Target molecules, assessing

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Biomarkers do not measure exposure directly, but are an indicator of absorbed dose. A biomarker of exposure is defined as a xenobiotic substance or its metabolite(s) or the product of an interaction between a xenobiotic agent and some target molecules(s) or cell(s) that is measured within a compartment of an organism and can be related to exposure. Urine, blood, nail, saliva, hair, and faeces are common media collected for biomarker measurements. Maternal biomarkers of exposure can also be measured in amniotic fluid and breast milk. These matrices can also provide a measure of exposure for children, both prenatally and postnatally. Biomarkers in first teeth have also been used to assess early childhood exposure, whereas biomarkers in meconium and cord blood have been used to assess in utero exposures. Biomarkers of genetic damage (e.g. DNA adducts) have been extensively used to assess exposure to genotoxic agents (Neri et al., 2006). 

The tedious purification of intermediates in classical solution synthesis produces pure compounds, which are then carried forward to the subsequent steps of the synthetic scheme. When a side product of the SP reaction remains attached to the resin, it becomes impossible to separate it from the desired intermediate/target molecule, thus irreversibly affecting the quality of the synthesis and the purity of the final product. This and other factors, which will be discussed in the following chapters, are considered during the so-called chemistry assessment phase of the synthesis in which the... 

The validation of a planned synthetic scheme in solution is the necessary starting point to design, refine, assess and carry out successfully the corresponding SPS. A synthetic scheme in solution must provide all the intermediates and the target molecule with good to excellent yields before being transferred onto SP. Sensitivity to changes of selected parameters (e.g., temperature, concentration, and solvent) should be known for the relevant reactions, and several experimental options for each step are desirable. Side reactions and side products formed at each step should be known and understood, and their dependence on reaction parameters should be determined. 

The term chemical assessment describes the process through which the reaction scheme to arrive at a target molecule is combinatorialized. This process may include the transfer of a reaction from solution onto SP and/or the adaptation of the reaction conditions to the use of many monomers with different reactivities and stabilities for library synthesis. Monomer rehearsal is an accurate check of the reactivity of a monomer set in the synthetic scheme for the buildup of the library so that the unreactive/difficult monomers are removed from the set. A model library is a small set of discretes, or a small pool, that is prepared using the planned synthetic route for the library and is fully characterized by the appropriate analytical tools Only if the results are satisfactory is the library synthesis carried out. Quality control determines the analytical profile of a library as a single entity, but data from each library individual, or a significant percentage of library individuals, are acquired. A library with 80% confirmed pure compounds is a good-quality library, but the 20% of samples that are... 

When the target molecule is similar to known structures, or when a sound synthetic scheme is drawn, the assessment of the best experimental conditions in solution is straightforward, while the transfer of a reaction sequence onto SP requires more theoretical (selection of the best support and linker) and experimental work to find appropriate reaction conditions. 

Reconstructive exposure assessment uses biological monitoring data, in conjunction with pharmacokinetic data and models, to estimate the levels of absorbed dose (e.g., systemic levels in plasma or whole blood), and in some cases, external exposure to a chemical that resulted in the measured levels in biological tissues and/or fluids. Biological monitoring consists of the measurement of the concentration of a chemical and/or its biotransformation products in biological tissues or fluids (e.g., adipose tissue, blood, urine) or the measurement of the amount of chemical bound to a target molecule (e.g., DNA-bound chemical). 

The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity, quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step. 

Analysis of the isotope distribution in target compounds can be, and frequently been, limited to measuring bulk specific activity (in the case of radioisotopes) or the overall abundance (in the case of stable isotopes) of the tracer isotope. Alternatively, the topological distribution of the isotope label can be narrowed down by chemical degradation in the case of radioactive tracers. In case of stable isotopes, the topology of isotope distribution in the target molecule can be assessed in considerable detail by NMR spectrometry, mass spectrometry, or a combination of these methods. 

Quantitative risk assessment depends on data that are reliable, sensitive and quantitative. It may well be that the numerical extrapolation from the current small scale (but manageable) laboratory tests can be substantially improved and moved downward to the effects of lower dose levels through the shrewd use of these isolated cell and biochemical test systems where the interplay of inactivation, activation and target molecule injury can be studied at concentrations well below those possible where one is looking at endpoints in relatively small groups of whole animals. 

During drug discovery, screening and candidate optimization, an expedient approach to target molecules is preferred to prepare small amounts of material for rapid assessment of in vitro activity. Accordingly, the initial preparation of 50 g of our drug candidate relied upon a direct adaptation of a literature procedure that employed a chiral auxiliary (Fig. 2) [3]. 

Weak, false positive nuclear staining may occur with some monoclonal or polyclonal antibodies. Careful assessment of the reaction pattern, correlated with the biology and the expected distribution pattern of the target molecule in normal cells, is always recommended. Nonspecific nuclear staining may be accentuated if slides are allowed to dry during the staining procedure. 

---