Introduction

We have developed Gassle, a strain of E. coli that can be used for testing Inflammatory Bowel Disease (IBD). One of the major challenges for practical application is delivering this Gassle strain to the colon while keeping it alive. This is a consideration not only for supplements and other health products but also for many bacteria, such as lactic acid bacteria, bifidobacteria, and E. coli. When administered orally, a large portion of these bacteria are destroyed by various factors, including stomach acid and bile acids.[1]

To prevent this and deliver more live Gassle to the colon, we are working on developing a capsule called "Colon-Y."

Colon-Y

Colon-Y consists of three layers surrounding a liquid medium containing E. coli: the Phase Master Coat (PMC), Colon Disintegrating Coat (CDC), and Enteric Coat (EC).

For detailed information on the creation methods and composition of these layers, please refer to the Experiments section.

Explanation of 3 Coating Layers

Phase Master Coat (PMC)

Colon Disintegrating Coat (CDC)

Entric Coat (EC)

What makes up each layer?

The completed Colon-Y

(Diameter: 13.0 mm, Fillable Volume: 65 μL)

Colon-Y Capsule Image

What Makes Colon-Y Amazing!

A "liquid-filled capsule" that is solid at room temperature and liquid at body temperature

In this capsule, we use a mixture of gelatin, agar, and sugar for the PMC (Phase Master Coat).

The melting point of gelatin varies depending on its concentration and quality, but it is generally around 30°C. What we focused on is that this melting point is higher than room temperature yet lower than body temperature.

By applying an additional coating on the outside, we created a very unique liquid-filled capsule that undergoes a phase transition and becomes liquid only inside the body. During manufacturing, sales, and storage, the capsule remains in a solid state with high strength. However, once inside the body, it quickly melts and turns into a liquid.

For Gassle, as mentioned earlier, it is desirable that the PMC remains solid at room temperature for strength and coating purposes. However, a solid spherical medium presents the following issues:

If the medium is liquid, then as soon as part of the CDC or EC is broken, the liquid PMC and E. coli inside will quickly be released. This was confirmed through The Disintegration Testing.

Thus, the issue is resolved by the PMC becoming liquid inside the body.

Furthermore, by varying the composition of the agar in the PMC and adjusting the drying time, we confirmed through Melting Test that the time until it starts melting at body temperature can be significantly controlled.

Although liquid-filled capsules are versatile in many ways, strength is a concern. In the case of our Gassle, the PMC turning into liquid in the stomach or small intestine, where release is not intended, would only result in a loss of strength, which is undesirable. However, as previously mentioned, by adjusting the composition of the agar and drying time, we enabled time-controlled release, ensuring the PMC remains a strong solid while passing through the stomach and becomes liquid only upon reaching the colon.

A capsule that can transport live bacteria

This allows the capsule to retain a sufficient number of viable bacteria upon release, ensuring the immediate efficacy and sensitivity of the diagnostic tool.

Generally, in functional foods using bifidobacteria, the bacteria are freeze-dried for transport. However, since we found few examples of freeze-drying in relation to E. coli, we referred to data on bifidobacteria.

To solve this problem, we considered incorporating a growth medium inside the capsule to allow the bacteria to proliferate within the capsule. Theoretically, this would prevent the bacterial count from decreasing after capsule production and even allow it to increase to a maximum.

A major reference for this approach was the "Bifina Bio" bio-capsule developed by Morishita Jintan Co., Ltd., a company known for producing many healthcare products in Japan. A key feature of their bio-capsule is that, as shown in Figure 1, the capsule is immersed in a nutrient medium.

Bio-Capsulation Using a Nutrient Medium
Figure 1) Bio-Capsulation Using a Nutrient Medium

Following this example, we also designed our Colon-Y capsule by using gelatin as the base material and encapsulating a liquid medium inside. The gelatin contains sugar as a nutrient, aiming to sustain live bacteria similar to Bifina Bio, and function as a bio-capsule where the capsule’s interior acts as a micro-culture chamber.

In fact, after First Viable Bacteria Count Test, we confirmed that by using a nutrient-rich PMC to hold the liquid medium, E. coli could be cultured within the capsule.

For our Gassle system, the presence of an abnormal odor, distinctly different from normal flatulence, serves as the standard for detection. When the capsule disintegrates, it can release the maximum number of viable bacteria, and in positive cases, it is expected to produce a stronger, faster abnormal odor than usual.

This innovation promises immediate efficacy and high sensitivity in the diagnostic tool.

A capsule is capable of site-specific release in the colon

Site-specific drug delivery (DDS) to the colon is crucial for enhancing the efficacy and stability of treatments for colon-related diseases and probiotics.

In our Gassle system, propionic acid is released in response to detecting NO (nitric oxide). However, if NO is detected outside the colon, false positives and potential side effects on the body become a concern. Therefore, the following innovations are necessary to prevent false positives.

The CDC and EC used in this capsule feature sustained-release characteristics.

Some capsules are designed to rupture and release their entire contents upon reaching the colon. However, this carries the risk of side effects and may only deliver the contents to a limited part of the colon. While this approach has some benefits, it poses significant drawbacks from a diagnostic perspective.

By employing sustained-release technology, E. coli can be evenly delivered throughout the colon. Even if only a small amount is initially released, the bacteria can multiply within the body and eventually detect inflammation throughout the entire colon. In other words, this innovation helps prevent false negatives.

Potential Applications

While many of these individual features have already been implemented in practical use, the novelty lies in combining them. Below are some potential applications where this novelty could be leveraged.

Leveraging the Phase Transition Feature

Expanding the Potential of Advanced Genetic Engineering Applications

Preventing Side Effects

Controlling the Time and Location of Release

Development Process and Performance Evaluation

From the prototype to the current capsule

We have been exploring the optimal materials and methods to create the capsule we envision, aiming for better efficiency and precision in production.

Enhancing the reproducibility of the manufacturing process is crucial to improving the reliability of performance evaluations. Additionally, iterating improvements using cost-effective and safe materials to enable mass production is essential as we aim for real-world implementation.

Prototype Creation

Evolution to Colon-Y

Melting Test

Reasons for Not Using the More Common Material, Chitosan

Coatings for colon-specific disintegration have already been studied and implemented. One of the most well-known materials is chitosan, which is used in chitosan-coated products [13].

However, HPMCP is now used instead of chitosan.

What happened?

Chitosan dissolves in acidic environments and reacts with polyanions to form a gel membrane. This property makes it suitable for encapsulating enzymes, microorganisms, and drugs, protecting them from the effects of stomach acid while enabling release in specific locations such as the colon. Its membrane permeability can be controlled, making it ideal for delivering E. coli to the colon while protecting it from stomach acid. It can also be designed to disintegrate under specific pH conditions, making it suitable for DDS (drug delivery systems) [14].

To explore whether chitosan coating would be optimal for our project, we consulted with experts from Aicello Chemical Co., Ltd. and Nissin Pharma Inc. We received extensive feedback, which can be found on the IHP page.

Through these discussions, we identified three major issues that led us to conclude that chitosan was not suitable for our approach of transporting live bacteria in a liquid medium:

  • The temperature required for drying the chitosan coating would kill the E. coli inside.
  • At the temperature necessary for drying the chitosan coating, the PMC would liquefy, making proper coating difficult.
  • Chitosan requires an acidic solvent, and the conditions for evaporating the acid are harsh, making production difficult and leading to the death of the E. coli inside.

While seeking an alternative material that could resolve these issues without losing the benefits of chitosan, we discovered HPMCP.

Although chitosan coatings leverage the biodegradability of gut microorganisms for colon-specific disintegration [15], HPMCP dissolves in response to the pH of the colon, which makes it less specific in targeting the disintegration location [16].

However, HPMCP offers similar advantages to chitosan, such as low toxicity, biodegradability, and moisture resistance, with the added benefit of very mild drying conditions. In our experiments, we confirmed that HPMCP dried completely after being left at room temperature for just 5 minutes.

Although HPMCP is not widely used, combining it with pH-sensitive polymers or water-insoluble polymers could potentially achieve a controlled, precise sustained-release profile [17], [18].

Focusing on these advantages, we decided to experiment with HPMCP.

Reasons for Not Using the More Common Material, Chitosan

Coatings for colon-specific disintegration have already been studied and implemented. One of the most well-known materials is chitosan, which is used in chitosan-coated products [13].

However, HPMCP is now used instead of chitosan.

What happened?

Chitosan dissolves in acidic environments and reacts with polyanions to form a gel membrane. This property makes it suitable for encapsulating enzymes, microorganisms, and drugs, protecting them from the effects of stomach acid while enabling release in specific locations such as the colon. Its membrane permeability can be controlled, making it ideal for delivering E. coli to the colon while protecting it from stomach acid. It can also be designed to disintegrate under specific pH conditions, making it suitable for DDS (drug delivery systems) [14].

To explore whether chitosan coating would be optimal for our project, we consulted with experts from Aicello Chemical Co., Ltd. and Nissin Pharma Inc. We received extensive feedback, which can be found on the IHP page.

Through these discussions, we identified three major issues that led us to conclude that chitosan was not suitable for our approach of transporting live bacteria in a liquid medium:

  • The temperature required for drying the chitosan coating would kill the E. coli inside.
  • At the temperature necessary for drying the chitosan coating, the PMC would liquefy, making proper coating difficult.
  • Chitosan requires an acidic solvent, and the conditions for evaporating the acid are harsh, making production difficult and leading to the death of the E. coli inside.

While seeking an alternative material that could resolve these issues without losing the benefits of chitosan, we discovered HPMCP.

Although chitosan coatings leverage the biodegradability of gut microorganisms for colon-specific disintegration [15], HPMCP dissolves in response to the pH of the colon, which makes it less specific in targeting the disintegration location [16].

However, HPMCP offers similar advantages to chitosan, such as low toxicity, biodegradability, and moisture resistance, with the added benefit of very mild drying conditions. In our experiments, we confirmed that HPMCP dried completely after being left at room temperature for just 5 minutes.

Although HPMCP is not widely used, combining it with pH-sensitive polymers or water-insoluble polymers could potentially achieve a controlled, precise sustained-release profile [17], [18].

Focusing on these advantages, we decided to experiment with HPMCP.

Viable Bacteria Count Test

We are evaluating two key aspects:

  • Whether the gelatin can function as a growth medium for E. coli.
  • Whether the outer coatings (CDC and EC) can protect the E. coli inside from stomach acid.

This is a crucial test to confirm the significance of the three coatings.

First Viable Bacteria Count Test

To evaluate the impact of the coatings on the E. coli inside, we conducted a viable bacteria count test under conditions that simulate passage through the stomach. We also measured samples without coating and those that were not exposed to stomach acid.

The test focused on two key aspects:

  • Whether the PMC can act as a growth medium for E. coli.
  • Whether the CDC and EC can protect the E. coli from stomach acid.

This is a crucial test to confirm the significance of the three coatings.

The test was performed under the following conditions:

  • Temperature: 25°C
  • Shaking Duration: 3 hours

Sample1… CDC, EC (-)、Acid Exposure (-)

Sample2... CDC, EC (-)、Acid Exposure(+)

Sample3... CDC, EC (+)、Acid Exposure (+)

To assess whether the PMC can protect the E. coli inside and act as a growth medium, we would typically compare the initial viable bacteria count with that of Sample 1 (PMC only). However, due to a lack of equipment, we were unable to measure the initial bacteria count in this test.

To evaluate the impact of acid exposure and determine whether protection from acid is necessary, we compared Sample 1 (no acid exposure) with Sample 2 (acid exposure).

Finally, to confirm whether the complete coating (CDC and EC) effectively protects the E. coli from acid, we compared Sample 2 (no coating, acid exposure) with Sample 3 (fully coated, acid exposure).

Result

Figure.8) Result

The results of the viable bacteria count test are shown in Figure 9. The test revealed that acid exposure led to a reduction in the number of live bacteria. This suggests the need for an acid-resistant coating to protect the bacteria from stomach acid.

Average Number of Colonies

Figure.9) Average Number of Colonies

The results highlight the following issues:

  • The inability to confirm that the PMC effectively suppressed the reduction in viable bacteria.
  • Due to a shortage of testing chips, we were unable to measure the number of viable bacteria at the time of encapsulation, so a comparison with Sample 1 could not be made. A second test needs to be conducted to measure the viable bacteria count at the time of encapsulation and to allow for proper comparison.
  • The loss of the liquid medium solvent.
  • The number of colonies in Sample 2 (which had no coating) was higher than in Sample 3 (which had a coating), suggesting that the coating had a negative effect. However, since the liquid medium in this experiment had evaporated, it was not possible to perform a quantitative recovery. As a result, the standard error was significantly larger, making the data unreliable.

To Obtain Reliable Capsule Evaluation Data

In our Gassle project with the Colon-Y capsule, the liquid medium inside will mix with the dissolved PMC once inside the body. Due to the thick coating applied, the internal moisture does not escape. Since gelatin gel has excellent water permeability, the moisture in the PMC is expected to distribute evenly, which means we do not need to worry about dehydration killing the internal E. coli.

Therefore, apart from the fact that viable bacteria count tests do not accurately evaluate capsule performance, the loss of the liquid medium solvent is not a major issue.

However, considering the following possibilities, we attempted improvements:

  • Long-term storage might negatively affect the product's shelf life.
  • In fields such as biotank or biotechnology research, or the development of fermented foods, this could become a significant issue.
  • If this capsule were applied to other fields where such thick coatings are not used, dehydration could result in bacterial function deterioration or death.

The likely cause of the liquid medium solvent loss is the osmotic pressure effect caused by mixing sugar and agar into the gelatin [19].

To address this, we experimented with incorporating a layer of sodium alginate or an oil lid between the PMC and the liquid medium.

Improvement Process

Sodium Alginate Layer

We utilized the membrane-forming ability of sodium alginate, which gels upon contact with divalent cations, a material we had used in our early prototypes. Since no melting temperature or drying is required, it is easier to manufacture and has minimal impact on the E. coli in the liquid medium. We used calcium lactate as the gelling agent for sodium alginate in the PMC.

After using calcium lactate, we observed (albeit subjectively) that the viscosity of the PMC solution and the hardness of the gel after drying had increased. We discovered that this was due to the addition of calcium ions to the gelatin, which also had the effect of raising the melting point [20]. Therefore, by incorporating calcium lactate as a variable, we believe that the melting time of the PMC can be further adjusted. However, due to time constraints, we could not measure these effects in the current test, so obtaining this data remains a task for future studies.

How to insert a layer of sodium alginate

Figure.10) How to insert a layer of sodium alginate

First, before pouring the PMC solution into the mold, calcium lactate is added to the PMC solution in advance, and sodium alginate is added to the liquid medium until saturation is reached. When the liquid medium is then poured into the molded PMC, a sodium alginate membrane forms between them.

After drying the PMC made this way and leaving it for 5 hours, cutting it open revealed that the alginate layer had formed successfully, and the solvent was still present inside the cavity, as confirmed visually (Figure 11).

The presence of a sodium alginate layer and moisture was confirmed

Figure.11) The presence of a sodium alginate layer and moisture was confirmed

After 2 hours, the PMC had absorbed the solvent and turned into a soft gel. By this point, the liquid medium had already been absorbed, and as shown in Figure 12, the hemispherical part, which acted as a lid, had bulged upward, filling the cavity.

PMC with protrusions due to absorption of Liquid Medium

Figure.12) PMC with protrusions due to absorption of Liquid Medium

The expansion of the PMC suggested that the sodium alginate membrane itself was permeable to water. Upon further investigation, we found that alginate gels, especially when crosslinked with calcium ions, exhibit strong hydrophilicity and possess a structure that allows water permeability and the diffusion of water-soluble substances [21].

Further research revealed that the water permeability of the gelatin-sodium alginate hydrogel, which caused issues in our test, has been applied in various fields [22][23][24].

Thus, exploring applications of sodium alginate-containing PMC remains one of the key challenges for future development.

Oil lid

As the next attempt, an oil layer was incorporated.

Before adding the liquid medium, a brush was used to apply oil to the cavity and the lid, in an attempt to physically prevent the absorption of the solvent.

This method allowed the liquid medium to be retained for about 7 hours, with only slight bulging occurring. However, after 10 hours, it took on the same shape as seen in Figure 12.

The likely cause of the solvent absorption was the application of melted PMC to the edges of the hemispheres to bond them together after the oil had been applied. Since the oil layer was incomplete, solvent diffusion likely occurred through this gap.

Inadequate oil lid

Figure.13) Inadequate oil lid

The adhesive used to join the hemispheres could be applied around the exterior after the two halves are joined, without applying it directly to the edges. This approach could prevent the solvent absorption issue, but due to time constraints, we were unable to implement it in this round.

The results are shown in Table 2. Both the sodium alginate layer barrier and the oil lid successfully reduced solvent absorption.

Currently, the main issue lies with the viable bacteria count test, which takes approximately 8 hours from the injection of the liquid medium to coating and shaking at 37°C. Therefore, we planned to incorporate the oil lid method and attempted the viable bacteria count test once again.

Table 2 Result

Table.2) Result

Second Viable Bacteria Count Test

We conducted a second viable bacteria count test, addressing the following two issues identified in the first test:

  • We were unable to obtain data showing that the PMC could suppress the reduction in viable bacteria.
  • The liquid medium solvent had evaporated.

First, we successfully measured the initial viable bacteria count, which we were unable to do in the first test.

Then, we incorporated the oil lid method and conducted the viable bacteria count test again. However, when attempting to retrieve the liquid medium from the capsule, no visible liquid medium remained. Despite this, the capsule appeared more moist compared to the previous test, indicating some improvement.

Condition of the Capsule after Destruction

Figure 14) Condition of the Capsule after Destruction

Samples are as follows:

Sample1… CDC, EC (+)、Acid Exposure (+)

Sample2... CDC, EC (-)、Acid Exposure(+)

Sample3... CDC, EC (-)、Acid Exposure (-)

Initial Value

Result

Figure.15) Result

The results of the viable bacteria count test are shown in Figure 16.

Average Number of Colonies

Figure.16) Average Number of Colonies

Compared to the initial value, it was confirmed that the number of viable bacteria in Sample 3 increased, as indicated by the light blue section. This suggests that culturing is possible within the PMC.

The increase in viable bacteria in Sample 2 compared to the initial value further supports the conclusion that culturing is possible within the PMC.

However, the viable bacteria count in Sample 2 was lower than in Sample 3, as shown by the blue section. This suggests that while culturing occurs inside the PMC, hydrogen ions (H+), with their small ionic radius and high permeability, passed through the gelatin-based PMC and reached some of the E. coli inside.

The significant increase in viable bacteria in Sample 1 compared to Sample 2 strongly supports the necessity of the CDC and EC coatings.

However, if the coatings were simply preventing H+ from passing through, the results for Sample 3 should have been similar. The unexpectedly higher count of viable bacteria suggests the following:

  • The coating likely helped retain moisture inside, aiding bacterial survival.
  • The sticky nature of the EC may have caused the sample to stick to the tube, physically protecting the E. coli from external damage.

However, due to the small number of samples, the reliability of this data is limited.

The sticky nature of the EC poses a challenge for in vivo applications as well, so further improvements and experiments are needed. Due to time constraints, the viable bacteria count test was concluded here, and we moved on to the disintegration Test to measure the capsule's strength.

The Disintegration Test

The primary concept of Colon-Y is to "deliver live E. coli to the colon," and this test is crucial for verifying the following:

  • The EC and CDC coatings should not dissolve in stomach acid during the 2-3 hours the capsule spends in the stomach.
  • The coatings should begin to dissolve at the pH of the small intestine.

What is The Disintegration Test

The disintegration test examines the timing of how capsules or tablets break down in the body. This ensures that the formulation works as planned inside the body and verifies whether the design is correct.

This test is described in the Japanese Pharmacopoeia (JP), the official standard for medicines in Japan. It is essential for the quality control of all medications and for ensuring patient safety. Passing the disintegration test increases the reliability of the product, moving it closer to approval and commercial sale [25].

Furthermore, the disintegration test adheres to international standards such as the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP). This allows the quality of medicines to be consistently evaluated across countries.

The primary concept of our Colon-Y capsule is to “deliver live E. coli to the colon.” To achieve this, the capsule must meet the following four criteria:

  • The EC and CDC should not dissolve in the stomach's acidic environment (pH ~1.2) for 2-3 hours.
  • The coating should start dissolving at the pH of the small intestine.
  • It should gradually disintegrate over approximately 7 hours as it passes from the small intestine to the colon.
  • The capsule should completely dissolve upon reaching the colon’s pH.

The disintegration test is designed to assess whether Colon-Y meets these criteria.

Verification of Criterion 1:

Using the first disintegration test fluid, which mimics stomach acid at pH 1.2, we performed the test at 37°C while shaking the samples. We observed the samples for 2 hours, and since none of them disintegrated, it was confirmed that the capsules would not break down in the stomach.

Verification of Criterion 2:

Using the second disintegration test fluid, which mimics the small intestine environment at pH 6.8, we performed the same procedure as with the first fluid for 1 hour.

Similar to the first test, we measured durability. The goal was to confirm that the EC dissolves due to the pH change when transitioning from the stomach to the small intestine, ensuring that it would not interfere with the release of E. coli upon reaching the colon.

As shown in Figure 17, the transit time from the small intestine to the colon is approximately 7 hours [26].

Therefore, it would be problematic if the EC were to completely dissolve after just 1 hour in the second fluid.

Gastrointestinal pH profile from a normal subject

Figure 17) Gastrointestinal pH profile from a normal subject. The position of the capsule in specific areas is labelled accordingly.

Thus, after confirming that none of the samples disintegrated during the test, we deem the test to have passed. Additionally, we verify that the EC (Enteric Coat) begins to peel, which is a positive indication of proper functionality, and we evaluate this as part of the performance assessment.

Verification of Criteria 3 and 4:

Due to the insufficient shaking time in the second disintegration test fluid, the test was not adequate to fully evaluate these criteria.

First Disintegration Test

The samples were subjected to the disintegration test, but unfortunately, they all developed holes and broke apart during the first fluid test, causing the disintegration test to fail. The same issue occurred with the second fluid, where the samples also developed holes.

All the capsules had collapsed

Figure.18) All the capsules had collapsed

Since we had recorded the test, upon reviewing the footage, we observed that in both experiments, the liquefied PMC began leaking through holes in the CDC and EC about 20-30 minutes after the test started (as shown in Figure 19).

Changes in the appearance of the First and Second liquid becoming colony

Figure.19) Changes in the appearance of the First and Second liquid becoming colony

From this observation, we can conclude that the PMC properly liquefies at body temperature, and once the outer CDC and EC coatings start to break down, the liquid gradually leaks out.

When comparing the first fluid (simulated gastric acid) to the second fluid (simulated small intestine), the second fluid became cloudy earlier and more intensely. This indicates that the shellac coating in the second fluid was gradually peeling off.

Since significant cloudiness was observed within about an hour of the leakage beginning, we also confirmed that the capsule can function as a sustained-release formulation. In this case, the sustained-release effect was not due to the gradual peeling of the coating but rather due to holes forming in the capsule. However, this suggests that by controlling the size of the holes and adjusting the coating strength, we may be able to develop a formulation that allows controlled sustained release in the stomach.

However, for our Gassle project, this strength issue is critical.

The 3-hour duration of this test represents a time when the capsule would likely still be in the stomach or early small intestine, and disintegration at this point would prevent the safe delivery of E. coli to the colon.

Therefore, we attempted improvements to address the capsule's strength. The weakness observed likely stemmed from shortcomings in our coating technique, so we made improvements and proceeded with the second disintegration test.

For details on the improvements, please refer to Experiments and Results.

Second Disintegration Test

We made improvements to the coating method and increased the number of EC layers by three, resulting in a smooth, spherical sample with an even coating (as shown in Figure 20).

Cleanly Spherical Sample

Figure.20) Cleanly Spherical Sample

As a result, the second disintegration test yielded the results shown in Table 3.

Results of the Second Disintegration Test

Table 3) Results of the Second Disintegration Test

The improved coating on this capsule proved to have sufficient strength.

After the test, we cut the sample in half to observe the state of the PMC, and it exhibited a sticky consistency, as shown in Figure 21.

Gel of PMC

Figure.21) Gel of PMC

After being exposed to body temperature for 1 hour, the PMC had become significantly more viscous, but it remained in a gel state, not a sol. In the first disintegration test, the solution began to turn cloudy after 20-30 minutes. This cloudiness was likely caused not by the PMC turning into a sol, but rather by the test fluid eroding the gel-like PMC inside, leading to the observed cloudiness.

Although time constraints prevented us from doing so in this test, taking samples every hour after exposure to body temperature, cutting them, and observing the PMC would likely confirm when it transitions into a sol. This would also allow us to monitor how the capsule's strength changes as the PMC transitions from a gel to a sol.

Open Issues and Next Steps

Further capacity gains

Current Status: At present, we can only create a cavity on one side of the capsule due to technical limitations. We have not yet been able to form a cavity on the other side.

Future Goal: In the future, we aim to increase the capsule’s capacity by creating cavities on both sides. While currently restricted by technical constraints, we believe that with advancements in machinery and techniques, it will be possible to form cavities on both sides.

Expected Benefits: By creating cavities on both sides, we anticipate the ability to carry more content, which will significantly improve the capsule’s performance. While this would be an exciting development, there is also a concern that it could lead to reduced strength. If the strength is compromised, the durability of the capsule may be an issue, necessitating further testing.

Next Steps: To confirm the capsule's strength, we plan to conduct a disintegration test. If the test is passed, it would be ideal to move forward with the production of capsules featuring cavities on both sides.

Considerations: However, reducing the PMC could decrease the total moisture content inside the capsule, which may impact both the shelf life and the activity of the E. coli contained within. This aspect will require careful consideration as well.

Details of the Impact of Gelatin Drying Time

Current Status: While we have confirmed that drying time significantly affects the composition of gelatin, due to time and sample number constraints, we have only been able to collect data for samples dried for up to two days.

Future Goal: The next challenge is to gather data on how extending the drying time impacts the melting time. In the second durability test, it was observed that samples dried for one week did not melt at all, even after being shaken for three hours at 37°C. Therefore, by extending the drying time, it may be possible to further adjust the melting time, which requires additional research based on this observation.

Improving the Inconsistencies Caused by Manual Work

Current Status: Since all samples were handmade, the lack of precision led to issues such as the cavity not being centered within the PMC hemisphere, resulting in reduced durability in areas with thinner walls. Additionally, the capsules were not perfectly spherical, and the coating was uneven.

Future Goal: By utilizing machinery, it is possible to produce capsules with consistent shape and higher precision, according to the design, allowing for large-scale production without individual variability. This would address the current issues related to the inconsistencies and reduced durability caused by manual work.

Predicting Behavior in the Intestine

Current Status: In the disintegration test, the shaking time in the second solution was insufficient, preventing accurate evaluation. Two White balls (half-day dried) and two Black balls (6-day dried) were placed in test tubes and observed every two hours, but due to time and location constraints, proper test equipment could not be used. As a result, shaking was not performed, and the temperature remained at 22°C, making the conditions inconsistent for evaluating behavior inside the body.

Future Goal: Moving forward, the challenge will be to observe under appropriate shaking and temperature conditions to obtain more accurate data. It will be essential to use a disintegration test machine that can simulate in vivo conditions to collect reliable data on how the capsule behaves in the body.

Social Implementation

There are a number of things to consider when putting this capsule into practical use.

Swallowability

The Japanese Pharmacopoeia does not have specific standards for the size of spherical capsules. Additionally, such spherical capsules are not commonly available on the market. Therefore, in terms of swallowability, we refer to traditional Japanese herbal medicine (Kampo) as a guide. Kampo is a traditional medicine that originated from ancient Chinese medicine but has evolved to meet the unique culture and needs of Japan. In Kampo, there is a spherical dosage form known as "maruzai", with sizes ranging from 8mm to 10mm, which are considered easy to swallow.

Therefore, our new drug, Colon-Y, will be marketed with a diameter of 12mm, which has been estimated as the ideal size through diameter and volume measurements. Future challenges include further reducing the size for better swallowability, as well as masking the taste and smell for a more pleasant user experience.

Cost

We plan to offer capsule manufacturing services at a price of $28 (3956 yen) per 100 capsules.

The process of cost estimate

In estimating the budget for producing 100 capsules under this contract, we found that the manufacturing cost is approximately $21.

Budget Estimate

Table.4: Budget Estimate

Therefore, assuming a 25% profit margin, we can reasonably estimate that the price per 100 capsules should be set at $28 (3956 yen). The assumptions used in this estimation are clearly outlined in the attached PDF.

Download PDF

At SUNAO Pharmaceutical Co., Ltd., the cost of manufacturing hard and soft capsules, when considering variations in batch size, is approximately $348 (50,000 yen) per 100 capsules.

(Source: https://sunao-seiyaku.com/oem/oem-supplement/)

Therefore, even after accounting for potential additional costs, our capsule manufacturing service remains significantly cheaper. Furthermore, increasing the batch size for each order could make it even more cost-effective.

References

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[2] Cetin Uyanikgil, Emel & Asikoglu, Makbule & Gundogdu, Evren & Orumlu, O. & Kirilmaz, Levent. (2011). In vitro and in vivo evaluation of hydroxypropylmethyl cellulose phthalate capsules. International Journal of Drug Delivery. 1. 279-285.

[3] Strich, S., Azehaf, H., Neut, C. et al. Film Coatings Based on Aqueous Shellac Ammonium Salt “Swanlac® ASL 10” and Inulin for Colon Targeting. AAPS PharmSciTech 24, 205 (2023).

[4] Luangtana-anan, M., Saengsod, S. & Limmatvapirat, S. Improvement of Bleached Shellac as Enteric Coating by Composite Formation. AAPS PharmSciTech 22, 241 (2021).

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