Patent Application
20240342234
The subject disclosure relates to a P2Et-loaded nanoparticulate formulation based on casein protein and its oral efficacy in a murine model of breast cancer.
Cancer is a significant public health problem and one of the major causes of Morbi- and mortality worldwide. World Health Organization (WHO) pointed out that at least 18 million new cases were diagnosed in 2018, and around 9 million deaths were caused by cancer during that year.
In this context, natural products have acquired great importance in tumor treatment due to their antioxidant, antiproliferative, antiangiogenic, and immunomodulatory abilities which may be related to the prevention of carcinogenesis, the destruction of tumor cells, the modulation of the tumor microenvironment, the activation of the specific anti-tumor immune responses, or the induction of epigenetic changes, but also the improvement of the quality of life of the patients.
Nevertheless, natural products in vitro proven health benefits do not show an apparent in vivo correlation. This phenomenon has been attributed to the physicochemical characteristics of natural molecules such as flavonoids or polyphenols. Generally, natural active ingredients show low water solubility, low instability in the gastrointestinal tract, and rapid metabolism. As a result, the oral bioavailability of this kind of substance is low. In addition, it must be noticed that the antitumoral activity of phytochemicals is estimated to occur through the combination of various phytochemicals acting synergistically rather than by isolated molecules.
To overcome this lack of in vivo efficacy, different strategies are being studied. In this regard, some efforts have explored the use of concomitant administration of the target phytochemical and metabolism inhibitors, such as piperine, or the use of synergistic combinations such as two different phytochemicals (e.g., quercetin-curcumin, resveratrol-curcumin or curcumin-genistein. Another alternative strategies are the modification of the chemical structure of the phytochemical or the design of innovative drug delivery systems. However, the adequacy of human use of the ingredients in this kind of formulations and their techno-economic feasibility still needs to be clarified. Caesalpinia spinosa also known as Dividivi, Tara, Guarango, Caesalpinia pectinate, Caesalpinia tara, Caesalpinia tinctoria, Coulteria tinctoria, Poinciana spinosa, and Tara spinosa, has been traditionally used by Latin American indigenous as a traditional medicine due to its antimicrobial, antioxidant, antitumor, and immunomodulatory properties. The standardized extract from C. spinosa, called P2Et, has shown great ability to decrease lipid peroxidation and tissue damage and induce complete autophagy in tumor or stressed cells. The antioxidant ability of P2Et is related to the high proportion of galloylquinic acid derivatives, and pentagalloyl glucose, among other gallic acid-containing compounds (gallates) in lower proportions. Thus, P2Et acts as a potent antioxidant with high cytotoxic activity against tumor cells, especially those expressing drug resistance pumps as PgP. This potential has been proved in different kinds of murine models of cancer, such as breast cancer and melanoma, in which P2Et promotes the activation of the immune system, particularly T cells and production of IFN-gamma, TNF-alpha, IL-4, and IL-5, which take part on primary tumor control.
The oral administration of P2Et is safe in healthy humans with a maximum tolerated dose of 600 mg/day, with no severe toxicity but some adverse events related to gastrointestinal symptoms without significant changes in safety parameters. The use of gastro-resistant capsules has made it possible to solve part of these effects and demonstrate the beneficial effect of this phytomedicine in patients with COVID-19. However, it is possible to improve the pharmaceutical form of this preparation to favor its better use in patients.
A P2Et-loaded nanoparticulate formulation based on casein protein and its oral efficacy in a murine model of breast cancer.
Sodium caseinate was purchased from Acros Organic (Belgium). L-lysine, mannitol, sodium hydroxide, hydrochloric acid, potassium phosphate monobasic, sodium chloride and Tween 20, were obtained from Sigma-Aldrich (Germany). Calcium chloride was from Merk (Germany). Acetonitrile, methanol, and formic acid were purchased from Panreac (Spain). Gallic acid and ethyl gallate standards were from Sigma-Aldrich (Germany). Ethanol was obtained from OPPAC (Spain). A water purification system prepared deionized reagent water (18.2 MO resistivity) (Wasserlab, Spain). Brilliant green bile broth (BGBB), violet red bile lactose agar, MacConkey Agar, Rappaport Vassiliads broth, Agar XLD, Agar cetrimide and Chapman Broth were purchased from Condalab (Spain). All reagents and chemicals used were of analytical grade. The brilliant green bile broth (BGBB), violet red bile lactose agar, MacConkey Agar, Rappaport Vassiliads broth, Agar XLD, Agar cetrimide and Chapman Broth were purchased from Condalab (Spain). Cell culture reagents (RPMI-1640, heat-inactivated fetal bovine serum (FBS), glutamine, penicillin, streptomycin, HEPES buffer, sodium pyruvate, trypsin/1×EDTA) were obtained from Eurobio Scientific, Paris, France.
Pods of C. spinosa (Feuillée ex Molina) Kuntze (Divi-divi or tara) were collected in Villa de Leyva, Boyaca, Colombia and identified by Carlos Alberto Parra of the National Herbarium of Colombia (copy number of voucher COL 588448). The P2Et is produced as described in Sandoval, T. A.; Urueña, C. P.; Llano, M.; Gómez-Cadena, A.; Hernández, J. F.; Sequeda, L. G.; Loaiza, A. E.; Barreto, A.; Li, S.; Fiorentino, S. Standardized Extract from Caesalpinia spinosa is Cytotoxic Over Cancer Stem Cells and Enhance Anticancer Activity of Doxorubicin. Am. J. Chin. Med. 2016, 44, 1693-1717, incorporated herein by reference, from the pods of C. spinose under GMP conditions. The quality control of P2Et is performed according to the regulatory guideline 1156 of 2018 of herbal drugs in Colombia, WHO guideline “Quality control methods for herbal materials” and FDA guideline “Botanical Drug Development Guidance for Industry” FDA, U. Botanical drug development guidance for industry. Pharm. Qual. CMC Revis. 2016, 1, 1-34, incorporated herein by reference.
Casein nanoparticles were prepared by simple coacervation procedure followed by a purification step by ultrafiltration and subsequent drying by Spray-drying (see
Briefly, 250 mg casein and 23 mg L-lysine were firstly dissolved in 19 ml of water type II under magnetic stirring at room temperature. Simultaneously, P2Et was dissolved in an ethanol 96% in order to obtain a 10 mg/mL solution. Then, different volumes of the P2Et solution were added to the casein-lysine water solution until a final concentration of 3%, 4.79% and 9.14% of P2Et/total solids was obtained. Then, the solution containing casein, L-lysine and the different P2Et concentrations was incubated under magnetic stirring at room temperature.
Casein nanoparticles were obtained by adding 12 mL of a calcium chloride solution (0.2% w/v) in purified water. Afterwards, and when needed, 2 mL of mannitol solution (100 mg/mL) was added to the already formed nanoparticles prior to the drying step in a Büchi Mini Spray Drier B-191 apparatus (Buchi Labortechnik AG, Flawil, Switzerland) under the following experimental conditions: (i) Inlet temperature of 100° C., (ii) Outlet temperature of 60-70° C., (iii) air pressure of 4-5 bar, (iv) pumping rate of 7.5 mL/min, (v) aspirator of 80% and (vi) air flow at 900 mL/h.
For the identification of the different formulations, the following abbreviations were used: NPC-P2Et-3% (casein nanoparticles containing a theoretical concentration of P2Et of 3% (w/w)), NPC-P2Et-5% (casein nanoparticles containing 5% (w/w) theoretical concentration of P2Et), and NPC-P2Et-9% (casein nanoparticles containing 9% (w/w) theoretical concentration of P2Et).
Empty nanoparticles were prepared following the protocol in the absence of P2Et. Those particles were identified as NPC (empty casein nanoparticles).
The particle size, polydispersity index (PDI) and zeta-potential (ζ) were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetasizer analyzer system (Brookhaven Instruments Corporation, Holtsville, NY, USA).
The diameter and polydispersity index (PDI) of the nanoparticles were determined after dispersion in ultrapure water (1/10) and measured at 25° C. by dynamic light scattering angle of 90°. The zeta potential was determined after diluting 2 mg of the sample in 2 ml of ultrapure water.
The yield of the preparative process of nanoparticles was calculated by gravimetry as shown in Arbós, P.; Wirth, M.; Arangoa, M. A.; Gabor, F.; Irache, J. M. GantrezANas a new polymer for the preparation of ligand-nanoparticle conjugates. J. Control. Release 2002, 83, 321-330, incorporated herein by reference.
The concentrations of P2Et were extrapolated from the gallic acid and ethyl gallate concentration. P2Et quantification was determined by reverse-phase high-performance liquid chromatography (HPLC) with UV detection. The chromatographic separation was performed using a liquid chromatographic system equipped with an Alliance 2695 system connected to a Waters 2998 photodiode array detector (Waters Corp., Mildford, MA, USA).
Detection was carried out at 274±4 nm and frequency of acquisition 2.5 Hz. Chromatographic separation was achieved using a Luna Omega C18 (150×2.1 mm; particle size 1.6 μm) column (Waters Corp., Mildford, MA, USA). The column temperature was 35° C., and samples were stored at 15° C. before injection. Injection volume was set at 10 μL.
The mobile phase, pumped at 0.37 mL/min, was a gradient mixture (Table 1) of formic acid 0.1% and acetonitrile (ACN).
Under these conditions, the peaks for gallic acid and ethyl gallate appear at run-times of 3.05 min and 11.80 min, respectively.
For the quantification of P2Et in the nanoparticles, 30 mg of the formulation was dissolved in 10 mL of a mixture of methanol:DMSO (4:1; v/v). Each sample was analyzed in triplicate, and the results are expressed as the amount of each compound per mg of the formulation.
Afterward, the encapsulation efficiency (E.E.) was calculated as follows:
Release experiments were conducted at 37° C. using simulated fluids for gastric (SGF; pH 1.2) and intestinal (SIF; pH 6.8) conditions prepared according to European Pharmacopoeia (EMA, European Pharmacopoeia 6.0, chapter 2.9.3 Dissolution Test for Solid Dosage Forms, 2008).
At different intervals, samples were collected and centrifuged at 10,000 rpm for 10 minutes (Centrifuge MIKRO 220, Hettich, Germany). For each specific time interval, 79 μg of P2Et, either free or entrapped into nanoparticles, was resuspended in 2 ml of the corresponding simulated fluid. The different formulations were kept in the SGF for 2 hours before being transferred to SIF for 20 hours. The amount of P2Et released was quantified by HPLC from the supernatants described above.
The microbiology evaluation was performed according to European regulation (EU Regulation 2073/2005 and 1441/2007) and following validated procedures from ISO standards. In addition, the acceptance criteria from USP <2023> were considered.
The total aerobic microbial count was performed according to ISO 4833:2013. The total coliforms were studied according to ISO 4832:2006 procedure, whereas fecal coliform was performed according to ISO 4832:2006. Total yeast and molds was performed following ISO 21527:2008. Escherichia coli study was done according to ISO 16654:2001. Salmonella sp was performed according to ISO 6579:2017, and Staphylococcus aureus was done according to ISO 6888 procedure.
4T1 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mML-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate and incubated in a humidified environment at 37° C. and 5% CO2. Cells were grown until 75% confluency and passaged using trypsin/1×EDTA, washed with PBS, and resuspended in supplemented RPMI-1640, as in Urueña, C.; Mancipe, J.; Hernandez, J.; Castañeda, D.; Pombo, L.; Gomez, A.; Asea, A.; Fiorentino, S. Gallotannin-rich Caesalpinia spinosa fraction decreases the primary tumor and factors associated with poor prognosis in a murine breast cancer model. BMC Complement. Altern. Med. 2013, 13, 74, incorporated herein by reference.
Young (6 to 12 weeks old) female BALB/c mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA) and housed at the animal facilities of the Pontificia Universidad Javeriana (PUJ, Bogotá, Colombia) following the established protocols of the Ethics Committee of the Faculty of Sciences, PUJ, and National and International Legislation for Live Animal Experimentation (Colombia Republic, Resolution 08430, 1993; National Academy of Sciences, 2010). The present study was approved by the ethics committee of the Faculty of Sciences, PUJ, on Aug. 9, 2018 (FUA-093-20). Mice were maintained in polyethylene cages with food and water provided ad libitum, on a 12-h light/dark cycle at 20-22° C. and 40-60% humidity.
For breast cancer murine model, 1×104 viable 4T1 cells were s.c. injected into the right mammary fat pad of BALB/c mice. To evaluate the effect of treatments on tumor growth, 3-5 days after tumor cell inoculation, 8 mice per group were treated with 18.7 mg/kg P2Et (Intraperitoneally I.P), 18.7 mg/kg P2Et (Oral), 168.3 mg/Kg NPC-P2Et 9%, 56.1 mg/Kg NPC-P2Et 9% (Oral) or PBS (negative control) two times per week. In all experimental settings, the size of the tumors was assessed three times per week with Vernier calipers, and the volume was calculated according to the formula V (mm3)=L (major axis)2×(minor axis)/2. Mice were euthanized by CO2 inhalation, and then spleen, tumor draining lymph nodes (TDLN), and tumor were removed and processed. Looking for sufficient statistical power adjusted to the standard deviation and to the proportion of losses in each model, eight mice were included for each treatment group.
For this purpose, nanoparticles and control samples were stored in 5 mL capacity glass containers in a climatic Test Cabinet TK 600 NUVE (2012) at 40° C. and 75% Relative Humidity (RH). The formulation's mean size, PDI and P2Et content was tested over time.
The physico-chemical characteristics of nanoparticles and the in vitro studies were compared using the Student's t test. Calculations were performed using OpenEpi, version 3, (open-source calculator t_testMean). For in vivo studies, comparisons were performed using the two-way analysis of the variance (ANOVA) and Dunnett's multiple comparison test. Data from in vivo analysis was analyzed using GraphPad Prism v9.3.1 for Mac OS X (GraphPad Software, La Jolla California USA, www.graphpad.com). Data are expressed as the mean±standard deviation (S.D.) of at least three experiments. In all cases p<0.05 was considered as a statistically significant difference.
This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.
Although casein nanoparticles can be easily prepared by simple coacervation in the presence of calcium the resulting nanoparticles display low stability in an aqueous environment. Thus, the basic amino acid lysine was added in order to increase the stability of casein nanoparticles following Penalva, R.; Esparza, I.; Agüeros, M.; Gonzalez-Navarro, C. J.; Gonzalez-Ferrero, C.; Irache, J. M. Casein nanoparticles as carriers for the oral delivery of folic acid. Food Hydrocoll. 2015, 44, 399-406.
Table 2 summarizes the main physico-chemical properties of P2Et loaded nanoparticles. When the phytotherapeutic P2Et was encapsulated into casein nanoparticles, a moderate decrease in the mean size of the resulting carriers was observed for those formulation containing lower amounts of P2Et (about 226 nm for empty nanoparticles vs. 140 nm for NPC-P2Et-3% and NPC-P2ET-5%). Whereas high payload formulation shows a slightly increase in the particle size (226 nm vs. 260 nm). Similarly, the zeta potential of the particles decreases with the increase on the payload. This result may reflect the presence of P2Et on the surface of the particles.
Finally, the P2Et concentration of the most promising prototypes (NPC-P2Et-5% and NPC-P2Et-9%) was quantified (Table 3):
P2Et release kinetics from casein nanoparticles was evaluated in two different media: simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).
When gallic acid is directly incubated (as P2Et free form) for two hours in SGF (pH 1.2) the molecule is stable. However, when the gallic acid is incubated into SIF (Ph 6.8) a high degradation phenomenon is observed. In fact, only about 2-3% of the initial gallic acid is quantify at this point.
When the particles are incubated for two hours in SGF, about 70% of the total gallic acid is released. Then, when added to SIF the nanoparticles still release gallic acid. It can be observed that the total estimation of gallic acid release from the nanoparticle formulation is higher than the initial amount. This increase is related to the ability of the particle to protect ethyl gallate and potentially, methyl gallate from degradation in the SGF.
The ethyl gallate concentration decreases by about 76% after two hours of incubation in SGF. Similarly, methyl gallate concentrations show a reduction of about 32% in these two hours (data not shown).
Conversely, the ethyl gallate release from the nanoparticles maintains a continuous pattern, in which about 27% of the total content is released from particles as ethyl gallate.
The microbiology evaluation was performed according to European regulation (EU Regulation 2073/2005 and 1441/2007), following validated procedures from ISO standards and considering the acceptance criteria from USP <2023>. Table 4 summarizes specifications according to the different ISO and the obtained result.
Escherichia coli
Staphylococcus
aureus (CFU/g)
Pseudomona aeruginosa
Salmonella spp.
2.2. In vivo NPC-P2Et treatment delays tumor growth and metastasis in breast cancer model We have previously described that P2Et I.P treatment post-tumor engraftment delays melanoma and breast cancer tumor growth. See, Gomez-Cadena, A.; Urueña, C.; Prieto, K.; Martinez-Usatorre, A.; Donda, A.; Barreto, A.; Romero, P.; Fiorentino, S. Immunesystem-dependent anti-tumor activity of a plant-derived polyphenol rich fraction in a melanoma mouse model. Cell Death Dis. 2016, 7, e2243; Urueña, C.; Gomez, A.; Sandoval, T.; Hernandez, J.; Li, S.; Barreto, A.; Fiorentino, S. Multifunctional T Lymphocytes Generated After Therapy With an Antitumor Gallotanin-Rich Normalized Fraction Are Related to Primary Tumor Size Reduction in a Breast Cancer Model. Integr. Cancer Ther. 2015, 14, 468-483; Lasso, P.; Llano Murcia, M.; Sandoval, T. A.; Urueña, C.; Barreto, A.; Fiorentino, S. Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model. Integr. Cancer Ther. 2019, 18, 1534735419848047, all incorporated by reference herein. We wanted to assess whether NPC-P2Et had the same antitumor effect in the metastatic breast cancer model or if an increased activity might be observed, and if oral administration might induce the same activity previously observed for P2Et-I.P inoculation. To answer this question, we treated mice with P2Et I.P, P2Et Oral, NPC-P2Et Oral or PBS (negative control) post-tumor engraftment and evaluated the tumor growth and macro-metastasis. The mice treatment with P2Et I.P, Oral P2Et or Oral NPC-P2Et significantly delayed tumor growth compared to mice treated with PBS (
The stability of nanoparticles was estimated over time considering modification on the mean size, Pdl and P2Et content of the formulation. The size and PDI of the formulation for 4.4 months show that the mean size of the particles was stable, and the polydispersity index was always below 0.3. This value was considered as acceptable limit for PDI (see table 5).
The P2Et concentration over time was estimated according to the gallic acid and ethyl gallate concentration in the formulation over time. During the stability test the concentration of ethyl gallate remained stable, whereas the concentration of gallic acid slightly varied as shown in
Orally administered of natural compounds such as P2Et need to face several biological barriers (pH, low water solubility, high degradation through the harsh conditions of the gastrointestinal tract) that make the oral bioavailability of the drug almost negligible. From a theoretical point of view, the use of nanoencapsulation of such compounds may be an adequate approach to reach a successful oral administration as the encapsulation will protect the loaded cargo against premature degradation, including the acidic conditions of the stomach, the presence of enzymes, as well as the mechanical stress in the lumen (e.g., osmotic pressure and peristalsis). See, Homayun, B.; Lin, X.; Choi, H. J. Challenges and Recent Progress in Oral Drug Delivery Systems for Biopharmaceuticals. Pharmaceutics 2019, 11, 129; Wu, S.; Bin, W.; Tu, B.; Li, X.; Wang, W.; Liao, S.; Sun, C. A Delivery System for Oral Administration of Proteins/Peptides Through Bile Acid Transport Channels. J. Pharm. Sci. 2019, 108, 2143-2152; all incorporated by reference herein.
From a general point of view, protein-based nanoparticles offer some advantages for drug delivery purposes, including their biodegradability and capability to accommodate a high variety of compounds in a non-specific way. See, Jain, A.; Singh, S. K.; Arya, S. K.; Kundu, S. C.; Kapoor, S. Protein Nanoparticles: Promising Platforms for Drug Delivery Applications. ACS Biomater. Sci. Eng. 2018, 4, 3939-3961, incorporated by reference herein. In this context, casein possesses some characteristics that may be considered as additional advantages for the formulation of nanoparticles as its regulatory status and low toxicity, see, Gil, A. G.; Irache, J. M.; Peñuelas, I.; González Navarro, C. J.; López de Cerain, A. Toxicity and biodistribution of orally administered casein nanoparticles. Food Chem. Toxicol. 2017, 106, 477-486, incorporated by reference herein.
In this work, casein-based nanoparticles were selected to evaluate and compare their ability to deliver P2Et by oral route. Both types of nanoparticles (empty and P2Et-loaded nanoparticles) displayed a mean size of around 250 nm (226 nm for empty nanoparticles and 260 nm for P2Et-loaded nanoparticles) and negative zeta potential (Table 2). Notably, the surface zeta potential of P2Et-loaded nanoparticles was remarkably less negative than that of empty nanoparticles, suggesting that, in the formation of P2Et-loaded nanoparticles, some structural changes may occur during P2Et encapsulation, and some P2Et molecules can adhere to the nanoparticle surface, inducing the observed changes with respect to the zeta potential. The most promising P2Et formulations (NPC-P2Et-9%) had the ability to entrapped with high encapsulation efficiency the both main components of P2Et (gallic acid and ethyl gallate). The release of P2Et from the nanoparticles appears to modify its own degradation pattern under gastrointestinal conditions. Thus, non-encapsulated gallic acid is highly degraded in simulated intestinal fluid, while encapsulation seems to protect it against this degradation, allowing its detection even after 2-6 h of incubation in this fluid. Similarly, ethyl gallate concentrations appear to be released in a continuous pattern over the time of the experiment. In fact, the benefit of casein nanoencapsulation in the preparation of new forms of drug delivery has been previously reported in Semwal, R.; Joshi, S. K.; Semwal, R. B.; Semwal, D. K. Recent Developments and Potential for Clinical Use of Casein as a Drug Carrier. Curr. Drug Deliv. 2023, 20, 250-260, and particularly with polyphenols, nanoencapsulation prolongs stability and improves release. See, Puligundla, P.; Mok, C.; Ko, S.; Liang, J.; Recharla, N. Nanotechnological approaches to enhance the bioavailability and therapeutic efficacy of green tea polyphenols. J. Funct. Foods 2017, 34, 139-151, incorporated by reference herein. In this sense, the efficacy of the formulation has been evidenced by an in vivo murine breast cancer model. Mice treated with both PC-P2Et preparations significantly reduce the size of the primary tumor, but the high concentration is more effective in reducing metastatic foci (
In addition, our formulation offers some interesting advantages that may enable a translational approach; particularly in those aspects related to the scale-up of a reproducible process (including the drying step) and the simplification of non-clinical toxicity assessments of the regulatory dossier. Among others, P2Et-loaded casein nanoparticles may be obtained in a simple and scalable preparative process that only requires the use of simple reagents and solvents (ethanol and water) and allows the generation of a powder formulation, easily dispersible in water. Furthermore, casein nanoparticles do not enter the systemic circulation minimizing a possible accumulation in the body and toxicological issues, and for the time studied the physico-chemical characteristics of the formulation (size and Pdl) are not modify over time, whereas active ingredient concentration variation is lower than 10%.
In this work, casein nanoparticles containing P2Et were satisfactorily prepared. These particles (NPC-P2Et) exhibited high EE for the active compounds of P2Et (gallic acid and ethyl gallate). According to the in vitro release study, this nanoparticles-based system modifies the pattern of release/absorption of the compounds in the gastrointestinal tract and thus, their bioavailability. Therefore, nanoencapsulated P2Et can improve the absorption profile of P2Et compounds, resulting in a greater therapeutic response. Our results agree that polyphenols, particularly P2Et, can be used within the framework of antitumor therapy, with an increase in the treatment efficiency when P2Et is nanoencapsulated.
These particles have a preparation procedure that only requires natural, non-toxic ingredients and are not subjected to nanomaterial restrictions. Finally, the results disclosed herein provide motivating elements to consider P2Et as a potential adjuvant in cancer treatment.
| Number | Date | Country | |
|---|---|---|---|
| 63459137 | Apr 2023 | US |
