Toxicity of 1,1′-alkyl-4,4′-Bipyridylium Salts in the Rat

ABSTRACT

Methyl (paraquat), propyl, isopropyl, butyl, methyl-pentyl, hexyl, octyl and benzyl viologens. (1,1′-Alkyl-4,4′-bipyridylium salts) were administered subcutaneously to female Sprague-Dawley rats to determine relative toxicities. These compounds all produce the spectrum of effects previously reported for paraquat and additionally produce a focal necrosis at the injection site, non-emptying of the stomach and adrenal enlargement. A lethal dose of propyl, hexyl or benzyl viologen often produced a yellow to red serous fluid in the pleural cavity. Many of the signs observed with viologen poisoning are similar to adrenal hormone effects and the suggestion is made that the adrenals may be contributing to toxicity.     

Promotion of Altered Hepatic Foci by 2,3′,4,4′,5-Pentachlorobiphenyl in Sprague–Dawley Female Rats

The tumor promotion potential of 2,3′,4,4′,5-pentachlorobiphenyl (PCB-118) was studied in a two-stage initiation/promotion bioassay in female Sprague–Dawley rats. The animals were initiated by intraperitoneal administration ofN-nitrosodiethylamine after partial hepatectomy. After 5 weeks of recovery, the promotion period commenced by once-weekly subcutaneous administrations of PCB-118 at six dose levels (10, 40, 160, 640, 2500, and 10,000 μg/kg body weight/week) for 20 weeks. In addition, three of these dose levels (40, 640, and 10,000 μg/kg body weight/week) were administered for 52 weeks. Evaluation of hepatic foci positive for glutathione S-transferase P demonstrated that the mono-orthochlorine substituted congener PCB-118 significantly increased the number of foci/cm³of liver in the two highest dose groups after 20 weeks, but did not significantly increase the percentage of the liver occupied by foci. After 52 weeks of treatment, both the percentage and the number of foci/cm³were significantly increased in the highest dose group. A toxic equivalency factor based on foci development during 20 weeks of treatment would be less than 0.00002. Altered relative liver and thymus weights were observed after treatment with both substances as well as an induction of methyl cholanthrene- and phenobarbital-inducible isoenzymes of cytochrome P450 monooxygenase. These results show that PCB-118 has a potency to enhance foci growth in rat liver, although the potency is low compared to that of structurally related compounds.     

Tissue distribution metabolism and excretion of 2,2′,4,4′, 5-pentachlorodiphenyl ether in the rat

The tissue distribution, metabolism and excretion of ¹⁴C-2,2,4,4,5-pentachlorodiphenyl ether (PCDE) were studied in the rat. Radioactivity was distributed in all tissues examined, with the highest concentrations being found in the fat followed by the skin, liver, kidney and muscle. Most of the radioactivity found in the tissues was due to unchanged PCDE. Decay of PCDE in the blood was fitted to a four-compartment pharmacokinetic model, and the last compartment had a half-life of 5.8 days. A total of 55% and 1.3% of an orally administered dose was excreted in feces and urine, respectively, in 7 days. More than 64% of the fecal radioactivity was due to unchanged PCDE, while hydroxylated PCDE accounted for 23%.     

Tissue disposition,excretion and metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) in the male Sprague-Dawley rat

1. A disposition, metabolism and excretion study of orally administered 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) was conducted in the conventional and bile duct-cannulated male rat. 2. In the conventional rat, >50% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition, i.e. adipose tissue, adrenals, gastrointestinal tract and skin. 3. Urinary excretion of BDE-99 was very low (<1% of dose), and glucuronidation of phenolic metabolites was suggested. 4. Biliary excretion of BDE-99 was slightly greater than observed in urine, i.e. 3.6% at 72h. 5. Over 43% of the dose in the conventional male rat and 86% in the bile duct-cannulated rat was excreted in the faeces, mainly as the unmetabolized parent compound. 6. Metabolites in bile and faeces were not conjugated. Mono- and di-hydroxylated pentabromodiphenyl ether metabolites were characterized by mass spectrometry. Two thiol metabolites were characterized in the bile. Oxidative debromination was also observed in the faecal metabolites. 7. Tissue BDE-99 was readily extractable, except for in the liver. The tissue ¹⁴C was not associated with lipids and was mainly the unmetabolized parent compound. 8. Total thyroxine (T4) plasma levels were elevated at 3 and 6 days, and returned to control levels by day 12.     

Reversal by 3,3′,5-triido-l-thyronine of the working memory deficit,and the decrease in acetylcholine,glutamate and γ-aminobutyric acid induced by ethylcholine aziridinium ion in mice

Summary The effect of 3,3,5-triiodo-l-thyronine (T₃) on working memory in ethylcholine aziridinium ion (AF64A)-treated mice was studied in a delayed nonmatching to sample task using a T-maze. After behavioural testing was completed, mice were killed by microwave irradiation and regional brain levels of acetylcholine, aspartate, glutamate, glutamine, glycine, taurine, and -aminobutyric acid (GABA) were measured by high-performance liquid chromatography with electrochemical detection. Treatment with AF64A (7 nmol, i. c. v.) produced a deficit in working memory performance in the non-matching to sample task at 30 s delay, and decreased acetylcholine, glutamate, and GABA levels in the hippocampus, but not in the septum and cerebral cortex. Administration of T₃ (0.3 mg/kg, p.o., once daily for 6 days) to AF64A-treated animals improved the deficit in working memory performance and reversed the decrease in acetylcholine, glutamate, and GABA levels in the hippocampus. These results indicate that the deficit in performance induced by AF64A can be improved by T₃ administration. Correspondence to: E. Abe at the above address     

Uranyl Nitrate: 28-Day and 91-Day Toxicity Studies in the Sprague–Dawley Rat

Although uranium (U) is a classic experimental nephrotoxin, there are few data on its potential long-term chemical toxicity. These studies were undertaken to derive a no-observed-adverse-effect level (NOAEL) in male and female Sprague–Dawley rats following 91-day exposure to uranium (as uranyl nitrate hexahydrate, UN) in drinking water. Following a 28-day range-finding study, five groups of 15 male and 15 female weanling rats were exposed for 91 days to UN in drinking water (0.96, 4.8, 24, 120, or 600 mg UN/L). A control group was given tap water (<0.001 mg U/L). Daily clinical observations were recorded. Following the study, animals were euthanized and exsanguinated, and multiple hematological and biochemical parameters were determined. Necropsies were conducted, and multiple tissues were sampled for histopathological examination. The hematological and biochemical parameters were not affected in a significant exposure-related manner. Although there were qualitative and slight quantitative differences between males and females, histopathological lesions were observed in the kidney and liver, in both males and females, in all groups including the lowest exposure groups. Renal lesions of tubules (apical nuclear displacement and vesiculation, cytoplasmic vacuolation, and dilation), glomeruli (capsular sclerosis), and interstitium (reticulin sclerosis and lymphoid cuffing) were observed in the lowest exposure groups. A NOAEL was not achieved in this study, since adverse renal lesions were seen in the lowest exposed groups. A lowest-observed-adverse-effect level of 0.96 mg UN/L drinking water can be reported for both the male and the female rats (average dose equivalent 0.06 and 0.09 mg U/kg body wt/day, respectively).     

Characterization of the Oral Absorption of β-Lactam Antibiotics. I. Cephalosporins: Determination of Intrinsic Membrane Absorption Parameters in the Rat Intestine In Situ

The oral absorption of five cephalosporin antibiotics, cefaclor, cefadroxil, cefatrizine, cephalexin, and cephradine, has been studied using a single-pass intestinal perfusion technique in rats. Intrinsic membrane absorption parameters, unbiased by the presence of an aqueous permeability (diffusion or stagnant layer), have been calculated utilizing a boundary layer mathematical model. The resultant intrinsic membrane absorption parameters are consistent with a significant carrier-mediated, Michaelis-Menten-type kinetic mechanism and a small passive component in the jejunum. Cefaclor colon permeability is low and does not exhibit concentration dependent behavior. The measured carrier parameters (±SD) for the jejunal perfusions are as follows: cefaclor, J _max ^* = 21.3 (±4.0), K _m = 16.1 (±3.6), P _m ^* = 0, and P _c ^*= 1.32 (±0.07); cefadroxil, J _max ^* = 8.4 (±0.8), K _m = 5.9 (±0.8), P _m ^* = 0, and P _c ^* = 1.43 (±0.10); cephalexin, J _max ^* = 9.1 (±1.2), K _m = 7.2 (±1.2), P _m ^* = 0, and P _c ^* = 1.30 (±0.10); cefatrizine, J _max ^* = 0.73 (±0.19), K _m = 0.58 (±0.17), P _m ^* = 0.17 (±0.03), and P _c ^* = 1.25 (±0.10); and cephradine, J _max ^* = 1.57 (±0.84), K _m = 1.48 (±0.75), P _m ^* = 0.25 (±0.07), and P _c ^* = 1.06 (±0.08). The colon absorption parameter for cefaclor is P _m ^* = 0.36 (±0.06, where J _max ^* (mM) is the maximal flux, K _m (mM) is the Michaelis constant, P _m ^* is the passive membrane permeability, and P _c*is the carrier permeability. Aminocephalosporin perfusion results indicate that jejunal absorption in the rat occurs by a nonpassive process, with some of the compounds possessing a small but statistically significant passive component, while the colon permeability is low and follows a simple passive absorption mechanism.     

Accumulation Kinetics of Propranolol in the Rat: Comparison of Michaelis– Menten-Mediated Clearance and Clearance Changes Consistent with the “Altered Enzyme Hypothesis”

(+)-Propranolol was infused at two rates into the pyloric vein (a portal vein tributary) of 15 male Sprague Dawley rats until apparent steady-state conditions were established (i.e., 8 hr at each rate). One group (n = 7) received the high dose (40 µg/min/kg) first, and in the other group (n = 8) the low dose (20 µg/kg/min) was used to initiate treatment. Free and total serum concentrations of propranolol were measured. When the low dose was given first, the apparent steady-state concentrations achieved during low- and high-rate infusion steps were 166 ± 37 and 774 ± 235 ng/mL, respectively. These data are consistent with a simple Michaelis–Menten kinetic model and the key parameters of such a model (V _max and K _m) were estimated. However, a crucial test of such a model (and one which should give insight regarding the relevance of an altered enzyme hypothesis) is to reverse the order of infusion steps since, in a system controlled by Michaelis–Menten kinetics, the same steady-state concentrations should be achieved regardless of the order in which infusion steps are given. When the sequence of infusion rates was reversed, steady-state concentrations were 492 ± 142 and 298 ± 79 ng/mL for the high and low infusion rates, respectively. Clearly, a history of high-dose exposure reduces the intrinsic clearance of total drug (CL_SS) during a subsequent low-dose exposure (i.e., the apparent steady-state levels during the low-dose pyloric vein infusions were significantly different; P < 0.001). When these data were corrected for plasma protein binding, the same trends emerged. For example, the intrinsic clearance of free drug (CL_Uss) during low dose treatment, when this treatment was given first, was 27.4 ± 7.3 L/min/kg. However, when the low dose was given as the last step, CL_Uss was only 14.4 ± 5.6 L/min/kg. This highly statistically significant decrease in CL_Uss (P < 0.002) is inconsistent with any simple Michaelis–Menten model, but it is consistent with an altered enzyme hypothesis.     

Teratological studies on the TCDD congener 3,3′,4,4′-tetrachloroazoxybenzene in sensitive and nonsensitive mouse strains: Evidence for direct effect on embryonic tissues

The teratogenicity of 3,3,4,4-tetrachloroazoxybenzene (TCAOB), a TCDD congener, was studied in Ah-responsive (C57BL and NMRI) and non-responsive (DBA/2J and AKR/NBom) strains of mice. In the responsive strains, the TCAOB produced cleft palate and hydronephrosis in 50–90% of the offspring at a dose level of 6–8 mg/kg b.w. in the absence of apparent maternal toxicity. Day 11 was shown to be the day of highest sensitivity (palatal closure occurs at day 14) in the C57BL strain. Higher doses (16 mg/kg b.w.) produced high rate of fetal death both in responsive (C57BL; 60%) and non-responsive (DBA; 40%) strains. These doses induced cleft palate in 95% of the surviving C57BL fetuses but failed to do so in the DBA strain. The non-sensitivity of the DBA and AKR strains appeared to segregate as a dominant trait. Backcrosses between NMRI x DBA F₁ generation and NMRI showed an intermediate sensitivity. It was shown that the genotype of the embryo was of ultimate importance for the development of cleft palate. There appeared however to be an additional host (maternal) factor as well, because the offspring of NMRI females mated with NMRI x DBA F₁ males showed a higher rate of cleft palate as compared to those of the crossing between NMRI x DBA F₁ females and NMRI males. Light and scanning electron microscopy indicated that the apical epithelial cells of the secondary palates failed to follow the normal pattern of programmed cell death, suggesting a similar mechanism of pathogenesis as previously described for TCDD.     

Repeated-Dose and Reproductive Toxicity of the Ultraviolet Absorber 2-(3′,5′-Di- tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole in Rats

2-(3′,5′-Di-tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole (DBHCB) is widely used as an ultraviolet (UV) absorber. In this study, the repeated dose and reproductive toxicity of DBHCB was evaluated in rats. Crj:CD(SD)IGS rats were given DBHCB by gavage at 0, 2.5, 25, or 250 mg/kg/d. Male and female rats were dosed beginning 28 d before mating, and each female rat was mated with a male rat of the same dosage group. Males were dosed for a total of 56–57 d, and females were dosed for a total of 55–69 d up to Day 3 of lactation throughout the mating and pregnancy periods. Ten males from each group were killed on the next day of the last administration, and 10 females were killed on Days 4–6 after parturition. Five rats/sex treated at 0 and 250 mg/kg/d for 56 d were then kept without treatment for 14 d (recovery period). No deaths were found in any group. No effects of DBHCB on general condition, body weight, food consumption, or reproductive/developmental parameters were observed. Significant increases in serum albumin and an albumin/globulin ratio at 25 mg/kg/d and higher and alkaline phosphatase levels at 250 mg/kg/d were noted in males. The absolute and relative weights of the liver were significantly increased in males at 25 mg/kg/d and higher. Significantly increased serum albumin and absolute and relative liver weight were also found in males at 250 mg/kg/d after the recovery period. No changes in these parameters were observed in females of any DBHCB-treated groups. No significant changes in organ histopathology were found in males or females. These findings indicated a sex difference in the toxicity of DBHCB in rats.     

Lack of Gender-Related Difference in the Toxicity of 2-(2′-Hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole in Preweaning Rats

In our previous toxicity studies using young rats, we showed that an ultraviolet absorber, 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole (HDBB), principally affected the liver, and male rats had nearly 25 times higher susceptibility to the toxic effects than females. In the present study, the toxicity of HDBB was investigated in preweaning rats. HDBB was administered by gavage to male and female CD(SD) rats from postnatal days 4 to 21 at a dose of 0, 0.1, 0.5, 2.5, or 12.5 mg/kg/day. No substance-related deaths, clinical signs of toxicity, or body-weight changes were observed. Increased levels of albumin, AST and ALP in both sexes, BUN in males, and LDH in females were found at 12.5 mg/kg. Liver weights increased at 2.5 mg/kg and above in both sexes. Histopathologically, hepatocellular findings, such as nucleolar enlargement, anisokaryosis, increased mitosis, and/or hypertrophy, were observed at 2.5 mg/kg and above in both sexes. These results indicate no gender-related differences in the susceptibility to the toxic effects of HDBB in preweaning rats.     

The influence of glutathione on the photoreaction of 3,4′,5-tribromosalicyl-anilide alone and in the presence of serum albumin

Glutathione is known to play a prominent detoxifying role in the organism. In this study attention has been paid to the possible occurrence of a detoxifying action of glutathione in the skin. For this reason we studied the influence of glutathione on the photoreaction of 3,4′,5-tribromosalicylanilide (Tbsa), which is notorious for causing photoallergy. It was found thatTbsa forms stable photoconjugates on irradiation with glutathione: 3-glutathyl-4′,5-dibromosalicylanilide and 5-glutathyl-4′-bromosalicylanilide. The rate of the photoreaction ofTbsa in the presence of glutathione, resulting in photostable products, is increased. Furthermore, covalent binding with serum albumin appeared to be decreased in the presence of glutathione.     

Gender-Related Difference in the Toxicity of Ultraviolet Absorber 2-(3′,5′-Di-tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole in Rats

2-(3′,5′-Di-tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole (DBHCB) is widely used as an ultraviolet absorber. Previously, we showed that male rats had more than a 100 times higher susceptibility to the toxic effects of DBHCB than females. In order to investigate the role of sex steroids in the mediation of this gender-related difference, DBHCB (0 or 250?mg/kg/day) was given to male and female young intact and castrated rats by gavage for 28 days in the current study. In intact rats, relative liver weight increased to more than two times that of the control in males, while the rate of change was less than 10% in females. On histopathology, hypertrophy of hepatocytes was observed in males but not in females. In castrated rats, an approximately 40% increase in the relative liver weight was found only in males, and no histopathological changes in the liver were detected in either sex. The gender-related difference was also determined in preweaning rats administered DBHCB at 0, 250, or 500?mg/kg/day by gavage from postnatal days 4 to 21. Blood biochemical changes, including increases in the levels of AST, ALT, and ALP, 80–95% increase in the relative liver weight and histopathological changes in the liver, such as hypertrophy and single cell necrosis of hepatocytes, were observed at both doses in both sexes. In conclusion, the gender-related difference in the toxicity of DBHCB, which was observed in young rats, was markedly reduced by castration and abolished in preweaning rats.     

Interactions of carbon tetrachloride and promethazine in the rat—I: Effects of promethazine on the concentrations of carbon tetrachloride in blood and liver,and on the production of chloroform

The effects of Promethazine (PM, 78 μmoles/kg body wt, i.p.) on the concentrations of CCl₄ in samples of blood and liver of male, fasted rats after oral dosing with CCl₄ have been determined. With an administered dose of CCl₄ of 13 moles/kg body wt the concentrations of CCl₄ in the blood and liver were measured using gas chromatography with a flame ionisation detector. It was found that Promethazine delayed absorption of CCl₄ from the gastro-intestinal tract by approximately 2 hr as judged by blood levels of CCl₄; the maximum blood concentration (C_max) and the total absorption of ccl₄ (assessed by the area under the plot of blood concentration vs time during the first 6 hr after administration of CCl₄) were not significantly changed by Promethazine treatment. Liver and blood measurements were carried out on each rat in this series and the ratio of the CCl₄-concentrations in liver: blood were found to lie within the range 8–12 when studied in the absence of Promethazine treatment.Gas chromatography with electron capture was used on serial samples of blood from the same rat to measure CCl₄ and CHCl₃ concentrations following oral administration of 13, 6.5 or 1.3 mmoles CCl₄/kg body wt. The increase in blood CCl₄ levels at all doses of CCl₄ administered was delayed by about 2 hr by administration of Promethazine. The maximum blood concentrations of CCl₄ and total amount absorbed (judged by area under the plot of blood concentration vs time) were dose-related to the amount of CCl₄ administered with or without Promethazine administration. Blood concentrations of CHCl₃ were relatively constant over the range of CCl₄-doses used indicating that the metabolic production rate of CHCl₃ is saturated at rather low doses of CCl₄ administered.     

Effects of Great Lakes Fish Consumption on the Immune System of Sprague–Dawley Rats Investigated during a Two-Generation Reproductive Study: I. Body and Organ Weights,Food Consumption,and Hematological Parameters

The effects of Great Lakes fish on food consumption, body and organ weights, and hematological parameters were investigated in the first- (F₁) and second- (F₂) generation Sprague–Dawley rats assigned to immunological studies. The parent- (F₀) generation rats were fed either a control diet or diets containing 5 or 20% lyophilized chinook salmon from Credit River (Lake Ontario, LO) or Owen Sound (Lake Huron, LH). The F₁and F₂pups were exposed to the fish dietin utero,through the dam's milk to 21 days of age and through the respective diets to 13 weeks of age. The study included an F₁-reversibility (F₁-R) phase in which rats at 13 weeks of exposure to fish or control diets were switched to the control diet for 3 months. Statistically significant effects included increased growth rates in the F₁male rats fed the LH fish diets compared to those fed the LO fish diets; increased liver weights in the F₂-generation male rats fed the LH-20% and LO-20% diets compared to those fed the 5% fish diets; reduced thymus weights in the F₁-R female rats fed the LO-20% fish diet compared to those fed the LO-5% or LH-20% fish diets and in the F₂male rats fed the LO diets compared to those fed the LH diets; increased kidney weights in the F₂male rats fed the LH-20% diet compared to those fed the LH-5% or LO-20% diets; reduced but reversible effects on red blood cell (RBC), white blood cell (WBC), neutrophil, lymphocyte, and monocyte numbers in the F₁-generation female rats fed the fish diets; reduced red blood cell (RBC), white blood cell (WBC), and lymphocyte numbers in the F₂male rats fed the LO diets compared to those fed the LH diets; and reduced WBC and lymphocyte numbers in the F₂female rats fed the LO-20% diet compared to those fed the LH-20% fish diet. These results suggested that long-term exposure to Great Lakes fish contaminants may have adverse effects on some immune-related parameters. The impact of such changes on the functional aspects of the immune system of rats and consequently on human health needs to be further investigated.     

Gonadal Influence on the Toxicity of 2-(2′-Hydroxy-3′,5′-di-tert-butylphenyl) benzotriazole in Rats

Previously, we showed that susceptibility of male rats to the toxicity of an ultraviolet absorber, 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole (HDBB), was nearly 25 times higher than that of females. In the current study, we investigated the role of sex steroids in the mediation of the gender-related difference using castrated rats. Male and female castrated CD(SD) rats were given HDBB by gavage at 0, 0.5, 2.5, or 12.5 mg/kg/day for 28 days. No deaths, clinical signs of toxicity, or changes in body weight or food consumption were found at any doses. Blood biochemical changes suggestive of hepatic damage, such as increased levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase, were detected at 12.5 mg/kg/day in males. Absolute and relative liver weight increased at 0.5 mg/kg/day and above in males and at 12.5 mg/kg/day in females. In the liver, histopathological changes, such as nucleolar enlargement, increased mitosis, hypertrophy in hepatocytes, and/or focal necrosis were observed at 0.5 mg/kg/day and above in males, and at 2.5 mg/kg/day and above in females. These findings indicate that castration markedly reduced the gender-related differences in toxicity of HDBB in rats.     

Tissue disposition,excretion and metabolism of 2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100) in male Sprague–Dawley rats

The absorption, disposition, metabolism and excretion study of orally administered 2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100) was studied in conventional and bile-duct cannulated male rats. In conventional rats, >70% of the radiolabelled oral dose was retained at 72?h, and lipophilic tissues were the preferred sites for disposition, i.e. adipose tissue, gastrointestinal tract, skin, liver and lungs. Urinary excretion of BDE-100 was very low (0.1% of the dose). Biliary excretion of BDE-100 was slightly greater than that observed in urine, i.e. 1.7% at 72?h, and glucuronidation of phenolic metabolites was suggested. Thiol metabolites were not observed in the bile as had been reported in other PBDE metabolism studies. Almost 20% of the dose in conventional male rats and over 26% in bile-duct cannulated rats was excreted in the faeces, mainly as the unmetabolized parent, although large amounts of non-extractable radiolabel were also observed. Extractable metabolites in faeces were characterized by mass spectrometry. Monohydroxylated pentabromodiphenyl ether metabolites were detected; mono- and di-hydroxylated metabolites with accompanying oxidative debromination were also observed as faecal metabolites. Tissue residues of [¹⁴C]BDE-100 in liver, gastrointestinal tract and adipose tissue contained only parent material. The majority of the 0–72-h biliary radioactivity was associated with an unidentified 79-kDa protein or to albumin.     

A Review of: “Interpreting and Reporting Clinical Trials: A Guide to the CONSORT Statement and the Principles of Randomised Controlled Trials,by A. Keech,V. Gebski,and R. Pike (eds.)”

Traditionally, Phase II trials have been conducted as single-arm trials to compare the response probabilities between an experimental therapy and a historical control. Historical control data, however, often have a small sample size, are collected from a different patient population, or use a different response assessment method, so that a direct comparison between a historical control and an experimental therapy may be severely biased. Randomized Phase II trials entering patients prospectively to both experimental and control arms have been proposed to avoid any bias in such cases. The small sample sizes for typical Phase II clinical trials imply that the use of exact statistical methods for their design and analysis is appropriate. In this article, we propose two-stage randomized Phase II trials based on Fisher’s exact test, which does not require specification of the response probability of the control arm for testing. Through numerical studies, we observe that the proposed method controls the type I error accurately and maintains a high power. If we specify the response probabilities of the two arms under the alternative hypothesis, we can identify good randomized Phase II trial designs by adopting the Simon’s minimax and optimal design concepts that were developed for single-arm Phase II trials.     

Solid-State Stability of Human Insulin I. Mechanism and the Effect of Water on the Kinetics of Degradation in Lyophiles from pH 2–5 Solutions

Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range. Methods. Solutions of 1.0 mg/mL insulin were adjusted to pH 2–5 using HC1, freeze-dried, then exposed to various relative humidities at 35°C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance. Results. Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH (pH) yielding, at any given water content, solid-state pH-rate profiles parallel to the solution pH-rate profile. This pH dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of 4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3–5 and at 35°C involves rate-limiting intramolecular nucleophilic attack of the Asn_A21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., Phe_B1, and Gly_Al) of another insulin molecule to generate either deamidated insulin (Asp_A21) or an amide-linked covalent dimer (e.g., [Asp_A21-Phe_B1] or [Asp_A21-Gly_A1]), respectively. The rate of insulin degradation in lyophilized powders at 35°C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20–50%) required to induce a glass transition. Conclusions. The decomposition of human insulin in lyophilized powders between pH 3–5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.     

New metabolites of the drug 5-aminosalicylic acid. I: N-β-D-glucopyranosyl-5-aminosalicylic acid

1. A new unstable metabolite of 5-aminosalicylic acid (5-ASA) was found in plasma from healthy volunteers dosed with 5-ASAi.v.

2. The metabolite was prepared by incubation of 5-ASA with rat liver homogenate, and isolated using preparative?h.p.l.c.

3. The metabolite was identified as N-β-D-glucopyranosyl-5-aminosalicylic acid by n.m.r. spectroscopy and by FAB mass spectrometry.

4. N-β-D-Glucopyranosyl-5-ASA was formed non-enzymically from 5-ASA and glucose in phosphate buffer pH7.4, and was unstable under weakly acidic conditions, decomposition increasing with temperature, i.e. decomposition was complete after 30?min at pH 5.0 and 23°C.