Safety Tests
|
Overview
Preliminary pre-clinical non-GLP toxicology studies have already been performed, so MA-8789 is expected to be non-toxic in the clinically-intended dose range in the pending GLP toxicology studies as well. Preliminary toxicity studies have shown that the median lethal dose (LD50) in male rats to be 31% concentration at 10 mL/kg body weight dose for male rats and 82% concentration for female rats. Preliminary tests have also shown that the maximum tolerated dose (MTD) may be more than 10% in rats, which is well above the 0.25% concentration dose that anecdotal evidence has shown efficacy for in cancer patients in South Korea. Preliminary local irritation tests, antigenicity tests, and genotoxicity studies have also all shown that MA-8789 may not produce any adverse effects in the clinically intended dose range.
I. Single Dose Acute Toxicity
- Acute toxicity: adverse changes occurring immediately or a short time following a single dose or short period exposure to a substance
- Adverse change: any effect that results in functional impairment and/or biochemical lesions that may affect the performance of the whole organism or that reduce the organ’s ability to respond to additional challenge
- Methods
- Clinical signs: observed every day
- Mortality: evaluated once every hour during the first six hours, then once every day after that
- Body weight: measured at initiation, then at 4, 7, and 14 days after treatment
- Autopsy: performed whenever an animal died and on all live animals after the 14-day observation period
- Animals Used
- 20 healthy male and female rats
- All rats were 7 weeks old at the time of acquisition
- Rats were quarantined for one week to be observed for clinical signs before initiation
- Dosage Groups
- 5 male and female rats in each group
- Oral injection: 10mL/kg
- High: 100% undiluted raw solution
- Middle: 50% concentration
- Low: 25% concentration
- Lowest: 13% concentration
- Conclusion
- LD50:
- Male: 31% concentration
- Female: 82% concentration
II. Repeated-Dose Toxicity
- 28-day daily-dose toxicity study in rats
- Methods
- Clinical signs: observed once a day
- Food uptake: measured once a week
- Water consumption: measured once a week
- Body weight: measured once a week
- Urinalysis: 5 rats from each group selected randomly
- Eye exam: 5 rats from each group selected randomly
- Hematological and serum biochemical findings
- Absolute and relative organ weights
- Histopathological findings
- Any statistical significant difference from control is at p<0.05
- Animals Used
- 50 healthy male and female rats
- 40 rats used in the study
- All rats were 4 weeks old at the time of acquisition
- Rats were quarantined for one week to be observed for clinical signs and checked for health before initiation
- Dosage Groups
- 10 male and female rats in each group
- Oral injection: 10mL/kg
- High dose: 10% concentration
- Middle: 1%
- Low: 0.1%
- Negative control
- Conclusion
- Suggested Maximum Tolerated Dose (MTD) may be more than 10% concentration of MA-8789
III. Local Irritation
- To evaluate the safety of local irritation of 0.1% MA-8789 on skin and eye of rabbit
- Skin irritation test:
- 6 male rabbits
- 0.5mL of 0.1% MA-8789 vs saline control
- One-time application, for 24 hours
- Eye irritation test:
- 9 male rabbits
- 0.1mL of 0.1% MA-8789 in left eye vs saline control in right eye
- Conclusion: 0.1% MA-8789 is non-irritant
IV. Antigenicity Tests
- Anaphylaxis: severe acute allergic reaction
- Active systemic anaphylaxis (ASA): generalized reaction caused by administration of a foreign antigen
- Passive cutaneous anaphylaxis (PCA): localized reaction transferred by local intradermal injection of antibody, followed by IV injection of corresponding antigen
- ASA: Treatment Groups
- Five male guinea pigs each in six groups
- Negative control: 0.9% saline
- Positive control: bovine serum albumin (BSA)
- Low: 0.3% MA-8789
- High: 3% MA-8789
- Low + adjuvant
- High + adjuvant
- Adjuvant: drug that has no effects on its own but may increase efficacy of other drugs
- ASA: Methods
- Sensitization phase:
- Treated 3 times a week for 3 weeks subcutaneously
- Negative control, low, and high dose groups
- Treated 3 times biweekly
- Positive control, low+adjuvant, high+adjuvant
- Challenge phase:
- High dose of MA-8789 (or control substance) injected IV through ear vein to observe anaphylactic shock on 10th and 12th day after last sensitization
- Homologous PCA: Dose Groups
3 guinea pigs each in six treatment groups - 1. anti-saline
- 2. anti-BSA
- 3. anti-MA-8789(low)
- 4. anti-MA-8789(high)
- 5. anti-MA-8789(low)+adjuvant
- 6. anti-MA-8789(high)+adjuvant
- Antisera (polyclonal antibodies) obtained from ASA test guinea pigs
- Homologous PCA: Methods
- Sensitization phase:
- Antisera diluted with saline and injected intradermally (100 microliter)
- Challenge phase:
- 24 hours after intradermal injection of antisera, IV injection of antigen (MA-8789 or control substance) into ear vein
- Heterologous PCA: Groups
- Sensitization (antisera) groups: 5 Mice each
- 1. Negative control: 0.9% saline
- 2. Positive control: BSA
- 3. Low dose MA-8789
- 4. High dose
- 5. Low + adjuvant
- 6. High + adjuvant
- Challenge groups: 2 Rats each
- 1. anti-saline
- 2. anti-BSA
- 3. anti-MA-8789(low)
- 4. anti-MA-8789(high)
- 5. anti-MA-8789(low)+adjuvant
- 6. anti-MA-8789(high)+adjuvant
- Heterologous PCA: Methods
- Sensitization phase:
- Antisera obtained from mice in similar fashion as the guinea pig homologous PCA test
- Challenge:
- 24 hours after injection of mouse antisera into rats, antigen (MA-8789 or control substance) injected IV into tail vein
- Antigenicity: Results
- ASA:
- No anaphylactic shock-like symptoms in all treatment groups (except for positive control)
- PCA:
- No significant change in all treatment groups compared to negative control (except for positive control)
- Thus MA-8789 at 0.3% and 3% concentration does no induce anaphylactic shock nor produce tissue-tropic IgE
V. Skin Sensitization Test
- Skin sensitizer: “a substance that will induce an allergic response following skin contact” (UNECE, 2004, p.151)
- Male guinea pigs
- First sensitization: intradermal injection
- Second sensitization: topical
- Challenge: topical
- Treatment Groups
- Negative control: 5 guinea pigs
- Sensitizations and challenges: 0.9% saline
- Positive control: 5 guinea pigs
- First sensitization: 15% benzocaine
- Second sensitization: 30% benzocaine
- Challenges: 20% benzocaine
- 3 Treatment groups: 10 guinea pigs each
- First sensitization (all groups): 1.5% MA-8789
- Second sensitization (all groups): 15% MA-8789
- Challenges:
- Low dose: 1% MA-8789
- Middle: 5% MA-8789
- High: 20% MA-8789
- Results
- MA-8789 concentration under 20% concentration does not have allergic potential
- Only 2 out of 10 guinea pigs in MA-8789 high dose group showed only slight erythema (redness of skin)
- MA-8789 did not induce experimental allergic contact dermititis
VI. Genotoxicity
- Reverse mutation (AMES) assay: tests whether the drug substance can mutate a histidine-dependent salmonella typhimurium strain into being able to grow in a histidine-free medium
- Chromosome aberration test: test for chromosome aberration in Chinese hamster lung cell line
- Micronucleus test: in vivo test in mice to measure ratio of micronucleated polychromatic erythrocytes
- Conclusion
- AMES assay: no significant differences between negative control and treatment groups
- Chromosome aberration test: ratios of aberrant chromosomes in treatment groups were not significantly different from control group
- Micronucleus: both male and female high dose (10% concentration) groups showed significant difference compared to negative control, but middle (5%) and low (2.5%) did not
- MA-8789 might not induce any genotoxic effect when applied at the clinically intended dose
Overall Conclusion
- Safety evaluation tests of MA-8789 conclude that MA-8789 does not show any toxic effect and might be safe to use within its clinically intended dose