Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies.

Green tea extract and its principal active ingredient, epigallocatechin gallate (EGCG), are gaining attention and increased usage due to their healthful properties. Despite the increasing demand for these products, few studies have examined their safety. The toxicity of purified green tea extracts containing high concentrations of EGCG have been evaluated in a series of studies in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. Topical EGCG preparations caused minor dermal irritation in rats and guinea pigs, but not rabbits, and was a moderate dermal sensitizing agent in the guinea pig maximization test. A rabbit eye irritation test produced a strong enough response to not warrant any further testing in this assay. An oral dose delivering 2000 mg EGCG preparation/kg was lethal to rats; whereas, a dose of 200 mg EGCG/kg induced no toxicity. The dietary administration of EGCG preparation to rats for 13 weeks was not toxic at doses up to 500 mg/kg/day. Similarly, no adverse effects were noted when 500 mg EGCG preparation/kg/day was administered to pre-fed dogs in divided doses. This dose caused morbidity when administered to fasted dogs as a single bolus dose, although this model was considered an unrealistic comparison to the human condition. From these studies a no-observed adverse effect level of 500 mg EGCG preparation/kg/day was established.

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 3: teratogenicity and reproductive toxicity studies in rats.

Green tea and its principal active ingredient, epigallocatechin gallate (EGCG), have been demonstrated to have anticancer properties through interactions with multiple biochemical processes. Since these processes are often crucial in normal fetal development it is important to evaluate the potential effects of EGCG on the fetus. EGCG preparations of >91% purity were administered to pregnant rats during organogenesis and development in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. In an initial preliminary study using subcutaneous and gavage routes, there was no evidence of any direct embryo-fetal toxicity, although some maternal toxicity was seen. In the main teratogenicity study, feeding pregnant rats diets supplemented at 1400, 4200 or 14,000 ppm during organogenesis was non-toxic to dams or fetuses. A two-generation study in rats fed 1200, 3600 or 12,000 ppm EGCG preparation showed no adverse effects on reproduction or fertility. The highest dose reduced the growth rate of offspring, and there was a slight increase in pup loss. A growth effect among pups was also seen at 3600 ppm, but in the second generation only. The lowest dose was considered the overall no-observed adverse effect level (NOAEL). As dams consumed twice the amount of feed during the crucial lactation period, the NOAEL was equivalent to 200 mg/kg/day EGCG preparation.

The cumulative effect of long-acting bronchodilators, exercise, and inspiratory muscle training on the perception of dyspnea in patients with advanced COPD.

BACKGROUND: Dyspnea is a common complaint during daily activities in patients with advanced COPD. The mechanisms underlying dyspnea and the appropriate treatment strategies to relieve it are still not totally understood. We hypothesized that the perception of dyspnea (POD) may be modified by the accumulative effect of bronchodilator therapy, exercise, and inspiratory muscle training (IMT). METHODS: Spirometry, submaximal exercise performance, inspiratory muscle strength and endurance, and the POD were assessed before and following three consecutive 6-week periods of therapy with a long-acting bronchodilator (LABD), the LABD plus exercise, and the LABD plus exercise plus IMT in 30 patients with moderate-to-severe COPD. RESULTS: There was a small, statistically insignificant, increase in FEV(1) in the study group (mean [+/- SEM] increase, 1.42+/-0.3 to 1.49+/-0.4 L) following the LABD therapy period, and no additional increase following the two other periods of therapy. There was a significant increase (p<0.05) in the 6-min walk distance following the therapy period with the LABD plus exercise (mean increase, 252+/-41 to 294+/-47 m) and an additional small increase following the therapy period with the LABD plus exercise plus IMT period (mean increase, 252+/-41 to 302+/-49 m). The decrease in the POD was small and statistically not significant following the therapy periods with the LABD and the LABD plus exercise. The major and statistically significant decrease in the POD was noted following the therapy period with the LABD plus exercise plus IMT. CONCLUSIONS: In patients with moderate-to-severe COPD, following sequential periods of therapy with the LABD, the LABD plus exercise, and the LABD plus exercise plus IMT, there is a cumulative benefit in the POD. The most significant improvement was associated with IMT and not with the LABD and exercise training. The FEV(1) was moderately increased following the therapy period with the LABD, and the addition of exercise has most affected the 6-min walk distance.

Specific inspiratory muscle training in patients with mild asthma with high consumption of inhaled beta(2)-agonists.

BACKGROUND: It has been known for many years that there are variations between asthmatic patients in terms of their perception of breathlessness during airway obstruction. STUDY OBJECTIVE: To investigate the relationship between beta(2)-agonist consumption and the score of perception of dyspnea, in mild asthmatics, and the relationship between the effect of specific inspiratory muscle training (SIMT) on the score of perception of dyspnea and beta(2)-agonist consumption in "high perceivers." METHODS: Daily beta(2)-agonist consumption was assessed during a 4-week run-in period in 82 patients with mild asthma. Patients with a mean beta(2)-agonist consumption of > 1 puff/d ("high consumers") then were randomized into two groups: one group of patients received SIMT for 3 months; the other group of patients was assigned as a control group and received sham training. Inspiratory muscle strength and perception of dyspnea were assessed before patients entered the study, following the 4-week run-in period, and after completing the training period. RESULTS: Following the 4-week run-in period, 23 high-consumer patients (mean [+/- SEM] beta(2)-agonist consumption, 2.7 +/- 0.4 puffs/d) were detected. The mean Borg score during breathing against resistance was significantly higher (p < 0.05) in the patients with high beta(2)-agonist consumption than in the subjects with low mean beta(2)-agonist consumption. Following SIMT, the mean maximal inspiratory pressure increased significantly from 94.1 +/- 5.1 to 109.7 +/- 5.2 cm H(2)O (p < 0.005) in the training group. The increase in inspiratory muscle strength was associated with a statistically significant decrease in the mean Borg score during breathing against resistance (p < 0.05) as well as in the mean daily beta(2)-agonist consumption. CONCLUSIONS: We have shown that patients with mild asthma, who have a high beta(2)-agonist consumption, have a higher perception of dyspnea than those with normal consumption. In addition, SIMT was associated with a decrease in perception of dyspnea and a decrease in beta(2)-agonist consumption.

Nocturnal cortisol secretion in asthmatic patients after inhalation of fluticasone propionate.

OBJECTIVES: This study was designed to assess the relationship between the degree of airflow obstruction and the suppression of the hypothalamic-pituitary-adrenal axis after inhalation of fluticasone propionate (FP) in asthmatic patients with varying degrees of airway obstruction. STUDY DESIGN: The nocturnal cortisol production (from 10:00 PM to 6:00 AM), defined as the integrated area under the curve of nocturnal plasma cortisol, was measured following inhalation of a placebo or a single dose of 500 microg FP at 8:00 PM in 28 patients with mild to moderate asthma, in a single, blind, 2-night study. RESULTS: The mean morning rise of cortisol decreased significantly following a single dose of inhaled FP. When the total nocturnal cortisol production after the second night (when the FP was inhaled) was compared to that after the first night (when the placebo was administered), it was found to have decreased by 29.4%. There was a statistically significant correlation between the FEV(1) and the fall in cortisol production just before the inhalation of FP (p < 0. 001). There was no correlation between baseline cortisol production and the fall in cortisol production. CONCLUSIONS: Our findings suggest that the degree of airway obstruction affects the systemic bioavailability of FP. FP is likely to induce a more severe decrease in nocturnal cortisol secretion in less obstructed patients. In order to reduce the risk for systemic side effects, the patient's degree of airway obstruction should be considered when planning inhaled FP treatment.

The perception of dyspnoea in patients with asthma, before and following treatment with inhaled glucocorticosteroids.

This study was designed in order to establish the perception of breathlessness during rest and while breathing against resistance, in patients with asthma, before and after 8 weeks of inhaled glucocorticoids (IGC) treatment and to compare these parameters in patients with and without improvement in FEV1. Sixty-seven asthmatic patients, with moderate asthma, attending the asthma clinic, and 20 normal subjects were studied. After a 2-week run-in period, in which the subjects were asked to use exclusively beta2-agonists as needed, the asthmatic patients were randomized to receive either treatment with IGC, 250 microg of fluticasone propionate (FP) twice a day, via a diskhaler (47 patients), or to receive placebo (20 patients) and to serve as a control group, for 8 weeks. Spirometry and measurements for the sensation of dyspnoea were performed before and at the end of the treatment period. The mean dyspnoea score during breathing against resistance was significantly lower (P<0.05) in the patients with asthma than in normal subjects, before entering the study. Following 8 weeks of inhaled FP, there was a significant improvement in the mean dyspnoea score during breathing against resistance in the asthmatics receiving IGCs but not in the control group (P<005). In the study group 32 patients had an improved FEV1 > 15% and 15 patients did not. There was a statistically significant difference in perception of dyspnoea (P<0.01), between the group of patients with a improved FEV1 and the group of patients that were under IGC treatment without improvement in their FEV1. There was also a difference in the mean beta2-agonists consumption between the two groups (P<0.01). Asthmatic patients have a significantly lower perception of dyspnoea compared to normal subjects. IGC treatment was associated with increased perception of dyspnoea. However, this improvement was noted only in patients with improved FEV1, while the patients without improvement remained with an equal degree of dyspnoea perception and beta2-agonists consumption.

Ammonia toxicity in cattle. V. Ammonia concentration of lymph and portal, carotid and jugular blood after the ingestion of urea.

Four rumen-fistulated Holstein steers were fitted with cannulas for the collection of portal, jugular and carotid blood. In addition, the thoracic duct of one steer was cannulated for the collection of lymph. Steers were given .125, .25 or .5 g urea/kg body weight 16 hr after a previous feeding. Within 5 min after the administration of the .5-g dose, rumen ammonia increased from 9.7 to 32.0 mg/100 ml, rumen pH from 6.47 to 7.87; portal blood ammonia from 1.02 to 8.01 mg/100 ml, carotid blood ammonia from .18 to 1.17 mg/100 ml and jugular blood ammonia from .13 to .36 mg/100 ml. Lymph ammonia increased from .22 to .32 mg/100 ml within 15 minutes. The .125- and .25-g doses or urea produced proportionate changes. In a second experiment, three Jersey cows were given .5 g urea/kg body weight, and the rates at which urea appeared in carotid and jugular blood were determined. Only small amounts of urea appeared in carotid and jugular blood during the first 5 min after dosing, but the concentrations then increased slowly but progressively. We concluded that because carotid blood ammonia concentration increased so rapidly after dosing with urea, ammonia must leak past the liver, and it is therefore unlikely, that there is a liver threshold for ammonia which must be exceeded before ammonia will reach the carotid artery. The marked difference in ammonia concentrations in carotid and jugular blood suggests that the brain takes up ammonia rapidly. While some ammonia is absorbed via the lymph, and thus bypasses the liver, the lymph does not appear to be a major contributor of ammonia to carotid blood.

Modification of synaptic efficiency, experiments in blatta orientalis.

The efficiency of synapt ic transmission in a nervous pathway--metathoracic ganglia from the cockroach--can be increased by various experimental situations, i.e., removal, immobilization or denervation of the forelegs of insects in ventral position. Leg immobilization with the insect in supine position by itself determines a minor increase which is not enhanced by removal or denervation of the forelegs. Under these conditions, the insects are able to hold their antennae with a midleg immediately after any of the leg treatments. On the other hand, if the insects are allowed to be in ventral position they can hold their antennae with a midleg only when they reach the ability to stand on the remaining three legs. During the time period needed to acquire this ability, the insects make repeated and spontaneous motor effects. The increase in synaptic efficiency observed and the achievement of the insect's ability to stand on three legs occur at about the same time. These observations suggest that the self-training determines the synaptic modifications which in turn could be related to the "learnt" behavioral reaction.

Hematological effects of 2',3'-dideoxycytidine in rabbits.

The antiviral nucleoside analogue 2',3'-dideoxycytidine (ddC) is a DNA chain terminator and/or inhibitor of human immunodeficiency virus (HIV) reverse transcriptase. We evaluated the effects of ddC in 36 New Zealand white rabbits. Three/sex were assigned to a control group and 5 treatment groups (10-250 mg/kg/day) for 13 or 18 weeks. Blood samples were taken 1 week prior to treatment and weekly thereafter to termination with the exception of the 2 highest dose groups, where blood sample collection was terminated at week 13. Selected hematological analytes were measured weekly with the exception of prothrombin time (PT) and activated partial thromboplastin time (APTT). PT and APTT and selected biochemical analytes were measured prior to treatment, at 7 weeks, and after 13 weeks of treatment. All rabbits were necropsied. Giemsa and hematoxylin and eosin sections were prepared from methacrylate-embedded marrow. Hematological effects included decreases in red blood cell count, hemoglobin, hematocrit, and white blood cell count and increases in mean corpuscular volume and red cell distribution width. Platelets, platelet volume, PT, APTT, and mean corpuscular hemoglobin concentration values were variable or unchanged. Effects were dose-related, most were seen at 1 week, and they persisted to term. Bone marrow histopathologic changes included megalocytosis, erythroid hypoplasia, bizarre erythroid nuclear morphology, nuclear-cytoplasmic asynchrony, and increased mitotic figures. Lymphopenia caused by ddC plateaued at 2 weeks and persisted until termination. Heteropenia (neutropenia) was sporadic. Biochemical values for serum analytes were unchanged by treatment. The principal hematological effect of ddC upon the erythron was characterized as a nonregenerative macrocytic anemia with erythroid hypoplasia and megaloblastic erythropoiesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral neuropathy induced by 2',3'-dideoxycytidine. A rabbit model of 2',3'-dideoxycytidine neurotoxicity.

The nucleoside analog 2',3'-dideoxycytidine (ddC) is a potent inhibitor of the reverse transcriptase of human immunodeficiency virus and a DNA chain terminator. In clinical trials in patients with acquired immunodeficiency syndrome, ddC treatment has been associated with a dose-limiting and dose-dependent, painful, sensorimotor peripheral neuropathy. In search of an animal model for ddC-induced neurotoxicity we studied 36 New Zealand White rabbits (3 males/3 females/group) given 0, 10, 50, 100, 150, or 250 mg/kg/day of ddC, by oral intubation, for 13 or 18 weeks. Rabbits in the 150 and 250 mg/kg/day groups were sacrificed at 13 weeks because of hematopoietic toxicity. After 16 weeks, rabbits in the 50 and 100 mg/kg/day groups showed hindlimb paresis and/or gait abnormalities. Nerve conduction velocities and amplitudes in the 100 mg/kg/day rabbits were reduced by 30 to 50%. The most prominent pathologic changes in peripheral nerve and ventral roots of ddC-treated rabbits were (a) myelin splitting and intramyelinic edema, (b) demyelination and remyelination of axons, and (c) axonal loss. Treatment-related histologic lesions were not observed in spinal cord, brain, or retina. The pathology in these ddC-treated rabbits is consistent with a peripheral myelinopathy and axonopathy. This represents the first clinical, electrophysiologic, and pathologic description of an animal model of a peripheral neuropathy induced by a nucleoside analog.

Aspirin-induced gastric mucosal damage in dogs: protection by the prostanoid Ro 22-1327.

Aspirin and other non-steroidal anti-inflammatory drugs are associated with gastritis and mucosal injury. The present study examined the efficacy of the synthetic prostanoid, Ro 22-1327, as a mucosal protectant against aspirin. Dogs were orally administered either vehicle followed by 650 mg of aspirin, or Ro 22-1327 followed one hour later by 650 mg of aspirin. Two hours later the dogs were endoscoped and lesions were scored. In separate dogs, with gastric pouches, acid secretion stimulated by food was monitored in the absence and presence of Ro 22-1327 for four hours. There were no gastric lesions in the dogs treated with the vehicle (polyethylene glycol 400; PEG) for Ro 22-1327 followed by vehicle (carboxymethylcellulose) for aspirin. Dogs dosed with PEG followed by aspirin had gastric lesions: mean (+/- S.E.) scores were 2.16 +/- 0.24 and 2.75 +/- 0.18 for the antrum and body, respectively. Ro 22-1327 provided protection from aspirin-induced injury. Significant (P less than 0.01) protection occurred at 1.0 mcg/kg of Ro 22-1327 in the antrum (0.60 +/- 0.40) and body (1.60 +/- 0.40). An oral dose of 10 mcg/kg did not inhibit peak or total food-stimulated gastric acid output. Ro 22-1327 is a potent mucosal protectant against aspirin-induced mucosal injury and this activity is not dependent upon inhibition of gastric acid secretion.

Angina-like cardiac disturbances of hypothalamic etiology in cat, monkey, and man.

Angina-like phenomena of CNS etiology were studied. Data were collected and analyzed from clinical cases of cerebral hematoma, concussion, and tumors in which cardiological disturbances were observed consequent to compressive brain lesions or surgical brain manipulations. The cases in which the disturbances were largely angina-like and without ectopic beats or arrhythmias were chosen for study. The brain region most significantly related to this phenomenon seemed to be the hypothalamus. Experiments conducted in cats and monkeys showed that stimulation of specific sites in the hypothalamus resulted in angina-like manifestations with no other cardiac changes. These data support our working hypothesis that a cerebrally induced syndrome that is conspicuously angina-like must be related to hypothalamic involvement. In the awake monkey hypothalamic stimulation caused angina-like ECG changes and behavior suggestive of referred pain. In some cases, repetitions of the stimulations or irritations in all of the species resulted in permanent pathological myocardial changes, mostly bleeding into the myocardium. In some animals there were minute hemorrhagic necroses that were subepicardial in distribution. It is significant that the most severe or permanent pathological changes were found in cases in which the angina-like ECG alterations were persistent. There were also cases, however, with persistent angina-like ECG changes that showed no such pathology.

Species differences in nucleotide pool levels of 2',3'-dideoxycytidine: a possible explanation for species-specific toxicity.

The nucleoside analogue, 2',3'-dideoxycytidine (ddC), a potent inhibitor of human immunodeficiency virus reverse transcriptase (in its anabolized triphosphorylated form), mediates virologic and immunologic improvements in AIDS patients. Clinical studies using ddC have shown various ddC-related toxicities, the most pronounced being a dose-limiting peripheral neuropathy. The dose responsiveness and manifestation of the ddC-related neuropathy vary among species, with greatest sensitivity in human > monkey > rabbit whereas mice and rats are insensitive to ddC-related neuropathy. This study has examined nucleotide pool sizes of ddCTP and its constituents (ddC, ddCMP, ddCDP) in cultured fibroblasts (human, rabbit, mouse) and freshly isolated peripheral lymphocytes (monkey, rabbit, rat, and mouse). Cells were treated with 10 microM [3H]ddC and nucleotide pool sizes analyzed by HPLC. The formation of nucleotide pools increased during the 24-hr assay period. Fibroblast pool formation of phosphorylated metabolites was significantly greater in human > rabbit > mouse. Lymphocytes demonstrated a similar pattern with monkey > rabbit > mouse = rat. Total ddC anabolite pools were also found to be significantly smaller (p < 0.05) in rodent lymphocytes than in those of rabbit or monkey, and rodent fibroblasts were smaller than those of human or rabbit (p < 0.05). These findings indicate that nucleoside phosphorylation and intracellular levels of phosphorylated metabolites may play an important role in determining species sensitivity and manifestation of ddC-related toxicity.

Administration of the oral antibiotic frenolicin-B selectively alters copper nutriture in male rats.

The polyketide antibiotic Frenolicin-B (FB) produces anorexia and esophageal epithelial hyperplasia (EH) in rats, findings that are characteristic of zinc deficiency. Because FB also chelates divalent cations in vitro, we conducted studies to determine whether FB modifies blood and organ concentrations of zinc and other essential metals (calcium, copper, iron and magnesium). Groups of male Sprague-Dawley rats ( approximately 250g; n = 20/group) consumed diets with adequate (40 microg/g), deficient (<2 microg/g) or fortified (100 microg/g) zinc concentrations ad libitum for 28 d. Two groups fed either Zn-adequate or Zn-fortified diets also were given 100 mg/(kg. d) of FB in diet, and 2 groups were pair-fed controls. Histopathology or metal analyses were performed on tissues from 10 rats/group. FB caused EH of the nonglandular stomach but not of other tissues. Of the metals evaluated, only copper concentrations were significantly reduced in all tissues examined except kidney. A broad range of kidney copper concentrations was found; these concentrations were associated with plasma copper and proteinaceous deposits within tubules. In rats, FB substantially and selectively depletes Cu in vivo, suggesting that drugs with structures that permit metal chelation should be evaluated for their potential to alter trace metal nutriture.

Mitochondrial schwannopathy and peripheral myelinopathy in a rabbit model of dideoxycytidine neurotoxicity.

BACKGROUND: The reverse transcriptase inhibitor, 2',3'-dideoxycytidine (ddC), causes a dose-limiting peripheral neuropathy in humans, the mechanism of which is unknown. Rabbits given ddC develop peripheral myelinopathy and axonopathy, but it has not been determined if either the myelin or axonal changes are primary or if they occur concurrently. EXPERIMENTAL DESIGN: To characterize sequential development of the ddC-induced neuropathy, 40 rabbits were given either vehicle or ddC by oral intubation at a dose of 35 mg/kg per day for 24 weeks. Electrophysiologic studies, pathologic examination of peripheral and central nervous system and skeletal muscle, and biochemical analysis of the sciatic nerve were performed at baseline (electrophysiology only) and after 8, 12, 16, 20, and 24 weeks of treatment. RESULTS: Neuropathologic changes in peripheral nerves were first evident at 16 weeks and were more pronounced at 20 and 24 weeks; onset of paresis occurred at week 20, whereas clear electrophysiologic deficits were seen only at week 24. Electrophysiologic changes were prolonged F-waves (measure of proximal motor conduction) and minor changes in distal conduction measurements. Pathologic changes included myelin splitting, intramyelinic edema, demyelination, and remyelination of the largest diameter nerve fibers in the ventral root and sciatic nerve. Axonal degeneration and reduction in axonal diameter were seen. Enlarged mitochondria with abnormal ultrastructure were present in Schwann cells of those animals with a myelinopathy. Mitochondrial abnormalities or other signs of degeneration were not seen in neurons of the dorsal root ganglia or in skeletal muscle. Significant changes were not present in myelin protein composition, myelin lipid composition, or activity of the myelin-specific enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase. Major reductions in levels of protein zero (P0, the homophilic adhesion protein of myelin) were not seen; however, the turnover rate of P0 was reduced as P0 messenger RNA expression in ddC-treated sciatic nerves decreased to 30 to 50% of control values. CONCLUSIONS: The peripheral neuropathy caused by ddC in rabbits is characterized as a myelinopathy of the proximal portion of the nerve fibers and as an axonopathy involving both proximal and distal fibers. The myelinopathy was associated with enlarged and abnormally shaped mitochondria in Schwann cells and is consistent with an effect of ddC on structure and function of Schwann cell mitochondria. Altered Schwann cell metabolism was evident by reduced levels of P0 messenger RNA, loss of homophilic myelin adhesion at the intraperiod line, and subsequent intramyelinic edema. Because axonal degeneration occurred concurrently with the myelin changes, it could not be determined if axonal changes were secondary to serve myelinic edema or if they represented a primary effect of ddC on neurons.