In this presentation include all the things like introduction, type, method of preparation,Formulation, Characterization, Application and Market Product.

1. SEMINAR
ON
INSITU GEL
PRESENTED BY :
DIGANT PATEL,
M.PHARM.
1

2. List of contents
 INTRODUCTION
 DEFINITION
 ADVANTAGES
 APPROACHES
 FORMULATION OF INSITU GEL
 EVALUATION
 APPLICATIONS
 MARKETED FORMULATIONS
 QUESTIONS
 REFERENCES
2

3.  In situ is a Latin phrase which translated literally
as ' In position’ .
 In situ gels are drug delivery systems that are in
solution form before administration in the body,
but once administered, undergo gelation in situ, to
form a gel .
 Administration routes for in situ gel:
- oral, ocular, rectal, vaginal, injectable and
intraperitoneal routes.
INTRODUCTION
3

4. ADVANTAGES
 Increased contact time.
 Improved local bioavailability.
 Reduced dose concentration.
 Reduced dosing frequency.
 Improved patient compliance and comfort.
 Its production is less complex and thus lowers the
investment and manufacturing cost.
4

5. 5
Weighed quantities of Timolol maleate, Benzalkonium
chloride, EDTA and sodium chloride are
dissolved in the pH 4 phosphate buffers under aseptic
conditions.
Polyacrylic acid (Carbopol 934p) is slowly added
with continuous stirring at a speed of 1,500-2,000 rpm
to minimize the formation of the lumps of undispersed
mass.
HEC is added with slow stirring to avoid foam
formation. Stirring is continued until a clear
dispersion is formed.
 General method of in situ gel :

6. APPROACHES
There are six approaches for the in situ gel :
1. Temperature-sensitive hydrogels
2. pH-sensitive hydrogels
3. Ion-sensitive hydrogels
4. Enzyme-sensitive hydrogels
5.Light-sensitive hydrogels
6. Dilution-sensitive hydrogels
6

7. 1) Temperature-sensitive hydrogels
 Temperature-sensitive hydrogels are probably the
most commonly studied class of environment-
sensitive polymer systems in drug delivery research.
 The use of biomaterial which transits from sol-gel is
triggered by increase in temperature, is an attractive
way to approach in-situ formation.
 The ideal critical temperature range for such system
is surrounding physiologic temperature, such that
clinical manipulation is facilitated and no external
source of heat other than that of body is required for
trigger gelation.
7

8. Temperature-sensitive hydrogels are classified into :
1) Negatively thermo-sensitive
2) Positively thermo-sensitive
3) Thermally reversible gels
1)Negatively thermo-sensitive
 Negative temperature-sensitive hydrogels have a
low critical solution temperature (LCST) and
contract upon heating above the LCST. Polymers
with low critical temperature (LCST) transition
between ambient and physiologic temperature is
used for this purpose.
 Polymers used in this type of hydrogel are
Poly(acrylic acid)(PAA), Poly(acrylamide)
(PAAm), Poly(acrylamide co- butyl methacrylate). 8

9. 9
 Poly (N,N-diethylacrylamide) (PDEAAm) is also
widely used because of its lower critical solution
temperature (LCST) in the range of 25°–32°C, close
to the body temperature.
 Block copolymers made of poly (ethylene oxide)
(PEO) and poly (propylene oxide) (PPO),Which
have LCST at around the body temperature and its
sol–gel phase conversion at the body temperature.
 A large number of PEO–PPO block copolymers are
commercially available under the names of Pluronics
(or Poloxamers) and Tetronics

10. 10

11. 2) Positively thermo-sensitive
 A positive temperature sensitive hydrogel has an upper
critical solution temperature (UCST), such hydrogel
contracts upon cooling below the upper critical solution
temperature.
 E.g. poly(acrylic acid) (PAA)
polyacrylamide ( PAAm )
poly( acrylamide -co-butyl methacrylate )
11

12. 12
Gelation mechanism.
1. At high temperatures a random coil conformation is
assumed.
2. With decreasing temperature, formation of double
helices that act as knots is observed.
3. The aggregation of such helices forms the physical
junctions of gels.

13. 13
(1)
(2)
(3)

14. 14
3) Thermally reversible gels
 The most commonly used thermoreversible gels are
prepared from polymer like….
 Poloxamer is triblock copolymer, which is used in
ophthalmic drug delivery. It is sol at 20°-25°C And
converted in to gel form at body temperature (35°-37°C).
 Other polymers are
- HPMC,
- Xyloglucan,
- poly(ethylene oxide)-b-poly(propylene oxide)-b
poly(ethylene oxide) (Pluronics®, Tetronics®,
poloxamer).

15.  In this type of formation gel is induced by pH
changes.
 All the pH-sensitive polymers contain pendant acidic
or basic groups that either accept or release protons in
response to changes in environmental pH.
 The Swelling of hydrogel increases as the external pH
increases in the case of weakly acidic (anionic)
groups, but decreases if polymer contains weakly
basic (cationic) groups.
 Polymers used in this type of gel are :
Chitosan, polyacrilic acid ( carbopol 940 ), etc.
2) pH-sensitive hydrogels
15

16. 16
 PAA is converted sol to gel when PH rise from 4.2 to 7.4
.
 At higher pH polymer form bond with mucin which lead
to formation of in situ gel.
 Chitosan sol form at pH 6.2 ,but when pH rise greater
than the 6.2 form gel.
 Other polymer are :
Hydroxy phthalate latex,
Cellulose acetate phthalate.

17. 3) Ion-sensitive hydrogels:-
 This system produce gel of the polymer when it get
specific ion in contact.
 e.g., Alginate (kelton) is used as the gelling agent
in combination with HPMC (Methocel E50Lv)
which acted as a viscosity enhancing agent.
 Gelrite gellan gum, a novel ophthalmic vehicle
that gels in the presence of mono or divalent
cations, present in the lachrymal fluid can be
used alone and in combinations with sodium
alginate as gelling agent.
17

18.  In situ formation catalysed by natural enzymes has not
been investigated widely but seems to have some
advantages over chemical and photochemical
approaches.
 For example, an enzymatic process operates efficiently
under physiologic conditions without need for
potentially harmful chemicals such as monomers and
initiators.
4) Enzyme sensitive hydrogel
18

19.  Cationic pH-sensitive polymers containing
immobilized insulin and glucose oxidase can swell in
response to blood glucose level releasing the entrapped
insulin in a pulsatile fashion.
 e.g P(MAA-g-EG) hydrogels are of particular interest
in this research.
 As described previously, glucose-sensitive hydrogels
can be produced by the incorporation of a pH-sensitive
hydrogel with immobilized GOD.
 A ‘‘squeezing gel’’ is expected when incorporating
P(MAA-g-EG) with GOD.
19

20. At high concentrations of glucose, the GOD–glucose reaction
produces GlucA, resulting in a decrease in the pH of the
environment.
The hydrogels are expected to collapse abruptly with the
decrease in pH. It is with this collapse that insulin
could be ‘‘squeezed out’’.
As the glucose concentration decreases by the action of the
released insulin, less GlucA is produced, resulting in
an increase in pH of the environment.
20

21. 21

22.  Adjusting the amount of enzyme also provides a convenient
mechanism for controlling the rate of gel formation, which
allows the mixtures to be injected before gel formation.
 Polymers used in this type of in situ gel are
• 4-hydroxymandelic acid
• Poly(acryl amide)
• Poly(ethylene glycol acryl amide), etc.
22

23.  Photo-polymerisation is commonly used for in situ
formation of biomaterials.
 A solution of monomers or reactive macromer and initiator
can be injected into a tissues site and the application of
electromagnetic radiation used to form gel.
 Acrylate or similar polymerizable functional groups are
typically used as the polymerizable groups on the
individual monomers and macromers because they rapidly
undergo photo-polymerisation in the presence of suitable
photoinitiator. Typically long wavelength ultraviolet and
visible wavelengths are used. Short wavelength ultraviolet
is not used often because it has limited penetration of tissue
and biologically harmful.
5) Light-sensitive hydrogels (Photo-polymerisat
23

24.  e.g A ketone, such as 2,2 dimethoxy-2-phenyl
acetophenone, is often used as the initiator for ultraviolet
photo polymerization, where as camphorquinone and ethyl
eosin initiators are often used in visible light systems.
 These systems can be designed readily to be degraded by
chemical or enzymatic processes or can be designed for
long term persistence in vivo.
 Photopolymerizable systems when introduced to the
desired site via injection get photocured in situ with the
help of fiber optic cables and then release the drug for
prolonged period of time.
 The photo-reactions provide rapid polymerization rates at
physiological temperature.
 Further more, the systems are easily placed in complex
shaped volumes leading to an implant formation.
24

25. 25
 UV-sensitive hydrogels :-
leuco derivative molecule
bis(4-di-methylamino)
phenyl methyl leucocyanide.
 Visible light-sensitive hydrogels :-
poly(N-iso-propyl acrylamide)

26. 6. Dilution-Sensitive Hydrogels
 This type of hydrogel contains polymer that undergoes
phase transition in presence of higher amount of water.
 By having a system undergoing phase transition as a
consequence of dilution with water, a higher amount of
polymer can be used.
 e.g lutrol
26

27.  For ophthalmic :-
 Drugs
-For ophthalmics in-situ gel suitable candidates are
moxifloxacin hydrochloride
Linezolid
Gatifloxacin
Polymers
- They are responsible for providing gelling properties.
-Examples:- 27

28. 28
HPMC E 50 LV
HPMC K 4M
Xanthan gum ,
Hydroxy ethyl cellulose ,
Carbopol 934P
 Preservatives:-
-These include Benzalkonium chloride
chlorhexidine acetate
 Solvents:-
-Distilled water

29. 29
 For Nasal :-
 Drugs
-For Nasal in-situ gel suitable candidates are
Vitamin B12
Chlorpheniramine maleate,Ondansentron
Flunarizine hydrochloride, Zolmitriptan
Salbutamol sulphate
 Polymers
-They are responsible for providing gelling properties.
-Examples:-sterculia foitida,
Pluronic F68 , Carbopol 934P
Pluronic F127 , Chitosan
Pluronic 407 , Gellan gum

30. 30
 Preservatives:-
-These include :- methyl paraben
propyl paraben
benzalkonium chloride
 Solvent :- Distilled water

31. 31
 For Parenteral insitu gel :-
 Drugs :-
Following drugs use in perentral in situ gel :-
e.g. Gatifloxacin
Doxycylin
Leuprolide etc.
 Polymers :-
They are responsible for providing gelling properties.
-Examples:- poly(n-isopropylacrylamide)
sodium alginate
poloxymer , Alginic acid
Gellan gum , Pluronic F127
Carbomer etc.

32. 32
 Buffer solution :-
Following buffers are used :
examles :- acetate buffer
citrophosphate buffer etc.
 Solvent :- Distilled water

33. For oral insitu gel
Drugs :-example Clotrimazole, Diltiazem
Ofloxacin, Clarithromycin
Nifedipine ,
Omeprazole
Polymers
They are responsible for providing gelling properties.
Examples:-Gellan and sodium alginate
-Chitosan
-Carbopol
-Gallan gum
-Xanthan gum, -Xyloglucan 33

34. Preservatives:-
These include
-Methyl paraben
-Propyl paraben
-Benzalkonium chloride
Flavoring agent:-
-Aspartem
-saccharine sodium etc.
Solvents:-
-Purified water
34

35. EVALUATION
 pH
 Clarity
 Texture analysis
 Gelling capacity
 Gel strength
 Rheological studies
 Sol-Gel transition temperature
 Fourier Transforms Infrared Spectroscopy
 Drug content estimation
 In vitro drug release studies
 Accelerated stability studies
35

36.  pH :
 pH of prepared in situ gel is measured with pH meter.
 Clarity :
 Clarity test is done by visual inspection of each container
under a good light, To check against reflection into the
eyes and viewed against a black and white background,
with the contents set in motion with a swirling action.
36

37. 37
 The firmness , consistency and cohesiveness of
formulation are assessed using texture analyzer,
which mainly indicates the syringeability of sol , so
the formulation can be easily administered
in-vivo.
 Higher values of adhesiveness of gels are needed to
maintain an intimate contact with surface like
tissues.
 Texture analysis :

38.  Gelling capacity :
 The gelling capacity of the prepared formulation is
determined by placing a drop of the formulation in a
vial containing 2 ml of freshly prepared simulated tear
fluid and visually observe. The time taken for its
gelling is noted down.
38

39. 39
 This parameter is evaluated using a Rheometer.
 Depending on the mechanism of the gelling of gelling
agent used, a specified amount of gel is prepared in a
beaker, from the sol form .
 This gel containing beaker is raised at a certain rate, so
pushing a probe slowly through the gel.
 The changes in the load on the probe can be measured
as a function of depth of immersion of the probe below
the gel surface.
 Gel-Strength :

40.  Rheological studies :
 The viscosity measurements are carried out using
Brookfield viscometer. The in situ gel formulations are
placed in the sampler tube.
 The samples are analyzed at 37°C ± 0.5°C by a
circulating bath connected to the viscometer adaptor
prior to each measurement. The angular velocity of the
spindle is increased 1 to 4 and the viscosity of the
formulation are measured.
40

41. 41
 Sol-Gel transition temperature :
 The sol-gel transition temperature may be defined as
that temperature at which the liquid phase makes a
transition to gel.
 Gelation point is considered as the temperature
where formulations would not flow when test tubes
are tilted to 90° angle as the temperature is gradually
increased.
 While in case of pH and ion dependant polymer
there is change in pH or contact with nasal fluid
they get change from sol to gel.

42. 42
 Fourier Transforms Infrared Spectroscopy:
Fourier-transform infrared (FT-IR) spectra are obtained
using an FT-IR spectrometer . The pure drug mixed
thoroughly with potassium bromide, an infrared
transparent matrix, at specified ratio. (usually 1:5 ratio).
 The KBr discs are prepared by compressing the
powders at a pressure of 5 tons for 5 min in a hydraulic
press.
Scans are obtained at a resolution of 4cm-1 ,from 4000 to
400 cm-1 .

43.  Drug content estimation :
 The drug content estimation is carried out by diluting 1
ml of prepared formulation in 100 ml of distill water and
analyzed using UV-visible spectrophotometer at
appropriate wavelength.
43

44.  In vitro drug release studies :
44
 In vitro release study of in situ gel solution is carried
out by using Franz diffusion cell.
 The formulation is placed in donor compartment
and freshly prepared simulated tear fluid in receptor
compartment.
 Between donor and receptor compartment dialysis
membrane is placed (0.22μm pore size).
 The whole assembly is placed on the thermostatically
controlled magnetic stirrer.
 Formulation is accurately pipetted into donor
chamber.

45. 45
 1ml of sample is withdrawn at predetermined time
interval of 1hr for 6 hr and same volume of fresh
medium is replaced.
 The withdrawn samples are diluted to 10ml in a
volumetric flask with respective solvent and analyze by
UV spectrophotometer at respective nm using reagent
as blank.
 The drug content is calculated using the equation
generated from standard calibration curve.
 The % cumulative drug release is (%CDR) calculated.

46. 46
 Accelerated stability studies :
 Optimized sterile formulation is subjected to stability
testing.
 Sterile optimized formulation is filled in glass vials,
closed with gray butyl rubber closures and sealed
with aluminium caps.
 The vials contain optimized formulation are kept in
stability chamber, maintained at 40°C ± 2°C and
75 % ± 5 % RH for one month.
 Samples are withdrawn weekly and estimated for
drug content, pH, visual appearance, gelling capacity
and in vitro drug release.

47. 47
 For Ophthalmic Formulation following tests are carried
out :-
 Antimicrobial Activity
 Ocular irritation studies
 Isotonicity Evaluation

48.  Antimicrobial Activity
 Antimicrobial activity is determined by agar diffusion
test employing cup plate technique. The drug is
allowed to diffuse through a solid agar medium.
 The standard minimum inhibitory concentration of
controlled and developed formulations containing drug
are prepared.
 A total of 60 ml of nutrient agar media is prepared and
sterilized at 15 lb/sq-inch pressure for 18 minutes in an
autoclave; 0.5 ml of microorganism suspension is
poured into the above medium which is maintained at
temperature of 52°C to 58°C.
 This will be done in an aseptic condition. Immediately
20 ml of the microbial agar suspension is poured into
each petriplate. 48

49.  After solidification of the media, sterile standard
solutions and the developed formulations are diluted
suitably with sterile distilled water (test solutions) and
poured in to the cup of sterile nutrient agar Petri plates.
 After allowing diffusion of the solutions for 2 hours, the
agar plates are incubated at 37°C for 24 hours.
 The Zone of inhibition (ZOI) is measured around each
cup and compare with that of control.
 The entire operation is carried out in a laminar airflow
unit. Each formulation solution is tested in triplicate.
 Both positive and negative controls are maintained
throughout the study. 49

50. 50
 Ocular irritation studies :
 The Draize-irritancy test is designed for the ocular
irritation potential of the ophthalmic product prior to
marketing.
 According to the Draize test, the amount of substance
applied to the eye is normally 100μl is placed into the
lower cul-de-sac with observation of the various
criteria made at a designed required time interval of
1hr, 24hr, 48hr, 72hr, and 1 week after administration
and rabbits are observed periodically for redness,
swelling, watering of the eye.

51. 51
 Isotonicity Evaluation
 Isotonicity is important characteristic of the
ophthalmic preparations. Isotonicity has to be
maintained to prevent tissue damage or irritation of
eye.
 Formulations are mixed with few drops of blood
and observed under microscope at 45X
magnification and compared with standard
marketed ophthalmic formulation

52. 52
 For Nasal formulation following test is carried out :-
 Determination of Mucoadhesive Strength
 The mucoadhesive strength is determined by using the
modified method.
 The mucoadhesive potential of formulation is
determined by measuring a force require to detach the
formulation from nasal mucosal tissue.
 A section of sheep nasal mucosa is fix on each of two
glass slides using thread .
 Gel is placed on first slide and this slide place below
the height adjustable pan. While another slide with
mucosal section is fix in inverted position to the
underside of the same pan.

53. 53
 Both the slides with gel formulation between them held
in contact with each other, for 2 min to ensure intimate
contact between them.
 Then weight is keep rising in second pan until slides get
detached from each other. The mucoadhesive force
express as the detachment stress in dyne/cm2
is determine from the minimal weight that detach the
mucosal tissue from surface of each formulation.
 Mucoadhesive Strength (dyne/cm2) = mg/A,
Where,
m = weight required for detachment in gram,
g = Acceleration due to gravity (980cm/s2),
A = Area of mucosa exposed

54. 54
 For Oral formulation (floating ) following test is
carried out :-
 In-vitro floating study :
 Floating study is carried out in 500 ml of 0.1 N HCl
(pH 1.2) in a beaker.
 Accurately measured 10 ml of solution is added to
HCl. Time required for immersing of gel on surface
after adding solution (floating lag time) and total
floating time are measured.

55. APPLICATIONS
A.Oral Delivery.
B.Parenteral Delivery
C.Ocular Delivery
D. Vaginal Delivery
E. Dermal and Transdermal Delivery
F. Nasal Delivery
55

56.  Pectin, xyloglucan and gellan gum are the natural
polymers used for in situ forming oral drug delivery
systems.
 The potential of an orally administered in situ gelling
pectin formulation for the sustained delivery of
paracetamol has been reported.
 The main advantage of using pectin for these
formulations is that it is water soluble,
 so organic solvents are not necessary in the
formulation.
 In situ gelling gellan formulation as vehicle for oral
delivery of theophylline is reported.
A. Oral-delivery
56

57.  The formulation consisted of gellan solution with
calcium chloride and sodium citrate complex.
 When administered orally, the calcium ions are
released in acidic environment of stomach leading to
gelation of gellan thus forming a gel in situ.
 An increased bioavailability with sustained drug
release profile of theophylline
 Its study in rats and rabbits was observed from gellan
formulations as compared to the commercial sustained
release liquid dosage form.
57

58. B. Parenteral Delivery
 One of the most obvious ways to provide sustained
release medication is to place the drug in a delivery
system and inject or implant the system into the body
tissue. Thermoreversible gels mainly prepared from
poloxamers are predominantly used.
 The suitability of poloxamer gel alone or with the
addition of hydroxy propyl methyl cellulose (HPMC),
sodium carboxy- methylcellulose or dextran was studied
for epidural administration of drugs in vitro.
 The compact gel depot acted as the rate-limiting step and
significantly prolonged the dural permeation of drugs in
comparison with control solutions.
58

59.  Pluronic F127 gels which contained either insulin or
insulin-PLGA nanoparticles can be useful for the
preparation of a controlled delivery system.
 Like, poloxamer gels were tested for intramuscular and
subcutaneous administration of human growth
hormone or with the aim to develop a long acting
single dose injection of lidocaine.
59

60. C. Ocular Delivery
 The efficacy of ophthalmic hydrogels is mostly based
on an increase of ocular residence time via enhanced
viscosity and mucoadhesive properties.
 Since resulted swollen hydrogel is aqueous based, it is
very comfortable in the human eye.
 Among these polymers, in situ gels are preferred since
they are conveniently dropped in the eye as a solution,
where it undergoes transition into a gel.
Thermosensitive, specific ion sensitive or pH-sensitive
hydrogels have been examined for their potential as
vehicles for ocular drugs.
60

61.  Formulations based on a thermoplastic graft copolymer
that undergo in situ gelation have been developed to
provide prolonged release of active ingredients such as
non oxynol-9, progestins, estrogens, peptides and
proteins.
 A mucoadhesive thermosensitive gel containing
combination of poloxamers and polycarbophil exhibited
increased and prolonged antifungal activity of
clotrimazole in comparison with conventional PEG-based
formulation.
D. Vaginal Delivery
61

62.  Thermally reversible gel of Pluronic F127 was
evaluated as vehicle for the percutaneous administration
of indomethacin. In-vivo studies suggest that 20% w/w
aqueous gel may be of practical use as a base for topical
administration of the drug.
 Poloxamer 407 gel was found suitable for transdermal
delivery of insulin.
 The combination of chemical enhancers and
iontophoresis resulted in synergistic enhancement of
insulin permeation.
E. DERMAL AND TRANSDERMAL DELIVERY
62

63.  Nasal formulations of chlorpheniramine maleate and
tetrahydrozoline hydrochloride were investigated.
 The findings suggest that liquid formulations of
chlorpheniramine maleate facilitate the instillation into
the nose and the hydrogel formed on the mucous
membrane provide controlled drug release.
F. NASAL DELIVERY
63

64. MARKETED FORMULATIONS
 It is a timolol maleate ophthalmic gel formulation of
Merck and Co.
 Which is supplied as a sterile, isotonic, buffered,
aqueous gel forming solution of timolol maleate.
 Dosage strengths 0.25% and 0.5% in market . Each ml
of Timoptic 0.25% contains 2.5 mg of timolol (3.4 mg
of timolol maleate ).
 pH of solution is approximately 7.0 Inactive
ingredients include gellan gum (Temperature and Ion
sensitive polymer ) , tromethamine , mannitol and water
for injection and the preservative used is
benzododecinium bromide 0.012%. 64
(1) Timoptic :-

65.  Oncogel is a frozen formulation of paclitaxel in
Regel.
 It is a free flowing liquid below room temperature
which upon injection forms a gel in-situ in response
to body temperature.
 hGHD-1 is a novel injectable depot formulation of
human growth hormone (hGH) utilizing Macromed's
Regel drug delivery system for treatment of patients
with hGH deficiency.
65
2) Oncogel

66. 66
 AzaSite is a marketed product of InSite Vision.
 AzaSite is a topical ophthalmic solution of
azithromycin formulated in DuraSite.
 AzaSite is supplied as a sterile aqueous ophthalmic
formulation designed for topical administration.
 The recommended initial dose of the drug is instill 1
drop in the affected eye(s) twice daily, eight to
twelve hours apart for the first two days and then in
still 1 drop in the affected eye (s) once daily for the next
five days.
(3) AzaSite:

67. 67
(4) Pilopine HS:
 Pilopine HS is a marketed product of Alcon
Laboratories. Pilopine HS (pilocarpine hydrochloride
ophthalmic gel) 4% is a sterile topical ophthalmic
aqueous gel which contains more than 90% water
and employs Carbopol-940 (pH Sensitive Polymer), a
synthetic high molecular weight cross linked polymer
of acrylic acid, to impart a high viscosity.

68. 68
(5) Akten™:
 Akten™ is an HPMC-based gel of lidocaine
hydrochloride for ocular surface anesthesia.
 Akten™ contains 35 mg of lidocaine hydrochloride
per mL as the active ingredient.
 Akten™ also contains Hypromellose, Sodium
Chloride, and Purified Water as inactive ingredients.
 The pH may be adjusted to 5.5 to 7.5 with
Hydrochloric Acid and/or Sodium Hydroxide.
 The recommended dose of Akten™ is 2 drops
applied to the ocular surface in the area of the
planned procedure. Akten™ may be reapplied to
maintain anesthetic effect.

69. 69