Use of preclinical and early clinical data for dose selection of a selective estrogen receptor modulator toremifene in treatment of breast cancer

In development of human medicines, it is important to predict early and accurately enough the disease and patient population to be treated as well as the effective and safe dose range of the studied medicine. This is pursued by using preclinical research models, clinical pharmacology and early clinical studies with small sample sizes. When successful, this enables effective development of medicines and reduces unnecessary exposure of healthy subjects and patients to ineffectice or harmfull doses of experimental compounds. Toremifene is a selective estrogen receptor modulator (SERM) used for treatment of breast cancer. Its development was initiated in 1980s when selection of treatment indications and doses were based on research in cell and animal models and on noncomparative clinical studies including small number of patients. Since the early development phase, the treatment indication, the patient population and the dose range were confirmed in large comparative clinical studies in patients. Based on the currently available large and long term clinical study data the aim of this study was to investigate how the early phase studies were able to predict the treatment indication, patient population and the dose range of the SERM. As a conclusion and based on the estrogen receptor mediated mechanism of action early studies were able to predict the treatment indication, target patient population and a dose range to be studied in confirmatory clinical studies. However, comparative clinical studies are needed to optimize dose selection of the SERM in treatment of breast cancer.

Cirurgia endoscópica transluminal por orifícios naturais: realidade atual

Natural orifice transluminal endoscopic surgery (NOTES) is an emerging experimental alternative to conventional surgery that eliminates abdominal incisions and incision-related complications by combining endoscopic and laparoscopic techniques to diagnose and treat abdominal pathology. Natural orifice transluminal endoscopic surgery refers to the method of accessing the abdominal cavity through a natural orifice under endoscopic visualization. Since its introduction in 2004, numerous reports have been published describing different surgical interventions. Recently, a group of expert laparoscopic surgeons and endoscopists outlined the limitations of this approach and issued recommendations for progress toward human trials. Transluminal surgery is a new method for accessing the abdomen under direct endoscopic visualization. Preliminary studies have demonstrated the feasibility of this technique in animal models; however, further research is warranted to validate its safety in humans.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT) led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC) offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed™) was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT) in an experimental model.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

Evaluation of a model for induction of periodontal disease in dogs

There are several methods for inducing periodontal disease in animal models, being the bone defect one of the most reported. This study aimed to evaluate this model, through clinical, radiographic, tomographic and histological analyzes, thus providing standardized data for future regenerative works. Twelve dogs were subjected to the induction protocol. In a first surgical procedure, a mucoperiosteal flap was made on the buccal aspect of the right third and fourth premolars and a defect was produced exposing the furcation and mesial and distal roots, with dimensions: 5mm coronoapical, 5mm mesiodistal, and 3mm buccolingual. Periodontal ligament and cementum were curetted and the defect was filled with molding polyester, which was removed after 21 days on new surgical procedure. Clinical and radiographic examinations were performed after the two surgeries and before the collection of parts for dental tomography and histological analysis. All animals showed grade II furcation exposure in both teeth. Clinical attachment level increased after induction. Defect size did not change for coronoapical and buccolingual measurements, while mesiodistal size was significantly higher than at the time of defect production. Radiographic analysis showed decreased radiopacity and discontinuity of lamina dura in every tooth in the furcation area. The horizontal progression of the disease was evident in micro-computed tomography and defect content in the histological analysis. Therefore, it is concluded that this method promotes the induction of periodontal disease in dogs in a standardized way, thus being a good model for future work.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Response threshold to aversive stimuli in stimulated early protein-malnourished rats

Two animal models of pain were used to study the effects of short-term protein malnutrition and environmental stimulation on the response threshold to aversive stimuli. Eighty male Wistar rats were used. Half of the pups were submitted to malnutrition by feeding their mothers a 6% protein diet from 0 to 21 days of age while the mothers of the other half (controls) were well nourished, receiving 16% protein. From 22 to 70 days all rats were fed commercial lab chow. Half of the animals in the malnourished and control groups were maintained under stimulating conditions, including a 3-min daily handling from 0 to 70 days and an enriched living cage after weaning. The other half was reared in a standard living cage. At 70 days, independent groups of rats were exposed to the shock threshold or to the tail-flick test. The results showed lower body and brain weights in malnourished rats when compared with controls at weaning and testing. In the shock threshold test the malnourished animals were more sensitive to electric shock and environmental stimulation increased the shock threshold. No differences due to diet or environmental stimulation were found in the tail-flick procedure. These results demonstrate that protein malnutrition imposed only during the lactation period is efficient in inducing hyperreactivity to electric shock and that environmental stimulation attenuates the differences in shock threshold produced by protein malnutrition

Aluminum induces lipid peroxidation and aggregation of human blood platelets

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 µM) and n-propyl gallate (NPG) (100 µM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 µM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

Molecular biology of the kallikrein-kinin system: from structure to function

The participation of the kallikrein-kinin system, comprising the serine proteases kallikreins, the protein substrates kininogens and the effective peptides kinins, in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The use of different experimental set-ups in concert with the development of potent and specific inhibitors and antagonists for the system has highlighted its importance but the results still lack conclusivity. Over the last few years, transgenic and gene-targeting technologies associated with molecular biology tools have provided specific information about the elusive role of the kallikrein-kinin system in the control of blood pressure and electrolyte homeostasis. cDNA and genomic sequences for kinin receptors B2 and B1 from different species were isolated and shown to encode G-protein-coupled receptors and the structure and pharmacology of the receptors were characterized. Transgenic animals expressing an overactive kallikrein-kinin system were established to study the cardiovascular effects of these alterations and the results of these investigations further corroborate the importance of this system in the maintenance of normal blood pressure. Knockout animals for B2 and B1 receptors are available and their analysis also points to the role of these receptors in cardiovascular regulation and inflammatory processes. In this paper the most recent and relevant genetic animal models developed for the study of the kallikrein-kinin system are reviewed, and the advances they brought to the understanding of the biological role of this system are discussed.

DNA vaccines for viral diseases

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

Bi-directional actions of estrogen on the renin-angiotensin system

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogen's inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the renin-angiotensin vasoconstrictor/vasodilator system is to promote the antihypertensive effect.

Effect of policosanol on cerebral ischemia in Mongolian gerbils

Policosanol is a mixture of higher aliphatic primary alcohols isolated from sugar cane wax, whose main component is octacosanol. An inhibitory effect of policosanol on platelet aggregation and cerebral ischemia in animal models has been reported. Thus, the objective of the present study was to evaluate the effect of policosanol on cerebral ischemia induced by unilateral carotid ligation and bilateral clamping and recirculation in Mongolian gerbils. Policosanol (200 mg/kg) administered immediately after unilateral carotid ligation and at 12- or 24-h intervals for 48 h significantly inhibited mortality and clinical symptoms when compared with controls, whereas lower doses (100 mg/kg) were not effective. Control animals showed swelling (tissue vacuolization) and necrosis of neurons in all areas of the brain studied (frontal cortex, hippocampus, striatum and olfactory tubercle), showing a similar injury profile. In the group treated with 200 mg/kg policosanol swelling and necrosis were significantly reduced when compared with the control group. In another experimental model, comparison between groups showed that the brain water content of control gerbils (N = 15) was significantly higher after 15 min of clamping and 4 h of recirculation than in sham-operated animals (N = 13), whereas policosanol (200 mg/kg) (N = 19) significantly reduced the edema compared with the control group, with a cerebral water content identical to that of the sham-operated animals. cAMP levels in the brain of control-ligated Mongolian gerbils (N = 8) were significantly lower than those of sham-operated animals (N = 10). The policosanol-treated group (N = 10) showed significantly higher cAMP levels (2.68 pmol/g of tissue) than the positive control (1.91 pmol/g of tissue) and similar to those of non-ligated gerbils (2.97 pmol/g of tissue). In conclusion, our results show an anti-ischemic effect of policosanol administered after induction of cerebral ischemia, in two different experimental models in Mongolian gerbils, suggesting a possible therapeutic effect in cerebral vascular disorders.

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Perinatal development and adult blood pressure

A growing body of evidence supports the concept of fetal programming in cardiovascular disease in man, which asserts that an insult experienced in utero exerts a long-term influence on cardiovascular function, leading to disease in adulthood. However, this hypothesis is not universally accepted, hence animal models may be of value in determining potential physiological mechanisms which could explain how fetal undernutrition results in cardiovascular disease in later life. This review describes two major animal models of cardiovascular programming, the in utero protein-restricted rat and the cross-fostered spontaneously hypertensive rat. In the former model, moderate maternal protein restriction during pregnancy induces an increase in offspring blood pressure of 20-30 mmHg. This hypertensive effect is mediated, in part, by fetal exposure to excess maternal glucocorticoids as a result of a deficiency in placental 11-ß hydroxysteroid dehydrogenase type 2. Furthermore, nephrogenesis is impaired in this model which, coupled with increased activity of the renin-angiotensin system, could also contribute to the greater blood pressure displayed by these animals. The second model discussed is the cross-fostered spontaneously hypertensive rat. Spontaneously hypertensive rats develop severe hypertension without external intervention; however, their adult blood pressure may be lowered by 20-30 mmHg by cross-fostering pups to a normotensive dam within the first two weeks of lactation. The mechanisms responsible for this antihypertensive effect are less clear, but may also involve altered renal function and down-regulation of the renin-angiotensin system. These two models clearly show that adult blood pressure is influenced by exposure to one of a number of stimuli during critical stages of perinatal development.