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PubMed1998

Construction of a quantitative PCR system to determine expression of tumor associated antigen.

Cancer immunotherapy is a strategy for cancer treatment by induction of anti tumor responses. The identification of candidate tumor associated antigens (TAA) suggested their potential use as immunogens for vaccination studies. Quantification of a TAA expression by cancerous cells is an important factor in determination of induced immune response efficiency against tumors and thus enables us to devise optimal immunotherapy protocol to cure cancer. The quantitative polymerase chain reaction (PCR) enables us to compare the TAA expression in highly metastatic tumor clones with that in less metastatic ones and in normal cells. It allows us to gain more insight into genome rearrangements that occur in malignant transformation as well as broaden our knowledge about tumor cell gene expression regulation. One of the peptides isolated previously in our lab from a murine lung carcinoma is the mutated Connexin 37 (cx37), a gap-junction protein. Research is underway to determine the expression level of the TAA in various Lewis lung carcinoma cells. This evaluation is achieved by means of quantitative PCR. A quantitative PCR experiment includes preparation of a control template, which is added in known amounts together with the target template in a series of amplification reactions. The control template uses the same primers as the target sequence, yet their PCR products differ in size so as to be distinguishable. Two methods were used to produce this control template. The first one included specific deletion of a sequence of approximately 100 bp that lay between the two primers, insertion of the new template into a plasmid vector, transformation of competent bacteria, detection of transformed bacterial colonies and isolation of the plasmid DNA in a large quantity. The non-mutated, deleted Connexin 37 cDNA was also isolated from bacteria and used for another experiment aimed at producing deleted, mutated Connexin 37 cDNA by means of primer mutagenesis. (Fig. 5, Ref 8.)

Animals↗
PubMed1999

Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis.

We examined codon usage in Bacillus subtilis genes by multivariate analysis, quantified its cellular levels of individual tRNAs, and found a clear constraint of tRNA contents on synonymous codon choice. Individual tRNA levels were proportional to the copy number of the respective tRNA genes. This indicates that the tRNA gene copy number is an important factor to determine in cellular tRNA levels, which is common with Escherichia coli and yeast Saccharomyces cerevisiae. Codon usage in 18 unicellular organisms whose genomes have been sequenced completely was analyzed and compared with the composition of tRNA genes. The 18 organisms are as follows: yeast S. cerevisiae, Aquifex aeolicus, Archaeoglobus fulgidus, B. subtilis, Borrelia burgdorferi, Chlamydia trachomatis, E. coli, Haemophilus influenzae, Helicobacterpylori, Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Pyrococcus horikoshii, Rickettsia prowazekii, Synechocystis sp., and Treponema pallidum. Codons preferred in highly expressed genes were related to the codons optimal for the translation process, which were predicted by the composition of isoaccepting tRNA genes. Genes with specific codon usage are discussed in connection with their evolutionary origins and functions. The origin and terminus of replication could be predicted on the basis of codon usage when the usage was analyzed relative to the transcription direction of individual genes.

Archaea↗
PubMed2006

Use of 65 kDa mannoprotein gene primers in Real Time PCR identification of Candida albicans in biological samples.

A method for the detection and quantification of Candida albicans in biological samples (blood, urine and serum) was developed with the use of Real-Time PCR utilizing CaMP65-specific primers. Two different systems were used for the detection in the LightCycler platform (Roche): the SYBR green fluorescent dye with melting peak analysis and the 5'nuclease fluorescent-probe detection. The amplification was highly specific for C. albicans, providing no cross-reaction on genomic DNA extracted from other Candida species or Aspergillus. The sensitivity in simulated biological samples was especially high (1 genome) when applied to sera and urine, and in blood samples the limit of detection was higher by ten-fold. Finally, the real-time PCR was employed in order to detect and quantify C. albicans in the sera from patients with invasive candidiasis.

Aspergillus↗
PubMed1993

Quantification of specific DNA O-alkylation products in individual cells by monoclonal antibodies and digital imaging of intensified nuclear fluorescence.

We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.

Alkylation↗
PubMed2026

Integrated phytochemical and bioactivity profiling of Xanthium strumarium fruits from Korea and China: Implications for origin-specific quality specification.

BACKGROUND: Geographic origin influences the phytochemical composition and biological activities of medicinal plant resources. Xanthium strumarium L. (XS) fruit is widely used in East Asian traditional medicine. However, current pharmacopeial standards primarily recognize Chinese-derived material, despite the availability and traditional use of XS in Korea. To address this gap and support origin-informed quality specification, we compared fruits from Korea (XS-K) and China (XS-C) using chloroplast genome sequencing, targeted phytochemical profiling (high-performance liquid chromatography (HPLC) for selected phenolics and gas chromatography-flame ionization detection (GC-FID) for fatty acids and phytosterols, and multivariate chemometric analysis. RESULTS: Chloroplast genome analysis revealed high overall similarity but localized divergence around the rpoC2 locus and a greater mutation burden in XS-C, supporting origin-associated genomic differentiation. Phytochemical profiling revealed distinct origin-dependent metabolic signatures. XS-K showed higher levels of phytosterols, chlorogenic acid, 4,5-dicaffeoylquinic acid (4,5-DCQ), and xanthatin was detected only in XS-K, whereas XS-C exhibited greater abundance of total fatty acids, particularly oleic acid. Unsupervised clustering and log2 fold-change ranking confirmed clear compositional separation, and variable importance in projection (VIP) analysis identified chlorogenic acid, β-sitosterol, oleic acid, 4,5-DCQ, and xanthatin as major discriminators between origins. Bioactivity assays demonstrated that XS-K exerted stronger antioxidant effects in ABTS, DPPH and FRAP assays, stronger skin-related enzyme inhibition, and greater antibacterial activity against Staphylococcus aureus, consistent with its enriched phenolic and sterol profile. CONCLUSION: Together, chloroplast sequence variation, targeted metabolite quantification, and screening bioassays consistently distinguished XS-K from XS-C. These findings support the use of candidate markers for the origin-based authentication and quality control of XS fruit-derived ingredients. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Fruit↗
PubMed2005

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms.

The repeat region of DC-SIGNR (CD209L) is polymorphic on the genomic level, and, in a separate study, we observed a correlation between the DC-SIGNR genotype and HIV-1 susceptibility during sexual contact. However, previous investigations using immunohistochemistry failed to detect membrane-bound DC-SIGNR on cells in the genital and rectal mucosa. We therefore explored the presence of DC-SIGNR in these compartments with a more sensitive limiting dilution RT-PCR, which also allowed for quantification of alternatively spliced mRNA isoforms. DC-SIGN (CD209) and DC-SIGNR mRNA transcript isoforms were found in all 12 vaginal and two rectal biopsies obtained from 14 healthy individuals. For DC-SIGNR, we detected significantly more isoform than full-length transcripts (mean copy numbers/mug RNA: 602 vs 26; P=0.0009). Four mucosal samples lacked full-length DC-SIGNR transcripts entirely. Cloning and sequencing of DC-SIGNR mRNA in three additional individuals revealed a diverse repertoire of DC-SIGNR isoforms, many of which encoded for proteins predicted to be soluble and secreted. Indeed, in one vaginal sample, we detected only soluble isoforms. In conjunction with our prior observation that the DC-SIGNR genotype has an effect on HIV-1 transmission in vivo, these findings emphasize that DC-SIGNR, in addition to DC-SIGN, should be considered as a cofactor in sexual HIV-1 transmission. Soluble isoforms, in particular, may modulate the efficiency of viral transmission and dissemination.

Amino Acid Sequence↗
PubMed1999

A comparison of reverse transcription-polymerase chain reaction and branched-chain DNA assays for hepatitis C virus RNA in patients receiving interferon treatment. Consensus Interferon Study Group.

Measurement of hepatitis C virus (HCV) RNA may be beneficial in managing the treatment of patients with chronic HCV infection. In a phase 3 study comparing consensus interferon (IFN) and IFN-alpha2b treatment in patients with chronic HCV infection, serum samples were assayed for HCV RNA using two different assays: a quantitative multicycle reverse transcription-polymerase chain reaction (RT-PCR) method and the Quantiplex branched-chain DNA (bDNA) method. Lower and upper detection limits were 100 copies ml-1 and 5 x 10(6) copies ml-1, respectively, for the RT-PCR method, and 3.5 x 10(5) and 4 x 10(7) genome equivalents ml-1, respectively, for the bDNA method. The two assays were generally concordant over the common range of detectability. The major discrepancy was where PCR still indicated detectable virus in the sample but the bDNA result was negative. Assessment of serum samples during IFN treatment demonstrated that 37% of samples were negative for HCV RNA by bDNA but positive by RT-PCR. Differences were also noted in the quantification of baseline HCV RNA by genotype. These data suggest that HCV patients could be categorized as treatment responders by the bDNA assay when the more sensitive RT-PCR assay indicates lack of complete viral response.

Antiviral Agents↗
PubMedDate not supplied

Digital image capture and quantification of subtle lens opacities in rodents.

A rapid, sensitive, and cost-effective method is reported for the subjective and objective documentation of subtle opacities in lenses of unanesthetized transgenic mice or selenite-injected rat pups as models for cataract formation. Animal eyes were dilated with eye drops and the animal was positioned in front of a Nikon FS2 photo slit lamp. Slit-lamp observations were recorded using a Canon Optura Pi digital video recorder. High-quality images of opacifying lenses were captured from the video and quantified using densitometry at progressive stages of opacification. In mice, targeted genomic deletion of the proteins CP49 (a lens-specific filament) or Six5 (a model for myotonic dystrophy) resulted in subtle cataracts that were easily recorded and quantified using this instrumentation. In rats, the early progressive changes leading to a dense nuclear opacity caused by selenite injection were easily documented using this instrumentation. Low-cost components combined with a conventional slit-lamp ophthalmoscope were used to capture high-quality images of selected stages of cataract formation for quantitative analysis using commercial software.

Animals↗
PubMed2004

Distribution and quantification of human herpesvirus 6 in multiple sclerosis and control brains.

Multiple sclerosis (MS) is thought to be precipitated by environmental factors, potentially including viruses, in genetically susceptible individuals and recently human herpesvirus 6 (HHV-6) has been associated with the disease. We have analysed post mortem brain for the presence, variant type and quantity of HHV-6 by PCR. A total of 124 samples from seven anatomically defined regions of brain were tested from MS cases and controls. HHV-6 DNA was detected in 41% and 44% of MS case and control samples. The median viral loads were 11 and 9 genome copies/microg DNA in cases and controls respectively and the viral load was not increased in lesions. Except in one instance, the HHV-6 DNA detected was variant B. There was no apparent difference in the distribution of HHV-6 DNA in the brains of MS cases versus controls, nor between normal appearing and lesional tissue in MS cases. Periventricular regions of the brain were more frequently positive for HHV-6 DNA in both MS cases and controls, although this difference was not statistically significant. These studies confirm the neurotropism of HHV-6 but do not demonstrate differences in the distribution, variant type or quantity of HHV-6 in brains from patients with MS versus controls.

Aged↗
PubMed2004

Detection of influenza B in clinical specimens: comparison of high throughput RT-PCR and culture confirmation.

Influenza virus is one of the major causes of worldwide respiratory tract infections during the winter season. Here we describe a high throughput (HTP) protocol for rapid diagnosis of influenza B that combines automated viral RNA extraction with detection and quantification by TaqMan-based PCR. Using this methodology, we tested 4176 nasal swabs collected from children enrolled in a European influenza vaccine trial during the winter of 2000 to compare our HTP PCR method to culture confirmation for detection of influenza B. Among these, 37 were positive by culture and 169 were positive by PCR irrespective of virus copy number. However, when specimens with fewer than 20 copies of the viral genome were disregarded, a good correlation between two methods was observed. At this cut-off, 34 specimens were positive and 4106 were negative by both methods. Statistical analysis of the data using culture confirmation as the standard indicated that the sensitivity of HTP RT-PCR for influenza B was 92% (95% CI, 0.83-1), and the specificity was 99% (95% CI, 0.99-1). In summary, HTP RT-PCR was proved to be more rapid and sensitive than culture confirmation, and it correlated significantly with culture confirmation for specimens containing more than 20 copies of the viral genome.

Animals↗
PubMed2003

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age.

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy beta-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 +/- 920, significantly higher than that in skeletal muscle, 3650 +/- 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.

Adolescent↗
PubMed2025

Pooled PPIseq: Screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform.

Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.

Humans↗
PubMed2006

Limitations and practical procedure in BclII-Ig heavy chain gene rearrangement real-time quantitative polymerase chain reaction.

Follicular lymphoma is characterized by the t(14;18)(q32;q21) translocation, which juxtaposes Ig heavy chain gene (IgH) sequences with the BclII gene. Several publications have highlighted the importance of molecular follow-up in follicular lymphoma, demonstrating that the detection of cells bearing the BclII-IgH rearrangement by real-time quantitative polymerase chain reaction (RQ-PCR) can anticipate a clinical relapse. In this context, we developed a BclII-IgH RQ-PCR. We began with SYBR Green I detection technology but observed that this system does not allow an accurate measurement of the tumor load when working with genomic DNA. While we were designing the assay using Taqman technology, Moppett et al (Moppett J, van der Velden VHJ, Wijkhuijs AJM, Hancock J, van Dongen JJM, Goulden N: Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 2003, 17:268-270) reported PCR inhibition problems in around 15% of blood and bone marrow samples, affecting the DNA quantification and thus the assessment of minimal residual disease. They demonstrated that this PCR inhibition could be partially resolved by adding nonacetylated bovine serum albumin. In our studies, we observed the same phenomenon in a single follicular lymphoma case and extended our study to other available cases. As a result, we suggest a new RQ-PCR procedure that is based on Taqman probe technology and that takes into account the PCR inhibition problems, making this assay more reliable in a routine molecular laboratory.

Aged↗
PubMed2004

A scalable high-content cytotoxicity assay insensitive to changes in mitochondrial metabolic activity.

Quantitative assessments of cell proliferation and cytotoxicity are key components of anticancer drug discovery. Advancements in genomics and combinatorial chemistry have increased the demand for cytotoxicity measurements, and almost all assays for cell proliferation that are compatible with high-throughput screening are single-parameter, homogeneous biochemical assays. Furthermore, the most popular methods currently in use are based on measurements of mitochondrial activity and therefore provide only an indirect measure of cell growth. Here we describe a high-content screening (HCS) methodology that permits the direct quantification of cell numbers as well as multiple measurements of drug activity in individual cells without the ambiguities of previously described homogenous metabolism-based assays. Using two human tumor cell lines, we compared our HCS method with a commonly used homogenous colorimetric assay that detects mitochondrial activity and found the colorimetric assay substantially underrepresented the cytotoxic effects of two mechanistically diverse, clinically used, anticancer drugs: a DNA-damaging agent, bleomycin, and a tubulin stabilizer, paclitaxel. Simultaneous evaluation of cell numbers, mitochondrial mass, and nuclear morphology by HCS provided an explanation for the differential toxicity. Furthermore, we expanded the scope of the HCS assay to include tubulin mass measurements in paclitaxel-treated cells as an example for a specific drug target. The data illustrate the utility of HCS as a powerful analysis tool compatible with the demands of high-throughput screening, and adaptable to include fluorescence-based markers of drug activity.

Antineoplastic Agents↗
PubMed2006

Development of a real-time PCR assay for detection and quantification of Francisella tularensis.

The facultative intracellular bacterium, Francisella tularensis, is an etiological agent of tularemia and is also considered to be a potential biological threat agent due to its extreme infectivity. We established a real-time PCR assay using the LightCycler (LC) system to detect a Francisella-specific sequence of the outer membrane protein (fopA) gene. Twenty-five F. tularensis strains including 16 Japanese isolates were subjected to this LC-PCR assay, and were tested positive, whereas Francisella philomiragia and other bacteria species did not show any specific fluorescent signal. A linear response was observed using F. tularensis genomic DNAs of between 20 fg and 2 ng, corresponding to 1.2 to 1.2 x 10(5) bacteria. The newly established real-time PCR allows the detection of the F. tularensis genome specifically, sensitively, and rapidly. This assay may contribute to the standardization of the laboratory diagnosis of tularemia.

Bacterial Outer Membrane Proteins↗
PubMed2002

Immobilisation of Semliki forest virus for atomic force microscopy.

Semliki Forest virus (SFV), an alphavirus, is a single-stranded positive-sense RNA virus. The RNA genome is surrounded by a protein shell known as the capsid which itself is surrounded by a lipid envelope of host cell origin. In this study, SFV strain L10 enveloped virus and its capsid were immobilised onto silicon wafer supports which had been pre-coated with a monolayer of the relevant anti-viral antibody. After drying, the samples were imaged in air, using non-contact mode atomic force microscopy (AFM). Quantification of the AFM images has revealed that both the strain L10 enveloped virus and capsid collapse when immobilised in this manner. The capsid undergoes more significant collapse compared to the enveloped virus. The dimensions of the immobilised enveloped virus and capsid have been compared to a model where the free spherical particles collapse into ellipsoids during immobilisation. For the immobilised capsid the dimensions are consistent with this model whereas for the enveloped virus the model is less effective. The dimensions of the enveloped virus appear to be affected by the antibody used for immobilisation.

Animals↗
PubMed2006

The DNA N-glycosylase MED1 exhibits preference for halogenated pyrimidines and is involved in the cytotoxicity of 5-iododeoxyuridine.

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k(st) values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20- to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity.

Animals↗
PubMed2003

Biomarkers, validation and pharmacokinetic-pharmacodynamic modelling.

Four elements are crucial to successful pharmacokinetic-pharmacodynamic (PK/PD) modelling and simulation for efficient and effective rational drug development: (i) mechanism-based biomarker selection and correlation to clinical endpoints; (ii) quantification of drug and/or metabolites in biological fluids under good laboratory practices (GLP); (iii) GLP-like biomarker method validation and measurements and; (iv) mechanism-based PK/PD modelling and validation. Biomarkers can provide great predictive value in early drug development if they reflect the mechanism of action for the intervention even if they do not become surrogate endpoints. PK/PD modelling and simulation can play a critical role in this process. Data from genomic and proteomics differentiating healthy versus disease states lead to biomarker discovery and identification. Multiple genes control complex diseases via hosts of gene products in biometabolic pathways and cell/organ signal transduction. Pilot exploratory studies should be conducted to identify pivotal biomarkers to be used for predictive clinical assessment of disease progression and the effect of drug intervention. Most biomarkers are endogenous macromolecules, which could be measured in biological fluids. Many exist in heterogeneous forms with varying activity and immunoreactivity, posting challenges for bioanalysis. Reliable and selective assays could be validated under a GLP-like environment for quantitative methods. While the need for consistent reference standards and quality control monitoring during sample analysis for biomarker assays are similar to that of drug molecules, many biomarkers have special requirements for sample collection that demand a well coordinated team management. Bioanalytical methods should be validated to meet study objectives at various drug development stages, and possess adequate performance to quantify biochemical responses specific to the target disease progression and drug intervention. Protocol design to produce sufficient data for PK/PD modelling would be more complex than that of PK. Knowledge of mechanism from discovery and preclinical studies are helpful for planning clinical study designs in cascade, sequential, crossover or replicate mode. The appropriate combination of biomarker identification and selection, bioanalytical methods development and validation for drugs and biomarkers, and mechanism-based PK/PD models for fitting data and predicting future clinical endpoints/outcomes provide powerful insights and guidance for effective and efficient rational drug development, toward safe and efficacious medicine for individual patients.

Biomarkers↗