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Biomedical subjects

M J Graham

Publications and source records attributed to M J Graham.

At least 19 recordsLinked to original sources
PubMed2003

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates.

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S-warfarin (CYP2C9 in humans) by dog liver microsomes (mean +/- SD from 12 (six male and six female) dogs = 10.8 +/- 1.9 fmol mg(-1) protein min(-1)) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S-mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 +/-1.5 pmol mg(-1) protein min(-1)) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R-enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K(m) or K(0.5) 15-22 micro M, V(max) 35-59 pmol mg(-1) protein min(-1), n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S-mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K(m) 42-64 micro M, V(max) 22-46 pmol mg(-1) protein min(-1), n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol mg(-1) protein min(-1) (p < 0.02); S-warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol mg(-1) protein min(-1) (p < 0.02); R-mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol mg(-1) protein min(-1) (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol mg(-1) protein min(-1) (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S-warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.

Algorithms↗
PubMed2001

Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration.

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3'-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5'-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.

Animals↗
PubMed2001

Comparative radiolabel and ATP analyses of adhesion of Pseudomonas aeruginosa and Staphylococcus epidermidis to hydrogel lenses.

PURPOSE: A comparative assessment of the relative primary adhesion of cells of Pseudomonas aeruginosa, its lux transformant, and of slime and non-slime producing strains of Staphylococcus epidermidis to various hydrogel lenses was conducted. METHODS: Hydrogel lenses were placed in cell suspensions with bacteria with or without a tritiated leucine label. After 2 hours exposure, the lenses were rinsed vigorously and densities of cells on the lenses were determined via scintillation counting or ATP analyses. RESULTS: The radiolabel procedure indicated greater numbers than the ATP analyses of adhered cells per lens per common inoculum of all strains. All strains exhibited greater primary adhesion to the 38% water content contact lens, with the lux transformant of P. aeruginosa showing the greatest degree of adhesion. Primary adhesion by P. aeruginosa was typically at least ten-fold greater per lens than that observed with S. epidermidis. CONCLUSIONS: Both a radiolabel-cell procedure and bioluminescent ATP analyses demonstrated similar patterns of primary adhesion of bacteria to hydrogel lenses. Generally the adhesion increased inversely to the water content of the lenses but the chemical composition of the lenses, particularly surface properties, altered this pattern for lenses of similar water content. The magnitude of primary adhesion varied with the species and strain of bacterium.

Adenosine Triphosphate↗
PubMed2000

Pharmacokinetic and toxicity profile of a phosphorothioate oligonucleotide following inhalation delivery to lung in mice.

Antisense oligonucleotides are currently being investigated for the treatment of a variety of diseases. Antisense drugs are being administered primarily by parenteral injection. To explore more convenient patient delivery methods, we have characterized the tissue kinetics and tolerability of an inhaled aerosol formulation of a phosphorothioate oligonucleotide in mice. Concentrations of oligonucleotide in bronchioalveolar lavage fluid, plasma, and tissue and immunohistochemical localization were used to assess deposition and pharmacokinetic parameters. Significant concentrations of oligonucleotide in lung, as well as systemic tissues, were measured following a pulmonary dose of 12 mg/kg. Doses as low as 1-3 mg/kg also produced significant concentrations of oligonucleotide (>50 microg oligonucleotide per gram of tissue), and these were maintained in the lung with a halflife of 20 hours or greater. Oligonucleotide was localized to bronchiolar epithelium and alveolar epithelium and endothelium. Toxicity was mild at the 12 mg/kg level and minimal to absent at doses of 3 mg/kg or below. Based on a favorable pharmacokinetic profile and a relative lack of toxicity, inhalation delivery appears to be a therapeutic option for antisense oligonucleotides.

Administration, Inhalation↗
PubMed2000

Variations in mRNA content have no effect on the potency of antisense oligonucleotides.

A fundamental question with regard to antisense pharmacology is the extent to which RNA content or transcription rate or both affect the potency of antisense drugs. We have addressed this by controlling RNA content and transcription rate using either an exogenous gene expressed after transfection or an endogenous gene induced with a cytokine. We have demonstrated that in both A549 and HeLa cells, varying RNA copy numbers from <1 to >100 copies per cell has no effect on the potency of RNase H-active antisense drugs transfected into cells, nor did variation in transcription rate have an effect on potency. We demonstrate that this is because the number of oligonucleotide molecules per cell is vastly in excess of the RNA copy number. These data further suggest that a significant fraction of cell-associated antisense drug molecules may be unavailable to interact with the target RNA, an observation that is not surprising, as phosphorothioate oligonucleotides interact with many cellular proteins. We suggest that these data may extrapolate to in vivo results.

Dexamethasone↗
PubMed2000

Metabolism of antisense oligonucleotides in rat liver homogenates.

Phosphorothioate antisense oligodeoxynucleotides are novel therapeutic agents designed to selectively and specifically inhibit production of various disease-related gene products. In vivo pharmacokinetic experiments indicate that these molecules are widely distributed in many species, with the majority of oligomers accumulating within liver and kidney. To better understand the metabolism of these agents, we studied the stability of several phosphorothioate oligodeoxynucleotides, their congeners, and second generation oligomer chemistries in rat liver homogenates. To examine metabolism, background nuclease activity was characterized in whole liver homogenates by using ISIS 1049, a 21-mer phosphodiester oligodeoxynucleotide. Nuclease activity could readily be detected in liver homogenates. Under optimized conditions, the predominant enzymatic activity was 3'-exonucleolytic and could be influenced by pH and ionic conditions. However, in addition to 3' exonucleases, 5' exo- and endonuclease activities were also observed. Our data indicate that metabolism of phosphorothioate oligodeoxynucleotides was more complex than that of phosphodiesters for many reasons, including phosphorothioate oligodeoxynucleotide inhibition of nucleases and the presence of R(p) and S(p) stereoisomers. The rate of phosphorothioate metabolism also appeared to be influenced by sequence, with pyrimidine-rich compounds being metabolized to a greater extent than purine-rich oligomers. Other factors affecting stability included oligomer chemistry and length. Concomitant experiments performed in rats dosed systemically with the same compounds mimic the activities seen in vitro and suggest that this liver homogenate system is a valuable model with which to study the mechanism of metabolism of antisense oligonucleotides.

Animals↗
PubMed2000

Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice.

It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.

Animals↗
PubMed1999

Secular trend in age at menarche in China: a case study of two rural counties in Anhui Province.

There is increasing evidence that age at menarche has decreased in Europe and the United States during the last century and in Japan over the last several decades. Data from a community-based survey conducted in two rural counties of Anhui Province in China indicate a similar, downward secular trend in age at menarche for Chinese women. The present study shows the mean age at menarche decreased by 2.8 years, from 16.5 to 13.7, over an approximate 40-year time interval. This rapid decrease in age at menarche may partly be due to better nutrition and living standards reflected by the improved socioeconomic standards experienced in China over the past few decades. To test this hypothesis, a number of determinants of age at menarche were assessed; year of birth, literacy status, county of residence, amount of physical labour, general health status, pesticide exposure before age at menarche, and drinking water source were all found to be associated with age at menarche.

Adolescent↗
PubMed1999

Oligonucleotide Therapeutics--IBC Sixth International Conference. 3-5 May 1999, La Jolla, CA, USA.

A wide array of strategies was presented for exploiting antisense oligonucleotides (AS ONs). Vitravene (ISIS Pharmaceuticals Inc), a first-generation phosphorothioate (PS) oligodeoxynucelotide (ODN) has been approved for use in the US and European markets for the treatment of CMV retinitis. A number of pharmaceutical companies introduced numerous compounds in both phase I and pivotal phase II clinical trials, for treatment of a wide range of diseases, including cancer, inflammation and viral agents. Advances in AS ON delivery were also described, including topical and oral routes of administration. New chemical modifications incorporated into second-generation oligonucleotides demonstrated superior potency and duration of action in a number of preclinical models. Finally, in response to the explosion in new genomic sequence information generated by the Human Genome Project, a number of companies are combining bioinformatics with high-throughput screening (HTS) to rapidly discover new drug targets. As a result, there was much excitement exhibited by researchers attending this meeting and a strong feeling that this new drug paradigm is delivering on its initial promise.

Journal Article↗
PubMed1998

Comparison of methods for the reduction of acute pain produced by rubber ring castration or tail docking of week-old lambs.

Behavioural and plasma cortisol changes were recorded for groups of eight Suffolk x Greyface lambs subjected to castration or tail docking using rubber rings with and without local anaesthetic treatment. Immediately after application of the rubber ring, local anaesthetic (2 x 0.2 ml 2% lignocaine) was administered either by needle and syringe or by high-pressure needleless injection into each side of the neck of the scrotum or tail at the site of the ring, or by high pressure needleless injection into the testes before ring application. In other groups, the innervation to the scrotum or tail was disabled by crushing with a powered bloodless castrator just proximal to the ring. Measurements were recorded in groups of control (handled) lambs, with and without local anaesthetic treatment. Application of local anaesthetic by high pressure needleless injection had little effect on either plasma cortisol values or behaviour of control lambs. For castration, application of the bloodless castrator and/or local anaesthetic at the ring site reduced the peak plasma cortisol concentration by 50% (P < or = 0.01), the incidence of active behavioural responses by 80 and 64% (P < or = 0.01) respectively, and the time spent in abnormal postures by 68 and 59% (P < or = 0.01) respectively. Both methods were effective in reducing pain for tail docking [active behavioural responses reduced by more than 80% (P < or = 0.01) and abnormal postures by 56% (P < or = 0.01)], although local anaesthetic was more effective, reducing the peak cortisol by 60% (P < or = 0.01) [crushing by 44% (P > 0.05)]. Injection of local anaesthetic into the testes was less effective than injection into the neck of the scrotum at the site of the ring [reduction in abnormal lying postures (P < or = 0.05), 45 vs 71%, respectively]. The rapid action, effectiveness, and ease of application of these experimental methods may provide the basis for commercially viable methods for reducing the acute pain produced by rubber ring castration and tail docking of lambs.

Anesthetics, Local↗
PubMed1998

In vivo distribution and metabolism of a phosphorothioate oligonucleotide within rat liver after intravenous administration.

In the rat, the liver represents a major site of phosphorothioate oligodeoxynucleotide deposition after i.v. administration. For this reason, we examined the intracellular fate of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, isolated from parenchymal and nonparenchymal cell types after systemic dosing using established perfusion and separation techniques followed by CGE. Isolated cells were further fractionated into nuclear, cytosolic and membrane constituents to assess the intracellular localization, distribution and metabolic profiles as a function of time and dose. After a 10-mg/kg i.v. bolus, intracellular drug levels where maximal after 8 hr and diminished significantly thereafter, suggesting an active efflux mechanism or metabolism. Nonparenchymal (i.e., Kupffer and endothelial) cells contained approximately 80% of the total organ cellular dose, and this was equivalently distributed between the two cell types, while the remaining 20% was associated with hepatocytes. Nonparenchymal cells contained abundant nuclear, cytosolic and membrane drug levels over a wide dose range. In contrast, at doses of less than 25 mg/kg, hepatocytes contained significantly less drug with no detectable nuclear-association. Doses at or above 25 mg/kg appeared to saturate nonparenchymal cell types, whereas hepatocytes continued to accumulate drug in all cellular compartments, including the nucleus. Our results suggest that although pharmacokinetic parameters vary as a function of hepatic cell type, significant intracellular delivery can be readily achieved in the liver after systemic administration.

Animals↗
PubMed1997

Effects of four analgesic treatments on the behavioural and cortisol responses of 3-week-old lambs to tail docking.

The behavioural and cortisol responses of groups of seven or eight lambs were used to determine which of three methods of tail docking (rubber ring, Burdizzo and rubber ring combined, or heated docking iron) produced the least signs of pain in the first 3 h after use and which of four analgesic treatments (1.0 ml bupivacaine subcutaneously, 0.5 ml bupivacaine epidurally, a topical cold analgesic spray or diclofenac 1.5 mg kg-1) was most effective in reducing these signs. Amputation with a heated docking iron produced levels of behaviour and cortisol responses which did not differ markedly from those of handled controls. The rubber ring method produced the greatest increase in all parameters (total active behaviour 110 +/- 91 counts; 51 +/- 23 min spent in abnormal postures; peak cortisol 93 +/- 51 nmol l-1). Subcutaneous bupivacaine, administered immediately prior to application of the ring, appeared to be the analgesic treatment most effective at reducing these responses (23 +/- 15 counts; 24 +/- 22 min.; 44 +/- 20 nmol l-1).

Administration, Topical↗
PubMed1997

Reduction in pain suffered by lambs at castration.

The acute pain produced by bloodless castrators was studied by comparing the behavioural and plasma cortisol changes in groups (n=8) of 3-week-old Dorset cross lambs after castration with a 22 cm (9") Burdizzo, a new power assisted castrator and by a combined method using a Burdizzo and elastrator ring. The time spent in abnormal postures (52-58 min) and the peak cortisol response (110-120 mmol l(-1)) were similar for the three methods, although the powered castrator produced a more sustained response. The Burdizzo method halved the incidence of active behaviours compared with the powered castrator and combined methods (16 versus 30, 32 counts). Intratesticular local anaesthetic administered 2 min before the Burdizzo castrator and combined method, or intramuscular injection of the non-steroidal anti-inflammatory drug diclofenac, 20 min before the application of a Burdizzo, significantly reduced the peak plasma cortisol response to 80 nmol l(-1). Diclofenac also significantly reduced the time spent trembling or in abnormal postures.

Anesthesia, Local↗
PubMed1996

Quantitation of phosphorothioate oligonucleotides in human plasma.

Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides.

Electrophoresis, Capillary↗
PubMed1996

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.

Alkylation↗
PubMed1996

Effect of antisense oligonucleotides on cytokine release from human keratinocytes in an in vitro model of skin.

ISIS 1082, a phosphorothioate oligonucleotide 21 nucleotides in length targeted to the translation initiation codon of herpes simplex virus (HSV) type 1 and 2 virion capsid protein, has been shown to inhibit HSV-1 replication in vitro. The effects of ISIS 1082, its phosphodiester congener, ISIS 1049, and analogs consisting of 2' methoxy and 2' propoxy phosphodiesters and phosphorothioates on IL-1 alpha release and viability were evaluated in a three-dimensional in vitro skin model consisting of neonatal keratinocytes and fibroblasts. This in vitro system displays many of the functional and metabolic properties of a differentiated epidermis and can be induced to specifically release IL-1 alpha in response to a mixture of lipopolysaccharide and phorbol myristate acetate. Incubation of the skin model with 250 to 1000 microM concentrations of ISIS 1082 and its 2' methoxy and propoxy phosphorothioate analogs resulted in a concentration-dependent increase of cytokine release with minimal effects on cellular viability, as measured by the Neutral Red assay. This response was confirmed in primary keratinocytes, which were also shown to secrete IL-1 alpha into media supernatants after incubation with phosphorothioate oligomers. These data suggest that the IL-1 alpha released from keratinocytes in response to ISIS 1082 may contribute to the inflammatory and immune cell response seen in vivo.

Antiviral Agents↗