National Toxicology Program

National Toxicology Program
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Abstract for IMM94007 - Pyrimethamine (CASRN 58-14-0) and AZT (CASRN30516-87-1)

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program (NTP). The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see http://ntp.niehs.nih.gov/ntp/htdocs/levels/09-3566%20NTP-ITOX-R1.pdf). The findings and conclusions for this study should not be construed to represent the views of the NTP or the US Government.

The Immunotoxicity of Pyrimethamine (CAS No. 58-14-0)
and AZT (CAS No.30516-87-1)

Dose Range-Finding Study in Female B6C3F1 Mice
NTP Report Number IMM94007

Summary

Pyrimethamine (5-[4-chlorophenyl]-6-ethyl-2,4-pyrimidinediamine,PYM) is a folic acid antagonist with antiparasitic activity commonlyused in combination with sulfadiazine to treat toxoplasmosis inAIDS patients also receiving 3'-azido-3'-deoxythymidine (AZT).At doses used in the treatment of toxoplasmosis, there may beleukopenia, thrombocytopenia, pancytopenia, and hypersensitivityreactions including erythema multiforme, skin eruptions, uticaria,anaphalactoid reactions, and several other allergic responses.In addition, previous studies by Luster et al.demonstrated that some of the anti-HIV drugs have adverse effectson the immune system. These adverse responses raise a concernthat PYM and AZT in combination may have adverse effects on theimmune system.

3'-Azido-3'-deoxythymidine and pyrimethamine were nominated tothe NTP for toxicological evaluation and were selected for immunotoxicitystudies by the chemical manager. Thus, the purpose of these studieswas to determine the potential effects of 3'-azido-3'-deoxythymidineand pyrimethamine on the immune system.

The study reported here is an initial range-finding study conductedin female B6C3F1 mice. The animals weretreated daily for 14 days by oral gavage alone or in combination.In-life phase of the study was conducted between 9 September 1994and 23 September 1994. PYM and AZT were prepared as solutionsin Maalox Therapeutic Concentrate (T.C.).

The baseline toxicology studies are summarized in Table ES-1.In this range-finding study of orally administered PYM at 2.5,5 and 10 mg/kg/day along with AZT at 200 mg/kg/day, AZT at 200mg/kg/day alone, or PYM at 10 mg/kg/day for 14 days, there wereno effects on body weight or rate of weight gain. PYM+AZT hadno effect on organ weights. The erythrocyte count, hemoglobin,and hematocrit were decreased by PYM+AZT and by AZT alone whilethe MCV and MCH were slightly increased. Reticulocyte and leukocytecounts were unaffected. The number of bone marrow cells per femurwas increased by PYM and AZT given alone, but in combination therewas no effect. DNA synthesis was reduced by PYM but not by AZT,and the high dose of PYM+AZT stimulated DNA synthesis.

Table ES-2 summarizes the immunology studies.PYM alonedid not significantly affect the AFC response. AZT at 200 mg/kgreduced the IgM AFC response by about one-third as did the samedose level of PYM+AZT. Neither PYM nor AZT altered the serum titerof anti-sRBC, but the high dose combination reduced the serumtiter by one-third. AZT increased the proliferative response ofresponder cells and responder+stimulator as did the high doseof PYM+AZT. Both PYM and AZT stimulated NK cell activity withPYM being 2-3 times as active as AZT at the doses used. PYM+AZTincreased NK cell activity 40-50%.

Overall, combined treatment with PYM and AZT in the dose rangeof about 10 mg/kg/day PYM with 200 mg/kg/day AZT for 14 days hadmoderate effects on the immune system without serious systemictoxicity. There was an impaired AFC response coupled with a one-thirdreduction in the anti-sRBC serum titer. NK cell activity per 107spleen cells was stimulated by combination treatment.



TABLE ES-1
SUMMARY TABLE FOR TOXICOLOGY STUDIES
AZT-PYM-14-1-PO

Parameter Result Maximum Effect Dose Comment


Body Weight        
Day 8 No Effect      
Day 15 No Effect      
Weight Changes        
Day 8-1 Decrease
Decrease
Decrease
-148%
-107%
-88%
5 mg/kg
200 mg/kg
10 mg/kg
PYM+AZT
AZT
PYM
Day 15-1 No Effect      



Pathology        
Gross Pathology        
Histopathology        
Organ Weights        
Liver No Effect      
Spleen Increase +14% 10 mg/kg PYM
Lungs Decrease
Decrease
-23%
-30%
200 mg/kg
10 mg/kg
AZT
PYM
Thymus No Effect      
Kidney No Effect      



Hematology        
RBCs Decrease
Decrease
Decrease
Decrease
-11%
-12%
-17%
-9%
2.5 mg/kg
5 mg/kg
10 mg/kg
200 mg/kg
PYM+AZT
PYM+AZT
PYM+AZT
AZT
Hemoglobin Decrease
Decrease
Decrease
Decrease
-7%
-7%
-14%
-5%
2.5 mg/kg
5 mg/kg
10 mg/kg
200 mg/kg
PYM+AZT
PYM+AZT
PYM+AZT
AZT
Hematocrit Decrease
Decrease
Decrease
Decrease
-8%
-9%
-15%
-7%
2.5 mg/kg
5 mg/kg
10 mg/kg
200 mg/kg
PYM+AZT
PYM+AZT
PYM+AZT
AZT
MCV Increase
Increase
Increase
Increase
+3%
+4%
+2%
+2%
2.5 mg/kg
5 mg/kg
10 mg/kg
200 mg/kg
PYM+AZT
PYM+AZT
PYM+AZT
AZT
MCH Increase
Increase
Increase
Increase
+4%
+5%
+3%
+4%
2.5 mg/kg
5 mg/kg
10 mg/kg
200 mg/kg
PYM+AZT
PYM+AZT
PYM+AZT
AZT
MCHC Increase +2% 200 mg/kg AZT
Reticulocytes No Effect      
Platelets No Effect      
Leukocytes No Effect      



Bone Marrow        
Cellularity Increase
Increase
+26%
+50%
200 mg/kg
10 mg/kg
AZT
PYM
DNA Synthesis Increase
Decrease
+35%
-17%
10 mg/kg
10 mg/kg
PYM+AZT
PYM
           

 



Table ES-2
SUMMARY TABLE FOR IMMUNOLOGY STUDIES
AZT-PYM-14-1-PO

Parameter Results Maximum Effect Dose Comment

IgM Humoral Immune Response to Sheep Erythrocytes
IgM AFC/106 Decrease -33% 10 mg/kg PYM+AZT
  Decrease -30% 200 mg/kg AZT
IgM AFC/Spleen Decrease -38% 10 mg/kg PYM+AZT
  Decrease -33% 200 mg/kg AZT
Serum Titer Decrease -37% 10 mg/kg PYM+AZT

Proliferation Assays, i.e. Mixed Leukocyte Response
MLR Responders +
Stimulators
Increase
Increase
+44%
+41%
10 mg/kg
200 mg/kg
PYM + AZT
AZT

NK Cell Activity
100:1 Increase
Increase
Increase
+44%
+14%
+44%
10 mg/kg
200 mg/kg
10 mg/kg
PYM+AZT
AZT
PYM
50:1 Increase
Increase
Increase
+50%
+13%
+44%
10 mg/kg
200 mg/kg
10 mg/kg
PYM+AZT
AZT
PYM
25:1 Increase
Increase
Increase
+39%
+20%
+41%
10 mg/kg
200 mg/kg
10 mg/kg
PYM+AZT
AZT
PYM

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