Structure-activity relationship homology (SARAH): a conceptual framework for drug discovery in the genomic era

Extension of the traditional pharmacological approach of protein target classification to whole target systems has the potential to relate elements of protein sequence to the structure-activity relationship (SAR) of small molecules that can modulate protein action. Grouping potential drug discovery targets into families based on the relatedness of their SAR provides a means to translate the information from genome-sequencing efforts into knowledge that will aid in the discovery of drugs.     

DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.     

An in vitro transcription assay for probing drug-DNA interactions at individual drug sites

In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.     

A bioassay for NSILA-S in individual serum samples and its relationship to somatotropin

A practical bioassay for the acid-ethanol soluble non-suppressible insulin-like activity (NSILA-S) of individual serum samples has been developed utilizing the incorporation of 14C-glucose into the lipid faction of isolated adipocytes. NSILA-S activity was correlated with somatotropin status. Thus, the mean potencies (+/-SD) relative to an extract of pooled normal human serum were: normal samples 1.11 +/- 0.14, acromegalic 2.91 +/- 0.72, and somatotropin deficient 0.13 +/- 0.06. This variation in NSILA-S was not due to variability in extraction recoveries. The within assay precision was 9% (coefficient of variation) and the between assay 23%. This method allows the simultaneous extraction and processing of relatively large numbers of samples, and compares favorably with other more complex methods. Because of the evidence that NSILA-S may be related to the somatomedins, the present method should provide a simpler and more reliable alternative to the cartilage bioassays used to measure somatomedin activity.     

Sparse sampling for assessment of drug exposure in toxicological studies

A dose of 200 cGy of total-body irradiation (TBI) is nonlethal in dogs: Following a granulocyte nadir in the third week post-TBI, peripheral blood cell counts recover to normal values by about 5 weeks. In the context of studies on a potential role of major histocompatibility (MHC) class II antigens in the regulation of stress hematopoiesis, we tested the effect of anti-MHC class II monoclonal antibodies (mAbs) on hematologic recovery after TBI. Thirteen dogs were given 200 cGy of TBI not followed by marrow infusion. Five received no additional treatment (concurrent controls) and eight were given daily intravenous (IV) injections of anti-class II mAbs H81.9 (anti-HLA-DR; n = 6) or B1F6 (anti-HLA-DR and -DP; n = 2) at 0.6 (n = 4) or 1.2 mg/kg/d (n = 4) on days 0-4 (n = 7) or days 0-9 (n = 1). One control dog died early from an intercurrent infection and four recovered uneventfully. Dogs given mAbs after TBI showed significantly different granulocyte and platelet kinetics. The granulocyte nadir was lower (p = 0.09) and was reached later (p = 0.005), the duration of neutropenia was longer (p = 0.08), and recovery occurred later (p = 0.02) than among controls. Similarly the platelet nadir was lower (p = 0.05), thrombocytopenia lasted longer (p = 0.02), and recovery occurred later (p = 0.02) than among controls. Four of eight mAb-treated dogs died with marrow aplasia. We propose that following irradiation, HLA-DR mediated signals result in terminal differentiation in more mature hematopoietic precursors but interfere with replication or differentiation in early hematopoietic precursors. These observations suggest a role for MHC class II molecules in the regulation of stress hematopoiesis.     

What changes drug metabolism in critically ill patients? Two preliminary studies in isolated human hepatocytes

The metabolism of many drugs is abnormal in the critically ill patient. The causes of this are unknown, and investigation in patients is difficult. We therefore used isolated, cultured human hepatocytes to study the effects of hypoxia and induction on cytochrome P450 3A, the cytochrome responsible for the metabolism of many drugs. When hepatocytes were exposed to 5% oxygen, the amount of 3A produced, after induction with rifampicin, was five to 10 times less than the amount produced in 21% oxygen. In another study, we exposed isolated hepatocytes for 4 days to serum from five critically ill patients or from volunteers. At the end of this time, the functional ability of the hepatocytes to glucuronidate 14C progesterone was measured. Four of the five patients had a substance in their plasma that reduced the ability of the hepatocytes to glucuronidate progesterone. The nature of this substance and the reason that the serum from the fifth patient did not affect metabolism are unknown. This model is able to simulate the abnormalities which occur in critically ill patients. Further studies are needed to explain our observations and to identify the substances in the serum from critically ill patients that alter drug metabolism.     

An alternative analysis for crossover studies that accounts for between-group disparities in drug response

A 3-year-old girl presented with epigastric pain and physical examination showed a hard right upper quadrant mass. Urine VMA and HVA were found to be raised. CT scan showed a large mass arising from the right adrenal gland, with necrotic areas and ring calcification There was midline extension. The diagnosis of neuroblastoma was confirmed by bone marrow aspiration biopsy. As the patient had inoperable stage IV neuroblastoma, she was treated with multi-drug chemotherapy. Follow-up CT scan showed excellent response to the chemotherapy, and the tumour was successfully resected. Harvesting of autologous peripheral bone marrow stem cells, megatherapy with Melphalan, and marrow rescue with the stem cells were effective. The child has been well for three years to date. The clinical and imaging features of neuroblastoma, particularly the role of imaging in staging, are emphasised.     

Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair

T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.     

Enantioselective analysis of N-hydroxymexiletine glucuronide in human plasma for pharmacokinetic studies

CO2 reactivity of cerebral hemoglobin concentration was studied in 16 healthy term neonates on days 1 and 4 after birth using the near infrared spectrophotometry (NIRS) technique. The aim was to establish data on the physiological range of CO2 reactivity in healthy newborns and to investigate the influence of postnatal age on it. The CO2 reactivity measured by NIRS is expressed as the change of the total cerebral hemoglobin concentration (tHbR) per change of CO2 tension in micromol/l/kPa. We evaluated CO2 reactivity during increases and decreases of transcutaneous CO2 partial pressure and found in our methodological setting the data of the increases more reliable. In all infants but 1 we found a tHbR on day 1 with a mean value of 8.19 micromol/l/kPa (-1.39 to 18.87), in all infants on day 4 with a mean value of 9.54 micromol/l/kPa (2.76-25. 88). There is a trend to higher values between day 1 and day 4 (difference = 2.25 micromol/l/kPa; p = 0.08). The noninvasive NIRS technique enabled us to test the cerebrovascular CO2 reactivity of the tHbR for the first time in healthy term newborns. Data on its physiologic range and variability are presented and compared to findings from ventilated infants and other age groups. As the CO2 reactivity might be an indicator for infants at risk of cerebral damage, it is necessary to have data on the physiological range of this parameter.     

Calcium responses in human fibroblasts: a diagnostic molecular profile for Alzheimer's disease

We have previously identified alterations of K+ channel function, IP3-mediated calcium release, and Cp20 (a memory-associated GTP binding protein) in fibroblasts from AD patients vs. controls. In the present study we introduce a scoring system based on these response alterations that integrates two or more alterations (and their degree) in AD vs. control fibroblasts. This scoring system generates an index that distinguishes AD patients from controls with both high specificity and sensitivity. We also show that low doses of bradykinin elicit intracellular calcium release almost exclusively in AD cell lines in an all or none fashion that provide a clear measurement of enhanced IP3-mediated function in AD vs. controls.     

A PC-based software test for measuring alcohol and drug effects in human subjects

A new software-based visual search and divided-attention test of cognitive performance was developed and evaluated in an alcohol dose-response study with 24 human subjects aged 21-62 years. The test used language-free, color, graphic displays to represent the visuospatial demands of driving. Cognitive demands were increased over previous hardware-based tests, and the motor skills required for the test involved minimal eye movements and eye-hand coordination. Repeated performance on the test was evaluated with a latin-square design by using a placebo and two alcohol doses, low (0.48 g/kg/LBM) and moderate (0.72 g/kg/LBM). The data on 7 females and 17 males yielded significant falling and rising impairment effects coincident with moderate rising and falling breath alcohol levels (mean peak BrALs = 0.045 g/dl and 0.079 g/dl). None of the subjects reported eye-strain or psychomotor fatigue as compared with previous tests. The high sensitivity/variance relative to use in basic and applied research, and worksite fitness-for-duty testing, was discussed. The most distinct advantage of a software-based test that operates on readily available PCs is that it can be widely distributed to researchers with a common reference to compare a variety of alcohol and drug effects.     

Persistent antibody responses but declining cytotoxic T-lymphocyte responses to multiple human immunodeficiency virus type 1 antigens in a long-term nonprogressing individual with a defective p17 proviral sequence and no detectable viral RNA expression

Long-term nonprogressor AD-18 has been infected with human immunodeficiency virus type 1 (HIV-1) for at least 16 years. During the past 5 years, he has had undetectable levels of plasma viremia, and HIV-1 cannot be isolated from him. Sequencing of proviral DNA indicates that the only HIV-1 sequences that can be identified in AD-18 have gross defects in the p17-encoding regions of the gag gene (Y. Huang, L. Zhang, and D. D. Ho, Virology 240:36-49, 1998). However, AD-18 has strong, sustained antibody responses to several HIV-1 antigens, including p17. Cytotoxic T-lymphocyte responses to Env and Gag antigens have gradually diminished over the past 4 years, at a time when the titers of antibodies to the same proteins have remained stable. We discuss what these observations might mean for the generation and maintenance of immunological memory.     

Levofloxacin population pharmacokinetics and creation of a demographic model for prediction of individual drug clearance in patients with serious community-acquired infection

Population pharmacokinetic modeling is a useful approach to obtaining estimates of both population and individual pharmacokinetic parameter values. The potential for relating pharmacokinetic parameters to pharmacodynamic outcome variables, such as efficacy and toxicity, exists. A logistic regression relationship between the probability of a successful clinical and microbiological outcome and the peak concentration-to-MIC ratio (and also the area under the plasma concentration-time curve [AUC]/MIC ratio) has previously been developed for levofloxacin; however, levofloxacin assays for determination of the concentration in plasma are not readily available. We attempted to derive and validate demographic variable models to allow prediction of the peak concentration in plasma and clearance (CL) from plasma for levofloxacin. Two hundred seventy-two patients received levofloxacin intravenously for the treatment of community-acquired infection of the respiratory tract, skin or soft tissue, or urinary tract, and concentrations in plasma, guided by optimal sampling theory, were obtained. Patient data were analyzed by the Non-Parametric Expectation Maximization approach. Maximum a posteriori probability Bayesian estimation was used to generate individual parameter values, including CL. Peak concentrations were simulated from these estimates. The first 172 patients were used to produce demographic models for the prediction of CL and the peak concentration. The remaining 100 patients served as the validation group for the model. A median bias and median precision were calculated. A two-compartment model was used for the population pharmacokinetic analysis. The mean CL and the mean volume of distribution of the central compartment (V1) were 9.27 liters/h and 0.836 liter/kg, respectively. The mean values for the intercompartmental rate constants, the rate constant from the central compartment to the peripheral compartment (Kcp) and the rate constant from the peripheral compartment to the central compartment (Kpc), were 0.487 and 0.647 h(-1), respectively. The mean peak concentration and the mean AUC values normalized to a dosage of 500 mg every 24 h were 8.67 microg/ml and 72.53 microg x h/ml, respectively. The variables included in the final model for the prediction of CL were creatinine clearance (CLCR), race, and age. The median bias and median precision were 0.5 and 18.3%, respectively. Peak concentrations were predicted by using the demographic model-predicted parameters of CL, V1, Kcp and Kpc, in the simulation. The median bias and the median precision were 3.3 and 21.8%, respectively. A population model of the disposition of levofloxacin has been developed. Population demographic models for the prediction of peak concentration and CL from plasma have also been successfully developed. However, the performance of the model for the prediction of peak concentration was likely insufficient to be of adequate clinical utility. The model for the prediction of CL was relatively robust, with acceptable bias and precision, and explained a reasonable amount of the variance in the CL of levofloxacin from plasma in the population (r2 = 0.396). Estimated CLCR, age, and race were the final model covariates, with CLCR explaining most of the population variance in the CL of levofloxacin from plasma. This model can potentially optimize the benefit derived from the pharmacodynamic relationships previously developed for levofloxacin.     

Aseptic isolation of human complement component C3 for in vivo studies

A procedure for aseptic isolation of human complement component C3 is described. The principles may be useful in the preparation of other proteins for in vivo studies.     

Rapid isolation of genomic clones for individual members of human multigene families: identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family

We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.     

Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.