Subjects

Fourteen healthy, young (mean age 25 years, range 20–31 years) male subjects participated after written informed consent; eight in Part 1 of the study, assessing emergence of brain MOR occupancy (0–60 min after dosing of IN naloxone), and six in Part 2 of the study, assessing its disappearance (300–360 min after dosing). Inclusion criteria included good general health, body weight 60–90 kg, and body mass index 18–28 kg m−2. Exclusion criteria included intake of any medication that could affect the outcome of the study, within 2 weeks prior to the tracer administration (2 months for enzyme-inducing drugs like rifampicin or carbamazepine), or <5 times the half-life of the medication, history of drug abuse or positive result in urine drug screen, high alcohol consumption, and regular smoking. Subject candidates with high scores (>2) on the Finnish version of the South Oaks Gambling Screen (SOGS-R) were excluded [21]. The study protocol was approved by the Ethics Committee of South-West Finland Hospital District and the Finnish Medicines Agency (Fimea). Relevant regulations and guidance for biomedical research involving human subjects, such as the Declaration of Helsinki and GCP were adhered to. The study was registered with the EudraCT registration number 2015-002681-23.

Study outline

The study was double blind and placebo controlled, consisting of two parts, each with different participants. Each participant received one IN dose of naloxone (2 mg, n = 7, or 4 mg, n = 7; the 4 mg doses were delivered by administering one 0.1 mL puff into each nostril; 2 mg into either the left or right nostril) and one placebo treatment in a balanced, randomized order. Doses (spray formulation of naloxone hydrochloride developed by Lightlake Therapeutics Inc., UK, 20 mg mL−1, or matching placebo) were given to supine subjects with a Pfeiffer Bidose liquid device. The two sessions were held at least 5 days apart in order to eliminate possible treatment-related effects on MOR availability. In Part 1 of the study, the participants received their IN doses of placebo and naloxone (2 mg, n = 4, or 4 mg, n = 4) during the PET scan, 20 min after the [11C]carfentanil tracer injection. PET scan data were collected until 60 min after the IN drug administration. In Part 2 of the study, placebo and naloxone (2 mg, n = 3, or 4 mg, n = 3) were administered 5 h before [11C]carfentanil and the start of PET scanning.

Structural magnetic resonance imaging (MRI) of the brain was conducted to exclude subjects with anatomical brain abnormalities and to collect anatomical reference data for PET data analysis. T1-weighted MRI scans were acquired using a 3 T scanner (Philips Ingenuity TF PET/MR, Philips Medical Systems, Cleveland, OH, USA).

PK assessments

Venous blood samples were collected for determination of naloxone concentrations in plasma. Samples were collected prior to dosing and at 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 80, and 100 min, as well as at 2, 3, 4, and 6 h after IN dosing (see Supplementary Methods for sample preparation and analysis). The lower limit of quantitation was 0.10 ng mL−1.

Non-compartmental analysis with WinNonlin® Phoenix 6.4 software (Pharsight Corporation, Mountain View, CA, USA) was used to determine the PK parameters peak concentration (C max ), time to peak (T max ), area under the concentration by time curve (AUC) to 6 h (AUC 0–6 ), AUC to the last measurable concentration (AUC 0–t ), elimination half-life (t 1/2 ), and AUC extrapolated to infinity (AUC 0–∞ ). Terminal time points were selected based on best-fit criteria and review of the selected points. The linear trapezoidal rule was used to estimate AUC.

PET assessments

The PET tracer, [11C]carfentanil, was produced [22] at the radiochemistry laboratory of Turku PET Center on the day of PET imaging. The target radioactivity of each administered bolus injection was 500 MBq, and the carfentanil mass was not allowed to exceed 2.0 µg per injected dose. PET data were acquired with a High-Resolution Research Tomograph (HRRT, Siemens Medical Solutions, Knoxville, TN, USA), and image reconstruction and preprocessing of the data were conducted as reported earlier [23] (see Supplementary Information for details).

Automated regions-of-interest (ROIs) were generated with the help of individual T1 MRI anatomical data and FreeSurfer software (version 5.3; http://surfer.nmr.mgh.harvard.edu/; [24]), and using Statistical Parametric Mapping (SPM8; Wellcome Institute, London, UK; [25]) to generate atlas-based subcortical ROIs, resulting in 60 ROIs [26]. An aggregate average ROI was generated over the regions listed in Table 1 to best reflect the global effect of naloxone in the brain. All analyzed ROIs are additionally reported as Supplementary Information. The lateral occipital cortex served as a reference region, being practically devoid of MORs [27].

Table 1 Fifteen most receptor-dense brain regions in a ROI-based analysis using MRTM (with the occipital cortex as reference) and all 14 PET scans with [11C]carfentanil and IN placebo administration Full size table

ROI-based binding potential as estimated using reference tissue models (BP ND ; [28]) was considered as the primary outcome parameter in PET modeling. Multilinear reference tissue modeling (MRTM) with fixed k 2 ′ [29, 30] was conducted for BP ND estimation in placebo scans and late (Part 2) naloxone scans, while an additional time-dependent term [31] was required for successful modeling of early (Part 1) naloxone scans, as indicated by model selection criteria (Supplementary Information). Model fits derived with the most compatible model were generally of high quality (cf. Figure 1c), whereas visually apparent deviations were observed when poorly complying models were applied for the early naloxone scans (data not shown). Subsequently, dynamic BP ND s were calculated in early naloxone scans as described before [32], yielding dynamic receptor occupancies (RO(t)) as defined in Eq. (1),

$${\mathrm{RO}}(t) = \left( {1 - \frac{{{\mathrm{BP}}_{{\mathrm{ND}}}^{{\mathrm{Naloxone}}}(t)}}{{{\mathrm{BP}}_{{\mathrm{ND}}}^{{\mathrm{Placebo}}}}}} \right) \times 100\% ,$$ (1)

where t is the time of BP ND estimation under the naloxone treatment. Dynamic receptor occupancies were analyzed to determine peak receptor occupancy (RO max ), and time to reach half of RO max (t RO/2 ). Late naloxone scans were analyzed for static receptor occupancy using Eq. (1) without time dependence.

Fig. 1 a Plasma naloxone concentrations (mean ± SEM) after intranasal (IN) naloxone administration in healthy male volunteers (n = 7 for 4 mg and n = 7 for 2 mg doses). b Mu-opioid receptor (MOR) occupancies (%) (mean ± SEM) after IN naloxone administration in healthy male volunteers (n = 4 (3) for 4 mg and n = 4 (3) for 2 mg doses in 0–60 min (300–360 min) time intervals) as estimated from [11C]carfentanil-derived BP ND data under the placebo and naloxone conditions (see Methods), using linearized-parametric neurotransmitter positron emission tomography (PET) (lp-ntPET) analysis (see Methods). c, d [11C]carfentanil PET-derived standardized time-activity course (TAC) data during the placebo and naloxone conditions in one individual subject (101) after 4 mg dosing of naloxone. Solid lines depict the radioactivity observations from the reference region (occipital cortex), and circles the ones from a relevant brain ROI with high MOR availability (the limbic striatum, average of both sides), and dashed lines depict the best lp-ntPET model fits to the data. Application of the nasal spray formulation occurred 20 min after [11C]carfentanil administration and initiation of PET data acquisition (vertical arrow). Appearance of MOR occupancy by naloxone is observable as a perturbation of the [11C]carfentanil signal pattern in the naloxone scan as compared to the expected pattern observed during the placebo scan Full size image

Relationships between PK and PD data

The relationship of the time courses of naloxone concentrations in plasma and brain MOR occupancy was investigated by plotting (GraphPad Software Inc., La Jolla, CA, USA) the log-linear receptor occupancy estimates and drug concentrations in plasma according to each in-scan PK sampling time point (3, 6, 9, 12, 15, 20, 25, 30, 40, 50, and 60 min). Departure of the plots of the early time points from the log-linear regression line of the last (60 min post naloxone) measurement was interpreted as hysteresis in brain MOR occupancy, while the earliest measurement time point exhibiting similar log-linear relationship with the 60 min data was deemed as the start time of equilibrium between plasma naloxone concentrations and brain MOR occupancy.

The drug concentration associated with half-maximal effect (EC 50 ) was estimated using GraphPad by fitting the plasma naloxone concentrations (C) at all in-scan measurement time points after equilibrium (see above) to the estimated occupancy (RO):

$${\mathrm{RO}} = {\mathrm{RO}}\prime \left( {\frac{C}{{C + {\mathrm{EC}}_{{\mathrm{50}}}}}} \right),$$

where RO′ represents the model-derived maximal occupancy. Theoretically, RO′ should be fixed at 100%, but to account for some uncertainty in the RO estimates, a non-fixed RO′ parameter was employed.

Statistical analysis

The small number of observations per naloxone dose level and time point precluded formal statistical testing of the primary PK and PET (PD) variables. The results of PK and PET data analysis are presented in terms of individual values and descriptive statistics including mean and standard deviation (SD) or median and range calculated with Microsoft Excel (Microsoft Office Professional Plus 2010, Version 14.0).