HK1038196A - Neuroprotective composition for the prevention and/or treatment of nervous and behavioural alterations due to anxiety states or depression - Google Patents

Description

For preventing and/or treating nervous and behavioral disorders due to anxiety states or depression Neuroprotective composition

The present invention relates to a composition for preventing and/or treating neurological and behavioral changes due to anxiety states or depression.

Thus, the composition may take the form of a dietary supplement or an effective drug and act, depending on whether the composition is supportive or prophylactic or strictly therapeutic in relation to the particular individual to which it is administered.

More particularly, the present invention relates to a composition comprising, in combination: (a) acetyl L-carnitine or a pharmaceutically acceptable salt thereof, optionally in combination with at least one other "carnitine", wherein "carnitine" means L-carnitine or an alkanoyl L-carnitine selected from the group consisting of propionyl L-carnitine, valeryl L-carnitine and isovaleryl L-carnitine, or a pharmaceutically acceptable salt thereof; and (b)1, 3, 4, 6, 8, 13-hexahydroxy-10, 11-dimethylphenanthro [1, 10, 9, 8-opqra ] perylene-7, 14-dione (hypericin) or a hypericum extract containing at least 0.3% by weight hypericin (hypericum perforatum, "Saint-John's-word)).

The novel compositions can be administered orally, parenterally, rectally or transdermally to humans and animals as pharmaceutical compositions, food additives or phytotherapeutic preparations.

The use of hypericum extract is well known in the medical community due to its ability to combat a range of pathological changes including conditions such as depression, anxiety, insomnia, neuralgia, migraine, dyspepsia and sciatica, as well as inflammatory and scarring processes.

Hypericum contains various active ingredients in its extract, the most interesting being naphthodianthrones, flavonoids, phloroglucinols, xanthones and various volatile oils.

The major naphthodianthrones are hypericin, pseudohypericin, and emodin anthrone.

The major flavonoids are proanthocyanidins present in trimers and tetramers or multimers of catechins and epicatechins.

The phloroglucinol compounds include prenylated derivatives of phloroglucinol, hyperforin and perforin.

In addition to caffeic acid, coumaric acid, ferulic acid, essential oils are present, which are mainly present in monoterpenes and sesquiterpenes.

Of all the components, hypericin has proved to be of interest compared to anything else, due to its ease of characterisation and its specific action.

In fact, the antidepressant, anxiolytic, scar healing and antiviral activity of hypericum extracts is mainly attributed to hypericin.

Recent studies have shown that hypericin inhibits the reuptake of monoaminooxidase and brain 5-hydroxytryptamine and reduces the expression of cytokines, particularly interleukin-6.

Carnitine-like compounds exhibit many types of activity, generally capable of activating processes necessary for ATP synthesis via fatty acid β -oxidation and of increasing the stabilization of cell membranes against oxidative processes of the cardiovascular and central systems.

Acetyl L-carnitine improves behavioral indices in rats and electroencephalographic abnormalities in elderly patients.

Nerve regeneration can also be improved by administering carnitine-based compounds.

Surprisingly, it has now been found that a composition containing characteristic components in combination with one another: (a) acetyl L-carnitine or a pharmaceutically acceptable salt thereof; and (b) hypericin, which is extremely effective in the prevention and/or treatment of neurological disorders associated with anxiety or depressive states due to the effective synergism of its ingredients.

It has further been found that component (a) may further comprise another "carnitine", which is selected from the group consisting of L-carnitine, propionyl L-carnitine, valeryl L-carnitine, isovaleryl L-carnitine or their pharmaceutically acceptable salts or mixtures thereof, and that component (b) may also consist of an extract of hypericum (hypericum perforatum, "john wort") containing at least 0.3% by weight of hypericin.

The weight ratio of the components (a) to (b) is 1: 0.01 to 1: 1. When component (b) of the composition may be present in the form of an extract of the plant product containing it, said plant product comprises the flowers, buds and apical leaves of the hypericum plant.

Toxicology test

In these tests, it was demonstrated that a high dose of a carnitine mixture, or acetyl L-carnitine, or hypericum extract or hypericin or a combination of the above ingredients, administered alone, could be well tolerated after a single dose administration and after 30 days of continuous administration. The tests were carried out in rats and mice, with oral administration of a dose of 2g/kg of a carnitine mixture (consisting of a combination of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same weight ratio to each other), or 2g/kg of acetyl L-carnitine, or 1g/kg of a hypericum extract containing about 0.3% hypericin, or 3mg/kg of hypericin, or a combination of these products, i.e. a mixture of 1g/kg of carnitine or a combination of 1g/kg of acetyl L-carnitine with 600mg/kg of hypericum extract or 1mg/kg of hypericin, with no signs of toxicity or mortality appearing in the animals thus treated. Even a combination of carnitine or acetyl L-carnitine with hypericum extract 500mg/kg or hypericin 1mg/kg for 30 days at a dose of 500mg/kg can be tolerated well and has no toxic effects. At the end of this treatment, no abnormal blood chemistry parameters were detected and histological tests carried out on the major organs (heart, lungs, liver, kidneys) failed to find any damaging effect of the composition to which the product was administered.

Protection against neurotoxic substance-induced abnormal brain 5-hydroxytryptamine concentrations

Since changes in the concentration of brain biogenic amines can modulate the state of excitatory and depressive emotional behavior, given the above-mentioned effects of hypericin and hypericum extracts at catecholamines and in particular 5-hydroxytryptamine-capable receptor levels and on the reuptake of 5-hydroxytryptamine itself, it was decided to evaluate the change in the concentration of brain 5-hydroxytryptamine after treatment with hypericum extracts, hypericin, carnitine mixtures or acetyl L-carnitine, alone or in various combinations, in combination with a substance such as fenfluramine, the neurotoxicity of fenfluramine also being manifested by a decrease in the concentration of 5-hydroxytryptamine. In fact, fenfluramine [ N-ethyl- α -methyl-m- (trifluoromethyl) phenethylamine ] is known to have neurotoxic effects on the brain, which are recognized histologically based on both a disturbance of the brain's 5-hydroxytryptamine energy structure and a complete depletion of the brain's 5-hydroxytryptamine concentration.

This test was carried out using a group of (Sprague-Dawley) male rats orally administered fenfluramine at a dose of 5mg/kg for 2 consecutive 5 days per day, separately or simultaneously over the same period, with a carnitine mixture (400mg/kg) (composition of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same weight ratio to each other), or acetyl L-carnitine (400mg/kg), or a hypericum extract (containing about 0.3% hypericin-300 mg/kg), or hypericin (1mg/kg), or a plurality of combinations of these products at the same dose.

After 2 weeks of treatment, animals were sacrificed and cerebral cortex was isolated, as per Wise (Wise, c.d).Biochemical annual newspaper(anal. biochem.), 18, 94, 1967) and ricaurate (ricaurate, g.a.,journal of principles of pharmacological testingImproved methods of (j. pharmacol. exptl. ther.), 261, 616, 1992) measure intracerebral concentrations of 5-hydroxytryptamine (5-HT) and hydroxy-indole-acetic acid (5-HIAA).

The results of these tests (Table 1) demonstrate that fenfluramine causes a severe reduction in the concentration of 5-HT and 5-HIAA in the brain as a consequence of its neurotoxic effects. The reduction in 5-hydroxytryptamine concentration can then be reversed by administering hypericum extract or hypericin, which is evident when hypericum extract or hypericin is administered in combination with either carnitine mixture or acetyl L-carnitine, almost resulting in the disappearance of the fenfluramine effect. While the positive effects of hypericum extract and hypericum anthrone on 5-hydroxytryptamine reuptake are well known, this effect of carnitine is unknown and these experiments demonstrate an effective synergistic protective effect at the neuronal level against the reduction of 5-hydroxytryptamine concentrations caused by neurotoxic substances such as fenfluramine.

TABLE 1With phenolsConcentration of intracerebral 5-hydroxytryptamine (5-HT) and hydroxy-indole-acetic acid (5-HIAA) in rats treated with mixtures of fluramine and carnitine or with acetyl L-carnitine, Hypericum extract, Hypericum anthrone, or different combinations of these products

Brain (cortex) concentration

(ng/mg tissue) treatment of 5-HT 5-HIAACO 0.37 ± 0.0180.28 ± 0.015FE 0.17 ± 0.0100.14 ± 0.011CC 0.36 ± 0.0230.30 ± 0.009AC 0.35 ± 0.0200.28 ± 0.012HE 0.38 ± 0.0260.30 ± 0.029HYP 0.36 ± 0.0190.30 ± 0.030CC + FE 0.20 ± 0.0150.16 ± 0.009AC + FE 0.18 ± 0.0110.15 ± 0.019HE + FE 0.26 ± 0.0230.24 ± 0.021HYP + FE 0.28 ± 0.0200.25 ± 0.018CC + HE + FE 0.36 ± 0.0290.25 ± 0.023CC + HYP + FE 0.38 ± 0.0250.29 ± 0.025AC + HE + FE 0.36 ± 0.0240.26 ± 0.016AC + HYP + FE 0.39 ± 0.0290.28 CO + hypericin acetyl hypericin-CO-hypericin-CO-hypericum perforatum extract-hypericum perforatum-hypericum-extract-hypericum-

Test for Activity in mice (Orifice plate test)

It has been demonstrated that in animals, in particular in mice, small amounts of amphetamine can cause an anxiety state, which corresponds to a decrease in locomotor exploration activity. This reduction in activity is not associated with the sedative effect induced by the drug in the animal and can be counteracted by the use of anxiolytic agents.

Using a bosisier (bosisier j.r.,physiology and behaviours(physiol. behav.), 2, 447.1967), the test (well plate test) was carried out in a group of mice in sequence in order to determine whether the decrease in locomotor exploratory activity induced by a low dose of amphetamine (1mg/kg, i.p.) in the animals could be related to an orally administered carnitine mixture (composition of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same weight ratio to each other), or acetyl L-carnitine, or a hypericum extract, or hypericin, or various combinations of these products. As can be seen from the data in table 2, while carnitine administration alone had no effect on the exploratory activity of the animals, hypericum extract and hypericin administration almost restored the exploratory activity to normal, the combination of carnitine plus hypericum extract or hypericin enhanced the exploratory activity to a degree similar to the effect of the higher amphetamine doses.

TABLE 2 In the administration of amphetamine (1 and 5mg/kg. i.p.) and carnitine mixture, acetyl L-carnitine, gold Mice 30 minutes after extraction of Hypericum perforatum, hypericin, or various combinations of these products Activity of the movement (orifice plate test)Treatment of changes compared with control- -amphetamine 1 mg/kg-45- -amphetamine 5mg/kg +55CC amphetamine 1 mg/kg-50 AC amphetamine 1 mg/kg-48 HE amphetamine 1 mg/kg-10HYPamphetamine 1 mg/kg-5 CC + HE amphetamine 1mg/kg +15CC + HYP amphetamine 1mg/kg +19AC + HE amphetamine 1mg/kg +25AC + HYP amphetamine 1mg/kg +20CC ═ carnitine mixture 400mg/kg AC ═ acyl L-carnitine 400mg/kg HE ═ hypericum extract 300mg/kg HYP ═ hypericum anthrone 1mg/kg

Platform test

Another behavioral test in mice is Burnell (Burnell, j.a.,comparison of physiology And psychology journal(J.Comp.Physiol.Psychol.), 1, 147, 1965). The test consisted of placing the animals on wooden platforms at different heights from the ground and counting the number of animals that were not hesitant to jump from the platform. Each group had 10 animals and the percentage of animals trying to get off the platform was calculated. As can be seen from the results in Table 3, although none of the animals in the control group fell from a platform 9cm in height from the ground and from a platform 5cm only about 50%, the behavior of the animals was improved by the administration of Hypericum extract (300mg/kg) or hypericin (1 mg/kg). By administering these products plus a carnitine mixture (composition of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same weight ratio as one another, for a total dose of 400mg/kg) or a composition of acetyl L-carnitine 400mg/kg, the behaviour can be improved more, without having an improving effect on the behaviour of the animals when they are administered alone.

The results of these tests clearly show that the action of hypericum extract (300mg/kg) and hypericin (1mg/kg) is significantly enhanced by combination with carnitine.

TABLE 3

Platform testing in micePercentage of mice treated for jumping down

Control 060 carnitine mixture from 5cm at 9cm, 400mg/kg 070 acetyl L-carnitine, 400mg/kg 070 hypericum extract, 300mg/kg 2090 hypericum anthrone, 1mg/kg 3090 carnitine mixture, 400mg/kg + hypericum extract, 300mg/kg 70100 carnitine mixture, 400mg/kg + hypericum anthrone, 1mg/kg 60100 acetyl L-carnitine, 400mg/kg + hypericum extract, 300mg/kg 60100 acetyl L-carnitine, 400mg/kg + hypericum anthrone, 1mg/kg 80100

Immobility test induced by forced swimming

One of the most well-established tests for evaluating the activity of antidepressant substances is the forced swim test in mice, which measures the change in the production of a number of test substances in swimming-induced immobility (Borsini, f.,psychopharmacology,94, 147, 1988). In these tests, persalt (r.d.,eur J Pharmacology，57，201，1979-Persolt，R.D.， International pharmacological literature(arch. int. pharmacology), 229, 327, 1977) using 10 animals per group. The animals were placed in a beaker 14cm high and having an internal diameter of about 12cm, the beaker was filled with water (20-22 ℃) to 7.5cm from the edge, and the animal was allowed to stay there for 6 minutes. The immobility time was calculated over the last 4 minutes. Mice are considered immobile when they move only when they have to float themselves on the water.

The test substance was administered orally in 2 portions 6 and 3 hours before the start of the test.

As is apparent from the results of table 4, the immobility time was reduced by the application of hypericum extract and hypericin, but this reduction was more limited when hypericum extract or hypericin was administered in combination with carnitine (composition of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same weight ratio as each other) or with acetyl L-carnitine, but not when they were administered alone.

TABLE 4

Immobility test treatment induced by forced swimming in mice immobility time to 210 + -8 carnitine mixture 220 + -7 acetyl L-carnitine 204 + -8 hypericum extract 190 + -9 hypericum anthracene 195 + -5 carnitine mixture + hypericum extract 170 + -6 carnitine mixture + hypericum anthracene 165 + -4 acetyl L-carnitine + hypericum extract 178 + -9 acetyl L-carnitine + hypericum anthracene 172 + -7

Isolation induced aggression test

Suitable for these tests are the Scott method (Scott, j.p.,physiology zoology(physiol. zoom.), 24, 273, 1951), modified by Sanchez (Sanchez, c.,psychopharmacology (Psychopharmacology),110, 53, 1993). The method involves making mice aggressive by segregating them in cages for 21 days and evaluating the latency required to elicit an attack by the segregated animal when another animal is placed in the cage after treatment. Only animals with a latency of less than 10 seconds prior to attack were enrolled in the test, and the attack time was the time that the isolated animal bites or attempts to bite other animals placed in the cage.

The observation time was 180 seconds and the test was started 30 minutes after the test product was administered. At 8 hours and half an hour before the test, all the segregating animals were treated with a carnitine mixture (400mg/kg) (composition of L-carnitine + acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine present in the same proportions as one another), or acetyl L-carnitine (400mg/kg), or hypericum extract (300mg/kg), or hypericin (1mg/kg), or various combinations of these products.

The results of these experiments (table 5) demonstrate that, although the carnitine compound alone does not modify the attack latency of the mice treated with it, its use in combination with hypericum extract or hypericin greatly enhances the latter's aggressiveness-reducing effect in mice.

These experiments also confirmed the unexpected and sudden synergy between carnitine-like compounds and hypericum extract or hypericin.

TABLE 5 Isolated induced challenge experiments. Extracting with carnitine mixture, acetyl L-carnitine, and fructus Rhodomyrti Male mice treated with hypericin, or various combinations of these products have potential for challenge

Volt timeTreatment latency (sec) control 8 + -2 Carnitine mixture, 400mg/kg 11 + -1 acetyl L-Carnitine, 400mg/kg 14 + -3 Hypericum extract, 300mg/kg 80 + -9 Hypericum Anthracene, 1mg/kg 100 + -9 Carnitine mixture, 400mg/kg + Hypericum extract, 300mg/kg 140 + -12 Carnitine mixture, 400mg/kg + Hypericum Anthracene, 1mg/kg 150 + -6 acetyl L-Carnitine, 400mg/kg + Hypericum extract, 300mg/kg 150 + -9 acetyl L-Carnitine, 400mg/kg + Hypericum Anthracene, 1mg/kg 160 + -11 Potassium carnitine

Illustrative, non-limiting examples of the formulations of the present invention are as follows: 1) carnitine mixture 600mg

(L-carnitine 150mg, acetyl L-carnitine 150mg, propionyl L-carnitine

150mg, isovaleryl L-carnitine 150mg)

Hypericum extract (designated 0.3% hypericin) 600mg2) Carnitine mixture 600mg

(L-carnitine 150mg, acetyl L-carnitine 150mg, propionyl L-carnitine

150mg, isovaleryl L-carnitine 150mg)

Hypericin 2mg3) acetyl L-carnitine 600mg

Hypericum extract (designated 0.3% hypericin) 600mg4) acetyl L-carnitine 600mg

Hypericin 2mg5) Carnitine mixture 300mg

(L-carnitine 75mg, acetyl L-carnitine 75mg, propionyl L-carnitine 75mg,

isovaleryl L-carnitine 75mg)

Hypericum extract (designated 0.3% hypericin) 300mg6) carnitine mixture 300mg

(L-carnitine 75mg, acetyl L-carnitine 75mg, propionyl L-carnitine 75mg,

isovaleryl L-carnitine 75mg)

Hypericin 1mg7) acetyl L-carnitine 300mg

Hypericum extract (designated 0.3% hypericum anthrone) 300mg8) acetyl L-carnitine 300mg

Hypericin 1mg9) Carnitine mixture 300mg

(L-carnitine 75mg, acetyl L-carnitine 75mg, propionyl L-carnitine 75mg,

isovaleryl L-carnitine 75mg)

Hypericum extract (designated 0.3% hypericum anthrone) 300mg

L-tyrosine 50mg

Histidine 50mg

Taurolactone 50mg

Glutamine 50mg

Valine 50mg

Tryptamine 50mg10) Carnitine mixture 300mg

(L-carnitine 75mg, acetyl L-carnitine 75mg, propionyl L-carnitine 75mg,

isovaleryl L-carnitine 75mg)

Hypericum extract (designated 0.3% hypericum anthrone) 300mg

Phosphoserine 100mg

Glycerol phosphocholine 100mg

Tryptophan 100mg

Tyrosine 100mg

CoQ10 10mg

Selenium 10mg

By pharmaceutically acceptable salts of L-carnitine or alkanoyl L-carnitine is meant any salt of these active ingredients with an acid which does not produce harmful toxicity or side effects. These acids are well known to those skilled in the art of pharmaceutical manufacture.

Non-limiting examples of suitable salts are as follows: a chloride; bromide; an iodide; aspartate, acid aspartate; citrate, acid citrate; a tartrate salt; phosphates, acid phosphates; fumarate, acid fumarate; a glycerophosphate salt; glucose phosphate; a lactate salt; maleate, acid maleate; orotate salts; oxalate, acid oxalate; sulfate, acid sulfate, trichloroacetate, trifluoroacetate and methanesulfonate.

The list of FDA-approved pharmaceutically acceptable salts is listedInternational journal of pharmacology(int.j.of pharm.)33, (1986), 201-217; the latter of which is incorporated herein by reference.

The composition of the present invention may further contain vitamins, coenzymes, inorganic substances and antioxidants.

Suitable excipients for the preparation of compositions suitable for a particular route of administration will be apparent to those skilled in the pharmaceutical and food industries.

Claims (15)

1. A composition comprising:

(a) acetyl L-carnitine or a pharmaceutically acceptable salt thereof, and

(b) hyperforanthone or a Hypericum extract containing at least 0.3% by weight of hypericin.

2. The composition of claim 1, wherein the component (a) further comprises a "carnitine" selected from the group consisting of L-carnitine, propionyl L-carnitine, valeryl L-carnitine, isovaleryl L-carnitine or their pharmacologically acceptable salts or mixtures thereof.

3. The composition of claim 1 or 2, wherein the weight ratio of (a) to (b) is from 1: 0.01 to 1: 1.

4. A composition as claimed in any one of the preceding claims wherein component (b) is in the form of a plant extract containing the component.

5. The composition of claim 4, wherein the plant extract comprises a flower, terminal bud, or leaf of a Hypericum plant.

6. The composition of any of the preceding claims, wherein the pharmaceutically acceptable salt of L-carnitine or alkanoyl L-carnitine is selected from the group consisting of: a chloride; bromide; an iodide; aspartate, acid aspartate; citrate, acid citrate; a tartrate salt; phosphates, acid phosphates; fumarate, acid fumarate; a glycerophosphate salt; glucose phosphate; a lactate salt; maleate, acid maleate; orotate salts; an acid oxalate; sulfate, acid sulfate, trichloroacetate, trifluoroacetate and methanesulfonate.

7. A composition according to any one of the preceding claims, further comprising vitamins, coenzymes, minerals and antioxidants.

8. The composition of any of the preceding claims, administered orally in the form of a food supplement.

9. A composition according to any preceding claim, in the form of a medicament for oral, parenteral, rectal or transdermal administration.

10. The food additive of claim 8, for preventing neuropathy caused by anxiety states, irritation or depression.

11. A medicament according to claim 9 for the treatment of neuropathy caused by anxiety states, irritation or depression.

12. A dietary supplement according to claim 8 or 10, in solid, semi-solid or liquid form.

13. The medicament of claim 9 or 11, in solid, semi-solid or liquid form.

14. The dietary supplement of claim 12, in the form of a pill, tablet, capsule, granule or syrup.

15. The medicament of claim 13, in the form of pills, tablets, capsules, granules, syrups, vials or drops.