DESC:FIELD OF THE INVENTION:
The present invention relates to a process for formulating acid stabilized oral bacterial vaccine, more particularly a combination of whole cell inactivated bacteria and acid stabilized rCTB (recombinant cholera toxin B) resulting in synergistic protection against diarrhea caused by various bacteria such as Vibrio cholerae, Enterotoxigenic Escherichia coli (ETEC). The present invention also relates to a novel process of preparation of a dosage form resulting in an easy and highly acceptable mode of administration.

BACKGROUND OF THE INVENTION:
Diarrhoea remains a global threat to public health. Major causes of diarrhea include certain bacteria, viruses or parasites, food intolerances etc. It is estimated that a mortality of 28000 to 142000 deaths and a morbidity of 1.4 to 4.3 million annual cases can be attributed to Cholera. (Cholera 2013: Weekly epidemiological record. WHO 2014, 89, 345-356)

Normally, large volumes of water are secreted into the small intestinal lumen, but a major percentage of this water is efficiently absorbed before reaching the large intestine. A condition when secretion of water into the small intestinal lumen exceeds absorption is termed as Diarrhea. It is a condition where a person suffers from abdominal cramps, bloating, nausea and loose-watery stools. In most cases, diarrhea signs and symptoms usually last a couple of days. But sometimes diarrhea can last for weeks and in these situations it is a sign of a serious disorder and requires early treatment.

Major causes of diarrhoea include certain bacteria, viruses or parasites, food intolerances etc. that affect the digestive system and bowel movement of a person. Infection with Enterotoxigenic Escherichia coli (hereinafter ‘ETEC’) is the leading cause of travellers' diarrhoea and a major cause of diarrheal disease in underdeveloped nations, especially among children. ETEC is transmitted by food or water contaminated with animal or human faeces. Other bacteria termed as Shigella, induces Shigellosis disease that includes diarrhea (often bloody), fever, and stomach cramps after exposure to the bacteria. Although Vibrio Cholera and other enterotoxigenic bacteria such as ETEC produce secretory diarrhea by exactly the same mechanism but cholera is much more severe form of diarrhea.

Cholera is a potentially epidemic and life-threatening secretory diarrhea induced by an enterotoxin secreted by Vibrio cholera, characterized by numerous, voluminous watery stools, often accompanied by vomiting, and resulting in hypovolemic shock and acidosis and lethal if the person is left untreated. Although there are many V. cholera serogroups that can produce cholera symptoms, but in 99% of the cases it is found that the most severe symptoms of cholera is caused by O1 serogroup and O139 serogroup.

Diarrhoea due to Cholera is most common among populations inhabiting in areas with poor sanitary condition and inadequate environmental management. Developing and under developed countries are at greater risks. Typical-at-risk areas include peri-urban slums, where basic infrastructure is not available and where minimum requirements of clean water and sanitation are not met.

Factors causing diarrhoea and cholera and other enteric infections are currently impossible to control, and in this context, vaccines have an important role to play. In the last few years attempts have been made to develop different types of Oral Cholera Vaccines (hereinafter ‘OCVs’), derived from either whole Vibrio cholerae inactivated cells or from live attenuated ones. World Health Organisation (WHO) recommends the use of OCVs for prevention of cholera epidemics.

There are number of patents and non-patents disclosures dealing with the subject matter. Non-patent disclosure in the articles, Stertman, L. 2004, titled as ‘Starch Microparticles as an Oral Vaccine Adjuvant with emphasis on the Differentiation of the Immune response’ dissertation from the Faculty of Pharmacy 317.56 pp Uppsala. ISBN 91-554-6101-8 discloses that the oral activity of rCTB can be improved by enteric coating the particles which could survive the transit through gastrointestinal tract but they do not suggest the synergistic activity of enteric coated rCTB and whole cell of V. cholerae bacteria. Another non-patent titled ‘Travel Medicine and Infectious Disease’ Volume 11, Issue 2, Pages 103–109, March–April, 2013 disclosing the effect of enteric coating of whole cell. The experimental data on animal studies suggest that the coating process of tablets do not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets. Thus the non-patent literature teaches away from the present invention.

In view of the above prior art, there is a need to formulate effective, acceptable, easy to administer and low cost OCVs to deal with the life threatening diarrhoea due to cholera and ETEC. One approach has been the development of oral killed whole cell vaccines. A licensed OCV vaccine is sold under the name of Shanchol™ (Sanofi Shantha) which comprises of whole cell inactivated Vibrio cholerae strains only and is administered as a liquid dosage form. Due to the absence of rCTB component it lacks the benefits such as short term ETEC cross protection, synergistic protective effect (anti-bacterial+anti toxic immunity), early and long term protection etc.
Dukoral® is a combination of rCTB and whole cell inactivated Vibrio cholerae which demonstrated high efficacy against cholera and also a significant efficacy against ETEC-induced diarrhoea. It comprises 3 different V. cholerae strains in four different formulations (two heat-killed and two formalin-killed) and in addition recombinantly produced cholera toxin B subunit (rCTB). The rCTB component contributes significantly to the efficacy against cholera and is solely responsible for the observed protection against ETEC diarrhoea due to its ability to induce cross-neutralizing antibodies against the cholera toxin (CT)-like E. coli heat-labile toxin (LT). However, rCTB is acid-labile so the vaccine has to be administered with 75-150mL of bicarbonate buffer prior to the administration of vaccine which makes the administration logistically difficult for children below 5 years of age and even for adults in epidemic settings where availability of clean drinking water is compromised.
For buffer preparation before administration of vaccine, sachet has to be opened and the contents have to be dissolved in requisite amount of water following addition and mixing of the vaccine from the glass vial. This additional dose preparation steps makes the vaccine inconvenient and cumbersome to use.
A real contribution in this field would be to formulate an acid stabilized OCV which elicits the benefits of both whole cell inactivated V. cholerae and rCTB. Thus formulated vaccine will have advantages with respect to ease of administration, logistic viability and affordability.

OBJECT OF THE INVENTION:
The main object of the present invention is to provide process for formulating acid stabilized oral bacterial vaccine.
Another object of the invention is to provide a process for formulating acid stabilized oral bacterial vaccine, more particularly oral cholera vaccine comprising of whole cell and acid stabilized rCTB.
Yet another object of the present invention is to provide an acid stabilized rCTB achieved by coating process.
Yet another object of the invention is to provide a novel dosage form with acceptable mode of administration.
Yet another object of the invention is to provide low cost oral cholera vaccine rendering improved and synergistic effects.
Yet another object of the invention is to provide an efficacious vaccine against cholera, ETEC diarrhoea and with simplified formulation which ideally produces protective immunity.
SUMMARY OF THE INVENTION:
Accordingly, the present invention relates to a process for formulating acid stabilized oral bacterial vaccine. The acid labile rCTB is enteric coated with a protective acid resistant polymer that dissolves and releases rCTB specifically in less acidic regions of the gastrointestinal tract (small intestine). This helps allowing the release of rCTB in the small intestine while protecting from the stomach acid environment irrespective of fed or fasting state. The method involves steps of rCTB layering on inert sugar pellet followed by seal coating and finally a coating of an enteric polymer over the rCTB to give gastric acid protection.
The present invention provides multiple ways of pharmaceutical presentation of said formulation depending on the target patient population. The said pharmaceutical presentation includes enteric coating of rCTB administrable after suspending in pre-determined volume of whole cell inactivated V. cholerae. The formulation is packaged and marketed in a frangible seal pouch or in a two compartment packing or packaged as an oral syringe or in the form of solid dosage form in blister/strip packing, for travellers or for patients above 5 years of age.
Therefore, the present invention describes a method for formulation of an Oral Cholera Vaccine which has improved acceptability, reduced dosage requirement, reduced footprint, low cost of manufacturing, enhanced stability, administration independent of gastric conditions, hence, highly cost effective vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: 1(A) SDS-PAGE of rCTB after rCTB coating and seal coating.
1(B) SDS-PAGE of rCTB after 40% and 50% enteric coating.
1(C) SDS-PAGE of rCTB after 60% enteric coating.
Figure 2: Amount of rCTB at different steps of coating.

DETAILED DESCRIPTION OF THE INVENTION:
In order to obviate the aforementioned drawbacks in the existing prior art, the present invention relates to a process for formulating acid stabilized oral bacterial vaccine. More particularly, the present invention relates to an oral cholera vaccine comprising of Vibrio cholerae whole cell and acid stabilized rCTB.
The whole cell is inactivated Vibrio cholerae bacteria of Hikojima MS1568 strain. This strain is obtained from Gotovax AB, Sweden.
Accordingly, the present invention provides an acid stabilized rCTB which is achieved by coating process including seal coating and enteric coating.
The said pharmaceutical presentation includes, but not limited to, enteric coating of rCTB administrable after suspending in pre-determined volume of whole cell inactivated V. cholerae, enteric coating of whole cell and rCTB together administrable with water, enteric coating of whole cell and rCTB together and compressed into tablets, enteric coating of rCTB compressed into tablets and coating of whole cell over compressed tablets.
Enteric coating is a polymer barrier coating performed on to drug/vaccines taken orally to prevent drug’s degradation by stomach’s acidic pH. The enteric polymer has the property of being stable at acidic pH (pH of around 3-4) mainly found in stomach and thereafter breaks down/dissolve in alkaline pH (pH 7-9) found in small intestine. This property of enteric coating enables to prevent the degradation of drug or vaccine from stomach’s acidic pH and allow maximum absorption of the drug in small intestine. Said maximum absorption is achieved irrespective of fed or fasting state. This whole process of coating involves three below-mentioned steps and is performed using Fluid bed Technology on a fluid bed/Glatt/Wurster coating machine.
Step 1: rCTB layering
The inert sugar pellets are subjected to first step of coating. Said process of coating is performed by layering a solution of rCTB and binding agent such as but not limited to Opadry 06A390050 (colorcon) over the sugar pellets. Dose relevant rCTB is layered onto inert sugar pellets using binder in appropriate concentrations. More specifically, said solution is in the concentration range of 2% w/w ± 1 wherein the ratio of concentration of rCTB to binding agent is 1:2.5 ± 0.5. The resultant rCTB layered sugar pellets have 200-300 micron size. More specifically, 1mg-2mg rCTB is layered on 150 mg-300mg of sugar pellets.
Step 2: Seal Coating:
A protective layer of a seal coating polymer is applied to smoothen the surface of the rCTB layered sugar pellet and to protect the acid sensitive rCTB from the acidic enteric coating polymer. Another solution of binding agent with requisite concentration more preferably, a solution of Opadry 03K19229 is prepared in a concentration range of 2% to 6%.
Thus, the seal coated pellet now proceeds for final step of coating.
Step 3: Enteric coating:
A final functional coating of enteric polymer is applied over the seal coated pellets. Enteric coating is performed by a solution of enteric polymers and plasticizer. An appropriate concentration of said solution of enteric polymers and plasticizers is coated. More preferably a solution of Eudragit L-30D 55 along with plasacryl plasticer HTP20 in a concentration range of 20% ± 2% is used. The said process of enteric coating resulted in enteric coated pellets.
At each step of coating enteric coated pellets are analysed quantitatively by SDS-PAGE as shown in figure 1.
The entire process resulted in 20 % - 30% loss of rCTB.
The level of acid protection is also checked by performing a dissolution test by suspending rCTB enteric coated pellets in 5ml of 0.1NHCl maintained at pH 3-4 and 5 ml of Phosphate buffer (PBS) maintained at pH 6.8-7.4. Samples of the dissolution media are checked at frequent intervals to determine the level of acid protection provided by different coatings.
The quantitatively analysed enteric coated pellets can be presented with the whole cell inactivated V. cholerae in the many ways and not limited to those as mentioned below:
1. Enteric coating of rCTB, administration after suspension in 2-5 ml of whole cell inactivated V. cholerae.
2. Enteric coating of whole cell and rCTB together, administration with water
3. Enteric coating of whole cell and rCTB together, compressed into tablets
4. Enteric coating of rCTB compressed into tablets and further coating of whole cells over the compressed tablets.
Thus the above said ways/options of presenting the acid stabilised oral vaccine are further packaged. The packaging can be carried in multiple ways including
• Packaging in a frangible seal pouch or a two compartment packaging and then administered using oral syringe
• Packaging in the form of the tablet in blister/strip packaging.

The enteric coated formulation so obtained has low administrative volumes and will have low manufacturing cost. The administration of said oral vaccine is independent of gastric conditions (fed or fasted) and has improved acceptability.
The above detailed description of process is illustrated by non-limiting examples:

EXAMPLES:
EXAMPLE 1: Enteric coating of recombinant Cholera toxin
Enteric coating is a polymer barrier applied on drugs/vaccines taken orally. This polymer barrier is stable at the highly acidic pH (3-4) found in the stomach but breaks down in the alkaline (pH 7-9) environment present in the small intestine. rCTB was entericaly coated with Eudragit L-30D 55 polymer to keep it stable in the stomach as it is to be administered orally along with the whole cell inactivated V.cholerae vaccine. Enteric coating was done in three steps: rCTB layering, seal coating and Enteric coating by polymer. The process parameters used for the three steps of enteric coating are listed in Table1.
Table 1 List of process parameters used for enteric coating
rCTB Coating
Seal Coating Enteric Coating
Inlet Temperature 42-44°C 42-44°C 30-32°C
Product Temperature 35°C 35°C 28°C
Atomized Air (bar) 0.8 bar 0.8 bar 0.8-0.9 bar
Column Height (mm) 15 15 15
Spray Rate (gm/min) 0.7 – 1.2 0.5 1.5
Spray Rate (RPM) 2-3 2 4

For rCTB layering, a coating solution 2% w/w was made by mixing rCTB and binder Opadry 06A390050 (Colorcon) in 1:2.5 ratio and layered on 250-300 micron sugar pellets so that 1mg of rCTB was layered on 150mg of sugar pellets. Seal coating is done by making a 4% coating solution containing Opadry 03K19229 (5% the weight of pellets, rCTB and binder).
Enteric coating is done by a 20% solution containing polymer Eudragit L-30D 55 and plastisizer Plasacryl HTP20 (60% the weight of pellets, rCTB, binder and seal coat). At each step SDS-PAGE of the samples are run as shown in Fig. 1. The samples are withdrawn at different coating weight gains and sent for rCTB ELISA, the results of which are shown in Fig. 2. One dose of vaccine has 150mg of sugar pellets coated with 1mg of rCTB. It is observed that each 150mg of sugar pellets is coated with 1mg of rCTB as is clearly evident from the ELISA at different percentage of coating. There was an overall 20%-30% loss of rCTB in the entire process.
The levels of acid protection is also checked by performing a dissolution test by suspending rCTB coated granules in 5ml of 0.1NHCl (pH 3-4) and 5ml PBS (pH 6-8-7.4) and mixed for 15-30 mins. In between, samples are withdrawn to check for rCTB release.
The dissolution test showed that the enteric coated rCTB could withstand acidic pH in the stomach and is released within minutes at alkaline pH.
,CLAIMS:We Claim:
1. A process for formulating an acid stabilized oral bacterial vaccine wherein said process comprises the steps of :
- Coating of a purified recombinant cholera toxin (rCTB) to obtain an acid stabilized rCTB;
- Combining said acid stabilized rCTB with a inactivated whole cell of Vibrio Cholerae
wherein said process provides said vaccine having synergistic protection against diarrhoea caused by various bacteria such as Vibrio cholera and Enterotoxigenic Escherichia coli (ETEC).
2. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 1 wherein said coating of said purified recombinant cholera toxin comprising of three steps using Fluidized bed Wuster coater:
a. rCTB layering at predetermined conditions;
b. seal coating at predetermined conditions; and
c. enteric coating at predetermined conditions.
3. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2 wherein said rCTB layering consists of a rCTB layering solution of 2% ± 1 w/w of a rCTB and a binding agent in a ratio of 1:2.5 ± 0.5 ratio; wherein said binding agent is a Opadry 06A390050 colorcon.
4. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 3 wherein said rCTB layering solution is layered on a sugar pellet such that 1 mg of said rCTB is layered on 150 mg of said sugar pellet.
5. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2 wherein said rCTB layering is performed at predetermined conditions of:
Process Parameter Value
Inlet Temperature 42-44°C
Product Temperature 35°C
Atomized Air 0.8 bar
Column Height 15mm
Spray Rate 0.7-1.2 (gm/min)
Spray Rate 2-3 (rpm).

6. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2 wherein said seal coating consist of a seal coating solution of 4% of a binding agent, preferably Opadry 03K19229.

7. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2 wherein said seal coating is performed at predetermined conditions of:
Process Parameter Value
Inlet Temperature 42-44°C
Product Temperature 35°C
Atomized Air 0.8 bar
Column Height 15mm
Spray Rate 0.5 (gm/min)
Spray Rate 2 (rpm).

8. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2 wherein said enteric coating consist of a enteric coating solution of a enteric polymer and a plasticizer;
wherein said enteric polymer is a Eudragit L-30D 55 and said plasticizer is a Plasacryl HTP20.

9. The process for formulating the acid stabilized oral bacterial vaccine as claimed in claim 2, said enteric coating is performed at predetermined conditions of:
Process Parameter Value
Inlet Temperature 30-32°C
Product Temperature 28°C
Atomized Air 0.8-0.9 bar
Column Height 15mm
Spray Rate 1.5 (gm/min)
Spray Rate 4 (rpm).

10. The process for formulating the acid stabilized oral bacterial vaccine wherein said vaccine comprises of a whole cell inactivated V.cholerae and an acid stabilised recombinant cholera toxin B (rCTB).