Safety Tests

  • I. Single dose (rats)
  • II. Repeated dose (rats)
  • III. Skin and eye local irritation (rabbits)
  • IV. Antigenicity / Immunogenicity
    • a. active systemic anaphylaxis (guinea pigs)
    • b. passive cutaneous anaphylaxis (mice and rats)
  • V. Skin sensitization (guinea pigs)
  • VI. Genotoxicity / Mutagenicity
    • a. reverse mutation (AMES) assay
    • b. chromosome aberration test
    • c. micronucleus test

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