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Team:Evry/Pill design
From 2013.igem.org
Capsule design
Abstract
Our engineered bacteria need to be delivered to the distal area of the duodenum and the proximal area of the jejunum in order to treat iron adsorption. We thus designed a polymer-based capsule that resists the low pH conditions in the stomach, but will dissolve at intestinal pH. We also improved its galenic formulation to optimize growth.
First, we create a novel capsule following the actual norms from the European Pharmacopeoa concerning gastro-enteric resistant formulations.
Second, we ensure that the bacteria survive following dissolution of the capsule.
Posology
The patient must receive a controlled dose of our engineered bacteria for them to function as as an effective medical treatment. This requires that the capsule be eaten on an empty stomach. In addition, the bacteria must also have enought time to grow once delivered in the duodenum and jejunum. Our strategy focuses on preparing the duodenum of the patient for the next meal and let the bacteria sufficient time to settle and produce sufficient chelators to reduce the iron absorption.
Capsule design requirements
We see three main challenges. First, the capsule must resist the low pH conditions in the stomach, which are lethal to our bacteria. Second, the capsule must rapidly dissolve in the duodenum to deliver its payload to the distal duodenum and the proximal jejunum. Third, the bacteria must have enough time to effectively uptake iron. We thus added HPMC (Hydroxypropylmethylcellulose) to the capsule interior. Once the capsule dissolves, the HPMC expands upon contact with water to slow nutrient movement in the intestine. Pharmaceutical research has evaluated HPMC to have the right viscosity to allow bacterial growth while also permitting the passage of food.
Capsule design, step by step
Which galenic formulation is best suited for our goals?
The first step in the design of our pill is to determine its galenic formulation. We want a per os administration for our bacteria and had the choice between either a tablet or a capsule. A tablet requires a heavy and dry compression and the bacteria in a lyophilized form, entailling high temperatures and pressures that would result in significant bacterial mortality. We decided a a capsule is more suited for our purpose because it offers the possibility to contain a non-compressed powder and avoids a lyophilization step, thus representing a more favorable environment for bacteria storage. In addition, a capsule avoids lyophilizing the bacteria, which would delay their metabolic activity after being released.
How to store the bacteria in the capsule?
The design of a capsule requires every component to be in a powder form. Thus, we tested which one was able to absorb the most LB medium saturated with bacteria. In galenic research, this is called a moisture absorbent and has the right chemical properties to keep our drug (here our bacteria in LB medium) in a dry environment. We experimented
For the next step, we want to increase the volume to 35 mL which is the sufficient amount to make 50 capsules (standard for one rack, figure 3). Thus, a diluent is required and we opted for HPMC (hydroxypropylmethylcellulose). The entire volume of powder has to be equally distributed in the capsules (figure 4).
Finally, the 50 capsules contain all together 11 ml of colloidal silica (where 4 mL of saturated bacteria have been dissolved in) and 24 mL of HPMC. The last step is the closing of the capsules (figure 5 and 6).
How to overcome the acidity of the stomach?
It is very common in the pharmaceutical world, and more precisely in galenical research, to overcome the acidity of the stomach and to target the duodenum/jejunum for the delivery of the capsule. By a simple saoking of one side of the capsule in an ethanol-based solution of methacrylic acid and followed by a drying process (hot air), we managed to surround our capsule with a double-enveloppe resistant that confers resistance to gastric acidity. The capsule has to resist as long as possible to the gastric content, but not to much otherwise we miss its dissolution in the duodenum.
How to deliver the bacteria in the jejunum?
The delivery in the duodenum/jejunum is already possible due to the gelatine-based composition of the capsule. In contact to water, the capsule dissolves and delivers its containment in the duodenum. This is only feasable when the capsule has already resisted to the gastric acidity. Moreover, we added a HPMC as a diulent. In the presence of water, right after the dissolution of the gelatine capsule, it is able to swell. Depending on its density, the polymere forms a more or less viscuous obstruction. This process creates an environment in the jejunum where bacteria can statically proliferate. Thus, HPMC, beside its properties as a diluent, is also called a bio-adhesive for its ability to stick to the membranes of the intestins and form an obstruction.
How to prove the real efficiency of our capsule?
As a proof of concept, we fulfilled the two basic requirements of the European Pharmacopeia to make a gastro-enteric resistant capsule which are as follows:
- No dissolution of the capsule after 2 hours of exposure to gastric acid (solution at pH = 2)
- Dissolution of the capsule within 1 hour of exposure to water (Phosphate Buffer solution, pH = 7) right afterwards
The dissolution machine is able to lift a basket up and down into a 800mL beaker (figure 9). For every experience, we were able to test 6 capsules spread into 6 apart columns (figure 10). Keep in mind that these columns have the possibility to be closed by a lid on the top to increase the dissolution and mimic different segments of the intestins.
We observe that after one hour of exposure to gastric acidity, the capsules are not dissolved. They kept their integrity (figure 11) and didn't deliver its contanment (figure 12).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that the water is very clear (figure 15), thus meaning that the first condition of the European Pharmacopeoa to create a gastro-enteric resistant capsule is fullfilled.
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
We observe that after two hours of exposure to gastric acidity, the capsule are still not dissolved. They kept their integrity (figure 13) and didn't deliver its contanment (figure 14).
