Team:IISER Bhopal/Implementation

Team:IISER Bhopal | Implementation

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Implementation

Overview

Our project BLAST aims to create an intravenous injectable product of transformed Bifidobacterium longum, so that it can locate sites of hypoxic solid tumors and destroy the cancer cells there. We aim to create a safe and effective drug with minimal side effects. Our proposed implementation takes our product from the lab to the consumers.

Our proposed implementation takes our project BLAST to the intravenous injectable known as Trasmax . For implementation purposes we have explored the following three sectors

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  • Drug Delivery
  • Pre-clinical and clinical trials
  • Entrepreneurship

Drug Delivery

For administering the recombinant bacteria we jotted down the options which were as follows

  • Administration in tumorospheres and thus an intratumoral injection.
  • Administration intravenously as intravenous injection
  • Oral administration in the form of a capsule.

After discussion we speculated that intratumoral administration using tumorospheres will be a problem in metastatic cancer, where the tumor cells leave the original tissue and then spread in other tissues. For oral administration, since we are administering a gram positive bacteria which is found in the human gut flora as well, this could cause disturbance in the gut microbiota.

So, we found the intravenous injectable as the most effective method of administering the bacteria. In the meeting with Doctor Padma Devrajan, a common concern was regarding the size of the bacteria implying the risk that it can create blockages in the vein. The concern was solved after we went through papers in which intravenous administration of bacteria was shown to be an effective and safe route for cancer therapy (Huicong Zhou et.al.) and studies in in mammary tumors in rats where Bifidobacterium longum was injected intravenously as demonstrated by Takayuki Sasaki et.al. and no associated risks were detected. After our stand for an IV injectable was supported we discussed formulating the bacterial injection.

Drug Delivery Steps At a Glance

As guided by Dr. Devrajan, we need to approach drug delivery in following steps:

Step 1 Preparing a solution with maintaining the osmolarity with blood: Blood or plasma has an osmotic pressure of about 0.3 Molar of solutes. The solution administered should be also of 0.3 Molar, so bacterial injection in 0.3 Molar aqueous solution with NaCl and dextrose with following relationships:

0.3 M NaCl is equivalent to 0.9% NaCl in H2O & 0.3 M Dextrose is equivalent to 5% Dextrose in H2O.

Step 2 Lyophilisation

Bacteria is known to be unstable in suspension medium, using freeze/drying technique is known to be effective for long term storage. Freeze drying is performed at triple point of water, which is about 0.01 degree Celsius and pressure of 4.6 mm of Mercury.

To put simply, freeze drying is a process where water surrounding the bacteria is converted to solid ice and ice is sublimed. For sublimation, the sample is put at a temperature less than 0.01 degree celsius, that is in the range of -15 degree celsius to -20 degree celsius.

Steps involved are as follows : 1) Sample preparation- The product before freeze drying needs to undergo some preparation for it to work effectively after mixing with water or other solvent. In our case the concentration of NaCl and Dextrose and Trehalose need to sum up to an overall concentration of 0.3 Molar, so it is isotonic with blood. Ice sublimation creates a stress which can damage the bacteria, so addition of cryoprotectants like Trehalose is done.

2) Freezing- The samples are loaded in the vials or flask and are then given thermal treatment at atmospheric pressure. Another process known as annealing is performed so as to obtain larger crystal growth and shorter drying times. The product temperature is normally cycled from -40 to -20 degree celsius and then to -40 degree back. But every product has a unique formulation so they also have a temperature fixed upto they can withstand the primary drying process without melting or collapsing, known as critical temperature.

3) Primary drying- This involves removal of crystallised water with sublimation performed under vacuum. This step is done safely below the product’s critical temperature.

4) Secondary drying- This involves removal of the remnant water which was not crystallised. After primary drying the temperature of the product can be increased without the risk of collapse or melting. The mostly accepted moisture content, which doesn’t deter the viability of the product, ranges from 0.5% to 3%. The final products are removed from the freeze-dryer and then a powder is prepared. Basic steps: Sample preparation-> Freezing-> Primary drying -> Secondary drying-> Final product

Step 3: Preparing the dosage Considering the weight of the bacteria and adaptive ability of the tumor we have to be careful with the amount of bacteria administered. As the weight of the bacteria is in Picogram , the concentration will be very less. So addition of diluent or filler like Mannitol is done with cryoprotectants like Trehalose in 1:1 ratio.

Weigh the final weight of the powder and divide it into 10 doses to collect aliquots and collect three samples of any amount but of fixed weight. These samples are then mixed with water and OD is checked for bacteria so as to ensure that the bacteria is homogeneously distributed across the powdered product. The final samples can be then mixed with water, as and when required to administer as injection.

Pre-Clinical Trials

We contacted Rodenta Bioserve, an organisation based in Hyderabad, Telangana for getting information over how to proceed towards the animal models from the cell lines. They suggested we use mice models for efficacy studies and rodent and non-rodent models for toxicity studies. They helped us formulate a protocol for the pre-clinical trials. The details of the protocol in brief are as follows: 

            I) Efficacy studies
            Animal Model: Female SCID mice,
            Age: 4 to 6 weeks old,
            Weight: 21-25g
            Number of groups: 3
            Number of mice per group: 6
Protocol

  1. A known number of human tissue specific adenocarcinoma (Colorectal/ lung) cells in growth phase will be implanted subcutaneously into the reach of the experimental mice.
  2. Tumor development is monitored using a calliper at regular intervals for 15-20 days and tumor volume is mathematically estimated.
  3. Animals with tumor volume greater than 130mm3 are randomised into 3 groups viz., Control, Experimental and reference
  4. Animals in the experimental groups are administered with TRAIL-Smac fusion protein (30 mg/kg/day) via tail vein injection on alternate days for six doses. While Control animals receive vehicle/saline, the reference group mice receive Bevacizumab (5 mg/kg/day) every third day by tail vein injection.
  5. Decrease in the size of the tumor volume in the experimental (administered TRAIL-Smac fusion) and reference mice is monitored after 14 days.
  6. Note: Animals will be euthanized when the tumor size exceeds 1.5 cm3 or became ulcerated as mandated by the Institutional Animal Ethical Committee recommendations.

II Toxicity Studies

Toxicity studies will be conducted on healthy small animal models as per OECD Guidelines and include the following studies.

  1. Maximum Tolerable Dose determination study– 14 days’ study in SD rats to identify the MTD
  2. Acute toxicity study: 14 days’ acute toxicity study done in 2 rodent models (Swiss mice & SD rats) as per OECD guidelines. The study parameters include general observations like behavioural alterations, regular health monitoring, mortality, clinical signs of toxicity and gross pathology. Generally, include 2 groups like vehicle control and High dose product.

Repeat dose toxicity study: The study is conducted for 28 days (weekly once administration for 4 weeks) followed by 2 weeks of recovery (14 days) as per OECD guidelines in one rodent (Swiss mice) and one non-rodent (Rabbit) model. The study parameters include general observations like behavioural alterations, regular health monitoring, mortality, clinical signs of toxicity, body weight, food consumption, clinical biochemistry in plasma & urine, haematology & Histopathology. Generally, include vehicle control groups, 3 product doses containing low, average & high doses, reference group and 2 recovery groups (vehicle control & high dose).

Clinical Trials

After successful studies in animal models we can go to clinical trials as suggested by Abiogenesis Clinpharm. As suggested by them we need to go ahead with phase I and phase II trials. To proceed to phase III the first two phase trials need to be approved by DCGI (Drugs Controller General of India). The trials need to proceed for one particular type of cancer (oral/breast etc.) first and then the phases can proceed to other types of cancers so as to check the effectiveness in various cancers and then understand possible adjuvant treatments which can be administered with the bacteriotherapy provided.

Phase I
Number of people :15-30
  1. Administering the prepared dosage to the patients.
  2. Waiting for a period of 1 week to 1 month to look for possible side effects post treatment.
Phase II
Number of people: less than 100

This phase helps understand if the therapy has effects on cancer and what side effects could manifest in a person's body and how they can be dealt with. People in this phase usually receive the highest dose which was seen harmless in phase I.

Phase I/II
Number of people: 25-100

This phase is basically a combination of both phase I and II and needs fewer people. It helps address problems much quickly and can help develop better formulations in the future.

Clinical Trials at a Glance

Provided below is a glance of how clinical trials normally proceed in diseases like cancers. Normally a lot of cancers are studied in initial phases but later on the studies can become more specific to a larger population which has been screened for one or two particular cancer types, making the studies coherent. Randomisation is done so as to divide the people into groups so as to administer different treatments to standardize and compare the efficacy and toxicity of the new treatment to the currently available ones.

Phase Number of Participants Cancer Type Trial Aim Randomization
0 10-20 Various Low Dose administration and looking for side effects No
I 20-50 Various Check for side effects in the body, post-treatment No
II Less than 100 More specific, one or two cancer type Administering the highest dose which was given in phase I and to look for side effects Sometimes
III Larger population, more than 100 Usually one cancer type or sometimes more Comparison of the new treatment to the standard treatment Yes
IV Medium to large, variable One type or sometimes more Finding long term benefits and possible side effects No


References