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
