Careers in the Pharmaceutical Sciences
Roshan Tolani asked:
The pharmaceutical sciences have saved millions of lives and improved quality of life by playing an important role in the discovery and development of new drugs and drug therapies. As science and medicine evolve and discoveries are made at an astonishing rate, the pharmaceutical industry continues to generate billions of dollars and employ top researchers and professionals.
With accelerating advances in science and technology, the pharmaceutical industry has entered its most promising period yet for new drug development. Pharmaceutical companies are using new knowledge and techniques to attack the root causes—rather than just the symptoms—of diseases and thus are revolutionizing the ways in which new drugs are discovered and developed.
The Disciplines of the Pharmaceutical Sciences
The pharmaceutical sciences can be broadly categorized according to the following disciplines:
* Drug discovery: This discipline deals with the design and synthesis of new drug molecules and includes medicinal chemistry, combinatorial chemistry, and biotechnology.
* Drug delivery: This discipline deals with designing the forms of drug dosages and their delivery to patients. Those involved in drug delivery work to determine the best concentrations of and schedules for drugs. Sciences related to this field include pharmaceutics, biomaterials, and pharmacokinetics.
* Drug action: This discipline examines the actions of drugs in living systems. Sciences dealing with drug action include molecular biology, pharmacology, pharmacodynamics, toxicology, and biochemistry.
* Clinical sciences: This discipline deals with the use of drugs to treat diseases. Drugs’ properties, such as efficacy, adverse effects, drug-to-drug interactions, and bioavailability, are tested in clinical trials.
* Drug analysis: This discipline deals with the separation, identification, and quantification of components of drugs.
* Cost effectiveness: This discipline deals with the economics of drug usage.
* Regulatory affairs: This discipline deals with the coordination of academia, industry, and regulatory bodies.
Careers in the Pharmaceutical Industry
The research-based pharmaceutical industry is one of the strongest components of the American economy and leads the world in discovering and developing innovative new life-saving medicines.
Almost half of the most important global drugs developed between 1975 and 1994 originated in the U.S. U.S. companies developed 370 new medicines to fight dreaded diseases during this period. In 2000, the market value of the industry was greater than $379 million. The field offers a myriad of opportunities to pharmaceutical scientists.
Pharmaceutical companies employ several hundred thousand professionals in a variety of jobs in the U.S. In view of the demand for well-trained professionals, the earning potential of pharmacists is very high. According to an American Pharmaceutical Association report, pharmacists’ salaries range from around $40,000 to $70,000.
Is a Career in Pharmaceutical Sciences Right for Me?
The pharmaceutical field is a good choice for those who:
* want to work in laboratories
* desire to contribute to the health and well-being of society
* love science and excel in the subject
* enjoy professional challenges
* enjoy finding solutions to medical problems baffling scientific communities
How Can I Become a Pharmaceutical Scientist?
Get an undergraduate or advanced college degree in pharmacy, chemistry, biology, medicine, or a related field. There are many who became pharmaceutical scientists after obtaining degrees in economics, marketing, business, or other non-scientific subjects. To work as a registered pharmacist, one needs to satisfy both national and state licensing requirements. Some states require fulfillment of a certain number of continuing education credits annually to stay abreast of developments in the field.
The pharmaceutical sciences have saved millions of lives and improved quality of life by playing an important role in the discovery and development of new drugs and drug therapies. As science and medicine evolve and discoveries are made at an astonishing rate, the pharmaceutical industry continues to generate billions of dollars and employ top researchers and professionals.
With accelerating advances in science and technology, the pharmaceutical industry has entered its most promising period yet for new drug development. Pharmaceutical companies are using new knowledge and techniques to attack the root causes—rather than just the symptoms—of diseases and thus are revolutionizing the ways in which new drugs are discovered and developed.
The Disciplines of the Pharmaceutical Sciences
The pharmaceutical sciences can be broadly categorized according to the following disciplines:
* Drug discovery: This discipline deals with the design and synthesis of new drug molecules and includes medicinal chemistry, combinatorial chemistry, and biotechnology.
* Drug delivery: This discipline deals with designing the forms of drug dosages and their delivery to patients. Those involved in drug delivery work to determine the best concentrations of and schedules for drugs. Sciences related to this field include pharmaceutics, biomaterials, and pharmacokinetics.
* Drug action: This discipline examines the actions of drugs in living systems. Sciences dealing with drug action include molecular biology, pharmacology, pharmacodynamics, toxicology, and biochemistry.
* Clinical sciences: This discipline deals with the use of drugs to treat diseases. Drugs’ properties, such as efficacy, adverse effects, drug-to-drug interactions, and bioavailability, are tested in clinical trials.
* Drug analysis: This discipline deals with the separation, identification, and quantification of components of drugs.
* Cost effectiveness: This discipline deals with the economics of drug usage.
* Regulatory affairs: This discipline deals with the coordination of academia, industry, and regulatory bodies.
Careers in the Pharmaceutical Industry
The research-based pharmaceutical industry is one of the strongest components of the American economy and leads the world in discovering and developing innovative new life-saving medicines.
Almost half of the most important global drugs developed between 1975 and 1994 originated in the U.S. U.S. companies developed 370 new medicines to fight dreaded diseases during this period. In 2000, the market value of the industry was greater than $379 million. The field offers a myriad of opportunities to pharmaceutical scientists.
Pharmaceutical companies employ several hundred thousand professionals in a variety of jobs in the U.S. In view of the demand for well-trained professionals, the earning potential of pharmacists is very high. According to an American Pharmaceutical Association report, pharmacists’ salaries range from around $40,000 to $70,000.
Is a Career in Pharmaceutical Sciences Right for Me?
The pharmaceutical field is a good choice for those who:
* want to work in laboratories
* desire to contribute to the health and well-being of society
* love science and excel in the subject
* enjoy professional challenges
* enjoy finding solutions to medical problems baffling scientific communities
How Can I Become a Pharmaceutical Scientist?
Get an undergraduate or advanced college degree in pharmacy, chemistry, biology, medicine, or a related field. There are many who became pharmaceutical scientists after obtaining degrees in economics, marketing, business, or other non-scientific subjects. To work as a registered pharmacist, one needs to satisfy both national and state licensing requirements. Some states require fulfillment of a certain number of continuing education credits annually to stay abreast of developments in the field.
Drug Development and Discovery in an Industrial Context
Niels Grønning Rasmussen asked:
Preface
A growing industrial demand for high throughput screening of new molecular entities (NME) has spurred great advances in various divisions of scientific development. Researchers has begun to combine various scientific methods to meet the competitive demands in modern drug development, and as a consequence optimized individual phases of drug development.
Technological improvements should ensure high quality expeditious drug development and a as a consequence a modern “fast track” from drug discovery to marketed product.
These scientific advances should however be viewed in sharp contrast to the increased economical expenditures observed in pharmaceutical companies worldwide. The price from discovery phase to actual marketing is estimated to be in the region of 0.8 billion to 1.7 billion dollars (11) and research has shown that investments in NME’s has increased 55 % over the last five year (12). These economical prospects are paradoxically followed by the fact that there has been observed a mere 7% increase in FDA-approved NME’s from 1993 to 2003. These combined factors are inherently linked to a new paradigm shift in drug development where technological advances outrun the collaborative marketing of new drugs.
This project will present the reader with three established analytical methods in drug development and link these methods with nine analytical properties proposed by M. Valcárel and A. Rios (2). Implementation of these methods in drug development and individual quality standards and guidelines will be presented and discussed and the project will be summarised with concluding remarks.
The nine analytical properties
The analytical properties proposed by M. Valcárel and A. Rios (2) should be regarded as a framework which efficiently introduces the students to the various aspects of analytical chemistry. They are a means of providing a hierarchical approach toward the analytical chemistry curriculum and should ensure an organized introduction to analytical chemistry.
The significance of introducing analytical properties hierarchically to students can be described in terms of the following three groups proposed by the authors. The most important group is the top or capital group. This group is defined by the need for accurate and representative data which in turn is the sole contributor towards the quality of the results.
The second group, the basic properties, ensure the quality of the analytical process and cover the more practical aspects of analytical chemistry. They include such properties as selectivity, precision, sensitivity and proper sampling. They are properties which during the course of an analytical approach can prove contradictory and could lead to a great deal of decision making in the process. The decisions which are made could alter the quality of the analytical processes and in turn influence the quality of the results. The scientist must reflect on these basic properties and establish a standard by which the analytical processes are conducted in order to facilitate high quality analytical processes. The various analytical factors their complementary and contradictory relationships (2) fig. 4) should be evaluated and the analytical research should be conducted accordingly.
The quality of the analytical process and the quality of the results are thus highly dependent on one another, and it should become apparent that the capital properties and the basic properties have a mutual relationship in obtaining high quality results meeting external quality standards. The accuracy and representativeness defined by the capital properties is only relevant if we ensure strict precision, sensitivity, selectivity and proper sampling in the laboratory.
The last group of properties, the accessory properties, are interesting in respects to their mutual influence and relationship with the basic properties. The accessory properties are represented by expeditiousness, cost-effectiveness and personnel safety and comfort. These are properties reflecting the competitive aspects of analytical chemistry and are mutually dependent on the basic properties. Thus, compromises regarding sensitivity, precision and selectivity could yield greater expeditiousness and improved productivity compared to competing companies or laboratories. There are however contradictory aspects of the accessory properties and reducing the basic properties could potentially influence various accessory properties thus leading to further challenges in the productivity and competitiveness.
The properties proposed by M. Valcárel and A. Rios should be thought of as a way to hierarchically introduce analytical chemistry. Each group represents a significant part of analytical chemistry and should be dealt with thoroughly by the teacher in order to facilitate a correct understanding by the individual student. The individual groups thus represent the framework by which we can introduce the student to analytical chemistry and rightfully cover the curriculum. It also reflects contradictory relationships in analytical chemistry and proposes some interesting aspects concerning competitiveness, expeditiousness and sensitivity in the laboratory. In industrial context the properties illustrate the paradox observed then assessing various pharmaceutical projects and they are an excellent way to graphically present various dilemmas in drug development.
Analytical techniques in drug design and discovery
Current initiatives in analytical chemistry propose some interesting aspects by combining various division of science towards high yield quality data. Some of these methods are successfully implemented in various phases of drug development and aid in shortening the timeline from lead compound to marketed drug. A collection of these methods are presented below and they should present the reader with a quick overview on some industrially applied analytical techniques. These methods equally show promising results towards scientific advances in analytical chemistry and they demonstrate the implementation of “lean” strategic guidelines in modern drug development and discovery.
“Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer” (3).
Boiscalir et. al (3) propose a new assay based on fluorescence resonance energy transfer (FRET) which successfully could lead to high throughput real time screening of the ubiquitination process in vitro. They furthermore challenge the novel DELFIA assay and suggest that the FRET assay shows lower data variability and due the simplicity of the FRET analysis, the tedious washing, binding and incubation steps in DELFIA are avoided.
Protein degradation by means of ubiquitination involves three enzymatic steps (E1 to E3). Each of these enzymes contributes to the ubiquitination process which leads to the covalent attachment of ubiquitin with ?-aminoacid lysine residues on the target protein (6). The binding of ubiquitin targets the protein for degradation by the proteasomes.
The suggested assay measures the transfer of ubiquitin between the E2 enzyme (Ubc4) and Rsc (human HECT protein). By coupling the Rsc protein to glutathione-S-transferase (GST) and using an anti-GST antibody labelled with Eu3+ it is possible to pre-label the Rsc-GST fusion protein.
The assay exploits the fact that the upon excitation of the compounds, the flourophores Eu3+ and APC exhibits energy transfer and the emission experienced is directly measurable at 665 nm (APC) and 615 nm (Eu3+). To introduce the APC flourophore, the authors have created an ubiquitin-biotin-Streptavidin-Apc complex. Eu3+ acts as the energy donor while APC acts as the energy acceptor, when these are in close proximity (less then 100 Å) to one another, emission is observed from Eu3+ at 615 nm and at 665 nm with APC. The signal intensity presents a means to quantify the ubiquitination process real-time.
The results presented in the paper and the sophisticated experimental set-up revels promising real time data acquisition of biological processes and the method could become a valuable tool in either lead optimization or could successfully be implemented in future HTS assays.
“Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry” (4).
Paul A. Wabnits and Joseph A. Loo propose a new method which successfully could improve the screening of active ligands in molecular binding studies. Micro-size exclusion chromatography offers a fast and simple way to separate the free ligand from the bound protein-ligand complex. Coupled with liquid chromatography and mass spectrometry the authors present a quick and efficient assay which potentially could lead to high throughput molecular binding quantification.
The metalloenzyme peptide deformylase (PDF) was chosen as the target protein, but in order to achieve sufficient spectroscopic data, the authors exchanged the native Fe2+ with the spectroscopically active Co2+. The binding assay was performed on PDF with several suspected inhibitory drugs (the complete structure was only revealed for actinonin due to commercial reasons).
Various ligands were incubated with PDF, and by means of micro-size exclusion chromatography and mass spectrometry, it was possible to obtain mass spectrums revealing the presence or absence of free ligands. The presence of free ligand should indicate weak/no binding and by performing this with various combinations of drugs the researchers where able to establish the binding rank order of the drugs ((4) table 3).
These data suggest the rank by which various drugs show inhibition of the PDF enzyme, and furthermore introduces a method which could yield fast screening of therapeutic leads towards potential macromolecular targets. The procedure shows high sensitivity and enables researcher to perform fast efficient binding studies of various protein and ligand combinations.
The proposed micro-size exclusion principle is furthermore a great example of the simplicity observed in recent advances in drug development and discovery.
“In vitro identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry” (5).
In order to perform quick efficient separation of novel drugs and their metabolites, a growing need for optimization of already established analytical techniques is required. A reduction in the time by which researchers can assess potential drugs and their metabolites could be proven valuable in the lead optimization stage of drug design. Even a small reduction in the identification time is of great interest economically and professionally, and could lead to an enhancement in this important phase of drug design.
Coupling fast gradient high liquid chromatography with tandem mass spectrometry could reduce the bottleneck observed in the lead optimization stage as proposed by Cornelis et. al.
The authors present a fast and efficient way to separate and identify isomeric drug metabolites as well as the parent compound, by use of already established analytical processes. Microsomal incubation of the novel drugs produces the suspected metabolites which by means of fast gradient high liquid chromatography are successfully separated. The separation process is proven by coupling the chromatographic process with tandem mass spectrometry. This enables the researcher to establish the presence or absence of various metabolites and indicates the metabolic fate of the investigated drug.
By reducing analysis time researchers could reduce this step and Cornelis et. al propose a technique which successfully could separate metabolites from parent compound in less than 2 minutes. Optimal conditions were obtained upon increasing the flow rate to 2000 µL/min, and enabled successful chromatographic separation of the metabolites and the pure compound ((6) fig. 2+3).
The method is thus of industrial significance due to the time reduction observed in the process and the relatively easy experimental set-up.
Lead discovery and optimization
High throughput screening (HTS)
Industrial high throughput screening (HTS) presents a multidisciplinary science protruding various scientific fields of study. HTS has become a standard in modern drug development signified by a new paradigm shift toward automatization in the lead discovery phase. It is no longer unusual, that pharmaceutical companies have huge screening libraries containing between 500.000 and 1.000.000 compounds (8), controlled by robotics and computational devices. These libraries provide an extensive database containing valuable compound information which linked to recombinant “humanized” in vitro tests could improve expeditiousness and thus as a consequence improve the drug development and discovery process. The libraries should however not be perceived as a clear cut improvement in the lead discovery phase. The expeditiousness of a lead finding is highly dependent on the development of independent assays modelled towards the suspected target. This could potentially produce a bottleneck in discovery phase and should thoroughly be debated in project decision making.
Criteria’s in HTS
The dawn of these enormous screening libraries has sequestered a demand for assays which are able to successfully screen thousands of compounds and furthermore provide sustainable data fast and efficient. This has forced researchers to optimize existing analytical techniques and implement various branches of natural science towards the creation of assays which fulfil specific standards.
The ideal HTS assay should exhibit the following properties (8):
High signal to background ratio (S/B) or signal to noise (S/N) High z´-factor (9) (values >0,5 represent a good assay) Fast screening time Low coefficient of variation (CV %) Large Stokes Shift
These properties are quantifiable measures aiding the researcher in producing high quality HTS assays modelled towards the proposed target. These criteria thus impose boundaries and guidelines which if followed properly optimistically results in ideal screening methods for future implementation in the drug development phases. Even the smallest improvement in one of the parameters could result in higher expeditiousness and potentially a shortening of the individual developmental phases.
As stated above, efficient screening of compounds is essential in locating the perfect lead candidate for future studies. Observing the parameters, it is evident that there are colliding interests between various aspects of analytical chemistry and economical interests. HTS could be grouped with the above mentioned accessory properties in the nine analytical properties (1,2), reflecting upon the balance between expeditiousness and cost effectiveness in terms of economical profitability. It stands in sharp contrast to the basic properties ensuring high precision, sensitivity and selectivity.
There is a fine balance between producing high throughput data and obtaining the adequate precision needed to produce quality results which live up to internal/external standards. As a consequence, the decisions we choose in these two groups highly reflect the accuracy and representativeness found in the capital properties and thus the quality of the results. These conflicting properties should be evaluated in the early drug development phases and it is the research and development team’s job in collaboration with the financial team to define internal standards for which they both can agree upon.
Implementation of quantitative techniques in drug development
HTS assays implemented in the early discovery phase are a powerful tool in establishing the lead compounds interaction with a suspected biological target. Once a suitable lead has been found quantitative studies are needed to further elucidate the leads biological and chemical characteristics. Quantitative studies following these screening assays provide direct quantifiable data concerning the leads metabolic, pharmacokinetic and toxicological fate. These studies provide valuable information concerning the leads biological properties, and the data obtained in the quantitative studies informs the researchers about potential problems such as toxicology, absorption, distribution excretion, pharmacodynamics and metabolism. Quantitative studies are thus often observed in lead optimization studies where they provide important experimental data.
These studies rely on analytical methods with great precision, expeditiousness, sensitivity and they inherit some of the same criteria as HTS assays (se above). Deviations to the above mentioned criteria’s exist and they include the following:
The need for an adequate internal standard Heightened Precision (of great importance in e.g. toxicological studies) High Selectivity Low matrix interference
When conducting these quantitative studies it is of great importance that the data obtained reflects the precise action of the drug and that the methods inherently refrains from producing false positives. It is obvious that quantification of a seemingly unknown drug is difficult. Purification and the construction of an internal standard is alone a tedious task which requires a great deal of experimental work. Introduction of the lead drug into the lead optimization stage furthermore requires the development of specific modelled analytical techniques which potentially could resolve in a bottleneck in the specific phase.
As stated above there is a clear cut difference between the quantitative studies and the screening methods employed upon implementing assays and procedures in drug design and development. Screening has obvious advantages in the lead discovery phase, and is a strong tool which efficiently and swiftly screens numerous compounds for desired characteristics. The screening assays are however restricted upon relying on a great deal of compound information concerning the proposed target and lead compounds. Structural information concerning pKa, lipphilicity, LogP and LogD is needed to develop an assay that follows the above mentioned success criteria. However, once developed, a successful assay generates valuable data for further use and is imperative when evaluating potential lead candidates in the lead discovery phase.
The quantitative study presented by Cornelis et. al (5) is an excellent example of a procedure which could be deployed in the lead optimization stage of drug development. The metabolic fate of the lead compound can be evaluated and quantification is possible in specific ADME/Tox studies. However, the procedure is dependent on extensive knowledge of the mass and structure of the metabolite or drug compound in order to adequately ascertain that we quantify the correct entity.
In conclusion
The drug development and discovery phases introduce some interesting possibilities for various implantation strategies in respects to scientific collaborations and technological advances. One recent advance in liquid chromatography is the dawn of ultra performance liquid chromatography (UPLC) which shows promising results toward quantitative studies (14). The current trends thus reveal great potential in implementation of these new analytical techniques and future studies may contribute to heightened quality in the lead discovery and optimization phases.
The increased governmental requirements for quantitative ADME/Tox studies has forced pharmaceutical companies to invest enormous amounts of money in order to fulfil requirements suggested by the FDA or other local medicines agencies. This has prolonged the development from lead compound to marketed drug and I suggest that this indeed has faltered a paradigm shift in modern drug development. New biotech companies and scientific entrepreneurs are now faced with tough decisions regarding financial aspects in order to ***** the sustainability of a suspected leads and they should rightfully evaluate the various properties presented by M. Valcárel and A. Rios. We are positioned in a technological vacuum where technological advances greatly exceed the introduction of new drugs towards patients and the industrial drug development is faced with exciting challenges for the future which successfully may increase the amount of drugs marketed per year.
References
1. Valcárcel M. A modern definition of analytical chemistry. Trends in Analytical Chemistry. 1997. 16. pp 124-131
2. Varcárcel M and Rios A. Teaching analytical properties. Fresenius Journal of Analytical Chemistry. 1997. 354. 202-205.
3. Michael D. Boisclair, Christopher McClure, Serene Josiah, Susan Glass, Steve Bottomley, Shubi Kamerkar and Ilkka Hemmilä. Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer. Journal of Biomolecular Screening, Vol. 5, 2000, pp. 328.
4. Paul A. Wabnitz and Joseph A. Loo. Drug Screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 85-91.
5. Cornelis E. C. A. Hop, Phillip R. Tiller and Leslie Romanyshyn. In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 212-219.
6. Jeremy M. Berg, John L. Tymoczko, Lubert Stryer. Biochemistry 5th edition. W. H. Freeman and Company.
7. György M. Keser? and Gergely M. Makara. Hit discovery and hit-to-lead approaches. Drug Discovery Today. Volume 5, Issue 7, 1 July 2000, Pages 286-293.
8. H P Rang. Drug Discovery and Development. Churchill Livingstone, Elsevier. 2006.
10. Alfonso Espada, Manuel Molina-Martin, Jeffrey Dage2 and Ming-Shang Kuo. Application of LC/MS and related techniques to high-throughput drug discovery. Drug Discovery Today. Volume 13, issue 9-10, May 2008, Page 417-423.
11. Tufts Center for the Study of Drug Development, Backgrounder: How New Drugs Move Through the Development and Approval Process, Boston: November 2001
12. Windhover’s In Vivo: The Business of Medicine Report, Bain drug economics model, 2003
13.
Preface
A growing industrial demand for high throughput screening of new molecular entities (NME) has spurred great advances in various divisions of scientific development. Researchers has begun to combine various scientific methods to meet the competitive demands in modern drug development, and as a consequence optimized individual phases of drug development.
Technological improvements should ensure high quality expeditious drug development and a as a consequence a modern “fast track” from drug discovery to marketed product.
These scientific advances should however be viewed in sharp contrast to the increased economical expenditures observed in pharmaceutical companies worldwide. The price from discovery phase to actual marketing is estimated to be in the region of 0.8 billion to 1.7 billion dollars (11) and research has shown that investments in NME’s has increased 55 % over the last five year (12). These economical prospects are paradoxically followed by the fact that there has been observed a mere 7% increase in FDA-approved NME’s from 1993 to 2003. These combined factors are inherently linked to a new paradigm shift in drug development where technological advances outrun the collaborative marketing of new drugs.
This project will present the reader with three established analytical methods in drug development and link these methods with nine analytical properties proposed by M. Valcárel and A. Rios (2). Implementation of these methods in drug development and individual quality standards and guidelines will be presented and discussed and the project will be summarised with concluding remarks.
The nine analytical properties
The analytical properties proposed by M. Valcárel and A. Rios (2) should be regarded as a framework which efficiently introduces the students to the various aspects of analytical chemistry. They are a means of providing a hierarchical approach toward the analytical chemistry curriculum and should ensure an organized introduction to analytical chemistry.
The significance of introducing analytical properties hierarchically to students can be described in terms of the following three groups proposed by the authors. The most important group is the top or capital group. This group is defined by the need for accurate and representative data which in turn is the sole contributor towards the quality of the results.
The second group, the basic properties, ensure the quality of the analytical process and cover the more practical aspects of analytical chemistry. They include such properties as selectivity, precision, sensitivity and proper sampling. They are properties which during the course of an analytical approach can prove contradictory and could lead to a great deal of decision making in the process. The decisions which are made could alter the quality of the analytical processes and in turn influence the quality of the results. The scientist must reflect on these basic properties and establish a standard by which the analytical processes are conducted in order to facilitate high quality analytical processes. The various analytical factors their complementary and contradictory relationships (2) fig. 4) should be evaluated and the analytical research should be conducted accordingly.
The quality of the analytical process and the quality of the results are thus highly dependent on one another, and it should become apparent that the capital properties and the basic properties have a mutual relationship in obtaining high quality results meeting external quality standards. The accuracy and representativeness defined by the capital properties is only relevant if we ensure strict precision, sensitivity, selectivity and proper sampling in the laboratory.
The last group of properties, the accessory properties, are interesting in respects to their mutual influence and relationship with the basic properties. The accessory properties are represented by expeditiousness, cost-effectiveness and personnel safety and comfort. These are properties reflecting the competitive aspects of analytical chemistry and are mutually dependent on the basic properties. Thus, compromises regarding sensitivity, precision and selectivity could yield greater expeditiousness and improved productivity compared to competing companies or laboratories. There are however contradictory aspects of the accessory properties and reducing the basic properties could potentially influence various accessory properties thus leading to further challenges in the productivity and competitiveness.
The properties proposed by M. Valcárel and A. Rios should be thought of as a way to hierarchically introduce analytical chemistry. Each group represents a significant part of analytical chemistry and should be dealt with thoroughly by the teacher in order to facilitate a correct understanding by the individual student. The individual groups thus represent the framework by which we can introduce the student to analytical chemistry and rightfully cover the curriculum. It also reflects contradictory relationships in analytical chemistry and proposes some interesting aspects concerning competitiveness, expeditiousness and sensitivity in the laboratory. In industrial context the properties illustrate the paradox observed then assessing various pharmaceutical projects and they are an excellent way to graphically present various dilemmas in drug development.
Analytical techniques in drug design and discovery
Current initiatives in analytical chemistry propose some interesting aspects by combining various division of science towards high yield quality data. Some of these methods are successfully implemented in various phases of drug development and aid in shortening the timeline from lead compound to marketed drug. A collection of these methods are presented below and they should present the reader with a quick overview on some industrially applied analytical techniques. These methods equally show promising results towards scientific advances in analytical chemistry and they demonstrate the implementation of “lean” strategic guidelines in modern drug development and discovery.
“Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer” (3).
Boiscalir et. al (3) propose a new assay based on fluorescence resonance energy transfer (FRET) which successfully could lead to high throughput real time screening of the ubiquitination process in vitro. They furthermore challenge the novel DELFIA assay and suggest that the FRET assay shows lower data variability and due the simplicity of the FRET analysis, the tedious washing, binding and incubation steps in DELFIA are avoided.
Protein degradation by means of ubiquitination involves three enzymatic steps (E1 to E3). Each of these enzymes contributes to the ubiquitination process which leads to the covalent attachment of ubiquitin with ?-aminoacid lysine residues on the target protein (6). The binding of ubiquitin targets the protein for degradation by the proteasomes.
The suggested assay measures the transfer of ubiquitin between the E2 enzyme (Ubc4) and Rsc (human HECT protein). By coupling the Rsc protein to glutathione-S-transferase (GST) and using an anti-GST antibody labelled with Eu3+ it is possible to pre-label the Rsc-GST fusion protein.
The assay exploits the fact that the upon excitation of the compounds, the flourophores Eu3+ and APC exhibits energy transfer and the emission experienced is directly measurable at 665 nm (APC) and 615 nm (Eu3+). To introduce the APC flourophore, the authors have created an ubiquitin-biotin-Streptavidin-Apc complex. Eu3+ acts as the energy donor while APC acts as the energy acceptor, when these are in close proximity (less then 100 Å) to one another, emission is observed from Eu3+ at 615 nm and at 665 nm with APC. The signal intensity presents a means to quantify the ubiquitination process real-time.
The results presented in the paper and the sophisticated experimental set-up revels promising real time data acquisition of biological processes and the method could become a valuable tool in either lead optimization or could successfully be implemented in future HTS assays.
“Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry” (4).
Paul A. Wabnits and Joseph A. Loo propose a new method which successfully could improve the screening of active ligands in molecular binding studies. Micro-size exclusion chromatography offers a fast and simple way to separate the free ligand from the bound protein-ligand complex. Coupled with liquid chromatography and mass spectrometry the authors present a quick and efficient assay which potentially could lead to high throughput molecular binding quantification.
The metalloenzyme peptide deformylase (PDF) was chosen as the target protein, but in order to achieve sufficient spectroscopic data, the authors exchanged the native Fe2+ with the spectroscopically active Co2+. The binding assay was performed on PDF with several suspected inhibitory drugs (the complete structure was only revealed for actinonin due to commercial reasons).
Various ligands were incubated with PDF, and by means of micro-size exclusion chromatography and mass spectrometry, it was possible to obtain mass spectrums revealing the presence or absence of free ligands. The presence of free ligand should indicate weak/no binding and by performing this with various combinations of drugs the researchers where able to establish the binding rank order of the drugs ((4) table 3).
These data suggest the rank by which various drugs show inhibition of the PDF enzyme, and furthermore introduces a method which could yield fast screening of therapeutic leads towards potential macromolecular targets. The procedure shows high sensitivity and enables researcher to perform fast efficient binding studies of various protein and ligand combinations.
The proposed micro-size exclusion principle is furthermore a great example of the simplicity observed in recent advances in drug development and discovery.
“In vitro identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry” (5).
In order to perform quick efficient separation of novel drugs and their metabolites, a growing need for optimization of already established analytical techniques is required. A reduction in the time by which researchers can assess potential drugs and their metabolites could be proven valuable in the lead optimization stage of drug design. Even a small reduction in the identification time is of great interest economically and professionally, and could lead to an enhancement in this important phase of drug design.
Coupling fast gradient high liquid chromatography with tandem mass spectrometry could reduce the bottleneck observed in the lead optimization stage as proposed by Cornelis et. al.
The authors present a fast and efficient way to separate and identify isomeric drug metabolites as well as the parent compound, by use of already established analytical processes. Microsomal incubation of the novel drugs produces the suspected metabolites which by means of fast gradient high liquid chromatography are successfully separated. The separation process is proven by coupling the chromatographic process with tandem mass spectrometry. This enables the researcher to establish the presence or absence of various metabolites and indicates the metabolic fate of the investigated drug.
By reducing analysis time researchers could reduce this step and Cornelis et. al propose a technique which successfully could separate metabolites from parent compound in less than 2 minutes. Optimal conditions were obtained upon increasing the flow rate to 2000 µL/min, and enabled successful chromatographic separation of the metabolites and the pure compound ((6) fig. 2+3).
The method is thus of industrial significance due to the time reduction observed in the process and the relatively easy experimental set-up.
Lead discovery and optimization
High throughput screening (HTS)
Industrial high throughput screening (HTS) presents a multidisciplinary science protruding various scientific fields of study. HTS has become a standard in modern drug development signified by a new paradigm shift toward automatization in the lead discovery phase. It is no longer unusual, that pharmaceutical companies have huge screening libraries containing between 500.000 and 1.000.000 compounds (8), controlled by robotics and computational devices. These libraries provide an extensive database containing valuable compound information which linked to recombinant “humanized” in vitro tests could improve expeditiousness and thus as a consequence improve the drug development and discovery process. The libraries should however not be perceived as a clear cut improvement in the lead discovery phase. The expeditiousness of a lead finding is highly dependent on the development of independent assays modelled towards the suspected target. This could potentially produce a bottleneck in discovery phase and should thoroughly be debated in project decision making.
Criteria’s in HTS
The dawn of these enormous screening libraries has sequestered a demand for assays which are able to successfully screen thousands of compounds and furthermore provide sustainable data fast and efficient. This has forced researchers to optimize existing analytical techniques and implement various branches of natural science towards the creation of assays which fulfil specific standards.
The ideal HTS assay should exhibit the following properties (8):
High signal to background ratio (S/B) or signal to noise (S/N) High z´-factor (9) (values >0,5 represent a good assay) Fast screening time Low coefficient of variation (CV %) Large Stokes Shift
These properties are quantifiable measures aiding the researcher in producing high quality HTS assays modelled towards the proposed target. These criteria thus impose boundaries and guidelines which if followed properly optimistically results in ideal screening methods for future implementation in the drug development phases. Even the smallest improvement in one of the parameters could result in higher expeditiousness and potentially a shortening of the individual developmental phases.
As stated above, efficient screening of compounds is essential in locating the perfect lead candidate for future studies. Observing the parameters, it is evident that there are colliding interests between various aspects of analytical chemistry and economical interests. HTS could be grouped with the above mentioned accessory properties in the nine analytical properties (1,2), reflecting upon the balance between expeditiousness and cost effectiveness in terms of economical profitability. It stands in sharp contrast to the basic properties ensuring high precision, sensitivity and selectivity.
There is a fine balance between producing high throughput data and obtaining the adequate precision needed to produce quality results which live up to internal/external standards. As a consequence, the decisions we choose in these two groups highly reflect the accuracy and representativeness found in the capital properties and thus the quality of the results. These conflicting properties should be evaluated in the early drug development phases and it is the research and development team’s job in collaboration with the financial team to define internal standards for which they both can agree upon.
Implementation of quantitative techniques in drug development
HTS assays implemented in the early discovery phase are a powerful tool in establishing the lead compounds interaction with a suspected biological target. Once a suitable lead has been found quantitative studies are needed to further elucidate the leads biological and chemical characteristics. Quantitative studies following these screening assays provide direct quantifiable data concerning the leads metabolic, pharmacokinetic and toxicological fate. These studies provide valuable information concerning the leads biological properties, and the data obtained in the quantitative studies informs the researchers about potential problems such as toxicology, absorption, distribution excretion, pharmacodynamics and metabolism. Quantitative studies are thus often observed in lead optimization studies where they provide important experimental data.
These studies rely on analytical methods with great precision, expeditiousness, sensitivity and they inherit some of the same criteria as HTS assays (se above). Deviations to the above mentioned criteria’s exist and they include the following:
The need for an adequate internal standard Heightened Precision (of great importance in e.g. toxicological studies) High Selectivity Low matrix interference
When conducting these quantitative studies it is of great importance that the data obtained reflects the precise action of the drug and that the methods inherently refrains from producing false positives. It is obvious that quantification of a seemingly unknown drug is difficult. Purification and the construction of an internal standard is alone a tedious task which requires a great deal of experimental work. Introduction of the lead drug into the lead optimization stage furthermore requires the development of specific modelled analytical techniques which potentially could resolve in a bottleneck in the specific phase.
As stated above there is a clear cut difference between the quantitative studies and the screening methods employed upon implementing assays and procedures in drug design and development. Screening has obvious advantages in the lead discovery phase, and is a strong tool which efficiently and swiftly screens numerous compounds for desired characteristics. The screening assays are however restricted upon relying on a great deal of compound information concerning the proposed target and lead compounds. Structural information concerning pKa, lipphilicity, LogP and LogD is needed to develop an assay that follows the above mentioned success criteria. However, once developed, a successful assay generates valuable data for further use and is imperative when evaluating potential lead candidates in the lead discovery phase.
The quantitative study presented by Cornelis et. al (5) is an excellent example of a procedure which could be deployed in the lead optimization stage of drug development. The metabolic fate of the lead compound can be evaluated and quantification is possible in specific ADME/Tox studies. However, the procedure is dependent on extensive knowledge of the mass and structure of the metabolite or drug compound in order to adequately ascertain that we quantify the correct entity.
In conclusion
The drug development and discovery phases introduce some interesting possibilities for various implantation strategies in respects to scientific collaborations and technological advances. One recent advance in liquid chromatography is the dawn of ultra performance liquid chromatography (UPLC) which shows promising results toward quantitative studies (14). The current trends thus reveal great potential in implementation of these new analytical techniques and future studies may contribute to heightened quality in the lead discovery and optimization phases.
The increased governmental requirements for quantitative ADME/Tox studies has forced pharmaceutical companies to invest enormous amounts of money in order to fulfil requirements suggested by the FDA or other local medicines agencies. This has prolonged the development from lead compound to marketed drug and I suggest that this indeed has faltered a paradigm shift in modern drug development. New biotech companies and scientific entrepreneurs are now faced with tough decisions regarding financial aspects in order to ***** the sustainability of a suspected leads and they should rightfully evaluate the various properties presented by M. Valcárel and A. Rios. We are positioned in a technological vacuum where technological advances greatly exceed the introduction of new drugs towards patients and the industrial drug development is faced with exciting challenges for the future which successfully may increase the amount of drugs marketed per year.
References
1. Valcárcel M. A modern definition of analytical chemistry. Trends in Analytical Chemistry. 1997. 16. pp 124-131
2. Varcárcel M and Rios A. Teaching analytical properties. Fresenius Journal of Analytical Chemistry. 1997. 354. 202-205.
3. Michael D. Boisclair, Christopher McClure, Serene Josiah, Susan Glass, Steve Bottomley, Shubi Kamerkar and Ilkka Hemmilä. Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer. Journal of Biomolecular Screening, Vol. 5, 2000, pp. 328.
4. Paul A. Wabnitz and Joseph A. Loo. Drug Screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 85-91.
5. Cornelis E. C. A. Hop, Phillip R. Tiller and Leslie Romanyshyn. In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 212-219.
6. Jeremy M. Berg, John L. Tymoczko, Lubert Stryer. Biochemistry 5th edition. W. H. Freeman and Company.
7. György M. Keser? and Gergely M. Makara. Hit discovery and hit-to-lead approaches. Drug Discovery Today. Volume 5, Issue 7, 1 July 2000, Pages 286-293.
8. H P Rang. Drug Discovery and Development. Churchill Livingstone, Elsevier. 2006.
10. Alfonso Espada, Manuel Molina-Martin, Jeffrey Dage2 and Ming-Shang Kuo. Application of LC/MS and related techniques to high-throughput drug discovery. Drug Discovery Today. Volume 13, issue 9-10, May 2008, Page 417-423.
11. Tufts Center for the Study of Drug Development, Backgrounder: How New Drugs Move Through the Development and Approval Process, Boston: November 2001
12. Windhover’s In Vivo: The Business of Medicine Report, Bain drug economics model, 2003
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