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. 2023 Mar 3;18(3):e0282593. doi: 10.1371/journal.pone.0282593

Effects of theophylline on ADCY5 activation—From cellular studies to improved therapeutic options for ADCY5-related dyskinesia patients

Dirk Tänzler 1,2, Marc Kipping 1,2, Marcell Lederer 3, Wiebke F Günther 1,2, Christian Arlt 1,2, Stefan Hüttelmaier 3, Andreas Merkenschlager 4, Andrea Sinz 1,2,*
Editor: Manuela Cabiati5
PMCID: PMC9983822  PMID: 36867608

Abstract

We show the effects of the three purine derivatives, caffeine, theophylline, and istradefylline, on cAMP production by adenylyl cyclase 5 (ADCY5)-overexpressing cell lines. A comparison of cAMP levels was performed for ADCY5 wild-type and R418W mutant cells. ADCY5-catalyzed cAMP production was reduced with all three purine derivatives, while the most pronounced effects on cAMP reduction were observed for ADCY5 R418W mutant cells. The gain-of-function ADCY5 R418W mutant is characterized by an increased catalytic activity resulting in elevated cAMP levels that cause kinetic disorders or dyskinesia in patients. Based on our findings in ADCY5 cells, a slow-release formulation of theophylline was administered to a preschool-aged patient with ADCY5-related dyskinesia. A striking improvement of symptoms was observed, outperforming the effects of caffeine that had previously been administered to the same patient. We suggest considering theophylline as an alternative therapeutic option to treat ADCY5-related dyskinesia in patients.

Introduction

Adenylyl cyclases (ADCYs) are central enzymes in all organisms [1]. The ADCY family comprises ten isoforms; nine of them are membrane-bound enzymes [2, 3]. ADCY5, which is most commonly expressed in brain and heart tissue, is one of the least studied isoforms [3]. As with all ADCYs, ADCY5 converts adenosine triphosphate (ATP) to cyclic adenosine-3’,5’-monophosphate (cAMP) and pyrophosphate (Fig 1) [3]. ADCY5 has been identified as the primary ADCY isoform that is responsible for up to 80% of the cAMP production in striatal medium spiny neurons [3, 4]. The complex system of initiating and controlling movement is dependent on a well-balanced signaling between G-Protein-Coupled-Receptors (GPCRs) and ADCYs [4]. Different neurotransmitters stimulate or inhibit hydrolysis of ATP to cAMP via ADCY-coupled GPCRs [5]. As such, adenosine receptor 2A (A2A) agonists increase intercellular cAMP levels [5].

Fig 1. Mode of action for caffeine, theophylline, and istradefylline on the reduction of cAMP production by ADCY5 via inhibiting the A2A receptor.

Fig 1

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ADCY5 contains an intracellular N-terminal domain, two membrane domains (M1 and M2) consisting of six helices, two cytoplasmic homologous domains (C1 and C2), and a short intracellular loop between the two catalytic domains (Fig 1) [2, 3, 6]. The formation of an ATP-binding pocket that brings the C1 and C2 domains into proximity is crucial for ADCY5’s catalytic activity is [2, 3]. Amino acid mutations in ADCY5 at different positions within the protein have been described, with the most prominent ones being R418W, R418G, R418Q and A726T [2, 4, 7]. The A726T mutant, in which an alanine is replaced by a threonine residue (Fig 1) in the C1b domain, influences ADCY5’s flexibility. This amino acid exchange, resulting from a single-point mutation, is suggested to alter the structure of the ATP-binding pocket by affecting the interaction strength between the C1 and C2 domains [2, 3]. The most frequently occurring variant of ADCY5 has a R418W exchange (Fig 1), where a positively-charged amino acid residue with a relatively long aliphatic side-chain (arginine) is replaced by a hydrophobic, aromatic amino acid residue (tryptophan). This exchange also results from a single-point mutation and is considered to alter the response as well as transmission of β-adrenergic receptor stimulation [2].

Gain-of-function ADCY5 mutants cause a significant increase in the catalytic hydrolysis of ATP in response to adrenergic stimulation [4, 8], resulting in increased intracellular cAMP concentrations in striatal cells [8]. All ADCY5 mutations result in kinetic disorders or dyskinesia that effect all muscles [2, 4, 9]. A combination of linkage analysis and exon sequencing provided strong evidence for mutations of ADCY5 being responsible for Familial Dyskinesia and Facial Myokymia (FDFM) [2, 3].

The inhibition of ADCYs impacts the conversion of ATP to cAMP in the gain-of-function mutants and therefore reduces intracellular cAMP concentrations [10, 11]. Increasing evidence suggests that A2A receptor antagonists exert positive effects on the symptoms of ADCY5-related dyskinesia [8]. Caffeine is an A2A antagonist that is hypothesized to alter cAMP levels in striatal neurons [12]. Theophylline showed a higher potency regarding different metabolic effects compared to caffeine [13], as well as resulting in improved ergogenic effects during whole body exercise [13]. Very recently, a report has been published that provides insight into the treatment of ADCY5-related dyskinesia patients with caffeine and which describes the effects on frequency and duration of paroxysmal movement disorders, baseline movement disorders, and other motor and no-motor features [14]. Overall, a consistent quality-of-life improvement was reported in 87% of patients. Only three out of 30 patients of this retrospective study reported a worsening of symptoms [14]. Based on these findings, the use of caffeine has been suggested as first-line treatment of ADCY5-related dyskinesia [14].

In the work reported here, we compare the effects of the three purine derivatives, caffeine [14], theophylline, and istradefylline [8], regarding their reduction of cAMP levels in ADCY5-overexpressing cell lines (ADCY5 wild-type and R418W mutant). We give a rationale for administering theophylline to ADCY5-related dyskinesia patients based on the highly promising results obtained for one patient. We envision that theophylline has the potential to complement or even replace the therapy with caffeine for treating patients with ADCY5-related dyskinesia.

Materials and methods

Chemicals and reagents

All chemicals and reagents were obtained from Roth or Sigma Aldrich at the highest purity available.

Cell culture

HEK293T/17 cells (ATCC, RRID: CVCL_1926) were stably transfected with vectors encoding GFP (for comparative purposes only), ADCY5 wild-type (ADCY5wt), and ADCY5 R418W mutant (ADCY5mut). Cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) for two days in six-well plates at 37°C and 5% CO2; 4.4 x 105 cells were cultivated in one well. Each treatment was performed in triplicate. Cells were incubated with caffeine, theophylline, and istradefylline at 37°C and 5% CO2 using different concentrations (caffeine and theophylline: 1 μM, 10 μM, 100 μM, 1 mM; istradefylline: 1 nM, 10 nM, 100 nM, 1 μM) for different time periods (10, 30, 60, 120, 240, and 480 min). Time-course experiments were performed in six replicates. Cells were harvested with PBS buffer, washed, and centrifuged at 1,500 x g for 5 min. A 200 μl aliquot of trizol reagent (9.5 g guanidinium thiocyanate, 3.1 g ammonium thiocyanate, 3.5 ml of 3 M sodium acetate, 5 g glycerol, 48 ml Roti Aqua-Phenol in 100 ml total volume) was added to the cell pellets to facilitate cell lysis and at the same time inactivate cAMP-degrading enzymes. Non-lysed cells and cell debris were removed by a centrifugation step using 30-kDa molecular weight cut-off filters (Amicon Millipore), which was performed at 14,000.0 x g for 10 minutes. Filtrates were stored at 4°C until they were analyzed by LC-MS/MS.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

LC separation of nucleotides (cAMP, AMP, ATP, cGMP, GMP, GDP, and GTP) was performed with a UPLC I-Class FTN system (Waters) equipped with an Atlantis Premier BEH C18 AX column (2.1 mm x 50 mm, 1.7μm, Waters). Separation was performed at a flow rate of 400 μl/min with the following LC gradient: 0–1 min: 3% B, 1–4 min: 3–20% B, 4–5 min: 20–85% B, and 5–5.5 min: 85% B; solvent A, 0.2% (v/v) formic acid in water; solvent B, 0.2% (v/v) formic acid in acetonitrile. The UPLC system was directly coupled to a Xevo TQ-XS mass spectrometer (Waters) with an electrospray ionization (ESI) source. Multiple-reaction monitoring (MRM) was performed using specific transitions for the nucleotides cAMP, AMP, ATP, cGMP, GMP, GDP, and GTP.

Ethics statement

The ethics committee of the University of Leipzig was informed and approved this study. The study has been registered at the DRKS (ID: DRKS00029154). Written consent was obtained from the parents.

Results

GFP, ADCY5wt, and ADCY5mut cells were compared for their ability to catalyze ADCY5-dependent cAMP production according to the workflow presented in Fig 2. Cells were incubated with caffeine, theophylline, and istradefylline at varying concentrations and samples were collected at different time points. All three drugs are purine derivatives that exert their effects by binding to the A2A receptor, resulting in inhibition of ADCY5 and reduced cAMP levels (Fig 1). As ADCY5-related dyskinesia is caused by gain-of-function ADCY5 amino acid-exchange mutations (Fig 1), reduction of cAMP levels will result in an improvement in movement disorders. This effect of caffeine on ADCY5-related dyskinesia patients has been impressively demonstrated in a recent report [14].

Fig 2. Analytical workflow.

Fig 2

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The main goal of the present study was to compare the effects of the three purines, caffeine, theophylline, and istradefylline, on cAMP concentrations at the cellular level. Caffeine, theophylline, and istradefylline differ in their Ki values (caffeine 23.4 μM, theophylline 1.7 μM, istradefylline 9.2 nM) at the A2A receptor and should therefore exhibit different effects on the mediated ADCY5-catalyzed cAMP production (Fig 1). After harvesting and lysing the cells, filtrates were collected and analyzed by LC-MS/MS (Fig 2). Based on MRM analyses, cAMP concentrations were quantified after treatment with the three purine drugs using cells that stably express GFP (control), ADCY5 wild-type, and ADCY5 R418W mutant. This allowed us to gain detailed insights into the ability of caffeine, theophylline, and istradefylline to reduce cAMP at the cellular level.

Comparison of caffeine, theophylline, and istradefylline regarding the reduction of cAMP levels

Consistent with the pivotal role of ADCY5 for cAMP production and its substantially increased catalytic activity caused by the R418W mutation, basal cAMP levels were upregulated ~5-fold in ADCY5wt while they were up-regulated ~30-fold in ADCY5mut cells. Effects of the three purines were found to be most pronounced for ADCY5mut cells, showing a drastic reduction of cAMP levels, even at concentrations of 100 μM caffeine and theophylline (Fig 3A). Already after ten minutes of drug treatment, initial reductions in cAMP levels were observed (Fig 3B); after one hour of drug treatment the effects on lowering cAMP concentrations became even more pronounced (Fig 3C). Interestingly, the cAMP concentrations observed after treatment of ADCY5mut cells with 100 μM theophylline attained roughly the same level as for non-treated ADCY5wt and GFP cells (Fig 3A). Apparently, treatment of ADCY5mut cells with a high concentration of theophylline results in basal cAMP levels that cannot be reduced any further. At the same time, rather large variations in cAMP levels were observed between different replicates in ADCY5mut cells (Fig 3A). This might be explained by the fact that the gain-of-function ADCY5 R418W mutant exhibits up to 30-fold increased basal cAMP levels compared to ADCY5 wild-type protein.

Fig 3. Influence of caffeine (red bars), theophylline (blue bars), and istradefylline (purple bars) at different concentrations on cAMP levels of different cell lines at different time points.

Fig 3

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Controls without drug treatment are shown as green bars. (A) GFP, ADCY5wt, and ADCY5mut cells, 1 h treatment. cAMP concentration of GFP cells (without ADCY5) are set to a value of 1.0. All cAMP concentrations of ADCY5mut cells are related to the GFP reference. (B) ADCY5mut cells, 10 min drug treatment (C) ADCY5mut cells, 1 h drug treatment. Experiments were performed in 3 replicates.

A comparison between the three purines under investigation revealed the most prominent reduction of cAMP concentrations in ADCY5mut cells for istradefylline, followed by theophylline and caffeine after already 10 minutes (Fig 3B). Apparently, the reduction of cAMP concentration correlates well with the respective Ki values (see above) of the three purines at the A2A receptor. After one-hour of treatment of the ADCY5mut cells with caffeine, theophylline, and istradefylline (Fig 3C), the reduction of cAMP concentrations essentially exhibited the same profile as that observed at 10 minutes of drug treatment.

Determination of cAMP concentration by LC-MS/MS

To determine ADCY5 activity, cAMP concentrations were determined by LC-MS/MS, quantifying different nucleotides by an LC-MRM-MS/MS approach to rule out any misassignments. Specific transitions were considered for cAMP, AMP, ATP, cGMP, GMP, GDP, and GTP (Fig 4A). For cAMP identification and quantification, the MS signal response showed a linear behavior in a concentration range up to 10 pmol/μl (Fig 4B). AMP, cAMP, GMP, cGMP reference compounds were used at a concentration of 100 fmol each, showing an LC baseline separation of all nucleotides with a total elution time of 3.5 minutes (Fig 4C). Based on our LC-MRM-MS/MS method, reliable and unambiguous cAMP quantification was performed using the cells’ supernatants (Fig 4D). As trizol reagent was used to stop all cellular reactions in our workflow, PDEs that are eventually present will also be inhibited during our analytical workflow (Fig 2), resulting in a lack of cAMP-to-AMP conversion.

Fig 4.

Fig 4

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(A) Selected transitions in triple-quadrupole MS/MS measurements for selected nucleotides. (B) Linearity of mass spectrometric response for cAMP in the concentration range between 0–10 pmol/μl. (C) LC-MRM-MS/MS analysis (100 fmol) of four selected nucleotides (AMP, cAMP, GMP, cGMP). The TIC (Total Ion Current) of the LC elution is shown in black, the LC gradient is shown in grey (%B). (D) LC-MRM-MS/MS of filtrates (see Fig 2) for selected nucleotides.

To confirm that an inhibition of PDEs by purines does not contribute to altered cAMP levels, we also measured GMP levels after treatment of cells with 100 μM caffeine and theophylline (Fig 5). Clearly, GMP levels do not change, verifying that PDEs are not involved in converting cGMP to GMP. It has to be noted in this context that our cGMP detection limit is at ~1 fmole. As we did not detect cGMP in any of our LC-MS/MS analyses, this again confirms that PDEs are not inhibited by the purines as this would result in an accumulation of cGMP. We are therefore confident that the cAMP concentrations determined by our workflow (Fig 2) present a correct reflection of how the three purines regulate ADCY5 activity via A2A receptor inhibition in the cellular systems studied herein.

Fig 5. Influence of caffeine (red bars) and theophylline (blue bars) at a concentration of 100 μM on GMP levels of different cell lines, 1 h drug treatment.

Fig 5

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Controls without drug treatment are shown as green bars. Experiments were performed in 3 replicates.

Time course of caffeine and theophylline treatment on ADCY5mut cells

Treatment of ADCY5mut cells with caffeine and theophylline (10 μM and 100 μM, each) over a time-course of 480 minutes revealed a reduction in cAMP levels at both concentrations (Fig 6). Strikingly, the reduction in cAMP levels was slightly more pronounced for theophylline compared to caffeine.

Fig 6.

Fig 6

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Time-dependence of (A) caffeine (10 μM: light red, 100 μM: red) and (B) theophylline (10μM: light blue, 100μM: blue) treatment of ADCY5mut cell lines. cAMP concentrations were monitored over a time-range of 8 hours. cAMP concentration of GFP cells (without ADCY5) was set to 1.0. All cAMP concentrations measured for ADCY5mut cells are related to the GFP reference. Experiments were performed in 6 replicates.

Theophylline treatment of a patient with ADCY5-related dyskinesia

Given the promising results of reducing cAMP concentrations by theophylline, especially in ADCY5mut cells, we hypothesized that theophylline might be an alternative to caffeine for treatment of patients with ADCY5-related dyskinesia [14]. Theophylline is available as a slow-release formulation and has been shown to exhibit only minor side effects in children with status asthmaticus that were treated with a low-dose of theophylline (5–7 mg/kg per day) [15]. Administering theophylline as a slow-release formulation might be favorable compared to caffeine and result in a more efficient reduction of cAMP levels in patients [16, 17]. Theophylline exhibits a narrow therapeutic window, which however can be efficiently met by a careful up-dosing of the drug and by regularly monitoring relevant blood parameters (see S1 Appendix).

A 6-year old female patient with ADCY5-related dyskinesia that had been successfully treated with 150 mg of caffeine (3x 50 mg daily) was treated with 400 mg of slow-release theophylline (2x 200 mg daily). The patient was diagnosed in 2018 with ADCY5 variant 1252 C>T, resulting in Arg418Trp, heterozygous and de novo mutation, no mosaicism. Initially, theophylline and caffeine were given in combination, but during the course of the therapy, the daily caffeine dose was reduced over a period of five months until theophylline was administered alone. The theophylline dose was gradually increased from ~6 mg/kg/day to ~23 mg/kg/day. After 6–8 weeks, theophylline blood levels ranged between 7.5 mg/l to 21.1 mg/l.

Already at a theophylline dose of ~12 mg/kg/day, divided into two single doses, the following effects were observed: The patient became more upright, showed increased muscle tone, was able to stand independently, and walk up to 50 meters without help. With caffeine alone, neither independent standing nor walking was possible. According to the physicians’ report during therapy, the patient was able to make less than 10 steps (with assistance only) and was in a wheelchair during the day. Targeted, slow gripping was possible. Speech was impaired as the patient could speak four to five words and was hard to understand.

Under theophylline treatment, the physicians’ report states an improvement in the quality of sleep as dyskinetic movements subsided completely when falling asleep. At the same theophylline dose (~12 mg/kg/day), the paroxysmal dyskinesia continued to fluctuate and recurrent administration to the patient led to a significant reduction of every symptom, such as the loss of independent standing. Bacterial or viral infection-associated deterioration of symptoms (see below) persisted despite increasing the daily dose of slow-release theophylline to ~18 mg/kg/day.

By increasing the theophylline dose to ~23 mg/kg/day, continuous improvements were observed. Supported by foot orthoses, the patient was able to walk ~50 meters hand-held and 7 meters freely, and climbed stairs independently. The paroxysmal dyskinesia showed a decrease in frequency and duration, and significantly improved dysarthria and functional hypersalivation.

On a scale from 0 (no improvement) to 10 (no symptoms), theophylline had a more pronounced influence in the afternoon (6–7), while its influence was ranked between 3–4 in the morning. The rating was done by the medical staff after consultation with the patient’s parents. The duration and frequency of episodes cannot be reliably quantified as different causes (light bacterial or viral infections, stress, joy, etc.) influence motoric conditions during theophylline treatment.

We did not observe any adverse side effects during theophylline dose titration; arterial blood pressure, pulse rate, and theophylline-related laboratory parameters were within the given reference ranges. Even at the maximum dose of ~23 mg/kg/day, sleep quality was greatly improved with no interruption of sleep for ten hours. Several patients are now being treated worldwide with theophylline. In none of the cases, side effects are observed for theophylline when the dosage is slowly increased as described in our dosage scheme and blood parameters are monitored regularly as described (see S1 Appendix).

Conclusions

We show that cAMP levels in ADCY5-overexpressing cells were reduced with all three purines, caffeine, theophylline, and istradefylline, under investigation. The effects were most prominent for the gain-of-function ADCY5mut cell line (R418W) and were in agreement with the Ki values of the three purines at the A2A receptor: The most prominent reduction of cAMP levels was observed for istradefylline, followed by theophylline and caffeine showing a less pronounced cAMP reduction. As istradefylline exhibits side-effects even at low-dose application it is currently not an approved drug in Germany [18]. Theophylline, on the other hand, is available as a slow-release formulation, showing only minor side-effects, even in pediatric applications despite its narrow therapeutic window. Theophylline was therefore administered as a slow-release formulation to a preschool-aged patient with ADCY5-related dyskinesia. Independent standing and walking, as well as sleep quality were substantially improved when treating the patient with theophylline compared to caffeine. We are aware that the cellular system used herein does not reflect the situation in the striatum. However, given the outstanding results in the patient described in this study—as well as in other patients that are being treated with theophylline worldwide now—this impressive improvement of dyskinesia underlines the results of our cellular studies. Based on our findings, the efficacy and safety profile of theophylline should now be evaluated for a larger patient cohort.

Supporting information

S1 Appendix. Dosage scheme for theophylline as slow-release formulation.

(DOCX)

Click here for additional data file. (33.2KB, docx)
S1 Dataset. LC/MS raw data underlying Figs 3 and 5.

The folder names (Fig 3A, Fig 3BC, and Fig 5) reflect the respective figure numbers in the main manuscript. The zip-file includes raw data, TargetLynx data and excel files of quantitation analysis.

(ZIP)

Click here for additional data file. (45.6MB, zip)

Acknowledgments

Dr. Wendy H. Raskind, University of Washington, Seattle, WA, is acknowledged for providing the ADCY5 plasmid. Dr. Frank Erdmann, Martin Luther University Halle-Wittenberg, is acknowledged for initial discussions; the Waters Corp. is acknowledged for providing the Xevo TQ-XS mass spectrometer. The authors are indebted to Prof. Gary Sawers, Martin Luther University Halle-Wittenberg, for critical reading of the manuscript.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

AS acknowledges financial support by the DFG (www.dfg.de) (RTG 2467, project number 391498659 “Intrinsically Disordered Proteins – Molecular Principles, Cellular Functions, and Diseases”, RTG 2751 “InCuPanC”, project number 449501615, INST 271/404-1 FUGG, INST 271/405-1 FUGG, and CRC 1423, project number 421152132), the Federal Ministry for Economic Affairs and Energy (www.bmwk.de) (BMWi, ZIM project KK5096401SK0), the region of Saxony-Anhalt (www.sachsen-anhalt.de), and the Martin Luther University Halle-Wittenberg (www.uni-halle.de) (Center for Structural Mass Spectrometry). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Alice Coles-Aldridge

14 Nov 2022

PONE-D-22-23902

Effects of Theophylline on ADCY5 Activation - From Cellular Studies to Improved Therapeutic Options for ADCY5-Related Dyskinesia Patients

PLOS ONE

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“AS acknowledges financial support by the DFG (www.dfg.de) (RTG 2467, project number 391498659 “Intrinsically Disordered Proteins – Molecular Principles, Cellular Functions, and Diseases”, RTG 2751 “InCuPanC”, project number 449501615, INST 271/404-1 FUGG, INST 271/405-1 FUGG, and CRC 1423, project number 421152132), the Federal Ministry for Economic Affairs and Energy (www.bmwk.de) (BMWi, ZIM project KK5096401SK0), the region of Saxony-Anhalt (www.sachsen-anhalt.de), and the Martin Luther University Halle-Wittenberg (www.uni-halle.de) (Center for Structural Mass Spectrometry).

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, Tänzler et al. report the results of theophylline and caffeine administration on cAMP concentrations in WT and ADCY5 mutant cells, as well as the results of theophylline treatment in a ADCY5 patient. It is overall well-written and easy to read. Even though both the cellular data and clinical report are of interest, there are issues with the way data are interpreted and what the authors conclude.

1) The first issue concerns the in vitro model that is used: both a non-neuronal (HEK) and non-integrated (cells) model. Given the complexity of striatal function, this limits greatly what can be inferred from the results – which should be mentioned in text.

2) The authors draw conclusions concerning caffeine and theophylline efficacy on cAMP lowering (“most prominent reduction of cAMP concentration for istradefylline, followed by theophylline and caffeine”, “strikingly, the reduction in cAMP level was more pronounced for theophylline compared to caffeine”) that are not supported by the data they present. Indeed, the same concentrations have been used for both molecules even though they are not equivalent, and the differences are clearly not statistically significant. This need to be rephrased, and limitations need to be clearly presented.

3) The authors present theophylline as a safe treatment, choosing to cite a paper reporting only minor side effects but at very low doses (5-7 mg/kg per day), even though they treat their ADCY5 patient with 23 mg/kg per day, and they do not cite any report on theophylline toxicity. Yet therapeutic window is known to be extremely narrow with this molecule, and there are several reports of sometimes fatal theophylline toxicity on the literature. So much so that theophylline is not prescribed for asthma if other medications are available (https://www.ncbi.nlm.nih.gov/books/NBK532962/). Such toxicity is only reported with extremely high doses of caffeine, making it the safest treatment of the two, which should be said in text. What’s more, the authors sate that theophylline was more effective than caffeine, but they used comparatively much higher doses of theophylline than caffeine in this patient. Even though doses up to 800 mg/day have been used in the recent report cited by the authors, they seem to have stopped at 150 mg a day in their patient. Have higher doses been tried? Have side effects prevented to raise the dose? In all likelihood, the benefits observed from high-dose theophylline treatment would have been observed with higher doses of caffeine, so that concluding here to the superiority of theophylline is quite misleading (the only advantage that could be put forward here is its slow-release formulation).

The case report part should therefore be rewritten to make room for all this additional (and crucial) information.

4) The authors state in the conclusion that istradefylline exhibits severe side effects. What are their sources? Most reports seem to conclude to limited side effects and acceptable tolerance with this drug. Most reported side effects appear to be dyskinesia, confusion and hallucinations, which are common in a population of Parkinson’s disease patients but cannot be generalized to other patients. Therefore, this needs to be rephrased.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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​

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Mar 3;18(3):e0282593. doi: 10.1371/journal.pone.0282593.r002

Author response to Decision Letter 0

25 Nov 2022

Responses to Reviewer’s Comments

Question: In this paper, Tänzler et al. report the results of theophylline and caffeine administration on cAMP concentrations in WT and ADCY5 mutant cells, as well as the results of theophylline treatment in a ADCY5 patient. It is overall well-written and easy to read. Even though both the cellular data and clinical report are of interest, there are issues with the way data are interpreted and what the authors conclude.

Response: We would like to thank this reviewer for his/her overall favorable comments on our work. I would like to point out that we performed all experiments in a diligent and careful manner and we believe that our conclusions are fully justified.

Question: The first issue concerns the in vitro model that is used: both a non-neuronal (HEK) and non-integrated (cells) model. Given the complexity of striatal function, this limits greatly what can be inferred from the results – which should be mentioned in text.

Response: We are aware that the cellular system used herein does not reflect the situation in the striatum. However, given the outstanding results in the patient described in this study (as well as in nine further patients treated with theophylline worldwide now) this impressive improvement of dyskinesia confirms the results of our cellular studies. We added a sentence addressing this issue in the “Conclusions” part on page 15: “We are aware that the cellular system used herein does not reflect the situation in the striatum. However, given the outstanding results in the patient described in this study - as well as in other patients treated with theophylline worldwide now - this impressive improvement of dyskinesia confirms the results of our cellular studies.”

Question: The authors draw conclusions concerning caffeine and theophylline efficacy on cAMP lowering (“most prominent reduction of cAMP concentration for istradefylline, followed by theophylline and caffeine”, “strikingly, the reduction in cAMP level was more pronounced for theophylline compared to caffeine”) that are not supported by the data they present. Indeed, the same concentrations have been used for both molecules even though they are not equivalent, and the differences are clearly not statistically significant. This need to be rephrased, and limitations need to be clearly presented.

Response: We agree with the reviewer that according to Figure 3B, the effects of cAMP reduction for caffeine and theophylline are similar to each other after 10 minutes of drug treatment. However, considering the effects of caffeine and theophylline at 60 minutes after drug treatment, the effect of theophylline in respect to cAMP level reduction is more pronounced at a concentration of 100 µM. We rephrased the respective sentences in the text: “The most prominent reduction of cAMP levels was observed for istradefylline, followed by theophylline and caffeine showing a less pronounced cAMP reduction.” “Strikingly, the reduction in cAMP levels was slightly more pronounced for theophylline compared to caffeine.”

Question: The authors present theophylline as a safe treatment, choosing to cite a paper reporting only minor side effects but at very low doses (5-7 mg/kg per day), even though they treat their ADCY5 patient with 23 mg/kg per day, and they do not cite any report on theophylline toxicity. Yet therapeutic window is known to be extremely narrow with this molecule, and there are several reports of sometimes fatal theophylline toxicity on the literature. So much so that theophylline is not prescribed for asthma if other medications are available (https://www.ncbi.nlm.nih.gov/books/NBK532962/). Such toxicity is only reported with extremely high doses of caffeine, making it the safest treatment of the two, which should be said in text.

Response: We are fully aware of the narrow therapeutic window of theophylline, which however can be efficiently met by a careful up-dosing of the drug. The reason why theophylline is not being used as first-line treatment for asthma bronchiale any more is that the development of ß2-sympathomimetics has advanced tremendously in the last 20 years. These drugs, such as salbutamol, salmeterol etc, exhibit a better efficacy for treating asthma bronchiale as theophylline.

During the last months, I have been approached by a number of parents of children suffering from ADCY5-related dyskinesia and I am advising neurologists and pediatricians on how to safely use theophylline. For this, I have assembled a guideline for medical doctors (and patients) regarding the dosage of theophylline as slow-release formulation for treating patients with ADCY5-related dyskinesia. We have now included this guideline in the Supporting Information of this manuscript.

Due to the spectacular success of theophylline treatment in the patient described in our manuscript who was confined to a wheelchair and is now able to walk without help, nine children with ADCY5-related dyskinesia are now being treated worldwide with theophylline. The videos the parents share with me are partially breathtaking. In none of the cases, side effects are observed for theophylline when the dosage is slowly increased as described in our dosage scheme and blood parameters are monitored regularly as described (see Supporting Information).

To clarify this important point, two sentences were added on pages 13, 14, and 15: “Theophylline exhibits a narrow therapeutic window, which however can be efficiently met by a careful up-dosing of the drug and by regularly monitoring relevant blood parameters (see Supporting Information).” “Several patients are now being treated worldwide with theophylline. In none of the cases, side effects are observed for theophylline when the dosage is slowly increased as described in our dosage scheme and blood parameters are monitored regularly as described (see Supporting Information).” “Theophylline, on the other hand, is available as a slow-release formulation, showing only minor side-effects, even in pediatric applications despite its narrow therapeutic window.”

Question: What’s more, the authors sate that theophylline was more effective than caffeine, but they used comparatively much higher doses of theophylline than caffeine in this patient. Even though doses up to 800 mg/day have been used in the recent report cited by the authors, they seem to have stopped at 150 mg a day in their patient. Have higher doses been tried? Have side effects prevented to raise the dose? In all likelihood, the benefits observed from high-dose theophylline treatment would have been observed with higher doses of caffeine, so that concluding here to the superiority of theophylline is quite misleading (the only advantage that could be put forward here is its slow-release formulation). The case report part should therefore be rewritten to make room for all this additional (and crucial) information.

Response: We assume that this reviewer is referring to the publication [Meneret A, Mohammad SS, Cif L, Doummar D, DeGusmao C, Anheim M, et al. Efficacy of Caffeine in ADCY5-Related Dyskinesia: A Retrospective Study. Mov Disord. 2022;37(6):1294-8. Epub 20220405. doi: 10.1002/mds.29006. PubMed PMID: 35384065] that we are also quoting in our manuscript (literature reference [14]). The reviewer is certainly aware that the caffeine dose of 800 mg/day was applied to a 17-year old male patient. Caffeine doses are however not given in mg/kg body weight, which is a deficiency of this publication. It seems obvious that the dose administered to a 17-year-old male patient has to be higher than that applied to a preschool-aged female patient. Therefore, a caffeine dose of 150 mg/day was absolutely sufficient for the patient described herein. Higher doses of caffeine have in fact been tried (up to 3 x 200 mg/day), but no improvement of dyskinesia symptoms was observed any more. Specifically, the patient was never able to walk under caffeine treatment, while under theophylline treatment the patient started walking independently.

The reviewer should also note that in that specific publication (Meneret et al, literature reference [14]) other examples are shown where caffeine was administered to a 15-year-old female patient at 100 mg/day with much improved symptoms and to a 13-year-old female patient at 225 mg/day with very much improved symptoms. This clearly demonstrates that the caffeine dose administered in the patient described herein is not too low.

Question: The authors state in the conclusion that istradefylline exhibits severe side effects. What are their sources? Most reports seem to conclude to limited side effects and acceptable tolerance with this drug. Most reported side effects appear to be dyskinesia, confusion and hallucinations, which are common in a population of Parkinson’s disease patients but cannot be generalized to other patients. Therefore, this needs to be rephrased.

Response: Istradefylline is currently being evaluated at the EMA for approval in the EU to treat Parkinson’s disease. It is known that istradefylline exhibits side effect that have so far hindered approval of this drug in the EU. It is not proven that the side effects of the drug can fully be attributed to common effects that are also observed in Parkinson’s patients. Nevertheless, the respective sentence was rephrased: ”As istradefylline exhibits side-effects even at low-dose application it is currently not an approved drug in Germany.”

Attachment

Submitted filename: Response_to_Reviewers.docx

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Decision Letter 1

Manuela Cabiati

23 Jan 2023

PONE-D-22-23902R1Effects of theophylline on ADCY5 activation - From cellular studies to improved therapeutic options for ADCY5-related dyskinesia patientsPLOS ONE

Dear Dr. Sinz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITORPlease indicate your revision and address the comments mainly of Reviewer 2.Best regards

Please submit your revised manuscript by Mar 09 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Manuela Cabiati, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Prof. Sinz

I apologize for the delay in the review, I hope you be able to answer the question and I am waiting for your revision!

Best regards Manuela cabiati

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have properly addressed some of the issues I raised, but not all of them. I still have concerns:

1) The addition of a sentence concerning the in vitro model is appreciated, but the authors make a statement that is not entirely supported by their results: the clinical results do not “confirm” the cellular results, they add another strong argument in favor of theophylline efficacy in ADCY5-related dyskinesia. It should be at least nuanced as a personal opinion: “we consider that…”

2) The authors have not addressed the second issue properly: they compare the effect of the same concentrations of different molecules that are not equivalent, and show at best tendencies, but no statistically significant differences between molecules. The figures indeed show very high variability of the results, and if we look at the effect of the highest doses used for all 3 molecules, they look about the same. Therefore, based on these results, it is false to draw conclusions on differential effects of these 3 molecules.

3) In addition to the supporting information that has been added, it would be helpful to clearly state in text what the potential side effects of theophylline are (and to cite a paper on the subject). I understand that the authors are themselves well aware of this, but it may not be the case of all readers. It would be very unfortunate if an accident happened due to insufficient monitoring by a prescribing physician who would be less aware of theophylline toxicity. As caffeine is safer, it should be recommended to try it first.

4) There is no weight-dose relationship with caffeine, so that a wide range of doses must be tried in patients before drawing conclusions. The authors have indeed tried higher doses of caffeine in their patient, without better results, which theophylline provided. That has to be said in text, as that information does provide a rationale to try theophylline in patients showing insufficient response to caffeine treatment.

5) As istradefylline has only been tried in Parkinson’s disease patients, its reported side effects may not be generalizable to other populations. The authors should therefore specify that the side effects they are mentioning were found in Parkinson’s disease patients.

Reviewer #2: This is an study reporting on cellular effects of caffeine, theophylline and istradefylline on cAMP production, and the response of a single patient with ADCY5 dyskinesia. The cellular studies are carefully done. The major conclusion is that there was a significant improvement on theophylline exceeding benefit from caffeine.

Given the authors’ emphasis on the clinical improvement of this patient, more care and details about the clinical report and treatment should be provided which are not included in the manuscript nor the supplementary file.

Major comments:

1) State the age of the patient, and report the ADCY5 variant detected in the patient. Was this heterozygous? inherited or de novo?

2) The baseline clinical status of the child prior to theophylline should be included in order to understand the difference with treatment. How far could the child walk with what type of assistance at baseline. What was the scale (0-10 used to assess the improvement?

3) Given the very slow caffeine taper over 5 months, this reviewer surmises at a theophylline dose of 12 mg/kg/day (when improvement is reported), the child was still on both medications. If so, please give the dose of both medications at that time.

4) Please differentiate clearly between parent reports of improvement and assessment made by trained physicians. Eg. Improvement of dyskinesia during sleep is reported by parents only.

5) Were the assessments done by same medical staff repeatedly or different staff over time? Were there attempts at inter-oberver reliability?

6) Was this assessment of the patient in clinic, or by evaluating videos? Were the staff blinded to the treatment (ie did not know the patient, or the dose).

Minor comments

7) In the supplemental file, it would be useful to have a graph tracking the doses of caffeine and theophylline over time, and with time points depicting the theophylline levels, and the improvements.

8) How long has the patient been followed at this point? Are there any long term side effects or waning of the therapeutic effect?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Mar 3;18(3):e0282593. doi: 10.1371/journal.pone.0282593.r004

Author response to Decision Letter 1

25 Jan 2023

Responses to Reviewers‘ Comments

Editor:

Question: Please indicate your revision and address the comments mainly of Reviewer 2.

Response: We appreciate the Editor’s suggestion to mainly address the questions raised by reviewer 2. Please note that we have already very carefully and thorougly addressed all comments raised by reviewer 1 in the previous revision of this manusript.

Reviewer 1:

Question: The addition of a sentence concerning the in vitro model is appreciated, but the authors make a statement that is not entirely supported by their results: the clinical results do not “confirm” the cellular results, they add another strong argument in favor of theophylline efficacy in ADCY5-related dyskinesia. It should be at least nuanced as a personal opinion: “we consider that…”

Response: We replaced the wording “confirms” by “underlines” in the Conclusions section on page 13.

Question: The authors have not addressed the second issue properly: they compare the effect of the same concentrations of different molecules that are not equivalent, and show at best tendencies, but no statistically significant differences between molecules. The figures indeed show very high variability of the results, and if we look at the effect of the highest doses used for all 3 molecules, they look about the same. Therefore, based on these results, it is false to draw conclusions on differential effects of these 3 molecules.

Response: We believe that our conclusions are fully justified and we have carefully addressed this issue in the previous revision of this manuscript. We agree with the reviewer that according to Figure 3B, the effects of cAMP reduction for caffeine and theophylline are similar to each other after 10 minutes of drug treatment. However, considering the effects of caffeine and theophylline at 60 minutes after drug treatment, the effect of theophylline in respect to cAMP level reduction is more pronounced at a concentration of 100 µM. We have already rephrased the respective sentences in the text accordingly: “The most prominent reduction of cAMP levels was observed for istradefylline, followed by theophylline and caffeine showing a less pronounced cAMP reduction.” “Strikingly, the reduction in cAMP levels was slightly more pronounced for theophylline compared to caffeine.” We believe that this is a correct description of the results obtained.

Question: In addition to the supporting information that has been added, it would be helpful to clearly state in text what the potential side effects of theophylline are (and to cite a paper on the subject). I understand that the authors are themselves well aware of this, but it may not be the case of all readers. It would be very unfortunate if an accident happened due to insufficient monitoring by a prescribing physician who would be less aware of theophylline toxicity. As caffeine is safer, it should be recommended to try it first.

Response: Again, this point has already been thoroughly addressed in the previous revision of this manuscript. We are fully aware of the narrow therapeutic window of theophylline, which however can be efficiently met by a careful up-dosing of the drug. For this, I have assembled a guideline for medical doctors (and patients) regarding the dosage of theophylline as slow-release formulation for treating patients with ADCY5-related dyskinesia. We have included this guideline in the Supporting Information of this manuscript. Every physician can refer to this guideline. I would like to refer in this context to Paracelsus’ prominent sentence “Dosis facit venenum” every physician learns in their first year of studies. Also caffeine will exhibit severe side effect caused by overdosing.

To clarify this important point, two sentences have already been added on pages 13, 14, and 15 to the previously revised version of this manuscript: “Theophylline exhibits a narrow therapeutic window, which however can be efficiently met by a careful up-dosing of the drug and by regularly monitoring relevant blood parameters (see Supporting Information).” “Several patients are now being treated worldwide with theophylline. In none of the cases, side effects are observed for theophylline when the dosage is slowly increased as described in our dosage scheme and blood parameters are monitored regularly as described (see Supporting Information).” “Theophylline, on the other hand, is available as a slow-release formulation, showing only minor side-effects, even in pediatric applications despite its narrow therapeutic window.”

Question: There is no weight-dose relationship with caffeine, so that a wide range of doses must be tried in patients before drawing conclusions. The authors have indeed tried higher doses of caffeine in their patient, without better results, which theophylline provided. That has to be said in text, as that information does provide a rationale to try theophylline in patients showing insufficient response to caffeine treatment.

Response: The reviewer should note that the patient described in our study was treated with caffeine in the first place, but the beneficial effects of theophylline have never been observed for caffeine – even when using high doses of caffeine. The same effects, mainly regarding the ability to walk, have in the meantime been observed for theophylline in other patients, too. So it is probably an effect related to theophylline itself that it can specifically improve some of the dyskinesia symptoms better than caffeine.

Question: As istradefylline has only been tried in Parkinson’s disease patients, its reported side effects may not be generalizable to other populations. The authors should therefore specify that the side effects they are mentioning were found in Parkinson’s disease patients.

Response: We have also addressed this point in the previous revision of our manuscript. Istradefylline is currently being evaluated at the EMA for approval in the EU to treat Parkinson’s disease. It is known that istradefylline exhibits side effect that have so far hindered approval of this drug in the EU. It is not proven that the side effects of the drug can fully be attributed to common effects that are also observed in Parkinson’s patients. Nevertheless, the respective sentence was rephrased: ”As istradefylline exhibits side-effects even at low-dose application it is currently not an approved drug in Germany.”

Reviewer 2:

Question: This is an study reporting on cellular effects of caffeine, theophylline and istradefylline on cAMP production, and the response of a single patient with ADCY5 dyskinesia. The cellular studies are carefully done. The major conclusion is that there was a significant improvement on theophylline exceeding benefit from caffeine.

Response: We appreciate the favorable evaluation of our manuscript by the reviewer.

Question: Given the authors’ emphasis on the clinical improvement of this patient, more care and details about the clinical report and treatment should be provided which are not included in the manuscript nor the supplementary file.

Response: We have now included information from the clinical report (made by physicians during rehab therapy). The reviewer should note that the report contains sensitive information that touches the patient’s personal rights. Therefore we selected several significant sentences to be included in this manuscript.

Question: State the age of the patient, and report the ADCY5 variant detected in the patient. Was this heterozygous? inherited or de novo?

Response: On page 11, we have now added additional information on the patient: “A 6-year old female patient with ADCY5-related dyskinesia that had been successfully treated with 150 mg of caffeine (3x 50 mg daily) was treated with 400 mg of slow-release theophylline (2x 200 mg daily).”

“The patient was diagnosed in 2018 with ADCY5 variant 1252 C>T, resulting in Arg418Trp, heterozygous and de novo mutation, no mosaicism.”

Question: The baseline clinical status of the child prior to theophylline should be included in order to understand the difference with treatment. How far could the child walk with what type of assistance at baseline. What was the scale (0-10) used to assess the improvement?

Response: We added some sentences from the confidential patient report (rehab report, written by physicians, in German) regarding this issue on page 12: “According to the report of physicians during therapy, the patient was able to make less than 10 steps (with assistance only) and was in a wheelchair during the day. Targeted, slow gripping was possible. Speech was impaired as the patient could speak four to five words and was hard to understand.”

The report was written by physicians (in German) during the rehab therapy of the patient before and during theophylline treatment. As the full report touches the personal rights of the patient it cannot be made available to the public. Please note that we extracted and translated those sentences that are indicative of the patient’s condition during theophylline therapy.

The reviewer should note that on page 12, we have already assessed the improvement of symptoms on a scale (0-10): “On a scale from 0 (no improvement) to 10 (no symptoms), theophylline had a more pronounced influence in the afternoon (6-7), while its influence was ranked between 3-4 in the morning. The rating was done by the medical staff after consultation with the patient’s parents. The duration and frequency of episodes cannot be reliably quantified as different causes (light bacterial or viral infections, stress, joy, etc.) influence motoric conditions during theophylline treatment.”

Question: Given the very slow caffeine taper over 5 months, this reviewer surmises at a theophylline dose of 12 mg/kg/day (when improvement is reported), the child was still on both medications. If so, please give the dose of both medications at that time.

Response: The exact dosage scheme is summarized in the Supporting Information (dosage of theophylline, slow-release formulation):

“The following dosage scheme has been applied by us:

- A preschool-aged patient with ADCY5-related dyskinesia that had been successfully treated with 150 mg of caffeine (3x 50 mg daily) was treated with 400 mg of slow-release theophylline (2x 200 mg daily).

- Initially, theophylline and caffeine were given in combination, but during the course of the therapy, the daily caffeine dose was reduced over a period of five months until theophylline was administered alone.

- The theophylline dose was gradually increased from ~6 mg/kg/day to ~23 mg/kg/day over a time course of 8 weeks. Theophylline blood levels ranged between 7.5 mg/l to 21.1 mg/l.

- At a theophylline dose of ~12 mg/kg/day (divided into two single doses), the following effects were observed: The patient became more upright, showed increased muscle tone, and was able to stand and walk independently. The quality of sleep improved as dyskinetic movements subsided completely when falling asleep.

- By increasing the theophylline dose to ~23 mg/kg/day, continuous improvements were observed.”

Question: Please differentiate clearly between parent reports of improvement and assessment made by trained physicians. Eg. Improvement of dyskinesia during sleep is reported by parents only.

Response: The physicians’ report contains also a statement on sleep quality, which has now been added on page 12: “Under theophylline treatment, the physicians’ report states an improvement in the quality of sleep as dyskinetic movements subsided completely when falling asleep.”

Question: Were the assessments done by same medical staff repeatedly or different staff over time? Were there attempts at inter-oberver reliability?

Response: The assessments of the patient were made by the same team of physicians.

Question: Was this assessment of the patient in clinic, or by evaluating videos? Were the staff blinded to the treatment (ie did not know the patient, or the dose).

Response: The assessments were always made during stays of the patient in the clinic by direct observation and interaction of the patient with physicians. The staff knew about the patient’s treatment.

Question: In the supplemental file, it would be useful to have a graph tracking the doses of caffeine and theophylline over time, and with time points depicting the theophylline levels, and the improvements.

Response: We believe that our exact dosage scheme given in the Supporting Information is sufficient:

“The following dosage scheme has been applied by us:

- A preschool-aged patient with ADCY5-related dyskinesia that had been successfully treated with 150 mg of caffeine (3x 50 mg daily) was treated with 400 mg of slow-release theophylline (2x 200 mg daily).

- Initially, theophylline and caffeine were given in combination, but during the course of the therapy, the daily caffeine dose was reduced over a period of five months until theophylline was administered alone.

- The theophylline dose was gradually increased from ~6 mg/kg/day to ~23 mg/kg/day over a time course of 8 weeks. Theophylline blood levels ranged between 7.5 mg/l to 21.1 mg/l.

- At a theophylline dose of ~12 mg/kg/day (divided into two single doses), the following effects were observed: The patient became more upright, showed increased muscle tone, and was able to stand and walk independently. The quality of sleep improved as dyskinetic movements subsided completely when falling asleep.

- By increasing the theophylline dose to ~23 mg/kg/day, continuous improvements were observed.”

Question: How long has the patient been followed at this point? Are there any long term side effects or waning of the therapeutic effect?

Response: The patient has been under theophylline treatment for 1.5 years, without any side effects being observed. Also other patients that are now being treated with theophylline did not experience any side effects so far. Please note that one paragraph addressing this issue has been added already during the previous revision of this manuscript: “We did not observe any adverse side effects during theophylline dose titration; arterial blood pressure, pulse rate, and theophylline-related laboratory parameters were within the given reference ranges. Even at the maximum dose of ~23 mg/kg/day, sleep quality was greatly improved with no interruption of sleep for ten hours. Several patients are now being treated worldwide with theophylline. In none of the cases, side effects are observed for theophylline when the dosage is slowly increased as described in our dosage scheme and blood parameters are monitored regularly as described (see Supporting Information).”

Decision Letter 2

Manuela Cabiati

21 Feb 2023

Effects of theophylline on ADCY5 activation - From cellular studies to improved therapeutic options for ADCY5-related dyskinesia patients

PONE-D-22-23902R2

Dear Dr. Sinz,

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Additional Editor Comments (optional):

All my doubts were clarified. the paper is accept in the present form.

Reviewers' comments:

Acceptance letter

Manuela Cabiati

24 Feb 2023

PONE-D-22-23902R2

Effects of theophylline on ADCY5 activation - From cellular studies to improved therapeutic options for ADCY5-related dyskinesia patients

Dear Dr. Sinz:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Appendix. Dosage scheme for theophylline as slow-release formulation.

    (DOCX)

    Click here for additional data file. (33.2KB, docx)
    S1 Dataset. LC/MS raw data underlying Figs 3 and 5.

    The folder names (Fig 3A, Fig 3BC, and Fig 5) reflect the respective figure numbers in the main manuscript. The zip-file includes raw data, TargetLynx data and excel files of quantitation analysis.

    (ZIP)

    Click here for additional data file. (45.6MB, zip)
    Attachment

    Submitted filename: Response_to_Reviewers.docx

    Click here for additional data file. (23.1KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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