Development and characterization of a novel peroral peptide drug delivery system.

Novel drug delivery systems were developed for peroral administration of peptide and protein drugs for site specific mechanical fixation at the gut wall and with specific release patterns. These so-called shuttle systems were designed by using superporous hydrogels (SPH) and SPH composite (SPHC) as the conveyor of a core which contained the model compound N-alpha-benzoyl-L-arginine ethylester (BAEE). Two different types of shuttle systems were evaluated: (a) core inside the shuttle system, and (b) core attached to the surface of shuttle system. Each of these systems was made of two parts: (1) the conveyor system made of SPHC which is used for keeping the dosage form at specific site(s) of the GI tract by mechanical interaction of the dosage form with the intestinal membranes, and (2) the core containing the active ingredient and incorporated in the conveyor system. The effect of formulation composition of the core on the release profile of BAEE was investigated by changing the type and amount of excipients in the formulations. In addition, the effect of various enteric-coat layers on the release profile and dissolving of the dosage form was investigated. The systems were also characterized for trypsin inactivation and Ca(2+) binding. The release profile of BAEE from the core formulation consisting of PEG 6000 microparticles or small tablets showed the desired burst release. When these core formulations were incorporated into the conveyor system made of SPH and SPHC, a suitable time-controlled release profile was obtained. Changing the type, concentration and thickness of the enteric-coat layer influenced the starting time of BAEE release from the dosage form, which indicates the necessary lag time for dissolving of the dosage form at any desired specific site of drug absorption in the intestine. Both SPH and SPHC were found to partly inhibit the activity of trypsin, due to two mechanisms: Ca(2+) binding and entrapment of the enzyme in these polymers. In conclusion, the presently developed delivery systems demonstrate suitable in vitro characteristics with an appropriate time-controlled release profile, making these systems very promising for effective peroral delivery of peptide and protein drugs.     

In vitro and in vivo characterization of a novel biocompatible polymer-lipid implant system for the sustained delivery of paclitaxel.

Recently, a novel chitosan-based implantable formulation (chitosan-ePC) was developed to provide controlled, local release of paclitaxel (PTX) for the treatment of ovarian tumors. Hence, the objective of this study was to evaluate this delivery system in vitro in human ovarian SKOV-3 cells and in vivo in mice with intraperitoneal implants of drug-free or 14C-PTX-chitosan-ePC films. In vitro, 14C-PTX-chitosan-ePC implants (10 mg) provided zero-order constant release of 0.92+/-0.03 pg/day PTX over 5 days. Released PTX retained dose-dependent activity; effectively inhibiting SKOV-3 proliferation with an ED50 of 211 ng/ml of released PTX. Drug-free implants did not affect cell viability or cell morphology of SKOV-3 cells. A sustained, zero-order release of PTX was also seen in vivo over a 2 week period in mice implanted with 14C-PTX-chitosan-ePC films. Correlations between the in vitro and in vivo release of PTX was highly significant (R2 = 0.975). After 2-4 weeks, mice with chitosan-ePC implants did not demonstrate any signs of encapsulation, inflammation or infection. Overall, our in vitro and in vivo results demonstrated zero-order drug release and biocompatibility of the novel chitosan-ePC film. This indicates potential usefulness of chitosan-ePC implants in the sustained and local delivery of anti-neoplastic agents.     

Physically bonded nanoparticle networks: a novel drug delivery system.

Monodispersed nanoparticles consisting of interpenetrating polymer networks (IPNs) of polyacrylic acid (PAAc) and poly(N-isopropylacrylamide) (PNIPAM) were prepared by a seed-and-feed method. The temperature-dependent viscosity measurement revealed that the IPN nanoparticle dispersions with polymer concentrations above 2.5 wt.% underwent an inverse thermoreversible gelation at about 33 degrees C. Dextran markers of various molecular weights as model macromolecular drugs were mixed with the IPN nanoparticle dispersion at room temperature. At body temperature, the dispersion became a gel. The drug release profiles were then measured using UV-Visible spectroscopy as a function of particle size and polymer concentration. The schematic structure of the nanoparticle network was proposed based on the experimental results. The drug delivery model presented here was significant because such a dispersion and a drug was mixed without chemical reaction at room temperature to form a drug delivery liquid. This liquid could be injected into a body to form in situ a gelled drug depot to release the drug slowly.     

微泡超声造影剂:一种新型的药物靶向载体

药物携载是目前重要的研究领域,如何使得药物安全、有效、靶向性地导入体内特定器官、组织并使其释放在靶细胞内是研究的重点.微泡超声造影剂作为一种新型的体内药物载体,受到国内外学者的广泛关注.本文对有关微泡超声造影剂作为药物载体的作用原理、制备要求、制备方法、特点和应用等方面的研究作了介绍.     

In vitro and in vivo evaluation of a matrix-in-cylinder system for sustained drug delivery.

A matrix-in-cylinder system for sustained drug delivery, consisting of a hot-melt extruded ethylcellulose (EC) pipe surrounding a drug containing HPMC-Gelucire 44/14 core, was evaluated in vitro and in vivo. In an aqueous medium, the HPMC-Gelucire core forms a gel plug, which releases the drug-through the open ends of the EC pipe--by means of erosion. The influence of hydrodynamic and mechanical stress and the effect of different 'physiologically relevant' dissolution media on the in vitro drug release were investigated. From these in vitro dissolution tests, it was concluded that the EC pipe has a protective effect on the drug containing HPMC-Gelucire core. It largely protects the core against hydrodynamics and mechanical stress. Furthermore, drug release from the matrix-in-cylinder system was only slightly affected by the composition of the dissolution medium. A randomised crossover in vivo study in dogs revealed that the matrix-in-cylinder system containing propranolol hydrochloride has an ideal sustained release profile with constant plasma levels maintained over 24 h. Moreover, administration of the matrix-in-cylinder system resulted in a 4-fold increase in propranolol bioavailability when compared with a commercial sustained release formulation (Inderal).     

纳米羟基磷灰石/硫酸庆大霉素缓释系统的制备及体内释放实验

目的:制备纳米羟基磷灰石/硫酸庆大霉素缓释系统(drug delivery system,DDS),观察其体内释药特性,为慢性骨髓炎的治疗寻找更好的方法。方法:实验于2004-05/2005-03在南方医科大学珠江医院中心实验室和华南理工大学生物工程与材料学院实验室完成。实验材料:纳米羟基磷灰石(nano-hydroxypatite,nano-HA)、聚羟基丁酸酯-羟基戊酸酯共聚物[poly(3-hydroxybutyrate-hydroxyvalerate),PHBV]、聚乙二醇(polyethylene glycol,PEG)、硫酸庆大霉素(gentamicin,GM)。新西兰大白兔24只。实验方法:①nano-HA/PHBV-PEG-GM-DDS的制备:以nano-HA为载药核心,外包裹生物相容性好且降解可调控的PHBV、PEG,承载GM制成。②采用SPSS10.0统计软件包绘制庆大霉素浓度与抑菌环直径的半对数标准曲线。③体内释放实验:在新西兰大白兔左股骨外上髁钻孔,刮除部分骨质和骨髓,将nano-HA-PHBV/PEG-GM微球自骨窗内植入,分别于术后1,3,5,7,10,14,21,28d8个时间点各选3只大白兔,麻醉后取干骺端松质骨及骨干皮质骨标本。用标准曲线计算出各标本药物浓度,用SPSS10.0统计软件包绘制nano-HA/PHBV-PEG-GM-DDS的释药浓度与时间关系曲线。实验评估:①扫描电镜下观察nano-HA/PHBV-PEG-GM-DDS的微球形态。②nano-HA/PHBV-PEG-GM-DDS的释药浓度与时间的关系。结果:纳入兔24只,均进入结果分析。①nano-HA/PHBV-PEG-GM-DDS微球形态:大小均匀规整,微球平均粒径为345μm,小微球粒径为78μm,微球表面形态一致,为多孔皱缩结构。②nano-HA/PHBV-PEG-GM-DDS释药浓度与时间的关系:术后第1天nano-HA/PHBV-PEG-GM-DDS周围皮质骨和松质骨中庆大霉素质量浓度分别为(110.10±11.70),(97.30±9.60)mg/L,其后逐渐下降,至术后第28天时皮质骨和松质骨中庆大霉素质量浓度仍可分别达(7.30±1.40),(6.80±1.10)mg/L,局部骨组织中庆大霉素质量浓度仍在金黄色葡萄球菌最小抑菌浓度(2mg/L)以上。结论:nano-HA/PHBV-PEG-GM-DDS具有较好的体内缓释作用。     

复合万古霉素海藻酸钠／壳聚糖缓释载体的体内释放实验

背景：研制一种生物相容性好、可降解、体内释药时间长、来源充分、使用安全的抗生素局部释药系统是目前国内外骨髓炎治疗顿域的一个重要发展方向。海藻酸钠和壳聚糖具备优良的生物特性,可以作为抗生素局部释药系统的良好载体。目的：观察海藻酸钠,壳聚糖作为万古霉素缓释载体局部用药的可行性与安全性,并与全身用药比较。设计、时间及地点：体内药物浓度测定,配对分组实验,于2008-08／2009-03在大连大学附属中山医院动物实验室、骨科实验室完成。材料：将海藻酸钠溶液与万古霉素溶液均匀混合后加入CaCO3和柠檬酸钠溶液凝胶化后,利用微胶囊制备仪将上述混合液制备成万古霉素海藻酸钙凝胶珠。取适量胶珠加入到配好的壳聚糖,万古霉素混合液中反应后收集微胶囊,加醋酸纤维素赋形制备成所需要的载药载体。方法：40只实验兔制作左侧股骨中段局部骨缺损模型,随机分为2组,每组20只。局部用药组于骨缺损中放入一定载药量的复合万古霉素海藻酸钠,壳聚糖缓释载体：全身用药组自兔耳缘静脉注射10％万古霉素（10mg／kg）。主要观察指标：局部用药组术后0．5,1,6,24,72h及1,2周,用高效液相色谱仪检测血清和骨组织中万古霉素浓度。全身用药组注射后10min,0．5,1,6,24,72h测定不同时间点血清、骨组织及肌肉组织中万古霉素浓度。制作局部用药组各重要脏器组织学切片以确定植入物的全身毒性反应。结果：全身用药组24h前备时间点的血药浓度均明显高于局部用药组,24h时全身用药组的血药浓度低于局部用药组：局部用药组的骨髂及肌肉组织药物浓度各时间点均显著超过全身用药组,局部用药组在2周时药物浓度仍然保持较高水平,而全身用药组在持续24h后即出现血药浓度低于最低抑菌浓度的情况。未发现全身毒性的组织学证据。结论：复合万古霉素海藻酸钠／壳聚糖缓释载体局部用药具有一定可行性及安全性,作用效果优于全身用药。     

复合万古霉素海藻酸钠/壳聚糖缓释载体的体内释放实验料

背景:研制一种生物相容性好、可降解、体内释药时间长、来源充分、使用安全的抗生素局部释药系统是目前国内外骨髓炎治疗领域的一个重要发展方向.海藻酸钠和壳聚糖具备优良的生物特性,可以作为抗生索局部释药系统的良好载体.目的:观察海藻酸钠/壳聚糖作为万古霉素缓释载体局部用药的可行性与安全性,并与全身用药比较.设计、时间及地点:体内药物浓度测定,配对分组实验,于2008-08/2009-03在大连大学附属中山医院动物实验室、骨科实验室完成.材料:将海藻酸钠溶液与万古霉素溶液均匀混合后加入CaCO_3和柠檬酸钠溶液凝胶化后,利用微胶囊制备仪将上述混合液制备成万古霉素-海藻酸钙凝胶珠.取适量胶珠加入到配好的壳聚糖/万古霉素混合液中反应后收集微胶囊,加醋酸纤维素赋形制备成所需要的载药载体.方法:40只实验兔制作左侧股骨中段局部骨缺损模型,随机分为2组,每组20只.局部用药组于骨缺损中放入一定载药量的复合万古霉素海藻酸钠/壳聚糖缓释载体;全身用药组自兔耳缘静脉注射10%万古霉素(10 mg/kg).主要观察指标:局部用药组术后0.5,1,6,24,72 h及1,2周,用高效液相色谱仪检测血清和骨组织中万古霉素浓度.全身用药组注射后10 min,0.5,1,6,24,72 h测定不同时间点血清、骨组织及肌肉组织中万古霉素浓度.制作局部用药组各重要脏器组织学切片以确定植入物的全身毒性反应.结果:全身用药组24 h前各时间点的血药浓度均明显高于局部用药组,24 h时全身用药组的血药浓度低于局部用药组;局部用药组的骨骼及肌肉组织药物浓度各时间点均显著超过全身用药组,局部用药组在2周时药物浓度仍然保持较高水平,而全身用药组在持续24 h后即出现血药浓度低于最低抑菌浓度的情况.未发现全身毒性的组织学证据.结论:复合万古霉素海藻酸钠/壳聚糖缓释载体局部用药具有一定可行性及安全性,作用效果优于全身用药.     

Optimization of iontophoretic transdermal delivery of a peptide and a non-peptide drug

In vitro iontophoretic transdermal delivery (ITD) of a tripeptide, enalaprilat (EP) and a non-peptide, cromolyn sodium (CS), across frozen hairless guinea pig (HGP) skin were investigated. Parameters for optimization of ITD included the influence of ionic strength (μ), buffer type and size, drug loading in the donor and the effect of pH. Drug permeation into the receptor compartment was monitored using HPLC assay methods developed for the study. An optimum μ of 6.66 mM in acetate buffer was found necessary for efficient ITD of CS. An exponential decrease in the flux of CS was observed with an increasing μ. Buffer ions larger than acetate ions inhibited the transport of CS ions. With an increase in the donor concentration of CS, a hyperbolic relationship for the increase in flux was observed. For EP, permeation was not detectable when μ was increased to greater than 31 mM in phosphate-buffered solution. With an increase in pH above the pK_a1 (3.55) for EP, a linear decrease in flux was observed. Higher drug loading of EP in the donor compartment provided better permeation. Effect of freezing of HGP skin on the iontophoretic delivery of EP and CS was also evaluated. Flux values for either of the drugs studied were similar when frozen or fresh skins were used. Reversibility studies indicated that no gross current induced permeation changes occurred with the HGP skin. Passive permeation of either of the drugs investigated was negligible.     

In vitro and in vivo evaluation of a novel mitomycin nanomicelle delivery system

Mitomycin C (MMC), naturally synthesized by Streptomyces caespitosus, is a potent antineoplastic antibiotic for the treatment of various solid tumors. However, the defects of conventional MMC injections have greatly limited its clinical application due to its toxic side effects and non-specific interactions. To solve this problem, the PEG_2k-Fmoc-Ibuprofen (PEG-FIbu) micellar nanocarrier was synthesized and the MMC-loaded micelles (PEG-FIbu/MMC) were prepared by thin film hydration method and characterized. Ibuprofen was used as a hydrophobic domain of PEG-FIbu nanocarrier, and we expect it to synergize with codelivered MMC in the overall antitumor activity. The in vitro release of PEG-FIbu/MMC was examined by dialysis method using MMC injection as a control. Our data suggested that PEG-FIbu/MMC micelles presented appropriate particle size, low CMC value, good stability, high drug loading efficiency and sustained release properties. In vitro cytotoxicity studies with several tumor cell lines showed that the carrier was effective in mediating intracellular delivery of MMC to tumor cells. In vivo pharmacokinetics, tissue distribution and therapeutic study proved that PEG-FIbu/MMC micelles prolonged blood circulation, significantly improved the tumor accumulation and therapeutic efficacy, and reduced undesirable side effect on normal tissues compared to MMC injection. In general, PEG-FIbu/MMC micelles represented an effective strategy to improve the performance for the delivery of MMC and safety of medication.

The introduction of a micellar delivery system of MMC increase efficiency, reduce toxicity and enhance specific interactions in tumor.     

Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole.

It was the purpose of this study to design and evaluate a new bioadhesive vaginal drug delivery system for clotrimazole. Chitosan, a cationic biopolymer derived by deacetylation of chitin, was modified by the introduction of thioglycolic acid (TGA). The modification was achieved by utilising a carbodiimide to link the carboxylic acid moieties of TGA covalently to the primary amino groups of chitosan. The amount of added carbodiimide was thereby varied, resulting in chitosan-TGA conjugates A and B with 160 microM (=micromol) and 280 microM thiol groups per gram polymer, respectively. In order to characterise the new polymers the water uptake, the disintegration behaviour, the bioadhesive properties utilising the rotating cylinder method, as well as the release of clotrimazole from tablets based on these derivatives were studied. The water uptake and cohesive properties of vaginal tablets consisting of these new conjugates could be significantly (p<0.05) improved. By adding clotrimazole the disintegration time of the conjugates was prolonged 1.6-fold for conjugate A and even 100-fold for conjugate B. Furthermore, the adhesion on vaginal mucosal tissue could be significantly improved. The addition of clotrimazole had also an impact on the adhesion time of chitosan-TGA conjugate B, which remained 26-times longer on vaginal mucosa than the corresponding unmodified polymer. The immobilisation of thiol groups guarantees a controlled drug release. Results of this study demonstrate that these new chitosan-TGA conjugates are very promising vehicles for the vaginal application of clotrimazole in treatment of mycotic infections.     

The design and evaluation of a novel targeted drug delivery system using cationic emulsion-antibody conjugates.

In an attempt to design a targeted drug delivery system to tumors' over-expressing H-ferritin specifically recognized by a monoclonal antibody, AMB8LK, a cationic emulsion - AMB8LK conjugate was prepared. A novel cross-linker molecule bearing maleimide group was synthesized and added to cationic emulsion formulation for AMB8LK Fab' fragment covalent coupling. NMR spectroscopy confirmed the cross-linker synthesis and the preservation of the active maleimide function. SDS gel-electrophoresis results corroborated the formation of the Fab' fragment. Different densities of Fab' fragments (10-200 Fab'/oil droplet) were conjugated to emulsion droplet interface and no changes in the physico-chemical properties were observed ( approximately 120 nm size and zeta potential of approximately +30 mV). The coupling efficiency ranged from 55% to 70% and was visualized by TEM showing gold particles attached to the droplet interface. Cell culture studies demonstrated specific binding to cells as confirmed by the occurrence of the marked reduction in binding when free AMB8LK Mab was incubated before adding the AMB8LK-emulsion conjugate to the cells. The coupling of AMB8LK Fab' fragment to the cationic emulsion increased the cells uptake by 50% as compared to non-conjugated respective cationic emulsion. Appropriate conditions were, thus, identified for coupling AMB8LK Fab' fragment to cationic emulsion without altering the specificity and affinity of the Mab fragment to the tumor antigen.     

Development of a multifunctional matrix drug delivery system surrounded by an impermeable cylinder.

A multifunctional drug delivery system based on hydroxypropyl methylcellulose (HPMC)-matrices (tablets) placed within an impermeable polymeric cylinder (open at both ends) was developed. Depending on the configuration of the device, extended release, floating or pulsatile drug delivery systems could be obtained. The release behaviour of the different devices was investigated as a function of HPMC viscosity grade, HPMC content, type of drug (chlorpheniramine maleate or ibuprofen), matrix weight, position of the matrix within the polymeric cylinder, addition of various fillers (lactose, dibasic calcium phosphate or microcrystalline cellulose) and agitation rate of the release medium. The drug release increased with a reduced HPMC viscosity grade, higher aqueous drug solubility, decreased HPMC content and increased surface area of the matrix. The release was fairly independent of the agitation rate, the position of the tablet within the polymeric cylinder and the length of the cylinder. With the pulsatile device, the lag time prior to the drug release could be controlled through the erosion rate of the matrix (matrix weight and composition).     

Controlled drug release from a novel liposomal delivery system. I. Investigation of transdermal potential

The in vitro release behavior of a novel liposome-based drug delivery device has been characterized. The system consists of a molded agarose matrix in which the model drug (progesterone) was dispersed either free or associated with one of four lipid formulations: egg-phosphatidylcholine (EPC) liposomes, EPC/cholesterol (2:1) liposomes, Intralipid^® emulsion, and dipalmitoylphosphatidylcholine (DPPC) liposomes. Drug release rates from the devices into aqueous buffer were measured at 37° C. The free progesterone release rate decreased rapidly over 24 h with over 90% delivered. The liposomal patches, on the other hand, imposed apparent zero-order kinetics: for example, both the EPC and DPPC systems delivered their progesterone payloads at about 1%/h over 24 h. Further, the EPC and DPPC patches significantly slowed transdermal drug delivery across excised hairless mouse skin. The EPC device retarded throughput to one-half the control value, the DPPC system reduced the transport kinetics by an order of magnitude. The results support two hypotheses: (a) the liposomal-based reservoir system can modulate drug input via the skin, (b) the zero-order release of progesterone from liposomes is determined by slow interfacial transport out of the bilayer into the surrounding aqueous medium.     

Pharmacokinetics of a self-microemulsifying drug delivery system of tacrolimus

This study evaluated the pharmacokinetics of tacrolimus (Tac) in a novel self-microemulsifying drug delivery system (SMEDDS) for improved oral administration. SMEDDS Tac consisted of ethyl oleate as the oily phase, Solutol HS 15 as the surfactant and glycofurol as the co-surfactant and contained 0.5 mg/mL tacrolimus. Blood and tissue concentrations of tacrolimus from two study groups (oral application of SMEDDS Tac and Prograf^®) were determined using ELISA technique following tacrolimus administration in rats. There was no difference between area under the whole blood concentration–time curve in the SEDDM Tac group and the Prograf^® group. Maximum concentrations of the drug were three times higher (P < 0.05) in the SEDDM Tac group accompanied by a 3-fold earlier peak time. Elimination half-life was significantly lower in the SEDDM Tac group. Application of SMEDDS Tac increased tissue accumulation. Already after 15 min, Tac levels of small intestine, liver, kidney, spleen, heart and bone marrow were significantly higher in the SMEDDS Tac group than in the Prograf^® group (P < 0.05). However, the Tac concentration in the kidney was significantly lower in the SMEDDS Tac group. Formulation of SMEDDS did not affect blood-brain barrier function. The SMEDDS is a potentially useful method for a local delivery of Tac to target organs. The selection of the optimum SMEDDS Tac composition might have advantage as an alternative oral dosage form for Tac.     

Construction and characterization of a t-PA mutant for use in ATTEMPTS: a drug delivery system for achieving targeted thrombolysis.

To resolve the bleeding risk associated with thrombolytic therapy, we have designed an approach, termed ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug Type Strategy), to deliver t-PA to the clot site in an inactive form and then trigger its conversion to the active form, so that it would selectively activate the clot bound plasminogen while alleviating the bleeding risk. This delivery system was composed of a large protein complex, consisting of two components: (i) a heparin-modified, negatively charged fibrin-targeting antibody; and (ii) a cationic peptide-modified, positively charged t-PA. Both in vitro and in vivo studies have confirmed the feasibility of this targeted drug delivery approach. A site-specific thrombolysis was observed in animals, without concomitant depletion of the coagulation factors -- the phenomenon in conventional thrombolytic therapy that contributes to the bleeding risk. Despite promise, the chemical conjugation method employed previously in the preparation of the cationic peptide-modified t-PA also revealed several major shortcomings. The primary drawback was that the number of the cationic peptides and the location at which these peptides were attached to a t-PA molecule could not be regulated by using the chemical conjugation method. As a consequence, the resultant modified t-PA possessed a wide range of heparin-binding strength, rendering the inhibition of t-PA activity by heparin binding ineffective. In this paper, we present a new strategy in producing the desired modified t-PA, utilizing the genetic engineering approach. A computer simulation-guided rational design strategy was adopted to identify the most desirable site in t-PA (i.e. the 37-loop) for incorporation of the heparin-binding peptide sequence. By altering the amino acid composition via mutation at three locations, i.e. Ser(300) to Cys, Gly(302) to Arg, and Glu(303) to Arg, a highly cationic nanomer sequence consisting of (297)KHRRCPRRR(304) and possessing a well-demonstrated heparin-binding domain was established within the 37-loop. To ensure the binding of heparin to this specifically modified domain, a cysteine residue (i.e. Cys(300)) was created to allow for site-specific conjugation of an additional heparin-binding peptide (i.e. the LMWP peptide previously developed in our laboratory) to this domain via the chemical conjugation method. In vitro fibrinolysis assays showed that both the t-PA mutant and the LMWP-attached t-PA mutant exhibited a fibrinolytic potency similar to that of the wild type t-PA. Inhibition studies using small chromogenic substrates demonstrated that the activity of mutant tPA-LMWP could be significantly inhibited by heparin binding. In conclusion, using computer simulation and molecular biology approaches, a mutated t-PA that meets the needs of the ATTEMPTS system, in providing a safe thrombolytic therapy, could be readily prepared.