A Novel Enhancer for Fe~(2 )-Catalyzed Light Emission Reaction of Luminol and Dissolved Oxygen

用ＥＤＴＡ可增强Ｆｅ２＋催化鲁米诺与溶解氧的发光反应，将ＥＤＴＡ加入鲁米诺溶液中可使流动注射分析测亚铁的检出限降低约１６０倍。在流动注射分析的试样分流路中使用新型的锌镀铜微还原柱，可同时测定Ｆｅ２＋和Ｆｅ３＋。该微还原柱可至少测定３０００个试样。测定Ｆｅ２＋和Ｆｅ３＋的线性范围是１×１０９～１×１０５ｍｏｌ·Ｌ１，检出限分别为２．７×１０１０和３．５×１０１０ｍｏｌ·Ｌ１，测定试样的速率为６０ｈ１。Ｃｒ３＋和Ｃｏ２＋有干扰。测定混合物中的Ｆｅ２＋和Ｆｅ３＋获得满意结果，测试样的结果同标准的分光光度法结果一致。实验表明，ＥＤＴＡ起增强剂的作用，Ｆｅ２＋是催化剂，而溶解氧是氧化剂。对反应机理进行了讨论。     

Inhibitory effects of α-methyl-4-(3-oxo-2H-1,2-benzoisoselenazol-2-yl)benzeneacetic acid on Cu~(2+) and Fe~(2+) induced oxidative modification of human low density lipoprotein

为观察α－甲基－４－（３－氧－２Ｈ－１，２－苯并异硒唑－２－基）苯乙酸（ＭＢＢＡ）对Ｃｕ２＋及Ｆｅ２＋氧化修饰低密度脂蛋白（ＬＤＬ）的保护作用及其作用机理，采用分光光度法测定ＬＤＬ中丙二醛（ＭＤＡ）和共轭双烯（ＣＤ）的产生量．ＭＢＢＡ（０．２－２μｍｏｌ·Ｌ－１）能以剂量依赖性抑制Ｃｕ２＋及Ｆｅ２＋诱导的ＭＤＡ和ＣＤ生成．２μｍｏｌ·Ｌ－１的ＭＢＢＡ对Ｃｕ２＋诱导ＬＤＬ产生ＭＤＡ和ＣＤ的抑制率分别为８９．７％和６０．３％．０．５ｍｍｏｌ·Ｌ－１ＧＳＨ对ＬＤＬ产生ＭＤＡ无影响，但能显著增强ＭＢＢＡ对ＭＤＡ生成的抑制作用．上述结果表明ＭＢＢＡ对ＬＤＬ氧化修饰的抑制作用可能依赖于其ＧＳＨ－Ｐｘ样活性的作用和（或）直接还原脂质氢过氧化物的作用．     

Distribution of the scavenger esterases against organophosphorus compounds in human tissues

测定了人体各种组织中与有机磷化合物代谢有关的二异丙基氟磷酸酯酶（ＤＦＰａｓｅ），对氧磷酶，乙酰胆碱酯酶（ＡＣｈＥ），丁酰胆碱酯酶（ＢＣｈＥ）及羧基酯酶（ＣａＥ）的活性水平．结果表明：在肾脏，脾脏和肝脏中含有丰富的ＤＦＰａｓｅ，可催化水解梭曼．其他组织该酶活性均很低．除人血清外，其他组织均未测到对氧磷酶活性．ＡＣｈＥ和ＢＣｈＥ主要分布于中脑，桥脑，延脑，纹状体及丘脑等脑干区域，另外肝脏中ＢＣｈＥ的比活性也较高．ＣａＥ主要分布于肝脏组织中，肺脏中也有较高的ＣａＥ活性．以上结果对了解有机磷化合物在人体中的中毒及代谢提供了一定的理论基础     

电位滴定法测定胰激肽释放酶活力测试条件探讨

本文用ＦＩＰ推荐的方法，以具有国际单位的参照品为标样，滴定液为０．０１ｍｏｌ／ＬＮａＯＨ标准溶液，考查了以ＢＡＥＥ为底物的电位滴定法测定胰激肽释放酶活力的实验条件，通过正交试验得出直观和方差分析的结果，依据酶活力单位高、ＳＤ和ＲＳＤ％较小的原则，求出酶活力测定的最适条件是：酶反应温度为２５℃；缓冲液为０．００１５ｍｏｌ／ＬＮａ２Ｂ４Ｏ７－０．２５ｍｏｌ／ＬＮａＣＬ－２×１０（－４）ｍｏｌ／ＬＥＤＴＡ，ｐＨ为８．００；反应液中酶浓度为０．４８ＩＵ／ｍｌ；底物浓度为５×１０（－３）ｍｏｌ／Ｌ；胰蛋白酶抑制剂浓度为１２５μｇ／ｍｌ。胰激肽释放酶活力测定的实验数据的相对标准偏差小于５％。     

亲和层析纯化的特异性免疫球蛋白及其片段对VX中毒的保护作用

用Ｓｅｐｈａｒｏｓｅ４Ｂ－６－氨基己酰－氨基ＶＸ亲和凝胶层析分离纯化兔抗ＶＸ抗血清中对ＶＸ特异的免疫球蛋白（Ｉｇｓ），再用胃蛋白酶裂解为Ｆ（ａｂ）２片段．用酶联免疫吸附试验法测定抗体活性，在吸光度１．０（４９２ｎｍ）时，抗血清，Ｉｇｓ和Ｆ（ａｂ）２的蛋白质浓度分别是１．７５，０．２５和０．１７ｍｇ·Ｌ－１．在试管内保护电鳐乙酰胆碱酯酶活性５０％所需的浓度，Ｉｇｓ和Ｆ（ａｂ）２只需抗血清的２．６％和３．８％．ｉｖ抗血清，Ｉｇｓ和风Ｆ（ａｂ）２对抗小鼠１×ＬＤ９５ＶＸ中毒死亡的最小全保护剂量分别为每鼠７００，３２和３７μｇ，在此剂量下对小鼠脑胆碱酯酶活性的保护申分别为６４％，６７％和７６％．     

糖尿病大鼠血中内源性一氧化氮合酶抑制物增高

目的：测定糖尿病大鼠血中内源性ＮＯ合酶抑制物二甲基精氨酸（ＤＭＡ）的含量，方法：在链佐星诱发的糖尿病大鼠测定血清ＤＭＡ的含量和乙酰胆碱（ＡＣｈ）诱导血管内皮依赖性舒张，结果：与对照组相比，糖尿病大鼠ＤＭＡ血清浓度显增加（５．４±１．０ｖｓ０．７±０．３μｍｏｌ．Ｌ＾－１，Ｐ〈０．０１）；丙二醛含量也高于对照组（２．５±０．３ｖｓ２１．５±０．１μｍｏｌ．Ｌ＾－１，Ｐ〈０．０１）；糖尿病大鼠ＡＣｈ     

4,5—二氢—6—[（苯乙酰基—哌嗪基）苯基]—5—甲基—3（2H）—哒…

４，５－二氢－６－［（苯乙酰基－哌嗪基）苯基］－５－甲基－３（２Ｈ）－达嗪酮（ＳＭⅡ４）０．１－２．５μｍｏｌ．Ｌ＾－＾１能使血小板激活因子（ＰＡＦ）及血栓素Ａ２类似物Ｕ４６６１９诱导的兔血小板聚集剂量一效应曲线左移且最大反应降低。其ｐＤ２分别为６．０±ｓ０．４及６．１±ｓ０．３．ＳＭⅡ４还能抑制ＡＤＰ，花生四烯酸（ＡＡ）及Ｕ４６６１９诱导的人血小板聚集，其ＩＣ５０分别是１．２，１．３及１．６．．     

RP─HPLC法测定兔血浆中安乃近及其活性代谢物的浓度

本文以２－萘磺酸钠为内标，建立了ＲＰ－ＨＰＬＣ法测定兔血浆中安乃近及其３种活性代谢物ＦＡＡ、ＡＡ、ＭＡＡ的浓度。Ｓｈｉｍ－ＰａｃｋＣＬＣ－ＯＤＳ（１５ｃｍ×６ｍｍＩＤ）为分析柱；ＯＤＳ预柱；流动相组成：甲醇－水－０．５ｍｏｌ／Ｌ磷酸二氢钠－三乙胺（３５：６３：２：０．０１），磷酸调节ｐＨ６．０±０．５；流速：１．０ｍｌ／ｍｉｎ；柱温：３５℃；检测彼长：２６０ｎｍ。本法简便、快速、准确、灵敏，适用于安乃近及其活性代谢物的血药浓度测定及药物动力学研究。     

利哌利酮

利哌利酮（Ｒｉｓｐｅｒｉｄｏｎｅ）３－［２－［４－（６－氟－１，２－苯并异唑－３－基）－１－哌啶基］乙基］－６，７，８，９－四氢－２－甲基－４Ｈ－吡啶并［１，２－ａ］嘧啶－４－酮Ｃ２２Ｈ２７Ｎ４Ｏ２Ｆ（３９５．５）ｍｐ１７０．０℃抗精神病药，对急、慢...     

四氢吡喃类药物对血小板激活因子诱导的脑微血管平滑肌细胞DNA合成及增殖的拮抗作用

研究了血小板激活因子（ＰＡＦ）对兔血小板聚集，牛脑微血管平滑肌细胞ＤＮＡ合成及增殖的影响及四氢吡喃类药物ｔｒａｎｓ－２，６－ｂｉｓ－（３，４－ｄｉｍｅｔｈｏｘｙ－ｐｈｅｎｙｌ）－ｔｅｔｒａｈｙｄｒｏ－（４Ｈ）ｐｙｒａｎ（ＳＺ－１）和ｔｒａｎｓ－２－（３，４，５－ｔｒｉｍｅｔｈｏｘｙｐｈｅｎｙｌ）－６－（２，４－ｄｉｆｌｕｏ－ｒｏｐｈｅｎｙｌ）－ｔｅｔｒａｈｙｄｒｏ－（４Ｈ）ｐｙｒａｎ（ＤＦＴＭ）的拮抗作用．结果表明：ＰＡＦ强烈刺激兔血小板聚集，在１．９１μｍｏｌ·Ｌ－１时，刺激的百分率为７１．７％．ＳＺ－１和ＤＦＴＭ剂量依赖性地抑制ＰＡＦ刺激的兔血小板聚集，ＩＣ５０分别为０．３９和０．８４ｎｍｏｌ·Ｌ－１．ＰＡＦ还刺激牛脑微血管平滑肌细胞增殖，在０．１ｎｍｏｌ·Ｌ－１时作用４８ｈ达最大效应．ＳＺ－１和ＤＦＴＭ显著抑制血小板激活因子的上述作用，在１ｎｍｏｌ·Ｌ－１时抑制率分别为２５．９％和３０．７％．ＳＺ－１和ＤＦＴＭ还抑制ＰＡＦ刺激的牛脑微血管平滑肌细胞ＤＮＡ合成，在１ｎｍｏｌ·Ｌ－１时抑制率分别为２９．１％和２４．４％．实验结果表明：ＰＡＦ可能通过促进血小板聚集，刺激脑血管平滑肌细胞增殖及ＤＮＡ合成而参与?     

Two enzymes for the detoxication of organophosphorus compounds—Sources,similarities, and significance

An enzyme in E. coli that hydrolyzes diisopropylphosphorofluoridate (DFP) has now been found to hydrolyze the nerve gas 1,2,2-trimethylpropylmethylphosphonofluoridate (soman) many times faster. With either substrate the E. coli enzyme is stimulated manyfold by 10⁻³ m Mn²⁺. These criteria are combined and applied to this, and to a superficially similar but distinctly different, enzyme found in squid nerve. The results suggest that while several tissues of the squid contain only this second kind of DFP hydrolyzing enzyme, termed squid type DFPase, many other sources including E. coli contain a mixture of squid type DFPase (the name not strictly indicative of source) and the other DFP hydrolyzing enzyme, now termed Mazur type DFPase. Procedures for the purification of Mazur type DFPase from hog kidney, while increasing the specific activity for DFP hydrolysis may actually have been enriching the purified material in the squid type DFPase. Because E. coli has the highest soman hydrolyzing capacity of any source so far examined, this organism is a promising source for the development of new purification procedures for Mazur type DFPase.     

In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase)

Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization--time of flight--mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD(50) dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose.     

Alteration of hepatic carboxylesterase activity by soman: inhibition in vitro and enhancement in vivo

1. Hydrolysis of the drug esters procaine, chloramphenicol succinate, and prednisolone succinate was studied. Addition of soman to guinea pig liver microsomes caused a dose-dependent inhibition of hydrolysis of all three substrates; at the highest soman concentration (1 microM), ester hydrolysis was totally abolished. 2. Ester hydrolysis was also measured in liver microsomes from guinea pigs pretreated with soman at a low dose (10% of LD50) or at a high dose (90% of LD50) either 1 h or 12 h before killing. Plasma-cholinesterase activity was decreased in all pretreated animals. Liver carboxylesterase activity, measured with the three drug substrates and by hydrolysis of 4-nitrophenyl acetate was increased by all pretreatments. 3. This enhancing effect varies with the substrate and increases with dose of soman. The 12 h pretreatment produced a greater increase in activity than did the 1 h pretreatment. 4. These studies indicate that soman is a potent inhibitor of carboxylesterase activity in vitro but increases the activity of the liver enzyme when administered in vivo.     

人 G类有机磷毒剂水解酶的性质(英文)

观察了部分纯化的人肝 G类毒剂水解酶 ( G酶 )的一些生化性质 .二硫苏糖醇 ( 5mmol·L-1)使G酶活性在 37℃ ,30 min内抑制 35% ;对氯汞苯甲酸 ( 1 - 1 0 0 0 μmol· L-1)不影响 G酶活性 .说明二硫键对酶分子的三维结构至关重要 ,而没有游离巯基参与酶的催化反应 .人肝 G酶专一性地水解带P- F键的有机磷化合物梭曼 ,但不催化带 P- O,P- C或 P- S键的有机磷化合物的反应 .     

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP)

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.     

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.     

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.     

Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge

The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20-95%. Additional perfusion with 10nM soman for 30min resulted in a residual activity of 1-5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon.     

Biomarkers of low-level exposure to soman vapor: comparison of fluoride regeneration to acetylcholinesterase inhibition

The nerve agent O-pinacolyl methylphosphonofluoridate, also known as soman or by its military designation GD, is a highly toxic organophosphorous compound that exerts its effects through inhibition of the enzyme acetylcholinesterase (AChE). In the present study, a fluoride ion based regeneration assay was developed to quantify the level of soman present in the blood of rats following a low-level whole-body inhalation exposure. It was hypothesized that the amount of regenerated nerve agent in the blood would be dose dependent in rats subjected to a whole-body inhalation exposure to a low-level dose of soman vapor, and that the fluoride ion-based regeneration method would be more sensitive for the detection of a low-level exposure to soman vapor than the measurement of whole blood AChE activity. Regenerated soman was dose-dependently detected in both the red blood cells (RBCs) and plasma of exposed rats at all concentrations tested (0.033-0.280 mg/m(3) for a 240-min exposure). Significant inhibition of whole blood AChE activity did not occur below a concentration of 0.101 mg/m(3), and was only depressed by approximately 10-25% at concentrations ranging from 0.101 mg/m(3) to 0.280 mg/m(3). This study is the first to utilize a fluoride ion-based regeneration assay to demonstrate the dose-dependent increases in soman in the blood following whole-body inhalation exposure to low levels of vapor. Additionally, the results of the present study demonstrate that the fluoride ion based regeneration assay was approximately threefold more sensitive than the measurement of AChE activity in the blood for the detection of exposure to soman, and also that miosis is a more sensitive marker of soman exposure than inhibition of AChE activity.     

In vivo protection against soman toxicity by known inhibitors of acetylcholine synthesis in vitro

Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of administration, dose-response relationships, and time to effect.