Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
  • A61K38/56—Protease inhibitors from plants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis

Definitions

• compositions for reducing post-prandial blood glucose in humans and, more specifically, to a proteinase inhibitor that delays gastric emptying and reduces post-prandial glycemia which may be beneficial in combating obesity and Type II diabetes.
• Both soybeans and potatoes are sources of proteinase inhibitors (Pi's), proteins that have been hypothesized to enhance the release of cholecystokinin (CCK), one of several gut peptides that regulate gastric emptying and satiety in humans (Liddle, R. A. (1995) Am JPhysiol 269, G319-27; Beglinger, C. (1994) Ann N YAcad Sci 713, 219-25; Beglinger, C. (2002) Curr Opin Investig Drugs 3, 587-8). Delayed gastric emptying, in turn, has been shown to result in a decreased rate of glucose absorption, and lower postprandial glucose levels (Lefebvre, P. J. & Scheen, A. J.
• Proteinase inhibitor II is a naturally occurring 21 kDa dimer and potent trypsin and chymotrypsin inhibitor present in white potatoes (Melville, J. C. & Ryan, C. A. (1972) JBiol Chem 247, 3445-53; Bryant, J., Green, T. R., Gurusaddaiah, T. & Ryan, C. A. (1976) Biochemistry 15, 3418-24).
• Previous studies using large doses of highly pure PI2 demonstrated increased CCK release and satiety in humans (Peikin, S. R., Springer, C. J., Dockray, G. J., Blundell, J.
• the invention consists of a method for reducing post-prandial glycogen levels in the blood of humans by the oral administration of a proteinase inhibitor or a combination of proteinase inhibitors.
• the proteinase inhibitor or combination is administered prior to the ingestion of a meal and reduces not only the initial rise in blood glucose following a meal ( ⁇ Glucose or AG) but also the integrated area under the blood glucose curve (AUC) following a meal.
• the proteinase inhibitor(s) is effective for helping to maintain healthy blood sugar levels and for treating persons, such as those with Type II diabetes, which have adverse health effects due to hyperglycemia. Further, the proteinase inhibitor(s) is expected to reduce the propensity for weight gain by reducing the glycemia experienced by the body.
• Proteinase inhibitors which exhibit the property include potato proteinase inhibitor II and soybean Bowman-Birk inhibitor, although other proteinase inhibitors with similar amino acid sequences and with similar proteinase inhibition properties may be used. While single proteinase inhibitors have been shown to be effective, combinations of two or more distinct proteinase inhibitors may also be used.
• a proteinase inhibitor product isolated from potatoes is administered orally prior to a meal.
• the potato proteinase inhibitor extract contains between about 15% and about 25% by weight PI2 and also contains other proteins, including a protein similar but not identical to soybean Bowman-Birk inhibitor.
• the potato proteinase inhibitor extract is present in an amount between about 1 mg and about 1000 mg per dose, and preferably between about 5 mg and about 100 mg per dose, and most preferably between about 7.5 mg and about 30 mg per dose.
• the potato proteinase inhibitor is effective to reduce the blood glucose spike following a meal by between about 5%o and about 30% and the AUC glucose by between about 5% and about 40%.
• Another preferred proteinase inhibitor is Bowman-Birk inhibitor, which is typically isolated from soybeans.
• the Bowman-Birk inhibitor is present in an amount between about 0.1 mg and about 5.0 mg per dose, and preferably between about 0.5 mg and about 2.0 mg per dose.
• the Bowman-Birk inhibitor is effective to reduce the blood glucose spike following a meal by between about 10% and about 25% and the AUC glucose by between about 5% and about 30%.
• Yet a further object of the invention is to combat Type II diabetes through the administration of one or more proteinase inhibitors either alone or in combination with other medications that are used in combating diabetes.
• Figs, la and lb are HPLC chromatograms of the potato PI2 extract used in the experiments and an authentic PI2 standard, respectively.
• Fig. 2 is a photograph of an SDS PAGE of the potato PI2 extract used in the experiments and an authentic PI2 standard.
• Fig. 3 is a graph showing the effect of an increasing PI2 dose on post-prandial integrated area under the blood glucose curve (AUC) after a test meal.
• Fig. 4 is a graph showing the effect of an increasing PI2 dose on the initial rise in blood glucose above the baseline ( ⁇ Glucose) thirty minutes after a test meal.
• Fig. 5 is a schematic diagram of the effects of chronic consumption of a high glycemic load.
• the composition for reducing post-prandial blood glucose levels in humans is based on a proteinase inhibitor that delays gastric emptying and reduces post-prandial glycemia which may be beneficial in combating obesity and in the therapeutic treatment of patients suffering from hyperglycemia.
• the proteinase inhibitor is believed to enhance the release of cholecystokinin (CCK), a peptide which regulates gastric emptying.
• CCK cholecystokinin
• the preferred proteinase inhibitors include potato proteinase inhibitor II and Bowman-Birk inhibitor.
• BioffectTM a proteinase inhibitor extracted from potatoes and available commercially from Kemin Consumer Care, L.C., Des Moines, Iowa, under the trademark BioffectTM was used in some of the examples. BioffectTM is also available in tablets formulated to contain 15 mg per dose and sold under the trademark SatiseTM.
• the invention is based on the surprising result that proteinase inhibitors administered orally before a meal have the effect of reducing the initial post-prandial glucose spike and also reduce the total integrated area under the curve blood glucose over more than three hours after a meal. Also surprising is that the proteinase inhibitors are effective when administered in a dose in the less than ten milligram range.
• Subjects were randomly allocated to receive placebo and two of the three following doses: 7.5, 15, or 30 mg PI2 extract.
• subjects arrived at 8.00 AM after a 10 hour fast. They were given breakfast and 500 ml of water to drink throughout the morning, but ate nothing further until the test meal. Height and weight of all subjects were recorded during their first visit. Three and a half hours after breakfast the first blood glucose measurement was made, and subjects were given the experimental capsule and 500 ml of water. Thirty minutes later the test lunch was served. As soon as each subject completed the meal, the timing for post-prandial glucose measurements began. Subjects recorded any adverse reactions at fifteen minute intervals for 200 minutes after eating the meal.
• test meal On each test day subjects were fed a breakfast of granola, skim milk, and orange juice that contained 330 kilocalories derived from 67 g of carbohydrate, 2.5 g of fat, and 12 g of protein. No other food was permitted until the test meal, which was consumed at noon on the test day.
• the test meal was Chicken Teriyaki (Boston Market) and contained no potato products.
• the nutritional content of the test meal is set out in Table 1. All subjects consumed all meals in their entirety.
• Finger-prick capillary blood samples were taken 30 minutes before the test meal (Baseline), and 30, 60, 90, and 120 minutes post-prandially. Glucose measurements were made with a Dex glucometer, Model # 3952E (Bayer Pharmaceuticals), in accordance with the manufacturer's instructions. Proteinase Inhibitor
• PI2 extract was provided by Kemin Consumer Care, L.C. (Des Moines, Iowa), and was supplied in 00 gelatin capsules containing 7.5, 15, or 30 mg, respectively. A mixture of dextrose and whey protein was used to bring all capsules to a uniform weight and volume and also served as a placebo. The doses in the present study were chosen based on previous studies demonstrating efficacy at 30 mg in liquid form (Spiegel, T. A., Hubert, C. & Peikin, S. R. (1999) University of Medicine and Dentistry of New Jersey; Vasselli, J. R., Greenfield, D., Schwartz, L. & Heymsfield, S. B. (1999) Obesity Research Center, St.
• the active material was produced from a single lot of potatoes (Russet Nuggets; Kemin lot 87289C, approximately 244.39 mg PI2 extract/kg).
• PI2 RP-HPLC Formulation of the active doses was based on quantitation by high performance liquid chromatography (HPLC). Reversed-phase HPLC (RP-HPLC) analyses were performed on a Hewlett Packard Model 1100 equipped with a diode array detector using a Microsorb C-l 8, 5 ⁇ m particle size, 300 Angstrom pore size, 4.6 X 250mm (Varian Analytical Instruments, Walnut Creek, CA). The chromatographic conditions were as follows: Isocratic elution for five mmutes of 80% of 0.1 % TFA in H 2 O (20% of 1% TFA in acetonitrile).
• SDS-PAGE To further characterize the PI2 extract, samples were analyzed by gel electrophoresis. SDS gels were prepared as 4% stacking, 15 % resolving with 1.5 M Tris, 0.5 M Tris, 10% SDS, 30% ammonium persulfate, TEMED, and 40%Acryl/Bis. Wells were loaded with pre-stained marker, PI2 standard, and PI2 extract. A current of 80 volts was applied for 1.5 hours. Gels were then stained with Coomasie blue staining. Pure PI2 standard was obtained by sequential RP-HPLC followed by gel filtration chromatography. Western blot using a rabbit polyclonal antibody developed by Kemin Foods, L.C. against PI2 protein, was used to further determine the identity of the major protein in the potato PI2 extract used in the current study.
• PROC MIXED function in SAS was used, as this allows a more general specification of the covariance matrix of the dependent variable, and allows random factors of both the model and error terms to be correlated ( Hongsen, Z. (2001) Proceedings of the 12th Annual Conference of the Midwest SAS Users Group, 132-140). All subjects received placebo on one visit, but only two of the three possible active treatments during the other visits, so an incomplete block design was used to evaluate the relative effectiveness of the doses.
• the strategy described by Wolfinger Wolfinger, R. D. (1993) Communications in Statistics, Simulation, and Computation 22, 1079-1106 was followed to select an appropriate variance-covariance structure for the ANOVA test.
• the Akaike's Information Criterion was used to select the appropriate variance-covariance structure for the model. Chi-square analysis was used to evaluate data obtained as discrete variables with p ⁇ 0.05 considered to be significant
• results of active PI2 extract were quantified by RP-HPLC.
• the integrated peak representing the PI2 extract co-eluted with a pure authentic PI2 standard, indicating that PI2 is contained in the extract and that it is the major protein (Figs. 1 a and lb).
• Results of gel electrophoresis further confirm the findings of the analysis by RP-HPLC and show that the PI2 in the extract is likely present as a monomer with a molecular weight of approximately ⁇ 12 kDa (Fig. 2).
• MALDI MS analysis of the purified PI2 protein demonstrated that this protein has a molecular weight of 12 kDa.
• PI2 protein Western blots of the separated proteins using a rabbit polyclonal antibody for PI2 protein demonstrated that the major protein band separated by SDS-PAGE is PI2.
• the actual amount of PI2 protein present in a given extract could vary and ranges from 17 — 20%.
• the PI2 extract was also characterized for its trypsin and chymotrypsin inhibition activity using an in vitro assay demonstrating both trypsin and chymotrypsin inhibition.
• PI2 extract product contained a ratio of 0.9 - 1.7:1 units of trypsin: chymotrypsin inhibition activity, respectively.
• Table 1 shows the nutrient value of the test meal and the mean glucose AUC following placebo.
• PI2 extract was administered, along with 27 of each of the 7.5 mg and 15 mg doses, and 26 of the 30 mg dose, respectively (one individual declined to provide blood samples and was included in determination of adverse events monitoring).
• Table 1 shows the nutrient value of the test meal and the mean glucose AUC following placebo.
• We first examined the effect of PI2 extract on AUC; the repeated measure ANOVA model used for this analysis showed a statistically significant effect of the experimental treatment (f 3.3, p ⁇ 0.05) but no statistically significant difference between the experimental blocks.
• Subjects given a dose of 7.5 mg PI2 extract before the test meal experienced no significant reduction in post-prandial glucose compared to placebo.
• the AUC of subjects receiving both 15 and 30 mg PI2 extract prior to the test meal was significantly reduced compared to placebo, but there was no significant difference in post-prandial AUC between the two higher doses (Fig. 3).
• the decrease in AUC for 15 and 30 mg was 29.8% and 24.5% respectively, each compared to placebo.
• the decrease in ⁇ glucose for the 15 and 30 mg doses was 25% and 20% respectively, each compared to placebo.
• Feeding 120 test meals resulted in 14 reports of an adverse reaction from subjects. These are summarized in Table 2.
• Gastrointestinal symptoms included nausea, cramping and diarrhea. Differences in occurrence rates of adverse reactions between the treatments and the placebo were not significant (p > 0.05, Chi square). Subjects experiencing symptoms rated them as mild, and frequently they were noted at only one of the recording times.
• lowering the glycemic load experienced by the body by diet or other means may be an effective way to reduce the post-prandial glycemia that can lead to weight gain and obesity.
• Findings of the present study suggest that it is possible to lower the glycemic load experienced by the body by ingesting a supplement containing a low dose of PI2 extract prior to a meal. Doses of either 15 mg or 30 mg taken 30 minutes before a test meal significantly reduced the subsequent rise in blood glucose (Figs. 3 and 4). A dose of 7.5 mg had no significant effect, indicating that under these test conditions the lowest effective dose lies between 7.5 and 15 mg. This study was limited to acute observations, and the effect of chronic oral administration of PI2 extract on blood sugar levels remains to be studied.
• PI2 has not previously been administered in solid form in an encapsulated supplement prior to the meal, and because a solid mixed meal was used for the first time.
• the dose used was substantially lower and less pure than that previously reported, and a larger cohort of subj ects was studied.
• a dose of 1.5 g PI2 (90 - 100% pure) by column chromatography (Clarence Ryan, Washington State University, Pullman, WA), administered in liquid form was used in two previous studies; in one study PI2 was added to soup and fed 8 minutes before a test meal, and in the other it was incorporated in a test beverage (Hill et al.; Schwartz et al.). In neither case was it encapsulated.
• Subjects consuming an isocaloric diets consisting of low glycemic index foods lose more weight or maintain their relative to those consuming high glycemic index foods ( Slabber, M., Barnard, H. C, Kuyl, J. JYL, Dannhauser, A. & Schall, R. (1994) Am J Clin Nutr 60, 48-53; Wolever, T. M., Jenkins, D. J., Vuksan, V., Jenkins, A. L., Wong, G. S. & Josse, R. G. (1992) Diabetes Care 15, 562-4; Clapp, J. R.
• PI2 extract is proposed to exert its effect on post-prandial glucose by enhancing the release of a well characterized peptide hormone, CCK, which is naturally secreted into the blood stream by enteroendocrine cells in response to a meal ( Crawley, J. N. & Corwin, R. L. (1994) Peptides 15, 731-55).
• CCK acts on various target tissues throughout the body including the gastrointestinal tract, where it delays gastric emptying leading to feelings of fullness, and the brain leading to feelings of satiety.
• PI2 is a pH, heat, and salt stable protein (Bryant et al), allowing it to be effective when administered orally, and making it unique among plant Pi's.
• the extract used in the present study contains PI2 (Figs. 1 and 2), and is derived from white potatoes using a method generally as described in United States Patent Application No. 09/900,555, incorporated herein by this reference. Although normally present in potatoes as a dimer, the PI2 separated from our extract appears to be in the monomeric form. The pure PI2 possess trypsin and chymotrypsin inhibition activities of 1.4 and 3.6 units and the PI2 extract possess trypsin and chymotrypsin inhibition activities of 22 and 13 units.
• the Bowman-Birk inhibitor used was obtained from Sigma-Aldritch and had a stated purity of greater than 80%. Bowman-Birk inhibitor has similar enzyme inhibiting properties as pPI2.
• the meal challenge was conducted at a breakfast meal instead of a lunch meal, as in the prior study, and consisted of 390 kcal with 100 kcal from fat and 53 g carbohydrate and was provided to individuals who had been fasting for at least 10 hours.
• Each participant made two visits to the research center and underwent two meal challenges - one for the placebo and one for an active (15 mg pPI2 or 0.8 mg Bowman-Birk inhibitor) and these treatments were provided in a double-blinded format.
• the randomization scheme also prevented the participants or the study personnel from knowing at which visit the placebo was given until the code was broken.
• the PI2 protein in the potato proteinase inhibitor extract and the trypsin/chymotrypsin inhibiting activity may be related to modulation of post-prandial glucose since the relative concentration of this activity in the pPI2 showed comparable glucose modulating activity as with the original potato proteinase inhibitor extract and the Bowman-Birk inhibitor which also contains an inhibitor or inhibitors of trypsin and chymotrypsin showed glucose modulating activity.

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