioselective degradation of thalidomide was observed in sodium chloride solution (0.9%) samples stored at 6 degrees C for nine days when hydroxypropyl-beta-cyclodextrin was employed as excipient. Therefore a thalidomide solution suitable for oral administration can be prepared by adding hydroxypropyl-beta-cyclodextrin at 10% (w/v).     

HPLC法测定沙利度胺片中沙利度胺的含量

目的：建立沙利度胺含量的质量控制方法。方法：采用Waters Nova-Pak C18柱（150 mm×3.9 mm,4μm）,以水-乙腈-磷酸（85∶15∶0.1）为流动相,流速为2 ml/min,检测波长为237 nm。结果：对照品在10.055～150.825μg/ml范围内呈良好线性关系（r=1.000）,回收率平均为100.7%,RSD为0.3%。结论：HPLC法准确、灵敏、可靠,专属性强,可用于测定沙利度胺片中沙利度胺的含量。     

Stereointegrity of thalidomide: gas-chromatographic determination of the enantiomerization barrier.

Enantioselective chromatographic methods, representing the most commonly used techniques for the determination of enantiomeric ratios, can also be used for the evaluation of stereochemical integrity. In the present study, two chromatographic methods, dynamic gas chromatography (DGC) and stopped-flow gas chromatography (SFGC) were used to determine the enantiomerization barrier of thalidomide. In the presence of a chiral stationary phase (CSP), the enantiomers of thalidomide produced characteristic elution profiles exhibiting plateaus and/or peak broadening which were observed between 190 and 220 degrees C in DGC. To obtain the enantiomerization barrier of thalidomide from experimental data, the fast and efficient simulation program ChromWin was used to simulate the elution profiles and obtain kinetic activation parameters. From temperature-dependent measurements the rate constants k(1) and k(-1) and the kinetic activation parameters DeltaG#, DeltaH# and DeltaS# of the enantiomerization of thalidomide were obtained by DGC. The enantiomerization barrier DeltaG# was determined to be 154+/-2 kJ/mol with DGC and 150+/-3 kJ/mol with the sfGC technique at 200 degrees C, respectively. The concept of the retention increment R' has been applied to separate the enantiomerization barrier of thalidomide in the dissolved and complexed state of the CSP.     

Hydroxylated metabolites of thalidomide: formation in-vitro and in-vivo in man

There is renewed interest in the clinical use of thalidomide, because of its unique immunomodulating action. Because data on the metabolism of thalidomide in man are very sparse, the aim of this study was to develop HPLC assays for the 5-hydroxy, 5,6-dihydroxy, 4,5-dihydroxy and 5'-hydroxy metabolites of thalidomide and to investigate their possible formation in man-in-vitro in liver homogenates and in-vivo in healthy volunteers. Reversed-phase HPLC assays with UV detection were developed for quantification of the metabolites in the low ng mL(-1) range in plasma and incubate samples. The stability of the metabolites was investigated and degradation was avoided by rapid chilling and acidification of the samples. After incubation of thalidomide with fraction S9 from human liver, formation of the 5-hydroxy and 5'-hydroxy metabolites could be demonstrated. The 5'-hydroxy metabolite was found, in low concentrations, in plasma samples from eight healthy male volunteers who had received thalidomide orally. The other three metabolites could not be found by HPLC with detection limits of 1-2 ng mL(-1). Thus the formation of two hydroxylated metabolites of thalidomide in the liver in man was demonstrated, but only one of these could be detected in the circulation.     

高效液相色谱法测定血浆中沙利度胺的浓度

目的建立测定人血浆中沙利度胺浓度的高效液相色谱法。方法采用蛋白沉淀法对血样进行预处理,以外标法计算浓度。色谱条件：Hypersil ODS色谱柱（4.6 mm×150 mm,5μm）,流动相为11 mmol.L-1四丁基溴化胺溶液-乙腈（84∶16,含2.5%三乙胺,冰醋酸调pH为3.12）,紫外检测波长为237 nm,柱温：流速为1.0 mL.min-1。结果沙利度胺保留时间为12.34 min,最低定量浓度为0.66μg.mL-1,线性范围为0.66～84.48μg.mL-1,绝对回收率〉67%。结论该方法专属性强、灵敏度高,适合沙利度胺的血浆浓度测定及药动学研究。     

Determination of thalidomide by high performance liquid chromatography: plasma pharmacokinetic studies in the rat

A sensitive and simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of thalidomide in rat plasma. Chromatography was accomplished with a reversed-phase Hypersil C18 column. Mobile phase consisted of acetonitrile-10 mM ammonium acetate buffer (pH 5.50) (28:72, v/v), at a flow rate of 0.8 ml/min. Thalidomide was monitored by ultraviolet detector at 220 nm and it gave a linear response as a function of concentration over 0.02-50 microM. The limit of quantitation in rat plasma was 0.50 ng (0.02 microM plasma concentration) with an aliquot of 20 microl. Results from a 3-day validation study indicated that this method allows for simple and rapid quantitation of thalidomide with excellent accuracy and reliability. Using this validated assay, the effect of coadministered irinotecan (CPT-11) on the plasma pharmacokinetics of thalidomide in rats was determined. Coadministration of CPT-11 (intravenously, 60 mg/kg) increased the maximum plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC(0-10h)) of thalidomide by 32.29 and 11.66%, respectively, as compared to the control, but none of the effect of CPT-11 was of statistical significance (P > 0.05). Concomitant CPT-11 also caused a 10.04% decrease in plasma clearance (CL) and 14.51% decrease in volume of distribution (V(d)) (P > 0.05). These results suggest that coadministered CPT-11 did not significantly alter the plasma pharmacokinetics of thalidomide in rats. Further studies are warranted to explore the pharmacokinetic and pharmacodynamic interactions between CPT-11 and thalidomide.     

Teratogen metabolism: thalidomide activation is mediated by cytochrome P-450

A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a type I spectral shift. Metabolite formation was reduced or eliminated by carbon monoxide, SKF-525A, metyrapone, and N-octylamine. Superoxide dismutase treatment had no effect. Metabolite formation required microsomes and NADPH and was dependent on the length of 37 degrees C incubation. The metabolite could be isolated by successive hexane and chloroform extractions. It is likely the inhibitory thalidomide metabolite was generated by a minor cytochrome P-450 species. Whether this thalidomide metabolite is involved in the drug's teratogenic activity remains to be shown.     

反应停的抗新生血管形成及抗肿瘤作用研究

目的　研究反应停的抗新生血管形成及抗肿瘤作用。方法　在HUVECs中MTT法检测细胞活性,电镜观察细胞死亡类型,流式细胞仪定量细胞凋亡率;用鸡胚尿囊膜模型观察新生血管形成;肉瘤S180小鼠体内模型观察反应停的抗肿瘤作用,并通过免疫组化观察反应停对肿瘤组织微血管数的影响。结果　反应停对HUVECs有直接抑制作用,IC50为(22 .91±1. 74) μmol·L-1;经反应停作用48h后的HU VECs,随剂量增加,电镜下可见核不规则,染色体浓集,空泡状内质网及凋亡小体等不同时期细胞凋亡现象;流式细胞检测显示反应停可依剂量诱导HUVECs凋亡或坏死;鸡胚尿囊膜新生血管形成模型中,反应停抑制新生血管形成的阳性率随剂量而增加;小鼠S180肿瘤移植模型中,反应停单独使用,虽可明显减少肿瘤血管密度, 无抑制肿瘤生长作用;与环磷酰胺联合使用,可减少环磷酰胺用量,与环磷酰胺有协同抗肿瘤作用。结论　反应停有抗新生血管形成作用,单独应用对小鼠S180肿瘤无抑制作用,但与环磷酰胺有协同作用。     

A new method for determination of both thalidomide enantiomers using HPLC systems

Although thalidomide was withdrawn due to teratogenicity and neuropathy, there is now growing clinical interest in this compound because of its immunomodulatory and anti-angiogenic properties. In 1998, thalidomide was approved by the U.S. Food and Drug Administration for the treatment of erythema nodosum leprosum (ENL), an inflammatory complication of Hansen's disease, through a restricted-use program. Thalidomide was approved for the treatment of relapsed or refractory multiple myeloma (MM) as an orphan drug in Japan. Direct deproteinization method was shown to be useful for quantitation of enantioselective thalidomide blood level. Stabilized blood was deproteinized with methanol and 2 M trichloroacetic acid. The supernatant was injected onto reverse-phase column (CHIRALPAK AD-RH). The mobile phase consisted of 10% acetonitrile, 70% methanol and 20% 0.025 m citrate buffer (pH 3.0), and the flow rate was 0.5 ml/min. Wavelength of detection was 220 nm. (-)-(S)-thalidomide and (+)-(R)-thalidomide were separated at 13.5 min and 17.6 min, respectively. The accuracy of this method was almost the same as that of the measurement technique with extraction and concentration. In clinical practice, MM patients usually take many kinds of drugs at the same time. Actually, this patient takes a lot of drugs with thalidomide. However, we found no interference of these drugs and thalidomide on the chromatogram. This simple and reliable HPLC determination method for both enantiomers of thalidomide is thought to be very useful for thalidomide studies.     

沙利度胺多晶型大鼠体内药代动力学研究

目的研究沙利度胺3种晶型在SD大鼠胃肠道吸收过程,评价其优势药物晶型,探讨药物晶型状态对临床用药的影响。方法 SD大鼠灌胃给予不同晶型沙利度胺固体原料,采用高效液相色谱法测定血浆沙利度胺浓度,计算其大鼠体内药动学参数并在不同晶型之间进行对比。结果大鼠经口给予沙利度胺晶型α、晶型β和晶型γ后,血液中Cmax分别为6.79、4.28和5.55 mg.L-1,晶型α的测定值最大;Tmax分别为8.33、8.00和7.33 h;达峰时间相差约1 h;AUC0→t分别为40.73、30.34和38.74 mg.h.L-1,最大差距约25%;T12分别为2.29、1.95和1.93 h。结论大鼠口服不同晶型沙利度胺后血药浓度存在差异,其中晶型α峰浓度最高,血药浓度维持时间长。     

Pharmacokinetics of thalidomide in patients with impaired renal function and while on and off dialysis

There is a renewed interest in thalidomide for use in malignancies and systemic inflammatory diseases. Reduced renal function is not uncommon among patients with these disease states but the pharmacokinetics has not been fully investigated. The aim of this study was to investigate the pharmacokinetics of thalidomide in haemodialysis patients while on and off dialysis and in myeloma patients with varying degrees of renal function. Two studies were performed. To establish the pharmacokinetics of thalidomide in patients with mild to moderate renal failure, blood samples were taken over 12 weeks from 40 patients with multiple myeloma. A second study was performed in six patients with end-stage renal disease both on a non-dialysis day and before and during a haemodialysis session. Thalidomide concentration was determined by HPLC. A one-compartment open model with first-order absorption and elimination was used to fit total thalidomide concentration to population pharmacokinetics and statistical models using the NONMEM program. Clearance and volumes were slightly below 10 L h-1 and 1 L kg-1, respectively, in both patient groups. The inter- and intra-patient variability was low. Clearance was doubled during dialysis. There was no correlation between thalidomide clearance and renal function. In conclusion, the pharmacokinetics of thalidomide in patients with renal failure are very similar to values reported by others for patients with normal renal function. Although clearance during dialysis is doubled, thalidomide dose need not be changed for patients with decreased kidney function. There is also no need for a supplementary dose due to haemodialysis.     

HPLC法测定法莫替丁的有关物质

目的建立高效液相色谱法测定法莫替丁有关物质的方法。方法采用C18色谱柱,流动相采用乙酸盐缓冲液（pH6．0）-乙腈,梯度洗脱,流速为1．5mL·min^-1,检测波长为270nm,柱温为35℃。结果在该色谱条件下,法莫替丁与有关物质分离度良好,能有效检出法莫替丁的降解产物。结论该方法能够检测出法莫替丁的降解情况,对药品质量进行有效控制。     

High performance liquid chromatographic determination of oxeladin citrate and oxybutynin hydrochloride and their degradation products

Two high performance liquid chromatographic (HPLC) methods are presented for the determination of oxeladin citrate (OL) and oxybutynin hydrochloride (OB) and their degradation products. The first method was based on HPLC separation of OL from its degradation product using a Nucleosil C(18) column with a mobile phase consisting of acetonitrile -0.1% phosphoric acid (60:40 v/v). The second method was based on HPLC separation of OB from its degradation product using a VP-ODS C(18) column with a mobile phase consisting of acetonitrile/0.01 M potassium dihydrogen phosphate/diethylamine (60:40:0.2). Quantitation was achieved with UV detection at 220 nm based on peak area. The two HPLC methods were applied for the determination of OL or OB, their degradation products, methylparaben and propylparaben in pharmaceutical preparations. The proposed methods were used to investigate the kinetics of acidic and alkaline degradation processes of OL and OB at different temperatures and the apparent pseudofirst-order rate constant, half-life and activation energy were calculated. The pH-rate profiles of degradation of OL and OB in Britton-Robinson buffer solutions within the pH range 2-12 were studied.     

High performance liquid chromatographic determination of thalidomide in patients affected by hepatocellular carcinoma

The present study developed a validate and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of thalidomide (T) in plasma, to quantify T in patients affected by hepatocellular carcinoma. Twelve male subjects aging from 62 to 82 years and weighting 66-88kg, were orally administered with single dose of T (200mg/BW). Two ml of stabilizer-solution (CH(3)OH/CH(3)CN, 1/1 (v/v)+CH(3)COOH 2%) were added to 1ml of human plasma and stoked to -80 degrees C until analyses. This moisture (1.38mul) was added with 20mul of CF(3)COOH and 100mul of phthalimide (IS) 1.75mug/ml, vortexed and centrifuged. Surnatant (800mul) was dried under vacuum at room temperature, added with 50mul of appropriate solution and injected onto HPLC. T and IS were detected at UV wavelength of 220nm with a run time of 10min. Mobile phase was 10mM pH 5.5NH(4)(+)CH(3)COO(-)/CH(3)CN, 75/25 (v/v) buffer at flow rate of 1.5ml/min. Inter-day and intra-day variation coefficient was <10% with an error of accuracy <10%. The present detection method was able to quantify T to every withdrawal time period (LOD 0.05mug/ml). The IS used in the present study had the same wavelength maximum absorption of T, differently from early UV detection methods reported in literature where phenacetin was used. Pharmacokinetic parameters belonging from the present study are not significantly different from those calculated in previously studies performed in human health subjects and patients affected by other pathology.     

Clinical pharmacokinetics of thalidomide

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions is being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200 mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (C(max)) of 1-2 mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUC( infinity )) of 18 mg. h/L, apparent elimination half-life of 6 hours and apparent systemic clearance of 10 L/h. Thalidomide pharmacokinetics are best described by a one-compartment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacokinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than its absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state C(max) (C(ss)(max)) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accumulation, with C(ss)(max) of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400 mg. Because of the low solubility of thalidomide, C(max) is less than proportional to dose, and t(max) is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokinetics are not expected to change in patients with impaired liver or kidney function.     

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods.     

Optimization and validation of conventional and micellar LC methods for the analysis of methyltestosterone in sugar-coated pills

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of methyltestosterone in sugar-coated pills using fluoxymesterone as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 45% water:acetonitrile 55% (v:v), a flow-rate 1 mlmin(-1) and a C(18) Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography the conditions were: mobile phase 40 mM sodium dodecyl sulfate: 10% propanol, flow-rate 0.5 mlmin(-1) and C(18) Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods, UV absorbance detection at 245 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required.     

淡豆豉药材的高效液相指纹图谱研究

目的建立淡豆豉药材高效液相色谱指纹图谱(HPLC-FPS),为其质量控制提供依据。方法应用高效液相色谱法(HPLC),利用反相色谱柱:Diamonsil C18(250 mm×4.6 mm,5μm),流动相:乙腈-3%冰乙酸梯度洗脱,流速:1 ml/min,柱温:30℃,检测波长:261 nm,进样量:10μl。结果不同产地淡豆豉在化学成分方面具有明显相似性,可建立淡豆豉的指纹图谱。结论建立的HPLC指纹图谱可用于淡豆豉的质量评价。     

Immunological effects of thalidomide. Inactivity of the drug and several of its hydrolysis products in mononucleocyte proliferation tests

In order to study the spontaneous degradation of thalidomide (N-phthalyl-glutamic acid imide), an HPLC method for the separation of the drug and its hydrolysis products was developed. The effect of these substances upon the proliferation of lectin- and allogeneically stimulated peripheral blood mononucleocytes was studied, using the incorporation of [3H]-thymidine as a marker. In contrast to optically evaluated experiments reported earlier, no inhibition of [3H] incorporation was found, regardless of the duration, mode, or timing of stimulation.