Safety, Tolerability, and Pharmacokinetics of Oral Nafithromycin (WCK 4873) after Single or Multiple Doses and Effects of Food on Single-Dose Bioavailability in Healthy Adult Subjects

Nafithromycin (WCK 4873), a novel lactone-ketolide, was administered to healthy adult subjects in 2 randomized, double-blind, placebo-controlled, phase 1 studies. In the first-in-human study, single ascending oral doses of nafithromycin (100 to 1,200 mg) were administered to subjects under fasted or fed conditions, with effects of food on bioavailability of nafithromycin studied at the dose levels of 400 and 800 mg.

ABSTRACT

Nafithromycin (WCK 4873), a novel lactone-ketolide, was administered to healthy adult subjects in 2 randomized, double-blind, placebo-controlled, phase 1 studies. In the first-in-human study, single ascending oral doses of nafithromycin (100 to 1,200 mg) were administered to subjects under fasted or fed conditions, with effects of food on bioavailability of nafithromycin studied at the dose levels of 400 and 800 mg. In the second study, multiple ascending oral doses of 600, 800, or 1,000 mg of nafithromycin were administered once daily for 7 days under fed conditions. Nafithromycin was generally well tolerated at all doses. No serious or severe adverse events were observed. The mean maximum plasma concentration (C_max) ranged from 0.099 to 1.742 mg/liter, and the area under the concentration-time curve from time zero to time t (AUC_0–t) ranged from 0.54 to 22.53 h·mg/liter. Nafithromycin plasma AUC_0–t increased approximately 1.2-fold under fed compared to fasted conditions. In the multiple-dose study, the day 7 nafithromycin C_max ranged from 1.340 to 2.987 mg/liter and the AUC over the final dosing interval (AUC_0–24) ranged from 13.48 to 43.46 h·mg/liter. The steady state was achieved after 3 days for the 600-mg and 800-mg-dose cohorts and after 4 days for the 1,000-mg cohort. Under both single- and multiple-dosing regimens, plasma exposure to nafithromycin appeared to increase more than dose proportionally. Nafithromycin showed moderate accumulation on day 7 of dosing. The human pharmacokinetic profile, safety, and tolerability data support further development of nafithromycin. (This study has been registered at ClinicalTrials.gov under registration no. NCT03926962 and NCT03979859.)

INTRODUCTION

Nafithromycin (previously known as WCK 4873) is a novel oral and intravenous lactone-ketolide optimized to provide coverage of multidrug-resistant (MDR) typical and atypical respiratory pathogens, including penicillin- and macrolide-resistant pneumococcal strains. Preclinical studies have shown that nafithromycin has pharmacodynamic action against macrolide- and ketolide-resistant strains of Streptococcus pneumoniae, in addition to other important respiratory pathogens such as Haemophilus influenzae, Moraxella catarrhalis, methicillin-susceptible Staphylococcus aureus, and group A streptococci. An antimicrobial surveillance study involving more than 4,500 clinical isolates collected worldwide during 2013 to 2014 showed nafithromycin MICs for 50% and 90% of isolates (MIC₅₀ and MIC₉₀) of 0.015 mg/liter and 0.06 mg/liter against S. pneumoniae, respectively (1). Nafithromycin was 2 to 8 times more potent in vitro than telithromycin, cethromycin, clindamycin, and clarithromycin against S. pneumoniae. Against erythromycin- and telithromycin-non-susceptible pneumococci, the nafithromycin MIC₉₀ was 0.12 mg/liter. Similarly, for methicillin-susceptible S. aureus, H. influenzae, M. catarrhalis, and Streptococcus pyogenes, the nafithromycin MIC₉₀s were 0.06, 4, 0.25, and 0.015 mg/liter, respectively (1). Nafithromycin also demonstrated potent activity against common atypical respiratory pathogens such as Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp., as well as intracellular killing of Legionella in an in vitro human macrophage cell line (2–4). Structurally, nafithromycin differs from solithromycin and telithromycin by having a lactone group at positions C11 and C12 of the ketolide nucleus instead of carbamate. Further, the lactone ketolide is attached with a side chain (2-pyridine-1,3,4-thiadiazole) through a relatively hydrophilic four-atom spacer containing a cis double bond and a chiral methyl group. This unique feature not only enabled nafithromycin to demonstrate potent activity against ermB-expressing as well as telithromycin-resistant S. pneumoniae strains (1) but also provided favorable oral absorption (5), high levels of lung penetration (possibly mediated through a transporter) (6), and low cytochrome P450 inhibition potential (7). The major N-desmethyl metabolite of nafithromycin is WCK 4978, observed across the species tested, including humans. WCK 4978 has antimicrobial activity but is 4 to 8 times less active than nafithromycin against macrolide-resistant pneumococci; however, the MICs of the two are comparable against wild-type susceptible strains.

Diverse in vivo preclinical pharmacokinetic (PK) and safety studies, including studies using a murine neutropenic lung infection model, have provided scientific evidence of the therapeutic potential of nafithromycin for treatment of community-acquired bacterial pneumonia (CABP) and other bacterial respiratory infections, including those difficult to treat, caused by MDR pathogens (7–10). As epithelial lining fluid (ELF) and alveolar macrophages (AM) are considered important sites for lower respiratory tract infections (RTIs) caused by extracellular and intracellular pathogens, intrapulmonary concentrations of nafithromycin in healthy adult subjects were studied and published (ClinicalTrials registration no. NCT02453529) (6). Similarly to other ketolide, azalide, and macrolide agents, the levels of ELF exposure and AM exposure for nafithromycin were over 10-fold and 100-fold higher, respectively, than the total plasma concentrations. Those ratios of exposure of ELF or AM to nafithromycin in plasma were found to be higher than those reported for solithromycin and telithromycin (6).

We present results of studies of the PK and tolerability of nafithromycin after administration of single and multiple oral doses in healthy adult subjects, as well as the food effect (FE) on oral bioavailability.

(These data were presented in part at the ASM [American Society for Microbiology] Microbe conference in 2016 [11, 12]).

RESULTS

Subject demographics and baseline characteristics.

A total of 144 subjects were screened for the single ascending dose (SAD)/FE study, among whom 41 were screen failures, 36 were not available or were reserve subjects, and 67 were randomized. Among the latter, 40 were randomized to receive nafithromycin or placebo in the SAD part of that study and 27 in the FE part. Subject demographics and baseline characteristics are presented in Table 1, and study cohorts are specified in Table 2. Demographic parameters were similar for all cohorts within the SAD part and for the 2 cohorts within the FE part, except the median age of subjects in the 200-mg dose cohort of the SAD part, which was higher (median 33 years) than that of the other dose cohorts (medians from 21 to 25 years) in that study part. For the entire SAD/FE study, all subjects completed the study per the protocol.

TABLE 1.

Subject demographics and baseline characteristics^a

Characteristic	Values
	SAD/FE study (N = 67)		MAD study (N = 30)
	SAD part (n = 40)	FE part (n = 27)
Sex
No. (%) of males	21 (52.5)	14 (51.9)	18 (60.0)
No. (%) of females	19 (47.5)	13 (48.1)	12 (40.0)
Race/ethnicity
No. (%) of Caucasian, non-Hispanic descent	35 (87.5)	26 (96.3)	26 (86.7)
No. (%) of mixed/Asian/black descent	5 (12.5)	1 (3.7)	4 (13.3)
Mean age in yrs (minimum–maximum)	27 (18–62)	30 (19–65)	40 (19–65)
Mean BMI (kg/m2) (minimum–maximum)	24.0 (19.8–30)	22.9 (18.2–29.9)	23.6 (18.5–29.4)

^aSAD = single ascending dose; FE = food effect; MAD = multiple ascending dose; BMI = body mass index.

TABLE 2.

Subject cohorts^a

Cohort/dosage		Design	No. of subjects		Nutritional state
			Nafithromycin	Placebo
SAD/FE study
SAD part	100 mg, single dose	Parallel	8	2	Fasted
	200 mg, single dose		8	2
FE part	400 mg, 2 single dosesb	Crossover	12	2	Fasted vs fedb
	800 mg, 2 single dosesb		11	2
SAD part	1,200 mg, single dose	Parallel	8	2	Fasted
	600 mg, single dose		8	2
MAD study
600 mg, o.d. for 7 days		Parallel	8	2	Fed
800 mg, o.d. for 7 days			8	2
1,000 mg, o.d. for 7 days			8	2

^aSAD = single ascending dose; FE = food effect; MAD = multiple ascending dose; o.d. = once daily. All cohorts were studied in randomized, double-blind fashion.

^bWith a 7-day washout period between drug administrations under fasting and fed conditions.

In the multiple ascending dose (MAD) study, 63 subjects were screened and 30 of them were randomized to receive nafithromycin or placebo. Subject demographics and baseline characteristics are presented in Table 1, and study cohorts are specified in Table 2. Demographic parameters were similar for all cohorts, except with respect to the median age of subjects (medians of 22 years in the 600-mg treatment cohort, 33 years in the 800-mg treatment cohort, and 57 years in the 1,000-mg treatment cohort). One subject from the 1,000-mg treatment cohort was withdrawn from the study on day 3 due to poor tolerability of the study drug (see “Safety and tolerability” below); the remaining 29 subjects completed the study per the protocol.

Safety and tolerability.

Single oral doses of nafithromycin were well tolerated at all dose levels tested. There were no deaths or treatment-emergent serious adverse events (SAEs) reported during the study and no withdrawals from the study due to an adverse event (AE). All treatment-emergent AEs (TEAEs) were of mild intensity, except for one event of vomiting of moderate severity (dosed at 1,200 mg, fed state). All TEAEs were transient and resolved without sequelae at follow-up (11 to 18 days postdose). The most common TEAEs were: dysgeusia (18 events in 18 [27%] subjects), headache (13 events in 13 [19%] subjects), nausea (11 events in 10 [15%] subjects), flatulence (9 events in 8 [12%] subjects), and dizziness (8 events in 8 [12%] subjects). Overall, no difference in tolerability was observed between the fed and fasted conditions. No clinically significant laboratory abnormalities or trends were seen in either (SAD or FE) part of the study, and the incidence of out-of-range laboratory values did not appear to be dose related. There were no findings of clinical relevance with respect to vital signs, 12-lead electrocardiography (ECG), and physical examination.

Multiple oral doses of nafithromycin administered for 7 days were generally well tolerated at all dose levels tested. There were no deaths or other treatment-emergent SAEs. One subject treated with 1,000 mg nafithromycin was withdrawn predose on day 3 due to poor tolerability of the study drug (reported symptoms were nausea with vomiting and diarrhea and abdominal pain with dyspepsia, along with dysgeusia, headache, and a flu-like illness). All TEAEs in all other subjects were of mild severity and were found to have resolved at the time of follow-up, except for one event of paresthesia of mild severity, considered to be not related to the study drug, reported for a subject treated with 800 mg nafithromycin. The numbers of TEAEs reported were similar for the 600-mg and 800-mg treatment groups (29 and 28 TEAEs reported, respectively) and higher in the 1,000-mg treatment group (44 TEAEs), mainly due to an increase in the number of gastrointestinal disorders. The most frequently reported TEAEs were dysgeusia (reported by 67% of the subjects), headache (40%), and dizziness (23%); no correlation between incidence and dose was observed. Both diarrhea and abnormal gastrointestinal sounds were reported by 20% of the subjects, and their incidence was likely dose dependent. Most of aforementioned TEAEs were considered drug related. In 2 subjects, an event of asymptomatic orthostatic hypotension of mild severity, considered possibly related to the study drug, was reported during the treatment period. There were no clinically significant findings with respect to clinical laboratory, ECG, physical examination, eye movement test, and visual acuity test. Three of 24 subjects on active drug treatment (1 in the 800-mg dose cohort and 2 in the 1,000-mg dose cohort) had mild (<2 times the upper limit of normal [ULN]) transient elevation of serum alanine and/or aspartate aminotransferase levels, all of which were found to have normalized at follow-up (at 10 to 17 days after the last dose).

Pharmacokinetics.

In the SAD/FE study, 55 of 56 subjects were analyzed for nafithromycin PK (1 enrolled subject was excluded upon admission to the clinic). In the MAD study, all 24 enrolled subjects with planned PK collection had analyses done.

(i) Single-dose pharmacokinetics. The mean values for PK parameters of nafithromycin in plasma after single doses of nafithromycin are shown in Table 3 (SAD part), with the mean plasma concentrations plotted in Fig. 1 (corresponding data for WCK 4978 are presented in Table S1 and Fig. S1 in the supplemental material). Following treatment with single ascending oral doses of 100 to 1,200 mg nafithromycin under either fasted or fed conditions, drug levels were detected in plasma within 0.5 to 2 h postdose (0.5 h was the first postdose sampling time point). The highest geometric mean plasma concentrations (T_max) were observed between 1 and 6 h postdose. The mean plasma concentration-time profiles displayed a clear dose-dependent increase. After reaching a maximum, the concentrations decreased rapidly by approximately 12 to 16 h postdose, followed by a slower decline. However, the mean of the combined individual profiles of the 1,200-mg fed treatment had 2 peaks, with the highest peak at approximately 6 h postdose. Consistently, most of the individual profiles for the 1,200-mg treatment showed 2 peaks up to several hours after dosing. Considerable interindividual differences (with about 2-fold to 8-fold differences in plasma concentrations) in PK profiles across all dose levels were observed. One subject in the 200-mg fasting cohort had low plasma concentrations (maximal concentration = 0.047 mg/liter) compared to the other subjects (maximal concentrations, 0.122 to 0.393 mg/liter). The low concentrations seen with this subject may have been due to incomplete absorption of the tablet, since the level of recovery of the drug from feces samples was relatively high (69.2%) in this subject.

TABLE 3.

Pharmacokinetic parameters for nafithromycin in plasma after single oral doses in fasted or fed subjects^a

Study part	Treatment cohort	Cmax (mg/liter)	Tmax (h)	AUC0–t (h·mg/liter)	AUC0–inf (h·mg/liter)	t1/2 (h)	CL/F (liters/h)	VZ/F (liters)
SAD	100 mg fasted (n = 8)	0.099 (46)	1.26 (0.50, 3.00)	0.540 (62)	0.662 (56)	5.15 (36)	151 (64)	1,123 (50)
	200 mg fasted (n = 8)	0.175 (54)	2.00 (1.00, 6.00)	1.307 (46)	1.508 (41)	7.08 (38)	133 (42)	1,355 (89)
	600 mg fed (n = 8)	0.911 (32)	4.00 (1.00, 6.00)	10.482 (24)	10.655 (23)	7.90 (10)	56.3 (25)	642 (20)
	1,200 mg fed (n = 8)	1.742 (23)	3.50 (1.00, 8.02)	22.530 (29)	22.925 (28)	8.09 (12)	52.3 (25)	611 (24)
FE	400 mg fasted (n = 12)	0.345 (46)	2.50 (1.50, 12.00)	3.416 (51)	4.106 (40)	7.87 (14)	97.4 (37)	1,106 (37)
	400 mg fed (n = 12)	0.444 (38)	3.00 (1.50, 6.00)	4.251 (34)	4.448 (33)	7.43 (13)	89.9 (30)	963 (39)
	800 mg fasted (n = 11)	0.932 (23)	4.00 (2.50, 6.02)	12.265 (36)	12.487 (36)	8.95 (10)	64.1 (37)	827 (42)
	800 mg fed (n = 11)	1.203 (23)	3.88 (1.50, 6.00)	14.618 (29)	14.848 (29)	8.56 (12)	53.9 (31)	665 (30)

^aGeometric means (% coefficient of variation [CV]) are presented, except for T_max, for which median (minimum, maximum) values are presented. SAD = single ascending dose; FE = food effect; CL/F = apparent clearance; V_Z/F, apparent volume of distribution during terminal phase.

FIG 1.

Mean plasma concentration-time profiles of nafithromycin following escalating single-dose nafithromycin administrations (linear scale). fas = fasting condition, fed = fed condition.

As for the WCK 4978 metabolite, the dose-dependent increase displayed in its concentration time profiles was similar to that described for the parent (nafithromycin) profiles. Also, the individual and combined individual WCK 4978 concentration time profiles displayed a pattern similar to that described for nafithromycin.

For dose proportionality, assessed for the full dose range and by nutritional status, the 95% confidence intervals (CI) and exponents of the power model corresponding to the exposure parameters (slopes of the regression lines) of both nafithromycin and WCK 4978 appeared to be higher than 1 for maximum plasma concentration (C_max) under fed conditions and for the area under the concentration-time curve from time zero to time t (AUC_0–t) and for AUC from time zero to infinity (AUC_0–inf) under both fasted and fed conditions. Results of the power analyses are presented in Table 4. Additionally, dose-normalized exposures (AUC_0–inf) versus the dose for nafithromycin are plotted in Fig. 2. Overall, plasma exposure to nafithromycin and WCK 4978 appeared to increase more than dose proportionally.

TABLE 4.

Results of power analyses for dose proportionality of nafithromycin and WCK 4978^a

Parameter	Nutritional state	Slope	95% CI
			Lower limit	Upper limit
Nafithromycin
Cmax	Fasted	1.08	0.83	1.32
	Fed	1.26	1.02	1.51
AUC0–t	Fasted	1.50	1.27	1.73
	Fed	1.54	1.29	1.80
AUC0–inf	Fasted	1.42	1.23	1.61
	Fed	1.51	1.27	1.76
WCK 4978
Cmax	Fasted	1.14	0.83	1.44
	Fed	1.30	1.03	1.57
AUC0–t	Fasted	1.97	1.51	2.43
	Fed	1.92	1.65	2.20
AUC0–inf	Fasted	1.97	1.20	2.74
	Fed	1.81	1.55	2.07

^aCI = confidence interval. Data represent results of estimation of the slope of logarithmically transformed parameter values (exponent of the power model) versus logarithmically transformed dose values. The model used is as follows: log(parameter) = slope * log(dose) + intercept + error. A slope of 1 (i.e., a 95% CI that includes a value of 1) means that no evidence of deviation from dose proportionality was found.

FIG 2.

Dose-normalized exposures of nafithromycin (AUC_0–inf) across single doses administered.

Across single-dose cohorts, between 10.0% and 19.5% of the administered dose was excreted via urine as unchanged nafithromycin and between 0.9% and 2.6% as the WCK 4978 metabolite. The majority of both analytes was excreted in urine at 48 h postdose, with the process being almost completed at 72 h postdose. The percentage of nafithromycin and WCK 4978 excreted in urine appeared to increase with increasing dose. As for excretion with feces, between 7.4% and 19.0% of the administered nafithromycin dose was recovered and between 9.8% and 13.5% as WCK 4978. The excretion data may have been incomplete at 72 h postdose (the last sampling time point). In contrast to the excretion in urine, the percentage of both analytes excreted in feces appeared to be independent of dose.

The mean level of plasma protein binding of nafithromycin across cohorts was 80.3% (±3.5%), with a mean plasma albumin level of 4.0 g/dl.

(ii) Food effect. The mean values for PK parameters for nafithromycin in plasma after single doses of nafithromycin in crossover fed/fasting cohorts of 400 and 800 mg nafithromycin are shown in Table 3 (FE part), with the mean plasma concentrations plotted in Fig. 3 (corresponding data for WCK 4978 are presented in Table S1 and Fig. S2). For the 400-mg dose cohort, nafithromycin exposure (AUC_0–t) was approximately 1.2-fold higher under the fed condition (percent fed/percent fasted [90% CI]: 124.5 [101.5 to 152.7]) than under the fasted condition. For that cohort, the remaining PK parameters of nafithromycin and the WCK 4978 metabolite did not differ between fed and fasted conditions. For the 800-mg dose cohort, the C_max of nafithromycin was approximately 1.3-fold higher under fed conditions (percent fed/percent fasted [90% CI]: 130.9 [109.5 to 156.5]), and AUC_0–t and AUC_0–inf were approximately 1.2-fold higher (percent fed/percent fasted [90% CI]: 121.3 [101.7 to 144.7] and 121.0 [101.9 to 143.7], respectively) than under the fasted condition. For the WCK 4978 metabolite in the latter cohort, C_max and AUC_0–t, but not AUC_0–inf, were approximately 1.2-fold higher under fed conditions than under fasted conditions (percent fed/percent fasted [90% CI] for C_max, 119.4 [102.9 to 138.5]; for AUC_0–t, 123.8 [105.0 to 145.9]). Thus, the overall exposure to nafithromycin and WCK 4978 metabolite appeared to be increased by the administration of food at the doses evaluated. This increased exposure did not appear to correlate with an increase in the incidence of AEs.

FIG 3.

Mean plasma concentration-time profiles of nafithromycin following fed/fasting crossover single-dose administrations (linear scale). fas = fasting condition, fed = fed condition.

Within the dose proportionality analysis using the power model, steeper slopes of the regression lines were noted for the fed cohorts than for the fasted cohorts for all 3 PK parameters of nafithromycin and for C_max of WCK 4978; however, there were no statistically significant differences (P values of >0.30) for any parameter (see Table 4).

(iii) Multiple-dose pharmacokinetics. The mean values corresponding to the PK parameters for nafithromycin in plasma on day 1 (after single doses) and on day 7 of multiple dosing of nafithromycin are shown in Table 5, with the mean plasma concentrations plotted in Fig. 4 (corresponding data for WCK 4978 are presented in Table S2 and Fig. S3). One subject (800-mg dose cohort) showed very low concentrations throughout the treatment period compared to the other subjects in the study (outlier). The PK data presented below are based on data from all subjects, including that outlier, unless specified otherwise. Overall, increases in C_max of nafithromycin on day 7 and in exposures over the final dosing interval (AUC_0–24) values of nafithromycin on both days 1 and 7 occurred at higher rates than would be anticipated from a dose-proportional relationship. A dose-proportional increase was observed for C_max of the metabolite WCK 4978 on both day 1 and day 7, whereas an increase in AUC_0–24 occurred at higher rates than would be anticipated from a dose-proportional relationship; the effect was more pronounced on day 7 than on day 1. After single and multiple dosing, the geometric mean elimination half-life (t_1/2) was between 9.16 and 14.4 h for nafithromycin and between 10.8 and 14.9 h for its metabolite WCK 4978 and tended to increase with increasing dose.

TABLE 5.

Pharmacokinetic parameters for nafithromycin in plasma on days 1 and 7 of multiple dosing^a

Treatment cohort	Cmax (mg/liter)	Tmax (h)	AUC0–24 (h·mg/liter)	t1/2 (h)	CL/F (liter/h)	Vz/F (liters)	Accumulation ratio
Day 1
600 mg o.d. (n = 8)	1.121 (23)	2.25 (1.00, 3.50)	9.418 (24)	ND	ND	ND	NA
800 mg o.d. (n = 8)	1.013 (35)	2.50 (2.00, 6.00)	10.346 (35)	ND	ND	ND	NA
800 mg o.d. (n = 7)a	1.207 (18)	2.50 (2.00, 6.00)	11.986 (22)	ND	ND	ND	NA
1,000 mg o.d. (n = 8)	1.968 (25)	2.49 (2.00, 4.00)	21.262 (30)	ND	ND	ND	NA
Day 7
600 mg o.d. (n = 8)	1.340 (19)	2.50 (2.00, 3.50)	13.478 (20)	9.16 (12)	44.5 (24)	588 (20)	1.43 (1.48)
800 mg o.d. (n = 8)	1.653 (33)	3.50 (2.50, 6.00)	19.857 (38)	11.0 (29)	40.3 (45)	642 (44)	1.92 (1.98)
800 mg o.d. (n = 7)b	1.888 (21)	3.50 (2.50, 6.00)	22.198 (30)	11.1 (31)	36.0 (23)	576 (21)	1.85 (1.93)
1,000 mg o.d. (n = 7)	2.987 (26)	3.50 (2.50, 4.00)	43.464 (37)	14.4 (31)	23.0 (34)	477 (13)	2.12 (1.99)

^ao.d. = once daily; ND = not determined; NA = not applicable; CL/F = apparent clearance; V_Z/F = apparent volume of distribution during terminal phase. Geometric means (%CV) are presented, except for T_max, for which median (minimum, maximum) values are presented, and accumulation ratio, for which geometric mean (median) values are presented.

^bPK parameters calculated without data from one subject (outlier).

FIG 4.

Mean plasma concentration-time profiles of nafithromycin following escalating multiple-dose administrations (linear scale).

For both nafithromycin and the WCK 4978 metabolite, steady state was achieved after day 3 for the 600-mg and 800-mg dose cohorts and after day 4 for the 1,000-mg cohort (see Fig. S4 for plots of combined nafithromycin concentrations at trough [C_trough]). For 2 subjects treated with the highest (1,000 mg) dose, steady state appeared to be reached later, i.e., at approximately day 7. Across the dose cohorts tested, geometric mean C_max and median T_max values observed on day 7 tended to be increased compared with day 1 for both nafithromycin and WCK 4978. Nafithromycin exposures (AUC_0–24) were approximately 1.4-fold to 2-fold higher (accumulation ratios) on day 7 than on day 1 (and 1.3-fold to 1.8-fold higher for WCK 4978).

The mean level of recovery of nafithromycin in urine (amount excreted in the dosing interval 0-τ, Ae_0–τ,urine) was between 10.7% and 13.0% on day 1 and increased to between 16.3% and 22.4% on day 7, with a tendency to increase with dose. For the WCK 4978 metabolite, the level of recovery in urine ranged between 1.4% and 1.7% on day 1 and increased to between 1.7% and 3.3% on day 7, with a tendency to increase with dose. The mean level of recovery of nafithromycin in feces (amount excreted in the dosing interval 0-τ, Ae_0–τ,feces) was between 0.6% and 1.7% on day 1 and increased to between 3.2% and 9.4% on day 7. The values for recovery in feces for the WCK 4978 metabolite were between 0.1% and 3.0% on day 1 and between 7.1 and 12.2% on day 7 and tended to increase with increasing dose. The aforementioned outlier subject with low plasma exposures had a considerably higher feces excretion of nafithromycin (29.5% between 144 and 168 h after the last dose and another 25.9% excreted between 168 and 192 h after the last dose).

DISCUSSION

The two phase 1 studies reported here explored safety, tolerability, and pharmacokinetics of single and multiple escalating oral doses of nafithromycin in healthy adult subjects, including its bioavailability following administration in fed and fasting states. Data collected from 97 healthy subjects exposed to oral nafithromycin show that its safety and tolerability are favorable. Administration of single ascending doses of oral nafithromycin ranging from 100 to 1,200 mg under fasted or fed conditions was safe and well tolerated by the healthy male and female subjects included in this study. Likewise, administration of multiple doses in the range of 600 mg to 1,000 mg once daily under fed conditions was safe and generally well tolerated by healthy male and female subjects. The AEs observed in the alimentary tract under conditions of multiple dosing (diarrhea and abnormal gastrointestinal sounds) are commonly observed for antibiotic treatments; their incidence was likely dose dependent. However, 1 subject in the MAD study was withdrawn due to diarrhea of moderate severity. No correlation between incidence and dose was found for the other most frequently reported adverse events, namely, headache, dizziness, and dysgeusia; all those events, along with two events of asymptomatic orthostatic hypotension, resolved without sequelae. There were no findings deemed clinically relevant with respect to clinical laboratory, ECG, and physical examination in each study or with respect to eye movement and visual acuity tests performed in the MAD study. The three events of mild (<2× ULN) asymptomatic transaminase elevation recorded in the MAD study were found to have been normalized at follow-up (within 10 to 17 days after the last dose).

The day 1 plasma concentration-time parameters observed in both studies reported here, in particular, at the comparable 800-mg dose level (single dose or daily for 7 days), are similar to the day 1 plasma PK parameters reported previously in a intrapulmonary PK study in healthy subjects following repeated dosing of oral nafithromycin at 800 mg daily for 3 days (6). The systemic exposures achieved in these SAD/FE and MAD studies were generally high, with mean C_max and AUC values for the 800-mg oral dose of nafithromycin achieving 0.9 mg/liter and 12.5 h·mg/liter (AUC_0–inf) after single dose in the (fasted) SAD regimen or 1.2 mg/liter and 12.0 h·mg/liter (AUC_0–24) after first dose in the MAD regimen, respectively (Tables 3 and 5). Taking into account that the AUC/MIC ratio has been suggested to be the pharmacokinetic-pharmacodynamic (PK-PD) parameter best correlated with the efficacy of macrolides and ketolides (13), those values can be expected to be therapeutically effective against a range of respiratory pathogens, based on in vitro MIC values as well as in vivo infection model results previously reported (1, 3, 4). Furthermore, results showing high penetration of nafithromycin into human pulmonary tissue, as evidenced in the study by Rodvold et al. with high ELF/plasma AUC_0–24 ratio values (13.8 and 10.9 for mean and median concentrations, respectively) and AM/plasma AUC_0–24 ratio values (527 and 364 for mean and median concentrations, respectively) (6), provide further support for the prediction of nafithromycin efficacy for RTIs. As discussed in detail by Rodvold et al., a further modeling and simulation study using data from these SAD and MAD studies and the intrapulmonary PK study (6), conducted by Bader et al. (14) using PK-PD targets (AUC/MIC) for S. pneumoniae, as well a study by Satav et al. (15) defining the ELF PK-PD target for H. influenzae from a neutropenic murine lung infection model, yielded high estimated AUC_0–24/MIC₉₀ ratios for ELF for strains of those extracellular pathogens.

In these SAD and MAD studies, the C_max values as well as exposures (AUC_0–inf and AUC_0–t, respectively) increased more than dose proportionally, which indicates nonlinear PK for nafithromycin (see Fig. 2 for the SAD/FE study data). Overall plasma exposure following single oral dose administration of 400 or 800 mg was increased by administration of food (1.2-fold). This increase in exposure is unlikely to affect safety of nafithromycin whether administered with or without food. In the MAD study, the C_max and exposure (AUC_0–24) values on day 7 were approximately 1.2-fold to 1.6-fold and 1.4-fold to 2-fold higher than the respective C_max and AUC_0–24 values on day 1 (Table 5; see also Fig. 4, as well as Table S2 and Fig. S3 in the supplemental material), indicating accumulation on multiple-dose administration. On average, steady state was achieved on day 3 for once-daily 600 mg and 800 mg nafithromycin and on day 4 for once-daily 1,000 mg nafithromycin. Plasma exposures of the WCK 4978 metabolite were 10% to 15% of those of nafithromycin. Up to 72 h following a single dose, urinary recovery of unchanged drug and its WCK 4978 metabolite across the doses was about 10% to 20% and fecal recovery was about 20% to 40%. Additionally, an absorption-metabolism-elimination study with radiolabeled nafithromycin (unpublished data) revealed that nafithromycin and its identified/characterized metabolites in feces and urine cumulatively accounted for 55% to 60% and 20% to 25% of the dose, respectively, suggesting metabolic clearance and, to a lesser extent, renal clearance as the main routes of elimination of nafithromycin, with a total recovery of 92% of the dose.

These data, taken together with the high pulmonary exposures observed during and within 48 h after 3 days of 800 mg once-daily oral dosing of nafithromycin (6), provide a rationale to consider dosing regimens with short treatment duration for treatment of CABP in future clinical trials.

MATERIALS AND METHODS

Study populations and designs.

Both the SAD/FE and MAD studies were conducted in Netherlands by Pharma Research Associates (PRA) Health Sciences. The protocols were approved by the Stichting Beoordeling Ethiek Bio-Medisch Onderzoek, Assen, the Netherlands, as well as by the national regulatory authority. The clinical conduct was in accordance with national regulations, international standards such as the Declaration of Helsinki by the World Medical Association, and good clinical practice guidelines from the International Conference on Harmonization, as well as with the U.S. Code of Federal Regulations, title 21.

Healthy male and female subjects aged 18 to 65 years (inclusive) were enrolled following written informed consent. The body mass index (BMI) value calculated for each subject was 18 to 30 kg/m² (inclusive). Female subjects of childbearing potential had to get a negative pregnancy test and were required to use protocol-defined acceptable birth control methods before and during nafithromycin administration and for 90 days following the last trial follow-up visit. Male subjects were also required to use protocol-defined birth control methods and were not allowed to donate sperm for 90 days after the study. Subjects with positive screen on viral hepatitis B or C virus or human immunodeficiency virus 1/2 antibodies were excluded. Subjects with medical history of any major pathology or any clinically relevant abnormality in screening 12-lead ECG or hematology or clinical chemistry tests of blood and urine were also excluded. Nonsmokers who had negative drug and alcohol screening results at enrollment and were willing to adhere to dietary and lifestyle restrictions of the protocol were eligible for enrollment.

No prospective calculations of statistical power were conducted for either study, but the number of proposed subjects was considered reasonable to limit the number of subjects exposed to the active agent while having a sufficient number of subjects to meet the objectives of the study. Each subject participated in only one cohort.

Single ascending dose and food effect study.

The SAD/FE study was a first-in-human, phase 1, single-center, randomized, double-blind, placebo-controlled, prospective study conducted in 67 healthy male and female subjects. Subjects in 6 cohorts were administered single escalating oral doses of 100 mg to 1,200 mg nafithromycin or matching placebo under fasted or fed condition. Details of the study cohorts are presented in Table 2. Doses were escalated only if the safety and tolerability of the previous dose level were acceptable to the investigator and after a statement of no objection of the ethics committee. For the first cohort (the 100-mg cohort), sentinel dosing design was used to ensure optimal safety. Initially, two subjects were dosed (one with nafithromycin and one with matching placebo). The subjects were closely observed for the first 24 h following drug administration. Subjects studied under fasted conditions were observed after an overnight fast of at least 10 h, with no fluids allowed from 2 h prior to drug administration until 2 h after drug administration apart from the 240 ml of tap water taken with the dose. In either of the 2 cohorts in the FE part of the study, subjects received a single dose of nafithromycin (400 mg or 800 mg) or placebo in a 2-sequence (fasted/fed or fed/fasted), 2-period crossover study. Subjects were dosed under fasting conditions in one period and under fed conditions in another period to examine the effect of food on the nafithromycin PK; the order of fasted or fed dose administration was determined by randomization. A washout period of at least 7 days was considered sufficient based on the projected t_1/2 of nafithromycin in human. Under the fed conditions, the study medication was administered 30 min after the start and within 10 min after consumption of a standard U.S. Food and Drug Administration (FDA)-compliant high-fat breakfast. One subject was determined not to be eligible for the study per the investigator and could not be replaced, resulting in a total of 13 subjects, instead of the planned 14 subjects, evaluated in the 800-mg cohort. Subjects in the next cohort (the 1,200-mg cohort), were randomly assigned to receive nafithromycin or placebo under fed conditions in parallel design. On the basis of interim analyses of tolerability, PK variability, and dose proportionality of the FE dose cohorts, it was decided to dose subjects in the last cohort to randomly receive a single dose of 600 mg nafithromycin or placebo under fed conditions (parallel design).

Subjects were screened within 3 weeks prior to check-in to the study center. Subjects stayed in the center for one period (parallel design cohorts) or two periods (crossover design cohorts). In each period, subjects stayed for 5 days (and 4 nights) and left the center after completion of the assessments scheduled for 72 h after drug administration. Subjects participating in the FE crossover cohorts (the 400-mg and 800-mg cohorts) stayed in the center for 10 days (8 nights) in total, divided over 2 periods, with a 7-day washout between periods, and were reassessed for continuing eligibility at the start of the second period. In each cohort, a follow-up medical examination was performed between 7 and 14 days after the last assessment (in case of FE crossover cohorts, after the second period) and consisted of physical examination, vital signs, ECG, and clinical laboratory tests.

Multiple ascending dose study.

The MAD study was a phase 1, single-center, randomized, double-blind, placebo-controlled, prospective, parallel design study conducted in 30 healthy male and female subjects. The dose levels in this study were not to exceed the dose levels which were well tolerated in the SAD/FE study. The increase from one dose level to the next one was not to be more than 3-fold. As an additional safety measure for a multiple-dose study, further eligibility criteria were required to be met such as bilirubin, aminotransferase, and alkaline phosphatase levels within the normal range; additional exclusion criteria were history of myasthenia gravis, hepatitis, and/or jaundice associated with the use of any antibacterial treatment, congenital prolongation of the QTc interval, and any ongoing proarrhythmic conditions such as uncorrected hypokalemia or hypomagnesemia or clinically significant bradycardia.

The study was conducted in 3 cohorts, with 10 subjects per cohort receiving nafithromycin or matching placebo for 7 consecutive days. Details of the study cohorts are presented in Table 2. On the basis of the safety, tolerability, and PK results of the SAD/FE study, the once-daily 600-mg dose was selected as the first dose to be evaluated in the MAD study. Ultimately, three cohorts were administered ascending multiple oral doses of from 600 mg to 1,000 mg nafithromycin or matching placebo once daily. Dosing was conducted under fed conditions on each dosing day. Doses were escalated only if the safety and tolerability of the previous dose level were acceptable to the investigator and after a statement of no objection of the ethics committee.

Subjects were screened within 3 weeks prior to check-in to the study center. Subjects stayed in the center for 11 days (10 nights) and left the center after completion of the assessments scheduled for 72 h after the last drug administration. In each cohort, a follow-up medical examination was performed between 7 and 14 days after the last assessment and consisted of physical examination and vital signs, ECG, and clinical laboratory tests.

Study medication.

Study medication was supplied by Wockhardt Limited, India, as 200-mg and 400-mg tablets (the 100-mg dose cohort in the SAD/FE study received half a 200-mg tablet). The tablets containing active drug and placebo were indistinguishable in appearance. All study drugs were stored in the study center pharmacy in a locked facility under the required storage conditions until dispensed by the pharmacist to the investigator. Dosing for each individual subject was at around the same time (±15 min) in the morning of each dosing day, under direct observation at the study site. The study drug tablets were swallowed in a sitting position with 240 ml of water of room temperature. Subjects did not lie down for 2 h after drug administration, except when required by assessments that were performed. Under fed conditions, the study medication was administered 30 min after the start and within 10 min after consumption of a standard FDA high-fat breakfast (SAD/FE study) or standard breakfast (MAD study).

Safety and tolerability assessments.

Assessments consisted of AE recording, vital signs, 12-lead ECG, physical examination, and clinical laboratory, including liver safety laboratory parameters. Assessments were performed prior to and immediately predose and/or following administration of study medication, through the follow-up visit conducted 7 to 14 days postdose in the SAD/FE study and 7 to 14 days after the last day of dosing in the MAD study. AEs were recorded from the time of obtaining informed consent of the subject until completion of the follow-up visit.

Sample collection for PK analyses.

In the SAD/FE study, blood samples were collected to measure drug concentration in plasma just before each dose administration and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 20, 24, 36, 48, and 72 h after drug administration (the 72-h postdose sampling was moved to 56 h postdose for the 800-mg crossover cohort and the 600-mg cohort and to 66 h postdose for the 1,200-mg cohort, as the 72-h time point was not likely to result in any analyzable levels). Samples for protein binding assessment were collected only from the 100-mg and 200-mg cohorts, as well as in each first period for the 400-mg and 800-mg-dose cohorts.

In the MAD study, blood samples for PK were collected just before each dose administration and at 0.5, 1, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 16, and 20 h postdose on days 1 and 7, at predose and 3 and 8 h postdose on days 2, 3, 4, 5, and 6, and at 24, 48, 60, and 72 h after the last dose on day 7.

In both studies, urine and feces samples were collected for PK of nafithromycin and its metabolite WCK 4978 at different intervals up to 72 h postdose in the SAD/FE study, up to 24 h on day 1, and up to 72 h after day 7 of the MAD study.

Bioanalytical procedures.

The analyses of nafithromycin and its metabolite (WCK 4978) in plasma, urine, and feces were performed in batches at the Bioanalytical Laboratory of PRA using a validated liquid chromatography-mass spectrometry (LC-MS) method. The range of quantification was 0.01 to 5.0 mg/liter, and clarithromycin was added as an internal standard during analysis. The lower limit of quantification was 0.01 mg/liter. For nafithromycin, accuracy ranged from −3.9% to 1.8% with precision of 2.7% to 11.5%. For WCK 4978, accuracy ranged from 4.6% to 12.1% with precision of 2.8% to 7.6%. Analysis of protein binding and albumin levels was performed by the Discovery Drug Metabolism and Pharmacokinetics Laboratory of Wockhardt Research Centre (India). The plasma samples and supernatant obtained after ultracentrifugation were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine total drug concentration and unbound drug, respectively. The plasma samples were also used to determine albumin levels using a biochemistry analyzer (Xpand; Dade Behring Inc., CA).

Analysis of pharmacokinetic parameters.

Plasma PK parameters for nafithromycin and its metabolite WCK 4978 were estimated from the plasma concentration-time profiles for all PK population subjects (all subjects who had received the study medication and providing the primary PK data considered to be sufficient and interpretable). PK parameters were calculated with noncompartmental methods using WinNonlin Professional software (Pharsight Corp., Mountain View, CA) versions 5.0.1 (SAD/FE study) and 6.3 (MAD study). Summaries of geometric mean concentrations versus nominal time were generated. In addition, plots by subject of the concentration versus actual time were depicted. Actual blood sampling times were used in the graphical presentation of individual data and PK calculations. For single-dose assessments, parameters calculated for plasma nafithromycin and its metabolite WCK 4978 included C_max, T_max, AUC_0–t, AUC_0–inf, t_1/2, and, for nafithromycin, also CL/F (apparent clearance) and V_Z/F (volume of distribution); these were summarized with descriptive statistics by treatment.

For multiple-dose assessments, parameters calculated for plasma nafithromycin and its metabolite WCK 4978 included C_max (with C_max at steady state at day 7), C_trough, AUC over a dosing interval (AUC_0–τ), T_max (with T_max at steady state at day 7), and, for nafithromycin, also CL/F and V_Z/F. The accumulation ratio was calculated by comparing the plasma exposures of nafithromycin and its metabolite WCK 4978 (AUC_0–τ) on day 7 to those on day 1.

Dose proportionality was evaluated for nafithromycin and its metabolite WCK 4978 by plotting individual and mean values for dose-normalized C_max, AUC_0–t, and AUC_0–inf (SAD/FE study) or C_max and AUC_0–τ (MAD study) parameters versus the dose. In addition, dose proportionality was explored using analysis of variance (ANOVA) on log-transformed parameters (C_max, AUC_0–t, and AUC_0–inf for the SAD/FE study and C_max and AUC_0–τ for the MAD study) using the power model.

For both studies, the PK parameters determined or calculated from urine and feces concentration time data for nafithromycin and its metabolite WCK 4978 were Ae_0–τ,urine and Ae_0–τ,feces.

Analysis of food effect.

In order to assess the food effect on the PK of nafithromycin and its metabolite WCK 4978 following a single oral administration (SAD/FE study), PK parameters (C_max, AUC_0–t, and AUC_0–inf) were logarithmically transformed and analyzed using an ANOVA model with dietary state, period, and sequence as fixed effects and subject within sequence as random effect. The ratios of least square means and corresponding CIs were obtained separately for the 400-mg and 800-mg cohorts from the ANOVA model for subjects in the relative bioavailability assessment.