First, in order to evaluate ZnBG’s inhibitory efficacy on HO activity, we orally administered vehicle or a range of ZnBG doses to 1-wk-old mice. We observed that ZnBG is orally absorbed by neonatal mice and can rapidly inhibit liver HO activity in a dose-dependent and organ-specific manner within 3h of oral administration, with an I50 of 4.0 μmol/kg BW.
Next, in order to evaluate the effects of ZnBG in heme-loaded newborn mice, we orally administered a higher dose of ZnBG in between two s.c. heme loads to 1-wk-old mice. A property necessary for ZnBG to be an effective anti-hyperbilirubinemia drug is its ability to reduce in vivo bilirubin production. We observed that peak bilirubin production in 1-wk-old mice receiving two heme loads and 15-μmol ZnBG/kg BW was 19% lower than peak production in heme-loaded mice not treated with ZnBG. Similarly, previous studies of adult mice treated with 30-μmol SnMP/kg BW at 3h before a 30-μmol/kg BW heme load showed a 20% reduction of peak bilirubin production as estimated from total body CO excretion (17
In addition to effectively reducing bilirubin production, 15-μmol ZnBG/kg BW completely eliminated heme-induced liver HO activity to baseline levels at 24h after a second heme load. Unlike in the liver, heme loading did not significantly increase spleen HO activity in the newborn mice. This result corroborates findings by Braggins et al (20
) and Maines et al (21
) that spleen HO-1 is already maximally upregulated in normal conditions, since it is a primary organ for red blood cell turnover. Under conditions of hemolysis, the liver can also act as another organ for processing excess heme, which results in the induction of both liver HO activity and transcription. When ZnBG was administered at the 15-μmol/kg BW dose between heme loads, we found that liver and spleen HO activity was reduced to baseline and to 30% below baseline levels, respectively. Inhibition to baseline or near-baseline levels of HO activity is desirable because under- or overmodulation of any key enzyme, such as HO, may be detrimental to a developing neonate (21
Our observed inhibition of HO activity by ZnBG in the liver and spleen, but not the brain, is most likely due to the delivery route of oral administration because we have shown that in vitro, ZnBG inhibits HO activity to comparable degrees in tissue sonicates of liver, spleen, and brain (4
). In addition, a recent in vivo study also showed that when ZnBG/kg BW was administered i.v. to adult mice, HO activity was inhibited in the liver, spleen, and brain within 1h after administration (Ronald J. Wong, unpublished observations). We have observed that orally-administered ZnBG to adult mice results in the inhibition of HO activity in the liver, spleen, and intestine (3
). In this study, we also found significant inhibition of liver and spleen HO activity after oral administration of ZnBG to 1-wk-old mice When a drug is administered orally, it is absorbed directly by the digestive and hepatic portal system – “first pass effect” – and therefore is not available to circulate to other tissues (22
), accounting for our observed lack of inhibition of brain HO activity.
At 6h post-administration of repeated heme loads (H-V-H), we found a 2.1-fold increase in spleen HO-1 mRNA. At this same timepoint, H-Zn-H treatment resulted in 2.3- and 2.2-fold increases in liver and spleen HO-1 mRNA, respectively, correlating with the increased HO-1 protein levels observed in these tissues 24h later. This upregulation of HO-1 mRNA appears to be transient, since by 24h post-administration of H-V-H or H-Zn-H, mRNA returned to baseline levels. In adult mice, we found a similar transient increase in HO-1 transcription 6h post-ZnBG administration, which returned to baseline by 24h (3
Even though HO-1 protein is induced 24h after ZnBG treatment, we found that by 2 wks after treatment, liver and spleen HO-1 protein (1.0±0.1 and 1.2±0.2-fold change, respectively) and HO activity (101±4% and 100±2%, respectively) protein levels have already returned to baseline levels. At 1 wk after treatment, liver HO-1 protein has already returned to baseline levels (1.1±0.1-fold), while spleen HO-1 protein (1.6±0.1-fold) is still increased. However, this induction was counteracted by a persistent inhibition of spleen HO activity to 77±5% of baseline. Taken together, the induction of HO-1 by ZnBG most likely has no long-term effects.
Our previous work in adult mice also showed that at 24h after two successive heme loads, liver HO-1 transcription and protein increased 7- and 2.3-fold over baseline, respectively (17
). That heme loads apparently have a more dramatic and sustained effect on HO-1 transcription in adult (17
) than in newborn mice may occur in part because HO is developmentally regulated in the liver, being expressed at a higher level in the neonate than the adult. This observation has been shown in the rat liver (23
) and the mouse cortex (24
). There may also be suppression of neonatal HO-1 inducibility in response to stresses such as heme loading. Previous reports by Kassovska-Bratinova et al (25
), Di Giulio et al (26
), and Lavrovsky et al (27
) have demonstrated reduced HO-1 inducibility in the neonatal rat because of increased levels of the known HO-1 re-pressor Bach1 in infancy (25
) or increased levels of the HO-1 inducer NF-κB in adulthood (27
). These mechanisms and their potential relevance to hemolysis merit further investigation.
It is possible that the induction of liver and spleen HO-1 in the H-Zn-H group occurs in response to not simply the exogenous heme from heme loads, but also the native heme that remains unmetabolized following the inhibition of HO by ZnBG. Kappas et al reported that following inhibition of HO by tin protoporphyrin, the unmetabolized heme is eventually excreted into the bile (28
), and we found that this excretion is proportional to the degree of HO inhibition (29
). It is likely that after inhibition of HO by ZnBG, unmetabolized heme (including any excess from heme loading) is eventually excreted through the same route.
Our lab (30
) and Bonkovsky’s (31
) have shown that various metalloporphyrins, including SnMP, upregulate HO-1 gene transcription by sequestering or causing downstream degradation of Bach1. However, we believe that ZnBG has much less interaction with Bach1 than CrMP or SnMP (Stephanie Schulz, personal communication), which suggests that ZnBG upregulates HO-1 through an as-yet-unknown mechanism.
Although ZnBG is similar in photoreactivity to SnMP, ZnBG’s high potency could make it clinically effective at much lower doses (11
). The present study showed the effectiveness of oral doses as low as 3.75-μmol ZnBG/kg BW in suppressing HO activity, and also showed the effectiveness of 15-μmol ZnBG/kg BW in our hemolytic newborn mouse model. Additionally, unlike SnMP, ZnBG has minimal effects on NOS and sGC (14
). To further examine the clinical utility of ZnBG, ongoing work includes light exposure studies comparing the relative photosensitivities of newborn mice under treatment with SnMP, CrMP, and ZnBG.
We have demonstrated that ZnBG is orally absorbable, inhibits both basal and heme-induced HO activity in the liver and spleen, and also reduces in vivo bilirubin production. Our data strongly suggest that even if ZnBG does upregulate HO-1 transcription and translation, the effect is a transient one. ZnBG is therefore an attractive compound for oral use in the treatment of neonatal jaundice caused by hemolytic disease, comparable or even preferable to SnMP.