Respirable aerosol exposures of nicotine dry powder formulations to in vitro, ex vivo, and in vivo pre-clinical models demonstrate consistency of pharmacokinetic profiles

Figures & data

Table 1. Blood and lung tissue sampling schedule in the rats exposed in vivo to the formulation containing 2.5% or 5% nicotine. All time points denote time after start of aerosol exposure (t = 0).

Treatment group	In live serial blood	Terminal blood	Lung and trachea
#1 (n = 3)	5,10,15 min	4 h	4 h
#2 (n = 3)	N/A	30 min	30 min
#3 (n = 3)	N/A	1 h	1 h

Figure 1. The upper panels show SEM images (two on the left) and a light microscope photo (right) of the 2.5% nicotine formulation. The lower panels show SEM images and a light microscope photo of the 5% nicotine formulation.

Figure 2. PK profiles of 2.5% and 5% nicotine powders tested in DissolvIt (mean ± SD). (A) Nicotine concentration in the perfusate over time. (B) Nicotine concentration normalized to the deposited dose. min: minutes.

Figure 3. The undissolved and/or unabsorbed fraction of the deposited nicotine dose (mean ± SD) remaining in the DissolvIt system as a function of time. min: minutes.

Table 2. PK parameters (mean ± SD) of the derived DissolvIt data for 2.5% and 5% nicotine powder aerosols.

			Mass balance
Exposure material	Cmax (ng/mL)	tmax (min)	Mperf (ng)	Msystem (ng)	Mdep (ng)	Fractionperf (peak), %	tmax (min)	Fractionret (at 1 min)	Fractionret (at 2 min)	T1/2α (0–1 min)
5% nicotine	121 ± 9	0.07 ± 0.05	32 ± 2	7 ± 2	39 ± 1	311 ± 25	0.07 ± 0.05	0.33 ± 0.032	0.17 ± 0.050	0.75 ± 0.05
2.5% nicotine	148 ± 16	0.04 ± 0.00	31 ± 4	14 ± 1	45 ± 3	325 ± 11	0.04 ± 0.00	0.31 ± 0.043	n/a	0.55 ± 0.07

C_max: the maximum concentration reached; t_max: the time point for C_max; M_perf: the total amount of nicotine accumulated in the perfusate during the experiment; M_system: the amount of nicotine undissolved and/or unabsorbed in DissolvIt at the end of the experiment; M_dep: the amount of nicotine initially deposited on the glass coverslip; Fraction_{perf (peak)}: the maximum value of the normalized concentration values; t_(peak): the time point for Fraction_{perf (peak)}; Fraction_ret: the fraction of M_dep remaining undissolved and/or unabsorbed in the DissolvIt system after 1 minute (2.5% nicotine) and after 1 and 2 minutes (5% nicotine); t_1/2α: the half-life of 2.5% and 5% nicotine assuming a first-order decay disappearance, as calculated during the first minute.

Table 3. Key parameters (mean ± standard deviation) of the inhalation exposures in the isolated perfused lung model ex vivo and in the intratracheally intubated rat in vivo.

Experiment (n)	Target dep. dose of nicotine (μg)	Target dep. dose of powder (μg)	Exposure tidal vol. (mL)	Exposure breathing rate (breaths/min)	Exposure duration (min)
5% nicotine, ex vivo (3)	33	660	1.9 ± 0.1	75 ± 0	2.2 ± 0.1
5% nicotine, in vivo (9)	33	660	1.3 ± 0.2	94 ± 25	2.3 ± 0.5
2.5% nicotine, ex vivo (3)	33	1320	2.1 ± 0.1	75 ± 0	1.9 ± 0.3
2.5% nicotine, in vivo (9)	33	1320	1.4 ± 0.2	73 ± 12	3.4 ± 0.8

Figure 4. Nicotine concentration (mean ± standard deviation, n = 3) as a function of time in the single-pass lung perfusate of the IPL exposed ex vivo to powder aerosols containing 5% or 2.5% nicotine. min: minutes.

Table 4. PK parameters of nicotine (mean, n = 3), in the rat IPL exposed ex vivo to powder aerosols containing 5% or 2.5% nicotine.

				Nicotine, calculated pharmacokinetic parameters
Formulation	Mproj (μg)	MDEP (μg)	Exp. Dur. (min)	Cmax (ng/mL)	tmax (min)	%Exp. Compl. at tmax	Cmax^(Norm) (ng/mL/μg)	tmax^(Norm) (min/μg)	%MDEP(peak) /mL	t^(Peak) (min)	FrMDEP^(120 min)	T1/2α0–4 min (min)	T1/2β0–120 min (min)
5% nicotine	33	48.403	2.2	817^#	1.7	76	17.01	0.03	1.55	2.0	0.0005	1.1	16.9
2.5% nicotine	33	40.522	1.9	573	1.0	55	14.05	0.02	1.28	2.0	0.0005	1.3	15.7

M_proj: target deposited nicotine dose in the lungs; M_DEP: attained initial deposited nicotine dose in the lungs; C_max: maximal concentration of nicotine in the lung perfusate; t_max: time from start of aerosol exposure (t = 0) at which the maximal concentration in the perfusate occurred; C_max ^(Norm) (equal to dose-normalized C_max): maximal concentration of nicotine in the perfusate, normalized to the initially deposited nicotine dose (M_DEP); t_max^(Norm) (equal to dose-normalized t_max): time from start of aerosol exposure (t = 0) at which the maximum concentration of nicotine in the perfusate occurred, normalized to the initially deposited nicotine dose (M_DEP); %M_DEP/mL ^(peak): percentage of the initially deposited nicotine dose cleared per milliliter of perfusate (the peak value); t^(peak): time from start of aerosol exposure (t = 0) at which %M_DEP/mL ^(peak) occurred; Fr M_DEP: fraction of the initially deposited dose (M_DEP) retained in the lung, calculated as 1−(cumulative mass cleared with perf/M_DEP); Fr M_DEP ^(120 min): fraction of the initially deposited dose (M_DEP) retained in the lung at the end of the perfusion period (i.e. 120 min post-exposure); t_1/2: half-time of retention in the lungs (α: 0–4 min and β: 4–120 min). The data for fraction of the initially deposited dose (M_DEP) retained in the lung was fitted to a first-order decay function for calculating the retention half-times exhibited by the compound.

^#: p < 0.05 vs. C_max 2.5% nicotine (t-test, 2-sided).

Figure 5. Nicotine concentration (normalized to the initially deposited dose according to LC-MS/MS) as a function of time in the single-pass lung perfusate of the rat IPL exposed ex vivo to powder aerosols containing 5% or 2.5% nicotine. %MDEP/mL, percentage of the initially deposited dose (MDEP) cleared per milliliter of lung perfusate. Data are presented as mean ± standard deviation (n = 3). min: minutes.

Figure 6. Nicotine retention (normalized to the initially deposited dose according to LC-MS/MS) in the lung as a function of time in the rat IPL exposed ex vivo to powder aerosols containing 5% or 2.5% nicotine. Fraction MDEP, fraction of the initially deposited dose (MDEP) retained in the lung. Data are presented as mean ± standard deviation (n = 3). min: minutes.

Figure 7. Blood plasma concentrations of nicotine and cotinine (mean ± standard deviation, n = 3) as a function of time in rats intratracheally exposed to powder aerosols containing 5% or 2.5% nicotine. h: hours.

Figure 8. Lung concentrations of nicotine and cotinine (mean ± standard deviation, n = 3) as a function of time in rats intratracheally exposed to powder aerosols containing 5% or 2.5% nicotine. h: hours.

Table 5. Plasma PK parameters for nicotine and cotinine (mean, n = 3) in rats intratracheally exposed to powder aerosols containing 5% or 2.5% nicotine.

		Nicotine plasma PK parameters											Cotinine plasma PK parameters
Formulation	Mproj (μg)	t1/2 (h)	tmax (h)	Cmax (ng/mL)	tlast (h)	Clast (ng/mL)	AUClast (h*ng/mL)	AUCinf (h*ng/mL)	AUCextr (%)	MRTlast (h)	MRTinf (h)		tmax (h)	Cmax (ng/mL)	tlast (h)	Clast (ng/mL)	AUClast (h*ng/mL)	MRTlast (h)
5% nicotine	33	2.38	0.083	71.9	4.0	18.5	137.0	205.8	31	1.62	3.46		4.0	17.4	4.0	17.4	39.6	3.18
2.5% nicotine	33	2.31	0.083	108	4.0	21.5	150.5	240.5	39	1.55	3.52		4.0	26.6	4.0	26.6	55.7	3.10

M_proj: target deposited nicotine dose; t_1/2: terminal elimination half-life; C_max: maximum concentration; t_max: time at which maximum concentration occurs; t_last: last sampling time point; C_last: concentration at the last sampling time point; AUC_last: area under the curve as a function of time up to the last sampling time point; AUC_inf: area under the curve as a function of time, calculated for t→∞; AUC_extr: area under the curve as a function of time extrapolated from t_last to t→∞ as a percentage of the total AUC, i.e. AUC_inf, MRT_last, MRT_inf, mean residence time (average time a drug spends in the accessible pool during all passages through the system before being irreversibly cleared).

Table 6. Lung PK parameters for nicotine and cotinine (mean, n = 3) in rats intratracheally exposed to powder aerosols containing 5% or 2.5% nicotine.

		Nicotine lung PK parameters											Cotinine lung PK parameters
Formulation	Mproj (μg)	t1/2 (h)	tmax (h)	Cmax (ng/g)	tlast (h)	Clast (ng/g)	AUClast (h*ng/g)	AUCinf (h*ng/g)	AUCextr (%)	MRTlast (h)	MRTinf (h)		tmax (h)	Cmax (ng/g)	tlast (h)	Clast (ng/g)	AUClast (h*ng/g)	MRTlast (h)
5% nicotine	33	–	0.5	100.9	4.0	44.1	262.8	–	–	1.87	–		4.0	16.5	4.0	17.4	39.6	2.84
2.5% nicotine	33	1.81	0.7	135.4	4.0	46.3	298.8	340.8	24	1.79	2.92		4.0	24.8	4.0	26.6	55.7	2.81

M_proj: target deposited nicotine dose; t_1/2: terminal elimination half-life; C_max: maximum concentration; t_max: time at which maximum concentration occurs; t_last: last sampling time point; C_last: concentration at the last sampling time point; AUC_last: area under the curve as a function of time up to the last sampling time point; AUC_inf: area under the curve as a function of time, calculated for t→∞; AUC_extr: area under the curve as a function of time extrapolated from t_last to t→∞ as a percentage of the total AUC, i.e. AUC_inf; MRT_last, MRT_inf, mean residence time (average time a drug spends in the accessible pool during all passages through the system before being irreversibly cleared).