Apolipoprotein E epsilon 4 allele is associated with increased atrophy in progressive mild cognitive impairment: a voxel-based morphometric study.

BACKGROUND: The apolipoprotein E (APOE) epsilon4 allele is a risk factor for Alzheimer's disease. Earlier studies have shown differences in brain structure according to the APOE epsilon4 status. OBJECTIVE: To assess possible differences in brain structure according to the APOE epsilon4 status in mild cognitive impairment (MCI) subjects in relation to conversion to dementia. METHODS: In a follow-up study of 56 MCI subjects, 13 MCI subjects progressed to dementia (PMCI) during a mean follow-up time of 31 months. Brain structure differences in both stable MCI (SMCI) and PMCI epsilon4 carriers and noncarriers in the baseline MRI scan were assessed with voxel-based morphometry. RESULTS: The SMCI epsilon4 carriers had atrophy in the amygdala and hippocampus compared to the SMCI noncarriers. The PMCI epsilon4 carriers revealed atrophy of the left inferior frontal gyrus and parietal cortex compared to the PMCI noncarriers. CONCLUSION: The rate of brain atrophy in certain brain areas may be increased in epsilon4-positive MCI subjects progressing to dementia.

Prediction of drug solubility from molecular structure using a drug-like training set.

Using a training set of 191 drug-like compounds extracted from the AQUASOL database a quantitative structure-property relationship (QSPR) study was conducted employing a set of simple structural and physicochemical properties to predict aqueous solubility. The resultant regression model comprised five parameters (ClogP, molecular weight, indicator variable for aliphatic amine groups, number of rotatable bonds and number of aromatic rings) and demonstrated acceptable statistics (r2 = 0.87, s = 0.51, F = 243.6, n = 191). The model was applied to two test sets consisting of a drug-like set of compounds (r2 = 0.80, s = 0.68, n = 174) and a set of agrochemicals (r2 = 0.88, s = 0.65, n = 200). Using the established general solubility equation (GSE) on the training and drug-like test set gave poorer results than the current study. The agrochemical test set was predicted with equal accuracy using the GSE and the QSPR equation. The results of this study suggest that increasing molecular size, rigidity and lipophilicity decrease solubility whereas increasing conformational flexibility and the presence of a non-conjugated amine group increase the solubility of drug-like compounds. Indeed, the proposed structural parameters make physical sense and provide simple guidelines for modifying solubility during lead optimisation.

Acyl chain and headgroup specificity of human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) is a plasma protein with two reported in vitro activities: transfer of phospholipids and modulation of HDL particle size. The mechanism of PLTP-mediated phospholipid transfer was studied by determining the acyl chain and headgroup specificity and comparing the results with those obtained with the non-specific lipid transfer protein (ns-LTP), a previously characterised intracellular transfer protein. To verify the results obtained with purified plasma PLTP, recombinant PLTP produced in COS-1 cells was used. The transfer rates were determined by monitoring the transfer of fluorescent, pyrene-labeled phospholipids from quenched donor phospholipid vesicles to HDL3 particles. When the length of the pyrene-labeled acyl chain was varied from 6 to 14 carbons, a fairly monotonous decrease in the transfer rate was observed. No difference in rate was observed for the isomers having the pyrene-labeled and unlabeled acyl chains in reversed positions. PLTP mediated equally the transfer of the various headgroup derivatives except phosphatidylethanolamine (PE), which was transferred 2-3-fold more slowly. In all experiments the plasma and recombinant PLTP behaved identically. The specificity patterns observed for PLTP and ns-LTP were very similar. No PLTP-phospholipid intermediate could be observed, indicating that PLTP, like ns-LTP, does not form a tight complex with the lipid substrate and may thus mediate the transfer of phospholipid via another, yet unspecified mechanism.

Oxidative modification of HDL3 in vitro and its effect on PLTP-mediated phospholipid transfer.

The oxidation of HDL3 by Cu(II) and its effect on the ability of these particles to act as phospholipid acceptors in human plasma phospholipid transfer protein (PLTP)-mediated lipid transfer were investigated. Oxidation of HDL3 was monitored by measuring the following parameters: (i) formation of conjugated dienes, (ii) production of thiobarbituric acid reactive substances (TBARS), (iii) decrease in reactive lysine and (iv) tryptophan residues, (v) change in particle charge and (vi) diameter, and (vii) oligomerisation of apoA-I and apoA-II. Formation of conjugated dienes was the parameter responding to the oxidative treatment with the fastest kinetics. The appearance of TBARS and modification of apolipoprotein tryptophan residues were detected simultaneously but required higher Cu(II) concentrations for maximal kinetics. Cross-linking of the major protein constituents of HDL3, apoA-I and apoA-II, represented later steps of the oxidation process. Further, the oxidative modification was accompanied by a progressive change in HDL3 particle charge and a minor increase in particle diameter. PLTP-mediated phospholipid transfer to the oxidized particles was investigated using an assay measuring the transfer of fluorescent, pyrene-labeled PC. The transfer was significantly inhibited, but only after extensive modification of the HDL proteins, suggesting that the HDL oxidative modifications occurring in vivo do not essentially impair its phospholipid acceptor function. A similar but less pronounced inhibition was observed when two other phospholipid transfer proteins, the nonspecific lipid transfer protein (ns-LTP) and the phosphatidylcholine transfer protein (PC-TP), were studied in parallel. This indicates that the inhibition was partly due to unspecific effects of the modification on acceptor particle surface properties, but included an aspect specific for PLTP.

Do more highly organized collagen fibrils increase bone mechanical strength in loss of mineral density after one-year running training?

The aim of this study was to evaluate the effects of long-term running training on the structural properties of bone. Ten beagle dogs ran according to a strenuous progressive program (up to 40 km/day) for 1 year. At the end of the training program, there was a significant reduction in bone mineral density (up to 9.7%) in the vertebrae of the runner dogs as compared with 10 sedentary control dogs. Polarized light microscopy of the vertebral trabecular bone, however, displayed proportionally higher retardation values of the collagen network of the runner dogs than of the sedentary dogs, suggesting a reorganization in a more parallel manner in the collagen fibrils. The concentration and cross-linking of collagen in the bones remained similar in both groups. No differences were observed in the force to failure of bones of the two groups nor in the histomorphometric analysis of the bones. We suggest that the collagen network in the bones accounted for the maintenance of the strength properties in the bones of the runner dogs despite the loss of mineral density.

Relation of sex hormones to bone mineral density in middle-aged men during a 4 year exercise intervention trial.

Recent studies have emphasized the symbiotic role of estradiol and testosterone on bone metabolism. Several anthropomorphic-, lifestyle-, and dual-energy X-ray (DXA)-derived parameters were measured with respect to estradiol (E(2)), testosterone (T), free T (fT), and sex hormone-binding globulin (SHBG) in 140 men (aged 53-62 years) participating in a controlled, randomized exercise intervention trial. After 4 years of intervention, 132 (94.3%) men remained as participants. During the period of study, aerobic threshold increased significantly in the exercise intervention group compared with the reference group (13.4% vs. -1.9%: p < 0.023). Serum E(2) and fT were not convincingly related to bone mineral density (BMD) or BMD change. Aerobic threshold or the change in aerobic threshold were not associated with sex hormone or SHBG levels. Body mass index was a significant determinant of T (beta = -0.337), fT (beta = -0.293), and SHBG (beta = -0.306), and smoking predicted T (beta = 0.231) and fT (beta = 0.245). Alcohol intake was a significant determinant of E(2) (beta = 0.213). Ultimately there was no convincing relation between sex hormone levels and BMD or BMD change in middle-aged men.

Aerobic exercise and bone mineral density in middle-aged finnish men: a controlled randomized trial with reference to androgen receptor, aromatase, and estrogen receptor alpha gene polymorphisms.

Our aim was to investigate associations of the polymorphic loci of androgen receptor (AR), aromatase CYP19, and estrogen receptor alpha (ERalpha) genes with bone mineral density (BMD) in a four-year controlled randomized exercise intervention trial in Finnish middle-aged men. Additionally, we studied whether the gene polymorphisms affect circulating testosterone (T), estradiol (E(2)), and sex hormone-binding globulin concentrations. The polymorphic CAG repeat of the AR gene, the TTTA repeat of the human aromatase gene, and the PvuII site of the ERalpha gene were analyzed. BMDs of the lumbar spine (L2-L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). In the exercise group, the subjects with the ERalpha gene PP or Pp genotypes showed an increase (+6.5 and +5.1%, respectively) in lumbar spine BMDs (P = 0.007; repeated measures ANOVA) during intervention, while there was no change in the subjects with the pp genotype. The long TTTA repeat (TTTA(9-12)) in aromatase gene was associated with greater height (P = 0.026) and lower BMI (P = 0.029) values than the short TTTA repeat (TTTA(6-8)). With regard to the AR gene, no statistically significant differences in bone properties were found between the genotypes. There were no significant associations of any analyzed polymorphic sites with the serum sex steroid hormone concentrations in the exercise or reference group. In conclusion, the Finnish middle-aged men with ERalpha PP or Pp genotypes appear to have increased BMD values in the lumbar spine. This increase may reflect a predisposition to age-related degenerative changes in the spine. In addition, the AR CAG repeat and aromatase TTTA repeat do not modify the effect of regular aerobic exercise on BMD.

The association of bone metabolism with bone mineral density, serum sex hormone concentrations, and regular exercise in middle-aged men.

Physical activity is an important factor in attaining bone mass. Our aim was to investigate if low to moderate intensity exercise affects bone resorption [serum tartrate-resistant acid phosphatase (TRAP) 5b activity] and formation (serum osteocalcin concentration) in a randomized controlled exercise intervention trial in Finnish middle-aged men. In addition, the relations of these bone turnover markers with bone mineral density (BMD) and serum sex hormone concentrations [circulating testosterone (T), estradiol (E2), and sex hormone-binding globulin (SHBG) concentrations] were evaluated. Serum TRAP 5b activity and osteocalcin concentration were measured at randomization and after 1 and 4 years of the exercise intervention. BMDs of the lumbar spine (L2-L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). At randomization, TRAP 5b activity was strongly correlated with the osteocalcin concentration (Spearman r = 0.541, P < 0.0001). In addition, TRAP 5b activity was significantly correlated with proximal femur BMD values (r = -0.201, P = 0.018) and osteocalcin concentration with femoral neck and proximal femur BMD values (r = -0.187, P = 0.028; r = -0.240, P = 0.005, respectively). Serum E2, free E2, and free T concentrations were inversely correlated with both bone turnover markers. After 1 year of exercise intervention, TRAP 5b activity was significantly lower in the exercise than reference group (P = 0.006). However, after 4 years of exercise intervention, the difference was no longer statistically significant. There were no differences in the osteocalcin concentrations between the study groups during the intervention. Our results show a connection between serum TRAP 5b activity and osteocalcin concentration. Furthermore, our results suggest that low to moderate exercise intervention and serum sex hormone concentrations may induce changes in bone metabolism in middle-aged men. However, exercise-induced effects on bone metabolism should be confirmed in other randomized controlled exercise trials taking into account exercise intensity and dose-response issues.

The impact of phospholipid transfer protein (PLTP) on HDL metabolism.

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, alpha-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.

Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.

A sensitive sandwich-type enzyme-linked immunosorbent assay (ELISA) for human plasma phospholipid transfer protein (PLTP) has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the accurate quantification of PLTP in the range of 25-250 ng PLTP/assay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample (n = 159) was determined to be 15.6 +/- 5.1 mg/l, the values ranging from 2.30 to 33.4 mg/l. PLTP mass correlated positively with HDL-cholesterol (r = 0.36, P < 0.001), apoA-I (r = 0.37, P < 0.001), apoA-II (r = 0.20, P < 0.05), Lp(A-I) (r=0.26, P=0.001) and Lp(A-I/A-II) particles (r=0.34, P<0.001), and negatively with body mass index (BMI) (r = -0.28, P < 0.001) and serum triacylglycerol (TG) concentration (r = -0.34, P < 0.001). PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer activity divided by PLTP mass, correlated positively with plasma TG concentration (r=0.568, P<0.001), BMI (r=0.45, P<0.001), apoB (r = 0.45, P < 0.001). total cholesterol (r=0.42, P < 0.001), LDL-cholesterol (r = 0.34, P < 0.001) and age (r = 0.36, P < 0.001), and negatively with HDL-cholesterol (r= -0.33, P < 0.001), Lp(A-I) (r= -0.21, P < 0.01) as well as Lp(A-I/A-II) particles (r = -0.32, P < 0.001). When both PLTP mass and phospholipid transfer activity were adjusted for plasma TG concentration, a significant positive correlation was revealed (partial correlation, r = 0.31, P < 0.001). The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates positively with parameters reflecting plasma high density lipoprotein (HDL) levels, but the protein appears to be most active in subjects displaying high TG concentration.

Estimation of aqueous solubility in drug design.

The solubility of drugs in water is of central importance in the process of drug discovery and development from molecular design to pharmaceutical formulation and biopharmacy. The ability to estimate the aqueous solubility and other properties of a promising lead compound affecting its pharmacokinetics is a prerequisite to rational drug design, although it has received much less attention than the prediction of drug-receptor interactions. In this review, methods for the estimation of aqueous solubility of organic compounds are described and limited to approaches, which might be used in the early stage of drug design and development.

Solid-phase bromination and Suzuki coupling of 2-carboxyindoles.

As part of an ongoing lead discovery project we have developed a convenient method for the modification and substitution of indole moieties at the 3-position. Selective bromination of three different 2-carboxyindoles was followed by Suzuki cross-coupling with aryl and heteroaryl boronic acids on a Merrifield resin solid-phase. After column chromatography, yields of the 3- substituted indoles ranged from 42-98%.

Synthesis and in vitro/in vivo evaluation of novel oral N-alkyl- and N,N-dialkyl-carbamate esters of entacapone.

Entacapone has a relatively low oral bioavailability which may, in part, be due to its low aqueous solubility at low pH and/or its hydrophilic character at neutral pH. Various novel N-alkyl and N,N-dialkyl carbamate esters of entacapone were synthesized as possible prodrugs of entacapone in order to increase its aqueous solubility at an acidic pH and to increase its lipophilicity at neutral pH. Oral bioavailability of entacapone and selected carbamate esters were investigated in rats. Both N-alkyl and N,N-dialkyl carbamate esters were relatively stable against chemical hydrolysis at pH 7.4 (t1/2 = 14.9-20.7 h), but hydrolyzed rapidly (t1/2 = 0.8-2.7 h) in human serum. However, in contrast to N-alkyl carbamates, N,N-dialkyl carbamates did not release entacapone in in vitro enzymatic hydrolysis (human serum) studies. N-Alkyl carbamates, 2a-c, showed increased aqueous solubility at pH 7.4, of which 2a and 2c also show increased aqueous solubility at pH 5.0, compared to entacapone. In addition to increased aqueous solubility, 2c showed increased lipophilicity at pH 7.4. However, two N-alkyl carbamates of entacapone did not increase the oral bioavailability of the parent drug in rats. Thus, it can be concluded that the relatively low lipophilicity of entacapone is not the cause of its low bioavailability.

Neural network modeling for estimation of the aqueous solubility of structurally related drugs.

The ability of neural network models to predict aqueous solubility within series of structurally related drugs was evaluated. Three sets of compounds representing different drug classes (28 steroids, 31 barbituric acid derivatives, and 24 heterocyclic reverse transcriptase inhibitors) were studied. Topological descriptors (connectivity indices, kappa indices, and electrotopological state indices) were used to link the structures of compounds with their aqueous solubility. Separate models were built for each class of drugs using back-propagation neural networks with one hidden layer and five topological indices as input parameters. The effect of network size and training time on the prediction ability of the network was studied by the leave-one-out (LOO) procedure. In all three compound groups a neural network structure of 5-3-1 was optimal. To avoid chance effects, artificial neural network (ANN) ensembles (i.e.; averaging neural network predictions over several independent networks) were used. The cross-validated squared correlation coefficient (Q2) for 10 averaged predictions was 0.796 in the case of the steroid set. The corresponding standard error of prediction (SDEP) was 0.288 log units. For the barbiturates, Q2 and SDEP were 0.856 and 0.383, respectively, and for the RT inhibitors, these parameters were 0.721 and 0.401, respectively. The results indicate that neural networks can produce useful models of the aqueous solubility of a congeneric set of compounds, even with simple structural parameters.

Prediction of partition coefficient based on atom-type electrotopological state indices.

The aim of this study was to determine the efficacy of atom-type electrotopological state indices for estimation of the octanol-water partition coefficient (log P) values in a set of 345 drug compounds or related complex chemical structures. Multilinear regression analysis and artificial neural networks were used to construct models based on molecular weights and atom-type electrotopological state indices. Both multilinear regression and artificial neural networks provide reliable log P estimations. For the same set of parameters, application of neural networks provided better prediction ability for training and test sets. The present study indicates that atom-type electrotopological state indices offer valuable parameters for fast evaluation of octanol-water partition coefficients that can be applied to screen large databases of chemical compounds, such as combinatorial libraries.

Prediction of aqueous solubility for a diverse set of organic compounds based on atom-type electrotopological state indices.

We describe robust methods for estimating the aqueous solubility of a set of 734 organic compounds from different structural classes based on multiple linear regression (MLR) and artificial neural networks (ANN) model. The structures were represented by atom-type electrotopological state (E-state) indices. The squared correlation coefficient and standard deviation for the MLR with 34 structural parameters were r(2) = 0.94 and s = 0.58 for the training set of 675 compounds. For the test set of 21 compounds, the equivalent statistics were r(2)(pred) = 0.80 and s = 0.87, respectively. Neural networks gave a significant improvement using the same set of parameters, and the standard deviations were s = 0.52 for the training set and s = 0.75 for the test set when an artificial neural network with five neurons in the hidden layer was used. The results clearly show that accurate models can be rapidly calculated for the estimation of aqueous solubility for a large and diverse set of organic compounds using easily calculated structural parameters.

Synthesis and in-vitro/in-vivo evaluation of orally administered entacapone prodrugs.

Entacapone is a new inhibitor of catechol-O-methyltransferase (COMT) that is used as an adjunct to L-dopa therapy in the treatment of Parkinson's disease. The bioavailability of orally administered entacapone is, however, relatively low (29-46%). In this study we have prepared more lipophilic acyl and acyloxyacyl esters, an acyloxy alkyl ether and an alkyloxycarbonyl ester of entacapone, and we have evaluated them as potential prodrugs to enhance the oral bioavailability of entacapone. All the derivatives fulfilled prodrug criteria and released entacapone in human serum in-vitro. The oral bioavailability of monopivaloyl (1a) and dipivaloyl (1b) esters of entacapone were investigated further in rats. The lipophilicity of 1b was high (log Papp 4.0 at pH 7.4) but its oral bioavailability was low (F = 0.6%), most probably due to its low aqueous solubility. The monopivaloyl ester of entacapone (1a) had a higher lipophilicity (log Papp 0.80) than entacapone (log Papp 0.18) at pH 7.4 while maintaining an aqueous solubility equal to entacapone. However, oral bioavailability was not increased when compared with the parent drug entacapone (F = 7.0% and 10.4%, respectively).

Prediction of biodegradation from the atom-type electrotopological state indices.

A group contribution method based on atom-type electrotopological state indices for predicting the biodegradation of a diverse set of 241 organic chemicals is presented. Multiple linear regression and artificial neural networks were used to build the models using a training set of 172 compounds, for which the approximate time for ultimate biodegradation was estimated from the results of a survey of an expert panel. Derived models were validated by using a leave-25%-out method and against two test sets of 12 and 57 chemicals not included in the training set. The squared correlation coefficient (r2) for a linear model with 15 structural parameters was 0.76 for the training set and 0.68 for the test set of 12 molecules. The model predicted correctly the biodegradation of 48 chemicals in the test set of 57 molecules, for which biodegradability was presented as rapid or slow. The use of artificial neural networks gave better prediction for both test sets when the same set of parameters was tested as inputs in neural network simulations. The predictions of rapidly biodegradable chemicals were more accurate than the predictions of slowly biodegradable chemicals for both the regression and neural network models.

Estimation of water solubility from atom-type electrotopological state indices.

Based on the atom-type electrotopological state (E-state) indices, a quantitative structure-property relationship model for the prediction of aqueous solubility for a diverse set of 745 organic compounds is presented. The multiple linear regression analysis was used to build the models. A training set of 674 compounds, containing 349 liquids and 325 solids and having a range of aqueous solubility (log S) values from 2.77 to 11.62, was obtained from the literature. For this set, the squared correlation coefficient and standard deviation for a linear model with 31 atom-type E-state indices and three simple correction factors were r2 = 0.94 and s = 0.58 (log units), respectively. The corresponding statistics for the test sets not included in the training set were, for a set of 50 pesticides, r2 = 0.79 and s = 0.81 and, for a set of 21 drug and pesticide compounds, r2 = 0.83 and s = 0.84, respectively. The contribution of melting points was also evaluated. The use of melting point increased the accuracy of the models in the fit of the training set but not in the prediction of the test sets. Hence, the proposed method offers fast and accurate estimation of aqueous solubility of organic compounds using atom-type E-state indices without the need of any experimental parameters like the melting points.

2(S)-Amino-3-[1H-imidazol-4(5)-yl]propyl cyclohexylmethyl ether dihydrochloride and 2(S)-amino-3-[1H-imidazol-4(5)-yl]propyl 4-bromobenzyl ether dihydrochloride.

(Cyclohexylmethyloxymethyl)(1H-imidazol-4-iomethyl)-(S)-ammonium dichloride, C(13)H(25)N(3)O(+).2Cl(-), and (4-bromobenzyl)(1H-imidazol-4-iomethyl)-(S)-ammonium dichloride, C(13)H(18)BrN(3)O(+).2Cl(-), are model compounds with different biological activities for evaluation of the histamine H3-receptor activation mechanism. Both title compounds occur in almost similar extended conformations.