European Journal of Cardiovascular Medicine

Sacubitril/valsartan represents a pioneering class of medications, combining neprilysin inhibition with angiotensin II receptor antagonism. This unique combination effectively enhances endogenous natriuretic peptides while inhibiting the renin–angiotensin-aldosterone system [1]. Sacubitril/valsartan is a cocrystal consisting of 6 sacubitril and 6 valsartan moieties in their anionic forms, 18 penta- and hexa-coordinated sodium cations, and 15 water molecules.

According to the PARADIGM-HF trial findings, sacubitril/valsartan demonstrated a significant advantage over enalapril in reducing cardiovascular mortality, heart failure hospitalization risk, heart failure symptoms, and physical limitations. The trial was prematurely stopped after a median follow-up of 27 months, revealing substantial advantages of sacubitril/valsartan [2]. The TRANSITION study further demonstrated the feasibility of achieving target doses within 10 weeks. This study revealed that initiating sacubitril/valsartan stabilized patients with heart failure with reduced ejection fraction (HFrEF) following an acute heart failure event, whether in the hospital or shortly after discharge [3].

Valsartan, classified as a Biopharmaceutical Classification System (BCS) Class II, exhibits low solubility and high permeability [4]. This classification suggests that bioavailability from oral dosage forms is dissolution rate limited. Sacubitril/valsartan are highly hygroscopic in nature and difficult to handle when used in amorphous (non-crystalline) form and can adversely affect it invitro and in vivo performance including storage concerns.  A drug's efficacy depends on various factors, including solubility, stability, dissolution rate, and hygroscopicity [5]. Ongoing research explores methods to enhance oral absorption for medications with low water solubility and/or limited permeability, with pharmaceutical cocrystals emerging as a promising tool for improving bioavailability [6].

As an emerging tool, pharmaceutical cocrystals can be utilized to modify the solubility, dissolution rate, and both the physical and chemical stability of drug substances, aiming to retain the drug's pharmacological effect without significant alteration [7].

The invention of the supramolecular complex, a cocrystal of sacubitril/valsartan, is a great boon to cardiovascular patients. In 2015, sacubitril/valsartan (Entresto, development code LCZ696) was approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as the first successful angiotensin receptor neprilysin inhibitor (ARNI) in the market [8]. The sacubitril/valsartan combination was developed by Novartis and marketed by JB Chemicals as Azmarda in India.  The polymorphic technology of Azmarda has been recognized and patented. This cocrystal formulation is the one that has been tried and proven effective in RCTs.  Many pharmaceutical companies have adopted alternative strategies to combine sacubitril/valsartan in their products, with different manufacturing process. One of these potential methods involves utilizing a fixed-dose combination (FDC) of sacubitril/valsartan.

Given the significance of the co-crystal form in sacubitril/valsartan tablets, this study aimed to assess various brands in the market for the presence of the co-crystal form, comparing them with the innovator product.

Study design and material

 The study involved the analysis of various marketed products containing sacubitril/valsartan tablets, including Azmarda manufactured by innovator company. The sacubitril/valsartan sodium salt complex in cocrystal form was acquired from Ami Life Sciences. Solid-state forms were identified using X-ray powder diffraction. The solvents and reagents used for co-crystallization, such as isopropyl alcohol, hydrochloric acid (HCl), sodium phosphate buffer, sodium hydroxide, nitric acid, and hydrogen peroxide, were of analytical grade from Merck Limited. Double-distilled water or high-performance liquid chromatography (HPLC) grade water was used as needed.

Physical mixture preparation method

Sacubitril/valsartan were mixed in a defined 1:1 stoichiometric ratio. This mixture was prepared and carefully stored away from light and moisture.

Differential Scanning Technology (DSC)

The thermal behaviour of the cocrystal was determined using the Perkin Elmer Model STA-8000 with DSC. Before measurement, the instrument was calibrated for temperature and heat flow using standard indium. A weighted sample (5 mg) was placed in a sealed non-hermetic aluminium pan, and a hold time of 1 minute at 45°C was maintained. The measurement was then conducted with an increasing step size of 10°C/min under a dry nitrogen atmosphere, running the sample from 45°C to 235°C. Subsequently, the peaks were estimated, and the resulting data were analyzed using the simultaneous thermal analyzer DSC from PerkinElmer.

Powder X-ray diffraction (PXRD)

The distinct peak of each compound was characterized using PXRD. Subsequently, various brands of marketed products in India were assessed against Azmarda tablets to investigate the presence of the innovative cocrystal in the formulation. All samples, within an uncertainty range of (5 mg - 20g), were weighed using an analytical balance (Mettler Toledo). The product underwent powder X-ray diffraction (PXRD) analysis, with samples exposed to Cu Kα radiation (0.15405 nm) at a tube voltage of 40 kV and a current of 30 mA. Data were collected within the 2θ range of 2° to 49.721°, employing a step size of 0.031 under atmospheric pressure. For sample preparation, the required quantity was taken into a mortar, and a fine powder was gently obtained using a pestle. The powdered sample was then placed on the sample holder, packed, and the surface was smoothed with a microscopic slide.

Sodium content determination

Sodium content was determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES; Agilent 5110 Instrument) for the sacubitril/valsartan sodium complex, Azmarda, and sacubitril/valsartan tablets. Sodium content of different marketed products of sacubitril/valsartan tablets was measured with parameters including radio frequency (RF) power ranging from 750W to 950W, atomizer airshed set at 1.00 to 1.25 L/min, assisted gas flow between 0.5 to 1.00 L/min, and a vertical observation height of 15 to 18 mm.

For sample preparation, 1.0 g of the sample was transferred into a 100.0 mL Teflon/PTFE vessel, followed by the addition of 1.0 mL of hydrogen peroxide and 10.0 mL of concentrated nitric acid. The Teflon/polytetrafluoroethylene (PTFE) vessel was then placed in a microwave digestion apparatus. After cooling the mixture to room temperature, the plasma was ignited and allowed to stabilize for approximately 15 minutes. The blank solution was aspirated, and the signal was zeroed. In triplicate cycles, the diluent, standard, and sample solutions were aspirated into the plasma, and the ionization intensity of different elements was measured.

Dissolution studies

The dissolution studies were conducted in 900 mL of 0.1N HCl and pH 6.8 phosphate buffers using a USP Type II dissolution apparatus (Lab India DS 14000 SMART) with a paddle speed set at 75 rpm. Various marketed products of sacubitril/valsartan tablets were subjected to dissolution studies. The temperature of the dissolution medium was maintained at 37°C ± 0.5°C throughout the specified time limit. Tablet samples of marketed products were subjected to the dissolution study. The 10 mL sample aliquots were collected at 15, 30, 45 min time points from the dissolution vessel and same were replaced with an equal volume of fresh medium. The sample aliquot solution was then filtered through a 0.45 μ polyvinylidene difluoride (PVDF) filter (Make: Millipore) for analysis at 242 nm using the HPLC.

A total of 16 brands of marketed product of sacubitril/valsartan tablets, including Azmarda were studied in the present analysis.

The DSC thermograms of marketed products was determined for 11 brands including Azmarda indicating exothermic peaks for sacubitril/valsartan between 131°C and 165°C and 72°C and 114°C, respectively (Table 1, Figure 1). A distinct melting pattern was observed in the reference product, with exothermic peaks of sacubitril/valsartan detected at 140°C and 102°C, respectively.

The PXRD analysis revealed that none of the products exhibited the same crystal lattice as Azmarda tablets. The prominent peaks of Azmarda were observed at position of 2θ, specifically at 4.125, 5.267, 12.482, and 22.496 (Figure 2 and 3). Among other comparators, brand-9 showed three peaks around 2θ value of 4.082, 5.047 and 12.420. Brand 5, 8, 11, 13 and 14 showed two peaks around 2θ value 4 and 5; brand 12 showed two peaks around 2θ value of 4.391 and 5.917; brand 2, 4, 7 and 10 showed two peaks around 2θ value of 4 and 22; however, brand 3, 15 and 16 showed only peak around 2θ value of 4. The prominent peaks of Azmarda in PXRD pattern confirmed the crystalline nature of compound; as compared to all other brands.

Sodium content was determined for 11 brands including Azmarda. Analysis showed that Azmarda had a sodium level of 1.72%, which was comparable to the sodium levels in brand 2 (1.61%), brand 4 (1.73%), and brand 7 (1.58%) (Figure 4). Other marketed products had a higher sodium level ranging from 3.164 to 3.951.

The innovator product, Azmarda, based on cocrystal technology, demonstrated a 64% release of sacubitril and a 57% release of valsartan within 30 minutes in 0.1N HCl (Table 2, Figure 5 and 6). Marketed products such as brand 7, brand 12, and brand 13 showed 52%, 57%, and 52% release of sacubitril respectively, and 45%, 52%, and 48% release of valsartan,

respectively, in 30 minutes. Most of the marketed products exhibited very negligible drug release in 0.1N HCl. Similarly, within 45 minutes there was 68% release of sacubitril and 61% release of valsartan which was much higher than all other products. Sacubitril/valsartan are freely soluble in pH 6.8 phosphate buffer and complete drug release is observed in 30 minutes during dissolution study in pH 6.8 phosphate buffer (Table 3).

Table 1: Results of DSC data of sacubitril/valsartan tablets of marketed products

Table 2: Dissolution data of marketed products in 0.1N HCl

Table 3: Dissolution data of marketed products in pH 6.8 phosphate Buffer

Figure 2. Powder X-ray diffraction profile of sacubitril/valsartan

Figure 3.  The characteristics powder diffraction peaks are described in terms of 2θ values for different marketed samples

Figure 4.  Sodium content of sacubitril/valsartan tablets of marketed product

Figure 5. Dissolution sacubitril/valsartan in 0.1N HCl

Figure 6. Dissolution profile of marketed products in 0.1 N HCl at 30 min

The study analyzed 16 brands of sacubitril/valsartan tablets, including the reference product Azmarda, using techniques such as Differential scanning calorimetry (DSC), Powder X-ray diffraction (PXRD), sodium content determination, and dissolution studies. Results revealed that Azmarda, utilizing a cocrystal form, exhibited better dissolution properties, unique crystal lattice structures, and optimized sodium content, highlighting the potential clinical advantages of this innovative formulation over other marketed products.

In Azmarda, the co-crystal complex is engineered with optimized sodium content to achieve better physical and chemical stability, hygroscopicity, handling and storage. The formation of the sacubitril/valsartan cocrystal holds substantial clinical significance, particularly in its impact on dissolution profiles. The better performance of the cocrystal, exemplified by Azmarda tablets, is consistent with studies emphasizing the crucial role of cocrystals in enhancing drug solubility and bioavailability [9, 10]. This enhanced solubility profile may translate to accelerated onset of therapeutic effects during acute cardiovascular events, aligning with findings from similar cocrystal-based formulations [11, 12].

A consistent and reproducible melting point is crucial for quality control. If a product consistently shows a higher melting point, it may indicate a more pure and well-defined material. In the present study, the distinct exothermic peaks observed in the DSC thermograms of sacubitril/valsartan in marketed products shed light on their thermal behavior. The reference product, Azmarda, displayed a unique melting pattern, corroborating the importance of thermal analysis in ensuring the stability and efficacy of pharmaceutical formulations [13, 14]. Such distinctive thermal characteristics contribute to the reliability of the innovative cocrystal technology, like Azmarda, which has demonstrated enhanced dissolution properties and could contribute to more predictable drug absorption in heart failure patients.

In PXRD analysis, prominent peaks correspond to specific crystallographic planes or lattice planes in the sample. These peaks provide information about the arrangement of atoms in the crystal lattice of the material being analyzed. The position, intensity, and shape of these peaks can be used to identify the crystalline phases present in the sample and to characterize its crystal structure [15].

The presence of well-defined, intense peaks suggests that the sample is relatively pure and that a single crystalline phase dominates. The absence of peaks or the presence of broad, diffuse peaks may indicate amorphous or less-crystalline material. Our findings highlight the unique crystal structure of Azmarda tablets and is of particular significance for patients with heart failure.

The absence of identical crystal lattice structures in generic products may have implications for their bioavailability and pharmacokinetics, emphasizing the need for careful consideration of crystal forms in drug development [16, 17].

This also indicates that many patients are deprived of the benefits of the original cocrystal and in comparison, to the innovative cocrystal utilized in clinical trials with Azmarda tablets, the chance of using a FDC or a distinct morphology of sacubitril/valsartan can impact the efficacy of treatment. In the context of heart failure management, where maintaining consistent drug levels is critical for optimal therapeutic outcomes, the reliance on formulations with a standardized and well-characterized crystal lattice structure, as demonstrated by Azmarda, becomes paramount.

The determination of sodium content in various brands, including Azmarda, provided valuable insights into the formulation's composition. In Azmarda, the Co-crystal complex is engineered with optimized sodium content to achieve better physical and chemical Stability, hygroscopicity, handling and storage. Azmarda, with a sodium level of 1.773%, was comparable to certain brands, such as brand 2 (1.61%), brand 4 (1.73%), and brand 7 (1.58%). However, several other marketed products displayed higher sodium levels ranging from 3.164% to 3.951%. The sodium content in sacubitril/valsartan formulations, as demonstrated in our study, is crucial for patients with heart failure, a population often sensitive to sodium intake. Elevated sodium levels can contribute to fluid retention and exacerbate heart failure symptoms. Monitoring and controlling sodium content in pharmaceutical formulations, such as Azmarda, aligns with the broader goal of managing sodium intake in heart failure patients to optimize their overall cardiovascular health and well-being [18].

The dissolution studies underscore the importance of solubility profiles in predicting drug performance under physiological conditions. The innovator product, Azmarda, exhibited a substantial release of both sacubitril/valsartan within 45 minutes, surpassing other products. Most of the marketed products exhibited very negligible drug release in 0.1N HCl, suggesting they might have been formulated as mere physical mixtures of sacubitril/valsartan. Products with comparable drug release might have surfactant-based formulations.

The superior solubility of Azmarda, as evidenced by dissolution studies in 0.1N HCl, holds clinical relevance for patients with heart failure. Improved solubility can potentially enhance drug absorption, leading to more predictable and consistent therapeutic effects. In heart failure patients, where maintaining stable medication levels is crucial, the enhanced solubility of Azmarda may contribute to better management of symptoms and overall cardiovascular health [9, 11, 19].

The absence of identical crystal lattice structures in generic products, as indicated by PXRD analysis, highlights the absence of co-crystal technology in the formulation and raises concerns about the interchangeability and equivalence of these formulations, particularly for patients with heart failure. Crystal structure variations may impact the bioavailability and efficacy of the medication, emphasizing the importance of ensuring consistent and reliable therapeutic outcomes.

These findings underscore the critical need for stringent quality control measures in generic formulations, aligning with regulatory considerations for generic drug approval and ensuring the reliable and standardized treatment of heart failure patients [20].

Strengths of our study include employment of comprehensive approach, utilizing various analytical techniques such as DSC, PXRD, sodium content determination, and dissolution studies. This multi-faceted analysis provided a thorough understanding of the sacubitril/valsartan formulations. The investigation of the cocrystal form in Azmarda tablets added an innovative dimension to the study, shedding light on the unique characteristics of this formulation. This emphasis on cocrystal technology is particularly relevant in the pharmaceutical field. Authors also acknowledge a few limitations of the study. First, the study was focused on a specific number of brands, and the generalizability of the findings to all sacubitril/valsartan formulations may be limited. Including a larger sample size could enhance the study's external validity. Second, the study discussed the clinical implications of certain characteristics, such as solubility, the absence of direct clinical data limits the ability to draw concrete conclusions about the real-world impact on patients with heart failure. Clinical studies with different formulations may provide additional insights.

The innovative cocrystal technology employed in Azmarda tablets has demonstrated superior dissolution properties, thermal stability, and unique crystal lattice structures compared to various marketed products. These attributes may have important implications for the clinical efficacy, safety, and regulatory considerations of sacubitril/valsartan-containing formulations in heart failure patients.

Disclosures

Acknowledgements: Authors would like to acknowledge Sneha Badgujar (Sqarona Medical Communications LLP, Pune) for providing medical writing support.

Lab Names:  Vimta Labs Ltd, Hyderabad and Accuprec Research Labs Pvt Ltd, Ahmedabad.

Funding: None

Conflict of Interest: No conflict of interest to declare

1. Sible AM, Nawarskas JJ, Alajajian D, Anderson JR. Sacubitril/Valsartan: A novel cardiovascular combination agent. Cardiol Rev. 2016;24(1):41-7.
2. McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014;371(11):993-1004.
3. Wachter R, Senni M, Belohlavek J, Straburzynska-Migaj E, Witte KK, Kobalava Z, et al. Initiation of sacubitril/valsartan in haemodynamically stabilised heart failure patients in hospital or early after discharge: primary results of the randomised TRANSITION study. Eur J Heart Fail. 2019;21(8):998-1007.
4. Thomas JE, Nayak UY, Jagadish PC, Koteshwara KB. Design and characterization of valsartan co-crystals to improve its aqueous solubility and dissolution behavior. Res J Pharm Technol. 2017;10(1):26-30.
5. Nanda A, Anand R. Formulation and evaluation of cocrystals of a BCS class II drug using glycine as coformer. Int J App Pharm. 2022;14(6):68-76.
6. Emami S, Siahi-Shadbad M, Adibkia K, Barzegar-Jalali M. Recent advances in improving oral drug bioavailability by cocrystals. BioImpacts. 2018;8(4):305-20.
7. Panzade P, Shendarkar G, Shaikh S, Balmukund Rathi P. Pharmaceutical cocrystal of piroxicam: design, formulation and evaluation. Adv Pharm Bull. 2017;7(3):399-408.
8. Zhang M, Zou Y, Li Y, Wang H, Sun W, Liu B. The history and mystery of sacubitril/valsartan: from clinical trial to the real world. Front Cardiovasc Med. 2023;10:1102521.
9. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46(1-3):3-26.
10. Aitipamula S, Banerjee R, Bansal AK, Biradha K, Cheney ML, Choudhury AR et al. Polymorphs, salts, and cocrystals: what's in a name? Cryst. Growth Des. 2012;12(5):2147-52.
11. Thakuria R, Delori A, Jones W, Lipert MP, Roy L, Rodríguez-Hornedo N. Pharmaceutical cocrystals and poorly soluble drugs. Int J Pharm. 2013;453(1):101-25.
12. Shan N, Zaworotko MJ. The role of cocrystals in pharmaceutical science. Drug Discov Today. 2008;13(9-10):440-6.
13. Kawakami K. Pharmaceutical applications of thermal analysis. Handbook of Thermal Analysis and Calorimetry. 2018;8:613-41.
14. Haleblian J, McCrone W. Pharmaceutical applications of polymorphism. J Pharm Sci. 1969;58(8):911-29.
15. Scrivens G, Ticehurst M, Swanson, JT. Strategies for improving the reliability of accelerated predictive stability (APS) studies. Accelerated Predictive Stability. 2018;175-206.
16. Jones W, Motherwell WDS, Trask AV. Pharmaceutical cocrystals: an emerging approach to physical property enhancement. MRS Bulletin. 2006;31(11):875-79.
17. Vishweshwar P, McMahon JA, Bis JA, Zaworotko MJ. Pharmaceutical co-crystals. J Pharm Sci. 2006;95(3):499-516.
18. Cogswell ME, Mugavero K, Bowman BA, Frieden TR. Dietary sodium and cardiovascular disease risk--measurement matters. N Engl J Med. 2016;375(6):580-6.
19. Bolla G, Nangia A. Pharmaceutical cocrystals: walking the talk. Chem Commun (Camb). 2016;52(54):8342-60.
20. Guidance for industry: Dissolution Testing of Immediate Release Solid Oral Dosage Forms. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), August 1997.