Erythropoietin: production by a particulate fraction of rat kidney.

An erythropoietic factor was extracted with hypotonic phosphate buffer from the kidneys of hypoxic rats. Normal rat serum enhanced the activity of this factor, which is associated with the light mitochondrial fraction. The data suggest that the renal factor is not physiologically active unless it interacts with a serum carrier or activator, or that the factor may be an enzyme which produces erythropoietin from some serum substrate.

Renal erythropoietic factor: role of ions and vasoactive agents in erythropoietin formation.

Evidence is provided for the existence of a renal erythropoietic factor, devoid of vasopressor activity, which upon interaction in vitro with normal serum yields erythropoietin. When undialyzed serum is used, erythropoietin inactivation develops in the incubation mixtures, and this inactivation appears to be dependent on an enzymatic component in preparations of the factor and on ions in serum.

Understanding genetic toxicity through data mining: the process of building knowledge by integrating multiple genetic toxicity databases.

ABSTRACT Genetic toxicity data from various sources were integrated into a rigorously designed database using the ToxML schema. The public database sources include the U.S. Food and Drug Administration (FDA) submission data from approved new drug applications, food contact notifications, generally recognized as safe food ingredients, and chemicals from the NTP and CCRIS databases. The data from public sources were then combined with data from private industry according to ToxML criteria. The resulting "integrated" database, enriched in pharmaceuticals, was used for data mining analysis. Structural features describing the database were used to differentiate the chemical spaces of drugs/candidates, food ingredients, and industrial chemicals. In general, structures for drugs/candidates and food ingredients are associated with lower frequencies of mutagenicity and clastogenicity, whereas industrial chemicals as a group contain a much higher proportion of positives. Structural features were selected to analyze endpoint outcomes of the genetic toxicity studies. Although most of the well-known genotoxic carcinogenic alerts were identified, some discrepancies from the classic Ashby-Tennant alerts were observed. Using these influential features as the independent variables, the results of four types of genotoxicity studies were correlated. High Pearson correlations were found between the results of Salmonella mutagenicity and mouse lymphoma assay testing as well as those from in vitro chromosome aberration studies. This paper demonstrates the usefulness of representing a chemical by its structural features and the use of these features to profile a battery of tests rather than relying on a single toxicity test of a given chemical. This paper presents data mining/profiling methods applied in a weight-of-evidence approach to assess potential for genetic toxicity, and to guide the development of intelligent testing strategies.

P2 purinergic receptors potentiate parathyroid hormone receptor-mediated increases in intracellular calcium and inositol trisphosphate in UMR-106 rat osteoblasts.

The PTH receptor has been cloned and shown to activate both adenylate cyclase and phospholipase C. Evidence exists that both signaling pathways are important for mediating the net physiological effects of this hormone on bone remodeling. We have shown previously that UMR-106 osteoblastic sarcoma cells express two calcium-signaling P2 purinergic receptors, a P2U and a unique P2T receptor. Neither receptor modulates PTH receptor-mediated activation of adenylate cyclase. We now report that stimulation of either P2 receptor will, however, potentiate the magnitude of the calcium signal observed after subsequent addition of human (h) PTH-(1-34) to fluo-3-loaded UMR-106 cells. Results from experiments with staurosporine and phorbol 12-myristate 13-acetate argue against a role for protein kinase C as a mediator of this potentiating effect of P2 receptor ligands. The P2 receptor-mediated intracellular calcium elevation itself cannot account for the potentiating mechanism, because addition of ionomycin will not replicate the effect of P2 receptor ligands on hPTH-(1-34) signaling. Addition of EGTA after exposure to P2 ligands does not prevent the potentiation of hPTH-(1-34), indicating that P2 ligands potentiate the release of intracellular calcium after PTH receptor stimulation. Inositol trisphosphate production is potentiated in response to hPTH-(1-34) after first priming [3H]inositol-labeled cells with a P2 agonist. We conclude that UMR-106 cells express PTH receptors that are capable of activating adenylate cyclase, but may be unable to activate phospholipase C until cells receive a signal as a consequence of P2 receptor activation. The nature of the signal is unclear, but appears not to be mediated by either calcium or protein kinase C.

Role for brain corticotropin-releasing factor in the weight-reducing effects of chronic fenfluramine treatment in rats.

Fenfluramine is an amphetamine derivative which is used as a weight-reducing agent in the treatment of obesity. It has been postulated that fenfluramine affects brain serotonin (5HT) neurons resulting in decreased food intake and altered autonomic outflow which, in turn, increases metabolism. CRF decreases food intake and, in addition, has been demonstrated to reduce body weight in genetically obese rats through selective activation of sympathetic and inhibition of parasympathetic outflows. Because 5HT is a potent CRF secretagogue, we tested the hypothesis that the weight-reducing effects of fenfluramine administration may be mediated, in part, through altered CRF secretion. Chronic fenfluramine treatment (1-24 mg/kg sc, twice daily, 4 days) resulted in a dose-dependent decrease in hypothalamic CRF concentration at 30 min after the final drug injection and was accompanied by a significant reciprocal increase in plasma corticosterone concentration. These data suggest that the decrease in hypothalamic CRF was a consequence of increased CRF secretion. These changes in hypothalamic CRF and plasma corticosterone correlated with brain fenfluramine levels. In contrast, high dose fenfluramine treatment significantly increased hippocampus, midbrain, and spinal cord CRF concentrations whereas levels in cerebral cortex, caudate putamen, thalamus, pons/medulla, and cerebellum were unaffected. There was no effect of this fenfluramine treatment protocol on regional brain TRH or neurotensin concentrations. In keeping with the well known development of tolerance to the weight-reducing effects of fenfluramine, chronic fenfluramine treatment resulted in lesser increases in corticosterone secretion than after acute treatment. Whereas weight loss observed after chronic fenfluramine treatment was associated with stimulation of hypothalamic-pituitary-adrenocortical hormone secretion, the weight-recovery phase after cessation of drug treatment was associated with decreased levels of plasma corticosterone. These data, demonstrating fenfluramine-induced alterations in brain CRF and plasma corticosterone, suggest that CRF may represent an important endogenous transmitter which mediates the weight-reducing effects of the drug.

In vivo transgenic bioassays and assessment of the carcinogenic potential of pharmaceuticals.

There is general agreement in the scientific community on the need to improve carcinogenicity testing and the assessment of human carcinogenic risk and to incorporate more information on mechanisms and modes of action into the risk assessment process. Advances in molecular biology have identified a growing number of genes such as protooncogenes and tumor-suppressor genes that are highly conserved across species and are associated with a wide variety of human and animal cancers. In vivo transgenic rodent models incorporating such mechanisms are used to identify mechanisms involved in tumor formation and as selective tests for carcinogens. Transgenic methods can be considered an extension of genetic manipulation by selective breeding, which long has been employed in science and agriculture. The use of two rodent species in carcinogenicity testing is especially important for identifying transspecies carcinogens. The capacity of a substance to induce neoplasia across species suggests that the mechanism(s) involved in the induction of the neoplasia are conserved and therefore may have significance for humans. Based on available information there is sufficient experience with some in vivo transgenic rodent carcinogenicity models to support their application as complementary second species studies in conjunction with a single 2-year rodent carcinogenicity study. The optional substitution of a second 2-year rodent carcinogenicity study with an alternative study such as an in vivo transgenic carcinogenicity study is part of the International Conference on Harmonization guidance S1B: Testing for Carcinogenicity of Pharmaceuticals. This guidance is intended to be flexible enough to accommodate a wide range of possible carcinogenicity assessment models currently under consideration or models that may be developed in the future. The use of an in vivo transgenic mouse model in place of a second 2-year mouse study will improve the assessment of carcinogenic risk by contributing insights into the mechanisms of tumorigenesis and potential human relevance not available from a standard 2-year bioassay. It is envisioned that this will stimulate the further development of more efficient and relevant methods for identifying and assessing potential human carcinogenic risk, which will benefit public health.

The duration of non-rodent toxicity studies for pharmaceuticals. International Conference on Harmonication (ICH).

At the present time, there are no uniform standards for the duration of non-rodent chronic toxicity studies. The European Union (EU) requires a 6-month non-rodent study. In Japan, a 6-month study is sufficient for most, but not all, compounds. The U.S. Food and Drug Administration (FDA) maintains its standard duration of 12 months for non-rodents, with 6-month studies accepted for some clinical indications on a case-by-case basis. To achieve harmonization on the duration of non-rodent toxicity studies, each member regulatory region (EU, U.S., and Japan) of the International Conference on Harmonization (ICH) collected non-rodent studies with significant new toxicological findings that had occurred after 6 months. An ICH expert working group was organized that included representatives from the regulatory authorities of each ICH region, to jointly review all available case studies for the purpose of arriving at a consensus on the best duration time for non-rodent toxicity studies. Eighteen case studies were identified and evaluated (16 original cases plus 2 additional FDA cases); most of the toxicities identified fell into the following categories: (1) toxicities identified at 6 months; (2) toxicities observed at 12 months, which were absent or considered isolated and not noteworthy findings at 6 months; (3) drug-related deaths or morbidity that occurred between 6 and 12 months, with a pattern of toxicity that permitted the interpolation of findings to an intermediate interval between 6 and 12 months; and (4) a shift in the dose response for toxicity with increasing duration of drug exposure. Of the 18 cases evaluated, 11 supported a study-duration of 9-12 months, 4 supported a duration of 12 months, and the 3 remaining cases indicated that a 6-month study would be adequate. The working group concluded that there was sufficient evidence to support a harmonized 9-month duration for non-rodent toxicity studies, which would be applicable for most categories of pharmaceuticals.

Altered state of cardiac sympathetic nerves during immunologically induced anemia.

Cardiac norepinephrine (NE) levels exhibit a marked reduction in rats suffering from hemolytic anemia induced with antibodies against rat red blood cells. Administration of antiserum via tail vein resulted in a highly reproducible 70% drop in hemoglobin levels by 72 h. At 96 h cardiac NE levels were decreased by 67%; NE levels in vas deferens and submaxillary gland were not decreased. Within 10 days, both hemoglobin and cardia NE returned to near control levels. Hearts from anemic rats showed a 68% decrease in their ability to accumulate 3H-NE administered in tracer doses at 72 h of anemia. Cardiac NE turnover rates were increased 88% in 72 h anemic animals. These results are consistent with an anemia-induced activation of cardiac sympathetic nerves. Cardiac monoamine oxidase and dopamine-beta-hydroxylase activities in whole heart homogenates were similar in control and anemic animals at 72 h. These results suggest that NE depletion is not the result of decreased synthetic capacity of the nerves or degeneration of existing terminals. The data suggest that cardiac NE depletion during anemic stress is associated with the combined effects of increased NE release and a decrease in the effective NE uptake or binding capacity of sympathetic nerves. Anemia-induced depletion may, therefore, be different from the depletion associated with other forms of cardiovascular stress.

Use of toxicological information in drug design.

This paper is an extension of the keynote address and another talk at the Symposium on the Use of Toxiciological Information in Drug Design. The symposium was organized by American Chemical Society's Chemical Information Division at the 220th National Meeting of the American Chemical Society in Washington, DC, August 20-24, 2000. We outline an approach for meeting the scientific information needs of the U.S. Food and Drug Administration (FDA). Ready access to scientific information is critical to support safety-related regulatory decisions and is especially valuable in situations where available experimental information from in vivo/in vitro studies are inadequate or unavailable. This approach also has applications for lead selection in drug discovery. A pilot electronic toxicology/safety knowledge base and computational toxicology initiative is underway in the FDA Center for Drug Evaluation and Research (CDER) that may be a prototype for an FDA knowledge base. The objectives of this effort are: (i) to strengthen and broaden the scientific basis of regulatory decisions, (ii) to provide the Agency with an electronic scientific institutional memory, (iii) to create a scientific resource for regulatory and applied research, and (iv) to establish an internal Web-based support service that can provide decision support information for regulators that will facilitate the review process and improve consistency and uniformity. An essential component of this scientific knowledge base is the creation of a comprehensive electronic inventory of CDER-regulated substances that permit identification of clusters of substances having similar chemical, pharmacological or toxicological activities, and molecular structure/substructures. Furthermore, the inventory acts as a pointer and link to other databases and critical non-clinical and clinical pharmacology/toxicology studies and reviews in FDA archives. Clusters of related substances are identified through the use of: (i) an extensive index of alternative names for each substance, (ii) a molecular structure key field consisting of a rudimentary or core structure represented as an ISIS.mol-file, (iii) global search terms (molecular group, chemical class, clinical indication, or pharmacologic activity), and (iv) molecular clustering using structure/sub-structure similarity indices. The information contained in a toxicology knowledge database has limited value unless means are available to extract information, identify relationships, and create and test hypotheses. One such means is computational toxicology, also called in silico toxicology, ComTox, or e-TOX. Computational toxicology is the application of computer technology and information processing (informatics) to analyze, model, and estimate chemical toxicity based upon structure activity relationships (SAR). A computational toxicology software package, MCASE, has been evaluated and successfully improved by CDER through the incorporation of data from FDA archives and concomitant alterations of the logic used in the interpretation of the results to reflect the data analysis and hazard identification practices and priorities of the Center. Our modifications and uses of the MCASE program are discussed in detail.

Intraventricular 6-hydroxydopamine lowers isolation-induced fighting behavior in male mice.

Male mice with high isolation-induced fighting tendencies were administered 200 mug 6-OHDA or vehicle intraventricularly and tested for fighting tendency for up to 10 weeks until sacrifice, and assayed for brain NE levels. A strong correlation was found between NE depletion and reduced fighting tendency after 6-OHDA treatment. The depressed fighting by mice with less than 200 ng. NE/g persisted throughout a series of test fights, indicating no recovery in fighting behavior throughout the survival time.

Comparison of the effects of repeated oral versus subcutaneous fenfluramine administration on rat brain monoamine neurons: pharmacokinetic and dose-response data.

The importance of the route of drug administration (oral vs. subcutaneous) on the neurochemical effects and pharmacokinetics of repeated d,1-fenfluramine administration in rats (1-24 mg/kg b.i.d., i.e., 2-48 mg/kg/day for 4 days) was examined. Overall, comparable dose-dependent alterations in brain monoamine markers were observed following repeated oral (PO) and subcutaneous (SC) administration of fenfluramine. Doses of 1 and 2 mg/kg fenfluramine were without significant effects on the density of 3H-paroxetine-labeled serotonin (5-HT) uptake sites. Higher doses of fenfluramine (4, 12 and 24 mg/kg) produced dose-dependent decreases in 5-HT, 5-hydroxyindoleacetic acid and 5-HT uptake sites with maximal decreases (80-90%) occurring at the 12 mg/kg dose. Fenfluramine administration produced dose-dependent and biphasic effects on brain dopamine markers with increases in homovanillic acid (HVA) observed at 2 hours, whereas decreases in the levels of dopamine, HVA and dihydroxyphenylacetic acid were evident at 18 hours posttreatment. Norepinephrine levels were only decreased at the highest dose of fenfluramine. Significantly higher levels of brain fenfluramine were observed following SC than following PO administration of the drug. On the other hand, comparable levels of its active metabolite norfenfluramine were present in the brain following the two routes of fenfluramine administration. These data suggest the importance of norfenfluramine levels in the brain in determining the high-dose neurotoxic effects of fenfluramine on brain 5-HT neurons in rats.

[Cerebral ultrasonography in children during the 1st year of life. A method for diagnosis and follow-up of ventricular dilatation]. / Ultra-sonografia cerebral em crianças no primeiro ano de vida. Método para o diagnóstico e acompanhamento das dilatações ventriculares.

The ultrasonography (Mode B--Real Time) experience was analyzed to evaluate neurological diseases in children during their first year of life. Forty-two examination were accomplished in twenty-eight children with the following diagnosis: hydrocephalus (22), normal (15), subdural hygroma (3), intracranial cyst and hydrocephalus (1), giant encephalocele (1). The technique consists of positioning the transducer in the coronal, sagital and axial direction and selecting dynamically the images to be photographed. In the coronal position, the height of the lateral ventricle and the width of the third ventricule were obtained. In the axial position, the ventricular ratio-lateral ventricle width cerebral hemisphere width was obtained. Although it was a small group of patients, those indexes can objectify the ventricular size variation in children with well or poor functioning shunts. The importance of this method was the possibility to follow the development of hydrocephalus in cases of myelomeningocele and to analyze the etiology and features of hydrocephalus with or without shunts. In conclusion, this test is very usefull, mainly because it is very brief (about 20 minutes), the patient does not need sedation, it is innocuous, very precise even when compared with computerized tomography and for its low cost.

Depletion of cardiac norepinephrine during two forms of hemolytic anemia in the rat.

Knowledge of the status of cardiac norepinephrine (NE) during anemia could lead to a better understanding of the role the sympathetic nervous system plays in cardiac function during anemia. Rats were made anemic by treatment with phenylhydrazine (PHZ). After the rapid onset of anemia, 60% of the stored NE in the heart was lost within 48 hours after treatment. Associated with the loss of cardiac NE was an increase in the wet weight of the heart, which reached a value 40% above control 48 hours after treatment. PHZ itself probably does not directly mediate this depletion of NE, since the vas deferens, brain and spleen had a normal store of NE at 48 hours. This contention was supported when rats, treated with PHZ, were transfused with normal rat red blood cells. This transfusion resulted in PHZ-treated rats which were not anemic. The hearts of these rats were not depleted of NE, but the hearts of the nontransfused, PHZ-treated controls were. Anemia also was induced by treating rats with anti-rat red blood cell serum. The hearts of these rats also were depleted of NE. These experiments show that during two forms of anemia there is a loss of NE from the sympathetic neurons innervating the heart. The effect of this on regulation of cardiac function remains to be determined.

A new highly specific method for predicting the carcinogenic potential of pharmaceuticals in rodents using enhanced MCASE QSAR-ES software.

This report describes in detail a new quantitative structure-activity relational expert system (QSAR-ES) method for predicting the carcinogenic potential of pharmaceuticals and other organic chemicals in rodents, and a beta-test evaluation of its performance. The method employs an optimized, computer-automated structure evaluation (MCASE) software program and new database modules which were developed under a Cooperative Research and Development Agreement (CRADA) between FDA and Multicase, Inc. The beta-test utilized 126 compounds with carcinogenicity studies not included in control database modules and three sets of modules, including: A07-9 (Multicase, Inc.), AF1-4 (FDA-OTR/Multicase, Inc.), and AF5-8 (FDA-OTR/proprietary). The investigation demonstrated that the standard MCASE(A07-9) system which had a small data-set (n = 319), detected few structure alerts (SA) for carcinogenicity (n = 17), and had poor coverage for beta-test compounds (51%). Conversely, the new, optimized FDA-OTR/MCASE(AF5-8) system had a large data-set (n = 934), detected many SA (n = 58) and had good coverage (94%). In addition, the study showed the standard MCASE(A07-9) software had poor predictive value for carcinogens and specificity for noncarcinogens (50 and 42%), detected many false positives (58%), and exhibited poor concordance (46%). Conversely, the new, FDA-OTR/MCASE(AF5-8) system demonstrated excellent predictive value for carcinogens and specificity for non-carcinogens (97%, 98%), detected only one false positive (2%), and exhibited good concordance (75%). The dramatic improvements in the performance of the MCASE were due to numerous modifications, including: (a) enhancement of the size of the control database modules, (b) optimization of MCASE SAR assay evaluation criteria, (c) incorporation of a carcinogenic potency scale for control compound activity and MCASE biophores, (d) construction of individual rodent gender- and species-specific modules, and (e) defining assay acceptance criteria for query and control database compounds.

Evidence for separate calcium-signaling P2T and P2U purinoceptors in human megakaryocytic Dami cells.

Recently (J Pharmacol Exp Ther 261:580, 1992), we have shown that K562 leukemia cells express a calcium-signaling purinoceptor with characteristics of the P2T receptor subtype for adenosine diphosphate (ADP) previously found only in platelets. Because these results suggested that the P2T receptor may be an early marker for megakaryocytic differentiation, we studied whether this calcium-signaling receptor is also expressed in Dami cells, a human megakaryocytic leukemia cell line. Here we report evidence that Dami cells express a P2T receptor for ADP. The calcium response EC50 values for ADP, 2-methylthioadenosine diphosphate (2-MeS-ADP), and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) in Dami cells are 0.4 mumol/L, 0.04 mumol/L, and 2 mumol/L, respectively, which approximate the potencies of these agonists in K562 cells and in platelets. The platelet P2T receptor antagonists 2-methylthioadenosine triphosphate (2-MeS-ATP), and 2-chloroadenosine triphosphate (2-Cl-ATP) were surprisingly potent agonists at the P2T receptor in both Dami and K562 cells. Dami cells, unlike K562 cells and platelets, also respond to adenosine triphosphate (ATP) and uridine triphosphate (UTP) with an increase in intracellular calcium. Adenosine monophosphate (AMP) is an effective antagonist of the response to ADP, 2-MeS-ADP, ADP beta S, 2-MeS-ATP, and 2-Cl-ATP, but not to ATP and UTP. The responses to maximal concentrations of UTP in combination with either ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP are additive. In contrast, ADP in combination with either 2-MeS-ADP, ADP beta S, 2-MeS-ATP, or 2-Cl-ATP are not additive. UTP desensitized Dami cells to ATP but not to ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP. Addition of ATP after UTP desensitization antagonized subsequent responsiveness to ADP. The data suggest that the receptor for ADP may be a unique P2T subtype, and the receptor for ATP and UTP is distinct from that of ADP and is most characteristic of the P2U (nucleotide) receptor subtype. Activation of either the P2T or P2U receptor causes a rapid generation of inositol trisphosphate in Dami cells.

Carcinogenicity testing and the evaluation of regulatory requirements for pharmaceuticals.

The results of rat and mouse carcinogenicity studies for 282 human pharmaceuticals in the FDA database were analyzed and compared as part of an International Conference on Harmonization (ICH) evaluation of rodent carcinogenicity studies and their utility for carcinogenicity testing. A majority of the carcinogenicity studies in the FDA database were carried out in Sprague-Dawley-derived rats and Swiss-Webster-derived CD-1 mice in contrast to Fisher 344 rats and B6C3F1 mice employed in National Toxicology Program (NTP) studies. Despite the differences in rodent strains, the relative proportion of compounds with positive findings (44.3%) and the degree of overall concordance between rats and mice (74.1%) in the FDA database were similar to the NTP rodent carcinogenicity database. Carcinogenicity studies in two rodent species are necessary primarily to identify trans-species tumorigens, which are considered to pose a relatively greater potential risk to humans than single species positive compounds. Two-year carcinogenicity studies in both rats and mice may not be the only means of identifying trans-species tumorigens. Sufficient experience is now available for some alternative in vivo carcinogenicity models to support their application as complementary studies in combination with a single 2-year carcinogenicity study to identify trans-species tumorigens. Our analysis of the rodent carcinogenicity studies supports such an approach for assessing carcinogenic potential without compromising the public health.