Ex vivo hepatotoxicity of 201a and 201b in precision cut liver slices

As shown in the previous section, toxicity of is low in different cancer cell lines. In addition to the in vitro results, the toxicity towards healthy cells or rather tissue is an important property concerning a possible use of the compound as drug. An attractive model is thereby the use of precision cut liver slices (PCLS), in which hepatotoxicity can be evaluated. Hepatotoxicity is thereby one of the major reasons for termination and for limitations by labelling medicines.^[270]

Precision cut tissue are viable explants of the tissue cultured ex vivo with all cell types in their natural environment, providing toxicity results which are expected to be more relevant than those obtained in cell cultures. Using this technology, all cells remain in their natural environment with maintenance of the original cell-cell and cell-matrix contacts, which is absent in classical 2D cell cultures in vitro.^[271–276] Notably, the technique is an FDA-approved model for drug toxicity and metabolism studies, and it has previously been used for the assessment of toxicity of metal-based compounds like cisplatin (kidney),^[277] experimental gold compounds (liver, kidney, and colon),^[278]

and amino-ferrocene-containing prodrugs (liver).^[279] The tissue in the experiment is sliced with a reproducible and well defined thickness, which is small enough to allow sufficient oxygen and nutrient supply to the inner cell layers, ensuring the viability of all cells. Slicing thereby allows a very efficient use of the organ tissue,^[280] since numerous compounds and conditions can be tested at the same time with only one organ. Precision cut tissue slices of the liver are frequently used to assess hepatotoxicity in healthy tissue.^[271–273] This is an enormous advantage compared for testing toxicity in single cell cultures, because drug-induced hepatotoxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells.^[281]

Based on previously discussed criteria, RR97a (201a) and RR97b (201b) were selected for hepatotoxicity investigations at the Rijksuniversiteit Groningen according to established procedures using rat precision cut liver slices (PCLS). Again, RR97b as ineffective kinase inhibitor serves as control compound, since any toxicity should be induced by other mechanisms than kinase inhibition. For the hepatotoxicity evaluation, PCLS were prepared and pre-incubated for 1 h before exposing to the rhodium containing complexes as described in the experimental section. The preincubation thereby is necessary to allow the tissue to recover from the cold ischemia and the slicing procedure. The treatment with RR97a and RR97b was performed at three single concentrations of the compounds (1 µM, 10 µM and 50 µM) with a final DMSO concentration of less than 0.5% and slices were incubated for 24 h. Afterwards PCLS were collected and their viability was determined measuring their ATP content using a bioluminescence assay and normalising it to their protein content, obtained by the LAWRY method, and thereby to the size of the tissue slice.

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Fig. 46: Viability of precision cut liver slices (PCLS) after 24 h incubation with the two diastereomeric complexes RR97a and RR97b after 1 h of preincubation.

Fig. 46 reports the viability of the PCLS upon treatment with different compound concentrations.

Based on the obtained results, both compounds are not toxic for the tissue at a concentration of 1 µM. At 10 µM concentration, some effects on tissue viability can be observed with respect to controls, with RR97a being markedly more toxic than RR97b (20% and 69% residual viability, respectively). The hepatotoxicity increases with the highest tested compound concentration (50 µM), RR97a leading to the most pronounced effect (9% residual viability compared to 30% for RR97b). Here again at high concentrations some precipitate occurred, maybe falsifying the data insofar that the compound might be more toxic than it seems in the experiment.

Notably, the inactive isomer RR97b presents reduced hepatotoxicity with respect to cisplatin in the same rat liver model. In fact, at 10 µM the anticancer Pt complex has been observed to induce a reduction of tissue viability up to 50% (unpublished data from Prof. Angela Casinis laboratory). In general anticancer agents are expected to show toxicity at therapeutically active doses, since the therapeutic effect and toxicities normally rely on the same mechanisms,^[189] so some overall toxicity of the more active KI is not surprising. As a general trend with KIs, the severity of organ toxicity seems to be associated with increased kinase promiscuity,^[22] which also aligns with the fact that RR97b does not inhibit kinases well, and RR97a targets more than one kinase.^[233]

Whether this toxicity is actually caused by inhibition of different kinases, or other tissue targets, is unclear. However the kinase Pim1, one of the preferred target kinases of RR97a, is known to be highly expressed in the fetal human liver but not in adult tissue,^[40] indicating that inhibition of Pim1 may not be the reason. To sum up, the different diastereomers seem to not only possess different KI properties, but also different toxicity effects, which seem not to be related to the Pim1 inhibition properties of RR97a, at least in the liver tissue, and may therefore arise from different physicochemical properties or off-target effects.

0 20 40 60 80 100 120 140

50 µM 10µM 1 µM

ATP/protein [%]

RR97b RR97a

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3.2.3.2.1. Metal uptake of PCLS after treatment with RR97a and RR97b

To investigate whether the amount of compound, respectively Rh, might influence the toxicity of the complexes RR97a and RR97b towards PCLS, the metal uptake after the incubation time was studied for the same concentration range. Therefore, PCLS were treated as described for the hepatotoxicity study until the end of the 24 h incubation time. The slices were then washed in Krebs medium and individually frozen at -80 °C and kept at this temperature until digestion with HNO3 as described in the experimental section. The content of Rh was measured via ICP-MS in the mass department of the Philipps-Universität Marbug (inductively coupled plasma mass spectrometry) and determined using an external Rh calibration curve (Fig. 47). Due to the previously shown high stability of the compounds in assay medium, the values are expected to reflect the true values for the complex.

0 10 20 30 40 50

-200 0 200 400 600 800 1000 1200

ng Rh (105 )/g tissue

Compound conc. [µM]

RR97a PCLS RR97b PCLS

0 10 20 30 40 50

0 400 800 1200 1600 2000

ng Rh/g medium

Compound conc. [µM]

RR97a medium RR97b medium

Fig. 47: Rh content of PCLS (left) and medium (right) after 24 h treatment. It seems that RR97b has a better uptake by the tissue compared to RR97a.

Taken from Fig. 47 it seems that RR97b is taken up to a higher degree compared to RR97a.

However, based on the high deviation these results are rather an estimation than a real value, as the samples were weighted in after thawing in its wet states, and not always all tissue was transferred. Additionally, similar amounts of Rh is found in the medium for both samples. Both compounds precipitated to a certain degree from the solution at high concentrations, so that the amount of compound in the medium might not be truly reflective. The trend of a higher uptake of RR97b is thereby a disagreement to the higher hepatotoxicity and cytotoxicity obtained in the previous assays.

91 3.2.3.2.2. Morphology of PCLS after treatment

During the treatment of PCLC with RR97a and RR97b some of the compound precipitated in wells with high concentrations, as previously described. In addition to that the colour of slices changed from its natural brownish colour^[280] to an intense pinkish red, indicating the confirmed uptake of the compound (see 3.2.3.2.1), as shown in Fig. 48.

Fig. 48: Left: PCLS after 24 h incubation with 10 µM RR97a. The intense pink colour reveals the uptake of the deep red complex.

Middle and right: Light microscope pictures of the freshly incubated slices showing deep red spots, which might be accumulations of RR97a.

Since the colour seemed to be distributed unevenly in the slices, the question arose whether the compound accumulates on the surface of the slice, in structural features like blood capillaries or inside the cells. Therefore, the tissue was first examined in its wet state using a standard light microscope (Fig. 48). The samples indeed showed deep red spots, which were not to be seen in untreated samples and which were distributed unevenly. To further verify whether the compound accumulates on the surface or penetrates the tissue, morphology studies with tissue cross-sections were conducted. Therefore, the slices were treated according to the hepatotoxicity experiment until the 24 h incubation with the compound. The slices were then fixed in 4% w/v formalin, dehydrated using a graded EtOH series and paraffin embedded. Cross sections of 4 µm thickness of the paraffin block were prepared and stained using haematoxylin and eosin. Haematoxylin thereby stains the nucleus (chromatin) blue and eosin stains the cytoplasm pink. The cover slips were scanned using an Aperio image capture device (LEICA BIOSYSTEMS). The treated sample and the 24 h reference control are show in Fig. 49.

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Fig. 49: Comparison of morphology of dried and stained PCLS. Left: After treatment with RR97a for 24 h. Right: 24 h control.

The cell nuclei were stained blue using haematoxylin, the cytoplasm was stained pink with eosin. No signs of compound accumulation can be seen.

As can be seen from Fig. 49 the positon of accumulation could not be verified using this method.

One possible explanation is, that accumulations were dissolved in one of the solutions used in the dehydration, embedding or staining process. This would indicate, that the accumulations were located extracellular, so that the solvent could reach and dissolve them. Keeping the results from np829 (80b) in mind, the previously observed toxicity for RR7a might be related to accumulations.

However, an effect like the crystallisation in cells, as it has been detected for 80b, was not seen in any experiment with RR97a, pointing to another reason for the observed hepatotoxicity and minor cytotoxicity in cancer cells.

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