Pharmaceutics (C&T) Flashcards

Question

How is DNA transferred naturally in the body

Answer 1

Transformation (uptake of free DNA) Transfection = human Competence = bacteria Conjugation -- transfer of DNA through cell-cell contact Transduction -- transfer of DNA mediated by virus Mobile genetic elements -- Plasmids -- Transposons and insertion seuqnce elements

Answer 2

Naturally occurring plasmids are not essential, but they encode for helpful genes -- antibiotic resistance/toxic metals -- metabolic functions (allowing growth on lactose or sucrose) -- production of virulence factors (haemolysin)

Answer 3

Produces multiple copies of DNA from a defined template in vivo. DNA sequence can be a gene, but can have non-coding elements (promotors)

Answer 4

Isolate the source DNA -- restriction enzyme used to cut out the required sequence and also used in the plasmid so that the DNA can be inserted to that area Insert the source DNA into the cloning vector -- DNA fragment enzymatically inserted into the DNA of a plasmid vector using DNA ligase -- Marker (ampicillin resistance gene and lacZ gene) also inserted so to identify which have taken up the DNA fragment (white colonies) and kill the hosts that have not taken it up (blue colonies). --\> Recombinant plasmid formed Introduce the recombinant plasmid into a host organism -- E. coli is mixed with the plasmid with CaCl2 -- heat pulse -- Culture the bacteria on nutrient agar plates impregnated with ampicillin --\> Cells that have been transformed survive, those that did not take up the plasmid die Plasmid replication then occurs to produce more of the inserted DNA ---\> Cell multiplication occurs producing a large quantity of cells containing the desired DNA

Answer 5

Polymerase chain reaction Method to produce a large quantity of the template DNA Reaction requires water, DNA template, nucleotides, primers (oligonucleotides, polymerase, and a buffer. There are 3 steps, which are repeated multiple time (25-35) Locate the target sequence to be cloned Primers are designed so that they are complementary just up and downstream of the target gene 1. ) Denature the DNA strands -- 30s at 94C 2. ) Anneal with primers -- 30s at 55-65C 3. ) Elongation with thermostable DNA polymerase (Taq polymerase - derived from thermophilic bacteria or archae) -- 1 min per kb at 72C

Answer 6

Palindromic sites are recognised by the enzyme (restriction sites) Typically 4-6 nt long Resstriction enzyme then cleaves these areas -- produces sticky or blunt ends. The enzyme then binds to the sticky ends of the sequence and is bound using DNA ligase.

Answer 7

A method using agarose in order to find the number of base pairs in a fragment. Requires... DNA Water Concentrated buffer Restriction enzyme An electric current A dye UV Light The size of the fragments can be determined by using a known set of reference bands.

Answer 8

An enzyme that ligates (binds) compatible DNA strands -- Sticky or blunt (though more efficient as linking compatible sticky ends) Usually plays a role in repairing DNA and replication \_\_\_\_ Requires... Vector for the DNA to be inserted into The insert DNA Concentrated buffer Water Buffer

Answer 9

Clones independent of DNA Available commercially or distributed freely Accepts inserts of 10kb\> (greater than this causes instability) Easy to use Narrow range of compatible hosts

Answer 10

The plasmid contains the lacZ gene -- encodes ß-galactosidase. This degrades lactose... MCS is part of the lacZ gene --\> If DNA is inserted. it inserts into the lacZ gene, causing inactivation of it. So, hosts that have taken up the DNA have ß-galactosidase inactivated, so that the artificial substrate x-gal is not degraded into the blue dye. ---\> Observe a white colony (desired) Hosts that have not taken up the DNA have the enzyme remaining active. This degrades the x-gal substrate, resulting in a blue dye being produced. ---\> Observe a blue colony (undesired)

Answer 11

Shuttle plasmids --\> Plasmid can replicate in a minimum of 2 different hosts Integration vectors --\> Cannot replicate, but integrates itself into the chromosome --\> Useful for knockouts ƛ Cloning vectors (spp to g(-) bacteria) --\> Based on bacteriophage lambda (ƛ) --\> Accomodates to larger inserts (up to 22kb) Artificial chromosomes --\> Construct is based off of bacterial (BAC) or yeasts (YAC) DNA --\> Can contain very large inserts (75-800kb) --\> Useful for cloning very large genes and genome mapping/sequencing

Answer 12

* Grows rapidly in inexpensive medium * Non-pathogenic * Easily takes up DNA * Is genetically stable * Allows replication of vector * Has many tools for genetic manipulation * Allows high level of expression of genes --\> High expression -\> more mRNA -\> more protein produced •N.B. Hosts for cloning and expression are not necessarily the same

Answer 13

Stimulus causes ER to produce preproinsulin via ribosomes. The protein produced from mRNA translation contains an A chain, B chain and C chain. A and B chain are linked via the C chain. As the protein is being made, it is transported into the lumen of the ER The signal peptide is then cleaved from the preprohormone, to produce proinsulin. A disulfide link is also produced between the A chain and B chain Proinsulin is transported to the golgi complex using vesicles As proinsulin is packaged into vesicles by the golgi complex, the C chain is cleaved, leaving the disulfide bond linking the A and B chain, as well as the internal disulfide bond in the A chain.

Answer 14

The peptide is short, therefore it is degraded in the cytoplasm of E. coli -- unstable. Thus it can be stabilized by fusion to a larger protein. Method 1 - A chain and B chain are produced separately in E. coli, as fusions with the gene encoding for ß-galactosidase. - ß-galactosidase is then cleaved from the fusion proteins, and purified. - A chain and B are combined and refolded in oxidising conditions to form the disulfide bonds Method 2 - Clone the gene for proinsulin - Attach ß-galactosidase encoding gene to proinsulin of E. coli - Extract the protein, purify and cleave off ß-galactosidase - Refold proinsulin - Enzymatic removal of the C chain to produce insulin

Answer 15

Initially produced using mRNA, and cloned using E. coli. However, factor VIII is glycosylated, so transfection into mammalian cell line is required. The plasmid is transfected into the genome. The cell line that produces the greatest number of Factor VIII genes is used for production.

Answer 16

Inactivates thrombin, Factor Xa and Factor IXa -\>regulates normal blood coagulation

Answer 17

•Gene linked to mammary gland-specific regulatory elements --\>gene is expressed only in milk * transgene is introduced into embryo by injection * embryos transferred to oviduct/uterus * transgenic offspring identified * lactation induced and production in milk is tested

Answer 18

Bacteria / yeast preferred when the protein does not need to be modified post-translation. Choice is made dependent on the biotherapeutic being produced. Proteins of mammalian origin (require glycosylation or are large) are produced in insects, animals, or animal cells.

Answer 19

Environmental conditions Excipients present Delivery route/vehicle

Answer 20

* Hydrophobic interactions (80% internal) * Electrostatic (repulsions, ion pairing) * H-bonding --\>Inter- and intramolecular * VDW forces * Steric effects * Hydration * Disulphide bridges

Answer 21

1. Conformational changes: * Formation of incorrect structures * Aggregation (partially hydrophobic residues interacting with another partialsly hydrophobic residue) 2. Chemical changes: e. g. Hydrolysis, oxidation, deamidation, glycation, disulphide bond rearrangment * Break peptide backbone * Modification of important residues * Change protein shape (i.e. conformational changes & aggregation!)

Answer 22

* Freezing/thawing * Agitation (interfaces) * Sonication (use of ultrasound to unfold proteins) * Contact with silicone oil * Low or high pH * Low or high salt * Specific salts * Chemical changes * Heat

Answer 23

* Altered solubility * Hypo-potency (most likely) * Hyper-potency * Off target binding -- Adverse events, faster clearance * Patient may generate neutralizing antibodies (ATAs) --Makes drug ineffective --May break tolerance --Cross react with endogenous protein

Answer 24

Deamidation Hydroylsis of peptide bonds Elimination Shuffling of disulfide bonds Oxidation Cross linking Thiol-disulphide exchange

Answer 25

* Solubility enhancers – e.g. surfactants, amino acids, sugars, polymers * Anti-absorption & anti-aggregation agents – e.g. surfactants, albumin * Buffering agents – usually citrate, phosphate or acetate * Preservatives & anti-oxidants – e.g. ascorbic acid, antimicrobials (repeated dosing) * Lyoprotectants/cake formers * Osmotic agents – NaCl, mono- or disaccharides

Answer 26

* Sugars, some amino acids - increased surface tension of water * Glycerol, polyols - repulsion between molecules, this maintains a water layer, and hence preserves the protein's structure * PEG - steric effects HSA -- human serum albumin (Indirect and direct stabilization of the protein) Polysorbates -- prevents aggregation and denaturing via.. * Preferential exclusion of solutes * Acting as a “chemical chaperone” aiding protein refolding * Binding to hydrophobic patches of proteins -- can reduce protein exposure to interfaces (can cause aggregation) -- forming of detergent film, in aqueous systems. This reduces protein exposure to air/water Methionine -- prevents oxidation of the protein * Interact with residues of opposite charge which may cause association of proteins * Aliphatic regions can cover exposed hydrophobic areas of proteins Cyclodextrins (prevents aggregation of proteins) -- HP-ß-CD is now approved for parenteral administration of leukine-enkephalin

Answer 27

Auto-oxidation/hydrolytic degradation can result in aggregation and denatuaring of enzymes. They produce... Reactive peroxides (antioxidants may help here) Aldehydes -- immunogenicity (even without aggregation) --Formaldehyde & acetaldehyde are potential carcinogens, and cause contact allergies. Extent is controlled by: * Identity of the protein * Protein and surfactant concentration * Other excipients * Temperature * Light exposure

Answer 28

Freeze drying --Restricts mobility --Reduces relative humidity --Allows storage at room temp. Requires reconstitution Takes time for full dissolution Agitation Air entrapped Potential for mistakes -- dilution? microbial contamination?

Answer 29

Volume can be directly withdrawn Less manipulation compared to lyophilization Agitation Shipping and storage between 2-8C Inadvertent freezing

Answer 30

Advantages... * Large doses can be administered with 100% bioavailability * Administration can be controlled/discontinued * Immediate access to the central compartment * Easy weight-based dosing Disadvantages * Additional manipulation * Patient inconvenience/compliance * Dose usually diluted into prefilled i.v. bag ----Adsorption leading to lower concentration * Multiple materials of construction (polyolefin, PVC, etc) * Agitation during transport may be significant * Risk of microbial exposure before use

Answer 31

Air-water (Vials, IV bags..) Oil-water (Silicone coated syringes) Hydrophobic surfaces (IV set and bag)

Answer 32

Normal saline is normally fine. Some hydrophobic proteins are less soluble/insoluble in high salt conc, it may be better to formulate lower salt content in these cases. Dextrose is usually NOT fine. Can react with lysine on protein surfaces to produce Schiff bases relatively quick. This can occur in vivo, esp in patients with uncontrolled diabetes

Answer 33

TNF-⍺ antagonist Consists of Fc region of IgG1 (prolongs the half life) fused with the extracellular domain of the TNF receptor Soluble protein given SC 50-100x better at binding to TNF-⍺ than the endogenous receptor Dimeric, so each molecule can bind to a maximum of 2 molecules

Answer 34

Monomers are formed at low conc Dimers are formed at high concs. Zinc ions promote the hexamer form of insulin Modification of quarternary structure --Long acting insulin promotes the hexamer form, has a reduced solubility and promoted binding to plasma proteins --Fast acting insulin prevents hexamer/dimer formation and increases solublity

Answer 35

Basal insulin Des(B30) human insulin C16 chain promotes the formation of multi-hexamers following injection Tresiba

Answer 36

PEGylation is a modification that can be performed on proteins. The apparent diameter is increased, and so clearance is reduced. Must not block the functional parts of the protein Hydrophilicity of PEG - Improved aqueous solubility - Reduced protein binding - Improved bioavailability Can allow attachment of other ligands or drugs -Avoids phagocytosis Flexibility of PEG - Shields antigenic sites (prevents its destruction by antibodies etc) - Reduced toxicity - Increased proteolytic resistance - Reduced clearance - Improved mechanical & thermal stability

Answer 37

A liquid form of the drug is produced, and an aerosol is formed in a drying chamber (filtered hot air). The droplets dry very quickly to produce micron sized particles of the protein and excipients. Particles that are too large are recirculated in the system. Benefits. * Continuous process (vs batch in lyophilization) * Gentler than freezing * Produces a powder -- no sieving or milling required * Particle size/area can be modified to change the dissolution rate * Can be performed aseptically * Can allow for room temp storage

Answer 38

Thrombin/Fibrinogen spray dried to produce a powder. This is used in surgery for clotting.

Answer 39

Method of drug delivery that prolongs the release of drug. As the polymer degrades, the drug is released (commonly used polymer is PLGA). Commonly used polymers are typically hydrophobic and only soluble in organic solvents. This is fine for most drugs -- not biologicals. Emulsions are usually required to get the hydrophilic drug into the polymer. Issues * Interfaces with the air, solvents and surfaces * Temperature * Degradation products at at release site

Answer 40

Implanted cells are coated with a semi-permeable membrane. This allows the flow of nutrients into the cell, but prevents damage to the cells from the host immune cells. --\> Prevents the requirement of immunosuppressants being used when external cells are implanted. --\> Potential for long term drug delivery

Answer 41

1. Poke and patch (Solid microneedle) 2. Coat and poke (Coated microneedle) 3. Poke and release (Dissolving microneedle) 4. Poke and flow (Hollow microneedles)

Answer 42

Bulk erosion Surface erosion Diffusion -\> Dependent on the polymer used.

Answer 43

Treatment of stenosis with stents. Restenosis is common after using stents. Drug eluting stents can be used that elicit its effects via diffusion/erosion of cell growth inhibiting drugs. e.g. Rapamycin (G1 cell cycle inhibition) Paclixatel (M phase arrest)

Answer 44

Drug release controlled by: * pH * Chemicals (including metabolites) * Enzymes * Ultrasound * Magnetism * Light * Electronics

Answer 45

PBA triggered release -- glucose competitively binds to PBA resulting in the polymer breaking apart and releasing insulin Glucose oxidase-triggered release -- increase of glucose concentration results in a decrease of pH. Polymer breaks apart. Glucose + water + oxygen ---(Glucose oxidase)---\> Gluconic acid + Hydrogen peroxide ConA triggered release -- glucose competitively binds to ConA, resulting in polymer breakdown PBA = phenylboronic acid

Answer 46

1. Cell isolation, purification/enrichment 2. Expansion of number of cells 3. Seeding on a suitable scaffold 4. Maturation of the tissue 5. Implantation into the patient

Pharmaceutics (C&T) Flashcards

(70 cards)