Exhibit 10.1


 


Modification to CRADA No. 26-97


July 2005


 


Modification
to CRADA No. 26-97


July 15,
2005


 


Appendix A


 


The following research objectives are added to those described in
Appendix A of CRADA No. 26-97.


 


A.                                    Targeting
of Interleukin-13 Receptor for pediatric brain tumor therapy:  In contrast to malignant glioma in adults
that are predominantly supratentorial, pediatric tumors are also localized in
posterior fossa and brain stem.  Because
of their location, Neurosurgcons are unable to resect most of the tumor
resulting in progressive disease with poor prognosis.  As there is not much option available for
these children, an urgent search for innovative therapeutic measures is needed.  Recent studies have demonstrated that 83% of
36 different pediatric brain tumor samples express IL-13R2 chain when
analyzed by immunohistochemical and in-situ hybridization studies (Kawakami et
al. Cancer 2004).  Although both assays
produced compatible results, quantitative difference in receptor density
between various samples is not known.  In
addition, it is not known whether IL-13 receptors in pediatric brain tumors are
sensitive to cytotoxic effect of IL-13-PE as adult glioma in vitro. 
Furthermore, it is not known whether IL-13-PE will be efficacious in
these tumors in animal models of brain tumors in
vivo.  Previous studies have
suggested that glioma cells expressing IL-13R are highly sensitive to IL-13-PE.  As IL-13R-targeted cytotoxin [IL13-PE38QQR]
is being tested in PRECISE Phase 3 clinical trial in adult patients with
recurrent glioma, it will be of importance to study IL-13-PE in not only in vitro and in vivo animal studies of pediatric brain tumors but also
tested in patients to address unmet medical need and to unravel antitumor
activity of this important molecule.


 


The following studies are planned to address important issues
identified above:


 


1.                                       Analysis of IL-13 receptor expression and type of
receptors in additional patient derived samples.  Previous study utilized limited number of
pediatric tumor samples including glioma, low-grade astrocytoma, giant cell
tumor, mixed glioma and ganglioglioma. 
Additional tumor samples will be acquired from Cooperative Human Tissue
Network (CHTN).  FDA IRB (RIHSC) has
approved our request.


 


2.                                       Identification on pediatric brain tumor cell lines and
isolation of primary cell cultures of tumors.  Tumor cell lines derived from pediatric
glioma, low-grade astrocytoma, giant cell tumor, mixed glioma and ganglioglioma
will be obtained from Commercial source or from investigators.  In addition, primary cell cultures from
surgical samples will be generated. 
These cells and cell lines will be tested for the expression of IL-13R
and sensitivity to IL-13-PE in vitro by
various techniques established in our laboratories.


 


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3.                                       Efficacy of IL-13-PE in Pediatric brain tumor models:  The efficacy of IL-13PE will be evaluated in
tumor bearing mice.  Tumor cell lines
derived from various tumors will be established as tumors in immunodeficient
mice.  These animals will be then treated
with IL-13-PE by various routes and schedules. 
Initially, animals with subcutaneous flank tumors will be treated and
later intracranial tumor models will be treated.  Various routes of administration of IL-13-PE
including CED through a burr hole will be evaluated.  Tumor shrinkage, response rate, and animal
survival will be analyzed.


 


These studies
will provide scientific support for a pediatric brain tumor study or tumors in
addition to malignant glioma.


 


B.                                    Evaluation
of synergistic therapeutic efficacy of IL-13-PE with radiation therapy or with
temozolamide:


 


Since radiation induced tumor cell stasis/death and IL-13-PE induced
cell death involve different mechanisms, it is possible that the combination of
radiation and IL-13-PE therapy may lead to combined or synergistic effect on
glioma cells.  As IL-13-PE is being
studied in initially diagnosed glioma along with radiation therapy to support
development, additional in vitro and animal studies should be performed to
determine the optimal schedule of therapy with these two modalities.  Similarly, since temozolomide (approved for
glioma therapy) and IL-13-PE will mediate therapeutic effect through different
mechanisms, it is also possible that the combination of both drugs may mediate
combined or synergistic effects.


 


Planned
studies include:


 


1.                                       In
vitro sublethal irradiation of glioma cell lines followed by culture with IL-13-PE
and conversely incubation with IL-13-PE followed by irradiation will be
performed and the growth, cell viability and cytotoxicity to IL-13-PE will be
assessed.  These studies have already
completed and the results show that prior irradiation although increase IL-13Ralpha2
mRNA expression but it did not enhance sensitivity to IL-13-PE.  Similarly, prior treatment with IL-13-PE did
not enhance sensitivity to irradiation.


 


2.                                       Concomitant
radiation and IL-13-PE incubation will also be performed in vitro and cell growth and protein
synthesis will be assessed.  Preliminary
studies indicate that the combined radiation and IL-13-PE incubation mediates
enhanced antitumor effect in vitro when compared to either modality alone.  Confirmatory studies are being performed and
need continued support to complete these studies, which are expected to be completed
by the end of the second quarter of 2005.


 


3.                                       Pre-treatment
irradiation of subcutaneous or intracranial glioma tumors followed by local
delivery of IL-13-PE are also planned that will be initiated in the beginning
of 2005 and expected to be completed by the end of third quarter of 2005.


 


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4.                                       Concomitant
effect of temozolomide and IL-13-PE in various glioma cell lines in vitro and
the effect of co-administration of temozolomide and IL-13-PE by various routes
in subcutaneous and intracranial tumors will continued to be evaluated.  Preliminary results suggest that temozolomide
synergies with IL-13-PE in mediating antitumor activity in vitro and in
vivo.  These studies are also expected to
be completed in 2005.


 


The following studies began in 2004 and are currently ongoing.  However, if funds remain at the level
proposed in Appendix C Revised. 2, they will not be completed in the year 2005.


 


3.                                      Study
of the mechanism of action of delayed effect of IL-13-PE in normal brain:


 


Clinical studies have demonstrated that IL-13-PE can induce cytotoxic
effect on tumors when administered intratumorally.  In addition, intra-parenchyma administration
of IL-13-PE in normal brain with infiltrating glioma is very well tolerated up
to the dosage of 0.5 microgram/ml.  At
1.0 microgram/ml dose limiting local toxicity has been observed.  In addition, in several patients appearance
of abnormal hyperintense signal on FLAIR images and new gadolinium enhancing
solid lesion with central hypointense area suggesting necrosis in normal or
infiltrated brain parenchyma have been observed between 4 and 8 weeks following
treatment.  This type of phenomenon has
not been observed previously as in all studies most rats or mice were
sacrificed only several days after IL-13-PE infusion.  No long term monitoring was performed after
intraparenchymal administration of IL-13-PE. 
The following studies have been initiated that are expected to lead to
an understanding of the mechanism of this effect and develop strategies to
avoid this delayed effect of IL-13-PE.


 


Planned studies:


 


a.                                       Develop
animal model in mice and rats to simulate human clinical situation by intra
parenchymal administration of IL-13-PE.  Part of
this study is completed and a manuscript has been accepted for publication
(Kawakami et al. J. Neurosurgery, December 2004).


 


b.                                      Study
of brain tissue sections by histology and immunohistochemisty harvested at
various time points of IL-13-PE administration. 
Part of this study has also completed and described in a manuscript
accepted for publication (Kawakami et al. J. Neurosurgery, December 2004).


 


c.                                       Identification
of local or systemic immune mechanism (if any) post IL-13-PE administration in
normal brain.  In some cases, animals
will be immunosuppressed by whole body sub lethal irradiation to determine the
role of chemotherapy on this process in the clinic.  These studies are yet to be initialed and
expected to begin in first quarter 2005.


 


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d.                                      Identification
of the role of cytokines or other factors in this mechanism will be evaluated
by microarray technology, which is available at CBER.


 


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Appendix
C-Revised.2


 


Financial and Staffing Contribution of the Parties:


 


This “Appendix C-Revised.2” replaces in full “Appendix C”, “Appendix
C-Revised” and “Appendix-C-Revised.1” that were attached to the FDA CRADA No. 26-97
entered into by FDA and NeoPharm, Inc. 
This Appendix C-Revised.2” will become effective upon signing below by
representatives of both FDA and NeoPharm, Inc.


 


Collaborator’s Contribution.


 


NeoPharm, Inc. will provide funding agreed upon in this “Appendix
C-Revised.2” at the level of $130,000. 
The term of CRADA No. 26-97 is hereby extended through April 30,
2006.


 


The funds will be used to recruit research personnel, purchase
equipment, laboratory supplies, animals, and reagents.  The PI will recruit one research fellow for
full-time participation in the CRADA project.


 


FDA will use the funds provided by NeoPharm for:


 


	
  Personnel:

  	
   

  	
   

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  One
  Post-Doctoral Research Fellow (including overhead charges)

  	
   

  	
  $

  	
  70,000

  	
   

  
	
  Annual
  Personnel Subtotal

  	
   

  	
  $

  	
  70,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Equipments:

  	
   

  	
   

  	
   

  
	
  Computer,
  software and printer

  	
   

  	
  $

  	
  5,000

  	
   

  
	
  Chromatography
  columns and lab supplies to purify IL-13-PE

  	
   

  	
  $

  	
  10,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Estimated
  Subtotal

  	
   

  	
  $

  	
  15,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Supplies:

  	
   

  	
   

  	
   

  
	
  Purchase of
  immunodeficient animals

  	
   

  	
  $

  	
  15,000

  	
   

  
	
  Tissue
  Histochemistry and Pathology & CBER Core Facility Services

  	
   

  	
  $

  	
  5,000

  	
   

  
	
  Chemicals,
  Reagents, antibodies, enzymes, test kits, cytokines

  	
   

  	
  $

  	
  20,000

  	
   

  
	
  Annual
  Supplies Subtotal

  	
   

  	
  $

  	
  40,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Travel:

  	
   

  	
   

  	
   

  
	
  Annual
  Travel Costs and Conference Fees for PI and a Fellow

  	
   

  	
  $

  	
  5,000

  	
   

  
	
   

  	
   

  	
   

  	
   

  
	
  Annual
  total for Personnel, Equipment, Supplies, and Travel

  	
   

  	
  $

  	
  130,000

  	
   

  



 


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FDA’s
Contribution


 


	
  FTE:

  	
  PI Time

  	
   

  	
  15

  	
  %

  
	
   

  	
  Technician
  Time

  	
   

  	
  5

  	
  %

  
	
   

  	
  Staff
  Scientist Time

  	
   

  	
  20

  	
  %

  
	
   

  	
  Post
  Doctoral Fellow Time

  	
   

  	
  100

  	
  %

  



 


FDA Supplies:                             Some of the supplies will
be purchased with intramural budget allotted to the Puri laboratory:


 


FDA Equipment:


 


Some of the equipment needed for the collaboration are available in the
laboratory or have been purchased with funds provided by the Collaborator
during the first six years of the collaboration.


 


PI will have laboratory space to house one post-doctoral fellow and
staff scientist.


 


FDA will provide the PI, who has expertise in the field of molecular
tumor biology.  The PI will also provide
onsite supervision of the fellows, laboratory technician and staff
scientist.  The PI will direct the work
of the postdoctoral fellow by planning experiments, assuring that the fellow
performs the required work in a professional manner, evaluate and interpret the
experimental results and prepare written progress reports for the Collaborator.


 


The Collaborator agrees that the PI has broad flexibility in the use,
transfer and disbursement of funds between the resource categories described
above.  Monies not spent in a given
fiscal year will be carried over into the following year.


 


SIGNATURES
APPEAR ON THE NEXT PAGE


 


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  FOR THE PHS:

  	
   

  
	
   

  	
   

  
	
   

  	
   

  
	
    /s/ JESSE L. GOODMAN

  	
   

  	
  8/8/05

  	
   

  
	
   

  	
  Date

  	
   

  
	
  Jesse L. Goodman, M.D., M.P.H.

  	
   

  
	
  Director

  	
   

  
	
  Center for Biologics Evaluation and
  Research

  	
   

  
	
  Food and Drug Administration

  	
   

  
	
   

  	
   

  
	
  Mailing Address for Notices:

  	
   

  
	
   

  	
   

  
	
  Food and Drug Administration

  	
   

  
	
  Center for Biologics Evaluation and
  Research (HFM-544)

  	
   

  
	
  1401 Rockville Pike

  	
   

  
	
  Rockville, MD 20892

  	
   

  
	
   

  	
   

  
	
   

  	
   

  
	
  FOR THE COLLABORATOR:

  	
   

  
	
   

  	
   

  
	
   

  	
   

  
	
    /s/ JEFFREY SHERMAN

  	
   

  	
  7/25/05

  	
   

  
	
   

  	
  Date

  	
   

  
	
  Dr. Jeff Sherman

  	
   

  
	
  Chief Medical Officer and Executive Vice
  President

  	
   

  
	
   

  	
   

  
	
   

  	
   

  
	
  /s/  LAWRENCE A. KENYON

  	
   

  	
  7/25/05

  	
   

  
	
   

  	
  Date

  	
   

  
	
  Larry Kenyon

  	
   

  
	
  Chief Financial Officer

  	
   

  



 


Mailing Address for Notices:


 


NeoPharm, Inc.


150 Field Drive, Suite 195


Lake Forest, IL  60045


 


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