EXHIBIT 10.7
Collaborative Research Agreement
This agreement dated as of November 1, 2001 between Pfizer Inc, a Delaware
corporation, having a principal place of business at 000 X 00xx Xxxxxx, Xxx
Xxxx, Xxx Xxxx 00000 and its Affiliates ("Pfizer") and MultiCell Technologies,
Inc., having a principal place of business at 00 Xxxxxx Xxxx, Xxxxx 000,
Xxxxxxx, XX 00000 ("MultiCell")
WITNESSETH;
Whereas, Pfizer wishes to identify hepatocyte cell lines with bile canaliculated
morphology and function and to identify cell lines predictive of CYP enzyme
induction in humans and cell lines that approximates metabolic response of liver
to glucocorticoids and to assess the functional carbohydrate metabolizing
activity of such cell lines; and
Whereas, MultiCell has a library of hepatocyte cell lines and the expertise to
test cell lines for morphology, function, induction and metabolic response; and
Whereas, Pfizer wishes to engage MultiCell to test their library of hepatocyte
cell lines for morphology, function, induction and metabolic response;
Now Therefore, in consideration of the promises and mutual covenants herein,
MultiCell and Pfizer agree as follows:
1. Definitions
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1.1 "Affiliate" shall mean any corporation, firm, partnership or
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other entity which directly or indirectly controls, is
controlled by, or is under common control with either of the
parties.
1.2 "Compound" shall mean a compound from Pfizer's chemical library
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and commercially available compounds provided to MultiCell for
testing in the Research Plan.
1.3 "Contract Period" means the period beginning November 1, 2001
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and ending October 31, 2002.
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1.4 "MultiCell Confidential Information" shall mean all information,
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including, but not limited to MultiCell Materials, which is
disclosed by MultiCell to Pfizer and designated "Confidential"
in writing by MultiCell at the time of disclosure to Pfizer or
within thirty (30) days following disclosure to the extent that
such information is not (i) known to Pfizer as of the date of
disclosure to Pfizer other than by virtue of a prior
confidential disclosure to Pfizer by MultiCell; or (ii) is not
disclosed in published literature, or otherwise generally known
to the public through no fault of omission of Pfizer; or (iii)
is not obtained from a third party free from any obligation of
confidentiality to MultiCell.
1.5 "MultiCell Material" shall mean any materials, including cell
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lines and/or progeny derived from cell lines, provided by
MultiCell to Pfizer for testing in the Research Plan.
1.6 "Pfizer Confidential Information" means all information,
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including, but not limited to Compounds and Pfizer Materials,
which is disclosed by Pfizer to MultiCell and designated
"Confidential' in xxxxxxx by Pfizer at the time of disclosure to
MultiCell or within thirty (30) days following disclosure to the
extent that such information is not (i) known to MultiCell as of
the date of disclosure to MultiCell other than by virtue of a
prior confidential disclosure to MultiCell by Pfizer; or (ii) is
not disclosed in published literature, or otherwise generally
known to the public through no fault or omission of MultiCell;
or (iii) is not obtained from a third party free from any
obligation of confidentiality to Pfizer.
1.7 "Pfizer Material" shall mean any materials provided by Pfizer to
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MultiCell for testing in the Research Plan.
1.8 "Research Plan" shall mean the written plan describing the
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research and testing of Compounds, Pfizer Materials and
MultiCell Materials to be carried out by MultiCell and Pfizer.
The Research Plan is appended to and made a part of this
Agreement as Exhibit A.
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1.9 "Results" shall mean all data generated from testing Compounds,
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Pfizer Materials and MultiCell Materials according to the
Research Plan.
2. Collaborative Research Program
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2.1.1 Purpose. MultiCell and Pfizer shall conduct the Research Plan
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throughout the Contract Period. The objective of the Research
Plan is to identify hepatocyte cell lines with bile canaliculi
morphology and function and to identify cell lines for
prediction of CYP enzyme induction in humans and cell lines that
approximate metabolic response of liver to glucocorticoids and
to assess the functional carbohydrate metabolizing activity of
such cell lines.
2.1.2 Modifications to Research Plan Modifications to the Research
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Plan shall be appended to Exhibit A and made part of this
Agreement. Any modifications to the Research Plan must be agreed
to in writing by both parties.
2.2 Research Committee
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2.2.1 Purpose. Pfizer and MultiCell each shall appoint, in their sole,
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unfettered discretion, members of the Research Committee, the
responsibility of which shall include:
(a) the review and evaluation of progress under the Research
Plan;
(b) the preparation of any amendments to the Research Plan;
(c) the coordination and monitoring of publication of research
results obtained from and the exchange of information and
materials that relate to the Research Plan. (This function
shall survive the termination of this Agreement).
2.2.2 Membership. Substitutes for Committee members may be appointed
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at any time. The members initially shall be:
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Pfizer: Xxxxxxx Xxxxx
------ Rob Poker
Xxx Xxxxxxxxxx
Xxxx Xxxxxxxx
MultiCell: Xxx Xxxxx
--------- Xxxxx Xxxxxxxx
Xxxxxxxxx Xxxxxx
Xxx Xxx
2.2.3 Chair. The Research Committee shall be chaired by two
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co-chairpersons, one appointed by Pfizer and the other appointed
by MultiCell.
2.2.4 Meetings. The Research Committee will meet at least once every
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quarter, in person, at places and on dates to be mutually agreed
by both parties. Representatives of Pfizer and MultiCell or
both, in addition to the Research Committee, may attend such
meetings at the invitation of both parties.
2.2.5 Minutes. The Research Committee shall keep accurate minutes of
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its deliberations which record all proposed decisions and
actions recommended or taken. Drafts of minutes shall be
delivered to all Research Committee members within ten (10)
business days after each meeting. Draft minutes shall be edited
by the co-chairpersons and shall be issued in final form with
their approval and agreement.
2.2.6 Decisions. All technical decisions shall be made by Pfizer and
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MultiCell, with final authority residing with Pfizer.
2.2.7 Expenses, Pfizer and MultiCell shall each bear all expenses,
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including reasonable travel, related to the participation of
theft designated members of the Research Committee,
respectively.
2.3 Reports and Materials.
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2.3.1 Reports. During the Contract Period, MultiCell shall furnish to
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the Research Committee a quarterly written report fifteen (15)
days prior to the Research
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Committee meeting, describing the progress under the Research
Plan and discussing and evaluating the results of such work.
2.3.2 Compounds and Pfizer Materials. During the Contract Period,
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Pfizer shall furnish to MultiCell Compounds and Pfizer Materials
(a) as a matter of course as described in the Research Plan, or
(b) upon MultiCell's' written request. MultiCell will not
elucidate the structures of any Compound or Pfizer Material,
shall use them only for testing under the terms and conditions
of this Agreement, shall supply to Pfizer but otherwise keep
confidential the Results of any tests conducted on such
Compounds and Pfizer Materials, and shall at Pfizer's election
either return or destroy any portion of such Compounds and
Pfizer Materials remaining after the conclusion of the testing.
2.3.3 MultiCell Materials. During the Contract Period, MultiCell shall
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furnish to Pfizer MultiCell Materials (a) as a matter of course
as described in the Research Plan, or (b) upon Pfizer's written
request. Pfizer shall use them only for testing under the terms
and conditions of this Agreement, shall supply to MultiCell but
otherwise keep confidential the Results of any tests conducted
on such MultiCell Materials, and shall at MultiCell's election
either return or destroy any portion of such MultiCell Materials
remaining after the conclusion of the testing.
2.4 Laboratory Facilities and Personnel. MultiCell and Pfizer each
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shall provide laboratory facilities, equipment and personnel, in
each case suitable to the need, for carrying out the Research
Plan.
2.5 Training. MultiCell will provide training as described in the
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Research Plan, at Pfizer's sole expense. The training shall
occur on mutually agreeable dates and Pfizer will pay actual
costs for supplies during the training, travel and lodging
expenses in connection with the training sessions. Pfizer
scientists may also seek MultiCell's advice by phone or email,
from time to time on specific questions.
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3. Funding the Research Plan.
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3.1. In consideration for its performance of the Research Plan,
Pfizer will pay MultiCell a total of seven hundred and twenty
four thousand five hundred dollars ($724,500.00) upon receipt of
invoice from MultiCell, payable as follows:
(a) Within thirty (30) days of the execution of this Agreement,
a fee of four hundred and seventy seven thousand five
hundred dollars ($477,500.00).
(b) On the first business day of each calendar quarter,
starting January 1, 2002, a payment of sixty one thousand
seven hundred and fifty dollars ($61,750.00) against
MultiCell's Invoice for such calendar quarter for work to
be performed and costs to be incurred in such calendar
quarter.
4. Treatment of Confidential Information
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4.1 Confidentiality
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4.1.1 Pfizer and MultiCell each agree that during the term of this
Agreement and for three (3) years thereafter, it will keep
confidential, and will cause its Affiliates to keep
confidential, all Pfizer Confidential Information or MultiCell
Confidential Information and Results, as the case may be, that
is disclosed to it, or to any of its Affiliates pursuant to this
Agreement. Neither Pfizer nor MultiCell nor any of their
Affiliates shall use such Confidential Information except as
expressly permitted in this Agreement.
4.1.2 Pfizer and MultiCell each agree that any disclosure of the
other's Confidential Information to any officer, employee or
agent of the other party or of any of its Affiliates shall be
made only if and to the extent necessary to carry out its
responsibilities under this Agreement and shall be limited to
the maximum extent possible consistent with such
responsibilities. Pfizer and MultiCell each agree not to
disclose the other's Confidential Information to any third
parties under any circumstance without written permission from
the other party. Each party shall take such action, and shall
cause its Affiliates to take such action, to preserve the
confidentiality of each other's Confidential Information as it
would customarily take to preserve confidentiality of its own
Confidential Information. Each party, upon the other's request,
will return all the Confidential Information disclosed to the
other party pursuant to this Agreement, I
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including all copies and extracts of documents, within sixty
(60) days of the request upon the termination of this Agreement
except for one (1) copy which may be kept for the purpose of
complying with continuing obligations under this Agreement.
4.1.3 Pfizer and MultiCell each represent that all of its employees;
and any subcontractors or consultants to such party,
participating in the Research Plan who shall have access to
Pfizer Confidential Information and MultiCell Confidential
Information are bound by agreement to maintain such information
in confidence.
4.2 Publication. Notwithstanding any matter set forth with
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particularity in this Agreement to the contrary, Results
obtained in the course of this Agreement may be submitted for
publication following scientific review and subsequent approval
by Pfizer's management, which approval shall not be unreasonably
withheld. After receipt of the proposed publication by Pfizer's
management, written approval or disapproval shall be provided
within thirty (30) days for a manuscript and within sixty (60)
days for an abstract for presentation at, or inclusion in the
proceedings of a scientific meeting, and for a transcript of an
oral presentation to be given at a scientific meeting and within
sixty (60) days for a transcript of an oral presentation to be
given at a scientific meeting in order for Pfizer to delete
Pfizer Confidential Information,
4.3 Publicity. No press releases or other statements in connection
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with this Agreement intended for use in the public or private
media shall be made by Pfizer or MultiCell without the prior
written consent of the other party. If either party is required
by law or governmental regulation to describe its relationship
to the other, it shall promptly give the other party notice with
a copy of any disclosure it proposes to make. In addition,
neither party shall use the other party's name in connection
with any products, promotion, or advertising without prior
written permission of the other party.
4.4 Permitted Disclosure.
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4.4.1 Disclosure Required by Law. If either party is requested to
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disclose the Confidential Information in connection with a legal
or administrative proceeding or is otherwise required by law to
disclose the Confidential Information, such party will give the
other party prompt notice of such request. The disclosing party
may seek an appropriate protective order or other remedy or
waive compliance with the provisions of this Agreement. If such
party seeks a protective order or other remedy, the other party
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will cooperate. If such party fails to obtain a protective order
or waive compliance with the relevant provisions of this
Agreement, the other party will disclose only that portion of
Confidential Information which its legal counsel determines it
is required to disclose.
5. Intellectual Property Rights. The following provisions relate to rights in
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the intellectual property developed by or for MultiCell or Pfizer, or both,
during the course of carrying out the Research Plan.
5.1 Ownership.
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(i) All MultiCell Confidential Information and MultiCell
Materials shall be owned by MultiCell.
(ii) All Pfizer Confidential Information, Compounds, Pfizer
Materials and Results shall be owned by Pfizer.
5.2. MultiCell acknowledges that Pfizer will use the Results as it
sees fit in its research and the discovery, development and
commercialization of chemical and biological products without
further compensation to MultiCell.
5.3. Pfizer acknowledges MultiCell will use the Results, pursuant to
the terms and conditions of this Agreement, as it sees fit in
its efforts to develop and commercialize MultiCell Materials
without further compensation to Pfizer, provided such use does
not require disclosure of Pfizer Confidential Information or
compete with development and commercialization of Pfizer
products.
6. Term, Termination and Disengagement.
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6.1 Term. Unless sooner terminated, as provided below, this
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Agreement shall expire on October 31, 2002.
6.2 Events of Termination. The following events shall constitute
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events of termination ("Events of Termination"):
(a) any written representation or warranty by MultiCell or
Pfizer, or any of its officers, made under or in connection
with this Agreement shall prove to hare been incorrect in
any material respect when made;
(b) MultiCell or Pfizer shall fail in any material respect to
perform or observe any term, covenant or understanding
contained in this Agreement or in any of the
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other documents or materials delivered pursuant to, or
concurrently with, this Agreement, and any such failure
shall remain unremedied for thirty (30) days after written
notice to the failing party.
6.3 Termination.
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6.3.1 Upon the occurrence of any Event of Termination, the party not
responsible may, by notice to the other party, terminate this
Agreement.
6.3.2 Termination of this Agreement for any reason shall be without
prejudice to:
(a) the rights and obligations of the parties provided in those
Sections of the Agreement which by virtue of their term and
condition extend beyond any termination of this Agreement;
(b) any other remedies which either party may otherwise have.
7. Representations and Warranties. MultiCell and Pfizer each represents and
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warrants as follows:
7.1 It is a corporation duly organized, validly existing and is in
good standing under the laws of Rhode Island and the State of
Delaware, respectively, is qualified to do business and is in
good standing as a corporation in each jurisdiction in which the
conduct of its business or the ownership of its properties
requires such qualification and has all requisite power and
authority, corporate or otherwise, to conduct its business as
now being conducted, to own, lease and operate its properties
and to execute, deliver and perform this Agreement,
7.2 The execution, delivery and performance by it of this Agreement
have been duly authorized by all necessary corporate action and
do not and will not (a) require any consent or approval of its
stockholders, (b) violate any provision of any law, rule,
regulations, order, writ, judgment, injunctions, decree,
determination award presently in effect having applicability to
it or any provision of its certificate of incorporation or
by-laws or (c) result in a breach of or constitute a default
under any material agreement, mortgage, lease, license, permit
or other instrument or obligation to which it is a party or by
which it or its properties may be bound or affected.
7.3 This Agreement is a legal, valid and binding obligation of it
enforceable against it in accordance with its terms and
conditions, except as such enforceability may be limited by
applicable bankruptcy, insolvency, moratorium, reorganization or
similar laws, from time to time in effect, affecting creditor's
rights generally.
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7.4 It is not under any obligation to any person, or entity,
contractual or otherwise, that is conflicting or inconsistent in
any respect with the terms of this Agreement or that would
impede the diligent and complete fulfillment-of its obligations.
7.5 It has good and marketable title to or valid leases or licenses
for, all of its properties, rights and assets necessary for the
fulfillment of its responsibilities under the Research Plan,
subject to no claim of any third party other than any relevant
lessors or licensors of which it is aware.
8. Indemnification. Pfizer and MultiCell will indemnify each other for damages,
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settlements, costs, legal fees and other expenses incurred in connection with a
claim by a third party (including without limitation by any employee of Pfizer
or MultiCell, as the case may be) against either party based on any action or
omission of the indemnifying party's agents, employees, or officers related to
its obligations under this Agreement; provided, however, that the foregoing
shall not apply (i) to the extent the claim is found to be based upon the
negligence, recklessness or willful misconduct of the party seeking
indemnification; or (ii) if such party fails to give the other party prompt
notice of any claim it receives and such failure materially prejudices the other
party with respect to any claim or action to which its obligation pursuant to
this Section applies. Notwithstanding the foregoing, Pfizer shall indemnify
MultiCell with respect to any and all claims arising from Pfizer's use of the
Results, to the extent the claim is found to be based upon the negligence,
recklessness or willful misconduct of MultiCell. Each party, in its sole
discretion, shall choose legal counsel, shall control the defense of such claim
or action and shall have the right to settle same on such terms and conditions
it deems advisable; provided however, it shall obtain the other party's prior
consent to such part of any settlement which requires payment or other action by
the other party or is likely to have a material adverse effect on the other
party's business.
9. Notices. All notices shall be in writing mailed via certified mail, return
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receipt requested, courier, or facsimile transmission addressed as follow, or to
such other address as may be designated from time to time:
If to Pfizer: Pfizer Global Research & Development
Xxxxxxx Xxxxx Xxxx
Xxxxxx, XX 00000
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Attention: Vice President Strategic
Alliances--PGRD
copy to: Assistant General Counsel--PGRD
If to MultiCell: MultiCell Technologies, Inc.
00 Xxxxxx Xxxx, Xxxxx 000
Xxxxxxx, XX 00000
Attention: Dr. Xxx Xxxxx
Copy to: Exten Industries
Notices shall be deemed given as of the date sent.
10. Governing Law. This Agreement shall be governed by and construed in
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accordance with the laws of the State of New York.
11. Miscellaneous,
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11.1 Binding Effect. This Agreement shall be binding upon and inure
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to the benefit of the parties and their respective legal
representatives, successors and permitted assigns.
11.2 Headings. Paragraph headings are inserted for convenience of
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reference only and do not form a part of this Agreement.
11.3 Counterparts. This Agreement may be executed simultaneously in
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two or more counterparts, each of which shall be deemed an
original.
11.4 Amendment. Waiver. This Agreement may be amended, modified,
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superseded or canceled, and any of the terms may be waived, only
by a written instrument executed by each party or, in the case
of waiver, by the party or parties waiving compliance. The delay
or failure of any party at any time or times to require
performance of any provisions shall in no manner affect the
rights at a later time to enforce the same. No waiver by any
party of any condition or of the breach of any term contained in
this Agreement, whether by conduct, or otherwise, in any one or
more instances, shall be deemed to be, or considered as, a
further or continuing waiver of any such condition or of the
breach of such term or any other term of this Agreement.
11.5 No Third Party Beneficiaries. No third party including any
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employee of any party to this Agreement, shall have or acquire
any rights by reason of this Agreement.
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Nothing contained in this Agreement shall be deemed to
constitute the parties partners with each other or any third
party.
11.6 Assignment and Successors. Unless the other party first consents
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in writing, neither party may assign this Agreement or any of
its duties under this Agreement, other that to (a) and
Affiliate; (b) any successor entity due to merger or
consolidation; or (c) any purchaser of all or substantially all
of its assets.
11.7 Force Majeure. Neither Pfizer nor MultiCell shall be liable for
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failure of or delay in performing obligations set forth in this
Agreement, and neither shall be deemed in breach of its
obligations, if such failure or delay is due to natural
disasters or any causes reasonably beyond the control of Pfizer
or MultiCell.
11.8 Severability. If any provision of this Agreement is or becomes
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invalid or is ruled invalid by any court of competent
jurisdiction or is deemed unenforceable, it is the intention of
the parties that the remainder of the Agreement shall not be
affected. IN WITNESS WHEREOF, the parties have caused this
Agreement to be executed by their duly authorized
representatives.
PFIZER INC
Vice President
Title: Strategic Alliances
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MultiCell Associates, Inc.
By:________________________
Title: Vice President Strategic Alliances
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cc: Pfizer Inc, Legal Division, Xxxxxx, XX 00000
Exhibit A
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Collaborative Research Proposal
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MultiCell Associates
and
Pfizer Inc
Part 1. Identification of an appropriate cell system for assessment of biliary
excretion.
Part 2. Identification and validation of appropriate cell lines for predictive
of CYP enzyme induction in humans.
Part 3. To identify an immortalized liver cell line and/or a standard source
and method for primary hepatocyte culture, that approximates the normal
metabolic response of liver to glucocorticoids (e.g. induction of gluconeogenic
enzymes).
Part 4. To identify an appropriate human hepatocyte cell line for utilization
in experiments to examine basal and hormone regulated carbohydrate and or lipid
metabolism.
MultiCell Research Proposal Part 1(X. Xx & X. Xxxxxx, CEO)
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Aim: Identification of an appropriate cell system for assessment of biliary
excretion.
Proposed Plan of Action:
1) Identification of appropriate cell or cell line for bile canaliculi
morphology and function. Goal: To identify a hepatocyte cell/cell line with bile
canaliculi morphology and function.
Preferred Approach: To use carboxydichlorofluorescein diacetate and
rhodamine-123 as fluorescent probes to assess bile canaliculi morphology and
function. Digital pictures of key images (both fluorescent and phase contrast)
should be provided from all studies. Expected timeframe is 3 to 6 months.
Description. Using phase contrast, fluorescent microscopy, and confocal
microscopy, Liu et al. Described and photographed the biliary excretion of
carboxydichlorofluorescein, a fluorescent MRP2 probe (Liu et al., AmericanJ. of
Physiology, Vol. 277, Issue 1, G12-G21, July 1999). Similarly, Sai et al..
Described and photographed the biliary excretion of rhodamine-123 into the bile
canaliculi of WIF-B cells, a polarized liver derived cell line (Sai et al., J.
of Cell Sci., vol. 112, 4535-4545, 1999). These two publications form the basis
of the studies proposed herein.
Part I. Fixed time-point screen (performed by MultiCell scientists at MultiCell
Associate). Hepatocyte monolayers are rinsed with buffer (e.g., MultiCell's
media F without phenol red). Carboxydichlorofluorescein diacetate (2 ug/ml final
is a suggested initial concentration) or rhodamine-123 (0.5 uM final is a
suggested initial concentration) is added to this buffer and incubation is
allowed at 37C for 10min. Thereafter, the monolayers are rinsed four times with
buffer to remove extracellular probe before viewing/photographing with a
fluorescent microscope. If a cell or cell line form tight cell junction and bile
canaliculi, a fluorescent network (as pictured by fluorescent microscope) will
co-localize with inter-cellular bile canaliculi (as pictured by phase contrast).
Part II. Time-course experiment (performed by MultiCell scientists at MultiCell
Associate). Hepatocyte or hepatocyte clones that passed Part I study are subject
to this time-course experiment. The goal of this experiment is to show that the
fluorescent network is indeed caused by probe uptake into hepatocytes followed
by efflux into bile canalicular (as opposed to direct bile canalicular uptake).
Basically, hepatocytes are rinsed with buffer. Carboxydichlorofluorescein,
diacetate or rhodamine-123 is added to this buffer and incubation is allowed at
37C for 2 min this time. The monolayers are quickly rinsed four times with
buffer to remove extracellular probe. Every 2 min afterwards, cells were
viewed/photographed with a fluorescent microscope. If a decrease in
intracellular fluorescence is observed with a concomitant increase of canaliculi
fluorescence, a good cell/cell line is identified.
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Expected Outcome. This study will be conducted by MultiCell Associates. For this
study, MultiCell scientists will also optimize culturing conditions for
multiwell plate formats (either the Permanox plastic plates or Glass-bottomed
multiwell plate).
If one or more cell or cell lines are identified that exhibits functional bile
canaliculi, Pfizer scientists will go to MultiCell or MultiCell will come to
Pfizer to learn/teach the culturing and experimentation with these cells. Pfizer
will also acquire the necessary material (cell or cell lines, media, buffer,
protocol, etc.) to carry out biliary excretion studies in-house.
Specific Aims and Timeline
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1. Set up and validate assay using shod-term primary hepatocyte cultures
(human) (4-6 week.).
2. Expand and screen top 100 human clones in monolayer culture using
carboxydichlorfluorescein and rhodamine-123. (3-4 months)
Project cost: $219,000
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MultiCell Research Proposal Part 2 (X. xx Xxxxxx & X. Xxxxx, CEG)
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Aim: Identification and validation of appropriate cell lines for predictive of
CYP enzyme induction in humans.
Proposed Plan of Action:
2) Identification and validation of an appropriate cell line for predictive of
CYP enzyme induction in humans.
Preferred Approach: To assess CYP3A4 induction in the human hepatocytes clones
and pooled cryopreserved hepatocytes. Induction should be consistent and
predictive of human in vivo. Studies for each of the cell lines are described.
Photographs of cell morphology should be provided in all studies.
Step 1: Quick assessment of calls for rifampin induction of CYP3A4
Human Hepatocyte Clones. MultiCell will culture human hepatocyte clones in the
presence of vehicle and 10 uM rifampin for 72 hours. Total RNA will be extracted
from cell lysates at MultiCell and shipped to Pfizer. Pfizer scientists will
quantitate levels of mRNA for CYP3A4 using the Invader(R) assay (Third Wave
Technologies, xxx.xxx.xxx). A clone will be deemed successful if the level of
CYP3A4 mRNA in the rifampin-treated sample is >5 fold higher than that seen in
vehicle-treated sample. If more than one clone meets this criteria, the clones
will be assessed based on 2 criteria: 1) the level of induction (increased
degree of induction is preferable) and 2) presence of other drug metabolizing
enzyme mRNAs (i.e. CYP1 A2).
Cryopreserved Human Hepatocytes. MultiCell will culture cryopreserved human
hepatocyte pooled from multiple donors in the presence of vehicle and 10 uM
rifampin for 72 hours. Assessment of CYP3A4 activity will be determined by
endpoint assay for the formation of 6-(beta) -hydroxytestosterone by MultiCell
scientists. Total RNA will be extracted from cell lysates at MultiCell and
shipped to Pfizer. Pfizer scientists will quantitate levels mRNA for CYP3A4 and
other drug metabolizing enzymes using the Invader(R) assay. Data for levels of
CYP3A4 mRNA and enzyme activity endpoint data will be compared for correlation.
This cellsystem will be deemed acceptable if the magnitude of CYP3A4 induction
is comparable to that seen in primary human hepatocytes, based on the literature
or from a parallel study. MultiCell will provide training to Pfizer scientists
under the terms of the Agreement.
Step 2: Assessment of induction with a panel of inducers on those cells that
showed optimum CYP3A4 Induction with rifampin.
MultiCell will culture cells in the presence of inducer for 72 hours (see Table
1). Assessment of CYP3A4 activity will be determined by endpoint assay for the
formation of 6-(beta)-hydroxytestosterone by MultiCell scientists. Cell lysates
should be frozen for future total RNA extractions. When possible, this
experiment should be repeated using fresh primary human hepatocyte cultures for
comparison. This cell line will be deemed acceptable if the rank order of
inducers is comparable to that seen in humans, based on the
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literature or from the parallel study in primary human hepatocytes. If the
results are positive, total RNA will be extracted from frozen cell lysates at
MultiCell and shipped to Pfizer. Pfizer scientists will quantitate levels of
mRNA for CYP3A4 using the Invader(R) assay. Data for levels of OYP3A4 mRNA and
enzyme activity endpoint data will be compared for correlation.
Inducer CYP3A4* Conc.
1 Reserpine P 10 uM
2 Rifampicin P 10 uM
3 Troglitazone P 10 uM
4 Mifepristone (RU-486) P 10 uM
5 Clotrimazole M-P l0 uM
6 Phenobarbital-Na M-P 1000 uM
7 Dexamethasone M 50 uM
8 Rifabutin W 10 uM
9 Midazolam W-none 10 uM
10 B-naphthotlavone none 10 uM
11 Chrysin none 25 uM
12 Vehicle none
* expected induction: P = potent; M = moderate; W = weak
2) Validation and Optimization of the Chosen Cell System(s).
Preferred Approach: Tao assess CYP3A4 and CYP1A2 induction in the cell system(s)
chosen from the initial studies. Induction should be consistent and predictive
of human in vivo. Photographs of cell morphology should be provided in all
studies. Expected timeframe is 3 to 6 months. Validation. The cell system(s)
chosen from the initial set of experiments will be validated by MultiCell
scientists repeating the study for the panel of inducers described previously.
This study will serve to assess reproducibility of results. In addition, the
study will involve assessment of earlier timepoints (16, 24 and/or 48 hours) and
Include CYP1A2 enzyme activity endpoint assay for some of the samples. Total RNA
will be extracted from cell lysates at MultiCell and shipped to Pfizer. Pfizer
scientists will quantitate levels of mRNA for CYP3A4 and other drug metabolizing
enzymes using the Invader(R) assay.
Optimization. Pfizer will work with Nunc (or comparable vendor) for the
development of Permanox multi-well plates. MultiCell scientists will optimize
culturing conditions for multiwell plate formats.
4
Specific Alms and timeline for conducting experiments (1 year)
1. Perform drug metabolism experiment with cryopreserved individual and
pooled human hepatocytes (6 weeks)
2. Prepare RNA from 100 human hepatocyte cell lines that had been exposed to
either vehicle or rifampin (3-4 months). It is highly recommended that
MCA also prepare cryopreserved cells from each clone that would be used
if further studies were warranted.
3. Perform testosterone assay and prepare RNA from top 5-10 clonal lines
exposed to a panel of CYP3A4 inducers using up to 10 different clonal
cell lines. (1-3 months)
4. Validation and optimization of "Induction bioassay" using top three MCA
clones (Human and porcine) (3 months) Optimize and validate culture
conditions for multiwell plate format (3-6 months)
Project Cost: $275,000
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MultiCell Research Proposal Part 3 (X. Xxxxxxxxxx, AI2.)
-------------------------------------------------------
Aims: 1) To Identify an immortalized liver cell line that approximates the
normal metabolic response of liver to glucocorticoids (e.g. induction of
gluconeogenic enzymes).
Approach 1) to identify one or more immortal cell clones from the MultiCell
hepatocyte collection that meets the criteria of glucocorticoid-regulated
expression of the gluconeogenic enzymes TAT and PEPCK. For the initial study,
MultiCell will culture human hepatocyte clones for ~18 hours in the absence or
presence of 50 and 500 nM dexamethasone and prepare total RNA (~5 ug). The RNA
will be transferred to Pfizer and Pfizer will test the RNA for the expression of
gluconeogenic enzymes (e.g. TAT [tyrosine amino transferase] and PEPCK) by
TaqMan analysis. All/selected cell clones with Inducible TAT/PEPCK expression
(>8 fold) will then be characterized by Pfizer Scientists for glucocorticoid
responsiveness. MultiCell will provide the candidate cell clones along with the
preferred growth media (minus glucocorticoid source). A clone will be deemed
successful if there is a significant and dose responsive increase (>8 fold) in
the level of TAT in response to glucocorticoids addition (e.g. a range of
dexamethasone between 10 nM and 10 uM) and which is competed- by-the addition of
the- glucocorticoid antagonist (RU486). -TAT enzyme activity will be-correlated
with RNA induction in follow-up assays conducted by Pfizer Scientists. Cell line
screening may occur by processing selected subsets of clones (10-25 individual
lines) until one or more suitable lines is/are identified. Screening of
additional cell lines will continue until one or more successful clones are
identified or until all immortal lines have been examined or if both parties
agree that reasonable and sufficient effort has been extended, Data developed
using reference compounds (e.g. cortisol, dexamethasone, RU486) will be shared
with MultiCell. Sharing of data generated with Pfizer compounds will be solely
Pfizer's discretion.
Expected time: frame: process and deliver specified RNA samples from 60 or more
clones each quarter.
Optional Assays and Publication: If the primary or immortal cell clones prove to
be a good model for metabolic responses, we may extend the cell line
characterization to include expression profiling using reference and/or Pfizer
compounds. If this expression analysis is pursued, we will share with MultiCell
the results obtained with reference compounds as indicated above. Additional
work may include assessing if the anti-inflammatory activity of glucocorticoids
in the chosen MultiCell clones. For example, we may assay for the upregulation
of cytokines, acute phase proteins, etc. in response to typical inducers (PMA,
IL-1, etc.).
Specific Aims and Timeline.
1. MCA will prepare and provide 5ug of RNA from 100 human clones treated
with vehicle, 50 and 500 nM dexamethasone. (Note: if done at the same
time as the experiments in Project 2, then this would entail only require
making an additional aliquot of RNA from the common vehicle control.
Otherwise, it will entail one more sample be prepared). It should be
noted that these samples will have been treated with methanol, (this
would be OK if we first validate the methanol vehicle has no adverse
effect on TAT induction in the primary hepatocytes), the vehicle control
for project 2 (3-6 months).
Cost of study: $150,000
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MultiCell Research Proposal (X.X Xxxxxxxx, X. X. XxXxxxxxx, and X. Xxxxxxxxxxx,
CVMD/Diabetes)
Aim: Identification of an appropriate human hepatocyte cell line for utilization
in experiments to examine basal and hormone regulated carbohydrate and or lipid
metabolism.
Part 1:
Goal: To assess the functional carbohydrate metabolizing activity of the cell
line(s) identified in Project 3.
Approach: Up to ten (10) cell lines identified from Project #3 will be provided
by MultiCell in a suitable format and tested by Pfizer in functional assays that
will assess their metabolic activity. The results will be compared to the
results of standardized hepatocyte preparations presently used in the lab (e.g.
freshly isolated rat hepatocytes and commercially-prepared primary human
hepatocytes). The following assays will be performed:
Glucogenesis: The ability of the cell line(s) to convert
[14C][14C]-labeled lactic acid to [][14C]-labeled glucose under basal
and hormone (e.g. glucagon) stimulated conditions will be assessed
using a routinely used assay that involves the separation of the
substrate from the product by mixed-bed ion exchange chromatography
(1).
Glucose Output: The cell line(s) will be tested for their ability to
release glucose into the incubation medium under basal and hormone
(e.g. glucagon) stimulated conditions, using an assay that involves
sequential sampling of the assay media for determination of glucose
concentration (2).
Glycogenolysis: Basal and hormone (e.g. glucoagon) regulated
glycogenolysis will be examined in either of two ways. One approach
involves prelabelling the cells with [14C]-labeled glucose (using
insulin to stimulate glycogenesis) and then examine the loss of
glycogen-associated radioactivity after stimulation with glucagon, The
second approach would be to directly measure the cellular glycogen
content in untreated and glucagon-treated cells (3).
References:
1. Xxxxxxxxx, X. X., XxXxxxxxx, X. X., and Xxxxxxxxx, X. X. Metabolism
42(12):1583-1587, 1993.
2. Xxxxxx, X. X., et al. Diabetes 47:1630-1638, 1998.
3. Xxxxxx, X. X., et al. PNAS 95:1776-1781, 1998.
Project Cost: $80,000.00
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