Pharmacological Evaluation of Respiratory Safety Following a Single Intravenous Administration of Theophylline in Sprague–Dawley Rats

Tea is one of the most commonly consumed beverages in the world and is a major component in many cultural traditions and social experiences. ¹ Tea consumption involves sensory factors, such as aroma and flavor, ² and it has acknowledged physiological benefits, such as reducing the risk of cardiovascular disease, ³ hypercholesterolemia, ⁴ and diabetes. ⁵ The physiological benefits of tea are primarily attributed to its richness in phenolic compounds, which enhance antioxidative capacity, and the inherent roles of polyphenols, such as catechin derivatives. ¹ In addition to phenolic compounds, methylxanthines are significant components of tea, which are produced naturally via plant metabolism. ⁶ Representative methylxanthines include caffeine, theobromine, and theophylline, which function biologically as stimulants that regulate the respiratory rate. ^{6 –8} Theophylline (1,3-dimethylxanthine) is a methylxanthine that is widely used therapeutically as a bronchodilator for patients with asthma and chronic obstructive pulmonary disease (COPD). ⁹ COPD is characterized by significant shortness of breath and is defined by the global initiative for chronic obstructive lung disease. ¹⁰ The chronic airflow limitation that is characteristic of COPD is caused by a combination of small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema), with the relative contributions varying from person to person. ¹¹

Theophylline has been used as a bronchodilator to help manage COPD for more than 90 years. ¹² Phosphodiesterase (PDE) breaks down a molecule called cyclic adenosine monophosphate (cAMP) within cells, and when theophylline inhibits PDE, the accumulation of intracellular cAMP increases. ¹³ This increase in cAMP relaxes the smooth muscles of the airways, which makes breathing easier. ¹³ Theophylline also exerts an anti-inflammatory effect by increasing the activity of histone deacetylases, which inhibit the activity of proinflammatory genes. ¹⁴ Thus, theophylline has anti-inflammatory effects in the respiratory system and may reduce overdistension and, consequently, dyspnea. ¹³ Specifically, in COPD, excess adenosine increases inflammatory responses and constricts smooth muscle, there narrowing the bronchi. ¹⁵ Furthermore, exposure to theophylline significantly blocks adenosine receptors, leading to bronchodilation. ¹⁶

However, theophylline has a narrow safe and effective therapeutic range, and consequently, even small deviations from the therapeutic dosage may have toxic effects. ¹⁷ The therapeutic index of theophylline, which represents the range of doses in which the medication is effective while minimizing the risk of unacceptable side effects, is relatively narrow, ranging from 10 to 20 mg/L. ¹⁸ Excessive amounts of theophylline can result in abnormal changes in the central nervous system, including excitement, anxiety, headache, insomnia, dizziness, tremor, tinnitus, and numbness of the hands, whereas abnormal changes in the respiratory system include tachypne. ¹⁹ Because of the narrow therapeutic window for efficacy and toxicity, safety evaluations of theophylline in the respiratory, cardiovascular, and central nervous systems are required.

The fundamental aim of this study was to evaluate the respiratory system’s pharmacological responses to theophylline. Respiratory system responses were systematically investigated in Sprague–Dawley rats to generate basic data to guide future studies and determine dose rates suitable for clinical use.

This study adhered to the International Conference on Harmonization (ICH) guidelines, specifically the Harmonized Tripartite Guideline S7A on Safety Pharmacology Studies for Human Pharmaceuticals, ²⁰ to standardize the evaluation of potential adverse effects of theophylline for a battery of pharmacological safety tests. All experimental animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of CentralBio Co., Ltd. (approval number: CBIACUC_22-0410SPR2). Initially, 32 healthy, 5-week-old male pathogen-free Sprague–Dawley rats (Samtako Bio Korea, Osan, Korea) were introduced to the animal facility and maintained for a week for acclimatization. All experimental rats were housed under controlled conditions of 150–300 lux, 45.3–55.7% humidity, and 21.0–23.5°C. During the acclimatization and experimental periods, all rats were fed a standard chow diet (1314 IRR; Altromin Spezialfutter GmbH & Co., Lage, Germany) and provided filtered and sterilized tap water ad libitum. ^{21 –23}

After a week of acclimatization, 32 Sprague–Dawley rats were selected based on body weight (183.1–194.1 g) and assigned to one of four groups (n = 8 per group). To evaluate the effects of theophylline (Sigma-Aldrich, St. Louis, MO, USA) on the respiratory system, 0 (vehicle control), 2.5, 5, and 10 mg/mL doses were sequentially prepared using sodium chloride (Dai Han Pharm, Seoul, Korea). Clinically, theophylline is administered orally at a dose of 200 mg, which is approximately 3.3 mg/kg for an average human (∼60 kg). To examine its toxicological effects, we selected concentrations approximately six times higher than the current clinical dosage. A stock solution of theophylline was initially prepared at a concentration of 10 mg/mL using sodium chloride as the solvent, and 5 and 2.5 mg/mL solutions were prepared subsequent to dilution with sodium chloride. The 5 mg/mL solution was prepared by diluting the 10 mg/mL stock solution with an equal volume of sodium chloride. Similarly, the 2.5 mg/mL solution was prepared by diluting the 5 mg/mL solution in a 1:1 ratio with sodium chloride. Pure sodium chloride solution was used as the vehicle control (0 mg/mL). All solutions were prepared freshly on the day of administration and stored in a refrigerator within two hours of formulation to maintain the stability until use. The highest concentration of theophylline (10 mg/mL) was selected based on a previous study on anti-nociception in a murine model, ²⁴ while the lower concentrations (5 mg/mL and 2.5 mg/mL) were determined by serial dilution, reducing the highest concentration by a factor of 2 for each subsequent dose. The administered volume was 5 mL/kg, and the volume for each individual experimental animal was calculated based on its body weight on the day of administration (before administration) after fasting. A single intravenous injection was administered into the caudal vein of each rat using a disposable syringe (Korea Vaccine, Ansan, Korea). Drinking water and feed were removed before the observation period and were made accessible after the 6-h observation period.

Each day, two experimental animals per group were administered the designated treatment between 9:00 AM and 10:00 AM, and their behaviors were recorded for four consecutive days. Respiratory measurements were taken at 0 (before administration), 0.25, 1.5, 4, and 6 h after administration, following a 0.5-h acclimatization period. All observations over the four consecutive days were conducted in a plethysmography chamber within a 5-min window of the planned time points (Buxco Electronics Inc., Wilmington, NC, USA). ²⁵ The average respiratory rate, tidal volume, and minute volume were calculated over a 10-minute interval using a plethysmography system (Buxco Electronics Inc.). After the final day of respiratory measurements, all experimental rats were humanely euthanized via thoracotomy following CO₂ exposure. ²⁵ All respiratory responses were statistically analyzed using SPSS (Version 25; IBM, Chicago, IL, USA), and data homogeneity was assessed using Levene’s test, confirming that all data had equal variance. After confirming equal variance, a one-way analysis of variance followed by Dunnett’s test as a post hoc analysis was used to determine statistical significance, with significance defined as P < .05. ²⁶ When homogeneity was not confirmed, Student's t-test was used to compare differences with the vehicle group (*P < .05).

The respiratory rate, also known as the breathing rate, is defined experimentally as the number of breaths taken within a 1-minute period. ²⁵ In the groups that received 5 and 10 mg/kg theophylline, the respiratory rate at 0.25 h post-administration showed a statistically significant increase (P < .05) compared with the vehicle control group (Table 1). At 0.25 h after intravenous theophylline exposure, the respiration rate for the vehicle control was 154.76 ± 46.15, whereas the rates for 5 and 10 mg/kg theophylline were 244.85 ± 26.23 and 292.38 ± 25.84, respectively.

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Table 1.

Effects of Theophylline on the Respiratory Rate in Male Sprague–Dawley Rats

Dose (mg/kg, i.v.)	n	Respiratory rate (unit: BPM)
		0	0.25	1.5	4	6
0 (vehicle)	8	142.22 ± 36.91	154.76 ± 46.15	123.87 ± 18.58	121.09 ± 9.03	222.43 ± 64.22
2.5	8	120.72 ± 12.95	187.67 ± 23.38	110.16 ± 7.77	126.51 ± 34.73	186.39 ± 70.41
5	8	125.55 ± 10.26	244.85 ± 26.23*	126.98 ± 18.44	133.65 ± 36.58	190.11 ± 52.02
10	8	153.00 ± 35.04	292.38 ± 25.84*	183.79 ± 61.95	160.96 ± 48.14	168.05 ± 64.71

Rats were divided into four groups and administered 0, 2.5, 5, or 10 mg/kg of theophylline intravenously. The respiratory rates were measured at 0, 0.25, 1.5, 4, and 6 h after intravenous theophylline injection. Values are means ± standard deviations (n = 8). Data were analyzed using one-way ANOVA when Levene’s test confirmed homogeneity of variances. For groups where homogeneity was not confirmed (5 and 10 mg/kg theophylline at 0.25 h post-administration), Student’s t-test was used to compare differences with the vehicle group (*P < .05).

ANOVA, analysis of variance; BPM, breaths per minute; i.v., intravenous administration.

Tidal volume is the amount of air that flows into or out of the lungs during each breathing cycle. ²⁷ In the control group (0 mg/kg), the tidal volume fluctuated between 0.96 and 1.00, with no statistically significant differences at any measured time point. Similarly, for all theophylline doses, the tidal volume ranged from 0.87 to 1.03. The consistency of the tidal volumes resulted in no statistically significant differences across different time points and theophylline doses (Table 2).

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Table 2.

Effects of Theophylline on the Tidal Volume in Male Sprague–Dawley Rats

Dose (mg/kg, i.v.)	n	Tidal volume (unit: mL)
		0	0.25	1.5	4	6
0 (vehicle)	8	1.00 ± .011	0.95 ± 0.08	0.98 ± 0.12	0.87 ± 0.08	0.91 ± 0.06
2.5	8	0.99 ± 0.10	0.95 ± 0.09	0.93 ± 0.10	0.98 ± 0.11	0.99 ± 0. 13
5	8	0.96 ± 0.08	0.93 ± 0.08	0.95 ± 0.04	0.96 ± 0.10	1.02 ± 0.08
10	8	0.99 ± 0.15	0.88 ± 0.10	0.92 ± 0.10	0.96 ± 0.17	1.03 ± 0.12

Rats were divided into four groups and administered 0, 2.5, 5, or 10 mg/kg of theophylline intravenously. The tidal volumes were measured at 0, 0.25, 1.5, 4, and 6 h after intravenous theophylline injection. Values are means ± standard deviations (n = 8). Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc test to determine statistical significance (*P < .05).

ANOVA, analysis of variance; i.v., intravenous administration.

The minute volume is the total amount of gas breathed in or out of the lungs each minute. ²⁸ In the groups that received 5 and 10 mg/kg theophylline, the minute volume at 0.25 h post-administration showed a statistically significant increase (P < .05) compared with the vehicle control group (Table 3). At 0.25 h after intravenous theophylline exposure, the minute volume for the vehicle control was 136.33 ± 30.21, whereas the values for 5 and 10 mg/kg theophylline were 205.11 ± 23.08 and 235.56 ± 29.32, respectively.

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Table 3.

Effects of Theophylline on the Minute Volume in Male Sprague–Dawley Rats

Dose (mg/kg, i.v.)	n	Minute volume (unit: mL/min)
		0	0.25	1.5	4	6
0 (vehicle)	8	136.36 ± 31.47	136.33 ± 30.21	119.65 ± 23.80	101.96 ± 4.72	180.16 ± 43.91
2.5	8	116.41 ± 15.77	162.45 ± 13.60	110.14 ± 6.39	118.13 ± 35.98	160.81 ± 41.84
5	8	116.62 ± 4.54	205.11 ± 23.08*	115.27 ± 16.27	121.56 ± 26.45	173.33 ± 45.45
10	8	141.82 ± 34.59	235.56 ± 29.32*	156.05 ± 48.16	146.55 ± 49.86	156.79 ± 52.63

Rats were divided into four groups and administered 0, 2.5, 5, or 10 mg/kg of theophylline intravenously. The respiratory rates were measured at 0, 0.25, 1.5, 4, and 6 h after intravenous theophylline injection. Values are means ± standard deviations (n = 8). Data were analyzed using one-way ANOVA when Levene’s test confirmed homogeneity of variances. For groups where homogeneity was not confirmed (5 and 10 mg/kg theophylline at 0.25 h post-administration), Student’s t-test was used to compare differences with the vehicle group (*P < .05).

ANOVA, analysis of variance; i.v., intravenous administration.

In this study, we observed a significant increase in respiratory rate and minute volume, whereas the tidal volumes remained unchanged following intravenous administration of theophylline at doses >2.5 mg/kg at the 0.25-h time point. The observed respiratory responses provide a clear understanding of the response of mammals to systemic theophylline exposure. Given the intravenous administration, theophylline is expected to be rapidly distributed throughout the circulatory system. Consequently, our findings may help elucidate respiratory responses in relation to the distribution, metabolism, and excretion of theophylline. However, it is important to note that our experimental design may not fully capture the complete effects of systemic theophylline exposure, as it does not consider the potential metabolites that result from oral intake, since intravenous administration was chosen as the exposure route.

In adults, approximately 90% of theophylline is metabolized in the liver. ²⁹ Theophylline primarily undergoes 8-hydroxylation to form 1,3-dimethyluric acid, which accounts for approximately 60–80% of the parent compound. Alternatively, N-demethylation serves as a metabolic pathway, producing 1-methylxanthine in 8–24% of cases and 3-methylxanthine in 5–15% of cases. ³⁰ Therefore, this study did not fully cover the respiratory responses caused by the metabolites of theophylline. Future follow-up experiments should focus on both intravenous metabolite exposure and oral administration of theophylline, which may provide a comprehensive understanding of how and at what concentrations theophylline exposure could alter respiratory responses.

This study consists solely of observational results, and a notable limitation is the lack of molecular indices, which would have provided deeper mechanistic insights. Furthermore, while adhering to widely accepted ICH guidelines, our measurements primarily focus on ventilation parameters and do not explore underlying mechanisms. To address these limitations in this study, future studies should include parameters such as inspiratory and expiratory times and flows, as well as the apneic time, to obtain a more comprehensive understanding of respiratory responses to other routes of exposure to theophylline or its metabolites, such as oral administration. At the very least, the selected dosages and exposure durations in this study will provide foundational scientific data to guide the generation of new hypotheses in subsequent pharmacological studies.