`
`Utility Patent Application Transmittal
`
`Docket Number
`
`Address To
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, Virginia 22313-1450
`
`First Named Inventor
`
`Name
`
`Charles M. Brenner
`
`Ma'l'”9 Address
`
`78 North Thetford Road
`
`City
`
`Country
`
`City of Residence
`
`Country of Residence
`
`Nicotinamide Riboside Kinase Compositions and Methods for Using the Same
`
`New Hampshire
`
`03768
`
`New Hampshire
`
`United States of America
`
`Title of Invention
`
`
`
`
`
`Assignee Information*
`Name—
`Com can
`Trustees of Dartmouth College
`
`
`
`
`Address
`
`11 Rope Ferry Road
`
`Hanover
`United States of America
`
`State
`Postal Code
`
`New Hampshire
`03755
`
`*Complete this section if applicant wants the patent application publication to include assignee information. Providing this information
`does not substitute for compliance with any requirement of Part 3 of 37 CFR to have an assignment recorded by the Patent Office.
`
`
`
`
`
`
`City
`Count
`
`
`
`
`
`1.
`2.
`
`3.
`
`4-
`
`5.
`
`Application Elements
`See MPEP chapter 600 concerning utility patent application contents.
`
`
`
`uspro Filing Fee (as calculated infra)
`Applicant claims small entity status
`See 37 CFR 12-,
`Specification
`70
`Total number of pages
`Both the claims and abstract must start on a new page
`(Forlnformation on the preferred arrangement, see MPEP 608.01(a))
`Drawing(s) (35 U.S,C. 173)
`
`6. El Application Data Sheet (37 CFR 1.76)
`7.
`'3 CD-ROM or CD-R in duplicate, large table or
`Computer Program (Appendix)
`B Landscape Table on CD
`Nucleotide and/or Amino Acid Sequence Submission
`(if applicable, items 3- ' C' are required)
`a m Computer Readable Form (CRF)
`b.
`Specification Sequence listing on:
`
`8.
`
`T°ta' ”umber °f Sheats
`
`1
`
`i.
`
`|:|
`
`CD-ROM or CD-R (2 copies). or
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Oath or Declaration
`ii. E Paper
`
`
` a. U Newly executed (original or copy), orD Unexecuted
`c. m Statements verifying identity ofabove copies
`
`b. m A copy from a prior application (37 CFR 1.63(d))
`
`
`(for Continuation or Divisional Application)
`i. D DELETION OFlNVENTOR(S)
`
`Signed statement attached deleting inventor(s)
`named in the prior application (see 37 CFR
`
`1.63(d) and 1.33(b).)
`
`
`Page 1 of4
`
`Elysium Health Exhibit 1004
`
`Page 1 of 186
`
`Elysium Health Exhibit 1004
`Page 1 of 186
`
`

`

`9. D Assignment
`a. D Recordation FormCoverSheet
`
`
`
`
`10.
`
`11.
`12.
`
`13.
`
`37 CFR 3730)) Statement (when there is an assignee)
`
`English Translation Document (if applicable)
`
`Information Disclosure Statement
`
`18. D CertificateofMailing
`a. D FirstCIassMall, or
`
`b. D Express Mail
`Label Number
`
`Docket Number Utility Patent Application Transmittal
`
`
`
`
`
`EDDEI
`
`
` Power of Attorney D No Power of Attorney
`19. D Other:
`
`
`Ijijijrj
`
` a. D Copies of citations attached
`
`Preliminary Amendment
`
`Return Receipt Postcard (MPEP 503)
`(Should be specifically itemized)
`Certified Copy of Priority Document(s)
`
`(if foreign priority is claimed)
`
`Nonpublication Request under 35 USC 122(b)(2)(B)(i)
`
`
`
`20.
`
`if a CONTINUING APPLICATION, check appropriate box, and supply the requisite information below and in the first
`sentence of the specification following the title, or in an Application Data Sheet under 37 CFR 1. 76:
`
`Continuation
`
`I] Divisional D Continuation-in-part(ClP) ofprior application No.
`
`11/912,400
`(XX/YYY,YYY)
`
`Prior application infonnation:
`
`Examiner
`
`Gebreyesus, Kagnew H.
`
`Art Unit:
`
`1656
`
`Request Not To Publish*
`
`21, El Applicant hereby requests that the attached application not be published under 35 U.S.C. 122(b).
`
`Applicant hereby certifies that the invention disclosed in the attached application has not and will not be the
`subject of an application filed in another country, or under a multilateral international agreement, that requires
`publication at eighteen months after filing.
`
`*Warning
`
`This request must be signed in compliance with 37 CFR 1.33(b) and submitted with the application upon filing.
`
`If applicant rescinds a request that an application not
`Applicant may rescind this nonpublication request at any time.
`be published under 35 U.S.C. 122(b), the application will be scheduled for publication at eighteen months from the
`earliest claimed filing date for which a benefit is claimed.
`
`If applicant subsequently files an application directed to the invention disclosed in the attached application in
`another country, or under a multilateral international agreement, that requires publication of applications eighteen
`months after filing, the applicant must notify the United States Patent and Trademark Office of such filing WIthm
`forty-five (45) days after the date of the filing of such foreign or international application. Failure to do so wull result
`in abandonment of this application (35 U.S.C. 122(b)(2)(B)(iii)).
`
`
`
`Request-To Publish Early
`22_ D Applicant hereby requests the publication of this patent application earlier than as set forth in 37 CFR 1.211(a)
`pursuant to 37 CFR 1.219 as provided in 37 CFR 1.215(0).
`
`Page 2 of 4
`
`Elysium Health Exhibit 1004
`
`Page 2 of 186
`
`Elysium Health Exhibit 1004
`Page 2 of 186
`
`

`

`Docket Number Utility Patent Application Transmittal
`
`Fee Calculation
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Utility Patent Application Fee
`Claims as Filed
`
`#Extra
`
`#Ailowed
`I
`ll
`N0
`
`Rate
`ll
`X 03 O
`>< i—o N UI
`
`||
`
`'l'l
`
`ee
`
`$95
`
`l
`
`
`or
`
`“H
`
`Total Claims
`
`#Filed
`
`I
`
`II
`
`00
`
`Total Indep. Claims
`l
`Multiple Dependent Claims (check if applicable)
`m E—Fiiino small entit must be selected to oet Filino Fee discount Basic Filino Fee
`
`Number of Specification Pages
`
`Total Pages
`
`70
`Number of Drawing Sheets
`|i—t
`
`Application Size Fee
`
`Pre-Grant Publication Fee
`
`Early publication fee as set forth in 37 CFR 1.18(d)
`
`Processing fee for publication or republication as set forth in 37 CFR 1170)
`
`El
`
`Assignment Recordal Fee
`
`Enter Number of Properties Affected by Recordal
`Examination Fee
`
`x 40
`
`Search Fee
`
`Examination Fee
`
`Search Fee
`
`TOTAL
`
`$125
`
`$310
`
`$530
`
`Method of Payment
`
`50-1619
`
`Deposit Account Number
`
`D Deposit Account m Credit Card D Check D Money Order D Other:
`I
`For the above-identified deposit account, the Director is hereby authorized to: (check all that apply)
`D Charge the fee(s) set forth above
`[Z] Charge any additional fee(s) or underpayments of fee(s) under 37 CFR 1.16 and 1.17
`D Charge fee(s) indicated above, except for the filing fee
`Credit any overpayments
`
`WARNING: Information on this form may become public. Credit card information should not be included
`on this form. Provide credit card information and authorization on form PTO-2038.
`
`Amount Grand Total
`
`$530
`
`Page 3 of 4
`
`Elysium Health Exhibit 1004
`
`Page 3 of 186
`
`Elysium Health Exhibit 1004
`Page 3 of 186
`
`

`

`
`
`
`Utility Patent Application Transmittal
`
`
`
`DC0317US.C1
`
`Docket Number
`
`Customer Number
`
`26259
`
`Correspondence Address
`
`Address
`
`City
`
`Country
`
`Phone Number
`
`E-mail Address
`
`
`
`Postal Code
`
`
`
`Certificate of Mailing by Express Mail
`
`
`
`
`
`
`
`
`(Name ofPerson Mailing Correspondence) (Signature ofPerson Mailing Correspondence)
`(Name of erson ransnnttmg Corresponden e) (Signature ofPerson Transmitting Correspondence)
`
`
`Patent Application,
`this
`that
`certify
`hereby
`l
`accompanying documents, and fee are being deposited
`With the United States Postal Service "Express Mail Post
`Office to Addressee" service under 37 CFR 1.10 in an
`envelope addressed to Commissioner for Patents, PO.
`Box 1450, Alexandria, Virginia 22313-1450 on the date
`indicated below:
`
`
`
`
`
`(Date ofMailing)
`
`
`
`(Typed or Primed Name ofPerson Mailing Correspondence)
`
`
`
`
`
` (Signature ofPerson Mailing Correspondence)
`
`(HExpress Mail" Mailing Label Number)
`
`
`Certificate of Mailing by First Class Mail
`
`i hereby certify that this Patent Application, accompanying documents. and fee
`are being deposited with the United States Postal Service with sufficient postage
`as first class mail in an envelope addressed to Commissioner for Patents, PO.
`Box 1450, Alexandria, Virginia 22313-1450 on the date indicated below:
`
`(Date ofMailing)
`
`Certificate of Transmission
`
`l hereby certify that this Patent Application, accompanying documents, and fee
`authorization are being facsimile transmitted to the United States Patent and
`Trademark Office on the date indicated below:
`
`.
`.
`(Date of Transmission)
`
`P
`
`T
`
`.
`
`.
`
`c
`
`Signature Instructions
`
`Select the name of the person who will electronically sign the Patent Application from the drop-down box below.
`
`ILthal.tpractitioner is not present in the drop-down list, you must close this form and select 'Add Practitioner...‘ in the Form Manager's
`i
`i y menu.
`i
`
`sign the submission.
`Verify that the signatory information is correct and press the 'eSi .n' button to electronicall
`If you prefer to Sign the form manually, Simply do not click the 'e ign' button; just print an manually Sign.
`
`Signatory Drop-Down Box
`
`I Kathleen A. Tyrrell
`
`}
`
`
`
`Kathleen A. Tyrrell
`
`Registration Number
`
`38,350
`
`Attorney for Applicant(s)
`
`E-mail Address
`
`ktyrrell@licataandtyrrell.com
`
`
`/Kathleen A. Tyrrell/
`
`
`Date Signed
`
`04/12/2012
`
`
`
`
`
`Page 4 of4
`
`Elysium Health Exhibit 1004
`
`Page 4 of 186
`
`Elysium Health Exhibit 1004
`Page 4 of 186
`
`

`

`
`
`Utility Patent Application Transmittal
`
`Docket Number
`
`Address To
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, Virginia 22313-1450
`
`First Named Inventor
`
`Name
`
`Charles M. Brenner
`
`Ma'l'”9 Address
`
`78 North Thetford Road
`
`City
`
`Country
`
`City of Residence
`
`Country of Residence
`
`Nicotinamide Riboside Kinase Compositions and Methods for Using the Same
`
`New Hampshire
`
`03768
`
`New Hampshire
`
`United States of America
`
`Title of Invention
`
`
`
`
`
`Assignee Information*
`Name—
`Com can
`Trustees of Dartmouth College
`
`
`
`
`Address
`
`11 Rope Ferry Road
`
`Hanover
`United States of America
`
`State
`Postal Code
`
`New Hampshire
`03755
`
`*Complete this section if applicant wants the patent application publication to include assignee information. Providing this information
`does not substitute for compliance with any requirement of Part 3 of 37 CFR to have an assignment recorded by the Patent Office.
`
`
`
`
`
`
`City
`Count
`
`
`
`
`
`1.
`2.
`
`3.
`
`4-
`
`5.
`
`Application Elements
`See MPEP chapter 600 concerning utility patent application contents.
`
`
`
`uspro Filing Fee (as calculated infra)
`Applicant claims small entity status
`See 37 CFR 12-,
`Specification
`70
`Total number of pages
`Both the claims and abstract must start on a new page
`(Forlnformation on the preferred arrangement, see MPEP 608.01(a))
`Drawing(s) (35 U.S,C. 173)
`
`6. El Application Data Sheet (37 CFR 1.76)
`7.
`'3 CD-ROM or CD-R in duplicate, large table or
`Computer Program (Appendix)
`B Landscape Table on CD
`Nucleotide and/or Amino Acid Sequence Submission
`(if applicable, items 3- ' C' are required)
`a m Computer Readable Form (CRF)
`b.
`Specification Sequence listing on:
`
`8.
`
`T°ta' ”umber °f Sheats
`
`1
`
`i.
`
`|:|
`
`CD-ROM or CD-R (2 copies). or
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Oath or Declaration
`ii. E Paper
`
`
` a. U Newly executed (original or copy), orD Unexecuted
`c. m Statements verifying identity ofabove copies
`
`b. m A copy from a prior application (37 CFR 1.63(d))
`
`
`(for Continuation or Divisional Application)
`i. D DELETION OFlNVENTOR(S)
`
`Signed statement attached deleting inventor(s)
`named in the prior application (see 37 CFR
`
`1.63(d) and 1.33(b).)
`
`
`Page 1 of4
`
`Elysium Health Exhibit 1004
`
`Page 5 of 186
`
`Elysium Health Exhibit 1004
`Page 5 of 186
`
`

`

`9. D Assignment
`a. D Recordation FormCoverSheet
`
`
`
`
`10.
`
`11.
`12.
`
`13.
`
`37 CFR 3730)) Statement (when there is an assignee)
`
`English Translation Document (if applicable)
`
`Information Disclosure Statement
`
`18. D CertificateofMailing
`a. D FirstCIassMall, or
`
`b. D Express Mail
`Label Number
`
`Docket Number Utility Patent Application Transmittal
`
`
`
`
`
`EDDEI
`
`
` Power of Attorney D No Power of Attorney
`19. D Other:
`
`
`Ijijijrj
`
` a. D Copies of citations attached
`
`Preliminary Amendment
`
`Return Receipt Postcard (MPEP 503)
`(Should be specifically itemized)
`Certified Copy of Priority Document(s)
`
`(if foreign priority is claimed)
`
`Nonpublication Request under 35 USC 122(b)(2)(B)(i)
`
`
`
`20.
`
`if a CONTINUING APPLICATION, check appropriate box, and supply the requisite information below and in the first
`sentence of the specification following the title, or in an Application Data Sheet under 37 CFR 1. 76:
`
`Continuation
`
`I] Divisional D Continuation-in-part(ClP) ofprior application No.
`
`11/912,400
`(XX/YYY,YYY)
`
`Prior application infonnation:
`
`Examiner
`
`Gebreyesus, Kagnew H.
`
`Art Unit:
`
`1656
`
`Request Not To Publish*
`
`21, El Applicant hereby requests that the attached application not be published under 35 U.S.C. 122(b).
`
`Applicant hereby certifies that the invention disclosed in the attached application has not and will not be the
`subject of an application filed in another country, or under a multilateral international agreement, that requires
`publication at eighteen months after filing.
`
`*Warning
`
`This request must be signed in compliance with 37 CFR 1.33(b) and submitted with the application upon filing.
`
`If applicant rescinds a request that an application not
`Applicant may rescind this nonpublication request at any time.
`be published under 35 U.S.C. 122(b), the application will be scheduled for publication at eighteen months from the
`earliest claimed filing date for which a benefit is claimed.
`
`If applicant subsequently files an application directed to the invention disclosed in the attached application in
`another country, or under a multilateral international agreement, that requires publication of applications eighteen
`months after filing, the applicant must notify the United States Patent and Trademark Office of such filing WIthm
`forty-five (45) days after the date of the filing of such foreign or international application. Failure to do so wull result
`in abandonment of this application (35 U.S.C. 122(b)(2)(B)(iii)).
`
`
`
`Request-To Publish Early
`22_ D Applicant hereby requests the publication of this patent application earlier than as set forth in 37 CFR 1.211(a)
`pursuant to 37 CFR 1.219 as provided in 37 CFR 1.215(0).
`
`Page 2 of 4
`
`Elysium Health Exhibit 1004
`
`Page 6 of 186
`
`Elysium Health Exhibit 1004
`Page 6 of 186
`
`

`

`Docket Number Utility Patent Application Transmittal
`
`Fee Calculation
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Utility Patent Application Fee
`Claims as Filed
`
`#Extra
`
`#Ailowed
`I
`ll
`N0
`
`Rate
`ll
`X 03 O
`>< i—o N UI
`
`||
`
`'l'l
`
`ee
`
`$95
`
`l
`
`
`or
`
`“H
`
`Total Claims
`
`#Filed
`
`I
`
`II
`
`00
`
`Total Indep. Claims
`l
`Multiple Dependent Claims (check if applicable)
`m E—Fiiino small entit must be selected to oet Filino Fee discount Basic Filino Fee
`
`Number of Specification Pages
`
`Total Pages
`
`70
`Number of Drawing Sheets
`|i—t
`
`Application Size Fee
`
`Pre-Grant Publication Fee
`
`Early publication fee as set forth in 37 CFR 1.18(d)
`
`Processing fee for publication or republication as set forth in 37 CFR 1170)
`
`El
`
`Assignment Recordal Fee
`
`Enter Number of Properties Affected by Recordal
`Examination Fee
`
`x 40
`
`Search Fee
`
`Examination Fee
`
`Search Fee
`
`TOTAL
`
`$125
`
`$310
`
`$530
`
`Method of Payment
`
`50-1619
`
`Deposit Account Number
`
`D Deposit Account m Credit Card D Check D Money Order D Other:
`I
`For the above-identified deposit account, the Director is hereby authorized to: (check all that apply)
`D Charge the fee(s) set forth above
`[Z] Charge any additional fee(s) or underpayments of fee(s) under 37 CFR 1.16 and 1.17
`D Charge fee(s) indicated above, except for the filing fee
`Credit any overpayments
`
`WARNING: Information on this form may become public. Credit card information should not be included
`on this form. Provide credit card information and authorization on form PTO-2038.
`
`Amount Grand Total
`
`$530
`
`Page 3 of 4
`
`Elysium Health Exhibit 1004
`
`Page 7 of 186
`
`Elysium Health Exhibit 1004
`Page 7 of 186
`
`

`

`
`
`
`Utility Patent Application Transmittal
`
`
`
`DC0317US.C1
`
`Docket Number
`
`Customer Number
`
`26259
`
`Correspondence Address
`
`Address
`
`City
`
`Country
`
`Phone Number
`
`E-mail Address
`
`
`
`Postal Code
`
`
`
`Certificate of Mailing by Express Mail
`
`
`
`
`
`
`
`
`(Name ofPerson Mailing Correspondence) (Signature ofPerson Mailing Correspondence)
`(Name of erson ransnnttmg Corresponden e) (Signature ofPerson Transmitting Correspondence)
`
`
`Patent Application,
`this
`that
`certify
`hereby
`l
`accompanying documents, and fee are being deposited
`With the United States Postal Service "Express Mail Post
`Office to Addressee" service under 37 CFR 1.10 in an
`envelope addressed to Commissioner for Patents, PO.
`Box 1450, Alexandria, Virginia 22313-1450 on the date
`indicated below:
`
`
`
`
`
`(Date ofMailing)
`
`
`
`(Typed or Primed Name ofPerson Mailing Correspondence)
`
`
`
`
`
` (Signature ofPerson Mailing Correspondence)
`
`(HExpress Mail" Mailing Label Number)
`
`
`Certificate of Mailing by First Class Mail
`
`i hereby certify that this Patent Application, accompanying documents. and fee
`are being deposited with the United States Postal Service with sufficient postage
`as first class mail in an envelope addressed to Commissioner for Patents, PO.
`Box 1450, Alexandria, Virginia 22313-1450 on the date indicated below:
`
`(Date ofMailing)
`
`Certificate of Transmission
`
`l hereby certify that this Patent Application, accompanying documents, and fee
`authorization are being facsimile transmitted to the United States Patent and
`Trademark Office on the date indicated below:
`
`.
`.
`(Date of Transmission)
`
`P
`
`T
`
`.
`
`.
`
`c
`
`Signature Instructions
`
`Select the name of the person who will electronically sign the Patent Application from the drop-down box below.
`
`ILthal.tpractitioner is not present in the drop-down list, you must close this form and select 'Add Practitioner...‘ in the Form Manager's
`i
`i y menu.
`i
`
`sign the submission.
`Verify that the signatory information is correct and press the 'eSi .n' button to electronicall
`If you prefer to Sign the form manually, Simply do not click the 'e ign' button; just print an manually Sign.
`
`Signatory Drop-Down Box
`
`I Kathleen A. Tyrrell
`
`}
`
`
`
`Kathleen A. Tyrrell
`
`Registration Number
`
`38,350
`
`Attorney for Applicant(s)
`
`E-mail Address
`
`ktyrrell@licataandtyrrell.com
`
`
`/Kathleen A. Tyrrell/
`
`
`Date Signed
`
`04/12/2012
`
`
`
`
`
`Page 4 of4
`
`Elysium Health Exhibit 1004
`
`Page 8 of 186
`
`Elysium Health Exhibit 1004
`Page 8 of 186
`
`

`

`DC0317US.C1
`
`-1-
`
`EATENT
`
`NICOTINAMIDE RIBOSIDE KINASE COMPOSITIONS AND METHODS FOR
`USING THE SAME
`
`Introduction
`
`This application is
`
`a
`
`continuation of U.S. Patent
`
`Application Serial No. 11/912,400 filed November 20, 2007,
`
`which.
`
`is the National Stage of
`
`International Application
`
`No.
`
`PCT/USZOO6/015495
`
`filed. April 20,
`
`2006, which. claims
`
`benefit of priority to U.S. Patent Application Serial No.
`
`11/113,701 filed April 25, 2005,
`
`the teachings of which are
`
`incorporated herein by reference in their entireties.
`
`This invention was made with government support under
`
`grant
`
`number
`
`CA77738
`
`awarded
`
`by
`
`the National Cancer
`
`Institute.
`
`The
`
`government
`
`has
`
`certain rights
`
`in
`
`the
`
`invention.
`
`Background of the Invention
`
`Nicotinic acid and nicotinamide, collectively niacins,
`
`are the vitamin forms of nicotinamide adenine dinucleotide
`
`(NAD+). Eukaryotes
`
`can synthesize NAD+
`
`de novo via the
`
`kynurenine pathway"
`
`from.
`
`tryptophan (Krehl, et al.
`
`(1945)
`
`Science 101:489—490; Schutz and Heigelson (1972)
`
`J. Biol.
`
`Chem.
`
`247:5327—5332)
`
`and niacin supplementation prevents
`
`the
`
`pellagra
`
`that
`
`can
`
`occur
`
`in
`
`populations with
`
`a
`
`tryptophan—poor diet. It is well—established that nicotinic
`
`acid is phosphoribosylated to nicotinic acid mononucleotide
`
`
`
`(NaMN), which is then adenylylated to form nicotinic acid
`
`adenine dinucleotide (NaAD), which in turn is amidated to
` form NAD+
`
`(Preiss
`
`and Handler
`
`(1958)
`
`J1 Biol.
`
`Chem.
`
`233:488—492; Preiss
`
`and Handler
`
`(1958b)
`
`J} Biol.
`
`Chem.
`
`233:493—50).
`
`NAD+ was
`
`initially characterized. as
`
`a
`
`co—enzyme
`
`
`for
`
`oxidoreductases.
`
`Though
`
`conversions
`
`between NAD+,
`
`NADH,
`
`Elysium Health Exhibit 1004
`
`Page 9 of 186
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`Elysium Health Exhibit 1004
`Page 9 of 186
`
`

`

`DCO317US.C1
`
`~2-
`
`PATENT
`
`NADP and NADPH would not be accompanied by a loss of total
`
`
`co—enzyme, it was discovered that NAD+ is also turned over
`
`in
`
`cells
`
`for
`
`unknown
`
`purposes
`
`(Maayan
`
`(1964) Nature
`
`204:1169—1170).
`
`Sirtuin
`
`enzymes
`
`such
`
`as
`
`Sir2
`
`of
`
`S.
`
`cerevisiae and
`
`its homologs deacetylate lysine residues
`
`with consumption of an equivalent of NAD+ and this activity
`
`is required for Sir2 function as a transcriptional silencer
`
` (Imai, et al.
`
`(2000) Cold Spring Harb. Symp. Quant. Biol.
`
`10
`
`15
`
`65:297—302).
`
`NAD+—dependent
`
`deacetylation
`
`reactions
`
`are
`
`required not only for alterations in gene expression but
`
`also for
`
`repression of
`
`ribosomal
`
`DNA
`
`recombination and
`
`extension of
`
`lifespan.
`
`in response to calorie restriction
`
`(Lin,
`
`et al.
`
`(2000) Science 289:2126—2128; Lin,
`
`et al.
`
`(2002) Abture 418:344—348).
`
`NAD+
`
`is consumed by Sir2 to
`
`produce a mixture of
`
`2’— and.
`
`3’ O—acetylatedv ADP—ribose
`
`plus nicotinamide and the deacetylated polypeptide (Sauve,
`
`et
`
`al.
`
`(2001) Biochemistry 40:15456—15463). Additional
`
`enzymes,
`
`including
`
`poly(ADPribose)
`
`polymerases
`
`and
`
`chDPribose
`
`synthases
`
`are also NAD+—dependent
`
`and produce
`
`nicotinamide and ADPribosyl products
`
`(Ziegler
`
`(2000) Eur.
`
`J. Biochem. 267:1550—1564; Burkle
`
`(2001) Bioessays 23:795—
`
`806).
`
`The
`
`non—coenzymatic properties of NAD+ has
`
`renewed
`
`interest
`
`in NAD+ biosynthesis.
`
`Four
`
`recent publications
`
`have
`
`suggested what
`
`is considered to be all of
`
`the gene
`
`products and pathways to NAD+ in S. cerevisiae (Panozzo, et
`
`al.
`
`(2002)
`
`FEBS Lett. 517:97—102; Sandmeier, et al.
`
`(2002)
`
`Genetics 160:877~889; Bitterman,
`
`et al.
`
`(2002)
`
`J. Biol.
`
`Chem.
`
`277:45099—45107; Anderson,
`
`et
`
`al.
`
`(2003) Nature
`
`423:181—185) depicting convergence of the flux to NAD+ from
`
`de novo synthesis, nicotinic acid import,
`
`and nicotinamide
`
`salvage at NaMN (Scheme 1).
`
`20
`
`25
`
`3O
`
`Elysium Health Exhibit 1004
`
`Page 10 of 186
`
`Elysium Health Exhibit 1004
`Page 10 of 186
`
`

`

`DC0317US.C1
`
`-3-
`
`PATENT
`
`o
`
`NaMN
`
`o
`
`NaAD+
`
`o
`
`NAD+
`
`DE NOVO
`Bna1—6
`’
`
`o—
`Nma1,2
`’
`
`‘
`
`\\\
`//
`N+
`Prbo
`
`ATP
`
`I
`
`\\‘
`//
`NVL
`ATP
`I
`lADPrbo Gln
`
`SALVAGE
`Nptl
`
`PrboPP
`
`Nicotinic acid
`
`0‘
`Qnsl
`
`’
`
`i
`
`\\\
`//
`N+
`ATP
`I
`PPi ADPrbo
`"
`
`a1
`
`NH2
`
`-
`LysAc
`Sir2
`
`nicotinamide
`
`\\\
`
`Pncl
`<——--
`I
`Nflgo /§+
`
`Lys +
`ADPrbOAC
`NHz
`
`IMPORT
`
`Tna1
`
`Plasma membrane
`
`Scheme 1
`
`Summary of the Invention
`
`It has
`
`now been shown
`
`that nicotinamide
`
`riboside,
`
`which was known to be an NAD+ precursor in bacteria such as
`
`Haemophilus
`
`influenza
`
`(Gingrich
`
`and Schlenk
`
`(1944)
`
`J.
`
`Bacteriol.
`
`47:535—550; Leder
`
`and Handler
`
`(1951)
`
`J3 Biol.
`
`Chem.
`
`189:889—899; Shifrine and Biberstein (1960) Nature
`
`187:623)
`
`that
`
`lack the enzymes of the de novo and Preiss—
`
`Handler
`
`pathways
`
`(Fleischmann,
`
`et
`
`a1.
`
`(1995)
`
`Science
`
`269:496—512),
`
`is an NAD+ precursor in a previously unknown
`
`but conserved eukaryotic NAD+ biosynthetic pathway. Yeast
`
`nicotinamide riboside kinase, Nrkl,
`
`and human Nrk enzymes
`
`with specific functions
`
`in NAD+ metabolism. are provided
`
`herein.
`
`
`The specificity of
`
`these enzymes
`
`indicates
`
`that
`
`they are the long—sought
`
`tiazofurin kinases
`
`that perform
`
`the
`
`first
`
`step
`
`in
`
`converting
`
`cancer
`
`drugs
`
`such
`
`as
`
`Elysium Health Exhibit 1004
`
`Page]].0f186
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Elysium Health Exhibit 1004
`Page 11 of 186
`
`

`

`DC0317US.C1
`
`-4—
`
`PATENT
`
`tiazofurin.
`
`and. benzamide
`
`riboside and,
`
`their analogs
`
`into
`
`toxic NAD+
`
`analogs. Further,
`
`yeast mutants
`
`of defined
`
`genotype were used to identify sources of nicotinamide
`
`riboside
`
`and
`
`it
`
`is
`
`shown
`
`that milk
`
`is
`
`a
`
`source
`
`of
`
`nicotinamide riboside.
`
`Accordingly,
`
`the present
`
`invention is
`
`an
`
`isolated
`
`nucleic acid encoding a eukaryotic nicotinamide riboside
`
`kinase
`
`polypeptide.
`
`A
`
`eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase nucleic acid encompasses
`
`(a)
`
`a rumfleotide sequence
`
`of
`
`SEQ
`
`ID NO:1,
`
`SEQ
`
`ID NO:2
`
`or
`
`SEQ ID NO:3;
`
`(b)
`
`a
`
`nucleotide
`
`sequence
`
`that
`
`hybridizes
`
`to
`
`a
`
`nucleotide
`
`sequence of SEQ ID NO:1,
`
`SEQ ID NO:2 or SEQ ID NO:3 or its
`
`complementary
`
`nucleotide
`
`sequence
`
`under
`
`stringent
`
`conditions, wherein
`
`said nucleotide
`
`sequence
`
`encodes
`
`a
`
`functional nicotinamide riboside kinase polypeptide; or
`
`(c)
`
`10
`
`15
`
`amino
`
`acid sequence
`
`a nucleotide
`
`sequence
`
`encoding
`
`an
`
`encoded by the nucleotide sequences of
`
`(a)
`
`or
`
`(b), but
`
`which
`
`has
`
`a
`
`different
`
`nucleotide
`
`sequence
`
`than
`
`the
`
`nucleotide sequences of
`
`(a) or
`
`(b) due to the degeneracy of
`
`the
`
`genetic
`
`code
`
`or
`
`the
`
`presence
`
`of
`
`non—translated
`
`nucleotide sequences.
`
`The present
`
`invention is also an expression vector
`
`containing an isolated nucleic acid encoding a eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide.
`
`In
`
`one
`
`embodiment,
`
`the expression vector is part of a composition
`
`containing’
`
`a
`
`pharmaceutically
`
`acceptable
`
`carrier.
`
`In
`
`another
`
`embodiment,
`
`the
`
`composition
`
`further
`
`contains
`
`a
`
`prodrug wherein the prodrug is
`
`a nicotinamide riboside—
`
`related analog that
`
`is phosphorylated by
`
`the
`
`expressed
`
`nicotinamide riboside kinase thereby performing the first
`
`step in activating said prodrug.
`
`The present
`
`invention is also an isolated eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide.
`
`In
`
`one
`
`20
`
`25
`
`30
`
`Elysium Health Exhibit 1004
`
`Page 12 of 186
`
`Elysium Health Exhibit 1004
`Page 12 of 186
`
`

`

`DC0317US.C1
`
`-5-
`
`PATENT
`
`embodiment,
`
`the
`
`isolated
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide has
`
`an
`
`amino acid sequence having at
`
`least
`
`about
`
`70% amino acid sequence similarity to an amino acid
`
`sequence of SEQ ID NO:4,
`
`SEQ ID NO:5 or SEQ ID NO:6 or a
`
`functional fragment thereof.
`
`The present
`
`invention is
`
`further
`
`a
`
`cultured cell
`
`containing an isolated nucleic acid encoding a eukaryotic
`
`nicotinamide riboside kinase polypeptide (n:
`
`a polypeptide
`
`encoded thereby.
`
`Still further,
`
`the present
`
`invention is a composition
`
`containing an
`
`isolated eukaryotic nicotinamide
`
`riboside
`
`
`
`kinase
`
`carrier.
`
` --
`
`in
`
`polypeptide
`
`and
`
`a
`
`pharmaceutically
`
`one
`
`embodiment,
`
`the
`
`composition
`
`acceptable
`.c
`further
`
`contains a prodrug wherein said prodrug is a nicotinamide
`
`riboside—related
`
`analog
`
`that
`
`is
`
`phosphorylated by
`
`the
`
`nicotinamide riboside kinase thereby performing the first
`
`
`
`step in activating said prodrug.
`
`The present
`
`invention is also a method for
`
`treating
`
`10
`
`15
`
`20
`
`25
`
`30
`
`.cancer by administering to a patient having or suspected of
`
`having
`
`cancer
`
`an
`
`effective
`
`amount
`
`of
`
`a
`
`nicotinamide
`
`riboside—related. prodrug in combination. with an isolated
`
`eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide
`
`or
`
`expression vector
`
`containing
`
`an
`
`isolated nucleic
`
`acid
`
`sequence
`
`encoding
`
`an
`
`eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase polypeptide wherein the nicotinamide riboside kinase
`
`polypeptide phosphorylates
`
`the prodrug"
`
`thereby performing
`
`the first step in activating the prodrug so that
`
`the signs
`
`or symptoms of said cancer are decreased or eliminated.
`
`The
`
`present
`
`invention
`
`is
`
`further
`
`a method
`
`for
`
`identifying a natural or synthetic source for nicotinamide
`
`riboside.
`
`The method
`
`involves
`
`contacting a
`
`first cell
`
`lacking a
`
`functional glutamine—dependent NAD+
`
`synthetase
`
`with
`
`an
`
`isolated extract
`
`from a
`
`natural
`
`source
`
`or
`
`Elysium Health Exhibit 1004
`
`Page 13 of 186
`
`Elysium Health Exhibit 1004
`Page 13 of 186
`
`

`

`DC0317US.C1
`
`-6-
`
`PATENT
`
`synthetic;
`
`contacting a
`
`second cell
`
`lacking functional
`
`glutamine—dependent
`
`NAD+
`
`synthetase
`
`and
`
`nicotinamide
`
`riboside kinase with.
`
`the isolated extract;
`
`and detecting
`
`growth. of
`
`the first cell
`
`compared.
`
`to the growth. of
`
`the
`
`second cell, wherein the presence of growth in the first
`
`cell and absence of growth in the second cell is indicative
`
`of
`
`the presence of nicotinamide riboside in the isolated
`
`extract.
`
`In one
`
`embodiment,
`
`the natural
`
`source is cow’s
`
`milk.
`
`10
`
`15
`
`Further,
`
`the present
`
`invention is a dietary supplement
`
`composition containing nicotinamide riboside identified in
`
`
`accordance with the methods of the present
`
`invention and a
`
`carrier.
`
`Moreover,
`
`the present
`
`invention
`
`is
`
`a method
`
`for
`
`preventing’ or
`
`treating‘
`
`a disease or condition associated
`
`with the nicotinamide
`
`riboside kinase
`
`pathway of
`
`NAD+
`
`a
`
`20
`
`25
`
`30
`
`biosynthesis.
`
`The method
`
`involves
`
`administering
`
`to
`
`patient having a disease or condition associated with the
`
`nicotinamide riboside kinase pathway of NAD+ biosynthesis
`
`an effective amount of a nicotinamide riboside composition
`
`so that
`
`the signs or
`
`symptoms of
`
`the disease or condition
`
`are
`
`prevented
`
`or
`
`reduced.
`
`In
`
`one
`
`embodiment,
`
`the
`
`nicotinamide
`
`riboside
`
`is
`
`neuroprotective.
`
`In
`
`another
`
`embodiment
`
`the nicotinamide riboside is anti—fungal.
`
`In a
`
`further
`
`embodiment,
`
`the
`
`nicotinamide
`
`riboside
`
`is
`
`administered in combination with tryptophan, nicotinic acid
`
`or nicotinamide.
`
`The present
`
`invention is also an in vitro method for
`
`identifying a nicotinamide
`
`riboside—related prodrug.
`
`The
`
`method involves contacting a
`
`rucotinamide riboside kinase
`
`polypeptide with a nicotinamide riboside—related test agent
`
`and determining whether said test agent
`
`is phosphorylated
`
`by said nicotinamide riboside kinase polypeptide wherein
`
`Elysium Health Exhibit 1004
`
`Page 14 of 186
`
`Elysium Health Exhibit 1004
`Page 14 of 186
`
`

`

`DC0317US.C1
`
`-7-
`
`PATENT
`
`phosphorylation of said test agent
`
`is indicative of said
`
`test agent being a nicotinamide riboside—related prodrug. A
`
`nicotinamide
`
`riboside—related prodrug identified by
`
`this
`
`method is also encompassed within the present invention.
`
`5
`
`
`The present
`
`
`invention is further a cell—based method
`
`for
`
`identifying a nicotinamide
`
`riboside—related prodrug.
`
`This method.
`
`involves contacting a first
`
`test cell which
`
`expresses a recombinant Nrk polypeptide with a nicotinamide
`
`ribosidesrelated test agent; contacting a second test cell
`
`10 which lacks a functional Nrk polypeptide with the same test
`
`agent;
`
`and determining the viability of
`
`the
`
`first
`
`and
`
`second test cells, wherein sensitivity of
`
`the first cell
`
`and. not
`
`the second cell
`
`is indicative of
`
`a
`
`Iqicotinamide
`
`riboside—related prodrug.
`
`A nicotinamide riboside—related
`
`15
`
`prodrug identified by
`
`this method
`
`is
`
`also encompassed
`
`within the context of the present invention.
`
`'The present
`
`invention is also a method for identifying
`
`an individual or
`
`tumor which is susceptible to treatment
`
`with a nicotinamide riboside—related prodrug. This method
`
`20
`
`involves detecting'
`
`the presence of mutations
`
`in, or
`
`the
`
`level of expression of,
`
`a nicotinamide riboside kinase in
`
`an individual or
`
`tumor wherein the presence of
`
`aa mutation
`
`or change in expression of nicotinamide riboside kinase in
`
`said
`
`individual, or
`
`tumor
`
`compared
`
`to
`
`a
`
`control
`
`is
`
`25
`
`indicative of said individual or
`
`tumor having an altered
`
`
`level of susceptibility to treatment with.
`
`a nicotinamide
`
`riboside—related prodrug.
`
`Brief Description of the Drawings
`
`Figure 1
`
`shows
`
`the amino acid sequence alignment and
`
`30
`
`consensus
`
`sequence
`
`(SEQ ID NO:34) of
`
`human. Nrkl
`
`(SEQ ID
`
`NO:5), human Nrk2 (SEQ ID NO:6), S. cerevisiae Nrkl
`
`(SEQ ID
`
`NO:4), S. pombe nrkl
`
`(SEQ ID N027), as compared to portions
`
`Elysium Health Exhibit 1004
`
`Page 15 of 186
`
`Elysium Health Exhibit 1004
`Page 15 of 186
`
`

`

`DC0317US.C1
`
`-8-
`
`PATENT
`
`of S. cerevisiae uridine/cytidine kinase Urkl
`
`(SEQ ID NO:8)
`
`and E. coli pantothenate kinase (SEQ ID NO:9).
`
`Detailed Description of the Invention
`
`A
`
`Saccharomyces
`
`cerevisiae
`
`QNSl
`
`gene
`
`encoding
`
`glutamine—dependent NAD+ synthetase has been characterized
`
`and mutation of either the glutaminase active site or
`
`the
`
`NAD+
`
`synthetase active
`
`site resulted in inviable cells
`
`(Bieganowski,
`
`et al.
`
`(2003)
`
`J} Biol.
`
`Chem.
`
`278:33049—
`
`33055). Possession of strains containing the qnsl deletion
`
`and a plasmid—borne QNSl gene allowed a determination of
`
`whether the canonical de novo,
`
`import and salvage pathways
`
`for
`
`NAD+ of
`
`Scheme
`
`1
`
`(Panozzo,
`
`et al.
`
`(2002)
`
`supra;
`
`Sandmeier, et al.
`
`(2002)
`
`supra; Bitterman, et al.
`
`(2002)
`
`supra; Anderson,
`
`et al.
`
`(2003)
`
`supra)
`
`are
`
`a
`
`complete
`
`representation of
`
`the metabolic pathways
`
`to NAD+
`
`in S.
`
`cerevisiae. The pathways depicted in scheme 1 suggest that:
`
`nicotinamide is deamidated to nicotinic acid. before the
`
`salvaged
`
`to make more
`
`NAD+,
`
`thus
`
`10
`
`15
`
`20
`
`25
`
`30
`
`pyridine
`
`ring
`
`is
`
`supplementation with
`
`nicotinamide may
`
`not
`
`rescue
`
`qnsl
`
`mutants
`
`by
`
`shunting
`
`nicotinamide—containing
`
`precursors
`
`through the
`
`pathway;
`
`and QNSl
`
`is
`
`common
`
`to the
`
`three
`
`pathways,
`
`thus there may be no NAD+ precursor that rescues
`
`qnsl mutants. However,
`
`it has
`
`now been.
`
`found that while
`
`nicotinamide does not
`
`rescue qnsl mutants even at
`
`l or 10
`
`mM, nicotinamide riboside functions as
`
`a. vitamin.
`
`forni of
`
`NAD+ at 10 uM.
`
`Anticancer agents
`
`such as
`
`tiazofurin (Cooney, et al.
`