*Article* **Salification Controls the In-Vitro Release of Theophylline**

**Laura Baraldi 1,2, Luca Fornasari 2, Irene Bassanetti 2, Francesco Amadei 2, Alessia Bacchi 1,3 and Luciano Marchiò 1,3,\***


**Abstract:** Sustained released formulation is the most used strategy to control the efficacy and the adverse reactions of an API (active pharmaceutical ingredient) with a narrow therapeutic index. In this work, we used a different way to tailor the solubility and diffusion of a drug. Salification of Theophylline with Squaric Acid was carried out to better control the absorption of Theophylline after administration. Salification proved to be a winning strategy decreasing the dissolution of the APIs up to 54% with respect to Theophylline. Most importantly, this was accomplished in the first 10 min of the dissolution process, which are the most important for the API administration. Two polymorphs were identified and fully characterized. Theophylline squarate was discovered as trihydrate (SC-XRD) and as a metastable anhydrous form. Indeed, during the Variable Temperature-XRPD experiment, the trihydrate form turned back into the two starting components after losing the three molecules of water. On the other hand, the synthesis of the trihydrate form was observed when a simple mixing of the two starting components were exposed to a high humidity relative percentage (90% RH).

**Keywords:** xanthines; theophylline; squaric acid; controlled release; dissolution; solubility

**1. Introduction**

Xanthines like theophylline, caffeine, and theobromine are a group of alkaloids, which act as mild stimulants and as bronchodilators. Theophylline in particular has an antiinflammatory effect in asthma and chronic obstructive pulmonary disease (COPD) at lower concentrations [1].

Theophylline's mechanism of action implies the inhibition of phosphodiesterase, which is responsible for the smooth muscle relaxation. However, theophylline is also known for its narrow therapeutic index (30–100 μM) caused by a remarkably low selectivity. Indeed, a concentration of 110 μM already leads to a wide range of adverse reactions such as nausea, vomiting, metabolic acidosis, and arrhythmias [2]. For this reason, despite being an effective API, theophylline is not considered the first choice in the treatment of asthma. It is usually administered orally in slow-release preparations for chronic treatment in combination with a short acting β2-agonist, long acting β2 agonist, or an inhaled corticosteroid [3–5].

Lots of significant work has been done in the formulation field to optimize the theophylline absorption profile. Pezoa et al. [6], Rodrigues et al. [7], and Jian et al. [8] are just a few examples of the huge effort that has been done so far aiming at a better in-vivo performance. Formulations were modified using excipients like Eudragit or involving nanoparticles to have a prolonged effect and better control on the adverse reactions [9]. While optimizing a formulation many aspects must be taken into account [10], such as the role of every single component and how they influence the final release or the scale up. The interactions between excipient-excipient and drug-excipient are also to be considered.

**Citation:** Baraldi, L.; Fornasari, L.; Bassanetti, I.; Amadei, F.; Bacchi, A.; Marchiò, L. Salification Controls the In-Vitro Release of Theophylline. *Crystals* **2022**, *12*, 201. https:// doi.org/10.3390/cryst12020201

Academic Editors: Changquan Calvin Sun and Junpei Yamanaka

Received: 6 December 2021 Accepted: 27 January 2022 Published: 29 January 2022

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Hydrolysis of the drug, ion interactions leading to new insoluble forms, or other physical interactions like adsorption of the API onto the surface of excipients might be significant drawbacks, resulting from the interactions between excipients and drugs [11].

In this work, we used a different strategy to tune the properties of this active pharmaceutical ingredient: a salification of theophylline with squaric acid was carried out to optimize its solubility, dissolution rate, and diffusion. Squaric acid is a strong acid belonging to the family of oxocarbons [12]. It has been studied extensively for its peculiar physical properties [13] and for its use in the synthesis of dyes [14,15]. In the pharmaceutical industry, squaric acid derivatives have attracted interest for their potential use as topical immunosensitizers [16], enzyme inhibitors, and receptor antagonists [17]. However, to the best of our knowledge, squaric acid has never been studied in the formation of API salts. From a crystal engineering perspective, the features that guided the selection of this acid as salt-former are its strong acidity (pKa1 = 0.6, pKa2 = 3.4) [18], its highly symmetric character, and the well-established tendency to form layered H-bonded assemblies [19,20], that should couple effectively with mostly planar molecules such as xanthines.

Usually, when looking for a new salt to improve the pharmacokinetic properties of an API, hydrate forms are unwelcome. Hydrates usually have a higher thermodynamic stability leading to a slower dissolution profile [21,22]. In this peculiar case, the hydrate form would turn useful, providing a better control of the released drug. Moreover, salification might turn to be easier and quicker with respect to the formulation development. Indeed, once the salt is synthesized already known manufacturing processes can be applied, yielding standard tablets or capsules for instance. Some theophylline salts have already been studied [23,24]. Furthermore, some systems have been designed with the aim of improving bioactivity [25] or stability [26], however, in both cases, the salts resulted more soluble than theophylline alone. Hence, salification has never been applied to theophylline with the purpose to ameliorate the therapeutic index issues
