Engineering of needle-free physical methods to target epidermal cells for DNA vaccination

A challenge in epidermal DNA vaccination is the efficient and targeted delivery of polynucleotides to immunologically sensitive Langerhans cells. This paper investigates this particular challenge for physical delivery approaches. The skin immunology and material properties are examined in the context of the physical cell targeting requirements of the viable epidermis. Selected current physical cell targeting technologies engineered to meet these needs are examined: needle and syringe; diffusion patches; liquid jet injectors; microneedle arrays/patches; and biolistic particle injection. The operating methods and relative performance of these approaches are discussed, with a comment on potential future developments and technologies. (c) 2005 Elsevier Ltd. All rights reserved.

Numerical analysis of gas and micro-particle interactions in a hand-held shock-tube device

A unique hand-held gene gun is employed for ballistically delivering biomolecules to key cells in the skin and mucosa in the treatment of the major diseases. One of these types of devices, called the Contoured Shock Tube (CST), delivers powdered micro-particles to the skin with a narrow and highly controllable velocity distribution and a nominally uniform spatial distribution. In this paper, we apply a numerical approach to gain new insights in to the behavior of the CST prototype device. The drag correlations proposed by Henderson (1976), Igra and Takayama (1993) and Kurian and Das (1997) were applied to predict the micro-particle transport in a numerically simulated gas flow. Simulated pressure histories agree well with the corresponding static and Pitot pressure measurements, validating the CFD approach. The calculated velocity distributions show a good agreement, with the best prediction from Igra & Takayama correlation (maximum discrepancy of 5%). Key features of the gas dynamics and gas-particle interaction are discussed. Statistic analyses show a tight free-jet particle velocity distribution is achieved (570 +/- 14.7 m/s) for polystyrene particles (39 +/- 1 mu m), representative of a drug payload.

Three-factor models versus time series models:quantifying time-dependencies of interactions between stimuli in cell biology and psychobiology for short longitudinal data

Signal integration determines cell fate on the cellular level, affects cognitive processes and affective responses on the behavioural level, and is likely to be involved in psychoneurobiological processes underlying mood disorders. Interactions between stimuli may subjected to time effects. Time-dependencies of interactions between stimuli typically lead to complex cell responses and complex responses on the behavioural level. We show that both three-factor models and time series models can be used to uncover such time-dependencies. However, we argue that for short longitudinal data the three factor modelling approach is more suitable. In order to illustrate both approaches, we re-analysed previously published short longitudinal data sets. We found that in human embryonic kidney 293 cells cells the interaction effect in the regulation of extracellular signal-regulated kinase (ERK) 1 signalling activation by insulin and epidermal growth factor is subjected to a time effect and dramatically decays at peak values of ERK activation. In contrast, we found that the interaction effect induced by hypoxia and tumour necrosis factor-alpha for the transcriptional activity of the human cyclo-oxygenase-2 promoter in HEK293 cells is time invariant at least in the first 12-h time window after stimulation. Furthermore, we applied the three-factor model to previously reported animal studies. In these studies, memory storage was found to be subjected to an interaction effect of the beta-adrenoceptor agonist clenbuterol and certain antagonists acting on the alpha-1-adrenoceptor / glucocorticoid-receptor system. Our model-based analysis suggests that only if the antagonist drug is administer in a critical time window, then the interaction effect is relevant.

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

DNA Mutation of the progranulin (GRN) gene in familial frontotemporal lobar degeneration (FTLD):a study of the pathology of nine cases

Frontotemporal lobar degeneration (FTLD) with transactive response (TAR) DNA-binding protein of 43kDa (TDP-43) proteinopathy (FTLD-TDP) is a neurodegenerative disease characterized by variable neocortical and allocortical atrophy principally affecting the frontal and temporal lobes. Histologically, there is neuronal loss, microvacuolation in the superficial cortical laminae, and a reactive astrocytosis. A variety of TDP-43 immunoreactive changes are present in FTLD-TDP including neuronal cytoplasmic inclusions (NCI), neuronal intranuclear inclusions (NII), dystrophic neurites (DN) and, oligodendroglial inclusions (GI). Many cases of familial FTLD-TDP are caused by DNA mutations of the progranulin (GRN) gene. Hence, the density, spatial patterns, and laminar distribution of the pathological changes were studied in nine cases of FLTD-TDP with GRN mutation. The densities of NCI and DN were greater in cases caused by GRN mutation compared with sporadic cases. In cortical regions, the commonest spatial pattern exhibited by the TDP-43 immunoreactive lesions was the presence of clusters of inclusions regularly distributed parallel to the pia mater. In approximately 50% of cortical gyri, the NCI exhibited a peak of density in the upper cortical laminae while the GI were commonly distributed across all laminae. The distribution of the NII and DN was variable, the most common pattern being a peak of NII density in the lower cortical laminae and DN in the upper cortical laminae. These results suggest in FTLD-TDP caused by GRN mutation: 1) there are greater densities of NCI and DN than in sporadic cases of the disease, 2) there is degeneration of the cortico-cortical and cortico-hippocampal pathways, and 3) cortical degeneration occurs across the cortical laminae, the various TDP-43 immunoreactive inclusions often being distributed in different cortical laminae.

Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.

Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

Molecular modelling assisted design and synthesis of cyclothialidine derivatives as DNA gyrase inhibitors

Since cyclothialidine was discovered as the most active DNA gyrase inhibitor in 1994, enormous efforts have been devoted to make it into a commercial medicine by a number of pharmaceutical companies and research groups worldwide. However, no serious breakthrough has been made up to now. An essential problem involved with cyclothialidine is that though it demonstrated the potent inhibition of DNA gyrase, it showed little activity against bacteria. This probably is attributable to its inability to penetrate bacterial cell walls and membranes. We applied the TSAR programme to generate a QSAR equation to the gram-negative organisms. In that equation, LogP is profoundly indicated as the key factor influencing the cyclothialidine activity against bacteria. However, the synthesized new analogues have failed to prove that. In the structure based drug design stage, we designed a group of open chain cyclothialidine derivatives by applying the SPROUT programme and completed the syntheses. Improved activity is found in a few analogues and a 3D pharmacophore of the DNA gyrase B is proposed to lead to synthesis of the new derivatives for development of potent antibiotics.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

Development and formulation of wax-based transdermal drug delivery systems

Topical and transdermal formulations are promising platforms for the delivery of drugs. A unit dose topical or transdermal drug delivery system that optimises the solubility of drugs within the vehicle provides a novel dosage form for efficacious delivery that also offers a simple manufacture technique is desirable. This study used Witepsol® H15 wax as a abase for the delivery system. One aspect of this project involved determination of the solubility of ibuprofen, flurbiprofen and naproxen in the was using microscopy, Higuchi release kinetics, HyperDSC and mathematical modelling techniques. Correlations between the results obtained via these techniques were noted with additional merits such as provision of valuable information on drug release kinetics and possible interactions between the drug and excipients. A second aspect of this project involved the incorporation of additional excipients: Tween 20 (T), Carbopol®971 (C) and menthol (M) to the wax formulation. On in vitro permeation through porcine skin, the preferred formulations were: ibuprofen (5% w/w) within Witepsol®H15 + 1% w/w T; flurbiprofen (10% w/w) within Witepsol®H15 + 1% w/w T; naproxen (5% w/w) within Witepsol®H15 + 1% w/w T + 1% C and sodium diclofenac (10% w/w) within Witepsol®H15 + 1% w/w T + 1% w/w T + 1% w/w C + 5% w/w M. Unit dose transdermal tablets containing ibuprofen and diclofenac were produced with improved flux compared to marketed products; Voltarol Emugel® demonstrated flux of 1.68x10-3 cm/h compared to 123 x 10-3 cm/h for the optimised product as detailed above; Ibugel Forte® demonstrated a permeation coefficient value of 7.65 x 10-3 cm/h compared to 8.69 x 10-3 cm/h for the optimised product as described above.

Studies on DNA methylation and mechanisms of hypomethylation

The methylation of cytosinc residues in DNA is thought to play an important role in the regulation of gene expression, with active genes generally being hypomethylated. With this in mind peptides were synthcsised to mimic the cytosine-5 methylation activity carried out by DNA mcthylase, which however, showed no ability to carry out this function. The imidazotetrazinoncs are a novel group of antitumour agents which have demonstrated good activity against a range of murinc tumours and human tumour xenografts, and hypomethylation of DNA has been implicated in the mechanism of action. Studies have been conducted on the mechanism by which such agents cause hypomethylation, using DNA methylase partially purified from murine L1210 leukaemia cells. Unmodified calf thymus DNA does not inhibit the transfer of methyl groups from SAM to M.lysodeikticus DNA by partially purified DNA methylase. However, if the calf thymus DNA is modified by alkylating agents such as imida-zotetrazinones or nitrosoureas, the treated DNA becomes an inhibitor of the methylation reaction. This has been correlated with the induction of DNA damage, such as single strand breaks, since X-ray treated DNA and deoxyribonuclease treatment produces a similar effect. The mechanism of inhibition by the drug treated or damaged DNA is thought to occur by binding of the enzyme to an increased concentration of non-substrate DNA, presumably by the occurrence of single strand breaks, since neither sonication nor treatment with the restriction enzyme Mspl caused an inhibition. Attempts were made to elucidate the strict structure activity relationship for antitumour activity observed amongst the imidazotctrazinones. The transfection of a murine colon adcnocarcinoma cell line (MAC 13) with DNA extracted from GM892 or Raji cells previously treated with either the methyl (temozolomide) or ethyl (ethazolastone) imidazotetrazinone was performed. X-irradiated DNA did not cause any suppression of cell growth, suggesting that it was not due to physical damage. Transfection of MAC 13 cells with DNA extracted from GM892 cells, was more effective at inhibiting growth than DNA from Raji cells. Temozolomide treated cellular DNA was a more potent growth inhibitor than that from ethazolastone treated cells. For both agents the growth inhibitory effect was most marked with DNA extracted 6h after drug addition, and after 24h no growth suppression was observed. This suggested that the growth inhibitory effect is due to a repairable lesion. .The methylation of M.lysodeikticus DNA by DNA methylase is inhibited potently and specifically by both hereto and homoribo and dcoxyri-bopolynucleotides containing guanine residues. The inhibitory effect is unaffected by chain length or sugar residue, but is abolished when the O-6 residue of guanine is substituted as in poly d(OGG)2o. Potent inhibition is also shown by polyinosinic and polyxanthylic acids but not by polyadenylic acid or by heteropolymers containing adcnine and thymine. These results suggest that the 6 position of the purine nucleus is important in binding of the DNA methylase to particular regions of the DNA and that the hydrogen bonding properties of this group are important in enzyme recognition. This was confirmed using synthetic oligonucleotides as substrates for DNA methylase. Enzymatic methylation of cytosine is completely suppressed, when O6 methylguanine replaces guanine in CG sites.

Antitumour imidazotetrazines: probes for the major groove of DNA

The antitumour imidazotetrazinones are believed to act as prodrugs for the triazene series of alkylating agents, showing a marked pteference for the alkylation of the middle guanine residue in a run of three or more contiguous guanines. However, the. exact nature of the interactions of imidazotetrazinones within the micro~environment of DNA are; as yet unknown. In order to examine such interactions a three pronged approach involving molecular modelling, synthetic chemistry and biological analysis has been undertaken during the course of this project. . Molecular modelling studies have shown that for the 8-carboxamido substituted imidazotetrazinones antitumour activity is dependent upon the. presence of a free NH group which can be involved in the formation of both intramolecular and intermolecular hydrogen bonds, and the presence of a non-bulky substituent with a small negative potential . volume. Modelling studies involving the docking of .mitozolomide into the major groove of DNA in the region of a triguanine sequence has shown that a number of hydrogen bonding interactions are feasible. A series of 8-substituted carboxamide derivatives of mitozolomide have been synthesised via the 8-acid chloride and 8-carboxylic acid derivatives including a number of peptide analogues. The peptide derivatives were based upon the key structural features of the helix-turn-helix motif of DNA-binding proteins with a view to developing agents that are capable of binding to DNA with greater selectivity. An examination of the importance of intramolecular hydrogen bonding in influencing the antitumour activity:of :the imidazotetrazinones has led to the synthesis of the novel pyrimido[4',5' :4,3]pyrazolo[5,1-d]-1,2,3,5-tetrazine ring system. In general, in vitro cytotoxicity assays showed that the new derivatives were less active against the TLX5 lymphoma cell line. than the parent compound mitozolomide despite an increased potential for hydrogen bonding interactions. Due to the high reactivity of the: tetrazinone ring system it is difficult to study the interactions between the imidazotetrazinones and DNA. Consequently a number of structural analogues that are stable under physiological conditions have been. prepared based upon the 1,2,3 triazin-4(3H)-one ring system fused with both benzene and pyrazole rings. Although the 3-methylbenzotriazinones failed to antagonise the cytotoxic activity of temozolomide encouraging results with a 3-methylpyrazolotriazinone may suggest the existence of an imidazotetrazinone receptor site within DNA. The potential of guanine rich sequences to promote the alkylating selectivity of imidazotetrazinones by acting as a catalyst for ring cleavage and thereby generation of the alkylating agent was examined. Experiments involving the monitoring: of the rate of breakdown of mitozolomide incubated in the presence of synthetic oIigonucleotides did not reveal any catalytic effect resulting from the DNA. However, it was noted that the breakdown of mitozolomide was dependent upon the type of buffer used in the incubations and this may indeed mask any catalysis by the oligonucleotides.

DNA hybridisation biosensor based on dual-peak long-period grating

Using an optical biosensor based on dual-peak long-period fibre grating, we demonstrate the detection of interactions between DNA biomolecules in real-time, showing a high sensitivity and reusability function.