A novel combination useful in treating micro- and macrovascular complications

Abstract

The present invention discloses a novel orally administered combination of biogold and biozinc, a method for its synthesis, pharmaceutical compositions containing the combination, and its use in micro- and macrovascular complications.

Description

[Technical field]

[0001] The present invention relates to the field of medicine. In particular, the present invention discloses a novel orally administered combination of biogold and biozinc, a method for its synthesis, pharmaceutical compositions containing the combination, and its use in micro- and macrovascular complications. [Background technology]

[0002] The macrovasculature and microvasculature play a vital role in the functioning of various vital organs. However, these vasculatures are at risk of being damaged by various systemic disorders that affect either the biochemical composition of the blood or the pressure exerted on the blood vessels. The microvasculature is made up of elastic large arteries. This elasticity is very important because it buffers the increased pulsatility caused by intermittent left ventricular contractions and the influence of more muscular arteries that act as conduits to deliver a steady flow of blood to the microvasculature. The microvasculature is defined as blood vessels with a diameter of <150 μm and includes arterioles, capillaries, and venules. In the presence of systemic diseases such as diabetes mellitus and hypertension, elastic aorta loses elasticity and thus buffering capacity is reduced. This can increase the deleterious effects of pulsatile transmission from the general circulation to the microcirculation, adversely affecting the vasculature. This can increase the propensity of many organs, including the heart, arterial system, brain, eyes, and kidneys, to develop adverse structural and functional changes (termed target organ damage).

[0003] Diabetes has been extensively studied in diabetic animals and humans with regard to endothelial dysfunction, an important cardiovascular risk factor, due to its critical and inductive nature in the development of vascular disease, being one of the main complications of such macrovascular and microvascular disorders. Atherosclerotic lesions (both morphological and functional) are signs of diabetic macrovascular disease. Diabetic microvascular disease is observed in patients in the form of retinopathy, neuropathy, nephropathy, and vascular abnormalities of the lower limbs. These diseases may further lead to renal failure, diabetic cardiomyopathy, stroke, and lower limb dysfunction, and visual impairment, etc. Chronic hyperglycemia is the main hazard that induces retinal microvascular endothelial dysfunction due to pericyte damage. If hyperglycemic damage is left untreated for a long period of time, other pathways (i.e., hexosamine, polyol, and advanced glycation) are activated in addition to glycolysis. It is recognized that these other pathways induce apoptosis and pericyte degeneration, gradually resulting in damage to the retina.

[0004] The current main treatment interventions for diabetic retinopathy (DR) or diabetic macular edema (DME) are laser photocoagulation and vitreoretinal surgery (anti-vascular endothelial growth factor (anti-VEGF)), but these are only applied in the advanced stages of the disease. Among them, vascular endothelial growth factor (VEGF) has been shown to be an important stimulus in the pathogenesis of macular edema associated with diabetes. Bevacizumab (Avastin) is a full-length antibody that inhibits all isoforms of the VEGF-A family. Bevacizumab has been approved by the US Food and Drug Administration (FDA) for the treatment of colorectal cancer. However, bevacizumab is widely used in the treatment of DME in the form of intravitreal injection. However, although macular edema after bevacizumab injection is well alleviated, if macular edema persists or remits and worsens repeatedly, it may damage the foveal photoreceptors and permanently impair vision. Intravitreal Avastin is ineffective in reducing macular thickness 4 weeks after injection. Injectables such as ranibizumab (Lucentis) and aflibercept (Eylea) are also available and used, especially in the EU, but they also suffer from similar problems as Avastin. FDA-approved implants such as Ozurdex (formerly Posurdex), based on a dexamethasone-loaded polyglycolic acid (PLGA) matrix, are used clinically to treat macular edema and non-infectious uveitis. However, complications have been reported during implantation, and migration of the implant from the vitreous to the anterior chamber has been reported. However, all ophthalmic interventions are uncomfortable for the patient and cause side effects when administered to a sensitive organ such as the eye. However, no oral compositions exist for intervention in diabetic retinopathy. In addition to diabetes, other diseases and their complications such as hypertension, heart disease, etc. cause micro- and macrovascular complications.

[0005] Gold, an inorganic material, is used in herbal compositions due to its biocompatibility, antioxidant and antiangiogenic properties, and ability to inhibit angiogenic molecules. Although gold has been used in several forms for various disorders, the functionalization of gold nanoparticles with an amorphous inorganic matrix and their activity in ocular diseases when administered orally have not been studied in detail. Another inorganic compound that is widely used in herbal and vitamin compositions is zinc. However, the activity of zinc in combination with other components of herbal compositions other than vitamins has not been extensively studied. Also, to the inventors' knowledge, surface functionalization of zinc is not known so far. Therefore, there is a need in the art for an oral composition based on natural products such as herbs and minerals for the treatment of micro- and macrovascular complications including diabetic retinopathy / diabetic macular edema that can overcome the problems of the prior art.

[0006] Object of the invention A novel orally administered combination of biogold and biozinc, a process for its synthesis, pharmaceutical compositions containing said combination and its use in micro- and macrovascular complications. Summary of the Invention

[0007] The present invention relates to an orally administered combination (hereinafter also referred to as "NTGX-10") useful for micro- and macrovascular complications, including diabetic retinopathy or diabetic macular edema. The present invention includes a combination of biogold and biozinc, particularly surface-functionalized nanoparticles. The biogold and biozinc of the present invention are structurally defined, safe, orally administered, and bioprocessed. The present invention also discloses a process for obtaining the biogold and biozinc of the present invention, the combination of the present invention, and a composition comprising the combination of the present invention together with a pharma- ceutically acceptable excipient. The present invention also discloses the utility of the combination and composition disclosed herein for efficacy in treating micro- and macrovascular complications, including diabetic macular edema (DME). [Brief description of the drawings]

[0008] [Figure 1] 1 shows an XRD pattern of gold treated according to the present invention. [Diagram 2] 1 shows FESEM images of gold-based samples at magnifications of (a) 1000×, (b) 10000×, and (c) 20000×. [Diagram 3] FETEM images of a gold-based sample at (a) low magnification, (b) medium magnification, and (c) high magnification are shown. [Figure 4] FETEM-STEM-HAADF-EDX elemental mapping images of gold-based formulation samples: (a) electron image, (b) Au Lα1, (c) C Kα1-2, (d) O Kα1, (e) Mg Kα1-2, (c) Ca Kα1, (g) layered image, and (h) EDX spectrum. [Diagram 5] XPS spectra of gold-based samples: (a) and (b) survey scan, and high-resolution scans corresponding to (c) C 1s, (d) Au 4f, (e) O 1s, (f) Si 2p, (g) N 1s, and (h) P 2p. XPS in (a) and (b) were acquired at SKKU and NCL, respectively. [Figure 6] 4 shows the XRD patterns of zinc-based compound samples. [Figure 7] 1 shows FESEM images of zinc-based samples at various magnifications: (a) 1000×, (b) 10000×, and (c) 20000×. [Figure 8] FETEM images of a zinc-based sample at (a) low magnification, (b) medium magnification, and (c) high magnification are shown. [Figure 9] FETEM-STEM-HAADF-EDX elemental mapping images of zinc-based formulation samples: (a) electron image, (b) Zn Kα1, (c) O Kα1, (d) Si Kα1, (e) layered image, and (f) EDX spectrum. [Figure 10] XPS spectra of zinc-based formulation samples: (a, b) survey scan and high-resolution scans corresponding to (c) C 1s, (d) Zn 2p, (e) O 1s, and (f) Si 2p. [Figure 11] Shows the effect on visual acuity following (a) oral administration of NTGX-10 and (b) intravitreal bevacizumab administration. [Figure 12] The effect of oral administration of NTGX-10 on contrast sensitivity is shown. [Figure 13] A baseline OCT image showing the presence of macular edema is shown (Case Study 1). [Figure 14] OCT images at day 180 showing resolution of macular edema are shown (Case Study 1). [Figure 15] 1 shows an OCT image of the left eye at baseline showing the presence of macular edema (Case Study 2). [Figure 16] Shown is an OCT image of the left eye on day 60 showing a reduction in macular edema (Case Study 2). [Figure 17] Shown is an OCT image of the left eye on day 120 showing resolution of macular edema (Case Study 2). [Figure 18] An OCT image of the left eye on day 180 showing resolution of macular edema is shown (Case Study 2). [Figure 19] 1 shows an OCT image of the left eye at baseline demonstrating the presence of macular edema (Case Study 3). [Figure 20] An OCT image of the left eye on day 60 showing resolution of macular edema is shown (Case Study 3). [Figure 21] Shown is an OCT image of the left eye on day 120 showing resolution of macular edema (Case Study 3). [Figure 22] An OCT image of the left eye on day 180 showing resolution of macular edema is shown (Case Study 3). [Diagram 23] Shown is an OCT image of the right eye at baseline showing the presence of macular edema (Case Study 4). [Figure 24] An OCT image of the right eye on day 120 showing a reduction in macular edema is shown (Case Study 4). [Diagram 25] 1 shows images of the YSM model captured 24 hours after treatment with selected doses of NTGX10, bio-processed gold, and bio-processed zinc. [Figure 26] A method for counting blood vessels is shown. [Figure 27] 4 shows YSMs 24 hours after treatment with selected doses of NTGX-10, bioprocessed gold and bioprocessed zinc. The vessel count results of individual YSMs are presented in the form of an Excel sheet. [Figure 28]A graphical representation of the results performed using sigma plot software is shown. [Figure 29] 1 shows a comparative study of the effects of bioprocessed gold, bioprocessed zinc, and NTGX-10 (a combination of bioprocessed gold and zinc) on angiogenesis in experimental chicken embryo YSM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to a novel orally administered combination of biogold and biozinc, a process for its synthesis, pharmaceutical compositions containing said combination and its use in micro- and macrovascular complications. The present invention relates to a combination of bio-gold and bio-zinc for oral administration useful in the alleviation of micro- and macrovascular complications.

[0010] Characteristics of the combination of the present invention The combination of the present invention has been broadly characterized, including the following parameters: The biogold of the present invention XRD results reveal the presence of highly crystalline face-centered cubic (FCC) phase of gold. No other significant crystalline peaks due to gold oxide or other metal impurities and / or their oxides are observed. The FESEM images show the formation of irregularly shaped agglomerates ranging in size from 500nm to 10μm, which are further composed of agglomerated particles ranging from 100nm to 500nm. Also, the gold nanoparticles are surrounded by an amorphous inorganic matrix. FETEM images show the formation of polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-shaped, and spherical particles and mixtures thereof composed of amorphous metal or amorphous metal oxide or mixtures. FETEM-STEM-HAADF-elemental mapping images show the formation of faceted irregular and regular shaped particles (triangular, rectangular, rod-like, etc.) and a matrix of spherical particles surrounding the predominantly spherical gold nanoparticles. XPS studies have shown that elemental gold (Au 0 ) as well as a significant proportion (almost 60%) of oxidized Au on the gold nanoparticle surface. 1+ Au in stable oxidation states, including species 1+ The peaks shown are due to partial surface oxidation of gold having a peak of

[0011] The combination of the present invention comprises biogold in the highly crystalline face-centered cubic (FCC) phase of gold. The biogold of the present invention exists as a nanoparticle system, with the biogold as spherical or modified spherical particles at the core of the system. The nanoparticle spherical or modified spherical biogold is surrounded by an amorphous inorganic matrix of polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-shaped, and spherical particles and mixtures thereof composed of amorphous metals or amorphous metal oxides or mixtures thereof in the particle size range of 500 nm to 10 μm. The biogold is composed of elemental gold (Au 0 ) and gold oxide Au 1+ It contains peaks related to in a ratio of 2:3.

[0012] Bio-Zinc of the Invention · XRD reveals the formation of hexagonal phase of zinc oxide (ZnO). No characteristic peaks of zinc metal are observed. FESEM images show the formation of aggregated heterogeneous polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-like, and spherical particles and mixtures thereof composed of amorphous metal or amorphous metal oxide or mixtures thereof in the size range of 10nM to 1000nM. The FETEM images also reveal the formation of submicron and nanoscale particles resulting in significant polydispersity. Most of the particles exhibit faceted growth with sheet-like, rod-like, and irregular morphological features, and there are very few spherical particles. XPS scans revealed the presence of peaks due to elements such as Zn, O, Mg, Si, C, K, and Ca, suggesting that MgO, SiO2, CaO, etc. form an amorphous matrix around the submicron-scale particles and nanoparticles of ZnO.

[0013] Bio-zinc exists as a hexagonal phase of zinc oxide (ZnO) with no free zinc present. Bio-zinc exists as agglomerated heterogeneous polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-shaped, and spherical particles and mixtures thereof composed of amorphous metals or amorphous metal oxides or mixtures thereof in the size range of 10 nM to 1000 nM. Bio-zinc includes additional elements selected from the group consisting of Zn, O, Mg, Si, C, K, Ca, or combinations thereof. Bio-zinc includes a coating of MgO, SiO2, CaO forming an amorphous matrix around submicron scale particles and nanoparticles of ZnO. The combination of the present invention comprises bio-gold in the range of 2-4% (w / w); bio-zinc in the range of 3-5% (w / w), in a ratio of 1:1 to 1:4.

[0014] The method of the present invention comprises the steps of obtaining biogold, which comprises the steps of: i. Obtaining metallic gold with a purity of 99.99% in the form of foil / powder / flakes; ii. heating the gold of step (i) until the gold is red hot; iii. quenching the red hot gold of step (ii) with a water extract of the rhizome of turmeric (Curcuma longa); iv. repeating steps (ii) and (iii) 10 to 100 times, preferably 15 to 50 times, more preferably 20 to 40 times, to obtain brittle gold; v. triturating the brittle gold of step (iv) with a water extract of turmeric rhizome in a ratio of 1:1 to 1:2 for 6 to 8 hours to obtain a powder; vi. placing the powder in a covered ceramic container and heating the mixture at 100°C to 1000°C, preferably 350°C to 850°C, more preferably 450°C to -750°C for 20 to 45 minutes, and then gradually cooling to room temperature; vii. Repeating steps (v) and (vi) for 10-500 times, preferably 50-250 times, more preferably 80-120 times to obtain a final biogold of finely divided pinkish color with particle size ranging from submicron to nanoscale. The method includes steps including:

[0015] The method of the present invention includes a step of obtaining biozinc, the step comprising: i. Obtaining metallic zinc with a purity of 99.99% in the form of foil / powder / flakes; ii. heating the zinc of step (i) until the zinc is completely liquefied; iii. quenching the liquefied zinc of step (ii) with an aqueous extract of the pulp of Phyllathus embellica fruit after removal of the seeds; iv. repeating steps (ii) and (iii) 10 to 100 times, preferably 15 to 75 times, more preferably 20 to 40 times, to obtain brittle zinc; v. triturating the brittle zinc of step (iv) with a water extract of the pulp of Emblica officinalis fruit in a ratio of 1:1 to 1:2 for 6 to 8 hours to obtain a powder; vi. placing the powder in a covered ceramic container and heating the mixture at 100°C to 1000°C, preferably 250°C to 750°C, more preferably 350°C to 600°C for 20 to 45 minutes, and then gradually cooling to room temperature; vii. Repeating steps (v) and (vi) 10-100 times, preferably 20-80 times, more preferably 30-60 times to obtain the bio-zinc fine powder of the present invention. The method includes steps including:

[0016] A method of obtaining the combination of the present invention by combining bionano gold and bionano zinc in a ratio of 1:1 to 1:4. In one aspect of the present invention, a composition is disclosed comprising an inventive combination as disclosed herein, together with a pharma- ceutically acceptable excipient. i. A composition comprising bio-gold in the range of 2-4% (wt / wt); bio-zinc in the range of 3-5% (wt / wt); and a pharma- ceutically acceptable excipient in the range of 91-95% (wt / wt). The combination or composition may be administered in an oral dosage form at a dose of 0.02 to 2.00 mg / Kg.

[0017] The composition of the present invention can be administered by oral dosage form, preferably in a suitable unit dosage form for easy administration of accurate dosage.Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.The above solid dosage forms can be prepared with coatings and shells, such as enteric coatings.Liquid dosage forms for oral administration include pharma-ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In one embodiment, the combination of the present invention may be administered orally for use in treating micro- and macrovascular complications including diabetes, diabetes-induced eye disorders such as diabetic retinopathy and diabetic macular edema.

[0018] The combinations and compositions comprising the combinations of the present invention can be used for the following complications which may result from macrovascular or microvascular complications: 1. Coronary heart disease 2. Myocardial ischemia 3. Myocardial infarction 4. Neuropathy 5. Nephropathy 6. Retinopathy 7. Dementia 8. Stroke 9.Chronic renal failure 10. Erectile Dysfunction 11. Peripheral vascular disease 12. The following vasculitis: o Large vessels: Takayasu arteritis, temporal arteritis Medium-sized vessels: Buerger's disease, Kawasaki disease, polyarteritis nodosa Small vessels: Behçet's syndrome, eosinophilic granulomatosis with polyangiitis, cutaneous vasculitis, granulomatosis with polyangiitis, Henoch-Schönlein purpura, and microscopic polyangiitis 13. The following venous diseases: Deep vein thrombosis o Superficial venous thrombosis or phlebitis Chronic venous insufficiency Varicose and spider veins

[0019] In other words, NTGX-10 aims to attenuate the deleterious effects of systemic diseases on blood vessels. The combination or composition of the present invention may be administered in an oral dosage form at a dose of 0.02-2.00 mg / kg, which is useful in alleviating micro- and macrovascular complications in diabetic macular edema (DME) and diabetic retinopathy. The combination or composition of the present invention can act in mammals regardless of pathology.

[0020] Advantages of the combinations and compositions of the present invention 1. The combination of the present invention (NTGX-10) has a neuroprotective effect against toxicity induced by anticancer drugs in vivo and, in addition to the ophthalmic action, has several beneficial effects: 2. NTGX-10 can be administered orally, making it easy to administer, unlike other drugs used to treat diabetic retinopathy that require intravitreal administration. 3.NTGX-10 is neuroprotective and helps protect the retina from the adverse effects of systemic diseases such as hypertension, diabetes, and metabolic syndrome. 4. NTGX-10 is well tolerated and safe for long-term use. All the biological resources of the present invention are readily commercially available and were procured from the local market. The present invention is illustrated by the following examples, which are for illustrative purposes only and are not to be construed as limiting. EXAMPLES

[0021] Example 1 Processes for the preparation of the compositions of the present invention Bioprocessing of Gold Step 1: Metallic gold of 99.99% purity is collected in the form of foil / powder / flakes. This pure metallic gold is exposed to heat till it becomes red hot and immersed in the aqueous extract of turmeric. The metal is cooled in the aqueous extract and then removed from the extract. This process is repeated in the same manner for a minimum of 21 times. This process makes the metal softer and more brittle than its original nature. Finally, metallic gold is removed from the aqueous extract of turmeric. Step 2: The material obtained in step 1 is again mixed with the water extract of turmeric. This mixing can be done by trituration for 6-8 hours or any other suitable method. After this, the mixture is kept in a ceramic vessel sealed with a muslin cloth. The ceramic vessel is then exposed to heat at a temperature of about 350°C to 600°C, but is not limited to this range only. The exposure to heat is gradually increased and held at about 600°C for 20-45 minutes, after which it is gradually cooled to room temperature. The entire process of trituration and heat exposure is repeated 80-120 times until a finely divided pinkish colored material is formed. This final material should not contain any traces of metallic gold and should have particle sizes in the submicron to nanoscale range.

[0022] Zinc bioprocessing Step 1: Zinc metal with 99.99% purity is collected in the form of foil / powder / flake. This pure zinc metal is exposed to heat until it liquefies and is immersed in the aqueous extract of Yukan. The metal is cooled in the aqueous extract and then removed from the extract. This process is repeated in the same manner for a minimum of 21 times. This process makes the metal softer and more brittle than its original nature. Finally, zinc metal is removed from the aqueous extract of Yukan. Step 2: The material obtained in step 1 is again mixed with the water extract of Emblica officinalis. This mixing can be done by trituration for 6-8 hours or any other suitable method. After this, the mixture is kept in a ceramic vessel sealed with a muslin cloth. The ceramic vessel is then exposed to heat at a temperature of about 400°C to 800°C, but is not limited to this range only. The exposure to heat is gradually increased and held at about 600°C for 20-45 minutes, after which it is gradually cooled to room temperature. The entire process of trituration and heat exposure is repeated 80-120 times until a finely divided material is formed. This final material should contain no traces of zinc metal and have particle sizes in the submicron to nanoscale range.

[0023] Example 2 Characterization of the products of the present invention Since the NTGX-10 based formulation is a biologically processed metal (Au, Zn) material, the present invention utilizes relevant characterization techniques that are non-destructive in nature: the characteristics of the material needed to be determined in its unprocessed state.

[0024] Example 2.1 Characterization of the Biogold of the Invention 2.1.1. X-ray diffraction (XRD) Using an X-ray diffractometer (Bruker, D8, ADVANCE, Germany), Ni-filtered CuK αThe XRD pattern of the bio-gold based formulation recorded using a 1.54 Å line (λ = 1.54 Å) is shown in Figure 1. The XRD pattern matches well with JCPDS file number 04-0784, which corresponds to the face-centered cubic (FCC) phase of gold. The peaks corresponding to the (111), (200), (220), and (311) (hkl) planes are clearly visible. The peaks are very sharp, which suggests that the sample is highly crystalline. Such behavior may be an obvious result of repeated process runs at high temperatures of around 700-800 °C. No other significant crystalline peaks due to gold oxide or other metal impurities and / or their oxides (arising from the matrix composition and other added components involved during the preparation) are observed in the sample.

[0025] 2.1.2 Field Emission Scanning Electron Microscopy (FESEM) FESEM images of the bio-gold based compounded sample were acquired using the instrument JSM6700F (JEOL, Japan). For this purpose, the powder samples were mounted on aluminum stubs. FESEM images of the bio-gold based compounded sample are shown in Figure 2 at different magnifications (1000×, 10000×, and 20000×). The low magnification images show the formation of irregularly shaped agglomerates of submicron to micron size (Figure 2a). The size of such agglomerates varies from 500 nm to 10 μm. The medium magnification image (Figure 2b) reveals that such agglomerates are composed of agglomerated particles. The high magnification image (Figure 2c) discloses that the size of the agglomerated particles is in the range of 100 nm to 500 nm. Morphological images in secondary electron (SE) mode and due to the charging effect that occurred in the case of this sample, not much significant information can be obtained. However, from this charging phenomenon it can be inferred that the metal nanoparticles are surrounded by a layer / coating / matrix of inorganic components, probably metal oxide particles.

[0026] 2.1.3 Field Emission Transmission Electron Microscopy (FETEM) Microstructural characterization at fine scale of bio-gold based formulation samples was performed using FETEM (JEOL, USA, JEM ARM 200F). For FETEM analysis, test samples were carefully prepared by dispersing the sample powder in deionized water, and then a drop of the dispersion was transferred onto a carbon-coated grid. Low, medium, and high magnification FETEM images of the bio-gold based formulation sample are shown in Figure 3a, b, and c, respectively. Very interestingly, the low magnification FETEM image (Figure 3a) reveals the formation of micron (occasionally), submicron, and nanoscale particles. Some of the particles show faceted growth, others show irregular shapes, and some spherical particles are also seen. Some of the particles have strong contrast in atomic number, which can be attributed to the presence of gold since gold is heavier than the other components of the bio-gold based formulation (see subsequent section). The medium magnification FETEM image (Figure 3b) reveals faceted irregular and regular shaped particles (triangles, rectangles, rods, etc.) as well as the formation of a matrix of spherical particles surrounding the gold nanoparticles. Most of the gold nanoparticles are spherical, but irregularly shaped particles are also intermittently observed. The high magnification FETEM image (Figure 3c) shows a representative gold nanoparticle surrounded by a matrix likely composed of the amorphous component of the formulation.

[0027] 2.1.4 Elemental mapping and STEM-HAADF tomography by EDX To obtain high imaging and spatial resolution for atom-to-atom chemical mapping of the materials, scanning transmission electron microscopy (STEM) imaging with a high-angle annular dark-field detector (HAADF) and energy dispersive X-ray spectroscopy (EDS) elemental mapping using a JEOL, JEM ARM200F instrument operating at 200 kV with a spherical aberration corrector were performed. The resulting STEM-HAADF tomography images of the bio-gold-based formulation samples are provided in Figure 4 along with the elemental mapping images and EDX data.

[0028] The STEM-HAADF images reveal nanoscale particles with dark atomic contrast embedded in a matrix of submicron and nanoscale particles with bright atomic contrast (Figure 4a). The dark particles can be attributed to gold (Figure 4b) and the bright particles to a matrix consisting of other major components identified as carbon, oxygen, magnesium, calcium, etc. (Figure 4c-e). Elemental mapping (Figure 4b-e) further supports this observation. Overlaying the calcium and magnesium mapping images with oxygen suggests the formation of MgO and CaO as major components of the matrix. However, their presence is not revealed by XRD, which may be due to their amorphous or poorly crystalline nature at the global scale. Surprisingly, oxygen is also seen to be present in the dark particles attributed to gold, albeit at a lower intensity. This may be due to partial oxidation of the gold surface, which may be a result of the harsh conditions of the bio-gold-based formulation preparation sequence. Table 1 shows the elemental distribution of the components of the bio-gold based compound samples obtained from EDS analysis.

[0029] [Table 1]

[0030] 2.1.5 X-ray photoelectron spectroscopy (XPS) EDS provides only the elemental composition of the compound, whereas XPS identifies the exact chemical composition of the compound, albeit limited to the surface / subsurface regions. To investigate the surface chemical composition of the bio-gold based formulation samples, 1 × 10 -9 X-ray photoelectron (XP) spectra were recorded at pressures above 1 Torr. Survey scans and C 1s, Au 4f, and O 1s core level spectra were obtained using a monochromated Mg K spectrometry with a pass energy of 50 eV and an electron take-off angle (angle between the electron emission direction and the surface plane) of 60 degrees. αThe XPS analysis was recorded using 1000 nm radiation (photon energy = 1253.6 eV). The overall resolution of the measurements is therefore approximately 1 eV. Core level spectra were background corrected using the Shirley algorithm and adjusted for the accidental C 1s BE of 285 eV (Table 2).

[0031] The surface composition of the prepared bio-gold based compound samples was examined by XPS. Figure 5 consists of the XPS survey scan and high-resolution spectra of carbon, gold, oxygen, silicon, nitrogen, and phosphorus. The XPS survey scan revealed the presence of carbon, gold, oxygen, silicon, magnesium, calcium, nitrogen, and phosphorus, etc. Figure 5d shows the high-resolution spectrum at the Au 4f core level. Two major double peak pairs are displayed in this spectrum. Each double pair is due to spin-orbit coupling (Au4f 7 / 2 and Au4f 5 / 2 The binding energy (BE) values ​​of 84.2 eV and 87.9 eV from the first and second peaks, respectively, can be attributed to elemental gold (Au 0 Interestingly enough, the peaks corresponding to binding energies of 85.4 eV and 89.1 eV are slightly stronger than those due to elemental gold. These peaks are related to the stable oxidation state of Au. 1+ The relative distribution (area under the curve) of the oxidized Au 1+ It is disclosed that the seeds are a significant proportion (approximately 60%) on the gold nanoparticle surface.

[0032] It is important to note that XPS is known as a technique applied to probe relatively thin near-surface layers (electrons emerge from the first few nanometers below the surface), while inner metal layers may not contribute to the integrated XPS signal. 1+ is mainly attributed to partial surface oxidation (at this point), which may suggest the formation of AuO. Bulk oxidation of nanoscale gold is possible. However, this can be ruled out considering the following facts: 1. Gold is reported to be an inert precious metal. Thus, gold is not chemically active, especially at the nanoscale, except at the surface. The surface chemical reactivity of gold at the nanoscale is well documented. Even partial oxidation of gold at the surface has been reported. However, this can only be determined by XPS and not by XRD techniques. 2. XRD, a bulk characterization technique, revealed that the samples contained peaks due to metallic gold only (phase purity). No diffraction peaks due to other impurities (especially those arising from matrix compounds) were seen in the XRD patterns corresponding to bio-gold.

[0033] [Table 2]

[0034] O1s is the most intense peak in both survey scans (Figures 5a and b). XPS analysis reveals an atomic percentage value of 43.95 for chemically active oxygen species, which is significantly higher than the atomic percentage value of oxygen species obtained from the EDS analysis of FETEM (31.8%). However, an opposite trend is observed in the mass percentage values ​​obtained by XPS (27.89%) and EDS (48%) (see Tables 1 and 2). The atomic percentage of Au is 2.27% for XPS but about 0.27% for EDS. The mass percentage values ​​of Au obtained by XPS (17.73%) and EDS (5%) show a similar trend.

[0035] [Table 3]

[0036] The XPS findings summarized in Table 3 are difficult to understand, which may be related to the presence of chemisorbed and / or subsurface adsorbed oxygen species on nano-Au and the high probability of partial oxidation of Au at the surface / subsurface level via chemisorbed / adsorbed oxygen species. It should be noted that XPS is a surface-sensitive technique with limitations in detecting bulk composition. It is speculated that chemically active oxygen may mask the bulk detection of Au in Au-based formulated samples. This may be applicable to the EDAX technique to some extent. Investigation of the partial oxidation of gold at the bulk level can be performed by He-ion microscopy with electron energy loss spectroscopy (EELS).

[0037] Example 2.2 Characterization of the Bio-Zinc of the Invention: Example 2.2.1 X-ray diffraction method (XRD) The XRD technique is a useful tool to identify the presence of crystalline phases within a sample. An X-ray diffractometer (Bruker, D8, ADVANCE, Germany) was used to measure the crystallographic properties of Ni-filtered CuK α The XRD pattern of the bio-zinc based formulation recorded using radiation (λ=1.54 Å) is shown in Figure 6. Identification by checking the d-spacing against the standard JCPDS database (CARD number 80-0075) revealed the formation of a hexagonal phase of zinc oxide (ZnO). The peaks at d=2.47 Å (2Θ=36.25°), d=2.81 Å (2Θ=31.75°), and d=1.62 Å (2Θ=56.58°) confirm the presence of ZnO (hexagonal) as the main crystalline phase in the sample. The seven most intense reflection peaks of the sample correspond to the (101), (100), (002), (110), (102), (103), and (110) planes of hexagonal ZnO (lines in the standard data). The presence of starting materials in the preparation of the Bio-Zinc based formulation (crystalline metallic zinc sheets) is not observed, with the characteristic peak of zinc metal appearing at d=2.09 Å (2Θ=43.23°). This result confirms the absence of crystalline zinc metal in the final product of the Bio-Zinc based formulation.

[0038] Example 2.2.2 Field Emission Scanning Electron Microscopy (FESEM) FESEM examination is an important step in determining the size and morphological characteristics of solid materials. FESEM images of bio-zinc based formulated samples were acquired using the instrument JSM6700F (JEOL, Japan). For this purpose, powder samples were mounted on aluminum stubs. Typical FESEM images of bio-zinc based formulated samples are shown in Figure 7. These images show the formation of aggregated particles (Figure 7a). The particles have various shapes such as sheets, rods, spheres as well as irregular particle shapes (Figure 7b). Faceted growth is observed for most of the particles, which is a general trend in the case of ZnO nanoparticles due to their unique hexagonal crystal structure. Although the maximum number of particles exhibit submicron particle size, a large number of nanoparticles with sizes below 100 nm have also been observed.

[0039] Example 2.2.3 Field Emission Transmission Electron Microscopy (FETEM) and Scanning Transmission Electron Microscopy (STEM) with Elemental Mapping Microstructural evaluation at fine scale of bio-zinc based formulation samples was carried out using FETEM (JEOL, USA, JEM ARM 200F). For FETEM analysis, test samples were carefully prepared by dispersing the sample powder in deionized water, and then a drop of the dispersion was transferred onto a carbon-coated Cu grid. Typical FETEM images obtained at low, medium, and high magnification are shown in Figure 8a, b, and c, respectively. Similar to the FESEM images, the FETEM images also disclose the formation of submicron and nanoscale particles resulting in significant polydispersity. Most of the particles exhibit faceted growth with sheet-like, rod-like, and irregular morphological features.

[0040] Intermittently, spherical particles are also seen. To obtain high imaging and spatial resolution for atom-to-atom chemical mapping of the materials, scanning transmission electron microscopy (STEM) with a high angle annular dark field detector (HAADF) and energy dispersive X-ray spectroscopy (EDS) elemental mapping using a JEOL, JEM ARM200F instrument operating at 200 kV with a spherical aberration corrector were performed. The resulting STEM-HAADF tomography images of the bio-zinc-based formulation sample are provided in Figure 9 along with the elemental mapping images and EDX data.

[0041] Example 2.2.4 Elemental mapping and STEM-HAADF tomography with EDX The STEM-HAADF image (Figure 9a) shows the atomic number contrast of the zinc atoms. However, this contrast is not very strong (as observed in the case of the Au-based formulation). This is expected from the fact that the difference in the atomic numbers of zinc, oxygen, and silicon is not very large. The elemental mapping images (Figures 9b and c) show that Zn K α1 Element map and OK α1 The precise overlap of the ZnO suggests the formation of ZnO in close agreement with the XRD data. Similarly, the presence of SiO2 is also occasionally detected (Fig. 9c and d). Also, unlike the Au-based blended sample, the bio-zinc-based blended sample has the highest mass percentage of zinc (Fig. 9f). Furthermore, the EDX mapping summary data shows the mass% values ​​of Zn and O of 75% and 25%, respectively (which corresponds to the atomic% values ​​of Zn and O of 16.5% and 22.3%, respectively).

[0042] Example 2.2.5 X-ray photoelectron spectroscopy (XPS) It may be noted that EDS provides only the elemental composition of the compound, whereas XPS identifies the exact chemical composition of the compound, albeit limited to the surface / subsurface region. To investigate the surface chemical composition of the bio-zinc based formulation samples, 1 × 10 -9X-ray photoelectron (XP) spectra were recorded at pressures above 10 Torr. Survey scans and C 1s, Zn 2p, and O 1s core level spectra were obtained using a monochromated Mg K ion beam with a pass energy of 50 eV and an electron take-off angle (angle between the electron emission direction and the surface plane) of 60 degrees. α The XPS analysis was recorded using 100 nm radiation (photon energy = 1253.6 eV). Thus, the overall resolution of the measurements is about 1 eV. The core level spectra were subjected to background correction using the Shirley algorithm and adjusted for the accidental C 1s BE at 285 eV. Chemically distinct species were resolved using a nonlinear least squares method. XP spectra of bio-zinc based formulation samples are shown in Figure 10. The XPS survey scan (Figure 10a) reveals the presence of peaks due to elements such as Zn, O, Mg, Si, C, K, and Ca. The XPS survey scans are well correlated / overlaid with each other. The Zn2+ peak in the XP spectrum of bio-zinc based formulation (shown in Figure 10a, d) p3 / 2 and Zn2 p1 / 2 The peak BE positions of are 1022.93 eV and 1045.4 eV, respectively. Table 4 summarizes the details of XPS BE, atomic and mass % values ​​of various elements present in the bio-zinc based formulation sample. Table 5 shows the putative BE assignments of prominent XPS peaks that indicate the formation of mainly ZnO with indication of Zn(OH)2. There is a close agreement between the atomic percentage value corresponding to Zn obtained from EDS analysis of FETEM (16.5%) and that obtained from XPS (15.7%). However, there is a discrepancy between the atomic percentage value corresponding to O obtained from EDS analysis of FETEM (22.3%) and that obtained from XPS (46.52%). This may be attributed to the chemisorption of O species on the ZnO surface, which can be detected with surface sensitive XPS tool.

[0043] [Table 4]

[0044] [Table 5]

[0045] Example 3 Compositions of the Invention NTGX-10 contains bio-processed gold and bio-processed zinc, synthesized using water extracts of Emblica officinalis and Curcuma longa. The combination of the present invention impacts the fundamental pathophysiological processes involved in the micro- and macrovascular complications of DM, HT, including diabetic retinopathy, by reducing disease-associated progression and complications. While most of the conventional treatment options for diabetic retinopathy are intended for use intravitreally, i.e., by injection into the eye, this particular product is intended for oral use.

[0046] Example 4 Biological testing of compositions of the invention. 4.1. Clinical Trials of the Combination of the Invention 4.1.1 Protocols for Clinical Trials In a 6-month clinical trial conducted in patients with diabetic retinopathy, study participants took one capsule of NTGX-10 orally twice daily. Patients underwent baseline evaluations, including ophthalmologic and safety evaluations. Hematological, biochemical, and ophthalmologic tests were performed according to the evaluation schedule to evaluate the efficacy and safety of the study drug. Visual acuity, contrast sensitivity, and slit-lamp evaluation were assessed at monthly intervals. Fundus fluorescein angiography and OCT were performed every 2 months to monitor macular edema, bleeding, and other ophthalmic complications. Hematological and biochemical evaluations were performed 90 and 180 days after the start of treatment, along with fundus examination, ophthalmoscopy, and intraocular pressure evaluation. Patient-reported outcomes were recorded monthly on the NEI Visual Function questionnaire. Patients were provided with a patient diary to monitor compliance. Adverse events were monitored throughout the study. The analysis assessed data on best corrected visual acuity (BCVA), contrast sensitivity, macular edema, and need for intravitreal injections (as efficacy measures), as well as all safety-related variables such as CBC, LFTs, RFTs, etc. In addition to this, secondary efficacy variables such as HbA1C and lipid profile will also be analyzed to assess whether the study formulation has any effect on these confounding variables.

[0047] 4.1.2 Best corrected visual acuity (BCVA) measurement BCVA was examined in patients using Snellen charts and converted to logMAR for statistical analysis. Improvement in visual acuity indicates improved visual function. After 6 months of NTGX therapy, a statistically significant improvement in BCVA was observed (from 0.42 to 0.31, difference 0.11 logMAR) (p<0.001) (Fig. 11b). Subset analysis of the population with decreased BCVA (>0.7 LogMAR) (n=26 eyes) suggests a statistically significant improvement in BCVA (1.01 to 0.77, difference 0.24 logMAR) (p<0.001) (Fig. 11b). In another study, the change in best corrected visual acuity (BCVA) at 1 month after intravitreal bevacizumab (Fig. 11c) was 0.90 to 0.76, difference was statistically significant (P<0.001). 4 .

[0048] 4.1.3: Patient contrast sensitivity Contrast sensitivity (CS) is an important outcome measure and can be considered as an adjunct to standard VA testing for a more complete assessment of visual function in DME patients. In the present study, contrast sensitivity was assessed at monthly intervals. A statistically significant (p<0.01) improvement in CS was observed 180 days after NTGX-10 treatment (Figure 12).

[0049] 4.1.4 Efficacy assessment in patients In a reported clinical study of 54 Irish patients with diabetic retinopathy, after one bevacizumab injection, 16 (29.6%) patients showed no improvement, 22 (40.7%) patients showed improvement with a reduction in macular thickness of less than 50 μm, 5 (9.2%) patients showed improvement with a reduction in macular thickness of more than 50 μm, and the remaining 11 (20.3%) patients showed a reduction in macular thickness of more than 100 μm. Other studies of bevacizumab injections have shown variable reductions in macular edema ranging from 36 μm to 110 μm. Ranibizumab, an FDA-approved drug for the treatment of DR, has also shown a mean reduction in central macular thickness (CMT) ranging from 118 μm to 196 μm. (Table 6)

[0050] [Table 6] To evaluate the effect of NTGX-10 in patients with macula, a subgroup analysis was performed including only eyes of patients with baseline macular edema, i.e., central macular thickness >300 μm (n=30). After 180 days of NTGX treatment in these patients, a statistically significant reduction in central macular thickness was observed (Table 7).

[0051] [Table 7] Various clinical trials conducted worldwide have demonstrated that patients with DR / DME require frequent intravitreal injections, up to 10 times per year.

[0052] [Table 8]

[0053] In the NTGX-10 clinical trial, only one patient required intravitreal therapy during the 180-day study period. Ingestion of NTGX-10 has also been shown to significantly improve visual acuity and prevent worsening macular edema. In addition to this, it has also been shown to significantly reduce central retinal thickness. Due to these therapeutic effects, NTGX-10 significantly reduces the need for intravitreal therapy in patients with diabetic retinopathy. This provides an important positioning for the formulation, considering the limitations of intravitreal anti-VEGF and anti-inflammatory therapies that cannot be administered repeatedly. Findings on fundus fluorescein angiography. FFA was performed to determine ocular vascular abnormalities that lead to hemorrhage. Grading of each eye was performed according to the ETDRS (Early Treatment Diabetic Retinopathy Study) severity scale for diabetic retinopathy. Scores were given according to the pathological features present in various locations of the eye, and a cumulative score was also given to each eye. The cumulative score remained constant throughout the treatment period. Thus, over the 6-month treatment period, no worsening of the ETDRS scale was observed in any eye (N=57), indicating that NTGX-10 effectively prevents the progression of diabetic retinopathy. No new bleeding events were observed that would require the administration of intravitreal injections.

[0054] Microaneurysms, hemorrhage, neovascularization, and other ophthalmic complications were evaluated by fundus fluorescein angiography (FFA) and fundusoscopic analysis (FA). Diabetic retinopathy is a progressive disease. Retinal microaneurysms (MA) are defined as small swellings of small blood vessels mainly located in the inner nuclear layer and deep capillary layer. The number and turnover rate of retinal MAs are considered indicators to evaluate the presence, severity, and progression risk of retinopathy. Proliferative diabetic retinopathy (PDR) is the main cause of vision loss in diabetic patients and is characterized by neovascularization (NV) occurring at the vitreoretinal interface and within the vitreous body. Neovascularization is clinically characterized by thin loops or vascular networks extending from the retinal surface into the vitreous cavity. All of these, if unchecked, lead to progressive vision loss. Therefore, it is important to prevent the progression of the disease.

[0055] In this study, the mean microaneurysms (baseline 49.24 ± 46.8, day 180 43.46 ± 42.2), disc neovascularization (baseline 0.43 ± 1.0, day 180 0.44 ± 1.2), other site neovascularization (baseline 0.34 ± 1.1, day 180 0.60 ± 1.5), speckle hemorrhage (baseline 12.09 ± 21.6, day 180 11.04 ± 19.4), and petechiae (baseline 2.59 ± 7.2, day 180 1.75 ± 5.3) remained stable throughout the study period, indicating a slowing of disease progression. The ETDRS scale is a disease severity scale for diabetic retinopathy. A worsening of 2 or more steps on the ETDRS scale is considered disease progression. In this study, ETDRS grading was performed and it was shown that there was no worsening on the ETDRS scale. This highlights that the study drug may prevent disease progression. All of the above improvements seen in study patients were without significant changes in glycemic control or lipid profile, thus ruling out the role of these confounding variables in the outcome of therapy.

[0056] 4.1.5: Studies on secondary efficacy variables Secondary efficacy variables were changes in lipid profile and glycosylated hemoglobin. NTGX-10 intake did not result in significant changes in HDL, triglycerides, total cholesterol, and glycemic control. However, a statistically significant change in LDL cholesterol was seen, but the change was small and not clinically important. There were no changes in any of the concomitant lipid-lowering or antidiabetic medications taken by any of the study participants.

[0057] [Table 9] NTGX-10 was well tolerated by all patients, no treatment-emergent adverse events were observed in any patient, and hematological and biochemical analyses performed on study participants showed no toxicity in any parameters after 180 days of study drug intake. NTGX-10 demonstrated excellent compliance in all patients.

[0058] [Table 10]

[0059] Safety Conclusion A total of 29 TEAEs were reported in 14 (35.0%) patients over the course of the study. No serious adverse events were reported during the study. Of the 29 TEAEs, 10 AEs reported by 8 patients were mild, 18 AEs reported by 9 patients were moderate, and 1 AE reported by 1 patient was severe. Of the 29 TEAEs, 1 AE reported by 1 patient was definitely related to the study drug, 26 AEs reported by 13 patients were unrelated, and 2 AEs reported by 2 patients were unlikely to be related.

[0060] Commonly (≥5%) reported adverse events were increased blood pressure (5%), increased blood urea (5%), decreased platelet count (5%), protein in the urine (5%), hyperkalemia (5%), increased blood creatinine (7.5%), and proteinuria (7.5%). In summary, based on the analysis of adverse events, NTGX-10 has been demonstrated to be safe and well tolerated.

[0061] 4.2 Case studies 4.2.1: Case study 1 A 54-year-old male patient with diabetes since 2007 presented to Shalakya OPD on 6 July 2019 with complaints of blurred vision. A topical ophthalmologic examination was performed and visual acuity was measured. Fundus examination and ophthalmoscopy revealed the presence of diabetic macular edema (DME), a hallmark of diabetic retinopathy. OCT was performed for evaluation of DME. The baseline central macular thickness (CMT) in the right eye was 456 μm and baseline central macular thickness in ETDRS visual field 2 was 467 μm. OCT images showed the presence of DME as observed in Figure 13. Treatment was initiated with one NTGX-10 capsule twice daily. The patient was instructed to mix the contents of the capsule in unequal proportions of honey and ghee and consume at least 30 minutes before meals. Treatment was continued for 6 months and OCT was performed every 2 months. Six months after treatment, a significant reduction in macular edema was observed and macular thickness returned to the normal range. The central macular thickness in the left eye was found to be 322 μm (a decrease of 134 μm) and the central macular thickness in the ETDRS visual field 2 was found to be 365 μm (a decrease of 102 μm). OCT images showed resolution of DME as observed in FIG. 14. The results are shown in Table 11.

[0062] [Table 11]

[0063] Case Study 2 A 54-year-old male patient with diabetes since 2014 presented to Shalakya OPD on 6 July 2019 with complaints of blurred vision. A topical ophthalmologic examination was performed to measure visual acuity (1.8 LogMAR units). Fundus examination and ophthalmoscopy revealed the presence of diabetic macular edema (DME), a hallmark of diabetic retinopathy. OCT was performed for evaluation of DME. The baseline central macular thickness of the left eye was 593 μm. OCT images showed the presence of DME as observed in Figure 15. Treatment was initiated with one NTGX-10 capsule twice daily. The patient was instructed to mix the contents of the capsule in unequal proportions of honey and ghee and consume at least 30 minutes before meals. Treatment was continued for 6 months and OCT was performed every 2 months. Macular thickness decreased after 2 months of treatment (CMT left eye 558 μm) and consistently decreased after 4 months of treatment (CMT left eye 267 μm) and 6 months of treatment (CMT left eye 221 μm). The overall decrease in macular thickness was 372 μm. OCT images showed a decrease in DME after 2 months of treatment (FIG. 16) and resolution of DME after 4 months of treatment (FIG. 17) and 6 months of treatment (FIG. 18). Visual acuity was found to be 0.8 LogMAR units after 6 months of treatment. The results are shown in Table 12.

[0064] [Table 12]

[0065] Case Study 3 A 62-year-old male patient with diabetes since 2006 presented to Shalakya OPD on 7 July 2019 with complaints of blurred vision. A topical ophthalmologic examination was performed to measure visual acuity (1 LogMAR units). Fundus examination and ophthalmoscopy revealed the presence of diabetic macular edema (DME), a hallmark of diabetic retinopathy. OCT was performed for evaluation of DME. The baseline central macular thickness (CMT) of the left eye was 571 μm. OCT images showed the presence of DME as observed in Figure 19. Treatment was initiated with one NTGX-10 capsule twice daily. The patient was instructed to mix the contents of the capsule in unequal proportions of honey and ghee and consume at least 30 minutes before meals. Treatment was continued for 6 months and OCT was performed every 2 months. Macular thickness decreased after 2 months of treatment (CMT left eye 355 μm), and consistently decreased after 4 months of treatment (CMT left eye 310 μm) and 6 months of treatment (CMT left eye 304 μm). The overall decrease in macular thickness was 267 μm. OCT images showed a decrease in DME after 2 months of treatment (FIG. 20), and resolution of DME after 4 months of treatment (FIG. 21) and 6 months of treatment (FIG. 22). Visual acuity was found to be 0.5 LogMAR units after 6 months of treatment. The results are shown in Table 13.

[0066] [Table 13]

[0067] Case Study 4 A 59-year-old female patient with diabetes since 2007 presented to Shalakya OPD on 10 April 2019 with complaints of blurred vision. A topical ophthalmologic examination was performed to measure visual acuity (0.8 LogMAR units). Fundus examination and ophthalmoscopy revealed the presence of diabetic macular edema (DME), a hallmark of diabetic retinopathy. OCT was performed for evaluation of DME. The baseline central macular thickness (CMT) of the right eye was 569 μm. OCT images showed the presence of DME (Figure 23). Treatment was initiated with one NTGX-10 capsule twice daily. The patient was instructed to mix the contents of the capsule in unequal proportions of honey and ghee and consume at least 30 minutes before meals. Treatment was continued for 6 months and OCT was performed every 2 months. Macular thickness decreased 6 months after treatment (CMT right eye 372 μm). The overall reduction in macular thickness was 197 μm. OCT images showed a reduction in DME 4 months after treatment (FIG. 24). Visual acuity was found to be 0.3 LogMAR units 6 months after treatment. The results are shown in Table 14.

[0068] [Table 14]

[0069] 4.3. Angiogenesis Screening of NTGX-10, Bioprocessed Gold, and Bioprocessed Zinc Using the In Ovo and In Vivo Chicken Embryo Yolk Sac Membrane (YSM) Model Introduction Angiogenesis is the process by which new blood vessels are formed from pre-existing vessels and is important for the growth and development of organisms. In adult organisms, physiological angiogenesis occurs periodically in the female reproductive system and in wound healing. Angiogenic disorders are seen in pathologies such as arthritis, diabetic retinopathy, macular degeneration, and cancerous tumors (Molecular Vision 2016;22:436-445; Nutrients 2016;8(4):200, BMC Cancer. 2015;15(1):1-7). On the other hand, antiangiogenesis - preventing the growth of new blood vessels - is of great importance in cancer treatment (Clinical Therapeutics. 2006;28(11):1779-1802;A Cancer Journal for Clinicians. 2010;60(4):222-243; Nature Reviews Clinical Oncology. 2009;6(8):465-477; Cancer Treatment Reviews 2011;37(1):63-74). Under normal physiological conditions, the balance between pro- and anti-angiogenic factors maintains a steady state in the microcirculation, allowing blood vessels to form only when and where they are needed (J Biol Chem. 1992;267:10931-10934). An excess of angiogenic factors upsets the balance, increasing the rate of migration and proliferation of capillary endothelial cells, leading to excessive angiogenesis (Journal of cellular biochemistry. 1985; 29: 275-28). Conversely, an excess of angiogenic inhibitors blocks various steps in the angiogenesis process, reducing the number of existing blood vessels. Drug development for the treatment of diseases such as cardiac and cerebral ischemia, as well as cancer and tumors, requires appropriate preclinical screening assays using animal models. Various model systems have been reported in the literature.In this study, angiogenesis screening of NTGX-10, bioprocessed gold, and bioprocessed zinc was performed using an in ovo-in vivo chick embryo yolk sac membrane (YSM) model system (Journal of cellular and molecular medicine. 2006;10(3):588-612; British journal of experimental pathology 1987;68(6):755; Mechanisms of development 2016;141:70-7; (CAM assay. 2013;3(8):1-7) was provided by the applicant as a positive control. Antiangiogenesis starves the tumor of all the necessary elements and kills the tumor. Therefore, it is the main treatment option in clinical oncology. Blood vessel quality plays an important role in the treatment of cancer via antiangiogenic pathways.

[0070] 4.3.2. Purpose: To identify the angiogenic / anti-angiogenic potential of NTGX-10 using an in ovo-in vivo chick embryo yolk sac membrane (YSM) model system.

[0071] Experimental procedure Test Item Item Codes: NTGX-10, Bio Processed Gold, Bio Processed Zinc Batch Number: NTGX-10-APPLICANT / SEP-17 / SUB-01;Bio Processed Gold-RB / BCNS-Au / 05 / 18 / 001;Bio Processed Zinc-RB / BCNS-Zn / 05 / 18 / 001 Supplier: Rasayani Biologics Pvt Ltd. Physical Appearance: NTGX-10: Greenish powder, Bio Processed Gold: Greyish powder and Bio Processed Zinc: Greyish powder Storage: Room temperature (27°C).

[0072] Test System In ovo-in vivo chicken embryo yolk sac membrane (YSM) model using freshly fertilized eggs incubated for 72 hours.

[0073] Rationale YSM is a well-vascularized membrane that is present during chick embryo development from day 3 to day 6 of incubation. During this period, the vascularized membrane area is used as a tissue to study the angiogenic / antiangiogenic response of biological or synthetic agents. Rosenbruch M, Holst A. The chick embryo yolk-sac blood vessel system as an experimental model for irritation and inflammation. (Toxicology in vitro. 1990;4:327-31, Murray JC. 2001 Angiogenesis Protocols, Methods in molecular medicine. New Jersey: Humana Press).

[0074] Dose Preparation and Administration To prepare stock solutions (A) at 1 mg / ml, NTGX-10, bio-processed gold, and bio-processed zinc were weighed out at 1 mg each and mixed into 1 ml of 0.9% sterile saline (isotonic). 81 ml of NTGX-10, bio-processed gold, and bio-processed zinc at 100 μg / ml were prepared from solution A (100 μl solution A + 900 μl saline). 200 μl solutions of selected concentrations of NTGX-10, bio-processed gold, and bio-processed zinc at 10 μg / ml (20 μl stock solution B + 180 μl 0.9% sterile saline) and 40 μg / ml (80 μl stock solution B + 120 μl 0.9% sterile saline) were prepared. A micropipette was used to directly deliver 40 μl of each dose into the angiogenesis area of ​​the YSM.

[0075] 4.3.6. Preparation of Avastin: A stock solution of Avastin (A) at a concentration of 25 mg / ml was provided by the applicant. A 10 μl working stock (B) of Avastin at a concentration of 2.5 mg / ml was prepared (1 μl stock B + 9 μl 0.9% saline). 1 ml of Avastin (15 μg / ml) was prepared from working stock B (6 μl B + 994 μl 0.9% saline). A 40 μl dose was delivered directly into the angiogenic area of ​​the YSM using a micropipette.

[0076] Protocol Fresh fertilized eggs of White Leghorn chickens weighing 58–60 g each, obtained from Venkateshwara Hatcheries Pvt. Ltd., Khadakwasala, Pune, Maharashtra, were washed in tap water to remove any mud or dirt and allowed to air dry. The eggs were then incubated for 72 hours at 37.5°C and 70–80% relative humidity. The developmental stage was checked by illuminating the eggs with a 40-watt light bulb. Embryos showing synchronous development were selected for the experiments. Eggs containing live embryos at HH stage 18–24 with well-vascularized YSM (Hamburger V, Hamilton HL. A series of normal stages in the development of chick embryos. Journal of Morphology. 1951;88:49-92 were surface sterilized with 70% alcohol in a laminar flow hood. 1-2 ml of thin albumen was aspirated through an opening in the eggshell. The opening was then sealed with Durapore tape. The shell was cut with a blunt end to expose the YSM. Drug concentrations of 10, 40, and 100 μg / ml were selected for the assay and diluted in 0.9% sterile saline. 40 μl of drug preparation was released directly into the vasculature using a micropipette, and the eggshell was closed using Durapore tape. Eggs were again incubated for 24 hours at 37.5°C and 70-80% humidity. In the master control, the YSM was not treated with any drug, and in the vehicle control, the YSM was treated with 0.9% saline. Eggs were opened by removing the Durapore tape, more thin albumen was removed with a micropipette, and the eggshell was cut to expose the YSM vasculature, which was then imaged and recorded on an Olympus D40 for comparative studies and quantification. Control and experimental (YSM) images were captured using an SLR camera. YSM images were cropped and resized to 992 x 992 pixels. A black rectangle of size 300 x 300 pixels was placed in the area where the effect of the test substance was observed. The number of primary, secondary, tertiary and quaternary vessels was counted manually using Image J software. Avastin (Yildiz et.al., 2013) was provided by RBPL as a positive control.

[0077] NTGX-10, bio-processed gold, and bio-processed zinc were screened to study their effect on the development of quaternary vessels using in ovo and in vivo chick embryo YSM models. Three different concentrations, namely 10 μg / ml, 40 μg / ml, and 100 μg / ml, were applied and observations were recorded after 24 hours of incubation. At all concentrations tested, namely 10 μg / ml, 40 μg / ml, and 100 μg / ml, NTGX-10 exhibited a significant reduction in the number of quaternary vessels in YSM compared to the master control (MC) and vehicle control (VC, i.e., 0.9% saline), bio-processed gold, and bio-processed zinc. It was observed that the number of quaternary vessels in YSM treated with all three concentrations was comparable to that of the positive control, i.e., Avastin (15 μg / ml). However, among the concentrations tested, NTGX-10 at 40 μg / ml, bio-processed gold at 100 μg / ml and 40 μg / ml, and bio-processed zinc at 100 μg / ml exhibited the greatest anti-angiogenic activity, suggesting that NTGX-10, bio-processed gold, and bio-processed zinc are products with anti-angiogenic potential at concentrations of 10 μg / ml, 40 μg / ml, and 100 μg / ml.

[0078] result Photographs of YSM models captured 24 hours after treatment with selected doses of NTGX10, bioprocessed gold and bioprocessed zinc (Figure 25). Blood vessels that develop next to the fetal heart are counted as primary vessels. Branches of primary vessels are secondary vessels, branches of secondary vessels are tertiary vessels, and branches of tertiary vessels are quaternary vessels. Angiogenesis is the process by which new blood vessels are formed from pre-existing vessels. Thus, quantification of angiogenesis is done in terms of an increase or decrease in the number of quaternary vessels (Figure 26).

[0079] Primary, secondary, tertiary, and quaternary vessels were counted in each YSM 24 hours after treatment with selected doses of NTGX-10, bio-processed gold, and bio-processed zinc. The results of the vessel counting in each YSM are presented in the form of an Excel sheet. The vessels that develop next to the fetal heart are counted as primary vessels. The branches of primary vessels are secondary vessels, the branches of secondary vessels are tertiary vessels, and the branches of tertiary vessels are quaternary vessels; primary, secondary, tertiary, and quaternary vessels were counted in each YSM 24 hours after treatment with selected doses of NTGX-10, bio-processed gold, and bio-processed zinc. The results of the vessel counting in each YSM are presented in the form of an Excel sheet in FIG. 27.

[0080] A graphical representation of the results, performed using sigma plot software, is attached as Figure 28. Statistical analysis of the results of counting the number of vessels of each type in the individual YSMs was performed using one-way analysis of variance using sigma stat software. The results sheet generated by the system 12 is attached as Figure 29. All the drugs studied showed antiangiogenic potential at their respective concentrations. Among all the drugs studied, NTGX-10, which consists of a combination of bio-gold and bio-zinc, showed higher antiangiogenic potential.

Claims

1. A combination of bio-gold and bio-zinc for oral administration useful in mitigating micro- and macrovascular complications.

2. 2. The combination of claim 1, wherein the biogold is a highly crystalline face-centered cubic (FCC) phase of gold.

3. 2. The combination of claim 1, wherein the bio-gold is present as a nanoparticle system, the bio-gold being at the core of the system as spherical or modified spherical particles.

4. 2. The combination of claim 1, wherein the nanoparticles of spherical or modified spherical biogold are surrounded by an amorphous inorganic matrix of polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-shaped, and spherical particles and mixtures thereof composed of amorphous metal or amorphous metal oxide or mixtures thereof in the size range of 0.5 μm to 10 μm.

5. The biogold is elemental gold (Au 0 ) and gold oxide Au 1+ 2. The combination of claim 1 , comprising peaks related to:

6. 2. The combination of claim 1, wherein the bio-zinc is present as a hexagonal phase of zinc oxide (ZnO) and no free zinc is present.

7. 2. The combination of claim 1, wherein said bio-zinc is present as aggregated heterogeneous polydisperse irregular or regular shaped particles selected from the group consisting of triangular, rectangular, rod-shaped, and spherical particles and mixtures thereof comprised of amorphous metal or amorphous metal oxide or mixtures thereof in the size range of 10 nM to 1000 nM.

8. 2. The combination of claim 1, wherein the bio-zinc comprises an additional element selected from the group consisting of Zn, O, Mg, Si, C, K, Ca, or combinations thereof.

9. The biozinc forms an amorphous matrix around submicron-scale particles and nanoparticles of ZnO, MgO, SiO 2 2. The combination of claim 1 comprising a coating of CaO.

10. 2. The combination of claim 1, wherein bio-gold is in the range of 2-4% (wt / wt); bio-zinc is in the range of 3%-5% (wt / wt), with a ratio of 1:1 to 1:

4.

11. 2. A method for preparing biogold according to claim 1, comprising the steps of: (i) obtaining metallic gold of 99.99% purity in the form of foil / powder / flake; (ii) heating the gold of step (i) until said gold is red hot; (iii) quenching the red hot gold of step (ii) with a water extract of the rhizome of Turmeric (Curcuma longa); (iv) repeating steps (ii) and (iii) 10-100 times, preferably 15-50 times, more preferably 20-40 times to obtain brittle gold; (v) triturating the brittle gold of step (iv) with a water extract of turmeric rhizome in a ratio of 1:1 to 1:2 for 6 to 8 hours to obtain a powder; (vi) placing the powder in a covered ceramic container and heating the mixture at 100° C. to 1000° C., preferably 350° C. to 850° C., more preferably 450° C. to −750° C., for 20 to 45 minutes, and then gradually cooling to room temperature; (vii) repeating steps (v) and (vi) for 10-500 times, preferably 50-250 times, more preferably 80-120 times to obtain a final finely divided pinkish coloured biogold with particle size in the submicron to nanoscale range.

12. 2. A method for preparing biozinc according to claim 1, comprising the steps of: i. Obtaining 99.99% pure zinc metal in foil / powder / flake form; ii. heating the zinc of step (i) until the zinc is completely liquefied; iii. Quenching the liquefied zinc of step (ii) with an aqueous extract of the pulp of Phyllathus embellica fruit after removal of the seeds; iv. Repeating steps (ii) and (iii) 10-100 times, preferably 15-75 times, more preferably 20-40 times to obtain brittle zinc; v. triturating the brittle zinc of step (iv) with the water extract of the pulp of the Emblica officinalis fruit in a ratio of 1:1 to 1:2 for 6 to 8 hours to obtain a powder; vi. placing the powder in a covered ceramic container and heating the mixture at 100°C to 1000°C, preferably 250°C to 750°C, more preferably 350°C to 600°C for 20 to 45 minutes, and then slowly cooling to room temperature; vii. Repeating steps (v) and (vi) 10-100 times, preferably 20-80 times, more preferably 30-60 times to obtain the bio-zinc fine powder of the present invention.

13. A method for obtaining the combination according to claim 1, comprising the step of combining the biogold according to claim 11 and the biozinc according to claim 12 in a ratio of 1:1 to 1:

4.

14. A composition comprising the combination of claim 1 together with a pharma- ceutically acceptable excipient.

15. 15. The composition of claim 14 comprising bio-gold in the range of 2-4% (wt / wt); bio-zinc in the range of 3-5% (wt / wt); and pharma-ceutically acceptable excipients in the range of 91-95% (wt / wt).

16. 15. The combination or composition of claim 1 or claim 14 in an oral dosage form at a dosage of 0.02 to 2.00 mg / Kg.

17. 15. A combination or composition according to claim 1 or claim 14 useful for alleviating micro- and macrovascular complications.

18. 15. The combination or composition of claim 1 or claim 14, useful for diabetic macular edema (DME), diabetic retinopathy.

19. 15. A combination or composition as claimed in claim 1 or claim 14 for action in a mammal, regardless of pathology.

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