Risorse bioinformatiche comunemente
utilizzate nel processo di “drug discovery”;
esperimento di screening farmaceutico.
Alessandro Provenzani
Lab of genomic screening
[email protected]
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Escursione storica
Drug discovery
Esempi “farmaci moderni”
Processo immissione nel mercato
Conclusioni
3000 a.c. Tavolette Sumere, indicazioni sulle operazioni galeniche, estratti acquosi o
oleosi. Nessuna indicazione sulle quantità, pesi, misure.
Tradizione orale ?
1550 a.c. papiro di Ebers, di Hearst, tutto rigorosamente misurato
estratti acquosi o oleosi. Medicamenti a base di birra, latte
vino.
600 a.c. Romani, pratica medica, influenzata dagli etruschi,
accompagnata da formule magiche e invocazioni alle divinità.
Casta sacerdotale era dedita alla Ars Divinatoria consiste nell’interpretazione di
segni e presagi offerti dalla natura
460 a.c. Ippocrate, ideatore della medicina razionale, distacco dalla figura
del medico-sacerdote. Inizio dell’osservazione clinica obiettiva.
“primum non nocere” : il medico deve essere parco nella somministrazione dei
medicamenti
teoria medica dei “quattro umori”: sangue (cuore), flemma (cervello), bile (fegato),
atrabile (milza)
Armamentario:
elleboro nero, coloquintide, veratro bianco, oppio, belladonna, scilla
Atropa Belladonna: pianta ricca di alcaloidi, più importante è l’atropina, anticolinergico
tutt’ora utilizzato in clinica
e anche improbabili come cervello di cane, sterco di capra, cuore di avvoltoio…
300 a.c. Arrivano a Roma i medici Greci professionisti
40 d.c. Roma imperiale, tentativi di catalogare e raccogliere la farmacologia sono ad opera
di Celso, classificazione delle forme farmaceutiche e medicamenti utilizzati in riferimento alle
patologie.
37 libri della Naturalis Historia di Plinio sono registrate 119 piante ad uso medicinale e
descrive la Teriaca del re Antioco.
129 d.c. Galeno medico di corte di Marco Aurelio, influenzò tutto il medioevo con i suoi
trattati
Nascono le prime forme di pubblicità per i medicamenti
I Cirenaici (Libia) impressero sulle loro monete la pianta del Silfio e veniva usato come
panacea per molte malattie
Estinzione della pianta
476-1492 d.c.
Il mantenimento del sapere in occidente è affidato ai monasteri che
mantengono gli scritti di Galeno, Ippocrate, Plinio…. Nasce l’Orto dei Semplici e l’Armarium
Pigmentarium (Spezeria del convento)
XV-XVII secolo
Philippus Aureolus Theophrastus Bombastus von Hohenheim detto
Paracelso, alchimista fondatore della iatrochimica “il vero scopo della chimica non consiste
nella fabbricazione dell’oro, ma nella preparazione delle medicine”
La malattia è uno squilibrio chimico del corpo che può essere ristabilito attraverso l’uso di
altre sostanze chimiche.
Scopre il Laudano
Nasce l’attenzione verso il principio attivo del preparato farmaceutico
Comincia la rivoluzione scientifica: momento di rottura con la
tradizione medievale
Newton, Keplero, Galilei, Pascal, Boyle
anche in medicina e biologia:
scoperta di batteri e protozoi (Van Leeuwenhoek)
dimostrazione della falsità della “generazione spontanea” (Francesco
Redi): nessun organismo si genera spontaneamente, ma ognuno
nasce da altri esseri viventi della sua stessa specie
Pasteur dovette ridimostrare, un paio di secoli dopo, la falsità della teoria
XVIII secolo
Lavoisier pone le basi della chimica moderna introducendo il metodo
scientifico e la necessità di determinazioni quantitative e riproducibili
Nasce la chimica farmaceutica con Scheele con l’identificazione di molecole di interesse
biochimico e farmaceutico, Withering individua nelle foglie della digitale una sostanza con
proprietà cardiotoniche
1775 Prieslty con il protossido di azoto gas ad azione esilalarante ed anestetica, che viene
ripreso il secolo successivo dal dentista Wells che però non ebbe seguito.
1846 Morton, amico e collega di Wells nonché studente, fece una
dimostrazione utilizzando etere per l’asportazione di un tumore
mandibolare
Nasce l’anestesia in camera operatoria
XIX secolo
Comincia il boom della chimica farmaceutica grazie all’isolamento e la
purificazione dalle piante medicinali dei principi attivi
Inizia un processo di ricerca e produzione di nuove molecole su vasta scala, nascono i legami con
la botanica, microbiologia, farmacologia
Vengono definiti gli alcaloidi, molecole con caratteristiche basiche presenti nelle piante
medicinali
Vengono isolati in rapida successione morfina, emetina, stricinina, atropina, caffeina, chinina,
codeina etc
Passaggio da preparati galenici classici, parzialmente efficaci e spesso tossici, a forme
farmaceutiche rigorose capaci di provocare un effetto costante e riproducibile
Nascono le aziende farmaceutiche
Drug definition
What are drugs
• Drugs are strictly defined as chemical substances that are used to prevent or cure
diseases in humans, animals and plants.
• The activity of a drug is its pharmacological effect on the subject, for example, its
analgesic or β-blocker action.
Drugs act by interfering with biological processes, so no drug is completely safe. All
drugs can act as poisons if taken in excess. For example, overdoses of paracetamol can
cause coma and death.
“All substances are poisons: there is none which is not a poison. The right dose
differentiates a poison and a remedy.”
Paracelsus (1493-1541)
• Furthermore, in addition to their beneficial effects, most drugs have non-beneficial
biological effects. Aspirin, which is commonly used to alleviate headaches, may also
cause gastric irritation and bleeding.
Lead compund
LEADS
Pharmaceutical chemists do not synthesise compounds at random. They usually
start with a so-called lead compound (ie a compound which leads them
onward), and make and test compounds with structures closely related to it.
This lead compound will have shown some activity as a drug and the chemists
hope that they can find a derivative of it which is better – is more effective as a
drug, has fewer side effects, is cheaper to make etc.
A good example of the lead compound approach is the development of the drug
aspirin:
receptors and ligands
John Langley in 1905 he proposed that so called receptive substances in the body could
accept either a stimulating compound, which would cause a biological response, or a
non-stimulating compound, which would prevent a biological response.
It is now universally accepted that the binding of a chemical agent, referred to as a
ligand, to a so called receptor sets in motion a series of biochemical events that result in
a biological or pharmacological effect: concept of drug molecolar target
structure complementarity and pharmacophores
Furthermore, a drug is most effective when all or a significant part of its molecular shape
and electron distribution (stereoelectronic structure), is complementary with the
stereoelectronic structure of the receptor responsible for the desired biological action.
The section of the structure of a ligand that binds to a receptor is known as its
pharmacophore.
It is now believed that side effects can arise when the drug binds to either the receptor
responsible for the desired biological response or to different receptors.
Lead identification: essential steps
The first problem in drug discovery is the drug target identification
Much of the effort of basic biomedical research is oriented towards the characterization
of a pathology, how and why it comes out, and which are its specific molecolar features
The most popular approach to drug design by synthesis is to start with the pathology of
the diseased state and determine the point(s) where intervention is most likely to be
effective.
Drug Design
Me-too drugs
The cost of introducing a new drug to the market is extremely high and continues to
escalate. One has to be very sure that a new drug is going to be profitable before it is
placed on the market. Consequently, the board of directors’ decision to market a drug
or not depends largely on information supplied by the accountancy department rather
than ethical and medical considerations.
One way of cutting costs is for companies to produce drugs with similar activities and
molecular structures to their competitors. These drugs are known as the ‘me-too
drugs’.
Serendipity and side effects
Chain and Florey’s choice turned out to be very fortunate. Because of its efficacy and
lack of toxicity, penicillin made the most compelling case for antibiotics in general
Lead identification: essential steps
After an appropriate molecular target is identified, the next
major task in the drug discovery process is generation and
optimization of lead compounds
Identification of lead compounds requires:
– Developing appropriate screening assays
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Binding affinity
Efficacy
Specificity
Toxicity
– Screening molecule libraries containing potential lead compounds
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Natural product libraries
Existing compound libraries
Combinatorial chemistry libraries
Virtual libraries
Lead identification
Today, many discoveries start with biological testing (bioassays or screening
programme) by pharmacologists of the potential sources in order to determine the
nature of their pharmacological activity as well as their potencies.
These screening programmes may be random or focused.:
In random screening programs all the substances and compounds available are tested
regardless of their structures. The random screening of soil samples, for example, led to
the discovery of the streptomycin and tetracycline antibiotics as well as many other
lead compounds.
Random screening is still employed, but the use of more focused screening procedures
where specific structural types are tested is now more common.
Lead identification: high-throughput screening
The goal of HTS (High-Throughput Screening) is to accelerate drug discovery by
screening large libraries often composed of hundreds of thousands of compounds (drug
candidates) at a minimal rate of 20,000 compounds per week.
For a number of years, HTS assays have been run in the standard 96-well microplate
(working volume of up to 250 µL). The current goal of most companies is to move
beyond this format to higher- density, lower-volume formats (e.g., 384- and 1536-well
microplates). There are two primary advantages of these formats: increased throughput
and lower volume, which translates into lower cost.
Lead identification: high-throughput screening
Generally speaking, first-pass HTS assays (the primary screen) are less quantitative than
traditional biological assays. Often, compounds are only tested in duplicate (an
increasing number of companies are using singlets), and usually at one concentration
(most often in the 1–10 µM range for combinatorial chemistry libraries). If a “hit”
(positive) is discovered, more accurate secondary assays are used for elucidating the
biological role and dose-response curve calculation.
Analogues are made of the most promising of these compounds and they in turn are
subjected to the screening procedure. This sequence of selective screening and
synthesis of analogues may be repeated many times before a potentially useful drug is
found.
Drug Design
The design cycle describes the optimization of a lead strucutre to one or several
development candidates. It is and iterative process with evolutionary character
Lead identification: High-throughput screening
Screening perhaps millions of compounds in a corporate
collection to see if any show activity against a certain
disease protein
Lead identification: advent of systematic screening
Development of automation and in vitro high-throughput biological screens
has had a dramatic impact on lead discovery.
Molecular biology has provided the tools for identification and validation of
therapeutic targets, cloning and expression of sufficient protein to
accommodate high-throughput screening, and determining the impact of
elimination of the therapeutic gene by knock-out mutations.
Lead identification: screening methodologies
Receptor screening methodologies can be based on either the determination of
a functional response (e.g. cell proliferation) or on the interaction of a ligand
with its receptor.
There is a trend towards the development of cell-based assays (e.g. to replace
animal studies), which has been facilitated by recombinant DNA technology
using reporter gene systems. Binding of a ligand (agonist or antagonist) to its
cognate receptor is the initial and indispensable step in the cascade of
reactions that finally cause a pharmacological effect and many successful and
widely used techniques are thus based on measuring ligand binding.
Sources of drugs
Once the ability to screen libraries developed, the pressure on medicinal chemists
increased to generate large quantities of compounds for screening.
It originally started with drugs and lead compounds derived from natural sources, such
as animals, plants, trees and microorganisms. Marine sources were not utilized to any
extent until the mid-20th century.
Today, natural sources are still important, but the majority of lead compounds are
synthesized in the laboratory. The nature of these synthetic compounds is initially
decided from a consideration of the biochemistry of the pathogenic condition.
Natural sources
Natural sources are still important sources of lead compounds and new drugs.
However, the large diversity of potential natural sources in the world makes the
technique of random screening a highly empirical process.
The screening of local folk remedies (ethnopharmacology) offers the basis of a more
systematic approach
powerful analgesic, about 200 times more
potent than morphine
potent neurotoxin
The principal alkaloid in
opium and the
prototype opiate analgesic
and narcotic.
A methylxanthine naturally occurring in some
beverages and also used as a pharmacological
agent
its primary
therapeutic use is in
the treatment of
gout
An alkaloid derived from the bark of the cinchona tree.
It is used as an antimalarial drug
An alpha- and beta-adrenergic agonist that may also
enhance release of norepinephrine
Lead compounds
broad-spectrum antibiotic produced by the streptomyces bacterium
Antibiotic
derived from the actinobacterium
Streptomyces griseus.
cholecistokinin
Natural sources
Once screening identifies a material containing an active compound, the problem
becomes one of extraction, purification and assessment of the pharmacological activity.
However, the isolation of useful quantities of a drug from its land or sea sources can
cause ecological problems.
The promising anticancer agent Taxol, for example, was originally isolated from the bark
of the Pacific yew tree. However, the production of large quantities of Taxol from this
source would result in the wholesale distruction of the tree (six 100 year old trees for a
patient), a state of affairs that is ecologically unacceptable.
Let’s try not to do as Cirenaici with Silfio
Anticancer agent
Lead identification: use of compound libraries
Synthetic Compound libraries
Using traditional methods of chemical synthesis the potential drug
substances are synthesised one at a time, purified, and then, after their
structures have been confirmed, are tested for their effectiveness as
drugs. So it could take many years to build up such a library.
Over the past few years several techniques called collectively
‘combinatorial chemistry’ have been developed which enable chemists to
prepare libraries of thousands of related compounds quickly. These
techniques use automated methods called ‘parallel synthesis’
Lead compounds
Figure 1 All new chemical entities, 01/1981 06/2006, by source (N = 1184).
Major Categories of Sources. The major categories used are as follows:
“B” Biological; usually a large (>45 residues) peptide or protein either isolated from an
organism/cell line or produced by biotechnological means in a surrogate host.
“N” Natural product.
“ND” Derived from a natural product and is usually a semisynthetic modification.
“S” Totally synthetic drug, often found by random screening/modification of an existing agent.
“S*” Made by total synthesis, but the pharmacophore is/was from a natural product.
“V” Vaccine.
Drug chemical space
Rational design
The MAGIC BULLET
The search for the magic bullet has inspired scientific drug discovery from its very beginnings
and has remained with this multidisciplinary endeavour ever since
In drug research, with the growth of chemistry, pharmacology and other disciplines, the ideal
of generating ‘magic bullets’ or achieving exclusive and absolute specificity has been a
constant aim.
The switch from mechanism- to target-oriented drug discovery brought about by molecular
genetics and genomics has recently re-established this ideal in a strict molecular context.
Ideally, an agent should interact with one selected target, elicit a premeditated or intended
response, disengage from the target and be metabolized and excreted without any further
effects on the organism in question.
The market
For the first time in decades, the oncology therapeutic area has overtaken cardiovascular
medicine as the leading revenue contributor
A major reason for the rise of oncology as a revenue contributor comes from the success of
bevacizumab (Avastin), rituximab (Mabthera/ Rituxan) and trastuzumab (Herceptin) and of
imatinib (Glivec/Gleevec)
Rational design
GLEEVEC case
Chronic Myeloid Leukemia (CML)
The Philadelphia (Ph) chromosome is an abbreviated chromosome 22 that was shortchanged
in an a reciprocal exchange of material with chromosome 9.
This translocation occurs in a cell in the bonemarrow cell and causes CML
On a molecular level the Philadelphia
chromosome translocation results in the
production of a fusion protein.
A large portion of a proto-oncogene, called
ABL, on chromosome 9 is translocated to the
BCR gene on chromosome 22.
The two gene segments are fused and
ultimately produce a chimeric protein that is
larger than the normal ABL protein.
Chronic Myeloid Leukemia (CML)
Modes of action
The core structure of the lead compound (black)
The addition of a 3'-pyridyl group (blue)
enhanced the cellular activity
An amide group (red) provided activity against
tyrosine kinases.
A 'flag methyl' (purple) abolished the undesirable
protein-kinase-C inhibitory activity
The attachment of an N-methyl piperazine moiety
(green) markedly increased the solubility and oral
bioavailability.
Docking of the molecule on the 3D structure of the
Abl kinase
Ribbon representation of the three-dimensional structure of Abl kinase domain in complex
with gleevec. The molecular surface of the inhibitor is shown. A central conserved region of
the kinase, the catalytic segment, is shown in green and the activation loop in magenta.
Clinically, CML is a chronic disease that evolves through three successive
stages, from the chronic phase to the end stage of blast crisis.
Overall, the median survival time of patients with newly diagnosed CML is
approximately 5–6 years.
The clinical development was particularly rapid, as can be seen by comparison with the
typical drug discovery and development times shown in the inset. An NDA for Glivec was
submitted just two years and nine months after treatment of the first patient with CML,
and FDA approval was given less than three months after application. CML, chronic
myelogenous leukaemia; GIST, gastrointestinal stromal tumour; NDA, new drug application;
PKC, protein kinase C.
Monoclonal antibodies:
Molecular biomedical research identifies potential drug target
EGFR and VEGFR signaling share common pathways. The network of both family receptors may
control pathways that affect cell proliferation, survival, differentiation, and migration.
Antibodies
Complex protein-based molecules produced by B lymphocytes that bind to and help to
eliminate foreign and infectious agents in the body.
Antibodies are Y shaped, having two sets of branches attached to a single stem.
The arms of the Y (Fab) are the so called variable regions, the tips of the arms contain
antigen-binding regions, and the stem (Fc) is a constant region.
The constant regions trigger effector functions (phagocytosis, cytolysis) by linking the
complex to other cells of the immune system.
Monoclonal antibodies (mAbs) can be designed to selectively target tumour cells and elicit a
variety of responses once bound.
206 therapeutic oncology mAbs entered clinical study sponsored by commercial organizations
between January 1980 and December 2005 (8.24/year).
To date, 12 of these anticancer mAbs have been approved for marketing in at least one
country
The success of mAbs is due to the rapid identification of new molecolar targets and to the
improvement of biotechnological methods for the production
Originally, mAbs were antibodies produced from a single B lymphocyte. mAbs of a defined
peptide sequence have identical antigen-binding regions and bind to the same site (the
epitope) of an antigen.
Modes of action
mAbs are potentially capable of multiple functions.
The mode of destruction can be
Direct: carrying toxic material to the target via conjugated radioactive isotopes or toxins,
or antibody triggered apoptosis
Indirect: by activation of immune system components, blockade of critical receptors,
sequestering growth factors or inducing apoptosis. In the case of soluble antigens, the
mAb should sequester the target.
Conjugated mAbs can increase the specificity of chemo- or radiation therapy and improve
the efficacy of immunotherapy, but have some drawbacks; they are more difficult to
manufacture and may have greater safety issues compared with their naked counterparts.
Immunoconjugates of various kinds constituted 44%
of the total anticancer mAbs in clinical study to date
Most immunoconjugates have been designed to
carry a radioactive isotope (radioimmunoconjugates) or a toxin (immunotoxins).
Immunotoxins composed of mAbs that are
conjugated to a wide variety of either protein or
small-molecule cytotoxins have also been studied.
3 of the 12 are radiolabeled,
1 conjugated with immunotoxin
For both type problems arise due to the lack of
potency at the doses delivered to the tumour site
Unconjugated mAbs can function through more than one mechanism, but a common primary
mode of action is the destruction of targeted cells through activation of components of the
human immune system.
For example, after binding to a target, mAbs can recruit effector cells such as natural killer cells
or macrophages to destroy the target
Although all eight currently approved unmodified anticancer mAbs potentially have the ability
to function through activation of the immune system, only two — rituximab (Rituxan; Biogen
Idec) and alemtuzumab (Campath; Genzyme) — are believed to use this way as their primary
mode of action.
The other six products function through alternative primary modes of action (blockade of
growth factor–receptor interaction, receptor downmodulation and inhibition of signalling).
mAbs are not necessarily restricted to a single mode of action.
For example, trastuzumab (Herceptin; Genentech) has been reported to induce immune system
modulation as a possible alternative or additional mechanism to human epidermal growth
factor receptor 2 (HER2) downmodulation
Efficacy
The use of monoclonal antibodies is diffuse in cancer therapy
Clinical responses varies from 15-50% of patients treated in clinical trials (partial or full
response).
Some MAbs have increased the efficacy of treatment of certain tumors, with acceptable safety
profiles.
Issues to be addressed include dosing strategies, timing, and schedule of antibody
administration; duration of treatment; need for tailoring; and further testing under specific
circumstances.
The discovery of effective combinations with other biologic agents would be very useful.
Multimodality approaches, based on synergistic effects observed with the combination of
antibodies with chemotherapeutic drugs and/or radiotherapy also merit further investigation
Tumor initiation
For decades, tumour initiation and development has
been regarded as a multistep process that is
reflected by the progressive genetic alterations that
drive the transformation of normal human cells into
highly malignant derivatives.
As cancers arise only after multiple mutagenic
events, long-lived cells are probably the most capable
of supporting such cumulative changes.
Cancer cells are genetically instable
It has been proposed that progressive genetic
alterations might occur at the level of tumour-initiating
cells, from which tumor originates (clonal
progression)
Stem Cell features
- undifferentiated
- self renewal by asymmetric division
-can divide without limit
Cancer Stem Cell features
(acquired by mutations)
- un (de) differentiated
- self renewal by asymmetric division
- can divide without limit
In normal tissues, the heterogeneity of cells reflects a hierarchical programme of
differentiation in which multiple mature cell types are derived from a common
multipotent stem cell through intermediate progenitors.
Heterogeneous populations of cancer cells at various differentiation stages could
be the result of both acquired mutations and aberrant but hierarchical
differentiation programmes.
Cancer is both a proliferation and a differentiation disease, and the 'clonal
evolution' and 'cancer stem cell' models might not be mutually exclusive, as initially
thought.
Owing to genetic instability, the tumour-initiating cells isolated from a clinically
detectable tumour would probably have a substantially different genetic profile
from the initial transformed cells that originated the tumour.
Drug development
• Over 90% of drugs
entering clinical trials fail
to make it to market
• On average, it takes 1015 years to create a new
drug
• The average cost to bring
a new drug to market is
estimated at $770 million
Drug discovery process
1 Drug
Molecule
patentable
nonnon-teratogenic
10,000 Drug
Candidates
Valid
Biomedical
Hypothesis?
Statistics show that out of each 10,000 or so new molecules generated, only about one will ultimately survive
the array of requirements necessary for a candidate molecule to become a drug that reaches patients
Drug development process
1 Drug
Launch
CostCost-effective manufacturing
Carcinogenicity studies
10 Drug
Molecules
The challenge continues in development
Drug development process
Identify disease
Isolate protein
involved in
disease (2-5 years)
Find a drug effective
against disease protein
(2-5 years)
Scale-up
Preclinical testing
(1-3 years)
Human clinical trials
(2-10 years)
Formulation
FDA approval
(2-3 years)
The number of new drug approvals is decreasing and the cost
for research and development is constantly increasing
- Drug discoverers have recently been able to generate molecules that
fulfill the demands of the ‘magic bullet’ concept to a considerable
degree
- Conventional cancer chemotherapy should be replaced step by step by
targeted therapy illustrates what has already been achieved and
indicates how drug therapy against cancer should develop over the
coming years
- In spite of a growing sophistication in describing the molecular details
of a particular target the ideal of designing drugs de novo or synthesizing
tailor-made chemicals that fulfill all requirements of efficacy, tolerance
and absorption, distribution, metabolism and excretion (ADME)
characteristics has remained elusive
The principle of the ‘magic bullet’ itself — or, as we would say today, the
concept of exclusivity in which a drug interacts with only one target
at least in some multifactorial diseases, a drug with multiple wellchosen points of attack might be preferable to a drug with only one
target, even if that target is crucial and its engagement by the drug in
question absolutely specific
Some authors have therefore advocated a return to biology-driven
strategies that expose drug candidates to more complex test systems
Approaches of this type might indeed help to select compounds that
address a typical combination of targets, rather than a single molecular
entity the so called ‘magic bullets of the second order’
Bibliografia:
Per la parte storico-introduttiva:
“Il progresso terapeutico” di Colapinto Leonardo, Annetta Antonino, Aboca Edizioni
Per la parte scientifica:
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DISCOVERY, JULY 2002 | 493
Jeffrey M. Rosen1 and Craig T. Jordan2, 26 JUNE 2009 VOL 324 SCIENCE, 1670
Janice M. Reichert and Viia E. Valge-Archer, NATURE REVIEWS | DRUG DISCOVERY, VOLUME 6 | MAY 2007 |
349
Jürgen Drews, NATURE REVIEWS | DRUG DISCOVERY, VOLUME 5 | AUGUST 2006 | 635
2008 FDA drug approvals, NATURE REVIEWS | Drug Discovery, VOLUME 8 | FEBRUARY 2009 | 93
David J. Newman* and Gordon M. Cragg, J. Nat. Prod. 2007, 70, 461-477
Sales trends by therapeutic area: 2008–2013°, nature reviews | Drug DiscovEry, volume 8 | september 2009
| 689
Sales of biologics to show robust growth through to 2013 nature reviews | Drug DiscovEry, volume 8 |
november 2009 | 837
Bin-Bing S. Zhou*, Haiying Zhang‡, Marc Damelin*, Kenneth G. Geles*,
Justin C. Grindley* and Peter B. Dirks§, nature reviews | Drug DiscovEry 806 | ocTober 2009 | VoluMe 8
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TEORIA 2 (4 dicembre 2009)