development of new therapeutical agents
TRANSCRIPT
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Development of new therapeutical agents
1. Pharmacometrics
1.1. Pharmacodynamic modelling
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1. Pharmacometrics
The main goal of pharmacometrics is to design the best chemical trials and build the
best models using a mathematical structure. It integrates many pharmaceutical sciences like
physiology, pharmacology, chemistry, pharmacokinetics and pharmacodynamics. Building
models with pharmacometrics allows
predicting results from a few experimental data. This
science became so important that FDA made a “critical path”, which are directions for the
drug development to make this process more efficient. One of this directions was to apply
pharmacometrics in this process.
Examples:
- We have experimental data for 2’5 mg/Kg and 5 mg/Kg doses. After building a model from
this data we’ll bioequivalencia able to predict the results for higher doses like 7’5 mg/Kg or 10
mg/Kg.
- In oncology: there are studies that relate biomarkers in blood with tumor size and survival rate of patients to know which is the best time to change the treatment before the patients
develop resistance to the drugs.
1.1. Pharmacodynamic modelling
Pharmacodynamics is what the drug does to the body, and it shouldn’t be confused
with pharmacokinetics, which is what does the body to the drug (distribution, metabolism,
excretion..). In cancer treatment, for example, a patient receives a cytostatic drug infusion for
6 hours (at this time there is maximum concentration). A good marker to observe the drug
effect is the neutrophils levels in blood, and the lowest levels are reached after 11 days. How
can this be explained? Maximum concentration of the drug in blood is reached after 6 hours,
but the effect appears after 11 days. This is because of the pharmacodynamic effects like
phosphorylation, DNA changes, etc. that need some time to perform. Another example are
the direct response models, where all the pharmacodynamic events happen very fast, and the
limitant step is the concentration of the drug in blood. In conclusion we can say that
pharmacometrics integrates pharmacokinetics, pharmacodynamics and system properties.
The most popular pharmacodynamic model y the Sigmoidal Emax model, it shows a
direct relationship between concentration and response (direct, not linear). We can define the
pharmacodynamic parameters:
- Emax: it's the maximum drug effect that we can achieve.
- C50: concentration of drug that gives a response that is half of Emax.
- n (gamma): sigmoidicity parameter. If n is very high, there's a threshold concentration, which
means that we go from no response to a very high response.
Other pharmacodynamic models are: Exponential model, linear model, combination of
Emax and linear models. [Ecuations][HW]
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Let's suppose that before taking a drug response is not = 0, which means there's a
baseline value. We can't use a PD model without considering the baseline condition (E0), it
has to be included and estimated during the analysis. We can even study a drug that lowers
this baseline condition, for example.
1.2. Drug interactions
If we administer a patient two or more active principles, we can have different
interactions:
- Additivity
21-octubre-2013
Oral/ mucosal administration of nanoparticlesPK modifications following oral drug delivery: Vincamicine, hydrocortisone, antigens,
insulin, indometacin, plasmids, human growth hormone, etc.
Oral absorption os nanoparticles:
- Prerequisite: direct contact or adhesion to the mucosal surface
- Reported mechanisms for uptake and translocation:
- Endocytosis by absorptive cells / instracellular uptake: very small particles (
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homogeneization.
- Developed to improve the dissolution rate (not the solubility) of highly lipophilic drugs and to
increase the specific surface. They can be administered iv or orally.
- Passive targeting: poor controlled release properties.
Polymer therapeutics - The term describes several distinct classes of agents:
a) Polymers
: drug conjugates and protein conjugates
- Drug/protein is covalently bounded to a water soluble polymer (MW
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d) Polyplexes
: complexes of cationic polymer with DNA (anionic). For non-viral vectors.
Lipid-based nanodrug delivery systems
a) Liposomes :
- Phospholipid bilayers with an entrapped aqueous volume containing cholesterol as stabilising agent.
- Types: MLV (multilamellar, >200nm), LUV (large unilamellar, 100-400nm), SUV
( CO2 + H2O).
- How are these polymeric particles prepared? By coacervation, salting out, ionic gelification,
multiple emulsification, solvent evaporation. A drying process like lyophilization protects from
erosion. the melting point of the nanoparticles should be higher than the body temperature.
Nanoparticles administered by IV route
- Adsorption/ deposition of blood opsonins with no anaphylactic reaction (no LPS).
- Captured by cells of the MMS (Monocyte macrophage system) of the liver or spleen. That
means there’s a transport after the adsorption.
- Factors affecting the rate of opsonization and clearance: size, shape and surface properties.
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Distribution of nanoparticles
a) Influence of the size :
- >200nm: activate rapidly the complement system and are sequestered by the MMS.-
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- Residual virulence: severe infections in immunocompromised patients.
- Sterility conditions, handling..
- When we use needles for vaccine inoculation if the mucosal routes offer security and
convenience?
- Reuse of syringes and needles in the absence of sterilization. Worldwide 40% -->
70%- Unsafe injections can transmit hepatitis B/C and HIV causing estimates 1.3 M early
deaths and annual burden uf USD 535 M in direct medical costs. Estimated annual deaths:
75.000
3. Adjuvants / immunopotentiators
Any substance, combination of substances or strategies, that enhance the immune
response against the antigen and biases the immunological response to either Th1 or Th2
style responses.
- Aluminium salts: polarize response towards Th-2
- MF59: approved in Europe. Firstly Th-2. Th0 adjuvant, which enhances whichever response is present, without biasing the profile.
- Antigen delivery systems (or facilitators of signal 1):
- Prolonged residency of the antigen and facilitate the presentation to and uptake by
APCs.
- Types: mineral adjuvants (alhydrogel, alum), emulsion, surfactants and derived
adjuvants (FIA, saponins), lipid particulate adjuvants (liposomes, virosomes), particulate
adjuvants (micro and nanoparticles)
-Facilitators of signal 2
- Ability to directly activate immune cells during antigen recognition and presentation.
- Types: pathogen-associated molecular patterns (PAMPs): LPS, murein, CpG, motifs, adhesins, cholera toxin B subunit.
- Lack for efficient mucosal adjuvants.
4. Rationale
- Some data about the mucosal immune system: about 60% of the total body lymphocytes are
in organized follicles (MALT).
- M-Cells: primary route by which antigens are transported in an unmodified form
(enterocytes).
- DCs (dendritic cells): sentinels in epithelium and PP that project dendrites to sample luminal
antigens.
- Toll-like receptors (TLR): Pattern-recognition receptor, proteins able to recognize molecular
markers in pathogens but absent in the host.
5. Nanoparticles for oral vaccination :
- Advantages: protection against degradation, controlled release properties.
- Drawbacks: failure to elicit immune response. Need of high and multiple doses, boost
strategy by s.c. injection.
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- Factors influencing the efficacy of NPs:
- Material: chitosan, PLGA, lipids, controlled release properties / bioadhesive
properties.
- Size: controversial: NPs rather than MPs more adapted for immune stimulation.
- Surface properties: biodistribution properties (ligands), slippery surface / bioadhesive
properties.- Other: methods of antigen loading (very important for plasmids).
6. PVM/MA
- Synthetic polyanhydrides.
- Low oral toxicity.
- Low cost.
- Pharmaceutical uses: denture adhesives, film coating agents, adjuvants for transdermal
patches. thickening and suspending agents.
- They can activate directly immune cells.
7. Biomimetic nanoparticles
To copy or imitate closely the bacteria and virus behavior (colonization and invasion)
- Bacterial colonization: first stage of microbial infection.
- Establishment of the pathogen at the portal of entry.
- Adhesion to a mucosal surface in two steps: reversible attachment by non specific
forces (hydrophobic, electrostatic), specific adherence or anchoring. Involves a receptor and a
ligand (Adhesins, glucoconjugates).
GENE DELIVERY SYSTEMS (Dra. Tros)We have two main groups of gene vectors used in gene therapy: viral and non viral
vector.
The main viral vectors are adenovirus, retrovirus, AAV and lentivirus. The main non-viral
vectors are made from peptides, polymers and polymers that form polyplexes. If we have
lipids instead of polymers we'll have lipoplexes.
1. Viral vectors
- Retrovirus:
- Advantages: High level of transfection, long-term expression, it can infect the most of
the cell types.
- Disadvantages: Instability of the retroviral particle, it doesn't infect non-proliferating
cells.
- Adenovirus:
- Advantages: Non-pathogenic
- Disadvantages: highly immunogenic
Disadvantages of viral vectors:
- Risk of tumorigenic mutations
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- Risk of oncogene activation
- Risk of generating viral particles
- Immunogenicity
-...
2. Non-viral transfection methods- DEAE- Dextran: it's the first transfection method, not very used nowadays. Principle: Charge
interaction. Advantages: Well.stablished method, high efficiency in some cell lines.
Disadvantages: Cytotoxicity, etc.
- Calcium phosphate
- Electroporation: high voltage leading to pore formation in cell membrane, but it can't be used
in humans.
- Cationic liposome: charge interactions between the liposome (+) and DNA (-), high
transfection efficiency. Citotoxicity and inhibition by serum.
3. Critical parameters for efficient transfection3.1. Method of transfection
It's important to choose the best one for each application.
3.2. Quality of DNA
It should be purified before it reached the cells.
3.3. Cell line
In the lab, a cell line is a cell type that multiplies continuosly, cancer cells for instance.
3.4. Media and quality of cell culture
The serum, the nutrients we have in the cell line that allow the cells to grow properly.
4. Ideal non-viral delivery system- Structurally well characterized, non-toxic, non-immunogenic.
- Efficient dissociation of DNA from the complex.
- Controlled duration and magnitude of expression.
- Protects DNA from degradation. (the serum degradates DNA)
- Stable in biofluids.
- Large-scale production.
5. delivery: everything before transcription
5.1. Pre-cellular (injection-->cell membrane)
- Interaction with serum
- Interaction with blood cells
- RES clearance
- Tissue competition
- Extravasation
5.2. Sub-cellular (cell membrane-->transcription)
- Binding and internalization
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- Endosomal release/cytoplasmic delivery
- Nuclear entry (the particle should reach the nucleus, its size can't be very big- 10-15nm)
- De-complexion
6. Lipidic systems
Lyposomes in gene delivery: liposomes are simply vesicles in whiche an aqueous volume is entirely enclosed by a membrane composed of lipid molecules.
6.1. Liposome composition
- Neutral lipids:
- Lecitins (Phophatidylcholines - PC)
- Shphingomyelins
- Cholesterol
- Negatively charged: PG
- Positively charged lipids
- Natural- Synthetic: DOTA.
6.2. Types of liposomes
- Multilamellar vesicles (MLVs) 100-1000nm
- Large unilamellar vesicles (LUVs) > 500 nm
- Intermediate-size unilamellar vesicles (IUVs) 100-500 nm
- Small unilamellar vesicles (SUVs) < 100 nm (Good systems for DNA)
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6.3. Preparation of liposomesLipids are dissolved in organic solvents (acetone, chloroforme), then the solvents are
dried and we obtain a dry lipid film. After that, water should be added to rehydrate, and finally
the film suffers swelling. After this point, depending on the size of the liposome we want to
get, we should use different techniques. Example: Sonication (for SUVs), extrusion (LUVs),
etc.
6.4. Advantages of liposomal vectors
- Targeting to specific cells or tissues. (ex: a particle that goes only to tumoral cells)
- Protection of DNA from degradation in biological milieu.
- Delivery of large pieces of DNA
- Non-immunogenic
- Safe relative to viral vectors
- Ease of large scale production
6.5. Current limitations
- Low efficiency compared to viral systems
- Toxicity
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- Inhibition by serum
- Relatively large particle size
6.6. Liposomes used in gene delivery
- Stable non-fusogenic
- Fusogenic pH-sensitive- Fusgenic cathionic
6.7. Mechanism of transfection
- Fusion: for fusogenic lipids, it fuses with the target cell membrane.
- Receptor-mediated endocytosis
7. Polymeric and peptidic systems
The main polymers are polylysine (PLL), polyethylenimine (PEI - toxic),
polyamidoamine dendrimers (PAMAM), poly-D-L-lactic-co-glycolic acid (PLGA). Others are
histones and protamine (very good to reduce the size of the particle because it's able to condense DNA).
7.1. Transfection by polyplexes
We have a positively charged particle, otherwise there's no way to interact with the
negatively charged membrane unless we have a ligand. The idea is to have an electrostatic
interaction with the cell membrane. When the polyplex is internalize, there's a change in the
osmotic pressure, so that the DNA can get our of the particle and then introduced in the
nucleus.
7.2. Strategies for the design of a ligand / DNA complex- Ligand directly binds to DNA:
- DNA binding antibodies
- DNA intercalating ligand complexes
- Ligand binds to a high affinity DNA binding polycation
7.3. Efficient gene delivery by transferrin-lipoplexes
Transferrin receptors are universal receptors because they are not specific.
[Dibujo Transferrin complexes]
Experimental conditions:
- HeLa cells (cervix carcinome - high level receptor expression) (80-90% confluency)
- 1 microgram of DNA/ well beta-galactosidate (pCMV LacZ) - marker, if expressed, bacteria
turn blue.
- DOTAP/DOPE liposomes (1:1 molar ratio)
- 32 microgram transferrin
- 4h transfection
- 48h for expression (cells lising and analyzing)
- It's very important to test the protection of DNA, so that it doesn't degradate. This is
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necessary before starting animal experimentation.
The mechanism of transferrin lipoplexes is receptor-mediated endocytosis, and there's
a competitive competition by free transferrin. It's really important to know how the system
works, so that we can improve it. It worked in culture cells, and after making the experiment in
mice the results were successful.
8. Antitumoral studies
Experiment: a colon carcinoma was induced to mice, and the treatment was with IL-12
gene administered subcutaneously. Tumor size decreased significantly in comparision with
controls. It also was studied why does the tumor disappear. IL-12 is able to stimulate the
immune system: that was the hypothesis. Then, IL-12 levels were measured in serum and it
was prooved that they were high.
9. Enhanced gene delivery to the liver by Asialofetuin-lipoplexes
These lipoplexes have a ligand that binds to the receptor in the liver, and they are
used to treat hepatocarcinoma. In HeLa cells we get no results because of the absence of this receptor.
It was really demonstrated that there was a change in the gene expression in the liver.
BIOLOGY AND BIOTECHNOLOGY MEDICINE
(Dra. Garrido)
Antibodies
The main cells that participate in immunity are macrophages and lymphocytes B and
T. The function of T-helper cells are:
- Antigen presenting cells (APCs) present antigen on their Class II MHC molecules (MHC2)
The antibodies are composed by a heavy chain and a light chain, where there's the epitope (it
recognizes the antigen). The heavy chain is able to induce the immune response.
Relative abundance:
-G: 1250 mg/100mL
- A: 210
- M: 150
- D: 3
- E: 0.03
IgG, IgA and IgM are called the "natural immunoglobulines". The main difference
between the immunoglobulines is the carbohydrate group on the heavy chain.
The immunoglobulin is prepared from pooled plasma from 3000-10000 healthy blood
donors. The large number of donorse makes more possible a contamination by virus. Natural
antibodies are produced by B lymphocytes in the absence of external antigen simulation.
1952: first use of immune globulines (Ig) from human plasma to treat immune deficiency.
1981 IVIG showed efficacy in autoimmune idiopathic thrombocytopenic purpura.
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Functions:
- Recognize self, altered self and foreign antigens, representing a first line defense.
- Can bind to pathogens.
- Tissue homeostasis, help to remove senescent or altered molecules cells, and tumors.
- Anti-tumorigenic function: by recognition of oligosaccharides expressed on tumor cells.
- Induce remyelination in the CNS- Inhibit the growth of autoreactive B-cell clones.
Production: from the plasma of approximately a thousand or more blood donors. (filtration)
Presentation: Intravenous immunoglobulines (IVIG), sterile and contain more than 95%
unmodified IgG with intact Fc-dependent effector functions and only trace of IgA or IgM. Half
life = 1 month. Before it was one week, before the modification of the purification process.
Some characteristics:
- All preparations of IgG have traces of IgA.
- IgA antibodies have also potent immunomodulatory properties, being able to both induce and suppress immune responses.
- IgA-mediated inhibitory function is able to inhibit several inflammatory diseases including
asthma and glomerulonephritis.
- IgM has shown promising results for multiple sclerosis treatment.
Applications of IVIG approved by FDA:
- Allogenic bone marrow transplantation.
- Chronic lymphocytic leukemia.
- Pediatric HIV type I infection...
- Obstetrics: helpful for recurrent pregnancy loss.- Pulmonology: asthma, chronic chest symptoms.
Spain: IG inespecíficas:
- Inmunodeficiencias primarias: para déficits congénitos o adquiridos: presentan infecciones
recurrentes, sobre todo respiratorias con complicaciones graves. Dosificación y posología se
basan en dos criterios: administración de dosis necesaria para impedir la aparición de
infecciones, y la monitorización sérica de las Ig.
- Inmunodeficiencias secundarias: transitorias o permanentes debidas a alteraciones
metabólicas, hematológicas o infeccionsas. Las hay que cursan con alteración en la
inmunidad humoral por bajos niveles de Ig. Déficit en la respuesta a Ag.
- Enfermedades autoinmunes: presentan eficacia en varias enfermedades aunque su
mecanismo inmunomodulador es muy complejo.
Indicaciones aprobadas en España: tratamientos de reposición y efecto modulador
(enfermedades autoinmunes).
IG Específicas:
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- Características: contienen un alto nivel de un solo antocuerpo.
- Anticuerpo monoclonal: Palivizumab. Prevencion de infecciones severas delt racto
respiratorio inferior causadas por el virus respiratorio sincitial (VRS).
- Production: hybridomas. Purification in column of chromatography.
Types of mAbs- omab: murins. Problems: immunogenicity response.
- ximab: quimerics. Variable part is from mice, and the heavy chain is from human.
- zumab: humanized. Some specific fragments of the variable part are murins. Rest is human.
- umab: fully humanized.
Oncology:
Several mechanisms are involved.
- We use monoclonal antibodies to destruct cancer cells by binding to a specific antigen
expressed in a specific antigen cells [CD20 (rituximab-breast cancer)-CD52].
- By binding to receptors in the surface inhibiting signals for cell proliferation (VEGF-(angiogenic process), EGFr)- trastuzumab, cetuximab (colon cancer), panitumumab.
- By stimulation of immune cells like cytotoxic Y lymphocytes (Ipilimumab - phase III)
- Immunoconjugates like immunotoxins or antibody drug conjugated.
Targeted therapies:
- Agents able to block the growth or spread of cancer by interfering with specific molecules
involved in tumor progression.
- Maint point is the identification of good or specific targets such as receptors or antigens.
- First molecular target for targeting was the "hormon estrogen receptor" involved in breast
cancers.- Mechanism for control breast cancer:
- Using receptor modulators, Tamoxifen only block the receptor.
- Destroying the receptor, Fulvestrant
- Aromatase inhibitors, by blocking the enzyme responsible to product estrogen,
Exemestane.
- Therapy focused on receptors: EGF, VEGF (Cetixumab, Bevacizzumab).
- Therapy to activate the immune system (Rituximab)
- Therapy to deliver toxic molecules (Ibritumomab tiuxetan)
Gene therapy:
- gene therapy show many obstacles nefore it can become a practical approach for trating
disease.
- At least four of these obstacles are: gene delivery tools, high costs, limited knowledge of the
functions of genes, and multigene disorders and efect of environment.
PD-1/PD-L1 BLOKADE CHECKPOINTS IMMUNOTHERAPY
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It's the newest strategy in cancer therapy, it's used to control the main checkpoints in
immunotherapy.
- Immune system under normal conditions: (to minimize collateral tissue damage)
- Maintenance of self-tolerance
- Modulation of physiological immune responses
- Tumors:- Immune-checkpoint pathways as a major mechanism of immune resistance:
tolerance. The tumor is like a chronic infection, finally it developes tolerance and it escapes
from the immune system. The tumor releases different inhibitory cytokine that provoke
lymphocytes apoptosis when the T-cell binds through the ligand. On the other hand, the
T-cells can bind to the ligand and produce cell killer cytokines (tumor death). If we use an
antibody that binds to the ligand of the tumor cells, the T-cell can continue to release cell killer
cytokines.
- Relevance:
- Expression: tumor growth + apoptosis of t-cells- Expression: prognosis and metastasis--> biomarker
- Immune inhibition
* An analysis of human tumor lines showed that there are baseline levels of PD-L1
It's observed that PD is dosis independent, there's a permanent block (irreversible)
PD-L1 or PD-1 blokade alone may be insufficent, so it's necessary to use a rational
combination of the blokage. Advantage: long lasting responses. Disadvantage:
immunogenicity.
Solution: combination of therapies.
Challenges:- the definition of potential biomarkers that can determine which immune-checkpoint pathway
or pathways dominate in particular tumos as well as the PK-characterization of monoclonal
AB.
- Combination therapies will need to be evaluated because of the hepatotoxicity.
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Targeting of EGFr with different ligands coupled to PEG-liposomes (Title)
They are liposomes pegylated in their surface. They have higher half life than
conventional liposomes, that means they are circulating in the blood for a longer time and that
they reach better the target tissue. It's possible to couple/link molecules in this polymer to
make targeting possible.
Introduction:
Colorectal cancer is the second cause of death in EU, the mortality is around 30% in the
developed world. The treatment is Capacitabine + Oxaliplatin + Leucouorin.
Oxaliplatin belongs to the third generation of platinum derivatives. It is in the first line of
chemotherapy in metastasic CRC within combined therapies (FOLFOX regimen). It has a
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good tolerability in patients with a manegeable side effects (neurotoxicity, reversible not like
others with irreversible ototoxicity). Disadvantages:
- Rapid distribution to the tissues.
- High partition coefficient with the plasmatic proteins and erythrocytes. (irreversible,
that makes it inactive)
- Rapid circulation clearance.Result: low clinical efficacy in monotherapy, that's why it's necessary to administrate a
high dose to get a good effect.
Nanotechnology gives a solution:
- Controlled drug release.
- Decrease the nonspecifical interactions.
- Increase the blood residence time.
- Increase the tumor drug accumulation.
*Effective anti-tumor activity of oxaliplatin encapsulated in transferrin-PEG-liposome* Because
tranferrin is a universal ligand. In 2009: *Oxaliplatin encapsulated in PEG-coated cationic
liposomes*. *Application of different methods to formulate PEG-liposomes of oxaliplatin: Evaluation in vitro and in vivo: summary of results:
- PEG-oxaliplatin liposome
- Better PK properties
- More anti-tumor effect than free drug. *Done in Xenograft animals: we inject human
cells in an animal model to induce tumor growth* *Singenic animal models: when we use
targeted therapy that induces immune regulation, we induce tumor with animal cells*
"Magic bullet" concept: a compound that selectively target a disease-causing organism
avoiding side effects in the patient. Targeted liposomes:
- Specific target for encapsulated compounds.
- Low incidence of side effects- Higher therapeutic effect.
Introduction to Epidermal Growth Factor receptor (EGFr)
This receptor is over expressed in many types of solid tumors, and also its variability is very
high (expression from 14-91%). The activation of this receptor induces phosphorylation, and
that induces the phosphorylation of different kinases to produce cell proliferation, inhibition of
apoptosis, angiogenesis, migration, adhesion, invasion (metastasis). Currently there are 2
types of treatments: intracellular inhibitors (Erlotinib, Gefitinib) that stop phorphorylation of the
receptor; and the use of monoclonal AB (Cetuximab) that bind to the extracellular part of the
receptor. It's a quimeric monoclonal AB. In the FOLFOX tratment, this antibody has
substituted Leucouorin.
Aims of this stydy: the development of different types of EGF receptor targeted
oxaliplatin liposomes and their iv-iv evaluation. We want to make liposomes with Oxaliplatin in
the inside, and Cetuximab in the surface.
Steps:
1. PEG-coated Liposome formulation (Stealth liposome). Film method (1978). PEG2000 will
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bind to Cetuximab and the polymer (covalent bond). Dil is a fluorescent proof to check the
distribution of the liposomes.
2. Fab fragment obtention: We get the Fab2 fragment from Cetuximab using pepsine. After
that, we divide the V fragment in a mono-valent fragment with beta-mercaptoethanol.
3. Ligand coupling to preformed liposomes. Thiolization process.
4. Ligand/Lipid ratio optimization. Quantification of the final quantity of ligand and the % of coupling, using Micro BCA. Internalization measurement with fluorescent liposomes, after 24
hours in HCT-116 CRC cell line.
5. Physicochemical characterization: the size and zeta potential were determined by laser
diffractometry. The % of ligand was measured by Micro BCA. Same size of liposomes, so
they can be used IV admon.
6. In vitro studies. Expression.
7. In vitro studies: citotoxicity and CE50. Uptake assay.
8. In vivo studies: efficacy evaluation (Xenograft model - it's an immunocompromised animal
model). Doses were injected at days 13 and 17.
It was seen that internalization of the liposome was better with the Fab fragment than with the entire AB. This happens because of the orientation of the molecule, and because it
was introduced a structural modification with SH. Monovalent is always linked in the same
orientation while the entire AB is not. Targeting with small molecules is better and possible
(afixbodies), and the main advantages are the orientation in the coupling and the antigenic
reactivity due to the constant fragment.
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