Document:

EX 4.01

    EXHIBIT
      4.01

    

    This
      Note
      is a Global Security within the meaning of the Indenture hereinafter referred
      to
      and is registered in the name of the Depository named below or a nominee of
      the
      Depository. This Note is not exchangeable for Notes registered in the name
      of a
      Person other than the Depository or its nominee except in the limited
      circumstances described herein and in the Indenture, and no transfer of this
      Note (other than a transfer of this Note as a whole by the Depository to a
      nominee of the Depository or by a nominee of the Depository to the Depository
      or
      another nominee of the Depository) may be registered except in the limited
      circumstances described herein.

    

    Unless
      this certificate is presented by an authorized representative of The Depository
      Trust Company, a New York corporation (the "Depository"), to the Company or
      its
      agent for registration of transfer, exchange, or payment, and any certificate
      issued is registered in the name of Cede & Co. or in such other name as is
      requested by an authorized representative of the Depository (and any payment
      is
      made to Cede & Co. or to such other entity as is requested by an authorized
      representative of the Depository), ANY TRANSFER, PLEDGE, OR OTHER USE HEREOF
      FOR
      VALUE OR OTHERWISE BY OR TO ANY PERSON IS WRONGFUL inasmuch as the registered
      owner hereof, Cede & Co., has an interest herein.

    

    CITIGROUP
      INC.

    5.10%
      Notes due September 29, 2011

    
      	
              REGISTERED

            	
              REGISTERED

            

    

    

    CUSIP:
      172967 DU 2

    ISIN:
      US172967DU25

    Common
      Code: 026993709

    

    
      	
              No.
                R-

            	
              $       
                

            

    

    

    CITIGROUP
      INC., a Delaware corporation (the "Company", which term includes any successor
      Person under the Indenture), for value received, hereby promises to pay to
      Cede
& Co., or registered assigns, the principal sum of $___________ on September
      29, 2011 and to pay interest thereon from and including September 29, 2006
      or
      from the most recent Interest Payment Date to which interest has been paid
      or
      duly provided for, semi-annually, on March 29 and September 29 of each year,
      commencing March 29, 2007, at the rate of 5.10% per annum, until the principal
      hereof is paid or made available for payment. The interest so payable, and
      punctually paid or duly provided for, on any Interest Payment Date will, as
      provided in the Indenture, be paid to the Person in whose name this Note is
      registered at the close of business on the Record Date for such interest, which
      shall be the March 15 and September 15 (whether or not a Business Day)
      immediately preceding such Interest Payment Date.

    
      
         

      

      
         

        
          

        

      

      
         

      

       

    

    Any
      such
      interest not so punctually paid or duly provided for will forthwith cease to
      be
      payable to the holder on such Record Date and may either be paid to the Person
      in whose name this Note is registered at the close of business on a subsequent
      Record Date, such subsequent Record Date to be not less than five days prior
      to
      the date of payment of such defaulted interest, notice whereof shall be given
      to
      holders of Notes of this series not less than 15 days prior to such subsequent
      Record Date, or be paid at any time in any other lawful manner not inconsistent
      with the requirements of any securities exchange on which the Notes of this
      series may be listed, and upon such notice as may be required by such exchange,
      all as more fully provided in the Indenture.

    

    Interest
      hereon will be calculated on the basis of a 360-day year comprised of twelve
      30-day months.

    

    If
      either
      an Interest Payment Date or the Maturity of the Notes falls on a day that is
      not
      a Business Day, such Interest Payment Date or Maturity will be the next
      succeeding Business Day. If a date for payment of interest or principal on
      the
      Notes falls on a day that is not a business day in the place of payment, such
      payment will be made on the next succeeding business day in such place of
      payment as if made on the date the payment was due. No interest will accrue
      on
      any amounts payable for the period from and after the due date for payment
      of
      such principal or interest. 

    

    For
      these
      purposes, “Business Day” means any day which is a day on which commercial banks
      settle payments and are open for general business in The City of New
      York.

    

    Payment
      of the principal of and interest on this Note will be made at the office or
      agency of the Trustee maintained for that purpose in The City of New
      York.

    

    Reference
      is hereby made to the further provisions of this Note set forth on the reverse
      hereof, which further provisions shall for all purposes have the same effect
      as
      if set forth at this place.

    

    Unless
      the certificate of authentication hereon has been executed by the Trustee or
      by
      an authenticating agent on behalf of the Trustee by manual signature, this
      Note
      shall not be entitled to any benefit under the Indenture or be valid or
      obligatory for any purpose.

    
      
         

      

      
        -2-

        
          

        

      

      
         

      

    

    IN
      WITNESS WHEREOF, the Company has caused this instrument to be duly executed
      under its corporate seal.

    

    Dated:
      September 29, 2006

    

    CITIGROUP
      INC.

    
 

    By:_________________________________

    Title:
      Chief Financial Officer

    

    

    

    ATTEST:

    

    By:___________________________

    Title:
      Assistant Secretary

    
      
         

      

      
        -3-

        
          

        

      

      
         

      

       

    

    This
      is
      one of the Notes of the series issued under the within-mentioned
      Indenture.

    

    Dated:
      September 29, 2006

    

    THE
      BANK
      OF NEW YORK,

    as
      Trustee

    

    

    By:_________________________________

    Name:

    Title:

     

     

    -or-

    

    

    CITIBANK,
      N.A.,

    as
      Authenticating Agent

    

    

    By:_________________________________

    Name:

    Title:

    
      
         

      

      
        -4-

        
          

        

      

      
         

      

       

    

    This
      Note
      is one of a duly authorized issue of Securities of the Company (the "Notes"),
      issued and to be issued in one or more series under the Indenture, dated as
      of
      March 15, 1987 (as amended and supplemented to date, the "Indenture"), between
      the Company and The Bank of New York, as Trustee (the "Trustee", which term
      includes any successor trustee under the Indenture), to which Indenture and
      all
      indentures supplemental thereto reference is hereby made for a statement of
      the
      respective rights, limitations of rights, duties and immunities thereunder
      of
      the Company, the Trustee and the holders of the Notes and of the terms upon
      which the Notes are, and are to be, authenticated and delivered. This Note
      is
      one of the series designated on the face hereof, initially limited in aggregate
      principal to $1,000,000,000.

    

    If
      an
      event of default (as defined in the Indenture) with respect to Notes of this
      series shall occur and be continuing, the principal of the Notes of this series
      may be declared due and payable in the manner and with the effect provided
      in
      the Indenture.

    

    The
      Indenture contains provisions for defeasance at any time of the entire
      indebtedness of this Note upon compliance by the Company with certain conditions
      set forth in Sections 11.03 and 11.04 thereof, which provisions apply to this
      Note.

    

    The
      Indenture contains provisions permitting the Company and the Trustee, without
      the consent of the holders of the Securities, to establish, among other things,
      the form and terms of any series of Securities issuable thereunder by one or
      more supplemental indentures, and, with the consent of the holders of not less
      than 66 2/3% in aggregate principal amount of Securities at the time outstanding
      which are affected thereby, to modify the Indenture or any supplemental
      indenture or the rights of the holders of Securities of such series to be
      affected, provided that no such modification will (i) extend the fixed maturity
      of any Securities, reduce the rate or extend the time of payment of interest
      thereon, reduce the principal amount thereof or the premium, if any, thereon,
      reduce the amount of the principal of Original Issue Discount Securities payable
      on any date, change the currency in which Securities are payable, or impair
      the
      right to institute suit for the enforcement of any such payment on or after
      the
      maturity thereof, without the consent of the holder of each Security so
      affected, or (ii) reduce the aforesaid percentage of Securities of any series
      the consent of the holders of which is required for any such modification
      without the consent of the holders of all Securities of such series then
      outstanding, or (iii) modify, without the written consent of the Trustee, the
      rights, duties or immunities of the Trustee.

    

    No
      reference herein to the Indenture and no provision of this Note or of the
      Indenture shall alter or impair the obligation of the Company, which is absolute
      and unconditional, to pay the principal of and interest on this Note at the
      times, place and rate, and in the coin or currency, herein
      prescribed.

    

    This
      Note
      is a Global Security registered in the name of a nominee of the Depository.
      This
      Note is exchangeable for Notes registered in the name of a person other than
      the
      Depository or its nominee only in the limited circumstances hereinafter
      described. Unless and until it is exchanged in whole or in part for definitive
      Notes in certificated form, this Note may not be

    
      
         

      

      
        -5-

        
          

        

      

      
         

      

    

    transferred
      except as a whole by the Depository to a nominee of the Depository or by a
      nominee of the Depository to the Depository or another nominee of the
      Depository.

    

    The
      Notes
      represented by this Global Security are exchangeable for definitive Notes in
      certificated form of like tenor as such Notes in denominations of $1,000 and
      whole multiples of $1,000 in excess thereof only if (i) the Depository
      notifies the Company that it is unwilling or unable to continue as Depository
      for the Notes or (ii) the Depository ceases to be a clearing agency registered
      under the Securities Exchange Act of 1934, as amended, or (iii) the Company
      in
      its sole discretion decides to allow the Notes to be exchanged for definitive
      Notes in registered form. Any Notes that are exchangeable pursuant to the
      preceding sentence are exchangeable for certificated Notes issuable in
      authorized denominations and registered in such names as the Depository shall
      direct. As provided in the Indenture and subject to certain limitations therein
      set forth, the transfer of definitive Notes in certificated form is registrable
      in the register maintained by the Company in The City of New York for such
      purpose, upon surrender of the definitive Note for registration of transfer
      at
      the office or agency of the registrar, duly endorsed by, or accompanied by
      a
      written instrument of transfer in form satisfactory to the Company and the
      registrar duly executed by, the holder thereof or his attorney duly authorized
      in writing, and thereupon one or more new Notes of this series and of like
      tenor, of authorized denominations and for the same aggregate principal amount,
      will be issued to the designated transferee or transferees. Subject to the
      foregoing, this Note is not exchangeable, except for a Global Security or Global
      Securities of this issue of the same principal amount to be registered in the
      name of the Depository or its nominee.

    

    No
      service charge shall be made for any such registration of transfer or exchange,
      but the Company may require payment of a sum sufficient to cover any tax or
      other governmental charge payable in connection therewith.

    

    Prior
      to
      due presentment of this Note for registration of transfer, the Company, the
      Trustee and any agent of the Company or the Trustee may treat the Person in
      whose name this Note is registered as the owner hereof for all purposes, whether
      or not this Note be overdue, and neither the Company, the Trustee nor any such
      agent shall be affected by notice to the contrary.

    

    The
      Company will pay additional amounts ("Additional Amounts") to the beneficial
      owner of any Note that is a non-United States person in order to ensure that
      every net payment on such Note will not be less, due to payment of U.S.
      withholding tax, than the amount then due and payable. For this purpose, a
      "net
      payment" on a Note means a payment by the Company or a paying agent, including
      payment of principal and interest, after deduction for any present or future
      tax, assessment or other governmental charge of the United States. These
      Additional Amounts will constitute additional interest on the Note.

    

    The
      Company will not be required to pay Additional Amounts, however, in any of
      the
      circumstances described in items (1) through (13) below.

    
      
         

      

      
        -6-

        
          

        

      

      
         

      

       

    

    (1) Additional
      Amounts will not be payable if a payment on a Note is reduced as a result of
      any
      tax, assessment or other governmental charge that is imposed or withheld solely
      by reason of the beneficial owner:

    

    
      	 	 	
              (a)

            	
              having
                a relationship with the United States as a citizen, resident or
                otherwise;

            

    

    
      	 	 	
              (b)

            	
              having
                had such a relationship in the past
                or

            

    

    
      	 	 	
              (c)

            	
              being
                considered as having had such a
                relationship.

            

    

    

    
      	 	
              (2)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld solely by reason of the beneficial
                owner:

            

    

    

    
      	 	
               

            	
              (a)

            	
              being
                treated as present in or engaged in a trade or business in the United
                States;

            

    

    
      	 	
               

            	
              (b)

            	
              being
                treated as having been present in or engaged in a trade or business
                in the
                United States in the past or

            

    

    
      	 	
               

            	
              (c)

            	
              having
                or having had a permanent establishment in the United
                States.

            

    

    

    
      	 	
              (3)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld in whole or in part by reason of the beneficial owner being
                or
                having been any of the following (as such terms are defined in the
                Internal Revenue Code of 1986, as
                amended):

            

    

    

    
      	 	
               

            	
              (a)

            	
              personal
                holding company;

            

    

    
      	 	
               

            	
              (b)

            	
              foreign
                personal holding company;

            

    

    
      	 	
               

            	
              (c)

            	
              foreign
                private foundation or other foreign tax-exempt
                organization;

            

    

    
      	 	
               

            	
              (d)

            	
              passive
                foreign investment company;

            

    

    
      	 	
               

            	
              (e)

            	
              controlled
                foreign corporation or

            

    

    
      	 	
               

            	
              (f)

            	
              corporation
                which has accumulated earnings to avoid United States federal income
                tax.

            

    

    

    
      	 	
              (4)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld solely by reason of the beneficial owner owning or having
                owned,
                actually or constructively, 10 percent or more of the total combined
                voting power of all classes of stock of the Company entitled to vote
                or by
                reason of the beneficial owner being a bank that has invested in
                a Note as
                an extension of credit in the ordinary course of its trade or
                business.

            

    

    

    For
      purposes of items (1) through (4) above, "beneficial owner" means a
      fiduciary, settlor, beneficiary, member or shareholder of the holder if the
      holder is an estate, trust, partnership, limited liability company, corporation
      or other entity, or a person holding a power over an estate or trust
      administered by a fiduciary holder.

    
      
         

      

      
        -7-

        
          

        

      

      
         

      

    

     

    
      	 	
              (5)

            	
              Additional
                Amounts will not be payable to any beneficial owner of a Note that
                is
                a:

            

    

    

    
      	 	
               

            	
              (a)

            	
              fiduciary;

            

    

    
      	 	
               

            	
              (b)

            	
              partnership;

            

    

    
      	 	
               

            	
              (c)

            	
              limited
                liability company or

            

    

    
      	 	
               

            	
              (d)

            	
              other
                fiscally transparent entity

            

    

    

    
      	 	 	
              or
                that is not the sole beneficial owner of the Note, or any portion
                of the
                Note. However, this exception to the obligation to pay Additional
                Amounts
                will only apply to the extent that a beneficiary or settlor in relation
                to
                the fiduciary, or a beneficial owner or member of the partnership,
                limited
                liability company or other fiscally transparent entity, would not
                have
                been entitled to the payment of an Additional Amount had the beneficiary,
                settlor, beneficial owner or member received directly its beneficial
                or
                distributive share of the payment.

            

    

    

    
      	 	
              (6)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld solely by reason of the failure of the beneficial owner
                or any
                other person to comply with applicable certification, identification,
                documentation or other information reporting requirements. This exception
                to the obligation to pay Additional Amounts will only apply if compliance
                with such reporting requirements is required by statute or regulation
                of
                the United States or by an applicable income tax treaty to which
                the
                United States is a party as a precondition to exemption from such
                tax,
                assessment or other governmental
                charge.

            

    

    

    
      	 	
              (7)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is collected
                or
                imposed by any method other than by withholding from a payment on
                a Note
                by the Company or a paying agent.

            

    

    

    
      	 	
              (8)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld by reason of a change in law, regulation, or administrative
                or
                judicial interpretation that becomes effective more than 15 days
                after the
                payment becomes due or is duly provided for, whichever occurs
                later.

            

    

    

    
      	 	
              (9)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is imposed
                or
                withheld by reason of the presentation by the beneficial owner of
                a Note
                for payment more than 30 days after the date on which such payment
                becomes due or is duly provided for, whichever occurs
                later.

            

    

    

    
      	 	
              (10)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any:

            

    

     

    
      
         

      

      
        -8-

        
          

        

      

      
         

      

       

    

    
      	 	
               

            	
              (a)

            	
              estate
                tax;

            

    

    
      	 	
               

            	
              (b)

            	
              inheritance
                tax;

            

    

    
      	 	
               

            	
              (c)

            	
              gift
                tax;

            

    

    
      	 	
               

            	
              (d)

            	
              sales
                tax;

            

    

    
      	 	
               

            	
              (e)

            	
              excise
                tax;

            

    

    
      	 	
               

            	
              (f)

            	
              transfer
                tax;

            

    

    
      	 	
               

            	
              (g)

            	
              wealth
                tax;

            

    

    
      	 	
               

            	
              (h)

            	
              personal
                property tax or

            

    

    
      	 	
               

            	
              (i)

            	
              any
                similar tax, assessment, withholding, deduction or other governmental
                charge.

            

    

    

    
      	 	
              (11)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment, or other governmental charge required to
                be
                withheld by any paying agent from a payment of principal or interest
                on a
                Note if such payment can be made without such withholding by any
                other
                paying agent.

            

    

    

    
      	 	
              (12)

            	
              Additional
                amounts will not be payable if a payment on a Note is reduced as
                a result
                of any tax, assessment or other governmental charge that is required
                to be
                made pursuant to any European Union directive on the taxation of
                savings
                income or any law implementing or complying with, or introduced to
                conform
                to, any such directive.

            

    

    

    
      	 	
              (13)

            	
              Additional
                Amounts will not be payable if a payment on a Note is reduced as
                a result
                of any combination of items (1) through (12)
                above.

            

    

    

    Except
      as
      specifically provided herein, the Company will not be required to make any
      payment of any tax, assessment or other governmental charge imposed by any
      government or a political subdivision or taxing authority of such
      government.

    

    As
      used
      in this Note, "United States person" means:

    

    
      	 	
              (a)

            	
              any
                individual who is a citizen or resident of the United
                States;

            

    

    
      	 	
              (b)

            	
              any
                corporation, partnership or other entity created or organized in
                or under
                the laws of the United States;

            

    

    
      	 	
              (c)

            	
              any
                estate if the income of such estate falls within the federal income
                tax
                jurisdiction of the United States regardless of the source of such
                income
                and

            

    

    
      	 	
              (d)

            	
              any
                trust if a United States court is able to exercise primary supervision
                over its administration and one or more United States persons have
                the
                authority to control all of the substantial decisions of the
                trust.

            

    

    

    Additionally,
      "non-United States person" means a person who is not a United States person,
      and
      "United States" means the states of the United States of America and the
      District of Columbia, but excluding its territories and its
      possessions.

    
      
         

      

      
        -9-

        
          

        

      

      
         

      

    

    Except
      as
      provided below, the Notes may not be redeemed prior to maturity.

     

    
      	
            	(1)	
              The
                Company may, at its option, redeem the Notes
                if:

            

    

    

    
      	 	 	
              (a)

            	
              the
                Company becomes or will become obligated to pay Additional Amounts
                as
                described above;

            

    

    
      	 	 	
              (b)

            	
              the
                obligation to pay Additional Amounts arises as a result of any change
                in
                the laws, regulations or rulings of the United States, or an official
                position regarding the application or interpretation of such laws,
                regulations or rulings, which change is announced or becomes effective
                on
                or after September 26, 2006 and

            

    

    
      	 	 	
              (c)

            	
              the
                Company determines, in its business judgment, that the obligation
                to pay
                such Additional Amounts cannot be avoided by the use of reasonable
                measures available to it, other than substituting the obligor under
                the
                Notes or taking any action that would entail a material cost to the
                Company.

            

    

    

    
      	 	
              (2)

            	
              The
                Company may also redeem the Notes, at its option,
                if:

            

    

    

    
      	 	 	
              (a)

            	
              any
                act is taken by a taxing authority of the United States on or after
                September 26, 2006, whether or not such act is taken in relation
                to the
                Company or any affiliate, that results in a substantial probability
                that
                the Company will or may be required to pay Additional Amounts as
                described
                above;

            

    

    
      	 	 	
              (b)

            	
              the
                Company determines, in its business judgment, that the obligation
                to pay
                such Additional Amounts cannot be avoided by the use of reasonable
                measures available to it, other than substituting the obligor under
                the
                Notes or taking any action that would entail a material cost to the
                Company and

            

    

    
      	 	 	
              (c)

            	
              the
                Company receives an opinion of independent counsel to the effect
                that an
                act taken by a taxing authority of the United States results in a
                substantial probability that the Company will or may be required
                to pay
                the Additional Amounts described under above, and delivers to the
                Trustee
                a certificate, signed by a duly authorized officer, stating that
                based on
                such opinion the Company is entitled to redeem the Notes pursuant
                to their
                terms.

            

    

    

    Any
      redemption of the Notes as set forth in clauses (1) or (2) above shall be in
      whole, and not in part, and will be made at a redemption price equal to 100%
      of
      the principal amount of the Notes Outstanding plus accrued interest thereon
      to
      the date of redemption. Holders shall be given not less than 30 days nor more
      than 60 days prior notice by the Trustee of the date fixed for such
      redemption.

    

    All
      terms
      used in this Note which are defined in the Indenture shall have the meanings
      assigned to them in the Indenture. The Notes are governed by the laws of the
      State of New York.

    
      
         

      

        -10-SECOND
      RESEARCH FUNDING AND INTELLECTUAL PROPERTY

    ASSIGNMENT
      AGREEMENT

    BETWEEN

    UNIVERSITY
      OF MELBOURNE

    AND

    PRANA
      BIOTECHNOLOGY LTD (ABN 37 080 699 065)

    

    

     

    

    

    

    

    
      
        
        

      

      
        1

        
          

        

      

      
        
        

      

    

    

    This
      Second Research Funding and Intellectual Property Assignment Agreement is made:
      

     

    BETWEEN

     

    THE
      UNIVERSITY OF MELBOURNE [ABN
      84
      002 705 224] of Parkville, Victoria 3010, a body politic and corporate pursuant
      to the provisions of the Melbourne
      The
      University
      Act
      1958
      ("the
      University").

    

    AND:

    

    PRANA
      BIOTECHNOLOGY LTD (ABN 37 080 699 065) having
      its principal office at Level 1, 100 Dorcas Street, South Melbourne, Victoria
      3205 ("Prana")

     

    RECITALS:

    

    
      	
              A.

            	
              Prana
                and the University are parties to an undated Research Funding and
                Intellectual Property Assignment Agreement, entered into on or about
                1
                December 2000 as amended from time to time, which expired on 1 December
                2003 ("The Research Agreement").

            

    

    

    
      	
              B.

            	
              Since
                the expiration of The Research Agreement, the parties have continued
                to
                conduct Projects and work together in accordance with the terms and
                conditions of the Original Research Agreement as if it continued
                to have
                full force and effect.

            

    

    

    
      	
              C.

            	
              The
                parties now wish to enter into this Second Research Funding and
                Intellectual Property Assignment Agreement ('Second Research Agreement')
                which is deemed to have come into effect on and from the date of
                expiration of The Research
                Agreement.

            

    

     

    NOW
      IT IS AGREED:

    

    
      	
              1.

            	
              DEFINITIONS
                & INTERPRETATION. Unless
                otherwise specified in this Second Research Agreement, all defined
                terms
                used in this Second Research Agreement shall have the same meaning
                as
                given to those terms in The Research
                Agreement.

            

    

    

    'Further
      Term'
      means a
      period of three years deemed to have commenced on and from the expiration of
      The
      Research Agreement and expiring on 1 December 2006.

    

    ‘The
      Research
      Agreement' means
      the
      undated Research Funding and Intellectual Property Assignment Agreement, entered
      into on or about 1 December 2000 as amended from time to time, which expired
      on
      1 December 2003.

    'Research
      Projects' has
      the
      meaning given to that term in The Research Agreement.

    'Second
      Research Agreement' means
      this Agreement.

    

    
      	
              2.
                

            	
              INCORPORATION
                OF TERMS AND CONDITIONS OF THE RESEARCH
                AGREEMENT

            

    

    

    
      
        
        

      

      
        2

        
          

        

      

      
        
        

      

    

    

    The
      terms
      and conditions of The Research Agreement are incorporated into this Second
      Research Agreement and are deemed to have had full force and effect as and
      from
      the expiration of The Research Agreement, save and except for any terms and
      conditions specifically amended, replaced or supplemented by this Second
      Research Agreement.

    

    
      

      
        	
                3.
                  

              	
                AMENDMENT
                  OF SCHEDULE

              

      

    

     

    The
      Parties agree that the Schedule to The Research Agreement shall be amended
      as
      provided by this Second Research Agreement.

    

    
      	4.	
              EFFECTIVE
                DATE OF THIS AGREEMENT AND EARLY
                EXPIRATION

            

    

    

    This
      Second Research Agreement shall be deemed to have come into effect on and from
      the date of expiration of The Research Agreement and shall remain in effect
      for
      a Further Term, unless the parties agree in writing to an earlier expiration
      date or termination occurs in accordance with clause 19 of The Research
      Agreement.

    

    

    SIGNED
      for
      and
      on behalf of THE
      UNIVERSITY OF 

    MELBOURNE

    

    In
      the
      presence of:)     ______________

    _______________     Authorised
      Officer

    Witness
      signature 

    

    _______________

    Name
      (printed) 

    

    

    

    

    SIGNED
      for and
      on behalf of PRANA
      

    BIOTECHNOLOGY
      LTD

    In
      the
      presence of:)     ______________

    _______________     Authorised
      Officer

    Witness
      signature 

     

    _______________

    Name
      (printed)

     

    
 

    
      
        
        

      

      
        3

        
          

        

      

      
        
        

      

    

    

    SCHEDULE
      A

    Replacement
      to Part B

    

    
      	1.	
              PROPOSED
                RESEARCH PROGRAM

            

    

    

    The
      Research comprises the following areas of development of possible therapeutic
      or
      diagnostic

    applications
      into Alzheimer's Disease and other neurodegenerative disorders and psychoses
      characterised by metal mediated oxidative stress and/or toxic gain of function
      by a protein.

    Reference
      to beta amyloid (AB) below is understood to include other functionally related
      proteins such as APP, a -synuclein, a -crystalline, polyglutamine expansions,
      SOD and Prion proteins.

    

    For
      clarity the following areas are defined:-

    

    
      	1.	
              The
                development of chelators or modulators of AB
                activity.

            

    

    

    
      	
              2.

            	
              The
                development of therapeutic or diagnostic agents arising from research
                into
                the structural and functional studies of AB metal binding sites,
                characterization of AB neurotoxic species that modulate A-B redox
                activity, and new molecular targets of AB defined by peptidomimetic
                and
                biosensor studies.

            

    

    

    
      	3.	
              The
                development of proteins that bind to and mediate the clearance of
                AB.

            

    

    

    
      	
              4.

            	
              The
                development of therapeutic or diagnostic applications arising from
                the
                elucidation of the behaviour of zinc, copper, iron and other metal
                ions
                with respect to redox activity at the
                synapse.

            

    

    

    
      	5.	
              The
                development of compounds with pharmaceutical potential which directly
                interfere with redox-mediated
                damage.

            

    

    

    
      	6.	
              The
                development of markers of biochemical oxidative stress and metal
                homeostasis in the human
                brain as a model for the development of neurodegenerative disorders
                or
                psychoses as above defined.

            

    

    

    
      	
              7.

            	
              The
                development of compounds and processes which modulate A-B membrane
                interactions, AB mitochondrial biology and other cellular biochemical
                neurotoxic processes mediated by
                AB.

            

    

    

    
      	
              8.

            	
              The
                development of any possible therapeutic or diagnostic applications
                arising
                from the characterization of mechanisms involved in transmembrane
                APP
                dimerization.

            

    

    

    
      	
              9.

            	
              The
                development of a rational based drug design programme arising from
                the
                elucidation of the biophysical characteristics and structure of
                AB.

            

    

    

    
      	
              10.

            	
              The
                development of possible therapeutic or diagnostic applications arising
                from studies of prion proteins in Creutzfeldt-Jakob
                Disease.

            

    

    

    
      	
              11.

            	
              The
                development of possible therapeutic or diagnostic applications arising
                from studies of SOD in Motor Neuron
                Disease

            

    

     

    
      	
              12.

            	
              The
                development of assays arising from studies of polyglutamine expansions
                in
                Huntington's
                Disease and other related
                disorders.

            

    

    

    
      	
              13.

            	
              The
                development of assays arising from studies of a-crystallin in
                neurodegenerative disorders and
                cataracts.

            

    

    

    
      	
              14.

            	
              The
                development of assays arising from studies of a-synuclein in Parkinson's
                Disease and related disorders.

            

    

    

    
      
        
        

      

      
        4

        
          

        

      

      
        
        

      

    

    
      	
              15.

            	
              The
                development of the technology, assay, cell line, animal model or
                patient
                database involved with the detection and screening of compounds developed
                under this Agreement for use in the above field of
                Research.

            

    

    

    
      	2.	
              STAFF/PROJECTS/BUDGET
                ESTIMATES

            

    

    

    Research
      Projects being conducted during the period 2 December 2003 to 31 December 2004.
      The
      details of each Research Project are provided in the Appendix to this Second
      Research Agreement.

    

    
      	4.	
              FUNDING
                FOR PERIOD 2 DECEMBER 2003 — 1 DECEMBER 2006:
                

            

    

    

    Funding
      for 2 December 2003 to 31 December 2004, all figures are ex GST: 

    
       

      
        	 Research
                Project Title.	
                Budget
                  Period.

              
	 	
                2
                  Dec 2003 — 31 March 2004.

              
	
              	 
	
                'AB
                  interactions with
                  mitochondrial                   
                  

                respiratory
                  chain complexes'*.

                (Trounce)

              	$   
                156,645
	 	 
	
                'AB
                  clearance mechanisms’**.

                (Cappai)

              	$   
                179,021
	 	 
	
                'AB
                  binding ligands for imaging
                  of                     
                  

                Alzheimer's
                  Disease’***. 

                (Masters)

              	$   
                134,266

      

    

     

    *Research
      Project formerly denoted as Project 7 in the Letter Amendment of 7 March 2003
      to
      the original Research Funding and Intellectual Property Assignment
      Agreement.

    

    **Research
      Project formerly denoted as Project 8 in the Letter Amendment of 7 March 2003
      to
      the original Research Funding and Intellectual Property Assignment
      Agreement.

    

    ***Research
      Project formerly denoted as Project 9 in the Letter Amendment of 7 March 2003
      to
      the original Research Funding and Intellectual Property Assignment
      Agreement.

     

    
      	 	
              2
                Dec 2003 — 2 Dec 2004.

            
	
              Project
                1.

            	 
	
              'Structure
                Based Drug Design'

            	
              $150,000

            
	 	 
	
              Project
                2.

            	 
	
              'Drug
                Screening & Development'

            	
              $300,000

            
	 	 
	
              Project
                3.

            	 
	
              'Cell
                Based Drug Discovery’

            	
              $150,000

            
	 	 
	
              Project
                4.

            	 
	
              'APP
                Metal Binding'

            	
              $12,000

            
	 	 
	
              Project
                5.

            	 
	
              'Blood
                Brain Barrier Studies'

            	
              $0
                to $24,000 invoice only on
                needs-be-basis.

            

    

     

    
      
        
        

      

      
        5

        
          

        

      

      
        
        

      

    

     

     

    
      	 	
              Commencing
                1 April 2004- 31 Dec 2004.

            
	 	 
	
              Project
                6.

            	 
	
              'Amyloid
                AB Imaging ligands:

            	
              $101,250

            
	
              high
                throughput screen approach

            	 
	
              —
                development of screening assays'

            	 
	 	 
	
              Project
                7.

            	 
	
              'Expression
                profiling of

            	
              $50,625

            
	
              AD
                imaging targets'

            	 
	 	 
	
              Project
                8.

            	 
	
              'Amyloid
                AB monoclonal antibodies

            	
              $50,625

            
	
              as
                imaging ligands'.

            	 
	 	 
	
              Project
                9.

            	 
	
              'AB
                binding proteins'

            	
              $151,875

            
	 	 
	
              Project
                10.

            	 
	
              'Amyloid
                imaging with PBT-1'

            	
              $174,300****

            
	 	 
	
              Project
                11.

            	 
	
              'AB
                binding metallocomplexes'

            	
              $101,250

            

    

    

    ****A
      12
      month
      budget for Project 10 of $275,000 (net GST) to be administered under the Austin
      Health Sub contract to The University of Melbourne commenced 1 January 2004
      under the Project 'AB binding ligands for imaging of Alzheimer’s Disease',
      accordingly the budget for the period 1 April 2004 to the final payment to
      31
      Dec 2004 is calculated: $275,000 -0.75($134,266) = $174,300.

    

    

    Funding
      for each subsequent calendar year 2005 and 2006 is to be set by the Management
      Committee one month before each calendar year in accordance with the funding
      for
      each annual Research Plan.

    

    

    
      	5.	
              UNIVERSITY
                REPRESENTATIVES

            

    

    

    Professor
      James Angus or a representative nominated by the University from time to
      time.

    

    
      	6.	
              PRANA
                REPRESENTATIVE

            

    

    

    Ms
      Dianne
      Angus, Prana or a representative nominated by Prana from time to
      time.

    

    

    
      	7.	
              MINIMUM
                PERFORMANCE LEVELS

            

    

    

    $2,000
      per annum for the period beginning on the date an amount first becomes payable
      under clause 12 of the Original Research Agreement until the end of the Further
      Term of this Agreement.

    

    
      
        
        

      

      
        6

        
          

        

      

      
        
        

      

    

    APPENDIX
      -TO SECOND RESEARCH AGREEMENT

    

    Project
      1. Research Prefect: ‘Structure Based Druq Design'

     

    
      	General Aim
              :              	
              Characterizing
                the biophysical interactions between metals and other co-factors
                with: a -
                synuclein with dopamine, APP and AB
                to:-

            

    

    
      	 	 	 

      	 	(i)	elucidate
              new therapeutic targets,

      	 	
              (ii)

            	
              study
                the effect of such biophysical interactions on the structure and
                potential
                oligomerisation of AB, APP and a -
                synuclein

            

    

    
      	 	
              (iii)

            	
              develop
                screens for PD and AD therapeutic
                agents.

            

    

    

    Proposed
      Objectives:

    (1) Develop
      a
      dopamine / a-synuclein / metal model to screen compounds for ability to moderate
      ROS production and ability of agent to compete with a-synuclein -
      dopamine.

    

    (2) Develop
      a
      PD-specic cell-based tox. assay (dopaminergic neurons) to screen
      for:

    -
      compounds positive in test (1),

    -
      to
      detect inhibition of toxicity otherwise induced by 6-OHDa +Dopamine +test
      agent

    -
      neuroprotection effects.

    

    (3) Develop
      a
      PD animal model to test compounds positive in test (2) via 6-OHDa model and
      transgenic PD models.

    

    (4) Investigate
      and characterise biophysical relationship of a-synuclein - dopamine interaction.
      [ie. determination of a-synuclein binding site by mutagenesis, effect on
      a-synuclein folding and aggregation of a-synuclein, effect on redox activity
      of
      a-synuclein] via:

    -
      protein
      production 

    -mitochondrial
      assays 

    -
      CD,
      ERR, NMR

    

    (5) Investigate
      and characterise biophysical relationship of APP and/or AB - metal interaction.
      [ie, determination of binding sites by mutagenesis, effect on protein
folding
      and aggregation and redox activity via;

    -
      protein
      production 

    -mitochondrial
      assays 

    -
      CD,
      ERR, NMR

    

    Budget:                  
      $150,000

    

    Personnel:              
      K
      Bamham:
      Project Leader 

                                     
      G.
      Kocak

    

    
      
        
        

      

      
        7

        
          

        

      

      
        
        

      

    

    Project
      2. Research Project: 'Drug Screening &
Development'

     

    General
      Aims :  To
      screen
      and characterize Prana compounds in models of (i) AD and (ii) PD related
      pathology and to develop new

                                
       screens based on the emerging knowledge of the underlying pathogenic
      mechanisms of these diseases. To characterize

                                 
      AB oligomers.

    

    

    Proposed
      Objectives:

     

    
      	
              ·

            	
              The
                hydrogen peroxide assay. To test the ability of candidate compounds
                to
                inhibit the copper catalysed generation of H2O2 by AB and a-synuclein
                via
                a 96 well format fluorometric
                assay.

            

    

    
      	
              ·

            	
              The
                brain amyloid solubilisation assay (BAS). To test the ability of
                candidate
                compounds to solubilise protein deposits in a sample of human brain
                tissue
                or development of an assay using synthetic AB oligomers to detect
                dissaggregation.

            

    

    
      	
              ·

            	
              Haemolysis
                assay: AB is incubated with red blood cells (RBC) in the presence
                of
                copper. Resultant lysis is assayed by UV absorption spectrometry.
                Candidate compounds are assayed for their ability to inhibit
                haemolysis.

            

    

    
      	
              ·

            	
              Cholesten
                4 assay. Assay development to identify the oxidation of cholesterol
                as a
                consequence of AB toxicity.

            

    

    
      	
              ·

            	
              Membrane
                perturbation assay. The insertion of protein into artificial lipid
                micelles is associated with pore formation. Candidate compounds will
                be
                assessed for their ability to inhibit pore
                generation.

            

    

    
      	
              ·

            	
              Acute
                toxicity assay for AD and PD. Incremental doses of a drug candidiate
                which
                has passed through the in
                vitro efficacy
                and toxicity screens are administered to mice to establish a tolerable
                dose range for in-vivo
                animal
                trials.

            

    

    
      	
              ·

            	
              Whole
                mouse plasma pharmocology assessment for PD and AD test
                agents.

            

    

    
      	
              ·

            	
              Animal
                trials. AD and PD Drug candidates to be administered to a cohort
                of the
                appropriate animal model for each disease. At completion of the trial,
                brain and other tissues may be collected and assayed for Target Protein
                content, changes to Target Protein and other markers, including
                metals.

            

    

    
      	
              ·

            	
              Characterisation
                of endogenous AB oligomeric species, incl. ADDLs and their role in
                AD
                pathology.

            

    

     

    Budget:              
      $300,000

    

    Personnel:          R.
      Cherny: Project Leader 

                                
      D.Carringtom,
      I. Volitakis

    
      
        
        

      

      
        8

        
          

        

      

      
        
        

      

    

     

    Project
      3. Research Project: ‘Cell Based Drug Discovery'

    

    General
      Aims:            
To
      screen
      and characterize Prana compounds in
      cell
      based systems that measure toxicity and cellular dysfunction

                                           
      associated with (i) AD & (ii) PD pathology and to develop new screens based
      on the emerging knowledge of the

                                            underlying
      pathogenic mechanisms of these diseases.

    Proposed
      Objectives:

     

    
      	
              ·

            	
              The
                screening/testing of therapeutic or diagnostic agents in cell based
                assays
                for (i) AD and (ii) PD that measure toxicity and cellular dysfunction.
                These assays will measure the agent's cellular
                toxicity

            

    

    
      	
              ·

            	
              Develop
                a PD-specific cell-based toxicity assay to screen
                for:

            

    

    -
      compounds which moderate ROS production,

    -
      to
      detect inhibition of toxicity otherwise induced by 6-OHDa +Dopamine

    +test
      agent

    -
      neuroprotection effects.

    
      	
              ·

            	
              The
                screening/testing of therapeutic or diagnostic agents in cell based
                AD
                assays that measure their ability to inhibit metal mediated oxidative
                stress interactions of AB, the ability to modulate cellular survival,
                the
                ability to modulate cellular survival following a toxic
                insult/stress.

            

    

    
      	
              ·

            	
              The
                screening/testing of therapeutic or diagnostic agents in cell based
                assays
                that measure the cell penetration and cell transport properties of
                the
                agents. These assays will provide a measure of the pharmacokinetic
                properties of the agents. The CaCo2 model and Menkes
                model.

            

    

    
      	
              ·

            	
              Screening/testing
                for toxicity induced by 6OHDa and Dopamine with PD test agents.
                

            

    

    
      	
              ·

            	
              Screening/testing
                of agents to determine effect on APP
                processing.

            

    

     

    Budget:  
            $150,000

    Personnel:   
      R.
      Cappai: Project Leader 

                          
      G.Kocak

    
      
        
        

      

      
        9

        
          

        

      

      
        
        

      

    

     

    Project
      4. Research Project: 'APP Metal Binding'

     

    General
      Aims:            
Adoption
      of an in
      silico screening
      methodology based on the NMR structure of the copper binding domain on

                                           
      APP to investigate this domain as an AD drug target.

    

    Proposed
      Objectives:

    
      	
              ·

            	
              Section
                of top ranked in
                silico screened
                putative binders to CuBD of APP. 

            

    

    
      	
              ·

            	
              Development
                of an in
                vitro fluorescence
                binding assay.

            

    

    
      	
              ·

            	
              Development
                of a secondary cell based assay to identify test agent(s) agonist
                activity. 

            

    

    
      	
              ·

            	
              Coordination
                of possible decision to undertake crystal structure analysis and
                medicinal
                chemistry.

            

    

     

    Budget:         
      $12,000*.

    Personnel:     
      R.
      Cappai
      and K.Barnham (Projects leaders),

                            
      Prana personnel: G Kok 

                            
      [SVIMR:
      M. Parker, G.
      Kong]

     

    *(Budget
      not inclusive of Prana payments to SVIMR in
      silico screening
      and crystal structure and Prana Medicinal Chemistry program).

    
      
        
        

      

      
        10

        
          

        

      

      
        
        

      

    

     

    Project
      5. Research Project: 'Blood Brain Barrier Studies'

     

    General
      Aims:        To
      assess
      the ability of new Prana test compounds to pass the Blood Brain Barrier in
      a rat
      model.

    

    Proposed
      Objectives:

    
      	
              ·

            	
              Work
                undertaken by M. Habgood on a "needs be” basis in the Department of
                Pharmacology as directed by Prana.

            

    

    
      	
              ·

            	
              Assay
                with a short duration direct perfusion of the rat brain with compounds
                at
                nominal 50uM concentrations in PBS buffered saline. Brain tissue
                sample
                collection and analysis of brain tissue
                uptake.

            

    

     

    Budget            
      Upto
      $24,000

    Personnel:      
      M.
      Habgood Dept. Pharmacology. Liasing with E. Gautier of Prana

    
      
        
        

      

      
        11

        
          

        

      

      
        
        

      

    

    

    Project
      6. Research Project: 'Amyloid AB imaging Iigands: a high throughput
      screen 

    approach
      - development of screening assays'

    
      
        

      

    

    General
      Aim:

    -Development
      of two assays used for HTS and medium throughput screen (MTS) to identify
      selective AB binding molecules for the imaging of Alzheimer's Disease.

    -In
      vivo
      imaging of AB in the brain for diagnosis and monitoring of therapy.

    
      
        

      

    

    Proposed
      Objectives: Research
      Plan for the coming 6 months: 

     

    A)
      High throughput screen (Melb.
      Uni., SAG)

    Two
      different assays will be evaluated for high throughput screening. Both assays
      are competition assays with fluorescent dye labeled AB1-42
      as
      ligand. One assay will use AB1-42
      synthetic plaque as target (developed by Melb. Uni, assay 1) and the other
      assay
      will use soluble oligomers of AB1-42
      as
      target (SAG, assay 2). Fluorescence Anisotropy (FP) will be use to detect the
      signal in both assays. Decision criteria for assay selection will be feasibility
      (costs per well), reproducibility, and stability under high throughput
      conditions (96 or 386 well format). If both assays fulfil these minimal
      HTS-decision criteria then HTS will be carried out with assay 2, because
      oligomers reflect the human situation in early stage AD better than synthetic
      plaques. Both assays will be transferred to Assay Development (SAG) for the
      assessment of the HTS suitability 3 month after project start. Decision will
      be
      taken 6 month after project start.

     

    Assay
      1:Competition
      assay with fluorescent labeled AB1-42
      on
      synthetic AB1-42
      plaques
      (conducted by Melb. Uni)

    
      	 	
              > 

            	
              Define
                condition for plaque formation to get as close as possible to the
                human
                situation (Zn-concentration, pH)

            

      	 	>	Determine the concentration of AB-binding sites
              in the
              plaque

      	 	> 	Assay:

    
      	 	1.	Formation
              of the AB1-42-plaques

      	 	2.	Fixation
              of the AB1-42-plaque
              to the well

      	 	3.	Incubation
              of compound with the AB1-42-plaque

      	 	
              4.

            	
              Incubation
                of Dye- AB1-42
                at
                the same time as the compound or after the incubation with the
                compound

            

      	 	5.	Detection
              via FP or fluorescence

    

    >  
      Positive
      control: unlabeled AB1-42

    >  
      Negative
      control: random aB1-42

    >  
      Transfer
      assay to SAG

    Assay
      2:Competition
      assay with fluorescent labeled AB1-42
      on
      soluble AB1-42
      oligomers (SAG) 

    
      
                       
          >   Define
          condition for formation of oligemer to get as close as possible to the
          human
          situation

      

    

    >   Determine
      the concentration of AB-binding sites at the oligomers

    >  
      Assay:

    1.  
      Formation
      of the AB1-42-oligomers

    2.   Incubation
      of compound with the AB1-42-oligomers

    3.  
      Incubation
      of Dye- AB1-42
      at the
      same time as the compound or after the incubation with the compound

    4.  
      Detection
      via FP

    >  
      Positive
      control: unlabeled AB1-42

    >  
      Negative
      control: random AB1-42

     

    B)
      Medium throughput screen (conducted by Melb. University)

    Medium
      throughput screen (MTS) will be carried out using a competition assay with
      S-35
      labeled AB1-42 on
      human
      late stage AD homogenates.

    >  
      Assay
      will be developed by Melb. Uni. and transferred to SAG (6
      month).

    
      
        
        

      

      
        12

        
          

        

      

      
        
        

      

    

     

    >  
      MTS
      will
      be carried out by SAG.

    >  
      Human
      late stage AD brain tissue samples will be provided by Melb. Uni. 

    >  
      S-35
      AB1-42
      will be
      produced by Melb. Uni. and delivered to SAG.

    >  
      Assay:

    1.  
      Incubate
      compound with the homogenate.

    2.  
      Incubation
      of S-35- AB1-42 at
      the
      same time as the compound or after the incubation
      with the compound.

    3.  
      Separate
      unbound from bound S-35 AB1-42
      by
      filtration.

    4.  
      Detection
      via radioactivity

    >  
      Positive
      control: unlabeled AB1-42

    >  
      Negative
      control: random AB1-42

     

      
        

      

    

    Milestones
      (month after project start):

     

    >   
      HTS
      assay
      development 3 month

    >   
      HTS
      assay
      transfer to assay development SAG 3 month

    >   
      MTS
      assay
      development 6 month

    >   
      MTS
      assay
      transfer to SAG 6 month

    >   
      Delivery
      of human late stage AD tissue sample (400 mg) and S-35 AB1-42
      12
      month

     

      

    

    Critical
      Issues:

     

    >   
      Difficulties
      in AB1-42
      handling
      -> shift to AB1-40

    >   
      Low
      signal to noise ratio in the MTS assay

    
      
        >   
          Set
          up
          stable and reproducible high throughput screen within 6 month. Identified
          hits
          may be not suitable for radio labeling/imaging.

      

    

    
      
        >   
          Legal
          issues regarding the use of homogenates from AD patients for drug
          characterization to be clarified by Melb.University.

      

    

     

      
        

      

    

    No
      -Go
      Criteria :

     

    No
      suitable HTS assay after 9 month. 

    No
      suitable MTS assay after 12 month.

     

    
      
        

      

    

     

    
      Budget:

    

    Personnel:
      R Cherny (Project Leader), [Sabine Krause (from Schering AG)]

    

    
      
        
        

      

      
        13

        
          

        

      

      
        
        

      

    

    

    Project
      7. Research Project: 'Expression profiling of AD imaging
      targets: 

    

    General
      Aims:

               

    
      	 	
              ·

            	
              To
                identify molecules which are differentially expressed in AD brains
                

            

    

    to
      use
      as novel AD specific imaging targets

    
      	 	
              ·

            	
              In
                vivo imaging of AB in the brain for diagnosis and monitoring of
                therapy.

            

    

       

    Proposed
      Objectives: Research
      Plan for the coming 24 months:

    

    The
      experimental plan is to use gene chip technology to identify gene(s) which
      are
      differentially expressed in AD. To come up with highly selective diagnostic
      markers different experimental settings reflecting different pathology relevant
      factors will be used and will allow a pathology driven selection process via
      calculation of the affymetrix data set.

    

    Resources
      (for the first research year):

    Melb.
      Uni: Tissue supply, Pathological characterisation, collection of patient data,
      animal model, 

    Validation
      by immunohistochemistry (0.5)

    

    [SAG:
      RNA
      extraction, real time PCR, Affymetrix analysis, Data calculation, Validation
      by

    immunohistochemistry
      (FTE to be allocated after project proposal approval)

    

    Experimental
      settings overview: Human patient samples

    ·     
      Disease
      vs non disease

    ·     
      Dementia
      AD type vs dementia non AD type (Lewy Body Dementia, fronto temporal
      dementia)

    ·     
      Brain
      vessels isolation will allow analysis of gene expression in vessel wall
      endothelial cells

    ·     
      MMSE
      (mini-mental state examination) will be taken and used for data
      calculation/correlation if available

    
      
        ·     
          Plaque
          load, soluble and insoluble AB fraction and NFT (neurofibrillary tangles)
          will
          be determined and used for data 

               
          calculation/correlation

      

    

    

    AD
      tissue
      will not only be compared versus control brain tissue but also versus tissue
      from other neurological diseases especially other dementia's to allow
      differential diagnosis. For each experimental settings 6 individual probes
      will
      be analysed, each on its own gene chip, to allow statistical evaluation of
      the
      results which is the absolute must in Affymetrix studies. In addition to RNA
      extraction from total brain tissue also brain vessels will be isolated to allow
      the analysis of differential expression in vessel endothelial cells. This is
      of
      especially importance because: 

    >
      Amyloid
      deposits in the vessel wall is one major pathological finding in AD

    >
      Significant differential expression in brain vessel endothelial cells are
      probably not visible without prior purification of the vessel wall

      
      because brain vessels represent less then 1 % of the total brain tissue and
      thus
      the signal will be dramatically diluted.

    >
      Cell
      surface proteins on the brain vessel wall are very attractive targets for brain
      imaging (no BBB)

     

    Experimental
      setting in detail: 

     

    Tissue
      probes

    

    Disease
      versus non disease

    Human
      brain tissue will be taken from

    >   
      non
      demented aged control; n=6

    >   
      AD
      dementia n=18 (6 early, 6 medium and 6 late disease stages)

    >   
      Non
      AD
      dementia n=9 (2 Lewy Body Demintia and 7 Fronto temporal
      demintia)

    
      
        
        

      

      
        14

        
          

        

      

      
        
        

      

    

    Affected
      area

    Different
      affected or non affected brain areas will be chosen: 

    >  
      frontal
      cortex (FL)

    >  
      temporal
      cortex (TL)

     

    Number
      of
      chips: (6+18+9) x 2 (FL/TL)= 66

    

    Brain
      parachym versus brain vessels

    From
      the
      non demented aged controls and the AD cases also brain vessels will be purified
      for RNA extraction

    >  
      non
      demented aged control; n=6

    >  
      AD
      dementia n=18 (6 early, 6 medium and 6 late disease
      stages)

    

    Number
      of
      chips: (6+18) x 2 (FL/TL)= 48 

     

    Total
      number of chips: 48 +
      66
      = 114

     

    For
      the
      human tissue the following assessments will be taken MMSE, Amyloid AB load
      (Plaque, soluble and insoluble AB) and NFT pathology.

    

    Data
      calculation and selection criteria

    Using
      all
      expression and patient information data, relevant AD imaging targets will be
      selected based on the following selection criteria:

    ·     
      Relevance
      to disease -> expressed only in AD

    ·     
      Relevance
      to phenotypic pathology -> correlation with amyloid AB load and/or NFT
      load

    ·     
      Relevance
      to functional outcome -> correlation with MMSE (early targets may not fit
      this criteria)

    
      
        ·     
          The
          differential expressed molecule has to have the potential as diagnostic
          marker
          (e.g. receptor, cell surface protein)

      

    

    

    Before
      starting the Affymetrix Experiment the quality of RNA extracted from AD post
      mortem tissue will be checked by PCR and a pilot Affymetrix experiment using
      4
      tissue probes.

    

    Data
      validation

     

    Selected
      targets will then be validated using real time PCR to get the real quantitative
      picture. Thereafter the differential expression of relevant AD imaging targets
      will be further validated on the protein and structural level by
      immunohistochemistry (depending on availability of antibodies) and in situ
      hybridisation using AD brain slices

    
      

    

    Milestones
      (times after month after start of project)

     

    >     
      Human
      Tissue sampling, collection of patient data and analysis of pathological
      parameter 4 month

    >     
      RNA
      extraction and quality control 6 month

    >     
      Affymetrix
      12 month

    >     
      Data
      calculation 13 month

    >     
      Validation
      of differential expressed targets 24 month

    

    Critical
      Issues :

    Get
      access to human tissue with appropriate post mortem time and thus RNA quality.
      

    Get
      capacity to perform Affymetrix analysis at Schering.

    Legal
      issues regarding the use of homogenates from AD patients for drug
      characterisation has to be clarified by Melb. University.

    
      
        
        

      

      
        15

        
          

        

      

      
        
        

      

    

    

    
      
        

      

    

    No
      -Go Criteria :

    Quality
      of human tissue does not allow extraction of intact RNA after 9 month

    Do
      not
      get necessary Affymetrix capacity to perform statistics.

    
      
        

      

    Budget

    Personnel:
      Colin Masters (Project Leader), [Thomas Dyrks (Schering AG)]

     

    
      
        
        

      

      
        16

        
          

        

      

      
        
        

      

    

    Project
      8. Research Project: 'Amyloid AB monoclonal antibodies as imaglnq
      ligands'.

    

    
      	
              General
                Aim:

            	
              -
                To develop and test radiolabelled Amyloid specific antibodies as
                AD
                imaging agents.

            

      	 	
              -
                In vivo imaging of AB in the brain for diagnosis and monitoring of
                therapy.

            

       

        

      

    

    Proposed
      objectives: Research
      Plan for the coming 15 months:

    

    >  
      Test
      already available monoclonal antibodies with regard to Amyloid AB
      pathology. 

    >   Set
      up
      strategies regarding PK questions: Antibody fragments, BBB-carrier
      systems

    >  
      Label
      antibody for proof of concept study in tg mice(biotin or fluorescent label
      may
      be chosen if 

       
 Antibody
      has to be send over to Melb. Uni.)

    >  
      Make
      proof of concept study with one selected already existing high affinity Antibody
      (anti AB42) using TG mice.

    

    Resources
      (for the first research year): 

     

    Melb.
      University: supply of human AD tissue, tissue of non-AD amyloidosis, reference
      tissue from healthy persons, for immunohistochemistry, production of paraffin
      sections of human AD tissue, supply of datab characterizing the pathologic
      material. SAG:
      Generation and labeling of antibodies, pathological characterization of
      monoclonal antibodies, antibody modification with regard to penetration of
      BBB. 

    
      

    

     

    Organizations
      aspects: Input from Melb.
      University
      

    Delivery
      of human patient tissue for Immunohistochemistry. 

    First
      delivery: June 2004

    

    >
      Tissue
      selection:

     

      
        

      

    

    · 
early,
      medium and late AD

    · 
non-AD
      amyloidosis (Lewy Body dementia, fronto temporal dementia)

    · 
healthy

    
      
        

      

    

    Milestones
      (month after project start)

    

    >
      PK
      strategy: 3 month

    >
      Antibody
      label: 6 month

    >
      Proof
      of
      concept with whole Antibody 9 month

    >
      Characterisation
      of already existing monoclonal Ab: 9 month

    >
      Development
      of new strategies: other Ab, Ab-fragment/ modified Ab 15 month

    >
      Label
      and
      proof of concept study with next generation antibodies (fragments/modified)
      24
      month

    

    Critical
      Issues :

    Legal
      issues regarding the use of brain tissue from AD patients for drug
      characterisation has to be clarified by Melbourne University (July
      04)

    
      
        

      

    

    No
      -Go
      Criteria :

    No
      proof
      of concept with existing Ab after 12 months.

    
      
        

      

    

     

      
        

      

    

     

    Budget:

    Personnel:
      Collin Masters, [Andrea Lippoldt (Schering AG)]

    

    
      
        
        

      

      
        17

        
          

        

      

      
        
        

      

    

    Project
      9. Research Project: 'AB binding proteins'

    

    

    General
      Aim:

    To
      identify proteins which bind to AB as novel AB imaging targets.

     

      
        

      

    

     

    
      

    

    

    Proposed
      Objectives: [Research
      Plan for the coming 6 months):

    

    The
      experimental plan is to identify specific brain proteins that bind to AB and
      not
      to control peptides and fulfil the criteria as drugable targets.

     

    Resources
      (for the first research year):

    Melb.
      Uni: Affinity isolation and identification of AB binding proteins.

    

    SAG:
      Assistance in
      proteomics
      for identification of AB binding proteins. 

     

    1.     
      Isolation
      of AB-complexes from human brain.

    

    1.1   
      AB-affinity
      capture of AB-complexes from human brain: AB will be coupled to Tosyl-activated
      magnetic beads and used to affinity purify AB binding proteins from brain
      homogenates. Bound proteins are separated by 2D-electrophoresis Control peptides
      will be coupled to the Tosyl-activated magnetic beads to determine specific
      AB
      binding proteins. Control peptides will include AB scrambled, AB-reverse,
      a-synuclein and/or Prion peptide 106-126. If specific spots (unique to the
      AB)
      are obtained they will be identified by mass spectrometry.

    

    1.2   
      AB-TAP
      protein affinity purification: An AB tandem affinity purification (TAP) tag
      will
      be used to affinity purify AB binding proteins from brain homogenates. A control
      TAP fusion protein will be used to determine specific AB binding proteins.
      The
      TAP controls will be TAP alone and a TAP-a-synuclein fusion protein. If specific
      spots (unique to the AB-TAP) are obtained they will be identified by mass
      spectrometry.

     

    2.    
      Verification
      of AB binding proteins.

    

    2.1   In
      vitro
      binding assay: In vitro binding of different AB peptides to recombinantly
      expressed candidate binding protein as measured by gel filtration or
      co-immunoprecipitation or pull-down assay or BiaCore.

    

    3.   
      Validation
      of AB binding proteins

    Immunohistochemistry
      to be performed against selected targets at the protein level by
      on AD
      and non-AD control brain slices to determine expression levels in AD vs
non-AD
      controls.

     

    
 

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    

    Data
      calculation and selection criteria

    Relevant
      AD imaging targets will be selected based on the following selection
      criteria:

    · 
Relevance
      to disease -> increased expression in AD

    · 
Relevance
      to affected area -> expressed only in affected brain area

    · 
Relevance
      to phenotypic pathology -> correlation with amyloid and/or NFT
      load

    · 
The
      AB
      binding molecule has to have the potential as diagnostic marker (e.g.
receptor,
      cell surface protein)

    

    Data
      validation

    Selected
      targets will then be validated as a relevant AD imaging target at the protein
      level by immunohistochemistry using AD brain slices (depending on availability
      of antibodies).

    

    Organisational
      aspects

    · 
Study
      to
      be performed by Melb. Uni.

    · 
Schering
      AG to assist if required with protein identification

    
      
        

      

    Milestones
      (times after month after start of project)

     

    1°
      milestone: Identification of specific AB-binding proteins which are unique
      in
      either the AB-affinity assay or the AB-TAP assay as compared to the control
      peptides on 2D-gels. Unique spots to be identified
      by mass spectrometry. (11/2004)).

    

    2°milestone:
      Verification (in vitro) of binding partner (12 month)

    

    3°
      milestone: Verification that binding protein binds to AB in tissue (24
      month)

    
      

    

    Critical
      Issues :

     

    Confirming
      that proposed AB binding proteins are physiologically/pathologically
      relevant.

    
      

    

    No
      -Go
      Criteria :

     

    No
      identified and verified (in vitro) binding partner by 11/2004

    Verification
      that the AB binding protein does not bind to AB in tissue (24
      month)

    
      
 

    

    

    Budget:

    Personnel:
      Roberto Cappai, [Thomas Dyrks (Schering AG)]

     

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    
 

    Project
      10. Research
      Project: ‘Amyloid imaging with PBT-1'

    

    General
      Aim:  - To test radiolabelled PBT1 as AD imaging agents.

                           
      -  In vivo imaging of AB in the brain for diagnosis and monitoring of
      therapy.

     

    
      

    

    
      

    

     

    Proposed
      Objectives:

    Research
      Plan for the coming 18 months:

    >  
      The
      experimental plan is to fully characterize radiolabeled PBT-1 (Clioquinol)
      binding to A[]. 4 month

    >  
      Radiotracers
      will be screened as A[] binding tracers through in vitro assays to determinate
      binding parameters (Kd, Bmax) 

        
      of high specific activity 125l-PBT1
      to
      synthetic A[] amyloid aggregates

    >  
      Biodistribution
      and subsequent ex vivo autoradiographic studies in tg and non tg mice,
      with and without competitor

         compounds
      (DTPA, Congo red, ThT).

    >  
      The
      competitor compound ThT will be used to evaluate the tg animal models.

    >  
      In
      vivo
      human SPECT studies with 125l-PBT1

    >  
      Based
      on
      full characterization of PBT1.

    

    Resources
      (for the first research year):

    Melb.
      Uni: Proof of concept study with A[] fibers, tg mice, and human SPECT
      studies.

    

    1. In
      vitro
      binding assays

    1.1  
      Synthetic A[] amyloid aggregates will be added to a mixture containing
      0.01-5

    pM
      of
125l-PBT1
      (2000 Ci/mmol) in buffer Tris-HCI (pH 7.35; final volume of 1

    ml).
      The
      final concentration of EtOH will be 10%. Nonspecific binding will be defined
      in
      the presence of 2 uM PBT1.

    1.2  
      Selectivity studies will be determined in the presence of different competitors
      (ThT, C red, DTPA, Zn(ll) and glycine). Ki value will be estimated for each
      competitor. The competitor concentration range will be ± 3 order of magnitude
      the concentration of 125I-PBT1.

    2. Ex
      vivo
      biodistribution studies

    2.1. 
      Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 125l-PBT1
      (1
      mCl). The animals will be sacrificed at 5, 15, 30, 60 and 120 min, and the
      organs will be dissected, weighted and each organ counted. Blood and urine
      samples will be also collected. The percentage of radioactivity in each organ
      (%ID/g) will be calculated.

    2.2  
      To estimate the specificity and saturability of the brain-uptake of 125l-PBT1,
      mice will be administered i.p. with cold PBT1, C red, DTPA, glycine,
      Thioflavin-t 30-60 min previous to 125l-PBT1
      i,v. injection.

    3. Autoradiographic
      studies

    3.1 
      In vitro autoradiography.

    3.1.1.
      Unfixed brain coronal slices of AD and AC brain tissue, as well as Tg2576 (15-20
      mm) and non-Tg mice (15-20 mm), will be adsorbed to gelatin-coated coverslip.
      The samples will be incubated with 125l-PBT1
      (100,000 cpm) in buffer Tris-HCI (pH 7.35) containing 10% EtOH for 1 h at 20°C.
      Samples will be submitted to 3 washing steps with the same buffer. The samples
      will be dried to 20° C for 2h. The regional distribution of 125l-PBT1
      will be analysed qualitatively by autoradiography using

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    autoradiographic
      films. Quantification of 125l-PBT1
      positive binding will be performed using radioactive strips
      standards.

    3.1.2.
      Competition studies will be performed in the presence of cold PBT1, DTPA, ThT
      glycine and Congo Red (1,000 times excess).

    3.1.3.
      To
      analyse co-localization of 125l-PBT1
      binding amyloid deposits, adjacent slices will be stained for amyloid with
      either Congo red, Thioflavin T or A[] Immunohistochemistry.

    3.2  
      Ex vivo autoradiography

    At
      the
      best time from TAC kinetics (5-15 mpi), brain coronal slides from Tg2576 and
      non-Tg mice i.v. injected with 125I-PBT1
      will be exposed to autoradiography film. Experiments will be performed as
      above.

    4. Radiometal
      binding.

    4.1.
       Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 64Cu.
      The animals
      will be sacrificed at 5 min and the organs will be

           
      dissected, weighted and
      each
      organ counted. Blood and urine samples will be also collected. The
      percentage of radioactivity in 

           
      each organ (%lD/g) will be calculate

    4.2. 
      Tg2576 (n=3) and non-Tg (n=3) mice will be injected i.v. with 64Cu-PBT1.
      The
      animals will be sacrificed at 5 min and the organs 

           
      will be dissected, weighted
      and each organ counted. Blood and urine samples will be also collected.
      The percentage of 

           
      radioactivity in each organ (%ID/g) will be calculated.

       
      4.3.  Comparison of results Prom. Radiometal binding with and without
      chelator transporter.

     

    5.
      SPECT
      studies

    Ten
      AD
      patients will be recruited to undergo 123l-PBT1
      SPECT brain imaging. The scans obtained will be compared with those of ten
      healthy, age-matched volunteers.

    The
      scans
      will undergo blinded analysis by two experienced nuclear medicine specialists
      who will assign each subject's scan to either a "diseased" or "normal" condition
      on the basis of visual analysis, normalized brain radioactivity (SUV) and
      kinetic analysis of cortical and subcortical regions of interest (ROI).
      Compartmental and graphical analysis using the cerebellum as reference region
      will be used.

    Formal
      whole-body radiation dosimetry calculations for 123l-PBT1
      will be performed on three of the control subjects.

    

    Organizations
      aspects: Input from Melb. Uni.

    Studies
      to be performed by Melb. Uni.

    Characterization
      of 123/125l-PBT1.

       
      Proof of concept study using tg mice. Biodistribution, In vivo Autoradiography
      /
      immunohistochemistry

    Proof
      of
      concept study in humans SPECT studies with 123I-PBT1

    Schering
      AG to assist if required with radiolabelling of BTA-1.

     

     

    
      
        

      

    

    Milestones
      (month after project start)

    >

    > 
       In
      vitro
      characterization PBT1 (completed):

    >  
      Ex
      vivo
      characterization PBT1 (ongoing): 1 month

    >  
      Ex
      vivo
      autoradiographic studies PBT1 (ongoing): 2 month

    >  
      Ex
      vivo
      characterization 64Cu
      and
64Cu-PBT1:
      3 month 

    >  
      Ex
      vivo
      autoradiographic studies BTA-1: 6 month

    >  
      Proof
      of
      concept with human SPECT studies (n=20) 12 month

    

    No
      -Go
      Criteria :

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    If
      IP
      situation regarding PBT1 and analogues thereof can not be solved after 4 month
      preclinical work will be closed. 08/2004

    Failure
      of proof of concept study with human SPECT after 12 month.
      (12/2004)

    
      
 

    Current
      Project Status:

    
      
 

    >  
      In
      vitro
      characterization PBT1 (ongoing)

    >  
      Ex
      vivo
      characterization PBT1 (ongoing)

    >  
      Autoradiographic
      studies PBT1 (ongoing)

    >  
      Proof
      of
      concept with human SPECT studies (n=20)

    

    
      

    

    Lead
      criteria:

    1.  Proven
      pharmacological efficacy in secondary in vitro (micro autoradiography
study
      using AD brain slices; potency 1-100nM)

    2.  Proven
      accumulation in AD plaques as determined by ex-vivo autoradiography after
      i.v. injection of I-125-PBT-1 into tg mice

    3.  Proven
      suitable pharmacokinetic properties (stability, plasma/brain level)

    4.  Compatible
      with PET/SPECT radiochemistry

    5.  Clear
      patent strategy

    6.  Primary
      structure binding relationship data

    

    

    

    Budget:

    Personnel:
      Victor L. Villemagne, R. Cherny, (Project leaders) [Matthias Friebe (Schering
      AG))

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    

    Project
      11. Research Project: 'AP binding metallocomolexes'

    

    

    General
      Aim:

    To
      test
      radiolabelled metallocomplexes as AD imaging agents. In vivo imaging of AB
      in
      the brain for diagnosis and monitoring of therapy.

    
      
 

    Proposed
      Objectives:                
[Research
      Plan for the coming 24 months]:

    The
      experimental plan is to identify, synthesize and characterize metallocomplexes
      that bind to A[] in vivo.

    

    Initial
      proof of concept (4 months):

    >   
      Synthesize a Pt(II) complex of a 1,10-phenanthroline derivative (Rphen) ligand
      suitable for radioiodination (Melb. Uni)

    >   
      Screen the cold metallocomplex in-vitro for binding affinity to ABfibers (Melb.
      Uni) 

    >   
      Derive procedures to radiolabel the Pt(Rphen)Cl2
      complex
      with l-125 (SAG) 

    >   
      Perform in-vitro autoradiography with 1-125-Pt(Rphen)Cl2
      on AD
      slices (Melb. Uni)

    

    Resources
      (for the first research year):

    Melb.
      Uni.: Chemistry and intial screening with AB fibers,

    SAG:
      Radiolabelling of selected metallocomplexes, toxicity screening

    
      

    

     

    General
      research plan (24 months):

    >  
      Candidate metallocomplexes will be synthesized and characterized (MS, NMR,
      HPLC)

    >  
      Candidate metallocomplexes will be screened as AB binding tracers through in
      vitro assays with AB fibers

    >  
      Selected metallocomplexes will be assessed for bioavailability: a) in vitro
      uptake in CaCo 2 cells; b) in vivo brain uptake in

         non-transgenic
      mice as determined by ICP-MS

    >   Selected
      metallodrugs will be assessed for radiolabeling potential (SAG)

    >  
      Radiolabelled metallocomplexes will be evaluated to ensure that they display
      similar
      binding profile (affinity, selectivity) as

        
      the cold
      complexes using the in vitro assays
      with AB fibers

    >  
      Ex
      vivo
      tissue binding, binding assays, dialysis studies, and autoradiography studies
      in
      tg and non tg mice, with and

         
      without competitors (DTPA, Congo red, ThT), and human AD/AC brain
      slices.

    >  
      Toxicity
      screen (SAG)

    

    Organizations
      aspects: Input from Melb. Uni.

    Synthetic
      chemistry and characterization of AB binding metallocomplexes. Bioavailability
      studies. Proof of concept study using AB fibers

     

    
      

    

    Milestones
      (month after project start)

     

    >  
      Proof
      of
      concept in vitro with l-125 Pt(Rphen)Cl2:
      4
      months

    >  
      Identification,
      synthesis, and characterization of 30 compounds: 12 months 

    >  
      Bioavailability
      evaluation (SAR): 5-24 months

    >  
      In
      vitro
      characterization (10-20/year): 7-24 months

    >  
      Ex
      vivo
      characterization (6-10/year): 9-24 months

    
      
        
        

      

      
        
        

        
          

        

      

      
        
        

      

    

    

    >  
      Radiolabeling:
      7-24 months

    >  
      Autoradiographic
      studies: 15-24 months 

    >  
      Toxicity
      screening: 9-24 months

    >  
      Proof
      of
      concept with human PET studies: > 24 months 

     

    No
      -Go
      Criteria :

    Failure
      of l-125 Pt(Rphen)Cl2
      proof of
      concept in-vitro after 4 months

    Failure
      to show binding of new metallocompiex to amyloid pathology in tg mice after
      20
      months

    
      
 

    Lead
      criteria:

    1  
      Proven pharmacological efficacy in secondary in vitro (micro autoradiography
      study using AD brain slices; potency 1-100nM)

    2  
      Proven suitable pharmacokinitic properties (stability, plasma/brain
      level)

    3  
      Compatible with PET/SPECT radiochemistry

    4  
      Clear patent strategy

    5  
      Primary structure binding relationship data

    

    

    Budget:

    Personnel:
      Kevin Bamham, John Cyr (Schering AG)

Source: [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00110-of-00352.parquet"}, [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00110-of-00352.parquet"}]]