A lymphatic-absorbed multi-targeted kinase inhibitor for myelofibrosis therapy

Abstract

Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.

Fig. 1. Multi-functional kinase inhibitor LP-182 exhibits selectivity and specificity.

a Targeting of downstream PI3K/mTOR and RAF/MEK signaling nodes by LP-182 in hematopoietic cells with constitutive JAK activation. b Single-point broad panel kinome screening against 2.5 μM LP-182. Average percent kinase inhibition from replicate data was input into Coral Human Kinome Visualization software, and analyzed as described in Methods^–. Scaling for branch color, node color, and node size represents percent inhibition as indicated. Source data are provided as a Source Data file. Kinase families: Tyrosine kinase, TK; Tyrosine kinase-like, TKL; Serine/threonine kinase, STE; Casein kinase 1, CK1; Protein kinase A/G/C, AGC; Ca²⁺/calmodulin-dependent protein kinase, CAMK; CDK/MAPK/GSK/CDK-like, CMGC; Phosphoinositol kinases, PI Kinases. c Docked structures of LP-182 at the PI3Kγ (PDB code 3L08), mTOR1 (PDB code 4JSX), and BRAF (PDB code 5HI2) catalytic sites, and MEK1 (PDB code 3ORN) allosteric pocket as indicated^{, –}. Molecular notation shows PI3K/mTOR inhibitor of LP-182 in blue, RAF/MEK inhibitor in orange, and polyethylene glycol linker in black. d Normalized fluorescence intensity values of phosphorylated AKT (pAKT; pS473) and ERK1/2 (pERK1/2; pT202/pY204) flow cytometry staining in SET-2 cells following treatment with indicated concentrations of LP-182 for 16 h. e Growth inhibition and Caspase activation values from SET-2 cells treated with indicated concentrations of LP-182 for 72 h or 48 h, respectively. Data represent the mean ± s.e.m., n = 3 independent experiments performed in replicate. Corrected data were normalized to vehicle treated control values and analyzed by non-linear regression where indicated. Source data are provided as a Source Data file.

Fig. 2. In vivo pharmacokinetics upon oral administration achieves sustained levels of LP-182 and potent biologically active metabolites.

a LP-182 in vivo metabolic degradation products. b In vivo pharmacokinetics of LP-182 and associated metabolites in mice following a single intravenous (15 mg kg⁻¹; top panel) or oral (400 mg kg⁻¹; bottom panel) dose of LP-182. Data represent the mean ± s.d., n = 3 animals sampled per time point per compound dosing. Source data are provided as a Source Data file. c Target kinase (PI3Kγ, mTOR, BRAF, MEK1) 10-point inhibition assays against LP-182, LP-527, or PD0316684 at the indicated concentrations. Data represent the mean ± s.e.m., LP-182 (n = 5, PI3Kγ; n = 3, mTOR; n = 3, BRAF; n = 5, MEK1; independent experiments performed in replicate), LP-527 (n = 5, PI3Kγ; n = 3, mTOR; independent experiments performed in replicate), PD0316684 (n = 1, BRAF; n = 1, MEK1; independent experiments performed in replicate). Calculated IC₅₀ values from replicate data are supplied in Supplementary Fig. 7. Source data are provided as a Source Data file. d Growth inhibition values from SET-2 cells treated with indicated concentrations of LP-182, PD0316684, LP-527, or LP-527 and PD0316684 for 72 h. Data represent the mean ± s.e.m., n = 3 independent experiments performed in replicate. Corrected data were normalized to vehicle treated control values and analyzed by non-linear regression where indicated. Source data are provided as a Source Data file.

Fig. 3. Absorptive lymphatic partitioning of LP-182 involves association with lipoproteins.

a Concentration of LP-182 in blood and mesenteric lymph nodes from mice 30 min following oral administration at the indicated doses. Data represent the mean ± s.e.m., n = 4 animals per dose. Statistical significance determined using multiple unpaired t-test corrected with Holm-Šidák multiple comparisons test (100 mg kg⁻¹, p = 0.039; 200 mg kg⁻¹, p = 0.034; 400 mg kg⁻¹, p = 0.000094; 600 mg kg⁻¹, p = 0.029; 800 mg kg⁻¹, p = 0.043; 1000 mg kg⁻¹, p = 0.036). b Concentration of LP-527, PD0316684, and LP-182 in blood and mesenteric lymph nodes from mice 4 h following oral administration at 100 mg kg⁻¹. Data represent the mean ± s.e.m., LP-527 (n = 9), PD0316684 (n = 10), and LP-182 (n = 9) animals per group. c Enterocyte schematic showing possible routes of drug access into the blood and intestinal lymphatics in an anesthetized rat mesenteric lymph cannulation model^,. d Kinetic profiles of LP-182 in lymphatic fluid from anesthetized rats following 1 h intra-duodenal infusion at 50 mg kg⁻¹. Data represent the mean ± s.e.m., n = 7 animals per time point. Source data are provided as a Source Data file. e Relative percent of total LP-182 measured within chylomicron, very low-density lipoprotein (VLDL), and low-density lipoprotein/high-density lipoprotein (LDL/HDL) fractions upon separation of pooled time course lymphatic fluid by ultracentrifugation from anesthetized rats following 1 h intra-duodenal infusion of LP-182 at 50 mg kg⁻¹. Data represent the mean ± s.e.m., n = 3 animals.

Fig. 4. Magnetic resonance imaging of LP-182 treatment response reveals reduced splenomegaly and tibia bone marrow cellularity in the MPL^W515L mouse model of myelofibrosis.

a Schematic for MPL^W515L myelofibrosis mouse model experimental design and daily oral treatment regimen with vehicle or LP-182 (400 mg kg⁻¹). b Representative coronal MR images highlighting spleen cross sections (purple) on the indicated day of treatment with vehicle or LP-182. c Spleen volume to body weight measurements on the indicated day of treatment with vehicle or LP-182. Data represent the mean ± s.e.m., vehicle (n = 2, day 21 timepoint; n = 4, all other timepoints), LP-182 (n = 5) animals per group. Source data are provided as a Source Data file. Statistical significance determined using two-tailed unpaired t-test (7 d, p = 0.0044; 14 d, p = 0.016). d Coronal and axial MR images through the tibia showing apparent diffusion coefficient (ADC) values as pseudocolored heat-maps (middle panels) overlaid on distal axial slices (hashed lines; top panels) at the indicated day of treatment with vehicle or LP-182. Tibia were manually segmented and ADC values within the bone marrow space averaged along lines perpendicular to the coronal plane yielding a two-dimensional perspective of percent ADC change relative to 0 d (color heat-maps; bottom panels). e Distal tibia bone marrow multi-slice average ADC measurements at the indicated day of treatment with vehicle or LP-182. Data represent the mean ± s.e.m., vehicle (n = 4), LP-182 (n = 5) animals per group. Statistical significance determined using two-tailed unpaired t-test (Vehicle 0 d vs. Vehicle 14 d, p = 0.19; Vehicle 0 d vs. LP-182 0 d, p = 0.82; Vehicle 0 d vs. LP-182 14 d, p = 0.0098; Vehicle 14 d vs. LP-182 14 d, p = 0.027; LP-182 0 d vs. LP-182 14 d, p = 0.0007).

Fig. 5. LP-182 attenuates signal transduction and restores immune cell balance to improve cellular disease phenotypes and survival in the MPL^W515L MF mouse model.

a Kaplan–Meier plot of MF mice treated as indicated. Data represent vehicle (n = 4), LP-182 (n = 5) animals per group. Statistical significance determined using Log-Rank (Mantel-Cox) test. b Mean fluorescence intensity values of pAKT (pS473) and pERK1/2 (pT202/pY204) in splenocytes from healthy control and MF mice treated as indicated. Data represent the mean ± s.e.m., healthy control (n = 5), vehicle (n = 4), LP-182 (n = 5) animals per group. Statistical significance determined using Two-way ANOVA corrected with Tukey’s multiple comparisons test (pAKT Healthy control vs. Vehicle, p < 0.0001; pAKT Vehicle vs. LP-182, p < 0.0001; pERK1/2 Healthy control vs. Vehicle, p < 0.0001; pERK1/2 Vehicle vs. LP-182, p < 0.0001). c Immune cell numbers from spleen of healthy control and MF mice treated as indicated. Data represent the mean ± s.e.m., healthy control (n = 5), vehicle (n = 3), LP-182 (n = 4) animals per group. Statistical significance determined using Two-way ANOVA corrected with Tukey’s multiple comparisons test (T cell Healthy control vs. Vehicle, p = 0.049; B cell Healthy control vs Vehicle, p < 0.0001; B cell Healthy control vs. LP-182, p < 0.0001; Neu Healthy control vs. Vehicle, p < 0.0001; Neu Vehicle vs. LP-182, p = 0.0002; MK Healthy control vs. Vehicle, p < 0.0001; MK Vehicle vs. LP-182, p < 0.0001). d Representative histological images and quantitation of MK cells per high-power field from spleen of MF mice treated as indicated. Hematoxylin & Eosin (H&E; 10x with 20x inset, scale 100 µm). Data represent the mean ± s.e.m., vehicle (n = 4), LP-182 (n = 5) animals per group. Statistical significance determined using two-tailed unpaired t-test (p = 0.015). e Representative immunohistochemistry images with quantitation of GFP-positive MK cells per high-power field from spleen of MF mice treated as indicated. Green Fluorescent Protein (GFP; 40x, scale 20 µm). Data represent the mean ± s.e.m., vehicle (n = 4), LP-182 (n = 4) animals per group. Statistical significance determined using two-tailed unpaired t-test (p = 0.0051). f Representative histological images with scored fibrosis grading from bone marrow of MF mice treated as indicated. Reticulin (40x, scale 20 µm). Data represent the mean ± s.e.m., vehicle (n = 4), LP-182 (n = 5) animals per group. Individual data sets and scoring details are provided in Supplementary Table 1. g Concentration of LP-182 and associated metabolites in serum and bone marrow from MF mice ~2 h following final oral administration. Data represent the mean ± s.e.m., n = 4 animals. h Immunoblot analysis and quantitation of ERK1/2 and pERK1/2 (pT202/pY204) from bone marrow of MF mice treated as indicated. Data were normalized to GAPDH and analyzed relative to ERK1/2 where indicated. Data represent the mean ± s.e.m., vehicle (n = 4), LP-182 (n = 4) animals per group. Statistical significance determined using two-tailed unpaired t-test (p = 0.0011). Source data are provided as a Source Data file. a-h MPL^W515L MF mice were treated p.o. daily with vehicle (14-21 d) or LP-182 (28 d) at 400 mg kg⁻¹.

Fig. 6. Physiochemical properties influence lymphatic uptake and release of lymphatropic kinase inhibitors into systemic circulation.

a Schematic model of the lymphatic absorption process for multi-targeted kinase inhibitors following oral administration. Prolonged drug release from mesenteric lymph into the systemic circulation supports sustained plasma levels to diminish compensatory and resistance mechanisms in human diseases such as myelofibrosis (MF). b Mesenteric lymph node to blood ratio of PD0316684, LP5-38, and LP5-37 in mice 4 h following oral administration at 100 mg kg⁻¹. Data represent the mean ± s.e.m., LP5-38 (n = 4), LP5-37 (n = 4), PD0316684 (n = 10; Fig. 3b) animals per group. In vivo pharmacokinetics of c LP-616 or d LP-65 and associated metabolites in mice following a single intravenous (15 mg kg⁻¹; top panel) or oral (200 mg kg⁻¹; bottom panel) dose of LP-616 or LP-65. Data represent the mean ± s.e.m., LP-616 (n = 6, 10 and 60 min timepoints, oral; n = 3, all other timepoints, intravenous and oral), LP-65 (n = 3, all timepoints, intravenous and oral) animals per group. Source data are provided as a Source Data file. e Concentration and f mesenteric lymph node to blood ratio of LP5-37, LP-182, LP-616, and LP-65 in mice 4 h following oral administration at 400 mg kg⁻¹. Data represent the mean ± s.e.m., LP5-37, LP-616, and LP-65 (n = 5), LP-182 (n = 9) animals per group. Statistical significance determined using two-tailed unpaired t-test (LP5-37, p < 0.0001; LP-182, p = 0.0019; LP-616, p = 0.042).