Lipophilicity Determines Routes of Uptake and Clearance, and Toxicity of an Alpha-Particle-Emitting Peptide Receptor Radiotherapy

Abstract

Lipophilicity is explored in the biodistribution (BD), pharmacokinetics (PK), radiation dosimetry (RD), and toxicity of an internally administered targeted alpha-particle therapy (TAT) under development for the treatment of metastatic melanoma. The TAT conjugate is comprised of the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), conjugated to melanocortin receptor 1 specific peptidic ligand (MC1RL) using a linker moiety and chelation of the ²²⁵Ac radiometal. A set of conjugates were prepared with a range of lipophilicities (log D _7.4 values) by varying the chemical properties of the linker. Reported are the observations that higher log D _7.4 values are associated with decreased kidney uptake, decreased absorbed radiation dose, and decreased kidney toxicity of the TAT, and the inverse is observed for lower log D _7.4 values. Animals administered TATs with lower lipophilicities exhibited acute nephropathy and death, whereas animals administered the highest activity TATs with higher lipophilicities lived for the duration of the 7 month study and exhibited chronic progressive nephropathy. Changes in TAT lipophilicity were not associated with changes in liver uptake, dose, or toxicity. Significant observations include that lipophilicity correlates with kidney BD, the kidney-to-liver BD ratio, and weight loss and that blood urea nitrogen (BUN) levels correlated with kidney uptake. Furthermore, BUN was identified as having higher sensitivity and specificity of detection of kidney pathology, and the liver enzyme alkaline phosphatase (ALKP) had high sensitivity and specificity for detection of liver damage associated with the TAT. These findings suggest that tuning radiopharmaceutical lipophilicity can effectively modulate the level of kidney uptake to reduce morbidity and improve both safety and efficacy.

Scheme 1. Synthetic Route of DOTA-Linker-MC1RL Compounds

Figure 1

Competitive binding assay for La³⁺-DOTA-linker-MC1RL compounds.

Scheme 2. Radiochemical Synthesis of ²²⁵Ac-DOTA-linker-MC1RL Compounds

Figure 2

BD results for (A) ²²⁵Ac-DOTA-MC1RL, (B) ²²⁵Ac-DOTA-Ahx-MC1RL, (C) ²²⁵Ac-DOTA-DLDL-MC1RL, and (D) ²²⁵Ac-DOTA-DLDG-MC1RL. Activities were calculated for tissues rendered from BALB/c mice (n = 6 per time point). The time courses vary among the different compounds due to differences in animal survival and liver clearance.

Figure 3

Radiation dosimetry of ²²⁵Ac and daughters following administration of (A) ²²⁵Ac-DOTA-MC1RL (no linker), (B) ²²⁵Ac-DOTA-Ahx-MC1RL, (C) ²²⁵Ac-DOTA-DLDL-MC1RL, and (D) ²²⁵Ac-DOTA-DLDG-MC1RLin BALB/c mice.

Figure 4

Gross appearance of kidneys (A, B) and histological appearance of kidney (C–F) and liver (G–J) from mice treated with different peptides in the toxicity study. (A, B) Gross appearance of kidneys from mice injected with 79.4 kBq of ²²⁵Ac-DOTA-DLDL-MC1RL and 305.4 kBq ²²⁵Ac-DOTA-MC1RL. (C) Kidney histology of a 135.4 kBq of ²²⁵Ac-DOTA-DLDL-MC1RL administered mouse, with acute tubular necrosis resulting in death at 11 days after administration. At necropsy, kidneys appeared grossly pale, pitted, small, and irregular in shape. Histologically, tubular cell necrosis was characterized by intense cytoplasmic eosinophilia with pyknotic nuclei (arrows), while other tubules appeared regenerative with cytoplasmic basophilia and nuclear crowding (arrowheads). (D) Kidney of a 293.4 kBq of the ²²⁵Ac–No linker administered mouse with nephropathy comprised of tubular epithelial cell degeneration with cytoplasmic vacuolization, necrosis, and regeneration with cytoplasmic basophilia and nuclear crowding, hypercellularity of glomerular tufts, and focal infiltration of mononuclear cells (arrow). (E) Kidney histology of a 84.2 kBq of ²²⁵Ac-DOTA-DLDG-MC1RL administered mouse euthanized 7 months after administration, with chronic progressive nephropathy. Little normal renal parenchyma remains due to extensive tubular cell necrosis (arrow), epithelial sloughing and cast formation, extensive tubular cell regeneration (arrowheads), diffuse interstitial edema and fibrosis with mild mononuclear inflammatory cell infiltrates, and hypercellular glomerular tufts. (F) Kidney of saline-treated mouse with normal eosinophilic cuboidal tubular epithelium, and normal glomerluli. (G) Liver of 292.2 kBq of the ²²⁵Ac-DOTA-Ahx-MC1RL administered mouse with hepatocellular eosinophillic cytoplasmic swelling, single hepatocellular apoptosis (arrows) with hypereosinophillic cytoplasm and pyknotic nuclei, and single hepatocellular necrosis (arrowheads) with pale eosinophilic cytoplasm and karyolysis. (H) Liver histology of a 135.4 kBq of ²²⁵Ac-DOTA-DLDL-MC1RL administered mouse, with focal hepatocellular eosinophillic cytoplasmic swelling, single hepatocellular apoptosis (arrows) with hypereosinophillic cytoplasm and pyknotic nuclei, and single hepatocellular necrosis (arrowhead) with pale eosinophilic cytoplasm and karyolysis. (I) Liver histology of a 36.0 kBq of ²²⁵Ac-DOTA-DLDL-MC1RL administered mouse, with hepatocellular eosinophillic cytoplasmic swelling, hepatocellular apoptosis (arrows), and hepatocellular necrosis (arrowhead). (J) Liver of saline administered mouse with normal, well-delineated hepatic cords and sinusoids, a portal triad (left), and a central vein (right).

Figure 5

Toxicity probability curves per injected activity for each radiopharmaceutical per (A) kidney pathology, (B) liver pathology [no toxicities were observed for the other two radiopharmaceuticals], (C) BUN, (D) ALKP, and (E) weight loss.