Tolerability in man following inhalation dosing of the selective TLR7 agonist, AZD8848

Abstract

Background: Many patients with asthma have a T-helper type 2 (Th2) driven inflammation of the lung, whereas toll-like receptor 7 (TLR7) agonists, by inducing type I interferons, inhibit Th2 responses. In man, oral or parenteral TLR7 agonists can induce influenza-like symptoms through systemic induction of type I interferons. Design of a TLR7 agonist that is only active in the lung could reduce the risk of side effects and offer a new means for treating asthma. We developed a TLR7 agonist antedrug, AZD8848, to determine its local and systemic effects in preclinical models and man.

Methods: In vitro cellular potencies for the TLR7 antedrug agonist, AZD8848, were determined along with pharmacokinetics and efficacy in a rat allergy model. Sputum and blood biomarkers were measured in single ascending and multiple ascending dose clinical studies following inhalation delivery of AZD8848 and tolerability assessed.

Results: AZD8848 was potent in cellular assays and pharmacokinetics confirmed lack of systemic exposure to AZD8848. Weekly lung dosing in an animal model showed efficacy 26 days beyond the final dose. In healthy volunteers, AZD8848 was initially well tolerated with target engagement being demonstrated by induction of CXCL10 in sputum. A second inhaled dose, given 1 week later, amplified the systemic interferon signal in more than half the participants and resulted in significant influenza-like symptoms.

Conclusions: The antedrug design restricted the direct actions of AZD8848 to the lung. However, the type I interferon induced locally by TLR7 spilled over systemically, limiting the utility of this inhaled antedrug approach.

Trial registration number: NCT01560234, NCT01818869.

Keywords: Allergic lung disease; Asthma; Asthma Mechanisms.

Figures

Figure 1

Structure of AZD8848 and potency in recombinant human TLR human embryonic kidney (HEK) reporter cells. (A) The chemical structure of AZD8848 and (B) the activity of AZD8848 and its acid metabolite, AZ12432045, in HEK cells stably expressing human toll-like receptor 7 (TLR7). Activity was calculated from the release of secretory alkaline phosphatase, whose expression was linked to an NF-κB promoter, with the data normalised to the maximal induction produced by the reference TLR7 agonist, R848 (set to 100%). Incubation of cells with compound was for 24 h. Data show the mean±SEM from nine separate experiments (AZD8848) and from six separate experiments (AZ12432045).

Figure 2

Pharmacokinetics and efficacy of AZD8848 in the Brown Norway rat. Brown Norway rats were dosed AZD8848 at 0.3 mg/kg intratracheally in a volume of 150 µL and blood samples taken at the indicated time points. Nine rats were dosed and at each time point samples from at least two animals were taken. Data for the plasma levels of compound (A) are presented as the mean of the determinations±SD or range and are representative of two such determinations. Following intratracheal administration, lung levels of AZD8848 and its acid metabolite were determined (B). At each time point, apart from the first, two animals were sacrificed and data are represented as the mean±range. The limits of detection for AZD8848 and AZ12432045 were 0.2 and 2 nmol/L, respectively. For the purposes of determining a representative value for each group of animals, any sample where the compound was undetectable was arbitrarily assigned a value of half that of the limit of detection. Data points including only such values are shown with a #. OVA-sensitised rats were dosed intratracheally with AZD8848, at the indicated doses (shown in brackets), 24 h prior to and 24 h after OVA challenge (C). Animals were sacrificed 48 h after the OVA challenge and the numbers of eosinophils or level of interleukin 13 in the BAL determined. The following rat numbers, in brackets, were used for each data point: sensitised only (5), intratracheally dosed (9). Data are representative of three studies. In further studies 14 days after OVA-sensitisation, Brown Norway rats were dosed topically with AZD8848 (1 mg/kg) or budesonide (1 mg/kg) via the intratracheal route on eight occasions at weekly intervals. Twenty-six days after the final dose of compound, animals were challenged with aerosolised OVA and 48 h later eosinophil number and IL-13 levels determined in the BAL (D). Ten animals were used for each data point. The effect of acute treatment with compound (24 h before and 24 h after OVA challenge) was also determined in groups of eight animals. The number of eosinophils or level of IL-13 in the BAL per animal was averaged across each treatment group and the result expressed as mean±SEM. All study groups were compared, with their respective OVA-stimulated control dosed with vehicle, using the Mann-Whitney U test; *p

Figure 3

Pharmacokinetics and biomarker induction following a single inhaled dose of AZD8848 in man.(A) Plasma concentration-time profiles of AZ12432045, the acid metabolite of AZD8848, following a single predicted lung-deposited dose of AZD8848 as indicated in the key. The predicted lung-deposited dose was assumed to be 63% of the dose delivered from the nebuliser. During sample preparation, any trace amounts of AZD8848 were hydrolysed by esterases in the plasma. All cohorts were administered by nebulisation taken in 10 breaths as described in Methods section. No data are shown for cohort 1 (0.15 µg) as levels of AZ12432045 were below the level of detection. Values shown are geometric means (±SD) from 3 to 5 individuals in each cohort. (B) CXCL10 was determined in induced sputum (black) and in plasma (grey) of healthy volunteers for the indicated predicted lung-deposited dose of AZD8848. The fold change at 24 h postdose from baseline for individual participants is shown. Geometric mean is shown in each cohort and significance relative to placebo (p

Figure 4

Plasma CXCL10 and blood lymphocyte counts following a second inhaled dose of AZD8848. CXCL10 in plasma (A) and lymphocytes in blood (B) were determined in healthy volunteers receiving two 30 μg doses of AZD8848 1 week apart. Data from individual participants are shown as separate lines, treatment as solid lines, and placebo as broken lines. Arrows indicate individual participants who experienced severe (black) or moderate (grey) influenza-like symptoms in the preceding period. Between-treatment and intrasubject comparisons for CXCL10 levels are described in the text.

Figure 5

Monitoring the induction of pro-inflammatory mediators and toll-like receptors (TLRs) following inhalation of AZD8848. The mediators (A) C reactive protein (CRP), (B) interferon γ (IFNγ), (C) interleukin 8 (IL-8) and (D) tumour necrosis factor α (TNFα) were measured in plasma as described in Methods section in healthy volunteers receiving two 30 μg doses of AZD8848 1 week apart. Data from individual participants are shown as separate lines, treatment as solid lines, and placebo as broken lines. Arrows indicate individual participants who experienced severe (black) or moderate (grey) influenza-like symptoms in the preceding period. Changes in (E) TLR7 and (F) TLR9 RNA levels in blood were determined in healthy volunteers receiving a 30 μg dose of AZD8848 and samples were taken at the time points indicated. Data from individual participants are shown, those receiving placebo as a broken line, those receiving AZD8848 as a solid line. In view of the small numbers of participants, no statistical testing was performed between treatment groups.