Patrizia Bottoni

Glycolytic enzyme inhibitors in cancer treatment

Background: The radio- and chemotherapeutics currently used for the treatment of cancer are widely known to be characterized by a low therapeutic index. An interesting approach to overcoming some of the limits of these techniques is the exploitation of the so-called Warburg effect, which typically characterizes neoplastic cells. Interestingly, this feature has already been utilized with good results, but only for diagnostic purposes (PET and SPECT). From a pharmacological point of view, drugs able to perturb cancer cell metabolism, specifically at the level of glycolysis, may display interesting therapeutic activities in cancer. Objective: The pharmacological actions of these glycolytic enzyme inhibitors, based primarily on ATP depletion, could include: i) amelioration of drug selectivity by exploiting the particular glycolysis addiction of cancer cell; ii) inhibition of energetic and anabolic processes; iii) reduction of hypoxia-linked cancer-cell resistance; iv) reduction of ATP-dependent multi-drug resistance; and v) cytotoxic synergism with conventional cancer treatments. Conclusion: Several glycolytic inhibitors are currently in preclinical and clinical development. Their clinical value as anticancer agents, above all in terms of therapeutic index, strictly depends on a careful reevaluation of the pathophyiological role of the unique metabolism of cancer cells in general and of Warburg effect in particular.

Nitric oxide donor drugs: an update on pathophysiology and therapeutic potential

The discovery of the multiple physiological and pathophysiological processes in which nitric oxide (NO) is involved has promoted a great number of pharmacological researches to develop new drugs that are capable of influencing NO production directly and/or indirectly for therapeutic purposes (i.e, NO-releasing drugs, NO-inhibiting drugs, and phosphodiesterase V inhibitors). In particular, the so-called NO donor drugs could actually have an important therapeutic effect in the treatment of many diseases such as arteriopathies (atherosclerosis and its sequelae, arterial hypertension and some forms of male sexual impotence), various acute and chronic inflammatory conditions (colitis, rheumatoid arthritis and tissue remodelling), and several degenerative diseases (Alzheimer’s disease and cancer). The old organic nitrates show some well-known pitfalls including the induction of tolerance and acute side effects related to abrupt vasodilation such as cephalea and hypotension, which limit their therapeutic indications. A low therapeutic index (i.e., peroxynitrite toxicity) has always characterised the sydnonimines class. A series of interesting new classes of NO donors are under intense pharmacological investigation and scrutiny (S-nitrosothiols, diazeniumdiolates and NO hybrid drugs), each characterised by a particular pharmacokinetic and pharmacodynamic profile. The most important obstacle in the field of NO donor drugs is represented by the difficulty in targeting NO release, and thereby its effects, to a particular tissue.

Pharmacological Modulation of Nitric Oxide Release: New Pharmacological Perspectives, Potential Benefits and Risks

Nitric oxide is becoming an increasingly important signalling molecule implicated in a growing number of physiological and pathophysiological processes. Moreover, with the recent advances in nitric oxide biochemistry, many well known drugs have been shown to act totally or partially by modulating NO metabolism with varying therapeutic results. The classic organic nitrates have been shown to exhibit beneficial therapeutic but suffer from some well known pitfalls (tolerability induction, abrupt cephalea and hypotension). Similarly, sydnonimines, another well known class of NO donor drugs, have a characteristically low therapeutic index (i.e., cyanide toxicity). At present, pharmacological researchers are designing and synthesising various chemical compounds capable of modulating NO metabolism for therapeutic purposes that also possess an optimal therapeutic index. Specifically, various new classes of NO donors are under intense pharmacological investigation (such as S-nitrosothiols, diazeniumdiolates, furoxans, zeolites and so on), each characterised by a particular pharmacokinetic and pharmacodynamic profile. To known the pharmacological development of these new NO donor drugs could help to ameliorate the use of these molecules in various therapeutic protocols. In fact, the pharmacologically modulated nitric oxide release showed to have an important therapeutic impact in the treatment of diseases such as arteriopathies, various acute and chronic inflammatory conditions, and several degenerative diseases. At present, the most important obstacle in the field of new NO donor drugs seems to be carefully targeting NO release to a particular tissue at an optimal concentration, so as to achieve a beneficial action and to limit possible toxic effects.

An update on pharmacological approaches to neurodegenerative diseases

Neurodegenerative diseases are now generally considered as a group of disorders that seriously and progressively impair the functions of the nervous system through selective neuronal vulnerability of specific brain regions. Alzheimer’s disease is the most common neurodegenerative disease, followed in incidence by Parkinson’s disease; much less common are frontotemporal dementia, Huntington’s disease, amyothrophic lateral sclerosis (Lou Gehrig’s disease), progressive supranuclear palsy, spinocerebellar ataxia, Pick’s disease and, lastly, prion disease. In this review, the authors intend to survey new drugs in different clinical phases but not in the preclinical or discovery stages nor already in the market, with new molecules aimed at interrupting or at attenuating different pathogenic pathways of neurodegeneration and/or at ameliorating symptoms. Drugs in different pharmacological phases are under study or are ready to be introduced into therapy for Alzheimer’s disease, which display anti-β-amyloid activity or nerve growth factor-like activity or anti-inflammatory properties. Other drugs possess mixed mechanisms of action, such as acetylcholinesterase inhibition and impairment of β-amyloid formation through inhibition of β-amyloid precursor protein synthesis and/or modulation of secretase activity. Other therapeutic approaches are based on immunotherapy, control of metal ions interactions with β-amyloid and ensuing oxidative reactions as well as metabolic or hormonal regulation. The symptomatic therapy of motor behaviour in Parkinson’s disease, based on l-DOPA, is registering adenosine A2A receptor antagonists, monoamine oxidase B inhibitors and ion channel modulators, as well as dopamine uptake inhibitors and glutamate AMPA receptor antagonists. There are also many other drugs involved, including astrocyte-modulating agents, 5-HT1A agonists and α2-adrenergic receptor antagonists, which are targeted at preventing or ameliorating Parkinson’s disease-related or l-DOPA-induced dyskinesias. Huntington’s disease therapy envisages a Phase III drug, LAX-101, which displays antiapoptotic properties by promoting membrane stabilisation and mitochondrial integrity. Other drugs with antioxidant and antiapoptotic steroid-like and neuroprotective activity are under investigation for the therapy of the less common neurodegenerative diseases.

Neuron-Specific Enolase as a Biomarker: Biochemical and Clinical Aspects

Neuron-specific enolase (NSE) is known to be a cell specific isoenzyme of the glycolytic enzyme enolase. In vertebrate organisms three isozymes of enolase, expressed by different genes, are present: enolase α is ubiquitous; enolase β is muscle-specific and enolase γ is neuron-specific. The expression of NSE, which occurs as γγ- and αγ-dimer, is a late event in neural differentiation, thus making it a useful index of neural maturation.NSE is a highly specific marker for neurons and peripheral neuroendocrine cells. As a result of the findings of NSE in specific tissues under normal conditions, increased body fluids levels of NSE may occur with malignant proliferation and thus can be of value in diagnosis, staging and treatment of related neuroendocrine tumours (NETs).NSE is currently the most reliable tumour marker in diagnosis, prognosis and follow-up of small cell lung cancer (SCLC), even though increased levels of NSE have been reported also in non-small cell lung cancer (NSCLC). The level of NSE correlates with tumour burden, number of metastatic sites and response to treatment.NSE can be also useful at diagnosis of NETs and gastroenteropancreatic (GEP)-NETs.Raised serum levels of NSE have been found in all stages of neuroblastoma, although the incidence of increased concentration is greater in widespread and metastatic disease. Moreover, NSE determination in cord blood offers an early postnatal possibility of confirming the diagnosis of neuroblastoma in newborns.NSE has been demonstrated to provide quantitative measures of brain damage and/or to improve the diagnosis and the outcome evaluation in ischaemic stroke, intracerebral hemorrhage, seizures, comatose patients after cardiopulmonary resuscitation for cardiac arrest and traumatic brain injury.Increased NSE serum levels have also been found associated with melanoma, seminoma, renal cell carcinoma, Merkel cell tumour, carcinoid tumours, dysgerminomas and immature teratomas, malignant phaechromocytoma, Guillain-Barré syndrome and Creutzfeldt-Jakob disease.

3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a New Class of Synthetic Histone Deacetylase Inhibitors. 2. Effect of Pyrrole-C2 and/or -C4 Substitutions on Biological Activity

Previous SAR studies (Part 1: Mai, A.; et al. J. Med. Chem. 2003, 46, 512-524) performed on some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C2 ethene chains of 1a-c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC50(2f) = 0.77 microM; IC50(1a) = 3.8 microM). IC50 values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C(4) position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC50 = 0.043 microM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 microM).

Mitochondrial dysfunction by synthetic ligands of peroxisome proliferator activated receptors (PPARs).

PPARs are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation and cell differentiation. This spectrum of biological activities stimulated pharmacological research to synthetize different molecules with PPARs binding activity with beneficial therapeutic effects. As a matter of fact, some synthetic PPAR‐ligands have been already employed in pharmacotherapy: PPAR‐α ligands, such as fibrates, are used in hyperlipidemias and thiazolidinediones, mainly PPAR‐γ ligands, are employed as insulin sensitizers. However, both classes of drugs showed pharmacotoxicological profiles which cannot be fully ascribed to activation of their specific receptors and which are causing a growing incidence of dramatic side effects (rhabdomyolysis, acute liver failure, heart failure, etc.). A re‐evaluation of the biological activities of PPAR synthetic ligands, in particular of the mitochondrial dysfunction based on a rotenone‐like Complex I partial inhibition and of its consequent metabolic adaptations, seems to explain some of the pathophysiologic aspects of PPARs allowing a better definition of the therapeutic properties of the so‐called PPAR‐ligands. IUBMB Life, 56: 477‐482, 2004

CA 19-9: Biochemical and Clinical Aspects

CA19-9 (carbohydrate antigen 19-9, also called cancer antigen 19-9 or sialylated Lewis a antigen) is the most commonly used and best validated serum tumor marker for pancreatic cancer diagnosis in symptomatic patients and for monitoring therapy in patients with pancreatic adenocarcinoma. Normally synthesized by normal human pancreatic and biliary ductal cells and by gastric, colon, endometrial and salivary epithelia, CA 19-9 is present in small amounts in serum, and can be over expressed in several benign gastrointestinal disorders. Importantly, it exhibits a dramatic increase in its plasmatic levels during neoplastic disease. However, several critical aspects for its clinical use, such as false negative results in subjects with Lewis (a-b-) genotype and false positive elevation, occasional and transient, in patients with benign diseases, together with its poor positive predictive value (72.3 %), do not make it a good cancer-specific marker and renders it impotent as a screening tool. In the last years a large number of putative biomarkers for pancreatic cancer have been proposed, most of which is lacking of large scale validation. In addition, none of these has showed to possess the requisite sensitivity/specificity to be introduced in clinical use. Therefore, although with important limitations we well-know, CA 19-9 continues being the only pancreatic cancer marker actually in clinical use.

Bezafibrate as differentiating factor of human myeloid leukemia cells

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.

Cancer stem cells: the development of new cancer therapeutics

Introduction: Cancer stem cells (CSCs) are a subpopulation of tumor cells with indefinite proliferative potential that drive the growth of tumors. CSCs seem to provide a suitable explanation for several intriguing aspects of cancer pathophysiology. Areas covered: An explosion of therapeutic options for cancer treatment that selectively target CSCs has been recorded in the recent years. These include the targeting of cell-surface proteins, various activated signalling pathways, different molecules of the stem cell niche and various drug resistance mechanisms. Importantly, approaching cancer research by investigating the pathogenesis of these intriguing cancer cells is increasing the knowledge of the pathophysiology of the disease, emphasizing certain molecular mechanisms that have been partially neglected. Expert opinion: The characterization of the molecular phenotype of these cancer stem-like cells, associated with an accurate definition of their typical derangement in cell differentiation, can represent a fundamental advance in terms of diagnosis and therapy of cancer. Preliminary results seem to be promising but further studies are required to define the therapeutic index of this new anticancer treatment. Moreover, understanding the pathogenetic mechanisms of CSCs can expand the therapeutic applications of normal adult stem cells by reducing the risk of uncontrolled tumorigenic stem cell differentiation.