Pharmacokinetic evaluation of C-3 modified 1,8-naphthyridine-3-carboxamide derivatives with potent anticancer activity: lead finding

Figures & data

Table 1. In vitro cytotoxicity of naphthyridine derivatives*.

	IC50 (50% growth inhibitory concentration, µM)
Compound	PA-1 (Ovary)	DU145 (Prostate)	KB (Oral)	SW620 (Colon)	HBL100 (Breast)	A549 (Lung)	Mean cytotoxicity† (Mean ± SD)	IC50 normal cell lines‡	Specificity to cell lines/ number of cell lines tested§
1	0.54	>10	>10	2.90	4.00	>10	2.48 ± 1.77	4.4	1/6
2	0.68	>10	4.9	2.10	2.00	6.1	3.15 ± 2.25	2.4	1/6
3	1.10	>10	>10	2.70	3.20	9.50	4.13 ± 3.69	4.4	1/6
4	0.50	0.60	1.10	1.40	>10	>10	0.9 ± 0.42	>10	4/6
5	9.10	2.90	>10	>10	5.90	6.09	6.00 ± 2.53	4.79	1/6
6	0.41	>10	3.70	1.40	4.10	3.06	2.53 ± 1.57	2.2	1/6
7	1.19	>10	>10	4.62	4.70	9.00	4.88 ± 3.2	5.0	1/6
8	3.55	>10	>10	2.79	>10	1.50	2.61 ± 1.04	>10	3/6
9	>10	>10	>10	>10	4.33	4.82	4.58 ± 0.35	>10	2/6
10	2.62	3.45	9.12	3.79	3.17	9.53	5.28 ± 3.16	6.86	3/6

*Data reported in (10, 11). Compounds 1, 2, 3 and 4 represent the compounds 20, 22, 19, and 34 respectively of ref [10] while compounds 5, 6, 7, 8, 9 and 10 represent the compounds 25, 29, 36, 47, 49, and 53, respectively, of ref [11].

†Mean cytotoxicity = [IC₅₀ (PA1) + IC₅₀ (DU145) + IC₅₀ (KB) + IC₅₀ (SW620) + IC₅₀ (HBL100) + IC₅₀ (A549)]/6.

IC₅₀> 10 µM was not taken in mean calculation.

‡NIH3T3 is used as normal cell lines except compound 8 and 9 where CHO is used as normal cell line.

§Specificity represents number of cell lines where the ratio of IC₅₀ normal cells/IC₅₀ cancer cells was greater than 2.

Table 2. Summary of analytical parameters of chromatographic methods used for sample analysis of various in vitro and in vivo ADME assays.

			Lower limit of	Variability range‡ (%CV, n = 3)
Assay	Matrix	Linearity range* (µg/ml)	quantification (LLOQ† (µg/ml)	Inter day	Intra day
Plasma stability	Plasma	0.1–50	0.1	3.0–5.8%	2.0–6.2%
Plasma protein binding	Plasma filtrate	0.1–50	0.1	2.8–6.7%	5.6–10.2%
Metabolic stability	Buffer	NA	NA	NA	NA
Caco-2 permeability	Buffer	0.05–100	0.05	3.2–10.4%	6.8–12.2%
Partition coefficient	Buffer	0.05–100	0.05	4.8–13.4%	7.5–9.2%
Pharmacokinetic study (rat)	Rat plasma	0.1–100	0.1	2.0 to 5.6%	2.3–5.2%
Pharmacokinetic study (TBM)	Mouse plasma	0.1–100	0.1	3.6–5.2%	1.2–6.7%
Tumor uptake study	Tumor homogenate	0.1–100	0.1	10.2–13.5%	7.8–11.0%

*Linearity range shown in the table represent the linearity range of each compound participated in the assay. Linearity range was kept same for all the compounds participated in a particular assay.

†LLOQ values reported in table is the value for each compound tested for that particular assay.

‡Variability range of the assay, shown in the table incorporates the maximum and minimum variation values shown by the compounds from the nominal values. Data was generated by performing the assay experiments in triplicate (n = 3) on the same day (intra day) and at three different days (inter day) at three concentrations; low quality control (LQC), mid quality control (MQC) and high quality control (HQC).

Table 3. In vitro ADME properties and structure correlation of naphthyridine derivatives.

					ADME properties
Cpd No	n	X	R1	R2	Metabolic stability (% un-metabolized)	Solubility (µM)	Permeability Log Pe
1	1	7-Cl	H		85.0 ± 1.3	10.2 ± 2.8	−5.70 ± 0.68
2	1	6-F,7-Cl	H		7.1 ± 0.55	9.9 ± 4.5	−5.60 ± 0.22
3	1	7-Cl	H		27.8 ± 1.3	4.2 ± 2.6	−5.89 ± 0.39
4	0	6-F,7-Cl	–		25.3 ± 0.74	15.5 ± 2.4	−5.66 ± 0.78
5	1	H	–OH		100.0 ± 0.35	148.7 ± 12.3	−5.01 ± 0.03
6	1	H	–OH		100.0 ± 1.06	202.4 ± 9.8	−4.90 ± 0.23
7	1	7-Cl	–OH		22.5 ± 1.04	45.9 ± 3.3	−6.08 ± 0.08
8	1	6-F,7-Cl	–OH		21.1 ± 0.97	13.8 ± 1.9	−5.52 ± 0.88
9	1	6-F,7-Cl	–OH		100.0 ± 1.00	78.6 ± 8.5	−5.28 ± 0.45
10	1	6-F,7-Cl	–OH		100.0 ± 0.82	82.6 ± 2.5	−5.37 ± 0.12

ADME properties like metabolic stability, solubility and permeability showed strong structure correlation. Properties like plasma stability, protein binding, CYP inhibition potential had optimum values across all the derivatives.

Table 4. Partition coefficient, plasma protein binding, Caco-2 permeability and plasma stability of naphthyridine derivatives.

Compound	Partition coefficient (Log P)	% Plasma protein binding	Caco-2 permeability Papp (nm/s)	Plasma stability*
1	5.41 ± 0.88	92 ± 4%	ND	Stable
2	5.96 ± 0.54	>97%	ND	Stable
3	>6	>97%	ND	Stable
4	6.22 ± 0.85	97 ± 2%	ND	Stable
5	4.25 ± 0.05	∼99%	ND	Stable
6	4.02 ± 0.82	∼97%	A–B: 9 ± 3; B–A: 198 ± 23	Stable
7	5.14 ± 0.23	100%	ND	Stable
8	5.82 ± 0.11	100%	ND	Stable
9	5.55 ± 0.02	>98%	ND	Stable
10	5.32 ± 0.56	>98%	ND	Stable

Data shown are mean ± SEM of three independent experiments. ND: Not Done.

*Derivative which showed the stability (% degradation <95.0) in plasma up to 8 h are considered stable.

Figure 1. Inhibition of cytochrome P450 enzyme isoforms (A) CYP1A2, (B) CYP2C9, (C) CYP2D6, (D) CYP3A4 and (E) CYP2C19. The ability of compounds at 10 µM to inhibit substrate of five specific CYP enzymes was analyzed using fluorometric microtiter plate assay. Data shown are mean ± SEM of three independent experiments. ** and *** represent p < 0.05, p < 0.01 and p < 0.001 when compared with other compounds in the study.

Figure 2. (A) Plasma concentration profile of derivatives 5 (squares) and 6 (diamond) following intravenous administration at 5.0 mg/kg dose in Wistar rat. Data shown in mean ± SD (n = 4). (B) Plasma and tumor concentration profiles of 5 and 6 following intravenous administration at 5.0 mg/kg dose in tumor-bearing mice (TBM). Data shown in mean ± SD (n = 4). Plasma and tumor concentration (µg/mL) determined by HPLC.

Table 5. Mean pharmacokinetic parameters of shortlisted naphthyridine derivatives following intravenous administration in Rat and Tumor bearing mouse (TBM) at intravenous dose of 5.0 mg/kg.*.

	Plasma data (Rat)†			Plasma data (TBM) ‡			Tumor data (TBM)
Cpd No.	C0 (µg/ml)	AUC (h×µg/ml)	T1/2 h	C0 (µg/ml)	AUC (h×µg/ml)	T1/2 h	C0t (µg/gm)	AUCt (h×µg/gm)	T1/2 h	TPI¶ (AUCt/AUC)
5	6.98 ± 2.18	11.58 ± 2.18	2.45 ± 0.27	3.67 ± 0.13	4.40 ± 0.38	3.22 ± 1.85	5.21 ± 0.91	11.00 ± 3.56	3.22 ± 0.24	2.50
6	6.61 ± 1.43	14.45 ± 0.61	7.02 ± 2.98	4.41 ± 1.55	5.15 ± 0.11	3.62 ± 2.48	6.09 ± 2.04	20.68 ± 2.06	3.01 ± 0.69	4.02
9	Plasma levels not detected				ND				ND
10	Plasma levels not detected				ND				ND

*The plasma and tumor time concentration data was analyzed using Winnonlin v5.0.1 non-compartmental model.

†Data shown is mean (n = 4) for rat.

‡Data shown is mean (n = 3) for tumor bearing mouse.

¶TPI: Tumour plasma index, ND: not done

Figure 3. In vivo antitumor activity of derivative 6. (A) Derivative 6 showed 33% tumor growth inhibition (TGI) against human ovarian xenograft model. Curve indicates the tumor volumes of ovary (PA1) tumor-bearing mice of vehicle-treated (circle) and derivative 6-treated (square) groups at i.v. dose of 5.0 mg/kg once daily for 14 day. **Represents p < 0.01 when compared with untreated group (B) Dotplot of derivative 6 showed mean tumor growth delay (TGD) of 4.3 days to reach the 4x tumor volume. Linear regression y = 45.41x + 56.45(R² = 0.998) and y = 26.88x + 84.24(R² = 0.99) was followed for time to reach 4x start volume for vehicle and treated group, respectively. *represents p < 0.05 when compared with untreated group (Mann–Whitney U test). Horizontal line (–) indicates the mean value. The Data shown are mean ± SD (n = 8). Arrow indicates start of the treatment.