EXHIBIT Amlmhelpdesk.com/wp-content/Docs/PruvIT_FG/Doc_0107-1.pdf · Preliminary Injunction Hearing scheduled for December 3-4, 2015 in the above-referenced case, I expect to testify

EXHIBIT A

Case 4:15-cv-00571-ALM-CAN Document 107-1 Filed 12/02/15 Page 1 of 36 PageID #: 1519

EXPERT DISCLOSURE OF DR. EDWIN FLORES FOR PRELIMINARY INJUNCTION HEARING Page 1 of 5

UNITED STATES DISTRICT COURT EASTERN DISTRICT OF TEXAS

SHERMAN DIVISION

PRUVIT VENTURES, LLC, Plaintiff, v. AXCESS GLOBAL SCIENCES, LLC, and FOREVERGREEN INTERNATIONAL, LLC, Defendants.

§ § § § § § § § § § § §

Civil Action No. 4:15-cv-00571-ALM-CAN

Honorable Judge Amos L. Mazzant

vs. PRUVIT VENTURES, INC., LACORE LABS, LLC, TERRY LACORE, BRIAN UNDERWOOD, CHRISTOPHER N. HARDING, BILLY FUNK, RAY DIETRICH, ROBERT DEBOER, MICHAEL RUTHERFORD, SAVIND, INC., AND KETOTECH, INC., Counterclaim and Third-Party Defendants.

§ § § § § § § § § § § §

EXPERT DISCLOSURE OF DR. EDWIN FLORES FOR PRELIMINARY INJUNCTION HEARING

My name is Edwin S. Flores, Ph.D., J.D. If permitted to appear as a witness at the

Preliminary Injunction Hearing scheduled for December 3-4, 2015 in the above-referenced case, I expect to testify regarding the following topics.

EDUCATION AND EXPERIENCE

1. Attached as Exhibit A is a copy of my resume.



MATERIALS REVIEWED 2. I have reviewed the following documents as part of my preparation for the preliminary

injunction hearing:

a. Emergency Joint Motion For Temporary Restraining Order, Expedited Discovery, And Preliminary Injunction By Global Sciences, LLC And Forevergreen International, LLC

b. Declaration of Dennis Jones, PhD

c. Declaration of Gary Millet d. U.S. Patent No. 6,613,356, and its file history

e. U.S. Patent No. 9,138,420, and its file history f. Petition for Inter Partes Review of U.S. Patent No. 6,613,356, and all exhibits thereto

g. U.S. Patent No. 6,884,454 h. U.S. Patent No. 6,380,244

i. PCT Published Application No. WO 2005/025322 j. Abstract of Fisler JS, Egawa M, Bray GA. “Peripheral 3-hydroxybutyrate and food

intake in a model of dietary-fat induced obesity: effect of vagotomy”, Physiol Behav. 1995 Jul; 58(1):1-7

k. Simkins et al., “Regulation of Food Intake in Ruminants. 4. Effect of acetate, propionate, butyrate, and glucose on voluntary food intake in dairy cattle”, 1635-1642 (Exhibit 1018 to Petition for IPR of ‘356 Patent)

DR. JONES DECLARATION 3. I reviewed the methodology, assumptions, and conclusions set forth in the Declaration of

Dennis Jones, PhD. Dr. Jones provides his “professional opinion about the contents of the KETO//OS product based on the description on the label” – specifically, “to determine how much BHB salt and how much MCT oil is included in the product.” In reaching his determination as to the amount of BHB salt and MCT oil in the product, Dr. Jones makes multiple assumptions, including at least:

a. “The source of the fat in the product is the MCT Powder, which is composed of a medium chain triglyceride, non fat milk, and adjunctive excipients such as small amounts of disodium phosphate and silicon dioxide.”

b. “To make an MCT Powder, the MCT oil can be emulsified with liquid non fat milk, whereupon the combination is dried.”

c. “Alternative processes may consist of blending the MCT oil with a dried non fat milk powder.”

d. “For example, the most likely form of MCT Powder used in the product is a MCT powder called ‘Captex 70 Powder,’ which is sold by ABITEC Corporation.”



e. “If, for example, Captex 70 Powder is used as the source of MCT Powder in KETO//OS, the product would contain 7 grams of MCT oil.”

f. “Based on this statement and the information on the product label, it is my opinion that the product likely includes both calcium betahydroxybutyrate and sodium beta-hydroxybutyrate, both of which are salts of BHB.”

g. “The KETO//OS label indicates that the product includes 85% of the recommended daily allowance of calcium. The recommended daily allowance of calcium is 1,000 mg. Thus, the label indicates that this product supplies 850 mg of calcium.”

4. If any of the assumptions made by Dr. Jones when reaching his determination as to the amount of BHB salt and MCT oil in the product are incorrect, then his determinations are unreliable.

INVALIDITY OF U.S. PATENT NO. 9,138,420 (“the ‘420 Patent”)

5. U.S. Patent No. 6,884,454 to Pimentel (“Pimentel”) (attached as Exhibit B) teaches: a. A diet bar that includes MCT (medium chain fatty acid) to suppress appetite when

ingested. Pimentel at col. 3, ll. 16-17. b. MCT’s are used in weight loss programs because MCT’s possess thermogenic effects

and suppress appetite. Pimentel at col. 1, l. 67 – col. 2, l. 3. 6. U.S. Patent No. 6,380,244 to Martin et al. (“Martin”) (attached as Exhibit C) teaches orally

administering, to mammals, compounds that include 3-hydroxyacids, wherein the preferred 3-hydroxyacids include 3-hydroxybutyric acid (also known as beta-hydroxybutyric acid (BHB)). Martin at Abstract; col. 4, ll. 20-23, 25-26.

7. The disclosure of Pimental in view of Martin made it obvious to a person of ordinary skill in the art to orally administer, in mammals, two well-known and tolerated appetite suppression aids – MCT and a salt of BHB acid, and that MCT leads to an increase of BHB (thereby teaching that an increase in BHB is inherent or obvious). Thus at least claim 1 of the ‘420 Patent is invalid as obvious under 35 U.S.C. Section 103.

8. The disclosure of Pimental in view of U.S. Patent No. 6,613,356 made it obvious to a person of ordinary skill in the art to combine their teachings against the ‘420 patent.

INVALIDITY OF U.S. PATENT NO. 6,613,356

9. Martin (US 6,380,244) teaches orally administering, to mammals, compounds that include 3-hydroxyacids, wherein the preferred 3-hydroxyacids include 3-hydroxybutyric acid (also known as beta-hydroxybutyric acid (BHB)). Martin at Abstract (“appetite suppression”); col. 4, ll. 20-23, 25-26. Martin thus anticipates at least claim 1 of the ‘356 Patent under 35 U.S.C. Section 102 because it discloses oral administration, to a mammal, of at least one of the compounds listed in such claim to increase ketone levels.

10. Abstract of Fisler JS, Egawa M, Bray GA. “Peripheral 3-hydroxybutyrate and food intake in a model of dietary-fat induced obesity: effect of vagotomy”, Physiol Behav. 1995 Jul;



58(1):1-7 (“Fisler”) (attached as Exhibit D) teaches that injections of 3-hydroxybutyrate cause reduction in food intake in rats. Fisler at Abstract. Oral administration, rather than infusions, would be obvious to a person of ordinary skill in the art since it is common to utilize infusions in animal studies, and subsequently transition to oral administration of therapeutic compounds with humans. See, e.g., Martin at Abstract (“orally administered … or intravenously”).

11. Thus at least claim 1 of the ‘356 Patent was anticipated or obvious since oral administration, to a mammal, of at least one of the compounds listed in the claim, for weight loss or avoidance of weight gain, is taught by Fisler.

12. With respect to claim 1 of the ‘356 Patent, the claim terms “causing weight loss, or avoidance of weight gain” and “derivatives” require construction by the Court as a prerequisite to a determination of infringement or invalidity.

a. If the preamble phrase “causing weight loss, or avoidance of weight gain” is given its plain and ordinary meaning, then such claim is indefinite. The phrase “causing weight loss, or avoidance of weight gain” is ambiguous subject to many potential meanings, including at least: loss of fat; loss of water weight; improved ratio of muscle to fat; among others.

b. If the preamble phrase “causing weight loss, or avoidance of weight gain” is construed, oral administration of butyric acid or salts of butyric acid (including BHB) is many decades old.

c. The claim term “derivatives” is ambiguous and renders the claim indefinite.

13. Simkins et al., “Regulation of Food Intake in Ruminants. 4. Effect of acetate, propionate, butyrate, and glucose on voluntary food intake in dairy cattle” (“Simkins”) (attached as Exhibit E) teaches providing cattle (a mammal), with an amount of butyrate, that results in the suppression of food intake. (page 1639). Simkins states “It is well established that the metabolism of butyrate by the rumen epithelium and liver results in the production of ketone bodies,” thus triggering ketosis in a mammal with butyrate was well known.

Date: December 2, 2015

Respectfully Submitted,

. Edwin S. Flores, Ph.D., J.D.


EXPERT DISCLOSURE OF DR. EDWIN FLORES FOR PRELIMINARY INJUNCTION HEARING Page 5

CERTIFICATE OF SERVICE

I hereby certify that on this 2nd day of December 2015, a true and correct copy of

Expert Disclosure Of Dr. Edwin Flores For Preliminary Injunction Hearing was served

upon all counsel of record via email delivery:

Charles L Roberts WASATCH- IP 2825 East Cottonwood Parkwy Suite 500 Salt Lake City, UT 84121 [email protected] Attorney for Axcess Global Sciences, LLC

Larry R Laycock Adam B Beckstrom MASCHOFF BRENNAN LAYCOCK GILMORE ISRAELSEN & WRIGHT 201 South Main Street, Suite 600 Salt Lake City, UT 84111 [email protected] [email protected]

Clay A. Hartmann THE HARTMANN FIRM, PC 6677 Gaston Avenue Dallas, Texas 75214 [email protected] Attorney for Third-Party Defendants Billy Funk, Ray Dietrich, Robert Deboer, and Michael Rutherford

Tyson K Hottinger MASCHOFF BRENNAN LAYCOCK GILMORE ISRAELSEN & WRIGHT 20 Pacifica, Suite 1130 Irvine, CA 92618 [email protected] Attorneys for ForeverGreen International, LLC

William M. Lawson OGLETREE, DEAKINS, NASH, SMOAK & STEWART, P.C. 7700 Bonhomme Avenue, Suite 650 St. Louis, MO 63105 [email protected] Attorney for Third-Party Defendant Savind, Inc.

/s/ Kelly J. Kubasta Kelly J. Kubasta


EXHIBIT A


Edwin S. Flores, Ph.D., J.D. Chalker Flores, LLP

14951 N. Dallas Parkway, Suite 400 Dallas, Texas 75254

[email protected] (214) 866-0001 T (214) 866-0010 F

EDUCATION:

The University of Texas School of Law, Austin, Texas J.D., December 1996 Chief Articles Editor-Texas Intellectual Property Law Journal 1996-1997 Texas Intellectual Property Law Journal 1995-1996 President Intellectual Property Law Society 1995-1996 Vice-President Intellectual Property Law Society 1994-95 Washington University, St. Louis, Missouri Ph.D., Molecular Immunology, 1993 NIH-NIGMS Grant Recipient Patricia Harris Minority Fellowship Award National Science Foundation Fellowship Award-Honorable Mention The University of Texas, Austin, Texas B.S., Microbiology, 1988 Undergraduate Research Assistant I-IV

ELECTED OFFICE:

Trustee, Dallas Independent School District, District 1, 2015-2018 EMPLOYMENT: July 2014 to Foret Plasma Labs (FPL), Chief Scientific Officer Present Lead research and development efforts of FPL for water treatment and

natural products processing, draft and oversee grants and grant funding, primary research contact for new ventures in Latin America and Asia.

September 2002 to Chalker Flores, LLP, Dallas, Texas Present Managing Partner. Determine patentability, research, draft and prosecute

patents in the fields of: recombinant biotechnology, pharmaceuticals, medical devices, nanotechnology, computer-aided imaging systems, nanobiotechnology, nanotechnology, integrated circuits, semiconductors and complex-mechanical patents for university and corporate clients. Areas of Specialization: Counseling start-ups, patent prosecution, litigation and licensing. Chalker Flores is a Certified DBE No. HMDB22517Y0405.

April 2001 to Assistant District Attorney, Dallas County, Dallas, Texas June 2001 Misdemeanor Prosecutor, Dallas County Lawyer-on-Loan Advanced Trial

Training Program. Participated in all aspects of trial, including: witness interviews, trial preparation, voir dire, opening statement, direct and cross


examination of Officers, lay witnesses and expert witnesses, closing argument and punishment phase.

May 1998 to Gardere, Wynne, Sewell, LLP, Dallas, Texas September 2002 Associate, Biotechnology Patent Group. Determined patentability,

researched, drafted and prosecuted patents of recombinant biotechnology, biopharmaceuticals, medical devices, computer-aided imaging systems and mechanical patents for university and corporate clients. Areas of Specialization: patent prosecution, litigation and licensing.

February 1996 to Warren & Perez, Dallas, Texas May 1998 Summer Clerk and Associate. Legal research and writing in the areas of

patent prosecution, litigation discovery, invalidity and infringement in a wide variety of intellectual property practice areas. Draft patents in the areas of biotechnology, electrical and mechanical devices, integrated circuits, semiconductor processing and packaging.

August 1993 to Arnold, White & Durkee, Austin, Texas August 1994 Scientific Advisor/Patent Agent-Biotechnology Patent Group. Determined

patentability, researched, drafted and prosecuted patents of recombinant biotechnology, biopharmaceuticals and mechanical patents for university and corporate clients. Areas of Specialization: Immunology, Recombinant Molecular Biology, Cancer Biology, Viral Gene Therapy and Growth Factors.

May 1995 to University of Texas School of Law, Austin, Texas May 1996 Research Assistant for Professor Charles Silver. Legal research and

writing in the areas of professional responsibility, insurance regulation, class actions and civil procedure in academic and consulting matters. Set-up internet forum homepage for legal discussions.

February 1994 to Informatec Translations, Inc., Austin, Texas 1996 Owner, Translator, Creative Writer, Editor. Spanish/English translations

including: legal documents, technical manuals, international business documents, and bilingual advertising campaigns. Wrote Spanish language advertising campaigns for the American Heart Association and the successful reelection campaign of a Texas State Senator.

BAR ADMISSIONS: State Bar of Texas United States Patent and Trademark Office Federal District Court for the Northern District of Texas COMMUNITY INVOLVEMENT: NIH Advisory Council - National Institute of General Medical Sciences (2006-2010). NIH - Council of Councils of the NIH (2008 – 2012).


SMU Tate Lecture Series (2006-2012), Good Shepherd Episcopal School (2006-2011), the Dallas Assembly (2006-present), Dallas Historical Society (2004-2006), the Southwestern Medical Foundation (2005), Teach for America-Dallas-Ft. Worth (2013-2015). Board member KIPP-DFW charter school (2013-2015), Board President, International Leadership of Texas Charter School (2014-present), Trustee, Dallas Independent School District (2005–2012). Trustee, The Museum of Nature and Science (2003 – present). The Tower Center Board (2010-present), the Boy Scouts Circle 10 Council (2006- present), Medical City Hospital Dallas (2009-present). LANGUAGES: First languages: Spanish and English - Bicultural (México/U.S.), beginner

Chinese. PUBLICATIONS:

Legal Edwin Flores, et al., Inequitable Conduct, Fraud and Your License to Practice Before the United States Patent and Trademark Office, 8 TEXAS INTELLECTUAL PROPERTY LAW JOURNAL 299 (Spring 2000) Edwin Flores, The Development of Modern Frameworks for Patent Prosecution: Mexico, A Model for Reform, 6 TEXAS INTELLECTUAL PROPERTY LAW JOURNAL 133 (Winter 1998) Charles Silver, with the assistance of Edwin Flores, Your Role in a Law Firm: Responsibilities of Senior and Junior Attorneys, 1996: A Guide to the Basics of Law Practice, The Texas Center for Legal Ethics and Professionalism, Austin, TX. pp. 103-115. Edwin Flores, Publish and Perish: The Patentability Aspects of Peer Review Misconduct, 5 TEXAS INTELLECTUAL PROPERTY LAW JOURNAL 47 Fall 1996. Edwin Flores, The Genetic Privacy Act: An Analysis of Privacy and Research Concerns, 25 JOURNAL OF LAW, MEDICINE & ETHICS 256 (1997).

Scientific Flores, E., Roy, G., Patel, D., Shaw A. and Thomas, M. L., PEP is a Nuclear Protein Tyrosine Phosphatase, MOL. CELL. BIO., Vol. 14, pp. 4938-46, 1994. Cahir McFarland E., Flores, E., Matthews, R. J., and Thomas, M. L., Protein Tyrosine Phosphatases Involved in Lymphocyte Signal Transduction, Samuelson L.E. (Ed.): Lymphocyte Activation, CHEM. IMMUNOL., Basel, Karger, Vol. 59, pp 40-61, 1994. Flores, E., Characterization of a Novel Hematopoietic Protein Tyrosine Phosphatase, PH.D. DISSERTATION, St. Louis, U.S.A., pp 1-128, August 1993.


Thomas M. L., Flores, E., Cahir McFarland, E., Matthews, R.J., Roy, G., Shaw, A., and Shenoi H., A Diversity of Protein Tyrosine Phosphatase Structures Expressed in T Lymphocytes, PROGRESS IN IMMUNOLOGY (Editors, J. Gergely, M. Benczur, A. Erdei, A. Falus, G. Fust, G. Medgyesi, G. Petranyi, & E. Rajnavolgyi) Springer-Verlog, Vol. 8, pp. 213-219, 1993. Matthews, R.J., Bowne, D., Flores, E., and Thomas M. L., Characterization of Hematopoietic Intracellular Protein Tyrosine Phosphatases: Description of a Phosphatase Containing an SH2 Domain and Another Enriched in PEST Sequences, MOL. CELL. BIO., Vol. 12, pp 2396-2405, 1992. Matthews, R.J., Flores, E., and Thomas, M. L., Protein Tyrosine Phosphatase Domains from the Protochordate Styela plicata, IMMUNOGENETICS, Vol. 33, pp 33-41, 1991.


EXHIBIT B


US006884454B2

(12> Ulllted States Patent (10) Patent N0.: US 6,884,454 B2 Pimentel (45) Date of Patent: Apr. 26, 2005

(54) APPETITE SUPPRESSING DIET BAR (56) References Cited

Inventor: JuliO LiOIlEl Pimelltel, Wll'ldgate Dr., Buford, GA (US) 30519-1941

4,423,072 A * 12/1983 Stahly ...................... .. 514/552

( * ) Notice: Subject to any disclaimer, the term of this 2002/0119915 A1 * 8/2002 Portman ...................... .. 514/8 patent is extended or adjusted under 35 U.S.C. 154(b) by 135 days. * Cited by examiner

(21) Appl. No.: 10/274,543

(22) Filed: Oct. 21, 2002

(65) Prior Publication Data

US 2004/0076719 A1 Apr. 22, 2004

Primary Examiner—Helen Pratt

(57) ABSTRACT

A method to decrease feed intake in humans by ingesting a diet bar comprising of Whole soybean and medium chain

(51) Int. Cl.7 ................................................ .. A23L 1/29 triglycerides and/0r medium chain fatty acids, said bar (52) US, Cl, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 426/634; 426/601 containing an adequate level of nutrients to serve as a meal.

(58) Field of Search .......................... .. 426/634, 72, 74, 426/601 7 Claims, N0 Drawings


US 6,884,454 B2 1

APPETITE SUPPRESSING DIET BAR

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention relates to a diet bar having a satiety

effect on humans When consumed prior to any meal. This diet bar is composed of Whole soybean containing trypsin inhibitor that increase CCK levels, Which suppress appetite; and medium chain fatty acids or medium chain triglycerides that increase the level of beta-hydroxy butyrate Which also suppress food intake.

2. Discussion of the Background There are not magic bullets for the reduction of body

Weight, all drugs, herbs and exercise programs Work because We reduce our food intake or make us expend more energy that the one We have ingested. All these procedures kept your body Weight doWn as long as you keep in the program. Almost 100% of all dieters come back to their pre-program Weight Within tWo years. The more effective Way to reduce your body Weight is by decreasing and maintaining a loW food intake. Obesity has become a leading cause of prevent able death, cardiovascular problems and diabetes been the major effects of obesity.

Medium-chain fatty acids (MCFA, 6—12 carbons) are naturally found in coconut oil, palm kernel oil, and milk. Medium-chain triglycerides (MCT) oil is comprised of primarily caprylic (C810) and capric (C1010) acids With a very small percentage of caproic (C610) and lauric (C1210) acids, Which are esteri?ed to a glycerol backbone. The most prevalent fatty acids found in food are oleic (C1811), palm itic (C1610), stearic (C1810), and linoleic (C1812). MCT oil is a light yelloW, translucent, odorless liquid at room tem perature. Although completely saturated, it is not athero genic (Blackburn et al ,1989) or solid in consistency like other saturated fats. The energy value of MCT oil is approxi mately 7—9 calories per gram, and this fat is metaboliZed differently than long-chain triglycerides (LCT). Complete hydrolysis to MCFA’s and small amounts of monoglycer ides occurs in the stomach With very little secretion of pancreatic lipase or bile acids. After MCFA’s are absorbed into the intestinal mucosal cells, they are not resynthesiZed into triglycerides and incorporated into chylomicrons as are long-chain fatty acids. MCFA’s bypass the lymphatic sys tem and are carried by the portal vein directly to the liver, Where they are metaboliZed to produce carbon dioxide, ketones, and acetate. MCFA enter the mitochondria inde pendently of the carnitine transport system and undergo preferential oxidation. They burn quickly and completely, producing huge amounts of energy (Bach and Babayan, 1982). Dietary fat and the fat stored on our body as adipose tissue are in the form of triglycerides, Which contain long chain fatty acids (14 carbons or more) (Babayan, 1987). Therefore MCT’s Will be not storage or accumulated in adipose tissues. MCT’s reduce the breakdoWn of muscle tissue When dieting due to the production of ketone bodies that burn preferentially to muscle tissue for energy. MCT’s improve the absorption of amino acids, Which are critical for muscle tissue repair. They also improve calcium and magnesium, minerals needed for the metabolism of carbo hydrates and amino acids (Kreb’s cycle)and for improving muscle contraction response time and energy. They also decrease the absorption of cholesterol in the intestine. Due to the unique properties of MCT’s, they are used as

a fat source in many diseases states. MCT oil can be used to add calories to a formula or diet in the case of malabsorption syndromes and people With intensive burns (Babayan, 1981), because it requires loWer concentrations of bile or pancreatic lipase for digestion and absorption. MCT is often

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2 used in Weight loss programs because it possesses ther mogenic effects and suppresses appetite When taken before meals.

It has been observed that supplementation of neWborn piglets With (MCT) decreased nitrogen excretion and break doWn of liver glycogen. These observations indicate neW born piglets utiliZe (MCT) as energy source rather than body reserves of glycogen and protein. Research Work at Illinois has demonstrated that the emulsi?cation of medium chain triglycerides further improves digestion and absorption. When MCT’s are substituted for long-chain triglycerides in the diet, animals gain less Weight, store less adipose, and experience an increase in metabolic rate (Hashim, S A, 1967; Baba, N et al, 1982 and Geliebter, A. et al, 1983). MCT-fed mice also have been shoWn to possess increased endurance over that of LCT-fed mice (Fushiki, T et al, 1995).

Rats fed 10% or 20% fat as MCT eat less than rats fed the same amount of food With LCFA (Furase et al, 1992). The intake of MCT produces beta-hydroxy butyrate, Which sup press food intake (Furase et al, 1997). MCT’s decreased food intake by a post-absorptive mechanism (Van Wymel beke V., et al, 1998). In long term MCFA feeding in animals, Weight accretion has been attenuated (Papamandj aris AA, et al, 1998). Infusion of MCT accelerates small-boWel transit time as compared to LCFA (long-chain fatty acids) or saline (Ledeboer M, et al. 1995). So the intake of MCT’s prior to a meal Will decrease meal siZe. Soybean contains antinutritional factors Which inhibit

animal performance, heat-labile antinutritional protease inhibitors are found in virtually every legume. Protease inhibitors are proteins that combine With the enZymes asso ciated With protein digestion such as trypsin and chymotrypsin, signi?cantly inhibit their function. This inhibition, if not inactivated, is accompanied by moderate to-severe depression in animal performance. Osborne and Mendel, in 1917, made the signi?cant discovery that soy beans had to be heated in order to support the groWth of rats. Soybean contains 61% polysaturates fatty acids, linoleic (c18-2) and linolenic. The extraction of oil from soybean removes sterols and

some saponins, Which are thought to help protect against colon cancer. Extracting soy ?our With ethanol to produce soy concentrate removes anticancer activity, presumably by extracting phytochemicals such as iso?avones (genestein), protease inhibitors and saponins. Soybean has iso?avones primarily genestein and daidZien and a minor called gly citein. The ingestion of raW soybean increase CCK release in

man and heat treatment reduces the trypsin inhibitor content of the ?our (Calam et al, 1987). TWo types of soybean protein inhibitors have been identi?ed, the KunitZ inhibitor (KSTI) and the BoWman-Birk inhibitor (BBI) both KSTI and BBI are inactivated during moist heat treatment. Hyper trophy of the pancreas is one the primary physiological effects, accompanied by a stimulation of its secretory activ ity. Neither raW soy ?our nor other soy product produced pancreatic enlargement in pigs or monkeys (Struthess and MacDonald, 1983). Feeding soybean extract containing the BoWman-Birk protease inhibitor or feeding the puri?ed protease inhibitor decreased chemically induced colon can cer in rats and mice (Thiagarajan, D et al, 1998).

It has been observed that feed intake and siZe of meal Was decreased for 6 hours after supplying trypsin inhibitors to Zucker rats. (Peiken 1985, McLaughlin 1983). The effect of trypsin inhibitor is to increase the concentration of CCK secretion producing satiety and resulting in a consequent decrease in food intake and over time decrease in body Weight. Feeding also heated or raW soybean ?ours had a signi?cant inhibitory effect on lipase digestive enZyme activities in the pancreas and in its secretion (Khalifa, et al 1994).


US 6,884,454 B2 3

In a study With fat pre-loads it Was observed that a one dose emulsion of long chain triglycerides (32 ml With 16 gr. lipids) containing:

c16=8%, c18-0=4%, c20-0=2%, c18-1=56% and c18-2= 23% (166 calories) evocked higher level of CCK than medium chain triglycerides (32 ml With 16 gr. lipids) containing: c6=1%, c8=81%, c10=16% and c12=2% (128 calories)(Isaacs, et al, 1987).

Suppression of appetite decreases caloric intake therefore a reduction of body Weight, a combination of medium chain fatty acids, long chain fatty acids and protease inhibitors in a complete nutritious health bar suppress appetite enough to have a reduce feed intake.

SUMMARY OF THE INVENTION

A diet bar containing medium chain triglycerides, Whole soybean that suppress appetite When ingested 15—120 min utes prior to a meal. The medium chain triglycerides produce beta-hydroxy-butyrate, Which has a satiety effect. The long chain fatty acids and the presence of trypsin inhibitors in Whole soybean increase the level of CCK Which have a satiety effect. The combination of these products When taken prior to a meal reduces meal siZe or otherWise produces satiety strong enough to be able to skip a meal (meal replacer).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Obesity is the major health risk for the development of several diseases including cardiovascular problems, arthritis, high blood pressure and diabetes. In a recent report it has been found that 65% of the US population is over Weight and that 10% are extremely obese. The major culprits of the increase in obesity are the high caloric intake and sedentary life, the role that genetic plays is minimal.

There are several treatments of obesity, they include surgical, appetite suppressant drugs, diets and exercise. Operation like stomach stapling is expensive and requires several hospitaliZation days. Liposuction takes out the fat but if the patient does not change his/her lifestyle, the fat Will accumulate in another areas. Appetite suppressant drugs and herbal products have secondary side effects and there have been cases that people have died due to the side effects. Recently a prescription drug had to be pulled of the market because of cardio-vascular problems. Inhibition of fat absorption by lipase inhibitor have also side effects, it produce steatorrhea and malabsorption of fat-soluble vita mms.

A better solution for the obesity problem is to reduce appetite by natural means, let our body control our appetite. This invention disclose a method to suppress food intake by increasing CCK level (appetite suppressant hormone) and beta-hydroxy butyrate after a consumption of a healthy diet bar containing Whole soybean and medium chain triglycer ides.

It is an object of the present invention to provide a natural mean to decrease appetite in humans.

Other object of the present invention is that other com pounds having trypsin inhibitor and beta-hydroxy butyrate, or puri?ed trypsin inhibitor or puri?ed beta-hydroxy butyrate can be utiliZed having the same results as the present invention.

Other object of the present invention is that it can be used as a Way to decrease body Weight due to decreased intake and the thermogenic effect of the medium chain fatty acids.

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4 EXAMPLE 1

The diet bar comprises of Whole soybean, soybean ?our, medium chain triglycerides, vitamins, minerals, ?avor coat ing and oats. The formulation can vary depending on the serving siZe and ingredients. The folloWing examples shoW some formulations but they can vary Without affecting the main objective of this invention.

(gr)

Ingredients

Oats 10 0 0 10 0 0 Soybean Whole 30 30 20 20 10 0 Soy Flour 0 0 10 10 20 30 MCT-oil 5 5 5 5 5 5 Vitamin & 1 1 1 1 1 1 Minerals Flavor Coating 4 4 4 4 4 4 Total Serving 50 40 40 50 40 40 (gr) Nutrients/Serving

Calories 202 165 166 200 168 169 Fat (gr.) 11.6 11 11 11.7 11.3 11.2 Protein (gr.) 12.6 11.5 11.2 12.4 10.9 10.7 Carbohydrates 15.8 9 9 15.6 8.7 8.6 (gr) Trypsin 1425 1425 1300 1300 1175 1050 Inhibitor (mg)

The ingredients are ?rst mixed, compressed to a bar form and ?nally coated With a ?avor coating and package. The diet bar is to be use before meal as a intake suppressant or as meal replacement. This bar has the required ingredients to replace a meal.

It Will be apparent for those skilled in the art that a number of modi?cations and variations may be made Without depart ing from the scope of the present invention as set forth in the appending claims. What I claim is: 1. A method for decreasing food intake in humans by

orally consuming a nutritive bar comprising of Whole or defatted soybean and medium chain triglycerides.

2. A method of claim 1, Wherein said nutritive bar is consumed 15 to 120 minutes prior to any of the three major daily meals.

3. A method of claim 1, Wherein Whole or defatted soybean consist of 50% to 80% of said bar total Weight.

4. A method of claim 1, Wherein medium chain triglyc erides consist of 5% to 25% of said bar total Weight.

5. A method of claim 1, Wherein the nutritive bar con taining Whole or defatted soybean and medium chain trig lycerides is supplemented With other feed ingredients, vitamin, minerals and ?avoring agents in order to formulate a nutrient-adequate and tasteful bar, said supplementation varies from 1% to 10% of said bar total Weight.

6. A method of claim 1, Wherein said decrease in feed intake is due to the appetite inhibition resulting from the increase in cholecystokinin (CCK) and beta-hydroxy butyrate.

7. A method of claim 6, Wherein the increase in CCK and beta-hydroxy butyrate can also result from the consumption of ingredients other than soybean or medium chain triglyc erides.


EXHIBIT C


(12) United States Patent Martin et al.

US006380244B2

(10) Patent N0.: (45) Date of Patent:

US 6,380,244 B2 *Apr. 30, 2002

(54) NUTRITIONAL AND THERAPEUTIC USES OF 3-HYDROXYALKANOATE OLIGOMERS

(75) Inventors: David P. Martin; Oliver P. Peoples, both of Arlington; Simon F. Williams, Sherborn; Luhua Zhong, Quincy, all of MA (US)

(73) Assignee: Metabolix, Inc., Cambridge, MA (US)

(*) Notice: This patent issued on a continued pros ecution application ?led under 37 CFR 1.53(d), and is subject to the tWenty year patent term provisions of 35 U.S.C. 154(a)(2).

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.

(21) Appl. No.: 09/359,086

(22) Filed: Jul. 22, 1999

Related US. Application Data (60) Provisional application No. 60/093,760, ?led on Jul. 22,

1998.

(51) Int. Cl.7 ..................... .. A61K 31/335; A61K 47/00

(52) US. Cl. ..................... .. 514/449; 514/450; 514/452; 514/460; 514/473; 514/546; 514/547; 514/509;

424/438; 424/439; 424/442 (58) Field of Search ............................... .. 514/546, 450,

514/449, 452, 547, 460, 473, 909; 424/438, 439, 442

(56) References Cited

U.S. PATENT DOCUMENTS

4,329,359 A 5/1982 Stahly 4,365,088 A * 12/1982 Vanlautem et al. ....... .. 562/579

4,423,072 A 12/1983 Stahly 4,563,354 A 1/1986 Chang et al. 5,093,044 A 3/1992 Wretlind et al. 5,126,373 A * 6/1992 Brunengraber et al. 514/547

FOREIGN PATENT DOCUMENTS

EP 0 316 993 A1 5/1989 EP 0321428 A1 6/1989 EP 0 780 123 A1 6/1997 JP 6/321778 11/1994 W0 WO 88/08301 A1 11/1988 W0 WO 90/02548 A1 3/1990 W0 WO 90/02549 A1 3/1990 W0 WO 90/11753 A1 10/1990 W0 WO 95/09144 A1 4/1995 W0 WO 95/18781 A1 7/1995 W0 WO 97/15681 A1 5/1997 W0 WO 98/41200 A1 9/1998 W0 WO 98/41201 A1 9/1998 W0 WO 99/34687 A1 7/1999

OTHER PUBLICATIONS

Beylot, et al., “Metabolic effects of a D—beta—hydroxybu tyrate infusion in septic patients: inhibition of lipolysis and glucose production but not leucine oxidation,” Crit. Care Med. 22(7):1091—98 (1994).

Birkhahn & Border, “Intravenous feeding of the rat With short chain fatty acid esters. II. Monoacetoacetin,” Am. J. Clin. Nutr. 31(3):436—41 (1978). Birkhaun, et al., “Monoglyceryl acetoacetate: a ketone body—carbohydrate substrate for parenteral feeding of the rat,” J. Nutr. 109(7):1168—74 (1979). Desrochers, et al., “R,S—1,3—butanediol acetoacetate esters, potential alternates to lipid emulsions for total parental nutrition,” Nutr. Biochem. 6:111—18 (1995).

Lammerant, et al., “Inhibitory effects of the D(—)isomer of 3—hydroxybutyrate on cardiac non—esteri?ed fatty acid uptake and oxygen demand induced by norepinephrine in the intact dog,” J. Mol. Cell Cardiol. 17(4):421—33 (1985). Muller, et al., “Bildung 12—bis 40—gliedriger oligolide aus enantiomerenreinen 3—Hydroxybuttersaure—Derivaten— Bausteine fiir eine 21—und eine 31 —Helix,” Chimia 45:376—79 (1991). PaWan & Semple, “Effect of 3—hydroxybutyrate in obese subjects on very—loW—energy diets and during therapeutic starvation,” Lancet. 1(8314—5):15—17 (1983). Riis & Mai, “Gas chromatographic determination of poly—[3—hydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis,” J. Chromatography 445:285—89 (1988).

(List continued on next page.)

Primary Examiner—Frederick Krass (74) Attorney, Agent, or Firm—Holland & Knight LLP

(57) ABSTRACT

Nutritional or therapeutic compositions are provided for increasing ketone body levels in the blood of mammals by providing a source of ketone bodies in the form of linear or cyclic oligomers and/or derivatives of 3-hydroxyacids. The 3-hydroxyacid can be in the form of a linear oligomer of 3-hydroxyacids other than linear homo-oligomers of 3-hydroxybutyric acid if administered in combination With acetoacetate, cyclic oligomers of 3-hydroxyacids, esters of the linear or cyclic oligomers, esters of 3-hydroxyacids other than 3-hydroxybutyric acid, and combinations thereof. An oligomer generally refers to a polymer of three or more hydroxyacids. Preferred 3-hydroxyacids include 3—hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, and 3-hydroxyheptanoate. Oligomers of odd-carbon number 3-hydroxyacids such as 3-hydroxyvalerate have advantages since they have a higher energy content than oligomers of 3-hydroxyacids having an even-number of carbons. The cyclic oligomers have advan tageous properties since they result in a sustained, and/or controlled, ketone blood level over a period of hours. The compositions can be administered orally, for example, as a nutritional or dietary supplement, or intravenously. Increas ing blood ketone levels is useful for seiZure control, meta bolic disease control, reduction of protein catabolism, appe tite suppression, parenteral nutrition, increasing cardiac ef?ciency, treatment of diabetes and insulin resistant states, and treatment of effects of neurodegenerative disorders and epilepsy.

12 Claims, 1 Drawing Sheet

Pruvit Ventures, Inc. & LaCore Enterprises, LLC IPR 2015-01798

Exhibit 1003 Page 1


US 6,380,244 B2 Page 2

OTHER PUBLICATIONS

Seebach, et al., “187. On the rnacrolactoniZation of [3—HydroXy Acids, Crystal structures of the pentolide and the heXolide frorn (R)—3—HydroXybutanoic acid. Molecular modeling studies of the tetrolide,” Helv. Chim. Acta. 72:1704—17 (1989). Seebach, et al., “The triolide of (R)—3—HydroXybutyric acid—Direct preparation forrn polyhydroXybutyrate and for rnation of a croWn estercarbonyl complex With Na Ions,” Angew Chem. Int. Eng. Ea'. 4:434—35 (1992). Seebach, et al., “18. High—yield synthesis of 20—, 24—, and 28—rnernbered rnacropentolide, —heXolide, and —heptolide, respectively, from (R)—or (S)—3—HydroXybutanoic acid under Yarnaguchi’s MacrolactoniZation Conditions1)” Helv. Chim. Acta. 71:155—67 (1988). Steinbuchel & Valentin, “Diversity of bacterial polyhy droXyalkanoic acids,” FEMS Microbiol. Lett. 128:219—28 (1995). Steinbuchel, et al., “Synthesis and production of poly(3—hydroXyvaleric acid) hornopolyester by Chromobac terium violaceum,” Appl. Microbiol. Biotechnol. 39:443—49 (1993).

Williams, et al., “Biodegradable plastics from plants,” CHEMTECH 26:38—44 (1996).

BroWn, et al., “Monitoring beta—hydroXybutyrate as an anticonvulsant level in the ketogenic diet,” Epilepsia 39:168—69 (1998).

Erecinska, et al., “Regulation of GABA level in rat brain synaptosornes: Fluxes through enzymes of GABA shunt and effects of glutamate, calcium, and ketone bodies,” J. Neu rochem. 67:2325—2334 (1996).

Seebach, et al., “176. Preparation and structure of oligolides frorn (R)—3—hydroXypentanoic acid and comparison With the hydroXybutanoic—acid derivatives:A small change With large consequences,” Helvetica Chimica Acta 77:2007—34 (1994). Tasaki, et al., “The dimer and trirner of 3—hydroXybutyrate oligorner as a precurser of ketone bodies for nutritional care,” Journal of Parenteral and Enteral Nutrition 23:321—325 (1999).

* cited by eXarniner


Exhibit 1003 Page 2


U.S. Patent Apr. 30, 2002 US 6,380,244 B2

0.4

0.35

0.3

0.25

@12 + R-BHB

O ]5_ + AcAc ‘ + 1010i KB

0.1

005 M / W

O T, I I I I 0 50 100 150 200 250

T TIME (MINUTES) BOLUS F/G. I


Exhibit 1003 Page 3


US 6,380,244 B2 1

NUTRITIONAL AND THERAPEUTIC USES OF S-HYDROXYALKANOATE OLIGOMERS

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to US. provisional application Ser. No. 60/093,760, ?led Jul. 22, 1998.

BACKGROUND OF THE INVENTION

The present invention is generally in the ?eld of nutri tional and therapeutic compositions for the modulation of ketone levels in humans and other mammals.

There are a number of conditions in human and animals in Which it is desirable to increase the levels of ketone bodies in the human or animal body. Examples include seiZure control, treatment of certain metabolic disorders, reduction of protein catabolism, appetite suppression during Weight loss, and parenteral nutrition. A number of treatments exist for seiZure control in

epileptic patients. Anti-seizure medications are popular; hoWever, they are not alWays effective and can cause unde sirable side-effects. Aketogenic diet has been used since the turn of the century, but lost favor With the development of anti-seiZure medications. The ketogenic diet recently has attracted neW interest for the treatment of certain forms of epilepsy, as Well as other medical conditions. The diet, Which typically is carefully controlled and doctor supervised, is very high in fat calories and loW in carbohy drates. The diet forces the body to metaboliZe fats instead of carbohydrates for energy, thereby elevating the level of acetoacetate and D-3-hydroxybutyrate in the blood. These compounds are referred to as “ketone bodies,” thus the term “ketogenic” is used to describe the diet.

While the exact mechanism of action of the ketogenic diet is not Well understood, it is believed that the elevated blood levels of ketone bodies have sedative effects Which help to prevent seiZures. In order to be effective for this purpose, hoWever, the patient must strictly observe the diet. Vitamin and mineral supplements are included in the diet to make it nutritionally complete, since the diet is very high in fat, loW in proteins, and requires the near elimination of carbohy drates. Each patient’s diet is mathematically calculated based on the age, siZe, and activity level of the patient. Patients normally folloW the diet for one to tWo years, at Which time the patient is sloWly Weaned onto a normal diet. The diet has been found to be particularly effective With epileptic children. Major draWbacks are that the diet is not very palatable and that patient compliance demands com plete commitment on the part of the patient and his or her family. Moreover, the diet’s high fat content can increase the risk of vascular diseases, such as atherosclerosis.

Special diets are also used When a person urgently needs to lose Weight for health reasons, for example prior to surgery or due to complications from obesity. In this situation, the doctor may prescribe a diet greatly restricting the person’s caloric intake. With the caloric intake reduced, the body is forced to metaboliZe storage reserves for energy. The body can derive energy from fat and skeletal tissue, such as muscle and proteins. It is preferable, hoWever, that fat tissue be used rather than protein, since the breakdoWn of proteins (i.e. “catabolism”) can undesirably result in mus cular atrophy, immuno-suppression, and reduced Wound healing. Supplementation of the diet With hydroxybutyric acid has been shoWn to reduce protein catabolism in subjects on loW energy diets (PaWan & Semple, Lancet 8:15 (1983)). It also has been reported that 3-hydroxybutyrate bene?cially suppresses the appetite.

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2 Total parenteral nutrition (“TPN”) is used to provide

nutrients to patients Who are unable to ingest food orally, such as in the case of intestinal failure. Common causes of this condition include in?ammatory disorders of the gas trointestinal tract (e.g., Crohn’s disease), radiation enteritis, and short boWel resulting from surgical resection of necrotic or diseased boWel. Approximately 22,000 outpatients and 150,000 inpatients currently receive TPN in the United States alone (PR Newswire: Orphan Medical Announcement, Jun. 9, 1995). Patients receive the nutrients, Which typically are concentrated fat emulsions, directly into their veins. The nutrient compositions are described, for example, in US. Pat. No. 4,563,354 to Chang et al.; EP 0321428 A1; US. Pat. No. 5,093,044 to Wretlind et al.; PCT WO 88/08301; PCT WO 90/02548; PCT WO 90/02549; and PCT W0 90/ 11753. Parenteral treatment With fat emulsions, hoWever, can have serious side effects, such as catheter obstruction, hyperlipemia, thrombopathy, fat overload syndrome, and fat embolism (Desrochers, et al.; J. Nutr. Biochem. 6:111—18 (1995)). It Would therefore be tremen dously bene?cial to develop high energy, Water soluble nutrients Which can be used for long-term intravenous feeding.

In principle, the ketone bodies R-3-hydroxybutyrate and acetoacetate, Which are natural constituents of human sera, could be used for intravenous feeding in lieu of fat emul sions. These compounds are good fuels for peripheral tissues, except during prolonged starvation and diabetic ketoacidosis, and are ultimately oxidiZed to carbon dioxide. Unfortunately, administration of these compounds in their acid form can cause vein irritation, and infusion of the compounds as sodium salts can result in a dangerous sodium overload (Desrochers, et al.; J. Nutr Bi0chem., 6:1 11—18 (1995)). To overcome these problems, researchers have explored the administration of R-3-hydroxybutyrate With other basic amino acid salts (Beylot et al.; Crit. Care Med. 22:1091—98 (1994); Lammerant, et al.,J. Mol. Cell. Cardiol. 17:421—33 (1985)). Such treatments, hoWever, may interfere With the transport of amino acids across the blood-brain barrier and/or harm patients With hepatic or renal patholo gies (Desrochers, et al.,J. Nutn Biochem. 6:111—18 (1995)). Others have described the use of sodium salts of 3-hydroxybutyric acid oligomers as nutrients, in order to decrease the ratio of salt to ketone body (Japanese Patent No. 94,321,778 to Hiraide, et al.).

Another approach utiliZing a ketone body as a nutrient focuses on the synthesis of a glycerol monoester of acetoacetate, Which is hydrolyZed in plasma and tissues to glycerol and acetoacetate (Birkhahn & Border, Am. J. Clin. Nutr. 3:436—41 (1978); Birkhahn, et al.; J. Nutr. 109:1168—74 (1979)). This composition Was ?rst to provide administration of large amounts of a ketone body Without a large sodium load.

Researchers also have explored using precursors to the ketone bodies. For example, R, S-1,3-butanediol is a Water soluble precursor, Which is metaboliZed in the liver to R, S-3-hydroxybutyrate (Desrochers, et al.; J. Nutr. Biochem. 6:111—18 (1995)). HoWever, the diol is unsuitable for use as an intravenous nutrient because it has a loW caloric density per osmol, and because its oxidation in the liver markedly increases the [NADH]/[NAD+] ratio, Which can induce alcoholic hypoglycemia. One effort to address these prob lems has focused on using an acetoacetate ester of R, S-1,3-butanediol, so that acetoacetate liberated by esterases can trap the reducing equivalents generated in the liver by the oxidation of the diol (Desrochers, et al.; J. Nutr Bio chem. 6:111—18 (1995)).


Exhibit 1003 Page 4


US 6,380,244 B2 3

Modulating ketone body levels also is useful in the production of animals for the meat industry. US. Pat. Nos. 4,329,359 and 4,423,072 to Stahly disclose feeding dihy droXyalkanols and triglycerides to pregnant soWs to improve the metabolic stability of neWborn pigs. These compositions function to increase the ketone body levels in the soW. The ketone bodies then are transferred across the placenta, providing a supplemental energy source to the developing fetus. PCT WO 98/41200 and PCT WO 98/41201 by British

Technology Group Ltd disclose the use of acetoacetate in combination With poly D-[3-hydroXybutyrate or esters or oligomers thereof, and/or a metabolic precursor or salt thereof in nutritional or therapeutic compositions to elevate the levels of ketone bodies in the blood for increasing cardiac ef?ciency, treatment of diabetes and insulin resistant states, and treatment of effects of neurodegenerative disor ders and epilepsy. Although these applications provide mechanisms by Which the ketone levels can be elevated for treatment of these disorders, the number of useful compo sition is limited to acetoacetate in combination With either a precursor of, or oligomer or ester of, D-[3-hydroXybutyrate.

It is therefore an object of the present invention to provide improved or alternative compositions for elevating ketone levels in the body of humans and other mammals, Which are suitable for oral or parenteral administration.

It is a further object of the present invention to provide compositions having better or longer bioavailability, or different metabolic products, and methods of use thereof for seiZure control, metabolic disease control, reduction of protein catabolism, appetite suppression, parenteral nutrition, increasing cardiac efficiency, treatment of diabetes, treatment of effects of neurodegenerative disorders or other conditions affecting or effected by ketone level in humans and other mammals.

SUMMARY OF THE INVENTION

Nutritional or therapeutic compositions are provided for increasing ketone body levels in the blood of mammals by providing a source of ketone bodies in the form of linear or cyclic oligomers and/or derivatives of 3-hydroXyacids. The 3-hydroXyacid can be in the form of a linear oligomer of 3-hydroXyacids other than linear homo-oligomers of 3-hydroXybutyric acid if administered in combination With acetoacetate, cyclic oligomers of 3-hydroXyacids, esters of the linear or cyclic oligomers, esters of 3-hydroXyacids other than 3-hydroXybutyric acid, and combinations thereof. An oligomer generally refers to a polymer of three or more hydroXyacids. Preferred 3-hydroXyacids include 3-hydroXybutyrate, 3-hydroXyvalerate, 3-hydroXyheXanoate, and 3-hydroXyheptanoate. Oligomers of odd-carbon number 3-hydroXyacids such as 3-hydroXyvalerate have advantages since they have a higher energy content than oligomers of 3-hydroXyacids having an even-number of carbons. The cyclic oligomers have advan tageous properties since they result in a sustained, and/or controlled, ketone blood level over a period of hours.

The compositions can be administered orally, for eXample, as a nutritional or dietary supplement, or intrave nously. Increasing blood ketone levels is useful for seiZure control, metabolic disease control, reduction of protein catabolism, appetite suppression, parenteral nutrition, increasing cardiac ef?ciency, treatment of diabetes and insu lin resistant states, and treatment of effects of neurodegen erative disorders and epilepsy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of plasma total ketone bodies (mn) from a dog given a single oral bolus of triolide at 5% of the daily

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4 caloric requirement, over time in minutes, for R-BHB (squares), acetoacetate (diamonds, control), and total ketone bodies (triangles).

DETAILED DESCRIPTION OF THE INVENTION

It Was discovered that certain hydroXyacids, derivatives, oligomers and esters thereof, can provide a source of ketone bodies to modulate ketone body levels in the blood of mammals, and that biologically produced polyhydroXyal kanoates are an eXcellent source for these hydroXyacids. These oligomers and/or derivatives of 3-hydroXyacids can be readily adapted to produce a variety of nutritional and therapeutic compositions, Without the draWbacks associated With knoWn methods and compositions for elevating ketone levels.

I. Nutritional and Therapeutic 3-HydroXyacids Compositions

The compositions include 3-hydroXyacids, linear or cyclic oligomers thereof, esters of the 3-hydroXyacids or oligomers, derivatives of 3-hydroXyacids, and combinations thereof. In one preferred embodiment, the compositions include the cyclic macrolide of R-3-hydroXyacids contain ing 3, 4, or 5 monomeric subunits. Preferred 3-hydroXyacids include 3-hydroXybutyric acid, 3-hydroXyvaleric acid, 3-hydroXyheXanoic acid and 3-hydroXyheptanoic acid. The preferred length of the oligomer must be such that the derivative has a suitable digestion rate for sustained release of monomer. In another preferred embodiment, the cyclic trimer (triolide) is used in a combination With other cyclic oligolides or linear esters and/or mixtures of both. The general formula for 3-hydroXyacids is:

0 R2 OH

Where: R1 is selected from hydrogen, methyl, alkyl, alkenyl, aryl,

arylalkyl, heteroalkyl, heteroaryl, thiol, disul?de, ether, thiolether, amine, amide, halogen,

R2 and R3 are independently selected from hydrogen, methyl, alkyl, alkenyl, aryl, arylalkyl, heteroalkyl, heteroaryl, thiol, disul?de, ether, thiolether, amine, amide, halogen, hydroXy, ester, nitrogen-substituted radicals, and/or oxygen-substituted radicals.

R4 is selected from hydrogen, alkyl, alkenyl, aryl, arylalkyl, heteroalkyl, heteroaryl, thiol, disul?de, ether, thiolether, amine, amide, halogen, hydroXy, ester, nitrogen substituted radicals, and/or oxygen-substituted radicals.

further, When R4 is not hydrogen or a halogen, R3 can be a direct bond to R4 and R4 can be methyl.

The folloWing de?nitions may be used through the speci ?cation. The term “alkyl” refers to C2_15 straight, branched or

cyclic alkyl groups. The term “alkenyl” refers to a branched or straight chain

C2—C77(15) hydrocarbon Which also comprises one or more carbon-carbon double bonds. The term “aryl” refers to a group a group containing one

or more aromatic rings. Aryl groups can be unsubstituted or substituted With substituents independently selected from alkyl, haloalkyl, alkoXy, amino, alkyl amino, dialkylamino, hydroXy, halo, and nitro.


Exhibit 1003 Page 5


US 6,380,244 B2 5

The term “arylalkyl” refers to an alkyl group (as de?ned above) to Which is appended an aryl group.

The term “heteroalkyl” refers to an alkyl group (as de?ned above) Wherein one or more of the carbon atoms is replaced With a non-carbon atom (such as, for example, oxygen, nitrogen, sulfur).

The term “heteroaryl” refers to a group containing one or more aromatic rings Wherein at least one of the atoms in an aromatic ring in not carbon. Heteroaryl groups can be unsubstituted or substituted With substituents independently selected from alkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo and nitro.

The term “thiol” refers to RSH Where R is alkyl, alkenyl, aryl, arylalkyl, heteroalkyl, or heteroaryl (as de?ned above).

The term “disul?de” refers to groups containing a sulfur sulfur bond.

The term “ether” refers to groups containing a C—O—C unit.

Hydroxyacid Oligomers In one preferred embodiment, the compositions include

linear oligomers of 3-hydroxyacids having from 5 to 10 carbon atoms. As used herein, the term “oligomer” means a polymer having a Weight average molecular Weight of less than about 2000 g/mol, preferably less than about 1000 g/mol, or having less than about 100 monomeric subunits. Representative examples include oligomers of 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, and combina tions thereof. As used herein, a homo-oligomer includes only one type of 3-hydroxyacid, While an oligomer can refer to either a homo-oligomer or hetero-oligomer including more than one type of 3-hydroxyacid.

In another preferred embodiment, the compositions include 3-hydroxyacids having an odd number of carbons, Which have a higher caloric value than 3-hydroxyacids having an even number of carbons. For example, oligomeric esters of 3-hydroxyvalerate (alone or mixed With other hydroxyalkanoates) can be used to deliver the odd numbered hydroxyacid 3-hydroxyvalerate.

In still another preferred embodiment, the compositions include cyclic oligomers of 3-hydroxyalkanoic acids or 3-hydroxyalkanoate oligomer esters, including 3-hydroxyacids of from 4 to 10 carbon atoms. The hydroxy acids are liberated as a result of digestion or metabolism of the ester form. By providing the hydroxyacids in ester form, these compositions can eliminate complications caused by delivery of the acid or salt forms of hydroxyalkanoic acids. As demonstrated by the folloWing examples, cyclic oli

gomers have the advantage that the ketone body levels remain elevated for a prolonged period of time of at least several hours after ingestion. For example, cyclic esters of 3-hydroxybutyrate, such as the triolide of 3-hydroxybutyrate, can provide sustained release of ketone bodies. SloW release provides a major advantage over prior art compositions, since the sloW release of monomers pro vides a more constant level of ketone bodies, such as 3-hydroxybutyrate, to the body over a prolonged period of time. This release pro?le reduces the frequency of doses required to maintain a speci?c ketone body concentration, Which is especially important during periods, such as during sleep, When it is dif?cult to administer the material.

DerivatiZed HydroxyAcids Since the family of PHAs contains a large variety of

hydroxyacids With varying side chain substituents, judicious selection of the type of 3-hydroxyacids provides a means to increase the caloric density on a per acid basis or to provide acids With odd number chain lengths. Preferred derivatives

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6 are Where the R groups on the formula shoWn above are ethyl or methyl.

Esters of 3-Hydroxyacids or Oligomers The compositions also can include esters of

3-hydroxyacids or esters of either linear or cyclic 3-hydroxyacid oligomers. In another preferred embodiment, the compositions include R-3-hydroxyalkanoate oligomers terminated With an ester linkage, for example, to 1,3 butanediol. The length of the oligomer preferably is such that the derivative has a solubility suitable for intravenous administration. The 1,3-butanediol may be coupled to the hydroxyacid oligomer by the primary alcohol, the secondary alcohol, and/or mixtures of both. FolloWing parenteral (e.g., intravenous) administration of the oligomer esters, the non R-3-hydroxyacid units should be readily tolerated and metaboliZed in the body after it is released from the oligomer derivative. The hydroxyacid oligomer also preferably is selected to

include desirable physical and nutritional properties, such as Water solubility and calori?c bene?ts.

Sources of the Hydroxyacid Compositions A useful source of hydroxyacids and hydroxyacid oligo

mers is the family of microbial storage polyesters, the polyhydroxyalkanoates, Which can be accumulated intrac ellularly by numerous microorganisms. Poly [(R)-3 hydroxyalkanoates] (PHAs) are biodegradable and biocom patible thermoplastic materials, produced from reneWable resources, With a broad range of industrial and biomedical applications (Williams & Peoples, CHEMTECH 26:38—44 (1996)).

In recent years, the PHA biopolymers have emerged from What Was originally considered to be a single homopolymer, poly-3-hydroxybutyrate (PHB), into a broad class of poly esters With different monomer compositions. To date around 100 different monomers have been incorporated into the PHA polymers (Steinbiichel & Valentin, FEMS Microbiol. Lett. 128:219—28 (1995)). As described herein, these natu rally occurring polyesters can be converted into derivatives suitable for nutritional and therapeutic uses. Methods for Making the Hydroxyacid Oligomers and

Derivatives Representative methods for preparing the hydroxyacid

oligomer derivatives described herein include direct degra dation of polyhydroxyalkanoates to oligomeric derivatives; ring-opening of cyclic oligomers of 3-hydroxyalkanoates; polymeriZation of hydroxyalkanoates or derivatives thereof; and, stepWise synthesis hydroxyalkanoate oligomers begin ning or ending With modi?cation of a terminal hydroxyal kanoate unit. Such syntheses can be readily carried out using methods knoWn in the art. In a preferred embodiment of the methods for synthesis of hydroxyacid oligomers terminated With an ester linkage to an alcohol, the process includes direct degradation of polyhydroxyalkanoate With the alco hol; ring-opening of a cyclic oligomer of hydroxyalkanoate With an alcohol; and, stepWise synthesis of hydroxyal kanoate oligomers beginning or ending With esteri?cation of a terminal hydroxyalkanoate unit by an alcohol. Such syn theses can be carried out using methods knoWn in the art.

Cyclic oligolides of (R)-3-hydroxybutyric acid can be prepared by a number of knoWn methods, Which are described, for example, in Seebach, et al.,Angew. Chem. Int. Eng. Ed., 4:434—35 (1992); Seebach, et al., Helv. Chim. Acta., 71:155—67 (1988); Seebach, et al., Helv. Chim. Acta. 72:1704—17 (1989); and Mueller, et al., Chimia 451376 (1991). These methods involve conversion from the bacterially-derived polyester, poly-(R)-3-hydroxybutyrate (PHB), or macrolide formation from the constituent acid


Exhibit 1003 Page 6


US 6,380,244 B2 7

(R)-3-hydroxybutyrate or esters thereof. The most direct route is degradation of PHB under acid catalyzed conditions to a mixture of linear oligomers and cyclic oligolides. Oligolides and oligomers can be isolated from the crude mixture via conventional Washing, extraction, and distilla tion steps to yield puri?ed materials.

II. Nutritional and Dietary Compositions

The compositions can be adapted for enteral or parenteral administration, for example, by combining the composition With the appropriate delivery vehicle. For enteral administration, the compositions can be added to food or drink, for example, as a dietary supplement. Alternatively, the compositions can be delivered parenterally, for example, by dissolving in a physiological saline solution for injection. Using genetic engineering techniques, plants can be engi neered to express the appropriate 3-hydroxyacids or oligo mers of 3-hydroxyacids. Suitable means and methods are described in WO97/15681 and PCT/US99/04999 by Metab olix.

The hydroxyacid formulations can be administered alone, in dry or poWdered form, in solution in a carrier such as Water, normal saline, or phosphate buffered saline, or mixed With other materials Which Will elevate blood ketones, such as free fatty acids, triglycerides alone or in combination With protein or carbohydrate. Traditional ketogenic diets, such as the diet recommended by the Marriott Corp. Health Care Services, Pediatric Diet Manual, Revised August 1987, contains from 3:1 to 4:1 g of fat for each g of combined carbohydrate and protein. Since the fat is metaboliZed to yield 3-hydroxyacid and acetoacetate, and desired levels are in the range of at least about 1 to 2 mM up to a maximum of about 7.5 mM (achieved during prolonged fasting of obese individuals), although ranges can be from 0.3 to 20 mM, the compositions containing the 3-hydroxyacids can be formulated to yield similar values to those of the traditional ketogenic diets, recogniZing that the yield Will be more ef?cient When the 3-hydroxy acids are administered directly.

These compositions can be mixed With meat or carbohydrate, as demonstrated in the examples, preferably maintaining an excess of 3-hydroxy acid relative to the amount of carbohydrate or protein.

III. Applications of the Compositions

The compositions described herein can readily be used in a variety of nutritional and therapeutic applications. One of skill in the art can readily select the appropriate hydroxyacid oligomer or derivative, as Well as amounts thereof, for administration. The particular composition used Will depend on the target ketone blood levels (required for a particular patient), as Well as the route and frequency of administra tion. In all cases, the digestion and metabolism of these compounds advantageously provides for the sloW release of ketone bodies.

Representative uses for the compositions described herein are provided beloW:

Using the hydroxyacid oligomer derivatives described herein, it is possible to sustain ketosis While overcoming draWbacks of the ketogenic diet. During normal digestion and metabolism of these compounds, ketone bodies (such as 3-hydroxybutyrate and acetoacetate) are released into the blood. The blood level of ketone bodies can be maintained at a level necessary to produce ketosis and reduce seiZures, Which for example, are associated With epilepsy.

The hydroxyacid oligomer derivatives described herein can also be administered to maintain the blood level of

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8 ketone bodies at a level necessary to reduce protein catabo lism and provide appetite suppression, to aid in Weight loss. Thus, addition of these ketogenic compounds to the diet functions to mimic some effects of a ketogenic diet. Pre ferred blood levels to be obtained are in the range of 2 to 3 mM 3-hydroxyacid. The caloric value of the ketone bodies is approximately 1.5 g of ketone/e g of fat. The hydroxyacid oligomer derivatives described herein can be administered parenterally to a mammal, typically a human, to maintain the blood level of ketone bodies at a level necessary to provide nutrients to the body. The compositions should be particu larly useful to patients Who are unable to digest food orally or otherWise require total parenteral nutrition. The compo sitions can be formulated to provide high energy, Water soluble nutrients, suitable for long-term intravenous feeding. The hydroxyacid oligomer derivatives described herein

can be administered to maintain the blood level of ketone bodies at a level necessary to overcome de?ciencies caused by metabolic disorders, such as insulin de?ciencies or insu lin resistant states. The hydroxyacid oligomer derivatives described herein can be administered to maintain the blood level of ketone bodies at a level necessary to treat insulin resistance in Which the normal insulin signaling pathWays is disordered and in conditions in Which cardiac (hydraulic Work) ef?ciency is reduced due to metabolic reasons, as described in PCT WO 98/41200 and PCT WO 98/41201, Which are incorporated herein by reference.

The hydroxyacid oligomer derivatives described herein can also be administered to a mammal, typically a human, to maintain the blood level of ketone bodies at a level neces sary to treat a variety of neurodegenerative diseases, par ticularly those involving neurotoxic plaques, such as amy loid plaques. Examples of neurodegenerative diseases Which the compositions described herein may aid in treating include AlZheimer’s disease, fronto-temperal degeneration associated With Pick’s disease, vascular dementia, senile dementia of LeWy body type, dementia of Parkinsonism With frontal atrophy, progressive supranuclear palsy and corticobasal degeneration, DoWns syndrome associated AlZheimer’s, myasthenia gravis, and muscular dystrophy. See, for example, PCT WO 98/41200 and PCT WO 98/41201 by British Technology Group, Ltd., Which dis closes that elevated levels of ketone bodies can improve nerve cell function and groWth, at least in part by enhancing cellular energy production. The preferred ketone blood level for treatment of neurodegenerative disorders is greater than for diet or seiZures, more typically in the range of 7.5 mM.

Supplemental Energy Source for Livestock The hydroxyacid oligomer derivatives described herein

can be administered to animals, such as pigs, particularly pregnant soWs, to provide a supplemental energy source and to possibly improve the metabolic stability of neWborn animals. For example, by increasing the ketone body levels in a pregnant soW, ketone bodies are transferred across the placenta, providing a supplemental energy source to the developing fetus. The compositions and methods described herein are fur

ther described by the folloWing non-limiting examples.

EXAMPLE 1

Preparation of (R,R,R)-4,8,12-Trimethyl-1,5,9 Trioxadodeca-2,6,10-Trione or Triolide of (R)-3

Hydroxybutyric Acid

PHB (20 g) Was dissolved in dioxane (700 mL) containing p-toluene sulfonic acid monohydrate (4 g) and concentrated


Exhibit 1003 Page 7


US 6,380,244 B2 9

sulfuric acid (5 mL). After re?uxing for 4 days, the reaction had achieved 40% conversion to the triolide, as determined by gas chromatography (GC) analysis (Riis & Mai, J. Chromatography 4:285—89 (1988)). The reaction mixture Was cooled to room temperature and quenched With satu rated sodium bicarbonate solution. Dioxane Was removed by rotary evaporation. The residue Was extracted into ethyl acetate (400 mL), Washed With brine, and concentrated to an oil. Vacuum distillation yielded puri?ed triolide (4 g).

EXAMPLE 2

Use of 3-Hydroxyalkanoic Acid Oligolide for Enteral Nutrition

Amongrel dog (21 kg) Was fasted overnight and given an oral bolus of triolide (R,R,R)-4,8,12-trimethyl-1,5,9 trioxadodeca-2,6,10-trione, 10 g) in gelatin. This amount of triolide is equivalent to 5% of the daily caloric requirement. Blood Was sampled at 0, 15, 30, 45, and 60 minutes and every half hour thereafter for a total of six hours.

The blood samples Were analyZed for glucose via enZy matic assay, and for acetoacetate and 3-hydroxybutyrate via GC-mass spectrometry (GC-MS) assay. As shoWn by FIG. 1, Within 90 minutes, the blood concentrations of 3-hydroxybutyrate and acetoacetate reached 0.3 and 0.05 mM, and the total ketone bodies in the blood Were 0.36 mM. After the fourth hour, the total ketone body concentration remained elevated at 0.24 mM. Glucose concentration in the blood dropped from 6.5 mM to 5 mM during the experiment. These results shoW that an oral dose of a

3-hydroxyalkanoic oligolide can elevate the ketone body concentration in the blood. A signi?cant ?nding is that the ketone body concentration remains elevated several hours after administration, demonstrating that the triolide is useful for the sloW release of ketone bodies.

EXAMPLE 3

Use of 3-Hydroxyalkanoic Acid Oligolide for Enteral Nutrition

A mongrel dog (25.5 kg) Was fasted overnight and fed a mixture of meat (111 g) and triolide ((R,R,R)-4,8,12 trimethyl-1,5,9-trioxadodeca-2,6,10-trione, 23.5 g). This amount of triolide is equivalent to 10% of the daily caloric requirement. Identical amounts of meat and triolide Were given at 0, 120, 360 and 540 minutes. Blood Was sampled at regular intervals for 12 hours.

Throughout the experiment, the dog exhibited no signs of distress; unusual behavior; or abnormal bodily functions, such as diarrhea, nausea, vomiting, or frequent urination. The blood samples Were analyZed for acetoacetate and 3-hydroxybutyrate. Within 30 minutes, the concentrations of 3-hydroxybutyrate and acetoacetate reached 0.85 and 0.15 mM, respectively. The total ketone bodies in the blood Were 1.0 mM. After the third feeding of triolide, total ketone body concentration remained elevated and steady at about 0.6 mM. Glucose concentration in the blood remained Within the normal range of 3.1 to 5.9 mM. Other clinical chemistry pro?les remained normal throughout the experiment. By the next morning, the ketone body concentration in the blood had returned to the normal value of 0.02 mM.

These results shoW that triolide is digested by the dog, resulting in a sustained increased in blood ketone body concentration. Signi?cantly, the ketone body concentration is Within the range achieved by the ketogenic diet used in the nutritional treatment of intractable epilepsy. Furthermore,

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10 the triolide Was found to be Well accepted by the dog, Which shoWed no sign of distress and no perturbation of clinical chemistry parameters. These results further demonstrate that the triolide is useful for the sloW release of ketone bodies.

EXAMPLE 4

Synthesis of an Alkyl Ester Terminated 3 Hydroxyalkanoate Oligomer

Oligomeric (R)-3-hydroxybutyrate Was prepared via con densation reaction of methyl (R)-3-hydroxybutyrate. Speci?cally, methyl (R)-3-hydroxybutyrate (250 pl) Was heated With dibutyltin oxide (2 mg) at 110° C. for 72 hours. The reaction vial Was left open to the atmosphere to permit removal of methanol. After cooling, the reaction formed a crystalline, White solid material, Which Was Washed With methanol and alloWed to air dry. NMR analysis shoWed formation of oligomeric (R)-3-hydroxybutyrate having an approximate molecular Weight of 1,700 g/mol. Gel perme ation chromatography (GPC) analysis con?rmed the MW at about 2,000 g/mol. NMR analysis also demonstrated the presence of a terminal methyl ester.

EXAMPLE 5

Synthesis of a Butanediol Ester Terminated 3 Hydroxyalkanoate Oligomer

Oligomeric (R)-3-hydroxybutyrate butanediol ester Was prepared via controlled transesteri?cation of the microbial polyester, poly[(R)-3-hydroxybutyrate] With 1,3-butanediol. Speci?cally, PHB (10 g, MW 600,000) Was dissolved With heating in 200 mL of dioxane and 1,3-Butanediol (2.1 mL). After dissolution, the reaction mixture Was cooled and concentrated sulfuric acid (1 mL) Was slowly added. The reaction mixture Was heated at re?ux for 48 hours. Samples Were removed periodically and precipitated into Water. After 6 hours, 95% of the product Was recovered, having a MW of 4,300 Da according to GPC analysis. After 45 hours, 52% of the product Was recovered, having a MW of 2,000 Da according to GPC analysis. NMR analysis demonstrated a 3-hydroxybutyrate oligomer of approximately 1,000 g/mol and demonstrated the presence of a terminal 1,3-butanediol ester.

EXAMPLE 6

Synthesis of a 3-Hydroxyalkanoate Oligomer

Oligomeric (R)-3-hydroxybutyrate Was prepared via con trolled hydrolysis of the microbial polyester, poly[(R)-3 hydroxybutyrate]. Speci?cally, PHB (150 g) Was dissolved With heating in 2 L of glacial acetic acid. Water (350 ml) Was sloWly added to the viscous solution to form a single phase. The reaction mixture Was heated at re?ux for 18 hours. After cooling to about 55° C., the mixture Was poured With rapid stirring into 9 L of Water. The White precipitate Was collected and Washed With Water to yield 92 g of 3-hydroxybutyrate oligomer after drying. NMR analysis demonstrated a 3-hydroxybutyrate oligomer of approximately 1,000 g/mol, With no terminal crotoniZation. GPC analysis con?rmed a molecular Weight of 1,000 g/mol. A similar process Was used, but With the addition of

hydrochloric acid, to produce 3-hydroxybutyrate oligomer of loWer molecular mass (approximately 200 g/mol). Oli gomeric (R)-3-hydroxyvalerate can be prepared using the same approach from poly(3-hydroxyvalerate) Which can be obtained by fermentation using Chromobacter violaceum (Steinbuchel, et. al.,Appl. Microbiol. Biotechnol. 39:443—49 (1993)).


Exhibit 1003 Page 8


US 6,380,244 B2 11

EXAMPLE 7

Use of 3-Hydroxyalkanoic Oligomers for Enteral Nutrition

Sprague-DaWley rats Were fed commercial rat chow for 10 days and then switched to a control diet containing 75% of the calories from starch, 20% as casein, and 5% as polyunsaturated oil, plus mineral mix and liver extract supplements. After 15 days, tWo groups of rats Were fed an experimental diet containing 25% of the calories from a 3-hydroxybutyrate oligomer. TWo different oligomers, short and medium, Were used With molecular masses of either the 200 g/mol or 1000 g/mol, respectively. A control group Was kept on the control diet Without oligomer.

The Weight of each rat Was measured daily. Urine samples Were collected daily and analyZed for 3-hydroxybutyrate by GC-MS. After 5 days on the experimental diet, the rats Were euthaniZed, and a blood sample Was collected and analyZed for 3-hydroxybutyrate and acetoacetate by GC-MS.

The Weight of the control group increased uniformly throughout the experiment, as did the Weight of rats fed the experimental diet containing the medium HB oligomer. The Weight of rats fed the experimental diet containing short HB oligomer decreased slightly While on the experimental diet.

The concentration of ketone bodies in the rat blood plasma collected at time of euthanasia Was measured by GC-MS. The control group shoWed normal concentrations of 3-hydroxybutyrate and acetoacetate, 0.07 and 0.02 mM, respectively. Rats fed the short HB oligomer had 3-hydroxybutyrate and acetoacetate concentrations of 0.65 and 0.05 mM, respectively, While rats fed the medium oligomer had concentrations of 0.15 and 0.04 mM, respec tively. These result shoW that the rats fed 3-hydroxybutyrate oligomers had increased levels of ketone bodies in their blood.

The concentration of 3-hydroxybutyrate in the urine of rats fed short and medium oligomers Was determined by GC-MS to be approximately 3.5 and 1.0 mM, respectively. 3-Hydroxybutyrate Was undetectable in the urine of the control rats. These results shoW that an oral dose of 3-hydroxybutyrate oligomers elevates the ketone body con centration in the blood and in the urine.

Modi?cations and variations of the present invention Will be obvious to those of skill in the art from the foregoing detailed description. Such modi?cations and variations are intended to come Within the scope of the folloWing claims. We claim: 1. A method of modulating blood ketone levels in a

mammal, comprising administering to a mammal in Which said modulation is intended a nutritional or therapeutic dietary composition comprising an effective and biocompat ible amount of a 3-hydroxyacid derivative selected from the group consisting of linear oligomers of 3-hydroxyacids, other than linear homo-oligomers of 3-hydroxybutyric acid; cyclic oligomers of 3-hydroxyacids; esters of

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12 3-hydroxyacids, other than 3-hydroxybutyric acid or linear homo-oligomers of 3-hydroxybutyric acid; esters of linear or cyclic 3-hydroxyacid oligomers other than linear homo oligomers of 3-hydroxybutyric acid; and combinations thereof; Wherein When the 3-hydroxyacid derivative is a linear oligomer of a 3-hydroxyacid or an ester of a 3-hydroxyacid, said 3-hydroxyacid derivative is adminis tered in combination With acetoacetate.

2. The method of claim 1 Wherein the blood ketone level is effective to control seiZures.

3. The method of claim 1 Wherein the blood ketone level is effective to control metabolic disorders of ketone body synthesis and metabolism.

4. The method of claim 1 Wherein the blood ketone level is effective to reduce protein catabolism in and/or suppress the appetite of the mammal.

5. The method of claim 1, Wherein the blood ketone level is effective to increase the cardiac efficiency of the mammal.

6. The method of claim 1, Wherein the blood ketone level is effective to treat diseases selected from the group con sisting of diabetes and other insulin resistant states in Which the normal insulin signaling pathWays are disordered, neu rodegenerative diseases, and epilepsy.

7. The method of claim 1, Wherein the mammal is a human or livestock animal.

8. The method of claim 1, Wherein the composition is administered parenterally.

9. The method of claim 1, Wherein the composition is administered orally as a dietary or nutritional composition.

10. A method of treating a neurodegenerative disorder in a mammal in need thereof, comprising administering to the mammal a nutritional or therapeutic dietary composition comprising an effective and biocompatible amount of a 3-hydroxyacid derivative selected from the group consisting of linear oligomers of 3-hydroxyacids other than linear homo-oligomers of 3-hydroxybutyric acid in combination With acetoacetate; cyclic oligomers of 3-hydroxyacids; esters of 3-hydroxyacids, other than 3-hydroxybutyric acid or linear homo-oligomers of 3-hydroxybutyric acid in com bination With acetoacetate; and esters of 3-hydroxyacid linear or cyclic oligomers other than linear homo-oligomers of 3-hydroxybutyric acid in combination With acetoacetate; and combinations thereof.

11. The method of claim 10, Wherein, folloWing said administration, the resulting blood ketone level is effective to increase the cardiac efficiency of the mammal.

12. The method of claim 10, Wherein the neurodegenera tive disorder is selected from the group consisting of AlZhe imer’s disease, fronto-temporal degeneration associated With Pick’s disease, vascular dementia, senile dementia of the LeWy body type, dementia of Parkinsonism With frontal atrophy, progressive supranuclear palsy and corticobasal degeneration, DoWn’s syndrome associated With AlZheimer’s, myasthenia gravis, and muscular dystrophy.


Exhibit 1003 Page 9


EXHIBIT D


1. Physiol Behav. 1995 Jul;58(1):1-7.

Peripheral 3-hydroxybutyrate and food intake in a model of dietary-fat inducedobesity: effect of vagotomy.

Fisler JS(1), Egawa M, Bray GA.

Author information: (1)Department of Medicine, School of Medicine, University of Southern California,Los Angeles 90033, USA.

We have examined the effect of peripheral 3-hydroxybutyrate injections on foodintake and the contribution of the vagus nerve in the resistance to dietaryfat-induced obesity in a rodent model. S 5B/Pl rats, which are resistant todietary-fat induced obesity, and Osborne-Mendel rats, which are sensitive, wereadapted to reverse light cycle. Food intake was measured for 24 h following theinjection of 3-hydroxybutyrate, lactate, or glycerol (all 5 mMol/kg0.75, SC) atthe onset of dark. Three-hydroxybutyrate reduced food intake (p < 0.0001) in S5B/Pl rats only. Lactate reduced food intake slightly (p < 0.009) in both strainsand glycerol had no effect on food intake. In a second experiment, S 5B/Pl andOsborne-Mendel rats were adapted to a high-fat diet and were then subjected toeither selective hepatic vagotomy or sham operation. Vagotomy had no effect onweight gain of Osborne-Mendel rats but allowed weight gain in S 5B/Pl rats (p <0.0001). Even in vagotomized S 5B/Pl rats, however, blood 3-hydroxybutyratelevels were inversely associated (r = -0.50) with food intake. These data suggestthat the hepatic vagus nerve may contribute to the resistance of S 5B/Pl rats to dietary-fat induced obesity, but the data do not rule out a strictly central rolefor the regulation of food intake by 3-hydroxybutyrate in this strain.

PMID: 7667404 [PubMed - indexed for MEDLINE]


Exhibit 1014 Page 1


EXHIBIT E


R E G U L A T I O N OF F O O D I N T A K E IN R U M I N A N T S . 4. E F F E C T OF A C E T A T E , P R O P I O N A T E , B U T Y R A T E , A N D G L U C O S E ON

V O L U N T A R Y F O O D I N T A K E IN D A I R Y C A T T L E 1, 2, a

£2. L. SIMKINS, JR., ~ J. W. SUTTIE, aND B. R. BAUMGARDT Departments of Dairy Science and Biochemistry, University of Wisconsin, Madison

ABSTRACT

Three dairy heifers received intraperitoneal infusions (one liter) of the following solutions in a 3 × 3 Latin-square-designed trial: (I) 0.9% sodimn chloride (control); ( I I ) 5.4% glucose; ( I I I ) 25% glucose. The heifers were fed a contpletely pelleted ration (60% alfalfa meal, 40% ground corn) ad libitum once daily for a 3-hr period, and the infusions were given immediately before food was offered. The food eonsmnption for Treatments I, II , and I I I was 62.6, 71.6, and 72.1 g/BWk~ '~, and jugular blood glucose concentration 1 hr after feeding was 50.5, 57.2, and 69.2 mg per 100 ml, respectively. I t was con- eluded that the glueostatie mechanism for food intake regulation is not important in ruminants.

Jsocalorie amounts of acetate, propionate, butyrate, and a VFA mixture (60, 20, and 20% of the calories from acetate, propionate, and butyrate, respectively) were infused intrarmninally into two cows fed a pelleted ration (75% alfalfa meal, 25% ground corn) and two cows fed a good-quality alfalfa hay. The rations were fed ad libitum from 10 AM to 1 P~ and from 4:30 PS~ to 8 a~. Three consecutive days of water infusion (control) preceded three days of metabolite infusion. The infusions were given from 8 A~f to 1 e~ each day, and the level of metabolite infused way equivalent to 15% of the estimated digestible energy requirement of the animals. The infusion of acetate, propionate, and the VFA mixture reduced (P < .05) the voluntary consumption of the pelleted ration during the 10 a_~ to 1 e~ feeding interval. Propionate and butyrate infusion caused a significant (P <:..05) reduction in the consumption of hay in the 10 A~r to 1 e_~ feeding interval. I t way concluded that acetate, propionate, and butyrate can act as satiety signal cempounds in the regulation of food intake. Blood and tureen metabolites were examined, and the possible mechanism of action of these metabolites in causing satiation was discussed.

l~eeent work (9) indicates tha~ ruminants will adjust their voluntary food intake in relation to physiological denmnd for energy if rmnen load does not limit their consumption. The mechanisms involved in food intake regulation are quite complex and encompass be-

Received for publication August 14, 1965. Published with the approval of the director of

the Wisconsin Agricultural Experiment Station. '~ Data presented are taken from a thesis pre-

sented by K. L. Simkins, Jr., in partial fulfillment of the requirements for the Ph.D. degree, Univer- sity of Wisconsin, June, 1965.

s This investigation was supported in part by a U.S. Public Health Serviee Grant, AM 07652-02, and in part by the ]~eseareh Committee of the Graduate School from funds supplied by the Wis- consin Alumni Research Foundation.

~Current address: Agricultural Research Cen- ter, American Cyanamid Company, Princeton, New Jersey.

havioral, metabolic, and neurologic areas of investigation. Several theories have been pro- posed to account for food intake regulation in monogastric animals (8), but the applicability of these theories to ruminants has not been extensively investigated.

In a previous paper (12) some of the metabolic events that accompany and possibly in- fluence hunger and satiety were exmnined. The objective of the studies to be reported in this paper was to determine the effect of some of the biochemical eonconfitants of hunger and satiety on food intake. In particular, the effects of glucose, acetate, propion~te, and butyrate on food intake were investigated.

E X P E R I S [ E N T A L PROCEDURE

Experiment 1. The effect of intraperitoneal glucose infusions on food intake was investigated in dairy heifers. A pelleted ration con-

1635


Exhibit 1018 Page 1


1636 K.L. SI~¢[KINS, JR., J. W. SUTTIE, AND B. R. BAU.~,IGARDT

taining 60% a l fa l fa meal and 40% ground corn was fed ad libitum to three Holstein heifers accustomed to being fed once daily f rom 8:30 A~ to 11:30 _~. A 3 × 3 Latin-square-designed trial was conducted and the three treatments were ( I ) 0.9% sodimn chloride, ( I I ) 5.4% glucose, and ( I I I ) 25% glucose. One l i ter of these solutions was infused intraper i toneal ly immediately before the animals were fed. The infusions were given through a 16-gauge, 2-hl. sterilized needle inserted through the body wall on the left side of the animal into the peri toneal cavity. A t least three days were allowed between t reatment periods.

Blood samples were taken f rom the jugular vein at 1 and 3 hr a f te r the infusions had been given. Blood sugar was determined, using glucose oxidase ( ]5) , and blood acetate was determined as previously described (12).

The animals were weighed at 11:30 A~ on three consecutive days before the experiment began. Each day the animals were turned out for exercise from 1 P~ to 2:30 P~. Wa te r and trace-mineralized salt were available at all times.

The data were analyzed statistically according to the procedures outlined by Steel and Torrie (:13) for a 3 × 3 Latin-square design.

Experiment 2. The effect of an in t raruminal

infusion of acetate, propionate, butyrate, and a V F A mixture (60, 20, and 20% of the calories f rom acetate, propionate, and butyrate, respectively) was investigated, using mature, rumen-fistulated dairy animals. Two animals (190J and 411H) were fed a pelleted rat ion (75% a l fa l fa meal, 25% ground corn), and two aninmls (184J and l102H) were fed a long-hay rat ion (good-quali ty a l fa l fa ) . The rations were fed ad libitmn from 10 :xyi to 1 P~ and f rom 4:30 t '~ to 8 .~t each day. Wa te r and trace- mineralized salt were available to the animals at all times. The cows were turned out each day for exercise f rom 1 e~[ to 2:30 P~[.

Isocalorie amounts of acetic acid, propionie acid, butyric acid, and the V F A mixture were infused. The acids were neutralized to p H 6.0 before infusion with an equilmolar mixture of Na0H and KOH. The level of metabolite infused was equivalent to :15% of the animal's estimated digestible energy requirement (II). A description of the aninmls and their estimated total digestible energy (DE) requirement is given in Table I.

The experimental design consisted of four t r ials ; the t reatment imposed in each trial is shown in Table 2. Three consecutive days of water (control) infusion preceded each three-

TABLE 1 Energy requirements of cows used in Experiment 2

Cow Weight D.E. re-

Description quirement " (kg)

190 Jersey 470 411 Holstein 617 184 Jersey 537

1102 Holstein 520

(therms) Ovariectomized, nonlactating 14.5 Nonpreguant, milking 18 lb daily 29.5 Ovariectomized, nonlactating 16.4 Six months pregnant, nonlactating 20.9

Estimated from National Research Council Publication 464 (1958).

TABLE 2 Experimental design and amount of VFA infused

Amount infused

Trial Days Treatment 190J 411H 184J 1102H (g)

I 3 Control 3 Propionie 439 893 496 632

I I 3 Control 3 Butyric 365 744 413 527

III 3 Control 3 VFA Mixture

Acetic 374 761 423 539 Propionlc 88 179 99 127 Butyric 73 149 83 105

IV 3 Control 3 Acetic 624 1269 705 899

a Cows 190J and 411H were fed pellets, whereas Cows 184J and 1102H were fed hay.


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R E G U L A T I N G FOOD I N T A K E IN RU~fINANTS 1637

day treatment infusion. The infusions began at 8 A~ and continued until 1 P~t each day. Four liters of solution were infused at constant rate (13.4 ml/min), using Beckman model 746 solution metering pumps. After each trial, three days were allowed for readjustment. During the readjustment period, intakes were recorded but nothing was infused.

Blood and rumen samples were taken on the first day of the control infusion and the last day of the metabolite infusion. The blood was taken from the jugular vein, and the tureen sample was obtained via the rumen fistula before and 2, 5, and 8 hr after the infusion began. The analytical procedures for blood and rumen metabolites were described previously (12). Ru- nlen p i t was determined on the 20-ml aliquot of rumen liquor used for VFA analysis.

In the statistical analysis, each trial was analyzed as a separate experiment, the results of the control infusion being compared to the treatment immediately following it. For the analysis of the food intake data, the fixed model of a factorial analysis of variance was employed (13). A random model for the factorial analysis of variance was used in analyzing the blood and rumen data.

t~ESULTS AND DISCUSSION

Experiment 1. The body weights (BW) of the three heifers were 385, 336, and 389 kg. The average consumption of pellets was 62.6, 71.6, and 72.1 g / B W ~ "~ control, 5.4% glucose, and 25% glucose infusions, respectively (Table 3). These differences were not statistically significant (P < .05). The effect of the intraperitoneal infusions on blood glucose and acetate concentrations is shown in Table 3. Blood glucose was increased by the intraperitoneal

glucose infusions; however, only the difference between the saline infusion and the 25% glueose infusion was statistically significant (P < .01). Blood acetate was similar on the three treatments, and the increase at 3 hr after feeding un- doubtedly reflects the fermentation of food in the rumen.

According to the glucostatie theory for food intake regulation in monogastric animals (8), an increase in glucose utilization in the ventro- medial nuclei of the hypothalamus (and perhaps other central and peripheral areas) causes satiety. Several workers (4, 5, 7) have shown that intravenous injections of glucose do not depress food intake in ruminants. In the present experiment, an intraperitoneal infusion of glueose was chosen, to simulate glucose absorption from the gastrointestinal tract. According to the work of Bergman (3), glucose utilization rate in ruminants tends to be proportional to the blood glucose concentration. Since the average blood glucose concentration was increased 18.7 mg per 100 ml by the 25% glucose infusion at 1 hr after feeding, there probably was an increased glucose utilization as a result of this treatment. However, there was no depression in food intake as a result of the increased glucose availability. I t was concluded that the glueo- static mechanism for food intake regulation is not important in ruminants. I t might be ex- pected that glucose would not be an important satiety signal compound in ruminants, because of their relatively low blood sugar levels, rela- tNe resistance to insulin, and lack of an ali- menta l , hyperglycemia following food intake.

Experiment 2. The effects of the various volatile fatty acid infusions on the voluntary consumption of pellets by two cows are shown in Table 4. The infusions of propionate and the

TABLE 3 Effect of intraperitoneal glucose infusions on food intake, blood glucose, and blood acetate

in dairy heifers Treatment

l~esponse 0.9% NaC1 5.4% Glucose 25% Glucose Food intake (g/BW~,g "75) 62.6 71.6 72.1

Blood glucose (rag~200 ml) 1 hr after infusion 50.5 57.2 69.2* 3 hr after infusion 47.7 58.7 ~ 59.1"

Blood acetate (rag~200 ml) 1 hr after infusion 2.66 2.74 2.50 3 hr after infusion 3.16 3.95 3.61

* Difference between 0.9% NaC1 and 25% glucose was significant at the 1% level of probability.

Difference between 0.9% NaCl and 5.4% glucose and the difference between the 0.9% NaC1 and 25% glucose approached significance at the 5% level of probability.


Exhibit 1018 Page 3


1638 K. L. S]MKINS, JR., J. W. SUTTIE, AND B. R. BAUMGARDT

TABLE 4 Effect of volatile fat ty acid infusions on pellet consumption

Trial

Dally feeding interval

10 AM 4 : 3 0 t ,m to to

Infused 1 I'M 8 AM Total

Pellets consumed ( g / B W ' 7 ~ ) - I Water 22.3 51.6 73.5

Propionate 9.7* 57.2 66.8

I I Water 28.7 52.4 81.1 Butyrate 22.5 56.6 79.1

I I I Water 28.0 57.8 85.5 YFA Mixture 19.32 57.7 76.9

IV Water 27.0 53.4 80.5 Acetate 8.72~ 47.9 56.6 ~

Difference between control (water) and treated observations (P < .05).

~* Statistically significant difference (P < .01).

was statistically significant

¥ F A mixture reduced (P < .05) the voluntary food intake by these cows. Acetate infusion resulted in a highly significant (P < .01) reduction in food intake. The infusion of butyrate depressed consumption, but the decrease was not statistically significant at the 5% probabil i ty level. The depressions in food intake occurred only during the 10 A~ to 1 P~ feeding interval when the V F A were being infused, and there was no carry-over effect f rom any of these infusions.

The infusion of propionate and butyra te significantly reduced the voluntary consumption of long hay by two cows (Table 5). Acetate and the V F A mixture infusions resulted in slight depressions in long-hay intake, but the decrease was not statist ically significant (P < .05). When cows were fed the long-hay ration,

i t is possible that rumen load was l imiting their intake. However , i t is doubtful that rumen load was l imit ing the voluntary consumption of the pelleted ration.

In general, all four of the t reatments tended to depress voluntary consumption. I t is clear that the animals, in some manner, monitored the t reatments infused. The obvious question raised by these experiments is, How did acetate, propionate, and butyra te act as satiety signal compounds in causing the reduction in food intake? Blood and rumen metabolites were examined, to see what compounds might be responsible for the decreased intake.

The effect of the propionate infusion on blood and rumen metabolites is shown in Table 6. The decreased food intake that occurred during the propiona te infusion was associated with an

TABLE 5 Effect of volatile fa t ty acid infusions on hay consumption

Daily feeding interval

10 AM 4:30 P~ to to

Trial Infused 1 ~'M 8 AM Total

Pellets consumed ( g / B W "7~) I Water 33.7 70.2 103.8

Propionate 23.8 ~ 72.8 96.4

I I Water 34.9 68.7 103.6 Butyrate 27.52 66.4 93.6

I I I Wa~er 25.6 73.5 99.0 VFA Mixture 23.5 66.4 89.9

IV Water 29.7 71.4 101.1 Acetate 25.6 73.1 98.7

Difference between control (water) and treated observations is statistically significant (P < .05).

~ Statistically significant difference (P ~ .01).


Exhibit 1018 Page 4


REGULATING FOOD INTAKE IN RUMINANTS

TABLE 6 Effect of propionate infusion on blood and rumen metabolites '~

1639

Pelleted ration Hay ration

Control Propionate Control Propionate Measurement infusion infusion infusion infusion

Blood Sugar (mg/lO0 ~l) 53.9 64.0** 53.3 57.4* Ketones (mg/lO0 cnl) 1.51 1.63 1.56 1.59 Total VFA (mg/lO0 mr) 4.39 3.34 5.48 3.89 Acetate (% of total) 94.6 91.0 97.3 92.1"* Propionate (% of total) 3.9 7.0 1.6 6.6 ~*

Rumen To~al VFA (mg/lO0 ml) 701 836 639 930** Acetate (% of total) 54.7 41.4 ~* 66.4 44.6** Propionate (% of total) 33.2 48.5** 18.6 43.2** Butyrate (% of total) 8.9 6.3** 11.0 7.7** Isobutyrate (% of total) 1.3 1.7"* 1.5 1.8"* Isovalerate (% of total) 0.6 1.4" 1.7 1.6 Valerate (% of total) 1.5 0.9** 0.9 1.1"* pH 6.03 6.64** 6.60 6.92**

Values represent the average of the samples taken before and at 2, 5, and 8 hr after infusion began.

* Propionate infusion values are significantly different from control (P < .05). **Propionate infusion values are signlficant]y different from control (P < .01).

increased blood sugar concentrat ion, increased blood and rumen propionate , and an increased rumen p H . F r o m the results in E x p e r i m e n t 1, it would appea r that the increased blood sugar was not responsible fo r the depression in food intake. Dowden and Jacobson (4) suggested that there could be chemoreceptors sensitive to increased blood propionate , and the increased blood prop iona te observed might have been causing the food intake depression in this trial.

Another explanat ion could be that there are chemoreceptors in the rumen wall, por ta l system, or liver that responded to the increased prop iona te concentrat ion to cause sat iety in these animals. I t is also possible that the heat released dur ing the assimilation of the infused p rop iona te was responsible for the food intake depression via a thermostat ic mechanism.

The effect of the butyra te infusion on blood and rumen metabolites is shown in Table 7.

TABLE 7 Effect of butyrate infusion on blood and rumen metabolizes '~


Control Butyrate Control Butyrate Measurement infusion infusion infusion infusion

Blood Sugar (mg/lO0 ~nl) 48.0 42.5* 46.5 41.3" Ketones (m#/lO0 ~nl) 1.24 4.11 *~ 1.55 5.91 *~ Total Vt~A (m#/190 ml) 3.76 4.38 3.76 4.98* Acetate (% of total) 90.5 82.4 ~ 94.2 87.2 ~* Propionate (% of total) 6.4 6.6 3.8 5.1 Butyrate (% of total) 2.2 4.3 1.0 3.4 *~

R umen To~al VFA (~ng/lO0 ml) 823 776 569 799** Acetate (% of total) 54.1 41.7 "~ 65.3 50.8 *~ Propionate (% of total) 32.4 18.4 ~~ 20.2 14.5"* Butyrate (% of total) 10.3 37.2 ~* 10.8 31.1"* Isobutyrate (% of total) 1.1 1.0 1.6 1.2 ~* Isovalerate (% of total) 0.8 1.0 1.4 1.4 Valerate (% of total) 1.5 0.9** 0.8 0.8 pH 5.90 6.27 ++ 6.81 6.70

aVMues represent the average of the samples taken before and at 2, 5, and 8 hr after infusion began.

* Butyrate infusion values are significantly different from control (P < .05). *~ Butyrate infusion values are significantly different from control (P < .01).


Exhibit 1018 Page 5


1640 K. L. SIMKINS, JR., J. W. SUTTIE, AND B. R. BAUMGARDT

Blood sugar concentration was decreased (P < .05) and blood ketones were increased (P < .01). I t is well established that the metabolism of butyra te by the rumen epithelium and l iver results in the production of ketone bodies. Ap- parent ly, the ketone bodies cause an increased insulin secretion (6), which could be responsible for the depression in blood sugar. The depression in food intake that occurred during the butyrate infusion could have been caused by the increased ketones via a chemostatic mechanism. Perhaps the heat released dur ing the metabolism of butyrate by the rumen wall, which is highly innervated, was responsible for the decreased intake. Another possible explanation fo r the food intake depression could be chemoreceptors sensitive to the increased butyrate pe r se.

The effect of the V F A maxture infusion on blood and rumen metabolites is shown in Table 8. Blood ketones appear to be lowered as a result of the V F A mixture infusion, but the control values for ketones are much higher than normal (Tables 5, 6, 8). Blood and tureen acetate increased, whereas blood and rumen propionate decreased as a result of the ¥ F A mixture infusion. The increased blood and rumen acetate may have been responsible for the decreased intake through a chemostatic or thermostat ic mechanism.

The in t ra l~minal infusion of acetate approximately doubled the acetate concentration in the blood and in the tureen (Table 9). I t is of interest to note that the blood and rumen changes

that occurred were similar for the animals fed the hay and pelleted rations. The reason that acetate significantly (P < .01) depressed the intake of pellets, but only slightly depressed hay consumption, is not clear. Several workers have suggested that acetate is an impor tant metabolite in food intake regulat ion by ruminants. Dowden and Jacobson (4) found that the intravenous inject ion of acetic acid or sodium acetate at a level equivalent to 12.5% of the cow's DE requirement significantly depressed food intake. Holder (5) repor ted that tile intravenous inject ion of sodium acetate did not depress food intake in sheep, but the level he infused was lower than that of Dowden and Jacobson (4). Several workers (2, 10, 14) have shown that the in t raruminal infusion of acetic acid will cause a depression in food intake in rmninants. The mechanism by which acetate causes a food intake depression is not known. Acetic acid does have a high heat increment (1), and the heat released dur ing the assimilation of acetic acid might be responsible for the food intake depression via a thernmstatie mechanism.

Baile and P fande r (2) suggested a chemosensitive feed intake regula tory mechanism per- ceptive to a change in rumeu fluid acetic acid concentration. Since rumen acetate concentration and blood acetate concentration are proportional (12), it could be that the changes in blood acetate concentrations are inducing satiety

When acetate was infused, the decreased food intake was associated with inereased blood and rumen acetate concentrations. However , i t is

TABLE 8 Effect of volatile fa t ty acid mixture infusion on blood and rumen metabolites ~


Control VFA Mixture Control YFA Mixture Measurement infusion infusion infusion infusion

Blood Sugar (mg/lO0 ml) 55.3 54.4 52.7 51.5 Ketones (mg/lO0 ml) 3.68 2.17 *~ 4.32 2.50 ~ Total VFA (mg/lO0 ml) 2.54 5.15 ~2 4.60 5.90 Acetate (% of total) 92.0 96.3 ~ 93.5 94.3 Propionate (% of total) 4.5 2.2 ~ 4.5 1.6 ~*

l~umen Total VFA (mg/lO0 ml) 717 874 719 900 ~ Acetate (v/o of total) 54.0 64.6 ~2 64.9 67.1 *~ Propionate (% of total) 32.7 21.9 ~ 19.3 17.3 ** Butyrate (% of total) 10.2 11.42 11.4 11.7 Isobutyrate (% of total) 1.2 1.0 1.7 1.322 Isovalerate (% of total) 0.3 0.6 ~ 0.6 1.4 ~2 Valerate (% of total) 1.7 0.6 ~ 2.2 1.22* pH 6.12 6.6622 6.75 6.77

"Values represent the average of samples taken before and at 2, 5, and 8 hr after infusion began.

VFA mixture infusion values are significantly differen~ from control (P < .05). 2~ VFA mixture infusion values are significantly different from control (P % .01).


Exhibit 1018 Page 6


REGULATING FOOD INTAKE IN RUMINANTS

TABLE 9 Effect of acetate infusion on blood and rumen metabolites ~

1641


Control Acetate Control Aeetatc Measurement infusion infusion infusion infusion

Blood Sugar (mg/lO0 cal) 52.3 53.2 53.1 52.1 Ketones (mg/lO0 ml) 1.81 1.75 2.24 2.82 ~ Total VFA (mg/lO0 ml) 3.31 6.03 ~ 5.32 10.32 ~ Acetate (% of total) 90.5 94.7 ~ 93.5 95.3 ~ Propionate (% of total) 3.6 1.3 ~ 2.0 1.3

Rumen Total VFA (mg/lO0 ml) 701 904 ~ 681 1,087 ~ Acetate (% of total) 55.6 81.5 ~ 63.6 77.8 ~ Propionate (% of total) 27.9 11.1 ~ 19.0 12.6 ~ Butyrate (% of total) 12.9 5.9 ~ 12.3 7.9 ~ Isobutyrate (% of total) 1.2 0.8 ~ 1.7 0.9 ~ Isovalerate (% of total) 0.8 0.5 2.0 0.6 ~ Valerate (% of total) 1.6 0.2 ~ 1.5 0.3 ~ pH 5.99 6.64 ~ 6.65 6.43 ~

a Values represent the average of the samples taken before and at 2, 5, and 8 hr after infusion began.

Acetate infusion values are significantly different from control (P < .05). ~ Acetate infusion values are significantly different from control (P < .01).

clear that other factors in addit ion to acetate are impor t an t in the regulat ion of food intake in ruminants . The infusions of p rop iona te and butyra te caused a reduction in food intake, but the decreased food intake dur ing these infusions was not associated with an increase in rumen or blood acetate concentrat ion.

SUMMARY

I t appears that acetate, propionate , and butyra te can act as satiety signal compounds in the regulat ion of food intake in ruminants . The mechanism by which these metabolites cause sat iat ion remains unknown. I f chemoreceptors are involved, then the receptors must be sensitive to all three of the volatile f a t t y acids investigated, since they all caused a reduction in food intake. I t would a p p e a r tha t chemoreceptors fo r p rop iona te and butyra te would have to be located in the rumen wall, in the por ta l system, or in the liver, since the concentrat ions of these two metaboli tes in the per iphera l circu- lation are very low. Acetate chemoreceptors could be located in the pe r iphe ry in addit ion to the three sites mentioned fo r p rop iona te and butyrate . There was no relat ion between the amounts of sodimn and potass ium infused with the volatile f a t ty acids and food intake, so it would appea r tha t the minerals added with the metabolites were not causing the decreased consumption. P e r h a p s the best explanat ion fo r the results obtained is that the heat released dur ing the assimilation of the infused V F A was responsible fo r the depression in food

intake. I t is believed that the thermostat ic theory fo r food intake regulat ion should be invest igated fu r the r in ruminants .

REFERENCES (1) Armstrong, D. G., and Blaxter, K. L. 1957.

The Utilization of Acetic, Propionic and Butyric Acids by Fattening Sheep. Brit. J. Nutrition, 11:413.

(2) Baile, C. A., and Pfander, W. tI. 1965. Chemosensitive Regulatory Mechanism of Ovine Feed Intake. Federation Froc., 24: 244.

(3) Bergman, E. N. 1964. Glucose Turnover R,ates in Pregnant and Non-pregnant Sheep. Nature, 202: 1333.

(4) Dowden, D. R., and Jacobson, D. ]K 1960. Inhibition of Appetite in Dairy Cattle by Certain Intermediate Metabolites. Nature, 188 : 148.

(5) Holder, J. M. 1963. Chemostatic Regulation of Appetite in Sheep. Nature, 200: 1074.

(6) Madison, L. L., Mebane, D., Unger, R. tI., and Lochner, A. 1964. The I-Iypoglycenlic Action of Ketones. II. Evidence for a Stimu]atory Feedback of Ketones on the Pancreatic Beta Cells. J. Clin. Invest., 43 : 408.

(7) Manning, R., Alexander, G. E., Krueger, H. M., and Bogart, R. 1959. The Effect of Intravenous Glucose Injections on Appe- tite ia Adult Ewes. Am. J. Vet. Research, 20 : 242.

(8) Mayer, Jean. 1964. Appetite and the Many Obesities. Australasian Ann. Med., 13: 279.

(9) Montgomery, M. J., and Baumgardt, B. R. 1965. Regulation of Food Intake in Rural-


Exhibit 1018 Page 7


1642 K. L. SIMKINS, JR., J. W. SUTTIE, AND B. R. BAUMGARDT

nants. I. Pelleted l~atlons Varying in En- ergy Concentration. J. Dairy Sci., 48: 569.

(10) Montgomery, M. J., Schultz, L. It., and Baumgardt, B. R. 1963. Effect of Intra- ruminal Infusion of Volatile Fa t ty Acids and Lactic Acid on Voluntary I-Iay Intake. J. Dairy Sci., 46: 1380.

(11) National Research Council, National Acad- emy of Sciences. 1958. Nutrient Require- ments of Dairy Cattle. Washington, D.C.

(12) Simkins, K. L., Jr., Suttie, J. W., and Baum- gardt, B. R. 1965. Regulation of Food

Intake in Ruminants. 3. Variation in Blood and Rumeu Metabolites in Relation to Food Intake. J. Dairy Sci., 48: 1629.

(13) Steel, R. G. D., and Torrie, J. H. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York.

(14) Ulyatt, M. J. 1964. Studies on Some Factors Influencing Food Intake in Sheep. Proc. New Zealand Soc. Animal Prod., 24:43.

(15) Worthington BiochemicM Corporation. 1963. Glueostat, a Prepared Enzymatic Glucose Reagent. Freehold, New Jersey.


Exhibit 1018 Page 8


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EXHIBIT Amlmhelpdesk.com/wp-content/Docs/PruvIT_FG/Doc_0107-1.pdf · Preliminary Injunction Hearing scheduled for December 3-4, 2015 in the above-referenced case, I expect to testify