Description:NANO-PHYTOPHOSPHOLIPID COMPOSITION AND THE METHOD OF PREPARATION THEREOF
TECHNICAL FIELD
[0001] The present invention relates to nano-liposomes and methods of preparation thereof. The present invention also relates to nano- phytophospholipid compositions. Further, the invention relates to methods for preparation of nano- phytophospholipid compositions. Further, the present invention specifically relates to nano-phytophospholipid composition/ formulation, method for preparing it and its application in enhancing the bioavailability or absorption of active agent(s).
REFERENCES
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BACKGROUND OF THE INVENTION
[0002] Liver is the body's primary glandular organ, controlling a diverse range of physiological and chemical processes and serves as the primary organ of metabolism and detoxification (Smilin Bell Aseervatham et al., 2018). The liver cells have the capacity to regenerate and recover quickly from acute and sporadic diseases (Oliva-Vilarnau et al., 2018). However, under pathological cases like hepatitis, non-alcoholic fatty liver disease, and other liver disorders, the regenerative potential of the hepatocytes diminishes, resulting in scarring, apoptosis, and cirrhosis (Forbes and Newsome, 2016; Oliva-Vilarnau et al., 2018).
[0003] Despite significant studies into the development of hepatoprotective medications that primarily work as pro-oxidant scavengers, just a few exhibit signs of recovery, but their long-term use induces inflammation (González-Ponce et al., 2018). As a result, herbal-based medicines for liver-specific diseases provide a cost-effective and low-toxicity alternative to the conventional regimen (Saha et al., 2019; Yarnell and Abascal, 2010).
[0004] Flavonoids such as quercetin, silybin, rutin, etc. are commonly used ingredients in the treatment or cure of liver diseases. The hepatoprotective action of flavonoids is thought to act by increasing the intracellular concentrations of glutathione and scavenge free radicals. Flavonoids inhibit lipid peroxidation process, promote ribosomal synthesis of proteins and cellular regeneration, and are inhibitor of stellate hepatocytes. Flavonoids also possess anti-inflammatory and anticarcinogenic effects. Such properties may contribute to the possible use of flavonoids in the treatment of diverse liver ailments (Fraschini et al., 2002; Loguercio and Festi, 2011). However, these flavonoids suffer from poor oral bioavailability due to poor permeability, aqueous solubility, instability at gastrointestinal pH, and rapid enzymatic metabolism (Ramaswamy et al., 2017).
[0005] Phytophospholipid have the potential to increase the therapeutic efficacy of active agent(s) by altering their solubility and release characteristics, leading to enhanced permeation through intestinal membranes. Phospholipids act as a bio-functional surfactant and are most commonly used for enhancing the dissolution and solubility characteristics of bioactive while also being hepatoprotective in nature (Semalty et al., 2010).
[0006] Phytophospholipid is formed due to the electron-accepting and donating nature of the oxygen and nitrogen atoms in phospholipids, respectively; hence a complex can be formed depending upon the structural profile of the phytochemical. Because the phenolic oxygen in flavonoids has a high tendency to accept electrons, hydrogen bonds with phospholipids are easily formed.
[0007] Even though the phospholipid can increase the lipid solubility of phytochemicals, they are difficult to disperse in water, particularly when the phospholipids are used at higher concentration. Hence, we developed a novel composition of nano-phytophospholipid which on addition in water disperses to form a dispersion or nanosuspension. Several attempts in the past were undertaken to enhance the oral bioavailability of the active agent(s) by producing phytophospholipid complex using the conventional method of preparation by solvent evaporation (Wang et al., 2015), salting out, antisolvent precipitation, spray drying (Pu et al., 2016) supercritical fluid technology, freeze-drying, thin-film hydration (Damle and Mallya, 2016). Since organic solvents are used, these conventional approaches pose problems for scale-up and commercialization. Furthermore, the presence of residual organic solvents in the finished product can cause stability and toxicity issues. As a result, a continuous manufacturing process without the use of organic or toxic solvents would overcome all the above disadvantages (Censi et al., 2018; Chi et al., 2020; Li et al., 2015; Serviddio et al., 2010).
[0008] The Extrusion/ Hot melt extrusion/ Twin-screw process/ Single Screw Process is a continuous manufacturing process that operates under defined conditions wherein the raw materials are forced through an orifice by controlling factors such as feed rate, barrel temperature, and screw speed. The premix forms a melt pool on entering the melting zone, thus intensifying the contact between the raw materials (Li et al., 2016). This process may be a solvent-free, scalable, and industrially feasible alternative to the conventional methods for the continuous production of phytophospholipid complex. It is possible to increase the stability by limiting hydrolysis and oxidation as well as minimizing residence time at elevated temperatures and screw rotation (Lee et al., 2021). However, the prior art references and processes are unbale to produce nano-phytophospholipid composition or formulation using Extrusion/ Hot melt extrusion (HME)/ Twin-screw process (TSP)/ Single Screw Process (SSP). Additionally, the prior art nano-phytophospholipid compositions/ formulations are unable to render promising bioavailability or absorption of active agent(s).
[0009] In the past, a few attempts have shown the development of a targeted liposome co-encapsulating two herbal drugs for the treatment of cancers using a novel composition that comprises a synthetic phospholipid namely DPPC and DSPE-mPEG2000, cholesterol, two herbal drugs namely silybin and glycyrrhizin acid, a monoclonal antibody. The liposomes were manufactured using a multistage thin film hydration method followed by sonication (Amoabediny et al., 2016). Another attempt was made by formulating lipid nanoparticles specifically nanospheres made of phospholipids and liquid lipids encapsulating cannabinoids for delivering precise microlitre dosage using conventional heating and ultrasonication methods (Kaufman, 2017). Another work exemplified the method to prepare sustained release nanoformulations of poorly bioavailable hydrophobic plant derived molecules/ extract using emulsification method without the use of phospholipids (Sripathy et al., 2015).
[0010] The present disclosure differs from the previous attempts to compose lipid nanoformulations by using phospholipid composition along with other excipients to form nano-phytophospholipid that causes the immediate dispersion of nano-phytophospholipid in nanometric/ micrometric dimension on agitation in an aqueous or mixed solution. This disclosure also stresses on the method of preparation thereof by [0008] Extrusion/ HME/ TSP/ SSP for a quick, solvent free continuous batch manufacturing of the nano-phytophospholipid. The nano-metric dimension has been considered up to 1000 nm in this disclosure (Goldshtein et al., 2005; Yu, 2019).
[0011] Based on the foregoing, it is believed that a need exists for an improved nano-phytophospholipid composition/formulation. Also, a need exists for a nano-phytophospholipid composition/ formulation, method for preparing it and its application in enhancing the bioavailability or absorption of active agent(s), as described in greater detail herein.
SUMMARY OF THE INVENTION
[0012] The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description.
[0013] The foremost objective of the invention is to produce nano-phytophospholipid composition or formulation using a defined ratio of active ingredient(s), phospholipid, surfactants, fatty acids, peptides/ proteins, polymers, bile acids or bile salts, super-disintegrants/ disintegrants, adsorbents, absorbents, diluents, lubricants, glidants, permeation enhancers etc.
[0014] It is yet another object of the present invention to produce particular mass or dispersion of the nano-phytophospholipid in a vehicle, mixture of vehicles or water.
[0015] It is yet another object of the present invention to enhance the oral bioavailability of active agent(s) using various formulation components.
[0016] It is an object of the present invention to produce nano-phytophospholipid using a solvent-free process using Extruder/ Hot melt extruder/ Twin screw processor/ Single screw processor.
[0017] It is yet another object of the present invention to produce nano-phytophospholipid in a continuous manufacturing process.
[0018] It is an object of the present invention to enhance or optimize the bioavailability of poorly soluble and/ or poor permeable or any other active agent(s) when administered orally.
[0019] It is an object of the present invention to enhance the oral bioavailability of active agent(s) by the use of phospholipid(s) along with other above-mentioned ingredients.
[0020] It is yet another object of the present invention to enhance the oral bioavailability of active agent(s) by improving its permeability or solubility or uptake.
[0021] These and other objects and features of the present invention will become readily apparent to one skilled in the art from the detailed description given hereafter.
[0022] Nano-phytophospholipid composition and the method of preparation thereof. The composition of nano-phytophospholipid comprises of active agent(s) and phospholipid(s) in a weight ratio of 1:0.001-20, a surfactant ranging from 0.001-60% by weight of the composition, a super disintegrant/ disintegrant at 0.001-25% by weight of the composition, an adsorbent at 0.1-75% by weight of the composition and bile acids or bile salt at 0.001-20% by weight of the composition. The nano-phytophospholipid composition/ formulation also comprises of fatty acid(s) at 0.001-50% by weight of composition, peptide(s)/ protein(s) at 0.001-20% by weight of composition, polymer(s) at 0.001-50% by weight of composition, absorbent(s) at 0.001-50% by weight of composition, diluent(s) at 0.001-75% by weight of composition, lubricant(s) at 0.001 to 10% by weight of composition, glidant(s) at 0.001 to 10% by weight of composition, permeation enhancer(s) at 0.001 to 30% by weight of composition, etc.
[0023] The nano-phytophospholipid composition is prepared using a continuous novel and solvent-free Twin Screw Processor and the compound is dispersed into an aqueous solution to form a nano-formulation (nanosuspension, nanoemulsion, or any other form) with particle size below 1000 nm.
[0024] The nano-phytophospholipid composition disclosed herein can increase the solubility, permeation, dissolution rate, and oral bioavailability or absorption of the active ingredient(s) for scale-up and large-scale manufacturing. The novel and solvent-free Twin Screw Processor disclosed herein provides optimized outcome wherein a person skilled in the art can prepare the nano-phytophospholipid composition with ingredient ratios mentioned above by using extrusion, single screw process or hot melt extrusion process as well as the conventional methods such as solvent evaporation, salting out, antisolvent precipitation, spray drying, freeze-drying, thin-film hydration, and supercritical fluid technology.
[0025] The invention disclosed herein is pertaining to the development of a novel nano-phytophospholipid formulation or composition to enhance solubility and/or oral bioavailability of active agent(s). In one embodiment of the present invention, the composition is prepared using a continuous manufacturing process involving a Twin Screw Processor or Hot melt extruder. The invention is based on the use of particular weight ratios of phospholipid(s) and active agent(s) along with other ingredients such as surfactant(s), super disintegrant(s)/ disintegrant(s), adsorbent(s), bile acid(s) or bile salt(s), fatty acid(s), peptide(s)/ protein(s), polymer(s), absorbent(s), diluent(s), lubricant(s), glidant(s), permeation enhancer(s), etc. to form particles or dispersions with less particle size (usually less than 1000 nm). The invention is also hereby teaching a novel method for eliminating the use of organic solvents using a Twin Screw Processor or Hot melt extruder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 illustrates a Multiview Fourier Transform Infrared spectrum of silybin, Soy phosphatidylcholine (SPC), Physical mixture (PM) (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition). Silybin had distinct peaks for –OH and –CH stretching between 2900 and 3400 cm-1, C-C stretching, and C=O stretching between 1000 and 1700 cm-1;
[0027] FIG. 2 illustrates Multiview Differential Scanning Calorimetry thermograms of silybin, SPC, PM (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition);
[0028] FIG. 3 illustrates Multiview Powder X-Ray Diffraction p-XRD diffractograms of silybin, SPC, PM (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition);
[0029] FIG. 4 illustrates Scanning Electron Microscopy images of silybin and H6 (nano-phytophospholipid). A1, A2, and A3 represent silybin images at 10 µm, 2 µm, and 200 nm, while B1, B2, and B3 represent H6 images at 10 µm, 2 µm, and 200 nm scale;
[0030] FIG. 5 illustrates In vitro dissolution profile of silybin and H6 at A) pH 1.2 and B) pH 6.8 (mean ± SD; n = 3). In pH 1.2 and pH 6.8, pure silybin exhibited a poor dissolution rate, with 34.22±1.93 % and 43.26±0.49% of the drug released at the end of a 10 h of dissolution;
[0031] FIG. 6 illustrates Ex vivo permeation studies of Silybin and H6. The Papp for silybin was 0.64 X 10-5 cm/min and H6 was 1.57 X 10-5 cm/min i.e., 2.45-fold increase in permeation; and
[0032] FIG. 7 illustrates Plasma concentration vs. time profile of plain silybin and H6 in preclinical in vivo pharmacokinetics study in rats.
DETAILED DESCRIPTION
[0033] The values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.
[0034] The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes all combinations of one or more of the associated listed items.
[0035] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0036] Nano-phytophospholipid composition and the method of preparation thereof. The composition of nano-phytophospholipid comprises of active agent(s) and phospholipid(s) in a weight ratio of 1:0.001-20, a surfactant ranging from 0.001-60% by weight of the composition, a super disintegrant/ disintegrant at 0.001-25% by weight of the composition, an adsorbent at 0.1-75% by weight of the composition and bile acids or bile salt at 0.001-20% by weight of the composition. The nano-phytophospholipid composition/ formulation also comprises of fatty acid(s) at 0.001-50% by weight of composition, peptide(s)/ protein(s) at 0.001-20% by weight of composition, polymer(s) at 0.001-50% by weight of composition, absorbent(s) at 0.001-50% by weight of composition, diluent(s) at 0.001-75% by weight of composition, lubricant(s) at 0.001 to 10% by weight of composition, glidant(s) at 0.001 to 10% by weight of composition, permeation enhancer(s) at 0.001 to 30% by weight of composition, etc.
[0037] The term 'active agent' refers to an agent, including but not limited to phyto-constituents, herbal extracts, small drug molecules, protein, peptide, nucleic acid (nucleotides, nucleosides, and analogs thereof) and/or prodrug, which may provide prophylactic action, nutritive effect, pharmacological or therapeutic action or treatment of disease upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components. The carrier may be selected from the class of natural, semi-synthetic, or synthetic phospholipids, lipids, fatty acids, ionic or non-ionic surfactants, and/ or their derivatives, polymers and other inactive ingredients as mentioned above.
[0038] The nano-phytophospholipid composition is prepared using a continuous novel and solvent-free Twin Screw Processor or Hot melt extruder and the compound is dispersed into an aqueous solution to form a nano-formulation (nanosuspension, nanoemulsion, or any other form) with particle size below 1000 nm. The nano-phytophospholipid composition or the product or the dispersion herein is termed as “SciSomes” and any method or technology used to prepare or manufacture these “SciSomes” is termed as “SciSome Technology”.
[0039] The nano-phytophospholipid composition disclosed herein can increase the solubility, dissolution rate, and oral bioavailability or absorption of the active ingredient(s) for scale-up and large-scale manufacturing. The novel and solvent-free Twin Screw Processor disclosed herein provides optimized outcome wherein a person skilled in the art can prepare the nano-phytophospholipid composition with ingredient ratios mentioned above by using Extruder, Hot melt extruder, single screw processor as well as conventional methods such as solvent evaporation, salting out, antisolvent precipitation, spray drying, freeze-drying, thin-film hydration, and supercritical fluid technology.
[0040] The invention disclosed herein is pertaining to the development of a novel nano-phytophospholipid formulation or composition to enhance solubility and oral bioavailability of active agent(s). In one embodiment of the present invention, the composition is prepared using a continuous manufacturing process involving a Twin Screw Processor or Hot melt extruder.
[0041] The invention is based on the use of particular weight ratios of phospholipid(s) and active agent(s) along with other ingredients such as surfactant(s), super disintegrant(s)/ disintegrant(s), adsorbent(s), bile acid(s) or bile salt(s), fatty acid(s), peptide(s)/ protein(s), polymer(s), absorbent(s), diluent(s), lubricant(s), glidant(s), permeation enhancer(s), etc. to form particles or dispersions with less particle size (usually less than 1000 nm). The invention is also hereby teaching a novel method for eliminating the use of organic solvents using a Twin Screw Processor or Hot melt extruder.
[0042] Best Method of Use of the Invention: In one embodiment, the phospholipid used was Soy phosphatidylcholine, and the active ingredient refers to Silybin, a hepatoprotective agent. The nano-phytophospholipid is prepared by composition/formation comprising Poloxamer P188 at 0.05-1% by weight of the composition; Sodium starch glycolate at 0.05-0.1% by weight of the composition; Microcrystalline cellulose at 10-40% by weight of the composition; Sodium deoxycholate at 0.5-4% by weight of the composition.
[0043] In another embodiment, the nano-phytophospholipid is prepared by composition/ formation comprising Poloxamer 188 at 0.1-5% by weight of the composition; Sodium starch glycollate at 1-3% by weight of the composition; Microcrystalline cellulose at 20-75% by weight of the composition; Sodium deoxycholate at 1-6% by weight of the composition.
[0044] The nano-phytophospholipid wherein the weight ratio of silybin and soy phosphatidylcholine is 1:0.1-10. The nano-phytophospholipid wherein the weight ratio of silybin and soy phosphatidylcholine is 1:0.5-5
[0045] The nano-phytophospholipid composition is prepared by weighing Silybin and soyabean phospholipid accurately and mixing in a mortar pestle wherein the mixture is passed through a #40 sieve to obtain uniform particles. Further, surfactant, super disintegrant/ disintegrant, bile salts or bile acids and adsorbent were individually weighed, added in the same sequence, and mixed geometrically. The above mixture was further mixed in a tumbler for 10 min, sieved again through a #40 sieve and then passed through Twin Screw Processor using a screw feeder at a screw speed of 30 to 50 rpm. A specific set of barrel temperatures in the four heating zones and an optimized screw speed of the Twin Screw Processor may be necessary to form nano-phytophospholipid (B1: 30 °C, B2: 100 °C±5 °C, B3: 140 °C±3 °C, B4: 100 °C±5 °C).
[0046] The prepared nano-phytophospholipid composition or formulation were characterized for the following tests- particle size, zeta potential, polydispersity using Malvern Zetasizer and the results are as follows: The nano-phytophospholipid showed a particle size ranging between 300-550 nm with a PDI value of 0.1-0.55. The zeta potential value was found to be between -30 to -40 mV, indicating that the particles are stable and have a low tendency to aggregate. The physicochemical interactions were studied by Differential Scanning Calorimetry (DSC), Fourier-transform infrared spectrometry (FTIR), powder X-ray diffraction spectroscopy (P-XRD) and Scanning Electron Microscopy (SEM). The results revealed a clear formation of nano-phytophospholipid composition.
[0047] The in vitro drug release, ex vivo intestinal permeation, and in vivo pharmacokinetic studies were also conducted. The results are as follows - pure silybin exhibited a poor dissolution rate, with 34.22±1.93% and 43.26±0.49% of the drug released at the end of a 10 h of dissolution in pH 1.2 and pH 6.8, respectively. In comparison to the plain silybin, the dissolution profile of the complex at the end of 10 h was 67.13±1.51% and 86.21±1.14% in pH 1.2 and pH 6.8, respectively. The dissolution profile of the nano-phytophospholipid showed an enhanced rate of dissolution ascribed to the improved wettability and solubility of the drug in the complex.
[0048] The ex vivo rat intestinal permeation study showed that the nano-phytophospholipid achieved higher permeation than that from passive diffusion of pure silybin at all the time points up to 3 h. The Papp for silybin was 0.64 X 10-5, and the nano-phytophospholipid was 1.57 X 10-5, i.e., a 2.45-fold increase in permeation.
[0049] The nano-phytophospholipid showed higher Cmax (435.1 ± 28.24 ng/mL), lower Tmax (0.5±0 h) and higher AUC0-t (2175.69 ± 148.57 h.ng/mL) compared to plain silybin (Cmax: 206.9 ± 12.01 ng/mL, Tmax: 2.00 ± 0 h; AUC0-t: 1693.52 ± 90.16 h.ng/mL). The nano-phytophospholipid exhibited improved in vivo pharmacokinetic profile in comparison with the drug alone.
[0050] Working of the Invention: FIG. 1 illustrates a Multiview Fourier Transform Infrared spectrum of silybin, Soy phosphatidylcholine (SPC), Physical mixture (PM) (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition). Silybin had distinct peaks for –OH and –CH stretching between 2900 and 3400 cm-1, C-C stretching, and C=O stretching between 1000 and 1700 cm-1. The difference observed in the FTIR pattern of H6 was identical to that of SPC, with no characteristic peaks of silybin between 1000 and 1700 cm-1, indicating that a new composite is formed. The presence of a new peak at 2924 cm-1 in the FTIR spectrum of H6 verified the establishment of H-bonding between the C=O groups of Soy phosphatidylcholine and free OH groups of silybin, while the disappearance of peaks at 1631 cm-1 (C-O stretching), due to shielding by the phospholipid molecule by the intermolecular coupling which indicated the formation of a new co-amorphous state.
[0051] FIG. 2 illustrates Multiview Differential Scanning Calorimetry thermograms of silybin, SPC, PM (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition). Silybin exhibited a sharp melting endotherm at 164.8 °C, signifying that the drug is crystalline and comparable to that reported in the literature (Bijak, 2017). The melting endotherm of Soy phosphatidylcholine showed a minor peak at 190 °C, which could be attributed to the movement of the polar head in the phospholipid molecular structure, and a sharp peak at 155 °C, which could be attributed to phosphatidylcholine’s phase transition from gel to the liquid crystalline state, and the phospholipid might have gone through melting, isomeric, or other crystalline changes (Bombardelli, 1991). On comparison of the DSC thermograms of the silibyn and soy phosphatidylcholine, the complex showed the absence of silybin peak, which may be attributed to encapsulation of silybin by soy phosphatidylcholine or conversion of the silybin's crystalline to amorphous form (Passerini et al., 2012). Hence the absence of key peaks, and lower phase transition temperature of the complex, conclude the formation of the nano-phytophospholipid through weak interactions, thus causing the hydrocarbon chain in soy phosphatidylcholine to rotate freely and envelop silybin (Chi et al., 2020).
[0052] FIG. 3 illustrates Multiview Powder X-Ray Diffraction p-XRD diffractograms of silybin, SPC, PM (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and H6 (nano-phytophospholipid composition). Clear diffraction peaks were detected for both pure silybin and the PM, suggesting the high crystalline nature of the drug in solid-state. However, SPC has no distinct diffraction pattern, indicating the amorphous nature of the compound. The low-intensity diffraction patterns observed in the H6 is different from that of silybin and physical mixture, which supports the hypothesis of complex formation.
[0053] FIG. 4 illustrates Scanning Electron Microscopy images of silybin and H6 (nano-phytophospholipid). A1, A2, and A3 represent silybin images at 10 µm, 2 µm, and 200 nm, while B1, B2, and B3 represent H6 images at 10 µm, 2 µm, and 200 nm scale. The surface of plain silybin was irregular and crystalline. In contrast, the H6 exhibited a major morphological change, with a rough and porous surface surrounding the active molecule, which may contribute to the increased dissolution rate and solubility of silybin.
[0054] FIG. 5 illustrates In vitro dissolution profile of silybin and H6 at A) pH 1.2 and B) pH 6.8 (mean ± SD; n = 3) In pH 1.2 and pH 6.8, pure silybin exhibited a poor dissolution rate, with 34.22±1.93 % and 43.26±0.49% of the drug released at the end of a 10 h of dissolution. In comparison to the plain silybin, the dissolution profile of the nano-phytophospholipid showed an enhanced rate of dissolution ascribed to the improved wettability and solubility of silybin in the complex. At the end of 10 h, 67.13±1.51% and 86.21±1.14% of silybin was released from H6 at pH 1.2 and pH 6.8, respectively. Dissolution rate of silybin in H6 was higher at both pH 1.2 and 6.8, indicating that the nano-phytophospholipid can be used for improved oral absorption.
[0055] FIG. 6 illustrates Ex vivo permeation studies of Silybin and H6. The Papp for silybin was 0.64 X 10-5 cm/min and H6 was 1.57 X 10-5 cm/min i.e., 2.45-fold increase in permeation. The permeation of silybin from nano-phytophospholipid across the rat ileum was found to be higher compared to silybin alone.
[0056] FIG. 7 illustrates Plasma concentration vs. time profile of plain silybin and H6 in preclinical in vivo pharmacokinetics study in rats. The maximal plasma concentration (Cmax) and time to obtain it (tmax) for H6 after a single oral dosage of silybin at 200 mg/kg were 435.1 ng/ml and 0.5 h, respectively. The Cmax observed with H6 was enhanced by 2.1-fold compared with the Cmax of pure silybin. The mean residence time (MRT) and t1/2 values of nano-phytophospholipid were greater than those of plain silybin, indicating that silybin in the nano-phytophospholipid form has a longer residence time in the body. The AUC values silybin in complex form were drastically improved (1.28-fold), hence suggesting the improvement in oral bioavailability.
[0057] Examples of Implementation:
[0058] Example-1: In this example, the phospholipid used was soy phosphatidylcholine, and the active ingredient refers to silybin, a hepatoprotective agent. The nano-phytophospholipid is made up of by weight using the following composition:
a. Poloxamer P188 is present in an amount ranging from 0.05-1% by weight of the composition
b. Sodium starch glycollate is present in an amount ranging from 0.05-0.1% by weight of the composition
c. Microcrystalline cellulose is present in an amount ranging from 10-40% by weight of the composition
d. Sodium deoxycholate is present in an amount ranging from 0.5-4% by weight of the composition
[0059] Example 2: In this example, the nano-phytophospholipid is made up of by weight using the following composition:
a. Poloxamer 188 is present in an amount ranging from 0.1-5% by weight of the composition
b. Sodium starch glycollate is present in an amount ranging from 1-3% by weight of the composition
c. Microcrystalline cellulose is present in an amount ranging from 10-75% by weight of the composition
d. Sodium deoxycholate is present in an amount ranging from 1-6% by weight of the composition
[0060] Example 3: In this example the weight ratio of silybin and soy phosphatidylcholine in the nano-phytophospholipid composition or formulation is 1:0.1-10 and 1:0.5-5.
[0061] Example 4: In this example, the fabrication of the nano-phytophospholipid formulation using the Twin screw processor or Hot melt extruder with a screw diameter ratio of 1.71 (Omicron 10P) is explained.
a. The silybin and soyabean phospholipid was weighed accurately and mixed in a mortar pestle
b. The mixture was passed through a #40 sieve to obtain uniform particles
c. Surfactant, super disintegrant/ disintegrant, bile salts or bile acids and adsorbent were individually weighed, added and mixed geometrically
d. The above mixture was further mixed in a tumbler for 10 min, sieved again through #40 sieve and then passed through Twin screw processor or Hot melt extruder using a screw feeder at a screw speed of 30 to 50 rpm
e. The screw speed of the barrel was set at 150-250 rpm
f. The temperature of the first heating zone was controlled at 20 to 40 °C., while the temperature of the second heating block was controlled to 70 to 110 °C., while the temperature of the third heating block was controlled to 120 to 160 °C., and the temperature of the fourth heating block was controlled to 70 to 110 °C. Zone B4 was regulated at 70 to 100 °C to ensure that the exit product was solid in nature rather than a liquified melt.
[0062] Example 5: In this example, the procedure for the characterization of the nano-phytophospholipid composition or formulation containing silybin as the active agent is described.
[0063] Particle size, polydispersity index (PDI) and zeta potential: After dispersing in water, the particle size, PDI, and zeta potential of the produced nano-phytophospholipid formulation were assessed using the Zetasizer (Malvern Instruments, UK) (Manikkath et al., 2020) and measurements were reported in the mean of triplicate.
[0064] Fourier transform infrared spectroscopy (FTIR) (Fernandes et al., 2019): The KBr pellet technique was used to determine IR spectra using an FTIR spectrophotometer (Shimadzu FTIR -8300, Kyoto, Japan)). Silybin, SPC, physical mixture (containing silybin, SPC, poloxamer188, sodium starch glycollate, sodium deoxycholate and microcrystalline cellulose) and nano-phytophospholipid composition were dried under a sodium lamp and then grinded with KBr in an agate mortar. The dry materials were then compressed into pellets under 5 tons of pressure for 5 minutes and scanned at 500 to 4000 cm-1.
[0065] Differential Scanning Calorimetry (DSC) (Fernandes et al., 2019): The DSC for silybin, SPC, physical mixture, and nano-phytophospholipid composition were performed using DSC (Shimadzu-TA-60 WS, Kyoto Japan). The samples were placed in an aluminum pan and heated at a rate of 10 °C/ min from 25 °C to 350 °C under continuous nitrogen flow. An empty aluminum pan was used as the reference for the analysis. Each sample had its heat flow measured as a function of temperature. For every sample, heat flow was measured as a function of temperature.
[0066] Powder X-ray Diffraction (PXRD) (Fernandes et al., 2019): The PXRD pattern of Silybin, SPC, physical mixture, and nano-phytophospholipid composition was obtained using a Rigaku-mini flex 600 X-ray diffractometer (Rigaku Co., Tokyo, Japan). The experiments were conducted at room temperature with a 2θ range from 5–80° using a 0.02 °/ min scanning rate. A typical scintillation counter recorded the X-ray-diffracted beam. The current and voltage were set at 15 mA and 40 kV, respectively.
[0067] Scanning Electron Microscopy (SEM) (Fernandes et al., 2019): Scanning electron microscope (SEM) was used to examine the surface morphology of the drug and nano-phytophospholipid composition. The instrument used was EVO MA18 with Oxford EDS(X-act), Zeiss, Germany. Pelletized samples were mounted on aluminum stubs using double-sided gold tape. To improve the conductivity of the samples, they were placed in a vacuum at 10 Torr. An electronic beam with a 20 kV acceleration potential was used to scan the samples. The obtained images were collected.
[0068] Example 6: In this example, the in vitro drug release study of Silybin and H6 formulation is described. The in vitro drug release test for silybin and nano-phytophospholipid composition was conducted using Type I USP dissolution test apparatus. The dissolution media were HCl solution (pH 1.2; with 0.5% Tween 80) and phosphate buffer pH 6.8 (with 0.5% Tween 80) to replicate the pH of the stomach and intestinal fluid. The dissolution flask of 1000 ml capacity was filled with 900 ml of dissolution medium and immersed in the water bath (37 °C ± 0.5 °C). The basket speed was set at 150 rpm. At the start of the study, silybin or nano-phytophospholipid (equivalent to 35 mg of silybin) was added to the dissolution media. The samples (10 mL) were removed from the flask at 0.5, 1, 2, 4, 6, 8, and 10 h and replenished with 10 mL of fresh buffer solution. The samples were filtered and analyzed spectrophotometrically by UV at 288 nm.
[0069] Example 7: In this example, the ex vivo permeation studies using everted rat ileum sac method of silybin and nano-phytophospholipid formulation are described (Managuli et al., 2019). The ileum portion of the rat was removed and flushed with pH 7.4 Ringer's solution. A glass rod was used to evert the ileum, with one end knotted with thread and the other end filled with 1 ml of Ringer's solution. The assembly was incubated in 50 ml of Ringer’s solution and was termed as the mucosal compartment; while the perfusion apparatus was termed as the serosal compartment wherein the silybin and nano-phytophospholipid formulation (equivalent to 5 mg/ml) was added in the beaker. The experimental solution was continuously aerated and maintained at 37 °C. The sample was withdrawn from the open end of the tissue at intervals up to 3 h. The extracted volume was replenished by fresh Ringer solution and analyzed using UV- visible spectroscopy to determine the concentration of the drug in the serosal compartment.
[0070] Example 8: In this example, the preclinical in vivo pharmacokinetics (Chi et al., 2020; Manne et al., 2021) study of silybin and H6 composition in rats are described.
[0071] Animals: The experiments were carried out on male Wistar rats weighing 200-250 g. The animals were housed at Central Animal Research Facility, Kasturba Medical College, Manipal. Animal handling was carried out in accordance with institutional and national norms for animal care and the use of animals.
[0072] Experimental Design: Male Wistar rats were subjected to fasting overnight before administering the drug and were divided into two groups.
[0073] Group I: Plain silybin (200 mg/kg; p.o.)
[0074] Group II: Nano-phytophospholipid composition (200 mg/kg; p.o.).
[0075] Plain silybin or nano-phytophospholipid composition (equivalent to 200 mg/kg of silybin) was dispersed in purified water and administered orally. At time intervals of 0.5, 1, 2, 4, 8, 12, and 24 h, about 200 µL of blood were drawn from retro-orbital plexus of rats and placed in a centrifuge tube containing 10% EDTA. After centrifuging at 10000 rpm for 10 min, about 100 μL of the plasma was separated and stored at -20°C until analysis.
[0076] The pharmacokinetic parameters such as maximum plasma concentration (Cmax), elimination half-life (Kel), absorption half-life (t1/2), area under the plasma concentration-time curve (AUC) and mean residence time (MRT) were calculated using PK Solutions software and the data was statistically analyzed using Graph Pad Prism software.
[0077] It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. , Claims:I/We Claim:
1. A nano-phytophospholipid composition, comprising
active agent(s) and phospholipid(s) in a weight ratio of 1:0.001-20;
a surfactant ranging from 0.001-60% by weight of the composition;
a super disintegrant/ disintegrant at 0.001-25% by weight of the composition;
an adsorbent at 0.1-75% by weight of the composition;
bile acids or bile salt at 0.001-20% by weight of the composition:
fatty acid(s) at 0.001-50% by weight of composition:
peptide(s)/ protein(s) at 0.001-20% by weight of composition;
polymer(s) at 0.001-50% by weight of composition;
absorbent(s) at 0.001-50% by weight of composition;
diluent(s) at 0.001-75% by weight of composition;
lubricant(s) at 0.001 to 10% by weight of composition;
glidant(s) at 0.001 to 10% by weight of composition;
permeation enhancer(s) at 0.001 to 30% by weight of composition, wherein the nano-phytophospholipid composition increases the solubility, dissolution rate, and oral bioavailability or absorption of the active ingredient(s) for scale-up and large-scale manufacturing. The said composition may or may not contain all the above listed ingredient(s)/ constituent(s) wherein the composition/formation is prepared using a continuous novel and solvent-free Twin Screw Processor, Hot melt extruder, Single screw processor, extruder or any method or technique of preparation involving the use of solvent(s) wherein the compound is dispersed into an aqueous solution/ dispersion/ vehicle to form a nano-formulation (nanosuspension, nanoemulsion, or any other form) with particle size below 1000 nm.
2. The nano-phytophospholipid composition as claimed in claim 1 wherein the active agent(s) is a flavonoid that is characterized into Flavanol (quercetin, kaempferol, isorhamnetin, rutin, myricetin, resveratrol), Flavanone (naringenin, hesperidin, naringin, eriodyctiol, silybin), Isoflavanone (genistein, daidzein, glycitein), flavone (apigenin, luteolin, tangeretin), Flavan-3-ols (epicatechin gallate, catechins) and Anthocyanins (delphinidin, malvidin, peonidin, pelargonidin, petunidin) or other constituents or herbal extract(s) or combinations of components or any other active component(s) or large molecule or bioactive compound which provide prophylactic or therapeutic or pharmacological or nutraceutical or cosmeceutical effect/ action upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components wherein Flavonoid belongs to the class of flavanone.
3. The nano-phytophospholipid composition as claimed in claim 2 wherein the flavanone is silybin
4. The nano-phytophospholipid composition as claimed in claim 1 the phospholipid is of natural, semisynthetic or synthetic origin or mixture of phospholipids or any other lipids like Sphingolipids; the phospholipid is soybean phospholipid, egg yolk phospholipid, or a mixture thereof; the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine; the phospholipid may also be a mixture of dipalmitoylphosphatidylcholine, dipalmitoyl phosphatidylethanolamine; the phospholipid may be mixed with other lipid material such cholesterol.
5. The nano-phytophospholipid composition as claimed in claim 7 wherein the phospholipid used was soyabean phospholipid.
6. The nano-phytophospholipid composition as claimed in claim 1 wherein the other inactive ingredients comprise of surfactants, fatty acids, peptides/ proteins, polymers, bile acids or bile salts, super-disintegrants/ disintegrants, adsorbents, absorbents, diluents, lubricants, glidants, permeation enhancers, and any other excipient or active which improves the performance of the formulation/ composition
7. The nano-phytophospholipid composition as claimed in claim 6 wherein
the surfactant is present in an amount ranging from 0.001-60% by weight of the composition;
the disintegrant is present in an amount ranging from 0.001-25% by weight of the composition;
the adsorbent is present in an amount ranging from 0.1-75% by weight of the composition;
the bile acids or bile salt is present in an amount ranging from 0.001-20% by weight of the composition
8. The nano-phytophospholipid composition as claimed in claim 6 and 7 wherein:
the surfactant is selected from the group consisting of sorbitan esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, poloxamers (polyethylene-polypropylene glycol block copolymers), sucrose esters, sodium lauryl sulfate, or any other surfactant and a combination thereof;
the disintegrant is chosen from sodium croscarmellose, crospovidone, sodium starch glycolate, any other disintegrant or surfactant and the combination thereof;
the adsorbent is chosen from silicon dioxide, crystalline cellulose, lactose, any other adsorbent and the combination thereof;
the bile acid or bile salt is chosen from cholic acid, a deoxycholic acid, a glycocholic acid, a glycodeoxycholic acid, a taurocholic acid, a glycocholate, a deoxycholate, a taurocholate, a taurodeoxycholate, a chenodeoxycholic acid, or a salt thereof, or any other bile acid/ bile salt or their derivatives or a combination thereof;
any type of fatty acids, peptides/ proteins, polymers, absorbents, diluents, lubricants, glidants, permeation enhancers, and any other excipient or active which improves the performance of the formulation/ composition
9. The nano-phytophospholipid composition as claimed in claim 8 wherein:
Surfactant is comprised of Poloxamer 188;
Disintegrant is comprised of Sodium starch Glycollate;
The adsorbent is comprised of Microcrystalline cellulose; and
Bile acid or bile salt is comprised of Sodium deoxycholate
10. The nano-phytophospholipid composition as claimed in claim 9 wherein the mixture is passed through the extruder which is having one or more heating zones with one or more screws (screw speed is set at 50 to 250 rpm) wherein the heating zones consist of a first, a second, a third, and a fourth heating zones; wherein the temperature of the first heating zone is controlled to 20 to 100 °C; wherein the temperature of the second heating zone is controlled to 30 to 180 °C; wherein a temperature of the third heating zone is controlled to 30 to 180 °C and wherein the temperature of the fourth heating zone is controlled to 30 to 180 °C.
11. The nano-phytophospholipid composition as claimed in claim 1 wherein the composition forms a dispersion or nanosuspension or any other form of mixture in any aqueous solution(s) including water or aqueous solution or organic solution.