In vitro activity (MICs and rate of kill) of AFN-1252, a novel FabI inhibitor, in the presence of serum and in combination with other antibiotics

Figures & data

Table 1. Determination of serum protein binding of AFN-1252

	% Binding
AFN-1252 concentration (μg/ml)	Mouse	Rat	Dog	Human
Equilibrium dialysis method
0·2	96·5	98·6	97·7	98·1
1	96·2	98·7	97·5	98·4
5	95·3	98·8	96·9	98·4
10	94·9	98·6	96·4	98·3
Ultrafiltration method
0·1	94·6	–	–	–
0·5	98·2	–	–	–
1·0	98·8	–	–	–
2·0	98·8	–	–	–
Ultrafiltration method
3·75	94·6	91·9	94·9	92·5

Figure 1. Rate of kill of AFN-1252 against MRSA 1659 in the presence and absence of 75% mouse and human serum.

Figure 2. Inhibitory effect models: change in log CFU/ml vs AFN-1252 concentrations at the 10-hour time point. (A) Human serum; (B) Mouse serum.

Table 2. In vitro activity of AFN-1252 and comparators against S. aureus in the presence and absence of pulmonary surfactant

	Range of MICs (μg/ml)
	AFN-1252 with			Vancomycin with			Daptomycin with
	surfactant (mg/ml)			surfactant (mg/ml)			surfactant (mg/ml)
	0	0·1	0·8	0	0·1	0·8	0	0·1	0·8
MSSA, n = 10	0·004–0·015	0·008–0·015	0·008–0·03	0·5–1	0·5–4	0·5–1	0·5–2	4–32	64
MRSA, n = 10	0·008	0·008	0·008–0·015	0·5–1	0·5–2	0·5–4	0·5–2	8–16	64

Note: CLSI QC range.

Table 3. Average PAE from four experiments

	AFN-1252		PAE (h)*
	× MIC	μg/ml	1-hour exposure	4-hour exposure
MSSA 29213	4	0·03	0	0
	32	0·25	0	0·1±0·1
	128	1·0	0·5	1·1±0·4
MRSA 43300	4	0·03	NT	0
	32	0·25	NT	0·2±0·1
	129	1·0	NT	1·2±0·7

Note: *average±SD.

Table 4. Checkerboard analysis of AFN-1252 with S. aureus

Combination antibacterial	Class	Target	Average FIC index
			MSSA 29213	MRSA 43300	Interaction
Gentamicin	Aminoglycoside	Protein synthesis	0·4	0·4	Synergy
Linezolid	Oxazolidinone		0·6	0·5	Additivity
Tetracycline	Tetracycline		1·0	0·8	Additivity
Erythromycin	Macrolide		2·0	NT	Indifference
Cloxacillin	Penicillin	Cell wall	1·0	0·8	Additivity
Nafcillin	Penicillin		0·6	0·6	Additivity
Cefuroxime	Cephalosporin		0·7	0·5	Additivity
Vancomycin	Glycopeptide		0·8	0·7	Additivity
Levofloxacin	Fluoroquinolone	DNA	0·9	1·0	Additivity
Rifampicin	Rifamycin	RNA	0·7	0·8	Additivity

Figure 3. Time-kill graphs to show synergy of AFN-1252 with gentamicin against MSSA 29213.

Table 5. Effect of AFN-1252 in combination with linezolid, gentamicin or vancomycin upon reduction in viable count (log CFU/ml) of MSSA 29213 after 24 hours

	Change in bacterial count after 24 hours (log CFU/ml)	Combination effect
Growth control	3·73	–
Linezolid (4× MIC)	−3·14	–
Linezolid (¼× MIC)	2·86	–
AFN-1252 (2× MIC)	−1·16	–
Linezolid (4× MIC)+AFN-1252 (2× MIC)	−2·98	Indifference
Linezolid (¼× MIC)+AFN-1252 (2× MIC)	−2·39	Indifference
Growth control	3·81	–
Gentamicin (2× MIC)	≧−4·572	–
Gentamicin (⅛× MIC)	2·88	–
AFN-1252 (2× MIC)	−1·10	–
Gentamicin (2× MIC)+AFN-1252 (2× MIC)	≧−4·572	Not evaluable*
Gentamicin (⅛× MIC)+AFN-1252 (2× MIC)	≧−4·572	Synergy
Growth control	3·22	–
Vancomycin (4× MIC)	≧−4·531	–
Vancomycin (¼× MIC)	3·32	–
AFN-1252 (2× MIC)	−1·53	–
Vancomycin (4× MIC)+AFN-1252 (2× MIC)	−2·49	Antagonism
Vancomycin (¼× MIC)+AFN-1252 (2× MIC)	−0·89	Indifference

Note: * – not evaluable because the viable cell count fell below the level of detection for both gentamicin alone and in combination with AFN-1252.