The oral administration of curcumin to humans, mice and rats has resulted in plasma levels typically not exceeding 1 μM concentrations and similarly low tissue levels (for recent reviews and references therein see: [26–28, 44]). Alternatively, curcumin has been delivered to mice or rats by intraperitoneal (i.p) injection at dosages of 6 to 100 mg/kg body weight [15, 45–47]. For this purpose, curcumin was either dissolved in DMSO [45–47] or in NaOH followed by neutralization . In those studies, peak curcumin plasma concentrations were within the range of approximately 3.5 to 25 μM and brain levels approximately 1 to 2 nmol/g. After i.v. injection in rats of 10 mg/kg curcumin solubilized in a cocktail containing DMA/PEG/dextrose, initial plasma concentrations of approximately 27 μM were reported . Similar results were reported in another study in which curcumin had been solubilized in glycerol formal and i.v. injected at a dosage of 40 mg/kg . By comparison, in the present study mice were i.v. injected with 0.1 ml of 24 mM curcuminoids solubilized in 10% HP-γ-CD. This represents a total dose of 0.84 mg or approximately 33 mg/kg curcuminoids. Under these conditions, initial curcuminoid plasma concentrations of about 100 μM were attainable. Adjusted for the total dose applied, these concentrations are similar to those observed for the rat , but they resulted in transient brain concentrations of approximately 47 nmol/g (Figure 4), which were higher than those reported in any other study. Similarly, a four-fold higher curcuminoid dose (approximately 134 mg/kg) administered by s.c. injection yielded maximal plasma concentrations of approximately 23 μM and brain levels of approximately 8 nmol/g. Although these amounts are lower than those achieved by i.v. injection, the parental curcuminoids were released gradually from the injection site and they persisted longer in both plasma and brain (Figure 5). These levels are also higher than those typically observed after i.p. injection (see above). However, in one study curcumin was administered at a much lower dose (3 mg/kg) and this yielded relatively high brain levels of approximately 3.2 nmol/g at four hours after intramuscular injection .
The injection protocol presented here combines the high solubility of curcuminoids in 10% HP-γ-CD with the relatively low level of toxicity of the carrier vehicle. In addition, HP-γ-CD does not cause apparent immune reactions that are associated with the use of heterologous serum to solubilize curcuminoids. However, at the highest doses used for i.v. injection (33 mg/kg), toxic reactions do develop and these may represent the maximum amount of curcuminoids tolerated before lethal effects occur. After a single i.v. injection, high plasma levels are exceedingly transient and the concentration of native curcuminoids drops to insignificant levels within 20 minutes (Figure 4). The decline in the level of circulating curcuminoids can be considered due to rapid metabolism combined with widespread binding/uptake to cells and tissues.
The binding of curcuminoids to cells in culture has been described in detail elsewhere . Those studies were expanded to include preparations employed here, where curcuminoids had been sequentially solubilized in mouse serum and 10% HP-γ-CD (Figure 8). This was done by first adding curcuminoids as a solid powder followed by the addition of DMSO-dissolved curcuminoids. This method results in a maximal solubility of curcuminoids and a relatively balanced composition . The apparent binding KDs in these preparations were higher for curcuminoids solubilized in mouse serum (14.88 μM) than in FCS (9.16 μM). Since mouse serum has the capacity to solubilize higher concentrations of curcuminoids than FCS (3 to 4 mM vs. approximately 1.7 mM ), it is likely that these differences in binding KDs reflect differences in affinities or concentrations between the curcuminoid interaction domains in serum components, as it may be reasonably assumed that the cellular binding affinities remain constant. It is further expected that curcuminoids solubilized in sera from different species or possibly different preparations from the same species, will produce different apparent cellular binding KDs due to their variable serum compositions. In addition, alternative solubilization vehicles may have differential affinities for individual curcuminoids. This is exemplified by the cellular binding of individual curcuminoids solubilized in HP-γ-CD (Figure 9). Although curcuminoids solubilized in either HP-γ-CD or serum have similar compositions (Figure 1), the primary curcuminoids bound to cells at saturating concentrations were BDMC and CUR, respectively. However, upon dilution with excess serum, both preparations converged to the same compositions of cellular-bound curcuminoids (Figure 9). Although these observations were made with cultured cells, it is likely that similar binding occurs in vivo. Indeed, the extensive binding of curcuminoids to brain (Figure 6) and other organs (not shown) shows a similar distribution pattern as that obtained with cultured cells incubated with curcuminoids and excess serum. Except for considerations relating to solubilization capacity, toxicity or immune reactions, it is in this case irrelevant whether curcuminoids are solubilized in serum or HP-γ-CD.
The metabolic conversion of curcuminoids after i.v. injection is rapid and it includes both conjugation and reduction products (Figure 4). Since conjugation takes place in the liver, intestines and kidneys, the resulting products are observed primarily in plasma but also at the sites of excretion [48–51]. In addition to conjugation products, the hexa- and octahydrocurcuminoid reduction products are also prominently represented in the circulation. It is likely that these are primarily contributed by curcuminoids that were taken up by peripheral cells and tissues, metabolized locally, and subsequently released into the bloodstream. In contrast, after s.c. injection the curcuminoid reduction products are not readily detected in plasma. This is most likely due to the more gradual release from tissues followed by rapid excretion. Although only the monoconjugates of sulfate and glucuronide were investigated here, it is likely that mixed diconjugates are also produced [48, 50, 51]. Some studies have reported the formation of tetrahydrocurcumin in mice [47, 49], which was not detected in this study. Instead, the hexa- and octahydrocurcuminoids predominated. In addition, different reduction products with different physical characteristics, here referred to as dihydrocurcuminoidols, were detected in the brain. These compounds were first identified as the final reduction products in the teratocarcinoma cell line NT2/D1 , while its presence in vivo has not been previously reported. However, dihydrocurcuminoidols are most prominently produced after perfusion under post-mortem conditions. Their time-dependent production in the brain following i.v. injection also seems to be correlated with the amount of unmodified curcuminoids present. It can, therefore, not be excluded that this is a post-mortem effect that rapidly occurs during the removal of tissues. The enzyme systems responsible for the reductive conversion of curcuminoids have not yet been conclusively identified. However, it appears that octahydrocurcuminoids are sequentially generated from hexahydrocurcuminoids. For example, during perfusion only hexahydrocurcuminoids together with smaller amounts of dihydrocurcuminoidols were generated and since the post-mortem conversion to octahydrocurcuminoids was blocked, there was a larger relative accumulation of hexahydro-BDMC compared to in vivo conditions (Figure 6). This indicates that the overall conversion to octahydrocurcuminoids requires distinct enzyme systems with different substrate specificities and metabolic requirements. The more efficient generation of octahydro-BDMC than octahydro-CUR is consistent with this notion, as is the gender-specific generation of reduction products in the rat . The curcumin reducing enzymes have also been found to be distributed between cytoplasmic and microsomal compartments . In addition, different cell lines in culture produce different reduction products. For example, in the astrocytoma cell line CCF-STTG1, reduction proceeds to the octahydrocurcuminoid stage, whereas in HeLa cells hexahydrocurcuminoids are the end products . These cell lines also produce varying amounts of dihydrocurcuminoidols . Based on these observations, possible alternative reduction pathways for curcuminoid reduction are proposed (Figure 7).