rDNA Drugs
Email Kathy

Recombinant DNA Drugs
August 30, 2007

 

First Warm UP Assignment Due Thursday8/30 at Noon -
Go to Oncourse Test and Survey Tool!

 

I. Recombinant DNA Technology: At the heart of most biotechnology techniques is...the gene. Manipulating genes and making gene-based products is what Biotechnology is all about! (at least until we understand more about the genome!!!) We won't be able to go too in depth about genes and molecular biology, but here are the basics:

 

1. A (real quick) review: What is a gene?

A. Anatomy of a Eukaryotic, Protein-encoding Gene (Figure; Print out)

B. Pre-mRNA processing and alternative splicing

2. Recombinant DNA Technology: the standard tools for cutting, pasting, and analyzing DNA

A. Plasmids - the "vehicle" for cloning Figure:

B. Restriction enzymes - "cut and paste" DNA Figure ; Animation

C. Host cells - to carry and replicate the cloned DNA Figure

 

II. Biotech's First Wave: Recombinant DNA Drugs - Homework!

 

The notes below are in OUTLINE form and require you to do a bit of homework (~2 hours of time before class). For each drug listed below, go to the link provided and read about the drug to determine:
(1) The Protein Product: what human protein is formulated as the drug?
(2) Indication: why is the drug prescribed and what it does it do in the body?
(3) Other notes: Alternate forms of this drug, competing products on the market, "the dark side" scandals, etc. More on this in class, but see if you can pick up any info beforehand.

It may be more difficult to get the info for some drugs than others - but get as much info as you can before class. This might be a great time to get to know your fellow students...split up the work...start thinking about a team project...!

 

Important Disclaimer: This is certainly not an inclusive list - but represents many of the best rDNA drugs on the market today. For an almost complete list of drugs in the pipeline, see List of biotech drugs in development Oct 2006 (rDNA and Monoclonals are in bold type). We will focus mainly on Drug Biotech companies (started as biotech firms) rather than the "Big Pharma" companies (Pfizer, J&J, Merck, Abbott, Wyeth, Bayer, Roche, etc) - many of which license biotech drugs and may even have developed a few of their own, but in general, aren't successful because of their biotech drugs (Lilly is an exception that we will discuss).

 

I. Amgen: (Nasdaq: AMGN)

 

 
Protein Product: what human protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, "Dark Side" etc)

1:Epogen /Procrit

rErythropoetin
[FDA approval 1989]

 

Epo binds the Epo receptor (Epo R) on bone marrow erythroid progenitors, inducing proliferation, maturation, and differentiation of red blood cells (MOA)

For patients with anemia due to Dialysis/ Chronic Kidney Disease / Renal Failure / Chemo / HIV

 

Stimulates production of RBCs. Truly revolutionized treatment of anemia!

Epo: For patients on dialysis with anemia.

Procrit: For non-dialysis use only = Cancer, Chronic Kidney Disease, HIV (anemia due to AZT treatment); some blood transfusions

Sales of ~$2.4 B annually

Increases the risk of thromboembolic complications (Stroke)


GOOD OR BAD FOR CANCER? Epo has pleiotropic effects

 

Epo Doping


2. Aranesp


rErythropoetin "+"
[FDA approval September 2001] (Darbepoetin)

For patients with anemia due to Dialysis/ Chronic Kidney Disease / Renal Failure / Chemo

2 additional N-linked carbohydrate chains creates a longer lasting effect (3X greater that Epo), requires fewer injections - 1 shot every 3 weeks, rather than once/week

Glycoengineering increased half life from 8.5 to 25 hours (see below)

 

Sales of $2.8 B annually
increases the risk of stroke

 

Muehlegg, Lazutina stripped of gold medals
February 24, 2002


3: Neulasta


rGranulocyte colony-stimulating factor (G-CSF) [FDA approvals 1991 for Neupogen, Feb 2002 for Neulasta]

For Neutropenia: low WBC count febrile neutropenia (low WBC count with fever/ infection) due to chemo, BMT, AML.

 

G-CSFs are glycoprotein cytokine hormones that stimulate proliferation and growth of granulocytes, particularly neutrophils (WBCs) but also eosinophils, and basophils

Tagline: "Are you ready to start Chemotherapy?" ... "Be Ready"

Neulasta:
Addition of a polyethylene glycol (PEG) molecule extends the half-life = only a single dose per chemotherapy cycle vs daily injections with Neupogen. Sales of $1.4 B annually

4. Infergen:


rInterferon alpha [FDA approval 1997]

A synthetic 'consensus' interferon based on the most common amino acid sequences in 12 natural interferons.


For patients with Chronic, non-responding, or relapsing hepatitis C viral (HCV) infection,

 

 

4 M Americans have Hepatitis C! -

MOA is unknown: "No one knows exactly how interferons work" but
Interferons are immunity-boosting proteins in WBCs with "antiviral, antiproliferative, and 'immunomodulatory' activities". Sales of ~$10 million/yr.

 


II. Biogen (Nasdaq: BGEN)
rDNA products on the Market

 
Protein Product: what human protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, etc)

1. Avonex

rInterferon beta-1a [FDA Approval May 1996]
Treatment of relapsing forms of MS. Slows the progression of MS by regulating the body's immune response against myelin.

Given as an IM injection (ouch!) once per week.
MOA: "Calms" or down-regulates the immune system*

Made in CHO cells. Sales of ~1 B annually

2. Intron A


rInterferon alpha-2b
[FDA Approval 1986
Over 20 indications,
* Malignant melanoma
* Non-Hodgkin's lymphoma
* Hairy cell leukemia
* Kaposi's sarcoma
* Chronic hepatitis B (HBV)
* Chronic hepatitis C (HCV)
* Condylomata acuminata
Interferons - see MOA Above. Sales exceeding $440 million in each of the last three years."

 

3. Engerix-B/
Recombiva
x

rHepatitis B vaccine
[FDA approval 8/99, 1/2000 for teens]

Infection with HBV
Prevention of 1° liver cancer

 

New HepB Law (2005) for all Indiana 9th and 12th graders

"The global HBV market exceeds $1 billion dollars annually. It will grow as more countries adopt WHO recommendations for the vaccination of newborns, teenagers, healthcare workers and other at-risk populations. "

4. Amevive


A recombinant fusion protein between IgG1 and the "leukocyte function-associated antigen-3" (LFA-3).
[FDA approval 1/31/03]

Moderate-to-severe chronic plaque psoriasis

 

Suppresses overactive T lymphocytes found in autoimmune diseases
(more on this when we get to mABs)

Injected by physician (15 mg IM, into the muscle) once a week for a total of 12 doses.


Because Amevive reduces T-cell counts (important for fighting off infections, etc), T-cell levels are monitored closely after
MOA



III. Chiron ( a Novartis partnership) recently acquired by Novartis, agreements with Bayer...

 
Protein Product: what human protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, etc)

1.
Betaseron


rInterferon beta-1b
[FDA approval July 93]
Multiple Sclerosis: Significantly delays the progression of secondary MS, including relapsing-remitting MS.

SubQ every other day.

 

Sales of ~$118 M annually


2.
Proleukin


Interleukin-2 - IL-2
[FDA approval 1992, 1998]

Cancer: Metastatic renal cell carcinoma, and metastatic melanoma.

 

IL-2 activates lymphokine-activated killer (LAK) cells, NK cells that normally destroy tumor cells.

MOA: IL-2 activates the immune system in several ways, but the major one is to stimulate T cell and natural killer (NK) cell proliferation, increasing and activating these immune system cells to find and destroy cancer cells. (See figure) Sales of ~$92 M annually; MOA


3.
Regranex

rPlatelet-derived growth factor. PDGF
[FDA approval 12/97]
Treatment of diabetic foot ulcers. PDGF is a cytokine (growth factor) that stimulates skin cell and blood vessel production

PDGF is involved in developing protective tissue and skin after a wound or ulcer (a process called granulation; MOA)

 

Sales of $48 M annually

 

OUCH!

6 October 2004: U.S. Will Miss Half Its Supply of Flu Vaccine "The British government has suspended the manufacturing license at the factory in Liverpool for three months because of concerns about sterility. As a result, the Chiron Corporation, the American biotechnology company that owns the factory, said it would not be able to supply any of the 46 million to 48 million doses it had planned to sell in the United States this year...."



IV. Genentech (NYSE: DNA)

rDNA products on the Market:

>
 
Protein Product: what human protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, etc)
1. Activase
1a.CathFlo Activase:
rTPA, tissue-plasminogen activator.
[FDA approval 4/95; CathFlo 9/00]

Thrombolytic:
Approved for treatment of AMI, cardiac ischemia, acute massive pulmonary embolism, and management of stroke.


CathFlo Activase: can be directly injected into an occluded central line catheter.

Converts plasminogen to plasmin, which activates fibrin, breaking down blood clot.


2. TNKase


rTPA, tissue-plasminogen activator "+"

 

TNKase can be administered over five seconds in a single dose, fastest administration of any thrombolytic

Thrombolytic: TNKase is bioengineered with 3 amino acid substitutions from natural t-PA: T, N and K (Thr, Asp, Lys).

 

=Increased Fibrin Specificity
=Long Plasma Half-Life
=Greater Resistance to Plasminogen Activator Inhibitor-1


Investigate the Molecule

"An advanced lytic by design"

 

1,000 variants of rt-PA were evaluated, using site-directed mutagenesis


3. Pulmozyme
rDeoxyribonuclease I (rhDNase)
[FDA approval 1994]

For management of cystic fibrosis in children 3 months - 5 years old. Inhalation Solution -


Pulmozyme hydrolyzes the DNA in sputum and airways of CF patients.

MOA: In CF patients, lungs and airways become clogged with mucous containing high concentrations of extracellular DNA released by degenerating leukocytes (neutrophils).

4: Neutropin


rHuman growth hormone (Somatropin; synthetic growth hormone)


[FDA approval 11/93]

What is a depot form?



Treatment of GHD in children and in adults

Identical to pituitary-derived hGH. Also indicated for growth failure associated with chronic renal insufficiency (CRI) prior to kidney transplantation, and short stature associated with Turner syndrome (45, X0)

Neutropin replaced Protropin [FDA approval October 1985; Genetech's first product];

Protropin stopped production in 2002

 

4a: Neutropin AQ: liquid formulation;
4b: Neutropin pen:
same stuff, single use throw-away 'pen' injection

 

 

Optional but interesting to scan (at least the beginning) if you are interested in how patents can be issued on human gene sequences and proteins!
1. US Patent 4,634,677, Issued January 6, 1987 to Goeddel and Genentech "Plasmid capable of expressing human growth hormone"
2. US Patent 4,670,393; Issued June 2, 1987 to Peter Seeburg and Genentech "DNA vectors encoding a novel human growth hormone-variant protein "
3. US Patent 5,424,199, Issued June 13, 1995 to Goeddel and Genentech "Human Growth Hormone"


V. Eli Lilly: (NYSE: LLY)
rDNA products on the Market: ( rDNA origin; there )

 
Protein Product: what human protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, etc)
1. Humulin
Chart comparing Time Activity Profiles (go here)
rInsulin
[FDA approval 1982]

Diabetes: Used by over 3.5 million people in the U.S. every day

Rapid-acting 'mealtime' form

Worldwide revenues of $2.5 B annually
1a: Humulin pens
1b: Humalog:
1c: Humalog Mix
rInsulin
1a: Single use injectable
1b. Rapid acting insulin
1c.Rapid acting + long acting 'basal' insulin

Chart comparing Time Activity Profiles

July 6, 2005 Lilly discontinues little-used insulins: Pork Insulin, and Humulin U and L
2. Humatrope

rHuman growth hormone (hGH) (Somatropin)
[FDA approval 8/96]
For Somatropin Deficiency Syndrome (SDS) in adults and GHD in children due to hypopituitarism, a pituitary tumor or other pituitary disorder, or Turner Syndrome

Annual revenues of ~$330 million.


July 25, 2003: FDA Approves Humatrope for Short Stature: (only the shortest 2-3% of children qualify).

3. Xigris

rActivated Protein C.
[FDA approval 11/01]

 

Activated Protein C acts inside the blood vessel as an anticoagulant / antithrombotic - reduces blood clots; controls inflammation. Activated by thrombin.

 

Treatment of severe sepsis, a fast-moving, dramatic, and often fatal acute response to infection that claimed 215,000 lives each year (pre-Xigris)

 

Costs associated with treatment of sepsis are $17B annually in the US.

 

Increases the odds of survival by over 38% (efficacy somewhat disputed)

FDA approved Xigris in a split vote by a 20-member advisory panel; its efficacy is somewhat disputed.

 

Standard treatments for sepsis (antibiotics, blood pressure drugs) usually cost less than $50 per day, while Xigris costs $6,800 per treatment.

 

Worldwide sales of ~$100M annually.

4. Forteo
rParathyroid hormone, N-terminal 34 amino acids (of 84)
[FDA Approval Nov 26, 2002]
Treatment of osteoporosis in women and men - Anabolic Therapy: stimulates new bone formation, osteoblasts, bone mineral density (BMD) and bone strength.

About 10 million people in the US suffer from osteoporosis.

 

Black box warning: Forteo may promote bone cancer (osteosarcoma) by stimulation of osteoblasts.

 

Worldwide sales of ~$400M annually.

5. Byetta

rGLP-1 (glucagon-like peptide-1) - "Exenatide"
[FDA Approval April 29, 2005]

 

Developed from Gila monster saliva! Patent 1995

Type II Diabetes: regulates glucose homeostasis and enhances glucose-dependent insulin secretion.

Byetta ia an incretin mimetic (extendin-4) similar to GLP-1, an incretin hormone that increases insulin release & decreases glucagon secretion from the pancreas; increases satiety

Downside: twice a day injection, but "an appropriate option to consider when Type II patients cannot control their blood sugar using oral medications."

 

Upside: Steady, significant appetite suppression and weight loss in overweight diabetic patients

 

Amylin (San Diego) is the developer - Lilly is partner

Projected sales of $60 million 2005 and $190 million in 2006


And one new one from Merck:



Protein Product: what protein is formulated as the drug
Indication: why drug is prescribed and what it does in the body
Other notes (Alternate forms of this drug, competing products on the market, etc)
1. Gardasil:

rQuadrivalent HPV (human papilloma virus) types 6, 11, 16, 18
[FDA Approval 6/8/06]

 

L1 capsid protein of all 4 viruses made individually in yeast cells and combined into one vaccine.

First Cancer Vaccine: Approved for the immunization of children aged 9 to 15 years and of adult females aged 16 to 26 years for the prevention of cervical cancer, high-grade cervical dysplasia (CIN 2/3), and warts caused by HPV s types 6, 11, 16 and 18
Currently 3,700 U.S. and 233,000 worldwide cervical-cancer deaths.

Virtually 100% effective in protecting against the HPV-16 and HPV-18 strains.

Downside: The wholesale price for Gardasil will be $120 per dose; $360 for all three doses.

 

OK, that's all!

 

A bit more detail on the Mechanism of Action (MOA) of some of the drugs can be found on the Case Studies page


 

Grumpy Complaint..."Why is it important to know the names of these drugs?"

Drug Name Confusion: Preventing Medication Errors

Also: From the WSJ, September 28, 2005 Agency Warns of Drug Mix-Ups (now archived)

 

The Food and Drug Administration and AstraZeneca PLC warned of drug-dispensing errors involving three brand-name medications.

  • Toprol, a beta blocker used to treat heart failure and hypertension.
  • Topamax, a J&J drug used to treat epilepsy and migraines
  • Tegretol, a Novartis AG drug used to treat seizures and neuralgia.

"In some cases, patients were hospitalized because of the medication errors. Most patients who were hospitalized suffered from a worsening of the underlying condition that wasn't treated properly as a result of receiving the incorrect medication. AstraZeneca said there were reports of recurrence of seizures, return of hallucinations, recurrence of hypertension and at least one suicide attempt, in its letter to health-care professionals.The company said most of the errors were the result of verbal and written prescriptions being incorrectly interpreted or incorrectly filled because of the similarity of the three drug names. Some of the drugs share overlapping strengths and they are typically stocked close together in pharmacies.

 



Objectives:
1. Know all the "parts" of a eukaryotic gene
2. Describe how restriction enzymes function to create recombinant DNA
3. Describe how bacteria can be used to produce eukaryotic proteins


For each of the companies studied, be able to list:

1. The Protein Product: what human protein is formulated as the drug?
2. Indication: why is the drug prescribed and what it does it do in the body?
3. Other notes we will discuss in class (Alternate forms of this drug, competing products on the market, "the dark side", etc)
4.You do not need to know who licenses what drug to what company, the annual revenues, the company history, or the dates of FDA approval...
5. Material on the readings
WILL end up as exam material - so read those carefully!


ARE YOU getting a feel for a life-saving drug that YOU and your fellow students just might develop in the near future? HOPE SO!

 

(back to Biol 540 schedule) ...........................................................Copyright Kathleen A. Marrs 2007