Genetic basis of inosine triphosphate pyrophosphohydrolase (ITPA) deficiency

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138GMA, 561GMA, 708GMA) and two associated with ITPase deficiency (94CMA, IVS2+21AMC). Homozygotes for the 94CMA missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94CMA heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21AMC homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94CMA (allele frequency: 0.06), 24 were heterozygotes for IVS2+21AMC (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21AMC heterozygotes and 94CMA/IVS2+21AMC compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

The prospects for tobacco harm reduction

Tobacco harm reduction (THR) policies aim to reduce the prevalence of tobacco-related harm by encouraging smokers who are unable or unwilling to quit to adopt less harmful ways of obtaining nicotine, such as pharmaceutical nicotine and oral tobacco snuff. Proponents of THR argue that the effects of tobacco control policies have reduced smoking as much as they reasonably can and that we can best reduce tobacco-related harm by encouraging smokers to use these methods, which substantially reduce the health risks of smoking. Critics argue that THR policies will undermine the two traditional tobacco control goals of preventing the uptake of smoking by young people and encouraging smokers to quit. I assess the main arguments and evidence advanced for and against THR.

Polytomous item response theory models

Polytomous Item Response Theory Models provides a unified, comprehensive introduction to the range of polytomous models available within item response theory (IRT). It begins by outlining the primary structural distinction between the two major types of polytomous IRT models. This focuses on the two types of response probability that are unique to polytomous models and their associated response functions, which are modeled differently by the different types of IRT model. It describes, both conceptually and mathematically, the major specific polytomous models, including the Nominal Response Model, the Partial Credit Model, the Rating Scale model, and the Graded Response Model. Important variations, such as the Generalized Partial Credit Model are also described as are less common variations, such as the Rating Scale version of the Graded Response Model. Relationships among the models are also investigated and the operation of measurement information is described for each major model. Practical examples of major models using real data are provided, as is a chapter on choosing an appropriate model. Figures are used throughout to illustrate important elements as they are described.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

The effect of protein binding on the hepatic first pass of O-acyl salicylate derivatives in the rat

In this work the in-situ perfused rat liver has been used to examine the effect of changing the protein content of the perfusate on the hepatic extraction of O-acyl esters of salicylic acid. The hepatic availability (F) of these solutes was studied at a flow-rate of 30 mt min(-1) with perfusate albumin concentrations of 0, 2, and 4% w/v. The hepatic availability of the esters was shown to decrease with increasing carbon-chain length in the O-acyl group; for all the esters the hepatic availability increased with increasing albumin concentration in the perfusate. The dispersion-model-derived efficiency number (R-N) Of the esters was shown to increase with increasing lipophilicity and decrease with increasing albumin concentration in the perfusate. The unbound fraction (f(u),) of the esters decreased with lipophilicity. R-N/f(u), for acetylsalicylic acid remained relatively constant as the albumin concentration was increased. However, R-N/f(u), for n-pentanoyl- and n-hexanoylsalicylic acids increased significantly as albumin concentration increased from 0% to 4%. Thus, for the more lipophilic solutes (n-pentanoyl- and n-hexanoylsalicylic acids) the presence of albumin apparently facilitates the uptake of unbound solute relative to acetylsalicylic acid.

Topical penetration of commercial salicylate esters and salts using human isolated skin and clinical microdialysis studies

Aims The penetration of active ingredients from topically applied anti-inflammatory pharmaceutical products into tissues below the skin is the basis of their therapeutic efficacy. There is still controversy as to whether these agents are capable of direct penetration by diffusion through the tissues or whether redistribution in the systemic circulation is responsible for their tissue deposition below the application site. Methods The extent of direct penetration of salicylate from commercial ester and salt formulations into the dermal and subcutaneous tissue of human volunteers was determined using the technique of cutaneous microdialysis. We also examined differences in the extent of hydrolysis of the methylester of salicylate applied topically in human volunteers and in vitro skin diffusion cells using full-thickness skin and epidermal membranes. Results The present study showed that whilst significant levels of salicylate could be detected in the dermis and subcutaneous tissue of volunteers treated with the methylsalicylate formulation, negligible levels of salicylate were seen following application of the triethanolamine salicylate formulation. The tissue levels of salicylate from the methylsalicylate formulation were approx. 30-fold higher than the plasma concentrations. Conclusion The absorption and tissue concentration profiles for the commercial methylsalicylate formulation are indicative of direct tissue penetration and not solely redistribution by the systemic blood supply.

Effect of metabolic inhibitors on cyclosporine pharmacokinetics using a population approach

Drugs known to inhibit the metabolism of cyclosporine are administered concomitantly to those who undergo cardiothoracic transplantation. The aim of this study was to examine in quantitative terms the relationship between cyclosporine oral dose rate and the trough concentration (Css(trough)) at steady state in patients who undergo cardiothoracic transplantation and are administered cyclosporine alone or in combination with drugs known to inhibit its metabolism. Dose and whole blood cyclosporine Css(tough) observations measured using the enzyme-multiplied immunoassay technique (EMIT) (396 observations) or the TDx assay (435 observations) were collected as part of routine blood concentration monitoring from 182 patients who underwent cardiothoracic transplantation. Data were analyzed using a linear mixed-effects modeling approach to examine the effect of metabolic inhibitors on dose-rate-Css(trough) ratio. The mean (and 95% confidence interval) dose-rate-Css(trough) ratio for cyclosporine generated from concentrations measured using EMIT was 94 (82.5-105.5) Lh(-1) for patients administered cyclosporine alone, 66.7 (58.1-75.3) Lh(-1) for patients administered concomitant diltiazem, 47.9 (15.4 -80.4) Lh(-1) for patients administered concomitant itraconazole, 21.7 (14.8-28.5) Lh(-1) for patients administered concomitant ketoconazole, and 14.9 (11.8-18.1) Lh(-1) for patients concomitantly administered diltiazem and ketoconazole. For patients administered concomitant cyclosporine, ketoconazole, and diltiazem, the dosage of cyclosporine, if it is administered alone, should be 20% to achieve the same blood concentrations. This will allow safer drug concentration targeting of cyclosporine after cardiothoracic transplantation.

Dendritic cells in rheumatoid arthritis - A model autoimmune inflammatory site

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to CD4+ T cells. The strong association of the disease with an epitope within the HLA-DR chain shared between various alleles of HLA-DR4 and DR1 emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and CD40-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.

Population pharmacokinetics and pharmacodynamics: An underutilized resource

A meeting was convened in Canberra, Australia, at the request of the Australian Drug Evaluation Committee (ADEC), on December 3-4, 1997 to discuss the role of population pharmacokinetics and pharmacodynamics in drug evaluation and development. The ADEC was particularly concerned about registration of drugs in the pediatric age group. The population approach could be used more often than is currently the case in pharmacokinetic and pharmacodynamic studies to provide valuable information for the safe and effective use of drugs in neonates, infants, and children. The meeting ultimately broadened to include discussion about other subgroups. The main conclusions of the meeting were: 1. The population approach, pharmacokinetic and pharmacodynamic analysis, is a valuable tool both for drug registration purposes and for optimal dosing of drugs in specific groups of patients, 2. Population pharmacokinetic and pharmacodynamic studies are able to fill in the gaps' in registration of drugs, for example, to provide information on optimal pediatric dosing. Such studies provide a basis for enhancing product information to improve rational prescribing, 3. Expertise is required to perform the population studies and expertise, with a clinical perspective, is also required to evaluate such studies if they are to be submitted as part of a drug registration dossier Such expertise is available in the Australasian region and is increasing. Centers of excellence with the appropriate expertise to advise and assist should be encouraged to develop and grow in the region, 4. The use of the population approach by the pharmaceutical industry needs to be encouraged to provide valuable information not obtainable by other techniques. The acceptance of population pharmacokinetic and pharmacodynamic analyses by regulatory agencies also needs to be encouraged, and 5. Development of the population approach to pharmacokinetics and pharmacodynamics is needed from a public health perspective to ensure that all available information is collected and used to improve the way drugs are used. This important endeavor needs funding and support at the local and international levels.

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.