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Abstract for IMM96013 - Pyrimethamine (CASRN58-14-0) and 3'-Azido-3'-Deoxythymidine (AZT) (CASRN 30516-87-1)

Abstract

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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see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 3'-Azido-3'-Deoxythymidine (AZT) (CAS No. 30516-87-1) n Female B6C3F1 Mice

NTP Report Number IMM96013

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.1demonstrated 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 studies were conducted in female B6C3F1mice. The animals were treated daily for 28 days by oral gavagealone or in combination. The in-life phase of the study was conductedbetween 26 October 1994 and 24 February 1995. AZT and PYM wereprepared as solutions in Maalox Therapeutic Concentrate (T.C.).

The baseline toxicology studies are summarized in Table ES-1.Oral administration of AZT at 50, 100 and 200 mg/kg/day alongwith PYM at 10 mg/kg/day, AZT at 200 mg/kg/day alone, or PYM at10 mg/kg/day for 28 days was without effect on body weight orrate of weight gain. There were few notable changes among thestandard toxicological parameters that were assessed. Spleen weightwas increased moderately in the high dose AZT+PYM group. Combinedhigh dose treatment appeared to interfere somewhat with hematopoesisand the reticulocyte count was increased. The leukocyte differentialwas unaffected by either AZT or PYM. The number of granulocyte/monocyteprogenitors was elevated by 30% in mice receiving the middle doselevel of AZT+PYM, and the number of macrophage progenitors wasincreased by 31% at the high dose of AZT+PYM. Serum SGPT and BUNwere modestly increased by the high dose combination treatment.In these experiments the no-effect level for combined treatmentof AZT+PYM as evaluated in standard toxicologic parameters appearsto be in the range of 50 to 100 mg/kg/day for 28 days.

Table ES-2 summarizes the immunology studies. The immunologic effects of AZT and PYM show a mixed profile.Combined treatment at the high dose of AZT reduced the numberof spleen B cells as did each agent given alone. The total numberof T cells was not altered. AZT and PYM alone each augmented thesplenocyte IgM AFC response per million cells but, in combination,treatment was without effect. AZT and PYM treatment had no effecton the anti-sRBC serum titer. Cytotoxic T cell activity per spleenwas not altered and sRBC clearance was unaffected by combinedtreatment. NK cell activity in terms of LU/107 cellswas increased at all three dose levels of AZT+PYM and by eachagent given alone, but combined treatment did not alter activityper whole spleen.

Overall, combined treatment with AZT and PYM in the dose rangeof 100 to 200 mg/kg/day AZT with 10 mg/kg PYM for 28 days hadlittle effect on the immune system parameters that were assessed.The most pronounced effect was a reduction in B cell numbers withouta reduction in the AFC response, anti-sRBC serum titer, nor inthe MLR or CTL responses. NK cell activity per 107spleen cells was stimulated by combination treatment, but totalNK activity per spleen was not affected.


Table ES-1
SUMMARY TABLE FOR TOXICOLOGY STUDIES
AZT-PYM-28-1-PO
Parameter Result Maximum Effect Dose Comment

Body Weight        
  Day 8 No Effect      
  Day 15 No Effect      
  Day 22 No Effect      
  Day 29 No Effect      

Weight Changes
       
  Day 8-1 No Effect      
  Day 15-1 Decrease -56% 10 mg/kg PYM
  Day 22-1 Decrease -37% 10 mg/kg PYM
  Day 29-1 Decrease -31% 10 mg/kg PYM

Pathology
       
  Gross Pathology No Effect      
  Histopathology Not Processed      
    Organ Weights        
      Liver No Effect      
      Spleen Increase
Increase
+22%
+11%
200 mg/kg
10 mg/kg
AZT+PYM
PYM
      Lungs No Effect      
      Thymus Increase +28% 50 mg/kg AZT+PYM
      Kidney No Effect      

Hematology
       
  RBCs Decrease
Decrease
Decrease
-16%
-10%
-3%
200 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
    Hemoglobin Decrease -7%
-3%
200 mg/kg
200 mg/kg
AZT+PYM
AZT
    Hematocrit Decrease -6% 200 mg/kg AZT+PYM
    MCV Increase
Increase
Increase
+12%
+9%
+3%
200 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
    MCH Increase
Increase
Increase
+11%
+8%
+2%
200 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
    MCHC Decrease -1% 10 mg/kg PYM
    Platelets Increase
Increase
Decrease
+6%
+6%
-16%
100 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
    Reticulocytes Increase
Increase
+53%
+21%
200 mg/kg
200 mg/kg
AZT+PYM
AZT
  Leukocytes No Effect      
    Leukocyte Diff        
      Lymphocytes (%) Increase +4% 10 mg/kg PYM
      Neutrophils No Effect      
      Eosinophils No Effect      

Serum Chemistries
       
  SGPT Increase +18% 200 mg/kg AZT+PYM
  Albumin No Effect      
  BUN Increase +21% 200 mg/kg AZT+PYM
  Glucose No Effect      
  Total Protein No Effect      
  Globulin No Effect      
  Alb/Glob Ratio No Effect      

Bone Marrow
       
  Cellularity No Effect      
  DNA Synthesis Decrease
Decrease
Decrease
-18%
-20%
-22%
200 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
  Stem Cells No Effect      
    CFU-GM Increase +30% 100 mg/kg AZT+PYM
    CFU-M Increase +31% 200 mg/kg AZT+PYM

Table ES-2
SUMMARY TABLE FOR IMMUNOLOGY STUDIES
AZT-PYM-28-1-PO

Parameter Results Maximum Effect Dose Comment

Surface Markers (Absolute Values)

Ig+ Decrease
Decrease
-18%
-17%
200 mg/kg
200 mg/kg
AZT+PYM
AZT
CD3+ No Effect      
CD4+CD8- Decrease -16% 200 mg/kg AZT
CD4-CD8+ No Effect      
CD4+CD8+ No Effect      

IgM Humoral Immune Response to Sheep Erythrocytes

IgM AFC/106 Cells Increase
Increase
+44%
+53%
200 mg/kg
10 mg/kg
AZT
PYM
Serum Titer No Effect      

Proliferation Assays, i.e. Mixed Leukocyte Response

MLR Index Decrease -46% 100 mg/kg AZT+PYM

Cytotoxic T Lymphocyte Activity

CTL Decrease -34% 200 mg/kg AZT+PYM

Reticuloendothelial System

RES: Lung Decrease -50% 200 mg/kg AZT

NK Cell Activity

200:1 Increase
Increase
Increase
+33%
+51%
+36%
50 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
100:1 Increase
Increase
Increase
+23%
+54%
+30%
100 mg/kg
200 mg/kg
10 mg/kg
AZT+PYM
AZT
PYM
50:1 Increase +56% 200 mg/kg AZT
25:1 No Effect      
12.5:1 Increase
Increase
+100%
+100%
200 mg/kg
10 mg/kg
AZT
PYM
6.25:1 Increase
Increase
+100%
+71%
200 mg/kg
10 mg/kg
AZT
PYM

Report Date: May 1996


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