Ciprofloxacin Hippurate Salt: Crystallization Tactics, Structural Aspects

Subscriber access provided by Northern Illinois University

Article

Ciprofloxacin hippurate salt: crystallization tactics, structural aspects and biopharmaceutical performance Renu Chadha, Pawanpreet Singh, Sadhika Khullar, and Sanjay K. Mandal Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00533 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Ciprofloxacin hippurate salt: crystallization tactics, structural aspects and biopharmaceutical performance Renu Chadha*1†, Pawanpreet Singh†, Sadhika Khullarф and Sanjay K. Mandal‡ †University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, INDIA. фDepartment of Chemistry, D.A.V. University, Jalandhar - Pathankot National Highway, Jalandhar-144012, Punjab, INDIA. ‡Department of Chemical Sciences, Indian Institute of Science Education and Research, Mohali, Sector 81, Manauli P.O., S.A.S. Nagar-140306, Punjab, INDIA. Abstract Ciprofloxacin (CP) is a widely prescribed fluoroquinolone antibacterial for the treatment of several types of bacterial infections; however, it suffers from unfavourable biopharmaceutical characteristics such as solubility as well as bioavailability. To improve dissolution and bioavailability characteristics, a salt of the drug has been prepared by treating hippuric acid with ciprofloxacin using the solvent assisted grinding technique. Furthermore single crystals of the salt were obtained by vapor diffusion technique. The new phase was analyzed using FTIR, DSC, PXRD and structural parameters were determined using single crystal X-ray diffraction. Solubility and intrinsic dissolution rate of the salt was measured in pharmaceutically relevant buffer solution with pH 1.2 and water (pH 6.3). In the aqueous solution, the salt demonstrated solubility improvement (22-fold) and faster dissolution rate than the parent form of the drug. Pharmacokinetic studies on rats showed double plasma AUC values in a single dose. The antibacterial efficacy (MIC) of the salt was found to be high even at low concentration as compared to the parent molecule. Thus the present work demonstrated the diverse pharmaceutically relevant properties, including dissolution, pharmacokinetic and antibacterial efficacy by new crystalline salt. Further, the vapour diffusion technique to attain single crystals of the prepared salt has also been assessed. Keywords: ciprofloxacin, vapour diffusion, pharmacokinetic, dissolution, antibacterial.

1

*Prof. Renu Chadha, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, INDIA. E-mail: [email protected]; Tel: +91-9316015096. 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ciprofloxacin hippurate salt: crystallization tactics, structural aspects and biopharmaceutical performance Renu Chadha*†, Pawanpreet Singh†, Sadhika Khullarф and Sanjay K. Mandal‡ †University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, INDIA. фDepartment of Chemistry, D.A.V. University, Jalandhar - Pathankot National Highway, Jalandhar-144012, Punjab, INDIA. ‡Department of Chemical Sciences, Indian Institute of Science Education and Research, Mohali, Sector 81, Manauli P.O., S.A.S. Nagar-140306, Punjab, INDIA. *Prof. Renu Chadha, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, INDIA. E-mail: [email protected]; Tel: +91-9316015096. Abstract Ciprofloxacin (CP) is a widely prescribed fluoroquinolone antibacterial for the treatment of several types of bacterial infections; however, it suffers from unfavourable biopharmaceutical characteristics such as solubility as well as bioavailability. To improve dissolution and bioavailability characteristics, a salt of the drug has been prepared by treating hippuric acid with ciprofloxacin using the solvent assisted grinding technique. Furthermore single crystals of the salt were obtained by vapor diffusion technique. The new phase was analyzed using FTIR, DSC, PXRD and structural parameters were determined using single crystal X-ray diffraction. Solubility and intrinsic dissolution rate of the salt was measured in pharmaceutically relevant buffer solution with pH 1.2 and water (pH 6.3). In the aqueous solution, the salt demonstrated solubility improvement (22-fold) and faster dissolution rate 2 ACS Paragon Plus Environment

Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


than the parent form of the drug. Pharmacokinetic studies on rats showed double plasma AUC values in a single dose. The antibacterial efficacy (MIC) of the salt was found to be high even at low concentration as compared to the parent molecule. Thus the present work demonstrated the diverse pharmaceutically relevant properties, including dissolution, pharmacokinetic and antibacterial efficacy by new crystalline salt. Further, the vapour diffusion technique to attain single crystals of the prepared salt has also been assessed. Keywords: ciprofloxacin, vapour diffusion, pharmacokinetic, dissolution, antibacterial.

3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction It is an untoward; that active pharmaceutical ingredients commonly do not exhibit the range of physical properties, which makes them directly suitable for development. A number of drug molecules available in the market suffer from formulation difficulties due to their poor solubility in water, which in turn results in the inconsistent bioavailability1. Hence efforts are being made to modify these drugs by preparing their multicomponent forms like salts2, cocrystals3, polymorphs4, hydrates and solvates5. Crystal engineering has contributed immensely towards the rational design of these solid forms mainly by exploiting supramolecular synthons. Salts have been shown to modulate the solubility, bioavailability and stability of APIs6–8. The term pharmaceutical salt is used to refer to an ionisable drug that has been combined with a counter-ion to form an ionic complex. The counter-ion is either from the GRAS9 (Generally Regarded as Safe) listed molecules or may be another drug molecule. In the present art, we report the pharmaceutical salt (CP-HA) form of ciprofloxacin (CP) with hippuric acid (HA) (Scheme 1). Ciprofloxacin10 is a second-generation fluoroquinolone antibiotic with a broad antibacterial spectrum that includes Gram negative as well as Grampositive bacteria; however it is known to have poor solubility and permeability particularly in basic media11. In recent past, CP has drawn great interest from crystal engineers, due to its tendency to form robust supramolecular architectures with compounds having carboxylic acid functional groups. A search of the CSD (Version 5.36) for ciprofloxacin results in 90 hits, out of which 21 belongs to multicomponent forms12–15 of CP like hydrates, drug-drug cocrystal and salts while the remaining structures belong to metal complexes. As far as cocrystals and salts are concerned they can be formulated further to improve the solubility as well as bioavailability of this poorly soluble drug. However, little emphasis has been put on these aspects and the biopharmaceutical parameters (pharmacokinetics and antibacterial efficacy) 4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


have not been reported by previous workers. The prime motive of this investigation is to make a new synergistic pharmaceutical solid form with improved pharmacokinetic and antibacterial action and we succeeded in obtaining a new salt form i.e. ciprofloxacin hippurate. HA is a benzoylaminoethanoic acid obtained from the urine of horses and other herbivores. A biological investigation of HA shows that it has limited antimicrobial activity16 at acidic pH values, and has synergistic potentiating effects on the selective toxicity of a mixture of 13 substances within the circulatory system17. The method of vapour diffusion is successfully applied and assessed in obtaining single crystals; to study various synthons and structural parameters.

Scheme 1 Chemical structures of ciprofloxacin and hippuric acid. 2. Experimental Section 2.1 Materials The anhydrous crystalline form of CP was obtained as a gift sample from Ranbaxy India Pvt. Ltd. The investigated coformer HA was purchased from Himedia Labs, India and used as received. All other solvents and chemicals were of analytical grade or chromatographic grade. 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.2 Sample preparation A 1:1 salt of CP-HA was obtained when an equimolar mixture of CP 331.3 mg (1 mmoL) and HA 179.1 mg (1 mmoL) were ground together along with 2 drops (0.2 mL) of ethanol in an agate mortar and pestle for approximately 15 min. The powder obtained was dried and stored in airtight vials in a desiccator. 2.3 Melting Point All mp determinations were carried out by the capillary tube method and are reported without correction. 2.4 Differential Scanning Calorimetry (DSC) Differential scanning calorimetry was conducted using DSC Q20 (TA Instruments, New Castle, Delaware, USA). The sample (2-4 mg) was placed in sealed non-hermetic aluminium pan and was scanned at ramping rate of 10°C/min under a dry nitrogen atmosphere (flow rate 50 cc/min). The data were managed by TA Q series Advantage software (Universal analysis 2000, New Castle, Delaware, USA). 2.5 Fourier Transform-Infra Red Spectroscopy (FTIR) A Spectrum RX I FTIR spectrometer (Perkin Elmer, UK) was employed in the KBr diffusereflectance mode (sample concentration 2 mg in 20 mg of KBr) for collecting the IR spectra of samples. Dry KBr (50 mg) was finely ground in mortar and sample (1-2 mg) was subsequently added and gently mixed in order to avoid trituration of the crystals. A manual press was used to form the pellet. The spectra were measured over the range of 4000-400 cm1

. Data were analyzed using Spectrum software.

2.6 Optical Microscopy


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Optical microscopy was performed using a Leica DM 3000 phase contrast microscope with polarizing attachment. 2.7 Powder X-ray Diffraction (PXRD) PXRD patterns were collected on X’Pert PRO diffractometer system (PANalytical, Netherlands) with a CuKα radiation (1.54060 Å). The tube voltage and current were set at 45 kV and 40 mA respectively. The divergence slit and anti-scattering slit settings were set at 0.48° for the illumination on the 10 mm sample size. Each sample was packed in an aluminium sample holder and measured by a continuous scan between 5 and 50° in 2θ with a step size of 0.017°. The experimental PXRD patterns were refined using X’Pert High Score software. 2.8 Crystallization technique: During the crystallization trials, a combination of solution creeping and vapour diffusion18 of antisolvent was successfully applied to obtain single crystal. The slow diffusion of antisolvent vapours in saturated solutions of salt reduced the solubility and facilitated crystallization. Simultaneously, the solution “crept” along the glass walls of the container, and crystalline material accumulated above the liquid−air surface. The experimental setup consists of inner amber colored 5 mL test tube with the salt to be crystallized in a solvent and an outer clear glass 18 mL test tube with an antisolvent. The outer test tube was closed with parafilm and the two liquids start to equilibrate via vapor diffusion. Both tubes share a common gas phase. The saturated solutions of the salt were prepared in different solvents like methanol, acetone, acetonitrile, dimethoxy ethane and tetrahydofuran by dissolving 4 mg (0.0078 mmol) of CP-HA in an appropriate volume i.e. 2-3 mL approximately and put them into small test tubes which were further placed in larger test tubes and covered. The different solvent combinations engaged in vapor diffusion crystallization trials are shown in Table 1. 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

Table 1. Solvents used during vapor diffusion crystallization trials of CP-HA salt and crystals outcome. Solvent in the inner tube Methanol

Solvent in the outer tube Hexane

Acetone

Hexane

Acetonitrile Tetrahydrofuran Dimethoxy ethane

Tetrahydropyran Cyclohexane Hexane

Observation Block shape crystals at the bottom Blocky crystals along side walls. No crystals No crystals Block shape crystals along side walls.

2.9 Single Crystal X-ray Diffraction (SCXRD) Crystal evaluation and data collection were performed on a Bruker AXS Kappa APEX II diffractometer equipped with a CCD detector (with the crystal-to-detector distance fixed at 60 mm) using sealed-tube monochromated MoKα

radiation (0.71073 Å) at 296 K. The

diffractometer was interfaced to a computer that controlled the crystal centring, unit cell determination, refinement of the cell parameters and data collection through the program APEX II19. By using the program SAINT19 for the integration of the data, reflection profiles were fitted, and values of F2 and σ(F2) for each reflection were obtained. Data were also corrected for Lorentz and polarization effects. The subroutine XPREP in the SHELXTL20 program was used for the processing of data that included determination of space group, application of an absorption correction, merging of data, and generation of files necessary for solution and refinement. All calculations were performed using the SHELXTL Version 11.0 suite of programs. All figures were drawn using MERCURY21 3.5.1, hydrogen bonding parameters were calculated using PLATON22 software. 2.10 Equilibrium solubility study The solubility of CP, and CP–HA were determined using shake flask method in purified water (pH 6.3) and acidic buffer (pH 1.2), at 37°C. Solid samples were sieved using a Gilson mesh sieve (no. 80) to obtain uniform particle size. An excess amount was added to 20 mL of 8 ACS Paragon Plus Environment

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the buffer and purified water contained in a flask, pre-equilibrated at 37°C, the resulting slurry was shaken in a water bath shaker. At predetermined time slots (i.e. 2 h and 12 h) aliquots of the slurry were withdrawn. The suspensions were filtered through a 0.45-µm nylon filter and assayed for drug content by HPLC at 278 nm. Finally, the concentrations of CP and CP-HA in each time slot were calculated using their respective calibration curves which were prepared in purified water (pH 6.3) and acidic buffer (pH 1.2) using HPLC. After a period of 24 hours, the residual solids were filtered, air-dried and analyzed by FTIR. The experiment was performed in triplicate and values were expressed as mean ± standard deviation. The identity of the undissolved material after the solubility experiment was determined by FTIR. 2.11 Intrinsic dissolution study Intrinsic dissolution study of drug and the salt was performed for 4 hr in purified water and for 30 min in acidic media (pH 1.2) on United States Pharmacopeia certified Dissolution testing apparatus Labindia DS 8000 (Thane, Maharashtra, India) using rotating disk method. Prior to IDR estimation, standard curves for all the compounds in the two media were obtained by HPLC at 278 nm. Samples were sieved by Gilson mesh sieve (no. 80), 100 mg of the compound was compressed in die cavity with the aid of a bench top carver press for 4-5 min at 2000 psi, and the base plate was then disconnected from the die to expose a smooth, compact pellet with a 0.5 cm2 surface area. A neoprene gasket was placed around the threaded shoulder of the die, which was then screwed onto the shaft holder, and the shaft was mounted on the stirring drive mechanism of the dissolution apparatus. After mounting the dies were lowered into the dissolution vessel containing 900 mL of pure water at 37 °C and stirred at 100 rpm. Aliquot (5 mL) were withdrawn at specific time intervals with replacement, filtered through 0.45 µm membrane filters and concentration of the aliquots was determined from the predetermined standard curves of the respective compounds. The linear 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

region of the dissolution profile was used to determine the IDR of the compound (as the slope of the amount dissolved divided by surface area of the disc) per unit time. 2.12 High-performance liquid chromatography HPLC analysis was performed using a Waters Alliance system, which includes a Waters 2695 separation module, a Waters 2996 Photodiode Array Detector. For solubility and dissolution study, a SunFire™ C18 5-µm column (4.6 mm×150 mm) was used. Pharmacokinetic study was performed using Hypersil GoldTM C18 5-µm column (4.6 mm×250 mm). 2.13 Pharmacokinetics study In order to determine blood level of the prepared salt, a pharmacokinetic study was performed. The animals used in the experiment were adult male Wistar rats (weighing 230– 250 g) kept under standard laboratory conditions, 4 rats per cage with free access to standard laboratory diet. The animals were divided into two groups of six each. Group I was given the pure drug (CP). Group II received the salt CP-HA. A single dose of all the preparations was suspended in 0.5% (w/v) carboxymethyl cellulose (CMC) and administered by oral gavage. Each animal was treated with a CP dose equivalent to 5 mg kg−1 BW. The dose volume for all administration was maintained at 5 mL kg−1 BW. Serial blood samples were collected from the retro-orbital venous plexus of the rats at 0(pre-dose), 0.25, 0.5, 1, 2, 4, 6 and 8 hours into heparinized plastic tubes. The blood samples were then centrifuged at 10,000 rpm for 10 min. The plasma were separated and stored at -20°C until drug analysis carried out by the HPLC method. Pharmacokinetic parameters such as Cmax, tmax AUC0–t and relative bioavailability of the pure drug and the developed salt were calculated by using noncompartmental analysis.


Page 10 of 28

Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.14 Antimicrobial activity The antimicrobial activity of the prepared salt was investigated with respect to the free drug activity by determining the minimal inhibitory concentration (MIC) on Pseudomonas

aeruginosa (MTCC 3542), Staphylococcus aureus (MTCC 1144), Escherichia coli (MTCC 1687) and Klebsiella pneumoniae (MTCC 1030). MIC values were determined by a broth dilution test in Muller-Hinton Broth (Himedia laboratories India Pvt. Ltd) using sterilized culture tubes. For each determination, 11 tubes at various ciprofloxacin and CP-HA concentrations were prepared (0.004-0.1 µg mL−1). Inoculation was done by adding a microorganism suspension of 10 µl (1×105 cfu mL−1; cfu = colony forming units) to all of them. Inoculated tubes were incubated for 24 h at 37oC, after which they were inspected for turbidity. MIC was defined as the lowest antibiotic concentration inhibiting visible growth after this incubation period. A positive control (growth) was formed by culture broth with microorganisms, a negative control (sterility) by broth without microorganisms. 3. Results and Discussion Preliminary characterization of the CP-HA dried powder after grinding the CP and HA was done by determining its melting point and comparing it with the parent molecules. As can be seen from Table 2, the mp of CP and HA agree well with the reported ones 24, whereas the mp of CP-HA was different to those of the API and coformer, suggesting the formation of a new solid phase. Table 2. Melting points (mp) determined by the capillary tube method for CP, HA, and CPHA. Sample CP

mp (oC) 269-272

HA CP-HA

186-189 213-217

Reported mp (oC) 255-25722 270-27523 187-18822 -



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.1 Differential Scanning Calorimetry (DSC) The DSC of CP-HA exhibits one sharp endotherm at 218.900C, corresponding to its melting point (onset 214.540C), followed by an exotherm at 280.370 C ascribed to drug decomposition as shown in Figure 1. The endothermic peak of CP-HA at 218.900C was different from both the drug i.e. 273.180C; onset 270.500C and coformer i.e. 190.080C; onset 188.350C, showing the formation of a new stable phase.

Figure 1. Overlaid DSC curves of CP, HA and CP-HA. 3.2 Fourier Transform-Infra Red Spectroscopy (FTIR) FTIR (Figure 2) shows remarkable increase in intensity for ‘N-H’ stretching frequency at 3432 cm-1. This is due to H-bonding between the CP and HA molecules. The broad IR absorption frequencies 2509-2535 cm-1 indicate the protonation of the piperazinyl ring N


Page 12 of 28

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


atom (NH2+). Furthermore, pKa values (Table S2) are consistent with deprotonation of the hippuric acid used (salt formation) compared with the COOH of drug molecule (zwitterions). The same is visualized in the ORTEP (Figure S1). Moreover, the appearance of two characteristic carboxylate IR absorption vibrations at 1542 and 1385 cm−1 due to asymmetric and symmetric O–C–O stretch, respectively, confirmed the proton transfer from the–COOH group in HA. The ‘C═O’ stretching frequency for HA is 1750 cm-1 where as it is 1723 cm-1 for CP-HA salt. This corresponds to the dimer formation of HA molecules within the crystal packing of CP-HA clearly visible from crystal structure of the same.

Figure 2. Comparison of FTIR vibrational frequencies between CP, HA and CP-HA. 3.3 Crystallization: The CP-HA crystallizes by diffusion of hexane vapours into the saturated solution at room temperature. After 1 week, well-formed colourless blocky crystals of salt appear close to the liquid−air surface in case of acetone:hexane (Figure 3C) and dimethoxy ethane:hexane 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(Figure 3E) combinations whereas colourless block shaped crystals (Figure 3D) exclusively at the bottom of the container in methanol:hexane combination. No crystals appear in rest of the combinations. The crystal habits were found to be identical in all the solvent combinations. Crystals of the salt appear first on the walls of the container, and the crystallization continues from the solution that creeps up the walls. The snapshots of crystallization assembly were given in Figure 3A and 3B. The crystals of salt were stable in solution for months; they do not redissolve upon standing and can easily be separated by decantation.The crystals from methanol:hexane combination were further used for structure determination through single crystal X-ray diffraction. The crystals from three combinations were identified and compared with each other by DSC (Figure S2 in supplementary information) and were found to be identical. The phenomenon of vapor diffusion is found to be an asset for the beginner in the field of single crystal growth when their usual single crystal growing methods fail to work. We have also listed a limited set of solvent combinations that we found useful for growing single crystals of CP salts and we think; it will also work on other set of CP multi-components.


Page 14 of 28

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Bench top setup for vapour diffusion (A) Single assembly (B) Multiple assemblies in a stand, amber coloured inside tube were used for protection against photo-chemical sensitization. Optical micrographs of CP-HA crystals from different solvent combinations (C) Acetone- hexane (D) Methanol-hexane (E) Dimethoxy ethane- hexane. All images are on the same scale i.e. 10X. 3.4 X-ray analysis 3.4.1 Powder X-ray Diffraction (PXRD) Every new crystalline material exhibits unique peaks indicative of reflections from specific atomic planes. Characteristic reflections at 2θ values of 8.5, 13.6, 14.7, 16.8, 20.9, 25.56 and 36.1° as shown in Figure 4 were observed for CP. PXRD pattern of CP–HA showed new peaks at 2θ values of 9.2°, 12.1°, 17.3°, 27.3° and 48.4° which were absent in both the drug



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and the coformer. In addition, peaks at positions 8.5°, 11.4°, 41.8°, 45.7° and 46.3° present in CP were absent in the case of CP–HA. The powder pattern of CP-HA differed clearly from those of the individual APIs, indicating the presence of a new crystalline phase.

Figure 4. PXRD patterns of CP, experimental CP-HA and simulated CP-HA. 3.4.2 Single Crystal X-ray Diffraction (SCXRD) The crystallographic data for CP-HA are listed in Table 3. The salt CP-HA crystallizes in the monoclinic space group C2/c with one molecule of each ionized drug in the asymmetric unit.


Page 16 of 28

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Transfer of proton from the acid group of HA (Figure 5) to the secondary N-atom on the piperazine ring of CP results in the ionization of molecules via N-H...O interactions (see below). Table 3. Crystallographic Data and Structure Refinement Parameters for CP-HA. Chemical Formula Formula weight Temperature (K) Wavelength (Å) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Dcalculated (g/cm3) µ (mm–1) θ range (°) F(000) range h range k range l Reflections collected Independent reflections Observed reflections No. of parameters R1 (I > 2σ(I))/ R1 (all data) wR2 (I > 2σ(I))/ wR2 (all data) GOF CCDC No.

C26 H27 F N4 O6 510.52 296 0.71073 Monoclinic C2/c 33.4838(19) 7.5637(5) 19.5174(12) 90 94.675(5) 90 4926.6 (5) 8 1.377 0.104 1.2 – 25.05o 2144 -39 to +39 -8 to +9 -23 to +23 17691 4341 2648 349 0.0465/0.0917 0.1224/0.1638 0.938 1402577



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Bonding interactions of CP-HA salt (a) Hydrogen bonded molecular unit in the CPHA crystal. (b) Tetrameric hydrogen bonding projections of CP and HA molecules within the salt. (c) Dimer formation between two HA molecules within CP-HA crystal packing. In the structure, piperazine ring of CP adopts a chair conformation as in the case of pure CP. The carboxylate group of HA interacts with one axial and one equatorial H–N(piperazine) group of two adjacent CP molecules via (N+(4)...O(1); d/Å, θ/°: 2.68, 171.0 and (N+(4)...O(2); d/Å, θ/°: 2.69, 169.0) forming a tetramer like assembly. Instead of this the HA molecules also form dimer using the amide-carboxylate synthon (N(1)...O(2); d/Å, θ/°: 2.91, 171.0). The carboxylic acid group of CP also forms an intramolecular hydrogen bond with the adjacent carbonyl group via O-H…O synthon (O(5) ...O(6); d/Å, θ/°: 2.54, 154.0). For full details of hydrogen bonding parameters see Table S1 in the Supporting Information. The HA molecules fits themselves as in a layering way (Figure 6) inside the pocket of CP molecules.


Page 18 of 28

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (a) Molecular packing projections for CP-HA viewed down the b-axis. (b) Layering of HA molecules within the pocket of CP molecules. The CP molecules are coloured in green. The HA molecules are coloured in blue. The H atoms are omitted. 3.5 Solubility and Intrinsic dissolution studies Solubility of CP and CP-HA were measured in pure water (pH 6.3) and 0.1 N HCl (pH 1.2) at 37°C. The equilibrium solubility values are provided in Table 4. The results showed a clear increase in the solubility of the drug as salt. The aqueous equilibrium solubility of the prepared salt was found out to be 22 times higher than pure ciprofloxacin, while in acidic medium (0.1 N HCl, pH 1.2); the salt was having lower solubility (7.4 mg/mL) as compared to the parent drug. Table 4: Solubility (in mg mL-1) of CP and its salt (n=3). Compound CP CP-HA

Pure water, pH 6.3 0.09 ± 0.01 1.98 ± 0.09

0.1 N HCl, pH 1.2 27.8 ± 1.05 14.6 ± 1.25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

The intrinsic dissolution rates (IDRs) of CP and CP-HA were also measured in both 0.1 N HCl and purified water (Figure 7) at 37°C as per the USP Pharmacopoeia guidelines for IDR calculation.

Figure 7. Dissolution profile of (a) CP and CP-HA in acidic medium (pH 1.2). (b) CP and CP-HA in pure water (pH 6.3). The IDR (Table 5) values were calculated up to 240 min of the dissolution curve in case of purified water and up to 30 min in case of 0.1 N HCl. Table 5: IDR (in mg cm-2 min-1) of CP and CP-HA. pH Pure water, pH 6.3 0.1 N HCl, pH 1.2

CP 0.04 5.2

CP-HA 0.33 1.3

The solubility of a salt may be affected by the factors such as solubility of the coformer, and particle morphology. In the first case, it is reasonable that the coformer (here HA) dissolves first in dissolution, followed by the transformation of the API into a higher energy amorphous phase through rearrangement of molecules caused by the voids created by dissolution of the coformers25 which further facilitates the dissolution process of CP-HA. Besides, morphology 20 ACS Paragon Plus Environment

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


may play a role in improving the dissolution rate of a solid form. Generally, block or plate morphologies provide higher surface area than an acicular morphology. This increases the exposure to the medium and hence the dissolution. In the present context, CP-HA has a blocky morphology (optical micrographs in Figure 3). This may be another reason for improved dissolution rate of the salt. The undissolved material analyzed at the end of the dissolution experiment by FTIR (Figure S3) was found to be stable to the dissolution conditions in pure water for 4 hr. The higher dissolution and solubility of the normally less soluble parent drug is due to protonation of the piperazine ring in acidic medium. 3.6 Pharmacokinetics study Plasma concentrations of CP and its salt were assessed by the sensitive HPLC method. The pharmacokinetic parameters (Table 6) for pure drug CP and CP–HA were calculated using the trapezoidal rule. Table 6. Relative pharmacokinetic parameters for CP and its salt. Drug/salt CP-HA CP

Cmaxa (ng mL-1) 453.05 256.29

AUC0-8b (ng h mL-1) 2530.3 1289.9

Rel. BA%c 1.96 -

a

Peak of maximum concentration. Area under the concentration–time profile curve until the last observation (up to 8 hours). c Relative bioavailability of prepared salt with reference to the pure drug. b

At a single administered dose of 5 mg/kg, salt showed a higher peak plasma concentration (453.05 ng/mL) compared to the pure drug (256.29 ng/mL, Figure 8), while AUC0–8 almost doubled for the salt. The higher plasma concentration of CP–HA than that CP justifies its enhanced in vivo absorption of the drug. By improving AUC values of the salt by a factor of 2, it is expected that the bioavailability of CP will go up.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

Figure 8. Mean plasma concentration−time profile of CP and its hippurate salt in rats. 3.7 Antimicrobial activity The antibacterial effectiveness of ciprofloxacin-hippuric acid salt against P. aeruginosa, E. coli, K. pneumoniea and S. aureus was assessed in comparison with an aqueous drug solution using a microbiological method. Table 7 shows the MIC values of the salt, determined after 24 hr and compared to free drug (CP in aqueous solution). In the parentheses of the first column of table, the exact amount of the API used was shown. Table 7. The antibacterial activity of CP and the reported salt. Compound CP (1) CP-HA (0.62)

S. aureus 0.3 ‹0.3

MIC (µg/mL) E. coli P. aeruginosa 0.06 0.3 ‹0.07 0.3

K. pneumoniea 0.3 0.4

It was clear from the readings that with lesser amount of drug either the same effect or better effect was observed, thus proving the salt to have enhanced pharmacological action.


Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. Conclusion The salt of an antibacterial drug, ciprofloxacin with hippuric acid has been successfully prepared and characterized by DSC, FTIR, PXRD and single crystal X-ray diffraction. The solubility (aqueous) and intrinsic dissolution rate of ciprofloxacin hippurate is superior to that of pure ciprofloxacin and have also been found to be stable. In addition, an increased oral bioavailability and synergistic pharmacological activity has also been observed. Thus the reported salt was found to be a promising candidate for next stage formulation development. Besides, the phenomenon of antisolvent vapour diffusion into saturated solutions could be an asset to the pharmaceutical chemist in the field of single crystal growth of salts. In conclusion, the design, synthesis and characterization of these kinds of salts are essential in order to further expand the scope of the available pre-formulation options beyond pure API forms. Associated content Supporting information Hydrogen bonding parameters and ORTEP view, IR spectra, DSC thermograms and PXRD patterns, pKa values and FTIR absorption bands. This material is available free of charge via the Internet at http://pubs.acs.org. Author information Corresponding Author *Phone: +919316015096, e-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgement and Disclosures The authors gratefully acknowledge the financial support provided by University Grants Commission (UGC), New Delhi, India; vide letter number F.5-94/2007(BSR) to Pawanpreet 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Singh as UGC-RFSMS fellow for accomplishing this work. The use of Powder X-ray facility at SAIF, Panjab University and single crystal X-ray facility at IISER, Mohali are gratefully acknowledged.


Page 24 of 28

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References 1. Datta, S.; Grant, D. J. W. Nat. Rev. Drug Discovery. 2004, 3, 42−57. (b) Meyer, M. C. Bioavailability of drugs and bioequivalence. In Encyclopedia of Pharmaceutical Technology; Swarbrick, J. Marcel Dekker Inc.: New York, 1998; Vol. 2, pp 33−58. 2. (a) Stahl, P. H.; Nakano, M. Pharmaceutical aspects of drug salt form. In Handbook of Pharmaceutical Salts: Properties, Selection, and Use; Stahl, P. H.; Wermuth, C. G, Eds.; Wiley-VCH Verlag: Weinheim, 2008; Chapter 4, pp 83-116. (b) Serajuddin, A. T. M. Adv. Drug Delivery Rev. 2007, 59, 603−616. (c) Childs, S. L.; Stahly G. P.; Park, A. Mol. Pharmaceutics. 2007, 4, 323−338. 3. (a) Duggirala, N. K.; Perry, M. L.; Almarsson, O.; Zaworotko, M. J. Chem. Commun. 2016, 52, 640-655. (b) Upadhyay, N.; Shukla, T. P.; Mathur, A.; Manmohana,; Jha, S. K. Int. J. Pharm. Sci. Rev. Res. 2011, 8, 144−148. (c) Brittain, H. G. Cryst. Growth Des. 2012, 12, 1046−1054. (d) Desiraju, G. R. Pharmaceutical salts and co-crystals: retrospect and prospects. In Pharmaceutical Salts and Co-crystals; Wouter, J., Quéré, L., Eds.; RSC Publishing: Cambridge, 2011; Chapter 1, pp 1−8. 4. (a) Byrn, S. R.; Pfeiffer, R. R.; Stowell, J. G. Polymorphs. In Solid-State Chemistry of Drugs; 2nd Eds.; SSCI, Inc.: West Lafayette, IN, 2002; Chapter 10, pp 256-829 (b) Bernstein, J. In Polymorphism in Molecular Crystals; 1st Eds, Oxford University Press: New York, 2002; (c) Lohani. S.; Grant D.J.W. Thermodynamics of polymorphs. In Polymorphism in the Pharmaceutical Industry; Hilfiker, R. 1st Eds, Wiley-VCH Verlag: Weinheim, 2006; Chapter 2, pp 21-42. (d) Brittain, H. G. In Polymorphism in Pharmaceutical Solids; 2nd Eds.; Informa Healthcare: New York, 2009; pp 192.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5. (a) Khankari, R. K.; Grant, D. J. W. Thermochim. Acta. 1995, 248, 61−79. (b) Kaushal, A.; Vangala, V. R.; Suryanarayanan, R. J. Pharm.Sci, 2011, 100, 1456−1466. 6. Pudipeddi, M.; Serajuddin, A. T. M.; Grant, D. J. W.; Stahl, P. H. In Handbook of Pharmaceutical Salts, Properties, Selection and Use; Stahland, P.H., Wermuth, C.G., Eds.; WileyVCH, Weinheim, Germany, 2002; pp 19-40. 7. Childs, S. L.; Chyall, L. J.; Dunlap, J. T.; Smolenskaya, V. N.; Stahly, B. C.; Stahly, G. P. Journal of the American Chemical Society. 2004, 126, 13335–13342. 8. Banerjee, R.; Bhatt, P. M.; Ravindra, N. V. Desiraju, G. R. Cryst. Growth Des. 2005, 5, 2299–2309. 9. GRAS and EAFUS (Everything Added to Food in the United States) materials list can be found at http://www.fda.gov/food/foodingredientspackaging/ucm115326.htm. 10. (a) Wise, R.; Andrews, J.M.; Edwards, L.J. Antimicrob Agents Chemother. 1983, 23, 559–564. (b) Roy, C.; Foz, A.; Segusa, C.; Tirado, M.; Tesvel, D. Infection. 1983, 11, 326–328. (c) Chin N.X.; Neu, H.C.; Antimicrob Agents Chemother. 1984, 25, 319– 326. (d) Smith, M.J.; Hodson, M.E.; Batten, J.C. Lancet. 1986, 327, 1103. 11. Breda, S. A.; Jimenez Kairuz, A. F.; Manzo, R. H.; Olivera, M. E. Int. J. Pharm. 2009, 371, 106. 12. Kruthiventi, A.K.; Roy, S.; Goud, R.; Javed, I.; Nangia, A.; Reddy, J. S. Synergistic pharmaceutical cocrystals., PCT/IN2009/000233. 13. Reddy, J. S.; Ganesh, S. V.; Nagalapalli, R. J. Pharm. Sci. 2011, 100, 3160–3176.


Page 26 of 28

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14. Vitorino, G. P.; Sperandeo, N. R.; Caira, M. R.; Mazzieri, M. R. Cryst. Growth Des. 2013, 13, 1050−1058. 15. Bag, P. P.; Ghosh, S.; Khan, H.; Devarapalli, R.; Reddy C. M. CrystEngComm. 2014, 16, 7393–7396. 16. Hamilton-Miller, J. M.; Brumfitt, W. Invest Urol. 1977, 14, 287–291. 17. Kulcsár, G. Cancer Detect Prev. 2000, 24, 485–495. 18. Spingler, B.; Schnidrig, S.; Todorova, T.; Wild F. CrystEngComm. 2012, 14, 751– 757. 19. APEX2, SADABS and SAINT, Bruker AXS Inc, Madison, WI, USA, 2008. 20. Sheldrick, G. M. Acta Crystallogr A. 2008, 64, 112–122. 21. Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edginton, P. R.; McCabe, P.; Pidocck, E.; Rodriguez-Monge, L.; Taylor, T.; Van de Streek, J.; Wood, P. A. J. Appl. Crystallogr. 2008, 41, 466. 22. Spek, A. L. PLATON, Version 1.62, University of Utrecht; 1999. 23. The Merck Index, XIV ed., Merck & Co. Inc., New Jersey, USA, 2006. 24. Ross, D.; Riley, C. Int. J. Pharm. 1990, 63, 237−250. 25. Guzmán, H. R.; Tawa, M.; Zhang, Z.; Ratanabanangkoon, P.; Shaw, P.; Gardner, C. R.; Chen, H.; Moreau, J. P.; Almarsson, Ö.; Remenar, J. F. J. Pharm. Sci. 2007, 96, 2686−2702.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only Ciprofloxacin hippurate salt: crystallization tactics, structural aspects and biopharmaceutical performance Renu Chadha*, Pawanpreet Singh, Sadhika Khullar and Sanjay K. Mandal

The present art describe the new salt form of ciprofloxacin with a GRAS status coformer (hippuric acid) by exploiting the technique of vapour diffusion to prepare single crystals. The work is fully supported by thermal, spectroscopic, diffraction, solubility and dissolution studies along with biological studies viz. pharmacokinetics and antibacterial activity (MIC).


Page 28 of 28

Ciprofloxacin Hippurate Salt: Crystallization Tactics, Structural Aspects

Recommend Documents