Preparation and characterization of co-crystals for improved physicochemical properties of poorly soluble drug

1
Presented By:
SANA ROOHI
170216886011
M. PHARMACY 2nd year
PHARMACEUTICS DEPT.
under the guidance of
Dr. MONIKA NIJHAWAN
M.PhArm, Ph.D
Department of Pharmaceutics

2
Introduction
Literature review
Materials and methods
Results and discussion
Conclusion
References

4
• Physicochemical properties of API are key parameters in developing acceptable
dosage form in determining the efficacy, activity of a drug.
• Co-crystals formation has emerged as a viable strategy towards improving the
solubility and bioavailability of poorly soluble drugs.
• Desiraju defined crystal engineering as ‘the understanding of intermolecular
interactions in the context of crystal packing and in the utilization of such
understanding in the design of new solids with desired physical and chemical
properties’.

• Aakeroy and Salmon defined co-crystals as structurally homogeneous crystalline
materials containing two or more components present in definite stoichiometric
amounts.
• “A stoichiometric multi-component system connected by non-covalent
interactions where all the components present are solid under ambient
conditions”.
• The FDA defines co-crystals as ‘solids that are crystalline materials composed of
two or more molecules in the same crystal lattice’.
• API-excipient molecular complex
5

6
Figure 1-1: Types of solid forms

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• It is assembly of 2 or more different molecules with superior physical properties
than individual component.
• Intermolecular interactions involved include van der waals contacts, stacking
interactions and the hydrogen bonding.
Figure 1-2: Adenine co-crystals

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Types of co-crystals
Anhydrates Hydrates (solvates)
Hydrates of co-
crystals of salts
Anhydrates of co-
crystals of salts
Figure 1-3: Types of co-crystals

9
• The coformer properties and interactions provide strategies to control co-crystal
solubility.
• Synthons are formed by the gathering of two molecules through molecular
functionalities that interact with each other in a predictable fashion by non-covalent
interactions.
• Intermolecular hydrogen bonding can be assessed using Cambridge Structural
Database, Hansen solubility parameters (HSPs), supramolecular synthon approach.
Figure 1-4: Common hydrogen bonded synthons used in crystal engineering

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Co-crystallization techniques
Classical/conventional techniques
Liquid-based methods
Solvent evaporation
technique
Slurry crystallization
Cooling crystallization
Anti-solvent addition
method
Solid-based methods
Neat grinding technique
Solvent-assisted grinding
technique
Thermal method
Advanced techniques
Microwave-assisted synthesis
Supercritical fluid technology
Ultrasound assisted co-
crystallization
High shear granulation
Hot-melt extrusion
Figure 1-5: Schematic representation of different co-crystallization techniques

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Solvent Evaporation: It involves super saturation of solution by evaporation, cooling
and addition of solubility changing solvent.
Eg: Fluoxetine hydrochloride-succinic acid, fumaric acid.
Slurry Crystallization: Equimolar proportion of the two coformers are dissolved in
small amount of different solvents at ambient temperature, evaporated and the solvent
is decanted and the material is dried.
Eg: Aspirin-4, 4-Dipyridil.
Solvent drop grinding: Grinding of two materials together with incorporation of small
quantity of solvent. Enhances the rate of co crystal formation, increased yield, control
polymorph production, better product crystallinity.
Eg: Caffeine-glutaric acid co-crystal.

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• Neat grinding: Mechanical grinding using ball mill, vibratory mill or by manual
grinding using motor and pestle. Polymorphic transition may occur.
Eg: Sulfadimidine –salicylic acid co-crystal.
• Supercritical fluid technology allows a single-step generation of particles. The
properties of different super critical fluids assist in generation of pure and dried co-
crystals.
• Antisolvent addition: It involves precipitation or recrystalization of the two co-
crystal former. Solvents consist of buffers and organic solvents.
Eg: Aceclofenac-chitosan (distilled water/sodium citrate).
• Hot melt extrusion: It involves highly efficient mixing and improved surface
contacts, co-crystals are prepared without use of solvent. The selection of this
method primarily depends on thermodynamic stability of compound.
Eg: Carbamazepine-nicotinamide co-crystals.

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• Pharmaceutical co-crystallization can be employed to all APIs and drugs lacking
ionizable functional groups (phenol) and compounds with sensitive group to
treatment of acids and bases.
• Enhances solubility and bioavailability of poorly soluble drugs.
• Improve the physicochemical properties of a drug without affecting its intrinsic
structure .
• Enhance other essential properties of the APIs such as flowability, chemical
stability, compressability and hygroscopicity.
• The existence of numerous potential counter molecules (food additives,
preservatives, pharmaceutical excipients) for co-crystal synthesis.
• Address intellectual property (IP) issues by extending the life cycles of old API.

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• In solid state grinding method, optimum temperature range should be known
• It is difficult to identify the structure.
• Phase separation of co-crystals into individual component on storage.
• Phase change may occur during formulation development of API.

15
Selection
and research
of APIs
co-crystal
formers
selection Empirical
and
theoretical
guidance
co-crystal
screening
co-crystal
characteri-
zation
co-crystal
performance
Figure 1-6: Steps for co-crystal design and preparation

• Supersaturation is used as a strategy to improve solubility and dissolution rate of
poorly soluble drugs.
• Two critical steps need to be maintained:
1. Generation of the metastable supersaturated state
2. Maintenance of the above state
16

17
Figure 1-7: The spring and parachute concept to achieve high apparent
solubility for insoluble drugs

18
Figure 1-8: Schematic representation of characterization of co-crystals

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• Melting point
• FTIR
• DSC (differential scanning colorimeter)
• Powder X-Ray Diffraction (PXRD)
• Saturation solubility studies
• Drug content determination
• Micromeritics
• In vitro studies

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• Pharmaceutical co-crystals of existing APIs exhibiting clinical advantages can be
developed as new drugs.
• Pharmaceuticals: The co-crystal formed from the chemotheraphy agent, tegafure
showed solubility much higher than that in pure crystalline phase.
• Cosmetics: co-crystals of 3-iodopropynyl butylcarbamate, an antifungal agent was
reported to have greater solubility in water, heat stability and better processability
properties.
• Agrochemicals: co-crystals were also used to raise the melting point of an
imidacloprid insecticide using oxalic acid with better shelf stability.
• Chromophores: co-crystals of titanyl fluorothalocyanine with titanyl fluorocyanine
have a novel spectrum with improved electrophotographic sensitivity.
• Alter electrical properties and shown to have potential as organic semi-conductors.

22
Title Authors
Year &
Journal
Conclusion
Synthesis of a
glibenclamide
cocrystal: full
spectroscopic and
thermal
characterization
Silva Filho SF
et al.
Journal of
Pharmaceutical
Sciences, 2018
Synthesised co-crystal of glibenclamide
using tromethamine (TRIS) by slow
solvent evaporation co-crystallization.
The co-crystal obtained was
characterized by XRD, DSC, Raman,
mid infrared and near-infrared
spectroscopy. The results showed the
formation of a co-crystal between API
and conformer with the synthons
corresponding to hydrogen bonding
between hydrogen in amines of
tromethamine and carbonyl and sulfonyl
groups in glibenclamide.

23
Title Authors
Year &
Journal
Conclusion
Solubility
enhancement of
lornoxicam by
crystal
engineering
D. D.
Gadade
et al.
Indian Journal
of
Pharmaceutical
Sciences,
March 2017
Co-crystals of lornoxicam were prepared by
neat grinding method with 19 different
coformers. The prepared co-crystals were
characterized by DSC, FTIR, XRD.
Maximum solubility and dissolution rate
were observed with co-crystal prepared
using saccharin sodium. Percent cumulative
drug release with marketed tablets
(Lofecam, Sun Pharma) was found to be
47.63±0.51% and 57.93±1.66% in distilled
water and phosphate buffer pH 7.4
respectively at the end of 60 min, while that
with optimized batch was 86.14±1.33% and
93.01±0.77% indicating improved
dissolution of lornoxicam by co-
crystallization

Title Authors
Year &
Journal
Conclusion
Enhancement of
solubility and
dissolution rate of
atorvastatin
calcium by co-
crystallization
Wicaksono
et al.
Tropical
Journal of
Pharmaceutical
Research, 2017
Co-crystallization of atorvastatin calcium
(AC) with isonicotinamide (INA) was
carried out by slow solvent evaporation
method using methanol. The solid obtained
was characterized by PXRD, DSC, FTIR,
SEM, and then further evaluated for
solubility and dissolution. The solubility of
ACINA co-crystal in distilled water (270.7
mg/L) was found to be significantly higher
than that of pure atorvastatin calcium
(140.9 mg/L). The dissolution rate of
ACINA co-crystal showed 2 - 3 times faster
drug release when compared to pure AC.
Formulation and
evaluation of
clarithromycin co-
crystals tablets
dosage forms to
enhance the
bioavailability
Pinki
Rajbhar et al.
The Pharma
Innovation
Journal, 2016
Clarithromycin co-crystals tablets were
prepared (solvent evaporation) using urea
co-crystals to improve the bioavailability.
Wet granulation method was attempted for
formulation of conventional tablets of
clarithromycin. It showed improved
solubility characteristics and in-vitro drug
release profile as compared to marketed
tablet (79.86%).
24

Title Authors
Year &
Journal
Conclusion
Three
pharmaceutical
co-crystals of
adefovir:
synthesis,
structures and
dissolution study
Xiaoming
Zhang et
al.
Journal of
Molecular
Structure, 2015
Three novel co-crystals of adefovir with PABA(1),
3,5-dihydroxybenzoic acid(2) and 2,6-
pyridinedicarboxlic acid(3). PXRD demonstrate
that co-crystal 1 and 2 form a strong hydrogen-
bond through the phosphoric acids of API with
water and carboxylic acids of CCF respectively.
co-crystal 3 is formed in which the phosphoric acid
groups of API are also held by the carboxylic acid
groups of CCF. The overall dissolution behavior
demonstrated that a complete release of co-crystal
3 was observed in 4 h, comparing 96.8%, 92.5%,
94.1% of co-crystal 1, 2 and API respectively
Solubility
enhancement of
nevirapine by
cocrystallisation
technique
Yogesh K.
Nalte et al.
Journal of
Pharmacy
Research,
2015
The co-crystals were prepared by neat grinding
method using maleic acid. Prepared co-crystals
were characterized by PXRD, DSC, FTIR.
Moreover they were studied for melting point
determination, flow property studies and
dissolution studies (0.1 N HCl, phosphate buffer
6.8). All the performed study revealed formation of
co-crystals, improvement in micromeritic
properties, dissolution. Drug solubility of
nevirapine was improved by 106 folds in 0.1N HCl
25

26
Title Authors
Year &
Journal
Conclusion
Utilization of
co-
crystallization
for solubility
enhancement of
a poorly soluble
antiretroviral
drug – ritonavir
Londhe et
al.
International
Journal of
Pharmacy and
Pharmaceutical
Sciences, 2014
Prepared co-crystals of ritonavir with different
co-formers succinic acid (SUC), adipic acid
(ADP), nicotinamide (NIC) and D-alanine (ALA)
in ratio of 1:5 (RTN : Co-former) using solvent
grinding method and methanol as a co-solvent.
The co-crystals characterized by melting point,
FTIR, DSC, XRD and solubility studies. Co-
crystals of drug with SUC, ALA and ADP
showed 6 folds increase in solubility and the co-
crystals of RTNSUC and RTNADP showed two
times faster drug release at initial time points as
compared to RTN alone but at the end of 1 hr,
only 15% increase in drug release was found.

Title Authors
Year &
Journal
Conclusion
Evaluation of
performance of
co-crystals of
mefloquine
hydrochloride in
tablet dosage
form
A. S. Shete et
al.
Drug
Development
and Industrial
Pharmacy, 2013
Co-crystals of MFL with different ratio of co-
crystal formers (benzoic acid, citric acid,
oxalic acid, salicylic acid, succinic acid) were
prepared by solution co-crystalliztion using
ethanol as a solvent and these co-crystals were
incorporated in tablet dosage form and
evaluated. Succinic acid co-crystal showed
superior dissolution in both the media (SGF,
SIF) and in both co-crystal form and tablet
form. Salicylic acid showed highest
dissolution at t15 and t45 in SGF i.e., 67.8%,
84.89% respectively as compared to that of
pure MFL tablet 39.4%, 58.76% respectively.
Novel approach
of pharmaceutical
co-crystals for
poorly soluble
drugs
Tejo
Vidyulata K.
et al.
International
Journal of
Pharmaceutical
Development &
Technology,
2012
Novel co-crystal of curcumin with methyl
paraben was obtained by liquid assisted
grinding method (1:1) and was evaluated for
anti-inflammatory activity. Low doses of pure
curcumin gave less inhibitory effect of 4.65%,
whereas prepared co-crystals showed
significant inhibition effect of 66.67%.
27

28
Title Authors
Year &
Journal
Conclusion
Coformer selection
in pharmaceutical
cocrystal
development: a
case study of a
meloxicam aspirin
cocrystal that
exhibits enhanced
solubility and
pharmacokinetics
Cheney et al.
Journal of
Pharmaceutical
Sciences, 2011
Targeted and prepared a co-crystal of
meloxicam and aspirin by solution, slurry, and
solvent drop grinding methods. In pH 7.4
phosphate buffer solution at 37 °C, the
solubility of meloxicam was found to be 0.005
mg/mL, whereas that of co-crystal was 0.22
mg/mL. Oral administration of co-crystal
exhibited an oral bioavailability of 69%
compared with 16% for meloxicam. Thus,
enabled an approximately 12-fold decrease in
the time required to reach a concentration of
0.51 µg/mL in rats compared with pure
meloxicam at an equivalent dose.
Improved
pharmacokinetics
of amg 517
through
co-crystallization
part 1: comparison
of two acids with
corresponding
amide co-crystals
Stanton et al.
Journal of
pharmaceutical
sciences,
2010
Studied the dissolution and pharmacokinetics
(PK) of AMG 517 co-crystals with cinnamic
acid and benzoic acid cinnamamide and
benzamide. The four co-crystals were found to
have faster intrinsic and powder dissolution
rates in FaSIF than the free base. This
correlated with a 2.4- to 7.1-fold increase in
the area under the concentration–time curve in
rat PK investigations.

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• Provided information regarding different GRAS listed coformers used for co-
crystal preparation.
• Based on the literature review it was concluded that Solvent evaporation, slurry
conversion and solvent drop grinding method are widely used for co-crystals
preparation. Out of this solvent drop grinding method was selected for co-crystal
preparation.
• An insight of literature review, furnished a glimpse of different analytical
techniques employed for characterization of co-crystals such as FTIR, DSC,
PXRD.

30
Aim:- The present study envisaged to prepare and evaluate co-crystals of BCS class II
drug.
Objectives:-
Select suitable drug candidate and coformers for altering the physicochemical
properties.
Prepare co-crystals with various coformers.
Characterize the co-crystals by using different techniques like melting point,
FTIR, DSC, PXRD, particle size.
Study the physicochemical properties of prepared co-crystals.
Perform in vitro dissolution studies with prepared co-crystals.

• Drug with low solubility belonging to BCS class II (low solubility and high
permeability) is selected for the study.
• Since there is no literature support for the formation of co-crystals, there is scope
for obtaining the co-crystals with selected drug. Based on the literature review and
objective of the investigations, suitable experimental methods were developed for
evaluation.
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Equipments Sources
UV – Visible – Spectrophotometer – 1800 Shimadzu Corporation Tokyo, Japan
IR spectrophotometer Shimadzu Corporation Kyoto, Japan
Differential scanning calorimeter Sicco DSC calorimeter Module 7020 Japan
Melting point apparatus Biotech India Melting apparatus, Mumbai
Dissolution test apparatus Electrolab USP XXII scientific, Mumbai
Orbital shaking Incubator Remi industries, Kerala
Electronic balance – AUX-220 Shimadzu Corporation Tokyo, Japan
pH meter Elico LI 613
PXRD Shimadzu module XRD 7000, Japan
Tapped density apparatus DBK tapped density apparatus
Particle size analyzer Nanotrac W3275, Microtrac USA
Table 3-1: List of the equipments and their sources

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Property Literature data
Description Solid, yellow crystalline powder,
BCS class II drug
Chemistry 3⁰ Nitrogen, 5 fused ring system
Chemical nature Basic
Molecular weight 336.4 g/mol
Dose 150 mg
pKa 2.47
Log P 2.1
UV data λmax- 345 nm
IR (cm-1) 1505, 1271, 1234, 1030, 1098, 1587
Table 3-2: Properties of drug

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Indications: The drug sample has significant antimicrobial activity towards a variety of
organisms It has also been reported to have a multitude of biological effects, including
anti-malarial, anti-hypertensive, anti-lipidemic, anti-arrhythmic, anti-hyperglycemic,
anti-tumor, anti-inflammatory, anti-fungal, anti-HIV, antifungal, cardioprotective,
immunoregulative, anti-oxidative, and cerebro-protective activities.

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Pharmacokinetic properties:
Absorption: The drug has poor oral bioavailability which is attributed to its poor
aqueous solubility, low gastrointestinal absorption and dissolution.
Distribution: The organ distribution of drug is rapid with maximum distribution
in liver, followed by kidneys, muscle, lungs, brain, heart, pancreas and with least
distribution in fat where it remains relatively stable for 48 h.
Metabolism: Drug is metabolized in the liver, undergoing demethylation in phase
I followed by conjugation with glucuronic acid or sulfuric acid to form phase II
metabolites.
Excretion: Oral administration of drug resulted in excretion of drug and its
metabolites in bile, urine and feces.

Coformer
(Chemical formula)
Molecular
weight (g/mol)
pKa
Melting point
(0C)
Structure
Hydroquinone
(C6H6O2)
110.11 10.9 170-171
Succinic acid
(C4H6O4)
118.09 4.2 185-1870C
Adipic acid
(C6H10O4)
146.14 4.43 152.1
Benzoic acid
(C7H6O2)
122.12 4.19 122.41
Boric acid
(H3BO3)
61.83 9.24 171
37
Table 3-3: List of the coformers used in preparing co-crystals

38
Coformer
(Chemical formula)
Molecular
weight (g/mol)
pKa
Melting point
(0C)
Structure
Nicotinic acid
(C6H5NO2)
123.11 4.8 236.6
Oxalic acid
(C2H2O4)
90 4.28 189.5
Catechol
(C6H6O2)
110.11 9.5 105
Glutaric acid
(C5H8O4)
132.11 4.34 97-98
Cinnamic acid
(C9H8O2)
148.16 4.46 134
Maleic acid
(C4H4O4)
116.07 3.44 138-139

Coformer
(Chemical formula)
Molecular
weight (g/mol)
pKa
Melting point
(0C) Structure
Tartaric acid
(C4H6O6)
150.08 1.5 206
L - Glutamic acid
(C5H9NO4)
147.13 2.23 213-224
D - Mannitol
(C6H14O6)
182.17 13.5 166-168
P - Amino Benzoic acid
(C7H7NO2)
137.14 2.38 188.5
3,5-Dihydroxybenzoic
Acid
(C7H6O4)
154.12 5.4 237
39

• Melting point: The melting point of the drug was determined using capillary
tubes. The sample was filled and placed in the melting point apparatus. The
observed melting point was noted .
• FTIR Studies: Drug was mixed with KBr in definite ratio and compacted. The
spectrum was recorded in the wavelength region of 4000–400 cm−1. The
characteristic bands were identified and compared with literature data.
• DSC Studies: Powder drug sample was weighed and taken into an aluminium
pan and analyzed at a rate of 10 ºC per min from 0 – 300 ºC with nitrogen
purging and empty aluminium pan was used as reference. DSC thermogram
was recorded.
40

UV Scan for determination of λmax of drug:
10 mg drug sample was dissolved in 10ml methanol (1000 µg/ml). From the
above stock solution, 1 ml solution was diluted and volume was made up to 100
ml with 0.1 N HCl solution (10 µg/ml) and was scanned in UV
spectrophotometer.
41

Calibration curve of drug in 0.1N hydrochloric acid solution:
• 10 mg of pure drug was accurately weighed and dissolved in 10 ml methanol (1000
µg/ml). [Primary stock solution]
• From the above stock solution, 1 ml solution was diluted to 10 ml with 0.1 N HCl
solution to give 100 µg/ml concentration. [Secondary stock solution]
• From secondary stock solution i.e., 100 μg/ml concentration solution - 2, 4, 6, 8, 10,
12 and 14 μg/ml concentrations were prepared by using 0.1 N HCl solution. The
absorbance of these solutions were measured at 345 nm.
42

43
- ∆pKa
Table 3-4: pKa, ∆pKa values of drug, coformers
Name
pKa of drug/
coformer
ΔpKa (pKa drug – pKa coformer)
Drug 2.47 N/A
Hydroquinone 10.9 -8.43
Succinic acid 4.20 -1.73
Benzoic acid 4.20 -1.72
Adipic acid 4.43 -1.96
Nicotinic acid 4.8 -2.33
Oxalic acid 4.28 -1.81
Catechol 9.5 -7.03
Glutaric acid 4.34 -1.87

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Name
pKa of drug/
coformer
ΔpKa (pKa drug – pKa coformer)
Cinnamic acid 4.46 -1.99
Maleic acid 1.93 0.54
Boric acid 9.24 -6.77
Tartaric acid 1.5 0.97
L - Glutamic acid 2.23 0.24
D - Mannitol 13.5 -11.03
p- Amino Benzoic acid 4.65 -2.18
3,5-Dihydroxybenzoic Acid 5.4 -2.93

Solvent drop grinding method: Drug (1mmol) and different coformers (1mmol)
were taken and mixed in a mortar pestle using ethanol (2-3 drops) as solvent. The
triturating process was carried out for 30-45 mins. The formation of new co-crystal
was confirmed by melting point, FTIR, PXRD and DSC.
45

46
10mg of drug in 10ml
volumetric flask. Make up
the volume with methanol.
(1000µg/ml)
Pipette 1 ml in 10ml
volumetric flask & make
up the volume with 0.1N
HCl. (100µg/ml)
HCl. (10µg/ml)
and scanned at 345 nm
10mg of coformer in 10ml
volumetric flask. Make up
to 10ml with 0.1N HCl.
(1000µg/ml)
HCl. (100µg/ml)
HCl. (10µg/ml) and
scanned at 345 nm
Pipette 1 ml each from
individual 100 µg/ml standard
stock solution of drug and
coformer in 10ml volumetric
flask & make up the volume
with 0.1N HCl. (100µg/ml)
Pipette1 ml in 10ml volumetric
flask & make up the volume
with 0.1N HCl. (10µg/ml) and
scanned at 345 nm
Figure 3-1: Schematic representation of preparation of standard stock solution (10 μg/ml) of
drug (a), coformer (b) and drug and coformer mixture (c) in 0.1 N HCl solution
(a) (b) (c)

47
•Melting point
•FTIR: The possible interaction between drug and coformers (catechol, mannitol) were
studied by IR spectroscopy.
•Powder X-Ray Diffraction (PXRD): PXRD gives a unique fingerprint diffraction
pattern characteristic of particular solid form. If a co-crystal has been formed between
two solid phases, the diffraction pattern of prepared co-crystal should be clearly
distinct from drug and coformer by the superimposition of PXRD pattern.
•Differential Scanning Colorimeter (DSC): DSC gives an accurate value for melting
onset temperature. DSC data is particularly valuable in constructing semi quantitative
energy-temperature relationship.
•Particle size
•Micromeritic Properties

• Saturation solubility studies: Excess of drug (pure drug) and drug co-crystals
were dissolved in 10 ml pH 1.2 buffer, and 10 ml water. The flasks were agitated in
orbital shaker at 25 ºC at 100 rpm for 24 h. After attainment of equilibrium, aliquots
were withdrawn, filtered and were diluted with pH 1.2 buffer, water accordingly
and were analyzed at 345 nm.
• Estimation of drug content in co-crystals: Drug content was determined by
dissolving 10 mg of co-crystal in 100 ml of 0.1N HCl. From the above solution 1ml
was pipette out and volume was made up to 10 ml using 0.1N HCl, which yields
sample of concentration 10 µg/ml. The samples were analyzed at 345 nm.
48

49
• Dissolution Studies: Pure drug and various co-crystals containing the drug
equivalent to 5.3 mg were taken and filled in hard gelatin empty capsules.
Dissolution studies were carried out in 900 ml of pH 1.2 buffer solution,
temperature was maintained at 37 ± 0.5 ºC and 50 rpm was used. Samples were
withdrawn at time intervals of 10, 20, 30, 40, 50, 60,70, 80, 90 and 120 minutes.
The samples were filtered through 0.45μm filter and analyzed
spectrophotometrically at 345 nm.
• Comparison of Dissolution Profiles:
• Dissolution Efficiency and Mean Dissolution Time: It is defined as the area
under the dissolution curve up to certain time, t, expressed as a percentage of the
area of the rectangle described by 100% dissolution in same time.
• Mean dissolution time:
• Mean dissolution time reflects the time for the drug to dissolve in vivo.

D.E. =
MDT in vitro =
Similarity Factor and Difference Factor: The factor f2 measures the closeness
between the two profiles, with emphasis on the larger difference among all time
points.
f2 = 50 x
Difference factor:
It measures the percent error between two curves over all time points.
f1 =
50

52
Melting Point
The melting point of the drug was determined using capillary tube. The drug
showed a melting range of 195-205 °C followed by decomposition. Similar
result was observed during DSC studies as shown in Figure 4-2.

53
Figure 4-1: FTIR spectrum of pure drug
Fourier Transform Infrared Spectroscopy

54
Characteristic bands
Literature values,
cm-1
Observed values,
cm-1
C=C Aromatic 1600-1475 1506.4
C-N stretching 1300-1080 1273,1230.5
C=N stretching 1400-1200 1363.6
C-H Aliphatic 3000-2850 2947.2
O-H Stretching 3600-3200 3387
C-O (Ether) 1200-1020 1105.2, 1037.7
C=O 1640-1550 1598.9
C-Cl 850-550 827.4
Table 4-1: Characteristic FTIR absorption bands of drug

55
Figure 4-2: DSC thermogram of pure drug at heating rate of 10 ºC per min
Differential Scanning Calorimetry

56
Figure 4-3: UV scan of drug solution (10 μg/ml) in 0.1 N HCl (max = 345 nm)

57
Concentration
(μg/ml)
Absorbance at 345 nm
(AM±S.D)*
0 0.000 ± 0.000
2 0.153 ± 0.015
4 0.319 ± 0.019
6 0.479 ± 0.032
8 0.601 ± 0.033
10 0.763 ± 0.048
12 0.930 ± 0.044
14 1.077 ± 0.054
Table 4-2: Data for standard plot of drug in 0.1 N HCl at 345 nm
*Mean of three determinations

Figure 4-4: Standard plot of drug in 0.1 N HCl solution at 345 nm
58
y = 0.076x + 0.003
R² = 0.999
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14 16
Concentration (μg/ml)
Absorbance

- ∆pKa
• The ΔpKa values were considered as a reference to predict, whether salts or co-
crystals will form. The pKa values of the drug and coformers were compiled
and ΔpKa values were calculated and summarized in Table 3-4.
• The ΔpKa values of formed adduct were less than 3 which implies salt or co-
crystal formation, but is not definitive.
59

60
Figure 4-5: Schematic representation of solvent drop grinding method

61
Figure. 4-6.1: UV absorption spectrum of drug,
mannitol and a mixture of drug-mannitol (10
µg/ml each) at 345 nm
catechol and a mixture of drug-catechol (10
µg/ml each) at 345 nm

62
Figure. 4-6.4: UV absorption spectrum of
drug, 3, 5 dihydroxy benzoic acid and a
mixture of drug- 3, 5 dihydroxy benzoic acid
(10 µg/ml each) at 345 nm
tartaric acid and a mixture of drug-tartaric acid
(10 µg/ml each) at 345 nm

63
cinnamic acid and a mixture of drug-cinnamic
acid (10 µg/ml each) at 345 nm
benzoic acid and a mixture of drug-benzoic
acid (10 µg/ml each) at 345 nm

Compound
Melting point, 0C
(Literature value)
Drug/co-crystal melting
range, 0C
(Observed value)
Drug
200.2
(Confirmed by DSC)
195-205 (followed by
decomposition)
Drug-mannitol 166-168 (Coformer)
150-158 (Red colour liquid
followed by decomposition)
Drug-catechol 105 (Coformer)
decomposition at 200)
Drug-tartaric acid 206 (Coformer)
decomposition at 210)
Drug-3,5 DHBA 237 (Coformer)
115-125 (orange colour liquid
followed by decomposition at
200)
Drug-benzoic acid 122.4 (Coformer)
95-100 (followed by
decomposition)
Drug-cinnamic acid 133 (Coformer)
90-100 (orange colour liquid
followed by decomposition at
183) 64
Table 4-3: Melting point values of drug, coformers and prepared co-crystals

65
Compound
Melting point, 0C
Literature value,
Drug/Coformer
Drug/co-crystal melting
range, 0C
Drug-succinic acid 185-188 (Coformer) 200-208
Drug-oxalic acid 189.5 (Coformer) 198-200
Drug-adipic acid 152.1 (Coformer) 224
Drug-nicotinic acid 236.6 (Coformer) 190-200
Drug-maleic acid 130-135 (Coformer) 200
Drug-glutaric acid 95-98 (Coformer) 204
Drug-boric acid 170.9-171 (Coformer) 205-210
Drug-hydroquinone 170-172.3 (Coformer) 198-205
Drug-PABA 187-187.5 (Coformer) 198-202
Drug-glutamic acid 213 (Coformer) 195-210

66
Figure 4-7.1: FTIR spectral comparison of drug, drug–mannitol co-crystal and mannitol

67
Figure 4-7.2: FTIR spectral comparison of drug, drug–catechol co-crystal and catechol

68
Figure 4-7.3: FTIR spectral comparison of drug, drug-tartaric acid co-crystal and
tartaric acid

69
Figure 4-7.4: FTIR spectral comparison of drug, drug-3, 5 dihydroxy benzoic acid co-
crystal and 3, 5 dihydroxy benzoic acid

70
Figure 4-7.5: FTIR spectral comparison of drug, drug-benzoic acid co-crystal and
benzoic acid

71
Figure 4-7.6: FTIR spectral comparison of drug, drug-cinnamic acid co-crystal and
cinnamic acid

72
Drug Characteristic bands, cm-1 Inference
Drug
C=C Aromatic – 1506.4
C-N (stretching) – 1273.0
C=N – 1363.6
C-O – 1037.7
C-Cl (bending) – 827.4
O-H (stretching) – 3387.0
Characteristic peaks have been
observed
Name of co-crystals Characteristic bands, cm-1 Inference
Drug-mannitol
C=N – 1363.6
C-O – 1037.7
O-H, C-Cl, C-N shift has been
observed. Co-crystals might have
formed
Drug-catechol
C=N – 1367.5
C-O – 1043.4
Shift has been observed in all
absorption bands. Co-crystals
might have formed
Table 4-4: FTIR bands for characteristic changes of drug co-crystals

73
Drug-tartaric acid
C=N – 1363.6
C-O – 1037.0
formed
Drug-3, 5 di hydroxy
benzoic acid
C=N – 1361.7
C-O – 1037.7
O-H, C-Cl, C=N shift has been
formed

74
Drug-benzoic acid
C=N – 1363.6
C-O – 1037.7
Slight shift has been observed.
Might be a physical mixture
Drug-cinnamic acid
C=N – 1363.6
C-O – 1037.7
formed

75
Figure 4-8.1: Overlay of the PXRD pattern of drug – mannitol co-crystal with its
individual components

76
Figure 4-8.2: Overlay of the PXRD pattern of drug – catechol co-crystal with its

77
Figure 4-8.3: Overlay of the PXRD pattern of drug- tartaric acid co-crystal
with its individual components

78
Figure 4-8.4: Overlay of the PXRD pattern of drug- 3, 5 dihydroxy
benzoic acid co-crystal with its individual components

79
Figure 4-8.5: Overlay of the PXRD pattern of drug- benzoic acid co-crystal with its

80
Figure 4-8.6: Overlay of the PXRD pattern of drug- cinnamic acid co-
crystal with its individual components

81
Name of co-
crystals
(Coformer 100%
intensity)
Peak 2θ value
Integrated
intensity of
Drug
Integrated
intensity of
Drug co-
crystals
Inference
Drug-mannitol
co-crystal
(Mannitol -18.79)
9.1
23.4
25.5
26.3
18.7
21.09
100
-
32.7
32.2
-
-
47.5
100
43.5
51
69.6
67
Change in
intensities and
formation of
new peaks was
observed
Drug-catechol co-
crystals
(Catechol-9.94)
5.8
8.2
9.1
25.5
26.3
-
-
100
32.7
32.2
93.6
100
-
92.6
45.5
Change in
intensities and
formation of
new peaks was
observed
Table 4-5: PXRD pattern comparison for characteristic changes of drug with drug co-
crystals

82
Name of co-crystals
(Coformer 100%
intensity)
Peak 2θ value
Integrated
intensity of
Drug
Integrated
intensity of
Drug co-
crystals
Inference
Drug- tartaric acid
co-crystal
(Tartaric acid-20.82)
9.06
6.8
20.65
25.47
26.27
25.73
100
23.4
13.1
32.7
32.2
-
80.2
63.4
72.5
100
82.8
66.7
Change in
intensities were
observed and
formation of
new peak were
observed
Drug- 3, 5
dihydroxy benzoic
acid co-crystal
(3, 5 dihydroxy
benzoic acid-21.45 )
26.21
13.08
25.75
11.37
9.1
25.39
32.2
-
-
5.3
100
32.7
100
48.9
33.8
28.6
4.3
-
Change in
intensities and
formation of
new peak were
observed

83
Name of co-
crystals
(Coformer 100%
intensity)
Peak 2θ value
Integrated
intensity of
Drug
Integrated
intensity of
Drug co-
crystals
Inference
Drug- benzoic
acid co-crystal
(Benzoic acid-
8.11)
8.08
9.097
17.16
26.29
6.84
-
100
-
32.2
23.4
100
66.6
58.2
52.8
38.8
Might be a
physical mixture
Drug- cinnamic
acid co-crystal
(Cinnamic acid-
9.75)
9.1
26.3
25.35
9.8
22.85
100
32.7
32.2
-
-
77.8
57.8
100
94.1
82.8
Change in
intensities were
observed and
formation of
new peak were
observed

84
Figure 4-9.1: DSC thermogram of drug – mannitol co-crystals

85
Figure 4-9.2: DSC thermogram of drug – catechol co-crystals

86
Figure 4-9.3: DSC thermogram of drug-tartaric acid co-crystals

87
Figure 4-9.4: DSC thermogram of drug-3, 5 dihydroxy benzoic acid co-crystals

88
Figure 4-9.5: DSC thermogram of drug- cinnamic acid co-crystals

89
S.no Drug Melting point (ºC)
(literature value)
Peak (ºC)
(Observed value)
Inference
1 Drug - 200.2 -
S.no Name of co-crystal Melting point (ºC)
(literature value)
Peak (ºC)
(Observed value)
Inference
1 Drug-mannitol 166-168 159.3 Co-crystal might
have formed
2 Drug-catechol 105 180.2 Co-crystal might
have formed
3 Drug-tartaric acid 206
162.6
Co-crystal might
have formed
4 Drug-3, 5 di hydroxy benzoic
acid
237 116.09 Co-crystal might
have formed
5 Drug-cinnamic acid 133
158.4
Co-crystal might
have formed
Table 4-6: DSC changes of drug co-crystals

90
Figure 4.10.1:Particle size distribution of the drug.
Figure 4.10.2:Particle size distribution of drug-mannitol co-crystals.

91
Figure 4.10.3:Particle size distribution of drug-catechol co-crystals.
Figure 4.10.4:Particle size distribution of drug-tartaric acid co-crystals.

92
Figure 4.10.5:Particle size distribution of drug-3,5 dihydroxy benzoic acid co-crystals.
Figure 4.10.6:Particle size distribution of drug-cinnamic acid co-crystals.

93
Table 4-7: Comparison of micromeritic properties of drug and prepared co-crystals
Name
Bulk density
Tapped
density
Carr’s index
Hausners
ratio
Angle of
repose Property
(AM±S.D)*
Drug 0.25±0.005 0.40±0.01 37.5±0.005 1.57±0.04 - Very poor
Drug- mannitol
co-crystal
0.39±0.001 0.44±0.01 13.48±1.9 1.16±0.02 31.11±0.55 Good
Drug-catechol
co-crystal
0.49±0.01 0.54±0.01 9.91±0.003 1.11±0.002 16.88±0.81 Excellent
Drug- tartaric
acid co-crystal
0.45±0.01 0.52±0.01 13.52±0.27 1.16±0.003 34.60±1.62 Good
Drug- 3,5
dihydroxy
benzoic acid co-
crystal
0.53±0.03 0.62±0.01 10.27±0.01 1.11±0.02 21.36±2.95 Excellent
Drug- cinnamic
acid co-crystal
0.37±0.003 0.43±0.005 14.27±0.81 1.16±0.01 31.39±0.75 Good

Drug/co-crystal
Solubility studies (mg/ml) (AM±S.D)*
pH 1.2 Fold increase
Drug 0.0053±0.001 -
Drug-mannitol 0.0107±0.004 2.01
Drug-catechol 0.00829±0.001 1.57
Drug-tartaric acid 0.0084±0.001 1.58
Drug-3,5 DHBA 0.0095±0.001 1.79
Drug-cinnamic acid 0.0082±0.0009 1.56
94
Table 4-8: Solubility data of drug and drug co-crystals in pH 1.2

95
0
0.002
0.004
0.006
0.008
0.01
0.012
1.2 pH
Co-crystals
Solubility(mg/ml)
Figure 4-10: Solubility profile of drug co-crystals in pH 1.2 at 25 ºC

Drug/co-crystal
% Drug Content
(AM±S.D)*
Drug-mannitol 34.53±3.2
Drug-catechol 54.26±1.3
Drug-tartaric acid 61.39±4.61
Drug-3,5 DHBA 64.34±2.37
Drug-cinnamic acid 59.0±3.96
96
Table 4-9: Concentration of drug present in drug co-crystals

Medium pH 1.2
Volume of the medium 900 ml
Apparatus Type USP –II (Paddle)
Rpm 50
Dose Equivalent to 5.3 mg
Run time 120 minutes
Temperature 37 ± 0.5 ºC
Time Points 10, 20, 30, 40, 50, 60, 70, 80, 90 and 120 minutes.
λmax 345 nm
97
Table 4-10: Dissolution conditions

98
Time
(mins)
Cumulative percentage drug dissolved, (AM±S.D)*
Pure drug
Drug-
mannitol co-
crystal
Drug-
catechol co-
crystal
Drug-
tartaric
acid co-
crystal
Drug-3,5
DHBA co-
crystal
Drug-
cinnamic
acid co-
crystal
0 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00
10 9.97±4.7 51.06±4.1 60.78±1.02 24.51±2.97 34.51±3.1 27.64±2.59
20 19.73±4.27 79.66±2.6 71.89±0.96 59.29±2.83 47.54±0.26 48.83±0.79
30 23.027±4.14 88.38±3.9 80.29±3.82 60.81±4.08 53.88±2.61 59.42±3.56
40 30.13±1.31 95.74±4.5 82.92±4.33 65.14±2.99 58.27±1.48 63.39±4.47
50 35.19±2.77 100.76±1.65 83.82±4.14 69.59±2.99 61.39±2.13 69.10±4.9
60 39.94±4.8 - 86.04±4.32 82.01±2.36 66.43±1.58 74.66±3.92
70 41.44±3.8 - 88.04±4.75 87.06±3.45 69.34±1.1 77.28±3.67
80 44.88±4.9 - 89.16±3.96 92.41±3.46 72.08±2.37 79.80±3.59
90 45.97±4.5 - 92.12±4.53 98.96±2.68 77.71±4.28 85.72±3.47
120 51.66±3.36 - 98.41±1.95 100.83±3.74 82.91±2.82 89.37±2.38
Table 4-11: Cumulative % dissolved-time profile of drug co-crystals with various coformers

99
Figure 4-11: Dissolution-time profile of drug and its co-crystals
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140
Pure drug
Tartaric acid
3, 5 DHBA
Mannitol
Catechol
Cinnamic acid
Cumulativepercentagedrugdissolved
Time (mins)

100
Drug/co-crystals
Drug dissolution analysis
Dissolution
efficiency (%)
Mean dissolution
time (min)
Similarity factor, f2 Difference factor, f1
Pure drug 34.48 39.91 - -
Drug- mannitol co-
crystal
73.04 13.75 11.0 71.60
Drug-catechol co-
crystal
81.23 20.94 15.35 59.0
Drug- tartaric acid
co-crystal
74.16 31.72 19.3 53.8
Drug- 3,5 dihydroxy
benzoic acid co-
crystal
61.93 30.35 27.4 45.2
Drug- cinnamic acid
co-crystal
67.13 29.85 23.5 49.4
Table 4-12: Dissolution efficiency, mean dissolution time (MDT) and similarity factor,
difference factor for drug and drug co-crystals.

• A UV spectrophotometric analytical method was developed for pure drug in 0.1 N
HCl. The λmax was found to be 345 nm, and Beer Lambert’s law was obeyed in the
range of 2 to 14 µg/ml (R2 = 0.999).
• UV spectral interference studies showed absence of interference at analytical
wavelength of the drug with coformer.
• Solvent drop grinding was opted for preparation of co-crystals with sixteen
different coformers.
• Based on ΔpKa and melting point co-crystals, the data suggested formation of five
co-crystals.
• IR studies showed slight changes in five drug co-crystals (catechol, mannitol,
cinnamic acid, tartaric acid and 3, 5 dihydroxy benzoic acid).
• The DSC exhibited the characteristic endothermic peak which was neither nearer to
the pure drug nor to the coformer in above five co-crystals.
101

102
• Powder X-ray diffraction pattern of drug co-crystals with catechol, mannitol,
cinnamic acid, tartaric acid and 3, 5 dihydroxy benzoic acid, indicated the presence
of additional peaks and shifting of peaks when compared to pattern of drug.
• The saturation solubility study of all five co-crystals showed higher solubility in pH
1.2 than drug. Drug-mannitol co-crystals showed two-fold increase in solubility at
25 ºC in pH 1.2 (0.0107 mg/ml) when compared to that of drug (0.0053 mg/ml).
• The order of the dissolution of the co-crystal forms after 90 minutes was found to
be mannitol > tartaric acid > catechol > cinnamic acid > 3, 5 dihydroxy benzoic
acid > drug (pure drug). The 100% drug release was observed for drug-mannitol co-
crystals in 50 mins, while the pure drug showed only 51.66%.

103
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• Blagden, N., de Matas, M., Gavan, P. and York, P. (2007). ‘Crystal engineering of active
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105

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