Pharmacokinetic profile and in vivo anticancer efficacy of anagrelide administered subcutaneously in rodents

Abstract

Anagrelide (ANA) is a phosphodiesterase 3A (PDE3A) inhibitor, commonly prescribed for essential thrombocythemia. It also functions as a molecular glue, inducing complex formation between PDE3A and Schlafen 12. This association either triggers apoptosis or inhibits proliferation in tumor cells, supporting its use in cancer therapy. Conventionally administered orally, ANA undergoes rapid metabolism and elimination, resulting in a short drug exposure time at the site of action. Here, we explored the pharmacokinetic profile of a subcutaneously (SC) injected ANA formulation in Sprague-Dawley rats by quantifying plasma ANA and metabolite concentrations using liquid-chromatography-tandem mass spectrometry. We further evaluated the in vivo tumor regression efficacy of orally and SC administered ANA in a patient-derived gastrointestinal stromal xenograft mouse model - UZLX-GIST2B - characterized by a KIT exon 9 driver mutation. The SC ANA exhibited extended-release plasma concentration-time profiles compared to intravenous and oral administrations. After a single administration in rats, plasma concentrations of ANA were detected up to 56 days later, and ANA metabolites up to 30 days later. The SC formulation also significantly reduced tumor volumes and demonstrated dose-dependent histological responses, nearly eradicating tumor tissue in 11 days with the highest dose. These findings suggest that the SC slow-release formulation maintains stable drug concentrations during treatment, potentially improving therapeutic efficacy at the target site.

Keywords: Anagrelide; GIST; pharmacokinetics; slow-release; subcutaneous.

Figure 1.

Plasma concentration–time profiles of (A) ANA and its metabolites, (B) 3-OH-ANA, and (C) RL603, after three different administration routes: IV, PO, or SC in rats. Metabolite concentrations quickly peaked after IV and PO administrations, but stayed relatively stable with SC administration. Data are presented as mean and standard deviation. RL603 was not detected above the quantifiable limit of 0.2 ng/mL after SC administration.

Figure 2.

Plasma concentration–time profiles of (A) ANA and its metabolites, (B) 3-OH-ANA, and (C) RL603, after SC administration to SD rats of two different concentrations, 7 mg/kg and 35 mg/kg. Data are presented as mean and standard deviation.

Figure 3.

Treatment effects of ANA administered via PO or SC in a patient-derived GIST xenograft. (A) SC ANA treatment resulted in best tumor size reductions. *Tumors that were already very small at the beginning of the experiment. (B) Tumor samples were stained with H&E for histological response evaluation. (C) Treatment efficacy was graded based on the amount of necrosis, myxoid degeneration, and fibrosis in the H&E-stained tumor tissues.

Figure 4.

Protein expression was investigated in tumor tissue. (A) PDE3A was strongly detected with IHC from formalin-fixed paraffin-embedded FFPE tumor tissue samples, although the SC ANA-treated tumors exhibited less PDE3A-positive cells. (B) KIT was also strongly expressed in the tumor cells, and KIT-positive cells were observed less in SC ANA-treated tumor samples. (C) SLFN12 was weakly visible with IHC from all tumor samples. (D) SC ANA treated tumors contained multiple HLA-A negative cells, most probably originating from the host animal.