Showing papers in "Current Pharmaceutical Design in 2000"

Antitumor properties of podophyllotoxin and related compounds.

Abstract: The lignan family of natural products includes compounds with important antineoplastic and antiviral properties such as podophyllotoxin and two of their semisynthetic derivatives, etoposide and teniposide. The latter are included in a wide variety of cancer chemotherapy protocols. Due to these biological activities, lignans, and especially cyclolignans, have been the objective of numerous studies focused to prepare better and safer anticancer drugs. The mechanism by which podophyllotoxin blocks cell division is related to its inhibition of microtubule assembly in the mitotic apparatus. However, etoposide and teniposide were shown not to be inhibitors of microtubule assembly which suggested that their antitumor properties were due to another mechanism of action, via their interaction with DNA and inhibition of DNA topoisomerase II. Other podophyllotoxin derivatives has also been reported which retained or even improved the cytotoxic activity, but these were weak inhibitors of topoisomerase II in vitro; the data revealed that such analogs exhibit a different, as yet unknown, mechanism of action. The main deficiency of these compounds is their cytotoxicity for normal cells and hence side effects derived from their lack of selectivity against tumoral cells. In this regard it is necessary to investigate and prepare new more potent and less toxic analogs, that is, with better therapeutic indices. It is well accepted from structure-activity studies in this field that the trans-lactones are more potent as antineoplastics than the cis-lactones. Not only the configuration of the D ring is an important factor for high cytotoxic activity, but also a quasi-axial arrangement of the E ring is necessary. On this basis, studies on lignans have been addressed to modify the lactone moiety and prepare analogs with heteroatoms at different positions of the cyclolignan skeleton. Our group has been working during the last few years on chemical transformations of podophyllotoxin and analogs and we have prepared a large number of cyclolignan derivatives some of which display potent antiviral, immunosuppressive and cytotoxic activities. We have reported several new cytotoxic agents with nitrogen atoms at C-7 or C-9 or at both C-7 and C-9: imine derivatives, oxime derivatives, pyrazoline-, pyrazo- and isoxazoline-fused cyclolignans. At present, we are preparing mainly new compounds by modifications of the A and E cyclolignan-rings. They are being tested on cultures of different tumoral cell lines (P-388 murine leukemia, A-549 human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human melanoma) and some of them have shown an interesting and selective cytotoxicity.

Cell Cycle Molecular Targets in Novel Anticancer Drug Discovery

Abstract: A number of potential molecular targets for novel anticancer drug discovery have been identified in cell cycle control mechanisms. Prominent among these are the regulatory proteins, cyclins and their effector counterparts the cyclin dependent kinases (CDKs). Aberrant expression of these proteins, particularly cyclins involved in the G1 phase of the cell cycle, namely the D and E cyclins, has been associated with a variety of human cancers, including breast and colorectal cancer, B-lymphoma, prostate and non-small cell lung cancer. Inhibition of CDK kinase activity has turned out to be the most productive strategy for the discovery and design novel anticancer agents specifically targeting the cell cycle. Other potentially useful cell cycle areas for exploration include cyclin-CDK interactions, Cdc25 activation of cyclin-CDK complexes, ubiquitin-mediated proteolysis of cyclins, cell cycle check point kinases like Chk1, and recently identified oncogenic cell cycle-related aurora and polo-like kinases. Potent specific inhibitors have been identified that bind to the ATP site of CDKs, mainly cyclin B-CDK1, cyclin A-CDK2, and cyclin D-CDK4 complexes, and inhibit kinase activity. X-ray crystallographic data of CDKs, and their complexes with inhibitors have played a major role in the success of drug discovery efforts. Combinatorial chemistry, highthroughput screening, functional genomics and informatics have also contributed. CDK inhibitors currently under investigation include flavopiridol, olomoucine, roscovitine, puvalanol B, the dihydroindolo[3,2-d][1]benzazepinone kenpaullone, indirubin-3 -monoxime and novel diaminothiazoles such as AG12275. The anticancer therapeutic potential of CDK inhibitors has been demonstrated in preclinical studies, and Phases I and II clinical trials in cancer patients are currently underway.

G-Quadruplex DNA as a target for drug design.

Abstract: G-rich DNA sequences can adopt unusual four-stranded DNA structures, called G-quadruplex DNA. Variations in the molecularity, topology, strand orientation, and glycosidic conformation of the G-quadruplex DNA provide a diverse array of structures. Although G-quadruplex structures have only been observed in vitro, strong indirect evidence for their existence in vivo comes from the characterization of G-quadruplex DNA binding proteins, helicases, and nucleases characterization of G-quadruplex DNA binding proteins, belicases, and n Telomeres are structures on the ends of chromosomes that are required for chromosomal stability. Telomeric DNA contains a single-stranded G-rich DNA overhang, which may adopt a G-quadruplex structure. Telomere shortening has been implicated in cellular senescence. Telomerase is an enzyme which synthesizes the G-rich strand of telomere DNA. Telomerase activity is highly correlated with cancer and may allow cancer cells to escape senescence. Based on these observations, telomerase has been proposed as a potential target for anticancer drug design, The targeting of telomerase is associated with potential problems, including the existence in some cancer cells of telomerase-independent mechanisms for telomere maintenance, and the long delay time between telomerase inhibition and effects on proliferation. One promising approach for inhibiting telomerase involves targeting the G-quadruplex DNA structures thought to be involved in telomere and telomerase function. Compounds that specifically bind G-quadruplex DNA may interact directly with telomeres, in addition to inhibiting telomerase, and produce more immediate antiproliferative effects. The diamidoanthraquinones, porphyrins, and perylene diimides have all been shown to bind G-quadruplex DNA and inhibit telomerase. Most of these compounds also bind double-stranded DNA and are cytotoxic at the concentrations required to inhibit telomerase; however, certain perylene diimides appear to be non-cytotoxic, G-quadruplex selective telomerase inhibitors. Biological characterization of such compounds may provide validation for the concept of the G-quadruplex as a target in drug design.

Astatine-211-Labeled Radiotherapeutics An Emerging Approach to Targeted Alpha-Particle Radiotherapy

Abstract: Targeted radiotherapy or endoradiotherapy is an appealing approach to cancer treatment because of the potential for delivering curative doses of radiation to tumor while sparing normal tissues. Radionuclides that decay by the emission of alpha-particles such as the heavy halogen astatine-211 (211At) offer the exciting prospect of combining cell-specific molecular targets with radiation having a range in tissue of only a few cell diameters. Herein, the radiobiological advantages of alpha-particle targeted radiotherapy will be reviewed, and the rationale for using 211At for this purpose will be described. The chemistry of astatine is similar to that of iodine; however, there are important differences which make the synthesis and evaluation of 211At-labeled compounds more challenging. Perhaps the most successful approach that has been developed involves the astatodemetallation of tin, silicon or mercury precursors. Astatine-211 labeled agents that have been investigated for targeted radiotherapy include [211At]astatide, 211At- labeled particulates, 211At-labeled naphthoquinone derivatives, 211At-labeled methylene blue, 211At-labeled DNA precursors, meta-[211At]astatobenzylguanidine, 211At-labeled biotin conjugates, 211At-labeled bisphosphonates, and 211At-labeled antibodies and antibody fragments. The status of these 211At-labeled compounds will be discussed in terms of their labeling chemistry, cytotoxicity in cell culture, as well as their tissue distribution and therapeutic efficacy in animal models of human cancers. Finally, an update on the status of the first clinical trial with an 211At-labeled targeted therapeutic, 211At-labeled chimeric anti-tenascin antibody 81C6, will be provided.

Immunology of VIP: A Review and Therapeutical Perspectives

Abstract: Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad distribution in the body that exerts very important pleiotropic functions in several systems. The present work reviews the immunology of VIP. Being daring, this neuropeptide could be included in the group of cytokines since it is produced and secreted by different immunocompetent cells in response to various immune signals, plays a broad spectrum of immunological functions, and exerts them, in a paracrine and/or autocrine way, through three different specific receptors. Although VIP has been classically considered as an immunodepressant agent, and its main described role has been as an anti-inflammatory factor, several evidences suggest that a better way to see this peptide is as a modulator of the homeostasis of the immune system. In the last decade, the pharmacology of VIP has spectacularly grown, and VIP itself, as well as more stable VIP-derived agents, have been used or proposed as efficient therapeutical treatments of several disorders, specially inflammatory and autoimmune diseases, such as septic shock, rheumatoid arthritis, multiple sclerosis, Crohn's disease and autoimmune diabetes. A broad field of perspectives is actually open, and further investigations will help us to definitively understand the immunology of this very important peptide.

Mechanisms of anti-cancer agents: emphasis on oxidative stress and electron transfer.

Abstract: A large body of evidence has accumulated indicating involvement of oxidative stress (OS) in the mode of action of various bioactive substances, including those of the immune system. The data for anticancer drugs (main and miscellaneous) are summarized herein. Although diverse origins pertain, reactive oxygen species (ROS) are frequently generated by redox cycling via electron transfer (ET) groups, such as quinones (or phenolic precursors), metal complexes (or complexors), aromatic nitro compounds (or reduced products) and conjugated imines (or iminium species). We believe it is not coincidental that these functionalities are frequently found in anticancer agents or their metabolites. Generally, the ET moieties display reduction potentials in the physiologically active range. Often ROS are also implicated in more traditional rationales, namely, enzyme inhibition, membrane or DNA insult, and interference with DNA or protein synthesis. A multi-faceted approach to mechanism appears to be the most logical. Significantly, the unifying theme of ET-OS also applies to other drug categories, as well as to toxins, carcinogens, hormones, and enzymes. Since this theoretical framework aids in our understanding of drug action, it can serve as a useful tool in the design of more active and safer pharmaceuticals.

Understanding and exploiting nature's chemical arsenal: the past, present and future of calicheamicin research.

Abstract: The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology, and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the critical research discoveries relevant to the clinically most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clinical studies are also highlighted.

Leptin transport across the blood-brain barrier: implications for the cause and treatment of obesity.

Abstract: Leptin has emerged as a major regulator of adiposity. Leptin is released into the blood from fat cells and circulates to the brain where it crosses the blood-brain barrier (BBB) to act at receptors within the central nervous system to affect appetite, thermogenesis, and a number of other actions. In humans and in many rodent models, resistance to leptin appears to be a chief cause of obesity. Determining the cause of leptin resistance is fundamental to developing strategies for the use of leptin in obesity. The literature characterizing the transport of leptin across the BBB is reviewed. This literature strongly suggests that the cause of leptin resistance is due a decreased transport of leptin across the BBB in obese humans and rodents. The main cause of this resistance appears to be an impairment in the activity of the transporter rather than just simply saturation at higher doses. Strategies to overcome impaired BBB transport are reviewed, including the use of allosteric regulators and the delivery of material by the intrathecal route.

Therapeutic applications for ligands of retinoid receptors.

Abstract: Synthetic retinoids, ligands for the RAR and RXR members of the steroid/thyroid superfamily of nuclear hormone receptors, are used for the treatment of psoriasis, acne, photoaging and cancer. Retinoid mechanisms of action for these conditions largely involve effects on epithelial differentiation and modulation of inflammation with some impact on the immune system. Retinoid medicinal chemistry in recent years has identified ligands highly specific for one of the three RAR subtypes (RAR-alpha) and for the RXR family of receptors, as well as antagonists for the RARs, RARalpha and the RXRs. Structure-activity relationships among the novel retinoid classes are reviewed along with potential therapeutic activities and side effects. RAR-alpha specific retinoids inhibit cancer cell growth but lack other retinoid toxicities, including skin irritation now ascribed to RAR-gama. RXR-specific retinoids lower blood glucose in animal models of type 2 diabetes albeit with a potential for mild hypothyroidism. Function-selective retinoids, especially a class of RAR antagonists called inverse agonists, have unexpected gene regulatory activity. Given the diverse properties and tissue distributions of the retinoid receptors, synthesis of additional classes of receptor-specific and function-selective ligands has the potential to produce novel therapeutic applications.

Cathepsin K and the design of inhibitors of cathepsin K

Abstract: Cathepsin K, a cysteine protease of the papain family, was identified by sequencing complementary DNA libraries derived from osteoclasts. Cathepsin K can cleave bone proteins such as Type I collagen, osteopontin, and osteonectin. The localization and maturation of cathepsin K in activated osteoclasts have been characterized. Furthermore, mutation of the gene expressing cathepsin K in humans results in pycnodysostosis, an autosomal recessive condition, resulting in osteoprosis and increased bone fragility. Knockout of cathepsin K in the mouse also results in retarded bone matrix degradation and osteopetrosis. Together, these data demonstrate that inhibition of cathepsin K should result in a dimunition of osteoclast-mediated bone resorption. Several novel classes of cathepsin K inhibitors have been designed from X-ray co-crystal structures of peptide aldehydes bound to papain. The convergence of the design of novel inhibitors and the discovery of cathepsin K has created opportunities to further understand bone and cartilage biology as well as provide new therapeutic agents for the treatment of disease states in man such as osteoporosis.

Seocalcitol (EB 1089) A Vitamin D Analogue of Anti-cancer Potential. Background, Design, Synthesis, Pre-clinical and Clinical Evaluation

Abstract: It is well established that the metabolically active form of vitamin D, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH)2 D 3 ) plays a key role in the establishment and maintenance of the calcium metabolism in the body. In addition to this classic effect of 1alpha,25(OH)2 D 3 , substantial evidence has emerged demonstrating that 1alpha,25(OH)2 D 3 is able to regulate cell growth and differentiation in a number of different cell types, including cancer cells. However, the clinical usefulness of 1alpha,25(OH)2 D 3 is limited by its tendency to cause hypercalcaemia. Much effort has therefore been directed to identifying new vitamin D analogues with potent cell regulatory effects, but with weaker effects on the calcium metabolism than those of 1alpha,25(OH)2 D 3 . One of these new synthetic analogues is Seocalcitol (EB 1089). Despite being 50-200 times more potent than 1alpha,25(OH)2 D 3 with respect to regulation of cell growth and differentiation in vitro as well as in vivo, EB 1089 displays a reduced calcaemic activity in vivo compared to that of 1alpha,25(OH)2 D 3 . These characteristics make EB 1089 a potentially useful compound for the treatment of cancer. Recent clinical evaluation of EB 1089 has focused mainly on establishing a maximum tolerated dose in cancer patients. Early results confirm that the low calcaemic activity observed in animals can be reproduced in the clinic. Furthermore, EB 1089 has been shown to induce regression of tumours, especially in hepatocellular carcinoma where complete remission has been obtained. In conclusion, the development of EB 1089 as an anti-cancer drug holds promise. However, its final evaluation must await the completion of ongoing controlled clinical trials.

MDM2 oncogene as a novel target for human cancer therapy.

Abstract: The MDM2 oncogene was first cloned as an amplified gene on a murine double-minute chromosome in the 3T3DM cell line, a spontaneously transformed derivative of BALB/c 3T3 cells. The MDM2 oncogene has now been shown to be amplified or overexpressed in many human cancers. It also has been suggested that MDM2 levels are associated with poor prognosis of several human cancers. The most exciting finding is the MDM2-p53 autoregulatory feedback loop that regulates the function of the p53 tumor suppressor gene. The MDM2 gene is a target for direct transcriptional activation by p53, and the MDM2 protein is a negative regulator of p53. The MDM2 oncoprotein binds to the p53 protein, inhibiting p53 functions as a transcription factor and inducing p53 degradation. The p53 tumor suppressor has an important role in cancer therapy, with p53-mediated cell growth arrest and/or apoptosis being major mechanisms of action for many clinically used cancer chemotherapeutic agents and radiation therapy. Therefore, the MDM2-p53 interaction may be a target for cancer therapy. In addition, the negative regulation of p53 by MDM2 may limit the magnitude of p53 activation by DNA damaging agents, thereby limiting their therapeutic effectiveness. If the MDM2 feed-back inhibition of p53 is interrupted, a significant increase in functional p53 levels will increase p53-mediated therapeutic effectiveness. Several approaches have now been tested using this strategy, including polypeptides targeted to MDM2-p53 binding domain and antisense oligonucleotides that specifically inhibit MDM2 expression. In addition to the interaction with p53, the MDM2 protein has been found to have interactions with other cellular proteins such as pRb and E2F-1. Although the exact function and significance of these interactions are not fully understood, the p53-independent functions of MDM2 may have a role in cancer etiology and progression, indicating that the MDM2 oncogene is a potential molecular target for cancer therapy.

Conotoxins-New Vistas for Peptide Therapeutics

Abstract: There are approximately 500 species of predatory cone snails within the genus Conus. They comprise what is arguably the largest single genus of marine animals alive today. It has been estimated that the venom of each Conus species has between 50 and 200 components. These highly constrained sulfur rich components or conotoxins represent a unique arsenal of neuropharmacologically active peptides that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes. Remarkable divergence occurs when cone snails speciate. Consequently, the complement of venom peptides in any one Conus species is distinct from that of any other species. Hence many thousands of peptides that modulate ion channel function are present within Conus venoms. Evolutionary pressures have afforded a "pre-optimized," structurally sophisticated library that has been "fine tuned" over 50 million years. The statistics associated with sampling such libraries bear testimony to the validity and feasibility of this strategy. Although approximately 100 conotoxin sequences have been published in the scientific literature, representing a mere 0.2 % of the estimated library size, this sample has already afforded a peptide of proven clinical utility and several pre-clinical leads for CNS disorders. Conus libraries represent a rich pharmacopoeia and the potential to "therapeutically mine" such a resource appears limitless. The paucity of synthetic methodologies necessary to achieve the regioisomeric folding patterns present in these native peptides precludes access to synthetic conotoxin libraries, further validating the overall "mining" strategy. In this article, we will present a pragmatic overview of the molecular diversity as well as the neurobiological mechanisms that define each major class of conotoxin.

Recent developments in receptor-selective retinoids.

Abstract: Natural (all trans-retinoic acid, RA) and synthetic retinoids exhibit potent anti-proliferative, normalization of differentiation and anti-inflammatory activities which appear to account for their therapeutic effects in acne, psoriasis, photoaging, precancerous lesions and established cancers. Although RA has shown considerable promise in dermatologic indications, certain side effects have restricted its use as a choice of agent for chronic administration. Systematic synthesis of receptor-selective retinoids has resulted in two topical drugs, Tazorac/Zorac (tazarotene) and Differin (adapalene). Tazorac is indicated for psoriasis and acne and Differin gel for the treatment of acne. These drugs bind to the retinoic acid receptor (RAR) family members. Various RAR subtype-specific and function-selective retinoids have been synthesized. These retinoids, which are in various stages of pre-clinical development for the treatment of cancers, psoriasis and as an antidote to Accutane-mediated mucocutaneous toxicity, will also be discussed in this review. Discovery of another retinoid receptor, retinoid X receptor (RXR), revealed that RXR-specific retinoids already existed in retinoid chemical libraries. Structure activity relationship studies based upon binding and transactivation assays led to the synthesis of RXR-specific ligands with high affinities for RXR subtypes. These compounds were found to be effective in the treatment of hyperglycemia in animal models of type II diabetes, The discovery of novel retinoids along with an increased understanding of the biological functions and mechanisms of action of retinoid receptors are likely to result in improved treatments for existing responsive indications and identification of new retinoid therapeutic targets.

Drugs with estrogen-like potency and brain activity: potential therapeutic application for the CNS.

Abstract: Numerous reports, ranging from molecular investigations to clinical studies, demonstrate the potency of estrogens to modulate brain function and their implications in schizophrenia and depression. Alterations of dopaminergic, cholinergic, GABAergic, glutamatergic and serotonergic neurotransmission through estrogen-mediated mechanisms have been consistently established. Moreover, studies using in vivo and in vitro models as well as epidemiological data suggest that estrogens provide neuroprotection of central nervous system (CNS) cells implicated in the etiology of neurodegenerative disorders such as Alzheimers (AD) and Parkinsons (PD) diseases. Numerous genomic or non-genomic mechanisms of actions of estrogens in the brain have been documented implicating classical nuclear estrogen receptors as well as possible estrogen membrane receptors, antioxidant activity of steroids, their effect on fluidity as well as on antiapoptotic proteins and growth factors. Selective estrogen receptor modulators (SERMs) have estrogenic and/or antiestrogenic activity depending on the target tissue. Hence, SERMs have the same beneficial effect as estrogen in skeleton and cardiovascular systems but act as antagonists in breast and uterus. The finding of beneficial side effects of SERMs in the CNS might improve their risk-benefit ratio in traditional indications. In this review, we will survey schizophrenia and depression as examples of mental diseases and AD and PD as neurodegenerative diseases. We will review brain effects of estrogens, steroids possibly acting as pro-drugs of estrogens such as testosterone and dehydroepiandrosterone (DHEA) and present novel findings with SERMs. Drugs with estrogen activity in the brain may have therapeutic potential either by modulating brain neurotransmitter transmission or through neuroprotective activity.

Fibroblast growth factors and their inhibitors.

Abstract: Fibroblast growth factors (FGFs) are members of a family of polypeptides synthesized by a variety of cell types during the processes of embryonic development and in adult tissues. FGFs have been detected in normal and malignant cells and show a biological profile that includes mitogenic and angiogenic activity with a consequent crucial role in cell differentiation and development. To activate signal transduction pathways, FGFs use a dual receptor system based on tyrosine kinases and heparan sulfate (HS) proteoglycans. Based on these considerations, a variety of inhibitors able to block the interactions between FGFs and their receptors have been designed and investigated for their biological properties related to antiangiogenesis and antitumor activity. In this paper, in addition to an extensive description of the FGF family members, we report several compounds acting as FGF inhibitors by direct interaction with the growth factors. Suramin and other diverse polyanionic polysulfated and polysulfonated compounds are described, with a particular focus on suradistas. For this class of molecules, by means of molecular modeling procedures, a binding model to FGF-2 has been proposed and the structure-activity relationships of suradistas have been analyzed on the basis of the computational model described.

The importance of vitamin A in nutrition.

Abstract: Preformed vitamin A (all-trans-retinol and its esters) and provitamin A (beta-carotene) are essential dietary nutrients that provide a source of retinol. Both retinyl esters and beta-carotene are metabolized to retinol. The retinol-binding proteins on binding retinol provide a means for solubilizing retinol for delivery to target tissues and for regulating retinol plasma concentrations. Oxidation of retinol provides retinal, which is essential for vision, and retinoic acid, a transcription factor ligand that has important roles in regulating genes involved in cell morphogenesis, differentiation, and proliferation. The observations that vitamin A can produce cell and tissue changes similar to those found during neoplastic transformation and that vitamin supplementation can reverse this process indicated a potential role for vitamin A in cancer prevention. Thus far, correlative epidemiological studies on vitamin A use and cancer prevention have produced mixed results, as this review indicates. Apparently, in populations deficient in vitamin A (caused by an inadequate diet or tobacco use), supplementation programs appear to be effective in reducing cancer incidence. In groups already having sufficient dietary or supplemental vitamin A, cancer prevention by added vitamin A may not be particularly effective. The most likely reason for the low efficacy in the latter groups is that feedback mechanisms that increase retinol storage in the liver limit retinol plasma levels; whereas, supplementation at higher doses causes toxicity. In addition to serving as a metabolic source of retinol, beta-carotene, along with other dietary carotenoids, function as antioxidants that can prevent carcinogenesis by decreasing the levels of the free-radicals that cause DNA damage.

Angiogenesis: new targets for the development of anticancer chemotherapies.

Abstract: Angiogenesis is the process by which new blood vessels are formed from preexisting microvasculature. To ensure an adequate blood supply, tumor cells release angiogenic factors that are capable of promoting nearby blood vessels to extend vascular branches to the tumor. In addition, larger tumors have been shown to release angiogeneic inhibitory factors that prevent blood vessels from sending branches to smaller, more distant tumors that compete for oxygen and nutrients. Angiogenesis is a complex multistep biochemical process, and offers several potential molecular targets for non-cytotoxic anticancer therapies. Strategies for exploiting tumor angiogenesis for novel cancer drug discovery include: (i) inhibition of proteolytic enzymes that breakdown the extracellular matrix surrounding existing capillaries; (ii) inhibition of endothelial cell migration; (iii) inhibition of endothelial cell proliferation; (iv) enhancement of tumor endothelial cell apoptosis. There is also a host of miscellaneous agents that inhibit angiogenesis for which the specific mechanisms are not clear. Several methods have been developed for measuring antiangiogenic activity both in vitro and in vivo. Although there has been intensive research efforts focused at the phenomena of angiogenesis, as well as the search for antiangiogenic agents for more than two decades, many questions remain unanswered with regard to the overall biochemical mechanisms of the angiogenesis process and the potential therapeutic utility of angiogenic inhibitors. Nevertheless potent angiogenic inhibitors capable of blocking tumor growth have been discovered, and appear to have potential for development into novel anticancer therapeutics. However there are still hurdles to be overcome before these inhibitors become mainstream therapies.

Cyclooxygenases in the Central Nervous System: Implications for Treatment of Neurological Disorders

Abstract: Recognition of two isoforms of cyclooxygenase and reports that nonsteroidal anti-inflammatory drugs may be beneficial in devastating neurological conditions such as Alzheimer's disease have led to increased interest in cyclooxygenase function in the nervous system. In the present paper we review current data on the multiplicity of cyclooxygenase and prostaglandin mediated effects in the central nervous system (CNS). We discuss CNS cells types, including neurons, glia, and cerebrovascular elements, where cyclooxygenases- 1 and -2 are expressed under normal conditions or can be induced by physiological or pathological stimuli. We also address physiological processes such as pain sensitization, CNS inflammation and fever induction that are regulated or modified by cyclooxygenase activity. Finally, we describe potential roles of cyclooxygenase in neurological diseases and rationales for nonsteroidal anti-inflammatory drug use in the treatment of neurodegenerative disorders, stroke and CNS injury.

GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects.

Abstract: In the mid seventies a drug design programme using the Amanita muscaria constituent muscimol (7) as a lead structure, led to the design of guvacine (23) and (R)-nipecotic acid (24) as specific GABA uptake inhibitors and the isomeric compounds isoguvacine (10) and isonipecotic acid (11) as specific GABAA receptor agonists. The availability of these compounds made it possible to study the pharmacology of the GABA uptake systems and the GABAA receptors separately. Based on extensive cellular and molecular pharmacological studies using 23, 24, and a number of mono- and bicyclic analogues, it has been demonstrated that neuronal and glial GABA transport mechanisms have dissimilar substrate specificities. With GABA transport mechanisms as pharmacological targets, strategies for pharmacological interventions with the purpose of stimulating GABA neurotransmission seem to be (1) effective blockade of neuronal as well as glial GABA uptake in order to enhance the inhibitory effects of synaptically released GABA, or (2) selective blockade of glial GABA uptake in order to increase the amount of GABA taken up into, and subsequently released from, nerve terminals. The bicyclic compound (R)-N-Me-exo-THPO (17) has recently been reported as the most selective glial GABA uptake inhibitor so far known and may be a useful tool for further elucidation of the pharmacology of GABA transporters. In recent years, a variety of lipophilic analogues of the amino acids 23 and 24 have been developed, and one of these compounds, tiagabine (49) containing (R)-nipecotic acid (24) as the GABA transport carrier-recognizing structure element, is now marketed as an antiepileptic agent.

Bioanalytical Applications of Accelerator Mass Spectrometry for Pharmaceutical Research

Abstract: Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14 C, 3 H, 41 Ca, and 36 Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (≤ 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (≤1 μg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

Anti-inflammatory Cytokines and Cytokine Antagonists

Abstract: Cytokines are critical mediators of protective host responses, including defense against microbial invasion and tumorigenesis. However, the production of specific proinflammatory cytokines must be tightly regulated and compartmentalized to prevent the overzealous expression of these molecules that can culminate in unabated inflammation and tissue injury. Cytokine production and/or biologic effects can be inhibited by a variety of endogenous molecules, including anti-inflammatory cytokines, soluble cytokine receptors, and receptor antagonist proteins. Additionally, synthetic molecules have been constructed to selectively block the synthesis, post-translation processing, or receptor binding of proinflammatory cytokines. Relevant anti-inflammatory cytokines and cytokine inhibitors (both endogenous and synthetic) will be the subject of this review, with a particular emphasis on those anti-inflammatory cytokines and cytokine inhibitors that have been used experimentally or clinically in the treatment of diseases that are believed to be mediated by excessive proinflammatory cytokine responses.

PET studies of brain monoamine transporters.

Abstract: Monoamine transporters are proteins mainly located on nerve terminals of dopaminergic, noradrenergic and serotonergic neurons They are members of a larger sodium dependent transporter family and represent a major mechanism terminating the action of released neurotransmitter in the synaptic cleft In addition to being important target molecules for many antidepressive drugs and substances of abuse, transporter proteins are good markers for the integrity of monoaminergic innervation Therefore, there is a growing interest for in vivo imaging studies using positron emission tomography (PET) or single photon emission computed tomography (SPECT) and ligands selective for monoamine transporters In this review, the use of monoamine transporter ligands (or tracers) for imaging studies of cocaine dependence, neurodegenerative diseases and mechanism of antidepressant drug action is discussed, with special focus on the use of PET for evaluating possible new pharmacological innovations

The search for selective ligands for the CB2 receptor.

Abstract: Following the identification of the CB2 receptor several groups explored the development of selective ligands for this receptor which occurs principally in the periphery. This led to the discovery that two cannabimimetic indoles, 1-(2, 3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholino]ethyl)-5-methoxyind ole (L768242) and 2-methyl-1-propyl-3-(1-naphthoyl)indole (JWH-015) have high affinity for the CB2 receptor with low affinity for the CB1 receptor. Shortly thereafter two 1-methoxy-delta8-THC analogues, 1-methoxy-delta8-THC-DMH (L759633) and 1-methoxy-delta9(11)-THC-DMH (L759656), were also found to have high affinity for the CB2 receptor and very little affinity for the CB1 receptor. Almost simultaneously two 1-deoxy-delta8-THC analogues, 1-deoxy-11-hydroxy-delta8-THC-DMH (JWH-051) and 1-deoxy-delta8-THC-DMH (JWH-057) were reported to have high affinity for the CB1 receptor, but even greater affinity for the CB2 receptor. These discoveries gave rise to a concerted effort by Huffman and co-workers to explore the structure-activity relationships (SAR) at CB2 of cannabimimetic indoles and 1-deoxy-delta8-THC analogues. These efforts have resulted in the synthesis and pharmacological evaluation of a number of derivatives of 3-(1-naphthoyl)indoles and 1-deoxy-delta8-THC analogues with various side chains. This review will describe the current status of the results of these studies and discuss the SAR for both these classes of ligands.

Mode of action of anti-infective agents: focus on oxidative stress and electron transfer.

Abstract: There is increasing evidence for involvement of oxidative stress (OS) in the mechanism of action of a wide variety of physiologically active materials. Often the reactive oxygen species (ROS) are generated by electron transfer (ET) or other routes mediated by free radicals. Principal ET functionalities are quinones (or precursors), metal complexes, aromatic nitro compounds (ArNO2), and conjugated imines. These moieties are commonly found in the structures of anti-infective agents or their metabolites. In most cases, the ET functionalities display reduction potentials in the physiologically active range, i.e. more positive than approximately -0.5 V. Though the focus of this review is on OS and ET, a mode of action which emulates the natural immune system of the host, in some cases, this mechanism also appears to be involved in more generally accepted approaches, such as enzyme inhibition, adverse effects on membranes and DNA, or interference with DNA or protein synthesis. OS-ET represents a broad understanding of drug action that can aid in the design of new anti-infective agents. It is significant that a relatively simple unifying theme can be applied not only to the action of the predominant groups of anti-infective agents, but also more generally to other drug classes, toxins, carcinogens, enzymes, and hormones.

Anti-tumor effects of 1,25-dihydroxyvitamin D3 and vitamin D analogs.

Abstract: The role of 1,25-dihydroxyvitamin D 3 (1,25-(OH)2 D 3 ) as a regulator of cell growth and differentiation is well recognized. Currently, 1,25-(OH)2 D 3 and vitamin D analogs are being evaluated for their therapeutic potential in the treatment of hyperproliferative disorders like cancer. In the present review, we will discuss several processes that might be involved in 1,25-(OH)2 D 3 - and vitamin D analog-mediated suppression of cancer cell growth. The effects on tumor cell proliferation, differentiation, apoptosis, angiogenesis, metastases, and parathyroid hormone-related peptide secretion will be highlighted. In addition, combination therapy with other tumor effective drugs will be addressed. Furtermore, we will focus on the potential drawbacks and the possible side effects of vitamin D compounds in the treatment of cancer.

What is the precise role of human MDR 1 protein in chemotherapeutic drug resistance

Abstract: Elucidating the molecular function of hu MDR 1 protein (also called P-glycoprotein or P-gp 1) and the precise role this protein plays in clinically relevant tumor drug resistance remains a perplexing problem. Hundreds of reports over the past decades summarize a dizzying array of observations relevant to hu MDR 1 protein function. A dominant model in the MDR literature that is used to explain many observations is the well known "drug pump" model first suggested by Keld Dano in 1973 [1]. Although this model has proved useful in conceptualizing additional experiments, it violates fundamental laws of biology and chemistry and in well over a decade of intense effort, active outward drug pumping via hu MDR 1 protein has still never been unequivocally measured. Also, in recent years it has become clear that the drug pump model cannot explain several important phenomena that are highly relevant to the cancer clinic. Thus, other models have also proved increasingly popular. One is the altered partitioning model, which does not violate fundamental laws, is consistent with the vast majority of available data, and has important predictive ability. This newer model has several novel facets that are relevant for cancer pharmacology, and that help explain phenomena not explained by the drug pump model. The basic principle of this model is that MDR proteins do not directly transport drugs, but that their altered expression leads to altered regulation of ion transport or signal transduction that is critical for setting key biophysical parameters of the cell (e.g. compartmental pH and membrane potentials) that dictate relative passive diffusion of drugs as well as important signal transduction linked to the cytotoxic actions of these drugs. Along with debate over the molecular details of hu MDR 1 function, additional controversy surrounds the precise role of hu MDR 1 in the clinic. Many investigators now debate the significance of its function (regardless of precise mechanism) with regard to "real" drug resistance phenotypes exhibited in the clinic. I believe that thorough debate on the pros and cons of various molecular models for hu MDR 1 function will help to address confusion over the clinical relevance of hu MDR1. In the current atmosphere of disappointment over the relative success of clinical trials based in large part on the logic of the drug pump model, it is important that we not lose sight of critical points. Namely, hu MDR 1 protein remains an extremely important window in on the complex pathways that lead to induced chemotherapeutic drug resistance. Exploring the rationale behind newer models for hu MDR 1 function leads to key predictions that can be tested.

Endocannabinoids: new targets for drug development.

Abstract: The possible therapeutic use of marijuanas active principles, the cannabinoids, is currently being debated. It is now known that these substances exert several of their pharmacological actions by activating specific cell membrane receptors, the CB 1 and CB 2 cannabinoid receptor subtypes. This knowledge led to the design of synthetic cannabinoid agonists and antagonists with high therapeutic potential. The recent discovery of the endocannabinoids, i.e. endogenous metabolites capable of activating the cannabinoid receptors, and the understanding of the molecular mechanisms leading to their biosynthesis and inactivation, opened a new era in research on the pharmaceutical applications of cannabinoids. Ongoing studies on the pathological and physiological conditions regulating the tissue levels of endocannabinoids, and on the pharmacological activity of these compounds and their derivatives, may provide a lead for the development of new drugs for the treatment of nervous and immune disorders, cardiovascular diseases, pain, inflammation and cancer. These studies are reviewed in this article with special emphasis on the chemical features that determine the interaction of endocannabinoids with the proteins mediating their activity and degradation.

The effect of drugs on the labeling of blood elements with technetium-99m.

Abstract: The influence of drugs on the labeling of red blood cells and plasma proteins with 99mTc has been reported. Any drug, which alters the labeling of the tracer, could be expected to modify the disposition of the radiopharmaceuticals. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. We have evaluated the effect of Thuya occidentalis, Peumus boldus and Nicotiana tabacum (tobacco) extracts on the labeling of RBC and plasma and cellular proteins with 99mTc. Blood was incubated with the drugs. Stannous chloride (SnCl2) solutions and 99mTc were added. Plasma (P) and blood cells (BC) were separated. The percentage of radioactivity (%ATI) bound to P and BC was determined. The %ATI on the plasma and cellular proteins was also evaluated by precipitation of P and BC samples with trichloroacetic acid (TCA) and isolation of soluble (SF) and insoluble (IF) fractions. The analysis of the results shows that there is a decrease in %ATI (from 97.64 to 75.89 percent) in BC with Thuya occidentalis extract. The labeling of RBC and plasma proteins can be decreased in presence of tobacco. This can be due either a direct or indirect effect (reactive oxygen species) of tobacco. The analysis of radioactivity in samples of P and BC isolated from samples of whole blood treated with Peumus boldus showed a rapid uptake of the radioactivity by blood cells in the presence of the Peumus boldus, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma. This study shows that extracts of some medicinal plants can affect the radiolabeling of red blood cells with 99mTc using an in vitro technique.

Structure-based design of compounds inhibiting Grb2-SH2 mediated protein-protein interactions in signal transduction pathways.

Abstract: Receptor protein tyrosine kinases are usually activated upon binding their growth factors, or other suitable ligands, to their extracellular domains. These activated receptors initiate cytoplasmic signalling cascades which, when aberrant, can result in different disease states, such as oncogenic transformation. Many receptor protein tyrosine kinases use Src homology 2 domains (SH2) to couple growth factor activation with intracellular signalling pathways to mediate cell control and other biological events. The characterization of the components involved in these signal transduction pathways has resulted in the identification of new attractive targets for therapeutic intervention. Such is the case for the protein-protein interactions involving the SH2 domain of growth factor receptor bound protein 2 (Grb2). Agents that specifically disrupt Grb2-SH2 binding interactions involved in aberrant signalling could potentially shut down these oncogenic pathways and thus block human malignancies. This paper reviews the structural characteristics of the Grb2-SH2 domain and the approaches which have been used to identify antagonists of the Grb2-SH2 domain. Examples have been selected from our own research to illustrate how the unique structural features of the ligand-bound Grb2-SH2 have been exploited to design potent and selective Grb2-SH2 antagonists.