B850A 11 50 AM Pg 1 Form 990 OMB No 1545-0047 Return of Organization Exempt From Income Tax 2004 Under section 501(c), 527, or 4947(a)(1) of the Internal Revenue Code (except black lung Open to Ic Department of the Treasury benefit trust or private foundation) Internal Revenue Service ► The oraanization may have to use a coot/ of this return to satisfy state reporting requirements hSP A For the 2004 calendar ear, or tax year beginning and ending B Check if applicable Please C Name of organization D Employer Identification no. use IRS Address change label or 59-1746396 Name change print or LIFE EXTENSION FOUNDATION, INC E Telephone number Initial return type. Number and street (or P O box if mail is not delivered to street address) Room/suite Final return See 3107 STIRLING ROAD 105 F Accounting method: Cash Specifl City or town, state or country, and ZIP + 4 a Accrual Other (specify) Amended return Instruc- Application pendin FT. LAUDERDALE FL 33312 ► .Section 501(c)(3) organizations and 4947(a)(1) nonexempt charitable H and I are not applicable to section 527 organizations trusts must attach a completed Schedule A (Form 990 or 990-EZ). H(a ) Is this a group return for affiliates? 11 Yes XX No G Website: ► N/A H(b) If "Yes," enter number of affiliates ► J Organization type H(c) Are all affiliates included? Q Yes D No check only one) ► IXI 501(c) ( 3 ) s (insert no) I 14947(a)(1) or 1 1 527 (If "No," att a list See mstr ) K Check here ► U if the organization's gross receipts are normally not more than $25,00 H(d) Is this a separate return filed by an The organization need not file a return with the IRS; but if the organization received a organization covered by a group ruling? F] Yes F] No Form 990 Package in the mail, it should file a return without financial data. Some states I Group Exemption Number ' require a complete return. M Check ► X if the organization is not required L Gross receipts: Add lines 6b, 8b, 9b, and 10b to line 12 ► 7,472,982 to attach Sch. B (Form 990, 990-EZ, or 990-PF) Part E Revenue expenses, ana cnan es in Net Assets or tuna Isalances oee page I o or me Instructions. I Contributions , gifts, grants, and similar amounts received. a Direct public support 1a 34 , 147 b Indirect public support lb c Government contributions (grants) Ic d Total (add lin through 1c) (cash $ 34, 147 noncash $ ) 1d 34 , 147 2 Program serv reve vernme ees and contracts (from Part Vll, line 93) 2 3 Membership ue y SEE STATEMENT 3 3,255,612 4 Interest on s sand tempora ry cas Inv ents 4 1,063,004 5 Dividends a h r 'trmme§jrTlJ6 5 53 , 540 6a Gross rents M1 v a 6a 3 4, 3 3 5 b Less : rental exper> EE STATEMENT 6b 24,139 c Net rental incoThe or (loss s'ub nacn fr m line 6a ) 6c 10,196 R 7 Other investment income (describd' 7 v 8a Gross amount from sales of assets other (A) Securities (B) Other de n than inventory 220,099 8a u b Less : cost or other basis and sales expenses 2 5 0 , 0 0 0 8b e c Gain or (loss) (attach schedule) -29,901 8c d Net gain or (loss) (combine line 8c, columns (A) and (B)) SEE STMT 8d -29,901 9 Special events and activities (attach schedule ). If any amount is from gaming, check here ► a Gross revenue (not including $ of contributions reported on line 1a) 9a b Less: direct expenses other than fundraising expenses 9b c Net income or (loss) from special events (subtract line 9b from line 9a) 9c 10a Gross sales of inventory, less returns and allowances 10a b Less, cost of goods sold 10b c Gross profit or (loss ) from sales of inventory (attach schedule )(subtract line 1Ob from line 10a) 10c 11 Other revenue (from Part VII, line 103 ) 11 2,812,245 12 Total revenue (add lines 1d 2, 3 4 5 6c, 7, 8d, 9c, 10c and 11) 12 7,198,843 E 13 Program services (from line 44, column (B)) 13 5,800,945 xp 14 Management and general (from line 44, column (C)) 14 5 8 6 , 9 2 2 15 en 15 Fundraising (from line 44, column (D)) s 16 Payments to affiliates (attach schedule) 16 e s 17 Total expenses (add lines 16 and 44, column (A)) 17 6,387,867 A 18 Excess or (deficit) for the year (subtract line 17 from line 12) 18 810,976 N s 19 Net assets or fund balances at beginning of year (from line 73, column (A)) 19 25,849,438 t Ite 20 Other changes in net assets or fund balances (attach explanation ) SEE STATEMENT 20 7 7 , 6 4 6 s 21 Net assets or fund balances at end of year (combine lines 18, 19, and 20) 21 26,738,060 1-or Privacy Act and Paperwork Keauctlon Act Notice , see the separate Form (2004) instructions. 990 DAA 11 ' FForm990(200004)9 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page2 Part It Statement of All organizations must complete column (A). Columns (B). (C), and (0) are required for section 501(cX3) and (4) organizations Functional Fmenses and s Jinn 4A471 a1/11 nnnaYAmnt chnrilahia fnists hart nntinnni fnr ntharc IRaa nano 99 of the Inctnleflnns Do not include amounts reported on line (B) Program (C) Management Total (D) Fundraising 6b, 8b, 9b, I Ob, or 16 of Part I. (� services and general 22 Grants and allocations (attach schedule) ,SEE. STMT 5 (cash$ 5,800,94 cash 22 5,800,945 5,800,945 23 Specific assistance to Individuals 23 24 Benefits paid to or for members 24 25 Compensation of officers, directors, etc. 25 26 Other salaries and wages 26 27 Pension plan contributions 27 28 Other employee benefits 28 29 Payroll taxes 29 30 Professional fundraising fees 30 31 Accounting fees 31 16 , 3 71 16 , 3 71 32 Legal fees 32 13 8 , 511 13 8 , 511 33 Supplies 33 34 Telephone 34 35 Postage and shipping 35 106,008 106,008 36 Occupancy ,. 36 37 Equipment rental and maintenance 37 38 Printing and publications 38 194,601 194,601 39 Travel 39 40 Conferences, conventions, and meetings 40 41 Interest 41 42 Depreciation, depletion, etc. (attach schedule) 42 26,667 26,667 43 Other expenses not covered above (itemize):a 43a b SEE STATEMENT 6 43b 104,764 104,764 c 43c d 43d e 43e 44 Total functi onal expenses (add lines 22 - 43) Organizati ons completing columns (121)4D), carry these totals to lines 13.15 44 6, 3 87 , 8 6 7 5, 8 0 0 , 9 4 5 586,9221 0 Joint Costs. Check ► U if you are following SOP 98-2. Are any joint costs from a combined educational campaign and fundraising solicitation reported in (B) Program services? ► Yes ❑X No If "Yes," enter (i) the aggregate amount of these joint costs$ (II) the amount allocated to Program services $ (IIi11 the amount allocated to Management and genera $ and (iv) the amount allocated to Fundraising$ Patt IN Statement of Program Service Accomplishments (See page 25 of the instructions.) What is the organization's prima ry exempt purpose? Program Service Expenses ► SEE NOTE A (Required for 501(c)(3) & All organizations must describe their exempt purpose achievements in a clear and concise manner. State the number* (4) orgs , & 4947(a)(1) of clients served, publications issued, etc. Discuss achievements that are not measurable. (Section 501(c)(3) and (4) trusts, but optional for or anizations and 4947(a)(1) nonexempt charitable trusts must also enter the amount of grants and allocations to others others.) a SEE NOTE A ......
(Grants and allocations $ b ...... (Grants and allocations $ c ...... (Grants and allocations $ d ...... (Grants and allocations $ e Other program services attach schedule Grants and allocations $ 5,800,945 5,800,945 f Total of Program Service Expenses (should equal line 44, column (B), Program services) ► 5,800,945 DAA Form 990 (2004) B850A 11.44 AM Pg 1
Form 990 (2004) LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 3 Part IV Balance Sheets (See page 25 of the instructions.)
Note : Where required, attached schedules and amounts within the description (A) (B) column should be for end-of-year amounts only. Beginning of year End of year 45 Cash-non-interest-bearing ...... 2,263,497 45 806,791 46 Savings and temporary cash Investments ...... 46
47a Accounts receivable ...... 47a b Less: allowance for doubtful accounts 47b 47c
48a Pledges receivable ...... 48a b Less- allowance for doubtful accounts 48b 48c 49 Grants receivable 49 50 Receivables from officers, directors, trustees, and key employees A (attach schedule) 50 $ 51a Other notes and loans receivable (attach s schedule) ...... 51a 15, 857, 869 e b Less: allowance for doubtful accounts . 51b 14,250,348 5i c 15,857,869 t 52 Inventories for sale or use ...... 52 s 53 Prepaid expenses and deferred charges , ,, ,, ,,,, , , , , 53 54 Investments-securities SEE STATEMENT ►❑ Cost n FMV 7,909,343 54 8,686,709 55a Investments-land, buildings, and equipment: basis ...... 55a 417,254 b Less: accumulated depreciation (attach schedule) SEE STATEMENT 55b 8 0 , 2 3 9 347,714 55c 337,015 56 Investments-other (attach schedule) ...... SEE STMT...., 450,038 56 450,038 57a Land, buildings, and equipment: basis 57a b Less: accumulated depreciation (attach schedule) 57b 57c 58 Other assets (describe ► SEE STATEMENT ) 643,271 58 6 0 2 , 7 7 4
59 Total assets (add lines 45 through 58) (must equal line 74) 25,864,211 59 26,741,196 L 60 Accounts payable and accrued expenses 8 , 9 8 7 60 61 Grants payable ...... 61 a 62 Deferred revenue ...... 62 b 63 Loans from officers, directors, trustees, and key employees (attach schedule) 63 64a Tax-exempt bond liabilities (attach schedule) 64a t b Mortgages and other notes payable (attach schedule) . 64b e 65 Other liabilities (describe ► SEE STATEMENT ) 5 , 7 8 6 65 3,136 s 66 Total liabilities (add lines 60 through 65) 14 , 7 7 3 66 3,136 Organizations that follow SFAS 117 , check here ► X and complete lines 67 through 69 and lines 73 and 74. NF 67 Unrestricted ...... 25, 849, 438 67 26, 738, 060 e u 68 Temporarily restricted 68 to d 69 Permanently restricted ...... 69 A Organizations that do not follow SFAS 117, check here ► fl and s B complete lines 70 through 74. s a 70 Capital stock, trust principal, or current funds 70 eI or capital surplus, or land, building, and equipment fund 71 t a 71 Paid-in s in 72 Retained earnings, endowment, accumulated Income, or other funds ... 72 c 73 Total net assets or fund balances (add lines 67 through 69 or lines o e 70 through rs 72; column (A) must equal line 19; column (B) must equal line 21) ...... 25,849,438 73 26,738,060 74 Total liabilities and net assets / fund balances add lines 66 and 73 25,864,21i 74 26,741,196 Form 990 is available for public Inspection and, for some people, serves as the primary or sole source of information about a particular organization . How the public perceives an organiza tion in such cases may be determined by the information presented on Its return . Therefore , please make sure the return Is complete and accurate and fully descri bes, In Part III , the organization's programs and accomplishments. DAA B850A 3 25 PM Pg 7 , T LIFE EXTENSION FOUNDATION -1746396 Past LV•A Reconciliation of Revenue per Audited Part 1V-8 Reconciliation of Expenses per Audited Financial Statements with Revenue per Financial Statements with Expenses per N/A Return (See page 27 of the instructions.) N/A Return a Total revenue, gains, and other support a Total expenses and losses per per audited financial statements ► a 0 audited financial statements ► a 0 b Amounts included on line a but not on b Amounts included on line a but not line 12, Form 990 on line 17, Form 990- (1) Net unrealized gains on (1) Donated services and use investments $ of facilities $ (2) Donated services and use (2) Prior year adjustments of facilities $ reported on line 20, (3) Recoveries of prior Form 990 $ year grants $ (3) Losses reported on line 20, (4) Other (specify) Form 990 $ (4) Other (specify)-
Add amounts on lines (1) through (4) ► b $ Add amounts on lines (1) through (4) ► b c Line a minus line b ► c 0 c Line a minus line b ► c 0 d Amounts included on line 12, d Amounts included on line 17, Form 990 but not on line a: Form 990 but not on line a: (1) Investment expenses (1) Investment expenses not included on line not included on line 6b, Form 990 $ 6b, Form 990 $ (2) Other (specify) (2) Other (specify).
$ $ Add amounts on lines (1) and (2) ► d Add amounts on lines (1) and (2) ► d e Total revenue per line 12, Form 990 e Total expenses per line 17, Form 990 (line c plus line d ► e 0 (line c plus line d ► e 0 Part V List of Officers, Directors, Trustees, and Key Employees (List each one even if not compensated, see page 27 of the instructions ) (B) Title and average ( C) Compensation (0) Contrib to (E) Expense (A) Name and address hours per week devoted to ( If not paid, enter plans account and other position _0., ,deferred allowances TINA EYTCHISON DIRECTOR FT. LAUDERDALE, FL 5 HRS.
WILLIAM FALOON DIRECTOR FT. LAUDERDALE, FL. 5 HRS.
SAUL KENT DIRECTOR FT. LAUDERDALE, FL. 5 HRS.
JIM HALPERN DIRECTOR FT. LAUDERDALE, FL. 5 HRS.
KEVIN BROWN DIRECTOR FT. LAUDERDALE, FL. 5 HRS.
PAUL GILNER DIRECTOR FT. LAUDERDALE, FL. 5 HRS.
75 Did any officer , director, trustee, or key employee receive aggregate compensation of more than $100,000 from your organization and all related organizations , of which more than $10 ,000 was provided by the related organizations ? ► Yes %X No If "Yes," attach schedule-see page 28 of the instructions
Form 990 (2004) OAA )OA325PMP9ji . / t m990(2004) ' LIFE EXTENSION FOUNDATION, INC 59-17463 'art VI Other Information (See page 28 of the in structions.) Yes No Did the organization engage in any activity not previously reported to the IRS? If "Yes," attach a detailed description of each activity 76 X Were any changes made in the organizing or governing documents but not reported to the IRS? 77 X If "Yes," attach a conformed copy of the changes. a Did the organization have unrelated business gross income of $1,000 or more during the year covered by this return? 78a X b If "Yes," has it filed a tax return on Form 990-T for this year? 78b Was there a liquidation, dissolution, termination, or substantial contraction during the year? If "Yes," attach a statement 79 X a Is the organization related (other than by association with a statewide or nationwide organization) through common membership, governing bodies, trustees, officers, etc., to any other exempt or nonexempt organization? 80a X b If "Yes, " enter the name of the organization ► and check whether it is exempt or 11 nonexempt. is Enter direct and indirect political expenditures See line 81 instructions 81a b Did the organization file Form 11 20-POL for this year? N/A 81b La Did the organization receive donated services or the use of materials, equipment, or facilities at no charge or at substantially less than fair rental value? SEE NOTE C 82a X b If "Yes," you may indicate the value of these items here. Do not include this amount as revenue in Part I or as an expense in Part II (See instructions in Part III) 82b 3a Did the organization comply with the public inspection requirements for returns and exemption applications? 83a X b Did the organization comply with the disclosure requirements relating to quid pro quo contributions? SEE NOTE B 83b 4a Did the organization solicit any contributions or gifts that were not tax deductible? 84a X b If "Yes," did the organization include with every solicitation an express statement that such contributions or gifts were not tax deductible? N/A 84b ;5 501 (c)(4), (5), or (6) organizations. a Were substantially all dues nondeductible by members? N/A 85a b Did the organization make only in-house lobbying expenditures of $2,000 or less? N/A 85b If "Yes" was answered to either 85a or 85b, do not complete 85c through 85h below unless the organization received a waiver for proxy tax owed for the prior year. c Dues, assessments, and similar amounts from members 85c d Section 162(e) lobbying and political expenditures 85d e Aggregate nondeductible amount of section 6033(e)(1)(A) dues notices 85e f Taxable amount of lobbying and political expenditures (line 85d less 85e) 85f g Does the organization elect to pay the section 6033(e) tax on the amount on line 85f? N/A 85 h If section 6033(e)(1)(A) dues notices were sent, does the organization agree to add the amount on line 85f to its reasonable estimate of dues allocable to nondeductible lobbying and political expenditures for the following tax year? N/A 85h 86 501(c)(7) orgs Enter. a Initiation fees and capital contributions included on line 12 86a b Gross receipts, included on line 12, for public use of club facilities 86b 87 501(c)(12) orgs Enter. a Gross income from members or shareholders 87a b Gross income from other sources (Do not net amounts due or paid to other sources against amounts due or received from them) 87b 88 At any time during the year, did the organization own a 50% or greater interest in a taxable corporation or partnership, or an entity disregarded as separate from the organization under Regulations sections 301.7701-2 and 301.7701-3? If "Yes," complete Part IX 5EE NQT p 88 X 89a 501(c)(3) organizations. Enter. Amount of tax imposed on the organization during the year under: section 4911 ► 0 ; section 4912 ► 0 section 4955 ► 0 b 501(c)(3) and 501(c)(4) orgs. Did the organization engage in any section 4958 excess benefit transaction during the year or did it become aware of an excess benefit transaction from a prior year? If "Yes," attach a statement explaining each transaction 89b X c Enter. Amount of tax imposed on the organization managers or disqualified persons during the year under sections 4912, 4955, and 4958 ► 0_ d Enter: Amount of tax on line 89c, above, reimbursed by the organization , . .. ► 0 )Oa List the states with which a copy of this return is filed ► .. FL b Number of employees employed in the pay period that includes March 12, 2004 (See instructions) J 90b )1 The books are in care of ► Telephone no. ► Located at ► ZIP + 4 ► 12 Section 4947(a)(1) nonexempt charitable trusts filing Form 990 in lieu of Form 1041- Check here 1 ( ► and enter the amount of tax-exempt interest received or accrued during the tax year .. ► 92 Form 990 (2004)
)AA 8850A232PM Form 9,qOf200V LIFE EXTENSION FOUNDATION, INC 59-1746396 Page6 Part-VII Analysis of Income-Producing Activities (See pane 33 of the instructions.) Note: Enter gross amounts unless otherwise Unrelated business incor 512, 513, or 51 (E) (D) Related or indicated code Amount exempt function 93 Program service revenue' Business Amount a b c d e f Medicare/Medicaid payments g Fees and contracts from government agencies 94 Membership dues and assessments 3,255,612 95 Interest on savings and temporary cash investments 13 1,063,004 96 Dividends and interest from securities 13 53,540 97 Net rental income or (loss) from real estate a debt-financed property b not debt-financed property 13 10,196 98 Net rental income or (loss) from personal property 99 Other investment income 100 Gain or (loss) from sales of assets other than inventory -29,901 101 Net income or (loss) from special events 102 Gross profit or (loss) from sales of inventory 103 Other revenue a b _ROYALTY INCOME - BUYER'S CLUB 13 2,812,245 c d e 104 Subtotal (add columns (B), (D), and (E)) 0 3,938,9851 3,225,711 105 Total (add line 104, columns (B), (D), and (E)) ► 7,164,696 Note Line 105 plus line 1d, Part I, should equal the amount on line 1 2, Part I Part VIII Relationship of Activities to the Accomplish ment of Exemot Purnoses (See naae 34 of the instructions.) Line No. Explain how each activity for which income is reported in column (E) of Part VII contributed importantly to the accomplishment T of the organization's exempt purposes (other than by providing funds for such purposes) 94 TO SUPPORT SCIENTIFIC AND MEDICAL RESEARCH AND GATHER AND PUBLISH INFORMATION RELATING TO PREVENTING DEGENERATIVE DISEASE.
Pa rt IX Information Reaardinn Taxable Subsidiaries and Disrenarded Entities (See nape 34 of the instructions) (A) (B) (C) (D) (E) Name , address, and EIN of corporation , Percentage of Nature of activities Total income End-of-year partnership, or disregarded entity ownership interest assets SEE NOTE D % U/
o� o� Pa rt X Information Regarding Transfers Associated with Personal Benefit Contracts (See page 34 of the instructions. (a) Did the organization, during the year, receive any funds, directly or indirectly, to pay premiums on a personal benefit contract'? Yes X No (b) Did the organization, during the year, pay premiums, directly or indlr Note : If "Yes" to (b), file Form 8870 and For instructions Under penalties of per decl a that I ve era ed this return, inc and belief, it is true, orre co ete Decl ion of preparer (oche Please Sign S1natur icer Here ' AU Type or print name and title
Preparer's Paid signature Preparer's Firm's name (or yours BURTON & COMPANY, Use Only if self-employed), 4310 SHERIDAN ST. address. and ZIP + 4 HOLLYWOOD, FL 33 DAA B850A 3 38 PM Pg 1
SCHE ULE I1 Organization Exempt Under Section 501 (c)(3) _MB No 1545-0047 (Form 990 or 990-EZ) ( Except Private Foundation) and Section 501(e), 501(f), 501(k), 501(n), or Section 4947( a)(1) Nonexempt Charitable Trust Supplementary Information-( See separate instructions.) 2004 ry Deerna RevenueService 10, MUST be completed by the above or anizations and attached to their Form 990 or 990-EZ Name of the organization Employer Identification number
LIFE EXTENSION FOUNDATION, INC 59-1746396 Part I Compensation of the Five Highest Paid Employees Other Than Officers, Directors, and Trustees (See Page 1 of the instructions. List each one. If there are none, enter "None.") (a) Name and address of each employee paid more (b) Title and average hours (d) Contributions to (e) Expense (c) Compensation empl ben plans & account and other than $50,000 per week devoted to position deferred comp allowances NONE
Total number of other employees paid over $50,000 ► Part II Compensation of the Five Highest Paid Independent Contractors for Professional Services (See page 2 of the instructions. List each one (whether individuals or firms). If there are none, enter "None.")
(a) Name and address of each independent contractor paid more than $50,000 (b) Type of service (c) Compensation
POWELL, GOLDSTEIN,LLP. 901 NEW YORK AVE., NW, WASHINGTON, D.C. LEGAL 72,922 BERNARD A. SINGER, P.A. 3107 STIRLING RD., FT. LAUDERDALE, FL. LEGAL 37,867 WIEBE & ASSOC. 377 N. CENTRAL AVE., UPLAND, CALIF. ACCOUNTING 15,678
Total number of others receiving over $50,000 for professional services ► 0 For Paperwork Reduction Act Notice, see the Instructions for Form 990 and Form 990-EZ. Schedule A (Form 990 or 990-EZ) 2004
DAA B850A 2 5¢ PM Pg 1
Schedul A (Form 990 or 990-EZ) 2004 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 2 Part III Statements About Activities (See page 2 of the instructions.) Yes No
1 During the year, has the organization attempted to influence national, state, or local legislation, including any attempt to influence public opinion on a legislative matter or referendum? If "Yes," enter the total expenses paid or incurred in connection with the lobbying activities ► $ (Must equal amounts on line 38, Part VI-A, or line I of Part VI-B.) E NQT E 1 X Organizations that made an election under section 501(h) by filing Form 5768 must complete Part VI-A. Other organizations checking "Yes" must complete Part VI-B AND attach a statement giving a detailed description of the lobbying activities 2 During the year, has the organization, either directly or indirectly, engaged in any of the following acts with any substantial contributors, trustees, directors, officers, creators, key employees, or members of their families, or with any taxable organization with which any such person is affiliated as an officer, director, trustee, majority owner, or principal beneficiary? (If the answer to any question is "Yes," attach a detailed statement explaining the transactions.)
a Sale, exchange, or leasing of property? 2a X b Lending of money or other extension of credit? SEE NOTE E 2b X c Furnishing of goods, services, or facilities? 2c X d Payment of compensation (or payment or reimbursement of expenses if more than $1,000)? 2d X
e Transfer of any part of its income or assets? 2e X 3a Do you make grants for scholarships, fellowships, student loans, etc ? (If "Yes," attach an explanation of how you determine that recipients qualify to receive payments ) 3a X b Do you have a section 403(b) annuity plan for your employees' 3b X 4a Did you maintain any separate account for participating donors where donors have the right to provide advice on the use or distribution of funds? . , 4a X b Do you provide credit counseling, debt management, credit repair, or debt negotiation se rvices? 4b X Part IV Reason for Non-Private Foundation Status (See pages 3 through 6 of the instructions.)
The organization is not a private foundation because it is (Please check only ONE applicable box.) 5 A church, convention of churches, or association of churches. Section 170(b)(1)(A)(i). 6 A school Section 170(b)(1)(A)(ii) (Also complete Part V.) 7 A hospital or a cooperative hospital service organization Section 170(b)(1)(A)(iii) 8 A Federal, state, or local government or governmental unit Section 170(b)(1)(A)(v). 9 A medical research organization operated in conjunction with a hospital Section 170(b)(1)(A)(iii). Enter the hospital's name, city,
and state ► 10 F1 An organization operated for the benefit of a college or university owned or operated by a governmental unit. Section 170(b)(1)(A)(iv) (Also complete the Suppo rt Schedule in Part IV-A.) 11a nX An organization that normally receives a substantial pa rt of its support from a governmental unit or from the general public Section 170(b)(1)(A)(vi) (Also complete the Support Schedule in Part IV-A ) 11b A community trust Section 170(b)(1)(A)(vi) (Also complete the Support Schedule in Part IV-A.) 12 An organization that normally receives ( 1) more than 33 1/3% of its suppo rt from contributions, membership fees, and gross H receipts from activities related to its charitable , etc , functions -subject to certain exceptions, and (2) no more than 33 1/3% of its support from gross investment income and unrelated business taxable income (less section 511 tax) from businesses acquired by the organization after June 30, 1975. See section 509(a )( 2) (Also complete the Suppo rt Schedule in Part IV-A.) 13 11 An organization that is not controlled by any disqualified persons (other than foundation managers ) and supports organizations described in: (1) lines 5 through 12 above ; or (2) section 501 (c)(4), (5), or (6), if they meet the test of section 509(a)(2). (See section 509(a)(3).) Provide the following information about the suooorted organizations. (See Dace 5 of the instructions.) (b) Line number (a) Name(s) of supported organization(s)
14 n An organization organized and operated to test for public safety. Section 509(a)(4) (See page 5 of the instructions.) Schedule A ( Form 990 or 990-EZ) 2004 DAA 8850A 12 01 PM Pg 1 12 1 Schedule A (Form 990 or 990-EZ) 2004 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 3 Part tV-A Support Schedule (Complete only if you checked a box on line 10, 11, or 12 .) Use cash method of accounting. Nnte: You may use the worksheet in the instrur.hnns for rnnvertinn from the acrnial to the rash methnri of arrniinhnn Calendar year (or fiscal year beginning in) ► (a) 2003 (b) 2002 (c) 2001 (d) 2000 (e) Total 15 Gifts, grants, and contributions received (Do not include unusual rants See line 28 32,224 57,394 4,225 10,000 103,843 16 Membership fees received 2,999,587 4,726,777 3,979,445 2,4901346 14,196,155 17 Gross receipts from admissions, merchandise sold or services performed, or furnishing of facilities in any activity that is related to the organization's charitable, etc , purpose 0 18 Gross income from interest, dividends, amounts received from payments on securities loans (section 512(a)(5)), rents, royalties, and unrelated business taxable income (less section 511 taxes) from businesses acquired by the organization after June 30, 1975 3,633,409 4,275,128 3,676,999 3,502,286 15, 087, 822 19 Net income from unrelated business activities not included in line 18 0 20 Tax revenues levied for the organization's benefit and either paid to it or expended on its behalf 0 21 The value of services or facilities furnished to the organization by a governmental unit without charge Do not include the value of services or facilities generally furnished to the pub ic without charge 0 22 Other income Attach a schedule Do not include gain or (loss) from sale of Capital assets 0 23 Total of lines 15 through 22 6,665,220 9,059,299 7,660,669 6,002,632 29, 387, 820 24 Line 23 minus lne17 6,665,220 9, 059, 299 7, 660, 669 6,002,632 29, 387, 820 25 Enter 1%of line 23 66,652 90,593 76,607 60, 026 26 Organizations described on lines 10 or 11: a Enter 2% of amount in column (e), line 24 ► 26a 587,756 b Prepare a list for your records to show the name of and amount contributed by each person (ether than a governmental unit or publicly supported organization) whose total gifts for 2000 through 2003 exceeded the amount shown in line 26a Do not file this list with your return. Enter the total of all these excess amounts ► 26b c Total support for section 509(a)(1) test Enter line 24, column (e) ► 26c 29,387,820 d Add Amounts from column (e) for lines 18 15,087,822 19 22 26b ► 26d 15,087,822 e Public support (line 26c minus line 26d total) ► 26e 14,299,998 f Public support percentage (line 26e (numerator) divided by line 26c (denominator)) ► 26f 48.6596% 27 Organizations described on line 12: a For amounts included in lines 15, 16, and 17 tnat were received from a "disqualified person," prepare a list for your records to show the name of, and total amounts received in each year from, each "disqualified person " Do not file this list with your return. Enter the sum of such amounts for each year- N/A (2003) (2002) (2001) (2000) b For any amount included in line 17 that was received from each person (other than "disqualified persons"), prepare a list for your records to show the name of, and amount received for each year, that was more than the larger of (1) the amount on line 25 for the year or (2) $5,000 (Include in the list organizations described in lines 5 through 11, as well as individuals ) Do not file this list with your return. After computing the difference between the amount received and the larger amount described in (1) or (2), enter the sum of these differences (the excess amounts) for each year. N/A (2003) (2002) (2001) (2000) c Add Amounts from column (e) for lines 15 16 17 20 21 ► 27c d Add Line 27a total and line 27b total ► 27d e Public support (line 27c total minus line 27d total) ► 27e f Total support for section 509(a)(2) test Enter amount from line 23, column (e) ► 27f g Public support percentage (line 27e (numerator) divided by line 27f (denominator)) ► 27 h Investment income percentage (line 18, column (e) (numerator) divided by line 27f (denominator)) ► 27h % 28 Unusual Grants: For an organization described in line 10, 11, or 12 that received any unusual grants during 2000 through 2003, prepare a list for your records to show, for each year, the name of the contributor, the date and amount of the grant, and a brief descrlotlon of the nature of the grant Do not file this list with your return. Do not include these grants in line 15. Schedule A (Form 990 or 990 -EZ) 2004 DAA 8850A 3 25. PM Pg 13
ScheduleA (orm 990 or 990-EZ) 2004 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 4 Part V Private School Questionnaire (See page 7 of the instructions.) (To be completed ONLY by schools that checked the box on line 6 in Part IV) 29 Does the organization have a racially nondiscriminatory policy toward students by statement in its charter, bylaws, N/A Yes No other governing instrument, or in a resolution of its governing body? 29 30 Does the organization include a statement of its racially nondiscriminatory policy toward students in all its brochures, catalogues, and other written communications with the public dealing with student admissions, programs, and scholarships? 30 31 Has the organization publicized its racially nondiscriminatory policy through newspaper or broadcast media during the period of solicitation for students, or during the registration period if it has no solicitation program, in a way that makes the policy known to all parts of the general community it serves? 31 If "Yes," please describe, if "No," please explain. (If you need more space, attach a separate statement )
32 Does the organization maintain the following. a Records indicating the racial composition of the student body, faculty, and administrative staff? 32a b Records documenting that scholarships and other financial assistance are awarded on a racially nondiscriminatory basis? 32b c Copies of all catalogues, brochures, announcements, and other written communications to the public dealing with student admissions, programs, and scholarships? 32c d Copies of all material used by the organization or on its behalf to solicit contributions? 32d
If you answered "No" to any of the above, please explain (If you need more space, attach a separate statement.)
33 Does the organization discriminate by race in any way with respect to:
a Students' rights or privileges?
b Admissions policies? 33b
c Employment of faculty or administrative staff?
d Scholarships or other financial assistance?
e Educational policies' 33e
f Use of facilities?
g Athletic programs?
h Other extracurricular activities?
If you answered "Yes" to any of the above, please explain (If you need more space, attach a separate statement.)
34a Does the organization receive any financial aid or assistance from a governmental agency?
b Has the organization's right to such aid ever been revoked or suspended? If you answered "Yes" to either 34a or b, please explain using an attached statement.
35 Does the organization certify that it has complied with the applicable requirements of sections 4.01 through 4.05 of Rev. Proc 75-50. 1975-2 C.B. 587, covennq racial nondiscrimination? If "No." attach an explanation Schedule A (Form 990 or 990-EZ) 2004
OAA B850A 12 05 PM Pg 1 I) Schedule +4 (Form 990 or 990-EZ) 2004 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 5 Part VI-A Lobbying Expenditures by Electing Public Charities (See page 9 of the instructions.) (To be completed ONLY by an eligible organization that filed Form 5768) N/A Check ► a if the organization belongs to an affiliated arouo. Check ► b if you checked "a" and "limited control" Drovisions anniv. (a) I (b) Limits on Lobbying Expenditures Affiliated group To be completed totals for ALL electing organizations (I he term "expenditures" means amounts paid or incurred 36 Total lobbying expenditures to influence public opinion (grassroots lobbying) 37 Total lobbying expenditures to influence a legislative body (direct lobbying) 38 Total lobbying expenditures (add lines 36 and 37) 39 Other exempt purpose expenditures 40 Total exempt purpose expenditures (add lines 38 and 39) 40 41 Lobbying nontaxable amount Enter the amount from the following table- If the amount on line 40 is- The lobbying nontaxable amount is- Not over $500,000 20% of the amount on line 40 Over $500,000 but not over $1,000,000 $100,000 plus 15% of the excess over $500,000 Over $1,000,000 but not over $1,500,000 $175,000 plus 10% of the excess over $1,000,000 41 Over $1 500.000 but not over $17,000,000 $225,000 plus 5% of the excess over $1,500,000 Over $17,000,000 $1,000,000 42 Grassroots nontaxable amount (enter 25% of line 41) 42 43 Subtract line 42 from line 36 Enter -0- if line 42 is more than line 36 44 Subtract line 41 from line 38. Enter -0- if line 41 is more than line 38
Caution : If there is an amount on either line 43 or line 44, you must file Form 4720 I 4-Year Averaging Period Under Section 501(h) (Some organizations that made a section 501(h) election do not have to complete all of the five columns below See th e in structi ons for line s 45 through 50 on page 11 of the instructions )
Lobbying Expenditures During 4-Year Averaging Period
Calendar year (or (a) (b) (c) (d) (e) fiscal year b innin in) ► 2004 2003 2002 2001 Total
45 Lobbying nontaxable amount 46 Lobbying ceiling amount (150% of line 45(e))
47 Total lobbying expenditures
48 Grassroots nontaxable amount 49 Grassroots ceiling amount (150% of line 48(e))
50 Grassroots lobbying expenditures Part VI-B Lobbying Activity by Nonelecting Public Charities (For reporting only by organizations that did not complete Pa rt VI-A (See page 11 of the instructions.) During the year, did the organization attempt to influence national, state or local legislation, including any Yes No Amount attempt to influence public opinion on a legislative matter or referendum, through the use of: a Volunteers X to Paid staff or management (Include compensation in expenses reported on lines c through h.) X c Media advertisements X d Mailings to members, legislators, or the public X e Publications, or published or broadcast statements X f Grants to other organizations for lobbying purposes X g Direct contact with legislators, their staffs, government officials, or a legislative body X h Rallies, demonstrations, seminars, conventions, speeches, lectures, or any other means X I Total lobbying expenditures (Add lines c through h.) If "Yes" to any of the above, also attach a statement giving a detailed description of the lobbying activities. Schedule A (Form 990 or 990-EZ) 2004
DAA , B850A 3 25•PM Pg 15
Schedule A (Form 990 or 990-EZ) 2004 LIFE EXTENSION FOUNDATION, INC 59-1746396 Page 6 Part VII Information Regarding Transfers To and Transactions and Relationships With Noncharitable Exempt Organizations (See page 11 of the instructions.) 51 Did the reporting organization directly or indirectly engage in any of the following with any other organization described in section 501(c) of the Code (other than section 501 (c)(3) organizations) or in section 527, relating to political organizations? a Transfers from the reporting organization to a nonchantable exempt organization of: Yes No (i) Cash 51a(l) X (ii) Other assets a(H) X b Other transactions (i) Sales or exchanges of assets with a noncharitable exempt organization b(l) X (ii) Purchases of assets from a noncharltabte exempt organization bit X (iii) Rental of facilities, equipment, or other assets b(III) X (iv) Reimbursement arrangements b(iv) X (v) Loans or loan guarantees b(v) X (vi) Performance of services or membership or fundraising solicitations b vI X c Sharing of facilities, equipment, mailing lists, other assets, or paid employees c X d If the answer to any of the above is "Yes," complete the following schedule. Column (b) should always show the fair market value of the goods, other assets, or services given by the reporting organization If the organization received less than fair market value in any transaction or shanno arranoement. show in column (dl the value of the goods. other assets. or services received. (a) I (b) I (c) I (d) Line no Amount involved Name of nonchantable exempt organization Description of transfers, transactions, and sharing arrangements
N/A
52a Is the organization directly or indirectly affiliated with, or related to, one or more tax-exempt organizations described in section 501(c) of the Code (other than section 501(c)(3 )) or in section 527? ► 11 Yes RX No b If "Yes ," complete the following schedule (c) Description of relationship
DAA Schedule A (Form 990 or 990-EZ) 2004 B850A 3 25 PM Pg 17 t' • Depreciation and Amortization 72 Form 4562 (Including Information on Listed Property) 2004 Department of the Treasu ry Internal Revenue Service ► See separate Instructions. ► Attach to your tax retu Name(s) shown on return Identifying number LIFE EXTENSION FOUNDATION, INC 59-1746396 Business or activity to which this form relates INDIRECT DEPRECIATION Part I Election To Expense Certain Property Under Section 179 Note : If you have any listed property, complete Pa rt V before you complete Pa rt I. 1 Maximum amount See page 2 of the instructions for a higher limit for certain businesses 1 102, 0 0 0 2 Total cost of section 179 property placed in service (see page 3 of the instructions) 2 3 Threshold cost of section 179 property before reduction in limitation 3 410,000 4 Reduction in limitation. Subtract line 3 from line 2. If zero or less, enter -0- 4 5 Dollar limitation for tax year Subtract line 4 from line 1 If zero or less, enter -0- If married filing separately,aratel , see page 3 of the instructions 5 (a) Description of property (b) Cost (business use onlvl (c) Elected cost
7 Listed property Enter the amount from line 29 7 8 Total elected cost of section 179 property Add amounts in column (c), lines 6 and 7 8 9 Tentative deduction Enter the smaller of line 5 or line 8 9 10 Carryover of disallowed deduction from line 13 of your 2003 Form 4562 10 11 Business income limitation Enter the smaller of business income (not less than zero) or line 5 (see instructions) 11 12 Section 179 expense deduction Add lines 9 and 10, but do not enter more than line 11 12 13 Carryover of disallowed deduction to 2005. Add lines 9 and 10, less line 12 ► I 13 Note: Do not use Part II or Part III below for listed property Instead, use Part V. Part II Special Depreciation Allowance and Other Depreciation (Do not include listed pro ert . 14 Special depreciation allowance for qualified prop (other than listed prop ) placed in service during the tax year (see pg 3 of the instruction 14 15 Property subject to section 168(f)(1) election (see page 4 of the instructions) 15 16 Other depreciation (including ACRS see page 4 of the instructions 16 Part III MACRS Depreciation (Do not include listed property.) (See page 5 of the instructions.) Section A 17 MACRS deductions for assets placed in service in tax years beginning before 2004 17 10,699 18 If you are electing under section 168(1)(4) to group any assets placed in service during the tax year n into one or more general asset accounts, check here ► F] Section B-Assets Placed in Se rv ice During 2004 Tax Year Using the General r)Pnreclafinn System (b) Month and (c) Basis for depreciation Recoveryry (a) Classification of property year placed in (business/investment use (d) (e) Convention (f) Method (g) Depreciation deduction service only-see instructions) period 19a 3-year property b 5-ear property c 7-ear property d 10 ear property 15-ear property f 20-ear property ear property 25 yrs S/L h Residential rental 27 5 yrs MM S/L property 27.5 yrs MM S/L I Nonresidential real 39 yrs MM S/L prope rty MM S/L Section C-Assets Placed In Se rvice During 2004 Tax Year Using the Alternative Denreciation System 20a Class life S/L b 12-year 12 yrs. S/L c 40-year 40 rs. MM S/L Part IV Summary (see page 8 of the instructions) 21 Listed property Enter amount from line 28 21 22 Total. Add amounts from line 12, lines 14 through 17, lines 19 and 20 in column (g), and line 21. Enter here and on the appropriate lines of your return. Partnerships and S corporations-see instr. 10,699 23 For assets shown above and placed in service during the current year, enter the oortion of the basis attributable to section 263A costs 23 For Paperwork Reduction Act Notice, see separate Instructions . Form 4562 (2004) DAA B850A 3 25 PM Pg 18
'LIPjE "EX!TENSION FOUNDATION , INC 59-1746396 Form 4562 (2004) Page 2 Part V Listed Property (Include automobiles, certain other vehicles, cellular telephones, certain computers, and property used for entertainment, recreation, or amusement.) Note : For any vehicle for which you are using the standard mileage rate or deductmg lease expense , complete only 24a, 24b, columns (a) through (c) of Section A. all of Section B, and Section C if applicable Section A-De reciation and Other Information (Caution: See pace 9 of the instructions for limits for passenger automobiles.) 24a Do you have evidence to support the business/investment use cla i med ? Yes No 24b If "Yes," is the evidence wri tten? Yes No (a) (g) (h) (I) (b) Business/(c) (d) (e) (f) Type of prop Date placed in investment Cost or other Basis for depreciation Recovery Method/ Depreciation Elected (list vehicles service use basis (business/investment period Convention deduction section 179 first) percentage use only) cost 25 Special depreciation allowance for qualified listed property placed in service during the tax year and used more than 50% in a qualified business use (see page 8 of the instructions) 25
27 Property used 50% or less in a cualtfied business use (see page 8 of the instructions)
S/L-
S/L- 28 Add amounts in column (h), lines 25 through 27 Enter here and on line 21, page 1
Section 113-Information on Use of Vehicles Complete this section for vehicles used by a sole proprietor, partner, or other "more than 5% owner," or related person. If you provided vehicles to your employees, first answer the questions in Section C to see if you meet an exception to completing this section for those vehicles 30 Total business/investment miles driven (a) (b) (c) (d) (e) (f) during the year ( do not include commuting Vehicle 1 Vehicle 2 Vehicle 3 Vehicle 4 Vehicle 5 Vehicle 6 miles-See page 2 of the instructions) 31 Total commuting miles driven during the year 32 Total other personal (noncommuting) miles driven 33 Total miles driven during the year Add lines 30 through 32 34 Was the vehicle available for personal Yes No Yes No Yes No Yes No Yes No Yes No use during off-duty hours? 35 Was the vehicle used primarily by a more than 5% owner or related person? 36 Is another vehicle available for personal use? Section C-Questions for Employers Who Provide Vehicles for Use by Their Employees Answer these questions to determine if you meet an exception to completing Section B for vehicles used by employees who are not more than 5% owners or related persons (see page 10 of the instructions). Yes No 37 Do you maintain a written policy statement that prohibits all personal use of vehicles, including commuting, by your employees? 38 Do you maintain a written policy statement that prohibits personal use of vehicles, except commuting, by your employees? See page 10 of the instructions for vehicles used by corporate officers, directors, or 1 % or more owners 39 Do you treat all use of vehicles by employees as personal use? 40 Do you provide more than five vehicles to your employees, obtain information from your employees about the use of the vehicles, and retain the information received? 41 Do you meet the requirements concerning qualified automobile demonstration use? (See page 10 of the instructions ) Note: If your answer to 37, 38, 39, 40, or 41 is "Yes," do not complete Section B for the covered vehicles Pa rt VI Amortization
(a) (b) (c) (d ) Amortization (f) Date amortization Amortizable Code period or Amortization for Description of costs begins amount section percentage this year 42
43 Amortization of costs that began before your 2004 tax year 26,667 12 26,667 DAA Form 4562 (2004) B8550A lrife Extension Foundation, Inc 3:25 PM 59'1746396 Federal Statements Page 1 FYE: 12/31/2004
Statement 1 - Form 990, Part I, Line 3 - Membership Dues and Assessments
Description Amount MEMBERSHIP INCOME $ 3,255,612 TOTAL $ 3,255,612
Statement 2 - Form 990, Part I, Line 6b - Rental Expenses
Descri Deduction
RENT EXPENSE - REDWOOD 13,440 DEPRECIATION EXP 10,699 TOTAL 24,139
1-2 B850A Life Extension Foundation, Inc 3:25 PM 59-1746396 Federal Statements Page 2, FYE: 12/31/2004
Statement 3 - Form 990, Part I, Line 8c - Sale of Assets Other Than Inventory - Securities Desc How Whom Date Date Sale Cost & Gain/ Recd Sold Acquired Sold Price Expense Deprec -Loss PUBLICLY TRADED SECURITIES $ 220,099 $ 250,000 $ $ -29,901 TOTAL $ 220,099 $ 250,000 $ 0 $ -29,901
3 B850A life Extension Foundation, Inc 3:25 PM 59-`1746396 Federal Statements Page 3 FYE: 12/31/2004
Statement 4 - Form 990, Line 20 - Other Changes in Net Assets or Fund Balances
Description Amount NET UNREALIZED GAINS (LOSS) $ 77,646 TOTAL $ 77,646
4 B850A Life Extension Foundation, Inc 3:25 PM 59-1746396 Federal Statements Page 4 FYE: 12/31/2004
Statement 5 - Form 990 , Pa rt II, Line 22 - Grants, Allocations and Contributions Name Relationship Class of Address to Org Activity Date of Description of Cash NonCash Book BV FMV Gift Property Contrib Contrib Value Explantn Explntn GRANTS - 21ST CENTURY MEDICAL $ 1,790,000 $ $ GRANTS-BIO-MARKER 1,617,756 GRANT-NUTRITIONAL INSTITUTE OF AMER 121,000 GRANTS-ALLIANCE FOR NATIONAL HEALTH 5, 000 GRANTS-IMMORTALITY INSTITUTE 5,000 GRANTS-UNIVERSITY OF NEBRASKA 82,717 GRANTS - CRITICAL CARE RESEARCH 911,500 GRANTS-STASIS FOUNDATION 300,000 GRANTS - REGENTS OF UNIV. OF CAL 12,972 GRANTS - SUSPENDED ANIMATION 955,000 TOTAL $ 5,800,945 $ 0 $ 0 B85QA Life Extension Foundation, Inc 3:25 PM 59-1746396 Federal Statements Page 5 FYE: 12/31/2004
Statement 6 - Form 990. Part II. Line 43 - Other Functional Expenses
Total Program Mgt & Fund- Description Expenses Service General Raising $ $ $ $ EXPENSES CHAR. CONTRIBUTIONS - ALCOR 89, 100 89,100 OFFICE EXPENSES 13,297 13,297 TAXES 2,367 2,367 TOTAL $ 104,764 $ 0 $ 104,764 $ 0
6 B850„A fife Extension Foundation, Inc 3:25 PM 59el1746396 Federal Statements Page 6 FYE: 12/31/2004
Statement 7 - Form 990. Part IV. Line 54 - Investments in Securities
Beginning End of Basis of Description of Year Year Valuation CORPORATE STOCK INVESTMENT - FERRIER 1,801,938 1,922,353 INVESTMENT-WELLS FARGO 1,314,184 1,303,310 INVESTMENT-BANQUE A LUXEMBOURG 111,189 109,170 INVESTMENT-CHARLES SCHWAB 1,022,386 686,339 INVESTMENTS - COUTTS, LONDON 315,369 372,388 INVESTMENTS - SMITHBARNEY 3,344,277 4,293,149 7,909,343 8,686,709
Statement 8 - Form 990 , Part IV, Line 55 - Investments in Land , Buildings, and Equip ment
Beginning Accum End of Accum Description of Year Deprec Year Deprec BUILDING $ 417,254 $ $ 417,254 $ ACCUM DEPRECIATION-BLDG 69,540 80,239 TOTAL $ 417,254 $ 69,540 $ 417,254 $ 80,239
Statement 9 - Form 990 , Part IV, Line 56 - Other Investments
Beginning End of Basis of Description of Year Year Valuation LIFE INSURANCE $ 450,038 $ 450,038 TOTAL $ 450,038 $ 450,038
Statement 10 - Form 990. Part IV. Line 58 - Other Assets
Beginning End of Description of Year Year ACCOUNTS RECEIVABLE-BUYERS CLUB $ 403,273 $ 389,443 GOODWILL 400,000 400,000 ACCUM AMORTIZATION-GOODWILL - 160,002 -186,669 TOTAL $ 643,271 $ 602,774
Statement 11 - Form 990 , Pa rt IV, Line 65 - Other Liabilities
Beginning End of Description of Year Year DEPOSIT PAYABLE $ 5,786 $ 3,136 TOTAL $ 5,786 $ 3,136
7-11 N 06
LIFE EXTENSION FOUNDATION, INC.. 2004-Form 990
NOTE A- PART III, STATEMENT OF PROGRAM SERVICE ACCOMPLISHMENTS
The mission of The Life Extension Foundation is to educate the public about and to support and fund scientific research to develop new methods of preventing and reversing age- related diseases , the aging process itself, and the processes by which we die. The following is a summary of the research and development conducted in the year 2004 by several of the research centers funded by The Foundation....
Critical Care Research, 9 page report attached.
BioMarker Pharmaceuticals, Inc., 16 page report attached.
21 S` Century Medicine, 52 page report attached.
NOTE B- PART VI-ITEM 83b, OTHER INFORMATION
The membership fee is $ 75.00 per year. Under Section 6115, no written notice is required regarding Quid Pro Quo contributions when the membership fee is $ 75.00 or less.
NOTE C- PART VI-ITEM 82 a
Life Extension Foundation Buyers Club, Inc. donated certain products and promotional material to Life Extension Foundation, Inc. for distribution by the Foundation their new and renewing members.
NOTE D- PART VI-ITEM 88 AND PART IX
Ownership Business Name Income EOY Assets
92.6% 21st Century Medicine, Inc. $ ( 1,516,253) $ 2,084,851 10844 Edison Court Rancho Cucamonga, Ca. 91730 # 33-0559567 80.6% Critical Care Research, Inc. S ( 608,315) $ 2,115,269 10743 Civic Center Drive Rancho Cucamonga, Ca. 91730 # 95-4750143 1' 1 . L
LIFE EXTENSION FOUNDATION, INC. 2004-FORM 990
100% California Advanced Biosciences , Inc. $ 998) $ 261 8460 Red Oak St. Rancho Cucamonga, Ca. 91730 # 33-0849929 69.0% Suspended Animation, Inc. $ ( 887,087) $ 371,590 1082 S. Rodgers Circle Boca Raton, Fla. # 46-0466303
NOTE E-SCHEDULE A-PART III, LINE 1 & 2b
Life Extension Foundation Buyers Club, Inc. ("Buyers Club"), a for-profit corporation, funds and publishes the Life Extension Magazine and distributes the Magazine to all Life Extension Foundation members. This Magazine serves as a means of describing the vitamin supplement products that Buyers Club sells to the public, in addition, this Magazine provides many informative articles that are carefully researched and presented to the Foundation members. Occasionally, the Magazine publishes articles highlighting legislation that affects the interests of the Foundation members. However, the Magazine is fully funded by Buyers Club and there are no lobbying expenditures funded by the Foundation. The Foundation is owed monies from Buyers Club as the result of a certain sale dated December 31, 1996. The receivable is being liquidated pursuant to an amortization schedule and all payments have been timely received. Foirrt 8868 Application for Extension of Time To File an (Rev. Deceni6er2004) Exempt Organization Return OMB No 1545-1709
Department of the Treasu ry I 10, File a separate application for each return. I Internal Revenue Service only • If you are filing for an Automatic 3-Month Extension, complete Part I and check this box juhl • If you are filing for an Additional (not automatic) 3-Month Extension , complete only Part II (on page 2 of this form). Do not complete Part II unless you have already been granted an automatic 3-month extension on a oreviously filed Form 8868. Part E. Automatic 3-Month Extension of Time- Only submit original (no copies needed)
Form 990-T corporations requesting an automatic 6-month extension-check this box and complete Part I only ...... ► ❑
All other corpora tions (including Form 990-C filers) must use Form 7004 to request an extension of time to file income tax returns. Partnerships, REMICs, and trusts must use Form 8736 to request an extension of time to file Form 1065, 1066, or 1041. Electronic Filing (e-file). Form 8868 can be filed electronically if you want a 3-month automatic extension of time to file one of the returns noted below (6 months for corporate Form 990-T filers). However, you cannot file it electronically if you want the additional (not automatic) 3-month extension, instead you must submit the fully completed signed page 2 (Part II) of Form 8868. For more details on the electronic filing of this form, visit www irs.gov/efile. Type or Name of FYpm' ^•^nw0--"-- Employer identification number 59-1746396 print File the by INC., LIFE EXTENSION FOUNDATION, due date for Number, tCt10nS. 3107 STIRLING RD filing your C/o BERNARD SINGER, ESQ. return See FL 33312 Fr. LAUD RD ALE, instructions City, tow E dress, see instructions.
Check type of return to be filed (file a separate application for each return)- Form 990 Form 990-T (corporation) Form 4720 Form 990-BL Form 990-T (sec. 401(a) or 408(a) trust) Form 5227 Form 990-EZ Form 990-T (trust other than above) Form 6069 Form 990-PF Form 1041-A Form 8870
• The books are in the care of ► M/4-/T S IN G£ A LS
Telephone No. ► ,f 98 Jlr- B 60.0 FAX No. ► I S yam- 98S 9 �-` 7 7 • If the organization does not have an office or place of business in the United States , check this box ► ❑ • If this is for a Group Return , enter the organization 's four digit Group Exemption Number ( GEN) . If this is for the whole group , check this box ► ❑ . If it is for part of the group , check this box ill ❑ and attach a list with the names and EINs of all members the extension will cover. I I request an automatic 3-month (6-months for a Form 990-T corporation ) extension of time until 5-10 to file the exempt organization return for the organization named above . The extension is for the organization 's return for: ► calendar year 2 0or 10. " tax year beginning . , . ,. , and ending
2 If this tax year is for less than 12 months , check reason : ❑ Initial return ❑ Final return ❑ Change in accounting period
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BY Director SEP .],.°9 ?005 Alternate Mailing Address - Enter the address if you want the copy of this application for an a, returned to an address different than the one entered above FIE,,t) DDIRFCTC( Name LIFE EXTE`SION FOUNDATION INC., �. I n Q a.^r n a ..l- 1 r v A e i4i4 c C/O BURTON & CO., P .,A., CPA .- Type or Number and street ( include suite , room, or apt. no.) Or a P.O. box nu print 4310 SHERIDAN STREET, SUITE 202 City or town, province or state, and count ry (Including postal or ZIP code) HOLLYWOOD, FL 33021 Form 8868 (12-2000) STF FED9056F 2 July 8, 2005
To: The Life Extension Foundation
Dear Sirs:
The purpose of this letter is to provide a summary of research conducted at 21st Century Medicine during 2004, and to request continued grant support for new and ongoing research projects for 2005. The primary mission of 21st Century Medicine remains to develop and deploy new technology and products in the fields of cryopreservation and ice control, and particularly to develop successful methods for the cryopreservation of complex biological systems.
The sections included in this grant application are as follows.
1. Summary of research completed during 2004. a. Brain Vitrification. i) Brain Slice Cryopreservation ii) Whole Brain Cryopreservation b. Whole Body Vitrification. c. Kidney Vitrification. d. Cornea Vitrification. e. Cryoprotectant Technology. f. Physical Science Research. i) Ice Control ii) Fracture Avoidance iii) Intermediate Temperature Storage g. Kidney Cold Storage (TransSend). h. Cardiac Cold Storage and Intermittent Perfusion. i. Basic Research. J.. Contract Research. k. Collaborative Research. 1. Discontinued projects. 2. Scientific publications. 3. Scientific presentations. 4. Patent awards, NIH grant awards. 5. Patent and NIH grant applications. 6. Proposed research and preliminary results for 2005. a. Brain Vitrification. i) Brain Slice Cryopreservation
10844 EDISON COURT • RANCHO CUCAMONGA, CALIFORNIA 91730 TELEPHONE 909.466-8633 • FAX 909.466-8618 • www.2[CM.com ii) Whole Brain Cryopreservation b. Whole Body Vitrification. c. Kidney Vitrification. d. Cornea Vitrification. e. Cryoprotectant Technology. f. Physical Science Research. i) Ice Control ii) Fracture Avoidance and ITS elaborations g. Kidney Cold Storage (TransSend). h. Cardiac Cold Storage and Intermittent Perfusion. i. Hepatocyte Cold Storage. j. Basic Research. k. Contract Research. 1. Collaborative Cryopreservation Research. 7. Physical Plant and Staff Considerations for 2005. 8. Non-LEF grant funding, 2004, 2005. 9. Summary of near term commercial opportunities. 10. Final Comments, budget structure and proposal. 11. List of attached appendices. 12. Proposed 2005 Operating Budget (attached spreadsheet).
1. Summary of research completed during 2004.
Note: For the convenience of the reader in rapidly finding information in this letter, bold text is used to highlight the central points being discussed in each paragraph. a
A) Brain Vitrification.
Brain Slice Cryopreservation
The overall aim of the brain slice project is to demonstrate optimal methods for the cryopreservation of rabbit hippocampal slices by vitrification. The availability of perfectly preserved brain slices would be of significant value to the pharmaceutical industry in facilitating drug screening and reducing the costs of drug development. In addition it will provide valuable insights on whole brain preservation and, ultimately, suspended animation. Electrophysiology recording station installation and slice testing method development. Although all the apparatus and parts for our neurophysiological testing station were ordered in December of 2003, the station was not ready to operate until June of 2004 due to delays in the arrival of some key parts and the need to renovate the electrophysiological laboratory. However, the electrophysiology recording system has been up and running ever since June, 2004. The optical recording system was installed and calibrated in December 15 of 2004. This system was assembled from numerous parts including some crafted in the 21 CM workshop. The recording station is state of the art, and it is capable of electrophysiology recording (intracellular and field potential recording), IR-DIC imaging and optical recording of electrical signals in brain tissue. While waiting for the equipment needed for electrophysiological testing, Dr. Tan established a rabbit hippocampal slice model and obtained a good baseline K/Na+ ratio
LEF Grant Application for the Year 2005 Page 2 of 52 as a first step. He then established a good baseline test of electrical responsiveness based on the measurement of evoked field potentials in the Schaefer collateral region in response to electrical stimulation of the CA3 dendrite field. We also successfully corrected our problems with the Vibratome and are now able to use this superior instrument on a routine basis in preference to the Mcllwain tissue chopper used initially. Establishment of a viable anesthetic method. We initially used an existing anesthesia protocol developed for the whole brain project (ketamine, 30mg/Kg) followed by Euthasol (0.3m1/Kg) for euthanasia just prior to removing the brain. Isolated hippocampi were chopped into 450um slices using our original Mcllwain chopper, and the slices were used for screening of carrier solutions, CPA protocols, and vitrification effects using the K/Na+ ratio assay. All seemed to be in order, but when, in June 2004, we were able to evaluate slices by electrophysiological means, we found to our surprise that the slices were not responding to electric stimuli. This was found to be due to the antagonistic effects of ketamine and Euthasol, presumably acting to inhibit excitatory receptors. A new protocol was therefore developed in which Nembutal was employed for anesthesia, cardiac fibrillation was employed for euthanasia, and the Vibratome was used for slicing. These changes in protocol overcame the suppression of electrical response and allowed us to obtain good results . The studies with the old protocol are still valid since good K+/Na+ ratio data were apparently obtained, and can be validly measured in the absence of electrical neuronal communication. Carrier solution evaluation . We evaluated 3 candidate "carrier solutions" (physiological support media used during the addition and removal of cryoprotectants) for their suitability for rabbit hippocampal slices at various temperatures (5, 10, and 15oC). The solutions chosen, RPS-2, LM5, and Krebs Ringer Solution (KRS), all relate to the balance between protecting biological viability on the one hand and maximizing vitrification tendency on the other. Slice viability was evaluated by K+/Na+ ratio and slice electrical responsiveness was evaluated by measuring the amplitude, time course, and shape of field excitatory post-synaptic potentials (fEPSPs) generated in response to electrical stimulation. No detrimental effects were shown after 2-hr incubation of slices in these three carrier solutions at the three different temperatures studied, a time sufficient for the addition and washout of cryoprotectants in this model. However, it seemed that LM5 may not be as protective against damage at 0°C as RPS-2, the carrier solution developed by Dr. Fahy for kidney cold storage and found by Yuri Pichugin found to provide good protection of brain tissue at 0°C. Cryoprotectant evaluation and vitrification method development. Because of our chilling injury result, we initially used RPS-2 as a carrier to introduce and remove the simplest of the new-generation vitrification solutions, VET, to gain experience with the rabbit hippocampus and provide a point of comparison with Yuri's rat results. The results showed that the rabbit hippocampus appears considerably more resistant to cryoprotectant toxicity than the rat hippocampus . The rabbit hippocampus tolerates higher concentrations of VEG at 15°C than the rat hippocampus tolerates at 10°C, and as a result of this, we were able to treat rabbit hippocampal slices with full- strength VEG at -3°C without damage. The rat hippocampus, by contrast, could not attain more than about 50% recovery of K+/Na+ ratio even when treated with VEG at -10°C. This result led us to the next logical step, which was to test the viability of rabbit hippocampal slices after vitrification in VEG. To our gratification, our very first experiment on the vitrification of rabbit brain slices resulted in the complete
LEF Grant Application for the Year 2005 Page 3 of 52 recovery of K+/Na+ ratio after vitrification and rewarming! Figure 1 shows the functional recovery of these slices, and Figure 2 shows typical cooling and warming profiles indicating that, after the 90 seconds of cooling used in this experiment, the slices were in fact below the glass transition temperature and therefore were truly vitrified. This demonstration shows that the ability to survive after vitrification and rewarming is not restricted to the rat hippocampus but is likely to be a general ability of all mammalian brains.
Recovery of Vitrified in 1.04X VEG (n=3) I Adult Rabbit Hippocampal Vitrified in VEG (n=3) Slices after Vitrification and Rewarming Untreated Controls (n=5)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 K+/Na+ Ratio
Figure 1: Recovery of adult rabbit brain slices after treatment with VEG alone (lower bar) or after vitrification in VEG or in a VEG concentrate (4% more concentrated than ordinary VEG.)
Rabbit Hippocampal Brain Slice Cooling and Warming Curve Figure 2 (left): Temperature trace of a 0 rabbit hippocampal slice -20 as measured by a small U �- 40 thermocouple inserted directly into the slice -60 during cooling and CL -80 warming. In Figure 1, the aEi -100 slices were cooled for a total of 90 seconds which, 0 -120 as shown in Figure 2, was CO -140 sufficient for vitrification. -160 0 20 40 60 80 100 120 Elapsed Time (Seconds)
After completing this experiment, we were able to finally set up and successfully
LEF Grant Application for the Year 2005 Page 4 of 52 use the equipment needed for testing brain slice electrical responsiveness, and we applied this more sensitive test to evaluate brain slices after the addition and washout of VEG and after vitrification. These experiments showed that, unlike the case when sodium and potassium transport were used to define viability, VEG addition and washout was not harmless, and vitrification was not successful in allowing recovery of electrical responses. The appearance of these slices under the microscope did not reveal any abnormalities that hinted at the reduced electrical responsiveness, in agreement with the fundamental viability of the brain tissue as indicated in Figure 1, but the more subtle property of electrical responsiveness was clearly inadequately preserved. Figure 1 showed slightly higher viability and considerable less variability of viability when the 1.04X VEG solution was used to vitrify rather than ordinary VEG, suggesting that part of the problem of recovering slice electrical responsiveness after vitrification might be related to the stability of the vitrification solution against ice formation. We therefore turned our attention to the more stable vitrification solution, VM3. As was true in the rat hippocampus using the K+/Na+ ratio assay, we found VM3 to be less toxic to rabbit hippocampal slice electrical responsiveness than VEG, even using LM5 as the carrier solution, as required for full activity of the ice blockers present in VM3. Further, rabbit hippocampal slices were vitrified in VM3 with full recovery of K+/Na+ ratio and almost full recovery of electrical responses in a couple of early experiments. However, for reasons that are not clear, we were not able to reproduce these initial very promising results. Later experiments showed that, in general, although 90-100% recovery of K+/Na+ ratio was attained in all slices treated with VEG and VM3, only about 60-80% recovery of electrophysiological response could generally be obtained, and then only in about 50% of the slices, with the rest showing no responses, with considerable variation from experiment to experiment. Fortunately , most of these problems were overcome in 2005 (see 2005 Preliminary Results, below). DSC measurements . We verified that rabbit hippocampal slice tissue, when cooled and warmed in a DSC pan, does not show freezing events when cooled and warmed at rates germane to our viability testing protocols.
Whole brain cryopreservation
Our first goal for 2004 was to pursue the possibility of opening the blood-brain barrier (BBB). We explored using the chemotherapeutic agent etoposide, osmotic pulsing with the sugar arabinose, or a combination of the two. Barrier opening was gauged by brain tissue staining by Evan's blue dye administered either systemically or after perfusion-cooling to below 10°C. The combination of etoposide and arabinose was examined both in sequential fashion, in which etoposide was given first according to literature methods and was then followed by arabinose, or in simultaneous fashion, in which etoposide was included in the arabinose solution. We found that BBB opening tended to cause rabbits to die if they were exposed to Evan's blue at normal body temperature, perhaps because dye penetration into the brain interfered with respiratory function. However, we found that using a pulse of arabinose containing etoposide at normal body temperature without exposure to dye allowed the animal to survive long enough for cephalic cooling to below -10°C. When the brain was perfused with dye at this temperature, we were gratified to find, for the first time, very good and uniform brain staining with dye, and in fact more uniform brain staining than we have seen reported in the literature. This is the first time anyone anywhere has shown that BBB
LEF Grant Application for the Year 2005 Page 5 of 52 opening initiated at normothermia persists after cooling to below 10°C. Even more gratifying, we found that brains subjected to BBB opening and then treated with M22 did not shrink, implying success in delivering M22 across the former BBB. Even more remarkably, brains subjected to BBB opening, M22 loading, and M22 unloading also weighed about as much as normal brains, indicating a lack of trapping of M22 or other perfusate constituents in the brain. However, as noted in last year's report, we were aware that results such as these could be misleading and needed to be followed up by ultrastructural observations. These follow-up studies were carried out and showed, unfortunately, that BBB opening followed by M22 loading causes unacceptably severe ultrastructural damage. Therefore, despite the very exciting brain weight data, we decided to abandon BBB opening until all alternatives to BBB opening had been exhausted. The next approach to adding cryoprotectants while minimizing brain shrinkage was, as proposed, to introduce the cryoprotectants at higher temperatures, i.e., at +10°C instead of our usual -3°C. Several experiments along these lines indicated that loading the first 5M cryoprotectant of the 9.3M total concentration of M22 at 10°C gave less brain shrinkage than our standard protocol involving initial loading at 3.5°C. When all 9.3M cryoprotectant was added at 10°C, the brain swelled rather than shrank, a fairly astonishing result that deserves more investigation but may represent failure of the BBB at that temperature. It was considered that further measures to promote cryoprotectant uptake beyond perfusion at probably damagingly high temperatures were desirable, but ultrastructural examination of brains treated at the higher temperature could not be completed in 2004 because of backlogs in viewing time on the electron microscope at USC. As noted in our report for 2003, we observed that rabbit brains perfused for 2 hours with B2C, a super-concentrated cryoprotectant formula that was our first formula to be licensed to Alcor, contained occasional cells (perhaps glial cells rather than neurons) that looked disintegrated or severely damaged. The first order of business was to repeat these experiments and determine whether the damage seen with B2C was a fluke or is a reliable finding. Our initial worrisome results were obtained after perfusion not with B2C proper but with a solution set to a total concentration of 110% of full-strength B2C and, as noted, perfused for 2 hours. Because B2C is a super-stable solution, such overkill measures should not be needed. We therefore repeated the experiment using a 1-hour perfusion at only 5% over the true concentration of B2C, bearing in mind that our experiments with M22 indicate that even 30 min of exposure at 0% over the true concentration of M22 is sufficient for vitrification in our system. The result was that 1 hour of perfusion with 105% of full-strength B2C did not replicate the damage seen in our original 2 hour B2C perfusions. Nevertheless, we remained concerned that B2C might be more damaging than necessary in order to achieve brain vitrification in view of its very high concentration. We therefore conducted, as proposed in last year's letter, several experiments devoted to determining whether M22 might be useful not only for rabbit brains, but also for human brains, including human brains with compromised perfusion. Three basic questions had to be addressed in order to confidently recommend a switch from M22 to B2C. First, do rabbit brains cooled at human neuropatient rates remain ice free? Second, if perfusion defects limit local concentrations of cryoprotectants to as little as 81 % of the full-strength arterial perfusate, can the brain still vitrify? And third, is M22 perfusion compatible with whole body perfusion and brain vitrification in situ?
LEF Grant Application for the Year 2005 Page 6 of 52 To do the first experiment, it was necessary to devise methods that can duplicate in a rabbit cephalon the cooling rates believed to be attained during human neurovitrification (0.3°C/min). For this, we found that placement of the cephalon inside a slightly larger beverage cooler, followed by placement of the cooler in a -135°C storage unit resulted in a remarkably faithful replication of Alcor neuropatient core cooling rates (mean 0.3°C per minute). Rabbit cephalons perfused with M22 and cooled at 0.3°C/min, rewarmed, fixed, and examined in the electron microscope were found to be apparently free of ice. `,. When the same experiment was repeated using only 81% of full strength M22, the same result was obtained, indicating that the rabbit brain is more resistant to ice formation than the solution used to perfuse it: this dilution of M22 by itself is not sufficient to fully vitrify in our typical testing procedures. This result shows that even for human patients with substantial perfusion deficits, M22 should be effective. We have also done an experiment to determine what ice looks like when brains freeze after perfusion with only 50% of full-strength M22. However, the results of that experiment are not yet in. This experiment is intended to examine the possible drawback of attempting vitrification in a situation where this attempt is likely to fail and freezing is likely to take place at unconventionally high concentrations of cryoprotectant. This is known to be hazardous due to the high likelihood of intracellular ice formation, and we would like to know more about the concentration at which the "safe zone" of concentration ends and the "damaging zone" of concentration begins. However, more information on this fourth question will have to be obtained in future research. To address the third question, about utility for attempted whole body vitrification, we included an additive in our cephalic perfusate that was used successfully in our previous whole body experiments to allow M22 to be used without the systemic edema observed without this additive. Use in the isolated cephalon showed that the additive reduced total flow somewhat, but still permitted good perfusion and did not prevent the cephalon from vitrifying when subsequently cooled and rewarmed, the brain not exhibiting any ice crystal damage. Collectively, these results and other observations indicate that M22 is a sufficiently stable, more perfusable, less toxic, and whole body-compatible replacement for B2C and is therefore approp riate for use on human subjects. Because hydroxyethyl starch from our traditional supplier, B. Braun, was discontinued and a substitute version, called N-HES, made by Ajinomoto, appeared to be damaging according to kidney perfusion experiments described below, we did a few experiments to determine whether RES could be replaced by more available polymers . Polyethylene glycol of similar molecular weight to HES was insoluble at the required concentrations, and PVP of similar molecular weight reduced brain flow by six- fold. Based on these findings, both agents were ruled out as possible replacements for B. Braun HES. In 2004 we began our first investigations on preventing histological and ultrastructural damage caused by warm ischemic injury and cold storage injury in the intact brain. For our whole brain studies to eventually result in viable brains, it will be necessary for the brains not to be killed by exposure to cold for the period of time required to introduce and remove cryoprotectants, which is about 5 hours. From the point of view of organizations like Alcor, stability over time scales of 24 to 48 hours is also relevant. Furthermore, cardiac arrest at normothermic temperatures is currently unavoidable for cryonics patients, and means of mitigating damage caused by this insult
LEF Grant Application for the Year 2005 Page 7 of 52 would be desirable. Two models were looked at: a) warm ischemia plus cold ischemia and b) cold ischemia only. In control experiments for series a), rabbits were heparinized, killed, packed in ice, and, 24 or 48 hours later, perfused free of blood with LM5 and then perfused with fixative. The experimental group in this series involved the same treatment except that, at 2 min before inducing cardiac arrest at normal body temperature, a protective additive was infused systemically. In series b), we simply followed our normal cephalic perfusion cooling protocol but using MHP-2 or TransSend-4 as the perfusate, with HES included as the colloid as usual, and stored the cephalons for 24 or 48 hours before perfusing them with fixative. In series a), the protective in vivo infusion appeared to confer considerable histological protection to the brain and improved the reperfusion and gross appearance of the brain. In series b), MHP-2 perfusion was surprisingly ineffective at protecting histology. TransSend-4 was better, but left considerable room for improvement. These experiments provide some basic orientation for much-needed future studies and support a patent application that we may file on the in vivo intervention in 2005. In 2003, we observed unsatisfactory ultrastructure in some control brains, which implied that our ability to preserve ultrastructure in treated brains might be limited by some basic artifact of fixation. We were able to resolve this problem in 2004, at least for the cortex and hippocampus, by fixing whole control brains in the usual way but then removing these two subregions and transferring them to electron microscopy grade glutaraldehyde for postfixing. Consequently, this modified fixation method was adopted as our standard method. We had planned to extend our understanding of brain cryopreservation by quantitating brain tissue concentrations of individual cryoprotectants using high performance liquid chromatography to visualize different peaks for different cryoprotectants in homogenized brain tissue after whole brain perfusion. This turned out to be more difficult than expected, and experiments on model CPA solutions led us to conclude that we would need different HPLC columns in order to adequately separate these agents, and these did not become available to us in 2004. We hope to acquire these columns in 2005. Unfortunately, although our own electron microscope was technically functional in 2004, the need for personnel training on its use and the difficulty of scheduling this training made it necessary to continue to rely on USC for expensive and time- consuming electron microscopy services for the balance of the year.
B) Whole Body Vitrification.
No experiments on whole body vitrification were completed in 2004. However, we exchanged emails with and had a conference call with individuals interested in providing us with funding for this project, and we agreed to submit a proposal for possible funding in 2005. We also realized that whole body vitrification research can likely be published if we describe the intent as suspended animation, which is, of course, our ultimate goal. Aubrey de Grey has asked us to submit a manuscript on vitrification studies on the brain and whole body before the end of 2005 for publication in Rejuvenation Research. This will probably not be submitted until 2006, but work will be done on it in 2005.
LEF Grant Application for the Year 2005 Page 8 of 52 C) Kidney Vitrification.
We evaluated a new cryoprotectant formula, designated as M10, that was successful in allowing similar or better viability in kidney slices at -10°C than we could achieve with M22 at -22°C. Unfortunately, although equilibration was somewhat improved, perfusion of whole kidneys with M10 at -10°C resulted in a mean peak creatinine level of over 15 and only a 50% survival rate, so we discontinued studies of this solution. �• In 2004, we were able to establish the following criteria for vitrifiability of the renal medulla . For the M22MX vitrification solution, the required urine refractive index (RI) is <_1.409 (1.409 being sufficient). For M22EG, the required RI is 1.404. For M22, the required urine RI is about 1.407. When kidneys were transplanted after deep cooling at lower urine RI values, varying degrees of medullary vascular damage were readily apparent 30 min after transplantation. Damage was manifested either as extravasation of medullary blood throughout the medulla or, when freezing was particularly severe, in the outer medulla only, blood never being able to even reach the inner medulla. We attempted to reproduce our previous successful results at -45°C, using M22MX but using 60 mmHg to promote equilibration . Unfortunately, only two of five kidneys survived , with creatinine peaks higher (>17) than in our original -45°C series with M22. The problem was not the M22MX, because when cooling with this formula was limited to -22°C to -28°C and pressure was confined to 40 mmHg, we obtained postoperative creatinine peaks averaging 9.0, which is normal. It therefore appears that either we needed more practice on cooling to -45°C, or perfusion at 60 mmHg sensitizes kidneys to subsequent cooling to below -22°C, most likely the former. An additional 5 kidneys perfused with M22 at -22°C gave an average peak creatinine of 7.3 mg/dl, which is excellent, confirming the integrity of our basic M22 baseline as of 8/6/2004. In response to a staff member's concern about our lack of use of intraoperative and postoperative pain medication, we began using Buprenex as a pain medication, and obtained a mean peak creatinine averaging 12.0 vs. 7.3 in the same M22 at -22°C protocol. We explored the possibility of reducing toxicity by using a reduced formamide, reduced DMSO version of M22 based on observations made as a result of our work with Genzyme and as a result of further contemplation of the qv* theory of cryoprotectant toxicity. Our first such solution, named M22B, seemed spectacularly successful, with a mean peak creatinine level after transplantation of only about 4 mg/dl, which is equal to what was reported for sham operated control rabbits in 1994. This was all the more remarkable considering that the total molar concentration of M22B is considerably higher than that of M22 (over 9.7M for M22B vs. 9.3M for M22). In addition, M22B incorporated our highly active, lower molecular weight form of PVP as well to provide extra medullary protection, and the low toxicity of this solution indicates that this form of PVP is, not itself toxic to any part of the kidney. Unfortunately, despite its other wonderful properties, M22B was much more viscous than M22, and when we checked the ice-forming tendency of kidneys perfused with M22B, frank medullary freezing was observed, presumably because the high viscosity reduced M22B delivery to the medulla.
LEF Grant Application for the Year 2005 Page 9 of 51 We attempted to correct this problem using a series of further modifications known as M22C-M22E, but got generally similar results: lower toxicity but also unacceptable medullary ice formation. From a practical point of view, it seemed that overcoming the equilibration issues of using these kinds of solutions would take too long and would represent a detour. Therefore, we decided to return to standard M22. Unfortunately, at this time we ran out of our traditional hydroxyethyl starch (HES) because the manufacturer, B. Braun, stopped making this critical component of our solutions, and it could not be re-ordered. We therefore had no choice but to begin searching for a form of HES from other sources that would have toxicity and perfusion characteristics comparable to or better than these features of B. Braun RES. We began with N-HES from Ajinomoto because it was of lower molecular weight and was expected to give higher flow rates and therefore better equilibration. At the time, we were judging equilibration largely on the basis of the arteriovenous (A-V) concentration differences at the end of M22 loading, and based on this measure, N-HES looked more favorable than B. Braun HES. Due to acute problems with our supply of 1,2-propanediol ("methoxyglycerol"), we tried N-HES for the vitrification of M22EG (a methoxyglycerol free version of M22), and using 100 mM mannitol to promote flow during renal blood washout and initial perfusion. This procedure allowed ice-free medullas to be obtained at both 40 mmHg and 60 mmHg using N-HES as the colloid. In these experiments, the urine refractive index reached 1 .0404 at both 40 and 60 mmHg. Unfortunately, when we transplanted a kidney perfused at 40 mmHg with this treatment (not including cooling below -22°C), the peak creatinine level went to nearly 20, so toxicity seemed to be a limiting factor. To overcome this problem, we turned to MEG, which was believed to be a less toxic solution than M22EG, and we deleted the use of 100 mM mannitol as a possibly damaging intervention. The first two perfusions with MEG gave a mean peak creatinine of 7.9 mg/dl, but with insufficient urine RI values. In one kidney with a urine RI of 1.405, serum creatinine peaked at 16 mg/dl. Raising perfusion pressure to 60 mmHg to get more reliable vitrification resulted in urine RI values of about 1.409 but also gave unsatisfactory results after transplantation. We decided to check our M22 baseline with the new N-HES to determine whether N-HES might be contributing to the problems encountered. Four transplants in this group gave urine RI values clustering at about 1.405, but one recipient died, and the mean peak creatinine for this group came to 13. This was a markedly worse result than we had obtained earlier in the year with B. Braun LIES (mean peak creatinine, 7.3, and no deaths), so we felt compelled to turn to other versions of HES. Three kidneys were perfused with a German form of HES, "H7". The RI values were the same as in the N-HES group, but postoperative creatinine peaks were too unreliable for this form of HES to be useful. Although it seemed that N-HES almost doubled the mean peak creatinine levels attainable with M22 perfusion in comparison to what was obtained earlier in the year with B. Braun HES, we decided to see whether a mean creatinine level of 13 might still be compatible with survival after cooling to -100°C. We increased perfusion pressure slightly, to 50 mmHg, and tried the experiment on two kidneys. Both failed to survive . The urine RI values were near 1.405 for both of these kidneys, which we now know is inadequate for medullary vitrification with M22. Raising N-HES levels to 5% at the beginning of the perfusion resulted in no u rine output during M22 perfusion, even
LEF Grant Application for the Year 2005 Page 10 of 52 when perfusion pressure was raised to 70 mmHg, and solid freezing of the medulla, tending to confirm a toxic effect of N-HES. In view of these results, we decided to re-investigate the German H7 HES. We perfused a kidney with M22 at 70 mmHg after initial use of H7 HES, cooled the kidney to -100°C, rewarmed it, transplanted it, and removed it after 30 min of blood reflow. In this kidney, the urine RI was about 1 .4075, and the medulla was free of ice damage. When this experiment was repeated using a perfusion pressure of 60 mmHg, the urine RI was 1.405 and the medulla became packed with trapped red blood cells, `�. indicating medullary freezing damage. This allowed us to understand that although a urine RI of 1.405 may be adequate for kidneys perfused with M22EG, an RI closer to 1.407 seems to be necessary for kidneys perfused with M22. Unfortunately , 70 mmHg seemed necessary for the prevention of medullary freezing, but when the 70 mmHg protocol was repeated and the kidney was transplanted, it did not survive despite having a presumably adequate urine RI of 1 .408. This failure could have been due to cooling injury, elevated pressure, or the H7 HES, so we chose to investigate the latter possibility by switching to a third form of HES, a second Japanese form called M-HES. Perfusion at 70 mmHg yielded unusually good urine RI values of 1.4085 and 1.411, but cooling to -100°C and rewarming did not result in life support after transplantation. It seemed necessary at this point to do a series of transplants involving M22 at -22°C and 2% M-HES at 40, 60, and 70 mmHg to determine whether our failure to survive cooling to -100°C was due to pressure or M-HES-related damage. Perfusion at 70 mmHg was compatible with survival although one kidney perfused at this pressure failed, and perfusion at 40 and 60 mmHg yielded comparable peak creatinine values, with an RI value of about 1.409 at 60 mmHg. The mean peak creatinine level for these experiments was 12.3. Unfortunately, it appeared that no substitute form of lIES tested in 2004 was as non-toxic as B. Braun HES.
D) Cornea Vitrification.
In preparation for receiving a Phase I SBIR grant for cornea vitrification, we decided to test a new supplier of human corneas in Alabama to make sure their transplant grade corneas would be suitable for transplantation into monkeys as called for in the grant. We also saw this as a way to fill in some of the gaps in our previous human-to-rabbit data set. One human cornea was vitrified, rewarmed, transplanted into a rabbit host, retrieved 10 days later, fixed, and sent to USC for evaluation by scanning electron microscopy . As we had observed before, junctions between the corneal endothelial cells were basically not visible. We are not sure what this means, since this cornea seemed to be perfectly normal clinically. A re-check of our live/dead cell staining results showed, as before, excellent viability and the lack of appreciable cell loss. Checking for cell junctions between endothelial cells using the Alizarin stain and light rather than electron microscopy demonstrated the persistence of cell-cell junctions as we had previously observed, but endothelial cell shapes were irregular, in contrast to our previous results. Perhaps this irregularity is related to the difficulty of visualizing these junctions by scanning electron microscopy. The grant stated that we would determine the critical cooling and warming rates
LEF Grant Application for the Year 2005 Page 11 of 52 for our vitrifiable corneas, within certain limits. One cornea was evaluated by DSC and did not yield any clear peaks indicative of either freezing or vitrification regardless of the cooling and warming rates used . We believe that is because the cornea equilibrates with its medium more by losing water than by taking up M22. Without water, even signs of a glass transition would be expected to be hard to see, as we observed. In handling this cornea, we noticed that it had the physical consistency of a thin piece of plastic, consistent with extreme dehydration.
E) Cryoprotectant Technology.
Our studies for Genzyme Biosurgery described below resulted in the discovery of a new cryoprotective agent. A new cryoprotective agent comes along about once every 30 years, so it was important to better define the possible uses of this new agent. We found that replacing either of two components of M22 with the new cryoprotectant resulted in lower toxicity in our rabbit renal cortical slice model. We pointed out in our 2004 proposal the need to run a toxicity neutralization curve for the new agent, determine its optimum concentration , its intrinsic toxicity, and its permeability to renal cells to enable us to craft a reasonably comprehensive patent application that appropriately defines the scope of utility of and optimal compositions containing this new agent. This remains to be completed. Our initial successes with the new agent progressed to the discovery of M22 replacements that could be used at temperatures as high as -5 to -15°C, in contrast to the need to use M22 at lower temperatures, with recoveries as high as or higher than that obtainable in our classical M22 at -22°C model. These studies resulted in the M10 solution (intended for use at -10°C) that was tried in the whole kidney model as a means of speeding equilibration by reducing viscosity and therefore increasing flow. It remains possible that this solution represents an advance over M22 for the whole kidney, despite our negative initial results discussed above. A second implication of our Genzyme results and of the qv* theory of cryoprotectant toxicity is that there might be a different way of combining our basic cryoprotectants so as to form still less toxic solutions. Following up on these possibilities resulted in a solution called M22B, which was later found to be dramatically less toxic to whole kidneys than M22, as noted above.
F) Physical Science Research.
Ice Control
Quantitating the effectiveness of ice control in M22. Our most useful solution to date, M22, is so stable that in the past we were unable to measure its critical cooling rate and its critical warming rate, or to define any condition in which it could be frozen. Partly in view of our proposal to discontinue B2C in favor of M22, we decided to attempt again to measure the critical cooling rate and the critical warming rate of M22, which are measures of the effectiveness of our total ice control measures (specific polymers plus colligative agents). Two novel methods were used for these studies. In the first, we took advantage of the ability of our intermediate temperature storage (ITS) units to double as controlled rate coolers and controlled rate warming devices. We vitrified 10 ml
LEF Grant Application for the Year 2005 Page 12 of 52 samples of M22 at 7°C/min in scintillation vials in a "corneastat" (an ITS unit built specifically for cornea storage in our lab) and found no devitrification after warming at a constant rate of 0.4°C per minute, but slight devitrification at 0.2°C/min, as determined by inspecting the samples at a temperature of -70°C (15°C below the melting point). In order to detect ice formation during cooling, a very large volume of M22 was cooled to allow even a few crystals to be observed within a vast excess volume with no ice formation. Vitrification of two liter volumes of M22 in glass Erlenmeyer flasks was studied. It was possible to vitrify this solution without visible ice crystals at a cooling rate of only 0.15°C per minute. (A cooling rate of 0.10°C per minute caused a small number of ice growth sites to appear in the solution.) Based on these results, we very conservatively set the critical cooling rate at 0.15°C/min and the critical warming rate at 0.4°C/min. An abstract describing these critical cooling rate and warming rate results has been accepted for oral presentation at the July, 2005 Annual Meeting of the Society for Cryobiology. Ice control by novel polymers. The polymer polyvinyl pyrrolidone (PVP) is used in 21CM vitrification solutions because of its low viscosity and extraordinary resistance to ice formation ("glass forming" ability). Specifically, low molecular weight PVP of the form PVP K12 (aka PVP5K) of a nominal molecular weight of 2,500 is used. (The internal company moniker "PVP5K" arose because the polymer vendor at one time erroneously told 21 CM that the polymer weight was 5,000.) Osmometer measurements revealed that the actual number average molecular weight of PVPSK is probably 1200. This is the lowest molecular weight PVP commercially available. A low molecular weight PVP is desirable because of low viscosity in solution, but also because of the unusual observation that the ice inhibiting (glass forming) ability of PVP increases with decreasing molecular weight. To push this frontier further in 2004, a 25% solution of PVP5K was passively dialyzed over 20 hours with Sigma D-0655 high retention dialysis tubing to isolate lower molecular weight PVP molecules. The resulting product, dubbed "PVP1K", had a molecular weight of 700 vs. the 1200 of PVP5K. At 30% w/w concentration, it had a 30% lower viscosity than PVP5K. As expected, it was also a better glass former than PVP5K. In parallel with the PVPIK studies, other water soluble polymers were also re- investigated as potential alternatives to PVP. In particular, poly(2-ethyl-2-oxazoline) (PEO), polyacrylamide (PAA), and various weights and terminations of polyethylene glycol (PEG) were studied. PEO and PAA were found in past studies to be poor glass formers, and were therefore regarded as potential low-toxicity cryoprotectants per the qv* theory of cryoprotectant toxicity. The data obtained are presented in Table 1. PEO was ruled out as too viscous. PAA might warrant more investigation as a novel poor glass forming (therefore low toxicity) polymer. However, unless it can be confirmed that the low molecular weight reading is due to an impurity that can be removed, it would seem to posses an undesirably high viscosity for its molecular weight. The glass forming ability of decaglycerol, PEG400, and PEGI000, and PEGDME were found to be the same. The glass forming ability of PEG3350 was decidedly better than any of the lower molecular weight PEGs, although still not as good as PVP5K. Apparently the glass forming ability of PEG increases with molecular weight, rather than decreasing like PVP.
LEF Grant Application for the Year 2005 Page 13 of 52 Table I ------Polymer Viscosity at 30% w/w Molecular Weight (by osmometer) ------PVPIK 5 cP 700 PVPSK 7 cP 1300 PEO 40 cP not measured ( 1000 nominal) (1500 nominal) PAA 7 cP 270 4• PEG3350* 15 cP 2000 PEGDME** 5 cP 500 PEG 1000* - 800 PEG600* 3.7 cP 500 Decaglycerol 3.7 cP 330 PEG400* 3.2 cP 340
*PEGn = PEG of nominal molecular weight n **PEGDME - 1000 nominal molecular weight PEG dimethyl ether
Sieving Characteristics of X-1000. The X-1000 ice blocker at 20% concentration in water is known to self-associate into particles on the order of one micron in diameter, making such solutions cloudy. Although the self-associating molecules of X-1000 represent a distinct subfraction of the product, there was concern that polymer aggregates might block renal glomeruli, thereby slowing cryoprotectant equilibration of kidneys being prepared for vitrification and slowing distribution of X- 1000 into the urinary space. In 2004, experiments were conducted to test whether X- 1000 at typical vitrification solution concentrations caused any blocking of pores in dialysis tubing. Aldrich D0655 dialysis tubing (which retains >99% of cytochrome c, M.W. 12400, over 10 hours) was filled with 50% ethylene glycol and 1% X-1000. Ethylene glycol was found to escape from the tubing at the same rate as a control ethylene glycol solution without X-1000. A similar experiment with 25% PVP5K also showed no inhibition of PVP polymer penetration of the tubing by X-1000. Kidney vitrification experiments with solutions excluding X-1000 initially seemed to indicate greater stability of the renal medulla without X-1000, but later observations suggested the opposite. When the jump is made to M22 in an actual kidney perfusion, the driving force for diffusion is a concentration gradient of about I molar, or 10.8% w/v, rather than 50%. Therefore, while we believe the above results are indicative, a more fully definitive result could be obtained by measuring the diffusion of M22 inside the dialysis bag into VMP outside the dialysis bag. Ice control in nature. We collaborated with Agrigenesis Biosciences, Ltd., of New Zealand, to search for synergy between our solutions and their newly discovered antifreeze proteins. The results to date and plans for future research as discussed below under Collaborative Research. We also collaborated with Jack Duman at Notre Dame University on the possibility that the Alaskan beetle, Cucujus, might spend the winter in a vitrified or deeply supercooled state. Those studies, also, are described under Collaborative Research. Contract DSC research on possible Alaskan microbial glass- forming proteins is described below under Contract Research.
LEF Grant Application for the Year 2005 Page 14 of 52 Fracture Avoidance
Previously it was found possible to cool 15 mL samples of M22 vitrification solution in polyethylene vials to the temperature of liquid nitrogen (-196°C) and successfully rewarm them without fracturing if they were held at ambient temperatures of -135°C, -150°C, and -185°C for one hour during both cooling and rewarming. In 2004 an attempt was made to cool a 100 mL volume of M22 solution in a 5 cm diameter polyethylene vial to -190°C without fracturing using similar techniques. The sample was maintained at -135°C for 12 hours, -150°C for three hours, and -190°C for 12 hours. Only one horizontal fracture appeared 1 cm above the vial bottom during the -190°C phase . This is an encouraging pilot result showing that human kidney-sized solution samples can be cooled well below the glass transition temperature (-124°C) without fracturing. To permit further study of slow cooling (annealing) protocols and fracturing, means are required to maintain solutions at controlled programmed temperature for longer periods of time. To achieve this, construction was begun on three containers capable of holding 100 mL solutions at controlled cryogenic temperatures for annealing study. The containers are being constructed using the same core technology used to build the Corneastat, Neuropod, and laboratory Intermediate Temperature Storage (ITS) devices developed at 21 CM. Our most dramatic result to date was a demonstration that it is possible to vitrify even 2 liters of M22 in an Erlenmeyer flask without fracturing. We used a monotonic (steadily decreasing temperature) protocol and showed (Figure 3) that if the
Figure 3: Large volume vitrification without freezing or fracturing. Two liters (5 pounds) of M22 solution cooled until vitrified as a solid at a temperature of - 124°C. The mass of this solution is about 60% larger than the mass of a typical human brain.
solution center is not allowed to descend significantly below the glass transition temperature of -124°C, fracturing is avoided. Cooling five degrees colder caused
LEF Grant Application for the Year 2005 Page 15 of 52 fracturing, but it must be remembered that this was a monotonic cooling protocol, not one in which particular annealing steps were introduced.
Intermediate Temperature Storage
Three intermediate temperature storage (ITS) product lines have been prototyped to date. The first ITS unit was designed as a neuropod in 2003. A second version of this prototype was constructed and sold to Alcor in 2004. Like the first i• prototype neuropod unit, the neuropod permits "fail safe" storage at an adjustable Q temperature by simple placement of the unit in a cryogenic dewar. The 2004 unit included a new controller with dual redundant temperature controllers and an audio alarm (Figure 4).
21CM Neuropod Controller Schematic 0312004
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0000 0000 the dual Heati ng Heati ng redundant ElementJaok 2 AUd10 ElementJadc I controller • Alarm • 500 ma N N N Audio Nann constructed 1 F use (internal) I for the Heater Heater Power Power more 8000 Select 8000 Select 25 W Switch 25 W Swito, advanced 3000 3000 neuropod 05W 05W 1000 1000 prototype 2W 2W delivered L.E.D L.E D to Alcor in 2004. N 0
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LEF Grant Application for the Year 2005 Page 16 of 52 The second ITS prototype version was a unit developed to store cryopreserved corneas (hence, referred to as a "corneastat"). It is still in use at 21 CM. The third ITS prototype version was a large unit built inside a cryogenic dewar, and designed to replace low temperature laboratory freezers . In early 2004, it replaced 21CM's CryoStar laboratory freezer and has functioned relatively smoothly ever since. The final version of 21 CM's patent application for intermediate temperature storage technology was filed in June, 2004, and the ITS concept will be presented at the 2005 annual meeting of the Society for Cryobiology.
G) Kidney Conventional Preservation (TransSend)
Our Small Business Innovation Research (SBIR) grant from the National Institutes of Health (NIH) continued to fund research on the development of our TransSend kidney preservation solution until April 15th, 2004. The research being completed until then was intended to show that TransSend (TS) has at least a 3-6 month shelf life before it becomes ineffective, and this goal was attained . In fact, our collaborative experiments involving the use of 3-month-old TransSend-4 to preserve dog kidneys at the University of Wisconsin were spectacularly successful , with stored TransSend-4 dramatically outperforming UW solution (Figure 5).
6 8 V uws t 5 • TS E 7 E 0 U+TFs 6 4 5 3 c 4 G 3 2 U) U U 2 E LM 1 a) ,a)^ VJ v/ 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 101214 Postoperative Day Postoperative Day
Figure 5: Effect of 4 days of storage at 0°C on dog kidney integrity as indicated by serum creatinine levels after transplantation into canine recipients. Left: TransSend (red circles) yielded dramatically lower mean creatinine levels at all postoperative time points in comparison to UW solution, the current world leader (blue triangles). The yellow diamonds show UW solutions plus protein "trophic factors" developed by Jon McAnulty that cost over $100 a liter to put into UW solution. By contrast, TransSend's components are dramatically cheaper than those of UW solution. Right: Individual results for UW solution (gray lines) and TransSend-4 (blue lines). All but the worst TransSend kidney did better than all but the best UW solution kidney.
Although we expressed concern in our previous grant letter that it might be necessary to fund studies on TransSend-9 in a canine model to further support our Phase
LEF Grant Application for the Year 2005 Page 17 of 52 II SBIR application, the results in this figure seemed to make that unnecessary. Based on Figure 5, TransSend is the best organ preservation solution in the world. All proposed experiments were completed successfully, and we wrote and submitted an extensive progress report to NIH. The conclusions were as follows. First, TransSend (TS) is superior to our predecessor solution, Renasol. Second, TS is superior to UW Solution in both the rabbit and the dog model. Third, the addition of McAnulty's "trophic factors" to TS has no significant effect in the rabbit, but may have a positive effect in the dog. Fourth, storage of TS for 3 months is not harmful to the solution, and storage for 6 months is probably acceptable as well, but more data are needed to confirm the innocuousness of 6 month storage. Fifth, urinary proteomic markers of renal injury as discovered using the Ciphergen mass spectrometer correlated with injury moderately well in the rabbit and in the dog, but different markers were observed in the two species, and marker levels rose with injury in the rabbit and fell with injury in the dog. A universal biomarker of renal cold storage injury was not found. When grant support ended for this project in 2004, we ceased further experiments on renal cold storage for the balance of the year.
H) Cardiac Cold Storage and Intermittent Perfusion.
Our Phase 2 SBIR grant on "Extended Cardiac Preservation" was funded on August 1st, 2004. After this date, we spent extensive time writing protocols for the procurement of human hearts by organ procurement agencies and in designing both the equipment for evaluating human hearts during normothermic blood perfusion and the laboratory space in which these experiments are to be carried out. As of the end of 2004, however, the new lab space was not completed and many components of the perfusion machine were still being identified and ordered. In addition, we experienced a lack of feedback from the organ procurement agencies that prevented any firm agreements from being signed before the end of 2004.
I) Basic Research.
A burning question for the long-term storage of mammalian systems near Tg is how rapidly damage accumulates with time as a function of temperature near and below Tg. If we could be sure it were safe to store M22-protected organs at, say, -135°C, for several hundred years, we would be able to reduce the risk of fracturing at lower temperatures very considerably. A mathematical model of injury rates developed by Fahy and based on biological damage following WLF kinetics implies that very long storage periods will be feasible at temperatures only 10-20°C below Tg, but other evidence makes this conclusion questionable. Actual experimental data are sparse and needed. Because damage rates may be too slow to conveniently measure below Tg, the best way to infer the temperatures that provide, for example, 100-1000 years of safe storage time may well be to measure injury development rates at higher temperatures and extrapolate them to lower temperatures. We therefore carried out the beginning of such a process, starting at the relatively convenient temperature of dry ice. Unfortunately, M22 stored for two days at dry ice temperature actually developed ice crystals, which is the first case in which we had been able to freeze M22. Although this ice seemed to develop from the surface and not the body of the solution, it precluded us from doing
LEF Grant Application for the Year 2005 Page 18 of 52 injury kinetics studies for the time being and undermined our conjecture that it might be feasible to store organs above the glass transition temperature . We may be able to prevent this ice formation by modifying the surfaces present in such experiments, but for the time being we have deferred these studies pending the solution of other more immediate problems. We also began to map the intensity of chilling injury in renal cortical slices as a function of temperature during cooling in M22, since this is the solution we are now using for the vitrification of both kidneys and brains. The results were very contrary to expectation. Damage at -40°C seemed to be greater than damage at -50 to -80°C, but damage then increased again between -80°C and -125°C (Tg) to a level similar to that seen at -40°C. These results contrast to those we have seen before in our slice model using other solutions, but are compatible with our whole kidney results. Additional studies are needed to either validate or invalidate these initial observations. In the course of the studies described under the Cryoprotectant Technology section above, indirect information was obtained on the effect of cooling renal cortical slices to -22°C at a tonicity of 1.4X. This tonicity has been little studied before but was known to be within a putatively optimal range between 1.2X and 1.5X. We have generally cooled to -22°C at a tonicity of 1.2X to avoid overshooting the 1.5X limit when increasing cryoprotectant concentration simultaneously with cooling, which can contribute to the apparent tonicity of the solution. However, in developing variants of M22 in the experiments described above, this approach was not appropriate to the symmetry of the experiments, and cooling instead took place at 1.4 X. The significance of this is that in many experiments, little or no injury was observed from the combination of cooling to -22°C and exposure to vitrification solution . This can only have been possible if both cooling injury and toxicity were both essentially eliminated at the 1.4X tonicity level chosen. This observation suggests that not only may it be possible to ignore permeating cryoprotectants when determining the tonicity to use for cooling, but it may also be that I.4X is the optimum tonicity for eliminating chilling injury. Dr. Fahy began modeling studies to predict the response of human oocytes to freezing under various conditions and to predict their shrink-swell responses to the addition of various cryoprotectants. Some of these results were used in his presentation at the First International Meeting on the Cryopreservation of the Human Oocyte, and some were used to prepare for possible collaborative studies with the clinic that sponsored this meeting.
J) Contract Research.
Genzyme Biosurgery
Based on our successful first phase of research to develop a formamide-free vitrification solution for use by Genzyme in 2003, the second phase of the project began in 2004 to select a single formula and a single addition and washout method for its use. We successfully completed these Phase 2 experiments and in the process developed a new solution, VeGD, that was actually less toxic than any of the three candidate solutions originally deemed to be satisfactory by Genzyme and, in fact, was even less toxic than VM3. This solution contains a novel cryoprotectant (see Cryoprotectant Technology section, above) that will form the basis of at least one forthcoming patent application.
LEF Grant Application for the Year 2005 Page 19 of 52 We sent Mr. John Phan, who conducts all of our kidney slice experiments, to Genzyme to transfer the technology of using the new solution to them to enable them to test our new solution and addition and washout protocol on porcine and human cartilage. This process went smoothly and to the satisfaction of all parties, and initial results with porcine cartilage were good (essentially zero percent cell death and cell loss and zero percent reduction in cell proliferative competence, a result 10 times better than required by Genzyme) despite temporary delays in adherence of liberated chondrocytes to their culture vessels. This supports the premise of our studies for Genzyme that findings in rabbit kidney slices based on the K+/Na+ ratio assay will convey to porcine and human cartilage based on cell recovery and proliferation. Genzyme progressed through its own internal experiments with VeGD for the balance of 2004, looking at both LM5 and its own tissue culture medium as alternative carrier solutions for the VeGD cryoprotectants. The next phase in the Genzyme studies will be to develop practical methods for actually vitrifying and rewarming cartilage samples . Experiments relating to this phase have already been completed, resulting in a vitrification protocol to be tested on site at Genzyme during 2005. The recoveries after vitrifying renal cortical slices in our preliminary studies with VeGD were not as good as would be expected for VM3, but were deemed to be good enough to meet Genzyme's needs.
Isolagen
Our communications with Isolagen did not result in a contract with them, in significant part due to internal realignments at Isolagen. This company would like us to do contract research for them on techniques for freezing skin cells that would allow the frozen skin cells to be thawed in a doctor's office and injected directly into the face of a patient. They have a strong need for our services, but it is up to them to make the decision to fund a project that would allow us to attempt to solve their problems.
Stem Cells
We were contacted by Dr. Michael West about collaborating on a grant that would result in contracts from the entity created by the California stem cell initiative. However, it became apparent that funding from this source is not likely for some time to come. We were contacted by Dr. Evan Lampieter about vitrifying cord blood stem cells in order to circumvent a troublesome patent on freezing cold blood stem cells. If vitrification works, it might give 21st Century Medicine an exclusive on the banking of cord blood stem cells in the USA. Ultimately, Dr. Lampieter solved his problem by confining his interests to Europe, and our attempts to pick up the trail and pursue this opportunity in the US were not successful. We were contacted by Thomas Bob, who requested on behalf of his company a proposal from us to cryopreserve stem cells derived from adolescent tooth primordia. These are basically embryonic stem cells available from adolescents , and could be highly lucrative (Eli Lily already pays $25,000 for 1 million of these cells). The deal would have also involved purchase of many of our CIVS units. Unfortunately, Organ Recovery Systems was on the advisory board of St. Jude Medical Center, a research
LEF Grant Application for the Year 2005 Page 20 of 52 partner of this group, and advised them to do business with Organ Recovery Systems rather than with us, which killed this particular deal. Our communications with Johns Hopkins University (Dr. Hankins) about cryopreserving tumors and tumor stem cells to facilitate chemotherapeutic susceptibility testing and the cure of cancer have stalled due to inaction on both sides.
Miscellaneous
We were contacted by Dr. John Garrisi, the Laborato ry Director of the Institute for Reproductive Medicine at the St. Barnabas Tertiary Care Facility in New Jersey about a collaborative project to vitrify human ova. Unfortunately, the perception on the part of the hospital administrators that 21 CM is linked to cryonics led to their emphatic decision not to work with us on this project. We were paid to perform a limited series of DSC studies on Alaskan environmental samples to determine whether micro-organisms in the samples might be secreting agents that facilitated vitrification. The results indicated that this is not the case. We were contacted by Dr. John Morris of Asymptote, Ltd., who became aware of us as a result of the Beijing meeting of the Society for Cryobiology in 2004, about a major opportunity in food preparation that would be brought about if consistent supercooling could be obtained. We sent samples of Z1000 and instructions on its use, but never received any feedback on the results. It is ironic that in the food industry, the major use of the Z1000 active ingredient is as a series of esters that might very well have greater anti-nucleation activity than Z1000 itself. Unfortunately, we have not yet tested such esters in our laboratory, though efforts are underway to find commercial sources for them. We were contacted by Dr. John Todd, a Canadian surgeon who has started a business to pursue cell, tissue, and organ preservation with a new cross-linked antifreeze glycoprotein preparation that appears to also allow red blood cells to survive freezing to -18°C without a permeating cryoprotectant. He may want to contract with us to perform some transplants of organs preserved using his protein complex.
K) Collaborative Research.
Protein Ice Blockers
On January 8t', 2004 Dr. Fahy was contacted by Paragon Development about an "IceControl" meeting being set up on February 3`d, 2004 on behalf of a client company that has discovered novel proteins from grass that powerfully inhibit recrystallization. Dr. Fahy attended the meeting on February 3`d and a collaborative relationship between 21st Century Medicine and the client company , Agrigenesis Biosciences , Ltd., of New Zealand, was established. Our main interest was to determine possible utility and ice blocking synergy of their proteins in combination with our solutions. 57% w/w ethylene glycol plus 0.01% antifreeze protein, X-1000, or polyethylene glycol were compared in a hanging vial devitrification assay. The antifreeze protein was found to inhibit devitrification to a comparable or slightly greater extent than X-1000. However 0.01% antifreeze protein added to dilutions of M22 vitrification solution (already containing close to 1% X-1000 and 2% Z-1000 ice blockers) provided no benefit, suggesting that
LEF Grant Application for the Year 2005 Page 21 of 52 any ice blocking provided by the antifreeze protein was swamped by 21 CM' s own ice blockers at the concentrations tested (300 times more synthetic ice blocker than natural ice blocker by weight). More studies are planned in 2005 to resolve this issue and the fundamental issue of synergy with our other ice blockers.
Vitrification as a Strategy in Nature
We were contacted by Dr. Jack Duman of Notre Dame University in Indiana about testing some Alaskan insects he recently discovered that may actually survive the bitter cold of the Alaskan winter by becoming vitrifiable. If they do, it would be a very significant scientific discovery. It may be that they naturally survive the winter in the vitrified state, or it may be that in nature they have the ability to vitrify but never experience temperatures low enough to vitrify them, so that they survive the winter in a supercooled or "rubbery" state. To learn more about these insects, Dr. Duman traveled to Alaska, retrieved these insects, and sent them to us in liquid nitrogen vapor using a commercial shipper we provided . We tested them using our differential scanning calorimeter to find out a) whether they vitrify naturally, and, if so, b) whether they survive after vitrification and rewarming in our DSC. Unfortunately, our initial results were negative, each insect showing melting endotherms at relatively high temperatures. Further studies were planned for 2005.
Drug Development
Inge deGraaf has reactivated her work on vitrifying kidney and liver slices using our VM3 solution to permit pharmaceutical companies to have ready access to drug metabolizing tissues for drug development research. We are providing her with VM3 and guidance and she is covering her other costs and is advising us of her progress. She has reproduced her positive results with the vitrification of rat kidney slices (e.g., freezing yielded 3% recovery of renal cortical ATP and 15% recovery of renal medulla ATP, but vitrification yielded 50% and 55% recovery, respectively), but had trouble repeating her positive results with the vitrification of rat liver slices with VM3 (vitrified slices being about the same as frozen slices). The main problem with the liver seems to be chilling injury. She is now modifying the introduction and washout methods for liver slices and is finding that trehalose is a better osmolyte for washout of cryoprotectants from liver slices than lower molecular weight agents, which is not surprising considering the liver's high permeability to solutes like mannitol. Avoiding chilling injury by adjustment of medium tonicity has not yet been tried.
Ovarian Tissue Vitrification
Near the end of 2004, we were approached by Armand M. Karow, Jr., the owner of Xytex, Inc., an international sperm banking company, about assisting him with the vitrification of ovarian tissue as a way for women to preserve their fertility without the requirement of learning how to freeze or vitrify their ova. Non-disclosure agreements were signed, protocols and solutions were provided to Dr. Karow, but no results became available by the end of 2004.
LEF Grant Application for the Year 2005 Page 22 of 52 Coral Organism Cryopreservation
Dr. Fahy accepted an invitation from Dr. Mary Hagedorn of the Smithsonian Institution and the National Zoo to join an international team to work on the problem of vitrifying the early larval stages of Hawaiian coral to provide a backup to the disappearance of coral reefs around the world in the face of global warming and increasing levels of atmospheric carbon dioxide. Several 21 CM vitrification solutions were used to attempt the vitrification of these organisms, but only partial success was obtained in the very short time available during the reproductive season of the coral organisms. The results of these efforts have been submitted for publication.
L) Discontinued projects.
We had taken steps to begin a project with HepaHope, Inc. circa November, 2004, but HepaHope balked at paying us $3,000 up front for one month of consulting services. It turned out that this company was financially unable to continue its operations, so we abandoned this project pending any further developments on the part of HepaHope.
2. Scientific publications.
2004 Scientific Publications:
Fahy, G.M., Wowk, B., Wu, J., and Paynter, S. "Improved vitrification solutions based on the predictability of vitrification solution toxicity." Cryobiology 48: 22-35, 2004.
Fahy, G.M., Wowk, B., Wu, J., Phan, J., Rasch, C., Chang, A., and Zendejas, E. "Cryopreservation of organs by vitrification: perspectives and recent advances." Cryobiology 48: 157-178, 2004.
Lemler, J., Harris, Platt, C., and Hufmann, T. "The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence." Annals of the New York Academy of Science 1019:559-563,2004. [This paper was ghost-written by Dr. Fahy.]
B. Wowk, Medical Time Travel, in: The Scientific Conquest of Death, Libros En Red, 2004, pp. 135-150.
Fahy, G.M. Vitrification of organs and tissues . Cryobiology 49: 293-294, 2004.
2005 Publications to date :
B. Wowk, Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker, Cryobiology 50, 325-331, 2005.
PendingPublications:
Pichugin, Y., Fahy, G. M., and Morin, R.M. "Successful cryopreservation of rat hippocampal slices by vitrification ." Cryobiology (in revision for publication in 2005).
LEF Grant Application for the Year 2005 Page 23 of 52 Hagedom, M., Pan, R., Cox, E., Hollingsworth, L., Krupp, D., Lewis, T.D., Leong, J., Mazur, P., Rall, W., MacFarlane, D., Fahy, G., and Kleinhaus, F.W. Coral larvae membrane physiology and cryobiology. Biol. Reprod. (submitted for publication), 2005.
B. Wowk, G.M. Fahy, Toward large organ vitrification : extremely low critical cooling and warming rates of M22 vitrification solution. (submitted as an abstract for the 2005 Annual Meeting of the Society for Cryobiology.)
B. Wowk, G.M. Fahy, Controlled temperature environments for maintenance and shipment of vitrified tissue. (submitted as an abstract submitted for the 2005 Annual Meeting of the Society for Cryobiology.)
Fahy, G. Vitrification as an approach to cryopreservation. (submitted as an abstract for the 2005 Annual Meeting of the Society for Cryobiology.)
Fahy, G.M. "The story of RPS-2." Cryobiology (in preparation for submission in 2005).
Fahy, G.M., Wowk, B., and Tan, Y. Prospects for the arrest of aging by physical means. (in preparation for submission to Rejuvenation Research).
3. Scientific presentations and media coverage during 2004 and early 2005.
Presentations:
Dr. Fahy spoke on possible applications of antifreeze proteins in agriculture and biology at a meeting held by Paragon Development on "IceControl" on February 3fd, 2004 in Los Angeles. (This initiated our collaborative work with Agrigenesis described above.)
Dr. Fahy delivered an invited keynote talk entitled "Vitrification of Organs and Tissues" at the 2004 meeting of the Society for Cryobiology in Beijing, China, on July 16th, 2004.
Dr. Fahy presented an invited seminar entitled "Life Extension by Brute Force: How Cryobiology Could Save Your Life" on life extension through the transplantation of cryopreserved tissue and organ replacements at the Kronos Longevity Research Institute in Phoenix, Arizona, on Sept. 10th, 2004.
Dr. Fahy gave an invited presentation entitled " Physicochemical Considerations for Oocyte Vitrification " at a meeting on "Cryopreservation of the Human Oocyte" on May 16`h, 2004 in Bologna, Italy.
Dr. Wowk will present two oral talks ("Toward large organ vitrification: extremely low critical cooling and warming rates of M22 vitrification solution" and "Controlled temperature environments for maintenance and shipment of vitrified tissue") at the 2005 Society for Cryobiology meeting in July.
LEF Grant Application for the Year 2005 Page 24 of 52 Dr. Fahy was invited to give a plenary lecture on vitrification at the 2005 annual meeting of the Society for Cryobiology in July at the University of Minnesota. (This will be his fifth consecutive invited major presentation at the annual cryo meetings, and seems to set a new record for the longest streak of such presentations in the history of the society.)
Dr. Fahy was invited to give a talk at SENS-2 (in September, 2005, in Cambridge in the UK) entitled "Cryopreservation: The Missing Link in the Regenerative Medicine Supply Chain." He will discuss the relationship of cryobiology to tissue engineering of replacement parts to be used to correct accumulated aging-related organ damage.
Media Coverage:
A press release on our discovery of methods capable of allowing the routine recovery of rabbit kidneys from -45°C was picked up by a number of media outlets.
A second press release on our award of an NIH grant for over $800,000 to develop advanced means of cardiac cold storage was also picked up by a number of financial news sites.
21st Century Medicine was mentioned in the Honolulu Star-Bulletin Hawaii News newspaper (see also http:%/starbuIletin.com12004/07/07ine%%s`storv 2.htm 1 ) on August 6, 2004, in connection with Dr. Fahy's participation in research at the University of Hawaii ' s Institute of Marine Biology on Coconut Island in Oahu' s Kaneohe Bay on the cryopreservation of coral organisms.
Dr. Fahy appeared on KITV (Channel 4, Hawaii) in July, 2004, in a news feature called "Coral Sea Dreaming" that was about coral organism cryopreservation (Gary Sprinkle, news anchor).
21st Century Medicine was featured in a news feature ("Life Extension Funds Research To Combat Aging & Death") announcing our NIH award in Life Extension Magazine in a special Winter 2004/2005 issue.
21 st Century Medicine was mentioned in an article in The National Geographic about cryonics, where Dr. Wowk corrected errors of fact presented by scientists working at or affiliated with Organ Recovery Systems, Inc.
Dr. Wowk was interviewed for a documentary, "Exploring Life Extension," produced by the Immortality Institute and released in 2005.
21st Century Medicine was featured in a documentary, "The Limitless Future," produced by WalshCOMM, an independent film making company. We were depicted as a cutting edge cryobiological biotechnology company not affiliated with cryonics or Alcor. Both Dr. Wowk and Dr. Fahy were filmed for this documentary, which was released in 2005.
LEF Grant Application for the Year 2005 Page 25 of 52 4. Patent awards, grant awards, contract awards.
Patents Issued:
"Methods of using ice-controlling molecules" U.S. Patent 6,773,877, August 10, 2004. G.M. Fahy.
"Advantageous carrier solution for vitrifiable concentrations of cryoprotectants, and compatible cryoprotectant mixtures" U.S. Patent 6,869,757 B2, March 22, 2005. G.M. Fahy
Patents Approved for Issue :
"Polyglycerol and lactose compositions for the protection of living systems from states of reduced metabolism" U.S. Patent application no. 10/066,285, filed 2/1/2002. G.M. Fahy and J. Wu. (This is a patent giving us very broad protection for TransSend.)
Grants Awarded in 2004:
A Phase II SBIR submission to NIH entitled "Extended Preservation of Hearts," G.M. Fahy, P.I., for a requested $957,587, from the National Heart, Lung, and Blood Institute (NHLBI), submitted to NIH on December 12th, 2003, was funded on August 1S`, 2004 at an $835, 104 level.
A Phase 1 SBIR submission to NIH entitled "Cornea Preservation by Vitrification," G.M. Fahy, P.I., for a requested $195,024, from the National Eye Institute, submitted to NIH on December 3rd, 2003, was funded for the requested amount on September 30th, 2004.
2004 Grant Support from Grants Awarded before 2004 :
Our Phase 1 SBIR award (1 R43 DK63806-01, "Improved Renal Preservation"), funded at the requested amount of $154,641 for renal cold storage solutions beginning in April, 2003 from the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), provided support through 4/15/04.
Our Phase I SBIR award (1 R43 HL66813-01A1, "Extended Cardiac Preservation"), funded at the requested amount of $164,852 for cardiac preservation by automated intermittent perfusion beginning in August, 2002 from the NIH, National Heart, Lung, and Blood Institute, remained in effect through 7/31/04.
5. Patent and NIH grant applications.
Patent Applications Filed:
"Methods and Compositions for the Cryopreservation of Organs," Patent Convention Treaty (PCT) Application PCT/US2004/030544, filed September 16`h, 2004. G.M. Fahy and B. Wowk.
LEF Grant Application for the Year 2005 Page 26 of 52 "Cryogenic Storage System" U.S. Patent Application 10/864,921, filed June 9, 2004. Brian Wowk and Michael larocci.
"Warm Intermittent Perfusion" U.S. Patent Application 10/871,504, filed June 17, 2004. G. Fahy and T. Wang.
"Extended Organ Preservation," PCT Application, filed June 17th, 2005. G.M. Fahy and T. Wang
"Improved Cryoprotectant Solutions" India Patent Application No. IN/PCT/2001 /00252/DEL, Request for Examination March 25th, 2004. G. Fahy and B. Wowk.
Grants Submitted in 2004 :
A Phase II SBIR submission to NIH entitled "Improved Renal Preservation," G.M. Fahy, P.I., for a requested $935,587, from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), was submitted on December 1st, 2004.
A Phase 1 SBIR submission to NIH entitled "Vitrification of Neural Tissue," Y. Tan, P.I., for a requested $125,000, from the National Institute of Mental Health (NIMH), was submitted on December 12`h, 2004.
A Phase I SBIR submission to NIH entitled "Resuscitation of Non-Heart-Beating Donor Hearts," G. Fahy, P.I., for a requested $150,000, was submitted to the NHLBI on December 12th 2004.
Grants Submitted in 2005:
A Phase 1 SBIR application to NHLBI entitled "Resuscitation of Non-Heart-Beating Donor Hearts," G. Fahy, P.I., for a requested $199,000, was resubmitted on April 1st, 2005.
Grants to be Submitted in 2005 :
A Phase I SBIR application to NIMN entitled "High throughput optical recording system," Y. Tan, P.I., for a requested $150,000, is planned to be submitted by the August 1 s`, 2005 deadline. This work will be in collaboration with Dr. Wu in Georgetown University, an expert in this field.
A Phase II SBIR submission to NIDDK entitled "Improved Renal Preservation," G.M. Fahy, P.I., for an estimated $950,000, will be resubmitted by the August 1 S`, 2005 deadline. (This grant missed funding by one slot on June 30th, 2005.)
LEF Grant Application for the Year 2005 Page 27 of 52 9. Proposed research and preliminary results for 2005.
A) Brain Vitrification.
Brain Slice Cryopreservation
Preliminary Results for 2005 . Studies have focused on recapturing the success initially observed in our VM3 series. A systematic series of experiments was devoted to "debugging" the protocol for adding and removing VM3, using recovery of electrical responsiveness as the key end-point, although we continued to measure the K+/Na+ ratio as a supplemental test. Lowering the temperature of exposure to VM3 from -10°C to -14°C seemed to be helpful, but was not sufficient. Surprisingly, basic investigations of the tolerance of these slices to osmotic changes indicated that they were far more sensitive to prolonged hypertonicity than to hypotonic exposures. This observation led us to try reducing the concentration of mannitol used in the VM3 washout steps. Again to our surprise, and in seeming contradiction to the Harbor-UCLA experiments, better results were obtained when mannitol was reduced from 300 mM to 100 mM, and still better results were obtained when no mannitol was used at all. This strongly confirmed that hippocampal slices are sensitive to prolonged shrinkage but are not particularly sensitive to swelling, the reverse of what holds true for most cells. Since each step of adding cryoprotectant involves transient shrinkage of the tissue, we sought to limit this shrinkage by using a continuous-gradient, pump-d riven system for part of the cryoprotectant addition and washout process rather than using discrete concentration steps. This change in procedure significantly improved our results, giving reasonable recovery of electrical activity and essentially complete recovery of K+/Na+ ratio. However, we were still frustrated by the fact that some slices failed to respond at all while others may be able to respond reasonably well but not entirely up to control standards. Further consideration of the method showed that the pumping system used, while better than stepwise methods, actually produced the worst possible curve of concentration vs time, concentration rising rapidly and then more slowly. To determine whether further gains could be obtained, we tested a program of manual re-setting of the pump speed according to a predetermined schedule so that concentrations would rise slowly at first and then more rapidly, and, on washout, would fall rapidly at first and then more slowly. The continuous gradient system not only reduced osmotic stress but also reduced the exposure of the slices to air and reduced slice handling variations. This final maneuver dramatically improved our results. Our VM3-treated slices are now responding electrically as well as control slices, with Xt/Na+ ratios also equivalent to controls . Both the quality of the electrical responses and the number of responding slices are now fully equal to 100% of control. These results demonstrate that damage caused by VM3 exposure to date has really been the result of avoidable osmotic stress. This provides us with very valuable and far from obvious proprietary information that will give us a competitive advantage over any possible competing brain slice vitrification group. Specific patent protection for this technique may be appropriate.
LEF Grant Application for the Year 2005 Page 28 of 52 Changes in apparatus were also made in order to carry out the continuous gradient system for adding and removing VM3. In our original non-gradient method, a slice carrier apparatus was using consisting of a single well 2 cm in diameter. Slices were positioned on a mesh floor at the bottom of the carrier and were transferred from container to container for loading and unloading. For the modified method, slices were held in 2-3 vertically stacked slice decks in a larger tube. Each deck carries 4 -6 slices.
Proposed Research for 2005. We must first repeat our current technique a few E• more times to obtain a publishable number of experiments (n=6 separate experiments) and make sure our comparison groups are also large enough to publish. We will also do some experiments to refine our method to make it faster without losing viability. The next step will be to go back to attempted vitrification and recovery. If we do not obtain immediate success, we will dissect the problems as we have dissected the problem of VM3 addition and washout until we identify what needs to be corrected and then attempt to correct it. The main potential problems are chilling injury, surface freezing, thermal shock, intracellular freezing, viscoelastic strain, and extracellular nucleation, each of which can be addressed as may be needed. As always, we hope, once we obtain good recovery of electrical responsiveness in vitrified/rewarmed rabbit hippocampal slices, that we will be able to go on to deeper issues involving the retention of biological memories and the ability to learn after vitrification and rewarming . Although the "learned" hippocampal responses of long term potentiation (LTP) and long term depression (LTD) do not represent the same kind of memory as remembering your name or riding a bicycle, they most certainly are biological memory processes, and asking whether they survive after vitrification and rewarming begins to answer the question of whether other kinds of memories can also survive. In the memory retention paradigm , the slices will be "trained," prior to cryoprotectant treatment and vitrification, by an appropriate conditioning series of electrical stimuli. This "training" will result in an exaggerated response to further shocks that can persist for long periods of time (LTP). After inducing LTP, the slices will be treated with VM3, vitrified, rewarmed, washed free of VM3, and tested to see if the LTP phenomenon still persists. If so, it means that a well-studied form of biological memory was successfully cryopreserved, so to speak. In the learning retention paradigm, the slices will not be modified prior to vitrification, but after rewarming and VM3 washout they will be given the same stimulus program as in the memory retention paradigm and then tested to see if LTP develops. If so, it means that the ability to "learn" has also been successfully cryopreserved, as it were. Other desirable endpoints for slice evaluation include LTD, which is similar to LTP but involves a learned reduced response rather than an increased response, and the use of voltage-sensitive dyes to visualize and map electrical responsiveness in a comprehensive manner. The use of these dyes will actually allow audiences to "see" the retrieval of memories in a vitrified-rewarmed slice and to "see" the formation of new memories in a vitrified-rewarmed slice. Even if our experiments with VM3 are filly successful, we will want to move on to studies of the best vitrification formulas developed for use on the whole brain. We would like to bring the whole brain methodology and the brain slice methodology into line with each other so we can gradually begin to apply techniques to the whole brain
LEF Grant Application for the Year 2005 Page 29 of 52 that are successful in the slice model. We will also continue to use the slice model to develop carrier solutions and thermal treatments that preserve electrical capabilities in the whole brain in the absence of cryoprotectants. Exactly how far we will get along the lines of testing baseline brain viability questions and solutions other than VM3 in the balance of 2005 is difficult to predict, but some progress forward will be made.
Whole Brain Cryopreservation
Preliminary Results for 2005 . Studies have focused on alternatives to M22, the origin of ultrastructural damage associated with cryoprotectant washout, freeze- substitution, mechanisms of observed tissue edema, and greatly expanded studies of brain cold storage by various means. Alternatives to M22. The most promising replacement for M22 at the moment is MEG, and another promising candidate is E22. These solutions are less toxic than M22 based on numerous kidney slice experiments, and it was hoped they might also better penetrate through the blood brain barrier. Both 100% and 81% of full-strength MEG were perfused, and 100% E22. The results are still pending. Because of the rather amazing observation that 81% of full-strength M22 allowed the brain to be vitrified and rewarmed without ice artifacts, we also considered the much more dilute VM3 solution currently being studied in the brain slice model. VM3 is an 8.4M solution, and 81 % M22 is a 7.6M solution like VS4, so VM3 should be both fully sufficient for brain vitrification and dramatically safer and more convenient to use than M22 and might produce less shrinkage injury than M22. Ultrastructural results after VM3 perfusion or perfusion plus vitrification and rewarming are pending. Some experiments were also carried out with 7.5M glycerol. This agent caused extreme shrinkage of the hippocampus because of the existence of the blood-brain barrier, but less shrinkage overall than M22. To study its possible use for vitrification in whole body cases, we devised a method for cooling rabbit cephalons at 0.15°C/min, the rate attainable in human whole body cases. Adding Dacron wool to the inside of the beverage cooler used in our 0.3°C/min studies resulted in a cooling rate of around 0.15°C per minute. Despite cooling to below Tg at this very low rate, only a small number of ice crystal artifacts were seen after rewarming and fixing these brains. Glycerol is known to be remarkably non-toxic for the brain, and deserves consideration as a future component of a brain vitrification solution. We have begun more detailed examination of the ability of 3-0-methyl-rac-glycerol and a relatively novel cryoprotectant we are further developing to cross the blood brain barrier and enter brain cells. We are also contemplating the design of a specialty cryoprotectant specifically tailored for rapid uptake by the brain. Origins of ultrastructural damage associated with cryoprotectant washout. The most obvious problem we currently see for recovering brain viability after cryoprotectant perfusion (other than the passage of time in the cold by itself, as discussed below) is the fact that we cannot currently remove vitrifiable levels of cryoprotectants without observing major ultrastructural damage. If we are to be able to address this problem, we must first find out its origin. If the damage is caused by re-expansion of the brain after shrinkage, how much shrinkage is permissible? Does it matter if permeating agents are present, or is the damage related to shrinkage regardless of the cause? Is the damage related to toxicity, so that lower concentrations don't produce the damage even if they cause almost the same amount of shrinkage as higher
LEF Grant Application for the Year 2005 Page 30 of 52 concentrations? If the injury is caused by re-expansion after shrinkage, how much re- expansion produces the injury, and would modifying the washout just before this point is reached allow the injury to be eliminated or reduced? To begin to answer some of these questions, the following experiments were done. First, some brains were perfused with low concentrations of cryoprotectant, then perfused free of cryoprotectant and fixed. Second, some brains were perfused with 0.3M concentrations of sucrose and glucose to verify that they also severely shrink the brain so that the results of washing out these agents can be compared to other shrinkage-re- expansion protocols. (Both glucose and sucrose did shrink the brain, but, interestingly, some glucose actually entered the brain and entered the brain cells based on the histological observation of shrinkage spaces surrounding neurons in brains perfused with sucrose but not with glucose. This observation suggests the possibility of using at least some glucose as a cryoprotectant for the brain.) Third, some brains were perfused with full-strength M22 and partially washed out and fixed. Fourth, some brains were perfused with VM3 and all of the VM3 was washed out, to see if the lower toxicity and lower concentration of VM3 might avoid the injury. As of this writing, ultrastructural results are not yet in for any of these experiments. Freeze-substitution studies . Freeze-substitution is a technique that allows ice to be visualized in frozen systems. It is of present interest for checking the ability of brains perfused with VM3 to remain truly ice free near dry ice temperature, versus simply being ice artifact free on warming. We assume the latter implies the former, but it is prudent to check this assumption. We have now used freeze substitution to check for the presence of ice in VM3-perfused brains under three different conditions, but as yet, ultrastructural results are not in. Mechanisms of edema development. When we washed VM3 out of rabbit cephalons, a surprisingly severe degree of edema was observed in the superficial tissues. Because VM3 contains 7% PVP and because VM3 was found to be much more damaging to rabbit kidneys than VMP, which lacks this PVP, we assumed it was probably the PVP that resulted in the observed edema. If so, this effect has to be produced by the simultaneous presence of the other components of VM3 because in the absence of cryoprotectant, cephalons perfused with LM5 plus PVP were no more edematous, based on their weight, than cephalons perfused with LM5 only. Therefore, the origin of the observed edema remains unexplained . If we decide to pursue studies of VM3, we will revisit this issue. Brain response to static cold storage. The problem of rapid brain deterioration in the cold, whether during static storage or during continuous perfusion, was investigated using histological, ultrastructural, and neurophysiological endpoints. Brains were perfused with RPS-2, LM5 + HES, RPS-TES, and B2, stored for 24 hours at 0°C, and fixed. Results for B2 are not available, but of the other solutions, RPS-TES was the best. Ultrastructural results just in for RPS-2 showed intact cell membranes but no intracellular contents after 24 hours of cold storage, although synapses and myelin appeared to remain intact. Other brains have been perfused with B2, stored one hour, and sliced so that the electrical responsiveness of the slices could be measured using Dr. Tan's standard method. One hour of cold storage in B2 was compatible with 74% recovery of control slice electrical activity and complete recovery of K+/Na+ ratio. However, 3 hours of cold storage reduced electrical activity to just 45 % of control, and 5 hours or 24 hours of storage resulted in no electrical responses. The texture of
LEF Grant Application for the Year 2005 Page 31 of 52 the brain was found to become progressively firmer in time with B2, making it hard to cut slices at 24 hours of storage. Brain response to cold perfusion at 3.5°C. Continuous perfusion preserved the ability of the hippocampus to respond electrically better than did simple cold storage in situ. Using a perfusate (B4) that did not permit any electrical response to be obtained after 3 hours of static cold storage, continuous perfusion at 3.5°C allowed electrical responses to be recovered after 5 hours of cold preservation . Evidently, continuous perfusion can usefully support brain metabolism even at 3.5°C, a result consistent with canine hypothermia experiments involving continuous perfusion versus circulatory arrest. Brain cold storage using Critical Care Research (CCR) neuroprotective cocktails. We perfused a rabbit cephalon with cold B5 brain perfusate containing CCR's water soluble protective agents until the temperature reached 5°C, and then the cephalon was stored statically for 3 hours and checked by Dr. Tan's slice assay. In a second experiment, most of CCR's water insoluble agents were tested in place of the water soluble agents in the first experiment. A third experiment was done the same way but without including any of CCR's agents in the cold perfusate. The results showed that without CCR's agents, perfuse-cooling with B5 allowed a small amount of activity to be observed, but both the water soluble agents and the water insoluble agents electrically silenced the ' brain . We think these agents take longer to wear off than the time scale of our experiments, which might in fact relate to the mechanism by which they protect. If this is the case, it may be impossible for us to truly test the efficacy of CCR's formulas in this model.
Proposed Research for 2005 . The first priority will be to complete our analysis of the origin of "washout injury." This should be straightforward and will proceed as described above. The next highest priority is to use the results of this analysis to design methods that reduce or eliminate "washout injury." The exact experiments that will be conducted in pursuit of this goal will depend upon the results of the preceding analytical experiments and cannot be predicted, nor can we predict how far we will be able to proceed by the end of 2005. However, we should have some good leads to follow and we stand ready to develop new cryoprotectant formulas, new techniques, and even to design new cryoprotectants if necessary to continue progress along these lines, particularly if we determine that the central problem is excessive brain shrinkage per se. The next priority will be the continued study of methods by which brain "electrical viability" can be consistently maintained for at least 5 hours, and by which brain "structural viability" can be consistently maintained for at least 24 hours . We will never be able to demonstrate brain viability after cryoprotectant treatment and after vitrification if control brains cannot be kept viable for the duration of such experiments. Our studies of the preservation solutions discussed in Preliminary Results above, as well as the solutions B3-B5, are gradually illuminating the principles we have to abide by for successful brain cold storage and cold perfusion, and we will continue to develop this knowledge as we are able to. Because isolated brain slices appear to be much less affected by cooling tolO°C than to 0°C, we would like to see whether perfusion of the whole brain at 10°C gives better results than perfusion at 3.5°C. There is an important detail that needs to be filled in, which is to complete our comparison of M22 and B2C. As noted in our report of 2004 results, our concerns over B2C were mostly derived from experiments in which B2C was perfused for 2 hours at 110% of full strength, a protocol that simulates what Alcor often does. We have not done
LEF Grant Application for the Year 2005 Page 32 of 52 the same experiment with M22, and if we do, we may not see an advantage of M22 over B2C. We will check this point in the balance of 2005. Our progress in all parts of the brain project has been significantly impeded by lack of access to electron microscope viewing time. This has been a chronic problem and must be solved. Although we were finally able to obtain training in the operation and maintenance of our electron microscope, we have still been unable to use it due to the lack of electron microscopy film and a film drying dessicator. Our electron microscope is old and does not have a built-in film dessicator, but commercially available `;. stand-alone dessicators are not designed for our application. It therefore took quite a while to decide on a solution to the seemingly simple problem of drying our film thoroughly enough for use in our scope, but with the aid of our electron microscopy consultant, we believe we now have a solution and will order a dessicator shortly. USC is capable of quickly and cost-effectively handling all aspects of tissue and film processing for us, but is chronically unavailable for actually viewing samples. Getting our own viewing capability will definitely make a difference, although we presently only have three film canisters available to hold desiccated film, and this may limit how many photos we can take of each brain.
B) Whole Body Vitrification.
We expect to submit a proposal to a wealthy family of Alcor members in 2005 that would fund a serious effort in whole body vitrification. We expect to ask for an initial $500,000 to establish fundamental methods for performing whole body vitrification procedures and, very importantly, developing practical measurements of their efficacy. Depending on the interest of the family, management at 21st Century Medicine is in favor of continuing this research over another few years. This would allow us to develop critical information on not only the possibility of vitrifying the body but also on reversing the process to move closer to true suspended animation. Of course, that would require considerably more funding, perhaps on the order of $3 million or more.
C) Kidney Cryopreservation.
Preliminary Results for 2005. As proposed, we tried improving the use of mannitol as an osmolyte to promote medullary perfusion by raising the mannitol concentration from 100 mM to 150 mM. Very surprisingly, kidneys treated in this way did not support life after transplantation. In last year's report, we proposed to look more closely at the effects of perfusion pressure to promote renal equilibration. Figure 6A shows that, based on data collected in both 2004 and 2005, there is a linear increase in urine refractive index with increasing perfusion pressure from 40 mmHg to 70 mmHg. There is also a trend for the rise to be faster with M-HES than with H7 HES or N-HES. Figure 6B shows the
LEF Grant Application for the Year 2005 Page 33 of 52 • BiBraun; R14, 40mmHg (n=9) • BiBraun (n=9,3=Buprenex) Fig • "H7" (45010.7,n=3,2) Fig. O "HT' (45010 7, n=3) 6A O N-HES (n=7,2,1) 6B 0 N-HES (n=5,1) • M-HES (n=1,22,2) • M-HES (n=1,4,1) © dG with VMP (n=5) O dG with VMP (n=5) VMP2 (n=2) o with VMP2 (n=3) O dG c C dG E N0 - - Minimum RI to Vitrify the Medulla CD N 1.4110 18 a- 1.4100 N 16 N rn 1.4090 a E 14 0 N C_ 1.4080 C 12 1.4070 N 1.4060 Y 10 M 2004-2005 d N for 25 minutes, 1.4050 data only a 8 (M22 at -22°C C 2004-2005 data only) ca U) 1 4040 m 2 40 50 60 70 I- 30 40 50 60 70 80 D Arterial Pressure during M22 Perfusion (mmHg) Arterial Pressure During M22 Perfusion (mmHg)
Figure 6 : Effect of perfusion pressure on A) renal equilibration with M22 (left) and B) renal injury (right). effect of pressure on postoperative mean peak creatinine level with the use of different forms of HES. Although it appeared that 60 mmHg was very damaging to kidneys in the M22B solution series (data not shown) and possibly when N-HES was used, 60 mmHg was relatively innocuous when both M-HES and M22 were used. As shown in Figure 6A, the latter condition also appeared sufficient for renal medullary vitrification (point above the horizontal dashed line). Unfortunately, injury at 60 mmHg was much higher than in the 40 mmHg B. Braun HES series (black point) due to elevation of damage even at 40 mmHg when HES forms other than B. Braun HES were used. We will discuss the data represented by the green boxes below. The lack of medullary ice formation at a urine RI value of about 1.408 (achieved using M-HES and 60 mmHg) was confirmed after cooling to -135°C in January of 2005. We therefore made 7 attempts to obtain survival after cooling these kidneys to -100°C. All of these attempts failed, despite appropriate urine RI values in most cases. These results are explicable on the basis of the following simple observation. In our original B. Braun M22 series, our peak creatinine level was about 9 mg/dl, and after cooling to -50°C, it rose to about 15 mg/dl, a change of 6 mg/dl. Our baseline peak creatinine level with physically vitrifiable kidneys and new forms of HES is currently about 14 mg/dl even without cooling below -22°C. If we were to experience only the same rise in peak creatinine level after cooling to -100°C as we previously observed after cooling to -50°C, the peak creatinine level should reach about 20 mg/dl. Unfortunately, this creatinine level is rarely survivable in our current model (no dialysis). If cooling from -50°C to -100°C were to add even another 1 mg/dl to the peak creatinine value, essentially all kidneys would become unable to support life without dialysis.
LEF Grant Application for the Year 2005 Page 34 of 52 Consequently, success after cooling to -100°C or below may be unlikely unless the elevated damage associated with the loss of our original supply of HES can be overcome . Unfortunately, there was no reason to believe that any untested form of HES that might be acquired would be any better than the B. Braun substitutes tested to date. To determine whether anything was wrong with our basic transplant method or with our perfusion machine, we perfused 5 rabbit kidneys with HES-free carrier solution for 5 hours at 3.5°C, transplanted them, and obtained excellent post-operative creatinine levels. We then repeated the experiment with the inclusion of 2% M-HES. Although no `,. statistically significant difference was obtained, we did find that there was a trend for M-HES-perfused rabbit kidneys to be more damaged than kidneys that were perfused for 5 hours in the absence of M-HES, other conditions being similar. This result is consistent with the possibility that M-HES and the other replacement forms of HES tested to date are damaging, and that this toxicity increases in the presence of M22. We concluded that a radical remedy to the HES problem was required. The remedy we chose to investigate was the replacement of HES with decaglycerol. Although we clearly showed that HES dramatically improves renal equilibration in comparison to no HES, and although decaglycerol is too low in molecular weight to serve as a colloid the way HES does, we postulated that, like mannitol, decaglycerol might be able to act as an osmolyte instead of as a colloid and thereby achieve the same goal of opening up the medullary circulation. Further, although this use of 100mM and especially 150 mM mannitol was found to be possibly toxic to the kidney in our model, we felt this might not pertain to decaglycerol in view of its known extraordinary non- toxicity and its higher molecular weight, which would avoid leakage into the renal cells. Consequently, we replaced 2% HES at the beginning of the perfusion with 2% decaglycerol (dG), and I% and 2% HES during the washout phase of the perfusion with 2% dG as well. The results were dramatic. As shown in Figure 7, using dG radically increased renal perfusate flow at all stages of introduction and washout of M22 and, as shown in Figure 6, dramatically improved renal equilibration with M22 at 40 mmHg. Figure 6A indicates that the use of decaglycerol at 40 mmHg (green boxes) gave equilibration similar to that obtained with M-HES at 60-70 mmHg but with lower peak creatinine levels (Figure 6B, Figure 7B). As this Letter is being written, we are beginning the next series of experiments intended to capitalize on the decaglycerol substitution effect further (the VMP2 method, described below). The results so far appear to be substantially better than our initial dG protocol: our peak creatinine levels are generally better despite urine RI values approximating 1.409, which is as high as we have ever seen. As shown in Figure 8, we have clearly uncoupled the often-observed relationship between increasing urine RI and increasing damage and are now in the most favorable situation for attaining successful vitrification we have ever experienced.
LEF Grant Application for the Year 2005 Page 35 of 52 Arterial Flow Rate During M22 Addition and Washout: B. Braun HES versus Decaglycerol 16 -f- M-HES, 60mmHg E 1.8 (RI = 1.4080, n=8) m 1.7 J 14 -o- dG, VMP, 40 mmHg (RI=1.4077, n=5) 1.6 B. Braun HES (n=11, 2004) rn VII E - -0- dG, VMP2, 40 mmHg y 1.5 Decaglycerol (n=5, 2005) 12 (RI=1.4090, n=2) 1.4 N -4-- B Braun HES, 40 mmHg w 1.2 • ;. (RI=1.4046,n=9) rt..4 m 1.2 c 10 E 1.1 1.0 8 0.9 U 0.8 > 0.7 6 E 0.6 4 3 0.5 0CL 0 0.4 Z FL 0.3 6 2 E 0.2 0- 0.1 M22 at -22°C for 25 minutes 1` 0.0 0 4 10 12 14 16 0 50 100 150 200 250 300 350 0 2 6 8 Time (min) Post-operative Day
Figure 7: Effect of HES replacement with decaglycerol on renal flow rate (A, left) and renal injury (B, right). Left: renal flow rate during the addition and washout of M22. M22 addition and washout with B. Braun HES was slightly faster than with dG. Data for dG are for the VMP method only. Gray=±1 sem. Right: Comparison between the effects of perfusion with B. Braun HES at 40 mmHg, dG at 40 mmHg, and M-HES at 60 mmHg on post-transplant creatinine levels. Also shown on the right are our first results from a second dG protocol involving the use of VMP2 vs. VMP (n=2).
20 • B. Braun HES, 40 mmHg Figure 8: • H7 HES, 40 mmHg Dissociation 18 • N-HES, 40 mmHg • M-HES, 40 mmHg of renal 16 ♦ M-HES, 60 mmHg injury from 0) © dG, VMP, 40 mmHg renal E 0 2dG, VMP2, 40 mmHg 14 equilibration using dG Y E 12 substitution for HES a) IL 10 (green boxes; for VMP2 8 method, n=4; M22, -22°C, 25 Minutes previous
6 I A 1 1 I I I 1 1 1 I I 1 1 1 figures not 1.4020 1.4030 1.4040 1.4050 1.4060 1.4070 1.4080 1.4090 yet updated). Urine RI
Proposed Research for 2005 / 2006. We will continue to develop the decaglycerol perfusion technique. The first dG technique described above used VMP as a transitional
LEF Grant Application for the Year 2005 Page 36 of 52 solution between no cryoprotectant and M22, but VMP has only 1% decaglycerol (dG). This means that the 2% dG used to improve flow was subsequently reduced to 1% prior to cooling to -22°C and then increased again after this cooling step. We will continue to investigate the use of VMP2, which contains 2% dG, to avoid the need to reduce and then re-introduce dG. This will raise the tonicity prior to cooling to 1.3X from the traditional 1.2X, which may reduce chilling injury based on the studies described under Basic Research above. It will also mean that the kidney is more equilibrated prior to the introduction of M22, and therefore should also be more equilibrated after 25 min of M22 perfusion, thus further reducing the risk of medullary freezing. Further, this maneuver should also reduce osmotic damage during M22 washout. If this method continues to be effective, it may prove able to reduce damage while at the same time improving equilibration. If so, we may investigate the use of 3% decaglycerol or 2% decaglycerol + 1-2% PVP K12 in the cooling step to raise the tonicity during cooling to 1.4X and thereby suppress cooling injury even more. If it is acceptable to do this with PVP K12, it will mean substantially increased equilibration at the end of M22 perfusion as well, and still greater osmotic protection during M22 dilution. Further reduction of damage may be possible by reducing the magnitude of the current % X dilution step. We will evaluate kidneys treated using the dG method for their ability to escape from medullary ice formation on cooling and warming. According to Figure 6, damage at 60 mmHg is not much greater than damage at 40 mmHg when M22 is used. This suggests that pressure elevation may have an additive effect to that of dG on medullary equilibration, without a substantial increase in injury. If this is the case, we should be able to solve any remaining medullary equilibration or devitrification problem by raising pressure to 50-60 mmHg.
The remaining issue will then be injury associated with cooling to below -22°C and rewarming. We will begin with cooling to -80°C and, if that works, we will vitrify and transplant kidneys equilibrated with the dG method once we are satisfied that they may be able to escape medullary freezing. If we do not obtain survival after vitrification and rewarming, or after cooling to -80°C, or if only a small proportion of kidneys survive, we will dissect the problem of chilling/cooling/warming injury more carefully. The first task will be to make sure our basic techniques are correct by checking our -45°C baseline results. Once that step has been successfully accomplished, we will examine the consequences of cooling to lower temperatures. Injury is likely to be affected by cooling rate, thermal gradient, renal core temperature upon reperfusion, temperature of reperfusion, and duration of warm perfusion prior to dilution of M22. We will analyze these features of the cooling and warming process as may be necessary to control injury sufficiently to obtain survival of vitrified/rewarmed/transplanted kidneys.
D) Cornea Vitrification.
Preliminary Results for 2005 . So far in 2005, we have been able to perform or cause to be performed transplants of vitrified human corneas into rabbits, monkeys, and human beings. To permit these experiments, we first developed, as proposed in our NIH grant and our last grant letter, a device for the maintenance of corneas near -145°C during
LEF Grant Application for the Year 2005 Page 37 of 52 Chinese (Human Clinical) Studies . As noted above, we shipped human corneas to China as part of a clinical pilot study arranged with the Department of Ophthalmology, the First Clinical College, China Medical University No.155, Shenyang, China. Because these corneas were lost in shipment as noted above, we were fortunate to be able to obtain a limited number of local human corneas, vitrify them in China, warm them back up, and transplant them. Unfortunately, all of the transplanted corneas failed. We believe these failures are due to local factors in China that are very different from the conditions prevailing in the United States, including the lack of baseline clinically used preservative solutions and the resulting inability to schedule surgeries electively. In addition, our first failure was associated with a severe eye infection that may have damaged the vitrified cornea. In addition to opening our eyes about the virtually non- existent nature of eye banking in China, we also learned of several economic and political infrastructure issues that make it difficult to do business in China. Monkey Studies . One vitrified cornea and two fresh human corneas were shipped to the island of St. Kitts and transplanted into two vervet monkeys by Dr. Wu at the Behavioral Sciences Foundation there. The vitrified cornea arrived at St. Kitts at about -196°C and was rewarmed and washed free of M22 by Dr. Wu. When transplanted into a female recipient, the vitrified cornea became cloudy, although not opaque as experienced in our human trial in China. After 1 -2 weeks in vivo, the appearance of the cornea appeared to improve, but specular microscopy was not successful in visualizing the endothelium even 7 weeks after transplant although, from one angle, half of this cornea appeared to be clear at 7 weeks post transplant. The two control corneas arrived in St. Kitts partially infiltrated, which made them basically opaque over about 50% of their surfaces. This infiltration has not resolved 7 weeks after transplantation into a male recipient, but we were able to get good endothelial images at about I month post- transplant as well as corneal thickness measurements. All corneas are clearly alive 5 weeks post-transplant and we have not observed either surgical complications or post-operative problems such as rubbing of the eyes, signs of inability to see, rejection, or infection. Rabbit Studies. Because of the problems we observed with corneal opacity or cloudiness in the Chinese and St. Kitts trials, we decided to repeat our basic human-to- rabbit model to verify that nothing has changed in our methodology that might be affecting our results. In the course of these studies, we transplanted a cornea that had been vitrified almost six months before as well as two corneas that were shipped to Texas and back, having gone to about -190°C in the process, as well as two freshly-vitrified corneas. Although we saw a little haziness in most corneas on days 1 and 2, all cleared up by day 3-4 except for the cornea preserved for longer than 5 months, which appeared somewhat cloudy a few days after transplantation and is still being observed. Some of the corneas in these studies were studied by vital staining, cell junction staining, and scanning electron microscopy, and seemed to be typical of what we had seen in the past by these measures. These studies suggest that although there does seem to be more damage in corneas transplanted recently than corneas transplanted prior to 2005, the damage is reversible and not reflective of the problems seen in China and St. Kitts. Therefore, our basic method still seems to work, and problems outside of our lab must be related largely to local issues rather than to the development of problems with our basic methodology.
LEF Grant Application for the Year 2005 Page 39 of 52 transcontinental cornea transportation . This device, the CorneaPorter, is shown in Figure 9. It was created by modifying a commercial "CryoPorter" liquid nitrogen "dry
v
Figure 9: CorneaPorter portable vitrified cornea shipper. Left: As seen from above, with the open lid shown on top and the open mouth of the shipper shown at the bottom. Right: Side view. shipper" by the addition of an internal "corneastat" of the kind used under non-transport storage conditions in our laboratory. The corneastat uses a battery-powered temperature controller to operate a heater that can maintain temperatures anywhere between -130°C and -190°C for up to four days during shipment in the CryoPorter. The CorneaPorter was used to ship human corneas one way to China and the island of St. Kitts in the eastern Caribbean, and two ways to Texas and back. The initial prototype used for shipment to China failed en route, apparently due to improper orientation during transport and the lack of adequate thermal insulation, arriving at its destination without liquid nitrogen. An improved prototype sent to St. Kitts survived shipment, arriving in St. Kitts in a trial run (no corneas) at the desired -145°C. Unfortunately, when it was shipped to St. Kitts a second time bearing one vitrified human cornea destined for transplantation into a monkey, it failed to control temperature but did retain liquid nitrogen, allowing the cornea to arrive near liquid nitrogen temperature. The latter failure seemed to be related to a tendency for batteries to vibrate loose during shipment. We therefore secured the batteries more strongly and shipped more corneas to Dean Barry's wife in Texas, who then shipped them back to 21 S` Century Medicine. In this test, the CryoPorter failed again , this time due to breakage of the heater wires due to impacts en route. The CryoPorter has since been "hardened" further, and is expected to survive its next test. We also plan to replace the heater wires with fiber-coated thermocouple wire that tolerates cryogenic temperatures better.
LEF Grant Application for the Year 2005 Page 38 of 52 Proposed Studies for 2005. We need to eliminate failure of the CorneaPorter to maintain temperature during corneal transport. We will soon repeat our Texan shipping experiment and will, if need be, repeat it again to ensure that we have the device fragility problems encountered to date solved. Once this is done, we will vitrify more fresh human corneas and ship them to St. Kitts for rewarming and transplantation. The trouble we had in St. Kitts might have been caused by a) Dr. Wu's warming technique back from the temperature of liquid nitrogen (this step was performed by Dr. Wowk in our in-house, human-to-rabbit transplant that suffered the same fate on shipping as did the cornea that was transplanted into a monkey in St. Kitts); b) Dr. Wu's cornea washout technique (performed to date by Dr. Ge); or c) the oppressive heat in St. Kitts, which may have damaged the vitrified cornea during M22 washout or during transplantation or both. Problem a) will be avoided if the corneas are never cooled to below -145°C in transit, and problem b) will be avoided if Dr. Ge is present to assist Dr. Wu as originally planned. Problem c) will be addressed by convincing St. Kitts personnel to turn on the air conditioning during M22 washout and cornea transplantation and by building a box for use in the refrigerator in St. Kitts that will prevent hot air from coming in contact with the corneas while they still contain cryoprotectant. Amy Beierschmidt, the attending veterinarian in St. Kitts and the person who works most closely with us, has agreed to the use of the air conditioner and the box. Although we tested one cornea by DSC as originally proposed, it is necessa ry to study a second cornea by DSC to fulfill the obligations of the NIH grant. This will be done.
E) Cryoprotectant Technology.
More basic studies are needed on the properties of our novel Genzyme cryoprotectant and another cryoprotectant we have found to be surprisingly non-toxic and largely overlooked. Both of these molecules are amides, and need to be evaluated by our toxicity titration test using DMSO to eliminate their toxicity in a dose-dependent fashion. These curves will better instruct us as to the optimal ways of incorporating these agents in vitrification solutions, and as to the maximum concentrations of each agent that are likely to be permissible. These studies on both agents are also very important for presenting limits necessary for the filing of patents for the use of both agents, individually or in combination with one another. The reason we have developed a novel cryoprotectant was due to Genzyme's concern over objections to the use of formamide. We will do some in vivo toxicity studies on the new cryoprotectant by perfusing kidneys with the new agent in vitro, transplanting them, and allowing the recipients to survive for one year or longer to check for any long-term carcinogenic effect. Other studies involving the injection of the new agent into rabbits may also be done.
F) Physical Science Research.
Ice Control
Preliminary Results for 2005 . 21 CM was contacted by two different research centers of the National Aeronautics and Space Administration (NASA) regarding the
LEF Grant Application for the Year 2005 Page 40 of 52 possible application of our ice blockers and cryoprotectant solutions in inhibiting ice formation on parts of spacecraft being filled with cryogenic fuels for launch. Although it was unlikely that our ice blockers and solutions as currently formulated would be useful, a reformulation was devised that may be useful. Specifically, a solution of propylene glycol, ice blockers, and surfactant in water was devised that forms a vitreous foam on cryogenic surfaces to protect them from ice formation.
Proposed Studies for 2005 . NASA. We will presumably continue the development of thermally insulating foams for NASA. The above-mentioned experimentally-tested foam will solidify at about -110°C. An ethylene glycol-based foam could go glassy at about -135°C, which might allow less foam to form or might allow formed foam to be more easily stripped from the spacecraft when it is launched. In addition, similar foams containing a mixture of ethanol and methanol might be better able to resist surface frosting by being able to dissolve more water. Further testing and development requires the execution of a non-disclosure agreement by NASA. M22 Stability Studies . Measurements of the critical cooling and warming rates of 21CM's most successful vitrification solution to date, M22, will be further refined. Time permitting, attempts will be made to measure the ice nucleation rate in M22 as a function of temperature also. Such studies are important to determine the most desirable long-term storage temperature for tissues vitrified with M22. The ice nucleation behavior of M22 may be quite different from other vitrification solutions because of its unprecedented low melting point and stability against ice formation. Low-molecular weight PVA. Polyvinyl alcohol (PVA) ice blockers are believed to start exhibiting ice blocking activity at a number of hydroxyls somewhere between 5 and 10. (In 2003 a custom-synthesized four hydroxyl version of PVA was found to be inactive as an ice blocker.) A better understanding of the minimum number of hydroxyls necessary for ice blocking is of considerable academic and practical interest. The practical interest is in developing ice blocker molecules that are smaller, and therefore less viscous and better able to penetrate small pores such as are found in the vascular endothelium and tubular cell junctions. We will pursue lower molecular weight (LMW) forms of PVA as time permits. We expect to use a scheme from The Chemistry Centre of Perth, Australia, which successfully synthesized the four-hydroxyl version of PVA for us two years ago. They developed a plan for synthesizing polyvinyl alcohol oligomers with 6, 7, or 8 hydroxyl groups by combining, for example 1,3,5,7-heptanetetrol and 1,3,5-pentanetriol, and 21CM has rights to use such molecules and will pursue various options for making them. We have contacted a chemical synthesis lab in northern California that may be able to make one or more of these oligomers for us, and we are pursuing this possibility. We already examined the use of simple dialysis of ordinary X-1000 to produce a lower molecular weight fraction that was active, but slightly less active than X1000. Unfortunately, we were unable to determine precisely what the mass of this fraction was, or the number of hydroxyls. Consequently, more precise studies using synthesized oligomers seems necessary to pin down the effect of molecular mass on ice blocking activity. Antifreeze molecules . New batches of antifreeze proteins provided by Agrigenesis will be also be studied to test for possible synergy or complementation with 21 CM's synthesis ice blockers. There may also be opportunity to study antifreeze
LEF Grant Application for the Year 2005 Page 41 of 52 proteins from Alaskan insects provided by John Duman of the University of Notre Dame. Recrystallization inhibition . It would be desirable to replicate and publish past literature claims of recrystallization inhibition by PVA. Since 21CM's version of PVA (X-1000) has been optimized for ice blocking in vitrification applications, it may also be an exceptionally strong recrystallization inhibitor in an appropriate assay. These possibilities will be investigated using the splat assay for recrystallization, time permitting, as will ideas for completely new ice blockers. Supercooling studies . We are in the process of redesigning our supercooling screening apparatus to eliminate problems of use that we encountered before. Once new apparatus is ready, we will resume our search for novel antinucleators that may be able to complement our existing X1000 and Z1000 products and produce stable and reliable supercooling of macroscopic volumes . Such studies may uncover new ice blockers that do not violate the ice blocker patents of our competitor, ORS.
Fracture Avoidance and ITS Elaborations
Small ITS (intermediate temperature storage) units intended for long-term fracturing studies will be completed in 2005. Development will include interfacing a computer control system to the temperature control units so that complex programmed temperature vs. time protocols can be tested for fracture avoidance. The fracturing tendency of different vitrification solutions will be compared, as will the effect of structural strengthening additives, such as macromolecules, fibers, and actual tissue.
G) Kidney Cold Storage (TransSend).
Preliminary Results for 2005 . Control rabbit kidney transplants were carried out involving 24 hours of storage with Renasol-2 and TransSend-9 because of concerns that our basic transplant baseline may have shifted to higher postoperative creatinine values. We found that, indeed, we were not able to replicate our original zero-damage baseline results after 24 hours of 0°C storage.
Proposed Studies for 2005 . We had planned to conduct canine kidney transplants at the University of Wisconsin and the University of Rochester as well as blood perfusion studies on human kidneys at 21st Century Medicine before the end of 2005. However, our NIH phase 2 grant was not funded (it was the next grant in line to be funded, had enough funds been available). NIH instructed us to resubmit the grant by the August 1't, 2005 deadline, and we will do this. However, although we are optimistic we will be funded the second time around, even if this happens we will not have funds in hand until April 1 s`, 2006.
H) Cardiac Cold Storage and Intermittent Perfusion.
Research for this project is being paid for by a phase 2 SBIR grant from NIH.
Preliminary Results in 2005. The Human Organ Lab (HOL) was successfully completed, and new office space to house staff displaced by the HOL was completed as
LEF Grant Application for the Year 2005 Page 42 of 52 well. Numerous components for the blood perfusion machine that will be used to evaluate preserved human hearts at 37°C as well as necessary new equipment such as a blood centrifuge and a clinical defibrillator were purchased. The perfusion machine components have been largely assembled and the largest uncompleted tasks for this machine are now in software development and computer interfacing with the machine components. A photograph of the human organ perfusion machine in an earlier stage of development on June 1St, 2005, is shown in Figure 10. In addition, design work and initial experiments have been done on the installation of a thermoelectric cooling system in the original intermittent perfusion device (CardioStat). An initial test thermoelectric device was purchased and tested on a model styrofoam box (Figure 11). Based on the performance of this unit, a larger unit that should be equal to the job of freezing the water in the CardioStat has been ordered for testing. The University of Rochester performed five practice experiments designed to convince their animal committee to allow them to proceed with the primary experiment, which is to preserve dog hearts for 24 hours using the University of Rochester Solution (URS), transplant them, and close the dogs and allow them to survive. The team will do one more control experiment and then proceed with the proposed studies.
Proposed Experiments for 2005 . We will complete the software and hardware improvements needed to make our human heart perfusion machine functional. If feasible, we will initially test our system using monkey hearts obtained from Primate Products, Inc., in Florida and preserved for transcontinental shipment to our lab using URS. We will initially obtain 1-3 dead human hearts and possibly a dead monkey heart as anatomical models to make sure our cannulae, are the right size and are well placed before we attempt to resuscitate any nominally living heart. We obtain human hearts preserved in a variety of ways from agencies that serve as intermediaries between organ procurement organizations (OPOs) and end research users such as ourselves. These hearts will be tested for life-sustaining functional capacity by normothermic perfusion with a blood-like solution to determine whether hearts preserved with URS recover better than hearts preserved with UW solution or with HTK solution. We will also complete development of the thermoelectric cooling technology needed to improve the automated intermittent perfusion device as required by our grant from NIH. The University of Rochester will proceed to transplant canine hearts preserved with URS or with UW solution and follow the ability of these hearts to maintain hemodynamically normal life over one month post-transplant.
11
LEF Grant Application for the Year 2005 Page 43 of 52 Figure 10: Beginning of the construction of the human heart perfusion apparatus. Computers and monitors on the left, 37°C cabinet with components partly assembled on the right. On the bottom of the cabinet is the blood reservoir (left) and the blood pump (right). On the top shelf of the cabinet are high-volume solenoid valves, a lift- table to create varying preloads, and the organ chamber (scarcely visible at the middle of the shelf).
Figure 11 (left): Engineering model of thermoelectric heat extraction from an ice chest. The thermoelectric device (TED) is shown penetrating the right wall of the styrofoam ice chest; the laptop running the TED and monitoring its results is shown at right.
I) Hepatocyte Cold Storage.
A variant of TransSend called TS-HC for "TransSend-Hepatocytes" will be tested by Tissue Transformation Technologies (TTT) for efficacy in preserving isolated hepatocytes for 24 and 48 hours of cold storage. If this solution beats a competing solution, Hypothermosol-FRS, it will be licensed to TTT for use. If it does not beat the competing solution, we will consider a further modification to be tested by TTT.
LEF Grant Application for the Year 2005 Page 44 of 52 J) Basic Research.
Computer analysis of cryoprotectant electronic structure. The biological and physical effects of different amides used in our laboratory and planned for use in our laboratory differ considerably. At present, the reason for these differences is not known, and may be significant for the prediction and discovery of more effective agents and agent combinations. HyperChem, currently installed in our laboratory, is capable of calculating the charge distribution of such small molecules. We will use Hyperchem to map amides of interest as well as DMSO, whose electronic structure blocks toxic effects of some but not all amides, to see if we can gain some insight into possible reasons for the effectiveness of DMSO. It is possible these studies could lead to DMSO-free vitrification solutions , which might have novel applications. Chilling injury. If time permits, we will re-examine the possibility of avoiding the freezing of M22 at -79°C for long enough to allow studies of the rate of biological deterioration at that temperature. This will be done by raising the air-liquid interface to a temperature above the melting point of M22, or eliminating the air-liquid interface altogether. We will also reinvestigate the temperature dependence of chilling injury in M22 in our slice model based on our recent dG method for eliminating HES toxicity. The renewed study of chilling injury in general is of heightened significance in view of our new-found ability to equilibrate the renal medulla with minimal renal toxicity and thereby circumvent most other obstacles to successful vitrification. Properties of higher molecular weight polyglycerol and polyglycerol esters. If we are able to obtain higher molecular weight polyglycerol or polyglycerol fatty acid esters, we will test them for ice blocking activity. Unlike X1000, in which maximum ice blocking activity is obtained at lower molecular masses, we believe polyglycerol may be more effective at higher chain lengths. Polyglycerol stearate has also been reported to be an extraordinarily good antinucleator in a now-expired Japanese patent. We would like to compare this molecule to our existing polyglycerol product and to higher- molecular weight polyglycerol and check for possible synergies between them. We are told there is a major opportunity in the food industry if such studies are successful. Mechanisms of cryoprotectant toxicity. We will attempt to set up two- dimensional (2-D) gel electrophoresis in our lab as part of a long-term, low-resource effort to pursue the mechanism of cryoprotectant toxicity and cryoprotectant toxicity reduction. Our past studies have identified proteins that change in response to formamide exposure and whose change is blocked by treatment with DMSO as a toxicity neutralizer, but we were unable to confirm these results due to the high cost of buying 2-D gel services from outside companies. We found that one-dimensional gels show no changes attributable to either formamide or DMSO plus formamide, so 2-D gels are necessary to identify proteins changed by amides. In addition, Arlan Richardson's lab in Texas has shown that ANS, which binds to denatured proteins, can be used to screen 2-D gels for denatured proteins. We will investigate the possibility of using this test to get extra information from 2-D gel analysis.
K) Contract Research.
Preliminary Studies in 2005 . We completed additional studies for our Genzyme contract in 2005 that gave critical information on the role of VeGD exposure time and the cooling protocol that gives the best results. We also assembled all data so
LEF Grant Application for the Year 2005 Page 45 of 52 far into four graphs that plot results as a function of cooling rate or warming rate when the data are normalized to either controls or VeGD treated slices that were not vitrified.
Proposed Studies for 2005 . We will conduct at least one more Genzyme experiment involving the testing of a custom-made apparatus designed by Dr. Faby for guaranteeing the correct cooling protocol based on our studies at 21 CM. We will also send Mr. Phan back to Genzyme at the completion of the 21 CM studies to transfer the cooling technology to Genzyme. Genzyme will then proceed to test the new cooling method on porcine and then human cartilage samples . This will hopefully be the final stage of providing our proof of principle for the feasibility of vitrifying human cartilage using a formamide-free solution, as originally desired by Genzyme. If the results are deemed to be satisfactory by Genzyme, the next stage will be to design and demonstrate a high-throughput version of our basic methodology under a further extension of our existing contract, and possibly carry out more in vivo toxicity tests. We are in receipt of antifreeze proteins supplied by Agrigenesis (now Genesis) and will be completing our tests of the effectiveness of these proteins alone and in combination with our ice blockers and M22. Other contract work might be conducted for other clients, but at the time of this writing, all other such projects remain to be established.
L) Collaborative Cryopreservation Research.
Preliminary Results for 2005 . Dr. Duman of Notre Dame University sent us hemolymph from the Alaskan beetle Cucujus after concentrating it up to 4 or 5 fold to simulate the dehydration of the insects that takes place as temperatures fall in the winter. The 4 x concentrate was found to vitrify at any cooling rate higher than 5°C/minute, and at 5°C/min froze with an energy of 3.76 J/g at -55°C. When the sample was cooled quickly to vitrify it and then warmed at different rates, it devitrified unless warmed at 160°C/min or faster. The thermodynamic melting point of the sample was -32°C, and the glass transition temperature was -98°C. Based on these results, these beetles will escape freezing in the winter only if they are concentrated to the 5 x level, 4 x being insufficient. If they make it to 5 x, they would never vitrify because the environmental temperature would never reach -98°C, but they would never freeze either, and would instead pass the winter in a supercooled state not far below their thermodynamic melting points.
Proposed Studies for 2005. We will continue to supply Inge de Graff of the Groningen University Institute for Drug Exploration (GUIDE) in the Netherlands with vitrification solutions to be tested on human tissues relevant to pharmaceutical drug development. She will continue to test our solutions at no cost to us, report her results to us, and publish her results with us. Very little effort will be required on our part for these experiments. It's possible that we might collaborate with Richard Caterini, a French businessman who has become interested in organ cryopreservation and whole body hypothermia as a result of our publications in 2004. We may also continue collaborative studies with John ("Jack") Duman of Notre Dame. Dr. Duman has agreed to test combinations of our vitrification solutions with his antifreeze proteins in his lab to check for possible synergy with his recombinant beetle antifreeze proteins and to confirm enhancement of thermal hysteresis by our ice blockers.
LEF Grant Application for the Year 2005 Page 46 of 52 7. Physical Plant and Staff Considerations for 2005.
Personnel changes. A new vivarium technician, Patty Wu, was hired to replace retiring vivarium technician Richard Infante. Eric Zendejas was replaced by Jesse Fuentes as the principal experimentalist on the whole brain cryopreservation project. Jacob Drainville was hired to support Mr. Fuentes' work on whole brain vitrification, and to assist Dr. Tan as well. He left to go to graduate school and was replaced with Joon Chang. As this Letter is being written, we have been informed that Patty Wu has accepted a job in an immunology lab and will need to be replaced as well. There are no net personnel changes planned currently. Any changes made will be in response to future NIH awards or private research contract requirements.
Facility changes. The build-out of our vivarium was completed and now allows our rabbits to enjoy maximum protection from potential microbial agent exposure while giving us enough carrying capacity to remove all housing restrictions on the number of experiments we can perform. The build-outs also make it convenient to show our vivarium to visitors through glass windows in the vivarium doors without disturbing or contaminating the rabbits. Construction was begun on a new laboratory, the Human Organ Lab, and on new office space for 4 or more investigators . The Human Organ Lab will house all experiments on the perfusion of viable human organs. The repairs to our newly-installed electron microscope and the installation of a new chiller unit to prevent it from overheating were all completed in 2004. Dr. Tan established new general access databases , first for his brain slice project data and then for most other projects at 21st Century Medicine. The first version database was programmed in early 2004, while Dr. Tan was waiting for his equipment to arrive. Data are entered into pop-up windows and automatically formatted into an excel spreadsheet, where necessary calculations are performed automatically. The application was revised in September 2004 to automatically generate a chart of the data labeled with the experiment date. Raw data and results are also automatically converted into html format to enable online sharing of the data in our own "intranet." The automated data processing features of these databases dramatically reduce the time and labor needed for data analysis and reduce the likelihood of human mistakes. In addition the data are stored electronically and presented in a standard format. Databases with an intranet web interface were developed for kidney cold storage experiments, kidney perfusion experiments, whole brain experiments, and brain slice experiments by September 2004. The databases also contain animal data and other information and are searchable by a variety of different criteria, which are customized depending on the database. Several building upgrades have been completed in 2004, including air conditioning, and electrical. No major capital equipment outlays are planned during the next budget period. Major equipment purchases during the previous budget period, including organ perfusion systems, centrifuge, microscopy, etc., have been funded as part of NIH grants.
LEF Grant Application for the Year 2005 Page 47 of 51 8. Non-LEF Grant Funding 2004, 2005.
During 2004, and into 2005, 21 t Century Medicine has received funding under two separate Small Business Innovative Research Grants, which had been submitted for approval at the end of calendar 2003. These grants are "Cornea Preservation," a Phase I grant, and "Extended Cardiac Preservation," a Phase 2 grant. The total award under these grants during the period of 2004 through September of 2005 is $785,083. This represents a 146% increase in government grant funding over the previous budget period. This trend is expected to continue with the anticipated award of two new Phase 1 grants, a new Phase 2 grant, and continuation of the current Phase 2 award during 2006. Additional avenues of funding from NIH are also being explored. These grants will continue to help in consolidation of costs associated with administration and physical plant operation, and further increase equipment utilization, and net staff productivity. The net result is more resources available for all research operations due to increased flexibility and economy of scale.
9. Summary of Near-term Commercial Opportunities.
21st Century Medicine continues to make progress on the commercialization projects funded through the NIH SBIR program . These include:
Cornea Preservation Hypothermic Cardiac Preservation Hypothermic Kidney Preservation Neural Tissue Preservation High Throughput metabolic data recording
The company will continue pursuit of government funding of these projects to the point where private commercialization is practical based on the demonstration of industrial efficacy.
The Genzyme research contract for the preservation of cartilage tissue is now into the final stage designed to demonstrate practical utility of the preservation technology. The next phase will involve engineering of the final product configuration(s) in accordance to specific customer requirements. This project holds the potential for significant revenue, however the timing and specifics of commercialization are yet to be determined.
21st Century Medicine continues to pursue additional privately supported research potentials in an effort to expand the range of potential applications for its technologies. These include the following.
T-cubed, a distributor of human hepatocytes for research uses, is interested in the the possibility of using TransSend to preserve these cells. The advantage of such an arrangement would be that no FDA clearance or liability issues would stand in the way of immediate sales. Limited work is currently underway (see Preliminary Results, 2005), and an up or down on this possibility may be possible before the end of 2005.
LEF Grant Application for the Year 2005 Page 48 of 51 In early 2005 Mr. Dean Barry attended a NASA sponsored conference in Cleveland, Ohio describing NASA's Office of Biological and Physical Research ongoing efforts and intended direction for future scientific investigation. Mr. Barry returned with information that the largest part of NASA's suspended animation investigations will not begin in earnest until late 2008 or early 2009. 21CM's name was added to a list of interested collaborators and contract researchers for both NASA and the ESA (European Space Administration). This addition has led to several communications and we have recently exchanged considerable information with NASA about a problem they are having with ice buildup on the liquid oxygen lines leading to the space shuttle rockets. They have apparently had this problem at least since the early 1980's and have no satisfactory solution to date. Scientists from both Redstone Arsenal and White Sands Test facility located 21 CM from the NASA list, and found VM3 as an ice control solution on our website. They each contacted us about helping them solve their problem using VM3. We have advised them that we can provide much more promising possible approaches than that, and we are expecting to hear back from them as this letter is being written.
Agrigenesis has provided us with antifreeze proteins to help them explore the commercial potential of these proteins . If helpful, we should be able to realize economic benefits from these proteins through a variety of possible mechanisms.
John Morris in the UK has informed 21st Century Medicine about an undefined but allegedly major opportunity in the food industry that might be obtained provided we can stably supercool a food product reliably by a few degrees. We sent Dr. Morris polyglycerol as an antinucleator, but did not hear back from him. Although we assume this means the tests failed, we might be able to rekindle interest in this project if our basic studies, discussed below, lead to more consistently successful supercooling.
We have been in touch with Hoag Hospital in Newpo rt Beach about the possibility of using TransSend for the short term preservation of blood vessels (saphenous veins) removed from patients' legs for use in coronary artery bypass graft surgery. Scheduling problems have prevented us from having a meeting with Hoag's thoracic surgeons to discuss this idea, so we plan to mail them hard copy of our TransSend results to catch their attention and gain their confidence.
Vitron, Inc., which performs drug screening services for pharmaceutical companies and is reviving the organ slice as the method of choice over isolated hepatocytes. Vitron is very interested in our vitrification technology for liver slices, and we feel Vitron may have strategic value as a proving ground and contact point for us to big pharma. In addition, our initial SBIR on the vitrification of neural tissue obtained an excellent priority score and should be funded upon resubmission with very minor changes. This effort is reinforced by our continued good relationship with the Groningen University Institute for Drug Education (GUIDE), where our solutions are being studied for the preservation of kidney, liver, and lung slices, and our forthcoming publication of the first demonstration of the survival of vitrified rat brain slices.
We are hoping for a significant opportunity to pursue human ova cryopreservation in conjunction with a fertility clinic, Tecnobios Procreazione, in
LEF Grant Application for the Year 2005 Page 49 of 51 Bologna, Italy run by Giovanni Coticchio, an MD fertility. Dr. Coticchio held the first international meeting on human ova cryopreservation and invited Dr. Fahy to participate. A good relationship between Dr. Fahy and Dr. Coticchio was established at that meeting. Dr. Coticchio reported to us in June, 2005 that he is working on avoiding a conflict of interest between working with 21st Century Medicine on ova vitrification and Cook, Inc. on ova freezing and definitely wants to work with us.
Xytex, Inc. has accepted our solutions and protocols for the vitrification of ovarian tissue, but we cannot speculate about whether there is any significant probability of commercial development of this line of work at this time.
HepaHope, Inc. was recently reorganized and may want to move forward with us on studies of the cryopreservation of their bioartificial livers.
Alcor Life Extention Foundation continues to contract with 21st Century Medicine for use of its formulations.
We find it encouraging that numerous businesses contacted us through our website in 2004 about -helping them develop marketable products. Our success in sustaining and building upon these opportunities depends on our ability to being these technologies to the point of demonstrated practical application, thus enhancing our credibility as a research and commercial partner.
10. Concluding Remarks
Commercialization of technologies developed internally, or licensed from other organizations, remains the overall goal and the most important means of sustaining the long-term research goals of 21st Century Medicine and the Life Extension Foundation. In the meantime, the research funding received by 21st Century Medicine from the federal government beginning in 2002 was continued in 2004 and 2005 , and we are optimistic this funding will grow to become an even more significant portion of our overall research support.
The attached operating budget reflects the total requirements for all research projects currently underway. However, 21st Century Medicine has not requested any funds from LEF for projects outside the scope of LEF directed research.
The results of the sponsored projects, including all relevant information, will continue to be published in such form as to be available to the interested public either currently, as developments in the project warrant, or within a reasonably short time after completion of the project. If patent rights are involved, publication will take place after such rights are secured, as promptly as is reasonably possible.
21st Century Medicine, Inc. is very grateful for the past support of the Life Extension Foundation, and for our ongoing relationship. We take seriously our responsibility to maintain a research focus that is in the public interest, and to make,
LEF Grant Application for the Year 2005 Page 50 of 51 At the end of the year, 21st Century Medicine will report to the Life Extension Foundation how it has complied with the requirements of the above paragraph. Sections 1, 2, 3, and 4 of this grant application are intended to serve this purpose with respect to calendar 2004 and the first half of calendar 2005. The support of the Life Extension Foundation is critical to this important research, and we will continue to make our best efforts to achieve our mutual scientific and commercial goals.
Vice President and Chief Scienti fic Officer
Chief Operating Officer
Brian owk, Ph. D., Senior Scientist
List of appended materials
A: Proposed budget for 2005 B: Reprints of papers published since the previous grant letter D: Grant applications submitted to NIH since the previous grant letter E: Patents awarded since the previous grant letter
LEF Grant Application for the Year 2005 Page 51 of 51 BI0111ARKERPHAR MACE UTICALS,INC.
Research & Development Progress Report
[2004]
BioMarker Pharmaceuticals, Inc. 900 E Hamilton Avenue, Suite 100 Campbell, CA 95008 Table of Contents
R&D Status and Progress for 2004 ...... 3
Establishment of Independent Laboratory and Operational Capability at the Oakland and Alameda Sites ...... 3
R&D Collaborative Activities and Opportunity Development ...... 6 Harvard University/Sirtris, Dr. David Sinclair - resveratrol ...... 6 Optigenex Inc. -AC-II ...... 7 National Cancer Institute, Division of Cancer Prevention, Dr. Stephen Hursting - graded obesity study .... 7 SRI International, Dr. Nurulain Zaveri - PPAR agonists ...... 7 University of California, Davis, Drs Ray Rodriguez and Kevin Dawson - Nutritional Genomics Center and High Performance Computing Center...... 7 University ofCalifornia, Riverside (UCR), Dr. Stephen Spindler - Research Agreement ...... 8
Small Business Innovation Grants (SBIR) ...... 8 Discovery ofdruggable protein targets in aging brain, Phase I ...... 8 Innovative technologies for the molecular analysis ofcancer, Phase I ...... 9
Acquisition of Patent Rights & Materials Under the License & Research Agreement...... 9
Research Publications in 2004 ...... 10
Citations and Recognition ...... 10 Fortune's "Chasing the Youth Pill "...... 10 Bio IT World "High-Tech Search for the Fountain of Youth ...... // Cambridge Healthtech Advisors "Model Animal Systems: Emerging Applications and Commercial Opportunities in Drug Discovery and Development ...... I/ Frost & Sullivan Excellence in Research Award - 2004 ...... 11
Development of Research Plan for 2005 ...... 11 BMP Project 1 01 - Profiling...... 12 BMP Project 102 - LEF grape extract/Sigma resveratrol ...... 13 BMP Project 103 - Cat's Claw ...... /4 BMP Project 104 - metformin ...... 14
Appendix A...... 15
Bio,arker Pharmaceuticals Inc, CONFIDENTIAL 2 R&D Status and Progress for 2004
In 2004, BioMarker has made significant strides in research and development, with several notable accomplishments. The company acquired access to a very high quality animal vivarium; mice were purchased and are now the subject of calorie restriction and other experiments. A location for a molecular biology lab was found and a fully functioning laboratory was established and staffed. The license to the principle technology was completed, allowing the transfer of technology and research materials to BioMarker and enabling the company to become independent and fully operational. Two highly significant research papers were published in 2004, advancing the technology and validating BioMarker's approach to develop calorie restriction mimetics and other therapeutic products based on our CR platform.
In recognition of its accomplishments in developing innovative R&D in the field of anti-aging, BioMarker was highlighted in several national media publications, and received the prestigious 2004 Frost & Sullivan Excellence in Research Award. BioMarker is now equipped to commercialize the technology, through aggressive service and product development strategies that are being implemented. We expect to have an even more impressive report at the end of 2005, as BioMarker now begins to do leading research in this field and undertake commercial services and product development.
A. Establishment of Independent Laboratory and Operational Capability at the Oakland and Alameda Sites
Animal vivarium - Children's Hospital Oakland Research Institute (CHORI)
In February 2004 BioMarker established a working relationship with CHORII, gaining access to a very high quality environment in which to maintain experimental animals which are required to conduct calorie restriction research. The CHORI vivarium is fully equipped with restricted access, infection and disease control, transgenic animal facilities, and surgical procedure rooms. Animals are housed in individual cages which are in turn attached to HEPA filters. An animal care technician at CHORI has been hired to provide daily care and feeding of animals, perform experimental observations and data collection for BioMarker's animals. All experiments that BioMarker plans to conduct are reviewed by the CHORI Animal Care and Use Committee, ensuring full compliance with federal and state law regarding the use of animals in experimental procedures. In addition to obtaining an excellent environment in which to maintain experimental animals, BioMarker also has an opportunity to establish working relationships with individual CHORI investigators, and has done so to advance collaborative research projects under the SBIR grant program (see below).
In April BioMarker purchased one-month-old mice from Harlan Breeders in order to establish the first set of experimental subjects. The first group of 230 male B6C3F1 animals was delivered and started on calorie restricted (CR) and control (CON) diets. In July this first set was supplemented with a second set of 207 animals (born around May 21, 2004) to bring the total
1 Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, California 94609 http:/hvww.chori.org/
BioMarker Pharmaceuticals Inc, CONFIDENTIAL number of experimental mice owned by BioMarker to 437. Shortly after arrival, the animals were placed on either a CON diet (93 kcal per week of chemically defined control diet - AIN- 93M, Diet No. F05312, Bioserv, Frenchtown, NJ) or CR diet (52.2 kcal per week of chemically defined CR diet - AIN-93M 40% restricted, Diet No. F05314, Bioserv). The CR animals were stepped down to the 40% restricted diet by first introducing them to a 77 kcal per week diet for two weeks prior to the final 52.2 kcal fully restricted diet. From the first set of animals, 70 were placed on CR and 160 maintained on CON. From the second set of animals, 63 were placed on CR and 144 maintained on CON. BioMarker thus has a total of 133 animals on CR and 304 on CON. The control diet-fed animals are available as subjects for experimental testing of drugs, foods, bioactives, etc. in order to identify CR mimetic candidates. Standard operating procedures (SOPs) have been established and rigorously applied to the housing, feeding, maintenance, observation, and data recording requirements as they apply to experimental animals.
In April 2004 BioMarker leased 600 sq. ft. of laboratory space at ACET (Advancing California's Emerging Technologies)2 at 851 W. Midway Avenue, Alameda, CA. This site is conveniently located approximately 15 minutes from CHORI. ACET is a non-profit business development accelerator for high-technology startups specializing in biotechnology. BioMarker has an arrangement with a tenant, the Montclair Group, to occupy laboratory space that is fully equipped with state-of-the-art instrumentation for molecular biology analysis, in addition to our own laboratory and office space.
The laboratory is headed by Dr. Joseph Dhahbi (Director of Research Program) and was staffed by four full time Research Associates at the end of December, 2004. In the past nine months BioMarker has completed the technology transfer acquired under the License Agreement with the University of California (see below), equipped the laboratory with essential instrumentation, acquired and trained a talented research staff, and has established working protocols for conducting the screening of CR mimetics as well as advancing basic research. Several important research projects have been planned and are underway.
In order to conduct high-quality microarray gene expression profile analysis, the principal underlying technology, BioMarker first needed to establish in-house capabilities in the following areas: high-quality animal husbandry, preparation of animal organs and tissues for molecular biological analysis, preparation of high-quality RNA suitable for microarray analysis, and a proficient bioinformatics and computational biology component sufficient to analyze the massive amounts of data that will be collected. In addition, a competent outsource needed to be identified that would provide access to Affymetrix array hybridization and scanning. BioMarker has made significant progress in establishing competency in all of the above areas and has identified a local and suitable source for performance of the Affymetrix arrays.
Animals have now been maintained on CR and CON diets for approximately 9 months since we established a high-quality animal environment through CHORI and successfully transferred the technology which was previously resident at Dr. Spindler's laboratory at the University of California at Riverside (UCR). During 2004, a few groups of test animals were sacrificed and necropsied and organs and tissues harvested in order to establish methods and procedures for
2 htt! ://%v%vw.giccnstart.ore/acc
BioMarker Pharmaceuticals Inc, CONFIDENTIAL high-quality RNA preparation. SOPs have been established and the methods have been evaluated for RNA preparation, RNA amplification, and labeling. BioMarker has completed the processing of tissues through the RNA labeling stage and has obtained the first test set of Affymetrix mouse gene chip array data for these samples. Arrays were hybridized and scanned through an arrangement with the Stanford Microarray Core Facility at the Stanford School of Medicine. Stanford has a well-recognized reputation as one of the leading innovators and developers of array technology. Organs and tissues processed by BioMarker have yielded suitable material for microarray analysis. This first set of test data has validated BioMarker's procedures and methods and represents a major milestone in establishing full competency in the performance of this highly sophisticated and technically demanding process.
In order to perform the above analysis, BioMarker has equipped the laboratory in Alameda with the necessary instrumentation. In addition to purchased equipment, we have access to equipment through the arrangement with the Montclair Group. The important acquisitions in 2004 include:
Agilent BioAnalvzer 2100: This is a microfluidics-based platform that uses lab-on-a-chip technology for the analysis of DNA, RNA, proteins and cells. It provides a rapid and efficient means of testing the quality of the RNA prepared from mouse tissues and organs, delivering high-quality digital data and electropherograms. Multiple samples can be processed simultaneously and in an automated fashion, ensuring high-performance output and large sample handling capability. The end-quality of the gene expression data from any microarray experiment is highly dependent on the quality of the input RNA.
NanoDrop ND-1000 UV-Vis Spectrophotometer : This highly accurate spectrophotometer is designed to handle extremely small samples (1-2 ul) and preserves most of the RNA for downstream processing. It enables BioMarker to maximize the use of the samples we prepare and also adds to efficiency since cycle time is also reduced while maintaining reproducibility.
Arcturus Veritas Microdissection - Automated Laser Capture Microdissection (LCM) and Laser Cutting System: This is a highly sophisticated and expensive system that gives BioMarker a unique capability - the ability to process samples and specimens with an extreme accuracy which has not yet been applied to calorie restriction experiments. While the current technology (whole organ processing) has been very useful in the preliminary correlation of gene expression results with cell-based physiology, the LCM system will enable a high degree of accuracy given its ability to isolate through UV cutting, single cells of a specific type. BioMarker is currently testing and establishing LCM SOPs and intends to fully employ this instrument in the second half of 2005.
ABI Prism 7700 Sequence Detection System: In addition to equipment purchased, BioMarker has access to a significant amount of standard and specialized laboratory equipment and instrumentation required for molecular biological analysis through an arrangement with the Montclair Group. This includes access to an ABI Prism 7700 Sequence Detection System, a complete, real-time PCR system that detects and quantitates nucleic acid sequences. Real-time PCR (cycle-by-cycle detection of accumulated PCR product) is an important validation step used in the quality control of microarray gene expression profile results.
BtoMarker Pharmaceuticals Inc, CONFIDENTIAL Informatics & statistical analysis software : BioMarker has upgraded its informatics core by the acquisition of commercial-grade software designed to support the statistical analysis of data generated from the Affymetrix microarrays. BioMarker purchased S-PLUS and ArrayAnalyzer with a desktop license (Insightful Inc.). This software package is designed for high-end microarray core facilities requiring leading-edge statistical and graphical methods for controlling the design and analysis of microarray experiments. We also obtained a powerful suite of software - Interaction Explorer Pathway Module (Stratagene) - which enables the identification of biological interactions among the genes of interest identified in the microarray experiments. The upgrade from previous "academic" freeware versions of analytical software gives BioMarker a significant depth in computational analysis. This area is essential since it represents the leading edge of research in this field and is most likely to generate intellectual property, research advances and secure a competitive advantage for BioMarker.
With purchased and leased equipment, and the installed bioinformatics capability, BioMarker is now fully operational and able to perform all of the steps necessary to screen compounds for CR mimetic activity and conduct an in-depth analysis of the resulting data. We now have an operational technology platform designed to provide both service as well as support product development.
B. R&D Collaborative Activities and Opportunity Development
BioMarker has been actively engaged in identifying partners that can provide access to technology, intellectual capital, resources or product development opportunities. R&D has worked closely with Business Development in the vetting of potential partners. Several meetings and on-site visits have been conducted in this area and the following highlight some of these activities:
Harvard University/Sirtris, Dr. David Sinclair - resveratrol
In February 2004 BioMarker met with Dr. David Sinclair, the founder of Sirtris Pharmaceuticals, a recently formed pharmaceutical company3 established to capitalize on Dr. Sinclair's discovery of the "anti-aging" effects of resveratrol. We have ongoing discussions with the scientific principal investigators at Sirtris and are moving towards a possible collaborative opportunity around the evaluation of Sirtris resveratrol derivatives using BioMarker's proprietary screening technology.
3 Sirtris Pharmaceuticals - 100 Beaver St. Suite 240 Waltham, MA 02453 http:/Iwww.sirtrisphanna.com/
B:oMarker Pharmaceuticals Inc, CONFIDENTIAL Optigenex Inc. - AC-11
BioMarker has advanced a contract Research Agreement to Optigenex4 , outlining the evaluation of AC- 1l, a proprietary extract of Uncaria tomentosa (common name-Cat's Claw), a plant indigenous to South America and used in traditional medicine primari ly for its anti-inflammatory properties. Optigenex holds patents on a process of manufacturing a defined extract, and the activity associated with DNA damage repair. BioMarker and Optigenex are in ongoing discussions around a revenue-generating experimental evaluation of AC-11. While the agreement is under consideration , BioMarker plans an independent evaluation of the active ingredients of Cat's Claw and other Uncaria extracts.
National Cancer Institute, Division of Cancer Prevention, Dr. Stephen Hursting - graded obesity study
BioMarker has had ongoing discussions with Dr. Stephen Hursting of the NCI and has planned a microarray analysis of subject animals that have been under study by Dr. Hursting. Animals that have been subjected to different levels of calories in diets resulted in groups of animals that can be segregated on the basis of weight including, calorie restricted (30%), calorie restricted (10%), normal weight, moderately obese and morbidly obese . These animals have already been evaluated and tissues have been collected. BioMarker will conduct microarray studies on adipose tissue and perform the bioinformatics analysis on tissue samples to be provided by Dr. Hursting . Affymetrix chips will be provided by Dr. Hursting . The study will be conducted under a cooperative research & development agreement (CRADA) with the NCI, giving BioMarker rights to negotiate a license should intellectual property be developed. In addition, the study will provide BioMarker with valuable research information on both the effect of fat tissue on the physiological effects of calorie restriction, as well as access to high level expertise in the field of cancer and obesity.
SRI International, Dr. Nurulain Zaveri - PPAR agonists
Dr. Zaveri is Drug Discovery Program Director at SRI International (formerly Stanford Research Institute)5. SRI has significant drug discovery and development capability which could be of downstream benefit to BioMarker. BioMarker gave a seminar and participated in discussions with Dr. Zaveri's group, which is focused on the development of PPAR ligands. PPARs (peroxisome proliferator activated receptors) serve as receptors for two very important classes of drugs: the hypolipidemic fibrates and the insulin sensitizing thiazolidinediones, and PPAR agonists have been identified as candidates for CR mimetics.
University of California, Davis, Drs. Ray Rodriguez and Kevin Dawson - Nutritional Genomics Center and High Performance Computing Center
4 Optigenex Inc., 750 Lexington Ave., 20'h Floor, New York, NY 10022 http://www.optiLcnex.coni/ s SRI International , 333 Ravenswood Avenue, Menlo Park, CA 94025-3493 http ://www.sri coin/
BioMarker Pharmaceuticals Inc. CONFIDENTIAL BioMarker has had meetings with the Director (Ray Rodriguez) and staff at the NIH-funded nutritional genomics center at UC Davis6. Discussions focused on a collaboration centered on access to advanced statistical analysis of microarray data, headed by Dr. Dawson. BioMarker also attended an informative nutritional genomics conference sponsored by the center. Nutritional genomics is an area that is highly complementary to calorie restriction and continuing to track developments and participants in this area will provide collaborative as well as business opportunities.
University ofCalifornia, Riverside (UCR), Dr. Stephen Spindler - Research Agreement
In October 2004 BioMarker completed a Research Agreement with UCR and Dr. Spindler as principal investigator for a study of mice that had been subjected to long-term calorie restriction and control diets. The Agreement gives BioMarker patent rights under the License Agreement (see below) as well as access to valuable biomaterials collected from the subject mice. B6C3F1 mice that had been on long-term diets (one month after birth in April 2002) were subjected to a diet-switching program intended to evaluate the effect of various diet time periods on liver gene expression. Aroximately '100 animals were divided into groups which included long-term control diet (LT-CON) animals switched to CR feeding for 2, 4, 8, and 16 week periods. In addition, CON and CR animals were maintained on their original diets, and a group of animals originally on CR were switched back to the CON diet for a period of 8 weeks. At the end of the respective time periods, all animals were sacrificed and all relevant target tissues and organs were harvested.
Under terms of the Research Agreement, BioMarker was provided with approximately half of all of the experimental subject tissues and materials. In addition, information on tumor incidence and pathology was collected on the experimental animals. A summary of the experimental data and material collected under the CR-control time course experiment has been completed and transferred to BioMarker.
C. Small Business Innovation Grants (SBIR)
BioMarker submitted two Phase 1 SBIR grant applications in 2004. The SBIR grant program is a federal government mandated program that sets aside 3.5% of all federal government expenditures in research for small businesses and is awarded through a competitive grant application process.
Discovery ofdruggable protein targets in aging brain, Phase 1
In April 2004 BioMarker submitted a Phase 1 SBIR grant application to fund the analysis of the potent neuroprotective effects of CR. The experimental model proposed was the identification of protein markers from hippocampal neurons obtained from mice subjected to CR. We proposed an analysis of specific neurons and their associated proteins, using the laser-assisted microdissection system recently acquired. The project was planned in collaboration with Dr.
6 http://nutrigenomics .ucdavis.edu/index.htm
BioMarker Pharmaceuticals Inc.. CONFIDENTIAL Jiankang Liu, a research investigator at CHORI and an expert in neuroscience. The total funds requested to support the study were $201,254. This proposal was reviewed by committee and in August 2004 received a priority score of 254. Although the score was not sufficient to obtain funding (the cut-off priority score was 250), the proposal can be resubmitted in an attempt to improve the priority score. { Innovative technologiesfor the molecular analysis ofcancer, Phase I
In June of 2004 BioMarker submitted a Phase I SBIR grant application in response to an RFA (request for applications) by the National Cancer Institute for the development of new technologies for the analysis of cancer. BioMarker's proposal focused on the development of an inducible transgenic mouse model for hepatocellular carcinoma (HCC). Such a model would be useful for studying the induction of HCC in both CR and control animals at an age before the appearance of spontaneous hepatoma would be observed (around 19 months). The model provides a specific approach for exploring the suppressive effects of CR on liver tumorigenesis. The study was planned and submitted in collaboration with Dr. Gordon Watson, CHORI.
D. Acquisition of Patent Rights and Materials Under the License and Research Agreement
In October 2004 BioMarker simultaneously concluded the principal License Agreement with The Regents of the University of California, and the Research Agreement with UCR to fund the project previously described. Under the License Agreement, BioMarker was provided access to valuable research materials in addition to the rights associated with the suite of patents. The biomaterial consists of Affymetrix gene chip data obtained from all of the CR related work conducted at Dr. Spindler's laboratory as well as access to tissues and specimens collected. BioMarker has received the transfer of the Affymetrix microarray data, and has made arrangements to receive tissues from selected animals in the first quarter of 2005. Notably, in addition to materials and patent rights to calorie restriction related work, the license agreement also provides access to the work performed in collaboration with Dr. Andzej Bartke (BioMarker Scientific Advisory Board member) on Ames dwarf mice, and for a method that may be useful for screening compounds for diabetogenic properties (see patent number 3 below).
7 1. U.S. Patent Number 6,406,853, entitled "Interventions to Mimic the Effects of Calorie Restriction," issued on June 18, 2002, by Stephen R. Spindler, UC Case Number 2000-178-2. 2. U.S. Patent Application Serial Number 10/056,749, entitled "Interventions to Mimic the Effects of Calorie Restriction," filed on January 22, 2002, by Stephen R. Spindler, UC Case Number 2000-178-3. 3. U.S. Patent Application Serial Number 10/177,958, entitled "Hepatic Gene Expression Profiling of Streptozotocin-Induced Diabetes," filed on June 21, 2002, by Stephen R. Spindler et al., UC Case Number 2000-345. 4. US Patent Application Serial Number 10/387,786, entitled "Methods of Analyzing Genes Affected by Caloric Restriction or Caloric Restriction Mimetics," filed on March 12, 2003, by Stephen R. Spindler et al., UC Case Number 2003-530. 5. US Patent Application Serial Number 10/387,743, entitled "Methods of Screening for Caloric Restriction Mimetics and Reproducing Effects of Caloric Restriction," filed on March 12, 2003, by Stephen R. Spindler et al., UC Case Number 2003-531. 6. US Patent Application Serial Number 10/622,160, entitled "Methods of Evaluating the Dynamics of Caloric Restriction and Identifying Caloric Restriction Mimetics," filed on July 16, 2003, by Stephen R. Spindler et al., UC Case Number 2004-052. 7. US Patent Application Serial Number 60/553,389 entitled "Genetic Networks Regulated By Attenuated GH/IGFI Signaling And Caloric Restriction," filed on March 16, 2004, by Stephen R. Spindler et al., and assigned to The Regents and Southern Illinois University, UC Case Number 2004-226. 8.Any patent application claiming any Elected Invention conceived and reduced to practice in the performance of work under the Research Agreement.
BioMarker Pharmaceuticals Inc., CONFIDENTIAL 9 E. Research Publications in 2004
BioMarker succeeded in publishing two significant papers this past year, both in April 2004, securing BioMarker's leadership in advancing microarray technology as it applies to the calorie restriction model. The first paper entitled "Temporal Linkage between the Phenotypic and Genomic Responses to Caloric Restriction" was published in the Proceedings of the National Academy of Sciences8. This landmark study showed that a cause-and-effect relationship is suggested by the tight linkage of the effects of calorie restriction on the rate of aging and the gene-expression biomarkers of CR. These effects were also seen in older animals (19 months) suggesting that a therapeutic benefit of a CR mimetic may be obtained at any age.
The second paper entitled "Additive Regulation of Hepatic Gene Expression by Dwarfism and Caloric Restriction" was published in the Journal of Physiological Genomics in collaboration with Dr. Andrzej Bartke9. The study was the first to compare the gene expression profiles of CR mice to dwarf mice, another well-accepted animal model of lifespan extension. The two groups of experimental animals showed overlapping yet independent gene expression changes. These results provide a novel and focused group of new genes associated with the regulation of lifespan in mammals.
In addition to the two papers published above, BioMarker has completed a manusc ript and submitted to the journal Circulation, an extensive gene expression profile analysis on the hearts of mice subjected to CR. The manuscript is currently in peer review (a patent application has been filed).
For its accomplishments in advancing research in the anti-aging field using gene expression biomarkers, BioMarker was awarded the Frost and Sullivan Excellence in Research Award for 2004 (see below).
F. Citations and Recognition
Based on progress and advancement in our research, and the increasing general interest in the molecular mechanisms involved in aging, BioMarker has already gained a position of prominence within the field. This is evidenced by several noteworthy media developments in 2004, some of which are listed below:
Fortune's "Chasing the Youth Pill "
In April BioMarker was mentioned in a Fortune article10 that covered the status of pharmaceutical development in the anti-aging field, stating that "Drugs that might extend human life are one of the hottest topics in biotech". The article included BioMarker as one of a few
8 Dhahbi J.M., Mote P.L, Kim H.J. & Spindler S R (2004) Temporal Linkage Between the Phenotypic and Genomic Responses to Caloric Restriction. Proc . Natl. Acad. Sci. U.S.A. 101:5524-5529 9 Tsuchiya T., Dhahbi J .M., Cui X., Mote P. L, Bartke A & Spindler S.R. (2004) Additive Regulation of Hepatic Gene Expression by Dwarfism and Caloric Restriction . Physiol. Genomics, 17 :307-315 10 Stipp D. Chasing the Youth Pill (April 19, 2004) Fortune
BioMarker Pharmaceuticals Inc., CONFIDENTIAL 10 companies actively engaged in the field, and reported on our finding that metformin induces gene expression changes in a manner similar to CR.
Bio IT World "High-Tech Search for the Fountain of Youth "
In January Bio IT World published an article on biotech development of drugs that slow agingt t. Bio IT is a widely read publication in the drug discovery & development field. BioMarker was extensively featured, and received as much attention as Elixir Pharmaceuticals, the well recognized leader in the field.
Cambridge Healthtech Advisors "Model Animal Systems: Emerging Applications and Commercial Opportunities in Drug Discovery and Development "
In June CHA published in its Advances in Life Sciences Report the use of model animal systems in drug discovery 12. This report was recently listed as one of their best sellers. In the report, BioMarker was identified as one of the "leading companies applying research on the biology of aging with model organisms to drug discovery". BioMarker was extensively profiled in the report, focusing on our development of the Intercept technology platform for the screening and identification of CR mimetics.
Frost & Sullivan Excellence in Research Award - 2004 3 BioMarker received the prestigious Frost & Sullivan's 2004 Excellence in Research Award, in the field of biomarker technology for our "development of innovative Intercept Approach which is instrumental in accelerating the discovery and development of drugs to treat diseases that are associated with aging". This award is presented to companies based on best practices in their field, after extensive analysis, comparison and ranking of companies within a field. BioMarker is honored to have received such recognition (a copy of the award is attached as Appendix A).
G. Development of Research Plan for 2005
After establishing the vivarium at CHORI and the build-out of the molecular biology lab in Alameda, BioMarker has devoted a considerable amount of time in planning and prioritizing research projects that take full advantage of the resources we have developed . A primary research objective is to establish a statistically significant data set on CR animals such that gene identification can be made with much higher accuracy than has been done to date (BMP Profiling project). For all planned projects , the most recent version of the Affymetrix mouse
11 Hopkin K. High-Tech Search for the Fountain of Youth - Dramatic advances may help biotechs develop drugs that slow aging (January 12, 2004) Bio IT World http://www.bio-itworld.com/ 12 Allan B. Haberman, Ph.D., Haberman Associates, The Biopharmaceutical Consortium, Cambridge Healthtech Advisors, Advances Life Sciences Reports: Model Animal Systems: Emerging Applications and Commercial Opportunities in Drug Discovery and Development, June 2004 httn:!/i+ww Chad' isors.com/services/AnimalModels'ovcrvicw.cfm 13 Shankar R. , F&A Excellence in Research Award, September 29, 2004 ham://wwnv.frost.com/prod/scrvlet/frost-liome.p ag
BioMarker Pharmaceuticals Inc, CONFIDENTIAL II gene microarray will be used : 430A 2.0 Array. This array interrogates approximately 39,000 transcripts from approximately 34,000 genes, which is a significant improvement over the previous version (U74). This will provide an opportunity to generate new intellectual property as well as to refine the experimental results. All experi ments will use a sufficiently large sample size so that results will be obtained with a high degree of statistical significance. Data will be analyzed using a more sophisticated informatics approach that will increase the statistical accuracy and will incorporate a molecular pathway analysis . All of these improvements, taken together, should give BioMarker a significant competitive advantage.
BioMarker has also developed screening programs for several compounds including single chemical entities (i.e., resveratrol, metformin) as well as complex extracts (i.e., LEF grape extract, Uncaria tomentosa extract). The current BioMarker projects are outlined below ("N" is the number of test animals used per group; organs to be collected, storage conditions, and schedule for processing are identified in the tables):
BMP Project 101 - Profiling
The Profiling project is intended to provide the definitive data set on calorie restricted mice vs. control-fed animals. Sample sizes have been selected to optimize statistical significance and accuracy. Initial microarrays will be conducted on liver and cells obtained from whole blood. Other organs will be collected and stored for subsequent analysis. Organs collected in OCT (a cryopreservative compound used to collect an organ that will later be thin-sectioned by cryostat) can be analyzed using the laser-assisted microdissection system.
Objective: CON (N=20) vs. CR (N=20) Mice: B6C3F1 DOB 02-07-04 CR start 04-07-04 Groups: 101CR, IOICON DNA Arrays: Affymetrix 430A 2.0 array Test groups: 101CR (N=15), 101CON (N=15) (total chips- 60 [15 101CON liver, 15 101CR liver, 15 101CON blood, 15 101CR blood]) Hybridization: Stanford Microarray Core Facility Organs to be collected: liver, whole blood, bone marrow, heart, spleen, lungs, muscle, brain, testis, kidney, fat Bioassays : blood glucose level (single time point at time of necropsy), every animal (N=40); white blood cell count 10I CON (N=20) RNA amplification and labeling: Liver and whole blood
Organ collection protocol: Organ �RNA Later OCT Liquid N2 Advance to RNA prep Liver j j Whole blood -�----- �------Bone marrow --•------Heart Spleen Lungs Muscle ------._...._---- B rain Testis Kidney Fat
BtoMarker Pharniaceulicals Inc., CONFIDENTIAL 12 2. BMP Project 102 - LEF grape extract/Sigma resveratrol
This project is designed to evaluate potential CR mimetic activity by gene expression profiling of the "LEF grape extract" and compare it to resveratrol obtained from Sigma-Aldrich (R5010 3,4',5-Trihydroxy-trans-stilbene, 99% by GC). The LEF grape extract (obtained from Life Extension Foundation) is Regrapex-R (whole grape extract enriched with resveratrol) originally supplied by Interpharma Praha, a.s. Regrapex-R is a complex dried powder formulation consisting of a whole grape (European grape Vitis vinifera) powdered extract, together with Polygonum cuspidatum in a ratio of 80:20. Known bioactive compounds include emodin, anthocyanins, piceid, polyphenols, and many uncharacterized compounds. Resveratrol (measured as a complex of trans-resveratrol plus its glycoside derivative (piceid/polydatin)) comprises 10% by weight of the material. Compounds have been formulated into food pellets by Bioserv and animals will be fed a single dose-containing food pellet as the first food of the day. The CR and CON animals from the Profiling project will serve as the basis for comparison.
Objective - Compare grape extract (N=20) and sigma resveratrol (N=20) to CR Mice: B6C3Fl DOB 02-07-04 CR start 04-07-04 Groups : 102LEF, 102SIGMA Dose : LEF grape extract (0.66 mg/mouse/day), sigma resveratrol (55 µg/mouse/day) Test period : 8 weeks DNA Arrays: Affymetrix 430A 2 .0 array . Test groups : 102LEF (N=15), 102SIGMA (N=15) (total chips 30 [15 102LEF liver, 15 102SIGMA liver]) Hybridization . Stanford Microarray Core Facility Tissues to be collected : liver, whole blood, bone marrow, heart, spleen, lungs, muscle, brain, testis, kidney, fat Bioassays : blood glucose level (single time point at time of necropsy), every animal (N=40) RNA amplification and labeling: Liver
Organ collection protocol: Organ RNA Later OCT Liquid N2 Advance to RNA prep Liver Whole blood Bone marrow Heart f--- - -_ Lungs ------Muscle Brain Testis Kidney Fat
BioMarker Pharmaceuticals Inc. CONFIDENTIAL 13 3. BMP Project 103 - Cat's Claw
A proprietary formulation of a Uncaria tomentosa extract obtained has demonstrated its anti- inflammatory and DNA repair properties. Microarray gene expression profiles will be obtained and compared to the CR and CON groups from the Profiling study. The extract has been formulated into food pellets by Bioserv and the animals will be fed a single dose-containing food pellet as the first food of the day. Uncaria extracts are made from various species and parts of the plant, in addition to being processed by a variety of methods (inner bark powder, water or alcoholic extraction, dialysis and filtration, etc.). Bioactive components include a variety of pentacyclic and tetracyclic oxindole alkaloids (i.e., pteropodine, isopteropodine, speciophylline, uncarine F, mytraphylline, isomytraphylline, ryncophylline, isoryncophylline, corynoxeine, isocorynoxeine), quinovic acid glyosides and triterpenes. In addition to evaluation of the gene expression response for similarity to CR, data will be analyzed for specific classes of genes known to be involved in DNA damage/repair.
BioMarker has obtained through other sources additional Cat's Claw material (e.g., U tomentosa inner vine bark powder from Peru - supplied by Raintree Nutrition Inc.) and can extend the studies to evaluate gene expression profiles that may be associated with specific classes of compounds or biological properties. This may serve as a useful demonstration project where microarray analysis might be found to be a useful technique to guide the further development of such complex extracts. BioMarker is also in business discussions with others (Rainforest Nutritionals Inc. / Santerra Pharmaceuticals Inc.) about evaluation of other proprietary Uncaria extracts that have shown efficacy in osteoarthritis of the knee.
4. BMP Project 104 - metformin
Extending the preliminary results obtained through collaboration with Dr. Spindler at UCR, BioMarker is independently conducting a high-quality analysis of metformin using the Affymetrix 430 microarray.
BioMarker Pharmaceuticals Inc, CONFIDENTIAL 14 BIOIIIARKER PH AR MACE UTICAL$.INC.
Appendix A.
BioMarker Pharmaceuticals Inc. receives the prestigious 2004 Frost & Sullivan Excellence in Research Award
September 29, 2004 By S. Ravi Shankar Research Analyst - Technical Insights Frost & Sul livan
Frost & Sullivan's 2004 Excellence in Research Award in the field of biomarker technology goes to BioMarker Pharmaceuticals Inc. for its development of the innovative "Intercept Approach" which is instrumental in accelerating the discovery and development of drugs to treat diseases that are associated with aging. Such drugs may also have the potential for prolonging the lifespan of human beings.
It is well documented that when animals are subjected to severe calorie restriction (CR) (where calorie intake is reduced by 30 to 40%) they experience a dramatic reduction in age-associated diseases, and experience a significantly longer lifespan. CR animals show a remarkable resistance to cancer, glucose abnormalities associated with diabetes, neurological diseases and heart disease.
BioMarker Pharmaceuticals Inc., a Campbell, California-based company, has developed a highly innovative approach that can screen for drugs that mimic the beneficial effects of CR. The approach is based on feeding drugs, foods, or neutraceuticals to mice, and then measuring how their genes respond using gene chips that can monitor virtually all of their genes at once. BioMarker calls this technique "Intercept" because it measures how all of the genes respond to the drug or food and then compares this pattern to that produced in mice subjected to CR. Interventions that show a significant overlap in the response of genes are prime candidates for further development as CR mimetics-drugs or foods that may prolong life and postpone the onset of common diseases of aging.
Until recently, aging research or biogerontology as it is now called, had not benefited from the significant advances made in the technology of genomics and the new tools that have become available as a result of mapping and sequencing genomes like that of mouse and man. With these tools come the ability to understand the role genes play in health and disease. BioMarker Pharmaceuticals Inc. is one of the first companies to focus on how genes that respond to a dietary intervention like CR affect the rate of aging, in the hope of developing compounds that have similar effects.
Recent scientific advances now suggest that aging is under the control of key genes, and that these genetic pathways might be targets for new drugs and interventions. BioMarker Pharmaceuticals has contributed directly to the scientific literature with two key publications in April 2004. The first study showed that gene changes occur relatively quickly in mice subjected to CR, in the same time frame that they begin to experience a deceleration in the rate of aging and a longer lifespan. Interestingly, BioMarker scientists observed that these changes occurred in older mice, the equivalent of 70 year-old humans. This finding was significant because up to then it was thought that either CR or an intervention might be required throughout the life of the animal.
15 BIOIIIARKER PHARMACEUTICALS.INC
The other study showed that genetically "dwarf' mice, which also live longer than normal mice, share many of the same gene responses found in CR animals. Together, these studies identify key genes and pathways involved in controlling the aging process, and suggest that interventions that modify the aging rate, and control susceptibility to a variety of age-related diseases, are possible.
BioMarker Pharmaceuticals intends to commercialize its proprietary technology by engaging pharmaceutical partners in the development of CR mimetics. Whereas long-term studies would be needed to prove the efficacy of such drugs in humans, BioMarker intends to also develop biomarkers as surrogate markers for use in human clinical trials in order to accelerate this process. Such biomarkers also afford diagnostic opportunities since they may well serve as predictors of health status, or for predisposition to certain age-related diseases.
BioMarker Pharmaceuticals Inc. has obtained an exclusive worldwide license from the University of California to U.S. patent 6,406,853 covering methods for identifying interventions for CR, as well as to several pending patent applications covering improvements in the technology as well as some initial CR mimetic candidates. BioMarker hopes to successfully use the technology to develop novel drugs that will help people live not only much longer but much healthier lives.
The Frost & Sullivan Award for Excellence in Research recognizes BioMarker Pharmaceuticals Inc. for its significant contributions to the drug discovery sector, and in particular for its efforts to accelerate the drug discovery process and enhance the productivity of the pharmaceutical industry by the use of this innovative biomarker technology.
The Frost & Sullivan team of industry experts present awards to companies demonstrating best practices in a variety of regional and global markets. These awards recognize the superior planning and execution of product launches, strategic alliances, distribution strategies, technological innovations, customer service and mergers & acquisitions. A host of other crucial marketing factors such as leadership, strategy, service, innovation, integration and development are also recognized. The companies that are commended as award recipients are those with the diligence, perseverance, and dedication required to develop a successful business plan and excel in the increasingly competitive global marketplace.
The methodologies for determining award recipients are uniform. Using interviews with all market participants and extensive secondary research of proprietary data, Frost & Sullivan analysts track competitor revenue and market share within the industry. The competitors are then compared and ranked. There are specific criteria used to ascertain the final competitor ranking in each Industry. For technology awards, the technologies and research projects are compared using customer base demands. In addition, factors such as feasibility of product launch, likelihood of customer acceptance and acceptance rates, and estimated time to market are taken into consideration.
16 Brief Annual Report for Critical Care Research for
2004
PROGRESS IN CALENDER YEAR 2004
Critical Care Research had a productive year in 2004.
ChiefProjects.
[1] Pursuit of Development of Portable Liquid Lung Cooling Device for Resuscitation, Jan-Dec 2004 and continuing.
12) Continued Work on Optimal Liquid Lavage Cooling Strategy Using Timing and Volume Variations with both Korr and SPECK devices
[3] Use of ice cartridge system for direct blood cooling: Pilot program.
[4] Work on a large scale Liquid Lung Cooling Device for use in Cryonics and possible deep human hypothermia applications.
[5] Continued dog breeding and experimental dog vivarium management. Availability of the dog model for other work. Discovery of MDR1 mutation homozygotes in our own inbred colony.
Project 1 : Lung Lavage Cooling Device Development (Development of All Peristalitic SPECK DEVICE).
The prototype lung lavage device which was reported in our RESUSCITATION article of 2001 used a PerFluoroCarbon (PFC) liquid cooled to near ice temperature, to lavage (wash in cycles) the lungs of a dog, resulting in very rapid cooling of the animal's brain and body core (-15 F in less than 20 minutes).
The purpose of cold lung lavage is to cool the brain very quickly. In 2003, trials of human brain cooling by means of venous cooling (requiring several hours) were reported by other authors, for the first time. These trials in general showed that medium-rapid cooling of the brain after injury (lack of oxygen or circulation, or even mechanical injury) inhibited the brain's automatic injury inflammatory response, which the brain exhibits like all other tissues of the body. This resulted in much less damage after cardiac arrest, and thus a much longer duration of cardiac arrest proved tolerable than had been previously possible. Cooling is the first treatment ever found to prove efficacious in preventing post resuscitation brain damage in human clinical trials.
The basic mechanism was thought to be that the brain's full-scale inflammatory response to several minutes of cardiac arrest, is inappropriate: it does not help heal brain damage, and in fact precipitates such damage. Evolution did not provide an appropriate "response" for the brain after cardiac arrest, because until recent development of technology, cardiac arrest was never a survivable injury. However, it has been demonstrated by very recent work that cooling helps to protect the brain from inflammation in the same basic way that application of cold to other parts of the body helps to inhibit swelling and damage, after sports injuries and bums. Although inflammation in the whole brain is unprogrammed by nature, it has proven controllable by application of cold, in the same way as inflammation in other tissues.
As detailed in last year's progress report (2003) we had engaged the services of Korr Medical (Salt Lake City, Utah) to redesign our lavage device to a portable configuration. This required the device pump module to be re-designed and computerized. This pump module utilized a double-chamber piston operation, weighed less than 35 lbs and was small enough to be transported by being carried in one hand, by a strap. This device was gas-powered from a small pressure tank and required a vacuum source. It used portable heat exchangers made from 10 pre-frozen "ice cartridges" which worked by routing PFC through bundles of thousands of hollow fibers, which were immersed and surrounded by water-ice. The amount of ice needed by this design was cut to only 8 lbs, with total heat exchanger assembly finally weighing less than 15 lbs, to cool the needed 7 degrees F (4 C) for a 50 lb dog. The amount of PFC in the circuit with this small heat-exchanger internal (liquid-containing) volume could be cut to about 4 liters (16 lbs).
In Summer of 2003, problems with Korr and a CCR search for a new engineering company to take on the lavage device development, produced a new partnership with SPECK Corporation, an engineering design firm based in the city of Orange, CA.
On looking at Korr's design, SPECK proposed a complete overhaul of the system, with a re-design which would use only "roller" type (peristaltic) pumps. This would allow direct electrical drive of both lavage liquid infusion and suction, and eliminate the need for both the vacuum and compressed air sources which had driven the Korr design.
A prototype SPECK all-peristalic pump breadboard device was built and successfully tested on a live dog in mid 2003, with the design showing only need for some modest improvement in suction performance.
Further development of this (basically) much simpler approach has occupied much of 2004. The prototype is expected to result in a smaller and more portable machine than could have been possible with the Korr compressed air/vacuum system. This SPECK design depends directly on electricity (local AC power or batteries) for function. This obviates the need for heavy compressed gas bottles, electrically operated compressors, or large electrically driven vacuum (suction) pumps.
However, the SPECK design in further testing in early 2004 revealed a number of problems which had not been suspected. Although the SPECK device was able to utilize the same novel ice-cartridge heat exchanger system as the KORR system, the simple SPECK design had some flaws. This design uses a continuously running peristaltic-pump with timed tubing occlusion system (using solenoid clamps) to stop fluid infusion (with bypass of the occlusion point by a pressure activated ball valve), and also to stop suction. This system's occlusion proved to be too high in electrical power consumption. A redesign using a knife-clamp driven by a leveraged geared solenoid proved much more thrift of power and less demanding on the DC power supply. It was thought to be adequate until further experiments using very low lavage volume cooling, showed that even minor leakage could influence the efficiency of the cooling cycle. These experiments, which were in continued search for improved lavage techniques, also occupied LVT research in 2004.
Ultimately, the problem of minor leaks past the SPECK occlusion system has not yet been completely solved, although progress has been made using softer tubing at the valve clamps, plus a new tubing clamp configuration. Further progress with development of the SPECK breadboard awaits a system of infusion and suction timing which has yet to be worked out by SPECK. The present system uses a simple ON-OFF design in which infusion is on while suction is off, and vice versa. Since some problems with the system happen during overlap of the two phases due to hysteresis in both infusion and suction functions, development of the machine will require a somewhat more complicated timing circuit which includes small delays between suction and infusion functions.
These have been proposed by SPECK to be handled by microprocessor and software control . After our difficulties with the overcomplicated KORR software and hardware design, however, we have requested a simpler timer, and ladder-logic design.
Project #2: Continued Work on Optimal Liquid Lavage Cooling Strategy Using Timing and Volume Variations with both Korr and SPECK devices. (See photo of Korr device, which can now be shown in the LEF magazine).
In 2004 we have continued our work to find the optimal volumes for lung lavage. In our initial work (RESUSCITATION 50/2, 187-203, 2001) and our patent, we had theorized that each volume of infused PFC fluid contains a thermal "dead space" analogous to dead space in an inhaled breath. This is a fractional volume of the original, from which no gas or heat is available for transfer, at the time scales of interest for a "breath." Presumably, this is due to the fact that the "dead space" fluid remains within the machine tubing, the trachea, or other areas of the lung (not the alveoli) where gas or heat transfer is able to take place in the time available for it.
An example of this concept is breathing behavior in an overheated dog, which is able to move a very large amount of gas in "panting," but can do this without hyperventilating, because most of the gas moved back and forth is in the form of very small "breaths" in which most of the gas is exposed only to the trachea, and doesn't participate in getting into or out of the blood.
We had thought that the thermal dead space would provide a limiting volume for lavages of liquid ventilation, below which we could not go because no heat would be transferred. Our studies had suggested this volume was about 3 to 5 mL/kg of animal--- a bit less than the dead-space volume seen in dogs while they are "panting."
As we actually began to try liquid PFC lavages of smaller volume, however, we were surprised that we did not see the very large expected fall-off in heat transfer that we had expected. Instead, heat transfer continued to remain at about 50%, even at very low lavage volumes. The only key was in HOW the fluid was delivered to the lungs. If a certain ventilation technique was used, even small volumes could make it to a portion of the lung in which their heat could be delivered.
These results are important, because it's critical to use as small volumes of liquid for heat transfer as possible. The smaller the volume of liquid used, the less pressure must be used to get it into the lung, and we now know from our experiments that the peak pressures used to ventilate the lung are more closely correlated with lung damaged than anything else. Typically, our dogs have no more than a slight case of the wheezes on the day after an experiment in which they've been cooled by 7 degrees F.--- enough to significantly increase survival in a human patient who had been recently subjected to potential brain injury from lack of oxygen after being in cardiac arrest for 5 to 10 minutes.
We expect that in the future, rapid cooling techniques such as we have demonstrated, which can cool this much in less than 20 minutes, will be critical in saving people who have experienced cardiac arrests in the critical "window" of 5 to 10 minutes, after which brain damage is almost always severe and (when it is closer to 10 minutes) is often enough to cause permanent vegetative states, such as in the unfortunate case of Terri Schiavo who suffered an estimated 10 minutes of cardiac arrest which permanently destroyed half of her brain.
By contrast, experimental dogs at Critical Care Research, Inc, subjected to cooling and given drugs AFTER resuscitation, have survived up to 16 minutes of total cardiac arrest without blood pressure, with no brain damage detectable at all. This is equivalent to a person with a heart attack going for more than 15 minutes with no heart beat, before the paramedics are able to restart the heart.
Adult dog brains are as sensitive to lack of oxygen as those of adult humans; this is well established in prior research. These dogs were NOT cooled before the arrest, as in cold- water drowning. It is important to understand that all the treatment they received came AFTER the arrest was already over, and their hearts had been restarted. We know the brain doesn't "die" after 5 minutes without oxygen, or even after 15 minutes! After 15 minutes there need not necessarily be any brain damage at all. Advanced resuscitation
4 techniques, which will certainly include rapid cooling, should one day return victims like Terri Schiavo to normal life, instead of leaving them with permanent severe brain damage, or even brain death.
Project #3. Use of ice cartridge system for direct blood cooling: Pilot program.
Because of the now-understood need to rapidly cool patients who have been recently resuscitated, perhaps even in the "field," before they reach the hospital, other'researchers have now begun to use direct techniques to cool resuscitated patients. These have had some success, and it is certain now that post-resuscitation cooling of the brain will become the first useful strategy to improve the survival outcomes of resuscitation and drowning patients. The only questions are how the cooling will be done. The brain cannot be cooled without the entire body being cooled, since the circulation of the brain is so large. And because of limitations with the insulation of the fat in the bodies of adults, cooling of adult resuscitation victims will probably need something more than just a bath of ice.
We are presently the only experimenters with the system of cold liquid lung lavage. Other workers have reported techniques involving insertion of a small heat exchanger into a major leg artery or vein, and running a solution of iced saline through this. This can produce cold temperatures faster than an ice bath, but not so fast as lung lavage. A final method of producing cold whole body temperatures involves removing the blood from an artery, running it though a heat exchanger, and returning it to a vein. This technique is actually the one used in resuscitating dogs after 15 minutes of cardiac arrest in the experiments described above. This technique has series dangers and difficulties, not least of which is the problem of how to gain rapid surgical access to the leg veins and arteries the needed time.
After that, comes the question of what kind of pump and heat exchanger are really needed for this work? In our work, we used a full Sarns 7000 cardiovascular bypass pump and membrane oxygenator and heat exchanger, of the sort that are already in commercial use to lower the temperature of heart bypass patients. But this machinery is cumbersome and not needed for a field rescue. For one thing, it requires a source of ice-water to run the heat exchanger, which is a separate system in itself.
We wondered, following the example of some more portable pump systems developed in Germany for resuscitation cooling, if the blood of an animal could be directly circulated over an ice-containing cartridge, using only the blood pressure of the animal itself. This experiment was tried as an example experiment for a patient application for blood compatible ice-containing cooling cartridges. We found that the system does work, dropping the temperature of the animal, about a third as quickly as lung lavage, for a total of -3 C = -5.4 F in 60 minutes (we had been achieving this amount of cooing in about 20 minutes with lung lavage). However, the circulatory volume loss and extra circulatory load from needing to drive blood though the the ice cartridge dropped the blood pressure to unacceptably low limits (60 mm Hg) during the experiment. Such a system will therefore probably will need some kind of pump assistance to be commercialized. Of note, however, is that our first and (to date) the only experimental subject of this direct ice cartridge blood-cooling technique, survived and did well.
Project #4: Very preliminary work on a large scale Liquid Lung Cooling Device for use in Cryonics and possible deep human hypothermia applications.
Hypothermic lung lavage at 4 C to a 37 C dog (gradient 33C) is capable of cooling alone at initial rates of > 20 C per hour (initially), implying a cooling time constant t_o = 33C gradient/ [20 deg/hr] = 1.65 hours. This is in the range of CPR+ice bath cooling. When added to cooling by CPR+whole body ice bath (measured time constant 1.7 hours), lavage cooling has the theoretical potential to lower the total cooling time constant to the sum of the reciprocal of these two time constants (1.65 hours and 1.7 hours), which is 0.8 hours. This is in the range of the rate of cooling possible with ice bath plus cardiac bypass cooling, which is the fastest possible rate in cryonics. Thus, it may be possible to cool cryonics patients at bypass rates, before bypass. For these reasons, it is quite desirable to add lung lavage cooling to cryonics cooling, so long as it can be utilized (intubated patients, CPR being done).
The heat power removed by lung lavage is substantial. A 70 kg patient has the heat capacity of 50 kg of water, and 70% of this thermal mass is cooled by lung lavage (the well-perfused thermal core). This is equivalent to 35 kg water, which requires 35 kg x 4184 J/kg/C x [20 C/3600 sec] = 800 watts initially. Thus, the heat exchanger keeping the perfluorocarbon (PFC) cold must be able to deal with at least I kw, and do it at internal heat exchanger temperature gradients less than about 4 C, since we wish to keep the PFC temperature below 5 C, and the presumed temperature of the icewater input to the heat exchanger is about I C (gradient of 4 C across the heat exchanger plates). Thus, the thermal performance of the heat exchanger H under theoretically best operating conditions (where flows are high enough on either side of the exchanger to not influence temperature of the fluids much from input to output, and the plate gradient is the same as the fluid temp gradient), is at least H = 800 watts/4 C = 200 watts/C.
We have been able to obtain a plate heat exchanger which passes 3 gal/min = 12 Umin of water when powered by a sump pump and put in series with a water-drip ice bath at I C. At 800 watts, the temperature change of this water flow will be less than [800 J/sec] / [12 kg x 4184 J/kg/C ] /60 sec = 1.0 C. This is close enough to zero to assume maximal heat exchanger performance for the cold side.
In initial tests, to test the actual heat removal capacity of the heat exchanger under these conditions, the exchanger was connected to pump and ice bath, and the other side circulated with 8 L of methanol standing in for an equal volume of fluorocarbon (total heat capacity of 8 L of methanol = "Cm" = 8 x 0.6 x 4184 J/C = 20,000 J/C). The temperature of the methanol fell to within 0.2 C of the 1.1 C limit of this system, within 3 minutes . From extrapolation of half-times in this cooling, the half-time of the cooling for the methanol was found to be close to 30 seconds . This implies a time constant for the system of 30 sec / ln2 = 43 sec.
Assuming Newtonian cooling in this system, so that the system heat removal rate is proportional to system temperature, and system temperature is proportional to system total heat, then:
The system cooling time constant t_o = total excess system heat at temp T/(heat removal rate at temp T)
= Q/[dQ/dt] = (Cm * deltaT)/power then solving for the heat exchanger performance H = (Power/deltaT) = Cm/t_o
In this case H = P/deltaT = Cm/t_o = (20,000 J/C) / (43 seconds) = 465 watts/degreeC
This is more than adequate performance, since 200 watts/C was the minimum needed.
The performance implies .that the PFC delivery will be at a maximum of
I degree C + (800 watts/465 watts/C) = 2.7 C.
In the next test, we delivered 1 . 15 kw to the "methanol" side of this circuit by immersion heater, until it came into equilibrium with the heat exchanger system (of course we won't use methanol). We had calculated this would happen at 1150 watts/(465 watts/C) = 2.5 C difference. This should be the equilibrium temperature difference between the warmed side of the circuit and the ice+water side . This temperature difference at an 1150 watt input should allow an independent check of heat exchanger performance H, since (assuming a relatively thermally isolated system , which is probably true at this power transfer rates) this T difference will be 1150 watts/H.
Tests of the above configuration with various sizes of ice+water containing cold reservoirs, from a 7 cubic foot Kirkland freezer with 7 inches of water and 40 lbs bagged ice, to a picnic ice-chest containing about 20 kg of ice and water. In all cases the much larger heat capacity of the ice side of the circuit caused the time constant to be determined by the 8 L of methanol, as above, and the halftime for cooling was about 30 seconds in both cases. These experiments suggest than in actual cooling, a cooler as small as a 30-40 L capacity can be used, so long as ice is added continuously during a suspension. The fact that such chests (- 10 gal) are used already with the Alcor portable perfusion pump proves the concept feasible. However, more ice will need to be obtained in the field, since it will still take in the range of 50 kg of ice to do full cryonics cooling.
Thus, we were also able to directly test heat exchanger performance with a heat load approximating a cryonics patient: the 1.15 kw electric immersion heater. For this test, we used two 10 gal ice chests were each filled with about 25 kg of H2O--- one of them containing liquid water, and the other mostly ice with enough water to run the large submersible pump (the heat sink). The other chest (the heat source) contained 22 kg
7 water (suitable for use with an immersion heater), with a heat capacity of (22 L /8 L) /0.6 = 4.6 times as large as the 8 L of methanol previously used as a fluorocarbon reservoir mockup. Consequently the water circuit was expected to have a half-time for equilibration 4.6 times as large as the methanol circuit, or about 2.3 minutes. This was the case, as it was found that 6 to 8 minutes was needed for equilibration to any new configuration (such as adding more ice or changing the position of the sump outlet tube). Consequently the full experiment using the 1150 W immersion heater required a run time of 35 minutes before becoming fairly stable. In the end we were able to obtain an approximately stable differential between the water reservoirs (heated water vs ice+water) of 2.4 C (2.7 C in the heated cooler vs. 0.4 C in the icewater cooler). This suggests heat exchanger performance of at least 1150 Watt/2.4 C = 480 watt/C. This is in line with predictions and is an independent test of heat exchanger performance, as above.
Thus, this plate heat exhanger should keep cooled flourocarbon (for example 8 L in our smaller reservoir) ready for lavage at no more than 2 or 3 degrees C above the temperature obtainable in the ice+water cold dump, which in turn should be less than 1 C, even under conditions of full cryonics cooling load.
Further plans involve testing the system above in the dog model.
Project #5: Dog Vivarium and Model Development, Breeding Program, and Community Outreach.
Our dependence on outside "class B" dog dealers as a source of experimental animals was ended in 2001 , as we began a program of complete in-house breeding of dogs, which were to be raised from puppies through adulthood at our facilities. At present, we maintain about 60 dogs in all stages of development, in complete indoor controlled environment kennel facilities overseen by a veterinarian and regularly inspected by the USDA. Our breeding program has generally been successful , and we have had good relations with the local veterinarians. This is partly as a result of using our colony as a source of donated dog blood to save injured dogs in a local vet practice . This does not harm our colony, and makes for better public relations. We benefit also from having our inspecting vet take time on his inspection days to do cat and kitten spay and neutering for a local cat adoption group . This benefits both the vet and the local cat-advocacy group, which knows that we don't use cats in any of our experimental programs.
In addition, we have cooperatively proposed research on a CCR heart preservation project, which will use some of our dogs as heart donors, if funded in fiscal year 2005.
In the process of collecting lungs from several of our dogs for our liquid ventilation project, we have used the anesthetized dogs before euthansia, as surgical training models for a Suspended Animation, Inc. (S.A.I, Boca Raton, Florida). These dogs never regained consciousness. This did not affect our scientific program, but allowed personnel from S.A.I. to practice large vessel artery and vein access in a way that they could not have done otherwise (not having access to large animals for experimental purposes). COLLABORATIONS
We are pleased that we have been able to establish a new collaboration with Dr. Katrina Mealey, a veterinarian and professor of small animal medicine working at Washington State University. Dr. Mealey's specialty is a mutant gene MDRI, which causes dogs with collie and herder backgrounds to be abnormally sensitive to certain pharmacologicals, notably the drug ivermectin (used to treat dogs for mites). See www.vetmed .wsu.edu/VCPL. In our inbred colony, we noted unusual ivermectin sensitivity in some of our animals, to the point of experiencing deaths in puppies from doses of ivermectin which should have been completely safe. Since our colony contains no dogs of the herder breeds that have classically been described with the gene (Collies, old English sheep dogs, Shelties, etc, we wondered if the MDRI gene could be the cause. But after contacting Dr. Mealey and doing extensive genetic testing of our colony, we found that indeed we had "accidentally" bred some pure MDRI homozygote animals against a mixed background of German Shepherd and hunting breeds . This does not affect our own research (there is no reason to think these dogs will behave any differently in our own experi ments, and we have genetically tested some of our resuscitation survivor dogs and found them free of the mutation). However, our colony may be helpful to Dr. Mealey's research and we are exploring ways to transfer several of our inbred animals to her facility. The existence of our animals is an example of a scientific opportunity which can come along just by making an observation and a connection. The dog MDRI gene no doubt has homologs in human the human genome also, and probably will eventually have some impact in the study of human drug metabolism.
The discovery of homozygote mutations in our own inbred and back-bred colony has underscored the need to bring fresh blood into our breeding system, and we have procured and raised an intact German Shepherd dog (who will be tested to insure freedom from the MDRI mutation) as the father of our next generation of puppies.
Steven B . Harris, MD Prepared for Critical Care Research, Inc.