Overcoming regulatory and economic challenges facing pharmacogenomics

The number of personalized medicines and companion diagnostics in use in the United States has gradually increased over the past decade, from a handful of medicines and tests in 2001 to several dozen in 2011. However, the numbers have not reached the potential hoped for when the human genome project was completed in 2001. Significant clinical, regulatory, and economic barriers exist and persist. From a regulatory perspective, therapeutics and companion diagnostics are ideally developed simultaneously, with the clinical significance of the diagnostic established using data from the clinical development program of the corresponding therapeutic. Nevertheless, this is not (yet) happening. Most personalized medicines are personalized post hoc, that is, a companion diagnostic is developed separately and approved after the therapeutic. This is due in part to a separate and more complex regulatory process for diagnostics coupled with a lack of clear regulatory guidance. More importantly, payers have placed restrictions on reimbursement of personalized medicines and their companion diagnostics, given the lack of evidence on the clinical utility of many tests. To achieve increased clinical adoption of diagnostics and targeted therapies through more favorable reimbursement and incorporation in clinical practice guidelines, regulators will need to provide unambiguous guidance and manufacturers will need to bring more and better clinical evidence to the market place.     

Statins accelerate the onset of collagen type II-induced arthritis in mice

Introduction

Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.

Methods

The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.

Results

Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFNγ production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.

Conclusion

These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.     

Naturally-occurring genetic variants in human DC-SIGN increase HIV-1 capture, cell-transfer and risk of mother-to-child transmission

Background

Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Dendritic cell–specific ICAM-3 grabbing-nonintegrin (DC-SIGN, also known as CD209) is an HIV-1 receptor that enhances its transmission to T cells and is expressed on placental macrophages.

Methods and Findings

We have investigated the association between DC-SIGN genetic variants and risk of MTCT of HIV-1 among Zimbabwean infants and characterized the impact of the associated mutations on DC-SIGN expression and interaction with HIV-1. DC-SIGN promoter (p-336C and p-201A) and exon 4 (198Q and 242V) variants were all significantly associated with increased risk of intrauterine (IU) HIV-1 infection. Promoter variants decreased DC-SIGN expression both in vitro and in placental CD163⁺ macrophages (Hofbauer cells) of HIV-1 unexposed infants but not of HIV-1 exposed infants. The exon 4 protein-modifying mutations increased HIV-1 capture and transmission to T cells in vitro.

Conclusion

This study provides compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection.     

Identification and analysis of the active phytochemicals from the anti-cancer botanical extract Bezielle

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it.     

Lessons learned developing a diagnostic tool for HIV-associated dementia feasible to implement in resource-limited settings: pilot testing in Kenya

Objective

To conduct a preliminary evaluation of the utility and reliability of a diagnostic tool for HIV-associated dementia (HAD) for use by primary health care workers (HCW) which would be feasible to implement in resource-limited settings.

Background

In resource-limited settings, HAD is an indication for anti-retroviral therapy regardless of CD4 T-cell count. Anti-retroviral therapy, the treatment for HAD, is now increasingly available in resource-limited settings. Nonetheless, HAD remains under-diagnosed likely because of limited clinical expertise and availability of diagnostic tests. Thus, a simple diagnostic tool which is practical to implement in resource-limited settings is an urgent need.

Methods

A convenience sample of 30 HIV-infected outpatients was enrolled in Western Kenya. We assessed the sensitivity and specificity of a diagnostic tool for HAD as administered by a primary HCW. This was compared to an expert clinical assessment which included examination by a physician, neuropsychological testing, and in selected cases, brain imaging. Agreement between HCW and an expert examiner on certain tool components was measured using Kappa statistic.

Results

The sample was 57% male, mean age was 38.6 years, mean CD4 T-cell count was 323 cells/µL, and 54% had less than a secondary school education. Six (20%) of the subjects were diagnosed with HAD by expert clinical assessment. The diagnostic tool was 63% sensitive and 67% specific for HAD. Agreement between HCW and expert examiners was poor for many individual items of the diagnostic tool (K = .03–.65). This diagnostic tool had moderate sensitivity and specificity for HAD. However, reliability was poor, suggesting that substantial training and formal evaluations of training adequacy will be critical to enable HCW to reliably administer a brief diagnostic tool for HAD.     

Extended access to cocaine self-administration results in reduced glutamate function within the medial prefrontal cortex

Previous studies have shown that brief access to cocaine yields an increase in D2 receptor binding in the medial prefrontal cortex (mPFC), but that extended access to cocaine results in normalized binding of D2 receptors (i.e. the D2 binding returned to control levels). Extended-access conditions have also been shown to produce increased expression of the NR2 subunit of the N-Methyl-D-aspartate receptor in the mPFC. These results implicate disrupted glutamate and dopamine function within this area. Therefore, in the present study, we monitored glutamate and dopamine content within the mPFC during, or 24 hours after, cocaine self-administration in animals that experienced various amounts of exposure to the drug. Na?ve subjects showed decreased glutamate and increased dopamine levels within the mPFC during cocaine self-administration. Exposure to seven 1-hour daily cocaine self-administration sessions did not alter the response to self-administered cocaine, but resulted in decreased basal dopamine levels. While exposure to 17 1-hour sessions also resulted in reduced basal dopamine levels, these animals showed increased dopaminergic, but completely diminished glutamatergic, response to self-administered cocaine. Finally, exposure to 17 cocaine self-administration sessions, the last 10 of which being 6-hour sessions, resulted in diminished glutamatergic response to self-administered cocaine and reduced basal glutamate levels within the mPFC while normalizing (i.e. causing a return to control levels) both the dopaminergic response to self-administered cocaine as well as basal dopamine levels within this area. These data demonstrate directly that the transition to escalated cocaine use involves progressive changes in dopamine and glutamate function within the mPFC.     

Quality control compartments coming of age

Maintenance of proteome integrity (proteostasis) is essential for cellular and organismal survival. Various cellular mechanisms work to preserve proteostasis by ensuring correct protein maturation and efficient degradation of misfolded and damaged proteins. Despite this cellular effort, under certain circumstances subsets of aggregation-prone proteins escape the quality control surveillance, accumulate within the cell and form insoluble aggregates that can lead to the development of disorders including late-onset neurodegenerative diseases. Cells respond to the appearance of insoluble aggregates by actively transporting them to designated deposition sites where they often undergo degradation. Although several protein aggregate deposition sites have been described and extensively studied, key questions regarding their biological roles and how they are affected by aging remained unanswered. Here we review the recent advances in the field, describe the different subtypes of these cellular compartments and outline the evidence that these structures change their properties over time. Finally, we propose models to explain the possible mechanistic links between aggregate deposition sites, neurodegenerative disorders and the aging process.