Ho - Midterm 1

Question 1

CDER vs. CBER

Answer 1

CDER: Center for Drug Evaluation and Research - chemical drugs and biopharmaceuticals (well-characterized)

CBER: Center for Biologics Evaluation and Research - vaccines

Question 2

Appendix III

Answer 2

• u- is human
• zu- humanized. mouse is the Fab fragment everything else is human.
• xi- human mouse chimera. more mouse than the humanized.
• o- mouse
• axo- rat mouse chimera

Question 3

Consequences on Pharmacy Practice

Answer 3

FDA approval based on risk/benefit consideration and the public has an expectation of “absolute” safety

Important to define the patient population who would benefit from the drug. Drug indication is incredibly important.

Question 4

Protein Glycosylation

Answer 4

1. Most glycoproteins contain NAN end glycosyl groups (sialated) which is a marker of enhanced circulation time in plasma/blood. Example is GM-CSF. Plasma concentrations are higher with increasing glycosylation. NAN is important for protection and prolongs half-life
2. De-sialation typically exposes galactose which is a marker for hepatocyte galactose receptor internalization/elimination.
3. Mannose receptors are found on macrophages.
4. When choosing a host cell for recombination keep in mind that E.coli prokaryote cells do not undergo post-translational glycosylations. Yet using E.coli is the least expensive route for recombinant protein expression on a pharmaceutical scale.

Question 5

Immunogenicity of Proteins

Answer 5

Proteins are more immunogenic than small molecular weight drugs.

Factors that can enhance protein immunogenicity are purity, aggregation, denaturation, incorrect or lack of glycosylation, homologue of other species, patient’s immune status and others.

Can decrease activity, is harmful and something FDA is concerned about. Always have to test the immunogenicity of proteins.

Question 6

Epo vs. Eprex

Answer 6

Seen before in manufacturing procedures affecting outcome of drug. Remember both were made from same master stock but different production cycles which caused production of antibody against both the endogenous erythropoietin as well as the recombinant drug. Example of neutralizing. Even though they came from same master stock the proteins were different.

Question 7

Neutralizing vs. Binding Antibody

Answer 7

neutralizing antibody blocks bioactivity. Example is the Eprex/Epo example. Long-term impacts. Eprex/Epo people had to take the drug forever because didn’t have the ability to make their own erythropoietin.

Binding antibody man not directly block function. Example is G-CSF

Question 8

Hybridoma Technology

Answer 8

monoclonal technology. Use B cell (cancerous) and myeloma cell hybrid from mouse for antibody against antigen with immortal characteristics. Allows for large pharmaceutical scale monoclonal antibody production.

Question 9

Fab fragments

Answer 9

Cleavage of IgG with papain forms two 50kDa Fab and cleavage with pepsin gives one Fab that is 100kDa.

Question 10

FcRN

Answer 10

Fc receptor to prevent intracellular degradation of IgG and to allow recirculation into blood (longer half-life). Recycles

biopharmaceuticals can take advantage of the FcRN to improve PK through fusion proteins.

Example: Enbrel TNF-alpha-receptor-Fc fusion protein. Normally TNF-alpha-receptor is cleared from blood in minutes however with binding to the fusion protein half life increases to 4.25 days.

Question 11

Antibody Functions

Answer 11

Receptor binding to block ligand-receptor interactions (steric hindrance)
neutralize circulating ligand and clear from system (example is inflammatory cytokine)
inactivate pathogen by Ab-Complement-mediated actions (antibody binds and attracts opsonization and complement).
ADCC: antibody-dependent cell-mediated cytotoxicity

Question 12

ADCC

Answer 12

antibody-dependent cell-mediated cytotoxicity
antibody binds to target then Fc receptor binds to Fc gamma. gets endocytose and is degraded through lysosomal pathway.
requires an Fc domain on the antibody.

Question 13

neutrophils and RBC overview

Answer 13

neutrophil half life is 6-8 hours. RBC half life is 100-120 days. Can not use neutrophil for transfusion but can do a blood transfusion.

Question 14

neutrophils and RBC overview

Answer 14

neutrophil half life is 6-8 hours. RBC half life is 100-120 days. Can not use neutrophil for transfusion but can do a blood transfusion.

Question 15

Balance erythropoiesis

Answer 15

Epoetin and the hyperglycosalted darbepoetin
90% produced by the kidneys
Increase RBC to reduce anemia.
Takes 10 days to detect an increase in RBC count. Immediate effects are increase in energy.

Question 16

PED and EPO

Answer 16

Use urine EPO test to determine if EPO is being injected as a PED. Stains based on MW. recombinant EPO more toward cathode (more positively charged) and darbepoitin is more toward anode with respect to endogenous. Western Blot. Endogenous will be lower (more negative than darbepoitin).

Question 17

Recombinant vs Exogenous G-CSF

Answer 17

Recombinant G-CSF produces greater numbers of fully functional neutrophils with a less time for maturation (1 day versus normal of 5). In total takes 13-18 days for recombinant to produce detectable neutrophils in circulation.

Question 18

Platelet Growth Factor

Answer 18

Deficiency in platelets can result from chemotherapy. Called chemotherapy-induced thrombocytopenia. IL-11 (oprelvekin) is a cytokine that is a platelet growth factor used in treatment. Stimulates growth of platelets.

Question 19

GPIIa/IIIb receptor

Answer 19

Drug binds to fibrinogen and mediates platelet aggregation.
Reopro (Abciximab) is an antibody.
integrillin (Eptifibatide) is a cyclic heptapeptide inhibitor.

Question 20

Hirudin

Answer 20

Hirudin is a 65 AA peptide produced in trace amounts by medicinal leeches.
MOA is to inhibit thrombin.
Leupirudin (Refludan) is a recombinant hirudin.
Bivalirudin (Angiomax) is an even smaller 20 AA optimized hirudin mimic.

Biotherapeutics from hirudin mimic leech hirudin.

Question 21

Cytokines

Answer 21

Hormone-like proteins. Function to regulate intensity and duration of immune response, cell-to-cell communication and mediate cell movement and growth
Cytokines include lymphokines, interleukins, and interferons.

Question 22

IFN production

Answer 22

made by live cells in response to heat-inactivated virus and confers cell resistance to infection by live virus. Example is that leukocytes, tumor-derived cells or virus-transformed cells can be exposed to lipopolysaccharides (LPS in bacterial membrane) and poly I:C to produce IFNs.

Question 23

IFN types

Answer 23

Two types. Type I are IFN produced by leukocytes or lymphoblastoid cells, IFN-alpha and also those produced by fibroblasts, IFN-beta. Type II IFN are produced by T lymphocytes, IFN-gamma. Families are similar in AA size but vary drastically in homology and thus have different functions.

Question 24

IFN-alpha2

Answer 24

as a therapeutic. May induce hairy-cell leukemia (B cells) to undergo differentiation. Combination with IL-2 is also shown to increase response rate in patients with metastatic melanoma. leaky capillary symptoms may occur prior to patients getting better and is a result of IFN-2 not the cancer. Flu-like symptoms.
Given in very small doses typically in the microgram range.

Question 25

IFN for MS

Answer 25

IFN-beta1 a and b.

Question 26

IFN for Hep C

Answer 26

IFN-2alpha a and b and con-1.

Question 27

IFN for osteopetrosis (abnormally dense bones)

Answer 27

IFN-gamma1b.

Question 28

IFN in Hep B treatment

Answer 28

Before the recombinant era 3 of 4 patients were treated with short course of low IFN and responded and 2 patients completely lost the hepatitis Be antigen. This antigen is not exposed on the Hep B viral particle. Now have an oral nucleoside called Lamivudine that may eventually replace subcutaneous IFN therapy.

Question 29

IFN in Hep C treatment

Answer 29

most common etiology for heptocellular carcinoma and often requires a liver transplant. IFN-alpha is effective treatment with a 41% response rate over a 6 month treatment course. Modification of IFN-alpha with the addition of PEG increases half life from 4-5 hours to 50-77 hours.

Question 30

IFN in MS treatment

Answer 30

IFN-beta-1alpha (Avonex and Rebif) and IFN-beta-1b (Betaseron) work by shutting down inflammation at the blood-brain barrier and thereby reducing the rate of relapse and decreasing the frequency and severity of symptoms
Both slow the progression of physical disability, reduce the rate of clinical relapses and reduce the development of brain lesions.

Question 31

IL-2 in treatment of Metastatic Renal Cell Carcinoma

Answer 31

IL-2 mediates antitumor effects through lymphokine-activated killer cells or LAK. These cells elicit cell-mediated tumor killing.
flu-like side effects but very impressive results.
Denileukin difitox (Ontack) is a fusion protein of IL-2 and diphtheria toxin for treatment of persistent or recurrent cutaneous T-cell lymphoma.

Question 32

rhGH

Answer 32

original recombinant human GH made in E. coli which added a methionine group on the N-terminus.

Used to increase levels of insulin like growth factor 1 in adults and increased growth rates in GH deficient children.

Somatrem: recombinant with methionyl (first produced)
Somatropin: recombinant GH without the methionine
Omnitrope: just recently approved.

Question 33

PTH

Answer 33

Forteo (Teriparatide): used for osteoporosis. Daily injections, expensive but very effective.
Initially synthesized as a single chain pre-peptide and is activated and processed down to the PTH form. Active part is N-terminal 24 AA. Smaller 11-mer fragment is sufficient to activate receptors.
functions to maintain Ca concentration in ECF through bone metabolism, renal tubular reabsorption and intestinal absorption. feedback mechanism. Also up-regulates osteoblasts that repair bone.

Question 34

PTH

Answer 34

Forteo (Teriparatide): used for osteoporosis. Daily injections, expensive but very effective.
Initially synthesized as a single chain pre-peptide and is activated and processed down to the PTH form. Active part is N-terminal 24 AA. Smaller 11-mer fragment is sufficient to activate receptors.
functions to maintain Ca concentration in ECF through bone metabolism, renal tubular reabsorption and intestinal absorption. feedback mechanism. Also up-regulates osteoblasts that repair bone.

Question 35

Somatostatin

Answer 35

Inhibits GH release and a number of GI and pancreatic hormones.

Sandostatin: octapeptide optimized for pharmaceutical properties. Recombinant pharmaceutical product is much smaller than the endogenous and has 2 D-AA for improved stability

Question 36

Adenosine Deaminase for SCID

Answer 36

SCID: severe combined immunodeficiency. autosomal recessive disease characterized by the absence of T and B cells. Linked to a deficiency of adenosine deaminase. Loss of T and B cells results in the inability to recover from even minor infections.

Intracellular enzyme, therefore is not found in the plasma.
initially developed a bovine ADA enzyme but it had a short half life and elicited antibodies

Modification of the bovine enzyme added on PEG polymers which decreased immunogenicity and increased hydration.

Question 37

Thrombolytic Enzymes

Answer 37

1. Streptokinase and Urokinase: nonfibrin-specific activation of plasmin through binding to plasminogen.
2. tPA: tissue plasminogen activator converts plasminogen to plasmin in a fibrin-dependent manner.
3. TNKase: sequence-modified version of tPA. Shown to produce even fewer major bleeding complications than tPA.
4. Streptokinase is a streptococci protein and immunogenicity is more of a concern compared to the recombinant tPA and TNKase

Question 38

DNAase

Answer 38

Cystic Fibrosis: genetic disease with a dysfunctional CFTR protein. Leads to poorly hydrated, thick mucous secretions in the airways that contain high concentrations of extracellular DNA.
DNAase is used to digest DNA polymers in mucus secretions in lung to decrease viscosity. The aerosolized version significantly decreases the risk of respiratory infection in CF patients.