SOP Chemotherapy Starr Lab
PI: Timothy K. Starr
Lab Location: 12-175 Moos Towers
Original issue date: 4/16/14
Revision dates:
Prepared by: Michelle Glasgow and Timothy K. Starr
Approved by: Timothy K. Starr
Table of Contents
Table of Contents ................................................................................................................................................ 1
Overview ................................................................................................................................................................ 1
Background........................................................................................................................................................... 1
Antimetabolites ............................................................................................................................................................... 2
Plant derivatives ............................................................................................................................................................. 2
Alkylating agents............................................................................................................................................................. 2
Targeted chemotherapeutics ..................................................................................................................................... 2
Procedures ............................................................................................................................................................ 2
General safety procedures........................................................................................................................................... 2
Preparation of chemotherapies................................................................................................................................. 3
Dosing of chemotherapies ........................................................................................................................................... 3
Intra-peritoneal (IP) injection of chemotherapies ............................................................................................. 3
Intra-venous (IV) injection of chemotherapies in tail vein ............................................................................. 4
Oral gavage administration of chemotherapies .................................................................................................. 4
Hazard Identification and Risk of Exposure to the Hazards: .............................................................. 6
Exposure Controls Specific to Above Risk of Exposure: .................................................................................... 6
Waste Generated and Disposal Methods .................................................................................................... 6
Spill and Accident Response Procedures ................................................................................................... 6
Further sources of information ..................................................................................................................... 7
References ............................................................................................................................................................. 8
Overview
This protocol covers procedures related to delivery of chemotherapeutic agents to mice.
Background
Chemotherapy agents fall into different classifications, including: antimetabolites, plant derivatives,
topoisomerase inhibitors, alkylating agents, and antibiotics, among others. The chemotherapeutic
agents covered by this protocol are discussed below based on their main mode of action.
Antimetabolites
5-fluorouracil (5-FU) is classified as an antimetabolite. Antimetabolites act by inhibiting essential
metabolic processes that are required for synthesis of purines, pyrimidines, and nucleic acids.
Antimetabolites are typically S phase-specific. Specifically, 5-FU acts through inhibition of
thymidylate synthase, which is required for synthesis of pyrimidine thymidine, a nucleoside
required for DNA replication. Following administration of 5-FU, there are insufficient levels of
thymidine monophosphate (dTMP) and rapidly dividing cancerous cells undergo cell death.
Plant derivatives
Taxol is a chemotherapy agent derived from a plant. Taxol was originally isolated from the bark of the
Pacific yew tree, however, it is now produced using plant cell fermentation technology. Its main
mechanism is to stabilize microtubules such that they cannot disassemble during cell division. As
a result, chromosomes are unable to achieve a metaphase spindle configuration and mitosis
cannot progress.
Alkylating agents
Carboplatin is one of the most commonly used alkylating agents. It prevents cell division by crosslinking strands of DNA. Cell death results because of continued synthesis of other cell
constituents, such as RNA and protein, which leads to unbalanced cell growth and ultimately cell
death.
Targeted chemotherapeutics
NVP-BKM120 is a compound that inhibits phosphoinosotide 3-kinase (PI3K), a lipid kinase that
consists of enzymatic subunits that are subdivided, on the basis of sequence homology and
substrate specificity, into class I, II, and III and the p85/p55 regulatory subunits. Activating and
transforming mutations in the PIK3CA gene of the p110α subunit (one of the subgroups of class
I) of PI3K are commonly found in cancer. NVP-BKM120 can inhibit all four Class I PI3K
isoforms, as well as the more common somatic PI3K mutations (H1047R, E542K and E545K).
Administration of NVP-BKM120 leads to inhibition of various Akt downstream signaling
pathway components. Studies have shown that this compound demonstrates significant,
concentration dependent, cell growth inhibition and induction of apoptosis in a variety of tumor
cancer cells, particularly for those harboring p110 mutants and/or over-expressing erbB2.
Antibodies targeting vascular endothelial growth factor (VEGF) are used to treat cancer. VEGFVEGFR signaling has been identified as a major regulator of endothelial cell growth and survival
and has been implicated in pathological angiogenesis. Monoclonal antibodies (mAbs) that bind
to VEGF and block this signaling have been developed for the purpose of inhibition of tumor
growth.
Procedures
General safety procedures
Working stocks of chemotherapeutics will be prepared inside a Biological Safety Cabinet. Always wear
PPE including lab coat, gloves, facemask and eye goggles when handling and preparing stock
solutions of chemotherapeutics.
RAR Area Supervisor must be informed of your intent to use chemotherapies.
PI must train RAR staff on hazards and proper handling procedures.
Cages containing animals injected with chemotherapies must be clearly labeled as such.
Preparation of chemotherapies
All chemotherapeutic agents will be prepared following manufacturer's guidelines. Stock solutions will
be sterilized when appropriate. In general, drugs will be delivered in the following vehicles:
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5-FU in 0.9% NaCl (saline solution)
Carboplatin in 0.9% NaCl (saline solution)
Taxol in 50% cremophor/50% ethanol
NVP-BKM120 in N-Methyl-2-pyrrolidone (NMP) and Polyethylene Glycol 300 (10%/90% v/v/)
Antibodies to VEGF in 0.9% NaCl (saline solution)
Dosing of chemotherapies
Dosing of chemotherapies is generally based on existing literature reporting use of the chemotherapies
in mice. In combination trials, experimental protocols will specify the dosing range and the
combination dosing range, generally based on in vitro assays previously used to determine
ED50s of the drugs.
5-FU: Multiple dosing strategies have been used in mice including: 1) 50 mg/kg for 4 consecutive days
(Farrell, Bready et al. 1998); 2) 40 mg/kg IP daily bolus for 2-week cycles of 5 days on followed
by 9 days off, continuing until euthanization or moribundity (Durkee, Shinki et al. 2009); 3)
Single IP bolus of 400 mg/kg (Boushey, Yusta et al. 2001); and 4) 12.5 mg/kg in HBSS IV daily
for 2 days in a xenograft model (Bunz, Hwang et al. 1999). The maximum tolerable dose in nude
mice is 100 mg/kg/week x 4 weeks (Azrak, Cao et al. 2004). In our studies we will start with 40
mg/kg administered IP daily for 2-week cycles of 5 days on followed by 9 days off, based on the
work by (Durkee, Shinki et al. 2009).
Taxol: Multiple dosing strategies have been used in mice including: 1) 12 mg/kg IV tail vein once every
2 days for a total of three doses (Roby, Niu et al. 2008); and 2) 10 mg/kg IP every 2 days
continuously until moribund or endpoint (Campbell, Greenaway et al. 2010). The maximum
tolerable dose in mice is 80 mg/kg in C3Hf/Kam mice (Li, Price et al. 1999) and 60 mg/kg
administered IV as 20 mg/kg per day for three days in nude mice (Huang, Lopez-Barcons et al.
2006). In our studies we will start with 10 mg/kg administered IP every 2 days based on the work
by (Campbell, Greenaway et al. 2010).
Carboplatin: Based on the work of (Farrell, Bready et al. 1998) we will start with 125 mg/kg
administered IV for one day for non-lethal treatment. The maximum tolerable dose in nude mice
is between 120 and 160 mg/kg when administered IV once per week for two weeks (Boven, van
der Vijgh et al. 1985) The LD(10) for mice is 165 mg/kg after a single IV bolus injection (van
Hennik, van der Vijgh et al. 1987).
NVP-BKM120: Based on the work of (Maira, Pecchi et al. 2012) we will use a single dose of 30-60
mg/kg administered by oral gavage.
Anti-VEGF antibodies: Based on the work of (Jiang, Engelbach et al. 2014) we will use the anti-VEGF
monoclonal antibody, B20-4.1.1 (Genentch), at 10mg/kg administered IP twice per week.
Intra-peritoneal (IP) injection of chemotherapies
Prepare working stock of chemotherapeutic by diluting stock solution to the appropriate concentration
using sterile vehicle solution. Load a 1 ml syringe (23 gauge needle) with appropriate volume of
working stock. Keep in sterile field.
Hold mouse firmly: Place mouse on a rough surface while holding the tail firmly (Note: Smooth
surfaces will frighten the mouse because it cannot get a foothold. This may cause it to turn
around and try to bite in its attempt to escape.) Grasp the nape gently and firmly with your free
hand and lift the mouse. Place the mouse's tail between the last two fingers of the hand that is
holding the nape. (See Figure below).
Insert needle 10 mm ventral to hip joint. Insert needle through
skin/peritoneum into stomach cavity. Avoid nipples and keep needle close
to skin to avoid perforating viscera. Inject up to 100 µl of working stock
solution. Dispose of needles immediately in sharps containers. Replace
mouse in cage.
Figure: Method of
immobilizing mouse for
IP injection
Intra-venous (IV) injection of chemotherapies in tail vein
Place mouse securely in harness.
Dip tail in warm water (45ºC) for 30 seconds. Wipe dry with sterile gauze
Insert needle into lateral tail vein and gently push plunger (see Figure below).
Remove needle, remove mouse from harness, and return mouse to cage.
A
B
Figure Proper technique for IV injections. A) Warm tail by dipping in 45ºC water for 30 seconds,
then wipe with sterile gauze. B) Insert needle in lateral tail vein.
Oral gavage administration of chemotherapies
The following procedure is based on the protocol described in (Bertola, Mathews et al. 2013). See figure
below as a guide.
Select the appropriate gavage needle gauge size and length based on the size and weight of animals. For
example, 15–20 g mice: 22-gauge (1–1.5 inches); 20–25 g mice: 20-gauge (1–1.5 inches); 25–30
g mice: 18-gauge (1.5–2 inches).
Attach gavage needle with ball tip to 1 ml syringe and draw up the appropriate volume of chemotherapy.
Volumes administered should not exceed 2 mls per 100 g body weight.
Gently grasp mouse as described in the IP injections above. Place needle in mouth between incisors and
molars and pass needle along roof of mouth while head is held in moderate extension.
Gently extend head and neck backwards using the needle as a lever and position the neck and esophagus
in a straight line.
Move the gavage needle smoothly along the upper palate into the esophagus. The mouse generally will
initiate a swallowing reflex as the needle nears the pharynx, which facilitates entry into the
esophagus.
Gently push the syringe plunger and expelling the liquid into the mouse's stomach
Slowly remove the needle by following the same motion used to insert the needle.
Figure: Oral gavage technique. a) measure length of the gavage needle against the animal's body. b) gently
insert needle through mouth and esophagus into stomach. c) gently expel chemotherapy using the syringe
plunger, then remove needle
Return the mouse to its cage and monitor the mouse for at least 10 minutes for any sign of labored
breathing or respiratory distress. If mouse displays any signs of respiratory distress, such as
frothing at the mouth or nose, or difficulty in breathing, the mouse may have aspirated or the
trachea or esophagus may have been perforated. If these signs persist for more than one to two
minutes after removing the needle, the mouse will be euthanized.
Tips for successful oral gavage:
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Place needle behind the base of tongue near the center of the back of the mouth. Avoid the side
of the mouth where mouse can bite needle.
Wait until the animal begins to swallow due to a swallowing reflex, which allows the needle to
"fall" down esophagus with little or no pressure.
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Make sure needle is in stomach, not trachea, by checking the depth of insertion. You can also
pull the plunger back a small distance and if you see air bubbles, that indicates the needle is in
the trachea, not the stomach.
Never use excessive pressure, which could perforate the mouth, or esophagus.
Hazard Identification and Risk of Exposure to the Hazards:
Sharps, especially needles used for injection. Never recap needles, but place directly in Sharps
container.
Exposure to chemotherapeutics via accidental spills, splashes, inhalation or needle sticks could cause
adverse health reactions.
Exposure Controls Specific to Above Risk of Exposure:
PPE - Lab coats, safety goggles, mask, gloves, sleeves and sharps containers.
Use Biologic Safety Cabinet during preparation and handling of chemotherapeutic agents.
All sharps and glass waste will be disposed of in an approved hard plastic sharps container (U Stores #
CX40245, MS07407 or similar), No Cardboard sharps pouches.
If exposure occurs:
Consult a physician. Show the MSDS to the doctor in attendance. Move out of dangerous area. If
breathed in, move person into fresh air. If not breathing, give artificial respiration. Consult a
physician. In case of skin contact wash off with soap and plenty of water. Consult a physician.
In case of eye contact flush eyes with water as a precaution. If swallowed never give anything by
mouth to an unconscious person. Rinse mouth with water. Consult a physician. In case of
accidental injection seek medical attention immediately.
Waste Generated and Disposal Methods
Chemotherapies are considered hazardous chemicals. Any unused chemotherapies that need to be
discarded should be placed in a properly labeled hazardous waste container and disposed of by
the University of Minnesota Chemical Waste Facility (part of the Division of Environmental
Health & Safety).
Sharps containers will be sealed when ¾ full and placed in designated waste area.
In addition to unused chemical waste, the following waste disposal measures should be followed:
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Sharps contaminated with trace carcinogens or hazardous drugs must be disposed of in a separate
sharps container that is labeled “Hazardous Sharps,” sealed, and placed in a yellow bag for
incineration
Residual and trace chemotherapeutic wastes, such as completely empty delivery devices, gowns,
gloves, tubing, etc., should be disposed of in yellow bags for incineration.
Bedding should be put in yellow bags for collection and managed as hazardous chemical waste
(only while being treated with chemotherapies)
Spill Response Procedures
Clean up waste regularly and abnormal spills immediately. Avoid breathing dust and avoid contact with
skin and eyes. Wear protective clothing, gloves, safety glasses and dust respirator. Use dry clean
up procedures and avoid generating dust. Vacuum up or sweep up. NOTE: Vacuum cleaner must
be fitted with an exhaust micro filter (HEPA type) (consider explosion-proof machines designed
to be grounded during storage and use). Dampen with water to prevent dusting before sweeping.
Place in suitable containers for disposal. If in liquid form, wipe up with paper towel. When
decontaminating is finished carefully place any and all materials in appropriate biohazard
bin/bag or autoclave bag.
Only attempt to clean up very small powder and liquid spills (e.g., very small amounts spilled around a
balance). In this situation, wear appropriate PPE as described, place wet paper towels over
powder or use absorbant pads for liquid, and place all materials in leak proof container. All spill
materials should be disposed of as hazardous waste through the U of M Chemical Waste
Program. Larger spills should be immediately reported to DEHS for cleanup (612-626-6002).
Accident Response Procedures
If Incident Results in a Hazard Exposure ( i.e. face or eye splash, cut or puncture with sharps, contact
with non-intact skin):
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Encourage needle sticks and cuts to bleed, gently wash with soap and water for 5 minutes; flush
splashes to the nose, mouth, or skin with water; and flush eyes at the nearest eyewash station
with clean water for 15 minutes.
Call 911 or seek medical attention.
o For urgent care employees may go to HealthPartners Occupational and Environmental
Medicine (M/F day time or Urgent Care after hours), or UMMC-Fairview Hospital (24
hrs). You may seek medical attention at the closest available medical facility or your own
healthcare provider.
o Follow-up must be done by HealthPartners Occupational and Environmental Medicine.
Report the incident to your supervisor as soon as possible, fill out the appropriate documentation.
o Employee First Report of Injury
o Supervisor Incident Investigation Report
Send Incident Report Form to the IBC if exposure has occurred during work on an IBC protocol.
Report all biohazard exposures to the Office of Occupational Health and Safety (612-626-5008)
or uohs@umn.edu.
Note: It is important to fill out all of the appropriate documents to be eligible to collect workers
compensation should any complications from the hazardous exposure arise in the future.
Further sources of information
For further information view the UMN DEHS website containing Bio Basic Fact Sheets at
http://www.dehs.umn.edu/bio_basicfacts.htm.
For general information on Biosafety, access the Biosafety in Microbiological and Biomedical
Laboratories (BMBL) 5th Edition from the CDC at BMBL
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
For Material Safety Data Sheets access the Public Health agency of Canada website MSDS
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php.
References
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McLeod and Y. M. Rustum (2004). "Therapeutic synergy between irinotecan and 5-fluorouracil against
human tumor xenografts." Clin Cancer Res 10(3): 1121-1129.
Bertola, A., S. Mathews, S. H. Ki, H. Wang and B. Gao (2013). "Mouse model of chronic and binge
ethanol feeding (the NIAAA model)." Nat Protoc 8(3): 627-637.
Boushey, R. P., B. Yusta and D. J. Drucker (2001). "Glucagon-like peptide (GLP)-2 reduces
chemotherapy-associated mortality and enhances cell survival in cells expressing a transfected GLP-2
receptor." Cancer Res 61(2): 687-693.
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(2009). "Longitudinal assessment of colonic tumor fate in mice by computed tomography and optical
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B. Wiemann, C. O. Starnes, A. M. Havill, Z. N. Lu, S. L. Aukerman, G. F. Pierce, A. Thomason, C. S.
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Menezes, G. Martiny-Baron, D. Fabbro, C. J. Wilson, R. Schlegel, F. Hofmann, C. Garcia-Echeverria,
W. R. Sellers and C. F. Voliva (2012). "Identification and characterization of NVP-BKM120, an orally
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