Article Figures & Data
Figures
- Fig. 1.
GGDPS inhibitors induce the UPR and apoptosis in MM cells. (A) Chemical structures of GGDPS inhibitors (VSW1198, RAM2093, and RAM2061). (B) Immunoblot analysis showing protein levels of apoptotic markers (cleaved caspases 3 and 8) and UPR markers [ATF4, phosphorylated-eIF2α (p-eIF2α) and eIF2α] in human MM cell lines treated with or without RAM2061 or RAM2093 for 48 hours. β-tubulin is shown as a loading control. Immunoblots are representative of three independent experiments. (C) quantitative real-time-polymerase chain reaction analysis of mRNA expression levels of UPR markers (ATF4, CHOP, IRE1α, and PERK) in human MM cell lines treated with or without RAM2061 or RAM2093 for 48 hours. Data represent fold change normalized to control (n = 3 biologic replicates). Data are displayed as mean ± S.E.M., *P < 0.05; **P < 0.01; ****P < 0.001, Statistical calculations were performed using the Bio-Rad CFX manager 3.1 software).
- Fig. 2.
The effects of single doses of RAM2061 and RAM2093 on liver morphology. Liver H&E sections (original magnification, 200×) from mice after intravenous injection with 0.3 mg/kg RAM2093, 0.5 mg/kg RAM2093, 0.5 mg/kg RAM2061, and 1.3 mg/kg RAM2061. A normal liver is shown as a control. Tissues were collected 14 days postinjection.
- Fig. 3.
The effects of repeat dosing of RAM2061 and RAM2093 on liver morphology. Liver H&E sections (original magnification, 200×) from mice after multiple intravenous injections with RAM2061 (0.1 mg/kg 2×/wk or 0.2 mg/kg 1×/wk) or RAM2093 (0.05 mg/kg 2×/wk or 0.15 mg/kg 1×/wk). A normal liver is shown as a control. Tissues were collected 35 days postinitiation of treatment.
- Fig. 4.
RAM2061 and RAM2093 disrupt protein geranylgeranylation in vivo. Immunoblot analysis of unmodified Rap1a in liver, spleen, and kidney tissues isolated from mice injected either twice weekly (A) or once weekly (B) with RAM2061 or RAM2093. β-tubulin is shown as a loading control. Lysates generated from U266 human myeloma cells treated with lovastatin were used as a positive control for unmodified Rap1a protein.
- Fig. 5.
Measurement of RAM2061 and RAM2093 in mouse plasma samples. Plasma concentrations (nanograms per milliliter) of RAM2061 and RAM2093 after a single dose (0.3 mg/kg i.v.) (mean ± S.D., n = 5 mice).
- Fig. 6.
Tissue distribution of RAM2061 and RAM2093. Tissue concentrations (nanograms per gram) over time for RAM2061 (A) and RAM2093 (B) after a single dose (0.3 mg/kg IV). (C) Concentration of RAM2061 and RAM2093 in the bone marrow (nanograms per milliliter). (D) Liver levels of RAM2061 and RAM2093 (nanograms per gram). Data are shown as mean ± S.D. (n = 5 mice, *P < 0.05; **P < 0.01; ****P < 0.001, two-tailed student t test with Bonferroni correction).
- Fig. 7.
RAM2061 and RAM2093 display metabolic stability in HLMs. Metabolic elimination profiles (% turnover or amount remaining vs. incubation time) for reaction with RAM2061, reaction with RAM2093, negative control (reaction without NADPH), and in-house positive control (testosterone). Data are shown as mean ± S.D. (n = 3).
- Fig. 8.
RAM2061 slows MM tumor growth in vivo. (A) Tumor growth curves for MM.1S flank xenografts. Mice were treated twice weekly with either solvent only control (PBS, n = 10 mice) or RAM2061 (n = 9 mice). Data are displayed as mean ± S.E.M. A linear mixed model was used for statistical analysis. (B) Kaplan-Meier curve for MM1.S flank xenografts showing time to sacrifice. Mice were euthanized when tumors reached 2000 mm3. (C) Immunoblot analysis of unmodified Rap1a in tumors collected from the treated animals. β-tubulin is shown as a loading control. Densitometric analysis of unmodified Rap1a (normalized to β-tubulin) in tumors of the treated animals normalized to untreated (control) is shown as relative intensity (Rel Int).
Tables
- TABLE 1
Blood chemistry panel for single dose testing of RAM2061 and RAM2093
Blood was collected at 7 and 14 days post-injection (mean ± S.D., n = 5 mice per group).
0.5 mg/kg RAM2061 0.3 mg/kg RAM2093 Day 7 Day 14 Day 7 Day 14 Normal Average Average Average Average BUN 9–33 17.8 ± 0.5 17.8 ± 2.9 14.8 ± 2.9 16.4 ± 4.8 CRE 0.2–0.9 0.4 ± 0.2 0.24 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 ALT 17–77 1320 ± 447 160 ± 76 503.5 ± 141 50.4 ± 8 ALP 35–222 300 ± 258 162 ± 83 221.8 ± 182 94.6 ± 21 AST 54–298 1322 ± 382 263.3 ± 64 580.5 ± 227 166.4 ± 54.5 TBIL 0–0.9 1.6 ± 2.6 0.25 ± 0.1 0.9 ± 0.8 0.3 ± 0.1 GLU 140–263 79.8 ± 52 194.8 ± 24 82.8 ± 30 231 ± 33 CA 6–13 12 ± 1 12.3 ± 0.6 11.6 ± 0.7 12 ± 0.5 TP 3.9–6.4 5.6 ± 0.9 5.3 + 0.2 5.42 ± 0.4 5.6 ± 0.2 ALB 2.5–4.6 4.4 ± 0.4 4.2 ± 0.3 4.1 ± 0.3 4.3 ± 0.4 GLOB 1.2–2.2 1.2 ± 0.5 1.0 ± 0.3 1.2 ± 0.2 1.1 ± 0.6 Na 110–195 117.5 ± 69 153.2 ± 2.2 149.4 ± 1.9 150.8 ± 2 K 4–10.5 7.9 ± 0.5 8.4 ± 0.1 7.5 ± 0.3 8.2 ± 0.8 Cl NR 103.8 ± 2.4 105.8 ± 2.2 105.4 ± 5 107.4 ± 3 TCO2 NR 24 ± 4.8 25.8 ± 3 22.5 ± 2.9 27.6 ± 2 LDH 64.5–288 ND 294 ± 22 ND 225.3 ± 71 CK 29.3–810 ND 96.3 ± 44.3 ND 97 ± 103 ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; CRE, creatinine; GLOB, globulin; GLU, glucose; LDH, lactate dehydrogenase; ND, not done; NR, not reported; TBIL, total bilirubin; TP, total protein.
- TABLE 2
Complete blood cell count analysis for single-dose testing of RAM2061 and RAM2093
Blood was collected 14 days postinjection (mean ± S.D., n = 5 mice per group).
0.5 mg/kg RAM2061 0.3 mg/kg RAM2093 Normal 14-day Average 14-day Average WBC 1.8–10.7 9 ± 4.1 6.5 ± 1.1 LYM (no.) 0.9–9.3 5.9 ± 2.8 6.3 ± 4.6 MON (no.) 0–0.4 1 ± 0.5 0.5 ± 0.2 GRA (no.) 0.1–3 2.4 ± 1.5 1.7 ± 0.4 LYM (%) 55.8–91.6 66.2 ± 15.7 66 ± 9.7 MON (%) 0–7.5 8 ± 1.7 8.5 ± 3.9 GRA (%) 6.6–45.8 25.8 ± 14.2 25.5 ± 5.8 RBC 6.36–9.42 8.7 ± 1.3 8 ± 0.8 HGB 11–15.1 13.1 ± 1.8 14.0 ± 0.9 HCT 35–45.4 48.5 ± 7.1 43.9 ± 4.2 MCV 45–60.3 56 ± 2.4 54.9 ± 2.1 MCH 14.1–19.3 15.2 ± 1.1 17.5 ± 2.0 MCHC 22.3–32 27.1 ± 1.9 32 ± 4.1 RDW 12.4–27 19.2 ± 1 16 ± 1.8 PLT 592–2872 963.6 ± 189 915 ± 254 MPV 5–20 5.0 ± 0.3 4.8 ± 0.5 GRA, granulocytes; HCT, hematocrit; HGB, hemoglobin; LYM, lymphocytes; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin count; MCV, mean corpuscular volume; MON, monocytes; MPV, mean platelet volume; PLT, platelets; RBC, red blood cell; RDW, red cell distribution; WBC, white blood cell.
- TABLE 3
Blood Chemistry panel for RAM2061 and RAM2093 multidose testing
Blood was collected after 4 wk of drug administration (mean ± S.D., n = 5 mice per group, with exception of RAM2093 once-weekly group--n=3).
RAM2061 RAM2093 0.2 mg/kg 1×/wk 0.1 mg/kg 2×/wk 0.15 mg/kg 1×/wk 0.05 mg/kg 2×/wk Normal Average Average Average Average BUN 9–33 15.3 ± 0.5 15.25 ± 2.6 13 ± 1.0 16.8 ± 2.4 CRE 0.2–0.9 0.3 ± 0.1 0.2 ± 0 0.3 ± 0.1 0.2 ± 0.04 ALT 17–77 1526 ± 325.9 823.5 ± 810.6 1452.3 ± 1151.8 1031.5 ± 483.4 ALP 35–222 255.8 ± 23.7 162.5 ± 65.6 186.3 ± 98.1 268.8 ± 70.1 AST 54–298 1721 ± 417.8 857.75 ± 760.2 1621 ± 1159.4 1329.5 ± 618 TBIL 0–0.9 0.5 ± 0.2 0.4 ± 0.1 0.2 ± 0 0.3 ± 0.05 GLU 140–263 127.3 ± 15.4 130 ± 11.4 111.3 ± 28 156.8 ± 16.8 CA 6–13 11.3 ± 0.3 10.7 ± 0.3 11.2 ± 0.2 11.7 ± 0.3 TP 3.9–6.4 5 ± 0.2 4.95 ± 0.1 5.1 ± 0.3 5 ± 0.2 ALB 2.5–4.6 4.3 ± 0.3 3.8 ± 0.2 3 ± 2.6 4.4 ± 0.3 GLOB 1.2–2.2 0.7 ± 0.1 1.1 ± 0.1 0.7 ± 0.1 0.6 ± 0.2 Na 110–195 150.8 ± 0.3 144.8 ± 1.5 151.7 ± 3.2 153.8 ± 2.6 K 4–10.5 8.5 ± 0.2 7.6 ± 0.3 9.3 ± 1.1 8.4 ± 0.25 Cl NR 115 ± 1.4 112.8 ± 2.2 109 ± 1.0 107.3 ± 2.5 TCO2 NR 29 ± 1.1 21.8 ± 2.6 27.7 ± 2.1 30 ± 1.9 LDH 64.5–288 377.6 ± 90.7 315.6 ± 82.1 317 ± 34.5 336.5 ± 100 CK 29.3–810 7.14 ± 0.7 21.3 ± 13.6 23.4 ± 22.8 58 ± 49 ALB, albumin; ALP, alkaline phosphatase; ALT, alanine amino-transferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; CRE, creatinine; GLOB, globulin; GLU, glucose; LDH, lactate dehydrogenase; NR, not reported; TBIL, total bilirubin; TP, total protein.
- TABLE 4
Complete blood cell count analysis for multidose testing of RAM2061 and RAM2093
Blood was collected after 4 wk of drug administration (mean + S.D., n = 5 mice per group, with exception of RAM2093 once-weekly group--n=3).
RAM2061 RAM2093 0.2 mg/kg 1× per week 0.1 mg/kg 2× per week 0.15 mg/kg 1× per week 0.05 mg/kg 2× per week Normal Average Average Average Average WBC 1.8–10.7 9.6 ± 2.1 11.7 ± 2.6 10.7 ± 3.6 9.2 ± 3.2 LYM (no.) 0.9–9.3 5.2 ± 2 10 ± 2.3 5.8 ± 1.9 5.6 ± 1.8 MON (no.) 0–0.4 0.5 ± 0.2 0.3 ± 0.1 0.8 ± 0.5 0.6 ± 0.3 GRA (no.) 0.1–3 3.0 ± 1.6 1.4 ± 0.7 4.1 ± 1.3 2.95 ± 1.2 LYM (%) 55.8–91.6 63.2 ± 15.8 85.9 ± 6.2 54.3 ± 3.2 61.7 ± 3.5 MON (%) 0–7.5 5.4 ± 1.6 2.7 ± 0.7 6.8 ± 2.3 6.5 ± 1.6 GRA (%) 6.6–45.8 32.9 ± 13.7 11.4 ± 6 38.8 ± 1.8 31.8 ± 4.1 RBC 6.36–9.42 8.8 ± 1.2 10.1 ± 0.4 10 ± 0.2 9.2 ± 0.7 HGB 11–15.1 12.7 ± 2.4 14.9 ± 1.3 14.2 ± 0.2 13.7 ± 1.3 HCT 35–45.4 49.4 ± 5.1 53.9 ± 2.7 55 ± 0.6 53 ± 3.6 MCV 45–60.3 56.6 ± 3 53.4 ± 1.1 55 ± 0.6 57.5 ± 1.6 MCH 14.1–19.3 14.5 ± 1.3 14.8 ± 0.7 14.2 ± 0.1 14.9 ± 1.1 MCHC 22.3–32 25.7 ± 2.2 27.7 ± 1 25.8 ± 0.1 25.9 ± 2.2 RDW 12.4–27 18.1 ± 0.5 17.5 ± 0.9 18 ± 0.3 17.9 ± 1.3 PLT 592–2872 642.6 ± 340.8 419.8 ± 180.5 493.5 ± 3.5 919.6 ± 701.7 MPV 5–20 5.6 ± 0.9 7.1 ± 0.7 5.3 ± 0.3 4.84 ± 0.2 GRA, granulocytes; HCT, hematocrit; HGB, hemoglobin; LYM, lymphocytes; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin count; MCV, mean corpuscular volume; MON, monocytes; MPV, mean platelet volume; PLT, platelets; RBC, red blood cell; RDW, red cell distribution; WBC, white blood cell.
Estimates (mean ± S.D.) Parameters (unit) RAM2061 RAM2093 C0 (ng/ml) 1518.4 ± 292.5 1230.7 ± 452.3 t1/2 (h) 29.2 ± 6.0 22.1 ± 4.2 AUC0-last (h*ng/ml) 920.5 ± 28.7 1681.2 ± 76.8 AUC0- ∞ (h*ng/ml) 946.1 ± 28.4 1828.3 ± 29.5 Vd (liters/kg) 13.4 ± 3.1 5.2 ± 1.0 Cl (liters/h per kilogram) 0.3 ± 0.01 0.2 ± 0.003 AUC, area under the curve from 0 to ∞ and last hr.; C0, concentration at time zero; Cl, clearance; t1/2, terminal half-life; Vd, volume of distribution.
- TABLE 6
Plasma protein binding of RAM2061 and RAM2093 in mouse plasma (n = 3, mean ± S.D.)
Parameters Mean ± S.D. RAM2061 RAM2093 % Remaining at 5 h (stability) 109.5 ± 8.9 106.3 ± 8.1 % Device recovery 116.3 ± 5.8 112.3 ± 6.3 % Plasma protein bound (at 1 μg/ml spiked sample) 94.8 ± 0.4 96.2 ± 0.4 % Plasma protein bound (at 10 μg/ml spiked sample) 93.6 ± 1.8 93.9 ± 0.9 % Plasma protein bound (2 h postinfusion (0.3 mg/kg i.v.)) 94.8 ± 0.2 92.8 ± 1.3 % Plasma protein bound (8 h postinfusion (0.3 mg/kg i.v.)) 96.3 ± 0.3 92.5 ± 1.8
Data Supplement
- Supplemental Data -
6 supplemental figures, 3 tables.
- Supplemental Data -
