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Predicting Drug Toxicity for Acute Cardiac Events

Posted in Advanced Drug Manufacturing Technology, Bio Instrumentation in Experimental Life Sciences Research, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Genomic Testing: Methodology for Diagnosis, Health Law & Patient Safety, International Global Work in Pharmaceutical, Molecular Genetics & Pharmaceutical, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Pharmacogenomics, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , on March 15, 2013| 5 Comments »

Predicting Drug Toxicity for Acute Cardiac Events

Reporter: Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/?p=10679/Predicting Drug Toxicity for Acute Cardiac Events

Pharmaceuticals Dilemma

The pharmaceutical industry has, as the clinical diagnostics industry, consolidated, and seen new entries that are at some time merged into an established giant, needing resources to grow.  In the past, it was considered essential for a scientific commercial entity to invest at least 8 percent of budget to R&D.   However, the cost of manufacturing has gone down, but a large part of the budget outside of manufacture has to be taken up with, maybe a few exceptions, development, validation in clinical trials, and marketing.  This leaves the situation precarious without a basic research base, and has lead to consortia between academic centers, the federal governmant, and the industries.  I can’t venture into the role of Wall Street Investment and Venture Capital in the process of innovation, proprietary rights to discoveries, and viability.  A large problem they encounter really comes down to complexity of the biomedical reality, that keeps peeling off layers like an onion, exposing new problems to deal with.  As a result, we have seen repeated recalls of drugs that were blockbusters, over the last 2 decades.  To date, every “miracle” drug to manage sepsis and the several cardiac related drugs have  resulted in unexpected toxicities.
One of the leading causes of drug attrition during development is cardiac toxicity, which has a serious impact on cost and can impact getting new drugs to patients. Detecting cardiovascular safety issues earlier in the drug development program

  • would produce significant benefits for pharmaceutical companies and, ultimately, public health, but
    • the reduction of therapeutic toxicities will not be easy and depends on the
    • emergence of genomic-based personalized medicine.

Comprehensive cardiovascular and electrophysiology assessments are routinely conducted in vivo and in vitro early in the preclinical or lead optimization phases of drug development. For example,

  • the isolated perfused guinea pig heart preparation (classically called the Langendorff preparation)
  • can be used to screen a series of related new chemical entities (NCE)

in the lead optimization phase for preliminary information on the relative effects on contractility and rhythm.
Additionally, intact animal non-GLP studies—generally conducted in anesthetized, non-recovery models—are designed to assess

  • effects of NCEs on a range of acute hemodynamic and cardiac parameters such as
    • heart rate,
    • blood pressure,
    • electrocardiogram (ECG),
    • ventricular contractility,
    • vascular resistance,
    • cardiac output, etc.

These studies employ small numbers of animals, but may allow termination of research into NCEs with obvious cardiovascular side effects. These preparations also provide information on the involvement of the

  • autonomic nervous system in the cardiovascular responses of the NCE.

Such effects can be important determinants in the total cardiovascular response to an NCE, and this information cannot be obtained with any known in vitro method.
But what if there are dangers that are not predictable in the short term because of the time span under which the effects can be viewed? The effects themselves are a result of interactions between

  • the drug,
  • endothelial cell receptors,
  • and/or imbalance in oxidative stress promoters and suppressors,
  • and involve signaling pathways.

That is a difficult challenge that may only be realized

  • by rapidly advancing knowledge at the molecular cell level.

The ICH S7A and ICH S7B guidelines provide

  • guidance on important physiological systems and
  • assessment of pharmaceuticals on
    • ventricular repolarization and
    • proarrhythmic risk.

The guidelines were designed to protect patients from potential adverse effects of pharmaceuticals. Since these guidelines were issued in 2000 and 2005, respectively,

  • cardiac safety study designs have been realigned
  • to identify potential concerns prior to administering the first dose to humans.

It is now routine for all NCEs to be evaluated using an

  • in vitro Ikr assay such as the hERG voltage patch clamp assay to assess for
    • the potential for QT interval prolongation.

Systems have evolved to screen large numbers of compounds

  • using automated high-throughput patch clamp systems early in the lead
  • optimization/drug discovery phase.

This is a cost effective method for determining an initial go/no-go gate. Once a compound has progressed to

  • the development phase, it can once again be assessed with the hERG assay
  • utilizing the gold standard manual patch clamp assay.

If the NCE under investigation is a cardiovascular therapy, then

  • pharmacological characterization should occur
  • early in the lead development process.

In addition to the techniques just discussed,

  • a variety of “disease models” are available to help determine
    • whether the NCE will be efficacious in a clinical setting.

However sound the in vitro data used in screening and selection process (e.g., receptor-binding studies),

  • NCEs that have been shown to be active in at least one in vivo model (e.g,. salt-sensitive Dahl rat model)
  • have a higher likelihood of clinical success.

Once a lead is identified, it should still go through the generalized safety characterization discussed earlier.
The in vivo study designs for NCEs reaching the development phase to support the Investigational New Drug (IND) application (just prior to the first human dose) require acquisition of

  1. heart rate,
  2. blood pressure, and
  3. ECG data
    • using an appropriate species
    • at and above clinically relevant doses.

The trend in the industry for these regulatory-driven studies has been to

  • utilize animals surgically instrumented with telemetry devices that
  • can acquire the required parameters.

The advantage of using instrumented animals over anesthetized animals is that

  • data can be acquired from freely moving animals over greater periods of time
  • without anesthetic in the test system,
    • which has the potential to confound and perturb results interpretation.

Appropriate dose selection relative to those used in the clinic provides valuable information about

  • potential acute cardiac events and
  • how they may impact trial participants.

The obvious limitation here is that the method of observation is essentially

  • the same or less than that which is used in clinical practice,
  • relying mainly on classical physiology to detect
    • inherently deep seated processes.

But it is not the same scale of issue as for the patient emergently presenting to the ED. Despite enormous efforts to reduce the development of and the complications of acute ischemia related cardiac events,

the accurate diagnosis of the patient presenting to the emergency room is still, as always, reliant on

  • clinical history,
  • physical examination,
  • effective use of the laboratory, and
  • increasingly helpful imaging technology.

and age, sex, diet, and ability to carry out the activities of daily living before treatment and 6 months to a year after discharge are relevant.

The main issue that we have a consensus agreement that PLAQUE RUPTURE is not the only basis for a cardiac ischemic event. There will be more to say about this.
Animal studies
Telemetry-instrumented animals can be used as screening tools earlier in the drug selection phase. Colonies of animals that can be reused, following a suitable wash-out period,
provide an excellent resource for screening compounds to detect unwanted side effects. The use of these animals

  • coupled with
  • recent advances in software-analysis systems allow for rapid data turnaround,
    • enables scientists to quickly determine if there are any potentially unwanted signals.

If any effects are detected on, for example, blood pressure or QT interval, then the decision to

  • either shelve the drug or
  • conduct additional studies

can be made before advancing any further in the developmental phase.   While this is very good for observing large effects, is it really sufficient for avoidance of late phase failure?

Interestingly, the experience that has been acquired since the approval of the ICH guidelines

  • has allowed pharmaceutical companies to temper their response to finding a potentially unwanted signal.
  • Rather than permanently shelve libraries of compounds that, for example, were
  • found to be positive in the hERG assay—common practice when the 2005 guidelines came into being—
    • companies can now determine a risk potential based on knowledge gained with the intact animal studies.

Similarly, if changes in hemodynamic parameters are detected, there are follow-up experiments employing anesthetized or telemetry models that include additional measurements like

left ventricular pressure.
These experiments can be utilized to further assess their potential clinical impact
by examining effects on
myocardial contractility,
relaxation, and
conduction velocity.
These techniques primarily address acute effects: those following a single exposure.
Chronic effects—those seen with long-term administration of the NCE to an intact organism—are difficult to obtain in early development, but are routinely monitored during safety studies,
are conducted non-clinically during Phase 1 and 2 of the development process.

  • ECGs typically are collected to evaluate the chronic cardiac effects in non-rodent species during these studies. It is recommended that
    • JET (jacketed external telemetry) techniques, which permit the recording of ECG’s—
    • but not blood pressure—

be applied in freely moving animals. If chronic effects are discovered,

  • follow-up experiments can be conducted with any of the techniques mentioned in this article.

As the focus on cardiac safety has matured over the last 10 years, the Safety Pharmacology Society has led efforts to establish an approach

  • to determine best practices for conducting key preclinical cardiovascular assessments in drug development.
  •  to provide sensitive preclinical assays that can detect high-probability safety concerns.

Parallel efforts have been made to more accurately assess the translation of preclinical cardiovascular data into

  • clinical outcomes and
  • to encourage collaborations
    • between preclinical and clinical scientists involved in cardiac safety assessment.

This has been conducted under the umbrella of the International Life Science Institute–Health and Environmental Services Institute (ILSI-HESI) consortium, which has bought together

  • industrial,
  • academic, and
  • government scientists
    • to discuss and determine what steps are necessary
    • to establish an integrated cardiovascular safety assessment program.

The goal is to provide better ways of predicting potential adverse events, allowing for earlier detection of cardiovascular safety issues and reducing the number of clinical trial failures.
http://www.dddmag.com/articles/2012/08/predicting-potential-cardiac-events?et_cid=2816494&et_rid=45527476&linkid=http%3a%2f%2fwww.dddmag.com%2farticles%2f2012%2f08%2fpredicting-potential-cardiac-events.

A recent poster presentation I think makes a good statement of advances that should move us forward:

http://www.biotechniques.com/multimedia/archive/00178/BTN_0311-March_Post_178205a.pdf

Another possibility is genetic testing to determine the likelihood of stroke, for example Corus CAD is

  • a shoebox-size kit that uses a simple blood draw to measure the RNA levels of 23 genes.
  •  it creates an algorrhytm-based score that determines the likelihood that a patient has obstructive coronary artery disease.

http://pharmaceuticalintelligence.com/2012/08/14/obstructive-coronary-artery-disease-diagnosed-by-rna-levels-of-23-genes-cardiodx-heart-disease-test-wins-medicare-coverage/
“By providing Medicare beneficiaries access to Corus CAD, this coverage decision enables patients to avoid unnecessary procedures and risks associated with cardiac imaging and elective invasive angiography, while helping payers address an area of significant healthcare spending,” CardioDx President and CEO David Levison said in a press release.
This discussion will be followed with a discussion of the evaluation of the patient acutely presenting with symptoms and signs that are suggestive of either acute pulmonary or cardiac disease, or both, that may be suggestive of a non ST elevation AMI. It becomes more difficult if ST depression or T-wave inversion is not detected.
Related articles
Obstructive Coronary Artery Disease diagnosed by RNA levels of 23 genes – CardioDx, a Pioneer in the Field of Cardiovascular Genomic Diagnostics
http://pharmaceuticalintelligence.com/2012/08/14/obstructive-coronary-artery-disease-diagnosed-by-rna-levels-of-23-genes-cardiodx-heart-disease-test-wins-medicare-coverage/

Related articles
English: QT interval corrected by heart rate.

English: QT interval corrected by heart rate. (Photo credit: Wikipedia)

Schematic diagram of normal sinus rhythm for a...

Schematic diagram of normal sinus rhythm for a human heart as seen on ECG (with English labels). (Photo credit: Wikipedia)

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Personalized Medicine: Clinical Aspiration of Microarrays

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, FDA Regulatory Affairs, Genome Biology, Health Economics and Outcomes Research, HealthCare IT, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, tagged , , , , , , , , , , , , , , , , , , , on March 13, 2013| 7 Comments »

Personalized Medicine: Clinical Aspiration of Microarrays

Reporter, Writer: Stephen J. Williams, Ph.D.

 In this month’s Science, Mike May (at http://www.sciencemag.org/site/products/lst_20130215.xhtml) describes some of the challenges and successes in introducing microarray analysis to the clinical setting.  Traditionally used for investigational research, microarray is now being developed, customized and used for biomarker analysis, prognostic and predictive value, in a disease-specific manner.

Challenges in data interpretation

      In an interview with Seth Crosby, director of the Genome Technology Access Center at Washington University School of Medicine in St. Louis, “the biggest challenge” in moving microarray to the clinical setting is data interpretation.  The current technology makes it possible to evaluate expression of thousands of genes from a patient’s sample however as Crosby describes is assigning clinical relevance to the data.  For example Crosby explains that Washington University had validated a panel of 45 oncology genes by next generation sequencing and are using these genes to develop diagnostic tests to screen patient tumors for the purpose of determining a personalized therapeutic strategy. Seth Crosby noted it took “hundreds of Ph.D. and M.D. hours” to sift through the hundreds of papers to determine which genes were relevant to a specific cancer type. However, he notes, that once we better understand which changes in the patient’s genome are related to a specific disease we will be able to narrow down the list and be able to produce both economical and more disease-relevant microarrays.

Is this aberration pathogenic or not?

     Microarrays are becoming an invaluable tool in cytogenetics, as eluded by Andy Last, executive vice president of the genetic analysis business unit at Affymetrix.  Certain diseases like Down syndrome have well characterized chromosomal alterations like additions or deletions of parts or entire chromosomes.  According to Affymetrix, the most common use of microarrays is for determining copy number variation.  However according to James Clough, vice president of clinical and genomic services at Oxford Gene Technology, given the hundreds of syndromes associated with chromosomal rearrangements, the challenge will be to determine if a small chromosomal aberration has pathologic significance, given that microarray affords much higher diagnostic yield and speed of analysis than traditional microscopic techniques.  To address this challenge, Oxford Gene Technologies, PerkinElmer, Affymetrix, and Agilent all have custom designed microarrays to evaluate disease specific copy number and SNP (single nucleotide polymorphism) microarrays.  For example PerkinElmer designed OncoChip™ to evaluate copy number variation in more than 1.800 cancer genes.  Agilent makes microarrays that evaluates both copy number variation such as its CGH (comparative genomic hybridization) plus SNP microarrays.  Patricia Barco, product manager for cytogenetics at Agilent, notes these arrays can be used in prenatal and postnatal research and cancer, and “can be customized from more than 28 million probes in our library”.

Custom Tools and Software to Handle the Onslaught of Big Data

     There is a need for FDA approved diagnostic tools based on microarrays. Pathwork Diagnostic’s has one such tool (the Pathwork Tissue of Origin test), which uses 2,000 transcript markers and a proprietary computational algorithm to determine from expression analysis, the tissue of origin of a patient’s tumor.  Pathwork also provides a fast, custom turn-around analytical service for pathologists who encounter difficult to interpret samples.  Illumina provides the Infinium HumanCore BeadChip family of microarrays, which can determine genetic variations for purposes of biological tissue banking.  This system uses a set of over 300,000 SNP probes plus 240,000 exome-based markers.

     Tools have also been developed to validate microarray results.  A common validation strategy is the use of quantitative real-time PCR to verify the expression changes seen on the microarray.  Life Technologies developed the TaqMan OpenArray Real Time PCR plates, which have 3,072 wells and can be custom-formatted using their library of eight million validated TaqMan assays.

Making Sense of the Big Data: Bridging the Knowledge Gap using Bioinformatics

          The use of microarray has spurned industries devoted to developing the bioinformatics software to analyze the massive amounts of data and provide clinical significance.  For example companies such as Expression Analysis use their bioinformatics software to provide pathway analysis for microarray data in order to translate the data into the biology.  Using such strategies can also validate the design of microarrays for various diseases.

Foundation Medicine, Inc., a molecular information company, provides cancer genomics test solutions. It offers FoundationOne, an informative genomic profile to identify a patient’s individual molecular alterations and match them with relevant targeted therapies and clinical trials. The company’s product enables physicians to recommend treatment options for patients based on the molecular subtype of their cancer.

The Canadian Bioinformatics Workshops series recently offered a course on using bioinformatic approaches to analyze clinical data generated from microarray approaches (http://bioinformatics.ca/workshops/2012/bioinformatics-cancer-genomics-bicg).   The course objectives are described below:

Course Objectives

Cancer research has rapidly embraced high throughput technologies into its research, using various microarray, tissue array, and next generation sequencing platforms. The result has been a rapid increase in cancer data output and data types. Now more than ever, having the bioinformatic skills and knowledge of available bioinformatic resources specific to cancer is critical. The CBW will host a 5-day workshop covering the key bioinformatics concepts and tools required to analyze cancer genomic data sets. Participants will gain experience in genomic data visualization tools which will be applied throughout the development of the skills required to analyze cancer -omic data for gene expression, genome rearrangement, somatic mutations and copy number variation. The workshop will conclude with analyzing and conducting pathway analysis on the resultant cancer gene list and integration of clinical data.

Successful Examples of Clinical Ventures Integrating Bioinformatics in Cancer Treatment Decision –Making

The University of Pavia, Italy developed a fully integrated oncology bioinformatics workflow as described on their website and at the ESMO 2012 Congress meeting:

http://abstracts.webges.com/viewing/view.php?congress=esmo2012&congress_id=370&publication_id=2530

ESMO

ONCO-I2B2 PROJECT: A BIOINFORMATICS TOOL INTEGRATING –OMICS AND CLINICAL DATA TO SUPPORT TRANSLATIONAL RESEARCH

Abstract:

2530

Congress:

ESMO 2012

Type:

Abstract

Topic:

Translational research

Authors:

A. Zambelli, D. Segagni, V. Tibollo, A. Dagliati, A. Malovini, V. Fotia, S. Manera, R. Bellazzi; Pavia/IT
  • Body

The ONCO-i2b2 project, supported by the University of Pavia and the Fondazione Salvatore Maugeri (FSM), aims at supporting translational research in oncology and exploits the software solutions implemented by the Informatics for Integrating Biology and the Bedside (i2b2) research centre, an initiative funded by the NIH Roadmap National Centres for Biomedical Computing. The ONCO-i2b2 software is designed to integrate the i2b2 infrastructure with the FSM hospital information system and the Bruno Boerci Biobank, in order to provide well-characterized cancer specimens along with an accurate patients clinical data-base. The i2b2 infrastructure provides a web-based access to all the electronic medical records of cancer patients, and allow researchers analyzing the vast amount of biological and clinical information, relying on a user-friendly interface. Data coming from multiple sources are integrated and jointly queried.

In 2011 at AIOM Meeting we reported the preliminary experience of the ONCO-i2b2 project, now we’re able to present the up and running platform and the extended data set. Currently, more than 4400 specimens are stored and more than 600 of breast cancer patients give the consent for the use of specimens in the context of clinical research, in addition, more than 5000 histological reports are stored in order to integrate clinical data.

Within the ONCO-i2b2 project is possible to query and merge data regarding:

• Anonymous patient personal data;

• Diagnosis and therapy ICD9-CM subset from the hospital information system;

• Histological data (tumour SNOMED and TNM codes) and receptor profile testing (Her2, Ki67) from anatomic pathology database;

• Specimen molecular characteristics (DNA, RNA, blood, plasma and cancer tissues) from the Bruno Boerci Biobank management system.

The research infrastructure will be completed by the development of new set of components designed to enhance the ability of an i2b2 hive to utilize data generated by NGS technology, providing a mechanism to apply custom genomic annotations. The translational tool created at FSM is a concrete example regarding how the integration of different information from heterogeneous sources could bring scientific research closer to understand the nature of disease itself and to create novel diagnostics through handy interfaces.

Disclosure

All authors have declared no conflicts of interest.

NCI has under-taken a similar effort under the Recovery Act (the full text of the latest report is taken from their website http://www.cancer.gov/aboutnci/recovery/recoveryfunding/investmentreports/bioinformatics:

Cancer Bioinformatics: Recovery Act Investment Report

November 2009

Public Health Burden of Cancer

Cancer is the second leading cause of death in the United States after heart disease. In 2009, it is estimated that nearly 1.5 million new cases of invasive cancer will be diagnosed in this country and more than 560,000 people will die of the disease.

To learn more, visit:

Cancer Bioinformatics Program Overview

Over the past five years, NCI’s Center for Biomedical Informatics and Information Technology (CBIIT) has led the effort to develop and deploy the cancer Biomedical Informatics Grid® (caBIG) in partnership with the broader cancer community.  The caBIG network is designed to enable the integration and exchange of data among researchers in the laboratory and the clinic, simplify collaboration, and realize the potential of information-based (personalized) medicine in improving patient outcomes. caBIG has connected major components of the cancer community, including NCI-designated Cancer Centers, participating institutions of the NCI Community Cancer Centers Program (NCCCP), and numerous large-scale scientific endeavors, as well as basic, translational, and clinical researchers at public and private institutions across the United States and around the world.  Beyond cancer research, caBIG capabilities—infrastructure, standards, and tools—provide a prototype for linking other disease communities and catalyzing a new 21st-century biomedical ecosystem that unifies research and care. ARRA funding will allow NCI to accelerate the ongoing development of the Cancer Knowledge Cloud and Oncology Electronic Health Records (EHRs) initiatives, thereby providing for continued job creation in the areas of biomedical informatics development and application as well as healthcare delivery.

The caBIG Cancer Knowledge Cloud: Extending the Research Infrastructure

The Cancer Knowledge Cloud is a virtual biomedical capability that utilizes caBIG tools, infrastructure, and security frameworks to integrate distributed individual and organizational data, software applications, and computational capacity throughout the broad cancer research and treatment community. The Cancer Knowledge Cloud connects, integrates, and facilitates sharing of the diverse primary data generated through basic and clinical research and care delivery to enable personalized medicine. The cloud includes information generated through large-scale research projects such as The Cancer Genome Atlas (TCGA), the cancer Human Biobank (caHUB) tissue acquisition network, the NCI Functional Biology Consortium, the NCI Patient Characterization Center, and the NCI Preclinical Development Pipeline, academic and industry counterparts to these projects, and clinical observations (from entities such as the NCCCP) captured in oncology-extended Electronic Health Records.  Through the use of the caBIG Data Sharing and Security Framework, the Cloud will support appropriate sharing of information, supporting in silico hypothesis generation and testing, and enabling a learning healthcare system.

A caBIG-Based Rapid-Learning Healthcare System: Incorporating Oncology-Extended Electronic Healthcare Records (EHRs)

The 21st-century Cancer Knowledge Cloud will connect individuals, organizations, institutions, and their associated information within an information technology-enabled cycle of discovery, development, and clinical care—the paradigm of a rapid-learning healthcare system. This will transform these disconnected sectors into a system that is personalized, preventive, pre-emptive, and patient-participatory.  To be realized, this model requires the adoption of standards-based EHRs. Presently, however, no certified oncology-based EHR exists, and fewer than 3 percent of oncologists with outpatient-based practices utilize EHRs. caBIG has recently established a collaboration with the American Society of Clinical Oncology (ASCO) to develop an oncology-specific EHR (caEHR) specification based on open standards already in use in the oncology community that will utilize caBIG standards for interoperability. NCI will implement an open-source version of this specification to validate the specification and to provide a free alternative to sites that choose not to purchase a commercial system. The launch customer for the caEHR will be NCCCP participating sites. NCI will work with appropriate entities to provide a mechanism for certifying that caEHR implementations are consistent with the NCI/ASCO specification.

Bards Cancer Institute has another clinical bioinformatics program to support their clinical efforts:

Clinical Bioinformatics Program in Oncology at Barts Cancer Institute at Barts and the London School of Medicine

http://www.bci.qmul.ac.uk/cancer-bioinformatics

BCI HomeCancer Bioinformatics

Bards

Why we focus on Cancer Bioinformatics

Bioinformatics is a new interdisciplinary area involving biological, statistical and computational sciences. Bioinformatics will enable cancer researchers not only to manage, analyze, mine and understand the currently accumulated, valuable, high-throughput data, but also to integrate these in their current research programs. The need for bioinformatics will become ever more important as new technologies increase the already exponential rate at which cancer data are generated.

What we do

  • We work alongside clinical and basic scientists to support the cancer projects within BCI.  This is an ideal partnership between scientific experts, who know the research questions that will be relevant from a cancer biologist or clinician’s perspective, and bioinformatics experts, who know how to develop the proposed methods to provide answers.
  • We also conduct independent bioinformatics research, focusing on the development of computational and integrative methods, algorithms, databases and tools to tackle the analysis of the high volumes of cancer data.
  • We also are actively involved in the development of bioinformatics educational courses at BCI. Our courses offer a unique opportunity for biologists to gain a basic understanding in the use of bioinformatics methods to access and harness large complicated high-throughput data and uncover meaningful information that could be used to understand molecular mechanisms and develop novel targeted therapeutics/diagnostic tools.

Developing Criteria for Genomic Profiling in Lung Cancer:

A Report from U.S. Cancer Centers

In a report by Pao et. al., a group of clinicians organized a meeting to standardize some protocols for the integration of microarray and genomic data from lung cancer patients into the clinical setting.[1]  There has been ample evidence that adenocarcinomas could be classified into “clinically relevant molecular subsets” based on distinct genomic changes.  For example EGFR (epidermal growth factor receptor) exon 19 deletions and exon 21 point mutations predict sensitivity to tyrosine kinase inhibitors (TKIs) like gefitinib, whereas exon 20 insertions predict primary resistance[2].

However, as the authors note, “mutational profiling has not been widely accepted or adopted into practice in thoracic oncology”.  

     Therefore, a multi-institutional workshop was held in 2009 among participants from Massachusetts General Hospital (MGH) Cancer Center, Memorial Sloan-Kettering Cancer Center (MSKCC), the Dana-Farber/Bingham & Women’s Cancer Center (DF/BWCC), the M.D. Anderson Cancer Center (VICC), and the Vanderbilt-Ingram Cancer Center (VICC) to discuss their institutes molecular profiling programs with emphasis on:

·         Organization/workflow

·         Mutation detection technologies

·         Clinical protocols and reporting

·         Patient consent

In addition to the aforementioned challenges, the panel discussed further issues for developing improved science-driven criteria for determining targeted therapies including:

1)      Including pathologists into criteria development as pathology departments are usually the main repositories for specimens

2)      Developing integrated informatics systems

3)      Standardizing new target validation methodology across cancer centers

 References

1.            Pao W, Kris MG, Iafrate AJ, Ladanyi M, Janne PA, Wistuba, II, Miake-Lye R, Herbst RS, Carbone DP, Johnson BE et al: Integration of molecular profiling into the lung cancer clinic. Clinical cancer research : an official journal of the American Association for Cancer Research 2009, 15(17):5317-5322.

2.            Wu JY, Wu SG, Yang CH, Gow CH, Chang YL, Yu CJ, Shih JY, Yang PC: Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clinical cancer research : an official journal of the American Association for Cancer Research 2008, 14(15):4877-4882.

Other posts on this website on Cancer and Genomics include:

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Imaging-biomarkers is Imaging-based tissue characterization

Posted in Advanced Drug Manufacturing Technology, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Ecosystems & Industrial Concentration in the Medical Device Sector, FDA Regulatory Affairs, FDA, CE Mark & Global Regulatory Affairs: process management and strategic planning - GCP, GLP, ISO 14155, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, HealthCare IT, Imaging-based Cancer Patient Management, International Global Work in Pharmaceutical, Medical Devices R&D and Inventions, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Pharmacotherapy and Cell Activity, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , on March 10, 2013| 3 Comments »

Imaging-biomarkers is Imaging-based tissue characterization

Author – Writer: Dror Nir, PhD

Article 11.1 Introduction by Dror Nir PhD

For everyone who is skeptical about the future role of imaging-based tissue chracterisation in the management of cancer, the following “Statement paper” ESR statement on the stepwise development of imaging biomarkers published online: 9 February 2013, by the European Society of Radiology (ESR), should provide substantial reassurance that this kind of technology will become a must! In support of this claim I quote the following information:

“The European Society of Radiology and its related European Institute for Biomedical Imaging Research (EIBIR) should have a relevant role in coordinating future developments of biomarkers and in the assessment and validation of imaging biomarkers as surrogate end points.

Acknowledgements

This paper was kindly prepared by the ESR Subcommittee on Imaging Biomarkers (Chairperson: Bernard Van Beers. Research Committee Chairperson: Luis Martí-Bonmatí. Members: Marco Essig, Thomas Helbich, Celso Matos, Wiro Niessen, Anwar Padhani, Harriet C. Thoeny, Siegfried Trattnig, Jean-Paul Vallée. Co-opted members: Peter Brader, Nicolas Grenier) on behalf of the European Society of Radiology (ESR) and with the help of Sabrina Doblas, INSERM U773, Paris, France.

It was approved by the ESR Executive Council in December 2012..”

According to ESR: “There is increasing interest in developing the quantitative imaging of biomarkers in personalised medicine”. In this perspective, “Biomarkers” are tissue properties that can be quantitatively and reproducibly measured by imaging devices. One example for a major unmet need, which I found to be most interesting is the imaging-based detection of tumor invasiveness.

Quoting from the paper: ” Biomarkers are defined as “characteristics that are objectively measured and evaluated as indicators of normal biological processes, pathological processes, or pharmaceutical responses to a therapeutic intervention” [1]. Broadly, biomarkers fall into two categories: bio-specimen biomarkers, including molecular biomarkers and genetic biomarkers, and bio-signal biomarkers or imaging biomarkers. Bio-specimen biomarkers are obtained by removing a sample from a patient. Examples of these molecular biomarkers are genes and proteins detected from fluids or tissue samples. Bio-signal biomarkers remove no material from the patient, but rather detect and analyse an electromagnetic, photonic or acoustic signal emitted by the patient [2]. These imaging biomarkers have the advantage of being non-invasive, spatially resolved and repeatable [3]. They are of particular interest if they can overcome the limitations of the established histological “gold standards”. Indeed, invasive reference examinations, such as biopsy, can be inconclusive, are non-representative of the whole tissue (which is a tremendous limitation when assessing malignant tumours, which are known to be heterogeneous) and possess non-negligible levels of mortality and morbidity.

Genetic biomarkers indicate whether a disease may occur, but they are usually inefficient to assess the presence and stage of a disease. Similar to molecular biomarkers, imaging biomarkers can be used for early detection of diseases, staging and grading, and predicting or assessing the response to treatment [3]. Accordingly, because of their relative lower cost compared with imaging, molecular biomarkers may be more appropriate for disease screening and early detection than imaging biomarkers. With their high sensitivity, molecular biomarkers could also detect subclinical stages of disease before any morphological or functional change is detectable on imaging. In contrast, imaging biomarkers are often more useful than molecular biomarkers for disease staging, and also grading and for assessing tumour response, because localised information is crucial.”

The main messages ESR wishes to deliver in this paper are that:

• Using imaging-biomarkers to streamline drug discovery and disease progression will drive a huge advancement in healthcare.

• The clinical qualification and validation of imaging biomarkers technology pose challenges, mainly in establishing the accuracy and reproducibility of such techniques. In that respect, agreements on standards and evaluation methods (e.g. clinical studies design) is imperative.

• There should be high motivation to pursue the development of imaging-biomarkers as the “clinical value of new biomarkers is of the highest priority in terms of patient management, assessing risk factors and disease prognosis.”

The paper deals to a great extent with the requirements on accuracy, reproducibility, standardization and quality control from the process of developing imaging-biomarkers:

“Accuracy: Before being routinely used in the clinic, imaging biomarkers must be validated. Determining the accuracy implies calculating the sensitivity and specificity of the biomarker when compared with a biological process, such as tumour necrosis, which can be assessed at histopathological examination… [6–9]  [10, 11]

Reproducibility: Repeatability (measurements at short intervals on the same subjects using the same equipment in the same centres) and reproducibility (measurements at short intervals on the same subjects using different facilities in the same and different centres) studies must be conducted for image acquisition and image analysis…. Reproducibility studies are now very often included in scientific papers, as advised by the “standards for reporting of diagnostic accuracy” (STARD) criteria and should ideally include Bland-Altman plots and results of coefficients of repeatability [16, 17].

Standardisation: Standardisation relates to the establishment of norms or requirements about technical aspects. In the development of imaging biomarkers, two main aspects should be considered: Standardisation of image acquisition and Standardisation of image analysis…  [18] [19–21]  [22] [27, 28] [31–33]

Quality control: Adequate phantoms could be used to validate, on a day-to-day basis, that the biomarker stays robust and to avoid any drift in the machine, acquisition or processing protocol….  [34] [30, 35] [36] [37] [23].”

The proposed development workflow:

“Similar to new drugs, the development of biomarkers has to pass along a pipeline going from discovery, through verification in different laboratories, validation and qualification before they can be used in clinical routine. Validation includes the determination of the accuracy and the precision (reproducibility) of the biomarker and standardisation concerns both acquisition and analysis. Qualification, defined as a “graded, fit-for-purpose evidentiary process linking a biomarker with biological processes and clinical end-points”, is a validation process in large cohorts of patients involving multiple centres, similar to phase III clinical trials, to obtain regulatory approval as surrogate endpoints [4]. A more extensive path to biomarker development has been reported [5]. The first step is the proof of concept, which defines any specific change relevant to the disease that can be studied using the available imaging and computational techniques. The relationship between this change and the presence, grading and response to treatment of the disease constitutes the proof of mechanism. The images needed to extract the biomarker must be appropriate (in terms of resolution, signal and contrast behaviour). Preparation of images relates to improving the data before the analysis (such as segmentation, filtering, interpolation or registration). The analysis and modelling of the signal by computational numerical adjustment of a mathematical model allow extracting the needed information (such as structural, physical, chemical, biological and functional properties). After this voxel-by-voxel computation, the spatial distribution of the biomarker can be depicted by parametric images, defined as derived secondary images which pixels represent the distribution values of a given parameter. Multivariate parametric images obtained by statistical modelling of the relevant parameters allow the reduction of data and a clear definition of the defined disease target. The abnormal values should be defined and measured through histogram analysis. A pilot test on a small sample of subjects, with and without the disease, has to be performed to validate the process—also called proof of principle—and to evaluate the influence of potential variations related to age, sex or any other source of biases. Finally, proofs of efficacy and effectiveness on larger and well-defined series of patients will show the ability of a biomarker to measure the clinical endpoint (Fig. 1).”

Steps for the development of imaging biomarkers (adapted from [5])

Steps for the development of imaging biomarkers (adapted from [5])

The authors admit that the requirement posed on development of imaging-biomarkers represents a huge challenge and they try to offer ideas, mainly taken from the “MRI experience” to overcome certain hurdles. There is one important point on which they do not discuss: the definition of appropriate reference test. It is my own experience, based on many study protocols I developed in the past decade, that without reaching an agreement on that point, the development of imaging-biomarkers will just move in circles. Note, that today’s most “acceptable” reference test is histopathology, which everyone admits (as well mentioned in this paper); suffers many limitations. When it comes to validating imaging-biomarkers, the need to accurately match imaging products with histopathology is an additional major hurdle.

This is why, I see as a necessary step, to develop “real-time” imaging based tissue characterization combined with in-situ imaging-based histology.

 

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Melatonin and its Effect on Acetylcholinesterase Activity in Erythrocytes

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Melatonin & Circadian Regulation, tagged , , , , , , , , , , on March 9, 2013| 5 Comments »

Melatonin and its effect on acetylcholinesterase activity in erythrocytes

Author: S. Chakravarty, PhD

 

Objective: The study was conducted to see the effect of melatonin on the activity of acetylcholinesterase in red blood cells.

Mammalian red blood cells contain membrane-bound acetylcholinesterase which acts as biomarkers of oxidative imbalance. Melatonin is a powerful free radical scavenger and upregulates several antioxidant enzymes to reduce oxidative stress. Being an effective antioxidant, it may initiate variation in erythrocyte acetylcholinesterase activity.

The study was carried out on twenty-nine subjects of both sexes who gave their informed consent for the use of their blood samples for the study (Chakravarty and Rizvi, 2011a). The red cells isolated from blood collected at two different timings of the day, viz., 10:00 a.m. and 10:00 p.m.,were subjected to in vitro treatment with melatonin in a dose-dependant manner followed by the assay of enzyme activity (Ellman et al., 1961).

Acetylcholinesterase (AChE) is also found on the red blood cell membranes, where it constitutes the Yt blood group of antigen, which is a blood-group determining protein. AChE has the features of a secreted rather than a transmembrane protein because it lacks long hydrophobic stretches, other than that which forms the signal peptide (Li et al., 1991). Besides, acetylcholinesterase activity in erythrocytes may be considered as a marker of central cholinergic status (Kaizer et al., 2008). AChE shows highest activity in the immature rat brain is at 6.00 a.m. and lowest after midnight, which undergoes a reversal after attaining maturity (Moudgil and Kanungo, 1973). The enzyme also exhibits annual changes in its activity (Lewandowski, 2008). Acetylcholinesterase activity has been used to for studying the activity pattern of human erythrocytes (Prall et al., 1998). Free radicals and increased oxidative stress have been found to reduce AChE activity (Molochkina et al., 2005). This indicates that melatonin may have some relation with the circadian rhythmicity of acetylcholinesterase activity.

The concentration-dependant assay of AChE activity in red cells bear close relation with the circadian rhythm in humans thus sharing a similar conclusion with that mentioned by Moudgil and Kanungo (Moudgil and Kanungo, 1973). The effect of melatonin on enzyme functions in erythrocytes follows rhythmic modulation with day/night cycle. The samples obtained in the morning exhibit significantly higher activity of acetylcholinesterase than those obtained during the night-time. The samples collected at two different timings of the day show different response to in vitro melatonin treatment. The rise in AChE activity is more pronounced at low doses of melatonin. Our results indicate significant increase in acetylcholinesterase activity in diurnal as well as nocturnal blood samples at different concentrations of exogenous melatonin (Rizvi and Chakravarty, 2011). At supraphysiological doses, the enzyme activity exhibits no significant change, owing to the prooxidative influence exerted by melatonin (Marchiafava and Longoni, 1999).

Acetylcholinesterase activity is affected by the hydrophobic environment of the cell membrane and depends on the plasma membrane fluidity and surface charge of the cell (Klajnert et al., 2004).  The activity of AChE depends largely on the biophysical features of membrane. Oxidative stress decreases the fluidity of membrane lipid bilayer, thus affecting its normal functions (Goi et al., 2005).  Such are the ill-effects of oxidative radicals that tend to increase with aging. The decrease in AChE correlates significantly with age-induced oxidative stress (Jha and Rizvi, 2009).  On the basis of our study we conclude that melatonin modulates acetylcholinesterase activity in erythrocytes. The rhythmicity observed in the activity of acetylcholinesterase in response to the melatonin confirms our opinion on the relationship between the enzyme function, pineal secretion and pharmacological dosage of the indole antioxidant.

References:

  1. Chakravarty S, Rizvi SI, Circadian modulation of sodium-potassium ATPase and sodium-proton exchanger in human erythrocytes: in vitro effect of melatonin. <a href=”80-6. “http://www.ncbi.nlm.nih.gov/pubmed/21366966
  2. Ellman GL, Courtney KD,      Andres Jr V, Featherstone RM, A new and rapid colorimeteric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7(2): 88–95.
  3. Goi G, Cazzola R,      Tringali C, Massaccesi L, Volpe SR, Rondanelli M, Ferrari      E, Herrera      CJ, Cestaro      B, Lombardo      A, Venerando      B, Erythrocyte membrane alterations during      ageing affect beta-D-glucuronidase and neutral sialidase in elderly      healthy subjects. Exp Gerontol 2005; 40(3): 219-25.
  4. http://www.ncbi.nlm.nih.gov/pubmed/?term=alterations+during++++++ageing+affect+beta-D-glucuronidase+and+neutral+sialidase+in+elderly++++++healthy+subjects.
  5. Jha R, Rizvi SI, Age-dependant  decline in erythrocyte acetylcholinesterase activity: correlation with oxidative stress. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009; 153(3):195–8.
  6. http://www.ncbi.nlm.nih.gov/pubmed/19851431
  7. Kaizer RR, Correa MC, Gris LR, Da Rosa CS, Bohrer D, Morsch VM, Schetinger MR, Effect of long-term exposure to aluminum on the acetylcholinesterase activity in the central nervous system and erythrocytes. Neurochem Res 2008; 33(11):2294-301.
  8. http://www.ncbi.nlm.nih.gov/pubmed/?term=Effect+of+long-term+exposure+to+aluminum+on+the+acetylcholinesterase+activity+in+the+central+nervous+system+and+erythrocytes.
  9. Klajnert B, Sadowska M,      Bryszewska M, The effect of polyamidoamine dendrimers on human erythrocyte membrane acetylcholinesterase activity. Bioelectrochem 2004; 65(1): 23-6.
  10. http://www.ncbi.nlm.nih.gov/pubmed/?term=The+effect+of+polyamidoamine+dendrimers+on+human+erythrocyte+membrane+acetylcholinesterase+activity.
  11. Lewandowski MH, Annual changes of circadian acetylcholinesterase activity in the brain stem compared to locomotor activity of the mouse under LD 12/12. J Interdisiplinary Cycle Res 1990; 21 (1): 25-32.
  12. http://www.tandfonline.com/doi/abs/10.1080/09291019009360023?journalCode=nbrr19
  13. Li Y, Camp      S, Rachinsky TL, Getman D, Taylor P, Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression. J Biol Chem 1991; 266(34): 23083–90.
  14. http://www.ncbi.nlm.nih.gov/pubmed/?term=Gene+structure+of+mammalian+acetylcholinesterase.+Alternative+exons+dictate+tissue-specific+expression
  15. Marchiafava PL, Longoni B, Melatonin as an antioxidant in retinal photoreceptors. J Pineal Res 1999; 26(3): 184-89.
  16. http://www.ncbi.nlm.nih.gov/pubmed/10231733
  17. Molochkina EM, Zorina OM, Fatkullina LD, Goloschapov AN, Burlakova EB, H2O2 modifies membrane structure and activity of acetylcholinesterase. Chem Biol Interact 2005; 157-158(1): 401-4.
  18. http://www.ncbi.nlm.nih.gov/pubmed/?term=H2O2+modifies+membrane+structure+and+activity+of+acetylcholinesterase.
  19. Moudgil VK, Kanungo MS, Effect of age on the circadian rhythm of acetylcholinesterase of the brain of the rat. Comp Gen Pharmacol 1973; 4(14):127-30.
  20. http://www.ncbi.nlm.nih.gov/pubmed/4770270
  21. Prall YG, Gambhir KK, Ampy FR, Acetylcholinesterase: an enzymatic marker of human red blood cell aging. Life Sci 1998; 63(3): 177-84.
  22. http://www.ncbi.nlm.nih.gov/pubmed/?term=Acetylcholinesterase%3A+an+enzymatic+marker+of+human+red+blood+cell+aging
  23. Rizvi SI, Chakravarty S, Modulation of acetylcholiesterase activity by melatonin in red blood cells. Acta Endocrinologica (Buc), 2011; 8(3): 311-16..

 

 

 

 

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Nanotechnology and Heart Disease

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, BioSimilars, Cardiac & Vascular Repair Tools Subsegment, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Frontiers in Cardiology and Cardiovascular Disorders, Medical Devices R&D Investment, Nanotechnology for Drug Delivery, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Stents & Tools, Valves & Tools, tagged , , , , , , on March 4, 2013| 3 Comments »

Nanotechnology and Heart Disease

Author and Curator:  Tilda Barliya PhD

Cardiovascular disease is the most common cause of death worldwide and will become even more prevalent as the population ages. New therapeutic targets are being identified as a result of emerging insights into disease mechanisms, and new strategies are also being tested, possibly leading to new treatment options. Improving diagnosis is also crucial, because by detecting disease early, the focus could be shifted from treatment to prevention (1).

Mortality rates for cardiovascular disease have improved, but there are inequalities across the UK

The World Health Organization estimates that more than 17 million people died from cardiovascular diseases in 2008. In the U.S., about 785,000 people will have new heart attacks this year and 470,000 will suffer recurrent ones. While more patients are surviving such events, about two-thirds don’t make complete recoveries and are vulnerable to heart failure (2).

Heart and vascular disease is the number one killer in most industrialized nations, and costs countries billions in health care, and lost wages. Nanotechnology, biotechnology, robotics, and stem cells are reinvigorating the development of artificial components of the cardiovascular system. We’ve seen hearts grown from stem cells in labs, artificial mechanical hearts, companies spending millions to develop artificial blood, and now even artificial vascular tubes which act more like the real thing. Combined with upcoming advances in robotic and micro-surgery, medicine could be on the path to conquering its public enemy number one.

Nanotechnology offers several tools and advantages in cardiovascular science which are in the areas of diagnosis, imaging, and tissue engineering.

including:

  • treating defective heart valves
  • detecting and treat arterial plaque
  • understanding at a sub-cellular level how heart tissue functions in both healthy  and damaged organs, which can help researchers design better treatments

Examples:

Robert Langer, Omid Farokhzad and colleagues have developed nanoparticles that can cling to artery walls and slowly release medicine, an advance that potentially provides an alternative to drug-releasing stents in some patients with cardiovascular disease. The particles, dubbed “nanoburrs” because they are coated with tiny protein fragments that allow them to stick to target proteins, can be designed to release their drug payload over several days (3, 4). The nanoburrs are targeted to a specific structure, known as the basement membrane, which lines the arterial walls and is only exposed when those walls are damaged. Therefore, the nanoburrs could be used to deliver drugs to treat atherosclerosis and other inflammatory cardiovascular diseases. In the current study, the team used paclitaxel, a drug that inhibits cell division and helps prevent the growth of scar tissue that can clog arteries

Prof. Erkki Ruoslahti and other researchers from UC Santa Barbara have developed a nanoparticle that can attack plaque –– a major cause of cardiovascular disease (5).  These lipid-based micelles target the p32 receptors known to overexpress in plaques. To accomplish the research, the team induced atherosclerotic plaques in mice by keeping them on a high-fat diet. They then intravenously injected these mice with the micelles, which were allowed to circulate for three hours.

Clinical Trials:

“Nanotechnology creates artificial artery for clinical trials”

Researchers at London Royal Free Hospital are hoping to save limbs and lives with the creation of their new artificial artery. Unlike current artery replacements, this grafting substance was created using nanotechnology and can pulse with the natural movements of the body. That pulsing will allow the polymer tube to be used in very small grafts (<8mm), giving hope that damaged arteries which would normally lead to amputations or heart attacks can now be treated (6). The clinical study should have started by the end of 2010. No further information is currently available on this clinical trial.

The new artificial artery material was developed by Professors George Hamilton (vascular surgery) and Alexander Seifalian (nanotechnology and tissue repair). The substance is a polymer which has been embedded with different types of special molecules. Some of these molecules aid circulation, others encourage stem cells to coat its walls. That coating is very important and may allow the artificial tissue to bond better with the body and promote long term health. Most importantly though, the design of the artificial vascular tissue is resistant to clotting and can pulse.

Summary:

Research of heart disease is progressing on several levels simultaniously. It is believed that nanotechnology may offer several advantages in detecting and treating several heart conditions, however, they have yet to progressed into the clinical trials.

Quoting Dr. Tal Dvir: ” Many current experimental approaches to heart attack involve supplying growth factors, drugs, stem cells and other therapeutic agents to the scarred, dying tissue. Some of these compounds, such as periostin and neuregulin, have been shown in animal models to enhance heart regeneration and improve cardiac function. But the existing delivery approaches are all invasive, involving direct injections into the heart, catheter procedures, or surgical placement of implants that release the necessary factors.

The ultimate goal is to have the particles release compounds that promote regeneration. One approach is to release factors that attract the patient’s own stem cells, avoiding the need for tissue-engineered patches. But to date, no one’s gotten stem cells to differentiate efficiently into cardiomyocytes”

REFERENCES

1. http://www.nature.com/nature/supplements/insights/cardiovascular/index.html

2. Novel Cure for Ailing Hearts. http://online.wsj.com/article/SB10000872396390443537404577577002440205144.html

3. Chan JM., Zhang L., Tong R., Ghosh D., Gao W., Liao G., Yuet KP., Gray D., Rhee JW., Cheng J., Golomb G., Libby P, Langer R and Farokhzad OC. Spatiotemporal controlled delivery of nanoparticles to injured vasculature. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2213-8.  http://www.pnas.org/content/107/5/2213.long

4. Chan JM., Rhee JW., Drum CL., Bronson RT., Golomb G., Langer R and Farokhzad OC. In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles. Proc Natl Acad Sci U S A. 2011 Nov 29;108(48):19347-52.

http://www.pnas.org/content/108/48/19347.long

5. Hamzah J., Kotamraju VR., Seo JW., Agemy L., Fogel V., Mahakian LM., Peters D., Roth L., Gagnon MK., Ferrara KW and Ruoslahti E. Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2011 Apr 26;108(17):7154-9. http://www.pnas.org/content/108/17/7154.long

6. Written By: : http://singularityhub.com/2010/01/05/nanotechnology-creates-artificial-artery-for-clinical-trials/

7. Ikaria® Commences Global Registration Trial for Bioabsorbable Cardiac Matrix. http://www.prnewswire.com/news-releases/ikaria-commences-global-registration-trial-for-bioabsorbable-cardiac-matrix-136581753.html.

8. Posted by: Prof. Lev-Ari :”Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel” http://pharmaceuticalintelligence.com/2013/02/27/arteriogenesis-and-cardiac-repair-two-biomaterials-injectable-thymosin-beta4-and-myocardial-matrix-hydrogel/

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DNA Sequencing Technology

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Genome Biology, Genomic Testing: Methodology for Diagnosis, International Global Work in Pharmaceutical, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , on March 3, 2013| 1 Comment »

DNA Sequencing Technology

Reporter: Larry H Bernstein, MD, FCAP

Focus on DNA Sequencing Technology
Nature Biotechnology  feb 2013; 31: 2.

 Editorial
Knocking on the clinic door  Nature Biotechnology 2012; 1009  http://dx.doi.org/10.1038/nbt.2428
The New York Genome Center – pp1021 – 1022  http://dx.doi.org/10.1038/nbt.2429
Direct-to-consumer genomics reinvents itself – pp1027 – 1029
Malorye Allison  By putting its foot in the door at the FDA, can 23andMe reinvigorate direct-to-consumer genomics?

Genomic DNA is fragmented into random pieces a...

Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.(click to expand) (Photo credit: Wikipedia)

Science Fellows

Science Fellows (Photo credit: AlphachimpStudio)

English: Created by Abizar Lakdawalla.

English: Created by Abizar Lakdawalla. (Photo credit: Wikipedia)

Related articles

 

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Virtual Biopsy – is it possible?

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Chemical Biology and its relations to Metabolic Disease, Ecosystems & Industrial Concentration in the Medical Device Sector, Health Economics and Outcomes Research, Imaging-based Cancer Patient Management, Medical Devices R&D and Inventions, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Personalized and Precision Medicine & Genomic Research, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , on March 3, 2013| 7 Comments »

Virtual Biopsy – is it possible?

Author and Curator: Dror Nir, PhD

Article 11.5.3 Virtual Biopsy is it possible

In a remark made to my last post: New envelopment in measuring mechanical properties of tissue, Dr. Aviva Lev-Ari, PhD, RN, Director and Founder of our Open Access Online Scientific Journal:  Leaders of Pharmaceutical Business Intelligence, asked whether OCT can be used for the purpose of performing biopsy. My answer to her question was “YES”. I thought that it will be worthwhile explaining why I am so “optimistic” about this:

A conventional biopsy is a process where a tissue sample is being cut out of the body and after being subjected to all kind of chemical processes a thin-film of tissue is trimmed and read under the microscope by a trained pathologist. Can imaging provide histological assessment of “thin-film” of tissue without cutting it out of the body? The answer would be positive if the imaging will result with high resolution reconstruction of a tissue sample identical in quality to a “live-sample” that is put under the microscope.

I was happy to find support to my optimism regarding the feasibility of constructing such device in the following article: Virtual skin biopsy by optical coherence tomography: the first quantitative imaging biomarker for scleroderma published on February 20th 2013 in Ann Rheum Dis doi:10.1136/annrheumdis-2012-202682

 This article reports an original, first study to perform histological comparison and explore Optical coherence tomography (“OCT”) as a potential imaging technique for the clinical assessment of patients presenting with systemic sclerosis (“SSc”). In their study the investigators used a device emitting low-intensity infrared laser beam, capable of producing high-contrast images of skin up to 2 mm deep with resolutions of 4–10 μm.

[START ORIGINAL PAPER]

ABSTRACT

Background

Skin involvement is of major prognostic value in systemic sclerosis (SSc) and often the primary outcome in clinical trials. Nevertheless, an objective, validated biomarker of skin fibrosis is lacking. Optical coherence tomography (OCT) is an imaging technology providing high-contrast images with 4 μm resolution, comparable with microscopy (‘virtual biopsy’). The present study evaluated OCT to detect and quantify skin fibrosis in SSc.

Methods

We performed 458 OCT scans of hands and forearms on 21 SSc patients and 22 healthy controls. We compared the findings with histology from three skin biopsies and by correlation with clinical assessment of the skin. We calculated the optical density (OD) of the OCT images employing Matlab software and performed statistical analysis of the results, including intraobserver/ interobserver reliability, employing SPSS software.

 Results

Comparison of OCT images with skin histology indicated a progressive loss of visualisation of the dermal–epidermal junction associated with dermal fibrosis. Furthermore, SSc affected skin showed a consistent decrease of OD in the papillary dermis, progressively worse in patients with worse modified Rodnan skin score (p<0.0001). Additionally, clinically unaffected skin was also distinguishable from healthy skin for its specific pattern of OD decrease in the reticular dermis (p<0.001). The technique showed an excellent intraobserver and interobserver reliability (intraclass correlation coefficient >0.8).

Conclusions

OCT of the skin could offer a feasible and reliable quantitative outcome measure in SSc. Studies determining OCT sensitivity to change over time and its role in defining skin vasculopathy may pave the way to defining OCT as a valuable imaging biomarker in SSc.

Virtual skin biopsy by OCT

The OCT images acquisition allowed the reconstruction of a virtual skin biopsy measuring 4×0.4×2 mm. The main structure of the healthy skin was easily recognisable by OCT (figure 1).

Virtual biopsy of forearm skin by optical coherence tomography. Representative 3D reconstruction from the tomography of healthy and systemic sclerosis (SSc) (site modified Rodnan skin score=3) skin scans. The keratin of the skin appears as a white line on the surface (k). The epidermis (ED) is quite visible in the healthy skin by the contrast with the increased optical density of the papillary dermis (PD). The dermal– epidermal junction (DEJ) is quite visible in the healthy skin between the ED and PD. On the contrary, neither clear distinction of ED and PD or DEJ is appreciable in the SSc skin. The vessels (*) are numerous and very well recognisable in healthy skin, whereas they appear less numerous and less distinct in the OCT image of SSc skin. Total depth of 3D reconstruction=1.2 mm. Scale bars are calculated by ImageJ.

Virtual biopsy of forearm skin by optical coherence tomography. Representative 3D reconstruction from the tomography of healthy and systemic sclerosis (SSc) (site modified Rodnan skin score=3) skin scans. The keratin of the skin appears as a white line on the surface (k). The epidermis (ED) is quite visible in the healthy skin by the contrast with the increased optical density of the papillary dermis (PD). The dermal– epidermal junction (DEJ) is quite visible in the healthy skin between the ED and PD. On the contrary, neither clear distinction of ED and PD or DEJ is appreciable in the SSc skin. The vessels (*) are numerous and very well recognisable in healthy skin, whereas they appear less numerous and less distinct in the OCT image of SSc skin. Total depth of 3D reconstruction=1.2 mm. Scale bars are calculated by ImageJ.

Some quantitative results  – in images:

Validation of optical coherence tomography (OCT) images by histology. (A and B) H&E staining (A) and corresponding OCT scan (B) from a healthy control (HC). The green line is the mean A-scan of the entire OCT image (100 scans) overlaid by matching the scale bars of OCT and histology. The green arrow indicates the nadir of the valley in the mean A-scan, which corresponds to the dermal–epidermal junction clearly visible on both images. The green arrowhead indicates the second peak of the mean OCT A-Scan which corresponds by the overlay to the most superficial region of the papillary dermis. (C and D) H&E staining (C) and corresponding OCT scan (D) from a systemic sclerosis (SSc) patient (site modified Rodnan skin score =3). The red line is the mean A-scan of the OCT image, overlaid by matching the scale bars in the two panels. The red arrow indicates the nadir in the valley of the mean A-scan, which in this case does not correspond to the dermal–epidermal junction. The red arrowhead corresponds to the second peak in mean A-Scan. (E) Overlay of HC and SSc. Scale bar=240 μm.

Validation of optical coherence tomography (OCT) images by histology. (A and B) H&E staining (A) and corresponding OCT scan (B) from a healthy control (HC). The green line is the mean A-scan of the entire OCT image (100 scans) overlaid by matching the scale bars of OCT and histology. The green arrow indicates the nadir of the valley in the mean A-scan, which corresponds to the dermal–epidermal junction clearly visible on both images. The green arrowhead indicates the second peak of the mean OCT A-Scan which corresponds by the overlay to the most superficial region of the papillary dermis. (C and D) H&E staining (C) and corresponding OCT scan (D) from a systemic sclerosis (SSc) patient (site modified Rodnan skin score =3). The red line is the mean A-scan of the OCT image, overlaid by matching the scale bars in the two panels. The red arrow indicates the nadir in the valley of the mean A-scan, which in this case does not correspond to the dermal–epidermal junction. The red arrowhead corresponds to the second peak in mean A-Scan. (E) Overlay of HC and SSc. Scale bar=240 μm.

Optical coherence tomography (OCT) of affected and not affected skin in plaque morphea. (A) OCT of not affected skin. Vertical scale represents depth in micrometre from the surface. The dermal–epidermal junction (DEJ) level is indicated by the white dotted line. Mean A-scan curve is overlaid and displayed in green. (B) OCT of affected skin in morphea patient. Mean A-scan curve is overlaid and displayed in red. Note the poorly visible DEJ and the valley of the curve below the DEJ (arrowhead). (C) Overlay of mean A-scan curves from the analysis of affected and unaffected skin in a morphea patient. Note that in the curves overlay graph both the difference depth of the first valley is clearly appreciable (arrowheads). Similarly the second mean A-scan peak (arrow) is subtle in the affected skin, similar to scleroderma affected skin.

Optical coherence tomography (OCT) of affected and not affected skin in plaque morphea. (A) OCT of not affected skin. Vertical scale represents depth in micrometre from the surface. The dermal–epidermal junction (DEJ) level is indicated by the white dotted line. Mean A-scan curve is overlaid and displayed in green. (B) OCT of affected skin in morphea patient. Mean A-scan curve is overlaid and displayed in red. Note the poorly visible DEJ and the valley of the curve below the DEJ (arrowhead). (C) Overlay of mean A-scan curves from the analysis of affected and unaffected skin in a morphea patient. Note that in the curves overlay graph both the difference depth of the first valley is clearly appreciable (arrowheads). Similarly the second mean A-scan peak (arrow) is subtle in the affected skin, similar to scleroderma affected skin.

DISCUSSION

The current gold standard for semiquantitative assessment of skin fibrosis, the mRSS, suffers from several shortcomings ranging from the subjectivity of skin palpation assessments and the high level of skill required from the clinical investigator. Even more importantly, a meta-analysis of three independent studies determined an overall within patient interobserver SD of five units independently of the mean skin score,[6 21] which represents an SE ranging from 20% to 26%. A primary outcome measure with 25% of SE entails the recruitment of a large number of patients to attain statistical validity in minimally significant changes, a task often difficult to accomplish given the comparatively low incidence of SSc.

A robust imaging biomarker for the assessment of skin fibrosis in SSc has not previously been reported. Herein we report the first study aimed to validate OCT for the quantitative assessment of skin involvement in SSc.

To date, the limited data on surrogate outcome measures for skin involvement are largely composed of histopathological or molecular changes in affected skin.[22 23] Despite conceptually very valuable, these studies, involving skin biopsies, are invasive and limited because of a site bias, referring to only one precise body area. Moreover, they are difficult to repeat in longitudinal manner and showed no sensitivity to change over time.[24] In this study, we evaluated OCT skin scanning as a reliable and quanti­tative tool that could be used as a surrogate marker of skin fibrosis. The technique requires minimal operator training, less than 10s per site examined, and offers the great advantage of saving image files for further or centralised operator independ­ent analysis. This latter is a particularly useful tool limiting the ‘hands on’ time in the clinic office and allowing a centralised, blinded assessment of results in clinical trials.

We observed an excellent correlation of OCT mean A-Scan curves and mRSS score at the site of analysis. More importantly, the corroboration of our OCT findings with pathological changes at the DEJ provides a robust construct validity for the technique. Of interest, we found that the changes of the OD of the dermis in SSc are similar to the ones observed in a case of plaque morphea, corroborating even further the potential value of OCT in measuring skin fibrosis.

Additional Comment

HFUS (High Frequensy Ultrasound) has been recently suggested to offer a quantitative assessment of skin thickness in SSc by several studies.8–10 In contrast with ultrasound, OCT does not require any use of gels, is able to give a higher resolution images and the analysis algo­rithm is automatic, not involving any operator interpretation. Nevertheless, since the penetration of OCT is limited to the first millimetre of skin, OCT and HFUS may be explored as comple­mentary imaging biomarkers in SSc.

REFERENCES

1     Jimenez SA, Derk CT. Following the molecular pathways toward an understanding
of the pathogenesis of systemic sclerosis. Ann Intern Med 2004;140:37–50.

2     Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder.
J Clin Invest 2007;117:557–67.

3     Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med
2009;360:1989–2003.

4     Clements PJ, Hurwitz EL, Wong WK, et al. Skin thickness score as a predictor and
correlate of outcome in systemic sclerosis: high-dose versus low-dose penicillamine trial. Arthritis Rheum 2000;43:2445–54.

5     Steen VD, Medsger TA Jr. Improvement in skin thickening in systemic sclerosis
associated with improved survival. Arthritis Rheum 2001;44:2828–35.

6     Pope JE, Baron M, Bellamy N, et al. Variability of skin scores and clinical
measurements in scleroderma. J Rheumatol 1995;22:1271–6.

Clements PJ, Lachenbruch PA, Seibold JR, et al. Skin thickness score in systemic

sclerosis: an assessment of interobserver variability in 3 independent studies. J Rheumatol 1993;20:1892–6.

   8     Akesson A, Hesselstrand R, Scheja A, et al. Longitudinal development of skin
involvement and reliability of high frequency ultrasound in systemic sclerosis. Ann Rheum Dis 2004;63:791–6.

   9     Moore TL, Lunt M, McManus B, et al. L. Seventeen-point dermal ultrasound scoring
system—a reliable measure of skin thickness in patients with systemic sclerosis. Rheumatology (Oxford) 2003;42:1559–63.

10     Kaloudi O, Bandinelli F, Filippucci E, et al. High frequency ultrasound

measurement of digital dermal thickness in systemic sclerosis. Ann Rheum Dis 2010;69:1140–3.

11     Aden N, Shiwen X, Aden D, et al. Proteomic analysis of scleroderma lesional skin
reveals activated wound healing phenotype of epidermal cell layer. Rheumatology (Oxford) 2008;47:1754–60.

12     Aden N, Nuttall A, Shiwen X, et al. Epithelial Cells Promote Fibroblast Activation via
IL-1alpha in Systemic Sclerosis. J Invest Dermatol 2010;130:2191–200.

13     Gambichler T, Jaedicke V, Terras S. Optical coherence tomography in dermatology:
technical and clinical aspects. Arch Dermatol Res 2011;303:457–73.

14     Marschall S, Sander B, Mogensen M, et al. Optical coherence tomography-current
technology and applications in clinical and biomedical research. Anal Bioanal Chem 2011;400:2699–720.

15     Coleman AJ, Richardson TJ, Orchard G, et al. Histological correlates of optical
coherence tomography in non-melanoma skin cancer. Skin Res Technol 2013;19: e10–9.

16     Preliminary criteria for the classification of systemic sclerosis (scleroderma).
Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum 1980;23:581–90.

17     Collins TJ. ImageJ for microscopy. Biotechniques 2007;43:25–30.

18     Clendenon JL, Phillips CL, Sandoval RM, et al. Voxx: a PC-based, near real-time

volume rendering system for biological microscopy. Am J Physiol Cell Physiol 2002;282:C213–18.

19     Bland JM, Altman DG. Statistical methods for assessing agreement between two
methods of clinical measurement. Lancet 1986;1:307–10.

20     LeRoy EC, Black C, Fleischmajer R, et al. Scleroderma (systemic sclerosis):
classification, subsets and pathogenesis. J Rheumatol 1988;15:202–5.

21     Merkel PA, Silliman NP, Clements PJ, et al. Patterns and predictors of change in
outcome measures in clinical trials in scleroderma: an individual patient meta-analysis of 629 subjects with diffuse cutaneous systemic sclerosis. Arthritis Rheum 2012;64:3420–9.

22     Farina G, Lafyatis D, Lemaire R, et al. A four-gene biomarker predicts skin disease
in patients with diffuse cutaneous systemic sclerosis. Arthritis Rheum 2010;62:580–8.

23     Milano A, Pendergrass SA, Sargent JL, et al. Molecular subsets in the gene
expression signatures of scleroderma skin. PLoS One 2008;3:e2696.

  24   Pendergrass SA, Lemaire R, Francis IP, et al. Intrinsic gene expression subsets of

diffuse cutaneou

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The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN

Posted in Atherogenic Processes & Pathology, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Frontiers in Cardiology and Cardiovascular Disorders, HTN, Human Immune System in Health and in Disease, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , on February 28, 2013| 14 Comments »

Heart Vasculature – Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle: A Concept-based Pharmacological Therapy of a Combination Three Drug Regimen including THYMOSIN 

Author & Curator: Aviva Lev-Ari, PhD, RN

 

ABSTRACT

A concept-based original pharmacological therapy was developed for the research results presented in Cell by Wu, Fujiwara, Cibulsky et al. (2006), Moretti, Caron, Nakano, et al. (2006) and for the research results in Nature by Smart, Risebro, Melville, et al. (2007). We propose the following concept-based original pharmacological therapy design for Preoperative and Postoperative management of cardiac injury to heart tissue, smooth muscle, to aorta and coronary artery disease. This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state while restoring its pluripotency.

VIEW VIDEO

What are Induced Pluripotent Stem Cells? (iPS Cells)

 http://www.youtube.com/watch?v=i-QSurQWZo0

Lasker Lecture: Dr. Shinya Yamanaka, 2 of 3:

Induced Pluripotent Stem Cells? (iPS Cells)

http://www.youtube.com/watch?v=DQNoyDwCPzM

Multipotent Embryonic Isl1^+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Alessandra A Moretti, Leslie L Caron, Atsushi A Nakano, Jason T JT Lam, Alexandra A Bernshausen,Yinhong Y Chen, Yibing Y Qyang, Lei L Bu, Mika M Sasaki, Silvia S Martin-Puig, Yunfu Y Sun, Sylvia M SM Evans, Karl-Ludwig KL Laugwitz, Kenneth R KR Chien
Cell 127(6):15 (2006), PMID 17123592

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1^+ precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1^+ cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1^+/Nkx2.5^+/flk1^+ defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1^+ cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

http://pubget.com/paper/17123592/Multipotent_Embryonic_Isl1___Progenitor_Cells_Lead_to_Cardiac__Smooth_Muscle__and_Endothelial_Cell_Diversification

 

Thymosin beta 4 (Tβ4)

is a highly conserved, 43-amino acid acidic peptide (pI 4.6) that was first isolated from bovine thymus tissue over 25 years ago. It is present in most tissues and cell lines and is found in high concentrations in blood platelets, neutrophils, macrophages, and other lymphoid tissues. Tβ4 has numerous physiological functions, the most prominent of which being the regulation of actin polymerization in mammalian nucleated cells and with subsequent effects on actin cytoskeletal organization, necessary for cell motility, organogenesis, and other important cellular events.

Recently,

  • Tβ4 was shown to be expressed in the developing heart and found to stimulate migration of cardiomyocytes and endothelial cells, promote survival of cardiomyocytes (Nature, 2004), and most recently
  • to play an essential role in all key stages of cardiac vessel development: vasculogenesis, angiogenesis, and arteriogenesis (Nature 2006).
  • These results suggest that Tβ4 may have significant therapeutic potential in humans to protect myocardium and promote cardiomyocyte survival in the acute stages of ischemic heart disease.

RegeneRx Biopharmaceuticals, Inc. is developing Tβ4 for the treatment of patients with acute myocardial infarction (AMI). Such efforts presented will include the formulation, development, and manufacture of a suitable drug product for use in the clinic, the performance of nonclinical pharmacology and toxicology studies, and the implementation of a phase 1 clinical protocol to assess the safety, tolerability, and the pharmacokinetics of Tβ4 in healthy volunteers.

http://onlinelibrary.wiley.com/doi/10.1196/annals.1415.051/abstract;jsessionid=BB7CC897572B7DDB60370EA64A81FC3F.d01t03?deniedAccessCustomisedMessage=&userIsAuthenticated=false

EXPLORATIONS with THYMOSIN beta4 for INDUCING ADULT EPICARDIAL PROGENETOR MOBILIZATION AND NEOVASCULARIZATION is presented in

Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

EXPLORATIONS with THYMOSIN beta4 for INDUCTION of ARTERIOGENESIS, Prevention and repair of damaged cardiac tissue post MI and other CVD related research projects are presented in

Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

http://pharmaceuticalintelligence.com/2013/02/27/arteriogenesis-and-cardiac-repair-two-biomaterials-injectable-thymosin-beta4-and-myocardial-matrix-hydrogel/

Recent research results with THYMOSIN beta4 in use for Cardiovascular Disease

appeared in 2010:

Annals of the New York Academy of Sciences, May 2010 Volume 1194 Pages ix–xi, 1–230

http://onlinelibrary.wiley.com/doi/10.1111/nyas.2010.1194.issue-1/issuetoc

appeared in 2012:

  • Thymosins in Health and Disease II: 3rd International Symposium on The Emerging Clinical Applications of Tymosin beta 4 in Cardiovascular Disease

Annals of the New York Academy of Sciences, October 2012 Volume 1270 Pages vii-ix, 1–121.

http://onlinelibrary.wiley.com/doi/10.1111/nyas.2012.1270.issue-1/issuetoc

Allan L. Goldstein, Enrico Garaci, Editors, Thymosins in Cardiovascular Disease, November 2012, Wiley-Blackwell (paperback)

http://www.wiley.com/WileyCDA/WileyTitle/productCd-1573319104.html?cid=RSS_WILEY2_LIFEMED

Selected for this article are the abstracts of the following research projects, all were presented at the 2nd International Symposium, May 2010:

Thymosin β4: structure, function, and biological properties supporting current and future clinical applications

Published studies have described a number of physiological properties and cellular functions of thymosin β4 (Tβ4), the major G-actin-sequestering molecule in mammalian cells. Those activities include the promotion of cell migration, blood vessel formation, cell survival, stem cell differentiation, the modulation of cytokines, chemokines, and specific proteases, the upregulation of matrix molecules and gene expression, and the downregulation of a major nuclear transcription factor. Such properties have provided the scientific rationale for a number of ongoing and planned dermal, corneal, cardiac clinical trials evaluating the tissue protective, regenerative and repair potential of Tβ4, and direction for future clinical applications in the treatment of diseases of the central nervous system, lung inflammatory disease, and sepsis. A special emphasis is placed on the development of Tβ4 in the treatment of patients with ST elevation myocardial infarction in combination with percutaneous coronary intervention, pp.179-189, May 2010.

  

Thymosin β4 and cardiac repair

Hypoxic heart disease is a predominant cause of disability and death worldwide. As adult mammals are incapable of cardiac repair after infarction, the discovery of effective methods to achieve myocardial and vascular regeneration is crucial. Efforts to use stem cells to repopulate damaged tissue are currently limited by technical considerations and restricted cell potential. We discovered that the small, secreted peptide thymosin β4 (Tβ4) could be sufficiently used to inhibit myocardial cell death, stimulate vessel growth, and activate endogenous cardiac progenitors by reminding the adult heart on its embryonic program in vivo. The initiation of epicardial thickening accompanied by increase of myocardial and epicardial progenitors with or without infarction indicate that the reactivation process is independent of injury. Our results demonstrate Tβ4 to be the first known molecule able to initiate simultaneous myocardial and vascular regeneration after systemic administration in vivo. Given our findings, the utility of Tβ4 to heal cardiac injury may hold promise and warrant further investigation, pp. 87-96, May 2010.

 

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans

http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html

After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC-derived endothelial-restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage, pp. 97-104, May 2010.

 

Thymosin β4: a key factor for protective effects of eEPCs in acute and chronic ischemia

Acute myocardial infarction is still one of the leading causes of death in the industrial nations. Even after successful revascularization, myocardial ischemia results in a loss of cardiomyocytes and scar formation. Embryonic EPCs (eEPCs), retroinfused into the ischemic region of the pig heart, provided rapid paracrine benefit to acute and chronic ischemia in a PI-3K/Akt-dependent manner. In a model of acute myocardial ischemia, infarct size and loss of regional myocardial function decreased after eEPC application, unless cell pre-treatment with thymosin β4 shRNA was performed. Thymosin ß4 peptide retroinfusion mimicked the eEPC-derived improvement of infarct size and myocardial function. In chronic ischemia (rabbit model), eEPCs retroinfused into the ischemic hindlimb enhanced capillary density, collateral growth, and perfusion. Therapeutic neovascularization was absent when thymosin ß4 shRNA was introduced into eEPCs before application. In conclusion, eEPCs are capable of acute and chronic ischemia protection in a thymosin ß4 dependent manner, pp. 105-111, May 2010.

Clinical Study Data of Thymosin beta 4 Presented

Published on October 3, 2009 at 5:10 AM

REGENERX BIOPHARMACEUTICALS, INC. (NYSE Amex:RGN) today reported on several clinical studies with Thymosin beta 4 (Tβ4) presented the Second International Symposium on Thymosins in Health and Disease, in Catania, Italy. The following are synopses of the presentations:

Myocardial Development of RGN-352 (Injectable Tβ4 Peptide)

David Crockford, RegeneRx’s vice president for clinical and regulatory affairs presented an overview of the biological properties that support Tβ4’s near term and long term clinical applications. Mr. Crockford noted that special emphasis is being placed on the development of RGN-352 for the systemic (injectable) treatment of patients with ST-elevation myocardial infarction (STEMI) in combination with percutaneous coronary intervention, the current standard of care in most western countries for this common type of heart attack. The goal with RGN-352 is to prevent or repair continued damage to cardiac tissue post-heart attack, when such tissue around the damaged site remains at risk.

Dr. Dennis Ruff, vice president and medical director of ICON, and principal investigator, presented the most current results on the Phase I safety study with RGN-352 entitled, “A Randomized, Double-blind, Placebo-controlled, Dose-response Phase I Study of the Safety and Tolerability of the Intravenous Administration of Thymosin Beta 4 and its Pharmacokinetics After Single and Multiple Doses in Healthy Volunteers.” Dr. Ruff discussed key aspects of the study and concluded with, “There were no dose limiting or serious adverse events throughout the dosing period. Synthetic Tβ4 administered intravenously up to 1260 mg, and for up to 14 days, appears to be well tolerated with low incidence of adverse events and no evidence of serious adverse events.”

http://www.news-medical.net/news/20091003/Clinical-study-data-of-Thymosin-beta-4-presented.aspx

RegeneRx Receives Notice of Allowance from Chinese Patent Office for Treatment and Prevention of Heart Disease

RegeneRx Receives Notice of Allowance from Chinese Patent Office for Treatment and Prevention of Heart Disease

February 7, 2013 — Rockville, MD

RegeneRx Biopharmaceuticals, Inc. (OTC Bulletin Board: RGRX) (“the Company” or “RegeneRx”) today announced that it has received a Notice of Allowance of a Chinese patent application for uses of Thymosin beta 4 (TB4) for treating, preventing, inhibiting or reducing heart tissue deterioration, injury or damage in a subject with heart failure disease. Claims also include uses for restoring heart tissue in those subjects. The patent will expire July 26, 2026 http://www.regenerx.com/wt/page/pr_1360265259

Theoretical treatment protocol differential between the Preoperative which may be between 3 to 6 month, and the Postoperative which may prolong to one year.

Proposal for Preoperative Treatment – Three drug combination involves

  • Drug # 1: Thymosin fraction 5 (a sublingual composition)
  • Drug # 2: Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID))
  • Drug # 3: Clevidipine (blood pressure lowering drug, (no effect on heart rate))

 

Proposal for Postoperative Treatment – Three drug combination consists of

  • Drug # 1: Thymosin fraction 5 (a sublingual composition)
  • Drug # 4: ACEI (Captopril (50mg))
  • Drug # 5: Beta Blocker and Diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor HCT

Unprecedented novel paradigm development in the scientific understanding of the origin of

  • (a) myocardial cells
  • (b) smooth muscle cells
  • (c) endothelial cells
  • (d) pace maker cells and
  • (e) heart vasculature: aorta, pulmonary artery and coronary arteries, occurred in 2006.

In a seminal article in Cell, “Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart” Wu, et al., (2006), described their discovery as follows:

“Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood.” To explore the relationship between developmental fate and potential.” They “isolated a cardiac-specific Nkx2.5+ cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5+ cells from in vitro differentiated murine embryonic stem cells and found ~28% of these cells expressed c-kit. These c-kit+ cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell.” They “confirmed these findings by isolating c-kit+Nkx2.5+ cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.”

Another breakthrough article in Cell, “Multipotent Embryonic Isl1+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification” Moretti, et al., (2006) described their discovery as follows:

“Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors.” They “use genetic fate-mapping studies to document that isl1+ precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells”, they “clonally amplified a cellular hierarchy of isl1+ cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1+/Nkx2.5+/flk1+ defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1+ cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.” (Moretti, et al., 2006).

Third scientific breakthrough was reported in Nature on the roles that Thymosin beta4 play in

  • (a) coronary vessel development
  • (b) induction of adult epicardial cell migration
  • (c) cardiomyocyte survival by vascularization which is dependent on Thymosin beta4 and
  • (d) identification of the pro-angiogenic tetrapeptide AcSDKP which is produced by endoproteinase activity of Thymosin beta4 (Smart, et al., 2007).

That new level of understanding has the potential to generate new pharmaco therapies to upregulate biological processes that underlie the function of the various compartments of the cardiovascular system, as new scientific explanations became available in 2006.

We have developed a methodology for discovery of concept-based original pharmacological therapy designs for combination of several drug regimens. We carry out two types of research strategy. Methodology Strategy Type One: we develop an original pharmacological therapy design specialized in addressing medical problems identified in targeted follow up studies on mortality and morbidity of cardiovascular patients. Methodology Strategy Type One is implemented in Lev-Ari & Abourjaily (2006a, 2006b, 2006c). We designed a specialized pharmaco therapy for the research results presented in NEJM, on “Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes” (Werner, Kosiol, Schiegl, et al., 2005a) and the editorial interpretation of these research results by Rosenzweig  (2005). We proposed the following concept-based original pharmacological therapy design for Endogenous Augmentation of circulating Endothelial Progenitor Cells for Reduction of Risk for Macrovascular Cardiac Events.

 

Proposal of Treatment – Three drug combination

  • Inhibition of ET-1, ETA and ETA-ETB (Bosentan)
  • Induction of NO production and stimulation of eNOS (Nebivolol)
  • Stimulation of PPAR-gamma (substitute to Rosiglitazone)

Our Methodology Strategy Type Two involves discovery of concept-based original pharmacological therapy design for combination of several drug regimens for underlying biological processes discovered in the pursuit of basic researchers conducted in wet lab experiments by vascular biologists and molecular cardiologists. Here, we developed a concept-based original pharmacological therapy for the research results presented in Cell by Wu, Fujiwara, Cibulsky et al. (2006), Moretti, Caron, Nakano, et al. (2006) and for the research results in Nature by Smart, Risebro, Melville, et al. (2007). We propose the following concept-based original pharmacological therapy design for Preoperative and Postoperative management of cardiac injury to heart tissue, smooth muscle and to aorta and coronary artery disease. This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency.

 

Proposal for Preoperative Treatment – Three drug combination

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 2:

Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID))

  • Drug # 3:

Clevidipine (blood pressure lowering drug, no effect on heart rate)

Proposal for Postoperative Treatment – Three drugs combination

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 4:

ACEI (Captopril (50mg))

  • Drug # 5:

HCTBeta Blocker and Diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor

 

Thymosin beta4 Induces Adult Epicardial Progenitor Mobilization and Neovascularization

 

Smart et al. (2007) implicate Thymosine beta4 (Tb4) with the following functions: (a) Tb4 in regulating all three key stages of cardiac vessel development: coronary vasculogenesis, angiogenesis and arteriogenesis – collateral growth; (b) identify the adult epicardium as a potential source of vascular progenitors which, when stimulated by Tb4, migrate and differentiate into smooth muscle and endothelial cells; (c) the ability of Tb4 to promote coronary vascularization both during development and in the adult, enhances cardiomyocyte survival and contributes significantly towards Tb4-induced cardioprotection.

The reaction in the scientific community to these investigative results was most favorable.

“These results are very exciting because most humans suffering from ischemic cardiac events, either acutely or chronically, do not develop the collateral vessel growth necessary to preserve and restore heart tissue. If, in humans, we see the same effects as seen in mice, TB4 would be the first drug to prevent loss of (heart) muscle cells and restore blood flow in this manner and provide a new and much needed treatment modality for these patients,”

commented Deepak Srivastava, M.D., Professor and Director, Gladstone Institute of Cardiovascular Disease, University of California San Francisco, CA. Dr. Srivastava and his colleagues published the first paper on TB4’s effects on myocardial infarction in Nature in November 2004.

http://phx.corporate-ir.net/phoenix.zhtml?c=144396&p=irol-newsArticle&ID=932573&highlight=

VIEW VIDEO

http://www.youtube.com/watch?v=Vjj7LSuSMAo

 

Review of the Chemistry and the Mechanism of action supporting the process by which, N-acetyl-seryl-aspartyl-lysyl- proline (Ac-SDKP) stimulates endothelial cell differentiation from adult epicardium, is presented in

Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

A Concept-based Pharmacologic Therapy of a Combined Three Drug Regimen for Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle.

This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency.

 

Preoperative Treatment – Three drugs

  • Drug # 1:
  • Thymosin fraction 5 (a sublingual composition)
  • Drug # 2:
  • Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID)) (25 mg PO bid)
  • Drug # 3:
  • Clevidipine (Blood pressure lowering drug, no effect on heart rate)

 

Postoperative Treatment – Three drugs

  • Drug # 1:
  • Thymosin fraction 5 (a sublingual composition)
  • Drug # 4:
  • ACEI (Captopril (50mg))
  • Drug # 5:
  • Beta Blocker and diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor HCT

Original Drug Therapy Combination Proposed

Drug # 1: Thymosin fraction 5

Drug # 2: Indomethacin

Drug # 3: Clevidipine

Drug # 1:

Sublingual compositions comprising Thymosin fraction 5

United States Patent:  6,733,791

http://www.pharmcast.com/Patents100/Yr2004/May2004/051104/6733791_Sublingual051104.htm

http://www.google.com/patents/US6733791

The compositions comprise a room temperature stable peptide or complex of peptides that may be administered in a dosage of between 0.0001 mg/ml or gm and 600 mg/ml or gm.

Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen (Huff et al. 2002). They suggest that Thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is co-released from stimulated platelets. This provides a mechanism to increase the local concentration of Thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses (Al-Nedawi, et al., 2004). The beta-Thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of Thymosin beta4. For example, Thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. They show that Thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, Thymosin beta10 (Huff et al. 2002).

Rationale for selection of Sublingual compositions comprising Thymosin fraction 5

The actin binding motif of Thymosin beta4 is an essential site for its angiogenic activity (Philip, et al. (2003). Thymosin beta4 is presented in Smart, et al. (2007) in the Nature article as a single factor that can potentially couple myocardial and coronary vascular regeneration in failing mouse hearts. They have shown that cells in the heart’s outer layer can migrate deeper into a failing organ to carry out essential repairs. The migration of progenitor cells is controlled by the protein Thymosin beta 4, already known to help reduce muscle cell loss after a heart attack.

http://news.bbc.co.uk/2/hi/health/6143286.stm

The discovery opens up the possibility of using the protein to develop more effective treatments for heart disease. Previously it was thought that cells within the adult heart are in a state of permanent rest and that any progenitor cells that can contribute to heart tissue repair travel into the heart from the bone marrow. See 150 references on that perspective on cEPCs origin and roles, which was the scientific frontier on this topic, prior to the publication of Smart et al., (2007), in Lev-Ari & Abourjaily (2006a, 2006b, 2006c).

However, researchers at University College London have demonstrated that beneficial cells actually reside in the heart itself (Smart et al. (2007). This approach would bypass the risk of immune system rejection, a major problem with the use of stem cell transplants from another source. Allogenic rejection was the main reason for the selection of an endogenous augmentation method for cEPCs using drug therapy by Lev-Ari & Abourjaily  (2006a, 2006b, 2006c). Closer examination revealed that without the Thymosin beta 4 protein, the progenitor cells failed to move deeper into the heart and change the cells needed to build healthy blood vessels and sustain muscle tissue.

http://www.irishhealth.com/clin/cholesterol/newsstory.php?id=10581

Drug # 2:

Indomethacin

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory effects and have been shown to have chemopreventive effects as well. NSAIDs inhibit cyclooxygenase (COX) activity to exert their anti-inflammatory effects, but it is not clear whether their antitumorigenic ability is through COX inhibition. Using subtractive hybridization, Jain et al. (2004) identified a novel member of the transforming growth factor- superfamily that has antitumorigenic activity from Indomethacin-treated HCT-116 human colorectal cancer cells. On further investigation of this library, they now report the identification of a new cDNA corresponding to the Thymosin beta-4 gene. Thymosin beta-4 is a small peptide that is known for its actin-sequestering function, and it is associated with the induction of angiogenesis, accelerated wound healing, and metastatic potential of tumor cells. However, only selective NSAIDs induce Thymosin beta-4 expression in a time- and concentration-dependent manner. For example,

Indomethacin and SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole] induce Thymosin beta-4 expression whereas sulindac sulfide does not.

They show that selective NSAIDs induce actin cytoskeletal reorganization, a precursory step to many dynamic processes regulating growth and motility including tumorigenesis. This is the first report to link Thymosin beta-4 induction with NSAIDs. These data suggest that NSAIDs alter the expression of a diverse number of genes and provide new insights into the chemopreventive and biological activity of these drugs (Jain et al. 2004).

Rationale for Indomethacin selection

 

Inhibitor of prostaglandin synthesis. Inhibits cyclooxygenase (COX) 1 selective.

Suggested dosage: 25 mg PO bid.

Jain et al. (2004) report a link between Thymosin beta-4 induction with NSAIDs. We selected both drugs (drug classes) and anticipate strong synergistic therapeutic effects.

Drug # 3:

Clevidipine

Clevidipine is the first third-generation calcium channel blocker, Dr. Papadakos said. It has what he called an “ultrashort” clinically relevant half-life of about one minute and then is rapidly metabolized. The effect on blood pressure is seen within one to two minutes.

http://www.medpagetoday.com/MeetingCoverage/SCCM/tb/5091

Clevidipine is an investigational agent undergoing late-stage clinical development to evaluate its potential as an innovative, targeted, fast acting intravenous product under investigation for lowering blood pressure before, during and after surgery.

http://www.themedicinescompany.com/products_Clevidipine.shtml

The Medicines Company entered into agreements with AstraZeneca PLC in March of 2002 for the development, licensing and commercialization of Clevidipine. If approved, the product could be an excellent fit with The Medicines Company’s emerging acute cardiovascular care franchise, which is led by Angiomax® (bivalirudin), an anticoagulant approved in the U.S. and other countries for use during coronary angioplasty procedures. If Clevidipine passes further clinical hurdles — phase III trials are under way — the drug may form a useful addition to the medications available to physicians in the perioperative setting

Mechanism of Action

Clevidipine belongs to a well-known class of drugs called dihydropyridine calcium channel antagonists. In vitro studies demonstrated that Clevidipine acts by selectively relaxing the smooth muscle cells that line small arteries, resulting in widening of the artery opening and reducing blood pressure within the artery (Levy, Huraux, Nordlander, 1997, 345-358).

Phase III Clinical Trials

The Medicines Company is currently sponsoring a Phase III clinical program of five studies to evaluate safety and efficacy of Clevidipine:

Early Development

The Medicines Company’s development program for Clevidipine follows upon the data sets generated by AstraZeneca, which completed clinical pharmacology, dose-finding and efficacy studies in almost 300 patients or volunteers. In clinical studies, Clevidipine has shown to provide the desired blood pressure lowering effect without causing an increase in heart rate (Kotrly, et al. 1984). Further studies demonstrate that reductions in blood pressure are dose-dependent, are not associated with an increase in heart rate and cease rapidly after stopping Clevidipine infusions (Ericsson, et al., 2000), (Schwieler, et al., 1999). In clinical studies Clevidipine was rapidly metabolized independent of the liver and the kidneys, allowing rapid clearance of the drug from the bloodstream (Ericsson, et al., 1999a), (Ericsson, et al., 1999b). Therefore, the effects of Clevidipine are short-lived, which translates into a rapid cessation of its effect on reducing blood pressure.

The two efficacy studies are known as ESCAPE-1 and ESCAPE-2. The primary objective of these studies is to determine the efficacy of Clevidipine injection versus placebo in treating pre-operative (ESCAPE-1) and post-operative (ESCAPE-2) high blood pressure. Three safety studies are collectively known as ECLIPSE. The primary objective is to establish the safety of Clevidipine in the treatment of perioperative high blood pressure, as measured by a comparison of the incidences of death, stroke, myocardial infarction and renal dysfunction between the Clevidipine and comparative treatment groups. The comparative treatments are nitroglycerin, sodium nitroprusside and nicardipine.  The ECLIPSE trial randomized 589 patients at 40 centers in the U.S. to get either sodium nitroprusside or Clevidipine. Sodium nitroprusside was administered according to institutional practice; Clevidipine was begun at 2 mg/kg and doubled every 90 seconds until blood pressure was lowered. The primary endpoint was the difference in major clinical events — death, myocardial infarction, stroke, and renal dysfunction 30 days after surgery. The secondary endpoint was blood pressure control during the first 24 hours after surgery.

The study showed no significant differences in the elements of the primary endpoint, except for mortality, Dr. Papadakos said, where 1.7% of Clevidipine patients died, compared with 4.7 of those getting sodium nitroprusside.  The difference was statistically significant at P<0.05, but Dr. Papadakos characterized the improvement as “slight.” On the other hand, the drug did show an important difference in blood pressure control over the first 24 hours, he said:

  • Patients on Clevidipine spent an average of 4.37 minutes per hour outside the desired blood pressure range.
  • Sodium nitroprusside patients spent, on average, 10.5 minutes per hour outside the desired range.
  • The difference was statistically significant at P<0.003.

Dr. Papadakos concluded that Clevidipine is a new drug that is effective, safe, and easy to use. On 2/20/2007, Dr. Deutschman, who moderated the late-breaking session at which Dr. Papadakos spoke, said that a better comparison, would be intravenous nicardipine (Cardene IV), a second-generation calcium channel blocker that is also in wide use and is considered the standard of care. “We don’t know yet if this drug is going to be better than nicardipine,” he said.

http://www.medpagetoday.com/MeetingCoverage/SCCM/tb/5091

Rationale for Clevidipine selection

Clevidipine is an acute care product. Blood pressure management is a major component of care during the 13.4 million inpatient surgeries conducted in the U.S. each year. Blood pressure control, which is managed by an anesthesiologist, is often important in patients with both normal and high blood pressure undergoing surgery or other interventional procedures. Some of these patients require rapid, precise control of blood pressure to avoid compromising key organ function such as the heart, brain and kidney.

CONCLUSION 

This is the first study to design a novel combination drug treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency. This treatment is based on the new three paradigms that were presented in Cell (2006) and Nature (2007). This combination drug therapy of three drugs, one in current use (Indomethacin), and two in clinical trials (Thymosin beta4 & Clevidipine), has not been proposed before. It represents an original concept drug combination design by Lev-Ari & Abourjaily (2007). This combination represents the cutting edge conceptualization of the field of treatment of cardiac injury based on a protein produced in the heart cells, Thymosin beta4, which function as a tissue and artery healer. Its upregulation by drug therapy will revolutionize cardiology and treatment for cardiovascular disease. The combination drug therapy consists of the following drugs:

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 2:

Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID)) (25 mg PO bid)

  • Drug # 3:

Clevidipine (Blood pressure lowering drug, (no effect on heart rate))

 

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Protein Discovered That Can Tell Human Heart to Heal Itself

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Retrieved 3/1/2007

http://www.pharmcast.com/Patents100/Yr2004/May2004/051104/6733791_Sublingual051104.htm

 

TMSB4X  Thymosin, beta 4, X-linked

Retrieved on 3/1/2007

http://www.ihop-net.org/UniPub/iHOP/gs/92756.html

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Wang, D., Oscar A. Carretero, O.A.,Yang, X-Y., Rhaleb, N-E., Liu, Y-H., Liao, T-D., Yang, X-P. (2004). “N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo.” Am J Physiol Heart Circ Physiol., 287, H2099-H2105.

Werner N, Junk S, Laufs L, Link A, Walenta K, Bohm M, Nickenig G., (2003).  Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res., 93, e17– e24.

Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Böhm M, Nickenig G. (2005a). Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes, NEJM, 353, 999-1007

Werner, N. & Nickenig, G. (2005b). Authors Reply to Correspondence to the Editor on Circulating Endothelial Progenitor Cells. NEJM, 353 (24), 2613-2616

Wu, S.M., Fujiwara, Y., Cibulsky, S.M., Clapham, D.E., Lien, C., Schultheiss, T.M., Orkin, S.H. (2006, December). “Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart.” Cell, 127, 1137-1150.

Other related articles on this Open Access Online Scientific Journal, include the following:

Saha, S. (2012b) Innovations in Bio instrumentation for Measurement of Circulating Progenetor Endothelial Cells in Human Blood.
http://pharmaceuticalintelligence.com/2012/07/08/innovations-in-bio-instrumentation-for-measurement-of-circulating-progenitor-endothelial-cells-in-human-blood/

 

Saha, S. (2012c) Endothelial Differentiation and Morphogenesis of Cardiac Precursor
http://pharmaceuticalintelligence.com/2012/07/17/endothelial-differentiation-and-morphogenesis-of-cardiac-precursors/

Saha, S. (2012e). Human Embryonic-Derived Cardiac Progenitor Cells for Myocardial Repair

http://pharmaceuticalintelligence.com/2012/08/01/human-embryonic-derived-cardiac-progenitor-cells-for-myocardial-repair/

Lev-Ari, A. 12/29/2012. Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles

http://pharmaceuticalintelligence.com/2012/12/29/coronary-artery-disease-in-symptomatic-patients-referred-for-coronary-angiography-predicted-by-serum-protein-profiles/

 

Bernstein, HL and Lev-Ari, A. 11/28/2012. Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

http://pharmaceuticalintelligence.com/2012/11/28/special-considerations-in-blood-lipoproteins-viscosity-assessment-and-treatment/

 

Lev-Ari, A. 11/13/2012 Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

http://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

 

Lev-Ari, A. 10/19/2012 Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

http://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

 

Lev-Ari, A. 10/4/2012 Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

 

Lev-Ari, A. 10/4/2012 Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

 

Lev-Ari, A. 8/28/2012 Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

http://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/

 

Lev-Ari, A. 8/27/2012 Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

http://pharmaceuticalintelligence.com/2012/08/27/endothelial-dysfunction-diminished-availability-of-cepcs-increasing-cvd-risk-for-macrovascular-disease-therapeutic-potential-of-cepcs/

 

Lev-Ari, A. 8/24/2012 Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

http://pharmaceuticalintelligence.com/2012/08/24/vascular-medicine-and-biology-classification-of-fast-acting-therapy-for-patients-at-high-risk-for-macrovascular-events-macrovascular-disease-therapeutic-potential-of-cepcs/

 

Lev-Ari, A. 7/30/2012 Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-intellectual-property-creation-and-protection-by-pioneer-and-by-biosimilar-manufacturers/

 

Lev-Ari, A. 7/29/2012 Biosimilars: Financials 2012 vs. 2008

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-financials-2012-vs-2008/

 

Lev-Ari, A. 7/29/2012 Biosimilars: CMC Issues and Regulatory Requirements

http://pharmaceuticalintelligence.com/2012/07/29/biosimilars-cmc-issues-and-regulatory-requirements/

 

Lev-Ari, A. 7/19/2012 Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

 

Lev-Ari, A. 4/30/2012 Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

Lev-Ari, A. 5/29/2012 Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

http://pharmaceuticalintelligence.com/2012/05/29/445/

 

Lev-Ari, A. 7/2/2012 Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

 

Lev-Ari, A. 7/9/2012 Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “powerhouse of the cell”

http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

 

Lev-Ari, A. 7/16/2012 Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Proginetor Cells endogenous augmentation

http://pharmaceuticalintelligence.com/2012/07/16/bystolics-generic-nebivolol-positive-effect-on-circulating-endothilial-progrnetor-cells-endogenous-augmentation/

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What is the Future for Genomics in Clinical Medicine?

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Infectious Disease & New Antibiotic Targets, International Global Work in Pharmaceutical, Interviews with Scientific Leaders, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , on February 17, 2013| 5 Comments »

What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

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WordCloud Image Produced by Adam Tubman

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.

Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Related articles

Other Related articles on this Open Access Online Scientific Journal, include the following:

http://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

http://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

http://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

http://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

http://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

http://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

http://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

http://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

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“Thymosin alpha1 and melanoma”

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, BioSimilars, CANCER BIOLOGY & Innovations in Cancer Therapy, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Health Economics and Outcomes Research, Human Immune System in Health and in Disease, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, tagged , , , , , , , , , , on February 15, 2013| 2 Comments »

Author, Editor: Tilda Barliya, PhD

Screen Shot 2021-07-19 at 7.36.50 PM

Word Cloud By Danielle Smolyar

Although melanoma accounts for only 4 percent of all dermatologic cancers, it is responsible for 80 percent of deaths from skin cancer; only 14 percent of patients with metastatic melanoma survive for five years (1). The incidence of melanoma is increasing worldwide, with a growing fraction of patients with advanced disease for which prognosis remains poor despite advances in the field (2). Treatment options are limited despite advances in immunotherapy and targeted therapy. For patients with surgically resected, thick (≥2 mm) primary melanoma with or without regional lymph node metastases, the only effective adjuvant therapy is interferon-α (IFN-α). However, because of the limited benefit upon disease-free survival and the smaller potential improvement of overall survival, the indication for IFN-α treatment remains controversial (2). A better understanding of melanoma immunosurveillance is therefore essential to enable the design of better, targeted melanoma therapies (4).

Risk factors (2):

  • Family history of melanoma, multiple benign or atypical nevi, and a previous melanoma
  • Immunosuppression
  • Sun sensitivity
  • Exposure to ultraviolet radiation

Each of these risk factors corresponds to a genetic predisposition or an environmental stressor that contributes to the genesis of melanoma and each factor is understood to various degrees at a molecular level. The Clark model of the progression of melanoma emphasizes the stepwise transformation of melanocytes to melanoma. The model depicts the proliferation of melanocytes in the process of forming nevi and the subsequent development of dysplasia, hyperplasia, invasion, and metastasis.

 

This Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene.

The choice of target antigens is key to the success of tumour vaccination or tumour immunotherapy. Melanoma candidate antigens include: (A) mutated or aberrantly expressed molecules (e.g. CDK4, MUM-1, beta-catenin) (B) cancer/testis antigens (e.g. MAGE, BAGE and GAGE) and (C) melanoma- associated antigens (MAA).

MAAs are self-antigens normally expressed during the differentiation of melanocytes and play a role in different enzymatic steps of melanogenesis. However, in transformed melanocytes (melanoma cells), MAAs are often overexpressed (4).

The main MAAs are tyrosinase, an enzyme that catalyses the production of melanin from tyrosine by oxidation, the tyrosinase-related proteins (TRP-1) and 2 (TRP-2), the glycoprotein (gp)100 (silver-gene) and MelanA/MART. It is thought that the specialized cell biology of melanin synthesis may favour the loading of MAA peptides into the antigen presentation pathway. 50% of melanoma patients have tumour-infiltrating lymphocytes (TILs) recognising tyrosinase and Melan A, indicating that these antigens are important in the natural melanoma immunosurveillance. Moreover, MAAs are well characterized in mice and humans, allowing the development of tetramers to detect antigen-specific immune responses.

Tα1 Mechanism of action

Tα1, a 28 amino acid peptide of ∼3.1 kDa, is endogenously produced by the thymus gland by the cleavage of its precursor pro-Ta1.  Although the fine immunologic mechanism(s) of action of T1 have not fully been elucidated, experimental evidence points to its strong immunomodulatory properties. In particular, it was reported that Ta1 enhances T cell–mediated immune responses by several mechanisms, including increased T cell production (i.e., CD4+, CD8+, and CD3+ cells), stimulation of T cell differentiation and/or maturation, reduction of T cell apoptosis, and restoration of T cell–mediated antibody production (5).

Furthermore, it was demonstrated that T1 acts on the immune system by modulating the release of proinflammatory cytokines (i.e., interleukin-2 (IL-2), interferon-gamma (IFN-)),12–14 and through the activation of natural killer and dendritic cells.12 In addition, T1 was also demonstrated to have direct effects on cancer cells by increasing the levels of expression of different tumor antigens and of components of the major histocompatibility complex class I, as well as by reducing cancer cell growth.

Together, these experimental findings bear relevance for cancer immunotherapy and suggest that T1 can activate innate and adaptive immune responses and modulate the immunophenotype of cancer cells, improving their immunogenicity and their recognition by the immune system.

Danielli R and colleagues have very nicely outlined the use of the Thymosin a1 in the clinical setting for treating melanoma (5) titled :”Thymosin a1 in melanoma: from the clinical trial setting to the daily practice and beyond”.  A large body of available preclinical in vitro and in vivo evidence points to thymosin alpha 1 (Ta1) as a useful immunomodulatory peptide,with significant therapeutic potential in metastatic melanoma in the absence of clinically meaningful toxicity.  The results emerging frominitial trials provide support of the ability of T1 to improve the clinical outcome of advanced melanoma patients through the activation of the immune system.

Ta1 and Clinical Trials in Melanoma

A large scale, randomized, phase II study was conducted at 64 European centers between 2002 and 2006 to investigate the efficacy of Ta1 administered in combination with DTIC (Dacarbazine) or with DTIC + IFNa, versus only DTIC + IFNa, in 488 previously untreated patients with cutaneous metastatic melanoma. The study was designed to evaluate the ability of Ta1 to potentiate the therapeutic efficacy of DTIC.

Patients were randomly assigned to five treatment groups: DTIC + IFNa and 1.6 mg of Ta1; DTIC + IFNa and 3.2 mg of T1; DTIC + IFN-a and 6.4 mg of Ta1; DTIC + 3.2 mg of Ta1; and DTIC + IFNa

Results:

The clinical rate (CBR), defined as the proportion of patients with a complete response, partial response, or stable disease, was significantly higher in patients who received Ta1 + DTIC than in those who received control therapy. Results in patients who received T1 (all groups combined) compared with those who received the control treatment

  • Improved progression-free survival (hazard ratio (HR): 0.80;
  • 95%confidence interval (CI): 0.63–1.01; P = 0.06) and
  • OS (median: 9.4 vs. 6.6 months)

These outcomes suggested to addition of Thymosin a1 to the treatment resulted in the reduction in the risk of mortality and disease progression in patients with metastatic malignant melanoma, and pointed to a poor effect of IFN- in the combination. More so, the poor results of the IFN group is not surprising due to the limited therapeutic activity of IFN observed in phase III clinical trials.

This study however have some limitations as standard assessment criteria, such as RECIST and WHO indications,  conventionally applied to cytotoxic agents, do not adequately capture some patterns of response observed in the course of immunotherapy; stemming from these considerations, immune-related response criteria (irRC) were developed to measure primary and secondary endpoints in immunotherapy clinical trials.

Therefore the above study might underestimate the therapeutic efficacy of Thymosin a1 since irRC criteria were not used.

In Summary:

A large scale phase III clinical trial should be designed to further explore the therapeutic activity of Thymosine a1 in melanoma patients with defined endpoints and irRC criteria. Moreover, combination studies should explore the activity of T1 in association with other approved agents, such as ipilimumab and vemurafenib or as maintenance therapy in melanoma patients who experience clinical benefit after treatment with these agents.

Also, because of the pleiotropic immunemechanism(s) of action of T1, including the upregulation of T cell–driven immune responses against specific tumor antigens, priming of immune responses and potentiation of antitumor T cell–mediated immune responses through the activation of Toll-like receptor 9 on dendritic cells, coupling Ta1 to cancer vaccines should be an additional useful therapeutic strategy to pursue. T1 could, in fact, prove helpful in overcoming the limited immunogenicity and the short-lived persistency of adequate immunologic antitumor responses frequently reported as potential causes of failure of therapeutic vaccines.

Ref:

1. Arlo J. Miller, M.D.,., and Martin C. Mihm, Jr. Mechanisms of disease: Melanoma. N Engl J Med 2006 (6); 355:51-65.

http://www.nejm.org.rproxy.tau.ac.il/doi/pdf/10.1056/NEJMra052166

http://www.nejm.org/doi/full/10.1056/NEJMra052166

2. Garbe C., Eigentler TK., Keilholz U., Hauschild A and Kirkwood JM. Systematic review of medical treatment in melanoma: current status and future prospects. Oncologist 2011;16(1):5-24.

http://theoncologist.alphamedpress.org/content/16/1/5.long

3. http://flipper.diff.org/app/items/info/1983

4.  Träger U, Sierro S, Djordjevic G, Bouzo B, Khandwala S, et al. (2012) The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens. PLoS ONE 7(4): e35005. doi:10.1371/journal.pone.0035005.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035005

5. Riccardo Danielli, Ester Fonsatti, Luana Calabr` o, Anna Maria Di Giacomo, and Michele Maio. Thymosin 1 in melanoma: from the clinical trial setting to the daily practice and beyond. Ann. N.Y. Acad. Sci. 1270 (2012) 8–12.

http://www.ncbi.nlm.nih.gov/pubmed/16822996

http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.2012.06757.x/abstract

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