Repurposing erectile dysfunction drugs tadalafil and vardenafil to increase bone mass

Abstract

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

Keywords: PDE5 inhibitor; computational modeling; cyclic GMP; osteoporosis; resorption.

Fig. 1.

Expression and in vitro actions of PDE5A inhibitors tadalafil and vardenafil. (A) TaqMan-based PCR using bone RNA showing the expression of 20 murine PDE isoforms, soluble guanylate cyclase isoforms (Gucy1a2 and Gucy1a3), protein kinase G isoforms (Prkg1 and Prkg2), as well as Tnfrsf11a and Tnfsf11 (controls). SYBR Green-based PCR using bone RNA from 10- and 40-wk-old mice showing the expression of Pde5a. Data are expressed as copy number per cell (normalized to Tubulin, Rps11, Actb, and/or Gapdh). Data are mean ± SEM. n = 3 biological replicates for TaqMan; n = 4 biological replicates for SYBR Green. (B) Data obtained from Human Genome U133 2.0 GeneChip Arrays (Affymetrix). The presence of transcripts was determined from the signal of perfect matched and mismatched probe pairs in each probe set, with statistical confidence (P value) indicated. Characteristic highly expressed osteoclastic and osteoblastic transcripts are also included as controls. (C) Photomicrographs showing immune labeling of PDE5A (blue, solid arrowhead) and RUNX2 (Left) or CSF1R (Middle) (brown, hollow arrowhead) in mouse bone marrow (colocalization is shown in purple, solid arrow), together with a control image without primary antibody (Right) (details in Methods). (D) Effect of tadalafil and vardenafil on Cfu-ob in 21-d cultures of bone marrow stromal cells isolated from marrow of 10-mo-old male mice in differentiating medium, shown as representative alizarin red-positive Cfu-ob and mean ± SEM absorbance of extracted dye per well (in triplicate). (E) Effect of tadalafil and vardenafil on tartrate-resistant acid phosphatase-positive (ACP5⁺) osteoclasts at 5 d following the incubation of bone marrow hematopoietic stem cells with RANKL and M-CSF, shown as representative plates and mean ± SEM ACP5⁺ osteoclast number per well (in triplicate). Statistics for D and E: unpaired two-tailed Student’s t test; *P < 0.05, **P < 0.01, or as shown.

Fig. 2.

Localization of PDE5A in sympathetic neurons in three brain regions. (A) Representative confocal photomicrographs showing the colocalization (yellow) of PDE5A (green) and DBH (red) immunolabeling in a number of neurons of the locus coeruleus (LC), raphe pallidus (RPa), and paraventricular nucleus of the hypothalamus (PVH). Also shown is the map of brain areas. (B) Retrograde transsynaptic virus-mediated tract tracing using a pseudorabies virus strain, PRV152, that expresses EGFP under control of the human cytomegalovirus immediate-early promoter. PRV152 was injected into the metaphysis or subperiosteally (shown as schematic) in live anesthetized mice at 6 d before sacrifice. Brain regions were dissected and processed for PDE5A (green) and EGFP (red) immunohistochemistry. The virus traversed from bone via the sympathetic nervous system to the three brain regions, LC, Rpa, and PVH, where it colocalized with PDE5A (yellow). Refer to SI Appendix, Fig. S1.

Fig. 3.

PDE5A inhibitors tadalafil and vardenafil increase bone mass. (A) Areal BMD of the total skeleton and of L4-6 vertebrae and right femur and tibia in female mice treated with vehicle (gray), tadalafil (green), or vardenafil (blue) measured at 0, 2, 4, and 6 wk by a PIXimus small animal densitometer (details and doses in Methods). (B) Representative images of 3D reconstructions of L5 vertebral trabecular bone at 6 wk following vehicle or drug treatment. Static quantitative morphometry from μ-CT, providing measures of vBMD, BV/TV, Tb.N, Tb.Th, Tb.Sp, and connectivity density (Conn.D). Data are shown as mean percent change ± SEM (vs. vehicle). Statistics: two-tailed Student’s t test; *P < 0.05, **P < 0.01, or as shown vs. vehicle; n = 4 to 5 mice per group.

Fig. 4.

PDE5A inhibitors tadalafil and vardenafil stimulate bone formation and reduce bone resorption. (A) Representative low-power photomicrographs (750-µm field) of calcein double labels with a magnified view (Bottom), as well as ACP5-stained surfaces in sections of vertebral trabecular bone at 6 wk following vehicle, tadalafil, or vardenafil treatment. (B and C) Histomorphometric analysis showing measured and calculated parameters of bone formation—MS, MAR, and BFR (5 to 24 sections from two or three mice) (B)—as well as number of osteoclasts (N.Oc) per bone surface (BS) or volume (BV) (9 to 20 sections from 3 to 5 mice) (C) (details and doses in Methods). Blinded measurements were made. Statistics: two-tailed Student’s t test; *P < 0.05, **P < 0.01, or as shown vs. vehicle. Osteoblastic gene signature (SYBR Green qPCR) consisting of a set of differentially regulated differentiation genes (Ogn, Bsp, Alp, Runx2, Tnfsf11, Col1a1, and Bmp2) (D and E), protein kinase G isoforms (Prkg1 and Prkg2) (F), and the unique sympathetic relay signature (39, 44) comprising Fos, Jun, Clock, Per1, Per2, Bmal1, Ccnd, and Myc (G), in differentiating osteoblast precursors from mice treated with tadalafil or vardenafil. (H) Schematic representation of the predicted roles of central and peripheral PDE5A inhibition and of reduced osteoclastic resorption in bone gain.

Fig. 5.

Structural modeling of tadalafil and vardenafil with mouse PDE5A. (A) Structures of tadalafil (blue) and vardenafil (orange) positioned in the mouse PDE5A catalytic domain. The positions of Zn²⁺ (gray) and Mg²⁺ (magenta) are highlighted in the pocket. Note that catalytic activity occurs via direct interactions between cGMP and the divalent cations Zn²⁺ or Mg²⁺ (52). (B) Close-up of interactions by tadalafil (Ta) and vardenafil (Va) in the catalytic pocket. (C) Two-dimensional plot of interactions of tadalafil and vardenafil in the binding site. Note the steric clash that vardenafil makes with the side chain of M806. SI Appendix, Fig. S2 provides validation details.