Evaluation of in vitro SARS-CoV-2 inactivation by a new quaternary ammonium compound: Bromiphen bromide.

The pneumonia (COVID-19) outbreak caused by the novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which unpredictably exploded in late December of 2019 has stressed the importance of being able to control potential pathogens with the aim of limiting their spread. Although vaccines are well known as a powerful tool for ensuring public health and controlling the pandemic, disinfection and hygiene habits remain crucial to prevent infection from spreading and maintain the barrier, especially when the microorganism can persist and survive on textiles, surfaces, and medical devices. During the coronavirus disease pandemic, around half of the disinfectants authorized by the US Environmental Protection Agency contained quaternary ammonium compounds (QACs); their effectiveness had not been proven. Herein, the in vitro SARS-CoV-2 inactivation by p-bromodomiphen bromide, namely bromiphen (BRO), a new, potent, and fast-acting QAC is reported. This study demonstrates that BRO, with a dose as low as 0.02%, can completely inhibit SARS-CoV-2 replication in just 30 s. Its virucidal activity was 10- and 100-fold more robust compared to other commercially available QACs, namely domiphen bromide and benzalkonium chloride. The critical micellar concentration and the molecular lipophilicity potential surface area support the relevance of the lipophilic nature of these molecules for their activity.

Micelles-in-Liposome Systems Obtained by Proliposomal Approach for Cannabidiol Delivery: Structural Features and Skin Penetration.

Deformable liposomes represent valuable drug carriers for cutaneous administration. Nevertheless, the fluid lipid membrane can favor the drug leakage during storage. Proliposomes may represent a suitable strategy to solve this issue. As an alternative, a novel carrier, which encloses hydrophobic drugs in the inner core of vesicles, namely, a drug-in-micelles-in-liposome system (DiMiL), has been proposed. In this work, we investigated the possible advantages of combining these two approaches to obtain a formulation able to enhance the skin penetration of cannabidiol (CBD). Proliposomes were prepared by spray-drying or slurry method testing lactose, sucrose, and trehalose as carriers at different sugar/lipid weight ratios. The ratio between soy-phosphatidylcholine (main lipid) and Tween 80 was instead fixed at 85:15 w/w. DiMiL systems were extemporaneously obtained by the hydration of proliposomes with a Kolliphor HS 15 micellar dispersion (containing CBD, when appropriate). Based on the technological properties, sucrose and trehalose at 2:1 sugar/lipid ratio resulted in the best carriers for spray-dried and "slurried" proliposomes, respectively. Cryo-EM images clearly showed the presence of micelles in the aqueous core of lipid vesicles and the presence of sugars did not alter the structural organization of DiMiL systems, as demonstrated by SAXS analyses. All formulations were highly deformable and able to control CBD release regardless of the presence of sugar. The permeation through human epidermis of CBD carried by DiMiL systems was significantly improved compared to that obtained loading the drug in conventional deformable liposomes with the same lipid composition or in an oil solution. Furthermore, the presence of trehalose led to a further slight increase of the flux. Altogether, these results demonstrated that proliposomes may be a valuable intermediate for the preparation of deformable liposome-based cutaneous dosage forms, improving the stability without compromising the overall performances.

Extraction Method and Analysis of Cannabinoids in Cannabis Olive Oil Preparations.

Recently, an increasing number of pharmacists had to supply medicinal products based on Cannabis sativa L. (Cannabaceae), prescribed by physicians to individual patients. Cannabis olive oil preparation is the first choice as a concentrated extract of cannabinoids, even though standardized operative conditions for obtaining it are still not available. In this work, the impact of temperature and extraction time on the concentration of active principles was studied to harmonize the different compounding methods, optimize the extraction process, and reduce the variability among preparations. Moreover, starting from the cannabis inflorescence, the effect of temperature on tetrahydrocannabinolic acid decarboxylation was evaluated. For the analysis, a GC/MS method, as suggested by the Italian Ministry of Health, and a GC/flame ionization detection method were developed, validated, and compared.

Investigation of the Effect of Different Emulsifiers on the Transdermal Delivery of EGCG Entrapped in a Polymeric Micelle System.

Epigallocatechin gallate, one of the most active antioxidant compounds, has a low chemical stability and ability to permeate the human epidermis. The encapsulation in polymeric micelles would be beneficial to improve both stability and permeation of epigallocatechin gallate and, therefore, to facilitate the pharmacological effects. Polymeric micelles containing epigallocatechin gallate were incorporated in O/W emulsions prepared by using different types of emulsifying systems. All emulsions were uniform in colour and aspect, without evidences of phase separation after centrifugation at the preparation time and over a 6-month period of storage at room temperature. Emulsions containing epigallocatechin gallate incorporated in polymeric micelles showed a colour variation, probably due to epigallocatechin gallate degradation, over the stability period. The skin permeability study evidenced a significant increase in epigallocatechin gallate permeation after encapsulation in micelles. Pure epigallocatechin gallate was not able to permeate the skin and only limited amounts were retained in the epidermis, while both permeated and retained amounts after 24 h were measured in the case of polymeric micelles containing epigallocatechin gallate. Moreover, the epigallocatechin gallate release and human skin permeability were affected by the type of emulsifier. The epigallocatechin gallate release in the presence of an emulsifier system based on cereal and fruit fibres never occurred. The best results in terms of release and skin permeability were obtained using glycerides of synthetic or semisynthetic origin or esters.

In vitro method to evaluate the barrier properties of medical devices for cutaneous use.

Barrier creams (BC) are marketed as cosmetic products or locally-applied medical devices to protect skin against damages induced by chemical agents or physical insults. However, the determination of the BC effectiveness is still a matter of discussion at both the clinical and the regulatory level. In this context, this work aimed at the development of a reliable, reproducible and easy-to-perform experimental protocol for the evaluation of BC performances. Preliminarily, an in vivo method based on the measurement of trans-epidermal water loss had been matter of investigation and was discarded: it required too much time and was not robust and sensitive enough. In vitro, reduction of the permeation of caffeine (used as a model of irritant), through an epidermal membrane mounted on a Franz cell or through a reconstructed 3D human epidermis model, was evaluated. Six BC among oil in water (O/W) or water in oil (W/O) creams were investigated with respect to the petrolatum, which is an efficient impermeable barrier against hydrophilic molecules. Despite minor differences, both methods could rate the effectiveness of the tested products in preventing caffeine exposure. Both methods enable to evaluate and quantify the BC effectiveness in a simple and fast manner. Their application may help regulatory agencies to prevent the marketing of ineffective products for the benefit of consumers.

Topical Treatment of Infantile Haemangiomas: A Comparative Study on the Selection of a Semi-Solid Vehicle.

BACKGROUND/AIM: Topical ß-blockers have recently been proposed as a valid alternative to oral drugs for treating cutaneous infantile haemangiomas, but clinical results in the literature are inconsistent due to the empirical choice of topical preparations. The current investigation aimed to rationalize the selection of a semi-solid vehicle for a locally applied drug product containing 1% w/w propranolol hydrochloride (PR-Cl). METHODS: A hydrophobic ointment of PR-Cl, two lipophilic creams, and a hydrophilic cream were prepared. In vitro release and skin permeation studies through human epidermis and full-thickness skin were performed by Franz diffusion cells. RESULTS: The overall results highlighted that PR-Cl was able to permeate the human epidermis, and its penetration pattern was strongly influenced by the composition of the semi-solid vehicle. PR-Cl release and permeation from lipophilic vehicles were extremely limited and influenced by their composition. Best results were obtained by using the hydrophilic cream. Furthermore, the retention study evidenced that epidermis acted as a reservoir, releasing the PR-Cl accumulated after preparation removal. CONCLUSION: The 1% w/w PR-Cl cream resulted the most suitable formulation for improving drug permeation through the human epidermis. On the contrary, the negligible permeation profile through full-thickness skin pointed out that PR-Cl cannot diffuse significantly to reach the deeper layers of human skin.

Formulation study of a patch containing propranolol by design of experiments.

OBJECTIVE: To evaluate the feasibility of a transdermal patch containing propranolol (PR). METHOD: Skin penetration enhancers (SPEs) able to improve the skin permeability of PR were selected and a quality by design approach was applied to the development of the patch by a 2(4) full factorial design. The permeation profile of PR from the formulations was assessed in in vitro permeation studies performed by using Franz diffusion cells and human epidermis as membrane. Finally, skin irritation was evaluated by the Draize test. RESULTS: N-methyl pyrrolidone (NMP) resulted as the best SPE: in addition, the critical factors influencing the PR diffusion through the human epidermis when loaded in the patch resulted in the matrix thickness (X1, p = 0.0957) and PR content (X3, p = 0.0004) which improved the flux; conversely, NMP lacked its enhancement effect when loaded in the patch and the increase in its concentration (X4, p = 0.006) affected the drug permeation through human epidermis. The flux of optimal formulation was 12.7 µg/cm(2)/h. On the basis of the steady-state concentration and clearance of PR, the estimated patch surface was 100-120 cm(2), since the activity of PR is related to its Senantiomer and no in vivo bioconversion occurs. CONCLUSION: A patch containing (S)-PR was prepared and the (S)-PR flux (13.3 µg/cm(2)/h) permitted to confirm the suitability of a transdermal administration of PR. In particular, the use of a 50 µm thick methacrylic matrix containing 8% (S)-PR and 15% NMP can allow to develop a patch non-irritating to the skin, in order to ensure a constant permeation flux of PR over 48 h.

Updating 'Data on the determination of human epidermis integrity in skin permeation experiments by electrical resistance' with Data on pig ear skin.

The data presented in this article are an update of the dataset provided by Musazzi et al. [1] and are related to the research article entitled "Equivalence assessment of creams with quali-quantitative differences in light of the EMA and FDA regulatory framework" [2]. In vitro permeation study (IVPT) is typically conducted using the method of Franz's diffusion cell for assessing the biopharmaceutical performance of topically applied products. While the human epidermis is considered the benchmark, various animal models (for instance, pig ear) have been accepted as a permeation membrane. Nonetheless, it is crucial to evaluate the integrity of the membrane to ensure the quality of the experiments. The methods employed for this assessment vary, and the outcomes are heavily reliant on the operational conditions, and the model membrane. The article contributes to the existing dataset by providing data on the electrical resistance values of pig ear skin samples and their correlation with the in vitro permeability fluxes of caffeine and benzoic acid. This data is utilized to determine a suitable cut-off for verifying the skin integrity of such an animal model. This information could be beneficial for facilitating critical or comprehensive analyses, contributing to the creation of a standard method.

Equivalence assessment of creams with quali-quantitative differences in light of the EMA and FDA regulatory framework.

EMA and FDA are upgrading guidelines on assessing the quality and the equivalence of topically applied drug products for developing copies of originator products and supporting post-marketing variations. For topical products having remarkably similar composition, both EMA and FDA accept the equivalence on the bases of the comparison of rheological properties and in vitro drug release constant (k) and skin permeation flux (J) values, instead of clinical studies. This work aims to evaluate the feasibility to expand this approach to variations of the composition of complex semi-solid preparations. Ibuprofen (IB) creams at two different strengths (i.e., 1 % and 10 %) were used as a model formulation. Two formulative changes were performed: (a) the addition of the humectant to simulate a minor post-marketing variation; (b) the substitution of the emulsifying system to simulate a major one. These variations impacted only in 1 % IB formulations where both the equivalences of rheological data and J-values failed. At the highest concentration, the presence of IB crystals broke down the differences in rheological patterns and lead the IB thermodynamic activity at the maximum figuring out an overlapping of the J-values. Such data suggest the combination of these studies, which are thought mainly for the development of copies, could be also applied to the management of post-marketing variations that involve product composition.

Crushing riluzole tablets: evaluation of loss of powder and active principle in a home-simulation experiment.

Objective: Swallowing difficulties cause patients with amyotrophic lateral sclerosis (ALS) to crush oral medications, falling outside the labeling instructions and entailing some risks. To date, there is no evidence about consequences of crushing riluzole tablets in a home setting. This simulation experiment evaluated the loss of powder and active principle ingredient (API) mimicking the home setting with two alternative crushing methods (A and B). Methods: The tests were carried out by 15 volunteers without experience in the preparation of medication. Each volunteer manually crushed 5 tablets with a meat tenderizer (method A) or two spoons pressed against each other (method B). Riluzole was weighed before (W1) and after crushing (W2). Then, a subsample of crushed tablets was analyzed by HPLC to measure API content. The loss of powder was calculated as a percentage of the intact tablet weight, and the loss of API as a percentage of the labeled API content. Results: The quantitative analysis showed a mean percentage loss of 6.27% corresponding to a mean (SD) loss of powder of 13(±13) mg. The API loss was directly related to the powder loss: overall the mean percentage of API loss was 8.53% (corresponding to a mean API loss of 4.27 ± 4.50 mg). The difference in powder and API loss was highly statistically significant. Conclusion: Crushing riluzole tablets in a simulated home setting determined a significant loss of powder and API. These results support neurologists to evaluate formulations that minimize the need to alter the product and can improve ALS patient journey.

Digital Technologies Applied to Control the One-Step Process of Cannabis Olive Oil Preparations.

The reproducibility of an extemporaneous preparation is an essential condition for guaranteeing the quality, efficacy, and safety of the medicinal product. This study aimed to develop a controlled one-step process for cannabis olive oil preparations by applying digital technologies. For this purpose, the chemical profile of cannabinoid contents in oil extracts of Bedrocan, FM2, and Pedanios varieties obtained with the already in use method, proposed by the Italian Society of Compounding Pharmacists (SIFAP), was compared with two new methods, specifically the Tolotto Gear® extraction method (TGE) and the Tolotto Gear® extraction method preceded by a pre-extraction procedure (TGE-PE). HPLC analyses showed that the concentration of THC using cannabis flos with a high THC content (over 20% w/w) was always higher than 21 mg/mL for the Bedrocan variety and close to 20 mg/mL for the Pedanios variety when applying TGE, while with TGE-PE, the THC concentration was higher than 23 mg/mL for the Bedrocan variety. For the FM2 variety, the amounts of THC and CBD in the oil formulations obtained using TGE were higher than 7 mg/mL and 10 mg/mL, respectively, and for TGE-PE, the concentrations of THC and CBD were higher than 7 mg/mL and 12 mg/mL, respectively. GC-MS analyses were performed to define the terpene contents in the oil extracts. The samples of Bedrocan flos extracted with TGE-PE displayed a distinctive profile, highly rich in terpenes and devoid of oxidized volatile products. Thus, TGE and TGE-PE allowed performing a quantitative extraction of cannabinoids and increasing the total mono-di-tri terpenes and sesquiterpene concentrations. The methods were repeatable and applicable to any quantity of raw material, preserving the phytocomplex of the plant.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives.

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.

Spotlight on Calcipotriol/Betamethasone Fixed-Dose Combination in Topical Formulations: Is There Still Room for Innovation?

Psoriasis is a lifelong disease which requires treatment adherence for successful management. Considering the complexity of this pathology, the combination of active pharmaceutical ingredients with a synergistic mechanism of action can improve the safety and efficacy of the treatment with respect to the conventional monotherapy. Moreover, a fixed dose of therapeutic agents in a topical formulation offers the possibility to simplify administration, reduce the doses of each active ingredient, and improve patient's compliance. Among the first-line treatments in mild to moderate psoriasis, the formulation of calcipotriol (Cal) and betamethasone dipropionate (BD) in a single vehicle is challenging due to their chemical incompatibility in an aqueous environment and the formation of degradation products. Based on these considerations, this review aims to provide an overview on the biopharmaceutical properties of Cal/BD fixed-dose combination products available on the market (namely ointment, oleogel, foam, and O/W cream), highlighting also the novel approaches under evaluation. The main differences among topical formulations are discussed considering the different features of the anatomic districts involved in psoriasis and the patient's adherence. Moreover, since in vitro experiments are fundamental to evaluate the skin permeation profile during the development of an efficacious medicinal product, special emphasis is given to models proposed to mimic psoriatic lesions.

Design of Liposomal Lidocaine/Cannabidiol Fixed Combinations for Local Neuropathic Pain Treatment.

The administration of drug fixed combinations by nanocarriers is a new attractive approach since it can allow improvements in both the skin penetration of cargo compounds and their synergistic effects. The cutaneous administration of lidocaine (LD) and cannabidiol (CBD) combination can be useful for the local treatment of neuropathic pain. In fact, these drugs might exert a complementary effect on pain acting on sodium and calcium channels. In this study, the feasibility to deliver this combination in the deeper layers of the skin using deformable liposomes was studied. Based on a study of the drug affinity for lipid components performed by DSC, CBD was loaded in the lipid bilayer for limiting the leakage, while LD was loaded in the inner core by a pH gradient method (G-liposomes) or after previous encapsulation in micelle (DiMiL). The effect of the presence of Tween 80 in the liposome membrane was also evaluated. DiMiL increased both the skin permeation and the retention in the dermis of CBD and LD with respect to G-liposomes (R24dermis: 11.52 ± 2.4 against 4.51 ± 0.8 µg/cm2 for CBD; 19.6 ± 2.9 against 3.2 ± 0.1 µg/cm2 for LD). Moreover, both DiMiL and G-liposomes were more efficient than control formulations carrying free drugs in improving drug skin permeation. Interestingly, in the presence of a drug exerting a fluidizing effect such as CBD, the removal of Tween 80 from the composition led to an improved control of drug release and a higher extent of drug retention in the dermis layer.

Ultrasound-Assisted Extraction of Cannabinoids from Cannabis Sativa for Medicinal Purpose.

Over the past 20 years, the interest in Cannabis oily extracts for medicinal use compounded in pharmacy has consistently grown, along with the need to have preparations of adequate quality. Hot maceration (M) is the most frequently used method to compound oily solutions. In this work, we systematically studied the possibility of using an ultrasonic homogenizer and a sonotrode (US) as an alternative extraction method. Oily solutions were prepared using two available varieties of Cannabis for medicinal use, called FM2 and Bedrocan. All preparations resulted with an equivalent content in CBD and THC, with the advantage of a faster process using US. In particular, 10 min sonication at the amplitude optimized for the sonotrode used (2 or 7 mm) provides not statistically different total Δ9-tetrahydrocannabinol (M-FM2: 0.26 ± 0.02 % w/w; US-FM2: 0.19 ± 0.004 % w/w; M-Bedrocan: 1.83 ± 0.17 % w/w; US-Bedrocan: 1.98 ± 0.01 % w/w) and total cannabidiol (M-FM2: 0.59 ± 0.04 % w/w; US-FM2: 0.58 ± 0.01 % w/w) amounts extracted in refined olive oil. It can therefore be confirmed that sonotrode is an efficient and fast extraction technique and its use is without negative consequence on the solvent properties. Despite DSC evidencing that both maceration and sonication modify the Tonset and enthalpy of the event at about -10 °C, the qualitative characteristics of the oil remained constant for the two treatments and similar to the starting material.

Critical Aspects in the Preparation of Extemporaneous Flecainide Acetate Oral Solution for Paediatrics.

The availability of liquid oral preparations compounded by pharmacists is essential to meet paediatric needs which remain unanswered by the pharmaceutical industry. Unfortunately, compendial monographs are often not available and, in many cases, pre-formulation studies (e.g., compatibility with other excipients and solubility evaluations) are not performed in-depth, leading, in some rare cases, to the inadvertent administration of a toxic dose. In this study, the preparation of an oral liquid formulation for paediatric use, containing flecainide acetate at different strengths, was considered, taking into account the possible effects of conventionally used excipients. First, the optimal vehicle was selected based on a solubility study, evidencing some unexpected formations of precipitates. As a matter of fact, the buffers commonly used for oral solutions significantly reduced flecainide solubility, and the concomitant presence of citrate buffer and methylparaben even caused the formation of non-resuspendable crystals. Then, chemical, physical, and microbiological stability were assessed. Solutions at strengths of 10 and 20 mg/mL flecainide acetate were stable up to 8 weeks when compounded by using a 40% sucrose solution as a vehicle. Microbiological data showed that the use of methylparaben was not necessary over this time period.

Stability of high concentrated triple intrathecal therapy for pediatrics and mitigation strategies.

Stringent formulation requirements are defined to intrathecally administer drug substances, avoiding neurological complications. In case of pediatric patients, these are further complicated due to the limited volumes of the celebrospinal fluid and, therefore, high concentrated solutions of methotrexate (MTX), cytarabine and corticosteroids (i.e., methylprednisolone or hydrocortisone) are prepared based on the patient's age. This work aims to assess the chemical and physical stability of triple intrathecal mixtures differing in volume and composition by a bracketing approach and to identify possible stress causes and mitigation strategies. Low solubility of MTX was the main factor limiting the physical stability of triple mixtures. Regarding solutions containing methylprednisolone, the amount of MTX remaining was about 95% in the solution at highest concentrations with the concomitant formation of a visible particulate sizing bigger than 1 µm after 24 h of storage at 25 °C. This behavior was mainly driven by the pH reduction due to the pH value of the cytarabine solution used; the shear stress also induced drug precipitation. In the case of the hydrocortisone based mixtures, the precipitate formation occurred at a slow rate. To improve the physical stability, a better control of the mixture pH (optimal value ≈ 7) is required or, as an alternative, the addition of the cytarabine solution to a pre-mixed binary mixture containing MTX and a corticosteroid should be preferred.