Claims:CLAIMS
I/We Claim:
A gastric resistant bioavailable formulation consisting of:
0.0052 g - 1.033 g of lactoferrin powder as an active ingredient;
0.004 g - 0.8 g of lactose, wherein the lactose is reducing sugar;
0.0023 g - 0.452 g of mannitol, wherein the mannitol is sugar alcohol;
0.00013 g - 0.025 g of polyvinylpyrrolidone k30;
0.00025 g - 0.05 g of silicone dioxide;
0.0003 g - 0.06 g of purified talc, wherein the purified talc is a hydrated magnesium silicate
0.0004 g - 0.08 g of magnesium stearate;
0.0005 g - 0.1 g of sodium starch glycolate;
0.0005 g - 0.1 g of croscarmellose sodium;
0.0005 g - 0.1 g of Polacrilin potassium;
0.0005 g - 0.1 g of L-arginine;
0.0015 g - 0.3 g of starch;
0.00010 g - 0.021 g of seal coat; wherein seal coat includes hydroxypropyl methylcellulose, polyethylene glycol 600, purified talc, polyethylene glycol 400
0.00224 g - 0.45 g of methacrylic acid based enteric coat;
0.0094 g - 1.9 g of isopropyl alcohol;
0.3425 g - 6.9 g of methyl dichloride;
0.00004 g - 0.01 g of yellow iron oxide;
0.00001 g - 0.002 of black iron oxide.
wherein, the formulation enhances bioavailability, absorption of the lactoferrin in intestine, stability and resistance from gastric acids in stomach.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the L arginine is used as pre-treatment to enhance the stability of lactoferrin.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the lactose, mannitol and starch are filler.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the polyvinylpyrrolidone k30 and isopropyl alcohol are binder.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the magnesium stearate is lubricant.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the sodium starch glycolate and croscarmellose sodium are disintegrating agent.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the Polacrilin potassium is dispersing agent.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the methyl dichloride and isopropyl alcohol are coating solvent.
The gastric resistant bioavailable formulation as claimed in claim 1, wherein the yellow iron oxide and black iron oxide are coloring agent.
A process for manufacturing a gastric resistant bioavailable formulation consisting steps of:
a. weighing, sieving and adding lactoferrin powder, lactose and mannitol in Rapid Mixer Granulator;
b. combining isopropyl alcohol and polyvinylpyrrolidone K30 in the mixture formed at step (a) and mixing properly in Rapid Mixer Granulator for 7 minutes;
c. discharging the combined granules formed at step (b) from Rapid Mixer Granulator and transferring to Fluidised bed dryer;
d. drying the mixture formed at step (c) at approximately 40 DEG C until 1.5 % to 2 % loss of drying is achieved and then sieving the mixture;
e. mixing granulate product formed at step (d) with starch, colloidal silicon dioxide, sodium starch gycolate, purified talc, Polacrilin potassium & L arginine;
f. sieving and blending the combined mixture formed at step (e) in the blender;
g. discharging the blended mixture formed at step (f) from the blender and transferring for compression;
h. compressing the blended mixture formed at step (g) under 3 ton pressure at 25 DEG C and 40% relative humidity to produce tablet cores;
i. dissolving hydoxypropyl methylcellulose, polyethylene glycol 600 in isopropyl alcohol and coating the seal coat on the compressed tablet formed at step (h) and allowing to dry;
j. dissolving methacrylic acid in isopropyl alcohol, methylene dichloride, yellow oxide of iron and black oxide of iron and coating the enteric coat on the seal coat coated compressed tablet formed at step (i) and allowing to dry. , Description:FIELD OF THE INVENTION
The present invention relates to the field of pharmaceutical products. More specifically the present invention relates to formulation and method for manufacturing a gastric resistant bioavailable formulation in the form of tablet containing lactoferrin with enhanced bioavailability, absorption and stability for optimum therapeutic effect. The present invention also relates to a formulation which enhances solubility resistance (insolubility under stomach) in an acidic condition, enables excellent enteric properties, and enhances bioavailability and stability of lactoferrin.

BACKGROUND OF THE INVENTION
This section is intended to provide information relating to the field and background of the invention and thus any approach/functionality described below should not be assumed to be qualified as prior art merely by its inclusion in this section.
Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is found in various secretory fluids, such as milk, saliva, tears, and nasal secretions. Lactoferrin is also present in secondary granules of PMNs and is secreted by some acinar cells. Lactoferrin can be purified from milk or produced recombinantly. Human colostrum ("first milk") has the highest concentration about seven times more (approximately 1.05 g/L), followed by human milk, then cow milk (approximately 0.15 g/L). The lactoferrin contained in mother's milk helps protect breast-fed infants against infections.
Lactoferrin is an important component of the immune system of the body; it has antimicrobial activity (bactericide, fungicide) and is part of the innate defense, mainly at mucoses. In particular, lactoferrin provides antibacterial activity and antiviral activity to human infants. Lactoferrin interacts with DNA, RNA, polysaccharides and demonstrating biological functions in complexes with these ligands. Lactoferrin is also found in fluids in the eye, nose, respiratory tract and gastro-intestinal tract.
Lactoferrin helps in the regulation of iron absorption in the body from the intestine. Research evidences demonstrate that lactoferrin protects against infections from bacteria, viruses, and fungi. Lactoferrin shows antibacterial activity by slowing down bacterial growth, starving bacteria of nutrients and rupturing bacterial walls.
Lactoferrin in sufficient strength acts on a wide range of human and animal viruses having DNA or RNA genomes, including the herpes simplex virus 1 and 2 (HSV-1 & 2), cytomegalovirus (CMV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), hantaviruses, rotaviruses, poliovirus (type 1), human respiratory syncytial virus (RSV), murine leukemia viruses (MLV) and Mayaro virus. The most studied mechanism of antiviral activity of lactoferrin is preventing the viral entry into target cells. Viruses bind to the lipoproteins of the cell membranes and gain cell entry. Lactoferrin binds to the same lipoproteins, preventing the viral particles from binding to these lipoproteins. In addition to interacting with the cell membrane, lactoferrin also directly binds to viral particles, such as the hepatitis viruses. This mechanism is also confirmed by the antiviral activity of lactoferrin against rotaviruses, which act on different human cell types. Lactoferrin also suppresses virus replication after the virus entry, which is a crucial process of the virus life cycle during viral infection. Such an indirect antiviral effect is achieved by lactoferrin’s effect on natural killer cells, granulocytes and macrophages cells, which play a crucial role in the early stages of viral infections, such as severe acute respiratory syndrome (SARS).
In order to take lactoferrin, which is known to have various actions, more effectively, it needs to be absorbed into the body instantaneously. In addition, it is useful to make tablets in order to make lactoferrin easy to carry, ingest and for its efficient absorption
As a protective film for allowing the active ingredient to reach the intestine, pharmaceuticals based on methacrylic acid are used, that do not dissolve under the pH condition (acidic) in the stomach but dissolve under the pH condition (neutral) of the small intestine. It is common to use shellac and zein for high molecular compounds and foods, but there is a demand for a coating composition that enables excellent enteric solubility.
Lactoferrin is hydrolysed due to the action of gastric pepsin in adults. This process of hydrolysis prevents LF from reaching the small intestine in a form that is efficient to bind to LF receptors. An enteric protective coating is an important requirement in a composition that contains a medicament, which is not sufficiently buffered and is unstable in an acidic environment such as stomach in the gastrointestinal tract to prevent its release prior to reaching the intestines. The composition thus increases bioavailability of gastric enzyme labile LF with less than 10% release in stomach and more than 90% release in intestinal fluid. Interest in products containing LF is increasing due to its potential for treatment of a wide variety of diseases. However, LF is a protein and thus it is prone to degradation, affecting the quality of products containing LF.
A Japanese patent number JP6642438B2 describes a coated preparation comprising: a substance to be coated; an under layer which is formed on the substance and contains a polymeric compound (A) having such a property that an aqueous 6 mass% solution has a viscosity of less than 300 mPa·s at 25°C; and an enteric coating layer which is formed on the under layer and contains an alginate salt (B) and a plasticizer (C). Here, pre coating is formed that has suppressed elution.
A PCT patent application number WO2015020186A1 describes tablet composition containing lactoferrin particles having an average particle size of 40 to 300 µm to improve the disintegration property of tablets and the elution property of lactoferrin while maintaining a low degree of wear.
A Japanese patent application number JP6589979B2 describes tablet composition comprising (A) 30% by mass or more of lactoferrin and (B) 0.12 to 1.0% by mass of a magnesium stearate which improves disintegrating properties and elution properties of tablet.
Based on aforesaid prior arts, there is a need to develop pharmaceutical formulation with an increased bioavailability of gastric enzyme labile lactoferrin in intestine.
OBJECT OF THE INVENTION
This section is intended to introduce certain objects of the disclosed systems in a simplified form, and is not intended to identify the key advantages or features of the present disclosure.
The main object of the present invention is to provide formulation with an enteric coating and high stability of lactoferrin.
Another object of the present invention is to provide formulation with an increased bioavailability and absorption of gastric enzyme labile lactoferrin in intestinal fluid.
A further object of the present invention is to provide formulation with increased chemical stability of gastric enzyme labile lactoferrin in stomach.
A still further object of the present invention is to provide formulation with lactoferrin having excellent resistance to disintegration at pH less than 3.
A still yet further object of the present invention is to provide formulation with lactoferrin having excellent drug release properties at pH greater than 5.5.
These and other objects, features and advantages of the present invention will become more apparent from the following description when taken in connection with the accompanying drawing which shows, for the purpose of illustration only, one embodiment in accordance with the present invention.
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention. This summary is not intended to identify key or essential inventive concepts of the invention, nor is it intended for determining the scope of the invention.
In order to overcome problems of conventional compositions or formulations with the bioavailability, absorption and stability of lactoferrin, the present invention discloses a gastric resistant bioavailable formulation in the form of tablet comprising 0.0052 g - 1.033 g of lactoferrin powder, 0.004 g - 0.8 g of lactose which is reducing sugar, 0.0023 g - 0.452 g of mannitol which is sugar alcohol, 0.00013 g - 0.025 g of polyvinylpyrrolidone k30, 0.00025 g - 0.05 g of silicone dioxide, 0.0003 g - 0.06 g of purified talc which is hydrated magnesium silicate, 0.0004 g - 0.08 g of magnesium stearate, 0.0005 g - 0.1 g of sodium starch glycolate, 0.0005 g - 0.1 g of croscarmellose sodium, 0.0005 g - 0.1 g of Polacrilin potassium, 0.0005 g - 0.1 g of L-arginine, 0.0015 g - 0.3 g of starch, 0.00010 g - 0.021 g of seal coat, 0.00224 g - 0.45 g of methacrylic acid based enteric coat, 0.0094 g - 1.9 g of isopropyl alcohol, 0.3425 g - 6.9 g of methyl dichloride, 0.00004 g - 0.01 g of yellow iron oxide, 0.00001 g - 0.002 of black iron oxide.
The protective coat, also known as seal coat of the gastric resistant bioavailable formulation of the present invention further includeshydroxypropyl methylcellulose, polyethylene glycol 600, purified talc, polyethylene glycol 400.
The lactose, mannitol and starch of the gastric resistant bioavailable formulation of the present invention are used as filler. The polyvinylpyrrolidone k30 and isopropyl alcohol of the gastric resistant bioavailable formulation of the present invention are used as binder. The magnesium stearate of the gastric resistant bioavailable formulation of the present invention is used as lubricant. The sodium starch glycolate and croscarmellose sodium of the gastric resistant bioavailable formulation of the present invention are used as disintegrating agent. The Polacrilin potassium of the gastric resistant bioavailable formulation of the present invention is used as dispersing agent. The methyl dichloride and isopropyl alcohol of the gastric resistant bioavailable formulation of the present invention are used as coating solvent. The yellow iron oxide and black iron oxide of the gastric resistant bioavailable formulation of the present invention are used as coloring agent.
A process for manufacturing a gastric resistant bioavailable formulation consisting steps of: (a) Weighing, sieving and adding lactoferrin powder, lactose and mannitol in Rapid Mixer Granulator; (b) Combining isopropyl alcohol and polyvinylpyrrolidone K30 in the mixture formed at step (a) and mixing properly in Rapid Mixer Granulator for 7 minutes; (c) Discharging the combined granules formed at step (b) from Rapid Mixer Granulator and transferring to Fluidised bed dryer; (d) Drying the mixture formed at step (c) at approximately 40 DEG C until 1.5 % to 2 % loss of drying is achieved and then sieving the mixture; (e) Mixing the granulate product formed at step (d) with starch, colloidal silicon dioxide, sodium starch gycolate, purified talc, Polacrilin potassium & L arginine; (f) Sieving and blending the combined mixture formed at step (e) in the blender; (g) Discharging the blended mixture formed at step (f) from the blender and transferring for compression; (h) Compressing the blended mixture formed at step (g) under 3 ton pressure at 25 DEG C and 40% relative humidity to produce tablet cores; (i) Dissolving hydoxypropyl methylcellulose, polyethylene glycol 600 in isopropyl alcohol and coating the seal coat on the compressed tablet formed at step (h) and allowing to dry; (j) Dissolving methacrylic acid in isopropyl alcohol, methylene dichloride, yellow oxide of iron and black oxide of iron and coating the enteric coat on the seal coat coated compressed tablet formed at step (i) and allowing to dry.

BRIEF DESCRIPTION OF DRAWING
In order to better understand the present invention of a gastric resistant bioavailable formulation, the characteristics of object of the present invention, will be better viewed from the detailed description hereinafter, which is only for a way of example, associated to the drawing referenced below, which are an integral part of this application. The parts in the drawing are not drawn to scale; the main objective is to understand the components, their arrangement and their working.
FIG. 1 is a flow chart of process for manufacturing a gastric resistant bioavailable formulation in accordance with the present invention;

DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and/or “comprising” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It will be understood that when an element is referred to as being “connected” or “coupled” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. Furthermore, the term “and/or” includes any and all combinations and arrangements of one or more of the associated listed items.
Unless otherwise defined, all 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.
Any headings provided herein are for convenience only and do not necessarily affect the scope or meaning of the claimed invention.
One of the embodiment of the present invention discloses a gastric resistant bioavailable formulation in the form of tablet comprising 0.0052 g - 1.033 g of lactoferrin powder, 0.004 g - 0.8 g of lactose which is reducing sugar, 0.0023 g - 0.452 g of mannitol which is sugar alcohol, 0.00013 g - 0.025 g of polyvinylpyrrolidone k30, 0.00025 g - 0.05 g of silicone dioxide, 0.0003 g - 0.06 g of purified talc which is hydrated magnesium silicate, 0.0004 g - 0.08 g of magnesium stearate, 0.0005 g - 0.1 g of sodium starch glycolate, 0.0005 g - 0.1 g of croscarmellose sodium, 0.0005 g - 0.1 g of Polacrilin potassium, 0.0005 g - 0.1 g of L-arginine, 0.0015 g - 0.3 g of starch, 0.00010 g - 0.021 g of seal coat, 0.00224 g - 0.45 g of methacrylic acid based enteric coat, 0.0094 g - 1.9 g of isopropyl alcohol, 0.3425 g - 6.9 g of methyl dichloride, 0.00004 g - 0.01 g of yellow iron oxide, 0.00001 g - 0.002 of black iron oxide.
The protective coat, also known as seal coat of the enteric coated formulation of the present invention further includes hydroxypropyl methylcellulose, polyethylene glycol 600, purified talc, polyethylene glycol 400.
The enteric coat used in the formulation of the present invention is methacrylic acid based coating, which is acidic in nature similar to most of the enteric coats used in the art. The interaction of acidic coatings and acid labile ingredients can cause chemical instability. The possibility of chemical instability increases further in high temperatures and humid conditions. A seal coat or a subcoat is usually included between the core containing the active ingredient and the enteric coat to reduce the chemical instability. The function of the seal coating is to improve stability of the formulation by physically separating the acid labile ingredient from the acidic enteric coat.
The lactose, mannitol and starch of the gastric resistant bioavailable formulation of the present invention are used as filler. The polyvinylpyrrolidone k30 and isopropyl alcohol of the gastric resistant bioavailable formulation of the present invention are used as binder. The magnesium stearate of the gastric resistant bioavailable formulation of the present invention is used as lubricant. The sodium starch glycolate and croscarmellose sodium of the gastric resistant bioavailable formulation of the present invention are used as disintegrating agent. The Polacrilin potassium of the gastric resistant bioavailable formulation of the present invention is used as dispersing agent. The methyl dichloride and isopropyl alcohol of the gastric resistant bioavailable formulation of the present invention are used as coating solvent. The yellow iron oxide and black iron oxide of the gastric resistant bioavailable formulation of the present invention are used as coloring agent.
In accordance with the present invention, an enteric coated, with a high stability of active ingredient formulation containing lactoferrin as the active medicament and a method of making the present composition is provided. The active ingredient in the formulation is lactoferrin. Lactoferrin is an 80 kDa protein, having immunological properties, and is both antibacterial and antiviral. Lactoferrin is hydrolysed due to the action of gastric pepsin in adults. This process of hydrolysis prevents lactoferrin from reaching the small intestine in a form that is efficient to bind to LF receptors. Additionally, LF is a protein and is thus prone to degradation affecting the quality of products containing LF. An enteric protective coating is an important requirement in a composition that contains a medicament, which is not sufficiently buffered and is unstable in an acidic environment such as stomach in the gastrointestinal tract to prevent its release prior to reaching the intestines. The composition thus increases bioavailability of gastric enzyme labile lactoferrin with less than 10% release in stomach and more than 90% release in intestinal fluid. The active ingredient lactoferrin is a multifunctional iron-binding glycoprotein in milk. Interest in products containing lactoferrin is increasing due to its potential for treatment of a wide variety of diseases. However, lactoferrin is a protein and thus it is prone to degradation, affecting the quality of products containing lactoferrin. Lactoferrin has been pre-treated with amino acid L-arginine for enhancing its stability. The enteric coated lactoferrin tablets have excellent resistance to disintegration at pH less than 3 but have excellent drug release properties at pH greater than 5.5. The novel enteric coated and stable lactoferrin tablet will provide for protection of the lactoferrin in acidic pH of the stomach making it bioavailable, and also enhance the stability of lactoferrin because of the presence of L-arginine.
A flow chart of process for manufacturing a gastric resistant bioavailable formulation of the present invention according to an embodiment of the present invention is shown in FIG. 1. As shown in FIG. 1, the flow chart 100 containing steps 101 to 110 of process for manufacturing a gastric resistant bioavailable formulation (a) Step 101: Weighing, sieving and adding lactoferrin powder, lactose and mannitol in Rapid Mixer Granulator; (b) Step 102: Combining isopropyl alcohol and polyvinylpyrrolidone K30 in the mixture formed at step (a) and mixing properly in Rapid Mixer Granulator for 7 minutes; (c) Step 103: Discharging the combined granules formed at step (b) from Rapid Mixer Granulator and transferring to Fluidised bed dryer; (d) Step 104: Drying the mixture formed at step (c) at approximately 40 DEG C until 1.5 % to 2 % loss of drying is achieved and then sieving the mixture; (e) Step 105: Mixing the granulate product formed at step (d) with starch, colloidal silicon dioxide, sodium starch gycolate, purified talc, Polacrilin potassium & L arginine; (f) Step 106: Sieving and blending the combined mixture formed at step (e) in the blender; (g) Step 107: Discharging the blended mixture formed at step (f) from the blender and transferring for compression; (h) Step 108: Compressing the blended mixture formed at step (g) under 3 ton pressure at 25 DEG C and 40% relative humidity to produce tablet cores; (i) Step 109: Dissolving hydoxypropyl methylcellulose, polyethylene glycol 600 in isopropyl alcohol and coating the protective coat on the compressed tablet formed at step (h) and allowing to dry; (j) Step 110: Dissolving methacrylic acid in isopropyl alcohol, methylene dichloride, yellow oxide of iron and black oxide of iron and coating the enteric coat on the protective coat coated compressed tablet formed at step (i) and allowing to dry.
The process for manufacturing tablets of the present invention in detail includes i. Granulation step, ii. Intermediate finishing step, iii. Compression step, iv. First coating step, and iv. Second coating step.
Granulation Step
Suitable quantities of lactoferrin powder, lactose & mannitol are first weighed and sieved and then combined in Rapid Mixer Granulator with isopropyl alcohol and Polyvinylpyrrolidone K 30. After this step, the combined granules are discharged from RMG and transferred to Fluidised bed dryer (FBD) for drying. The mixture is dried at approximately 40° C until 1.5 % loss of drying is achieved. The dried mixture is then sieved through #18 sieve.
Intermediate Finishing Step
The product obtained after granulation is mixed with with starch, colloidal silicon-dioxide, sodium starch gycolate, purified talc, Polacrilin Potassium & L-Arginine. The combined mixture is then sifted in #40 mesh size and blended. After blending, magnesium stearate is added to the material. The blended mixture is discharged from the blender and transferred for compression.
Compression Step
The mixture from the intermediate finishing step is then compressed under 3 ton pressure at 25 DEG C and 40% relative humidity to produce tablet cores. The pressed cores are stored in a dry place in a double bag and silica gel and protected from light.
First Coating Step:
(Seal Coat) Hydoxy propyl methylcellulose, Polyethylene Glycol 600 is dissolved in isopropyl alcohol. The solution is then coated on the previously prepared compressed tablet until the desired weight increase is achieved and allowed to dry.
Second Coating Step:
Suitable quantities of enteric coat EN is dissolved in isopropyl alcohol and methylene -dichloride with colours (yellow oxide of iron and black oxide of iron). The solution is then coated on the previously prepared compressed tablet until the desired weight increase is achieved and allowed to dry.
The manufactured enteric coated tablet of the present invention has undergone various tests to provide sufficient evidence for the properties of disintegration, dissolution and microbial growth.
The disintegration test of enteric coated tablet is performed in two stages as below:
(1) Acid Stage: Tablet was added into each tube, and the assembly was suspended in the beaker containing 0.1 M Hydrochloric acid and operated without the disc for 2 hours. After 2 hours there was no damaged tablet showing the evidence of disintegration, cracking or softening.
(2) Buffer Stage: The assembly was removed from 0.1 M HCl, which was replaced with mixed phosphate Buffer pH 6.8 in the beaker. A disc was added to each tube and the apparatus was operated. The assembly was removed and checked for residue at regular intervals. Time was noted when all the tablets had disintegrated and no residue remained on the screen of the tubes. If the tablet failed to comply because of adherence to the disc, the test was repeated further on 6 tablets without the disc. The tablets pass the test if all six have disintegrated.
The dissolution test of enteric coated tablet in 0.1M Hydrochloride Acid and measured by liquid chromatography described as below:
Medium - 900 ml of 0.1M Hydrochloride Acid
RPM - 100 rpm
Time - 120 minutes
Apparatus - Paddle
Temperature - 37.0±0.5 DEG C
Limit - Not Less Than 85% Drugs should retain in 0.1N HCl after 2 hours
Standard Preparation: Approximately 55 mg of lactoferrin W.S. was weighed and added in a 100 ml volumetric flask, 10 ml of dissolution medium was added to it and the solution was sonicated for 15 min. This solution was diluted to volume with dissolution medium. 5 ml of this solution was taken and diluted to 50 ml volumetric flask volume was made up with dissolution medium. Filtrate was obtained with 0.45µ membrane filter.
Test Solution: 1 tablet was placed in six dissolution vessels containing 900 ml of medium and was equilibrated to 37.0±0.5 DEG C, and the apparatus was started immediately. The sample was collected after the specified time. The paddle was rotated at 100 RPM for 120 minutes. After the completion of the process the solution was withdrawn within 45 sec and filtered.
Chromatographic Condition: Column - Mab SCX - 10, 50mm X 4.0 mm, 3 µm
Mode: Gradient
Flow Rate: 0.5 ml/min
Column Temperature: 25 DEG C
Detector: (UV) 280 nm
Injection Volume: 100 µL
Gradient Composition:
Reagents:
1. Sodium DiHydrogen Orthophosphate Monohydrate
2. Sodium Phosphate Dibasic Heptahydrate
3. Sodium Chloride
4. Hydrochloric Acid
5. Sodium Hydroxide

Time (Min) %B
0 0
20 100
25 100
27 0
37 0

Preparation of Mobile Phase (A) Solution containing 0.5998 g Sodium Di Hydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate 1 Liter distilled water was prepared. pH was adjusted to 6.8 with 1M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Preparation of Mobile Phase (B) A solution containing 87.66 g Sodium Chloride, 0.5998 g Sodium DiHydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate was prepared in 1 Liter water. pH was adjusted to 6.8 with 1 M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Standard solution and test solution were injected and concentration of lactoferrin was calculated using the formula:
(Aspl×Ws×5×900×P×100)/(Astd×100×50×1Tab×100)
Aspl = Area of Sample
Astd = Area of Standard
Ws = Weight of working standard in mg
P = Potency
The dissolution test of enteric coated tablet in phosphate buffer and measured by liquid chromatography is described as below:
Medium - 900 ml of phosphate buffer pH 7.4
RPM - 75 rpm
Time - 45 minutes
Apparatus - Paddle
Temperature - 37.0±0.5 DEG C
Limit - Not Less Than 70.0% of the stated amount of lactoferrin
Standard Preparation: 55 mg of a lactoferrin W.S. was weighed and added to 100 ml volumetric flask, 10 ml of Dissolution Medium was added to it and the solution was sonicated for 15 min. The solution was then diluted to volume with Dissolution medium. 5 ml of this solution was taken and diluted to 50 ml volumetric flask. The volume was made up with Dissolution medium. Filtrate was obtained with 0.45µ membrane filter.
Test Solution: 1 tablet was placed in six dissolution vessels containing 900 ml of medium which was equilibrated to 37.0±0.5 DEG C and the apparatus was started immediately. The sample was collected after a specified time. The paddle was rotated at 100 RPM for 120 minutes, and after completion of the process, the solution was withdrawn within 45 seconds and filtered.
Chromatographic Condition: Column - Mab SCX - 10, 50mm X 4.0 mm, 3 µm
Mode: Gradient
Flow Rate: 0.5 ml/min
Column Temperature: 25 DEG
Detector: (UV) 280 nm
Injection Volume: 100 µL
Gradient Composition:
Reagents:
1. Sodium DiHydrogen Orthophosphate Monohydrate
2. Sodium Phosphate Dibasic Heptahydrate
3. Sodium Chloride
4. Hydrochloric Acid
5. Sodium Hydroxide

Time (Min) %B
0 0
20 100
25 100
27 0
37 0

Preparation of Mobile Phase (A) Solution containing 0.5998g Sodium Di Hydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate 1 Liter distilled water was prepared. pH was adjusted to 6.8 with 1M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Preparation of Mobile Phase (B) A solution containing 87.66 g Sodium Chloride, 0.5998 g Sodium DiHydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate was prepared in 1 Liter distilled water. pH was adjusted to 6.8 with 1 M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Standard solution and test solution were injected and concentration of lactoferrin was calculated using the formula:
(Aspl×Ws×5×900×P×100)/(Astd×100×50×1Tab×100)
Aspl = Area of Sample
Astd = Area of Standard
Ws = Weight of working standard in mg
P = Potency
Assay:
Standard Preparation: 100mg of Lactoferrin Working standard was accurately weighed in a 100 ml Volumetric flask. 20 ml of Mobile phase A was added to the solution followed by sonication for about 20 minutes. Volume was adjusted with mobile phase A & filtered through 0.45µ syringe filter.
Test Preparation: 20 tablets were weighed & crushed powder (eqv. to 100mg) of Lactoferrin was taken in a 100 ml Volumetric flask. 20 ml of Mobile phase A was added to it and the solution was sonicated for about 20 minutes. The volume was adjusted with mobile phase A and filtered through a 0.45µ syringe filter.
Reagents:
1. Sodium DiHydrogen Orthophosphate Monohydrate
2. Sodium Phosphate Dibasic Heptahydrate
3. Sodium Chloride
4. Hydrochloric Acid
5. Sodium Hydroxide
Preparation of Mobile Phase (A) Solution containing 0.5998g Sodium Di Hydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate 1 Liter distilled water was prepared. pH was adjusted to 6.8 with 1M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Preparation of Mobile Phase (B) A solution containing 87.66 g Sodium Chloride, 0.5998 g Sodium DiHydrogen Orthophosphate Monohydrate and 1.34 g of Sodium Phosphate Dibasic Heptahydrate was prepared in 1 Liter distilled water. pH was adjusted to 6.8 with 1 M Hydrochloric acid Solution or Sodium Hydroxide Solution.
Chromatographic Condition: Column - Mab SCX - 10, 50mm x 4.0 mm, 3 µm
Mode: Gradient
Flow Rate: 0.5 ml/min
Column Temperature: 25 DEG
Detector: (UV) 280 nm
Injection Volume: 100µL(1mg/ml Lactoferrin)
(Please note, Inject 50µL (Twice) on fresh column)
Gradient Composition:
Time (Min) %B
0 0
20 100
25 100
27 0
37 0

Standard solution and test solution were injected and concentration of lactoferrin was calculated using the formula:
(Aspl×Ws×100×P)/(Astd×100×Wt.×100)×Avg.weight×100/LabelClaim
Aspl = Area of Sample
Astd = Area of Standard
Ws = Weight of working standard in mg.
Wt= Weight of test in mg.
P = Potency