Diffuse alveolar hemorrhage (DAH) is a fatal complication in patients with lupus. DAH can be induced in B6 mice by an intraperitoneal injection of pristane. Since human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein, we investigated the protective effect of hAAT against pristane-induced DAH in B6 mice and hAAT transgenic (hAAT-Tg) mice. We first showed that hAAT Tg expression lowers TNF-α production in B cells, as well as CD4+ T cells in untreated mice. Conversely, the frequency of regulatory CD4+CD25+ and CD4+CD25-IL-10+ cells was significantly higher in hAAT-Tg than in B6 mice. This confirmed the anti-inflammatory effect of hAAT that was observed even at steady state. One week after a pristane injection, the frequency of peritoneal Ly6Chi inflammatory monocytes and neutrophils in hAAT-Tg mice was significantly lower than that in B6 mice. Importantly, pristane-induced DAH was completely prevented in hAAT-Tg mice and this was associated with a modulation of anti- to pro-inflammatory myeloid cell ratio/balance. We also showed that treatment with hAAT decreased the severity of DAH in B6 mice. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH.

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

Background Tamoxifen is frequently used in the adjuvant treatment of hormone sensitive breast cancer. Unfortunately, tamoxifen can lead to bothersome side effects resulting in non-adherence in 40% of patients. Patients searching for relief from these side effects are increasingly turning to cannabinoids such as CBD. However, since tamoxifen is mainly metabolised by CYP2D6, and CBD is suggested to be an inhibitor of CYP2D6, the use of CBD might affect tamoxifen pharmacokinetics (PK). Since the effect of CBD on both tamoxifen PK as tamoxifen-related side effects has never been investigated, the aims of this study were to determine the pharmacokinetic interaction between CBD and tamoxifen, and to subsequently investigate whether there is a beneficial influence of CBD on tamoxifen-related side effects. Methods Patients had to be treated with tamoxifen for at least 3 months, have steady-state endoxifen levels >16 nM (conservative threshold) and experience tamoxifen-related side effects. PK sampling was done at initiation of CBD-oil and 28 days thereafter. Bio-equivalence could be concluded if the 90% confidence interval (CI) for the difference in endoxifen area under the curve (AUC) fell within the [-20%; +25%] interval (n = 15, two-sided α 0.05, β 0.20). In addition, endoxifen PK was analyzed for each CYP2D6 phenotype separately. The effect of CBD on side effects was evaluated with the FACT-ES questionnaire (n = 25, two-sided α 0.05, β 0.20). An improvement > 0.5 times standard deviation (SD) of baseline score was considered clinically relevant. Last, potential side effects of CBD were assessed. Results In this study 15 patients were included for PK analysis and 24 patients for side effect analysis. Endoxifen AUC decreased after CBD by 12.6% (90% CI -18.7%, -6,1%) but remained within bio-equivalence boundaries. The decrease seemed more pronounced in patients with intermediate (IM) CYP2D6 phenotype (-20.8%, 90% CI -26.4%, -14.8%, n = 8) compared to normal CYP2D6 phenotype (-2.2%, 90% CI -11.1%, 7.6%, n = 7). There was no difference in tamoxifen AUC (with or without CBD). On average, the endocrine sub-scale of the FACT-ES improved with a clinically relevant improvement of 8.3 points (95% CI 4.9 – 11.7) after using CBD (baseline SD = 12.8). CBD itself has a mild toxicity profile with few side effects in 10 of 24 patients. Side effects were headache (n=2), dry mouth (n=3), fatigue (n=3), gastroesophageal reflux (n=1), abdominal pain (n=1) and nausea (n=1) and all graded CTCAE 1. Conclusions As endoxifen levels with or without CBD remained within bio-equivalence boundaries and CBD-oil might have a positive effect on tamoxifen-related side effects, it could be considered in case of treatment-related side effects. However, caution is needed in patients with IM or poor metabolizer CYP2D6 phenotypes. Citation Format: Sanne Buijs, Louwrens Braal, Stefan Buck, Noud F. van Maanen, Lonneke van der Meijden-Erkelens, Heleen Kuijper-Tissot van Patot, Esther Oomen-de Hoop, Lotte Saes, Sophia van den Boogerd, Liesbeth Struik, Quirine van Rossum-Schornagel, Ron Mathijssen, Stijn Koolen, Agnes Jager. CBD-oil: a potential solution in case of severe tamoxifen-related side effects [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-02-08.