Radioiodination and preliminary biological tests of aniline-mustard and its glucuronide conjugate as a potential anticancer prodrug

Aniline-mustard and its glucuronide conjugate were radioiodinated with ¹³¹I. The preliminary dynamic tests were carried out on rabbits. The scintigrams showed clearly that the glucuronide conjugate of aniline-mustard was very quickly cleared from the metabolism, accumulating in the bladder in about 15 minutes. The clearance time of radioiodinated aniline-mustard-glucuronide was considerably longer (about 45 min.). The results obtained from the biodistributional studies have represented interesting differences between the metabolic details of radioiodinated compounds, and indicated that the glucuronide conjugate of aniline-mustard may be a promising radioiodinated prodrug, if verification of its selective accumulation in some kinds of tumor cells can be obtained.     

Iodination of Peptide Hormones and Purification of Iodinated Peptides by HPLC

Abstract

Hexagastrin, Arg⁸-vasopressin, oxytocin, Tyr¹-somatostatin, and ACTH 1-39 were iodinated in order to yield precursors for tritium labelling or radioiodinated tracers for radioimmunoassay, respectively. The heterogeneous mixture of iodination products was purified via reversed-phase high-performance liquid chromatography. Iodination of peptides resulted in a marked increase in retention time on the reversed-phase adsorbent. A simple and quick method was applied for purification of radioiodinated peptides on a Sep-pak® C-18 cartridge for rapid sample preparation.     

A new poly(2-hydroxy-3-phenoxypropylacrylate, 4-hydroxybutyl acrylate, diethyl maleate) membrane controlled clonidine linear release in the transdermal drug delivery system

Oral clonidine, used as an antihypertensive, can result in some side effects such as dry mouth, drowsiness, dizziness and sedation; thus, clonidine transdermal drug delivery (TDD) was considered. Use of the controlled release membrane was one of the methods in TDD systems to regulate the permeation properties. A new type of copolymer membrane that controlled clonidine linear release in TDD system was synthesized by UV radiation. This membrane consisted of three monomers: 2-hydroxy-3-phenoxypropylacrylate, 4-hydroxybutyl acrylate and diethyl maleate. The membrane had both fine permeation properties and perfect physical properties when three monomers were in the weight ratio of 4:4:2; this type of membrane was chosen as an optimized membrane. It was found that the membrane controlled clonidine zero-order release, the permeation rates decreased with the thicknesses of membranes increasing, and the permeation rates were linearly dependent on the square root of the concentration of clonidine. Furthermore, the optimized membranes were characterized by FTIR, DSC and SEM.     

Preparation and characterization of125I labeled progesterone derivatives for the development of a radioimmunoassay for progesterone

Preparation and purification of radioiodinated progesterone derivatives for the development of a radioimmunoassay of progesterone is described. We have standardized two procedures for preparing radioiodinated progesterone conjugate. In the first procedure¹²⁵I labeled histamine was conjugated to progesterone 11 hemisuccinate by the mixed anhydride method. In the secod procedure, tyrosyl methyl ester was conjugated to progesterone 11 hemisuccinate and iodination of the conjugate was carried out. Purification of the iodinated products was carried out by solvent extraction and thin layer chromatography techniques. The radiochemical purity of the tracers prepared by both methods were more than 95%. Labeled progesterone derivatives prepared were used for developing a radioimmunoassay procedure. The non-specific binding of the tracer was about 2–3%, while up to 80% binding could be obtained in the presence of excess antibody. The radioiodinated tracer could be used up to four months in the assay.     

Some Antihypertensive Drugs Clonidine (Clofelin) and Guanfacine - New Nitric Oxide Donors

The presence of in vitro NO-donor characteristics in the antihypertensive drugs clonidine and guanfacine, the structure of which contains a guanidine grouping that relates these drugs to arginine (the main substrate of the enzyme NO-synthase, producing NO in vivo) was established by chemical oxidation of the drugs followed by polarographic detection of the produced nitric oxide in the form of nitroprusside ion. It was shown that under the investigated conditions in vitro the NO-donor activity of clonidine and guanfacine is comparable in magnitude with the NO-donor activity of arginine. It was also found that guanfacine and clonidine (the latter in the presence of NAD⁺) activate the soluble guanidatecyclase enzyme, which is a characteristic indication of NO donors. It is suggested on the basis of the obtained data that clonidine and guanfacine can undergo enzymatic oxidation to NO in vivo and that the pharmacologica l action of clonidine and guanfacine may be due to some extent to the NO-donor characteristics of these drugs.     

A new copolymer membrane cured by 2‐hydroxy‐3‐phenoxypropylacrylate, 4‐hydroxybutyl acrylate,and isobutyl methacrylate controlled clonidine linear release in the transdermal drug delivery system

The main objective of this present study was to synthesize a new type of copolymer membrane that presented a linear release property in clonidine transdermal drug delivery system. Three monomers, 2‐hydroxy‐3‐phenoxypropylacrylate, 4‐hydroxybutyl acrylate, and isobutyl methacrylate were treated under strong power UV radiation to prepare a new type of copolymer membrane. The effects of monomers' ratios, membrane thickness, and clonidine concentration on the permeation rates were investigated. It was discovered that the membrane controlled clonidine near zero‐order release. Furthermore, the membrane was characterized by FTIR, DSC, and SEM. Copyright © 2007 John Wiley & Sons, Ltd.     

Separable Forms of Radioiodinated Ovine Lutropin (oLH) Subunits,Fractionated by Anion Exchange High Performance Liquid Chromatography and Analyzed by Radioimmunoassay

Abstract

Radioiodinated oLH α and oLH ß subunits were fractionated with the aid of high performance liquid chromatography (HPLC) using a Waters Protein Pak DEAE 5PW anion exchange column. The content of these subfractions differed in their binding maxima to their respective subunit antisera. An increase of the pH from 6.5 to 7.5 and 8.5 affected the chromatographic profile of 8-week-old radioiodinated α-subunit. Overall, material from the various radioactive peaks exhibited binding to ß-subunit antiserum in the range of 32.0% - 81.0%, depending on the storage time of the tracer and the pH. Shifting strategies, we either applied the labelled subunits to a Pharmacia gel filtration column or subjected them to cellulose adsorption prior to HPLC. The radioiodinated α- and β-subunits subjected to HPLC after gel filtration were both eluted in only one peak with respective immunoreactivities of 46.6% and 73.2%.

When radioiodinated β-subunit was applied first to a cellulose column and then to HPLC, the chromatographic profile showed two radioactive peaks with retention times of 5 min (73.2% immunoreactivity) and 7.5 min (43.0% immunoreactivity), respectively.

It was concluded that an 8-week-old-tracer i s useful in such studies, owing to its highly stable immunoreactivity after repurification on an anion exchange HPLC.     

Validation of a HPLC/MS method for simultaneous quantification of clonidine,morphine and its metabolites in human plasma

A high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of morphine, morphine's major metabolites morphine‐3‐glucuronide and morphine‐6‐glucuronide, and clonidine, to support the pharmacokinetic analysis of an ongoing double‐blinded randomized clinical trial that compares the use of morphine and clonidine in infants diagnosed with neonatal abstinence syndrome. Plasma samples were processed by solid‐phase extraction and separated on an Inertsil ODS‐3 (4 μm) column using an 0.1% formic acid in water–0.1% formic acid in methanol gradient. Detection of the analytes was conducted in the positive multiple reaction monitoring mode. The range of quantitation was 1–1000 ng/mL for morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide, and 0.25–100 ng/mL for clonidine. Intra‐day and inter‐day accuracy and precision were ≤15% for all analytes across the quantitation range. Extraction recovery rates were ≥94% for morphine, ≥90% for M3G, ≥87% for M6G and ≥ 79% for clonidine. Matrix effect ranged from 85–94% for clonidine to 101–106% for M3G. The method fulfilled all predetermined acceptance criteria and required only 100 μL of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.     

Development of 15-(p-123I-phenyl)-pentadecanoic acid for in-vivo diagnosis of the myocardium

Radioiodinated ω-phenylfatty acids were recently proposed as radiopharmaceuticals for determining myocardial metabolic alterations. Therefore uptake and elimination of different radiohalogenated phenylfatty acids were determined in blood and heart muscle of mice. The structure activity dependence i.e. the effect of length of the carbon chain, position of the substituent at the benzene ring and type of radiohalogen was studied. Highest myocardial accumulation was found in case of a phenylfatty acid with 15 carbon atoms in the alkylgroup and the radiohalogen attached to the benzene ring in the para position. No difference was observed between the radiobrominated and radioiodinated substrates. In contrast to aliphatic radioiodinated fatty acids, the radioactivity in the stomach remained almost constant (i.e. below 1% dose/organ). Thus 15-(¹²³I-phenyl)-pentadecanoic acid (IPPA) could be brought into clinical application with success. Blood clearance and urine excretion of the radioactivity were determined and the results found to agree with the expectations based on the principal metabolic path of phenylfatty acids.     

可乐定分子构象异构和互变异构的理论研究

采用HF/6-31G*方法, 对氨基型和亚胺型可乐定中性分子以及质子化的可乐定分子进行了构象分析. 之后采用B3LYP/6-31G**方法对处于势能面上的能量极小构象异构体进行全自由度几何优化和频率分析. Onsager反应场溶剂模型用于水相的计算. 结果表明, 在气相和水相中可乐定中性分子主要以亚胺型存在, 这同已有实验结果一致. 进一步, 寻找构象异构化过渡态和氨基型亚胺型互变异构化过渡态, 探讨质子化和水溶剂化效应对异构化过程的几何结构和能量的影响. 为了考察氯苯的共轭效应对可乐定互变异构体稳定性的影响, 在B3LYP/6-31G**水平上, 研究了2-氨基-2-咪唑啉的互变异构化反应机理.     

Liquid chromatography tandem mass spectrometry method for the quantification of clonidine with LLOQ of 10 pg/mL in human plasma

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of clonidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 230 to 44 for clonidine and m/z 254 to 44 for the internal standard. The assay exhibited a linear dynamic range of 10-2000 pg/mL for clonidine in human plasma. The lower limit of quantification was 10 pg/mL with a relative standard deviation of less than 6.8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Radioimmunoassay kit formulation and its validation for serum progesterone using progesterone radiotracer purified by gel filtration

Purification of the radioiodinated progesterone tyrosine methyl ester conjugate by gel filtration and the development, optimization and clinical validation of a direct radioimmunoassay (RIA) of progesterone using this radiotracer are described. High purity radiotracer is essential for the error free performance of any RIA. Progesterone 11 hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mixed anhydride method and this conjugate was then radioiodinated by the chloramine-T method. Purification of the radioiodinated product was carried out by gel filtration. About 12 batches of the radiotracer were prepared and purified. The purification by gel filtration gave reproducible elution pattern and purity. The radiotracer thus purified was found to have consistent quality as compared to that of any other purification methods. Non-specific binding of the radiotracer was found to be <5% in second antibody-polyethylene glycol based separation. 'Zero standard' binding was found to be consistently 50-60% in all the batches of radiotracer. The radiochemical purity of all the batches of radiotracer was >95% as checked by paper electrophoresis. The stability (retention of the immunoreactivity) of the radiotracer was two to three months. No appreciable changes in the assay characteristics were observed during this period. The assay involved 3 hours incubation of progesterone antibody with individual standards or sample and radiotracer at room temperature. The optimized assay was then validated for internal and external quality control parameters. A RIA kit was then formulated with this radiotracer for estimation of progesterone in serum. The assay kit consisted of lyophilized individual standards ranging from 0.25 to 50 ng/ml. The clinical performance of the developed kit was compared with that of a commercial ELISA kit and a correlation of 0.94 was observed.     

Synthesis and bioevaluation of novel radioiodinated PEG-modified 2-nitroimidazole derivatives for tumor hypoxia imaging

Journal of Radioanalytical and Nuclear Chemistry - Two novel radioiodinated PEG-modified 2-nitromidazole derivatives were prepared. The two radiotracers demonstrate good stability in vitro. In the...     

Development of an LC‐MS/MS method for determining the pharmacokinetics of clonidine following oral administration of Zhenju antihypertensive compound

Zhenju antihypertensive compound (ZJAHC) is a combined Chinese–Western medicine formula including clonidine (CLO), hydrochlorothiazide (HCT), rutin, Chrysanthemum indicum extract and pearl powder. Compared with CLO preparations, ZJAHC shows improved activities and decreased adverse effects. It is believed that the side effects of CLO are caused by its high peak plasma concentration. Hence, study of the influence of ZJAHC on the pharmacokinetic behaviors of clonidine seems essential. In present study, the plasma concentrations of CLO were determined with a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method. The MS/MS transitions monitored for clonidine and internal standard were 230.2 → 213.1 and 152.2 → 110.2, respectively. The analyte was quantified in a single run within 3 min. The pharmacokinetic study showed that the area under the plasma concentration–time curve of CLO in ZJAHC (60 µg/kg CLO) was similar to that of CLO‐HCT‐high (120 µg/kg CLO) but the peak concentration was much lower than that in CLO‐HCT‐high. ZJAHC could enhance the bioavailability without greatly increasing peak concentration of clonidine. This comprehensive effect of enhancing the bioavailability and avoiding the high peak plasma concentration for CLO might mainly result from the co‐contribution of Western medicine and traditional Chinese medicine (TCM), while the effect of TCM was stronger than that of Western medicine. Copyright © 2014 John Wiley & Sons, Ltd.     

Radioiodinated magnetic targeted carriers (<Superscript>131</Superscript>I-MTC)

Magnetically targeted drug delivery by particulate carriers is an efficient method of delivering drugs to localized disease sites, such as tumors. Thus, high concentrations of carrier molecules such as therapeutic radiopharmaceuticals can be achieved near the target site without any toxic effects to normal surrounding tissue. In this study, magnetic targeting carriers (MTC) were radioiodinated with ¹³¹I using three different methods (1) ¹³¹I was directly bound onto MTC, (2) an iron complex of 8-hydroxyquinoline (8-OHQ) has been absorbed onto magnetic microspheres. The iodogen method was performed for the iodination of the complexes. (3) 8-OHQ was radioiodinated before chelating with Fe. Reaction parameters were investigated in order to optimize the final properties of the labeled MTC. The best labeling yield and the best stability were obtained when 8-OHQ was chelated before the radioiodination. Binding efficiency was found to be 99.58%. The labeling of the MTC with ¹³¹I was undertaken to allow for therapy with ¹³¹I-labeled MTC with simultaneous imaging.     

Synthesis and in vitro evaluation of iodinated derivatives of piperazine as a new ligand for sigma receptor imaging by single photon emission computed tomography

A new series of radioiodinated analogues of 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) was synthesized and evaluated as a potential brain sigma-1 receptor imaging ligands by single photon emission computed tomography (SPECT). Iodinated analogues of SA4503 (4a-c) were prepared from piperazine in a high yield. The in vitro competition binding studies using [3H] DTG (sigma-1, 2), [3H] (+)-pentazocine (sigma-1), and [3H] DTG in the presence of carbetapentane (sigma-2) as sigma receptor selective radioligands were revealed that iodinated analogues 4a-c possess high affinities to sigma receptors (IC50: 4a=7.1, 4b=31.0, and 4c=77.3 nM). In particular, the affinity of 4a, bearing iodine at ortho position on the phenyl ring, was 4.4 times greater than SA4503, and 3 times greater than that of haloperidol. The meta-iodo analogue 4b was the same to SA4503, the lead compound. The radioiodinated derivatives, [125I] 4a, 4b were synthesized no-carrier-added from the corresponding tributyltin precursors by the iododestannylation reaction with high yields. The binding of [125I] 4a, 4b have been characterized in the rat brain membranes. These compounds were indicated single population binding to sigma receptor with high affinity (4a: Kd=1.86+/-0.34 nM, Bmax=205+/-28.9 fmol/mg protein, 4b: Kd=3.30+/-0.51 nM, Bmax=231.5+/-13.8 fmol/mg protein). In vitro blocking studies were confirmed that the high specificity of 4a, 4b. These results suggest that radioiodinated 4a and 4b are promising sigma receptors imaging ligand for pursuing further in vivo studies.     

NEW RADIOHALOGENATED ALKENYL TELLURIUM FATTY ACIDS

Abstract

Radiolabeled long-chain fatty acids have diagnostic value as radiopharmaceutical tools in myocardial imaging. Some applications of these fatty acids are limited due to their natural metabolic degradation in vivo with subsequent washout of the radioactivity from the myocardium. The identification of structural features which will increase the myocardial residence time without decreasing the heart uptake of long-chain fatty acids is of interest. Fatty acids containing the tellurium heteroatom were the first modified fatty acids developed and show unique prolonged myocardial retention and low blood levels. Our detailed studies with radioiodinated vinyliodide substituted tellurium fatty acids demonstrate that heart uptake is a function of the tellurium position. New techniques of tellurium and organoborane chemistry have been developed for the synthesis of a variety of radioiodinated iodoalkenyl tellurium fatty acids.     

Synthesis and alpha-adrenergic binding ligand affinities of 2-iminoimidazolidine derivatives

In order to obtain possible veinotonic drugs acting through alpha2 receptor activation, we prepared clonidine analogues in which the 2-imino-imidazolidine was attached to various aliphatic or aromatic heterocycles. Among them, the two benzopyranic derivatives 16 and 22 exhibited interesting affinities (19 and 95 nM respectively on [3H]rauwolscine binding, compared to 35 nM for clonidine). Their affinity for alpha1 receptors was found to be much lower: 7570 and 5030 nM for 16 and 22 respectively, suggesting 16 to be 400 times more selective for alpha2 than for alpha1-adrenoceptors.     

Synthesis of 4-(4-iodophenyl)piperazine and the 1-carboxamidino derivative

The previously unreported 4-(4-iodophenyl)piperazine ( 2 ) was synthesized in a 70% yield and characterized. The reaction of 2 with S-methylthiouronium sulfate gave the corresponding carboxamidino derivative 4 in a 61% yield. The tlc properties of 4 were identical with those of the radioiodinated material that had been prepared and evaluated as a potential myocardial imaging radiopharmaceutical.     

Radiopharmaceuticals

For studying the metabolism of fatty acids in the heart muscle in-vivo, ω-phenylpenta-decanoic acid (PPA) was labelled with radioiodine to the ortho and para position of the benzene ring with a radiochemical yield of 60%. The products were obtained in a relative isomer distribution of 29%ortho and 71%para radioiodinated PPA. The radiopharmaceutical quality control was performed by sequential radio high pressure liquid chromatography.