Inhibition of cAMP-phosphodiesterase 4 (PDE4) potentiates the anesthetic effects of Isoflurane in mice

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Abstract

Despite major advances, there remains a need for novel anesthetic drugs or drug combinations with improved efficacy and safety profiles. Here, we show that inhibition of cAMP-phosphodiesterase 4 (PDE4), while not inducing anesthesia by itself, potently enhances the anesthetic effects of Isoflurane in mice. Treatment with several distinct PAN-PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast, and RS25344, significantly delayed the time-to-righting after Isoflurane anesthesia. Conversely, treatment with a PDE3 inhibitor, Cilostamide, or treatment with the potent, but non-brain-penetrant PDE4 inhibitor YM976, had no effect. These findings suggest that potentiation of Isoflurane hypnosis is a class effect of brain-penetrant PDE4 inhibitors, and that they act by synergizing with Isoflurane in inhibiting neuronal activity. The PDE4 family comprises four PDE4 subtypes, PDE4A to PDE4D. Genetic deletion of any of the four PDE4 subtypes in mice did not affect Isoflurane anesthesia per se. However, PDE4D knockout mice are largely protected from the effect of pharmacologic PDE4 inhibition, suggesting that PDE4D is the predominant, but not the sole PDE4 subtype involved in potentiating Isoflurane anesthesia. Pretreatment with Naloxone or Propranolol alleviated the potentiating effect of PDE4 inhibition, implicating opioid- and β-adrenoceptor signaling in mediating PDE4 inhibitor-induced augmentation of Isoflurane anesthesia. Conversely, stimulation or blockade of α1-adrenergic, α2-adrenergic or serotonergic signaling did not affect the potentiation of Isoflurane hypnosis by PDE4 inhibition. We further show that pretreatment with a PDE4 inhibitor boosts the delivery of bacteria into the lungs of mice after intranasal infection under Isoflurane, thus providing a first example that PDE4 inhibitor-induced potentiation of Isoflurane anesthesia can critically impact animal models and must be considered as a factor in experimental design. Our findings suggest that PDE4/PDE4D inhibition may serve as a tool to delineate the exact molecular mechanisms of Isoflurane anesthesia, which remain poorly understood, and may potentially be exploited to reduce the clinical doses of Isoflurane required to maintain hypnosis.

Introduction

Inhalation anesthetics, particularly the halogenated ethers Isoflurane, Sevoflurane, and Desflurane, are widely used in the clinic to induce and/or maintain general anesthesia during surgical procedures [1], [2], [3]. They are often the preferred choice as they allow for precise fine-tuning of the anesthetic state, are low cost, easy to use, and provide fast induction and short recovery times. Isoflurane, the most potent of the halogenated ethers, is also widely used in animal research; its application extending far beyond surgeries to various procedures that require immobilization of the animal, even if for short times, such as for the delivery of reagents (e.g. tail vein or intracerebroventricular injections) or in vivo imaging (e.g. ultrasound).

The clinical success of Isoflurane and related ethers is in part owed to the fact that they are generally well tolerated. Common adverse effects associated with these drugs are cardiac and/or respiratory depression [1], [4], [5] which, however, are characteristic of any form of systemic anesthesia. In addition, upon recovery from Isoflurane anesthesia, ~30% of patients experience post-operative nausea and vomiting (PONV) [1], [6], [7], which is commonly treated with pre- and/or post-operative antiemetics. In addition, shivering or effects resembling allergic reactions such as rash, hives, itching, swelling, trouble breathing, dizziness, or passing out have been reported [1]. These can sometimes be intense, thus limiting the utility of the halogenated ethers for susceptible patients. While very rare, fluranes may also cause severe, potentially life-threatening, side effects including malignant hyperthermia [8], liver and kidney toxicity, or arrhythmias [1].

Lab research and animal studies have indicated potential concerns regarding the long-term effects of inhalation anesthetics, such as an impairment of neurocognitive function [1], [9], [10], [11] or an increased risk of tumor recurrence/progression [12], [13]. Inhalation anesthetics, including Isoflurane, have been demonstrated to cause neuronal cell death as well as long-term neurocognitive dysfunction [9], [10], [11] in neonatal/juvenile rodents and juvenile non-human primates. Furthermore, Isoflurane and other halogenated ethers facilitate the formation of β-amyloid and tau oligomers which may have implications for the neuropathogenesis of Alzheimer’s disease [14], [15]. However, it remains unclear if, and to which extent, any of these findings may apply to humans. This subject is complex and remains under investigation. For example, animal studies reported that fetal/neonatal or juvenile rodents are significantly more prone to neurodevelopmental changes resulting from flurane exposure compared to adult animals. Conversely, inhalation anesthetics are widely used in children, and no evidence confirming inhalation anesthesia as a risk factor for neurodevelopmental deficits has emerged [10]. In a subset of adult patients undergoing surgeries, the procedure per se can lead to temporary cognitive impairments, and so may general anesthesia produced by any agent. But it remains to be determined whether the specific use of Isoflurane or related ethers represents an independent risk factor for cognitive impairment [9]. Similarly, no evidence has emerged that continuous, low-level exposure to waste anesthetic gases represents a unique risk for healthcare workers [16]. Nevertheless, given the significant side effects experienced by some patients, and the potential risk of long-term effects uncovered in animal studies, any reduction in the doses required to induce or maintain anesthesia may potentially be beneficial to both patients and healthcare workers.

The second messenger cAMP has been shown to modulate many functions of the central nervous system ranging from cognition and memory formation [17], [18], mood and emotions [19], to psychosis [20] or nociception [21]. The cellular concentration of cAMP is determined by the equilibrium between the rate of its synthesis by adenylyl cyclases, and the rate of its hydrolysis and inactivation by cyclic nucleotide phosphodiesterases (PDEs) [22]. PDEs comprise a superfamily of isoenzymes that are grouped into 11 PDE families based on sequence homology as well as their substrate kinetics and pharmacologic properties [23]. The PDE4 family is the largest, comprising four genes, PDE4A-D, that together generate likely over 25 protein variants via use of alternate promoters and alternative splicing [24], [25]. PAN-selective PDE4 inhibition produces numerous therapeutic effects [26], [27], [28], [29] including potent anti-inflammatory effects [30], memory and cognition improvement [17], [18], as well as cardiovascular [31], metabolic [32] and antineoplastic [33] effects. However, adverse effects, particularly nausea and emesis, have constrained their clinical utility and commercial success until now.

While exploring potential anti-inflammatory benefits of PDE4 inhibition in a mouse model of bacterial lung infection, we noticed that pre-treatment with a PDE4 inhibitor augmented the intensity and duration of Isoflurane anesthesia that we employed for the intranasal delivery of the bacteria. As a role of cAMP signaling in general, and an effect of PDE4 inhibition in particular, on Isoflurane anesthesia has not been reported before, and to delineate any potential impact of altered Isoflurane anesthesia on our animal model, we have further explored this observation.

Section snippets

Drugs

Piclamilast (RP73401; 3-(Cyclopentyloxy)-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide), Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-one), Roflumilast (3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide), Cilostamide (N-cyclohexyl-N-methyl-4-[(2-oxo-1H-quinolin-6-yl)oxy]butanamide), Prazosin ([4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-(furan-2-yl)methanone), Clonidine (N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine) and

Treatment with PAN-PDE4 inhibitors potentiates the anesthetic effects of Isoflurane in mice

While exploring the potential anti-inflammatory benefits of PDE4 inhibition in a mouse model of bacterial lung infection, we noticed that mice pretreated with the PDE4 inhibitor Piclamilast/RP73401 (5 mg/kg, i.p.; 1 h prior) appeared to lose consciousness faster upon exposure to Isoflurane anesthesia (e.g. loss of righting), and appeared deeper asleep (e.g. no muscle movement in response to moving or horizontal displacement of the anesthesia chamber). Subsequent to intranasal Pseudomonas

Potentiation of Isoflurane anesthesia is a class effect of PAN-PDE4 inhibitors in mice

We report here for the first time that inhibition of PDE4, but not inhibition of PDE3, induces a dose-dependent potentiation of Isoflurane anesthesia as detected using time-to-righting assays in mice (Fig. 1, Fig. 2). Several structurally-distinct PAN-PDE4 inhibitors, including Piclamilast, Rolipram, Roflumilast and RS25344 (all 1 mg/kg, i.p.; Fig. 1), all delay recovery from Isoflurane anesthesia, suggesting this is a class effect of PDE4 family-selective inhibitors. Conversely, YM976, a

CRediT authorship contribution statement

Ileana V. Aragon: Investigation, Writing - review & editing. Abigail Boyd: Investigation, Writing - review & editing. Lina Abou Saleh: Investigation, Writing - review & editing. Justin Rich: Methodology, Investigation, Writing - review & editing. Will McDonough: Methodology, Investigation, Writing - review & editing. Anna Koloteva: Resources, Writing - review & editing. Wito Richter: Conceptualization, Methodology, Investigation, Resources, Writing - original draft, Visualization, Project

Acknowledgements

We are grateful to the entire staff of the Department of Comparative Medicine at the University of South Alabama for providing excellent care of the animals, their advice on experimental design, and help with experimentation. This work was supported in part by funds from the Cystic Fibrosis Foundation (RICHTE16GO), the National Institutes of Health (HL076125, HL141473, HL066299) and a Research and Scholarly Development Grant from the University of South Alabama Office of Research and Economic

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