Pharmacophore‐based discovery of inhibitors of a novel drug/proton antiporter in human brain endothelial hCMEC/D3 cell line

Background and Purpose

An influx drug/proton antiporter of unknown structure has been functionally demonstrated at the blood–brain barrier. This transporter, which handles some psychoactive drugs like diphenhydramine, clonidine, oxycodone, nicotine and cocaine, could represent a new pharmacological target in drug addiction therapy. However, at present there are no known drugs/inhibitors that effectively inhibit/modulate this transporter in vivo.

Experimental Approach

The FLAPpharm approach was used to establish a pharmacophore model for inhibitors of this transporter. The inhibitory potency of 44 selected compounds was determined against the specific substrate, [³H]‐clonidine, in the human cerebral endothelial cell line hCMEC/D3 and ranked as good, medium, weak or non‐inhibitor.

Key Results

The pharmacophore model obtained was used as a template to screen xenobiotic and endogenous compounds from databases [Specs, Recon2, Human Metabolome Database (HMDB), human intestinal transporter database], and hypothetical candidates were tested in vitro to determine their inhibitory capacity with [³H]‐clonidine. According to the transporter database, 80% of the proton antiporter inhibitor candidates could inhibit P‐glycoprotein/MDR1/ABCB1 and specificity is improved by reducing inhibitor size/shape and increasing water solubility. Virtual screening results using HMDB and Recon2 for endogenous compounds appropriately scored tryptamine as an inhibitor.

Conclusions and Implications

The pharmacophore model for the proton‐antiporter inhibitors was a good predictor of known inhibitors and allowed us to identify new good inhibitors. This model marks a new step towards the discovery of this drug/proton antiporter and will be of great use for the discovery and design of potent inhibitors that could potentially help to assess and validate its pharmacological role in drug addiction in vivo.

Abbreviations

ADE

absorption, distribution and elimination

BBB

blood–brain barrier

DPBS

Dulbecco''s PBS

DPH

diphenhydramine

G‐I

good inhibitor

Glob‐Prod flap

global product similarity score

Glob‐Sum flap

global sum similarity score

KH

Krebs–HEPES buffer

M‐I

medium inhibitor

N‐I

non‐inhibitor

PCA

principal component analysis

P‐gp

P‐glycoprotein

PK

pharmacokinetics

W‐I

weak inhibitor

     

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.     

Daytime sleepiness and EEG spectral analysis in apneic patients before and after treatment with continuous positive airway pressure

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is characterized by recurrent apneas during sleep, resulting in repetitive hypoxemic episodes and interruptions of the normal sleep pattern. A previous study showed EEG slowing (ie, a higher ratio of delta + theta frequencies to alpha + beta frequencies on EEG) during rapid eye movement (REM) sleep and wakefulness in untreated OSAS patients. STUDY AND OBJECTIVES: To determine whether EEG slowing is reversible with continuous positive air pressure (CPAP) treatment and to verify whether the persistence of excessive daytime sleepiness (EDS) is correlated with residual slowing of the EEG. PATIENTS: Ten healthy subjects (9 men and 1 woman) and 14 patients with moderate-to-severe OSAS (13 men and 1 woman) were studied before and after 6 months of treatment with CPAP. RESULTS: Untreated OSAS patients showed EEG slowing in frontal and central cortical regions during both wakefulness and during REM sleep compared to healthy control subjects. This EEG slowing was found to be independent of time spent with arterial oxygen saturation < 90%, severity of OSAS, or mean sleep latency as determined by the multiple sleep latency test. CPAP treatment was found to correct the EEG slowing for both REM sleep and wakefulness. Daytime sleepiness also greatly improved with treatment, but some degree of somnolence remained. CONCLUSION: CPAP treatment was found to correct the EEG slowing that was observed in untreated OSAS patients. Persistent EDS may be related to persistent obesity after CPAP treatment.     

Epidemiological and economic burden of pneumococcal diseases in Canadian children

BACKGROUND:

With the arrival of a new conjugate pneumococcal vaccine, it is important to estimate the burden of pneumococcal diseases in Canadian children. The epidemiological data and the economic cost of these diseases are crucial elements in evaluating the relevance of a vaccination program.

METHODS:

Using provincial databases, ad hoc surveys and published data, age-specific incidence rates of pneumococcal infections were estimated in a cohort of 340,000 children between six months and nine years of age. The costs of these diseases to the health system and to families were also evaluated using data from Quebec and Manitoba.

RESULTS:

Cumulative risks were one in 5000 for pneumococcal meningitis, one in 500 for bacteremia and one in 20 for pneumonia, leading to 16 deaths in the cohort. About 262,000 otitis media episodes and 32,000 cases of myringotomy with ventilation tube insertion were attributable to Streptococcus pneumoniae. Societal costs were estimated at $125 million, of which 32% was borne by the health system and 68% was borne by families. Invasive infections represented only 2% of total costs, while 84% were generated by otitis media.

CONCLUSION:

Pneumococcal infections represent a significant burden for Canadian children and society that could be significantly reduced through immunization. Key Words: Canada, Economics, Epidemiology, Pneumococcal infectionsStreptococcus pneumoniae is an important cause of serious illness in young children in developed countries (1). A 7-valent pneumococcal conjugate vaccine (PCV-7) has recently been licensed in Canada. This vaccine is now being used in publicly funded programs for all children in Alberta and Nunavut. Some Canadian provinces (Quebec, Prince Edward Island and Saskatchewan) have implemented programs limited to some high-risk groups and others are still considering its use in their provincial programs (2,3). Evaluation of the epidemiological and economic burden of pneumococcal disease is an important criterion in decision making and is a prerequisite for the analysis of the cost-effectiveness of routine and catch-up immunization programs. Epidemiological data collected in the United States may not be valid for Canada because of possible differences in the prevalence of risk factors. Extrapolation is even more problematic for economic parameters because of variations in the composition of families, availability of natural caregivers, employment rates, incomes and costs of health services. The aim of this study is to estimate the incidence and societal costs of pneumococcal disease in Canadian children between six months and nine years of age.