Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idrd20 Download by: [St Louis College of Pharmacy Library] Date: 24 January 2017, At: 10:29 Drug Delivery ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20 Intranasal brain-targeted clonazepam polymeric micelles for immediate control of status epilepticus: in vitro optimization, ex vivo determination of cytotoxicity, in vivo biodistribution and pharmacodynamics studies Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M. Rashed & Ahmed B. Ibrahim To cite this article: Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M. Rashed & Ahmed B. Ibrahim (2016) Intranasal brain-targeted clonazepam polymeric micelles for immediate control of status epilepticus: in vitro optimization, ex vivo determination of cytotoxicity, in vivo biodistribution and pharmacodynamics studies, Drug Delivery, 23:9, 3681-3695, DOI: 10.1080/10717544.2016.1223216 To link to this article: http://dx.doi.org/10.1080/10717544.2016.1223216 Published online: 20 Sep 2016. Submit your article to this journal Article views: 64 View related articles View Crossmark data
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Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=idrd20
Download by: [St Louis College of Pharmacy Library] Date: 24 January 2017, At: 10:29
Intranasal brain-targeted clonazepampolymeric micelles for immediate control ofstatus epilepticus: in vitro optimization, exvivo determination of cytotoxicity, in vivobiodistribution and pharmacodynamics studies
Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M.Rashed & Ahmed B. Ibrahim
To cite this article: Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M.Rashed & Ahmed B. Ibrahim (2016) Intranasal brain-targeted clonazepam polymeric micellesfor immediate control of status epilepticus: in vitro optimization, ex vivo determination ofcytotoxicity, in vivo biodistribution and pharmacodynamics studies, Drug Delivery, 23:9,3681-3695, DOI: 10.1080/10717544.2016.1223216
To link to this article: http://dx.doi.org/10.1080/10717544.2016.1223216
Drug Deliv, 2016; 23(9): 3681–3695! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/10717544.2016.1223216
RESEARCH ARTICLE
Intranasal brain-targeted clonazepam polymeric micelles for immediatecontrol of status epilepticus: in vitro optimization, ex vivo determinationof cytotoxicity, in vivo biodistribution and pharmacodynamics studies
Samia A. Nour1, Nevine S. Abdelmalak1, Marianne J. Naguib1, Hassan M. Rashed2, and Ahmed B. Ibrahim2
1Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt and 2Labeled Compounds Department, Hot Lab. Center,
Egyptian Atomic Energy Authority, Cairo, Egypt
Abstract
Clonazepam (CZ) is an anti-epileptic drug used mainly in status epilepticus (SE). The drugbelongs to Class II according to BCS classification with very limited solubility and highpermeability and it suffers from extensive first-pass metabolism. The aim of the present studywas to develop CZ-loaded polymeric micelles (PM) for direct brain delivery allowing immediatecontrol of SE. PM were prepared via thin film hydration (TFH) technique adopting a centralcomposite face-centered design (CCFD). The seventeen developed formulae were evaluated interms of entrapment efficiency (EE), particle size (PS), polydispersity index (PDI), zeta potential(ZP), and in vitro release. For evaluating the in vivo behavior of the optimized formula, bothbiodistrbution using 99mTc-radiolabeled CZ and pharmacodynamics studies were done inaddition to ex vivo cytotoxicty. At a drug:Pluronic� P123:Pluronic� L121 ratio of 1:20:20 (PM7), ahigh EE, ZP, Q8h, and a low PDI was achieved. The biodistribution studies revealed that theoptimized formula had significantly higher drug targeting efficiency (DTE¼ 242.3%), drugtargeting index (DTI¼ 144.25), and nose-to-brain direct transport percentage (DTP¼ 99.30%)and a significant prolongation of protection from seizures in comparison to the intranasallyadministered solution with minor histopathological changes. The declared results reveal theability of the developed PM to be a strong potential candidate for the emergency treatmentof SE.
Keywords
Brain, micelles, clonazepam, central compos-ite, intranasal
History
Received 6 June 2016Revised 1 August 2016Accepted 8 August 2016
Introduction
Status epilepticus (SE) represents a medical emergency that is
associated with high morbidity and mortality (Manno, 2003).
It is defined as continuous or intermittent seizures lasting
more than 5 min, without full recovery of consciousness
between seizures (Chen & Wasterlain, 2006). It requires
immediate intervention (Brophy et al., 2012) as the longer the
seizures, the greater the risk of cerebral damage (Macri,
2010). Treatment involves intravenous administration of a
central nervous system (CNS) depressant, namely, of benzo-
diazepine (BDZ) class (Lockey, 2002).
Clonazepam (CZ) is a potent, long-acting nitrobenzodia-
zepine derivative with anticonvulsant, muscle-relaxant, and
anxiolytic properties. It increases the effects of d-aminobu-
tyric acid (GABA) via modulation of the GABA receptor
(Nardi et al., 2013). Furthermore, CZ offers advantages over
other BDZ due to longer duration of action (Rey et al., 1999).
Clinical studies also revealed that clinical symptoms resolved
more completely with CZ (Lockey, 2002).
Oral or intravenous administration of CZ releases the drug
directly into the peripheral circulation that results into both
limited uptake across the blood-brain barrier (BBB) (Vyas
et al., 2006) and distribution to non-targeted sites which leads
to a number of side effects including palpitation, hair loss and
anorexia (Roche, 2009).
In addition, oral or intravenous administration of the drug
to patients suffering acute SE might be impractical or
inconvenient. From one side, intravenous administration
requires a qualified personnel or a near hospital facility.
From the other side, SE may impair the ability of the patient
for swallowing tablets (Anon, 2015). Thus, intranasal drug
delivery would present a competitive pathway for drug
targeting. It protects the drug from first-pass elimination
(Illum, 2003), circumvents the obstacles of BBB via olfactory
region allowing direct delivery to the CNS (Pires et al., 2009).
Moreover, intranasal delivery is considered to be simple,
convenient, and cost-effective (Marx et al., 2015).
CZ has been previously formulated as intranasal mucoad-
hesive microemulsion for brain targeting (Vyas et al., 2006), it
has been formulated also as solid lipid nanoparticles for
parental administration. To our knowledge, CZ has not been
formulated as polymeric micelles (PM) nanocarriers for
intranasal administration. Thus, herein, mixed PM wereAddress for correspondence: Marianne J. Naguib. Email:[email protected]
developed and optimized as another potential system for brain
targeting of CZ.
PM are nanoscopic structure formed by amphiphilic block
copolymers composed of hydrophilic and hydrophobic chains
that self-assemble in water, above a certain concentration
named the critical micelle concentration (CMC) (Chiappetta
& Sosnik, 2007). They consist of an inner core of assembled
hydrophobic segments capable of solublizing lipophilic
substances and an outer hydrophilic corona serving as a
stabilizing interface between the hydrophobic core and the
external aqueous environment (Francis et al., 2004).
PM have the advantage of by-passing the P-glycoprotein
(P-gp) efflux since they are transported into the cells via
receptor-mediated endocytosis in contrast to the typical free
drug diffusion (Srivalli & Lakshmi, 2012). P-gp are drug
efflux protein that hinders distribution of many drugs to the
brain, intestine, and multidrug-resistant (MDR) tumors (Amin,
2013). However, such systems have the drawbacks of forma-
tion of aggregates with a large size, which falls outside the
apparent preferred size range for drug delivery systems using
nanoscale particles and lack of stability in aqueous dispersion
leading to phase separation (Oh et al., 2004).
So, the aim of the present study was to formulate and
optimize stable PM for rapid brain targeting of CZ. PM are
expected to provide rapid nose-to-brain delivery with greater
transport and resident of the drug in the brain. This can help
to increase drug efficacy, reduce side effects, and decrease the
dose and dosing frequency. The performance of the prepared
micelles was evaluated in vitro using different criteria, ex vivo
for cytotoxic properties and in vivo in mice using biodistribu-
tion of 99mTc-clonazepam and appropriate pharmacodynam-
ics models.
Materials and methods
Materials
CZ was a kind gift from Amoun Pharmaceuticals (Elabour
city, Egypt), poly(ethylene glycol)-block-poly(propylene
showed the highest blood accumulation of the drug due to
direct delivery of the drug to the blood, followed by intranasal99mTc-CZ solution and then intranasal 99mTc-PM7. These
differences were proved to be statistically significant
(p50.05).
Brain/blood ratios computed for different radiolabeled
preparations (Table 5) were obtained by dividing brain
reading by blood reading for each mouse at each time
interval. Statistically higher brain/blood ratios of intranasal99mTc-PM7 (p50.05), in comparison to the intranasally
administered solution and to the intravenously administered
PM7 formula, indicates the brain targeting ability of the
optimized PM formula.
The pharmacokinetic behavior of the three administered
preparations were mathematically evaluated by the calcula-
tion of Cmax, Tmax, and AUC0–1 for brain and blood (Table 6).
For the brain, the values were (0.24, 4.28, 0.29) %ID/g, (0.25,
0.25, 0.5) h and (0.32, 2.68, 0.80) h%ID/g for intranasal99mTc-CZ solution, intranasal 99mTc-PM7 and intravenous99mTc-PM7, respectively (Table 6). The significantly higher
Figure 3. TEM photomicrographs of mixedPluronic� L121/P123 polymeric micelles(PM7).
DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3689
Cmax and AUC0–1 values of the intranasal 99mTc-PM7
confirm direct delivery of the drug to the brain in comparison
to the other two administered radiolabeled preparations.
This is further proved by the relative bioavailability (Table 6)
which was found to be 812.96% and 11.83% for brain and
blood, respectively.
Drug administered intranasally can reach brain using
mainly two different pathways: (i) either through reaching
Figure 4. Photomicrographs of the anterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positivecontrol, b), and CZ-loaded polymeric micelles (c) (100�).
Figure 5. Photomicrographs of the posterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positivecontrol, b), and CZ-loaded polymeric micelles (c) (100�).
3690 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695
the systemic circulation then crossing BBB into the brain or
(ii) direct nose-to-brain transport from the nasal mucosa
through the olfactory region and the trigeminal nerve
bypassing the BBB (Illum, 2003). Based on the results
of the biodistribution study, DTE, DTI, and DTP values
were calculated for both intranasal 99mTc-CZ solution and
intranasal 99mTc-PM7 (Table 7). DTE% represents time
average partitioning of drug between brain and blood
(Haque et al., 2014), while DTI is a measure of the
differential targeting between intranasal and intravenous
delivery (Taylor et al., 2010) and DTP% represents the
percent of drug directly transported to the brain by the
olfactory and trigeminal neural pathway (Haque et al., 2014).
Their values were 242.39%, 5.78%, 144.25, 3.46, and 99.3,
70.07 for intranasal 99mTc-PM7 and intranasal 99mTc-CZ
solution, respectively.
Figure 6. Variation of the radiochemical yield of 99mTc-clonazepam as a function of clonazepam amount (a), Na2S2O4 amount (b), pH (c), reactiontemperature, (d) and time (e).
DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3691
These results are in accordance with Jain et al. (2010) and
Kanazawa et al. (2011) who found that intranasal PM have a
very high potential for brain targeting of zolmitriptan and
coumarin, respectively.
Pharmacodynamic studies
The ability of the preparations to protect mice from PTZ-
induced seizures after intravenous and intranasal administra-
tions was evaluated to compare the preparations and their
delivery routes (Florence et al., 2011). PTZ was administered
after predefined intervals of 15-, 30-, and 45-min posttreat-
ment with CZ preparations. The onset of seizures in animals
treated with different preparations and routes is shown in
Table 8. The saline-treated control group produced
convulsions with an onset of 58 secs, on average, at the
three time intervals. CZ solution (CZS) was administered
intravenously 15, 30, and 45 min prior to PTZ challenge. It
offered protection against PTZ-induced convulsions by
delaying the onset significantly (p50.05) for 30
(160.6 ± 7.02 s) and 45 min (139.0 ± 8.18 s) treatment group
in comparison with the control group (less than 60 s).
However, intravenous CZS failed to induce significant
protection after 15 min (p40.05).
Although intranasal CZ solution offered prolongation of
the onset of PTZ-induced seizures at all time intervals in
comparison to control groups (Table 8), these differences
were found to be statistically insignificant. This may be due to
the limited ability of the i.n. solution to deliver the drug in
adequate conc. to the brain. On the other hand, the offered
Table 5. Brain/blood distribution of CZ administration as intranasal 99mTc solution, intranasal 99mTc-PM7, and intravenous 99mTc-PM7 in male Swissalbino mice (mean ± SD, n¼ 3).
Time
Formulation/route of administration Organ or tissue 0.25 0.5 1 2 4 8
Figure 7. CZ concentration in brain atdifferent time intervals following adminis-tration of intranasal 99mTc-CZ solution,intranasal 99mTc-PM7 and intravenous 99mTc-PM7, mean ± SD, n¼ 3, in male Swiss albinomice.
0
1
2
3
4
5
0 1 2 3 4 5 6 7 8
CZ
Con
c. in
Bra
in (
%/g
)
Time (hr)
99mTc-CZ(i.n) 99mTc- PM7 (i.n) 99mTc-PM7(i.v)
Figure 8. CZ concentration in blood atdifferent time intervals following adminis-tration of intranasal 99mTc-CZ solution,intranasal 99mTc-PM7 and intravenous 99mTc-PM7, mean ± SD, n¼ 3, in male Swiss albinomice.
0
2
4
6
8
10
0 1 2 3 4 5 6 7 8
CZ
Con
c. in
Blo
od (
%/g
)
Time (hr)
99mTc-CZ(i.n) 99mTc- PM7 (i.n) 99mTc-PM7(i.v)
3692 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695
protection produced by intranasal PM7 is significantly higher
(p50.05) than all treatment groups and the control at all time
intervals. It reached 424.33 ± 31.5, 332.33 ± 41.1, and
314.66 ± 24.58 after 15, 30, and 45 min, respectively. This
confirms the ability of the PM to directly deliver the drug to
the brain in high concentration depending on the ability of the
Pluronics� to overcome the P-gp efflux mechanism, in
addition to offering a solubilized form of the drug that
allows its immediate uptake and improved efficacy.
Conclusion
Kinetically and thermodynamically stable PM were success-
fully developed using TFH technique. The ability of the
optimized polymeric micelle formula (PM7) with an accept-
able PS range and ZP, the lowest PDI and the highest EE for
incorporation of the drug was confirmed by TEM and DSC
results. PM7 produced minor histopathological changes
without affecting the integrity of the sheep nasal mucosa. In
addition, the biodistribution and pharmacodynamics studies
demonstrated the rapid and effective brain uptake of CZ in
mice following intranasal administration of the suggested
formula. This may represent an alternative to intravenous
administration in the management of acute SE especially
when oral administration is not feasible or it is clinically not
possible to treat the patient before hospitalization. However,
clinical benefits to risk ratio of the developed formulation
have to be established for its appropriateness in the clinical
practice.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
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DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3695