JP2002520280A - Compositions and methods for treating and preventing bone disease using tocotrienol - Google Patents

Abstract

  (57) [Summary] The present invention relates to the treatment and prevention of bone diseases using tocotrienol. In particular, the present invention relates to the use of individual tocotrienols, mixtures of tocotrienols and mixtures of one or more tocotrienols with other bone-enhancing substances and compositions containing them. The compositions and methods of the present invention are particularly well suited for the treatment and prevention of osteoporosis and other degenerative bone disorders such as Paje's disease and Gaucher's disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to the treatment and prevention of bone diseases using tocotrienol. In particular, the invention relates to the use of individual tocotrienols, mixtures of tocotrienols, and mixtures of one or more tocotrienols with other bone-enhancing substances, and compositions comprising them. The compositions and methods of the present invention are particularly well-suited for the treatment and prevention of osteoporosis and other degenerative bone disorders such as Paget's disease and Gaucher's disease.

BACKGROUND OF THE INVENTION Osteoporosis is a progressive disorder characterized by reduced bone mass and increased susceptibility to fracture. Osteoporosis is the leading cause of morbidity in postmenopausal women and elderly men. There are numerous risk factors for the development of osteoporosis,
These risk factors mediate these effects through regulation of bone remodeling, bone metabolism and bone recovery (WB Ershler et al., Dev. Comp. Immunol).
. , 21 (6), 487-99 (1997)).

[0003] The goal of pharmacological intervention in osteoporosis is to increase bone mass by inhibiting bone resorption and / or increasing bone formation. Bone resorption is associated with increased osteoclast activity. Recent studies indicate that increased secretion of increased bone-active cytokines (eg, interleukin-6 (IL-6)) from osteoblasts and stromal cells is central to increased osteoclast activity and the pathogenesis of osteoporosis. Have been suggested to play a role (GS Kim et al., J. Bone Miner. Res.
12 (6), 896-902 (1997); Sporeno et al., Bloo
d. 87 (11), pp. 4510-19 (1996)). IL-6 is also believed to enhance osteoclast recruitment and bone resorption (E. Bornefalk et al., J. Bone Miner. Res., 12 (2) 228-33 (1997).
)). In addition to its osteoclastogenic properties,
IL-6 also appears to promote osteoblast differentiation directed to a more mature phenotype (T. Bellido et al., Endocrinology, 138).
(9) 3666-76 (1997)). A number of well-recognized bone enhancers affect IL-6 production. For example, estrogens may cause IL- in bone marrow cells.
6 is known to be important in regulating the expression of (HK Vaanane).
n and p. L. Harkonen, Maturitas, 23 Addendum 565-9
P. (1996)), Calcium concentration in plasma affects IL-6 secretion from mononuclear blood cells via a direct calcium sensing mechanism (E
. Bornefalk et al., Supra), and bisphosphonates have
It appears to inhibit L-6 production (N. Giuliani et al., Scand.
Rhematol. 27 (1) p. 3841 (1998)).

[0004] Primary preventive therapies for osteoporosis often involve altered behavior and diet. Secondary prevention in women who have undergone menopause often involves estrogen replacement therapy (JY Reginstar et al., Rev. Rhum. Ed. F.).
r. 61 (10) 1555S-64S (1994)). In addition to estrogens, there are numerous other drugs used to prevent and treat osteoporosis and other degenerative bone disorders. These prevention and treatment options for osteoporosis include:
In general, they can be divided into two subsets: those that inhibit bone resorption and those that stimulate bone formation. The first category includes: calcium (including calcium salts), antiestrogens (eg, tamoxifen), environmental estrogens (eg, cumestrol, methoxychlor),
Bisphenol A and zeranol), vitamin D (including analogs and metabolites), gonadal and anabolic steroids, calcitonin, flavonoids (eg, pentahydroxyflavone, naringenin (
narringenin, hesperetin, tangeretin, nobiletin, quercetin,
Apigenin, isoflavone genestein (geneste)
in) and daidzein, and isoflavone derivatives, ipriflavone), tyrosine kinase inhibitors (eg, herbimycin, protein kinase C)
Modulators (eg, phorbol esters) and bisphosphonates (eg, alendronate, risedronate (rise)
dronate) and etidronate). While some of the drugs in this first category also fall into the second category, the most widely used drugs to stimulate bone formation are fluoride (usually in the form of sodium fluoride). ). Synthetic parathyroid hormone, vitamin K (including K1 and K2), and cyclic bisphosphonates are also used as bone enhancers (C. Genera et al.,
Drug Sert, 11 (3) pp. 179-95 (1994); We
ber, Int. J. Vita. Nut. Res. 67 (5) 350-56 (
1997)).

[0005] Despite the numerous prevention and treatment options that exist, osteoporosis and other bone diseases remain a serious health threat. Thus, there remains a well-recognized and unmet need for new agents for preventing and treating osteoporosis and other bone diseases.

SUMMARY OF THE INVENTION The present invention satisfies the need for new prophylactic and therapeutic agents that are effective in preventing and treating osteoporosis and other bone diseases.

One embodiment of the present invention provides a method for preventing osteoporosis and other bone diseases in a patient, the method comprising administering to the patient a prophylactically effective amount of a composition comprising: Including: tocotrienol, a mixture of tocotrienols, or a combination of one or more tocotrienols with one or more additional bone enhancing substances.

[0008] Another embodiment of the present invention provides a method of treating osteoporosis and other bone diseases in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition comprising: Including: tocotrienol, a mixture of tocotrienols, or 1
A combination of one or more tocotrienols with one or more additional bone enhancers.

[0009] Yet another embodiment of the present invention provides a combination of one or more tocotrienols with one or more additional bone enhancers that are particularly well suited for treating and preventing osteoporosis and other bone diseases. A novel composition comprising:

DETAILED DESCRIPTION OF THE INVENTION As used herein, the following definitions apply (unless explicitly stated to the contrary); "bone-enhancing substances"
A factor that has a beneficial effect on bone status. Preferably, these bone enhancing substances are
It inhibits bone resorption and / or stimulates new bone formation, and preferably, these bone augmenting agents are selected from the individual bone augmenting agents mentioned herein. Markers and tests for measuring these activities are well-established in the art (eg, M Gambacciani et al., Calcif. Tiss.
ue Int, 61S1, S15-18 (1997)).

[0011] As used herein, "composition" refers to a preparation for administration via any acceptable route known to those of skill in the art. Such routes include, but are not limited to, oral, nasal, inhalation, parenteral, and topical administration. "Composition" includes pharmaceutical compositions, as well as dietary supplements, foodstuffs, food additives, and the like.

“Patient” refers to warm-blooded mammals and, preferably, humans. Patients in need of prophylactic treatment to prevent osteoporosis and other bone diseases, including Paget's disease and Gaucher's disease, may have one or more risk factors for developing osteoporosis (postmenopausal women, elderly men (ie, about 50 Aged over 1 year old) and patients with insufficient calcium uptake) or one or more other risk factors for bone disease. Patients in need of treatment for osteoporosis and other bone disorders are treated with bone loss and fractures or other bone disorders (
For example, patients who have one or more symptoms of osteoporosis, including an increased prevalence of symptoms of Paget's disease, i.e., pain and deformity due to enlargement of skeletal segments. “P ₁₈ tocotrienol” refers to a tocotrienol having the formula:

[0013]

Embedded image P ₁₈ tocotrienol and P ₁₈ are available from Bionutrics. Inc. (Pho
enix, Arizona). “P ₂₅ tocotrienol” is tocotrienol 3,4-dihydro-2- (4,8,12-trimethyltrideca-3 ′ (E), 7 ′ (E), 1 having the following formula:
1-trienyl) -2H-benzopyran-6-ol):

[0014]

Embedded image This compound is also known as didesmethyl-tocotrienol in some of the references cited herein. P _{25-tocotrienol} and P ₂₅ is, Bionutrics. Inc. (Phoenix, Arizona).

A “prophylactically effective amount” prevents the development of one or more clinical aspects of osteoporosis or other bone disease in a patient with one or more risk factors associated with osteoporosis or other bone disease. Amount of active ingredient sufficient to treat osteoporosis or other bone disorders
A sufficient amount of the active ingredient to prevent one or more symptoms. "Prophylactically acceptable means" refers to a means effective to provide a prophylactic effect.

“Tocotrienol”, as used with respect to certain compounds, refers to compounds 3,4-
Dihydro-2-methyl-2- (4,8,12-trimethyltrideca-3 '(E)
, 7 '(E), 11 "-trienyl) -2H-1-benzopyran-6-ol. In general, when used to describe a class of a compound, the definitions given below apply.

“Tocotrienol”, as used in a general sense, refers to a compound having three structural features: (1) a hydrogen donor group attached to an aromatic ring system (or hydrolyzed to a hydrogen donor group; (2) a side chain attached to an aromatic ring system comprising one or more isoprenoid or isoprenoid-like units, and (3) a methylene unit, or a side chain attached to the aromatic ring system. A functional group (preferably CH ₂ C ＝ O, CHOH, O, S or NH) having at least one lone pair of electrons adjacent to the atom, wherein the electron is conjugated to an aromatic ring system ). Particular isoprenoid-like units, shortened isoprenoid, and partly may be also be saturated not be saturated, and optionally OH, NH ₂ and C ₁ -
C ₆ Includes truncated or full length isoprenoids substituted with alkyl or alkoxy, branched or unbranched. Preferred tocotrienol for use in the method of the invention, tocotrienols naturally occurring a (tocotrienols, alpha-tocotrienol, beta-tocotrienol, .gamma.-tocotrienol, .delta.-tocotrienol, including P ₂₅ and P _18), and individually or It can be used in combination. These naturally occurring tocotrienols can be conveniently isolated from biological material or synthesized from commercially available starting materials.
Preferably, tocotrienol for use in the method of the present invention is stabilized and extracted from biological material, for example, from PCT Publication WO 91/17.
985, the entire disclosure of which is incorporated herein by reference. Examples of preferred biological materials, tocotrienols, and methods for obtaining tocotrienols synthetically and from biological materials are described in co-owned US Pat. No. 5,591,772 and PCT Publication WO 91/17985.
(The entire disclosures of which are incorporated herein by reference). Preferred biological materials from which some preferred tocotrienols useful in the compositions and methods of the present invention can be obtained include conifers, legumes, asteraceae,
Ingredients from the Grasses and Palmaceae, and more particularly stabilized bran (especially stabilized rice bran), psyllium, barley, pine nuts, sunflowers, peanuts, palm nuts, millet, avocado, Rat berries, cedar leaves, mango, pine needles, gum tree leaves, tocotrienol-enriched extracts from tomato and amaranth are mentioned. In addition to the above tocotrienols, the term "tocotrienol" also refers to the farnesylated tetrahydro- as described in U.S. Patent Nos. 5,204,373 and 5,393,776 (both generally and specifically). Includes naphthalenol and monocyclic tocotrienol analogs.

[0018] Particular preferred tocotrienols of the invention include the following tocotrienols of formula (I):

[0019]

Embedded imageWhere R₁And R_ThreeIs independently H, halogen, OH, OCH_Three, And C₁~ C ₆ Selected from the group consisting of branched or unbranched alkyl of (preferably,
H, halogen, and C₁~ C_ThreeBranched or unbranched alkyl, and
More preferably H and methyl); R_TwoIs halogen (preferably chlorine), as well as OH, NHR₈, CO_TwoY also
Is C (R₈)_TwoCO_TwoH and C₁~ C₈Branched or unbranched alkyl (
OH, NHR₈, CO_TwoY or C (R₈)_TwoCH_TwoH substituted for H)
Selected from the group consisting of hydrogen donor groups (preferably OH and C ₁ ~ C_ThreeBranched or unbranched alkyl (substituted with OH), and more
Preferably OH); R_FourIs O, NH, CH-R₉, C = O and CH-OH.
(Preferably O, CH_Two, And C = O); R_FiveIs CH_Two, C = O, CHOH, O, S, and NH
(Preferably O, CH_TwoAnd C = O, and more preferably O and C =
O); R₆Are H and C₁~ C₆From the group consisting of branched or unbranched alkyl of
Selected (preferably H and C₁~ C_ThreeBranched or unbranched alkyl of
And more preferably, H and methyl); R₇Is selected from the group consisting of isoprenoids and isoprenoid-like side chains;
And more preferably selected from the group consisting of the side chains of the following formulas (a) to (c)
,

[0020]

Embedded image

[0021]

Embedded image And also comprises a combination of one or more of the recurring units shown in side chains (a)-(c), and if the recurring unit shown in side chain (a) is present, one or more of its unsaturated analogs Or, if there is a repeating unit in side chain (c), including one or more oxidized analogs thereof, including its keto and dione analogs, wherein
Each R ₁₀ is independently, H, OH, NH _2, and branched C ₁ -C _6, or is not a branch is selected from the group consisting of alkyl or alkoxy, and R ₁₁ is H
, C ₁ -C ₆ branched or unbranched alkyl or alkoxy, CH ₂ O
H, CO ₂ H (and its C ₁ -C ₆ alkyl esters), and OH (preferably, R ₇ is a side chain of formula (a), wherein R ₁₀ and R ₁₁ is each independently selected from the group consisting of H and C ₁ -C ₆ branched or unbranched alkyl, and more preferably is H and methyl);
Each R ₃ and R ₉ is independently selected from the group consisting of H and C ₁ -C ₆ branched or unbranched alkyl (preferably H and C ₁ -C ₃ branched of,
Or unbranched alkyl, and more preferably H and methyl); Y is H or C ₁ -C ₁₈ branched or unbranched alkyl (preferably H and C ₁ -C ₆ ) Z is H, halogen, OH, CH ₂ OH, CH ₇ , branched or unbranched alkyl, and more preferably H and C ₁ -C ₄ branched or unbranched alkyl); Selected from the group consisting of OCH ₃ and COCH ₃ (preferably H and CH ₃ ); n is an integer selected from the group consisting of 0, 1, 2, 3, and 4 (preferably 0 And 1); and m is an integer selected from the group consisting of 1 to 30 (preferably 1 to 20,
More preferably, 3-10, and most preferably, 3-7).

More preferred tocotrienols of the present invention are tocotrienol, α-tocotrienol, tocotrienol, γ-tocotrienol, δ-tocotrienol,
P _{18-tocotrienol,} and a P _{25-tocotrienol.}

The present invention specifically encompasses prodrug forms of tocotrienol. Upon administration to a patient, such prodrugs will undergo biotransformation into their active forms. Prodrugs include the esterified forms of tocotrienol used in the present invention that include a carboxylic acid functionality.

The tocotrienols for use in the compositions and methods of the present invention may be in their isomerically pure form, or may exist as a mixture of isomers. For example, the tocotrienols of the present invention can exist as the d- or l-isomer or as a d, l-racemic mixture. Its naturally occurring isomer (usually
d isomer) and d, l-racemic mixtures are preferred.

“TRF” refers to the tocotrienol-rich fraction obtained by stabilization and extraction of a biological source. The TRF typically comprises varying amounts of tocotrienol, α-tocotrienol, β-tocotrienol, γ-tocotrienol,
And δ-tocotrienol, and may contain large amounts of P ₁₈ and P ₂₅ tocotrienol. Most commonly, the TRF comprises at least about 25% to about 90% tocotrienol w / w (preferably, at least about 35% to about 90%, and more preferably, at least about 50% to about 90%.
). “TRF ₂₅ ” refers to TR containing a significant weight percent of P ₂₅ tocotrienol
Refers to F. Preferably, TRF ₂₅ is at least about 5% P ₂₅ , more preferably at least about 10% P ₂₅ , and even more preferably at least about 15%
Of P ₂₅ w / w. Examples of the preparation of particular TRF ₂₅ are described in A. See Kureshi et al., Nutr. Biochem. 8.290-98 (1997). T
RF ₂₅ is a preferred component of the compositions and methods described herein. TR
F ₂₅ is, Bionutrics, Inc. (Phoenix, Arizona)
Is a trademark. TRF and TRF ₂₅ can be used in any of the methods and compositions described herein for individual tocotrienols or mixtures thereof.

All documents cited herein are incorporated by reference in their entirety, including any drawings.

[0027] Without wishing to be bound by any particular theory, tocotrienols are useful in preventing and treating osteoporosis and other bone disorders due to their unique combination of antioxidant and anti-inflammatory properties. Seems to be effective for In contrast to conventional therapeutic agents that target a single mechanism, tocotrienol targets multiple mechanisms that cause and spread osteoporosis and other bone disorders. Tocotrienol is
Inhibits the production of various cytokines (including IL-6) that are involved in the development and histology of osteoporosis and other bone disorders. In addition, tocotrienol mediates superoxide levels and possibly nitric oxide production. These factors are involved in the process of bone resorption of osteoclasts (PM Gallop et al., Connect. Tissue Res. 29 (2) 153-61 (199).
3) and D.I. M. Evans and S.M. H. Ralston, J .; Bone
Miner. Res. 11 (3), p. 30005 (1996). In particular, the bone-enhancing effect of ipriflavone is its major metabolite (7-isopropoxy-3- (4-hydroxyphenyl) -4H-1-benzopyran, which inhibits liposaccharide (LPS) -induced nitric oxide release. -4-one) (S. Koncz and EJ Horvath, Acta Ph.
ysiol. Hung. 84 (3), pp. 223-38 (1996)). Tocotrienol has also been associated with a progressive decline in glycation end product (AGE). AGE production has recently been associated with osteoclast-induced bone resorption (T.
Myata et al. Am. Soc. Nephral. 8 (2), pp. 260-70 (1997)). As a result of a unique combination of their properties, tocotrienols can fight the root cause of osteoporosis and bone disorders and reverse the degenerative effects.

The compositions of the present invention are prepared by combining one or more tocotrienols and an acceptable carrier. For the pharmaceutical compositions of the present invention, the carrier must be pharmaceutically acceptable (ie, a carrier that is non-toxic to the patient at the level administered and that does not disrupt the activity of the active ingredients of the composition). Must.
Acceptable carriers, including pharmaceutically acceptable carriers, are well known to those skilled in the art.

[0029] The compositions of the present invention may be used or administered by any therapeutically or prophylactically acceptable means for patients in need of treatment and prevention of osteoporosis.
For example, the pharmaceutical compositions of the present invention can be administered orally, nasally, topically, transdermally, or parenterally. These compositions may be formulated so as to provide a time release benefit. Oral compositions can take the form of tablets, capsules, caplets, emulsions, liposomes, suspensions, powders, and the like. Topical compositions include, but are not limited to, gels, lotions, and creams. Parenteral compositions take such forms as sterile solutions and emulsions. Intravenous compositions include, but are not limited to, sterile solutions. The preferred route of administration is oral.

[0030] Dosage levels and requirements are well-recognized in the art and can be selected by those skilled in the art from publicly available sources. Typically, dosage levels range between about 0.1 to about 10,000 mg of tocotrienol or tocotrienol mixture per dose. Preferably, this range is 1
Between about 0.1 and about 5,000 mg per dose (more preferably between 0.1 and 100 mg)
(Between 0 mg). Sustained doses may be required over time to obtain maximum benefit. The particular dosage and treatment regime will depend on factors such as the patient's overall health, the severity and course of the patient's disorder or predisposition thereto, and the judgment of the attending physician. Higher or lower doses may be used as needed.

[0031] Tocotrienol and mixtures thereof can be used in combination with bone augmenting agents in the methods described herein. These additional therapeutic and prophylactic agents may be administered separately from the tocotrienol or mixture thereof, or they may be formulated together in a single dosage form. Such combination therapy may reduce or avoid the toxicity that may be incurred when the bone augmenting agent is used as a monotherapy, or produce other doses of lower doses that may produce an additive or synergistic effect. A bone augment is advantageously used. In addition to the conventional drugs used in the treatment and prevention of osteoporosis, the tocotrienols used in the methods of the present invention may also be used in combination with any conventional anti-inflammatory or antioxidant. These conventional drugs preferably have independent bone enhancing properties. In a particularly preferred embodiment, one or more tocotrienols may be used in combination with one or more of the following bone enhancing materials:
Calcium (including calcium salts), estrogens, antiestrogens (eg,
Tamoxifen), environmental estrogens such as cumestrol, methoxychlor, bisphenol A, and zeranol, vitamin D (including analogs and metabolites such as calciferol and 1α-hydroxycholecalciferol), gonadal steroids and anabolic steroids , Calcitonin, flavonoids (e.g., pentahydroxyflavone, naringenin, hesperitin, tangeretin, nobiletin, quercetin, apigenin, isoflavone genestein (ge)
neustein, and daidzen, and isoflavone derivatives, ipriflavone (including its metabolites), tyrosine kinase inhibitors (eg, herbimycin, protein kinase C)
Modulators (eg phorbol esters), bisphosphonates (cyclic bisphosphonates and alendronate, risedronate and etidronate)
e)), fluoride (eg, sodium fluoride), synthetic thyroid hormone,
Vitamin K (including K1 and K2), other cytokine mediators (eg, IL-1 inhibitor-like probucol and lipoic acid, IL-6 inhibitor, IL-11 inhibitor, TNF inhibitor, INF-γ inhibitor, tocopherol (eg, α -Tocopherol (vitamin E)) and selenium. A preferred combination is one or more tocotrienols selected from calcium (including calcium salts), flavonoids (more preferably ipriflavone), vitamin D, vitamin K, lipoic acid and tocopherol. Together with one or more drugs.

EXAMPLES The following examples are provided so that the invention might be more fully understood. These examples are for illustrative purposes only, and should not be construed as limiting the scope of the invention in any way.

The methods used to obtain and purify tocotrienols and mixtures thereof (including TRF) useful in the methods of the invention are described in the Examples section of US Pat. No. 5,591,772. It is described in. Stabilization of rice bran is in accordance with Example 1 of US Patent No. 5,591,772. TRF, purification of P ₂₅ tocotrienols and P ₁₈ tocotrienols, according to Examples 2-4 of U.S. Patent No. 5,591,772.

The following specific protocol can be used to produce rice bran oil from which tocotrienols and their mixtures can be isolated: (Protocol I: Drv heat stabilization) Extruder: Wenger Model X-25

[0035]

[Table 1] (Protocol II: Stabilization of wet heat after drying and heating) Dry heat stage: Protocol I Wet heat stage: Extruder: Anderson 4 inch Screw barrel arrangement: Standard cut flight Die setting: Diameter: 0.1875 inch Length: 0. 75 inches Operating conditions: Feeding speed: 378 lb / hr Shaft speed: 279 rpm Steam Injection: 36 lb / hr (32 psi with # 8 holes) Mechanical pressure: 750 psi (ast.) Humidity Feeding: 11.4% Discharge humidity: 15% Discharge speed : 450 lb / h Discharge temperature: 121 ° C (Protocol III: drying / cooling procedure) The wet heat stabilized product of Protocol II (15% humidity) is discharged onto an aluminum tray and the humidity is reduced to 8-10% Until (about 1.5 hours) 101.
Placed in tray oven at 1 ° C. The tray was then placed on a tray rack and allowed to cool at ambient temperature (about 20 ° C).

[0036]

[Table 2] Hexane was removed from the extract by gentle heating (40 ° C.) under light vacuum.

(Protocol V: Dewaxing) 20 lb of the crude was cooled at -15.6 ° C. for 24 hours. The supernatant (including the dewaxed oil) was decanted from the solidified wax. The wax is then centrifuged and the entrained oil (
entrained oil) and 0.59 Ib of wax and 19.41 I
A dewaxed oil of b was obtained.

Evaluation of Tocotrienol Activity Example 1 The effect of tocotrienol (in the form of TRF mixture and individual tocotrienol) on the plasma levels of thromboxane B ₂ and platelet factor 4 in chickens was determined. These levels can be controlled by inflammatory cytokines (eg, I
It is known to correlate with the level of L-6). The following feeding conditions were used: Each group of 6 chickens (6 week old female White Leghorn) received a chicken feed control diet or a control diet containing one or more additives. The amount of food consumed by all groups was comparable to the control group. The feeding period was 4 weeks. The birds were fasted for 14 hours before slaughter.

The chicken feed control diet contained the following ingredients:

[0040]

[Table 3] Mineral mixture is based on 1 kg of diet: zinc sulfate H ₂ O, 110 mg; manganese sulfate 5H ₂ O, 70 mg; ferric citrate H ₂ O, 500 mg;
5H ₂ O, 16 mg; sodium selenite, 0.2 mg; DL-methionine,
2.5 g; choline chloride (50%), 1.5 g; ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), 125 mg; and 1.8 mg thiamine HCl. . Vitamin mixture per 1 kg of diet;
Vitamin A, 1500 units Vitamin D _3, 400 units; vitamin E, 10 units; riboflavin, 3.6 mg; calcium pantothenate, 10 mg; niacin, 25 mg; pyridoxine HCl, 3 mg; folacin, 0.55 mg; biotin, 0. 15 mg; vitamin B ₁₂ , 0.01 mg; and vitamin K ₁ , 0.5
Contains 5 mg.

The results are recorded as the mean ± standard deviation. The percentage of control is recorded in parentheses. The following results were obtained: Experiment 1

[0042]

[Table 4] Significant reductions in plasma levels of thromboxane B ₂ and platelet factor 4 are due to TR
This was observed in chickens fed a control diet supplemented with F.

Experiment 2

[0044]

[Table 5] Significant reductions in plasma levels of thromboxane B ₂ and platelet factor 4 are due to TRF
And a control diet supplemented with individual tocotrienols was observed in the fed chickens.

Example 2 The effect of tocotrienol (in the form of a TRF mixture and individual tocotrienol) on the plasma levels of porcine thromboxane B ₂ and platelet factor 4 was determined. These levels are known to correlate with the levels of inflammatory cytokines (including IL-6). Were used following feeding conditions: 3 pigs (Lpd ⁵ and Lpu ¹ age carrying mutant alleles 5 months pig) each group to the control diet or one or more control additive was supplemented, Diet was given. Immediately after 12 hours, plasma samples were collected at 42 days from the beginning of the feeding period. The porcine control diet contained the following ingredients:

[0046]

[Table 6] The mineral mixture contained the following per kg of feed: zinc sulfate-H ₂ O, 1
10 mg; manganese sulfate · 5H ₂ O, 70 mg; iron citrate · H ₂ O, 500 mg
Copper sulfate 5H ₂ O, 16 mg; sodium selenite, 0.2 mg; DL-methionine, 2.5 g; choline chloride (50%), 1.5 g; ethoxyquin (1,
2-dihydro-6-ethoxy-2,2,4-trimethyquinoline (trimethhine)
yquinoline)), 125 mg; and thiamine HCl, 1.8 mg.
The vitamin mixture contained the following per kg of feed: Vitamin A, 1,500
Units Vitamin D _3, 400 units; vitamin E, 10 units; riboflavin, 3.
6 mg; calcium pantothenate, 10 mg; niacin, 25 mg; pyridoxine HCl, 3 mg; folacin, 0.55 mg; biotin, 0.15 mg; vitamin B ₁₂ , 0.01 mg; and vitamin K ₁ , 0.55 mg. Weight gain in all groups was compared to controls.

The results are reported as the mean ± standard deviation. The percentage of control is reported in parentheses. The following results were obtained:

[0048]

[Table 7] Significant reductions in plasma levels of thromboxane B ₂ and platelet factor 4 are due to TR
A control diet supplemented with F and individual tocotrienols was observed in fed chickens.

Example 3 The effect of γ-tocotrienol on superoxide release from neutrophils in human peripheral blood was measured. Superoxide amplifies the inflammatory response, thereby increasing bone resorption.

The neutrophils tested were compared to Ficoll-Hypaque using conventional methods.
The gradient was isolated by density centrifugation (E. Serbinova et al., Free R).
ad. Bio. and Med. , 10, 263-75 (1991)).
. These neutrophils were then placed in 96-well plates. γ-tocotrienol and phorbol myristate acetate were added simultaneously to the wells. Superoxide secretion was measured as superoxide dismutase-inhibited reduction of ferricytochrome C. The results of this study are shown in FIG.

The amount of superoxide released was determined to be 19.7 nmol (5
(× 10 ⁵ cells / hour) to 8.0 and 0.0 nmol (γ-tocotrienol concentrations of 10 ⁻⁶ and 10 ^{−5, respectively} ).

Example 4 The following model can be used to assess the effects of tocotrienol on the prevention and treatment of osteoporosis and other bone diseases: Civiteli et al., Calcif Tissue Int. 56, 21
Pages 5-19 (1995) describe a rat model for measuring bone biomechanics, impact strength, and mineral bone density. K. Notova et al., Calcif Tissue Int. 58 (2), 88
-94 (1996) describe a rat model for measuring bone repair in immobilized rats after unilateral sciatic nerve transection. N. Giuliani et al., Supra, describe an in vitro model for measuring IL-6 production in a human osteoblast cell line (MG-63).

While the invention has been described using specific embodiments and examples, it is to be understood that changes to the embodiments and examples shown can be made without departing from the scope and spirit of the invention. It is clear to the trader.

[0054] Other embodiments are within the following claims.

[Brief description of the drawings]

FIGS. 1A and 1B show the effect of γ-tocotrienol (GT301) on superoxide production in human peripheral blood neutrophils.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 33/00 A61K 33/00 38/22 A61P 19/08 A61P 19/08 19/10 19/10 A61K 37 / 24 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG) , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, N, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF term (reference) 4C084 AA02 BA44 DB31 DC32 MA02 MA52 NA05 ZA971 ZA972 ZC751 4C086 AA01 AA02 BA08 BA09 DA09 DA15 DA16 HA04 HA08 HA09 MA03 MA03 MA52 NA05 ZA97 ZC75 4C206 AA01 AA02 CA16 CB28 FA23 MA03 MA04 MA72 NA05 ZC75

Claims (17)

[Claims] 1. A composition comprising one or more tocotrienols and one or more bone enhancers selected from the group consisting of calcium, calcium salts, ipriflavone, vitamin D and vitamin K. 2. The composition of claim 1, wherein said bone enhancer is ipriflavone. 3. The composition according to claim 1, wherein said composition is TRF or TRF ₂₅ . 4. A dietary supplement containing the composition according to claim 1 or 3. A foodstuff comprising the composition according to claim 1 or 3. 6. A pharmaceutical composition comprising the composition according to claim 1 or 3. 7. A method for preventing osteoporosis or other bone disease in a patient, comprising administering to the patient a prophylactically effective amount of a composition comprising one or more tocotrienols. Including, methods. 8. The method of claim 7, wherein said composition contains TFR or TFR ₂₅ . 9. The method according to claim 7, wherein the composition contains one or more additional bone enhancing substances. 10. The method of claim 9, wherein said additional bone augmenting substance is selected from the group consisting of a bone resorption inhibitor and an osteogenic stimulant. 11. The additional bone enhancing substance is calcium, estrogen,
Consists of antiestrogens, environmental estrogens, vitamin D, gonadal steroids, anabolic steroids, calcitonin, flavonoids, tyrosine kinase inhibitors, protein kinase C modulators, bisphosphate, fluoride, synthetic parathyroid hormone, vitamin K, cytokine mediators and selenium 11. The method of claim 10, wherein the method is selected from a group. 12. The additional bone-enhancing substance may be a calcium salt, estrogen, tamoxifen, cumestrol, methoxychlor, bisphenol A, zeranol, calciferol, 1-α-hydroxycholecalciferol, pentahydroxyflavone, naringenin, hesperetin, tangeretin, Nobiletin,
Quercetin, apigenin, genestein and daidzein, ipriflavone,
Herbimycin, phorbol ester, cyclic bisphosphate, alendronate,
11. The method of claim 10, wherein the method is selected from the group consisting of risedronate, etidronate, sodium fluoride, vitamin Kl, vitamin K2, probucol, lipoic acid, and alpha tocopherol. 13. The method of claim 10, wherein said additional bone augmenting substance is selected from the group consisting of calcium salts, ipriflavone, genestein, vitamin D, vitamin K, lipoic acid and tocopherol. 14. The composition according to claim 1, wherein the composition is tocotrienol, α-tocotrienol,
β-tocotrienol, gamma-tocotrienol, [delta] tocotrienol, containing one or more tocotrienol selected from the group consisting of P ₁₈ tocotrienols and P ₂₅ tocotrienols The method of claim 7. 15. The method of claim 7, wherein said composition is administered orally. 16. The composition of claim 1, wherein said composition comprises about 0.1 mg and about 5000 mg per dose.
The method according to claim 7, comprising tocotrienol between 17. The method of claim 7, further comprising the step of administering to the patient one or more additional bone enhancing drugs.