New liquid oral formulations of hydroxychloroquine: a physicochemical stability study

Introduction

Hydroxychloroquine (HCQ) is an old molecule from the class of 4-aminoquinolines. It was originally developed as an antimalarial drug [1]. Many additional drug properties of HCQ increase its therapeutic use. There are, indeed, different research pathways in numerous autoimmune, inflammatory, and infectious diseases, as well as in cancerology, with variable outcomes depending on the disease [1], [2], [3]. It is a weak base that interacts with phagocytic functions by raising the intracellular pH, leading to an alteration in the selective autoantigens presentation. HCQ inhibits the production and the release of cytokines by T cells: IL-1, IL-2, IL-6, IL-18, TNF-α, and IFN-γ. These effects are achieved by inhibiting the activation of the toll-like receptor involved in innate immunity and autoimmune diseases [1], [2], [3]. HCQ is associated with multiple metabolic benefits. It improves lipid profiles and lowers blood glucose levels [1], [2], [3]. It also decreases platelet aggregation. Furthermore, HCQ is a chloroquine derivate that accumulates in lysosomes, which leads to neutralization of the lysosome and inhibition of autophagy [2], [4], [5]. In light of these data, in clinical practice HCQ is recommended for the management of systemic lupus erythematosus [5], [6], [7], [8], [9] and rheumatoid arthritis [10], [11]. Numerous studies report its efficacy more or less established in other diseases such as scleroderma [12], sarcoidosis [13], Gougerot–Sjorgren’s syndromes [14], antiphospholipid syndrome [15], disseminated interstitial lung disease [16], idiopathic thrombocytopenic purpura [17], preeclampsia, repeated spontaneous abortions [18] and diabetes [19]. HCQ is also evaluated in infectious diseases such as Q fever and Whipple’s disease [20], [21]. The combination of HCQ with chemotherapies is being tested in oncology, particularly as a strategy for modulating autophagy in order to reduce chemoresistance [22], [23].

HCQ pharmacokinetics properties are characterized by a good oral bioavailability of about 75%, a good diffusion into tissues with very important volumes of distribution (up to 44,000 L) with long mean residence time (1,300 h), and a long plasma elimination half-life of approximately 40 days with a high interindividual variation [1], [3]. HCQ is mainly eliminated by the kidneys (40–60%), with a very long tissue half-life. There is no evident therapeutic concentration threshold. For example, on the suppressive treatment of malaria, suggested plasma or serum concentrations should be up to10 ng/mL, while on systemic lupus erythematosus, target concentrations should be above or equal to 500 ng/mL [24].

Because of this variability, pharmacological monitoring is recommended in order to obtain effective plasma concentrations and to prevent possible overdosage which may contribute to the occurrence of adverse events, in particular ocular events [1], [2], [3]. HCQ reaches its steady-state concentration by about 6 months of therapy [1].

Recommended adult doses range from 100 to 600 mg per day. In pediatrics, the doses for the different diseases vary from 2 to 13 mg/kg/day divided into 1 or 2 doses according to the indications [25], [26].

In all countries where HCQ is currently marketed, the drug is available as HCQ sulfate in film-coated or coated tablets for oral use. No other pharmaceutical form is currently available on the market. For pediatric therapies, HCQ tablets can be crushed but still have an off-flavor bitter taste [27]. In fact, tablets are not indicated for children younger than 6 years, and old persons are sometimes not able to swallow solid pharmaceutical forms. HCQ is a compound belonging to class 1 of the Biopharmaceutics Classification System (BCS) proposed by L.G Amidon in 1995 to predict the bioavailability of oral drugs [28]. According to BCS, class 1, is the most favorable situation corresponding to high permeability and solubility drugs. Thus, modification of HCQ oral formulation is unlikely to change its bioavailability [29]. In light of the various points discussed, the formulation of an oral liquid pharmaceutical form of HCQ and its stability evaluation is essential. This must ensure the safety of HCQ administration in adult or pediatric patients who cannot swallow the tablets. An oral liquid solution with a concentration of 50 mg/mL allows covering both adult and pediatric dosages with adapted volumes. The availability of an oral liquid form would also allow adjustment of doses in light of drug monitoring, which is necessary considering the interindividual variation of plasmatic dosage of HCQ and to prevent some adverse events.

As explained above oral liquid forms are not commercially available, but HCQ stability in Oral Mix^® and Oral Mix SF^® bases, at a concentration of 25 mg/mL is already reported [29]. Oral Mix^® and Oral Mix SF^® are suspending bases products of Medisca Pharmaceutique Inc, mainly available in North America, while in Europe, ORA^® and Syrspend^® products, for instance, are available. All of these products’ composition and physicochemical properties are summarised in Table 1.

As compared to Syrspend^® SF PH4, ORA^® and Oral^® ranges are more similar in term of qualitative composition, but they differ from their antifoaming agent: dimethicone for ORA^® and simethicone for Oral^®. Also, ORA-Plus^® contains calcium sulfate and trisodium phosphate as buffering agents, while Oral Mix^® contains sodium citrate. They also differ from their osmolality, 3,200 mOsm/kg for ORA-Sweet^® while Oral Mix^® osmolarity is 1,231 mOsm/kg. Furthermore, no data concerning the quantitative composition of these bases are available.

For all these reasons, extrapolating HCQ stability from Oral^® Mix [31] to ORA^® is, according to us, not possible. Thus, there was a need to study the stability of HCQ in alternative ready-to-use bases. We here propose stability studies in two different bases at a concentration of 50 mg/mL of HCQ: 50/50 mix of ORA-Plus^® – ORA-Sweet^®, and in liquid Syrspend^® SF PH4, with the idea to offer maximum solutions to pharmacists for compounding preparations.

Material and methods

Results

Assay chromatographic characteristics

The retention time of HCQ was found to be 6.20 min (Figure 1).

Figure 1:

Chromatogram of HCQ reference solution in water at 0.5 mg/mL.

The three standard curves were very similar and the mean equation of the relationship between HCQ concentration (y) and peak area (x) was: y=0.00006x + 5.4875. The correlation coefficient (r²) was found greater than 0.999. The accuracy of the analytical method was 4.87, 3.41 and 3.59% respectively for 0.3, 0.45 and 0.625 mg/mL concentrations. The repeatability of the method was systematically inferior to 5% (2.25, 0.9 and 0.49% for the day 1, 2 and 3 respectively) and the intermediate precision was evaluated to be 1.21%. Limit of quantification determination is not necessary in the case of a stability study whose objective is to quantify near 100% of HCQ concentration.

Degradations were observed just for three conditions (alkaline, oxidation, and light) with approximatively 10–30% of HCQ degradation, and the apparition of degradation products on chromatogram (Figure 2). Conversely, we observed no modification of chromatogram for acid or heat stress degradation conditions.

Figure 2:

Examples of the chromatogram of HCQ standard solution after degradation in different conditions: oxidation condition (H₂O₂ 30 V, 24 h) (A) and alkaline stress condition (NaOH 1 M, 3 h) (B).

For forced degradation of oral suspension vehicles alone, we can see for ORA-Plus^®/ORA-Sweet^® and Syrspend^® SF PH4 the chromatogram in Figure 3. These results demonstrated the degradation product peaks are separated from the HCQ peak.

Figure 3:

Chromatograms of fresh HCQ suspensions on ORA-Plus/ORA-Sweet^® (A), and Syrspend^® SF PH4 (B). Chromatograms of HCQ suspension on ORA-Plus/ORA-Sweet^® (C, E) and Syrspend^® SF PH4 (D, F), after stress degradation alkaline (NaOH, 1 M) associate with heat (80 °C) respectively after 1 h (C, D) and 4 h (E, F).

Relative HCQ concentrations were measured and presented in Table 2. The diminution of the area under the curve of HCQ peak with the progressive appearance of degradation products was observed.

Table 2:

Relative concentrations of HCQ (%) in each oral vehicle suspension after NaOH (1 M) and Heat (80 °C) stress condition during 1 or 4 h(%).

	HCQ suspension ORA-Plus®/ORA-Sweet® NaOH (1 M)/80 °C		HCQ suspension Syrspend® SF PH4 NaOH (1 M)/80 °C
	1 h	4 h	1 h	4 h
Sample 1	88.3	44.68	89.6	53.6
Sample 2	92.78	47.06	87.6	66.9
Sample 3	95.04	47.70	81.4	74.2
Mean	92.0	46.5	86.2	64.9
Standard deviation	3.4	1.6	4.3	10.5

The results of sampling preparation to check the matrix effect after the different steps (US, centrifugation, dilution) are presented in Table 3. The difference between the reference solution and each HCQ suspension is less than 2%. Thus, there is no influence of oral vehicle suspension.

Table 3:

Area under the curve (AUC) of different samples after preparation before analytical assays for each hydroxychloroquine suspension in comparison with the reference solution.

	HCQ 0.5 mg/mL ORA-Plus/ORA-Sweet®	HCQ 0.5 mg/mL Syrspend® SF PH4	Reference solution HCQ 0.5 mg/mL
Sample 1	8.09 10E6	8.16 10E6	8.38 10E6
Sample 2	8.17 10E6	8.20 10E6	8.15 10E6
Sample 3	8.25 10E6	8.30 10E6	8.28 10E6
Mean	8.17 10E6	8.22 10E6	8.27 10E6
Standard deviation	8.23 10E5	4.75 10E5	1.16 10E5
CV, %	1.2	0.6	0.1

The comparison between all standard curves, either prepared with HCQ solution or HCQ suspension (Ora-Plus/Ora-sweet and Syrspend SF pH 4), shows that equation curves are very similar. Respectively, y = 6.20E-08x+0.01 or y = 5.86 E−8x+0.01 and y = 5.91E-08x+0.01. These results are confirmed with the graphical representation because standard curves are totally overlaid (Figure 4). Thus, in this case, it was considered not necessary to perform specific statistical analysis. These results add supplementary proof for the absence of the matrix effect of oral suspension vehicles. For the realization of working standard solutions for method validation, it is thus possible to avoid using an oral suspension vehicle.

Figure 4:

Comparison of HCQ standard curve with and without oral vehicle suspension. (square: HCQ solution, without oral vehicle suspension; round: HCQ suspension, with oral vehicle suspension)

Suspensions stability

The pH remained stable at about 4.2 for all formulations during 90 days (Table 4). A slight alkalinization (<to 0.1 pH unit) of suspensions was observed for each tested condition. This result is in the range of the pH meter precision.

Table 4:

Mean of pH and osmolality of eight measures during 90 days of each HCQ suspension in different storage conditions.

		pH		Osmolality
		Mean ± SD	CV, %	Mean ± SD, mOsm/kg	CV, %
HCQ 50 mg/mL ORA-Plus/ORA-Sweet®	5 ± 3 °C	4.24 ± 0,03	0.76	2,796 ± 110.6	3.96
	30 ± 2 °C	4.29 ± 0,04	0.88	2,826 ± 150.7	5.33
HCQ 50 mg/mL Syrspend® SF PH4	5 ± 3 °C	4.23 ± 0,04	0.89	184 ± 10.7	5.84
	30 ± 2 °C	4.27 ± 0,06	1.34	195 ± 13.5	6.92

Osmolality remained stable during 90 days, at about 2,800 mOsm/kg for 50 mg/mL ORA suspensions and 190 mOsm/kg for 50 mg/mL SYR suspensions (Table 4).

No modification of the visual aspect of all HCQ suspensions was observed during the stability study: the cloudy suspensions remained white without crystallization after 90 days whatever the storage conditions. Slight reversible sedimentation occurred, but suspensions easily resuspended when shaking the flask. SYR suspension kept its cherry odor while ORA suspension gave a faint sweet smell.

As shown in Figure 5, after 90 days, the 50 mg/mL HCQ suspension in OraSweet^®/Ora Plus^® remained stable for both storage conditions (5 and 30 °C).

Figure 5:

Representation of relative HCQ concentration during 90 days of stability study of HCQ suspension (50 mg/mL), in two different oral vehicles suspension ORA (OraSweet^®/OraPlus^®) and SYR (Syrspend^®SF PH4), at both ICH conditions 5 and 30 °C. (Triangle: ORA HCQ at 30 °C; cross: ORA HCQ at 5 °C; square: HCQ SYR at 5 °C; diamond: HCQ SYR at 30 °C).

For HCQ suspension in Syrspend^® SF PH 4, stability is only approved at 5 °C during 90 days, indeed at 30 °C at 90 days concentration measured reaches 112% of initial relative concentration.

Discussion

The HPLC stability-indicating method with forced degradation has demonstrated its suitability for testing the stability of HCQ suspensions in OraSweet^®/Ora Plus^® and Syrspend^® SF PH 4. Indeed, to be able to distinguish degradation products from HCQ and oral suspension vehicles, we have added to the forced degradation of HCQ a forced degradation of HCQ in oral suspension vehicles. Because oral vehicles are certified stable for a long time (until one year) we choose just one extreme condition, and we applied simultaneously alkaline (NaOH 1 M) and heat (80 °C) stress conditions during two exposition times i.e. one or 4 h. In these conditions, no degradation was observed for both oral suspensions.

All chromatographic parameters meet the acceptance criteria for the HCQ assay. In contrast to the study of Singh et al. [33], it was decided to performed reversed-phase HPLC with gradient method instead of an isocratic method to allow elution in short runtime of impurities or probable degradations products like HCQ-O-Acetate (HCA), HCQ-O-Sulphate (HCS), or dichloroquinoline (DCQ) as cited by Dongala et al. [37]. Indeed, gradually increasing organic mobile phase proportion permits to elute these impurities, which are more apolar. Concerning the degradation products, Saini et al. performed a specific study on HCQ degradation product in ICH conditions. We obtained similar results. HCQ is susceptible only to alkaline, photolysis, and oxidative conditions. To avoid this reaction of photodegradation, the use of amber bottles is justified.

Among the parameters monitored in this study, osmolality remained stable for 90 days whatever the formulations and storage conditions. A strong difference exists between ORA and SYR osmolality, ORA being much more hyperosmolar than SYR. To avoid side effects like bloating, diarrhea, a hypo osmolar oral vehicle suspension will often be preferred.

Whatever the chosen oral base, the presence of carboxymethyl cellulose sodium or sodium citrate, coupled with a sweetener and flavor in ORA-Plus^® or Syrspend^® SF PH4 is attempted to reduce the bitter taste of HCQ [30].

Finally, the obtained results allow an optimal organization of the compounding process due to the extended stability of the formulation. Indeed, except for the final point of HCQ suspension in Syrspend SF PH4 at 30 °C, all other assay values were within 10% of the initial concentration without detectable signs of decomposition after 90 days.

Concerning the last point at 90 days for HCQ suspension in Syrspend SF PH 4^® at 30 °C, we suspect that this result is due to slight evaporation. Indeed, for Syrspend SF PH4^®, better preservation was observed at the refrigerated condition. We hypothesize that this result is due to solvent loss because of evaporation which was more important at ambient temperature. We do not think HCQ degrades in this condition, especially since (1) HCQ is really hard to degrade (as seen in Table 2) and (2) no degradation products appeared at this time of analysis.

The formulations of HCQ suspensions at 50 mg/mL are stable for at least 90 days at 4 °C. At 30 °C, these HCQ suspensions are stable, for 90 and 60 days respectively, in ORA and SYR. These stability data are comparable to the results obtained by Henry et al. [31], although the study was performed in different oral suspension vehicles.

This study constitutes the first step of the development of oral suspensions of HCQ. It could be improved by a microbiological stability study, as recommended by the GERPAC guidelines [38].

Conclusion

We propose two novel easy-to-handle formulations of HCQ in oral suspensions. The suspension in the ORA vehicle remained stable for 90 days at 4 and 30 °C. For HCQ suspension in SYR, it remained stable for 90 days at 4 °C and 60 days at 30 °C. For all preparations, no significant physical or chemical modification was noticed during the period of the study.