Topically applied novel TRPV1 receptor antagonist, ACD440 Gel, reduces evoked pain in healthy volunteers, a randomized, double-blind, placebo-controlled, crossover study.

BACKGROUND: The TRPV1 receptor is a key molecule in pain generation. Previous development of oral TRPV1-antagonists was halted due to systemic heat insensitivity and body temperature alterations. The present Phase 1b study investigated the efficacy, safety and plasma exposure of a topically administered TRPV1-antagonist (ACD440 Gel) in healthy subjects. METHODS: The study comprised two parts. In part 1, 24 healthy subjects were included in this randomized double-blind, placebo-controlled, crossover trial. ACD440 Gel or Placebo was applied once daily and wiped off after 1 h, for 5 consecutive days. Assessments were done in normal skin, skin optimized for penetration (by stripping and occlusive gel application) and UVB-irradiated skin. Pain induced by thermo-nociceptive CO2 laser impulses generated laser-evoked potentials (LEPs), with readouts of peak-to-peak (PtP) amplitude in vertex-EEG and pain assessments by VAS (0-100). Endpoints include effects at 1 hour post-dose, AUC(Days 1-5) and AUC(0-24, Day 4). In UVB-irradiated skin, also pain on pinprick and skin redness were assessed. Part 2 explored the plasma pharmacokinetics of ACD440. RESULTS: ACD440 Gel reduced LEP PtP amplitude and VAS pain, p < 0.001, in all skin conditions, versus placebo. In UVB-irradiated skin, pinprick pain was also reduced, p = 0.047. Effects were significant after 1 h, maintaining for at least 9 h. There were no adverse events or drug-induced erythema. Plasma exposures of ACD440 were too low to establish an elimination half-life of ACD400. CONCLUSIONS: Topical ACD440 Gel demonstrated a significant analgesic effect on LEP, VAS score and pinprick pain, with low systemic exposures, supporting further clinical development. SIGNIFICANCE: This study demonstrates that the topical administration of a TRPV1-antagonist, ACD440 Gel, has potential as a new treatment for painful conditions affecting the skin, such as chronic peripheral neuropathic pain, without any local or systemic side effects.

Kinetics of melittin induced pore formation in the membrane of lipid vesicles.

We have investigated the permeabilization of POPC unilamellar vesicle bilayers upon the addition of melittin. This process was measured in an early time range of a few minutes by means of monitoring the release of an entrapped marker, the self-quenching fluorescent dye carboxyfluorescein. Pore formation is indicated by an apparent 'all-or-none' efflux out of individual vesicles and a higher than linear dependence on melittin concentration. Applying a recently developed evaluation procedure, the data are readily converted into the gross number of pores per vesicle formed within the elapsed measuring time t. The results can be generally described in terms of a fast initial rate of pore formation that slows down to a much lower value after a period of about 1 to 2 minutes, following a single exponential time course. The three rate parameters involved are shown to be power functions of the concentration of melittin that is actually associated with the vesicle membrane. These findings are in excellent quantitative agreement with a proposed scheme of reaction steps where the formation of lipid associated peptide dimers becomes rate determining once an initial fast deposit is exhausted.

Dilute blood clot lysis time and electrophoretic lipoprotein fractions in a population sample of healthy Romanians.

Serum total lipids and cholesterol, electrophoretically determined lipoprotein concentrations, serum pseudocholinesterase and dilute blood clot lysis time were determined in 630 healthy subjects (287 men and 343 women) aged 20-60, working in the food industry. A high incidence of over-weight was noted ranging from 22.4% in women aged 20-40 to 58.7% in men aged 41-60. Over-weight subjects presenting higher levels of serum cholesterol, total lipids and of the pre-beta electrophoretic fraction also had a higher pseudo-cholinesterase activity and a more delayed clot lysis time than normal-weight subjects matched as to age and sex. When the material was divided into quintiles for pre-beta- and beta-lipoproteins, a highly significant delay of fibrinolysis was noted in the fourth and fifth quintiles for pre-beta-lipoproteins, but no significant changes of lysis time occurred with increasing concentrations of beta-lipoproteins. Possible explanations of the abovementioned findings are briefly discussed.

Serum pseudocholinesterase and ceruloplasmin in various types of hyperlipoproteinemia.

Starting from previous observations emphasizing an increased pseudocholinesterase (PCE) activity in obese and hyperlipemic subjects, the behaviour of this enzyme and of ceruloplasmin was studied in connection with changes of serum lipids and lipoproteins in various types of hyperlipoproteinemia. When compared to values detected in 67 middle-aged normal weight normolipemic subjects, PCE activity was found to be significantly greater (smaller than 0.001) in the 49 overweight subjects without obvious hyperlipemia but presenting a moderate increase of the prebeta electrophoretic fraction. PCE activity was much higher in lean or overweight subjects with endogenous hypertriglyceridemia (68 patients with type IV and 86 patients with mixed hyperlipemia). The slight increase of mean values of PCE activity in the 53 subjects with type II-a was due mainly to overweight subjects, while this enzyme's activity was not significantly changed in lean subjects with pure hypercholesterolemia. PCE activity was positively correlated with serum triglyceride (r equals 0.540; p smaller than 0.001) and the prebeta electrophoretic fraction (r equals 610; p smaller than 0.001). The correlation with beta-lipoproteins was not significant. Ceruloplasmin levels were not significantly changed. It is suggested that elevation of PCE activity could be connected to mechanisms leading to an increased secretion rate of lipoproteins.

A nonclassical radiation process (Comorosan effect) studied by a photographic technique.

Unconventional and highly unexpected results in the Comorosan Effect have been obtained in a photographic study using N-methyl-4-aminophenol (metol) as the developer. Before dissolving the developer was irradiated with green light (lambda = 546 nm) for various distinct periods of time (5, 10 and 15 seconds). A significant difference was observed in the rates of the developing process for different samples of the developer which had been irradiated for 5 seconds compared to non-irradiated controls, but no differences were observed for samples irradiated for 10 or 15 seconds, respectively. This unusual radiation phenomenon is similar to the Comorosan Effect by which rates of enzymatic reactions are manifested and significantly influenced by prior irradiation of the enzyme substrates, but only for times which are certain multiples of 5 seconds.

In vitro toxicity evaluation of Ti(4+)-stabilized γ-Bi2O3 sillenites.

We report results regarding the in vitro toxicology of γ-Bi2O3 represented by its isomorphous phase Bi12TiO20 (γ-BTO). The γ-BTO microparticles were synthesized by two methods: coprecipitation from a bismuth nitrate-tetrabutyl titanate solution and solid state reaction of Bi2O3 and TiO2 oxides. The structural and morphological characteristics of the obtained materials were determined using X-ray diffraction (XRD), selected area electron diffraction (SAED), transmission (TEM) and scanning (SEM) electron microscopy. The elemental composition was investigated using energy dispersive spectrometry (EDS). The cytotoxicity and oxidative/nitrosative stress (intracellular reactive oxygen species (ROS) and nitric oxide (NO) release) induced by the studied microparticles in HepG2, SH-SY5Y and 3T3-L1 cell cultures were determined using the MTT, DCF-DA (2',7'-dichlorfluorescein-diacetate) and Griess methods respectively. Depending on the cell type and γ-BTO concentration, results showed only weak cytotoxic effects after 24h of γ-BTO exposure and cell proliferation effects for longer treatment times. Only reduced NO release increases (corresponding to high γ-BTO concentrations) were detected in case of SH-SY5Y and 3T3-L1 cells. The intracellular ROS production (higher for HepG2 cells) appeared inversely proportional to the γ-BTO concentration. The obtained results indicated a promising in vitro biocompatibility of γ-BTO and encourage further studies regarding its potential for biomedical applications.

The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays.

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO2, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO2 nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO2 nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO2 concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO2 concentrations generally higher than 50µg/ml), induced by the TiO2-MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.

Sci-Fri PM: Delivery - 01: Approach to evaluate the accuracy of individual control-points of a VMAT delivery measured by an EPID.

A unique approach that uses Monte Carlo (MC) methods to validate time-resolved measured dose by an electronic portal imaging device (EPID) during delivery of volumetric modulated arc therapy (VMAT) treatments was investigated. Time-resolved dose is simultaneously scored in both the patient and EPID geometries. A RapidArc© verification plan was generated and delivered on a homogeneous cylindrical phantom. Portal images were collected using an aS1000 amorphous-silicon EPID attached to a Varian Clinac 2100ix. The images were acquired in the continuous acquisition mode and in-house analysis software was used to obtain images for each control point. We performed MC simulation and dose calculation of the verification plan using the EGSnrc MC package. The MC phantom file contains both the EPID model and the cylindrical phantom derived from the CT data sets. For the individual control point simulation, we rotate the phantom only using the gantry angle information. For this work, we calculated dose distributions for five control points (out of a total of 177) in the verification plan. The normalized measured and calculated data were compared using the chi comparison (computationally efficient implementation of gamma). The chi comparison between measured and calculated doses for the five control points using criteria of 3% and 3mm revealed an average of 88.7% of all pixels having χ<1. The agreement will improve when the measured portal images are corrected for flood field and support-arm backscatter effects. This approach allows us to obtain both the EPID dose and the phantom/patient dose. These preliminary results demonstrate promising accuracy. With further improvements, this approach will be useful for benchmarking of time-resolved EPID dose applications intended for rotational IMRT QA and adaptive radiation therapy.

A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV.

A novel effect-positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities-is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others.

Serum electrophoretic lipoprotein factors and pseudocholinesterase activity in thyroid disease.

Decreased levels of serum cholesterol and beta-lipoproteins occurring in hyperthyroidism were found to be accompanied by an enchanced activity of pseudocholinesterase, while in patients with myxedema, the pathologically increased levels of serum cholesterol and beta-lipoproteins were associated with diminished serum pseudocholinesterase activity. The percentage of the pre-beta fraction was found to be increased in both hypo- and hyperthyroid patients, but mechanisms leading to this change are probably different in the two pathological conditions. The behaviour of cholesterol and pseudocholinesterase activity and especially the ratio between these paramameters might be used for the diagnosis of thyroid disease and for the control of therapeutic efficiency.

The relationship between systemic lupus erythematosus and tuberculosis.

Investigation of 280 cases of systemic lupus erythematosus (SLE) admitted during 15 consecutive years to the "N. Gh. Lupu" Institute of Internal Medicine, Bucharest, revealed association of this disease with tuberculosis (tb) in 51 cases (19%). In most of these patients, tb onset preceded that of SLE; in the others it was either concomitant or it occurred later, in the course of SLE evolution. It is assumed that tb is favouring SLE development, probably by antigenic oversollicitation in patients with genetic immune deficiencies. When it occurs in the late stages of SLE, association of tb is of severe prognosis.

Mutation spectrum and phenylalanine hydroxylase RFLP/VNTR background in 44 Romanian phenylketonuric alleles.

The mutation spectrum and polymorphic haplotype background in 22 Romanian families have been analysed in this study using the restriction digestion of phenylalanine hydroxylase (PAH) regions specifically amplified or the DGGE/direct sequencing methods. Eleven PAH mutations specifically associated with six mutant haplotypes were detected. In spite of the relative heterogeneity of the molecular defects in the PAH gene, three mutations covered almost 70% of all alleles: R408W, 47.72%, 21/44; K363fsdelG 13.63%, 6/44; and P225T 6.81%, 3/44. Among these, R408W, the most frequent mutation in our population, represented 50% of all the phenylketonuric (PKU) chromosomes. Splice mutation IVS12nt1g-->a affected two PAH alleles (4.54%); the remaining seven mutations were rare, each having an effect on just one chromosome (1/44), resulting in a relative frequency of 2.27%. A high frequency was observed in our PKU samples for the relatively uncommon mutations, K363fsdelG and P225T mutation, suggesting a possible founder effect at origin. Within the investigated panel, these mutations, both very rare among other Caucasians were exclusively linked to haplotype 5.8 and 1.7, respectively. These results provide a basis for the development of a routine molecular analysis of Romanian PKU families.

Long-term study of the risk factors in atherosclerosis.

A study of the risk factors in atherosclerosis carried out in 595 subjects over a period of 10 years showed an increased prevalence of arterial hypertension, of high serum cholesterol values and of overweight in 1970 as compared to 1960. This rise was significant for all the parameters studied and was particularly evident in serum cholesterol values and overweight, beginning with the 21 to 30 age decade. The frequency of subjects with risk factors increased with age. During the period of study there occurred certain changes within the population such as an increase in the total consumption of calories and in the lipid contents of the food, as well as of the cultural level of the population. The authors show that the increase in the number of subjects with risk factors and the natural history of the various diseases or disturbances depend on the changes of living conditions and of environment.

Serum pseudocholinesterase activity during experimental fattening.

The exact role of serum pseudocholinesterase (PSCE) is not known. Its main role probably consists in the degradation of butyril-choline, an intermediate of lipid metabolism. Decreased values have been described in liver cirrhosis as an index of diminished proteosynthetic function. An increased activity of this enzyme was reported in obesity, diabetes mellitus, nephrosis, hyperthyroidism and in hyperlipemic subjects without obesity. A significant correlation was found between serum cholesterol, triglycerides and PSCE, as an expression of lipid metabolism. In view of assessing the possible change of this enzyme during fattening, the authors investigated PSCE activity during fattening in pigs (the same breed). The results indicate a statistically significant increase of PSCE during fattening, from 30.4 +/- 1.7 to 43.6 +/- 1.7 (p less than 0.001). These results together with those reported in a previous paper concerning blood lipids and cholesterol, show a positive correlation between these parameters and PSCE during fattening in pigs, which might be due to an adaptative increase of the hepatic synthesis of this enzyme in response to the increased lipid metabolism.

Essential arterial hypertension and polymorphism of angiotensinogen M235T gene.

Several candidate genes, chosen from the renin- angiotensin system, were examined for their association with essential hypertension. The genes of the renin- angiotensin system (RAS) are good candidates for such an approach because this system is well known to be involved in the control of blood pressure. One of these candidate genes is the gene encoding for angiotensinogen (the most important gene of the RAS associated with essential hypertension in the most population, is the gene for angiotensin-converting enzyme- ACE). One DNA polymorphism within exon 2- with threonine instead of methionine at position 235 (M235T) was found to be significantly associated with hypertension. The objective of this study is the analysis of M235T polymorphism in angiotensinogen gene in Romanian patients with essential hypertension as well as controls. We examined 38 patients with essential hypertension and 21 normotensive patients. In order to identify the M235T angioteninogen variant, we used the following methods: DNA extraction, PCR amplification and enzymatic digestion of the PCR product using Tth 111I restriction endonuclease enzyme. In the study groups, the M235T variant (Met?Thr in aminoacid position 235) was found more frequently in hypertensive patients (81,57%), than in control subjects (66,66%). We identified 52,63% M235T heterozygotes in the hypertensive group compared with 47,61% in the control group, and 28,94% T235T homozygotes in the hypertensive group compared with 19,04% in the control group. The results of our study suggest an association of the M235T polymorphism in the gene encoding angiotensinogen with essential hypertension.

Increased plasma level of clotting factors VII and X in hyperlipoproteinemia.

When compared to values obtained in healthy normal-weight normolipidemic controls, the combined activities of clotting factors VII and XI, as well as serum cholinesterase were found to be significantly higher in hyperlipidemic and especially in hypertriglyceridemic subjects. The levels of the vitamin K-dependent clotting factors are positively correlated with the concentration of serum cholesterol, the logarithm of serum triacylglycerol level and serum cholinesterase activity. Based on these data as well as on previous observations concerning an increased plasma level of other liver secretion enzymes in endogenous hypertriglyceridemia, a possible interrelationship between accelerated turnover of lipoproteins, enhanced hepatic protein synthesis and thromboatherosclerosis is suggested.

Influence of pregnancy, birth and abortion on the evolution of systemic lupus erythematosus.

The effect of pregnancy, birth and abortion on systemic lupus erythematosus (SLE) was studied in 225 patients with or without a history of pregnancy and abortion. It was observed that pregnancy, birth and abortion aggravate the evolution of disease in patients with active SLE. The tissue autoantibodies studies presented a low titer in patients in whom pregnancy had occurred in the course of disease. Antimeasles antibodies presented a high titer in patients with a history of gestation. These data suggest that gestation induces immune changes which aggravate SLE.

Behaviour of gamma-glutamyltransferase in chronic consumers of alcohol.

Results obtained in three successive investigations involving various groups of population are presented. In a first study gamma-glutamyltransferase (gammaGT) activity was found to exceed the upper limit of the normal in 70% of the 36 subjects from a rural community in Northern Transylvania who were known by the local physician as being addicted to alcohol since at least five years. In a subsequent study gammaGT activity was determined in 86 patients seeking psychiatric help for a drinking problem. Only 55% of these patients were presenting increased gammaGT values and this finding could be explained by the fact that some of the psychiatric patients were admitted to the clinic for pathological bouts of dipsomania and not because of longstanding chronic intake of alcohol. When studied in connexion with the behaviour of serum triglyceride and alanineaminotransferase (GPT) levels, gammaGT activity was found to be particularly high in hypertriglyceridemic alcoholics and in those presenting a moderate increase of serum transaminases. Population studies emphasized that gammaGT activity exceeded the upper normal limit in 49.2% of the men and in 21.6% of the women working for more than five years in the industry of alcoholic beverages, while an increased activity of this enzyme was detected only in 19% of the men and in 1.7% of the women in a random population.