Developing a novel dual PI3K–mTOR inhibitor from the prodrug of a metabolite

Figures & data

Figure 1 Structures of NVP-BEZ235 and compounds 1–3.

Notes: (A) NVP-BEZ235 is a dual PI3K–mTOR inhibitor and a positive control in this study; (B) compound 1 was the provisional candidate for screening and structure–activity relationship study from NVP-BEZ235; (C) compound 2 was the metabolite of compound 1; (D) compound 3 was a prodrug of compound 2.

Figure 2 In vivo efficacy of compound 1 and NVP-BEZ235.

Notes: Dosed orally in tumor-bearing immunocompromised mice at approximately 15–60 mg/kg and 30 mg/kg, respectively. PC-3 cells were injected subcutaneously into nude mice (n=8 per group), and the observation groups were treated orally once daily for 14 days. Tumor volumes were measured twice weekly; antitumor activity is expressed as treatment/control (T/C) and tumor-growth inhibition (TGI). The dose–effect relationship showed perfect linearity. Furthermore, TGI increased from 15 mg/kg to 60 mg/kg for compound 1, while compound 1 and NVP-BEZ235 showed broadly similar effects, both at 30 mg/kg.

Figure 3 Plasma-concentration data of compounds 1 and 2 following a 60 mg/kg oral dose of compound 1 in BALB/c nude mice.

Notes: Nude mice were dosed orally with compound 1 at 60 mg/kg (n=9 per group). Blood was collected from different animals and prepared by centrifugation. Concentrations of compounds 1 and 2 in plasma were determined by LC-MS/MS. As compound 2 was a dominant metabolite in mice, its AUC was about 50-fold higher than compound 1, and the half-life of compound 2 was longer than compound 1.
Abbreviations: LC, liquid chromatography; MS, mass spectrometry; AUC, area under the curve.

Figure 4 Drug concentrations of compound 1 and 2 in the liver microsomes at 60 minutes.

Notes: Compound 1 was incubated with liver microsomes of nude mice, Sprague Dawley rats, beagles, and humans. Concentrations of compounds 1 and 2 were quantified by LC-MS/MS. Compound 1 was metabolized rapidly into compound 2, and compound 2 was the dominant metabolite only in mouse microsomes. However, the metabolic profiling of compound 1 in microsomes of rats, dogs, and humans showed low levels of compound 2 in the products.
Abbreviations: LC, liquid chromatography; MS, mass spectrometry.

Table 1 PK/PD exploration in BALB/c nude-mouse xenograft model of PC3 cell line (n=8)

	Body weight (g)		Tumor volume (mm^3)	T/C (%)	TGI (%)	AUC0–24 in plasma (ng·h/mL)	Concentration in tumor tissue (ng/g)c,d
	Before treatmentd	After treatmentd
Control	23.1±0.5	22.8±0.5	1,026±68	–	–	–	–
Compound 1 (15 mg/kg)	23.1±0.5	22.4±0.4	643±40	62.7	37.3a	30	5.49±1.86
Compound 1 (30 mg/kg)	23.2±0.4	21.5±0.4	412±38	40.3	59.7b	111	5.33±1.97
Compound 1 (60 mg/kg)	23.2±0.4	19.4±0.3	185±21	17.9	82.1b	328	15.3±4.29
NVP-BEZ235 (30 mg/kg)	23.2±0.5	20.8±0.3	331±16	32.4	67.6b	4,779	310±61.8

Notes:

a P<0.05 (vs vehicle control using one-way ANOVA);

b P<0.001 (vs vehicle control using one-way ANOVA);

c at 4 hours after the last dose;

d data presented as mean ± standard deviation. PC-3 cells were injected subcutaneously into nude mice (n=8 per group), and observation groups were treated orally once daily for 14 days. Tumor volumes were measured twice weekly, and antitumor activity is expressed as T/C (%) and TGI (%). The dose–effect relationship showed perfect linearity, TGI increased from 37.3% to 82.1% as doses ascended from 15 mg/kg to 60 mg/kg for compound 1 (compared with vehicle control, P<0.05), while compound 1 and NVP-BEZ235 showed broadly similar effect, both at 30 mg/kg.

Abbreviations: T/C, treated/control; TGI, tumor growth inhibition; AUC, area under the curve; ANOVA, analysis of variance; PK/PD, pharmacokinetic/pharmacodynamic.

Table 7 Pharmacokinetics in rats of compound 2 after IV and PO dosing of compound 3

	IV				PO
	Cl (L/h/kg)	Vss (L/kg)	AUC0–t (ng·h/mL)	t½ (hours)	Cmax (ng/mL)	AUC0–t (ng·h/mL)	t½ (hours)	F (%)
Compound 2a	0.23±0.03	0.85±0.05	8,687±1,209	3.24±0.22	143.3±25.9	1,695±1,033	9.04±4.33	12.2±9.4

Notes:

a Dosing compound 3 (dosage conversion calculated as compound 2) and detecting compound 2; PO at 4 mg/kg, IV at 2 mg/kg (n=3). Compound 3 was dosed in rats via IV (2 mg/kg) and PO (4 mg/kg) routes, with three animals per group; blood was collected and prepared by centrifugation. The concentration of compounds 2 and 3 was separately quantified by LC-MS/MS. Pharmacokinetic parameters were calculated by WinNonlin software with a noncompartmental model. The bioavailability of compound 2 was only 12.2%, which was too low to be administered as an oral drug via the strategy of the prodrug. Data presented as mean ± standard deviation.

Abbreviations: IV, intravenous; PO, per os (oral); Cl, clearance; V_ss, volume of distribution at steady state; AUC, area under the curve; t_½, half-life; C_max, maximum concentration; LC, liquid chromatography; MS, mass spectrometry.

Figure 5 Synthetic route of compound 1.

Figure 6 Synthetic route of compound 2.

Figure 7 Synthetic route of compound 3.

Table 2 Pharmacokinetic parameters of compound 1 and its metabolite compound 2 after oral dosing of compound 1 in nude mice (60 mg/kg)

	Animal	t½ (hours)	tmax (hours)	Cmax (ng/mL)	AUC0–24 (ng·h/mL)
Compound 1a	BALB/c nude mice	NAb	0.5	77.6	61.3
Compound 2a	BALB/c nude mice	10.1	0.5	448	3,404

Notes:

a Dosing compound 1 and detecting compound 1 and compound 2. Nude mice were dosed orally with compound 1 at 60 mg/kg (n=9 per group);

b not available. Blood was collected from different animals and prepared by centrifugation. The concentration of compounds 1 and 2 in plasma was determined by LC-MS/MS. The AUC of compound 2 was about 50-fold higher than compound 1, and the half-life (t_½) of compound 2 was much longer than compound 1.

Abbreviations: C_max, maximum concentration; t_max, time to C_max; AUC, area under the curve; LC, liquid chromatography; MS, mass spectrometry; NA, not available.

Table 3 In vitro enzyme and cellular activity of compounds 1 and 2 and NVP-BEZ235 (IC₅₀, nM)

Assay	NVP-BEZ235	Compound 1	Compound 2
Enzyme
PI3Kα	33	8.5	7.8
mTOR	0.85	16	2.5
Cell line
U87MG	3.7	104.8	29.48
BT474	10.7	127.3	33.66
A549	11.8	265.9	63.16
SKOV-3	6.7	98.22	51.17
PC-3	8.3	103.6	33.73
HCT116	50	217.9	100.2
786-0	6.5	264.5	65.65

Notes: Inhibitory activity of compounds 1 and 2 and NVP-BEZ235 on PI3Kα and mTOR was assessed and cellular assay performed. Compound 2 showed similar inhibitory activity against PI3K and mTOR to compound 1 and NVP-BEZ235. It also exerted good inhibitory activity on the proliferation of several tumor-cell lines, eg, U87MG, SKOV-3, and PC-3.

Table 4 Liver-microsome stability of compound 1 in mouse, rat, dog, and human species at 1 μM

Time point, minutes	0 (nM)	5 (nM)	10 (nM)	20 (nM)	30 (nM)	60 (nM)
Nude mouse
Compound 1	804	30.85	13.27	24.55	26.15	18.95
Compound 2	0	13.15	34.7	77.65	114.5	180.5
Rat
Compound 1	776	527	356	198	118	33
Compound 2	0	0	0	0	0	2.5
Dog
Compound 1	764	662	515	327	257	86
Compound 2	0	0	0	0	0	0
Human
Compound 1	847	598	530	256	142	29
Compound 2	0	0	0	0	0	5

Notes: Compound 1 was incubated with liver microsomes of the nude mice, Sprague Dawley rats, beagles, and humans. Concentration of compounds 1 and 2 were quantified by LC-MS/MS. Compound 1 was metabolized rapidly into compound 2, and compound 2 was the dominant metabolite only in mouse microsomes. However, the metabolic profiling of compound 1 in rat, dog, and human microsomes showed small amounts of compound 2 in the products.

Table 5 Pharmacokinetics of compound 2 in rats with different preformulations

	Dose (mg/kg)	Cmax (ng/mL)	tmax (hours)	AUC0–t (ng·h/mL)	F (%)
Formulation 1a	5	50.8±17.5	4	449±55.6	2.02±0.28
Formulation 2b	5	34.5±11.7	4	281±73.6	1.33±0.34
Formulation 3c	4	129±11	6	1,568±58.3	8.45±0.32
Formulation 4d	4	205±32.6	6	2,549±352	14±1.93

Notes:

a 0.5% MC (n=3);

b 0.5% MC + 0.5% SDS (n=3);

c solid-dispersion technology (n=3);

d 30% DMF + 50% PEG₄₀₀ + 20% (0.9% saline) (n=3). Compound 2 was dosed orally in rats at 4 or 5 mg/kg (n=3 per group). Blood was collected and the concentration of compound 2 determined by LC-MS/MS. Pharmacokinetic parameters were calculated by WinNonlin software with a noncompartmental model. The bioavailability of compound 2 was particularly poor in routine formulations. Data presented as mean ± standard deviation.

Abbreviations: C_max, maximum concentration; t_max, time to C_max; AUC, area under the curve; MC, methylcellulose; SDS, sodium dodecyl sulfate; DMF, dimethylformamide; PEG, polyethylene glycol; LC, liquid chromatography; MS, mass spectrometry.

Table 6 Plasma concentration of compounds 3 and 2 at the same time points after dosing compound 3 in rats (intravenous injection, 2 mg/kg)

Time (hours)	Compound 3 (ng/mL)	Compound 2 (ng/mL)
0.083	719±226	2,383.3±255.4
0.25	47.5±5.86	2,230±212.8
0.5	BQL	1,996.7±160.7
1	BQL	1,733.3±184.8
2	BQL	1,193.3±141.5
4	BQL	685.7±98
6	BQL	387.7±95.5
8	BQL	196.3±51.6
24	BQL	8.4±4.2

Notes: Compound 3 was dosed as intravenous bolus in rats at 2 mg/kg (n=3 per group), and blood was collected and prepared by centrifugation. The concentration of compounds 2 and 3 was separately quantified by LC-MS/MS. After dosing, compound 3 disappeared, while the concentration of compound 2 decreased slowly with time. Data presented as mean ± standard deviation.

Abbreviations: BQL, below quantifiable limit; LC, liquid chromatography; MS, mass spectrometry.