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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5636_Библиотеки_им_академика_М_И_Перельмана
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10 Microparticles as Drug Delivery Systems for the Back of the Eye
MODULATION OF THE RELEASE RATE
OF ACTIVE SUBSTANCES
Without additive
0
0510 15 20
Time (days)
Released Ganclclovlr (%)
25 30 35 40 45
20
40
60
80
100
FSiO
Migliol
Vit E
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Fig. 10.8 Released ganciclovir profiles from PLGA microspheres as a function of molecular
weight (0.39 dl/g and 0.65 dl/g) and particle size (106–212 and 212–300)
Fig. 10.9
Influence of different additives on the release profile of ganciclovir PLGA microspheres
(1:10 ganciclovir:polymer) prepared by the O/O emulsion technique. Size of particles (212–
300 mm). Additives: Fluorosilicone oil (nonbiodegradable); Vit E (a-tocopherol), Vitamin E
(biodegradable); Migliol (biodegradable). Adapted from Barcia et al. (2005)
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Additives such as pluronic F68, polyethylene glycol (PEG) 30%, and migliol
have been used to improve the release rate of the drug and diminish the initial burst
(He et al. 2006). Addition of an appropriate amount of gelatin in the external phase
of the emulsion of PLGA microspheres loaded with acyclovir allowed diminishing
the dose of microspheres to be administered by intraocular injection (MartinezSancho et al. 2003b).
A significant challenge in intravitreal drug delivery is the use of additives with
therapeutic activity. This is the case of oils such as retinoic acid (RA) (vitamin A)
and a-tocopherol (vitamin E) that have antiproliferative and antioxidant properties.
Vitamin A was added to the acyclovir PLGA 50:50 microspheres resulting in a more
prolonged release of the acyclovir. In this formulation, RA improved the release
of acyclovir and potentially prevents the adverse effects of intravitreal injections
(Martinez-Sancho et
A novel concept of “combo” microspheres in which more than one active substance is encapsulated has been recently introduced in intraocular drug delivery for
the treatment of glaucoma. Checa et al. (2011) have prepared microspheres loaded
with a neurotrophic factor GDNF and vitamin E for glaucoma treatment. Under the
technological point of view, the addition of the oil produces an increase of GDNF
encapsulation efficiency and prolongs its release rate up to 19 weeks. Furthermore,
vitamin E is released from the microparticles. Microspheres loaded with GDNF and
Vitamin E have been injected in humans (Fig. 10.10).
al. 2006).
10.4 Sterilization of Microparticles
Sterility is a critical factor for the intraocular systems. A final sterilization is preferred over aseptic conditions. Nevertheless, PLGA particles are sensitive to most
sterilization methods usually employed (heat and ethylene chloride). Gamma irradiation has a high capacity for penetration. The dose required to assure sterilization
of a pharmaceutical product is 25
sterilize microparticles. However, gamma-irradiation of bioresorbable polyesters
induces dose-dependent chain scission as well as molecular weight reduction,
affecting the properties of the final product (Nijsen et al. 2002). The reduction of
the polymer molecular weight accelerates degradation of the polyester. Furthermore,
the degradation rate of polymeric biomaterials as PLGA due to gamma-irradiation
has been linked to radical formation (Sintzel et al. 1998) and a decrease of Tg values of PLGA favoring subsequent reactions of free radicals due to a higher mobility of the polymer chains (Bittner et al. 1999). This technological problem can be
solved by using low temperatures during the exposure time of the microparticles to
gamma-radiation. Sterilization by gamma radiation at low temperature has presented optimal results with formulations including ganciclovir, acyclovir, and celocoxib for intravitreal injection ((Herrero-Vanrell et al. 2000; Martinez et al. 2004;
kGy. This procedure has been employed to
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Fig. 10.10 Intravitreal injection of sterilized PLGA microspheres loaded with GDNF and vitamin E
in humans 1 day after injection (a) and 7 days after injection (b). Courtesy of Dr. Daniel Lavinsky
and Jose Cardillo
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Fig. 10.11 Percentage of ganciclovir released in PBS (1.5 ml) from 10 mg of PLGA microspheres.
Size of particles (300–500 mm). Sterilized (filled triangles) nonsterilized (filled cirle). Adapted
from Herrero-Vanrell et al. (2000)
Amrite et al. 2006). In the case of ganciclovir, the release rate of the drug from
PLGA 50:50 microspheres was not significantly affected by gamma radiation exposure at low temperature. In this work, the release of the drug was compared before
and after sterilization by gamma radiation (25 kGy). Release profiles before and
after sterilization were compared using the similarity factor (f2). The values of this
factor range from 0 to 100 with a higher similarity factor value indicating higher
resemblance between two release curves. In the case of the reported work the
release rate of the active substance was not significantly affected by the sterilization
procedure with a value of f2 higher than 85 (Fig. 10.11).
Change in particle size due to aggregation after gamma irradiation exposure can
be avoided with low temperatures. Martinez et al. (2004) reported similar mean
diameters of sterilized (45.47 ± 13.36 mm) and nonsterilized (46.38 ± 12.79 mm)
microspheres. The authors reported no morphological change in acyclovir microspheres after gamma-irradiation treatment because samples were protected with dry
ice during irradiation exposure.
Microparticles loaded with celecoxib (14.93%) were sterilized by gamma radiation at 25 kGy at low temperature. The sterilization process was not significantly
affected by the release profile of the active substance from celecoxib PLGA loaded
microspheres. The release was slightly lower at a few intermediate time points for
the nonsterilized microspheres although the differences were not significant (Amrite
et al. 2006).
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10 Microparticles as Drug Delivery Systems for the Back of the Eye
0 ss
K C Cl=´
ss
C
1/ 2
0.693
,
d ed
Cl V K V
t
=´ =´
e
K
ss
C
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10.5 Calculation of the Dose of Microparticles for Injection
The amount of microspheres to be injected in the vitreous can be theoretically
calculated according to the following mathematical equation:
where
is the effective drug concentration that has to be maintained in the vitreous
and Cl is the drug clearance in the vitreous. These two parameters allow calculating
K0 that is the theoretical zero-order drug release rate from the microspheres to
achieve therapeutic levels in the vitreous.
An estimation of the drug clearance from the vitreous can be calculated according
to the general equation:
in which Vd is the volume of the vitreous (i.e., 1.5 ml in rabbits, 4.5 ml in humans,
5 ml in rats, etc.) and
is the drug intravitreal elimination rate constant which can
be easily derived from the half-life of the drug.
Once calculated, K0 is employed to determine the minimum amount of microspheres necessary to provide effective concentrations in the vitreous and represents
the minimum amount of drug per time released from the microspheres to achieve
. Generally, the release rate is expressed in mg/day.
10.6 Injectability Studies
Injectability of microspheres is an important criterion because it allows testing the
minimum needle diameter for a successful intravitreal injection. Application of a
maximum ejection force of 12 Newtons over 10 s can be considered as suitable for
a properly intraocular injection. Tests are carried out on a suspension of microspheres in an aqueous vehicle employed in the clinical practice to inject the particles
(e.g., saline solution, BSS, or phosphate buffer pH 7.4). Then, suspensions of
microparticles are injected through different needle diameters. Injectability values
lower than 12 s indicate neither partial nor complete blockage of the suspension
flow.
Martinez et al. (2004) evaluated the injectability of sterilized acyclovir PLGA
50:50 (15,000 Da) size 20–40 mm. To this, particles were injected through different
needle diameters (27G, 25G, and 21G). Data (12.5, 8.4, and 5.5 N, respectively)
indicated neither partial nor complete blockage of the suspension flow. Thus, the
developed microspheres were considered suitable for intraocular injection through
a 27G needle.
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10.7 In Vivo Studies
10.7.1 In Vivo Injection of Microparticles
Local administration of micro- and nanoparticles has been carried out by intravitreal and periocular injections (Herrero-Vanrell and Refojo 2001; Kompella et al.
2003; Amrite and Kompella 2005; Ranta and Urtti 2006).
Injection of microparticles suspension has been usually made using conventional
needles: 33, 30, 27, 26, 25, 23, 20, and 18-gauge. In some cases, the needle has been
connected to a glass micropipette (tip diameter of 40 mm) (Herrero-Vanrell and
Refojo 2001). Needles most frequently employed are 25–30 G for sizes 1–106 mm
and 18 G for particle sizes up to 500 mm (Veloso et al. 1997; Urata et al. 1999; Lee
et al. 2002; He et al. 2006).
The microspheres are suspended in a physiological solution that acts as the vehicle for the injection of the particles into the eye. The most frequent vehicles used to
suspend microspheres for intraocular or periocular injections are isotonic phosphate
buffer solution (PBS) or balanced salt solution (BSS) (pH 7.4). Because a significant proportion of the dose of microparticles suspended in PBS or BSS tended to
adhere and remain in the syringe after its injection, some investigators have employed
viscous vehicles to retain the particles in suspension better than the less viscous
PBS and BSS (Veloso et al. 1997). Microparticles have been suspended in physiological solutions of hyaluronic acid (HA) or hydroxypropylmethyl cellulose
(HPMC). These vehicles are commonly used as surgical aids in ophthalmology
(Chan et al. 1984). Furthermore, these polymers form solutions that are transparent
and biocompatible, and are rapidly diluted in the intraocular fluids and are eventually eliminated from the eye (Chan et al. 1984; Tolentino et al. 1989).
10.7.2 Ocular Disposition and Cellular Uptake
PLGA microparticles suffer aggregation after their intravitreal injection, diminishing the surface area and prolonging the drug release time. This effect has been
already observed in animal models (rabbits) and humans (Giordano et al. 1995;
Cardillo et al. 2006).
One concern of intravitreal injection of microspheres is regarding the behavior of
impairing and/or vitreous haze following a single injection. However, preliminary
investigation using triamcinolone PLGA microspheres for the treatment of diabetic
macular edema in 25 human eyes has showed the opposite. In fact, in contrast to
initial fears, the tendency of the microspheres to aggregate and condensate at the
site of the injection and leave a free visual axis was clinically observed in all patients
(Cardillo et al. 2006; Herrero-Vanrell and Cardillo 2010). However, when used in
the eye, care should be taken to inject the microspheres such that they do not interfere with the visual pathway.
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The movement of microspheres (7–10 mm size) was studied in phakic and
aphakic eyes. The intravitreous injected microparticles were retained in the vitreous
cavity in phakic and aphakic eyes of rabbits. On the contrary, some particles moved
to the anterior chamber although most of them remained in the vitreous for long
periods of time (Algvere and Martini, 1979).
In the case of periocular injection, the size of particles affects their ocular
distribution.
Subconjunctival injection of nanoparticles (20 and 200 nm) and microparticles
mm) demonstrated that particles higher than 200 nm are retained in the site of
(2
injection up to 60 days (Amrite and Kompella 2005). The study was carried out
with non biodegradable fluorescent polystyrene particles. The results obtained in
this work have suggested the potential use of particulate systems of 200 nm and
above for sustained drug delivery to the retina after periocular injection. On the
contrary, if delivering the entire particle is desired, the subconjuctival administration is not a good approach.
Moritera et al. (1994) studied the cellular uptake of PLGA micro- and nanoparticles by RPE. The authors demonstrated that PLA and PLGA microparticles up to
1–2 mm size are susceptible to suffer phagocytosis by RPE cells.
10.7.3 Tolerance of Microparticles
Several reactions have been described after intraocular administration of PLGA
microspheres. Khooebi et al. (1991) have pointed out the presence of whitish material in the vitreous cavity during the 10 days after injection of fluorescein PLGA
microparticles that disappeared at 20–25 days after injection in rabbits (Veloso et al.
1997). However, histologically retinal and choroidal damage were not reported after
35 days of administration.
Regarding to other ocular reactions to microparticles, Veloso et
described a mild localized foreign body reaction surrounding partially degraded
ganciclovir loaded microspheres after their injection in rabbits. Histopathologic
analysis at 4 and 8 weeks, after injection of microparticles, showed mononuclear
cells and multinucleated giant cells with no involvement of the retina or other ocular
structures. In the last case, biodegradation was virtually completed by day 63. In
general, the foreign body reaction, associated with these polymers in the eye as well
as intramuscular, gradually decreased with time. According to some authors, 12
weeks after surgery only degraded pieces of microparticles could be recognized
remaining at the implantation site (Gould et al. 1994; Moritera et al. 1992).
Signs of inflammation have been described after intravitreal injection of microparticles in rabbits. These signs were similar to the ones reported for sutures and disappeared 2–4 weeks after administration (Giordano et al. 1995). Moreover, the reaction
is similar to that described for microspheres injected intramuscularly in rabbits
(Visscher et al. 1985; Park and Park, 1996). On the other hand, Amrite et al. (2006)
described no signs of inflammation in the retina at 60 days after administration of the
PLGA microspheres loaded with celecoxib. Furthermore, no significant changes were
al. (1997)
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observed in the thickness of retinal layers between untreated normal rats and normal
animals treated with celecoxib microparticles. In this study, the visual inspection of
the site of action (periocular tissue) did not reveal the presence of inflammation
including redness and edema. The difference in terms of inflammation due to PLGA
microparticles can be attributed to the different animals tested. It is well known that
rabbits are more susceptible to inflammation than other animal species.
PLGA microspheres loaded with triamcinolone as potential treatment for diabetic
macular edema have already been injected intravitreally in humans. Preliminary results
have reported good tolerance after microparticle injection (Cardillo et
Periocular injection of poly(d,L lactide-co-glycolide) glucose microspheres nonloaded and loaded with 25% (42.5 mm) or 50% (67.7 mm) PKC412 caused mild conjunctival injection that resulted similar among the three groups. The authors reported
discernible signs of inflammation or irritation. Gross pathologic examination of the
eyes showed microspheres outside of sclera (Saishin et al. 2003).
Nonloaded PLGA microspheres and dexamethasone microspheres have been
administered by periocular injection in rabbits. An amount of 5 mg suspended in
BSS was administered by yuxtaescleral injection. IOP remained unchanged before,
and 24 h, 1, 2, and 4 weeks after blank MP and Dx-MP administration. No adverse
signs were observed after injection of formulations in terms of conjunctival discharge, conjunctival swelling, aqueous flare, light reflex, iris involvement, cornea,
surface of cornea cloudiness, pannus, fluorescein stain, lens, vitreous opacity, vascular congestion, vitreal and retinal hemorrhage, and retinal detachment (RD). The
only sign observed was a conjunctival congestion at the injection site at 24 h and 2
weeks postinjection for unloaded microspheres and 24 h and 1 week for dexamethasone microspheres. Authors concluded that PLGA microparticles unloaded and
loaded with dexamethasone are suitable for juxtascleral injection with no adverse
effects (Barbosa et al. 2010).
In terms of intraocular tolerance, the nature of the polymer is critical. Rincon
et al. (2005) studied the response to microparticles prepared from an elastin derivative poly (valine-proline-alanine-valine-guanine) (VPAVG). Although no inflammatory response was observed after subcutaneous injection in the hind-paw of the
rat and only a few eyes (2/11) of the experimental group presented inflammation
signs after intravitreal injection of 2.5 mg of poly (VPAVG) microparticles, 45%
(5/11) of the animals showed tractional retinal detachment (TRD). This adverse
effect was related to certain fibroblastic activity induced by the polymer.
al. 2006).
10.7.4 In Vivo Degradation of PLA and PLGA Microparticles
Experience has demonstrated that the PLA and PLGA polymers are biocompatible.
Their biodegradation products, lactic acid and glycolic acid, are also biocompatible
and easily eliminated from the body (Colthrust et al. 2000).
As cited previously, the rate of polymer biodegradation mainly depends on the
polymer composition (Thomas et al. 1993; Robinson 1993) and its molecular
weight (Miller et al. 1977). Others factors such as particle size (total surface area)
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(Herrero-Vanrell and Refojo 2001; Grizzi et al. 1995) and the type and amount of
drug contained in the formulation are also critical. Acidic and basic drugs encapsulated in microparticles might enhance the hydrolytic degradation of PLA and PLGA
polymers (Maulding et al. 1991; Delgado et al. 1996; Li 1999). Beck et al. (1983)
studied the biodegradation of DL-PLGA microcapsules loaded with norethisterone
and observed a faster biodegradation of the microcapsules as the ratio of glycolide
in the copolymers increases. On the other hand, Delgado et al. (1996) reported that
drug released from the microspheres was higher when the molecular weight of the
polymer or the amount of the encapsulated drug increases. The reported data suggest
that the presence of drug, as reported by other authors, may affect degradation time
of the polymer (Visscher et
al. 1985; Maulding et al. 1991).
Vitrectomy is an important issue regarding to microparticle clearance. Moritera
et al. (1991) reported the degradation of PLA microspheres loaded with 5FU after
intravitreal administration. Particles gradually become smaller and finally disappeared from the normal rabbit vitreous in 48 ± 5.2 days. However, the clearance
from the vitreous cavity was accelerated to 14 ± 2.4 days in the eyes that had undergone vitrectomy. On the other hand, when Giordano et al. (1995) evaluated the biodegradation and clearance time of unloaded PLGA microspheres of a relatively low
molecular weight (inherent viscosity 0.2 dl/g) from the vitreous cavity in rabbits
after gas vitrectomy, they found evidence of the microparticles up to 24 weeks
postinjection (Giordano et al. 1995).
Other factors that affect the rate of degradation of microspheres after intravitreal
injection are the amount and the size (total surface area) of the microspheres injected,
the properties of the polymer (polymer crystallinity, lactic acid and glycolic acid ratio
and molecular weight). For example, the amorphous 50:50 PLGA has shorter half-life
than the 75:25 PLGA, and this one shorter than the crystalline PLA (Li 1999).
For the same polymer composition, the lower molecular weight is the faster
is the degradation time. Smaller size microparticles degrade faster than larger sizes
(Grizzi et al. 1995).
10.8 In Vitro and In Vivo Correlation
There are many published examples of drugs with dissolution data that correlate
well with drug absorption in the body in the oral route. These studies are not so
frequent for the intraocular route. Good correlations between in vitro and in vivo
data of the released drug resulted useful to understand the in vivo behaviour of a
drug delivery system.
He et al. (2006) injected 0.1 ml containing 10 mg of PLGA (75:25) microspheres
loaded with cyclosporine in rabbits. Cyclosporine was quantified from samples of
blood, aqueous humor, conjunctiva, iris/ciliary body, sclera, lens, vitreous, and
retina\choroids. A good correlation was observed between in vivo AUC
(area under the drug concentration vs. time points for the total period of 65 days)
expressed in percentage for retina and choroids and the in vitro cumulative release
percent (%) for the corresponding time points (up to 65 days).
\AUC65
t
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10.9 Microparticles for the Treatment of Posterior Segment
Diseases. Animal Models and Human Studies
Microparticles intended for the treatment of posterior segment diseases have been
mainly injected by periocular or intravitreal injection. Although several studies have
been conducted employing topical routes there is no evidence of effective concentrations in the vitreous with this administration.
PLGA microparticles have been prepared with different drugs, such as adriamycin,
5-fluorouracil (5-FU), and RA for proliferative retinopathy, dexamethasone and
cyclosporine for uveitis, anti vascular endothelial growth factor (VEGF) for age macular degeneration (AMD), budesonide and celecoxib for diabetic retinopathy, TA for
macular edema, acyclovir for herpes infection, ganciclovir for cytomegalovirus retinitis, neurotrophic factors for neuroprotection, an inhibitor of protein kinase C
(PKC412) for choroidal neovascularization (CNV), triamcinolone for macular edema,
neuroprotective agents for glaucoma and retinitis pigmentosa, and a combination of
steroids (TA) and antibiotic agents (ciprofloxacin) to prevent ocular inflammation and
infection after cataract surgery. Finally, co-transplantation of MMP2-microspheres
and RPCs ha been reported as a practical and effective strategy for retinal repair.
10.9.1 Proliferative Vitreoretinopathy (PVR)
Antiproliferative drugs have demonstrated to be therapeutically active in the treatment
of the PVR in which contractile cellular membranes are formed mainly by retinal
pigment epithelium (RPE) cells (Pastor 1998). Microparticles employed for the treatment of PVR have been loaded with active agents with antiproliferative activity
(Moritera et al. 1991, 1992). Adriamycine was encapsulated in PLA (3,400 Da).
Microspheres (50
normal rabbit eyes and in a rabbit model of PVR and compared with the administration of the active substance alone. The authors found a significant decrease in the
retinal toxicity of the single injection of 10 mg of adriamycine in comparison with
the administration of 10 mg of PLA microspheres containing 10 mg of the drug with
neither histological abnormalities nor electrophysiologic changes in the eye. Regarding
to antiproliferative properties, the RD was decreased from 50 to 10% after 4 weeks of
the administration. On the contrary, a dose of 3 mg of PLA microspheres containing
3 mg of adriamycine did not decrease the rate of RD.
Moritera et al. (1991) demonstrated the influence of the polymer composition
and the molecular weight of the polymer on the release in vitro and in vivo
of 5-fluorouracil (5-FU) from microspheres 50 mm size. The polymers employed
were two low molecular weight PLAs (3,400 and 4,700 Da) and PLGA (70:30,
3,300 Da). PLGA microspheres showed an in vitro release of almost the whole
5FU (98%) in only 2 days while the PLA took 7 days to release 85% of the encapsulated drug. Microspheres prepared from PLGA 4,700 daltons released 70% of
5FU over 7 days.
mm size) containing 1% of the active substance were injected in
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