WO2012103472A1

WO2012103472A1 - Granularized particular thermostable rotovirus vaccine preparation

Info

Publication number: WO2012103472A1
Application number: PCT/US2012/022962
Authority: WO
Inventors: Brian Pulliam
Original assignee: Brian Pulliam
Priority date: 2011-01-28
Filing date: 2012-01-27
Publication date: 2012-08-02
Also published as: WO2012103464A2; WO2012103464A3

Abstract

Disclosed herein are methods for thermostable Rotovirus vaccine preparation for oral administration. The preparation of the present invention provides an effective Rotovirus vaccine preparation that has a shelf-life. The thermostable Rotovirus vaccine is prepared as a granular particulate which can be packaged and delivered to remote areas where sources of electricity and refrigeration are cost prohibitive.

Description

[001]. GRANULARIZED PARTICULAR THERMOSTABLE ROTAVIRUS VACCINE PREPARATION

[002]. BACKGROUND AND FIELD OF THE PRESENT INVENTION; [003]. 1. Field of the Present invention

[004]. The present invention relates to vaccines or antigenic compositions and methods of preparation thereof. More specifically, the invention relates to a method of preparing a vaccine composition which is orally digestible and suitable for the administration of a Rotavirus vaccine.

[005]. 2. Background of the Invention

[006]. Rotovirus (or Rotavirus) is a common viral infection among young children. It is the most common cause of diarrhea in U.S. children and is a leading cause of death among children in developing countries. The virus works by attacking the lining of the small intestine, causing often copious loss of fluids and electrolytes. The virus is spread through oral contact to fecal material, and is common in child care environments.

[007]. While some medications may be prescribed to help treat symptoms such as nausea, there is currently no drug that is prescribed to treat the virus itself. Antibiotics are not effective since it is a viral infection as opposed to a bacterial infection. Two vaccines are currently being given with success. One is called RotaTeq and the other is called Rotarix. In developing world, the distribution and effectiveness of these vaccines is extremely limited because neither vaccine is thermostable at room temperature. Currently, each vaccine is liquid based and must be refrigerated.

[008]. 3. Description of Related Art

[009]. Rotarix is a human, live attenuated rotavirus vaccine containing a rotavirus strain of G1P specificity. Rotarix is indicated for the prevention of rotavirus gastroenteritis caused by Gl and non-Gl types (G3, G4, and G9) when administered as a 2-dose series in infants and children. The licensure of Rotarix is in process in the European Union. In March 2010 FDA officials urged pediatricians to temporarily stop using GlaxoSmithKline's Rotarix because they found it contaminated with fragments of genetic material— broken pieces of DNA— from what's called porcine circovirus. Even though it is believed to be benign, vaccines are supposed to be sterile, and because there is a competing vaccine against diarrhea-causing rotavirus that has tested clean, Merck's RotaTeq, the FDA decided to err on the side of caution. In May 2010 the suspension of the vaccine was lifted.

[0010], RotaTeq is a live, oral pentavalent vaccine that contains five rotaviruses produced by reassortment. The rotavirus A parent strains of the reassortants were isolated from human and bovine hosts. Four reassortant rotaviruses express one of the outer capsid, VP7, proteins

(serotypes Gl, G2, G3, or G4) from the human rotavirus parent strain and the attachment protein VP4 (type P7) from the bovine rotavirus parent strain. The fifth reassortant virus expresses the attachment protein VP4, (type P1A), from the human rotavirus parent strain and the outer capsid protein VP7 (serotype G6) from the bovine rotavirus parent strain. In February 2006, the U.S. Food and Drug Administration approved RotaTeq for use in the United States. In August 2006, Health Canada approved RotaTeq for use in Canada. Merck is working with a range of partners including the Rotavirus Vaccine Program, PATH, (Program for Appropriate Technology in Health) and other governmental and non-governmental organizations to develop and implement mechanisms for providing access to this vaccine in the developing world.

[0011]. Regardless of their individual effectiveness, both current Rotavirus vaccines are limited in use because of their lack of thermostability. Because of this, the great majority of children around the world and particularly in developing countries have no realistic prospect of becoming vaccinated.

[0012]. Summary of the Present Invention

[0013]. To overcome the limitations of the prior art, the current invention proposes to create a thermostable digestible granular particulate for the administration of a Rotavirus vaccine that includes an antacid component, has good digestibility and which allows for good dosing of effective levels of vaccine. More specifically, the present invention includes the use of acid neutralizing formulations of ionic salts which are combined with the vaccine particles in a layer fashion and a Hydroxypropyl cellulose (HPC) for a digestible granular particulate which is more easily transportable and thermostable. [0014]. Description of the Preferred Embodiments

[0015]. In a preferred form, the method of the present invention comprises granular particulate ionic salts, a sweetener and cellulose with an antigenic viral culture providing a thermostable, inexpensive and easily packaged vaccine preparation. The present invention is particularly suitable for the preparation of any of the five species of rotavirus pathogens and specific strains of the rotavirus or a combination thereof.

[0016]. The method of the present invention is particularly suitable for preparation of viral pathogens which can be grown in a culture medium. The present invention does not expose the viral culture to extremes in heating or stress that would denature the proteins of the virus.

[0017]. Mannitol is a sugar alcohol in a class of polyols including Maltitol, sorbitol, xylitol and isomalt. Polyols are commonly added to foods because their caloric content is lower than sugars. For the purposes of the present invention any one of the polyols could be used as a substitute or in some combination thereof with Mannitol as is well-known to one skilled in the art.

[0018]. Monosodium phosphate (NaH₂PO₄), also known as anhydrous monobasic sodium phosphate is a chemical compound of sodium with a phosphate counterion. It is used as a laxative and, in combination with other sodium phosphates, as a pH buffer. The pKa is 6.8-7.20 (depending on the physicochemical characteristics during pKa determination). The sodium chloride equivalent value, or E- Value, is 0.49. It is soluble in 4.5 parts water. It is most commonly available as colorless crystals or a white powder. It is insoluble in alcohol. For the purposes of the present invention, it may be assumed that the pH buffers found in Example 1 below may be used as a substitute separately or in some combination thereof with the

Monosodium phosphate as is well-known to one skilled in the art.

[0019]. Hydroxypropyl cellulose (HPC) is a derivative of cellulose with both water solubility and organic solubility. It is used as a topical ophthalmic protectant and lubricant. HPC is an ether of cellulose in which some of the hydroxyl groups in the repeating glucose units have been hydroxypropylated forming -OCH2CH(OH)CH3 groups using propylene oxide. The average number of substituted hydroxyl groups per glucose unit is referred to as the degree of

substitution (DS). Complete substitution would provide a DS of 3. Because the hydroxypropyl group added contains a hydroxyl group, this can also be etherified during preparation of HPC. When this occurs, the number of moles of hydroxypropyl groups per glucose ring, moles of substitution (MS), can be higher than 3.

[0020]. Because cellulose is very crystalline, HPC must have an MS of about 4 in order to reach a good solubility in water. HPC has a combination of hydrophobic and hydrophilic groups, so it has a lower critical solution temperature (LCST) at 45 °C. At temperatures below the LCST, HPC is readily soluble in water; above the LCST, HPC is not soluble. HPC forms liquid crystals and many mesophases according to its concentration in water. Such mesophases include isotropic, anisotropic, nematic and cholesteric. The last one gives many colors such as violet, green and red. For the purposes of the present invention, it may be assumed that HPC can be substituted or used in combination with other substances with similar physical and chemical properties as HPC as is well-known to one skilled in the art.

[0021]. Example 1 provides a method for yielding the acid neutralization capacity of preferred buffers which act to neutralize the gastric acids of the stomach creating an environment in which the live, attenuated rotavirus vaccine can pass through entering the intestines where an immune response may be elicited.

[0022]. Further features of the vaccine preparations of the present invention and the processing for the preparation thereof will be apparent from the following non-limiting examples. The following examples are provided to illustrate various aspects of the present invention, but are not intended to be limiting in any way.

Γ0023Ί. Examples

[0024]. Example 1: Acid Neutralization Capacity (ANC/lOOmg)

[0025].

[0026]. Example 2: Preparation of granular particulate

[0027]. In a preferred form of the present invention, the materials used are comprised of an equivalent of 30 mg of Sodium Phosphate monobasic monohydrate, 115 mg of Sodium Citrate tribasic dehydrate, 326 mg of Mannitol, 25 mg of HPC, and a rotavirus grown in a cultured media mass per dose.

[0028]. To yield a batch of a total mass of 50 doses of the vaccine preparation 1.5 grams of Sodium Phosphate monobasic monohydrate were placed in a mortar and stirred for five minutes with a pestle until forming a powder. Then 5.75 grams of Sodium Citrate tribasic dihydrate were added into the above powder and mixed for 5 minutes. Then 1 .3 grams of Mannitol was added to the powders mixture in the mortar and mixed for 5 additional minutes. Then the powders mixture was transferred into a Invitrogen Mixer (a hula mixer) and mixed for 15 minutes at a speed of 100 rpm. After which the mixture was transferred into a Proctor Silex #5 beaker. In another beaker a 26.31% (w/w) HPC solution was prepared by adding 1.25 grams of HPC into 4.75 grams of water. The 0.2 grams of Rotavirus from the culture media was added to the HPS solution. The HPC/Rotavirus solution was poured onto the dry mixture of Mannitol and Antacid while mixer was constantly stirring. Once mixture was uniformly mixed, the wet mass was passed through a #18 sieve to form uniform granules. The granules were put in a VWR

Scientific Forced Air oven, 1350 FM, at 35 degrees Celsius for 2 hours.

[0029]. The final weight of the granules was 19.000 gm which reflects a net weight loss 24 %. The granular product produced a visible crystal form with an even particle shape and even surface texture. The granular product was tested for dissolution in 25 ml of water at a temperature of 37 degrees Celsius at 100 rpm resulting in an average dissolution time of 187 seconds or 3:07 minutes. Titration results for a Karl Fischer Titration were 4.40% water content with an average mEq Titration of 0.78mEq/500mg of granules.

[0030]. Example 3: Preparation of granular particulate [0031]. In a preferred form of the present invention, the materials used comprised of an equivalent of 30 mg of Sodium Phosphate monobasic monohydrate, 115 mg of Sodium Citrate tribasic dehydrate, 331 mg of Mannitol, 20 mg of HPC, and a rotavirus grown in a cultured media mass per dose.

[0032]. To yield a batch of a total mass of 50 doses of the vaccine preparation 1.5 grams of Sodium Phosphate monobasic monohydrate were placed in a mortar and stirred for five minutes with a pestle until forming a powder. Then 5.75 grams of Sodium Citrate tribasic dihydrate were added into the above powder and mixed for 5 minutes. Then 16.55 grams of Mannitol was added to the powders mixture in the mortar and mixed for 5 additional minutes. Then the powders mixture was transferred into a Invitrogen Mixer (a hula mixer) and mixed for 15 minutes at a speed of 100 rpm. After which the mixture was transferred into a Proctor Silex #5 beaker. In another beaker a 21.05% (w/w) HPC solution was prepared by adding 1.00 grams of HPC into 4.75 grams of water. The 0.2 grams of Rotavirus from the culture media was added to the HPS solution. The HPC/Rotavirus solution was poured onto the dry mixture of Mannitol and Antacid while mixer was constantly stirring. Once mixture was uniformly mixed, the wet mass was passed through a #18 sieve to form uniform granules. The granules were put in a VWR Scientific Forced Air oven, 1350 FM, at 35 degrees Celsius for 2 hours.

[0033]. The final weight of the granules was 18.250 gm which reflects a net weight loss 27 %. The granular product produced a visible crystal form with an even particle shape and even surface texture. The granular product was tested for dissolution in 25 ml of water at a temperature of 37 degrees Celsius at 100 rpm resulting in an average dissolution time of 178 seconds or 2:58 minutes. Titration results for a Karl Fischer Titration were 4.31% water content with an average mEq Titration of 0.86mEq/500mg of granules.

[0034]. Example 4: Preparation of granular particulate

[0035]. In a preferred form of the present invention, the materials used are comprised of an equivalent of 30 mg of Sodium Phosphate monobasic monohydrate, 115 mg of Sodium Citrate tribasic dehydrate, 336 mg of Mannitol, 15 mg of HPC, and a rotavirus grown in a cultured media mass per dose. [0036]. To yield a batch of a total mass of 50 doses of the vaccine preparation 1.5 grams of Sodium Phosphate monobasic monohydrate were placed in a mortar and stirred for five minutes with a pestle until forming a powder. Then 5.75 grams of Sodium Citrate tribasic dihydrate were added into the above powder and mixed for 5 minutes. Then 16.80 grams of Mannitol was added to the powders mixture in the mortar and mixed for 5 additional minutes. Then the powders mixture was transferred into a Invitrogen Mixer (a hula mixer) and mixed for 15 minutes at a speed of 100 rpm. After which the mixture was transferred into a Proctor Silex #5 beaker. In another beaker a 15.8% (w/w) HPC solution was prepared by adding 0.75 grams of HPC into 4.75 grams of water. The 0.2 grams of Rotavirus from the culture media was added to the HPS solution. The HPC Rotavirus solution was poured onto the dry mixture of Mannitol and Antacid while mixer was constantly stirring. Once mixture was uniformly mixed, the wet mass was passed through a #18 sieve to form uniform granules. The granules were put in a VWR Scientific Forced Air oven, 1350 FM, at 35 degrees Celsius for 2 hours.

[0037]. The final weight of the granules was 18.070 gm which reflects a net weight loss 27.72%. The granular product produced a visible crystal form with an even particle shape and even surface texture. The granular product was tested for dissolution in 25 ml of water at a temperature of 37 degrees Celsius at 100 rpm resulting in an average dissolution time of 177 seconds or 2:57 minutes. Titration results for a Karl Fischer Titration were 4.28% water content with an average mEq Titration of 0.90mEq/500mg of granules.

[0038]. Example 5: Preparation of granular particulate

[0039]. In a preferred form of the present invention, the materials used comprised of an equivalent of 30 mg of Sodium Phosphate monobasic monohydrate, 115 mg of Sodium Citrate tribasic dehydrate, 341 mg of Mannitol, 10 mg of HPC, and a rotavirus grown in a cultured media mass per dose.

[0040]. To yield a batch of a total mass of 50 doses of the vaccine preparation 1.5 grams of Sodium Phosphate monobasic monohydrate were placed in a mortar and stirred for five minutes with a pestle until forming a powder. Then 5.75 grams of Sodium Citrate tribasic dihydrate were added into the above powder and mixed for 5 minutes. Then 17.05 grams of Mannitol was added to the powders mixture in the mortar and mixed for 5 additional minutes. Then the powders mixture was transferred into a Invitrogen Mixer (a hula mixer) and mixed for 15 minutes at a speed of 100 rpm. After which the mixture was transferred into a Proctor Silex #5 beaker. In another beaker a 10.55% (w/w) HPC solution was prepared by adding 0.50 grams of HPC into 4.75 grams of water. The 0.2 grams of Rotavirus from the culture media was added to the HPS solution. The HPC/Rotavirus solution was poured onto the dry mixture of Mannitol and Antacid while mixer was constantly stirring. Once mixture was uniformly mixed, the wet mass was passed through a #18 sieve to form uniform granules. The granules were put in a VWR Scientific Forced Air oven, 1350 FM, at 35 degrees Celsius for 2 hours.

[0041]. The final weight of the granules was 17.700 gm which reflects a net weight loss 29.20%. The granular product produced a visible crystal form with an even particle shape and even surface texture. The granular product was tested for dissolution in 25 ml of water at a temperature of 37 degrees Celsius at 100 rpm resulting in an average dissolution time of 169 seconds or 2:49 minutes. Titration results for a Karl Fischer Titration were 4.13% water content with an average mEq Titration of 0.85mEq/500mg of granules.

[0042]. Example 6: Preparation of granular particulate

[0043]. In a preferred form of the present invention, the materials used comprised of an equivalent of 30 mg of Sodium Phosphate monobasic monohydrate, 115 mg of Sodium Citrate tribasic dehydrate, 346 mg of Mannitol, 5 mg of HPC, and a rotavirus grown in a cultured media mass per dose.

[0044]. To yield a batch of a total mass of 50 doses of the vaccine preparation 1.5 grams of Sodium Phosphate monobasic monohydrate were placed in a mortar and stirred for five minutes with a pestle until forming a powder. Then 5.75 grams of Sodium Citrate tribasic dihydrate were added into the above powder and mixed for 5 minutes. Then 17.30 grams of Mannitol was added to the powders mixture in the mortar and mixed for 5 additional minutes. Then the powders mixture was transferred into a Invitrogen Mixer (a hula mixer) and mixed for 15 minutes at a speed of 100 rpm. After which the mixture was transferred into a Proctor Silex #5 beaker. In another beaker a 5.26% (w/w) HPC solution was prepared by adding 0.25 grams of HPC into 4.75 grams of water. The 0.2 grams of Rotavirus from the culture media was added to the HPS solution. The HPC/Rotavirus solution was poured onto the dry mixture of Mannitol and Antacid while mixer was constantly stirring. Once mixture was uniformly mixed, the wet mass was passed through a #18 sieve to form uniform granules. The granules were put in a VWR Scientific Forced Air oven, 1350 FM, at 35 degrees Celsius for 2 hours.

[0045]. The final weight of the granules was 17.394 gm which reflects a net weight loss 30.42%. The granular product produced a visible crystal form with an even particle shape and even surface texture. The granular product was tested for dissolution in 25 ml of water at a temperature of 37 degrees Celsius at 100 rpm resulting in an average dissolution time of 121 seconds or 2:01 minutes. Titration results for a Karl Fischer Titration were 3.99% water content with an average mEq Titration of 0.86mEq/500mg of granules.

[0046]. The % HPC concentration was tested from 5% to 1 % in which results varied from dissolution time of 187 seconds to 121 seconds respectfully and it can be concluded that dissolution time decreases with respect to a decrease in % HPC concentration. However, based on the effect of the % HPC concentration with respect to % weight loss and dissolution time, final conclusions showed that the 5% HPC concentration, as shown in Example 2 above, is the best formulation out of the other HPC concentration formulation. Results further concluded that the Acid Neutralization Capacity and % water content are not affected with change in the % of HPC concentration.

Claims

What is claimed is:

1. A thermostable Rotavirus vaccine preparation forming a granular particulate, wherein the vaccine preparation comprises a water-soluble thermoplastic binder, a pH buffer, a polyol sweetener, and a Rotovirus culture.

2. The thermostable Rotovirus vaccine preparation of claim 1, wherein the water-soluble thermoplastic binder is hydroxypropyl cellulose (HPC).

3. The thermostable Rotovirus vaccine preparation of claim 1, wherein the pH buffer is selected from the following group of pH buffers: Sodium phosphate, Sodium Citrate, Disodium phosphate or Sodium monobasic phosphate.

4. The thermostable Rotovirus vaccine preparation claim 1, wherein the pH buffer is a mixed ratio of Sodium citrate and Sodium phosphate.

5. The thermostable Rotovirus vaccine preparation of claim 1, wherein the polyol sweetener added is selected from the following group of polyols: malitol, sorbitol, xylitol or isomalt.

6. A thermostable Rotavirus vaccine preparation forming a granular particulate, wherein the vaccine preparation comprises Sodium Phosphate monobasic monohydrate, Sodium Citrate tribasic dehydrate, Mannitol, HPC, and a rotavirus grown in a cultured media.