Recent Developments in Mass Spectrometry to Support Next-Generation Synthesis and Screening.

The complexity of new therapeutics continues to increase and the timeline for the discovery of these therapeutics continues to shrink. This creates demand for new analytical techniques to facilitate quicker discovery and development of novel drugs. Mass spectrometry is one of the most prolific analytical techniques that has been applied across the entire drug discovery pipeline. New mass spectrometers and the associated methods for sampling have been introduced at a rate that keeps pace with new chemistries, therapeutic types, and screening practices used by modern drug hunters. This microperspective covers application and implementation of new mass spectrometry workflows that enable current and future efforts in screening and synthesis for drug discovery.

Herpes Zoster and Its Prevention by Vaccination.

Herpes zoster (HZ; shingles) results from reactivation of varicella-zoster virus (VZV) after primary infection as varicella (chicken pox). It affects mainly older adults and people with immunocompromising diseases or treatments. The most common complication is postherpetic neuralgia (PHN), which has significant adverse effects on quality of life and activities of daily living. Since PHN cannot be prevented once HZ has occurred, and treatment is only modestly successful and is associated with significant side effects, the recent introduction of an effective vaccine is an important achievement. This new vaccine, which combines a single VZV glycoprotein (gE) and a multicomponent adjuvant, is superior to the previously available live attenuated VZV vaccine. The recombinant adjuvanted vaccine is remarkably effective in restoring the protective T cell-mediated immunity required to prevent HZ. Its clinical efficacy is much greater than that observed with other vaccines for older individuals affected by immune senescence, and its safety profile is very acceptable. It has been recommended in the USA and Canada for people who are 50 years of age and older. The immunogenicity and safety of this vaccine in severely immunocompromised individuals, such as after chemotherapy for malignancy, after solid organ or stem cell transplant, and in people with HIV are being studied.

Medical conditions at enrollment do not impact efficacy and safety of the adjuvanted recombinant zoster vaccine: a pooled post-hoc analysis of two parallel randomized trials.

In two pivotal efficacy studies (ZOE-50; ZOE-70), the adjuvanted recombinant zoster vaccine (RZV) demonstrated >90% efficacy against herpes zoster (HZ).Adults aged ≥50 or ≥70 years (ZOE-50 [NCT01165177]; ZOE-70 [NCT01165229]) were randomized to receive 2 doses of RZV or placebo 2 months apart. Vaccine efficacy and safety were evaluated post-hoc in the pooled (ZOE-50/70) population according to the number and type of selected medical conditions present at enrollment.At enrollment, 82.3% of RZV and 82.7% of placebo recipients reported ≥1 of the 15 selected medical conditions. Efficacy against HZ ranged from 84.5% (95% Confidence Interval [CI]: 46.4-97.1) in participants with respiratory disorders to 97.0% (95%CI: 82.3-99.9) in those with coronary heart disease. Moreover, efficacy remained >90% irrespective of the number of selected medical conditions reported by a participant.As indicated by the similarity of the point estimates, this post-hoc analysis suggests that RZV efficacy remains high in all selected medical conditions, as well as with increasing number of medical conditions. No safety concern was identified by the type or number of medical conditions present at enrollment.

Tissue Imaging by Mass Spectrometry: A Practical Guide for the Medicinal Chemist.

Understanding the tissue distribution of therapeutic molecules is often critical for assessing their efficacy and toxicity. Unfortunately, standard methods for monitoring localized drug distribution are resource-intensive and are typically performed late in the discovery process. As a result, early development efforts often progress without detailed information on the effect that changes in structure and/or formulation have on drug localization. Recent innovations in mass spectrometry (MS) provide new options for mapping the spatial distribution of drug in tissue and allow parallel detection of endogenous species. These advances are improving access to drug distribution data early in discovery and provide insight into local biochemical changes that are directly related to drug activity. The literature on these topics is voluminous, and the technology is advancing rapidly, offering a bewildering array of options for researchers who are new to the field. To guide medicinal chemists who wish to apply these methods in their research, this technology perspective provides our views on practical applications that are currently enabled by various MS imaging (MSI) approaches, along with recommendations for how best to implement these methods in pharmaceutical R&D.

Quality of Life Impact of an Adjuvanted Recombinant Zoster Vaccine in Adults Aged 50 Years and Older.

BACKGROUND: To determine the efficacy of an adjuvanted recombinant zoster vaccine in reducing the herpes zoster (HZ) burden of illness, HZ burden of interference with activities of daily living, and HZ impact on quality of life. METHODS: The assessments were integrated in two Phase III trials, ZOE-50 (NCT01165177) and ZOE-70 (NCT01165229). HZ burden of illness and HZ burden of interference with activities of daily living were assessed by the Zoster Brief Pain Inventory (ZBPI) instrument and quality of life by the EuroQol-5 Dimension (EQ-5D) utility index and the SF-36 health survey. We report the ZOE-50 results and a pooled analysis of patients aged 70 years and older from the trials combined. RESULTS: The estimated vaccine efficacy in reducing HZ burden of illness and HZ burden of interference was greater than 90% in both the ZOE-50 and the pooled ZOE-70 analysis. In confirmed HZ cases, adjuvanted recombinant zoster vaccine reduced the maximal ZBPI worst-pain score in the pooled ZOE-70 analysis (p = .032) and the maximal ZBPI average-pain scores in both the ZOE-50 (p = .049) and the pooled ZOE-70 analysis (p = .043). In breakthrough HZ cases, trends for diminished loss of quality of life compared with placebo-recipient HZ cases were observed, with differences up to 0.14 on the EQ-5D index at time points during the 4 weeks following HZ onset. CONCLUSIONS: Adjuvanted recombinant zoster vaccine reduced the HZ burden of illness significantly, particularly due to its very high vaccine efficacy in preventing HZ. For breakthrough HZ cases, the results suggest that the adjuvanted recombinant zoster vaccine mitigated severity of HZ-related pain, burden of interference with activities of daily living, and recipients' utility loss.

Complications of herpes zoster in immunocompetent older adults: Incidence in vaccine and placebo groups in two large phase 3 trials.

BACKGROUND: An adjuvanted herpes zoster (HZ) subunit vaccine, HZ/su, demonstrated high efficacy against HZ and postherpetic neuralgia (PHN) in two randomized, observer-blind, placebo-controlled trials in adults aged ≥50 and ≥70 years (ZOE-50 and ZOE-70, respectively). METHODS: Data from ZOE-50 and ZOE-70 trials were analyzed to evaluate the efficacy of HZ/su against mortality, hospitalizations, and non-PHN complications of HZ including HZ-associated vasculitis, stroke, and disseminated, ophthalmic, neurologic, and visceral diseases. RESULTS: In the pooled ZOE-50/ZOE-70 analysis, 1 of 32 HZ/su recipients (3.1%) and 16 of 477 placebo recipients (3.4%) with a confirmed HZ episode had complications other than PHN. Efficacy against HZ-related complications was 93.7% (95% confidence interval, 59.5-99.9%) in adults aged ≥50 years and 91.6% (43.3-99.8%) in adults ≥70 years. Five HZ-related hospitalizations, all in placebo recipients, and no HZ-related deaths were reported. CONCLUSIONS: HZ/su reduces the risk of HZ-associated complications in older adults (NCT01165177; NCT01165229).

Herpes zoster epidemiology, management, and disease and economic burden in Europe: a multidisciplinary perspective.

Herpes zoster (HZ) is primarily a disease of nerve tissue but the acute and longer-term manifestations require multidisciplinary knowledge and involvement in their management. Complications may be dermatological (e.g. secondary bacterial infection), neurological (e.g. long-term pain, segmental paresis, stroke), ophthalmological (e.g. keratitis, iridocyclitis, secondary glaucoma) or visceral (e.g. pneumonia, hepatitis). The age-related increased incidence of HZ and its complications is thought to be a result of the decline in cell-mediated immunity (immunosenescence), higher incidence of comorbidities with age and social-environmental changes. Individuals who are immunocompromised as a result of disease or therapy are also at increased risk, independent of age. HZ and its complications (particularly postherpetic neuralgia) create a significant burden for the patient, carers, healthcare systems and employers. Prevention and treatment of HZ complications remain a therapeutic challenge despite recent advances. This is an overview of the multidisciplinary implications and management of HZ in which the potential contribution of vaccination to reducing the incidence HZ and its complications are also discussed.

Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues.

For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily adaptable to large-scale clinical and pharmaceutical quantification of biomarkers or biotherapeutics.

Effects of calibration approaches on the accuracy for LC-MS targeted quantification of therapeutic protein.

LC-MS provides a promising alternative to ligand-binding assays for quantification of therapeutic proteins and biomarkers. As LC-MS methodology is based on the analysis of proteolytic peptides, calibration approaches utilizing various calibrators and internal standards (I.S.) have been developed. A comprehensive assessment of the accuracy and reliability of these approaches is essential but has yet been reported. Here we performed a well-controlled and systematic comparative study using quantification of monoclonal-antibody in plasma as the model system. Method development utilized a high-throughput orthogonal-array-optimization, and two sensitive and stable signature-peptides (SP) from different domains were selected based on extensive evaluations in plasma matrix. With the purities of all protein/peptide standards corrected by quantitative amino acid analysis (AAA), five calibration approaches using stable-isotope-labeled (SIL) I.S. were thoroughly compared, including those at peptide, extended-peptide, and protein levels and two "hybrid" approaches (i.e., protein calibrator with SIL-peptide or SIL-extended-peptide I.S.). These approaches were further evaluated in parallel for a 15 time point, preclinical pharmacokinetic study. All methods showed good precision (CV% < 20%). When examined with protein-spiked plasma QC, peptide-level calibration exhibited severe negative biases (-23 to -62%), highly discordant results between the two SP (deviations of 38-56%), and misleading pharmacokinetics assessments. Extended-peptide calibration showed significant improvements but still with unacceptable accuracy. Conversely, protein-level and the two hybrid calibrations achieved good quantitative accuracy (error < 10%), concordant results by two SP (deviations < 15%), and correct pharmacokinetic parameters. Hybrid approaches were found to provide a cost-effective means for accurate quantification without the costly SIL-protein. Other key findings include (i) using two SP provides a versatile gauge for method reliability; (ii) evaluation of peptide stability in the matrix before SP selection is critical; and (iii) using AAA to verify purities of protein/peptide calibrators ensures accurate quantitation. These results address fundamental calibration issues that have not been adequately investigated in published studies and will provide valuable guidelines for the "fit for purpose" development of accurate LC-MS assays for therapeutic proteins and biomarkers in biological matrices.

Similar herpes zoster incidence across Europe: results from a systematic literature review.

BACKGROUND: Herpes zoster (HZ) is caused by reactivation of the varicella-zoster virus (VZV) and mainly affects individuals aged ≥50 years. The forthcoming European launch of a vaccine against HZ (Zostavax®) prompts the need for a better understanding of the epidemiology of HZ in Europe. Therefore the aim of this systematic review was to summarize the available data on HZ incidence in Europe and to describe age-specific incidence. METHODS: The Medline database of the National Library of Medicine was used to conduct a comprehensive literature search of population-based studies of HZ incidence published between 1960 and 2010 carried out in the 27 member countries of the European Union, Iceland, Norway and Switzerland. The identified articles were reviewed and scored according to a reading grid including various quality criteria, and HZ incidence data were extracted and presented by country. RESULTS: The search identified 21 studies, and revealed a similar annual HZ incidence throughout Europe, varying by country from 2.0 to 4.6/1 000 person-years with no clearly observed geographic trend. Despite the fact that age groups differed from one study to another, age-specific HZ incidence rates seemed to hold steady during the review period, at around 1/1 000 children <10 years, around 2/1 000 adults aged <40 years, and around 1-4/1 000 adults aged 40-50 years. They then increased rapidly after age 50 years to around 7-8/1 000, up to 10/1 000 after 80 years of age. Our review confirms that in Europe HZ incidence increases with age, and quite drastically after 50 years of age. In all of the 21 studies included in the present review, incidence rates were higher among women than men, and this difference increased with age. This review also highlights the need to identify standardized surveillance methods to improve the comparability of data within European Union Member States and to monitor the impact of VZV immunization on the epidemiology of HZ. CONCLUSIONS: Available data in Europe have shortcomings which make an accurate assessment of HZ incidence and change over time impossible. However, data are indicative that HZ incidence is comparable, and increases with age in the same proportion across Europe.

Vaccination against herpes zoster in developed countries: state of the evidence.

Although progress has been made in the treatment of herpes zoster (HZ) and postherpetic neuralgia (PHN), available therapeutic options are only partially effective. Given evidence that a live-attenuated varicella-zoster-virus vaccine is effective at reducing the incidence of HZ, PHN and the burden of illness, policymakers and clinicians are being asked to make recommendations regarding the use of the zoster vaccine. In this report, we summarize the evidence regarding the: (1) burden of illness; (2) vaccine efficacy and safety; and (3) cost-effectiveness of vaccination, to assist evidence-based policy making and guide clinicians in their recommendations. First, there is general agreement that the overall burden of illness associated with HZ and PHN is substantial. Second, the safety and efficacy of the zoster vaccine at reducing the burden of illness due to HZ and the incidence of PHN have been clearly demonstrated in large placebo-controlled trials. However, uncertainty remains about the vaccine's duration of protection. Third, vaccination against HZ is likely to be cost-effective when the vaccine is given at approximately 65 y of age, if vaccine duration is longer than 10 y.

Analysis of chloroquine and metabolites directly from whole-body animal tissue sections by liquid extraction surface analysis (LESA) and tandem mass spectrometry.

The rapid and direct analysis of the amount and spatial distribution of exogenous chloroquine (CHQ) and CHQ metabolites from tissue sections by liquid extraction surface sampling analysis coupled with tandem mass spectrometry (LESA-MS/MS) was demonstrated. LESA-MS/MS results compared well with previously published CHQ quantification data collected by organ excision, extraction and fluorescent detection. The ability to directly sample and analyze spatially resolved exogenous molecules from tissue sections with minimal sample preparation and analytical method development has the potential to facilitate the assessment of target tissue penetration of pharmaceutical compounds, to establish pharmacokinetic/pharmacodynamic relationships, and to complement established pharmacokinetic methods used in the drug discovery process during tissue distribution assessment.

Implementation of proteomic biomarkers: making it work.

While large numbers of proteomic biomarkers have been described, they are generally not implemented in medical practice. We have investigated the reasons for this shortcoming, focusing on hurdles downstream of biomarker verification, and describe major obstacles and possible solutions to ease valid biomarker implementation. Some of the problems lie in suboptimal biomarker discovery and validation, especially lack of validated platforms with well-described performance characteristics to support biomarker qualification. These issues have been acknowledged and are being addressed, raising the hope that valid biomarkers may start accumulating in the foreseeable future. However, successful biomarker discovery and qualification alone does not suffice for successful implementation. Additional challenges include, among others, limited access to appropriate specimens and insufficient funding, the need to validate new biomarker utility in interventional trials, and large communication gaps between the parties involved in implementation. To address this problem, we propose an implementation roadmap. The implementation effort needs to involve a wide variety of stakeholders (clinicians, statisticians, health economists, and representatives of patient groups, health insurance, pharmaceutical companies, biobanks, and regulatory agencies). Knowledgeable panels with adequate representation of all these stakeholders may facilitate biomarker evaluation and guide implementation for the specific context of use. This approach may avoid unwarranted delays or failure to implement potentially useful biomarkers, and may expedite meaningful contributions of the biomarker community to healthcare.

Prevention of herpes zoster pain.

SUMMARY Herpes zoster (HZ; shingles) is frequently accompanied by significant pain that may precede rash appearance and persist as postherpetic neuralgia (PHN). PHN results in detriment to quality of life and has healthcare cost implications. Treatment for PHN often provides poor relief, with up to half of patients achieving less than 50% reduction of pain and significant treatment-related side effects. Vaccination against varicella reduces the number of individuals in the population carrying latent wild virus who can develop HZ. Enhancing zoster-specific cell-mediated immunity of older adults by HZ vaccination has been shown to reduce the incidence of both HZ and PHN. Treatments applied during the acute phase of HZ that may reduce pain duration are discussed, including antiviral drugs, anti-inflammatory steroids, effective analgesia and nerve blocks.

Characterization of cis-regulatory elements controlling repo transcription in Drosophila melanogaster.

The glial cells missing (gcm) gene has been identified as a "master regulator" of glial cell fate in the fruit fly Drosophila. However, gcm is also expressed in and required for the development of larval macrophages and tendon cells. Thus, the Gcm protein activates the transcription of different sets of genes in different developmental contexts. How the Gcm protein regulates these different outcomes is not known. Our goal is to identify proteins that collaborate with Gcm to promote the transcriptional activation of Gcm target genes specifically in glial cells, or prevent their activation in the other tissues in which Gcm is expressed. To address this, we have focused on the transcriptional regulation of a well-characterized glial-specific Gcm target gene, the transcription factor reversed polarity (repo). We aim to understand how the transcription of the glial-specific Gcm target gene repo is regulated by Gcm and other factors. Previously we defined a 4.3 kb cis-regulatory DNA region that recapitulates the endogenous Repo expression pattern dependent on multiple Gcm binding sites. We proposed that there may be multiple cis-regulatory sub-regions that drive cell-specific expression independent of Gcm binding sites. Here, using lacZ reporter activity in transgenic lines, we have characterized three cis-regulatory elements: 1) a distal element that promotes expression in dorsolateral epidermis; 2) a repressor element that suppresses expression in the epidermis; and, 3) a proximal element that promotes expression in a subset of cell body glia. Most significantly, we have defined a minimal cis-regulatory element that recapitulates the endogenous repo expression pattern dependent on a single Gcm binding site.

A quantitation method for mass spectrometry imaging.

A new quantitation method for mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) has been developed. In this method, drug concentrations were determined by tissue homogenization of five 10 µm tissue sections adjacent to those analyzed by MSI. Drug levels in tissue extracts were measured by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The integrated MSI response was correlated to the LC/MS/MS drug concentrations to determine the amount of drug detected per MSI ion count. The study reported here evaluates olanzapine in liver tissue. Tissue samples containing a range of concentrations were created from liver harvested from rats administered a single dose of olanzapine at 0, 1, 4, 8, 16, 30, or 100 mg/kg. The liver samples were then analyzed by MALDI-MSI and LC/MS/MS. The MALDI-MSI and LC/MS/MS correlation was determined for tissue concentrations of ~300 to 60,000 ng/g and yielded a linear relationship over two orders of magnitude (R(2) = 0.9792). From this correlation, a conversion factor of 6.3 ± 0.23 fg/ion count was used to quantitate MSI responses at the pixel level (100 µm). The details of the method, its importance in pharmaceutical analysis, and the considerations necessary when implementing it are presented.

A prospective study of the herpes zoster severity of illness.

OBJECTIVE: MASTER, a multicenter prospective study, was conducted to provide a thorough understanding of the burden of herpes zoster (HZ) and postherpetic neuralgia (PHN). Objectives are to: (1) describe the herpes zoster severity-of-illness (HZSOI), a composite measure of pain duration and severity; and (2) to identify the characteristics at recruitment predictive of greater HZSOI at the different phases of HZ. METHODS: From October, 2005 to July, 2006, 261 outpatients with HZ, aged more than equal to 50 years, were recruited within 14 days of rash onset across Canada. The pain was measured by the Zoster Brief Pain Inventory at recruitment and 7, 14, 21, 30, 60, 90, 120, 150, and 180 days later. The HZSOI represents the area under the curve of pain severity over time and ranges from 0 (no pain) to 1800 (pain=10 for 180 d). RESULTS: Median pain duration was 32.5 days. The predictors of greater HZSOI varied according to the different phases of HZ. Higher pain severity at recruitment, more lesions, lower income, and being immunocompromised were the predictors of a greater acute HZSOI. Higher acute pain severity, lower income, being immunocompromised, older age, and not receiving antivirals were the predictors of greater postherpetic HZSOI. DISCUSSION: Using an informative measure capturing simultaneously the burden caused by pain duration and severity, we identified subgroups that suffer most during the different phases of HZ. It is interesting to note that, younger participants were as likely to suffer as the older ones during the acute phase of HZ. This information should aid in optimizing the treatment and prevention of HZ.

The impact of herpes zoster and post-herpetic neuralgia on quality-of-life.

BACKGROUND: The potentially serious nature of herpes zoster (HZ) and the long-term complication post-herpetic neuralgia (PHN) are often underestimated. One in four people will contract herpes zoster in their lifetime, with this risk rising markedly after the age of 50 years, and affecting one in two in elderly individuals. Pain is the predominant symptom in all phases of HZ disease, being reported by up to 90% of patients. In the acute phase, pain is usually moderate or severe, with patients ranking HZ pain as more intense than post-surgical or labour pains. Up to 20% of patients with HZ develop PHN, which is moderate-to-severe chronic pain persisting for months or years after the acute phase. We review the available data on the effect of HZ and PHN on patients' quality-of-life. DISCUSSION: Findings show that HZ, and particularly PHN, have a major impact on patients' lives across all four health domains--physical, psychological, functional and social. There is a clear correlation between increasing severity of pain and greater interference with daily activities. Non-pain complications such as HZ ophthalmicus can increase the risk of permanent physical impairment. Some elderly individuals may experience a permanent loss of independence after an acute episode of HZ. Current challenges in the management of HZ and PHN are highlighted, including the difficulty in administering antiviral agents before pain becomes established and the limited efficacy of pain treatments in many patients. We discuss the clinical rationale for the HZ vaccine and evidence demonstrating that the vaccine reduces the burden of the disease. The Shingles Prevention Study, conducted among >38,000 people aged >or=60 years old, showed that the HZ vaccine significantly reduces the burden of illness and the incidence of both HZ and PHN. In the entire study population, zoster vaccination reduced the severity of interference of HZ and PHN with activities of daily living by two-thirds, as measured by two questionnaires specific to HZ. SUMMARY: A vaccination scheme may positively impact the incidence and course of HZ disease, thereby improving patients' quality-of-life.