Though shown to be a potent anti-tumor platinum compound, CDPCP was never tested clinically. Because of its positive charge, the drug may have been thought to have poor pharmacokinetic characteristics. In recent studies, OCT1 was shown to play a role in the cellular uptake and consequent cytotoxicity of CDPCP in cell lines over-expressing OCT1 (25
). Accordingly, we hypothesized that OCT1 mediates the uptake of CDPCP in vivo
in various tissues, particularly the intestine and liver, and that OCT1-mediated transmembrane flux may result in a favorable pharmacokinetic and toxicity profile for CDPCP. To test our hypothesis, we performed a comprehensive pharmacokinetic analysis using genetically modified Oct1-/-
mice in this study. The results suggest that deletion of Oct1 not only decreased the uptake of cellular platinum but also reduced the level of platinum-DNA adduct formation in hepatocytes after CDPCP exposure (Fig. ). These results are in agreement with those in cells transfected with human OCT1 (25
), although the fold difference in platinum levels between hepatocytes harvested from Oct1+/+ versus Oct1-/-
mice was much less than that between HEK293 cells over-expressing human OCT1 and control cells (25
). These differences were not due to species differences between mOct1 and hOCT1, since our data suggest that CDPCP is a comparable or better substrate of mOct1 than of hOCT1 (Fig. ). Drug- metabolizing enzymes and drug transporters normally are lost by liver cells in culture as a consequence of the adaptation of cells to the in vitro
environment. Lecureu et al.
reported that hepatocytes maintained in primary culture for several days were known to not only lose expression of albumin and cytochrome P-450 but also displayed low levels of OCT1 expression and activity (31
). Studies from our laboratory also confirmed that the expression level of Oct1 in primary hepatocytes cultured from mouse liver was considerably lower than that in mouse liver (data not shown), suggesting that the isolation and culture procedures likely resulted in a reduced expression level of the Oct1 protein and therefore a reduction in the observed effect of Oct1 in the isolated hepatocytes.
The significantly higher hepatic and intestinal accumulation of CDPCP in Oct1+/+
mice was consistent with the abundance of Oct1 in liver and intestine of rodents (12
). Normalization of tissue platinum levels to plasma concentrations of CDPCP excluded the effects of different plasma exposure on the uptake of CDPCP into the various tissues. Although there is strong Oct1 expression in the kidney of mice (12
), it was notable that no differences were observed in the normalized levels of platinum in the kidneys of Oct1+/+ versus Oct1-/-
mice. This may be explained by the redundant function of Oct1/2 in the rodent kidney, and the Oct2 strongly expressed in the kidney can completely compensate for the loss of Oct1. Previously, CDPCP was shown to be a substrate of OCT2 (25
). It is notable that enhanced expression of Oct2 and Oct3 in the liver of Oct1
-/- mice may have resulted in a reduction in the observed effect of Oct1 on CDPCP uptake in the liver (Fig. ).
Consistent with the high hepatic accumulation in Oct1+/+ mice, the hepatotoxicity of CDPCP was more prominent in these mice, as demonstrated by the increased liver-to-body-weight ratio, decreased total serum protein levels, increased serum glucose levels and histological changes at 90 mg/kg in the Oct1 wild-type mice. The low BUN in the wild-type mice treated with CDPCP is also consistent with hepatotoxicity in these mice. A low BUN usually has little significance, but its causes include liver problems, malnutrition (insufficient dietary protein), or excessive alcohol consumption. Although there was greater hepatic toxicity in Oct1+/+ mice, the level of toxicity was considered mild and reversible. If CDPCP is developed as an anti-cancer drug, hepatic toxicity may not be a concern.
Recently, use of oxaliplatin has been associated with development of hepatic lesions, which include sinusoidal alteration, portal hypertension, increase in transaminases, gamma glutamyltranspeptidase and alkaline phosphatase, and steatohepatitis (6
). In our study, the role of Oct1 in the tissue accumulation and the pharmacokinetics of oxaliplatin was proven to be minimal, and oxaliplatin was not hepatotoxic under the conditions used in this study. However, total plasma platinum levels may not reflect platinum complexes available for DNA and protein binding, anti-tumor effects and toxicity of the oxaliplatin. The fact that oxaliplatin administration resulted in a greater reduction in body weight in the wild-type mice in comparison to the Oct1
knockout mice may suggest that Oct1 plays a role in the systemic toxicities of oxaliplatin.
In this study, we made an unexpected observation that Oct1
deletion markedly increased the renal toxicity of CDPCP in Oct1-/-
mice, which was revealed by much greater Kim-1 mRNA expression level and histological changes of tubular cast formation and degeneration of tubular cells in Oct1-/-
mice (Fig. .II, and c.III). The differential localization of organic cation transporters Oct1 and Oct2 in the basolateral membrane of rodent kidney proximal tubules may account for the greater kidney toxicity in Oct1-/-
mice. In particular, Oct1 was shown to be concentrated in the early proximal tubules in the renal cortex, whereas Oct2 was abundant in the late proximal tubules in the outer stripe of the outer medulla (34
). In the report by Jonker et al
. of the relative impact of Oct1 and Oct2 on the clearance of TEA (36
), they observed that elimination of neither Oct1 nor Oct2 resulted in a decrease in renal TEA clearance, whereas elimination of both transporters eliminated TEA secretion. These data suggest that there is sufficient Oct1 transport capacity in the early renal proximal tubules to effectively clear the blood of substrate before it reaches the downstream Oct2-expressing cells of the late renal proximal tubules. CDPCP is an excellent substrate of both OCT1 and OCT2 (25
). In Oct1-/-
mice, CDPCP will be secreted by Oct2-expressing cells in late renal proximal tubules. The greater exposure of the late proximal tubule to CDPCP may contribute to the enhanced toxicity of CDPCP observed in the Oct1-/-
mice. Further, Oct2 is likely to localize in the outer strip of the outer medulla and may produce a localized accumulation of CDPCP in this region. The outer medulla was the region most damaged by CDPCP (Fig. .II and III).
The pharmacokinetics of CDPCP were consistent with the pharmacokinetics observed previously for oxaliplatin and picoplatin, suggesting that the drug has a pharmacokinetic profile similar to clinically used anti-cancer platinum agents. Deletion of Oct1 resulted in a reduction in the clearance and volume of distribution of CDPCP, which may be explained by the differences in the distribution of CDPCP to the tissues with high Oct1 expression, in particular, liver and intestine. The higher plasma platinum concentrations and AUC in Oct1-/- mice in comparison to Oct1+/+ mice can be attributed to the lower clearance and volume of distribution in Oct1-/- mice. Since the renal clearance of CDPCP was similar between Oct1+/+ and Oct1-/- mice, the lower total clearance of CDPCP in the Oct1-/- mice is likely due to a reduction in the uptake and subsequent covalent binding of CDPCP in the liver and intestine, organs that accumulate high levels of platinum.
Though other transporters may affect the disposition of CDPCP, this study clearly demonstrates that Oct1 plays a significant role in the pharmacokinetics of this drug. The lack of an important role of Oct1 in the pharmacokinetics of oxaliplatin may suggest a lower specificity of oxaliplatin for Oct1 and that other transporters (or diffusion) may contribute to its tissue distribution and pharmacokinetics. In this study, CDPCP exhibited excellent pharmacokinetic properties, comparable to other clinically used platinum analogs. Moreover, CDPCP appears to possess a favorable toxicity profile, and Oct1 plays an important role in determining the toxicity profile of CDPCP. Our study supported the concept that by targeting drug influx transporters, off-target toxicities, such as renal, hematological toxicities, can be spared. Whether CDPCP can be used for targeted therapy of various cancers is worthy of further investigation. Further, this study provides another example of a drug distinct from metformin for which OCT1 may play a profound role in its pharmacokinetics and toxicities (4