Improving the solubility and dissolution of poorly soluble drugs by salt formation and the consequent effect on mechanical properties

The bioavailability of BCS II compounds may be improved by an enhanced solubility and dissolution rate. Four carboxylic acid drugs were selected, which were flurbiprofen, etodolac, ibuprofen and gemfibrozil. The drugs were chosen because they are weak acids with poor aqueous solubility and should readily form salts. The counterions used for salt formation were: butylamine, pentylamine, hexylamine, octylamine, benzylamine, cyclohexylamine, tert-butylamine, 2-amino-2-methylpropan­2-ol, 2-amino-2-methyl propan-1,3-ol and tromethamine. Solubility was partially controlled by the saturated solution pH with the butylamine counterion increasing the solution pH and solubility and dissolution to the greatest extent. As the chain length increased, solubility was reduced due to the increasing lipophilic nature of the counterion. The benzylamine and cyclohexylamine counterions produced crystalline, stable salts but did not improve solubility and dissolution significantly compared to the parent compound. The substitution of hydroxyl groups to tert-butylamine counterions produced an increase in solubility and dissolution. AMP2 resulted in the most enhanced solubility and dissolution compared to the parent drug but using the tris salt did not further improve solubility due to a very stable crystal lattice structure. The parent drugs were very difficult to compress due to orientation effects and lamination. Compacts were prepared of each parent drug and salt and their modulus of elasticity values were measured using a three-point bend (Young’s modulus, E0) were extrapolated to zero porosity and compared. Compressibility and E0 were improved with the butylamine, tert-butylamine, cyclohexylamine and AMP2 counterions. The most significant improvement in compression and E0 was with the AMP2 salts. Mechanical properties were related to the hydrogen bonding within the crystal lattice structure for the gemfibrozil salt series.

Structural studies on antifolate drugs

Single crystal X-ray structure determinations are reported for eleven compounds all of which are either biologically active or potentially biologically important. The compounds fall into two distinct classes:- 1. Substituted diaminopyrimidines 2. Substituted aminopyrimidinones The first class of compounds were all selected on the basis of their common diaminopyrimidine nucleus which has been demonstrated to be a vital requirement for antifolate activity. They may all be described as non-classical or small molecule lipophilic dihydrofolate reductase (DHFR) inhibitors, as opposed to the classical folate analogues, having the ability to cross the blood-brain barrier, enter cells via a rapid passive diffusion process, and achieve high intracellular concentrations. Thus they are an excellent choice in the search for crystallography in the solid state, providing geometrical and distance data not available from any other analytical techniques to date; supporting and enhancing data obtained in the lower resolution studies of protein crystallography. The biological importance of these compounds is discussed and an attempt is made to relate/predict their pharmacological activity to observed structural features in the crystalline environment. Special attention is focussed on hydrogen bonding, confirmational flexibility and hydrophobicity of substituents; each of which appear to make contributions to tight binding in the enzyme active site. Chapter 9 describes the use of data from the literature and the solid state modelling of an observed enzyme-substrate interaction in an attempt to define it more accurately in terms of its geometric flexibility. Of the second class, one compound (ABPP) is reported; studies in two different crystal forms. In demonstrating both antiviral and high interferon inducing activity it is possible that this compound could be useful against cancer and also viral infections.

Novel biological roles for pyrimidines

The development of classical and lipophilic inhibitors of dihydrofolate reductase (DHFR) as antitumour agents is reviewed and the advantages and problems associated with each class are discussed. The antitumour activity, pharmacokinetics and metabolism of m-azido-pyrimethamine (MZP), a novel lipophilic inhibitor, are considered and compared with metoprine, the prototype lipophilic antifolate. Evidence for a folate-independent target for lipophilic DHFR inhibitors is presented. Synthetic studies centred on three principal objectives. Firstly a series of structural analogues of MZP were prepared encompassing alkoxy, chloro and alkylamino substituents and evaluated, as the ethanesulphonate salts, for activity against mammalian DHFR. Inhibitory constant (KI) determinations were conducted by a Zone B analysis, the corresponding 4'-azido isomer of MZP proving more potent than the parent compound. Secondly, to facilitate metabolism and stability studies on MZP, a range of possible reference compounds were synthesised and characterised. Finally, a series of diaminopyrimidine derivatives were synthesised embracing structural features incompatible with DHFR inhibitory activity, in order that such compounds may serve as biochemical probes for the unidentified folate-independent target for lipophilic diaminopyrimidines discussed previously. Inactivity against DHFR was achieved via introduction of an ionic or basic group into a normally hydrophobic region of the molecule and compounds were screened against a mammalian DHFR and thymidylate synthase to confirm the abolition of activity. Several derivatives surprisingly proved potent inhibitors of DHFR exhibiting KI values comparable to that of methotrexate. Analogues were screened for antitumour activity in vitro and in vivo against murine leukaemia cell lines in order to identify potential lead compounds. Several derivatives virtually inactive against DHFR exhibited a disparate cytotoxicity and further biochemical studies are warranted. The nobreak hitherto unreported debenzylation of 2,4-diamino-5-(N-alkyl-benzylaminophenyl) pyrimidines was discovered during the course of the synthetic studies, treatment of these compounds with nitrous acid affording the corresponding benzotriazoles.

Delivery of phosphonoformate prodrugs

AIDS dementia complex is a common neurological syndrome thought to result from the invasion of the CNS by HIV. Phosphonoformate has anti-HIV activity but due to its charged nature is excluded from the CNS by the blood-brain barrier. Lipophilic triesters of phosphonoformate designed to improve transport properties are unsuitable prodrugs due to their rapid and complicated hydrolysis, involving competitive P-O and P-C bond cleavage. Diesters, though hydrolytically stable, are considered too polar to passively diffuse into the CNS. Hydrophilic drugs mimicking endogenous nutrients are known to be actively transported across the blood-brain barrier. In this thesis the possibility that diesters of phosphonoformate may be actively transported is investigated. Triesters of phosphonoformate with labile aryl carboxyl esterrs were synthesised and their hydrolysis followed by 31P NMR spectroscopy. The triesters were found to undergo rapid hydrolysis via P-C bond cleavage to the phosphite. Phosphonoformate diesters designed to be analogues of actively transported -keto acids have been synthesised and fully characterised. Tyrosine-phosphonoformate and lipid-phosphonoformate conjugates have also been synthesised and characterised. An in vitro model of the blood-brain barrier utilising confluent monolayers of porcine brain microvessel endothelial cells grown on a permeable support has been established. The presence of enzyme and antigen markers specific to the blood-brain barrier has been demonstrated for the endothelial cells and the diffusional properties of the model investigated with hydrophilic and lipophilic compounds. Active transport systems for -keto acids and large amino acids have been identified in the endothelial cell monolayers using 14C-pyruvate and 3H-L-tyrosine respectively. Temperature and concentration dependence of the two systems have been demonstrated and transport constants calculated. Competition with 14C-pyruvate transport was shown with other monocarboxylic acids including the anti-epileptic drug valproate. Stereospecificity was shown in that L-lactate inhibited pyruvate transport while D-lactate did not. Sodium methyl methoxycarbonylphosphonate, a phosphonoformate diester was shown not to compete for 14C-pyruvate transport indicating that this compound has no affinity for the carrier. Competition with 3H-L-tyrosine transport was shown with other large amino acids, including the anti-Parkinsonian agent L-dopa. Stereospecificity was shown using L- and D-tyrosine and L- and D-dopa. The tyrosine-phosphonoformate conjugate, which was stable under the experimental conditions, was shown to compete with 3H-Ltyrosine transport indicating that it may be actively transported at the blood-brain barrier. Thirty two triesters, diesters and monoesters of phosphonoformate, showed no activity in an anti-HIV screen above that attributable to hydrolysis to the parent compound.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Skin adhesive hydrogels for the topical delivery of active agents

This thesis illustrates the development of tailor-made, partially hydrated skin adhesive hydrogels as a vehicle for the topical delivery of moisturising agents. Maintaining an optimum hydration level of the stratum corneum ensures that the barrier properties of the skin are preserved. An unsaturated ionic monomer 2-acrylamido-2-methylpropanesulfonic acid sodium salt, glycerol, water, a photoinitiator Irgacure 184 and crosslinker Ebacryl II facilitated the production of monophasic sheet skin adhesives using photopolymerisation. The exploration and modification of the hydrogel components coupled with their influence on the adhesive and dynamic mechanical behaviour led to the development of novel monophasic and biphasic hydrogels. Biphasic pregels comprising of a hydrophobic monomer (epoxidised soybean oil acrylate, lauryl acrylate or stearyl acrylate) micellised with a non ionic surfactant Tween 60 allowed a homogeneous distribution throughout a predominantly hydrophilic phase (2-acrylamido-2-methylpropanesulfonic acid sodium salt, 4-acryloylmorpholine, glycerol and water). Further development of biphasic hydrogel technology led to the incorporation of preformed commercial O/W emulsions (Acronal, Flexbond 150, DM137 or Texicryl 13056WB) allowing the hydrophobic component to be added without prior stabilisation. The topical release of moisturising agents 2-pyrrolidone-5-carboxylic acid, lactobionic acid and d-calcium pantothenate results in the deposition onto the skin by an initial burst mechanism. The hydration level of the stratum corneum was measured using a Comeometer CM 825, Skin Reader MY810 or FT-ATR. The use of hydrophilic actives in conjunction with lipophilic agents for example Vitamin E or Jojoba oil provided an occlusive barrier, which reduced the rate of transepidermal water loss. The partition coefficients of the release agents provided invaluable information which enabled the appropriate gel technology to be selected. In summary the synthetic studies led to the understanding and generation of transferable technology. This enabled the synthesis of novel vehicles allowing an array of actives with a range of solubilities to be incorporated.

The synthesis and application of room temperature ionic liquids

This research project is concerned with the development and use of eco-friendly reaction media for a variety of organic transformations in the preparation of organic chemicals with potential pharmaceutical applications. These chemicals will then be investigated for their anti-cancer, anti-bacterial and anti-inflammation properties. In this project, different methods were used to synthesize various kinds of ionic liquids. Some new ionic liquids were prepared. In addition, Knoevenagel condensation reactions were investigated in RTILs. For the first time, some neutral ionic liquids such as [BMIM]+[BF4]-, [MeOEtMIM]+[CF3COO]- acted as both catalysts and solvents to promote Knoevenagel reactions. All these experiments indicated that RTILs have a great potential as alternative solvents in synthetic chemistry. Furthermore, nucleoside chemistry is an important research area in drug discovery. Various chemical modified nucleosides have therapeutic activities. However, these compounds usually have poor solubility in common organic solvents. RTILs such as [MeOEtMIM]+[CH3SO3]- have good dissolving capability for these chemicals. A range of thio-substituted nucleobases and nucleosides with potential pharmaceutical applications have been synthesized in several RTILs. These chemicals will then be investigated for their anti-cancer properties.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Interrogation of a sonogashira cross-coupling of 8-bromoguanosine with phenylacetylene on amberlite:evidence for Pd/Cu ion binding and propagation of Pd/Cu nanoparticles

The reactivity of Amberlite (IRA-67) base "heterogeneous" resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and postreaction Pd and Cu species. The Pd2Cl3(PPh)2 precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ∼2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications. Copyright © Taylor and Francis Group, LLC.

Preparation of 6-thioguanosine phosphoramidite for oligoribonucleotide synthesis

6-Thioguanosine phosphoramidite was prepared, using 2,4-dinitrophenyl as protection group for thio-function, and its stability towards conditions of RNA synthesis was investigated. The results show that the monomer was stable under the conditions of RNA synthesis and suitable for incorporation of thioguanine into oligoribonucleotides.

Biomimetic synthetic lubricants (Biosurfactants)

Poly(styrene-co-maleic anhydride) (PSMA) based copolymers are known to undergo conformational transition in response to environmental stimuli. This smart behaviour makes it possible to mimic the behaviour of native apoproteins. The primary aim of this study was to develop a better understanding of the structure-property relationships of various PSMA-based copolymers sought. The work undertaken in this thesis has revealed that the responsive behaviour of PSMA-based copolymers can be tailored by varying the molecular weight, hydrophobic (styrene) and hydrophilic (maleic acid) balance, and more so in the presence of additional hydrophobic, mono-partial ester moieties. Novel hydrophilic and hydrophobic synthetic surfactant protein analogues have successfully been prepared. These novel lipid solubilising agents possess a broad range of HLB (hydrophilic-lipophilic balance) values that have been estimated. NMR spectroscopy was utilised to confirm the structures for PSMA-based copolymers sought and proved useful in furthering understanding of the structure-property relationships of PSMA-based copolymers. The association of PSMA with the polar phospholipid, 2-dilauryl-sn-glycero-3- phosphocholine (DLPC) produces polymer-lipid complexes analogous to lipoprotein assemblies present in the blood plasma. NMR analysis reveals that the PSMA-based copolymers are not perfectly alternating. Regio-irregular structures, atactic and random monomer sequence distribution have been identified for all materials studied. Novel lipid solubilising agents (polyanionic surfactants) have successfully been synthesised from a broad range of PSMA-based copolymers with desired estimated HLB values that interact with polar phospholipids (DLPC/DPPC) uniquely. Very low static and dynamic surface tensions have been observed via the du Noϋy ring method and Langmuir techniques and correlate well with the estimated HLB values. Synthetic protein-lipid analogues have been successfully synthesised, that mimic the unique surface properties of native biological lubricants without the use of solvents. The novel PSMA-DLPC complexes have successfully been combined with hyaluronan (hyaluronic acid, HA). Today, the employment of HA is economically feasible, because it is readily available from bacterial fermentation processes in a thermally stable form - HyaCare®. The work undertaken in this thesis highlights the usage of HA in biolubrication applications and how this can be optimised and thus justified by carefully selecting the biological source, concentration, molecular weight, purity and most importantly by combining it with compatible boundary lubricating agents (polar phospholipids). Experimental evidence supports the belief that the combined HA and PSMA-DLPC complexes provide a balance of rheological, biotribological and surface properties that are composition dependent, and show competitive advantage as novel synthetic biological lubricants (biosurfactants).