|
AACF Home
Submission Info
Screening Agreement
Compound Submission Form
Screening Assays
-Biodefense Virus Panel
-BK Virus
-Hepatitis B Virus
-Hepatitis C Virus
-Herpes Viruses
-Human
Papilloma Virus
-Orthopoxviruses
-Respiratory Virus Panel
-SARS CoV
Data Interpretation
Medicinal Chemistry
Contact Info
|
Orthopoxviruses Assays
M. Prichard, Ph.D., University of
Alabama Birmingham
General Approach for
Determining Antiviral Activity and Toxicity for Orthopoxviruses
Our approach for
determining antiviral efficacy and toxicity of antiviral agents is to gain
enough definitive information such that a compound can be taken into
animal efficacy and toxicology studies and then into Phase I/II Clinical
Studies. The experimental approach is based upon the following: 1) in our
previous experience with this screening contract, we have found that a
high percentage of samples submitted will not have activity or will be too
toxic to evaluate. Therefore, an inexpensive, rapid assay such as a CPE-inhibition
assay that is semi-automated needs to be used initially to screen out the
negatives; 2) all screening assays are conducted in low-passaged human
cells; 3) each assay system contains a positive control (CDV) and a
negative control (ACV); 4) efficacy should be demonstrated by at least two
different assay systems that detect functional biologic activity; 5)
efficacy against VV and CV should be confirmed using other isolates; 6)
toxicity is determined using both resting and proliferating human
fibroblast cells; and 7) for selected compounds, toxicity in rodent
myeloid and erythroid progenitor cells is assessed.
A. Screening Assay
Systems for Determining Antiviral Activity Against VV and CV
Compounds are initially screened for activity using the CPE assay
in HFF cells. Further testing in two other cells lines, Vero and MRC-5,
and against other strains of virus is possible for compounds that
demonstrate activity in other assay systems. All the screening assay
systems utilized have been selected to show specific inhibition of a
biologic function, i.e., cytopathic effect (CPE) in susceptible human
cells. In the CPE-inhibition assay, drug is added 1 hr prior to infection
so the assay system will have maximum sensitivity and detect inhibitors of
early replicative steps such as adsorption or penetration as well as later
events. To rule out non-specific inhibition of virus binding to cells all
compounds that show reasonable activity in the CPE assay are confirmed
using a classical plaque reduction assay in which the drug is added 1 hr
after infection. These assay systems also can be manipulated by
increasing the pre-treatment time in order to demonstrate antiviral
activity with oligodeoxynucleotides and/or peptides. By delaying the time
of addition of drug after infection, information regarding which step in
the virus life cycle is inhibited (i.e., early vs. late functions) can be
gained. Upon request by the program officer, a direct inactivation assay
is employed to determine the virucidal activity of selected compounds.
1. Efficacy.
In all the assays used for primary screening, a minimum of six drug
concentrations was used covering a range of 100mg/ml
to 0.03mg/ml,
in 5-fold increments. These data allow us to obtain good dose response
curves. From these data, we calculated the dose that inhibited viral
replication by 50% (effective concentration 50; EC50) using the
computer software program MacSynergy II by M.N. Prichard, K. R. Asaltine,
and C. Shipman, Jr., University of Michigan, Ann Arbor, Michigan.
2. Toxicity.
The same drug concentrations used to determine efficacy were also used on
uninfected cells in each assay to determine toxicity of each experimental
compound. The drug concentration that is cytotoxic to cells as determined
by their failure to take up a vital stain, neutral red, (cytotoxic
concentration 50; CC50) was determined as above. We have
utilized a neutral red uptake assay and found it to be reliable and
reproducible and allows quantitation of toxicity based on the number of
viable cells rather than cellular metabolic activity.
It is important also to
determine the toxicity of new compounds on dividing cells at a very early
stage of testing. We have found that a cell proliferation assay using HFF
cells is a very sensitive assay for detecting drug toxicity to dividing
cells and the drug concentration that inhibits cell growth by 50% (IC50)
was calculated as described above. In comparison with four human diploid
cell lines and Vero cells, HFF cells are the most sensitive and predictive
of toxicity for bone marrow cells.
3. Assessment
of Drug Activity. To determine if each compound has sufficient
antiviral activity that exceeds its level of toxicity, a selectivity index
(SI) was calculated according to CC50/EC50. This
index, also referred to as a therapeutic index, was used to determine if a
compound warrants further study. For these studies, a compound that had
an SI of 10 or greater was evaluated in additional assay systems.
B. Confirmation of
Antiviral Activity and Toxicity for VV and CV
1.
Antiviral Activity. Compounds
that show activity in the CPE-inhibition assay will be confirmed using the
plaque reduction assay. Susceptibility of additional virus strains of VV
and activity in other cell types will also be determined for selected
compounds.
2.
Toxicity. In
addition to the toxicity component incorporated into each assay system, a
standardized cell cytotoxicity assay using a vital stain uptake (Neutral
Red) will be performed using 7 days of drug exposure to confluent
non-dividing cells. This assay measures direct cell cytotoxicity (CC50).
For the past 15 years, we have utilized a neutral red uptake assay and
found it to be reliable and reproducible and allows quantitation of
toxicity based on the number of viable cells rather than cellular
metabolic activity. It is important also to determine the toxicity of new
compounds on dividing cells at a very early stage of testing. We have
found that a cell proliferation assay using HFF cells is a very sensitive
assay for detecting drug toxicity to dividing cells and the drug
concentration that inhibits cell growth by 50% (IC50) was
calculated as described above.
C.
Laboratory Procedures for Determining Antiviral Efficacy and Toxicity
1.
Preparation of compounds for in vitro testing. The
letter of agreement with the drug sponsor included in the RFP indicates
that the sponsor will provide pertinent information regarding structure,
molecular weight, solubility, toxicity and any handling precautions the
sponsor is aware of. After receipt of the compound, they are entered into
a log book and into the drug screening inventory data base, and are stored
according to the location assigned. The compounds are then weighed using
an analytical balance and reconstituted in the appropriate vehicle. It is
critical at this point that solubility data be provided by the sponsor so
drug is not wasted determining the solubility. If the compound is water
soluble, it will be dissolved in tissue culture medium without serum at 1
mg/ml and diluted for use as indicated below in the description of the
assay system. If the compound is not water soluble, then it is
automatically dissolved in DMSO at a concentration of 10 mg/ml and diluted
for use in each assay. This has worked very well for the assays in HFF
cells as DMSO is not toxic for these cells at the concentrations utilized
(1.0%). When DMSO or other solvents are used, control cultures receive
media containing the same concentration of solvent as test cultures.
Although a compound that is not soluble would not be developed as an
antiviral agent, it is important to determine if these compounds have
activity so they can be modified chemically to increase their solubility.
2.
Screening and Confirmation Assays for VV and CV
a.
Preparation of Human Foreskin Fibroblast (HFF) Cells. Newborn
human foreskins are obtained as soon as possible after circumcision and
placed in minimal essential medium (MEM) containing vancomycin, fungizone,
penicillin, and gentamicin at the usual concentrations, for 4h. The
medium is then removed, the foreskin minced into small pieces and washed
repeatedly with phosphate buffered saline (PBS) deficient in calcium and
magnesium (PD) until red cells are no longer present. The tissue is then
trypsinized using trypsin at 0.25% with continuous stirring for 15 min at
37 C in a CO2 incubator. At the end of each 15-min period the
tissue is allowed to settle to the bottom of the flask. The supernatant
containing cells is poured through sterile cheesecloth into a flask
containing MEM and 10% fetal bovine serum. The flask containing the
medium is kept on ice throughout the trypsinizing procedure. After each
addition of cells, the cheesecloth is washed with a small amount of MEM
containing serum. Fresh trypsin is added each time to the foreskin pieces
and the procedure repeated until all the tissue is digested. The
cell-containing medium is then centrifuged at 1000 RPM at 4 C for 10 min.
The supernatant liquid is discarded and the cells resuspended in a small
amount of MEM with 10% FBS. The cells are then placed in an appropriate
number of 25 cm2 tissue culture flasks. As cells become
confluent and need trypsinization, they are expanded into larger flasks.
The cells are kept on vancomycin and fungizone to passage four, and
maintained on penicillin and gentamicin. Cells are used only through
passage 10.
b. Cytopathic Effect Inhibition Assay
Low
passage HFF cells are seeded into 96 well tissue culture plates 24h prior
to use at a cell concentration of 2.5 x 105 cells per ml in 0.1
ml of MEM supplemented with 10% FBS. The cells are then incubated for 24h
at 37 C in a CO2 incubator. After incubation, the
medium is removed and 125
ml
of experimental drug is added to the first row in triplicate wells, all
other wells having 100ml
of MEM containing 2% FBS. The drug in the first row of wells is then
diluted serially 1:5 throughout the remaining wells by transferring 25ml
using the BioMek 2000 Laboratory Automation Workstation. After dilution
of drug, 100 ml
of the appropriate virus concentration is added to each well, excluding
cell control wells, which received 100
ml
of MEM. The virus concentration utilized is 1000 PFU’s per well. The
plates are then incubated at 37 C in a CO2 incubator for 7
days. After the incubation period, media is aspirated and the cells
stained with a 0.1% crystal violet in 3% formalin solution for 4h. The
stain is removed and the plates rinsed using tap water until all excess
stain is removed. The plates are allowed to dry for 24h and then read on
a BioTek Multiplate Autoreader at 620 nm. The EC50 values are
determined by comparing drug treated and untreated cells using a computer
program.
c. Plaque
Reduction Assay using Semi-Solid Overlay
Two days
prior to use, HFF cells are plated into 6 well plates and incubated at 37
C with 5% CO2 and 90% humidity. On the date of assay, the drug
is made up at twice the desired concentration in 2X MEM and then serially
diluted 1:5 in 2X MEM using 6 concentrations of drug. The initial starting
concentration is usually 200
mg/ml
down to 0.06 mg/ml.
The virus to be used is diluted in MEM containing 10% FBS to a desired
concentration which will give 20-30 plaques per well. The media is then
aspirated from the wells and 0.2 ml of virus is added to each well in
duplicate with 0.2 ml of media being added to drug toxicity wells. The
plates are then incubated for 1h with shaking every 15 min. After the
incubation period, an equal amount of 1% agarose will be added to an equal
volume of each drug dilution. This gives final drug concentrations
beginning with 100mg/ml
and ending with 0.03
mg/ml
and a final agarose overlay concentration of 0.5%. The drug/agarose
mixture is applied to each well in 2 ml volume and the plates are
incubated for 3 days, after which the cells are stained with a 0.01%
solution of neutral red in phosphate buffered saline. After a 5-6h
incubation period, the stain is aspirated, and plaques counted using a
stereomicroscope at 10X magnification.
3.
Screening and Confirmation Assays for Toxicity
a. Neutral
Red Uptake Assay
Twenty-four h prior to assay, HFF cells are plated into 96 well plates at
a concentration of 2.5 x 104 cells per well. After 24h, the
media is aspirated and 125
ml
of drug is added to the first row of wells and then diluted serially 1:5
using the BioMek 2000 Laboratory Automation Workstation in a manner
similar to that used in the CPE assay. After drug addition, the plates
are incubated for 7 days in a CO2 incubator at 37 C. At this
time the media/drug is aspirated and 200 ul/well of 0.01% neutral red in
PBS is added. This is incubated in the CO2 incubator for 1h.
The dye is aspirated and the cells are washed using a Nunc Plate Washer.
After removing the PBS, 200
mg/well
of 50% ETOH/1% glacial acetic acid (in H2O) is added. The
plates are rotated for 15 min and the optical densities read at 540 nm on
a plate reader. The EC50 values are determined by comparing
drug treated and untreated cells using a computer program.
b. Cell
Proliferation Assay
Twenty-four h prior to assay, HFF cells are seeded in 6-well plates at a
concentration of 2.5 x 104 cells per well in MEM containing 10%
FBS. On the day of the assay, drugs are diluted serially in MEM
containing 10% FBS at increments of 1:5 covering a range from 100
mg/ml
to 0.03 mg/ml.
For drugs that have to be solubilized in DMSO, control wells receive MEM
containing 1% DMSO. The media from the wells is aspirated and 2 ml of
each drug concentration is then added to each well. The cells are
incubated in a CO2 incubator at 37 C for 72h. At the end of
this time, the media-drug solution is removed and the cells washed. One
ml of 0.25% trypsin is added to each well and incubated until the cells
start to come off of the plate. The cell-media mixture is then pipetted
up and down vigorously to break up the cell suspension and 0.2 ml of the
mixture is added to 9.8 ml of Isoton III and counted using a Coulter
Counter. Each sample is counted 3 times with 2 replicate wells per
sample.
c. Bone
Marrow Clonogenic Assays.
In vitro toxicity to
bone marrow progenitor cells can be determined by inhibition of myeloid
[colony-forming units granulocyte/macrophage (CFU-GM)] and erythroid
[burst-forming unit-erythroid (BFU-E)] colony formation in soft agar
clonal assays (36, 37). Using a 21-23 gauge needle attached to a syringe,
rodent bone marrow cells are collected from the leg bone of rats or mice
by flushing with Isocoves’ Modified Dulbecco’s medium (IMDM). A single
cell suspension is obtained by repeated aspiration through the needle.
Nucleated cells are enumerated with a hemacytometer and adjusted to the
desired cell concentration in IMDM. Murine CFU-GM assays are prepared with
2.5 x 105 nucleated cells/ml, 20% FBS, 10 ng/ml rmGM-CSF, and
0.2% agarose. BFU-E cultures include 30% FBS, 1% deionized BSA, 0.1 mM
2-ME, 4 U/ml rhEpo, 10 ng/ml rmIL-3, 2.5 x 105 nucleated
cells/ml and 0.2% agarose. Triplicate wells (in 6 well plates) containing
0.1ml of drug (10X) receive 1 ml of either culture mixture for each
concentration group and slowly mixed. The cultures are allowed to gel at
4 C and then incubated for 7 (CFU-GM) or 9 (BFU-E) days at 37 C in a
humidified atmosphere of 5% CO2 in air. Colonies are counted
using an inverted microscope. CFU-GM colonies are identified as cell
clones containing at least 40 cells. BFU-E cultures are stained with
dianisidine, and aggregates of greater than 60 hemoglobin-containing cells
are counted as erythroid colonies. The median inhibitory concentration
(IC50) and the 90% inhibitory concentration (IC90)
are derived from linear regression analysis of the logarithm of drug
concentration versus CFU-GM or BFU-E survival fraction.
4.
Reporting of results
a.
Screening and confirmation of efficacy and toxicity
A monthly
reporting form for screening and evaluation of antiviral efficacy and
toxicity is used to transmit results monthly to the Program Officer.
Included on the form are the month and year of report, the profile, dates
of testing, EC50 and EC90 data for efficacy of the
test and control compounds, the CC50 and IC50 for
the toxicity tests, the SI for each test and a space for comments and
recommendations.
|
