Cyclophosphamide, 100 mg/kg, and Adriamycin, 5 mg/kg, were synergistic in treating L1210 leukemic mice that were inoculated with 1 × 10⁵ L1210 cells 4 days prior to treatment. Only one course of treatment was given. There was a dramatic circadian variation in response as monitored by mean survival time or cure rate. The variation in cure rate (mice alive and apparently free of disease 75 days post-tumor inoculation) as a function of treatment timing ranged from 8 to 68% in male animals standardized to 12 hr of light alternating with 12 hr of darkness. Similarly, in female mice standardized to 8 hr of light alternating with 16 hr of darkness, the cure rate ranged from 0 to 56% depending on when the drugs were injected during a 24-hr span. No cures were obtained with either drug alone.

The maximal cure rate was recorded when the two drugs were administered during the early part of the dark portion of the light-dark cycle (whether 12 hr of light and 12 hr of darkness or 8 hr of light and 16 hr of darkness), whereas maximal mortality occurred following treatment early in the light span.

Extensive evidence also documents that maximal therapeutic advantage was obtained when the two drugs were separated by 2- or 3-hr intervals and that this effect of drug sequencing was strongly circadian stage dependent. The results indicate the need for information on endogenous temporal organization in studies of order and intervals in the administration of combination therapy.