Novel use of 3,3'-diindolylmethane

The present invention relates to a novel use of 3,3'-diindolylmethane (CAS 1968-05-4) and/or its derivatives; in particular it relates to the use of 3,3'-diindolylmethane and/or its derivatives in the form of a pharmaceutical and/or nutritional composition to retard or delay typical signs of aging and/or age-related pathological changes.

3,3'-diindolylmethane (DIM) is a known plant indol derivative which can be found in cruciferous vegetables including cabbage, broccoli, Brussels sprouts, and cauliflower. DIM is formed from a condensation reaction of two molecules of its precursor, indole-3-carbinol (I3C).

DIM has been shown to have several beneficial properties such as cardio and brain protective activities, cancer prevention, and benefits for perimenopausal women in premenstrual syndrome (PMS), endometriosis, and cervical dysplasia. Women on estrogen replacement (HRT) also benefit from DIM supplementation, as well as men with estrogen-related conditions, including prostate hypertrophy. I3C and DIM have also been shown to have multiple anti-cancer effects both in in-vivo and in-vitro models.

Some of the most important hallmarks of aging are described in the following paragraphs.

1. General phenotypic changes

Mammalian aging is phenotypically characterized by hair loss, graying, wrinkles, general weight loss and abnormal spinal curvature (kyphosis). General underlying mechanisms are the loss of the ability of specific cell division and apoptosis.

2. Oxidative damage

A large body of evidence indicates that oxidative stress and other toxic chemical insults support pathological findings, such as e.g. degenerative processes and/or age related diseases. Reactive oxygen species (ROS) are one of the causes of age-related cellular degeneration throughout the organism. Free radicals or ROS, a product of cellular respiration, normally exist in three forms: the superoxide anion (O Y), hydrogen peroxide (H ₂ O ₂ ) and the hydroxyl radical (OH). These species are very unstable, reacting with and thereby damaging proteins, lipids and DNA. Endogenous, cellular enzymes, such as

catalase, copper/zinc and manganese superoxide dismutase (SOD1 and SOD2, respectively) and peroxidase convert ROS to neutral, non-reactive molecules. Accordingly these substances are important contributors to cellular stability and survival. The importance of these endogenous antioxidants in cochlear cell survival was demonstrated in SOD1 transgenic mice in which early hearing loss and degeneration of auditory neurons and hairs cells was observed (McFadden et al., 1999a, McFadden et al., 1999b, McFadden et al., 2001 and Keithley et al., 2005). The stria vascularis is much less affected, so it seems that each oxidative tissue may use different antioxidant mechanisms to clear ROS. Although the absence of SOD1 results in a great deal of degeneration, half as much SOD1 was sufficient to maintain hearing and cochlear morphology (Keithley et al., 2005). Mitochondrial DNA (mtDNA), located near the site of oxidative phosphorylation in the inner membrane of mitochondria, is especially vulnerable (Miquel et al., 1980, Barja, 2004 and Sastre et al., 2003).

3. Loss of mitochondrial function

Loss of mitochondrial function has been described as a major causal factor in aging (Loeb et al., PNAS 102, 18769-18770, 2005). All factors that lead to mitochondrial damage, e. g. oxidative stress, accumulation of mutations, also contribute to aging. Mitochondrial outer membrane permeabilization and cytochrome C release trigger apoptotic pathways (Green & Kroemer, Science 305, 626-629, 2004). Mice carrying a homozygous mutation leading to a defect in the proofreading function of mitochondrial DNA polymerase-γ show typical hallmarks of accelerated aging (Trifunovic et al., Nature 429, 417-423, 2004; Kujoth et al., Science 309, 481-484, 2005; Mouse model for aging, US Patent No. 60/663930, 2005). It has been suggested that - instead of reactive oxygen induced damage - mitochondrial respiratory chain dysfunction is the primary inducer of premature aging in these mice (Trifunovic et al., PNAS 102, 17993-17998, 2005).

4. Age related hearing loss

Approximately one third of the American population in the age of 65 to 75 suffers from hearing loss and 50 % of people above 75 have some degree of hearing loss, as indicated by the National Institute of Deafness and Other Communication Disorders (NIDCD). Age- related hearing loss, or presbycusis, affecting many species, is characterized by an age- related progressive decline of auditory function, and is an irreversible process. A hearing loss (also hearing impairment) exists when an individual is not sensitive to the sounds

normally heard by its kind. It is not yet fully understood why hearing loss occurs, and many factors have been demonstrated to be the risk factor. Hearing loss arises mainly from the degeneration of hair cells or spiral ganglion (SG) cells in the cochlea. When the tiny hairs inside the cochlea are damaged or die, which often happens as people age, hearing loss occurs. Structural changes in the inner ear, such as the degeneration of sensory cells, auditory neurons and cells of the stria vascularis, also contribute to hearing loss. In humans, the cochlear degeneration can be attributed to the accumulated effects of numerous insults including exposure to acoustic trauma and ototoxic drugs. However, hearing loss is a common phenomenon in mammals, regardless of noise and toxin exposure. In addition, hereditary factors, blood supply dysfunction due to heart disease, high blood pressure, diabetes, or other circulatory problems are all risk factors leading to hearing loss.

Currently, no dietary or medical interventions exist which have proved efficacy on treatment or prevention of hearing loss. Magnesium was used to protect ears from noise-induced hearing loss. It is not clear how magnesium might protect hearing and studies in animals suggest that magnesium deficiency can increase the stress on cells involved with hearing and thereby make them more susceptible to damage caused by intense noise. However, human magnesium deficiency is rare, so it is possible that supplemental magnesium acts in some entirely different way.

Ginkgo extract was used to treat sudden hearing loss, or so called unilateral idiopathic sudden hearing loss. Ginkgo extract, which is shown to improve circulation, has been tested as treatment for the sudden hearing loss. In a double-blind study, ginkgo was compared to pentoxifylline, a circulation-enhancing drug used in Germany for the treatment of sudden hearing loss and the results indicate that ginkgo was at least as effective as the medication.

Antioxidant supplementation was suggested as promising measures for hearing loss prevention, and commonly used antioxidants are citrus bioflavonoids, coenzyme Q10, lipoic acid, lutein, lycopene, oligomeric proanthocyanidins (OPCs), vitamin C and vitamin E. However, there is lack of scientific evidence on the effect of antioxidants in preventing hearing loss.

5. Sarcopenia

Sarcopenia is the degenerative loss of skeletal muscle mass and strength in senescence. About a third of muscle mass is lost in old age. The level of sarcopenia can be so severe that it prevents an elderly person from living an independent life and they require constant assistance and care. Sarcopenia is an important independent predictor of disability in population-based studies, linked to poor balance, gait speed, falls, and fractures.

It was an object of the following invention to provide a composition to retard or delay typical signs of aging and/or age-related pathological changes.

It has surprisingly been found that the object of the present invention is achieved by a composition for the retard or delay of typical signs of aging and/or age-related pathological changes in mammals characterized in that the composition contains 3,3'-diindolylmethane.

The terms "3,3'-diindolylmethane" and "DIM" as used herein also include derivatives of 3,3'-diindolylmethane according to the following chemical structure:

wherein the residues R1 , R2 and R3 are independently chosen from the following groups:

• R1 : H, OCH ₃ , CH ₃ with the provisio that at least 3 of the 5 residues R1 are H;

• R2: CH ₃ -(CH ₂ ) _n -CH ₃ wherein n = 0 - 16;

• R3: H, CH ₃ -(CH ₂ ) _n -R4 wherein n = 0 - 16 and R4 = H or COOH;

According to the present invention 3,3'-diindolylmethane may be of synthetic origin or it may be isolated from plant extracts, such as extracts of cruciferous vegetables (including their seeds and/or sprouts). It may also be advantageous to use extracts containing 3,3'-

diindolylmethane, for example extracts obtainable from cruciferous vegetables. Based on the particular extraction procedure the amount of phenolic compounds in a plant extract can be easily adjusted by a person skilled in the art. It is preferred if the amount of 3,3'- diindolylmethane in a plant extract according to the present invention is in the range of from 20 to 80 % by weight, more preferred from 30 to 50 % by weight, each based on the total weight of the extract.

The terms "typical signs of aging" and "age-related pathological changes" as used herein include besides the most important hallmarks of aging as described in the paragraphs 1. to 5. as described above also the following symptoms:

• Loss of balance:

Balance, or one's sense of equilibrium, is controlled through the vestibular system that is also contained in the inner ear. The vestibular organs share the temporal bone space with the cochlea. These organs also share the same fluid that is in the cochlea. Balance and equilibrium help us stay erect when standing, know where we are in relation to gravity, and help us walk, run, and move without falling. The functioning of the vestibular system depends on information from many systems, hearing as well as vision and muscle feedback. Problems with balance occur whenever there is a disruption in any of the vestibular, visual, proprioceptive or cognitive systems. Symptoms of a balance disorder may include dizziness, vertigo (spinning), disequilibrium (off balance and falls), pre-syncope (light headedness). Many different terms are often used to describe what is collectively known as dizziness. Common descriptions include words such as lightheaded, floating, whoozy, giddy, confused, helpless or fuzzy. Vertigo, Disequilibrium and Pre-syncope are the terms in use by most doctors.

Thus, in one aspect, the present invention relates to the use of 3,3'-diindolylmethane to hinder and/or reduce (age-related) loss of balance and/or dizziness, and to the use of 3,3'- diindolylmethane in the manufacture of a composition such as a food or beverage, or a supplement for food or beverage, for hindering and/or reducing (age-related) loss of balance and/or dizziness.

• Loss of wellness and/or mobility:

Wellness denotes the general state of physical and mental health of an organism, especially when maintained by proper diet; exercise and habits (see The American Heritage® Stedman's Medical Dictionary, publ. Houghton Mifflin Co, 2002). However, the pre-conditions for maintaining the wellness state are not always fulfilled in today's population. The term "wellness state" as used herein denotes to the actual state of physical and mental health of an individual and may therefore vary from being completely healthy to being completely ill and can accordingly be promoted any time when it differs from being completely healthy. The wellness state can be determined, inter alias, by evaluating parameters (body functions) such as blood pressure, heart rate, body fat composition, pulmonary function, liver function, brain function and levels of physiologically vital components in body fluids etc. as e.g. disclosed in US patent 5,692,501. Mobility denotes the general state of being able to move and accordingly live an independent life without the need of constant assistance and care.

Thus, in a further aspect, the present invention relates to the use of 3,3'-diindolylmethane to promote the wellness state of a mammal, and to the use of 3,3'-diindolylmethane in the manufacture of a composition such as a food or beverage, or a supplement for food or beverage, for promoting the wellness state of a mammal. Such promotion of the wellness state is especially desirable in the status of convalescence, i.e. during recovery of health and strength after illness.

The composition according to the invention is preferably a dietary supplement, a food or feed additive, a functional food or feed, a food or feed premix, and/or a beverage.

While for the purpose of the present invention mammals are preferably humans, the invention is not limited to humans but includes other mammals, such as dromedaries, camels, elephants, and horses and pets such as dogs, cats and/or small animals.

For the purposes of the invention, 3,3'-diindolylmethane (DIM) may be administered to, e.g., a human adult (weighing about 70 kg) in an amount of up to about 10 to 1000 mg/day in one or several dosage units or servings. In a particular embodiment of the invention, the

dosage for a human adult (weighing about 70 kg) is up to about 500 mg/day, especially up to about 30 to 300 mg/day. If administered in a food or beverage the amount of DIM contained therein is suitably no less than about 100 mg per serving. If DIM is administered as a pharmaceutical formulation such formulation may contain up to about 500 mg per solid dosage unit, e.g. per capsule.

The term "serving" as used herein denotes an amount of food and/or beverage normally ingested by a human adult with a meal at a time and may range, e.g., from about 100 g to about 500 g food and/or beverage.

The composition according to the present invention can preferably be a nutraceutical composition. The term "nutraceutical" as used herein denotes usefulness in both, the nutritional and pharmaceutical field of application. Thus, "nutraceutical compositions" according to the present invention can serve as supplements to food, feed and beverages, dietary supplements and as pharmaceutical formulations which may be solid - such as capsules - or liquid - such as solutions or suspensions. It is evident from the foregoing that the term "nutraceutical composition" also comprises food, feed and beverages containing DIM.

A multi-vitamin and mineral supplement may be added to the compositions according to the present invention, e.g. to maintain a good balanced nutrition or to obtain an adequate amount of an essential nutrient missing in some diets. The multi-vitamin and mineral supplement may also be useful for disease prevention and protection against nutritional losses and deficiencies due to lifestyle patterns and common inadequate dietary patterns sometimes observed in diabetes.

The composition according to the present invention can be a food or beverage composition. Beverages can be e.g. sports drinks, energy drinks or other soft drinks, or any other suitable beverage preparation, e.g. yoghurt drinks, hot beverages or soups. In a preferred embodiment of the present invention the beverage is an "instant beverage", i.e. a beverage which is produced - normally by the consumer themselves - by stirring a powder into a liquid, usually milk or water.

By "sports drink" a beverage is meant which is consumed before, during and/or after physical exercise, mainly to hydrate as well as to (re-) store electrolytes, sugar and other nutrients. Sports drinks are usually isotonic, meaning they contain the same proportions of nutrients as found in the human body.

Energy drinks are beverages which contain (legal) stimulants, vitamins (especially B vitamins) and/or minerals with the intent to give the user a burst of energy. Common ingredients include caffeine, guarana (caffeine from the Guarana plant) and/or taurine, various forms of ginseng, maltodextrin, inositol, carnitine, creatine, glucuronolactone, coenzyme Q10 and/or ginkgo biloba. Some may contain high levels of sugar, or glucose, whereas others are sweetened completely or partially with a sugar alcohol and/or an artificial sweetener like Ca-cyclamate or Aspartame. Many such beverages are flavored and/or colored.

A soft drink is a drink that does not contain alcohol. In general, the term is used only for cold beverages. Hot chocolate, tea, and coffee are not considered soft drinks. The term includes carbonated and non-carbonated drinks, e.g. mineral water or so-called "near water drinks", i.e. water-based beverages which have an additional benefit, e.g. a special flavor and/or further (functional) ingredients. One simple example for a "near water drink" is a mixture of water with very little juice.

If the composition is prepared in form of one of the following food articles it is according to the present invention advantageous if the amount of DIM in the composition is selected from the ranges given in the following table:

If the composition is prepared in form of a capsule it is according to the present invention advantageous if the amount of DIM is selected from the ranges given in the following table:

The invention is further illustrated by Figures 1 to 4:

Figure 1 shows the effect of DIM on total body weights in D257A mice. Each bar represents the average of at least 4 independent mice + SEM. The y-axis shows the body weight in g. Black bars represent male, grey bars female mice. Values are shown for: I: Control; II: DIM;

Figure 2 shows the effect of DIM on femur bone mineral density in female D257A mice (y- axis in mg/cm ² ). Each bar represents the average of 4 independent mice + SEM. Values are shown for: I: Control; II: DIM;

Figure 3 shows the effect of DIM on gastrocnemius weights in D257A mice (y-axis in mg). The male control represents the average of 2 mice, the female control of 3 mice + SEM; DIM treatment represents the average of 6 male mice + SEM and 2 female mice. Values are shown for:

I: Control;

II: DIM;

Figure 4 shows the effect of several compounds on age related hearing loss (presbycusis) in D257A mice, an accelerated ageing mouse model, which exhibit significantly reduced hearing function.

The auditory-evoked brain stem response threshold (hearing threshold) in decibels (dB) 8 kHz is shown on the y-axis. Black circles represent male, grey circles female D257A mice. ∞ means that no response was obtained. Values are shown for: I: Control;

II: DIM;

The invention is further illustrated by the following examples.

Examples:

Example 1

The efficacy of DIM was demonstrated as shown by tests set forth below. The test system was D257A-/- (D257A) mice that express a proofreading-deficient version of the mitochondrial DNA polymerase γ (POLG). These mice display features of accelerated aging including a decrease in body weight, hair graying, decrease in bone mineral density (BMD), muscle loss and age related hearing loss (Kujoth et al., Science 309, 481-484, 2005).

Compound feeding

Heterozygous D257A (-/+) mice were bred to generate homozogous D257A (-/-) mice, denoted D257A hereafter. Animals were identified with specific numbering and ear clippings. D257A mice were placed in various dietary regimens following PCR based genotyping and weaning at 3 weeks of age. Mice received a diet based on AIN-93M maintenance purified diet (TestDiet, Richmond, USA), with neither no supplementation (Control group), or supplementation with DIM (40 mg/kg, BioResponse LLC Boulder, CO).

Body weight

The body weight of all surviving D257A mice was determined at 9 months of age.

Gastrocnemius weight

Gastrocnemius weights were determined from the surviving D257A mice at 9 months of age following euthanasia to a standard necropsy protocol.

Assessment of hearing function

Hearing function was tested in 9 month-old D257A mice receiving the various compounds. Auditory brainstem responses (ABRs) were measured with a tone burst stimulus (8, 16 and 32 kHz) using an ABR recording system (Intelligent Hearing System, Miami FL). Animals were anesthetized with a mixture of xylazine hydrochloride (10 mg/kg, i.m.) and ketamine hydrochloride (40 mg/kg, i.m.), and needle electrodes were placed subcutaneously at the vertex (active electrode), beneath the pinna of the measured ear (reference electrode), and beneath the opposite ear (ground). The stimulus duration, presentation rate, and rise/fall time was 3 ms, 19.3/s, and 1 ms respectively. Responses of 1024 sweeps were averaged at each intensity level (5 dB steps) to access the hearing threshold. The hearing threshold was defined as the lowest intensity level at which a clear reproducible waveform was visible in the trace. Means of ABR thresholds were compared at 8, 16 and 32 kHz.

Results

Weight loss is a general hallmark of the late stages of aging in mammalians. Therefore, we first looked for changes in total body weights of the D257A mice treated with DIM and other compounds in comparison to untreated control. DIM treated male D257A mice tended to have a higher total body weight compared to controls treated mice (Fig. 1 ). Aging in rodents and humans is characterized by loss of muscle mass (sarcopenia).

D257A mice displayed age related loss of skeletal muscle (Kujoth et al., Science 309, 481- 484, 2005). Therefore, we tested whether DIM could inhibit age related muscle loss. We found that gastrocnemius weight in male D257A mice fed with DIM tended to be higher as compared to control treated mice (Fig.3). The gastrocnemius weight of these mice was close to wild-type.

Age-related loss of auditory function and cochlear degeneration in D257A mice at the age of 9 months were described (Kujoth et al., Science. 2005 JuI 15;309(5733):481-4). Based on these data we tested whether DIM could help to prevent presbycusis. With a frequency of 8 kHz no deaf mice were present of the 8 mice treated with DIM. In the untreated control group 3 out of 8 mice were deaf. In the DIM treated group the hearing threshold was approximately 10 dB. In the control group it was approximately 40 dB. At 32 kH the percentage of deaf mice was also significantly lower in the DIM group compared to the control group. Taken together, DIM showed a protective effect on age related hearing loss.

Pharmaceutical compositions may be prepared by conventional formulation procedures. Example 1 Soft gelatin capsule

Soft gelatin capsules are prepared by conventional procedures containing as active ingredient 100 to 200 mg of DIM per capsule.

Example 2 Hard gelatin capsule

Hard gelatin capsules are prepared by conventional procedures containing as active ingredient 100 to 200 mg of DIM per capsule.

Example 3 Food items may be prepared by conventional procedures containing DIM in an amount of 10 mg to 400 mg per serving. Examples of such food items are soft drinks, bread, cookies, yoghurt, ice cream, and sweets.