pharmaceutics - biotech, NDDs II, research Flashcards
what is different between biologics and biotech
biologics: can kill (or live attenuate which means to make the cell no longer pathogenic) and inject cells
biotech: remove cells and reinject living cells into a patient
gene therapy
what does it look for and what does it do
what disease is this used for
look for genetic defects and correct them (use virus as a vector and DNA as drug)
like hemophilia
Biotechnology
what kind of technique is it
what may it include
what does invitro mean
what does in vivo mean
Any technique that uses living organisms in the production or modification of products.
- May include tissue culture, living cells, or cell enzymes to make a defined product
- in vitro: in cells, cell culture work in a petri dish
in vivo: in the organism
Questions for reflection
Do genetically modified organisms used in production facilities pose unknown risks for an ecosystem and for the human race itself?
Furthermore, is it right to modify the genetic structure of living organisms?
Recombinant DNA
when was DNA first isolated and when was the chemical composition determined
what was discovered in the 1940’s.
what did Watson, Crick and Franklin do
now what do we understand about DNA
a typical gene has what and what can we do to them
DNA first isolated in 1869
Chemical composition determined in1900s
The determination that genes in the chromosome were made up of DNA in the 1940s
James D. Watson and Francis H.C Crick discovered DNA structure is a double helix. Rosalind Franklin who took the images
- Now we know and understand the role of Adenine, Thymine, Guanine, and Cytosine in base pairing.
- A typical gene has hundreds of bases that are always arranged in pairs. Can correct the sequences (gene therapy and recomb. DNA promises)
Recombinant DNA
to create a new cell, what must happen to the DNA
how is this done
what are the steps to protein production
how many proteins can a single E.coli cell make
why are Endonucleases & DNA Ligase important
To create a new cell or new organism, DNA must be able to replicate (clone) itself.
Unwinding and separation of the two DNA strands, then follow attachment to new bases within the cell.
Protein Production:
- DNA is translated to messenger RNA (ribonucleic acid) from the most common amino acids from which all proteins are made.
- A single E.coli bacterium can produce about 2000 proteins.
- Endonucleases, DNA Ligase, Why important?
——–For the first time, we could now join two different DNA molecules together at specific sites
Welcome to BioTechnology.
Advantages of Recombinant DNA technology
what kind of cells can now be used to manufacture proteins in large quantities
now it is easier for scientists do to what
for example: how many many cadaver pituitary glands were required to treat a how many growth-hormone deficient chldren for how many years. Were the glands pure?
today, is the biosynthetic product comtaminated with viral products for the cadaver soruce of the hormone
what were the 2 first products of rDNA
Nonhuman cells identical to those produced in human cells can now be used to manufacture proteins in large quantities.
- Now easier for scientists to produce molecules that were once difficult to obtain from human sources
For example,
- 50 Cadaver pituitary glands were required to treat a single growth hormone-deficient child for 1 year. Well also, how pure are the glands?
- TODAY: The biosynthetic product is free of viral contamination from the cadaver source of the hormone
Human growth hormone and insulin were the first recombinant DNA products available for patient use - they serve as a model for future products too
with rDNA tech, now we can use nonhuman cells to produce human proteins!! much better than using lots of cadavers
Polymerase Chain Reaction
what does it do
what are the 3 steps
each cycle is repeated until when
what is an example
what is the directionality of the primer and DNA polymerase
This technology amplifies nucleic acid sequences
A three-step process:
1- DNA is denatured to separate the 2 strands
2- Nucleic acid primer is hybridized to each strand
3- DNA polymerase extends the primer along the DNA strand to copy the nucleic acid sequence
Each cycle is repeated until DNA material is copied
For example:
- 20 cycles with a 90% success rate will yield a 375,000 amplification of a DNA sequence
primer 5’ to 3’ , DNA polymerase from 3’ to 5’
Gene Therapy
what does it introduce into the target cell population
defintion of gene therapy
- is the genetic material endogenous or exogenous
- into what kind of cells
- what is the purpose of this
what are some of the diseases that are used for this
this is a good option so far as what can be deleivered to the apporpriate target cell or tissue
what are the two things that gene therapy must be. Where do we want to deliver and what do we wnat to spare
what cells are preferred over another and why
what is the difference between stem and somatic cells
This technology introduces a new function or property into a target cell population
gene therapy:
“A process in which exogenous genetic material is transferred into somatic cells to correct an inherited or acquired genetic defect” – but I thought they like stem cells better?
- For some common and life-threatening diseases such as cystic fibrosis, hemophilia, sickle cell anemia, and diabetes.
A viable option so long as the transferred genetic material can be delivered to the appropriate target cell or tissue.
Should be SAFE and EFFECTIVE. Want to deliver to the target cell and spare healthy tissue from damaging effects
__stem cells___________are preferred over __somatic cells__________ because stem cells can self renew. Also, an inserted gene can remain in place for subsequent generations of differentiated cells or tissue populations
stem cells replicate over and over again. Around a lot longer than somatic cells
somatic cells have a definite lineage, so they live and then die. We’re already programmed and then die
Gene Therapy
what happens to the patient’s cells
what is the basic process:
1- what do the cells receive
2- what do cells start to produce
3- what is returned to the patient
4- what is produced and how does it affect the disease
WHAT Conditions should be OPTIMIZED (in the basic process) in order to achieve the greatest success?
1- what must the area be
2- what must the needle be
3- what do we want to make sure of the virus
4- what can we use micelles for
5- what should the broth medium be
6- should you change everything at once
Patient’s cells (i.e., T lymphocytes) are harvested and grown within the laboratory
The BASIC PROCESS
- Cells receive the gene from a viral carrier (i.e., Moloney murine leukemia virus),
- Cells start to produce the missing protein required to correct the deficiency
- The newly engineered cell is then returned to the patient
- The normal protein is produced and released, alleviating the disease WHAT Conditions should be OPTIMIZED (in the basic process) in order to achieve the greatest success?
- the area is not contaminated, no lotions
- right size needle and type
- want to make sure viruses cannot cause problems
- do we have a naked gene? Can use micelles or liposomes and use the right ratio of lipids to get more into a virus. Can use mivroinejction. Look for the best way to incorporate genetic material into the virus
- broth medium, want to keep cells alive, make sure conditions for cells are optimal for cell survival. Do not use phenol or heat, can use slaine, or centrifuge. Want floating cells. 37*C
- change one thing at a time
remove cells from body,
The BASIC PROCESS of gene therapy
1- what happens to cells
2- what happens to the virus
3- where is the gene inserted
4- what is the altered virus mixed with
5- what happens to the cells of the patient
6- where is it injected into
7- what do the genetically altered cells produce
1 - cells are removed from the patient
2- in the lab. a virus is altered so that it cannot reproduce
3- a gene is inserted into the virus
4- the altered virus is mixed with cells from the patient
5- the cells from the patient become genetically altered
6- the altered cells are injected into the patient
7- the genetically altered cells produce the desired protein or hormone
Gene therapy- Hemophilia
what is gene therapy used for, what do it have to do with hemophilia
for gene therapy for hemophilia, what is the drug and is the delivered
what is injected into a large group of cells
what is the hope of this
what is the virus used as
Gene therapy is being thoroughly researched as a cure for several genetic diseases.
- Out of all genetic disorders, hemophilia has the most favorable characteristics for this potential cure.
Gene therapy works in hemophilia by using DNA as the drug and viruses as the deliverer.
A virus containing the gene that produces Factor VIII or Factor IV (in the case of Hemophilia B) is injected into a large group of cells in the patient.
The hope of gene therapy is to have the cell produce more of the cured cells and spread throughout the rest of the body.
using a virus as a vector
grow cells, maintain cells, (antibiotics are usually not used)
bayer
what can it fund
what are they developing
Fabrizio Bensch /Reuters
“Bayer will fund clinical trials and handle regulatory submissions in a deal with Dimension Therapeutics.
Dimension Therapeutics, a Cambridge biotechnology startup, has struck a deal worth up to $252 million with the pharmaceutical giant Bayer HealthCare to develop a gene therapy to treat hemophilia, a rare disease that prevents blood from clotting.”
Nucleotide Blockade/ Antisense therapy
what does it focus on
what is the sense sequence
what is the anti-sense sequence
what do anti-sense drugs recognize and bind to
what does that prevent
This technology focuses on the study of the function of specific proteins and intracellular expression.
The sequence of the nucleotide chain containing the information for protein synthesis is called the ‘_sense___’ sequence.
The nucleotide chain complementary to the sense sequence is called the ‘_anti-sense___’ sequence.
Antisense drugs both recognize and bind to the sense sequence of specific mRNA molecules.
This prevents the synthesis of unwanted proteins and destroys the ‘_sense______’ molecules in the process
Nucleotide Blockade/ Antisense therapy
what has been problematic
what is being explored to overcome this obstacle
animals treated with what get side effects and what is the result of some
what will not occur
Problems, and Why the Side Effects?
Delivery of antisense oligonucleotides into target cells or the cell nucleus has been problematic.
A variety of viral and non-viral delivery systems are currently being explored to overcome this obstacle.
Animals treated with antisense oligonucleotides have had significant side effects, some of which have been lethal.
translation (which goes on to make protein and then causes disease) will not occur
Problems, and Why the Side Effects?
- anti-sense prevents
- you may have a different sequence very similar to the intended sequence and as a result the antisense will bind that one. This is an off-target effect
What is an off-target effect?
Off-target effects“
- A secondary effect of the antisense strand of a specific siRNA or shRNA where it has the appropriate homology to knockdown the expression of another related gene that carries the same sequence.”
How can antisense technology be used for today?
what does it discover about a protein
what can it be used for for dysfunctional mRNA or DNA and what is this called
what does it focus on and because of that, where is it applied
A new way to discover how proteins, whose expression has been selectively repressed in a cell, function in that cell.
To stop the expression of dysfunctional messenger RNA or DNA and control the disease process. A new process aka~ Reverse Genetics
Since antisense technology focuses on preventing gene expression, it has been most widely applied to cancer gene therapy.
Products of Biotechnology
what can it be classified into
what are the different classes
how are Biotechnological peptides divided
what are examples of each of the divided groups
“May be classified into major classes
(i.e.,
antisense,
clotting factors,
hematopoietic factors,
hormones,
interferons,
monoclonal antibodies
vaccines).”
Biotechnological peptides are further divided into physiological & Non-physiological peptides.
Example of Physiological Peptides:
- Those for Substitutions: Clotting factors, insulin, human growth hormones
Example of non Physiological Peptides:
- Mutants of physiological peptides, vaccines, thrombolytic Agents and antithrombics.
Colony stimulating factors
what are they and what do they bind to
what do they control and into what groups
what are patients with low amounts of endogenous CSFs more prone to
CSFs are four glycoprotein regulators that bind to specific surface receptors.
Control proliferation and differentiation of bone marrow cells into,
- macrophages, neutrophils, basophils, eosinophils, platelets, or erythrocytes
Patients with low amounts of endogenous CSFs are prone to secondary infections
Granulocyte Colony-Stimulating Factor (Filgrastim)
how is it produced and what does it do
what is it approved for
how is it suppliied
should it be frozen
Produced by rDNA technology, this drug stimulates the production of neutrophils in the bone marrow.
Approved for chemotherapy-related neutropenia
Filgrastim is supplied in boxes containing
- 10 glass vials, packaged in gel–ice insulating container with a temp. indicator to detect freezing.
- should not be frozen!!!
Antisense Drugs
what is an example of one
what is it used for
who is CMV uncommon for
CMV retinitis is the most common cause of what in people who have what
Fomivirsen Sodium - The very first antisense drug product on the market.
Approved in 1988. Discontinued in early 2000s
(vitravene) injectable, an antisense drug approved for the local treatment of cytomegalovirus (CMV) in patients with AIDS, who were intolerant of, or have a contraindication to, other treatments for CMV retinitis.
– Uncommon in individuals with functional immune systems, but can be serious in those individuals with impaired immune systems.
– CMV retinitis is the most common cause of blindness in persons with AIDS or other immuno-suppressed
Fomivirsen Sodium,
what kind of drug is it
where is it injected
what is targeted and what happens
how often is it injected
this treatment might avoid what
is it fewer or more frequent injections than other intravitreal injection treatments
what can it be a suitable adjunt to
an anti-sense drug
Administered by direct injection into the vitreous body of the eye.
The oligonucleotide is targeted specifically to the CMV genetic information, shutting down the CMV virus without interfering with the normal function of human DNA
Induction dose on days 1,15 followed by monthly injection of 330 µg.
Treatment approach might potentially avoid surgery, IV therapies and their associated complications.
Fewer injections compared to other intravitreal injection treatments
May be suitable adjunct to oral ganciclovir therapy
Erythropoietins
what is it contain
what does it enhance by stimulating the formation of what and the release of what from the bone marrow
what is it secreted by, in response to what and how it is transported
where is it primarily produced when what happens to blood oxygen levels
how is human erythropoietin manufactured, using what kind of DNA technology and what is it used for the treatment of
A sialic acid-containing glycoprotein
enhances erythropoiesis by stimulating the formation of proerythroblasts and the release of reticulocytes from bone marrow.
- Secreted by the kidney in response to hypoxemia and transported to the bone marrow through the plasma.
Erythropoietin is primarily produced by the kidney when a drop in blood oxygen level is perceived
- Human erythropoietin is manufactured commercially using recombinant DNA technology and is used for the treatment of anemia (low red blood cell count) in human dialysis and cancer patients.
so this product is an rDNA technology product that is used to stimulate the production of RBCs when the blood has low levels of oxygen
DNA probe technology
what is it used for
what is a small piece of DNA used for
what can we now locate and monitor and what can it lead to
what can be easily performed
what has it been used to identify
what is the level of accuracy
are the results easy to read
This technology is used to diagnose disease.
Small piece of DNA is used to search a cell for viral infection or genetic defect, Cancer, infectious disease, genetic defects, gene susceptibility
We can now locate and monitor disease causing-genes that can lead to development of gene replacement therapies
Easy-to-perform moderately-complex DNA probe technology that takes about 45 minutes.
Conclusive identification of Candida species, Gardnerella vaginalis and Trichomonas vaginalis from a single vaginal sample.
Accuracy up to 30% higher than slide microscopy.
Results are easy-to-read too.
so DNA probe technology: use a small piece of DNA to search a cell for infection and can be used to locate and monitor a gene causing a disease!
erythropoietin
how long does one dose last
how long do the effects last
for Epoetin Alpha (EPO):
where is it administered
how often is it administered and how many times per week
what happens if the therapy is >8weeks
what did EPO enter the market and as the first biopharm. product to treat what
biologically, is EPO similar to endogenous erythropoietin
what kind of drugs are these and what does that mean
A dose of erythropoietin lasts about a day,
- but its effect is seen approximately 5 days later (when the red cell proliferation it has induced is mature enough for release into the circulation).
Epoetin Alpha:
- IV or subcutaneous administration
- 50-100 IU/kg body weight 3 times per week
- If >8 weeks of therapy the hematocrit has not increased to at least 5 to 6 points (below 30-33%) the dose is increased.
- In April 1993, epoetin alfa (EPO) first entered the U.S. market as the only biopharmaceutical product approved by the FDA for the treatment of *CIA.
- EPO is biologically indistinguishable from endogenous erythropoietin
*CIA = Chemotherapy-Induced Anemia
anemia because this a recombinanat DNA technology product that is used to stumultae the production of RBCs
Monoclonal antibody
“Monoclonal antibodies are purified antibodies produced by a single source of clone of cells. They are engineered to recognize and bind to a single specific antigen.”
Examples:
- Rituximab (Rituxan)- The first monoclonal antibody approved for the treatment of cancer in 1997.
- Trastuzumab (Herceptin)-The second monoclonal antibody approved for the treatment of cancer in 1998.
what are they used for, what cancers know for the exam
on final exam
Rotate sites for injection
how many sites
what is the location for each
ayyyy macarena
which side do you start and end with
area 1 - right abdomen - leave about 2” on the right side of naval
area 2 - right thigh - low
area 3- left abdomen (leave about 2” on left side of navel)
area 4- left thigh
area 5- left arm (upper back portion)
area 6- left buttock
area 7- right arm (upper back portion)
area 8- right buttock
start and end with right side!
FDA Office of Biotechnology Products
The office was established in 1989.
Was created to serve as a central coordinating, problem-solving, and advisory role within the Office of the Commissioner.
Effective point of contact with the FDA for those outside of the agency on issues related to new biotechnology
The office is no longer in existence:
- Replaced by six divisions relating to biotechnological products
- Division of Monoclonal Antibodies created under the Centers for Biologics Evaluation and Research of the FDA, Office of Therapeutics Research and Review.
Patient Information from the Pharmacist
instruct on what.
should reinforce what
who should do the first injection
some products come with what
what should they be educated on
they should be shown how to dispose of what
they must understand what about the product
what must they emphasize
Pharmacist should instruct patients on parenteral administration
Pharmacists should reinforce the printed information sheet
The first injection should be performed by a health care professional to assure patient comprehension and understanding of techniques
Some products (e.g., Betaseron) come with training videos demonstrating reconstitution and self-administration techniques
Patients should be educated on how to prepare, give injections, and how to rotate injection sites
Patients should be shown how to dispose of needles and syringes.
The patient must understand that the product should not be agitated or shaken.
Pharmacists must emphasize the need for compliance with dosage regimens.
- For example, Betaseron is administered every other day. A calendar reminder system might be helpful for patients using medication.
Briefly discuss the differences between a Biotechnologic Product and a more conventional Finished Pharmaceutical Product.
biotech. product has live/living organisms - the organisms are still alive
while more conventional pharmaceutics products have killed or attenuated organisms (such as biologics) or no organisms at all
also
Biotech companies research, develop, and market products derived from living organisms.
The products of pharma companies tend to be derived from chemicals and artificial sources.
A beneficial protein product could result when a gene is successfully inserted into a human cell. Briefly describe the process starting from when cells are removed from a patient to when they are returned to the patient.
1 - cells are removed from the patient
2- the virus is altered so that it will not replicate/reproduce
3- A gene is inserted into the virus that codes for a protein that will help cure the genetic defect
4- the virus is mixed with the patient’s cells
5- patient cells become genetically altered due to their exposure to the viral cells
6- cells are reinjected into the patient
7- the genetically altered cells produce the desired protein or hormone
As a follow-up to question#2, discuss two reasons why the process failed to live up to expectations, and what would YOU do to improve the outcome?
make sure I use the right broth when growing and keeping the viral or human cells alive
making sure that I am not intorudcing anything like phenol or heat that could kill the cells
Tumor microenvironment characteristics contributing toward MDR
what are there increased levels of
what are there decreased levels of
Increased levels
*Oncogenes
*Growth factors/receptors
*Nutrient importers
*ABC transporters
*Aerobic glycolysis
*Interstitial fluid pressure
*DNA repair
*Detoxification enzymes - altar the structure of the drug to get eliminated faster
Decreased levels
*Tumor suppressors
*Oxidative phosphorylation
*pH - specifically the center; so very acidic!
*Cell cycle regulation
*Increased apoptosis
looking for ways to overcome tumors being resistant to drugs
Overcoming barriers limiting the entry of drugs into target cells
Drug efflux pumps
altered apoptotic pathway, if Bcl-2 is altered then cells do not die and live on
decrease drug uptake as a result of the drug efflux pumps
altered drug targets
altered DNA repair
delivered to a specific tumor
not only to get to the tumor but also to get absorbed etc
Bcl-2
Magnetic Drug Targeting
what is a nano drug delivery system loaded with
what are the 3 parameters to consider
- [ ]
- magnet
- time
what is the point of NDDs II?
In this approach, a nano drug delivery system is loaded with a ferrofluid (attracted to an externally applied magnet) or drug and ferrofluid alone.
The parameters to consider are as follows:
- Concentration of ferrofluid must be sufficient to get attracted to a magnetic field. (If too low, nanoparticles are not as responsive, and will not be able to retain much because it is as if the ferrofluid is not even there. sufficient amount)
- The magnetic strength of the external magnet should be sufficient to attract the magnetic particle. You can use permanent magnets.
- The duration of magnetic field application should be the Optimum amount of time for ferrofluid to get attracted to the magnet. length of time you are going to apply the magnet to the tumor surface
so basically
- concentration of ferrofluid in the drug
- magnetic strength of external magnet
- duration of time for the external magnet to be applied
the purpose of NDDs II
1 - add ferrofluid (has magnetic properties) to liposome with the drug (usually in the aqueous layer especially for Doxorubicin)
2 - give drug to patient
3 - apply external magnet to tumor site for the ferrofluid within the drug to be attracted to it. This allowed better drug accumulation of 15% as compared to 5% without any magnet applied
goal: improve the accumulation of drug to the tumor site
hmm okay :)
How do you increase the amount of drug incorporated in a liposome?
1) Increase drug-to-lipid ratio (1% vs 10%), want to be potent so you will not need as much. The drug should be compatible with ferrofluid
2) change the lipid composition, bilayer
now ferrofluid is already there and will take up some space, so need to make space for drugs as well
use appropriate lipids
drug and Ferro (if water-soluble) can be in the bilayer or the aqueous layer
before, want to increase the drug in the bilayer: DMPC vs DSPC, DSPC is larger and incorporates more drugs – this works out if the bilayer is where you want to put the drug
Ferro Fluid (Magnetic Material)
Core has magnetite (Fe3O4)
Matrix has Citric Acid
Zeta Potential: -35 mv, negatively charged, (anionic liposome). If +35 mv it will be positively charged (cationic liposome)
ferro has
- magnetite in the core
- citric acid matrix on the outside
citric acid matrix on the outside
- needed otherwise magnetite will aggregate. With it, magnetite will not aggregate
Zeta potential
- is the charge
- the electric potential along the double membrane surface
- For example: if we introduce phosphatidylglycerol which is negatively charged, we will have negatively charged liposomes. We can take a vesicle and measure the surface charge
Some physicochemical properties of magnetic liposomes
MAG-C = Ferrofluid
MAGnetite & Citric acid
phase transition temp.
- when it is reached: 50% gel, 50% lipo crystalline phase
particle size and zeta potential characteristics shown in two graphs
- both are important for targeting efficiency
- both are determinants of targeting
MAG-C = ferrofluid
graph for particle size and [MAG-C]
- particle size as a function of concentration MAG-C
- we see a signification increase in particle size as we increase [MAG-C]
graph for zeta potential (or surface charge)
- zeta potential as a function of concentration MAG-C
- zeta potential at 0 [MAG-C]: almost +60 mv
- as we increase the concentration of MAG-C, the zeta potential decreases
- the Zeta potential decreases, which tells us at 2.5 it is saturated. Levels off, MAG-C goes inside and not on the surface anymore, saturates on the surface as much as possible and the rest goes inside
graph for phase transition temp. and [MAG-C]
- phase transition temp as a function of concentration MAG-C
- melting temp. is 55 degrees Celsius
- stays relatively the same from 0 to 0.5 then the temp. drops when it reaches 2.5, (from 0 to 2.5 it increases just a little bit) because MAG-C does not alter the properties deeper in the bilayer. If you want to change the melting temp then you need to change some of the properties deeper inside the bilayer. If it stays on the surface then there is not much change (to do that, you need higher amounts of MAG-C).
- at 2.5, it starts to go deeper inside the bilayer because 2.5 is a high concentration of MAG-C
so:
based on size, zeta potential, and melting temp.,
2.5mg was probably used
2.5mg had the biggest particle size, lowest zeta potential and lowest phase transition temp.
Magnetic
take any of the drug delivery systems: liposomes, micelles
administer through iV
want to accumulate the drug to one site for example to a tumor and reduce reuptake by healthy tissue
we can accumulate a lot more to the target site as compared to the free drug
magnetic: the drug is attracted to an externally applied magnetic field, because delivery systems have something attracted to other magnet the drug goes to the tumor and is attracted to the tumor rather than the healthy tissue
Magnetic liposomes picture
- can see ferrofluid inside
- these are melanoma cells
- can see plasma membrane and nucleus
- get a perinucleus accumulation formation
- the drug is probably around the nucleus, will target the nucleus, and kill cells
green picture
- a tumor with blood vessels on the top left
- take the mouse anesthetized and can see blood vessels
- inject magnetic cation liposomes with ferrofluids
- apply magnetic to the window of the tumor for one hour
- then we looked at the extent to which the nanoparticles will accumulate in the tumor with and without the magnet
- no magnetic, also wait just as long
no magnet: NDD part 1
magnet: NDD part 2- can see the advantage: can accumulate more drug to the tumor site
these are vascular targeting
tumorous blood vessels have an overexpression of negatively charged functional groups, these accumulate in the tumor and accumulate in the blood vessels
forcing tumors to be retained
magnetic cationic liposome
- + charge
- liposome vesicle
- ferrofluid
- chemotherapy agents
the negative charge of the tumor is attracted to the + charge of the cationic liposome
endothelial cells…
- can accumulate more drugs!!!! wow
exam: regarding physiochemical changes
primary tumor is not the issue but the issue is when the tumor starts metastasizing - when it goes to lymph nodes to move onto secondary site and cannot control that
happens through blood vessels, must go through blood vessels
- removed organs
primary tumor metastasizing to liver, lung, spleen
Proof of concept
- remove mice organs
- euthanized
- all black: melanoma
- apply magnetic: no evidence at all of the metastasizing tumor!
- following IV administration
- inject a million cells subq
- inject IV magentic liposomes that have chemotherapy agent
- wait 2 weeks and wait for the tumor to respond
- apply magnet for 1 hour then remove for an hour - so we were able to retain enough of the liposome in the tumor to cause destruction to the blood vessels that it did not allow for disseminating of the cancer cell to a secondary site like the lungs. we damaged the blood vessels so much that we were not able to get transport to distant sites. that is why the mice with the magnet are clear. there were more uptake of the nanoparticle by using the magnet
- we were able to retain enough chemotherapy drug liposomes in the tumor and cause dissemination to the blood vessels which was so severe that did not allow dissemination to a secondary site
- take a mouse, inject a million cells, and wait for the tumor to grow, you will see it. It naturally metastasizes to an area outside the lungs. - this is the control group
- then for a free drug: chemotherapy agent without magnetic liposome. We see there is less perinuclear.
- with magetic with liposome (+ charge), ferrofluid. We had 3 out of 6 mice had melanoma but when we applied the magnet to the tumor, all of the mice did not have any more cells
green picture (from lecture video) - melanoma
what charge is the ferrofluid and why does it attract the vessels
- did surgery for the mouse and implanted a small tumor piece into the center of the window so it would grow
- once it grows, place the mouse anesthetized look for blood vessels, and inject the magnetic cationic liposomes. Applied the magnet to the window of the tumor for 1 hour
- now we look for the extent to which the nanopartciles (has the drug) will accumatte within the blood vessels in tumro with or without the magnet
- with magnet: 15% was targeted, but 5% was targetted without the magnet
- this is all following the IV administration of the magnetic cationic liposomes
chart:
- natural accumulation: 5% - this would be NDD part 1
- accumulation with magnet: 15% - this would be NDD part 2 - the magnetic component, we can see the advantage. Accumulate more nanoparticles are the blood vessels
why is this important:
- these are magnetic cationic liposomes
- tumorous blood vessels have an overexpression of negatively charged functional groups, these accumulate in the tumor and accumulate in the blood vessels. the charge is more than what you would find in healthy tissue
- if + charge liposomes with ferrofluid, it will accumulate the tumor along the blood vessel wall
- if you have a magnet, in addition to the natural accumulation, you are also forcing the magnetic cationic liposomes to be retained (and be attracted to the - charged tumor and blood vessels)
clinical implication
- magnetic liposomes have a chemotherapy agent
- chemotherapy agent in the vesicle accumulates along the wall of the vessel: will exert its action against endothelial cells and kill them :(
- the vessel has endothelial cells
- if you kill the blood vessel of the tumor
mice
tumor
- control:
- free drug:
- no magnet
- magnet
liver
- control:
- free drug:
- no magnet:
- magnet
spleen
- control:
- free drug:
- no magnet:
- magnet
lung
- control:
- free drug:
- no magnet:
- magnet
what was the drug that was used and how was it given to the mice
which had better results, no magnet or free drug
tumor
- control: metastasize to the liver
- free drug: metastasize to the liver
- no magnet
- magnet
liver
- control: metastasize to spleen
- free drug: metastasize to spleen
- no magnet: less metastasize
- magnet
spleen
- control: metastasize to the lung
- free drug: metastasize to the lung
- no magnet: less metastasize
- magnet
lung
- control: metastasize
- free drug: nothing here
- no magnet: less metastasize
- magnet
drug: vinblastine sulfate IV to mice
free drug: FDA approved
very qualitative study
will the free drug, drug with no magnet or drug with magnet prevent the metastasis of the tumor to the liver, lung and spleen
result: no magnet: had better results than the free drug and caused less metastasis
Liver Metastases from Colorectal Cancer: Drug delivery with liposome-encapsulated doxorubicin
what do we want to know about doxorubicin-loaded liposomes
To determine where doxorubicin-loaded liposomes accumulate in liver tumors following injections by 3 different routes of administration
Depending on the hydrophilic or hydrophobic nature of the encapsulated agent:
Drugs, contrast agents, genes, and peptides can be trapped within where of what
Drugs, contrast agents, genes, and peptides can be trapped within the aqueous or lipid phases of liposomes for delivery to various biological targets
– Doxorubicin can be encapsulated in liposomes and this formulation has been used to target tumors in various experimental animal models.
Doxorubicin???
DOXIL - commercial product of doxorubicin
Formulations incorporating doxorubicin have been developed to decrease the incidence of severe toxicity seen with the conventional formulation of doxorubicin while taking advantage of the unique delivery properties of liposomes and the cytotoxic effects of doxorubicin.
Approved for treatment of
Aids-related Kaposi Sarcoma,
Ovarian cancer and many other
Solid tumor varieties
Commercial product
using stealth (limits uptake by liver, lung, and spleen so should be available long enough to circulate in the blood so the tumor can take up more of it) liposomes - in the vesicle and is surrounded by PEG
Doxil itself is not more effective (reducing tumors) than the free drug (active drug). So then why use it, if you load Doxil into the liposome because it is not taken up long enough then it turns out that it reduces cardiotoxicity
Doxorubicin hydrochloride’
what is there activity against
what are the mechanisms of action
what are the side effects
Activity against:
Lymphomas
Leukemias
Sarcomas
Carcinomas
read the article to distinguish one from the other
Mechanism(s) of action:
- Directly cytotoxic to cells without entry by cancer cells
- Intercalate with DNA in cancer cells & inhibit DNA synthesis
- Can inhibit topoisomerase I and II activity and interfere with DNA strand separation - interfere with DNA replication
Side effects:
- Severe cardiotoxicity (which is why stealth is used because it reduces this) is associated with treatment but liposomes have been shown to reduce the toxicity of the drug.
so this drug prevents DNA replication of the cancer cells and if it is put into a liposome then it will reduce the cardiotoxic effects
Doxorubicin Pharmacokinetic profile
for the bolus injection,
Bolus injection- drug undergoes biotransformation in the liver to active and inactive metabolites
Drug binds extensively to plasma proteins and tissues (about 70%)
Drug is excreted primarily in the bile (40 to 50%)
Drug clears slowly from blood (half-life- 18 to 32 hrs).
Drug does not cross blood brain barrier in clinically relevant concentrations
Limited data suggest that doxorubicin can cross the placenta into fetal tissue, but fetal damage is rare
Preclinical studies: Efficacy experiments
establish if the drug is effective, carry them out in vivo
4 groups
have 6-8 mice with tumor (inject a million cells sub Q and the tumor will grow on the surface so we can monitor and measure the size, we cannot do this if we did this parenterally
A- is a placebo. We inject a million cells and the tumor has to grow. Not all tumor cells will grow in mouse models but this tumor will.
Graph: tumor volume as a function of days. Inject cells and wait and see how long it will take the tumor to grow
D- takes 30 days to grow
another took 20 days
take a human cell line and mice,
tumors are heterogeneous, because of genetic, cellular, and molecular reasons. If injected, half may move to one area of the mouse and some may split into other areas. So the amount of cells that will participate in a tumor varies because they go in different directions
B vs A- B is delayed in growth and takes a longer time to grow. To achieve 30 days - the tumor growth rate was suppressed
C - drug into liposome now,
D- the tumor is not able to grow, it never gets to the full amount. It looks like the tumor is responding to the therapy. They applied to the tumors probably. Applied heat, the tumor is not affected by heat. It does not kill the tumor but it makes it more susceptible to the celsion’s doxo.
Tumors of the liver
liver matters
routes of admin: hepatic artery, hepatic portal vein
Liver Metastases from Colorectal Cancer: Drug delivery with liposome-encapsulated doxorubicin
what is the purpose of this study?
what is the title and the authors?
To determine where doxorubicin-loaded liposomes accumulate in liver tumors following injections by 3 different routes of administration (3 routes are important)
Liver Metastases from Colorectal Cancer: Drug delivery with liposome-encapsulated
doxorubicin(
Osman Cay, MD, Jonathan B. Druskal, MD, PhD, Imad Nasser, MD, Peter Thomas, PhD, Melvin E. Clouse, MD., Radiology 1997; 205:95-101) volume and then page numbers ***
Purpose of the research study
- 3 routes
- what is administered
- what animal model is used
Why is this important?
To compare Intra-venous, Intra-portal, and Intra-arterial administration of liposomal doxorubicin, by using an established mouse model of hepatic colorectal cancer metastasis
Why is this important?
- Improve drug access to target sites, and
- It’s a way to circumvent drug barriers
what are the 3 Routes of administration used in this experiment?
what does each route do
IV
- what vein in the mouse
IA
Intravenous:
mouse tail vein
Intra-portal:
carries all blood from the abdominal GI tract, spleen, pancreas, and gall bladder to the heart through the liver
Intra-arterial:
Carrier of oxygen-rich blood to the liver from the heart
the liver is the eventual organ site, we want to target tumors in the liver
Animal Study
what animal was used
- are they babies or adults
where were they contained
Studies were performed with adult nude mice (Mean weight 20 grams) - immunocompromised
Animals were housed under controlled temperature and humidity and were allowed access to water and laboratory chow
Animal tumor model
Cells maintained in RPMI 1640 medium (like a broth) with 10% FBS (fetal bovine serum)
Anesthesia: 30 mg of sodium pentobarbital per/ kilogram of body weight (mg/kg) was injected intra-peritoneally
5,000,000 human CX-1 (colorectal cancer, metastases to the liver) cells in a solution of 0.5 ml of phosphate-buffered saline (pH 7.4) (will not kill cells, isotonic) were injected into the spleen.
want to inject 5 million cells.
they could have injected the tumor cells into the liver itself but they took colorectal cancer cells into the spleen because the liver tumors will develop more naturally as opposed to forcing them to grow in the liver
Techniques used to evaluate liposome accumulation in tumors
what are the 5 techniques?
what does invitro mean
In vivo Video Microscopy
In vivo Biologic Distribution of Doxorubicin
In vitro uptake studies
Electron Microscopy
Statistical Analyses
_______________________________________
In vitro: studies conducted in culture
In vivo: studies conducted in small animal models, usually rats or mice
pay attention to this question, will be on an exam lol
Some Procedures
what happened after Intrasplenic injection of CX-1 human colorectal cancer cells into mice and within what time frame
in each mouse, on average how many tumors (of what ranges) were observed in the liver
now what do we have
Intrasplenic injection of CX-1 human colorectal cancer cells into mice resulted in spontaneous growth of multiple (avascular) metastatic tumors in all livers within 7 days of injection.
In each mouse, an average of 5 tumors (range 3-6) were observed in the liver - good to know because when you inject the 5 million cells and they develop in the liver, we will not open the mouse to look at the tumor.
Now we have a clinically relevant tumor model that can be used to carry out overall research
study now begins
Routes of injection - give an injection of liposomal doxorubicin
after 5 minutes, was there fluorescence, and in what areas
Five minutes after injection of the hepatic, portal vein, or tail vein no visible fluorescent activity was observed in the:
- Hepatic intravascular compartment
- Parenchymal (normal tissue areas in the liver)
- Tumors
- Hepatocytes or
- Sinusoidal endothelial cells
why expect to see fluorescent activity, doxorubicin hydrochloride is red and is naturally fluorescent
detect presence in the liver: video microscopy
Routes of injection
what happened 30 minutes after intrarterial or intraportal injection,
30 minutes after intrarterial or intraportal injection, what about tail vein injection (ut has been dropped off for some reason)
Focal spots of fluorescence activity (evidence of liposomal doxorubicin) were observed in areas corresponding to the tumor nodule and to the peritumoral (outside of the tumor mass) (regions immediately surrounding the tumor). Able to see some of the red dots
Routes of injection
after60 minutes of injection, what was observed and what does it correspond to
were there differences between intra-arterial and intra-portal injections
were there differences between tumors of different sizes and in different zones of the liver lobules
were there differences noted between accumulation of liposomes by arterial and intra-portal injections
60 minutes after intra-arterial or intra-portal injection, fluorescent spots were observed in all microscopy fields and these spots correspond to the metastatic tumors.
No differences between intra-arterial and intra-portal injections
No differences between tumors of different sizes and in different zones of the liver lobules
No differences were noted between the accumulation of liposomes by arterial and intra-portal injections
3 routes: IV, IA, IP
Summary of research
fluorescein (the fluorescence channel that is used to detect the presence of doxorubicin) intensity after intraarterial or intraportal injection of doxorubicin in Mice:
tumorous
at 30 minutes, they see 81
at 60 minutes, they see 119
nontumorous
at 30 minutes, they see 62
at 60 minutes, they see 82
the higher the fluoresce intensity the more drug is there
Results of tail vein injection
minimal accumulation was noted where
no significant differences were noted where
is this enough evidence
Minimal accumulation was noted in normal liver and in liver with tumors
No significant differences were observed between tumor and background liver fluorescent activity. Uptake for the tumor and normal area were all similar which is what we do not want
- This offers evidence to support insufficient concentration levels of doxorubicin in tumors relative to controls.
Conclusions of research
what can Doxorubicin be used for
what is needed now in regards to humans
Doxorubicin can be selectively targeted to hepatic metastases from colorectal cancer via the hepatic artery or the portal vein by using a novel delivery vehicle (i.e., Liposomes) - so through the hepatic artery and the portal, colorectal tumor cells that metastatsize in the liver (so liver tumor can be targetted) can be targeted by doxorubicin through 3 routes of admin - really just 2 routes of admin that had very similar results of accumulation overall
An evaluation of the efficacy of this agent in the treatment of hepatic metastases related to colorectal cancer (and hepatic metastases) in humans is needed
oooh so we injected the CX-1 (colorectal cells) into the spleen (intrasplenic injection) and it metatsized to the liver naturally.
then we injected teh drug throguh 3 different routes: IV, IP, IA to find out which route would give the more accumulation of the drug to the tumor site!
Liposome-cell or Liposome-tissue interaction
want to know liposome cell interaction, the extent to which the cells interact with the liposome
the formulation accumulates in the tumor, well are they taken up by the cells
well plates,
cell growth ( brain cancer, breast)
put the cell in a well
expose cells in various concentrations to different liposomal amounts
do not load in the first column so we can compare
3000 cells per well, exposed to fluorescently labeled. liposomal vehicle (no doxorubicin yet)
Are the liposomes taken up by cells in the liver?
what did the graph show
- what was the question that this graph was attempting to answer
- did the availability of oxygen affect how fast the drug was taken up by the liposomes, what was the rate of the uptake
- maximum uptake of the drug in tumor cells was achieved after how long
- what was the rate of uptake in Kupffer cells compared to tumor cells
the graph shows:
- the % of uptake of liposome-encapsulated doxorubicin (drug) in CX-1 cells under normoxic and hypoxic conditions in Kupffer cells - so how much of the drug was taken up by the Kupffer cells in different conditions
- there was a gradual uptake of liposomes in tumor cells and in Kupffer cells (macrophages in the liver so they will take up anything that is introduced to it), regardless of the availability of oxygen in the environment
- maximum uptake was achieved in tumor cells at 60 minutes of incubation
- the rate of uptake in Kupffer cells was less than that in the tumor cells
taken up by the tumor in the liver
cells taken up liposomes as a function of the duration of incubation
good news because the CX-1 cells in the tumor
hypoxic: do not worry about
Did the experimental design support the main hypothesis?Why, or why not?
Off target effect:
antisense tries to target sense strand but you end up targeting a different sequence than the sense sequence. So you knock down the expression of a sequence that you did not mean to target
Fomivirsen sodium:
antisense drug, FDA approved for treatment of CMV in patients with AIDS. This is a drug that treats that virus. Also used for CMV retinitis (a type of CMV)
Erythropoietins:
enhances erythropoiesis (production of red blood cells). It does this by stimulating proerythroblasts which produce red blood cells. This is not antisense drug but an rDNA technology drug! :)
Epoetin alpha is
the brand name of erythropoietin, can be IV or subQ. Dose increased if treatment has occured >8 weeks of therapy the Hct has not increased. Used to stimulate the erythropoiesis for anemic patients (got anemia from chemotherapy)
Side effects of antisense technology:
Off-target effects. It recognizes and bind a specific sense sequence of an mRNA molecule. And this prevents the synthesis of proteins that sense sequence will code for.
Monoclonal antibodies are
a different class of biotech drugs, purified antibodies produced by a single source of clonal cells. Examples of Rituximab, and Trastuzumab. T
he trick to remembering the examples is they all end in “mab” = monoclonal antibodies.
Rituximab is used for non-Hodgkin lymphoma & lymphocytic leukemia
Trastuzumab is used for early-stage breast cancer.
process of mab
Patient has a virus (antigen), they’re bodies produce antibodies for that antigen. Those antibiotics are extracted and cloned and so 100s of 1000s are cloned and are now fused and cloned into the patient. And now a patient has the antigen and their body is not producing antibodies then the patient will be given monoclonal antibodies to recover.
Metastases
patients have colon cancer but now the cancer spreads to the liver. Drug delivery of Doxorubicin in liposomes. Long travel of the tumor
Determine where doxorubicin loaded injection will accumulate following injection
Main question: Which route of administration is the most effective
intra-portal and intra-arterial are more effective than intravenous because at least they prodcued different results compared to the control
but the portal and arterial injections had similar results and accumulated at the liver tumor site and so these were the best roue of admins
while the IV route had the same result as the control
Where in the liver is the drug accumulating, which method of administration is most effective.
portal and arterial are most effective
In vivo vs In vitro
In vivo: studies conducted in small animal models, usually rats or mice
In vitro: studies conducted in culture
Inject cells and watch growth of tumors (which is heterogenous). Once the tumor grows then you inject the drug
Results for tail vein injection:
small [ ] in normal liver and liver with tumor - there was no difference between the 2. So the control and the experiment looked the same - no big differences so that route of admin was dropped
Best results for research
actually they all (except intravenous through the tail vein) had very similar results
