Design, Synthesis, Pharmacokinetics, and Biodistribution of a Series of Bone-Targeting EP4 Receptor Agonist Prodrugs for Treatment of Osteoporosis and Other Bone Conditions

Abstract

A series of bone-targeting EP4 receptor agonist conjugate prodrugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Singly and doubly radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream, and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up biodistribution and elimination studies. Doubly radiolabeled conjugate 4 was found to partition largely to the liver and bones, and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from bone-bound 4 with a half-time of about 7 days. When dosed orally, radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged, and only traces of radiolabeled 4 were found in the liver, blood, or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate, forming a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

Figure 1

Prostaglandin E₂ (1), EP4 receptor agonist 2 and the conjugate drug C1 (3).

Figure 2

Conjugate C1 (3) and its conversion to liberate components.

Figure 3

Conjugate prodrugs designed to target bone and liberate EP4 agonist and an inactive bisphosphonate which remains bound to bone. The red triangle triads indicate the ¹⁴C-labeled sites, and the green triangle triads indicate the ³H-labeled sites.

Figure 4

Target conjugates and their putative liberated bisphosphonate components.

Scheme 1. Synthesis of Prodrug Conjugates 4–6 and Their Tritiated Analogs

Scheme 2. Synthesis of the Conjugate Fragments 8–10

Scheme 3. Synthesis of Conjugate 7 (C6)

Scheme 4. Synthesis of Conjugate Fragment 11

Scheme 5. Synthesis of [¹⁴C]-4

Figure 5

Blood levels of tritium (±SEM) after intravenous dosing of [³H]-4, [³H]-5, and [³H]-6 (to 6 h) (average ± half differential for 4).

Figure 6

Tissue levels of tritium (±SEM) after intravenous dosing of [³H]-4, [³H]-5, and [³H]-6 (after 6 h) (average ± half differential for 4).

Figure 7

Release of tritium label from bones over 28 days after dosing with [³H]-4.

Figure 8

Percentage of originally dosed radiolabel in blood after intravenous dosing of [³H]-4 and [¹⁴C]-4 at 5 mg/kg.

Figure 9

Percentage of originally dosed radiolabel found in long bones after intravenous dosing of [³H]-4 and [¹⁴C]-4 at 5 mg/kg. Data for tritium levels at 7 and 14 days is from the previous experiment where [³H]-4 was dosed alone.

Figure 10

Percentage of originally dosed radiolabel found in liver after intravenous dosing of [³H]-4 and [¹⁴C]-4 at 5 mg/kg.

Figure 11

Neutrophil chemotaxis (nm/min) measured in a Zigmond chamber induced by fMLP (1 μM) and blank or with fMLP plus compound. Murine bone marrow neutrophils were pretreated at 37 °C for 30 min with compound (at 50 μM) or blank buffer in the case of fMLP alone. Each experiment is the average of measure of tracked movement of at least 30 cells over 15 min.