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. 2024 Jun 5;9(7):890-902.
doi: 10.1016/j.jacbts.2024.04.006. eCollection 2024 Jul.

Novel Tryptophan Hydroxylase Inhibitor TPT-001 Reverses PAH, Vascular Remodeling, and Proliferative-Proinflammatory Gene Expression

Ekaterina Legchenko  1 , Philippe Chouvarine  1 , Fatimunnisa Qadri  2 , Edgar Specker  3   4 , Marc Nazaré  3 , Radoslaw Wesolowski  2   4 , Susann Matthes  2 , Michael Bader  2   4   5   6   7 , Georg Hansmann  1   8
Affiliations

Novel Tryptophan Hydroxylase Inhibitor TPT-001 Reverses PAH, Vascular Remodeling, and Proliferative-Proinflammatory Gene Expression

Ekaterina Legchenko et al. JACC Basic Transl Sci. .

Abstract

The serotonin pathway has long been proposed as a promising target for pulmonary arterial hypertension (PAH)-a progressive and uncurable disease. We developed a highly specific inhibitor of the serotonin synthesizing enzyme tryptophan hydroxylase 1 (TPH1), TPT-001 (TPHi). In this study, the authors sought to treat severe PAH in the Sugen/hypoxia (SuHx) rat model with the oral TPHi TPT-001. Male Sprague Dawley rats were divided into 3 groups: 1) ConNx, control animals; 2) SuHx, injected subcutaneously with SU5416 and exposed to chronic hypoxia for 3 weeks, followed by 6 weeks in room air; and 3) SuHx+TPHi, SuHx animals treated orally with TPHi for 5 weeks. Closed-chest right- and left heart catheterization and echocardiography were performed. Lungs were subject to histologic and mRNA sequencing analyses. Compared with SuHx-exposed rats, which developed severe PAH and right ventricular (RV) dysfunction, TPHi-treated SuHx rats had greatly lowered RV systolic (mean ± SEM: 41 ± 2.3 mm Hg vs 86 ± 6.5 mm Hg; P < 0.001) and end-diastolic (mean ± SEM: 4 ± 0.7 mm Hg vs 14 ± 1.7 mm Hg; P < 0.001) pressures, decreased RV hypertrophy and dilation (all not significantly different from control rats), and reversed pulmonary vascular remodeling. We identified perivascular infiltration of CD3+ T cells and proinflammatory F4/80+ and CD68+ macrophages and proliferating cell nuclear antigen-positive alveolar epithelial cells all suppressed by TPHi treatment. Whole-lung mRNA sequencing in SuHx rats showed distinct gene expression patterns related to pulmonary arterial smooth muscle cell proliferation (Rpph1, Lgals3, Gata4), reactive oxygen species, inflammation (Tnfsrf17, iNOS), and vasodilation (Pde1b, Kng1), which reversed expression with TPHi treatment. Inhibition of TPH1 with a new class of drugs (here, TPT-001) has the potential to attenuate or even reverse severe PAH and associated RV dysfunction in vivo by blocking the serotonin pathway.

Keywords: drug discovery; pulmonary arterial hypertension; serotonin; tryptophan hydroxylase inhibitor.

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Conflict of interest statement

This study was funded by the Federal Ministry of Education and Research (01KC2001B and 03VP08053 to Dr Hansmann; 01KC2001A, 03VP08051, and 16GW0298 to Dr Bader). Dr Hansmann also receives funding from the German Research Foundation (DFG KFO311 grant HA4348/6-2) and the European Pediatric Pulmonary Vascular Disease Network. Dr Nazaré has received funding from the Federal Ministry of Economic Affairs (ZIM grant 16KN073251). Drs Specker, Nazaré, Matthes, and Bader hold patents on the novel class of TPHi. Drs Specker, Wesolowski, and Bader are founders of Trypto Therapeutics GmbH. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

None
Graphical abstract
Figure 1
Figure 1
TPHi Attenuates Pulmonary Arterial Hypertension in the SuHx Rat Model: Invasive Hemodynamic Measurements (A) Experimental design. Three age-matched groups: 1) control (ConNx); 2) SuHx (injected with SU5416, 20 mg/kg per dose, subcutaneous, and then exposed to hypoxia for 3 weeks, followed by 6 weeks of room air); and 3) SuHx+TPHi (treated orally with TPHi [TPT-001, 25 mg/kg /day] for 5 weeks). (B to G) Invasive hemodynamic measurements performed 6 weeks after the end of hypoxia to assess the right ventricular systolic pressure (RVSP), right ventricular end-diastolic pressure (RVEDP), systolic blood pressure (SAP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and heart rate (HR). Mean ± SEM, n = 7-11, ANOVA–Bonferroni post hoc test. ∗∗∗P < 0.001.
Figure 2
Figure 2
TPHi Attenuates Pulmonary Arterial Hypertension in the SuHx Rat Model: Noninvasive Echocardiographic Measurements (A) Representative pulsed-wave Doppler images for pulmonary artery acceleration time (PAAT) measurement in the 3 groups at the end of the study. The SuHx group has a mid-systolic notch, which is no longer present in the group treated with TPHi. (B to G) PAAT as a surrogate of mean pulmonary arterial pressure and pulmonary vascular resistance, RV end-diastolic diameter (RVEDD) and end-diastolic anterior wall thickness (RVAWD), left ventricular end-diastolic diameter (LVEDD) and end-diastolic posterior wall thickness (LVPWD), and heart rate (HR). Mean ± SEM, n = 7-14, ANOVA–Bonferroni post hoc test. ∗∗P < 0.01; ∗∗∗P < 0.001.
Figure 3
Figure 3
TPHi Attenuates Muscularization of Small Pulmonary Arteries and Cardiac Fibrosis in the SuHx Rat Model (A to C) Representative images of small peripheral pulmonary arteries in hematoxylin and eosin (H&E), Masson’s Trichrome, and α-smooth muscle actin (SMA) staining, respectively. Scale bars: 25 μm. Bar graphs: (B) percentage of perivascular collagen. n = 20-40 vessels per animal; n = 7-8 animals. (C): Peripheral pulmonary artery muscularization. n = 20-40 vessels per animal; n = 7-8 animals. (D) Masson’s Trichrome staining of interstitial collagen in RV tissue. n = 4-7 individual animals. Mean ± SEM, ANOVA–Bonferroni post hoc test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
Figure 4
Figure 4
Immunofluorescent Evaluation of Key Targets in the Rat Lung (A) Representative photographs showing proliferating cell nuclear antigen (PCNA) staining. PCNA signal in red, 6-diamino-2-phenylindole (DAPI) in blue, and red blood cells in yellow, quantification to the right. (B) Representative photographs showing accumulation of CD3+ T cells in the lungs of SuHx animals, which is not present in the treated group. CD3 signal in red, DAPI in blue, and red blood cells in yellow, quantification to the right. (C and D) Representative photographs showing accumulation of F4/80+ and CD68+ macrophages in the lung of SuHx animals, which is absent after the treatment. CD68 signal in red, DAPI in blue, quantification to the right. Mean ± SEM, ANOVA–Bonferroni post hoc test. ∗∗∗P < 0.001.
Figure 5
Figure 5
Gene Expression Analysis (RNA-seq) of Rat Whole Lung Samples Reveals Key DEGs Likely Contributing to Beneficial Effects of TPHi Therapy (A) Heatmap of DEGs whose differential expression was reverted by the TPHi therapy. These DEGs are likely involved in biological processes mediated by serotonin signaling and associated with pulmonary arterial hypertension (PAH); however, the roles of many of these genes remain unclear. (B) Heatmap of selected treatment DEGs (SuHx+TPHi vs SuHx) and their corresponding expression in the PAH model (SuHx vs ConNx). The selection is relevant to PAH based on the literature. (C) Proposed model of TPHi therapy based on observed gene expression (RNA-seq, no additional validation) indicates likely beneficial effects, ie, decrease in: 1) vasoconstriction; 2) pulmonary arterial smooth muscle cell (PASMC) proliferation; 3) vascular fibrosis; and 4) inflammation. Targets indicated in bold were significantly differentially expressed (false discovery rate <0.05 and fold changes >2 or <0.5). The arrows next to targets indicate the observed up-regulation (↑) or down-regulation (↓). The arrows and T-bars in the models indicate the effects (induction or suppression, respectively) known from the literature. The arrows next to biological processes (↑ or ↓) follow from the observed regulation of the upstream targets. Sample sizes: ConNx, n = 4; SuHx, n = 6; SuHx+TPHi, n = 4. 5-HT = 5-hydroxytryptamine (serotonin); cGMP = cyclic guanosine monophosphate; ConNx = control group in normoxia; Hx = hypoxia; NO = nitric oxide; PKG = protein kinase G; ROS = reactive oxygen species; SuHx = Sugen 5416 in hypoxia; TPH, tryptophan hydroxylase.
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