Note: Descriptions are shown in the official language in which they were submitted.
W O 94/17840 PCTAEP94/00294
~ $51~
Pharmaceutical compositions comprising a spongy material consisting
of ester derivatives of hyaluronic acid combined with other
pharmacologically active substances
Field of the invention
The present invention relates to new pha~ -^eutical compositions
comprising a spongy material consisting of' total or partial ester
derivatives of hyaluronic acid, either !;ingly or as a mixture
thereof, co-lyophilized with a solution containing other
pharmacologically active substances, the process for their
production, and the use of same in surgery and in particular in of
microsurgery.
Description
As known, hyaluronic acid plays a major role in tissual repair
processes, especially in the early granulation tissue formation
phases, by serving several functions: it stabilizes the coagulum
matrix and controls the degradation of same, helps response of
inflammatory cells, e.g. polymorphonucleates and monocytes, of
mesenchymal cells, e.g. fibroblasts and endothelial cells, and
oriènts the successive migration of epith~ 1 cells.
As known, the ~' ;ni~tration of hyaluronic acid solutions speeds up
the recovery of patients suffering from decubitus ulcers, wounds and
burns.
The role of hyaluronic acid (HA) during the various tissual repair
process phases was illustrated through a theoretical model by Weigel
P.H. et al., "A model for the role of hyaluronic acid and fibrin in
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2~S~8 ''
the early events during the inflammatory response and wound
he~ling", J. Theor. Biol., 119, 219, 1986.
The main problem still de Anding solution is that repeated HA
~min;ctrationS are required, whatever the vehicle used, HA being
very rapidly elirin~ted from the lesion site.
Should HA solutions be directly applied, no drug release control
would be possible. This would cause short times of drug retention by
the lesion and, consequently, repeated ~m;nictrations resulting in
the treated area moistening and maceration, would be required.
Furthermore, should non-perfectly biocompatible inert matrices be
used, local phlogistic reactions and cicatrix adhesions would
develop.
It has now been found that the new pharmaceutical compositions
forming the object of the present invention - compared with the
compositions already known - represent a significant technological
impl-vve~ t in that they do not raise the same problems and give
better results.
The compositions of this invention are made of a spongy material
consisting of total or partial ester derivatives of hyaluronic acid,
wherein a solution cont~inin~ a compatible active ingredient is
absorbed and later co-lyophilized.
Said new compositions acquire greater flexibility and softness by
addition of glycerin or appropriate plasticizers.
In another embodiment of the present invention, a pierced
biocompatible membrane capable of favouring cell growth adheres to
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one or both sides of the colyophilized pharnaceutical composition.
Other objects of the present invention are a process for the
production of said compositions and the use of same in surgical and
in particular microsurgical practice.
The cl~ compositions represent a great technological progress,
being capable of acting as a mechanical guide for re-epithelization
thanks to the chemo-physical characteristics of the spongy material
and to the presence of active ingredients l~bsorbed therein and, at
the same time, of providing a controlled drug release at the site
of treatment. Consequently, high local drug concentrations and slow
release of same are guaranteed.
Due to the presence of hyaluronic acicl in the absorbed and
colyophilized solution, the new compos:Ltions combine, in one
product, the capability of HA to induce a rapid and complete tissual
lS repair process and the characteristics of applicability,
elasticity, and tolerability of hyaluronic acid ester derivatives,
which are excellent mechanical guides for the tissual repair
process.
Furthermore, the biocompatibility of the ,spongy material and the
pharmacological activity of the hyaluronic acid absorbed therein
suggest that the new compositions are an ideal biomaterial for use
in various surgical fields, such as for example otologic and
otoneurologic microsurgery, functional, post-traumatic and
endoscopic rhinosinusal microsurgery, plastic and reconstructive
surgery, and any other surgical practice envisaging the use of non-
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reabsorbable materials, such as controlled release systems of
pharmacologically active substances.
The new compositions allow maint~-ning high local concentrations of
the active ingredient, e.g. hyaluroniç acid, at the site of
treatment and offer the great advantage of a single a~;n;~tration,
which results in a reduction in the number of physicians'
interventions, dispensary controls, and hospitalizations.
The new compositions have a constitution guaranteeing a solid
matrix of optimal elastic and biocompatible properties, capable
therefore, of acting as a mechanical guide for tissual repair
processes in general and for the ~y ~n;c membrane repair process
in particular.
Addition of glycerin or other appropriate plasticizers to the
~ d compositions gives a flexible and soft spongy material,
which offers two further advantages:
- ease of h~n~ling and application to the site of treatment, the
material softness making the application less painful;
- highly increased hydration, the spongy material absorbing in
water about lO times its original weight in 3 to 4 seconds.
In another embodiment of the present invention, a pierced
biocompatible membrane capable of favouring cell growth on its
surface, e.g. fibroblasts and keratinocytes, is applied to the
surface of the pharmaceutical compositions to be placed in contact
with the lesion.
The pharmaceutical compositions of this invention are made of a
W O 94/17840 21 5~i 51~ PCT~EP94/00294
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spongy material consisting of total or partial ester derivatives of
hyaluronic acid, either singly or as a mixture thereof, in
particular HA ethyl ester (HYAFF-7) and HU~ benzyl ester (HYAFF-ll),
which is caused to absorb a solution cont~ining hya;Luronic acid or
another pharmacologically active ingredient (e.g. growth factors,
fungicides, antibiotics, bacteria-fighting compounds, steroid and
non-steroid anti-inflammatory agents, etc.) and in particular
pharmacologically active hyaluronic acid derivatives as are
illustrated in European patent applications EPA 0216453 and EPA
lO 0197718 filed in the name of Fidia S.p.A., which are then subjected
to lyophilization.
With a view to obt~inin~ an end product of improved elasticity and
softness, glycerin or an appropriate plast:icizer may be added before
final lyoph; 1 i ~tion.
The characteristics of the end product may vary depending on the HA
ester derivatives solution used to produce the spongy material and
on the absorbed solutions.
Said characteristics are summarized in Tab:Le 1.
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TABLE 1
Description Unit Lower limit Upper limit
of measurement
Aspect: Odourless white sponge
Dry weight mg/cm3 3O 200
Water absorption % (w/w) 5OO 1500
IR identification - positive positive
Esterification % 5O 102
HA content % (w/w) 3 5O
LAL test UE/mg - 0.2
Glycerin (optional) % (w/w) 5 3O
In another embodiment of this invention, the pierced membranes
applied to one surface of the spongy material are biocompatible and
made of materials of natural, synthetic or semisynthetic origin,
preferably of HA benzyl ester, and favour the growth on their
surface of cells, such as for example fibroblasts and
keratinocytes.
In particular, the membranes that may be used are lO to 5OO ~ thick
and pierced with a regular series of holes of a definite and
constant size between lO and lOOO ,u, separated from each other by a
constant distance of between 5O and lOOO ,u, as are illustrated in
European patent application EPA 91108654.4 filed on 28th May, 1991,
in the name of Fidia S.p.A.
With a pierced membrane applied to the surface of the spongy
material, the new compositions combine their aforementioned
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advantages with the specific action of pierced membranes, i.e. they
also favour re-epithPli~tion.
The products of this invention are obtained on the basis of the
following process.
1) Solubilization
The starting material consisting of total or partial ester
derivatives of hyaluronic acid, either singly or as a mixture
thereof, is completely solubilized in an appropriate solvent to a
concentration of 20 to 50 mg/ml, preferab]y 35 mg/ml. The solution
obtained is filtered through a filter with 40 ,um pores.
2) Coagulation
The resulting solution is poured into apprc,priate containers, later
placed in a chamber with relative humidity of 60 to 100%, preferably
85%, until evident coagulation of the material.
3) ~ashing
The solid panels obtained are cut into 1UDIPS of appropriate size,
which are placed in a NaCl solution at a concentration of 80 to 120
g/l, preferably 100 g/l. Said solution is periodically renewed.
4) Lyophilization
Lyophilization is carried out as follows:
4.1) Lumps are placed on freeze-dryer plates.
4.2) Starting from room temperature, plates temperature lowering is
set to -45 C. The temperature lowering rate is the maximum admitted
by the system.
4.3) Plates are cooled to the freezing temperature and maintained at
WO 94/17B40 PCT~EW4/00294
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said temperature for a period of 3 hrs so the lumps can be cooled to
said temperature.
4.4) In-chamber pressure is set to 3 x 10 1 to 2 x lO 1 bar and
heating is started. The heating temperature is -12 C; said
temperature has to be reached gradually over a period of 4 hrs and
maintained at said value for 35 to 55 hrs, preferably 48 hrs, i.e.
the time required for complete sublimation.
4.5) Temperature rise is then set to 20 C, which temperature is
reached over a period of 12 hrs and maintained at said value for 3
hrs.
5) Washing
The resulting panels are placed in a ~ in~ralized and apyrogenous
water bath and washed for at least 16 hrs; during said step, baths
are periodically renewed every 2 or 4 hrs.
6) Imbibition with active ingredient solution
Lumps are imbibed with the solution containing drug at a
concentration of from 0.1% to the limit of solubility of the solute.
Wishing to obtain soft and flexible sponges, glycerin or an
appropriate plasticizer is added to the solution in an amount of 5
to 30% by wt., preferably 20%.
7) Drying by lyophilization
An additional lyophilization cycle as described under 4) is carried
out.
The products obtained may be sterilized by gamma-rays or equivalent
systems.
WO 94/17840 PCT~EP94/00294
The following examples illustrate the proce!ss for the preparation of
the products of this invention. These examples are illustrative
only; in no event are they to be regarded as limiting the scope of
the invention.
Exnmple 1
Method for the preparation of HYAFF-7 ovcid spongy tampons having
diameters of 15 mm x 10 mm and thickness of' 4 mm, each cont~;n~ng 10
mg hyaluronic acid
40 g of HYAFF-7 were solubilized in DMSO (1142 ml) in a reactor
equipped with agitator, thermostabilized at 25 C.
Once the product solubilization was completed, i.e. after 8 hrs
approx., the solution was filtered through a membrane with pores of
40 ,um. The solution was poured onto a 30 x 45 cm stainless steel
tray.
The tray was placed in a chamber under 25 C temperature control,
saturated with steam acting as coagulating solvent. Coagulation
lasted 60 hrs approx.
A gelatinous HYAFF-7 cake was obtained. For ease of h~n~ling, it was
cut into 100 x 150 mm lumps, which were pl~ced in a saline solution
(2000 ml) at a concentration of 100 g/l of NaCl for a period of 3
days.
The saline solution baths were renewed every 4 hrs.
Lumps were placed on the plates of a pre-set freeze-dryer to be
subjected to a lyophilization cycle.
Lyophilization was carried out as follows:
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- starting from room temperature, plates temperature lowering was
set to -45 C at the maximum lowering rate admitted by the system;
- plates were cooled to the freezing temperature and maintained at
said temperature for 3 hrs so the lumps could be cooled to said
temperature;
- in-chamber pressure was set to 3 x lO 1 to 2 x 10~1 bar and
heating was started. The heating temperature was -12 C; said
temperature had to be reached gradually over a period of 4 hrs and
maintained at said value for 48 hrs approx. until sublimation
completion;
- temperature rise was then set to 20 C, which temperature was
reached over a period of 12 hrs and maintained at said value for 3
hrs.
The spongy product thus obtained was washed 6 times with distilled
apyrogenous water (1000 ml) for NaCl elimination. Each washing
lasted 4 hrs approx.
Lumps having diameters of 15 cm x 10 cm and thickness of 5 mm were
hollow punched to obtain approx. 300 oval tampons with diameters of
15 mm by 10 mm.
A hyaluronic acid solution (150 ml) at a concentration of 24 mg/ml
was prepared in an appropriate reactor.
Each tampon was wrung out to remove most wash water. Then 412 ~1 of
the previously prepared solution, corresponding to lO mg of
hyaluronic acid, were distributed on one tampon side by a dosing
system.
W O 94/17840 21~55 ~ ~ PCT~EW4/00294
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The time taken for the solution complete absorption and spreading
inside the spongy structure was 3O min.
The soaked tampons were further lyophilized as per the parameters of
the previous cycle until obtAining the end product.
F ,le 2
Method for the preparation of HYAFF-7 ovoid spongy tampons having
diameters of 15 mm x lO mm and thickness of 4 mm, each cont~;nin~ lO
mg of hyaluronic acid, whereto an adhes:ive HYAFF-11 film pierced
with constantly spaced (80 ,um) holes of 4O um size is applied
Some tampons prepared as per Example 1 Wl !re made to adhere to a
film pierced with constantly spaced (8O ,um) holes of 4O ,um size
according to the following procedure.
Pierced film sheets (120 x 120 mm) were cut into pieces of 20 x 25
mm size. Meanwhile, a solution of HYAFF-7 in hexafluoro isopropanol
(HFIP) at a concentration of 20 mg/ml was prepared in an appropriate
reactor. Once the solubilization was completed, the solution was
filtered through a membrane with pores of 4O ,um.
Five 15 ,ul drops of a HYAFF-7 solution in HFIP were distributed on
one tampon side by a suitable dosing system as follows: 4 drops at
the end points and 1 drop at the central point.
The tampon side where the five drops were distributed was caused to
adhere to the centre of the pierced film by applying a slight
pressure.
Fifteen minutes later, i.e. the time required for the low-boiling
solvent to evaporate, a perfect adhesion between film and tampon was
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obtained.
Once cohesion was completed, tampons were allowed to stand in an
oven at a temperature of 3O C and at a pressure of 1 x lO 2 mbar for
a period of 24 hrs.
F le 3
Method for the preparation of HYAFF-7 sponFy tampons, flexible and
dry-mouldable in ovoid form, having diameters of 15 mm x 10 mm and
thickness of 4 mm, each cont~;nin~ 10 mg of hyaluronic acid
No. 6 lumps of spongy material having dimensions of 150 mm x 100 mm
and 5 mm thickness were prepared as per Example 1 until the stage of
material washing with NaCl, after the first lyophilization cycle.
lOOO ml of glycerin in distilled and apyrogenous water at a
concentration of 8% were prepared separately.
Once the w~h;ngq were completed, the 6 lumps were wrung out by a
mechanical system to remove most of the absorbed water and placed in
the glycerin solution previously prepared. Spongy lumps were allowed
to stand in said solution for approx. 60 min.
The process proceeds as per Example 1.
A glycerin content of 20% was detected by chemical analysis.
~ le 4
Preparation of a spongy material consisting of 60X HYAFF-7 and 40%
HYAFF-11, cont~;n;ng 10 mg hyaluronic acid
A solution of HYAFF-7 (24 g) and HYAFF-11 (16 g) in DMSO (1142 ml)
was obtained by mixing in a reactor e~uipped with a vacuum/pressure
system and agitator, and thermostabilized at .25 C.
W O 94/17840 21 S S 5 ~ 8 PCTAEW4/00294
Once solubilization was completed, the solution was filtered through
a membrane with pores of 40 ,um.
The process proceeds as per Example 1.
Some in vivo tests were carried out with a view to proving the
efficacy of the compositions of the invention.
The results of a test made to evaluate the efficacy of the new
compositions in favouring the tympanic membrane repair process in
the rat are reported below.
TEST 1
With a view of evaluating the efficacy of the new compositions in
favouring the tympanic membrane repair prccess in the rat and the
time of repair, a test was conducted usin~, the diabetic rat as an
experimental model.
Eight mature rats (T, D, C, TD, TC, TDC, B, GAD) aged 8 months and
weighing 250-350 g, with six-months' diabetes induced by treatment
with streptozotocine (STZ, 60 mg/kg i.p.) were subjected to
bilateral tympanic membrane perforation.
The upper-posterior quadrant of the tympanic membrane (TM) of the
left ear was bilaterally perforated by means of a lanceolate
bistouri with the aid of an operating microscope.
The TM was dressed with a tampon obtained as per Example 1 and
soaked with one drop of physiological saline solution. The tampon
was fixed therein by a cross stitch sewn on the external acoustic
meatus.
The TM of the right ear was not treated and was used as a control. A
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cross stitch was sewn also on the external acoustic meatus of the
right ear.
Tampons were left in situ for a period of 6 days; during said period
two external observations were conducted to make sure that
dressings and stitches were regularly in place. All dressings were
removed on the 6th day.
TM controls with a microscope were made on the 6th, 8th, 10th, 12th,
and 15th day.
Complete repair of the left TM was observed on the 6th day in rats
D, TC and B; on the 8th day in two further rats, i.e. C and GAD; on
the 10th day in the three rF ~in;ng rats, i.e. T, TD and TDC. Always
on the 10th day, complete repair of the right ear TM was observed in
rats D and C; on the 12th day in rats TC and B; on the 15th day in
the l~ ~ining four rats T, TD, TDC, and GAD.
The results obtained are recapitulated in Table 2.
TABLE 2
rat T D C TD TC TDC B GAD
ear L R L R L R L R L R L R L R L R
day
6th * * *
8th * *
10th * X X * *
12th X X
15th X X X X
W O 94/17840 PCT~EP9410029~
~ 2I ~55i~ 8
* = complete repair of the left tympanic membrane (TM)
X = complete repair of the right tympanic membrane (TM)
Briefly, the control made on the 10th day showed that all TM's
treated with the new compositions were repaired , while only two
untreated TM's showed the same result. ~urthermore, on the last
observation through an operating microscope, the TM's repaired with
the new compositions showed improved characteristics of gloss and
transparency, no tympanic retraction, dyschromia, and dysmorphism.
To conclude, the new compositions proved to be effective in
favouring an improved TM repair in much shorter times than required
by spontaneous repair.
The animal model selected for the experiment, i.e. the rat aged 8
months and with 6 months' diabetes, implied the hardest
experimental conditions: as known, in fact, said ~n; ~1~ exhibit
noticeably slowed down tissual repair processes as a consequence of
the induced dysmetabolic pathology. Said hard experimental
conditions were even more evident by the long diabetic condition (6
months).
Therefore, the results obtained provide evidence that the new
compositions are highly effective in indl~c;n~ a complete and very
rapid tissual repair, even by a single ~mi ni stration and with a few
days' contact with the damaged TM. The experimental results obtained
by us suggest that the new compositions can be profitably used in
surgery and, in particular, microsurgery as well as in the treatment
of tympanic membrane perforations.
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16
~s~l~
Furthermore, the biocompatibility characteristics of the spongy
material and the pharmacological activity of the hyaluronic acid
absorbed therein make the new compositions an ideal biomaterial for
use in various surgical fields, such as for example otologic and
otoneurologic microsurgery, functional, post-traumatic and
endoscopic rhinosinusal microsurgery, plastic and reconstructive
surgery, and any other surgical practice envisaging the use of non-
reabsorbable materials, such as controlled release systems of
pharmacologically active substances suitable for favouring the
tissual repair process.
Furthermore, since the spongy material can absorb solutions
cont~;n;ng pharmacologically active ingredients, either singly or as
a mixture with HA or in the form of HA salts or esters, such as e.g.
antibiotics, fungicides, bacteria-fighting compounds, growth
factors, corticosteroids, non-steroid anti-inflammatory agents, as
are e.g. illustrated in European patent applications EPA 0216453 and
EPA 0197718 in th~n ? of Fidia S.p.A., it is possible to obtain a
wide range of highly interesting products to be used in external
dressings, endocavitary and post-operative dressings.
Some examples of the possible applications of the compositions of
the invention are conveyed hereinbelow by way of indication, not of
limitation.
- A product capable of releasing HA and an antibiotic at the site of
treatment can be used, e.g., for dressing infected wounds,
cutaneous ulcers and surgical wounds and for treating external
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otitides, bacterial vaginites, etc.
- A combined release of HA and a fungicide is greatly advantageous
in the treatment of skin mycoses in general and of external
acoustic duct mycoses in particular, an adequate ad hoc local
treatment being possible.
- A combined release of HA and a cort;icosteroid is greatly
advantagesous in the treatment of eczematous dermatitises and of
all dermatologic pathologies favourably aff'ected by local treatment
with corticosteroids. A particular application concerns the
eczematous dermatitises of the external acoustic duct.
- A combined release of HA and growth factors finds application in
plastic and reconstructive surgical practices, whenever cellular
growth and superficial and deep tissues reconstruction are to be
favoured and enhanced.
