Exhibit 10.2

 

Research Agreement dated January 28, 2004

between Medlyte, Inc.

and San Diego State University

together with Amendments No. 1 and No.2

 

--------------------------------------------------------------------------------

 

AMENDMENT NO. 2 TO
RESEARCH AGREEMENT BETWEEN
SAN DIEGO STATE UNIVERSITY AND MEDLYTE, INC.
SDSU AGREEMENT # 40031030

 

Original agreement dated January 28, 2004, and amended By Amendment #1, dated
January 28, 2005, is hereby further amended as follows:

 

Effective August 25, 2004 — Change – Name of Company from Medlyte, Inc. to Lpath
Therapeutics, Inc.

 

Extend the term of the lease to December 31, 2005.

 

Delete Exhibit A Budget dated 1/1/04 to 12/31/04

 

Add Revised Exhibit A Budgeted dated 1/1/05 to 12/31/05

 

Change authorized representative for the University from Joe Vasquez, Associate
Vice President of Business Enterprises to Scott Burns, Associate Vice President,
Enterprise Operations.

 

Delete – University and Sponsor each agree to indemnify and to hold harmless the
other party from damage to person or property resulting from any act or omission
on the part of itself, its employees, its agents, or its officers. For purposes
of this section, any person employed by University who is also an officer,
director or shareholder of Sponsor shall be considered only an employee of
University.

 

Add - Sponsor shall hold harmless, indemnify and defend the State of California,
the Trustees of the California State University, the San Diego State University
and the officers, employees, volunteers and agents of each of them from and
against any and all liability, loss, damage, expense, costs of every nature, and
causes of actions arising out of or in connection with the use by the Sponsor of
said property. For purposes of this section, any person employed by University
who is also an officer, director or shareholder of Sponsor shall be considered
only an employee of University.

 

All other terms and conditions of the original agreement shall remain the same.

 

Agreement of University and Sponsor in the terms stated above is indicated by
signatures affixed below.

 

For University: SAN DIEGO STATE UNIVERSITY

For Sponsor: Lpath Therapeutics, Inc.

 

 

 

By:

/s/ Scott Burns

2/4/05

By:

/s/ Scott Pancoast

 

 

Scott Burns

 

 

 

Associate VP, Enterprise Operations

Title:

Secretary

 

 

 

 

 

By:

/s/ Thomas R. Scott

2/28/05

 

 

 

Thomas Scott

 

 

 

Dean, College of Sciences

 

 

 

 

 

 

 

By:

/s/ Christopher Glembostki

 

 

 

 

Christopher Glembostki

 

 

 

Chair, Department of Biology

 

 

 

 

 

 

 

By:

/s/ Roger Sabbadini

 

 

 

 

Roger Sabbadini

 

 

 

Principal Investigator

 

 

 

--------------------------------------------------------------------------------

 

Exhibit A

 

SDSU DETAILED BUDGET FOR INITIAL BUDGET PERIOD
DIRECT COSTS ONLY

 

FROM
01/01/05

 

THROUGH 12/31/05

 

PERSONNEL

 

TYPE

 

%

 

 

 

DOLLAR AMOUNT REQUESTED (omit cents)

 

NAME (RT or salary)

 

ROLE ON
PROJECT

 

APPT.
(months)

 

EFFORT PROJ.

 

INST. BASE
Annual SALARY

 

SALARY
REQUESTED

 

FRINGE
BENEFITS

 

TOTALS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No Employees

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBTOTALS

 

 

 

$

0

 

$

0

 

$

0

 

 

CONSULTANT COSTS Note: Forcasted expenditures for Sabbadini Laboratory Only (not
7993 acct) student stipends to reimburse SDSU Biology Department (e.g., Nicole
Gellings)

 

$

24,000

 

 

 

 

 

EQUIPMENT (Itemize) Note: Forcasted expenditures for Sabbadini Laboratory Only
(not 7993 acct) no equipment may be purchased through SDSU

 

 

 

 

 

 

 

SUPPLIES (Itemize) Note: Forcasted expenditures for Sabbadini Laboratory Only
(not 7993 acct) appoximately $10k per month

 

$

120,000

 

 

 

 

 

TRAVEL Note: Forcasted expenditures for Sabbadini Laboratory Only (not 7993
acct)

 

$

0

 

 

 

 

 

PATIENT COSTS

INPATIENT

 

$

0

 

(includes patient payment)

OUTPATIENT

 

$

0

 

BIOLOGY DEPARTMENT RENTAL AND SERVICES FEE (biosafety, radiation safety, etc.)
$2.50/ft2 x 820 ft2 = $2,050/mo x12 mo = $24,605

 

$

24,605

 

SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD

 

$

168,605

 

FACILITIES FEES TO UNIVERSITY (Business Affairs)

 

$

7,400

 

CONSORTIUM/CONTRACTUAL

CONTRACTUAL:

 

 

 

ATTORNEY FEES

ATTORNEY:

 

 

 

TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD

 

$

176,005

 

INDIRECT COSTS FOR INITIAL BUDGET PERIOD (6% DC)

 

10,560

 

TOTAL COSTS FOR INIITIAL BUDGET PERIOD

 

$

186,565

 

 

--------------------------------------------------------------------------------

 

RESEARCH AGREEMENT BETWEEN SAN DIEGO STATE UNIVERSITY AND MEDLYTE, INC.

 

This Agreement dated January 28, 2004 is by and between San Diego State
University, a public institution of higher education (hereinafter referred to as
“University”), and Medlyte, Inc., a Delaware corporation with offices in San
Diego County, California (hereinafter referred to as “Sponsor”).

 

Whereas, Sponsor is interested in sponsoring research at University concerning
the role of sphingolipids in human disease in the form of a fee-for-service
research agreement;

 

Therefore, University and Sponsor hereby agree to the terms stated below.

 

1.             Scope of Work. The scope of work (“Project”) shall be as
described in the research proposal entitled “Role of Sphingolipids in Human
Disease” (the “Proposal”), by Dr. Roger Sabbadini, Ph.D. of Department of
Biology. It is understood that the Proposal is an “instructional research
project” whereby undergraduate and graduate students may have the opportunity to
use this project as an “incubator” for gaining practical laboratory experience
in the industry-related biotechnology arena.

 

2.             Services. University will provide facilities for use by
University volunteers, adjunct faculty associated with Medlyte (Life Sciences
Rooms 308 and 308B and other space totaling at least 820 ft2), as well as
equipment, accounting and other services described in the Proposal and related
budget that are necessary or appropriate to perform and complete the work
contemplated in the Proposal. Sponsor shall have the right to purchase, or
direct University to purchase on its behalf, equipment (defined as
non-disposable items costing more than $500) and to place such equipment at
University. However, Sponsor will own all right, title and interest to such
equipment and will have the right to remove such equipment at any time.

 

3.             Key Personnel. The project director/principal investigator will
be Dr. Roger Sabbadini, Ph.D., who may select, supervise and terminate project
staff as needed. No other person will be substituted for the project director
except with Sponsor’s approval. Sponsor may exercise the Termination provision
of this Agreement if a satisfactory substitute is not identified. Any employees
conducting research at SDSU will receive direct salary compensation by the
Sponsor only and will be recognized by the university either as ‘volunteer
workers’ or ‘adjunct faculty’ members under special appointment by the Biology
Department.

 

4.             Control of Research. Control of the research will rest entirely
with University. However, it is agreed that University, through the project
director, will maintain continuing communication with Sponsor. University’s
project director and Sponsor will mutually define the frequency and nature of
these communications.

 

5.             Funding and Payment. Sponsor will pay a monthly administrative
fee for

 

1

--------------------------------------------------------------------------------

 

utilities and maintenance and a fee equal to 6% of overhead costs to the
University, as well as a fee to the Biology Department for rental and services,
as set forth in the budget (“Budget”) attached as Exhibit A.  Sponsor will pay
the amounts on the schedule as set forth in the Budget on the condition that the
Project Director submits a written Progress Report to the Sponsor on a quarterly
basis. The Project Director may transfer funds from one category to another on
the Budget after written notice to Sponsor of such reallocation. Any amounts not
spent on the Project shall be refunded to Sponsor at the end of the Project.

 

6.             Project Period. The Agreement will be effective for twelve (12)
months beginning January 1, 2004 and ending December 31, 2004. This period may
be amended by mutual written agreement by authorized representatives of
University and Sponsor.

 

7.             Invention Rights. Title to any discovery, invention, finding,
data or conclusion, whether or not patentable, derived from the Project or
research under this Agreement will be owned solely by Sponsor. University agrees
to take all actions necessary or appropriate, at Sponsor’s request and cost, to
effectuate such sole ownership.

 

8.             Publication. University will be free to publish the results of
research conducted under this Agreement within a reasonable time. Prior to
submission for publication of a manuscript or outline/notes for a symposium, the
University agrees to send the Sponsor a copy of the manuscript/outline to be
submitted, and shall allow the Company thirty (30) days from receipt to
determine whether the manuscript/outline contains subject matter for which
patent protection should be sought prior to publication or public disclosure.
Should the Sponsor believe the subject matter of the manuscript contains a
patentable invention to which it may have rights under this Agreement, the
Sponsor shall have until the end of such 30-day period to notify the University
that it wishes to either seek to obtain patent protection or that it wishes to
keep the information non-public, and University agrees to provide reasonable
cooperation to the Sponsor and to take no action inconsistent with the Sponsor’s
decision (including withholding publication for a reasonable period of time upon
request in order to file for patent protection). In order to fully protect the
rights of University and Sponsor, any contemplated publication containing
details of an invention, whether or not patentable, will be withheld until a
patent application is filed or other appropriate steps to protect commercial
value have been completed.

 

9.             Hold Harmless. University and Sponsor each agree to indemnify and
to hold harmless the other party from damage to persons or property resulting
from any act or omission on the part of itself, its employees, its agents, or
its officers. For purposes of this section, any person employed by University
who is also an officer, director or shareholder of Sponsor shall be considered
only an employee of University.

 

10.           Use of Names. University and Sponsor each agree that they will not
use the name, trademark, or other identifier of the other for any advertising,
promotion, or other commercially related purpose except with advance written
approval.

 

11.           Assignment. Sponsor shall have the right to assign or transfer any
rights

 

2

--------------------------------------------------------------------------------

 

or obligations arising from this Agreement without the prior written notice to
the University.

 

12.           Disclaimer of Warranties. All information received from or
technology developed with the University is experimental in nature and the
University makes no express or implied warranties or representations with
respect to its utility, safety, merchantability, or fitness for a particular
purpose. All warranties, express or implied arising out of or in connection with
the furnishing, performance, or use of any University technology are hereby
disclaimed.

 

13.           Applicable Law and Venue. This Agreement shall be governed by and
enforced according to the laws of the State of California without giving effect
to the conflict of laws provisions thereof. Exclusive jurisdiction and venue of
any dispute under this Agreement shall lie with the United States District Court
for the Southern District of California or the Superior Court of California for
San Diego County.

 

14.           Nonperformance. Nonperformance by the University shall not operate
as a breach of the terms of this Agreement if due to strikes or other labor
disputes or to prevention or prohibition by law, the loss or injury to products
in transit, an act of God, or war or other cause beyond the control of the
University.

 

15.           Severability. If any of the provisions of this Agreement shall be
determined to be illegal or unenforceable by a court of competent jurisdiction,
the other provisions shall remain in full force and effect.

 

16.           Notices. Unless otherwise indicated elsewhere in this Agreement,
all notices and communications in connection with this Agreement will be
addressed to University/Sponsor officials who sign this Agreement.

 

17.           Termination. Either University or Sponsor may terminate this
Agreement by giving thirty (30) days written notice to the other. In the event
of such termination, University will cease further obligation of project funds
and will take all reasonable steps to cancel or otherwise reduce outstanding
obligations. Sponsor will be obligated to pay actual costs and firm obligations
as reduced by diligent efforts of University. In the event of a termination by
the University, the Sponsor shall have the right to technology pursuant to
Section 7 resulting from the research as defined in Section 1.

 

18.           Amendments. Any amendment to this Agreement must be in writing and
signed by authorized representatives of University and Sponsor. No waiver of
this Agreement shall be valid and enforceable unless in writing and signed by
the authorized representative for the party granting the waiver. The waiver by
any party of a breach of any of the provisions of this Agreement shall not
operate or be construed as a waiver of any subsequent breach by any party or a
breach of the entire Agreement. The authorized representative for the University
is Joseph Vasquez, Associate Vice President of Business Enterprises, San Diego
State University. Faculty members and other research personnel are not
authorized to bind the University.

 

19.           Entire Agreement. This Agreement expresses the entire agreement

 

3

--------------------------------------------------------------------------------

 

between the parties. All prior negotiations, understandings, promises and
agreements, oral or written, are superseded hereby.

 

Agreement of University and Sponsor in the terms stated above is indicated by
signatures affixed below.

 

For University:

For Sponsor:

SAN DIEGO STATE UNIVERSITY

MEDLYTE, INC.

 

 

By:

 

/s/ Joseph W Vasquez

 

By:

 

/s/ Scott Pancoast

 

(signature)

 

 

(signature)

 

 

 

 

 

Name:

Joseph Vasquez

 

Name:

Scott Pancoast

 

 

 

 

 

Title:

Assoc. VP for Business Enterprises

 

Title:

Director of the Company

 

 

 

 

 

 

 

 

By:

 

/s/ Thomas R. Scott

 

 

 

 

 

(signature)

 

 

 

 

 

 

 

 

Name:

Thomas Scott

 

 

 

 

 

 

 

 

Title:

Dean, College of Sciences

 

 

 

 

 

 

 

 

 

 

 

By:

 

/s/ Christopher Glembostki

 

 

 

 

 

(signature)

 

 

 

 

 

 

 

 

Name:

  Christopher Glembostki

 

 

 

 

 

 

 

 

Title:

  Chair, Department of Biology

 

 

 

 

 

 

 

 

 

 

 

By:

 

/s/ Roger Sabbadini

 

 

 

 

 

(signature)

 

 

 

 

 

 

 

 

Name:

  Roger Sabbadini

 

 

 

 

 

 

 

 

Title:

  Principal Investigator

 

 

 

 

4

--------------------------------------------------------------------------------

 

Exhibit A

 

SDSU DETAILED BUDGET FOR INITIAL BUDGET PERIOD
DIRECT COSTS ONLY

 

FROM
01/01/04

 

THROUGH 12/31/04

 

PERSONNEL

 

TYPE

 

%

 

 

 

DOLLAR AMOUNT REQUESTED (omit cents)

 

NAME (RT or salary)

 

ROLE ON
PROJECT

 

APPT.
(months)

 

EFFORT
PROJ.

 

INST. BASE
Annual SALARY

 

SALARY
REQUESTED

 

FRINGE
BENEFITS

 

TOTALS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nicole Gellings, PhD student

 

student

 

 

 

 

 

$

18,962

 

$

18,962

 

$

5,689

 

$

24,651

 

John Vekich, MS student

 

student

 

 

 

 

 

$

8,696

 

$

9,600

 

$

0

 

$

8,696

 

PhD student TBN

 

student

 

 

 

 

 

$

18,962

 

$

13,552

 

$

0

 

$

13,552

 

Brad Sibald, MS students

 

student

 

 

 

 

 

$

8,696

 

$

9,600

 

$

0

 

$

8,696

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUBTOTALS

 

 

 

$

51,714

 

$

5,689

 

$

55,595

 

 

CONSULTANT COSTS Note: Forcasted expenditures for Sabbadini Laboratory Only (not
7993 acct)
Miscellaneous Consulting and external services conducted at SDSU

 

$

30,000

 

 

 

 

 

EQUIPMENT (Itemize) Note: Forcasted expenditures for Sabbadini Laboratory Only
(not 7993 acct)
Misc. small equip., columns, etc.

 

$

20,000

 

 

 

 

 

SUPPLIES (Itemize) Note: Forcasted expenditures for Sabbadini Laboratory Only
(not 7993 acct)

 

$

120,000

 

 

 

 

 

 

TRAVEL Note: Forcasted expenditures for Sabbadini Laboratory Only (not 7993
acct)

 

$

5,000

 

 

 

 

 

 

PATIENT COSTS

INPATIENT

 

$

0

 

(includes patient payment)

OUTPATIENT

 

$

0

 

BIOLOGY DEPARTMENT RENTAL AND SERVICES FEE (biosafety, radiation safety, etc.)
$2.50/ft2 x 820 ft2 = $2,050/mo x12 mo = $24,605 + $5,000 in fees

 

$

29,605

 

SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD

 

$

260,200

 

FACILITIES FEES TO UNIVERSITY (Business Enterprises)

 

$

7,400

 

CONSORTIUM/CONTRACTUAL

CONTRACTUAL:

 

 

 

ATTORNEY FEES

ATTORNEY:

 

 

 

TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD

 

$

267,600

 

INDIRECT COSTS FOR INITIAL BUDGET PERIOD (6% DC)

 

16,056

 

TOTAL COSTS FOR INIITIAL BUDGET PERIOD

 

$

283,656

 

 

--------------------------------------------------------------------------------

 

Explanation of EXHIBIT A

 

Rental fees: The Sponsor agrees to reimburse the Department of Biology for
occupied space at a fee of $2.50 ft2.

 

Facilities fees: The Sponsor agrees to reimburse SDSU for utilities, janitorial
and other services at a fixed cost of $7,400 to be paid to the Office of
Business Enterprises. These fees include $200 per month for utilities plus a
blanket $5,000 in compensation for other services such as janitorial,
landscaping, etc.

 

Other fees: The Sponsor agrees to pay for other services on a fee-for-service
basis, including fees for vivarium use, health and safety issues, protection of
human subjects, etc at a commercially acceptable rates with the exception of
services provided to students performing thesis work at the university under the
sponsorship of the Sponsor.

 

Student stipends: The Sponsor agrees to support PhD and MS students according to
Biology Department reimbursement policies.

 

5

--------------------------------------------------------------------------------

 

EXHIBIT B: ‘THE PROPOSAL’

THE ROLE OF SPHINGOLIPIDS IN HUMAN DISEASE

 

RESEARCH PLAN PROJECT I: The Role of Sphingolipids in Cardiac Ischemia

 

A.            SPECIFIC AIMS

 

The identification of signaling components involved in cardiac ischemia
reperfusion (IR) injury is a critical step toward discovering therapeutic
interventions for reducing the size of cardiac infarcts and improving patient
outcomes. Research has demonstrated sphingolipid signaling molecules are key
components that participate in the cell death and loss of function associated
with cardiac IR injury, and furthermore, that induction of this pathway is one
of the earliest events during this process. Preliminary studies in our
laboratory have suggested that activation of the sphingolipid-signaling pathway
by IR occurs through the neutral form of sphingomyelinase (nSMase1) and its
adaptor protein, FAN (factor associated with neutral SMase activation).

 

With this in mind, we have the following project goal: The development of a
small molecule inhibitor of the sphingomyelinase signaling system that will
provide protection from cardiac cell death and loss of inotropy during acute
ischemic insults such as during an acute myocardial infarction (AMI).

 

•                 PHASE I: Validation of FAN and nSMase1 as targets for
therapeutics intervention. This validation is required before embarking on a
drug discovery program (Phase II).

 

•                 PHASE II: Demonstration in pre-clinical trials that drug
candidates against nSMase1 and/or FAN are effective in preventing cardiac cell
death and loss of inotropy associated with IR injury.

 

The proposed Phase I research plan will include preliminary studies that reveal,
for the first time, that FAN is a critical mediator in the cardiomyocyte cell
death process induced by IR. These studies will additionally provide evidence
that nSMasel is the specific isoform of nSMase involved in this process.

 

Accordingly, the Phase I validation of nSMase1 and FAN as targets for
therapeutic intervention will include the following specific aims:

 

•                 Specific aim 1: Determine whether infarct sizes are reduced
and left ventricular pressure improved after an acute ischemic event in the
absence of the FAN/nSMase signaling system by producing reversible cardiac
infarcts using our in-house FAN knockout mouse model.

 

•                   Milestone 1: Completion of surgeries and measurements of
infarct size and left ventricular developed pressure (LVDP). Expectation of 20 –
50% decrease in infarct size and significant increase in LVDP.

 

•                 Specific aim 2: Determine whether infarct sizes are reduced
and left ventricular pressure improved after an acute ischemic event in the
absence of the FAN/nSMase signaling system by producing reversible cardiac
infarcts using a currently available nSMase1 knockout mouse model.

 

6

--------------------------------------------------------------------------------

 

•                   Milestone 1: Establishment of breeding colony.

 

•                   Milestone 2: Completion of surgeries and measurements of
infarct size and left ventricular developed pressure (LVDP). Expectation of 20 –
50% decrease in infarct size and a significant increase in LVDP.

 

The anticipated success of accomplishing these aims will demonstrate that the
sphingomyelinase signaling system is a major participant in ischemia/reperfusion
injury by validating nSMase1 and FAN as targets for drug discovery. This target
validation will enable us to identifying small molecule inhibitors of this
signaling system that can used in pre-clinical trials that we will propose in
PHASE II. The ultimate goal of the research is to develop lead compounds that
can be taken to Phase 1 Clinical Trials. The proposed work will form the basis
of discovering novel medical therapeutics that will reduce the cell death
associated with AMI and other acute ischemic events in the clinical setting.

 

RESEARCH PLAN PROJECT II: Sphingolipids as Markers of Cardiac Ischemia

 

A.            SPECIFIC AIMS

 

The over all project goal is to develop a sphingolipid-based diagnostic for
cardiac ischemia that can be used to evaluate patients suffering from acute
cardiac ischemia.

 

PHASE I: Validation that sphingolipids are sensitive and specific markers of
cardiac ischemia in a trial designed to provoke ischemia in stable angina
patients. This validation is required before embarking on a chest pain Emergency
Department trial (Phase II).

 

PHASE II: Conduct a follow-on clinical trial using sphingolipids as biomarkers
for the evaluation of acute coronary syndrome (ACS) in the Emergency Department.
Develop a simple antibody-based test platform that would be used in clinical
laboratory instruments and point-of-care triage (POCT) determinations of cardiac
ischemia.

 

It is now known that coronary artery disease is aggravated by inflammation. It
has been suggested that sphingolipid signaling molecules are mediators of
inflammation, particularly in response to pre-inflammatory cytokines such as
TNFa and IL2. In addition to release by these molecules as part of the
inflammatory response, sphingolipids are possibly produced as a direct result of
ischemia. In this case, one may expect to observe increased sphingolipid
production either as a consequence of the TNFa produced by ischemic cardiac
tissue or directly from heart cells. It is also possible that the sphingolipids
are responsible for the poor outcomes observed in acute coronary syndrome
patients who have elevated serum levels of TNFa. Thus, we reasoned that
sphingolipids produced either by inflammation or ischemia would correlate with
coronary disease. Consequently, we initiated a clinical study designed to test
the hypothesis that blood levels of key sphingolipids are predictive of
obstructive CAD.  In an open trial of all patients sent to the catheterization
lab for angiography, serum sphingosine-1-phosphate (S1P) was found to be a
robust predictor of coronary disease. Subsequent subgroup analyses demonstrated
that the predictive power of serum S1P was dramatically improved for patients
hospitalized for evaluation of cardiac ischemia. Animal and cell culture
experiments demonstrated that ischemic hearts produce significant amounts of
sphingolipids and release it into the extracellular compartment. These recently
published studies suggest that serum sphingolipids could be used

 

7

--------------------------------------------------------------------------------

 

to predict cardiac ischemia independent of the inflammatory response. A clinical
trial is required with clear ischemia endpoints to determine the predictive
power of serum sphingolipids as ischemic markers.

 

Accordingly, we have designed a trial with the following PHASE I specific aims:

 

1)              Conduct a clinical trial designed to provoke active ischemic in
patients presenting with angina who undergo exercise treadmill testing (ETT).
Serial blood samples will be obtained before and several times after treadmill
testing for the determination of serum sphingolipid levels.

 

Milestone: Consent 200 patients into the trial

 

2)              Compare serum sphingolipid levels with inflammatory biomarkers,
CRP and TNFa, MI markers (TnI) and standard assessments of CHD, including stress
ECG, stress echo, nuclear myocardial scanning and coronary angiography

 

Milestone 1: Establish the specificity and sensitivity of the putative ischemic
markers with a yes/no evaluation of active ischemia

 

Milestone 2: Confirm yes/no for ischemia based on positive stress ECGs, stress
echos and nuclear myocardial scans with CAD determined by coronary angiography.
Ischemia-negative patients are those with a negative stress ECGs, stress echos
and nuclear myocardial scans.

 

Milestone 3: Serum sphingolipids as putative ischemic markers should better
correlate with clinicial endpoints than other serum analytes used in the study
(inflammatory markers and TnI) in patents not experiencing MI

 

3)              Develop monoclonal antibodies suitable for developing a serum
assay for testing of patients suspected of cardiac ischemia

 

Milestone 1: Obtain stable hybridoma cell lines with high titers (>100,000
dilution)

 

Milestone 2: Purify antibodies and determine Kds for each hybridoma culture

 

The anticipated success of accomplishing these aims will demonstrate that serum
sphingolipids are ischemic biomarkers that can form the basis of a simple blood
test for ischemia that will be developed in PHASE II. Thus, the overall goal of
this work is to develop a test for active ischemia suitable for clinical
laboratory instruments and point-of-care triage (POCT) applications. This test
could be used to improve the sensitivity and specificity of ETT and could be
used as a point of care triage in the emergency department to demonstrate
non-cardiac causes of chest pain. Patients presenting with potential cardiac
complaints could be screened initially with sphingolipid analyte testing in
addition to standard assays. A second use for sphingolipid ischemic markers may
be as an additional component of risk stratification. Given that published work
from our laboratory indicates that elevated serum S1P levels are independent of
traditional factors, it may improve the accuracy of algorithms designed to
identify patients who need more aggressive diagnostic evaluations or medical
interventions (12).

 

The proposed studies will demonstrate for the first time that serum
sphingolipids are powerful and novel markers of cardiac ischemia. Positive
results from the proposed work will suggest additional clinical trials and
laboratory investigations needed to elucidate the underlying mechanisms linking
sphingolipids with ischemia and provide guidance on how sphingolipids can be
best used to improve patient care and outcomes.

 

8

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RESEARCH PLAN PROJECT III: Anti-sphingolipid Antibodies in the Treatment of
Cancer

 

A.            SPECIFIC AIMS

 

The identification of signaling components that promote tumor growth is a
critical step toward discovering therapeutic interventions for reducing cancer’s
morbidity and mortality and generally improving patient outcomes. Research has
demonstrated that sphingosine kinase (SPHK) is a recently validated oncogene
that produces an extracellular sphingolipid signaling molecule,
sphingosine-1-phosphate (S1P), that promotes tumor growth. Tumor growth is
promoted both directly and indirectly by S1P’s growth factor actions related to
tumor cell proliferation and metastasis, as well as S1P’s pro-angiogenic
effects. Medlyte has produced a biospecific monoclonal anti-S1P antibody
(anti-S1P Mab) that could be used as a therapeutic molecular sponge to
selectively absorb S1P, thus lowering extracellular concentrations of this tumor
growth factor with the anticipated reduction in tumor volume and metastatic
potential as well as simultaneously blocking new blood vessel formation that
would, otherwise, feed the growing tumor. The anticipated success of the
molecular absorption concept will represent an innovative approach to the
treatment of cancer.

 

Accordingly in Phase I of the proposed work, our project goal is to evaluate the
therapeutic potential of our anti-S1P monoclonal antibodies. In accomplishing
this goal, we have the following Specific Aims:

 

•                  Specific Aim 1: In vitro testing of anti-S1P Mab’s ability to
reduce S1P-dependent cell proliferation and metastatic potential.

 

•                 Milestone 1: Test ability of anti-S1P Mab to reduce
S1P-dependent proliferation in selected tumor cell lines. Determine if
Mab-reduced cell count is due to removal of S1P’s anti-apoptotic protective
effects.

 

•                  Milestone 2: Test ability of anti-S1P Mab to reduce
S1P-dependent cell migration in an in vitro Matrigel assay for metastatic
potential.

 

•                  Milestone 3: Test ability of anti-S1P Mab to reduce
S1P-dependent angiogenesis in an in vitro Matrigel angiogenesis assay using
HUVECs.

 

•                  Specific Aim 2: In vivo testing of anti-S1P Mab’s ability to
reduce tumor growth and associated angiogenesis.

 

•                  Milestone 1: Test Mab’s ability to reduce volume of
Xenografted tumors in SCID mice.

 

•                  Milestone 2: Use Matrigel plugs implanted subcutaneously in
C57B1/6N mice to test the anti-angiogenic effects of the anti-S1P Mab.

 

By successfully accomplishing these aims, we will demonstrate that Medlyte’s
anti-S1P Mab is an effective therapeutic in the treatment of cancer in animal
models. The anticipated success of the proposed initial pre-clinical work will
lead to more intensive pre-clinical testing for safety and efficacy planned for
Phase II. The ultimate goal of the Phase II research is to develop humanized
anti-S1P Mabs that can be taken to Phase 1 Clinical Trials for comparison with
the

 

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anthracycline anti-neoplastic agents. Further, the anti-S1P Mab will be
developed as an in vitro diagnostic tool to define the minimum dosage for
optimum efficacy. While therapeutic antibodies are not generally regarded as
toxic, the theranostics approach will improve patient safety in our planned
clinical trials. Importantly, the pathway for getting our putative therapeutic
antibody to market will be much shorter than the conventional small molecule
approach.

 

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