Robert Sabin

Patent Trials and Appeals Board

Sep 12, 2019

12661964 - (D) (P.T.A.B. Sep. 12, 2019)

APPLICATION NO. FILING DATE 12/661,964 03/26/2010 4988 7590 ALFRED M. WALKER 225 OLD COUNTRY ROAD MELVILLE, NY 11747-2712 09/12/2019 FIRST NAMED INVENTOR Robert Sabin UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONERFORPATENTS P.O. Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov ATTORNEY DOCKET NO. 100101 Sabin AD CONFIRMATION NO. 8431 EXAMINER STONE, CHRISTOPHER R ART UNIT PAPER NUMBER 1628 MAIL DATE DELNERYMODE 09/12/2019 PAPER Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte ROBERT SABIN Appeal2019-002986 Application 12/661,964 Technology Center 1600 ERRATUM The Decision on Appeal for the above-identified application mailed September 12, 2019 referenced an article cited by Appellant (Thimmappa S. Anekonda et al., Phytic Acid as a Potential Treatment for Alzheimer's Pathology: Evidence from Animal and in vitro Models, J. of Alzheimer's Disease, vol. 23, 21-35 (2011)). A review of the record indicates this document was inadvertently omitted. Accordingly, a copy of Anekonda et al. is appended to the Decision. All other portions the Decision on Appeal remain unchanged. Any confusion caused regarding this matter is regretted. If there any questions pertaining to this Erratum, please contact the Patent Trial and Appeal Board at 571-272-9797. BAR UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte ROBERT SABIN Appeal2019-002986 Application 12/661,964 Technology Center 1600 Before ULRIKE W. JENKS, TIMOTHY G. MAJORS, and MICHAEL A. VALEK, Administrative Patent Judges. VALEK, Administrative Patent Judge. DECISION ON APPEAL Pursuant to 35 U.S.C. § 134(a), Appellant submits this appeal under 35 U.S.C. § 134(a) involving claims to methods for treating Alzheimer's Disease and related conditions. 1 Examiner rejected claims 6, 21-23, and 28-37 as obvious and for obviousness-type double patenting ("ODP"). We have jurisdiction under 35 U.S.C. § 6(b ). We AFFIRM. 1 Herein we refer to the Non-Final Office Action mailed February 21, 2018 ("Non-Final Act."); Appeal Brief filed June 11, 2018 ("App. Br."); Examiner's Answer mailed Jan. 8, 2019 ("Ans."); and Reply Brief filed March 7, 2019 ("Reply Br."). Appeal2019-002986 Application 12/661,964 STATEMENT OF THE CASE Claims 6, 21-23, and 28-37 are on appeal and can be found in the Claims Appendix of the Appeal Brief. App. Br. 18-19. Claim 6 is representative of the claims on appeal. It reads as follows: 6. A method for the treatment of Alzheimer's disease and related amyloid plaque development and reduction of amyloid plaque, protein aggregation and amyloidosis consisting essentially of the steps of orally administering to a patient afflicted with Alzheimer's disease an effective amount of a compound selected from the group consisting of phytic acid, a phytate salt, an isomer or hydrolysate of phytic acid or a phytate salt, or a mixture of any combination thereof, being administered in an amount from about 0.5 grams to about 18.75 grams per day to effectively treat Alzheimer's disease and related amyloid plaque development and reduction of amyloid plaque, protein aggregation and amyloidosis. App. Br. 36 (Claims Appendix). Claims 23, 31, and 32 are also independent and similarly recite administration of a compound from the same Markush group within the same dosage range. Dependent claims 30 and 36 additionally recite that the compound "is administered to a patient on an empty stomach. Id. at 38-39. Appellant seeks review of the following rejections: I. Claims 6, 21-23, and 28-37 under 35 U.S.C. § 103 as obvious over Sabin2 and Frey3 ("Rejection I"). II. Claims 6, 21-23, and 28-37 under 35 U.S.C. § 103 as obvious over Sabin, Frey, and Grases4 ("Rejection II"). 2 US 4,847,082, issued July 11, 1989 ("Sabin"). 3 US 2006/0014716 Al, published Jan. 19, 2006 ("Frey"). 4 F. Grases et al., Intracellular and Extracellular myo-Inositol Hexakisphosphate (InsP6),from Rats to Humans, Anticancer Research, Vol. 2 Appeal2019-002986 Application 12/661,964 III. Claims 6, 21-23, and 28-37 for ODP over claims 1-7 of Sabin in view of Frey and Grases ("Rejection III). IV. Claims 6, 21-23, and 28-37 for ODP over claims 1-3 of Sabin II5 in view of Frey and Grases ("Rejection IV"). The issue for each of these rejections is whether Examiner's conclusion of obviousness is supported by the preponderance of the evidence. Appellant does not argue claims 21-23, and 28, 29, 31-35, and 37 separately from claim 6 for any of the above rejections so those claims stand or fall with claim 6. 37 C.F.R. § 41.37 (c)(l)(iv). Appellant offers some additional arguments regarding Grases and the "empty stomach" limitation of claims 30 and 36 (see App. Br. 26-29) that we address separately in our analysis below. Findings of Fact FF 1. Sabin teaches the administration of "an effective symptom-alleviating amount of a compound selected from the group consisting of phytic acid, phytate salt, an isomer or hydrolysate of phytic acid or phytate salt, or a mixture of any combination thereof' to treat Alzheimer's Disease. Sabin, Abstr. Sabin teaches the "preferred method of administration is by oral dosages of about Yz to 3 grams/kilogram bodyweight per day." Id. According to Sabin, such a dosage "will usually be effective," but that the "dosage to be administered will vary with the severity of the diseased condition" and is left to the discretion of the attending physician. Id. at 4:4--12. 25, 2593-98 (2005) ("Grases"). 5 US 4,758,430, issued July 19, 1988 ("Sabin II"). 3 Appeal2019-002986 Application 12/661,964 FF2. Frey teaches the administration of "pyrophosphate analogs," including phytic acid, to treat or prevent, inter alia, Alzheimer's Disease. Frey ,r,r 10, 12, 119. According to Frey, "[d]oses for humans and other mammals can range from about 0.001 mg/kg to about 100 mg/kg" and can be orally administered. Id. ,r,r 80, 82. Frey teaches an embodiment in which the pyrophosphate analog is administered alone to protect a muscarinic acetylcholine receptor (mAChR) as well as embodiments in which it is administered to increase the efficacy of another agent. Id. ,r 5. FF3. Grases reports results from a "study of different stomach conditions before InsP6 [i.e., phytic acid] administration (empty stomach, empty with an alkalinizing agent and full stomach)" in humans that "demonstrated that no differences in the excretion profiles between the three different conditions were produced at 8 hours." Grases 2595. According to Grases, this data suggests "that the overall InsP 6 absorption took place independently of the stomach state, full or fasted stomach." Id. Thus, Grases teaches that to "maintain optimum InsP 6 levels" phytic acid supplements "can be taken either during or between meals with the same efficacy." Id. Analysis Obviousness We analyze Rejection I and Rejection II together because they are premised on the same references and present common issues. Examiner found that "Sabin teaches a method of treating Alzheimer's disease ... consisting essentially of orally administering phytic acid," albeit at a somewhat higher dosage range than that recited in Appellant's claims. Non-Final Act. 4. Examiner determined Frey teaches administering phytic acid at an oral dosage of about 100 mg/kg, "which would correspond to 7 g 4 Appeal2019-002986 Application 12/661,964 for an average 70kg adult." Id. ( citing Frey ,r,r 80, 82). According to Examiner, "it would have been prima facie obvious ... to administer the therapy of Sabin at the dosages[ in Frey] since Sabin teaches that the frequency and dosage ... varies with the severity of the disease as needed and discretionarily determined by the attending physician ... and the instantly claimed dosage ranges were known to be appropriate for Alzheimer's therapy." Id. at 3--4. We agree that Examiner's obviousness rejections are supported by the preponderance of the evidence. Sabin teaches all of the limitations of claim 6 other than the dosage range. FF 1. Frey teaches the administration of the same compound (i.e., phytic acid) to treat the same disease at a dosage range that substantially overlaps6 with the "about 0.5 grams to about 18.75 grams per day" range recited in claim 6. FF2. That overlap is sufficient to establish a prima facie case of obviousness. See In re Peterson, 315 F.3d 1325, 1329 (Fed. Cir. 2003) ("[W]e and our predecessor court have consistently held that even a slight overlap in range establishes a prima facie case of obviousness."). Such a prima facie case may be rebutted "by establishing that the claimed range is critical, generally by showing that the claimed range achieves unexpected results relative to the prior art range." Id. at 1330 ( citations omitted). But, as explained further below, Appellant has not shown that the claimed range achieves unexpected results as compared to the dosages taught in Frey or Sabin or otherwise established 6 Examiner found that a 100 mg/kg dose (i.e., the upper end of Frey's range) in a 70 kg person overlaps with the claimed range. See Non-Final Act. 4. We note that the overlap extends down from 100 mg/kg to at least 10 mg/kg, which would equate to a dose of about O. 7 grams in the same person. 5 Appeal2019-002986 Application 12/661,964 that the claimed range is critical. Accordingly, the record supports Examiner's obviousness determination and, as explained below, we are not persuaded by Appellant's arguments to the contrary. Appellant argues that Frey fails to teach a dose within the claimed range because it discloses "an infinite/endless number of combinations" of pyrophosphate compounds with an "infinite/endless number of doses, with thousands of diseases, with different genders, with different ages." See App. Br. 8-14. We disagree. Frey teaches that the pyrophosphate analog "employed in the appropriate embodiment of the method of the invention" may be "an inositol hexaphosphate," i.e., phytic acid. Frey ,r,r 7-10. And it teaches that "according to the method of the invention" such compounds are administered in a dosage range "from about 0.001 mg/kg to about 100 mg/kg." Id. ,r,r 80, 82. Moreover, Frey teaches that "[p ]referably, the method of the invention ... can treat or prevent Alzheimer's Disease." Id. ,r 12. Therefore, Frey ties these disclosures to the same "method of the invention" and Examiner is not merely "picking and choosing" from different embodiments, as Appellant contends. See App. Br. 11-12. The mere fact that Frey discloses a multitude of combinations "does not render any particular" combination less obvious. See Merck & Co., Inc. v. Biocraft Labs., Inc., 874 F.2d 804, 807 (Fed. Cir. 1989) (explaining that a reference's disclosure of "a multitude of effective combinations does not render any particular formulation less obvious"). Moreover, the teachings in Sabin further demonstrate that it would be obvious to select this particular combination, i.e., to administer phytic acid orally for the treatment of and alleviation of symptoms associated with Alzheimer's Disease. See FFl. 6 Appeal2019-002986 Application 12/661,964 We are also not persuaded by Appellant's argument that Frey's disclosure is so broad that it is not enabled. See App. Br. 12-14. "[A] prior art reference need not enable its full disclosure." In re Antor Media Corp., 689 F.3d 1282, 1290 (Fed. Cir. 2012). Rather, "[e]nablement of prior art requires that the reference teach a skilled artisan to make or carry out what it discloses in relation to the claimed invention." Id. (emphasis added). Appellant does not argue, nor is there evidence in the record to suggest, that undue experimentation would be required to orally administer phytic acid to treat Alzheimer's Disease at the dosage taught in Frey. Accordingly, Appellant has not overcome the presumption that "both the claimed and unclaimed disclosures in [Frey] ... are enabled." Amgen Inc. v. Hoechst Marion Roussel, Inc., 314 F.3d 1313, 1355 (Fed. Cir. 2003). Nonetheless, Appellant urges that Frey "admitted during prosecution that their disclosure was overly broad and non-enabled." App. Br. 15. We disagree. First, the fact that claims that ultimately issued from Frey, a published patent application, are limited to intranasal administration to treat meningitis does not constitute "a disavowing of other forms of administration for other diseases," as Appellant contends. Id. at 15-16. As our reviewing court has explained, "[t]he scope of a patent's claims determines what infringes the patent; it is no measure of what it discloses." In re Benno, 768 F.2d 1340, 1346 (Fed. Cir. 1985). Indeed, as a printed publication, Frey would be prior art for the teachings in that publication even if no patent had ultimately issued from that application. See 35 U.S.C. § 102. Second, and contrary to Appellant's assertions, there are no "admissions in the [Frey] file history" that Frey's teachings as they relate to 7 Appeal2019-002986 Application 12/661,964 the Appellant's claims are not enabled. App. Br. 17 (citing 132 Declaration of Robert Sabin dated March 29, 2017 ("Sabin Deel.")). The Sabin Declaration quotes passages from the prosecution of an application related to Frey, describing the "primary advantages of the present invention, as it is claimed," i.e., intranasal delivery as opposed to other routes, to try to distinguish those claims over the cited prior art. Sabin Deel. 2. At most this passage, and the others cited in the Sabin Declaration, suggest that the Frey inventors preferred intranasal delivery. See Sabin Deel. 2---6. None of the statements Appellant relies upon suggest that undue experimentation is required to practice Frey's other teachings, much less do they evidence that a skilled artisan would somehow be dissuaded from administering phytic acid orally, as expressly taught by both Frey and Sabin. See FF1-FF2. Appellant also argues that Sabin "teaches away from using the lower dosages claimed" in Appellant's present claims. App. Br. 20 (emphasis omitted). Again, we disagree. "A reference may be said to teach away when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction divergent from the path that was taken by the applicant." In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). Appellant here points to Sabin's teaching that "oral administration of from Yz to 3 grams of phytic acid ... per kilogram of body weight in the diet per day will usually be effective." App. Br. 20 ( quoting Sabin 4:4--10). But the statement that a somewhat higher dose "will usually be effective" does not discourage a skilled artisan, nor does it lead away, from pursuing a lower dose. This is particularly so given that Sabin acknowledges that the dosage will "vary with the severity 8 Appeal2019-002986 Application 12/661,964 of the diseased condition" and is left to discretion of the attending physician. FPL Finally, Appellant has not shown that the claimed dosage range of "about 0.5 grams to about 18.75 grams per day" is critical, or otherwise provides unexpected benefits, compared to the ranges taught in Sabin and Frey. The law is replete with cases in which the difference between the claimed invention and the prior art is some range or other variable within the claims. See, e.g., In re Peterson, 315 F .3d at 1329 ( citing cases). These cases have consistently held that the Appellant must show that the particular range is critical, generally by showing that the claimed range achieves unexpected results relative to the prior art range. Id. The burden rests with Appellant to establish that ( 1) the comparisons are to the disclosure of the closest prior art, (2) the evidentiary showing is commensurate in scope with the claimed subject matter, and (3) the alleged unexpected results are, in fact, unexpected over the prior art. See In re Klosak, 455 F.2d 1077, 1080 (CCP A 1972). All three of those requirements are lacking here. First, Appellant has not provided evidence comparing the claimed dosage range to the ranges taught in Sabin and Frey. The data in the Specification purports to show "a reduction in Abeta plaque [in mice] after treatment with 2% phytic acid, compared to the control vehicle without phytic acid." Spec. 11 (emphasis added). Appellant further relies on data in an article on which he is a co-author, Anekonda. 7 That data similarly involves mice treated with 2% phytic acid in their drinking water. See App. 7 Thimmappa S. Anekonda et al., Phytic Acid as a Potential Treatment for Alzheimer's Pathology: Evidence from Animal and in vitro Models, J. of Alzheimer's Disease, vol. 23, 21-35 (2011). 9 Appeal2019-002986 Application 12/661,964 Br. 24--25, 31-32; Anekonda 24. 8 According to Appellant, the 2% phytic acid solution in these tests would equate to a daily dose in humans that falls within the claimed range. Id. at 31-32. But even then, these data merely compare the claimed dose to a "control vehicle without phytic acid." Spec. 11. They do not purport to compare the results achieved at the claimed dose to either the somewhat higher dose reported in Sabin or the 100 mg/kg dose taught in Frey. Thus, the data and statements in the Specification and in Anekonda9 do not show that the claimed invention, i.e., administration of phytic acid within the claimed range, achieves unexpected results as compared to the closest prior art, i.e., administration of the same compound at the somewhat higher dosage in Sabin or at the dosage range in Frey. In re Baxter-Travenol Labs., 952 F.2d 388,392 (Fed. Cir. 1991) ("[W]hen unexpected results are used as evidence of nonobviousness, the results must be shown to be unexpected compared with the closest prior art."). Indeed, Anekonda evidences that the opposite is true. It states that "the administered PA dose (2%) was likely too mild" and postulates that "a much higher dose of PA starting as early as 2-3 months of animal age is expected to provide preventative effect on the accumulation of AB plaques." Anekonda 30. 8 Both the Specification and Anekonda report data from in vitro tests in MC65 cells. See Spec. 9; Anekonda 26-28. There is, however, no evidence demonstrating how the concentration of phytic acid applied to the cells in those tests relates to the claimed dosage range. Accordingly, this data does not evidence that Appellant's present claims are non-obvious over the prior art. 9 Appellant additionally cites a July 29, 2011 email from Thimmappa Anekonda with the subject line "Re: Novel findings of our PA study in AD." See App. Br. 24--25 and 31. This email does not appear to present any additional information beyond the Anekonda article itself. 10 Appeal2019-002986 Application 12/661,964 Second, there is no evidence that the results Appellant relies upon are reasonably commensurate with the full breadth of the claimed dosage range. According to Appellant's calculations, giving mice water containing 2% phytic acid equates to a daily dose in humans near, or slightly exceeding, the upper end of the claimed range (18.75 grams per day). App. Br. 31-32. But the lower end of the range in claim 6 is only 0.5 grams per day. There is no evidence suggesting that a dose at or near 0.5 grams per day, i.e., more than 35 times less than the 18.75 gram upper end of the range, would achieve results similar to the studies Appellant relies upon involving 2% phytic acid. Indeed, as noted above, Anekonda affirmatively evidences the contrary by stating that the 2% dose in those studies "was likely too mild." Anekonda 30. Third, Appellant has not shown that the relied-upon results were unexpected over the prior art. Sabin teaches that phytic acid is useful to treat and alleviates symptom of Alzheimer's Disease. See Sabin 2:23--44. Appellant's mouse data supports Sabin's conclusion. What Appellant has not shown is how that data and the literature cited in the Appeal Brief (see App. Br. 24--26) demonstrate a result that is unexpectedly different in kind from the prior art, which already taught that phytic acid was effective to alleviate symptoms of Alzheimer's Disease. At most, Appellant's evidence elucidates the mechanisms by which phytic acid may be effective in alleviating symptoms of Alzheimer's Disease, e.g., by providing "protection against APP-C99-mediated toxicity," "[ e ]nhanc[ing] cytochrome oxidase (mitochondrial function)," or "[s]uppress[ing] malondialdehyde or Lipid peroxidation." See id. at 24--25. The question before us is not whether such findings are scientifically significant, but whether they are probative of non- 11 Appeal2019-002986 Application 12/661,964 obviousness because they show unexpected results that are "'different in kind and not merely in degree from the results of the prior art.'" See Galderma Labs., L.P. v. Tolmar, Inc., 737 F.3d 731, 739 (Fed. Cir. 2013) (quoting Iron Grip Barbell Co. v. USA Sports, Inc., 392 F.3d 1317, 1322 (Fed. Cir. 2004)). As our reviewing court has explained, even when unexpected if "in efficacy is measured by a small percentage ... and the evidence indicates that skilled artisans were capable of adjusting the percentage, the result constitutes a difference in degree, not kind." Id. Here, there is no evidence that the claimed dosage increases efficacy or reduces toxicity as compared to the prior art dosages taught in Sabin and Frey, much less the percentage of any such change. For all these reasons, Appellant has not persuasively demonstrated that the claimed dosage range is critical or that it otherwise exhibits unexpected results over the dosages taught in Sabin and Frey. Thus, when weighing the record as a whole in light of Examiner's comparatively strong prima facie case, we determine that the preponderance of the evidence supports Examiner's rejections as to claims 6, 21-23, and 28, 29, 31-35, and 37. Regarding the "empty stomach" limitation of claims 30 and 36, Appellant additionally argues that Grases "teaching of administration on an empty stomach is lacking sufficient evidentiary support" to show obviousness because it involved healthy subjects and did not include the number of subjects required for a Phase 1 or Phase O clinical study. See App. Br. 27-28. We disagree. "Conclusive proof of efficacy is not necessary to show obviousness. All that is required is a reasonable expectation of success." Hoffman-La Roche Inc. v. Apotex Inc., 748 F.3d 12 Appeal2019-002986 Application 12/661,964 1326, 1331 (Fed. Cir. 2014). Grases teaches that there were "no differences in the [phytic acid] excretion profiles" when administered on an empty versus a full stomach and concludes that optimum phytic acid levels can be achieved by taking supplements "either during or between meals with the same efficacy." FF3. That teaching is sufficient to support a reasonable expectation that phytic acid could be successfully administered to a patient on an empty stomach, as recited in claims 30 and 36. While additional data may be required for FDA approval, "absolute certainty for success" is not required to show obviousness. PAR Pharm., Inc. v. TWI Pharms., Inc., 773 F.3d 1186, 1198 (Fed. Cir. 2014) (citation omitted). Accordingly, the preponderance of the evidence also supports Examiner's rejections of claims 30 and 36. We therefore affirm Rejection I and Rejection II. ODP Rejections With respect to Rejection III and Rejection IV, Appellant incorporates the same arguments it presents for the obviousness rejections. See App. Br. 34. As explained above, we are unpersuaded by those arguments. Since Appellant does not present any additional argument as to why the claims of Sabin and Sabin II, in view of Frey and Grases, do not render his present claims obvious, we affirm. See 37 C.F.R. § 4I.37(c)(l)(iv) ("any arguments or authorities not included in the appeal brief will be refused consideration by the Board for purposes of the present appeal"). SUMMARY We affirm the rejection of claims 6, 21-23, and 28-37 under 35 U.S.C. § 103 as obvious over Sabin and Frey. 13 Appeal2019-002986 Application 12/661,964 We affirm the rejection of claims 6, 21-23, and 28-37 under 35 U.S.C. § 103 as obvious over Sabin, Frey, and Grases. We affirm the rejection of claims 6, 21-23, and 28-37 for ODP over claims 1-7 of Sabin in view of Frey and Grases. We affirm the rejection of claims 6, 21-23, and 28-37 for ODP over claims 1-3 of Sabin II in view of Frey and Grases. TIME PERIOD FOR RESPONSE No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). AFFIRMED 14 Application/Control No. Applicant(s)/Patent Under 12/661,964 Reexamination Robert Sabin Notice of References Cited Examiner Art Unit Page 1 of 1 U.S. PATENT DOCUMENTS * Document Number Date Country Code-Number-Kind Code MM-YYYY Name Classification 1 A US- 1 B US- C US- D US- E US- F US- G US- H US- I US- J US- K US- L US- M US- FOREIGN PATENT DOCUMENTS * Document Number Date Country Code-Number-Kind Code MM-YYYY Country Name Classification N 0 p Q R s T NON-PATENT DOCUMENTS * Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages) u Thimmappa S. Anekonda et al., Phytic Acid as a Potential Treatment for Alzheimer's Pathology: Evidence from Animal and in vitro Models, J. of Alzheimer's Disease, vol. 23, 21-35 (2011). V w X *A copy of this reference is not being furnished with this Office action. (See MPEP § 707.05(a).) Dates in MM-YYYY format are publication dates. Classifications may be US or foreign. U.S. Patent and Trademark Office PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No. 1 Journal of A:zhcimer',c, DJsea,c,e L\ (2011) ,~ l 35 DOI l0.3LB/JAD-2J)J 0-l OJ 287 IOS Press 21 Pl1ytic Acid as a Potential Treatn1ent for Alzhein1er's Pathology: Evidence from Ani1nal and in vitro Models Thimmappa S. Anekonda",b-*, Teri L. Wadsworih",b, Roberi Sabin", Kate Prahl er", Christopher Harrisb, Babeu Petrikoa, Martina Railed, Randy Woltjer" and Joseph F. Quinna,b "Departmenr cf Neurology, Oregon Health & Science University, 1-'onland, OR, USA b Portland W'teran Administration Medical Cente1; Oregon Health & Science UniFersirv, Portland. OR, USA csupe1J'ox 'l'.herapeutics Inc.; Aiill 1Veck, i\TY, l]SA d Department of Biochemistry and Molecular Bioiogy Oregon Healrl, & Science University Portland, OR, USA "Depamnent of Pathology. Oregon Health & Science Universiry. Portland, CJR, USA Accepted 3 Sep tern ber 20 l 0 Ab5tract. Alzhetmer's disease (AD) causes progressive, age-dependent cortical and hippocampal dysfunction kadrng to abnor- 1nal inteHectual capacity and rnen1ory. \Ve propose a novel protective treatment for AJ) pathology with phytic add (inositol hexakisphosphate), a phytochemical found in food grains and a key signaling molecule in mammalian cells. We evaluated the protective and beneficial effects of phytic acid against amy loid--f1 (AP,) pathology in MC65 cells and the Tg2576 mouse model. ln MC65 cells, 48-72-hour treatment with phytic acid prov,ded complete protection against amyloid precursor protein-C-terminal fragment-induced cytotoxicity by attenuating levels of increased intracellular calcium, hydrogen peroxide, supernxide, Af1 oligomers. and moderately up regulated the expression of amophagy (bedin--1) protein_ In a tolerance paradigm, wild type mice were trnated with 2% phydc acid in drinking water for 70 days. Phytic actd was well tolerated. Cernloplasmin activ1ty, brain copper and iron JeveJs~ and brajn superoxjde disn1utase and ATP JeveJs \:Vere unaffected by the treatrnent. There ;vas a signjficant increase in brain levels of cytochrome oxidase and a decrease in lipid peroxidalion with phytic acid administration. In a lreatment paradigm, 12--month old Tg2576 and wild type mice were treated with 2%, phytic acid or vehicle for 6 months. Brain levels of' copper. iron, and zinc were unaffected, The effects of phytic acid were modest on the expression of Af:'.PP trafficking-associated protein APJ80, autophagy-associated protems (bedin-1, LC3B). simun l, the ratio of phosphorylated AMP-activated protein kinase fPAMPK) lo AMPK, soluble Ai31 40 , and insoluble Afl,1_42 • These results suggest lhat phytic acid may provide a viable treatment option for AD. Keywords: Amylmd-p, amyloid-p protein precursor. antioxidant, autophagy, beclin-J, phosphorylated AMP-activated protein kinase. sirtuin l INTRODUCTION Alzheimer's disease (ADj causes progressive cor- tical and hippocampal dysfunctions with age and impacts both intellec1 and memory in affected pa1ients. iCon::e:'.)pondence to: Thimmappa S. An,;:;konda, Departmcni of Neurology, 11aH Code. R&D 67, Oregon HeaHh & Sdence Uni- versity. 3181 Sam J;;ckson Park Road, Portland, OR 97239, USA; Td.: +1 503 220 8262 ext 52233; Fax: +l 503 494 7499; E-mail: ;rnckondt (i_t'l ohsu.,;:;d u, Tbe arnyloid cascade bypotbesis suggests 1ha1 a 4-kDa amyloid-f, (/\~1) peptide is tbe fundamental cause of AD [l-7], ,vith selective loss of neuronal subpopu- lations including cholinergic fibers, proliferation of reactive astrocy1es and rnjcroglia~ all progressively leading to mild cognitive impairment (MCI) and ulti- mately AD [1-3]. Oxidative stress has been suggested as one ot' the earliest manifesta1ions ot' MCI and AD pathology [8]. The mainstream research in AD appears to focus on two main therapeutic strategies ISSN 1387-2877/ll/$27.50 ,g 2011 - IOS Press and the authors. All rights reserved 22 TS. A..nekonda et al. l P.lzytic Acid as a Potential Treatnwnt ,fof' Alzheimer·.~ Pathology --- prevention of Af3 accumulation and promotion ot' A[3 clearance [8---1 OJ. CutTently there are four Food and Drug Administration-approved drugs available for use in patients with AD, but only as symptomatic treatments. There is a huge unmet medical need for therapies that are safe and effective. Jn this study, we investigated a novel protective treatment for AD pathology with phytic acid {PA, inositol hexakispbosphate). PA is strncturally a myo- inosirol sugar ring attached to 6 phosphate molecules. lt is found naturally and ubiquitously, as a phosphate- storage phytocbemical in unprocessed whole food grains~ vegetables~ and fruits~ and as a key signa1-- ing molecule in mammalian cells. The Ca/Mg form of PA found in most plan1s is known as "Phytin" with its salt form known as "Phytate". Although PA is often described as a metal chclator [1 lJ, grow- ing literature indicates that PA influences multiple processes, including antioxidant fonctions 111--13 ], anti--apoptotic effects [12], clathrin--coated endocyto-- sis [l4-l61, DNA repair [17], and mRNA export from the nucleus 118]. Phytic acid also lowers serum choles- terol and triglycerides [19]. These studies suggest that PA possesses much broader functions than simply the originally-presumed metal binding properties. We hypothesized that some of the properties of PA may have a favorable effect on brain aging and AD pathology, by way of: l) mimicking caloric restriction, 2) promoting autophagy, and 3) modulating c:lathrin- coated endocytosis of amyloid{s protein precursor (Ai3PP) and its cleavage products. Caloric restriction (CR) mimetics like resveratrol {which, like PA, is an antifungal in plants) are under study for anti--aging effects [20--22], and resveratrol is already in clini-- cal trials for AD. We hypothesized that PA is a CR mimetic because it has protective functions similar to resveratrol in plants [23--25]. Both CR and resvera- trol act to increase levels and activation of Sirtuin l (SIRT l ), an NAD-dependent class III his tone and non- bistone protein deacetylase wbicb is decreased in AD and other pathologies and extends lifespan in yeasts, worms, flies, and mice [24, 26-39], Since rcsveratrol acts by way of SIRT 1 activation, we tested the effect of PA upon SIRT-l in cell culmre and in animal models of AD. Autophagy is a self-cleaning cellular housekeeping mechanism tbat plays an important role in numer-- ous pathologies [40]. A heterozygous deletion of the autophagy marker beclin-1 in Tg:2576 mice increases intraneuronal Ai3 accumulation, extracellular Ai3 deposits, and neurodegeneration [4lJ, suggesting that autophagy plays a key protective role against AD. lndeed one of the main functions of autophagy is to regulate mitochondrial function by enzymatic degra- dation of dysfunctional mitochondria and by clearing misfolded proteins within tl1e cell [42-44;. Our pre- vious s1udy in Tg2576 mice provides strong evidence for mitochondrial deposition of Ai3 species and gener- ation of free radicals [45,461. SIRT l loss parallels the loss of proteins associated with autophagy (beclin-1 and LC3B) and oxidative stress [47, 48], strongly sug- gesting that SIRTl regulates autophagy in the brain, and this regulation may be a pathogenic mechanism and therefore a therapeutic target in AD [40, 48]. Some recent studies, however, question the ability of resveratrol ro activate SIRTl [49, 50]. A SIRT l- independent mechanism mediated predominantly by the metabolic sensor protein phospborylated AMP- activated protein kinase (PAMPK) in neurons has been suggested to play an important role in Ai3 peptide metabolism L51, 52]. Therefore, ,ve inves1igated the effects of PA on the PAlvlPK to AMPK ratio in Tg2576 mice. Furt.l-1ermorc, it has been previously shown that PA suppresses APl 80/AP3 protein [ 14, 15], a clathtin- coated vesicle protein, implicated in the regulation of Ai3PP trafficking and Ai3 generation [16]. Thus, we sought to clarify the effect of PA on this protein in Tg2576 mice. The main objective of this study was to clarify the beneficial or harmful effects of PA in the con- text of AD pathology and determine if clinical trials of phytic acid for AD are justified. The mechanisms considered include metal chelation, SIRTl activation, autophagy activation, PAivIPK activation, and regula- tion of endocy1osis. \Ve employed one in vitro (bum an neuroblastoma./l\1IC65 model of A~1 neurotoxicity [53-- 581) and one animal model (Tg2576 [59]) of AD to test these possibilities. Our in vitro model, MC65 cells, are stably transfected with an inducible Aj3PP-C99 construct and conditionally express a fusion protein composed of the amino-17 and carboxyl-99 residue- containing fragment of Af3PP [55, 56]. Expression is comrolled by a tetracycline-responsive promoter whose activity is repressed in the presence of tetracy- cline, Removal of tetracycline first leads to expression of the C--terminal Ai3PP fragment and subsequent pro- cessing of this fragment into Ai3 species follmved by accumulation of intracellular Ai3 oligomers, and precipitous cell dea.tb in 3--4 days [53, 55, 56]. Our darn indicates tbat PA protected MC:65 cells against intra.neuronal Ai3 oligomcrs while up regulating SIRTl and the autophagy-associated protein beclin- L In the animal model, chronic PA administration bad no effect on brain levels of metals (copper, iron, zinc), J:S. ilnekonda et al. l Phytic Acid as C! Porential TreC!rrnent for lll::}u:itnef''s Pat.lwlogy but modu la1ed oxidative stressirni,ocbornitial function, autophagy (beclin- l, LC3BJ), SIRTl, PAMPK, and endocytosis associated protein AP180 protein, ,vhile producing a modest reduction in Af:3 and plaques in Tg2576 mice. MATERIALS AND METHODS lv!aterials Cell culture media, L-glutamine, and ,rypsiniEDT.i\ were obtained. from lnvitrogen (Carlsbad., CA); fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA); 6El0 monoclonal antibody was obtained from Covance Laboratories (Princeton, NJ); Phytic acid sodium salt hydrate from rice and anti-- assembly protein AP180 were from Sigma-Aldrich (St. Louis, MO). Anti-f:3-tubulin, an,i-beclin-1, anti- LC3B, anti-pbospho-AM l-'K(t, and anti--AMPI(o, were from Cell Signaling Technologies, inc. ( Danvers, !VIA); Anti-SIRT l was from Santa Cruz Biotechnology, Inc. (Sama Cruz, CA\ Unless otherwise mentioned all other chemicals were obtained from Sigma. (~ell culture MC65 cells were cultured in 75-cm2 TC flasks or l 00 x 20 mm pe1ri dishes containing tv1EMa.lpha growth medium supplemented with fetal bovine serum (10% ), L-glutamine (2 mM) and tetracycline (1 µg/ml), and maintained in a tissue culture incubator at 37"C and in the presence of 5% CO2 as described [57, 60, 61]. We limited our assavs to early cell passages. Cell survival assay Cell survival was determined by tbe IVITS assay (Promega) simultaneously with calcium assays (des- cribed below) on the same cell passages, following manufactmer's protocol and published metbods [58]. Cells were harvested from TC flasks at 90'fo confluence using 0.05% trypsiniEDTA, washed twice with PBS, and plated into 96-well plates at a density of 15 x 103 cellsiwell in OPTIMem medium in tbe presence (Tet+) or absence (Tet-) of tetracycline ( l µg/mlj and vehi- cle, or phytic acid (100 µJv1). Cells grm.vn under Tet- condition usually die after approximately 3--4 da.ys in culmre in association with expression of endogenously produced Af:3 oligomers, but the cells grown under Tet+ condition fully survive during ,his period [58]. Tbe wells containing growth medium without cells served as background. control, and 'let+ wi1h vehicle served as a positive con1rol. Each treatment was replicated in 3-6 wells and treatments were applied for periods of 72 h in each experiment. Absorption was measured a, ,vave leng1h 490 n.t\1 using a Spec1ra .Max PLUS plate reader (Molecular Devices inc.). Percent cell survival in different treatments was determined relative to cell survival in Tel+ treatment. Experiments were repeated al least three times. Inrracellular calciun1 assay Intracellular Ca2+ levels 'Were determined using the Fluo-4 l'<\V Ca'+ Assay Kit following the man- ufacturer's protocol (Molecular Probes) and published methods [ 62]. Brielly, .MC65 cells at a density of 15 x l03 cells/well were grown in black, 96-well cul- mre plates with (Tet+) and without (Tet-) tetracycline in eitber vehicle or pbytic acid (100 p.Jvl). Tbe same set of treatments presented. in the MTS assay described above were included in the ca=·.+ assay, and both assays were performed in parallel from tl1e same batch of cells. Af1er ,he treatment period, Fluo--4 N\V dye {100 p.l) containing 2.5 mM probenecid was added directly irno each well to prevent the dye efflux from the cells, and tbe plate was incubated. in a TC cbarnber a1 37'C for 30 min, followed. by another 30 min at the room tem- perature. Intracellular Ca2+ levels for each treatment and control were measured in terms of relative fluo- rescence units in live cells (RFU) for 10 min at room temperature using tbe Victor3 i 420 Multilabel Counter (Perkin Elmer) with specific excitation (485/20nM) and emission (535/20 nM ). ·n,e fluorescence values of different cultures may be variable depending on the cell density, the time of exposure to the probe, cell loading with the fluorescent dye, and fluctuations in room temperatures. Therefore, special care was taken 10 calibrate fluorescent values of all other treatments within each experiment relative to the Tet+ treatment; this strategy eliminated errors associated with absolute measurements of fluorescence and assigned relative values meaningful for comparing treatments across different experiments. l)eter,nination oj~free radicals Hydrogen peroxide (H202) levels and intracellular superoxid.e levels were measured following published methods Briefly, H2 02 levels were determined in the cell media collected from cells treated with Tet+, '['et---, and 'let-PA for 48 h using 1he Bioxytech H202- 560TM Quantitative Hydrogen Peroxide Assay (Oxis 24 TS. A..nekonda et al. l P.lzytic Acid as a Potential Treatnwnt ,fof' Alzheimer·.~ Pathology International, Inc., foster City, CA) as per manufo.c- turer's instructions. intracellular superoxide radicals were determined in the cell lysates from the same treat- ments using the nitroblue tetrazolium (NBT) assay as previously described [63]. Briefly, NBT (Sigma, St. Louis, MO) was added to the cell media at a final concentration of l mg/ml and incubated for 3 h at 37'C in a humidified atmosphere containing 5% CC\. The NBTcontaining medium was removed, the cells washed t,vice ,vith warm PBS followed by methanol, ,hen air-dried. Intracellular blue formazan pa.rticles ,vere dissolved by adding 120 p.J/well 2 M potassium hydroxide followed by 140 µli-well DMSO. The absorbance of dissolved NBT was measured at 620 nm using a Spectra Max Plus. Cell harvesring, protein qucmtijzcation, and western blotting We followed previously published methods for pro- tein quantification and western blotting [58 1. Briefly, MC65 cells were grown under 'let-- and 'let+ con- dition and Tct-PA ( 100 pJ'v1) in 100 x 20 mm petri dishes in 10 ml OPTIMcm. After 72 h treatment, the medium was removed, and ,he eel ls were wasbed with PBS. Cells were lyscd in 300 µl of lysis buffer (Tris, 62.5 mM, pH 6.8; sodium dodecyl sulfate, 2%; glyc- erol, 10'7a) containing protease inhibitor cocktail set VJ [ (Calbiochem, San Diego, CA) without DTT or bromophenol blue and then scraped. and collected into l .5 ml Eppendorf tubes. The cells were sonicated on ice for 10 s, boiled at 95°C for 5-10 min, allowed to cool 10 room temperature, and centrifuged at 14,000 ,1lm for 5 min. TI1e supernatant was transfcn-ed to a new tube, the protein quantity in each sample was deter- mined. using BCA Assay (Pierce), and tben lysates were stored at ---20 C until further use. Cell lysates from different treatments were normalized to the same final concentration of total protein using the lysi.s buffer, and adjusted to a fi.nal concentration of 50 nu\JI DTT and 0.01 % bromophcnol blue. A 20 I-Lg protein \Vas loaded onto a 10-20% or 161?0 acrylami.de gels containing tricine (Bio- Rad) for frac1iona,ion and tbe proteins transferred to nitrocellulose membranes. The membrane ,vas probed with 6El O mouse MAb, anri- AP180 mouse MAb, anti-SLR.Tl or anti.-beclin-1 rabbit polyclonal antibodies; or the loading control :mti{S·· mbulin antibody. Following treatment with appropriate secondary antibody, the membrane was exposed to enhanced cbemilurninescence solutions {Invi,rogen), and visualized using the l\folecular imager Gel Docu- mentation System and quantified using Quantity One Software (Bio-Rad). Animal studies T\vo cohorts of mice were used in this smdy. Jn the tolerance cohort, all mice were C57BU6J wild types. ln the treatment cobort, Tg2576 mice were generated. from a breeding pair originally provided by Dr. Karen Hsiao-Ashe (Mayo Clinic, l'vIN). The transgcne was caJ.Tied on a genetic background of C57BU6J X SJL. Following weaning, litters ·were genotypcd and group housed at 4-5 animals per cage until the commence- ment of experiments. All of the mice in this study were female, because males are prone to marked aggres- sion towards cage mates. Mice were maintained in a climate-controlled environment wit.1-i a 12-h lighr/12-h dark cycle, and fed AIN-93 l'vl Purified Rodent Diet (Dyels Inc., Bethlehem, PA). Diel and waler were sup- plied ad libirnm. All procedures were conducted in accordance with the NlH Guidelines for the Ca.re and Use of Laboratory Animals and were approved by the institutional Animal Ca.re and Use Committee of the Portland VA Medi.cal Center. 1i-ea/ments In the initial tolerance paradigm, female wild type mice (age 9 months) ·were treated. for 70 days ·with either vehiclcNEH (di.stilled drinking water, n = lO) or 21?0 PA dissolved in drinking water (n=lO). PA was well tolerated as indicated by tbe fact that average weekly body weights (an indirect measurement of tox- icity) were simil3.1· for vehicle and PA-treated animals. ln the second treatment paradigm, Tg2576 and wild type mice were randomized to receive either 2% PA or vehicle in their drinking water. PA doses were adjusted on a weekly basis in order to assure that body weight did not drop more than 1 !)'fa. ln tbe treatment paradigm, i 2 mon1h-old. mice were treated for six momhs as fol-- lows: 22 wild type mice (vehicle n = l2, PA n = 10 j and 15 Tg2576 mice (vehicle n=7, PA n:::8). Again, PA was well tolerated in these animals as indicated by their average monthly body weights. (~eruloplas:nin ((]]) assay In the tolerance paradigm, blood ,vas collected from the saphenous vein every 2-3 weeks from 4-6 ani- mals per treatment group and plasma Cp activity measured using a modified version of the oxidasc method j 64, 65 ]. Briefly, for each mouse, 7.5 µl of plasma and 75 pJ of 100 mM sodium acetate (pH 6.0) ·were added in duplicate to a 96-well plate and incubated 5 min at 37"C. Aqueous o-dianisidinc dihy- drochloridc solution (30 ~d, 2.5 mg/ml, pre-incubated J:S. ilnekonda et al. l Phytic Acid as C! Porential TreC!rrnent for lll::}u:itnef''s Pat.lwlogy 25 to 37c,c:) \Vere added 10 eacb Vv'ell and 1he plate incu- bated al 3TC for 5 min. An aliquot (56.3 f.ll) of the mixture was immediately transferred to empty wells. The solution remaining in the original wells (t::: 5 min) was quencbed with l50pJ 9M sulforic acid. The plate was incubated for an additional 15 min at 3TC, then the secondary ,vells (t= 20 min) were quenched. Absorbance was read at 540 nm and the enzyrna.tic activity of Cp determined from the following equation: Activity (U/ml) = (Abs1"" 2.o-Abs1"" 5) (416). 1iss11e homogenates and ELISA Following behavioral analysis, animals were euth- anized by CO2 inbalation and cervical dislocation. Blood and liver samples were collected and frozen and processed as described [65]. Brains were quickly removed and divided. Tbe anterior 3 mm of bilateral frontal cortex and a l mm section behind the fromal cortex ("frontal slice") were dissected and frozen for beta amyloid ELlSA and brain metal analysis, respec- tively. The rigbt hemisphere ,vas immersion fixed in 4'fo formaldehyde in phosphate buffered saline for his- tochemical analysis. The contralareral hemisphere was frozen for determination of copper-dependent enzyme activity. Frontal cortex tissue was homogenized in tris buffered saline (TBS) containing protease inhibitor cocktail (EMD Chemicals, (]ibbs1own, NJ) and l 'fo Triton X--100 (Roche, Indianapolis, IL), then centrifuged at 40,000 xg for 30 min at 4 °C. The super- natant from this step was labeled '"soluble fraction". The pellet generated in this step was homogenized in TBS, protease inhibitors, and l % Triton X-100, and then centrifuged at 40,000xg for 30min at 4°C. The supernatant from this step was discarded as a wash. Tbe pellet was homogenized in 70':7<:, formic acid (Sigma), incubated for l h at room temperature, and cemrifuged at 40,000 xg 30 min. The resulting supernatant was collected and labeled "insoluble fraction". Af31.-40 and Ai3:-42 were measured in the soluble membrane and insoluble fractions using commercial ELISA (Invitro- gen, Camarillo, CA) according to the manufacturer's instructions. 1lssue nu:tal assays Brain iron and copper levels in the tolerance cohort were measured using atomic absorption spectroscopy (AAS). Plasma and brain copper, iron, and zinc levels in the treatmem paradigm were measured using both AAS and inductively coupled plasma mass spectroscopy (lCP-MS), respectively. Tissues (approx- imately 20--100 mg wet weight) were digested. using a modifi.ed version of previously published method [66]. Briefly, samples were incubated overnight in 0.5- 2 ml of pure HN03 (metals grade, Thermo-Fisher), centrifuged. at 12,000xg for 5 min and the super- natams sequentially diluted to yield 8 ml of solutions containing 35% HN03. AAS measurements were can-ied out using an AA6650 spectrometer (Sbirna.dzu, Columbia, IVID) equipped with a graphite furnace and ASC6100 autosampler. Samples were diluted with 267c nitric acid to be in the linear absorption range of the calibration c11tve (l---10 ppb (ng/L, generally between l 00--1000)). ·r\,enty µl of each sample was injected up to 3 rimes depending to achieve average da.ta with a low standard deviation. Copper and iron concentrations were derived by comparing the absorp-- rion of the samples with a 6 point calibration curve (0, 2, 4, 6, 8, lOng/g). ICP MS analysis was performed using :rn Agilent 7700 :,: system equipped witb an ASX 250 aulosampler. The system was operated at a radio frequency pmver of 1550 \V, an argon flow rate of 15 L/min, can-ier gas tlow rate of l .02 Umin, and helium gas flow rate of 4.3 ml/min (when applicable). Data were quantified using a 5-point calibration curve (0, 1, 10, 100, 1000 ngi g) with external standards for Fe, Cu, and Zn. For 1he ana.lysis of plasma samples, 30 µl of each sample was diluted in 1.5 ml l ':7<:, HN03 (Metals grade, Fisher) and measured in He mode to remove interferences. For the analysis of tissue samples, 100 µl of digested tissue solution was diluted into 4 ml of l 'Tc HN03 (Metals grade, Fisher). To prevent protein deposition and minimize nebulizer clogging as well as cross contamination the sample probe was washed with 1 % I-IN03 for 30 s followed by 30 s normal wash after each sample. Assays for copper·-dependenl proteins, ATP and lipid peroxidation Copper-dependent enzyme activity was measured in left hemisphere homogenates (soluble fraction) from the tolerance cohort following our published meth- ods [60, 67--69]. Cytochrome c oxidase activity was determined by measuring the oxidation of fetTicy- rochrome c using the Cytochrome c Oxidase Assay Kit (Sigma, St. Louis, MO) following manufacturer's instructions. Cy10chrome c oxidase and its inhibitor sodium azide were, respectively, used as positive and negative controls. Superoxide dismutase (SOD) activ- ity was determined using the N\VL.ssTM Superoxid.e Dismutase Activity Assay (Northwest Life Science Specialties, LLC, Vancouver, WA) according to manu- facturer's instrnctions. This assay is specific for Cu/Zn, 26 TS. A..nekonda et al. l P.lzytic Acid as a Potential Treatnwnt ,fof' Alzheimer·.~ Pathology Mn and Fe isoforrns of SOD, however Cu/Zn SOD is tbe most common cytoplasmic form of this enzyme. ATP levels were measured in the brain homogenates of mice in the tolerance cohort using the ATPlite 1 step Luminescence ATP Detection Assay System (Perkin Elmer, Boston, MA) as per manufacturer's instrnc-- rions and our published methods [60]. Briefly, brain homogenate was mixed witb an equal volume of 2.0% trichloroacetic acid (TCA) and centrifuged for 10 min at 10,000xg at 4"C. The supernatant was diluted 50% with phosphate buffered saline before adding equal volume of i\:TP--lite substrate solution. The plate was sba,.l.cen for 2 min and luminescence read on a Victor3 Multilabel Reader. ATP levels were quantified from an ATP standard curve. ATP levels were normalized to total protein in the homogenate. Oxidative stress in the brain homogenates of the tolerance cohort was measured by Malondialdehyde (JVIDA) Colorimetric Assay Kit, an indicator of lipid peroxidation following the manufacturer's protocol (Oxis International, Inc.) [60]. Western blor analysis 1.7.f tissue samples Protein expression in the soluble fraction from the treatment cohort \Vas determined by \Vestem blot analysis. Briefly, protein quantity in ea.ch sample ·was determined using the BCA assay (Pierce). Pro-- rein (20 I-Lg) was loaded onto 4-12% Bis-Tris gels, then separated and transferred onto nitrocellulose using 1he NuPage® electrophoresis system ([nvitro- gen). The membrane ·was probed with antibodies to anti-AP180, anti-beclin-1, anti-LC3B, ami-SIRTl, anti--phospho-AII/IPKo:, and anti- AMPKn, and anti-13-- mbulin (Sigma), treated ·with appropriate secondary antibody, exposed IO enhanced chemiluminescence solutions (Invitrogen), visualized using the Molecu- lar lma.ger Gel Documentation System and quantified using Quantity One Software (Bio--Rad). 5'ratistical analysis Sta1istical significance of the treatments, treatment durations, and their interaction effects were deter-- mined for cell survival and intracellular calcium using one·-\vay or t\V(}-\Vay analysis of variance, and \V]tb appropriate /--tests. \Ve also conducted the Bonfetrnni post-hoc test (for comparisons between two samples at a time_) The treatment difference is considered signifi- cant if p _s 0.05. All statistical analyses were performed using GraphPad Prism 5 software. RESULTS Celi culture studies l~ffects ;:_~f phytic acid on _free radicals. intracellular calciutn, and sur1Jival in A1(~65 cells Figure l shows levels of hydrogen peroxide (H,02), and superoxide (02· ), intracellular calcium, and cell s11rvival in tv1C65 cells cul1ured in 1he presence (Tet+) or absence of tetracycline (Tet-), and in the presence of phytic acid in Tet- conditions (Tet-PA; 100 j.LM). In all instances tbe measurement in Tet+ was considered i 00%, and IO which all other treat- ments were compared. At 48-hour treatment, both II202 (15% increase;p _s 0.05; Fig. lA) and 02· (2.4- fold increase; p _s 0.001; Fig. l B) levels increased significantly in Tet- compared IO Tet+. PA treat- ment significantly decreased H2 02 levels (p _s 0.00 l; Fig. lA) and maintained superoxide a.t Tel+ levels (Fig. l B). After 72 h treatment, intracellular cal- cium levels increased by nearly 5-fold (p :::-; 0.01, Fig. l C) and cell survival decreased significantly (80G!a decrease; p s (J.01; Fig. l D) in 'let-- com- pared to Tet+ treatment. Phytic acid (Tet-PA) provided near-complete protection against Af3PP-C99-mediated toxicity as sbown by greatly attenuated intracel- lular calcium (Fig. i C) and superior cell survival (Fig. lD). Effect of phytic acid on A/3 oligomers in lv!C65 cells The effect of pbytic acid on A(3 oligomer expression was determined using western blot analysis of MC65 cells 72 h after tetracyline withdrawal (Fig. 2.A, B.L Products of the transfected C99 construct are located at the low-molecular weight portion of the gel image, especially in Tet-treatment (Fig. 2A). Endogenous Af3PP appears to be present in all three treatments in tbe higher-molecular weigh1 range ot" the blot. Ai3 oligomers between approximately i 6 and 44 kDa (4--, 6-, and 8-mers as shown in Fig. 2B) \Vere atten- uated significantly {p :S 0.05) by PA treatment. The C-terminal 99 amino acid fragment (C99) and an 8-kDa band previously defined as either a dimer (P8) [58] or a combination of unknown protein species with some Af3 species [53], were unaffected by PA treatment. FJ.fect of phyric acid on autophagy and sirtuin proteins in lVIC.65 cells Figure 2C and D show expressions of the autophagy protein beclin -1 and SIRTl protein, respectively, in MC65 cells following 48-h exposure to tbe follow- J:S. ilnekonda et al. l Phytic Acid as C! Porential TreC!rrnent for lll::}u:itnef''s Pat.lwlogy (A) _ 125 0 .;; 100 i::: 8 75 (B) (D) 175 - 150· ~ 125 i1~~ I 'o~ 50 H 2~-- ----•----T;it+ Tet- r.s 'l'et•?A 11S Tut-PA 27 Fig. l. Protective effects of phytic acid (PA) in human neuroblastoma/MC6S cells, which •,;ere grown in the presence (Tet+) or absence (Tet- of tetracycline and under Tel-- with Fi\ (100 µM) for 48 or 72h, and assessed for: (A) H202 prnduct1on, (B) superoxide production, (C) intraceHufr.ff cakimn, and (D,1 susvivaL H202 prod.~1cticm and superoxjde productjon vvas asse:--,sed after 43-h treatsnent perjod. Cell :;undval and th,;:; frH.raceHular calcimn kvcls in live cells "Nere ass.;:;,ssed as rdafr,,re f!.uorescenc,;:; unjt.:, (Rf~1J,1_._i·ve cdls) aft.er 72-h treatn1.;:;rH. per_iocl _Each treatment was replicated on a\rerage 3-6 times in each of 3 experi..'Tients, and the error bars represent an average percentage of cell survival for the ex_perJments. Pair-\vise cornparison with Bonferroni post-hoc tests \Vere made on percent survival betv/een Tet+ and other treatments. Statistical significance levds: n.s, not significant. *p :::~ 0.05. **p :S 0.01, ***p :S 0.001. !A) (f3) $ Ohi- llli§ T~!-PA i4 ,:: ·Ai3f'f' ;l .. -i, 2 .. .lJ: .. mer : 1 -6-mer 0 4-mer P8i2.;n~r C99 4-;i;tr o--mer 3-mer ..C99 Oilgomfrs and olher bands .f'8 jloffier?) (D) Fig, 2. Th,;:; "Nest,;:;rn blot expn;ss_ion of AP oligomers, b,;:x:ljn-1, and SlRTt protein:, in J\tiC65 cells grown under Tet+ and Tet-jn the presence of vehicle (Veh) or phytic acid (&\, 100 µ:tvI). Resveratrol (RSV, 5 p.MJ served as a positive control. (A) Ai3 oligomers, (B) Ai3 oligomer d.ensJtometry an~lysis, (C) beclin-1, ~nd (D) SlRT'l. Aµ ohgon1ers 'Nere fractionated on J 69(, tricine gels. and. beclin-l and SIR.Tl on J 0-20%: bi.s-tris gd and tran.sferred to nitrocellulose membranes. The 1ne.::11hrane vvas probed with 6El0 .rnonodonal antibody to Af~ oligrnners, antj- becHn-1, and anti-SI RT 1, APPP= amy]oid-[3 proiein _precur:,or. AP oligomers appear in th,;:; range of] 6 and 44kDa. C99 = C-terminal 99 amjno acid·-fragment, and P8 = an 8·-kDa Aµ domain containing fragment. The bars represent average for each band rdativ·e to Tet+ Veh band and adjusted for the loading control rJ -tnbulm. Results are summarized from 3 experiments and the error b;;rs = SE±. Statistical significance levels: 'p ~' 0.05. 28 TS. A..nekonda et al. l P.lzytic Acid as a Potential Treatnwnt ,fof' Alzheimer·.~ Pathology ing four treatments: Tet+, Tet--, Tet--- with resvera.trol (RSV; 5 [-LM), and Tet--with PA ( l 00 µ.!VI). Resveratrol, a phytochemical known to upregulate SIRTl expres- sion and promote longevity in numerous organisms [24, 70], served as a posi1ive control. Beclin--1 expres-- sion in RSV and PA-treated cells increased by 1.4 and 1.8-fold, respectively, over the base-level expression in Te1+ cells and the effect was significant (p:::: (l.05) in tbe latter (Fig. 2C). SIRT1 expression in RSV and PA- treated cells remained similar to Tet+ levels (Fig. 2D). Anirnal studies Tolerance paradigm E.f]Ccts of phyric acid on anirnal body }Veight, cent- loplasmin (Cp) activity and meral ions in WT mice. Animal body weights taken before and during the 10- week treatment period are shown as weekly averages for each group of vehicle (VEH) and 2% phytic acid (PA) treated wild type female mice in Fig. 3A. Body weights for VEH and PA-treated groups remained sim- ilar during most of the treatmem period. Figure 3B shows average Cp activity in the serum of mice treated with VEH or 2% PA. Cp is the main copper transporter in the serum and Cp activi1y represents an indirec1 measure of freely available copper [71 J. (A) 30 E: :E 23 01 J 26 >- ~ 24 ill -,,. VEH ~- PA Cp activity was similar in vehicle and PA-treated animals (Pig. 3B ). In addition, copper and iron lev- els were measured in frontal cortex tissue using atomic absorption spectroscopy. There was no sig- nificant difference in brain copper and iron levels between vehicle and PA-treated animals (Fig. 3C). The activity of the copper-dependent anti-oxidant enzyme CuiZn superoxide disrnuta.se (SOD) levels also did not differ significantly between vehicle and PA-treated animals (Fig. 3D). These results suggest that when animals a.re fed a nutritional diet, PA may not have any ad verse effects on copper-· or iron--dependent metabolisms. E]fects of phyric acid on AT!~ COX and MDA. leveis in WT mice. ATP levels did not differ sig- nificantly between vehicle and PA-treated animals (Fig. 3D). Levels of cytochrome oxidase (COX), an index of mitochondrial function that declines in AD and aging pathologies [67], significantly (p<0.01) increased (Fig. 3D), and levels of malondialdehyde (MDA), an indicatoroflipid peroxidation, significantly reduced (Fig. 3E) in the frontal cortex homogenates of PA-treated animals.111ese results suggest that PA treat- ment promoted mitochondrial fonction and attenuated free radical formation in mouse brains. (C) 40 g 11111111 WTVEH _;, .:,30 lil!III WTPA ol! II. i 11'20 a.·- :l. g, 8-rn 0 22,..,,..~~~~~~~~~ o 2 3 <1 5 s 7 a 9 10 Weaks oftraaimen! WT\IEH WT?A Copper !ion Treatment (E) 1018 WTVEH l '""'" Ii mi al II 0Jl0-'--""""',,,.........__...,.....,,.., """'"-- SOD AT? cox MDA Fig. J. Effects of phytic and. (PA} in 'Nild··ty11e fe111ale mice. Nine··n1011th-0Jd v1iJd type (\VT) n11ce 'Nere treated with d1stilled dnnkmg water (VEH; n = l 0) or 1.9% phytk acid (PA; n = l G) for 70 days. (A) Body weights (g) of mke were measured 27 tfrnes during the treatsnent pedod and the week]y .-Nerag,;:;s are presented for V.EH and J?A treaied anirn.ab. (B) Celopla:'.)min activity (Cp) was measured six iimcs in th,;:; ph1sma samples and the average values are presented for each animal group. In addition. the folloviing assessments were made in the frontal cortex (FC) homogenates: (C) Copper/Cu & Iron/Fe, (D) Superoxide dismut;;se/SOD & /1'.rP, and /E) cytochrome oxidase/COX and melondehaldehyde (tvIDA). StatisticaJ :;ignificances: ~ct:p < O.Gl; ***p <0.001. J:S. ilnekonda et al. l Phytic Acid as C! Porential TreC!rrnent for lll::}u:itnef''s Pat.lwlogy 29 l}eatrnenr paradigrn 1-})Ccrs i~f'phytic acid on cerulopfasmin (C'p) activ- ity, rnetal ions and ~4,B species in Tg2576 rnice. Body weights (g) of mice measured over 30 times just before and during the treatment period are presented as monthly averages for VEH and PA treated animals in Fig. 4A. No consistent differ- ence exists between animal groups trea1ed with either VEH or PA. Figure 4 B shows terminal plasma Cp activity for four groups of mice in the treatment paradigm- Cp activity was significantly lower in both wild type (mean difference::: 32.4; p s (J.05) and Tg2576 (mean difference::: 31. l, p ::; Cl.OJ) mice treated with phytic acid than vehicle-treated animals. Figure 4C and D show ICP-MS measurements of copper, iron, and zinc in plasma and frontal cor- tex homogenates, respectively, from the same four groups of mice. Plasma copper levels \Vere signifi- cantly lower (t\1ean difference::: 144 _-.f.c 57; p s (J.05) in PA-treated Tg:2576 mice than vehicle-treated mice and iron levels were significamly higher (Mean differ- ence::: 3770 ,J_- 1577; p :s 0.05) in PA-trea1ed wild type mice than vehicle-treated animals (Fig. 4C). All other plasma comparisons between vehicle and phytic acid- treated animals \Vere non-significant. Interestingly, the levels of all three metal ions (Cu, Fe, Zn) remained sirn-- ilar across the four animal groups in the from al cortices (Fig. 4D). These results further suggest that PA did not affect the status of brain metal ions when animals were fed a nutritionally balanced diet. Figure 4E, F, and C) shmv soluble Ai3 t-40, insoluble Ai3:-40 and insoluble Af3J-42, respectively, in frontal cortex homogenates of the same four animal groups_ Aj3 levels in tbe \VT mice were negligible and soluble A[31.42 was undetectable in Tg:2576 mice; hence the data are not presented. Tg2576 mice treated. with PA showed. lower but non-significant levels of soluble and insoluble Aj3,.4o (Fig. 4E, F) and insoluble Aj:\1.,:2 (Fig. 4G) levels in the frontal cortices relative to vehicle-treated mice. ~{/i'cts of phyric acid on A.P 180, autophagy, SIR.Tl, AMPK. and PAA1PKpmteins in wild type and Tg2576 rnice. Molecular effects of PA treatment on the expres- sion ot' AP180, beclin-1, LC3Bl & [I, SJ RTl, AMPK, and PAlvlPK proteins in four animal groups from the rreatmem paradigm were not statistically significant, hence tbe results are not presented. Although statis- tically non--significarn, the western blot expression of AP180, an adapter vesicle protein indicative of Ai3PP trat1icking, showed a slight trend of decline in the PA-treated. animals compared. to YEH-treated groups. There was a trend towards increased. expression of two autophagy proteins, beclin- l and LC3B (LC3B-II/I ratio), SJ RTl and PAMPKJ AMP[( ratio in 1he cortices of animals treated with PA. These results appear to sug- (A) (8) 11111 M \/EH ~ Tg257o \/EH (CJ !1111111 WT \IEH ia::3 Tg,575 VEK !lli:II Wf I'll !l!lll Tg2576 PA l6 §34 t 32 l ,., ~o '8 Ill is +WTVEM .Q.- T~15-?SV~ ._ WTPA -+ T~:?5i9:FA. T LJ: 2 3 4 5 6 7 Monllis or treatmem !1111111 WT \iEH ~ T;2576 VEH 1!11!1 Wi PA illrrJ T;;2&76 PA iron Zinc il!iill \'vi !'A l!!ll Tg2S76PA 0 :f~1 :i m i ~l 11i~ ilillll l1H 0 ~ ~ ~ Coppar iron Zlnc Treatment (E) (F) (C:i) Fjg. 4. Protective effects of phytic add (PA) fr1 'Tg2.576 mou:;e .modeJ of AD. Twelve-n1onth-old \VT and Tg2576 tnice were treated wHh VEH (n = l9) or 2% PA (n-.::: 18) for 6 months. Their body weights and terminal assessments are prf:sent,;:;d. (A) Body weights (g) of mfr:e \Vere measured over 30 times