Non-clinical studies in the process of new drug development - Part II: Good laboratory practice, metabolism, pharmacokinetics, safety and dose translation to clinical studies.

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

Genetic variability affecting Exserohilum turcicum resistance in popcorn lines grown under high and low phosphorus conditions.

Northern leaf blight (NLB), caused by Exserohilum turcicum, is one of the main foliar diseases that affect popcorn culture. Farmers use many control measures to minimize damage caused by this disease, among which, the use of cultivars with genetic resistance is the most effective and economical. The aim of this study was to investigate genetic variability influencing resistance to NLB in 25 popcorn maize lines grown under high and low phosphorus conditions in relation to foliar fungal disease caused by E. turcicum. We evaluated the disease incidence and severity, by analysis of variance and cluster test (Scott-Knott). There was sufficient genetic variability between strains for resistance traits. Genotypic variance was higher than environmental variance, and had more discriminatory power. We conclude that new progenies could be selected for the establishment of future populations. P-7, P-9, L-59, L-71, and L-76 progenies possess promising characteristics that simultaneously reduce the severity and the incidence of NLB in popcorn plants.

Non-clinical studies required for new drug development - Part I: early in silico and in vitro studies, new target discovery and validation, proof of principles and robustness of animal studies.

This review presents a historical overview of drug discovery and the non-clinical stages of the drug development process, from initial target identification and validation, through in silico assays and high throughput screening (HTS), identification of leader molecules and their optimization, the selection of a candidate substance for clinical development, and the use of animal models during the early studies of proof-of-concept (or principle). This report also discusses the relevance of validated and predictive animal models selection, as well as the correct use of animal tests concerning the experimental design, execution and interpretation, which affect the reproducibility, quality and reliability of non-clinical studies necessary to translate to and support clinical studies. Collectively, improving these aspects will certainly contribute to the robustness of both scientific publications and the translation of new substances to clinical development.

Non-clinical studies in the process of new drug development - Part II: Good laboratory practice, metabolism, pharmacokinetics, safety and dose translation to clinical studies

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

Ação de extratos de plantas medicinais sobre a motilidade do trato gastrointestinal / Action of medicinal plant extracts on the gastrointestinal tract motility

Muitas plantas são utilizadas pela população para o tratamento e a cura de doenças. Entre elas encontram-se a Persea major Kopp, Piper mollicomum Kunth. e Serjania erecta Radlk. as quais são utilizadas para diversas enfermidades, inclusive para tratar distúrbios do trato gastrointestinal. O objetivo deste trabalho foi estudar os efeitos dos extratos dessas três plantas sobre a motilidade gastrointestinal. Camundongos Swiss foram tratados com os extratos pela via oral 1 hora antes da administração de uma solução semisólida de carboximetilcelulose 1,5% e vermelho de fenol 0,05% e, após 15 minutos, o esvaziamento gástrico e o trânsito intestinal avaliados. O extrato hidroalcoólico da P. major (100 a 1000 mg Kg-1, p.o.) e o extrato hidroalcoólico da P. mollicomum (100 e 300 mg Kg-1, p.o.) aumentaram o trânsito intestinal. No entanto, somente o extrato da P. major (100 e 300 mg Kg-1) também aumentou o esvaziamento gástrico. O extrato etanólico da S. erecta (100 a 1000 mg Kg-1, p.o.) não alterou a motilidade gastrointestinal. Estes resultados sugerem que a Persea major e a Piper mollicomum mereçam estudos mais aprofundados em busca de princípios ativos ou matéria vegetal efetiva para o tratamento de distúrbios do trato gastrointestinal como a constipação.

Many plants are popularly used for the treatment and healing of diseases. The Persea major Kopp, Piper mollicomum Kunth. and Serjania erecta Radlk. are used in several illnesses, including the treatment of disorders of the gastrointestinal tract. The aim of this study was to evaluate the effects of the extracts of these plants on the gastrointestinal motility. Swiss mice were orally treated with extracts one hour before the administration of a semisolid solution of 1.5% carboxymethylcellulose and 0.05% phenol red. After 15 minutes, the gastric emptying and intestinal transit were determined. The hydroalcoholic extract of P. major (100 to 1000 mg Kg-1, p.o.) and the hydroalcoholic extract of P. mollicomum (100 and 300 mg Kg-1, p.o.) increased the intestinal transit. However, only the P. major extract (100 and 300 mg Kg-1) increased the gastric emptying. The ethanolic extract of S. erecta (100 to 1000 mg Kg-1, p.o.) did not alter the gastrointestinal motility. These results suggest that Persea major and Piper mollicomum can be of interest for further studies in the search of active principles or effective plant material for the treatment of disorders of the gastrointestinal tract, such as constipation.

A 3-O-methylated mannogalactan from Pleurotus pulmonarius: structure and antinociceptive effect.

A polysaccharide (Mw 2.39x10(4)g/mol) was extracted with cold water from the basidiomycete Pleurotus pulmonarius, and its antinociceptive and anti-inflammatory properties were evaluated. It was a mannogalactan (MG), whose structure was characterized using mono- and two-dimensional NMR spectroscopy, methylation analysis, and a controlled Smith degradation. It had a main chain of (1-->6)-linked alpha-D-galactopyranosyl and 3-O-methyl-alpha-D-galactopyranosyl units, both of which are partially substituted at O-2 by beta-D-mannopyranosyl non-reducing ends. The MG was tested for its effects on the acetic acid-induced writhing reaction in mice, a typical model for inflammatory pain, causing a marked and dose-dependent inhibition of the nociceptive response, with ID50 of 16.2 (14.7-17.7)mg/kg and inhibition of 93+/-3% at a dose of 30mg/kg. An inflammatory response was not inhibited.

Bioactive glass as a drug delivery system of tetracycline and tetracycline associated with beta-cyclodextrin.

The aim of this study was to evaluate the physical-chemical properties, in vivo biocompatibility and antimicrobial activity of bioactive glasses (BG) used as a controlled release device for tetracycline hydrochloride and an inclusion complex formed by tetracycline and beta-cyclodextrin at 1:1 molar ratio. The BG as well as their compounds loaded with tetracycline (BT) and tetracycline:beta-cyclodextrin (BTC) were characterized by FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry and by scanning electron microscopy and energy dispersive spectroscopy. The in vivo test was carried out with female mice split into three groups treated with bioactive glass either without drugs, or associated with tetracycline, or with tetracycline:beta-cyclodextrin by subcutaneous implantation. The histological examination of tissue at the site of implantation showed moderate inflammatory reactions in all groups after 72 h. The bacterial effect was tested on A. actinomycetemcomitans suspended in BHI broth, with or without bioactive particles. A considerable bacteriostatic activity was found with BT and BTC glasses, as compared to plain glass. The presence of cyclodextrin was important to slow down the release of tetracycline for a long period of time and it was verified that the presence of tetracycline or its inclusion complex, tetracycline:beta-cyclodextrin, did not affect the bioactivity of the glass.

The "early' CD4+CD8+ stage of thymocte differentiation hallmarks the end of a strong positive correlation between extracellular matrix receptor expression and protein tyrosine phosphorylation.

We used irradiation-induced thymic regression/reconstitution to study phosphotyrosine (PTyr) levels and expression of extracellular matrix receptors in thymocyte subsets by flow cytometry. High PTyr levels (PTyr(hi)) characterized cells from the CD4-CD8-(DN)CD25in/hi to the "early" CD4-CD8+(DP)CD25- stage. Correlation indexes (R) between the percentages of these PTyrhi cells and cells with up-regulated expression of alpha4 integrin (alpha4hi) were strongly positive (R= 0.91, P= 0.002, for DN; R= 0.98, P= 0.0001 for DP). At the "early" DP stage, R between PTyrhi cells and cells with up-regulated expression of alpha5 integrin and L-selectin (alpha5hi and L-sel(hi)) also rendered strongly positive (R>0.95, p<0.0003). "Late" expanding DP cells exhibited intermediate PTyr levels (PTyr(in)), associated with a down-regulation of the adhesion receptors assessed. Triple-labeling suggested that in most early CD3-/lo cells, alpha4hi and alpha5hi, but not L-sel(hi) expression preceded a PTyr(hi) content. CD3in/hi-enriched CD8+ cells were also PTyr(hi), but conversely to the immature ones exhibited a tendency for a negative R between PTyr(hi) and alpha4hi (R = -0.93, P = 0.067, n= 4) or alpha5hi cells (R = -0.77, P = 0.23, n = 4). CD4+ cells were either PTyr(hi) or PTyr(in), exhibiting a tendency for a positive R (R = 0.59, P = 0.124, n= 8) between PTyr(hi) and L-sel(hi) cells only. In conclusion, our results associate an up-regulation of alpha4 and alpha5 chains expression with PTyr(hi) levels and, as elsewhere published, with increased adhesion to fibronectin up to the "early" DP stage, but not afterwards.

Automated Talairach atlas labels for functional brain mapping.

An automated coordinate-based system to retrieve brain labels from the 1988 Talairach Atlas, called the Talairach Daemon (TD), was previously introduced [Lancaster et al., 1997]. In the present study, the TD system and its 3-D database of labels for the 1988 Talairach atlas were tested for labeling of functional activation foci. TD system labels were compared with author-designated labels of activation coordinates from over 250 published functional brain-mapping studies and with manual atlas-derived labels from an expert group using a subset of these activation coordinates. Automated labeling by the TD system compared well with authors' labels, with a 70% or greater label match averaged over all locations. Author-label matching improved to greater than 90% within a search range of +/-5 mm for most sites. An adaptive grey matter (GM) range-search utility was evaluated using individual activations from the M1 mouth region (30 subjects, 52 sites). It provided an 87% label match to Brodmann area labels (BA 4 & BA 6) within a search range of +/-5 mm. Using the adaptive GM range search, the TD system's overall match with authors' labels (90%) was better than that of the expert group (80%). When used in concert with authors' deeper knowledge of an experiment, the TD system provides consistent and comprehensive labels for brain activation foci. Additional suggested applications of the TD system include interactive labeling, anatomical grouping of activation foci, lesion-deficit analysis, and neuroanatomy education.

Upregulated expression of fibronectin receptors underlines the adhesive capability of thymocytes to thymic epithelial cells during the early stages of differentiation: lessons from sublethally irradiated mice.

A 250-cGy whole-body gamma-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3(-)CD25(hi)CD44(+) and CD3(-)CD25(in/hi)CD44(-) cells occurred. Enhanced expression of L-selectin, alpha4, and alpha5 integrin chains (L-selhi alpha4(hi) alpha5(hi)) was also exhibited by these cells. This pattern of expression was maintained until the CD4(+)CD8(+) (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4(+) or CD8(+) single positive (SP) thymocytes (L-selin alpha4(in) alpha5(in)). In some recently generated SP cells, alpha4 expression was downregulated before the alpha5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the alpha6 integrin chain was downregulated only in maturing CD4(+) cells. Importantly, the increased expression of L-selectin and alpha4 and alpha5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the alpha4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for alpha5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the beta1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25(+) precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Epidermal growth factor modulates fetal thymocyte growth and differentiation.

In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44/CD25-. Some developed into gammadeltaTCR+ cells with a mature (CD3+) phenotype, but not into alphabetaTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for alphabetaTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity.

Epidermal growth factor (EGF) modulates fetal thymocyte growth and differentiation: partial reversal by insulin, mimicking by specific inhibitors of EGF receptor tyrosine kinase activity, and differential expression of CD45 phosphatase isotypes.

We have recently reported that epidermal growth factor (EGF) modulates thymocyte development in fetal thymus organ cultures. Exogenously added EGF arrested thymocyte growth and differentiation, acting at the transition from the CD4-CD8- (double-negative (DN)) to the CD4+CD8+ (double-positive (DP)) phenotype. In this study, we further investigate some molecular aspects of this blockade. This inhibitory effect could be mimicked by tyrphostins, which are selective inhibitors of EGF receptor kinase activity. An attempt to use insulin (INS) as a synergizing effector resulted in partial restoration of lobe cellularity, leading to expansion of the CD44-CD25+ DN subset. However, INS did not overcome the EGF-driven blockade of the thymocyte DN --> DP transition. Analysis of CD45 phosphatase showed that this transition was preceded by a rise in CD45RB isotype expression. At the end of a 7-day culture, the remaining DN cells from both EGF- and EGF+INS-treated fetal thymus organ cultures showed a CD45RB- phenotype and were negative for the EGF-immunoreactive molecule described previously on the fetal thymocyte surface. This finding implies that neither molecule is related to the growth capability of cells at this early developmental stage; it is more likely that the molecules are related to subsequent events in the thymocyte pathway to the DP phenotype. Thus, our data support the concept that EGF receptor-related circuitry may be relevant in thymus ontogeny. Additionally, evidence is provided for the duality between growth and differentiation at this particular early stage of thymocyte development.

Radio- and chemoprotection of bone marrow cells by opposite cell cycle-acting cytokines.

Cytokines such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), stem cell factor (SCF), and interleukin-12 (IL-12), among others, are presently known to exert a radioprotective effect on bone marrow (BM) precursor cells. IL-1, TNF-alpha, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1alpha) exert a chemoprotective effect on BM cells, while a putative role of IL-12 in this effect is still unknown. IL-1, SCF, and IL-12 are known to promote BM precursor cell cycling. Conversely, TNF-alpha, MIP-1alpha, and TGF-beta, the latter a radiosensitizer, induce cycle arrest in these cells. Cycling increases radioprotection, while arrest reduces chemical damage. IL-1 and TNF-alpha are unique in their ability to induce detoxifying mechanisms. The present communication overviews these effects. It also proposes a model, based on the induction of biochemical detoxifying mechanisms, aiming to explain BM cell radio- and chemoprotection by opposite cell cycle-acting cytokines.

Automated labeling of the human brain: a preliminary report on the development and evaluation of a forward-transform method.

A forward-transform method for retrieving brain labels from the 1988 Talairach Atlas using x-y-z coordinates is presented. A hierarchical volume-occupancy labeling scheme was created to simplify the organization of atlas labels using volume and subvolumetric components. Segmentation rules were developed to define boundaries that were not given explicitly in the atlas. The labeling scheme and segmentation rules guided the segmentation and labeling of 160 contiguous regions within the atlas. A unique three-dimensional (3-D) database label server called the Talairach Daemon (http://ric.uthscsa.edu/projects) was developed for serving labels keyed to the Talairach coordinate system. Given an x-y-z Talairach coordinate, a corresponding hierarchical listing of labels is returned by the server. The accuracy and precision of the forward-transform labeling method is now under evaluation.

In vivo and in vitro expression of tenascin by human thymic microenvironmental cells.

Increasing evidence reveals that extracellular matrix components can be regarded as a group of mediators in intrathymic T-cell migration and/or differentiation. Yet, little is known about the expression and putative function of one particular extracellular matrix protein, namely, tenascin in the thymus. Herein we investigated, by means of immunocytochemistry, tenascin expression in normal infant and fetal human thymuses, as well as in cultures of thymic microenvironmental cells. In situ, tenascin distribution is restricted to the medulla and cortico-medullary regions of normal thymuses. This pattern thus differed from that of fibronectin, laminin and type IV collagen, in which subseptal basement membranes were strongly labeled. Interestingly, tenascin did not co-localize with the cytokeratin-defined thymic epithelial cell network. This was in keeping with the in vitro data showing that tenascin-bearing cells were nonepithelial (and probably nonfibroblastic) microenvironmental elements. Studies with fetal thymuses revealed a developmentally regulated expression of tenascin, with a faint but consistent network labeling, in thymic rudiments as early as 12 weeks of gestational age, that progressed to a strong TN expression at 18 weeks of fetal development, which was similar to the distribution pattern observed thereafter, including postnatally. Our results clearly indicated that tenascin is constitutively expressed in the human thymus, since early stages of thymic ontogeny, and suggest that the cell type responsible for its secretion is a nonepithelial microenvironmental cell.

Interleukin-1 and tumor necrosis factor-alpha as radio- and chemoprotectors of bone marrow.

Administration of interleukin 1 (IL-1) or tumor necrosis factor-alpha (TNF alpha) protects bone marrow precursor cells (BMPC) from ionizing radiation and antineoplastic drugs. The time of injection is critical: the best protective results being obtained when cytokines are given around 24h prior to the induced injury. Multiple daily cytokine injections that precede irradiation or drug administration are more effective than single ones although single doses are quite effective at increasing survival in mice. Protection is positively correlated with both rapid granulocyte recovery and BMPC survival. Mechanisms involved in BMPC radioprotection include: (1) push to the S/G2 + M or arrest in the G0 phases of the cell cycle by IL-1 or TNF alpha, respectively, and (2) induction of mitochondrial manganous superoxide dismutase synthesis. For BMPC chemoprotection, proposed mechanisms are: (1) increase of aldehyde dehydrogenase synthesis, and (2) modulation of multiple-drug resistant gene expression. Stimulation of glutathione synthesis in BMPC could be operating in both radio- and chemoprotection. These findings point to the relevance of IL-1 or TNF alpha in cancer therapy as a means of reducing BMPC sensitivity to cytoreductive drugs or irradiation (including radioimmunotherapy) as well as in in vitro tumor cell purging with drugs in autologous BMT. Prior administration of these cytokines should be also considered for people in imminent danger of exposure to radiation.

A modified assay for measuring thymocyte co-stimulatory activity.

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays.