CHROMIUM-CONTAINING COMPOSITIONS FOR IMPROVING ENDOTHELIAL FUNCTION AND CARDIOVASCULAR HEALTH
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I claim:
This application claims the benefit of earlier filed U.S. Provisional Application No. 62/001,438, filed on May 21, 2014, which is hereby incorporated by reference herein.
The present invention relates to a method for improvement of endothelial function and cardiovascular health by using compositions containing a chromium complex (Crominex®3+), prepared by complexing chromium with a standardized extract of Phyllanthus emblica and Shilajit, and combinations thereof.
Cardiovascular disease (CVD) is the number one cause of death globally. More people die annually from CVD than from any other cause. Smoking, hypertension, high LDL cholesterol, low HDL cholesterol and diabetes mellitus (DM) are the five major risk factors for CVD. Diabetes is associated with an increased risk of atherosclerosis, which may result in coronary artery disease (CAD) (A. Pandolfi, et al., “Chronic hyperglycemia and nitric oxide bioavailability play a pivotal role in proatherogenic vascular modifications,” Genes & Nutrition (2007) 2 (2): 195-208). Physiological impairments that link DM with a marked increase in atherosclerotic vascular disease include platelet hyper-reactivity, a tendency for negative arterial remodeling, impaired fibrinolysis, increased inflammation, and endothelial dysfunction.
Endothelial dysfunction, present at disease onset, may be the cause of atherogenesis that is present throughout the course of DM and associated with late-stage adverse outcomes (Panwar, et al., “Atherothrombotic risk factors & premature coronary heart disease in India: A case-control study,” Indian J. Med. Res. (July 2011) 134: 26-32). The endothelial dysfunction results from reduced bioavailability of the vasodilator nitric oxide (NO) mainly due to accelerated NO degradation by reactive oxygen species (J. A. Beckman, “Pathophysiology of Vascular Dysfunction in Diabetes,” Cardiology Rounds (December 2004) Volume 8, Issue 10). A currently favored hypothesis is that oxidative stress, through a single unifying mechanism of superoxide production, is the common pathogenic factor leading to insulin resistance, β-cell dysfunction, impaired glucose tolerance (IGT) and ultimately to Type 2 DM (T2DM). Furthermore, this mechanism has been implicated as the underlying cause of both the macrovascular and microvascular complications associated with Type 2 DM. It follows that therapies aimed at reducing oxidative stress would benefit both patients with T2DM and those at risk for developing diabetes (Potneza, et al., “Endothelial Dysfunction in Diabetes: From Mechanism to Therapeutic Targets,” Current Medicinal Chemistry (2009) 16: 94-112; S. E. Inzucchi, “Oral Antihyperglycemic Therapy for Type 2 Diabetes. Scientific Review and Clinical Applications,” Journal of American Medical Association (Jan. 16, 2002—Vol 287, No. 3, pp. 360-372; and Wright, et al., “Oxidative stress in type 2 diabetes: the role of fasting and postprandial glycaemia,” Int. J. Clin. Pract. (2006 March) 60(3): 308-314).
Many natural product compositions possess potent antioxidant, anti-inflammatory and cardio-protective properties and are used by patients with increased risk of cardiovascular morbidity and mortality in order to treat or prevent disease and/or reduce symptoms. Among them, Phyllanthus emblica, syn. Emblica officinalis Gaertn., the Indian gooseberry (PE, “Amla”) is widely used in Indian medicine for the treatment of various diseases. There are studies which show significant anti-hyperglycaemic and lipid lowering effects of PE in diabetic patients. In in-vitro and animal studies, PE demonstrates potent antioxidant effects against several test systems such as superoxide radical and hydroxyl radical scavenging action, and in systemic augmentation of antioxidant enzymes in animals (Antony, et al., “A pilot clinical study evaluate the effect of Emblica officinalis extract (Amlamax™) on markers of systemic inflammation and dyslipidemia,” Indian J. Clin. Biochemistry (2008) 23(4): 378-381).
In addition, Shilajit is an herbo-mineral drug, which oozes out from a special type of Himalayan mountain rocks in the peak summer months. It is found at high altitudes ranging from 1000-5000 meters. The active constituents of Shilajit contain dibenzo-alpha-pyrones, fulvic acids and related metabolites, small peptides (constituting non-protein amino acids), some lipids, and carrier molecules (fulvic acids). See, Ghosal, S., et al., “Shilajit Part 1—Chemical constituents,” J. Pharm. Sci. (1976) 65:772-3; Ghosal, S., et al., “Shilajit Part 7—Chemistry of Shilajit, an immunomodulatory ayurvedic rasayana,” Pure Appl. Chem. (IUPAC) (1990) 62:1285-8; Ghosal, S., et al., “The core structure of Shilajit humus,” Soil Biol. Biochem. (1992) 23:673-80; and U.S. Pat. Nos. 6,440,436 and 6,869,612 (and references cited therein); all hereby incorporated by reference herein.
Shilajit (PrimaVie®) finds extensive use in Ayurveda, for diverse clinical conditions. For centuries people living in the isolated villages in Himalaya and adjoining regions have used Shilajit alone, or in combination with, other plant remedies to prevent and combat problems with diabetes (Tiwari, V. P., et al., “An interpretation of Ayurvedica findings on Shilajit,” J. Res. Indigenous Med. (1973) 8:57). Moreover being an antioxidant it will prevent damage to the pancreatic islet cell induced by the cytotoxic oxygen radicals (Bhattacharya S. K., “Shilajit attenuates streptozotocin induced diabetes mellitus and decrease in pancreatic islet superoxide dismutase activity in rats,” Phytother. Res. (1995) 9:41-4; Bhattacharya S. K., “Effects of Shilajit on biogenic free radicals,” Phytother. Res. (1995) 9:56-9; and Ghosal, S., et al., “Interaction of Shilajit with biogenic free radicals,” Indian J. Chem. (1995) 34B:596-602). It has been proposed that the derangement of glucose, fat and protein metabolism during diabetes, results in development of hyperlipidemia. In one study, Shilajit produced significant beneficial effects on lipid profile in rats (Trivedi N. A., et al., “Effect of Shilajit on blood glucose and lipid profile in alloxan-induced diabetic rats,” Indian J. Pharmacol. (2004) 36(6):373-376). Further, Chromium 3+ (Cr 3+) helps insulin metabolize fat, turn protein into muscle, and convert sugar into energy. Chromium-activated insulin considerably increases the amount of blood sugar available for energy production. Some of the other chromium compounds available as supplements are Chromium chloride, Chromium picolinate, Chromium polynicotinate, and Chromium dinicocysteinate.
Crominex®3+, which is a complex of chromium with the polyphenolic compounds of Phyllanthus emblica and Shilajit, as described herein in embodiments of the present invention surprisingly exhibited improvement in endothelial function and blood lipid profile in type 2 diabetics as well as individuals with symptoms of metabolic syndrome, although it contains very small amounts of Phyllanthus emblica and Shilajit (3 mg of each per 200 mcg dose of chromium, and 6 mg each per 400 mcg dose of chromium, respectively). Both Phyllanthus emblica and Shilajit are usually effective in doses of 250 or 500 mg per dose. For example, U.S. Pat. No. 8,962,576 describes a composition and method of improving endothelial function and cardiovascular health using Phyllanthus emblica extract at 250 mg and 500 mg twice a day dosing (which is equivalent to 500 mg and 1000 mg per day). Similarly, U.S. Patent Application Publication US2014/0079729A1 describes a method of improving endothelial function and decreasing cardiovascular morbidity using Shilajit at 250 mg twice a day dosing (which is equivalent to 500 mg per day). However, a combination of Chromium, Phyllanthus emblica extract and Shilajit surprisingly exhibited effectiveness in improving endothelial function and cardiovascular risk parameters, although the concentrations of Phyllanthus emblica extract and Shilajit are very low as discussed above and described herein.
Thus, it would be advantageous to have a composition with small amounts of chromium and small amounts of Phyllanthus emblica and Shilajit which would significantly improve endothelial function and cardiovascular risk factors.
An objective of the present invention is to develop a method of using chromium-three cation in combination with Phyllanthus emblica extract and Shilajit for improving endothelial function and cardiovascular health in patients with Type 2 diabetes mellitus as well as in healthy subjects with pre-metabolic syndrome symptoms.
A method of treating or preventing endothelial dysfunction is provided including administering to an individual in need thereof an effective amount of a composition comprising chromium-three in combination with Phyllanthus emblica extract and Shilajit, wherein endothelial function and cardiovascular risk factors are improved or mitigated.
Other embodiments are contemplated for the effective treatment of human patients having type 2 diabetes mellitus (T2DM). In one embodiment, a method of treating a diabetic individual suffering from type 2 diabetes mellitus includes administering to an individual in need thereof an effective amount of a composition comprising chromium-three cation in combination with Phyllanthus emblica extract and Shilajit, wherein endothelial function, as measured in the levels of one or more markers of oxidative stress and/or inflammation, and cardiovascular risk factors, such as total cholesterol, HDL, LDL, triglycerdies, hsCRP and HbA1c, are improved significantly.
In another embodiment, a method of treating a diabetic individual suffering from type 2 diabetes mellitus includes administering to an individual in need thereof an effective amount of a composition comprising chromium-three cation in combination with Phyllanthus emblica extract and Shilajit, wherein a blood lipid parameter is improved, or one or more cardiovascular risk parameters are improved or mitigated.
In another embodiment, a method of treating an individual with metabolic syndrome symptoms includes administering to an individual in need thereof an effective amount of a composition comprising chromium-three cation in combination with Phyllanthus emblica extract and Shilajit, wherein endothelial function is improved as measured in the levels of one or more markers of oxidative stress and/or inflammation.
In yet another embodiment, a method of treating an individual with metabolic syndrome symptoms includes administering to an individual in need thereof an effective amount of a composition comprising chromium-three cation in combination with Phyllanthus emblica extract and Shilajit, wherein a blood lipid parameter is improved, or one or more cardiovascular risk parameters is improved.
A chromium complex (Crominex®3+), prepared by complexing chromium with small amounts of standardized extract of Phyllanthus emblica and Shilajit, has been clinically studied in type 2 diabetic patients as well as subjects with metabolic syndrome symptoms in two separate studies, and it was surprisingly discovered that Crominex®3+, despite the fact that it contains very small doses of Phyllanthus emblica extract and Shilajit, has significantly reduced total cholesterol, LDL cholesterol, triglycerides, highly sensitive C-reactive protein (hsCRP), and HbA1c levels, and improved endothelial function and HDL Cholesterol levels in the blood.
In another aspect, the present invention reveals the usefulness of chromium-three cation in combination with Phyllanthus emblica extract and Shilajit for improvement of endothelial function and cardiovascular health in a human patient or in an animal.
One suitable composition used herein is an extract blend which is isolated in stable form from the fruit of the Phyllanthus emblica plant, as described in detail in U.S. Pat. No. 6,124,268. The extraction process includes treating the finely-pulped fruit with a dilute aqueous or alcoholic-water salt solution, e.g. a 0.1 to 5% (w/w) sodium chloride solution, or the like, preferably at about 70° C.±5° C., or with a buffer solution, e.g. 0.1 to 5% (w/w) of sodium citrate/citric acid, or the like, filtering and drying, to provide the extract in powder form.
The extract includes the active constituents Emblicanin-A and Emblicanin-B, which are gallic/ellagic acid derivatives of 2-ketoglucono-δ-lactone, in an amount by weight of about 35-55%, as well as: Punicagluconic acid, also named punigluconin (about 4-15% by weight), Pedunculagin (about 10-20% by weight), Rutin (about 5-15% by weight), and low-to-medium molecular weight tannoids of gallic/ellagic acid (about 10-30% by weight), gallic acid (up to about 5% by weight), and ellagic acid (up to about 5% by weight). Note that taken together, the overall extracted mixture or blend comprises an isolatable, identifiable, and purifiable group of components comprising a group of low molecular weight hydrolyzable tannoids (“LMwHTs”), generally excluding free gallic acid and ellagic acid. The composition may further include a nutraceutically or pharmaceutically acceptable carrier.
In one suitable embodiment, the amount of LMwHTs contained in a purified and/or enriched extract of Phyllanthus emblica is at least about 60% by weight. In another embodiment, the amount of LMwHTs contained in a purified and/or enriched extract of Phyllanthus emblica is greater than about 60% by weight. In another embodiment, the amount of LMwHTs contained in a purified and/or enriched extract of Phyllanthus emblica is greater than about 70% by weight. CAPROS® (available from Natreon, Inc., New Brunswick, N.J. is one such exemplary extract of Phyllanthus emblica.
Shilajit (PrimaVie®, available from Natreon, Inc., New Brunswick, N.J.) is a standardized dietary supplement ingredient extracted and processed from Shilajit bearing rocks, containing not less than about 50% to 60% by weight fulvic acids (FAs), at least about 10% by weight dibenzo-α-pyrone chromoproteins, and at least 0.3%, or more, by weight total dibenzo-α-pyrones (DBPs). Water content is about 6%, or less, by weight. Water-soluble extractive value is about 80% (w/w), or greater.
In an embodiment, a suitable daily dose of each of an extract of Phyllanthus emblica and Shilajit is in a range of from about 3 mg to about 100 mg, and the daily dose of trivalent chromium in a range from about 100 mcg to about 1000 mcg. In another embodiment, the daily dose of each of an extract of Phyllanthus emblica and Shilajit is in a range of from about 3 mg to about 100 mg, and the daily dose of trivalent chromium in a range from about 200 mcg to about 500 mcg.
Patients with diabetes have vascular complications and endothelial dysfunction is one of the early prognostic markers of atherosclerosis which may eventually result in cardiovascular disease. Studies have reported that endothelial dysfunction occurs in patients with diabetes much earlier than clinical manifestations of diabetic vascular complications (Schalkwijk, et al., “Vascular complications in diabetes mellitus: the role of endothelial dysfunction,” Clinical Science (2005) 109: 143-159). Diabetes is associated with accelerated atherosclerosis and microvascular complications are a major cause of morbidity and mortality, as discussed above. Endothelial cell dysfunction is emerging as a key component in the pathophysiology of cardiovascular abnormalities associated with diabetes mellitus.
Increased arterial stiffness, as measured by pulse wave analysis, is associated with cardiovascular risk factors and established coronary artery disease. Pulse wave analysis is simple and reproducible to stratify cardiac risk in diabetes. Whilst arterial compliance is determined predominantly by structural factors, the vascular endothelium is also involved. The vascular endothelium contributes to vascular tone and endothelial dysfunction is implicated as an early functional alteration predating structural changes of the vasculature. Conventional cardiac risk factors such as dyslipidemia, hypertension, smoking, and Type 2 diabetes are associated with impaired endothelial function. The intact endothelium promotes vasodilatation principally via the release of NO—originally also called endothelium derived relaxing factor. Endothelium dependent vasodilators reduce pulse wave velocity suggesting nitric oxide (NO) plays a role in determining arterial distendability. Free radical NO has emerged as a fundamental signaling device regulating virtually every critical cellular function and is a potent mediator of cellular damage in many conditions. Nitric oxide is produced in endothelial cells from the substrate L-Arginine via endothelial Nitric oxide synthatase (eNOS). Elevated asymmetric dimethylarginine levels cause coupling, a mechanism which leads to decreased NO bioavailability. The endothelial dysfunction associated with diabetes has been attributed to lack of bioavailable nitric oxide due to reduced ability to synthesize NO from L-Arginine. New basic research insights provide possible mechanisms underlying the impaired NO bioavailability in Type 2 diabetes.
Use of herbs for the treatment of cardiovascular diseases and diabetes in Ayurveda, Chinese and Unani systems of medicine has provided a new lead to understanding the pathophysiology of these diseases. Therefore, it is rational to use our natural resources for identifying and selecting inexpensive and safer approaches for the management of cardiovascular disease along with current therapy. For example, Shilajit provides potential benefits for the diabetic patient (Bhattacharya S. K., “Shilajit attenuates streptozotocin induced diabetes mellitus and decrease in pancreatic islet superoxide dismutase activity in rats,” Phytother. Res. (1995) 9:41-4), and may also provide significant beneficial effects in lipid profile (Trivedi N. A., et al., “Effect of Shilajit on blood glucose and lipid profile in alloxan-induced diabetic rats,” Indian J. Pharmacol. (2004) 36(6):373-376).
Oxidative stress induced by reactive oxygen species (ROS) also plays an important role in the etiology of atherosclerosis and coronary heart disease. Recent research has suggested that oxidative stress is one of the mechanisms involved in endothelial dysfunction. Wide spread attention has been focused on involvement of oxygen free radicals in pathogenesis of diabetes. Cellular enzymatic (e.g., superoxide dismutase or “SOD”) and non enzymatic antioxidants (glutathione or “GSH”) act as primary line of defense to cope with the deleterious effect of these radical species. Earlier studies showed the beneficial effects of Amla on atherosclerosis and dyslipidemia (Antony, et al.).
Hypercholesterolemia is a major risk factor for the development of atherosclerosis and is associated with coronary and peripheral vascular disease. Several lines of evidence show that the improvement and incidence of coronary artery disease (CAD) is associated with lowering hypercholesterolemia. To treat hypercholesterolemia, extensive interventions are recommended including diet control, exercise and the use of hypocholesterolemic drugs. However some patients cannot tolerate the adverse events from these drugs, such as liver damage which necessitates the use of other safer and efficacious alternative medications. One research group evaluated the anti-hyperglycemic and lipid lowering properties of Phyllanthus emblica in normal and diabetic human volunteers (Akhtar, et al., “Effect of Amla fruit (Emblica officinalis Gaertn.) on blood glucose and lipid profile of normal subjects and type 2 diabetic patients,” Int. J. Food Sci. Nutr. (2011) 62(6): 609-616). In this study, significant decreases were observed in total cholesterol (TC) and triglycerides (TG), and increases were observed in high density lipoprotein-cholesterol (HDL-C) in normal and diabetic volunteers receiving 2 or 3 g of Phyllanthus emblica powder per day. In another study of Phyllanthus emblica significant reduction in TC, LDL (low density lipoprotein) and TG was reported, whereas there was significant elevation of HDL (high density lipoprotein) (Antony, et al.). The results of their study at doses of 500 mg/day and 1000 mg/day brought significant reduction in the level of risk factors arising from dyslipidemia and inflammation. The exact mechanism by which the fruit of P. emblica exerts a beneficial effect is presently not clear. Without intending to be bound by theory, it is believed that P. emblica, like statins, possesses HMG CoA reductase inhibitory activity. Thus, P. emblica is believed to exert beneficial effects on cardiovascular parameters (Anila, et al., “Flavonoids from Emblica Officinalis and Mangifera indica-effectiveness for dyslipidemia,” J. Ethnopharmacol. (2002) 79:81-7).
A precursor formulation for Crominex® 3+ was evaluated and published with title “Effects of adjunct therapy of a proprietary herbochromium supplement (HCrS) in type 2 diabetes: randomized clinical trial” (Biswas, et al., Int J Diab Dev Ctries, July-September 2010, Volume 30, Issue 3, pp. 153-161). In this study, the precursor formulation was studied as adjunct therapy at 400 mcg per day chromium dose in type 2 diabetics who are being treated with oral anti-diabetic drugs metformin, glipizide and pioglitazone. The results indicate that none of the cardiovascular parameters tested showed any statistically significant improvement compared to the placebo group. This formulation had the same ingredients in the same proportions as the Crominex® 3+ formulation, but was processed differently. After the ingredients were mixed in water, the dispersion was dried under vacuum at 45° C. in a Rotovap®, possibly destroying the bioactive polyphenolic compounds in Phyllanthus emblica. In distinct comparison, Crominex® 3+ product is manufactured by mixing the ingredients in water and spray drying the dispersion, which exposes the bioactives to heat for only a few seconds, thus preserving their activity. This preservation of the bioactives by spray drying may explain the significant clinical efficacy of Crominex® 3+ compared to the precursor formulation of Biswas, et al. In an alternative embodiment, the Crominex® 3+ product is manufactured using freeze drying or lyophilization. In general, the manufacturing processes employed minimize exposure to heat to prevent loss of efficacy in the finished products.
A published patent application, U.S. 20050085454, titled “Phenolic antioxidant chromium complexes for treatment or prevention of type 2 diabetes or glucose intolerance” describes a phenolic antioxidant-chromium complex for treating, preventing or maintaining a condition in primates, especially humans, particularly Type 2 diabetes or glucose intolerance, and more particularly, it relates to chromium complexed with agents which are low molecular weight hydrolyzable tannins of plant origin and/or purified Shilajit containing oxygenated dibenzo-.alpha.-pyrone (DBP) or its conjugates, including dimers and oligomers and fulvic acids, obtained by extraction of native Shilajit, and pharmaceutical and nutritional compositions thereof, useful for supplementing dietary chromium, lowering blood glucose and serum lipid, including the lowering of undesirably high blood serum LDL-cholesterol levels and the raising of blood serum HDL-cholesterol levels and increasing lean body mass. However, the disclosures of this application are not based on any clinical data.
The clinical efficacy of Crominex® 3+ described above may be further understood by the following Examples of clinical studies in type 2 diabetics who are already on treatment with metformin and in subjects with metabolic syndrome symptoms, which were conducted in the Department of Clinical Pharmacology and Therapeutics, Nizam's Institute of Medical Sciences, Hyderabad, India.
In the present studies the formulation Crominex (Natreon, Inc., New Brunswick, N.J. used contains Chromium chloride (CrCl3.6H2O), Phyllanthus emblica fruit extract, processed Shilajit and microcrystalline cellulose in a proportion of 1:3:3:3 (wt. ratio).
Study Design.
The present clinical study is a prospective, randomized, double blind, placebo-controlled study. Patients of either sex, aged 30-65 years, fasting plasma glucose of 110-126 mg/dL, a glycosylated hemoglobin (HbA1c) between 6.5% and 8% and taking stable dose of anti-diabetic treatment (metformin 1500-2000 mg/day) for the past 8 weeks prior to the screening visit; and having endothelial dysfunction defined as ≦6% change in reflection index (RI) on post salbutamol challenge test were included in the study. Patients with severe uncontrolled hyperglyceamia, uncontrolled hypertension, cardiac arrhythmia, impaired hepatic or renal function, history of malignancy or stroke, smoking, chronic alcoholism, any other serious disease requiring active treatment and treatment with any other herbal supplements, were excluded from the study.
After screening, all the eligible subjects were randomized to receive either one of the three treatments orally for duration of 12 weeks—Group 1-one capsule of Crominex®3+, 200 mcg once daily, Group 2-one capsule Crominex®3+, 400 mcg once daily; Group 3-one capsule of placebo once daily.
Subjects were reviewed for follow up at 4, 8 and 12 weeks of therapy. At each visit they were evaluated for efficacy and safety. Pharmacodynamic evaluation for endothelial function was conducted at every visit. Blood samples were collected for evaluation of biomarkers before and at the end of treatment. Safety lab investigations for hematological, hepatic and renal biochemical parameters were conducted before and at the end of the study and also as and when required (in case of any adverse drug reaction (ADR)). Subjects were enquired for the presence of ADR and the same was recorded in the case report form. Compliance to therapy was assessed by pill count method.
The active ingredients used in the capsules have the following compositions.
Crominex®3+ is a useful supplement containing Cr 3+ commercially available from Natreon, Inc. (New Brunswick, N.J., USA). Crominex®3+ may be described broadly as a complex of Chromium with the polyphenols in Phyllanthus emblica, such complex (called Chromium emblicate) being incorporated into the fulvic acid structure of Shilajit to improve bioavailability. Cr 3+ is >=200 mcg in 10-12 mg. Crominex®3+ is a chromium supplement available commercially from Natreon, Inc., New Brunswick, N.J., and is a combination of trivalent chromium, Phyllathus emblica and Shilajit. For every 200 mcg dose of trivalent chromium, Crominex® 3+ contains 3 mg of Phyllanthus emblica extract and 3 mg of Shilajit extract and less than 3 mg of microcrystalline cellulose as a filler.
Capros® (available from Natreon, Inc., New Brunswick, N.J.) is a standardized extract of Phyllanthus emblica containing at least 60%, and up to about 70%, low molecular weight hydrolysable tannins, including Emblicanin-A, Emblicanin-B, Punigluconin and Pedunculagin as active ingredients.
Procedure for Assessment of Endothelial Function by Determination of Reflection Index (RI).
A salbutamol (albuterol) challenge test employing digital volume plethysmography was used to assess endothelial function as reported by Chowienczyk et al., “Photoplethysmographic assessment of pulse wave reflection: blunted response to endothelium dependant beta 2-adrenergic vasodilation in type 2 diabetes mellitus,” J. Am. Coll. Cardiol. (1999 December) 34(7):2007-14; and Naidu, et al., “Comparison of two β2 adrenoceptor agonists by different routes of administration to assess human endothelial function,” Indian J. Pharmacol. (2007) 39:168-9. The patients were examined in supine position after 5 minutes of rest. A digital volume pulse (DVP) was obtained using a photo plethysmograph (Pulse Trace PCA2, PT200, Micro Medical, Gallingham, Kent, UK) transmitting infrared light at 940 nm, placed on the index finger of the right hand. The signal from the plethysmograph was digitized using a 12 bit analogue to digital converter with a sampling frequency of 100 Hz. DVP waveforms were recorded over 20 second period and the height of the late systolic/early diastolic portion of the DVP was expressed as a percentage of the amplitude of the DVP to yield the reflection index (RI), per the procedure described in detail by Millasseau et al., “Determination of age related increases in large artery stiffness by digital pulse contour analysis,” Clinical Science (2002) 103: 371-377. Three DVP recordings were taken, and measurements of reflection index (RI) were calculated and the mean value was determined. Patients were then administered 400 μg of salbutamol by inhalation. After 15 minutes three measurements of RI were obtained again and the difference in mean RI before and after administration of salbutamol was used for assessing endothelial function. A change of ≦6% in RI post-salbutamol was considered as endothelial dysfunction.
Biomarker Evaluation.
Nitric oxide, MDA, Glutathione and levels were estimated spectrophotometrically as follows. Malondialdehyde (MDA) levels were determined as described in Vidyasagar, et al., “Oxidative stress and antioxidant status in acute organophosphorous insecticide poisoning,” Indian J. Pharmacol. (April 2004) 36(2): 76-79. Glutathione levels were determined as described in G. L. Ellman, Arch. Biochem. Biophys. (1959) 82: 70-77 (original determination). Nitric oxide levels were estimated spectrophotometrically as described in Miranda, et al., “A Rapid, Simple Spectrophotometric Method for Simultaneous Detection of Nitrate and Nitrite,” NITRIC OXIDE: Biology and Chemistry (2001) Vol. 5, No. 1, pp. 62-71. hsCRP (high sensitivity C-reactive protein) was determined by ELISA method.
Safety Assessments.
All the subjects had undergone complete physical examination, safety lab evaluations at baseline and at the end of the treatment. Samples were collected after an overnight fast of 12 hrs after the last dose of medication for determination of hemoglobin, glycosylated hemoglobin (HbA1c), blood urea, serum creatinine, liver function, lipid profile (Total cholesterol, High density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) using appropriate standard techniques.
Primary and Secondary Efficacy Parameters.
The primary efficacy measure was a change in endothelial dysfunction as assessed by more than 6% change in reflection index at 12 weeks. Secondary efficacy parameters include change in oxidative stress markers, serum levels of nitric oxide at 12 weeks in all the treatment groups. Additionally, safety and tolerability assessment of the test medications were also conducted.
Data Analysis.
Data are expressed as mean±SD. ANOVA and paired and unpaired t-test were performed for within group and between groups analysis respectively. A p-value <0.05 was considered to be statistically significant. All statistical analyses were performed using the Prism Graphpad 4 (GraphPad Software, Inc., La Jolla, Calif., USA).
Results of Study.
Total of 72 subjects were screened and 60 eligible subjects completed the study. Twenty subjects in each group completed the study. Detailed demographic characteristics of the three study groups are shown in Table 1. There was no significant difference between treatment groups in baseline characteristics including age, weight and body mass index.
Table 2 above shows the effect of treatments on Reflection Index (RI). Table 2 indicates that treatment with Crominex®3+ 200 mcg and 400 mcg showed significant reduction in RI, suggesting improvement in endothelial function.
As shown in Table 3 and
It can be seen from Table 4 that, treatment with Crominex®3+ 200 mcg and 400 mcg showed significant increase in NO and GSH and significant decrease in MDA and hsCRP levels, whereas no significant changes were observed in the placebo group.
As shown in Table 4A, there was significant change observed in absolute change in the biomarkers of oxidative stress when compared to baseline and placebo.
As shown in Table 5 and
As shown in Table 6, treatment with Crominex®3+ 200 mcg and 400 mcg showed significant reduction in TC, LDL-C, TG, VLDL-C plasma levels, and increase in HDL-C compared to baseline. In placebo group no significant changes were found in all parameters compared to baseline.
The above Table 6A indicates that, there was significant difference observed in absolute change in lipid parameters when compared among the three treatments.
The above Table 6B (and
As shown in Table 7, treatment with Crominex®3+ 200 mcg and Crominex®3+400 mcg showed significant reduction in HbA1c levels compared to baseline.
As shown in Table 7A there was significant difference observed in absolute change when compared among the three treatments.
As shown in the above Tables, the present study treatment with Crominex®3+ 200 mcg and 400 mcg produced significant improvement in mean RI index compared to baseline and placebo (see
Study Design.
Patients included in the study, were of either gender, aged 30-68 years, having endothelial dysfunction defined as ≦6% change in reflection index (RI) on post salbutamol challenge test and central obesity as defined by The International Diabetes Federation guidelines, dated 2006, and any two of the following conditions:
(1) Raised triglycerides >150 mg/dL (1.7 mmol/L) or specific treatment for this lipid abnormality;
(2) Reduced HDL cholesterol: <40 mg/dL (1.03 mmol/L) in males, <50 mg/dL (1.29 mmol/L) in females, or specific treatment for this lipid abnormality;
(3) Raised blood pressure: systolic BP>130 or diastolic BP>85 mmHg, or treatment of previously diagnosed hypertension; and
(4) Raised fasting plasma glucose of ≧100 mg/dL, previously diagnosed type 2 diabetes. If FPG is >5.6 mmol/L or 100 mg/dL, an oral glucose tolerance test is strongly recommended, but it is not necessary to define presence of syndrome.
If BMI is >30 kg/m2, central obesity can be assumed and waist circumference does not need to be measured.
Patients with severe uncontrolled hyperglyceamia, uncontrolled hypertension, cardiac arrhythmia, impaired hepatic or renal function, history of malignancy or stroke, smoking, chronic alcoholism, any other serious disease requiring active treatment and treatment with any other herbal supplements, were excluded from the study.
After screening, all the eligible subjects were randomized to receive either one of the three treatments orally for duration of 12 weeks: group 1-one capsule of Crominex®3+ 200 mcg once daily; group 2-one capsule 400 mcg once daily; group 3-one capsule of Placebo once daily.
Subjects were reviewed for follow up at 4 weeks, 8 and 12 weeks of therapy. At each visit they were evaluated for efficacy and safety. Pharmacodynamic evaluation for endothelial function was conducted at baseline and end of treatment. Blood samples were collected for evaluation of biomarkers before and at end of treatment. Safety lab investigations for hematological, hepatic and renal biochemical parameters were conducted before and at the end of the study and also as and when required (in case of any adverse drug reaction (ADR)). Subjects were enquired for the presence of ADR and the same was recorded in the case report form. Compliance to therapy was assessed by pill count method.
Assessment of endothelial function, biomarkers, safety parameters and primary and secondary efficacy parameters and data analysis were performed as described under the study with type 2 diabetic subjects in Example 1.
Results of Study.
Total of 75 subjects were screened and 61 eligible subjects completed the study. Twenty subjects in group 1 receiving Crominex®3+ 200 mcg, 21 subjects in group 2 receiving Crominex®3+ 400 mcg, and 20 subjects in group 3 receiving Placebo completed the study.
Detailed demographic characteristics of the three study groups are shown in Table 8. There was no significant difference between treatment groups in baseline characteristics including age, weight, and body mass index.
Table 10 above indicates that baseline RI was nonsignificant between the three treatments. Treatment with Crominex®3+ 400 mcg showed significant reduction in RI, suggesting improvement in endothelial function. No significant change was found in RI on treatment with placebo, although there was a minor apparent response with Crominex®3+ 200 mcg dose.
As shown in Table 10A and
As shown in Table 11, treatment with Crominex®3+ 400 mcg showed significant increase in NO and GSH levels and significant decrease in MDA and hsCRP levels, whereas no significant changes were observed in Crominex®3+ 200 mcg and placebo groups.
Table 11A and
As shown in Table 12, treatment with 400 mcg showed significant reduction in TC, LDL-C, TG levels and an increase in HDL-C compared to baseline. In the Crominex®3+ 200 mcg group except for TC no significant improvement was observed in the lipid parameters. In the placebo group no significant changes were found in all parameters compared to baseline.
Table 12A (and
In this study with metabolic syndrome subjects, treatment with Crominex®3+ 400 mcg produced significant improvement in RI compared to baseline (see
It is proposed that additional studies in accordance with the Examples may be undertaken in other, larger patient groups.
The nutraceutical compositions of the present invention may be administered in combination with a nutraceutically acceptable carrier. The active ingredients in such formulations may comprise from 1% by weight to 99% by weight, or alternatively, 0.1% by weight to 99.9% by weight. “Nutraceutically acceptable carrier” means any carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the user. In accordance with one embodiment, suitable nutraceutically acceptable carriers can include ethanol, aqueous ethanol mixtures, water, fruit and/or vegetable juices, and combinations thereof.
The pharmaceutical compositions of the present invention may be administered in combination with a pharmaceutically acceptable carrier. The active ingredients in such formulations may comprise from 1% by weight to 99% by weight, or alternatively, 0.1% by weight to 99.9% by weight. “Pharmaceutically acceptable carrier” means any carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the user.
Delivery System
Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums, and confectionaries. Sublingual delivery systems include, but are not limited to, dissolvable tabs under and on the tongue, liquid drops, and beverages. Edible films, hydrophilic polymers, oral dissolvable films or oral dissolvable strips can be used. Other useful delivery systems comprise oral or nasal sprays or inhalers, and the like.
For oral administration, a chromium-containing composition, or Phyllanthus emblica extract and/or Shilajit may be further combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable dosage forms. For example, the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents, absorbents, or lubricating agents. Other useful excipients include magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, and the like.
The components of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof. Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use. Such pharmaceutical compositions and unit dosage forms thereof many comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
The components of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a chemical compound of the invention or a pharmaceutically acceptable salt of a chemical compound of the invention.
For preparing pharmaceutical compositions from a chemical compound of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from five or ten to about seventy percent of the active compound(s). Suitable carriers are magnesium carbonate, magnesium state, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethlycellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. The chemical compound according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose for in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
Compositions suitable for topical administration in the mouth includes lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in single or multi-dose form. In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenges itself, or it can be the appropriate number of any of these in packaged form.
Tablets, capsules and lozenges for oral administration and liquids for oral use are preferred compositions. Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.
Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).
Solid nutritional compositions for oral administration may optionally contain, in addition to the above enumerated nutritional composition ingredients or compounds: carrier materials such as corn starch, gelatin, acacia, microcrystalline cellulose, kaolin, dicalcium phosphate, calcium carbonate, sodium chloride, alginic acid, and the like; disintegrators including, microcrystalline cellulose, alginic acid, and the like; binders including acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose, ethyl cellulose, and the like; and lubricants such as magnesium stearate, stearic acid, silicone fluid, talc, waxes, oils, colloidal silica, and the like. The usefulness of such excipients is well known in the art.
In one preferred embodiment, the nutritional composition may be in the form of a liquid. In accordance with this embodiment, a method of making a liquid composition is provided.
Liquid nutritional compositions for oral administration in connection with a method for preventing and/or treating inflammation, colds and/or flu can be prepared in water or other aqueous vehicles. In addition to the above enumerated ingredients or compounds, liquid nutritional compositions can include suspending agents such as, for example, methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, polyvinyl alcohol, and the like. The liquid nutritional compositions can be in the form of a solution, emulsion, syrup, gel, or elixir including or containing, together with the above enumerated ingredients or compounds, wetting agents, sweeteners, and coloring and flavoring agents. Various liquid and powder nutritional compositions can be prepared by conventional methods. Various ready-to-drink formulations (RTD's) are contemplated.
Routes of Administration
The compositions may be administered by any suitable route, including but not limited to oral, sublingual, buccal, ocular, pulmonary, rectal, and parenteral administration, or as an oral or nasal spray (e.g. inhalation of nebulized vapors, droplets, or solid particles). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical, or subcutaneous administration. Also contemplated within the scope of the invention is the instillation of a pharmaceutical composition in the body of the patient in a controlled formulation, with systemic or local release of the drug to occur at a later time. For example, the drug may be localized in a depot for controlled release to the circulation, or for release to a local site.
Pharmaceutical compositions of the invention may be those suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebal, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflations, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped artices, e.g. films or microcapsules.
The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. ±10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. ±5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. ±2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. ±1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
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