WO1999017761A1 - Use of nordihydroguaiaretic acid to lower serum triglycerides, blood pressure and to treat syndrome x - Google Patents

Abstract

The present invention is directed to formulations comprising nordihydroguaiaretic acid (NDGA) and an amphiphilic vehicle. The present invention is also directed to pharmaceutical compositions comprising a formulation of the present invention and a pharmaceutically acceptable carrier. The present invention further provides methods for using NDGA, including but not limited to, the novel NDGA containing formulations and compositions of the present invention as agents to lower serum triglyceride, free fatty acid or glycerol level in animals. NDGA containing formulations and compositions can also be used as agents to lower free fatty acid levels in animals that have normal levels of glucose, triglycerides and cholesterol. The NDGA formulations can be used to lower blood pressure. The methods of the present invention can also be used to treat or ameliorate the characteristic manifestations of Syndrome X in a non-diabetic animal with normal serum glucose levels. This includes lowering of one or more of the following: serum insulin level, blood pressure, serum triglycerides and free fatty acid levels. The methods entail administering, to an animal in need of such treatment, an effetive amount of a composition whose active ingredient consists essentially of NDGA. The invention is also directed to methods of treatment using NDGA in conjunction with another hypolipidemic agent.

Description

USE OF NORDIHYDROGUAIARETIC ACID TO LOWER SERUM TRIGLYCERIDES. BLOOD PRESSURE AND TO TREAT SYNDROME X

This application is a continuation-in-part of Application Serial No. 08/944,848 the entire disclosure of which is incorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

This invention relates to formulations and pharmaceutical compositions comprising ultra-low, therapeutically effective amounts of nordihydroguaiaretic acid, and methods for using the same for lowering serum triglyceride, free fatty acid or glycerol levels in animals. The invention also relates to formulations and methods for treating and/or ameliorating symptoms of Syndrome X, including insulin resistance, hypertension, high triglyceride level, etc.

2. BACKGROUND OF THE INVENTION 2.1. NORDIHYDROGUAIARETIC ACID

Nordihydroguaiaretic acid ("NDGA") is a phenolic lignan found in several Larrea spp . , including in Larrea divaricata Cav. (C. . Waller et al., J . Am . Pharm . Assn . _.14:78 (1945)) and Larrea tridentata (M. Winkelman, J. Ethnopharmacol . Ij3:109 (1986)). NDGA is also known as (R*, S*) -4, 4 '-(2, 3-dimethyl-l,4-butanediyl)bis[l, 2-benzendiol] ; meso-4 , 4'- (2 , 3 ' -dimethyletramethylene) dipyrocatechol; 2,3- bis(3 , 4 '-dihydroxybenzyl) butane; β , γ-dimethyl- , 5-bis (3,4- dihydroxyphenylbutane) ; masoprocol; CHX-100; and ACTINEX®.

NDGA

2.2. ETHNOBOTANY OF LARREA SPP.

Larrea spp . (Zigophyllaceae ) have been used as botanical medicines in several parts of the New World. In the United States, L . tridentata was reportedly used traditionally as a tonic and as a treatment for numerous ailments, including sores, wounds, rheumatism, and bowel cramps (C.W. Waller, supra) . This plant still has widespread medicinal use today among several groups of traditional peoples in the Southwest United States and in Mexico (F. Brinker, Brit . J . Phytother . 2(l):10-29 (1993/4); R.E.

Dimayuga, J . Ethnopharm . ):209-222 (1987); and M. Winkelman, J. Ethnopharm . JL8_.:109-131 (1986)). Oral decoctions and hot water extracts of dried branches or dried leaf or dried root of Larrea tridentata have been used in Baja California to treat diabetes (Dimayuga, et al., 1987, supra ; Winkelman, Medicinal Anthropology, 11:255-268 (1989)).

Dried leaf decoctions of Larrea tridentata have also been taken as a therapy for kidney problems and urinary tract infections, rheumatism and arthritis, wounds and skin injuries, and paralysis (Winkelman (1986) , supra) . Today in Baja California Sur, some of the local people reportedly utilize Larrea tridentata in treatments for rheumatism, stomach ache, and ulcers. Several elder local informants of Baja California Sur recalled that Larrea tridentata was employed to treat foot infections, kidney pain, diabetes, high blood pressure, and headache (Dimayuga, et al. (1987) , supra) . These informants claimed that their knowledge of medicinal plants was passed down to them by their forebears, the Pericues , one of several indigenous tribes inhabiting the area that now constitutes the southernmost part of Baja California (R.E. Dimayuga et al., J. Ethnopharm . 17:183-193 5 (1986); A.W. North, A.W. , American Anthropologist . 10:236-250 (1908; Reprint, 1962; New York: Kraus Reprint Corp.)).

In Argentina, a dried leaf decoction of L. divaricata (jarilla in vernacular) is used externally for the treatment of inflammation (A.L. Bandoni et al., Lloydia

10 3.5(1) :69-77 (1972)) and orally as a therapy for urinary tract infections (C. Perez et al., Fitoterapia , 65 (2) : 169-172 (1994)). In the United States, oral hot water extracts of L . divaricata have been administered for the treatment of arthritis and inflammation (J.R. Christopher, School of

15 Natural Healing (1976)).

In the mid-nineteenth century, a surgeon documented the medicinal use of Larrea tridentata by Native Americans in New Mexico (V.J. Vogel, American Indian Medicine (1970) ; and A. Clapp, 5 Transactions of the American Medical Association

20 750-751 (1852)). Indigenous Amerindian populations throughout the Southwest, certain areas of the Southeast, and possibly Mexico, historically utilized this species in traditional remedies for numerous ailments. Among Southwest tribes, the Pi a and Maricopa reportedly used fresh twigs and

25 branches of Larrea tridentata in therapies for rheumatic and body pain (V.J. Vogel (1970) , supra ; and A. Hrdlicka, Bureau of American Ethnology 242, 244-245 (1908)). Another Southwest Indian people, the Papago, treated contusions with topical solutions of a hot decoction of boiled leaves of

30 Larrea tridentata mixed with salt (V.J. Vogel (1970) , supra ; and A. Hrdlicka (1908) , supra) .

2.3. ISOLATION AND SYNTHESIS OF NDGA NDGA occurs naturally as the meso-form in the 35 leaves and small stems of the creosote bush, Larrea divaricata, Zygophyllaceae (Covillea tridentata) . NDGA was first isolated, in its meso form, from Larrea divaricata , the creosote bush of the Southwestern United States (C.W. Waller et al. (1945), supra) .

The preparation of NDGA from guaiaretic acid dimethyl ether was reported independently (G. Schroeter et al., Ber. .51:1587 (1918); and R.D. Haworth et al. J. Chem .

Soc . 1423 (1934)). The stereochemistry of NDGA in its naturally occurring form has been assigned (Perry et al., J.

Org . Chem . , 2:4371 (1972)). Other syntheses of NDGA have been reported (C.W. Perry et al., J. Org. Chem . 37 (26) :4371 (1972); A.W. Schrecker, J. Am . Chem . Soc . 19:3823 (1957);

Lieberman et al., J. Am . Chem . Soc , 619.: 1540 (1947); EP

Publication No. 247 035 to Chemex Pharmaceuticals, Inc.; U.S.

Patent No. 4,562,298 to Parkhurst et al.; U.S. Patent No.

3,906,004 to Perry; U.S. Patent No. 3,843,728 to Perry; U.S. Patent No. 3,769,350 to Perry; U.S. Patent No. 2,644,822 to

Pearl; and U.S. Patent No. 2,456,443 to Mueller et al.).

2.4. BIOLOGICAL ACTIVITIES OF NDGA NDGA has been reported to be useful for treating tumors (U.S. Patent No. 5,409,690 to Howell et al.; U.S. Patents No. 4,77,229, 4,880,637 and 5,008,294 to Jordan et al.; and PCT Publication No. WO 88/03805 to Chemex Pharmaceutical, Inc.); for treating HIV (J.N. Gnabre et al., Journal of Chromatography A 719 : 353 (1996); and J. Gnabre et al., Tetrahedron 51(45): 12203 (1995)); as an antioxidant (U.S. Patent No. 2,373,192 to Lauer) ; as an agent to lower glucose absorption (G.L. Kellett et al., Biochemical Pharmacology 4_5(9):1932 (1993)); as an agent to inhibit glucose-induced insulin release (J. Turk et al., Biochimica et Biophysica Acta 834:23 (1985); S. Yamamoto et al., J.

Biol . Chem . 2_58 (20) : 12149 (1983); and S.A. Metz et al. , Life Sciences 12_.:903 (1983)); as an agent to retard the rate of insulin denaturation (H. Thurow, Insulin, Chemistry, Structure and Function of Insulin and Related Hormones 215 (1980)); as a lipoxygenase inhibitor (A.M. Band et al.. Pharmacology Communications 4.(4) :285 (1994); M. Miiller et al.. Biochemistry 31:4656 (1992); G. Pieper et al.. Am . J. Physiol . H825 (1988)); and U.S. Patent No. 4,708,964 to Allen); as an anti-aging agent (U.S. Patent No. 4,695,590 to Lippman) ; as a thromboxane A₂ synthetase inhibitor (U.S. Patent No. 4,495,357); for treating multi-drug resistance (PCT Publication WO 95/00129 to Chemex Pharmaceuticals,

Inc.); for treating actinic keratoses (Olsen et al., 1991, J. Am . Acad . Dermatol . _.14:738 (1991); D.E. Wilson et al., J. Neurosurg. 21:551 (1989); and Wilkinson et al.. Int . J. Dermatol . 2J>:660 (1987)); as an agent for inhibiting platelet-derived growth factor-stimulated DNA synthesis (J. Do in et al., J . Biol . Chem . 169(11): 8260 (1994)); as an inhibitor of eicosaniod metabolism (T. Bahr et al., Biomed . Biochim . Acta 42(10/11) : S289 (1988)); as an inhibitor of blood vessel response to prostaglandin F_2α (I. Kumura et al., Jpn . J . Pharmacol . _.64_:65 (1994)); and as a "metabolism stimulator" (French Patent Publication No. 3.866M).

In addition, the effect of NDGA on contractile responses of rat urinary bladder (K. Kamata et al.. Gen . Pharmac . 24.(3) :547 (1993)); arachidonic acid uptake (C. Perez, Diabetes Research and Clinical Practice 2:69 (1989)), reduction of N-methyl-D-aspartate toxicity (S.M. Rothman, Neuropharmacology 12.(11) :1279 (1993)) and arachidonic acid- mediated progesterone production (R.E. Ciereszko et al., Prostaglandins _.50:103 (1995)), have also been reported. NDGA has also been reported to be useful for lowering triglycerides. PCT Publication No. WO 93/14751 discloses the use of vanadium and niobium complexes of NDGA for treating hypertriglyceridemia. U.S. Patent No. 3,934,034 to Manning discloses the use of NDGA, at a daily dose of from about 2 to about 500 milligrams per kilogram of body weight p.o., to treat lipidemia. In addition, United Kingdom Patent Publication Nos. 1,361,856 and 1,427,411 to Sandoz, Ltd. disclose a genus of compounds, encompassing NDGA, useful as hypolipide ic agents at a daily dosage of from about 150 mg to about 4000 mg.

NDGA has been shown to be cytotoxic in hep2 and Vero cell lines, as a likely result of its catechol groups (J.M. Za ora et al., Journal of the Tennessee Academy of Sciences 62(4) :77 (1992)).

2.5. SYNDROME X The ability of insulin to stimulate glucose uptake varies widely amongst individuals with normal glucose tolerance, and the defect in insulin action in about 25% of these individuals does not differ substantially from that of patients with either impaired glucose tolerance (IGT) or non- insulin-dependent diabetes mellitus (NIDDM) . The degree to which glucose tolerance deteriorates in insulin resistant individuals varies as a function of both the magnitude of the loss of in vivo insulin action and the capacity of the pancreas to compensate for this defect. Insulin resistant individuals with normal glucose tolerance are hyperinsulinemic (exhibit high insulin levels) when compared with an insulin-sensitive control group (Reaven,G.M. Pathophysiology of Insulin Resistance in Human Disease. Physiol . Rev . 25:473-486, (1995)). It appears that the increase in plasma insulin concentration permits these individuals to overcome the defective insulin action. As long as insulin-resistant individuals are capable of increasing their insulin secretory response, gross decompensation of glucose homeostasis can be prevented. The compensatory hyperinsulinemia necessary to overcome insulin resistance can prevent the onset of NIDDM. However, insulin resistant individuals are at greater risk to be dyslipedemic, e.g., exhibit a high plasma triglyceride (TG) and/or a low high-density lipoprotein (HDL) -cholesterol concentration, and hypertensive, all of which increases the risk of coronary heart disease (CHD) . Thus a whole cluster of interrelated symptoms are within Syndrome X (Reaven G.M. , Syndrome X Past, Present and Future in Clinical Research in Diabetes and Obesity, Vol. II: Diabetes and Obesity, Draznin B., Rizza R. eds. Humana Press 357-382, (1997)).

Presently, conventional CHD treatment plans utilize multiple therapeutic agents to treat these multiple symptoms. It would be beneficial to reduce the risk of CHD by treating this multi-symptomatic syndrome with a single therapeutic agent. Concurrent treatment of multiple symptoms would eliminate drug-drug interactions, reducing exposure to undesirable side-effects and decreased efficacy. Patient compliance would be improved since it would be easier to take just one therapeutic agent. There would be a cost benefit with the use of one therapeutic agent to treat this multiple- symptomatic syndrome. Thus the use of a single therapeutic agent would offer a unique approach to treating the multiple symptoms of Syndrome X.

2.6. HYPERTENSION

U.S. Patent No. 5,559,105 to Bryan (PCT Publication No. WO 94/02474) discloses a genus of compounds similar to, but not specifically including, NDGA that are useful as antagonists of the endothelin receptor. These compounds are stated to be useful in the treatment of a variety of cardiovascular including hypertension and renal diseases. Cathecols (also known as pyrocathecols; 1,2- benzenediols; o-dihydroxybenzenes) have been reported to have anti-hypertensive activity. For example, a dihydroxyphenylalanine (DOPA) derivative, methyldopa (also known as a methyldopa; L-3- (3 , 4-dihydroxyphenyl) -2 methylalanine; and aldomet) has been reported to have centrally acting hypotensive activity. (Wolff, F. , J. Chronic Dis . 12(8) :721 (1964)). A synthetic cathecol, U-0521 (3' ,4'-dihydroxy-2-methylpropriophenone) reduced blood pressure in spontaneously hypertensive rats (SHR) . (Lloyd, T, et al., Life Sci . 11(19) :2121 (1982)).

Citation or identification of any reference in Section 2 or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention. 3. SUMMARY OF THE INVENTION

The present invention is directed to novel formulations comprising nordihydroguaiaretic acid (NDGA) and an a phiphilic vehicle. The present invention is also directed to pharmaceutical compositions comprising a formulation of the present invention and a pharmaceutically acceptable carrier. The formulations and pharmaceutical compositions of the present invention can be used as agents to lower serum triglyceride, free fatty acid and/or glycerol levels in animals, preferably in mammals, including humans. The present invention is also directed to methods for use of the novel formulations to lower serum triglyceride, free fatty acid or glycerol levels in animals, preferably in mammals, including humans in which such treatment is desired, for example, in animals having hyperlipidemic conditions. The present invention is further directed to methods comprising administration of a pharmaceutical composition of the present invention to lower serum triglyceride, free fatty acid or glycerol levels in animals, preferably in mammals, including humans in which such treatment is desired, for example, in animals having hyperlipidemic conditions.

The present invention is also directed to methods for lowering serum free fatty acid levels comprising administering NDGA to an animal having an increased level of serum free fatty acids and but having normal levels of serum triglycerides, glucose and cholesterol.

Compositions containing NDGA, including the novel formulations and compositions of the present invention can be used in the treatment of or amelioration of symptoms of any disease or disorder in which the lowering of serum triglyceride, free fatty acid and/or glycerol levels is desired. Such diseases or disorders include, but are not limited to, hyperlipidemia, hypertriglyceride ia, high levels of free fatty acids, coronary heart disease, arteriosclerosis, atherosclerosis and atherosclerotic heart disease. The present invention is further directed to methods of lowering blood pressure. This method comprises administering NDGA to a non-diabetic animal, including a human, in an amount effective to lower pressure. The present invention is further directed to methods to ameliorate or reduce the symptoms of Syndrome X and also to treat Syndrome X. This method comprises administering NDGA to a non-diabetic animal, including a human, having one or more of the following symptoms: increased levels of serum insulin, increased serum triglycerides, increased free fatty acids, inappropriate HDL- cholesterol level or elevated blood pressure.

Compositions containing NDGA, including the novel formulations and compositions of the present invention can also be used to treat Syndrome X or ameliorate or reduce its symptoms in non-diabetic animals, including humans. According to the present invention, NDGA, including the novel formulations of the invention containing the same, advantageously ameliorates or treats at least two symptoms of Syndrome X including insulin resistance, hypertension, elevated triglyceride level, etc.

The present invention may be understood more fully by reference to the following figures, detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.

4. DESCRIPTION OF THE FIGURES

Fig. 1 is a line graph showing mean triglyceride levels in hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle or 0.5, 1.0 or 2.5 mg of NDGA in GELUCIRE™ per kg of body weight, b.i.d. —o— represents GELUCIRE™ vehicle; -■- represents NDGA in GELUCIRE™ administered at 0.5 mg of NDGA per kg of body weight, b.i.d.; —♦— represents NDGA in GELUCIRE™ administered at 1.0 mg per kg of body weight, b.i.d.; and ^■— — represents NDGA in GELUCIRE™ administered at 2.5 mg per kg of body weight, b.i.d. *P < 0.05 (GELUCIRE™ vehicle vs. 1.0 mg, 2.5 mg) . #P < 0.05 (GELUCIRE™ vehicle vs. 0.5 mg, 1.0 mg, 2.5 mg) . N is all cases is 6.

Fig. 2 is a line graph showing mean serum glucose levels in hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle or 0.5, 1.0 or 2.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —O— represents GELUCIRE™ vehicle; ^■—■— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d.; —♦— represents NDGA in GELUCIRE™ administered at 1.0 g per kg of body weight, b.i.d.; and —x— represents NDGA in GELUCIRE™ administered at 2.5 mg per kg of body weight, b.i.d. N in all cases is 6.

Fig. 3 is a line graph showing mean body weights of hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle or 0.5 , 1.0 or 2.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —O— represents GELUCIRE™ vehicle; — 9— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d.; —♦— represents NDGA in GELUCIRE™ administered at 1.0 mg per kg of body weight, b.i.d.; and —x— represents NDGA in GELUCIRE™ administered at 2.5 mg per kg of body weight, b.i.d. N in all cases is 6. *P < 0.05 (GELUCIRE™ vehicle vs. 2.5 mg) . Fig. 4 is a line graph showing mean triglyceride levels in hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —O- - represents GELUCIRE™ vehicle; —•— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d. *P < 0.05. N for GELUCIRE™ vehicle only = 6; N for 0.5 mg of NDGA in GELUCIRE™ per kg of body weight, b.i.d. =

5.

Fig. 5 is a line graph showing mean triglyceride levels in hypertriglyceridemic (fructose-fed) rats administered with CMC vehicle only or 0.5 mg of NDGA (formulated in sodium carboxymethylcellulose (CMC) ) per kg of body weight, b.i.d. —D— represents CMC vehicle; —■— represents NDGA in CMC administered at 0.5 mg per kg of body weight, b.i.d. N in both cases is 6.

Fig. 6 is a line graph showing mean serum glucose levels in hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —o-

- represents GELUCIRE™ vehicle; —•— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d. N for GELUCIRE™ vehicle only = 6; N for 0.5 mg of NDGA in GELUCIRE™ per kg of body weight, b.i.d. = 5.

Fig. 7 is a line graph showing mean serum glucose levels in hypertriglyceridemic (fructose-fed) rats administered with CMC vehicle only or 0.5 mg of NDGA (formulated in sodium carboxymethylcellulose (CMC) ) per kg of body weight, b.i.d. —D— represents CMC vehicle; —■— represents NDGA in CMC administered at 0.5 mg per kg of body weight, b.i.d. N in both cases is 6.

Fig. 8 is a line graph showing mean body weight of hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —O— represents GELUCIRE™ vehicle; —•— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d. N for GELUCIRE™ vehicle only = 6; N for 0.5 mg of NDGA in GELUCIRE™ per kg of body weight, b.i.d. = 5.

Fig. 9 is a line graph showing mean body weight of hypertriglyceridemic (fructose-fed) rats administered with CMC vehicle only or 0.5 mg of NDGA (formulated in sodium carboxymethylcellulose (CMC)) per kg of body weight, b.i.d. —D— represents CMC vehicle; —■— represents NDGA in CMC administered at 0.5 mg per kg of body weight, b.i.d. N in both cases is 6.

Fig. 10 is a line graph showing mean food consumption of hypertriglyceridemic (fructose-fed) rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA (formulated in GELUCIRE™) per kg of body weight, b.i.d. —o-

- represents GELUCIRE™ vehicle; —•— represents NDGA in GELUCIRE™ administered at 0.5 mg per kg of body weight, b.i.d. N for GELUCIRE™ vehicle only = 6; N for 0.5 mg of NDGA in GELUCIRE™ per kg of body weight, b.i.d. = 5.

Fig. 11 is a line graph showing mean food 5 consumption of hypertriglyceridemic (fructose-fed) rats administered with CMC vehicle only or 0.5 mg of NDGA (formulated in (formulated in sodium carboxymethylcellulose (CMC)) per kg of body weight, b.i.d. —D— represents CMC vehicle; —■— represents NDGA in CMC administered at 0.5 mg 10 per kg of body weight, b.i.d. N in both cases is 6.

Fig. 12 is a line graph showing triglyceride levels in diabetic rats treated with PTP vehicle or NDGA formulated in PTP at 80 mg/kg. —o— represents PTP vehicle; —■— represents NDGA in PTP administered at 80 mg per kg of body 15 weight, b.i.d. N in both cases is 7.

Fig. 13 is a line graph showing glucose levels in diabetic rats treated with PTP vehicle or NDGA at 80 mg/kg formulated in PTP. —O— represents PTP vehicle; —■— represents NDGA in PTP administered at 80 mg per kg of body 20 weight, b.i.d. N in both cases is 7.

Fig. 14 is a line graph showing free fatty acid levels in diabetic rats treated with PTP vehicle or NDGA at 80 mg/kg formulated in PTP. —O— represents vehicle; —■— represents NDGA in PTP administered at 80 mg per kg of body 25 weight, b.i.d. N in both cases is 7.

Fig. 15 is a line graph showing triglyceride response to Triton™ injection in diabetic rats treated with PTP vehicle or NDGA at 80 mg/kg formulated in PTP. —o— represents vehicle; —■— represents NDGA in PTP administered 30 at 80 mg per kg of body weight, b.i.d. N in both cases is 7.

Fig. 16 is a line graph showing triglyceride levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA at 80 mg/kg formulated in GELUCIRE™. —O— represents GELUCIRE™ vehicle; —■— represents NDGA in GELUCIRE™ administered at 35 80 mg per kg of body weight, b.i.d. N in both cases is 6.

Fig. 17 is a line graph showing glucose levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA at 80 mg/kg formulated in GELUCIRE™. ^■—O— represents GELUCIRE™ vehicle; —■— represents NDGA in GELUCIRE™ administered at 80 mg per kg of body weight, b.i.d. N in both cases is 6. Fig. 18 is a line graph showing free fatty acid 5 levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA at 80 mg/kg formulated in GELUCIRE™. —O— represents GELUCIRE™ vehicle; —■— represents NDGA in GELUCIRE™ administered at 80 mg per kg of body weight, b.i.d. N in both cases is 6.

10 Fig. 19 is a line graph showing triglyceride response to Triton injection in diabetic rats treated with GELUCIRE™ vehicle or NDGA at 80 mg/kg formulated in GELUCIRE™. —O— represents GELUCIRE™ vehicle; —■— represents NDGA in GELUCIRE™ administered at 80 mg per kg of

15 body weight, b.i.d. N in both cases is 6.

Figs. 20A-20B are line graphs showing triglyceride levels in diabetic rats treated with vehicle or NDGA. Fig. 20A, --•— represents CMC vehicle; —D— represents NDGA in CMC at 40 mg/kg; and —O— represents NDGA in CMC at 80

20 mg/kg. Fig. 20B, —•— represents water; —■— represents GELUCIRE™ vehicle; —o— represents NDGA in GELUCIRE™ at 40 mg/kg; and —Δ— represents NDGA in GELUCIRE™ at 250 mg/kg.

Figs. 21A-21B are line graphs showing glucose levels in diabetic rats treated with vehicle or NDGA. Fig.

25 21A, —•— represents CMC vehicle; —□— represents NDGA in CMC at 40 mg/kg; and —o— represents NDGA in CMC at 80 mg/kg. Fig. 2IB, —•— represents water; —■— represents GELUCIRE™ vehicle; —O— represents NDGA in GELUCIRE™ at 40 mg/kg; and —Δ— represents NDGA in GELUCIRE™ at 250 mg/kg.

30 Figs. 22A-22B are line graphs showing free fatty acid levels in diabetic rats treated with vehicle or NDGA. Fig. 22A, —•— represents CMC vehicle; —D— represents NDGA in CMC at 40 mg/kg; and —o— represents NDGA in CMC at 80 mg/kg. Fig. 22B, —•— represents water; —■— represents

35 GELUCIRE™ vehicle; —o— represents NDGA in GELUCIRE™ at 40 mg/kg, and —Δ— represents NDGA in GELUCIRE™ at 250 mg/kg. Fig. 23 is a line graph showing serum insulin levels in diabetic rats treated with vehicle or NDGA. —•— represents water; —■— represents GELUCIRE™ vehicle; —o— represents NDGA in GELUCIRE™ at 40 mg/kg; and ^■—Δ— represents NDGA in GELUCIRE™ at 250 mg/kg.

Fig. 24 is a bar graph showing liver glycogen levels in diabetic rats treated with CMC vehicle (left bar) or NDGA formulated in CMC at 40 (middle bar) or 80 mg/kg (right bar) . Fig. 25 is a line graph showing serum glucose levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA formulated in GELUCIRE™. —•-- represents GELUCIRE™ vehicle control; —□— represents NDGA in GELUCIRE™ at 150 mg/kg. Fig. 26 is a line graph showing serum glucose levels in diabetic rats having starting glucose concentrations greater than 350 mg/dl treated with GELUCIRE™ vehicle or NDGA. —•— represents GELUCIRE™ vehicle control; —D— represents NDGA (in GELUCIRE™) at 150 mg/kg. Fig. 27 is a line graph showing serum triglyceride levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA formulated in GELUCIRE™. —•— represents GELUCIRE™ vehicle control; —D— represents NDGA (in GELUCIRE™) at 150 mg/kg. Fig. 28 is a line graph showing serum insulin levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA formulated in GELUCIRE™. —•— represents GELUCIRE™ vehicle control; —D— represents NDGA (in GELUCIRE™) at 150 mg/kg. Fig. 29 is a line graph showing serum free fatty acid levels in diabetic rats treated with vehicle or NDGA. —•— represents GELUCIRE™ vehicle control; —D— represents NDGA (in GELUCIRE™) at 150 mg/kg.

Fig. 30 is a line graph showing serum glycerol levels in diabetic rats treated with GELUCIRE™ vehicle or NDGA formulated in GELUCIRE™. —•— represents GELUCIRE™ vehicle control; —D— represents NDGA (in GELUCIRE™) at 150 mg/kg.

Fig. 31 is a bar graph showing liver glycogen concentrations in diabetic rats treated with GELUCIRE™ 5 vehicle or NDGA formulated in GELUCIRE™ at 150 mg/kg.

Figs. 32A-32C are line graphs showing mean serum glucose levels in NIDDM model rats. Fig. 32A, —•— represents CMC vehicle; —D— represents non-micronized NDGA in CMC at 150 mg/kg; and — — represents non-micronized NDGA

10 in CMC at 250 mg/kg. Fig. 32B, —•— represents CMC vehicle; —D— represents micronized NDGA in CMC at 150 mg/kg. Fig. 32C, —•— represents CMC vehicle; —D— represents non- micronized NDGA in CMC at 250 mg/kg; —Δ— represents micronized NDGA in CMC at 200 mg/kg; and —O— represents

15 micronized NDGA in CMC at 250 mg/kg.

Figs. 33A-33C are line graphs showing mean serum triglyceride levels in NIDDM model rats. Fig. 33A, —•— represents CMC vehicle; —D— represents non-micronized NDGA in CMC at 150 mg/kg; and —Δ— represents non-micronized NDGA

20 in CMC at 250 mg/kg. Fig. 33B, —•— represents CMC vehicle; —D— represents micronized NDGA in CMC at 150 mg/kg. Fig. 33C, —•— represents CMC vehicle; —D— represents non- micronized NDGA in CMC at 250 mg/kg; —Δ— represents micronized NDGA in CMC at 200 mg/kg; and —0— represents

25 micronized NDGA in CMC at 250 mg/kg.

Fig. 34 is a bar graph showing mean blood pressure in a non-diabetic animal model; left panel, animals treated with vehicle only; right panel, animals treated with NDGA (a/k/a masoprocol) . See, Section 11 for details.

30

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. FORMULATIONS OF NDGA/AMPHIPHILIC VEHICLE COMPOSITIONS

The present invention is directed to novel

__ formulations comprising nordihydroguaiaretic acid (NDGA) and an a phiphilic vehicle. The present invention is also directed to pharmaceutical compositions comprising a formulation of the present invention and a pharmaceutically acceptable carrier. The formulations and compositions of the present invention can be used as agents to lower serum triglyceride, free fatty acid or glycerol levels in animals, preferably in mammals, including humans or to treat or ameliorate one or more of the symptoms of Syndrome X. Advantageously, NDGA is used to treat or ameliorate at least two of the symptoms of Syndrome X.

The present invention is also directed to methods for use of compositions containing NDGA, including , but not limited to, the novel NDGA formulations to lower serum triglyceride, free fatty acid or glycerol levels in animals, preferably in mammals, including humans in which such treatment is desired, for example, in animals having hyperlipidemic conditions. The present invention is further directed to methods comprising administration of a pharmaceutical composition containing NDGA to lower serum triglyceride, free fatty acid or glycerol levels in animals, preferably in mammals, including humans in which such treatment is desired, for example, in animals having hyperlipidemic conditions.

The invention is based, at least in part, on the discovery that NDGA can lower free fatty acid levels in animals. The invention is also based, at least in part, on the discovery that when NDGA is contained in a formulation with an amphiphilic vehicle, the amount of NDGA required for efficacious administration to reduce triglyceride or glycerol levels can be substantially lower than that needed for NDGA formulations having an aqueous vehicle. The formulations and compositions of the present invention can be used in the treatment of or amelioration of symptoms of any disease or disorder in which the lowering of serum triglyceride, free fatty acid or glycerol levels is desired. Such diseases or disorders include, but are not limited to, hyperlipidemia, hypertriglyceridemia, high levels of free fatty acids, coronary heart disease, arteriosclerosis, atherosclerosis and atherosclerotic heart disease.

NDGA containing compositions, including but not limited to, the novel NDGA formulations and compositions of the present invention, can be used to lower blood pressure in non-diabetic animals, including humans. Advantageously, NDGA compositions can be used to treat or ameliorate hypertension including essential hypertension, in non-diabetic animals, including humans. NDGA containing compositions, including but not limited to, the novel NDGA formulations and compositions of the present invention, can be used in the treatment of or amelioration of symptoms of Syndrome X in non-diabetic animals, including humans. Advantageously, NDGA compositions can be used to treat or ameliorate at least two symptoms of Syndrome X, including insulin resistance, elevated blood pressure, elevated triglycerides, etc. This is based on the discovery of the ability of NDGA to lower the levels of serum insulin, triglycerides, free fatty acids and blood pressure in animals with normal levels of serum glucose.

The novel formulations of the present invention comprise NDGA and an amphiphilic vehicle. NDGA is well known in the art and can be obtained by a variety of known methods, such as isolation from natural sources or by chemical synthesis, see Section 2, supra . Further, all stereoisomers of NDGA can be employed (D-, L-, racemic mixture of D- and L- , and meso form of NDGA) .

As used in the present application, an "amphiphilic vehicle" is defined as a composition which has a Hydrophile- Lipophile Balance (HLB) value of about 12 to about 18, preferably from about 13 to about 15, more preferably of about 14 (see generally, "Remington's Pharmaceutical Sciences" by E.W. Martin) . In one embodiment of the present invention, a suitable amphiphilic vehicle is a water dispersible blend of glycerides and polyethylene glycol esters of fatty acids. In another embodiment, a suitable amphiphilic vehicle is a water dispersible blend composed of glyceril and polyethylene glycol esters.

In yet another embodiment, the amphiphilic vehicle is a composition having a HLB value of about 12 to about 18, preferably from about 13 to about 15, more preferably of about 14 and in which NDGA can be dissolved.

Additional examples of suitable amphiphilic vehicles include, but are not limited to, glycerides, such as a monoglyceride of a fatty acid, a diglyceride of a fatty acid, a triglyceride of a fatty acid, e . g . , a triglyceride of a fatty acid having 12 to 18 carbons, a triglyceride of a vegetable fatty acid and mono- and di-glycerides, a glyceride and an emulsifier, an eutectic mixture of mono, di, and triglycerides, saturated polyglycolized glycerides, such as GELUCIRE™ (Gattefosse Corp., Westwood, New Jersey) and ethoxylated glycerides. Other examples of suitable amphiphilic vehicles include partially hydrogenated and hydrogenated plant, vegetable, and animal fats, such as corn oil, beeswax and cocoa butter; alkylene glycol fatty acid esters; polyalkylene glycol fatty acid esters, such as

EMULPHOR™ (Rhone Poulenc, Monmouth Junction, NJ) ; petroleum- based food-grade waxes, and the like. Other examples of suitable amphiphilic vehicles include polyoxyethylene fatty acid esters, e . g. , polyoxyethylene castor oil derivatives, such as CREMAPHOR™ (BASF Corporation, Wyandotte, Michigan) ; and saturated and unsaturated fatty acids such as palmitic, stearic, oleic, linoleic, linolenic and arachidonic acid. More than one amphiphilic vehicle can be employed in the formulations and compositions of the present invention. A preferred amphiphilic vehicle is a derivative of polyoxyethylene castor oil.

A more preferred amphiphilic vehicle is any of a family of saturated polyglycolized glycerides that comprise a mixture of mono-, di-, and tri-glycerides and mono- and di- fatty acid esters of polyethylene glycol, and are obtained by polyglycolysis of natural hydrogenated vegetable oils with polyethylene glycols. These compounds are non-aqueous inert semi-solid waxy materials, amphiphilic in character, and available with varying physical characteristics. They are surface active in nature and disperse in aqueous media forming micelles, microscopic globules or vesicles. They are identified by their melting point/HLB value, where the HLB (Hydrophile-Lipophile Balance) value is a measure of their hydrophobic or hydrophilic nature. The lower the HLB value, the more hydrophobic is the material. The higher the HLB value, the more water-soluble it is. The polyglycolyzed glycerides are commercially available and are sold under the trademark GELUCIRE™ (Gattefosse Corp. , Westwood, New Jersey) . They are mixtures of monoesters, diesters and/or triesters of glycerides of long chain (C,₂ to C₁₈) fatty acids, and PEG (mono- and/or di- esters of long chain (C₁₂ to C₁₈) fatty acids with controlled hydrophilic-lipophilic properties (HLB values) as well as melting points. The large family of GELUCIREs™ is characterized by a wide range of melting points from about 33 °C to about 55 °C, and by a variety of HLB values of from about 1 to about 14. The first number in the nomenclature of GELUCIREs™ denotes its melting point, while the second number denotes the HLB value. In the present invention all grades of GELUCIRE™ may be employed. A preferred GELUCIRE™ is GELUCIRE™ 44/14. Other GELUCIREs™ suitable for use herein include GELUCIRE™ 42/12 and 50/13 alone or in combinations. One or a mixture of different grades of GELUCIRE™ material may be chosen to achieve the desired characteristics, e . g . , melting point or HLB value.

In an alternative embodiment, a formulation of the present invention comprises NDGA in an oil together with an aqueous carrier and an emulsifier or a surfactant. The formulation of this alternative embodiment is obtained by dissolving NDGA in an oil, adding an aqueous carrier and an emulsifier or surfactant, and mixing sufficiently to combine the components to form a uniform, stabilized suspension.

Suitable oils for this embodiment include but are not limited to those of petroleum oil such as mineral oil; vegetable oil such as peanut oil, corn oil, cottonseed oil, soybean oil, and sesame oil; animal oil; or oil of synthetic origin. Suitable emulsifiers or surfactants include but are not limited to sorbitan trioleate (1.8) , sorbitan tristearate (2.1), propylene glycol monostearate (3.4), sorbitan sesquioleate (3.7), glycerol monostearate (non-self- emulsifying) (3.8), sorbitan monooleate (4.3), propylene glycol monolaurate (4.5), sorbitan monostearate (4.7), glyceryl monostearate (self-emulsifying) (5.5), sorbitan monopalmitate (6.7), sorbitan monolaurate (8.6), polyoxyethylene-4-lauryl ether (9.5), polyethylene glycol 400 monostearate (11.6), polyoxyethylene-4-sorbitan monolaurate (13.3), polyoxyethylene-20-sorbitan monooleate (15.0), polyoxyethylene-20-sorbitan monopalmitate (15.6), polyoxyethylene-20-sorbitan monolaurate (16.7), polyoxyethylene-40-stearate (16.9), sodium oleate (18.0) and sodium lauryl sulfate (40.0) (where numbers in parenthesis equal the HLB) . Suitable aqueous carriers include but are not limited to water, aqueous solutions such as saline, etc. The present invention also provides pharmaceutical and therapeutic compositions comprising NDGA, an amphiphilic vehicle and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, or excipient with which the formulation is administered. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil; vegetable oil such as peanut oil, corn oil, cottonseed oil, soybean oil, and sesame oil; animal oil; or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as pharmaceutically acceptable carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, magnesium stearate, microcrystalline cellulose, stearic acid, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The formulations and compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, preservatives, anti-oxidants or pH buffering agents. These formulations and compositions can take the form of solutions, suspensions, emulsion, tablets, capsules, powders, sustained- release formulations and the like. The formulations and compositions can be formulated with traditional binders, carriers and preservative agents, such as antioxidants, e.g., Vitamin C (ascorbic acid) , Vitamin E (α-tocopherol) . Examples of suitable pharmaceutically acceptable carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such formulations and compositions contain a therapeutically effective amount of NDGA, together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to a subject. The formulation should suit the mode of administration. Suitable pharmaceutical salts of NDGA according to the present invention include but are not limited to sodium, potassium, calcium, lithium, magnesium, barium, ammonium salts, salts of amino acids, such as aspartate and glutamate and combinations thereof. NDGA can be in the form of mono-, di-, tri- or tetra-salts. Due to the nature of some amphiphilic vehicles, the amphiphilic vehicle can act also as the pharmaceutically acceptable carrier. Further, mixtures of any of the foregoing pharmaceutically acceptable carrier compounds can be used in the formulations and compositions of the present invention.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients, i .e . , NDGA, amphiphilic vehicle, of the pharmaceutical compositions of the invention. 5.2. METHODS FOR USE OF CONTAINING NDGA COMPOSITIONS

In one embodiment, the methods of the present invention comprise administering to a subject in need thereof an effective amount of an NDGA containing composition, including, but not limited to, formulation of the present invention, i .e . , NDGA and an amphiphilic vehicle, to a subject to lower serum triglyceride, free fatty acid or glycerol levels.

In one mode of this embodiment, an effective amount of a therapeutic formulation comprising NDGA, an amphiphilic vehicle and a pharmaceutically acceptable carrier is administered systemically to a subject to lower serum triglyceride, free fatty acid or glycerol levels. The methods of the present invention further comprise administering another hypolipidemic agent in combination with the formulation or composition of the present invention. The subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably a human. In another embodiment, the methods of the present invention comprise administering to a subject in need thereof an effective amount of an NDGA containing composition to lower blood pressure or to treat or ameliorate hypertension. The subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably a human.

In another embodiment, an effective amount of a therapeutic formulation comprising NDGA, and a pharmaceutically acceptable carrier is administered to a subject to treat or ameliorate one or more of the symptoms of Syndrome X, including but not limited to lowering blood pressure, decreasing serum insulin, triglycerides or free fatty acids. In a preferred embodiment, an effective amount of NDGA is administered to a subject to treat or ameliorate at least two symptoms of Syndrome X. The subject is a non- diabetic animal, preferably a mammal, and most preferably a human. The methods, formulations and pharmaceutical compositions of the present invention are used in the treatment of or amelioration of symptoms of any disease or disorder in which the lowering of serum triglyceride, free fatty acid or glycerol levels is desired. Such diseases or disorders include, but are not limited to, hyperlipidemia, hypertriglyceridemia, high levels of free fatty acids, coronary heart disease, arteriosclerosis, atherosclerosis and atherosclerotic heart disease. The methods, formulations and pharmaceutical compositions of the present invention are also used in the treatment of or amelioration of the symptoms of Syndrome X.

Various delivery systems are known and can be used to administer a composition containing NDGA as the sole active component, including a novel NDGA formulation or composition of the present invention. For example, an NDGA containing pharmaceutical composition can be administered systemically by, e.g., intravenous or intramuscular injection. In another example, the NDGA containing pharmaceutical composition can be introduced by any suitable route including intravenously, sub-cutaneously, orally, rectally, trans-cutaneously, topically, intramuscularly, intraarticularly, retrobulbarly , subconjunctivally , by inhalation, etc. For veterinary purposes the composition may be administered intraperitoneally.

While the preferred mode of administration is through the oral mode, the precise mode of administration is left to the discretion of the practitioner. The NDGA containing compositions are advantageously effective when administered orally. Compositions for oral administration may be in the form of tablets, troches, lozenges, granules or powders, emulsions, capsules, syrups or elixirs. Orally administered compositions may contain one or more agents, such as, sweetening agents such as fructose, aspartame or saccharin, flavoring agents such as peppermint, oil of wintergreen, or cherry, coloring agents and preserving agents to provide a pharmaceutically palatable preparation. Moreover, compositions in tablet form may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable oral administered compositions. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used.

In yet another embodiment, a formulation or pharmaceutical composition can be delivered in a vesicle, in particular, a liposome see Langer, 1990, Science 249: 1527-1533 ; Treat et al., 1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid . , pp. 317-327; see generally ibid . ) .

In yet another embodiment, the formulation or pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra ; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88.:507; Saudek et al. , 1989, N. Engl. J. Med. 321:574) . In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise, 1974, (eds.), CRC Pres. , Boca Raton, Florida; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), 1984, Wiley, New York; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. H:61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105) . Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533. The amount of NDGA in the formulation or pharmaceutical composition of the invention which is effective in the lowering of blood pressure, serum triglyceride, free fatty acid or glycerol levels or in the 5 treatment of a particular disease or disorder will depend, for example, on the pre-treatment levels of blood pressure, serum triglycerides or the nature of the disease or disorder and, in light of the teaching of this application, can be determined by standard clinical techniques. In general, the 0 dosage of NDGA ranges from about 0.001 mg/kg body weight to about 350 mg/kg body weight per day. Preferably, the dosage of NDGA ranges from about 0.01 mg/kg to about 150 mg/kg per day. More preferably, the dosage of NDGA ranges from about 0.1 mg/kg to about 10 mg/kg per day. Most preferably, the 5 dosage of NDGA ranges from about 0.5 mg/kg to about 1.5 mg/kg per day. In addition, in vitro or animal assays may optionally be employed to help identify optimal dosage ranges.

The formulations and compositions described herein 0 can be used for research purposes, for example, to investigate the mechanism and activity of hypolipidemic agents and agents which lower glycerol or free fatty acids. The following series of examples are presented by way of illustration and not by way of limitation on the scope 5 of the invention.

6. EXAMPLE: TRIGLYCERIDEMIC LOWERING EFFECTS OF ULTRA-LOW DOSES OF NDGA

The following experiments demonstrate that ultra- _ low doses of NDGA significantly lower serum triglycerides in hypertriglyceridemic, non-diabetic rats, which is an art- recognized model useful to predict the effects of hypotriglyceridemic and hypolipidemic compounds.

5 6.1. MATERIALS AND METHODS

Male CD rats, weighing 126-175 g on arrival from Charles River Laboratories, Hollister, CA, were fed a high fructose (60% by weight) diet (TD 78463) obtained from Harlan Teklad, Madison, WI, which was stored at 4°C prior to use. Administration of a high fructose (~60%) diet is a well-known method of inducing hypertriglyceridemia in rats (L.H. Storlien et al.. Diabetes 41:457 (1993); and I. Zavaroni et al., Metabolism 31:1077 (1982)).

NDGA was obtained from Western Engineering and Research Corp., El Paso, TX; GELUCIRE™ 44/14 vehicle was obtained from Gattefosse Corp., Westwood, New Jersey; and sodium carboxymethylcellulose (CMC) , triglyceride standard and reagent, and glucose standard and reagent were obtained from Sigma Chemical Co., St. Louis, Missouri.

GELUCIRE™ 44/14 (GELUCIRE™) vehicle was warmed to 48-50°C prior to addition of the appropriate amount of NDGA. The resulting mixture was vortexed and then sonicated at 48- 50°C until the NDGA completely dissolved. Prior to administration, GELUCIRE™ vehicle and NDGA/GELUCIRE™ were kept in a water bath at 48-50 °C to prevent solidification. NDGA/carboxymethylcellulose was prepared as a suspension by vortexing a mixture of NDGA and an 0.25% solution of carboxymethylcellulose (CMC) in water prior to use, and mixing intermittently during the administration procedure.

NDGA formulations were prepared fresh daily. The appropriate amount of NDGA in GELUCIRE™ or CMC was administered by oral gavage at a volume of 2.5 ml/kg of body weight.

Blood samples from tail snip bleeds were collected into MICROTAINER™ brand (Becton Dickinson, Franklin Lakes, NJ) serum separator tubes, and centrifuged at 12,000 rpm for 10 minutes. Serum was removed, and triglyceride levels were measured using enzymatic colorimetric methods (M.W. McGowan et al., Clin . Chem . 19.: 538 (1983) and P. Trinder, Ann . Clin . Biochem . 6_.:24 (1969)) using Sigma Diagnostic kits, Sigma Chemical Co., St. Louis, Missouri.

Triglyceride levels are expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) , followed by post-hoc Fisher's protected least- squares difference (PLSD) tests. Statistical significance was defined as p < 0.05.

6.2. EXPERIMENTAL PROTOCOL After administering the high fructose diet for at least two weeks to the rats, basal (pre-treatment) blood samples from tail snip bleeds were collected, and serum triglyceride (TG) levels were measured. A computer sorting program was used to distribute the animals into groups having equivalent initial mean TG levels. Subsequent treatment with vehicle alone or NDGA in the appropriate vehicle was twice per day (b.i.d.) .

Blood samples were collected, by tail snip, three hours after the first dose. In Study #1, an additional blood sample was collected each day, prior to the first dose.

Serum was collected from each blood sample, and triglyceride levels were measured.

The treatments used for Study #1 and Study #2 were as follows:

GROUP DOSE SCHEDULE ROUTE NO. OF BLEEDS (mg/kg) RATS STUDY #1

Vehicle NA b.i.d. oral 6 2/day (GELUCIRE™) NDGA 0.5 b.i.d. oral 6 2/day

(in

GELUCIRE™)

NDGA 1.0 b.i.d. oral 6 2/day

(in

GELUCIRE™)

NDGA 2.5 b.i.d. oral 6 2/day

(in

GELUCIRE™) STUDY #2

Vehicle NA b.i.d. oral 6 1/day

(GELUCIRE™)

NDGA 0.5 b.i.d. oral 5 1/day

(in

GELUCIRE™) Vehicle NA^' b.i.d. oral 6 1/day (CMC) NDGA 0.5 b.i.d. oral 6 1/day (in CMC) NA = none administered

6.3. RESULTS; STUDY #1 Serum triglyceride (TG) levels observed over the course of treatment with GELUCIRE™ vehicle or NDGA (0.5, 1.0 and 2.5 mg/kg of body weight, b.i.d.) in GELUCIRE™ vehicle are shown in Fig. 1. When the data are collapsed (pooling all the daily data values from a given treatment group to produce a single mean value for that treatment group) across the 10 days of treatment and sampling, mean TG levels (mg/dl ± SEM) were: GELUCIRE™ vehicle = 485 ± 22; NDGA (0.5 mg) = 398 ± 14; NDGA (1.0 mg) = 330 ± 13; and NDGA (2.5 mg) = 342 ± 17. At each of the NDGA dosage levels, TG levels were highly significantly lower (p < 0.01) than those obtained after administration of vehicle only. While TG level lowering was significant even at an administration dose of 0.5 mg of NDGA/kg body weight, b.i.d., TG levels obtained from the administration of 1.0 mg and 2.5 mg of NDGA/kg body weight, b.i.d., were significantly lower than those of the 0.5 mg group. This finding indicates that the TG-lowering effect of NDGA is dose-dependent, even at such ultra-low dose levels.

When compared at individual sampling times, the data indicate that the treatment groups that received 1.0 mg and 2.5 mg/kg of body weight, b.i.d., of NDGA, had mean TG levels that were significantly lower than those of the

GELUCIRE™ vehicle treatment group from day 5 through the end of treatment. The treatment group that received 0.5 mg/kg of body weight, b.i.d., of NDGA, had a mean TG level that was significantly lower than that of the GELUCIRE™ vehicle treatment group on day 9. As shown in Fig. 2, serum glucose levels remained normal for all treatment groups throughout the treatment period.

Daily mean body weights are shown in Fig. 3. On days 5 and 6, the treatment group that received 2.5 mg/kg of body weight, b.i.d., of NDGA, had a significantly lower mean body weight than those groups administered with vehicle only. Apart from these two instances, the gain in rat body weight over the course of the treatment was normal and similar among the treatment groups and it does not appear that there was any biologically significant difference in body weight.

6.4. RESULTS; STUDY #2

Serum triglyceride (TG) levels that occurred over the course of treatment with GELUCIRE™ vehicle or NDGA administered at 0.5 mg/kg of body weight, b.i.d., in GELUCIRE™ vehicle, are shown in Fig. 4; serum triglyceride (TG) levels that occurred over the course of treatment with CMC vehicle or NDGA administered at 0.5 mg/kg of body weight, b.i.d., in CMC vehicle, are shown in Fig. 5. When the data are collapsed (pooling all the daily data values from a given treatment group to produce a single mean value for that treatment group) across the 10 days of treatment and sampling, mean TG levels (mg/dl ± SEM) were: GELUCIRE™ vehicle only = 511 ± 26; NDGA (0.5 mg) in GELUCIRE™ vehicle = 376 ± 14; CMC vehicle only = 426 ± 18; and NDGA (0.5 mg) in CMC vehicle = 449 ± 30. The TG levels were significantly lower in the treatment group that was administered with 0.5 mg of NDGA/kg body weight, b.i.d., in GELUCIRE™ vehicle, relative to the treatment group that was administered with GELUCIRE™ vehicle only. There was no significant difference in TG levels between the treatment group that was administered with CMC vehicle only and the treatment group that was administered with 0.5 mg of NDGA/kg body weight, b.i.d., (0.5 mg) in CMC. Accordingly, NDGA, when administered at an ultra-low dosage of 0.5 mg/kg of body weight, b.i.d., was effective at significantly reducing serum triglyceride concentration when formulated in GELUCIRE™, but not in CMC.

In addition, as shown in Fig. 4, when compared at individual sampling times, the treatment group that was administered with 0.5 mg of NDGA/kg body weight, b.i.d., in GELUCIRE™ had significantly lower mean TG levels than the treatment group that was administered with GELUCIRE™ vehicle only, on day 7. As shown in Fig. 6 and Fig. 7, serum glucose levels remained normal throughout the treatment period. Daily mean body weights of fructose-fed rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA/kg body weight, b.i.d., in GELUCIRE™; or with CMC vehicle only or with 0.5 mg of NDGA/kg body weight, b.i.d., in CMC, are shown in Fig. 8 and Fig. 9, respectively. As shown in Fig. 8 and Fig. 9, the gain in body weight over the course of treatment was normal, and was not significantly affected by NDGA treatment.

Daily mean food consumption of fructose-fed rats administered with GELUCIRE™ vehicle only or 0.5 mg of NDGA/kg body weight, b.i.d., in GELUCIRE™; or with CMC vehicle only or with 0.5 mg of NDGA/kg body weight, b.i.d., in CMC, are shown in Fig. 10 and Fig. 11, respectively. As shown in Fig. 10 and Fig. 11, there were no significant effects of NDGA treatment on food consumption.

7. EXAMPLE: EFFECTS OF NDGA TREATMENT ON SERUM TRIGLYCERIDE LEVELS AND SECRETION OF TRIGLYCERIDE BY THE LIVER IN DIABETIC ANIMALS

The following experiments demonstrate that NDGA treatment lowers serum triglyceride (TG) levels and may slightly lower TG secretion by the liver in the NIDDM animal model.

7.1. MATERIALS AND METHODS

Male CD rats, weighing 151-175 g on arrival from Charles River Laboratories, Hollister, CA, were fed a high fructose (60% by weight) diet (TD 78463) obtained from Harlan Teklad, Madison, WI , which was stored at 4°C prior to use, and after at least one week the rats were injected with streptozotocin (STZ) at 40-50 mg/kg.

NDGA was obtained from Western Engineering and Research Corp., El Paso, TX; GELUCIRE™ 44/14 vehicle was obtained from Gattefosse Corp. , Westwood, New Jersey; and carboxymethyl cellulose, triglyceride standard and reagent. and glucose standard and reagent were obtained from Sigma

Chemical Co., St. Louis, Missouri.

GELUCIRE™ 44/14 (GELUCIRE™) vehicle was warmed to

48-50°C prior to addition of the appropriate amount of NDGA. The resulting mixture was vortexed and then sonicated at 48-

50°C until the NDGA completely dissolved. Prior to administration, GELUCIRE™ vehicle and NDGA/GELUCIRE™ were kept in a water bath at 48-50°C to prevent solidification.

NDGA/polyethyleneglycol : tween:propanediol (85 : 10 : 5) (NDGA/PTP) was prepared by vortexing a mixture of NDGA/PTP and then sonicated at 50-55 °C until the NDGA completely dissolved.

The appropriate amount of NDGA, 80 mg/kg, in

GELUCIRE™ or PTP was administered by oral gavage at a volume of 2.5 mL/kg of body weight.

Triton™ WR 1339 was diluted to a concentration of

300 mg/ml in saline and then vortexed and sonicated with heat

(60-70°C) in order to produce an homogenous solution.

Triton™ WR 1339 at 600 mg/kg was injected into a tail vein at a volume of 2 ml/kg body weight.

Blood samples from tail snip bleeds were collected into MICROTAINER™ brand (Becton Dickinson, Franklin Lakes,

NJ) serum separator tubes, and centrifuged at 12,000 rpm for

10 minutes. Serum was removed, and triglyceride and glucose levels were measured using enzymatic colorimetric methods

(M.W. McGowan et al., Clin . Chem . 2j9:538 (1983) and P.

Trinder, Ann . Clin . Biochem . J5:24 (1969)) using Sigma

Diagnostic kits, Sigma Chemical Co., St. Louis, Missouri.

Free fatty acid levels were measured in previously frozen and thawed serum samples by an enzymatic colorimetric method using kits from Wako Chemicals, Richmond, VA.

The data are expressed as the mean ± SEM. Data were analyzed by one-way or two-way analysis of variance

(ANOVA) as appropriate, followed by post-hoc Fisher's protected least-squares difference (PLSD) tests. Statistical significance was defined as p < 0.05. 7.2. EXPERIMENTAL PROTOCOL

After at least one week on the high fructose diet and 3-4 days after STZ injection, basal (pre-treatment) serum triglyceride and glucose levels were measured. After eliminating any animals that were not overtly diabetic (serum glucose <300 mg/dl) , the remaining animals were sorted into groups having equivalent initial mean TG levels using a computer sorting program. Subsequent treatment was with vehicle control or with NDGA twice daily (b.i.d.). Food was removed from the animals' cages 90-120 minutes before the first blood sample was taken during the study so that serum TG and glucose measurements would not fluctuate with food injection. The treatments used for Study #1 and Study #2 were as follows:

GROUP DOSE SCHEDULE ROUTE NO. OF (mg/kg) RATS

STUDY #1

Vehicle NA b.i.d. oral 7 (PTP)

NDGA 80 b.i.d. oral 7 (in PTP)

STUDY #2

Vehicle NA b.i.d. oral 6 (GELUCIRE™)

NDGA 80 b.i.d. oral 6 (in

GELUCIRE™)

NA = none administered

On the last day of treatment, the detergent Triton™ WR 1339, which blocks the tissue uptake of triglycerides, was administered as an indirect method for determining the secretion rate of TG by the liver (Donnelly et al., 1994, J. Pharmacol. Exp. Therap. 270:809-813 ; Mondon et al., 1993, Hypertension 21:373-379; Otway and Robinson, 1967, J. Physiol. 190:321-332) . Triton™ WR 1339 at 600 mg/kg was injected into a tail vein approximately three hours after the first dose of NDGA or vehicle and blood was collected by tail snip 60 and 120 minutes after Triton injection.

7.3. RESULTS; STUDY #1

Serum TG levels in diabetic animals over the course of vehicle (PTP) or NDGA (80 mg/kg) treatment are shown in Figure 12. The NDGA treated animals had significantly lower levels of serum TG than the vehicle treated controls. Serum glucose levels were elevated in both vehicle and NDGA treated animals and there were no significant differences between the two groups, as shown in Figure 13. Serum free fatty acid levels (Figure 14) were significantly lower with NDGA treatment. The TG response to Triton™ is shown in Figure 15.

The degree of accumulation of TG after intravenous injection of Triton appeared to be fairly similar between the NDGA and vehicle treatment groups, with a trend towards reduced accumulation in the NDGA treated animals.

7.4. RESULTS; STUDY #2

Serum TG levels in diabetic animals over the course of vehicle (GELUCIRE™) or NDGA (80 mg/kg) treatment are shown in Figure 16. In this particular experiment, there was no significant difference in TG levels between the NDGA treated and vehicle treated animals, although the NDGA treated animals tended to have lower serum TG levels. Any difference between the groups was obscured by the rather large variability in individual TG levels observed in this study. In both groups there was one outlying animal with exceedingly high (>1000 mg/dl) levels of serum TG.

Serum glucose levels (Figure 17) were consistently elevated in both treatment groups and there was no significant difference between the two groups.

Serum free fatty acid levels (Figure 18) tended to be lower in the NDGA treated group after several days of treatment.

The TG response to Triton administration is shown in Figure 19. There was a trend toward reduced TG accumulation in NDGA treated animals, indicating that secretion of TG by the liver may be reduced in the NDGA treated animals. This may be a consequence of reduced free fatty acids, which serve as a substrate for liver production of TG.

All of the animals used in these two studies appeared healthy and normal. No animal health problems were associated with vehicle or NDGA/vehicle treatment.

Comparison of Results of Study #1 and #2

The effect of treatment with NDGA at 80 mg/kg b.i.d. in fructose-fed/STZ injected diabetic animals to decrease serum triglyceride levels was clearly shown in Study #1 and indicated in Study #2. It is not clear whether this was due to an effect on liver secretion of TG since blunting of the TG response to Triton administration was observed in NDGA treated animals in Study #2 but not in Study #1. This reduction in secretion of TG by the liver may be a consequence of reduced serum free fatty acid levels observed in the NDGA treated animals, since free fatty acids released from adipose tissue can serve as a substrate for liver production of TG.

Different vehicles were used for these two studies which may account for some of the differences observed in the TG response to NDGA. However, these two vehicles are fairly similar in terms of chemical composition, and moreover, there are several other potentially confounding factors that make direct comparison of these two studies very difficult. The animals in Study #2 were more severely diabetic and slightly older than the animals in Study #1 at the start of the study. The end result was that at least one animal in the vehicle treated group and in the NDGA treated group had exceedingly high TG levels, which remained high during the experiment.

8. EXAMPLE: EFFECTS OF NDGA TREATMENT ON SERUM TRIGLYCERIDE LEVELS IN A RODENT MODEL OF NON INSULIN DEPENDENT DIABETES MELLITUS

The following experiments demonstrate that NDGA is a safe and effective treatment for reducing serum triglyceride (TG) and free fatty acid (FFA) levels in diabetic animals. In addition, NDGA is more effective when administered in an amphiphilic vehicle.

8.1. MATERIALS AND METHODS Male Sprague Dawley rats, 250 g body weight, obtained from Charles River Laboratories, Hollister, CA, were fed a 20% fat diet obtained from Harlan Teklad, Madison, WI. for two weeks and then injected with 45 mg/kg streptozotocin

(STZ) by intravenous injection.

NDGA/carboxymethylcellulose ("CMC") was prepared as a suspension by vortexing a mixture of NDGA and an 0.25% solution of carboxymethylcellulose in water at a concentration of 60-100 mg/ml prior to use, and mixing intermittently during the administration procedure.

GELUCIRE™ 44/14 (GELUCIRE™) vehicle was warmed to 48-50°C prior to addition of NDGA. The resulting mixture was vortexed and then sonicated at 48-50°C until the NDGA completely dissolved. Prior to administration, GELUCIRE™ vehicle and NDGA/GELUCIRE™ were kept in a water bath at 48- 50°C to prevent solidification. The concentration of NDGA in either CMC or

GELUCIRE™ was adjusted so the dose could be delivered in a volume of 5-10 ml/kg body weight.

Blood samples from tail snip bleeds were collected and analyzed as described above. Data are expressed as the mean ± SEM. Data were analyzed by analysis of variance with a Fisher's Protected Least Significant Difference post-hoc test. A p value of less than 0.05 is considered significant.

8.2. EXPERIMENTAL PROTOCOL

In two experiments, male Sprague Dawley rats were fed a high fat diet and injected with STZ to make them diabetic and insulin resistant. Animals were pre-screened by blood sampling and analysis for serum glucose levels. Animals with hyperglycemia (>250 mg/dl) were sorted into either control vehicle or NDGA treatment groups.

In experiment #1, animals were orally gavaged with an aqueous vehicle (0.25% CMC, 10 ml/kg) or NDGA formulated in CMC at 40 or 80 mg/kg once a day for four days. In experiment #2, animals were orally gavaged with an amphiphilic vehicle (GELUCIRE™, 10 ml/kg for days 1 and 2 and 5 ml/kg for days 3-5) or NDGA at 40 or 250 mg/kg formulated in GELUCIRE™ once a day for 5 days. Another group of animals in experiment #2 was dosed with water as a control for any effects of the GELUCIRE™ vehicle.

Blood samples in both experiments were taken before dosing and at three hours post dosing. Blood samples were analyzed for serum glucose, triglycerides (TG) and free fatty acid (FFA) levels. In experiment #1, liver samples were taken at sacrifice at the end of the study for measurement of glycogen (Lo et al., 1970, J. Appl. Physiol. 18:234-236). In experiment #2, serum insulin levels were also measured to determine if NDGA stimulates insulin secretion (RIA, Linco Research, St. Charles, MO) . Body weight and food consumption were measured daily as gross indicators of animal health and appetite.

8.3 RESULTS AND DISCUSSION Triglyceride data for the combined two experiments are shown in Figures 20A-20B. NDGA significantly lowered TG concentrations at a dose of 80 mg/kg when administered in the aqueous vehicle (CMC) and at 40 and 250 mg/kg when administered in the amphiphilic vehicle (GELUCIRE™) . NDGA did not lower serum TG levels at 40 mg/kg when administered in the aqueous vehicle. Therefore, the amphiphilic vehicle appeared to enhance the TG lowering effect of NDGA. The amphiphilic vehicle by itself did not alter TG levels when compared to water. This indicates that TG lowering effects of NDGA are specific to NDGA and are enhanced when NDGA is formulated in GELUCIRE™.

Glucose data for the two experiments are shown in Figures 21A-21B. NDGA when administered in the q.d. dosing regimens employed in the present study did not have a significant effect on glucose levels. In addition, GELUCIRE™ vehicle alone did not alter glucose levels in comparison to water. Free fatty acid (FFA) data are shown in Figures

22A-22B. NDGA significantly lowered FFA levels at a dose of 80 mg/kg when delivered in the aqueous vehicle and at 40 and 250 mg/kg when delivered in the amphiphilic vehicle. NDGA did not lower FFA levels at 40 mg/kg when delivered in the aqueous vehicle. Therefore, as with the TG data, the amphiphilic vehicle appeared to enhance the FFA lowering effect of NDGA. The ability of NDGA to suppress FFA levels indicates that NDGA suppresses lipolysis in the adipose tissue. The reduced release of FFA from adipose tissue provides a potential mechanism for the TG lowering effects, for if the supply of FFA available to the liver to re- esterify into TG is reduced, it is likely that TG production by the liver will decline.

Insulin data for experiment #2 are shown in Figure 23. Over the course of the experiment, NDGA did not have a consistent effect on serum insulin concentrations. This indicates that the apparent suppression of lipolysis in the adipose tissue was not due to an increase in insulin concentration .

Liver glycogen concentrations for experiment #1 are shown in Figure 24. The data show that NDGA at doses of 40 and 80 mg/kg q.d. does not inhibit liver glycogen storage. This is potentially due to the fact that blood glucose concentrations in the present study were not reduced, and the liver, therefore, did not have to break down glycogen to maintain blood glucose. NDGA did not have a significant effect on body weight in either experiment, nor did the amphiphilic vehicle alter body weights in comparison to water. In addition, all the animals appeared healthy and there were no deaths in either experiment. NDGA dosed in CMC had no effect on food consumption, but NDGA dosed in GELUCIRE™ initially suppressed food consumption (days 1-2) . It was also apparent that GELUCIRE™ by itself suppressed food consumption as compared to water. This effect, however, was completely eliminated when the dosing was reduced from 10 ml/kg to 5 ml/kg. This indicates that large oral doses of GELUCIRE™ inhibits food consumption, potentially due to the caloric contribution of fats in the vehicle itself. 9. EXAMPLE: EFFECTS OF NDGA TREATMENT ON SERUM GLUCOSE, TRIGLYCERIDE, FREE FATTY ACID AND

GLYCEROL LEVELS IN A RODENT MODEL OF NON INSULIN DEPENDENT DIABETES MELLITUS

The following experiments demonstrate that NDGA is

5 a safe and effective glucose, triglyceride, free fatty acid and glycerol lowering agent in a NIDDM animal model.

9.1. MATERIALS AND METHODS

Male Sprague Dawley rats, 250 g body weight, 10 obtained from Charles River Laboratories, Hollister, CA, were fed a 20% fat diet obtained from Harlan Teklad, Madison, WI . for two weeks and then injected with 45-55 mg/kg streptozotocin (STZ) by intravenous injection.

GELUCIRE™ 44/14 (GELUCIRE™) vehicle was warmed to 15 48-50°C prior to addition of NDGA. The resulting mixture was vortexed and then sonicated at 48-50°C until the NDGA completely dissolved. Prior to administration, GELUCIRE™ vehicle and NDGA/GELUCIRE™ were kept in a water bath at 48-

50°C to prevent solidification. 20 Blood samples from tail snip bleeds were collected and analyzed as described above.

Data are expressed as the mean ± SEM. Data were analyzed by using an unpaired t-test. A p value of less than

0.05 is considered significant. 25

9.2. EXPERIMENTAL PROTOCOL

In four experiments, male Sprague-Dawley rats were fed a high fat diet for two weeks and then injected with STZ to make them diabetic and insulin resistant. Animals were

30 pre-screened by blood sampling and assaying for serum glucose concentration. Animals with hyperglycemia (>250 mg/dl) were sorted into vehicle and NDGA treatment groups. The animals were orally gavaged with vehicle (GELUCIRE™, 2.5 mg/kg) or NDGA formulated in GELUCIRE™ at a concentration of 150 mg/kg

35 twice a day for four days. Blood samples were taken before the first daily dose and three hours post first daily dose. Blood and liver samples were collected and analyzed for serum glucose, triglyceride, free fatty acid levels and glycogen content as described above. Serum glycerol levels were measured by the method of McGowan et al., 1983, Clin. Chem. 19:538 using a kit from Sigma Diagnostics, St. Louis, MO.

9.3. RESULTS AND DISCUSSION Glucose data for the combined four experiments is shown in Figure 25. Treatment with NDGA at 150 mg/kg twice a day significantly lowered serum glucose levels compared to vehicle control. When the 3 hour post first daily dose values are collapsed across all time points, the average difference compared to control is 42 mg/dl. When only the data for animals with starting glucose concentrations >350 mg/dl, the difference between control and NDGA treatment is greater, 54 mg/dl (Figure 26) , indicating that NDGA lowers blood glucose more effectively when the starting glucose concentration is higher.

Triglyceride (TG) data for the four experiments are shown in Figure 27. Treatment with NDGA dramatically lowered TG with a mean decrease of 50%. Control group TG levels tended to increase during the course of the study, a response believed to be due to handling stress in the animals. This pattern is often seen in this animal model, and is not believed to affect the conclusion that NDGA lowers serum TG levels.

Insulin data are shown in Figure 28. Treatment with NDGA had no consistent effect on insulin concentrations, indicating that the glucose lowering effects of NDGA is not due to an increase in insulin production. Free fatty acid (FFA) and glycerol data are shown in Figures 29 and 30, respectively. Treatment with NDGA reduced FFA and glycerol concentrations 35% and 55%, respectively. This strongly indicates that NDGA suppressed adipose tissue lipolysis. Liver glycogen levels are shown in Figure 31. Four days of treatment with NDGA reduced liver glycogen concentrations approximately 50%. Treatment with NDGA had no effect on body weight and only a slight effect on initial food consumption. By day 3 of dosing, food consumption in the NDGA treatment group was equivalent to control. In the high fat, low-dose STZ rat model of NIDDM,

NDGA lowers serum glucose, triglyceride, free fatty acid and glycerol levels at a concentration of 150 mg/kg without any apparent negative health effects. These data indicate that NDGA is a safe and effective anti-diabetic and hypolipidemic agent.

10. EXAMPLE: TRIGLYCERIDE AND GLUCOSE LOWERING ACTIVITY OF MICRONIZED AND NON-MICRONIZED NDGA IN AN AQUEOUS VEHICLE

The following experiments demonstrate that NDGA, either micronized or non-micronized in an aqueous vehicle, was able to lower serum triglyceride and glucose levels in rats with non-insulin dependent diabetes mellitus (NIDDM) .

10.1. MATERIALS AND METHODS Male Sprague Dawley rats obtained from Charles River Laboratories, Hollister, CA, were fed a 20% fat diet obtained from Harlan Teklad, Madison, WI . for two weeks and then injected with 50 mg/kg streptozotocin (STZ) by intravenous injection.

NDGA/carboxymethylcellulose ("CMC") was prepared as a suspension by vortexing a mixture of NDGA and an 0.25% solution of carboxymethylcellulose in water at a concentration of 60-100 mg/ml prior to use, and mixing intermittently during the administration procedure. Micronization was performed by Oread, Palo Alto, CA.

Blood samples from tail snip bleeds were collected into MICROTAINER™ brand (Becton Dickinson, Franklin Lakes, NJ) serum separator tubes, and centrifuged at 12,000 rpm for 10 minutes. Serum was removed, and triglyceride and glucose levels were measured using enzymatic colorimetric methods (M.W. McGowan et al., Clin . Chem . 19:538 (1983) and P. Trinder, Ann . Clin . Biochem . 6_.: 24 (1969)) using Sigma Diagnostic kits, Sigma Chemical Co., St. Louis, Missouri.

Data are expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance or using an unpaired t-test as appropriate. A p value of less than 0.05 is considered significant.

10.2. EXPERIMENTAL PROTOCOL In three experiments, the rats were fed a high fat diet for two weeks and then injected with STZ to make them diabetic and insulin resistant. Rats with hyperglycemia (>250 mg/dl) were sorted into two groups, vehicle control and NDGA, and were orally gavaged with vehicle (0.25% CMC, 2.5 mg/kg) or NDGA twice a day b.i.d. In experiment 1 the rats were administered non-micronized NDGA at 150 mg/kg or 250 mg/kg b.i.d.; in experiment 2, micronized NDGA at 150 mg/kg; and in experiment 3, non-micronized NDGA at 250 mg/kg or micronized NDGA at 200 mg/kg or 250 mg/kg. Blood samples were taken on the first day of the experiment (pre-dosing sample) and at three hours after the first daily dose thereafter. Body weight and food consumption were measured daily as gross indicators of animal health and appetite.

10.3. RESULTS AND DISCUSSION Glucose data for the three experiments are shown in

Figures 32A-32C. Treatment with non-micronized NDGA significantly lowered blood glucose levels at 150 and 250 mg/kg (p<0.05). Micronized NDGA also lowered blood glucose levels at 250 mg/kg. Although micronized NDGA appeared to lower blood glucose levels at 150 mg/kg and 200 mg/kg, these differences were not statistically significant. These results demonstrate that NDGA treatment affectively lowers blood glucose levels in diabetic rats when the NDGA is formulated in an aqueous vehicle, and that micronization appears not to improve this effect.

Triglyceride (TG) data for the three experiments are shown in Figures 33A-33C. Treatment with either micronized or non-micronized NDGA dramatically lowered blood TG levels at all doses tested. Control group TG levels tended to increase gradually over the course of the experiments which is believed to be due to handling stress in the diabetic animals. This pattern is often observed and it is not believed to affect the conclusion that NDGA lowers serum TG levels.

With regard to body weight and food consumption, treatment with NDGA had no effect on body weight and tended to have only a transient effect on food consumption.

However, micronized NDGA at 250 mg/kg b.i.d. consistently lowered food consumption to approximately 50% of vehicle values (p<0.05) .

NDGA formulated in an aqueous vehicle administered to high fat, low dose STZ NIDDM model rats lowers glucose and triglyceride levels without negative effects. Micronization appears to have little or no effect on the efficacy of NDGA. These data indicate that NDGA is a safe and effective anti- diabetic and anti-glyceridemic agent.

11. EXAMPLE: NDGA LOWERS BLOOD PRESSURE, SERUM INSULIN, TRIGLYCERIDE AND FREE FATTY ACID LEVELS IN A NON-DIABETIC RODENT MODEL OF SYNDROME X

The following experiment demonstrates the ability of NDGA to lower blood pressure in an animal model of Syndrome X. This experiment uses a non-diabetic rodent model where blood insulin levels, blood pressure and serum triglycerides were elevated but serum glucose levels were within normal limits. Results show that NDGA lowers blood pressure, blood insulin, free fatty acid and triglyceride levels in a non-diabetic rat model where blood glucose levels remain normal.

11.1. MATERIALS AND METHODS Male, Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) , initially weighing 175-199 g were used for all experiments. Prior to dietary manipulation, all rats were fed Purina Rat Chow (no. 5012; St. Louis, MO) and water ad libitum and maintained on a 12-h (0600-1800 h) light-dark cycle. The rats were then placed on a diet (TD 78463; Harlan Teklad, Madison, WI) which provided 60% of total calories as fructose. The fructose-enriched diet was given for 11 days, during which time the rats were acclimated to the procedure of blood pressure measurement.

Ambient temperature was kept at 30°C. The equipment used includes magnetic animal holders connected with manual scanner (model 65-12, IITC, Inc., Woodland Hills, CA) , pulse amplifier (model 59, IITC, Inc.), and dual-channel recorder (model 1202, Linear Intrs. Corp., Reno, Nevada). Results are expressed as means + SEM, and statistical significance of differences between the two groups were compared by one-way analysis of variance.

11.2. EXPERIMENTAL PROTOCOL At the end of the initial dietary period, blood pressure was determined, and rats randomly divided into two groups. Both groups were maintained on the fructose-enriched diet, but one group was gavaged with NDGA (80 mg/kg, b.i.d.), dissolved in GELUCIRE™, whereas the other was treated in the same manner with vehicle alone. Blood pressure was measured before and after doses of either NDGA or vehicle (3-1/2 days of treatment) . In both instances, the general procedure was similar. Rats were removed from the animal room and taken to the laboratory at 0900 h. They were allowed free access to water and were kept in a quiet area before the blood pressure was measured at

1300 h. The tail-cuff method, without external preheating, was used to measure the systolic blood pressure.

The systolic blood pressure was measured in the conscious state and has been shown with this technique to be similar to that obtained by direct arterial cannulation. The mean of consecutive readings was used as the measurement of the systolic blood pressure of each rat for that day. The final blood pressure determinations were performed on the afternoon following the last morning dose of NDGA or vehicle.

In approximately half of the rats studied, tail vein blood was removed at 1300 h (four hours after removal of 5 food) , centrifuged, frozen, and later assayed for plasma glucose, insulin, and triglyceride concentrations. Plasma free fatty acid concentration was assayed enzymatically by the ACS-ACOD method using a commercial kit (Waro Chemicals Inc. , Richmond, VA) . 10

11.3. RESULTS AND DISCUSSION Both groups of rats tolerated the intervention without any obvious problems, and their weight was essentially identical at the end of the treatment period, 15 258+4 vs 250+13, for the vehicle and NDGA treated groups, respectively.

Blood pressure showed a marked response to treatment with NDGA. Results are illustrated in Fig. 34. Although the baseline blood pressures were similar in the two 20 groups, they diversed dramatically once the intervention began. Blood pressure continued to increase in vehicle- treated rats, whereas blood pressure actually decreased in rats treated with NDGA. At the end of the study the blood pressure averaged 44 mm Hg lower in NDGA-treated rats 25 compared to rats treated with vehicle only. (p<0.0001). Plasma glucose, insulin, free fatty acid, and triglyceride concentrations following treatment with either NDGA or vehicle are shown in Table 1 below.

30 Variable Vehicle Masoprocol B

Glucose (mg/dl) 135±6 140±7 NS Insulin (μU/ml) 44±4 30+5 p<0.05 Free Fatty Acid (μEq/L) 692+30 551+20 p<0.05 Triglyceride (mg/dl) 219±32 79+5 p<0.001

35 Plasma glucose concentrations were similar in the two groups and were within the normal range. However the NDGA-treated rats had significantly lower plasma insulin, free fatty acid, and triglyceride concentrations. The animal model used in this example has many of the features of Syndrome X. Fructose fed rats do not have increased blood glucose and therefore this is not a diabetic model. However, these rats do show increased serum insulin, increased triglycerides and free fatty acid concentration and increased blood pressure. Thus, this animal model is fundamentally different from the previous examples of non- insulin dependent diabetes mellitis induced by streptozotocin (STZ) administration and is the animal model for Syndrome X. In this protocol NDGA treatment was able to improve the characteristic cluster of symptoms associated with Syndrome X.

The results of this experiment show that NDGA can simultaneously lower blood pressure, blood insulin levels, triglyceride and free fatty acid concentration in a non- diabetic rodent model where blood glucose remains normal. This demonstrates the efficacy of NDGA treatment for the clinical manifestations of Syndrome X. This protocol also demonstrates the effect of NDGA treatment in an animal model fundamentally different from the streptozotocin (SZT) induced NIDDM model.

The invention claimed and described herein is not to be limited in scope by the specific embodiments herein disclosed since these embodiments are intended as illustrations of several aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. A number of references are cited herein, the entire disclosures of which are incorporated herein, in their entirety, by reference.

Claims

WHAT IS CLAIMED IS:

1. A formulation which lowers serum triglyceride, free fatty acid, or glycerol level comprising nordihydroguaiaretic acid (NDGA) and an amphiphilic vehicle, in which the amphiphilic vehicle is a mixture of saturated polyglycolyzed glycerides.

2. A pharmaceutical composition comprising the formulation according to claim 1 and a pharmaceutically acceptable carrier.

3. A formulation which lowers serum triglyceride, free fatty acid, or glycerol level comprising nordihydroguaiaretic acid (NDGA) in an oil together with an aqueous carrier and an emulsifier or a surfactant.

4. The formulation according to claim 8 in which the oil is selected from the group consisting of petroleum oil; vegetable oil; animal oil; and oil of synthetic origin and the aqueous carrier is sterile saline or water.

5. A method for treatment of or amelioration of a disease or disorder in which lowering of serum triglyceride, free fatty acids or glycerol level is desired comprising administering to a subject in need thereof an effective amount of a formulation comprising nordihydroguaiaretic acid (NDGA) and an amphiphilic vehicle.

6. The method according to claim 5 in which the formulation further comprises a pharmaceutically acceptable carrier.

7. The method according to claim 5 in which the formulation further comprises another hypolipidemic agent.

8. The method according to claim 5 in which the amphiphilic vehicle is selected from the group consisting of glycerides; partially hydrogenated and hydrogenated plant, vegetable, and animal fats; alkylene glycol fatty acid esters; polyalkylene glycol fatty acid esters; petroleum- based food-grade waxes; polyethylene glycols; saturated and unsaturated fatty acids; and combinations thereof.

9. The method according to claim 5 in which the amphiphilic vehicle is a mixture of saturated polyglycolyzed glycerides.

10. The method according to claim 5 in which the amphiphilic vehicle is a polyoxyethylene castor oil derivative.

11. The method according to claim 5 in which NDGA is administered at a dose of 0.001-350 mg/kg body weight per day.

12. The method according to claim 11 in which NDGA is administered at a dose of 0.5-1.5 mg/kg body weight per day.

13. The method according to claim 5 in which the formulation is administered orally, parenterally, rectally or by inhalation.

14. A method for treatment of or amelioration of a disease or disorder in which the lowering of free fatty acid level is desired comprising administering to an animal in need thereof an effective amount of a formulation comprising nordihydroguaiaretic acid (NDGA) , in which the animal has normal blood levels of glucose, triglycerides, and cholesterol.

15. A method for lowering blood pressure comprising administering to an animal in need thereof an effective amount of a formulation comprising nordihydroguaiaretic acid (NDGA) , in which the animal is a non-diabetic animal.

16. A method for treatment of or amelioration of hypertension comprising administering to a non-diabetic animal in need thereof an effective amount of nordihydroguaiaretic acid (NDGA) .

17. A method for treatment of or amelioration of at least two symptoms of Syndrome X comprising administering to a non-diabetic animal suffering from Syndrome X an effective amount of a formulation comprising an effective amount of a formulation comprising nordihydroguaiaretic acid (NDGA) .

18. The method according to claim 17, in which the amount of NDGA is effective to lower blood pressure and blood insulin level.

19. The method according to claim 17, in which the NDGA is administered in combination with an amphilic vehicle.

20. The method according to claim 19, in which the amphiphilic vehicle is selected from the group consisting of glycerides; partially hydrogenated and hydrogenated plant, vegetable, and animal fats; alkylene glycol fatty acid esters; polyalkylene glycol fatty acid esters; petroleum- based food-grade waxes; polyethylene glycols; saturated and unsaturated fatty acids; and combinations thereof.

21. The method according to claim 19, in which the amphiphilic vehicle is a mixture of saturated polyglycolyzed glycerides.

22. The method according to claim 19, in which the amphiphilic vehicle is a polyoxyethylene castor oil derivative.

23. The method according to claim 17, in which the formulation is administered orally, parenterally, rectally or by inhalation.

24. A method according to claim 17, in which the formulation comprises nordihydroguaiaretic acid (NDGA) in an oil together with an aqueous carrier and an emulsifier or a surfactant.

25. The method according to claim 15, 16 or 17 in which NDGA is administered at a dose of 0.001-350 mg/kg body weight per day.

26. The method according to claim 15, 16 or 17 in which NDGA is administered at a dose of 0.5-1.5 mg/kg body weight per day.