Should personalised dosing have a role in cancer treatment?

Almost all pharmaceutical products are approved on the basis of their effect in patients representing the "average" of the population studied in registrational trials, with most drug labels allowing, at most, for empirical dose reduction in the case of toxicity. In this perspective article we explore some of the evidence that supports the use of personalised dosing in cancer treatment and show how we have been able to build on existing models linking dose, exposure and toxicity to demonstrate how dose optimisation, including increasing the dose, has the potential to significantly improve efficacy outcomes. We also explore, through the lens of our own experience of developing a personalised dosing platform, some of the hurdles that stand in the way of implementing a personalised approach to dosing in real world settings. In particular, our experience is illustrated by the application of a dosing platform for docetaxel treatment in prostate cancer.

In silico strategy to rationally engineer metabolite production: A case study for threonine in Escherichia coli.

Genetic engineering of metabolic pathways is a standard strategy to increase the production of metabolites of economic interest. However, such flux increases could very likely lead to undesirable changes in metabolite concentrations, producing deleterious perturbations on other cellular processes. These negative effects could be avoided by implementing a balanced increase of enzyme concentrations according to the Universal Method [Kacser and Acerenza (1993) Eur J Biochem 216:361-367]. Exact application of the method usually requires modification of many reactions, which is difficult to achieve in practice. Here, improvement of threonine production via pyruvate kinase deletion in Escherichia coli is used as a case study to demonstrate a partial application of the Universal Method, which includes performing sensitivity analysis. Our analysis predicts that manipulating a few reactions is sufficient to obtain an important increase in threonine production without major perturbations of metabolite concentrations.

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling.

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum.

Ketopantoate reductase catalyzes the second step of the pantothenate pathway after ketoisovalerate, common intermediate in valine, leucine and pantothenate biosynthesis. We show here that the Corynebacterium glutamicum ilvC gene is able to complement a ketopantoate reductase deficient Escherichia coli mutant. Thus ilvC, encoding acetohydroxyacid isomeroreductase, involved in the common pathway for branched-chained amino acids, also exhibits ketopantoate reductase activity. Enzymatic activity was confirmed by biochemical analysis in C. glutamicum. Furthermore, inactivation of ilvC in C. glutamicum leads to auxotrophy for pantothenate, indicating that ilvC is the only ketopantoate reductase- encoding gene in C. glutamicum.

Metabolic network analysis during fed-batch cultivation of Corynebacterium glutamicum for pantothenic acid production: first quantitative data and analysis of by-product formation.

A first generation genetically modified strain of Corynebacterium glutamicum has been assessed for its potential to synthesise and accumulate the vitamin pantothenic acid in the medium using fed-batch cultivation technology, with biomass concentration controlled by isoleucine limitation. Kinetic analysis of specific rates throughout the process has been used to model carbon flux through both central metabolism and the specific pathways involved in product formation. Flux towards pantothenic acid is potentially high but much of this flux is dissipated as by-products within associated pathways, notably linked to amino acid synthesis. The major limitation of vitamin production in this strain is linked to the tenfold higher flux of keto-isovalerate towards valine rather than pantothenic acid. Attempts to modify this ratio by imposing nitrogen limitation provoked carbon overflow as unidentified non-nitrogenous compounds. The observed accumulation of glycine suggests that the flux towards pantothenate production may by limited by the rate of the pathway intermediate (5,10-methylene-tetrahydrofolate) regeneration.

Dynamic simulation of pollutant effects on the threonine pathway in Escherichia coli.

The enzymatic activities of threonine pathway in Escherichia coli are sensitive to pollutants such as cadmium, copper and mercury, which, even at low concentration, can substantially decrease or even block the pathway at several steps. Our aim was to investigate the complex effects on a metabolic pathway of such general enzyme inhibitors with several sites of action, using a previously developed computer simulation of the pathway. For this purpose, the inhibition parameters were experimentally determined and incorporated in the model. The calculation of the flux control coefficient distribution between the five steps of the threonine pathway showed that control remains shared between the three first steps under most inhibition conditions. Response coefficient analysis shows that the inhibition of aspartate semialdehyde dehydrogenase is quantitatively dominant in most circumstances.

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain.

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO2 via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.

Dependence of control coefficient distribution on the boundaries of a metabolic system: a generalized analysis of the effects of additional input and output reactions to a linear pathway.

Both experimental and theoretical studies of metabolism are likely to relate to a segment that has been isolated for analytical purposes. In practice, it will be embedded in the whole of cellular metabolism. Thus, it is necessary to consider how conclusions about the control of an isolated pathway may be modified in this wider context where the input and output metabolites are considered as variables of cellular metabolism. Here, we analyse the effect of expanding a linear metabolic pathway by adding an extra input or an extra output. In particular, we analyse the effect of the elasticities of the extra steps on control coefficients. We derive matrix algebraic relationships for obtaining flux and concentration control coefficients from expressions depending on these extra elasticities and on parameters (elasticities and control coefficients) of the original pathway. These equations can be shown in certain cases to be generalized versions of earlier rescaling relationships and to be related to top-down analysis, but also apply where the new variable metabolite of the expanded pathway is an effector of more than one step of the original pathway. We use our relationships to analyse the dependence or independence of control coefficients upon these extra elasticities for the published analyses of the pathway of mammalian serine biosynthesis (Fell & Snell, 1988) and Escherischia coli threonine biosynthesis (Chassagnole et al., 2001). The same analysis can be applied to determine whether the transport reactions of substrates and products of a pathway in and out of a cell need to be included in estimations of the control coefficients of the enzymes.

Dynamic modeling of the central carbon metabolism of Escherichia coli.

Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.