Efficacy and tolerability on melasma of a topical cosmetic product acting on melanocytes, fibroblasts and endothelial cells: a randomized comparative trial against 4% hydroquinone.

BACKGROUND: Recent data demonstrated that an altered basal membrane, activated melanocytes and secreted factors from keratinocytes but also fibroblasts and endothelial cells are involved in the pathophysiology of melasma. OBJECTIVES: To evaluate the efficacy and tolerability on melasma of a new topical skin-lightening cosmetic product combination (CCP) targeting several factors identified to be involved in melasma pathogenesis compared to 4% hydroquinone (HQ). METHODS: Forty-three women with melasma were enrolled in a 12-week double-blind, randomized, parallel-group trial and treated with CCP or 4% HQ cream. Efficacy was evaluated with the modified Melasma Area Severity Index (mMASI) score and colorimetric change. Cutaneous tolerability and patient satisfaction were also investigated. RESULTS: The mMASI score decreased for both products from baseline and over the study period. At week 12, 90% of the subjects who received the combination products had an improvement in pigmentation vs. 79% with HQ. Similarly, both products significantly increased Individual Typological Angle parameters. For both measures, no statistically significant difference was observed between CCP and HQ in terms of change from baseline. CPP was very well tolerated. CONCLUSIONS: Cosmetic product combination is as effective as HQ in the management of facial dyspigmentation and represents a safe alternative.

The functional similarity and structural diversity of human and cartilaginous fish hemoglobins.

Although many descriptions of adaptive molecular evolution of vertebrate hemoglobins (Hb) can be found in physiological text books, they are based mainly on changes of the primary structure and place more emphasis on conservation than alterations at the functional site. Sequence analysis alone, however, does not reveal much about the evolution of new functions in proteins. It was found recently that there are many functionally important structural differences between human and a ray (Dasyatis akajei) Hb even where sequence is conserved between the two. We have solved the structures of the deoxy and CO forms of a second cartilaginous fish (a shark, Mustelus griseus) Hb, and compared it with structures of human Hb, two bony fish Hbs and the ray Hb in order to understand more about how vertebrate Hbs have functionally evolved by the selection of random amino acid substitutions. The sequence identity of cartilaginous fish Hb and human Hb is a little less than 40 %, with many functionally important amino acid replacements. Wider substitutions than usually considered as neutral have been accepted in the course of molecular evolution of Hb. As with the ray Hb, the shark Hb shows functionally important structural differences from human Hb that involve amino acid substitutions and shifts of preserved amino acid residues induced by substitutions in other parts of the molecule. Most importantly, beta E11Val in deoxy human Hb, which overlaps the ligand binding site and is considered to play a key role in controlling the oxygen affinity, moves away about 1 A in both the shark and ray Hbs. Thus adaptive molecular evolution is feasible as a result of both functionally significant mutations and deviations of preserved amino acid residues induced by other amino acid substitutions.

Sero-prevalence study of IgE responses to allergens from Malaysian house dust (HDM) and storage mites (SM).

Allergens of Dermatophagoides and Blomia species are well-characterized but not for other species. This study was conducted to determine the prevalence of allergic sensitization to house dust (HDM) and storage mites (SM). One hundred adult subjects (aged ≥ 18) were recruited. The mite specific IgE of all allergic subjects were higher compared with healthy subjetcs despite being not statistically significant except for D. farinae and G. malaysiensis. The mean serum IgE levels against HDM and SM for allergic subjects were significantly higher compared with those in healthy subjects. They were mainly sensitized to Dermatophagoides farinae (35%) and Glycycometus malaysiensis (37%). Immunoblots revealed not all allergic subjects showed positive immuno-reactivity against the mites tested. Single or multiple bands were observed for different species. The subjects were commonly sensitized to Group 2 (9-12 kDa), 10 (38 kDa) and 18 (40-48 kDa) allergens. Twenty-one out of 60 allergic subjects were sensitized to either one or more species. The majority of them (71%) were sensitized to single species. The allergic subjects were mainly sensitized to D. pteronyssinus, followed by Tyrophagus putrecentiae and Aleuroglyphus ovatus. Seven were solely sensitized to HDM while 10 were solely sensitized to SM. Four subjects were sensitized to both. Pre-adsorption study revealed no cross-reactivity. There was difference between the prevalence and reactivity to allergens of HDM and SM in these subjects. Both ELISA and immunoblot did not correlate well but can complement each other in improving the detection of mite allergens to the species level.

Structure-based design of novel HIV protease inhibitors: carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent nonpeptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover nonpeptidic HIV protease inhibitors had previously identified compound II (phenprocoumon, K(i) = 1 muM) as a lead template. Crystal structures of HIV protease complexes containing the peptide-derived inhibitor I (1-(naphthoxyacetyl)-L-histidyl-5(S)-amino-6-cyclohexyl-3 (R),4(R)-dihydroxy-2(R)-isopropylhexanoyl-L-isoleucine N-(2-pyridylmethyl)amide) and nonpeptidic inhibitors, such as phenprocoumon (compound II), provided a rational basis for the structure-based design of more active analogues. This investigation reports on the important finding of a carboxamide functionally appropriately added to the 4-hydroxycoumarin and the 4-hydroxy-2-pyrone templates which resulted in a new promising series of nonpeptidic HIV protease inhibitors with improved enzyme-binding affinity. The most active diastereomer of the carboxamide-containing compound XXIV inhibited HIV-1 protease with a K(i) value of 0.0014 muM. This research provides a new design direction for the discovery of more potent HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Structure-based design of nonpeptidic HIV protease inhibitors from a cyclooctylpyranone lead structure.

Recently, the novel cyclooctylpyranone HIV protease inhibitor 1 was identified in our labs, and an X-ray structure of this inhibitor complexed with HIV-2 protease was obtained. This crystal structure was used to develop two strategies for creating derivatives of 1 with enhanced enzyme inhibitory activity. The first strategy, substitution on the cyclooctyl ring, met with limited success, but provided some interesting information about the conformationally-flexible cycloocytyl ring on the inhibitors. The second strategy, substitution at the meta position of the aryl ring, was far more successful and generated compounds, such as the carboxamide derivatives 41 (Ki = 3.0 +/- 0.4 nM) and 36 (Ki = 4.0 +/- 0.8 nM), which were significantly more active than the corresponding unsubstituted cycloocytlpyranone 2 (Ki = 11.7 +/- 4.7 nM). An X-ray crystal structure of 36 complexed with HIV-1 protease indicated the increase in binding affinity is most likely due to the additional interactions between the amide substituent and the S3 region of the protease.

Evaluation of a vitamin-cloaking strategy for oligopeptide therapeutics: biotinylated HIV-1 protease inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha psi[CH-(OH)CH(OH)]Val- core inhibitory insert, three particularly potent inhibitors (K(i) < or = 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(1-methylethyl)-5-[[3-methyl-1-oxo-2-[[5- (hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1- oxopentyl]amino]butyl]amino]-N-[2-methyl-1-[[(2- pyridinylmethyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta (1R*,2R*,3R*),6 alpha]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide,6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-ox o-1H- thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinyl- methyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta(1R*,2R*),6a alpha]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Cycloalkylpyranones and cycloalkyldihydropyrones as HIV protease inhibitors: exploring the impact of ring size on structure-activity relationships.

Previously, 3-substituted cycloalkylpyranones, such as 2d, have proven to be effective inhibitors of HIV protease. In an initial series of 3-(1-phenylpropyl) derivatives with various cycloalkyl ring sizes, the cyclooctyl analog was the most potent. We became interested in exploring the influence of other structural changes, such as substitution on the phenyl ring and saturation of the 5,6-double bond, on the cycloalkyl ring size structure-activity relationship (SAR). Saturation of the 5,6-double bond in the pyrone ring significantly impacts the SAR, altering the optimal ring size from eight to six. Substitution of a sulfonamide at the meta position of the phenyl ring dramatically increases the potency of these inhibitors, but it does not change the optimal ring size in either the cycloalkylpyranone or the cycloalkyldihydropyrone series. This work has led to the identification of compounds with superb binding affinity for the HIV protease (Ki values in the 10-50 pM range). In addition, the cycloalkyldihydropyrones showed excellent antiviral activity in cell culture, with ED50 values as low as 1 microM.

Structure-based design of novel HIV protease inhibitors: sulfonamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent non-peptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover non-peptidic HIV protease inhibitors previously identified compound I (phenprocoumon, Ki = 1 microM) as a lead template. Structure-based design of potent non-peptidic inhibitors, utilizing crystal structures of HIV protease/inhibitor complexes, provided a rational basis for the previously reported carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones. The amino acid containing compound V (Ki = 4 nM) provided an example of a promising new series of HIV protease inhibitors with significantly improved enzymatic binding affinity. In this report, further structure-activity relationship studies, in which the carboxamide is replaced by a sulfonamide functionality, led to the identification of another series of nonamino acid containing promising inhibitors with significantly enhanced enzyme binding affinity and in vitro antiviral activity. The most active diastereomer of the sulfonamide-containing pyrone XVIII (Ki = 0.5 nM) shows improved antiviral activity (IC50 = 0.6 nM) and represents an example of a new design direction for the discovery of more potent non-peptidic HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-hydroxy-2-pyrones as effective, nonpeptidic inhibitors.

From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2-pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S1' and S2' subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (Ki = 15 nM) and showed antiviral activity (IC50 = 5 microM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.

Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class.

A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.

Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyramones.

Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.

Inhibition of human immunodeficiency virus type 1 infection in vitro by combination of delavirdine, zidovudine and didanosine.

Delavirdine (DLV), a non-nucleoside reverse transcriptase inhibitor (RTI) of human immunodeficiency virus type 1 (HIV-1), was evaluated in two and three-drug combinations against acute and co-culture infections of HIV-1JRCSF in human peripheral blood mononuclear cells. DLV combined with didanosine (DDI) at 1:10 and 1:30 ratios were statistically synergistic (combination indices (CI) < 1) at > 75% inhibition levels. However, at 1:100 ratio, the interaction appeared to be additive. Three-drug combinations of zidovudine (ZDV), DLV, and DDI (at a ratio of 1:2:333) were synergistic at 50-99% inhibition levels. The three-drug group also showed significantly (P < 0.01) lower p24 levels in acute cultures than two-drug combination groups (DLV + ZDV, DLV + DDI, ZDV + DDI). In co-culture studies, the extent of viral inhibition was dependent on drug dose and the duration of treatment. Combination of DLV, ZDV, and DDI at IC95 concentration of the individual drugs showed complete inhibition of viral growth in co-culture after 19 days, but not after 7 or 12 days of treatment. The combinations studied did not show additive or synergistic drug toxicity. These data provide an in vitro basis for beneficial use of DLV in combinations with DDI and ZDV in HIV-1 infected patients.

Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing.

Traditional ways of identifying slow growing bacteria is slow and often difficult. In this study, a small, Gram-negative, facultative anaerobic, slow growing bacillus was isolated from the blood culture of a 7-year old traffic accident victim. The bacterium was non-hemolytic, catalase and oxidase positive. An attempt to use the Vitek system (GNI+) and the API system (20NE) to identify the strain was unsuccessful as the growth controls showed negative results. 16S ribosomal RNA gene sequencing showed that there was 1 base difference between the isolate and Arcobacter cryaerophilus (GenBank Accession no. U25805), 1 base difference between the isolate and A. cryaerophilus (GenBank Accession no. U34387), 10 base differences between the isolate and A. cryaerophilus (GenBank Accession no. L14624), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34386), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34387), and 38 base differences between the isolate and A. butzleri (GenBank Accession no. L14626), indicating that the isolate most closely resembled a strain of A. cryaerophilus. Identification of the isolate in our case by conventional methods was difficult, as the absence of a curved morphology has made it confused with other Gram-negative non-fermentative bacteria, and the slow growth rate has made it unidentifiable by both the Vitek and API systems. Although the exact source of infection and route of transmission in our case remains elusive, we speculate that the bacteria were transmitted through the respiratory tract while the boy was suffocated in the mud. The present report represents an example of showing the usefulness of 16S rRNA gene sequencing for identification of slow growing bacteria.

Application of the recurrent multilayer perceptron in modeling complex process dynamics.

A nonlinear dynamic model is developed for a process system, namely a heat exchanger, using the recurrent multilayer perceptron network as the underlying model structure. The perceptron is a dynamic neural network, which appears effective in the input-output modeling of complex process systems. Dynamic gradient descent learning is used to train the recurrent multilayer perceptron, resulting in an order of magnitude improvement in convergence speed over a static learning algorithm used to train the same network. In developing the empirical process model the effects of actuator, process, and sensor noise on the training and testing sets are investigated. Learning and prediction both appear very effective, despite the presence of training and testing set noise, respectively. The recurrent multilayer perceptron appears to learn the deterministic part of a stochastic training set, and it predicts approximately a moving average response of various testing sets. Extensive model validation studies with signals that are encountered in the operation of the process system modeled, that is steps and ramps, indicate that the empirical model can substantially generalize operational transients, including accurate prediction of instabilities not in the training set. However, the accuracy of the model beyond these operational transients has not been investigated. Furthermore, online learning is necessary during some transients and for tracking slowly varying process dynamics. Neural networks based empirical models in some cases appear to provide a serious alternative to first principles models.

Cystic hygroma in adulthood.

The majority of cystic hygromas present in childhood, usually below the age of two years. A case report of adult presentation is presented, with discussion of development, presentation, pathology and management of cystic hygroma.

Permanent cardiac pacing in Malaysia. An update.

Updated data on permanent cardiac pacing in Malaysia is presented. Over the past 3 1/2 years (1976-1980), 75 patients underwent insertion of pacemakers giving an annual incidence of about 20 cases as compared with a total of 21 cases in the previous 8 years (1968-1977). Many of the features reported in an earlier paper in 1977 viz mode of presentation, age and sex distribution and indications for pacing remain unchanged. Over this period only 4 patients required lead replacement. Since concentrating mainly on the use of epicardial leads implanted via a subxiphoid approach, complications have been remarkably low. The problem of availability of pacemakers has been averted. Cost remains a major consideration when recommending one pacemaker in preference over another. The details concerning clinical features, indications for pacing, complications and other problems encountered in the management of these patients are discussed.