Muscular irritation study of gadobenate dimeglumine formulation (E7155) in rabbits

To examine the local muscular irritation potency of gadobenate dimeglumine formulation (E7155), E7155 was injected into the right vastus lateralis muscle of male Kbl:JW rabbits, and saline as the negative control was injected into the left muscle. Half of the animals were subjected to necropsy at 2 or 14 days after administration. The muscles were examined macroscopically and histopathologically. Also, 0.425 w/v% and 1.7 w/v% acetic acid solutions were used as a positive control. In macroscopic observation, hemorrhage with white or brown coloration was seen in the muscles treated with E7155 at 2 days after administration, and white coloration was seen in one case at 14 days after administration. In histopathological examination, slight or moderate hemorrhage, edema, cellular infiltration, degeneration of muscle fibers and necrosis of muscle fibers were seen in the muscles treated with E7155 at 2 days after administration, and very slight to slight cellular infiltration, degeneration of muscle fibers, fibrosis, calcification of muscle fibers and foreign body giant cells were seen in the muscles treated with E7155 at 14 days after administration. The changes in the muscle caused by E7155 were definitely less than those caused by the 1.7 w/v% acetic acid solution at both 2 and 14 days, and slightly less and definitely less than those caused by the 0.425 w/v% acetic acid solution at 2 days and 14 days after administration, respectively. The changes caused by E7155 were more severe than those caused by saline. It was concluded that the local muscular irritation potency of E7155 could be classified at Grade 2.     

Mutagenicity study of gadobenate dimeglumine formulation (E7155) (3)--Micronucleus test in rat bone marrow cells

The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the micronucleus test in rats. Single intraperitoneal injection of E7155 to Sprague Dawley rats at the dose of 5295.2 mg/kg (5 mmol/kg) did not induce any statistically significant increase in the frequency of micronucleate cells in the bone marrow sampled after 18, 42 and 66 hr from time of administration.     

Mutagenicity study of gadobenate dimeglumine formulation (E7155) (2)--Chromosome aberration test with human lymphocytes in culture

The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the chromosome aberration test in cultured human lymphocytes. Human lymphocytes were exposed to E7155 at 0.078-10 mM both in the presence and absence of S9 mix derived from rat livers. Three dose levels (2.5-10 mM) were selected for the metaphase analysis. E7155 induced no increase in the incidence of aberrant cells or polyploid cells in any treatments both in the presence and absence of metabolic activation. Thus, it is concluded that E7155 has shown no evidence of clastogenic or polyploidy-inducing activity under these experimental conditions.     

General toxicity study of gadobenate dimeglumine formulation (E7155) (1)--single dose intravenous and intracisternal toxicity study in rats

Gadobenate dimeglumine formulation (E7155) was evaluated for its general toxicity potential following a single intravenous and intracisternal administration to rats. Dosage levels tested were 3.3, 4.5, 6.0 and 8.0 mmol/kg at the injection rate of 6 ml/min and 7.50, 8.89, 10.54 and 12.50 mmol/kg at 1 ml/min for the intravenous administration route, and 0.15, 0.21, 0.29 and 0.40 mmol/kg for the intracisternal administration route. Parameters measured during the 14-day observation period were mortality, clinical signs and macroscopic examination. After intravenous administration at the injection rate of 6 ml/min, twitches, respiratory blocking and prostration were observed at 6.0 mmol/kg, and dyspnoea and sedation at 3.3 and 4.5 mmol/kg. Deaths occurred within 1 min after administration at 6.0 mmol/kg and above. LD50 values were 7.97 mmol/kg in males and 6.22 mmol/kg in females. After intravenous administration at the injection rate of 1 ml/min, shallow breathing, twitches and sedation were observed at 7.50 mmol/kg and above and respiratory arrest at 8.89 mmol/kg. Deaths occurred within 1 min after administration at 8.89 mmol/kg and above. LD50 values were 9.0 mmol/kg in males and 9.7 mmol/kg in females. After intracisternal administration, symptoms consisted of sedation, staggering gait, dyspnoea, twitches and ataxia at 0.15 mmol/kg and above, prostration, paralysis of forelimbs, and/or hind limbs and chromodacryorrhea at 0.21 mmol/kg, and convulsions at 0.29 mmol/kg and above. Deaths occurred within 7 days after administration at 0.21 mmol/kg and within 5 min at 0.29 mmol/kg and above. LD50 values were 0.42 mmol/kg in males and 0.25 mmol/kg in females.     

General toxicity study of gadobenate dimeglumine formulation (E7155) (2)--Single dose intravenous toxicity study in dogs

Gadobenate dimeglumine formulation (E7155) was given by single intravenous injection to 4-5 month-old beagle dogs at doses of 2 or 6 mmol/kg. Treatment was followed by a 14-day observation period in order to evaluate the test article's toxicity. The male and female dogs at 6 mmol/kg vomited and showed reddened gums and ears as clinical signs. One male dog at 6 mmol/kg was euthanized approximately 23 hr after administration due to its very poor clinical condition, which included an unwillingness to move, pale gums and weak pulse. Body weight was decreased at 6 mmol/kg, and also slightly at 2 mmol/kg. Decreased food consumption was noted both at 2 and 6 mmol/kg. Hematology for the euthanized male at 6 mmol/kg showed increases in the total white blood cell count, packed cell volume, hemoglobin and red cell count and a decrease in the platelet count. Biochemistry showed a dose-related increase in alkaline phosphatase, GPT and GOT at 2 and 6 mmol/kg. Males and females at 6 mmol/kg showed increases in bilirubin, calcium and urea, and a reduction in glucose. Females at 6 mmol/kg also showed a reduction in total protein. Urinalysis showed an increase in pH at 2 mmol/kg and above. For females at 6 mmol/kg, an increase in urine volume and a decrease in specific gravity and osmolality were noted. An increase in relative liver and kidney weights was recorded for males and females dosed at 6 mmol/kg. For the euthanized male at 6 mmol/kg, postmortem examination revealed a pale liver with rounded edges and an accentuated lobular pattern, and dark material on the gastro-intestinal mucosal surface. In macroscopic pathology, the male at 6 mmol/kg revealed single liver cell necrosis, minimal early hyperplasia in small biliary ductules, inflammatory cells in the sinusoidal and portal tracts, centrilobular inflammatory cells, diffuse vacuolation of the hepatocytes and sinusoidal dilatation in the liver, and cortical tubular vacuolation in the kidneys. In the female dog treated at 6 mmol/kg, hyperplasia in the small biliary ductules, inflammatory cells in the portal tracts, diffuse vacuolation of hepatocytes and sinusoidal dilatation were seen in the liver, and increases in the severity of cortical tubular basophilia, cortical tubular dilatation and cortical tubular casts were detected in the kidney. Based on these results, the lethal dose of E7155 was set at 6 mmol/kg. It is also concluded that a dose of 2 mmol/kg was tolerated in the beagle dog after a single injection followed by a 14-day observation period.     

Reproductive and developmental toxicity study of gadobenate dimeglumine formulation (E7155) (1)--Fertility study in male rats by intravenous administration

The influence of gadobenate dimeglumine formulation (E7155) on general reproductive performance and fertility in male rats of the Sprague-Dawley strain was assessed in this study. E7155 was administered by intravenous injection at a dosage of 0.3, 1.0, or 2.0 mmol/kg/day to groups of 22 male rats for 13 weeks. Control animals received 0.9% sterile physiological saline throughout the same period. After four weeks of treatment, each male was paired with an untreated female of the same strain. Each male was paired again after 10 weeks of treatment with another untreated female of the same strain. All females were killed on Day 14 of gestation for examination of pregnancy status. No significant toxicological signs associated with systemic exposure to E7155 were observed. There were no effects of treatment with E7155 on body weight gain, food consumption, macroscopic findings, reproductive organ weights and sperm count or sperm motility in male rats. Mating performance after pairing at Weeks 4 and 10 of treatment as well as litter size and number of survival embryos on Day 14 of gestation were not affected by paternal treatment with E7155. From these results, the No Observed Adverse Effect Level (NOAEL) of E7155 was 2.0 mmol/kg/day for general and reproductive toxicity parameters in male rats treated with E7155 and for development in their embryos.     

Reproductive and developmental toxicity study of gadobenate dimeglumine formulation (E7155) (3)--Study of embryo-fetal toxicity in rabbits by intravenous administration

Gadobenate dimeglumine formulation (E7155) was daily administered by intravenous injection at 0.3, 0.9 or 2.0 mmol/kg/day to mated NZW female rabbits (20/group) to assess the effect on embryo-fetal development. Treatment with 2.0 mmol/kg/day caused initial, notable loss of body weight and reduction in food consumption. Slightly reduced body weight gain and food intake were recorded at 0.9 mmol/kg/day. There were no obvious adverse effects in dams given E7155 at 0.3 mmol/kg/day. There was a slightly higher incidence of early intrauterine deaths at 0.9 and 2.0 mmol/kg/day. Morphological examination of fetuses at 2.0 mmol/kg/day revealed small eye/microphthalmia and/or retinal irregularities in three fetuses from three separate litters. There was also an increase in the incidence of additional and/or fused sternebral centres and 20-thoracolumbar vertebrae at this dosage. From these results, the No Observed Adverse Effect Level (NOAEL) for general toxicity of dams and embryo-fetal development was 0.3 mmol/kg/day.     

Reproductive and developmental toxicity study of gadobenate dimeglumine formulation (E7155) (2)--Combined study of effects on fertility and embryo-fetal toxicity in female rats by intravenous administration

The influence of gadobenate dimeglumine formulation (E7155) on fertility and general reproductive performance and embryo-fetal development was assessed in female Sprague-Dawley rats. E7155 was administered by intravenous injection at a dose of 0.3, 1.0 or 2.0 mmol/kg/day to groups of 22 female rats for 15 days before pairing. Treatment was continued throughout mating and up to Day 17 of gestation. Control animals received 0.9% sterile physiological saline throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents. There were no toxic clinical signs of treatment. The body weight and food consumption of females before pairing and during gestation were not affected by treatment. Estrous cycles, mating performance, litter size and fetal weight, survival and development were also not affected by treatment. Based on the above results, the No Observed Adverse Effect Level (NOAEL) of E7155 was 2.0 mmol/kg/day for general toxicologic effects and reproduction of female rats and the development of their fetuses.     

General toxicity study of gadobenate dimeglumine formulation (E7155) (4)--4-week repeated dose intravenous toxicity study followed by 4-week recovery period in dogs

Gadobenate dimeglumine formulation (E7155), at doses of 0 (physiological saline), 0.25, 0.5, 1 and 2 mmol/kg/day of body weight, was administered intravenously to male and female beagle dogs once daily for 4 consecutive weeks in order to evaluate the subacute toxicity of the test article. Reversibility of toxicity was evaluated during a 4-week recovery period at 1 and 2 mmol/kg/day. No toxicologically significant changes were observed at 0.25 and 0.5 mmol/kg/day. In animals receiving 1 or 2 mmol/kg/day, transient swelling and redness of the facial and eye areas, lethargy, decreased activity, emesis, retching, watery or unformed stool, decreased body weight or body weight gain, decreased food consumption, decreased hematocrit and hemoglobin concentration, increased APTT, increases in plasma ALP, GPT or gamma-GT, decreased plasma inorganic phosphorus, total protein or albumin, increased liver or kidney weight, subacute inflammatory infiltrates, loss of centrilobular hepatocytes or hepatocellular cytoplamic vacuolation in the liver, vacuoles in the epithelial cells of the renal tubles and/or hypocellularity in the bone marrow were seen. The results of toxicokinetic analysis showed that systemic exposure was similar in males and females, and there was no accumulation of the test material over the treatment period, although AUC tended to be enhanced by slightly more than the proportionate dose increase. These effects were recovered or tended to be reversed after a post-dosing period for 4 weeks. In conclusion, the No Observed Adverse Effect Level (NOAEL) was 0.5 mmol/kg/day.     

General toxicity study of gadobenate dimeglumine formulation (E7155) (3)--4-week repeated dose intravenous toxicity study followed by 4-week recovery period in rats

A 4-week repeated dose toxicity study of gadobenate dimeglumine formulation (E7155) was conducted in Sprague-Dawley rats to assess its non-clinical safety. E7155 was administered intravenously at doses of 0.3, 1.0 and 3.0 mmol/kg/day to male and female rats once a day during 4 weeks. The reversibility of toxicity was evaluated during a 4-week recovery period at 3.0 mmol/kg/day. At 0.3 mmol/kg/day and higher, vacuolation of the cortical epithelium was seen in the kidneys and an increase in the incidence of local damage at the injection sites. In the 1.0 and 3.0 mmol/kg/day male and female groups, scabbing/ulceration of the tail at the injection sites, macroscopic pale/thickened fundic mucosa in the stomachs, vacuolation of the urinary bladder, and mucosal mineralization with epithelial hyperplasia of the glandular stomach were found. In the 1.0 and 3.0 mmol/kg/day male group and 3.0 mmol/kg/day female group, increases of water consumption and urinary potassium excretion, increased kidney weight and enlargement of the kidneys were observed. In the 3.0 mmol/kg/day male and female group, hepatocyte necrosis with inflammatory cells in the liver and epithelial degranulation in the interlobular ducts of the salivary glands were found. In addition, in the 3.0 mmol/kg/day male group, increases in plasma sodium and decreases of urinary sodium and chloride excretion, and degenerative changes in the testes and epididymides were observed. After the 4-week recovery period, except for an increase in urinary potassium excretion, increased kidney weights and changes in the testes and epididymides, all of the above findings had complete or partial recovery. Vacuolation of renal tubular cells was common, expected, and known as an adaptive change of treatment with hypertonic solutions, and an increase in the incidence of local damage at the injection sites was due to irritation by repeated intravenous dosing with hypertonic solutions. Therefore, these changes were not toxic changes. In conclusion, the dose level of 0.3 mmol/kg/day should be regarded as the No Observed Adverse Effect Level (NOAEL) after repeated administration of E7155 in rats.     

Mutagenicity study of nine monoalkyl phthalates and a dialkyl phthalate using Salmonella typhimurium and Escherichia coli

Nine monoalkyl (C1-C8) phthalates and di-(2-ethylhexyl) phthalate (DEHP) were assayed for mutagenicity in two strains of Salmonella typhimurium (TA98 and TA100) and two strains of Escherichia coli WP2 try- (uvrA+ and uvrA-) with and without metabolic activation with S-9 mix. The procedure of Ames et al. (Mutation Res. 1975, 31, 347) was used, with minor modifications. None of the compounds tested showed any mutagenic activity, but all the monoalkyl phthalates showed some lethality towards the S. typhimurium strains, the most toxic being monoheptyl phthalate. A marginally lethal effect on the Salmonella strains was shown by DEHP, but only at the highest concentration tested (2000 micrograms/plate) and in the absence of S-9 mix.     

Inhibition of the mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine and 9-Aminoacridine by indenopyridines in the Salmonella typhimurium tester strain 1537 and E. coli

Abstract

The goal of the present research was to determine the protective potential of five newly synthesized indenopyridine derivatives against N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 9-aminoacridine (9-AA) induced mutagenesis. MNNG sensitive Escherichia coli WP2uvrA and 9-AA sensitive Salmonella typhimurium TA1537 were chosen as the bacterial tester strains. All of the test compounds showed significant antimutagenic activity at various tested concentrations. The inhibition rates ranged from 25.6% (Compound 2 - 1?mM/plate) to 68.2% (Compound 1 - 2.5?mM/plate) for MNNG and from 25.7% (Compound 4 - 1?mM/plate) to 76.1% (Compound 3 - 2.5?mM/plate) for 9-AA genotoxicity. Moreover, the mutagenicity of the test compounds was investigated by using the same strains. None of the test compounds has mutagenic properties on the bacterial strains at the highest concentration of 2.5?mM. Thus, the findings of the present study give valuable clues to develop new strategies for chemical prevention from MNNG and 9-AA genotoxicity by using synthetic indenopyridine derivatives.     

Mutagenicity of 4-aminobiphenyl and 4-acetylaminobiphenyl in Salmonella typhimurium strains expressing different levels of N-acetyltransferase.

4-Aminobiphenyl (4-ABP), an aromatic amine present in tobacco smoke, is an animal and human carcinogen. 4-ABP can undergo several biotransformation reactions to yield DNA-binding species. The role of acetylation in the biotransformation of 4-ABP to reactive intermediates was investigated by determining mutagenicity in Salmonella typhimurium strains expressing various levels of acetyltransferases (NAT/OAT). Strain YG1029, which has multiple copies of the NAT/OAT gene, was the most sensitive to 4-ABP. With rat S9 activation, 4-ABP (5 micrograms/plate) induced 789 +/- 98 revertants/plate. At that concentration, an average of 200 revertants/plate was seen in both TA100, which has a single copy of the NAT/OAT gene, and in TA100/1,8DNP6, which is NAT/OAT deficient. This pattern was also present when the bacteria were exposed to the acetylated derivative, 4-acetylaminobiphenyl (4-AABP). At 10 micrograms/plate, 4-AABP induced 855 +/- 47 revertants/plate in YG1029 while 169 +/- 39 and 149 +/- 28 revertants/plate were observed in strains TA100 and TA100/1,8DNP6, respectively. The mutagenic profiles of 4-ABP and 4-AABP observed with the mouse S9 activating system were similar to that seen with the rat. These data establish a correlation between increased bacterial NAT/OAT activity and increased mutagenicity of 4-ABP. Results with both 4-ABP and 4-AABP support acetylation of the oxygen to be a key step in activation.     

Mutagenicity of cytochrome P450 2E1 substrates in the Ames test with the metabolic competent S. typhimurium strain YG7108pin3ERb5

Poor metabolic competence of in vitro systems was proposed to be one of their major shortcomings accounting for false negative results in genotoxicity testing. For several "low molecular weight cancer suspects" this was specifically attributed to the lack of cytochrome P450 2E1 (CYP2E1) in conventional in vitro metabolising systems. One promising attempt to overcome this problem is the transfection of "methyltransferase-deficient"S. typhimurium strains with the plasmid pin3ERb5. This plasmid contains DNA encoding for a complete electron transport chain, comprising P450 reductase, cytochrome b5 and cytochrome P450 2E1. In order to answer the question if CYP2E1 substrates that yield negative or inconclusive results in the Ames test can be activated by metabolic competent bacterial strains, we used YG7108pin3ERb5 to investigate the following compounds: acetamide, acrylamide, acrylonitrile, allyl chloride, ethyl acrylate, ethyl carbamate, methyl-methacrylate, vinyl acetate, N-nitrosopyrrolidine, trichloroethylene and tetrachloroethylene. N-Nitrosodiethylamine served as a positive control. In addition to these known or proposed CYP2E1 substrates, we investigated the polycyclic aromatic hydrocarbon benzo[alpha]pyrene and the heterocyclic aromatic amines 2-aminofluorene and 2-aminoanthracene. RESULTS: The extensive metabolic competence of the transformed strain is underlined by results showing strong mutagenicity between 10 and 500 micro g N-nitrosopyrrolidine per plate. Unexpectedly, 2-aminoanthracene was mutagenic at a concentration range between 25 and 250 micro g per plate using YG7108pin3ERb5. Moreover, we demonstrate for the first time a clear response of sufficiently characterised allyl chloride in the Ames test at a reasonably low concentration range between 300 and 1500 micro g per plate. We achieved similar results in the parent strain YG7108 with conventional metabolic activation. Without metabolic activation less pronounced mutagenicity occurred, suggesting a contribution of a direct alkylating effect. Propylene oxide is usually contained in allyl chloride as stabilizer at amounts up to 0.09%. Though YG7108 revealed to be very sensitive towards propylene oxide, allyl chloride dissolved in water was not mutagenic, showing that no water soluble compounds contribute to its mutagenicity. None of the remaining compounds showed mutagenic effects using YG7108pin3ERb5. CONCLUSION: YG7108pin3ERb5 and its parent strain YG7108 are sensitive for compounds which are negative in conventional tester strains including N-nitrosodiethylamine, N-nitrosopyrrolidine, propylene oxide and allyl chloride.     

Mutagenicity of N-nitrosodiethylamine in the Ames test with S. typhimurium TA1535 is due to volatile metabolites and is not dependent on cytochrome P4502E1 induction

N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.     

重组人PD-L1在大肠杆菌BL-21中的表达、纯化和鉴定

目的 分离纯化人程序死亡蛋白-1(PD-LI)基因,制备重组人PD-L1蛋白,为进一步研究PD-L1在肿瘤免疫逃逸中的作用和机制建立基础.方法 通过RT-PCR分离纯化人PD-L1基因,并将其克隆到原核表达质粒pGEX-4T一1中,经酶切,测序鉴定分析后,转化大肠杆菌BL21(DE3),经IPTG诱导表达.产物经GST蛋白纯化系统进行纯化后,用10%SDS-PAGE及Western Blot分析鉴定.结果 经RT-PCR分离纯化的PD-L1 cDNA分子由645 bp构成,编码相对分子量约25 KD的蛋白,并与GST形成融合蛋白.GST-PD-L1融合蛋白相对分子量约46 KD.Western Blot结果显示,该蛋白能被兔抗GST和抗PD-L1抗体识别.结论 成功构建了PD-L1原核表达载体,并在大肠杆菌BL21中获得高效表达.     

汉坦病毒S基因在大肠埃希菌中的克隆及表达

目的：在大肠埃希菌中克隆和表达汉坦病毒SEO型及HTN型S基因。方法：PCR扩增汉坦病毒SEO型L99株及HTN型Z10株S基因读码区，前者经EcoRI及XhoI双酶切，后者经SacI及XhoI双酶切，将两者定向克隆入pET32a（＋），转入感受态E．coli Top10。获取重组质粒，经PCR、双酶切及序列分析鉴定后，转入E．coli BL21感受态。经IFTG诱导表达，其产物经SDS-PAGE和Western-blot检测。结果：SEO型L99株及HTN型Z10株S基因正确克隆于pET32a（＋），目的蛋白的分子质量约为67．1ku，表达量占菌体总蛋白的40％以上，Western-blot有特异性条带。结论：SEO型及HTN型汉坦病毒S基因在大肠埃希菌中获得表达，为其后的应用奠定了基础。     

A chromatographic method for the quantification of prostaglandin E(1) and prostaglandin A(1) encapsulated in an intravenous lipid formulation

Peripheral vascular disease is a common ailment of the aged and diabetic communities. As the numbers of these individuals increase, the need for therapeutic interventions will continue to grow. One of the possible therapies is the use of prostaglandins (PGE(1), prostacyclin and Iloprost) to decrease the vascular tone and increase vascular blood flow. Due to the hydrophobicity of the prostaglandins and prostaglandin analogues, various vehicles have been utilized to maintain the active pharmaceutical ingredient in a stable solution, e.g. alpha-cyclodextrin (Alprostadil, Edex) or emulsified lipid vehicles. In our laboratory, we designed a method for separating and assaying lipid-encapsulated PGE(1). Utilizing organic extraction, automated solid-phase extraction and precipitation techniques, we validated the measurement of the PGE(1) and PGA(1) content of the clinical drug formulation in the microgram per milliliter concentration range with an high performance liquid chromatography (HPLC) assay.