Drug Screening Methods SK Gupta
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Newer Tools for Drug ScreeningCHAPTER 1

 
INTRODUCTION
The last quarter of the century has seen transformation of the pharmaceutical industry. The advances in the field of information technologies have driven basic research to evolve into fast-track drug discovery and development program that is target oriented while still beating the clock. There is competition within the industry to continuously generate the next billion-dollar selling compound. Market estimates project that the journey of a new molecule from laboratory to market takes well over seven years at an average cost of over $600 m. Consequently there is thrust on developing advances in technologies aiming to reduce the time and the costs involved in bringing a new drug to market. This has lead to the introduction of revolutionary technological advances such as combinatorial chemistry, biochemical assays, genomics, proteomics, miniaturization, automation, robotic systems and computerization, which have cumulatively increased the speed of lead generation manifold.
Just to cite a case, by using way of traditional drug development techniques, it took nearly half-a-century to tap the cholesterol biosynthesis pathway and develop statin drugs, as cholesterol lowering agents. On the other hand, molecular-revelations regarding the role of the HER-2 receptor in breast cancer led to the development of the chemotherapeutic agent, Herceptin® within three years. The advanced techniques of in silico molecular modeling, high throughput screening and genomic and proteomic databases, were instrumental in the quick discovery and development of Herceptin.1
Some of the screen paradigms that are being rigorously adopted by pharmaceutical companies to speed up the development of next blockbuster drug are discussed in this chapter.
 
CASSETTE DOSING
As compared to generation of 100 leads that were produced a decade ago, combinatorial chemistry now enables the chemist to produce thousands of leads each year.2 The problem is no longer of too few drug candidates emerging from the discovery process – in fact the number of quality lead compounds that emerge is much higher. It is being understood that rather than drug discovery, it is drug development that is proving to be the bottleneck (Fig. 1.1). The battery of preclinical tests such as toxicity, bioavailability and pharmacokinetics are extremely critical, time-consuming and costly. Prioritization of leads with regard to 2these studies becomes a stumbling block in the selection of viable targets from millions of compounds.
zoom view
Figure 1.1: Bottleneck at drug development stage reducing the speed of generation of novel drugs
Due to several constraints, classical methods are not high throughput and have often impeded the quick development of new drug candidates. This has generated need for high throughput drug development program to be set-up and started in full throttle.3
Alth ough pharmacokinetic evaluation is an essential component of drug discovery program, it has proved to be a serious bottleneck during the ‘hit-to-lead’ and lead optimization phases of drug discovery and development program. The prioritization of leads with regard to drug metabolism and pharmacokinetics (DMPK) assumes importance as it determines the selection of viable targets from millions of compounds. A compound with favorable pharmacokinetics is more likely to be efficacious and safe. Early elimination of pharmacokinetically ineligible candidates helps to sift grain from chaff. For this critical juncture in drug development program, absolute reliance on in vitro tests cannot be advocated. As in vitro tests can never replicate the complex biological system, they serve as confirmatory tests rather than preliminary tests.4
Rodent species (mice, rats) have been classically used for in vivo pharmacokinetic studies. As there is a limitation to the amount of blood samples that may be withdrawn per animal, different animals have to be used for each time point, leading to high animal usage. In addition, inter-individual variation of various pharmacokinetic parameters is commonly encountered (up to two-fold) due to the differences in the expression of drug metabolism enzymes and genetic polymorphism. These further complicate data interpretation and slow the progress.3
Advances in drug development technology have provided the solution to the problems by providing alternate methods that help the discovery scientists to predict pharmacokinetics within the constraints of real physiological environment, but at the pace of in vitro or in silico methods. Cassette dosing is an elegant, inexpensive, nonlabor/time intensive novel technique that has been developed with the aim to rapidly assess pharmacokinetics of a large number of compounds (Table 1.1). Scientists at Glaxo Wellcome have been the pioneers in its development.3
3In theory, cassette dosing or CD or ‘N-in-one dosing’ involves the simultaneous administration of several compounds (5-10) to a single animal followed by rapid sample analysis for the compounds and their metabolites by liquid chromatography/tandem mass spectrometry. It is a highly recommended technique as it enhances the efficiency in terms of time, money, manpower while reducing animal usage.3
Table 1.1   Estimated reduction in preclinical screening time after adopting cassette dosing
Stage of drug discovery program
Currently time taken (months)
Estimated time taken with cassette dosing (months)
Preclinical
15
12
Phase I
18
14
Phase II
22
12
Phase III
31.5
17.5
Total estimated time
7.2 years
4.5 years
Cassette dosing is based on the concept of serial bleeding that involves withdrawal of blood samples from the same animal for all the time points, which is estimated to not only dramatically reduce the number of animals used but also increased the quality of the kinetic data for compounds compared in that animal. Furthermore, the volume of blood samples that are withdrawn/animal/time point is miniscule. Literature search brings to fore descriptions of analytical methods that have used only 10-20 μl of whole blood using capillary LC/MS/MS. The onus for the success of this approach has to be granted to the superlative technique of LC/MS/MS, as it would not have been possible with other classical methods of analysis (spectrophotometry and chromatography).
Cassette dosing poses an analytical challenge as it involves simultaneously assaying many compounds in a single sample. In the absence of theoretical guidance, a set of intuitive assumptions has developed regarding the nature of the errors and how to avoid them.3 These assumptions are:
  1. Drug-drug interactions only occur when one of the dosed compounds is a potent inhibitor of drug-metabolizing enzymes.
  2. One may guard against competitive inhibition of a shared metabolic enzyme by keeping doses small.
  3. The size of the cassette (n) is limited only by the sensitivity of the assay and the solubility of the compounds.
  4. Errors can be detected by including a benchmark compound with known pharmacokinetic characteristics.
  5. Drug-drug interactions can lead only to false positives, which will be discovered later, and
  6. Even if the absolute values are wrong, the correct rank order will be observed.
Although cassette dosing is an advantageous technique in terms of resources and throughput, there are possible complications associated with this approach. The technique of cassette dosing has been under critical review and is subject to high level validation. While using the technique of cassette dosing, inherent limitations of the technique have to be accounted for. Firstly, the potential for compound interactions is increased manifold.5
4Although cassette dosing has been reported to yield useful results when used as a screen, especially to rank-order drug candidates, it has been shown to be fraught with both theoretical and experimental large errors. Consequently, under no circumstances can the pharmacokinetic parameters derived from cassette dosing be accepted as accurate. Potentially affected parameters include F, CL, AUC, t½, mean residence time, Vd. High-clearance compounds have the greatest potential for screening errors (i.e., false-positive, false-negative). To detect errors, a second dosing episode could be opted, that may in itself defeat the productivity gained from cassette dosing.5
A better way to detect errors is to include a benchmark compound with known in vivo pharmacokinetics. To minimize the potential for errors, one should use the smallest doses detectable and keep the total number of co-administered compounds small.4
Even the looming limitation of drug-drug interactions entailed with cassette dosing is being overcome. With advances in IT and availability of predictive databases, approaches such as structure-metabolism relationship (SMR) are gaining importance. Knowledge about ligand structure, ligand-active site interactions and stereoelectronic factors involved in metabolic transformations, the metabolic pathways that may be involved and corresponding potential metabolites formed can be predicted. Recently, METAPRINT, a metabolic fingerprint has been developed to facilitate in the design of cassette dosing experiments. These approaches will supplement cassette dosing and go a long way in reducing any confounding information especially with regard to drug-drug interactions in cassette dosing.6
In another development, a novel method of serial bleeding has been developed to withdraw blood samples from the same animal for all the time points in a pharmacokinetic study. This helps to not only dramatically reduce the number of animals used, but also increased the quality of the kinetic data for compounds compared in that animal. Following protocol has been developed with the approval of Animal Use and Care Committee for conducting in vivo experiments.
Male Swiss Webster mice, 7 weeks old (body weight 28-36 g), are used for pharmacokinetic studies. Using a stratified randomization procedure the animals are either administered vehicle, standard or test drug. The route of drug administration may be oral, intraperitoneal or subcutaneous, as per the protocol. After administration, serial tail bled blood samples (5 μl) are collected using heparinized tip at various time points (5 min to 24 h). The samples are transferred to a microcentrifuge tube, weighed with an analytical balance and vortexed with purified water and internal standard.7
The samples are extracted with organic solvent (ethyl acetate, methanol or acetonitrile). The organic layer is transferred to a microcentrifuge tube, and dried under nitrogen. The residues can be reconstituted in minimum volume (up to 25 μl) of appropriate solvent (methanol). Aliquots are injected onto LC/MS/MS system for analysis.7
The technique has the following distinct advantages:
  • Minimizes the number of animals used
  • Significantly reduces trauma to the animal that is associated with sample withdrawal
  • Marked reduction in inter-individual variation in pharmacokinetic parameters
  • Reduces the amount of drug used
  • Sample processing time is markedly minimized.
5However, care has to be taken, that the small animals are handled with care as serial bleeding may alter the physiological state of the animal. For example, micro sampling increases the amount of inflammatory eicosanoids in blood and may decrease the proportion of cellular components in a sample.7
 
VIRTUAL SCREENING
The rate of synthesis of compounds has increased exponentially owing to the advances in combinatorial chemistry. These compounds have been housed in huge virtual libraries. Numerous such databases have been created, each housing over 109 compounds, in each. The obvious question, which arises, is how can this enormous database be filtered to bring forth compounds of utility? To achieve this goal, miniaturized and automated assays have been developed that limit cost, material, time and manpower requirement. As a natural extension high speed computer systems running specialized softwares have been developed that are capable of screening the molecules from the libraries against identified targets.8
As the first step the appropriate technique of X-ray crystallography, Nuclear Magnetic Resonance (NMR) are used to determine the 3-D structure of the macromolecular target. This is followed by application of 2D QSAR (2-Dimensional Quantitative Structure Activity Relationship), wherein, the chemical structure is quantitatively correlated against a biological activity so as to predict its biological activity. The compounds are superpositioned on the target site as a function of energy and potential. On the basis of this screen, compounds exhibiting favorable kinetics are selected for “fine tuning” and the rest are eliminated.
While searching a virtual library, maximum output can be generated, if information under following heads is available:
  • Information about other known ligands (substrates, agonists, antagonista, etc.) that are bioactive at the target.
  • Detailed structural and functional information about the target site on the site, binding thermodynamics, etc.
  • Lastly, a thorough knowledge in rules of conformational analysis and a medicinal chemistry ‘instinct’ proves to be beneficial.8
 
MICROASSAYS
Modern chemical and biological techniques have revolutionized the synthesis of new chemical entities, and have set a pace that was unimaginable with traditional methods of synthesis.
With the advancements in modern science and DNA recombinant biotechnology,9-12 various novel and sensitive procedures have been developed to determine the side effects, pharmacological action, toxicity, and efficacy of biologically active and clinically important compounds, derived from herbal origin or prepared synthetically. This has provided a much needed impetus to drug development program. Conventional bioassays have been replaced by sensitive ELISA, reverse transcriptional polymerase chain reaction (RT-PCR), ribonuclease protection assays, cDNA microarrays, etc. some of which are briefly described below.6
 
Radioimmunoprecipitation
This elegant procedure is employed to quantitatively estimate the gene expression at the translation level using radiolabeled 35S-methionine.13-15 The labeled sample is generated as radioimmunoprecipitate which is counted above background using liquid scintillation counter.8
 
Immunoblotting
SDS-polyacrylamide gel electrophoresis is performed to study protein, enzyme, neurotransmitter or hormone expression of the immunoprecipitated lysates. Slab gel electrophoresis is performed and the autoradiograms are densitometrically analyzed.13
 
RNA Extraction
Cellular monolayer is trypsinized to detach from the bottom of the flask and the cell pellet is obtained by centrifugation. The pellet is suspended in guanidine isothiocyanate (GITC) solution (composition: 0.1 M dithiothretol, 4 M guanidine isothiocyanate, 0.5% (v/v) N-lauryl sarcosine, 20 mM sodium acetate, pH 4.0) and treated according to standard protocol. RNA is pelleted by centrifugation. One μl of the purified RNA sample is diluted to 1 ml. Readings are taken at 260 nm and 280 nm to determine the A260/A280 ratios (pure RNA provides a ratio between 1.6 and 1.8). RNA is resolved in 1% agarose gel containing ethidium bromide at a current strength of 30 mA for 2-3 h (80 volts for 1 h). The gels are visualized on UV eluminator.
 
Reverse Transcription (First Strand cDNA Preparations)
Reverse transcription of 1 μg of RNA is conducted using either 50-100 ng of poly(A) mRNA or 5-10 μg of total RNA. The volume is adjusted to 38 μl with DEPC-treated water. Three μl of oligo-dT primers (100 ng/μl) or 3 μl of random primers (100 ng/μl) are added and the contents are mixed gently. Both control and experimental tubes are incubated at 65°C. The tubes are cooled slowly at room temperature (10 min) to allow primers to anneal to RNA. First strand cDNA is synthesized by adding the following reagents in the control and experimental tubes in sequence. Five μl (10X) first strand buffer, 1 ml of RNase block (Ribonuclease inhibitor, 40 U/μl), 2 μl of 100 mM dNTPs. One μl of MMLV-RT (50 U/μl). The tubes are mixed gently and incubated at 37°C for 1 h followed by incubation at 90°C for 5 min. The first strand cDNA is kept on ice for use in PCR amplification protocol.
 
Amplification of First Strand cDNA
One to five μl of first strand cDNA is transferred in autoclaved 500 μl PCR tubes. In control PCR amplification reaction tubes, 10 μl of Taq-DNA polymerase buffer is added along with 0.8 μl of 100 mM dNTPs, 3 μl of control primer set (100 ng/μl) and double distilled water to adjust the final volume to 99.5 μl. In the experimental PCR reaction mixture following reagents are administered in a sequence, 10 μl of 10X Taq DNA polymerase buffer, 0.8 μl of 100 mM dNTPs, 2 μl of 10 μM oligonucleotide (primer 1: Forward: and 2 μl of 10 μl of oligonucleotide) (primer 2. Reverse) primers are added. The final volume of the reaction mixture is adjusted to 99.5 μl. Both control and experimental amplification reaction mixture tubes are placed in a DNA Thermal cycler. Each PCR amplification reaction is heated to 91°C for 5 min and 7then immediately cooled at 54°C for 5 min. This step is essential to maximize thermal cycling performance. The control and experimental PCR amplification reaction tubes are removed from the Thermal cycler, briefly microcentrifuged and 0.5 μl of Taq-2000 (DNA polymerase) (5 U/μl) to each reaction tube is added. The reaction tubes are briefly centrifuged again. The PCR amplification reaction mixture is overlaid with a drop of mineral oil to prevent evaporation of reaction components during thermal cycling. The PCR amplification reaction tubes are placed in the thermal cycler and processed for amplification. For PCR amplification, programmed thermal cycler is used and experimental parameters are established that are optimal for the oligonucleotide primer set employed. Amplification is usually done depending on the primer length, GC content, and its sequence (usually 25-40 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 54°C, and extension of 2 min at 72°C). The final reaction is done using 72°C for 10 min to complete the amplification. The reaction products are kept at 6°C before the analysis. Ten μl of each PCR amplification reaction is taken from below the mineral oil layer into separate lanes of 1.2% agarose. One Kb DNA ladder is used as molecular weight marker. The amplified products are analyzed densitometer.
 
Cell Transfection
Cell transfection studies are conducted on healthy cells at subconfluent stage. Usually we have used the cells between 4-5th passage. One μg of antisense oligonucleotide to μ-synuclein: 5’-CCT-TTT-CAT-GAA-CAC-ATC-CAT-GGC-3’, Reverse Sense: 5’-GCC-ATG-GAT-GTG-TTC-ATG-AAA-GG-3’; Scrambled: 5’-TAG-CTC-GCT-ACG-TAA-TCA-CCA-CT-3’. Metallothionein-1 antisense: CAC-AGC-ACG-TGC-ACT-TGT-CCG-CCG-CCG-CTT-TGC-AGA-CAC-AGC-C, MT-1 Forward: GTT-CGT-CTC-ACT-GGT-GTG-AGC, MT-1 Reverse: AAA-AGA-AAT-CGA-GGA-AAT-GGC (GIBCO/BRL Life Technologies, USA), mixed with 8 μl of enhancer, and 25 μl of Effectine transfection reagent as per manufacturer's recommendations. The transfected cells are authenticated using Radioimmunoprecipitation, immunoblotting, and RT-PCR using specific primer sets of genes. Spontaneous and drug-induced apoptosis is studied using heat shock, staurosporine (1 μM), serum deprivation, ceramide or other apoptogens including toxic drugs.
 
Multiple Fluorochrome Comet Assay
This sensitive assay is performed to determine mitochondrial and nuclear DNA damage simultaneously in a single cell in response to various environmental neurotoxins or physiological stress. It can provide basic information regarding condensed, partially condensed, partially fragmented and fully fragmented DNA based on the charge associated with each molecular species of DNA. Thus, this procedure provides information regarding single cell apoptosis. Multiple fluorochrome Comet assay provides more quantitative information regarding the extent of genotoxicity of a compound and that can be determined by quantitatively estimating the Comet tail length, tail intensity, and tail diameter. This procedure is particularly useful to determine the levels of DNA damage in an alkaline medium and is employed particularly in the field of genotoxicology. It is a convenient and more sensitive method for multiple processing and drug screening. It is an assay at the inter-phase between molecular biology and cellular biology and is little more sensitive and specific as compared to conventional DNA fragmentation assay performed on agarose gels. This method is very useful in comparative pharmacological analysis of excitoneurotoxins as well as drugs.148
 
Triple Fluorochrome Analysis
Triple fluorochrome analysis is conducted to assess various stages of apoptosis at the plasma membrane and cytoplasmic level, using 100 nM acridine orange, which stains specifically RNA and proteins, and is very useful to detect apoptotic bodies; to estimate mitochondrial membrane potential and mitochondrial apoptosis, JC-1 and decifer are employed, whereas nuclear apoptosis is detected by fluorochrome.
The neurons are grown in eight-chambered microscopic slides, exposed to either toxins or drugs overnight for seven days, and incubated at 37°C for 45 min in a mixture of fluorochromes. The monolayer is washed with Dulbecco's phosphate buffered saline and the cellular monolayer is mounted, air-dried in the dark chamber, and observed under Fluorescence microscope, equipped with immunofluorescence imaging system. The fluorescence images are digitized using Digital camera and analyzed. For obtaining a detailed analysis of apoptosis and its intermediary events, images captured with different filters are combined and plotted.15,16
 
Multiprobe Ribonuclease Protection Assay (MRPA)
MRPA is a highly sensitive procedure to simultaneously quantify several mRNA species in a single sample of total RNA and can be used for comparative analysis of different mRNA species, which can be compared between samples. MRPA can be performed on total RNA preparations by standard methods from either frozen tissue or cultured cells without further purification of polyA+ RNA. It is highly sensitive procedure for the detection and quantification of gene transcripts, induced in response to neurotoxic insult. It was discovered based on the knowledge about DNA-dependent RNA polymerase from bacteriophage SP6, T7 and T3 and information of their promoter sequences. To synthesize high specific activity RNA probes from DNA templates as they have high degree of fidelity for the promoters, polymerize RNA at high rates, efficiently transcribe long segments, and do not require high concentrations of rNTPs. So a cDNA fragment of interest can be subcloned into a plasmid that contains bacteriophage promoters and the construct can be used as a template for the synthesis of radiolabeled and antisense RNA probes. We use T7 polymerase-directed synthesis of high specific activity 32P labeled anti-sense RNA probe set. The probe set is hybridized in excess to target RNA in solution after which free probe and other single stranded RNA are digested with RNase. The remaining RNase protected probes are purified, and resolved on denaturing PAGE and quantified by autoradiography or phosphor imaging. Quantity of each mRNA species in the original RNA sample can be determined based on the intensity of the properly sized-protected probe segment. The procedure takes usually three days.
Day 1: For probe synthesis and overnight hybridization, Day 2: RNase treatment, purification of protected probe, and Gel Electrophoresis, and Day 3: Autoradiography or phosphor imaging.
 
cDNA Microarrays for Differential Gene Expression
This is a relatively new research procedure employed to study differential multiple gene expression under the influence of drugs, neurotransmitters, enzymes, or hormones, etc. under investigation. As many as 30,000 genes can be analyzed with this procedure. cDNA Microarray scanning requires cDNA microarray scanner with computer software to investigate the role of various genes involved in drug-induced apoptosis and antioxidants-induced antiapoptosis. 9The procedure works hand in hand with DNA sequencing for high throughput screening for exploring point mutations of nuclear and/or mitochondrial origin. Plastic microarrays are relatively economical, and they can be utilized to investigate as low as 1200 genes of interest from the biological samples. cDNA microarray scanning is relatively sensitive procedure and can pinpoint minor yet subtle changes in the gene expression in response to environmental neurotoxins as well as pharmacological drugs of clinical importance (such as anticarcinogenic or antiapoptotic agents). The main objective of these technical procedures is to develop cDNA chips for clinical diagnosis, better prognosis, and effective treatment of various diseases with low undesirable effects.
 
APPLICATION OF MODERN ANALYTICAL TECHNIQUES IN BIOLOGICAL SYSTEMS
 
Atomic Absorption Spectroscopy
This method is employed to estimate the concentration of various metal ions of physiological significance, such as Na, K, Ca, Cl, Fe, Cu, and Zn from the biological fluids, tissue extracts, and from the microdialysates. Various atomic absorption spectrometers equipped with graphite furnace and computer software are now available which can analyze more than four metal ions from as small as 20 μl biological sample. Tissue or cell extracts are prepared in 0.3 N perchloric acid by sonication at low wattage and microcentrifuged at 14,000 rpm at 4°C. The supernatants are filtered through the syringe tip filters and the filtered extracts are directly utilized in the atomic absorption spectrometer or high performance liquid chromatography with electrochemical, UV, or fluorescence detector capabilities. Perkin-Elmer Atomic Absorption Spectrometer is equipped with Graphite furnace (which is operated in an argon environment), an autosampler, and computer software for the online data analysis and preparing concentration reports. Usually, pyrolysis at 1,300-1,700°C, and atomization of samples at 2,400-2,800°C, is employed, depending on the metal ion under investigation.13-15
 
Coulter Counting
This procedure is very simple and requires a photocell, which estimates the number of particles present in the photocell. Beckman-Coulter Company (USA) has developed this procedure to determine total number of cells following treatment with a drug. Although it is a single step method and requires only 1:20 dilution of a sample, it does not decipher between live and dead cells. Therefore, Coulter counting is supplemented with hemocytometer reading made using a microscope and Trypan blue exclusion method (the live cells exclude trypan blue, while dead cells are stained with trypan blue).
 
Fluorescence Activated Cell Sorting (FACS)
This biophysical equipment is utilized to determine differential expression of as many as 6 genes with a capability of estimating 6 more physical parameters of physiological interest, such as number of cells undergoing apoptosis and necrosis, and the number of live cells 10simultaneously based on the side scatter, forward scatter, and granularity. The FACS machine (flow cytometer) employs lasers (such as He-Ne, Argon lasers, Ruby lasers and Cadmium lasers) microbeams for the determination of fluorescence properties of cells. For measuring intracellular free ionized calcium, UV detectors are employed. The equipment can also sort out genetically engineered cells by a turbo sorter facility. Fluorescence activated cell sorting (FACS) machines are now being utilized to prepare stable transfectants using vectors encoding for green, red, or yellow fluorescence proteins. This approach eliminates the need to perform in vitro reporter gene analysis using either X-Gal or luciferase reporter gene assays. pEGFP-N-1 vectors are thus very convenient to study the behavior of various pharmacological agents on genetically engineered cell lines. This approach is being utilized particularly in gene therapy labs. FACS machine is also utilized to detect the efficacy of anticancer drugs based on the extent of DNA damage (apoptosis and/or necrosis) they produce in vitro. This machine is also utilized to determine at which phase of the DNA cycle (G1-S, G2-M), the drug might have induced its maximum effect. The cells can be synchronized using chemicals influencing the DNA cell cycle at a particular phase. In addition, this machine is being utilized to study mitochondrial membrane potential, and to determine the production of free radicals in response to a particular drug/agent. Although very useful, this equipment is very costly. Moreover, its maintenance cost is also quite expensive. In addition, the cost of fluorochromes adds to its limited use in many labs all over the world. This equipment requires qualified and trained persons to handle and interpret the experimental data.
 
Positron Emission Scanning
With the advancements in linear accelerators, cyclotrons, and targets, the preparation of short-lived positron emitters has been facilitated. This procedure is noninvasive and provides information regarding brain regional physiology and biochemistry such as synthesis of DNA, RNA, and proteins in vivo. Furthermore, cyclotron-generated positron emitters (15O, t½ 120 sec, 13N t½ 10 min, 11C t½ 20 min, and 18F t½ 110 min) are now being utilized in basic research and clinical practice to study brain regional metabolism and molecular neuroimaging of genes of interest. 18F-DOPA is being employed to examine nigrostriatal dopamine transporter activity, and to discover pleasure centers involved in drug addiction for substances of abuse. 18F-deoxyglucose is being employed for detecting the stages of malignancies. High-resolution microPET scanners (Concorde Microsystems Inc, Knoxville, TN, USA) are now being used to discover new and clinically effective neuroprotective drugs and for an early diagnosis of diseases. CTI Corporation (USA), Physics for Medicine (USA), Seimens-Gamma-Med (German), and Digi-Rad have recently developed high-resolution CT-SPECT fusion scanners for research purpose as well as for clinical applications.
 
Magnetic Resonance Imaging and Spectroscopy
High-resolution magnetic resonance imaging is utilized in basic and applied research. In particular high-resolution magic angle spinning nano-NMR probes require as small as 40 μl of biological samples in 750 MHz magnets. This equipment can analyze and quantitate biological samples with microgram to nanogram concentrations. Running cost of this equipment could be as high as $800 per hr. This equipment is used to determine the concentration, purity, and structural formula of as many as 80 metabolites from the aliphatic and 11aromatic regions. Biological compounds including DNA, RNA and proteins contain carbon, hydrogen, and nitrogen atoms. Their nuclei spin at a particular frequency, which can be picked up and detected to evaluate their clinical significance. Usually, brain regional metabolism of amino acid neurotransmitters and metabolites can be explored by these advanced and sophisticated techniques. Various resonances are picked up and computer-analyzed using Fast Fourier transformation (FFT) analysis. From the position of the resonance peak, we identify the compound/metabolite, and from the peak height, we determine the concentration of the compound/metabolite. These advanced research tools are also utilized to determine the structure and purity of various unknown compounds and molecular designing of drugs. Various pharmaceutical industries are interested to determine the structural formula of their product before evaluating its therapeutic potential. High-resolution magnetic resonance imaging is performed on small animals (rats, mice) to determine any space-occupying lesion (such as cyst, infarct, tumor, edema). This equipment measures regional proton density per unit area, which is altered during edema or during water accumulation in the brain in response to neuronal injury or following neurotoxic insult. Thus, MRI is used to correlate and confirm information derived from PET and computerized axial tomography conducted employing soft X-rays.
 
CELL FREE ASSAYS
Cell-free assays include simple to very complex systems. These biochemical assays include enzyme assays, protein-protein interactions and membrane receptor ligand and soluble receptor-ligand binding assays. The advantages of this kind of assay system includes ready accessibility of the compounds to the target, easy identification of the target of the compound without any ambiguity, a well-defined mechanism of action, the possibility of developing inexpensive screens, the easy adaptability to newer technologies, amenability to miniaturization, and ready automation.17 These assays can be classified as heterogeneous and homogeneous assays. Heterogeneous assays are multistep assays that include steps of incubations, washings, filtrations, reading of signals etc. as in ELISA. On the other hand, one pot assays that do not involve any transfer or wash steps are known as homogeneous assays, such as, chromogenic, absorbance or fluorescence based assays.18
 
MICROBE-BASED SCREENING ASSAYS
Microbe-based screening has been used to screen antibacterial agents and cytotoxic anti-cancer agents. Advance biotechnology techniques are used to clone and express target proteins. The mammalian proteins are expressed in microbial cells and stored therein inclusion bodies. These proteins are not useful for the microbial cells. The insoluble aggregates are isolated, dissolved and refolded and if needed, subjected to post-translational modifications. The advantages of this type of assay systems include low cost, simple technique and high yield. Commonly used microbial systems include E. coli, Saccharomyces and Yeast. Microbial systems have been adapted to identify agonists and antagonists of GPCRs, detail the mechanism of action of immunosuppressants such as cyclosporin and FK506, screen for K+ channel openers and blockers and many more.19-2112
 
RECEPTOR SCREENS
Langley and Ehrlich described the concept of receptor-ligand interaction.22 The concept of receptor has been overhauled since then, to include cell membrane, nuclear, ion, voltage gated, tyrosine kinase, tyrosine phosphatase, hematopoietic cytokine, peptide, extracellular calcium sensing, cAMP receptors. The receptors as ligand target represent more than 60% of all drug discovery targets.23
Receptor-ligand binding assays using high affinity radiolabeled ligand provide a direct screening approach for detecting specific/non-specific agonists/antagonists. This technique also finds application for quantitation of the potency of competing agents, investigating functional signal transduction pathways, monitoring molecular changes within a single cell, determine functions of orphan receptors.24
 
NANO SCREENS
The face of drug discovery process has been imparted a dramatic lift by combinatorial chemistry, robotics, miniaturization. At the screening rates enabled by ultra-high throughput screening (uHTS) applications, reagent consumption poses as the limiting factor. This calls for means to reduce the cost by reducing the volume of reagent required. This form of miniaturization has given birth to nano screens, wherein assay protocols have been validated using nano volumes (including pipetting, dispensing, and compound retrieval).
To achieve this modular platforms have been specially designed to handle high precision liquid handling and sensitive detection. In these operation systems, piezo technology is used to focus the liquid droplets into accurate volume and well. Moreover, the inherent advantage of amplification by the fluorescence detection systems is utilized at the read-out. NanoStore, EVOscreen, EVOTEC are some of the examples of systems adept at microseparation, detection and analysis.25
 
Patch Clamp Technique: Single Channel Recording
In vitro techniques are crucial to understand the normal physiological processes of synthesis and release, which are ongoing in individual cells. The pivotal patch clamp technique being widely applied today was developed from a series of experiments in frog muscle. Current was passed through individual ion channels activated by ACh and measured with high resolution. This was shown to be a discrete pulse-like event with duration of a few milliseconds. Fluctuation and relaxation measurements of end-plate currents led to the conclusion that the rate of channel opening increases with agonist concentrations, and that the channel, once open, closes spontaneously.26
The patch clamp technique has revolutionized cellular physiology since its introduction in the early 1980s. It allows investigators to assess: (i) ionic currents of a whole cell, including the molecular level of single ion channels, (ii) cell membrane potential and (iii) including fusion of a single secretory vesicle.27 When combined with microfluorimetry and digital imaging techniques, the method can be used to measure the spatio-temporal aspects of intracellular levels and distribution of ions such as calcium, sodium, chloride, changes in pH, production of signaling molecules and movement of these molecules. These techniques help to investigate 13signaling pathways at the cellular or molecular level in (cell lines, primary and transformed tissue cultures, brain slice preparations transient and/or stable transfections of cells and in primary cells derived from transgenic animals.
Conventional fine tip microelectrodes impale the cell in order to measure potential across the cell membrane, while patch clamp electrodes are too large to be inserted into a cell. The patch pipette is stuck onto the surface of a cell membrane instead of piercing it (Fig. 1.2). If a patch pipette is placed onto the cell surface and gentle suction is applied, a bubble shape of membrane is drawn into the patch pipette. The edges of this patch of membrane adhere tightly to the glass of the patch pipette. The electrical resistance of this seal between pipette glass and membrane is so high (a giga-ohm seal, or gigaseal) that the small patch of membrane underneath the patch pipette is by comparison a low resistance pathway and thus the favored route for current flow. This small patch of membrane may be voltage clamped to a series of potentials and the conductance of the patch calculated from the amount of current required to move from one potential to another. The patch of membrane under the pipette is very small. If the radius of a patch clamp pipette is 1@m, then the area of the patch under the pipette will be about 3 square picometres. Therefore, opening or closing of a single ion channel will cause a significant alteration in the overall conductance of the patch. Mostly, ion channels are either open or closed and they switch very rapidly from one state to the other. Therefore, the opening of a single ion channel causes an abrupt increase in the conductance of the patch of membrane beneath the pipette. The patch clamp technique involves a step-like increase in current. At a given voltage and ionic environment, the size of the current deflection is directly proportional to the conductance of this channel; the larger the deflection, the greater the conductance. If two channels open simultaneously, then the current is exactly twice as large. Ion channels may be distinguished from one another on the basis of this characteristic unit conductance, the duration of each opening (open time) and on the probability of the channel being open (open probability) under specific experimental conditions.
A patch of membrane from the cell can be removed without breaking the gigaseal and thus measure ion channel openings in an isolated patch of membrane. Besides, the single channel recording modes, the patch clamp technique may be applied to measure the currents that result from ion movements across the membrane of the whole cell. This mode of operation is known as the whole cell configuration. The first step in achieving this configuration is to obtain a high resistance contact between the pipette and the cell membrane (gigaseal). However, the patch of membrane under the pipette, which was the focus of attention in the single channel experiments is, in whole cell experiments, ruptured by application of a short pulse of negative pressure.
zoom view
Figure 1.2: Patch electrode on cell membrane
14The tight seal between pipette glass and cell membrane persists and the low resistance route for current flow is now into the cell and across entire cell surface membrane. A second feature of the whole cell configuration is that, following disruption of the patch of membrane under the pipette, the interior of the patch pipette is continuous with the cell interior. Thus, the solution filling the patch pipette will enter into and equilibrate with the cell interior. Small ions equilibrate within seconds of breaking through into the whole cell configuration.28
The application of the patch clamp technique has provided so many insights into cellular physiology that its originators, Bert Sakmann and Erwin Neher were awarded the Nobel Prize for Physiology and Medicine in 1991.
 
Advances in Patch Clamp Technique
More than ten years have passed since the slice-patch-clamp technique was established as a powerful method for the analysis of central synaptic transmission. Although this technique was earlier restricted only to young animal preparations, we can now even apply it to slices obtained from adult animals.
In addition, the advances have made paired whole-cell recording from two or more synaptically connected neurons, recording from dendrites or some presynaptic terminals, possible. Whole-cell patch-clamp technique can help to achieve complete biophysical characterization of an individual neuron, intrinsic, synaptic and spiking properties of cells in either superficial or deep structures, determining the synaptic receptive fields of single cells in anesthetized or awake head-fixed and even freely moving preparations so that synaptic activity may be linked directly to sensory processing and behavior.29
Future trends include developments facilitating microscopic analysis such as investigating glutamatergic sensitivities of single dendritic spines in combination with two-photon photolysis of a caged-glutamate compound and physiological function-oriented manner such as investigation of pain perception mechanisms using in vivo patch clamp technique.30
 
Microdialysis Technique
The neurobiologists wish to follow moment-by-moment, the sequence of biochemical events in various parts of the brain during a behavior. In this regard, various in vitro methods such as incubation of tissue slices and subcellular components have been very successful. Nevertheless, there is a definite need for a chemical technique comparable to the techniques of physiology where functional events can be followed closely over time. So far, the most successful in vivo “chemophysiological” techniques have been ventricular perfusions, cup perfusions on the surfaces of the brain, and push-pull perfusions carried out under stereotactic control in various parts of the nervous system.
The technique of microdialysis is a very important tool for in vivo studies in neuropsychopharmacology, toxicology, drug delivery, pharmacokinetics and endocrinology. Microdialysis is an extension of the push-pull technique because the perfusion fluid is circulating inside a semipermeable membrane instead of freely in the tissue. Substances in the extracellular fluid will diffuse into the perfusate, while substances included in the perfusate will diffuse into the tissue. This idea was first applied by Delgado and then by Ungerstedt who introduced the 15use of hollow fibers continuously perfused by a physiological liquid. Physiological processes may be closely followed in anesthetized as well as awake animals.31
 
Principle
Principle of dialysis has been applied for sampling the extracellular fluid of brain, thereby circumventing the problems associated with perfusion solution coming into direct contact with brain tissue. This technique is based on the principle of an artificial blood vessel surgically inserted into the tissue. The diffusion of chemical substances will occur in the direction of the lowest concentration. In this way, substances may be recovered from the organ or added to the organ depending upon their relative concentration in the perfusion fluid. There will be bidirectional molecular and ionic traffic between the interior of the microdialysis probe and the surrounding tissue. This provides the unique possibility of carrying out an entire pharmacological experiment within less than a cubic millimeter of tissue.31
 
Factors Affecting the Microdialysis
A unique feature of microdialysis is the possibility to compare in vitro experiments with in vivo experiments. The in vitro experiments may be performed on a solute contained in a simple beaker. The recovery of individual substances may be studied by determining the relative concentration in the perfusate in comparison with the concentration in the outside medium. The concentration will depend upon the properties of the membrane (most notably its molecular cut-off and its thickness), the speed of the perfusion, and the initial concentration of the compound in the perfusate. By using an appropriate size membrane and a sufficiently low perfusion speed, it is possible to reach 100% recovery.6
The dialysis membrane also acts as a filter to prevent the diffusion of large molecules from extracellular fluid into the perfusion medium. This provides certain advantages for the analysis of transmitter content in the dialysate. First, the membrane can prevent large molecules such as enzymes from entering the perfusion solution and thereby halt the continuous enzymatic degradation of neurotransmitters once they have entered the perfusion solution. Also, by virtue of its ability to exclude molecules from the perfusion solution, the membrane partially purifies samples prior to their analysis.32
 
Dialysis Probe
The development of the loop probe provided a means of reducing the extent of surgically induced injury. This probe consists of a loop of dialysis membrane, which is implanted vertically into the brain via a single hole in the skull (Fig. 1.3). Still less damage is produced by a vertical concentric style dialysis probe. This probe consists of a single piece of dialysis tubing blocked off at one end with glue; the inlet and/or outlet portions of the probe pass down into the dialysis tubing.
Adaptations in microdialysis probe designs have made it possible to obtain samples from the extracellular fluid of a variety of tissues with high temporal resolution. The resulting small volume samples, often with low concentration of the analyte(s) of interest, present a particular challenge to the analytical system. Rapid separations can be coupled online with microdialysis to provide near real-time data.3316
zoom view
Figure 1.3: Dialysis probe
 
Analysis of Sample
In vivo microdialysis in itself is only a sampling technique. The ability to measure compounds within dialysate is entirely dependent upon the sensitivity of an appropriate analytical method. In addition to the postoperative time at which samples are collected, other variables include the ionic composition of the dialysate, and the rate of perfusion affect sample content in the dialysate. Pharmacological tools have been used to compensate for inadequate sensitivity by increasing the level of substance to be analyzed. For example, acetylcholinesterase inhibitors have been used to enable detection of ACh in dialysate.34 Another approach has been to prelabel neurons by infusing isotopes of the transmitter or precursors and assaying the radiolabeled compounds.35
 
Duration of Experiment
Implantation of the dialysis probe results in several reactions within the CNS tissue. Knowledge of the time course of these events is critical in determining the interval during which microdialysis experiments can be performed with minimal interference from tissue reactions. In general it is thought that dialysis experiments should not be performed either very soon (< 10 h) or very long (several days to weeks) after probe implantation. The optimal interval for performing microdialysis experiments is approximately 16-48 h after implantation of the dialysis probe. Efforts have been made to develop methods whereby sampling can be carried out over many days in a single subject using either chronic implantation of a dialysis probe or implantation of a guide cannula followed by multiple insertions of a probe over days. However, these have generally been unsuccessful.17
 
Microdialysis versus Other Techniques
This technique has advantages over the blood sampling since it provides protein free samples ready to analyze without any loss of blood, permits more frequent sampling and offers an option of simultaneous drug delivery at the same site. Microdialysis can be considered to be superior to biosensors as more than one chemical system can be analyzed while circumventing the electrode contamination.
 
Present Status of Microdialysis Technique
Microdialysis is now used extensively for the study of several neurotransmitters in the CNS. The two main areas of application of microdialysis are the recovery of endogenous substances (neurotransmitters, catecholamines, neurotrophic factors, cAMP) and the infusion of drugs through the microdialysis cannula (retrodialysis). Clinical applications of microdialysis includes monitoring of ischemic injury, subarachnoid hemorrhage, trauma and epilepsy.36
Permutation and combination of novel membrane sampling techniques, ultrafiltration procedures and functional imaging (PET, MRI) have allowed numerous applications of the basic technique in pharmacokinetics, metabolism and/or pharmacodynamics.37,38 Semi-invasive techniques like microdialysis can be used to measure concentrations of the free, active drug or endogenous compounds in tissues and organs, determine transdermal drug distribution, tissue pharmacokinetics. Thus it gains importance as a widely used sampling technique in clinical drug monitoring, drug development, therapy and disease follow-up. It can play pivotal role in rationalizing drug dosing regimens and influencing the clinical decision-making process.39
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