Amide Codrug Approach: Synthesis and Biopharmaceutical Evaluation of Nonsteroidal Anti-Inflammatory Drug and Calcium Channel Blocker Conjugates

Introduction

The co-drug, also known as a drug-drug conjugate or mutual prodrug, is linked to one another or pro-drugs thereof, via a labile covalent bond. These drugs are usually selected to target multiple receptors of comorbidities or to provide synergistic treatment of the target disease or disorder. The co-drug exchanges a dormant group for an active one that is either directly or indirectly connected. It must remain stable in the gastrointestinal tract before being hydrolyzed to form two (or more) distinct medications that are well absorbed and both the components are released simultaneously and quantitatively after absorption without compromising therapeutic affinity and efficacy. Co-morbidity is the presence of one or more disorders (or diseases) in addition to primary disease or disorders. It is linked to a slower rate of recovery and a higher risk of death and long-term impairment. The major comorbidity of osteoarthritis (OA) patients is hypertension.^1,2 Osteoarthritis usually begins after 40 years of age and symptoms begin in the middle age. Its prevalence after the age of 65 years is about 60% in men and 70% in women.^3-6 Hypertension, the most common cardiovascular disease can result in target organ damage, increased incidence of renal and cardiac failure as well as stroke. As per report, 40% of patients with osteoarthritis have concomitant diagnosis of cardiovascular disease, particularly hypertension. Hence, co administration of nonsteroidal anti-inflammatory drug (NSAID) inhibitors with antihypertensive agents is common in clinical practice.^5,7

Various codrug techniques have been inspired by rationale and have shown as potential as drugs against a plethora of diseases including pain, inflammation, cancer, bacterial infections, Alzheimer’s disease, and others. Hepatoprotective and antimycobacterial isoniazide-alpha-lipoic acid amide codrug,⁸ azo-linked sulphonamide scaffold to the salicylamide pharmacophore strategy,⁹ secnidazole and ciprofloxacin codrug approach,¹⁰ dexamethasone and fumaric acid ester conjugate codrug for inflammatory disorders,¹¹ 5-FU/heme oxygenase inhibitor based codrugs therapy for anticancer,¹² paracetamol conjugate with ibuprofen and naproxen for antiinflammatory and anticoagulant effects¹³ and azidothymidine (AZT) with dihydroartemisinin (DHA) for HIV/malaria combination therapy¹⁴ are some recent approaches in codrug design. These techniques are often utilized to improve physicochemical, biological and drug transport properties and polypharmacy in patients.¹⁵

In this context, the present study aimed on the synthesis and evaluation of the novel amide based codrugs of various Non-steroidal anti-inflammatory agents (NSAIDs) and Amlodipine (Calcium channel blockers (CCB), first line antihypertensive), to eliminate the adverse gastrointestinal effects, to improve delivery, therapeutic efficacy to address comorbid conditions of hypertension, osteoarthritic pain and inflammation in geriatric patients with significant reduction of polypharmacy.

Material and Methods

Chemical and Reagents

NSAIDs and Amlodipine besylate were obtained from Micro Labs Ltd. & Strides Pharma Science Ltd, Bangalore. The reagents and solvents were purchased from Sigma Aldrich, CDH, Merck chemicals. The reactions were monitored by thin layer chromatography (TLC) on precoated silica G plates using visualization chamber. REMI thermos-controlled magnetic stirrers (5 MLH plus) were used for stirring of the reaction mixtures. Solvent recovery was done using Rotavapor R-300 (Buchi). Melting point was measured using the paraffin bath. IR spectra were obtained using Fourier-transform infrared spectroscopy (FT-IR) Shimadzu model IR-Affinity-1 spectrophotometer using VARIAN., 1 H,13C NMR spectra were recorded on Brucker WM 400 spectrometer (Bruker AG) was conducted at Sophisticated Analytical Instrumentation Facility (SAIF), Punjab. Mass spectra were performed using WATERS-XEVO C2-XS-QToF High-Resolution mass spectrometer (HRMS) equipped with a custom-made electrospray interface (ESI) at School of Advanced Science (SAS) Vellore Institute of Technology, Tamil Nadu and the hydrolysis study was monitored using USP dissolution apparatus, USP type II apparatus (paddle type).

Chemistry

General Synthesis Procedure

To a mixture of 0.002 mol of acid (NSAIDs) and 0.002 mol of amine (Amlodipine) in DCM (dichloromethane), 0.001 mol DMAP (dimethyl amino pyridine) was added with continuous stirring. After 30 min of stirring at 0°C, the cooled solution of 0.002 mol of DCC (N,N`dicyclohexylcarbodiimide) was added in to the above reaction mixture and allowed to stir at room temperature under the inert environment for 15-18 h. The reaction was monitored using TLC using Chloroform: methanol (8:2 w/w) as the solvent system. After completion of the reaction, the product was extracted using ethyl acetate. Repeated washing with 1N HCl, 10% sodium bicarbonate and 5% NaOH solution was continued. The product was purified using silica column using suitable solvent (Figure 1).^16,17 The Ibuprofen-Amlodipine (IBAM), Aspirin-Amlodipine (ASAM) and Naproxen-Amlodipine (NPAM) codrugs were synthesized using the described method.

Spectral Analysis

The spectral analyses of Codrug derivatives were carried out. The IR, Mass, 1H NMR and 13C NMR spectral interpretation were done and explained in results and discussion sections.

Biopharmaceutical Evaluation

Solubility

Approximately 10 mg of IBAM, ASAM and NPAM conjugate was dissolved in different solvents measured by saturation shake flask method. The excess amount of drug was placed in contact with various solvents in the Eppendorf tube. In a horizontal shaker, the samples were shaken for 48 hours at 37 ͦ C. The resultant sample was centrifuged, filtered and the supernatant was passed through a syringe filter. The amount of drug solubilized was analyzed spectrophotometrically.^18,19

Partition Coefficient

The n-Octanol phases were saturated with distilled water for at least 24 hours before the experiment. A solution of codrug conjugate (10−4M) was prepared with distilled water. Then, 2 mL of this solution was transferred to 10 mL test tubes containing 2 mL of the organic phase. The tubes were then stoppered and agitated for 24 hours at room temperature. After centrifugation at 3500 U/ min for 15 minutes, the concentration of the drug in the water phase was analyzed spectrophotometrically. The concentration of the drug in n-octanol was calculated from the difference between the initial and final concentrations in the water phase. Three replicates were used for the concentrations of n-octanol–distilled water solutions for partition coefficient calculations.¹⁹

In Vitro Hydrolysis Study

Methods

The Absorption maxima of the pure drugs Ibuprofen, Aspirin, Naproxen, Amlodipine besylate and Codrugs were determined using UV/Visible spectroscopy in hydrochloric acid buffer (pH 1.2), and phosphate buffer (pH 7.4). Solutions ranging from 5-200 µg/mL were scanned from 200-400 nm using UV spectrophotometer. The Detection wavelength was selected by overlaying the spectra of all components depending on their isobestic points.¹⁹

Hydrolysis studies

The hydrolytic studies of codrugs of IBAM, ASAM and NPAM were carried out in simulated gastric fluid (SGF) at pH 1.2, simulated intestinal fluid (SIF) at pH 7.4, maintained at a temperature of 37±0.5ºC.

Procedure

A solution of 10 mg of codrug was prepared in 90 mL of Simulated Gastric Fluid, (SGF) (pH 1.2) and Simulated Intestinal Fluid (SIF) (pH 7.4). An aliquot of 15 mL of this solution was withdrawn repeatedly and placed in test tubes maintained at 37±0.5ºC. Samples were withdrawn at 15 min interval of time up to 2 h for the SGF solution and up to 5 h for the SIF. Each aliquot was withdrawn from different test tubes and was transferred to micro centrifuge tubes followed by addition of methanol to make up the volume. The tubes were placed in freezing mixture in order to arrest further hydrolysis, followed by vortexing at high speed for 5 min. After vortexing, the tubes were centrifuged at high speed (3000 rpm) for 5 min. A 5 mL of clear supernatant obtained from each tube was measured on UV spectrophotometer for the amount of free drug released after the hydrolysis of IBAM, ASAM and NPAM in SGF at 237, 240 and 251 nm, respectively and 234, 272 and 306 nm, in SIF, respectively.²⁰

Results and Discussion

The reaction used for synthesis is shown in the Scheme 1 (Figure 1). Amide linked NSAIDs and CCB codrugs IBAM, ASAM and NPAM were synthesized by one pot amidation reaction using DCC and DMAP as coupling agent. The synthesized codrugs were characterized on the basis of molecular weight, color, melting-point and thin-layer chromatography (Table 1) and FTIR, 1H NMR, 13C NMR, MASS spectroscopy. Their solubility (Table 2), partition coefficient (Table 3), and hydrolytic rates (Figure 2) were determined.

Spectral data of IBAM codrugs: IR (cm^-1): 3283 (NH), 1691 (amide I), 1613 (amide II), 1443(C=N ring str.); 1H NMR (δ, ppm) (CDCl3): 7.72 (s, 1H, NH), 7.36- 7.11(m, 4 H, ArH), 7.26 & 7.09 (t,1H, CONH), 4.92 (2H, CH=CH), 3.37 (2H, OCH3), 2.50 (1H, CH in ring); 1.8 (m, 1H, CH), 0.90(d,6H,CH3); 13C NMR (δ, ppm) (CDCl3): 17.36, 39.14, 39.23, 39.37, 39.51, 39.65, 39.71, 39.93, 40.05, 48.03, 61.79, 65.23, 112.92, 115.73, 116.49, 117.24, 128.57, 128.16, 129.22, 129.37, 129.72, 131.62, 132.64, 133.44, 134.12, 134.63, 134.71, 137.30, 158.83, 168.10; Mass (m/z): 617(M⁺ ).

Spectral data of ASAM codrugs: IR (KBr, cm^-1): 3374 (NH str), 1640 (CO str Amide I), 1590 (NH Amide II),1340-1363cm^-1(C-N str), 1H NMR (δ, ppm) (CDCl3): 7.38 (s, 1H, NH), 7.19-7.08 (m, 4 H, ArH), 7.22 & 7.08 (t,1H, CONH), 4.92 (2H, CH=CH), 3.37 (s, 2H, OCH3), 2.50 (1H, CH in ring), 1.8 (m, 1H, CH), 0.90(d,6H,CH3); 13C NMR (δ, ppm) (CDCl3): 17.21, 39.09, 39.23, 39.37, 39.51, 39.65, 39.71, 39.93, 40.05, 48.03, 61.79, 65.23, 112.92, 115.73, 116.49, 117.24, 128.57, 128.16, 129.22, 129.37, 129.72, 131.62, 132.64, 133.44, 134.12, 134.63, 134.71, 158.15 , 168.12; Mass (m/z): 551(M⁺ )

Spectral data of NPAM codrugs: IR (KwBr, cm^-1): 3424 (NH), 1686 (amide I), 1626 (amide II), 1270 (CONH str.), 1491 (C=N ring str.); 1H NMR (δ, ppm) (CDCl3): 7.46 (s,1H, NH),7.24-7.11 (m, 4 H, ArH), 7.24 & 7.04 (t,1H, CONH), 4.92 (2H, CH=CH), 3.37 (2H, OCH3), 2.50 (1H, CH in ring); 1.8 (m, 1H, CH), 0.90(d,6H,CH3); 13C NMR (δ, ppm) (CDCl3): 17.21, 39.09, 39.23, 39.37, 39.51, 39.65, 39.71, 39.93, 40.05, 48.03, 61.79, 65.23, 112.92, 115.73, 116.49, 117.24, 128.57, 128.16, 129.22, 129.37, 129.72, 131.62, 132.64, 133.44, 134.12, 134.63, 134.71, 137.30, 169.23, 171.44, 176.54; Mass (m/z): 620.6 (M⁺ )

The IR spectra of synthesized codrugs predicted distinctive singlet peaks between 3424-3283cm^-1 due to N-H str of the secondary amide linkage, C=O (amide I) stretching bands between 1691-1640 cm^-1 whereas, amide II (w) band was between 1626-1590 cm^-1. 1H NMR spectra of Codrugs showed chemical shift (ppm) at 1.12-2.5 (CH3 terminal peaks), 3.23-3.51 (CONH), 8.18-9.77 (NH) and 7.28-7.82 (multiplicity of relative number of different proton of benzene ring), The 13C NMR spectra of range 168-176, and MASS spectrum corresponds to molecular ion peak establishing the formation of amide linked codrugs, respectively.

Solubility and Partition coefficient

The synthesized codrugs were insoluble in water and 0.1 N HCl, slightly soluble in Phosphate buffer pH 7.4, but showed moderate-to-high solubility in methanol, chloroform and acetone indicating that they are lipophilic in nature. Partition coefficient study of codrugs showed that the major fraction of the codrugs was partitioned towards the organic phase. It indicates enhancement in the lipophilic property, which might be favorable to biological absorption.

The in vitro hydrolysis studies were designed to mimic the entire gastrointestinal tract pH. The minimum reversion was observed at gastric pH (SGF, pH 1.2) suggesting the stability of synthesized codrugs in gastric pH. However, at higher pH values i.e., in SIF representing intestine, the percentage reversion was significantly good, thereby making the free drug available for absorption in the intestine. Also, the process of reversion increases almost linearly with time at intestinal pH.

The patterns of hydrolysis of IBAM, ASAM and NPAM codrugs are shown in Figure 2. The amount of free drug regenerated (% drug release) on hydrolysis of codrugs in SGF (pH 1.2) was found as 3.14%, 5.96% and 4.78% and SIF (pH 7.4) was found as 12.64%, 16.3% and 13.13%, respectively. The hydrolysis studies of codrug showed that their release was dependent on pH of the medium. The codrug showed negligible hydrolysis in pH 1.2 which might be useful in minimizing the GIT disturbances. They also demonstrated slightly better enhanced hydrolysis in pH 7.4 due to enhancedment in lipophilicity, resulting in a and showed controlled and delayed release pattern,. This level of hydrolysis was sufficient for easy absorption however enough to get absorbed easily through the GIT, and produceleading to the desired biological effect. Further, evaluation of enzymatic hydrolysis studies of the codrugs in SIF+80% human plasma to estimate the bioavailability and dose of codrug is essential.

Conclusion

In conclusion, the synthesis and evaluation of the amide-based codrugs have demonstrated their potential as innovative drug delivery systems. The codrug synthesis proved chemical conjugation's ability to develop new drugs. These codrugs exhibit better physicochemical characteristics and prolonged active moieties release. This sustained release characteristic may reduce dosing frequency and enhance patient compliance relative to parent medicines. These findings suggest that the amide-based codrug may increase drug distribution and therapeutic effects. This research emphasises prodrug-based approaches in pharmaceutical research and suggests the amide-based codrug for drug distribution and therapeutic efficacy. Future research could optimise the codrug formulation, conduct more in vivo tests, and apply it to specific therapeutic targets. This could pave the way for revolutionary drug delivery systems in the pharmaceutical industry.

Conflict of Interest

None

Acknowledgments

Authors are thankful to the Management, Krupanidhi Group of Institutions, Dr. Raman Dang, Principal, Krupanidhi College of Pharmacy, Dr. Amit Kumar Das, Former project guide, and to Dr. Kuntal Das, Research Director & Professor, Mallige College of Pharmacy, Bangalore for their constant support and for providing necessary infrastructure and resources for the successful completion of the study.