Pharmaceutical R+D

What is a drug?

In Pharmacology, a drug is a natural or synthetic substance able to produce biological or functional effect in a living organism. A medication is a drug used for the treatment, cure, prevention, or diagnosis of a disease as well as in some condition to enhance physical or mental well-being.

Preclinical phase

After the drug discovery, its biological activity is analysed in computational models, cell cultures or animals (from less to more sensitive according to their phylogenetic scale) following the 3 R's principle of Russell and Burch. The protocols must be submitted and approved by the Ethical Committee of Experimental Animal Research of the university or research centre where the research is going to be carried out. 

Preclinical studies are also required to determine the efficacy of the medicament via the pharmacokinetic (the response of the organism to the drug) and pharmacodynamic (how the drug acts in the organism) analyses. In addition, the security of the drug needs to be evaluated by toxicological studies after chronic and acute exposure.  An improper testing of the efficacy or security of the drugs can cause serious health problems in the clinical stage, as happened in the middle of the XX century with Thalidomide, a drug prescribed as a sedative and antiemetic for morning sickness. Thousands of pregnant women took Thalidomide to relieve their symptoms. In the late 50's and early 60's reports of children born with deformities such as phocomelia (rare congenital disorder involving malformations of the limbs, see picture in the right) as a consequence of Thalidomide use saw the light. The effects of Thalidomide raised the issue of safety of pharmaceutical drugs with testing and approval of toxins becaming more important.

After preclinical testing, the drug can progress to the clinical phase following the positive evaluation of the National (AEMPS: Agencia Española de Medicamentos y Productos Sanitarios in Spain), European (EMA: European Medicines Agency) or American (FDA: Food and Drug Administration) authorities.

Clinical phase 

The clinical phase includes a serial of tests to ensure the efficacy and security in humans as well as to generate data of the adverse drug reactions or adverse effects with other treatments. All the investigations must conform to the Declaration of Helsinki and the protocols must be submitted and approved by the Ethical Committee of Clinical Research. The clinical developmental phases are:

• Phase I: the drug is administered, for the first time, to 20-100 healthy individuals in order to evaluate the safety of the dosage and how the drug should be given. Usually this stage lasts from 6 to 12 months.
• Phase II:  the biological activity and the optimal dosage of the drug is tested in 100-200 patients and healthy individuals involved in blind clinical trials. Information about the drug's safety, side effects and potential risks are also collected. This phase of the clinical trial lasts between 1-2 years.
• Phase III trials enroll a large number of individuals (1,000-3,000 patients) in order to compare the efficacy of the new drug with other existing medicaments in a double-blind, randomized study. Additional information about the safety and side effects of the drug is gathered during a period of 2-3 years. Once the drug has been proven succesful in the Phase III clinical trial, the researchers can submit an application to AEMPS, EMA or FDA approval. If data from the clinical trial meet the AEMPS's, EMA's or FDA's standards of safety and efficacy, the drug is approved for a specific use.
• Phase IV or post-marketing surveillance: the safety surveillance is designed to detect any rare or long-term adverse effects over a large population. Harmful effects can result in a drug being withdrawn or restricted to certain uses. A recent example include rofecobix (Vioxx), a nonsteroidal antiinflammatory drug used for the treatment of osteoarthritis, acute pain and dysmenorrhoea, that was withdrawn from the market because it caused serious secondary cardiovascular effects.

The entire process of a drug from the lab to this point may take approximately 12-18 years. In the following video, you can see a summary of the 4 stages of the clinical phase:

You will have to know all the phases of the pharmaceutical R&D process as well as their main characteristics:

 

Scientific Writing

Today we have addressed the topic of how to present data in written form, focusing mainly on writing papers for biomedical journals. Why is writing important in science? Scientists need to communicate their findings and this is usually achieved via publishing their work in scientific journals. Thus, a critical aspect of the scientific process is to share your information and experience with the scientific community. Reporting your new results in biomedical journals is a quick way to disseminate the information to the specialized community contributing to increase the knowledge within a discipline as well as to help others interpret their own results.

Types of articles

We have mentioned in class the different types of articles and their main characteristics. While most articles published are original research papers, there are also reviews, editorials, commentaries, perspectives, case reports or letters to the editor, among others.



Fundamental style considerations

In the International Committee of Medical Journal Editors - ICMJE) you can find the Uniform Requirements for Manuscripts. Most biomedical journals have their own format, structure and writing styles. Therefore, it is important that you are familiar with the Instructions for Authors of the specific journal you aim to submit your manuscript.

Your text should conform to the conventions of standard written English (sentence form, grammar, spelling, etc.). Structure your sentences carefully using the basic division into subject, verb, and complement. In addition, the structure of the sentence will convey emphasis, draw attention to the most important part at the same time as help readers to interpret your meaning correctly. Moreover, you should structure your sentences and paragraphs to flow, conferring your article cohesion and coherence.
 
Your ideas will have little impact, no matter how good the research is, if they are not communicated effectively!

Keep in mind that scientific terminology has precise meaning. Thus, be certain you select your words correctly and adequately; write clearly and concisely. Writing and thinking are very closely linked, "fuzzy writing reflects fuzzy thinking". When people have difficulties in translating their ideas into words, it generally reflects that they may not be fully aware of what they want to transmit.

• Adapt to the special requirements of your selected audience, journal and type of article.

• Be precise and accurate: Scientific terminology carries specific meaning; learn to use it appropriately and consistently. In fact, it takes a deeper understanding to explain a complex topic simply and succinctly. 

• Be clear and concise: Write briefly and to the point. Say what you mean simply and clearly avoiding embellishment with unnecessary words or phrases. Brevity is very important. Do not use colloquial speech or slang; do not use contractions like for example "don't" or "isn't".

"Brevity is the soul of wit" (W. Shakespeare)

Considerations for the preparation of a research article

One of the initial considerations is to choose the appropriate readership for your article. Usually you will be writing to your peers. Knowing your audience will help you to decide what information to include. Obviously, you write a very different article for a highly specialized, technical, disciplinary journal as opposed to a more general publication that covers a broad range of disciplines.  Another relevant aspect to take into account is the impact factor of the journal. 

The first step in the process of writing is to order and organize the information you wish to present. Another critical point when preparing your first draft is to decide the authors and their specific order. The choice of a good title is also extraordinarily important; it should be informative at the same time as attractive and objective. Some people work well from an outline, others do not. Whatever system you follow, be aware that scientific writing requires special attention to order and organization. Because the paper will be divided into sections, you need to know what information will go into each.

The following video very nicely summarizes the main sections of an article and their content.

• You need to know the different types of articles (original research papers, reviews, editorials, commentaries, perspectives, case reports and letters to the editor, among others) together with their main characteristics and usefulness.

• In addition, you have to be familiarized with the main sections of a research article, how to structure and develop them in order to communicate effectively. 

 

"Omics" technologies

The complete sequencing of the human genome has introduced us in a new era of research methodology referred as omics technologies. This term comprises high-throughput experimental technologies characterized by automation, miniaturized assays and large-scale data analysis. These technologies are increasingly generating massive and complex genomic data sets so that interpreting functional consequences of millions of discovered genetic variants is one of the biggest challenges. Over the past few years omics technologies have revolutionized research projects.

There are five major types of high-throughput measurements that are commonly performed:

• Genomic: analysis of the DNA sequence (i.e., SNP).
• Transcriptomic: analysis of transcribed RNA (i.e., the simultaneous measurement of gene expression values in a cell or tissue type).
• Proteomic: determination of proteins present in a sample.
• Metabolomic:  identification and quantification of all metabolites in a sample.
• miRNAomics: regulatory mechanisms underlying control of transcription.

Schematic representation of omics technologies, their corresponding analysis targets, and assessment methods. Taken from Wu RD et al. JDR 2011; 90:561-572.

Below is a list of the various types of information obtained from some of the omics technologies (adapted from Sawyers CL. Nature 2008; 452: 548-52):

• DNA copy-number assessment: comparative genome hybridization to DNA microarrays.
• Mutation screening: DNA sequencing,  mass-spectrometry-based genotyping, mutation-specific PCR.
• Gene-expression profiling: microarrays, multiplex PCR, microRNA-expression profiling. 
• Proteomic profiling: mass spectrometry, phosphoproteomic profiling, mass spectrometry after immunoprecipitation with specific antibodies. 
• Metabolomic profiling: mass spectrometry.

Tissue microarrays (TMAs) are an ideal platform for validating the results obtained from large-scale transcriptomic and proteomic studies. The most commonly performed assays on tissue microarrays are immunohistochemistry for protein expression, in situ hybridization for RNA expression, and fluorescence in situ hybridization for DNA copy number.

TMAs accrue value over time and allow investigators to build u

p IHC

profiles for individual tumours, as demonstrated by the expression of six different biomarkers in a single tumour core. Taken from Brennan DJ et al. Cancer Genomics Proteomics 2007; 4:121-134.

2-D eletrophoresis is an important step in proteomics analysis. In the following video you can learn more about how to prepare 2-D gels:

This video shows microarray technology and applications:

1. Study the main omics technologies and identify their differences.
2. Know the methodology used by each omic subdiscipline.
3. Advantages and limitations related to omics technologies.
4. Be able to relate the omics methodologies with the distint type of research.
5. Familiarize yourself with the diverse types of analysis in proteomics.
6. Know what epigenomics referes to.

 

 

 

Practical session 2 - Group 3

OPHTHALMOLOGY WORKSHOP

The sense of sight is fundamental to human well-being. Because of this, ophthalmologic research has been growing exponentially in recent years, but it has grown even more so because of the peculiarities of the visual system. The fact that the visual system is relatively isolated makes it an ideal model to test treatments that would otherwise be confounded by more variables in other body systems. While you are already familiar with some of the main characteristics of the eyes the ophthalmology course will take place in 4th Med School year, during which the anatomy, function, and pathologies of the eye will be explained. In this ophthalmology research workshop, we provide you a rough outline of the visual system as well as of the most common ocular diseases (from refractive problems such as myopia to severe visual disabilities such as glaucoma and age-related macular degeneration).

Transverse cut of the eye and its anatomy. Details of the retina and its layers. Source: eMedicine

Simulation of the diferent levels of severity of viusal alteration in patients with Age related Macular Degeneration. Source: Oftalmología Clínica, JJ Kanski 2009

The most practical part of the workshop consists of trying different smartphone/tablet applications (for both iPhone and Android) as useful tools to help in diagnosing ocular diseases.

Applications that are used to test visual function: Vision Test and Federopticos. 

These tools can be useful to test your own visual function as well as to test others that may have more serious visual dysfunction such as the aforementioned age-related macular degeneration.

Additionally, we will try another application, Cataract Surgery, that you can download for free from the Appstore for iPhone or from Google-Play for Android. As you´ll see, this app allows us to practice real ophthalmologic surgical techniques such as the membrane peel (capsulorhexis) or lens emulsification (Phaco).

The App Cataract Surgery allows us to practice cataract surgery techniques.

Many more useful ophthalmology applications are available, but go beyond the scope of this practical session. (Eye Handbook, Ophthalmology i-pocketcards, eyetube, among others).

Some of the most used apps in ophthalmology:  Eye Hanbook, Ophthalmology i-pocketcards, eyetube.

1. Learn the basic anatomy of the eye and the functions of its distinct parts.  
2. Differentiate the characteristics of patients with diseases related to optical defects and severe visual pathologies.  
3. Test the Smartphone/Tablet apps related to visual function with your classmates through various tests (visual acuity, color test, astigmatism test and dominant eye, as indicated in the practical session).  
4. Perform a simulation of cataract surgery through the app cataract surgery with all the required steps (capsulorhexis and phacoemulsification).

Basic molecular biology in biomedical research

Milestones of molecular biology

The major advances in genetic engineering have occured during the last 6 decades, but the history of molecular biology started at the end of the XIX century with the research of Darwin and Mendel. On his book "On The Origin of Species", Charles Darwin explained that heredity is the mechanism that perpetuates variations, since the traits in offspring result from a blending of the traits of each parent. The Austrian friar Gregor Mendel conceived the idea of heredity units, which he called "factors" (later known as "genes"), by studying Pisum sativum, or the common pea plant. Mendel proposed that "factors" normally occur in pairs and these two "particles" of the factors (later known as "alleles") segregate from each other in the formation of gametes. Therefore, Mendel stated that each individual inherits two "particles" for each trait, one from each parent.  In 1869, Friedrich Miescher first isolated what he called "nuclein" (later known as "deoxyribonucleic acid" or "DNA" inside the nuclei of human white blood cells. 

 

In 1953, Dr. James Watson and Dr. Francis Crick deduced the three dimensional, double-helical model for the structure of DNA, based on some crucially important X-ray chrystallography work of Dr. Rosalind Franklin and Dr. Maurice Wilkins. Watson, Crick and Wilkins received the Nobel Prize in Medicine for the double-helix model of DNA in 1962, but the scientific work of Rosalind Franklin was not recognized. The picture on the right pannel illustrates the schematic respresentation of the double-helix model of DNA proposed by Watson and Crick and published in the prestigious Nature journal (view the manuscript here).

In 1990 the second major milestone in the genetics of the XX century started with the sequentiation of the human genome in the "Human Genome Project", that concluded in 2003. A working draft of the Human Genome was completed and simultaneously published in Nature and Science on February 15, 2001.

New challenge in molecular biology: the ENCODE project

ENCODE (acronym of ENCyclopedia Of DNA Elemens) is a project funded by the National Human Genome Research Institute to identify all regions of transcription, transcription factor association, chromatin structure and histone modification in the human genome sequence. Thanks to the identification of these elements, 80% of the components of the human genome now have at least one biochemical function associated with them, in particular, the non-encoding regions. In this link to the ENCODE project, the most relevant results obtained in relation to the organization and regulation of the genes are shown.

The identification of new elements of the human genome has revealed new insights into biomedical research, as you can see in this video:

The central dogma of molecular biology consists of three main processes: DNA replication, RNA transcription and protein translation. Some of the basic techniques used in biomedical research are shown below:

Molecular techniques for the study of DNA:

• Analysis of numeric chromosomal anomalies: cariotype.
• Analysis of structural chromosomal anomalies: FISH 
• Detection of mutations: RFLP.
• Study of gene function: transgenic animals.

Molecular techniques for the study of RNA:

• Conversion of RNA to cDNA: reverse transcription.
• Gene expression analysis: conventional PCR, real-time PCR and microarrays.

Molecular techniques for the study of proteins:

• Quantification of proteins: ELISA, western-blot and proteomics.
• Localization of proteins: immunohistochemistry.
•  Study of protein function: stimulation of cell cultures, inoculation in experimental animals or patients.

Research Grants - Evaluation of Scientific Activity

The experiments needed to carry out scientific studies usually involve high economic expenses in reagents and consumables, equipment, and personnel, among other things. Therefore, it is necessary to apply to competitive grant programs in order to get funding for research. There are sources of research funding at local (for example our University), regional, national or international (e.g. FP7 and Horizon 2020, or the NIH) levels.

Besides the scientific interest and originality of the study, one of the main aspects that determines the possibility of success of the requested grant is the curriculum vitae (CV) of the principal investigator (PI) and the other team members (co-applicants). One of the most valued aspects of a given CV is the record of scientific publications relative to the topic of the application. A way to quantify the relevance of scientific articles is the impact factor (IF) of the journal in which it was published. The IF is a numeric value that produces the Journal of Citation Reports of the Institute for Scientific Information (ISI, Thomson Teuters)  (accessible online from the University). The IF is published annually (the most recent update available is from July 2014 and refers to citations within 2013) and indicates the impact of a journal regarding the times its articles are cited (the math is explained in class and is available at ADI or the ISI webpage).

However, the value of a researcher’s publication record considering the quantity and the IF of the journals in which they have been published have received a lot of criticism in recent years. This is because an article can be published in a high IF journal, but may not have much "real" impact. For this reason, more importance is given to the number of citations of works of an author, information that can be also found on the ISI website (Web of Science). It may happen that an author has received many citations globally, but it is because he/she has an article cited many times (for example if the author has published an innovative technique). To combine both aspects, number of publications (productivity) and the times they are cited (impact), Jorge Hirsch suggested in 2005 the h-index. The h-index may be found on the ISI website (Web of Science) and can be obtained as shown in the following video:

The h-index is increasingly used to evaluate the CV of a researcher when applying for a grant or a position, or when promoting in academia or the scientific career. However, for calculating the h-index, once a paper is selected to belong to the top h papers, this paper is not “used” anymore in the calculation of the h-index and, therefore, the h-index is insensitive to outstandingly highly cited papers. To overcome this “problem” the g-index was suggested by Leo Egghe in 2006. Whilst keeping the advantages of the h-index, an author has a g-index g if g is the highest rank such that the top g papers have, together, at least g² citations.

Funny Science

A funny cartoon showing the difference between journals with high versus low IF:

Practical session 2 - Group 1

BODY COMPOSITION WORKSHOP

Body composition analysis can be approached from five different levels: atomic (hydrogen, carbon, oxygen and other atoms), molecular (water, lipids, proteins, carbohydrates and minerals), cellular (cells, extracellular fluids and extracellular solids), tissular (adipose tissue, skeletal muscle, bone and visceral organs) and bicompartimental (fat mass and fat-free mass).

In the last years, rapid improvements in body composition determination have been developed due to the high increase in the prevalence of obesity all over the world. The most commonly used methods for the evaluation of body composition are shown below:

1. Skinfold measurements estimate the percentage of body fat by measuring skinfold thickness at specific anatomic regions (see the picture below) using the skinfold calipers. The results of skinfold thickness are entered into an equation to estimate the percentage of body fat according to a person's age and gender. This is a non-invasive, inexpensive and easy method to estimate the body composition, but with a high inter-observer variability.

2. Biolectrical impedance is a commonly used bicompartimental method for estimating body composition based on the resistance to a small electric current through body tissues. Fat free mass is a good conductor since it contains the majority of corporal fluids and electrolytes, while fat mass is a bad one. 

3. Dual energy X-ray absorptiometry (DEXA) is a non-invasive technique based on the attenuation of two X-ray beams with different energy levels depending on the organic tissue they pass through. DEXA provides whole-body and regional estimates of bone, fat free mass and fat mass. The advantages of DEXA include a good accuracy and reproducibility. The main disadvantage of DEXA includes a small amount of radiation with this method and the underestimation of body fat in obese individuals.

4. Air displacement plethysmography (Bod-Pod®) measures the body volume by the air displaced by an individual in a sealed chamber; body volume is combined with body mass in order to determine body density. Additionally, the system discounts the pulmonary residual volume air. This non-invasive, easy, non-radiactive bicompartimental method calculates fat free mass and fat mass with excellent accuracy and reproducibility.

 

PHYSICAL ACTIVITY WORKSHOP

Two main methods for assessing physical activity levels are available:

Subjective methods: are related to your own perceptions of physical activity. They include:

• Self reports: diaries, interviews, questionnaires, logs.

• Recalls: remembering what activities you have done.

Advantages of subjective methods:

1. Suitable for small groups or individuals, as well as for population groups or subgroups.

2. Easy to complete.

3. Provides information on the context of an activity.

Disadvantages of subjective methods:

1. Heavy subject burden (entries completed throughout the day).

2. Less reliable than objective methods due to potential misinterpretation or inaccurate recording.

3. Invasion of study participant privacy.

Below you have an example of a physical activity questionnaire:

Objective methods: These methods rely on solid data and observations. They are predominately used for application in individuals and include the following:

1.- Direct observation. This method uses trained observers to systematically record physical activity behaviours of participants in a restricted environment, such as a physical education class, school playground, or in the workplace. Usually a prepared form or application is used to record the type and intensity of the activity. The biggest advantage of direct observation is that it provides more detailed information than any other methods. Moreover, trained observers also are able to evaluate physical activity more objectively than participants themselves. However, the accuracy depends on the ability of the observer.

2.- Doubly-labelled water. This method consists in a non-invasive procedure involving the intake of a quantity of water labelled with a known concentration of stable isotopes of hydrogen and oxygen. As energy is expended in the body, carbon dioxide and water are produced, and the differences between the isotope elimination rates are used to calculate total energy expenditure.

3.- Heart rate monitoring allows to measure the heart rate in real time as well as to record the heart rate for later study. The maximum heart rate (HRmax) is the highest heart rate a person can safely achieve without severe problems through exercise stress.

 

4.- Calorimetry. Direct calorimetry involves the measurement of heat produced by the body during physical activity, whereas indirect calorimetry involves the measurement of respiratory gas exchange (oxygen consumption and carbon dioxide production) during a variety of controlled conditions.

 5.- Motion sensors. Motion sensors can be divided into two main types, pedometers and accelerometers.

• Pedometers measure total physical activity by counting the number of steps an individual takes. When walking is the predominant type of activity, pedometers are quite effective as a measurement device. As much as 10,000 steps are recommended per day!

 

• Accelerometers provide more detailed information regarding intensity and direction of movement. The accelerometer monitoring system that we have used in the session with your classmates is the ActicalTM, which provides calculated energy expenditure values for energy expenditure in kilocalories and total energy expenditure in Metabolic Equivalents per Time (METs) in kilocalories/min/kg. Software reports values and average values per minute of activity count, energy expenditure and duration at each intensity level (sedentary, light, moderate and vigorous) every minute, hour, day and for total recording period.

 Below you can observe an example of the physical activity as well as the energy expenditure report of a 19-year-old male (accelerometer worn on wrist band). Studying this actigraph:

1.- How many kilocalories are consumed on Saturday September 29th?

2.- Explain the physical activity of this day.

Answers:

1.- The total energy expenditure of Saturday  September 29th is 4,247 kcal: 2,402 kcal associated to the physical activity and 1,845 kcal needed to maintain the normal body functions.

2.- The accelerometer registered an average of 212 counts, so physical activity is fine. In fact, he did 48 minutes of vigorous activity which is quite an accomplishment!

How to make a home-made accelerometer with everyday objects



Where do you have a natural accelerometer?

The inner ear consists of the cochlea, the semicircular canals and the auditory nerve. The cochlea and the semicircular canals are filled with a water-like fluid, the endolymph. The fluid and nerve cells of the semicircular canals do not participate in the task of hearing; they serve as miniature accelerometers that maintain the sense of balance by responding to gravity and changes in acceleration.