Using several in vitro technologies performed in isolated tissues, we have previously shown that Ca2+, a primary ligand that activates the Ca2+-sensing receptor, can act on intestinal epithelium and enteric nervous system and reverse cholera toxin-induced fluid secretion. In the present study, using the cholera toxin-pretreated C57BL/6 mice as a model, we show that the anti-diarrheal effect of Ca2+ can also occur in vivo. Our results raise a question of whether this divalent ion also needs to be replaced in diarrhea management. Perhaps, an ideal rehydration therapy would be solutions that contain both monovalent ions, which reduce diarrhea mortality, and divalent minerals, which reduce diarrhea morbidity.Diarrhea like cholera remains a leading cause of mortality and morbidity globally. Oral rehydration solution (ORS) that developed in 1970s significantly decreases diarrhea mortality; yet, it does not reduce diarrhea morbidity and its usage has reduced persistently. Patients with diarrhea lose not only monovalent ions Na Despite recent advances in research from molecular biology to public health initiatives, few advancements have been made in the realm of diarrhea therapeutics, and oral rehydration solution (ORS) developed in the early 1970s has remained the mainstay therapy. Although use of ORS has decreased the diarrheal mortality, it does not reduce its morbidity. ORS neither reduces disease severity nor duration and its usage has declined persistently. Currently, only one third of cases are used for many reasons but most notably due to perceived failure to reduce patient’s stool output. Therefore, for decades there has been continuous effort searching for new therapeutics that are able to halt diarrhea and alleviate disease symptoms–7, particularly those simple, widely available and inexpensive therapeutics, 8–10.Diarrhea disease such as cholera remains a leading cause of mortality and morbidity among the most vulnerable populations+, K+, Cl− and HCO3−) but also divalent minerals , 12. While losses of monovalent ions are replaced via ORS, losses of divalent minerals are not. Studies now suggest that despite divalent minerals contributing relatively few osmoles to diarrhea stool, their effects on intestinal functions are large due to the presence of divalent mineral-sensing receptors  in the gut and their amplifying effects. As a result, losses of Zn2+ from diarrheal stools without replacement will significantly increase the magnitude and duration of diarrhea symptoms; replacements of Zn2+ losses will reduce the severity of diarrheal illnesses and accelerate their recovery. This is well documented and today Zn2+ replacement has been adopted as policy in all countries–18. In 2019, World Health Organization has added user-friendly Zn2+/ORS co-pack to the Essential Medicine List.Patients with diarrhea lose not only monovalent ions . We employed CTX mouse model of secretory diarrhea not only because most of our prior preclinical studies in vitro were performed using this model, including CaSR knockout mouse studies–28, but also because this well characterized enterotoxin secretagogue has been widely used as a human alternative to provide proof of concept of whether an antidiarrheal agent is therapeutic or not –37. We did not use the bacteria Vibrio cholerae as a model primarily for concerns of known confounding effects of the Ca2+ on bacterial colonization and biofilm formation that would complicate result interpretation. To make certain that the anti-diarrheal effect was specific to the Ca2+, not due to the associated anion of Ca2+ compounds, we tested three different sources of Ca2+, i.e., Ca2+ citrate, Ca2+ chloride, and Ca2+ carbonate. A drug effect is a function of time and dose; to further demonstrate the specificity of the effect of the Ca2+, we also studied Ca2+ time and dose responses. Our results show that Ca2+ supplemented ORS is indeed reducing diarrheal morbidity, at least in cholera toxin-pretreated mice.Therefore, this present study compared ORS with 2+ citrate (intervention) for 3 days, and changes in disease activity were monitored and diarrhea severity scored according to vs. ORS containing Ca2+.In Experiment 1, cholera toxin (10 μg/mouse) was gavaged into the stomach of 6–8-week-old young adult C57BL/6 mice to first induce diarrheal disease. Ninety minutes later when diarrhea formed, mice were randomly assigned to receive ORS alone (control) or ORS with 6 mM Ca2+ also developed diarrheal disease within 2 hours and peaked at 6–8 hours, they developed a significantly less severe and less pronounced disease. The disease activity scores mean ± SE (n), expressed as AUC (area under curve) control vs. intervention: 81.2 ± 1.6(7) vs. 14.3 ± 1.9(7) (P < 0.05). The disease duration (hours): 34.6 ± 1.2(7) vs. 14.5 ± 1.4(7) (P < 0.05). Ca2+ reduced diarrhea severity and duration by 82% and 58%, respectively. Thus, while in ORS treated mice the disease activity score had remained significantly elevated above baseline until 36–48 hours post CTX exposure, in ORS + Ca2+ treated mice a close to normal activity score had been achieved at least 20 hours earlier at 12–24 hours post CTX treatment , 2.5, 5 & 20 mM mean ± SE (n) : 4.3 ± 0.2(5) (control), 3.0 ± 0.3(5) (P > 0.05), 2.5 ± 0.3(5) (P < & 0.8 ± 0.3(5) (P < 0.05). Thus, diarrhea reduced by 35% at low [Ca2+] of 2.5 mM, 50% at medium [Ca2+] of 5 mM, and 80% at high [Ca2+] of 20 mM.To be certain that the observed anti-diarrheal effect was specific to the Ca2+ effect, in Experiment 3, we tested Ca2+ carbonate using the same disease model and treatment protocol. vs. with 5 mM Ca2+ carbonate. Compared to mice who drank ORS without Ca2+, mice who drank ORS with Ca2+ had a significantly less severe disease.To further confirm the Ca2+ to ORS, regardless of whether the Ca2+ added is Ca2+ citrate, Ca2+ chloride or Ca2+ carbonate, all reduces diarrhea.Taken together, these results demonstrate that adding Cain vivo demonstration, showing that Ca2+ fortified ORS is more effective than standard ORS in reducing diarrheal morbidity. This includes reducing not only the severity but also the duration. The finding is in keeping with the findings in Zn2+ research, suggesting that both mono- and di-valent ions lost in diarrheal stools should be promptly replaced during rehydration therapy. Inadequate replacement of these divalent ion losses may lead to a more severe and more prolonged course of the diarrhea.This is the 1st +, K+, Cl−, and HCO3−) and divalent minerals  are lost that can impact diarrhea outcomes. Without replacing monovalent ions, patients with diarrheal stools may develop hypovolemia, hyponatremia, hypokalemia, hypochloremia and metabolic acidosis, increasing risk of diarrheal mortality; without replacement of lost divalent minerals, the body will develop Ca2+ deficit/CaSR hypofunction and Zn2+/ZnSR hypofunction, leading to increase in diarrheal morbidity, making the disease more severe and more protracted. The proposed solution for the former is ORS rehydration/replacement therapy, which is well recognized and has been standard of care. Whereas the clinical solution for the latter is to add back Ca2+/Zn2+, which is still not fully recognized, particularly for replacing of Ca2+.2+ replacement in their drinking ORS bottles, mice developed a significantly more severe and more protracted diarrheal disease when exposing to cholera toxin provocation; with Ca2+ replacement, the disease severity was significantly reduced so was the time to diarrhea recovery  and indirectly by activating enteric nervous system (ENS), 41. According to our previous work, Ca2+ can act via CaSR and reverse the diarrhea-producing pathways provoked by CTX. For example, while CTX activates adenylyl cyclase and cyclic AMP generation, Ca2+/CaSR activates phosphodiesterase and cyclic nucleotide destruction. Similarly, while CTX reduces claudin-2, opening up tight junctions, Ca2+/CaSR enhances claudin-2, closing down tight junctions. Finally, while CTX activates the ENS-mediated secretion, Ca2+/CaSR inhibits it. However, considering that Ca2+/CaSR enhances degradation but does not prevent generation of the 2nd messengers induced, Ca2+ is not anticipated to prevent occurrence anticipated to prevent occurrence of diarrhea although this latter was not explored in the present study.How does Ca2+ and ORS. We did it both for physiological and clinical considerations. Physiologically, this combined approach would maximally increase the Ca2+ bioavailability thus greatest stimulates the CaSR in the intestine. Diarrhea with high stool output will speed up the passage of substances through the digestive tract, particularly if they were delivered in tablets or boluses. This will inevitably reduce the residence and contact time of the substances with the intestine unless they are delivered frequently and or in high doses. Otherwise, their anticipated drug effect on diarrhea may be compromised or not produced as evidenced in a recent study with an oral CFTR inhibitor. Continuously or frequently administering Ca2+ via ORS in a drinking bottle that was adopted in the present study could avoid or minimize this risk.The present study used simultaneous provision of the two effective interventions Ca2+ and ORS together will also have clinical significance, particularly in remote areas of developing countries, where there are health system obstacles to widespread use of a separate Ca2+ as well as Zn2+ during acute diarrhea. These obstacles include the need for Ca2+/Zn2+ suspension, syrup or tablets to be made available at health facilities and prescribed by health care providers in addition to ORS. Thus, if Ca2+ and ORS can be co-delivered or co-packaged together, it could considerably increase treatment prescription, administration, compliance, and adherence of the both interventions. In addition, it could simplify the logistics of incorporating Ca2+ into treatment guidelines for diarrhea, as does for Zn2+ and ORS.The provision and administration of the two effective interventions Ca2+ to ORS might be a solution to help reducing diarrheal morbidity. If this is verified in humans, this simple approach could benefit hundreds of millions of diarrhea patients worldwide. Randomized controlled trials in humans are under way.In summary, diarrhea remains a leading cause of mortality and morbidity globally. While ORS helps reduce diarrheal mortality, it does not reduce diarrheal morbidity. We show that adding Ca2 inhalation and by cervical dislocation. The use of animals as well as the protocols for cholera toxin and ORS treatments was approved by the Institutional Animal Care and Use Committee (IACUC# 201807567) at University of Florida.Experiments were performed using 6–8 weeks of age male/female C57BL/6 mice that were bred and maintained in-house at the University of Florida Communicore Animal Facility in accordance with the Animal Welfare Act and the Public Health Policy on Humane Care. Animals were fed and maintained on regular chow (Harlan) with free access to water before experiment. After completion of the experiment, animals were sacrificed with standard CO3 buffer containing 10μg CTX. In these experiments, 10μg CTX per mouse was dosed to induce a moderate severity of diarrheal disease. After CTX gavage, animals continued fasted for additional 90 minutes before they were allowed access to drinking water  and regular chow in order to avoid their possible interferences on toxin binding and action. Our pilot studies showed that diarrhea started ~½ hour and peaked ~ 1½ hours post cholera toxin gavage consistent with a previous report. To reduce bias, animals were assigned, randomly, to receive ORS with vs. without Ca2+ by one investigator and then monitored for progression of disease, blindly, by different investigators. Previously, we scored disease activity using stool consistency score; while it is simpler, stool consistency score was often found difficult to correctly evaluate the diarrhea severity, particularly when the stool was stuck to the bedding chips. Also, it was hard to appropriately determine the exact grade for the parameter owing to the acute nature of watery diarrhea induced and the overlapping ambiguous boundaries between grades. Consequently, in this present study, we employed the disease activity score that uses sum of stool consistency, appearance and activity as an indicator of health  75 Na+, 20 K+, 65 Cl−, 10 citrate and 75 glucose with total osmolarity of 257 mOsm/kg H2O.Cholera toxin was obtained from Sigma-Aldrich, and 5 mg/ml stock solutions were prepared in water. Sodium chloride, potassium chloride, sodium citrate, citric acid, CaP < 0.05 was considered significant.Values are given as means ± SEM. Statistical comparisons between two means were performed by Studenťs t-test, whereas comparisons among multiple means were by one-way ANOVA with Tukey’s post hoc tests.


Recently, non-covalent reactions have emerged as approaches to improve the physicochemical properties of active pharmaceutical ingredients (API), including antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). This review aimed to present and discuss the non-covalent reaction products of antibiotics, including salt and neutral multi-component solid forms, by framing their substituents and molar ratios, manufacturing techniques, characterization methods, benefits, potency changes, and toxicity, and is completed with an analysis of the development of computational models used in this field. Based on the data, NSAIDs are the most-developed drugs in multi-component system preparations, followed by antibiotics, i.e., antituberculosis and fluoroquinolones. They have reacted with inorganic elements, excipients, nutraceuticals, natural products, and other drugs. However, in terms of treatments for common infections, fluoroquinolones are more frequently used. Generally, NSAIDs are acquired on an over-the-counter basis, causing inappropriate medication. In addition, the pKa differences between the two groups of medicine offer the potential for them to react non-covalently. Hence, this review highlights fluoroquinolone–NSAID multi-component solid systems, which offer some benefits. These systems can increase patient compliance and promote the appropriate monitoring of drug usage; the dual drug multi-component solids have been proven to improve the physicochemical properties of one or both components, especially in terms of solubility and stability. In addition, some reports show an enhancement of the antibiotic activity of the products. However, it is important to consider the possibility of activity changes, interaction, and toxicity when using drug combinations. Hence, these aspects also are discussed in this review. Finally, we present computational modeling, which has been utilized broadly to support multi-component system designs, including coformer screening, preparation methods, and structural modeling, as well as to predict physicochemical properties, potency, and toxicity. This integrated review is expected to be useful for further antibiotic–NSAID multi-component system development. Antibiotics have been some of the most used drugs in the last decades. Consequently, microorganism resistance has occurred due to their longtime usage. In addition, antibiotics must target microbes, but should be safe for human cells; hence, their toxicity must also be considered. On the other hand, low solubility and instability are typical problems for some antibiotics, which may cause the drug’s content not to reach the appropriate levels. Next, chemical degradation also may produce toxicity and allergens. Thereafter, efforts have been made to find more selective, potent, safe, and effective antibiotics. However, the complexity of finding new drugs, from modeling and synthesis to clinical testing and formulation, makes the process slow .The development of new drugs requires reagents, i.e., starting chemicals, catalysis, solvent organics, etc. In addition, the synthesis of new drugs requires optimization to obtain a high percentage of yield or purity. Besides, the process may create chemical waste and side products. After its synthesis, a new synthetic drug should be evaluated by more extended pharmacological testing, including toxicity, mutagenicity, clinical testing Phase I, II, and III, etc., before it can be accepted as a suitable active pharmaceutical ingredient. These, often long, steps of drug discovery are costly because they consume a significant amount of resources and time.In addition to traditional drug development strategies, combining existing drugs with other compounds to create multi-component solids is becoming an increasingly interesting approach to improving drug performance. This technique requires less solvent and is easier to perform than other methods, making it an attractive option. The process involves low-energy, non-covalent reactions that don’t affect the drug–receptor binding, making it ideal for use in the body. By using established drugs to create multi-component systems, certain clinical tests may be avoided, making the solid engineering technique even more desirable. In addition, no reagents are used, high yields are obtained, and almost no waste or residue is yielded. However, it’s important to carefully consider the interaction cases when combining APIs in a multi-component solid.Computational structure–activity relationship modeling has been progressively developed to predict antibiotics’ structure, potency, resistance, mechanism of action, and toxicity ,3. One sOn the other hand, aside from antibiotics, infected patients usually consume anti-inflammatory agents to overcome inflammation symptoms. NSAIDs , which have relatively few adverse effects, have become first-choice drugs, and are available widely in various dosage forms. However, they generally have low solubility. Furthermore, some are physically unstable, i.e., they show polymorphism and pseudopolymorphism, which change their solid characters. In addition, they may also be chemically unstable. NSAIDs also have exhibited non-covalency with various counterparts in the multi-component solids, which could explain their lack of physicochemical properties. Furthermore, combining both medicines to enhance patient convenience and compliance and improve their physicochemical properties without changing their activity is an exciting prospect. This effort is supported by the fact that many NSAIDs tend to be acidic ; inverseThe first and crucial step in creating multi-component structures is determining the coformer or stoichiometric ratio. Once this is established, appropriate methods can be used to react with the components, including slow evaporation, fast evaporation, neat grinding, liquid-assisted grinding, slurry, and co-precipitation ,10,11. AHowever, only powder X-ray diffraction (PXRD) can precisely identify the new solid phase formation based on the diffractogram pattern. The specific distance and intensity represent the unique electron position of the atoms in the new crystal lattice. Hence, different diffractogram patterns from the starting materials indicate the multi-component crystal structure formation. Next, a solid analysis should be conducted to fix the structure and kind of interaction. Various methods can be used, including vibrational spectroscopy techniques such as Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and Terahertz spectroscopy, as well as nuclear magnetic resonance (NMR) ,25.This review aims to present and discuss various antibiotic multi-component structures and their preparation methods, challenges, and benefits. First, we highlight antituberculosis agents as the first antibiotic multi-component systems. Then, we discuss fluoroquinolone multi-component solids, which are the currently most-reported structures. However, these drugs can be poorly soluble and unstable in light and humid conditions. It has been found that N-methyl-amine-piperazine is responsible for photodegradation and requires protection ,27,28,29Finally, the in-silico method has been recognized. It is currently preferred, as a green, efficient, and effective tool for designing new multi-component solids and calculating their physicochemical properties ,36,37,38Based on their chemical structure and mechanism of action, antibiotics are divided into six groups: (1) cell wall synthesis inhibitors, including the beta-lactam class  and dissimilar agents, such as cycloserine, vancomycin, and bacitracin ; (2) thoMeanwhile, based on indications, antibiotics are divided into sulfonamides, trimethoprim, sulfamethoxazole, quinolones , penicillin, cephalosporins, aminoglycosides, macrolides, clindamycin, quinupristin/dalfopristin, linezolid, vancomycin, teicoplanin, daptomycin, bacitracin, polymyxin, and mupirocin ,48. ThosA multi-component crystal is a solid phase consisting of more than one kind of molecule stoichiometrically. This solid phase comprises intermolecular bonds between the components, which are also influenced by their intramolecular chemical bonding. Based on the type of interactions, the multi-component solids are divided into three groups: salts, cocrystals, and hydrate/solvate. The APIs can be an acid, base, or neutral compound, and can combine with another drug, nutraceutical, excipient, or substituent to form a multi-component crystal. Salt or cocrystal formation depends on the “rule of pKa”, which states that components with a pKa value of less than three will produce cocrystals; salt requires a pKa value higher than three. In addition, salt cocrystals can also be produced as a result of the reaction between neutral multi-component systems, acids or Schiff bases, and metal atoms . Multi-component solids may be built by combining a non-soluble component with a water-soluble substituent via hydrogen bonding to attract the water molecules, increasing the solubility. The solid-state arrangement may improve a wide range of APIs’ physicochemical properties, including their solubility, stability, flowability, and permeability. Many also enhance activities and performances, as recently reported ,53,54,55Both ionic and non-ionic reactions of the fluoroquinolone group, i.e., ciprofloxacin and levofloxacin, with some substituents increase the solubility of these compounds ,9, as weSalt is formed by the different ionic/charge compounds, which transfer hydrogen atoms between acid and base moieties, and may consist of inorganic and organic counterparts to the host molecule. This structural derivation has been applied for a long time, significantly increasing drug solubility and stability, physically and chemically. In addition, the changes observed in antibiotics’ physicochemical properties after salt formation can improve in terms of antibacterial potency, as shown by levofloxacin in combination with antioxidants, citric acid , and dihAlthough it was discovered in 1844 and characterized in 1958, the term ‘cocrystal’ was first used in 1963 by Lawton and Lopez. This term refers to a combined system of solid compounds in a crystal lattice, formed due to non-ionic intermolecular bonding with at least a neutral organic counterpart, referred to as a “co-former”. A coformer, particularly non-API, should be pharmacologically neutral, non-toxic, and considered a GRAS  compound. The API and the paired combination must have different polarities to form cocrystals, which then interact in the binding site, known as a “synthon”. Supramolecular synthons may be a homo-synthon  or hetero-synthons (various moieties). Drug–drug, drug–nutrient, drug–vitamin, and drug–excipient combinations can cause the formation of a neutral multi-component structure. The cocrystals may involve hydrogen or halogen bonding in two or more different neutral compounds. Furthermore, combining an ionic component with a cocrystal can form an “ionic-cocrystal” with a charge . However, antibiotics may also form neutral and ionic multi-component solids, i.e., ciprofloxacin with dicarboxylic acids ,21 and sAnother type of multi-component solid is formed when a molecule compound interacts with an organic solvent or water molecule and builds a stoichiometrically neutral structure, producing a solvate and hydrate, respectively. Solvate and hydrate variations depend on concentration, temperature, humidity, and pressure. Naturally, many antibiotics have hydrate forms, which are commonly more stable than solvates, with different physicochemical properties, such as density, the molecular weight of the crystal, solubility, stability, hygroscopicity, etc. ,26,56. MFluoroquinolones have many sites with the potential to form hydrogen bonds and, often, more than one lattice hydrate forms. For example, levofloxacin and ciprofloxacin may be observed in hemihydrate and monohydrate phases ,19,26,56Several methods can be used to create a new solid structure, which can be categorized into three techniques: dissolving, grinding, and heating. The dissolution method commonly produces single crystals and comprises slow evaporation, rapid evaporation, cooling, chemical reactions, spraying, and freeze-drying ,9. In thAnother evaporation method is a cooling technique at a low temperature to accelerate the saturated condition and produce more crystals spontaneously. Subsequently, the freeze-drying methods involve dispersing an unsaturated solvent into a nozzle using nitrogen gas. These processes aim to remove the unsaturated solution of drugs and co-formers through extreme conditions, such as the use of high pressure and low temperature, to cause the other parts become very saturated; as such, the evaporation process runs faster ,9. Next, grinding methods are divided into neat and solvent-assisted grinding. First, neat grinding is done in a mortar to mix and press the component thoroughly, without any solvent. This method produces large numbers of multi-component systems and is quicker than evaporation. However, the homogeneity of this method could be better in large-scale production. The second method is liquid-assisted grinding, using a small amount of solvent in the milling process. The solvent acts as a catalyst without waste production . Other rHeating methods include hot melt extrusion (HME) , microwaNext is the microwave method, which differs from HME in its energy source. HME uses a “stove” apparatus, heat conductance, and pressure; a microwave uses the radio-electromagnetic wave, which vibrates and excites the atoms and molecules. Microwaving accelerates this process and maximizes the multi-component solid yield significantly. For example, the diclofenac-L-proline cocrystal can be produced using a microwave in 30 s . Last, tBesides conventional methods, some developed techniques exist, such as the supercritical process , sublimaMeanwhile, sublimation is a process that facilitates the formation of multi-component systems by providing the necessary energy to transform a mixture of solid components into a gas and subsequently condensing it into a multi-component solid form. Commonly, an oven is used simply in the hot melt extrusion and sublimation methods. However, this technique has been used to successfully isolate 4-fluorophenol–p-benzoquinone cocrystals, with different polymorphs than those in the conventional method’s yield, showing that sublimation determines the phase space of cocrystals .The standard instruments and techniques used to confirm the new solid phase formation are microscopes , thermal analysis , vibrational spectrophotometry , NMR, and X-ray diffractometry (PXRD AND SCXRD). Thermal analysis observes the thermal properties of solid compounds. Next, structural analysis methods, such as vibrational spectroscopy  and NMR, analyze structural changes that occur after a successful reaction. Finally, SCXRD determines the 3D conformation to show the atoms/molecules’ position, distance, and angle position of atoms and molecules. In this subsequent, we present the solid instrumentations used to characterize the new multi-component phases.Every material has a unique energy that is reflected in its physicochemical properties. Thermal analysis, which observes the phase transitions of a substance under heating, such as melting point, recrystallization, oxidation, and degradation, is one of the oldest methods used for material characterization. In the past, conventional apparatuses such as a melting block, Thiele apparatus, or bomb calorimeter have been used to observe those parameters. A simple thermal method in solid-state observation is thermal microscopy, published by Koffler many years ago , but it A DSC is a calorimeter that measures the heat flow rate versus the temperature increase. This method is used to observe the thermal characteristics of a solid material in detail, such as the glass transition, water/solvent release from a pseudopolymorphism, fusion/melting point, recrystallization at high temperature, and decomposition, as well as to observe subtle physical changes, such as the glass transition temperature and eutectic point ,23,63,64DSC and DTA thermograms will show endothermic and exothermic curves, representing the energy change during the phase transformation. For example, the glass transition is demonstrated by an endothermic blunt curve, a broad endothermic peak indicates water or solvent molecules release, the melting point shows a sharp endothermic curve, a sharp exothermic curve indicates recrystallization, and oxidation/degradation is indicated by some irregular curves ,21,22,23Depending on their lattice thermodynamics, a multi-component crystal may have a lower or higher melting point/degradation temperature than the starting compounds. One can observe the energetical changes resulting from the interaction energy present in the new multi-component solid phase. For instance, the formation of the levofloxacin–phthalimide cocrystal and levofloxacin–caffeic acid salt led to a reduction in melting point, with values of 172–176 °C and 150–152 °C, respectively, compared to levofloxacin and the coformers, which all have melting points above 200 °C . This in−1); the infrared wave spectrum has a wave number region of 14,000 cm−1 to 10 cm−1, but the region used for compound analysis is only the middle region, with a wavelength of 2.5–50 µm or wave number 400–4000 cm−1. FTIR is commonly used in the characterization and identification of solid states. This method can recognize functional groups involving various bonds, including ionic, covalent, van der Waals, and hydrogen bonds. The working principle of this instrument is to measure the interaction of infrared waves with the bond energy between atoms. The transition of intramolecular vibrational energies (vibrations in the bonds between atoms) will be measured in terms of the amount of radiation contained in the infrared waves emitted by the source. Molecular vibrations are classified into two main groups: stretching and bending. The frequency of infrared waves is expressed in units of wave number . NMR spectroscopy interacts with the nuclei of atoms with radio electromagnetic (REM) waves, then profiles the resonance frequency yielded. Solid-state NMR may identify and quantify the multi-component solids, of both a crystalline and amorphous nature, by using an irradiation frequency of 500–1000 MHz, with the C and H positions and their bindings represented by the chemical shift in the spectra. Hence, SSNMR deals with solid-structure elucidation, amorphous and crystal characterization, dynamics, and stability. The structure can be solved accurately by reading the exact position of the resonance of the distinct nuclei. NMR has been used in solid-state development to ensure the formation of new interactions. For example, the reaction of levofloxacin with citric acid  and dihyThe X-ray diffractometry observes the change of atomic or molecular order and position, represented by their distance and angle in the lattice structure. X-ray diffraction is an analytical method based on the ability of a crystal to diffract X-rays and produce a characteristic pattern, which allows for an in-depth study of the solid phase structure. This diffraction method utilizes the constructive interference event of monochromatic X-rays. When X-ray radiation reaches atoms in the crystal structure, the high energy of the radiation will cause electrons to move to a higher level. However, this transfer of electrons does not last long because the state is unstable, so the electrons will return to their ground state and release light with identical wavelengths. This phenomenon is known as Rayleigh scattering. X-ray diffractometry is specific to analyzing crystalline samples because the amorphous forms will not produce a regular diffraction pattern ,69. InstNext, X-ray diffractometry is classified into PXRD (powder X-ray diffraction) and SCXRD . PXRD differs from SCXRD in terms of sample preparation and instruments. PXRD measures the diffractogram of the powder sample, and SCXRD is used to determine a single crystal system’s new solid structure three-dimensionally. Hence, PXRD analysis is relatively faster than SCXRD, due to its more straightforward sample preparation steps. Practically, isolating single crystals with an appropriate size to be analyzed by SCXRD is challenging. However, PXRD requires a homogenous particle size with a dimension of less than ~10 μm. The small and homogenous crystal’s size can be randomly oriented, but the large variations in the particle size cause the broadening of the diffractogram and complicate structural assignments. In addition, PXRD requires an appropriate number of samples; meanwhile, SCXRD only needs one crystal but should be physically stable, with a size of about 0.1–0.5 mm . SCXRD dMercury, in order to match the structure based on diffraction data, as measured by the goniometer [SCXRD observes single or discrete diffraction peaks and transforms the peak positions into coordinates to recover the underlying crystal lattice dimensions or orientations commonly occurring in powder diffraction. As a result, the interpretation of single crystal diffraction is much less ambiguous than powder diffraction methods. Still, preparing the appropriate single crystal samples is challenging, laborious, and time-consuming. However, even though the crystal structure data are easier to interpret, the spatial properties of a single crystal may not represent the bulk solid, or appropriately describe the bulk properties of interest for a given application . In thisniometer ,69,70. Grinding with a mortar and pestle can be used for PXRD sample preparation, but it cannot be used for SCXRD, which requires a clear crystal. Therefore, only solvent-evaporation products are commonly used for SCXRD analysis. However, when using solvent evaporation, some hydrates and solvates may be formed ,18,69. TAfter a multi-component structure was obtained, some testing was conducted to evaluate the solid characters and properties, such as solubility, stability, hygroscopicity, etc. Moreover, some pharmacological aspects were checked, including activity, potency, and toxicity, to ensure the drug’s safety and efficacy. Antibiotics have been reacted using various counterions/coformers to produce multi-component solid systems and have become the most developed compounds in multi-component solid form after NSAIDs. That fact is probably due to the high feasibility of their structure to react with other compounds. Secondly, there are several issues regarding the lack of physicochemical properties, such as solubility and stability, that need to be addressed. Fluoroquinolones are the second most studied group of antibiotics for multi-component solids after antituberculosis, as shown in 11–N12–H13, with the -OH of the carboxylic acid of the conformer; this improved the physicochemical characteristics of the active pharmaceutical ingredient [gredient .Additionally, from the cephalosporine group, cefixime was combined with nicotinamide to improve its solubility, dissolution, and stability . Next, cIsoniazid has been combined with some acid substituents  and plant metabolites . This group of acid–coformer agents exhibited better solubility and stability and lower hepatotoxicity than the parent drug and the second group, which was a combination of isoniazid with some base substituents. Afterward, the combinations of prothionamide and phloroglucinol produced a new solid phase , which wThe multi-component systems comprising isoniazid–acid substituents and natural resources were the following: isoniazid combined with cinnamic acid, fumaric acid ,82,85,86After the tuberculosis drugs, fluoroquinolones have become the most developed antibiotics, formed by non-covalent interactions with other components. Hereafter, the fluoroquinolone multi-component solid systems will be discussed in the following section, but we will still refer to the listing in Escherichia, Salmonella, Shigella, Enterobacter, Campylobacter, Neisseria, and Staphylococcus [Neisseria gonorrhea sp., which has been resistant to other antibiotics. Moreover, fluoroquinolones are also used to treat resistant Salmonella infections [Fluoroquinolones are highlighted in this review because they are currently the most used antibiotics in common infectious bacterial diseases. Various fluoroquinolone derivatives show high effectiveness in killing bacteria. Structurally, their origin compound is nalidixic acid, with fluorine (F) added to the quinolone site, which produces a strong antibacterial effect. Ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, lomefloxacin, moxifloxacin, pefloxacin, and sitafloxacin are the fluoroquinolones used clinically . Those alococcus . In addifections . However, this antibiotic group generally has poor and pH-dependent solubility, besides exhibiting hydrate transformation, and is unstable in light ,5. PhotoAnother report explains that levofloxacin protected from daylight remained stable in 0.9% NaCl, 5% dextrose, and Ringer’s solution ,7,26,30.2O) and then to a monohydrate (levofloxacin H2O). Inversely, in particular conditions, the hemihydrate could transform into anhydrous forms with various polymorphs , or an amorphous form [Next, fluoroquinolone antibiotics are reported to have some hydrate forms, i.e., ciprofloxacin, levofloxacin, and sitafloxacin hydrates. The anhydrous levofloxacin absorbs some water molecules quickly and changes to a hemihydrate  resulted in more stable levofloxacin. The antimicrobial potency test results showed the same activity as free-form levofloxacin . However17H18FN3O3) crystallizes with 2-thiobarbituric (H2tba) and barbituric acid (H2ba) in the aqueous solution to yield the salt CfH2(Htba)·3H2O, the salt cocrystal CfH2(Hba)(H2ba)·3H2O, and the salt CfH2(Hba)·H2O. These three salts were reported to have a more stable structure than the original compound [Another combination system is the three-salt multi-component solid, with various hydrate molecules inserted. Ciprofloxacin  ,70,108. Hereafter, next, we focus on fluoroquinolone–NSAID combinations for some reasons. First, it has been known that NSAIDs are essential substituents for clinical purposes, including in treating infectious diseases. Infected patients commonly use antibiotics to kill bacteria, and NSAIDs are used to overcome pain due to inflammation symptoms. Vice versa, inflammation also may trigger microbial infections. These drugs are available over the counter; therefore, NSAID usage is commonly not well-monitored, even though many adverse effects have been reported. Combining antibiotics with NSAIDs in a multi-component system will be better for increasing patient compliance ,30. The second supporting reason is structural; fluoroquinolones are amphoteric or tend to form bases ,7, and aOn the other hand, the salt reaction has limitations, because it cannot be applied to compounds that have difficulty forming ions. For example, an ibuprofen derivative, zaltoprofen, used to treat acute pains , is a noAs mentioned earlier, NSAIDs and antibiotics are frequently used and have been found to react non-covalently with other compounds, which can offer both advantages and challenges ,109. A rComparing drug–excipient combinations, not many multi-component dual drugs, including antibiotic–NSAID combinations, have been developed. This phenomenon is predicted, due to the complex factors affecting the feasibility of host–coformer interactions, safety, dose issue, and toxicity. Additionally, the data show that ciprofloxacin and levofloxacin are the most-developed fluoroquinolones, in the context of creating multi-component solids with NSAIDs. Ciprofloxacin is in the first rank, followed by levofloxacin because they are commonly used to treat broad infection cases and are easy to react with a counterion or coformer. Reports have widely explained that their main advantages comprise enhancing NSAID solubility and improving fluoroquinolone antibiotic stability ,31. For example, the ciprofloxacin-salicylic acid reaction produced various molar ratios of crystal, solvate, and hydrate forms of muli-component systems . A studyIn addition, as shown in Briefly, the multi-component system of fluoroquinolones and NSAIDs has the potential to improve stability and maintain antibiotic potency. In the future, improved potency may also be achieved. However, as a dual drug, multi-component solid, interactions between APIs, mainly NSAIDs, should be considered thoroughly ,115,116.Referring to other cases, apigenin, a bioflavonoid compound with anti-inflammatory activity against chronic aging diseases, has been combined with 4,4′-bipyridine, enhancing its efficacy and lowering toxicity . In addiAfter discussing the antibiotic-NSAID multi-component solid, the following sections will focus on toxicity and computational studies. These studies aim to provide insights into selecting the appropriate host–coformer/counterion, preparation methods, structural determination, physicochemical properties, activity, and safety measures. Every drug has certain side effects, including antibiotics and NSAIDs, which may affect the gastrointestinal tract, nervous systems, and elimination organs, such as the liver and kidneys. For example, fluoroquinolones kill microbes by the inhibition of bacterial topoisomerases II and IV, and resistance arises from these target enzymes’ mutations. In addition, they have rare adverse effects, namely tendinopathy and tendon rupture, peripheral neuropathy, and aortic aneurysm . FluoroqOn the other side, fluoroquinolones show many adverse effects on the gastrointestinal tract and the central nervous system, namely, phototoxicity and dermal toxicity, including allergenicity. Levofloxacin has favorable adverse reaction profiles compared to other fluoroquinolones. Among the reported cases of dermal toxicity, there are few reports of toxic epidermal necrosis (TEN) and stomatitis associated with levofloxacin usage. Stomatitis is characterized by pain, inflammation, and ulceration in the oral cavity . There are mild to moderate side effects of fluoroquinolones, as shown when treated with ciprofloxacin, levofloxacin, and sitafloxacin groups. However, reports also reveal the severe side effects of temafloxacin, trovafloxacin, grepafloxacin, and clinafloxacin. For example, temafloxacin causes immune hemolytic anemia, trovafloxacin causes hepatotoxicity, grepafloxacin causes cardiotoxicity, and clinafloxacin causes phototoxicity. Therefore, those compounds are no longer used clinically. Additionally, regulatory agencies such as the FDA have warned about the inappropriate use of fluoroquinolone antibiotics, due to their severe side effects, such as tendon rupture, neuropathy, or heart valve regurgitation. Although the molecular principles underlying these side effects are not yet fully understood, there is evidence that they inhibit human mitochondrial topoisomerase II . Mycobacterium tuberculose, including pre-XDR tuberculosis (TB) and MDR TB, which resist fluoroquinolones and are classically considered risk factors for treatment failure [S. aureus strains tested were MDR, showing resistance to fluoroquinolones [Pseudomonas auroginosa that causes antibiotic resistance [Another problem in antibiotic usage is bacterial resistance, which is divided into three categories, namely, multi-drug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) microbes. These categories illustrate the level of global complexity in the issue of antibiotic resistance and the many factors needed to be considered in solving it. Gram-negative bacteria are primarily XDR, followed by PDR and MDR. Recently, a study found resistant  failure . In thes failure . Also, minolones ,127,128.sistance .On the other hand, side effects and toxicity of NSAIDs have also been reported. Most commonly, severe GI adverse effects increase with NSAID medication, such as ulceration, bleeding, or perforation . The occAside from the singular NSAIDs, there are concurrent adverse effects related to other drug usage forms. Regarding their pharmacokinetics, NSAIDs may interact with other plasma protein-bound drugs and increase the free serum concentration of these medicines. In addition, NSAIDs decrease renal perfusion and inhibit the glucuronidation process. Therefore, their usage may increase the toxicity of drugs, in terms of renal clearance (such as lithium) or hepatic metabolism . FurtherChlorella sp. and Desmodesmus spinosus also was reported [The combination of fluoroquinolone with NSAIDs has been reported to increase the risk of seizure disorders, particularly in the central nervous system . Howeverreported . Anotherreported . The nanreported .Escherichia coli and Staphylococcus aureus. Additionally, NSAIDs were combined with kanamycin (KAN) and tetracycline (TET). ASA, DCF, and PXC reduced metabolic activity and culturability. Meanwhile, PXC reduced biofilm mass. Additive interactions were obtained for most of the combinations between NSAIDs and KAN or TET. Hence, A benefit of NSAIDs is the control of biofilms, as they are more effective than conventional antibiotics [Interestingly, a study reported that NSAIDs might increase the antibiotic’s strength, as shown by azithromycin–paracetamol co-crystal . Next, tibiotics . In addiibiotics , as demoibiotics .The exploration and investigation of toxicity and activity improvement by combining antibiotics and NSAIDs in a multi-component system is still a challenging task. However, the interactions between the two must be given attention, as they have shown similar adverse effects on the gastrointestinal tract based on available data. Therefore, for the next experiment, observing the possible pharmacokinetics and pharmacodynamic interactions and their impact on the neural, bloodstream, hepatic, and renal systems will be valuable for ensuring the safety and suitability of their multi-component solids. Furthermore, improvements in antibiotic activity and potency can be expected to be related to solubility and penetrability increases. As well as other synthetic drugs’ development, the relationship between structure and activity has been modeled in many ways, including by studying the activity and toxicity of antibiotic–NSAID multi-component solid systems, which should be confirmed by in vitro and in vivo testing. Computational simulations have become a popular and valuable technique for designing stable chain conformations and packing structures, predicting molecular interaction, etc. The computational approach in developing multi-component systems involves principles and specific calculations of the binding energy between multiple substituents. Molecular dynamics, mechanism, and quantum mechanics are some principles of multi-component solids’ design ,33,39. In line with experimental research, multi-component crystal building needs a suitable coformer, solvent, and preparation methods. Using a database, i.e., CSD –CCDC , and mathematical calculation, the thermodynamic pattern of the multi-component system’s interaction can be predicted . Based oMercury, Hyperchem, COSMO-RS, COSMOtherm software, etc. [We have selected several multi-component systems as examples that have been modeled computationally and can be used as a basis for the development of antibiotics-NSAIDs multi-component systems. It is important to note that using in silico modeling is a more environmentally friendly and efficient approach compared to traditional trial-and-error experiments. Firstly, some programs have been used for solvent and coformer screening, such as re, etc. .For example, in the HME process for ciprofloxacin–isonicotinic acid multi-component development, a design of experiment (DoE) was used to evaluate the factors that determined the yield, and researchers found three factors: the effect of temperature, the screw speed, and screw configuration . FurtherA feasibility study was conducted using thermodynamic modeling, regarding the non-covalent reaction process, using perturbed-chain statistical associating fluid theory (PC-SAFT). This method can predict pharmaceutical cocrystal behavior by calculating the single-crystal solubility of any solvent at any temperature. Based on that calculation, the cocrystal’s solubility in other solvents and different temperatures also can be predicted without additional measurements. Using this mathematical approach, the properties of (+)-mandelic acid/(-)-mandelic acid (1:1), caffeine/glutaric acid (1:1), and carbamazepine/nicotinamide (1:1) cocrystal systems were in excellent agreement with the experimental data . Developed in the early 1990s, the liquid phase thermodynamics theory of conductor-like screening model for real solvents (COSMO-RS) has been used in solubility prediction, solvent screening, excipient ranking, pKa prediction, redox potentials calculation, and partitioning coefficients’ determination . In detaAfterward, COSMO-RS was coupled with the COSMOtherm software to screen common solvents and co-formers for CL-20. The excess enthalpy and solvent cavity volume determined the solvate system’s arrangement. However, the COSMO method was challenging in predicting cocrystal formation, and it still needs entropy and solid-state interactions as the essential data. Meanwhile, solvent selection should maximize entropy. Calculating entropy is a critical factor in predicting cocrystal formation, and an accurate method is essential for screening cocrystal formation. This enables the design of an intelligent approach to multi-component crystal development . Additionally, the supercooling liquid phase can be determined by in silico calculation . A recenNext, an antibiotic, linezolid (LIN), was reported to produce many crystals named LIN_II, LIN_III, LIN:BA cocrystal, LIN:PHBA cocrystal hydrate, and LIN:2,6-DHBA cocrystal. Those crystal systems were obtained from some methods, including neat grinding, liquid-assisted grinding, and solvent evaporation, which fits with the in silico study. The virtual cocrystal-screening tools optimized the molecular complementarity, hydrogen bond propensity, and molecular electrostatic potential maps. However, the molecular electrostatic potential maps approach is closer to the experimental results than molecular complementarity and hydrogen bond propensity calculations. This work shows that the contribution of the total energy of the coformer crystal systems determines the cocrystal formation feasibility. Mercury 4.3.1 software provides data to analyze molecular complementarity . Another modeling application in multi-component research is the study of spatial charge descriptors, which can predict cocrystal formation using machine learning algorithms . Two modModeling software for designing multi-component systems is continuing to grow in number. Recently, Mswahili et al. developed a model to predict cocrystal formation by extracting the descriptor values from the simplified molecular-input line-entry system (SMILES) of compounds. They demonstrated that the calculated values of half of the selected descriptors using feature selection algorithms showed comparable results with experimental data from 1476 instances . FurtherFurthermore, new software prototypes have been developed to arrange multi-component systems by considering both thermodynamic and spatial aspects to fit the host compounds with their counterions/coformers. For example, a multi-component crystal may involve non-covalent bonds with low energy only, including hydrogen, van der Waals, and dipole–dipole bonds, until an ionic reaction, which forms relatively higher-energy salts Still, the space conformation and the environment also should be considered ,99,143.Modeling studies have played a significant role in the development of antibiotic multi-component crystals ,37,99. A2O3/BiFeO3, supported by modeling, showed photocatalytic degradation [3 catalyst [Another multi-component computational study was performed, combining antibiotic fluoroquinolone with metal by a dynamic and porin-mimetic approach. The metal–antibiotic system was predicted to improve solubility, which was then confirmed by the wet experiment . Furtherradation . In siliradation . Similarradation  and lamiradation  cocrystacatalyst . Finally, in the present, graphing a molecular structure three-dimensionally is usually assisted by SHELXT, and is refined using SHELXL software, combined with Mercury’s data and modeling ,149. AllBesides resistance cases, antibiotics’ main problem is their lack of physicochemical properties, such as low solubility and stability. Therefore, one of the fruitful approaches in antibiotic development is solid-state engineering, i.e., the formation of multi-component systems composed to improve the stability and solubility of antibiotics. This strategy is growing in popularity and is supported by solid instrument development, straightforward preparation methods, and computational modeling, making it more efficient and environmentally friendlier. The most developed antibiotics in the multi-component solids are the antituberculosis group (isoniazid and pyrazinamide) and fluoroquinolones (levofloxacin and ciprofloxacin). Multi-component solid systems have several synthons that interact structurally to improve solubility and stability. However, based on the reports, many antituberculosis multi-component systems only result in new crystal structures without affecting stability and solubility. Meanwhile, fluoroquinolones react with their counterion or coformer at their amine piperazine moiety, which protects them from oxidative degradation. Moreover, fluoroquinolones’ multi-component solid systems may also increase their potency by re-arranging the polarity, supporting its capability to pass through the cell membrane of bacteria after the solid rearrangement. In conclusion, the development of the fluoroquinolone-NSAID multi-component solid system holds great promise and presents a challenging opportunity. This combination has the potential to leverage the advantages of both components and may lead to increased patient compliance. In general, multi-component solids overcome the low solubility and stability issues of NSAIDs. Moreover, some experiments focusing on improving the stability and polarity of these components may push antibiotic potency polarity. However, we also must pay attention to their safety factors by studying the toxicity, which may be assessed simultaneously by computational and laboratory studies.


Many of intracellular processes depend on continuous ionic permeation, including exocytosis, contraction, metabolism, transcription, fecundation, proliferation, and apoptosis. These mechanisms are precisely regulated, for instance, through hormonal activity. The lipophilic nature of steroidal hormones allows their free transit into the cell where, in most cases, they occupy their cognate receptor to generate genomic actions. In the sense, estrogens can stimulate development, proliferation, migration, and survival of target cells, including in lung physiology. Non-genomic actions on the other hand do not imply estrogen’s intracellular receptor occupation, nor do they initiate transcription and are mostly immediate to the stimulus. Among estrogen’s non genomic responses regulation of calcium homeostasis and contraction and relaxation processes play paramount roles in ASM. On the other hand, disruption of calcium homeostasis has been closely associated with some ASM pathological mechanism. Thus, this paper intends to summarize the effects of estrogen on ionic handling proteins in ASM. The considerable diversity, range and power of estrogens regulates ionic homeostasis through genomic and non-genomic mechanisms.To preserve ionic homeostasis (primarily Ca Homeostasis is a state of cellular equilibrium maintained through regulatory mechanisms resistant to change that preserve the constant physiological conditions. The composition of the cytosol, the optimal internal cellular environment, differs significantly from that of the extracellular space, and these spaces are separated by a semipermeable lipid bilayer called cellular membrane that functions not only as a barrier between these two spaces but also prevents charged particles from crossing freely from one side to the other. Several cellular functions, as for instance the action potential in the nerves, depend on ions permeating through the cellular membrane. Conceivably, the ionic composition on either side of the cell membrane is different and many cellular mechanisms work in accordance to maintain a constant concentration of them and consequently, an electrochemical gradient ,2. In orProtein ion channels allow specific ions to flow across the cell membrane in favor of their electrochemical gradient, consequently generating transmembrane electric currents . Ion chaOn the other hand, ion pumps do require a source of energy achieved through the hydrolysis of ATP, to move the ions against their electrochemical gradient; this process is referred to as active transport . Pumps aThe third type of transporters are exchangers. They transfer different ions in or out of the cytosol simultaneously, they do not require ATP as an energy source, and instead exploit the electrochemical gradient by moving the ions downhill of the concentration gradient. This type of transport is also known as facilitated transport. Three types of exchangers have been identified. Uniporters transport a single type of ion in one direction in favor of the concentration gradient. The second type are symporters, where two different types of ions are moved simultaneously across the membrane in the same direction. The last type are called antiporters, proteic structures apt to move different types of ions across the membrane in opposite directions  3,5].,5.3,5].To maintain ionic homeostasis and provide a stable electrophysiological status in cells, these proteins are strictly regulated by numerous pathways, both extrinsic  and intrinsic cellular mechanisms and the electrochemical gradients . Ion hanIn this sense, hormones have been recognized as one of the main homeostatic regulatory systems, and in recent years sex steroid hormones (SSHs) gained increasing interest for their role outside of reproductive physiology. SSHs have been implicated in regulating many critical physiological processes along every stage of life, including growth, regulation of homeostasis, reproduction, and aging ,8. SpeciHere, we review emerging evidence suggesting estrogens influence airway smooth muscle function at the cellular level, modifying processes involved in intracellular ion homeostasis on different physiological and pathological conditions.Estrogens belong to the family of SSHs derived from cholesterol, and they share a common structural base consisting of four rings called cyclopentanoperhydrophenanthrene ,8,12. InDuring the reproductive lifetime of women, three main estrogens are physiologically synthesized: estrone (E1), 17β-estradiol (E2), and estriol (E3); E2 has the highest production representing the most abundant and potent estrogen during a woman’s reproductive lifetime. In non-pregnant women, physiological circulating levels ranging from 80 pM–1.5 nM and going up to 150 nM during pregnancy ,12. In pEstrogens exert regulatory actions via long-term genomic and acute non-genomic actions ,19. Clas2+) homeostasis is maintained by a finely tuned Ca2+ signaling system made-up of numerous Ca2+ transporters  regulating the influx and efflux of this cation from the cytoplasm to preserve its balance. Ca2+ homeostasis is essential for the cell. As a second messenger, Ca2+ signaling regulates various cellular processes that depend on the time of the Ca2+ and concentration. It is well known that Ca2+ regulates exocytosis, contraction, protein phosphorylation, dephosphorylation, metabolism, gene transcription, fecundation, cell proliferation, and even apoptosis [2+ homeostasis keeps intracellular basal Ca2+ concentrations (b[Ca2+]i) at around 100–150 nM, while Ca2+ concentrations in the intracellular stores and extracellular space are higher  creating a large chemical gradient in favor of Ca2+ influx into the cytosol [2+]i, numerous proteins exists to facilitate the cellular influx and efflux of Ca2+. Among the calcium-handling proteins, we can include the voltage-dependent Ca2+ channels (VDCCs), store-operated Ca2+ channels (SOCCs), receptor-operated Ca2+ channel (ROCCs), transient receptor potential channels (TRPs), and the Na+/Ca2+ exchanger in its reverse form (NCXREV) as influx mechanisms located in the cellular membrane. On the other hand, the Na+/Ca2+ Exchanger (NCX) and the plasma membrane Ca2+ ATPase (PMCA) are efflux mechanisms located in the cellular membrane. Meanwhile, the sarcoplasmic reticulum (SR) functions as the main internal Ca2+ store in the ASM. On the SR membrane, we can find the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptors (RyR) as Ca2+ efflux mechanisms. In contrast, the sarcoplasmic reticulum Ca2+ ATPase (SERCA) acts as an intracellular Ca2+ extrusion mechanism  h2+ entry is primordial in Ca2+ signaling, and its influx is primarily driven by an electrochemical gradient. In ASM, both L- type VDCCs (Long Lasting Currents voltage-dependent Ca2+ channels) and T-type VDCCs (Transient Currents voltage-dependent Ca2+ channels) are expressed, but L-VDCC is the predominant type in human ASM and various other species [2+]i. This phenomenon seems to depend on the type of receptor activated. An ERα-specific agonist -THC) augmented the [Ca2+]i response induced by histamine in both asthmatic and non-asthmatic hASM cells. Meanwhile, an ERβ-specific agonist (DPN), decreased the [Ca2+]i response induced by histamine in asthmatic and non-asthmatic hASM cells. The effect observed on the [Ca2+]i response with activation of ERβ signaling appears to implicate the inhibition of L-VDCC . When SARAF interacts with STIM1, it prevents spontaneous activation or the interaction between STIM1-Orai1 i. E2, through Orai1, positively regulated cellular and migration , and suppressed cellular proliferation apoptosis through caspase3 expression regulation i. The membrane-bound protein CD38 synthesizes or degrades cADPR, which functions as a ligand for the protein FKBP-12.6 (12.6 kDa FK506-binding protein). In turn, FKBP112.6 binds to the RyR, providing stabilization and reducing the opening probability of the channel. When cADPR binds to the regulatory protein, this causes a conformational change in the RyR, activating Ca2+ release to the cytosol [2+]i concentration is increased further after Gq protein coupled receptor (GPCR) activation i of 100 nM. This effect caused by the addition of ERβ1 could indicate a synergic interaction with Ca2+ and RyR that increases the open probability of the channel and could potentiate RyR activity of Ca2+-induced Ca2+-release i via the PLCβ-IP3 pathway [2+]i through an ER interaction with type 1a metabotropic glutamate receptors (mGluR1a), activating the PLCβ-IP3 pathway in female rat astrocytes [3R1 expression is suppressed after 48h exposure to E2 (10 nM) in human g-292 osteosarcoma cells and rat osteoblasts [2+]i in response to TAS2R agonists i increase and contraction induced by carbachol (CCh 100 µM) ,,2+]i buf 100 µM) . The Ca2KB-R7943 . rev also has an important function during oscillatory contractions in mouse ASM induced by potassium channel blockade with tetraethylammonium chloride (TEA). In this experiment, two pattern changes of [Ca2+]i were induced. One was a high-frequency oscillation, and the other a low-frequency rhythmic oscillation. Both types of Ca2+ changes participate in triggering ASM contraction, and they might participate in other physiological processes in the ASM. Remarkably, these oscillations augment [Ca2+]i, an increase that activates NCX, initiating the relaxation phase by extruding Ca2+ , an, an20], d by ERs . Contrard by ERs ,99. Liked by ERs ,100. Mord by ERs .2+]i in male mice but not in female mice. Male mice cardiomyocytes, when exposed to E2 (nanomolar range), decreased [Ca2+]i post-MI in a concentration-dependent manner [2+]i overload, indicating the protective role of E2 to compensate for the greater activity of NCX i overload. These findings suggest a protective role of estrogen over the NCX activity during ischemic/reperfusion injury i at lower levels, preventing the activation of Ca2+-dependent apoptosis. These mechanisms could be especially useful to counteract the cytotoxicity induced by glutamate through NMDA or AMPA receptor activation i to basal levels after increases induced by agonist stimulation, Ca2+ can be pumped across the membrane and out of the cell against its electrochemical gradient, expending ATP during the process [2+]i and favor AHR i and restoring depleted Ca2+ internal stores [2+]i, a phenomenon that has been linked to an asthmatic ASM phenotype that contributes to airway remodeling and hyperresponsiveness i decay in the response induced by histamine was higher in the group treated with TNF-α or IL-13. This response was reversed by a treatment with WAY but not by E2 or PPT , voltage-activated potassium channels (Kv), and ATP-regulated potassium channels (KATP), although the latter channels have been identified in ASM cells, they do not seem to play a significant role in regulating airway contraction or relaxation ..+ electr2+-activated K+ channels (KCa) are constituted by three families: the large-conductance Ca2+-activated K+ channels (BKCa), the intermediate conductance (IKCa), and the small conductance (SKCa). BKCa have the highest importance in ASM cells ..2+-activ2-adrenergic agonists are the main therapeutic option for bronchodilation (in asthma and chronic obstructive pulmonary disease [COPD]), and BKCa channel activation is one of the key mechanisms of action of these drugs . D. D+ moletraction ,156,157. and NCX ,120. TheThe effects of E2 on NKA activity has not been explored in ASM cells. In H9C2 rat cardiomyocytes, E2 enhanced the expression of NKA in a concentration-dependent manner ,159. In +/K+/Cl− cotransporter (NKCC) facilitates the movement of Na+, K+, and Cl− ions across the membrane in an electrically neutral way  contraction in a gender-specific manner. In female rats, inhibition of NKCC1 did not modify the maximal contraction with PE, while OVX increased NKCC1 activity and E2 treatment blunted this effect . The mod2+]i is necessary, although it is not the only contributor to ASM contraction regulation. In the extracellular space, Na+ concentration is the highest (140 mM), while the intracellular concentration is normally much lower, about 4–16 mM. This electrochemical gradient is usually utilized by excitable cells to generate action potentials and to facilitate the transport of energetically unfavorable solutes coupled to Na+ i) activates the NCXREV, increasing [Ca2+]i  are some of the first channels to consider. In ASM cells, VGSCs have already been identified to be functionally expressed in humans and rabbits, including the isoforms Nav1.2, 1.5, and 1.7 ,174,175. and 1.7 ,175. Add and 1.7 . Further [Ca2+]i  i increase induced by LVDCCs currents activate CACCs, and Cl− ions participate in the contractile phase by favoring membrane depolarization and enhancing the contraction by further activating LVDCCs i by activation of LVDCCs or Ca2+ release from the internal stores . C. C− chanectively . The GAB subunit . In hASM subunit , and selh muscle ,211. Whe WT mice . The selA receptor in ASM cells, some effects have been observed in other tissues. In female rat brains, E2 treatment after OVX showed a significant increase of the α2 and the γ1 subunits only in regions that also expressed ERs, suggesting that the effect depends on the ER signaling [A receptor in the OVX vs. OVX + E2 treated groups [A receptor expression is also relevant in the developmental stages. In neonatal female rats, β-estradiol 3-benzoate (EB) treatment upregulated the expression of extrasynaptic α4/δ subunits but decreased the expression of synaptic α1/α4/γ2 subunits [Although no reports have been made concerning the effect of estrogens on the modulation of the GABAignaling . The effignaling . Throughignaling . Conversd groups . E2 regusubunits .In summary, a wide range of evidence from different cell types and animal models indicate that estrogens have a promiscuous nature, being able to interact with a variety of regulatory proteins essential in the homeostasis of intracellular ions. The estrogenic effects are not clearly cut but are instead subject to the conditions and variables that surround them. The effects of estrogens can vary greatly depending on the concentration, time of exposure, type of estrogen, requirement or independence of ER signaling, and even cell type. In this context, the effects of estrogens in some proteins such as IP3 receptors, PMCA, SERCA, NKA, NKCC, VGSCs, and CACCs on ASM cells have not been explored in-depth. There is not a single regulatory protein in which at least one study of E2 modulation is reported. Thus, the necessity still exists to better understand the possible effects that estrogens could have in physiological and pathological conditions in ASM cells. In this sense, it seems that single-cell genomics studies and spatial transcriptomics analysis may be quite useful in the following years to unravel the punctual functions of estrogens, particularly on the surrounding tissues where they are synthesized.