Use of compounds that are able to increase the serum IGF-1 level for the preparation of a therapeutical composition for treatment of various disease states associated with a reduced IGF-1 serum level in humans and animals

Abstract

The present disclosure relates to the use of one or more compounds that are capable of increasing the serum level of insulin-like growth factor 1 (IGF-1) for the preparation of a therapeutical composition, in particular, in the form of a food supplement, for the treatment of subjects suffering from serious fatigue and exhausting symptoms, burn-out and chronic fatigue syndrome. The same composition can also be used by patients suffering from depression, Alzheimer disease, irritated bowel syndrome, osteoporosis, type 2 diabetes, or for anti-aging, immune therapy and recovery after exercise. The composition also has a use in veterinary applications for increasing the growth and immunity in animals.

Claims

1. A method of administering a compound to a subject, the method comprising: identifying the subject as suffering from irritable bowel syndrome; and administering to the subject a compound comprising purified and/or isolated indole acetic acid (IAA), 4-hydroxy-IAA, 4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxyIAA, 6-hydroxy-IAA, 6-methoxy-IAA, 7-hydroxy-IAA, or 7-methoxy-IAA, thereby treating the irritable bowel syndrome in the subject.

2. The method according to claim 1, wherein the compound further comprises at least one amino acid.

3. A method of administering a compound to a subject, the method comprising: identifying the subject as suffering from irritable bowel syndrome; and administering to the subject a compound comprising 1 to 100 mg of indole acetic acid (IAA), 4-hydroxy-IAA, 4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxyIAA, 6-hydroxy-IAA, 6-methoxy-IAA, 7-hydroxy-IAA, 7-methoxy-IAA, or a combination thereof, thereby treating the irritable bowel syndrome in the subject.

4. The method according to claim 1, wherein the compound is in the form of a capsule.

5. The method according to claim 3, wherein the compound comprises 10 to 90 mg of indole acetic acid (IAA), 4-hydroxy-IAA, 4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxyIAA, 6-hydroxy-IAA, 6-methoxy-IAA, 7-hydroxy-IAA, 7-methoxy-IAA, or a combination thereof.

6. The method according to claim 3, wherein the compound comprises 40 mg of indole acetic acid (IAA), 4-hydroxy-IAA, 4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxyIAA, 6-hydroxy-IAA, 6-methoxy-IAA, 7-hydroxy-IAA, 7-methoxy-IAA, or a combination thereof.

7. The method according to claim 3, wherein the compound is in the form of a capsule.

8. The method according to claim 3, wherein the compound further comprises at least one amino acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present disclosure will be further illustrated in the following examples that are not intended to limit the disclosure. Reference is made to the following figures:

(2) FIG. 1: The course of plasma IGF-1 and IGF-BP3 level in real time in eight patients suffering from CFS. The patients were treated with the food supplement of the disclosure.

(3) FIG. 2: IGF-1 levels in nmol/l in type 2 diabetic patients during medication with IAA.

(4) FIG. 3: The ratio of IGF-BP3/IGF-1 in type 2 diabetic patients during medication with IAA.

(5) FIG. 4: The HbA1c (hemoglobin A1c) level in type 2 diabetic patients during medication with IAA.

(6) FIGS. 5A-5C: IGF-1 levels in individuals with general anti-aging problems during medication with a composition of the disclosure.

(7) FIGS. 6A-6C: Levels of complaints in individuals with general anti-aging problems during medication with a composition of the disclosure.

(8) FIGS. 7A-7C: Levels of activity in individuals with general anti-aging problems during medication with a composition of the disclosure.

(9) FIG. 8: IGF-1 level in athletes taking IAA or a placebo.

(10) FIGS. 9A-9C: Complaints questionnaire for Chronic Fatigue Syndrome.

DETAILED DESCRIPTION

Examples

Example 1Use of the Composition of the Disclosure in the Treatment of Chronic Fatigue Syndrome

(11) 1. Introduction

(12) Chronic fatigue syndrome (CFS) is clinically defined by feelings of fatigue that exist for longer than 6 months in the absence of a proper medical diagnosis. In CFS patients, the symptomatology is very wide and consists of neuro-cognitive problems, muscle pains, multiple joints pains, headache, non-refreshing sleep, post-exertional malaise, adenopathy and sore throat. Because of the relatively high prevalence (117/100,000 adults in the Netherlands) and unavailability of a successful treatment, the conditions are a burden to society both, with respect to medical care and the economy. This example gives the effects of the composition of the disclosure on the course of CFS. The supplement has auxin as the active ingredient and increases the plasma IGF-1 concentration.

(13) 2. Material and Methods

(14) Eight women varying in age from 25 to 53 years having severe complaints of fatigue that made it impossible to function normally, both at work and in free time. All reported an excessive need for sleep, which was never refreshing even though it was prolonged (more than 12 hours). After excluding all other diseases and infections, they were all diagnosed for CFS. Physical examination did not reveal any abnormalities in heart and lungs.

(15) Blood samples of 20 ml were taken and liver and kidney functions, the red and white hemogram, IGF-1 and IGF-BP3 determined. The patients took 1000 mg/day (corresponding to 40 mg IAA and 960 mg salt and WPC 70) of the composition of the disclosure for 4 weeks. After this, the dosage was reduced by 250 mg (corresponding to 10 mg IAA) every 2 weeks to 750 mg/day, 500 mg/day 250 mg/day, respectively. Check-ups were performed and blood samples taken (for determining IGF-1 and IGF-BP3) after 1, 3 and 5 to 7 months of therapy.

(16) At the start of the study and at each check-up point, the work participation and physical activity pattern were scored on a scale from 1 to 10 and plotted as the percentage of the patient's normal activities. Hormone assays for IGF-1 were performed by means of an immunoassay (DSL-5600 ACTIVE, DSL Deutschland GmbH, Germany) having an intra-assay variance of 3.0% and an inter-assay variance of 4.9%. IGF-BP3 was determined by means of an immunoassay (DSL-5600 ACTIVE, DSL Deutschland GmbH, Germany) having an intra-assay variance of 4.4% and an inter-assay variance of 6.6%.

(17) 3. Statistics

(18) All values are presented as meanSD. The Wilcoxon signed rank test for paired observations was applied to show differences between hormone values on each point of time. In case a p value<0.05 was found, the differences were considered to be significant.

(19) 4. Results

(20) All patients had very severe restrictions resulting in a high non-attendance at work. The participation in work and the physical activity level of all eight women was very low before the treatment. Only two patients worked 1 or 1.5 days/week. Normal activities like climbing stairs and going for a walk were considered to be impossible or a heavy burden. However, already after 1 month of treatment, five of eight women worked for 1 or more days, while all stated that their capacity to perform normal physical work was highly increased.

(21) After 5 months of therapy, the participation in work was increased to 69.4% (50-100%), while the placebo group was the same as at the start of the study.

(22) The plasma IGF-1 level (FIG. 1) of the patients was at the first visit in only three patients higher than the standard value of 20 g/1. The plasma IGF-1 values increased significantly after 1 month of treatment with the composition of the disclosure (p<0.001), a trend that persisted after 3 months of treatment (p<0.01), after which the values did not significantly increase.

(23) The plasma IGF-BP3 value (FIG. 1) decreased significantly in time. This means that the free IGF-1 was increased.

(24) 5. Discussion

(25) The composition of the disclosure appears to positively influence the course of the CFS. This is accompanied by a significant increase in plasma IGF-1 level and a decrease in the major binding protein, IGF-BP3. This means that the free IGF-1 concentration (biologically active fraction) in blood is increased.

Example 2Double-Blind Study with a Group of 20 Patients

(26) The next study had a double-blind, placebo-controlled design. Twenty subjects known with CFS were included and treated either with the composition (10 mg IAA, 100 mg NaCl, 140 mg WPC 70) of the disclosure, for 3 months, or with a placebo. After this period, 54% of the subjects receiving the composition of the disclosure improved in mood state as indicated by an adapted profile of mood state (POMS) questionnaire, against 15% of the placebo group. IGF-1 values increased with 40% in the treated group, and remained essentially unchanged in the placebo group.

Example 3A Double-Blind, Placebo-Controlled Study of the Composition of the Disclosure with Amino Acids in Patients with Chronic Fatigue Syndrome

(27) Ninety individuals suffering from CFS (according to the Holmes and/or Fukuda definition) entered the study. They were not allowed to take any other medication other than minor pain relievers and homeopathic medications. At baseline, week 4 and week 8, IGF-1, IGF-BP3 and safety parameters, such as liver tests and hematology, were determined. The treatment protocol consisted of 4 weeks 250 mg composition (10 mg IAA, 100 mg NaCl and 140 mg WPC 70) of the disclosure four times a day in combination with the amino acid preparation All-Amino S (Optipharm) (Table 1) and subsequently 4 weeks 250 mg of the composition of the disclosure twice a day in combination with the amino acid preparation. The primary efficacy variable was the change from baseline in CFS as measured by the Clinician's Global Impression Scale (CGI) after 4 and 8 weeks of treatment.

(28) TABLE-US-00001 TABLE 1 Composition of All-Amino S Amino acid mg per capsule mg/gr powder L-Lysine HCl 50 43.2 L-Histidine base 30 25.9 L-Arginine base 125 108 L-Asparagine mono 74 63.9 L-threonine 41 35.4 L-Serine 53 45.8 L-Proline 110 95 L-Alanine 34 29.4 L-Cystine 9.4 8.1 L-Valine 71.5 61.7 L-Methionine 32 27.6 L-Isoleucine 59 50.9 L-Tyrosine 16 13.8 L-Leucine 102 88.2 L-Phenylalanine 22 19 L-Tryptophane 13 11.2 L-Cysteine mono 10 8.6 L-Glutamine 15.3 13.2 L-Taurine 10.7 9.2 L-Glutamic acid 230 199 L-Carnithine 50 43.2

(29) In the test group, the IGF-1 levels increased significantly at week 4 and week 8 compared to the placebo group (p<0.0002). In the test group, 54% noted an improvement in the symptoms, compared to 16% in the placebo group. The symptom change according to the CGI yielded an improvement in the group receiving the composition of the disclosure at week 4 (p<0.004) and at week 8 (p<0.0003). No significant changes were noted in the placebo group.

(30) The composition of the disclosure in combination with amino acids gives an improvement in CFS symptoms. No major adverse effects were noted.

Example 4Use of the Composition of the Disclosure in Type 2 Diabetes Patients

(31) 1. Introduction

(32) The system of IGF-I and its binding proteins is a complex system with many physiological functions including metabolic regulations. This example aimed to describe changes of its particular components in type 2 diabetic patients. Clinical examination and estimation of serum concentrations of IGF-I and IGF-BP3 influenced by supplementation with IAA were performed. A relationship has been proven between glycemic control and serum IGF-I levels, with worse control being associated with lower IGF-I levels.

(33) 2. Material and Methods

(34) The Patients

(35) Five male type 2 diabetic patients took part in the study. They were all very difficult to regulate. When the study started, the average HbA1c was to be approximately 9.0%. The patients had no other clinical problems. In addition, the IgF-BP3/IgF-1 ratio had to be over 10. The age of the subjects were between 45 years and 75 years with an average of 58. The period of the investigation was 6 months. At the start of the study, after 2 month, 4 months and at the end at 6 months, blood was taken. The following parameters were checked: IGF-1 and IGF-PB-3. Also, at the start, safety parameters (liver tests, hematology) were determined. Every 8 weeks, HbA1c was determined. The HbA1c test is a lab test that reveals average blood glucose over a period of two to three months.

(36) All samples from one subject were analyzed in the same run to avoid intra-assay variability. Quantification of IGF-1 was performed using an immunoradiometric assay (IRMA) (DSL-5600 ACTIVE, DSL, Germany GmbH, Germany). Intra- and inter-assay variance was: 4.0% and 9.2% and 1.5% for IGF-1. The Hba1c was analyzed on HPLC Menarine HA 81-60.

(37) Calculations and Statistics

(38) The total work output of every subject was calculated with a 2-way ANOVA for repeated measures and the hormone values were analyzed with the Wilcoxon test. In all cases, a p value of <0.05 was accepted as significant.

(39) TABLE-US-00002 TABLE 2 Medication with IAA 1 Capsule IAA contained 10 mg IAA, 100 mg NaCl, and 140 mg WPC 70 Month 1 and 2 IAA in the morning 1 mg (1 capsule) in the afternoon 1 mg (1 capsule) In the evening 1 mg (1 capsule) Month 3 and 4 IAA in the morning 1 mg (1 capsule) in the evening 1 mg (1 capsule) Month 5 and 6 IAA in the morning 1 mg (1 capsule)
3. Results

(40) With all subjects, the average IgF-1 level increased from 10.8 nmol/l to 14.1 after 6 months (Table 3-1). All subjects started with a ratio above 10. At the start, the average was 10.6. After 6 months, the ratio was 6.8 (Table 3-2). The HbA1c decreased from 9.0 to 6.7% (Table 3-3).

(41) TABLE-US-00003 TABLE 3-1 IgF-1 level in nmol/l Month 0 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Subject 1 11.1 12.5 13.1 13.8 14.3 14.9 15.2 Subject 2 9.6 10.1 10.6 11.1 11.8 12.1 12.9 Subject 3 10.4 10.8 11.6 12.1 12.6 13.3 13.9 Subject 4 11.2 11.6 11.9 12.3 12.8 13.3 14.4 Subject 5 11.3 12.0 12.6 13.0 13.5 13.9 14.3 Average 10.7 11.4 12.0 12.5 13.0 13.5 14.1

(42) TABLE-US-00004 TABLE 3-2 Ratio IgF-BP3/IgF-1 Month 0 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Subject 1 10.2 9.7 9.0 8.6 8.0 7.2 6.6 Subject 2 10.6 10.1 8.6 8.3 7.6 6.8 6.3 Subject 3 10.8 9.9 9.4 8.9 8.3 7.6 7.2 Subject 4 11.3 10.8 9.2 8.8 7.8 7.3 6.9 Subject 5 10.0 8.9 8.5 8.1 7.6 7.2 6.9 Average 10.6 9.9 8.9 8.5 7.9 7.2 6.8

(43) TABLE-US-00005 TABLE 3-3 HbA1c Week 0 Week 8 Week 16 Week 24 Subject 1 9.6 8.9 8.0 7.4 Subject 2 9.0 8.2 7.1 6.4 Subject 3 9.1 8.3 7.4 6.6 Subject 4 8.6 7.6 6.9 6.3 Subject 5 8.8 8.3 7.6 6.8 Average 9.0 8.3 7.4 6.7
4. Discussion

(44) The patients felt better after the treatment.

Example 5Use of the Disclosure for the Treatment of Individuals with General Aging Problems

(45) 1. Methods

(46) 180 Individuals suffering from fatigue, depression, concentration problems, stress (burn-out) and bad shape entered the study. Other medication was excluded from the research. The period of the treatment is 6 months. They were not allowed to take any other medication. At baseline, at the start, after 1 month, 2 months and 3 months, IGF-1, IGF-BP3 and safety parameters (liver tests, hematology) were determined. Every month, the IGF-1 and the IGF-BP3 were checked. The treatment protocol consisted of 3 months 1 sachet (75 mg acclydin, corresponding to 3.75 mg IAA) of the preparation of the disclosure either in the morning or in the evening.

(47) TABLE-US-00006 TABLE 4 Composition of an Anti-Aging Sachet According to the Present Disclosure. Vitamins vitamin A 1.833 I.E. -carotene 2.333 I.E. vitamin B1 (thiamine) 16.6 mg vitamin B2 (riboflavin) 12.5 mg vitamin B6 12.5 mg vitamin B12 15 g vitamin C 600 mg vitamin E (D--tocopherol) 58.3 mg vitamin D3 31 I.E. folic acid 125 g niacin 25 mg niacinamid 25 mg vitamin B5 (panthothene acid) 75 mg biotin 50 g bioflavonioc complex 16.6 mg PABA 8.3 mg inositol 12.5 mg choline 25 mg Minerals magnesiumoxide 33.3 mg potassium 16.6 mg Trace elements chromium 33.3 g zinc 8.3 mg Iodine 25 g Manganese 1.6 mg selenium 33.3 g molybdenum 16.6 g boron 330 g Amino acids L-cysteine 33.3 mg DL-methionine 11.6 mg glutamine 500 mg betaine 30 mg Remaining pinecone nut extract 20 mg grape nut extract 80 mg acclydine (3.75 mg IAA, 75 mg 30 mg NaCl, 41.25 mg WPC 70)

(48) The primary efficacy variable was the change from baseline in CFS as measured by the questionnaire (activities and complaints, see FIGS. 9A-9C and Table 19) and the IGF-1 and IGF-BP3 values.

(49) TABLE-US-00007 TABLE 5 The individuals were divided in the following groups: 30 Sachets of the invention A 30-45 Years 30 Placebo Test group A Male Female 14 16 Placebo group A Male Female 12 18 30 sachets of the invention B 46-60 Years 30 Placebo Test group B Male Female 17 13 Placebo group B Male Female 15 15 30 sachets of the invention C >61 Years 30 Placebo Test group C Male Female 14 16 Placebo group C Male Female 16 14

(50) Before the study started, all individuals filled out the questionnaire (FIGS. 9A-9C and Table 19, respectively). This was repeated every month when blood was also taken. The questionnaire is divided in two parts (called herein part 1 and part 2).

(51) Part 1 gives an insight into the development of which complaints occur and how serious they were over a period of 3 months. This part had a scale of 0 to 4.

(52) Part 2 gives an insight into the development of activities that were performed over a period of 3 months. This part had a scale of 0 to 4.

(53) Explanation Scale Part 1.

(54) 4. The individual had serious/major complaints

(55) 3. The individual had considerable complaints

(56) 2. The individual had moderate complaints

(57) 1. The individual had little complaints

(58) 0. The individual had no complaints at all

(59) Explanation Scale Part 2.

(60) 4: The individual performed almost no activities

(61) 3: The individual performed little activities

(62) 2: The individual performed moderate activities

(63) 1: The individual performed considerable activities

(64) 0: The individual can perform all activities he/she wants

(65) 2. Materials

(66) All samples from one subject were analyzed in the same run to avoid intra-assay variability. GH was assayed with a chemiluminescence kit (Nichols Institute Diagnostics, San Juan Capistrano, Calif., USA). Quantification of IGF-1 and IGF-BP3 was performed using an immunoradiometric assay (IRMA) (DSL-5600 ACTIVE, DSL, Germany GmbH, Germany). Intra- and inter-assay variance was: 4.0% and 9.2% for GH; 3.0% and 1.5% for IGF-1 and 4.4% and 6.6% for IGF-BP3.

(67) Calculation and Statistics

(68) The total work output of every subject was calculated with a two-way ANOVA for repeated measures and the hormone values were analyzed with the Wilcoxon test. In all cases, a p value of <0.05 was accepted as significant.

(69) 3. Results

(70) IgF-1 Concentrations

(71) Group A

(72) In the group that was given the composition of the disclosure, the IgF-1 level significantly increased statistically over a period of 3 months with ca. 19% (Table 6-1). At point 0 (start of the study), the level had an average value of 25.1 nmol/l. At point 3 (end of the study), the IgF-1 level had an average of 29.9 nmol/l.

(73) The level of IgF-1 in the placebo group shows no significant difference. At point 0 (start of the study), the level had an average value of 26.0 nmol/l. At point 3 (end of the study), the IgF-1 level had an average of 25.9 nmol/l.

(74) Group B

(75) In the group that was given the composition of the disclosure, the IgF-1 level significantly increased statistically over a period of 3 months with ca. 24% (Table 6-1). At point 0, the level had an average value of 20.6 nmol/l. At point 3, the IgF-1 level had an average of 25.6 nmol/l.

(76) The level of IgF-1 in the placebo group shows no significant difference. At point 0, the level had an average value of 19.9 nmol/l. At point 3, the IgF-1 level had an average of 20.4 nmol/l.

(77) Group C

(78) In the group that received the composition of the disclosure, the IgF-1 level significantly increased statistically over a period of 3 months with ca. 35% (Table 6-1). At point 0, the level had an average value of 14.9 nmol/l. At point 3, the IgF-1 level had an average of 20.3 nmol/l.

(79) The level of IgF-1 in the placebo group shows no significant difference. At point 0, the level had an average value of 14.5 nmol/l. At point 3, the IgF-1 level had an average of 14.4 nmol/l.

(80) TABLE-US-00008 TABLE 6-1 IgF-1 Level in nmol/l Time in months 0 1 2 3 Group A Average Test group 25.1 28 29.4 29.9 SD Test group 3.44 3.12 3.22 3.08 Average placebo group 26 27.1 25.4 25.9 SD placebo group 3.6 3.42 3.32 3.51 Group B Average Test group 20.6 24.1 25 25.6 SD Test group 3.03 3.29 3.1 3.21 Average placebo group 19.9 20.7 19.2 20.4 SD placebo group 2.99 3.4 3.22 3.25 Group C Average Test group 14.9 17.9 19.4 20.3 SD Test group 1.94 2.14 1.88 2.38 Average placebo group 14.5 14 15.1 14.4 SD placebo group 1.96 1.67 2.02 1.97

(81) Complaints

(82) Group A

(83) In the group that was given the composition of the disclosure, the complaints significantly decreased statistically over a period of 3 months (Table 6-2). At point 0, the level had an average value of 2.88. At point 3, it had an average of 0.62.

(84) The level the complaints in the placebo group showed no significant difference. At point 0, the level had an average value of 2.90. At point 3, it had an average of 2.79.

(85) Group B

(86) In the group that received the composition of the disclosure, the complaints significantly decreased statistically over a period of 3 months (Table 6-2). At point 0, the level had an average value of 3.18. At point 3, it had an average of 0.75.

(87) The level the complaints in the placebo group showed no significant difference. At point 0, the level had an average value of 3.15. At point 3, it had an average of 3.10.

(88) Group C

(89) In the group that was given the composition of the disclosure, the complaints significantly decreased statistically over a period of 3 months (Table 6-2). At point 0, the level had an average value of 3.56. At point 3, it had an average of 0.72.

(90) The level of the complaints in the placebo group showed no significant difference. At point 0, the level had an average value of 3.60. At point 3, it had an average of 3.56.

(91) TABLE-US-00009 TABLE 6-2 Month 0 Month 1 Month 2 Month 3 Group A Average Test 2.88 2.02 1.11 0.62 group SD Test group 0.31 0.26 0.21 0.15 Average Placebo 2.9 2.69 2.88 2.79 group SD Placebo 0.31 0.24 0.29 0.32 group Group B Average Test 3.18 2.28 1.34 0.75 group SD Test group 0.32 0.25 0.23 0.12 Average Placebo 3.15 2.99 2.97 3.1 group SD Placebo 0.3 0.28 0.34 0.31 group Group C Average Test 3.56 2.41 1.27 0.72 group SD Test group 0.27 0.22 0.25 0.2 Average Placebo 3.6 3.44 3.48 3.56 group SD Placebo 0.3 0.24 0.29 0.31 group

(92) Activities

(93) Group A

(94) In the group that received the composition of the disclosure, the activities significantly decreased statistically over a period of 3 months (Table 6-3). At point 0, the level had an average value of 2.11. At point 3, it had an average of 0.77.

(95) The level of the activities in the placebo group showed no significant difference. At point 0, the level had an average value of 2.04. At point 3, it had an average of 2.02.

(96) Group B

(97) In the group that received the composition of disclosure, the activities significantly decreased statistically over a period of 3 months (Table 6-3). At point 0, the level had an average value of 2.60. At point 3, it had an average of 1.11.

(98) The level of the activities in the placebo group showed no significant difference. At point 0, the level had an average value of 2.66. At point 3, it had an average value of 2.70.

(99) Group C

(100) In the group that was given the composition of the disclosure, the activities significantly decreased statistically over a period of 3 months (Table 6-3). At point 0, the level had an average value of 3.28. At point 3, it had an average of 1.17.

(101) The level of the activities in the placebo group showed no difference. At point 0, the level had an average value of 3.30. At point 3, it had an average of 3.32.

(102) TABLE-US-00010 TABLE 6-3 Month 0 Month 1 Month 2 Month 3 Group A Average Test 2.11 1.44 0.9 0.77 group SD Test group 0.28 0.33 0.29 0.3 Average Placebo 2.04 1.76 1.88 2.02 group SD Placebo 0.29 0.3 0.27 0.29 group Group B Average Test 2.6 1.88 1.4 1.11 group SD Test group 0.44 0.4 0.45 0.44 Average Placebo 2.66 2.59 2.61 2.7 group SD Placebo 0.4 0.44 0.41 0.45 group Group C Average Test 3.28 2.4 1.6 1.17 group SD Test group 0.6 0.69 0.63 0.65 Average Placebo 3.3 3.2 3.25 3.32 group SD Placebo 0.67 0.54 0.6 0.65 group
4. Discussion

(103) The purpose of this Example was to investigate whether the composition of the disclosure could increase work output. The results show that the composition has an ergogenic effect. It does not increase physical performance acutely, but enhances the ability to perform repeated bouts of strenuous exercise. The placebo groups show no significant changes in performance and the psychical condition. The composition of the disclosure gives an improvement on all symptoms. The individuals of the groups who use the composition have more energy and feel better. Also, it can be concluded from the questionnaire that the test group feels younger, more optimistic, not depressed and happier.

Example 6Food Supplement for Athletes

(104) 1. Introduction

(105) This example was performed to investigate the influence of IAA on the level of IgF-1 with intensive training. The experiment was performed for 12 weeks.

(106) 2. Methods

(107) Twenty athletes entered the study. Other medication was excluded from the research. The period of the treatment was 12 weeks. They were not allowed to take any other medication or food supplements. They continued their level of training. After 2 weeks, the training was intensified by 25%. At baseline, at the start, after 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks, IGF-1 and safety parameters (liver tests, hematology) were determined. The group was divided into two groups of ten athletes. Group A received IAA; group B got a placebo.

(108) TABLE-US-00011 TABLE 7 Group A 6 male 4 female Group B 5 male 5 female

(109) All samples from one subject were analyzed in the same run to avoid intra-assay variability. GH was assayed with a chemiluminescence kit (Nichols Institute Diagnostics, San Juan Capistrano, Calif., USA). Quantification of IGF-1 was performed using an immunoradiometric assay (IRMA) (DSL-5600 ACTIVE, DSL, Germany GmbH, Germany). Intra- and inter-assay variance was: 4.0% and 9.2% for GH; 3.0% and 1.5% for IGF-1.

(110) Calculations and Statistics

(111) The total work output of every subject was calculated with a two-way ANOVA for repeated measures and the hormone values were analyzed with the Wilcoxon test. In all cases, 1 p value of <0.05 was accepted as significant.

(112) IAA

(113) The following schedule was used for the supplements. 1 IAA capsule contained 10 mg IAA, 100 mg NaCl, 140 mg WPC 70. One Glutamine capsule contained 500 mg glutamine. The amino acids that were taken are as described in Table 1.

(114) TABLE-US-00012 TABLE 8 Week 1 and 2 IAA in the morning 8 mg (2 capsules) In the evening 8 mg (2 capsules) Amino Acids in the morning 5200 mg (6 capsules) In the evening 5200 mg (6 capsules) Glutamine in the morning 500 mg (1 capsule) In the evening 500 mg (1 capsule) Week 3 until 12 IAA in the morning 4 mg (1 capsule) In the evening 4 mg (1 capsule) Amino Acids in the morning 2700 mg (3 capsules) In the evening 2700 mg (3 capsules) Glutamin in the morning 500 mg (1 capsule) In the evening 500 mg (1 capsule)
3. Results

(115) In group A, who were given IAA, the IgF-1 level significantly increased statistically over a period of 8 weeks with ca. 47.7% (Table 9). At point 0 (start of the study), the level had an average value of 27.9 nmol/l. At the end of the study, the IgF-1 level had an average of 41.2 nmol/l.

(116) In group B, who were given placebo, the level of IgF-1 shows a decrease. At point 0 (start of the study), the level had an average value of 27.3 nmol/l. At the end of the study, the IgF-1 level had an average of 17.8 nmol/l.

(117) TABLE-US-00013 TABLE 9 IgF-1 Level in nmol/l 0 1 2 3 4 5 6 Group A Average Test 27.9 35.8 38.9 42.5 41.7 41.4 41.2 group SD Test group 3.41 3.45 3.34 3.51 3.44 3.48 3.44 Group B Average placebo 27.3 27.1 24.1 22.6 20.5 19.2 17.8 group SD placebo group 3.42 3.51 3.47 3.43 3.45 3.39 3.24
4. Conclusions

(118) In the end, the group that received IAA performed better; they were more alert and focused. For the placebo group, the performances decreased over 12 weeks. The intensive training caused fatigue with this group, whereas the active group was still fit. IAA seams to be an effective product to increase the performance of athletes.

Example 7Treatment of Poor Growing Piglets with IAA

(119) 1. Introduction

(120) This Example is for testing the theory of the impaired functioning of the hypothalamic-pituitary-adrenal (HPA) axis by infection pressure and the influence of the treatment on health and growth.

(121) The trial was done in a well-managed farm with 1000 sows of the Dutch Landrace. Although the farm is well run, the technical performance is not optimal. There were latent problems with mortality and with growth rates of the piglets. There are too many poor-growing piglets, without fully understanding the technical or veterinary reason. There is no clear pathology to be seen on the farm.

(122) 2. Methods

(123) The farmer selected the poor-growing piglets for this weaning period. The piglets were weaned for two days at the start of the trial. There were three groups: the normal well-growing piglets, which served as a control group; the B group with 78 poor growers that are treated with 12.5 mg/kg/LW (Life Weight) IAA; and the X group with 52 poor growers that are not treated.

(124) Blood samples for IGF-1 measuring were taken from each group at the beginning of the trial and at the end of the trial. Two pens (13 piglets) of the B and X groups were weighed at the beginning and at the end of the trial. The active ingredient IAA was mixed in dextrose. Five grams of this mixture per piglet per day was added to the feed, which resulted in a dosage of 12.5 mg/kg/LW. The B group received the treatment five days after the first IGF-1 measuring.

(125) 3. Results

(126) Two days after weaning, there is no difference in IGF-1 levels between the three groups. IGF-1 levels are low, due to the stress of weaning. But already after one week, the farmer notices a clear difference between group B and group X. The piglets in group B are looking better, the bellies were better filled and the general appearance of the piglets started to look better than in group X.

(127) This phenomenon gets more obvious as the treatment continues. There are less poor growers in the B group, skin and hair is looking much better, less medical treatment is needed in group B.

(128) TABLE-US-00014 TABLE 10-1 IGF-1 levels in nmol/L IGF-1 measurement 1.sup.st 2.sup.nd Control group 1 0.8 14 2 4.3 16.6 3 7.9 25.4 4 6.1 30.3 5 3.9 31.7 total 23 118 average 4.6 23.6 Treated group B 1 3 25.2 2 2.7 28.9 3 6.1 25.5 4 5.3 29.9 5 5.7 16.9 total 22.8 126.4 average 4.56 25.28 Non-treated group X 1 2.9 11.3 2 4.3 21.6 3 1.6 died 4 13.1 20.8 5 1.3 15.1 total 23.2 68.8 average 4.64 17.2

(129) After 21 days of treatment, IGF-1 levels were measured again. The B group was on the level of the healthy control group (25.3 vs. 23.6 nmol/l) but was clearly higher than the non-treated group X (17.2 nmol/l).

(130) TABLE-US-00015 TABLE 10-2 Weight gain Weighing 1 Weighing 2 (kg) (kg) av. Weight Pen nr. Total average Total average gain B 3L 86 (n = 13) 6.62 150 (n = 11) 13.64 7.02 B 3R 88 (n = 13) 6.77 167 (n = 13) 12.85 6.08 X 4L 80 (n = 12) 6.67 141 (n = 11) 12.82 6.15 X 4R 92 (n = 13) 7.08 171 (n = 13) 13.15 6.08

(131) Pen B 3 L(eft) grew, on average, 850 grams more then their neighbors from pen X 4 L(eft). Pen B 3 R(ight) gained, on average, as much weight as their neighbors from pen X 4 R(ight), but weighed 310 grams less at the start of the trial. On average, the treated group gained (for the pens that were weighed) almost 0.5 kg more over a period of 21 days.

(132) After the treatment is stopped, the piglets from group B continue to perform better than group X.

(133) Poor-growing piglets start to look and grow better than the non-treated group. These characteristics are clearly coupled with a higher IGF-1 level in the treated groups. The results of this trial confirm that treatment for 14-21 days with 12.5 mg/kg/LW IAA effectively restores IGF-1 levels in poor-growing piglets. There is enough evidence to believe that once this level is restored to normal, these piglets will catch up the lost growth and will do well during the fattening period, without continuing the treatment.

Example 8Treatment of Growing Laying Hens with IAA

(134) 1. Introduction

(135) Following the successful treatment of poor-growing piglets in Example 6, the same treatment was tested on poor-growing laying hens, since this is a big problem in rearing laying hens. The flocks do not grow uniformly, and about 10-15% of the birds have a too poor growing performance. The same logic as for the poor-growing piglets (infections raise the cytokine levels and lower the IGF-1, see also Example 8) can be applied to rearing laying hens.

(136) 2. Methods

(137) Growing laying hens of 10 weeks old were selected in the flock to create four groups of ten hens: Group GB: Ten normally growing hens, not treated Group GNA: Ten normally growing hens, treated with 10 mg/kg/LW IAA (LW=live weight) Group SB: Ten poorly growing hens, not treated Group SNA: 10 poorly growing hens, treated with 10 mg/kg/LW IAA.

(138) In this example, not only the poor-growing birds were treated, but also normally growing animals. The birds receive daily forced oral treatment with a capsule that contains IAA. The different groups are weighed every week. Treatment is continued at least until the hens start laying, to see if the period of onset of laying is influenced by the treatment.

(139) 3. Results

(140) From the first week on, a better weight gain is seen in both the treated groups. The difference in weight gain is consistent over the first four weeks of the trial. The group GNA has now about 2 weeks advanced over the normal rearing schedule and the SNA group has caught back up to schedule. The last couple of weeks of the experiment were very stressful for the birds, since it was very hot, and the birds were vaccinated against ILT.

(141) TABLE-US-00016 TABLE 11 Group no. weight weight gain weight weight gain GB GNA 1 690.5 740 2 802 111.5 883 143 3 878 76 996 113 4 995 117 1095 99 5 1098 103 1222 127 total weight 407.5 482 gain SB SNA 1 498.5 489.5 2 585 86.5 598 108.5 3 685 100 718 120 4 790 105 885 167 5 907 117 995 110 total weight 408.5 505.5 gain

(142) The weight gain already after one week gave a clear indication that IAA has the same effect as seen in piglets and calves. Therefore, it is assumed that the mode of action is the same in chickens as in mammals. The group SNA shows the highest weight gain in this test period. There seems to be a phenomenon of catching up of lost growth, after the IAA has normalized the function of the HPA-axis.

(143) It can also be concluded that IAA has an action in normal birds, and that the IAA does not seem to induce a resistance to its mode of action at the dosage of 10 mg/kg/LW.

(144) So the results of this example indicate that the use of IAA can bring poor-growing hens back to the normal rearing schedule and prevent loss of animals for normal production, can speed up the rearing process and deliver much stronger birds at the end of the rearing period.

Example 9Treatment of Survivors of Porcine Reproductive and Respiratory Syndrome Virus with IAA

(145) 1. Introduction

(146) From experience with treatment of humans with Chronic Fatigue Syndrome (CFS), it follows that efficacy of the treatment is closely linked to the restoration of normal IGF-1 serum levels. Growth rates in most animals, including pigs, are also correlated with IGF-1 status. It is known that IGF-1 concentrations are affected by disease and immunological challenge. Some of the piglets surviving PRRSV infections show resembling symptoms to patients with CFS (no drive to move or to eat, looking sad, etc.).

(147) Therefore, IGF-1 concentrations were tested in PRRSV piglets and they were treated with IAA.

(148) 2. Methods

(149) The experiment was performed with Belgian Land Race piglets that had a history of PRRSV. The piglets were weaned at four weeks and relocated, twelve piglets per pen. IGF-1 levels from three different groups were tested at the age of five weeks.

(150) Group P

(151) These are the pigs with the big problems. They are looking poor, have a low weight, bad color and some of them have Staphylococcus infections. It was the firm belief of the farmer and the veterinarian that these piglets would not reach the end of the fattening cycle. Blood samples were taken at random from five of the twelve piglets.

(152) TABLE-US-00017 TABLE 12 Ref. IGF-1 nmol/l Piglet 1 2.3 Piglet 2 <0.5 Piglet 3 <0.5 Piglet 4 2.0 Piglet 5 1.8

(153) The results indicate a severe affection of the IGF-1 levels and a clear correlation between poor growth and health and IGF-1 concentration.

(154) Group R

(155) These piglets were looking the best. They received a special pre-starter feed before weaning. The IGF-1 concentration of five piglets was checked at random.

(156) TABLE-US-00018 TABLE 13 Ref. IGF-1 nmol/l Piglet 1 4.3 Piglet 2 1.0 Piglet 3 7.3 Piglet 4 22.2 Piglet 5 14.7

(157) The special pre-starter seems to boost the IGF-1 levels in this group.

(158) Group T

(159) These were normal looking piglets. They received a regular piglet starter feed before weaning

(160) TABLE-US-00019 TABLE 14 Ref. IGF-1 nmol/l Piglet 1 2.7 Piglet 2 <0.5 Piglet 3 0.8 Piglet 4 0.5 Piglet 5 3.5

(161) These piglets have normal growth, but not as good as the R group. This is also reflected in the lower IGF-1 levels.

(162) 3. Treatment

(163) After seeing the low IGF-1 levels in group P, it was decided to give them the same treatment as in humans with CFS. The average weight of the piglets would have been around seven kilos at five weeks of age. They were treated with 125 mg of the composition comprising 5 mg IAA and 120 mg NaCl and WPC 70 for ten days. The product was fed in a trough, mixed in liquid feed. All the piglets ate from the same trough.

(164) 4. Results

(165) After ten days of treatment, random blood samples were taken from the different groups. A code was added for the size of the animals (K=small; N=normal; Z=heavy).

(166) Group P

(167) All twelve pigs were still alive and their condition had dramatically improved. They all had a nice pink color, no more stiff hair, all the ears were in normal position, no piglets suffered from Staphylococcus infection anymore. They had gained very much in weight and in muscle growth and had caught up very much to the average of the rest of the normal litter mates. All external signals for good health were now present.

(168) At random, IGF-1 concentrations of five piglets of this group were measured.

(169) TABLE-US-00020 TABLE 15 Ref. IGF-1 nmol/l Piglet PN 0.8 Piglet PK <0.5 Piglet PZ 4.2 Piglet PK <0.5 Piglet PN <0.5

(170) The IGF-1 levels do not reflect the dramatic improvement in health, weight and condition of the piglets. The IGF-1 levels did not rise. The same is seen in CFS patients. The patient feels much better after a couple of weeks of treatment, but there is no increase yet in IGF-1 concentration. This increase in IGF-1 occurs only a couple of weeks later. It is assumed that all the extra IGF-1 production is used in the process of the extra-growth. Still, there is a clear link between individual weight and IGF-1 levels.

(171) Group R

(172) These piglets are now on a normal commercial piglet starter diet, so no longer on pre-starter formula. This group is growing normally. No particular signs are present. Blood samples were taken at random from six piglets.

(173) TABLE-US-00021 TABLE 16 Ref. IGF-1 nmol/l Piglet RK 11.4 Piglet RN 11.4 Piglet RN 32.6 Piglet RZ 18.7 Piglet RK 9.8 Piglet RZ 10.4

(174) The average IGF-1 concentration rose in two weeks from 9.9 nmol/l to 15.72 nmol/l.

(175) Group T

(176) These piglets continue to be on their original piglet starter feed. This group is also growing normally without any particular symptoms. Five blood samples were taken at random for the IGF-1 concentration.

(177) TABLE-US-00022 TABLE 17 Ref. IGF-1 nmol/l Piglet TZ 10.5 Piglet TN 6.0 Piglet TN 3.1 Piglet TN 10.9 Piglet TZ 16.3

(178) The average IGF-1 concentration rose in two weeks from 1.6 to 9.36 nmol.

(179) Three weeks later, blood samples were again taken, but only from the problem group.

(180) TABLE-US-00023 TABLE 18 Ref. IGF-1 nmol/l Piglet 1 30.0 Piglet 2 23.4 Piglet 3 65.1 Piglet 4 13.7 Piglet 5 60.1 Piglet 6 36.9 Piglet 7 55.6

(181) These results clearly show that the piglets' IGF-1 production was increased, even though the treatment was stopped.

(182) 5. Discussion

(183) The treatment with IAA dramatically improved the condition of the problem piglets. Their immune system eliminated the Staphylococcus infections. This improvement is not immediately reflected in the IGF-1 serum concentration, but three weeks after the treatment was stopped, the IGF-1 level had risen to what is believed to be almost normal levels.

(184) It is known that multiple infections raise TNF-alfa, IL-1 and IL-6 six to much higher levels than single infections. It is also known that in severe infections, TNF-alfa, IL-1 and IL-6 modulate reactions that prevent the transformation of GH into IGF-1. This leads to high serum concentrations of GH and low serum concentrations of IGF-1. These high levels of GH will give a feedback reaction to the HPA-axis to produce less GH-RH. For some animals, as for human CFS patients, after the source of infection is gone, the HPA axis stays in a sleeping mode, induced by the feedback of the high GH-levels. IAA provides the necessary stimulus to put the HPA-axis again in the active mode and to restore normal IGF-1 levels, which in their turn lead to a normally functioning immune system and a normal growth, or for CFS patients normal levels of energy.

(185) Please tell us if the below-mentioned activities cause you nuisance, yes or no, and how far during the last seven days of your illness, or if you cannot do them at all. Please do not pass over any symbol.

(186) TABLE-US-00024 TABLE 19 Annex 2 Activity 0 1 2 3 4 toilet (at least one a day) Brush one's teeth 0 1 2 3 4 Hair comb/brush 0 1 2 3 4 Shaving/put on make-up 0 1 2 3 4 Take a bath (at least 2 a week) Getting in/out of the bath 0 1 2 3 4 Manipulate of the taps 0 1 2 3 4 To wash the under limbs 0 1 2 3 4 To wash the back 0 1 2 3 4 Hair washing 0 1 2 3 4 toilet Manipulate clothes 0 1 2 3 4 Getting up and down the toilet 0 1 2 3 4 To use the toilet paper 0 1 2 3 4 To dress (at least 1 a day) Manipulate the clothes 0 1 2 3 4 manipulate underwear//bra 0 1 2 3 4 Manipulate socks/shoes 0 1 2 3 4 Manipulate shirt/blouse 0 1 2 3 4 Manipulate pants/panty 0 1 2 3 4 Manipulate coat 0 1 2 3 4 Prepare a meal (at least once a day) To open packings 0 1 2 3 4 To use tin opener (manual) 0 1 2 3 4 To clean/cut vegetables 0 1 2 3 4 To stit with resistance 0 1 2 3 4 To hold an electric mixer 0 1 2 3 4 To open an oven door 0 1 2 3 4 To carry a big pot of water 0 1 2 3 4 To pour milk out off full cardboard 0 1 2 3 4 To cover the table 0 1 2 3 4 To clean (at least once a week) To pick up subjects 0 1 2 3 4 To clean mess products 0 1 2 3 4 To make the bead ready 0 1 2 3 4 To dust 0 1 2 3 4 To mop 0 1 2 3 4 To brush 0 1 2 3 4 To vacuum a room 0 1 2 3 4 To clean the bath 0 1 2 3 4 To scrub the floor 0 1 2 3 4 Window cleaning 0 1 2 3 4 To clean the oven 0 1 2 3 4 Dish washing 0 1 2 3 4 To put the dishes in the cupboard 0 1 2 3 4 Take away the garbage 0 1 2 3 4 Washing (at least once a week) To carry a full bucket with washings 0 1 2 3 4 To use the washing machine 0 1 2 3 4 Get off wet clothes 0 1 2 3 4 To fold clothes 0 1 2 3 4 To put the clothes on a coat hanger 0 1 2 3 4 To iron 0 1 2 3 4 Garden maintenance To cut the grass 0 1 2 3 4 To rake 0 1 2 3 4 To dig 0 1 2 3 4 To weed 0 1 2 3 4 To replace lamps 0 1 2 3 4 To hammer nails 0 1 2 3 4 To use a screwdriver 0 1 2 3 4 transport Getting in/out of a car 0 1 2 3 4 Getting in/out bus/train 0 1 2 3 4 To understand traffic signs 0 1 2 3 4 Mobility/activity Walk a little 0 1 2 3 4 Getting up the stairs 0 1 2 3 4 To stand for one hour 0 1 2 3 4 To sit for 2 hours 0 1 2 3 4 To bend forwards 0 1 2 3 4 To bend for picking up something 0 1 2 3 4 To kneel 0 1 2 3 4 To crawl 0 1 2 3 4 Household tasks To push a shopping cart 0 1 2 3 4 Visit a supermarket 0 1 2 3 4 To pay the bill 0 1 2 3 4 To put away the shopping 0 1 2 3 4 To carry the shopping 0 1 2 3 4 To take away the shopping 0 1 2 3 4 Physical manipulations To open door locks 0 1 2 3 4 To use door handles 0 1 2 3 4 To open/close the curtains 0 1 2 3 4 To open/close the blinds 0 1 2 3 4 To open/close the window 0 1 2 3 4 Communication Understands every communication 0 1 2 3 4 Understands the newspaper 0 1 2 3 4 Understands television 0 1 2 3 4 To write a letter (a4) 0 1 2 3 4 Computer tasks for 30 minutes 0 1 2 3 4 Telephone conversation 0 1 2 3 4