Nasal melatonin gels using pluronic PF-127 for chronobiological

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Nasal melatonin gels using pluronic PF-127 for chronobiological Powered By Docstoc
					Indian Journal of Biotechnology
Vol 3, July 2004, pp 369-377




    Nasal melatonin gels using pluronic PF-127 for chronobiological treatment of
                                   sleep disorder
                                  S S Pisal, P Reddy, A R Paradkar, K R Mahadik* and S S Kadam
                            Department of Pharmaceutics, Poona College of Pharmacy and Research Centre
                                    Bharati Vidyapeeth Deemed University, Pune 411 038, India
                                             Received 5 May 2003; accepted 24 June 2003

            Melatonin, a neurohormone, is formulated as a thermoreversible pluronic gel for nasal administration as an ‘overlap
      dosage form’ for chronobiological treatment of sleep disorders. Aqueous PF gels containing drug (0.5 mg & 1 mg/0.1 ml),
      PEG 400 and PEG 15,000 were prepared by cold method. Pluronic gels were evaluated for gelation and gel melting. Gela-
      tion temperature (T1) decreased with pluronic concentration while gel melting temperature (T2) increased. Melatonin shifted
      gelation range to higher temperature while PEG narrowed the gel range. Flux of diffusion decreased with PF concentration.
      Drug flux decreased in higher drug strength gels due to more partitioning in micellar phase. Pluronic gel (20%w/w, 1 mg/0.1
      ml) showed bimodal pattern with a desired second peak flux (0.248μg/min/cm2) at 300 min. Flux pattern changed invariably
      with PEG. Bioadhesion time and strength to sheep nasal mucosa were more for gels containing melatonin and PEG 400. Na-
      sal gels produced fast onset of action and induced sleep within fifteen minutes. The low intensity and rounded α-EEG wave
      pattern was observed for sleep duration of 5 hrs. Good correlation was observed in sleep pattern and low intensity α-EEG.
      The results are encouraging and nasal melatonin gels have potential in the treatment of circadian cycle sleep disorders.
      Keywords: pluronic gels, melatonin, nasal, chronobiological release, sleep pattern
      IPC Code: Int. Cl.7 A 61 K 38/00, 47/36

Introduction                                                          very slow onset of action and require higher dose for
   Melatonin (MT) is an indolamine neurohormone                       therapeutic effect. These are intended to facilitate the
secreted by pineal gland. The phase shifting effect of                sleep (5 mg), regulate the body clock (propounding or
circadian rhythm by melatonin is mediated by MT2                      postponing sleep, 5-10 mg), supplement in jet lag and
receptors1. Pineal gland functions as biological clock                major depression4-6. Researchers have established
by secreting melatonin according to circadian cycle.                  clinical usefulness of melatonin in the treatment of
Normal plasma level of melatonin during daytime                       oxidative stress, breast cancer and Alzheimer's dis-
hours is 10pcg/ml; after sunset the level rises continu-              ease7-12. The conventional tablet, solution and tea of
ously eventually peaking around 2 A M (50 pcg/ml),                    melatonin have been marketed as a supplementary
then gradually declines, reaching minimum in the                      nutrient in USA. Transdermal films of melatonin con-
morning2. The disturbed melatonin secretion leads to                  taining propylene glycol showed poor systemic
changes in sleep-wake up cycle. Impairment in mela-                   bioavailability (18 %). Transdermal administration
tonin production contributes to well-known inci-                      does not stimulate the nocturnal release pattern. A
dences of insomnia and delayed sleep syndrome.                        sugar spheres loaded melatonin with additional aqua-
   Lerner was the first to characterize the sleep en-                 coat, and HPMC matrix tablet showed sustained re-
hancing properties of melatonin3. Sleep, after the ad-                lease for 8 hrs13. Oral formulations showed large in-
ministration of melatonin, resembles natural sleep                    tersubject variability in plasma profiles. Steady state
unlike currently available hypnotic agents. Oral mela-                plasma levels were also achieved after oral TMD
tonin administration undergoes extensive hepatic-first                patch.
pass (70%) and has a biological half-life of 45 min.                     Exogenous nasal melatonin formulation could be
Many conventional formulations of melatonin have                      used as circadian rhythm synchronizer in humans.
                                                                      Crucial for successful treatment of chronological dis-
_________________                                                     orders with melatonin is its correct timing according
*Author for correspondence:
Tel: 91-20-25437237; Fax: 91-20-25439383
                                                                      to phase response curve. An 'overlap' dosage regimen
E-mail: krmahadik@rediffmail.com                                      in which exogenous and endogenous melatonin peaks
370                                     INDIAN J BIOTECHNOL, JULY 2004


overlap is desired14,15. Recently, an attempt to formu-   [equivalent to 1, 3 & 5% (w/w)], and PEG 15,000
late nasal melatonin using ethanol caused serious mu-     [0.5, 1 & 1.5% (w/w)] each were prepared separately.
cosal irritation, painful administration and hence poor   Gels containing 1 % w/w melatonin were prepared by
patient compliance16. Transnasal drug administration      dissolving drug in ethanol and following the cold
avoids first pass metabolism, gastric degradation of      method. In case of PEG 400 and PEG 15,000 contain-
peptide and gives rapid drug absorption e.g. calci-       ing PF 127 gels, the required amount of PEG was dis-
tonin, propranolol and gentamycin17. However, short       solved in distilled water, prior to polymer addition.
nasal residence of solutions results in non-uniform
absorption profiles. Attempts have been made to pro-      Evaluation of Gels
long the nasal residence by designing powder sprays,
                                                          Gelation and Gel Melting
microspheres, and erythrocyte based bioadhesive sys-
                                                             A modified Millar et al technique using 2 ml of gel
tems18-21.
                                                          in test tubes sealed with aluminum foil, immersed in
   Pluronic PF127 (a poloxamer block copolymer) has
                                                          water bath at 4ºC was used27. Water bath (Haake C25
been accorded GRAS status and has high solubilizing
                                                          P) temperature was increased by 1ºC and left to
capacity22. Aqueous pluronic dispersions (20-35%)
                                                          equilibrate at each new setting. Gelation (T1) occurred
are solutions at low temperature and are converted
                                                          when the meniscus would no longer move upon tilting
into semisolid gel at higher (or body) temperature.
                                                          through 90º. The gel melting temperature (T2) was
These useful physico-chemical properties of pluronic
                                                          recorded when the gel started flowing.
have been evaluated for controlled release of drugs
for topical, rectal and ophthalmic routes23-25. Nasal     Permeation Studies
melatonin gels are aimed to obtain the release resem-        A water-jacketed nasal diffusion cell having, 60 ml
bling the nocturnal pattern. The effect of formulation    total capacity, flanged top (3 mm) and donor chamber
variables like pluronic concentration, PEG (400,          (10 cm long, 1.13 cm i.d.) was fabricated with glass28.
15,000), drug, solvent (ethanol) on thermal properties    Sheep nasal mucosa separated from sublayer bony
of gels and drug release has been studied. The optimal    tissues and blood was mounted onto donor chamber
nasal gel and marketed tablets are clinically compared    with serosal surface towards receptor chamber and the
using four-channel EEG recorder and Biopack soft-         mucosa just touching water. Receptor chamber with
ware.                                                     40 ml distilled water was agitated magnetically. The
                                                          diffusion cell wrapped in aluminium foil was equili-
Materials and Methods
                                                          brated at 37 ± 2°C. Melatonin gel sample, 0.1 ml, was
   Pluronic PF127 (Lutrol, Batch No. 62/0441) was a
                                                          placed on dorsal surface of nasal mucosa and lowered
gift sample from BASF Corporation, New Jersey. Me-
                                                          to touch diffusion medium. The 0.5 ml of diffusion
latonin (Batch No. 20/2001) was provided by Aristo
                                                          samples collected in amber coloured ampoules each at
Pharmaceuticals (MP), India. Polyethylene glycol
                                                          15 to 480 min was diluted to 1 ml with methanol. The
(PEG 400 and PEG 15,000) were obtained from
                                                          drug content was estimated by RP-HPLC method29.
Merk-Schuchardt, Hohenbrunn, Beimunchen. Metha-
                                                          Release profiles and fluxes were calculated. In vitro
nol    (HPLC      grade)    was     purchased  from
                                                          dissolution of oral marketed melatonin tablets (3 mg)
E Merck (India) Ltd., Mumbai. All chemicals of high-
                                                          was performed in triplicate using USP paddle appara-
est purity were used throughout the research.
                                                          tus (900 ml DW, 100 rpm, 37°C, 1 ml sample, LOD
Preparation of Gels                                       and LOQ were 5 and 10 ng/ml, respectively).
   Aqueous gels containing 20, 25 and 30% w/w of
PF127 were prepared by cold method described by           Bioadhesion Strength
Schmolka et al26. The method involved slow addition          Bioadhesion of gels was determined by Hang-Gon
of polymer in cold water with continuous agitation.       Choi method30. The modified balance technique using
The mixture was stored overnight at–4°C. PF127 gels       two-glass vials and sheep nasal mucosa was used. The
containing melatonin (0.5%w/w) and 1%w/w (with            0.5 ml of the gel sample was placed between the two
5% w/w ethanol) were prepared separately in 20, 25        mucosal membranes attached to the bottom of the vi-
and 30% w/w each of PF 127 in deionized water.            als. Weights were added on other side of the balance
Similarly, gels containing 20% (w/w) PF127, 1%            after 30 min. The minimum weight required to break
(w/w) melatonin, 5% (w/w) ethanol with PEG 400            the mucosal adhesion was measured (g/sq cm).
                                 PISAL et al: THERMOREVERSIBLE NASAL MELATONIN GELS                                             371

In vivo Sleep Pattern with Optimized Nasal Gel                           gels increased sufficiently and hence outside the ex-
    Optimized melatonin gel (20% w/w PF-127, 1mg /                       perimental range (above 95°C). The effect of ethanol,
0.1ml nasal dose) was subjected for in vivo studies                      used to incorporate the drug, was predominant in low
using the Biopack software and α EEG pattern was                         strength of melatonin gels. These gels containing, 0.5
recorded in a isolated dark room. Five healthy volun-                    % w/w drug, showed higher values of T1 as compared
teers participated in a crossover study after the study                  to respective plain gels, but slightly less than only
protocol was explained and a written consent was ob-                     melatonin containing gels, signifying decrease in T1
tained from the drug and ethical committee of the in-                    due to ethanol. In case of 1 % w/w melatonin gels (all
stitute. Three electrodes were attached to scalp sur-                    three PF concentration) the ethanol effect of decrease
face after removing the hairs as per the EEG experi-                     in both T1 and T2 has been masked by melatonin ef-
mental procedure31. For each volunteer, normal EEG                       fect of increase in T1 and T2. Gel forming ability de-
was recorded for six hrs. Oral tablet (3 mg) was simi-                   creased (T1 increase) in concentration dependent
larly evaluated after a washout period. The sleep onset                  manner with PEG 400 and PEG 15,000. Here, gel
time and duration of the sleep was recorded simulta-                     melting was promoted and occurred at lower tempera-
neously. The volunteers were asked to fill sleep logs                    ture (T2). This effect on T1 and T2 was more pro-
indicating sleep characteristics.                                        nounced for higher molecular weight PEG 15,000
                                                                         (combined data in Fig.1).
Results and Discussion                                                      The surface-active poloxamer (PF127) consisted of
Gelation and Gel Melting of Pluronic PF127 Gels                          water insoluble polyoxypropylene (30%) portion
   Aqueous pluronic gels containing melatonin                            sandwiched between two polyoxyethylene chains
(0.5%,1% w/w), release and gel point modifiers (PEG                      (70%) with an average molecular weight 12,500.
400, PEG 15,000) and solvent for drug were prepared                      Concentrated aqueous solutions of PF127 (above 20%
in 20-30% w/w PF-127 concentration. This affected                        w/w) exhibited reverse thermal gelation32. As the
gel formation and gel melting temperature of PF127                       temperature increased, micellar entanglement was
gels, in a manner depending upon the physico-                            promoted, leading to gel formation and an overall in-
chemical nature of additive and their interaction with                   crease in bulk viscosity. Temperature played an im-
polymer during phase transitions. The sol-gel phase                      portant role in the micelle formation process through
transition temperature of nasal melatonin gels is im-                    hydration of the ethylene oxide units. At low tempera-
portant for understanding the drug release kinetics and                  ture water was a good solvent for polyoxyethylene
stability. The observed T1 and T2 for gels under inves-                  and a good solvent for polyoxypropylene also. At
tigation are shown in Table.1. Gel formation tempera-                    higher temperature the solubility of polyoxypropylene
ture T1 decreased and gel melting temperature T2 in-                     was reduced and micelle formation occurred.
creased with increasing concentration of PF127,                             Ultrasonic velocity, light scattering and small angle
therefore, gel range broadened with concentration of                     neutron scattering studies have reported gelation of
polymer. Gelation temperature increased in the pres-                     PF127 due to body-center-cubic packing of spherical
ence of melatonin in dose dependent manner. Similar                      micelles33,34. The 13C NMR studies have concluded
trend was observed at gel melting temperature for                        that increased temperatures produced conformational
20% w/w PF gels. The T2 for 25 and 30% w/w PF                            changes in the methyl group of the polyoxypropylene
                            Table 1⎯Effect of drug and ethanol on gelation and melting temperature of PF gels

PF Conc.        Phase change temp.        Plain PF gel                              Melatonin conc. (% w/w)
                                                                  0.5                0.5 + ethanol               1+ ethanol

20 % w/w               T1 →                27.12 °C             30.12°C                28.16°C                    31.10°C
                       T2 →                66.66°C              82.66°C                74.45°C                    80.12°C

25 % w/w               T1 →                 21.34°C            24.85°C                 22.46°C                    26.21°C
                       T2 →                 88.30°C          Solid at 95°C           Solid at 95°C              Solid at 95°C

30 % w/w               T1 →                17.21°C             20.85°C                 18.26°C                    22.15°C
                       T2 →              Solid at 95°C       Solid at 95°C           Solid at 95°C              Solid at 95°C
T1 and T2 are gel formation and gel melting temperatures, respectively
372                                          INDIAN J BIOTECHNOL, JULY 2004


                                                                 temperature of PF127 solutions. The hydrophilic end
                                                                 chains of PF127 comprises the same polyoxyethylene
                                                                 chains that are present in PEG. It is suggested that the
                                                                 esters bind to these chains, promoting dehydration
                                                                 and causing an increase in entanglement of adjacent
                                                                 micelles. The results are in agreement with the effect
                                                                 of solutes and polymers on gelation of PF12738. The
                                                                 effect of PF concentration and PEG on temperature-
                                                                 viscosity profile of the gels (data not shown) shows a
                                                                 Newtonian behaviour at low temperature and an
                                                                 abrupt increase in viscosity in the vicinity of gelation
                                                                 onset temperatures (non-Newtonian). Thus, the con-
                                                                 firmed micellar gelation was dependent on polymer
                                                                 concentration. The gels strengthening and weakening
Fig. 1⎯Gelation and gel melting of PF 127 gels-PEG Effect (-Δ-   effects (as revealed by phase change temperatures) of
T1-PEG 400, -▲- T2-PEG 400, -■- T1-PEG 15,000, -□- T2-PEG        viscosity coincides well with the gel range widening
15,000)                                                          and narrowing, respectively39,40.
within the hydrophobic micellar region, and in the                   Melatonin is amphoteric in nature and capable of
motion of the hydrophilic end chains. The subsequent             hydrogen bonding through oxygen atom (C=O, C-O-
dehydration increased chain friction and caused gela-            CH3 groups) with polyoxyethylene chains of PF127.
tion35,36. According to a thermodynamic model, there             Pluronic PF127 hydrates with water absorption and
existed a local higher order of water molecules around           swells extensively. The –OH and ethereal oxygen of
the hydrophobic unit of the polymer in solution37. As            the polymer structure is capable of hydrogen bonding
gelation occurred, the interaction between the hydro-            with the like molecules. Such an additive binding of
phobic units of polymer molecules squeezed out these             solutes with PF127 has been reported41. This in-
ordered water molecules into the bulk solution of                creased T1 to a significant extent compared to plain
lower order. This resulted in an overall disorder,               gels. Melatonin increased T2 due to entanglement of
which was the driving force for hydrophobic associa-             large size molecules in the outer polyoxyethylene
tion.                                                            chains favouring hydration. However, in a similar
                                                                 study performed in our laboratory (unpublished re-
   These fundamental physico-chemical approaches                 sults), water soluble vitamin B12 (0.52% w/w) incor-
reported above can be used to illustrate and explain             porated in PF127 gels (20-20% w/w) showed decrease
the results presented. The decrease in the gelation              in gel formation temperature (T1) and increase in T228.
temperature with increase in PF127 concentration                 Ethanol used as solvent (5% w/w) to incorporate me-
may be attributed to the higher number and volume                latonin in micellar gel of PF127 is expected to cause
occupied by micelles at lower temperatures. As the               dehydration and a subsequent decrease in gelation
concentration of PF127 increases, the gel structure              temperature (T1). However, in the presence of drug,
becomes more closely packed with the arrangement in              melatonin effect of increase in T1 is predominant and
a lattice pattern. Gel melting increases with PF127              hence the gels showed decreased gel-forming ability
concentration because weight fraction of PF127 mi-               (i.e. increase in T1).
celles increase. Micelles in gel phase become more
tightly packed leading to increase in T2 with PF127.             Nasal Melatonin Permeation Studies
Thus, the disruption of the lattice melting of gel oc-              Pluronic PF127 gels containing melatonin were
curs at higher temperatures. Addition of water-soluble           analyzed by RP-HPLC method using an equation of
polymer PEG produces increase in the gel-sol transi-             standard curve, y=94.732+262.28 (R2=0.998, LOD
tion temperature of PF127 depending on the concen-               and LOQ were 5 and 10 ng/ml, respectively). The
tration of PEG. The phenomenon may be mediated                   melatonin content was identical (98-100%) with 2%±
through modification of the process of micellar asso-            S.D. The formulation additives did not interfere with
ciation of the PF127 molecules. In addition, the PEG             the melatonin estimation. With increasing concentra-
molecules may form mixed micelles with PF127. Ad-                tion of the polymer the release of the drug decreased
dition of PEG causes an increase in sol-gel transition           significantly i.e. for 20, 25 and 30%w/w gels amounts
                            PISAL et al: THERMOREVERSIBLE NASAL MELATONIN GELS                                         373

released were 88, 79, and 58%, respectively in 8 hrs
(Fig. 2) A 20 %w/w PF 127gel, 0.5 mg without sol-
vent, has shown initial high flux (0.67
mcg/min/sq.cm, 15 min) because of the resultant
weak gel structure. The flux of drug diffusion was
lowered with increase in PF127 concentration. Peak
flux (0.312 and 0.28 mcg/min/sq cm) and peak flux
time (80-90 min) was decreased for 25 and 30 % gels,
because of increase in pluronic concentration (Fig. 3).
The decrease in release may be due to increase in
number and size of micelles, and increase in overall
and micro viscosity of aqueous channels.
   Peak flux of melatonin from similar gel (0.5 mg/0.1
ml, 20%w/w PF) prepared using ethanol (5%w/w)              Fig. 2⎯Plot of % drug release Vs Time-Effect of PF127 (-□-20
was (0.88 mcg/min/sq cm), as the presence of ethanol       %PF(0.5 mg/0.1 ml), -■-25 %PF(0.5 mg/0.1 ml), -▲-30 %PF(0.5
increased the membrane mobility and hence, the dif-        mg/0.1 ml)
fusion rate42. However, the release rate of melatonin
with and without solvent in 25 and 30 %w/w PF gels
was identical, indicating that the penetration en-
hancement effect of ethanol had been masked by re-
lease retardant effect of higher PF concentration.
Also, partitioning of the drug into micellar core of PF
127 was responsible for retard release.
   The higher strength melatonin gels (1%w/w)
needed ethanol compulsory to incorporate drug in
micellar gel system. A trend in drug release, similar to
low drug strength gels, was observed for 1 mg/0.1 ml
gels (20, 25, 30% w/w PF-127, Fig. 4). These gels
with increasing PF127 concentrations showed de-
creased drug release. Compared to 0.5 mg/0.1ml gels,
1 mg/ 0.1 ml gels at all the three PF127 concentra-        Fig. 3⎯Effect of pluronic concentration on flux of melatonin (-□-
tions have shown decreased flux of release with cor-       20 % PF(0.5 mg/0.1 ml), -■-25 %PF(0.5 mg/0.1 ml), -▲-30
responding peak flux of 0.354, 0.283, 0.263                %PF(0.5 mg/0.1 ml)
mcg/ml/sq.cm, respectively (Fig. 5). However, mela-
tonin gel (20% w/w PF127, 5% w/w ethanol, and 1
mg/0.1 ml drug) showed a desired second peak flux
(0.248 mcg/min/sq cm) at 300 min of diffusion. The
diffusion results of 1 mg/0.1 ml gels revealed that
more drug was partitioned into the micellar core, lead-
ing to decrease in flux. The optimized nasal melatonin
gel showed a second peak flux at 300 min. This indi-
cated that the pluronic 127 gel (20%w/w PF127,
5%w/w ethanol, and 1 mg/0.1ml drug) required 300
min to absorb sufficient water from nasal secretions to
break the gel structure. This released higher amount
of drug and hence, the second peak flux.
   The drug release pattern of 20%w/w PF127 gels (1-
%w/w drug) increased in the presence of PEG 400
and PEG 15,000. Increase might be due to interfer-         Fig. 4⎯Plot of % drug release Vs time - Effect of PF127 (1 mg
ence of PEG with micellar association thereby in-          dose) (-□-20 % PF, -■-25 %PF, -▲-30 %PF)
374                                      INDIAN J BIOTECHNOL, JULY 2004


creasing gel fluidity and decreasing the release retar-
dant effect of PF. This has been reflected in decreased
gel formation ability of PF127 (increase in T1 propor-
tional to PEG concentration) in the presence of PEG.
Insignificant difference in the drug release pattern and
amount diffused (85-95%) in 8 hrs was observed for
all the formulations containing PEG. The flux of me-
latonin diffusion decreased in the order of 5% > 3%>
1% w/w PEG 400. In the presence of PEG the gel in-
tegrity was compromised and PEG also acted as co-
solvent for drug, producing faster drug release. Peak
flux of 1.5 % PEG-15,000 gel was high (0.44
mcg/min/sq cm, 15 min), which diffused out quickly.        Fig. 5⎯Effect of pluronic concentration on flux of melatonin (1
The 1 and 0.5 % PEG 15,000 gel gave an initial peak        mg) (-□-20 % PF, -■-25 %PF, -▲-30 %PF)
flux of 0.382 and 0.345 in about 15 min. Thereafter,
flux of melatonin in all the PEG 15,000 containing
gels changed invariably throughout the diffusion pe-
riod. The increased polarity and fluidity of the gels,
and continuous changes in state and structure of gels
on the nasal mucosa might be responsible. The con-
ventional oral tablets release 60% drug in 15 min and
the release was complete in 1.5 hrs indicating rapid
drug release from tablets.
Bioadhesion of Gels
   The force with which PF127 gels bound to sheep
nasal mucosa, obtained by modified balance method,
is shown in Fig. 6. The bioadhesion of PF127 gels
containing PEG-400 (1%, 3% and 5%w/w) and PEG-             Fig. 6⎯Bioadhesion of PF of gels (-□- Plain PF gel, -■- Drug (0.5
15,000 (0.5, 1 and 1.5 % w/w) were found to be 2.81,       %w/w), -▲-Drug (0.5 %w/w with solvent), -Δ- Drug (1 % w/w
                                                           with solvent)
2.90, 3.01 and 6.406, 6.169, 3.716 g/sq cm, respec-
tively. Nasal membrane consisted of glycoproteins,         dence during in vivo sleep pattern study.
capable of interacting with diverse materials43. PF127
gels possessed moderate bioadhesive force through          In vivo Sleep Pattern with Optimized Nasal Melatonin Gels
hydrogen bonding and chain penetration effect in mu-          Most of the formulation researches of melatonin
cosa. Bioadhesion increased with polymer concentra-        are mainly based on the in vitro release characteris-
tion due to extensive bonding with glycoproteins. Al-      tics. Analysis of sleep pattern and EEG will provide
cohol decreased the adhesion due to dehydration ef-        direct indication of the drug availability. Melatonin
fect. Melatonin increased the gel strength by hydro-       undergoes extensive gastric metabolism and hence it
gen bond formation (effect on T1) with number of           is difficult to rely on in vitro drug release performance
functional groups of polymer but reduced the chances       of the formulation. The spontaneous, rhythmic varia-
of bond formation with mucosal membrane. The in-           tions in the voltage originating from human brain can
creased PEG concentration showed little increase in        be recorded and analyzed systemically using electro-
adhesion. Bioadhesion of PEG 15,000 gels was sig-          encephalogram (EEG). Bipolar recording of EEGs
nificantly higher to PEG 400 but declined with higher      (most accepted method) involves the measurement of
concentration. This may be attributed for more             voltage generated between two active electrodes
spreading and increase in hydrophobic interactions.        placed upon the skull. EEG reflects alterations in elec-
The increased fluidity and hence flexibility of poly-      tric potential and provides information of interest to
mer chains above optimum value with PEG 15,000             clinicians as an index of function of brain, especially
(1 and 1.5%w/w) caused less mucosal adhesion. The          its functioning during the waking and sleeping
optimized melatonin gel showed desired nasal resi-         states44. The intensity and pattern of electrical activity
                                 PISAL et al: THERMOREVERSIBLE NASAL MELATONIN GELS                                                   375

of α rhythms originating from thalamus has a fre-                       their sleep latency was 15 to 20 min. Furthermore,
quency and amplitude of 8-13 cycles/sec and 50 μv,                      subjects fell asleep more easily, their sleep was deeper
respectively. α Waves are present in the EEG of all                     and felt more rested. However, in a crossover study
normal individuals when they are awake and disap-                       with oral melatonin tablets, subjects showed extended
pear entirely during sleep. High frequency waves of                     sleep latency of 50-60 min, the acrophase of shorter
short duration indicate fast activity (excitation),                     duration (up to 2 hrs) and experienced difficulty in
whereas low frequency waves of long duration re-                        waking up or becoming fully alert.
flects slow activity.
   The α-EEG waves obtained after administration of
melatonin as oral tablet and optimized nasal gel at
different time periods post administration has been
presented in Fig.7. Intensity and pattern of waves af-
ter oral administration of tablet does not show any
significant change in first 30 min. The voltage (μv)
and frequency decreased after 50 min. The effect on α
waves lasted up to 150 min. Normal wave recovery
was attained in 180 min. Reduction in the amplitude
and frequency of α waves occurred within 15 min
after nasal administration of 1 mg/0.1ml melatonin
gel. The characteristic low voltage spindle wave pat-
tern indicating sleep onset was observed at 30 min.
The effect of transnasal melatonin on α-EEG waves
i.e. reduction in intensity to 10-15μv and frequency
(4-5 cycles/sec) lasted up to 300 min. The low inten-
sity α wave pattern between 15 to 300 min was in-
variably observed in all the five volunteers. The re-
covery of normal α wave pattern started appearing at
300 min post-nasal dosing. The self-rated sleep char-
acteristics in subjects with the treatment of melatonin
oral tablet (3 mg) and optimized nasal gel (1 mg) are
reported in Table 2. The two formulations produced
significantly different effects on sleep quality. The                   Fig. 7⎯Representative α-EEG wave pattern, a. normal with tablet
nasal melatonin administration produced the best                        (3 mg) at b.30 c. 60 d. 120 e. 180 min, and nasal gel (1 mg) at f.
overall sleep quality. The total sleep time was longest                 15 g. 30 hrs. 60 i. 120 j. 300 k. 360 min, respectively.
(299.4±11 min) with subjects taking nasal gel and                          The comparative EEG of melatonin effect from

                                 Table 2⎯Sleep pattern with oral tablet and optimized nasal melatonin gel
  Volunteer        Dosage form/          Sleep latency (min)      Acrophase of sleep (min)      Self-rated sleep quaity   Post sleep effect
                   Melatonin dose
                  Oral tablet / 3mg               50                         104                      Superficial                ++
  I               Nasal gel / 1mg                 20                         298                        Deep                    +++
                  Oral tablet / 3mg               60                         118                      Superficial                ++
  II              Nasal gel / 1mg                 20                         287                        Deep                    +++
                  Oral tablet / 3mg               55                         125                      Superficial                ++
  III             Nasal gel / 1mg                 20                         306                        Quit                    +++
                  Oral tablet / 3mg               55                         100                      Superficial                ++
  IV              Nasal gel / 1mg                 20                         295                        Deep                    +++
                  Oral tablet / 3mg               50                         117                      Superficial                ++
  V               Nasal gel / 1mg                 15                         311                      Very good                 +++
  ++ drowsiness, +++ refreshing feeling, sleep quality = deep (5) , very good (4 ), Quit (3), Superficial (2), bad (1)
376                                            INDIAN J BIOTECHNOL, JULY 2004


tablet and nasal gel revealed that nasal absorption of                   shifts human circadian rhythms according to a phase re-
melatonin was faster and the sleep produced resem-                       sponse curve, Chronobiol Int, 9 (1992) 380-392.
                                                                    11   Beom J L, Seung G R & Jing H C, Formulation and release
bled to one during nocturnal chronobiological mela-                      characteristics of hydroxypropyl methylcellulose matrix tab-
tonin secretion. The optimized formulation has pro-                      let containing melatonin, Drug Dev Ind Pharm, 25 (1999)
vided bimodal drug release extending over 5 hrs at                       493-501.
significantly low dose i.e.1 mg as compared to 3 mg                 12   Benes L, Claustrat B, Horriere F, Geoffriau M, Konsil J et al,
oral dose. Good correlation was observed in sleep                        Transmucosal, oral controlled-release and drug administra-
                                                                         tion in human subjects: A crossover study with melatonin, J
pattern and low intensity α-EEG indicating suitability
                                                                         Pharm Sci, 86 (1997) 1115-1119.
of technique. The thermoreversible pluronic gel had                 13   Beom J L, Keith A P, Ayres J W & Sack R L, Development
desired bioadhesion and showed reproducible sleep                        and characterization of an oral controlled release delivery
characteristics. The formulation does not show any                       system for melatonin, Drug Dev Ind Pharm, 22 (1996) 269-
sensitizing effect in subjects and is expected to im-                    274.
prove patient acceptance. In conclusion, observed                   14   Lewy A J & Sack R L, Human melatonin production de-
                                                                         creases with age, J Pineal Res, 3 (1986) 379-388.
EEG changes and sleep patterns reveal that the opti-
                                                                    15   Lewy A J & Sack R L, Exogenous melatonin’s phase-
mized thermoreversible nasal melatonin gels are                          shifting effects on the endogenous melatonin profile in
suited in the treatment of circadian cycle sleep disor-                  sighted humans: A brief review and critique of the literature,
ders and has excellent commercial potential.                             J Biol Rhythms, 12 (1997) 588-594.
                                                                    16   Merkus F W H M, Nasal melatonin composition, US Pat
Acknowledgement                                                          6007834 (to Kasterlee, BE), 26 March 1998.
                                                                    17   Chein Y W & Su K S E, Nasal systemic drug delivery (Mar-
   SSP generously acknowledges All India Council
                                                                         cel Dekker, New York) 1989, 2-10.
for Technical Education, New Delhi, for providing                   18   Pontiroli A, Alberetto M & Pozza G, Intranasal calcitonin
financial assistance in the form of Career Award For                     and plasma calcium concentration in normal subjects, Br
Young Teachers (2002). The expertise provided by Dr                      Med J, 290 (1985) 1390-1395.
S L Bodhankar in conducting the sleep pattern study                 19   Farraj N F, Johansen B R, Davis S S & Illum L, Nasal ad-
is highly appreciated.                                                   ministration of insulin using bioadhesive microspheres as a
                                                                         delivery system, J Controlled Release, 13 (1990) 253-261.
                                                                    20   Bjork E & Edman P, Degradable starch microspheres as na-
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