[A short history of anti-rheumatic therapy--VII. Biological agents]. / Piccola storia della terapia antireumatica--VII. I farmaci "biologici".

The introduction of biological agents has been a major turning-point in the treatment of rheumatic diseases, particularly in rheumatoid arthritis. This review describes the principle milestones that have led, through the knowledge of the structure and functions of nucleic acids, to the development of production techniques of the three major families of biological agents: proteins, monoclonal antibodies and fusion proteins. A brief history has also been traced of the cytokines most involved in the pathogenesis of inflammatory rheumatic diseases (IL-1 and TNF) and the steps which have led to the use of the main biological drugs in rheumatology: anakinra, infliximab, adalimumab, etanercept and rituximab.

Peri-operative management of percutaneous fetoscopic spina-bifida repair: a descriptive review of five cases from the United Kingdom, with focus on anaesthetic implications.

We present a case-based review of the first five percutaneous fetoscopic in-utero spina bifida repair procedures undertaken in the UK. Our focus is on implications of anaesthesia and analgesia for the mother and fetus, provision of uterine relaxation and fetal immobilisation while providing conditions conducive to surgical access. Minimising risks for fetal acidosis, placental and fetal hypoperfusion, maternal and fetal sepsis and maternal fluid overload were the foremost priorities. We discuss optimisation strategies undertaken to ensure fetal and maternal well-being under anaesthesia, shortcomings in the current approach, and possible directions for improvement.

Nucleic acid aptamers based on the G-quadruplex structure: therapeutic and diagnostic potential.

Aptamers are short DNA- or RNA-based oligonucleotides selected from large combinatorial pools of sequences for their capacity to efficiently recognize targets ranging from small molecules to proteins or nucleic acid structures. Like antibodies, they exhibit high specificity and affinity for target binding. As a result, they may display effective interference in biological processes, which renders them not only valuable diagnostic tools, but also promising therapeutic agents. In fact, one aptamer that inhibits human VEGF already received approval for the treatment of age-related macular degeneration, while several others are undergoing clinical trials. Aptamers display a large number of structural arrangements, which accounts for their binding efficiency and selectivity for unrelated targets. Among several architectures, the G-quadruplex (G-4) is adopted by several aptamers, the most popular of which shows inhibitory properties against thrombin, a pharmacologically relevant protein. G-4 structures consist of planar arrays of four guanines, each guanine pairing with two neighbours by Hoogsteen bonding. Recent work shows that G-4 arrangement is highly polymorphic and therefore represents a large family of stable structures with a common overall fold, but with well differentiated recognition elements that allow prominent diversity to be explored. Conformational plasticity consents fine tuning of target recognition as obtained by aptamer selection. Here, we will review the present knowledge on aptamers based on the G-4 structures and assess their diagnostic and therapeutic potential as biotech drugs for the detection and treatment of severe pathologies including vascular, cancer and viral diseases.

The topoisomerase II poison clerocidin alkylates non-paired guanines of DNA: implications for irreversible stimulation of DNA cleavage.

Clerocidin, a diterpenoid with antibacterial and antitumor activity, stimulates in vitro DNA cleavage mediated by mammalian and bacterial topoisomerase (topo) II. Different from the classical topoisomerase poisons, clerocidin-stimulated breaks at guanines immediately preceding the sites of DNA cleavage are not resealed upon heat or salt treatment. To understand the mechanism of irreversible trapping of the topo II-cleavable complex, we have investigated the reactivity of clerocidin per se towards DNA. We show here that the drug is able to nick negatively supercoiled plasmids. DNA cleavage by clerocidin in enzyme-free medium is due to the ability of the drug to form covalent adducts with guanines. Indeed, clerocidin was able to specifically react with short oligonucleotides when the guanines were unpaired and exposed as in bulges or in the single-strand form. The clerocidin epoxy group attacks the nitrogen at position 7 of guanines, leading to strand scission at the modified site. Our findings also demonstrate that trapping of topoisomerases by clerocidin is specific for type II enzymes. The guanine-alkylating ability of clerocidin suggests an unprecedented mechanism of topo II poisoning, according to which the enzyme renders the drug reactive toward DNA by distorting the double-helical structure of the nucleic acid at the cleavage site.

Biologics targeted at TNF: design, production and challenges.

Several biotech-derived drugs aimed at Tumor Necrosis Factor (TNF) have been licensed in the last years, profoundly changing the therapy of several autoimmune diseases based on inflammation, affecting the life of patients and bringing to the market attention the growth potentials of biologics directed at cytokines. The proof of principles that led to the design of these compounds dates back from the nineties, when the involvement of TNF in rheumatoid arthritis was proved by the ability of specific anti-TNF proteins to modulate the inflammatory response in animal models. Monoclonal antibodies aimed at neutralizing the excess TNF were developed with therapeutic purposes, and a chimeric and a full human antibody are now approved for several clinical indications. The design of soluble receptors able to bind and neutralize human TNF paralleled the development of antibodies as therapeutics, and the clinical success of these drugs was achieved by the clever design of a novel recombinant dimeric protein, consisting of the extracellular portion of human TNF receptor linked to the constant portion of a human immunoglobulin. All approved biologics designed to bind and neutralize TNF were obtained through the power of biotechnological methods: the development of these important biopharmaceutical products, their means of production and the challenges they face will be analyzed here in details.

Identification of topoisomerase I as the cytotoxic target of the protoberberine alkaloid coralyne.

Protoberberine alkaloids (coralyne and its derivatives), which exhibit antileukemic activity in animal models, have been shown to be potent inducers of topoisomerase (topo) I-DNA cleavable complexes using purified recombinant human DNA topo I. Different from the structurally similar benzophenanthridine alkaloid nitidine (a dual poison of both topos I and II), coralyne and its derivatives have marginal poisoning activity against DNA topo II. Yeast cells expressing human DNA topo I are shown to be specifically sensitive to killing by coralyne derivatives and nitidine, suggesting that cellular DNA topo I is their cytotoxic target. Two human camptothecin-resistant cell lines, CPT-K5 and A2780/CPT-2000, which are known to express highly camptothecin-resistant topo I, are only marginally resistant to coralyne derivatives and nitidine. Purification of human topo I from Escherichia coli cells overexpressing CPT-K5 recombinant topo I has demonstrated similar marginal cross-resistance to nitidine. It seems possible to develop coralyne and nitidine derivatives as new topo I-targeted therapeutics to overcome aspects of camptothecin-related resistance.

On the mechanism of action of quinolone drugs.

Antibacterial quinolones are thought to inhibit DNA gyrase by trapping the enzyme as a complex with the DNA substrate. The precise molecular details of drug-DNA and drug-enzyme interactions remain controversial. Here, a model is proposed that accounts for the influence of magnesium ions on quinolone-DNA binding.

Drugs acting on the beta isoform of human topoisomerase II (p180).

Topoisomerase II is the target of several anticancer agents. The discovery of a second enzyme, called topoisomerase II beta, genetically distinct from alpha, prompted the investigation on the different functional roles of the two isoforms. Whereas the first recognized isozyme is essential for life due to its role in chromosome condensation and segregation, beta functions remained elusive, although its importance in neural development is appearing clearer. Topoisomerase II beta is regulated differently than alpha, and its level of expression does not change significantly during cell cycle. The presence of this isoform in non-proliferating cells suggests that drug preferentially aimed at beta may be active in slow growing tumors. Topoisomerase II poisons were hence evaluated in light of their selectivity toward one or the other isozyme, indicating how the beta isoform may represent an important target for selected classes of drugs. Newer compounds were also synthesized and tested for their potential antitumor activity and their topoisomerase II beta poisoning. The literature dealing with "old" and "new" drugs targeted at topoisomerase II is reviewed trying to link, whenever possible, selective poisoning and cytotoxic effects to chemical structures, in the hope to indicate new lead compounds that will contribute to unveil molecular determinants of selectivity.

Drugs acting on DNA topoisomerases: recent advances and future perspectives.

DNA-topoisomerases, a family of DNA-processing enzymes, represent the pharmacological target of major clinically useful chemotherapeutic agents. These drugs essentially act by trapping a topoisomerase-DNA cleavable complex, an intermediate in the enzyme s catalytic cycle. Research activity in this field continues to grow exponentially, resulting in a wealth of new information on the functional role and the biochemical and structural properties of the enzymes. In addition, the drug pharmacophores have been further characterized, along with their sequence preferences, and key interactions with the target macromolecules are being unveiled. This review will discuss the recent advances in elucidating the mode of action of DNA-topoisomerases and of topoisomerase-targeted anticancer agents.

Synthesis and evaluation of terbenzimidazoles as topoisomerase I inhibitors.

The synthesis and pharmacological activity of a series of terbenzimidazoles are described. The ability of these derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These analogs were also assayed for their cytotoxicity in RPMI 8402 cells and the camptothecin-resistant CPT-K5 cells. In addition the potential for these compounds to serve as substrates for MDR1 was also determined. Several terbenzimidazoles exhibited similar cytotoxicity against variants of human tumor cells that either overexpress MDR1 or are camptothecin-resistant.

Substituted 2,5'-Bi-1H-benzimidazoles: topoisomerase I inhibition and cytotoxicity.

Several 2'-aryl-5-substituted-2,5'bi-1H-benzimidazole derivatives were synthesized and evaluated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell line RPMI 8402. This study focused on 18 2,5'-bi-1H-benzimidazole derivatives which contained either a 5-cyano, a 5-(aminocarbonyl), or a 5-(4-methylpiperazinyl) group. Among these bibenzimidazoles, the pharmacological activity of 2'-phenyl derivatives and the influence of the different positional isomers of either a 2'-tolyl group or a 2'-naphthyl moiety on cytotoxicity and topoisomerase I inhibitory activity were determined.

Peptidyl anthraquinones as potential antineoplastic drugs: synthesis, DNA binding, redox cycling, and biological activity.

A series of new compounds containing a 9,10-anthracenedione moiety and one or two peptide chains at position 1 and/or 4 have been synthesized. The amino acid residues introduced are glycine (Gly), lysine (Lys), and tryptophan (Trp), the latter two in both the L- and D-configurations. The peptidyl anthraquinones maintain the ability of intercalating efficiently into DNA, even though the orientation within the base-pair pocket may change somewhat with reference to the parent drugs mitoxantrone (MX) and ametantrone (AM). The interaction constants of the mono-, di-, and triglycyl derivatives are well comparable to those found for AM but 5-10 times lower than the value reported for MX. On the other hand, the glycyl-lysyl compounds bind DNA to the same extent as (L-isomer) or even better than (D-isomer) MX. As for the parent drugs without peptidyl chains, the new compounds prefer alternating CG binding sites, although to different extents. The bis-Gly-Lys derivatives are the least sensitive to base composition, which may be due to extensive aspecific charged interactions with the polynucleotide backbone. As far as redox properties are concerned, all peptidyl anthraquinones show a reduction potential very close to that of AM and 60-80 mV less negative than that of MX; hence, they can produce free-radical-damaging species to an extent similar to the parent drugs. The biological activity has been tested in human tumor and murine leukemia cell lines. Most of the test anthraquinones exhibit cytotoxic properties close to those of AM and considerably lower than those of MX. Stimulation of topoisomerase-mediated DNA cleavage is moderately present in representatives of the glycylanthraquinone family, whereas inhibition of the background cleavage occurs when Lys is present in the peptide chain. For most of the test anthraquinones, the toxicity data are in line with the DNA affinity scale and the topoisomerase II stimulation activity. However, in the lysyl derivatives, for which lack of cytotoxicity cannot be related to poor binding to DNA, the steric and electronic properties of the side-chain substituent must impair an effective recognition of the cleavable complex.

6-Aminoquinolones as new potential anti-HIV agents.

A series of 6-aminoquinolone compounds were evaluated for their in vitro activity against human immunodeficiency virus type 1 (HIV-1). Compound 12a, bearing a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at the C-7 position, was the most active in inhibiting HIV-1 replication on de novo infected C8166 human lymphoblastoid cell lines. The 12a EC(50) value was 0.1 microM, a 7-20-fold lower concentration relative to that for compounds 8a and 7a containing a cyclopropyl and tert-butyl substituent at the N-1 position, respectively. When the C-6 amino group was replaced with a fluorine atom, a decreased antiviral effect was observed. The observed effects are selective, since potency is substantially reduced when testing the compounds against the herpes simplex virus type 1 (HSV-1). Active quinolone derivatives very efficiently interact with TAR RNA, which suggests a nucleic acid-targeted mechanism of action.

Preferred interaction of D-peptidyl-anthraquinones with double-stranded B-DNA.

The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone-peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine-glycine dipeptide in the side chains. The D enantiomer binds right handed double stranded DNA more efficiently than the L form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the D-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The D enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development.

An unusual case of pneumoperitoneum: nephrocolic fistula due to a giant renal staghorn calculus.

Gastrointestinal perforations usually lead to pneumoperitoneum and peritonitis. Rarely, if ever described, a complete giant staghorn renal stone might cause a nephrocolic fistula with sigmoid impaction and perforation similar to gallstone ileus. Few nephrointestinal fistulae have been described in the literature and none of them were presented as an acute abdomen with pneumoperitoneum and pneumoretroperitoneum. To our knowledge, this is the only case showing CT and radiographic findings of a pathology not yet described in the literature. We named the sigmoid perforation by a renal stone ileus "Lorenzi's syndrome" after the physician who hypothesized this rare differential diagnosis based only on history and clinical examination.

In vitro selection of DNA aptamers that bind L-tyrosinamide.

We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design.

Pyrrolo-quinoline derivatives as potential antineoplastic drugs.

Some novel pyrrolo-quinoline derivatives have been synthesized as potential antineoplastic agents. They contain an angular aromatic tricyclic or tetracyclic system, to which the methanesulfon-anisidide side chain typical of amsacrine as such, or lacking the m-methoxy substituent, is connected. A methyl group can be present at position 7 of the pyrrolo-quinoline ring. The novel compounds exhibit interesting cell growth inhibitory properties when tested against the NCI panel of cell lines, in particular those obtained from solid tumors like CNS-, melanoma- and prostate-derived cells. The mechanism of cytotoxic action does not seem to be related to topoisomerase II poisoning ability. Most active proved to be compound 4a, which lacks both methyl and methoxy substituents, followed by 5a, having the methoxy group only. Biological activity is less pronounced in the tetracyclic family of derivatives 6 and 7.

Novel pyrrolo[3,2-f]quinolines: synthesis and antiproliferative activity.

Novel pyrrolo[3,2,f]quinoline derivatives have been synthesized and tested as antiproliferative agents. They are characterized by an angular aromatic tricyclic system, to which a methyl group can be bound at position 7, and by a methanesulfon-anisidide side chain as such, or lacking the m-methoxy substituent at position 1. The novel compounds were shown to exhibit cell growth inhibitory properties when tested against the NCI panel of cell lines, in particular those obtained from leukemias. Although the compounds are able to stimulate topoisomerase II poisoning at high concentration, the cell growth inhibition properties do not appear to rest principally on this mechanism of action. Overall, the most active proved to be compound 9, having the m-methoxy substituent typical of amsacrine, followed by the 7-methyl derivative 10 and by the unsubstituted compound 8. Comparison with previously investigated regioisomers shows modulation of activity dictated by the position and conformational freedom of side-chain groups.

DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors.

A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a pharmacological target of MGBLs. In the presence of calf thymus Topo I, MGBLs induced limited but highly specific single-strand DNA breaks. The 3' ends of the broken DNA strands are covalently linked to Topo I polypeptides. Protein-linked DNA breaks are readily reversed by a brief heating to 65 degrees C or the addition of 0.5 M NaCl. These results suggest that MGBLs, like camptothecin, abort Topo I reactions by trapping reversible cleavable complexes. The sites of cleavage induced by MGBLs are distinctly different from those induced by camptothecin. Two of the major cleavage sites have been sequenced and shown to be highly A + T-rich, suggesting the possible involvement of a Topo I-drug-DNA ternary complex at the sites of cleavage. Different MGBLs also exhibit varying efficiency in inducing Topo I-cleavable complexes, and the order of efficiency is as follows: Hoechst 33342 and 33258 >> distamycin A > berenil > netropsin. The lack of correlation between DNA binding and cleavage efficiency suggest that, in addition to binding to the minor grooves of DNA, MGBLs must also interact with Topo I in trapping Topo I-cleavable complexes.

Quantitation of camptothecin and related compounds.

Camptothecin and congeners represent a clinically very useful class of anticancer agents. Proper identification and quantitation of the original compounds and their metabolites in biological fluids is fundamental to assess drug metabolism and distribution in animals and in man. In this paper we will review the recent literature available on the methods used for separation and quantitative determination of the camptothecin family of drugs. Complications arise from the fact that they are chemically labile, and the pharmacologically active lactone structure can undergo ring opening at physiological conditions. In addition, a number of metabolic changes usually occur, producing a variety of active or inactive metabolites. Hence, the conditions of extraction, pre-treatment and quantitative analysis are to be carefully calibrated in order to provide meaningful results.