H-ras (IC50 = 1.9 nM); K-ras (IC50 = 5.2 nM); N-ras (IC50 = 2.8 nM)[1]

Lonafarnib (Sch66336) suppresses the transformed growth properties of human tumor cell lines carrying activated Ki-Ras proteins and potently inhibits Ha-Ras processing in whole cells[1]. When compared to the control treatment, all treatment groups that contained Lonafarnib (10 μM) had a noticeably greater amount of unfarnesylated H-Ras (116–137%)[2].

Lonafarnib (Sch66336) exhibits good oral bioavailability and pharmacokinetic characteristics in the mouse, rat, and monkey systems. Lonafarnib exhibits strong oral efficacy in a variety of human tumor xenograft models in the nude mouse, including tumors originating from the colon, lung, pancreatic, prostate, and urinary bladder[1]. In comparison to vehicle-treated control mice (T/C of 0.67), lionafarnib alone (80 mg/kg by oral gavage, once day) had a limited capacity to suppress orthotopic U87 tumors. The intended outcome of XRT/Tem (2.5 Gy/day for 2 days; 5 mg/kg by oral gavage 90 min before XRT) is a moderate in vivo suppression of tumor growth (T/C of 0.42). The strongest growth reduction (T/C of 0.02) and significant superiority over XRT/Tem (p<0.04) is achieved by concurrent administration of Lonafarnib/XRT/Tem (Lonafarnib 80 mg/kg by oral gavage, once daily, XRT 2.5 Gy/day for 2 days, and Tem 5 mg/kg by oral gavage 90 minutes prior to XRT). Most animals show a decrease in tumor volume (p<0.05) after 2 weeks and the effect persists after 4 weeks (p<0.05)[2].

SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336.
FPTactivity is determined by measuring the transfer of [3H]farnesyl from [3H]farnesyl PPi to trichloroacetic acid-precipitable Ha-Ras-CVLS. GGPT-1 activity is similarly determined using [3H]geranylgeranyl diphosphate and Ha-Ras-CVLL as substrates[1].

Non-Radioactive MTS Cytotoxicty Assay[2]
Assays were performed under manufacturer’s instructions with 5000 cells/well in a 96-well tissue culture plate. Plates were irradiated 24 h after drug exposure and assayed 96 h after XRT, with fresh drug treatments applied each day. For quantification, dye was added directly to each well, plates were washed as per the manufactures recommendation and cell viability determined by optical density. Significance was analyzed using the Student’s T-test.

---

Proliferation Assay[2]
12-well plates were seeded with 100,000 cells/well. Drug treatments were initiated 24 h after plating, and media was replaced every 24 h for a total of 96 h of drug exposure. Plates were irradiated after 24 h of drug exposure. Cells from triplicate sets of treatments were trypsonized and counted 48 h after irradation using a Z1 series coulter counter, and compared to cell numbers from wells counted on Day 1 (the day drug treatment was initiated). Proliferation after drug treatments were normalized to the control wells and expressed as % of the control treatment. Significance was analyzed using the Student’s T-test.

---

Downstream Pathway Analysis[2]
2.5 ×106 cells per 100mm3 dish were seeded, and drug treatments initiated 24 h after plating. Plates were irradiated after 24 h of drug exposure, and cells were lysed after 48 h of drug exposure (24 h after XRT). Total protein was extracted with ice-cold T-Per supplemented with protease and phosphatase inhibitors, and quantitated using the BCA protein assay kit. 500ug of total protein was used to probe different Human Phospho-RTK Human Phospho-MAPK Arrays. Arrays were washed and developed according to manufacturer’s instructions, and exposed to film. Films were scanned using a flatbed scanner, and dots were quantitated using ImageJ. Relative changes between treatment groups were expressed as % of control, with significance assessed by Student’s T-test.

---

Western Blotting of H-Ras[2]
2.5 ×106 cells per 100mm3 dish were seeded, and drug treatments initiated 24 h after plating. Plates were irradiated after 24 h of drug exposure, and cells were lysed after 48 h of drug exposure (24 h after XRT). Total protein was extracted with ice-cold T-Per supplemented with protease and phosphatase inhibitors, and quantitated using the BCA protein assay kit. Samples (20 µg total protein) were run on 4–15% Tris HCl SDS-PAGE Criterion gels (Biorad, Hercules, CA) and probed for H-Ras and α-tubulin as an internal loading control. Blots were exposed to film, and films were scanned using a flat bed scanner. Bands were quantitated using ImageJ (NIH, Bethesda, MD), and graphed using Excel. H-Ras was normalized to the loading control and expressed as a % of the control treatment. Significance was assessed using the Student’s T-test.

Formulation: lonafarnib (SCH66336, Sarasar®) and Temozolomide were reconstituted in 4% DMSO in 20% (2-hydroxypropyl)-beta-cyclodextrin in PBS. Lonafarnib was given once daily at 80mg/kg with twice weekly weightings to ensure accurate dosing.

---

Tumor Cell Line Xenografts[2]
Tumor cell lines were harvested in mid-logarithmic growth phase and resuspended in PBS. Homozygous NCR nude mice were anesthetized with Ketamine/Xylazine before exposure of the cranium and removal of the periosteum with a size 34 inverted cone burr. Mice were fixed in a stereotactic frame, and 5×104 cells in 10 ul of PBS were injected through a 27-gauge needle over 5 min at 2 mm lateral and posterior to the bregma and 3 mm below the dura. The incision was closed with staples. Animals were observed daily for signs of distress or development of neurologic symptoms at which time the mice were sacrificed.

---

In Vivo Imaging[2]
Mice were anesthetized with Ketamine/Xylazine, injected with D-luciferin at 50 mg/kg i.p., and imaged with the Xenogen IVIS 100 Imaging System for 10–120 s, bin size 2 as previously published. To quantify bioluminescence, identical circular regions of interest were drawn to encircle the entire head of each animal, and the integrated flux of photons (photons per second) within each region of interest was determined by using the Xenogen LIVING IMAGES software package. Data were normalized to bioluminescence at the initiation of treatment for each animal. Statistical significance was assessed using the Student’s T-test.

---

Glioma Neurosphere Assay[2]
Collection and use of fresh and discarded human tumor tissue was approved by the Brigham and Women’s Hospital Institutional Review Board. After frozen section diagnosis of malignant glioma by the attending neuropathologist, tumor material was grossly dissected from the tissue sample. Portions of the tumors were collected in chilled media for the studies described here and other portions were allocated for paraffin embedding for histological diagnosis and for genotyping. Expansion of tumor material and propagation was accomplished by subcutaneous implantation in Icr SCID mice (cells were never grown on plastic). When tumors reached ~1 cm, tumors were disaggregated, cells were counted and then grown in serum-free media with EGF, FGF and LIF as described previously to form tumorspheres [25, 26]. Drugs (SCH 5uM, TMZ 100uM) were added immediately after plating cells into 24 well plates and radiation given at 24hrs after plating and tumor neurospheres were counted in triplicate 10 days after plating.

---

Dissolved in 20% (w/v) HPβCD; 50 mg/kg; Oral gavage

NOD/SCID mice between 6–12 weeks of age

Absorption, Distribution and Excretion
The absolute oral bioavailability of lonafarnib is unknown; in healthy subjects administration of either 75 or 100 mg of lonafarnib twice daily resulted in mean peak plasma concentrations (%CV) of 834 (32%) and 964 (32%) ng/mL, respectively. Twice daily administration of 115 mg/m2 lonafarnib in HGPS patients resulted in a median tmax of 2 hours (range 0-6), mean Cmax of 1777 ± 1083 ng/mL, mean AUC0-8hr of 9869 ± 6327 ng\*hr/mL, and a mean AUCtau of 12365 ± 9135 ng\*hr/mL. The corresponding values for a dose of 150 mg/m2 are: 4 hours (range 0-12), 2695 ± 1090 ng/mL, 16020 ± 4978 ng\*hr/mL, and 19539 ± 6434 ng\*hr/mL, respectively. Following a single oral dose of 75 mg in healthy subjects, the Cmax of lonafarnib decreased by 55% and 25%, and the AUC decreased by 29% and 21% for a high/low-fat meal compared to fasted conditions.
Up to 240 hours following oral administration of 104 mg [14C]-lonafarnib in fasted healthy subjects, approximately 62% and <1% of the initial radiolabeled dose was recovered in feces and urine, respectively. The two most prevalent metabolites were the active HM21 and HM17, which account for 14% and 15% of plasma radioactivity.
In healthy patients administered either 75 or 100 mg lonafarnib twice daily, the steady-state apparent volumes of distribution were 97.4 L and 87.8 L, respectively.

---

Metabolism / Metabolites
Lonafarnib is metabolized _in vitro_ primarily by CYP3A4/5 and partially by CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, and CYP2E1. Formation of the primary metabolites involves oxidation and subsequent dehydration in the pendant piperidine ring.

---

Biological Half-Life
Lonafarnib has a mean half-life of approximately 4-6 hours following oral administration of 100 mg twice daily in healthy subjects.

Hepatotoxicity
In the small prelicensure clinical trials conducted in children with progeria, serum aminotransferase elevations occurred in 35% of lonafarnib treated subjects but were usually mild and self-limited, rising to above 3 times the upper limit of normal (ULN) in only 5%. There were no liver related serious adverse events and no patient had a concurrent elevation in serum aminotransferase and bilirubin levels. Since approval of lonafarnib, there have been no published reports of drug induced liver injury associated with its use, although clinical experience with the drug, particularly with long term therapy, has been limited.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

---

Protein Binding
Lonafarnib exhibits _in vitro_ plasma protein binding of ≥99% over a concentration range of 0.5-40.0 μg/mL.

[1]. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 1998 Nov 1;58(21):4947-56.

[2]. Lonafarnib (SCH66336) improves the activity of temozolomide and radiation for orthotopic malignant gliomas. J Neurooncol. 2011 Aug;104(1):179-89.

[3]. Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial. Lancet Infect Dis. 2015 Oct;15(10):1167-1174.

Pharmacodynamics
Lonafarnib is a direct farnesyl transferase inhibitor that reduces the farnesylation of numerous cellular proteins, including progerin, the aberrantly truncated form of lamin A that accumulates in progeroid laminopathies such as Hutchinson-Gilford progeria syndrome. Treatment with lonafarnib has been associated with electrolyte abnormalities, myelosuppression, and increased liver enzyme levels (AST/ALT), although causation remains unclear. Also, lonafarnib is known to cause nephrotoxicity in rats and rod-dependent low-light vision decline in monkeys at plasma levels similar to those achieved under recommended dosing guidelines in humans; patients taking lonafarnib should undergo regular monitoring for both renal and ophthalmological function. In addition, based on observations from animal studies with rats, monkeys, and rabbits with plasma drug concentrations approximately equal to those attained in humans, lonafarnib may cause both male and female fertility impairment and embryo-fetal toxicity.

C27H31BR2CLN4O2

638.82

636.05

C, 50.76; H, 4.89; Br, 25.02; Cl, 5.55; N, 8.77; O, 5.01

193275-84-2

(Rac)-Lonafarnib;193275-86-4

148195

White to off-white solid powder

1.5±0.1 g/cm3

710.4±70.0 °C at 760 mmHg

214.5-215.9° (monohydrate); mp 222-223°

383.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.630

5.03

1

3

3

36

790

1

C1CN(CCC1CC(=O)N2CCC(CC2)[C@@H]3C4=C(CCC5=C3N=CC(=C5)Br)C=C(C=C4Br)Cl)C(=O)N

DHMTURDWPRKSOA-RUZDIDTESA-N

InChI=1S/C27H31Br2ClN4O2/c28-20-12-19-2-1-18-13-21(30)14-22(29)24(18)25(26(19)32-15-20)17-5-9-33(10-6-17)23(35)11-16-3-7-34(8-4-16)27(31)36/h12-17,25H,1-11H2,(H2,31,36)/t25-/m1/s1

4-[2-[4-[(2R)-6,15-dibromo-13-chloro-4-azatricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-yl]piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide

Lonafarnib; SCH66336; Sarasar; Sch 66336; Sch66336; Sch-66336; Zokinvy; lonafarnibum; Trade name: Sarasar; SCH 66336; SCH-66336;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)