In vitro and ex vivo studies on diltiazem hydrochloride-loaded microsponges in rectal gels for chronic anal fissures treatment

Article abstract of DOI:10.1016/j.ijpharm.2018.12.039

Diltiazem hydrochloride, topically applied at 2% concentration, is considered effective for the treatment of chronic anal fissures, although it involves several side effects among which anal pruritus and postural hypotension. To test the hypothesis that a sustained delivery system of diltiazem hydrochloride may be helpful for the treatment of chronic anal fissures, in the present study we evaluated the potential of gels containing diltiazem hydrochloride entrapped in microsponges. Such microsponges were based on Eudragit RS 100 and the effect of some formulation variables was assessed by a 2³ full factorial screening design. An optimized formulation of diltiazem hydrochloride microsponges was dispersed in Methylcellulose 2% or Poloxamer 407 20% and the resulting gels (micro-l-diltiazem hydrochloride 2%) were subjected to in vitro drug release, ex vivo permeability and drug deposition after application on porcine rectal mucosa. The results showed a prolonged release up to 24 h from micro-l-diltiazem hydrochloride at 2% in the gels. The permeation tests revealed up to 18% higher drug retention on the mucosal tissue after 24 h by the micro-l - diltiazem hydrochloride 2% gels compared to conventional diltiazem hydrochloride gels at 2%. These results suggest that diltiazem hydrochloride-loaded microsponges dispersed in rectal gels may be useful to overcome some limitations of conventional local chronic anal fissure therapy.

Full text of DOI:10.1016/j.ijpharm.2018.12.039

Accepted Manuscript
In vitro and ex vivo studies on Diltiazem hydrochloride-loaded microsponges in
rectal gels for chronic anal fissures treatment
Nadezhda Antonova Ivanova, Adriana Trapani, Cinzia Di Franco, Delia
Mandracchia, Giuseppe Trapani, Carlo Franchini, Filomena Corbo, Giuseppe
Tripodo, Iliyan Nikolov Kolev, Georgi Stoyanov Stoyanov, Kameliya
Zhechkova Bratoeva
PII:
S0378-5173(18)30955-4
https://doi.org/10.1016/j.ijpharm.2018.12.039
IJP 18008
DOI:
Reference:
International Journal of Pharmaceutics
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 September 2018
17 December 2018
18 December 2018
Please cite this article as: N.A. Ivanova, A. Trapani, C.D. Franco, D. Mandracchia, G. Trapani, C. Franchini, F.
Corbo, G. Tripodo, I.N. Kolev, G.S. Stoyanov, K.Z. Bratoeva, In vitro and ex vivo studies on Diltiazem
hydrochloride-loaded microsponges in rectal gels for chronic anal fissures treatment, International Journal of
Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.12.039
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1
In vitro and ex vivo studies on Diltiazem hydrochloride-loaded microsponges in rectal gels for
chronic anal fissures treatment
Nadezhda Antonova Ivanova1, Adriana Trapani2*, Cinzia Di Franco3, Delia Mandracchia2, Giuseppe
Trapani2, Carlo Franchini2, Filomena Corbo2, Giuseppe Tripodo4, Iliyan Nikolov Kolev1 Georgi
,
Stoyanov Stoyanov5,6, Kameliya Zhechkova Bratoeva6
1Faculty of Pharmacy, Medical University, "Prof. Dr. Paraskev Stoyanov", 84 Tsar Osvoboditel str., Varna,
Bulgaria
2Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, Via Orabona, 4, 70125 Bari, Italy
3CNR-IFN Bari, Bari Via Amendola 173 – 70126 Bari, Italy
4Department of Drug Sciences, University of Pavia, Viale Taramelli, 12, 27100 Pavia, Italy
5Department of General and Clinical Pathology, Forensic Medicine and Deontology, Faculty of Medicine,
Medical University, "Prof. Dr. Paraskev Stoyanov", 55 Marin Drinov str., Varna, Bulgaria
6Faculty of Medicine, Medical University, "Prof. Dr. Paraskev Stoyanov", 55 Marin Drinov str., Varna,
Bulgaria
*Corresponding author:
Adriana Trapani
Dept. of Pharmacy-Drug Sciences
University of Bari “Aldo Moro” –70125 Bari, Italy
E-mail address: adriana.trapani@uniba.it
Tel: +39 0805442114, Fax: +39 0805442724

2
Abstract
Diltiazem hydrochloride, topically applied at 2% concentration, is considered effective for the
treatment of chronic anal fissures, althought it involves several side effects among which anal pruritus
and postural hypotension. To test the hypothesis that a sustained delivery system of diltiazem
hydrochloride may be helpful for the treatment of chronic anal fissures, in the present study we
evaluated the potential of gels containing diltiazem hydrochloride entrapped in microsponges. Such
microsponges were based on Eudragit RS 100 and the effect of some formulation variables was
assessed by a 23 full factorial screening design. An optimized formulation of diltiazem hydrochloride
microsponges was dispersed in Methylcellulose 2% or Poloxamer 407 20% and the resulting gels
(micro-l-diltiazem hydrochloride 2%) were subjected to in vitro drug release, ex vivo permeability
and drug deposition after application on porcine rectal mucosa. The results showed a prolonged
release up to 24 h from micro-l-diltiazem hydrochloride at 2% in the gels. The permeation tests
revealed up to 18% higher drug retention on the mucosal tissue after 24 h by the micro-l diltiazem
hydrochloride 2% gels compared to conventional diltiazem hydrochloride gels at 2%. These results
suggest that diltiazem hydrochloride-loaded microsponges dispersed in rectal gels may be useful to
overcome some limitations of conventional local chronic anal fissure therapy.
Keywords: Diltiazem hydrochloride; Chronic anal fissures; Microsponges; Factorial screening
design; Rectal gels; Permeation.
Diltiazem hydrochloride (PubChemCID: 62920);
Dichloromethane (PubMedCID: 6344);
Polyvinyl alcohol (PubChem CID: 11199);
Methylcellulose (PubChem CID: 6850753);
Poloxamer 407 (PubMedCID: 24751);
Disodium hydrogen phosphate (PubChem CID: 24203);
Potassium dihydrogen phosphate (PubChem CID: 516951);
Sodium hydrogen carbonate (PubChem CID: 516892).
Abbreviations
c-diltiazem hydrochloride gel
Diltiazem
conventional gel;
hydrochloride-loaded
hydrochloride-loaded
Mc-based c-diltiazem hydrochloride
Diltiazem
conventional
gel
prepared
with
Methylcellulose (Mc);

3
Px-based c-diltiazem hydrochloride
Diltiazem
hydrochloride-loaded
conventional
gel
prepared
with
Poloxamer 407 (Px);
Micro-l-diltiazem hydrochloride gel
Diltiazem hydrochloride-entrapped in
microsponge formulation (F 9)-loaded
gel;
F9 loaded Mc-based micro-l-diltiazem hydrochloride
F9 loaded Px-based micro-l-diltiazem hydrochloride
Diltiazem hydrochloride-entrapped in
microsponge formulation (F 9)-loaded
gel prepared with Methylcellulose (Mc);
Diltiazem hydrochloride-entrapped in
microsponge formulation (F 9)-loaded
gel prepared with Poloxamer 407 (Px).

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1. Introduction
Chronic anal fissure is recognized as a common and painful proctologic disease which can bring about
even serious impairment of quality of life (Altomare et al., 2011). This pathological condition is very
probably related to hypertonia at the internal anal sphincter (IAS) and, therefore, the objective of
chronic anal fissures clinical treatment is focused to reduce such IAS pressure (Altomare et al., 2011;
Canelles et al., 2015). Although lateral internal sphincterotomy still remains the gold standard of
treatment for the disease, it is associated to potential complications including fecal incontinence
(Motie and Hashemi, 2016; Vaithianathan and Panneerselvam, 2015). Among the various medical
therapeutic alternative approaches, the use of calcium channel blockers, as diltiazem hydrochloride
(Figure 1S) or nifedipine topically applied, is considered effective enough even though the patients
often show a high risk for recurrence after the end of therapy (Altomare et al., 2011; Canelles et al.,
2015). However, a comparative study of medical versus surgical approaches clearly showed that,
despite good response to medical treatment, surgical procedure is more effective (Motie and Hashemi,
2016). It follows that the medical approach, particularly that with diltiazem hydrochloride could be
of choice in patients who are not willing to undergo surgery. On the other hand, according to the
Guidelines from the American Society of Colon and Rectal Surgeons, medical approaches of anal
fissures are recommended as first line therapy, specifically with pharmacological agents as calcium
channel blockers topically formulated (Sahebally et al., 2017; Brady et al., 2017). Moreover, there
are studies suggesting that the use of diltiazem hydrochloride at the concentration of 2% for treatment
of chronic anal fissures involves headache, anal pruritus and postural hypotension as frequent side
effects (Canelles et al., 2015; Hashmi and Siddiqui, 2009). These adverse effects lead to patient non-
compliance and it is further heightened from the needing of frequent administrations (2-3 times per
day) of the calcium channel blocker drug, likewise due to diltiazem hydrochloride short half-life
(3.2±1.3 h) and its high water solubility (Stepanovs et al., 2016). In an attempt to increase patient
compliance and improve the results of diltiazem hydrochloride based medical approach, we
hypothesized that a modified delivery system able to release the calcium channel blocker in a
sustained manner may be helpful to reduce both side effects and administration frequency. We
reasoned, indeed, that a drug sustained delivery system may be useful both to avoid the achievements
of drug levels corresponding to adverse effects and to reduce the administration number.
To accomplish a sustained delivery system of diltiazem hydrochloride, there are several options
including the prodrug or conjugate approach as well as the use of micro- or nano-particulate systems.
Among all, microencapsulation is recognized as a valuable strategy because several advantages can
be attained including biocompatibility, stability, better compliance, and sustained release pattern that
allows to reduce the toxicity and dosage frequency (Bale et al., 2016). Several types of
microparticulate delivery systems have been to date developed such as microspheres, microcapsules
and microbeads and their feasibility for diltiazem hydrochloride delivery has also been well
documented in the literature (Al-Zoubi et al., 2016; Cetin et al., 2012). In this context, a notable step
forward was reached in 1987 by Won who introduced the so-called microsponge delivery systems
(MDS) to overcome some disadvantages exhibited by classic microspheres (Won, 1987). In
particular, unlike classic microspheres, MDS are constituted by spherical cross linked spongy
particles which possess a porous surface and have been used mostly for topical applications and even
for oral administration (Srivastava and Pathak, 2012). Several preparative methods of microsponges

5
have been reported in literature among which the liquid-liquid suspension polymerization and the
quasi emulsion solvent diffusion method are the most employed.
The aim of the present study was to evaluate the potential of gels containing diltiazem hydrochloride
entrapped in microsponges intended for the treatment of chronic anal fissures. To the best of our
knowledge, this approach based on the non-dihydropyridine calcium channel blocker diltiazem
hydrochloride and on microsponge technology is unprecedented in the literature. For this purpose,
we designed microporous diltiazem hydrochloride-loaded microsponges based on Eudragit RS 100
prepared by the quasi emulsion solvent diffusion method (Srivastava and Pathak, 2012; Kumari et
al., 2016). Since this method requires poor water solubility of the drug to be loaded in particles
(Srivastava and Pathak, 2012; Kumari et al., 2016), the following considerations were taken into
account: diltiazem hydrochloride is commercially available as hydrochloride salt and belongs to Class
I of Biopharmaceutical Classification System (BCS), possessing high solubility and high permeability
(Stepanovs et al., 2016; Trapani et al., 2004). Instead, diltiazem free base belongs to Class II of BCS
with limited water solubility and high permeability (Stepanovs et al., 2016; Trapani et al., 2004) and,
therefore, a temporary conversion of the hydrochloride salt to free base may be used as an approach
to incorporate this calcium channel blocker into modified-release microsponges via the
aforementioned technique in a satisfactory way.
The quasi emulsion solvent diffusion method is technologically simple (Figure 1S), but the choice of
formulation variables is crucial for the characteristics of the resulting particles. Among the different
Eudragit polymers, Eudragit RS 100 was selected since it is well known that this copolymer leads to
microsponges of smaller size and satisfactory release rate control (Devrim and Canefe, 2006).
According to manufacturer information, Eudragit® RS 100 is a copolymer of ethyl acrylate, methyl
methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups
(trimethylammonioethyl methacrylate chloride). To evaluate the effect of the drug:polymer weight
ratio, the volume of the organic solvent [dichloromethane (DCM)] employed and the surfactant
Poly(vinyl alcohol) (PVA) used on the production yield, drug entrapment efficiency and particle size,
a 23 full factorial screening design was carried out taking into account the relevance of such statistical
approach in the development of microsponges as drug carriers (Crcarevska et al., 2015). The
compatibility of diltiazem with formulation components was established by Fourier transform infra-
red (FT-IR) spectroscopy and differential scanning calorimetry (DSC). Shape and surface
morphology of the microsponges were examined using scanning electron microscopy (SEM). The in
vitro drug release kinetics from selected microsponges were studied in phosphate buffer at pH 5.5
and pH 7.4. Finally, an optimized formulation was used to obtain diltiazem hydrochloride-loaded
microsponges dispersed in two types of hydrogel bases containing Methylcellulose 2% or Poloxamer
407 20% as gelling agents. These gels were compared to conventional diltiazem hydrochloride 2%
gel (c-diltiazem hydrochloride 2%) for in vitro drug release, ex vivo permeability and drug deposition
after application on porcine rectal mucosa.
2.
Materials and methods
2.1.
Materials

6
Diltiazem hydrochloride 99.9% was purchased from Puho Pharmaceuticals Co. Limited, China,
Ammonio methacrylate copolymer (type B) (Eudragit RS 100) was a kind gift from Evonik Industries
AG (Germany). Poloxamer 407 (Lutrol F-127) was a gift from BASF (Germany). Other reagents and
materials were purchased as follows: Poly(vinyl alcohol, Mw 49.000 Da) – (Sigma Aldrich, USA);
Methylcellulose high viscosity (Metolose 90SH – 30000) – Synthapharm Ges, F. - Germany;
Dichloromethane (DCM, HPLC grade > 97.8%) and anhydrous Ethanol 99.5% were purchased from
(Fisher Chemical, Germany); Disodium hydrogen phosphate dehydrate, Sodium chloride, Potassium
dihydrogen phosphate and Sodium hydrogen carbonate were purchased from Thermo Fisher
Scientific (USA); Sulfuric acid 96% was provided by Panreac, Spain; Dialysis membranes were
provided from a local hospital (10 kDa molecular weight cutoff); Polyethersulfone (PES) 0.22 µm pore
size syringe filter were provided by Filters Fioroni (Spain).
2.2.
Methods
2.2.1. Quantitative determination of diltiazem
Calibration curves for the quantitative determination of diltiazem hydrochloride and diltiazem base
were obtained in distilled water, in two buffer media (pH values equal to 5.5 and 7.4) as well as in
anhydrous ethanol, by spectrophotometric analysis on UV/Vis spectrophotometer Rayleigh UV-9200
(China). Absorption maxima were found for all above-mentioned media in the wavelength range of
238 nm-244 nm (ethanol: 244 nm, distilled water: 240 nm, buffer pH 5.5: 240 nm, buffer pH 7.4: 238
nm). The curves were linear in the concentration range of 1.5 – 45.0 g/mL (R2 > 0.999).
2.2.2. Diltiazem hydrochloride conversion from hydrochloride salt to base
Diltiazem hydrochloride was converted to the corresponding free base by treatment with NaHCO3 as
previously described (Ivanova et al., 2017). Briefly, 10 g of diltiazem hydrochloride were dissolved
in distilled water (100 mL) under magnetic stirring. To this solution, NaHCO3 was slowly added in
excess to the stoichiometrically calculated amount (1.85 g) and precipitation of the water insoluble
diltiazem base occurred. The resulting slurry was stirred for further 15 min and then diltiazem base
was isolated through filtration and washed with distilled water.
The structural assignment of diltiazem base was confirmed by: i) Gas Chromatography-Mass
spectroscopy (GC-MS, Hewlett-Packard 5995c GC-MS low-resolution spectrometer); ii) comparison
of the melting point with the one from an authentic sample (106.6 °C), being the melting point of
diltiazem hydrochloride of 213.1 °C (https://www.drugbank.ca); iii) comparison of FT-IR functional
groups with the ones from an authentic sample. Diltiazem base was kept in desiccator for at least 24
h until further use.
2.2.3. Solubility studies
o
For diltiazem hydrochloride and free base, water solubility tests were carried out at 37 C. Test
compound was added to 10 mL distilled water or PBS pH7.4 under continuous stirring. The mixture
was kept under stirring for 24 h. Undissolved fractions were detected at the end of the test for both
diltiazem forms. Samples from the supernatant were withdrawn, filtrated and analyzed
spectrophotometrically for diltiazem content as reported in Section 2.2.1.

7
2.3. Preparation of diltiazem-loaded microparticles with microsponge-like morphology
The microsponges containing diltiazem were prepared by the quasi emulsion solvent diffusion
method (Ivanova et al., 2017). Formulations were prepared with variable drug:polymer ratios, organic
phase volume (dichloromethane, DCM) and surfactant concentrations (PVA). Briefly, PVA was
dissolved in 100 mL distilled water at 80 oC to stabilize the external aqueous phase. Diltiazem base
and polymer Eudragit RS100 were dissolved in DCM and added dropwise to the cooled external
phase while kept under continuous stirring at 650 rpm. The mixture was stirred for 3 h in a laboratory
hood and then attached to а vacuum system for another hour to eliminate residues of DCM.
Microsponges were isolated by centrifugation (5 min at 4000 rpm), following filtration through
stainless steel wire cloth, 400 mesh, and multiple washing with distilled water. Drug unloaded
microsponges were also prepared and taken as control. Each formulation was repeated three times
and all samples were dried in desiccator at room temperature for 24 h. Characterization and analysis
of particles was done at this stage. In the case of microsponges from the optimized formulation F 9,
the entrapped diltiazem base was converted back to the corresponding hydrochloride salt using the
HCl generating chamber (Figure 2S) according to the procedure reported in Supplementary Material.
Once this conversion was made, these diltiazem hydrochloride loaded microsponges were dispersed
in Methylcellulose 2% or Poloxamer 407 20% hydrogel bases for the studies on porcine rectal mucosa
(see below).
2.3.1. Production yield
After 24 h stay in desiccator, each sample of microsponges was accurately weighted. Production
yield (P.Y.) was presented as percent, using Equation 1:
Actual weight of microsponges
P.Y.% = Theoretical total weight of microsponges (drug + polymer).100
Eq. 1
2.3.2. Drug entrapment efficiency
To determine diltiazem entrapment efficiency in microsponges, test solutions were prepared
according to the following procedure. An appropriate amount of microsponges, theoretically
containing 10 mg of polymer Eudragit RS 100, was accurately weighted, transferred to a 100 mL
volumetric flask and dissolved in 5 mL of DCM. Further dilution with anhydrous ethanol up to the
mark was performed and, then, 1 mL of stock solution was transferred to 10 mL volumetric flask and
again diluted to up the mark with anhydrous ethanol to obtain a final concentration of diltiazem
between 3-30 mg/L.
Reference solution was prepared as follows: 10 mg of Eudragit RS 100 were accurately weighed,
transferred to a 100 mL volumetric flask, dissolved in 5 mL of DCM and further diluted up to the
mark with anhydrous ethanol. One mL of stock solution was transferred to 10 mL volumetric flask
and diluted up to the mark with anhydrous ethanol.
Diltiazem concentration was measured by UV spectrophotometry at λ = 244 nm of test solution
against reference solution, using calibration curve of diltiazem in anhydrous ethanol.
Entrapment efficiency (E.E.) was presented as percent, using Equation 2:

8
Actual drug content in particles
E.E.% = Theoritical drug content in particles .100
Eq. 2
Each measurement was carried out in triplicate.
2.3.3. Experimental design
Three formulation factors including drug:polymer w:w ratio (X1), DCM volume (X2) and PVA
concentration (X3) were evaluated for their effects on Production Yield (P.Y.%, Y1), Drug
Entrapment Efficiency (E.E.%, Y2), and particle size (P.S., Y3) of the prepared microparticles by a 23
full factorial screening experimental design using Statgraphics Centurion XVI.II software (StatPoint
Technologies, Inc., Warrenton, VA, USA). Table 1 shows the independent variables, their levels and
studied responses. The dependent variables (i.e., P.Y.% (Y1), E.E.% (Y2), and P.S. (Y3)) were
evaluated and polynomial equations were generated by applying one-way analysis of variance
(ANOVA) (p < 0.05). To demonstrate graphically, the influence of each factor on responses, three-
dimensional response surface plots were generated. Multiple regression analysis was used to assess
the responses employing the following Equation 3:
Y = b0 + b1X1 + b2X2 + b3 X3 + b12X1 X2 + b13X1 X3 + b23X2 X3
Eq. 3
Eq. 3 shows the quantitative effects of the independent variables (X1, X2 and X3) and their interaction
(X1 X2 , X1 X3 and X2 X3) on the selected dependent variable (Y); b0 is the intercept and b1, b2 and b3
represent the regression coefficients for the polynomial equation. Significance was set at p < 0.05 in
the model for the effects of all factors. A factor is considered to significantly influence a selected
response if the p value differs from 0 and is < 0.05. To graphically show the independent variables
which significantly affect a dependent variable, standardized Pareto charts were used. In this charts a
vertical reference line at p value of 0.05 is showed for which an effect exceeding this line is
statistically significant. Moreover, to graphically show the effect of changing two independent
variables on the selected variable Y when the third one is kept at an intermediate value, 3D response
surface plots were built.
[Insert Table 1]
2.4.Characterization of microparticles
2.4.1. Thermal analysis by Differential Scanning Calorimetry (DSC)
DSC studies were carried out with a Mettler Toledo DSC 822e STARe 202 System equipped with a
DSC Mettler STARe Software. About 5 mg of solid sample were placed in an aluminium pan and
hermetically sealed. The scanning rate was of 5 °C/min under a nitrogen flow of 20 mL/min, and the
temperature range was from 25 to 250 °C. The calorimetric system was calibrated by using indium
(purity 99.9%) and following the procedure of the Mettler STARe Software. Each experiment was
carried out in triplicate.
2.4.2. FT-IR spectroscopy
FT-IR spectroscopic analyses were performed using a Perkin-Elmer 1600 FT-IR spectrometer
(Perkin-Elmer, Milan, Italy) on samples dispersed in KBr and compressed in pellets by hydraulic
press. The range examined was 4000–400 cm−1 with a resolution of 1 cm−1.

9
2.4.3. Scanning electron microscopy (SEM)
The size, morphology and surface characteristics of the microparticles were evaluated by SEM
analysis. All the samples were stuck on double-face carbon adhesive stubs and coated with a 10 nm
palladium. The morphological reproducibility was assessed by means of replicate analyses for
each sample. The electron microscope images were taken by a Sigma Zeiss FE-SEM at 5-10 kV,
30 µm aperture. The statistical analysis of the microsponges was made by ImageJ Software.
2.5. In vitro drug release study from diltiazem containing microsponges
Diltiazem containing microsponges were subjected to in vitro drug release test as described in
European Pharmacopoeia Dissolution Apparatus 2 (Paddle apparatus), using 7-station Compact
Dissolution Tester PT-DT 70 (Pharma test, Germany). An accurately weighed amount of
microparticles equivalent to 5 mg o f diltiazem was placed in a dialysis bag and dispersed in 1
mL of dissolution medium constituted by PBS pH 7.4 at 37 oC and rotation speed of 30 rpm. The
dialysis bag was then attached to the paddle and immersed into 400 mL dissolution medium. In
vitro drug release behavior was also investigated in phosphate buffer at pH 5.5, temperature of 37
oC and rotation speed of 30 rpm. Samples from dissolution medium were withdrawn at scheduled
times and diltiazem released was spectrophotometrically determined. Each experiment was carried
out in triplicate.
2.6.
Preparation of microsponge-loaded and conventional diltiazem hydrochloride 2% gels
For these studies microsponges from the optimized formulation F 9, after the treatment with the HCl
generating chamber (Supplementary Material) were used.
Two common gel bases were used – Methylcellulose high viscosity 2% (Mc) and Poloxamer 407
20% (Px). Methylcellulose was added to hot water (90oC) under continuous stirring to obtain
homogeneous dispersion and then left overnight to dissolve in refrigerator at 5oC. Poloxomer 407
was added to the water for gelation at room temperature and left overnight to dissolve in refrigerator
at 5oC.
Diltiazem hydrochloride-entrapped in microsponge formulation (F 9)-loaded 2% gels (micro-l-
diltiazem hydrochloride 2% gels) were prepared as follows. Microsponges were sieved through pore
size stainless steel sieve (d=250 µm) and an appropriate amount of microsponges, corresponding to
0.2 g diltiazem hydrochloride, was added to each of the gel bases (up to 10 g) and homogenized. In
such a way, both F9 loaded Mc-based micro-l-diltiazem hydrochloride 2% gel and F9 loaded Px-
based micro-l-diltiazem hydrochloride were prepared using Methylcellulose (Mc) and Poloxamer 407
(Px) gel bases, respectively.
Conventional diltiazem hydrochloride 2% (c-diltiazem hydrochloride 2%) gels were prepared as
follows. At first, diltiazem hydrochloride (0.2 g) was dissolved in a sufficient amount of water for
gelation. The remaining amount of water for gelation was used for the preparation of each of the gel
bases. Diltiazem hydrochloride solution was added in small aliquots via slow manual stirring to the
corresponding gel base to obtain 10 g gel. Thus, both Mc-based c-diltiazem hydrochloride and Px-
based c-diltiazem hydrochloride were prepared using Methylcellulose (Mc) and Poloxamer 407 (Px)
gel bases, respectively.

10
2.7. In vitro drug release from gels
Drug release studies from the above mentioned gels were carried out on Franz diffusion cell trough
cellulose acetate membrane. The cell possessed exposed membrane surface area of 0.98 cm2 and
receptor volume of 8 mL. Receptor volume was filled almost to the top with PBS pH 7.4 and the cell
was set at 37±0.5oC with the aid of circulating thermostatic bath. About 400 mg of each test
formulation (corresponding to 8 mg diltiazem hydrochloride) were carefully applied on the surface
of the cellulose membrane through the open cell cap, adhesion to the membrane was ensured with the
aid of a tiny piston, after which the open cell cap was covered with Parafilm® to prevent evaporation
from the dosage form and release medium during the test. Receptor volume was adjusted to the top
and stirring was set at 300 rpm. Sufficient volumes (from 0.1 to 0.5 mL) of release medium were
withdrawn at selected time-point, analyzed spectrophotometrically for drug content and replaced with
fresh buffer medium. Data for cumulative drug release were obtained for the period of 12 h for c-
diltiazem hydrochloride 2% and 24 h for micro-l-diltiazem hydrochloride 2%.
2.8.
Drug permeation through porcine rectal mucosa of diltiazem hydrochloride from rectal gels
2.8.1. Mucosal tissue preparation
A fresh specimen of a pig rectum received from a local slaughterhouse of 10 cm in length was
harvested, cut longitudinally to open the lumen and observed for any abnormalities, thoroughly
washed with PBS pH 7.4 and frozen at -80 C°. Following a 24 h freezing time at -80C°, the rectum
was left to defrost at room temperature and placed in cooled PBS pH 7.4 (15 C°).
The specimen was manually dissected under a stereomicroscope with a magnification of 15 x with
cold steel instruments. The instruments used were a sharp tipped dissection forceps, for grasping the
lamina propria, tela submuscosa, tela muscularis and the adventitia and a De Bakey forceps for
grasping and non-traumatic mechanical peeling of the mucosa. After the dissection, the separated
mucosa was further observed under the same magnification of the stereomicroscope for removal of
any attached blood vessels left and remaining non-mucosal tissue. The separated mucosa was
measured to be 0.080±0.005 cm thick. After being thoroughly washed in PBS pH 7.4, the mucosal
tissue was cut in sections measuring 2 x 2 cm with a cold steel scalpel. The prepared sections were
placed in cooled PBS pH 7.4 (Amores et al., 2014).
2.8.2. Procedure
Ex vivo permeation study was performed on Franz diffusion cell following the same procedure as
described above (Section 2.7), using mucosa, isolated from pig rectum, as membrane. Mucosa was
placed so that the smooth mucosal surface acts as the donor compartment. The percentage of
cumulative drug permeated was acquired at 2, 4, 8, 12 and 24 h of the experiment.
2.8.3. Data analysis
To evaluate the results from ex vivo permeation studies, the linear region of each permeation profile
(representing the steady state diffusion) was used by applying the Fick’s first law for steady state

11
.
diffusion (Jss= Kp C, where Jss is the steady state flux, Kp is the permeability coefficient and C is
the concentration gradient representing the difference between the drug concentration in the donor
compartment (Cd) and that in the receptor compartment (Cr). Permeation parameters were obtained
from the cumulative drug permeated versus time plots. The steady state flux (Jss) was calculated from
the slope of the linear tract of permeation curve and presented as mg.cm-2 h-1. The permeability
coefficient (Kp) was calculated as Kp = Jss/ C, assuming that the concentration gradient (C = Cd-
Cr) is constant and thus C can be replaced with initial drug concentration in the dosage form Cd)
[mg.cm-3] (C ≈ Cd, and Kp ≈ Jss/Cd). The lag time (TL) represents the time necessary to establish
steady state diffusion after the initial contact of the dosage form with the mucosal tissue. TL was
calculated for y = 0 using the linear regression of the flux (Brodin, et al., 2009; Khan, et al., 2012;
Herkenne, et al., 2007). These experiments were carried out in duplicates.
2.9.
Permeation through porcine rectal mucosa or dialysis membrane of diltiazem hydrochloride
from diltiazem hydrochloride 2% aqueous solutions
To estimate the impact of the membrane on drug permeation, in vitro permeation study was performed
on diltiazem hydrochloride 2% aqueous solution through porcine rectal mucosa or dialysis membrane
using a Franz diffusion cell. A stock solution of diltiazem hydrochloride was prepared at
concentration 2% in distilled water corresponding to the concentration used in pharmaceutical gels.
Samples from the release media (PBS 7.4, volume – 8 mL) were withdrawn and analyzed
spectrophotometrically at scheduled times. Cumulative graphs, describing the permeation profile of
diltiazem hydrochloride through both types of membrane were built.
2.10. Drug deposition of diltiazem hydrochloride after application on porcine rectal mucosa
Deposition of diltiazem hydrochloride on the mucosal tissue 24 h after application was determined
by two methods:
Method I: At the end of the drug permeation assay, mucosal tissue was carefully detached from the
receptor compartment’s surface, washed thoroughly with PBS pH 7.4, poured in absolute ethanol (10
mL) and shattered in small pieces with the aid of scissors. The so prepared mucosa was kept in ethanol
at room temperature, protected from the light, for 24 h. Ethanol was then decanted in 25 mL
volumetric flask and replaced with fresh portion solvent (4-5 mL) every 2 h. This procedure was
repeated several times until final volume of 25 mL was obtained in the volumetric flask. The ethanol
extract was filtered through 0.22 µm pore size PES syringe filter and analyzed spectrophotometrically
for diltiazem hydrochloride content.
Method II: At the end of the drug permeation study, all cell parts having had contact with the test
dosage form, including the mucosal tissue, were thoroughly washed with absolute ethanol in a 250
mL beaker and then removed from the glass. The mixture was well homogenized as remaining gel
parts were visible and then transferred to 25 mL volumetric flask. Volume was adjusted up to the
mark with absolute ethanol, filtered as above and analyzed spectrophotometrically for drug remained
unreleased. Drug deposition in mucosa was calculated as (Equation 4):
퐷푒푝표푠푖푡푖표푛 푖푛 푚푢푐표푠푎 = 푡표푡푎푙 푎푚표푢푛푡 표푓 푑푟푢푔 푎푝푝푙푖푒푑 ‒ 푑푟푢푔 푢푛푟푒푙푒푎푠푒푑
Eq. 4
‒ 푑푟푢푔 푟푒푙푒푎푠푒푑 푖푛 푟푒푐푒푝푡표푟 푐표푚푝푎푟푡푚푒푛푡

12
For both methods drug deposition was presented as mg.cm-3, considering the mucosal tissue volume
of 0.078 cm3 (exposed area 0.98 cm2 x thickness 0.08 cm = 0.078 cm3 volume). These experiments
were carried out in duplicates.
2.11. Statistical analysis
The results are shown as means ±standard deviation (SD). Data were statistically analyzed by one-
way analysis of variance (ANOVA) and the Bonferroni's “post hoc” test for multiple comparison
using GraphPad Prism v.4 software. Differences were considered statistically significant when
p<0.05.
3. Results and Discussion
The aim of the present work was to evaluate gels containing diltiazem hydrochloride-loaded
microsponges as a modified delivery system of this calcium channel blocker in an attempt to
overcome some drawbacks showed by the use of conventional topical diltiazem hydrochloride
formulations at the concentration of 2%. for the treatment of chronic anal fissures. Such microsponges
were based on Eudragit RS 100 and prepared by the quasi emulsion solvent diffusion method
(Srivastava and Pathak, 2012; Kumari et al., 2016) which involves formation of an emulsion whose
internal phase is constituted by a drug/polymer solution in volatile organic solvent (DCM) and the
external one by an aqueous solution of surfactant (PVA) (Figure 1S). Moreover, since several factors
may influence the results in the development of microsponges as drug carriers (Crcarevska et al.,
2015), the most satisfactory combination of factor levels has been investigated using a 23 full factorial
screening design. It must be noted that, in the search of an optimized formulation, in any case we
used diltiazem base applying the quasi emulsion solvent diffusion method to prepare the Eudragit RS
100 based microsponges. In fact, being diltiazem hydrochloride freely water soluble (453 mg/mL in
distilled water 37°C or 58.23 mg/mL in PBS pH 7.4 37°C), a considerable diffusion in water of the
salt should take place and, consequently, a limited encapsulation efficiency should result, a limitation
that cannot be presented by the poorly water soluble diltiazem base.
3.1. Factorial design
Three formulation parameters (i.e., Drug:polymer weight:weight ratio (X1), DCM volume (X2) and
PVA conc (X3)) were evaluated for assessing their effects on Production Yield (Y1), Entrapment
Efficiency (Y2) and Particle size (Y3) using a 23 full factorial screening experimental design where
X1-X3 constitute the independent variables and Y1-Y3 the dependent ones. The goals to be achieved
are maximization of Y1 and Y2 and minimization of Y3. From the design polynomial equations were
generated which relate the independent variables with each of the dependent ones. The data obtained
were statistically examined to gain information on the optimum values of the independent variables
to accomplish the goals of the study. Table 2 shows the results of regression analysis for Y1-Y3 and
the full model polynomial equations are presented in Eq 5-7, respectively.

13
Y1 (P.Y. %) = 70.41 – 2.43 X1+ 0.79 X2 – 39.05 X3 – 0.12 X1 X2 + 4.18 X1 X3 – 0.06 X2 X3
5
Eq.
Y2 (E.E.%) = 79.09 + 3.58 X1 – 0.16 X2 + 41.82 X3 + 0.017 X1 X2 – 15.66 X1 X3 – 0.41 X2 X3 Eq.
6
Y3 (P.S.) = -14.67 – 0.82 X1+ 23.36 X2 – 153.86 X3 - 6.82 X1 X2 + 125.46 X1 X3 – 20.63 X2 X3 Eq.
7
[Insert Table 2]
The goodness of fit is substantiated by the correlation coefficient R2 values and even more by the
adjusted R2 (Adj R2) values which, if are near to 1, indicate an optimal-excellent correlation between
the independent variables and the selected response. As reported in Table 2, the R2 and Adj R2 values
for P.Y. and E.E. are greater than 0.99 and it means that the corresponding models are highly
significant and explain more than the 99% of variation around the mean value. In contrast, the R2 and
Adj R2 values for P.S. are lower than 0.99 suggesting that the significance of the corresponding model
is worse compared to the previous ones. The coefficients of the equations evaluate the effect of a
particular independent variable from a quantitative point of view and the statistical significance of
the coefficients is assessed by the p-value (Table 2) or graphically by the Pareto charts and response
surface plots.
3.1.1. Effect of independent variables on production yield (P.Y.)
P.Y.% of the particles studied in the factorial design varied in the range from 58.30% to 76.80%. The
effect of independent variables on P.Y. is shown by the polynomial Eq. 5 and data reported in Table
2 as well as graphically by Figure 1. Based on these findings, it is evident that decrease of
drug:polymer ratio or PVA concentration led to a significant increase in P.Y.% of particles prepared
using the quasi emulsion solvent diffusion method (Srivastava and Pathak, 2012; Kumari et al., 2016).
In contrast, an increase of DCM volume brought about a less significant increase in P.Y.%. All these
effects of independent variables on P.Y.% have been already described in literature and possible
rationalizations have been presented (Srivastava and Pathak, 2012; Jain and Singh, 2010). Further
information which can be deduced from the reported findings is that the interaction terms (i.e., the
effect of two factors simultaneously) did not have significant influence and it has been not previously
described in the literature.
[Insert Figure 1]
3.1.2. Effect of independent variables on entrapment efficiency (E.E.)
E.E.% of microsponges ranged from 79.63% to 88.95% and again the effect of independent variables
on E.E.% can be deduced from the polynomial equation (i.e., Eq. 6) data reported in Table 2 and from
Figure 2. All the results clearly show that decrease of drug:polymer ratio or PVA concentration
caused a significant increase in E.E.% and once again possible explanations have been reported
(Srivastava and Pathak, 2012; Jain and Singh, 2010). However, the most significant effect on E.E.%
was shown by the interaction between drug:polymer ratio and PVA concentration (X1X3) and, more
specifically, decrease of this interaction term brought about a significant increase in E.E.%. To the

14
best of our knowledge, the effect of this interaction term has been not previously shown in the
literature.
[Insert Figure 2]
3.1.3. Effect of independent variables on particle size (P.S.)
As above mentioned, no independent variables or their interactions had significant effect on particle
size (p > 0.05) of the microsponges prepared. This insignificant effect of the independent variables
chosen, in our opinion should be due to the wide standard deviation observed in the size measure of
the particles studied in the factorial design by SEM analysis (Table 1). However, it can be useful to
evaluate from a qualitative point of view the corresponding polynomial equation (i.e., Eq. 7), data
reported in Table 2 and Figure 3S. Focusing our attention to the main independent variables of the
model, it can be deduced that increasing the drug:polymer ratio results in small particles as reported
in literature (Cetin et al., 2012). A similar effect should be played by the interaction between
drug:polymer ratio and DCM volume (X1X2).
By using the “response optimization” option of the Statgraphics Centurion XVI.II software, it was
calculated the optimum desirability in order to identify an optimized formulation which accomplishes
an optimum combination of factor levels. It was found that a total optimum desirability value of
72.66% corresponds to the following selection of factor levels: drug:polymer ratio X1 = 3, DCM
volume X2 = 12; PVA concentration X3 = 0.05. Using this combination of factor levels, it results a
predicted P.Y. % of 66.83 (confidence interval 60.94% - 72.71%) and a predicted E.E.% of 87.98%
(confidence interval 84.91% - 91.05%). However, we noted that, in general, using high drug/polymer
mass ratio X1 ≥ 3 the work-up and isolation procedure resulted somewhat difficult and substantial
losses of final product were observed. Therefore, further experimental runs were carried out in order
to find possible additional experimental conditions with good optimum desirability value and the
amount of each component used in these supplementary formulations together with the corresponding
responses are reported in detail in Table 1. As seen, spherical microsponges were obtained in high
yield (63.10% - 86.25 %) and high entrapment efficiency (79.63% - 91.50%) as well as with mean
diameters ranging from 33 m to 239 m. In particular, using a drug:polymer ratio of 0.33, a DCM
volume of 8 mL and a PVA concentration of 0.15 % w/v, spherical microsponges endowed with P.Y.
% of 82.90% and E.E.% greater than 90% were obtained (F 9, Table 1) and such experimental
conditions seem a good combination of factor levels for a satisfactory optimized formulation. Under
such circumstances spherical microsponges were obtained while in other cases, irregular spherical
microsponges or even rods were observed (Table 1).
3.2. Physicochemical characterization of microsponges
The DSC thermogram of diltiazem base showed a sharp endothermic peak at 106°C which
corresponds to the melting of the drug in crystalline form, while the thermogram of the polymer alone
displayed a less intense sharp endothermic peak at 198°C (Figure 3). Blank microsponges showed
the endothermic peak attributable to the polymer, although at lower temperature (i.e., at 173°C) while,
in the case of F 9, the endothermic peak of the polymer occurs at very low intensity and at intermediate
temperature (i.e., at 182°C) without the characteristic endothermic peak due to the drug melting. This

15
finding may suggest a change in the drug crystallinity in the Eudragit RS 100 matrix of the optimized
formulation involving the presence of the drug in an amorphous state or disordered crystalline phase
(Ahmed and Aljaed, 2017; Sun et al., 2015).
The FT-IR spectrum of pure diltiazem base showed a characteristic peak of the acetate stretching at
1743 cm-1 as well as the C=O stretching of the amide group at 1680 cm-1(Figure 3). The spectrum of
blank microsponges displayed a large peak centered at 1732 cm-1 due to the C=O stretching of the
carbonyl groups of the Eudragit RS 100 matrix, while in the F 9 the characteristic drug stretching of
the amide group at 1680 cm-1 can be detected besides to the large peak centered at 1732 cm-1 of the
polymeric matrix. Such result should suggest that no interaction between drug and polymer occurs
and, hence, they should be compatible in the formulations studied.
.
[Insert Figure 3]
The particle size of the microspheres was determined by SEM analysis and it resulted below enough
250 m. Furthermore, SEM analysis allowed to evaluate the surface and the whole morphology of
the particles (Figure 4 and Figure 4S) which resulted porous enough and predominantly spherical,
although in some cases irregular spherical microsponges or even rods were obtained. It has been
proposed that the pores are brought about by diffusion of solvent from surface of microsponges during
their preparative procedure (Srivastava and Pathak, 2012). Morever, using the Energy Dispersive X-
ray Analysis (EDX) option of the SEM-EDX microscope, it was found that the particles were
constituted by a very low amount of chlorine atom on the surface (Figure 4) suggesting so that DCM
may be essentially removed by the experimental work-up followed by us. Therefore, it can be
concluded that the quasi-emulsion solvent method we used allows the preparation of microsponges
without residual traces of organic solvents and, consequently, without concerns from a toxicological
point of view. Moreover, SEM analysis allowed us to gain also information on the surface chemical
nature of the microparticles since elemental analysis of the surface components can be deduced.
Appreciable sulfur content is indicative of surface location. The sulfur content of the unloaded
microsponges reflected the amount of this element as impurity of the polymer used.
At this regard, EDX analysis showed that the diltiazem hydrochloride-loaded microparticles were
constituted by an amount of sulfur atom on the surface greater than that found for blank microsponges
(i.e., 0.21±0.07 weight percent) and it suggests that diltiazem hydrochloride may be in part present
on the surface of the drug-loaded particles. Thus, for instance, in the case of F 9, it was found a surface
sulfur content of 1.68±0.99 weight percent, different enough from that occurring for blank
microsponges (Figure 4).
[Insert Figure 4]
3.3. In vitro drug release studies on microsponges from Formulation 9
To evaluate the potential of the studied MDS for sustained delivery of diltiazem, the release kinetic
of selected formulations was evaluated in PBS at pH 7.4 and pH 5.5. It should be noted that the
colonic pH has been reported to range by values fluctuating from pH 5 to pH 8 depending on the
transit times and composition of food intake (Koziolek et al., 2014; Marques et al., 2011). Of course,

16
such features are highly important for the understanding of drug release from dosage forms containing
ionizable drugs as diltiazem hydrochloride. The selected pH values employed in drug release studies
in this paper can be considered near to the two above mentioned limits of the pH range accepted for
colon (i.e., pH 7.4 and pH 5.5) and the obtained results for the representative Formulation 9 (F 9) are
shown in Figure 5.
[Insert Figure 5]
Results showed that after 6 h at pH 5.5 about 72% of diltiazem was released from F 9. Instead, the
same formulation at pH 7.4 only after 18 h released about 61% of the calcium channel blocker drug,
demonstrating that under the latter conditions a sustained release of the occurs.
3.4. Ex vivo permeability experiments through porcine rectal mucosa
As above mentioned, prior to proceed with release studies from hydrogels as well as with those of ex
vivo permeability through and drug deposition on porcine mucosa, the entrapped diltiazem base of F
9 was converted to the corresponding diltiazem hydrochloride by using the HCl generating chamber
(Supplementary Material). This treatment on microsponges from F 9 was made before their dispersion
in hydrogel bases and it was necessary to increase the therapeutic potential of diltiazem base which
should be limited due to the poor water solubility of this compound (0.202 mg/mL in PBS pH 7.4,
37°C). Such conversion method is unprecedented and non-destructive and it can be envisaged as an
approach to incorporate water soluble substances as hydrochloride salts in microsponges through the
quasi emulsion solvent diffusion method.
The two gel bases were used to prepare the micro-l-diltiazem hydrochloride at 2% and they are well-
known for their peculiar features as the temperature-sensitive properties of Poloxamer 407 and the
mucoadhesive ones of Methylcellulose (Singh et al., 2009).
As shown in Figure 6 A, the drug release from conventional c-diltiazem hydrochloride 2% gels
resulted practically constant after 6-12 h, whereas micro-l-diltiazem hydrochloride 2% gels exhibited
a prolonged release up to 24 h. In addition, it was established that the use of microsponges in diltiazem
hydrochloride hydrogels reduces the initial “burst effect”. Using methylcellulose as gelling agent, for
both c-diltiazem hydrochloride 2% and micro-l-diltiazem hydrochloride 2% gels, a faster and more
complete diltiazem hydrochloride release compared to Poloxamer 407 occurred. It seems that these
results can be probably related to different viscosity between the two gel types.
The permeability profiles through porcine rectal mucosal are reported in Figure 6 B and the
corresponding permeation parameters are shown in Table 3. The highest permeation rate of diltiazem
hydrochloride was observed from Mc-based c-diltiazem hydrochloride, whereas the lowest from F9
loaded Mc-based micro-l-diltiazem hydrochloride. A possible explanation accounting for the greater
permeation rate of Methylcellulose gels compared to the Polaxamer 407 ones might be related to
better mucoadhesive properties of the former polymer. An almost bimodal trend in the permeation
profiles was observed for most formulations, in particular, changes in the permeation course occurred
between 1-2 h for conventional gels and between 3-6 h for microsponge loaded gels. This
phenomenon is likely observed in the time required for the permeation of diltiazem hydrochloride to
establish an equilibrium between the dosage form and the mucosal tissue. As a consequence of these

17
bimodal trends, in the calculation of steady state diffusion flux (Jss) values, low regression coefficients
were obtained (Table 3). As can be seen in Table 3, the flux values observed for the conventional
formulations were greater than those entrapping diltiazem hydrochloride in microsponges, as
expected taking into account that the latter formulations constitute sustained delivery systems of the
calcium channel blocker drug. Next, our attention was focused on the negative values of the lag time
(TL). From the plots in Figure 6 B it is evident that a substantial “delay” in the steady-state permeation
establishment after application of the dosage form does not occur for none of the formulations tested.
However, such negative values are also probably related to the aforementioned deviations from
linearity observed in these permeation curves (Table 3).
An in vitro permeation study through rectal mucosa or synthetic membrane using diltiazem
hydrochloride 2% solution, evidenced the marked impact of the former membrane in drug
permeation. When artificial cellulose acetate membrane was used, indeed, equilibrium in drug
concentration between the donor and receptor compartment of Franz cell was established within 2.0-
2.5 h with a drug permeation of about 70% after 5h (Figure 6 C). In contrast, using rectal mucosa a
slower transport resulted with a drug permeation of 39% after 24 h. This result should be likewise
ascribed to the fact that equilibrium between three compartments (i.e., donor- membrane- receptor-
compartment) of rectal mucosa occurs much more slower than that through cellulose acetate
membrane. Moreover, the solubility of diltiazem hydrochloride decreases substantially under the
slightly alkaline physiological pH in the rectum (pH 7.4) due to a partial conversion to diltiazem base.
[Insert Figure 6]
The drug deposition on porcine rectal mucosa is an important indication of the local chronic anal
fissures therapy efficacy. In this regard, we used two experimental procedures referred to as Method
I and Method II, respectively (Section 2.9), both characterized by some limitations which should be
taken into account. Thus, a shortcoming of Method I is that the complete drug extraction from the
mucosa is not feasible and the interference on the assay caused by lipids extracted from the mucosa
may occurs. On the other hand, a drawback of Method II is represented by a higher risk of error.
However, the results from both Methods clearly showed that micro-l-diltiazem hydrochloride 2% gels
provide a diltiazem hydrochloride concentration twofold higher than that displayed by conventional
formulations (Table 3 and Figure 7). Similarly, the percentages of drug deposited on rectal mucosa
resulted more than twofold higher than those of conventional formulations (Figure 7). Altogether,
these results confirmed our expectations that a modified-release diltiazem hydrochloride semi-solid
dosage form should be advantageous compared to the conventional one by lowering the release and
permeation rate. Micro-l-diltiazem hydrochloride 2% gels showed 15.39% and 17.95% higher
diltiazem hydrochloride retention on mucosa for Poloxamer 407 and Methycellulose gels,
respectively.
[Insert Table 3 and Figure 7]
Conclusions

18
In this work, gels containing diltiazem hydrochloride-loaded microsponges were evaluated as an
approach to overcome some drawbacks showed by the conventional topical diltiazem hydrochloride
formulations for the treatment of chronic anal fissures. Such microsponges were based on Eudragit
RS 100 and prepared by the quasi emulsion solvent diffusion method. The optimized microsponge
formulation used for these studies was selected on the basis of a 23 full factorial screening design.
The in vitro drug release resulted faster for conventional formulations and essentially complete within
6-12 h, while the microsponges loaded gels showed a prolonged release up to 24 h. Ex vivo
permeability studies demonstrated that the highest drug permeation rate was observed for the
conventional diltiazem hydrochloride 2% (c-diltiazem hydrochloride 2%) formulations, whereas the
lowest occurred from Poloxamer 407 20% diltiazem hydrochloride loaded microsponges 2% gel (F9
Px-diltiazem hydrochloride). From drug deposition after application on porcine rectal mucosa studies,
it was observed that micro-l-diltiazem hydrochloride 2% gels provide a diltiazem hydrochloride
concentration twofold higher than that displayed by conventional formulations. Overall, the results
obtained lend support for the hypothesis that a sustained delivery system of diltiazem hydrochloride
may be helpful to enhance patient compliance and improve the results of the treatment of chronic anal
fissures using diltiazem hydrochloride based medical approach. In particular, the use of microsponge
loaded formulations, characterized by a lower permeation rate combined with a higher deposition on
mucosa compared to the conventional ones, may be considered as a promising approach to overcome
the side effects and patient non-compliance showed by the conventional diltiazem hydrochloride
formulations employed in the treatment of chronic anal fissures. In this context, the performance of
the Poloxamer 407 20% microsponge loaded- diltiazem hydrochloride 2% gel (F9 Px-diltiazem
hydrochloride) appears particularly interesting and its further development with in vivo studies is
worthy to be carried out.
Declarations of interest
None.
Acknowledgments
NAI, AT, CF would like to acknowledge the recipient of Erasmus Plus Programme 2014-2015 for
financing NAI’s research mobility.
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